Plant in Vitro Culture for the Production of Antioxidants — a Review Biotechnology Advances

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 Matkowski

Department Pharmaceutical Biology, Medical University in Wroclaw, Al Jana Kochanowskiego 10, 51-601 Wroclaw, Poland article info abstract

Article history: Plant in vitro cultures are able to produce and accumulate many medicinally valuable secondary metabolites. Received 4 April 2008 Antioxidants are an important group of medicinal preventive compounds as well as being food additives inhibiting Received in revised form 1 July 2008 detrimental changes of easily oxidizable nutrients. Many different in vitro approaches have been used for Accepted 10 July 2008 increased biosynthesis and the accumulation of antioxidant compounds in plant cells. In the present review some Available online 16 July 2008 of the most active antioxidants derived from plant tissue cultures are described; they have been divided into the

Keywords: main chemical groups of polyphenols and isoprenoids, and several examples also from other chemical classes are ﬁ Antioxidant presented. The strategies used for improving the antioxidants in vitro production ef ciency are also highlighted, In vitro culture including media optimization, biotransformation, elicitation, Agrobacterium transformation and scale-up. Medicinal plants © 2008 Elsevier Inc. All rights reserved. Biosynthesis

Contents

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

1. Introduction Cultured plant cells synthesize, accumulate and sometimes exude many classes of metabolites. Medicinal compounds are of particular The use of plant cell and tissue culture methodology as a means interest and much effort has been devoted to obtaining some of the of producing medicinal metabolites has a long history (Rout et al., most active and precious therapeutics. Numerous alkaloids, saponins, 2000; Verpoorte et al., 2002). Since plant cell and tissue culture cardenolides, anthraquinones, polyphenols and terpenes have been emerged as a discipline within plant biology, researchers have reported from in vitro cultures and reviewed several times (Misawa, endeavored to utilize plant cell biosynthetic capabilities for obtaining 1994; Verpoorte et al., 2002; Vanisree and Tsay, 2004). useful products and for studying the metabolism (Misawa, 1994; In recent decades, interest in chemopreventive plant natural Verpoorte et al., 2002). 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 E-mail address: [email protected]. antioxidants (Halliwell, 1995; Aruoma, 2003; Soobrattee et al., 2005).

The present review summarizes the achievements of plant cell their activity will create the demand for more specific products that and tissue culture technology for the production of secondary could be used in accurately defined therapeutic and nutritional metabolites which have significant potential as antioxidants. The situations. Finally, as was inherent in plant in vitro cultures from the focus will be on the major chemical classes of antioxidants and on the very beginning, investigation into the biosynthetic routes advances approaches used to improve the in vitro methods for producing these our understanding of the function and regulation of plant metabolites. compounds. Moreover, it is still barely known what the actual roles are of many antioxidant secondary metabolites in a real plant (Grassmann et al., 1.1. What do we call an antioxidant? 2002; Misawa, 1994).

Broadly defined, an antioxidant is a compound that inhibits or 1.3. Methods used for assessment of antioxidant properties delays the oxidation of substrates even if the compound is present in a significantly lower concentration than the oxidized substrate In a majority of tissue culture papers reporting the production of (Halliwell, 1995; Halliwell and Gutteridge, 2007). The scavenging of secondary metabolites, their antioxidant activity has not been actually reactive oxygen species (ROS) is one of possible mechanism of action. determined. The knowledge of their properties usually comes from Others include the prevention of ROS formation by metal binding or studies involving dietary antioxidants and compounds isolated from enzyme inhibition. Chain breaking antioxidants prevent damage by ethnomedicinal plants with both food conservation and chemopre- interfering with the free radical propagation cascades. vention in mind. Thus, in most cases the antioxidant power of many The antioxidant compounds can be recycled in the cell or are secondary metabolites is so well established, that real time monitor- irreversibly damaged, but their oxidation products are less harmful or ing of activity during the culture seems to be redundant. On the other can be further converted to harmless substances (Halliwell and hand, the enormous variability among antioxidants, and their Gutteridge, 2007). At the cellular and organism level the antioxidant complex structure–activity relationships suggest that antioxidant protection is provided by numerous enzymes and endogenous small and any other biological activities should be paid more attention molecular weight antioxidants such as ascorbic acid, uric acid during research. There are a number of simple, slightly more complex, glutathione, tocopherols and several others. Many compounds contain and quite sophisticated methods for antioxidant testing. The matter antioxidant activity in addition to their specialized physiological has been reviewed by many authors with respect to different aspects function, and their importance as antioxidants in vivo is sometimes of the problem (Halliwell, 1995; Aruoma, 2003; Sanchez-Moreno, ambiguous (Azzi et al., 2004). The antioxidant activity of plant 2002; Matkowski, 2006). secondary metabolites has been widely established in in vitro systems The antioxidant testing can reveal various mechanisms of action, and involves several of the above mentioned mechanisms of action. depending on features of the particular assay. Simple methods Therefore, in this review, only those classes of compounds with include free radical scavenging with use of colored, artificial stable established antioxidant properties will be considered. free radicals such as 2,2′-azinobis-(3-ethylbenzothiazoline- 6-sulfonate) (ABTS used in the TEAC assay — Trolox equivalent an- 1.2. Why hunt for antioxidants in vitro? tioxidant capacity) (Re et al., 1999) and DPPH&z.rad; (1,1-diphenyl- 2-picrylhydrazyl free radical) (Molyneux, 2004), as well as transition The natural resources of both potential and established antiox- metal reduction that can be monitored by colorimetry. The metal- idants are vast. Some antioxidant compounds are extracted from based methods include the reduction of ferric ions: FRAP — (ferric easily available sources, such as agricultural and horticultural crops reducing ability of plasma) and ferric thiocyanate assays (Halliwell, (maize, buckwheat, grapevine, carrots, beetroot, citrus hesperidia, 1995; Aruoma, 2003), or molybdenum ion — phosphomolybdenum hops, apples, berries, tea leaves etc.), or medicinal plants such as pine, (P-Mo) assay (Prieto et al., 1999). These tests are easy and afford- skullcap, sage, rosemary, tormentil, and many others. Recently, the use able and can be used in high throughput screening. Their main of wine and olive industry waste products has been considered as a drawback is that their relevance to the real oxidizing life is some- potential source of dietary or food preserving antioxidants (González- what limited. The first issue is the chemical context of the assays, Paramás et al., 2004; Obied et al., 2005). This leads to the question: which use artificial compounds or are conducted in unrealistic con- why is the complex and rather expensive in vitro technology needed ditions. This problem is eliminated in methods based on naturally for products that may be obtained from abundant and cheap sources? occurring reactive oxygen species, but the fate of a free radical is The necessary conditions that make using biotechnological methods observed either indirectly with chromophore reagents or with more for the production of secondary plant metabolites economically viable expensive ESR techniques (electron spin resonance). The scavenging have been firmly established: high economic value, insufficient of superoxide radical anion, hydroxyl radical, or nitric oxide can be abundance in intact plants, limited availability from natural sources observed (Darmon et al., 1992; Aruoma, 2003; Sreejayan and Rao, (rare/endangered/overexploited species), and difficult cultivation 1997). (Misawa, 1994; Verpoorte et al., 2002). When taking these factors Several assays based on a substrate degradation inhibition are into consideration, in vitro technology offers some or all of the available which can be used to find out whether the tested compound following benefits: simpler extraction and purification from interfer- can really protect biomolecules from oxidative damage. Polyunsatu- ing matrices, novel products not found in nature, independence from rated lipids, proteins, components of nucleic acids, cellular mem- climatic factors and seasons, more control over biosynthetic routes for branes, microsomal fractions from different organs can serve as model obtaining the most desired variants (e.g. enantiomers or glycosides) or systems to be protected. The oxidation can be initiated by various a proportion of given compounds, shorter and more flexible produc- chemical, physicochemical or biological approaches. The degradation tion cycles, easier fulfillment of the high-profile pharmaceutical products are monitored by spectrophotometry (thiobarbituric reac- production demands (such as GLP and GMP), and last but not least, tive substances — TBARS, carotene bleaching), fluorescence (ORAC the exploitation of the genetic engineering potential for avoiding legal assay — for oxygen radical absorption capacity) or chromatography restrictions against GMO introduction into the natural environment. (Halliwell, 1995; Matkowski, 2006; Aruoma, 2003; Sanchez-Moreno, As will be shown in this paper, some of the listed conditions are likely 2002). to be fulfilled with respect to antioxidants, whereas others will not, Some of the described methods have been used for testing the due to the ubiquitous presence of the main classes of antioxidants and antioxidant activity of compounds from in vitro cultures of several the abundance of the aforementioned inexpensive sources. On the plants and will be referred to later in this paper as well as being other hand, growing knowledge about the specific mechanisms of specified in Table 1. 550 A. Matkowski / Biotechnology Advances 26 (2008) 548–560

