Biotechnology Advances 26 (2008) 548560

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Biotechnology Advances
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Research review paper

Plant in vitro culture for the production of antioxidants A review

a r t i c l e

i n f o

a b s t r a c t
Plant in vitro cultures are able to produce and accumulate many medicinally valuable secondary metabolites. Antioxidants are an important group of medicinal preventive compounds as well as being food additives inhibiting detrimental changes of easily oxidizable nutrients. Many different in vitro approaches have been used for increased biosynthesis and the accumulation of antioxidant compounds in plant cells. In the present review some of the most active antioxidants derived from plant tissue cultures are described; they have been divided into the main chemical groups of polyphenols and isoprenoids, and several examples also from other chemical classes are presented. The strategies used for improving the antioxidants in vitro production efciency are also highlighted, including media optimization, biotransformation, elicitation, Agrobacterium transformation and scale-up. 2008 Elsevier Inc. All rights reserved.

Article history: Received 4 April 2008 Received in revised form 1 July 2008 Accepted 10 July 2008 Available online 16 July 2008 Keywords: Antioxidant In vitro culture Medicinal plants Biosynthesis

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. What do we call an antioxidant? . . . . . . . . . . . . . 1.2. Why hunt for antioxidants in vitro? . . . . . . . . . . . . 1.3. Methods used for assessment of antioxidant properties . . 2. Chemical classes of antioxidant secondary metabolites from in vitro 2.1. Polyphenols . . . . . . . . . . . . . . . . . . . . . . . 2.2. Isoprenoids . . . . . . . . . . . . . . . . . . . . . . . 2.3. Other structures . . . . . . . . . . . . . . . . . . . . . 3. Strategies for increased production . . . . . . . . . . . . . . . 3.1. Optimization of biosynthesis by culture conditions . . . . . 3.2. Production in differentiated tissues . . . . . . . . . . . . 3.3. Selection of high-producing cell lines . . . . . . . . . . . 3.4. Precursor feeding and biotransformation . . . . . . . . . 3.5. Elicitation and stress induced production . . . . . . . . . 3.6. Transformation with Agrobacterium rhizogenes hairy roots 3.7. Scale-up or the never-ending bioreactor story . . . . . . . 3.8. Concluding remarks and future perspective . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 548 549 549 549 550 550 553 553 555 555 555 555 556 556 557 557 557 557

1. Introduction The use of plant cell and tissue culture methodology as a means of producing medicinal metabolites has a long history (Rout et al., 2000; Verpoorte et al., 2002). Since plant cell and tissue culture emerged as a discipline within plant biology, researchers have endeavored to utilize plant cell biosynthetic capabilities for obtaining useful products and for studying the metabolism (Misawa, 1994; Verpoorte et al., 2002).

E-mail address: [email protected]. 0734-9750/$ see front matter 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2008.07.001

Cultured plant cells synthesize, accumulate and sometimes exude many classes of metabolites. Medicinal compounds are of particular interest and much effort has been devoted to obtaining some of the most active and precious therapeutics. Numerous alkaloids, saponins, cardenolides, anthraquinones, polyphenols and terpenes have been reported 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 natural products has grown rapidly. The etiology of several degenerative and aging-related diseases has been attributed to oxidative stress, and numerous studies have been undertaken to search for the most effective antioxidants (Halliwell, 1995; Aruoma, 2003; Soobrattee et al., 2005).

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The present review summarizes the achievements of plant cell and tissue culture technology for the production of secondary metabolites which have signicant potential as antioxidants. The focus will be on the major chemical classes of antioxidants and on the approaches used to improve the in vitro methods for producing these compounds. 1.1. What do we call an antioxidant? Broadly dened, an antioxidant is a compound that inhibits or delays the oxidation of substrates even if the compound is present in a signicantly lower concentration than the oxidized substrate (Halliwell, 1995; Halliwell and Gutteridge, 2007). The scavenging of reactive oxygen species (ROS) is one of possible mechanism of action. Others include the prevention of ROS formation by metal binding or enzyme inhibition. Chain breaking antioxidants prevent damage by interfering with the free radical propagation cascades. The antioxidant compounds can be recycled in the cell or are irreversibly damaged, but their oxidation products are less harmful or can be further converted to harmless substances (Halliwell and Gutteridge, 2007). At the cellular and organism level the antioxidant protection is provided by numerous enzymes and endogenous small molecular weight antioxidants such as ascorbic acid, uric acid glutathione, tocopherols and several others. Many compounds contain antioxidant activity in addition to their specialized physiological function, and their importance as antioxidants in vivo is sometimes ambiguous (Azzi et al., 2004). The antioxidant activity of plant secondary metabolites has been widely established in in vitro systems and involves several of the above mentioned mechanisms of action. Therefore, in this review, only those classes of compounds with established antioxidant properties will be considered. 1.2. Why hunt for antioxidants in vitro? The natural resources of both potential and established antioxidants are vast. Some antioxidant compounds are extracted from easily 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, and many others. Recently, the use of wine and olive industry waste products has been considered as a potential source of dietary or food preserving antioxidants (GonzlezParams et al., 2004; Obied et al., 2005). This leads to the question: why is the complex and rather expensive in vitro technology needed for products that may be obtained from abundant and cheap sources? The necessary conditions that make using biotechnological methods for the production of secondary plant metabolites economically viable have been rmly established: high economic value, insufcient abundance in intact plants, limited availability from natural sources (rare/endangered/overexploited species), and difcult cultivation (Misawa, 1994; Verpoorte et al., 2002). When taking these factors into consideration, in vitro technology offers some or all of the following benets: simpler extraction and purication from interfering matrices, novel products not found in nature, independence from climatic factors and seasons, more control over biosynthetic routes for obtaining the most desired variants (e.g. enantiomers or glycosides) or a proportion of given compounds, shorter and more exible production cycles, easier fulllment of the high-prole pharmaceutical production demands (such as GLP and GMP), and last but not least, the exploitation of the genetic engineering potential for avoiding legal restrictions against GMO introduction into the natural environment. As will be shown in this paper, some of the listed conditions are likely to be fullled with respect to antioxidants, whereas others will not, due to the ubiquitous presence of the main classes of antioxidants and the abundance of the aforementioned inexpensive sources. On the other hand, growing knowledge about the specic mechanisms of

