Eng. Life Sci. 2014, 00, 1–10
Ana Gallego1 Karla Ramirez-Estrada2 Heriberto Rafael Vidal-Limon2 Diego Hidalgo2 Liliana Lalaleo2 Waqas Khan Kayani2,3 Rosa M. Cusido2 Javier Palazon2 1
Departament de Ciencies Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona, Spain
2
Laboratori de Fisiologia Vegetal, Facultat de Farmacia, Universitat de Barcelona, Barcelona, Spain
3
Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan
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Review
Biotechnological production of centellosides in cell cultures of Centella asiatica (L) Urban Centella asiatica (L.) Urban plants have been used since ancient times for their medicinal properties, and their extracts have proven antioxidant, wound healing, sedative, and neuroprotective activities, among others. The natural compounds responsible for C. asiatica bioactivity are triterpene saponins formed from the dammarene branch of the triterpene biosynthetic pathway, collectively known as centellosides, with madecassoside and asiaticoside and their aglycones, madecassic acid and asiatic acid being the most important. Several biotechnological approaches have been developed for the bioproduction of centellosides, based on cell, hairy root, and in vitro plant cultures. This review summarizes the main therapeutic properties of these compounds, as well as their biosynthetic pathways, referring to genetic studies that have identified genes involved in their formation. The biotechnological production of centellosides from a small scale to bioreactor level is also covered. Finally, we summarize the most effective strategies for increasing centelloside yield, including recent transcriptomic, proteomic, and metabolomic studies that have gained new insights into the centelloside biosynthetic pathway and its control. Keywords: Biotechnological production / Centella asiatica / Centellosides / In vitro cultures / Triterpene saponins Received: April 2, 2014; revised: May 7, 2014; accepted: May 22, 2014 DOI: 10.1002/elsc.201300164
1
Introduction
Centella asiatica (L.) Urban is a perennial species of the Umbelliferae native to tropical and subtropical regions. It contains potent anti-inflammatory agents that have been used in traditional medicine and exploited commercially for wound healing [1], nervine complaints, and skin ailments [2]. Centella asiatica is reported to have neuroprotective [3], antioxidant [3, 4], antidiabetic and antimicrobial [3, 5], antitumor [4, 6], and antidepressant [7] properties. Its secondary metabolites of therapeutic importance, known as centellosides, include asiaticoside, madecassoside, brahmoside, brahminoside, thankuniside, sceffoleoside, and centellose, and sapogenins such as asiatic, brahmic, Correspondence: Prof. Javier Palazon (
[email protected]), Laboratori de Fisiologia Vegetal, Facultat de Farmacia, Universitat de Barcelona, Av. Joan XXIII sn., 08028 Barcelona, Spain Abbreviations: α/βASs, α/β-amyrin synthases; CabAS, Centella asiatica putative β-amyrin synthase; CaDDS, C. asiatica dammarenediol synthase; CAE, Centella asiatica extracts; CYS, cycloartenol synthase; DMAPP, dimethylallyl diphosphate; DW, dry weight; FPP, farnesyl diphosphate; FPS, farnesyl diphosphate synthase; IPP, isopentyl diphosphate; MeJA, methyl jasmonate; OSCs, oxidosqualene cyclases; SQS, squalene synthase; TDZ, thidiazuron; TTFCA, total triterpenoid fraction of Centella asiatica; UGTs, UDP-glucosyltransferases
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centellic, and madecassic acids [8, 9]. The therapeutic potential of C. asiatica extracts (CAE) with specific proportions of centellosides has been extensively studied [8]. Interest in CAE and the compounds they contain has led to an over-exploitation of their host plants, and they are currently under threat of extinction due to excessive uprooting [10]. On the other hand, the chemical synthesis of centellosides is either extremely difficult or economically unfeasible. Consequently, attention has been focused on the potential offered by plant cell culture technology for efficient plant secondary metabolite production [11–13]. Several groups around the globe have established C. asiatica cell cultures with enhanced centelloside production, but none of the biotechnological procedures designed so far have proved suitable for commercialization [14]. This is probably due to the still scarce knowledge of the specific steps of centelloside biosynthesis and their regulation in vitro. The use of elicitors is a well-established strategy to enhance secondary metabolite production and has been effectively employed in centelloside biosynthesis. Centelloside production in C. asiatica cell suspension cultures has been increased by the addition of 100 µM methyl jasmonate (MeJA), which stimulated the expression level of Centella asiatica putative β-amyrin synthase (CabAS) and CaSQS genes [15]. Other elicitors and treatments used to enhance centelloside biosynthesis include MeJA with Cu ions, Cu ions alone, pectin, salicylic acid, yeast
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Figure 1. Chemical structure of the main centellosides found in C. asiatica extracts.
