Trends in Food Science & Technology 89 (2019) 26–44

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Trends in Food Science & Technology

journal homepage: www.elsevier.com/locate/tifs

Review

Different techniques for extraction and micro/nanoencapsulation of saffron T

bioactive ingredients
Farhad Garavanda,b, Somaye Rahaeec, Nooshin Vahedikiad, Seid Mahdi Jafarie,∗
a
Bioprocess Engineering Laboratory (BPEL), Department of Food Science and Engineering, University of Tehran, Karaj, Iran
b
The Center of Research & Development, Mihan Dairy City, Tehran, Iran
c
Department of Biotechnology, Amol University of Special Modern Technologies, Amol, Iran
d
Department of Food Technology, Institute of Chemical Technologies, Iranian Research Organization for Science & Technology (IROST), Tehran, Iran
e
Department of Food Materials and Process Design Engineering, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran

A R T I C LE I N FO A B S T R A C T

Keywords: Background: Saffron is an enriched pool of bioactives including crocins, crocetin, safranal, picrocrocins, essential
Saffron oils, minerals and trace amounts of B1 and B2 vitamins. Obtaining any valuable ingredients like bioactive
Bioactive compounds compounds which are naturally present in plants is completely depending on the extraction and purification
Extraction procedures. An efficient extraction method of bioactives should meet the green chemistry aspects including
Encapsulation
safety, environment-friendly, run-down or at least little impurities, efficiency, and economic requirements. On
Protection
Separation
the other hand, efficient entrapment of saffron bioactive compounds into the protected carriers is indispensable
owing to their liability to the operational (process), environmental, and body digestive conditions.
Scope and approach: This review will present recent advances on the extraction and encapsulation of saffron
bioactive components which could result in value-added products from the so-called red gold (saffron) as the
most expensive spice in the world for different purposes such as food, pharmaceutical, and cosmetic formula-
tions.
Key findings and conclusions: Many different extraction methods, alone or in combination, have been exploited to
produce the bioactives from saffron such as convectional routes (e.g. maceration and solvent extraction) and the
modern routes (e.g. supercritical fluids, pulsed electric field, emulsion liquid extraction, microwave, sonication,
etc.). Encapsulation techniques are strongly proposed to shelter the bioactive compounds found in saffron, over
the other protective techniques. In this regard, microencapsulation (spray drying, freeze drying, extrusion,
emulsion systems) and nanoencapsulation (nanoemulsions, solid lipid nanoparticles and nanodispersions, na-
nohydrogels, electrospinning, nano spray drying) procedures have been investigated for saffron bioactives.

1. Introduction
Saffron (Crocus sativus L.) is one of the valuable ancient herbs mostly
cultivated and produced in Iran, Italy, Turkey, etc. Saffron components
including corm, flower and stigma have regarded as flavour, colour and
taste-introducing natural additives which are extensively used for
producing food and nutraceutical products, especially due to the pre-
sence of bioactive ingredients (Jafari, Mahdavee Khazaei, & Assadpour,
2019; Sarfarazi, Jafari, & Rajabzadeh, 2015). However, most of the
antioxidant, anti-carcinogenic, anti-inflammatory, anti-tumor and anti-
depressant functions of saffron is attributed to the role of bioactive
compounds found in stigma (Moghaddam, Garavand, Razavi, &
Talatappe, 2018). Saffron is an enriched pool of nutraceuticals in-
cluding crocins, crocetin, safranal, picrocrocins, essential oils, minerals
and trace amounts of B1 and B2 vitamins (Shahi, Assadpour, & Jafari,
2016). All of the above-mentioned bioactives should be delivered to the
body in a tact form to emerge their action well. Successful and guar-
anteed delivery of bioactive compounds to the target tissues in human
body is mainly influenced by both external (i.e. processing, storage,
drying, and extraction), and internal (exposure of bioactive compounds
to other food complexes and human digestion system) conditions. Ac-
cordingly, the way of cultivation, storage, processing, packaging and
use of these ingredients should be controlled and monitored over the
time, until they are delivered to the right site in human body (Rahaiee
et al., 2015; Urbani, Blasi, Simonetti, Chiesi, & Cossignani, 2016).
Obtaining any valuable ingredients like bioactive compounds which
are naturally present in plants is completely depending on the extrac-
tion and purification procedures. An efficacious extraction method of

∗
Corresponding author.
E-mail address: smjafari@gau.ac.ir (S.M. Jafari).

https://doi.org/10.1016/j.tifs.2019.05.005
Received 7 December 2018; Received in revised form 1 May 2019; Accepted 12 May 2019
Available online 15 May 2019
0924-2244/ © 2019 Elsevier Ltd. All rights reserved.
F. Garavand, et al. Trends in Food Science & Technology 89 (2019) 26–44

Table 1
Chemical characteristics of key saffron bioactive compounds.
Saffron CAS Molecular structure Molecular Chemical Melting Sensory Biological activity References
bioactive number weight formula point (°C) effect
compound (g.mol−1)

Crocins 42553- 976.96 C44H64O24 186 Colour Anti- Mousavi, Bathaie,

65-1 Schizophrenia; Fadai, & Ashtari, 2015;
anti- Alzheimer Finley & Gao, 2017

Crocetin 27876- 328.41 C20H24O4 285 Colour Neuro-protective; Nam et al., 2010;
94-4 anti-fatigue Mizuma et al., 2009

Picrocrocin 138-55- 330.37 C16H26O7 156 Taste Anti-proliferate; Kyriakoudi,

6 anti-cancer O’Callaghan, Galvin,
Tsimidou, & O’Brien,
2015; Hoshyar,
Bathaie, & Ashrafi,
2008
Safranal 116-26- 150.21 C10H14O < 25 Aroma Anti-depressant; Gout, Bourges, &
7 anti-convulsant Paineau-Dubreuil,
2010; Hosseinzadeh &
Talebzadeh, 2005
Lycopene 502-65- 536.87 C40H56 172 Colour Anti-tumor; Bolhassani, Khavari, &
8 cardiovascular Bathaie, 2014;
protection Kamalipour &
Akhondzadeh, 2011
Zeaxanthin 144-68- 568.88 C40H56O2 215 Colour Anti-oxidant Bolhassani et al., 2014;
3

β-carotene 7235- 536.87 C40H56 180 Colour Neuro-protective; Ochiai et al. (2007)
40-7

α-carotene 7488- 536.87 C40H56 187 Colour Anti-oxidant Bhargava (2011)

99-5

Isophorone 78-59-1 128.31 C9H14O −8 Aroma Anti-oxidant Zarghami and Heinz

(1971)

bioactive compounds should meet safety, environment-friendly, run-
down or at least little impurities, efficiency, and economic requirements
(Garavand, Madadlou, & Moini, 2017). The extraction of bioactive
compounds is also a critical step in their further isolation and fractio-
nation. It is stated that extraction procedure plays an important role in
the quality and quantity of saffron bioactive ingredients. Accordingly,
many different extraction methods, alone or in combination, have been
exploited to separate the big-ticket bioactives from saffron sections
(Nerome, Ito, Machmudah, Kanda, & Goto, 2016). Up-to-now, extrac-
tion and purification methods from convectional routes (e.g. macera-
tion and solvent extraction) to the modern routes (e.g. supercritical
fluids, pulsed electric field, emulsion liquid extraction, microwave,
sonication, etc.) have been applied to separate and purify the plant-
based bioactive compounds especially saffron compartments (Heydari
& Haghayegh, 2014).
On the other hand, efficient entrapment of bioactive compounds
into the protected pools is indispensable owing to their liability to the
operational (process) and environmental conditions (Khazaei, Jafari,
Ghorbani, Kakhki, & Sarfarazi, 2016). Encapsulation techniques are
strongly proposed to shelter the bioactive compounds found in food and
nutraceutical items, over the other protective techniques. In these
strategies, fine particles of bioactive compounds are surrounded by wall
materials to organize round-shape glumes of different sizes named
“capsules” (Jafari, Mahdavi-Khazaei, & Hemmati-Kakhki, 2016; Rajabi,
Ghorbani, Jafari, Mahoonak, & Rajabzadeh, 2015). Protection of valu-
able bioactive targets such as micronutrients followed by their con-
trolled release in gastrointestinal system is accomplished by en-
capsulation approaches. There is a wide-range of encapsulation
techniques from convectional (e.g. spray drying and freeze drying) to
supplementary modern techniques (e.g. emulsion systems, liposomes,
coacervation, and supercritical fluids) to protect the volatile com-
pounds (mainly safranal) and other bioactive compounds of saffron
(e.g. crocins and crocetin) from oxidation and degradation (Rajabi
et al., 2015). Each of these techniques has its own features in case of
utilities, the way of preparation, suitability for especial means and also
efficiency. Accordingly, well-advised pick out of encapsulation techni-
ques plays a key role in acquiring an efficient, and sustained delivery of
bioactive compounds and micronutrients (Khazaei, Jafari, Ghorbani, &
Kakhki, 2014).
In the recent years, various review notes on saffron agronomy
(Kumar et al., 2008), clinical applications (Moshiri, Vahabzadeh, &
Hosseinzadeh, 2015), biomedical aspects (Abdullaev & Espinosa-
Aguirre, 2004), extraction and microextraction (Heydari & Haghayegh,
2014), and bioactive compounds (Rahaiee et al., 2015; Rios, Recio,
Giner, & Manez, 1996; Shahi et al., 2016) have been published. In the
current work, a comprehensive review on recent advances in extraction
routes, quality control measures and encapsulation techniques of saf-
fron bioactive compounds will be discussed.
2. Bioactive compounds of saffron
Saffron is basically consisted of stigma, anther, tepal and corm.
Saffron compartments, especially stigma, are worthwhile sources of
more than 160 bioactive ingredients, in which the vitamins, pigments,
flavonoids, anthocyanins and volatile aromatic essences are of high
importance (Shahi et al., 2016). In case of abundance, crocins, crocetin,
picrocrocin and safranal are the most important bioactives avail in
saffron, followed by lycopene, α-carotene, β-carotene, zeaxanthin,

