tesefrazao esteva

UNIVERSIDADE DE TRÁS-OS-MONTES E ALTO DOURO
Valorisation of Cistus ladanifer L.: From production to evaluation of

raw materials
PhD Thesis
- Agricultural Production Chains - From Fork to Farm -
DAVID PASSOS MORGADO FRANCO FRAZÃO
Supervisors
Professor Doctor Amélia Maria Lopes Dias da Silva (UTAD)

Professor Doctor Fernanda Maria Grácio Delgado Ferreira de Sousa (IPCB)
Professor Doctor José Carlos Dias Duarte Gonçalves (IPCB)
Vila Real, 2022
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UNIVERSIDADE DE TRÁS-OS-MONTES E ALTO DOURO
Valorisation of Cistus ladanifer L.: From production to evaluation of

raw materials
PhD Thesis
- Agricultural Production Chains - From Fork to Farm -
DAVID PASSOS MORGADO FRANCO FRAZÃO
Supervisors:

Professor Doctor Fernanda Maria Grácio Delgado Ferreira de Sousa (IPCB)
Professor Doctor José Carlos Dias Duarte Gonçalves (IPCB)
Jury members
Professor Doctor José Luis Teixeira de Abreu de Medeiros Mourão (UTAD)
Professor Doctor Ana Paula Coelho Duarte (UBI)
Professor Doctor Carlos Manuel Freire Cavaleiro (UC)
Professor Doctor Maria Margarida Branco de Brito Tavares Tomé (ULisboa)
Professor Doctor Alberto Carlos Pires Dias (UMinho)
Professor Doctor Alfredo Augusto de Carvalho Aires (UTAD)
Vila Real, 2022
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This work was produced as original thesis for the degree of
Doctor in AgriChains – Agricultural Production Chains: From Fork
to Farm at the University of Trás-os-Montes and Alto Douro in
accordance with Decree-Law No. 74/2006, of March 24, as amended
by Decree-Law No. 107/2008, of June 25, and Decree-Law No.
230/2009 of 14 September.
The doctrines presented in this PhD thesis are from the exclusive
responsibility of the author.
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The funding was provided by national funds through FCT-Portuguese Foundation for
Science and Technology (PD/BD/135332/2017), under the Doctoral Program
“Agricultural Production Chains – from fork to farm” (PD/00122/2012).
Additionally, this Doctoral work was partially funded by CENTRO-01-0247-FEDER-

033815 under the Project InovEP - “Inovação com extratos de plantas: na senda de
produtos farmacêuticos disruptivos e de base tecnológica”, and by CENTRO-01-0145-
FEDER-000020 under the project CULTIVAR,
The research developed within this Doctoral work was part of the activities of the Centre
for the Research and Technology of Agro-Environmental and Biological Sciences
(CITAB, UIDB/04033/2020), of the Plant Biotechnology Centre of Beira Interior
(CBPBI), and of the Polytechnic Institute of Castelo Branco (ESA-IPCB)
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Acknowledgements
I would like to present my acknowledgements to the Agrichains doctoral program, to
University of Trás-os-Montes and Alto Douro (UTAD, Vila Real, Portugal), to Centre for
the Research and Technology of Agro-Environmental and Biological Sciences (CITAB,
Vila Real Portugal), to Plant Biotechnology Centre of Beira Interior (CBPBI, Castelo
Branco, Portugal), to the Agrarian School of Polytechnic Institute of Castelo Branco
(ESA-IPCB, Castelo Branco, Portugal), to Sociedade Agrícola de Couto de Penha Garcia
(Idanha-a-Nova, Portugal), to University of Minho (UM, Braga, Portugal), to Karlsruhe
Institute of Technology (KIT, Karlsruhe, Germany) for providing the academic
conditions, infrastructures and means to carry out the research work of this doctoral thesis.
I am truly grateful to all my supervisors, Prof. Doctor Amélia Silva, Prof. Doctor
Fernanda Delgado, Prof. Doctor José Carlos Gonçalves for the promptly given
supervision, advice, and resources.
I am grateful to Prof. Doctor Fátima Peres, Prof. Doctor Carmo Horta, Prof. Doctor
Margarida Ataíde, Prof. Doctor Conceição Mesquita, Prof. Doctor Teresa Marta Lupi,
Prof. Doctor Cristina Pintado (ESA-IPCB) for the interest in my work and in my person
and for providing high quality scientific advice, help, and means to perform the research
work. I am equally grateful to Engª Graça Diogo, Engª Helena Silva, Engª Conceição
Vitorino, Engª Manuela Goulão, Engª Telma Tavares, Engª Cecília Gouveia, and Engª
Fátima Palma for all the interest and assistance in my work and progress.
I am grateful to Prof Doctor Mirko Bunzel, Doctor Judith Keller, and Doctor Jan L. Steck
for willingly accepted to help me in their Institution (KIT), despite the interruption.
I am grateful to Prof. Doctor Teresa Sosa Dias (CITAEX, Badajoz, Spain) for welcoming
me and for teaching me the technique to fractionate the resin and for keeping contact,
help and willingness to collaborate.
I am grateful to my PhD colleague Rita Martins for the friendship, interest and help during
these years, to my colleague Celina Barroca for the friendship, interest, help in field work
and for her botanical knowledge, and to Carlos Martins Gomes for the friendship, interest,
and help in the laboratory and interpreting results of cell viability, anti-inflammatory, and
enzymatic inhibition assays. A warm thanks to Madalena d’Oliveira and Catarina Mateus
who during their internship helped me in the laboratory.
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I thank to all my friends who helped me at any way during these four years of intense
work, but namely those who helped in the field work: Adriana Gouveia, Ana Mangana,
Raquel Almeida, Dona Neusa, Dona Belarmina, David Flores, and João Filipe Santos.
I thank Débora for all that she means to me and for the expertise help in the laboratory
and GC-MS (her thing).
Finally, I thank my parents, Margarida and Francisco, my sister Laura, and my brother
Rodrigo for always being there to support me at any way.
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Abstract
Cistus ladanifer L. (Cistaceae) is a widespread underexplored resource, occupying, as
dense rockrose shrublands, extensive areas in Iberian Peninsula. Because of its
abundance, Cistus ladanifer cultivation was never reported within its endemic area but it
may be relevant for production, phytoremediation, soil cover and recuperation,
ornamental use, and research purposes. To reimburse natural shrubland management
practices and cultivation, valorisation of its products, as raw materials for several
industries, and development of a sustainable value chain is a demand. Currently, only
essential oil and labdanum resin are valued at some extent, for their aromatic properties
in the perfumery and fragrance sectors. Recently, other extracts and biomass have been
proposed to be valued for several purposes but there is a gap of scientific works
addressing the potential use of labdanum resin for cosmetic/medicinal purposes and of
seeds for human consumption despite their traditional use. To contribute for an integrative
exploitation of C. ladanifer and a circular economy, this work intended to experiment a
non-destructive harvest as management practice for a natural shrubland to reduce fuel
load, to experiment cultivation with the same management practice, to use in vitro tissue
culture for clonal propagation of C. ladanifer and secondary metabolite production, to
assess the nutritional value of C. ladanifer seeds, to evaluate the labdanum resin
extraction processes, and to confirm the potential of labdanum resin as a cosmetic and
medicinal ingredient. Natural and cultivated shrublands could be properly managed, by
harvesting during summer, using a cut at 0.5 m height, inhibiting the production of fire
fuel wood biomass, and yielding a photosynthetic biomass yearly production of around
1500 kg/ha (natural shrubland) and 5500 kg/ha (cultivated shrubland) and resin yearly
production of around 150 kg/ha (natural shrubland) and 550 kg/ha (cultivated shrubland)
if harvest is done later in the Summer. Flowering, capsules, and seeds production needed
a two-year growth to be significant and reach production of around 270 kg/ha of capsules
and 80 kg/ha of seeds in both natural and cultivated shrublands. Internodal stem segments
and basal leaf segments from C. ladanifer showed to be suitable explants for in vitro shoot
regeneration (38% using stem explants and 6-benzylaminopurine, BAP) and induction of
rhizogenic calli (100% using both explants and 2,4-dichlorophenoxyacetic acid, 2,4-D,
and BAP) or calli with potential for shoot regeneration (100% using stem explants and
BAP and 1-naphthaleneacetic acid, NAA) that may be used for in vitro production of
secondary metabolites. Seeds from C. ladanifer showed to be a source of protein (16.2
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%), calcium (238 mg/100g), and zinc (3.22 mg/100g), and a high source of fibre (20.9%),
unsaturated fatty acids (74%, fat at 13%), phosphorous (452 mg/100g), iron (5.18
mg/100g) and magnesium (232 mg/100g). To extract labdanum resin, the Andalusian
process (2-11% weight dry resin/fresh plant) was more efficient than the Zamorean
process (0-2% weight dry resin/fresh plant) but both produce an effluent with a high
content of phenolic compounds (1-12 gGAE/L) worth to be valorised. Both resins showed
an absolute extraction yield of around 70 %. Andalusian resin absolute was mainly
composed by labdane-type diterpenoids and methylated flavonoid aglycones whereas
Zamorean resin absolute was mainly composed by phenylpropanoids, fatty acids and
diterpenoids. Andalusian resin absolute showed UV protection potential (5 Sun
Protection Factor) and anti-inflammatory (95% inhibition of NO production from LPS-
stimulated RAW 264.7 cells, at 15 µg/mL) activities, mostly associated to flavonoids,
relevant for its use in cosmetic, dermocosmetics or topical medicines however, weak
elastase inhibition and antioxidant (DPPH, FRAP, and ABTS) activities and low
spectrum antimicrobial (only for Staphylococcus aureus ATCC-25923, MIC 1.2 mg/mL)
activity were observed. Andalusian absolute showed 70% inhibition of
acetylcholinesterase at 0.5 mg/mL, diterpenoid fraction showed 30% inhibition of α-
amylase at 0.5 mg/mL, and Andalusian absolute showed cytotoxic activity (IC50, after
24h exposure, 77 µg/mL and 53 µg/mL for HepG2 e Caco-2 cells). These results are
relevant for valorisation as medicinal ingredient or source of pharmaceuticals. Cistus
ladanifer shrublands may be properly managed to yield valuable raw materials/products
such as labdanum resin (e.g., for cosmetic and pharmaceutical industries), phenolic rich
extracts (e.g., as food and feed additives), seeds (for the food industry), photosynthetic
biomass (e.g., for animal feed purposes), and some capsules remains and wood biomass
(e.g., as energy source), while decreasing fire load and risk and maintaining soil cover
and recover.
Keywords
Cistaceae; Cosmetic Labdanum; Medicinal Labdanum; Rockrose edible seeds; Rockrose

shrubland management; Rockrose tissue culture.
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Resumo
A esteva, Cistus ladanifer L. (Cistaceae), é um recurso subvalorizado largamente
distribuído na Península Ibérica. Devido à sua abundância, a esteva nunca foi cultivada
na sua área de distribuição endémica, mas poderá ser relevante em termos de produção,
fito-remediação, ocupação e recuperação de solos, uso ornamental, e investigação. A
valorização dos produtos de esteva e a criação de uma cadeia de valor sustentável são
necessárias para compensar a gestão dos seus matos naturais ou do seu cultivo.
Atualmente, só o óleo essencial e a resina lábdano são valorizados nos setores das
fragrâncias e perfumaria, mas, outros extratos e a biomassa de esteva têm sido estudados
para serem valorizados. Embora a resina e as sementes de esteva, tradicionalmente,
tenham sido usadas medicinalmente e como alimento, respetivamente, não existem
estudos que abordem essa valorização. Este trabalho teve como objetivos: testar uma
prática de gestão do esteval natural de modo a reduzir a biomassa combustível; testar o
cultivo da esteva aplicando a mesma prática de gestão; usar técnicas de cultura de tecidos
vegetais in vitro para a propagação clonal de esteva e produção de metabolitos; avaliar o
valor nutricional das sementes e avaliar o potencial do lábdano como ingrediente
cosmético e medicinal, de modo a contribuir para uma valorização integrada da esteva e
contribuir para uma economia circular. O corte a 0,5 m efetuado no verão, no esteval
silvestre e no cultivado, mostrou ser adequado para a gestão destes dois tipos de campos
de esteva, inibindo a acumulação de biomassa lenhosa combustível e rendendo uma
produção anual de biomassa verde fotossintética de 1500 kg/ha (esteval silvestre) e de
5500 kg/ha (esteval cultivado) e uma produção anual de lábdano, para uma colheita no
final do verão, de 150 kg/ha (esteval silvestre) e 550 kg/ha (esteval cultivado). Para uma
produção significativa de cápsulas e sementes foi necessário um crescimento por dois
anos consecutivos tendo, os dois estevais, atingido uma produção de 270 kg/ha de
cápsulas e 80 kg/ha de sementes. Por técnicas de cultura in vitro, a partir de segmentos
caulinares internodais e segmentos foliares foi possível induzir a regeneração de rebentos
(38%: explantes caulinares e 6-benzilaminopurina, BAP), a formação de callus
rizogénicos (100%: dois explantes e ácido diclorofenoxiacético, 2,4-D, e BAP) ou a
formação de callus com potencialidade para a regeneração de rebentos (100%: explantes
caulinares e BAP e ácido 1-naftalenoacético) que poderão ser usados para a produção de
metabolitos in vitro. As sementes de esteva mostraram ser uma fonte de proteína (16,2
%), cálcio (238 mg/100g) e zinco (3,22 mg/100g) e uma alta fonte de fibra (20,9%),
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ácidos gordos insaturados (74%, gordura: 13%), fósforo (452 mg/100g), ferro (5,18
mg/100g) e magnésio (232 mg/100g). A extração de lábdano pelo processo Andaluz (2-
11% peso resina seca/material fresco) foi mais eficiente que pelo processo Zamoreano (0-
2% peso resina seca/material fresco), ambos produzindo um efluente aquoso com grande
quantidade de fenóis (1-12 gGAE/L). As duas resinas renderam cerca de 70% do seu peso
em extrato absoluto. O absoluto de lábdano obtido pelo processo Andaluz mostrou ser,
principalmente, constituído por diterpenoides e flavonoides enquanto o absoluto de
lábdano obtido pelo método Zamoreano mostrou ser constituído, principalmente, por
fenilpropanoides, ácidos gordos e diterpenoides. O absoluto Andaluz mostrou atividades
de proteção UV (Fator de proteção solar 5) e anti-inflamatória (95% de inibição de
produção de NO em células RAW 264.7, a 15 µg/mL), devido principalmente aos
flavonoides, o que é relevante para o seu uso como ingrediente em produtos cosméticos,
dermocosméticos e medicamentos tópicos, mas mostrou fraca inibição da elastase e
atividades antioxidante (DPPH, FRAP, and ABTS), e atividade antimicrobiana
unicamente para Staphylococcus aureus ATCC 25923 (MIC 1,2 mg/mL). Este absoluto
revelou 70% de inibição da acetilcolinesterase a 0,5 mg/mL, a fração de diterpenos
revelou 30% de inibição da α-amilase a 0,5 mg/mL, e o absoluto revelou atividade
citotóxica (IC50 (24h de exposição): 77 µg/mL e 53 µg/mL em células HepG2 e CACO2)
o que é relevante para sua valorização como ingrediente medicinal e fonte de fármacos.
Resumidamente, os estevais podem ser geridos de modo a obter produtos de valor
acrescentado como lábdano (p.e., ingrediente cosmético e medicinal), extratos ricos em
compostos fenólicos (p.e., aditivos alimentares), sementes (como alimento humano),
biomassa verde (p.e., forragem) e restos de cápsulas e biomassa lenhosa (p.e., fonte de
energia), ao mesmo tempo que se reduz a produção de biomassa combustível e se mantém
a cobertura e recuperação de solos.
Palavras-chave
Cistaceae; Cultura de esteva in vitro; Lábdano cosmético; Lábdano medicinal; Gestão do

esteval; Sementes comestíveis de esteva.
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Index
Acknowledgements ......................................................................................................... ix
Abstract ............................................................................................................................ xi
Resumo .......................................................................................................................... xiii
Index ............................................................................................................................... xv
Figures Index ................................................................................................................. xxi
Tables Index ................................................................................................................. xxv
List of abbreviations .................................................................................................... xxix
Chapter 1 .......................................................................................................................... 1
The rockrose Cistus ladanifer L.: a neglected Mediterranean natural and valuable
resource. ........................................................................................................................ 1
1.1. Cistus ladanifer L. .......................................................................................... 1
1.1.1. Taxonomy ............................................................................................... 1
1.1.2. Distribution ............................................................................................. 5
1.1.3. Morphology and phenology .................................................................... 6
1.1.4. Ecology ................................................................................................... 9
1.1.4.1. Reproduction ...................................................................................... 9
1.1.4.2. Nutritive demands ............................................................................ 11
1.1.4.3. Shrublands ........................................................................................ 12
1.1.4.4. Labdanum resin exudate .................................................................. 14
1.1.4.5. Pests and diseases............................................................................. 16
1.2. Cistus ladanifer L. raw materials (products). ............................................... 17
1.2.1. Essential oil ........................................................................................... 17
1.2.2. Labdanum resin ..................................................................................... 19
1.2.2.1. Extraction ......................................................................................... 19
1.2.2.2. Chemical characterisation ................................................................ 22
1.2.3. Polar extracts ......................................................................................... 27
1.2.4. Biomass ................................................................................................. 29

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1.3. Integrative exploitation of Cistus ladanifer L. ............................................. 32
1.4. Research objectives ...................................................................................... 43
Chapter 2 ........................................................................................................................ 45
Rockrose Production ................................................................................................... 45
Subchapter 2.1 ................................................................................................................ 45

Rockrose land management: contribution of periodic harvesting to increase value and
control Cistus ladanifer L. shrublands. ....................................................................... 45
2.1.1. Introduction .............................................................................................. 47
2.1.2. Material and methods ............................................................................... 50
2.1.2.1. Characterisation of the study area ..................................................... 50
2.1.2.2. Experimental plots delimitation and evaluation ................................ 51
2.1.2.3. Soil analysis....................................................................................... 51
2.1.2.4. Soil seed bank evaluation .................................................................. 51
2.1.2.5. Experimental design .......................................................................... 52
2.1.2.6. Phenological evaluation .................................................................... 53
2.1.2.7. Meteorological data ........................................................................... 53
2.1.2.8. Harvest evaluation ............................................................................. 53
2.1.2.9. Labdanum resin extraction ................................................................ 54
2.1.2.10. Statistical analysis ............................................................................. 54
2.1.3. Results and discussion .............................................................................. 55
2.1.3.1. Initial characterisation ....................................................................... 55
2.1.3.2. Phenological evaluation .................................................................... 56
2.1.3.3. Production ......................................................................................... 62
2.1.4. Conclusion ................................................................................................ 68
Subchapter 2.2 ................................................................................................................ 69

Cistus ladanifer L. cultivation: first results. ............................................................... 69
2.2.1. Introduction .............................................................................................. 71
2.2.2. Material and Methods ............................................................................... 72
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2.2.3. Results and discussion .............................................................................. 73
2.2.4. Conclusion ................................................................................................ 76
Chapter 3 ........................................................................................................................ 77
Rockrose tissue culture: potential of leaf and stem explants from Cistus ladanifer L.
.................................................................................................................................... 77
3.1. Introduction .................................................................................................. 79
3.2. Material and methods ................................................................................... 80
3.2.1. Chemicals.............................................................................................. 80
3.2.2. Leaf and stem explants establishment and callogenesis ....................... 80
3.2.2.1. Callus index (CI) .............................................................................. 81
3.2.3. Callus propagation and evaluation ....................................................... 81
3.2.3.1. Callus subculture mass propagation ................................................ 81
3.2.3.2. Dry weight (dw) ............................................................................... 82
3.2.3.3. Histologic evaluation ....................................................................... 82
3.2.3.4. Shoot organogenesis ........................................................................ 83
3.2.4. Statistical analysis ................................................................................. 83
3.3. Results .......................................................................................................... 83
3.3.1. Establishment of leaf and internodal stem explants .............................. 83
3.3.2. Callus propagation and evaluation ....................................................... 87
3.4. Conclusion.................................................................................................... 90
Chapter 4 ........................................................................................................................ 91
Cistus ladanifer L. seeds: from ancient snack to novel and sustainable food
ingredient. ................................................................................................................... 91
4.1. Introduction .................................................................................................. 93
4.2. Materials and Methods ................................................................................. 94
4.2.1. Chemicals.............................................................................................. 94
4.2.2. Capsule collection and processing ........................................................ 94
4.2.3. Moisture content ................................................................................... 95
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4.2.4. Ash content ........................................................................................... 95
4.2.5. Protein Content ..................................................................................... 95
4.2.6. Lipid content and fatty acid composition ............................................. 95
4.2.7. Crude Fibre content .............................................................................. 96
4.2.8. Carbohydrate content and energetic value ............................................ 96
4.2.9. Mineral analysis .................................................................................... 96
4.2.10. Statistical Analysis ............................................................................ 96
4.3. Results and discussion.................................................................................. 97
4.4. Conclusion.................................................................................................. 103
Chapter 5 ...................................................................................................................... 105

Labdanum resin from Cistus ladanifer L.: evaluation of residual water vs. extraction
yield. ......................................................................................................................... 105
5.1. Introduction ................................................................................................ 107
5.2. Materials and Methods ............................................................................... 108
5.2.1. Chemicals............................................................................................ 108
5.2.2. Plant material and labdanum extraction (reference method) .............. 108
5.2.3. Experimental design of method variations ......................................... 109
5.2.4. Residual water analytical methods ..................................................... 109
5.2.5. Statistical analysis ............................................................................... 111
5.3. Results and Discussion ............................................................................... 111
5.4. Conclusion.................................................................................................. 117
Chapter 6 ...................................................................................................................... 119

Labdanum resin valorisation ..................................................................................... 119
Subchapter 6.1 .............................................................................................................. 119

Labdanum resin from Cistus ladanifer L.: a natural and sustainable ingredient for skin
care cosmetics with relevant cosmeceutical bioactivities. ........................................ 119
6.1.1. Introduction ............................................................................................ 121
6.1.2. Material and Methods ............................................................................. 124
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6.1.2.1. Chemicals ........................................................................................ 124
6.1.2.2. Labdanum absolute ......................................................................... 124
6.1.2.3. Diterpenoid and flavonoid fractions ................................................ 125
6.1.2.4. Chemical characterisation (UPLC-DAD-ESI-MS) ......................... 125
6.1.2.5. UV radiation absorption .................................................................. 126
6.1.2.6. Antioxidant activity ......................................................................... 126
6.1.2.7. Elastase inhibition activity .............................................................. 127
6.1.2.8. Anti-inflammatory activity.............................................................. 128
6.1.2.9. Antimicrobial activity ..................................................................... 129
6.1.2.10. Statistical analysis ........................................................................... 129
6.1.3. Results and discussion ............................................................................ 130
6.1.3.1. Extraction and purification yields ................................................... 130
6.1.3.2. Chemical profile .............................................................................. 131
6.1.3.3. UV radiation protection activity ...................................................... 136
6.1.3.4. Antioxidant activity ......................................................................... 137
6.1.3.5. Elastase inhibition activity .............................................................. 138
6.1.3.6. Anti-inflammatory activity.............................................................. 138
6.1.3.7. Antimicrobial activity ..................................................................... 140
6.1.4. Conclusion .............................................................................................. 143
Subchapter 6.2 .............................................................................................................. 145

Labdanum resin from Cistus ladanifer L. as a source of compounds with medicinal
related bioactivities. .................................................................................................. 145
6.2.1. Introduction ............................................................................................ 147
6.2.2. Materials and methods ............................................................................ 149
6.2.2.1. Chemicals ........................................................................................ 149
6.2.2.2. Labdanum resin extraction and purification.................................... 150
6.2.2.3. Chemical characterisation (GC-EI-MS). ......................................... 151
6.2.2.4. α-Amylase, α-glucosidase, and AChE inhibition ............................ 151

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6.2.2.5. CACO-2 and HepG2 cytotoxicity ................................................... 152
6.2.2.6. Statistical Analysis .......................................................................... 153
6.2.3. Results and discussion ............................................................................ 154
6.2.3.1. Extraction and purification yield and chemical profile ................... 154
6.2.3.2. Enzymatic inhibition activities ........................................................ 160
6.2.3.3. Cytotoxic activity ............................................................................ 163
6.2.4. Conclusion .............................................................................................. 167
Chapter 7 ...................................................................................................................... 169

7.1. General discussion...................................................................................... 169
7.2. General conclusion ..................................................................................... 175
7.3. Future perspectives ..................................................................................... 179
List of references .......................................................................................................... 183
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Figures Index
Chapter 1.
Figure 1.1: “Distribution map and number of Cistus species.”.. ................................................ 1
Figure 1.2: “Distribution of C. ladanifer in its native range”. ................................................... 6
Figure 1.3: Cistus ladanifer morphology. .................................................................................. 8
Figure 1.4: “Phenology of Cistus ladanifer”. ............................................................................. 8
Figure 1.5: Ecological functions of the labdanum resin........................................................... 14
Figure 1.6: Labdane diterpene skeleton. .................................................................................. 25
Figure 1.7: Kaempferol flavonol and apigenin flavone skeletons.. ......................................... 26
Figure 1.8: Schematic representation of C. ladanifer products and economic sector of possible
valorisation pathways. .............................................................................................................. 33
Chapter 2.
Subchapter 2.1.
Figure 2.1.1: Phenological evaluation of C. ladanifer throughout the natural shrubland

management study period......................................................................................................... 60
Figure 2.1.2: Cistus ladanifer vegetative growth, mean temperature, and mean daily rainfall
(line graphics) at each observation period throughout the study. ............................................ 61
Subchapter 2.2.
Figure 2.2.1: Cistus ladanifer cultivation................................................................................. 73
Chapter 3.
Figure 3.1: Morphological evaluation of the leaf and shoot establishment and callus culture.
.................................................................................................................................................. 87
Figure 3.2: Microscopic photographs of callus histological slides stained with toluidine blue.
................................................................................................................................................ 889
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Chapter 4.
Figure 4.1: Principal Component Analysis and projection of the nutritional components of C.
ladanifer and other nuts and seeds. ......................................................................................................... 103
Chapter 5.
Figure 5.1: Labdanum resin and labdanum methanol absolute extraction yields in relation to
plant material weight, for the various extraction conditions. ................................................. 113
Figure 5.2: Sulphate estimated content of the residual waters resulted from labdanum resin
extraction with different extraction conditions. ..................................................................... 116
Figure 5.3: Sodium estimated content of the residual waters resulted from labdanum resin
extraction with different extraction conditions. ..................................................................... 116
Figure 5.4: Total phenol content of the residual waters resulted from labdanum resin extraction
with different extraction conditions. ...................................................................................... 117
Figure 5.5: pH of the residual waters resulted from labdanum resin extraction with different
extraction conditions. ............................................................................................................. 117
Chapter 6.
Subchapter 6.1.
Figure 6.1.1: DAD and MS chromatograms obtained in the UPLC-DAD-ESI-MS analysis of

labdanum absolute. ................................................................................................................. 133

diterpenoid fraction from labdanum absolute. ....................................................................... 133

“flavonoid” fraction from labdanum absolute. ....................................................................... 133
Figure 6.1.4: UV absorption spectral scan of labdanum absolute, flavonoid fraction, and
diterpene fraction related to a concentration of 100 µg/mL. .................................................. 136
Figure 6.1.5 RAW 264.7 cells viability, as percentage in relation to the control, of labdanum
absolute and fractions at concentrations between 6.25 and 100 µg/mL. ............................... 140
Figure 6.1.6: Anti-inflammatory activity of labdanum absolute and fractions. ..................... 140
xxii
Subchapter 6.2.
Figure 6.2.1: GC-EI-MS chromatograms of the TMCS + BSTFA derivatized absolute of the
labdanum resin obtained by the Andalusian process. ............................................................ 155
Figure 6.2.2: GC-EI-MS chromatograms of the TMCS + BSTFA diterpenoid fraction of the
Figure 6.2.3: GC-EI-MS chromatograms of the TMCS + BSTFA flavonoid fraction of the
Figure 6.2.4: GC-EI-MS chromatograms of the TMCS + BSTFA derivatized absolute of the
labdanum resin obtained by the Zamorean process. .............................................................. 157
Figure 6.2.5: GC-EI-MS chromatograms of the non-derivatized diterpenoid fraction of

labdanum resin obtained by the Andalusian process. .......................................................... 1577
Figure 6.2.6: Effect of labdanum resin extracted by the Andalusian process, its purified
flavonoid and diterpenoid extracts, and labdanum resin extracted by the Zamorean process on
Caco-2 and HepG2 cells viability after 24 and 48 h incubation. .......................................... 164
Figure 6.2.7: Four-parameter symmetric sigmoidal curves of Caco-2 cell viability versus Adl
labdanum absolute, and its flavonoid and diterpenoid fractions, concentration at 24h and 48h
incubation. .............................................................................................................................. 166
Figure 6.2.8: Four-parameter symmetric sigmoidal curves of HepG2 cell viability versus Adl
labdanum absolute, and its flavonoid and diterpenoid fractions, concentration at 24h and 48h
incubation. .............................................................................................................................. 166
xxiii
xxiv
Tables Index
Chapter 1.
Table 1.1: Recognized Cistus species and their division by subgenera or by phylogenetic
groups based on plastids and/or nuclear DNA sequencing analysis. ............................... 3
Table 1.2: Exudate yield, extracted with CHCl3, of some Cistus species. ....................... 4
Table 1.3: Most representative chemical compounds identified in C. ladanifer essential

oil, reported in more than 50% of 22 essential oils reported in literature. ..................... 18
Table 1.4: Most representative chemical compounds identified in C. ladanifer labdanum

resin, reported more than three times within the 31 references. ..................................... 23
Table 1.5: Most representative phenolic compounds identified in C. ladanifer polar

extracts, reported more than three times within the 7 references. .................................. 27
Table 1.6: Total aboveground biomass and biomass fractions productivity of C. ladanifer
shrublands according to age, reported in literature......................................................... 29
Table 1.7: Cistus ladanifer biomass and biomass fractions general chemical composition
data, available in literature.............................................................................................. 30
Table 1.8: Resume of scientific reports evaluating biological activities of polar extracts
from C. ladanifer, from 2010 to 2021. ........................................................................... 37
Chapter 2.
Subchapter 2.1.
Table 2.1.1: Main physiscal and chemical properties of the natural C. ladanifer shrubland
soil.. ................................................................................................................................ 50
Table 2.1.2: Vegetation parameters of the selected C. ladanifer shrubland. .................. 55
Table 2.1.3: Cistus ladanifer seed bank characetrisation of the natural C. ladanifer
shrubland soil, seed density and seed germination parameters. ..................................... 56
Table 2.1.4: Spearman’s rank order correlation between C. ladanifer mean vegetative
growth and mean temperature and mean daily rainfall throughout the study periods. .. 62
xxv
Table 2.1.5: Biomass production and percentage in relation to total biomass of
experimental plots in a natural C. ladanifer shrubland at the beginning of study (2019)
and at the end of study (2021) subjected to different harvest modalities. ...................... 63
Table 2.1.6: Labdanum resin yield at the three years and seasons of C. ladanifer harvest.
........................................................................................................................................ 66
Subchapter 2.2.
Table 2.2.1: Biomass production and percentage in relation to total biomass of C.

ladanifer cultivation, for the first two years, upon different harvest periodicities, done at
mid-summer. ................................................................................................................... 75
Chapter 3.
Table 3.1: PGRs supplementation in MS medium used for C. ladanifer leaf and stem
explants establishment. ................................................................................................... 81
Table 3.2: PGRs supplementation in MS medium used for C. ladanifer callus culture.
........................................................................................................................................ 82
Table 3.3: PGRs supplementation in MS medium used for C. ladanifer shoot

organogenesis. ................................................................................................................ 83
Table 3.4: Percentage of contamination, survival, and induction responses observed for
the C. ladanifer leaf and stem explants establishment. .................................................. 84
Table 3.5: Callus Index for C. ladanifer leaf and stem explants establishment, in relation
to survivor explants. ....................................................................................................... 85
Table 3.6: Correlation test between initial mass of transferred C. ladanifer calli and the
percentage of growth of all calli lines, for the 5 subcultures or for the last 3 subcultures.
........................................................................................................................................ 87
Table 3.7: Callus evaluation for the 3 subcultures of C. ladanifer callus proliferation.
........................................................................................................................................ 88
Table 3.8: Percentage of morphological evaluations for the three subcultures of C.

ladanifer callus proliferation. ......................................................................................... 88
xxvi
Chapter 4.
Table 4.1: Nutritional composition and energetic value of C. ladanifer seeds flour from
early, middle, and late summer collection during two consecutive years. ..................... 90
Table 4.2: Fatty acids composition of C. ladanifer seeds flour from early, middle, and late
summer collection during two consecutive years. ........................................................ 100
Table 4.3: Mineral composition of C. ladanifer seeds flour from early, middle, and late
summer collection during two consecutive years. ........................................................ 102
Chapter 5.
Table 5.1: Labdanum Absolute yield, in relation to resin, for the various combinations of
extraction conditions. ................................................................................................... 113
Table 5.2: Water quality parameters of the residual water from the reference method used
to extract labdanum resin, assessed by analytical methods or by estimation considering
the quantity of reagents used. ....................................................................................... 114
Chapter 6.
Subchapter 6.1.
Table 6.1.1: Labdanum resin yield in relation to plant material, and labdanum absolute
and column chromatography fractions yields in relation to labdanum resin. ............... 130
Table 6.1.2: UPLC-DAD-ESI-MS peaks in the chromatograms of labdanum absolute and

fractions. ....................................................................................................................... 134
Table 6.1.3: Spectrophotometric Sun Protection Factor, and UVB and UVA total
absorbance of 100 µg/mL solutions of labdanum absolutes and fractions. .................. 136
Table 6.1.4: Folin-Ciocalteu test, DPPH scavenging activity, FRAP, and ABTS free
radical scavenging activity of labdanum absolutes and fractions................................. 138
Table 6.1.5: Inhibition of elastase activity by the labdanum absolute and fractions at
concentrations of 1 and 0.5 mg/mL. ............................................................................. 138
Table 6.1.6: Antimicrobial activity: Minimum Inhibitory Concentration (MIC) and

Minimum Microbicidal Concentration (MMC), of labdanum absolute and fractions in
relation to selected microorganisms. ............................................................................ 142
xxvii
Subchapter 6.2.
Table 6.2.1: Dry labdanum resin yield in relation to the initial C. ladanifer plant material
fresh weight and dry labdanum absolute yield in relation to the dry resin weight. ...... 154
Table 6.2.2: GC-EI-MS relevant peaks in the chromatograms of the labdanum absolutes
and fractions. ................................................................................................................ 158
Table 6.2.3: Inhibition of α-amylase activity by the labdanum absolutes and fractions at
concentrations of 1 and 0.5 mg/mL. ............................................................................. 161
Table 6.2.4: Inhibition of α-glucosidase activity by the labdanum absolutes and fractions
at concentrations of 1 and 0.5 mg/mL. ......................................................................... 161
Table 6.2.5: Inhibition of acetylcholinesterase activity by the labdanum absolutes and

fractions at concentrations of 1 and 0.5 mg/mL. .......................................................... 163
Table 6.2.6: IC50 of labdanum absolutes and fractions in relation to HepG2 and Caco-2
cell viability at 24h and 48h incubation........................................................................ 165
Table 6.2.7: R-squared and Hill slope of the best fitted symmetric sigmoidal curves to the
HepG2, and Caco-2 cell viability versus labdanum absolutes, and its Flv and Dit fraction,
concentration data at 24h and 48h incubation. ............................................................. 166
xxviii
List of abbreviations
1Y: one year periodicity (yearly) FID: Flame ionization detector
2Y: two years periodicity (every two Flv: Flavonoid
years)
FRAP: Ferric Reducing Antioxidant
2iP: isopentenyl adenine Power
2,4-D: 2,4-Dichlorophenoxyacetic acid fw: Fresh weight
%Growth: Percentage of growth GAE: Gallic acid equivalents
%TB: Percentage of total biomass GC-MS: Gas chromatography – mass
spectrometer
ABTS: 2,2'-azino-bis(3-
ethylbenzothiazoline-6-sulfonic acid IAA: Indole-3-acetic acid
ACE: Angiotensin-converting enzyme IBA: Indole-3-butyric acid
AChE: Acetylcholinesterase IC50: 50% inhibition concentration
Adl: Andalusian (process) Kin: Kinetin
BAP: 6-Benzylaminopurine LD50: 50% lethal dose
CC50: 50% cytotoxic concentration LPS: Lipopolysaccharide
CC: Column chromatography LS: Late Summer
CI: Callus index MBC: Minimum bactericidal
concentration
CVC: Central vascular cylinder
MGT: Mean germination time
DAD: Diode array detector
MIC: Minimum inhibitory
Dit: Diterpenoid concentration
DMEM: Dulbecco's Modified Eagle MMC: Minimum microbicidal
Medium concentration
DMSO: Dimethyl sulfoxide MS: Middle Summer
DPPH: 2,2-diphenyl-1-picrylhydrazyl MS medium: Murashige and Skoog
dw: Dry weight medium
EC50: 50% effective concentration MTT: 2,5-diphenyl-2H-tetrazolium
bromide
ECHA: European Chemicals Agency
N: neutralization of carbonates
EI: Electron ionization
NAA: 1-Naphthaleneacetic acid
ES: Early Summer
NCI: National Cancer Institute (US)
ESI: Electro-spray ionization
NMR: Nuclear magnetic resonance
Ext: Extract
OM: Organic matter
FBS: Foetal bovine serum
ORAC: Oxygen Radical Absorbance
FGP: Final germination percentage Capacity
xxix
PBS: Phosphate buffer saline
PGPR: Plant growth promoting
rhizobacteria
PGR: Plant growth regulator
ROS: Reactive Oxygen Species
RP: Reducing power
SPF: Sun Protection Factor
s/: no acidification
T50: Time to reach 50% germination
T: temperature
TAA: Total antioxidant activity
TBARS: Thiobarbituric acid reactive
substances
TDZ: Thidiazuron
TE: trolox equivalents
TEAC: Trolox equivalent antioxidant
capacity
TLC: Thin-layer liquid
chromatography
TMS: Trimethylsilyl
UPLC-MS: Ultra-performance liquid
chromatography – mass spectrometer
UV: Ultraviolet radiation
Vis: Visible radiation
w3: omega-3 fatty acid
w6: omega-6 fatty acid
Zam: Zamorean (process)
xxx
Chapter 1
The rockrose Cistus ladanifer L.: a neglected Mediterranean natural

and valuable resource.
1.1. Cistus ladanifer L.
1.1.1. Taxonomy
Cistaceae family comprises 8 genera and 180 species of heliophyte shrubs, subshrubs and
herbs present in open areas and poor soils, distributed in temperate and subtropical
regions of the northern hemisphere. Widely present in the Mediterranean flora, 5 of the 8
genera (Cistus, Fumana, Halimium, Helianthemum, Tuberaria) are native to the region,
while Crocanthemum, Hudsonia and Lechum are present in temperate regions of America
(Guzmán and Vargas 2009).
Cistus genus is thought to be constituted by 23 species of which the highest diversity of

them occurs in the Western Mediterranean (Guzmán and Vargas 2005; Civeyrel et al.
2011) (Figure 1.1).
Figure 1.1: “Distribution map and number of Cistus species. Pie diagrams include proportion of white-
flowered (white) and purple-flowered (grey) species in every country. Notice the highest species diversity
in Western Mediterranean. The Mediterranean region shown in grey”. In Guzmán and Vargas (2005).
Based on morphological characters Cistus species are divided into three subgenera:
Cistus, Leucocistus and Halimioides, as proposed by Demoly and Montserrat (1993)
(Table 1.1). Cistus subgenus is constituted by purple-pink flowered species and the other
two subgenera (Leucocistus and Halimioides) by white flowered species. Halimioides
1
Chapter 1 The rockrose Cistus ladanifer L.: a neglected Mediterranean natural and valuable resource.
species share more morphological features with Halimium species (e.g., leaf shape,
number of sepals and pollen type). Demoly and Montserrat (1993) characterized only
species from Iberian Peninsula (12 Cistus spp.) and only morphological characteristics
such as number of sepals, style/stamens length, number of capsule valves and pollen were
considered for the subgenera division.
Since morphological features do not show to be taxonomic consistent within Cistus genus
and even between species of Cistus and Halimium genera, phylogenetic studies based on
molecular analysis have been performed:
• Phylogenetic analysis of 20 Cistus species based on plastid (trnL-trnF, matK)

and nuclear (ITS) DNA sequencing data (Guzmán and Vargas 2005);
• Phylogenetic analysis of 21 Cistus species and 8 Halimium species based on
plastid (trnL-trnF, trnK-matK, trnS-trnG, rbcL) and nuclear (ncpGS, ITS) DNA
sequencing data (Guzmán et al. 2009);
• Phylogenetic analysis of 47 Cistaceae species based on plastid (rbcL, trnL-trnF)
DNA sequencing data (Guzmán and Vargas 2009);
• Phylogenetic analysis of all available Cistus (23) and Halimium (10) species
based on plastid (trnL-trnF, trnS-trnG) DNA sequencing data (Civeyrel et al.
2011).
Based on these phylogenetic studies, it is evidenced the phylogenetic proximity of Cistus

and Halimium species. In fact, based on morphological characteristics, the separation of
these two genera is not widely accepted. All phylogenetic studies also lead to a simple
division, in Cistus genus, between a pink-flowered group and a white flowered group and,
interestingly, C. parviflorus (pale-pink flower) appears always at the side of white-
flowered species. Finally, when crossed with morphological features, the white-flowered
group (Leucocistus + Halimioides + C. parviflorus) shows to be very heterogenic.
Guzmán et al. (2009) suggest an adaptive radiation of the white-flowered group

(excluding C. clusii and C. munbii) resulting in a rapid ecological/morphological
diversification, in which diverse ecological conditions of Mediterranean habitats may
have played a key role. In addition, successful hybridisations between Cistus species and
even between Cistus and Halimium species shows their wide range of genetic
compatibility and may be a mode of species evolution in the Cistus genus (Guzmán and
Vargas 2005). Self-incompatible reproduction of Cistus species promotes outcrossing
2
which favours inter-individual and inter-specific production of hybrids (Guzmán and

Vargas 2005). It’s worth to note that Cistus and Halimium species share the same
chromosome number (2n = 18) different from all the other genera of Cistaceae (Demoly
and Montserrat 1993; Civeyrel et al. 2011; Guzmán and Vargas 2009)
Table 1.1: Recognized Cistus species and their division by subgenera1 or by phylogenetic groups based on
plastids and/or nuclear DNA sequencing analysis2.
23 Cistus species Subgenera division1 Phylogenetic grouping2
C. albidus
C. creticus
C. heterophillus
C. asper
C. chinamadensis
Pink-Purple flowered
C. ochreatus
Cistus subgenus species
C. palmensis
C. symphytifolius
C. osbekiifolius
C. horrens
C. crispus
C. parviflorus
C. inflatus (C. psilosepalus)
C. ladanifer
C. salviifolius
C. laurifolius
Leucocistus subgenus
C. monspeliensis White and Whitish flowered
C. pouzolzii species
C. sintenisii (C. albanicus)
C. populifolius
C. libanotis
C. clusii Halimioides subgenus
C. munbyi
1
(Demoly and Montserrat 1993)
2
(Guzmán and Vargas 2005; Guzmán et al. 2009; Guzmán and Vargas 2009; Civeyrel et al. 2011).
Separation of Cistus subgenus from Leucocistus and Halimioides subgenera and the
proximity of these later two, is supported by polyphenolic profile analyses of water
extracts from Cistus species showing that Cistus subgenus species are richer in flavonoids
and deprived of ellagitannins and species from the other subgenera are richer in
ellagitannins, specially Leucocistus species, and poor in flavonoids (Barrajón‐Catalán et
al. 2011).
The exudation of a resin, called labdanum, is a common trait within the Cistus genus. In
Table 1.2, labdanum yields are given per Cistus species based on Vogt et al. (1987) and
Gülz et al. (1996) studies. Vogt et al. (1987) identified methylated flavonoid aglycones
in all studied species but at a different profile involving kaempferol, apigenin, quercetin,
myrcetin and luteolin derivatives. The same authors also found that the presence of
coumarins in labdanum resin, scopoletin and ayapin, also differed between Cistus species.
3
Scopoletin was present in labdanum from all species except from C. ladanifer and C.
salviifolius. Ayapin was only present in labdanum resin from C. monspeliensis, C.
laurifolios, C. symphytifolius, and C. psilosepalus. Gülz et al. (1996) observed the
presence of secretory glandular trichomes (glands) and of non-secretory hair trichomes
on the leaf surface of all the Cistus species studied. However, the morphology
combination of such trichomes was also found to be a distinctive character between the
species.
Only 14 species of the 23 recognized (Table 1.1), are included in the Table 1.2. It is well
known that C. creticus exudes resin (Demetzos et al. 1994; Rauwald et al. 2019),
however, no information on this respect was found for the other species.
Table 1.2: Exudate yield (% dw/dw of leaf), extracted with CHCl3, of some Cistus species.
14 Cistus species Vogt et al. (1987) Gulz et al. (1996)
C. albidus 1.6-2.3 2.2
C. symphytifolius 13.4 10.1
C. osbekiifolius 1.4 -
C. crispus 4.0 1.6
C. parviflorus 2.0 1.2
C. inflatus (C. psilosepalus) 4.7 2.0
C. ladanifer 10.8-13.3 11.5-12.5
C. salviifolius 0.5 0.5
C. laurifolius 8.1 13.5
C. monspeliensis 15.5 10.7
C. sintenisii (C. albanicus) 6.9 1.0
C. populifolios 9.0 5.6
C. libanotis 7.3 6.1
C. clusii 10.2-10.8 6.0
Cistus ladanifer belongs to the Leucocistus subgenus, Ladanium section (Demoly and
Montserrat 1993) and to the white-flowered lineage group supported by the phylogenetic
studies. Within this species, three subspecies are recognized: C. ladanifer ladanifer, C.
ladanifer sulcatus (former C. palhinhae Ingram) and C. ladanifer africanus (sometimes
reported as subsp. mauritianus or petiolatus). All above mentioned phylogenetic studies
support this subspecies division. Within C. ladanifer two varieties are reported: albiflorus
and maculatus.
Gülz et al. (1984) analysed essential oils from C. ladanifer (most likely subsp. ladanifer)
and C. ladanifer subsp. sulcatus (referred as C. palhinhae Ingram), identifying mono-,
sesqui- and diterpenes and alkanes. Gülz et al. (1984) observed that the monoterpenes α-
pinene and camphene were the major constituents of C. ladanifer essential oil whereas
4
the major constituents of C. palhinhae essential oil were alkanes (tricosane, heptacosane
and nonacosane).
1.1.2. Distribution
Cistus ladanifer have a western Mediterranean distribution (south of France, Iberian

