Medicinal plant cell suspension cultures: pharmaceutical applications and high-yielding strategies for the desired secondary metabolites.

Critical Reviews in Biotechnology

ISSN: 0738-8551 (Print) 1549-7801 (Online) Journal homepage: http://www.tandfonline.com/loi/ibty20

Medicinal plant cell suspension cultures: pharmaceutical applications and high-yielding strategies for the desired secondary metabolites Wei Yue, Qian-liang Ming, Bing Lin, Khalid Rahman, Cheng-Jian Zheng, Ting Han & Lu-ping Qin To cite this article: Wei Yue, Qian-liang Ming, Bing Lin, Khalid Rahman, Cheng-Jian Zheng, Ting Han & Lu-ping Qin (2016) Medicinal plant cell suspension cultures: pharmaceutical applications and high-yielding strategies for the desired secondary metabolites, Critical Reviews in Biotechnology, 36:2, 215-232, DOI: 10.3109/07388551.2014.923986 To link to this article: http://dx.doi.org/10.3109/07388551.2014.923986

Published online: 25 Jun 2014.

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Date: 08 October 2016, At: 18:14

http://informahealthcare.com/bty ISSN: 0738-8551 (print), 1549-7801 (electronic) Crit Rev Biotechnol, 2016; 36(2): 215–232 ! 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, 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 secondary metabolites has been underway for more than three decades. Plant cell cultures with the production of high-value secondary metabolites are promising potential alternative sources for the production of pharmaceutical agents of industrial importance. Medicinal plant cell suspension cultures (MPCSC), which are characterized with the feature of fermentation with plant cell totipotency, could be a promising alternative ‘‘chemical factory’’. However, low productivity becomes an inevitable obstacle limiting further commercialization of MPCSC and the application to large-scale production is still limited to a few processes. This review generalizes and analyzes the recent progress of this bioproduction platform for the provision of medicinal chemicals and outlines a range of trials taken or underway to increase product yields 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.

Application, medicinal plant, pharmaceutical, plant cell culture, secondary metabolite

Introduction Higher plants can produce a mass of substances that have always been an excellent source of pharmaceuticals, insecticides, flavorings, fragrances and food colorants (Kieran et al., 1997). These substances are traditionally obtained from naturally grown whole plants, e.g. ginsenoside is obtained from Panax quinquefolium for pharmaceutical use through large-scale crop cultivation, which usually takes 4–6 years (Zhong et al., 1996). There are regional and environmental restrictions, which can also limit the commercial production of natural compounds. A few important natural products with simple chemical structures such as aspirin can be produced via chemosynthesis, whereas other complex structures are hard to be synthesized or the cost of their synthesis outweighs their commercial availability (Danishefsky et al., 1995; Stevenson & Szczeklik, 2006). For some of these pharmaceutical compounds, medicinal plant cell suspension culture (MPCSC) can provide another alternative method besides traditional cultivation methods and chemical synthesis routes.

*Wei Yue and Qian-liang Ming contributed equally to this work. Address for correspondence: Dr. Cheng-Jian Zheng, Ting Han and LuPing Qin, Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, Shanghai 200433, China. Tel/Fax: +86 021-81871305 (C.-J. Zheng); +86 021-81871306 (T. Han); + 86 02181871300 (L.-P. Qin). E-mail: [email protected] (C.-J. Zheng); [email protected] (T. Han); [email protected] (L.-P. Qin)

History Received 12 September 2013 Revised 22 January 2014 Accepted 02 March 2014 Published online 19 June 2014

Plant tissue and cell suspension cultures have been investigated by biotechnological methods and provide a promising bioproduction platform for desired natural products. Tissue cultures of shoots or roots display undifferentiated metabolite characteristics compared to their parent plants, whereas similar cultures often accumulate target compounds to a less level (Kolewe et al., 2008). Hairy root culture obtained from Agrobacterium rhizogenes’ Ri T-DNA-mediated transformation is another technology, which is genetically stable and its growth is not restricted to additional growth regulators. This can also provide an increased capacity for secondary metabolite accumulation (Giri & Narasu, 2000). However, the major obstacle limits the commercial use of hairy roots to produce valuable plant secondary metabolites due to challenges in cultivating hairy roots in an industrial system (Giri & Narasu, 2000; Hansen et al., 1989). In contrast, MPCSC, which is simple and cost effective, can overcome the problems of large-scale production and has been extensively used. 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 metabolites for pharmaceutical use (Moscatiello et al., 2013). This technology offers an alternative attractive potential to whole plant for the production of high-value natural products such as paclitaxel (Li & Tao, 2009), resveratrol (Cai et al., 2012a,b), artemisinin (Baldi & Dixit, 2008), ginsenosides (Jeong et al., 2008) and ajmalicine