extract, thidiazuron (TDZ), α-amyrin, and sugars. The most recent attempts to boost centelloside production have focused on metabolic engineering. An important drawback of hairy root cultures of C. asiatica is that centellosides form and accumulate mainly in the aerial part of the plant, but various research groups have tackled this problem by designing hairy root systems coupled with elicitation [16, 17]. This review, after a brief summary of the bioactive compounds found in C. asiatica and their biological activities and biosynthesis, is focused on the current progress in the biotechnological production of centellosides in plant cell cultures. Described are new developments in scaled-up bioreactors and emerging research on expression analysis of genes involved in centelloside biosynthetic pathways, together with a short discussion on the role of elicitors.
2
Plant description and bioactive compounds
The Centella genus, which includes more than 50 species, belongs to the Order Umbelliferae and the family Apiaceae. The most ubiquitous species in this genus is Centella asiatica (L.) Urban (C. asiatica), previously known as Hydrocotyle asiatica L., and traditionally referred to as gotu-kola (Europe and America), Indian navelwort, Indian pennywort, pegaga (Malasia), pegagan or kaki kuda (Indonesia), luei gong gen or tung chain (China) [18,19]. Centella asiatica is distributed in tropical and subtropical regions up to an altitude of 1800 m, favoring moist habitats and tolerating dense shade. It is consequently found in Southeast Asia, Sri Lanka, parts of China, the western South Sea Islands, Madagascar, South Africa, the southeast of the United States, Mexico, Venezuela, and Colombia and also in eastern regions of South America [3, 18, 20]. A perennial plant, C. asiatica can grow up to 40 cm tall [21]. The leaves, with long reddish petioles about 2–6 cm long and 1.5–5 cm wide, are thin and soft with palmate nerves, hairless or with only a few hairs, and with crenate margins. It is a creeping and stoloniferous plant with glabrous and striated stems, rooting at the nodes. Centella asiatica has sessile pale violet or pink flowers in simple umbels arising from the leaf axils. Each umbel bears two to five small oval fruits, which are densely reticulated. The seeds are pumpkin-shaped nutlets and are embedded within the pericarp [3, 18]. Centella asiatica has been used for medicinal purposes since prehistoric times, and features in Ayurvedic medicine. In many 2
Asian countries, it is applied to enhance wound healing and to revitalize the nerves and brain cells [22, 23]. It has also been used to treat asthma [24], ulcers, leprosy, psoriasis, lupus, and vein diseases, and as a memory-enhancing, antidepressant, antibacterial, antifungal, antiviral, and anticancer agent [3, 18, 25]. Centella asiatica is commonly consumed as a plant food, and is used to make infusions or juice drinks [4]. The bioactive compounds of C. asiatica are different kinds of secondary metabolites, including the pentacyclic triterpenoid saponins and sapogenins collectively known as centellosides, which act in the plant as phytoanticipins due to their antimicrobial activities and protective role against pathogen infections. About 25 triterpenoid compounds of C. asiatica are reported in the literature, although there exist duplicate names, synonyms, and contradictory findings. These terpenoid saponins include asiaticoside, centelloside, madecassoside, brahmoside, thankuniside, sceffoleoside, and centellose, and the most abundant sapogenins are asiatic, brahmic, centellic, and madecassic acids [8] (Fig. 1). However, the content and proportion of triterpene components in C. asiatica vary according to the location and environmental conditions of the plant [4, 8, 18]. The most biologically active of these triterpenes are asiatic acid, madecassic acid, asiaticoside, and madecassoside. Centella asiatica also contains other bioactive secondary compounds, including volatile oils, flavonoids, tannins, phytosterol, mucilages, resins, free amino acids, fatty acids, and sugars [9]. Recent pharmacological, biochemical, and clinical studies of C. asiatica have been performed with titrated extracts and total triterpenoid fractions of Centella asiatica (TTFCA), both containing asiatic acid (30%), madecassic acid (30%), and asiaticoside (40%), as well as total triterpenic fractions, which contain asiatic acid + madecassic acid (60%) and asiaticoside (40%). However, all the known extracts have the same components and are used commercially in the pharmaceutical preparation R R R , Centellase⃝ , or Blastoestimulina⃝ [8,18]. known as Madessol⃝ These extracts are used in oral pharmaceutical forms (tablets and drops), topical medication (ointments and powder), and injections, as well as homeopathic preparations [9].