2. Chemical classes of antioxidant secondary metabolites from in increase in cell-wall bound and oxidized forms is observed in callus vitro cultures cultures (Lopez Arnaldos et al., 2001), which reflects the natural processes during plant–pathogen interactions (e.g. hypersensitive 2.1. Polyphenols response) (Hammerschmidt, 2005). Polyphenols in plants derive mainly from the shikimic acid It is ironic from the point of view of the tissue culturist, that the pathway through aromatic carboxylic acids — cinnamic or benzoic. easily oxidizable phenolics, which are often regarded as nuisance Most of them possess antioxidant properties, albeit to varying degrees. and the reason for experiment failure, are at the same time highly The most powerful of the flavonoids are the flavanols and their desirable as dietary and therapeutic free radical scavengers. Consider- oligomeric forms called condensed tannins or proanthocyanidins, able attention has been paid to minimizing the “phenolization” of flavones and flavonols and anthocyanins. Some isoflavones are also cultures, frequently by adding antioxidants, such as ascorbic acid or known as antioxidants. Among other groups of antioxidant poly- glutathione (GSH), or phenol absorbing polymers like PVP (polyphenols there are: phenolic acids (caffeic acid derivatives), lignans, vinylpyrrolidone). The excessive production of polyphenols results hydrolysable tannins (gallotannins and ellagitannins), stilbenes, and from unfavorable or suboptimal culture conditions. At the onset of xanthones. Their physiological functions in plants are versatile and senescence, the decrease in free phenolics and a simultaneous have been reviewed recently (Harborne, 2001; Matkowski, 2006).

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

Species Compounds Culture system Antioxidant testing References for in vitro culture References for antioxidant activity Ajuga reptans Anthocyanins Flower cell culture β-carotene bleaching, lipid Callebaut et al., 1990; Terahara et al., 2001 peroxidation Terahara et al., 2001 Anchusa officinalis Rosmarinic acid Cell suspension Many in vitro chemical assays De-Eknamkul and Ellis, 1984; e.g. Petersen and Simmonds, Su et al., 1993 2003; Soobrattee et al., 2005 Anthoceros agrestis Rosmarinic acid and its Cell suspension Many in vitro chemical assays Vogelsang et al., 2006 e.g. Petersen and Simmonds, glucosides 2003; Soobrattee et al., 2005 Arachis hypogea Piceatannol (a stilbene) Callus Oxidative DNA damage in cell culture Ku et al., 2005 Ovesna and Horvathova-Kozics, 2005 Artemisia judaica Flavonoids Shoot cultures in bioreactor DPPH Liu et al., 2004 Liu et al., 2004 Beta vulgaris Betalains Cell suspension, hairy roots DPPH Pavlov et al., 2005a,b; Pavlov et al., 2005a,b Hunter and Kilby, 1999 Carthamus tinctorius Kinobeon A Cell suspension Cell membrane peroxidation, Kanehira et al., 2003; Kanehira et al., 2003; XOD/NBT, singlet oxygen quenching Kambayashi et al., 2005 Kambayashi et al., 2005 Cistanche deserticola Phenylethanoid glycosides Cell suspension DPPH Cheng et al., 2005 Cheng et al., 2005 Crocus sativus Crocin Callus Alleviation of oxidative stress in Chen et al., 2003, 2004 Ochiai et al., 2004 cultured mammalian cells Cynara cardunculus Cynarin, chlorogenic acid Callus TBARS Trajtemberg et al., 2006 Trajtemberg et al., 2006 Daucus carota Anthocyanins Callus, cell suspensions Lipid peroxidation Ravindra and Narayan, 2003 Ravindra and Narayan, 2003 Fagopyrum esculentum Rutin Hairy roots Many in vitro chemical assays Lee et al., 2007 e.g. Hinneburg et al., 2006 Glehnia littoralis Anthocyanins Callus, cell suspension Many in vitro chemical assays Miura et al., 1998; e.g. Kahkonen and Heinonen, Kitamura et al., 2002 2003 Hemidesmus indicus Rutin Callus, shoot culture Many in vitro chemical assays Misra et al., 2005 e.g. Ravishankara et al., 2002 Hyssopus officinalis Rosmarinic acid, Hairy roots Many in vitro chemical assays Murakami et al., 1998; e.g. Petersen and Simmonds, lithospermic acid B Kochan et al., 1999 2003; Soobrattee et al., 2005 Ipomoea batatas Anthocyanins Callus, cell suspension DPPH Konczak-Islam et al., 2000; Konczak-Islam et al., 2003; Terahara et al., 2004 Terahara et al., 2004 Lavandula officinalis Rosmarinic acid Callus, cell suspension, Superoxide radical scavenging Georgiev et al., 2006a,b; Kovacheva et al., 2006 bioreactor Kovacheva et al., 2006; Pavlov et al., 2005a,b Ocimum basilicum Rosmarinic acid Bioreactor culture of nodal Many chemical in vitro assays Kintzios et al., 2004 e.g. Petersen and Simmonds, explants and cell suspension 2003; Soobrattee et al., 2005 Passiflora quadrangularis Flavone-C-glycosides UV irradiated callus DPPH Antognoni et al., 2007 Antognoni et al., 2007 Petroselinum sativum Flavonols and flavones Cell suspension In vivo (rats) Hempel et al., 1999 Hempel et al., 1999 Rosmarinus officinalis Carnosic acid Callus, shoot culture Oxidative stress reduction in living Caruso et al., 2000 e.g. Wijeratne and Cuppett, cells and many chemical in vitro assays 2007 Salvia officinalis Rosmarinic acid, abietane Callus, shoot culture, hairy DPPH, P-Mo, lipid peroxidation Grzegorczyk et al., 2006, 2007 Grzegorczyk et al., 2007 diterpenoids roots, cell suspension Salvia miltiorrhiza Lithospermic acid B, Callus, regenerated plantlets, DPPH Morimoto et al., 1994; Chen Chen et al., 1999b rosmarinic acid hairy roots et al., 1999a; Yan et al., 2006