their activity will create the demand for more specic products that could be used in accurately dened therapeutic and nutritional situations. Finally, as was inherent in plant in vitro cultures from the very beginning, investigation into the biosynthetic routes advances our understanding of the function and regulation of plant metabolites. Moreover, it is still barely known what the actual roles are of many antioxidant 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 of secondary metabolites, their antioxidant activity has not been actually determined. The knowledge of their properties usually comes from studies involving dietary antioxidants and compounds isolated from ethnomedicinal plants with both food conservation and chemoprevention in mind. Thus, in most cases the antioxidant power of many secondary metabolites is so well established, that real time monitoring of activity during the culture seems to be redundant. On the other hand, the enormous variability among antioxidants, and their complex structureactivity relationships suggest that antioxidant and any other biological activities should be paid more attention during research. There are a number of simple, slightly more complex, and quite sophisticated methods for antioxidant testing. The matter has been reviewed by many authors with respect to different aspects of 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 methods include free radical scavenging with use of colored, articial stable free radicals such as 2,2′-azinobis-(3-ethylbenzothiazoline6-sulfonate) (ABTS used in the TEAC assay Trolox equivalent antioxidant capacity) (Re et al., 1999) and DPPH&z.rad; (1,1-diphenyl2-picrylhydrazyl free radical) (Molyneux, 2004), as well as transition metal reduction that can be monitored by colorimetry. The metalbased methods include the reduction of ferric ions: FRAP (ferric reducing 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 affordable and can be used in high throughput screening. Their main drawback is that their relevance to the real oxidizing life is somewhat limited. The rst issue is the chemical context of the assays, which use articial compounds or are conducted in unrealistic conditions. This problem is eliminated in methods based on naturally occurring reactive oxygen species, but the fate of a free radical is observed either indirectly with chromophore reagents or with more expensive ESR techniques (electron spin resonance). The scavenging of superoxide radical anion, hydroxyl radical, or nitric oxide can be observed (Darmon et al., 1992; Aruoma, 2003; Sreejayan and Rao, 1997). Several assays based on a substrate degradation inhibition are available which can be used to nd out whether the tested compound can really protect biomolecules from oxidative damage. Polyunsaturated lipids, proteins, components of nucleic acids, cellular membranes, microsomal fractions from different organs can serve as model systems to be protected. The oxidation can be initiated by various chemical, physicochemical or biological approaches. The degradation products are monitored by spectrophotometry (thiobarbituric reactive substances TBARS, carotene bleaching), uorescence (ORAC assay 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 the antioxidant activity of compounds from in vitro cultures of several plants and will be referred to later in this paper as well as being specied in Table 1.

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2. Chemical classes of antioxidant secondary metabolites from in vitro cultures 2.1. Polyphenols It is ironic from the point of view of the tissue culturist, that the easily oxidizable phenolics, which are often regarded as nuisance and the reason for experiment failure, are at the same time highly desirable as dietary and therapeutic free radical scavengers. Considerable attention has been paid to minimizing the phenolization of cultures, frequently by adding antioxidants, such as ascorbic acid or glutathione (GSH), or phenol absorbing polymers like PVP (polyvinylpyrrolidone). The excessive production of polyphenols results from unfavorable or suboptimal culture conditions. At the onset of senescence, the decrease in free phenolics and a simultaneous

increase in cell-wall bound and oxidized forms is observed in callus cultures (Lopez Arnaldos et al., 2001), which reects the natural processes during plantpathogen interactions (e.g. hypersensitive response) (Hammerschmidt, 2005). Polyphenols in plants derive mainly from the shikimic acid pathway through aromatic carboxylic acids cinnamic or benzoic. Most of them possess antioxidant properties, albeit to varying degrees. The most powerful of the avonoids are the avanols and their oligomeric forms called condensed tannins or proanthocyanidins, avones and avonols and anthocyanins. Some isoavones are also known as antioxidants. Among other groups of antioxidant polyphenols there are: phenolic acids (caffeic acid derivatives), lignans, hydrolysable tannins (gallotannins and ellagitannins), stilbenes, and xanthones. Their physiological functions in plants are versatile and have been reviewed recently (Harborne, 2001; Matkowski, 2006).

Table 1 Selected examples of production of antioxidants from plant in vitro cultures Species Ajuga reptans Anchusa ofcinalis Anthoceros agrestis Arachis hypogea Artemisia judaica Beta vulgaris Carthamus tinctorius Cistanche deserticola Crocus sativus Cynara cardunculus Daucus carota Fagopyrum esculentum Glehnia littoralis Hemidesmus indicus Hyssopus ofcinalis Ipomoea batatas Lavandula ofcinalis Compounds Anthocyanins Rosmarinic acid Rosmarinic acid and its glucosides Piceatannol (a stilbene) Flavonoids Betalains Kinobeon A Phenylethanoid glycosides Crocin Cynarin, chlorogenic acid Anthocyanins Rutin Anthocyanins Rutin Rosmarinic acid, lithospermic acid B Anthocyanins Rosmarinic acid Culture system Flower cell culture Cell suspension Cell suspension Callus Shoot cultures in bioreactor Cell suspension, hairy roots Cell suspension Cell suspension Callus Callus Callus, cell suspensions Hairy roots Callus, cell suspension Callus, shoot culture Hairy roots Callus, cell suspension Callus, cell suspension, bioreactor Bioreactor culture of nodal explants and cell suspension UV irradiated callus Cell suspension Callus, shoot culture Callus, shoot culture, hairy roots, cell suspension Callus, regenerated plantlets, hairy roots Hairy roots overexpressing CHI under camv35s Hairy roots, cell suspension Antioxidant testing -carotene bleaching, lipid peroxidation Many in vitro chemical assays Many in vitro chemical assays Oxidative DNA damage in cell culture DPPH DPPH Cell membrane peroxidation, XOD/NBT, singlet oxygen quenching DPPH Alleviation of oxidative stress in cultured mammalian cells TBARS Lipid peroxidation Many in vitro chemical assays Many in vitro chemical assays Many in vitro chemical assays Many in vitro chemical assays DPPH Superoxide radical scavenging References for in vitro culture Callebaut et al., 1990; Terahara et al., 2001 De-Eknamkul and Ellis, 1984; Su et al., 1993 Vogelsang et al., 2006 Ku et al., 2005 Liu et al., 2004 Pavlov et al., 2005a,b; Hunter and Kilby, 1999 Kanehira et al., 2003; Kambayashi et al., 2005 Cheng et al., 2005 Chen et al., 2003, 2004 Trajtemberg et al., 2006 Ravindra and Narayan, 2003 Lee et al., 2007 Miura et al., 1998; Kitamura et al., 2002 Misra et al., 2005 Murakami et al., 1998; Kochan et al., 1999 Konczak-Islam et al., 2000; Terahara et al., 2004 Georgiev et al., 2006a,b; Kovacheva et al., 2006; Pavlov et al., 2005a,b Kintzios et al., 2004 Antognoni et al., 2007 Hempel et al., 1999 Caruso et al., 2000 Grzegorczyk et al., 2006, 2007 Morimoto et al., 1994; Chen et al., 1999a; Yan et al., 2006 Li et al., 2006 Morimoto et al., 1998; Nishikawa et al., 1999; Stojakowska and Malarz, 2000 Tadhani et al., 2007 Orihara et al., 2002 Meyer et al., 2002 Decendit and Mrillon, 1996; Fauconneau et al., 1997; Tassoni et al., 2005 Kumar et al., 2005 References for antioxidant activity Terahara et al., 2001 e.g. Petersen and Simmonds, 2003; Soobrattee et al., 2005 e.g. Petersen and Simmonds, 2003; Soobrattee et al., 2005 Ovesna and Horvathova-Kozics, 2005 Liu et al., 2004 Pavlov et al., 2005a,b Kanehira et al., 2003; Kambayashi et al., 2005 Cheng et al., 2005 Ochiai et al., 2004 Trajtemberg et al., 2006 Ravindra and Narayan, 2003 e.g. Hinneburg et al., 2006 e.g. Kahkonen and Heinonen, 2003 e.g. Ravishankara et al., 2002 e.g. Petersen and Simmonds, 2003; Soobrattee et al., 2005 Konczak-Islam et al., 2003; Terahara et al., 2004 Kovacheva et al., 2006