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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. (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 Pa´dua 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 biochemistry, the culture of protoplast and plant somatic hybridization. From an expert opinion, MPCSC has more of an immediate potential for commercial application compared to plant tissue or organ cultures (Ramachandra Rao & Ravishankar, 2002; Xu et al., 2011a,b). Meanwhile, there are still inevitable problems, including the instability of cell lines, slow growth and scale-up obstacles, which results in lower

Crit Rev Biotechnol, 2016; 36(2): 215–232

production of metabolites in MPCSC. Accordingly, most research efforts have been directed towards the commercialization of MPCSC. This review will introduce the MPCSC in detail, including its application to obtain important plant medicinal components, generalize systematic strategies to increase the production 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 analyzing 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, flavorings 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

Figure 1. Schematic depiction of the medicinal plant cell suspension culture (MPCSC) process for producing natural medicine.

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 notoginseng 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 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 metabolites of pharmaceutical potential, is shown in Table 1. Seeking desired or new active compounds through biotransformation Biotransformation has been widely applied in the process of herbal fermentation. Cultivated plant cells show the biochemical capability of transforming exogenously supplied compounds, 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 stereoselective, 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 biotransformation 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

217

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-hydroxybufotalin, 3-epi-gamabufo-talin and 3-dehydro-D1-gamabufotalin, 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-dihydroxy5a-pregnan-20-one-3b-O-b-glucoside (de Pa´dua et al., 2012).

Strategies to increase secondary metabolite yields Although the basic techniques for MPCSC are well established, 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 highyielding 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 conditions. 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 accumulated 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

Antitumour

Antimicrobial

Antihypertensive

Antimalarial

Antioxidative

Antimicrobial

Antifungal

Intestinal ailment

Antitumour

Antitumour Antimalarial Counterirritant

Regulation of cell growth

Calcium absorption Antimicrobial Antioxidative

Sedative Antitumour Anticoagulant

Alkaloids

Ajmalicine

Artemisinin

Anthocyanin

Anthraquinones

Antifungal monoterpene

Berberine

Camptothecin

Canthinone alkaloids Capsaicin

Cerebroside

Cholecalciferol Chlorogenic acid

Codeine Colchicine Coumarins

Main use

Abietane diterpenoids

Product

Papaver somniferum Colchium autumnale Ammi majus

Solanum malacoxylon Eucommia ulmoides

Lycium chinense

MS supplemented with 2 mg/L 2,4-D, 0.1 mg/L kinetin, and 30 g/L sucrose. MS supplemented with1 mg/L 2,4-D, 0.1 mg/L kinetin and 50 g/L sucrose. MS supplemented with 1 mg/L IAA,1 mg/L NAA, 0.1 mg/L kinetin and 40 g/L sucrose. MS supplemented with 30 g/L sucrose, 0.1 mg/L of each of NAA and kinetin. MS supplemented with 0.2 mg/L 2,4-D, 0.5 mg/L 6-BA and 30 g/L sucrose. LS supplemented with 30 g/L sucrose, 1 mg/L 2,4-D and 0.1 mg/L BA. B5 supplemented with 20 g/Lsucrose, 2 mg/L 2,4-D, 0.5 mg/L NAA, 0.5 mg/L IAA and 0.2 mg/L kinetin. MS supplemented with 30 g/Lsucrose, 1 mg /L IAA, 0.2 mg/L NAA and kinetin. MS supplemented with 1.0 mg/L 2,4-D and 0.1 mg/L kinetin. MS supplemented with 1 mg/L NAA and 1 mg/L BAP. LS supplemented with 10 mM NAA and 0.1 mM 6-BA. LS supplemented with 10 mM 2,4-D and 100 mM BAP. B5 supplemented with 1 mg/L 2,4-D, 0.5 mg/L kinetin and 40 g/L sucrose. Not investigated MS supplemented with 7.6 mmol/L 2,4-D and 2.3 mmol/L kinetin. MS supplemented with 1.0 ppm 2, 4-D and 0.1 ppm kinetin. B5 MS supplemented with 30 g/L sucrose, 300 mg/L LH and 2 mg/L 2,4-D. LS Not investigated B5supplemented with 30 g/L sucrose.

