plant in vitro culture for the production of antioxidants — a review

Biotechnology Advances 26 (2008) 548–560

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Biotechnology Advances

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Research review paper

Plant in vitro culture for the production of antioxidants — A review

Adam MatkowskiDepartment Pharmaceutical Biology, Medical University in Wroclaw, Al Jana Kochanowskiego 10, 51-601 Wroclaw, Poland

E-mail address: [email protected].

0734-9750/$ – see front matter © 2008 Elsevier Inc. Aldoi:10.1016/j.biotechadv.2008.07.001

a b s t r a c t
a r t i c l e i n f o
Article history:
Plant in vitro cultures are ab Received 4 April 2008Received in revised form 1 July 2008Accepted 10 July 2008Available online 16 July 2008
Keywords:AntioxidantIn vitro cultureMedicinal plantsBiosynthesis

le to produce and accumulate many medicinally valuable secondary metabolites.Antioxidants are an important groupofmedicinal preventive compounds aswell as being food additives inhibitingdetrimental changes of easily oxidizable nutrients. Many different in vitro approaches have been used forincreased biosynthesis and the accumulation of antioxidant compounds inplant cells. In the present review someof the most active antioxidants derived from plant tissue cultures are described; they have been divided into themain chemical groups of polyphenols and isoprenoids, and several examples also from other chemical classes arepresented. The strategies used for improving the antioxidants in vitro production efficiency are also highlighted,including media optimization, biotransformation, elicitation, Agrobacterium transformation and scale-up.

© 2008 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5481.1. What do we call an antioxidant? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5491.2. Why hunt for antioxidants in vitro? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5491.3. Methods used for assessment of antioxidant properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 549

2. Chemical classes of antioxidant secondary metabolites from in vitro cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5502.1. Polyphenols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5502.2. Isoprenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5532.3. Other structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 553

3. Strategies for increased production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5553.1. Optimization of biosynthesis by culture conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5553.2. Production in differentiated tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5553.3. Selection of high-producing cell lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5553.4. Precursor feeding and biotransformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5563.5. Elicitation and stress induced production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5563.6. Transformation with Agrobacterium rhizogenes — hairy roots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5573.7. Scale-up or the never-ending bioreactor story . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5573.8. Concluding remarks and future perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557

1. Introduction

The use of plant cell and tissue culture methodology as a meansof producing medicinal metabolites has a long history (Rout et al.,2000; Verpoorte et al., 2002). Since plant cell and tissue cultureemerged as a discipline within plant biology, researchers haveendeavored to utilize plant cell biosynthetic capabilities for obtaininguseful products and for studying the metabolism (Misawa, 1994;Verpoorte et al., 2002).

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Cultured plant cells synthesize, accumulate and sometimes exudemany classes of metabolites. Medicinal compounds are of particularinterest and much effort has been devoted to obtaining some of themost active and precious therapeutics. Numerous alkaloids, saponins,cardenolides, anthraquinones, polyphenols and terpenes have beenreported from in vitro cultures and reviewed several times (Misawa,1994; Verpoorte et al., 2002; Vanisree and Tsay, 2004).

In recent decades, interest in chemopreventive plant naturalproducts has grown rapidly. The etiology of several degenerative andaging-related diseases has been attributed to oxidative stress, andnumerous studies have beenundertaken to search for themost effectiveantioxidants (Halliwell, 1995; Aruoma, 2003; Soobrattee et al., 2005).

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The present review summarizes the achievements of plant celland tissue culture technology for the production of secondarymetabolites which have significant potential as antioxidants. Thefocus will be on the major chemical classes of antioxidants and on theapproaches used to improve the in vitromethods for producing thesecompounds.

1.1. What do we call an antioxidant?

Broadly defined, an antioxidant is a compound that inhibits ordelays the oxidation of substrates even if the compound is presentin a significantly lower concentration than the oxidized substrate(Halliwell, 1995; Halliwell and Gutteridge, 2007). The scavenging ofreactive oxygen species (ROS) is one of possible mechanism of action.Others include the prevention of ROS formation by metal binding orenzyme inhibition. Chain breaking antioxidants prevent damage byinterfering with the free radical propagation cascades.

The antioxidant compounds can be recycled in the cell or areirreversibly damaged, but their oxidation products are less harmful orcan be further converted to harmless substances (Halliwell andGutteridge, 2007). At the cellular and organism level the antioxidantprotection is provided by numerous enzymes and endogenous smallmolecular weight antioxidants such as ascorbic acid, uric acidglutathione, tocopherols and several others. Many compounds containantioxidant activity in addition to their specialized physiologicalfunction, and their importance as antioxidants in vivo is sometimesambiguous (Azzi et al., 2004). The antioxidant activity of plantsecondary metabolites has been widely established in in vitro systemsand involves several of the above mentioned mechanisms of action.Therefore, in this review, only those classes of compounds withestablished antioxidant properties will be considered.

1.2. Why hunt for antioxidants in vitro?

The natural resources of both potential and established antiox-idants are vast. Some antioxidant compounds are extracted fromeasily available sources, such as agricultural and horticultural crops(maize, buckwheat, grapevine, carrots, beetroot, citrus hesperidia,hops, apples, berries, tea leaves etc.), or medicinal plants such as pine,skullcap, sage, rosemary, tormentil, andmany others. Recently, the useof wine and olive industry waste products has been considered as apotential source of dietary or food preserving antioxidants (González-Paramás et al., 2004; Obied et al., 2005). This leads to the question:why is the complex and rather expensive in vitro technology neededfor products that may be obtained from abundant and cheap sources?The necessary conditions that make using biotechnological methodsfor the production of secondary plant metabolites economically viablehave been firmly established: high economic value, insufficientabundance in intact plants, limited availability from natural sources(rare/endangered/overexploited species), and difficult cultivation(Misawa, 1994; Verpoorte et al., 2002). When taking these factorsinto consideration, in vitro technology offers some or all of thefollowing benefits: simpler extraction and purification from interfer-ing matrices, novel products not found in nature, independence fromclimatic factors and seasons, more control over biosynthetic routes forobtaining themost desired variants (e.g. enantiomers or glycosides) ora proportion of given compounds, shorter and more flexible produc-tion cycles, easier fulfillment of the high-profile pharmaceuticalproduction demands (such as GLP and GMP), and last but not least,the exploitation of the genetic engineering potential for avoiding legalrestrictions against GMO introduction into the natural environment.As will be shown in this paper, some of the listed conditions are likelyto be fulfilled with respect to antioxidants, whereas others will not,due to the ubiquitous presence of the main classes of antioxidants andthe abundance of the aforementioned inexpensive sources. On theother hand, growing knowledge about the specific mechanisms of

their activity will create the demand for more specific products thatcould be used in accurately defined therapeutic and nutritionalsituations. Finally, as was inherent in plant in vitro cultures from thevery beginning, investigation into the biosynthetic routes advancesour understanding of the function and regulation of plant metabolites.Moreover, it is still barely known what the actual roles are of manyantioxidant secondary metabolites in a real plant (Grassmann et al.,2002; Misawa, 1994).