27
F. Garavand, et al.
kaempferol and quercetin (Melnyk, Wang, & Marcone, 2010). The well-
known beneficial characteristics of saffron i.e. colour, aroma and taste
are attributed to crocins, crocetin and picrocrocin, respectively, all of
them originated from stigma section (Melnyk et al., 2010). Accordingly,
most of the studies have been focused on stigma bioactive compounds.
Crocins (particularly α-crocin) which generating a stable water-so-
luble golden red colour, cause a wide range of applications in the food
industry. Crocins are also act as antioxidant compounds and exhibit
some therapeutic effects including anti-Schizophrenia, anti-Alzheimer
and memory boosters. Crocetin and its derivatives, which are mainly
presented in stigma and act as colorant, also possess a wide range of
therapeutic and health-promoting features in the area of medicine,
pharmaceutical and food (Rahaiee et al., 2015). Picrocrocin, the col-
ourless compartment of stigma, is undertaken as saffron bitter taste and
pacemaker of saffron aroma components, showed anti-proliferate and
anti-cancer attributes. Lastly, safranal and isophorone, the unstable
volatile components widely found in stigma, are derived from picro-
crocin which result in the distinctive aroma of saffron. These compo-
nents are valuable in light of their anti-depressant and anti-convulsant
properties (Heydari & Haghayegh, 2014). Therefore, almost all bioac-
tive components in saffron are bestowing exquisite effects on human
health through their antioxidant, anticancer, anti-depression, lowering
the insomnia and anxiety, memory enhancing, and antitumor effects
(Shahi et al., 2016). Table 1 represents some chemical features and
biological effects of main saffron bioactive compounds.
3. Extraction methods of saffron bioactives
Extraction of bioactive compounds from different plant compart-
ments is very crucial in their further implementations in health-pro-
moting food and nutraceutical products. It is declared that proper ex-
traction route can collect the target bioactives up to 5 times, compare to
the traditional methods. Extraction techniques from traditional to the
advanced ones should be optimized according to the type of bioactive
compounds available in the investigated plant and intrinsic attributes of
the plant itself, e.g. tissue complexity, heat sensibility, etc. (El Asbahani
et al., 2015). Hence, considering the variety of plants and the relevant
tissues, followed by selecting the best extraction method can guarantee
the successful extraction of bioactives from plant materials. Generally,
conventional extraction methods which mainly include Soxhlet ex-
traction, vapor or hydro distillation and maceration or solvent extrac-
tion use huge amounts of organic hazardous solvents, are not selective,
take long extraction times and in some cases extirpate thermo-liable
compounds such as bioactives (Heydari & Haghayegh, 2014). To sur-
mount such obstacles, the novel extraction methods are developed as
proposed as alternatives for out-of-date methods. These modern ex-
traction techniques which regarded as “green methods” are safer, effi-
cient, eco-friend, fast and accurate. The milestones in the extraction
procedures for extracting saffron bioactive compounds are chron-
ologically depicted in Fig. 1. Emulsion liquid membranes (ELMs), en-
zyme assisted extraction (EAE), supercritical fluid extraction (SFE),
ultrasound assisted extraction (UAE) or sonication, microwave assisted
extraction (MAE), and pulsed electric field (PEF) extraction are ex-
amples of green extraction techniques, which exhibit appropriate po-
tentials to extract saffron bioactive compounds. Generally, the effec-
tiveness of extraction routes essentially relies upon the selecting proper
solvents, considering solvent-solute affinity and employing com-
plementary or co-extraction methods (Azmir et al., 2013). The ad-
vantages and disadvantages of various extraction methods employed in
saffron compartments are listed in Table 2.
3.1. Conventional methods for extraction of saffron bioactives
3.1.1. Soxhlet extraction
Soxhlet extraction method which is regarded as the reference
method for extraction of fats, oils, essential oils and bioactive
28
Trends in Food Science & Technology 89 (2019) 26–44
compounds was developed for the first time by German chemist Soxhlet
in 1879. To do this, a little part of dry specimen is put into a thimble.
The thimble is then set in distillation flask which contains the desired
solvent(s). In the wake of coming to an overflow level, the thimble-
holder containing solvent-solute mixture is suctioned by a siphon.
Siphon empties the solution again into the distillation flask. This sol-
vent conveys separated solutes into the bulk solvent. Solute is stayed in
the distillation reservoir and the solvent goes back into the plant tissue
matrix. The procedure runs over and over until the point when the
extraction is finished (Azmir et al., 2013).
The organic or aqueous-based solvent extraction of saffron bioactive
compounds via Soxhlet extractor was reported by Corti, Mazzei, Ferri,
Franchi, and Dreassi (1996), Feizzadeh et al. (2008), Parizadeh,
Ghafoori Gharib, Abbaspour, Tavakol Afshar, and Ghayour-Mobarhan
(2011) and Samarghandian, Borji, Farahmand, Afshari, and Davoodi
(2013) mainly to investigate therapeutic and anti-cancer attributes of
saffron compartments. The main drawbacks of Soxhlet extractor com-
pared to novel or complementary extraction methods are prolonged
extraction times, high solvent and energy consumption, impurity of the
extract, low safety, and low extraction efficiency (Gupta, Naraniwal, &
Kothari, 2012). So, modification of soxhlet procedure by combination
with advanced extraction routes can boost its application for extraction
of bioactive compounds from plant materials.
3.1.2. Solvent extraction or maceration/soaking
The maceration approach was utilized in hand crafted planning of
tonic from quite a while. It turned into a well-known and reasonable
approach to get fundamental bioactive ingredients and essential oils. In
order to extract at laboratory and pilot plant scale, maceration is ba-
sically comprised of a few stages. Initially, crushing of dried plant
materials into little pieces is done to build an extended surface area to
expose plant compartment onto the solvent. Also, in maceration pro-
cess, a solvent named menstruum is poured into a shut vessel. Finally,
the liquid is stressed off; however, the marc which is the solid part of
extraction procedure is squeezed to recover extensive levels of impeded
solutions. The collected strained section and the fluid resulted from
press are blended and filtrated to omit impurities (Azmir et al., 2013).
In many cases, the target plant tissues are exposed to the heat-acid
treatments or some chemical adjustments (El Asbahani et al., 2015).
An extensive range of solvents including water, organic solvents and
their combination have been engaged to extract saffron bioactive
compounds (Sarfarazi et al., 2015). For example, water-soluble car-
otenoids (i.e. crocin and its derivatives) which are abundantly found in
saffron can be effectively separated from stigma exploiting the aqueous
or aqueous-based extraction strategies. Water, methanol, ethanol and
petroleum ether are frequently used solvents to extract saffron bioactive
compounds (Kakouri et al., 2017; Rahaiee et al., 2015). The ethanol-
water solvent at different ratios (e.g. 50:50 and 70:30) was found to be
the best media to extract polyphenols and other bioactive compounds
from saffron plant (Ferrara, Naviglio, & Gallo, 2014; Masi et al., 2016;
Sánchez-Vioque et al., 2016).
It should be pointed out that, the bioactives acquired by organic
solvents may contain trace amounts of residues and contaminates,
made them hazardous for pharmaceutical and nutraceutical applica-
tions. Hence, to overcome these drawbacks, the use of water or organic
solvents with a low boiling point (e.g. n-pentane) followed by a steam
distillation is recommended (El Asbahani et al., 2015).
3.1.3. Hydro or steam distillation
Bioactives and essential oils can also be recovered from plant tissues
by hydro/steam-distillation technique. It deals with water, steam or the
combination bare use of organic solvents and it should be performed
before drying of plant materials. In hydrodistillation, the plant mate-
rials are stuffed in a still compartment; the adequate amounts of water
are included in flask and afterwards heated up to boiling point. On the
other hand, hot water and steam are regarded as the fundamental
F. Garavand, et al.
Fig. 1. The milestones in extraction of bioactive compounds of saffron by various methods.
persuasive means to free bioactive compounds from plant metrics. It
should be considered that high extraction temperature makes some
unpredictable lost in bioactive contents. This disadvantage restrains its
widespread use in extraction of thermo-sensible bioactive compounds
(Azmir et al., 2013).
The benefit of water in different states (liquid, vapor or steam) is
that it doesn't affect the bioactive compounds chemically and conse-
quently, after extraction, bioactives could be strenuously isolated from
water by a straightforward decantation. This strategy is reasonable for
the extraction of bioactive compounds from various parts of plants such
as petals and blooms to the stem and root (Yang, Jiang, Shi, Chen, &
Ashraf, 2011). This technique has, in any case, a few disadvantages
including a long processing time, some chemical changes such as cy-
clization and hydrolysis, in terpenic atoms by drawn out contact with
bubbling water and loss of some polar molecules due to the over-
heating. The turbo-distillation which is considered as an updated ver-
sion of hydrodistillation, permits to acquire a high efficiency by reusing
the water source in a sweeping low extraction time. Turbo-distillation
provides the full recuperation of bioactive compounds present in the
vapor by using the plate column. Affordable instrumentation, ease of
methodology and high selectivity has made hydrodistillation up till
now as a suitable extraction method for industrial and semi-industrial
applications (El Asbahani et al., 2015).
In a research conducted by Tarantilis and Polissiou (1977), steam
distillation, microsteam distillation, and vacuum head space distillation
techniques employed to extract saffron volatile compartments. The GC-
MS outputs confirmed the successful extraction of safranal, isophorone
and their isomers particularly by microsteam and vacuum head space
distillation methods (Tarantilis and Polissiou, 1977). Zareena, Variyar,
Gholap, and Bongirwar (2001) also investigated the alterations in vo-
latile compounds and coloring agents of saffron after γ-irradiation by
steam-distillation. The results showed no considerable changes in saf-
fron bioactive as affected by γ-irradiation, excluding 5 kGy dosages
(Zareena et al., 2001).
3.2. Novel methods for extraction of saffron bioactives
3.2.1. Emulsion liquid membrane (ELM)
ELMs are designed for extracting metal ions from hazardous ef-
fluents, wastewater treatment and recovery of biotechnological and
bioactive compounds. Actually, ELM systems (Fig. 2) are double
emulsions (W1/O/W2) in which W1 is the internal aqueous phase con-
taining stripping agents, O is organic membrane phase containing di-
luent, surfactant, carrier and in some cases co-surfactant, and W2 is the
external aqueous phase (feed phase) enriched with the target
29
Trends in Food Science & Technology 89 (2019) 26–44
ingredients e.g. metal ions, phenolic compounds, organic acids, etc.
ELM possess key advantages over conventional solvent extraction
techniques such as accelerated mass transfer, high selectivity, high
extraction efficiency using green solvents, recyclability, extraction and
purification at the same time and low investment costs (Garavand &
Madadlou, 2014). Beside the reported advantages, ELM usually works
at low agitation speeds of 300–500 rpm, introducing a qualitative solute
with least damage or degradation.
Recently, Mokhtari and Pourabdollah (2013) developed an ELM
system to extract saffron bioactive compounds from aqueous solution of
dried saffron stigmas. The extraction optimization by one factor-at-a-
time method exhibited the following conditions: Span 80 at con-
centration of 2.5% as surfactant, n-decane as diluent in membrane
phase, phase ratio (internal aqueous phase to membrane phase) of 0.8,
treat ratio (emulsion to external aqueous phase) of 0.3, and agitating
speed of 300 rpm. They reported that under the optimized conditions,
more than 90% of saffron bioactives (i.e. safranal, picrocrocin and
crocins) were collected into the aqueous phase of emulsion globules
(Mokhtari and Pourabdollah, 2013).
3.2.2. Microwave-assisted extraction (MAE)
The dispersal of electromagnetic waves at a frequency range of
300 MHz to 300 GHz in the illuminated perimeter can induce micro-
wave heating. Nevertheless, the frequently used frequencies in mag-
netrons (i.e. microwave ovens used in domestic and laboratory) for
food, medical, pharmaceutical, and nutraceutical means are 0.915 GHz
and 2.45 GHz (Hao, Han, Xue, & Deng, 2002). Opposed to classical
solvent extractions in which the mass and heat transfer happens from
the inside to the outside, and vice versa, respectively, the both heat and
mass transfer of MAE occurs in a same direction from inside of plant
material to the solvent medium. One pot heat-mass transfer phenom-
enon results in an accelerated solute transfer, collecting more bioactive
compounds in extremely short times. Relying on the applied microwave
power and characteristics of plant material (water content, texture
complexity and its loss factor), the heat transfer and temperature rise
will be different (Mason, Chemat, & Vinatoru, 2011). The extraction of
bioactive compounds by conventional methods may introduce many
unwelcome components to the extracted solutes which affect its quality,
purity and appearance. In contrast, novel extraction practices like MAE
underrate the presence of undesired by-products, save time and energy,
and cause minimum solvent consumption (Chemat, Abert-Vian, &
Fernandez, 2012).
Solvent supported MAE which is developed based on the solvent
polarity, greatly influences the release of bioactive compounds en-
trapped within the plant matrix. In solvent assisted microwave recovery
 Table 2
Advantages and limitations of different extraction methods applied for saffron.
F. Garavand, et al.