Medium

Continuous light Not investigated 16 h light

16 h light

Dark

Shake flask Not investigated Shake flask

Shake flask Shake flask

Shake flask

Not investigated Shake flask

Shake flask

16 h light Not investigated Continuous light

Shake flask

Shake flask

Shake flask

Shake flask

Dark

Dark

Not investigated

Dark

Shake flask

16 h light

Shake flask

16 h light

Shake flask

Shake flask

16h light

16 h light

Shake flask

Darkness

Shake flask

Shake flask

Continuous light

Continuous light

Shake flask

Culture type

Darkness

Photoperiod

Culture conditions

John Tam et al. (1980) Yoshida et al. (1988) Staniszewska et al. (2003)

Aburjai et al. (1997) Wang et al. (2003)

Jang et al. (1998)

Roberts (1994) Sudha & Ravishankar (2003)

Pasqua et al. (2006)

Nakagawa et al. (1984)

Hara et al. (1988)

Saad et al. (2000)

Nazif et al. (2000)

Perassolo et al. (2011)

Chiang & Abdullah (2007)

Zhang et al. (1997b)

Wang et al. (2004)

Baldi & Dixit (2008b)

Ten Hoopen et al. (2002a)

Crespi-Perellino et al. (1986)

Xu et al. (2011)

Reference

W. Yue et al.

Brucea spp. Capsicum frutescens

Camptotheca acuminate

Thalictrum minus

Coptis japonica

Piqueria trinervia

Cassia acutifolia

Rubia tinctorum

Morinda elliptica

Fragaria ananassa

Vitis vinifera

Artemisia annua

Catharanthus roseus

Ailanthus altissima

Cephalotaxus fortune

Species

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

218 Crit Rev Biotechnol, 2016; 36(2): 215–232

Antioxidative Antimicrobial

Heart stimulant

Steroidal precursor

Anti-HIV Analgesic Anti-inflammatory Antipyretic Diuretic Antitumour Antiparasitic

Bronchial asthma Antitumour Antioxidative

Antitumour Antimicrobial Health tonic

Health tonic

Antidiabetic

Antitumor

Antimicrobial Antitumor

Antidepressive

Diabetics

Antitumour Antioxidative Antitumour

Anti-Parkinson

Sedative Arthritis Digestive disorders Rheumatism Venereal diseases

Cryptotanshinone

Digoxin

Diosgenin

Dipyranocoumarins Eleutheroside

Forskolin Furanocoumarin

Furoquinoline alkaloids Ginkgolides

Ginsenosides

Gymnemic acid

Hispidulin

Homoisoflavonoids

Hypericin

Inulin

Isoquinoline alkaloids

L-dihydroxyphenylalanine

Morphine Phenolic compound

Jaceosidin

Ellipticine Emetine

Anticancer

Crocin

Papaver somniferum Larrea divaricata

Mucuna pruriens

Saussurea medusa

Fumaria capreolata

Helianthus tuberosus

Hypericum perforatum

Caesalpinia pulcherrima

Saussurea medusa

Gymnema sylvestre

Panax ginseng

Choisya ternata Ginkgo biloba

Coleus forskolii Glehnia littoralis

Calophyllum inophyllum Eleutherococcus sessiliflorus Ochrosia elliptica Cephaelis ipecacuanha

Dioscorea deltoidea

Digitalis lanata

Salvia miltiorrhiza

Crocus sativus

MS supplemented with 30 g/L sucrose, 10 g/L glucose, 100 mg/L myo-inositol, 0.5 mg /L BA and 2 mg/L NAA. MS supplemented with 1mg/L IAA, 1 mg/L BA and 40 g/L saccharose. LS MS supplemented with 9 mM 2,4-D and 5 mM BA.

DAM MS supplemented with IBA, IAA and 60 g/L sucrose. B5 supplemented with 30 g/L sucrose. LS supplemented with 1 mg/L 2,4-D and 1 mg/L kinetin. Not investigated MS supplemented with 30 g/L sucrose and 3.5 mg/L NAA. MS supplemented with 1 mg /L 2,4-D, 0.1 mg/L kinetin and 30 g/L sucrose. MS supplemented with 0.5mg/L BA and 1.5 mg/L IAA. MS supplemented with 30 g/L sucrose, 10 g/L glucose, 100 mg/L myo-inositol, 0.5 mg/L BA and 2 mg/L NAA. MS supplemented with 10 mM 2,4-D and 1 mM 6-BA. MS supplemented with 0.90 mM 2,4-D, 0.11 mM kinetin and 30 g/L sucrose. MS supplemented with 1 mg/L NAA and 1 mg/L BA. LS

B5 supplemented with 300 mg/L casein hydrolysate, 2 mg/L NAA, 2 mg/L IAA and 0.5 mg/L BA. MS medium supplemented with 30 g/L sucrose. MS supplemented with 340 mg/L KH2PO4, 4 mg/L glycine and 33 g/L glucose. MS supplemented with 0.1 mg/L 2,4D. WPM with 20g/L sucrose. MS supplemented with 1mg/L 2,4-D.