Saussurea involucrata Apigenin Hairy roots overexpressing H2O2-induced cell damage Li et al., 2006 Fan and Yue, 2003 CHI under camv35s Scutellaria baicalensis Baicalin, wogonoside Hairy roots, cell suspension Many in vitro chemical assays Morimoto et al., 1998; e.g. Huang et al., 2006 Nishikawa et al., 1999; Stojakowska and Malarz, 2000 Stevia rebaudiana Flavonoids Callus FRAP, DPPH Tadhani et al., 2007 Tadhani et al., 2007 Torreya nucifera Abietane diterpenoids Cell suspension LDL oxidation, nitric oxide inhibition Orihara et al., 2002 Lee et al., 2006 Vaccinium pahalae Anthocyanins Cell and aggregate Many in vitro chemical assays Meyer et al., 2002 e.g. Kahkonen and Heinonen, suspension 2003 Vitis vinifera Stilbenes, procyanidins Cell suspension DPPH, lipid peroxidation Decendit and Mérillon, 1996; Fauconneau et al., 1997 Fauconneau et al., 1997; Tassoni et al., 2005 Withania somnifera Withanoloids Hairy roots DPPH, carotene-linoleic acid oxidation, Kumaretal.,2005 Kumaretal.,2005 brain lipid peroxidation, hydroxyl radical scavenging A. Matkowski / Biotechnology Advances 26 (2008) 548–560 551

Phenolic acids occur in plants in free form as glycosides and can be 1985; Su and Humphrey, 1990; Su et al., 1993), Eritrichum sericeum integrated into larger molecules in an ester form. They are common as (Fedoreyev et al., 2005), Lithospermum erythrorrhizon (Yamamoto depsides — the intermolecular ester of two or more units composed of et al., 2000) (Boraginaceae), and Coleus blumei (Petersen et al., 1993), the same or different phenolic acids such as: caffeic, coumaric, ferulic, Lavandula vera (Pavlov et al., 2005a,b; Georgiev et al., 2006a,b), Oci- gallic, and syringic. mum basilicum (Kintzios et al., 2004), Salvia officinalis (Hippolyte et al., Depsides are, for example, a ubiquitous chlorogenic as well as 1992), Zataria multiflora (Mohagheghzadeh et al., 2004) (Lamiaceae). isochlorogenic, ellagic, lithospermic and rosmarinic acids. Due to the Depending on the species, the yield of rosmarinic acid from presence of a high number of hydroxyl groups and a carboxyl moiety, suspension cultures can exceed 10% of cell mass and 6 g/l of medium. their antioxidant properties are very pronounced (Rice-Evans et al., An interesting example is also the production of rosmarinic acid of 1996; Sroka, 2005). over 5% of cell dry mass in Anthoceros agrestis (Vogelsang et al., 2006). Special attention should be paid to rosmarinic acid (RA), a depside Another system used for rosmarinic acid production are transformed composed of two caffeic acid molecules (Fig. 1), found in many La- roots of Salvia miltiorrhiza (Chen et al., 1999a,b, 2001) and S. officinalis miaceae and Boraginaceae species. Its biochemistry and pharmacology (Grzegorczyk et al., 2006, 2007). In the second species, the accumula- has been extensively reviewed by Petersen and Simmonds (2003). The tion was much higher reaching 4.5% of dry weight and its antioxidant antioxidant properties of rosmarinic acid are also well established. activity has been confirmed with several assays. Extracts from Several plants and various techniques have been used for in vitro rosmarinic acid accumulating lavender cells also have superior radical production of this compound (Shetty, 1997). Rosmarinic acid can scavenging properties (Kovacheva et al., 2001, 2006). Even larger accumulate in cell cultures to amounts greater than those in intact amounts (over 7% dw) of rosmarinic acid accumulated in Agrobacter- plants. The suspension cultures producing rosmarinic acid were ium transformed callus of C. blumei (Bauer et al., 2004) and trans- generated from Anchusa officinalis (De-Eknamkul and Ellis, 1984, formed roots Hyssopus officinalis, but only when cultured on WP