Ocimum basilicum Passiora quadrangularis Petroselinum sativum Rosmarinus ofcinalis Salvia ofcinalis Salvia miltiorrhiza Saussurea involucrata Scutellaria baicalensis

Rosmarinic acid Flavone-C-glycosides Flavonols and avones Carnosic acid Rosmarinic acid, abietane diterpenoids Lithospermic acid B, rosmarinic acid Apigenin Baicalin, wogonoside

Many chemical in vitro assays DPPH In vivo (rats) Oxidative stress reduction in living cells and many chemical in vitro assays DPPH, P-Mo, lipid peroxidation DPPH H2O2-induced cell damage Many in vitro chemical assays

e.g. Petersen and Simmonds, 2003; Soobrattee et al., 2005 Antognoni et al., 2007 Hempel et al., 1999 e.g. Wijeratne and Cuppett, 2007 Grzegorczyk et al., 2007 Chen et al., 1999b Fan and Yue, 2003 e.g. Huang et al., 2006

Stevia rebaudiana Torreya nucifera Vaccinium pahalae Vitis vinifera

Flavonoids Abietane diterpenoids Anthocyanins Stilbenes, procyanidins

Callus Cell suspension Cell and aggregate suspension Cell suspension

FRAP, DPPH LDL oxidation, nitric oxide inhibition Many in vitro chemical assays DPPH, lipid peroxidation

Tadhani et al., 2007 Lee et al., 2006 e.g. Kahkonen and Heinonen, 2003 Fauconneau et al., 1997

Withania somnifera

Withanoloids

Hairy roots

DPPH, carotene-linoleic acid oxidation, brain lipid peroxidation, hydroxyl radical scavenging

Kumar et al., 2005

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Phenolic acids occur in plants in free form as glycosides and can be integrated into larger molecules in an ester form. They are common as depsides the intermolecular ester of two or more units composed of the same or different phenolic acids such as: caffeic, coumaric, ferulic, gallic, and syringic. Depsides are, for example, a ubiquitous chlorogenic as well as isochlorogenic, ellagic, lithospermic and rosmarinic acids. Due to the presence 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 depside composed of two caffeic acid molecules (Fig. 1), found in many Lamiaceae and Boraginaceae species. Its biochemistry and pharmacology has been extensively reviewed by Petersen and Simmonds (2003). The antioxidant properties of rosmarinic acid are also well established. Several plants and various techniques have been used for in vitro production of this compound (Shetty, 1997). Rosmarinic acid can accumulate in cell cultures to amounts greater than those in intact plants. The suspension cultures producing rosmarinic acid were generated from Anchusa ofcinalis (De-Eknamkul and Ellis, 1984,

1985; Su and Humphrey, 1990; Su et al., 1993), Eritrichum sericeum (Fedoreyev et al., 2005), Lithospermum erythrorrhizon (Yamamoto et al., 2000) (Boraginaceae), and Coleus blumei (Petersen et al., 1993), Lavandula vera (Pavlov et al., 2005a,b; Georgiev et al., 2006a,b), Ocimum basilicum (Kintzios et al., 2004), Salvia ofcinalis (Hippolyte et al., 1992), Zataria multiora (Mohagheghzadeh et al., 2004) (Lamiaceae). Depending on the species, the yield of rosmarinic acid from suspension cultures can exceed 10% of cell mass and 6 g/l of medium. An interesting example is also the production of rosmarinic acid of over 5% of cell dry mass in Anthoceros agrestis (Vogelsang et al., 2006). Another system used for rosmarinic acid production are transformed roots of Salvia miltiorrhiza (Chen et al., 1999a,b, 2001) and S. ofcinalis (Grzegorczyk et al., 2006, 2007). In the second species, the accumulation was much higher reaching 4.5% of dry weight and its antioxidant activity has been conrmed with several assays. Extracts from rosmarinic acid accumulating lavender cells also have superior radical scavenging properties (Kovacheva et al., 2001, 2006). Even larger amounts (over 7% dw) of rosmarinic acid accumulated in Agrobacterium transformed callus of C. blumei (Bauer et al., 2004) and transformed roots Hyssopus ofcinalis, but only when cultured on WP

Fig. 1. Structures of some phenolic antioxidant metabolites from plant in vitro cultures.