3
Biological activity
As already mentioned, C. asiatica has been used in traditional Ayurvedic medicine to treat skin illnesses, nervous disorders, and venous insufficiency, and also as an antipyretic, analgesic, ⃝ C
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and anti-inflammatory agent. Other applications have been described, although without experimental evidence, including treatment of albinism, anaemia, asthma, bronchitis, cholera, dysentery, hepatitis syphilis, and urethritis [26]. To date, C. asiatica plants and their extracts (CAE) have been shown to exhibit a wide range of biological activities, notably: (i) Antioxidant activity. CAE show considerable free radical scavenging capacity, their antioxidant activity being comparable to vitamin C and grape seed extracts and other important antioxidants. However, this activity is mainly due to a high phenolic content, including flavonoids, tannins, vitamin E, etc., as well as the high amounts of vitamin C, carotenes, and xanthophylls found in methanolic extracts [3]. (ii) Antimicrobial, antifungal, and antiviral effects. Centella asiatica is one of the plants with highest antibacterial activity against a wide range of enteric pathogens and other bacteria (both gram + and gram −) [3, 5, 24, 27]. (iii) Anti-inflammatory and antirheumatoid activity, which is mainly due to the therapeutic action of asiatic acid and madecassic acid [25,28]. (iv) Important anticancer and cytotoxic activity, mainly due to the presence of asiatic acid [6, 25]. (v) Sedative, anxiolytic, and neuroprotective properties, which Ayurvedic medicine attributed to asiaticoside and the brahmoside and brahminoside constituents of C. asiatica [7]. (vi) Wound-healing activity. Centella asiatica increases the percentage of collagen in cell layer fibronectin, thereby promoting and accelerating wound healing [3, 29]. It functions by increasing hydroxyproline-rich peptide levels, which remodels collagen synthesis in wounds. R , is used to Asiatic acid, the active ingredient in Madecassol⃝ treat keloids and the proliferation of connective tissues and hypertrophic scars. In burnt skin tissue it promotes fibroblast proliferation, increasing the infiltration of inflammatory cells and stimulating the epithelisation process [4]. Comprehensive reviews about the biological activities of C. asiatica plant extracts and their main bioactive components have been published by several authors [8, 30–32].
4
Biosynthesis of centellosides
Centellosides are triterpene saponins and sapogenins bearing an ursane or oleane skeleton (Fig. 1). Triterpenes are terpenoids with 30 carbon atoms that arise via dimerization of two farnesyl diphosphate (FPP) units to produce the intermediate compound, squalene. FPP is obtained after the head-to-tail union of dimethylallyl diphosphate (DMAPP) and two isopentyl diphosphate (IPP) molecules [33, 34]. In plants, IPP and DMAPP are formed either through the mevalonate pathway in the cytosol, or from pyruvate and phosphoglyceraldeyde via the plastidial methylerythritol phosphate pathway [8,33–37]. Farnesyl diphosphate synthase (FPS) catalyses the union of two molecules of IPP with DMAPP (obtained in this case through the mevalonate route) to give FPP (Fig. 2), the common precursor of the majority of sesquiterpenes produced by plants [37]. The cDNA of the FPS enzyme of C. asiatica (CaFPS) (EC2.5.1.1/EC2.5.1.10) was isolated, sequenced, and expressed in Escherichia coli [38]. The FPS enzyme was found to be encoded by a single gene, FPS, which is closely related with the FPS in other plants such as Artemisia annua, Arabidopsis thaliana, and Oryza sativa [38]. ⃝ C