Peninsula, North Marrocos and Argelia) (Figure 1.2). Subspecies ladanifer typical
shrublands grow in the meridional half of the Iberian Peninsula over siliceous soils and
in the occidental half over shales and granitic soils. Subspecies africanus is distributed
over the north of Africa (Argelia and Marrocos) and in the south of Iberian Peninsula
(Málaga), preferring serpentine and ultrabasic soils. Subspecies sulcatus grows
exclusively in windy and sandy places over limestone rock of the extreme south-west of
Portugal (Demoly and Montserrat 1993).
According to Demoly and Montserrat (1993), subsp. ladanifer has been cultivated and
possibly naturalised outside of its endemic area. In South Africa (South-West, Fynbos)
C. ladanifer is classified as an invasive species, posing a significant threat to local
endemic vegetation (Du Plessis et al. 2018). In Australia, C. ladanifer and C. x purpureus
(C. ladanider x C. creticus) have been recently cultivated to produce essential oils
(EOPAA 2017). Dias et al. (2019) cite some references regarding the presence of C.
ladanifer in some California counties, USA. Cistus ladanifer is reported to had been
cultivated in China for research purposes (Li et al. 1991).
In 2005, C. ladanifer dense shrublands, characterized by a widely open tree layer of

essentially cork-holm oaks and a very dense shrub/herb layer of C. ladanifer associated
with Lavandula spp. and other shrub species, was the 5th forest typology occupying more
land (249.382 ha), representing 7.5% of the Portugal mainland forest area and
concentrated mainly in the south (Godinho-Ferreira et al. 2005). Furthermore, although
not dominant, this species was present on many other forest typologies, such as cork oak,
pine, and eucalyptus forests, which together accounted for 79.9% of the Portugal
mainland forest area which in turn occupied approximately 37% of the mainland.
A more recent estimation reports that C. ladanifer occupies 2.450.857 ha in Spain

mainland widely associated to Quercus spp. and Pinus spp. forests, 460.088 ha of those
are occupied by C. ladanifer dominated shrublands often accompanied by Genista
hirsuta, Thymus zygis and Astragalus (Montero et al. 2020).
5
Most studies regarding C. ladanifer are not clear about the subspecies in question.
However, it may be inferred that most of them study subsp. ladanifer because of the
geographic area of study. Therefore, from now on, C. ladanifer, when not specified the
subspecies, refers to its subspecies ladanifer.
Figure 1.2: “Distribution of C. ladanifer in its native range, using georeferenced data from Global
Biodiversity Information Facility—Cistus ladanifer L. in GBIF Secretariat (2017). GBIF Backbone
Taxonomy Checklist Dataset https://doi.org/10.15468/39omei accessed via GBIF.org on 2017-11-06. Inset
presents the geographic context of C. ladanifer distribution area in southwestern Europe. Red square in the
inset marks the area zoomed in the detail map” in Frazão et al. (2018).
1.1.3. Morphology and phenology
Cistus ladanifer (Figure 1.3) is an evergreen erect shrub (50-200 cm), however sometimes
found in creeping form, with a hard-lignified wood and a brown-reddish bark. Leaves and
young stems are usually impregnated with an aromatic resin, called labdanum. Leaves are
sessile or shortly petiolate, welded together at the base, generally lanceolate, coriaceous,
and somewhat revolute margin. Upper leaf surface is glabrous and dark green with 1-3
nerves. Terminal solitary flowers are of 5-8 cm diameter with a short peduncle (5-16 mm),
bracts and 3 sepals. The 5 petals (30-55 mm) are white with a small yellowish spot at the
base, and sometimes an overlapping purple blot (var. maculatus). Stamens vary in size
and are longer than the pistil which in turn is characterized by a sessile but large stigma.
6
Seed capsules are globular loculicidal with 6-12 locules and valves and are densely
covered by peltate hairs. Seeds (1 mm) have a globular-polyhedric format. (Demoly and
Montserrat 1993).
Pollen is medium sized (2.8-4.2 µm) with a prolate spheroidal shape and a (large)
reticulate exine surface (Civeyrel et al. 2011).
Non-secretory trichomes (hairs) are found in a very dense arrangement and unicellular
stellate form (6-20 branches) on the lower leaf surface (Gülz et al. 1996). Single hairs are
also found in stems (Demoly and Montserrat 1993). Secretory trichomes (glands),
multicellular structures of 20-30 µm short stalked small heads are found in leaves and
photosynthetic stems (Gülz et al. 1996) and are responsible for labdanum secretion.
Subspecies africanus has petiolate leaves while the other two subspecies have sessile
ones. Although variable in their format, subsp. sulcatus presents more elliptic leaves with
visible nerves while the other two present more lanceolate ones with nerves only visible
at light. Subsp. sulcatus is more prostrated than the other two (Demoly and Montserrat
1993).
Two varieties of C. ladanifer are recognized based on the presence of a purple blot in a
white flower (var. maculatus) or absence (var. albiflorus) (Demoly and Montserrat 1993).
Along the year, the shrub may have three phases of vegetative growth separated by hard
summer conditions, hard winter conditions and flowering period (Figure 1.4) although
some authors report a continuous vegetative growth since autumn until late spring but
much more intensely marked during autumn and spring (Correia et al. 1992; Simões et
al. 2008; Simões et al. 2012). Reduced net photosynthesis has been demonstrated in
several populations during winter due to low temperatures but more drastically during
summer drought (Núñez-Olivera et al. 1996). Two types of shoots grow simultaneously
in this plant: dolichoblasts (elongated shoots) and brachyblasts (small shoots) (Simões et
al. 2008).
7
Figure 1.3: Cistus ladanifer morphology. A) Personal photograph of C. ladanifer ladanifer var. maculatus
in central-east Portugal (Idanha-a-Nova, Penha Garcia) and magnification of flower; B) SEM micrographs
of prolate spheroidal C. ladanifer pollen grain (left to right): grain in polar view, grain in equatorial view,
largely reticulate exine (Civeyrel et al. 2011); C) Photograph of C. ladanifer mature closed capsule (left)
and opened 6 valve-capsule (right) (Narbona et al. 2010); D) Personal photograph of C. ladanifer ladanifer
globular-polyhedric seeds; E) SEM photograph of C. ladanifer upper leaf surface after washing with CHCl3
showing numerous trichomes with short stalked small heads shape, most collapsed (Gülz et al. 1996); F)
SEM photograph of C. ladanifer lower leaf surface after washing with CHCl3 with a very dense
arrangement of stellate hairs with 8-20 branches (Gülz et al. 1996); G) SEM photograph of a single short
stalked small head trichome collapsed (Gülz et al. 1996).
Figure 1.4: “Phenology of Cistus ladanifer, and schematic representation of a branch in successive
phenophases. a flowering; b fruiting; c during seed dispersal; d flowering” in Talavera et al. (1993).
8
C. ladanifer is considered a semi-deciduous plant because it shows some seasonal

dimorphism. It produces larger leaves (winter leaves) on dolichoblasts during autumn,
which fall in spring, and smaller leaves (summer leaves) on brachyblasts in spring, falling
in autumn (Simões et al. 2008; Simões et al. 2012).
Flowering phenophase only starts to happen at 3-4 years-old plants and scarcely at 2
years-old (Talavera et al. 1993; Luiscalabuig et al. 1996). Flower buds initiate during
winter in brachyblasts (Simões et al. 2008), at the apex of vigorous branches produced in
the previous year, and flowering period lasts two months from mid-March to mid-May
(Talavera et al. 1993). Anthesis lasts 1 to 3 days with the reclosing of petals around the
ovary at the end of each day and reopening at sunrise. When flowers drop their petals,
sepals reclose around the ovary. During the first day of anthesis only small quantity of
nectar is produced with maximum yield during the second day when flowers are pollen-
less (Talavera et al. 1993).
Capsules are mature and exposed by early summer, starting seeds dispersal mostly below
the parent plant canopy for an 8-10-month period. In autumn, alternation between dry and
wet periods, causing a greater opening of capsules, as well as the shaking of branches by
wind and rain promote a faster seed dispersal (Talavera et al. 1993; Bastida and Talavera
2002). Narbona et al. (2010) showed that the number of valves as of seeds per capsule
varies between 5-12 and 300-1300, respectively.
After the first autumn rains seedlings from the current year seed-crop start to emerge at
the time the vegetative growth of C. ladanifer plants also initiates (Talavera et al. 1993).
1.1.4. Ecology
1.1.4.1. Reproduction
To yield fruits (capsules), cross pollination by insect vectors is mandatory (Talavera et al.
1993). In fact, these authors found a negative correlation between the number of fruits
per individual as well as the number of seeds per valve and capsule and the distance
between them. The number of valves as of seeds per capsule vary a lot at the inter-
population, intra-population, intra-individual and inter-annual levels (Narbona et al.
2010). Number of seeds per capsule was shown to increase with a higher number of valves
per capsule and, in turn, number of valves per capsule was shown to have a negative
relationship with altitude and a positive one with precipitation (Narbona et al. 2010). Co-
9
ancestry (based on phylogeographic and phylogenetic outputs) was also argued, by the
latter authors, to influence this fruit variation.
In autumn, alternation between dry and wet periods, causing a greater opening of
capsules, as well as the shaking of branches by wind and rain promote a faster seed
dispersal (Talavera et al. 1993; Bastida and Talavera 2002). Although seed dispersal in
this species is much higher in time than in space, seed predators (deer and ants) are
thought to promote a wider seed dispersal (Bastida and Talavera 2002).
Cistus ladanifer seeds are thought to have a physical dormancy characterized by an “hard”
coat which makes them impermeable to water and which is usually broken down by heat
or abrasion, a mechanism known as hardseededness (Thanos et al. 1992). This hard coat
confers seeds a long longevity (Pérez-García 1997). Heat treatments, smoke and
nitrogenous salts, simulating natural fire conditions, have shown to increase seed
germination (Valbuena et al. 1992; Pérez-García 1997; Delgado et al. 2001; Pérez-
Fernández and Rodríguez-Echeverría 2003). However, more recently Dias et al. (2019)
could not find evidence that seeds dormancy was physical. Seeds dispersed by deer
ingestion (faeces) showed to have higher proportion of rapid germinable ones than those
gathered from the plants (Malo and Suárez 1996).
However, in all the latter studies, germination was observed when no treatment/event was
applied/observed. According to Delgado et al. (2010) that fraction is composed of lighter
seeds (in weight) which don’t present dormancy (i.e., soften coat). In fact, early released
seeds may germinate into seedlings in the same year after the first rains of autumn
(Talavera et al. 1993), without a dormancy breaking event.
The production of seeds with different degrees of dormancy allows C. ladanifer to

propagate under different environments or scenarios. On the one hand, soft coated seeds
may help to constitute a dense population germinating in open spaces with no competition
and in favourable environments if the large dispersal period is considered. On the other
hand, the large number of hard seeds released, their longevity and small size help to create
a persistent soil seed bank waiting to germinate after a perturbation in the following years
when conditions are suitable (e.g., a fire). Environmental conditions, such as rainfall
patterns, have shown to modulate germination and shrubland recovery (Moreno et al.
2011; Chamorro et al. 2016).
10
1.1.4.2. Nutritive demands
Cistus ladanifer root systems are reported to be dense and shallow (Bolaños and Guinea
1949; Simões et al. 2012) but this shrub is thought to produce deep roots, which may
constitute an advantage during drought periods (Simões et al. 2012). The latter authors
reported a belowground: aboveground ratio of annual biomass production of 0.8 which
reflects the effort made by this shrub on root system development compared to the aerial
parts. Dense and shallow roots allow a fast water uptake in transient wet periods (e.g.
occasional rain during a summer drought) (Núñez-Olivera et al. 1996) but deeper roots
enhance water exploitation from deeper soil layers. An auxin producing Plant Growth
Promoting Rhizobacteria (PGPR) strain was isolated from C. ladanifer rhizosphere,
showing the possible role of PGPRs in root development (Solano et al. 2007).
Cistus ladanifer low nutritive demands are directly inferred by the soils (of nonspecific
type and pH between 3.5 and 6) where it grows which are, generally, poor in nutrients
(Demoly and Montserrat 1993).
Simões et al. (2012) studied the nutrient dynamics during 2 years in a C. ladanifer
population, obtaining values of nutrient (N, P, K, Mg, and Ca) concentrations in leaves
close to values observed by other authors for evergreen sclerophylls and lower than values
for Cistus salvifolius and other seasonal dimorphic plants. However, despite the lower
concentration, C. ladanifer leaves held higher pool of nutrients than those of C. salvifolios
mainly because of higher biomass productivity and overall, more efficient biomass-to-
nutrient ratios. In terms of nutrient portioning, upper parts of the plant but particularly
winter leaves showed the highest storage of all studied nutrients except for Mg, which
was mostly stored in roots. Moreover, the authors observed a strong nutrient internal
translocation from winter leaves to summer ones and reproductive organs, suggesting that
a great proportion of the annual nutrient requirement may derive from internal sources.
This is an important strategy because of the decline of nutrient absorption at the root level
in the spring-summer period.
In addition to the low edaphic demands, subsp. ladanifer and africanus have been shown
to bare metalliferous substrates, colonizing serpentine areas and mine tailings sometimes
as the dominant species (Frazão et al. 2018; Raimundo et al. 2018). Raimundo et al.
(2018) highlights C. ladanifer as a Mn and Zn accumulator and as As, Cu, Ni, and Pb
excluder.
11
PGPR, predominantly nutrient mobilizing strains (siderophore producers and phosphate

solubilizers), were isolated from C. ladanifer rhizosphere (Solano et al. 2007). In
addition, several ectomycorrhizal fungi symbioses have been reported in this species
which may have a critical role in its tolerance to environmental stresses (Comandini et al.
2006).
1.1.4.3. Shrublands
Cistus ladanifer shrubland is one of the most widespread shrub systems in the Iberian
Peninsula, very poor in other plant species and a tendence to perpetuate instead of to
develop to mature vegetation stages (Mendes et al. 2015). Núñez-Olivera et al. (1995)
argues that the presence of some sclerophyllous evergreens such as Quercus rotundifolia
and Q. suber derives from an ancient flora rather than a successional stage.
Cistus ladanifer shrublands are relatively short-lived and when they are 18-20 years-old,
and consist of highly lignified and dried biomass shrublands, natural conditions are
gathered for the ignition and spread of fire (Oria-de-Rueda et al. 2008). In fact, the
production of non-woody photosynthetic biomass is increased when neighbour plants are
removed (Delgado et al. 2011) which means that higher shrubland cover, typical of older
shrublands, is associated to higher proportion of woody biomass. There is a general idea
that the exuded labdanum resin, covering principally the photosynthetic biomass, is
flammable and thus helps the spreading of fire. Although no specific references can be
found about it, labdanum gum (resin) is registered in ECHA (EC number 946-963-9) as
a non-flammable solid, a statement supported by approved testing methods.
Rather than a pyrophyte species, Trabaud (1995) classifies this species as opportunistic,
occupying open disturbed areas without intense vegetative cover and strong competitors.
C. ladanifer communities are known to comprise an early secondary succession stage of
areas after perturbations such as typical Mediterranean fires or human activity, for
instance, the cleaning of areas through cutting and ploughing and then their abandonment
(Alonso et al. 1992; Tárrega et al. 1995; Luiscalabuig et al. 1996; Tárrega et al. 2001).
Reproductive success, low nutritive demands, and resilience/tolerance to stress factors
explains the regeneration and perpetuation of such systems.
According to Simões et al. (2009) these C. ladanifer shrubs improve soil quality in the 0-
5 cm top-soil layer, through litter-fall, by enhancement of organic matter and nutrient
content beneath their canopies. According to the authors, this soil recuperation after a
12
perturbation may favour the posterior establishment of more demanding woodland

species such as Quercus species, following a typical secondary succession mechanism.
However, the resilience of these populations is well demonstrated by their auto-
succession process potentiated by the persistent soil seed bank, allelopathy, high plant
cover and relatively short period perturbations.
Besides preventing a more advanced land degradation, C. ladanifer shrublands have

recently been regarded as important carbon sinks, which is important because a key
mitigation strategy for climate changes is the sequestration of greenhouse gases such as
CO2 (Ruiz-Peinado et al. 2013; Alías et al. 2015; Carrión-Prieto et al. 2017a). Therefore,
these shrublands must be carefully managed either to reduce or explore them.
The agro-forestry-pasture system known as “montado” in Portugal or “dehesa” in Spain

is a “multi-layered dryland system” where evergreen oaks associate with shrubs, such as
C. ladanifer, and native pastures or crops, and from which several benefits and services
are supposed to be achieved in a sustainable manner, such as animal raising, hunting, and
firewood production (Ruiz-Peinado et al. 2013). In a 20-year study, Mendes et al. (2015)
observed that continuous cutting and grazing were able to modify the C. ladanifer
shrubland in order to progressively and simultaneously reverse land degradation and
biodiversity improvement, pointing out grazing as the more economically viable solution
because of the cutting costs with no profit.
Based on Godinho-Ferreira et al. (2005) work about Portugal’s forest typologies and on
insights of C. ladanifer shrubland dynamics, three general types of C ladanifer shrublands
may be proposed:
• Dense shrublands (7.5% of Portugal forest area in 2005): most likely abandoned
areas with no management and profit with a high probability of shrubland
perpetuation due to fire events.
• Shrublands associated to oak, pine, and eucalyptus forests (41.9% of Portugal
forest area in 2005): most likely managed areas because of the expected profit
(e.g., periodic total clearings of the shrub layer). Open oak woodlands would
include the “montado” agro-forestry-pasture system.
• Shrublands associated to a diverse forest (33.2% of Portugal forest area in 2005):
most likely poorly managed and no profit and a probability of dense C. ladanifer
shrublands reclamation.
13
1.1.4.4. Labdanum resin exudate
Cistus ladanifer exudes a phenolic and terpenoid resin from its photosynthetic tissues
which is comercially but wrongly called labdanum gum. In fact, a gum is a plant exudate
composed by complex chains of hydrophilic polissacharides (Langenheim 2003). This
resin possesses important ecological functions, protecting the plant against both abiotic
and biotic stresses, as suggested by Dias and Dias (1987) (Figure 1.5).
Figure 1.5: Ecological functions of the labdanum resin as proposed by and adapted from Dias and Dias
(1987).
There is a general belief that C. ladanifer exudes more resin in the summer months when
temperature rises and rainfall decreases, supported by the traditional exploitation of such
resource, mainly in Spain, which is done in summer months, and demonstrated by Chaves
et al. (1993) and Sosa et al. (2005).
Dias and Dias (1987) hypothesized that labdanum solar reflectance and volatilisation of
terpenic compounds, increasing the density of the foliar gas phase and boundary layer,
would add resistance to transpiration of the plant tissues. UV damage protection was
discussed by Chaves et al. (1993) who observed labdanum resin absorption between 260-
400 nm, concluding that the resin would function as an UV-A and UV-B filter enabling
however the photosynthesis that needs higher wavelength visible radiation. The authors
further discuss that flavonoid aglycones would be the resin components responsible for
that feature. In fact, in a later study Chaves et al. (1997a) concluded that flavonoid
aglycones content in the resin is increased by the UV radiation stress and synergistically
but not alone by hydric stress. High temperature and hydric stress did however increase
14
the content of more methylated flavonoid aglycones which are more hydrophobic and
may be related to the resistance to drought, reducing water vapor exchanges.
Some diterpenes are produced in lower amounts than flavonoids during all year (Valares
et al. 2016b). However, their exudation follows an inverse pattern than that of the
flavonoids, increasing in winter, regardless the decrease on the overall exudation at the
season (Alías et al. 2012). In fact, Alías (2006) demonstrated that, at controlled
conditions, low temperatures stimulated the synthesis of 3 diterpenoids. Valares et al.
(2016b) discuss that these compounds may have a role in wound induced responses and
in cell membrane stability. It is not yet well understood if these compounds provide
resistance or tolerance to abiotic stresses such as chilling or frost. It is however
demonstrated the allo-allelopathy (Alías 2006) and auto-allelopathy (Chaves et al. 2019),
regulating the germination and growth of either other plant species or of the own species,
respectively. Alías (2006) observed that the quantity of diterpenes in young leaves is
higher in winter but their quantity in fallen leaves and in the soil is higher in
spring/summer which means an interesting mechanism where diterpenes are produced in
winter to act in spring/summer.
Other compounds such as phenolic acids, fatty acids and volatile terpenes present in
labdanum resin were found to have alo-allelopathic activity against several plant species
seed germination and development, some acting synergistically but others
antagonistically (Chaves and Escudero 1997; Chaves et al. 2001a; Chaves et al. 2001b;
Chaves et al. 2002; Dias and Moreira 2002; Dias et al. 2004; Alías 2006; Herranz et al.
2006). The allelopathic activity of some compounds was demonstrated to vary differently
with photoperiod and temperature conditions, indicating a broad allelopathic activity
under several environmental conditions (Chaves et al. 2002; Alías 2006). Despite the
persistence of C. ladanifer flavonoid aglycones in its soils (Sosa et al. 2010), those
compounds do not appear to have strong allelopathic effects when compared to other
compounds (Chaves and Escudero 1997; Chaves et al. 2019).
In what regards feeding avoidance, Sosa et al. (2004) demonstrated that C. ladanifer
exudate, principally the one produced in summer months, and its flavonoid aglycones are
potent inhibitors of sarcoplasmic reticulum Ca2+-ATPase activity of rabbit mouth skeletal
muscle. Labdanum resin, by the action of its flavonoid aglycones, consumed more with
more herbaceous plant material, causes an avoidance anti-feeding reaction in vertebrate
15
and insect herbivores, at low quantities, by relaxation of the mouth skeletal muscle (Sosa
et al. 2004).
Antimicrobial activity of labdanum resin has been poorly documented. Greche et al.
(2009) observed that labdanum resin extracts presented some inhibitory activity and poor
microbicidal activity against some fungi and bacterial strains, and Rauwald et al. (2019)
observed no inhibitory activity of C. ladanifer labdanum resin against Borrelia
burgdorferi contrasting to the labdanum resin from C. creticus. However, C. ladanifer
essential oil and other extracts have consistently been showing a broad-spectrum
antimicrobial activity (Raimundo et al. 2018). Perhaps the major compounds of labdanum
resin, labdane diterpenoids and flavonoid aglycones, may not be as good antimicrobials
such as minor or other non-exuded compounds.
1.1.4.5. Pests and diseases
Cistus ladanifer is not frequently reported to have pests and diseases. But this could be
underestimated because this species is not considered a crop and thus diseases and pests
reducing plant productivity may not be regarded as an important issue.
Pre-dispersal seed predation by Lepidoptera larvae (Noctiodae) and Coleopteran larvae

(Bruchidae, Scarabeidae, Curculionidae) is known to be the major cause of fruit and seed
loss (Serrano et al. 2001; Bastida and Talavera 2002; Delgado et al. 2007; Narbona et al.
2010). Deer is also reported to feed on capsules (Malo and Suárez 1996). When capsules
were open, an intense activity of granivorous ants (Goniomma hispanicum and G. kugleri)
was observed by collecting seeds directly from the opened capsules and carrying them
into their nests (Bastida and Talavera 2002).
Botryosphaeria dothidea is considered a primary pathogen of C. ladanifer shrubs

responsible for foliar wilting and death of branches, possibly leading to death of a healthy
shrub in just one growing season (Sánchez‐Hernández et al. 2002). This pathogen does
not require wounds or a stressed status to infect plants and may behave as a saprophyte,
persisting in dead or necrotic plant material. This disease incidence was shown to be
higher in increasing plant densities and young plants lacking lignified branches were
shown to be free of disease.
Phytophtora cinnamomi is reported to highly infect C. ladanifer roots causing severe

chlorosis, dieback of branches, brown leaves and death (Moreira and Martins 2005).
16
1.2. Cistus ladanifer L. raw materials (products).
1.2.1. Essential oil
Essential oil is extracted by hydro- or steam-distillation of the photosynthetic plant

material. Yield of essential oil from C. ladanifer is relatively low. In relation to the fresh
weight, the plant renders between 0.07 and 0.6% v/w of essential oil (Proksch and Gülz
1980; Mariotti et al. 1997; Lawrence 1999; Oller-López et al. 2005; Greche et al. 2009;
Guimarães et al. 2010; Verdeguer et al. 2012; El Karkouri et al. 2021).
According to Cocker et al. (1956), the identification of acetophenone and of 2,2,6-

trimethylcyclohexanone in C. ladanifer essential oil was documented in 1912, more than
100 years ago. In total, twenty studies were consulted from 1980 to 2021 regarding the
chemical characterisation of twenty-two C. ladanifer essential oil and eighteen studies
presented at least a relative quantification of compounds from twenty essential oils.
The chemical characterisation of the essential oils was typically done by GC, using the
EI-MS spectra and the retention indexes based on a n-alkane series for identification and
the peak area from the FID chromatogram detector for relative quantification. More,
rarely standards were used for both identification and quantification. NMR spectroscopy
was used for structure elucidation (identification) mainly by the older studies. Strack et
al. (1980) developed, however, an HPLC method for the analysis of C. ladanifer essential
oil showing good separation of diterpenes, sesquiterpenes, and oxygenated monoterpenes.
The essential oil composition showed to be highly variable, and, in total, 268 different
compounds were identified.
From the 84 monoterpenes (including the oxygenated monoterpenoids), 56 compounds

were reported more than once and only 9 compounds were reported in more than 50% of
the references (Table 1.3): α-pinene, bornyl acetate, camphene and p-cymene were most
reported (82%) followed by borneol and terpinene-4-ol which were respectively reported
in 73 and 68% of the essential oils. Regarding sesquiterpenes, 65 compounds were
identified of which 38 were reported more than once and 2 were reported in more than
50% of the essential oils (Table 1.3): viridiflorol (73%), δ-cadinene (64%). Only 2
diterpenes and 3 diterpene alcohols were reported amongst the various references, 15-
nor-labdan-8-ol was reported in 19% of the essential oils. Lastly, 114 non terpenic
compounds were identified. Many of them are minor components analysed on purpose
17
by Guy and Vernin (1996), including 32 fatty acids, 3 aromatic and 14 phenolic
compounds. In this group, only 18 compounds were reported more than once, half
constituted by alkanes, most probably epicuticular waxes. 2,2,6-trimethylcyclohexanone
was reported in 64% of the essential oils. In a previous review, this ketone was
hypothesized as an authenticity and taxonomic marker of C. ladanifer (Frazão et al.
2018).
Table 1.3: Most representative chemical compounds identified in C. ladanifer essential oil, reported in more
than 50% of 22 essential oils reported in literature.
Class Compound reporting (%) References reporting*
α-pinene 82 [2,4,5,7-18,20]
bornyl acetate 82 [1,4,5,7,8,10-20]
camphene 82 [2,4,5,7-19]
p-cymene 82 [2,4,5,7-18,20]
Monoterpenes/oids borneol 73 [4,7-10,12-20]
terpinen-4-ol 68 [5,7-15,17,18,20]
verbenone 64 [4,7-17]
limonene 59 [2,4,5,7,9-12,15,16,18]
pinocarveol 54 [5,7-12,14,15,20]
viridiflorol 73 [5,7-15,17-20]
Sesquiterpenes/oids
δ-Cadinene 64 [2,5,7,8,10-15,17,19,20]
other 2,2,6-trimethylcyclohexanone 64 [1,4-14,17,19]
*[1]Proksch and Gülz (1980); [2]Gülz et al. (1984); [3]Regino et al. (1987); [4]Li et al. (1991); [5]Vernin
et al. (1992); [6]Guy and Vernin (1996); [7]Mariotti et al. (1997); [8]Mrabet et al. (1997); [9]Robles et al.
(2003); [10]Gomes et al. (2005) (3 essential oils); [11]Oller-López et al. (2005); [12]Costa et al. (2009);
[13]Greche et al. (2009); [14]Viuda-Martos et al. (2011); [15]Verdeguer et al. (2012); [16]Rincón et al.
(2007); [17]Zidane et al. (2013); [18]Vieira et al. (2017); [19]Benali et al. (2020); [20]El Karkouri et al.
(2021).
The number of compounds reported only a few times demonstrates the variability of
composition of C. ladanifer essential oil. Regino et al. (1987) studied the composition of
C. ladanifer essential oils from different locations in Portugal, along the year and during
storage, concluding that although the overall composition varied, the major compounds
remained the same: α-pinene, 2,2,6-trimethylcyclohecanone, and ledol. Mariotti et al.
(1997) found appreciable variations within essential oils between individual plants of a
same population principally in what regards the major component which shifted between
α-pinene and viridiflorol. Mrabet et al. (1997) also found variation between the two
varieties of C. ladanifer from Marrocos, maculatus and albiflorus. However, viridiflorol,
bornyl acetate, and δ-cadinene were the most representative compounds in both essential
oils.
18
1.2.2. Labdanum resin
1.2.2.1. Extraction
Traditionally, labdanum resin is extracted, using water, by two different processes: The
Zamorean process and the Andalusian process (Lawrence 1999; Morgado et al. 2005;
Burguer 2016). The Zamorean process consists in a physical extraction where labdanum
resin is removed from the surface of C. ladanifer plant material using boiling water and
then the resin at the surface of the water is continuously separated using a skimmer. The
Andalusian process is a chemical extraction where labdanum resin is extracted from the
surface of the plant material with warm alkaline water and afterwards precipitated by
lowering the pH with acid and finally separated with a skimmer or by decantation.
Nowadays, this last process is used as the industrial process (Biolandes n.d.) most
probably because of its higher yields.
Burguer (2016) could not manage to separate the resin obtained by the Zamorean process
because of the very small quantity at the surface of the boiling water. In contrast the author
could obtained labdanum resin by the Andalusian process with a yield between 7.79-
8.86% (dw/dw) in relation to the plant material. Morgado et al. (2005) obtained a
labdanum extraction yield of between 7-18% (dw/dw) in relation to usable biomass
(photosynthetic plant material, which also includes soft lignified biomass). However,
labdanum yield, in relation to both usable and total biomass, was shown to vary with plant
age, reaching its maximum in 2–3-year-old plants for usable biomass yield and in 1-year-
old plants for total biomass yield. Considering the total biomass collected, maximum
yields of between 400 and 450 kg/ha were obtained for 3-4-year-old and 12-15-year-old
plants, respectively.
Burguer (2016) extracted the labdanum resin by the Andalusian process with an aqueous
solution of Na2CO3 at 25 g/L for 90 min at 50oC, using 200 g of plant material and 1 L of
extracting solution. Afterwards the solution was filtrated and cooled overnight. Then it
was acidified until pH 1.5 - 2.0 with H2SO4 (90%, v/v) and the resin separated by
filtration. In contrast, Greche et al. (2009) extracted 750 g of plant material with 10 L of
an aqueous solution of NaHCO3 at 8.4 g/L for 10 min at 60 - 70oC. Then, they just
neutralised the solution with HCl at 0.2 M and removed the resin with a perforated
skimmer. Morgado et al. (2005) extracted the resin with a solution of Na2CO3 (10 g/L)
19
at 50oC, precipitating the gum with H2SO4 93% (v/v) which was left to rest for 24 h and
separated by decantation and dried at room temperature.
For analytical purposes of the labdanum resin, some authors have extracted the exudate
from the surface of the plant material with organic solvents, such as hexane (de Pascual
et al. 1974; de Pascual et al. 1982; de Pascual et al. 1983; de Pascual et al. 1984; Greche
et al. 2009), acetone (Wollenweber and Mann 1984), ethanol (Chaves et al. 1993) and
chloroform (Vogt et al. 1987; Chaves et al. 1997b; Sosa et al. 2005; Alías et al. 2012;
Valares et al. 2016a). Burguer (2016) compared hexane, toluene, and ethanol to prepare
such extracts and concluded that toluene was by far the best solvent with a yield of 12.9%
(dw/dw). Similar or even higher yields were obtained with chloroform (Sosa et al. 2005).
These extracts would be called Cistus concretes if done with hydrocarbon solvents such
as hexane and toluene (Handa 2008). Other authors prepared extracts from the already
extracted labdanum resin using ether (Cocker et al. 1956; de Pascual et al. 1968; Tabacik
and Bard 1971; de Pascual et al. 1972), which, because they were done with an
hydrocarbon solvent, would be called labdanum resinoids (Handa 2008).
Several of these labdanum extracts have been subjected to a dewaxing process based on
the dissolution of the dried extracts or resin in warm methanol or ethanol and then cooling
down to negative temperatures to precipitate and separate, by centrifugation, waxes (de
Pascual et al. 1984; Vogt et al. 1987; Chaves et al. 1997b; Sosa et al. 2005; Greche et al.
2009; Alías et al. 2012; Burguer 2016). According to Burguer (2016), methanol presents
a much higher efficiency in separating waxes (38% dw/dw) than ethanol (8.5% dw/dw).
In fact, Greche et al. (2009) precipitated waxes from the labdanum absolute prepared with
ethanol and even so, waxy compounds appeared as significant compounds in the extract.
Waxes separation yield of between 10-16% (dw/dw) were reported for C. ladanifer
concretes prepared with hexane (de Pascual et al. 1974; de Pascual et al. 1982; de Pascual
et al. 1983; de Pascual et al. 1984). The alcoholic extract would be called Cistus absolute
or Labdanum absolute depending on the starting material (Handa 2008).
To characterize labdanum components, from 1956 to 1984 labdanum resin was subjected
to acidic fractionation. The acidic fraction was first obtained by Cocker et al. (1956)
washing the ether extract with the alkali salt KHCO3. The alkaline solution was acidified
until pH 5 and the liberated acids taken up (washed) again in ether, giving the acidic
fraction (25%, dw/dw). The authors further washed the labdanum ether solution with
NaOH to obtained what they called the phenolic fraction (55%, dw/dw). The remaining
20
labdanum ether extract was considered the neutral fraction (17.5%, dw/dw). de Pascual
et al. (1968) performed a similar fractionation of the labdanum resin obtaining different
fraction yields: acidic fraction, 60.1% (dw/dw), phenolic fraction, 14.1% (dw/dw), and
neutral fraction, 25.8% (dw/dw). In a later work, de Pascual et al. (1974) obtained
different acidic fractions washing the labdanum ether solution with aqueous solutions of
KHCO3, Na2CO3, and NaOH successively, meaning that those solutions are successively
stronger to extract the acids. Tabacik and Bard (1971) obtained just one acidic fraction
(68% dw/dw), by washing with Na2CO3, and a neutral fraction (29% dw/dw). Later,
Greche et al. (2009) applied the same method as Cocker et al. (1956), after extracting the
volatiles, to three different resin extracts (resinoid, concrete and absolute). The
fractionation yielded 3.1-4.6% (dw/dw) of volatile fraction, 22.3-27.8% (dw/dw) of
acidic fraction (KHCO3), 23.8-37.5% (dw/dw) of phenolic fraction (NaOH), and 39.4-
48.9% (dw/dw) of neutral fraction. These yields were also quite different from the ones
cited previously. In a different manner, Burguer (2016) washed an ether labdanum
resinoid first with NaOH, obtaining the neutral fraction in the ether. Afterwards, lowered
the pH to 8 with CO2, washing the phenolic fraction with ether, and lowered again to pH
1.5 with HCl to wash the acidic fraction with ether. Burguer (2016) obtained a neutral
fraction with a yield of 41.1% (dw/dw), a phenolic fraction with a yield of 4 % (dw/dw)
and an acidic fraction with a yield of 19.9% (dw/dw). Interestingly, the later author reports
a loss of about 31% (dw/dw) when dissolving labdanum resin in ether. In conclusion, if
the phenolic fraction is considered an acidic fraction, the acidic fraction was consistently
the major fraction, accounting to more than a half of the resin weight.
After separating the waxes from the chloroform exudate extract, Vogt et al. (1987) loaded
the extract directly on Sephadex LH-20 to separate terpenoids from flavonoids, by
molecular sizing column chromatography, using methanol as eluent. Terpenoids eluted
first and, according to Vogt and Gülz (1991), they could be detected by TLC on silica gel
60, using toluene: ethyl acetate (9:1) as eluent, when sprayed with concentrated sulphuric
acid and heated at 100oC for 10 min. Flavonoids were also detected in the TLC using an
UV lamp (350 nm). This method would be used by most of the subsequent works to obtain
labdanum fractions for quantification and isolation of labdanum compounds.
Similar to the essential oil, labdanum oil is extracted by distillation of the labdanum resin
instead of C. ladanifer plant material (Moyler and Clery 1997; Weyerstahl et al. 1998).
Contrasting to C. ladanifer essential oil, a lower number of studies, 4, were found
21
regarding labdanum oil. Cocker et al. (1956) obtained labdanum oil with a yield of 1.5%
(dw/dw). Labdanum oil yield is expected to be higher than for the essential oil considering
the different starting material. However, if labdanum gum yield is considered (e.g., 5%,
dw/fw) and if the labdanum oil yield was to be reported in relation to the original plant
material, the yield would decrease to values near the ones reported for the essential oil
(0.08%, dw/dw). No more references were found reporting labdanum oil yield. From a
labdanum absolute (without waxes), de Pascual et al. (1982) steam-distilled an oil with a
yield of 2.6%, however this labdanum resin was extracted with hexane from the plant
material and not by the traditional processes. Greche et al. (2009) obtained volatile oils
with yields between 3.1-4.6% (dw/dw) from a concrete, resinoid and absolute.
Nevertheless, these oils would be considered more a labdanum oil rather than a C.
ladanifer essential oil.
1.2.2.2. Chemical characterisation
At early studies, individual compounds were isolated from the fractionated/purified

extracts mostly by Column Chromatography (CC) and/or Thin Layer Chromatography
(TLC), and their structure was elucidated by UV-Vis, Infrared, and NMR spectroscopy,
mass spectrometry, and degradation/synthesis methods or identified using standards
(Cocker and Halsall 1956; Cocker et al. 1956; Halsall and Moyle 1960; de Pascual et al.
1968; de Pascual et al. 1972; de Pascual et al. 1974; de Pascual et al. 1977; de Pascual et
al. 1982; de Pascual et al. 1983; de Pascual et al. 1984; Proksch and Gülz 1984;
Wollenweber and Mann 1984).
In later studies, GC systems coupled to MS and FID detectors were mostly used to analyse
labdanum oils (volatiles) but also some labdanum extracts (Moyler and Clery 1997;
Weyerstahl et al. 1998; Chaves et al. 2001a; Greche et al. 2009; Burguer 2016). However,
acidic components of labdanum extracts had to be derivatized, methylated, with
diazomethane (Greche et al. 2009) or Trimethylsilyldiazomethane (Burguer 2016) to turn
them more volatile. Methyl esters of the acidic fraction compounds were also obtained
with diazomethane by de Pascual et al. (1982) and de Pascual et al. (1983), however not
for the purpose of GC analysis. Reversed phase HPLC analysis with UV and MS detectors
were used to identify and quantify non-volatile components of the labdanum resin, such
as diterpenoids and flavonoids (Chaves et al. 1997a; Chaves et al. 1997b; Chaves et al.
1998; Sosa et al. 2005; Alías et al. 2012; Valares et al. 2016a; Valares et al. 2016b).
22
In total, 31 studies were consulted from 1956 to 2016 regarding the chemical
characterisation of labdanum resin from C. ladanifer, resulting in a list of 277 compounds.
More than a half of all the compounds identified are terpenes/oids (181 compounds): 78
monoterpenes/oids, 56 sesquiterpenes/oids, and 47 diterpenes/oids. Other compounds
include free fatty acids, waxes, flavonoids, phenylpropanoids, and other low molecular
weight compounds. Table 1.4 gathers the most representative compounds present in
labdanum resin based on number of reports, more than 3.
Labdanum resin has a volatile fraction very similar to C. ladanifer essential oil.
Labdanum oil is mostly composed by monoterpenes/oids, sesquiterpenes/oids, and other
low molecular compounds such as the authenticity marker 2,2,6-trimethylcyclohexanone
as demonstrated by Boelens (1984) and Ohloff (1994), Weyerstahl et al. (1998), and
Moyler and Clery (1997). Regarding these compounds, Table 1.4 is very similar to table
1.3 which report the most representative compounds of the essential oil.
Table 1.4: Most representative chemical compounds identified in C. ladanifer labdanum resin, reported
more than three times within the 31 references.
Nº
Compound Group Compound identified References reporting*
References
Bornyl acetate 4 [12,17,22,29]
Borneol 4 [12,17,22,29]
α-Terpineol 3 [12,22,29]
Pinocarveol 3 [17,22,29]
Monoterpenes/oids p-Cymene 3 [12,20,29]
Pinocarvone 3 [12,22,29]
Verbenone 3 [12,22,29]
Camphene 3 [12,17,20]
α-Pinene 4 [12,17,20,29]
Ledol 6 [8,17,20,22,26,29]
Palustrol 3 [12,22,29]
Viridiflorol 7 [8,12,13,17,20,22,29]
Allo-Aromadendrene 4 [12,17,22,29]
Sesquiterpenes/oids
Isoledene 3 [22,26,29]
Ledene 4 [17,20,22,29]
α-Copaene 3 [20,22,29]
δ-Cadinene 3 [20,22,29]
labdan-8,15-diol 3 [5,10,13]
Ladenol (Labd-8(17)-en-15-ol) 3 [8,10,22]
Oxocativol (6-oxo-labd-7-en-15-ol) 3 [8,10,13]
Ambroxide 4 [12,20,22,29]
Labdanolic acid (labdan-8-ol-15-oic
8 [1,2,9,10,13,25,26,29]
acid)
Diterpenes/oids
6-Oxocativic acid (6-oxo-labd-7(8)-
8 [2,3,13,25,26,28,30,31]
en-15-oic acid)
Labdenic acid (Labd-8(17)-en-15-oic
4 [6,10,22,26]
acid)
Cativic acid (Labd-7-en-15-oic acid) 4 [10,22,26,29]
7-Oxo-labd-8(9)-en-15-oic acid 6 [9,13,25,28,30,31]
23
6β-Acetoxy-7-oxo-labd-8(9)-en-15-
6 [9,13,25,28,30,31]
oic acid
8α--Methoxy-labda-15-oic acid 3 [9,10,13]
8α-Hydroxy-labd-13(E)-en-15-oic
3 [9,10,13]
acid
Cinnamic acid (3-phenylpropenoic
3 [12,23,26]
acid)
Phenylpropanoids
Dihydrocinnamic acid (3-
7 [2,10,12,22,23,26,29]
phenylpropanoic acid)
other 2,2,6-trimethylcyclohexanone 3 [12,17,22]
Palmitic acid (Hexadecanoic acid) 3 [22,26,29]
Linoleic acid (9-12-octadecadienoic
Fatty acids 3 [22,26,29]
acid)
Arachidic acid (eicosanoic acid) 3 [2,26,29]
Isokaempferide (kaempferol-3- [11,14-
11
methyl ether) [16,18,19,21,24,27,30,31]
Jaranol (kaempferol-3,7-dimethyl [4,7,11,14-
13
ether) 16,18,19,21,24,27,30,31]
Kaempferol-3,4’-dimethyl ether 8 [11,15,16,18,19,21,24,27]
Metiljaranol (Kaempferol-3,7,4’-
8 [11,13,15,16,18,19,21,27]
trimethyl ether)
Flavonoids
[11,14-
Apigenin 11
16,18,19,21,24,27,30,31]
[11,14-
Acacetin (apigenin-4’-methyl ether) 11
16,18,19,21,24,27,30,31]
[11,13-
Genkwanin (apigenin-7-methyl ether) 12
16,18,19,21,24,27,30,31]
Apigenin-7,4’-dimethyl ether 7 [11,13,15,16,18,19,21]
* [1]Cocker and Halsall (1956), [2]Cocker et al. (1956), [3]Halsall and Moyle (1960), [4]de Pascual et al.
(1968), [5]Tabacik and Bard (1971), [6]de Pascual et al. (1972), [7]de Pascual et al. (1974), [8]de Pascual
et al. (1977), [9]de Pascual et al. (1982), [10]de Pascual et al. (1983), [11]Wollenweber and Mann (1984),
[12]Boelens (1984), [13]de Pascual et al. (1984), [14]Proksch and Gülz (1984), [15]Vogt et al. (1987),
[16]Chaves et al. (1993), [17]Ohloff (1994), [18]Chaves et al. (1997a), [19]Chaves et al. (1997b),
[20]Moyler and Clery (1997), [21]Chaves et al. (1998), [22]Weyerstahl et al. (1998), [23]Chaves et al.
(2001a), [24]Sosa et al. (2005), [25]Alías (2006), [26]Greche et al. (2009), [27]Viuda-Martos et al. (2011),
[28]Alías et al. (2012), [29]Burguer (2016), [30]Valares et al. (2016a), [31]Valares et al. (2016b).
Diterpene/oids are representative compounds in labdanum resin not only because of the
total number of compounds identified (47) but also because of the number of reporting
references (Table 1.4). Most of these compounds are labdane-type (bicyclic core, Figure
1.6) but labdane-related diterpenes/oids bearing more complex ring systems have been
identified, such as kaur-16-ene (Greche et al. 2009; Burguer 2016) and manoyl oxide
(Weyerstahl et al. 1998; Burguer 2016).
24
Figure 1.6: Labdane diterpene skeleton. Image adapted from https://en.wikipedia.org/wiki/Labdane

(03/01/2022) and C-numbering according to Demetzos and Dimas (2001).
Within diterpenoids, Labdane-type diterpenoid acids, with a carboxylic group at position

15, are the most consistently reported compounds. Labdanolic acid, 6-oxocativic acid, 7-
oxo-labd-8(9)-en-15-oic acid, and 6β-acetoxy-7-oxo- labd-8(9)-en-15-oic acid are the
most reported. Labdanolic and 6-oxocativic acid were the first to be reported by Cocker
and Halsall (1956) and Halsall and Moyle (1960), respectively. More Labdane-type
diterpenoid acids, including the two previously mentioned, would be later identified by
de Pascual et al. (1972), de Pascual et al. (1982), and de Pascual et al. (1983) in the
exudate of C. ladanifer ladanifer and C. ladanifer sulcatus (former C. palinhae).
According to Alías et al. (2012), 6-oxocativic acid, 7-oxo-labd-8(9)-en-15-oic acid, and

6β-acetoxy-7-oxo- labd-8(9)-en-15-oic acid, together, accounted to 21 mg/g (dw/dw) of
mature leaves in winter and dropped to approximately 10 mg/g (dw/dw) in the other
seasons. Using the exudate yields from Sosa et al. (2005), these three diterpenes alone
would account to between 6 - 16% (dw/dw) of the resin. Labdanolic acid was not
quantified most probably because it cannot be detected by UV-Vis detectors, as indicated
in Alías (2006). However, labdanolic acid is a major compound in labdanum resin as
shown by Alías (2006), Greche et al. (2009), and Burguer (2016), and other
diterpenes/oids were not quantified as well. In conclusion, the diterpenoid yield in
labdanum resin of 6 - 16% (dw/dw) is an underestimation.
Flavonoid aglycones (non-glycosylated) are also significantly present in labdanum resin

as shown in Table 1.4 as the most reported compounds and constitute the only
polyphenolic group so far identified in labdanum resin. These compounds are represented
by methoxylated derivatives of the flavonol kaempferol and by the flavone apigenin and
methoxylated derivatives (Figure 1.7).
25
The most reported flavonoid, jaranol, was the first to be identified by de Pascual et al.
(1968). Later, metiljaranol, genkwanin and 7,4’-dimethyl-apigenin were additionally
identified by de Pascual et al. (1974). Proksch and Gülz (1984), Wollenweber and Mann
(1984) and Vogt et al. (1987) would complete the identification of the eight flavonoids
present in the exudate of C. ladanifer.
Figure 1.7: Kaempferol flavonol (left) and apigenin flavone (right) skeletons. Image adapted from
https://en.wikipedia.org/wiki/Kaempferol and https://en.wikipedia.org/wiki/Apigenin (05/01/2022) and C-
numbering according to Alam et al. (2020).
According to Chaves et al. (1997a) and Sosa et al. (2005) these flavonoids account to
between 14 -28% (dw/dw) of the resin in summer and to between 6 - 16% (dw/dw) in
winter. Jaranol, kaempferol-3,7-methyl ether, was consistently reported as the major
compound within flavonoids (Sosa et al. 2005; Viuda-Martos et al. 2011; Valares et al.
2016a; Valares et al. 2016b). Apigenin was also consistently reported as the minor
flavonoid by the same authors, but care must be taken because most of them did not
quantify the eight identified flavonoids.
Some free fatty acids are reported to be present in labdanum resin. However, studies about
the fatty acid composition of labdanum resin are scarce, as it is shown in Table 1.4. In
total, 15 free fatty acids were identified but only three were consistently reported (Cocker
et al. 1956; Weyerstahl et al. 1998; Greche et al. 2009; Burguer 2016): palmitic acid (C16,
hexadecenoic acid), linoleic acid (C18, 9,12-octadecadienoic acid) and arachidic acid
(C20, eicosanoic acid). In a recent work done by Jerónimo et al. (2020), these three fatty
acids were also found to be the major fatty acids present in leaves and stems. The study
was however not considered for the labdanum resin characterisation because milled plant
extracts were used instead of exudate or labdanum resin extracts.
26
1.2.3. Polar extracts
Polar extracts made from milled aerial parts of C. ladanifer have shown to be rich in
polyphenolic compounds (Table 1.5). These extracts were prepared using water
(Barrajón-Catalán et al. 2010; Fernández‐Arroyo et al. 2010; Barrajón‐Catalán et al.
2011; Tomás-Menor et al. 2013), ethanol (Lekbach et al. 2018), methanol (Gaweł-Bęben
et al. 2020), ethanol/water (Tomás-Menor et al. 2013), and methanol/water (Barros et al.
2013; Gaweł-Bęben et al. 2020), most using heating but at temperatures below 65oC and
some using ultrasound-assisted extraction. Barrajón-Catalán et al. (2010) report an
extraction yield of 7 - 10% (dw/fw) of aerial plant material. Similarly, Tomás-Menor et
al. (2013) report an extraction yield of 3 - 12% (dw/fw) of non-specified plant material,
depending on the solvent and drying process applied. Within the seven references
mentioned, a total of 46 phenolic compounds were identified, most of them being
polyphenols (flavonoids and tannins) and phenolic acids. Of those 46 compounds, 17
were reported more than three times within the seven references mentioned and are shown
in Table 1.5.
Table 1.5: Most representative phenolic compounds identified in C. ladanifer polar extracts, reported more
than three times within the 7 references.
Nº References
Compound Group Compound Identified
references reporting*
Apigenin 3 [2,5,6]
Apigenin-methyl ether 4 [2,5-7]
Flavonoid aglycones Kaempferol-dimethyl ether 4 [2,5-7]
Kaempferol-methyl ether 3 [2,4,6]
Epigallocatechin 3 [2,4,7]
Kaempferol diglucoside 5 [2,4-7]
Flavonoid glycosides
Quercetin diglucoside 3 [2,4,6]
Hexahydroxydiphenoyl-D-glucose 3 [3,5,6]
Punicalagin 5 [1-5]
Punicalagin gallate 3 [3-5]
Hydrolysable tannins Punicalin 4 [1-3,5]
Ducheside A 3 [2,6,7]
Glucogallin 3 [2,5,6]
Uralenneoside 4 [2,5-7]
Ellagic acid 4 [2,4,6,7]
Phenolic acids
Gallic acid 4 [1-3,7]
other 3,4´-Dihydroxypropiophenone-3-β-D-glucoside 4 [2,4-6]
*[1]Barrajón-Catalán et al. (2010), [2]Fernández‐Arroyo et al. (2010), [3]Barrajón‐Catalán et al. (2011),
[4]Barros et al. (2013), [5]Tomás-Menor et al. (2013), [6]Lekbach et al. (2018), [7]Gaweł-Bęben et al.
(2020).
One of the most reported compounds was kaempferol diglucoside, which was reported to
be present in two different glycosylation patterns and both as major compounds in
methanol and hydro-methanol extracts by Gaweł-Bęben et al. (2020). Apigenin-methyl
27
ether and Kaempferol-dimethyl ether were also significantly reported. Two forms of these
compounds and the other flavonoid aglycones, except epigallocatechin, are reported to be
present in significative amounts in the exudate (Table 1.5). It is important to note that
none of the references mentioned a removal of the exudate prior extraction and, therefore,
its components may be present in these extracts. In fact, Tomás-Menor et al. (2013)
identified labdanolic acid, a labdanum diterpenoid acid, in the polar extracts. 3,4´-
Dihydroxypropiophenone-3-β-D-glucoside and the hydrolysable ellagitannins
punicalagin and punicalin stands also as the most reported compounds.
Other works report polar extracts from C. ladanifer biomass despite the fact that they did
not perform a compound identification analysis. Alves-Ferreira et al. (2019b) extracted
36.9% (dw/dw) of the C. ladanifer biomass with a successive ethanol (23.3%) and water
(13.6%) Soxhlet extraction. Tavares et al. (2020b) extracted the biomass with a successive
ethanol (13.3% dw/dw) and 70% (v/v) acetone (11% dw/dw) ultrasound-assisted
extraction at 30oC. Benali et al. (2020) report similar extraction yields, between 5.67-
6.64% (dw/dw), using water, ethanol and methanol in an extraction done at ambient
temperature. Alves-Ferreira et al. (2020) report differences in ethanol/water (50/50 v/v)
ultrasound-assisted extraction yields (30oC), depending on the biomass type. Stems
rendered the lowest yield (5.8% dw/dw) and leaves the highest (37.8% v/v).
Condensed tannins are reported to be present in C. ladanifer polar extracts, quantified by