Extraction procedure Advantages Disadvantages Considerations Reference

Conventional methods (Soxhlet,
maceration and distillation)
Emulsion liquid membrane
Microwave assisted extraction
Commonly used extraction routes
Simple operation
Best suited for small-scale industries
Rapid extraction and high efficiency
High selectivity
Capability of scaling up Least use of toxic organic
solvents
Recyclability of membrane components
One pot extraction procedure
Low energy consumption
Selectivity and provide high quality extracts
Short extraction time and high extraction yield
Cost-effective compared to traditional extraction
techniques
Simply operable and economically feasible for
scale-up
Time-consuming methods Preferred for preliminary extraction of bioactives, oils, essential oils etc. Sagar, Pareek, Sharma, Yahia,
Not eco-friendly and consume large or use them as reference methods and Lobo (2018), Sarfarazi et al.,
quantities of solvents 2015
Not suitable for heat-sensitive
ingredients
Generate byproducts and degrade
some bioactive compounds
Low efficiency
Emulsion instability Optimization of emulsion system including type and amount of career, Garavand and Madadlou (2014)
Emulsion leakage and swelling surfactant, stripping agent etc. is very important in high efficiency
extraction of bioactive compounds
Not suitable for heat-sensitive It can be used for rapid extraction of bioactive compounds (especially Sagar et al. (2018)
bioactive compounds polyphenols)
Apparatus and equipment are
expensive
Difficulty of operation
Less eco-friendly because of using
organic solvents
Poor extraction yield for nonpolar

30
Ultrasound assisted extraction
High hydrostatic pressure
extraction
Supercritical fluid extraction
Enzyme assisted extraction
Pulsed electric field extraction
compounds
Ease of use due to simplicity of technique Dilution of extract in case of Careful experimentation should be considered to select optimum level of Ameer, Shahbaz, and Kwon
Fast extraction rate dynamic sonication solvents to achieve high efficiency (2017)
Appropriate for thermo-sensitive bioactives Generation of free radicals at high
Economic-clean method sonication powers
Ease of coupling with other extraction routes A solvent needed procedure
Least interaction with bioactives Difficulty of scale-up for industrial
Facilitate solvent penetration to the core of plant uses
tissue
Doesn't need any heating process High price of facilities High hydraulic pressure extraction performed at the range of Shinwari and Rao (2018)
Accelerated mass transfer Difficulty of maintenance of constant 100–800 MPa
Can be applied to the extraction of strongly polar, processing pressure
weakly polar and non-polar compounds using
different solvents
High yield and extract purity
Low energy approach
Follow green chemistry principals
Reduction in organic solvents and extraction time Not suitable for most drug and Highly recommended for volatile compounds like safranal Nerome et al. (2016)
Higher mass transfer pharmaceutical samples
Eco-friendly and ability to recycle the supercritical Polar molecules cannot be dissolved
fluid Costly system thermodynamics
Applicable at ambient temperature complicated
Green route owing to use water instead of Expensive price of enzyme The chemical composition, moisture content and particle size of plant Sagar et al. (2018)
common chemical solvents Enzyme sensitivity tissue should be considered
Appropriate to separate bound ingredients toward Difficult to scale-up for industrial The reaction time and concentration of enzyme should be carefully
the plant matrix applications monitored
Considerable extraction rate
Capability of scale-up (10000 kg/h) Best for extraction of phytosterols and various polyphenols Pourzaki et al. (2013)
High efficiency at short extraction time
(continued on next page)
Trends in Food Science & Technology 89 (2019) 26–44
F. Garavand, et al. Trends in Food Science & Technology 89 (2019) 26–44

Reference

Fig. 2. A schematic of the extraction of bioactive compounds from saffron by

ELM system.

of bioactive compounds, the polar extractants are heated up to boiling

point, penetrate into the plant structure and dissolve the bioactive so-
lutes (Chemat et al., 2012). In the recent years, MAE technique has been
used to separate saffron bioactive compounds in various operational
conditions. The irradiation frequency and time intervals, the operation
temperature, the type of plant material and the applied solvent are
main variables studied by researchers on recovery of saffron bioactives.
As can be seen from Table 3, crocins, safranal, picrocrocin and antho-
Considerations

cyanins are successfully obtained by MAE method. The superiority of

MAE over the maceration, sonication, supercritical CO2, and Soxlhet
extraction in extraction of saffron bioactive compounds has been ac-
credited by numerous studies (Gallo, Ferracane, Graziani, Ritieni, &
Fogliano, 2010; Muzaffar, Khan, Riaz, Mir, & Ahmed, 2015; Sobolev
et al., 2014). Keeping the integrity of extracted bioactives is the further
advantage of MAE compared to classical extraction methods (Sobolev
High maintenance is needed

et al., 2014).
Require accurate control of
processing parameters

3.2.3. Ultrasound-assisted extraction (UAE)

Ultrasound or sonication is defined as a tandem compression and
Disadvantages

expansion of sound waves at the frequency range of 20 kHz–100 MHz.

Sonication implies generation, amplifying and collapse of micro bub-
bles as a result of cavitation phenomenon. The conversion of motion
energy into the heating can create a huge quantity of energy which acts
as an activation energy to initiate some physicochemical reactions.
Usually, the generated bubbles possess the temperature of ca. 4726 °C
and pressure of 101.3 MPa (Chemat et al., 2017). Based on this theory,
extraction using ultrasound waves has been developed to liberate the
entrapped organic and inorganic components from plant materials by
exacerbating mass transfer and speeding up the contact of solvent to the
Yield highly purified extract

target compounds (Adam, Abert-Vian, Peltier, & Chemat, 2012;

Low energy consumption
Environment compatible

Garavand et al., 2017). For this purpose, the plant materials should be
extracted or liquefied in a proper medium at the beginning. The op-
erational variables of UAE including the applied frequency, tempera-
ture and time of ultrasound source, the features of plant material from
Advantages

particle size to the moisture content and the properties of carrier sol-
vent are important notes which should be attended to achieve an effi-
cient extraction (Chemat et al., 2011). In some cases, complementary
extraction procedures such as pressurized solid-liquid extraction
(Ferrara et al., 2014), micro-simultaneous hydro distillation extraction
(Kanakis, Daferera, Tarantilis, & Polissiou, 2004), rapid solid-liquid
dynamic extraction (Ferrara et al., 2014), and dispersive liquid-liquid
micro-extraction (Sereshti, Heidari, & Samadi, 2014) have been com-
Extraction procedure
Table 2 (continued)

bined with UAE to improve the bioactive recovery and reduce the ex-
traction time. Rapid extraction, high selectivity, advanced mass and
energy transfer, high purity finish product, cost effective, and capability
of scaling up are accounted as some superiorities of UAE technique over
conventional extraction methods (Kyriakoudi, Chrysanthou,
Mantzouridou, & Tsimidou, 2012).

31
 Table 3
Microwave assisted extraction of saffron bioactive compounds.
Saffron bioactive compound Radiation details Identification procedure Findings Reference
F. Garavand, et al.

Bioactive metabolites
Crocin 1, safranal and picrocrocin
Total polyphenols and total
anthocyanins
Safranal
Antioxidant activity of phenolic
compounds
0–300 W at 40 °C by single mode microwave Nuclear magnetic resonance (NMR) Microwave assisted extraction didn't degrade saffron metabolites and was more Sobolev et al. (2014)
reactor using deuterated solvent (CD3OD) efficient than conventional methods with reduced extraction times and solvent
volume
0–300 W at 40 °C for 10 min HPLC-DAD and Near-infrared The best solvent was ethanol: water (50:50) for safranal and picrocrocin, and Nescatelli et al. (2017)
spectroscopy (NIR) methanol: water (50:50) for crocin 1. Microwave extraction reduced the extraction
time considerably
800 W at 66 °C by water: ethanol (41:59) Spectrophotometric assay More than 98% of polyphenols and anthocyanins extracted by solid to liquid ratio Da Porto and Natolino
followed by filtration using acetate cellulose of 1:50 at optimum situation (2018)
membrane
1000 W for 4 min, methanol used as solvent High performance liquid The significant increase in the safranal contents at high temperatures were Muzaffar et al. (2015)
chromatography obtained because of thermal conversion of picrocrocin to safranal.
200 W at 50 °C for 18 min with 50% of nominal ABTS, DPPH and FRAP assays Extracts obtained using microwaves were rich in antioxidant metabolites Gallo et al. (2010)
power, ethanol/water (50:50 v/v) as solvent compared to the ultrasound assisted extraction

32
Table 4
Ultrasound-assisted extraction of saffron bioactive compounds in combination with solvent extraction and complementary methods.
Saffron bioactive compound Sonication frequency Solvent (s) Complementary method Results Reference