1.3. Methods used for assessment of antioxidant properties

In a majority of tissue culture papers reporting the production ofsecondarymetabolites, their antioxidant activity has not been actuallydetermined. The knowledge of their properties usually comes fromstudies involving dietary antioxidants and compounds isolated fromethnomedicinal plants with both food conservation and chemopre-vention in mind. Thus, in most cases the antioxidant power of manysecondary metabolites is so well established, that real time monitor-ing of activity during the culture seems to be redundant. On the otherhand, the enormous variability among antioxidants, and theircomplex structure–activity relationships suggest that antioxidantand any other biological activities should be paid more attentionduring research. There are a number of simple, slightly more complex,and quite sophisticated methods for antioxidant testing. The matterhas been reviewed by many authors with respect to different aspectsof the problem (Halliwell, 1995; Aruoma, 2003; Sanchez-Moreno,2002; Matkowski, 2006).

The antioxidant testing can reveal various mechanisms of action,depending on features of the particular assay. Simple methodsinclude free radical scavenging with use of colored, artificial stablefree radicals such as 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS used in the TEAC assay — Trolox equivalent an-tioxidant capacity) (Re et al., 1999) and DPPH&z.rad; (1,1-diphenyl-2-picrylhydrazyl free radical) (Molyneux, 2004), as well as transitionmetal reduction that can be monitored by colorimetry. The metal-based methods include the reduction of ferric ions: FRAP — (ferricreducing ability of plasma) and ferric thiocyanate assays (Halliwell,1995; Aruoma, 2003), or molybdenum ion — phosphomolybdenum(P-Mo) assay (Prieto et al., 1999). These tests are easy and afford-able and can be used in high throughput screening. Their maindrawback is that their relevance to the real oxidizing life is some-what limited. The first issue is the chemical context of the assays,which use artificial compounds or are conducted in unrealistic con-ditions. This problem is eliminated in methods based on naturallyoccurring reactive oxygen species, but the fate of a free radical isobserved either indirectly with chromophore reagents or with moreexpensive ESR techniques (electron spin resonance). The scavengingof superoxide radical anion, hydroxyl radical, or nitric oxide can beobserved (Darmon et al., 1992; Aruoma, 2003; Sreejayan and Rao,1997).

Several assays based on a substrate degradation inhibition areavailable which can be used to find out whether the tested compoundcan really protect biomolecules from oxidative damage. Polyunsatu-rated lipids, proteins, components of nucleic acids, cellular mem-branes, microsomal fractions from different organs can serve as modelsystems to be protected. The oxidation can be initiated by variouschemical, physicochemical or biological approaches. The degradationproducts are monitored by spectrophotometry (thiobarbituric reac-tive substances — TBARS, carotene bleaching), fluorescence (ORACassay — for oxygen radical absorption capacity) or chromatography(Halliwell, 1995; Matkowski, 2006; Aruoma, 2003; Sanchez-Moreno,2002).

Some of the described methods have been used for testing theantioxidant activity of compounds from in vitro cultures of severalplants and will be referred to later in this paper as well as beingspecified in Table 1.

550 A. Matkowski / Biotechnology Advances 26 (2008) 548–560

2. Chemical classes of antioxidant secondary metabolites from invitro cultures

2.1. Polyphenols

It is ironic from the point of view of the tissue culturist, that theeasily oxidizable phenolics, which are often regarded as nuisanceand the reason for experiment failure, are at the same time highlydesirable as dietary and therapeutic free radical scavengers. Consider-able attention has been paid to minimizing the “phenolization” ofcultures, frequently by adding antioxidants, such as ascorbic acid orglutathione (GSH), or phenol absorbing polymers like PVP (poly-vinylpyrrolidone). The excessive production of polyphenols resultsfrom unfavorable or suboptimal culture conditions. At the onset ofsenescence, the decrease in free phenolics and a simultaneous

Table 1Selected examples of production of antioxidants from plant in vitro cultures

Species Compounds Culture system Antioxidan

Ajuga reptans Anthocyanins Flower cell culture β-caroteneperoxidatio

Anchusa officinalis Rosmarinic acid Cell suspension Many in vit

Anthoceros agrestis Rosmarinic acid and itsglucosides

Cell suspension Many in vit

Arachis hypogea Piceatannol (a stilbene) Callus Oxidative D

Artemisia judaica Flavonoids Shoot cultures in bioreactor DPPHBeta vulgaris Betalains Cell suspension, hairy roots DPPH

Carthamus tinctorius Kinobeon A Cell suspension Cell membXOD/NBT, s

Cistanche deserticola Phenylethanoid glycosides Cell suspension DPPHCrocus sativus Crocin Callus Alleviation

cultured mCynara cardunculus Cynarin, chlorogenic acid Callus TBARSDaucus carota Anthocyanins Callus, cell suspensions Lipid peroxFagopyrum esculentum Rutin Hairy roots Many in vitGlehnia littoralis Anthocyanins Callus, cell suspension Many in vit

Hemidesmus indicus Rutin Callus, shoot culture Many in vitHyssopus officinalis Rosmarinic acid,

lithospermic acid BHairy roots Many in vit

Ipomoea batatas Anthocyanins Callus, cell suspension DPPH

Lavandula officinalis Rosmarinic acid Callus, cell suspension,bioreactor

Superoxide

Ocimum basilicum Rosmarinic acid Bioreactor culture of nodalexplants and cell suspension

Many chem

Passiflora quadrangularis Flavone-C-glycosides UV irradiated callus DPPHPetroselinum sativum Flavonols and flavones Cell suspension In vivo (ratRosmarinus officinalis Carnosic acid Callus, shoot culture Oxidative s

cells and mSalvia officinalis Rosmarinic acid, abietane

diterpenoidsCallus, shoot culture, hairyroots, cell suspension

DPPH, P-M

Salvia miltiorrhiza Lithospermic acid B,rosmarinic acid

Callus, regenerated plantlets,hairy roots

DPPH

Saussurea involucrata Apigenin Hairy roots overexpressingCHI under camv35s

H2O2-indu

Scutellaria baicalensis Baicalin, wogonoside Hairy roots, cell suspension Many in vi

Stevia rebaudiana Flavonoids Callus FRAP, DPPHTorreya nucifera Abietane diterpenoids Cell suspension LDL oxidatiVaccinium pahalae Anthocyanins Cell and aggregate

suspensionMany in vi

Vitis vinifera Stilbenes, procyanidins Cell suspension DPPH, lipid

Withania somnifera Withanoloids Hairy roots DPPH, carobrain lipidradical scav

increase in cell-wall bound and oxidized forms is observed in calluscultures (Lopez Arnaldos et al., 2001), which reflects the naturalprocesses during plant–pathogen interactions (e.g. hypersensitiveresponse) (Hammerschmidt, 2005).