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medium which was much more superior to both MS and B5 (Murakami et al., 1998). Razzaque and Ellis (1977) report a yield of 811% of rosmarinic acid in C. blumei cell cultures. An extremely high level of RA production was reported by Hippolyte et al. (1992) in S. ofcinalis reaching 6.4 mg/l of suspension culture, although the result has not appeared in any other reference. A closely related antioxidant is lithospermic acid B, a tetrameric depside (Fig. 1), accumulating especially in the aforementioned hairy root cultures of H. ofcinalis and elicited S. miltiorrhiza as well as in Lithospermum erythrorhizon suspension cultures (Yamamoto et al., 2000, 2002). Oligomeric caffeic acid depsides such as DCQAs (dicaffeoylquinic acids) potent antioxidants and antivirals, accumulate also in cell cultures of sweet potato storage roots, where they are accompanied by anthocyanins (Konczak et al., 2004). Chlorogenic acid and cynarin (another DCQA) are present in cardoon (Cynara cardunculus) callus in quantities two-times greater than in the leaves the usual medicinal crude drug. The antioxidant capacity of the cardoon callus was conrmed by the TBARS method (Trajtemberg et al., 2006). Flavonoids are one of the largest groups of secondary metabolites based on a common structure. Chemically they possess a tricyclic phenylbenzopyran (with the exception of chalcones) structure but biosynthetically come from phenylalanine and malonyl-CoA in the phenylpropanoid pathway. Subclasses of avonoid include avonols, avones, isoavonoids, avanons, avanols, proanthocyanidins (catechins), and anthocyanidins. Substitution patterns include the hydroxylation of rings A and B, methylation of hydroxyl groups, prenylation, and glycosylation. Other modications can form biavonoids, lignans, oligomeric proanthocyanidins, glycoside esters with other phenolics etc. Antioxidant properties of avones depend on the hydroxylation pattern. Substitution with hydroxyl, methoxyl or other groups, and glycosylation also inuences hydrophilicity and many biological activities. Among the vast variety of structures, several avones from the genus Scutellaria (Lamiaceae) are distinguished by their unsubstituted B-ring and common occurrence in the form of glucuronides (Fig. 1). Scutellaria baicalensis, the favorite natural drug of traditional Oriental medicines, contains in the roots probably the highest known amount of avones (up to 20% dw) of which the strong lipophilic antioxidants: baicalin, baicalein, wogonoside and wogonin are the most abundant. In cell suspension and the hairy root cultures of S. baicalensis the accumulation of these compounds was reported (Morimoto et al., 1998; Nishikawa et al., 1999; Stojakowska and Malarz, 2000). The in vivo antioxidant function of baicalein in metabolizing hydrogen peroxide has been proposed as a result of experiments using cell cultures (Morimoto et al., 1998). Flavone C-glycosides are of particular interest because of their limited occurrence in plants and they have important therapeutic properties, including the prevention of cardiovascular disorders related to oxidative stress. In tissue cultures of Passiora quadrangularis several C-glycosylated avones such as isoorientin, orientin (Fig. 1), vitexin and isovitexin have been induced in varied amounts, and their antioxidant activity determined by DPPH assay (Antognoni et al., 2007). Crataegus monogyna is another source of these C-glycosides and its long-term callus cultures have been shown to produce several types of phenolic antioxidants assayed with various methods (Rakotoarison et al., 1997; Bahorun et al., 2003). Oligomeric proanthocyanidins (also known as condensed tannins) are considered to be the most powerful antioxidants of all the avonoids owing to their structure (procyanidin B1 as an example in Fig. 1) (the chemical mechanisms have been reviewed by Bors and Michel, 2002). They are responsible for the antioxidant activity of wines, teas and fruits as well as of popular nutraceuticals made from grape seeds or pine bark. The thorough genomic and metabolic engineering 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 oligomeric proanthocyanidins have been reported in the cell cultures of Vaccinium pahalae (Kandil et al., 2000) and Vitis vinifera (Decendit and Mrillon, 1996). Another example of avonoid polyphenols frequently studied for in vitro production is anthocyanins. Pigments are useful as products because their accumulation can easily be visually evaluated. Moreover, their benecial health promoting properties as well as their growing importance as bioactive and natural food colorants additionally increases their attractiveness for plant biotechnology. The versatile values of anthocyanins have been reported and reviewed in countless publications. One of their most pronounced features is their substantial antioxidant capacity determined in various assays, but their biological activity varies greatly, due to their structural diversity. Analogously to other polyphenols, more hydroxylated compounds express larger free radical scavenging capacity, whereas methylation of the hydroxyl groups decreases this effect partially (Stintzing and Carle, 2004). Glycosylation can also reduce the ability to scavenge free radicals, but on the other hand enhances the stability of the molecule. Acylation of the sugar moieties with carboxylic, usually hydroxycinnamic acids (an example of a glycosylated and acylated cyanidin is shown in Fig. 1) additionally boosts the antioxidant potential and color stability. For that reason, not all anthocyanins are equally desirable as natural medicines. Cyanidin and delphinidin glycosides acylated with phenol carboxylic acids are preferred over less substituted versions and petunidin, pelargonidin, peonidin or malvidin derivatives. This is one of the main reasons for conducting tissue culture experiments. In abundant natural sources, the anthocyanin prole is rarely optimal and the raw material often requires complex extraction and purication procedures. In several species listed below the level of anthocyanins in vitro equals or exceeds the natural sources and their metabolic prole is more useful. Thus, even if anthocyanins are not so expensive, the ability to obtain products with useful structural and functional properties may become economically feasible (Konczak et al., 2005). Anthocyanins are obtained from in vitro cultures of Prunus sp. (Durzan et al., 1991; Blando et al., 2005), Daucus carota (Ravindra and Narayan, 2003), Glehnia litoralis (Miura et al., 1998), V. pahalae (Meyer et al., 2002), Ipomaea batatas (Konczak-Islam et al., 2000; Terahara et al., 2004) Fragaria ananassa (Edahiro et al., 2005), V. vinifera (Decendit and Mrillon, 1996), Rudbeckia hirta (Luczkiewicz and Cisowski, 2001) and Ajuga reptans (Callebaut et al., 1990; Terahara et al., 2001) and others. Interestingly, two species used medicinally due to their anticancer alkaloids, Catharanthus roseus and Camptotheca acuminata, produce signicant amounts of anthocyanins of over 200 g/g fresh weight in cell cultures (Filippini et al., 2003; Pasqua et al., 2005). In C. roseus, p-coumaroyl glucosides of highly methoxylated malvidin or hirsutidin predominated, unlike in eld-grown owers. In C. acuminata a rare cyanidin galactoside accounted for over 95% of all anthocyanins. In A. reptans ower-derived cell cultures the anthocyanin composition was altered from delphinidin-based in owers towards stronger antioxidants di-acylated cyanidin glycosides (Callebaut et al., 1997; Terahara et al., 2001). This observed simplication and alteration of the metabolic prole, when compared to natural conditions, is typical for in vitro produced anthocyanins. In the extensively studied I. batatas, in the tissue cultures induced from storage roots the accumulation of anthocyanins can even exceed the tubers by several times. The production of valuable di-acylated cyanidin glycosides from callus and cell suspension as well as their regulation towards overproduction and their more desirable prole has also been reported (Terahara et al., 2000, 2004; Konczak et al., 2005). The superior stability and antioxidant activity of anthocyanin-rich extracts have also been conrmed. In this species the extraordinary high amount of anthocyanins was recorded in selected cell lines along with a greater proportion, in comparison to the tubers, of cyanidin-based to peonidin-based