Crocetin esters 33 kHz Ethanol: water Pressurized solid-liquid extraction (PSLE) and ca. 4 times higher phenolics and antioxidant activity in PSLE compare Ferrara et al. (2014)
(50:50) rapid solid-liquid dynamic extraction to RSLDE
(RSLDE)
Crocin, safranal and picrocrocin 30 kHz, with pulsed and Distilled water – Both sonication methods significantly increased the saffron main Kadkhodaee and
continuous sonication bioactives at a short extraction time Hemmati-Kakhki (2006)
Saffron volatile compounds 50–60 kHz Methanol: Dispersive liquid-liquid microextraction Facilitated and shortened extraction at ambient temperature Sereshti et al. (2014)
(mainly safranal) acetonitrile (38:62)
Safranal 35 kHz Diethy ether – The changes in safranal and other bioactive compounds of various Maggi et al. (2010)
saffron samples monitored over the storage time
Saffron bioactive glycosides (i.e. 20 kHz Water: methanol – The optimized methanol percentage, the sonication duration and duty Kyriakoudi et al. (2012)
crocins and picrocrocin) cycles combination for the recovery of crocins and picrocrocin were
50%, 30 min, 0.2 s and 0.44%, 30 min, 0.6 s, respectively
Tepal bioactives 35 KHz Petroleum ether – Saffron tepals are rich in antioxidant and antimicrobial compounds Kakouri et al. (2017)
because of the presence of phenolic compounds especially kaempherol,
quercetin and myricetin
Safranal and HTCC 35 KHz Diethyl ether Micro-simultaneous hydro distillation The quantity of safranal isolated by MSDE ranged between 288.1 and Kanakis et al. (2004)
extraction (MSDE) 687.9 mg/100 g of saffron, whereas in UAE of safranal and HTCC it was
40.7–647.7 mg/100 g of saffron and 41.7–397.7 mg/100 g of saffron,
respectively.
Trends in Food Science & Technology 89 (2019) 26–44
F. Garavand, et al.
The extraction of bioactive compounds using UAE has been devel-
oped for recovery of aroma compounds, pigments, micronutrients, and
antioxidant agents from various animal and plant sources (Chemat
et al., 2017). As described in Table 4, the UAE of crocetin esters, crocin
derivatives, saffron volatile compounds (safranal and isophorone), pi-
crocrocin and bioactives like kaempherol, quercetin and myricetin from
saffron plant has been investigated. From Table 4, various aqueous and
organic solvents or their combination can be used at the frequency
range of 20–60 kHz to extract saffron bioactive compounds. The most
prominent attributes of UAE for saffron bioactives are higher yield,
short extraction time and working at ambient temperatures. Verma and
Middha (2010) concluded that due to no severe heat generation during
the operation of UAE, no significant alteration of bioactive compounds
happened. Maggi et al. (2009) investigated the UAE of saffron volatile
compounds from numerous commercial saffron varieties with diethyl
ether and cyclohexane as solvents and dynamic headspace desorption.
Their findings revealed that UAE can produce a greater extent of vo-
latile ingredients, while dynamic headspace method was faster and
needed less amount of saffron samples to trace the saffron volatile
criterion.
3.2.4. High hydrostatic pressure (HHP) extraction
The HHP extraction is a non-thermal method which has been pro-
posed to enhance the yield and quality of bioactive compounds from
plant materials through mass transfer created by ultra-high hydraulic
pressure of 1000–8000 bars. The mass transfer induced by the force of
applied high pressure, is generating an accelerated transfer of bioac-
tives from plant cell wall. Unlike thermal extractions which influence
covalent bonds, HHP extraction method impacts non-covalent associa-
tions (e.g. hydrogen, ionic, and hydrophobic bonds). Thus, covalently
attached bioactive compounds are saved by HHP extraction (Shinwari
& Rao, 2018). Prasad, Yang, Yi, Zhao, and Jiang (2009) stated that the
high molecular weight compartments of plants (carbohydrates, pro-
teins, etc.) contain non-covalent bonds, and are denatured or deformed
by applying ultra-high pressure; so they do not release into the solvent,
and as a result, a pure solute will be achieved. The higher permeability
of solutes along with rapid extraction times in HPP extraction compared
to other extraction methods is originated from deprotonation of
charged groups; by this means, upsetting the salt bridges and hydro-
phobic links in cell membranes is prompting permeability of solutes.
The advantages of HHP extraction method are: minimum use of sol-
vents, time and energy saving, eco-friendly, high purity extraction and
obtaining a wide range of valuable compounds (Shouqin et al., 2005).
Recently, Shinwari and Rao (2018) studied the HPP extraction of
saffron bioactive compounds. The applied pressure range was
1000–6000 bars in association with ascended extraction temperatures
of 30–70 °C. The HPLC analysis demonstrated that increase in the ap-
plied pressure caused a drastic increase in crocin, picrocrocin, and sa-
franal content by nearby 52–63%, 54–85%, and 55–62%, respectively.
Though, the elevated temperatures caused a considerable drop
(25–36%) in crocin content. The operation parameters of 5800 bar at
50 °C for 5 min were regarded as the optimum parameters for HHP
extraction of saffron bioactive compounds. The HHP extracted bioac-
tives were more efficient (28%) in quenching cancer cells compared to
the bioactives collected by classical methods (Shinwari & Rao, 2018).
3.2.5. Supercritical fluid extraction (SFE)
SFE is an extraction strategy which utilizes supercritical fluids as
extraction solvents. The common supercritical solvent for SFE is carbon
dioxide (CO2) which is known as a green solvent without any toxicity or
side effects. Its similar density as liquids, low viscosity and high diffu-
sivity has turned CO2 as a selective fluid in SFE. In general, less polar
segments such as safranal in saffron can be separated by CO2-mediated
SFE. Aqueous or organic solvents (e.g. ethanol) are employed initially
to remove polar segments (crocin, crocetin, etc.) from saffron
(Goleroudbary & Ghoreishi, 2016). Generally, reduced amount of
33
Trends in Food Science & Technology 89 (2019) 26–44
solvent, shortened extraction time, facilitated automation, selectivity,
capability of combination with other separation methods, and the su-
perior purity extracts are mentioned as the main advantages of SFE
(Heydari & Haghayegh, 2014).
Ahmadian-Kouchaksaraie and Niazmand (2017) carried out a
survey on CO2-mediated SFE of antioxidant agents of saffron industrial
deposits. The extraction time, pressure and temperature picked out as
extraction variables for response surface methodology (RSM) optimi-
zation. The maximum amounts of radical quenching compounds (i.e.
phenolic content, flavonoids and anthocyanins) were obtained by the
optimal SFE findings as follow: pressure of 16.4 MPa at 62 °C for
47 min. They also compared the antioxidant potent of the extract ac-
quired at optimized conditions through CO2-mediated SFE to the clas-
sically generated extract via maceration by water-ethanol mixture. The
results showed a fast-tracked mass transfer, shortened extraction time,
and some extract rich in bioactive compounds and antioxidant activity
in CO2-mediated SFE. As well, separation of chief bioactive compounds
of saffron, namely crocin and safranal performed by CO2-mediated SFE
coupled with methanol, and CO2-mediated SFE, respectively
(Goleroudbary & Ghoreishi, 2016). The extraction time, carbon dioxide
volume velocity, gas pressure and temperature were regarded as op-
erational parameters in order to optimize extraction conditions of saf-
fron bioactives. According to the results, the optimized conditions for
extraction of around 33% of crocin was reported as follow: the flow rate
of 1.0 cm3/min and pressure of 19.3 MPa at 44 °C for 110 min. Al-
though, more than 90% of safranal recovered from saffron at 92 °C,
pressure of 21.3 MPa, and flow rate of 0.9 cm3/min for 122 min
(Goleroudbary & Ghoreishi, 2016). As mentioned above, the high affi-
nity of supercritical CO2 to non-polar components caused a consider-
able recovery of safranal compared to crocin.
In another study, Nerome et al. (2016) extracted saffron phyto-
chemicals using CO2-mediated SFE in combination with methanol and
aqueous solutions as entrainers, compared to conventional extraction
with methanol and aqueous solutions. The HPLC results confirmed a
higher productivity of saffron bioactive compounds (safranal, HTCC (4-
hydroxy-2,6,6-trimethyl-4-hydroxy-1-carboxproducts), picrocrocin,
and some crocin derivatives) in SFE contrasted with conventional sol-
vent extraction method. The highest productivity of crocin derivatives
and picrocrocin were obtained at the pressure of 30 MPa and 80 °C,
while the optimal conditions were 40 MPa at 80 °C for safranal and
HTCC. The aqueous solvent was chosen as the best entrainer for re-
covery of crocin derivatives and methanol entrainer was more efficient
in extraction of HTCC, safranal and picrocrocin.
3.2.6. Enzyme assisted extraction (EAE)
An accelerated release of hydrophilic or hydrophobic adhered pig-
ments, phenolics, volatile compounds and other bioactive materials
from plant matrix can be provided by the action of different enzymes as
extraction pre-treatments (Puri, Sharma, & Barrow, 2012). The de-
struction of plant cell walls and hydrolyzing the bounded bioactive
compounds attached to the carbohydrate and lipid chains can take
placed in EAE (Fig. 3). The EAE approach is able to reduce the time and
energy consumption of classic extraction methods and their further
filtration and purification processes. Employing some naturally occur-
ring enzymes including cellobiase, cellulase, pectinesterase, hemi-
cellulase, fructosyltransferase, pectinase, α-amylase and protease in
solvent extraction and pressurized hot water extraction (PHWE) is
proven to enhance the yield of extract with premium quality (Lin,
Chang, & Deng, 2009; Puri et al., 2012). Basically, the EAE is preceded
by using solvents (i.e. water) or cold press based on the intended pur-
pose. EAE is classified as ‘’green extraction technique” due to the nat-
ural origin of enzymes and water, in lieu of using hazardous organic
solvents and some toxic co-extractors. For better extraction of bioactive
compounds by EAE, the following points should be monitored: the
moisture content and particle size of plant material, the chemical
composition of the plant matrix, the type, dosage and the required
F. Garavand, et al.
Fig. 3. A schematic drawing on the enzyme mode of action in extraction of saffron bioactive compounds.
conditions for enzyme, solvent to solid ratio, and time-temperature
pattern of the extraction process (Azmir et al., 2013).
Hereupon, Lotfi, Kalbasi-Ashtari, Hamedi, and Ghorbani (2015)
developed an enzyme mediated solvent extraction of saffron antho-
cyanin contents. A commercial enzyme blend consisted of cellulase,
hemicellulase, and pectinase at various levels was used to separate
saffron anthocyanins from the plant matrix. For this purpose, a com-
mercial enzyme blend named Pectinex was dissolved in distilled water
at the levels of 1–10% to separate anthocyanins at 40 °C for various
intervals of extraction times. The results revealed that ca. 40% more
recovery of anthocyanins from saffron tepal achieved by the optimized
EAE (enzyme level of 5% at extraction time of 1 h) in comparison with
solvent extraction by ethanol (as a reference method). The quality of
enzyme assisted extract was more stable and the colour was dis-
tinguished, while the solvent extracted product was pale and unstable
against degradation. It seems that the presence of higher amounts of
cyaniding-3-glucosides and lower levels of pelargonidin-3,5-glucosides
in enzyme mediated extract is responsible for its sharp hue and con-
siderable stability against degradation and polymerization (Lotfi et al.,
2015).
The capability of uncomplicated scaling-up is also one of the im-