the vanillin assay (Dentinho et al. 2005; Dentinho et al. 2007; Alves-Ferreira et al. 2020),
However, specific condensed tannins were not identified and the monomeric
epigallocatechin (a flavan-3-ol with a single bond at 2,3 position, Figure 1.7) is quantified
by the vanillin assay (Versari et al. 2013). The flavonoid is consistently reported in
literature (Table 1.5) in significative amounts in polar extracts from C. ladanifer (Barros
et al. 2013; Gaweł-Bęben et al. 2020). Dimeric epigallocatechin (procyanidin) was
reported by Gaweł-Bęben et al. (2020) but at lesser amounts then the monomeric.
According to Alves-Ferreira et al. (2020) leaves yield more tannins than stems because
of the overall higher extraction yield but stem polar extracts are more concentrated in
those compounds. According to Guerreiro et al. (2016) condensed tannins are present at
higher quantities in soft stems and leaves during summer and autumn.
28
1.2.4. Biomass
Total aboveground biomass of C. ladanifer increases with age (Table 1.6) with a tendency
to stabilize at 10/11-years-old (Hernández-Rodríguez et al. 2017). Hernández-Rodríguez
et al. (2017) report a total biomass of 12-13 ton/ha between 11 and 25-year-old
shrublands. Nuñez et al. (1989) and Morgado et al. (2005) report a total biomass weight
of 16 ton/ha for 12-15-year-old shrublands. These old plants were characterized by around
80% (dw/dw) of woody biomass (Nuñez et al. 1989; Morgado et al. 2005; Alves-Ferreira
et al. 2019a).
Biomass fractions weight also changes with age. Usable biomass (towards resin) weight
ratio is higher in younger plants, but its quantity increases with age however, stabilizing
in old plants (Morgado et al. 2005). According to Patón et al. (1998) the annual production
of forage biomass is higher in 1 to 3 years-old plants. Accordingly, Morgado et al. (2005)
observed that 1 and 3-years-old shrubland presented the higher percentage of productive
biomass (Table 1.6) but the highest absolute productivity of this kind of biomass was
achieved in 6 to 9-years-old shrublands. Resin productive or usable biomass (i.e., covered
by resin) comprise both soft/herbaceous shoots and leaves and semi-lignified biomass.
For the purpose of this work, this biomass is called photosynthetic biomass, as in Nuñez
et al. (1989).
Table 1.6: Total aboveground biomass and biomass fractions (photosynthetic biomass; wood biomass; and
capsules/fruit biomass) productivity (ton/ha and % of total biomass) of C. ladanifer shrublands according
to age (years-old), reported in literature.
Biomass Shrubland age (years-old)
fractions 1 3 4 5 12-15 15 10-18
Total biomass 0.94 1.9 5.8 6.5 3.64 16 16.06 17.7
Photosynthetic 0.76 1.65 3.48 3.18 1.22 3.2 1.61 4.58
biomass (81%) (87%) (60%) (49%) (34%) (20%) (10%) (26%)
0.18 0.25 2.32 3.32 2.39 12.8 14.66 12.36
Wood biomass
(19%) (13%) (40%) (51%) (66%) (80%) (91%) (70%)
Capsules 0.1 0.3 0.65
0 (0%) - - - -
biomass (3%) (2%) (4%)
Reference* A B B B A B A C
*A: Nuñez et al. (1989); B: approximate values extracted from a graphic in Morgado et al. (2005); C: Alves-
Ferreira et al. (2019a).
(Capsules productivity is included in woody biomass from Morgado et al. (2005)).
The different biomass fractions differ in their composition (Table 1.7), meaning that the
overall constitution of C. ladanifer plants or shrublands biomass also differ with age,
given the different biomass fraction ratios.
29
Extracts yield is different when using different solvents and overall higher extraction
yield are obtained by successive Soxhlet extraction with pure solvents (Alves-Ferreira et
al. 2020). Leaves rendered the highest extraction yield, nearly 50% (dw/dw) of the
biomass weight but branches and capsules also rendered a high extraction yield of about
30% (dw/dw). As shown for other similar solvents in other studies, dichloromethane,
ether and acetone extracts must be principally composed by labdanum resin and lipids.
Ethanol and water extracts were characterized by the authors to be rich in phenolic
compounds such as phenolic acids, flavonoids, and tannins. All solvent extracts presented
the highest yields when leaves were used, except for water extracts in which the highest
yield was found for capsules.
Lignin, mostly insoluble, and polysaccharides, mainly cellulose, contents were highest
for stems and then for branches and capsules, following an opposite pattern than that of
the extractives. Guerreiro et al. (2016) determined the insoluble fibre in acid and neutral
detergents. Fibre, principally the neutral detergent fibre includes polysaccharides,
excluding pectin, and lignin (Van Soest 1994). Given the material tested by Guerreiro et
al. (2016), soft stems and leaves, results for acid detergent insoluble lignin and for neutral
detergent fibre are near to the Klason lignin and total lignin + polysaccharides,
respectively, reported by Alves-Ferreira et al. (2020) in C. ladanifer leaves.
Table 1.7: Cistus ladanifer biomass and biomass fractions general chemical composition data, in %
(dw/dw), available in literature.
Soft
Parameter Stems Branches Leaves Capsules Whole plant
stems/leaves
Ash 2.2 4.1 5.2 3.9 3.1 3.1 4.0-5.3
Extracts 10.8 31.9 53.7 33.5 6.2 7.4 5.7-9.1
Acetone - - - - 6.2 - -
Ether - - - - - - 5.7-9.1
Dichloromethane 1.5 6.4 14.8 4.2 - - -
Ethanol 6.1 16.5 24.1 12.3 - - -
Water 3.3 9.0 14.9 17.1 - - -
Protein - - - - 9.2 - 5.5-10
Total lignin 21.2 18.6 15.4 15.8 - 34.2 -
Klason lignin 18.2 16.8 13.6 14.0 15.6 32 -
Acid detergent - - - - - - 8.6-12.5
Soluble lignin 3.0 1.7 1.8 1.8 - 2.2 -
Fibre - - - - - - -
Neutral detergent - - - - - - 32.1-41.0
Acid detergent - - - - - - 27.7-33.9
Polysaccharides 59.5 40.4 23.3 42.4 58.6 - -
Cellulose - - - - 34.9 - -
Hemicellulose - - - - 6.6 - -
Pectin and others - - - - 17.1 - -
Reference* A A A A B C D
*A: Alves-Ferreira et al. (2020); B: Ferro et al. (2015); C: Ferreira et al. (2009); D: Guerreiro et al. (2016)
30
In conclusion, wood biomass, present in higher ratios in older plants and shrublands, is
richer in lignin and polysaccharide content (fibre). In contrast, photosynthetic biomass,
more present in younger plants and shrublands, is richer in extractives and ashes.
Regarding lipids, fatty acids content, mostly saturated, was shown to be in the range of
0.5 - 0.9% (dw/dw) in soft leaves and stems (Guerreiro et al. 2015). The same authors
observed some variability in fatty acid content and composition with season, increasing
the overall content and the saturated fatty acids ratio in summer and autumn months. In a
later study, Jerónimo et al. (2020) found that within biomass fractions, capsules biomass
was the one with higher fatty acid content (1.0 - 2.3% dw/dw) and mostly composed by
unsaturated fatty acids. In contrast leaves and stems showed a fatty acid content of 1.4 -
1.8 and 0.3 - 0.4% (dw/dw), respectively, and mostly composed by saturated fatty acids.
Capsules bear seeds and those seeds may have influenced the results.
Krollmann and Gülz (1983) reported a lipid content of seeds of about 10% (dw/dw),
mostly composed by triacyclglycerols (78% dw/dw), sterols (8.9% dw/dw) and
phospholipids (4.1% dw/dw). Most of the derived free fatty acids were unsaturated,
mainly represented by linoleic acid (w6, 18:2) and linolenic acid (w3, 18:3). The main
saturated free fatty acid was palmitic acid (16:0). Jerónimo et al. (2020) also found those
free fatty acids as the major representatives of capsules fatty acids.
31
1.3. Integrative exploitation of Cistus ladanifer L.
In a review on the potentiality of C. ladanifer as a valuable resource, two different

approaches on production systems were proposed: long cycle silviculture exploitation of
natural shrubland and short cycle agriculture exploitation to recover oligotrophic and use
trace metal contaminated soils (Raimundo et al. 2018). These production systems are
intended to manage environmental problems such as fire risk and desertification.
Valorisation of C. ladanifer products may figure a way to reimburse the costs of or even
create profit from such management practices.
There are different types of C. ladanifer shrublands (section 1.1.4.3). Some shrublands
are continuously cleared to recover productive spaces and decrease fire risk (e.g., oak,
pine, and eucalyptus forests). In this context, it is relevant to mention the use of a blade
harvester and baler system (Biobaler WB55) coupled to a tractor to collect total
aboveground rockrose biomass of C. laurifolios L. (Bados et al. 2020) and C. ladanifer
(Mediavilla et al. 2021). Other shrublands do not have an economic value (e.g., dense
rockrose shrublands) and thus are poorly managed and constitute a high fire risk.
However, short term periodic clearings, reducing C. ladanifer population, of such
marginal areas may enhance desertification. A harvesting system, simulating a non-plant-
destructive harvesting, at a specific height, as it is traditionally done for labdanum
collection would most likely control accumulated biomass in the shrubland, decreasing
fire risks, while maintaining the cover of unused soils. This type of mechanical harvest
would be even more relevant in the context of cultivation of C. ladanifer to recover
oligotrophic and use contaminated soils.
It is possible to utilize whole C. ladanifer plants in several economic sectors such as food,
feed, cosmetic, pharmaceutical, agriculture and energy. A schematic representation of C.
ladanifer valorisation pathways based on traditional knowledge and recent research is
presented in Figure 1.8. Some valorisation pathways involve a direct application of plant
products (e.g., seeds, essential oil, labdanum resin) others may need further conversion
processes in the context of biorefining (e.g., capsules, photosynthetic, and wood
biomass).
32
Figure 1.8: Schematic representation of C. ladanifer products and economic sector of possible valorisation
pathways.
González et al. (2018) gathered information from several ethnobotanical studies on

traditional uses of C. ladanifer, highlighting a variety of possible applications for several
economic sectors of all parts and products from the plant:
• Food and feed sector: flowers and seeds used to be consumed as human food.
Goats and sheep feed directly from soft stems, leaves, floral buds, and capsules.
Goats were feed with flowers to increase milk production. Burn of branches was
used to smoke cheeses.
• Cosmetic sector: labdanum products have been used in perfumery and fragrance
sectors for their capacity to fixate aromas.
• Pharmaceutical/medicinal sector: Decoctions from C. ladanifer labdanum,
shoots, or flower buds were used to treat several human illnesses related to the
circulatory system (haemorrhoids and varicose veins), digestive system
(toothache, diarrhoea, stomach-ache, peptic ulcers, and liver disorders),
genitourinary system (vaginal infections and cervical polyps), respiratory system
(colds and whooping cough), endocrine-metabolic system (hyperglycaemia),
musculoskeletal system (as analgesic for rheumatism, sprains, and contusions),
integumentary system (hair loss, calluses, cracks, lesions, burns, chilblains,
eczema), nervous system (neuralgia), and others (mental disorders, tumours,
fever, antiseptic). Veterinary uses of such decoctions include wounds and
contusions healing and treatment of goats and sheep viral dermatitis (contagious
ecthyma).
33
• Energy sector: woody parts were used as high calorific power firewood and to
produce charcoal for home use and forges.
Essential oil from C. ladanifer has potential to be used in the perfumery (cosmetic) and
fragrance (other products such as candles and detergents) industries because of its
aromatic property (Frazão et al. 2018). In addition, it has preservative properties, due to
its antimicrobial and antioxidant activities, that may be use in perishable products such
as food (Luís et al. 2020) and cosmetic (Ramos 2019) products or even for plant/crop
protection purposes in the context of agriculture.
El Karkouri et al. (2021) reported a broad-spectrum antimicrobial activity of C. ladanifer

essential oil against several bacteria and fungi with MIC and MMC values below 100
µl/mL: 10 µl/mL for bacteria and 16-64 µl/mL for yeasts and moulds. Benali et al. (2020)
reported essential oil’s MIC values of 0.19 and 0.78 mg/mL for two phytopathogenic
bacteria. Ramos (2019) evaluated the antimicrobial activity of two essential oils against
skin cosmetics relevant bacteria and fungi, reporting MIC values between 0.5 and 128
µL/mL and MBC values between 8 and > 128 µL/mL, and also evaluated the antioxidant
activity of the essential oils by the DPPH method, reporting an EC50 of near 10 µL/mL.
Similarly, Zidane et al. (2013) evaluated the antioxidant activity of the essential oil by the
DPPH method at concentrations between 1 and 15 mg/mL, reporting always higher
radical inhibition compared to ascorbic acid. However, Guimarães et al. (2010) reported
lower antioxidant activity of C. ladanifer essential oil compared to methanol extracts
regarding DPPH radical inhibition (IC50 = 36.28 and 0.13 mg/mL, respectively) and
reducing power (EC50 = 4.00 and 0.19 mg/mL, respectively), but similar lipid
peroxidation inhibition (IC50 = 0.12 mg/mL). Tavares et al. (2020a) demonstrated a broad
antimicrobial activity and significant antioxidant activity of C. ladanifer essential oils
and, in addition, the by-product of the steam-distillation process (hydrolate) showed to
have potential anti-inflammatory activities.
Seed germination inhibition and phytotoxic activities of essential oil was demonstrated
by Verdeguer et al. (2012) which could then find uses in agriculture as a natural herbicide.
In general, essential oils may be explored for pharmaceutical uses such as antioxidant,
antimicrobial, anti-inflammatory, wound healing, and anxiolytic (Cimino et al. 2021).
Yarovaya and Salakhutdinov (2021) discuss the relevance of essential oils, principally of
monoterpenes herein, as building blocks for the synthesis of compounds with antiviral
34
activity but highlight the problem of their low and unstable availability to sustain a
production process. Given the low extraction yields of C. ladanifer essential oil (0.07 -
0.60%, v/w, section 1.2.1) compared to other plant species (Guimarães et al. 2010), such
so far non-specific applications may not be feasible. However, it may be used for
combined purposes as for its specific aromatic and overall preservative functions.
Labdanum resin has higher extraction yields than essential oil (7 - 18% dw/dw, Chapter
1, section 1.2.2.1). Labdanum resin and derivative products are used in perfumery and
fragrance industries because of its fixative and aromatic properties (Biolandes n.d.). It is
not only a substitute of ambergris, a restricted raw material that used to be obtained from
the protected sperm whale Physeter catodon, but also a source of precursor compounds
(e.g. labdanolic acid) for the synthesis of Ambrox®, the prototype compound of ambergris
derived compounds (Raimundo et al. 2018).
Valorisation pathways for labdanum resin may be considered based on traditional uses
but also based on its ecological functions (Figure 1.5, this chapter, section 1.1.4.4).
Labdanum resin is worth to be considered as an ingredient for skin care cosmetics or
dermocosmetics, satisfying the recent trends for the natural, organic, and sustainable,
because of documented traditional use on human and animal skin. Ecological function
such as UV protection, solar reflection, hydrophobic barrier, and air density increase are
relevant in the context of skin care products.
Labdanum resin may also figure an abundant ingredient or source of high valuable
compounds for pharmaceutical, and for agriculture uses. Regarding medicinal uses and
thus pharmaceutical applications, references from González et al. (2018) report more the
use of plant material water extracts than specifically of labdanum resin. Those extracts
are, however, done with the intact planta material, in a process very similar do the
Zamorean process used to extract labdanum resin. Within scientific literature, studies
evaluating bioactivities relevant to medicinal applications of labdanum resin are scarce
and concerns only antimicrobial activity (Greche et al. 2009; Rauwald et al. 2019). In
what regards agriculture applications, weed and pest control is possible given the
demonstrated allelopathic and anti-feeding activity of labdanum resin constituents
(Chapter 1, section 1.1.4.4).
Literature is vast regarding the evaluation of polar extracts from mostly C. ladanifer
photosynthetic plant material (leaves and soft stems). Those extracts are made from
35
milled plant material using a variety of processes and solvents and their chemical
composition is different from labdanum resin but may share some compounds (e.g.,
flavonoid aglycones), as discussed in section 1.2.3. Some scientific literature, from 2010
to 2021, regarding polar extracts bioactivities evaluation, are summarized in Table 1.8.
Extraction yields varied between 7 and 41% (dw/dw). Relevant biological activities such
as antioxidant, antimicrobial, cytotoxicity, antidiabetic, anti-hyperpigmentation, UV
protection, and hypotensive activities were confirmed at some extent using in vitro assays.
In vivo assays have shown the potential of water extracts for antidepressive,
immunomodulatory, analgesic and anti-inflammatory activities. Most of these activities
are related to traditional medicinal uses of water extracts from C. ladanifer and thus, they
are relevant for the pharmaceutical industry, either using these extracts as an ingredient
or a source of active compounds, and for skin care products, such as cosmetics (UV
protection, antioxidant, antimicrobial) or dermocosmetics/topical medicines.
Their potential use in food or feed sectors may be related mostly to its preservative
functions (antioxidant and antimicrobial). However, as feed additive, condensed tannins
extract from C. ladanifer soft biomass have shown to be beneficial to improve feed
protein utilisation during ruminant digestion, increasing their flux into the post ruminal
compartment and subsequently increasing the uptake of amino acids by the animal
(Dentinho et al. 2005; Dentinho et al. 2007; Dentinho et al. 2014).
In agriculture, proven feeding inhibition activity of flavonoids (section 1.1.4.4.) and UV

protection (section 1.1.4.4, and Table 1.8) indicate the possible use for plant protection
purposes. In addition, phytotoxicity of water-soluble compounds from C. ladanifer shoots
have been shown to inhibit early root growth of subterranean clover (Dias and Moreira
2002) and of wheat (Dias et al. 2004). Seed germination inhibition by C. ladanifer
aqueous extracts against some shrub and herbaceous species of the same habitat as C.
ladanifer is reported by Herranz et al. (2006). Phytotoxic and germination inhibition
activities may indicate a possible use of these extracts for weed control which is relevant
in the context of agriculture.
Essential oils, labdanum resin, and polar extracts compete for the same type of biomass
(soft, photosynthetic, or herbaceous). However, from labdanum resin a labdanum oil, very
similar to the essential oil regarding chemical profile and yield as discussed in section
1.2.2.2, may be obtained. In addition, traditional processes to obtain labdanum resin also
yields an aqueous supernatant that may be considered a polar extract. Perhaps a
36
consolidated extraction may be designed, yielding three products each one more suitable
to specific applications.
Table 1.8: Resume of scientific reports evaluating biological activities of polar extracts from C. ladanifer,
from 2010 to 2021.
Reference Extract Bioactivity test Main results
Barrajón- Water extract of In vitro antioxidant: -TEAC = 35.85
Catalán et al. milled aerial parts -ABTS (TEAC) mmolTE/100g
(2010) -TBARS -TBARS = 72.13%
Yield = 7 - 10% -ORAC inhibition (0.375 mg/mL)
dw/fw -FRAP -ORAC = 3329 µmolTE/g
-FRAP = 117.72
In vitro antimicrobial (dilution mmolFe2+/100g
method)
-Staphylococcus aureus MIC50:
-Escherichia coli -Staphylococcus aureus
(0.154 mg/mL) similar to
In vitro cytotoxicity (MTT) neomycin
-SKBr3 (breast carcinoma) -Escherichia coli (0.900
-MCF-7 (breast cancer) mg/mL)
-JIMT-1 (breast cancer)
-M186 (pancreatic CC50:
adenocarcinoma) -SKBr3 (16.10 mg/mL)
-M220 (pancreatic -MCF-7 (0.53 mg/mL)
adenocarcinoma) -JIMT-1 (1.69 mg/mL)
-HS-766T (pancreatic -M186 (6.34 mg/mL)
carcinoma metastasis) -M220 (0.49 mg/mL)
-HT29 (colon cancer -HS-766T (4.37 mg/mL)
carcinoma) -HT29 (8.27 mg/mL)
Guimarães Methanol extract of In vitro antioxidant: -DPPH (EC50) = 0.13
et al. (2010) freeze-dried -DPPH mg/mL
powdered leaves -RP (Prussian blue) -RP (EC50) = 0.19 mg/mL
-Lipid peroxidation (β- -LP (EC50) = 0.12 mg/mL
Yield = 41.24% carotene bleaching inhibition)
dw/dw
Ferreira et Acetone/water, In vitro antimicrobial (disk No activity against:
al. (2012) ethanol 95%, diffusion and dilution -Escherichia coli
methanol, and method): -Salmonella typhimurium
ethanol extracts of -Bacillus cereus -Candida albicans
stalks wood, bark, -Staphylococcus aureus MIC 0.625-2.5 mg/mL:
and leaves powder -Enterococcus faecalis -Bacillus cereus
-Pseudomonas aeruginosa -Staphylococcus aureus
-Klebsiella pneumoniae -Klebsiella pneumoniae
-Escherichia coli MIC > 2.5 mg/mL:
-Salmonella typhimurium -Enterococcus faecalis
-Helicobacter pylori -Pseudomonas aeruginosa
-Candida tropicalis
-Candida albicans
Barros et al. Methanol/water In vitro antifungal (dilution MIC < 0.5 mg/mL (strong):
(2013) extract of freeze- method): -Candida albicans
dried leaves -Candida albicans -Candida glabrata
-Candida tropicalis -Candida parapsilopsis
-Candida glabrata MIC = 0.625 mg/mL
-Candida parapsilopsis (médium):
-Candida tropicalis
Bousta et al. Water extract of In vivo (rats): LD50 = 450 mg/kg

(2013) leaves powder -Acute toxicity (LD50)
150-200 mg/kg:
37
Yield = 21% dw/dw -Antidepressant (swimming -no mortality

test). -reduction of immobility.
-Immunomodulatory (blood -reduction of lymphocytes
sample lymphocytes, flow
cytometry).
Tomás- Water and In vitro antimicrobial (dilution MIC50:
Menor et al. ethanol/water extracts method): -Staphylococcus aureus:
(2013) of milled plant -Staphylococcus aureus 0.144-0.569 mg/mL
material -Escherichia coli -Escherichia coli: 0.113-
0.612 mg/mL
Zidane et al. Water, ethanol, In vitro antioxidant: -Concentrations tested: 1-15
(2013) ethanol/water, -DPPH mg/mL.
methanol, -DPPH % inhibition similar
methanol/water to ascorbic acid.
extracts of Fruits, -Water extracts less active at
stems, leaves, and lower concentrations.
flowers powder.
Guerreiro et Acetone 70% extract In vitro antioxidant: -Higher antioxidant activity
al. (2016) of dried and -DPPH of plants collected in
grounded aerial parts -RP (Prussian Blue) Summer.
Lourenço Water extract of dried In vitro antioxidant: -TAA: 0.498
(2016) pawdered aerial parts -TAA (phosphomolybdenum gAscorbicAcid/gExtractdw
method) -RP: 0.361 gTE/gExtractdw
-RP (Prussian blue) -FRAP: 0.603 gTE/gExtract
-FRAP dw
-DPPH -DPPH (IC50) = 19.47
-ABTS µg/mL
Antidiabetic: -ABTS (IC50) = 102.82
-α-Amylase inhibition µg/mL
-α-Glucosidase inhibition
-α-Amylase: 0.674-0.947
mgAcarbose/gExtractdw
-α-Glucosidase: > 4.029
mgAcarbose/gExtractdw
Youbi et al. Water extract of In vivo (rats): Analgesic Index:
(2016) leaves powder -Central analgesic activity (hot -150 mg/kg: 70.30%
plate method) -175 mg/kg: 74.55%
Yield = 21% dw/dw -Anti-inflammatory activity -200 mg/kg: 93.33%
(carrageenan induced acute -Tramadol (6.66 mg/kg):
inflammation) 44.46%
Edema inhibition:
-150 mg/kg: 66.67%
-175 mg/kg: 67.65%
-200 mg/kg: 77.78%
-Indomethacin (2mg/kg):
66.67%
Köse et al. Ethanol/water extract In vitro antimicrobial (dilution MIC:
(2017) of leaves powder method): -Escherichia coli (25
-Escherichia coli mg/mL)
Yield = 19.05% -Staphylococcus aureus -Staphylococcus aureus
dw/dw -Staphylococcus epidermidis (1.56 mg/mL)
-Staphylococcus epidermidis
(0.58 mg/mL)
Chaves et al. Methanol extract of In vitro antioxidant: -DPPH: 0.030
(2020) dried grounded leaves -DPPH mgQuercetin/mgExtract dw
-ABTS -ABTS: 0.010
-RP (Prussian blue) mgQuercetin/mgExtract dw
-FRAP -RP: 0.022
mgQuercetin/mgExtract dw
38
-FRAP: 0.071
mgFeSO4/mgExtract dw
Gaweł- Methanol and In vitro antioxidant: -DPPH (IC50) = 4.08 - 10.20
Bęben et al. methanol/water -DPPH µg/mL
(2020) extract of dried In vitro Cytotoxicity (neutral
leaves and stems (no red): -A375 (IC50) = 95.30-199.01
indication of milling -A375 (malignant melanoma) µg/mL
or grounding) -SCC-15 (squamous cell -SCC-15 (IC50) > 500
carcinoma) µg/mL
-HaCaT (immortalised -HaCaT (IC50) > 500 µg/mL
keratinocytes)
Antihyperpigmentation: -At maximum concentration
-Mushroom tyrosinase tested (200 µg/mL)
inhibition. inhibition between 0-50%
In vitro SPF (Kojic acid near 100%)
-At 100 µg/mL, SPF of

3.33-4.37
Boy et al. Ethanol 90% extract In vitro antioxidant: -DPPH = 2.4 gTE/100g
(2021) of plant material (no -ABTS fresh plant
indication of milling -DPPH -ABTS = 0.5-0.8 gTE/100g
or grounding) Hypotensive: fresh plant
-inhibition of angiotensin-
converting enzyme, ACE -IC50(ACE) = 5.8-11.3
In vitro antimicrobial (drop µg/mL
diffusion):
-Staphylococcus aureus Diameter inhibition zones
-Bacillus cereus -S. aureus (3.22 mm)
-Listeria monocytogenes -B. cereus (3.00 mm)
-Listeria innocua -L. monocytogenes (1.85
-Salmonella choleraesuis mm)
-Escherichia coli -L. innocua (3.23 mm)
-Candida boidinii -S. choleraesuis (7.82 mm)
-Priceomyces carsonii -E. coli (2.86 mm)
-Kregervanrija fluxuum -C. boidinii (7.55 mm)
-Zygosacharomyces bailii -P. carsonii (8.11 mm)
-K. fluxuum (7.83 mm)
-Z. bailii (7.69 mm)
Bakrim et al. Successive In vitro antioxidant: -DPPH (IC50) = 0.6-0.81
(2021) extractions with -DPPH mg/mL
hexane,
dichloromethane, In vitro antimicrobial (disk MIC/MBC (methanol
methanol, and water diffusion, dilution, kinetics extract):
of dried powdered and cell adhesion): -S. aureus (8.75/ > 40
leaves. - Enterococcus faecalis mg/mL)
-Listeria monocytogenes - no activity against other
Yield: -Staphylococcus aureus bacteria
-Methanol: 15.84% -Pseudomonas aeruginosa -C. albicans (8.75 / 8.75
-Water: 21.24% -Salmonella typhimurium mg/mL)
-Escherichia coli - not performed against
-Candida albicans other fungi
-Botrytis cinérea - no activity of water extract
-Fusarium oxysporum
Anti-biofilm effect (4.38-
17.5 mg/mL):
-S. aureus: 26.9-51.9%
-C. albicans: 45.1-74.3%
-Not tested against other
microorganisms
39
Lukas et al. Water extract of dried In vitro antioxidant: -DPPH = 0.3 gTE/gExtract
(2021) powdered leaves -DPPH dw
The biorefinery concept and biorefinery platforms aim to convert renewable biomass into
a variety of products such as chemicals, fuels, food/feed ingredients, and materials,
minimizing waste (Castilla-Archilla et al. 2019). According to the authors, first and third
generation biorefineries use crops and microalgae as feedstock, respectively, while
second generation biorefineries use industrial, agro-industrial, and forestry wastes as
feedstock. The main advantage of second generation biorefineries is the non-competition
for farmland and with the food and feed sectors but the main drawback is the seasonal
availability of raw materials and, in the case of forest wastes, the complex lignocellulosic
raw materials which require pre-treatments before conversion to simple chemicals.
After extracting essential oil, labdanum resin or polar extracts from photosynthetic
biomass, a soft biomass is left and may be used as roughage for ruminants. In fact, goats
and sheep use to eat leaves, soft stems, and flowers of C. ladanifer during pasture
(González et al. 2018). At some extent, this soft biomass may substitute a portion of
dehydrated lucerne, in lamb feeding, without compromising their growth and even
increasing meat oxidative stability (Jerónimo et al. 2012; Francisco et al. 2015).
An integrated fractionation process of C. ladanifer biomass leftover of essential oil

distilleries, i.e., after essential oil extraction, was proposed by Alves-Ferreira et al.
(2019b). Firstly, the authors obtained a polar extract at a near 40% (dw/dw) yield by
successive ethanol and water extract. Afterwards, the extracted biomass was subjected to
a hydrothermal process to yield a liquid phase composed of hemicellulose
oligosaccharides (15%, dw/dw yield) and a solid phase composed of lignin and cellulose
(Alves-Ferreira et al. 2019a). The solid phase was then delignified by several methods
(highest yield of 87% using an alkali process) resulting in lignin extract rich in phenolic
compounds and a cellulose enriched solid (Alves-Ferreira et al. 2021). Finally, both
hemicellulose oligosaccharides hydrolysate and the cellulose enriched solid was
fermented to yield D-lactic acid which has several applications in food, cosmetic, and
pharmaceutical sectors and for biopolymers (Alves-Ferreira et al. 2022). Otherwise,
Carrión-Prieto et al. (2019) report that in addition to C. ladanifer biomass being rich in
cellulose the crystallinity results of the fibre is within the range of values for wood pulp,
discussing its cellulose-derived application such as packaging and as reinforcement in
40
composite materials. The initial biomass was reported to be composed of leaves, bark,
and branches. However, this proposed fractionation process could be designed to be used
just on wood biomass or photosynthetic biomass which most likely would mean different
processes and outputs (e.g., photosynthetic biomass would yield none or less lignin).
Biomass from C. ladanifer may also be valorised as a renewable and carbon neutral
energy source. According to Carrión-Prieto et al. (2017b), biomass from C. ladanifer may
be used to make wood pellets for industrial and home heating, however foliage biomass
(i.e., photosynthetic biomass) must be avoided to meet requirements of ash percentage
and heating value. After a pre-treatment to separate lignin and hemicelluloses, cellulose
may be used to produce the liquid fuel bioethanol. Ferreira et al. (2009) report a study on
optimisation of the saccharification process of C. ladanifer biomass after a pre-treatment
with diluted acid. Ferro et al. (2015) report that steam explosion pre-treatment and
simultaneous saccharification and fermentation increased the velocity and efficiency of
bioethanol production from 10-years old shrubs (high wood biomass). According to
González et al. (2018) biomass from C. ladanifer was traditionally used to make charcoal
with high heating power. In this context, Nuñez et al. (1989) obtained charcoal with a
yield of 32 - 45% and a heating value comparable to other trees, reporting a similarity
between photosynthetic and wood biomass and discussing the value of other products
from pyrolysis such as liquids (acetic acid, methanol, and acetone) and gaseous (methane,
hydrogenous, ethylene, and carbon dioxide). Encinar et al. (2020) report the production
of high heating value gases (H2 majority, CO, and CO2) from C. ladanifer charcoal using
a catalytic steam gasification process.
Capsules of the year are produced in flowering shoots of C. ladanifer and thus are usually
harvested together with photosynthetic biomass in the following Summer. These fruits
are reported to be eaten by goats and sheep during pasture (González et al. 2018) and deer
(Malo and Suárez 1996) and, therefore, they may be used for animal feed purposes. As
discussed in section 1.2.4, capsules represent the biomass fraction with higher fatty acids
content however it may be because of the seeds they bear. Those seeds alone are reported
to be traditionally eaten by humans (González et al. 2018) but studies regarding this
application are inexistent. If seeds are isolated, a significant amount of capsules material
are left. El Farissi et al. (2017) report the production of bio-oil as the main output but also
of solid residue (charcoal) and gas from capsules material using a slow pyrolysis process,
41
discussing the application of the bio-oil as a renewable fuel or a source of chemicals.

Nevertheless, the authors did not report to have separated seeds from the capsules.
42
1.4. Research objectives
The general objective of this doctoral thesis was to contribute with knowledge for Cistus
ladanifer exploitation and valorisation, addressing existent gaps from production to
added-value applications. For this purpose, specific objectives were purposed:
• Evaluate a natural C. ladanifer shrubland management practice, simulating a non-

destructive mechanized harvest process based on the traditional way to harvest,
regarding biomass and labdanum resin yields.
• Evaluate a cultivation experiment of C. ladanifer regarding biomass yields.
• Evaluate the potential of in vitro plant tissue cultures for clonal propagation and
secondary metabolite production.
• Evaluate the nutritional composition of C. ladanifer seeds.
• Evaluate traditional processes to extract labdanum resin regarding extraction
yields and effluent quality.
• Evaluate the potential use of labdanum resin as a cosmetic/cosmeceutical
ingredient by assessing relevant in vitro bioactivities such as antioxidant,
antimicrobial, elastase inhibition, UV protection, and anti-inflammatory
activities.
• Confirm some medicinal uses of labdanum resin and evaluate it as a potential
source of pharmaceuticals by assessing relevant in vitro bioactivities using
standard enzymatic assays (to assess antidiabetic and neuroprotective potential)
and in vitro human cell cultures (cytotoxic activity).
43
44
Chapter 2
Rockrose Production
Subchapter 2.1
Rockrose land management: contribution of periodic harvesting to

increase value and control Cistus ladanifer L. shrublands.
Abstract
Cistus ladanifer L (Cistaceae) occupies, as dense shrublands, extensive areas and is also
present in other major forest typologies in Iberian Peninsula. Cistus ladanifer shrublands
are mostly present in oligotrophic lands with little valorisation and management and, as
they grow old (up to 20 years-old), they promote the ignition and perpetuation of fire. To
contribute for proper management and valorisation practices of such systems, a 5-years-
old dense shrubland was evaluated for its biomass, labdanum resin, and capsules/seeds
yields using different non-destructive harvest periodicities (every year and after two
years) and seasons (early, mid-, and late summer), in a two-year experiment. Every-year
harvest modality accumulated the highest photosynthetic biomass production (2902 ± 705
kg/ha) but negligible capsules production after two years. Harvest after two-years of
growth yielded half of the photosynthetic biomass from the every-year harvest modalities
but higher capsules production (259 ± 115 kg/ha), like the initial 2019 harvest, and higher
wood biomass production (750 ± 345 kg/ha), although lower than the initial 2019 harvest.
In this study we propose two modalities of periodic harvest to be considered as proper
long cycle management practices of rockrose lands minimizing fire risks and increasing
profit from such forest systems based on three direct outputs with a myriad of applications
and valorisation pathways.
Keywords
Cistus biomass production; Cistus phenology; Cistus seeds production; Labdanum

production; Shrubland management.
This subchapter was submitted for publication as:
Frazão DF, Gonçalves JC, Silva AM, Delgado F Rockrose land management: contribution of periodic
harvesting to increase value and control Cistus ladanifer L. shrublands. (Under review)
45
Subchapter 2.1 Rockrose land management: contribution of periodic harvesting to increase value and
control Cistus ladanifer L. shrublands.
46
2.1.1. Introduction
Cistus ladanifer L. belongs to the Leucocistus subgenus, Ladanium section (Demoly and
Montserrat 1993) and to the white-flowered lineage group supported by the phylogenetic
studies (Guzmán and Vargas 2005; Guzmán et al. 2009; Guzmán and Vargas 2009;
Civeyrel et al. 2011). Within this species, three subspecies are recognized: C. ladanifer
subsp. ladanifer, C. ladanifer subsp. sulcatus (former C. palhinhae Ingram) and C.
ladanifer subsp. africanus (sometimes reported as C. mauritianus or C. petiolatus).
Within C. ladanifer two varieties are reported: albiflorus and maculatus. Cistus ladanifer
have a western Mediterranean distribution (south of France, Iberian Peninsula, North
Marrocos and Argelia). Subspecies ladanifer shrublands grow in the meridional half of
the Iberian Peninsula over siliceous soils and in the occidental half over shales and
granitic soils. Subspecies africanus is distributed over the north of Africa (Argelia and
Marrocos) and in the south of Iberian Peninsula (Málaga), preferring serpentine and
ultrabasic soils. Subspecies sulcatus grows exclusively in windy and sandy places over
limestone rock of the extreme south-west of Portugal (Demoly and Montserrat 1993).
According to the latter authors, subsp. ladanifer has been cultivated and possibly
naturalised outside of its endemic area. In South Africa (South-West, Fynbos) C.
ladanifer is classified as an invasive species, posing a significant threat to local endemic
vegetation (Du Plessis et al. 2018). In Australia, C. ladanifer and C. x purpureus (C.
ladanider x C. creticus) have been recently cultivated to produce essential oils (EOPAA
2017). Dias et al. (2019) cite some references regarding the presence of C. ladanifer in
some California counties, USA. Cistus ladanifer is reported to had been cultivated in
China for research purposes (Li et al. 1991).
In 2005, C. ladanifer dense shrublands, characterized by a widely open tree layer of

essentially cork-holm oaks and a very dense shrub/herb layer of C. ladanifer associated
with Lavandula spp. and other shrub species, was the 5th forest typology occupying more
land (249.382 ha), representing 7.5% of the Portugal mainland forest area and
concentrated mainly in the south (Godinho-Ferreira et al. 2005). Furthermore, although
not dominant, this species was present on many other forest typologies, such as cork, pine
and eucalyptus forests, which together accounted for 79.9% of the Portugal mainland
forest area which in turn occupied approximately 37% of the mainland. A more recent
estimation reports that C. ladanifer occupies 2.450.857 ha in Spain mainland widely
associated to Quercus spp. and Pinus spp. forests, 460.088 ha of those are occupied by C.
47
ladanifer dominated shrublands (Montero et al. 2020). The agro-forestry-pasture system

known as “montado” in Portugal or “dehesa” in Spain is a “multi-layered dryland system”
where evergreen oaks associate with shrubs, such as C. ladanifer, and native pastures or
crops, and from which several benefits and services are supposed to be achieved in a
sustainable manner, such as animal raising, hunting, and firewood production (Ruiz-
Peinado et al. 2013).
In fact, C. ladanifer shrubland is one of the most widespread shrubland in the Iberian
Peninsula, very poor in other plant species and a tendence to perpetuate instead to develop
into mature vegetation stages, according to Mendes et al. (2015). Rather than a pyrophyte
species, Trabaud (1995) classified this species as opportunistic, occupying open disturbed
areas without intense vegetative cover and strong competitors. Cistus ladanifer
communities comprise an early secondary succession stage of areas after perturbations
such as typical Mediterranean fires or human activity, for instance, the cleaning of areas
through cutting and ploughing and then their abandonment (Alonso et al. 1992; Tárrega
et al. 1995; Luiscalabuig et al. 1996; Tárrega et al. 2001). Reproductive success, low
nutritive demands, and resilience/tolerance to stress factors explains the regeneration and
perpetuation of such systems. According to Simões et al. (2009) C. ladanifer shrubs
improve soil quality in the 0 - 5 cm topsoil layer, through litter-fall, by enhancement of
organic matter and nutrient content beneath their canopies. According to the authors, this
soil recuperation after a perturbation (e.g., fire) may favour the posterior establishment of
more demanding woodland species such as Quercus species, following a typical
secondary succession mechanism. However, the resilience of these populations is well
demonstrated by their auto-succession process potentiated by the persistent soil seed
bank, allelopathy, high plant cover and relatively short period perturbations.
Besides preventing a more advanced land degradation and protecting wildlife

biodiversity, C. ladanifer shrublands have recently been regarded as relevant carbon
sinks, which is important because a key mitigation strategy for climate changes is the
sequestration of greenhouse gases such as CO2 (Ruiz-Peinado et al. 2013; Alías et al.
2015; Carrión-Prieto et al. 2017a). In addition, C. ladanifer potential role to occupy trace-
elements contaminated soils producing heavy metal free biomass or for phytoextraction
of Zn and Mn has been discussed (Raimundo et al. 2018).
Hernández-Rodríguez et al. (2017) proposed long cycle total clearings (24 years) as a
suitable management practice of C. ladanifer natural shrublands based on incomes just
48
from biomass harvesting and mushroom (Boletus edulis) picking. In fact, C. ladanifer
biomass, mainly the wood fraction, may be considered for pellets (Carrión-Prieto et al.
2017b), biochar and biogas (Encinar et al. 2020), and bioethanol (Ferro et al. 2015)
production. Biomass may also be transformed into lignin-derivatives and glucan rich
solids to be used in bioconversion processes (Alves-Ferreira et al. 2021), such as lactic-
acid production (Alves-Ferreira et al. 2022) and valorised as a source of cellulose with
wood pulp like crystallinity (Carrión-Prieto et al. 2019). Leaves and soft stems as well as
condensed tannin extracts have been pointed out for ruminant feeding at some extent
(Jerónimo et al. 2012; Dentinho et al. 2014; Francisco et al. 2015) However, such
management practice constitute a high fire risk as pointed out by Oria-de-Rueda et al.
(2008). In addition, indirect economic activities such as hunting and beekeeping, and
direct outputs such as labdanum resin (or even essential oil) and capsules have not been
considered.
C. ladanifer seeds nutritional value is addressed in this work (chapter 4) and capsules
material may be valorised as biofuel (El Farissi et al. 2017) or considered for ruminant
and monogastric feed in “montados” extensive animal production systems (Rodrigues et
al. 2021).
Labdanum is the resin exuded and covering photosynthetic biomass of C. ladanifer (Sosa
et al. 2005). It is nowadays mostly valorised in the perfumery and fragrance industry but
has potential to be used in the cosmetic and pharmaceutical industries given its known
bioactivities (Raimundo et al. 2018). During summer, one of the traditional and most used
collection practices involves the harvest of resin productive biomass from which the resin
is then extracted using alkaline warm water and acidic precipitation, a process known as
the Andalusian process. This harvest practice is non-destructive of the shrubland and may
reconcile with the management practice proposed by Hernández-Rodríguez et al. (2017).
We proposed, in this study, to evaluate the effect of the traditional harvesting practice for
resin collection but simulating a machinery operation with a constant cut height, in a
natural C. ladanifer shrubland to contribute for a best management practice of rockrose
lands based on biomass, labdanum resin, and capsules/seeds productivity. Harvest
periodicity (one or two years) and season (early, mid-, and late summer) were set as
independent factors and compared regarding the raw material’s productivity.
49
2.1.2. Material and methods
2.1.2.1. Characterisation of the study area
The natural C. ladanifer shrubland, selected to carry out the study, was located in Penha
Garcia, Idanha-a-Nova, Castelo Branco, Portugal (GPS coordinates in DMS: 40º01’01’’N
6º59’06’’W), land property of Sociedade Agrícola de Couto de Penha Garcia, Lda.
According to the agricultural operations records, the field selected was carved clean in
2014 during spring, meaning that at the beginning of the study (2019) it was a 5-year-old
shrubland. According to the soil chart of the region (Agroconsultores 2004) the selected
field is located in Dystri-Epileptic Regosols. The soil is loamy, acidic, with low level of
organic matter (OM) and available phosphorous, medium level of available potassium,
and low levels of exchangeable bases (Table 2.1.1). The climatic conditions of the region
(climatological normal 1986-2015) are characteristic of the Mediterranean climate:
annual mean temperature of 15.0oC with a mean maximum temperature of 21.5oC and
mean minimum temperature of 9.4oC, total annual mean rainfall of 735 nm concentrated
in autumn, winter, and spring months, with a dry season during summer months (June -
August) when monthly mean rainfall is lowest and monthly mean temperature is highest
(Monteiro 2016). According to the biogeographic typology of Portugal mainland (Costa
et al. 1998) the study area presents a Genisto hirsutae-Cistetum ladaniferi climatophilous
vegetation (Ocidental Mediterranean region, Ibero-atlantic super-province, Luso-
Extremadurence province, Cacerence super-district), dominated by Cistus ladanifer and
Genista hirsute, but Labiatae species (e.g., Lavandula Sect. Stoechas) were also
representative.
Table 2.1.1: Main physiscal and chemical properties of the natural C. ladanifer shrubland soil.
Parameters Values
Sand 56.4
Texture class Silt 24.7
(%) Clay 18.9
OM 2.36
Available phosphorus (P2O5 mg/kg) 7
Available potassium (K2O mg/kg) 99
Calcium 280
Exchangeable bases Magnesium 51.4
(mg/kg) Potassium 57.4
Sodium 8.86
pH (H2O) 5.4
50
2.1.2.2. Experimental plots delimitation and evaluation
The natural shrubland was divided in 10 x 10 m (approx.) square plots using a 4 m wide
disc harrow coupled to a farm tractor to clean the space between and delimit the plots. A
total of 48 plots were delimited in excess for posterior selection. Five 2 x 2 m squares in
each plot (near the 4 vertices and in the middle of plot) were evaluated, considering the
plants with the main trunk inside the square. At the height where each plant was wider,
maximum and its perpendicular diameters were measured and used, as a mean diameter,
to estimate the cover area considering it a circle. After that, cover areas of all plants were
added up to a total cover area which was then expressed as percentage of cover (% cover)
in relation to the 2 x 2 square area. A mean % cover was calculated for each plot using
the values from the five sampling squares. Other data such as plants height and density
were also recorded.
2.1.2.3. Soil analysis
A composite soil sample was collected at 20 cm depth, using a soil sampling probe, in
October of 2019, representing the study polygon with a total area of 0.83 ha. Soil sample
was analysed for its granulometry (Robinson's Pipette Method), pH (H2O) (ISO
10390:2005), organic matter (modified Walkley and Black method), available
phosphorous and potassium (Égner method), and exchangeable bases (extraction with an
ammonium acetate solution buffered at pH 7.0 and quantified by atomic absorption
spectrophotometry) in the soil laboratory of the School of Agriculture of Polytechnic
Institute of Castelo Branco.
2.1.2.4. Soil seed bank evaluation
Soil seed bank was evaluated based on Ferrandis et al. (1999) method. In 2019, at the
time of plots delimitation, 3 soil samples were collected, with an undisturbed soil sample
probe with 4.5 cm diameter and 6.0 cm height, at 9 spots along the experimental polygon,
making up 9 samples. Soil samples were dried at 30oC in a ventilated chamber and gently
disaggregated. Afterwards, samples were sieved at 0.5 cm to remove coarse material and
at 0.05 cm to retain C. ladanifer seeds which was also used to wash the sample with water.
The washed samples were dried and weighted. Five sub-samples with 2 g were used to
count and isolate C. ladanifer seeds under a magnifying glass and the mean of the 5 sub-
samples was used to estimate seed density (count/m2) from each sample.
51
Isolated seeds were mixed and separated in four batches of fifty seeds, two of which were
placed in a 100oC pre-heated ventilated oven for five minutes. Afterwards, all seeds were
disinfected by immersion in a 10% v/v of sodium hypochlorite solution for five minutes
followed by washing with sterilised distilled water. Seeds of each batch were placed over
a glass Petri dish above a sheet of filter paper kept moistened with sterilised distilled
water during the experiment. Seeds were placed in the culture room with a 16 h
photoperiod (“cool white” fluorescent lamps, 45±5 µmol/m2s PPFD). Culture room
temperature was maintained at 25oC/day and 22oC/night. Seeds showing radical
emergence were recorded as germinated and removed from the dishes every day until no
response was observed during two consecutive weeks and at least after one month.
Average accumulated percentage of germinated seeds was plotted against the duration, in
days, of the trial to obtain the average germination curves for each group of seeds. Final
germination percentage (FGP), mean germination time (MGT), and the time to reach 50%
of germination (T50) were calculated according to the following equations (Dastanpoor
et al. 2013):
𝐹𝐺𝑃 (%) = 𝑛⁄𝑁 × 100 (1)
Where, n is the number of germinated seeds and N is the number of total seeds tested.
𝑀𝐺𝑇 = ∑ 𝐷𝑛⁄∑ 𝑛 (2)
Where, n is the number of seeds germinated on D day and D is the number of days counted
from the beginning of the experiment.
(𝑁⁄2 − 𝑛𝑖 ) × (𝑡𝑖 − 𝑡𝑗 ) (3)

𝑡50 = 𝑡𝑖 +
𝑛𝑖 − 𝑛𝑗
Where, N is final number of germinated seeds, and ni and nj are the most adjacent
cumulative number of germinated seeds at times ti and tj, respectively, regarding ni < N/2
< nj.
2.1.2.5. Experimental design
The experiment was designed with two combined harvest factors (harvest modalities):
periodicity and season of the harvest. The first factor comprised two levels (periodicities):
every year (1Y) and at the end of the study, two years (2Y). The second factor comprised
three levels (seasons): harvest at early summer (late June/early July, ES), mid-summer
(August, MS) and late summer (late September/early August, LS). Four plots were
52
assigned to each harvest modality (4 x 2 x 3 = 24 plots) and four additional plots were
assigned to the control group, used only for phenological evaluation. In total, 28 plots
were needed. Based on the evaluation of the initial 48 plots (section 2.1.2.2.), all plots
with a % cover higher than 60% were included in the study and distributed randomly
between harvest modalities: plots over 80% cover area were randomly distributed and,
afterwards plots with a 60 - 80% cover were again randomly distributed.
2.1.2.6. Phenological evaluation
Three plants at each plot, meaning twelve plants per harvest modality and control, were
selected for continuous monitoring of the phenophase, once a month. Vegetative growth
was monitored by measuring the length of a brachyblast shoot, labelled during summer,
from the base to the top of the selected shoot. Flowering was monitored as the presence
or absence in each plant. Seed dispersion was considered when most of the capsules of
the monitored plant were opened and considered finished when all plants were in seed
dispersion phenophase. Observation periods (once a month) were transformed in periods
of 30 days, considering a constant dairy vegetative growth within that observation period.
2.1.2.7. Meteorological data
Meteorological data (daily mean temperature and daily total rainfall), since the beginning
of the study (August 2019) until its end (September 2021), were kindly provided by
Instituto Português do Mar e Atmosfera (IPMA), recorded by their nearest station: Main
Automatic Station nº 570, Castelo Branco (GPS coordinates in DMS: 39°50'22.00"N
7°28'43.30" W). Daily total rainfall data within each observation period was used to
calculate the mean daily rainfall of that period.
2.1.2.8. Harvest evaluation
The effect of the harvest factors was evaluated in relation to phenology, biomass yield,
and resin yield. At each plot, biomass was harvest using a hedge trimmer (STIHL HS 45)
to cut the plants at 50 cm high which were then collected by hand and joined as bales to
be weighted in a semi-industrial balance (BW-M, Libra Weighing Machines, Ltd.). After
weighting, three biomass samples consisting of 3 - 5 branches each were divided in type
of biomass: wood biomass, photosynthetic biomass (which includes herbaceous shoots
and stems with some degree of lignification), and capsules. Those samples were weighted
using an analytical balance (d = 0.001, Sartorius, model ENTRIS323I-1S). Afterwards,
53
wood biomass was discarded whereas photosynthetic biomass was stored in the freezer
until labdanum extraction. Photosynthetic biomass comprises both soft/herbaceous shoots
and leaves and semi-lignified biomass, covered by resin/exudate. Capsules were smashed
to release seeds which were separated by sieving with a metal mesh of 850 µm aperture
and weighted.
2.1.2.9. Labdanum resin extraction
Based on Burguer (2016) slightly modified method, simulating the traditional Andalusian
method, 25 g of photosynthetic biomass, cut into small pieces, were used for extraction
with 200 mL of Na2CO3 (Biochem, Frilabo, Portugal) aqueous solution (25 g/L). The
extraction was done inside a 250 mL shot flask for 1 h at 60oC using a water bath.
Afterwards, the solution was filtered through a stainless-steel mesh (106 µm aperture)
and left to rest and cool down overnight. The extraction solution was then subjected to
acidification to precipitate the whole resin. For that purpose, H2SO4 (H2SO4 97% Chem-
Lab, JMGS, Portugal) solution (5 M) was added drop by drop to the solution, under
agitation, until pH = 2 was reached. The precipitated solution was homogenized and
separated in 16 mL centrifuge tubes for centrifugation at 3030 g for 15 min (Mega Star
600R, VWR). Labdanum resin was removed from the centrifuge tubes using distilled
water and joined in a previous weighted flask. The resin was frozen at -80oC for 24 h and
then freeze-dried to remove the water at 0.3 mbar for 72 h (Zirbus VaCo 10-II-D). After
the drying process, the flasks were weighted again, and labdanum yield was assessed by
finding the reason between the total resin dry weight (dw) and the plant material fresh
weight (fw). Labdanum was extracted from four samples of different plots collected at
each season and year (n = 4). Mean values and standard deviation were used to calculate
productivity of each plot given the productivity values of photosynthetic biomass.
2.1.2.10. Statistical analysis
Data sets were evaluated for normality (Shapiro-Wilk’s test, ρ ≥ 0.05) and homogeneity
of variances (Levene’s test, ρ < 0.05). Some data sets were transformed using ln(x),
2
ln(x+1), and √𝑥 to improve the homogeneity of variances between data sets. ANOVA, t-
test, two-way-ANOVA, post-hoc Tukey’s tests were used to compare mean values
between data sets normally distributed and with homogeneity of variances. Welch’s
ANOVA and post-hoc Dunnett’s T3 tests were used to compare mean values between
data sets normally distributed and with homogeneity of variances not assumed. Kruskal-
54
Wallis non-parametric test was used to compare mean values between data sets not
normally distributed. Spearman´s rank order was used to test correlation between non
normally distributed variables. Statistical analyses were performed for a confidence level
of 95% (α = 0.05) using the IBM SPSS Statistics 27 Software.
2.1.3. Results and discussion
2.1.3.1. Initial characterisation
Cistus ladanifer vegetation parameters of the 24 experimental plots, selected for testing
the harvest modalities, at the beginning of the study are presented in Table 2.1.2. No
significant differences were found between plots assigned to each harvest modality
(ANOVA or Welch’s ANOVA ρ-value > 0.05). Using the non-linear model for total
biomass estimation of C. ladanifer shrublands and the mean root to shoot biomass ratio
of 0.56 reported by Ruiz-Peinado et al. (2013), in 2019 mean total aboveground biomass
of experimental plots was estimated to be 9389 ± 3405 kg/ha. For 4/5-years old
shrublands, Nuñez et al. (1989) reported a total dry aboveground biomass of 3.6 ton/ha,
Morgado et al. (2005) reported a total aboveground biomass 6.5 ton/ha, and Hernández-
Rodríguez et al. (2017) estimated a total aboveground biomass of 6.6 ton/ha.
Table 2.1.2: Vegetation parameters of the selected C. ladanifer shrubland.