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pigments (Konczak et al., 2005). Given the aforementioned ability of cell culture from this species to accumulate medicinally active depsides, this is one of the most promising systems for the biotechnological production of health-promoting polyphenols. Silybum marianum, milk thistle, the famous hepatic drug contains in the achenes an isomeric avonoid lignans complex called silymarin that is a strong antioxidant (e.g. silybin in Fig. 1). The untreated cell cultures contain low and variable amounts of avonolignans, but after they have been elicited their production can be increased 6-fold (Sanchez-Sampedro et al., 2005a). In another study, the same authors reveal the pivotal role of calcium deprivation in stimulating silymarin biosynthesis (Sanchez-Sampedro et al., 2005b). The role of peroxidases in both the synthesis by oxidative coupling of precursors and the degradation of silymarin compounds has also been explored (Sanchez-Sampedro et al., 2007a). Stilbenes are bicyclic polyphenols represented by the well-known resveratrol (Fig. 1), a phytoalexin and the red wine antioxidant. Although present in a considerable amount of natural sources like grapes and some Polygonaceae species, it has been also induced in elicited hairy root cultures of peanut (Arachis hypogea) and recovered efciently from the liquid medium (Medina-Bolivar et al., 2007). The same species was used to enhance production of a rare stilbene piceatannol in cell culture (Ku et al., 2005). This approach seems quite promising for the production of a puried single antioxidant which the pharmaceutical industry prefers to use. In cell cultures of V. vinifera, the inuence of elicitors on the stilbene biosynthesis and the stilbene synthase activity has been studied (Tassoni et al., 2005) as well as the antioxidant capacity of resveratrol and astringin from cultured cells (Fauconneau et al., 1997). Other examples of recently reported antioxidant activities of in vitro cultured, polyphenol-rich plants include: Curcuma longa mass propagation on a thin-lm rocker system where signicant and clone dependent DPPH scavenging was noted (Cousins et al., 2007); Cistanche deserticola cell suspensions producing antioxidant phenylethanoid glycosides also assayed by a DPPH test (Cheng et al., 2005); Stevia rebaudiana the herbal sweetener callus extracted with water and methanol had higher antioxidant activity than the leaves of eldgrown plants, which correlated with the higher levels of avonoids and total polyphenols (Tadhani et al., 2007). 2.2. Isoprenoids Isoprenoids are produced in plant cells from isopentynyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). These basic units are synthesized via cytoplasmic mevalonate or plastidic deoxyxylulose (or methylerythritol) phosphate pathways. IPP and DMAPP are subsequently fused by sets of prenyltransferases and terpene synthases and a variety of modifying enzymes to eventually yield enormously variable compounds in numerous groups of terpenoids (mono, sesqui, di, and triterpenes and their derivatives), carotenoids, and steroids (Bouvier et al., 2005; Tholl, 2006). Each of these groups contains metabolites of different chemical characters such as alcohols, acids, aldehydes, or a combination of these; they can also be aromatic, glycosylated and form more complex structures. The most powerful antioxidants among them are carotenoids and abietane diterpenoids (some of them containing a phenolic structure within a molecule). In comparison to most phenylpropanoid polyphenols, isoprenoids are generally more lipophilic, hence their important role in protecting membrane lipids. Both -carotene and lycopene are recognized as dietary chemopreventive agents, protecting against cancerogenesis. The participation in an antioxidant cascade is likely to contribute to these prophylactic properties. Crocin derived from saffron (stigmata of Crocus sativus) has been conrmed as a powerful antioxidant (Ochiai et al., 2004), stronger than -tocopherol, and being 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 by means of microbial (Bhosale and Bernstein, 2005) and fungal biotechnology (Bhme et al., 2006), it is reasonable to obtain crocin from the plant tissue cultures (Chen et al., 2003, 2004) of C. sativus. In this case, the in vitro production could be protable due to the high cost of the raw material (about 200 000 owers are needed to get 1 kg of dried stigma). Abietane diterpenes are present in many Lamiaceae medicinal plants as well as in some gymnosperms. Some of these compounds demonstrate substantial antioxidant potential in various systems. Carnosic acid (Fig. 2) and carnosol, rosmanol and ferruginol (Fig. 2) are responsible for the antioxidant activity of sage (Salvia sp.) and rosemary (Rosmarinus ofcinalis) along with the phenolic rosmarinic and salvianolic acids, but the organ distribution differs between the two mentioned chemical classes. However, the published tissue culture studies in Lamiaceae aimed at enhancing the rosmarinic acid production rather than at abietane diterpenoids. The tissue cultures as a source of abietane diterpenes have been established for R. ofcinalis (Caruso et al., 2000), S. ofcinalis (Grzegorczyk et al., 2007) and Salvia sclarea (Kuma et al., 2006). The antioxidant activity of in vitro cultures of common sage, especially in lipophilic systems such as the inhibition of lipid peroxidation, can be attributed to abietane diterpenes rather than to phenolics (Grzegorczyk et al., 2007). Light plays an important role in stimulating the diterpene biosynthesis in vitro (Caruso et al., 2000; Kuma et al., 2006) even though in natural conditions they can only be found in the underground parts of plants. Volatile terpenoids responsible for fragrant properties of plant organs serve as a multipurpose chemical defense weapon against fungal and bacterial pathogens as well as a means of long distance chemical communication e.g. attracting pollinators. Moreover, the role of a monoterpene -thujaplicin in the alleviation of oxidative damage in cell cultures of cypress has been postulated (Zhao et al., 2005), but this does not imply that this is its direct function as an antioxidant. The antioxidant properties of several monoterpenes, such as -terpinene and limonene have been reported. This suggests that this group of compounds may receive more attention although at present they are underestimated as antioxidants. Tissue cultures are able to produce volatile constituents of essential oils and many reports have been produced on that subject (Mulder-Krieger et al., 1988; Hrazdina, 2006). However, in vitro production of essential oils has not been aimed at the antioxidant properties of the products. 2.3. Other structures Tocopherols, widely used in human nutrition as vitamin E and in food conservation, are powerful free radical scavengers and protect plant cells against oxidative damage in a lipophilic environment (Kruk et al., 2005). Plants are the primary source of natural tocopherols isolated from vegetable oils or maize embryos. Their biosynthesis integrates the aromatic aminoacids (shikimic acid) and plastidic isoprenoid deoxyxylulose phosphate (pentose) pathways. The rst pathways create the aromatic (phenolic) head while the second gives rise to the hydrophobic tail of a tocochromanol skeleton (DellaPenna, 2005). -Tocopherol (Fig. 2) is the most active form of vitamin E, while the extraction from plant oils usually yields a mixture of , , and tocopherols and tocotrienols. Therefore there is a demand for alternative sources of pure -tocopherol from plant tissues. Various culture systems of Helianthus annuus (sunower) have been established for this purpose. A 100% increase in cell suspensions has been achieved by media optimization, elicitation and precursor feeding (Caretto et al., 2004; Gala et al., 2005). A photomixotrophic cell line has also established a cultured on sucrose-deprived medium (Fachechi et al., 2007). An attempt has been also made to select the most efcient genotype of hazel (Corylus avellana) for the production of tocopherols in bioreactors (Sivakumar et al., 2005).