perative features of EAE which is not found in the other non-thermal
extraction methods (Puri et al., 2012). In addition, EAE can be coupled
with other extraction procedures such as SFE. Mushtaq, Sultana, Anwar,
Adnan, and Rizvi (2015) used an extraction system based on the com-
bination of EAE and SFE to separate phenolic compounds from pome-
granate peel. The pomegranate peels experienced an enzyme pre-
treatment (pectinase, protease, cellulase, alcalase, and viscozyme)
succeeded by supercritical CO2 with ethanol as solvent, to collect
bioactive antioxidant ingredients. Such trend caused a duplicated
content of phenolic contents (vanillic acid, ferulic acid and syringic
acid) and antioxidant properties. They concluded that EAE-SEF com-
bination can be introduced as a potent eco-friendly technique to re-
trieve bioactive ingredients from plant materials in an ample yield
(Mushtaq et al., 2015).
34
Trends in Food Science & Technology 89 (2019) 26–44
3.2.7. Pulsed electric field (PEF) extraction
Transient electrical pulses which last around a few seconds at
electric power of 20 kV/cm to 80 kV/cm can generate PEF. PEF can act
as a non-thermal extraction technique with the least alteration in nu-
tritional components, bioactive compounds and product appearance. In
PEF extraction route, especially at low electric powers, the plant tissue
membrane is perforated by electric fields, in turn; a permanent or
temporary loss of membrane semi-permeability is occurred in the plant
interface, encouraging the liberation of bounded bioactive compounds
(Barba et al., 2015). Extraction of valuable bioactive compounds (e.g.
phenolic compounds, dietary fibers, pigments, etc.) by PEF assisted
extraction technique, alone or in combination with sonication or su-
percritical fluids, has been extensively employed in several plant ma-
terials as an alternative to the routine extraction practices (Vorobiev &
Lebovka, 2016). The PEF is able to stimulate the “electroporation” of
eukaryotic cell membranes in order to increase the release of bioactive
compounds at room temperature. Such non-thermal extraction avoids
any degradation, hydrolysis and polymerization of the target com-
pounds. Accordingly, PEF extraction of plant originated materials is a
promising way for the recovery of phenolic compounds, vitamins, and
other value-added components (Puértolas, Cregenzán, Luengo, Álvarez,
& Raso, 2013). Promoted mass transfer and extraction efficiency, low
extraction time and processing temperature, supporting sensitive
bioactives, energy saving, and negligible environmental hazards are the
emerging advantages of PEF extraction (Barba et al., 2015). Applying
PEF is also recommended to avoid the growth of some spoilage and
pathogenic microbial organisms (Vorobiev & Lebovka, 2016).
Pourzaki, Mirzaee, and Hemmati Kakhki (2013) used PEF for ex-
traction of saffron major bioactive ingredients. At first, an aqueous
extract of saffron ingredients was prepared at ambient temperature,
then a PEF treatment at variable voltages, pulse-width, pulse intervals
and pulse powers was performed. The results revealed that the em-
ployed pulses at 1–5 kV/cm are adequate to generate a proper per-
meation routes through the saffron tissue. The PEF time intervals and
electric field power were also considered as main parameters which can
induce the release of saffron bioactives from plant matrix. Approxi-
mately 14, 15.5 and 10.2% increase in crocin, safranal and picrocrocin
F. Garavand, et al.
content of stigma and also, 5.76, 7.5% and 5.9% increase in crocin,
safranal and picrocrocin content of pomace were achieved at the opti-
mized PEF treatment (pulse number of 35, pulse-width of 100 μs and
electric power of 5 kV), compared to the solvent extraction.
4. Quality attributes of saffron
The severe manual labor and insignificant amount of saffron com-
partments makes the saffron industry susceptible to various adultera-
tions such as addition of comparable filaments or powders of other
plants, colourants and blending of low quality saffron with premium
one. Such unlawful activities made some concerns to end-users who are
looking for healthy products, and food and pharmaceutical industries
(Ordoudi, de los Mozos Pascual, & Tsimidou, 2014). Nowadays, inter-
national authorities have laid down some regulations as ISO/TS 3632 to
prevail over these frauds in saffron products and guarantee the quality
control of saffron products. Severe inspections and quality assurance
measures are deliberated by the mentioned standard to assess saffron
quality which greatly affects the market price as well as consumer's
preference. In general, the presence and quantity of crocin, picrocrocin
and safranal as main bioactives of saffron determine the grade of saf-
fron. Of them, crocin is the first compartment which is considered as
quality index and identified by spectroscopic and chromatographic
tools. Lowliest to the premium saffron filaments and/or powders are
categorized by I to IV indications. Spanish standard is somewhat dif-
ferent from ISO in which the superior saffron is classified and labelled
as “Coupe” and the lowest quality saffron is labelled as “Sierra”. Ac-
cording to the Iranian standard, the first class saffron or “Sargol” pos-
sess the highest quality in terms of appearance, crocin content, high
light absorbance like Coupe and excellent colour attributes (Zougagh,
Ríos, & Valcárcel, 2006).
Since a wide range of bioactive components are responsible for
saffron quality, comprehensive quality assurance measures should be
addressed prior to collecting the plant sections and also over the pro-
cessing procedures. Previous studies have demonstrated that the quality
of saffron is critically influenced by the origin, harvest conditions,
transportation, processing, packaging, etc. in the worldwide market;
that's why such information are fundamental in identification of
bioactive compounds and any probable frauds in saffron. The identifi-
cation methods for characterizing saffron ingredients need validation
analytical assays to meet the ISO or reference protocols (Aghaei, et al.,
2018, 2019). Accordingly, numerous analytical methods and validation
assays have been employed to identify saffron ingredients by rapid,
high accuracy and trusted approaches, in the recent years (Table 5).
For instance, Sereshti, Poursorkh, Aliakbarzadeh, and Zarre (2018)
applied a thin layer chromatography combined with image analysis and
chemometrics procedures to rapid identify and validate saffron bioac-
tive ingredients. To do that, some pre-processing corrective sequences
were used before image analysis of the obtained data from thin layer
chromatography outlines of saffron samples. Afterwards, multivariate
data analysis and chemometric techniques were engaged to validate the
truthfulness of Iranian saffron samples and categorize some kinds of
common frauds in saffron. They reported that the above-mentioned
procedure can be applied in a short time without any special equipment
(just a camera or smartphone) to determine and validate saffron
bioactives and possible adulterations. Nenadis, Heenan, Tsimidou, and
Van Ruth (2016) used an analytical method based on proton transfer
reaction mass spectrometry (PTR-MS) technique in combination with
chemometrics in order to assess quality control aspects of saffron vo-
latile organic compounds (VOCs) over the storage time. The obtained
data revealed that the fingerprint of VOCs altered over the storage time
so that the amount of safranal dropped increasingly. Such strategy
could be used to monitor the presence of inferior quality saffron in the
commercial products in a short time.
35
Trends in Food Science & Technology 89 (2019) 26–44
5. Green analytical chemistry (GAC) and saffron extraction
GAC concepts have been presented with the aim to reduce the
amount of reagents and solvents during sample preparation, extraction
process and analyte purification. Mechanization of analytical proce-
dures and minimal use of chemical agents caused a significant reduc-
tion in reagents and extraction byproducts. GAC supports all idea in
preparation, extraction and purification of bioactives from various
sources without any considerable impact on environment including
non-solvent, remote, non-destructive methodologies (Armenta,
Garrigues, & de la Guardia, 2008). 12 concepts of GAC are including:
inhibit byproduct and waste production, use of nontoxic solvents and
reagents, boost energy efficiency, elude derivative products, use of in
situ measurements, work with high safety, use of least specimen size,
use of green solvents, use of the combination of extraction approaches
and/or solvents, automation and remote control, use of direct analytical
methods and use of integrated eco-friendly analytical approaches
(Gałuszka, Migaszewski, & Namieśnik, 2013).
As GAC focuses on developing the economic and fast approaches
with the least reagent and solvent consumption, reduction in generated
byproducts and low energy consumption, high demand on GAC-based
procedures as alternatives of the traditional analytical techniques arose
during the last years. Accordingly, highly automated, non-destructive,
eco-friendly, sensor-based, on-line, and remote sensing strategies re-
commended meeting the requirements of GAC. Furthermore, image
processing systems, smartphone applications and other easy-access in-
direct methods are employed to monitor the samples quality in a safe,
fast and chemical-free way (Armenta et al., 2008). Direct analytical
approaches like spectrometric and electrochemical methods are also
mandatory to track some trace elements including bioactives in dif-
ferent plant materials. In the recent years, some advanced analytical
methods including Raman and near or middle infrared (NIR or MIR)
spectrophotometric, proton transfer reaction mass spectrometry, atte-
nuated total reflectance Fourier transform infrared spectrometric (ATR-
FTIR), NMR metabolite fingerprinting, tandem mass spectrometry, and
X-ray are exploited to monitor the bioactive compounds and adultera-
tions in saffron (Lee, Htar, & Akowuah, 2015; Nenadis et al., 2016;
Petrakis, Cagliani, Polissiou, & Consonni, 2015; Rubert, Lacina,
Zachariasova, & Hajslova, 2016).
Use of minimum sample for analysis can reduce the saffron wastes,
decrease the amounts of solvents and reagents and enhance the energy
efficiency. For example, the traditional solvent based extraction pro-
cedures could be substituted by solid phase extraction or solid phase
microextraction systems with a high selectivity, least chemical con-
sumption, short extraction time and easy combination with chromato-
graphic measurements. The automated extraction systems can reduce
the touch of workers with samples and reagents, develop safety, facil-
itate the monitoring and minimize the use of reagents, solvents and
energy (Armenta et al., 2008; Rubert et al., 2016). The automated ro-
botic-based novel extraction systems like microwave, ultrasound, en-
zyme or supercritical fluid assisted techniques are highly recommended
extraction procedures for saffron ingredients in accordance with GAC
principals. Green analytical chemistry aspects in extraction of bioactive
compounds from saffron are illustrated in Fig. 4.
6. Protection of saffron bioactives by microencapsulation
techniques
There are some benefits related to encapsulation of bioactive in-
gredients including preservation of functional properties, improvement
of stability, masking undesirable flavours, enhancement of bioactive
properties of compounds, increase of bioavailability and controlled
release and targetability of bioactive compounds (Davidov-Pardo, Joye,
& McClements, 2015; Kayitmazer, Seeman, Minsky, Dubin, & Xu,
2013). Saffron as a functional food is widely used for enhancing
healthcare by possessing various properties like anti-carcinogenic, anti-