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Squalene synthase (SQS) is a single microsomal polypeptide enzyme that controls the head-to-head condensation of two molecules of FPP to obtain squalene. SQS in C. asiatica was characterized [39] and its full length of 1573 bp encodes 416 amino acids, with a molecular mass of 47.3 kDa and a 39–86% homology with other species. After eliciting C. asiatica cell cultures with MeJA, the observed increase in terpene saponin concentration was accompanied by higher mRNA levels of the CaSQS gene, suggesting that the latter plays an important role in saponin production [40]. Squalene is converted to (3S)-squalene 2,3-epoxide, the common intermediate in the biosynthesis of steroids and triterpenoids, by the action of the microsomal squalene epoxidase in the presence of O2 and NADPH [8, 33–36]. The family of 2,3-oxidosqualene cyclases (OSCs) is enzymes involved in the cyclization of squalene to cyclic triterpene alcohols with various conformations, giving rise to a great diversity of structures and molecules bearing different numbers of rings. The branching point in the biosynthesis of sterols and triterpenoid saponins is the cyclization of 2,3-oxidosqualene to the protosteryl cation or dammarenyl cation, controlled by OSCs (Fig. 2). This step starts with the breaking of the oxirane ring in acid media, and after different rearrangements two conformations are obtained: chair-boat-chair-boat (protosteryl cation) or chair-chair-chair-boat (dammarenyl cation). In plants, the protosteryl cation, which is the precursor of sterols, is converted by cycloartenol synthase (CYS) to cycloartenol. The latter is the precursor of the phytosterols, which are important structural membrane constituents. Full-length cDNA corresponding to the CYS gene, CaCYS, was obtained from the mRNA of C. asiatica leaves [41], presenting a molecular mass of 86.3 kDa, with a very high level of sequence identity (98%) with the CYS of ginseng, and also of Glycyrrhiza glabra, A. thaliana, and Avena strigosa. Northern blot analysis showed that CaCYS was expressed in all C. asiatica tissues except the roots, with the highest transcript levels found in leaves. The transcript accumulation for this gene increased with growth, but clearly decreased when MeJA was added to the medium [41]. Since asiaticoside production increased after elicitation with MeJA and the addition of the antisenescent cytokinin TDZ, it can be inferred that it was due to the growth of shoots, the main site of asiaticoside biosynthesis, rather than a genetic stimulation of secondary metabolite production. Following the centelloside biosynthetic pathway, the dammarenyl cation is converted to different cationic intermediates: baccharenyl, lupenyl, and olenayl cations. The dammarenyl cation may be converted to dammarene-like sapogenins by dammarendiol synthase (a type of OSC). Lupeol synthase uses the lupenyl cation, formed from the dammarenyl cation, to biosynthesize lupeol and lupine-type sapogenins. And finally, the most abundant sapogenins, the oleanes, are generated by α/β-amyrin synthases (α/βASs), which transform the oleanyl cation, also arising from the lupenyl cation, to α-amyrin (ursane skeleton) or β-amyrin (oleane skeleton) [8,33–36]. A full-length cDNA obtained from the mRNA of leaves of MeJA-elicited C. asiatica plants producing high amounts of asiaticoside was identified and cloned [42], and the cDNA was named CabAS (GenBank accessionno.: AY520818) and encoded a 761 amino acid protein of around 86.7 kDA. The structure of the protein 3
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Figure 2. Biosynthetic pathway of centellosides.
and its homology with other βASs suggested that CabAS may be a novel βAS resembling A. strigosa βAS encoded by only one copy of the CabAS gene. The high transcript accumulation of this gene found in leaves, particularly after MeJA treatment together with an enhanced asiaticoside production, suggested that the CabAS gene encodes a βAS [42]. However, some years later, the same authors, after functionally expressing the CabAS gene in a 4
lanosterol-synthase-deficient yeast mutant, showed that rather than a βAS, it encodes a dammarenediol synthase. The CabAS gene was consequently renamed C. asiatica dammarenediol synthase (CaDDS) [43]. In C. asiatica, asiatic acid and madecassic acid apparently arise from β-amyrin, although their chemical structure (ursane skeleton) points to an α-amyrin origin. This remains an unknown aspect of centelloside biosynthesis. ⃝ C
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Figure 3. Steps for obtaining plant cell cultures of C. asiatica. (A) Plantlet in vitro culture; (B) stem explants; (C, D) explants with calli; (E) callus culture; (F) cell suspension culture.
