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Medicinal plant cell suspension cultures: Pharmaceutical applications and

high-yielding strategies for the desired secondary metabolites

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DOI: 10.3109/07388551.2014.923986 · Source: PubMed

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ISSN: 0738-8551 (print), 1549-7801 (electronic)

Crit Rev Biotechnol, Early Online: 1–18

! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/07388551.2014.923986

REVIEW ARTICLE

Medicinal plant cell suspension cultures: pharmaceutical applications

and high-yielding strategies for the desired secondary metabolites
Wei Yue1,2*, Qian-liang Ming1*, Bing Lin1, Khalid Rahman3, Cheng-Jian Zheng1, Ting Han1,4, and Lu-ping Qin1
1
Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, Shanghai, China, 2School of Life Science, East China
Normal University, Shanghai, China, 3Faculty of Science, School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University,
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Byrom Street, Liverpool, UK, and 4School of Forestry and Biotechnology, ZheJiang Agriculture & Forestry University, Lin’an, Hangzhou, China

Abstract Keywords
The development of plant tissue (including organ and cell) cultures for the production of Application, medicinal plant, pharmaceutical,
secondary metabolites has been underway for more than three decades. Plant cell cultures with plant cell culture, secondary metabolite
the production of high-value secondary metabolites are promising potential alternative sources
for the production of pharmaceutical agents of industrial importance. Medicinal plant cell History
suspension cultures (MPCSC), which are characterized with the feature of fermentation with
plant cell totipotency, could be a promising alternative ‘‘chemical factory’’. However, low Received 12 September 2013
productivity becomes an inevitable obstacle limiting further commercialization of MPCSC and Revised 22 January 2014
the application to large-scale production is still limited to a few processes. This review Accepted 02 March 2014
generalizes and analyzes the recent progress of this bioproduction platform for the provision of Published online 19 June 2014
medicinal chemicals and outlines a range of trials taken or underway to increase product yields
For personal use only.

from MPCSC. The scale-up of MPCSC, which could lead to an unlimited supply of
pharmaceuticals, including strategies to overcome and solution of the associated challenges,
is discussed.

Introduction Plant tissue and cell suspension cultures have been

investigated by biotechnological methods and provide a
Higher plants can produce a mass of substances that have
promising bioproduction platform for desired natural prod-
always been an excellent source of pharmaceuticals, insecti-
ucts. Tissue cultures of shoots or roots display undifferenti-
cides, flavorings, fragrances and food colorants (Kieran et al.,
ated metabolite characteristics compared to their parent
1997). These substances are traditionally obtained from
plants, whereas similar cultures often accumulate target
naturally grown whole plants, e.g. ginsenoside is obtained
compounds to a less level (Kolewe et al., 2008).
from Panax quinquefolium for pharmaceutical use through
Hairy root culture obtained from Agrobacterium rhizogenes’
large-scale crop cultivation, which usually takes 4–6 years
Ri T-DNA-mediated transformation is another technology,
(Zhong et al., 1996). There are regional and environmental
which is genetically stable and its growth is not restricted to
restrictions, which can also limit the commercial production
additional growth regulators. This can also provide an
of natural compounds. A few important natural products with
increased capacity for secondary metabolite accumulation
simple chemical structures such as aspirin can be produced
(Giri & Narasu, 2000). However, the major obstacle limits the
via chemosynthesis, whereas other complex structures are
commercial use of hairy roots to produce valuable plant
hard to be synthesized or the cost of their synthesis outweighs
secondary metabolites due to challenges in cultivating hairy
their commercial availability (Danishefsky et al., 1995;
roots in an industrial system (Giri & Narasu, 2000; Hansen
Stevenson & Szczeklik, 2006). For some of these pharma-
et al., 1989). In contrast, MPCSC, which is simple and cost
ceutical compounds, medicinal plant cell suspension culture
effective, can overcome the problems of large-scale produc-
(MPCSC) can provide another alternative method besides
tion and has been extensively used.
traditional cultivation methods and chemical synthesis routes.
MPCSC can be interpreted as free cells or small groups of
cells from the medicinal plant callus cultured in a liquid
medium scattered which can produce a mass of secondary
*Wei Yue and Qian-liang Ming contributed equally to this work. metabolites for pharmaceutical use (Moscatiello et al., 2013).
Address for correspondence: Dr. Cheng-Jian Zheng, Ting Han and Lu- This technology offers an alternative attractive potential
Ping Qin, Department of Pharmacognosy, School of Pharmacy, Second to whole plant for the production of high-value natural
Military Medical University, Shanghai 200433, China. Tel/Fax: +86 products such as paclitaxel (Li & Tao, 2009), resveratrol
021-81871305 (C.-J. Zheng); +86 021-81871306 (T. Han); + 86 021-
81871300 (L.-P. Qin). E-mail: zhengchengjian@smmu.edu.cn (C.-J. (Cai et al., 2012a,b), artemisinin (Baldi & Dixit, 2008),
Zheng); than927@163.com (T. Han); qinsmmu@126.com (L.-P. Qin) ginsenosides (Jeong et al., 2008) and ajmalicine
 2 W. Yue et al.
(Ten Hoopen et al., 2002). Plant cell is biosynthetically
totipotent, which implies that each cell of the plant is capable
of producing the chemicals, which are identical to those
present in the parent plant under suitable conditions. Over the
last 15 years, considerable progress has been extended in
plant molecular biology, and this has helped MPCSC to
emerge as an available alternative production platform for
plant-derived pharmaceuticals. In addition, this natural
therapeutic meets regulatory and safety standards.
Compared with conventional cultivation methods, the
exceeding superiorities of MPCSC are as follows. (a) It is
not subject to seasonal and geographical restrictions and other
various environmental variations. (b) MPCSC is considered as
a stable production platform, which ensures the consecutive
production of natural products of uniform quality and yield.
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(c) It is dominant for providing homogeneity and a higher
efficiency of propagation of cultured cells in parallel with
callus cultured on solidified medium (Xu et al., 2011a,b).
(d) Cultured cells, possibly to synthesize novel products that
do not normally exist in the native plant (de Pádua et al.,
2012; Ye et al., 2003; Zhang et al., 2011).
Processes of MPCSC for producing natural products are
shown in Figure 1. MPCSC is used not only for obtaining
plant-derived pharmaceuticals, but also as a platform for
researchers to investigate plant cell physiology and biochem-
istry, the culture of protoplast and plant somatic hybridization.
From an expert opinion, MPCSC has more of an imme-
For personal use only.
diate potential for commercial application compared to plant
tissue or organ cultures (Ramachandra Rao & Ravishankar,
2002; Xu et al., 2011a,b). Meanwhile, there are still inevit-
able problems, including the instability of cell lines, slow
growth and scale-up obstacles, which results in lower
Figure 1. Schematic depiction of the medicinal plant cell suspension culture (MPCSC) process for producing natural medicine.
Crit Rev Biotechnol, Early Online: 1–18
production of metabolites in MPCSC. Accordingly, most
research efforts have been directed towards the commercial-
ization of MPCSC.
This review will introduce the MPCSC in detail, including
its application to obtain important plant medicinal compo-