Table 5
Evaluation of different assays to investigate the quality control aspects of saffron ingredients.
Investigated ingredients
Common bulking agents in
commercial saffron
Saffron stigma compartments
Safranal contents
36
Main crocetin esters and picrocrocin
Chemical composition and
geographical origin
Quality control of traded saffron by
measuring major apocarotenoids
Analysis method
Sequence characterized amplified
regions (SCARs)
HPLC method on RP18 2.5 μm and
monolithic RP18 material
Ultrasound assisted- non-polar solvent
extraction followed by UV–Vis analysis
Spectrophotometric data followed by
HPLC method
Near-infrared reflectance (NIR)
spectroscopy
Fourier-transform mid-infrared (FT-MIR)
spectroscopy and chemometrics
Validation assay
Random amplified polymorphic DNA marker (RAPD)
amplicon (OPA11Cs791) obtainable from the
amplification with the primer OPA11 on C. sativus DNA
ICH-compliant protocol using UV spectra by co-injection
with reference material
Intra-laboratory method validation
Modified Kennard−Stone algorithm
UV–vis and HPLC-DAD measurements
ISO 3632 specifications as reference method
F. Garavand, et al.
Results Reference
Proper SCAR markers may represent a fast, sensitive, reliable, and Marieschi, Torelli, and
low-cost screening method for the authentication of dried Bruni (2012)
commercial saffron material
A specification for high-quality saffron of > 20% crocins, > 6% Lechtenberg et al.
picrocrocin and not less than 0.3% of volatiles, calculated as sum of (2008)
safranal, isophorone and ketoisophorone, was developed. Saffron
extracts and crocetin had a clear binding capacity at the PCP binding
side of the NMDA receptor and at the σ1 receptor, while the crocins
and picrocrocin were not effective
Intra-laboratory validation of the optimized conditions and analysis Maggi et al. (2011)
by UV–Vis spectrophotometry showed satisfactory results in
linearity, repeatability, intermediate precision and recovery. The
limit of detection was 1 mg safranal/kg saffron and the limit of
quantification was 3 mg safranal/kg saffron
The best predictions were obtained with the sum of crocetin esters Sánchez et al. (2008)
model, followed by the model for cis-crocetin (β-D-glucosyl)-(β-D-
gentiobiosyl) ester, trans-crocetin di-(β-D-gentiobiosyl) ester,
and trans-crocetin (β-D-glucosyl)-(β-D-gentiobiosyl) ester, whereas
the worst predictions were found with the picrocrocin and trans-
crocetin (β-D-gentiobiosyl) ester models
The method is appropriate to determine moisture and volatile Zalacain et al. (2005)
content of different saffron. NIR can be used to estimate the
compounds responsible for saffron colour including: trans-crocetin
di-(β-D-gentibiosyl) ester, trans-crocetin (β-D-glucosyl)-(β-D-
gentibiosyl) and picrocrocin
The band at 1028 cm−1, associated with the presence of glucose Ordoudi et al. (2014)
moieties, along with intensities in the region 1175–1157 cm−1,
linked with breakage of glycosidic bonds, were the most useful for
diagnostic monitoring of storage effects on the evaluation and test
set samples. FT-IR was found to be a promising, sensitive and rapid
tool in the fight against saffron fraud
Trends in Food Science & Technology 89 (2019) 26–44
F. Garavand, et al.
Fig. 4. Green analytical chemistry and extraction of bioactive compounds from saffron. (For interpretation of the references to color in this figure legend, the reader
is referred to the Web version of this article.)
depressant, antioxidant and anti-inflammatory activities (Shahi, 2016).
In recent years, investigating the application of natural pigments in-
stead of chemical colorant is an interesting subject of many studies due
to development of allergy and risk of cancer (Inomata, Osuna, Fujita,
Ogawa, & Ikezawa, 2006). In this regard, there are most tendencies to
use carotenoids because of their anticancer and antioxidant activities.
Saffron pigments including its water soluble carotenoids or crocins are
suitable for a wide range of uses such as culinary, bakery and bev-
erages, but there are some limitations such as fast oxidation and iso-
merization that cause destruction of their coloring strength and nu-
tritive value. Crocin, an important bioactive compound found in
saffron, is related to coloring property of saffron. This pigment is widely
used as a natural coloring agent due to quickly dissolving in water to
create an orange colour solution (Esfanjani, Jafari, Assadpoor, &
Mohammadi, 2015; Grumezescu & Holban, 2017).
The bioactive compounds of saffron have some drawbacks including
degradation during processing, untargeted release and low bioavail-
ability in freely form which poses the restrictions for expanding their
application; so further studies are needed on new delivery systems to
overcome the above mentioned problems (Esfanjani, Jafari, &
Assadpour, 2017). Many researches have overcome this problem by
using various bottom-up encapsulation techniques (Table 6) such as
spray drying (Mahdavi, Jafari, Ghorbani, & Assadpoor, 2014), freeze
drying (Lim et al., 2012), and nanoencapsulation (Dehcheshmeh &
Fathi, 2019; Rahaiee, Hashemi, Shojaosadati, Moini, & Razavi, 2017).
However, the choice of the encapsulation agent or encapsulation
technique depends on the physicochemical properties of core and wall
materials, carrier matrix stability during shelf-life, loss of reactivity of
wall material with core compound and availability and reasonable price
of carrier materials (Özkan and Bilek, 2014; Galland, 2013). There are
suitable encapsulating agents for successful encapsulation of bioactive
37
Trends in Food Science & Technology 89 (2019) 26–44
compounds but from the health and safety point of view, carrier agents
must be approved as “generally recognized as safe” (GRAS) substances
in food industries (Robin & Sankhla, 2013).
There have been different studies in the area of encapsulation for
bioactive compounds of saffron as shown in Table 6.
Nowadays, microencapsulation of bioactive compounds by spray
drying and supercritical CO2 are the most commonly used techniques
(Janiszewska-Turak, 2017). Supercritical micronization is a novel
method for encapsulation of bioactive compounds due to applying low
temperatures and non-toxicity of supercritical fluids. In this green
process, microencapsulation of compounds is mostly taking place under
supercritical CO2 conditions (Santos, Albarelli, Beppu, & Meireles,
2013). Many studies have been focused on supercritical anti solvent
(SAS) method as a simple process for the micronization because this
approach can provide better results and higher levels of carotenoid
protection. However, future researches are needed about the me-
chanism of particle generation especially in its scaling up process
(Janiszewska-Turak, 2017). To the best of our knowledge, there has
been no basic study on the encapsulating of saffron carotenoids by
supercritical micronization.
6.1. Spray drying encapsulation
Spray drying is the most popular encapsulation method to increase
the protection and encapsulation efficiency of carotenoids due to its
simplicity and flexibility. Although it has numerous limitations in-
cluding determination of optimal conditions like type and amount of
encapsulating agent, inlet and outlet temperature, and feed speed for
each carotenoid sample (Janiszewska-Turak, 2017). In many re-
searches, different wall materials such as maltodextrin, sodium algi-
nate, pectin, modified starch, cyclodextrins, cellulose, carboxymethyl
F. Garavand, et al.
Table 6
Summary of microencapsulation methods for saffron bioactive compounds.
Microencapsulation methods Advantages
Spray drying Increase of stability & bioavailability
Better encapsulation efficiency
Freeze drying Colour stabilizing of saffron
Extrusion High encapsulation efficiency & storage
stability
cellulose, pullulan and others have been used in microencapsulation
technique. These carrier agents provide a good protection against heat
and oxidation depending to their molecular and physicochemical
characteristics (Shishir, Xie, Sun, Zheng, & Chen, 2018). Recently,
cellulose nanocrystals (CNCs) and nanofibrillar cellulose (NFC) have
been applied in bioactive compounds microencapsulation in order to
increase the encapsulation efficiency and core agent stability (Valo
et al., 2011; Wang, Jung, & Zhao, 2017; Wang, Yu et al., 2017).
Ahmad, Ashraf, Gani, and Gani (2018) studied the stability and
bioavailability of microencapsulated saffron anthocyanins in β-glucan
and β-cyclodextrin by spray drying technique and found that combi-
nation of these wall materials resulted in the protection of anthocyanins
from adverse conditions of gastrointestinal environments and its sta-
bility in different processing conditions.
Factors influencing encapsulation efficiency are operating condi-
tions of spray dryer including inlet and outlet temperatures, nuzzle size,
air flow, humidity and feed flow rate (Jafari, Assadpoor, He, &
Bhandari, 2008; Janiszewska-Turak, 2017). The microencapsulation of
blueberry anthocyanins through ultrasonic assisted spray drying en-
hanced the retention and storage stability of these pigments. Ultrasonic
assisted system could reduce the mechanical degradation of the
bioactive compounds (Shishir et al., 2018). Esfanjani et al. (2015) re-
ported spray-drying encapsulation of saffron extract nanoparticles ob-
tained by W/O/W multiple emulsions and found smooth surface par-
ticles with sizes less than 100 nm, resulting in a better encapsulation
efficiency for bioactive compounds (crocin, safranal and picrocrocin) of
saffron extract due to using WPC and pectin as stable wall materials.
Similar results have been also reported by Rajabi et al. (2015) and
Rodríduez-Huezo et al. (2004).
6.2. Emulsion systems
In recent years, different types of emulsions have been investigated
as delivery systems for the food bioactive compounds. Microemulsions
are stable systems with droplets sizes less than 100 nm which can
supply a high solubility and bioavailability of bioactive compounds
especially for hydrophilic compounds (Assadpour, Maghsoudlou, Jafari,
Ghorbani, & Aalami, 2016). Although water in oil in water (W/O/W)
multiple emulsions could entrapped hydrophilic compounds within the
inner aqueous phase resulting in a greater stability and controlled re-
lease of these agents but these emulsions are thermodynamically un-
stable (Schuch 2013).
6.3. Extrusion
Extrusion is a simple and convenient process which is used to obtain
granules encapsulating bioactive compounds for food applications with
a long shelf life. Extrusion process does not need high temperatures or
using of organic solvents. This method consists of mixing the core into
carrier followed by extrusion through a nozzle (Silva et al., 2018).
Various biopolymers have been evaluated for the encapsulation of
Trends in Food Science & Technology 89 (2019) 26–44
Saffron bioactive Wall material Reference
compounds
Anthocyanin β-glucan & β-cyclodextrin Ahmad et al. (2018)
Saffron extract WPC & pectin Esfanjani et al. (2015)
Saffron extract Starch, gum Arabic, maltodextrin, Chranioti et al. (2015)
chitosan
Anthocyanin Cress seed gum Jafari et al. (2016)
Saffron petal's extract Maltodextrins & Arabic gum Khazaei et al. (2014)
Crocin, safranal & Alginate & chitosan Shakoori and Krasaekoopt
picrocrocin (2015)
Shakouriizeiee (2014)
bioactive compounds via extrusion or in fact, co-extrusion process. For
this reason, encapsulation of saffron bioactives as an additive using
copolymer complexes and extrusion method has been investigated by
Shakoori and Krasaekoopt (2015). Their results showed that alginate
and chitosan biopolymers were a good complex for encapsulating saf-
fron compounds. Encapsulation efficiency of crocin, safranal and pi-
crocrocin by this copolymer was 52.9, 86.2 and 66.3%, respectively.
The encapsulation of saffron bioactive compounds with alginate-chit-
osan biopolymers was performed by extrusion technique. It was found
that extrusion method led to the highest encapsulation efficiency as
well as storage stability (Shakouriizeiee, 2014). In recent years, na-
noextrusion process has been introduced as a suitable and promising
alternative to improve the stability of bioactive compounds (Li,
Ioannidis, Gogos, & Bilgili, 2017).
6.4. Freeze drying
Freeze drying as a simple technique in encapsulating bioactive
compounds is based on co-lyophilized core materials and matrix solu-
tion to make dried assembly. Freeze drying encapsulated ingredients
have good sensory properties and maintained biofunctionality because
of using low temperatures in the process. Chranioti, Nikoloudaki, and
Tzia (2015) used different agents such as starch, gum Arabic, mal-
todextrin, and chitosan to microencapsulate saffron extract by freeze
drying. Their results showed that the application of this method caused
a great protection of saffron extract in a chewing gum preparation as a
model that suggested a promising encapsulation technique for colour
stabilizing of saffron in food industry. Anthocyanins, found in saffron
petals, are vulnerable natural colors which are influenced by different
factors including pH, temperature, oxygen, and light. These natural
dyes are good substitutes to synthetic dyes applicable in food and
pharmaceutical industries (Fang & Bhandari, 2011). Some researchers
have introduced freeze drying method for encapsulating anthocyanins
by various wall materials (Jamenez-Aguilar et al., 2011; Kaushik &
Roos, 2007; Khazaei et al., 2014). Jafari et al. (2016) investigated an
efficient approach for stabilizing the anthocyanin pigments found in
saffron petals by microencapsulation followed by freeze drying. Their
results showed that cress seed gum as a natural hydrocolloid was a
suitable wall material for encapsulating anthocyanins. It has been
suggested that microencapsulated saffron anthocyanins by freeze
drying could be used as a suitable technique for colour stabilizing of
saffron in addition to as a promising method for natural colorant pro-
duction (Chranioti et al., 2015). Recently, spray freeze drying has been
introduced as a better approach for the encapsulation of bioactive
compounds due to overcoming the limitations of freeze drying by en-
capsulating in a large surface-to-mass ratio (Shishir et al., 2018).
7. Nanoencapsulation of saffron bioactives
Although microencapsulation is an effective method for sensitive
compounds but low bioavailability and uncontrolled release issues is
38
F. Garavand, et al. Trends in Food Science & Technology 89 (2019) 26–44