Polyphenols in plants derive mainly from the shikimic acidpathway through aromatic carboxylic acids — cinnamic or benzoic.Most of them possess antioxidant properties, albeit to varying degrees.The most powerful of the flavonoids are the flavanols and theiroligomeric forms called condensed tannins or proanthocyanidins,flavones and flavonols and anthocyanins. Some isoflavones are alsoknown as antioxidants. Among other groups of antioxidant poly-phenols there are: phenolic acids (caffeic acid derivatives), lignans,hydrolysable tannins (gallotannins and ellagitannins), stilbenes, andxanthones. Their physiological functions in plants are versatile andhave been reviewed recently (Harborne, 2001; Matkowski, 2006).

t testing References for in vitro culture References for antioxidantactivity

bleaching, lipidn

Callebaut et al., 1990;Terahara et al., 2001

Terahara et al., 2001

ro chemical assays De-Eknamkul and Ellis, 1984;Su et al., 1993

e.g. Petersen and Simmonds,2003; Soobrattee et al., 2005

ro chemical assays Vogelsang et al., 2006 e.g. Petersen and Simmonds,2003; Soobrattee et al., 2005

NA damage in cell culture Ku et al., 2005 Ovesna andHorvathova-Kozics, 2005

Liu et al., 2004 Liu et al., 2004Pavlov et al., 2005a,b;Hunter and Kilby, 1999

Pavlov et al., 2005a,b

rane peroxidation,inglet oxygen quenching

Kanehira et al., 2003;Kambayashi et al., 2005

Kanehira et al., 2003;Kambayashi et al., 2005

Cheng et al., 2005 Cheng et al., 2005of oxidative stress inammalian cells

Chen et al., 2003, 2004 Ochiai et al., 2004

Trajtemberg et al., 2006 Trajtemberg et al., 2006idation Ravindra and Narayan, 2003 Ravindra and Narayan, 2003ro chemical assays Lee et al., 2007 e.g. Hinneburg et al., 2006ro chemical assays Miura et al., 1998;

Kitamura et al., 2002e.g. Kahkonen and Heinonen,2003

ro chemical assays Misra et al., 2005 e.g. Ravishankara et al., 2002ro chemical assays Murakami et al., 1998;

Kochan et al., 1999e.g. Petersen and Simmonds,2003; Soobrattee et al., 2005

Konczak-Islam et al., 2000;Terahara et al., 2004

Konczak-Islam et al., 2003;Terahara et al., 2004

radical scavenging Georgiev et al., 2006a,b;Kovacheva et al., 2006;Pavlov et al., 2005a,b

Kovacheva et al., 2006

ical in vitro assays Kintzios et al., 2004 e.g. Petersen and Simmonds,2003; Soobrattee et al., 2005

Antognoni et al., 2007 Antognoni et al., 2007s) Hempel et al., 1999 Hempel et al., 1999tress reduction in livingany chemical in vitro assays

Caruso et al., 2000 e.g. Wijeratne and Cuppett,2007

o, lipid peroxidation Grzegorczyk et al., 2006, 2007 Grzegorczyk et al., 2007

Morimoto et al., 1994; Chenet al., 1999a; Yan et al., 2006

Chen et al., 1999b

ced cell damage Li et al., 2006 Fan and Yue, 2003

tro chemical assays Morimoto et al., 1998;Nishikawa et al., 1999;Stojakowska and Malarz, 2000

e.g. Huang et al., 2006

Tadhani et al., 2007 Tadhani et al., 2007on, nitric oxide inhibition Orihara et al., 2002 Lee et al., 2006tro chemical assays Meyer et al., 2002 e.g. Kahkonen and Heinonen,

2003peroxidation Decendit and Mérillon, 1996;

Fauconneau et al., 1997;Tassoni et al., 2005

Fauconneau et al., 1997

tene-linoleic acid oxidation,peroxidation, hydroxylenging

Kumar et al., 2005 Kumar et al., 2005


Phenolic acids occur in plants in free form as glycosides and can beintegrated into larger molecules in an ester form. They are common asdepsides— the intermolecular ester of two or more units composed ofthe same or different phenolic acids such as: caffeic, coumaric, ferulic,gallic, and syringic.

Depsides are, for example, a ubiquitous chlorogenic as well asisochlorogenic, ellagic, lithospermic and rosmarinic acids. Due to thepresence of a high number of hydroxyl groups and a carboxyl moiety,their antioxidant properties are very pronounced (Rice-Evans et al.,1996; Sroka, 2005).

Special attention should be paid to rosmarinic acid (RA), a depsidecomposed of two caffeic acid molecules (Fig. 1), found in many La-miaceae and Boraginaceae species. Its biochemistry and pharmacologyhas been extensively reviewed by Petersen and Simmonds (2003). Theantioxidant properties of rosmarinic acid are also well established.Several plants and various techniques have been used for in vitroproduction of this compound (Shetty, 1997). Rosmarinic acid canaccumulate in cell cultures to amounts greater than those in intactplants. The suspension cultures producing rosmarinic acid weregenerated from Anchusa officinalis (De-Eknamkul and Ellis, 1984,

Fig. 1. Structures of some phenolic antioxidan

1985; Su and Humphrey, 1990; Su et al., 1993), Eritrichum sericeum(Fedoreyev et al., 2005), Lithospermum erythrorrhizon (Yamamotoet al., 2000) (Boraginaceae), and Coleus blumei (Petersen et al., 1993),Lavandula vera (Pavlov et al., 2005a,b; Georgiev et al., 2006a,b), Oci-mum basilicum (Kintzios et al., 2004), Salvia officinalis (Hippolyte et al.,1992), Zataria multiflora (Mohagheghzadeh et al., 2004) (Lamiaceae).Depending on the species, the yield of rosmarinic acid fromsuspension cultures can exceed 10% of cell mass and 6 g/l of medium.An interesting example is also the production of rosmarinic acid ofover 5% of cell dry mass in Anthoceros agrestis (Vogelsang et al., 2006).Another system used for rosmarinic acid production are transformedroots of Salvia miltiorrhiza (Chen et al., 1999a,b, 2001) and S. officinalis(Grzegorczyk et al., 2006, 2007). In the second species, the accumula-tion was much higher reaching 4.5% of dry weight and its antioxidantactivity has been confirmed with several assays. Extracts fromrosmarinic acid accumulating lavender cells also have superior radicalscavenging properties (Kovacheva et al., 2001, 2006). Even largeramounts (over 7% dw) of rosmarinic acid accumulated in Agrobacter-ium transformed callus of C. blumei (Bauer et al., 2004) and trans-formed roots Hyssopus officinalis, but only when cultured on WP

t metabolites from plant in vitro cultures.


mediumwhichwasmuchmore superior to bothMS and B5 (Murakamiet al., 1998). Razzaque and Ellis (1977) report a yield of 8–11% ofrosmarinic acid in C. blumei cell cultures. An extremely high level of RAproduction was reported by Hippolyte et al. (1992) in S. officinalisreaching 6.4 mg/l of suspension culture, although the result has notappeared in any other reference.

A closely related antioxidant is lithospermic acid B, a tetramericdepside (Fig. 1), accumulating especially in the aforementioned hairyroot cultures of H. officinalis and elicited S. miltiorrhiza as well as inLithospermum erythrorhizon suspension cultures (Yamamoto et al.,2000, 2002). Oligomeric caffeic acid depsides such as DCQAs(dicaffeoylquinic acids) — potent antioxidants and antivirals, accumu-late also in cell cultures of sweet potato storage roots, where theyare accompanied by anthocyanins (Konczak et al., 2004). Chlorogenicacid and cynarin (another DCQA) are present in cardoon (Cynaracardunculus) callus in quantities two-times greater than in the leaves—the usual medicinal crude drug. The antioxidant capacity of the car-doon callus was confirmed by the TBARS method (Trajtemberg et al.,2006).

Flavonoids are one of the largest groups of secondary metabolitesbased on a common structure. Chemically they possess a tricyclicphenylbenzopyran (with the exception of chalcones) structure butbiosynthetically come from phenylalanine and malonyl-CoA in thephenylpropanoid pathway.

Subclasses of flavonoid include flavonols, flavones, isoflavonoids,flavanons, flavanols, proanthocyanidins (catechins), and anthocyani-dins. Substitution patterns include the hydroxylation of rings A and B,methylation of hydroxyl groups, prenylation, and glycosylation. Othermodifications can form biflavonoids, lignans, oligomeric proantho-cyanidins, glycoside esters with other phenolics etc.