nents, generalize systematic strategies to increase the pro-
duction of desired compounds and the scale-up of plant cell
suspension culture. Approaches to overcome and solve the
associated challenges of this culture system will also be
discussed. A systematic summary and knowledge of future
prospects are necessary to facilitate further studies for
efficiently obtaining useful compounds via MPCSC.
Peer reviewed investigation was accomplished by analyz-
ing worldwide accepted scientific databases (Pubmed, Scopus
and Web of Science, SciFinder) and was scrutinized for the
available information on MPCSC.
Application for obtaining pharmaceutical secondary
metabolites
Obtaining plant secondary metabolites
Plant secondary metabolites such as terpenes, phenols,
alkaloids and cyanogenic glycosides can be utilized as
pharmaceutics, cosmetics, agrochemical biopesticides, flavor-
ings or food additives, fragrances and natural pigments
(Chiang & Abdullah, 2007). MPCSC has been extensively
used to produce these beneficial secondary metabolites for
pharmaceutical applications. A prestigious anticancer drug
paclitaxel (Taxol) produced by Taxus species has been
extensively studied for many years and is a very important
example of a drug produced by cell culture methods (Onrubia
et al., 2012). It has been successfully extracted from
 DOI: 10.3109/07388551.2014.923986
suspended Taxus chinensis cells and considerable advances
have been made to improve its accumulation by MPCSC
(Malik et al., 2011). Saponin obtained from Panax notogin-
seng cell suspension culture was identified as the most
effective inhibitor of tumor promoters (Zhang et al., 1996).
Resveratrol extracted from Vitis vinifera cell culture exhibited
a wide range of important biological and pharmacological
properties (Upadhyay et al., 2008). Terpenoid indole
alkaloids, including catharanthine, vindoline, ajmalicine,
bisindoles vinblastine and vincristine with high medicinal
and economic values, can be produced by suspending
Catharanthus roseus cells (Zhao et al., 2001). Shikonin
derivatives, which display antimicrobial, anti-inflammatory,
wound-healing and anti-tumor activities, can be acquired
successfully from Lithospermum erythrorhizon cell
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suspension cultures (Yamamoto et al., 2000). Anthocyanin
generated from cell suspension cultures of wild carrot,
Strobilanthes dyeriana (Smith et al., 1981), Vitis hybrida,
Hibiscus sabariffa and strawberry has been used not only as
food additives but also for cancer therapy (Zhang et al.,
1997). A large amount of high-value natural products have
generated considerable interest due to their utilization.
MPCSC, used for obtaining plant-derived secondary metab-
olites of pharmaceutical potential, is shown in Table 1.
Seeking desired or new active compounds through
biotransformation
For personal use only.
Biotransformation has been widely applied in the process of
herbal fermentation. Cultivated plant cells show the biochem-
ical capability of transforming exogenously supplied com-
pounds, which offers a broad potential and an interesting
contribution towards the modification of natural and new
synthetic products. Bioconversion purposes can be basically
achieved due to the enzymatic potential of plant cells. Plant
enzymes show the abilities to catalyze regio- and stereo-
selective, hydroxylation, oxido-reduction, hydrogenation,
glycosylation and hydrolysis for various organic compounds
as well as microorganisms (Giri et al., 2001). Therefore, plant
enzymes can be considered as useful tools for the production
of desired natural pharmaceuticals or new chemicals (Ishihara
et al., 2003). MPCSC has also been touted as a model system
for the study of biosynthetic pathways in plant cells.
The cultured cells of Eucalyptus perriniana are able to
convert aroma compounds involving thymol, carvacrol and
eugenol into glycosides, which have accumulated in the cells
(Shimoda et al., 2006). It is reported that the biotransform-
ation of hyoscyamine into scopolamine has been carried out
in transgenic tobacco cell cultures (Moyano et al., 2007) and
the biotransformation of glycosylation of capsaicin and
8-nordihydrocapsaicin has been investigated in
Catharanthus roseus cell cultures (Shimoda et al., 2007).
Biotransformation has been used for the synthesis of new
active components. The biotransformation of cinobufagin by
cell suspension cultures of Catharanthus roseus and
Platycodon grandiflorum were investigated, a new compound
was identified as 1b-hydroxyl desacetylcinobufagin and other
compounds showed cytotoxic activities against HL-60 cell
lines (Ye et al., 2003). An alkaloid biosynthetic gene with
re-engineered substrate specificity was transformed into
Medicinal plant cell suspension cultures 3
Catharanthus roseus, which resulted in that a variety of
unnatural alkaloid compounds was synthesized when
co-cultured with precursors (Runguphan & O’Connor,
2009). In Saussurea involucrata cell suspension culture,
three bioactive bufadienolides have been transformed to 11
products, six of which were first reported as 3-epi-bufotalin,
3-epi-desacetylbufotalin, 3-O-b-D-glucoside, 1b-hydroxybu-
fotalin, 3-epi-gamabufo-talin and 3-dehydro-D1-gamabufota-
lin, respectively (Zhang et al., 2011). In another study,
biotransformation of 21-O-acetyl-deoxycorticosterone by cell
suspension cultures of Digitalis lanata was reported, three
new compounds were isolated and the structures were
elucidated as 2b,3b,21-trihydroxy-4-pregnen-20-one,
2b,3a,21-trihydroxy-4-pregnen-20-one and 3b,21-dihydroxy-
5a-pregnan-20-one-3b-O-b-glucoside (de Pádua et al.,
2012).
Strategies to increase secondary metabolite yields
Although the basic techniques for MPCSC are well estab-
lished, their application to large-scale production is still
limited to a few processes. Numerous approaches have been
taken to develop production of the desired natural products,
such as a selection of high-producing cell lines, optimizations
of culture conditions, addition of elicitors or precursors, using
a two-phase culture system, absorption techniques and
metabolic engineering. The systematic enhancement of
secondary metabolites is shown in Figure 2.
Screening of high-yielding cell line
Screening highly productive cell line is based on the theory of
biochemical heterogeneity in plant cells. Statistically high-
yielding cell lines originate from high-producing plants, but
the production levels of cells originating from high-producing
plants also display variability. Variability leads to a reduction
in metabolite productivity with sub-culturing and has been
attributed to genetic changes by mutation in the culture, or
epigenetic changes, which are due to physiological condi-
tions. MPCSC is composed of low-yielding, high-yielding
and non-producing cells. Production of paclitaxel was
reported to be affected more easily by differences in
biosynthetic activity among the cultured Taxus baccata
suspension cell lines than by any other factor (Bonfill
et al., 2006). Techniques such as HPLC (High Performance
Liquid Chromatography) and RIA (Radioimmunoassay) were
used to screen for high-yielding cell lines (Chen & Chen,
2000).
Cell cloning methods are supposed as a credible way of
selecting high-yielding cell lines from the suspension culture,
which is similar to the monocolony isolation of bacteria
(Smetanska, 2008). A cell line of Euphorbia milli accumu-
lated about 7-fold the level of anthocyanins produced by the
parent culture after 24 selections (Stafford, 2002). In cultures
of Vitis vinifera, extensive screening of a number of clones
resulted in a 2.3- to 4-fold anthocyanin increase in production
(Curtin et al., 2003).
Mutation strategies and the use of selective agents have
also been employed in order to obtain overproducing cell
lines. This technique can be described as that a large number
of cells are exposed to a toxic inhibitor or environmental
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For personal use only.
4