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

Betalains the purple betacyanins and yellow betaxanthines pigments functionally replace anthocyanins in 13 taxons grouped in Caryophyllales (previously Centrospermae) order, such as the families: Chenopodiaceae, Amaranthaceae, Portulacaceae, Cactaceae, Phytolaccaceae and others. These nitrogen containing compounds (Fig. 2) although widely used as non-toxic food colorants, remain understudied in terms of their antioxidant potential (Gliszczyska-Swigo et al., 2006; Kanner et al., 2001). These properties of betalains have been recently reviewed by Stintzing and Carle (2004). The major advantages of betalains as dietary antioxidants are their bioavailability, which is greater than most avonoids, 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 metabolic in vitro studies on their biosynthesis. They possess the useful properties of pigments and are therefore preferred as a model system despite their low commercial value. The production of beetroot pigments has been performed using a variety of techniques such as cell suspensions and transformed roots in bioreactors (Hunter and Kilby, 1999; Pavlov et al.,

2002, 2005b). Being ideal metabolites for direct observation they have also been used for compound recovery experiments using various permeabilization and extraction methods (Hunter and Kilby, 1999). Red beet betalains from hairy roots have been shown to be efcient free radical scavengers (Pavlov et al., 2002, 2005b). The growing knowledge of their therapeutic and preventive properties is likely to further increase the research interest in their in vitro biosynthesis. Indole glucosinolates are a tryptophane containing group from the diverse class of thioglycoside compounds typical to the Brassicaceae. Upon hydrolysis by myrosinase they generate biologically active antioxidants and carcinogenesis inhibitors such as indole-3-carbinol derived from glucobrassicin (Fig. 2). In cruciferous plants they are thought to contribute to the regulation of auxin biosynthetic routes (Bak et al., 2001). Although they are present in common cabbage related vegetables (broccoli in particular) their instability and uneven or unpredictable distribution would make their biotechnological production an interesting option. Yet, to date there has been very little data available on that topic. The horseradish (Armoracia rusticana) cells, both in suspension and immobilized in polyurethane

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foam, produce indole-3-methylglucosinolate and 4-hydroxyindole-3methylglucosinolate (Mevy et al., 1999). Kinobeon A a novel, red-colored compound isolated from Carthamus tinctorius cell cultures uniquely combines the quinoid aromatic character with several conjugated double bonds (Fig. 2). It has therefore been demonstrated as being a remarkably strong antioxidant. Kinobeon A efciently quenches various reactive oxygen species, inhibits lipid peroxidation and supports the survival of mammalian cells during oxidative stress (Kanehira et al., 2003; Kambayashi et al., 2005). To date, little is known about kinobeon A physiological functions and biosynthesis. Whether it occurs also in other organisms or is an example of an exclusive cell-culture product remains to be determined. 3. Strategies for increased production The continuous monitoring of a chosen metabolite is a prerequisite for the successful development of production technology and can be carried out using a number of different methods. For accurate measurements, chromatographic methods such as HPLC and GC with the appropriate detection are sufcient. Laboratories that lack phytochemical facilities need other equivalent substitution methods. For operation on a large scale, time saving is also a consideration. These faster and less elaborate techniques involve spectrophotometry that can be used for some groups of compounds, while for other types it is rather unsuitable. The rst group contains colored compounds avonoid, carotenoid, and quinoid pigments or UV absorbing phenolics. Anthocyanins are commonly estimated by this technique, which can also be applied non-destructively (Durzan et al., 1991; Smith, 2000). On the other hand, this approach is only adequate if there is just one dominant antioxidant, differing in the absorption maxima from any interfering compounds. The rapid progress in phytochemical instrumentation development and the trend towards a metabolomic approach and high throughput screening is likely to also inuence the monitoring of secondary metabolites production in vitro. Indeed, in recent publications the chromatographic determination of antioxidants predominates, frequently accompanied by bioactivity guided fractionation, spectral structure elucidation of isolated chemicals by MS or NMR. The use of hyphenated techniques such as LC-MS and LCNMR or 2D-NMR is considered to be the best means for structure determination when novel compounds are present in vitro that have not 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 to inuence the biosynthetic pathways of secondary metabolites. Light induction of anthocyanin pigments is a well documented example (Harborne, 2001; Stintzing and Carle, 2004). In cultured cherry (Prunus cerasus) callus and suspensions, the light exposure causes a rapid change of the tissue color from white to purple (Blando et al., 2005). The anthocyanidin prole was different both from intact leaves and fruits with cyanidin-3-glucoside as the single dominant compound, absent delphinidin (unlike in fruits) and only a small amount of cyanidin-3rutoside (dominant in the leaves). This is a good example of a distinct metabolic prole under in vitro conditions. However, the irradiation of cultures can lead to an increase in cost and can generate undesirable temperature gradients in the culture vessels. Therefore, several examples of constitutive, light-independent systems for anthocyanin production were established such as Glehnia 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 nal 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 of biosynthetic pathways, unless we deal with a hormone-independent transformed root culture. In anthocyanin producing G. littoralis cultures, NAA at 1 mg/l was the preferred auxin, superior to IAA and 2,4-D and the addition of 0.1 mg/l kinetin further improved cell growth and pigment biosynthesis (Miura et al., 1998). The higher concentration of NAA (5 mg/l) was optimal for R. hirta (Luczkiewicz and Cisowski, 2001). In the same study, the highest accumulation was achieved by adding cystein, which is one of the constituents of CoA, a crucial factor in avonoid biosynthesis. A two or three-stage growth system can be also useful for optimizing metabolite production. The rst stage is used for the initiation and/or the maintenance of a culture with the aim of obtaining the highest percentage of induction and the fastest biomass growth. The second phase aims at the accumulation of a product. It has been successfully applied to anthocyanins in R. hirta (Luczkiewicz and Cisowski, 2001) where the tissues in the optimized second stage media contained up to 5% anthocyanins in dry mass. In C. sativus cultures the two-stage system leads to a substantial accumulation of crocin (430 mg/l) simply by changing the hormone composition from NAA at 2 mg/l, BAP at 1 mg/l for biomass growth to IAA 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 for producing a considerable amount of secondary products, especially if the undifferentiated cell culture is either unable to or barely accumulating antioxidant compounds. This has been reported for several classes of metabolites, especially for alkaloids, and has also been published for some antioxidant compounds. Ellagic acids present in Rubus chamaemorus plants was 3 times lower in shoot cultures and over 10 times lower in callus (Thiem and Krawczyk, 2003). Similarly in O. basilicum cell cultures (Kintzios et al., 2004) the accumulation of rosmarinic acid was markedly lower than in regenerated plantlets. In S. ofcinalis (Grzegorczyk et al., 2007) and rosemary (Caruso et al., 2000) in vitro cultures, the abietane diterpene antioxidants (carnosol and carnosic acid) were present only in shoot cultures and not in callus, suspension or hairy roots. On the other hand, undifferentiated cell suspensions are able to accumulate great amounts of phenolic acids. Moreover, the cells cultured in suspension tend to form larger aggregates upon differentiation, therefore increasing their shear stress resistance. The potential of fast growing somatic embryos cultures can be also utilized for the production of medicinal metabolites, but reports on that topic are scarce and deal chiey with alkaloid production such as paclitaxel (Lee and Son, 1995). For example, phenol glycosides, lignans and avonoid accumulation has been mentioned in Siberian ginseng somatic embryos (Shoahel et al., 2006). Even in dedifferentiated cells, some biosynthetic potential typical for the developed organs from which they were initiated, can be conserved. In Pueraria lobata callus cultures, the bioactive isoavonoid content depended on the source organ, reecting relations in the mature plant (Matkowski, 2004). The selection of proper donor plants and organs should already be considered when starting the culture, unless it can be overcome by a suitable treatment. However, in most circumstances the dedifferentiation apparently also involves some of the biochemical properties of the cells. The modication of relative biosynthesis to degradation ratios of a desired product can also inuence the nal levels of a desired compound in the culture. 3.3. Selection of high-producing cell lines The super-efcient clones of cultured cell or tissues can be selected by monitoring the level of the compound of interest, or can be complemented by a selecting agent facilitating the process. The