Mean plant Mean plant
Plant density Cover Estimated biomass
height cover area
2 (plants/m2) (%) (kg/ha)*
(m) (m )
1.10 ± 0.29 0.872 ± 0.730 1.62 ± 1.17 76.9 ± 28.9 9389 ± 3405
Values presented as mean ± standard deviation (n = 120)
*Calculated according to Ruiz-Peinado et al. (2013).
The soil of the natural shrubland showed to have a very dense and viable seed bank able
to germinate with or without a typical fire event (Table 2.1.3). Seed densitiy in the 0 - 6
cm top soil layer (57.7 x 104 seeds/m2) was much higher than the one reported by
Ferrandis et al. (1999) in a pastureland 0 - 10 cm top soil layer located in Castiilla-La
Mancha, Spain (< 1000 seeds/m2). These authors reported a near 100% viability of seeds
and a germination, without any dormancy breaking treatment, of between 4.5 and 8.0%
wich is also much lower than the 38% FGP reported in this study.
55
Table 2.1.3: Cistus ladanifer seed bank characetrisation of the natural C. ladanifer shrubland soil, seed
density and seed germination parameters: Final Germination Percentage (FGP), Mean Germination Time
(MGT), and the time to reach 50% of germination (t50).
Seed density (seeds/m2, x 104)
57.7 ± 44.1
Seed germination
Treatment FGP (%) MGT (days) t50 (days)
None 38.00 ± 6.00b 15.56 ± 1.96 11.67 ± 1.58
100oC, 5 minutes 93.00 ± 3.32a 13.87 ± 2.36 12.92 ± 0.60
Values are presented as mean ± standard deviation (n = 9 seed density, n = 4 seed germination parameters)
In each column, within mean values of seed germination parameters, coefficient letters report the t-test
results in which different letters reflects a significant difference (α = 0.05).
Results for seed germination in this study match the results obtained by Dias et al. (2019)
who reported a germination of 21.6 - 37.9% for untreated seeds and a germination of near
100% for seeds treated for 15 min at 90oC. In addition, this study revealed the similar
germination kinetics of non-dorment and dorment seeds. Seeds evaluated by Dias et al.
(2019) were collected directly from capsules atached to the plant whereas, in this study,
seeds were collected from the soil. This means that C. ladanifer seeds may preserve their
germination properties in the soil. However, soil samples were collected in summer when
C. ladanifer is in seed dispersion phenophase and therefore, most of the seeds were
possibly recently released.
2.1.3.2. Phenological evaluation
Mean vegetative growth of non-flowering shoots throughout the study period,

discriminated by harvest modalities, is presented in the bar graphic of Figure 2.1.1. The
same parameter is also presented in two distinct tables in Figure 2.1.1 for each period and
for each harvest modality, which are considered two independent factors. Vegetative
growth values higher or equal to three times the standard deviation from the mean values
were considered outliers and omitted from each data group defined by the two factors:
period of the year and harvest modality. Most of these data groups (91%, 136 groups out
of 149) showed a normal distribution of the data (Shapiro-Wilk’s test ρ-value ≥ 0.05),
which was an improvement compared to when outliers were not omitted (81%, 120
groups out of 149). Data organised only by period of the year factor showed 42% of the
data groups (14 out of 24) with a normal distribution. In contrast, data organized only by
the harvest modality factor showed none of the seven data groups with a normal
distribution. Regarding variances, data sets organised by the two factors and organised
by the period of the year factor did not presented homogeneity of variances (Levene’s test
ρ-value = 0.000 for both). In conclusion, the classical ANOVA and two-way ANOVA
56
were not used to analyse the vegetative growth data given that the assumption of equal
variances was not true. The Welch’s test and the post-hoc Dunnett’s T3 test were used to
test if the period of the year had a significant effect on mean vegetative growth,
independently of the harvest modality, and to test significant differences between periods,
respectively. Regarding the harvest modality factor, the non-parametric Kruskal-Wallis’s
test was used to test the significant effect of the factor and significant differences between
harvest modalities, since data groups did not present a normal distribution of the data.
Throughout the study period, mean vegetative growth was highest mostly during spring,
registering the highest values between March and May of 2020. Lowest vegetative
growth, near zero, was observed during summer, between July and September. Talavera
et al. (1993) reports three phases of vegetative growth for C. ladanifer, separated by hard
summer conditions, winter conditions, and flowering period. In this study, mostly
between 2019 and 2020, vegetative growth was pronounced also in autumn, between
September and November. However, the flowering period matched the period of highest
vegetative growth. Simões et al. (2008) observed a similar pattern in a two-year
monitoring experiment between 1993 and 1995: vegetative growth was continuous from
the beginning of autumn to the end of spring, more pronounced between February and
March and between May and June. Interestingly, in this study, vegetative growth
significantly decreased just before seed dispersion (capsules opening), between May and
June. Regarding this fact, Simões et al (2008) presents a sigmoid-type plot of shoot length
versus time, throughout the year, starting at autumn and ending at late summer, with an
upper plateau (no growth) during summer months but a less pronounced growth just
before it. This reduction of vegetative growth may be a result of acclimation strategy to
cope with upcoming summer stress conditions and/or a result of resources allocation for
fruit setting/maturation.
Regarding the effect of the harvest modality factor, mean vegetative growth was clearer
lowest for plants not harvested (control plots). Plants harvested every year showed higher
mean vegetative growth than plants harvested after a two-year period. In fact, mean
vegetative growth of the 1YMS harvest modality was the highest and significantly
different from the mean vegetative growth observed fo the 2Y harvest modalities. Simões
et al. (2008) differentiates two types of shoots in C. ladanifer branch based on the
internode lenght, dolichoblasts (> 3 cm) and brachyblasts (< 3 cm), that may differ in
vegetative growth throughout a growing season. Brachyblasts develop from axilary buds
57
of a dolichoblast and may differentiate into flowers or into a dolichoblast in the following
year. Simões et al. (2008) observed a higher brachyblast vegetative growth compared to
the dolichoblast’s growth within a branch (or whole shoot) at one of the years of
observation but, given that brachyblast vegetative growth was a sum of all brachyblasts
grown within that branch during the year, the growth per shoot was most likely smaller
for brachyblasts than for the dolichoblast. In this study, vegetative growth was evaluated
on previous year brachyblasts that did not developed flowers. Those shoots developed
into dolichoblasts and did not develop flowers from axilary buds. This observation is
consistent with Talavera et al. (1993) who reported that flowers only developed in
vigorous branches (dolichoblast and brachyblast assemblage) produced in the previous
year. Harvested plants lost most of the vigourous branches in the following year and
therefore developed much less flowers (some did not develop any flower) compared to
the control plants. However, plants harvested at the beginning and at the end of the study
(2Y periodicity) showed vigorous branches in the second/last year and therefore higher
development of flowers; but no difference was found regarding vegetative growth
compared to plants harvested every year (1Y periodicity), except when compared to the
1YMS modality.
Mean temperature and mean daily rainfall at each observation period are plotted alongside
mean vegetative growth in Figure 2.1.2. Spearman’s rank order correlation was used to
evaluate the relationship between vegetative growth and the two meteorological variables
for each harvest modality (Table 2.1.4). Overall, mean vegetative growth had a significant
but low negative correlation with mean temperature. The correlation was observed
isolately for the control plants but not for the harvested plants. However, excluding
observation periods between May and September, mean vegetative growth showed
significant positive and moderate correlation (correlation coefficient between 0.5 and 0.7)
with mean temperature. The same held true for the mean vegetative growth per harvest
modality which was even high (correlation coefficient between 0.7 and 0.9) for the 1YES
and 1YLS modalities. Mean daily rainfall was only significantly and negatively correlated
to mean vegetative growth of 2YES modality, when periods between May and September
were not considered. Between May and September, the Mediterranean climate is
characterised by drought and high temperatures, which are known abiotic stresses that
influence plant physiology, including vegetative growth. Regarding the highest
correlation between mean vegetative growth and mean temperature for 1Y harvest
58
modalities, it may be explained by the fact that harvesting reduce the development of
other phenological stages such as flowering and fruit production which happened in 2Y
and control harvest modalities.
59
Subchapter 2.1 Rockrose land management: contribution of periodic harvesting to increase value and control Cistus ladanifer L. shrublands.
Figure 2.1.1: Phenological evaluation of C. ladanifer throughout the natural shrubland management study period: Shoot vegetative growth from individual plants in relation to
harvest modalities and observation periods (bar graphic, mean ± 95% confidence interval); Flowering (grey shadow) and seed dispersion (purple shadow) phenophases. Shoot
vegetative growth (mean ± standard deviation) at each observation period and Welch’s ANOVA and Dunnett’s T3 post-hoc results (Table below the graphic); Shoot vegetative
growth (mean ± standard deviation) for each harvest modality and kruskal-Wallis’s test and post-hoc results (Table on the upper right corner). Different post-hoc letters represent
significant differences between mean values. Different colours in the table below the graphic represent groups of means that are significantly different from each other.
60
Subchapter 2.1 Rockrose land management: contribution of periodic harvesting to increase value and control Cistus ladanifer L. shrublands.
Figure 2.1.2: Cistus ladanifer vegetative growth (bar graphic, mean ± 95% confidence interval), mean temperature, and mean daily rainfall (line graphics) at each observation
period throughout the study.
61
Table 2.1.4: Spearman’s rank order correlation between C. ladanifer mean vegetative growth (overall and
for each harvest modality) and mean temperature and mean daily rainfall throughout the study periods.
Mean temperature Mean daily rainfall
treatment Correlation Coefficient ρ-value Correlation Coefficient ρ-value
Overall -0.441 0.031 0.335 0.109
Control -0.407 0.048 0.284 0.179
1YES -0.159 0.502 0.198 0.403
2YES -0.135 0.569 0.230 0.329
1YMS -0.279 0.220 0.378 0.092
2YMS -0.101 0.662 0.018 0.940
1YLS -0.100 0.666 0.121 0.600
2YLS -0.339 0.122 0.210 0.349
Overall 0.554 0.035 -0.311 0.259
Control* 0.496 0.062 -0.439 0.103
1YES* 0.704 0.005 -0.225 0.419
2YES* 0.654 0.010 -0.243 0.382
1YMS* 0.879 0.000 -0.159 0.604
2YMS* 0.529 0.045 -0.539 0.041
1YLS* 0.521 0.049 -0.396 0.145
2YLS* 0.433 0.124 -0.393 0.165
* Without observation periods between May and September
2.1.3.3. Production
Mean biomass production results (kg/ha) or mean percentage of the total harvested
biomass (%TB) at fresh weight basis, of the initial harvest and of the two-year harvesting
modalities are presented in Table 2.1.6. All data sets used for statistical analysis showed
normality (Shapiro-Wilk test, α ≥ 0.05), with a very few exceptions. For statistical
analysis, data sets that showed equal variances (Levene’s test, α ≥ 0.05) were compared
by ANOVA, two-way ANOVA, and the post-hoc Tukey’s test. In cases of
2
heteroscedasticity, data transformations such as ln(x), ln(x+1), and √x were attempted
to improve the homogeneity of variances within data sets while maintaining normality,
to allow the use of two-way ANOVA. In cases where data sets could not achieve similar
variances, even as transformed data, Welch’s ANOVA and the post-hoc Dunnett’s T3 test
were used to compare means.
At the beginning of the experiment (2019) biomass yield results are presented as the mean
of the 24 plots. In fact, no significant differences between yield means, of each set of four
plots assigned to the different harvesting modalities in the following years, were found.
The only exception was the capsules biomass percentage for which the two-ANOVA
demonstrated that mean %TB decreased along summer (season factor ρ-value: 0.008;
Tukey’s test: ES 10.12 ± 1.34%a; MS 8.51 ± 0.89%b; and LS 7.66 ± 1.96%c; α = 0.05).
62
However, the same trend was not reflected for capsules mean biomass yield (kg/ha). In
conclusion, plots to be assigned to different harvest modalities were considered to be at
the same productivity conditions at the beginning of the study.
Table 2.1.5: Biomass production (kg/ha) and percentage in relation to total biomass (fw/fw, in brackets) of
experimental plots in a natural C. ladanifer shrubland at the beginning of study (2019) and at the end of
study (2021) subjected to different harvest modalities.
Total Wood Photosynthetic Capsules
Harvest treatment
biomass biomass biomass biomass
Initial and accumulated final harvests
2019 1860 ± 766a 1522 ± 416b 322 ± 117a
3704 ± 1134a
(Initial harvest) (49.3 ±7.6a) (42.0 ±7.1b) (8.76 ± 1.78a)
b b
2021 750 ± 345 1747 ± 346 259 ± 115a
b b
(2Y harvest) (26.7 ± 10.2 ) (64.1 ± 12.2 ) (9.22 ± 3.16a)
2953 ± 624b c a
2020/2021 216 ± 97 2902 ± 705 14.3 ± 12.5b
(1Y accumulated harvest) (7.17 ± 5.10c) (92.3 ± 5.3a) (0.494 ± 0.629b)
0.000 0.000 0.000
Welch’s ANOVA p-value 0.009
(0.000) (0.000) (0.000)
Post-hoc test - Dunnett’s T3 Dunnett’s T3 Dunnett’s T3
Accumulated final harvests by harvesting modalities
257 ± 83bc 2861 ± 563ab 15.4 ± 9.6b
1YES 3133 ± 613
(7.91 ± 3.67c) (91.6 ± 3.8ab) (0.483 ± 0.505b)
ab c
738 ±320 1544 ± 203 306 ± 152a
2YES 2587 ± 460 ab bc
(27.5 ± 7.2 ) (61.1 ± 11.1 ) (11.39 ± 4.08a)
c abc
225 ± 117 2479 ± 492 12.6 ± 15.0b
1YMS 2716 ± 502
(8.25 ± 6.93c) (91.2 ± 7.3ab) (0.591 ± 0.853b)
a bc
1067 ± 227 1667 ± 350 306 ± 80a
2YMS 3040 ± 363 a bc
(35.6 ± 8.6 ) (54.5 ± 8.3 ) (9.89 ± 1.49a)
168 ± 61c 3368 ± 732a 14.8 ± 12.1b
1YLS 3550 ± 737 c ab
(5.34 ± 3.40 ) (94.2 ± 3.6 ) (0.406 ± 0.432b)
abc bc
442 ± 55 1979 ± 290 176 ± 40a
2YLS 2597 ± 307
(17.1 ± 1.5bc) (76.0 ± 3.3bc) (6.93 ± 1.89a)
f(x) = ln(x) None f(x) = ln(x+1)
Data transformation None 2
(None) (None) (f(x) = √x )
ANOVA / Welch’s 0.000 0.002 0.000
0.246
ANOVA p-value (0.000) (0.000) (0.000)
Tukey
Post-hoc test None Tukey Tukey
(Dunnett’s T3)
Harvest (at 0.5 m height) modalities: Periodicity: every year, 1Y, and after two-years, 2Y; Season: early
summer, ES, mid-summer, MS, and late summer, LS.
Values are presented as the mean ± standard deviation. Values for plots harvested every year (1Y) are
accumulated values from 2020 and 2021 harvest.
Within each column for the “Initial and final harvests” and the “Final harvests by harvesting modalities”,
coefficient letters report the multiple comparison post-hoc tests results in which similar letters reflects a
homogeneous subset.
In this study, only the biomass above the 50 cm were harvested, which explains the lower
production compared to the estimated total aboveground biomass for 2019. Furthermore,
for a five years-old shrubland, Morgado et al. (2005) reports 51% of wood biomass,
including capsules, and 49% of photosynthetic biomass, and Nuñez et al. (1989) reports
63
66% of wood biomass, 34% of photosynthetic biomass, and 3% of capsules biomass.

Capsules biomass percentage reported in this study for the five-years old shrubland
(2019) is much higher than the percentage reported by Nuñez et al. (1989). Regarding
wood and photosynthetic biomass, their %TB are between the two studies by Nuñez et
al. (1989) and Morgado et al. (2005). However, because most of the biomass below 50
cm is non-productive wood biomass, wood biomass %TB of total aboveground biomass,
if evaluated in this study, would be higher.
After the initial harvest in 2019, total biomass production was similar between all harvest
modalities, regardless the harvest season and periodicity, in the following two years.
Within the plots harvested every year, total yearly mean biomass productivity (1567 ±
438 kg/ha.year) was similar regardless the year and season of collection (ANOVA, ρ
value = 0.202). Total biomass production of the two-year harvest (2Y) and of the two-
year accumulated harvest (1Y) were similar but lower than the production obtained at the
beginning of the study (2019).
Wood biomass is the hard lignified biomass from C. ladanifer which do not exude
labdanum resin. Two-way ANOVA performed on yield data (kg/ha), transformed using
the natural logarithm function, revealed that periodicity was the only factor affecting
mean yield (ρ-value = 0.000), higher for 2Y plots. The same result of two-way ANOVA
was obtained for total biomass percentage data, which did not need transformation to
achieve equal variances. Evaluating 1Y plots, the mean yearly productivity of wood
biomass was 108 ± 86 kg/ha.year with a mean total biomass percentage of 7.17 ± 5.10%,
not statistically different within the two harvest years and the three seasons: ANOVA ρ-
value for mean productivity (original data) was 0.171 and for total biomass percentage
was 0.156. Wood biomass yield and percentage of total biomass at the initial harvest
(2019) was more than two-fold higher than for 2Y harvesting modality which in turn was
three-fold higher than for 1Y harvesting modality, considering the two-years accumulated
harvested biomass.
Photosynthetic biomass includes herbaceous shoots and stems with some degree of
lignification, that are covered by labdanum resin. Like for wood biomass, the two-way-
ANOVA revealed that harvest periodicity was the only factor affecting mean
photosynthetic biomass yield (ρ-value = 0.000), however in this case it was higher for 1Y
plots. Regarding %TB, variances were not similar between data sets, even with logarithm
and root square transformations, but Welch’s ANOVA revealed that mean differences
64
exist between all data sets and the post-hoc Dunnett’s T3 test grouped, as similar mean
percentages, the three season data sets of each harvest periodicity. Within 1Y plots, no
significant differences were found between data sets, including the two years of collection
and seasons, regarding mean %TB (ANOVA ρ-value = 0.215) and productivity (1451 ±
434 kg/ha.year, ANOVA ρ-value = 0.179). Even though accumulated photosynthetic
biomass from 1Y plots is significantly higher than from 2Y plots and from the initial
harvest (2019), no such difference was found when comparing the photosynthetic
biomass harvested at each year (ANOVA ρ-value = 0.164).
In this study, after harvest, most of the photosynthetic biomass produced in one year of
growth developed into wood biomass during the second year while producing a similar
amount of photosynthetic biomass: i) there was no difference in photosynthetic biomass
production per year at plots harvested every year and photosynthetic biomass production
at plots harvested after two years; ii) There was an increase in wood biomass in plots
harvested after two years compared to plots harvested every year; and iii) there was a
similar vegetative growth of 1Y and 2Y harvested plants according to the phenological
evaluation (section 2.1.3.2).
Without experimental data, it is difficult to predict if a 3-years or more harvest periodicity

would maintain the same behaviour and if that behaviour would hold true for shrublands
with different ages and structures. According to Nuñez et al. (1989) and Morgado et al.
(2005) the photosynthetic biomass percentage of total biomass tends to decrease as total
biomass increases, however that total biomass increase may compensate the percentage
decrease and result in higher photosynthetic biomass yield. In contrast, Hernández-
Rodríguez et al. (2017) estimates that total biomass productivity starts to increase less
above the 8-years-old and remains practically constant above the 10-years-old and
Morgado et al. (2005) not only reported a decrease in photosynthetic biomass percentage
of 12/15-years-old shrublands compared to 6-9-year-old shrublands as also reported a
decrease in production yields. Other interesting factor to study is how long the harvesting
modalities of this study may be used before the productivity starts to decay. According to
Oria-de-Rueda et al. (2008) C. ladanifer shrublands are short-lived, 18/20-years old,
because the amount of highly lignified and dried biomass promote the ignition and spread
of fire. However, Alías et al. (2015) report to have evaluated plants up to 55-years-old.
These harvesting modalities may serve as rejuvenation pruning preventing the
65
accumulation of wood biomass and therefore reducing the risk of fire and increasing the
shrubland lifetime.
Labdanum resin extraction yields from photosynthetic biomass (% dw/fw) and labdanum
resin production (kg/ha) at the three years and seasons of harvest are presented in Table
2.1.7. All data sets showed normality and equal variances. Two-way ANOVA revealed
the significant effect of the harvest year (ρ-value = 0.000), harvest season (ρ-value =
0.000), and of the interaction between harvest year and season (ρ-value = 0.037) on
labdanum yield.
Table 2.1.6: Labdanum resin yield (% dw/fw) at the three years and seasons of C. ladanifer harvest.
ES MS LS
Resin yield (% dw/fw)
2019 7.41 ± 0.59bc 8.54 ± 1.43ab 10.50 ± 0.82a
c bc
2020 6.02 ± 0.82 7.49 ± 0.71 7.35 ± 0.65bc
c bc
2021 6.48 ± 0.41 6.11 ± 0.91 7.10 ± 0.39bc
Resin production (kg/ha)
2019 (initial harvest) 108 ± 18bc 115 ± 26bc 184 ± 55ab
c bc
2021 (2Y harvest) 90 ± 12 119 ± 25 134 ± 20bc
2020/2021
169 ± 33abc 181 ± 36ab 230 ± 50a
(1Y accumulated harvest)
Seasons: early summer, ES, mid-summer, MS, and late summer, LS.
Values are presented as mean ± standard deviation (n = 4).
For each parameter (resin yield and productivity), including values in rows and columns, coefficient letters
report the multiple comparison post-hoc Tukey’s test results in which similar letters reflects a homogeneous
subset.
Labdanum resin yield was higher for the initial harvest (2019) and at late summer (LS).
The post-hoc Tukey’s test revealed the significantly highest labdanum resin extraction
yield from the biomass collected at late summer of 2019, except when compared to the
one obtained at midsummer of the same year. Excluding the 2019 harvest from the
analysis, only the season factor had a significant effect (ρ-value = 0.004). The post-hoc
Tukey’s test revealed that the resin extraction yield was significantly highest at LS season
and lowest at ES season, but that the yield at MS season was not significantly different
from the yields at LS and ES seasons. It is known for a fact that labdanum extraction yield
varies with season and type of photosynthetic biomass as shown by Sosa et al. (2005)
who reported that labdanum resin yield is higher in summer and autumn compared to
winter and spring seasons, and in mature leaves compared to stems. The authors report a
extraction yield between 5 - 7% and 12 - 15% (dw/dw) for photosynthetic stems and
leaves, respectively, in autumn and summer, but using a chloroform extraction. Using the
Andalusian process, Burguer (2016) report a 7.79 - 8.86% (dw/dw) extraction yield and
Morgado et al. (2005) report a 7 - 18% (dw/dw) extraction yield. Regarding resin
66
production (kg/ha), two-way ANOVA revealed that both periodicity and season factors
had a significant effect (ρ-value = 0.000 and 0.0001, respectively). Higher resin
production was obtained in 1Y plots and at LS season. Resin production is both dependent
on resin extraction yield and on production of photosynthetic biomass which was higher
for 1Y plots because of the harvest accumulation (Table 2.1.7). Within those plots, mean
resin production was 85, 95, and 115 kg/ha per year at ES, MS, and LS, respectively, with
no significant difference between the two years of collection (ANOVA ρ-value = 0.175).
Using resin productivity (per year) of 1Y modalities, a two-way ANOVA once again
revealed the effect of both factors and no interaction, but in this case higher resin
productivity was achieved at the initial harvest in 2019 at LS season, similarly to what
happened to extraction yield. Morgado et al. (2005) report a resin production of 400 - 450
kg/ha in 3/4-years-old shrubland which is two-fold higher than the productivity obtained
at the initial harvested in a 5-years-old shrubland.
Capsules are the globular loculicidal fruits of C. ladanifer. The two-way ANOVA
performed on capsules production data, transformed using the log(x-1) function to include
null/zero values, revealed that capsules mean production was only affected by the harvest
periodicity (ρ-value = 0.000) where 2Y plots presented much higher values of capsules
production than 1Y plots. The same held true for capsules %TB, transformed using the
root square function. Like the other biomass fractions, capsules mean production and
mean %TB was not significantly different at both years of harvest for 1Y plots. Capsules
production and %TB of 2Y plots was similar to the mean values of the initial harvest in
2019. However, even though the harvest season influenced capsules percentage of total
biomass at the initial 2019 harvest, the same was not true for the other two years of
collection at any harvest modality. In C. ladanifer plants, capsules develop after the cross-
pollination of flowers by insect vectors (Talavera et al. 1993). The production of capsules
in 1Y plots was residual and that is explained by the fact that practically all photosynthetic
biomass was harvested at 50 cm height, including dolichoblasts that would develop into
brachyblasts that, in turn, some would develop flower buds during the next year (Simões
et al. 2008). This two-year period to develop flowers and capsules is well demonstrated
by the fact that plots harvested after two years of growth reached capsules production
values of the original 5-years-old shrubland in 2019. Nuñez et al. (1989) reported 3 and
2% capsules percentage of total biomass and 100 and 300 kg/ha of capsules production
for a 5 and 12/15-years old shrublands, respectively. Alves-Ferreira et al. (2019a) reported
67
that capsules represented 4% of total biomass and a production of 650 kg/ha for
shrublands between 10 and 18-years old. Excluding the data sets of 1Y plots, mean seed
production was 89 ± 39 kg/ha and mean percentage weight in capsules was 29.1 ± 4.4%.
Seed production and percentage in capsules was similar regardless the season and time
(2019 or 2021 in 2Y plots) of collection.
2.1.4. Conclusion
Starting with a 5-years old shrubland for a 2-year period harvest study, the first harvest
yielded higher total and woody biomass than the accumulated harvest during the
following two years but the same did not happened regarding photosynthetic (resin
productive) and capsules biomass. Every-year accumulated biomass yielded the highest
photosynthetic biomass production but negligible capsules production after two years.
Two-year harvest biomass matched the initial photosynthetic biomass and capsules
production. summer seasons (early, mid-, and late summer) had no overall effect on
harvest productivities but affected resin yield and production which was higher from mid-
to late summer. Labdanum extraction yield varied between 6.02 and 10.5% (dw/fw) and
its production reached a maximum of 230 kg/ha at the end of two years when harvested
was done yearly at late summer. Seeds yield and production was also not affected by the
season of harvest. Seeds yield from capsules was 29.1% (fw/fw) and mean seed
production was 89 kg/ha. Most of the plants did not die after the harvest and rejuvenated
by producing less amount of woody and fire fuel biomass and therefore, this management
practice may be used together with a long cycle management of rockrose lands to yield
mushrooms, biomass, labdanum resin, and capsules/seeds. However, it is relevant to
replicate this study on shrublands with different ages and densities and longer periods of
harvest periodicities such as 3 and 4-years may be considered.
Based on this study, the periodic harvest at 0.5 cm must be considered a good
management practice for C. ladanifer shrublands, reducing fire risk, maintaining soil
cover and enrichment, and increasing profit for forest producers. If the goal is to extract
resin the harvest must be done every year. If the goal is to obtain seeds and extract resin
the harvest must be done every two years.
68
Subchapter 2.2
Cistus ladanifer L. cultivation: first results.
Abstract
Cistus ladanifer cultivation was never reported within its endemic area because of its
abundance in the wild. However, it is relevant for production, phytoremediation,
ornamental use, and research purposes. In this work Cistus ladanifer was planted at a 0.5
x 1.0 m spacing in autumn with 1-year-old greenhouse germinated and grown plants. The
culture was evaluated during the first two years regarding plants behaviour and fresh
biomass yield at different harvest periodicity modalities (harvest done every year, 1Y,
and after the two years, 2Y). At the end of the experiment, 3-years-old plants started to
form a continuous edge with a marked flowering in plant lines harvested only at the end
of the study (2Y). Accumulated photosynthetic biomass production was similar for the
two harvest modalities (5678 ± 1205 and 5325 ± 972 kg/ha for 1Y and 2Y modalities,
respectively) and capsules production, correlated to flowering, was significant only for
2Y modality (284 ± 37 kg/ha) with a seed yield of 26.7 ± 2.4% in relation to capsules
weight. At least during the first two years after cultivation, a two-year interval before
harvest is ideal to maximize production (photosynthetic biomass, labdanum resin, and
capsules) and ornamental attributes (flowering). However, in the following years the two
different harvest periodicities will most likely maximize different products and attributes.
Keywords
Ornamental shrub; Rockrose biomass production; Rockrose cultivation.
Frazão DF, Gonçalves JC, Silva AM, Delgado F Cistus ladanifer L. cultivation trial: first results. (Under
review)
69
Subchapter 2.2 Cistus ladanifer L. cultivation trial: first results.
70
2.2.1. Introduction
The rockrose Cistus ladanifer is an abundant wild resource in the Iberian Peninsula
(Godinho-Ferreira et al. 2005; Montero et al. 2020) and it is currently exploited, by
collection in the wild, for its aromatic labdanum resin and essential oil (Biolandes n.d.).
Labdanum resin, essential oil, and extracts from this plant have been considered to have
value for the cosmetic and pharmaceutical industries and for agriculture (Raimundo et al.
2018). In addition, its seeds are reported to be a source of nutrients for human
consumption (chapter 4) and its soft biomass may be used as forage (Jerónimo et al. 2012;
Francisco et al. 2015).
Due to the vast amount of this resource, cultivation has never been considered for this
plant species within its endemic area. However, several purposes such as production,
phytoremediation, ornamental use, and research, awakes the need for it. In fact, C.
ladanifer and C. x purpureus (C. ladanider x C. creticus) have been recently cultivated
in Australia to produce essential oils (EOPAA 2017) and C. ladanifer is reported to had
been cultivated in China for research purposes (Lawrence 1999).
The amount of the resource in the wild surpasses the need for it regarding its products,
for now. However, shrubland deforestation practices to reduce fire risk and to increase
pastureland affects the constant local availability of the resource. In addition, C. ladanifer
shrublands comprise other plant species and a mechanical harvest will collect a mixture
of plants, increasing biomass selection costs or reducing the final quality and yield of the
products. Cistus ladanifer cultures would have low fire risks consequences because of the
continuous management and easier mechanical harvest.
Raimundo et al. (2018) discuss the use of C. ladanifer, as a viable alternative to civil
engineering methods, for phytoremediation of metal-rich soils either by phytostabilisation
or phytoextraction of mainly magnesium and zinc. However, as an excluder of many trace
elements (such as arsenic, copper, nickel, and lead) C. ladanifer may be cultivated in such
soils and produce heavy metal-free biomass and products.
This plant may also be thought for ornamental use such as plant hedges because it is an
evergreen shrub, it has a certain glossiness due to the exuded resin, it presents big white
flowers during a 1-2-month period, and it releases an amber aroma to the surrounding
environment.
71
The selection and improvement of genotypes suitable to specific purposes (e.g., biomass,
resin, and fruits production, phytoremediation, and ornamental attributes) justifies
research on propagation and cultivation practices which are scarce when regarding C.
ladanifer.
This work presents a first attempt to culture C. ladanifer, evaluating the biomass
production upon a non-destructive harvest practice using two different harvest
periodicities.
2.2.2. Material and Methods
Cistus ladanifer capsules were randomly collected in October of 2018 from three capsules
of ten different plants in a Penha García’s natural shrubland (GPS coordinates in DMS:
40º01’01’’N 6º59’06’’W) separated by 10 m in a transect. Seeds were obtained from the
capsules and were treated for 5 min at 100oC, inside a glass petri dish, in a ventilator
chamber prior germination. This treatment to break dormancy was selected based on
insights from Pérez-García (1997) and Delgado et al. (2001), more recently discussed by
Dias et al. (2019). In October, seeds were seeded in universal substrate (Nutrofertil –
Nutrição e Fertilizantes, Lda, Portugal) using germination trays and distributing 1-3 seeds
per alveolus. Germination trays were placed on a heated blanket at 26oC inside the
greenhouse for 2 months. Afterwards, the small plants were transferred to 0.5 L cotton
bags filled with the same substrate. Plants grew in the greenhouse until the end of summer
(September of 2019) when they were transferred to the outside environment before
planting. Grown plants were planted without removing the cotton bag because of its
biodegradability and to not hurt the roots.
The field (Figure 2.2.1) was cultivated with 1-year-old plants in 7 lines at a spacing of
0.50 m in the line and 1.00 m between lines in October of 2019 (plant density of 2
plants/m2). Three lines (14 m) were assigned to each harvest modality and one line was
excluded. To exclude edaphic effects due to the slope of the field, harvest modalities were
evenly applied to the lines, excluding one line interrupted by a tree (line 5). Harvest
modalities comprised two periodicities of harvest: every year (1Y) and after two years
(2Y), starting at the summer after planting (2020). The effect of harvest modalities was
evaluated in relation to total biomass, wood biomass, photosynthetic biomass, and
capsules/seeds production. Biomass was harvested using a hedge trimmer (STIHL HS
45) at 50 cm height, was collected by hand, was separated by type of biomass (wood,
72
photosynthetic, and capsules biomass), and was weighted in a semi-industrial balance

(BW-M, Libra Weighing Machines, Ltd.). Photosynthetic biomass comprised both
soft/herbaceous shoots and semi-lignified biomass, covered by resin/exudate. Capsules
were weighted using an analytical balance (Sartorius, model ENTRIS323I-1S). The only
operation done during cultivation was weeding the space between lines at the beginning
of summer.
Figure 2.2.1: Cistus ladanifer cultivation field. A: Photograph in spring in the first-year after planting. B:
Satelite image obtained from Google Earth Pro Software (Google Inc.) 05/2021; Study field is marked by
a square and amplified (not at scale). Lines 1, 3 and 6 (dashed) were assigned to the 2-Year periodicity
treatment and Lines 2, 4, and 7 (dashed) were assigned to the 1-Year periodicity treatment. Line 5 was
excluded from the study.
Statistical differences (α = 0.05) between mean biomass production (kg/ha) and mean
percentage of the total harvested biomass (%TB) at fresh weight basis were analysed by
ANOVA and Tukey’s test (if Shapiro-Wilk test, ρ-value ≥ 0.05 and Levene’s test, ρ-value
≥ 0.05), Welch’s ANOVA and Dunnett’s T3 test (if Shapiro-Wilk test, ρ-value ≥ 0.05
and Levene’s test, ρ-value < 0.05), and Kruskal-Wallis non-parametric test (if Shapiro-
Wilk test, ρ-value < 0.05), using the IBM SPSS Statistics 27 Software.
Regarding plant propagation, at the end of the first month, germination achieved 89.2%
(1842 plants) and the number of plants dying after transplantation to bags, growth in the
greenhouse, acclimatation, and transplantation to the soil was negligible. No irrigation
was applied to the plants, even after harvest and during summer, which were able to
73
survive. Only after 2 years of growth, a continuous hedge started form within each line
and more intensely in lines harvested every year (1Y). Flowering was marked, during
spring, only at the end of the experiment (3 years-old plants) in lines harvested after two
years of continuous growth (2Y). During spring and summer, the characteristic aroma of
C. ladanifer was notorious near the field.
Production results are presented in Table 2.2.1. In the first year of harvest (2020), 1Y
lines yielded 500 ± 154 kg/ha of total biomass which was 100% photosynthetic biomass.
In the second year of harvest (2021), 1Y lines yielded 5335 ± 1030 kg/ha of total biomass,
mostly photosynthetic (96.9 ± 4.0%TB), which is ten-fold higher than the harvest of the
previous year. In 2021, 2Y lines were harvest for the first time, yielding 7403 ± 1242
kg/ha of total biomass, composed by 71.7 ± 1.1% of photosynthetic biomass, 24.4 ± 3.3%
of wood biomass, and 3.9 ± 0.2% of capsules.
For a 3 years-old natural shrubland, Morgado et al. (2005) report a yield of near 6000
kg/ha of total biomass, of which near 60% was resin productive biomass (photosynthetic
biomass). Total biomass and total photosynthetic biomass production, in this study, from
2Y lines are slightly higher than the values reported by Morgado et al. (2005). However,
in this study, biomass was harvested at a certain height contrary to Morgado et al. (2005),
who harvested all aboveground biomass. This means that at the same conditions as
Morgado et al. (2005), in this study, total biomass would increase and %TB of
photosynthetic biomass would decrease because wood biomass is more representative at
the lower parts of the plant.
Photosynthetic biomass is the biomass from which labdanum resin can be extracted and
therefore its production is an indirect measurement of labdanum production. Results from
this experiment reveal similar photosynthetic biomass accumulated production after two
years, for both harvest periodicities. In fact, for 1Y lines, the two-year accumulated
harvest is similar to the second-year harvest because the 2020 harvest was very low.
Interestingly, the same amount of photosynthetic biomass was harvested in 2021 for the
two harvest modalities even though the %TB of photosynthetic biomass was significantly
lower for the 2Y modality. From the study of Morgado et al. (2005) photosynthetic
biomass production was similar between 3, 4, and 12/15 years-old shrublands despite the
fact that total biomass increased with age. Considering a stabilisation of photosynthetic
biomass production, the 1Y harvest modality may accumulate more photosynthetic
biomass in upcoming years.
74
Table 2.2.1: Biomass production (kg/ha) and percentage in relation to total biomass (fw/fw, %TB) of the C. ladanifer culture, for the first two years, upon different harvest
periodicities done at mid-summer.
Harvest Total biomass Photosynthetic biomass Wood biomass Capsules
Harvest year
Periodicity kg/ha kg/ha % TB kg/ha % TB kg/ha % TB
2020 1Y 500 ± 154b 500 ± 154b 100.0 ± 0 0 ± 0b 0.0 ± 0.0b 0 ± 0b 0.0 ± 0.0b
2021 1Y 5335 ± 1030a 5178 ± 1090a 96.9 ± 4.0a 130 ± 183b 2.6 ± 3.7b 28 ± 17b 0.5 ± 0.3b
a a a b b b
2020 + 2021 1Y 5835 ± 1161 5678 ± 1205 97.2 ± 3.6 130 ± 183 2.3 ± 3.3 28 ± 17 0.5 ± 0.3b
2021 2Y 7403 ± 1242a 5325 ± 972a 71.7 ± 1.1b 1794 ± 233a 24.4 ± 3.3a 284 ± 37a 3.9 ± 0.2a
ANOVA ANOVA Welch’s ANOVA Kruskal- Kruskal- ANOVA ANOVA
Statistical tests (α = 0.05)
Tukey Tukey Dunnett T3 Wallis Wallis Tukey Tukey
Harvest (0.5 m height) periodicities: every year, 1Y, and after two-years, 2Y.
Values are presented as mean ± standard deviation (n = 3).
a,b
Different coefficient letters mean statistical differences between means by the respective post-hoc test (indicated in the table) at 95% confidence (α = 0.05).
75
Capsules production was around 284 ± 37 kg/ha for plant lines harvested after two years
and negligible for lines harvested every year. Those capsules rendered around 26.7 ±
2.4% of their weight in seeds which would convert to a seed production of around 76
kg/ha. Those seeds may be used as human food (chapter 4) and the whole capsules, or the
leftover, may be thought to be used for animal feeding (Bastida and Talavera 2002;
González et al. 2018). Capsules are a direct product of flowering and cross pollination
which starts to happen at 3 years-old plant (Talavera et al. 1993) which explains the
marked flowering only at the end of this study. In addition, flower buds initiate during
winter in brachyblasts at the apex of dolichoblasts produced in the previous year (Simões
et al. 2008), which explains the absence of a marked flowering and much lower capsules
production in plant lines harvested every year in which at the least the upper part of
dolichoblasts produced in one year (from September to June) were cut.
2.2.4. Conclusion
Cistus ladanifer showed to be very easy to germinate and to plant at autumn season
without irrigation or fertilisation in the following years. This plant showed to form
continuous hedges at 3 years-old using a planting space of 0.5 m (line spacing) but not at
1.0 m space (between lines spacing). After planting, at the least a two-year interval before
harvest is ideal to maximize production (photosynthetic biomass, labdanum resin, and
capsules) and ornamental attributes (flowering). After the first years of cultivation, a
choice most likely must be done if it is intended to maximize photosynthetic biomass and
labdanum resin production (harvest done every year) or if it is intended to maximize
ornamental attributes and capsules production (harvest or cut done every two-years).
Research works intended to evaluate C. ladanifer genotypes individually must safeguard
at least a 1.0 m x 1.0 m spacing between plants, if done until the 3 years of age.
76
Chapter 3
Rockrose tissue culture: potential of leaf and stem explants from Cistus
ladanifer L.
Abstract
Cistus ladanifer L. exudes compounds such as methylated flavonoids and labdane-type

diterpenes from its leaves and stems with interesting medicinal properties. Despite the
high abundance in the wild, this plant has been proposed to be cultivated as crop to
recuperate oligotrophic and trace-elements contaminated soils or for ornamental, and
research purposes. Plant tissue culture may be used to produce specific metabolites and
for clonal propagation of specific genotypes for cultivation. Regarding this
biotechnology, only one work has attempted in vitro propagation of C. ladanifer from
adult plant material. The goal of this work was to evaluate the suitability of leaf and
internodal stem explants from C. ladanifer for in vitro tissue culture using three plant
growth regulators: 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP),
and 1-naphtaleneacetic acid (NAA). Stem explants presented a survival rate of 77.4%
(and 60% in a second establishment) and leaf apical and basal explants presented a
survival rate of 13.0 and 94.0%, respectively. Shoots were regenerated from the explants
under the influence of BAP (30.8 and 37.6% using stem explants with or without NAA,
respectively, and 16.7% using leaf explants with NAA) and two types of compact calli
were induced: dark green calli were induced under the influence of BAP (75-100% using
stem explants and 50-75% using leaf explants, with or without NAA) and light green calli
were induced under the influence of 2,4-D (100% using stem explants and 100 and 11.1%
using leaf explants with and without BAP, respectively). Light green calli were able to
grow between 559 and 709% during subsequent subcultures and showed rhizogenic
capacity when the amounts of BAP were lower than of 2,4-D. However, they showed no
potential for shoot organogenesis. Dark green calli may be suitable for shoot
organogenesis but further experiments need to be done. The suitability of the two calli
lines to produce important metabolites and transposition to liquid cultures is relevant to
be studied and compared to organ in vitro cultures.
Keywords
Cistus callogenesis; Cistus organogenesis; Cistus propagation.
77
Chapter 3 Rockrose tissue culture: potential of leaf and stem explants from Cistus ladanifer L.
This chapter was submitted for publication as:
Frazão DF, Barroca C, Silva AM, Delgado F Gonçalves JC Rockrose tissue culture: potential of leaf and
stem explants from Cistus ladanifer L. (Under review)
78
3.1. Introduction
Cistus ladanifer L. is an endemic and abundant plant/resource in Iberian Peninsula