Table 1. Plant secondary metabolites of pharmaceutical use obtained via medicinal plant cell suspension cultures.

Culture conditions
Product Main use Species Medium Photoperiod Culture type Reference
Abietane diterpenoids Antitumour Cephalotaxus fortune MS supplemented with 2 mg/L 2,4-D, Darkness Shake flask Xu et al. (2011)
W. Yue et al.

0.1 mg/L kinetin, and 30 g/L

sucrose.
Alkaloids Antimicrobial Ailanthus altissima MS supplemented with1 mg/L 2,4-D, Continuous light Shake flask Crespi-Perellino et al. (1986)
0.1 mg/L kinetin and 50 g/L
sucrose.
Ajmalicine Antihypertensive Catharanthus roseus MS supplemented with 1 mg/L Darkness Shake flask Ten Hoopen et al. (2002a)
IAA,1 mg/L NAA, 0.1 mg/L kinetin
and 40 g/L sucrose.
Artemisinin Antimalarial Artemisia annua MS supplemented with 30 g/L 16h light Shake flask Baldi & Dixit (2008b)
sucrose, 0.1 mg/L of each of NAA
and kinetin.
Anthocyanin Antioxidative Vitis vinifera MS supplemented with 0.2 mg/L 16 h light Shake flask Wang et al. (2004)
2,4-D, 0.5 mg/L 6-BA and 30 g/L
sucrose.
Fragaria ananassa LS supplemented with 30 g/L sucrose, Continuous light Shake flask Zhang et al. (1997b)
1 mg/L 2,4-D and 0.1 mg/L BA.
Anthraquinones Antimicrobial Morinda elliptica B5 supplemented with 20 g/Lsucrose, 16 h light Shake flask Chiang & Abdullah (2007)
2 mg/L 2,4-D, 0.5 mg/L NAA,
0.5 mg/L IAA and 0.2 mg/L
kinetin.
Rubia tinctorum MS supplemented with 30 g/Lsucrose, 16 h light Shake flask Perassolo et al. (2011)
1 mg /L IAA, 0.2 mg/L NAA and
kinetin.
Cassia acutifolia MS supplemented with 1.0 mg/L Dark Shake flask Nazif et al. (2000)
2,4-D and 0.1 mg/L kinetin.
Antifungal monoterpene Antifungal Piqueria trinervia MS supplemented with 1 mg/L NAA Not investigated Shake flask Saad et al. (2000)
and 1 mg/L BAP.
Berberine Intestinal ailment Coptis japonica LS supplemented with 10 mM NAA Dark Shake flask Hara et al. (1988)
and 0.1 mM 6-BA.
Thalictrum minus LS supplemented with 10 mM 2,4-D Dark Shake flask Nakagawa et al. (1984)
and 100 mM BAP.
Camptothecin Antitumour Camptotheca acuminate B5 supplemented with 1 mg/L 2,4-D, 16 h light Shake flask Pasqua et al. (2006)
0.5 mg/L kinetin and 40 g/L
sucrose.
Canthinone alkaloids Antitumour Antimalarial Brucea spp. Not investigated Not investigated Not investigated Roberts (1994)
Capsaicin Counterirritant Capsicum frutescens MS supplemented with 7.6 mmol/L Continuous light Shake flask Sudha & Ravishankar (2003)
2,4-D and 2.3 mmol/L kinetin.
Cerebroside Regulation of cell growth Lycium chinense MS supplemented with 1.0 ppm 2, 4-D Dark Shake flask Jang et al. (1998)
and 0.1 ppm kinetin.
Cholecalciferol Calcium absorption Solanum malacoxylon B5 16 h light Shake flask Aburjai et al. (1997)
Chlorogenic acid Antimicrobial Antioxidative Eucommia ulmoides MS supplemented with 30 g/L Shake flask Wang et al. (2003)
sucrose, 300 mg/L LH and 2 mg/L
2,4-D.
Codeine Sedative Papaver somniferum LS Continuous light Shake flask John Tam et al. (1980)
Colchicine Antitumour Colchium autumnale Not investigated Not investigated Not investigated Yoshida et al. (1988)
Coumarins Anticoagulant Ammi majus B5supplemented with 30 g/L sucrose. 16 h light Shake flask Staniszewska et al. (2003)
Crit Rev Biotechnol, Early Online: 1–18
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Crocin Anticancer Crocus sativus B5 supplemented with 300 mg/L Darkness Shake flask Chen et al. (2003)
casein hydrolysate, 2 mg/L NAA,
2 mg/L IAA and 0.5 mg/L BA.
Cryptotanshinone Antioxidative Antimicrobial Salvia miltiorrhiza MS medium supplemented with Dark Shake flask Tsutomu et al. (1983)
30 g/L sucrose.
Digoxin Heart stimulant Digitalis lanata MS supplemented with 340 mg/L Continuous light Shake flask Kreis & Reinhard (1992)
KH2PO4, 4 mg/L glycine and
33 g/L glucose.
Diosgenin Steroidal precursor Dioscorea deltoidea MS supplemented with 0.1 mg/L 2,4- Dark Shake flask Tal et al. (1984)
D.
DOI: 10.3109/07388551.2014.923986

Dipyranocoumarins Anti-HIV Calophyllum inophyllum WPM with 20g/L sucrose. Continuous light Pawar & Thengane (2009)
Eleutheroside Analgesic Anti-inflammatory Eleutherococcus MS supplemented with 1mg/L 2,4-D. Dark Shake flask Shohael et al. (2008)
Antipyretic Diuretic sessiliflorus
Ellipticine Antitumour Ochrosia elliptica DAM Not investigated Not investigated Kouamo et al. (1985)
Emetine Antiparasitic Cephaelis ipecacuanha MS supplemented with IBA, IAA and Not investigated Not investigated Jha et al. (1991)
60 g/L sucrose.
Forskolin Bronchial asthma Coleus forskolii B5 supplemented with 30 g/L sucrose. Dark Shake flask Mukherjee et al. (2000)
Furanocoumarin Antitumour Antioxidative Glehnia littoralis LS supplemented with 1 mg/L 2,4-D Dark Shake flask Kitamura et al. (1998)
and 1 mg/L kinetin.
Furoquinoline alkaloids Antitumour Antimicrobial Choisya ternata Not investigated Not investigated Not investigated Creche et al. (1993)
Ginkgolides Health tonic Ginkgo biloba MS supplemented with 30 g/L sucrose Dark Shake flask Kang et al. (2009)
and 3.5 mg/L NAA.
Ginsenosides Health tonic Panax ginseng MS supplemented with 1 mg /L 2,4-D, Dark Shake flask Zhong et al. (1996)
0.1 mg/L kinetin and 30 g/L
sucrose.
Gymnemic acid Antidiabetic Gymnema sylvestre MS supplemented with 0.5mg/L BA 16 h light Shake flask Praveen et al. (2011)
and 1.5 mg/L IAA.
Hispidulin Antitumor Saussurea medusa MS supplemented with 30 g/L Continuous light Shake flask Zhao et al. (2005)
sucrose, 10 g/L glucose, 100 mg/L
myo-inositol, 0.5 mg/L BA and
2 mg/L NAA.
Homoisoflavonoids Antimicrobial Antitumor Caesalpinia pulcherrima MS supplemented with 10 mM 2,4-D Dark Shake flask Zhao et al. (2004)
and 1 mM 6-BA.
Hypericin Antidepressive Hypericum perforatum MS supplemented with 0.90 mM Dark and light Shake flask Walker et al. (2002)
2,4-D, 0.11 mM kinetin and 30 g/L
sucrose.
Inulin Diabetics Helianthus tuberosus MS supplemented with 1 mg/L NAA Not investigated Not investigated Taha et al. (2012)
and 1 mg/L BA.
Isoquinoline alkaloids Antitumour Fumaria capreolata LS Not investigated Not investigated Rueffer (1985)
Antioxidative
Jaceosidin Antitumour Saussurea medusa MS supplemented with 30 g/L Continuous light Shake flask Zhao et al. (2005)
sucrose, 10 g/L glucose, 100 mg/L
myo-inositol, 0.5 mg /L BA and
2 mg/L NAA.
L-dihydroxyphenylalanine Anti-Parkinson Mucuna pruriens MS supplemented with 1mg/L IAA, Continuous light Shake flask Pras et al. (1993)
1 mg/L BA and 40 g/L saccharose.
Morphine Sedative Papaver somniferum LS Continuous light Shake flask Huang & Kutchan (2000)
Phenolic compound Arthritis Digestive disorders Larrea divaricata MS supplemented with 9 mM 2,4-D 16 h light Shake flask Palacio et al. (2012)
Rheumatism and 5 mM BA.
Venereal diseases
(continued )
Medicinal plant cell suspension cultures
5
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6

Table 1. Continued

Culture conditions
Product Main use Species Medium Photoperiod Culture type Reference
Phenylethanoid Anhrodisiacs Cistanche salsa MS supplemented with 40 g/L glucose Dark Shake flask Liu et al. (2007a)
W. Yue et al.

glycosides and 3.0 mg/L IAA.