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selected line should exceed both the levels of production obtained in unselected cell cultures and the natural biosynthetic productivity of an intact organism. The addition of a phenylalanine analogue to the culture medium causes damage of the majority of cells except those who express a high degree of activity of PAL (phenylalanine ammonia lyase). The overexpression of PAL in selected variants results in elevated phenylpropanoid compounds, many of which are desired antioxidants. Rosmarinic acid producing cells of lavender (Georgiev et al., 2006b) and shikonin producing cells of L. erythrorhizon (Bulgakov et al., 2001) have been selected with the use of uorinated phenylalanine. The selected lines appear to be stable over time and can synthesize twice as much of the desired compounds as the control cultures. If the desired product is colored as in the case of anthocyanins, a simple visual selection with the mechanical dissection of accumulating cells can be performed with good results. An impressive example is the origin of the stable (over a period of several years), lightindependent anthocyanin accumulating cell line of G. litoralis selected from a single purple spot in a white, dark grown callus (Miura et al., 1998; Kitamura et al., 2002). Similarly, a selected cell line of purple sweet potato (PL) accumulates a large amount of acylated anthocyanins 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 its intact form accumulates substantial amounts of valuable products, it sometimes happens that a dedifferentiated cell mass is unable to complete the biosynthesis. This is due to the uncoupling of the enzymatic machinery, or an insufcient expression of developmentally regulated biosynthetic genes, or a lack of environmental stimuli. If biosynthetic enzymes are expressed in part of the pathway, the exogenous delivery of precursors can solve the problem. It can be also helpful in omitting the metabolic channeling that directs the precursors to a pathway other than that desired. In a Rhodiola rosea compact callus aggregate culture no secondary products were detected until the medium was supplemented with cinnamyl alcohol that was efciently transformed into a pharmacologically active rosin and rosavin (Gyrgy et al., 2004). However, neither are antioxidants. Salidroside, the antioxidant present in the plant, was not found in that study, but other authors have succeeded in antioxidant production in this species (Furmanowa et al., 1998). In contrast, the feeding of naringenin or phenylalanine signicantly increased anthocyanins in Rudbeckia (Luczkiewicz and Cisowski, 2001) and strawberry (Edahiro et al., 2005) cells, respectively. In Cistanche salsa cell cultures, their enrichment with a mixture of phenylethanoid glycosides precursors results in a doubling of production 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 the successful enhancement of in vitro antioxidant production. Curcumin is a yellow colored, potent polyphenolic antioxidant and cholagogue from the roots of turmeric (C. longa). Its chief weakness is its low water solubility, which limits its intestinal absorption and hence its pharmaceutical usefulness (Sharma et al., 2007). In another surprising application of C. roseus cell cultures, they were modied by cellular enzymes into unnatural glycosides that are much more soluble in water (Kaminaga et al., 2003). 3.5. Elicitation and stress induced production One of the best established roles of secondary metabolites is the involvement in stress responses (Grassmann et al., 2002). Phytoalexins are synthesized in reaction to a pathogen attack, other compounds are associated with abiotic disturbances.

The upregulation of biosynthesis of secondary metabolism upon exposure to stress factors has been used to obtain a high yield of many medicinal compounds (Verpoorte et al., 2002; Vanisree et al., 2004). Exposure to stress factors results in an excessive level of oxidizing molecules such as ROS due to the disturbances in redox state equilibrium. The increased ROS production may help to combat the invasive pathogens but can also be triggered by harmful environmental factors such as UV radiation, xenobiotics, heavy metals, drought, and mechanical damage. Therefore, antioxidant properties of some metabolites along with antioxidant enzymes can aid the plant in restoring the redox homoeostasis (Matkowski, 2006). Apart from direct antioxidant activity, some compounds e.g. avones can serve as substrates for cellular peroxidases (Morimoto et al., 1998). In vitro cultured material can be elicited by bacterial or fungal lysates or stress response mediators such as salicylate or jasmonic acid/methyl jasmonate and hydrogen peroxide as well as by environmental factors like metals or irradiation. The stress related growth regulators, jasmonic acid and its esters, especially methyl jasmonate (MeJa) have been widely used in promoting the biosynthesis of both inducible and constitutive secondary metabolites, including medicinal compounds such as anticancer alkaloids (Vanisree and Tsay, 2004; Verpoorte et al., 2002). With respect to the antioxidants, jasmonic acid enhanced tocopherol production by sunower and A. thaliana cell cultures (Gala et al., 2005) and cyanidin glucosides in cherry callus (Blando et al., 2005). On the other hand, in the resveratrol producing grapevine cell cultures (Tassoni et al., 2005), jasmonic acid proved to be a weaker elicitor than its methyl ester. Besides, MeJa promoted the accumulation of trans-resveratrol, whereas JA resulted in a modest increase of the less desirable cis-resveratrol. MeJa and salicylic acid also stimulated the curcumin glycosylation by C. roseus cell cultures (Kaminaga et al., 2003). Using natural biotic elicitors also results in enhanced secondary metabolism. Yeast extracts (YE also read as yeast elicitor), but neither chitin nor chitosan, have a positive impact on the production of silymarin, the effect being potentiated when combined with JA. However, it proved more effective when supplemented with MeJA in this system (Sanchez-Sampedro et al., 2005a). Chitin elicited the production of several new avonoids 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 the accumulation of rosmarinic and lithospermic B acids, preceded by a signicant rise in tyrosine aminotransferase activity (Chen et al., 2001; Yan et al., 2006). Compared to the effect of an abiotic elicitor Ag+, YE was superior (Yan et al., 2006). Rosmarinic acid production was also enhanced in O. basilicum hairy roots by Phytophtora cell walls, while salicylic and jasmonic acids or chitosan suppressed it (Bais et al., 2002). In peanut hairy roots (Medina-Bolivar et al., 2007) sodium acetate and chitosan were the most effective of the ve tested elicitors causing a 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 in lavender cell culture nearly three-fold (Georgiev et al., 2006a), while in Vitis cells only the cis isomer of resveratrol was induced (Tassoni et al., 2005). Rare earth elements such as La3+ and Ce3+ can also serve as elicitors to promote crocin (Chen et al., 2004), phenylethanoids from C. deserticola (Ouyang et al., 2003), and silymarin (Sanchez-Sampedro et al., 2005b) production. The mechanism of these lanthanides actions involves interference with cellular calcium signaling (SanchezSampedro et al., 2005b). Irradiation with UV has been also used for making the plant cells synthesize more antioxidants. In particular, avonoids and other polyphenols are known to protect plants from harmful UV impact. In P. quadrangularis callus subjected to UV-B treatment, the irradiation