Continuous light 16 h light


Shake flask

Shake flask

Continuous light

Continuous light

Not investigated

Not investigated

Not investigated

Shake flask

Dark and light Not investigated

Shake flask

Shake flask

Continuous light

Dark

Shake flask

Shake flask

Dark 16 h light

Not investigated Shake flask


Not investigated Not investigated

Shake flask

Not investigated Dark

Dark Dark

Not investigated Not investigated

Continuous light Dark

Shake flask

Shake flask

Continuous light Dark

Shake flask

Shake flask

Dark

Darkness

(continued )

Huang & Kutchan (2000) Palacio et al. (2012)

Pras et al. (1993)

Zhao et al. (2005)

Rueffer (1985)

Taha et al. (2012)

Walker et al. (2002)

Zhao et al. (2004)

Zhao et al. (2005)

Praveen et al. (2011)

Zhong et al. (1996)

Creche et al. (1993) Kang et al. (2009)

Mukherjee et al. (2000) Kitamura et al. (1998)

Kouamo et al. (1985) Jha et al. (1991)

Pawar & Thengane (2009) Shohael et al. (2008)

Tal et al. (1984)

Kreis & Reinhard (1992)

Tsutomu et al. (1983)

Chen et al. (2003)

DOI: 10.3109/07388551.2014.923986

Medicinal plant cell suspension cultures 219

Antitumour

Hpyerglycemic Antimicrobial Antiphlogistic

Antihypertensive

Health tonic

Antimalarial

Antioxidative

Antiplaque

Antibacterial

Podophyllotoxin

Polyphenols

Reserpine

Resveratrol

Robustaquinones

Rosmarinic acid

Sanguinarine

Shikonin

Quassin

Anticancer Antimicrobial Antifertil

Olea europaea

Antioxidative Anti-inflammatory Antihypertensive Health tonic

Lithospermum erythrorhizon

Papaver somniferum

B5 supplemented with 20 g/L glucose, 1.0 mg/L 2,4-D and 0.5 mg/L kinetin. LS supplemented with 1 mM 2,4-D, 1 mM kinetin and KNO3 instead of NH4NO3. B5 supplemented with 30 g/L sucrose and 250 mg/L casein hydrolysate, 0.1 mg/L NAA and 0.2 mg/L kinetin. B5 supplemented with 20 g/L sucrose, 1mg/L 2,4-D, 0.2 mg/L kinetin and 50 mg/L cysteine. B5 supplemented with 30 g/L sucrose, 1mg/L 2,4-D, 0.1 mg/L kinetin. MS supplemented with 30 g/L sucrose, 0.5 mg/L 2,4-D, 0.5 mg/L kinetin and vitamins. CB2 Not investigated LS supplemented with 1 mM 2,4-D, and 1 mM kinetin. MS supplemented with 0.1 mg/L 2,4-D, 0.5 mg BA and 30 g/L sucrose. MS supplemented with 0.1 mg/L kinetin, 2 mg/L NAA and 30 g/L sucrose. LS supplemented with 10 mM IAA and 10 mM kinetin.

MS supplemented with 40 g/L glucose and 3.0 mg/L IAA. MS supplemented with 30 g/L sucrose, 1 mg/L 2,4-D, 0.1 mg/L kinetin and K+ instead of NHþ 4. MS supplemented with 5.7 mM IAA and 4.40 mM BAP. Heller supplemented with 0.25 mg/L NAA, 5 mg/L IBA and 0.05 mg/L BAP. MS supplemented with 30 g/L glucose, 30 g/L sucrose, 11.4 mL IAA, 1.5 mg/L pectinase and 10 g/L polyvinylpyrrollidone. MS supplemented with 2,4-D and BA.