Antioxidant properties of flavones depend on the hydroxylationpattern. Substitution with hydroxyl, methoxyl or other groups, andglycosylation also influences hydrophilicity and many biologicalactivities. Among the vast variety of structures, several flavones fromthe genus Scutellaria (Lamiaceae) are distinguished by their unsub-stituted B-ring and common occurrence in the form of glucuronides(Fig. 1). Scutellaria baicalensis, the favorite natural drug of traditionalOriental medicines, contains in the roots probably the highest knownamount of flavones (up to 20% dw) of which the strong lipophilicantioxidants: baicalin, baicalein, wogonoside and wogonin are themost abundant. In cell suspension and the hairy root cultures ofS. baicalensis the accumulation of these compounds was reported(Morimoto et al.,1998; Nishikawa et al.,1999; Stojakowska andMalarz,2000). The in vivo antioxidant function of baicalein in metabolizinghydrogen peroxide has been proposed as a result of experiments usingcell cultures (Morimoto et al., 1998).

Flavone C-glycosides are of particular interest because of theirlimited occurrence in plants and they have important therapeuticproperties, including the prevention of cardiovascular disordersrelated to oxidative stress. In tissue cultures of Passiflora quadrangularisseveral C-glycosylated flavones such as isoorientin, orientin (Fig. 1),vitexin and isovitexin have been induced in varied amounts, and theirantioxidant activity determined by DPPH assay (Antognoni et al.,2007). Crataegusmonogyna is another source of these C-glycosides andits long-term callus cultures have been shown to produce several typesof phenolic antioxidants assayed with various methods (Rakotoarisonet al., 1997; Bahorun et al., 2003).

Oligomeric proanthocyanidins (also known as condensed tannins)are considered to be the most powerful antioxidants of all theflavonoids owing to their structure (procyanidin B1 as an example inFig. 1) (the chemical mechanisms have been reviewed by Bors andMichel, 2002). They are responsible for the antioxidant activity ofwines, teas and fruits as well as of popular nutraceuticals made fromgrape seeds or pine bark. The thorough genomic and metabolicengineering studies have been recently initiated, too (Dixon et al.,2005). Conversely, they are rarely studied for in vitro production by

plant cells. The successful induction and isolation of oligomericproanthocyanidins have been reported in the cell cultures of Vacci-nium pahalae (Kandil et al., 2000) and Vitis vinifera (Decendit andMérillon, 1996).

Another example of flavonoid polyphenols frequently studied forin vitro production is anthocyanins. Pigments are useful as productsbecause their accumulation can easily be visually evaluated. Moreover,their beneficial health promoting properties as well as their grow-ing importance as bioactive and natural food colorants additionallyincreases their attractiveness for plant biotechnology. The versatilevalues of anthocyanins have been reported and reviewed in count-less publications. One of their most pronounced features is theirsubstantial antioxidant capacity determined in various assays, buttheir biological activity varies greatly, due to their structural diversity.Analogously to other polyphenols, more hydroxylated compoundsexpress larger free radical scavenging capacity, whereas methylationof the hydroxyl groups decreases this effect partially (Stintzing andCarle, 2004). Glycosylation can also reduce the ability to scavenge freeradicals, but on the other hand enhances the stability of the molecule.Acylation of the sugar moieties with carboxylic, usually hydroxycin-namic acids (an example of a glycosylated and acylated cyanidin isshown in Fig.1) additionally boosts the antioxidant potential and colorstability. For that reason, not all anthocyanins are equally desirable asnatural medicines. Cyanidin and delphinidin glycosides acylated withphenol carboxylic acids are preferred over less substituted versionsand petunidin, pelargonidin, peonidin or malvidin derivatives. This isone of the main reasons for conducting tissue culture experiments.In abundant natural sources, the anthocyanin profile is rarely opti-mal and the raw material often requires complex extraction andpurification procedures. In several species listed below the level ofanthocyanins in vitro equals or exceeds the natural sources and theirmetabolic profile is more useful. Thus, even if anthocyanins are not soexpensive, the ability to obtain products with useful structural andfunctional properties may become economically feasible (Konczaket al., 2005).

Anthocyanins are obtained from in vitro cultures of Prunus sp.(Durzan et al., 1991; Blando et al., 2005), Daucus carota (Ravindra andNarayan, 2003), Glehnia litoralis (Miura et al., 1998), V. pahalae (Meyeret al., 2002), Ipomaea batatas (Konczak-Islam et al., 2000; Teraharaet al., 2004) Fragaria ananassa (Edahiro et al., 2005), V. vinifera(Decendit and Mérillon, 1996), Rudbeckia hirta (Luczkiewicz andCisowski, 2001) and Ajuga reptans (Callebaut et al., 1990; Teraharaet al., 2001) and others.

Interestingly, two species used medicinally due to their anticanceralkaloids, Catharanthus roseus and Camptotheca acuminata, producesignificant amounts of anthocyanins of over 200 µg/g fresh weightin cell cultures (Filippini et al., 2003; Pasqua et al., 2005). In C. roseus,p-coumaroyl glucosides of highly methoxylated malvidin or hirsutidinpredominated, unlike in field-grown flowers. In C. acuminata a rarecyanidin galactoside accounted for over 95% of all anthocyanins.

In A. reptans flower-derived cell cultures the anthocyanin composi-tion was altered from delphinidin-based in flowers towards strongerantioxidants — di-acylated cyanidin glycosides (Callebaut et al., 1997;Terahara et al., 2001). This observed simplification and alteration of themetabolic profile, when compared to natural conditions, is typical forin vitro produced anthocyanins. In the extensively studied I. batatas,in the tissue cultures induced from storage roots the accumulation ofanthocyanins can even exceed the tubers by several times. Theproduction of valuable di-acylated cyanidin glycosides from callusand cell suspension aswell as their regulation towards overproductionand their more desirable profile has also been reported (Teraharaet al., 2000, 2004; Konczak et al., 2005). The superior stability andantioxidant activity of anthocyanin-rich extracts have also been con-firmed. In this species the extraordinary high amount of anthocyaninswas recorded in selected cell lines along with a greater proportion, incomparison to the tubers, of cyanidin-based to peonidin-based


pigments (Konczak et al., 2005). Given the aforementioned ability ofcell culture from this species to accumulate medicinally active dep-sides, this is one of the most promising systems for the biotechnolo-gical production of health-promoting polyphenols.

Silybum marianum, milk thistle, the famous hepatic drug containsin the achenes an isomeric flavonoid lignans complex called silymarinthat is a strong antioxidant (e.g. silybin in Fig. 1). The untreatedcell cultures contain low and variable amounts of flavonolignans, butafter they have been elicited their production can be increased 6-fold(Sanchez-Sampedro et al., 2005a). In another study, the same authorsreveal the pivotal role of calcium deprivation in stimulating silymarinbiosynthesis (Sanchez-Sampedro et al., 2005b). The role of perox-idases in both the synthesis by oxidative coupling of precursors andthe degradation of silymarin compounds has also been explored(Sanchez-Sampedro et al., 2007a).

Stilbenes are bicyclic polyphenols represented by the well-knownresveratrol (Fig. 1), a phytoalexin and the red wine antioxidant.Although present in a considerable amount of natural sources likegrapes and some Polygonaceae species, it has been also induced inelicited hairy root cultures of peanut (Arachis hypogea) and recoveredefficiently from the liquid medium (Medina-Bolivar et al., 2007). Thesame species was used to enhance production of a rare stilbene —

piceatannol in cell culture (Ku et al., 2005). This approach seemsquite promising for the production of a purified single antioxidantwhich the pharmaceutical industry prefers to use. In cell cultures ofV. vinifera, the influence of elicitors on the stilbene biosynthesis andthe stilbene synthase activity has been studied (Tassoni et al., 2005) aswell as the antioxidant capacity of resveratrol and astringin fromcultured cells (Fauconneau et al., 1997).