Antioxidative Olea europaea MS supplemented with 30 g/L Dark Shake flask Orihara & Ebizuka (2010)
Anti-inflammatory sucrose, 1 mg/L 2,4-D, 0.1 mg/L
Antihypertensive kinetin and K+ instead of NHþ 4.
Phytoestrogens Health tonic Psoralea corylifolia MS supplemented with 5.7 mM IAA 16 h light Shake flask Shinde et al. (2009)
and 4.40 mM BAP.
Plumbagin Anticancer Drosophyllum lusitanicum Heller supplemented with 0.25 mg/L 16 h light Shake flask Komaraiah et al. (2003)
Antimicrobial Antifertil NAA, 5 mg/L IBA and 0.05 mg/L
BAP.
Podophyllotoxin Antitumour Podophyllum MS supplemented with 30 g/L glu- 16 h light Bioreactor Chattopadhyay et al. (2002)
cose, 30 g/L sucrose, 11.4 mL IAA,
1.5 mg/L pectinase and 10 g/L
polyvinylpyrrollidone.
Polyphenols Hpyerglycemic Cornus kousa MS supplemented with 2,4-D and BA. Not investigated Not investigated Ishimaru et al. (1993)
Antimicrobial
Quassin Antiphlogistic Picrasma quassioides B5 supplemented with 20 g/L glucose, Continuous light Shake flask Scragg & Allan (1986)
1.0 mg/L 2,4-D and 0.5 mg/L
kinetin.
Reserpine Antihypertensive Rauwolfia serpentina LS supplemented with 1 mM 2,4-D, Dark Shake flask Yamamoto & Yamada (1986)
1 mM kinetin and KNO3 instead of
NH4NO3.
Resveratrol Health tonic Vitis vinifera B5 supplemented with 30 g/L sucrose Dark Shake flask Yue et al. (2011)
and 250 mg/L casein hydrolysate,
0.1 mg/L NAA and 0.2 mg/L
kinetin.
Robustaquinones Antimalarial Cinchona robusta B5 supplemented with 20 g/L sucrose, Continuous light Shake flask Schripsema et al. (1999)
1mg/L 2,4-D, 0.2 mg/L kinetin and
50 mg/L cysteine.
Rosmarinic acid Antioxidative Anchusa officinalis B5 supplemented with 30 g/L sucrose, Continuous light Shake flask De-Eknamkul & Ellis (2007)
1mg/L 2,4-D, 0.1 mg/L kinetin.
Salvia officinalis MS supplemented with 30 g/L Continuous light Shake flask Hippolyte et al. (1992)
sucrose, 0.5 mg/L 2,4-D, 0.5 mg/L
kinetin and vitamins.
Coleus blumei CB2 Not investigated Not investigated Szabo et al. (1999)
Orthosiphon aristatus Not investigated Not investigated Not investigated Sumaryono et al. (1991)
Lithospermum LS supplemented with 1 mM 2,4-D, Dark Shake flask Mizukami et al. (1992)
erythrorhizon and 1 mM kinetin.
Sanguinarine Antiplaque Sanguinaria canadensis MS supplemented with 0.1 mg/L Continuous light Shake flask Archambault et al. (1996)
2,4-D, 0.5 mg BA and 30 g/L
sucrose.
Papaver somniferum MS supplemented with 0.1 mg/L Continuous light Shake flask Holková et al. (2010)
kinetin, 2 mg/L NAA and 30 g/L
sucrose.
Shikonin Antibacterial Lithospermum LS supplemented with 10 mM IAA and Dark Shake flask Yamamoto et al. (2000b)
erythrorhizon 10 mM kinetin.
Crit Rev Biotechnol, Early Online: 1–18
 DOI: 10.3109/07388551.2014.923986 Medicinal plant cell suspension cultures 7

Sánchez-Sampedro et al. (2005)

stress, and only cells that are able to resist the selection
procedures will survive (Ramachandra Rao & Ravishankar,

Nagella & Murthy (2010)

Orihara & Furuya (1990)
2002). p-Fluorophenylalanine (PFP) was extensively applied
in selecting high-phenolics-yielding cell lines, which is an

Endo et al. (1987)

Chen et al. (1997)
Pan et al. (2000)
analogue of phenylalanine. A selection of cell aggregates of

Ho et al. (2010)
BK-39 callus culture in a medium containing PFP yields a cell
line possessing a higher resistance to the inhibitor. However,
the shikonin derivative content was two times higher than that
of the control, reaching 12.6% of DW cell biomass (Bulgakov
et al., 2001).
Shake flask

Shake flask
Shake flask

Shake flask

Shake flask

Shake flask

Shake flask
Optimization of MPCSC conditions
Culture conditions play an important role in the quality and
quantity of the material obtained through MPCSC.
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Optimization of the culture condition is effective in improving

the accumulation of the desired product. External factors such
as carbon source, nitrogen source, growth regulators, medium
16 h light

pH, temperature, light and oxygen are considered easy to

regulate the expressions of plant secondary metabolite
Dark

Dark
Dark

Dark

Dark

Dark

pathways. Constituents in plant cell culture medium are

determinants of growth and production of secondary metab-
sucrose, 0.5 mg/L NAA and 0.5 mg/
sucrose,1 mg/L 2,4-D and 0.5 mg/L

mins, 30 g/L sucrose, 10 mM 2,4-D,

B5 supplemented with 30 g /L sucrose.

sucrose, 2 mg/L IBA and 0.1 mg/L

olites (Zhang & Zhong, 1997).

MS supplemented with 2.5 mg/L 6-
B5 supplemented with 2  B5 vita-

Several types of growth and production medium are put

4 mM kinetin and 1 mM GA3.

BA, 1 mg/L NAA and 30 g/L

MS supplemented with 30 g/L

MS supplemented with 30 g/L

MS supplemented with 30 g/L

MS supplemented with 30 g/L

into use, such as Murashige and Skoog Stock (MS) liquid

media, Gamborg (B5) liquid media, Linsmair & SKoog (LS)
liquid media, N6 liquid media and other improved liquid
For personal use only.

medium according to the growth behavior of plant cells

(Coste et al., 2011; Schenk & Hildebrandt, 1972). These
mediums supplemented with the required amount of sucrose
sucrose.

sucrose.
kinetin.

and plant growth regulators are appropriate for MPCSC (Han

L BA.
BA.

& Zhong, 2003). The differences among them were the

nutrient levels of carbon, nitrogen, phosphate and inorganic
mineral (Gamborg et al., 1968; Hockin et al., 2012).
Research towards the influence on cell growth and expression
of secondary metabolite are extensively in process. The total
Catharanthus roseus
Eriobotrya japonica

Withania somnifera
Silybum marianum

Salvia miltiorrhiza

triterpene production was optimal, when Eriobotrya japonica

Camellia Sinensis
Taxus brevifolia

cells were cultured in MS medium instead of B5 or N6

medium (Ho et al., 2010). Higher levels of phosphate were
found to enhance the cell growth, whereas it had negative
influence on secondary metabolite accumulation (Chandler &
Dodds, 1983). In Gymnema sylvestre cell culture, the macro
elements concentration (2.0  strength KH2PO4) and nitrogen
source supply (0.5  concentration of NH4NO3, NHþ 
4 =NO3
Antitumor Antidiabetes

ratio of 7.19/18.80) were investigated to reach the highest

accumulation of biomass and gymnemic acid (11.35 mg/g
Antiinflammation
Cardiac disorders

DW; Praveen et al., 2011). The maximum production of

Antihypertensive

daidzein (2.20% DW) and genistein (0.29% DW) was

Liver ailment

Antileukemic

Antistress
Health tonic

obtained when medium comprised with NHþ 

Anticancer

4 =NO3 at ratio
20:40 mM in suspension culture of Psoralea corylifolia
(Shinde et al., 2009). Pawar and Thengane (2009) optimized
the culture conditions for obtaining highest yields of different
desired products in cell suspension culture of Calophyllum
inophyllum. The pH of the culture conditions is regulated
between pH 5.0–6.2 before autoclaving (Ouyang et al., 2005;
Pasqua et al., 2006; Pawar & Thengane, 2009). The initial
Withanolide A