Medium

Shake flask

Continuous light

Shake flask

Shake flask

Continuous light

Dark

Not investigated Not investigated Shake flask

Not investigated Not investigated Dark

Shake flask

Continuous light

Shake flask

Continuous light

Shake flask

Shake flask

Dark

Continuous light

Shake flask

Dark

Shake flask

Continuous light

Bioreactor

16 h light

Not investigated

Shake flask

16 h light

Not investigated

Shake flask

Shake flask

Dark 16 h light

Shake flask

Culture type

Dark

Photoperiod

Culture conditions

Yamamoto et al. (2000b)

Holkova´ et al. (2010)

Archambault et al. (1996)

Szabo et al. (1999) Sumaryono et al. (1991) Mizukami et al. (1992)

Hippolyte et al. (1992)

De-Eknamkul & Ellis (2007)

Schripsema et al. (1999)

Yue et al. (2011)

Yamamoto & Yamada (1986)

Scragg & Allan (1986)

Ishimaru et al. (1993)

Chattopadhyay et al. (2002)

Komaraiah et al. (2003)

Shinde et al. (2009)

Orihara & Ebizuka (2010)

Liu et al. (2007a)

Reference

W. Yue et al.

Coleus blumei Orthosiphon aristatus Lithospermum erythrorhizon Sanguinaria canadensis

Salvia officinalis

Anchusa officinalis

Cinchona robusta

Vitis vinifera

Rauwolfia serpentina

Picrasma quassioides

Cornus kousa

Podophyllum

Drosophyllum lusitanicum

Psoralea corylifolia

Cistanche salsa

Species

Anhrodisiacs

Main use

Plumbagin

Phytoestrogens

Phenylethanoid glycosides

Product

Table 1. Continued



Chen et al. (1997) Pan et al. (2000)

Orihara & Furuya (1990)

Ho et al. (2010)

Endo et al. (1987)

Nagella & Murthy (2010)


Shake flask

Shake flask

Shake flask

Shake flask

Dark Dark

Dark

Dark

Dark

16 h light

Sańchez-Sampedro et al. (2005)

Health tonic Antistress Withanolide A

Withania somnifera

Antileukemic Vinblastine

Catharanthus roseus

Antiinflammation Antitumor Antidiabetes Triterpene

Eriobotrya japonica

Antihypertensive Theamine

Camellia Sinensis

Cardiac disorders Anticancer Tanshinone Taxol

Salvia miltiorrhiza Taxus brevifolia

Liver ailment Silymarin

Silybum marianum

MS supplemented with 30 g/L sucrose,1 mg/L 2,4-D and 0.5 mg/L BA. B5 supplemented with 30 g /L sucrose. B5 supplemented with 2 B5 vitamins, 30 g/L sucrose, 10 mM 2,4-D, 4 mM kinetin and 1 mM GA3. MS supplemented with 30 g/L sucrose, 2 mg/L IBA and 0.1 mg/L kinetin. MS supplemented with 2.5 mg/L 6BA, 1 mg/L NAA and 30 g/L sucrose. MS supplemented with 30 g/L sucrose, 0.5 mg/L NAA and 0.5 mg/ L BA. MS supplemented with 30 g/L sucrose.

Dark

Shake flask

DOI: 10.3109/07388551.2014.923986

221

stress, and only cells that are able to resist the selection procedures will survive (Ramachandra Rao & Ravishankar, 2002). p-Fluorophenylalanine (PFP) was extensively applied in selecting high-phenolics-yielding cell lines, which is an analogue of phenylalanine. A selection of cell aggregates of 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). Optimization of MPCSC conditions Culture conditions play an important role in the quality and quantity of the material obtained through MPCSC. 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 pH, temperature, light and oxygen are considered easy to regulate the expressions of plant secondary metabolite pathways. Constituents in plant cell culture medium are determinants of growth and production of secondary metabolites (Zhang & Zhong, 1997). Several types of growth and production medium are put 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 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 and plant growth regulators are appropriate for MPCSC (Han & 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 triterpene production was optimal, when Eriobotrya japonica 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 ratio of 7.19/18.80) were investigated to reach the highest accumulation of biomass and gymnemic acid (11.35 mg/g DW; Praveen et al., 2011). The maximum production of daidzein (2.20% DW) and genistein (0.29% DW) was obtained when medium comprised with NHþ 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 medium pH of 5.8 was found feasible in cell suspension cultures of Withania somnifera for the production of withanolide A (Nagella & Murthy, 2010) and the extremes of pH should be avoided.