Other examples of recently reported antioxidant activities of in vitrocultured, polyphenol-rich plants include: Curcuma longa mass prop-agation on a thin-film rocker system where significant and clonedependent DPPH scavenging was noted (Cousins et al., 2007); Cis-tanche deserticola cell suspensions producing antioxidant phenyletha-noid glycosides also assayed by a DPPH test (Cheng et al., 2005); Steviarebaudiana – the herbal sweetener – callus extracted with water andmethanol had higher antioxidant activity than the leaves of field-grown plants, which correlated with the higher levels of flavonoidsand total polyphenols (Tadhani et al., 2007).

2.2. Isoprenoids

Isoprenoids are produced in plant cells from isopentynyl pyropho-sphate (IPP) and dimethylallyl pyrophosphate (DMAPP). These basicunits are synthesized via cytoplasmic mevalonate or plastidicdeoxyxylulose (or methylerythritol) phosphate pathways. IPP andDMAPP are subsequently fused by sets of prenyltransferases andterpene synthases and a variety of modifying enzymes to eventuallyyield enormously variable compounds in numerous groups ofterpenoids (mono, sesqui, di, and triterpenes and their derivatives),carotenoids, and steroids (Bouvier et al., 2005; Tholl, 2006). Each ofthese groups contains metabolites of different chemical characterssuch as alcohols, acids, aldehydes, or a combination of these; they canalso be aromatic, glycosylated and formmore complex structures. Themost powerful antioxidants among them are carotenoids and abietanediterpenoids (some of them containing a phenolic structure within amolecule). In comparison to most phenylpropanoid polyphenols,isoprenoids are generally more lipophilic, hence their important rolein protecting membrane lipids. Both β-carotene and lycopene arerecognized as dietary chemopreventive agents, protecting againstcancerogenesis. The participation in an antioxidant cascade is likely tocontribute to these prophylactic properties. Crocin derived fromsaffron (stigmata of Crocus sativus) has been confirmed as a powerfulantioxidant (Ochiai et al., 2004), stronger than α-tocopherol, andbeing a glycoside of an apocarotenoid acid, crocetin, it is water soluble(Fig. 2). Whereas other carotenoids are readily available from

abundant natural sources such as carrots or tomatoes as well as bymeans of microbial (Bhosale and Bernstein, 2005) and fungalbiotechnology (Böhme et al., 2006), it is reasonable to obtain crocinfrom the plant tissue cultures (Chen et al., 2003, 2004) of C. sativus. Inthis case, the in vitro production could be profitable due to the highcost of the rawmaterial (about 200000 flowers are needed to get 1 kgof dried stigma).

Abietane diterpenes are present in many Lamiaceae medicinalplants as well as in some gymnosperms. Some of these compoundsdemonstrate substantial antioxidant potential in various systems.Carnosic acid (Fig. 2) and carnosol, rosmanol and ferruginol (Fig. 2) areresponsible for the antioxidant activity of sage (Salvia sp.) androsemary (Rosmarinus officinalis) along with the phenolic rosmarinicand salvianolic acids, but the organ distribution differs between thetwo mentioned chemical classes. However, the published tissueculture studies in Lamiaceae aimed at enhancing the rosmarinic acidproduction rather than at abietane diterpenoids. The tissue cultures asa source of abietane diterpenes have been established for R. officinalis(Caruso et al., 2000), S. officinalis (Grzegorczyk et al., 2007) and Salviasclarea (Kuźma et al., 2006). The antioxidant activity of in vitrocultures of common sage, especially in lipophilic systems such as theinhibition of lipid peroxidation, can be attributed to abietanediterpenes rather than to phenolics (Grzegorczyk et al., 2007). Lightplays an important role in stimulating the diterpene biosynthesisin vitro (Caruso et al., 2000; Kuźma et al., 2006) even though innatural conditions they can only be found in the underground parts ofplants.

Volatile terpenoids responsible for fragrant properties of plantorgans serve as a multipurpose chemical defense weapon againstfungal and bacterial pathogens as well as a means of long distancechemical communication e.g. attracting pollinators. Moreover, the roleof a monoterpene β-thujaplicin in the alleviation of oxidative damagein cell cultures of cypress has been postulated (Zhao et al., 2005), butthis does not imply that this is its direct function as an antioxidant. Theantioxidant properties of several monoterpenes, such as γ-terpineneand limonene have been reported. This suggests that this group ofcompounds may receive more attention although at present they areunderestimated as antioxidants. Tissue cultures are able to producevolatile constituents of essential oils and many reports have beenproduced on that subject (Mulder-Krieger et al., 1988; Hrazdina,2006). However, in vitro production of essential oils has not beenaimed at the antioxidant properties of the products.

2.3. Other structures

Tocopherols, widely used in human nutrition as vitamin E and infood conservation, are powerful free radical scavengers and protectplant cells against oxidative damage in a lipophilic environment (Kruket al., 2005). Plants are the primary source of natural tocopherolsisolated from vegetable oils or maize embryos. Their biosynthesisintegrates the aromatic aminoacids (shikimic acid) and plastidicisoprenoid deoxyxylulose phosphate (pentose) pathways. The firstpathways create the aromatic (phenolic) head while the second givesrise to the hydrophobic tail of a tocochromanol skeleton (DellaPenna,2005).α-Tocopherol (Fig. 2) is themost active form of vitamin E, whilethe extraction from plant oils usually yields a mixture of β, γ, δ and α-tocopherols and tocotrienols. Therefore there is a demand foralternative sources of pure α-tocopherol from plant tissues. Variousculture systems of Helianthus annuus (sunflower) have been estab-lished for this purpose. A 100% increase in cell suspensions has beenachieved by media optimization, elicitation and precursor feeding(Caretto et al., 2004; Gala et al., 2005). A photomixotrophic cell linehas also established a cultured on sucrose-deprived medium(Fachechi et al., 2007). An attempt has been also made to select themost efficient genotype of hazel (Corylus avellana) for the productionof tocopherols in bioreactors (Sivakumar et al., 2005).

Fig. 2. Structures of antioxidant metabolites from plant in vitro cultures from isoprenoid and other chemical classes.


Betalains — the purple betacyanins and yellow betaxanthinespigments functionally replace anthocyanins in 13 taxons grouped inCaryophyllales (previously Centrospermae) order, such as the families:Chenopodiaceae, Amaranthaceae, Portulacaceae, Cactaceae, Phytolacca-ceae and others. These nitrogen containing compounds (Fig. 2) althoughwidely used as non-toxic food colorants, remain understudied in termsof their antioxidant potential (Gliszczyńska-Swigło et al., 2006; Kanneret al., 2001). These properties of betalains have been recently reviewedby Stintzing and Carle (2004). The major advantages of betalains asdietary antioxidants are their bioavailability, which is greater thanmostflavonoids, and their superior stability in comparison to anthocyanins(Kanner et al., 2001; Stintzing and Carle, 2004).