(Frazão et al. 2018). Extracts from these plants have been evaluated regarding their
bioactivities and they have been showing to have potential to be valorised for their
medicinal, cosmetic, preservative and plant protection properties (Raimundo et al. 2018).
This plant was suggested to be used to recuperate oligotrophic soils and cultivated in
heavy metal contaminated soils by Raimundo et al. (2018) which highlights the need to
select specific plant lines for that purpose.
In vitro plant tissue culture is an emergent biotechnology alternative either for plant
propagation and/or for metabolite production (Espinosa-Leal et al. 2018).
Propagation of C. ladanifer is important once specific genotypes are selected for

cultivation purposes. This plant propagates in nature by seeds resulting from cross
pollination (Talavera et al. 1993). Using temperature (e.g., 90oC) during a short time (e.g.,
10 min) to break dormancy it is possible to obtain near 100% of germination (Dias et al.
2019), however due to the cross-pollination process seeds are not ideal to propagate
specific genotypes. Micropropagation has the advantage of producing large numbers of
homogeneous plants at any time, generating disease-free and aseptic plants, and
enhancing multiplication rates. Iriondo et al. (1995) developed a micropropagation
method for several Cistus species from node segments starting from in vitro germinated
seedlings. Boukili et al. (2018) were able to induce shoot formation from nodal segments,
of wild grown plants, pre-treated with an antioxidant solution. Iriondo et al. (1995) and
Boukili et al. (2018) report the advantage of using BAP over Kin or 2iP for shoot
multiplication, the use of IBA over NAA to increase rooting, and achieving a survival
rate of between 62 and 70%, respectively, in the acclimatisation phase.
Production of labdanum resin by in vitro systems may not be competitive with wild
collection because of the abundant resource. However, the need to produce specific
compounds, since some may show interesting individual bioactive properties, can justify
in vitro culture systems in which secondary metabolism could be easier to manipulate.
Plant tissue culture advantages over traditional field culture or natural exploitation for
metabolite production are the independence from geographical, seasonal and
environmental variations, uninterrupted production in uniform yield and quality, no need
for pesticide and herbicide application, and the shorter growing cycles (Espinosa-Leal et
79
al. 2018). However, a significant disadvantage is the higher costs involved (Espinosa-
Leal et al. 2018).
Callus culture may be used as a source material for protoplast isolation (ideal for the
production of hybrids and genetic and cytoplasmatic manipulation), as a source of cells
for genetic manipulation, as inoculum for the initiation of suspension cultures (ideal for
the production of metabolites), to provide information on the regeneration potentiality of
a plant (Bhatia et al. 2015) and for propagation purposes. In fact, according to Efferth
(2019) callus cultures and cell suspension cultures have high potential to be used for
pharmaceuticals production and for plant breeding. Callus culture and secondary
metabolite production by plant tissue culture using C. ladanifer is not yet reported in
literature. Nevertheless, Madesis et al. (2011) regenerated shoots from Cistus creticus leaf
explants through organogenesis of callus using a combination of TDZ and NAA, and
Skorić et al. (2012) were able to obtain extracts rich in labdane-type diterpenoids with
cytotoxic activity against human cancer cell lines from C. creticus shoots in vitro cultures
but the same did no happened with C. creticus roots in vitro cultures.
The aim of this preliminary work was to evaluate the potential of leaf and internodal stem
explants from C. ladanifer to obtain in vitro tissue cultures for clonal propagation and
secondary metabolite production.
3.2. Material and methods
3.2.1. Chemicals
Ethanol 99% (Honeywell, USA), tween 20, saccharose, gum agar (VWR, USA), toluidine
blue, HgCl2, formaldehyde 37%, glacial acetic acid ≥ 98%, 2,4-D, BAP, NAA (Merck,
Germany), hydroxyethylmethacrylate (LKB Historesin, Leica Biosystems, Germany) and
DMSO (Invitrogen, Thermo Fisher Scientific, USA), Mancozan (80% Mancozeb, Bayer,
Germany)
3.2.2. Leaf and stem explants establishment and callogenesis
Non-flowering, semi-woody/herbaceous, dolichoblast shoots of C. ladanifer were

collected from plants in a natural shrubland in Penha Garcia, Castelo Branco, Portugal
(GPS 40o01’43.5’’N 6o59’35.4’’W) in April 2018.
Leaves and leafless shoots were washed with 70% ethanol for 1 min and with tap water
under constant flow for 20 min. Afterwards, explants were washed with distilled water +
80
tween 20 (6 drops/L) for 2 min and disinfected with fungicide (Mancozeb solution, 0.5
g/L) for 10 min and then with a mercury chloride solution (0.08% m/v) for 4 min. Finally,
they were washed 5 times with sterilised distilled water. Internodal segments and leaf
segments (both basal and apical with 2 cm length) were cut and used as explants on MS
(Murashige and Skoog 1962) basal medium with sacharose (20 g/L) and agar (8 g/L).
Explants were placed in tubes with 12 mL medium and incubated in a 16 h photoperiod
(“cool white” fluorescent lamps, 45 ± 5 µmol/m2s PPFD) and at 25 ± 2oC during 45 days.
Explants were established using different plant growth regulators (PGRs) combination
(Table 3.1). Twenty-four leaf and stem explants were used per treatment (leaf explants
were divided in twelve basal and twelve apical explants per treatment). During 45 days,
events such as contaminations, death/survival, and induction responses (without, callus
and organ formation) were registered. Contamination percentage was calculated in
relation to the total number of explants used, viability percentage was calculated in
relation to the number of explants not contaminated, and induction responses percentage
was calculated in relation to the number of viable explants.
Table 3.1: PGRs supplementation in MS medium used for C.ladanifer leaf and stem explants establishment.
Treatment
PGR
T0 T1 T2 T3 T4 T5
2,4-D - 0.5 mg/L - - 0.5 mg/L -
NAA - - 0.5 mg/L - - 0.5 mg/L
BAP - - - 0.5 mg/L 0.5 mg/L 0.5 mg/L
3.2.2.1. Callus index (CI)
CI was calculated based on Wakhlu and Barna (1989) method to evaluate the treatments
at the callogenesis stage. Calli were visually rated (G) at a scale from 1 to 3 (1: callus
initiation; 2: callus covering partially the explant; 3: callus covering completely the
̅), the number
explant). CI was calculated, per treatment, based on the average calli rate (G
of explants with callus (n) and the total number of explants, according to the formula: CI
̅ (1/N)
= 100.n.G.
3.2.3. Callus propagation and evaluation
3.2.3.1. Callus subculture mass propagation
Non-flowering, semi-woody/herbaceous, dolichoblast shoots of C. ladanifer were

collected from plants in a natural shrubland in Penha Garcia, Castelo Branco, Portugal
81
(GPS 40o01’43.5’’N 6o59’35.4’’W) in April 2019. Leafless shoots were washed and
disinfected as previously described (section 3.2.2) and 30 internodal segments were used
as explants to induce callus in T4 (T4B) medium.
Ten calli lines obtained in T4 medium were evaluated during five consecutive 30-day
subcultures, in medium supplemented with 2,4-D and different concentrations of BAP
(Table 3.2) to find the best combination for callus proliferation. Four non-
necrotic/oxidized and non-differentiated pieces of calli were placed, in a flask, on 50 mL
of the same medium of origin, corresponding to one callus line. Remaining portions were
saved for dry weight (dw) and histological analysis. Fresh weight (fw) of the initial and
final calli mass per calli line was monitored and reported as percentage of growth. Calli
growth was morphologically evaluated regarding texture, colour, necrosis/oxidation, and
differentiation. Callus growth from T0 was excluded due to the lack of sufficient calli
material after the first subculture.
Table 3.2: PGRs supplementation in MS medium used for C. ladanifer callus culture.
Treatment
PGR
(T0)* T1 T4A T4B
2,4-D - 0.5 mg/L 0.5 mg/L 0.5 mg/L
BAP - - 0.25 mg/L 0.5 mg/L
*Excluded after the first subculture.
3.2.3.2. Dry weight (dw)
After each subculture a small portion of each calli line was initially weighted, dried in a
ventilated chamber at 105oC for 1 day and finally weighted to assess the dry weight (dw)
content.
3.2.3.3. Histologic evaluation
From the fourth subculture, callus portions without any differentiation from the 3
treatments and callus portions with differentiated structures (Figure 1 g-j) were
immediately immersed in FAA solution (formaldehyde 37% v/v: glacial acetic acid:
ethanol: water 2:1:10:7 v:v:v:v), for fixation, during 24 h. Then samples were rinsed twice
with 50% ethanol during 2 h and after that dehydrated with subsequent higher
concentrations of ethanol (60, 70, 80, 90, 99%) for 1 h each and 24 h for the last.
Infiltration was done with hydroxyethylmethacrylate under vacuum, firstly in a 50%
solution during 2 h and finally in the pure resin during 3 h. Inclusion was done using the
same resin and DMSO as a polymerisation agent during 2 h at ambient temperature. Cuts
82
were done at 7 µm thickness using a rotative microtome (American Optical Company,

model 820). Staining was done with 0.05% (w/v) toluidine blue for 1 minute. Slides were
visualised in a Motic BA 310 Digital microscope and photographs were taken using Motic
Images Plus 2.0 software.
3.2.3.4. Shoot organogenesis
Five calli lines (n = 20) obtained in T4B medium were evaluated during two consecutive
30-day subcultures, in medium supplemented with different NAA and BAP combinations
(Table 3.3) to find the best combination to induce shoot organogenesis. Four non-
necrotic/oxidized and non-differentiated pieces of calli were placed, in a flask, with 50
mL of the same medium of origin, corresponding to one callus line. During the two
subcultures, induction responses (differentiation into shoots) were registered.
Table 3.3: PGRs supplementation in MS medium used for C. ladanifer shoot organogenesis.
Treatments
PGR
T0 T3A T3B T5A T5B
NAA - - - 0.5 mg/L 0.5 mg/L
BAP - 0.5 mg/L 1.0 mg/L 0.5 mg/L 1.0 mg/L
3.2.4. Statistical analysis
Statistical analysis, namely Pearson’s correlation test, ANOVA and post-hoc Tukey’s
test, and the non-parametric Kruskal-Wallis’s test were performed using IBM SPSS
Statistics 25 software.
3.3. Results
3.3.1. Establishment of leaf and internodal stem explants
Percentage of contaminations, survival, and induction responses of leaf and stem explants
establishment are reported as percentage of the events in Table 3.4. The explant
preparation method showed a disinfection efficiency of about 70%. In addition, the
method does not seem to influence the explants survival because of the high survival rates
of leaf basal segments (94%) and internodal segments (77.4%). However, leaf apical
segments showed a very low survival rate (13%).
83
Table 3.4: Percentage of contamination, survival, and induction responses observed for the C. ladanifer
leaf and stem explants establishment.
Response (%)
Contamination Survival
Explants Treatment Callus and
(%) (%) Without Callus shoots
shoots
T0
100 0 0 0
(n = 12)
T1
0 100 0 0
(n = 9)
T2
81.8 18.2 0 0
Internodal segments 35.4 77.4 (n = 11)
(n = 144) (n = 51) (n = 72) T3
6.2 56.2 18.8 18.8
(n = 16)
T4
0 100 0 0
(n = 12)
T5
0 69.2 30.8 0
(n = 13)
T0
100 0 0 0
(n = 6)
Basal
94.0 T1
Segments 88.9 11.1 0 0
(n = 47) (n = 9)
(n = 72)
T2
Leaf 100 0 0 0
33.3 (n = 8)
segments
(n = 48) T3
(n = 144) 45.5 54.5 0 0
(n = 11)
Apical
13.0 T4
Segments 0 100 0 0
(n = 6) (n = 7)
(n = 72)
T5
25.0 58.3 16.7 0
(n = 12)
Twenty-four explants (internodal segments or leaf segments) were used per treatment. Leaf segments in
each treatment were composed by twelve basal segments and apical segments.
The combination of 2,4-D with BAP in equal amounts (T4) performed better than the
three growth regulators alone or the combination between NAA and BAP, inducing
compact light green calli on 100% of the survivor explants (Table 3.4). The same held
true analysing the CI values (Table 3.5). Even though 2,4-D alone (T1) induced callus
formation as well as the T4 using internodal segments, calli proliferated less, as shown
by the CI (100 compared to 200), and 2,4-D alone could only induce callus on 11.1% of
the leaf segments. NAA alone showed to be weaker than 2,4-D at the same concentration
in inducing callus, and BAP alone or in combination with NAA showed to induce dark
green very compact callus and shoot differentiation in contrast to the light green compact
callus when 2,4-D was used (Figure 3.1 a-d). Even though, all induced calli were
compact, the ones induced in the presence of 2,4-D (T1 and T4) were less rigid, easier to
separate, and in higher quantity (especially in T4 treatment) than the ones induced in the
presence of BAP and absence of 2,4-D. Given the higher amount of calli, the ones induced
in T4 medium were selected for further experiments on callus propagation.
84
Table 3.5: Callus Index (CI) for C. ladanifer leaf and stem explants establishment, in relation to survivor
explants.
Explants Treatment CI Explants Treatment CI Explants Treatment CI
T0 0.0 T0 0.0 T0 0.0
Internodal T1 55.6 T1 100.0 T1 11.1
segments T2 9.1 T2 18.2 T2 0.0
Internodal Leaf
and leaf
T3 64.8 segments T3 75.0 segments T3 54.5
segments
(all) T4 200.0 T4 200.0 T4 200.0
T5 83.0 T5 107.7 T5 58.3
In this work we demonstrated, for the first time, that both leaf and internodal stem
explants may be used for propagation of specific C. ladanifer genotypes under the
influence of BAP in the culture medium with or without NAA and without 2,4-D.
However, internodal segments seem to be more suitable for that purpose. Regarding this
plant species, only the work from Boukili et al. (2018) report shoot induction in 50% of
nodal segment explants of wild grown plants under the influence of BAP (0.2 mg/mL)
and IBA (0.1 mg/mL). Shoot induction percentage reported by Boukili et al. (2018) is
slightly higher than the higher shoot regeneration (organogenesis) percentage found in
this study for internodal segments under the influence of BAP at 0.5 mg/mL (37.6%,
Table 3.4). However, Boukili et al. (2018) used an antioxidant pre-treatment step to
prevent explants from browning which was not needed in this work. Shoot regeneration
from leaf explants compact calli of C. creticus using TDZ with or without NAA is
reported by Madesis et al. (2011). In this work it is not possible to conclude if
organogenesis happened from callus mass or directly from the explant because in some
internodal explants shoots regenerated without a visible callus mass and the dark green
compact callus were not isolated, subcultured, and evaluated for that feature. In this work,
shoot multiplication, rooting and acclimatisation to field conditions were not evaluated
but two works report protocols for those steps:
• Iriondo et al. (1995) developed a micropropagation process for six Cistus species
using nodal segments from germinated seedlings and microshoots. Solid MS basal
medium with 3% of sucrose was used for all in vitro procedures. Shoot
proliferation in non-supplemented media (2.8 shoots per explant after 4 weeks)
was similar to shoot proliferation with BAP at near 0.2 mg/mL (2.7
shoots/explant) and higher than shoot proliferation with Kin at near 0.2 mg/mL
(2.0 shoots/explant). Microshoots percentage of rooting was possible without
media supplementation but increased with IBA supplementation, varying between
85
75 and 92%. Number of roots per microshoot was around 5 which was not
significantly affected by IBA supplementation. In addition, C. ladanifer was one
of the Cistus species with higher rooting ability. Rooted plants were transferred to
ex vitro conditions and acclimatisation survival rates of C. ladanifer was 62%. For
that purpose, roots were sprayed with fungicide and transferred to pots containing
peat and vermiculite (5:2), plants were covered with glass for six weeks in a
greenhouse. Afterwards glass was removed, and plants maintained in the
greenhouse for one week and transferred to the field.
• Using nodal segments of germinated seedlings, Boukili et al. (2018) report the
advantage of using BAP instead of 2iP for shoot proliferation, finding an optimal
concentration of 0.2 mg/mL of BAP which resulted in 62.5% of response, 1.5
shoots/explant and 2.0 cm shoot length. Regarding rooting, IBA performed better
than NAA however the formation of roots in IBA supplemented medium was
accompanied by callus formation. After 4-5 weeks, rooted shoots were transferred
to sterilised vermiculite: soil (1:2) and acclimatised for 15 days in the same
conditions of in vitro experiments but at 90% relative humidity. Afterwards,
plants were transferred to the glasshouse and after 30 days they were planted in
the field. Using this procedure, more than 70% of plants survived.
In this study, two types of calli were induced using both types of explants. Dark green
compact calli were induced using BAP with or without NAA and were associated to shoot
regeneration. Light green, with white and yellow areas, compact calli, however less rigid
and in high amount than dark green calli were induced under the influence of 2,4-D with
or without BAP. This work reports, also for the first time, callus induction from C.
ladanifer explants despite the fact that callus formation during rooting with IBA is
reported by Boukili et al. (2018). Nevertheless, Pela et al. (2000) reported callus induction
from shoot-tips and lateral bud explants of C. creticus using BAP and which were
enhanced when combined with IAA and Zygomala et al. (2003) reported callus induction
from internodal segments of C. creticus using NAA alone. Callus may be used for
propagation purposes, genetic manipulation, and production of secondary metabolites in
liquid cultures.
86
Figure 3.1: Morphological evaluation of the leaf and shoot establishment and callus culture. a: shoot
differentiation from a stem explant in T3 medium; b: Compact dark green callus plus shoot differentiation
from a stem explant in T5 medium; c: Compact light green callus from a stem explant in T4 medium; d:
Compact green callus from a leaf basal explant in T5 medium; e: Compact yellow/white callus from T4A
subculture; f: Compact green callus from T4A subculture; g and h: Compact green callus with root-like
differentiation from T1 subculture; i: Callus with true roots, result from a side experiment were callus from
T4A were subculture in T4A medium without removing differentiation structures; j: Root-like
differentiation detail using a magnifying glass from a T4A callus.
3.3.2. Callus propagation and evaluation
Light green compact calli were obtained from internodal stem segments using T4 (Table
3.1) or T4B (Table 3.2) medium which was the medium that induced higher amount of
callus mass in the previous experiment (Table 3.5). This time contamination happened in
16.6% (n = 5), 60% (n = 18) of the explants survived and callus were induced in 100%
of those viable explants. Comparing to the first establishment (Table 3.4), contamination
rate was lower and so it was the survival rate.
Callus propagation was evaluated using 2,4-D and different amounts of BAP (Table 3.2)
during five subcultures. The initial mass of transferred callus showed to be inversely
correlated to the percentage of growth, i.e., smaller quantities of callus grew more (Table
3.6). Due to this feature only the three last subcultures were used for data analysis because
they did not show correlation between growth and initial mass, meaning that the initial
mass transferred on those subcultures was stabilised (approximately 0.5 cm in Ø or 70
mg).
Table 3.6: Correlation test between initial mass of transferred C. ladanifer calli and the percentage of
growth (% Growth) of all calli lines, for the 5 subcultures or for the last 3 subcultures.
%
Subcultures % Growth Subcultures
Growth
Pearson's Correlation -0.489** Pearson's Correlation -0.030
Significance (2
All 5 Significance (2 extremes) 0.000 Last 3 0.781
extremes)
n 150 n 90
n = 10 x 5 or 10 x 3 repetition for the 5 subcultures or for the last 3 subcultures, respectively.
Correlation is significant for **α = 0.01; * α = 0.05
87
Regarding callus mass propagation, no significant difference was found between the
different proportions of 2,4-D and BAP (Table 3.7). However, when 2,4-D was used
alone, calli presented a significantly higher dry weight. Morphologically, the three
treatments continued to produce light green calli with some exceptions of uncoloured
(yellow) calli (or parts of it) and some very green calli from T1 (Table 3.8 and Figure 3.1
e-h). However, T1 and T4A promoted the formation of differentiated structures erupting
from some calli (Figure 3.1 g, h, j). Necrotic tissues (brown, black/oxidized parts), mostly
inside de callus, appeared in calli from all treatments but less in accordance with the
degree of dedifferentiation. The degree of dedifferentiation may be also related to calli
dry weight.
Table 3.7: Callus evaluation for the last three subcultures of C. ladanifer callus proliferation.
Treatment Growth fw (%) Growth dw (%) dw (%)
T1 558 ± 262 577 ± 292 6.26 ± 1.05a
T4A 650 ± 299 677 ± 323 5.63 ± 0.62b
T4B 708 ± 342 686 ± 355 5.34 ± 0.76b
Values presented as mean ± standard deviation (n = 30).
a, b
Different coefficient letter means statistically significant difference (α = 0.05) by the post-hoc Tukey’s
HSD test.
Table 3.8: Percentage of morphological features for the last three subcultures of C. ladanifer callus
proliferation.
Colour (%)
Treatment Necrosis (%) Differentiation (%)
Yellow and green Green Yellow
T1 40 ± 14 50 ± 0 10 ± 14 97 ± 5 80 ± 8a
T4A 67 ± 21 23 ± 26 10 ± 8 70 ± 22 53 ± 12ab
T4B 47 ± 5 40 ± 8 13 ± 5 53 ± 31 3 ± 5b
Values presented as mean percentage of happening of the events ± standard deviation (n = 30).
a, b
Different coefficient letter means statistically significant difference (α = 0.05) by the non-parametric
Kruskal-Wallis’ test.
In a side experiment where calli with root-like differentiation were transferred to fresh
medium containing equal amounts of 2,4-D and BAP, some started to develop roots
(Figure 3.1 i). Histologic observation also suggests that these structures are roots, with a
defined central vascular cylinder (CVC) and a cortex (Figure 3.2 a1-2 and c1-3), although
not completed perhaps because of the initial stage of development. In longitudinal
sections, phloem sieve tubes with transversal cell walls are observed in the CVC
periphery (better observed as green stripes under the microscope than in the photographs)
and xylem tracheary-type cells are also observed in the centre (Figure 3.2 a2). A pattern
of primary xylem (metaxylem) is observed in transversal sections (Figure 3.2 a3). In
longitudinal and transversal sections of such differentiated structures, large cells are
88
observed at the cortex. An epidermal layer was also sometimes observed (Figure 3.2 b)
and endodermis assembling the CVC may be observed in Figure 3.2 (a2). However, a
root cap pattern was not well observed. Non-differentiated calli parts were also
histologically examined showing, overall, no tissue organisation (Figure 3.2 e). However,
a supposed non-differentiated part from a callus grown in T1 showed some tissue
organisation in what seems to be an external meristematic organisation due to a line of
highly stained small cells (Figure 3.2 d1-2). Studies regarding Arabidopsis thaliana calli
induction with auxin alone or in association with cytokinin showed that the calli initiated
in the pericycle (or analogous in embryo-apical organs) were very similar to root lateral
meristems but not exactly the same either morphologically (e.g. lack of apical
organisation) or in expressed genes, with the potential to form true roots or even shoots,
depending on the growth regulator and also on a time window of that meristem
development (Atta et al. 2009; Sugimoto et al. 2010). That rhizogenic ability is observed
in these calli.
Figure 3.2: Microscopic photographs of Callus histological slides stained with toluidine blue: a and b:
Callus from T4 (a) and T1 (b) with differentiated structures; a1: longitudinal section of the differentiated
structure (x10); a2: longitudinal section of the differentiated structure (x40), arrows pointing to sieve tubes;
c: Callus from T4 with differentiated structures (more deep slide than a and b); c1: several differentiated
structures (x4); c2: longitudinal and transversal sections of several differentiated structures; c3: transversal
section of a differentiated structure; d1 and 2: Callus T4 without differentiated structures however with
some degree of tissues specialisation; e1: Callus T4 without differentiated structures.
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Using the NAA and BAP combinations (Table 3.3) for the subculture of T4B callus, the
only organogenic response was the formation of roots in one callus subcultured in T2 and
one callus subcultured in T4. Necrosis appeared in almost all calli regardless the
treatment, and dead (full necrosis) happened in 30% of the calli subcultured in T0 and in
15 and 20% of the first calli subculture in T5A and T5B, respectively. In this conditions,
light green calli showed no potential for shoot organogenesis. In fact, those associated
with shoot regeneration were the dark green calli.
In sum, light green compact calli, obtained either from basal leaf or internodal stem
segments under the influence of the auxin 2,4-D, are rhizogenic. However, that rhizogenic
ability may be reduced by increasing the amount of BAP. Using other auxin, NAA, or
BAP alone or in combination also reduced the rhizogenic ability but did not induce shoots
formation from light green calli.
This type of calli is not suitable for propagation purposes at the conditions tested and they
also may not be suitable as starting material for cell suspension liquid cultures for the
synthesis of secondary metabolites because of their compact texture. In addition, root
cultures may not be ideal for the synthesis of important secondary metabolites because
root cultures of C. creticus showed to be unable to produce resin compounds such as
diterpenes (Skorić et al. 2012).
3.4. Conclusion
Leaf and internodal stem explants from Cistus ladanifer showed to be suitable starting
material for shoot regeneration under the influence of BAP and for callus production.
Two types of compact calli were obtained. Light green rhizogenic calli were obtained
under the influence of 2,4-D, yet root differentiation was inhibited by including BAP in
the culture media, mostly in equal amounts. Those calli showed not to be suitable for
shoot regeneration under the influence of BAP and NAA. Dark green calli were induced
in the same conditions as the regeneration of shoots under the influence of BAP alone
with or without NAA but further studies need to be done to evaluate their organogenic
ability and ultimately their utility to increase shoot multiplication rates. In addition, the
ability to synthetize specific compounds with pharmacological interest should be
evaluated for the two types of calli as cell cultures or even as starting material for organ
(e.g., roots, shoots) cultures.
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Chapter 4
Cistus ladanifer L. seeds: from ancient snack to novel and sustainable
food ingredient.
Abstract
Cistus ladanifer is a persistent, abundant, and widespread underexplored resource in the

Iberian Peninsula. Its seeds have been used as food for centuries, although their nutritional
value and potential as food ingredient have not been exploited until now. In this study
seeds from a natural shrubland were collected at three seasons during summer for two
consecutive years. Analytical evaluation of macronutrients content, fatty acids, and
mineral composition was performed. Regarding macronutrients, seeds showed a
carbohydrate content of 46.1 ± 1.6%, a fibre content of 20.9 ± 1.4%, a protein content of
16.2 ± 0.4%, a lipid content of 13.0 ± 1.1%, and an ash content of 3.87 ± 0.16%. Fatty
acid composition was found to be mostly unsaturated (74.1 ± 0.6%). Within mineral
composition, potassium was the most abundant (975 ± 53 mg/100g) followed by
phosphorous, magnesium and calcium. In conclusion, several nutrient related label claims
may be used for C. ladanifer seeds as food ingredient. Compared to common cereals, nuts
and seeds, C. ladanifer seeds stand close to flax and chia seeds in relation to macronutrient
and fatty acid composition, and to pine nuts in relation to mineral composition.
Keywords
Cistaceae; Fatty acids; Nutrients; Minerals; Rockrose
This chapter is published as:
Frazão DF, Paulo L, Peres F, Resende M, Espírito Santo C, Barroca C, Moitinho A, Delgado F (2022)
Cistus ladanifer seeds: From ancient snack to novel and sustainable food ingredient. Journal of Food
Composition and Analysis 109: 104503.
91
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Chapter 4 Cistus ladanifer L. seeds: from ancient snack to novel and sustainable food ingredient.
4.1. Introduction
Cistus ladanifer L., from the Cistaceae family, is a wild evergreen, usually erect shrub,
present principally in the South Iberian Peninsula (Portugal and Spain) and North
Morocco (Demoly and Montserrat 1993). In Portugal, dense C. ladanifer shrublands
occupy 7.5% (249,382 ha) of Portugal mainland forest area which in turn occupies almost
40% of the country mainland (Godinho-Ferreira et al. 2005). Even though this vast
resource holds no value for most agricultural and forestry activities, in recent years some
research contributed to its valorisation. Inclusion of C. ladanifer soft stems and leaves
and their extracts in ruminant diets has proven to be beneficial (Jerónimo et al. 2012;
Dentinho et al. 2014). Herbaceous biomass is used for essential oil and labdanum resin
extraction which until now are essentially used by the fragrance and perfume industry
(Raimundo et al. 2018). Lignified biomass can be used for the bio-fuel industry (Carrión-
Prieto et al. 2017b; Fernandes et al. 2018; Alves-Ferreira et al. 2019a). Together, these
uses are starting to draw a biorefinery approach for C. ladanifer valorisation which,
combined to its vast distribution, density, and resilience makes it a sustainable resource
worth investigating.
Cistus ladanifer fruits are globular loculicidal capsules bearing globular-polyhedric 1mm
seeds (Demoly and Montserrat 1993). Capsules are mature and exposed at early summer,
starting seed dispersal mostly below the parent plant canopy for an 8–10-month period
(Bastida and Talavera 2002). Additional to deer and ants, pre-dispersal seed predation by
Lepidoptera larvae (Noctiodae) and Coleopteran larvae (Bruchidae, Scarabeidae,
Curculionidae) is known to be the major cause of fruit and seed loss (Serrano et al. 2001;
Bastida and Talavera 2002; Delgado et al. 2007; Narbona et al. 2010).
The edible use of C. ladanifer seeds, eaten raw with a walnut-like taste snack or ground
into flour for bread and cake making, is documented through ethnobotanical studies and
surveys across the Iberian Peninsula (Tardío et al. 2005; Tardío et al. 2006; Aceituno
Mata 2010), with no indications of unsafety for human health. However, currently on the
EU market there is no evidence of the use of such seeds as food. According to Regulation
(EU) 2015/2283 concerning novel foods, a food produced or isolated from plants, or their
parts, is not considered a novel food if there is evidence of safe use and if it is produced
by traditional propagation practices used within the Union before May of 1997. Thus, C.
ladanifer seeds may not need an authorisation procedure or to be included in the novel
food list.
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According to the European Commission Health Promotion knowledge Gateway

(https://ec.europa.eu/), most EU countries recommend the consumption of nuts and seeds
as a part of a healthy diet. Although distinct in composition, nuts are important in diets
because they have been linked to a reduced risk of type 2 diabetes and cardiovascular
diseases (Kim et al. 2017).
Upcoming environmental changes such as low availability and high salinity of water will
reduce fruit, nut and seed yields, which could have a negative impact on populations
health, thus justifying the need for an adaption where sustainable and resilient food
systems are supported and safeguarded (Alae-Carew et al. 2020). In this regard, given the
potential biorefinery like valorisation and the abundance of C. ladanifer, its seeds are
worth evaluating as food and as an additional valorisation path.
This work aims to characterize the nutritional composition of C. ladanifer seeds collected
at three harvest seasons during two consecutive years.
4.2. Materials and Methods
4.2.1. Chemicals
Kjeltabs Cu-3.5 (FOSS, Micro Atomo, Portugal); sulphuric acid (Prolabo, VWR),
hydrochloric acid 0.1 M, hydrochloric acid 37%, potassium hydroxide, hydrogen
peroxide 30%, nitric acid 67-69%, petroleum ether, n-hexane, methanol (Normapur,
VWR, Portugal); FAME Mix C8-C24 (CRM 18918, Supelco, Sigma-Aldrich, USA).
4.2.2. Capsule collection and processing
C. ladanifer subsp. ladanifer capsules were collected from a natural shrubland located in
Penha Garcia, Idanha-a-Nova, Castelo Branco, Portugal (GPS: N 40o1’43.4’’ W
6o59’34.8’’) for two years (2019 and 2020) at three different times: Early Summer (ES,
end of June), Midsummer (MS, middle of August), and Late Summer (LS, beginning of
October). After each collection, capsules were smashed to release seeds. Seeds were
separated from the capsule parts by sieving with a metal mesh (> 1 mm). Seeds were
stored in a dark and dry place until further analysis. Before evaluation, seeds were ground
to a fine powder.
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4.2.3. Moisture content
Moisture content was determined using a gravimetric method. Samples (5.0 g) were
dried, under vacuum, at 70 ± 3oC for 4 hours, until constant weight. Moisture
corresponded to the weight loss during drying, reported in relation to the initial weight of
the samples.
4.2.4. Ash content
Ash content was determined using a gravimetric method. Samples (3.0 g) were
incinerated at 550 ± 25oC overnight. Ash content corresponded to the weight left after the
incineration, reported in relation to the initial weight of the samples.
4.2.5. Protein Content
Protein content was determined by the Kjeldahl’s method. Samples were weighted (1.0 g)
in a tared nitrogen free weighing paper. Two kjeltabs Cu-3.5 catalyst tablets and 12 mL
of 95% H2SO4 were added. Mixture tubes were placed in the digestion block (Tecator
Digestor, FOSS) at 420 ºC during 1h. Then, tubes were left to cool for 15 min. Samples
were automatically analysed in Kjeltec (8400, FOSS). Nitrogen was quantified by
titration with HCl (0.1 M) and protein content was obtained using the conversion factor
of 6.25.
4.2.6. Lipid content and fatty acid composition
Samples (2.0 g) were hydrolysed with HCl (6 N), on a Soxcap System extraction (2047,
FOSS). Samples were dried at 65 ± 3oC overnight. Pre-treated samples were subjected to
extraction with petroleum ether (90 mL) in Soxtec (2055, FOSS). Lipid content
corresponded to the weight of the extract, dried at 103 ± 2 °C for 1h.
Fatty acids composition was assessed by methylation of the lipid extract by dissolving
the dried extract (100 mg) in n-hexane and methanolic potassium hydroxide 2 N. Fatty
acids were analysed on GC SSL-FID (7890A, Agilent), HP5 column (30 m × 0.32 mm
i.d.× 0.25 µm), helium flow rate 0.8 mL/min, temperature detector 300 °C and
temperature injector 260 °C with split ratio of 1:100. Temperature program: initial
temperature 50 °C for 3 min, increased by 10 °C/min to 170 °C, increased by 2 °C/min to
200 °C for 10 min, increased by 10 °C/min to 250 °C for 15 min. Their relative
composition was determined by integration of the peak’s chromatogram. FAME Mix C8-
C24 was used for identification.
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4.2.7. Crude Fibre content
Because total lipid content of seeds was higher than 5% (w/w) it was necessary to remove
excess of sample fat. Crude fat was removed by extraction from the dried sample using
as organic solvent petroleum ether b.p. 40–60 °C in a Soxtec System HT1043 Extraction
unit apparatus (Tecator, Hoganas, Sweden). After lipid extraction, the powder was dried
in a ventilated chamber set at 103 °C.
Crude fibre was determined by the Weende method (Fibertec System1020, Tecator).
During the acid digestion the feed sample was boiled in 1.25% (v/v) H2SO4 (extraction
of free sugar and starch) followed by alkaline digestion with 1.25% (w/v) NaOH (to
remove proteins, some hemicelluloses and lignin). After each digestion, crucibles were
washed with hot distilled water to remove acid and alkaline residues and neutralised.
Finally, the samples where defatted again with acetone and the crucibles were placed at
a muffle furnace for ash determination.
4.2.8. Carbohydrate content and energetic value
Carbohydrate content and the energetic value of the samples were calculated according
to EU regulation No 1169/2011.
4.2.9. Mineral analysis
Minerals were analysed by first digesting the samples (0.5 g) with 6 mL nitric acid 67-
69% and 2 mL hydrogen peroxide 30% in a microwave digestion system (Ethos One,
Milestone). Minerals were quantified by atomic absorption spectroscopy (ICE3000,
Thermo Scientific) or Inductively Coupled Plasma – Atomic Emission Spectrometry
(Activa M, Horiba Jobin Yvon). Flame atomic spectrometric measurements using the
optimal instrumental parameters for each element with the following wavelengths: 589.0
nm (Na), 766.5 nm (K) and 422.7 nm (Ca). The operating conditions of the inductively
coupled plasma atomic emission spectrometry equipment were as follows: 1000 W
plasma power, 15 L/min plasma gas flow, 0.02 L/min nebulizer Air flow and 1.0 bar air
pressure. The analytical wavelengths (nm) were: 327.395 (Cu), 259.940 (Fe), 257.610
(Mn), 213.857 (Zn), 213.618 (P) and 279.553 (Mg).
4.2.10. Statistical Analysis
One- and two-way-ANOVA and the Tukey’s post-hoc test were performed using the IBM
SPSS Statistics 27 Software. All the analysis were performed in duplicate (n = 2).
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Multivariate data analysis, namely Principal Component Analysis (PCA) was performed
on datasets considering nutritional and mineral composition of C. ladanifer and other
seeds and nuts (almond, peanut, hazelnut, chestnut, walnut, pine nut, wheat, rye, oat, corn,
pumpkin seeds, chia seeds, flax seeds, sesame seeds) using STATISTICA 7 Software.
Data was compiled from the Portuguese Platform of Food Information (INSA 2021) and
compared to mean values of C. ladanifer seeds, as absolute values.
4.3. Results and discussion
The overall nutritional composition of C. ladanifer seeds are shown in Table 4.1.
Carbohydrates and fibres stand as the major macronutrients representing, respectively,
46.1 ± 1.6% and 20.9 ± 1.4% of the seeds weight. Although not very clear, carbohydrate
content showed to be highest at the MS season and fibre content showed to be lowest, in
both years. Ash content (3.87 ± 0.16%) and the energetic value of seeds (384 ± 9 kcal/100
g) did not show a consistent season variability. In contrast, lipid (13.0 ± 1.1%) and protein
(16.2 ± 0.4%) decreased from the beginning of summer to its end, even at the dry weight
basis, removing the effect of the water content variation (5.88 ± 0.53%) during summer.
The lipid content of C. ladanifer seeds was already reported to be 13% elsewhere
(Krollmann and Gülz 1983; Borges 1986).
According to EFSA (2010a), total carbohydrates intake should range between 45 to 60%
of the total energy intake for children and adults, regardless of gender and lifestyle. Total
carbohydrates of C. ladanifer seeds, excluding fibres, comprises 45% of the total energy,
making it an equilibrated source of carbohydrates. Fibre adequate intake stands for 25
g/day for adults and lower for children, meaning that a portion of 132 g of C. ladanifer
seeds is enough. Indeed, according to Regulation (CE) No 1924/2006, regarding food
nutritional and health claims, these seeds are considered high in fibre (≥ 6 g of fibre per
100 g). Fibre is a general term for indigestible polysaccharides for humans that, generally,
influence gut microbiota and nutrient absorption by different mechanisms, with generally
accepted health benefits (Dhingra et al. 2012; Fuller et al. 2016; Qi et al. 2018). Proteins
contribute to 16% of the total energy of C. ladanifer seeds, which means that according
to Regulation (CE) No 1924/2006, as food, those seeds can be considered as a source of
protein. According to EFSA (2012), although several health outcomes are associated to
protein intake, data is insufficient to estimate a dietary reference value, pointing out a
population reference intake of 0.83 g/kg body weight. Total lipids comprise 29% of the
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seeds’ total energy, which is within the reference intake range for adults (20 - 35%)
proposed by EFSA (2010b). Thus, C. ladanifer seeds are a balanced source of lipids.
Cistus ladanifer seeds’ fatty acid profile is presented in Table 4.2. Unsaturated fatty acids
represent 74.1 ± 0.6% of the total fatty acids, distributed in 58.9 ± 2.3% for
polyunsaturated and 15.2 ± 2.4% for monounsaturated. Specifically, oleic acid (C18:1,
ω9), linoleic acid (C18:2, ω6) and linolenic acid (C18:3, ω3) are the major fatty acids
comprising 14.9 ± 2.3, 46.1 ± 1.5, and 12.8 ± 1.1%, respectively. Results obtained for the
main fatty acids are in accordance with the values reported by Krollman and Gülz (1983)
and very similar to the composition of some walnuts cultivars for oleic and linolenic acids,
and lower for linoleic acid that are usually higher than 50% for this nut (Martínez et al.
2010; Tapia et al. 2013). Therefore, according to Regulation (CE) No 1924/2006, claims
such as “High omega-3 fatty acids”, “high polyunsaturated fat” and “high unsaturated
fat” may be used for C. ladanifer seeds as food. Additionally, the ω3 to ω6 ratio was
observed to be 0.27 which matches the 1:4 recommendation (Kamal‐Eldin and Pickova
2008). Despite the season variability observed for total lipid content (Table 4.1), for the
year of 2020 a similar decrease was observed for linoleic acid accompanied by an increase
in oleic acid. Erucic acid (C22:1), reported as toxic (Knutsen et al. 2016), was not detected
in C. ladanifer seeds. Krollmann and Gülz (1983) report that approximately 10% of the
seed lipids are free sterols, mainly β-sitosterol, which according to Bin Sayeed et al.
(2016), although in need for extensive research, may be a health promoting molecule for
consumers.
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Table 4.1: Nutritional composition (% dw/dw) and energetic value of C. ladanifer seeds flour from early, middle, and late summer collection (ES, MS, LS, respectively) during
two consecutive years (2019 and 2020).
Energetic value
Year Season Water Content Protein Lipid Ash Fibre Carbohydrate
(kcal)/100 g
ES 5.89 ± 0.00c 16.7 ± 0.1a 14.2 ± 0.1a 3.83 ± 0.03b 21.9 ± 0.3a 43.3 ± 0.3d 387 ± 1b
d b,c b b c a
2019 MS 5.55 ± 0.02 16.0 ± 0.2 14.0 ± 0.0 3.74 ± 0.02 18.4 ± 0.7 47.8 ± 0.5 395 ± 1a
e c c b b b
LS 5.22 ± 0.01 15.8 ± 0.3 13.5 ± 0.0 3.73 ± 0.01 20.3 ± 0.4 46.7 ± 0.1 390 ± 1b
ES 5.57 ± 0.07d 16.7 ± 0.1a,b 12.7 ± 0.0d 4.01 ± 0.01a 21.9 ± 0.0a 44.7 ± 0.1c 381 ± 1c
2020 MS 6.36 ± 0.15b 16.1 ± 0.2a,b,c 11.7 ± 0.0e 4.13 ± 0.05a 21.0 ± 0.0a,b 47.1 ± 0.0a,b 374 ± 1d
LS 6.70 ± 0.07a 15.7 ± 0.0c 11.6 ± 0.0e 3.78 ± 0.05b 22.2 ± 0.0a 46.8 ± 0.0b 371 ± 0d
Overall 5.88 ± 0.53 16.2 ± 0.4 13.0 ± 1.1 3.87 ± 0.16 20.9 ± 1.4 46.1 ± 1.6 383 ± 9
Values presented as mean ± standard deviation (n = 2 for season x year and n = 6 for overall).
Different upper-case letters represent significant (α < 0.05) statistical difference of means by the Tukey’s post-hoc test, for each column parameter.
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Table 4.2: Fatty acids composition (%) of C. ladanifer seeds flour from early, middle, and late summer collection (ES, MS, LS, respectively) during two consecutive years
(2019 and 2020).
Year Season C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 (w6) C18:3 (w3) C20:0
ES 0.124 ± 0.003 19.4 ± 0.1a,b 0.0773 ± 0.0022c 4.56 ± 0.01 12.6 ± 0.0c 47.6 ± 0.3a 13.8 ± 0.1a 0.434 ± 0.005c
2019 MS 0.118 ± 0.002 20.1 ± 0.2a 0.0753 ± 0.0026c 4.54 ± 0.08 13.2 ± 0.2c 46.9 ± 0.5a 13.2 ± 0.3a 0.455 ± 0.008c
LS 0.123 ± 0.001 19.4 ± 0.1a,b 0.0603 ± 0.0021d 4.50 ± 0.05 12.7 ± 0.1c 47.4 ± 0.0a 14.0 ± 0.1a 0.454 ± 0.002c
ES 0.113 ± 0.002 18.2 ± 0.2b 0.0777 ± 0.0010c 4.46 ± 0.06 15.5 ± 0.2b 46.0 ± 0.3a,b 12.6 ± 0.2a,b 0.526 ± 0.000b
2020 MS 0.126 ± 0.001 18.3 ± 0.6b 0.101 ± 0.001b 4.76 ± 0.21 17.8 ± 0.5a 44.5 ± 0.9b,c 11.6 ± 0.6b 0.570 ± 0.016a
LS 0.120 ± 0.003 18.6 ± 0.1a,b 0.117 ± 0.003a 4.84 ± 0.01 17.8 ± 0.0a 44.0 ± 0.4c 11.5 ± 0.1b 0.587 ± 0.004a
Overall 0.121 ± 0.005 19.0 ± 0.8 0.0846 ± 0.0186 4.61 ± 0.16 14.9 ± 2.3 46.1 ± 1.5 12.8 ± 1.1 0.504 ± 0.060
Year Season C20:1 C22:0 C24:0 Saturated Unsaturated Monounsat. Polyunsat. w3/w6
c d c
ES 0.119 ± 0.029 0.0575 ± 0.0047 0.0832 ± 0.0012 24.6 ± 0.2 74.3 ± 0.4 12.8 ± 0.1 61.4 ± 0.4a 0.290 ± 0.001a
c c,d c
2019 MS 0.130 ± 0.028 0.0908 ± 0.0025 0.0862 ± 0.0004 25.4 ± 0.5 73.5 ± 0.7 13.4 ± 0.2 60.1 ± 1.0a 0.281 ± 0.04a,b
LS 0.125 ± 0.002 0.0480 ± 0.0059c 0.0998 ± 0.0104b,c,d 24.6 ± 0.2 74.2 ± 0.3 12.8 ± 0.1c 61.4 ± 0.1a 0.296 ± 0.002a
ES 0.157 ± 0.057 0.595 ± 0.007a 0.121 ± 0.003a,b,c 24.0 ± 0.3 74.4 ± 0.4 15.8 ± 0.1b 58.7 ± 0.7a,b 0.275 ± 0.003a,b
b a,b a
2020 MS 0.244 ± 0.010 0.369 ± 0.035 0.131 ± 0.011 24.2 ± 1.0 74.3 ± 1.1 18.1 ± 0.5 56.1 ± 1.7b 0.260 ± 0.009b
LS 0.271 ± 0.001 0.442 ± 0.012b 0.147 ± 0.002a 24.7 ± 0.2 73.6 ± 0.4 18.1 ± 0.0a 55.4 ± 0.5b 0.260 ± 0.000b
Overall 0.175 ± 0.067 0.267 ± 0.213 0.112 ± 0.024 24.6 ± 0.6 74.0 ± 0.6 15.2 ± 2.4 58.9 ± 2.6 0.277 ± 0.014
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Regarding mineral composition, like ash content, no clear season and year content
variability trend was observed for the individual minerals (Table 4.3). According to
Regulation (CE) No 1924/2006 and to Annex XIII of Regulation (CE) No 1969/2011, C.
ladanifer seeds may be claimed to be a source of calcium, and a high source of potassium,
phosphorous, magnesium, copper, iron, manganese, and zinc, besides “sodium-free”.
Despite these claims, potassium was the major mineral, found in higher quantity (974.50
mg/100 g). Minerals are micronutrients and therefore are needed in low quantities in the
human diet for the normal physiological function of the human body. However, many
global food systems fail to provide them resulting in alarming deficiency disorders as
demonstrated by Welch (2002). Palmer and Clegg (2016) discuss the benefits of high-
potassium diets, namely blood pressure reduction, stroke risk decrease, and bone health
improvement. In this regard, C. ladanifer seeds may stand as a fortifying food worth
including in human diet. However, risks associated to a high intake of micronutrients
should be evaluated (Engle‐Stone et al. 2019).
To find a position for C. ladanifer seeds among other nuts and cereals, Principal
Component Analysis (PCA) was performed, comparing nutrient composition values
extracted from the Portuguese Platform of Food Information (INSA 2021,
www.portfir.insa.pt) (Figure 4.1). By PCA, it was possible to reduce the initial 9-
dimension hyperspace to a plane defined by the first two principal components (new axis),
containing 75.08% of the variance of the original data matrix. The projection of the
original variables and of the nut/cereals on these planes is shown in Figure 4.1. A clear
separation between cereals and nuts was observed, the first ones higher in carbohydrates
and water and the others higher in protein and lipid content. C. ladanifer, flax and chia
seeds were however separated from those groups by the higher ash and fibre content. C.
ladanifer seeds additionally showed an intermediate carbohydrate, protein, and lipid
content in relation to the two groups clearly formed by nuts and cereals, once more
showing a balanced composition in what regards those macronutrients. The second PCA
aimed to group the several nuts and cereals in relation to the mineral composition and
explained 75.93% of the total variance using two components. From this analysis, two
groups were clearly formed, this time clustering the seeds (chia, sesame, flax, and
pumpkin seeds) in one group with higher general mineral content and clustering nuts,
cereals, and C. ladanifer seeds in another group. Within the latter group C. ladanifer and
pine nuts are at extreme points mainly because of their high potassium content.
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Table 4.3: Mineral composition (mg/100 g, dw/dw) of C. ladanifer seeds flour from early, middle, and late summer collection (ES, MS, LS, respectively) during two consecutive
years (2019 and 2020).
Year Season Sodium Copper Iron Manganese Zinc Phosphorous Magnesium Calcium Potassium
ES 1.27 ± 0.04b 1.48 ± 0.00a 4.55 ± 0.09c,d 9.88 ± 0.09a 3.37 ± 0.00a 452 ± 3a,b 287 ± 57 221 ± 0 940 ± 4b,c
a b a c a,b b
2019 MS 2.15 ± 0.42 1.41 ± 0.01 6.26 ± 0.35 6.88 ± 0.04 3.35 ± 0.03 437 ± 7 237 ± 23 226 ± 2c 931 ± 19b,c
a a a,b b a a,b
LS 2.33 ± 0.22 1.48 ± 0.01 5.97 ± 0.00 9.25 ± 0.00 3.41 ± 0.04 445 ± 13 216 ± 5 223 ± 2c 924 ± 9c
ES 2.33 ± 0.12a 1.38 ± 0.02b 5.28 ± 0.12b,c 6.48 ± 0.09d 3.15 ± 0.00a,b,c 442 ± 6b 221 ± 0 252 ± 3a,b 972 ± 17b
2020 MS 2.39 ± 0.13a 1.38 ± 0.01b 4.60 ± 0.18c,d 5.54 ± 0.18e 3.00 ± 0.15c 458 ± 14a,b 202 ± 16 259 ± 2a 1059 ± 6a
a a d b b,c a
LS 2.93 ± 0.01 1.49 ± 0.01 4.41 ± 0.18 8.90 ± 0.03 3.08 ± 0.06 479 ± 3 245 ± 25 248 ± 1b 1021 ± 1a
Overall 2.23 ± 0.54 1.44 ± 0.05 5.18 ± 0.77 7.82 ± 1.67 3.22 ± 0.17 452 ± 16 232 ± 35 238 ± 16 974 ± 53
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Figure 4.1:Principal Component Analysis of nutritional Projection of the nutritional components of C.

ladanifer and other nuts and seeds. Projection of variables (1) and values (2) on the two-factor plot obtained
by the Principal Component Analysis of the general nutritional composition (A, 75.08% of the variance)
and of the mineral composition (B, 75.93% of the variance). Values for C. ladanifer seeds were obtained
from Tables 4.1, 4.2, and 4.3 as the mean overall value, whereas values for the other nuts and seeds (whole
grain, whole flour, or kernel) were obtained from the Portuguese Platform of Food Information (INSA
2021).
4.4. Conclusion.
To our knowledge, this study is a first step to understand the nutritional composition of
C. ladanifer seeds. In general, these seeds have an intermediate compostion between the
carbohydrate rich cereals and lipid rich nuts. They are also a source of protein and
calcium, and a high source of fiber, unsaturated fatty acids, and of several minerals. The
fatty acid profile gives these seeds a portential value as a health promoting food
ingredient. Regarding valorisation as a food, future research should focus on amino acids,
103
vitamins, and polyphenolics profile, and evaluation of other lipid compounds such as
sterols. A toxicological evaluation could also be benneficial to safeguard consumers.
Cultivation/collection practices to maximize the efficiency and reduce harvest costs
would be highly relevant.
104
Chapter 5
Labdanum resin from Cistus ladanifer L.: evaluation of residual water
vs. extraction yield.
Abstract
Cistus ladanifer L. (Cistaceae) exudes an aromatic resin nowadays valued in the

perfumery and fragrance industry. Traditional processes for the extraction and isolation
of such resin use boiling water or alkaline water followed by acidic precipitation. A
concern raised about the effluents resulting from these extraction processes. Labdanum
resin was extracted with Na2CO3 solution (25 g/L) at 60oC and precipitated with sulphuric
acid (5 M). The residual water was evaluated regarding total phenolic content, suspended
solids, electric conductivity, and sulphate, sodium, magnesium, and calcium content. The
effluent of the reference Andalusian process was characterized by a total phenolic content
of 1245 ± 455 mgGAE/L, 1338 ± 101 mg/L of suspended solids, pH of approximately 2,
electric conductivity of 34.8 ± 0.7 mS/cm, 22284 ± 710 mg/L of sulphate, 9696 ± 1072
mg/L of sodium, 3.97 ± 0.24 mg/L of magnesium, 3.52 ± 0.80 mg/L of calcium, and a
SAR of 876 ± 112. Because the values were far from the limit values set by the Portuguese
decree-law 236/98 for residual waters discharge and irrigation waters, it was concluded
that efforts should be made to optimise the extraction process. In that regard, a factorial
designed experiment was done to evaluate the effect of Na2CO3 concentration (0; 2.5; and
25 g/L), extraction temperature (60 and 100oC) and acidification extent (pH2,
neutralisation, and none) on the residual water quality and on the yield of labdanum resin
extraction. Alkalinisation and acidification are important to obtain high resin extraction
yields (Andalusian vs. Zamorean process), but mostly Na2CO3 concentration may be
reduced to meet sulphate criteria for discharge without significantly affect resin extraction
yields. Despite that, to meet salinity criteria for irrigation waters a higher reduction in
Na2CO3 concentration is needed for the Andalusian process. An advantage of the
Zamorean process is that effluent salinity is low but so it is labdanum extraction yield.
Phenolic content, although lower for extractions done at 60oC, was far from the limit
values for discharge, regardless experimental conditions. Given the high phenolic content
(2 – 12 gGAE/L), the residual water from labdanum extraction by both traditional
processes must be treated before discharge. If separated, phenolic compounds may be
valorised as a by-product.
105
Chapter 5 Labdanum resin from Cistus ladanifer L.: evaluation of residual water vs. extraction yield.
Keywords
By-product, Cistaceae, effluent, rockrose, sustainability

This chapter is published as:
Frazão DF, Silva H, Silva AM, Gonçalves JC, Mesquita C, Delgado F (2022) Labdanum resin from Cistus
ladanifer L.: evaluation of residual water vs. extraction yield. Actas Portuguesas de Horticultura 37:73-
84.
106
5.1. Introduction
Cistus ladanifer (Cistaceae) is an evergreen shrub abundantly distributed in the western