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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 highdensity 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-dichlorophenoxyacetic acid (2,4-D), a-naphthaleneacetic acid (NAA), indole3-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 differentiation (Coste et al., 2011; Komatsuda et al., 1992). In the suspension culture of Eriobotrya japonica cells, supplied with

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 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 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 Cha´vez et al., 2000; Orbań et al., 2008). Table 2 shows the biotic elicitors, signal molecules and abiotic elicitors used to stimulate pharmaceutical secondary metabolite 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 metabolites (Aly et al., 2010; Aschehouget al., 2012). Some endophytic 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 metabolism has have been undertaken in MPCSC extensively.


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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 (Do¨rnenburg & 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 maximum anthocyanin accumulation per culture was 30 and 81% higher than those in a single Phe-feeding culture and nonfeeding 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 promoted 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 compartments. The biochemical synthesized by suspension cultured

Nerium oleander Rubia tinctorum

Perilla frutescens Perilla frutescens Perilla frutescens Calendula officinalis

Cistanche deserticola

Papaver somniferum Hypericum perforatum

Vitis vinifera Vitis vinifera

Cocos nucifera Taxus media

Ammi majus

Teucrium chamaedrys

Teucrium chamaedrys

Taxus cuspidate

Taxus cuspidate

Morinda citrifolia Rubia tinctorum

Vitis vinifera

Silybum marianum

Calendula officinalis Papaver somniferum Taxus media

Anthocyanin Oleanolic acid Ursolic acid Oleanolic acid

phenylethanoid glycosides

Sanguinarine Xanthone

3-O-glucosyl-resveratrol 4-(3,5-dihydroxyphenyl)-pheno Total phenolics Taxane

Umbelliferone

Teucriosides

Teucriosides

Paclitaxel

10-Deacetyl- baccatin III

Anthraquinone

trans-resveratrol

Silymarin

Oleanolic acid Sanguinarine Taxane

Plant species

Oleandrin Anthraquinone

Product

DW DW DW DW DW Cells: 2.01 mg/g DW Medium: 0.105 mg/50 ml 0.089 mg/g DW 15.6 mg/g DW 13.61 mg/L 13.61 mg/L

3.92 mg/g 3.81 mg/g 9.17 mg/g 74.6 mg/g 74.6 mg/g 0

2.32 mg/L

3.14 mg/L

Not published

Not published

0.1 mg% of DW

0.38 mg/g FW 8.14 mg/L

Not published Not published 0.84 mg/g DW 169.5 mg/g DW 38.41 mg/L 50.80 mg/L

12 mg/g DW 6.65 mg/g DW 26.6 mg/g DW 108.3 mg/g DW 82.8 mg/g DW 2666.7 mg/L

25.86 mg/L

5.84 mg/L

Not published

Not published

9.6 mg% of DW

1.3 mg/g FW 77.46 mg/L

1.18 mg/g DW 1.53 mg/g DW

107 mg/g DW 10.2% DW 19 mg/L 27 mg/L 0.37 mg/g DW 0.22 mg/g DW 0.12 mg/g DW 237.7 mg/L 317.8 mg/L 288.0 mg/g DW Not published

74.6 mg/g DW 0.9 g/L 13 mg/L 22 mg/L 0.074 mg/g DW 0.088 mg/g DW 0.067 mg/g DW 129 mg/L 129 mg/L 15.6 mg/g DW Not published 0.17 mg/g DW 0.43 mg/g DW

3.164 mg/L 262 mg/g DW

Yield after elicitation

0.35 mg/L 74.6 mg/g DW

Yield of control

572 315 9.4 10.8 2.82 3.73

3.06 1.75 2.9 1.452 1.11

11

1.8

1.9

1.7

96

3.4 9.5

7.0 3.6

1.46 1.24 5.00 2.50 1.80 1.84 2.46 18.4 12.0

1.43

8.8 3.51

Fold of control

Wiktorowska et al. (2010) Holkova´ et al., (2010) Bonfill et al. (2003) Bonfill et al. (2003)

Sańchez-Sampedro et al. (2005)

Komaraiah et al. (2005) Komaraiah et al. (2005) Chong et al. (2005) Orbań et al. (2008) Orbań et al. (2008) Yue et al. (2011)

(Li & Tao (2009)

Li & Tao (2009)

Antognoni et al. (2012)

Antognoni et al. (2012)

Staniszewska et al. (2003)

Chakraborty et al. (2009) Onrubia et al. (2012)

Cai et al. (2012b) Cai et al. (2012b)

Holkova´ et al. (2010) Conceiçaõ et al. (2006)