Fig. 1. Structures of some phenolic antioxidant metabolites from plant in vitro cultures. 552 A. Matkowski / Biotechnology Advances 26 (2008) 548–560 medium which was much more superior to both MS and B5 (Murakami plant cells. The successful induction and isolation of oligomeric et al., 1998). Razzaque and Ellis (1977) report a yield of 8–11% of proanthocyanidins have been reported in the cell cultures of Vacci- rosmarinic acid in C. blumei cell cultures. An extremely high level of RA nium pahalae (Kandil et al., 2000) and Vitis vinifera (Decendit and production was reported by Hippolyte et al. (1992) in S. officinalis Mérillon, 1996). reaching 6.4 mg/l of suspension culture, although the result has not Another example of flavonoid polyphenols frequently studied for appeared in any other reference. in vitro production is anthocyanins. Pigments are useful as products A closely related antioxidant is lithospermic acid B, a tetrameric because their accumulation can easily be visually evaluated. Moreover, depside (Fig. 1), accumulating especially in the aforementioned hairy their beneficial health promoting properties as well as their grow- root cultures of H. officinalis and elicited S. miltiorrhiza as well as in ing importance as bioactive and natural food colorants additionally Lithospermum erythrorhizon suspension cultures (Yamamoto et al., increases their attractiveness for plant biotechnology. The versatile 2000, 2002). Oligomeric caffeic acid depsides such as DCQAs values of anthocyanins have been reported and reviewed in count- (dicaffeoylquinic acids) — potent antioxidants and antivirals, accumu- less publications. One of their most pronounced features is their late also in cell cultures of sweet potato storage roots, where they substantial antioxidant capacity determined in various assays, but are accompanied by anthocyanins (Konczak et al., 2004). Chlorogenic their biological activity varies greatly, due to their structural diversity. acid and cynarin (another DCQA) are present in cardoon (Cynara Analogously to other polyphenols, more hydroxylated compounds cardunculus) callus in quantities two-times greater than in the leaves — express larger free radical scavenging capacity, whereas methylation the usual medicinal crude drug. The antioxidant capacity of the car- of the hydroxyl groups decreases this effect partially (Stintzing and doon callus was confirmed by the TBARS method (Trajtemberg et al., Carle, 2004). Glycosylation can also reduce the ability to scavenge free 2006). radicals, but on the other hand enhances the stability of the molecule. Flavonoids are one of the largest groups of secondary metabolites Acylation of the sugar moieties with carboxylic, usually hydroxycin- based on a common structure. Chemically they possess a tricyclic namic acids (an example of a glycosylated and acylated cyanidin is phenylbenzopyran (with the exception of chalcones) structure but shown in Fig. 1) additionally boosts the antioxidant potential and color biosynthetically come from phenylalanine and malonyl-CoA in the stability. For that reason, not all anthocyanins are equally desirable as phenylpropanoid pathway. natural medicines. Cyanidin and delphinidin glycosides acylated with Subclasses of flavonoid include flavonols, flavones, isoflavonoids, phenol carboxylic acids are preferred over less substituted versions flavanons, flavanols, proanthocyanidins (catechins), and anthocyani- and petunidin, pelargonidin, peonidin or malvidin derivatives. This is dins. Substitution patterns include the hydroxylation of rings A and B, one of the main reasons for conducting tissue culture experiments. methylation of hydroxyl groups, prenylation, and glycosylation. Other In abundant natural sources, the anthocyanin profile is rarely opti- modifications can form biflavonoids, lignans, oligomeric proantho- mal and the raw material often requires complex extraction and cyanidins, glycoside esters with other phenolics etc. purification procedures. In several species listed below the level of Antioxidant properties of flavones depend on the hydroxylation anthocyanins in vitro equals or exceeds the natural sources and their pattern. Substitution with hydroxyl, methoxyl or other groups, and metabolic profile is more useful. Thus, even if anthocyanins are not so glycosylation also influences hydrophilicity and many biological expensive, the ability to obtain products with useful structural and activities. Among the vast variety of structures, several flavones from functional properties may become economically feasible (Konczak the genus Scutellaria (Lamiaceae) are distinguished by their unsub- et al., 2005). stituted B-ring and common occurrence in the form of glucuronides Anthocyanins are obtained from in vitro cultures of Prunus sp. (Fig. 1). Scutellaria baicalensis, the favorite natural drug of traditional (Durzan et al., 1991; Blando et al., 2005), Daucus carota (Ravindra and Oriental medicines, contains in the roots probably the highest known Narayan, 2003), Glehnia litoralis (Miura et al., 1998), V. pahalae (Meyer amount of flavones (up to 20% dw) of which the strong lipophilic et al., 2002), Ipomaea batatas (Konczak-Islam et al., 2000; Terahara antioxidants: baicalin, baicalein, wogonoside and wogonin are the et al., 2004) Fragaria ananassa (Edahiro et al., 2005), V. vinifera most abundant. In cell suspension and the hairy root cultures of (Decendit and Mérillon, 1996), Rudbeckia hirta (Luczkiewicz and S. baicalensis the accumulation of these compounds was reported Cisowski, 2001) and Ajuga reptans (Callebaut et al., 1990; Terahara (Morimoto et al.,1998; Nishikawa et al.,1999; Stojakowska and Malarz, et al., 2001) and others. 2000). The in vivo antioxidant function of baicalein in metabolizing Interestingly, two species used medicinally due to their anticancer hydrogen peroxide has been proposed as a result of experiments using alkaloids, Catharanthus roseus and Camptotheca acuminata, produce cell cultures (Morimoto et al., 1998). significant amounts of anthocyanins of over 200 µg/g fresh weight Flavone C-glycosides are of particular interest because of their in cell cultures (Filippini et al., 2003; Pasqua et al., 2005). In C. roseus, limited occurrence in plants and they have important therapeutic p-coumaroyl glucosides of highly methoxylated malvidin or hirsutidin properties, including the prevention of cardiovascular disorders predominated, unlike in field-grown flowers. In C. acuminata a rare related to oxidative stress. In tissue cultures of Passiflora quadrangularis cyanidin galactoside accounted for over 95% of all anthocyanins. several C-glycosylated flavones such as isoorientin, orientin (Fig. 1), In A. reptans flower-derived cell cultures the anthocyanin composi- vitexin and isovitexin have been induced in varied amounts, and their tion was altered from delphinidin-based in flowers towards stronger antioxidant activity determined by DPPH assay (Antognoni et al., antioxidants — di-acylated cyanidin glycosides (Callebaut et al., 1997; 2007). Crataegus monogyna is another source of these C-glycosides and Terahara et al., 2001). This observed simplification and alteration of the its long-term callus cultures have been shown to produce several types metabolic profile, when compared to natural conditions, is typical for of phenolic antioxidants assayed with various methods (Rakotoarison in vitro produced anthocyanins. In the extensively studied I. batatas, et al., 1997; Bahorun et al., 2003). in the tissue cultures induced from storage roots the accumulation of Oligomeric proanthocyanidins (also known as condensed tannins) anthocyanins can even exceed the tubers by several times. The are considered to be the most powerful antioxidants of all the production of valuable di-acylated cyanidin glycosides from callus flavonoids owing to their structure (procyanidin B1 as an example in and cell suspension as well as their regulation towards overproduction Fig. 1) (the chemical mechanisms have been reviewed by Bors and and their more desirable profile has also been reported (Terahara Michel, 2002). They are responsible for the antioxidant activity of et al., 2000, 2004; Konczak et al., 2005). The superior stability and wines, teas and fruits as well as of popular nutraceuticals made from antioxidant activity of anthocyanin-rich extracts have also been con- grape seeds or pine bark. The thorough genomic and metabolic firmed. In this species the extraordinary high amount of anthocyanins engineering studies have been recently initiated, too (Dixon et al., was recorded in selected cell lines