The last biosynthetic steps of the centelloside pathway have still not been elucidated, but probably involve enzymes and reactions commonly found in plants [8,35,36]. Following cyclization, further diversity is conferred by modification of the products by oxidation, hydroxylation, glycosylation, and other substitutions mediated by cytochrome P450-dependent monooxygenases, glycosyltransferases, and other enzymes. Little is known about the enzymes required for these chemical transformations. In the case of saponins, the oligosaccharide chains are likely synthesized by the sequential addition of single sugar residues to the aglycone, but triterpenoid glycosylation remains unexplored [37]. Both saponin and triterpenoid formation are catalyzed by cytochrome P450 enzymes. Asiatic acid and madecassic acid are glycosylated by UDP-glucosyltransferases (UGTs), which link the trisaccharide (Rha+Glc+Glc) at the C-28 position to obtain asiaticoside and madecassoside [17]. Cytrochrome P450 enzymes are encoded by a gene family involved in numerous primary and secondary metabolic processes and their function indicates an important role in the final modifications of the centelloside pathway. P450 genes are known to be involved in triterpenoid saponin synthesis: CYP93E1 in Glycine max [44], CYP51H10 (Sad2) in A. strigosa [45], and CYP88D6 and CYP9E3 in G. uralensis [46]. Recently, a cDNA library was constructed from MeJA-treated C. asiatica hairy roots to establish an expressed sequence tag database [17], from which six P450 genes were analyzed by RT-PCR. It was concluded that MeJA elicitation, which is known to promote triterpene metabolism [47] increased the transcription level of the CYT50B04, CYT47G03, and CYT46B07 genes, suggesting they play an important role in triterpenoid biosynthesis in C. asiatica [17]. Additionally, RT-PCR analysis of four UGTs from this database revealed an increase in the expression level of the 35F01 gene after MeJA treatment, pointing to its role in linking glucose to centelloside production.
5
Biotechnological centelloside production
5.1
Small scale
The structural complexity of most plant-derived pharmaceuticals makes their chemical synthesis a difficult and economically unfeasible process, and most of the plants producing valuable natural products are endangered or grow in inaccessible habitats. Consequently, biotechnological production constitutes an alternative and efficient system for obtaining a sustainable and ⃝ C
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reliable source of target secondary compounds, ensuring a rational utilization of biodiversity. The depletion of the wild stock of C. asiatica by unrestricted exploitation and high industrial demand has prompted the micropagation of C. asiatica to maintain a continuous stock of plants and the development of centellosideproducing in vitro cultures.
5.1.1
Callus and cell cultures
Calli and cell suspensions of C. asiatica, established by several researchers, have proved to be an efficient system for obtaining centellosides, albeit not highly productive. The first report in this field was published in 1987, when Bister-Miel [48] used C. asiatica cell suspensions for biotransformation. In a more recent study, it was found that asiaticoside production decreased after the application of a range of auxins and cytokinins in B5 liquid media; only 0.025 mg/L TDZ had an enhancing effect [49]. Other plant growth regulators at different concentrations have been tested by different authors in solid and liquid media [50,51]. The production of the main centellosides in calli and cell cultures of two C. asiatica phenotypes from South Africa was studied, obtaining values of 0.15–0.25% dry weight (DW) of asiatic acid, 0.15–0.30% DW of madecassic acid, 1.4–2.9% DW asiaticoside, and 1.30–2.84% madecassoside [52]. Total centelloside levels were higher (1.5–1.8 times) in calli than in the corresponding cell suspensions, and only low amounts were excreted by cells into the media. The fact that production in an undifferentiated system was always much lower than in the leaves of the original plants indicates the importance of cell differentiation for a high production of centellosides. Centella asiatica cell suspensions derived from callus cultures obtained from young leaves and petioles were established [53] (Fig. 3) and centelloside production was determined after optimizing the callus culture conditions for a high biomass formation. The main product was