Cheng et al. (2005, 2006)

Orbań et al. (2008) Wang et al. (2004) Wang et al. (2004) Wang et al. (2004) Wiktorowska et al. (2010) Wiktorowska et al. (2010)

Ibrahim et al. (2007) Orbań et al. (2008)

References

W. Yue et al.

Jasmonic acid Methyl jasmonate Methyl jasmonate Arachidonic acid

Chitosan Coronatine of Pseudomonas syringae Autoclaved lysate of Enterobacter sakazaki. Mycelial extracts fromTrichoderma viridae Mycelial extracts fromFusarium moniliforme Paclitaxel-producing fungal endophyte Paclitaxel-producing fungal endophyte Signal molecules NO Methyl jasmonate Jasmonic acid Jasmonic acid Salicylic acid Combination of salicylic acid and methyl jasmonate Methyl jasmonate

Biotic elicitors Aspergillus niger extract Coriolus versicolor derived polysaccharide Botrytis cinerea derived elicitor Yeast elicitor Yeast elicitor Yeast elicitor Chitosan Yeast extract Trichoderma viride homogenate Yeast elicitor Chitosan Botrytis cinerea homogenate Combination of Methyl jasmonate and Colletotrichum gloeosporioides homogenate Saliva of Manduca sexta larvae Saliva of Manduca sexta larvae

Elicitors

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


Vitis vinifera

Taxus media Ammi majus

Teucrium chamaedrys Catharanthus roseus Catharanthus roseus Catharanthus roseus

Rubia tinctorum

Vitis vinifera

Vitis vinifera

Taxus media Saussurea medusa

Saussurea medusa

Teucrium chamaedrys Teucrium chamaedrys Teucrium chamaedrys

Catharanthus roseus Crocus sativus

Taxane Umbelliferone

Teucriosides Ajmalicine Catharanthine Total alkaloids

Anthraquinone

Anthocyanin

Stilbenes

Taxane Jaceosidin

Hispidulin

Teucriosides Teucriosides Teucriosides

Ajmalicine Crocin

Eleutherococcus sessiliflorus Eleutherococcus sessiliflorus Eleutherococcus sessiliflorus Eleutherococcus sessiliflorus Vitis vinifera Panax ginseng Vitis vinifera Vitis vinifera

Eleutheroside E Eleutheroside E1 Chlorogenic acids Resveratrol Ginsenoside Anthocyanins 4-(3,5-dihydroxyphenyl)-phenol 3-O-glucosyl-resveratrol

Eleutheroside B Methyl jasmonate Methyl jasmonate Methyl jasmonate Methyl jasmonate N, N0 -dicyclohexylcarbodiimide Indanoyl-isoleucine Indanoyl-isoleucine N-linolenoyli-glutamine N-linolenoyl-i-glutamine Indanoyl-isoleucine Methyl jasmonate benzo(1,2,3)-thiadiazole-7-carbothionic acid S-methyl ester (BIONÕ ) Methyl jasmonate Nitric oxide Nitric oxide Nitric oxide Abotic elicitors Combination of Proline and aminoindan-2-phosphonic acid Combination of methyl jasmonate and red light Combination of methyl jasmonate and red light Vanadyl sulfate Combination of silver nitrate and glutathione (GSH) Combination of silver nitrate and GSH Proline Hydroxyproline Combination of proline and hydroxyproline Cadmium 3+ La and Ce3+

Methyl jasmonate

7.96 mg/L 12.68 mg/L

5.08 mg/g FW 5.08 mg/g FW 5.08 mg/g FW

3.11 mg/L

13.61 mg/L 32.01 mg/L

Not published

Not published

Not published

36.5 mg/L 90 mg/L

30.18 mg/g FW 73.16 mg/g FW 51.05 mg/g FW

7.9 mg/L

39.14 mg/L 84.3 mg/L

Not published

Not published

Not published

50 mg/g FW 20.1 mg/L 24.2 mg/L 51.3 mg/L

174 mg/g DW 184 mg/g DW 6.9 mg/g DW 150.8 mg/L 3.362 mg/g DW 4.6 mg/g DW 0.33mg/g DW 1.49 mg/g DW 3.59 mg/g DW 3.16 mg/g DW 21.48 mg/L 0.4 mg% of DW