medium pH of 5.8 was found feasible in cell suspension

Tanshinone

Vinblastine
Triterpene
Theamine
Silymarin

cultures of Withania somnifera for the production of with-

Taxol

anolide A (Nagella & Murthy, 2010) and the extremes of pH

should be avoided.
 8 W. Yue et al.
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Nyu Medical Center on 06/29/14
For personal use only.
Figure 2. Systematic strategies to increase secondary metabolites production with plant cell suspension cultures.
Conditioned medium has been used to cultivate plant cells
to improve secondary metabolites production (Sakurai &
Mori, 1996). It was found that the addition of conditioned
medium effectively promoted cell growth and accumulation
of ginseng polysaccharide in both conventional and high-
density Panax notoginseng cell cultures in a bioreactor
(Woragidbumrung et al., 2001). Investigations have revealed
that cultured plant cells produce and release conditioning
factors into the culturing medium, which could promote cell
growth, cell division and secondary metabolism at low or high
cell density (Sakurai & Mori, 1996). Another possibility is
that the artificial medium supplied to the cells may not be
optimal, but cells can improve or convert the medium during
culturing by providing optimum conditions for secondary
metabolites synthesis, changes in amounts and ratio of
ingredients, pH and osmotic pressure of the medium
(Stachel et al., 1986). Conditioned medium is often used as
a superior technique in obtaining secondary metabolites for
pharmaceutical use.
Plant growth regulators, including 2,4-dichlorophenoxya-
cetic acid (2,4-D), a-naphthaleneacetic acid (NAA), indole-
3-acetic acid (IAA), indole-3-Butytric acid (IBA), cytokinin,
ethylene, 6-benzyladenine (BA) and kinetin regulate plant cell
growth processes by regulating cell division and cell differ-
entiation (Coste et al., 2011; Komatsuda et al., 1992). In the
suspension culture of Eriobotrya japonica cells, supplied with
Crit Rev Biotechnol, Early Online: 1–18
the medium with 2.5 mg/L of BA, 1 mg/L of NAA reached
high level of total triterpene production (Ho et al., 2010).
It has been reported that cytokinin and ethylene can
up-regulate the alkaloid accumulation in Catharanthus
roseus cells through independent pathways (Yahia et al.,
1998). There are a large number of studies on plant growth
regulators and manipulation towards obtaining high levels of
valuable natural products involving paelitaxel (Pan et al.,
2000), ginsenosides (Zhong et al., 1996), ginkgolides (Kang
et al.,2009), resveratrol (Yue et al., 2011) and so on.
Culture temperature also affects the cell cultures and
should be optimized. Most plant cells are cultured at room
temperature or at the temperature ranged between 20 and
28  C. The production of paclitaxel was increased to
137.5 mg/L by a temperature shift (24–29  C) a suspension
culture of Taxus chinensis (Choi et al., 2000).
The cell culture of Ammi majus was carried out in the
optimal temperature of 20–22  C (Staniszewska et al., 2003).
The growth temperature of Catharanthus roseus plant cells
for the production of ajmalicine was found to be optimal at
27.5  C (Ten Hoopen et al., 2002).
Light condition is determined by the photoperiod emerged
in characteristics of plant growth. Main types of light
conditions in MPCSC are darkness, 12 h photoperiod, 16 h
photoperiod and continuous light. Plant cells can adapt to
different types of light conditions. Table 1 summarizes the
 DOI: 10.3109/07388551.2014.923986
photoperiods of MPCSC. Actually, ultraviolet (UV) and red
light are normally used as a means of enhancing the
production of secondary metabolites. The production of
catharanthine in Catharanthus roseus cell suspension cultures
was increased by UV-B (Ramani & Chelliah, 2007).
Productions of stilbene and anthocyanins were improved
with the treatment of red light in Vitis vinifera cell suspension
culture (Tassoni et al., 2012).
Plant cells can be cultured either in shake flasks or in
large-scale bioreactors. The effects of oxygen partial pressure
(pO2) play an essential role in growth and second metabolism
of cultured cells. Low pO2 is unfavorable to the plant cell due
to less oxygen supply, whereas high pO2 inhibits cell growth
and reduces the production of the desired compounds due to
the detrimental effect of the oxidative burst. In the bioreactor
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suspension culture of Panax notoginseng cells, a pO2 of
21.3–29.3 kPa was found to be optimal for the cell mass and
the production of ginseng saponin and polysaccharide (Han &
Zhong, 2003). A 40% oxygen supply was found to be optimal
for the production of both cell mass and saponin yielding
(4.5 mg/g DW) in Panax ginseng cells cultured in a 5 L
balloon-type bubble bioreactor (Trung Thanh et al., 2006).
Induction of secondary metabolite pathways by
elicitors
Elicitors usually refer to the extracellular signal compounds of
For personal use only.
plant cells that trigger or initiate plant defense responses and
phytoalexin synthesis (Ebel & Cosio, 1994). The formation of
secondary metabolites can be triggered by biotic elicitors,
signal molecules and abiotic elicitors (Wang & Wu, 2013).
Biotic elicitors of live bacteria, yeast, fungal polysaccharides,
lipid and glycoproteins have a positive effect on enhancing the
accumulation of desired natural products. Signal molecules,
including ROS, NO and jasmonic have been investigated to
increase the yield of pharmaceutical compounds. Abiotic
elicitors such as heavy metal ions, light and UV, temperature
shift and osmotic stress have also been applied extensively
(Sharma & Shahzad, 2013; Wang & Wu, 2013). Fungal
elicitor is widely used in increasing secondary metabolites
(Mandujano Chávez et al., 2000; Orbán et al., 2008). Table 2
shows the biotic elicitors, signal molecules and abiotic
elicitors used to stimulate pharmaceutical secondary metab-
olite production in MPCSC over the last 10 years.
Plants synthesize secondary metabolites in nature, which is
also considered as a defense against pathogens (Zhao et al.,
2005a,b). Plants have been found to elicit the same response
as the pathogen itself when challenged by elicitors, which are,
signals triggering the biosynthesis of the secondary metabol-
ites (Aly et al., 2010; Aschehouget al., 2012). Some endo-
phytic fungi isolated from the wild plant or the chemicals
produced by these fungi can also induce the defense response
as fungal elicitor (Porras-Alfaro & Bayman, 2011). As a
result, some particular secondary metabolites of the host plant
cells are induced to achieve a high accumulation. A new
antifungal monoterpene was isolated from suspended cells of
Piqueria trinervia by the induction of fungi isolated from
wild Piqueria trinervia (Saad et al., 2000). The relationship
between elicitors and the change of plant secondary metab-
olism has have been undertaken in MPCSC extensively.
Medicinal plant cell suspension cultures 9
An increasing number of studies have focused on the signal
transduction, gene expression and enzyme activity involved in
the biosynthesis of important pharmaceutical compounds
involving salicylic acid, paclitaxel, resveratrol, etc., after
elicitation (Belch et al., 2012; Mustafa et al., 2009; Nims
et al., 2006). These are potential steps in the pathway of
targeted metabolic engineering, which can lead to an increase
in the accumulation of medicinal secondary metabolites in
MPCSC.
Precursors feeding
Precursor feeding has been a normal and a popular approach
to increase the yield of medicinal compounds in MPCSC.
This method is based on the theory that any compound, which
is an intermediate involved in the biosynthetic pathway,
stands a good chance of increasing the accumulation of the
final product (Dörnenburg & Knorr, 1995). A great deal of
attempts is being expended to induce or increase the
production of the desired products with the addition of
precursors or intermediate compounds have demonstrated to
be effective in many cases.
Paclitaxel production was 2-fold higher than that without
optimization when Taxus chinensis cells were cultured with
phenylalanine and glycine (Luo & He, 2004). In the
L-phenylalanine (Phe) repetitive feeding culture, the max-
imum anthocyanin accumulation per culture was 30 and 81%
higher than those in a single Phe-feeding culture and non-
feeding culture, respectively (Edahiro et al., 2005). The
production of phenylethanoid glycosides (PeG) was enhanced
by feeding precursors in cell cultures of Cistanche deserticola
and phenylalanine were found to be the optimal precursor
(Ouyang et al., 2005). Feeding cis-farnesol, the precursor of
patchouli alcohol, to suspension cultured Pogostemon cablin
cells resulted in patchouli alcohol being increased from 19.5
to 25.5 mg/L (Bunrathep et al., 2006). The addition of
precursors such as tyrosine, phenylalanine, caffeic acid and
cucumber juice at the right concentrations could improve the
total accumulation of phenylethanoid glycosides in Cistanche
salsa cell cultures (Liu et al., 2007). Qu et al. (2011) found a
combination of phenylalanine and methyl jasmonate pro-
moted the highest level of anthocyanin biosynthesis, resulting
in 4.6- and 3.4-fold increases in anthocyanin content and yield
over the control respectively. Another study reported that
phenolic compound production was improved by varying
degrees after four precursor (L-phenylalanine, cinnamic acid,
ferulic acid and sinapic acid) feeding to Larrea divaricata cell
cultures (Palacio et al., 2012).
There are problems related to feeding in that a few
precursors a certain level is toxic to plant cells and can inhibit
cell growth instead of promoting it. The desired compound
synthesis and improper level of precursor is also liable to
inhibit secondary metabolism by a feedback mechanism. As a
result, screening of non-toxic precursors and determining
their safe level is extremely important.
Two-phase culture system and absorption culture
Ordinarily, the process of synthesis and storage of secondary
products in plant cells often takes place in separate compart-
ments. The biochemical synthesized by suspension cultured
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10

Table 2. Biotic elicitors, signal molecules and abiotic elicitors used to stimulate pharmaceutical secondary metabolite production in medicinal plant cell suspension cultures.

Yield after
Product Plant species Elicitors Yield of control elicitation Fold of control References
Biotic elicitors
Oleandrin Nerium oleander Aspergillus niger extract 0.35 mg/L 3.164 mg/L 8.8 Ibrahim et al. (2007)
W. Yue et al.