madecassoside, followed by madecassic acid, asiaticoside, and asiatic acid, the total production being approximately 900 µg/g DW. The low expression level of CabAS and CaSQS genes in calli compared to leaves could explain the lower centelloside production of these cultures. Feeding experiments have also been carried out to increase centelloside production in C. asiatica cell suspension cultures [54]. The capacity of C. asiatica cells to convert α-amyrin into centellosides was shown when, 7 days after supplementing the cell culture with this potential precursor, 84% had been transformed into centellosides. Total centelloside contents achieved by the treated cultures increased more than sevenfold. 5
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Table 1. Effect of the elicitors on centelloside production in different plant in vitro systems. Culture system
Elicitor
Calli and cells
Production
Reference
AA: 0.15–0.25% DW
[52]
Whole plant
Trichoderma harzianum extracts (3%)
In leaves: A: 9.63 mg/g DW
[63]
Whole plant
MeJA (100 µM)
In leaves: A: 1.4 mg/g DW M: 0.6 mg/g DW AA: 0.3 mg/g DW MA: 0.25 mg/g DW
[47]
A: 3.98 mg/g DW 4.2 mg/g DW 5.91 mg/g DW 6.74 mg/g DW
[61]
In leaves: M: 17 mg/g DW A: 28 mg/g DW MA: 0.55 mg/g DW AA 1.1 mg/g DW
[62]
TC: 1.7 mg/g DW M: 10.6 µg/g WW A: 20.9 µg/g WW MA: 8.0 µg/g WW AA: 4.9 µg/g WW
[53]
[51]
MeJA (50 µM) MeJA (100 µM) MeJA (200 µM) Copper ion (10 µM) Copper ion (25 µM)
494.6 mg/g DW A: 99% compared to control 165% compared to control 243% compared to control 224% compared to control 676% compared to control
Salicylic acid (100 µM) Yeast extract (4 g/L)
A: 102.6 mg/g DW 165.4 mg/g DW
Whole plant CdCl2 (5 mM) CuCl2 (5 mM) Yeast extract (0.1 g/L) MeJA (0.01 mM) Whole plant
MeJA (100 mM) + TDZ (0.025 mg/L)
Whole plant Cell suspensions
MeJA (0.2 mM)
Cell suspensions Cell suspensions
Cell suspensions
[58]
[56]
[57]
Cell suspensions
MeJA (100 µM)
A+M: 1.1 mg/g DW
[15]
Calli
IBA (0.1 mg/L) + 4PU-30 (2 mg/L)
TC: 0.85 mg/g DW
[53]
Hairy roots
MeJA (0.1 mM) MeJA (100 µM)
A: 7.12 mg/g DW M: 0.17% DW A: 0.38% DW
[16]
Hairy roots (PgFPS gene)
[69]
A = Asiaticoside; M = Madecassoside; AA = Asiatic acid; MA = Madecasic acid; TC = Total centellosides; WW = Wet weight; DW = Dry weight.
Supplementing the culture medium with different elicitors is a well-established strategy to increase secondary metabolite production in plant cell cultures (see Table 1), indicating that many of these target compounds are involved in plant defense mechanisms. Studies carried out by our research group [55] showed that centelloside production in a C. asiatica cell suspension increased seven-fold with the addition of 100 µM MeJA, the most abundant being madecassoside followed by asiaticoside. The clear increase in CabAS and CaSQS gene expression on the first day of elicitation could explain the enhanced centelloside production. In contrast, the expression level of the CaCYS gene (involved in the formation of phytosterol) decreased after elicitation. Recent studies [56] have confirmed the enhancement of asiaticose production in elicited C. asiatica callus and cell cultures. 6
Pectin at 0.05, 0.1, and 0.2% w/v was added to the callus cultures, whereas 50, 100, and 200 µM of MeJA and 10 and 25 µM Cu ions were used for cell suspensions. Asiaticoside production in calli treated with pectin increased by up to 31% and in cell cultures elicited with MeJA up to 171, 125, and 494% with respect to leaves, control cell cultures, and 8-week-old callus cultures, respectively. Similar or even better results were obtained with copper ion elicitation (25 µM). In another elicitation study, the greatest enhancement of asiaticoside production in C. asiatica cell cultures was achieved with 100 µM salicylic acid, followed by yeast extract, both being supplied to the medium at day 10 of culture [57]. A metabolomic study of C. asiatica cell cultures, using chromatographic techniques and multivariate statistical models, was recently carried out after the addition of 200 µM MeJA to the
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medium [58]. Metabolomic profiles of the four main centellosides, asiatic acid, madecassic acid, asiaticoside, and madecassoside, showed that after the fourth day of elicitation the amount of these compounds was 20–40 times higher than in untreated cultures, asiaticoside being predominant, followed by madecassoside. Other metabolic changes suggested that MeJA treatment reprogrammed both the terpenoid and flavonoid pathways.