Mediterranean: south of France, Iberian Peninsula, North Marrocos and Argelia (Demoly
and Montserrat 1993; Godinho-Ferreira et al. 2005; Montero et al. 2020). Like many
Cistus species (Gülz et al. 1996), this plant exudes an aromatic terpenoid and phenolic
resin called labdanum. This resin is commercially traded by the name of “labdanum gum”
principally for the perfumery and fragrance industry sectors (Raimundo et al. 2018).
Traditionally, labdanum resin is extracted, using water, by two different processes: the
Zamorean process and the Andalusian process (Lawrence 1999; Morgado et al. 2005;
Burguer 2016). The Zamorean process consists in a physical extraction where labdanum
resin is removed from the surface of C. ladanifer plant material using boiling water and
then the resin at the surface of the water is continuously separated using a skimmer. The
Andalusian process is a chemical extraction where the labdanum resin is extracted from
the surface of the plant material with warm alkaline water and afterwards precipitated by
lowering the pH with acid and finally separated with a skimmer or by decantation.
Andalusian process is currently used as the industrial process and annually between 100
and 1000 tons of labdanum gum from C. ladanifer, according to European Chemicals
Agency (ECHA) registrations.
Given the abundance of the plant resource and the use of water as solvent, labdanum gum
extraction process lines up with “green extraction principles” which in turn are derived
from the “green chemistry” and “green engineering” principles (Chemat et al. 2019).
However, this “green” approach also highlights the importance of the solvent recovery
and the production of by-products instead of waste. Recovery of water is facilitated either
by nature or, if needed, by water treatment plants. Little is known, within scientific
literature, about the residual water of the labdanum extraction processes.
This study aims to assess the effluent by evaluating some water quality parameters
relevant not only for residual water discharged in the environment but also for the use as
irrigation water, according to the guidelines defined by the Portuguese Decree-Law
236/98 which regulates quality criteria and rules for the various uses of water, including
human consumption, irrigation and residual water discharge in natural waters and soils.
107
5.2. Materials and Methods
5.2.1. Chemicals
Sodium carbonate (Na2CO3, Biochem, Frilabo, Portugal), Methanol 99.9% (Fisher

Scientific, EUA), Sulfuric acid 97% (H2SO4 Chem-Lab, JMGS, Portugal), Folin-
Ciocalteu reagent and Gallic acid-1-hydrate (Panreac, LaborSpirit, Portugal), BaCl2H2O,
1000 mg/L standard solution of Na, Mg, and Ca, and LiCl (Merck, Darmstadt, Germany),
LaCl37H2O (VWR, Avantor, International)
5.2.2. Plant material and labdanum extraction (reference method)
Herbaceous and semi-woody plant material was collected from a 5-year-old C. ladanifer
shrubland, located in in Penha Garcia, Portugal (GPS: N 40o1’43.4’’ W 6o59’34.8’’), in
August 2018. Labdanum resin was extracted by the method described in Burguer (2016),
simulating the Andalusian process, with some modifications. Briefly, 25 g of plant
material were immersed in 200 mL of Na2CO3 solution (25 g/L) for 1 h at 60oC. The
solution was cleared from the plant material through filtration and left to rest overnight.
Then, H2SO4 (5 M) was added slowly to the stirring solution at room temperature to
decrease its pH until it reached 2. The precipitated resin was separated from the
supernatant by centrifugation (3030 xg for 15 min, Mega Star 600R, VWR). The
supernatant is the residual water, which was saved for analysis. The solution initially
started as a strong black solution and after the resin precipitation it turned transparent
yellow/orange. The precipitated resin was freeze-dried at 0.3 mbar for 72 h (Zirbus VaCo
10-II-D). Labdanum absolute was obtained by dissolving 1 g of the resin in 20 mL of
methanol under an ultrasonic bath, and waxes were precipitated at -20oC overnight and
separation by centrifugation (3030 xg at -5oC for 15 minutes). The procedure was
repeated three times and the three methanolic solutions were joined as the absolute
extract, which was finally dried using a rotatory evaporator operating at 30oC.
To evaluate the possibility of extraction saturation, using the same starting material, this
method was performed, three times, using 15 g, 25 g, and 35 g of plant material. The same
resin extraction yield (6.42 ± 1.23% dw/fw) was obtained for the three starting amounts
of plant material (ANOVA ρ-value = 0.415) while the quantity of resin extracted
increased with higher starting amount of plant material (ANOVA ρ-value = 0.043).
108
5.2.3. Experimental design of method variations
To evaluate the effect of extraction conditions in extraction yields and residual water
quality parameters, using the reference method (section 5.2.2) three independent factors
were studied: i) Na2CO3 concentration: 0 (simulating Zamorean process), 2.5, and 25 g/L
(simulating Andalusian process); ii) Acidification extent with H2SO4 (5 M): no
acidification (s/), neutralisation of carbonates i.e. until CO2 stops to be release (N), and
until pH = 2; iii) temperature of extraction: 60 and 100oC. All methods were done in
triplicate (n = 3). Resin and resin absolute yields were recorded, and the supernatants
were saved for analysis.
5.2.4. Residual water analytical methods
The residual water obtained by the reference extraction method was characterized by
standard methods for the examination of water and wastewater (Bridgewater L 2012) for
the following parameters: total suspended solids; total sulphate concentration was
determined using a gravimetric method; total sodium (Na+), total calcium (Ca2+), and total
magnesium (Mg2+) concentrations were determined by flame atomic absorption
spectrometry (Thermo Scientific ice 3500); electrical conductivity was measured using a
conductivity meter (WTW inoLabCond Level 1); pH was monitored using a pH meter
(Consort C3060).
Total suspended solids were determined by filtration of the residual water (< 200 mL)
through a filter paper of 45 µm porosity and dryness of the filter paper at 105oC until
constant weight. This method was done only for the residual waters of the reference
method
Sulphates were quantified on filtered samples by gravimetry. Briefly, 20 mL of

BaCl2.H2O (100 g/L) were added dropwise to 250 mL (Vsample) boiling sample solution
(at further 1:100 dilution), under constant stirring. After resting for 24h, the solution was
filtered using a filter paper (45 µm porosity) placed over a P1 glass filter, previously dried
and weighted (P2). The glass filter + filter paper was dried at 105oC until constant weight
(P1). Sulphate concentration was calculated according to equation 1:
(𝑃2 − 𝑃1 ) × 411.5
𝑆𝑂42− (𝑚𝑔/𝐿) = × 1000 𝑥 𝐷𝑖𝑙. 𝐹𝑎𝑐𝑡𝑜𝑟 (1)
𝑉𝑠𝑎𝑚𝑝𝑙𝑒 (𝑚𝐿)
109
This method was used to analyse the residual waters of the reference method. Sulphate
content of the residual waters obtained with other experimental conditions was estimated
directly from the volume of H2SO4 5 M solution spent.
Sodium (Na+), magnesium (Mg2+), and calcium (Ca2+) ions were quantified on filtered
samples by flame atomic absorption spectrometry. Sodium was read at emission mode
(289 nm) and quantified using standard solutions of 1.2, 3.6, 5.4, 7.2, and 9.0 mg/L. For
sodium quantification LiCl solution (61.09 g/L) was added to standards and samples at
1:9 proportion, to avoid ionisation interferences. Magnesium and calcium were read at
absorption mode (285 and 422 nm, respectively) using Mg and Ca hollow-cathode lamps,
respectively, and deuterium lamp was used for background correction. Magnesium was
quantified using standard solutions of 0.2, 0.4, 0.6, 0.8, and 1.0 mg/L. Calcium was
quantified using standard solutions of 1.4, 2.8, 4.2, 5.6, and 7.0 mg/L. For Magnesium
and calcium quantification, LaCl3 solution (20.05) was added to standards and samples at
1:9 proportion, to avoid ionisation interferences. Air-acetylene burner at a flow rate of
1.1 L/min was used for atomisation. Samples were diluted 1:1000 for Na+ quantification,
1:5 for Mg2+ quantification and no dilution for Ca2+ quantification.
The potential degree of sodification of soils, due to the use of residual water for irrigation,
was evaluated by the sodium absorption ratio (SAR): ionic strength ratio of Na+
concentration to the Ca2+ and Mg2+ concentration, calculated according to equation 2:
[𝑁𝑎 + ]
𝑆𝐴𝑅 =
2+ 2+ (2)
√[𝐶𝑎 ] + [𝑀𝑔 ]
2
Sodium content of the residual waters obtained with other experimental conditions was
estimated directly from the quantity of Na2CO3 spent.
Total phenolic content was done on filtered residual waters from all experimental
conditions by the Folin-Ciocalteu method. Briefly, 2.5 mL of diluted sample (1:10) was
joined with 0.5 mL of Na2CO3 solution (7.5% w/v), 1.75 mL of distilled water and 0.25
mL of Folin-Ciocalteu reagent. The reactive mixture was incubated at room temperature,
in the dark, for 2 h and the absorbance was read at 725 nm (Specord 200 plus,
Analytikjena). Sample blanks were prepared by changing the Folin-Ciocalteu reagent for
distilled water and their absorbances were subtracted. Standard solutions were prepared
110
with gallic acid at concentrations between 0.1 and 0.003 mg/mL. Results are expressed
as gallic acid equivalents (mgGAE/mL).
5.2.5. Statistical analysis
Independent t-tests for equal variances not assumed (Welch’s correction) were used to
evaluate significant differences from the reference method (α = 0.05) for all the
parameters evaluated, using the IBM SPSS Statistics 27 Software.
5.3. Results and Discussion
Labdanum resin and absolute yields, as dry weight in relation to plant material fresh
weight, obtained with all combinations of extraction conditions are shown in Figure 5.1.
The extraction conditions based on Burguer (2016) were considered the reference.
In Figure 5.1, statistical differences are reported in relation to the reference method. Resin
and absolute extraction yields did not differ significantly when Na2CO3 concentration was
reduced to 2.5 g/L, when acidification extent was reduced to neutralisation, or when
temperature of extraction was increased to 100 ºC. However, the combination of those
method variations resulted in a significant decrease in extraction yields. The only
exception was the combination of extraction at 100 ºC and acidification until
neutralisation in resin extraction yield. Extraction with distilled water rendered lower
yields of resin and absolute compared to the reference method regardless other conditions,
and the same happened for when acidification was not done (s/). Extraction with water
alkalinised with Na2CO3 at least at 2.5 g/L and acidification at least until neutralisation
of CO32- and CO3H- are thus important to obtain high yields of labdanum resin and
absolute, like the reference method yields. A temperature of 100oC during extraction did
not enhance extraction yields.
Burguer (2016) obtained labdanum resin by a process similar to the reference method of
this study with a yield between 7.79-8.86% (dw/dw) in relation to the plant material which
is slightly lower given the fact that in this study results are reported in relation to the fresh
weight of plant material. The author used filtration to recover the precipitated resin
instead of centrifugation. In fact, filtration was attempted in preliminary experiments but
excluded because of the difficulty to recover the resin from the filter. Morgado et al.
(2005) obtained a labdanum extraction yield of between 7-18% (dw/dw) in relation to
usable biomass. Authors extracted the resin with a solution of Na2CO3 (10 g/L) at 50oC,
111
precipitating the gum with H2SO4 93% (v/v) which was left to rest for 24 hours and
separated by decantation and dried at ambient temperature. Greche et al. (2009) extracted
750 g of plant material with 10 L of an aqueous solution of NaHCO3 at 8.4 g/L for 10 min
at 60 - 70oC. Then the authors neutralised the solution with HCl at 0.2 M and removed
the resin with a perforated skimmer, not reporting extraction yields.
Labdanum absolute yields in relation to the resin weight are presented in Table 5.1. In
contrast to both resin and absolute extraction yields in relation to the plant material, there
was not a clear effect of each extraction factor. However, the ten-fold reduction of
Na2CO3 concentration significantly increased the absolute proportion in the resin and the
higher temperature of extraction reduced it significantly. Overall, it seems to exist a
decrease of that absolute proportion related to a decrease in acidification extent and an
increase in extraction temperature, and the other way around for a decrease of Na2CO3
concentration. Several authors extracted labdanum absolute from labdanum resin by
dissolving the dried extracts or resin in warm methanol or ethanol and then precipitating
and removing waxes using negative temperatures (de Pascual et al. 1984; Vogt et al. 1987;
Chaves et al. 1997b; Sosa et al. 2005; Greche et al. 2009; Alías et al. 2012; Burguer 2016).
According to Burguer (2016) methanol extracts more waxes (38% dw/dw) by this process
than ethanol (8.5% dw/dw). In fact, Greche et al. (2009) precipitated waxes from the
labdanum absolute prepared with ethanol and even so, wax compounds appeared as
significant compounds in the ethanol extract.
112
Figure 5.1: Labdanum resin (dark grey) and labdanum methanol absolute (light grey) extraction yields in relation to plant material weight (mean, n = 3, % dw/fw), for the
various extraction conditions (base to top: Na2CO3 (g/L), temperature (oC), Acidification extent) . Error bars correspond to the 95% confidence interval. (*) statistical difference
in relation to the reference (marked with rectangle), found by performing independent t-tests for equal variances not assumed (α = 0.05).
Table 5.1: Labdanum Absolute yield, in relation to resin (% dw/dw), for the various combinations of extraction conditions.
Na2CO3 (g/L) 0 2.5 25
T (oC) 60 100 60 100 60 100
Acidification
s/ pH 2 s/ pH 2 s/ N pH 2 s/ N pH 2 s/ N pH 2 s/ N pH 2
extent
Yield (% 69.7 ± 65.0 ± 71.2 ± 77.2 ± 50.1 ± 70.8 ± 90.7 ± 46.2 ± 53.4 ± 71.9 ± 34.4 ± 79.0 ± 78.2 ± 24.6 ± 44.5 ± 59.8 ±
dw/dw) 6.8 5.8* 2.1 3.2 1.7* 6.5 0.8* 0.9* 3.3* 3.0 1.8* 1.7 5.3 3.5* 14.2* 1.1*
Values presented as mean ± 95% confidence interval (n = 3).
(*) Statistical difference absolute yields and the yields obtained with the conditions (marked with rectangle), found by performing independent t-tests for equal variances not
assumed (α = 0.05).
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Using validated methods, the residual water from the reference labdanum resin extraction
method was evaluated regarding relevant water quality parameters for residual water
discharge to the environment and/or use as irrigation water, according to Portuguese
decree-law 263/98 (Table 5.2). Residual water presented values far from the limit values
for all the parameters. High suspended solids may be overcome by enhancing the
separation process (higher centrifugation or filtration), reducing the parameter to
acceptable limits, and also recovering more labdanum resin, increasing extraction yields.
Table 5.2: Water quality parameters of the residual water from the reference method used to extract
labdanum resin, assessed by analytical methods or by estimation considering the quantity of reagents used.
Residual Water Irrigation water
Parameter Discharge (VLE)
Method Estimated (VMA)
Suspended solids
1338 ± 101 - 60 60
(mg/L)
Sulphate (SO42-,
22284 ± 710 24019 ± 959 2000 575
mg/L)
+
Sodium (Na ,
9696 ± 1072 11709 ± 261
mg/L)
Magnesium 640 (total
3.97 ± 0.24 - -
(Mg2+, mg/L) dissolved salts)
Calcium (Ca2+,
3.52 ± 0.80 -
mg/L)
SAR (meq/L) 876 ± 112 - - 8
Electric
conductivity 34.8 ± 0.7 - - 1
(mS/cm)
pH 2.02 ± 0.01 - 6.0-9.0 4.5-9.0
Phenols
1245 ± 455 - 0.5 -
(mgGAE/L)
Values presented as mean ± 95% confidence interval (n = 3).
Emission limit value (VLE) for residual water discharge and allowable maximum limit (VMA) for
irrigation waters as regulated by the Portuguese decree-law 236/98.
Sulphate and sodium ions are present in high amounts in the residual water of the
reference method, contributing significantly for the high salinity (total dissolved
salts/electric conductivity and SAR). In fact, sulphate content was ten or forty-fold higher
than the sulphate limit values for residual water discharge or irrigation water,
respectively. Similarly, sodium content alone is more than fifty-fold higher than the total
dissolved salts limit for irrigation water, to which sulphate content should also be
considered. Those two ions are a direct product of the alkalinisation and acidification
steps with Na2CO3 and H2SO4, respectively, during the extraction of the resin. Therefore,
their quantity in the residual water is dependent on the quantity of reagents used, i.e., on
the Na2CO3 concentration during extraction and acidification extent. In addition, the
quantity of acid needed to neutralise carbonates is directly proportional to Na2CO3
concentration. Extractions with 2.5 g/L of Na2CO3 needed much less quantity of H2SO4
114
to neutralise carbonates and thus the estimated sulphate content of the residual waters
dropped to near or even below the limit values for wastewater discharge (Figure 5.2).
Acidification extent logically affected sulphate content of the residual water and when
acidification was not done, sulphate content is expected to be zero. Sodium content in the
residual water is also dependent from Na2CO3 concentration of the extraction solution.
Using a concentration of 2.5 g/L in the extraction solution sodium content in the residual
waters is estimated to be near the maximum recommended value for total dissolved salts
in irrigation waters (Figure 5.3). Both sulphate and sodium ions contribute to water
salinity and even at conditions such as 2.5 g/L of Na2CO3 and neutralisation with H2SO4
the residual water would not be recommended for irrigation. Regarding this issue,
extraction conditions simulating the Zamorean process are advantageous because
alkalinisation (Na2CO3 0 g/L) and acidification (s/) are not done and therefore do not
contribute with sodium and sulphate ions.
Total phenolic content of the reference method (1245 ± 455 mgGAE/L) is far from the
limit emission value for wastewater discharge (0.5 mg/L, Table 5.2). In fact, this
parameter is far from the limit value regardless of the extraction conditions and showed
to be mainly increased by higher extraction temperature since, compared to the reference
method, all extractions done at 100oC presented a significant higher value, between two
and six-fold higher (Figure 5.4). Folin-Ciocalteu method is not amongst the reference
analytical methods to quantify phenols defined by Decree-law 236/98. This method
usually indicates higher phenolic content than the Decree-Law reference 4-
aminoantipyrine method because it reacts with more types of phenolic compounds but
also more with interferents such as ascorbic acid and reducing sugars (Stratil et al. 2007).
Water extracts of C. ladanifer milled plant material had shown to be rich in phenolic
compounds such as phenolic acids, flavonoids aglycones and glycosylated, and tannins
(Barrajón-Catalán et al. 2010; Fernández‐Arroyo et al. 2010). Given the high content in
phenolic compounds, residual waters from labdanum extraction are not in conditions to
be discharged by any means. Phenolic compounds are regarded as toxic pollutants to
humans, animals and aquatic life and several methods are available to reduce their content
in residual waters by means of separation or degradation (Villegas et al. 2016). If
separated, polyphenolic compounds may be regarded as by-products and find applications
for food and biological systems (Barrajón-Catalán et al. 2010). In fact, phenolic
115
compounds from olive mill residual waters have been showing potential as food additives
and preservatives (Galanakis 2018).
Figure 5.2: Sulphate (SO42-) estimated content of the residual waters resulted from labdanum resin
extraction with different extraction conditions (base to top: Na 2CO3 (g/L), temperature (oC), Acidification
extent). Error bars correspond to the 95% confidence interval. (*) statistical difference in relation to the
reference (marked with rectangle), found by performing independent t-tests for equal variances not assumed
(α = 0.05). Limit emission value for residual water discharge (2000 mg/L, dot line) and maximum
recommended value for irrigation water (575 mg/L, straight line) defined in Portuguese decree-law nº
236/98.
Figure 5.3: Sodium (Na+) estimated content of the residual waters resulted from labdanum resin extraction
with different extraction conditions (base to top: Na 2CO3 (g/L), temperature (oC), Acidification extent).
Error bars correspond to the 95% confidence interval. (*) statistical difference in relation to the reference
(marked with rectangle), found by performing independent t-tests for equal variances not assumed (α =
0.05). Maximum recommended value for total dissolved salts in irrigation water (645 mg/L, dot line)
defined in Portuguese decree-law nº 236/98.
Residual water pH is a direct consequence of the acidification extent: compared to the

reference method, pH of the residual waters is higher for methods with none or less
acidification (Figure 5.5). Neutralisation rendered residual waters with a pH within the
upper and lower limit emission values for residual water discharge and admissible values
for irrigation waters. Extraction with 2.5 g/L of Na2CO3, until neutralisation, also
rendered residual waters with pH within the upper and lower limits. Compared to the
reference method, both conditions did not significantly affect labdanum resin and
116
absolute extraction yields but when done together those conditions significantly decrease
them (Figure 5.1). As discussed above, neutralisation produces a very turve residual water
which should mean higher suspended solids and thus less resin recovery, however not
clearly showed by this study.
Figure 5.4: Total phenol content (Gallic acid equivalents) of the residual waters resulted from labdanum
resin extraction with different extraction conditions (base to top: Na 2CO3 (g/L), temperature (oC),
Acidification extent). Error bars correspond to the 95% confidence interval. (*) statistical difference in
relation to the reference (marked with rectangle), found by performing independent t-tests for equal
variances not assumed (α = 0.05). Limit emission value for residual water discharge is defined at 0.5 mg/L
in Portuguese decree-law nº 236/98.
Figure 5.5: pH (Sorensen scale) of the residual waters resulted from labdanum resin extraction with
different extraction conditions (base to top: Na2CO3 (g/L), temperature (oC), Acidification extent). Error
bars correspond to the 95% confidence interval. (*) statistical difference in relation to the reference (marked
with rectangle), found by performing independent t-tests (α = 0.05). Lower limit emission value for residual
water discharge (pH = 5, dot line), minimum admissible value for irrigation water (pH = 4.5, straight line),
and upper limit emission value for residual water discharge and maximum admissible value for irrigation
water (pH = 9, trace line), defined in Portuguese decree-law nº 236/98.
5.4. Conclusion
Regarding aqueous labdanum resin extraction, alkalinisation of the water followed by

acidification (Andalusian process) renders higher resin and resin absolute yields in
comparison to the simulated Zamorean process. However, overall salinity, sulphate
117
content, and phenolic content do not allow the residual water of the Andalusian process
to be used as irrigation water or to be discharged as a wastewater before any treatment,
according to the Portuguese Decree-Law 263/98. Salinity and sulphate content of the
residual water are significantly reduced using 2.5 g/L instead of 25 g/L of Na2CO3 for
alkaline extraction without significantly reducing yields, however not enough for the
residual water to be recommended for irrigation. In this regard Zamorean process
conditions do not present a problem but at the cost of lower extraction yields. Total
phenolic content of the residual water is far above limits regardless the extraction
conditions, significantly lower when using 60oC instead of 100oC, limiting its discharge
to the environment. Extraction parameters may still be optimized to render less pollutant
residual waters, principally in what regards salinity for Andalusian processes, but a pre-
treatment to remove phenolic compounds from the residual water, regardless the
traditional process, will still be needed before discharging in the environment. If those
phenolic compounds are recovered, they may be considered a by-product worth to
valorise.
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Chapter 6
Labdanum resin valorisation

Subchapter 6.1
Labdanum resin from Cistus ladanifer L.: a natural and sustainable

ingredient for skin care cosmetics with relevant cosmeceutical
bioactivities.
Abstract
Labdanum resin from Cistus ladanifer L. (Cistaceae) is an abundant natural resource in

the Iberian Peninsula worth and possible to be explored in a sustainable manner. This raw
material is already used in the cosmetic industry, specifically by the fragrances/perfumery
sector. However, given the highest market share and traditional uses, labdanum resin has
potential to be also used and valorised as a skin care cosmetic ingredient. Aiming to
evaluate that potential, labdanum methanolic absolute and fractions purified by column
chromatography were characterized by UPLC-DAD-ESI-MS and were evaluated for UV
protection, antioxidant, elastase inhibition, anti-inflammatory, and antimicrobial
activities. Labdanum absolute represented near 70% of the resin, and diterpenoid and
flavonoid fractions represented near 75% and 15% of the absolute, respectively. Labdane-
type diterpenoids and methylated flavonoid aglycones were detected as the main
compounds in the labdanum absolute and in diterpenoid and flavonoid fractions,
respectively. Labdanum absolute showed a spectrophotometric sun protection factor
(SPF) near 5, which is mainly due to flavonoids, as this fraction shows SPF of 13. A low
antioxidant activity was observed, being the scavenging of the ABTS radical the most
significant (0.142 ± 0.017, 0.379 ± 0.039 and 0.010 ± 0.003 mgTE/mgExt, for absolute
and flavonoid and terpene fractions, respectively). Anti-aging and anti-inflammatory
activity of labdanum absolute and its fractions is here reported for the first time, by
inhibition of elastase activity (22% and 13%, by absolute and flavonoid extract at 1
mg/mL), and by inhibition of nitric oxide production in LPS-induced RAW 264.7 cells
(84% to 98% at 15 µg/mL, being the flavonoid fraction the most active), respectively.
Antimicrobial activity was evaluated against relevant skin and cosmetic products
microorganisms: Staphylococcus aureus ATCC® 25923TM, Pseudomonas aeruginosa
ATCC® 27853TM, Candida albicans ATCC® 10231TM, and Escherichia coli ATCC®
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Subchapter 6.1 Labdanum resin from Cistus ladanifer L.: a natural and sustainable ingredient for skin
care cosmetics with relevant cosmeceutical bioactivities.
25922TM. Only S. aureus showed susceptibility to the inhibitory activity of labdanum

absolute (MIC: 1.2 mg/mL) and its fractions (MIC: < 0.3 mg/mL). In conclusion,
labdanum resin showed potential to be used in sunscreen cosmetics, and anti-
inflammatory skin care cosmeceuticals or medicines, but low potential as a cosmetic
product preservative given the low antioxidant and narrow-spectrum antimicrobial
activities.
Keywords:
Cistus ladanifer L.; Anti-inflammatory; Antimicrobial; Antioxidant; Diterpene;

Flavonoid; Sunscreen effect; Rockrose.
This subchapter is published as:
Frazão DF, Martins-Gomes C, Steck JL, Keller J, Delgado F, Gonçalves JC, Bunzel M, Pintado CMBS,
Dias TS, Silva AM (2022) Labdanum resin from Cistus ladanifer L.: a natural and sustainable ingredient
for skin care cosmetics with relevant cosmeceutical bioactivities. Plants 11: 1477.
120
6.1.1. Introduction
The rockrose Cistus ladanifer L. (Cistaceae) is an abundant and widespread plant resource
in the Iberian Peninsula, also present in the South of France and North Africa (Demoly
and Montserrat 1993; Godinho-Ferreira et al. 2005). From its photosynthetic leaves and
stems (Valares et al. 2016b), C. ladanifer exudes the commonly called “labdanum gum”,
which is in fact a resin instead of a gum (Langenheim 2003). Industrially and traditionally,
this resin is usually extracted from the plant material with alkaline water followed by acid
neutralisation, but it may also be extracted using just boiling water but with lower
extraction yields (Morgado et al. 2005; Greche et al. 2009; Burguer 2016). This resin is a
complex mixture of terpenoid and phenolic compounds, which are represented mainly by
labdane-type diterpenes and methylated flavonoid aglycones (Alías 2006; Valares et al.
2016b). Although waxes may not be considered as constituents of the resin, they are
extracted together either with the alkaline water process (Burguer 2016) or with organic
solvents such as chloroform (Valares et al. 2016b). To remove waxes, labdanum must be
dissolved in warm methanol and then cooled down to negative temperatures to precipitate
and separate, by centrifugation, waxes (de Pascual et al. 1984; Sosa et al. 2005; Greche
et al. 2009; Burguer 2016), rendering the labdanum absolute. Labdanum absolute may be
separated into diterpenoid and flavonoid fractions by molecular sizing column
chromatography (Vogt et al. 1987; Sosa et al. 2005; Alías et al. 2012; Valares et al.
2016b).
A current application of labdanum resin and its extracts is in the perfumery/fragrance

industry (Raimundo et al. 2018). The global market of cosmetics in 2020 was estimated
to be higher than 200 billion euros (L'ORÉAL 2020). Although fragrances/perfumery
products have a significant market share, skin care products dominate the cosmetics
market (L'ORÉAL 2020; CosmeticsEurope 2022). Interestingly, labdanum resin is
documented to be traditionally used on human and animal skin to treat wounds (González
et al. 2018). In addition, labdanum resin and labdanum absolute are already registered in
the European Chemicals Agency (ECHA) according to Regulation (CE) 1907/2006,
which establishes a registration system for chemical products (REACH) to be used or
traded within the European Union. In fact, those registrations predict the end use of the
“substances” in fragrance and cosmetic products, between other uses. A consumer-driven
trend for cosmetic products made from natural and sustainable ingredients (Bom et al.
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2019; CBI 2020) justifies even more the evaluation of labdanum resin/absolute as an
ingredient for skin care cosmetics.
Skin topical cosmetics are mainly used to restore or maintain skin barrier properties by
including moisturizers, antioxidants, anti-inflammatory agents, or even UV-protection
agents (Pilae et al. 2016). Skin exposure to UV radiation leads mainly to skin photoaging
damage (premature skin aging) but can also result in erythema (sunburn),
immunosuppression, and carcinogenesis (Minkis et al. 2016). Given the harmful effects
of such radiation to the skin, the use of sunscreens is widely advised (Linden 2018). Sun
Protection Factor (SPF) is a measure of sunscreen protection against UVB (280-320 nm)
radiation based on the erythema symptom, and currently only in vivo methods are
accepted to determine it (Young et al. 2017). However, for prospection purposes, a simple
spectrophotometric method for SPF determination was proposed by Mansur et al. (1986)
and implemented by Dutra et al. (2004) and Gaweł-Bęben et al. (2020).
Reactive Oxygen Species (ROS) generation by both UVA and UVB radiation is a known
mechanism that leads to skin damage by direct chemical modification of nuclear and
mitochondrial DNA, cell lipids, and dermal matrix proteins, such as collagen produced
by dermal fibroblasts, or damage of the cornified envelop in the epidermis stractum
corneum that functions as a permeability and mechanical barrier (Minkis et al. 2016).
Antioxidants can scavenge ROS to prevent the harmful effects of oxidative stress in the
skin. In addition, antioxidant ingredients in a cosmetic contributes to extent its shelf-life.
There are several methods to evaluate the antioxidant activity of substances or mixtures,
some based on the transfer of hydrogen atoms (ORAC test), others based on the transfer
of electrons (FRAP and Folin-Ciocalteu tests) and also based on the transfer of hydrogen
atoms and/or electrons (ABTS and DPPH tests) (Munteanu and Apetrei 2021).
The activation of transduction pathways directly by UVB radiation compiles a general

mechanism that leads to skin damage (Minkis et al. 2016). A variety of pro-inflammatory
mediators are induced by UVB radiation (Mohania et al. 2017). Sunburns induced by
UVB radiation are characterized by acute inflammatory response causing skin structure
damage (Mohania et al. 2017). In addition, UV-induced chronic inflammation is
associated to cancer development (Zhong et al. 2021). Macrophages are crucial for
inflammatory response and once stimulated produce pro-inflammatory mediators (e.g.,
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cytokines, nitric oxide) and have been extensively used to evaluate the anti-inflammatory
activity of various substances (Silva et al. 2020).
Elastases are enzymes responsible for the breakdown of elastin and fibronectin, and other
proteins responsible for the elasticity of the connective tissue (Imokawa and Ishida 2015).
Under physiological conditions the activity of elastase is controlled (inhibited) by serpins,
however this balance can be disturbed by several mechanisms, such as ROS, UV-
radiation and lead to skin barrier disturbance, contributing to skin inflammation, loss of
skin elasticity and wrinkling formation (Takeuchi et al. 2010). Compounds capable of
inhibiting elastase activity would therefore have a beneficial property for cosmetic skin
care ingredients, preventing an additional pathway of skin photoaging.
Skin microflora (or skin microbiome) is important to prevent but may also contribute to
skin diseases. Gram-positive bacteria staphylococci, in particular Staphylococcus aureus
and Staphylococcus epidermis are well-known and prominent opportunistic
microorganisms that cause skin infection inhibiting healing and promoting further
inflammation of skin lesions (Coates et al. 2014; Weyrich et al. 2015). Gram-negative
bacteria Pseudomonas aeruginosa (Wu et al. 2011) and the yeast Candida albicans
(Kashem and Kaplan 2016) are also recognized as skin colonizers and opportunistic
pathogens, although not so frequent. Cosmetic ingredients with antimicrobial activity
may thus help to prevent skin infections and, like antioxidants, to increase the final
product shelf-life. In fact, according to European Standard EN ISO 17516:2014 the
microbiological quality/safety of a cosmetic must be evaluated regarding total aerobic
mesophilic microorganisms, Escherichia coli, S. aureus, P. aeruginosa and C. albicans.
Thus, responding to the high demand for using natural-based products and alternative
sources, the main aim of this study was to evaluate the potential of labdanum resin as a
natural and sustainable ingredient for skin care cosmetics, by evaluating its ability in
maintaining skin health, through evaluation of labdanum resin anti-oxidant, anti-
inflammatory and UV-protection potential, and also by exploring its antimicrobial
activities in order to promote skin care but also to act as a preservative in the final
cosmetic product.
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6.1.2. Material and Methods
6.1.2.1. Chemicals
Sodium carbonate (Na2CO3, Biochem, Frilabo, Portugal). Methanol LC-MS grade 99.9%
(Fisher Scientific, EUA), Acetonitrile HPLC (Honeywell Fluka, Fisher Scientific, EUA).
2,4,6-Tris(2-pyridyl)-S-triazine (TPTZ, Tokyo Chemical Industry, JMGS, Portugal).
Folin-Ciocalteu reagent; Gallic acid-1-hydrate (Panreac, LaborSpirit, Portugal). Acetic
acid glacial (CH3COOH); Iron(III) chloride hexahydrate (FeCl3.6H2O); Sulfuric acid
97% (H2SO4); sodium acetate (CH3COONa) (Chem-Lab, JMGS, Portugal). 2,2-
Diphenyl-1-picrylhydrazyl (DPPH); 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic
acid) diammonium salt (ABTS); potassium persulfate (K2S2O8); Dimethyl Sulfoxide
anhydrous (DMSO); Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl); N-
(methoxysuccinyl)-ala-ala-pro-val-4-nitroanilide; Phosphate buffered saline tablet
(PBS); elastase (Sigma Aldrich, Merck, Germany). Versene, trypsin-EDTA, sodium
pyruvate, L-glutamine, penicillin, streptomycin, fetal bovine serum (FBS) and Modified
Eagle Medium (DMEM) (Alfagene, Invitrogen, Portugal). Alamar Blue® (Life
Technologies Invitrogen, Portugal). Resazurin tablets (VWR, Leuven, Belgium); Potato
Dextrose Broth, PDB (VWR, Leuven, Belgium); McFarland standard (BioMérieux,
Marcy-L´Étoile, France); Potato Dextrose Agar, PDA (HIMEDIA, Nashik, India);
Müeller-Hinton Broth, MHB (OXOID, Basingstoke, United Kingdom) and Müeller-
Hinton Agar, MHA (OXOID, Basingstoke, United Kingdom).
6.1.2.2. Labdanum absolute
Herbaceous/semiwoody terminal leafy stems (10-30 cm) were collected from a Cistus
ladanifer subsp. ladanifer shrubland in Penha Garcia, Portugal (GPS: N 40o1’43.4’’ W
6o59’34.8’’), in August 2018. Plant material was stored fresh in the freezer (-20oC) until
use. Subspecies was identified using “Flora Ibérica” by Demoly and Montserrat (1993)
and two exemplars were deposited in the herbarium collection of School of Agriculture
of Polytechnic Institute of Castelo Branco, under the code 004 ESACBA1 CIS CIS LAD
02.
Labdanum was extracted based on the method described by Burguer (2016). Twenty-five
grams of plant material were extracted in 200 mL of Na2CO3 aqueous solution (25 g/L),
for 1 h at 60oC (water bath), inside a flask. The solution was filtered through a stainless-
steel sieve (mesh no. 140, 106 µm aperture). After cooling, H2SO4 solution (5 M) was
124
added until pH 2. The precipitated resin was separated from the supernatant by
centrifugation, at 3030 g for 15 min (Mega Star 600R, VWR), and finally freeze-dried at
0.3 mbar for 72 h (Zirbus VaCo 10-II-D).
Labdanum absolute was obtained by removing “waxes” from the labdanum resin based
on the method described by Vogt et al. (1987) and used in recent works (Sosa et al. 2005;
Alías et al. 2012; Valares et al. 2016b). Briefly, one gram of resin, maximum, was
dissolved in 20 mL methanol using an ultrasound bath, left at -20oC overnight, and then
centrifuged at 3030 g at -5oC for 15 minutes. The procedure was repeated three times,
and the supernatants were joined. Three independent extractions were done.
6.1.2.3. Diterpenoid and flavonoid fractions
Labdanum absolute was subjected to fractionation by molecular-sizing column

chromatography as described by Vogt and Gülz (1991) and used in recent works (Sosa et
al. 2005; Alías et al. 2012; Valares et al. 2016b). Absolute was directly eluted on
SephadexTM LH-20 (GE Healthcare Biosciences AB, Sigma Aldrich, USA) compacted in
a 25 cm long and 1.5 cm width column using methanol as eluent. Fractions of about 2-
3mL were continuously collected and analysed by thin layer chromatography using silica
gel TLC plates (F254s 0.2mm 10x20cm) (Merck, Germany), as described by Vogt and
Gülz (1991). Toluene: ethyl acetate (9:1) was used as eluent and, after drying, plates were
visualised at 312 nm using a transilluminator to detect flavonoid bands. To detect
diterpene bands, plates were sprayed with 10 M H2SO4 solution and heated at 100oC for
10 minutes in a ventilated chamber. Purple/red bands were assigned to the diterpenes and
yellow bands to the flavonoids because they matched the bands visualised at 312 nm.
Fractions presenting purple bands were joined as the diterpene fraction (Dit) and fractions
presenting flavonoid bands were joined as the flavonoid fraction (Flv). This procedure
was done for the three independent labdanum absolutes.
6.1.2.4. Chemical characterisation (UPLC-DAD-ESI-MS)
Labdanum absolute and fractions were characterized by UPLC-DAD-ESI-MS (Nexera

X2 system; LC-30AD pump, Shimadzu). The chromatographic separation and analysis
were performed, using a Kinetex C18 column (150 mm x 4.6 mm, 2.7 µm particle size)
from Phenomenex (Torrance, CA, USA). Mobile phase comprised solvent A (ultrapure
water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid).
Separation was carried out at 0.5 mL/min flow rate, 30oC oven temperature, and at the
125
following elution gradient: 0 min, 20% solvent B; 60 min, 100% solvent B; 65 min, 100%
solvent B; 67 min, 20% solvent B; 70 min, 20% solvent B. Data were acquired using
DAD (190-700 nm) and ESI-MS detection (positive and negative mode, m/z ions 100-
2000, interface 4.5 kV, interface temperature 350oC, desolvation line temperature 250 oC,
heat block 200oC, 1.5 L/min nebulizing gas (Nexera X2 SPD-M30A + LCMS 2020,
Shimadzu), 20 L/min drying gas. Labdanum absolutes were injected at the following
concentrations: Absolute and Dit: 0.266 mg/mL; Flv: 0.066 mg/mL. Injection volume
was 20 µL. Compounds were identified based on their pseudo-molecular ions and
literature comparison.
6.1.2.5. UV radiation absorption
Spectrophotometric SPF (sun protection factor) was determined by the method described
by Gaweł-Bęben et al. (2020). UV absorbance scan (200-400 nm) of the labdanum
absolute and fractions was measured on a spectrophotometer in a 0.5 cm quartz cell using
proper dilutions to obtain absorbance values below 1. UV absorbance scan was read in
duplicate for each extract replicate and methanol was used as reference. Absorbance
values for 100 µg/mL solutions were read at 290, 295, 300, 305, 310, 315, 320 nm, and
SPF was calculated by the Mansur equation (1) (Mansur et al. 1986):
320
𝑆𝑃𝐹 = 𝐶𝐹 × ∑ 𝐸𝐸(𝜆) × 𝐼(𝜆) × 𝐴𝑏𝑠(𝜆) (1)

290
Where EE (erythemal effect spectrum) x I (solar intensity spectrum) at a given λ

(wavelength) is a normalised constant proposed by Sayre et al. (1979). CF is a correction
factor which was set at 10 for comparison purposes to Gaweł-Bęben et al. (2020) work.
In addition, UVB (280-315 nm) and UVA (315-400 nm) total absorbance (arbitrary units,
a.u.) of 100 µg/mL solutions was obtained by integration (area) of the UV absorption
spectra.
6.1.2.6. Antioxidant activity
Stock solutions of labdanum absolute and fractions were prepared to obtain proper
colorimetric readings. All methods were performed in duplicate for each extract replicate.
126
For the Folin-Ciocalteu method, extracts were further diluted (1:10) with distilled water
to obtain 5% v/v methanol in the final reaction mixture, as suggested by Cicco et al.
(2009). Total phenolic content was determined by mixing 200 µL of standard/sample
solution, 650 µL of distilled water, 100 µL of Na2CO3 (7.5% m/v), and 50 µL of Folin-
Ciocalteu reagent. Mixtures were incubated for 2 h, in the dark at room temperature, and
absorbance was read at 725 nm. Gallic acid (0.01-0.2 mg/mL) was used as standard
compound and the results were expressed as gallic acid equivalents (GAE, mg) per
sample dry weight (mg).
ABTS radical was prepared by mixing equal volumes of ABTS (7 mM) and K2S2O8 (2.5
mM), the solution was allowed to react for 16 h in the dark. Then, the radical solution
was diluted in acetate buffer (20 mM, pH 4.5) until absorbance of 0.7 ± 0.02 nm at 734
nm. Scavenging activity was determined by mixing 200 µL of radical solution and 20 µL
of standard/sample solution prepared in 10% (v/v) methanol, or acetate buffer (control).
Mixtures were left to react for 15 min, in the dark at room temperature, absorbance was
read at 734 nm on a microplate reader. DPPH•+ scavenging activity was assessed by
mixing 1300 µL of 80 µM DPPH solution, in methanol, with 150 µL of standard/sample
solution or methanol (control). Mixtures were left to react in the dark for 30 minutes and
the absorbance was read at 517 nm in a spectrophotometer. For both ABTS and DPPH
methods, absorbance values were transformed in percentage of inhibition in relation to
the controls.
Ferric reducing antioxidant power (FRAP) was assessed by mixing 1500 µL of FRAP
reagent (2.5 mL of 10 mM TPTZ in 40 mM HCl and 2.5 mL of 20 mM FeCl3.6H2O in a
25 mL solution made up with acetate buffer 0.3 M, pH 3.6), 150 µL of methanol and 50
µL of standard/sample solution or methanol (control). Mixtures were allowed to react for
30 minutes at 37oC (water bath) in the dark, and absorbance was read at 595 nm using a
spectrophotometer.
For ABTS, DPPH, and FRAP methods Trolox (0.05-0.3 M) was used as standard
compound, and results were expressed as Trolox equivalents (TE, mg) per sample weight
(mg).
6.1.2.7. Elastase inhibition activity
Elastase inhibition assay was performed in a 96-well microplate and absorbance was read
using a microplate spectrophotometer. The assay was performed as described in Taghouti
127
et al. (2018). Labdanum absolute and fractions, tested at 0.5 and 1 mg/mL, were diluted
in PBS from stock solutions prepared in DMSO. The final concentration of DMSO was
always less than 5% and a control with 5% DMSO was used to exclude solvent-induced
enzyme inhibition. Elastase inhibition was assessed by adding 160 µL of Tris-HCl buffer
(0.2 mM, pH 8) and 20 µL of N-(methoxysuccinyl)-ala-ala-pro-val-4-nitroanilide (0.8
mM, in 0.2 mM Tris-HCl, pH 8) to 50 µL of the extracts. The mixtures were incubated
for 10 minutes at room temperature, and 20 µL of elastase (0.4 U/mL, in 0.2 M Tris-HCl,
pH 8) was added. After incubation for 20 minutes, at room temperature, absorbance was
read at 410 nm. Percentage of inhibition (%inhibition) was calculated according to
equation 2:
(𝐴𝑏𝑠𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑏𝑠𝑠𝑎𝑚𝑝𝑙𝑒 )
%𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = × 100 (2)
𝐴𝑏𝑠𝑐𝑜𝑛𝑡𝑟𝑜𝑙
Where Abs is the absorbance value of the control (Abscontrol) and of sample (Abssample).
6.1.2.8. Anti-inflammatory activity
Anti-inflammatory activity of labdanum absolute and fractions was evaluated as the

inhibition of lipopolysaccharide (LPS)-induced nitric oxide production in Raw 264.7 cells
(mouse macrophages, Abelson murine leukaemia virus-induced tumour, acquired from
Cell Lines Service CLS, Eppelheim, Germany), as previously described by Silva et al.
(2020). Briefly, Raw 264.7 cells were cultured in complete culture media (Dulbecco’s
Modified Eagle Media (DMEM), supplemented with 10% (v/v) foetal bovine serum
(FBS), 1 mM L-glutamine, and antibiotics (penicillin 100 U/mL, streptomycin 100
g/mL) in an incubator (5% CO2; 37 °C, controlled humidity). For experiments, cells
seeded in 96-well plates (5  104 cells/mL; 100 L per well) were exposed to non-
cytotoxic concentrations of labdanum absolutes (prepared in FBS-free culture media) in
the presence and in the absence of LPS (1 µg/mL). After a 24 h incubation, 50 µL of
supernatant of each well was transferred to a new 96-well microplate, to which 50 µL of
Griess reagent, prepared as described by Silva et al. (2016), were added, followed by a
15 min incubation in the dark, at room temperature. The absorbance was read at 550 nm,
and a sodium nitrite (NaNO2) standard curve was used for nitric oxide quantification.
Results were expressed as % of control (LPS-stimulated cells not exposed to extracts).
The experiment was repeated three times with four repetitions.
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6.1.2.9. Antimicrobial activity
Reference microorganism strains used in susceptibility tests were Escherichia coli

ATCC® 25922TM, Pseudomonas aeruginosa ATCC® 27853TM, Staphylococcus aureus
ATCC® 25923TM, and Candida albicans ATCC® 10231TM, acquired from ielab (Alicante,
Spain).
Labdanum absolute and fractions were prepared at 10 mg/mL directly in culture medium
with 10% (v/v) DMSO. Five 1:2 serial dilutions were done for each extract, resulting in
five solutions with twice the extract concentration to test and twice the DMSO percentage
tolerated (5% v/v, found by a preliminary test with all microorganisms used). In 96-well
plates, 50 µL of extract was mixed with 50 µL of microbial suspension, resulting in a 1:2
dilutions for both. A control was prepared by using 50 µL of culture medium and 50 µL
of microbial suspension. Other control was prepared by using 50 µL of culture medium
and 50 µL of each sample solution. Müeller-Hinton Broth was used for E. coli, P.
aeruginosa, and S. aureus and Potato Dextrose Broth was used for C. albicans. Strains,
from -80ºC storage, were grown overnight on Potato Dextrose Agar or Müeller-Hinton
Agar and sub-cultured three times before use to prepare the microbial suspensions in 0.9%
(w/v) NaCl saline solution. Bacterial suspensions were adjusted to a 0.5 McFarland
standard and yeast suspensions to a 1.0 standard. From the initial suspension, a 1:100
dilution, in broth medium, was done to obtain twice the concentration needed for the
susceptibility test. Microplates were incubated for 24 h at 37oC. Afterwards, 5 µL from
each well were inoculated on solid medium plates, which were again incubated for
additional 24 h to determine the Minimum Microbicidal Concentration (MMC).
Meanwhile, 20 µL of 0.01% (m/v) resazurin was added to each well of the microplate
and then incubated for further 2 h to determine the Minimum Inhibitory Concentration
(MIC). This method was designed based on CLSI M07-A9 (2012) standard and was
performed in duplicate for each extract replicate.
6.1.2.10. Statistical analysis
Statistical analysis was used to find significant variable effects and significant differences
between means of different treatments (p<0.05) and was performed using IBM SPSS
Statistics 25 and GraphPad Prism 9 software. Shapiro-Wilk’s test was used to test
normality of data and Levene’s test to test homogeneity of variance. ANOVA, post-hoc
Tukey’s test, and t-test were used for normal data with homogeneous variance. Welch’s
129
ANOVA, Welch’s t-test, and Dunnett’s T3 post-hoc tests were used for normal data with
equal variances not assumed.
6.1.3.1. Extraction and purification yields
In this study, labdanum resin was extracted based on the Andalusian process, yielding
7.44 ± 0.41% (dw/fw) of labdanum resin in relation to plant material (Table 6.1.1). The
Andalusian process was chosen in contrast to the Zamorean process because of its higher
resin yields (Burguer 2016). The difference between the two methods is that the latter
uses boiling water to physically remove, by melting, the resin from the plant material
instead of the alkalinisation and, when cooled down, the resin precipitates. The yield
obtained in this study is similar to the one obtained by Burguer (2016). In fact, the same
process was used to extract the resin, only differing in the workup processes such as
centrifugation, and the final freeze-drying step. The Na2CO3 solution (25 g/L, 0.24 M)
was considered a buffer solution as it was able to keep the pH, at approximately 11, during
extraction. Greche et al. (2009) used a similar process because albeit using a lower
concentration of NaHCO3 (0.1 M), which is about half the one used in this study, they
used a lower proportion of fresh plant material in relation to the extraction solution, 0.075
g/L, compared to the 0.125 g/L used in this study. Greche et al. (2009) did not report resin
yields, but the extraction process differed. In this study, the acidification was done until
pH 2 instead of neutralisation. However, neutralisation was attempted in this study but it
was further excluded because i) the pH was very instable near neutralisation ii) after
neutralisation, albeit getting a precipitate, the supernatant was not as clear as the one
obtained at pH 2, and iii) the volume of acid used between neutralisation and acidification,
to reach pH 2, was little (~1 - 1.5 mL) compared to the volume needed for neutralisation
(~6 - 7 mL) of initial extract (at pH 11).
Table 6.1.1: Labdanum resin yield (% dw/fw) in relation to plant material, and labdanum absolute and
column chromatography fractions yields (% dw/dw) in relation to labdanum resin or absolute, respectively.
Resin Absolute Dit Flv Remainder
7.44 ± 0.41 73.3 ± 0.9 74.2 ± 1.8 15.6 ± 4.6 10.1 ± 2.9
According to Handa (2008), a resinoid is defined as the extract obtained from a natural
resin using a hydrocarbon solvent. An absolute is an alcohol extract of a hydrocarbon
extract, usually done at 40 - 60oC and then cooled to negative temperatures, to separate
130
waxes. Among literature, these terms are not consistent. For instance, Greche et al. (2009)
named an ethanol extract of the resin as a resinoid. In this study, labdanum absolute is
considered the best term for a direct methanolic extract of the crude labdanum resin. The
yield of labdanum absolute was 73.3 ± 0.9% (dw/dw) from labdanum resin (Table 6.1.1).
According to Burguer (2016), methanol is the best solvent to separate long chain
hydrocarbons from the crude resin compared to hexane or ethanol, obtaining a methanol
absolute yield of 62.3% (dw/dw), a value slightly lower than the one here obtained (Table
6.1.1). Other authors also used methanol to separate waxes from chloroform extracts of
the plant exudate (Sosa et al. 2005; Alías et al. 2012; Valares et al. 2016b).
Fractionation of labdanum absolute by molecular sizing column chromatography (Table