along with a greater proportion, in 2005). Conversely, they are rarely studied for in vitro production by comparison to the tubers, of cyanidin-based to peonidin-based A. Matkowski / Biotechnology Advances 26 (2008) 548–560 553 pigments (Konczak et al., 2005). Given the aforementioned ability of abundant natural sources such as carrots or tomatoes as well as by cell culture from this species to accumulate medicinally active dep- means of microbial (Bhosale and Bernstein, 2005) and fungal sides, this is one of the most promising systems for the biotechnolo- biotechnology (Böhme et al., 2006), it is reasonable to obtain crocin gical production of health-promoting polyphenols. from the plant tissue cultures (Chen et al., 2003, 2004)ofC. sativus.In Silybum marianum, milk thistle, the famous hepatic drug contains this case, the in vitro production could be profitable due to the high in the achenes an isomeric flavonoid lignans complex called silymarin cost of the raw material (about 200 000 flowers are needed to get 1 kg that is a strong antioxidant (e.g. silybin in Fig. 1). The untreated of dried stigma). cell cultures contain low and variable amounts of flavonolignans, but Abietane diterpenes are present in many Lamiaceae medicinal after they have been elicited their production can be increased 6-fold plants as well as in some gymnosperms. Some of these compounds (Sanchez-Sampedro et al., 2005a). In another study, the same authors demonstrate substantial antioxidant potential in various systems. reveal the pivotal role of calcium deprivation in stimulating silymarin Carnosic acid (Fig. 2) and carnosol, rosmanol and ferruginol (Fig. 2) are biosynthesis (Sanchez-Sampedro et al., 2005b). The role of perox- responsible for the antioxidant activity of sage (Salvia sp.) and idases in both the synthesis by oxidative coupling of precursors and rosemary (Rosmarinus officinalis) along with the phenolic rosmarinic the degradation of silymarin compounds has also been explored and salvianolic acids, but the organ distribution differs between the (Sanchez-Sampedro et al., 2007a). two mentioned chemical classes. However, the published tissue Stilbenes are bicyclic polyphenols represented by the well-known culture studies in Lamiaceae aimed at enhancing the rosmarinic acid resveratrol (Fig. 1), a phytoalexin and the red wine antioxidant. production rather than at abietane diterpenoids. The tissue cultures as Although present in a considerable amount of natural sources like a source of abietane diterpenes have been established for R. officinalis grapes and some Polygonaceae species, it has been also induced in (Caruso et al., 2000), S. officinalis (Grzegorczyk et al., 2007) and Salvia elicited hairy root cultures of peanut (Arachis hypogea) and recovered sclarea (Kuźma et al., 2006). The antioxidant activity of in vitro efficiently from the liquid medium (Medina-Bolivar et al., 2007). The cultures of common sage, especially in lipophilic systems such as the same species was used to enhance production of a rare stilbene — inhibition of lipid peroxidation, can be attributed to abietane piceatannol in cell culture (Ku et al., 2005). This approach seems diterpenes rather than to phenolics (Grzegorczyk et al., 2007). Light quite promising for the production of a purified single antioxidant plays an important role in stimulating the diterpene biosynthesis which the pharmaceutical industry prefers to use. In cell cultures of in vitro (Caruso et al., 2000; Kuźma et al., 2006) even though in V. vinifera, the influence of elicitors on the stilbene biosynthesis and natural conditions they can only be found in the underground parts of the stilbene synthase activity has been studied (Tassoni et al., 2005)as plants. well as the antioxidant capacity of resveratrol and astringin from Volatile terpenoids responsible for fragrant properties of plant cultured cells (Fauconneau et al., 1997). organs serve as a multipurpose chemical defense weapon against Other examples of recently reported antioxidant activities of in vitro fungal and bacterial pathogens as well as a means of long distance cultured, polyphenol-rich plants include: Curcuma longa mass prop- chemical communication e.g. attracting pollinators. Moreover, the role agation on a thin-film rocker system where significant and clone of a monoterpene β-thujaplicin in the alleviation of oxidative damage dependent DPPH scavenging was noted (Cousins et al., 2007); Cis- in cell cultures of cypress has been postulated (Zhao et al., 2005), but tanche deserticola cell suspensions producing antioxidant phenyletha- this does not imply that this is its direct function as an antioxidant. The noid glycosides also assayed by a DPPH test (Cheng et al., 2005); Stevia antioxidant properties of several monoterpenes, such as γ-terpinene rebaudiana – the herbal sweetener – callus extracted with water and and limonene have been reported. This suggests that this group of methanol had higher antioxidant activity than the leaves of field- compounds may receive more attention although at present they are grown plants, which correlated with the higher levels of flavonoids underestimated as antioxidants. Tissue cultures are able to produce and total polyphenols (Tadhani et al., 2007). volatile constituents of essential oils and many reports have been produced on that subject (Mulder-Krieger et al., 1988; Hrazdina, 2.2. Isoprenoids 2006). However, in vitro production of essential oils has not been aimed at the antioxidant properties of the products. Isoprenoids are produced in plant cells from isopentynyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). These basic 2.3. Other structures units are synthesized via cytoplasmic mevalonate or plastidic deoxyxylulose (or methylerythritol) phosphate pathways. IPP and Tocopherols, widely used in human nutrition as vitamin E and in DMAPP are subsequently fused by sets of prenyltransferases and food conservation, are powerful free radical scavengers and protect terpene synthases and a variety of modifying enzymes to eventually plant cells against oxidative damage in a lipophilic environment (Kruk yield enormously variable compounds in numerous groups of et al., 2005). Plants are the primary source of natural tocopherols terpenoids (mono, sesqui, di, and triterpenes and their derivatives), isolated from vegetable oils or maize embryos. Their biosynthesis carotenoids, and steroids (Bouvier et al., 2005; Tholl, 2006). Each of integrates the aromatic aminoacids (shikimic acid) and plastidic these groups contains metabolites of different chemical characters isoprenoid deoxyxylulose phosphate (pentose) pathways. The first such as alcohols, acids, aldehydes, or a combination of these; they can pathways create the aromatic (phenolic) head while the second gives also be aromatic, glycosylated and form more complex structures. The rise to the hydrophobic tail of a tocochromanol skeleton (DellaPenna, most powerful antioxidants among them are carotenoids and abietane 2005). α-Tocopherol (Fig. 2) is the most active form of vitamin E, while diterpenoids (some of them containing a phenolic structure within a the extraction from plant oils usually yields a mixture of β, γ, δ and α- molecule). In comparison to most phenylpropanoid polyphenols, tocopherols and tocotrienols. Therefore there is a demand for isoprenoids are generally more lipophilic, hence their important role alternative sources of pure α-tocopherol from plant tissues. Various in protecting membrane lipids. Both β-carotene and lycopene are culture systems of Helianthus annuus (sunflower) have been estab- recognized as dietary chemopreventive agents, protecting against lished for this purpose. A 100% increase in cell suspensions has been cancerogenesis. The participation in an antioxidant cascade is likely to achieved by media optimization, elicitation and precursor feeding contribute to these prophylactic properties. Crocin derived from (Caretto et al., 2004; Gala et al., 2005). A photomixotrophic cell line saffron (stigmata of Crocus sativus) has been confirmed as a powerful has also established a cultured on sucrose-deprived medium antioxidant (Ochiai et al., 2004), stronger than α-tocopherol, and (Fachechi et al., 2007). An attempt has been also made to select the being a glycoside of an apocarotenoid acid, crocetin, it is water soluble most efficient genotype of hazel (Corylus avellana) for the production (Fig. 2). Whereas other carotenoids are readily available from of tocopherols in bioreactors (Sivakumar et al., 2005). 554 A. Matkowski / Biotechnology Advances 26 (2008) 548–560