65 mg/g DW 72 mg/g DW 2.1 mg/g DW 0 1.136 mg/g DW 1.78 DW 0.05 mg/g DW 0.43 mg/g DW 1.3 mg/g DW 1.3 mg/g DW 8.14 mg/L 0.1 mg% of DW 10 mg/g FW 12.6 mg/L 8.3 mg/L 28.5 mg/L

138 mg/g DW

38 mg/g DW

4.6 7.1

5.9 14.4 10.1

2.5

2.88 2.6

1.5

1.9

1.5

5 1.6 2.9 1.8

2.6 4

2.8 2.4

3.0 2.6 6.4 3.5

2.6 2.5 3.2

3.5

Zheng & Wu (2004) Chen et al. (2004)

Zhao et al. (2005a,b) Antognoni et al. (2012 Antognoni et al. (2012)

Zhao et al. (2005a,b)

Bonfill et al. (2003) Zhao et al. (2005a,b)

Tassoni et al. (2012)

Tassoni et al. (2012)

Perassolo et al. (2007)

Antognoni et al. (2012) Xu & Dong (2005a,b) Xu & Dong (2005a,b) Xu & Dong (2005a,b)

Onrubia et al. (2012) Staniszewska et al. (2003)

Cai et al. (2012b)

Donnez et al. (2011) Huang et al. (2013) Cai et al. (2012b) Cai et al. (2012b)

Shohael et al. (2008)

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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 pharmaceutical 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 metabolites 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). 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 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 ajmalicine (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


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 perforatum cell suspension cultures through a jasmonicacid-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 metabolites. 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 specificity and efficiency of genetic modification using transcription 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 nicotine content in Nicotiana sylvestris, whereas suppression of endogenous PMT activity severely decreased the nicotine content, ectopic expression of (S)-scoulerine 9-O-methyltransferase (SMT) caused the accumulation of benzylisoquinoline 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 transformation 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 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 JAmediated genetic reprogramming of metabolism, which 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 products 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 (Rodrı´guez-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 (Do¨rnenburg & 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 microorganisms, 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.


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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 mammalian 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 feasibility 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 sanguinarine 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 productivity 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 advantages: (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

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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 stirredtank 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 pharmaceutical use. The main advantage of this technology is that it can be carried out under controlled conditions and various 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 biotechnology. In addition, many new compounds have been isolated from suspended cells of medicinal plants combined with biotransformation. The proposed research directions of biotransformation should lie in exploring the pharmacological 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 shikonin 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 production 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


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 bioreactors 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 technology 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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Progesterone biotransformation by plant cell suspension cultures.

Improvement of hairy root cultures and plants by changing biosynthetic pathways leading to pharmaceutical metabolites: strategies and applications.

Electrospray differential mobility analysis for nanoscale medicinal and pharmaceutical applications.

Nitration of plant apoplastic proteins from cell suspension cultures.

Cell-cycle arrest of plant suspension cultures by tunicamycin.

Endotransglycosylation of xyloglucans in plant cell suspension cultures.

Light-induced fluctuations in biomass accumulation, secondary metabolites production and antioxidant activity in cell suspension cultures of Artemisia absinthium L.

A method for the rapid production of fine plant cell suspension cultures.

A generic approach for "shotgun" analysis of the soluble proteome of plant cell suspension cultures.

[Possibilities of using tissue cultures (= cell cultures) in medicinal plant research].

Cell division and plant development from protoplasts of carrot cell suspension cultures.

A new micro-method for testing plant growth retardants in cell suspension cultures.

Plant cell suspension cultures as model systems for investigating growth regulating compounds.

Putting the Spotlight Back on Plant Suspension Cultures.

The effects of cadmium chloride on secondary metabolite production in Vitis vinifera cv. cell suspension cultures.

On the role of S: Sulfotransferases in assimilatory sulfate reduction by plant cell suspension cultures.

Expression of the plant sulphite reductase in cell suspension cultures from Catharanthus roseus L.

Comparison of secondary product accumulation in photoautotrophic, photomixotrophic and heterotrophic Nicotiana tabacum cell suspension cultures.

Direct somatic embryogenesis and plant regeneration from single cell suspension cultures of Elymus giganteus Vahl.

Production, maintenance and plant regeneration from cell suspension cultures of Stevia rebaudiana (Bert.) Bertoni.

Human and plant fungal pathogens: the role of secondary metabolites.

Evidence for biological denitrification inhibition (BDI) by plant secondary metabolites.

Yield improvement strategies for the production of secondary metabolites in plant tissue culture: silymarin from Silybum marianum tissue culture.

Plant regeneration from long-term suspension cultures of white clover.