Table 7
Summary of nanoencapsulation methods for saffron bioactive compounds.
Nanoencapsulation methods Advantages Saffron bioactive Wall material Reference
compounds

Nanoparticles pH-dependent release Crocin PEG-functionalized selenium NPs Mary et al. (2016)
Anticancer activity
Anticancer activity Crocin Magnetic NPs El-Kharrag et al., 2017
Low cytotoxicity & excellent stability Saffron Chitosan NPs Ntohogian et al. (2018)
Nanoemulsions and Nanodroplets High storage stability & Controlled Crocin Polyglycerol polyrecioleate Mehrnia et al. (2016)
release Crocin, safranal & WPC-pectin-maltodextrin Esfanjani et al. (2017)
picrocrocin complexes
Nanostructured lipid dispersions High encapsulation efficiency Crocin Mono-olein Esposito et al. (2017)
Anticancer activity
Enhanced skin hydration & UV Safranal solid lipid nanoparticles (SLNs) Khameneh et al. (2015)
protection
Nano-hydrogels Anticancer activity & storage stability Saffron extract Chitosan & alginate Rahaiee, Moini, et al. (2015)
Rahaiee et al. (2017)
Electrospinning Thermal stability Saffron extract Tragacanth/zein nanofibers Dehcheshmeh and Fathi (2019)

Fig. 5. Different approaches for nanoencapsulation of saffron bioactive compounds.

positively associated with the large particle size of microcapsules.
Nanoencapsulation is a promising approach for encapsulating many
different bioactive compounds by surrounding them in different wall
materials (Jafari, 2017). There are various nanoencapsulation methods
(Table 7) for carotenoids including nanoparticles, nanoemulsions, na-
nodroplets and nanohydrogels as shown in Fig. 5. Selection of above
mentioned approaches depends on different parameters including
physicochemical characteristics, particle size, and required rate of re-
lease and delivery of bioactive compound. Economic considerations of
final product are essential so many researchers are interested in finding
inexpensive and efficient carrier agents (Jafari, 2017). Biomaterials like
polysaccharides (pectin, starch, cellulose and glucans) and proteins
(polypeptone, soy and milk proteins, gelatins, cereal proteins) are
widely used for food compounds as encapsulating agents. Proteins as
GRAS materials are good candidates for encapsulation due to their in-
solubility in acidic conditions and excellent functionalities such as
gelling and emulsification properties (Shishir et al., 2018). Oancea et al.
(2017) applied β-lactoglobulin protein for encapsulation of anthocya-
nins. Their results depicted that β-lactoglobulin in a pre-heat treated
state has the highest antioxidant activity. Nanoencapsulation
techniques could be effective candidates to enhance bioactivity and
bioaccessibility of bioactive compounds as well as to increase their
storage stability against harsh treatments due to nano size of particles.
Also, this approach could provide proper absorption and bioavailability
of nanoencapsulated ingredients (Ahmad et al., 2019; Hu et al., 2002).
7.1. Nanoemulsions and nanodroplets
Emulsification is a suitable approach for encapsulation of bioactive
compounds because of small-sized droplets and their good stability.
There are different approaches for emulsion production that can affect
on physiochemical properties, encapsulation and release behavior of
emulsions (Shishir et al., 2018). In this regard, different studies have
been reported on lipid-based nanocarriers for carotenoids encapsulation
due to their hydrophobic nature. In this regard, oil-in-water (O/W)
nanoemulsions serve as carriers of carotenoids such as β-carotene. On
the other hand, water-in-oil (W/O) nanoemulsions and double nanoe-
mulsions (W/O/W) could be used to encapsulate hydrophilic car-
otenoids such as crocin (Jafari, 2017). Recently, the protein-poly-
saccharide complexes are good candidates to produce stable emulsions