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

Betalains — the purple betacyanins and yellow betaxanthines 2002, 2005b). Being ideal metabolites for direct observation they have pigments functionally replace anthocyanins in 13 taxons grouped in also been used for compound recovery experiments using various Caryophyllales (previously Centrospermae) order, such as the families: permeabilization and extraction methods (Hunter and Kilby, 1999). Red Chenopodiaceae, Amaranthaceae, Portulacaceae, Cactaceae, Phytolacca- beet betalains from hairy roots have been shown to be efficient free ceae and others. These nitrogen containing compounds (Fig. 2) although radical scavengers (Pavlov et al., 2002, 2005b). The growing knowledge widely used as non-toxic food colorants, remain understudied in terms of their therapeutic and preventive properties is likely to further of their antioxidant potential (Gliszczyńska-Swigło et al., 2006; Kanner increase the research interest in their in vitro biosynthesis. et al., 2001). These properties of betalains have been recently reviewed Indole glucosinolates are a tryptophane containing group from the by Stintzing and Carle (2004). The major advantages of betalains as diverse class of thioglycoside compounds typical to the Brassicaceae. dietary antioxidants are their bioavailability, which is greater than most Upon hydrolysis by myrosinase they generate biologically active flavonoids, and their superior stability in comparison to anthocyanins antioxidants and carcinogenesis inhibitors such as indole-3-carbinol (Kanner et al., 2001; Stintzing and Carle, 2004). derived from glucobrassicin (Fig. 2). In cruciferous plants they are Red beetroot (Beta vulgaris) is a rich natural source of betacyanins thought to contribute to the regulation of auxin biosynthetic routes (with betanin predominating — Fig. 2) and is also an object of metabolic (Bak et al., 2001). Although they are present in common cabbage in vitro studies on their biosynthesis. They possess the useful properties related vegetables (broccoli in particular) their instability and uneven of pigments and are therefore preferred as a model system despite their or unpredictable distribution would make their biotechnological low commercial value. The production of beetroot pigments has been production an interesting option. Yet, to date there has been very performed using a variety of techniques such as cell suspensions and little data available on that topic. The horseradish (Armoracia transformed roots in bioreactors (Hunter and Kilby, 1999; Pavlov et al., rusticana) cells, both in suspension and immobilized in polyurethane A. Matkowski / Biotechnology Advances 26 (2008) 548–560 555 foam, produce indole-3-methylglucosinolate and 4-hydroxyindole-3- Auxins and cytokinins are crucial for the proper stimulation of methylglucosinolate (Mevy et al., 1999). biosynthetic pathways, unless we deal with a hormone-independent Kinobeon A — a novel, red-colored compound isolated from transformed root culture. In anthocyanin producing G. littoralis Carthamus tinctorius cell cultures uniquely combines the quinoid cultures, NAA at 1 mg/l was the preferred auxin, superior to IAA and aromatic character with several conjugated double bonds (Fig. 2). It 2,4-D and the addition of 0.1 mg/l kinetin further improved cell has therefore been demonstrated as being a remarkably strong growth and pigment biosynthesis (Miura et al., 1998). The higher antioxidant. Kinobeon A efficiently quenches various reactive oxygen concentration of NAA (5 mg/l) was optimal for R. hirta (Luczkiewicz species, inhibits lipid peroxidation and supports the survival of and Cisowski, 2001). In the same study, the highest accumulation was mammalian cells during oxidative stress (Kanehira et al., 2003; achieved by adding cystein, which is one of the constituents of CoA, a Kambayashi et al., 2005). To date, little is known about kinobeon A crucial factor in flavonoid biosynthesis. physiological functions and biosynthesis. Whether it occurs also in A two or three-stage growth system can be also useful for opti- other organisms or is an example of an exclusive cell-culture product mizing metabolite production. The first stage is used for the initiation remains to be determined. and/or the maintenance of a culture with the aim of obtaining the highest percentage of induction and the fastest biomass growth. The 3. Strategies for increased production second phase aims at the accumulation of a product. It has been successfully applied to anthocyanins in R. hirta (Luczkiewicz and The continuous monitoring of a chosen metabolite is a prerequisite Cisowski, 2001) where the tissues in the optimized second stage for the successful development of production technology and can media contained up to 5% anthocyanins in dry mass. be carried out using a number of different methods. For accurate In C. sativus cultures the two-stage system leads to a substantial measurements, chromatographic methods such as HPLC and GC with accumulation of crocin (430 mg/l) simply by changing the hormone the appropriate detection are sufficient. Laboratories that lack phyto- composition from NAA at 2 mg/l, BAP at 1 mg/l for biomass growth to chemical facilities need other equivalent substitution methods. For IAA at 2 mg/l and BAP at 0.5 mg/l for crocin production (Chen et al., operation on a large scale, time saving is also a consideration. These 2003). faster and less elaborate techniques involve spectrophotometry that can be used for some groups of compounds, while for other types it is rather 3.2. Production in differentiated tissues unsuitable. The first group contains colored compounds — flavonoid, carotenoid, and quinoid pigments or UV absorbing phenolics. In some cases, full development in natural conditions is useful for Anthocyanins are commonly estimated by this technique, which producing a considerable amount of secondary products, especially if can also be applied non-destructively (Durzan et al., 1991; Smith, the undifferentiated cell culture is either unable to or barely 2000). On the other hand, this approach is only adequate if there is accumulating antioxidant compounds. This has been reported for just one dominant antioxidant, differing in the absorption maxima several classes of metabolites, especially for alkaloids, and has also from any interfering compounds. The rapid progress in phytochemical been published for some antioxidant compounds. Ellagic acids present instrumentation development and the trend towards a metabolomic in Rubus chamaemorus plants was 3 times lower in shoot cultures and approach and high throughput screening is likely to also influence the over 10 times lower in callus (Thiem and Krawczyk, 2003). Similarly in monitoring of secondary metabolites production in vitro. Indeed, in O. basilicum cell cultures (Kintzios et al., 2004) the accumulation of recent publications the chromatographic determination of antiox- rosmarinic acid was markedly lower than in regenerated plantlets. In idants predominates, frequently accompanied by bioactivity guided S. officinalis (Grzegorczyk et al., 2007) and rosemary (Caruso et al., fractionation, spectral structure elucidation of isolated chemicals by 2000) in vitro cultures, the abietane diterpene antioxidants (carnosol MS or NMR. The use of hyphenated techniques such as LC-MS and LC- and carnosic acid) were present only in shoot cultures and not in NMR or 2D-NMR is considered to be the best means for structure callus, suspension or hairy roots. On the other hand, undifferentiated determination when novel compounds are present in vitro that have cell suspensions are able to accumulate great amounts of phenolic not been known from intact plants (Tian et al., 2005; Farag et al., 2007; acids. Moreover, the cells cultured in suspension tend to form larger Sanchez-Sampedro et al., 2007b). aggregates upon differentiation, therefore increasing their shear stress resistance. The potential of fast growing somatic embryos cultures can 3.1. Optimization of biosynthesis by culture conditions be also utilized for the production of medicinal metabolites, but reports on that topic are scarce and deal chiefly with alkaloid A range of environmental and nutritional factors are known to production such as paclitaxel (Lee and Son, 1995). For example, influence the biosynthetic pathways of secondary metabolites. Light phenol glycosides, lignans and flavonoid accumulation has been induction of anthocyanin pigments is a well documented example mentioned in Siberian ginseng somatic embryos (Shoahel et al., 2006). (Harborne, 2001; Stintzing and Carle, 2004). In cultured cherry (Prunus Even in dedifferentiated cells, some biosynthetic potential typical cerasus) callus and suspensions, the light exposure causes a rapid change for the developed organs from which they were initiated, can be of the tissue color from white to purple (Blando et al., 2005). The conserved. In Pueraria lobata callus cultures, the bioactive isoflavonoid anthocyanidin profile was different both from intact leaves and fruits content depended on the source organ, reflecting relations in the with cyanidin-3-glucoside as the single dominant compound, absent mature plant (Matkowski, 2004). The selection of proper donor plants delphinidin (unlike in fruits) and only a small amount of cyanidin-3- and organs should already be considered when starting the culture, rutoside (dominant in the leaves). This is a good example of a distinct unless it can be overcome by a suitable treatment. However, in most metabolic profile under in vitro conditions. However, the irradiation of circumstances the dedifferentiation apparently also involves some of cultures can lead to an increase in cost and can generate undesirable the biochemical properties of the cells. The modification of relative temperature gradients in the culture vessels. Therefore, several biosynthesis to degradation ratios of a desired product can also examples of constitutive, light-independent systems for anthocyanin influence the final levels of a desired compound in the culture. production were established such as Glehnia littoralis (Miura et al.,1998), I. batatas (Konczak et al., 2005)orAralia cordata (Kobayashi et al., 1993). 3.3. Selection of high-producing cell lines