Red beetroot (Beta vulgaris) is a rich natural source of betacyanins(with betanin predominating— Fig. 2) and is also an object of metabolicin vitro studies on their biosynthesis. They possess the useful propertiesof pigments and are therefore preferred as amodel system despite theirlow commercial value. The production of beetroot pigments has beenperformed using a variety of techniques such as cell suspensions andtransformed roots in bioreactors (Hunter and Kilby, 1999; Pavlov et al.,

2002, 2005b). Being ideal metabolites for direct observation they havealso been used for compound recovery experiments using variouspermeabilization and extraction methods (Hunter and Kilby, 1999). Redbeet betalains from hairy roots have been shown to be efficient freeradical scavengers (Pavlov et al., 2002, 2005b). The growing knowledgeof their therapeutic and preventive properties is likely to furtherincrease the research interest in their in vitro biosynthesis.

Indole glucosinolates are a tryptophane containing group from thediverse class of thioglycoside compounds typical to the Brassicaceae.Upon hydrolysis by myrosinase they generate biologically activeantioxidants and carcinogenesis inhibitors such as indole-3-carbinolderived from glucobrassicin (Fig. 2). In cruciferous plants they arethought to contribute to the regulation of auxin biosynthetic routes(Bak et al., 2001). Although they are present in common cabbagerelated vegetables (broccoli in particular) their instability and unevenor unpredictable distribution would make their biotechnologicalproduction an interesting option. Yet, to date there has been verylittle data available on that topic. The horseradish (Armoraciarusticana) cells, both in suspension and immobilized in polyurethane


foam, produce indole-3-methylglucosinolate and 4-hydroxyindole-3-methylglucosinolate (Mevy et al., 1999).

Kinobeon A — a novel, red-colored compound isolated fromCarthamus tinctorius cell cultures uniquely combines the quinoidaromatic character with several conjugated double bonds (Fig. 2). Ithas therefore been demonstrated as being a remarkably strongantioxidant. Kinobeon A efficiently quenches various reactive oxygenspecies, inhibits lipid peroxidation and supports the survival ofmammalian cells during oxidative stress (Kanehira et al., 2003;Kambayashi et al., 2005). To date, little is known about kinobeon Aphysiological functions and biosynthesis. Whether it occurs also inother organisms or is an example of an exclusive cell-culture productremains to be determined.

3. Strategies for increased production

The continuous monitoring of a chosen metabolite is a prerequisitefor the successful development of production technology and canbe carried out using a number of different methods. For accuratemeasurements, chromatographic methods such as HPLC and GC withthe appropriate detection are sufficient. Laboratories that lack phyto-chemical facilities need other equivalent substitution methods. Foroperation on a large scale, time saving is also a consideration. Thesefaster and less elaborate techniques involve spectrophotometry that canbe used for some groups of compounds, while for other types it is ratherunsuitable. The first group contains colored compounds — flavonoid,carotenoid, and quinoid pigments or UV absorbing phenolics.

Anthocyanins are commonly estimated by this technique, whichcan also be applied non-destructively (Durzan et al., 1991; Smith,2000). On the other hand, this approach is only adequate if there isjust one dominant antioxidant, differing in the absorption maximafrom any interfering compounds. The rapid progress in phytochemicalinstrumentation development and the trend towards a metabolomicapproach and high throughput screening is likely to also influence themonitoring of secondary metabolites production in vitro. Indeed, inrecent publications the chromatographic determination of antiox-idants predominates, frequently accompanied by bioactivity guidedfractionation, spectral structure elucidation of isolated chemicals byMS or NMR. The use of hyphenated techniques such as LC-MS and LC-NMR or 2D-NMR is considered to be the best means for structuredetermination when novel compounds are present in vitro that havenot been known from intact plants (Tian et al., 2005; Farag et al., 2007;Sanchez-Sampedro et al., 2007b).

3.1. Optimization of biosynthesis by culture conditions

A range of environmental and nutritional factors are known toinfluence the biosynthetic pathways of secondary metabolites. Lightinduction of anthocyanin pigments is a well documented example(Harborne, 2001; Stintzing and Carle, 2004). In cultured cherry (Prunuscerasus) callus and suspensions, the light exposure causes a rapid changeof the tissue color from white to purple (Blando et al., 2005). Theanthocyanidin profile was different both from intact leaves and fruitswith cyanidin-3-glucoside as the single dominant compound, absentdelphinidin (unlike in fruits) and only a small amount of cyanidin-3-rutoside (dominant in the leaves). This is a good example of a distinctmetabolic profile under in vitro conditions. However, the irradiation ofcultures can lead to an increase in cost and can generate undesirabletemperature gradients in the culture vessels. Therefore, severalexamples of constitutive, light-independent systems for anthocyaninproductionwere established such asGlehnia littoralis (Miura et al.,1998),I. batatas (Konczak et al., 2005) or Aralia cordata (Kobayashi et al., 1993).In the latter species, several factors had been optimized (e.g. CO2

supplementation) to obtain a final yield of over 17% of the dry mass.The media composition has to be optimized for both an intensive

biomass increase and the accumulation of the desired metabolite.

Auxins and cytokinins are crucial for the proper stimulation ofbiosynthetic pathways, unless we deal with a hormone-independenttransformed root culture. In anthocyanin producing G. littoraliscultures, NAA at 1 mg/l was the preferred auxin, superior to IAA and2,4-D and the addition of 0.1 mg/l kinetin further improved cellgrowth and pigment biosynthesis (Miura et al., 1998). The higherconcentration of NAA (5 mg/l) was optimal for R. hirta (Luczkiewiczand Cisowski, 2001). In the same study, the highest accumulation wasachieved by adding cystein, which is one of the constituents of CoA, acrucial factor in flavonoid biosynthesis.

A two or three-stage growth system can be also useful for opti-mizing metabolite production. The first stage is used for the initiationand/or the maintenance of a culture with the aim of obtaining thehighest percentage of induction and the fastest biomass growth. Thesecond phase aims at the accumulation of a product. It has beensuccessfully applied to anthocyanins in R. hirta (Luczkiewicz andCisowski, 2001) where the tissues in the optimized second stagemedia contained up to 5% anthocyanins in dry mass.

In C. sativus cultures the two-stage system leads to a substantialaccumulation of crocin (430 mg/l) simply by changing the hormonecomposition from NAA at 2 mg/l, BAP at 1 mg/l for biomass growth toIAA at 2 mg/l and BAP at 0.5 mg/l for crocin production (Chen et al.,2003).

3.2. Production in differentiated tissues

In some cases, full development in natural conditions is useful forproducing a considerable amount of secondary products, especially ifthe undifferentiated cell culture is either unable to or barelyaccumulating antioxidant compounds. This has been reported forseveral classes of metabolites, especially for alkaloids, and has alsobeen published for some antioxidant compounds. Ellagic acids presentin Rubus chamaemorus plants was 3 times lower in shoot cultures andover 10 times lower in callus (Thiem and Krawczyk, 2003). Similarly inO. basilicum cell cultures (Kintzios et al., 2004) the accumulation ofrosmarinic acid was markedly lower than in regenerated plantlets. InS. officinalis (Grzegorczyk et al., 2007) and rosemary (Caruso et al.,2000) in vitro cultures, the abietane diterpene antioxidants (carnosoland carnosic acid) were present only in shoot cultures and not incallus, suspension or hairy roots. On the other hand, undifferentiatedcell suspensions are able to accumulate great amounts of phenolicacids. Moreover, the cells cultured in suspension tend to form largeraggregates upon differentiation, therefore increasing their shear stressresistance. The potential of fast growing somatic embryos cultures canbe also utilized for the production of medicinal metabolites, butreports on that topic are scarce and deal chiefly with alkaloidproduction such as paclitaxel (Lee and Son, 1995). For example,phenol glycosides, lignans and flavonoid accumulation has beenmentioned in Siberian ginseng somatic embryos (Shoahel et al., 2006).