6.1.1), rendered the diterpenoid fraction as the major fraction (≈ 75% dw/dw) eluting first
and then the flavonoid fraction with a yield around 15% (dw/dw). The remainder,
calculated by difference, accounted up to 10% (dw/dw). A part of the remainder was
collected after flavonoids, during a long period and with a large dispense of eluent,
recovering only 6.72 ± 0.79% (dw/dw) of the absolute. According to Chaves et al. (1997a)
and Sosa et al. (2005) flavonoids account to between 14 - 28% (dw/dw) of the resin, for
plants harvested in summer, and to between 6 - 16% (dw/dw), for plants harvested in
winter, which is in accordance to the fraction yield obtained in this study (plants harvested
in summer, see methods). According to Alías et al. (2012), 6-oxocativic acid, 7-oxo-labd-
8(9)-en-15-oic acid, and 6β-acetoxy-7-oxo- labd-8(9)-en-15-oic acid, together, accounted
to 21 mg/g (dw/dw) of mature leaves in winter and dropped to approximately 10 mg/g
(dw/dw) in the other seasons. Considering the exudate yields from Sosa et al. (2005),
these three diterpenes alone would account to between 6 - 16% (dw/dw) of the resin which
is far below the diterpenoid fraction yield obtained in this study. However, labdanum
resin is known to be composed by more labdane-type diterpenes such as labdanolic acid
(de Pascual et al. 1982; Greche et al. 2009). Labdanolic acid is a major compound in
labdanum resin as shown by Alías (2006), Greche et al. (2009), and Burguer (2016)
therefore the diterpenoid yield in labdanum resin of 6 - 16% (dw/dw) is an
underestimation.
6.1.3.2. Chemical profile
The chemical profile of labdanum absolute and fractions is presented in Table 6.1.2 which
summarises the information obtained by UPLC-DAD-ESI-MS analysis. Chromatograms
of labdanum absolute and its fractions are presented in Figures 6.1.1, 6.1.2, and 6.1.3.
131
The three extracts present distinct chromatographic profiles which in turn confirmed the
composition of the purified fractions.
The chromatogram of flavonoid fraction (Figure 6.1.3) revealed 7 peaks that could be
addressed to flavonoid aglycones described in literature, namely apigenin-4’-methyl ether
(peak 3, 24.7 min) and apigenin-7-methyl ether (peak 4, 24.9 min) were distinguished
based on their order of elution from other reversed-phase chromatographic methods
(Valares et al. 2016b). Despite two different kaempferol-dimethyl ethers are described in
literature, only one peak (5, 26.6 min) was observed that could be assigned to both
compounds, however kaempferol-3,7-methyl ether (jaranol) not only is it more frequently
reported in literature (section 1.2.2.2), but it is also reported as a major flavonoid in
labdanum resin (Sosa et al. 2005)
Diterpene fraction revealed 14 peaks in the chromatogram (Figure 6.1.2) but only 5 could
be assigned to compounds described in literature. All of those matches are labdane-type
diterpenoids, which have also been shown to be a major group of compounds in labdanum
resin (de Pascual et al. 1982; Greche et al. 2009; Alías et al. 2012; Chaves et al. 2019).
Peaks 13 (35.4 min) and 15 (37.4 min) were the major peaks in the diterpenoid fraction
chromatogram and were assigned to compounds already described as major diterpenoid
compounds, oxo-labdenoic acid (most likely 6-oxo-cativic acid) and labdanolic acid,
respectively. Labdanolic acid do not absorb UV radiation and because of that it cannot be
detected using classical HPLC methods using DAD or UV detectors.
Labdanum absolute chromatogram (Figure 6.1.1) showed a chemical profile in which the
same flavonoid aglycones present in the flavonoid fraction were predominantly detected
but also showed some major non-identified peaks of compounds with longer retention
times such as peak 20 (42.3 min) and peak 23 (45.9 min). These non-identified
compounds were lost when obtaining the purified diterpenoid and flavonoid fractions by
molecular sizing chromatography. Some diterpenes were detected as lower intensity
peaks and interestingly the major peak of the diterpene fraction (labdanolic acid, peak 15,
37.4 min) was not detected in the absolute chromatogram.
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Figure 6.1.1: DAD (250 and 350nm, absolute intensity) and MS (TIC, m/z - and m/z+, relative intensity)
chromatograms obtained in the UPLC-DAD-ESI-MS analysis of labdanum absolute. Numbers represent a
peak at a retention time as presented in Table 6.1.2.
Figure 6.1.2: DAD (250 and 350nm, absolute intensity) and MS (TIC, m/z- and m/z+, relative intensity)
chromatograms obtained in the UPLC-DAD-ESI-MS analysis of diterpenoid fraction from labdanum
absolute. Numbers represent a peak at a retention time as presented in Table 6.1.2.
Figure 6.1.3: DAD (250 and 350nm, absolute intensity) and MS (TIC, m/z - and m/z+, relative intensity)
chromatograms obtained in the UPLC-DAD-ESI-MS analysis of “flavonoid” fraction from labdanum
absolute. Numbers represent a peak at a retention time as presented in Table 6.1.2.
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Subchapter 6.1 Labdanum resin from Cistus ladanifer L.: a natural and sustainable ingredient for skin care cosmetics with relevant cosmeceutical bioactivities.
Table 6.1.2: UPLC-DAD-ESI-MS peaks in the chromatograms of labdanum absolute and fractions. Considering the four detection signals (absorbance at 250 and 350 nm and
total ion count at positive and negative modes), +: presence and ++: major peak. Peak order number, retention time (min), ESI ions weight (m/z- and m/z+), literature compound
match (considering pseudo-molecular ions [M-H]- and [M+H]+), matched compound molecular weight, and literature references of the matched compound are presented.
ESI ions Literature compounds match
Retention
Peak [M-H]- [M+H]+ MW
time (min) Absolute Dit Flv Compound Reference*
(m/z) (m/z) (g/mol)
1 16.8 269 271 + + Apigenin 270 [4, 5, 15, 16, 18-21, 23]
2 18.4 299 301 ++ ++ Kaempferol-3-methylether (isokaempferide) 300 [4, 5, 15, 16, 18-21, 23]
3 24.7 283 285 + + Apigenin-4’-methylether (acacetin) 284 [4, 5, 15, 16, 18-21, 23]
[4, 5, 10, 15, 16, 18-21,
4 24.9 283 285 + + Apigenin-7-methylether (genkwanin) 284
23]
Kaempferol-dimethylether (3,7, jaranol, or [4, 5, 8, 9, 15, 16, 18-
5 26.6 313 315 ++ ++ 314
3,4’) 21, 23]
6 28.6 335 337 + - (336)
363
7 30.9 379 + - (380)
(292/303/321)
8 32.0 381 - + - (382)
9 32.2 - - + -
[1, 2, 7, 10, 13, 14, 18,
10 33.1 319 321 + Oxo-labdenoic acid 320
19]
11 33.5 309 (379) - + Labdandiol (p.e. 8,15) 310 [10, 11, 17]
12 33.9 - 299 + + Apigenin-7,4’-dimethylether 298 [4, 5, 10, 21, 23]
[1, 2, 7, 10, 13, 14, 18,
13 35.4 319 321 + ++ Oxo-labdenoic acid 320
19]
Kaempferol-3,7,4’-trimethylether
14 35.7 - 329 + + 328 [4, 5, 10, 20, 21, 23]
(methyljaranol)
15 37.4 323 - ++ Labdanolic acid 324 [2, 3, 6, 7, 10, 11, 13]
Labdenoic acid (8(17) ladenic, 7 cativic
16 37.9 - 307 + 306 [3, 11-13, 22]
acids)
17 38.4 - 257/351 (375) + 8α-methoxy-labdan-15-oic acid 256 [7, 10, 11]
18 39.6 - 257 + - (256)
19 40.1 371 338 (398/241) + - (372)
20 42.3 - 456 (393) ++ - (455)
335
21 43.6 381/433 + - (382/434)
(376/435/269)
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Subchapter 6.1 Labdanum resin from Cistus ladanifer L.: a natural and sustainable ingredient for skin care cosmetics with relevant cosmeceutical bioactivities.
22 44.4 - 384 + - (383)

23 45.9 - 335 (376) ++ + - (334)
24 47.1 - 271 + - (270)
25 48.1 - 398 (165) + - (397)
ESI ions and matched compound weight numbers rounded down
Light grey: matched flavonoids; Dark grey: diterpenoids; Colourless: no match.
*[1]Alías et al. (2012); [2]Alías (2006); [3]Burguer (2016); [4]Chaves et al. (1997b); [5]Chaves et al. (1998); [6]Cocker and Halsall (1956); [7]de Pascual et al. (1982); [8]de
Pascual et al. (1968); [9]de Pascual et al. (1974); [10]de Pascual et al. (1984); [11]de Pascual et al. (1983); [12]de Pascual et al. (1972); [13]Greche et al. (2009); [14]Halsall
and Moyle (1960); [15]Proksch and Gülz (1984); [16]Sosa et al. (2005); [17]Tabacik and Bard (1971); [18]Valares et al. (2016a); [19]Valares et al. (2016b); [20]Viuda-Martos
et al. (2011); [21]Vogt et al. (1987); [22]Weyerstahl et al. (1998); [23]Wollenweber and Mann (1984).
135
6.1.3.3. UV radiation protection activity
Sun Protection Factor (SPF) as well as the integral of the absorption spectra at UVB (280
- 315 nm) and UVA (315 - 400 nm) regions of labdanum absolute, diterpenoid fraction,
and flavonoid fraction are presented in Table 6.1.3. UV absorption spectra of the extracts
are shown in Figure 6.1.4. Regarding all three parameters, flavonoid fraction showed the
highest values followed by labdanum absolute. Diterpenoid fraction showed very low UV
absorption. Labdanum absolute showed UVB SPF value (4.98 ± 0.19) that is very similar
to the values obtained by Gaweł-Bęben et al. (2020) for C. ladanifer methanolic extracts
(between 3.33 ± 0.15 and 4.37 ± 0.42). In this work we further demonstrate the significant
absorbance of UVA radiation by the labdanum absolute and that flavonoid aglycones are
responsible for that feature whereas the diterpenoid fraction contribution is very low or
even negligible. This directly correlates with the chemical structures of compound
classes. Our results show that, at least partially due to flavonoid aglycones, labdanum
absolute possesses a broad-spectrum of UV protection by absorption, and therefore
labdanum resin may be considered as a natural ingredient with potential to be included in
sunscreen formulations.
Table 6.1.3: Spectrophotometric Sun Protection Factor (SPF), and UVB (280-315 nm) and UVA (315-400
nm) total absorbance (area under the curve, in a.u.) of 100 µg/mL solutions of labdanum absolutes and
fraction.
Total absorbance (a.u.)
Extract
SPF UVB UVA
Absolute 4.98 ± 0.19b 9.05 ± 0.36b 16.7 ± 0.6b
Dit 0.736 ± 0.015c 1.44 ± 0.02c 1.67 ± 0.03c
a a
Flv 13.0 ± 1.4 22.0 ± 2.3 47.1 ± 5.3a
Different coefficient letters mean statistical differences by the post-hoc Dunnett’s T3 test (α = 0.05)
Figure 6.1.4: UV absorption (250-400 nm) spectral scan of labdanum absolute, flavonoid fraction (Flv),
and diterpene fraction (Dit) related to a concentration of 100 µg/mL (n=3, mean curve).
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6.1.3.4. Antioxidant activity
Antioxidant activity results of labdanum absolute and its purified fractions are presented
in Table 6.1.4. Results are expressed as equivalents to well know antioxidant compounds,
gallic acid or trolox. DPPH and FRAP methods showed a very low antioxidant activity
of all the extracts in relation to trolox standard. For these two methods, all the extracts
presented values below 0.1 mgTE/mgExt (i.e., 0.4 mmolTE/gExt) meaning that they have
an antioxidant activity more than 10 times lower than the standard. In contrast ABTS and
Folin-Ciocalteu methods showed more prominent antioxidant activity in relation to trolox
and gallic acid, respectively, for labdanum absolute and flavonoid fraction extracts.
Flavonoid fraction showed an ABTS antioxidant activity around 3 times lower than trolox
and a Folin-Ciocalteu antioxidant activity around 5 times lower than gallic acid.
Labdanum absolute showed an ABTS and Folin-Ciocalteu antioxidant activity less than
10 times lower than the standards. Despite the differences between methods, labdanum
absolute and flavonoid fraction antioxidant activities were not statistically different, for
each of the methods used (Table 6.1.4). Labdanum absolute has much less amount of
flavonoid aglycones than the purified flavonoid fraction. This would mean that although
flavonoid aglycones contribute to the antioxidant activity of the labdanum absolute, other
compounds in the labdanum absolute not included in the diterpenoid fraction must also
contribute to the antioxidant activity. In fact, labdanum absolute was shown to have
significant compounds not present in both purified fractions but that could not be assigned
to a known component of the labdanum resin described in literature (Table 6.1.2). C.
ladanifer methanol extract done by Chaves et al. (2020) contained a lower Folin-
Ciocalteu antioxidant activity than the labdanum absolute of this study, but according to
the authors, among different plant species of the Iberian Peninsula, C. ladanifer is in the
group of plants with higher antioxidant activity. In sum, in vitro antioxidant activity of
the labdanum resin is demonstrated, which seems to be related but not only to the typical
flavonoid aglycones of the resin. This property may confer an additional activity of the
potential ingredients to be used in skin care cosmetics, conferring antioxidant activity and
thus mitigating/scavenging ROS generation, which can be induced by several factors such
as UV radiation, causing skin damage or photo-aging. Other potential applications
include the use of these ingredients as preservatives in cosmetic products.
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Table 6.1.4: Folin-Ciocalteu test, DPPH scavenging activity, FRAP, and ABTS free radical scavenging
activity of labdanum absolutes and fractions, expressed as gallic acid (GAE) or as trolox (TE) equivalents,
in the dried extract (Ext).
Folin-Ciocalteu DPPH FRAP ABTS
Extract
(mgGAE/ mgExt) (mgTE / mgExt)
Absolute 0.128 ± 0.015a,b,c 0.031 ± 0.008a,b,c 0.038 ± 0.008a,b,c 0.142 ± 0.017a,b,c
a,b a,b a,b
Flv 0.203 ± 0.044 0.054 ± 0.005 0.049 ± 0.004 0.379 ± 0.039a,b
b,c b,c b,c
Dit 0.011 ± 0.001 0.005 ± 0.001 0.020 ± 0.003 0.010 ± 0.003b,c
Different coefficient letters mean statistical differences between extracts for each method by the post-hoc
Dunnett’s test (α = 0.05).
6.1.3.5. Elastase inhibition activity
Elastase inhibition was observed only for labdanum absolute at the highest concentration
used, 1 mg/mL (Table 6.1.5). At 0.5 mg/mL none of the extracts showed elastase
inhibition activity. Inhibition of elastase activity is shown for several Mediterranean
plants, including representatives of Cistus spp. (Chiocchio et al. 2018). Inhibition of
elastase activity and thus the breakdown of skin elastin and other proteins responsible for
connective tissue elasticity reduces the skin aging process and would be an interesting
property for a skin care cosmetic ingredient. In this study, the weak inhibition activity
regarding this enzyme by labdanum absolute is demonstrated. To the best of our
knowledge, this study is the first to report the elastase-inhibiting activity of C. ladanifer
labdanum. Gaweł-Bęben et al. (2020) demonstrated that C. ladanifer methanolic extracts
inhibited, at some extent, tyrosinase activity, which is a target enzyme in the treatment of
hyperpigmentation, a result of skin photoaging.
Table 6.1.5: Inhibition of elastase activity (%) by the labdanum absolute and fractions at concentrations of
1 and 0.5 mg/mL.
Elastase inhibition (%)
Extract
1 mg/mL 0.5 mg/mL
Absolute 22.1 ± 0.3* n.a.
Dit n.a. n.a.
Flv 13.7 ± 0.3* n.a.
“n.a.”: no activity considered when inhibition mean values of the extracts were not statistically different
from the mean value of the control, by the post-hoc Dunnett’s test. Symbol (*) means statistical significant
difference according to t-test. (α = 0.05).
6.1.3.6. Anti-inflammatory activity
The anti-inflammatory activity of labdanum absolute and fractions was assessed based on
the quantification of the pro-inflammatory mediator, nitric oxide (NO), released by
lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Extracts concentrations to be
tested were selected based on RAW 264.7 cells viability assay results (Figure 6.1.5).
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Flavonoid fraction (Figure 6.1.6, grey bars) produced the highest anti-inflammatory
activity at a concentration dependent manner, inhibiting near 90% of NO release in
relation to the control, at the lowest tested concentration (5 µg/mL). At 15 µg/mL,
flavonoid fraction and absolute anti-inflammatory activity was similar (p < 0.05),
reaching near 98% inhibition of NO release. Both extracts showed a concentration
dependent activity. Although, diterpene fraction, at 25 µg/mL slightly affected cell
viability (≈ 80% of control; Figure 6.1.5) and, at 12.5 µg/mL, cell viability was ≈ 95% of
control, the NO production by LPS-stimulated cells exposed to 10 and 15 µg/mL of
diterpene fraction is presented, which was on average higher than that produced by
exposure to the other extracts (at same concentrations), indicating a lower anti-
inflammatory action (Figure 6.1.6). However, comparing with control cells, labdanum
resin diterpenoid fraction showed significant anti-inflammatory activity at the lowest
tested concentration (5 µg/mL), reducing ≈ 40% of NO release (Figure 6.1.6, white bars)
and at this concentration RAW 264.7 cells show 100% viability (Figure 6.1.5). In sum,
labdanum absolute presents anti-inflammatory activity, property given prominently by
flavonoid aglycones (the most active fraction) but also by some components of the
diterpenoid fractions (Figure 6.1.6).
To the best of our knowledge, this is the first work reporting the in vitro anti-inflammatory
activity of the labdanum resin (and of its fractions). Nevertheless, Youbi et al. (2016)
reported anti-inflammatory effect of C. ladanifer aqueous extracts in an in vivo rat model
of inflammation (Wistar rats; carrageenan-induced paw oedema). Phenolic rich extracts
of other Portuguese Mediterranean plant, Thymus zygis subsp. zygis, showed NO release
inhibition of 48% and of 89%, by aqueous and hydroethanolic extracts at 50 µg/mL,
respectively (Silva et al. 2020), using the same testing method. However, when looking
at results of absolute and flavonoid fraction at 5µg/mL (Figure 6.1.6) the same NO release
inhibition was observed but with a ten-fold lower concentration, indicating that labdanum
resin has a high anti-inflammatory activity. Additional to the SPF, antioxidant, and
elastase inhibition activities, the anti-inflammatory activity may give an added-value to
the labdanum resin as a cosmetic ingredient worth to be incorporated in skin care products
since inflammation (erythema) is an UV-mediated skin damage effect as discussed
earlier.
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Figure 6.1.5 RAW 264.7 (murine macrophage) cells viability, as percentage in relation to the control (0.00
µg/mL), of labdanum absolute and fractions at concentrations between 6.25 and 100 µg/mL (mean values
± standard deviation, 3 independent experiments of n = 4) . Symbol (*) mean significant difference (α =
0.05) in relation to the control by the post-hoc Tukey’s test.
Figure 6.1.6: Anti-inflammatory activity of labdanum absolute and fractions. Nitric oxide (NO) release by
LPS-stimulated RAW 264.7 cells in relation to the control (%), when exposed to labdanum absolute (left
bars, blue colour), and to flavonoid (middle bars, white colour) and terpenoid (right bars; grey colour)
fractions, expressed as percentage of control (black bars). Results are expressed as mean ± standard
deviation (3 independent experiments of n = 4). Different coefficient letters mean statistical differences
between values by the post-hoc Dunnett’s T3 test or Welch’s t-test (α = 0.05).
6.1.3.7. Antimicrobial activity
Antimicrobial activity results of labdanum absolute and fractions against reference strains
relevant for skin care and cosmetics are shown in Table 6.1.6. Labdanum absolute and
fractions were only active against the Gram-positive bacteria S. aureus ATCC® 25923TM,
a clinical isolate indicated for infectious diseases research. Labdanum absolute showed
only S. aureus inhibition at 1.2 mg/mL (MIC); however, the purified fractions showed a
much lower MIC value (< 300 µg/mL), indicating that the diterpenes and the flavonoids
are the main responsible for the S. aureus growth inhibitory activity. Concerning the
microbicidal activity, diterpene and flavonoid fraction showed bactericidal activity
against S. aureus at 2.5 mg/mL, and the labdanum absolute MMC could not be achieved
140
within the range of concentrations tested. We may assume that the components of each
purified are the ones with higher antimicrobial activity; and that the other components
(other than diterpenes and flavonoids) in absolute may have antagonism or none
antimicrobial activity, which highly reduces the labdanum absolute MIC and MMC
activity. To our knowledge, only two works report the antimicrobial activity of the
labdanum resin, the ones done by Greche et al. (2009) and by Rauwald et al. (2019).
Rauwald et al. (2019) only tested growth inhibition of Borrelia burgdorferi and showed
no susceptibility for labdanum resins from C. ladanifer (at 0.2 mg/mL). Greche et al.
(2009) showed a broad-spectrum growth inhibition for labdanum absolute and similar
extracts (concrete and resinoid) ), at concentrations between 1 and 10 mg/mL. Labdanum
absolute showed growth inhibitory activity against strains also tested in this work,
however with higher MIC, for example S. aureus (MIC: 2-4 mg/mL) and E. coli (MIC: >
5 mg/mL), but no microbicidal activity of the absolute, for any of the tested
microorganisms, was observed for the higher concentrations (10 mg/mL) (Greche et al.
2009). Thus, the labdanum absolute extracted in this work has higher antimicrobial
activity, and it can be potentiated by fractionating it into terpenoid and flavonoid fractions
(Table 6.1.6). Excluding essential oils, several works evaluating the antimicrobial activity
of C. ladanifer extracts are published. However, those extracts usually use milled plants
and the whole extract is tested which makes them different from the labdanum resin here
studied. Boy et al. (2021) showed a broad-spectrum antimicrobial activity, by disk
diffusion, of 90% ethanol (v/v) extracts obtained from C. ladanifer intact leaves (a
phenolic rich extract), at concentrations comparable to this study (0.5-2 mg/mL). In fact,
other works showed P. aeruginosa, S. aureus, E. coli, and Candida spp. susceptibility for
ethanolic/aqueous extracts of C. ladanifer rich in phenolic acids and tannins (Barrajón-
Catalán et al. 2010; Barros et al. 2013; Tomás-Menor et al. 2013; Lekbach et al. 2018).
Methanol, ethanol, and acetone/water extracts from milled C. ladanifer material are also
reported to have a broad-antimicrobial activity, however higher in relation to Gram-
positive bacteria (Ferreira et al. 2012; Köse et al. 2017). Comparing our results to the
literature leads to the hypothesis that terpenoids, flavonoids, tannins, and other phenolic
compounds are the compounds responsible for the broad-spectrum antimicrobial activity
found for C. ladanifer extracts. During labdanum extraction, some of those compounds
most probably are retained in the residual water of the extraction process because of their
hydrophilic character or are extracted at low amounts compared to other components.
141
The four microorganisms tested in this study are important regarding the quality and
safety microbiological criteria for final cosmetic products, which must be absent in 1 mg
or mL of product according to the European Commission guidelines. In addition,
Staphylococcus aureus, P. aeruginosa, and C. albicans are important because they can
cause opportunistic skin infections. Although labdanum resin does not show a high
potential preservative application, the high inhibition susceptibility showed against S
aureus is an important property because it causes the most frequent skin opportunistic
infections as discussed earlier.
Table 6.1.6: Antimicrobial activity: Minimum Inhibitory Concentration (MIC) and Minimum Microbicidal
Concentration (MMC), of labdanum absolute and fractions in relation to selected microorganisms.
Microorganisms
Extracts E. coli ATCC P. aeruginosa ATCC S. aureus ATCC 25923 C. albicans ATCC
25922 27853 MIC MMC 10231
Absolute n.a. n.a. 1.2 n.a. n.a.
Flv n.a. n.a. ≤0.3 2.5 n.a.
Dit n.a. n.a. ≤0.3 2.5 n.a.
Values presented as mode (n = 3).
MIC and MMC values in mg/mL. “n.a.”: no activity in relation to the control.
Within the European Union, regulation (EC) nº 1223/2009 establishes standard rules for
cosmetic products. Although the cosmetics regulation of the European Commission
allows some active ingredients (e.g., sunscreens) to “protect or keep the human body in
good conditions” if those ingredients are used to “restore, correct or modify physiological
functions” by means of a “pharmacological, immunological or metabolic action” the final
product must be considered medicinal. It is thus a question of the intent of the product in
question, however Directive 2001/83/EC clearly states that when in doubt regarding
definitions, the product would be considered a medicinal product. In fact, there is no clear
official definition and regulation in the world about cosmeceuticals which would be
products that are between cosmetic and pharmaceutical/medicinal products (Pandey et al.
2019). Elastase inhibition and anti-inflammatory activities would be properties to classify
a skin care product as a medicinal product. UV radiation protection/filters ingredients
may be included in cosmetics according to EU regulation but must be listed in annex IV
of the regulation and used according to specifications. In addition, a complete
toxicological profile of cosmetic ingredients according to the regulation is also
mandatory, particularly photo-induced toxicity for UV filter substances. There are three
ECHA registrations (REACH regulation) regarding labdanum resin with a complete
chemical safety assessment: i) “Gum of Cistus ladaniferus (Cistaceae) obtained from
stems and leaves by extraction with alkaline solution”, raw labdanum resin; ii) “Concrete
142
of Cistus ladaniferus (Cistaceae) obtained from stems and leaves by organic solvents
extraction”; iii) “Resinoid of Cistus ladaniferus (Cistaceae) obtained from labdanum gum
by ethanol extraction” which is in fact an ethanol labdanum absolute. All these registered
substances are reported to have no physical, environmental, and human health hazards,
based on a complete toxicological assessment.
6.1.4. Conclusion
Labdanum absolute represents about 70% (dw/dw) of labdanum resin and is principally
composed by labdane-type diterpenes and methylated flavonoid aglycones. Labdanum
absolute showed to possess relevant UV protection and anti-inflammatory activities
mainly because of the flavonoids fraction. These properties are relevant for skin care
cosmetics regarding the damage effects that UV radiation has on the constantly exposed
human skin. However, at least within the UE, labdanum resin needs to be included in a
list of substances approved to be used as UV filters of the cosmetics regulation and if used
for its anti-inflammatory activity labdanum resin must be considered a medicinal product
and most likely must comply with regulatory dispositions for medicinal products under a
different regulation. Labdanum absolute showed low antioxidant and narrow spectrum
antimicrobial activity meaning it is not suitable as a cosmetic preservative. Despite that,
the extract showed selective and high inhibitory activity against Staphylococcus aureus,
compared to other cosmetic and skin relevant microorganisms, which is the most common
cause of opportunistic skin infections.
143
144
Subchapter 6.2
Labdanum resin from Cistus ladanifer L. as a source of compounds

with medicinal related bioactivities.
Abstract
Labdanum resin or “gum” may be obtained from Cistus ladanifer L. (Cistaceae) by two
different extraction procedures: The Zamorean and the Andalusian processes. This
product is currently traded for the fragrance/perfumery industries but, traditionally, it had
been used as medicine to control hyperglycaemia and mental illnesses. The yield of
labdanum resin extracted by the Andalusian process was 25-fold higher the Zamorean
one. Both resins were purified as absolutes and the Andalusian absolute was purified into
diterpenoid and flavonoid fractions. GC-EI-MS analysis confirmed the presence of
phenylpropanoids, labdane-type diterpenoids, and methylated flavonoids which are
already described in literature, and revealed other compounds that remained unidentified.
All extracts presented different chemical profile. Potential antidiabetic activity, by
inhibition of α-amylase and α-glucosidase, and potential neuroprotective activity, by
inhibition of acetylcholinesterase was performed to all extracts. Diterpenoid fraction
showed the highest α-amylase inhibitory activity (40.0 ± 2.8%, at 1 mg/mL). Zamorean
absolute showed the highest α-glucosidase inhibitory activity (24.1 ± 4.6%, at 1 mg/mL).
Andalusian absolute showed the highest acetylcholinesterase inhibitory activity (75.1 ±
0.9%, at 1 mg/mL). Using Caco-2 and HepG2 cell lines, Andalusian absolute and its
purified fractions showed high cytotoxic activity (IC50 30 - 80 µg/mL), whereas
Zamorean absolute did not (IC50  200 µg/mL). Here we show for the first time that
Andalusian labdanum resin and its fractions are composed of phytochemicals with
potential anti-diabetic, neuroprotective and anti-proliferative activities which are worth
investigating for the pharmaceutical industry, however toxic side-effects must also be
addressed when using these products by ingestion.
Keywords
α-amylase inhibition; α-glucosidase inhibition; Acetylcholinesterase inhibition;

Cytotoxic activity; Labdanum resin; Labdane-type diterpenoids; Methylated flavonoids.
145
Subchapter 6.2 Labdanum resin from Cistus ladanifer L. as a source of compounds with medicinal related
bioactivities.
Frazão DF, Martins-Gomes C, Dias TS, Delgado F, Gonçalves JC, Silva AM Labdanum resin from Cistus
ladanifer L. as a source of compounds with medicinal related bioactivities. (Under review)
146
Subchapter 6.2 Labdanum resin from Cistus ladanifer L. as a source of compounds with anti-diabetic,
neuroprotective and anti-proliferative activity.
6.2.1. Introduction
The rockrose Cistus ladanifer L. (Cistacea family, subgenus Leucocistus, Ladanium

section), namely the subspecies ladanifer, is an abundant (dense shrublands) and wide-
spread plant resource in the Iberian Peninsula, including Portugal and Spain (Demoly and
Montserrat 1993; Godinho-Ferreira et al. 2005; Montero et al. 2020). Cistus ladanifer
dominated shrubland is very poor in other plant species and has a natural tendency to
perpetuate instead of to develop to mature vegetation stages (Mendes et al. 2015),
registering their occupation mostly on abandoned and degraded areas. It is therefore an
abundant not endangered plant resource worth to be exploited in a sustainable manner.
Currently, the most significant valorisation of such resource is the extraction of its exuded
resin, which has the commercial name of labdanum “gum”, for the fragrance/perfumery
industry because of its aromatic and fixative properties, replacing ambergris used to be
obtained from the protected sperm whale Physeter catodon (Raimundo et al. 2018). In
fact, according to Biolandes SA, one of the most important producers of C. ladanifer
extracts, around 300 - 350 tons of raw labdanum resin are extracted each year from 6000-
7000 tons of manually harvested plant (Biolandes n.d.). In order to diversify and increase
the economy around this plant, in addition to perfumery, it is necessary to find other
applications for the valorisation of labdanum resin which is also extremely relevant to
maintain a sustainable management of agro-forestry systems.
Ethnobotanical studies report that tisanes or infusions of labdanum or C. ladanifer shoots

and leaves were traditionally ingested as human medicine to heal disorders of the gastro-
intestinal system (e.g. toothache, stomach ache, peptic ulcer, colon pain and liver
disorders), respiratory system (e.g. colds and whooping cough), nervous system and
mental illness (e.g. sedative for anxiety, insomnia, and digestive spasms, and neuralgia),
and to control blood sugar spikes and rheumatism, besides the extensive use to heal skin
disorders (Martínez-Lirola et al. 1996; Papaefthimiou et al. 2014; Raimundo et al. 2018;
Vieira et al. 2019; Bencheikh et al. 2021; Zalegh et al. 2021). The traditional use of C.
ladanifer shoot and leaves “aqueous extracts” is much more reported than the use of
labdanum.
Traditionally, labdanum resin is extracted, using water, by Zamorean process or the

Andalusian process (Lawrence 1999). The Zamorean process is a physical extraction
where labdanum resin is removed from the surface of C. ladanifer plant material using
147
boiling water and then the resin at the surface of the water is continuously separated using
a skimmer (Lawrence 1999). The Andalusian process is a chemical extraction where the
labdanum resin is extracted from the surface of the plant material with warm alkaline
water and afterwards precipitated by lowering the pH with acid and finally separated with
a skimmer or by decantation (Lawrence 1999). Labdanum resin is mostly composed by
labdane-type diterpenes and methylated flavonoid aglycones (Sosa et al. 2005; Alías et
al. 2012; Valares et al. 2016a) (section 1.2.2.2). To separate epicuticular lipids, mostly
waxes, from the labdanum resin and to obtain purified diterpenoid and flavonoid fractions
a cold methanol precipitation followed by molecular sizing column chromatography has
been done (Sosa et al. 2005; Alías et al. 2012; Valares et al. 2016a) (section 1.2.2.1).
High anti-inflammatory activity of labdanum resin mainly due to the flavonoid aglycones,
based on inhibition of nitric oxide release by LPS-stimulated RAW 264.7 macrophages,
and antioxidant properties were observed (subchapter 6.1). This activity may explain
several traditional medicinal applications enumerated above. However, other bioactivities
and their mechanisms of action, as well as toxicity, are relevant to be studied principally
when this substance or preparations from it are intended to be ingested.
Given the traditional use of Cistus spp. to control hyperglycaemia, aqueous and
hydromethanolic extracts from the Moroccan Cistus salviifolius L. and Cistus
monspeliensis L., were reported to moderately inhibit α-amylase and strongly inhibit α-
glucosidase activity (Sayah et al. 2017), these enzymes are relevant in the management
of Diabetes Mellitus type 2 by lowering intestinal carbohydrate breakdown and
subsequently lowering glucose absorption (Vieira et al. 2019). Thus, it would be worth to
study Cistus ladanifer labdanum inhibitory activity on these enzymes.
Given the documented traditional use of Cistus spp. to control nervous system and mental
disorders (Martín et al. 2009; González et al. 2018), studying the inhibitory action of
Cistus ladanifer labdanum on acetylcholinesterase (AChE) activity is relevant. In fact,
cholinesterase, AChE and at some extent BChE (butyrilcholinesterase), inhibition is the
current therapeutic strategy to manage neurodegenerative disorders such as Alzheimer’s
disease by directly improving cholinergic tone (i.e., increasing acetylcholine
neurotransmitter levels) and by the less understood long-term anti-neurodegenerative
activity (Moss 2020).
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In the possibility of being ingested for medicinal purposes, the gastrointestinal and
hepatic toxicity of the labdanum resin must be evaluated. As a preliminary approach to
evaluate it, the assessment of in vitro cytotoxic activity using relevant cell lines, such as
the Caco-2 (human colorectal adenocarcinoma) and the HepG2 (human hepatocyte
carcinoma) cell lines could be used to screen potential toxicity of the resin, or of its
extracts, to the gastrointestinal epithelial barrier and to the hepatocyte cells, respectively.
Thus, the main aim of this work is to study the potential anti-diabetic and neuroprotective
effect of the labdanum resin, and of its extracts, by assessing the inhibition of relevant
enzymes and to evaluate the cytotoxic/safety profile of these extracts, using relevant cell
lines.
6.2.2. Materials and methods
6.2.2.1. Chemicals
Sodium carbonate (Na2CO3, Biochem, Frilabo, Portugal). Methanol LC-MS grade 99.9%
(Fisher Scientific, EUA). C7-C30 saturated alkanes (Supelco, LaborSpirit, Portugal).
Acetic acid glacial (CH3COOH); Sulfuric acid 97% (H2SO4); BSTFA (N,O-
bis(trimethylsilyl)trifluoroacetamide) + 10% TMCS (trimethylsilyl chloride); Dimethyl
Sulfoxide anhydrous (DMSO); 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB);
Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl); Acetylthiocholine iodide;
Phosphate buffered saline tablet (PBS); starch; acarbose; potassium iodide (KI); diiodide
(I2); Rats intestinal acetone powder; p-nitrophenyl-α-D-glucopyranoside;
Acetylcholinesterase (AChE); α-amylase; α-glucosidase (Sigma Aldrich, Merck,
Germany). Trypsin-EDTA, sodium pyruvate, L-glutamine, penicillin, streptomycin, fetal
bovine serum (FBS) and Modified Eagle Medium (DMEM) (Alfagene, Invitrogen,
Portugal). Alamar Blue® (Life Technologies Invitrogen, Portugal). Labdanolic acid and
6-oxocativic acids (Merck, Darmstadt, Germany); apigenin, kaempferol, kaempferol-3-
methyl ester, kaempferol-3,4’-dimethyl ester, apigenin-4’-methyl ester, apigenin-7-
methyl ester, apigenin-7,4’-dimethyl ester, kaempferol-3,7-dimethyl ester, and
kaempferol-3,7,4-trimethyl ester (Carbosynth Lda, UK).
149
6.2.2.2. Labdanum resin extraction and purification
Cistus ladanifer subsp. ladanifer shoots were collected from a shrubland in Penha Garcia,
Portugal (GPS: N 40o1’43.4’’ W 6o59’34.8’’), in August 2018. Plant material was stored
fresh in the freezer (-20°C) until use.
To simulate the Andalusian process (Adl), based on the method described by Burguer
(2016), twenty-five grams of plant material were extracted in 200 mL of Na2CO3 aqueous
solution (25 g/L) for 1 h at 60°C (water bath), inside a flask. The solution was filtered
through a stainless-steel sieve (mesh n. 140, 106 µm aperture). After cooling, H2SO4
solution (5 M) was added dropwise to the extract, under agitation, until reaching pH 2.
The precipitated resin was separated from the supernatant by centrifugation, at 3030 xg
for 15 min (Mega Star 600R, VWR), and finally freeze-dried. To simulate the Zamorean
process (Zam) (Lawrence 1999), labdanum resin was extracted by using the same
procedure described above but using distilled water instead of Na2CO3 aqueous solution.
All extractions were done in triplicate, resulting on three similar resins (n=3). Resin yield
of the extractions was calculated by the ratio between the total dry weight (dw) of the
resin and the fresh weight (fw) of the plant material (presented in %).
Labdanum absolute was obtained by removing “waxes” from the resins by dissolving 1 g
(maximum) of the resins in 20 mL of methanol under an ultrasonic bath. Then, the
solution was left at -20oC overnight, and afterwards centrifuged at 3030 xg at -5oC for 15
minutes. The procedure was repeated three times and supernatants were joined as the
absolute. This method was first described by Vogt et al. (1987) and used in recent works
(Sosa et al. 2005; Alías et al. 2012; Valares et al. 2016b).
Diterpenoid (Dit) and Flavonoid (Flv) purified fractions were obtained by eluting the Adl
labdanum resin on SephadexTM LH-20 (GE Healthcare Biosciences AB, Sigma Aldrich,
USA) compacted in a 25 cm long and 1.5 cm width column, using methanol as eluent (the
Zam labdanum resin was not fractionated due to low yield) Sub-fractions of 2-3 mL were
continuously collected and analysed by thin layer chromatography using silica gel TLC
plates (F254s 0.2mm 10x20cm) (Merck, Germany). Sub-fractions were eluted with
toluene: ethyl acetate (9:1). Flavonoid bands were visualised at 312 nm using a trans-
illuminator. After sprayed with 10 M H2SO4 solution and heated at 100oC for 10 minutes
in a ventilated chamber, diterpenoid bands were visualised as purple/red bands. Sub-
fractions were joined to make the purified fractions.
150
6.2.2.3. Chemical characterisation (GC-EI-MS).
Labdanum absolutes and fractions were prepared at 10 mg/mL. A volume of 20 µL of

BSTFA + 10% TMCS was added to 100 µL of sample for derivatisation, at room
temperature, during 30 min, according to Azemard et al. (2016). A volume of 1 µL of the
reaction mixture was injected at a split ratio of 1:50 in the Gas Chromatography coupled
to an Electron Ionisation Mass Spectrometer system (GC-EI-MS, Scion-SQ 456 GC,
Bruker) equipped with a HP5-MS column (30 m x 0.25 mm x 0.25 µm, Agilent J&W GC
columns). Helium was used as carrier gas at 1 mL/min flow rate. Temperature gradient
was set as follows: 50oC - 175oC (10oC/min), 175 oC - 300oC (3oC/min). Data was
acquired through electron impact ionisation mass spectrometry (EI-MS, scan mode of
positive m/z ions 50 - 650, 70 eV ionisation potential). Injector and detector port
temperatures were set at 250oC. The major peaks of the sample chromatogram were
tentatively identified using the NIST library (2017) mass spectra and Kovats retention
index database and by matching their retention times and mass spectra to standards:
labdanolic acid, 6-oxocativic acid, apigenin, kaempferol, kaempferol-3-methyl ester,
kaempferol-3,4’-dimethyl ester, apigenin-4’-methyl ester, apigenin-7-methyl ester,
apigenin-7,4’-dimethyl ester, kaempferol-3,7-dimethyl ester, and kaempferol-3,7,4-
trimethyl ester.
6.2.2.4. α-Amylase, α-glucosidase, and AChE inhibition
All enzymatic inhibition assays were performed in 96-well microplates and absorbances
were read using a microplate spectrophotometer (MultiskanGo, Thermo Scientific).
Methods were performed as described in Taghouti et al. (2018). Labdanum absolutes,
tested at 0.5 and 1 mg/mL, were diluted in PBS from stock solutions prepared in DMSO.
The final concentration of DMSO was always less than 5% and a control with 5% DMSO
was used in each assay to exclude solvent-induced inhibition. All the assays were done
in triplicate (n=3).
α-Amylase inhibition assay, 35 µL of PBS and 35 µL of starch solution (0.05% m/v, pH

7) were added to 50 µL of the extracts. The mixtures were incubated for 2 minutes at
37oC, and 20 µL of α-amylase (50 µg/mL in 10 mM phosphate buffer, pH 6.9) was added.
After incubation for 10 minutes at 37oC, 50µL of HCl (0.1 M) was added to stop the
reaction. Afterwards, 150 µL of Lugol solution (0.5 mM I2, 0.5 mM KI, in water) was
added and, after a 15-minute incubation, absorbance was read at 580 nm.
151
α-Glucosidase inhibition was assessed by first obtaining a crude enzyme extract by

diluting 250 mg of rat´s intestinal acetone powder in 10 mL of phosphate buffer (0.1 M,
pH 7), centrifuging for 20 minutes at 4oC (5000 g), and recovering the supernatant.
Then, 100 µL of the supernatant was joined to 50 µL of the extracts and left to incubate
for 10 minutes at room temperature. Afterwards, 50 µL of p-nitrophenyl-α-D-
glucopyranoside (5 mM in phosphate buffer 0.1 M, pH 7) was added and, after a 30-
minutes incubation at 37oC, absorbance was read at 405 nm.
Acetylcholinesterase (AChE) inhibition was assessed by first adding 125 µL of Ellman’s

reagent (0.3 mM DTNB, prepared in 50 mM Tris-HCl, pH 8.0), and 25 µL of
acetylthiocholine iodide (1.5 mM, in 20 mM Tris-HCl, pH 7.5) to 50 µL of the extracts.
The mixtures were incubated for 2 minutes, and then 25 µL of AChE (0.026 U/mL in 20
mM Tris-HCL, pH 7.5) was added. After incubation for 10 minutes at 25oC, absorbance
was read at 405 nm.
Blanks including samples without the enzymes, controls including the enzymes without
any sample, and positive control including acarbose (α-glucosidase and α-amylase; 1
mg/mL acarbose inhibited 100% of α-amylase and 80% α-glucosidase activity, at
indicated experimental conditions) were performed. Blanks’ absorbance values were
subtracted from the sample value. Percentage of inhibitions were calculated according to
equation (1), except for α-amylase which was calculated according to Torres-Naranjo et
al. (2016) (2).
(𝐴𝑏𝑠𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑏𝑠𝑠𝑎𝑚𝑝𝑙𝑒 ) (1)

𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 (%) = × 100
𝐴𝑏𝑠𝑐𝑜𝑛𝑡𝑟𝑜𝑙
𝐴𝑏𝑠𝑏𝑙𝑎𝑛𝑘1 − 𝐴𝑏𝑠𝑠𝑎𝑚𝑝𝑙𝑒 (2)

𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 (%) = (1 − ) × 100
𝐴𝑏𝑠𝑏𝑙𝑎𝑛𝑘2 − 𝐴𝑏𝑠𝑐𝑜𝑛𝑡𝑟𝑜𝑙
Where Absblank1 or Absblank2 are the absorbance of incubated solution containing sample
and starch or only starch, respectively.
6.2.2.5. Caco-2 and HepG2 cytotoxicity
The cytotoxicity/anti-proliferative assay was performed in Caco-2 (Human epithelial

colorectal adenocarcinoma; Cell Lines Service (CLS), Eppelheim, Germany) and in
HepG2 (Human hepatocellular carcinoma; ATCC® N.: HB-8065TM, ATCC, USA).
Cells maintenance and handling were performed as described by Silva et al. (2020).
152
Briefly, cell cultures were maintained in DMEM (Dulbecco’s Modified Eagle Medium)
supplemented with 10% (v/v) foetal bovine serum (FBS), antibiotics (100 U/mL of
penicillin and 100 μg/mL of streptomycin) and 1 mM L-glutamine in an atmosphere of
5% (v/v) CO2/95% (v/v) air, at 37oC with controlled humidity. Cells were detached from
the culture flasks with trypsin-EDTA and, after counting (TC10™ Automatic counter,
BIORAD) were diluted to a density of 5 x 104 cells/mL (in complete culture medium),
and then seeded in 96-well microplates (100 µL/well; 5 x 104 cells/mL). After 48h culture
(to adhere and stabilize), the culture media was removed and 100 µL of extracts solutions,
diluted in FBS-free culture media (range 6.25-200 µg/mL, prepared from stock solutions
in 100% DMSO), were added. After 24 h or 48 h exposure, the test solutions were
replaced with 100 µL of 10%(v/v) Alamar Blue® solution, and the absorbance was read,
at 570 nm and 620 nm, after a 5 h incubation, using a microplate reader (Multiskan EX;
MTX Lab Systems, Inc., Bradenton, FL, USA). Alamar Blue® reduction was calculated
as described in Andreani et al. (2014), and results were expressed as percentage of cell
viability, in relation to untreated control. DMSO final concentration in the extracts
solutions was lower than 1% (v/v), at the highest concentration tested. Previous results
showed that, at 1% (v/v), DMSO induced no cytotoxicity. The concentration of extracts
needed to inhibit 50% of cell growth/proliferation (or the cytotoxic concentration at which
50% viability is achieved), the IC50, was calculated as previously describe (Silva et al.
2019) and is presented as mean ± standard deviation (3 independent experiments of n =
4). The extracts´ activity against the studied cell lines was categorized according to
previously established criteria by the National Cancer Institute (NCI) guidelines (Geran
et al. 1972): highly active IC50 < 0.02 mg/mL; moderately active IC50 0.02 - 0.20 mg/mL;
weakly active IC50 0.20 - 0.50 mg/mL and inactive IC50 > 0.50 mg/mL.
6.2.2.6. Statistical Analysis
Statistical analysis to find significant variable effects and significant differences between
means of different treatments (α = 0.05), was performed using IBM SPSS Statistics 25
and GraphPad Prism 9 software. Shapiro-Wilk’s test was used to test normality of data
and Levene’s test to test homogeneity of variance. ANOVA and post-hoc Tuckey’s test
was used for normal data with homogeneous variance. Welch’s ANOVA, Welch’s t-test,
and Dunnet’s T3 post-hoc tests were used for normal data with equal variances not
assumed. Four-parameter symmetric sigmoid curves were fitted as the regression dose-
153
response model for cell viability data (as % in relation to the control) versus extracts
concentration, using GraphPad Prism 9 software, to extract IC50 values.
6.2.3.1. Extraction and purification yield and chemical profile
In this study, labdanum resin, extracted based on the Andalusian process (Adl), yielded
5.79 ± 0.52% (dw/fw) (Table 6.2.1). The Zamorean process (Zam) was performed as a
control to evaluate the importance of alkaline extraction of labdanum resin. In fact, the
Zam extraction yielded only 0.23 ± 0.07%, which is approximately 25 times lower than
the yield of Adl resin. In this study, the Zam resin was successfully isolated, however
using acidification to precipitate it like it was done for the Adl resin. Despite the
difference of extraction yields, both resins rendered similar absolute yields, around 70%
(dw/dw). Diterpenoid and flavonoid fractions were obtained by column chromatography
from the Adl labdanum absolute, rendering 79.9 ± 0.8 and 10.6 ± 2.2% (dw/dw) yields,
respectively. A remainder fraction, lost during the purification process, represented 9.48
± 1.87% (dw/dw) of the absolute. As the yield of Zam labdanum resin was too low it was
not fractionated.
Table 6.2.1: Dry labdanum resin yield in relation to the initial C. ladanifer plant material fresh weight and
dry labdanum absolute yield in relation to the dry resin weight.
Extraction process Resin yield (% dw/fw) Absolute yield (% dw/dw)
Adl 5.79 ± 0.63a 72.61 ± 1.52a
b
Zam 0.227 ± 0.081 71.03 ± 4.18a
Different coefficient letters mean statistical differences by t-test, with Welch’s correction to compare resin
yields (α = 0.05).
The chemical profile of the labdanum resins and purified fractions was assessed by GC-
MS with a prior TMS derivatisation reaction of the mixtures (see methods). Fragment ion
73 m/z was used to confirm TMS derivatives (Harvey and Vouros 2020). Overall, extracts
chromatograms revealed 37 peaks (Figure 6.2.1, 6.2.2, 6.2.3, and 6.2.4) with a peak area
above 2% in relation to the major peak which depended on the extract (Table 6.2.2). Using
standards, eight peaks could be identified as typical labdane-type diterpenoids or
flavonoid aglycones. Using the NIST library, only 3-phenylpropanoid acid compound
could be identified as a TMS derivative with an excellent fragmentation pattern Match of
921 (peak 2, 9.64 min). Other compounds were identified using NIST library with a good
match (800-900) however most as methyl ester derivatives. Those compounds, identified
as methyl esters derivatives, presented ion 73 m/z which means that they are most likely
154
TMS derivatives. Interestingly, carboxylic acids (phenylpropanoids, fatty acids and

diterpenoid acids) presented ion at m/z 73 but alcohols (rhododendrol and flavonoids) did
not which is inconsistent with what is described by Harvey and Vouros (2020). In this
work methanol was used as solvent for the reaction which is not ideal because it is a protic
solvent and most probably reacted with the derivatizing agent. Therefore, the
derivatisation treatment of the extracts may have not be complete. Nevertheless, using
this procedure a well-resolved chromatogram was obtained in contrast to the
chromatogram of a non-derivatized extract, as it is shown for the Dit fraction (Figure
6.2.5 compared to 6.2.2). Furthermore, the most frequently reported labdane-type
diterpenes and flavonoid aglycones (Sosa et al. 2005; Alías et al. 2012) (section 1.2.2.2),
were successfully identified using standards.
In sum (Table 6.2.2), diterpenoid fraction (Figure 6.2.2) showed to be mostly composed
by labdane-type diterpenoid acids, flavonoid fraction (Figure 6.2.3) showed to be mostly
composed by methylated flavonoid aglycones with apigenin and kaempferol skeleton,
and Adl labdanum absolute (Figure 6.2.1) showed to be mostly composed by labdane-
type diterpenoid acids, fatty acids and jaranol. Zam labdanum absolute (Figure 6.2.4)
showed to be mostly composed by phenylpropanoids, fatty acids, and several non-
identified compounds, and to have a different chemical profile from the Adl labdanum
absolute.
Figure 6.2.1: GC-EI-MS chromatograms of the TMCS + BSTFA derivatized absolute of the labdanum resin
obtained by the Andalusian process (peak numbers according to Table 6.2.2).
155
Figure 6.2.2: GC-EI-MS chromatograms of the TMCS + BSTFA diterpenoid fraction of the labdanum resin
Figure 6.2.3: GC-EI-MS chromatograms of the TMCS + BSTFA flavonoid fraction of the labdanum resin
156
Figure 6.2.4: GC-EI-MS chromatograms of the TMCS + BSTFA derivatized absolute of the labdanum resin
obtained by the Zamorean process (peak numbers according to Table 6.2.2).
Figure 6.2.5: GC-EI-MS chromatograms of the non-derivatized diterpenoid fraction of labdanum resin
obtained by the Andalusian process.
157
Subchapter 6.2 Labdanum resin from Cistus ladanifer L. as a source of compounds with anti-diabetic, neuroprotective and anti-proliferative activity.
Table 6.2.2: GC-EI-MS relevant peaks in the chromatograms of the labdanum absolutes and fractions: relative quantification in relation to the major peak area (+: 2-10%; ++:
10-50%; +++: 50-90%; M: 90-100%), tentative identification based on Kovats retention index (RI) and fragmentation m/z- ions pattern compared to NIST2017 database
(Match), and identification using standards (grey).
Experimental NIST library / Standard Identification
Extract TMS
Peak RT (min) RI (i.u.) NIST Match Compound NIST RI (i.u.)
Adl Zam Flv Dit (73)
1 7.46 ± 0.02 1133 ± 1 + yes 886 ± 48 3-Acetoxy-3-hydroxypropionic acid, methyl ester
2 9.64 ± 0.03 1286 ± 2 + ++ yes 921 ± 48 3-Phenylpropanoic acid (hydrocinnamic acid), TMS 1279/1246
3 9.72 ± 0.02 1291 ± 1 ++ yes 814 ± 78 3-Methyl-hexanedioic acid, dimethyl ester 1285/1253
4 10.37 ± 0.00 1341 ± 0 + -
5 12.42 ± 0.10 1499 ± 8 ++ -
6 13.36 ± 0.02 1571 ± 2 ++ 872 ± 15 4-Hydroxy-α-methyl-benzenepropanol (rhododendrol) 1585
7 14.20 ± 0.07 1630 ± 4 ++ -
8 14.98 ± 0.13 1681 ± 8 +++ yes -
9 16.02 ± 0.28 1742 ± 16 M yes -
10 18.58 ± 0.02 1875 ± 1 + yes -
11 19.80 ± 0.03 1932 ± 1 ++ yes 885 ± 18 Hexadecanoic acid, methyl ester 1926/1909
12 24.58 ± 0.04 2135 ± 2 ++ + + yes 884 ± 40 Octadecanoic acid, methyl ester 2128/2110
13 26.48 ± 0.02 2212 ± 1 ++ yes -
14 26.59 ± 0.04 2216 ± 1 + -
15 26.81 ± 0.04 2225 ± 2 + -
16 27.00 ± 0.04 2233 ± 2 M ++ + + yes 862 ± 32 Labd-8(20)-en-15-oic acid, methyl ester
17 27.21 ± 0.05 2242 ± 2 +++ ++ + yes -
18 27.51 ± 0.04 2254 ± 2 + - -
19 27.91 ± 0.05 2269 ± 2 +++ ++ + + yes -
20 29.65 ± 0.04 2339 ± 1 ++ + + yes 837 ± 56 Eicosanoic acid, methyl ester 2329/2310
22 31.08 ± 0.01 2395 ± 0 + + M yes Labdanolic acid, TMS (standard)
23 32.87 ± 0.04 2467 ± 1 ++ ++ + ++ yes 6-oxo-labd-7-en-15-oic acid / 6-oxocativic acid, TMS (standard)
24 33.46 ± 0.01 2492 ± 0 + yes -
25 33.83 ± 0.01 2506 ± 0 + -
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Subchapter 6.2 Labdanum resin from Cistus ladanifer L. as a source of compounds with anti-diabetic, neuroprotective and anti-proliferative activity.
26 34.11 ± 0.00 2515 ± 0 + yes -