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 super-efficient clones of cultured cell or tissues can be selected The media composition has to be optimized for both an intensive by monitoring the level of the compound of interest, or can be biomass increase and the accumulation of the desired metabolite. complemented by a selecting agent facilitating the process. The 556 A. Matkowski / Biotechnology Advances 26 (2008) 548–560 selected line should exceed both the levels of production obtained in The upregulation of biosynthesis of secondary metabolism upon unselected cell cultures and the natural biosynthetic productivity of exposure to stress factors has been used to obtain a high yield of many an intact organism. medicinal compounds (Verpoorte et al., 2002; Vanisree et al., 2004). The addition of a phenylalanine analogue to the culture medium Exposure to stress factors results in an excessive level of oxidizing causes damage of the majority of cells except those who express a high molecules such as ROS due to the disturbances in redox state degree of activity of PAL (phenylalanine ammonia lyase). The equilibrium. The increased ROS production may help to combat the overexpression of PAL in selected variants results in elevated invasive pathogens but can also be triggered by harmful environ- phenylpropanoid compounds, many of which are desired antiox- mental factors such as UV radiation, xenobiotics, heavy metals, idants. Rosmarinic acid producing cells of lavender (Georgiev et al., drought, and mechanical damage. Therefore, antioxidant properties 2006b) and shikonin producing cells of L. erythrorhizon (Bulgakov of some metabolites along with antioxidant enzymes can aid the plant et al., 2001) have been selected with the use of fluorinated in restoring the redox homoeostasis (Matkowski, 2006). Apart from phenylalanine. The selected lines appear to be stable over time and direct antioxidant activity, some compounds — e.g. flavones can serve can synthesize twice as much of the desired compounds as the control as substrates for cellular peroxidases (Morimoto et al., 1998). cultures. In vitro cultured material can be elicited by bacterial or fungal If the desired product is colored as in the case of anthocyanins, a lysates or stress response mediators such as salicylate or jasmonic simple visual selection with the mechanical dissection of accumulat- acid/methyl jasmonate and hydrogen peroxide as well as by environ- ing cells can be performed with good results. An impressive example mental factors like metals or irradiation. is the origin of the stable (over a period of several years), light- The stress related growth regulators, jasmonic acid and its esters, independent anthocyanin accumulating cell line of G. litoralis selected especially methyl jasmonate (MeJa) have been widely used in from a single purple spot in a white, dark grown callus (Miura et al., promoting the biosynthesis of both inducible and constitutive 1998; Kitamura et al., 2002). Similarly, a selected cell line of purple secondary metabolites, including medicinal compounds such as sweet potato (PL) accumulates a large amount of acylated anthocya- anticancer alkaloids (Vanisree and Tsay, 2004; Verpoorte et al., nins without light exposure (Konczak et al., 2005). 2002). With respect to the antioxidants, jasmonic acid enhanced α- tocopherol production by sunflower and A. thaliana cell cultures (Gala 3.4. Precursor feeding and biotransformation et al., 2005) and cyanidin glucosides in cherry callus (Blando et al., 2005). On the other hand, in the resveratrol producing grapevine cell When starting an in vitro culture of a medicinal plant, that in its cultures (Tassoni et al., 2005), jasmonic acid proved to be a weaker intact form accumulates substantial amounts of valuable products, it elicitor than its methyl ester. Besides, MeJa promoted the accumula- sometimes happens that a dedifferentiated cell mass is unable to tion of trans-resveratrol, whereas JA resulted in a modest increase of complete the biosynthesis. This is due to the uncoupling of the the less desirable cis-resveratrol. MeJa and salicylic acid also enzymatic machinery, or an insufficient expression of developmen- stimulated the curcumin glycosylation by C. roseus cell cultures tally regulated biosynthetic genes, or a lack of environmental stimuli. (Kaminaga et al., 2003). If biosynthetic enzymes are expressed in part of the pathway, the Using natural biotic elicitors also results in enhanced secondary exogenous delivery of precursors can solve the problem. It can be also metabolism. Yeast extracts (YE — also read as yeast elicitor), but helpful in omitting the metabolic channeling that directs the neither chitin nor chitosan, have a positive impact on the production precursors to a pathway other than that desired. of silymarin, the effect being potentiated when combined with JA. In a Rhodiola rosea compact callus aggregate culture no secondary However, it proved more effective when supplemented with MeJA in products were detected until the medium was supplemented with this system (Sanchez-Sampedro et al., 2005a). Chitin elicited the cinnamyl alcohol that was efficiently transformed into a pharmaco- production of several new flavonoids in cactus (Cephalocereus senilis) logically active rosin and rosavin (György et al., 2004). However, cell cultures (Liu et al., 1993). YE applied to the hairy root culture of S. neither are antioxidants. Salidroside, the antioxidant present in the miltiorrhiza improved both the growth of the roots and the plant, was not found in that study, but other authors have succeeded accumulation of rosmarinic and lithospermic B acids, preceded by a in antioxidant production in this species (Furmanowa et al., 1998). significant rise in tyrosine aminotransferase activity (Chen et al., 2001; In contrast, the feeding of naringenin or phenylalanine significantly Yan et al., 2006). Compared to the effect of an abiotic elicitor — Ag+,YE increased anthocyanins in Rudbeckia (Luczkiewicz and Cisowski, was superior (Yan et al., 2006). Rosmarinic acid production was also 2001) and strawberry (Edahiro et al., 2005) cells, respectively. In enhanced in O. basilicum hairy roots by Phytophtora cell walls, while Cistanche salsa cell cultures, their enrichment with a mixture of salicylic and jasmonic acids or chitosan suppressed it (Bais et al., phenylethanoid glycosides precursors results in a doubling of 2002). production when compared to feeding individually with tyrosine, In peanut hairy roots (Medina-Bolivar et al., 2007) sodium acetate phenylalanine, caffeic acid or cucumber juice (Liu et al., 2007). and chitosan were the most effective of the five tested elicitors causing Hence, the choice of an appropriate precursor is essential for the a 60-fold increase in resveratrol accumulation. successful enhancement of in vitro antioxidant production. The use of metal-based elicitors gives positive results as well. Curcumin is a yellow colored, potent polyphenolic antioxidant and Vanadium salts increase the accumulation of rosmarinic acid in cholagogue from the roots of turmeric (C. longa). Its chief weakness is lavender cell culture nearly three-fold (Georgiev et al., 2006a), while its low water solubility, which limits its intestinal absorption and in Vitis cells only the cis isomer of resveratrol was induced (Tassoni hence its pharmaceutical usefulness (Sharma et al., 2007). In another et al., 2005). surprising application of C. roseus cell cultures, they were modified by Rare earth elements such as La3+ and Ce3+ can also serve as elici- cellular enzymes into unnatural glycosides that are much more tors to promote crocin (Chen et al., 2004), phenylethanoids from soluble in water (Kaminaga et al., 2003). C. deserticola (Ouyang et al., 2003), and silymarin (Sanchez-Sampedro et al., 2005b) production. The mechanism of these lanthanides actions 3.5. Elicitation and stress induced production involves interference with cellular calcium signaling (Sanchez- Sampedro et al., 2005b). One of the best established roles of secondary metabolites is the Irradiation with UV has been also used for