Anthraquinone Rubia tinctorum Coriolus versicolor derived 74.6 mg/g DW 262 mg/g DW 3.51 Orbán et al. (2008)
polysaccharide
Botrytis cinerea derived elicitor 74.6 mg/g DW 107 mg/g DW 1.43 Orbán et al. (2008)
Anthocyanin Perilla frutescens Yeast elicitor 0.9 g/L 10.2% DW Wang et al. (2004)
Oleanolic acid Perilla frutescens Yeast elicitor 13 mg/L 19 mg/L 1.46 Wang et al. (2004)
Ursolic acid Perilla frutescens Yeast elicitor 22 mg/L 27 mg/L 1.24 Wang et al. (2004)
Oleanolic acid Calendula officinalis Chitosan 0.074 mg/g DW 0.37 mg/g DW 5.00 Wiktorowska et al. (2010)
Yeast extract 0.088 mg/g DW 0.22 mg/g DW 2.50 Wiktorowska et al. (2010)
Trichoderma viride homogenate 0.067 mg/g DW 0.12 mg/g DW 1.80
phenylethanoid glycosides Cistanche deserticola Yeast elicitor 129 mg/L 237.7 mg/L 1.84 Cheng et al. (2005, 2006)
Chitosan 129 mg/L 317.8 mg/L 2.46
Sanguinarine Papaver somniferum Botrytis cinerea homogenate 15.6 mg/g DW 288.0 mg/g DW 18.4 Holková et al. (2010)
Xanthone Hypericum perforatum Combination of Methyl jasmonate Not published Not published 12.0 Conceição et al. (2006)
and Colletotrichum gloeospor-
ioides homogenate
3-O-glucosyl-resveratrol Vitis vinifera Saliva of Manduca sexta larvae 0.17 mg/g DW 1.18 mg/g DW 7.0 Cai et al. (2012b)
4-(3,5-dihydroxy- Vitis vinifera Saliva of Manduca sexta larvae 0.43 mg/g DW 1.53 mg/g DW 3.6 Cai et al. (2012b)
phenyl)-pheno
Total phenolics Cocos nucifera Chitosan 0.38 mg/g FW 1.3 mg/g FW 3.4 Chakraborty et al. (2009)
Taxane Taxus media Coronatine of Pseudomonas 8.14 mg/L 77.46 mg/L 9.5 Onrubia et al. (2012)
syringae
Umbelliferone Ammi majus Autoclaved lysate of Enterobacter 0.1 mg% of DW 9.6 mg% of DW 96 Staniszewska et al. (2003)
sakazaki.
Teucriosides Teucrium chamaedrys Mycelial extracts Not published Not published 1.7 Antognoni et al. (2012)
fromTrichoderma viridae
Teucriosides Teucrium chamaedrys Mycelial extracts fromFusarium Not published Not published 1.9 Antognoni et al. (2012)
moniliforme
Paclitaxel Taxus cuspidate Paclitaxel-producing fungal 3.14 mg/L 5.84 mg/L 1.8 Li & Tao (2009)
endophyte
10-Deacetyl- baccatin III Taxus cuspidate Paclitaxel-producing fungal 2.32 mg/L 25.86 mg/L 11 (Li & Tao (2009)
endophyte
Signal molecules
Anthraquinone Morinda citrifolia NO 3.92 mg/g DW 12 mg/g DW 3.06 Komaraiah et al. (2005)
Rubia tinctorum Methyl jasmonate 3.81 mg/g DW 6.65 mg/g DW 1.75 Komaraiah et al. (2005)
Jasmonic acid 9.17 mg/g DW 26.6 mg/g DW 2.9 Chong et al. (2005)
Jasmonic acid 74.6 mg/g DW 108.3 mg/g DW 1.452 Orbán et al. (2008)
Salicylic acid 74.6 mg/g DW 82.8 mg/g DW 1.11 Orbán et al. (2008)
trans-resveratrol Vitis vinifera Combination of salicylic acid and 0 2666.7 mg/L Yue et al. (2011)
methyl jasmonate
Silymarin Silybum marianum Methyl jasmonate Cells: 2.01 mg/g DW Not published 572 Sánchez-Sampedro et al. (2005)
Medium: 0.105 mg/50 ml Not published 315
Oleanolic acid Calendula officinalis Jasmonic acid 0.089 mg/g DW 0.84 mg/g DW 9.4 Wiktorowska et al. (2010)
Sanguinarine Papaver somniferum Methyl jasmonate 15.6 mg/g DW 169.5 mg/g DW 10.8 Holková et al., (2010)
Taxane Taxus media Methyl jasmonate 13.61 mg/L 38.41 mg/L 2.82 Bonfill et al. (2003)
Arachidonic acid 13.61 mg/L 50.80 mg/L 3.73 Bonfill et al. (2003)
Crit Rev Biotechnol, Early Online: 1–18
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Eleutheroside B Eleutherococcus Methyl jasmonate 38 mg/g DW 138 mg/g DW 3.5 Shohael et al. (2008)
sessiliflorus
Eleutheroside E Eleutherococcus sessiliflorus Methyl jasmonate 65 mg/g DW 174 mg/g DW 2.6
Eleutheroside E1 Eleutherococcus sessiliflorus Methyl jasmonate 72 mg/g DW 184 mg/g DW 2.5
Chlorogenic acids Eleutherococcus sessiliflorus Methyl jasmonate 2.1 mg/g DW 6.9 mg/g DW 3.2
Resveratrol Vitis vinifera Methyl jasmonate 0 150.8 mg/L Donnez et al. (2011)
Ginsenoside Panax ginseng N, N0 -dicyclohexylcarbodiimide 1.136 mg/g DW 3.362 mg/g DW 3.0 Huang et al. (2013)
Anthocyanins Vitis vinifera Indanoyl-isoleucine 1.78 DW 4.6 mg/g DW 2.6 Cai et al. (2012b)
4-(3,5-dihydroxy- Vitis vinifera Indanoyl-isoleucine N-linolenoyl- 0.05 mg/g DW 0.33mg/g DW 6.4 3.5 Cai et al. (2012b)
phenyl)-phenol i-glutamine 0.43 mg/g DW 1.49 mg/g DW
DOI: 10.3109/07388551.2014.923986