27 34.49 ± 0.05 2528 ± 2 + + yes -
28 34.62 ± 0.05 2532 ± 2 + yes 884 ± 1 Docosanoic acid, methyl ester
29 38.39 ± 0.03 2696 ± 2 + yes - -
30 38.97 ± 0.03 2723 ± 2 ++ yes 826 ± 4 Octadecanoic acid, 2,3-hydropropyl ester
31 39.99 ± 0.00 2767 ± 0 + -
32 43.72 ± 0.00 2934 ± 0 ++ Apigenin-4',7-dimethyl ether
33 44.40 ± 0.06 2963 ± 0 +++ Kaempferol-4',3,7-trimethyl ether (methyljaranol)
34 45.40 ± 0.01 * + Apigenin-4'-methyl ether (acacetin)
35 45.66 ± 0.01 * ++ Apigenin-7-methyl ether (genkawin)
36 46.10 ± 0.03 * ++ + M Kaempferol-3,7-dimethyl ether (Jaranol)
37 47.83 ± 0.01 * ++ Apigenin
Retention time (RT), experimental Kovats RI, and NIST Match presented as mean values ± standard deviation of all the extracts.
Relative quantification based of the mean values of triplicates for each extract.
*Retention time higher than for triacontane (C30).
159
6.2.3.2. Enzymatic inhibition activities
Concerning the inhibition of the enzymes involved in the starch breakdown which reduces
glucose availability to intestinal absorption was assessed. Inhibition of α-amylase and α-
glucosidase activity by labdanum absolutes and purified fractions is presented in Tables
6.2.3 and 6.2.4, respectively. Diterpenoid fraction was the extract with the highest α-
amylase inhibitory activity (around 40 and 30% inhibition at 1 and 0.5 mg/mL,
respectively) followed by Adl labdanum absolute. Flavonoid fraction presented low α-
amylase inhibitory capacity (≈ 4% at 1 mg/mL), and Zam labdanum absolute did no
inhibited the enzyme (Table 6.2.3). In contrast (Table 6.2.4), Zam labdanum absolute
produced the highest inhibitory effect on α-glucosidase activity (around 25% at 1
mg/mL), followed by Adl labdanum absolute (≈ 14% inhibition at 1 mg/mL) and
flavonoid fraction which showed similar inhibitory effect. Diterpenoid fraction showed
the lowest ability to inhibit α-glucosidase activity (≈ 6.5% inhibition, at 1 mg/mL). A
variety of fungi- and plant-based metabolites belonging to several classes of compounds,
including flavonoids and terpenoids, have shown α-amylase and α-glucosidase activity
(Taghouti et al. 2018; Vieira et al. 2019; Papoutsis et al. 2020). Acarbose is currently one
of the drugs used to control hyperglycaemia in individuals with type-2 diabetes by
competitively and reversibly inhibiting intestinal α-glucosidases, a family of enzymes
that hydrolyses oligosaccharides into glucose and other monosaccharides, and non-
reversibly inhibiting α-amylase, secreted by salivary glands and exocrine pancreas, that
cleaves complex polysaccharides (e.g., starch) into oligosaccharides (Standl et al. 2014;
Vieira et al. 2019). We observed that, acarbose inhibitory effect was higher against α-
amylase (77.17 ± 1.28% and 100% inhibition at 0.5 mg/mL and 1 mg/mL, respectively)
than against α-glucosidase (70.14 ± 2.57% and 80% inhibition at 0.5 mg/mL and 1
mg/mL, respectively), this inhibitory trend is reported by other authors (e.g. (Poovitha
and Parani 2016)). As observed, labdanum absolutes and fractions are not so effective in
inhibiting α-amylase (Table 6.2.3) or α-glucosidase (Table 6.2.4) as acarbose, however
this extract and fractions have potential as anti-diabetic agents, since the inhibitory effect
against α-amylase and α-glucosidase is much higher than that of medicinal and aromatic
plants commonly reported to have antidiabetic activities. These extracts (Table 6.2.3 and
6.2.4) showed higher or similar enzymatic inhibition than Thymus pulegioides L. aqueous
or hydroethanolic extracts (10% inhibition of α-glucosidase and none effect at α-amylase,
at 0.5 mg/mL) (Taghouti et al. 2018) or orange thyme extracts that at 0.5 mg/mL inhibited
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10% of α-amylase and 12% of α-glucosidase activity (Silva et al. 2021). Concerning
Cistus spp., hydromethanolic extracts obtained from Cistus salviifolius (L.) and Cistus
monspeliensis (L.) aerial parts showed very high α-glucosidase inhibitory activity (IC50 ≈
0.01 mg/mL, for both plants) and high α-amylase inhibitory activity (IC50 ≈ 0.6 and 0.7
mg/mL, for C. salviifolius and C. monspeliensis, respectively) (Sayah et al. 2017).
Aqueous infusion extracts of C. ladanifer were reported to inhibit α-amylase (IC50 ≈ 1.3
mg/mL), however these inhibitory assays (Lourenço 2016) were performed at different
temperature conditions from the ones here reported. These results indicate that labdanum
resin, in particular the diterpenoid fraction, has compounds with α-amylase activity
(Table 6.2.3) while the flavonoid fraction has compounds with higher activity against α-
glucosidase (Table 6.2.4).
Table 6.2.3: Inhibition of α-amylase activity (%) by the labdanum absolutes and fractions at concentrations
of 1 and 0.5 mg/mL.
α-Amylase inhibition (%)
Extract
1 mg/mL 0.5 mg/mL
Adl* 19.8 ± 0.9b 12.5 ± 1.6b
Zam n.a. n.a.
Dit* 40.0 ± 2.8a 29.8 ± 0.9a
Flv* 3.39 ± 0.05c 4.62 ± 0.10c
Dit and Flv are fractions of Adl absolute.
Different coefficient letters mean statistical differences between values within each column by the post-hoc
Dunnett’s T3 test (α = 0.05). Symbol (*) mean statistical difference between the two tested concentrations
for each extract by unpaired t-test with Welch’s correction (α = 0.05). n.a. means no activity compared to
the control.
Table 6.2.4: Inhibition of α-glucosidase activity (%) by the labdanum absolutes and fractions at
concentrations of 1 and 0.5 mg/mL.
α-Glucosidase inhibition (%)
Extract
1 mg/mL 0.5 mg/mL
Adl 14.0 ± 0.9a 13.1 ± 2.2a,b,c
Zam 24.1 ± 4.6a 13.7 ± 0.5a,b
Dit 6.4 ± 1.4b 4.3 ± 0.6c,d
a
Flv 15.4 ± 0.9 11.8 ± 5.3a,b,c
for each extract by unpaired t-test with Welch’s correction (α = 0.05).
AChE inhibition activity of labdanum absolutes and purified fractions is shown in Table
6.2.5. Adl labdanum absolute showed the highest inhibition effect (≈ 70% and 75%
inhibitory activity, at 0.5 and 1 mg/mL, respectively), but Zam labdanum absolute only
produced ≈ 22% and 7% AChE inhibitory effect at 1 and 0.5 mg/mL, respectively. Adl
labdanum fractions showed moderate and similar AChE inhibitory activities (Table
161
6.2.5), indicating that compounds present in both fractions have AChE target ability.
Around 10% (dw/dw) of the Adl labdanum absolute was lost during the purification of
diterpenoid and flavonoid fractions and GC-MS analysis revealed that there are
compounds present in significant amounts in Adl labdanum absolute and which are not
present in both purified fractions (between peak 13, at 26.48 min, and peak 31, at 39.99
min, Table 6.2.2), which may explain the higher activity of the absolute comparing to the
sum of fractions activity (Table 6.2.5). Several plant extracts and plant-derived
compounds have been shown to possess AChE inhibition activity, mostly alkaloid
compounds (Santos et al. 2018), and phenolic rich extracts, such as the aqueous and
hydroethanolic extracts of T. pulegioides that, at 0.5 mg/mL, inhibited ≈ 82% and 89%
AChE, respectively (Taghouti et al. 2018). Concerning Cistus spp., essential oils of five
species (Cistus creticus, Cistus salvifolius, Cistus libanotis, Cistus monspeliensis and
Cistus villosus) were reported to have AChE inhibitory activity with wide range of
activity (at 0.5 mg/mL ≈ 12% and 90% inhibition for C. creticus and C. libanotis,
respectively) (Loizzo et al. 2013). In this work, we report for the first time the inhibition
of AChE by a C. ladanifer extract, the labdanum resin (absolute and fractions), and thus
its neuroprotective potential (Table 6.2.5). Furthermore, since alkaloids are not reported
for this species, this activity may be associated to compounds belonging to the flavonoid
or terpenoid classes. In fact, compounds belonging to these classes of phytochemicals
have shown promising potential as AChE inhibitors (Khan et al. 2018; Lai Shi Min et al.
2022). Research on inhibitors of AChE is important for the development of drugs with
neuroprotective activity, since decreased or altered cholinergic function is observed in
many neurodegenerative disorders, such as Alzheimer’s disease, senile dementia,
Parkinson’s disease, and others (Moss 2020). Rivastigmine (based on physostigmine
chemical structure, from Physostigma venenosum) and galantamine (an alkaloid from
Galanthus woronowii) are among the most prescribed cholinergic enhancers, acting as
AChE inhibitors, and are derived from natural sources (Howes and Houghton 2012), thus
finding new AChE inhibitors is essential to new pharmaceutical drug development. AChE
inhibitors are also important, but to a lesser extent, for development of toxins useful for
agriculture, principally those selectively toxic for arthropods and not to vertebrates
(Pohanka 2012; Moss 2020).
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Table 6.2.5: Inhibition of acetylcholinesterase activity (%) by the labdanum absolutes and fractions at
concentrations of 1 and 0.5 mg/mL.
AChE inhibition (%)
Extract
1 mg/mL 0.5 mg/mL
Adl* 75.1 ± 0.9a 69.8 ± 0.6a
b,c,d
Zam 21.2 ± 6.9 7.6 ± 1.3c,d
b,d
Dit* 35.3 ± 0.4 24.1 ± 0.5b,c
c,d
Flv 22.9 ± 2.6 22.9 ± 5.1b,c,d
for each extract by unpaired t test with Welch’s correction (α = 0.05).
6.2.3.3. Cytotoxic activity
Anti-proliferative or cytotoxic activity of labdanum absolutes and fractions was

performed against two relevant cell lines, HepG2 (human hepatocellular carcinoma cell
line) and Caco-2 (human colorectal adenocarcinoma cell line), namely if medicinal
preparations are aimed for ingestion purposes, as Caco-2 cells represent the absorptive
enterocytes and HepG2 cells the first pass hepatocytes. Caco-2 and HepG2 cells were
exposed to different concentrations of both labdanum absolutes (Adl and Zam absolutes)
and of Adl fractions (Dit and Flv fractions) for 24 h and 48 h, and results as shown in
Figure 6.2.6 and the IC50 values, i.e., concentration that inhibits 50% of cell
viability/proliferation, is shown in Table 6.2.6. Cell viability data versus extracts
concentration were plotted and four-parameter symmetric sigmoid curves were fitted as
the regression model (Figures 6.2.7 and 6.2.8 for Caco-2 and HepG2 cells, respectively).
Top and bottom plateau parameters of the sigmoid curves were fixed at 100 and 0%,
because of the use of sample blanks and of a control. The Hill slope parameter and the R2
of the sigmoid curves are presented in Table 6.2.7. The mid-range point parameters,
which corresponds to the IC50 of the assays, are presented in Table 6.2.6. M3P absolute
presented no cytotoxicity at the concentrations tested (25-200 µg/mL) so the sigmoid
regressions could not be projected. Regarding the other extracts, sigmoid regressions
presented a R2 higher than 0.9 at all conditions, considering them a good fit to extract the
IC50 parameter.
163
Caco-2 HepG2
#
Cell viability (% of control)

#
100 100
* #
*
*
#
Adl
#
50 * 50 *
*
* #
#
24 h * 24 h
* *
* * 48 h
* 48 h
0 0
Cont. 25 50 75 100 200 Cont. 25 50 75 100 200
Concentration (g/mL) Concentration (g/mL)
* * * #
*

#
100 * 100
*
#
*
*
#
Flv
50 * 50
* *
#
24 h 24 h
* *
* 48 h * * * 48 h
0 0
Cont. 25 50 75 100 200 Cont. 25 50 75 100 200
# #
#
100 * 100
* *
* *
*
Dit
50 #
50
#
* #
* #
* 24 h * 24 h
* *
* 48 h * * * 48 h
0 0
Cont. 25 50 75 100 200 Cont. 25 50 75 100 200
100 100
Zam
50 * 50 *
* *
24 h 24 h
* *
48 h 48 h
0 0
Cont. 25 50 75 100 200 Cont. 25 50 75 100 200
concentration (g/mL) Concentration (g/mL)
Figure 6.2.6: Effect of labdanum resin extracted by the Andalusian process (Adl), its purified flavonoid
(Flv) and diterpenoid extracts (Dit), and labdanum resin extracted by the Zamorean process (Zam) on Caco-
2 (Human epithelial colorectal adenocarcinoma) and HepG2 (Human hepatocellular carcinoma) cells
viability (% of control) after 24 and 48 h incubation (3 independent experiments of n = 4 each). * Means
statistical differences in relation to the control by the post-hoc Tukey’s test (α = 0.05). # Means statistical
differences between incubation times by the post-hoc Tukey’s test (α = 0.05).
As observed in Figure 6.2.6, Zam labdanum absolute did not show cytotoxic activity
against both cell lines at the maximum tested concentration (200 µg/mL). On the other
164
hand, Adl labdanum resin and its purified diterpenoid and flavonoid fractions presented
dose-dependent cytotoxicity against both cell lines (Figure 6.2.6, 6.2.7, and 6.2.8). At 100
µg/mL, Adl labdanum resin and fractions, reduced cell viability to values below 30% of
control, independently of the cell line, incubation time, and extract (Figure 6.2.6).
Regarding the IC50 parameter, it is evident that Caco-2 cells are more susceptible than
HepG2 cells to all extracts (Table 6.2.6). In general, at 24 h exposure the diterpenoid
purified fraction presented on average higher cytotoxicity against Caco-2 and HepG2
cells, as indicated by the lower IC50 values. Although, at 48 h incubation, the cytotoxic
activity of Adl labdanum resin was on average higher (lower IC50), against both cell lines,
the cytotoxic activity was not statistically different (ρ ≥ 0.05) from Flv and Dit, for HepG2
cells, or from the Dit, for Caco-2 cells, fractions. Considering that Caco-2 cell line is
commonly used as model of the intestinal epithelial barrier, because it retains typical
properties of absorptive enterocytes (Lea 2015), and that HepG2 cell line is used as model
to predict hepatotoxic potential of substances, because hepatocyte features and properties
are maintained (Tham et al. 2019), given the results obtained (Figure 6.2.6 and Table
6.2.6), labdanum resin is potentially toxic to the gastrointestinal tract if ingested and to
the liver if absorbed. However, having in account that these are cancer cell lines, this
cytotoxic/anti-proliferative activity could be regarded as a cytostatic action, and thus
these extracts and their individual compounds (mainly composed of methylated flavonoid
aglycones and labdane-type diterpenoids) are worth to be studied in the future as anti-
cancer agents.
Table 6.2.6: IC50 (µg/mL) of labdanum absolutes and fractions in relation to HepG2 and Caco-2 cell
viability at 24h and 48h incubation.
HepG2 Caco-2
Extract
24h 48h 24h 48h
Adl 77.4 ± 3.9b C 48.3 ± 1.2a B 52.8 ± 4.3a B 36.7 ± 2.0a A
Zam > 200 > 200 > 200 > 200
Dit 60.3 ± 2.3a B 53.8 ± 4.1a B 44.6 ± 4.5a A 38.7 ± 2.0a A
Flv 76.7 ± 3.0b C 52.8 ± 1.2a A 69.5 ± 2.1b B 48.3 ± 2.7b A
Value presented as mean ± standard deviation (3 independent experiments of n = 4).
Different coefficient small or capital letters mean statistical differences between mean values within each
column or row, respectively, by the post-hoc Tukey’s test (α = 0.05).
165
Figure 6.2.7: Four-parameter symmetric sigmoidal curves of Caco-2 cell viability, as percentage in relation
to the control (0.00 µg/mL), versus Adl labdanum absolute, and its flavonoid (Flv) and diterpenoid (Dit)
fractions, concentration at 24h and 48h incubation (Regression parameters in Table 6.2.7) (3 independent
experiments of n = 4).
Figure 6.2.8: Four-parameter symmetric sigmoidal curves of HepG2 cell viability, as percentage in relation
to the control (0.00 µg/mL), versus Adl labdanum absolute, and its flavonoid (Flv) and diterpenoid (Dit)
fractions, concentration at 24h and 48h incubation (Regression parameters in Table 6.2.7) (3 independent
experiments of n = 4).
Table 6.2.7: R-squared and Hill slope of the best fitted symmetric sigmoidal curves to the HepG2, and
Caco-2 cell viability versus labdanum absolutes, and its Flv and Dit fraction, concentration data at 24h and
48h incubation.
Cell Extract Incubation R2 Hill Slope
24h 0.9574 -3.020 ± 0.706
Adl
48h 0.9883 -3.246 ± 0.419
24h 0.9464 -4.216 ± 1.533
Caco-2 Dit
48h 0.9904 -5.223 ± 0.849
24h 0.9843 -6.576 ± 1.168
Flv
48h 0.9755 -6.034 ± 2.667
24h 0.9335 -5.951 ± 1.858
Adl
48h 0.9942 -6.001 ± 1.221
24h 0.9817 -7.776 ± 1.422
HepG2 Dit
48h 0.9178 -9.707 ± 8.163
24h 0.9465 -8.959 ± 3.892
Flv
48h 0.9934 -10.110 ± 3.440
Values presented as mean ± standard deviation (3 independent experiments of n = 4)
At each data point, top and bottom plateaus were set at 100 and 0%, respectively.
Zam resin without cell viability reduction activity.
To the best of our knowledge, this is the first work reporting the anti-proliferative activity
of Cistus ladanifer labdanum resin absolute and of its purified fractions. However,
Barrajón-Catalán et al. (2010) reported the cytotoxicity effect of hot-water extract from
milled shoots of C. ladanifer against several human cancer cell lines, showing IC50 values
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between 0.49 and 16.10 mg/mL, which are much higher than those obtained in this study
(Table 6.2.6) which were lower than 0.1 mg/mL. These differences might be correlated
with the extract composition, as hot-water extract is rich in polyphenols, flavonoids and
tannins (Barrajón-Catalán et al. 2010), and labdanum is also rich in diterpenoids (Table
6.2.2) with cytotoxic activity (Table 6.2.6, and Figure 6.2.6). Gaweł-Bęben et al. (2020)
reported that C. ladanifer aerial parts extracted with methanol (at different proportions
with water) gave extracts with cytotoxic activity against A375 (human malignant
melanoma), with IC50 values between 100 - 200 µg/mL, depending on the extract, but
were ineffective against ACC-15 (human squamous cell carcinoma) cells, IC50 > 500
µg/mL, which were higher than the results shown in this study (Table 6.2.6). Cistus
ladanifer extracts (acetone/water and ethanol) only slightly affected skin fibroblast
viability (Andrade et al. 2009). Extracts from other Cistus spp. were reported to have anti-
proliferative activity, as is the case of the ethanol extract of Cistus creticus subsp. creticus
L. shoots that was cytotoxic on HeLa (cervix carcinoma), MDA-MB-453 (breast apocrine
carcinoma) and FemX (melanoma) cancer cells with IC50 reaching 80.83 g/mL, 76.18
g/mL, and 87.52 g/mL, respectively (Skorić et al. 2012), values that are identical to
those obtained in HepG2 cells at 24 h exposure (Table 6.2.6). Cistus incanus L. extracts,
obtained with different organic solvents, rich in polyphenols were tested against HCT116
(human colon adenocarcinoma) cells showing IC50 values between ≈ 140 g/mL and 400
g/mL, depending on the extract (Szeremeta et al. 2018), that although not so effective
as the extract here obtained (Table 6.2.6) corroborates the anti-proliferative/cytotoxic
activity against colon cancer. This comparison with literature suggests that Adl labdanum
absolute, but not Zam labdanum absolute, has higher cytotoxic activity than C. ladanifer
whole plant extracts (water or organic solvent extracts). It is worth to emphasize that the
IC50 values here obtained for Adl resin absolute and its fractions, according to NCI
guidelines, are considered moderately active IC50 0.02 - 0.20 mg/mL, as anti-cancer
agents. However, to evaluate the activity against cancer proliferation, besides the
cytotoxic activity against cancer cell lines it should be also evaluated such bioactivity
against normal cells as shown in Andrade et al. (2009) and Gaweł-Bęben et al. (2020)
studies.
6.2.4. Conclusion
From this work we conclude that the Andalusian process of labdanum resin extraction
rendered higher labdanum resin yields than the Zamorean process. In addition, the two
167
resins showed different chemical profile by GC-EI-MS analysis, which indicates the loss
of relevant bioactive compounds during the Zamorean process. Labdane-type
diterpenoids and methylated flavonoid aglycones, already reported in literature, were
identified using standards (mostly in the Andalusian labdanum resin), but several
compounds remained unidentified for both resins. Using relevant enzymatic inhibition
assays to confirm traditional medicinal uses, such as hyperglycaemia control and the
treatment of mental disorders, labdanum resin, mainly the one extracted by Andalusian
process, is worth to be further studied as a medicinal product or a source of
pharmaceuticals ingredients with potential for anti-diabetic and neuroprotective
activities, as the labdane-type diterpenoids revealed potential as α-amylase inhibitors and
the overall Andalusian labdanum resin revealed potential as a source of
acetylcholinesterase inhibitors. Given the high cytotoxic activity against Caco-2 and
HepG2 cells, the gastrointestinal and hepatic toxicity is worth to be considered for
labdanum resin and its products. However, this cytotoxic activity may also indicate that
the labdanum diterpenoids and flavonoids have potential anti-proliferative activity that
are worth to explore for pharmaceutical applications.
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Chapter 7
7.1. General discussion
In Chapter 2, the same management practice was applied, for two consecutive years, to a
5-years-old natural C. ladanifer shrubland (subchapter 2.1) and to a younger 1-year-old
cultivated C. ladanifer shrubland (subchapter 2.2). Regarding harvest periodicity
modalities, the studies revealed different but complementary conclusions. After the initial
cut of the 5-years-old shrubland, every year harvest was ideal to maximize photosynthetic
biomass and labdanum resin production whilst a harvest after two years was ideal to
maximize capsules/seeds production. After cultivation, the 1-years-old plants, harvest
after two years was ideal to maximize photosynthetic biomass, labdanum resin, and
capsules/seeds production. As shown by Nuñez et al. (1989) and Morgado et al. (2005)
the production of aboveground photosynthetic biomass tends to stabilize after a certain
age of the shrubland and although its percentage in relation to total aboveground biomass
may be higher for young shrublands its total aboveground production is lower. In this
study, the shrubs were cut at a height of 0.5 m and for the 5-years-old natural shrubland
the production of photosynthetic biomass per year, in 1Y, and after two years (2Y) was
similar. It means that the photosynthetic biomass produced in one year of growth
generated wood biomass during the next year of growth if not harvested. In the first year
of harvest of the 5-years-old shrubland a significant amount of wood biomass was
harvested but in the next year it was not. In contrast, for the 1-year-old cultivated
shrubland wood biomass was only harvested after two years of continuous growth (3-
years-old shrubland) but photosynthetic biomass production was similar between
shrubland lines harvested also at the first year of growth and shrubland lines harvested
after two years of growth. In addition, the photosynthetic biomass production after the
first year of growth in the field, despite representing 100% of the total harvested biomass,
was ten-fold lower than the production of the following year. It was also on the second
year of growth that shrubland lines started to form a continuous hedge, meaning that
individual plants reached at least a 0.5 m of cover diameter (0.20 m2 of cover area). To
harvest at 0.5 m height, the plant must then reach a certain age and development, such as
3-years-old or when wood biomass reaches the 0.5 m height.
A management practice through harvesting of natural shrublands is justified to reduce

environmental problems such as fire risks and, at the same time, to generate value or
169
Chapter 7 General discussion
income from such abandoned forestry areas. An advantage of exploiting natural

shrublands is the low cost “installation” of the productive field, however a disadvantage
is the contamination of harvest biomass with other plant species, increasing post-harvest
separation costs.
The cultivated shrubland presented a photosynthetic biomass production at 3 years-old of

around 5500 kg/ha, whereas the 5 years-old natural shrubland presented a production of
around 1500 kg/ha at the initial and subsequent harvests (non-accumulated). Capsules
production at the end of two years of growth was 280 kg/ha for the cultivated shrubland
and was around 322 kg/ha for the natural shrubland at the initial harvest and around 259
kg/ha after two subsequent years of growth. The cultivated shrubland had a plant density
of 2 plants/m2 whereas the natural shrubland had a close plant density but highly variable
(1.62 ± 1.17 plants/m2) which can justify in part the different productivities. Cultivated
shrubland have the advantage to facilitate mechanized harvesting with low contamination
of harvest biomass with other plant species, the higher productivities, and the possibility
to plant genotypes with specific traits. However, cultivation involves higher initial costs,
namely plant propagation and nursery and planting. Cistus ladanifer cultures may be
useful for ornamental purposes or to recuperate oligotrophic soils and/or produce non-
contaminated biomass in heavy metals contaminated soils. Vallejo and Alloza (2019)
discuss strategies for post-fire land restoration in the Mediterranean Basin, highlighting
the advantage of sclerophyllous resprout species (such as Quercus species) compared to
obligate seeders, as it is the case of Cistus ladanifer, to provide protection to soil shortly
after a fire. According to Vallejo and Alloza (2019) never cultivated oak forests are
reference ecosystems for restoration whereas seeders shrubland communities are often
associated to short-term fire cycles and ecosystem degradation loops. However, in this
work we have shown that proper management of C. ladanifer shrubland reduce the
production of wood biomass, fuel biomass for fire ignition, while maintaining soil cover
and generating income. In addition, Quercus species such as cork and holm oaks species
were present within the shrubland, which means that if properly managed that shrubland
may succeed into an oak forest.
Fort the purpose of Cistus ladanifer cultivation, the selection of genotypes with improved
characteristics (e.g., biomass production, metal excluder, resin productivity,
capsules/seeds productivity) is advantageous and therefore there is a need for efficient
clonal propagation practices. To obtain plants for the cultivation experiment, a first
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preliminary attempt on C. ladanifer clonal multiplication was made by vegetative

propagation (data is not shown within this thesis) of terminal 3/4 nodes cuts with or
without auxin (IBA) supplementation during spring of 2018, but, after two months, high
mortality (above 50%) was registered, although roots formation was high in viable cuts
(above 75%). Given the high mortality in the first experiment of vegetative propagation
and the absence of sufficient literature regarding vegetative propagation of this plant
species, plants for the cultivation experiment were later obtained by seed germination and
growth directly on substrate in the greenhouse for one year. At the same time of vegetative
propagation experiment, basal leaf and internodal stem explants from C. ladanifer were
demonstrated to be suitable for C. ladanifer clonal propagation through shoot
organogenesis from the explants under the influence of the cytokinin BAP (Chapter 3).
However, shoots were regenerated only in around one third of viable stem explants. In
addition, leaf and stem explants were also able to produce two types of calli, rhizogenic
light green compact calli and dark green more compact calli most likely with potential
for shoot regeneration, that can be used for in vitro metabolite manipulation and
production systems and to increase C. ladanifer multiplication rates via calli mass
propagation and organogenesis. Micropropagation is more expensive than vegetative
propagation of plant cuts, however it has the advantages of being able to produce larger
numbers of homogeneous plants at any time, to generate disease-free and aseptic plants
and to enhance multiplication rates.
In this thesis, an integrative exploitation of C. ladanifer is proposed (section 1.3) and the
evaluation of the nutritional value of seeds and cosmetic/pharmaceutical value of
labdanum resin were considered an obvious gap.
Capsules production was estimated to reach 259 ± 115 kg/ha for a 5-years-old shrubland
cut at 0.5 m and grown for two consecutive years and 284 ± 37 kg/ha for a 3-years-old
cultivated shrubland with close plant density. Seeds represented between 25 and 30% of
capsules weight meaning a production of around 80 kg/ha for both shrublands. Those
seeds are here proposed to be valued as food because of their traditional use and their
high nutritional value. Cistus ladanifer may have a role and contribute to resilient food
systems given its abundance as a resource and resilience to unfavourable conditions for
plant growth and ultimately to upcoming environmental changes (Alae-Carew et al.
2020). The remaining weight of capsules (70 – 75%) is significant and should be valued.
In (section 1.3) it is discussed the use of capsules as feed, for energy and for chemicals
171
extraction, but studies regarding these applications are scarce and missing in what regards
seedless remaining portions of capsules.
Photosynthetic biomass production was estimated to reach 1747 ± 346 kg/ha for a 5-
years-old shrubland cut at 0.5 m and grown for two consecutive years, 2902 ± 705 kg/ha
for a 5-years-old shrubland cut at 0.5 m and harvested during two consecutive years, and
5325 ± 902 kg/ha for a 3-years-old cultivated shrubland with close plant density. From
photosynthetic biomass harvested in the natural shrubland, labdanum resin extraction
yield was between 6.02 and 10.50% (dw/fw), reaching a maximum production of 230
kg/ha at the end of two years, when harvest was done yearly at late summer, and half that
value, when harvesting was done after two years of growth or considering yearly
production. Using the same labdanum resin yields, cultivated shrubland would produce
between 320 and 559 kg/ha of labdanum resin at the third year.
For simplification purpose, in this study, resin extraction processes using alkaline
extraction, regardless temperature of extraction and acidification extent, were considered
Andalusian processes (although the typical Adl process involves extraction at around 50-
60oC and acidification until neutralisation of carbonates), and resin extraction processes
using water extraction, regardless temperature of extraction and acidification extent, were
considered Zamorean processes (although typical Zam process involves boiling water,
i.e. 100oC, and no acidification). Using the same method (extraction with 25 g/L Na2CO3
at 60oC and acidification until pH 2), the Adl process yielded 6.02 – 10.50% (dw/fw,
subchapter 2.1), 8.82% (dw/fw, chapter 5), 7.44% (dw/fw, subchapter 6.1), and 5.79%
(dw/fw, subchapter 6.2) along this thesis (6 – 11% dw/fw). As demonstrated in chapter 5,
the typical Zam process yields much lower quantity of labdanum resin (around 1%
dw/fw). In subchapter 6.2, Zam resin was extracted at 60oC with acidification until pH 2,
yielding 0.2% (dw/fw) of resin which is consistent to the yield obtained in chapter 5 for
the same method (0.3% dw/fw) but lower than the typical Zam process yield.
Additionally, Adl resin (constituted mostly by flavonoid aglycones and labdane
diterpenoids) showed a different chemical profile than Zamorean resin (constituted
mostly by phenylpropanoids, fatty acids, and labdane diterpenoids) by GC-EI-MS
analysis (subchapter 6.2)
Andalusian resin is nowadays commercialised for its aromatic and aroma fixative
properties in cosmetics and other products (Biolandes n.d.) but, in this thesis, based on
traditional medicinal uses and ecological functions, it is proposed to be further valorised
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as a cosmetic ingredient, mainly because of the UV protection activity and as a

dermocosmetic or topical medicine, mainly because of the anti-inflammatory activity.
Zamorean resin was also evaluated for some relevant properties for cosmetics
valorisation, but results are not presented in subchapter 6.2. Regarding SPF, Zam resin
presented a highest value (7.60 ± 1.59 SPF) than Adl resin (4.98 ± 0.19 SPF) but lower
than the Flv fraction (13.0 ± 1.4 SPF). Regarding elastase inhibition, Zam resin presented
a similar value to Adl resin at 1 mg/mL (20.09 ± 3.30 and 21.96 ± 0.66% inhibition,
respectively) but higher value at 0.5 mg/mL (9.81 ± 0.66 and no activity, respectively).
Regarding antioxidant activity, Zam resin presented higher values than Adl resin, and its
Flv and Dit fractions, namely for Folin-Ciocalteu (0.27 ± 0.06 mgGAE/mgExt), DPPH
radical scavenging (1.32 ± 0.14 mgTE/mgExt), FRAP (0.85 ± 0.20 mgTE/mgExt), and
ABTS radical scavenging (0.73 ± 0.07 mgTE/mgExt). However, Zam resin showed no
anti-inflammatory activity in the range of concentrations tested (5 – 15 µg/mL) contrary
to Adl resin and its purified fractions. These results confirm the different chemical
constitution of Zam resin compared to Adl resin. Zam resin showed superior properties
for a cosmetic ingredient (SPF and antioxidant activity) but inferior anti-inflammatory
activity (cosmeceuticals). In addition, the lower extraction yield is a disadvantage
compared to Adl resin. In fact, Zam resin phytochemicals (phenylpropanoids and fatty
acids) may constitute an active fraction also present in Adl resin however diluted by the
presence of other phytochemicals (such as flavonoids and diterpenoids). In subchapter
6.2, both resins were evaluated for the medicinal properties (others than anti-
inflammatory) and as a source of pharmaceutical compounds, mainly because of the
potential anti-hyperglycaemia, neuroprotective, and anti-cancer activities. Only regarding
α-glucosidase inhibition and safety (no cytotoxicity), Zam resin was superior compared
to Adl resin.
In section 1.3, it is discussed that labdanum resin, labdanum oil, and a polar extract could
be obtained simultaneously. In fact, it is shown in this thesis that the residual water of the
labdanum extraction process is rich in phenolic compounds (Folin-Ciocalteu: 2-12
gGAE/L, depending on extraction conditions) that most probably matches the compounds
of polar extracts described in literature. Labdanum oil may be extracted directly from
labdanum resin by distillation. However, such process may deteriorate labdanum
components, because of the high temperatures, and lead to the loss of active properties.
173
To overcome this problem, supercritical CO2 extraction may be an alternative as it has

been already used to extract C. ladanifer essential oil (Rincón et al. 2007).
After extraction of labdanum resin (and other extracts), photosynthetic biomass remains
as a by-product which may be used as forage for ruminants, source of feed additives,
source of high-grade cellulose and a source of chemicals for several industries after
bioconversion (section 1.3). If harvesting of C. ladanifer is done as proposed in this thesis
(Chapter 2), at a defined height every year, photosynthetic biomass will be the only by-
product. In contrast, if harvesting of C. ladanifer is done once every two years at a defined
height or destructively harvested (total clearings at the soil level) a wood biomass must
also be considered. This biomass may be used as a feedstock for biorefinery processes to
extract chemicals or used directly to produce energy, for example, in the form of heat
through pellets and charcoal (section 1.3) which in turn is necessary to extract labdanum
resin.
174
7.2. General conclusion
In this work, several topics of different scientific areas were studied around Cistus
ladanifer. The goal was to address existing gaps regarding C. ladanifer exploitation, from
production to valorisation of products or raw materials, in order to contribute with
knowledge for an integrative exploitation of such resource and for the development of a
sustainable value chain based on such abundant resource. The main conclusions of this
work are:
• Cistus ladanifer shrublands, natural or cultivated, may be properly managed in a

non-destructive way to yield photosynthetic biomass, labdanum resin, and
capsules/seeds.
• For young shrublands, natural or cultivated, the initial harvest at a more mature
stage (3-years-old in this study) is ideal to maximize production (photosynthetic
biomass, labdanum resin, and capsules) and ornamental attributes (alternative
hedges with native flowering).
• For young mature C. ladanifer shrublands (5-years-old), natural or cultivated, the

periodic harvest at 0.5 cm must be considered a good management practice,
reducing fire risk, maintaining soil cover and enrichment, and increasing profit
for producers. If the goal is to extract resin the harvest must be done every year.
If the goal is to obtain seeds, extract resin, and increase ornamental attributes
(flowering) the harvest must be done every two years, and in this case some wood
biomass will also be harvested.
• Harvest must be done between middle and late Summer, to maximize labdanum
resin production.
• Leaf and internodal stem explants from C. ladanifer showed to be suitable starting
material for clonal propagation of selected genotypes for cultivation, once shoots
were regenerated from the explants under the influence of BAP.
• Leaf and internodal stem explants from C. ladanifer showed to be suitable for cell
in vitro cultures that may be used to increase propagation rates and to produce
secondary metabolites, once two types of calli most likely with different features
were produced under the influence of 2,4-D or BAP.
175
Chapter 7 General conclusion
• Seeds from C. ladanifer, intended for human consumption, have an intermediate

macronutrient composition between the carbohydrate rich cereals and lipid rich
nuts, constituting a source of protein and calcium, and a high source of fibre,
unsaturated fatty acids, and several minerals.
• Regarding macronutrient composition, C. ladanifer seeds stand close to flax and

chia seeds and, regarding mineral composition, C. ladanifer seeds stand close to
pine nuts.
• The Andalusian labdanum resin extraction process (alkaline extraction followed

by acidic precipitation) renders higher labdanum resin yields than the Zamorean
process (aqueous extraction followed by decantation).
• The Andalusian process produces a residual water with higher salinity but with
similar phenolic content compared to the Zamorean process.
• Regardless the extraction process (Andalusian or Zamorean), higher temperatures

of extraction resulted in higher phenolic content in the residual water.
• Labdanum absolute represents around 70% of labdanum resin weight, extracted

by both Andalusian and Zamorean processes.
• Andalusian labdanum absolute is mainly composed by labdane-type diterpenes

(around 75%) and flavonoids (around 15%).
• Zamorean labdanum absolute is mainly composed by phenylpropanoids and fatty

acids. However, labdane-type diterpenes and flavonoids were also identified.
• Andalusian absolute showed to possess bioactive properties relevant for

formulation of cosmetics and/or dermocosmetics/topical medicines products,
such as anti-inflammatory, anti-aging and neuroprotective effects, but showed
weak preservative activity.
• Andalusian absolute showed some antioxidant activity and narrow antimicrobial

activity towards Staphylococcus aureus.
• Flavonoid aglycones are the most responsible compounds for the UV-protection
and anti-inflammatory activity of labdanum resin.
176
• Labdane-type diterpenes are the most responsible compounds for the α-amylase
inhibition activity (antidiabetic/antihyperglycemic property).
• Andalusian labdanum absolute showed high acetylcholinesterase inhibition

(neuroprotective property) but such activity could not be clearly addressed to
labdane-type diterpenoids or flavonoid aglycones.
• Andalusian labdanum absolute showed potential gastrointestinal and hepatic

toxicity and potential anticarcinogenic activity. Both labdane-type diterpenoids
and flavonoid aglycones seemed to be responsible for such activities.
• Labdanum resin and its fractions are composed of relevant molecules that could
be exploited by cosmetic and pharmaceutical industry and thus is worth of
investment.
177
178
7.3. Future perspectives
Future perspectives on management of Cistus ladanifer shrublands:
• It is relevant to replicate the management study (periodic harvests) on shrublands

with different ages and plant densities and longer harvest periodicities such as 3
and 4-years may be considered to evaluate if survival, rejuvenation, and
production occurs similar to the 5-years-old shrubland used in this study.
• Periodic harvests should be done for a longer period of study to evaluate when
production stabilizes (cultivation experiment) and if it is maintained (natural
shrubland experiment).
• Selection C. ladanifer genotypes with specific traits such as biomass production,

resin production and quality, capsules/seed production, and heavy metal
excluding and extracting ability is relevant for cultivation purposes.
• Transposition of the cultivation experiment to oligotrophic and trace-element

contaminated soils, assessing soil recuperation and protection, productivity, and
quality of products.
• Evaluation of the possibility to associate C. ladanifer management and

exploitation with the stimulation and maintenance of forestry or agro-forestry
systems succession and maintenance.
• Development or experimentation of suitable mechanized harvest systems for

cultures and for natural shrublands.
Future perspectives for in vitro tissue culture of Cistus ladanifer:
• Evaluation of the effect of other cytokinins than BAP and different concentrations
in shoot organogenesis from basal leaf and internodal stem explants.
• Optimisation of propagation/maintenance of dark green compact callus and

evaluation of their ability to regenerate into shoots via organogenesis, as it was
done for the rhizogenic light green calli.
• Evaluation of the suitability of the two calli lines for liquid cultures and synthesis
of secondary metabolites.
Future perspectives to the study of the nutritional value of Cistus ladanifer seeds:
179
Chapter 7 Future perspectives
• Evaluation of amino acids, vitamins, and phenolic profile and content, and of
other fat compounds such as sterols.
• Although they had been traditionally consumed by humans and animals,

toxicological studies could be beneficial and relevant to safeguard consumers.
• The remaining part of capsules after seeds separation should be studied for its
application for energy generation, for feed utilisation and for chemicals extraction
to valorise it as a by-product of seeds production.
Future perspectives to improve labdanum resin extraction:
• Chemical characterisation of the residual water to evaluate if it has a similar

profile to polar extracts reported in literature, to valorise it as a by-product and
minimize treatment costs of the aqueous effluent.
Future perspectives on labdanum resin quality assessment:
• Further characterize the chemical composition of labdanum by compounds

isolation and structure elucidation through more advanced techniques because
several compounds remained to be identified in this study.
• Improve GC-MS analysis of the labdanum resin through derivatisation of low

volatile compounds.
• Improve HPLC and/or HPLC-MS analysis since the method used in this study
failed to identify phenylpropanoids and fatty acids identified by GC-MS.
• Evaluate the biological activities of the major isolated compounds and their
potential synergism and antagonism.
• Study the stability, degradation/transformation, and behaviour of labdanolic acid,

a major compound in labdanum resin since its content was inconsistent when
comparing labdanum absolute to the purified diterpene fraction.
Future perspectives on labdanum resin valorisation:
• Formulate a topical product with labdanum resin, perform skin toxicity studies
and evaluate cosmetic/dermocosmetics relevant properties with more suitable and
approved methods.
180
Future perspectives on overall Cistus ladanifer valorisation:
• Development and analysis of a complete value chain for Cistus ladanifer, using
the current knowledge on its exploitation.
181
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UNIVERSIDADE DE TRÁS-OS-MONTES E ALTO DOURO

Valorisation of Cistus ladanifer L.: From production to evaluation of

- Agricultural Production Chains - From Fork to Farm -

DAVID PASSOS MORGADO FRANCO FRAZÃO

Professor Doctor Amélia Maria Lopes Dias da Silva (UTAD)

Vila Real, 2022

Valorisation of Cistus ladanifer L.: From production to evaluation of

- Agricultural Production Chains - From Fork to Farm -

DAVID PASSOS MORGADO FRANCO FRAZÃO

Professor Doctor Amélia Maria Lopes Dias da Silva (UTAD)

Vila Real, 2022

Additionally, this Doctoral work was partially funded by CENTRO-01-0247-FEDER-

Cistaceae; Cosmetic Labdanum; Medicinal Labdanum; Rockrose edible seeds; Rockrose

Cistaceae; Cultura de esteva in vitro; Lábdano cosmético; Lábdano medicinal; Gestão do

1.1. Cistus ladanifer L. .......................................................................................... 1

1.1.1. Taxonomy ............................................................................................... 1

1.1.2. Distribution ............................................................................................. 5

1.1.3. Morphology and phenology .................................................................... 6

1.1.4. Ecology ................................................................................................... 9

1.1.4.1. Reproduction ...................................................................................... 9

1.1.4.2. Nutritive demands ............................................................................ 11

1.1.4.3. Shrublands ........................................................................................ 12

1.1.4.4. Labdanum resin exudate .................................................................. 14

1.1.4.5. Pests and diseases............................................................................. 16

1.2. Cistus ladanifer L. raw materials (products). ............................................... 17

1.2.1. Essential oil ........................................................................................... 17

1.2.2. Labdanum resin ..................................................................................... 19

1.2.2.1. Extraction ......................................................................................... 19

1.2.2.2. Chemical characterisation ................................................................ 22

1.2.3. Polar extracts ......................................................................................... 27

1.2.4. Biomass ................................................................................................. 29

1.4. Research objectives ...................................................................................... 43

Subchapter 2.1 ................................................................................................................ 45

2.1.1. Introduction .............................................................................................. 47

2.1.2. Material and methods ............................................................................... 50

2.1.2.1. Characterisation of the study area ..................................................... 50

2.1.2.2. Experimental plots delimitation and evaluation ................................ 51

2.1.2.3. Soil analysis....................................................................................... 51

2.1.2.4. Soil seed bank evaluation .................................................................. 51

2.1.2.5. Experimental design .......................................................................... 52

2.1.2.6. Phenological evaluation .................................................................... 53

2.1.2.7. Meteorological data ........................................................................... 53

2.1.2.8. Harvest evaluation ............................................................................. 53

2.1.2.9. Labdanum resin extraction ................................................................ 54

2.1.2.10. Statistical analysis ............................................................................. 54

2.1.3. Results and discussion .............................................................................. 55

2.1.3.1. Initial characterisation ....................................................................... 55

2.1.3.2. Phenological evaluation .................................................................... 56

2.1.3.3. Production ......................................................................................... 62

2.1.4. Conclusion ................................................................................................ 68

Subchapter 2.2 ................................................................................................................ 69

2.2.1. Introduction .............................................................................................. 71

2.2.2. Material and Methods ............................................................................... 72

2.2.4. Conclusion ................................................................................................ 76

3.1. Introduction .................................................................................................. 79

3.2. Material and methods ................................................................................... 80

3.2.2. Leaf and stem explants establishment and callogenesis ....................... 80

3.2.2.1. Callus index (CI) .............................................................................. 81

3.2.3. Callus propagation and evaluation ....................................................... 81

3.2.3.1. Callus subculture mass propagation ................................................ 81

3.2.3.2. Dry weight (dw) ............................................................................... 82

3.2.3.3. Histologic evaluation ....................................................................... 82

3.2.3.4. Shoot organogenesis ........................................................................ 83

3.2.4. Statistical analysis ................................................................................. 83