making the plant cells involvement in stress responses (Grassmann et al., 2002). Phytoalex- synthesize more antioxidants. In particular, flavonoids and other ins are synthesized in reaction to a pathogen attack, other compounds polyphenols are known to protect plants from harmful UV impact. In are associated with abiotic disturbances. P. quadrangularis callus subjected to UV-B treatment, the irradiation A. Matkowski / Biotechnology Advances 26 (2008) 548–560 557 stimulated a large increase in the amount of flavonoid, corroborating 3.7. Scale-up or the never-ending bioreactor story the higher radical scavenging activity (Antognoni et al., 2007). Similarly, the callus of A. hypogea was induced by UV radiation to The ultimate goal of the biotechnology of medicinal plants is the accumulate much greater amounts of stilbenes (Ku et al., 2005). industrial production of useful natural products. However, most of the The use of a downstream mediator of elicitation such as MeJa may published work deals with laboratory experiments on a small scale, be more effective than biotic elicitors. The latter bring about a broad even if there are liquid cultures involved. When thinking of industrial spectrum of responses, not all of which are necessarily desirable. On exploitation of the potential of plant cell factory, systematic scale-up the other hand, biotic elicitors are of some use due to their lower cost studies must be carried out. However, the literature on that is and are particularly appropriate at the initial stage of research when relatively limited. One of the few established commercial systems is the pathways of induction for a particular compound are not yet the well-known shikonin technology using L. erythrorhizon (Misawa, determined. In addition, different elicitors can diversely act on the 1994; Bulgakov et al., 2001). Additional papers have been produced on biosynthetic competence of the cells, as illustrated by V. vinifera experimental bioreactor systems in which cells, aggregates or cultures resveratrol isomers production (Tassoni et al., 2005). differentiated organs are sources of antioxidant compounds. A few pilot-scale studies have been published on the production of α- 3.6. Transformation with Agrobacterium rhizogenes — hairy roots tocopherol from hazel (Sivakumar et al., 2005), rosmarinic acid from lavender (Pavlov et al., 2005a) and basil (Kintzios et al., 2004), This is a commonly used method to enhance the production of betalains from B. vulgaris (Pavlov et al., 2007). Several types of vessels secondary metabolites. Thousands of species have been transformed are applied, but the most popular are classic designs like stirred, air- with A. rhizogenes with the aim of achieving a transformed roots lift or mist bioreactors (Pavlov et al., 2007). Temporary immersion induction. If the intact plant is known to synthesize large amounts of systems are also used, e.g. for betalains (Pavlov and Bley, 2006) and antioxidant compounds, one can expect a substantial increase in their semi-continuous perfusion high density cultures for rosmarinic acid level in the cultured roots and sometimes also in plantlets regenerated production by A. officinalis (Su et al., 1993; Su and Humphrey, 1990). from transformed roots. The subject of transformed root methodology, The scale-up study on V. pahalae cell cultures proved the importance its recent developments and the prospects for production of medicinal of physical factors such as illumination of the bioreactor interior for compounds have been addressed in several reviews (Georgiev et al., sustained production of anthocyanins (Meyer et al., 2002). Similarly, in 2007; Guillon et al., 2006; Uozumi, 2004). The manipulation and the aforementioned A. cordata cultures, the upscale in 500 l. fermentor optimization of transformed roots productivity are generally the same allowed the production of over 500 g of mixed anthocyanins during a as those of other culture systems with perfect culture conditions, cell 16 day period (Kobayashi et al., 1993). line selection, precursor feeding and elicitation. Thus, only a few Nonetheless, the bioreactor technology in relation to above men- examples that specifically report the production of antioxidant tioned antioxidants, though sometimes remarkably efficient in terms of compounds by hairy roots will be mentioned in this chapter. production (as in the case of rosmarinic acid), does not seem likely to Several species from the Lamiaceae have been transformed with approach factory-scale industrial application in the near future. Hope- A. rhizogenes for the production of polyphenolic antioxidants. The very fully, at least some of the mentioned research on antioxidants popular rosmarinic acid is again a good example of this. In S. officinalis biotechnology will finally result in market implementation. hairy roots, the antioxidant activity and RA level were much higher than in untransformed organs (Grzegorczyk et al., 2006, 2007). This 3.8. Concluding remarks and future perspective depside has also been efficiently produced by the hairy roots of other species such as: S. miltiorrhiza,(Chen et al., 2001; Yan et al., 2006), Despite the immense variety of antioxidant compounds ob- C. blumei (Bauer et al., 2004), O. basilicum (Bais et al., 2002), H. officinalis tained from in vitro cultured plant, their cells, tissues and organs, (Murakami et al., 1998; Kochan et al., 1999). not many examples of practically applicable technologies can be named. Baikal skullcap (S. baicalensis) is one of the richest natural sources The few that are available include rosmarinic acid, α-tocopherol from of flavones reaching up to 20% in the root. Transformed roots produced sunflower cells and improved anthocyanins from several sources. baicalein and wogonin glucuronides, but also a phenylethanoid There are, however, numerous inspiring and promising reports acteoside, a compound unknown from plants growing in natural on such compounds as resveratrol, kinobeon A, lithospermic acid B and conditions (Nishikawa et al., 1999; Stojakowska and Malarz, 2000). In other oligomeric caffeic acid depsides or crocin, which should provoke S. sclarea hairy roots some abietane diterpenoids are also reported, and encourage more research focused on the regulation of biosynthesis although not in large enough amounts for them to be considered as a and its increased economic feasibility. Finally, the burgeoning source of antioxidants (Kuźma et al., 2006). An effective system for metabolomic and metabolic engineering approaches may add a new rutin production has been established with the hairy roots of fascinating dimension to the possibilities of antioxidant-making plant buckwheat (Fagopyrum esculentum) whose leaves are a rich natural cell factories that have been presented in this paper. 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). References An innovative transgenic approach with respect to antioxidant Antognoni F, Zheng S, Pagnucco C, Baraldi R, Poli F, Biondi S. Induction of flavonoid production has been applied to Saussurea involucrata transformed roots production by UV-B radiation in Passiflora quadrangularis callus cultures. Fitoterapia overexpressing heterologous chalcone isomerase from a related species 2007;78:345–52. (Li et al., 2006). This has led to the enhanced production of the flavone Aruoma OI. 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Root specific elicitation and boraginaceous species: E. sericeum and L. erythrorhizon, having antimicrobial activity of rosmarinic acid in hairy root cultures of sweet basil introduced the rolC gene exhibited greatly reduced levels of the two (Ocimum basilicum L.). Plant Physiol Biochem 2002;40:983–5. Bak S, Tax FE, Feldmann KA, Galbright DW, Feyereisen R. CYP83B1, a cytochrome P450 at antioxidants: rabdosiin and rosmarinic acid in comparison to the lines the metabolic branch point in auxin and indole glucosinolate biosynthesis in without the introduced rolC (Bulgakov et al., 2005). Arabidopsis. Plant Cell 2001;13:101–11. 558 A. Matkowski / Biotechnology Advances 26 (2008) 548–560

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