3-O-glucosyl-resveratrol Vitis vinifera N-linolenoyl-i-glutamine 1.3 mg/g DW 3.59 mg/g DW 2.8 2.4 Cai et al. (2012b)
Indanoyl-isoleucine 1.3 mg/g DW 3.16 mg/g DW
Taxane Taxus media Methyl jasmonate 8.14 mg/L 21.48 mg/L 2.6 Onrubia et al. (2012)
Umbelliferone Ammi majus benzo(1,2,3)-thiadiazole-7-car- 0.1 mg% of DW 0.4 mg% of DW 4 Staniszewska et al. (2003)
bothionic acid S-methyl ester
(BIONÕ )
Teucriosides Teucrium chamaedrys Methyl jasmonate 10 mg/g FW 50 mg/g FW 5 Antognoni et al. (2012)
Ajmalicine Catharanthus roseus Nitric oxide 12.6 mg/L 20.1 mg/L 1.6 Xu & Dong (2005a,b)
Catharanthine Catharanthus roseus Nitric oxide 8.3 mg/L 24.2 mg/L 2.9 Xu & Dong (2005a,b)
Total alkaloids Catharanthus roseus Nitric oxide 28.5 mg/L 51.3 mg/L 1.8 Xu & Dong (2005a,b)
Abotic elicitors
Anthraquinone Rubia tinctorum Combination of Proline and ami- Not published Not published 1.5 Perassolo et al. (2007)
noindan-2-phosphonic acid
Anthocyanin Vitis vinifera Combination of methyl jasmonate Not published Not published 1.9 Tassoni et al. (2012)
and red light
Stilbenes Vitis vinifera Combination of methyl jasmonate Not published Not published 1.5 Tassoni et al. (2012)
and red light
Taxane Taxus media Vanadyl sulfate 13.61 mg/L 39.14 mg/L 2.88 Bonfill et al. (2003)
Jaceosidin Saussurea medusa Combination of silver nitrate and 32.01 mg/L 84.3 mg/L 2.6 Zhao et al. (2005a,b)
glutathione (GSH)
Hispidulin Saussurea medusa Combination of silver nitrate and 3.11 mg/L 7.9 mg/L 2.5 Zhao et al. (2005a,b)
GSH
Teucriosides Teucrium chamaedrys Proline 5.08 mg/g FW 30.18 mg/g FW 5.9 Zhao et al. (2005a,b)
Teucriosides Teucrium chamaedrys Hydroxyproline 5.08 mg/g FW 73.16 mg/g FW 14.4 Antognoni et al. (2012
Teucriosides Teucrium chamaedrys Combination of proline and 5.08 mg/g FW 51.05 mg/g FW 10.1 Antognoni et al. (2012)
hydroxyproline
Ajmalicine Catharanthus roseus Cadmium 7.96 mg/L 36.5 mg/L 4.6 Zheng & Wu (2004)
3+
Crocin Crocus sativus La and Ce3+ 12.68 mg/L 90 mg/L 7.1 Chen et al. (2004)
Medicinal plant cell suspension cultures
11
 12 W. Yue et al.
plant cells are excreted into the culture medium and have a
negative effect due to the feedback inhibition on the cell
growth and on their own synthesis. Therefore, the pharma-
ceutical compounds cannot be produced continuously as
expected. A two-phase system employs the use of a
partitioning system to redistribute extracellular product into
a second, generally non-polar phase, which can effectively
avoid the effect of feedback inhibition (Collins-Pavao et al.,
1996; Roberts, 2007). Another advantage may be the
enhancement of releasing pharmaceutical secondary metab-
olites from the medium or the initiation of releasing
molecules normally stored within the cells. In a two-phase
system of Morinda citrifolia cell suspension culture,
the production of anthraquinone was 2-fold higher than that
observed in the control (Bassetti & Tramper, 1995).
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The productions of azadirachtin-related limonoids,
b-thujaplicin and paclitaxel were enhanced obviously by
two-phrase culture and the phenomenon of feedback was
effectively avoided (Choi et al., 2001; Raval et al., 2003;
Yamada et al., 2002)
The supply of artificial materials for the accumulation of
secondary products has been studied to be an advancing tool
for promoting biosynthesis in MPCSC. The formation of
secondary metabolites tends likely to be subject to feedback
inhibition or intracellular degradation, the removal and
sequestering of the product in an artificial site holds a
promise for total metabolite yield improvement. Activated
For personal use only.
charcoal (AC), cork tissue, liquid paraffin and XAD have
been reported to be ideal adsorbents (Cai et al., 2012a,b; Zare
et al., 2010). Addition of 1 g/L AC resulted in maximum
extracellular taxol (5.584 mg/L) which was 2.19-fold higher
than that in the control in suspension culture of Taxus baccata
(Kajani et al., 2010). It has been demonstrated that the
addition of cork tissue to Sophora flavescens suspension cell
culture stimulated the production of sophoraflavanone G and
most of the sophoraflavanone G was recovered from the
added cork tissue (Zhao et al., 2003). AmberliteÕ XAD-7HP
resin was used to improve ajmalicine removal rate of
Catharanthus roseus cell culture and avoid the effect of
feedback, which increased the overall production of ajmali-
cine (Wong et al., 2004).
Metabolic engineering
The goal of improving productivity of secondary metabolites
can be achieved by metabolic engineering, which requires the
knowledge of various metabolic pathways in plant cell
detailed. This methodology is also applied in exploring
novel plant derived compounds of biological activity. The
information is concluded towards the studies associated with
the enzymes involved in the secondary metabolic pathway,
their characterization, measuring their activity and their
regulation of metabolism (Hughes & Shanks, 2002).
The current state that a series of plant genes have been
cloned, which are used in gene expression and regulation,
enable metabolic engineering as a reliable technique to
increase target compound yields. Metabolic engineering
involves overexpressing the target pathway; overcoming
rate-limiting steps; suppressing of catabolism of the product
of interest; blocking other pathways competing with the target
Crit Rev Biotechnol, Early Online: 1–18
pathway; or any feasible combination of the above.
Despite of lacking the thorough knowledge related
with metabolic pathways and genes involved, metabolic
engineering is quite promising, which increases the yields
of known compounds, produces new metabolites for a plant
species or even biosynthesizes novel molecules for the plant
possibly.
Elicitation which is regarded as an effective approach to
increase the yield of secondary metabolite, closely related to
the signal transduction within plant cells. It is important to
focus on upstream signal transduction pathways regulating
expression of biosynthetic genes and transcription factors.
Some investigation of the current stage indicates Nitric oxide
(NO) plays a signal role in the elicitor-induced defense and
secondary metabolism activities (Wang & Wu, 2004; Xu &
Dong, 2005a,b). NO was investigated to mediate the fungal
elicitor-induced hypericin production of Hypericum perfor-
atum cell suspension cultures through a jasmonic-
acid-dependent signal pathway (Xu & Dong, 2005a,b).
Signaling molecules as salicylic acid (SA), jasmonates (JAs)
and ethylene jasmonate (ET) display as regulators in plant
defense responses against microbial pathogens (Memelink
et al., 2001). Cytosolic Ca2+ spiking is taking place normally
in fungal elicitor-induced jasmonate biosynthesis (Yazaki,
2005). Among the classes of metabolites that are induced by
JAs are free and conjugated forms of polyamines, quinones,
terpenoids, alkaloids, phenylpropanoids, glucosinolates and
antioxidants (Toubiana et al., 2012). Increasing studies prove
that genetic manipulation of the JAs signaling pathway can be
employed to enhance the accumulation of secondary metab-
olites. It has been reported that ectopic expression of a
JA-responsive transcription factor (ORCA3) increased the
yield of terpenoid indole alkaloids in Catharanthus roseus
cells (Van der Fits & Memelink, 2000, 2001). Studying on
signal transduction thoroughly will crucially improve speci-
ficity and efficiency of genetic modification using transcrip-
tion factors or biosynthetic genes, and it will further improve
the performance of manipulation of the metabolic flux
towards certain secondary metabolites and lead to optimal
production of medicinal products (Zhao et al., 2005a,b).
It is feasible to improve the production of desirable
compounds by the overexpression of genes controlling the
limiting steps or by suppressing the undesired product
biosynthesis. Several genes in the biosynthetic pathways for
plant alkaloids involving scopolamine, nicotine and berberine
have been cloned, making the metabolic engineering of these
alkaloids possible (Hughes & Shanks, 2002). Overexpression
of putrescine N-methyltransferase (PMT) increased the nico-
tine content in Nicotiana sylvestris, whereas suppression of
endogenous PMT activity severely decreased the nicotine
content, ectopic expression of (S)-scoulerine 9-O-methyl-
transferase (SMT) caused the accumulation of benzylisoqui-
noline alkaloids in Eschscholzia californica (Sato et al.,
2001). Overexpression of Petunia chi-a gene encoding
chalcone isomerase resulted in that transgenic tomato lines
produced an increase of up to 78-fold in fruit peel flavonols
(5–10 mg/kg FW; Muir et al., 2001). Overexpression of
cyp80b3 cDNA resulted in an up to 45% increase in
the amount of total alkaloid in Papaver somniferum (Frick
et al., 2007).
 DOI: 10.3109/07388551.2014.923986
Genes that are involved in the biosynthesis and transport of
secondary metabolites are important for systematic metabolic
engineering aimed at increasing the productivity of valuable
secondary metabolites (Yazaki, 2005). The process of genetic
engineering consists of isolation, characterization, reordering
of genetic material and its transfer to foreign organisms
(Kleckner et al., 1977). Efforts to apply genetic engineering
to increase the accumulation of a desired product include
exploring key biosynthetic genes and integrating target genes
into the plant cell genome. Agrobacterium-mediated trans-
formation with the merits of effective, cheap and simple to use
is regarded as the most common vector to transform numerous
plants. Wild-type Agrobacterium transfers T-DNA from its Ti
plasmid through the plant membranes and integrate it into the
genomic DNA of plant cells adjacent to a wound site (Lessard
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Nyu Medical Center on 06/29/14
et al., 2002). A Nerium oleander cell suspension culture was
derived from Agrobacterium tumefaciens-transformed calli
and the accumulation (3.164 mg/L) of oleandrin was 8.8-fold
higher than that of control (Ibrahim et al., 2007).
Phenylpropanoid pathway genes were activated by expression
of the maize C1 and R transcription factors in soybean, which
enhanced the accumulation of isoflavones in Glycine maxseed
(Yu & McGonigle, 2005). Technology of cDNA-amplified
fragment length polymorphism (AFLP) was applied to