5.1.2
Hairy root cultures
Hairy root cultures of C. asiatica obtained by genetic transformation of plant material with Agrobacterium rhizogenes do not produce large amounts of centellosides, since these compounds form and accumulate in the aerial part of the plant, and the genes encoding enzymes associated with triterpene saponins, such as α/βAS (cytochrome P450-independent carboxylase/hydroxylase [P450] and glucosyltransferase are probably poorly expressed in root tissues). Asiaticoside and madecassoside were not detected in C. asiatica hairy roots transformed with A. rhizogenes R1601 [59]. In contrast, when C. asiatica calli were transformed with A. rhizogenes ATCC 15834, an increase in asiaticoside levels of up to 172% was observed in transformed roots compared to the untransformed calli [56]. It has been demonstrated that A. rhizogenes rol genes can increase the production of plant secondary metabolites in the transformed tissues [60], and in this case the enhanced asiaticoside levels in the C. asiatica transformed roots could be attributed to the presence of rol genes in their genome. On the other hand, the possibility of increasing the centelloside production in C. asiatica hairy roots after supplementing cultures with MeJA has been reported. Asiaticoside production in hygromycin-resistant hairy roots was significantly enhanced after the addition of MeJA (100 µM) to the culture medium [16]. These results were correlated with an increased CabAS expression after elicitation, confirming the role of this gene in asiaticoside biosynthesis. In the same biological model, several transcripts with significant sequence similarities to P450s and UGTs were identified, and may be involved in triterpene saponin biosynthesis [17]. Among the unique sequence tags identified, the authors selected six corresponding to P450s and four to UGTs and determined their expression pattern by quantitative RT-PCR. The results indicated that three P450 genes (35F09, 46B07, and 50B04) and one UGT (35F01) are strong candidates for genes involved in the transformation of α-amyrin to asiatic and madecassic acids and of these two acids to the corresponding saponins. To elucidate the contribution of FPS to triterpene biosynthesis, C. asiatica hairy root lines overexpressing Panax ginseng FPS (PgFPS) were established. Quantitative analysis of squalene, phytosterols, madecassoside, and asiaticoside, and mRNA levels of genes encoding SQS (CaSQS), dammarenediol synthase (CaDDS), and CYS (CaCYS) showed that all these parameters were higher in hairy root lines overexpressing the FPS gene compared with the controls. However, no differences were found in the expression level of the CaSQS gene. The production of total sterols in the hairy roots was approximately three times higher than in the controls, indicating that FPS performs a regulatory function in phytosterol biosynthesis. The madecassoside and ⃝ C
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asiaticoside contents also increased 1.15-fold after 14 days of 100 µM MeJA treatment, whereas in the controls the addition of the elicitor surprisingly enhanced the studied centelloside production up to 1.39-fold after 28 days of elicitation. It was presumed that the overexpression of FPS resulted in a feedbackinduced suppression of the downstream gene. All these results suggest that FPS plays a role in the regulation of phytosterol and triterpene biosynthesis. Taken as a whole, these results confirm that hairy root cultures, despite their low centelloside production, constitute an excellent platform for the study of centelloside metabolism. Green hairy roots [60] are transformed roots, which turn green when grown in the light, and are frequently able to produce plant secondary metabolites that are produced/accumulated in plant aerial parts. As most experiments carried out to date with C. asiatica hairy roots were performed in the dark, transferring them to light is a potential system to improve their centelloside production.
5.1.3
In vitro plant cultures
Whole C. asiatica plant cultures have proved suitable for studying the centelloside biosynthetic pathway and its regulation, but there are few reports on their use for the biotechnological production of centellosides. The effect of a range of elicitors (CdCl2 , CuCl2 , MeJA, and yeast extract) on asiaticoside production in in vitro plant cultures of C. asiatica was studied [61], but only MeJA and yeast extract had a positive action. The highest content of asiaticoside was found in leaves (82% of the total) after supplementing the medium with MeJA (100 µM) together with the cytokinin TDZ (0.025 mg/L). These results were later confirmed [62]. When testing the effect of MeJA (100 µM) on centelloside production in C. asiatica cultured plantlets, the descending order of product concentration was asiaticoside, madecassoside, asiatic acid, and madecassic acid, found mainly in leaves [47]. The positive action of MeJA as an inducer of enzymes involved in triterpenoid biosysnthesis downstream from 2,3-oxidosqualene was especially patent after 4 weeks of culture, although plant growth was negatively affected. The action of different fungal elicitors in asiaticoside and biomass production of C. asiatica shoot cultures has been recently studied [63]. The best results were obtained with 3% v/v Trichoderma harzianum culture filtrate, which clearly enhanced both asiaticose production (2.35-fold) and biomass growth (1.24-fold). Although generally centellosides accumulation in plants is higher than in cell suspension or hairy root cultures, to date, as this review makes clear few approaches have been developed to produce centellosides in in vitro plant cultures, probably because the most important problem is to scale up the process. The bioprocessing of differentiated plant in vitro systems has been recently reviewed [64] and the challenge of scaling up the process has been met by using bioreactors well adapted to differentiated cultures such as adapted stirred tank and rotating drum bioreactors. But probably more suitable are the temporary immersion systems such as the twin-flask system or r´ecipient a` immersion temporaire automatique which could prevent asphyxia of the tissues and hyperhydricity [64]. 7