Even in dedifferentiated cells, some biosynthetic potential typicalfor the developed organs from which they were initiated, can beconserved. In Pueraria lobata callus cultures, the bioactive isoflavonoidcontent depended on the source organ, reflecting relations in themature plant (Matkowski, 2004). The selection of proper donor plantsand organs should already be considered when starting the culture,unless it can be overcome by a suitable treatment. However, in mostcircumstances the dedifferentiation apparently also involves some ofthe biochemical properties of the cells. The modification of relativebiosynthesis to degradation ratios of a desired product can alsoinfluence the final levels of a desired compound in the culture.

3.3. Selection of high-producing cell lines

The super-efficient clones of cultured cell or tissues can be selectedby monitoring the level of the compound of interest, or can becomplemented by a selecting agent facilitating the process. The


selected line should exceed both the levels of production obtained inunselected cell cultures and the natural biosynthetic productivity ofan intact organism.

The addition of a phenylalanine analogue to the culture mediumcauses damage of themajority of cells except thosewho express a highdegree of activity of PAL (phenylalanine ammonia lyase). Theoverexpression of PAL in selected variants results in elevatedphenylpropanoid compounds, many of which are desired antiox-idants. Rosmarinic acid producing cells of lavender (Georgiev et al.,2006b) and shikonin producing cells of L. erythrorhizon (Bulgakovet al., 2001) have been selected with the use of fluorinatedphenylalanine. The selected lines appear to be stable over time andcan synthesize twice as much of the desired compounds as the controlcultures.

If the desired product is colored as in the case of anthocyanins, asimple visual selection with the mechanical dissection of accumulat-ing cells can be performed with good results. An impressive exampleis the origin of the stable (over a period of several years), light-independent anthocyanin accumulating cell line of G. litoralis selectedfrom a single purple spot in a white, dark grown callus (Miura et al.,1998; Kitamura et al., 2002). Similarly, a selected cell line of purplesweet potato (PL) accumulates a large amount of acylated anthocya-nins without light exposure (Konczak et al., 2005).

3.4. Precursor feeding and biotransformation

When starting an in vitro culture of a medicinal plant, that in itsintact form accumulates substantial amounts of valuable products, itsometimes happens that a dedifferentiated cell mass is unable tocomplete the biosynthesis. This is due to the uncoupling of theenzymatic machinery, or an insufficient expression of developmen-tally regulated biosynthetic genes, or a lack of environmental stimuli.If biosynthetic enzymes are expressed in part of the pathway, theexogenous delivery of precursors can solve the problem. It can be alsohelpful in omitting the metabolic channeling that directs theprecursors to a pathway other than that desired.

In a Rhodiola rosea compact callus aggregate culture no secondaryproducts were detected until the medium was supplemented withcinnamyl alcohol that was efficiently transformed into a pharmaco-logically active rosin and rosavin (György et al., 2004). However,neither are antioxidants. Salidroside, the antioxidant present in theplant, was not found in that study, but other authors have succeededin antioxidant production in this species (Furmanowa et al., 1998).In contrast, the feeding of naringenin or phenylalanine significantlyincreased anthocyanins in Rudbeckia (Luczkiewicz and Cisowski,2001) and strawberry (Edahiro et al., 2005) cells, respectively. InCistanche salsa cell cultures, their enrichment with a mixture ofphenylethanoid glycosides precursors results in a doubling ofproduction when compared to feeding individually with tyrosine,phenylalanine, caffeic acid or cucumber juice (Liu et al., 2007).Hence, the choice of an appropriate precursor is essential for thesuccessful enhancement of in vitro antioxidant production.

Curcumin is a yellow colored, potent polyphenolic antioxidant andcholagogue from the roots of turmeric (C. longa). Its chief weakness isits low water solubility, which limits its intestinal absorption andhence its pharmaceutical usefulness (Sharma et al., 2007). In anothersurprising application of C. roseus cell cultures, they were modified bycellular enzymes into unnatural glycosides that are much moresoluble in water (Kaminaga et al., 2003).

3.5. Elicitation and stress induced production

One of the best established roles of secondary metabolites is theinvolvement in stress responses (Grassmann et al., 2002). Phytoalex-ins are synthesized in reaction to a pathogen attack, other compoundsare associated with abiotic disturbances.

The upregulation of biosynthesis of secondary metabolism uponexposure to stress factors has been used to obtain a high yield of manymedicinal compounds (Verpoorte et al., 2002; Vanisree et al., 2004).Exposure to stress factors results in an excessive level of oxidizingmolecules such as ROS due to the disturbances in redox stateequilibrium. The increased ROS production may help to combat theinvasive pathogens but can also be triggered by harmful environ-mental factors such as UV radiation, xenobiotics, heavy metals,drought, and mechanical damage. Therefore, antioxidant propertiesof somemetabolites along with antioxidant enzymes can aid the plantin restoring the redox homoeostasis (Matkowski, 2006). Apart fromdirect antioxidant activity, some compounds — e.g. flavones can serveas substrates for cellular peroxidases (Morimoto et al., 1998).

In vitro cultured material can be elicited by bacterial or fungallysates or stress response mediators such as salicylate or jasmonicacid/methyl jasmonate and hydrogen peroxide as well as by environ-mental factors like metals or irradiation.

The stress related growth regulators, jasmonic acid and its esters,especially methyl jasmonate (MeJa) have been widely used inpromoting the biosynthesis of both inducible and constitutivesecondary metabolites, including medicinal compounds such asanticancer alkaloids (Vanisree and Tsay, 2004; Verpoorte et al.,2002). With respect to the antioxidants, jasmonic acid enhanced α-tocopherol production by sunflower and A. thaliana cell cultures (Galaet al., 2005) and cyanidin glucosides in cherry callus (Blando et al.,2005). On the other hand, in the resveratrol producing grapevine cellcultures (Tassoni et al., 2005), jasmonic acid proved to be a weakerelicitor than its methyl ester. Besides, MeJa promoted the accumula-tion of trans-resveratrol, whereas JA resulted in a modest increase ofthe less desirable cis-resveratrol. MeJa and salicylic acid alsostimulated the curcumin glycosylation by C. roseus cell cultures(Kaminaga et al., 2003).

Using natural biotic elicitors also results in enhanced secondarymetabolism. Yeast extracts (YE — also read as yeast elicitor), butneither chitin nor chitosan, have a positive impact on the productionof silymarin, the effect being potentiated when combined with JA.However, it proved more effective when supplemented with MeJA inthis system (Sanchez-Sampedro et al., 2005a). Chitin elicited theproduction of several new flavonoids in cactus (Cephalocereus senilis)cell cultures (Liu et al., 1993). YE applied to the hairy root culture of S.miltiorrhiza improved both the growth of the roots and theaccumulation of rosmarinic and lithospermic B acids, preceded by asignificant rise in tyrosine aminotransferase activity (Chen et al., 2001;Yan et al., 2006). Compared to the effect of an abiotic elicitor— Ag+, YEwas superior (Yan et al., 2006). Rosmarinic acid production was alsoenhanced in O. basilicum hairy roots by Phytophtora cell walls, whilesalicylic and jasmonic acids or chitosan suppressed it (Bais et al.,2002).