understand the secondary metabolism of JA-elicited tobacco
cells and transcriptome analysis suggested an extensive JA-
mediated genetic reprogramming of metabolism, which
For personal use only.
correlated well with the observed shifts in the biosynthesis
of the metabolites investigated (Goossens et al., 2003). These
studies demonstrated the power of genetic engineering to
understand the structures of complex alkaloid natural prod-
ucts in MPCSC.
Scale-up of plant cell suspension culture
Features of plant cell cultured in bioreactors
MPCSC in bioreactors have been considered as an alternative
technology to obtain natural compounds for use in the
pharmaceutical industries (Rodrı́guez-Monroy & Galindo,
1999). Unlike microbial cells, plant cells exhibit some
drawbacks such as less stable in productivity, high shear
sensitive, low oxygen requirements, slow growth rate and they
often occur as cell clumps (Dörnenburg & Knorr, 1995).
Reduction of the impeller speeds of mechanically
agitated bioreactors is able to prevent shear damage to the
plant cells, but the gas bubbles cannot be dispersed effectively
by the impeller to some extent. Compared with microorgan-
isms, plant cells require less oxygen due to their slow
metabolism. Under some circumstances, a high oxygen
concentration is regarded as toxic to the metabolic activities
of plant cells, whereas high impeller speeds may strip
nutrients such as CO2 from the medium (Eibl & Eibl,
2008). Some high-yielding cell lines do not provide a
continuous high yield. Mixing evenly cannot be neglected
in MPCSC, especially when high cell concentrations and
large-volume bioreactors are used (Chattopadhyay et al.,
2002). For moderate cell concentrations, pneumatically
agitated bioreactors are befitting. As a result, different
configurations of bioreactors have been utilized according
to the nature of cells.
Medicinal plant cell suspension cultures 13
MPCSC has been more suitable for scale-up than tissue or
organ culture, meanwhile, the configuration of bioreactors
should be designed appropriately for different cell types. The
key endpoint is not only to increase the biomass, but also to
enhance the yield of the natural pharmaceuticals in an
economical way.
Application of different bioreactors in
pharmacological secondary metabolites production
Various types of bioreactors are extensively used for mam-
malian cell culture and microbial fermentation, which can
also be applied with proper modifications to MPCSC for
natural compound production (Eibl & Eibl, 2008). Adequate
oxygen mass transfer, low shear stress to cells, adequate
nutrient supply and product removal from cells should be
considered for choosing a suitable bioreactor and bioreactor
design (Eibl & Eibl, 2002, 2008). ain types of bioreactors
applied in plant cell fermentation are stirred-tank bioreactor,
bubble column bioreactor and airlift bioreactor.
Stirred-tank bioreactors are commonly used due to the
following advantages: (1) ease of large-scale production,
(2) use for high viscously cell culture, (3) high oxygen mass
transfer ability and (4) good fluid mixing andalternative
impellers (Huang & McDonald, 2009). An investigation of
Azadirachta indica suspension culture established the feasi-
bility of large-scale azadirachtin production in stirred tank
bioreactors (Prakash & Srivastava, 2007). A cell line of
Papaver somniferum was cultivated in bioreactors using
elicitation of plant pathogenic fungi, and optimal sanguinar-
ine production (300 mg/L) was achieved, which showed the
possibility of commercial production of sanguinarine using
large-scale MPCSC of Papaver somniferum (Park et al.,
1992). Production of podophyllotoxin by a suspension of
cultured Podophyllum hexandrum cells in a 3 L stirred tank
bioreactor represented a 27% increase in volumetric product-
ivity compared to shake flask cultivation after optimizations
of the carbon source, light condition and agitation speed
(Chattopadhyay et al., 2002). However, the disadvantages of
high shear stress around the impeller, high operational cost,
heat generation due to mechanical mixing, high energy cost
due to mechanic agitation and contamination risk with
mechanical seal should not be ignored.
Bubble column bioreactors possess the following advan-
tages: (1) favorable for plant cells, (2) easy to manipulate and
scale up, (3) cost effective and low shear stress. Some
problems such as poor oxygen mass transfer ability and poor
flow mixing in high density cultures and serious foaming
under high aeration conditions are usually improved (Smart &
Fowler, 1984). Suspension cultivation of a novel cell derived
from the microscopic filamentous gametophyte life phase of
the complex brown alga Laminaria saccharina was found to
be feasible in an illuminated bubble column bioreactor at
13  C with CO2 in the air as the sole carbon source for growth
(Zhi & Rorrer, 1996).
Airlift or modified airlift bioreactors also have some
advantages: (1) multiple-choice of internal draft tubes,
(2) better oxygen supply (Breuling et al., 1985). Cell culture
of Saussurea medusa in a 2 L periodically submerged airlift
bioreactor (PSAB) was investigated and this study concluded
 14 W. Yue et al.
that PSAB had advantages in improving cell growth of
Saussurea medusa and the production of total flavonoids
(501 mg/L; Yuan et al., 2004). As a result, an obstacle of
poor oxygen mass transfer ability compared with stirred-
tank bioreactor should be overcome. Other issues such as
poor fluid mixing of highly viscous culture and serious
foaming under high aeration conditions should not be
overlooked.
Conclusions and perspectives
MPCSC is a reliable model system for plant science research
and in vitro production of secondary metabolites for pharma-
ceutical use. The main advantage of this technology is that it
can be carried out under controlled conditions and various
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Nyu Medical Center on 06/29/14
elicitors can be utilized for increasing the accumulation of the
metabolites. It is independent of diverse geographical,
seasonal and environmental conditions and contributes a
stable production system, which ensures the continuous
accumulation of products with uniform quality and yield.
Numerous natural medicinal products with various activities
of anticancer, antitumor, antimalarial, antioxidative and
antidiabetic, etc., have been obtained through this biotech-
nology. In addition, many new compounds have been isolated
from suspended cells of medicinal plants combined with
biotransformation. The proposed research directions of bio-
transformation should lie in exploring the pharmacological
For personal use only.
activity of the new substances and enabling them to show
pharmacological activities by modifying their chemical
structures.
Systematic strategies involving selection of high-yielding
cell lines, optimizations of culture condition, addition of
elicitors and precursors, employing a two-phase culture
system absorption techniques and metabolic engineering are
utilized to enhance the accumulation of desired products.
Studies focusing on the signal transduction, gene expression
and enzyme activity in the biosynthesis of important
pharmaceutical compounds after elicitation display a good
tendency for further development.
Hundreds of MPCSCs can produce large amounts of
natural pharmaceutical compounds, however, there are few
successful cases of production on a commercial scale. Only
scopolamine, protoberberines, paclitaxel, rosmarinic acid,
ginsenoside saponins, echinaceae polysaccharides and shiko-
nin are commercially available via plant cell culture for
pharmaceutical applications (Cai et al., 2012a,b; Georgiev
et al. 2009; Wu & Zhong, 1999). The primary challenges,
from an expert perspective, lie in the area of process scale-up,
the reasons for these are stated below.
(1) The low proliferative effect and the unstable production
of natural compounds results in the high cost of the
industrial production.
(2) Due to the particularities of the plant cell presented,
selection of a suitable bioreactor for large-scale produc-
tion is a key problem.
(3) One of the main problems encountered is lacking basic
knowledge of the biosynthetic pathway, and mechanisms
responsible for the production of plant metabolites.
The prospect of producing high-value and low-yield
products such as anti-cancer compounds are necessary, thus
Crit Rev Biotechnol, Early Online: 1–18
focusing this technology on being able to make a few
potentially favorable pharmaceuticals.
A series of methods have also been applied to enhance the
accumulation of the desired natural products. MPCSC is still
one of the most potentially useful technologies that can be
utilized for the production of natural products. In addition, the
research emphasis of MPCSC should be placed on:
(1) Study of the pharmaceutical secondary metabolites which
are in short supply in clinical use, or difficult to obtain
from the natural plant and hard to synthesize.
(2) Clarify the biosynthetic routes of natural pharmaceutical
substances to exploit various methods to regulate the
biosynthetic process.
(3) Employ membrane permeabilization, cell immobilization
and in situ product removal techniques to improve the
secretion of the desired products into the extracellular
medium (Cai et al., 2012a,b).
(4) Combine effective strategies such as addition of elicitors,
precursors feeding, two-phase culture system and bio-
reactors enhance the production of desired compounds.
(5) (5) Explore appropriate selection of bioreactor design
and advanced bioreactor culture strategies.
In conclusion, this review summarizes the pharmaceutical
applications and high-yielding strategies of MPCSC technol-
ogy to date and focuses on the scale-up of plant cell culture, in
which lie the primary challenges of this unlimited potential
technology for producing more pharmaceuticals. MPCSC has
an extraordinary potential for providing natural products for
pharmaceutical and clinical use for the future.
Declaration of interest
This article was supported by the Young Scientist Special
Project of the National High Technology Research and
Development Program of China (No. 2014AA020508) and
Outstanding Youth Program of Shanghai Medical System
(No. XYQ2013100).
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