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Scale up to bioreactors
When scaling up plant cell suspension cultures to bioreactor level for commercial purposes, the growth, production behavior, morphology, and shear tolerance of the plant culture should be studied to choose the best bioreactor for each bioprocess. In general, a bioreactor should have a good mixing system, adequate oxygen transfer, and low shear stress [65, 66]. Optimum cell growth and metabolite production requires that the cells are provided with appropriate nutrients and that there is a correct balance between the mixing and shear sensitivity of the plant cells to minimize cell damage. This parameter depends on each species and cell line [67]. Also, the operating parameters of temperature, pH, oxygen concentration, and substrate concentrations should be easy to control and monitor. To date, only one report describes culturing C. asiatica cell suspensions at bioreactor level [22]. The optimized conditions for asiaticoside production in C. asiatica cells in a 5-L bioreactor are explained, paying particular attention to the agitation speed, aeration rate, and inoculum size. Cell biomass and asiaticoside production peaked at day 24 (116.17 g fresh weight and 12.24 g of DW, and 56.21 mg/g DW of asiaticoside), with an inoculum size of 30 g, agitation speed of 120 rpm, and aeration rate of 2 L/min. Cell proliferation in C. asiatica suspension cultures and asiaticoside production depend on agitation but too high speeds can destroy the cell walls [68] and subsequently reduce asiaticoside biosynthesis. The optimum speed tested was 150 rpm, when the culture achieved a growth index 4.27-fold higher compared with other speeds and an asiaticoside production of 59.43 mg/g DW [22]. The optimum aeration rate was 2.5 L/min, which increased asiaticoside concentration to 62.14 mg/g DW. Three different inoculum sizes were compared in the 5-L bioreactor, ranging from 30 to 100 g. The largest size (100 g) achieved the maximum biomass but it resulted in the lowest growth index and also a very low yield of asiaticoside, demonstrating that inoculum size is a crucial factor in bioreactor culturing of C. asiatica.
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Conclusions and perspectives
Interest in C. asiatica has increased over the years due to its medicinal properties and cosmetic applications. Extracts of this plant in a variety of pharmaceutical forms are being used in the treatment of several diseases. However, uncontrolled collection of this species, which requires specific environmental conditions for proper growth, means it is now endangered in the wild. Biotechnology offers an opportunity to sustainably exploit plant cells, tissues, organs, and the entire plant organism, using in vitro cultures and genetic manipulation, for the production of plant secondary metabolites such as centellosides. Besides their benefits for human health and use in pharmaceutical products, these important triterpene compounds are also currently attracting considerable attention in the field of cosmetology and several cosmetic companies are using C. asiatica cell suspensions in their products, including Centella Stems GXTM Plant Stem Cells by Sederma (France), Centella Asiatica Stem GTM by Schmelzkopf 8
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Cosmetics (Australia), Plant Stem Cells Science (containing C. asiatica stem cells) by Intelligent Nutrients (USA), etc. Improvement of secondary compound production depends on knowledge of complex biosynthetic pathways and their genetic manipulation. In the case of centellosides, their biosynthetic pathway has been characterized up to the formation of 2,3-oxidosquelene, but the latter’s subsequent cyclization by different OSCs and the final metabolic steps remain unclear. Recent transcriptomic studies of C. asiatica have identified several genes that are overexpressed under elicitation. Also, different C. asiatica genes have been sequenced and cloned, although their application in in vitro systems using metabolic engineering techniques remains limited. The availability of powerful new tools for functional genomics studies (transcriptomics, proteomics, and metabolomics) will allow researchers to further explore the centelloside pathway, identify the genes that encode the key enzymes, and modify their expression. Metabolomic studies can also ascertain how the secondary metabolite pattern in a culture is altered by elicitation. In this way, the key metabolic steps that regulate the carbon flux to the compounds of interest can be determined and efficiently targeted by metabolic engineering to establish highly productive biotechnological systems.
Practical application This review reports on the therapeutic activity of Centella asiatica extracts that could be of practical interest for technical departments of pharmaceutical and cosmetic companies. Additionally, the details given for optimizing centelloside biotechnological production may be useful for biotechnology laboratories interested in developing and commercializing C. asiatica cell suspensions whose application as a cosmetic ingredient is currently growing.
Work in the Plant Physiology Laboratory (University of Barcelona) was financially supported by the Spanish MEC (BIO2011-29856C02-01) and the Generalitat de Catalunya (2009SGR1217). The authors have declared no conflict of interest.
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