39
F. Garavand, et al.
which could be applied in food formulations. Proteins and carbohy-
drates increase the stability of emulsions by a viscoelastic adsorbed
layer onto oil droplets and an increase in aqueous-phase viscosity, re-
spectively. Also, the use of complex biopolymers can provide high
stability, high encapsulation efficiencies and controlled release of
loaded bioactive compounds (Jimenez-alvarado, 2009). The good
properties of nanoemulsions such as their high stability and bioavail-
ability make them as an interesting protective approach. Spontaneous
emulsification has many benefits including low energy method, low
need of surfactant, thermal stability and co-surfactant free; therefore, it
can be used as a suitable method for encapsulation of saffron bioactive
compounds. In this regard, Mehrnia, Jafari, Makhmal-Zadeh, and
Maghsoudlou (2016) investigated the effect of parameters such as
crocin content, surfactant to water ratio and preparation conditions for
encapsulating crocin in W/O microemulsions. Their results showed that
the smallest droplet size of prepared microemulsions was achievable by
polyglycerol polyrecioleate (PGPR) and higher surfactant concentra-
tions. Moreover, PGPR was a good and stable surfactant for preparing
nanoemulsions for a long time (Mehrnia et al., 2016).
Nanodroplets with sizes in the nanoscale range are attractive and
can be provided via microemulsions or nanoemulsions which lead to a
thermodynamically stable system or metastable system respectively, for
high bioavailability and solubility of bioactive compounds (Mehrnia
et al., 2016). Such characteristics of final products such as droplet size,
colloidal stability, and solubilization is dependent on appropriate for-
mulation of nanodroplets (Rodríguez-Abreu & Vila, 2014). Esfanjani
et al. (2017) investigated the ability of whey protein concentrate
(WPC)-pectin-maltodextrin complexes for loading of bioactive com-
pounds of saffron (crocin, picrocrocin and safranal) in nanodroplets
(< 100 nm). They examined two parameters including dispersed phase
content and biopolymer-based films on the stability and release content
of nanodroplets. They observed that produced multiple emulsions by
WPC-pectin-maltodextrin with 5% dispersed phase content had a high
level of stability during 22 days storage and in vitro controlled release of
encapsulated compounds (Esfanjani et al., 2017).
Recently, sun protection creams or emulsions have been formulated
by nanoparticles as carrying nanosized natural bioactive agents such as
pigments and antioxidants. The applicability of chitosan nanoparticles
loaded with saffron was studied in the preparation of sunscreen emul-
sions. The findings demonstrated that nanoparticles had a spherical
shape with 150–250 nm size. Also, all prepared emulsions based on
saffron loaded nanoparticles had a low cytotoxicity and excellent sta-
bility during 90 days storage as well as minimum sunscreen protection
was obtained with low SPFs (Ntohogian et al., 2018).
7.2. Nanoparticles
Nanoparticles are a good delivery system for bioactive compounds
due to enhanced bioavailability, targetability and controlled release of
natural compounds across the gastrointestinal tract (Kim et al., 2006).
Recently, there has been a great interest to the use of polymeric na-
noparticles as encapsulating agents for natural compounds because they
could improve bioavailability, water solubility, storage stability and
targeted delivery due to small size of nanoparticles (Sheth, Nagane,
Bahadur, & Bahadur, 2017; Yoon et al., 2015). Food proteins have a
high nutritional value and functional properties so they are suitable
materials in designing nano delivery systems. Some studies have shown
that zein-composite nanoparticles are good nanocarriers to encapsulate
various bioactive compounds due to an increase in encapsulation effi-
ciency and better protection of loaded compounds against unfavorable
environmental factors (Zhang et al., 2014). However, more researches
should be carried out to examine the stability retention and bioavail-
ability of saffron bioactive compounds loaded into nanoparticles for the
functional foods.
Nanoparticels can be loaded with bioactive compounds due to a
high surface area and high loading capacity. Mary, Shanthi, Vimala,
40
Trends in Food Science & Technology 89 (2019) 26–44
and Soundarapandian (2016) designed and prepared PEG functiona-
lized selenium nanoparticles to carry crocin as a drug delivery system
for anticancer targeting. The obtained nanoparticles were spherical in
shape with a size of 40–50 nm and showed a pH-dependent release
delivery vehicle for potential cancer treatment. The in vitro cytotoxicity
of nanoparticles conjugated crocin clearly confirmed that the powerful
synergistic effects between crocin and Se nanoparticles as well as there
was a great selectivity between cancer and normal cell lines. Their re-
sults depicted that prepared nanoparticles could inhibit the prolifera-
tion of lung cancer cell line in a dose dependent manner in A549 xe-
nografts nude mice (Mary et al., 2016). To enhance cancer therapy,
magnetic nanoparticles (MNPs) are suitable anticancer agents due to
their high surface, biocompatibility and biodegradability properties. In
a study by El-Kharrag, Amin, Hisaindee, Greish, and Karam (2017) in
vivo and in vitro anticancer activities of crocin coated MNPs were in-
vestigated. Treatment of HepG2 cells with crocin coated MNPs caused a
significant inhibition of their growth probably due to stimulation of
caspase-mediated apoptosis. Their results showed that MNPs could in-
crease anticancer activities and biological effect of crocin using in vitro
and in vivo models.
7.3. Nanostructured lipid dispersions
Nanostructured lipid dispersions (NLDs) are dispersion of mono-
glycerides along with an emulsifier agent that results in complex lyo-
trophic liquid crystalline nanostructures. These nano-carriers are able
to solubilize water insoluble compounds that lead to their sustained
delivery and enhanced protection (Puglia et al., 2013). In the last few
years, some researches have been focused on the anticancer properties
of saffron due to inhibitory effect of crocin as a potent water soluble
carotenoid found in saffron. Human cells are subjected to oxidative
stresses, which may lead to DNA damage. Carotenoids such as crocin
have shown chemoprotective properties by inhibition of tumor pro-
motion but the precise inhibitory mechanisms are still unclear
(Marnewick et al., 2005). Esposito et al. (2017) investigated the em-
bedded of crocin in NLDs prepared by dispersing mono-olein in excess
water. Natural biocompatible stabilizers such as Na-cholate and Na-
caseinate were used to satisfy sustainability requirements. Their results
revealed that the peculiar molecular architecture of NLDs caused the
formation of lamellar phases. Although previous studies demonstrated
that NLDs are a possible method for encapsulating hydrophilic mole-
cules but in this research, it has been shown that hydrophilic compo-
nents like crocin can be efficiently encapsulated within NLDs with a
high encapsulation efficiency. Also, the antioxidant potential of NLDs
has shown its interesting capability to prolong crocin antioxidant ac-
tivity. Their data demonstrated that increase of crocin stability may
give rise to enhanced anticancer activity of NLDs containing crocin
(Esposito et al., 2017). Safranal, as a bioactive compound of saffron, has
great pharmacological activities including sunscreen activity.
Khameneh, Halimi, Jaafari, and Golmohammadzadeh (2015) evaluated
the sunscreen and moisturizing properties of solid lipid nanoparticles
(SLNs) loaded with safranal preparations. The encapsulation efficiency
of safranal was about 72–85% dependent on the applied concentrations.
The SLNs loaded with safranal formulations enhanced the skin hydra-
tion properties and they observed that UV light and the absorption
content increased at higher safranal contents in formulations so this
finding suggested that safranal as an herbal UV blocking agent could be
a promising agent in UV blocking cosmetic formulations (Khameneh
et al., 2015).
7.4. Nanohydrogels
Nanohydrogels are promising delivery systems for bioactive com-
pounds due to prolonged in vivo circulation times and ability for en-
capsulating hydrophilic bioactive compounds (Douglas et al., 2005).
Recently, the development of protein nanohydrogels has been
F. Garavand, et al.
commonly being used due to attractive properties like a large surface
area and good ability to encapsulate different compounds. Bourbon,
Cerqueira, and Vicente (2016) suggested lactoferrin-glycomacropeptide
nanohydrogel as a bioactive and non-toxic nanocarrier system for en-
capsulation and controlled release of bioactive compounds in food and
medical applications. Crocin has a low bioavailability due to disin-
tegration of crocin into crocetin and elimination of glucose moieties
during intestinal absorption that leads to its poor absorption (Asai,
Nakano, Takahashi, & Nagao, 2005; Chryssanthi, Lamari,
Georgakopoulos, & Cordopatis, 2011). Rahaiee, Moini, et al. (2015) and
Rahaiee, Shojaosadati, et al. (2015) investigated the stability of crocin
encapsulated by chitosan and alginate hydrogels via ionic gelation
method. They optimized fabrication process by response surface
methodology (RSM) to determine the optimum values of parameters
(chtiosan and alginate concentrations and pH) influencing nano-
particles production. The obtained spherical nanoparticles with a size of
160–230 nm provided better stability of crocin-loaded nanoparticles
under unfavorable environmental conditions including pH, temperature
and light. Also, crocin-loaded nanoparticles showed a better controlled
release, favorable antioxidant property and anticancer activity that
strongly suggested as a nutraceutical for application in food industry
(Rahaiee et al., 2017).
7.5. Electrospinning
Electrospinning is a simple, economical and emerging technology to
produce nanofibers and nanocomposites with interesting characteristics
including a large surface area and high porosity for potential applica-
tions (Wang et al., 2017; Wang, Yu et al., 2017; Xu et al., 2017). This
technique does not need heat treatment and thereby it is a suitable
encapsulation approach for heat sensitive compounds such as car-
otenoid pigments (Shishir et al., 2018). Core-shell nanofibers prepared
by polymers with different solubility have shown potential applications
for the delivery of bioactive compounds. Coaxial electrospinning
method could be used to produce fibers from non spinnable biopoly-
mers (Yao et al., 2017). Encapsulation of aqueous extract of saffron was
performed by core/shell (tragacanth/zein) nanofibers using coaxial
electrospinning and their physicobiochemical properties were ana-
lyzed. FTIR data revealed that nanofibers could appropriately en-
capsulate saffron extract without any chemical reactions between core
and shell. Also, increase in thermal stability of encapsulated saffron was
seen by thermal analysis. The release content of saffron extract was
about 44% and 16% in the gastric and intestinal media, respectively.
So, it was suggested that thermostable nanofibers of saffron could be
used as saffron nano-carriers in various food applications especially
products that are processed in high temperature (Dehcheshmeh & Fathi,
2018). Although electrospinning is a versatile and efficient encapsula-
tion technique, but its large scale application is limited due to its low
throughput (Yang et al., 2017).
7.6. Nano spray drying
The saffron extracts loaded within maltodextrin by nano spray-
drying were characterized in the study of Kyriakoudi and Tsimidou
(2018) and physicochemical properties such as their stability and
controlled release were examined. The particles were smooth surface
spherical regarding the spray cap mesh size employed. Their findings
depicted that encapsulation efficiency of compounds was heavily as-
sociated with core: wall ratio. Also, their results showed that mal-
todextrin as a nanocarrier seemed to reinforce stability of saffron
bioactive compounds (with emphasis on crocin and picrocrocin) under
thermal treatment. The nanoencapsulated saffron extract improved
bioavailability of saffron compounds under in vitro gastrointestinal
conditions (Kyriakoudi & Tsimidou, 2018).
41
Trends in Food Science & Technology 89 (2019) 26–44
8. Conclusion and future prospective
Saffron has been widely used in different industries including food,
cosmetics and pharmaceutical because of its valuable properties due to
its bioactive compounds. Saffron bioactive compounds are rather vul-
nerable to environmental factors such as pH, light, oxygen, enzyme,
heat and metallic ions. Selection of apposite extraction routes based on
new technologies such as HHP, PEF and EAE are highly recommended
ways to maximize the extraction efficiency and keep the target com-
pounds away from detrimental environmental conditions. It could be
predicted that some strategies for one-pot extraction and entrapment of
saffron bioactive compounds will be the subject of future investigates.
Novel validation assays to identify the bioactive compounds of saffron
and advanced quality control measures to detect the adulterations in
saffron like NIR, FTIR and modified chromatographic methods should
be rapid, accurate and non-destructive. It is concluded that na-
noencapsulation offers protection of saffron bioactive compounds from
gastric environment which can provide an increased half-life during
digestion process and sustained release profile. Nanoencapsulated saf-
fron bioactive compounds with biopolymers significantly suppressed
the cancer cells viability. Therefore, chemoprevention using natural
nutraceuticals is suggested to ameliorate the cancer complications.
Encapsulation is a promising strategy for the conservation of distinctive
coloring and flavoring agents of saffron in food and medicinal appli-
cations. Although different micro and nano-encapsulation techniques
have been investigated in the literature for encapsulation of saffron
bioactive compounds, there is no unique method that could be re-
cognized as a faultless and scale-up applicable for the development of
encapsulation systems. Despite doing some investigations related to the
encapsulation of saffron compounds, limited practical applications in
commercial products have been reported. However, studies should be
emphasized on the innovative encapsulate systems (niosomes, proli-
posomes, phytosomes and nano-hydrogels) for encapsulation of saffron
bioactive compounds to overcome the limitations of above-mentioned
methods and improving overall functionality as well as provide re-
quirements for large-scale production. In other words, in order to have
added-value in saffron industry, it is necessary to design and implement
novel extraction and encapsulation techniques for production of highly-
efficient saffron bioactives applicable in different industries such as
food, pharmaceutical and cosmetic industries.
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