In peanut hairy roots (Medina-Bolivar et al., 2007) sodium acetateand chitosanwere themost effective of the five tested elicitors causinga 60-fold increase in resveratrol accumulation.

The use of metal-based elicitors gives positive results as well.Vanadium salts increase the accumulation of rosmarinic acid inlavender cell culture nearly three-fold (Georgiev et al., 2006a), whilein Vitis cells only the cis isomer of resveratrol was induced (Tassoniet al., 2005).

Rare earth elements such as La3+ and Ce3+ can also serve as elici-tors to promote crocin (Chen et al., 2004), phenylethanoids fromC. deserticola (Ouyang et al., 2003), and silymarin (Sanchez-Sampedroet al., 2005b) production. The mechanism of these lanthanides actionsinvolves interference with cellular calcium signaling (Sanchez-Sampedro et al., 2005b).

Irradiation with UV has been also used for making the plant cellssynthesize more antioxidants. In particular, flavonoids and otherpolyphenols are known to protect plants from harmful UV impact. InP. quadrangularis callus subjected to UV-B treatment, the irradiation


stimulated a large increase in the amount of flavonoid, corroboratingthe higher radical scavenging activity (Antognoni et al., 2007).Similarly, the callus of A. hypogea was induced by UV radiation toaccumulate much greater amounts of stilbenes (Ku et al., 2005).

The use of a downstream mediator of elicitation such as MeJa maybe more effective than biotic elicitors. The latter bring about a broadspectrum of responses, not all of which are necessarily desirable. Onthe other hand, biotic elicitors are of some use due to their lower costand are particularly appropriate at the initial stage of research whenthe pathways of induction for a particular compound are not yetdetermined. In addition, different elicitors can diversely act on thebiosynthetic competence of the cells, as illustrated by V. viniferacultures resveratrol isomers production (Tassoni et al., 2005).

3.6. Transformation with Agrobacterium rhizogenes — hairy roots

This is a commonly used method to enhance the production ofsecondary metabolites. Thousands of species have been transformedwith A. rhizogenes with the aim of achieving a transformed rootsinduction. If the intact plant is known to synthesize large amounts ofantioxidant compounds, one can expect a substantial increase in theirlevel in the cultured roots and sometimes also in plantlets regeneratedfrom transformed roots. The subject of transformed rootmethodology,its recent developments and the prospects for production of medicinalcompounds have been addressed in several reviews (Georgiev et al.,2007; Guillon et al., 2006; Uozumi, 2004). The manipulation andoptimization of transformed roots productivity are generally the sameas those of other culture systems with perfect culture conditions, cellline selection, precursor feeding and elicitation. Thus, only a fewexamples that specifically report the production of antioxidantcompounds by hairy roots will be mentioned in this chapter.

Several species from the Lamiaceae have been transformed withA. rhizogenes for the production of polyphenolic antioxidants. The verypopular rosmarinic acid is again a good example of this. In S. officinalishairy roots, the antioxidant activity and RA level were much higherthan in untransformed organs (Grzegorczyk et al., 2006, 2007). Thisdepside has also been efficiently produced by the hairy roots ofother species such as: S. miltiorrhiza, (Chen et al., 2001; Yan et al., 2006),C. blumei (Bauer et al., 2004), O. basilicum (Bais et al., 2002), H. officinalis(Murakami et al., 1998; Kochan et al., 1999).

Baikal skullcap (S. baicalensis) is one of the richest natural sourcesof flavones reaching up to 20% in the root. Transformed roots producedbaicalein and wogonin glucuronides, but also a phenylethanoidacteoside, a compound unknown from plants growing in naturalconditions (Nishikawa et al., 1999; Stojakowska and Malarz, 2000). InS. sclarea hairy roots some abietane diterpenoids are also reported,although not in large enough amounts for them to be considered as asource of antioxidants (Kuźma et al., 2006). An effective system forrutin production has been established with the hairy roots ofbuckwheat (Fagopyrum esculentum) whose leaves are a rich naturalsource of rutin (Lee et al., 2007). A high yield of antioxidant betalainscan be achieved from the transformed roots of B. vulgaris (Pavlov et al.,2002, 2005b).

An innovative transgenic approach with respect to antioxidantproduction has been applied to Saussurea involucrata transformed rootsoverexpressing heterologous chalcone isomerase from a related species(Li et al., 2006). This has led to the enhanced production of the flavoneapigenin, even in comparison to the selected wild type cell lines ortransformed roots not bearing foreign chalcone isomerase gene.

Besides the numerous positive results of transformed rootsproducing secondary metabolites, there is one example that achievedthe opposite result in relation to antioxidants. Specifically, in twoboraginaceous species: E. sericeum and L. erythrorhizon, havingintroduced the rolC gene exhibited greatly reduced levels of the twoantioxidants: rabdosiin and rosmarinic acid in comparison to the lineswithout the introduced rolC (Bulgakov et al., 2005).

3.7. Scale-up or the never-ending bioreactor story

The ultimate goal of the biotechnology of medicinal plants is theindustrial production of useful natural products. However, most of thepublished work deals with laboratory experiments on a small scale,even if there are liquid cultures involved. When thinking of industrialexploitation of the potential of plant cell factory, systematic scale-upstudies must be carried out. However, the literature on that isrelatively limited. One of the few established commercial systems isthe well-known shikonin technology using L. erythrorhizon (Misawa,1994; Bulgakov et al., 2001). Additional papers have been produced onexperimental bioreactor systems in which cells, aggregates ordifferentiated organs are sources of antioxidant compounds. A fewpilot-scale studies have been published on the production of α-tocopherol from hazel (Sivakumar et al., 2005), rosmarinic acid fromlavender (Pavlov et al., 2005a) and basil (Kintzios et al., 2004),betalains from B. vulgaris (Pavlov et al., 2007). Several types of vesselsare applied, but the most popular are classic designs like stirred, air-lift or mist bioreactors (Pavlov et al., 2007). Temporary immersionsystems are also used, e.g. for betalains (Pavlov and Bley, 2006) andsemi-continuous perfusion high density cultures for rosmarinic acidproduction by A. officinalis (Su et al., 1993; Su and Humphrey, 1990).The scale-up study on V. pahalae cell cultures proved the importanceof physical factors such as illumination of the bioreactor interior forsustained production of anthocyanins (Meyer et al., 2002). Similarly, inthe aforementioned A. cordata cultures, the upscale in 500 l. fermentorallowed the production of over 500 g of mixed anthocyanins during a16 day period (Kobayashi et al., 1993).

Nonetheless, the bioreactor technology in relation to above men-tioned antioxidants, though sometimes remarkably efficient in terms ofproduction (as in the case of rosmarinic acid), does not seem likely toapproach factory-scale industrial application in the near future. Hope-fully, at least some of the mentioned research on antioxidantsbiotechnology will finally result in market implementation.

3.8. Concluding remarks and future perspective

Despite the immense variety of antioxidant compounds ob-tained from in vitro cultured plant, their cells, tissues and organs,not many examples of practically applicable technologies can be named.The few that are available include rosmarinic acid, α-tocopherol fromsunflower cells and improved anthocyanins from several sources.

There are, however, numerous inspiring and promising reportson such compounds as resveratrol, kinobeonA, lithospermic acid B andother oligomeric caffeic acid depsides or crocin, which should provokeandencouragemore research focused on the regulation of biosynthesisand its increased economic feasibility. Finally, the burgeoningmetabolomic and metabolic engineering approaches may add a newfascinating dimension to the possibilities of antioxidant-making plantcell factories that have been presented in this paper.

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