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stimulated a large increase in the amount of avonoid, corroborating the higher radical scavenging activity (Antognoni et al., 2007). Similarly, the callus of A. hypogea was induced by UV radiation to accumulate much greater amounts of stilbenes (Ku et al., 2005). The use of a downstream mediator of elicitation such as MeJa may be more effective than biotic elicitors. The latter bring about a broad spectrum of responses, not all of which are necessarily desirable. On the other hand, biotic elicitors are of some use due to their lower cost and are particularly appropriate at the initial stage of research when the pathways of induction for a particular compound are not yet determined. In addition, different elicitors can diversely act on the biosynthetic competence of the cells, as illustrated by V. vinifera cultures 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 of secondary metabolites. Thousands of species have been transformed with A. rhizogenes with the aim of achieving a transformed roots induction. If the intact plant is known to synthesize large amounts of antioxidant compounds, one can expect a substantial increase in their level in the cultured roots and sometimes also in plantlets regenerated from transformed roots. The subject of transformed root methodology, its recent developments and the prospects for production of medicinal compounds have been addressed in several reviews (Georgiev et al., 2007; Guillon et al., 2006; Uozumi, 2004). The manipulation and optimization of transformed roots productivity are generally the same as those of other culture systems with perfect culture conditions, cell line selection, precursor feeding and elicitation. Thus, only a few examples that specically report the production of antioxidant compounds by hairy roots will be mentioned in this chapter. Several species from the Lamiaceae have been transformed with A. rhizogenes for the production of polyphenolic antioxidants. The very popular rosmarinic acid is again a good example of this. In S. ofcinalis hairy roots, the antioxidant activity and RA level were much higher than in untransformed organs (Grzegorczyk et al., 2006, 2007). This depside has also been efciently produced by the hairy roots of other 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. ofcinalis (Murakami et al., 1998; Kochan et al., 1999). Baikal skullcap (S. baicalensis) is one of the richest natural sources of avones reaching up to 20% in the root. Transformed roots produced baicalein and wogonin glucuronides, but also a phenylethanoid acteoside, a compound unknown from plants growing in natural conditions (Nishikawa et al., 1999; Stojakowska and Malarz, 2000). In S. sclarea hairy roots some abietane diterpenoids are also reported, although not in large enough amounts for them to be considered as a source of antioxidants (Kuma et al., 2006). An effective system for rutin production has been established with the hairy roots of buckwheat (Fagopyrum esculentum) whose leaves are a rich natural source of rutin (Lee et al., 2007). A high yield of antioxidant betalains can be achieved from the transformed roots of B. vulgaris (Pavlov et al., 2002, 2005b). An innovative transgenic approach with respect to antioxidant production has been applied to Saussurea involucrata transformed roots overexpressing heterologous chalcone isomerase from a related species (Li et al., 2006). This has led to the enhanced production of the avone apigenin, even in comparison to the selected wild type cell lines or transformed roots not bearing foreign chalcone isomerase gene. Besides the numerous positive results of transformed roots producing secondary metabolites, there is one example that achieved the opposite result in relation to antioxidants. Specically, in two boraginaceous species: E. sericeum and L. erythrorhizon, having introduced the rolC gene exhibited greatly reduced levels of the two antioxidants: rabdosiin and rosmarinic acid in comparison to the lines without 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 the industrial production of useful natural products. However, most of the published work deals with laboratory experiments on a small scale, even if there are liquid cultures involved. When thinking of industrial exploitation of the potential of plant cell factory, systematic scale-up studies must be carried out. However, the literature on that is relatively limited. One of the few established commercial systems is the well-known shikonin technology using L. erythrorhizon (Misawa, 1994; Bulgakov et al., 2001). Additional papers have been produced on experimental bioreactor systems in which cells, aggregates or differentiated organs are sources of antioxidant compounds. A few pilot-scale studies have been published on the production of tocopherol from hazel (Sivakumar et al., 2005), rosmarinic acid from lavender (Pavlov et al., 2005a) and basil (Kintzios et al., 2004), betalains from B. vulgaris (Pavlov et al., 2007). Several types of vessels are applied, but the most popular are classic designs like stirred, airlift or mist bioreactors (Pavlov et al., 2007). Temporary immersion systems are also used, e.g. for betalains (Pavlov and Bley, 2006) and semi-continuous perfusion high density cultures for rosmarinic acid production by A. ofcinalis (Su et al., 1993; Su and Humphrey, 1990). The scale-up study on V. pahalae cell cultures proved the importance of physical factors such as illumination of the bioreactor interior for sustained production of anthocyanins (Meyer et al., 2002). Similarly, in the aforementioned A. cordata cultures, the upscale in 500 l. fermentor allowed the production of over 500 g of mixed anthocyanins during a 16 day period (Kobayashi et al., 1993). Nonetheless, the bioreactor technology in relation to above mentioned antioxidants, though sometimes remarkably efcient in terms of production (as in the case of rosmarinic acid), does not seem likely to approach factory-scale industrial application in the near future. Hopefully, at least some of the mentioned research on antioxidants biotechnology will nally result in market implementation. 3.8. Concluding remarks and future perspective Despite the immense variety of antioxidant compounds obtained 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, -tocopherol from sunower cells and improved anthocyanins from several sources. There are, however, numerous inspiring and promising reports on such compounds as resveratrol, kinobeon A, lithospermic acid B and other oligomeric caffeic acid depsides or crocin, which should provoke and encourage more research focused on the regulation of biosynthesis and its increased economic feasibility. Finally, the burgeoning metabolomic and metabolic engineering approaches may add a new fascinating dimension to the possibilities of antioxidant-making plant cell factories that have been presented in this paper.

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