Pilot-scale culture of somatic embryos of Eleutherococcus senticosus in airlift bioreactors for the production of eleutherosides

Purpose of work

To establish pilot scale bioreactor cultures of somatic embryos of Siberian ginseng for the production of biomass and eleutherosides. Somatic embryos of Eleutherococcus senticosus were cultured in airlift bioreactors using Murashige and Skoog medium with 30 g sucrose l^?1 for the production of biomass and eleutherosides. Various parameters including the type of bioreactor, aeration volume, and inoculum density were optimized for 3 l capacity bioreactors. Balloon-type airlift bioreactors, utilizing a variable aeration volume of 0.1–0.3 vvm and an inoculum of 5 g l^?1, were suitable for biomass and eleutheroside production. In 500 l balloon-type airlift bioreactors, 11.3 g dry biomass l^?1, 220 µg eleutheroside B l^?1, 413 µg eleutheroside E l^?1, and 262 µg eleutheroside E1 l^?1 were produced.     

HPTLC-profiling of eleutherosides,mechanism of antioxidative action of eleutheroside E1, the PAMPA test with LC/MS detection and the structure–activity relationship

Human body is constantly generating free radicals, which causes oxidative stress. Despite naturally occurring antioxidant systems in human body, free radicals cause lipid, proteins and DNA oxidation. New antioxidants are still urgent as well as their mechanisms of action should be explained. In this study, we investigated the mechanism by which eleutherosides B, E and E1 may act as antioxidants, identified eleutherosides in Eleutherococcus lasiogyne and Eleutherococcus giraldii, and explained in vitro the absorption of eleutheroside E1 based on passive transport. The DPPH¹ and DB-HPTLC tests were used to assess the antioxidant activity. Of the three eleutherosides, only eleutheroside E1 exhibited a strong anti-DPPH¹ activity (EC₅₀ 37.03 μg/mL; 63 mMol) compared to the raw extracts (EC₅₀ 170 and 180 μg/mL for E. lasiogyne and E. giraldii). This activity was also confirmed by the DB-HPTLC autography technique. According to Za?uski’s hypothesis, the antioxidant mechanism of eleutheroside E1 is based on the complexation of DPPH¹ molecule with its aryl radical. During this reaction, the aryl radical of eleutheroside E1 (E1¹) and DPPHH are created. Next, the aryl radical (E1¹) is complexed with another DPPH¹ molecule. Additionally, the aryl radical can be stabilized by the presence of the methoxy groups in the aromatic ring, which increases its antioxidative action. The HPTLC-identification of extracts showed the presence of eleutherosides B, E and E1 in both species. The PAMPA test coupled with LC/MS detection showed a low permeability of eleutheroside E1 across artificial membrane. Because eleutherosides belong to the polyphenols, the TPC and TFC were quantified. The TPC and TFC varied from 51.4 to 49.3 mg/g dry extract for TPC, and from 5.73 to 4.91 mg/g dry extract for TFC, for E. giraldii and E. lasiogyne, respectively. In conclusion, eleutheroside E1 in its pure form could be a chemopreventive ingredient of new pharmacological or dietary products, stimulating the GALT. These findings can explain partially the adaptogenic activity of eleutheroside E1 on the GALT, which has been still unknown.     

Biotechnological production of caffeic acid derivatives from cell and organ cultures of Echinacea species

Caffeic acid derivatives (CADs) are a group of bioactive compounds which are produced in Echinacea species especially Echinacea purpurea, Echinacea angustifolia, and Echinacea pallida. Echinacea is a popular herbal medicine used in the treatment of common cold and it is also a prominent dietary supplement used throughout the world. Caffeic acid, chlorogenic acid (5-O-caffeoylquinic acid), caftaric acid (2-O-caffeoyltartaric acid), cichoric acid (2, 3-O-dicaffeoyltartaric acid), cynarin, and echinacoside are some of the important CADs which have varied pharmacological activities. The concentrations of these bioactive compounds are species specific and also they vary considerably with the cultivated Echinacea species due to geographical location, stage of development, time of harvest, and growth conditions. Due to these reasons, plant cell and organ cultures have become attractive alternative for the production of biomass and caffeic acid derivatives. Adventitious and hairy roots have been induced in E. pupurea and E. angustifolia, and suspension cultures have been established from flask to bioreactor scale for the production of biomass and CADs. Tremendous progress has been made in this area; various bioprocess methods and strategies have been developed for constant high-quality productivity of biomass and secondary products. This review is aimed to discuss biotechnological methods and approaches employed for the sustainable production of CADs.     

Production of eleutherosides in in vitro regenerated embryos and plantlets of <Emphasis Type="BoldItalic">Eleutherococcus chiisanensis</Emphasis>

High frequency somatic embryogenesis of Eleutheorcoccus chiisanensis was achieved through suspension culture of embryogenic cells in hormone-free Murashige and Skoog liquid medium supplemented with 30 g sucrose l⁻¹. Cotyledonary somatic embryos were germinated and converted into plantlets using 20 μM gibberellic acid which were then grown in a 10 l airlift bioreactor. HPLC analysis revealed the accumulation of eleutheroside B, E and E1 in the embryos and plantlets. Thus mass production of embryos and plantlets of E. chiisanensis can be achieved in liquid cultures and the biomass produced may become an alternative source of eleutherosides.     

Effect of temperature on secondary metabolites production and antioxidant enzyme activities in Eleutherococcus senticosus somatic embryos

Somatic embryos of Eleutherococcus senticosus were exposed at 12, 16, 24 and 30 °C for duration of 45 days in bioreactor. The effects of such treatments on the growth, eleutheroside B, E, E₁, total phenolics, flavonoids, chlorogenic acid concentrations and antioxidant enzymes activities were investigated. The results revealed that low (12 and 18 °C) and high (30 °C) temperature caused significant decrease in fresh weight (FW), dry weight (DW), total phenolics, flavonoids and total eleutheroside accumulation, while low temperature increased eleutheroside E accumulation in somatic embryos. Low temperature significantly increased superoxide dismutase (SOD), catalase (CAT), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) activities whereas a strong increase in ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDHAR) activity was obtained at 12 °C grown somatic embryos. In contrast, high temperature significantly decreased antioxidant enzymes activities and even guaiacol peroxidase (G-POD) activity also decreased at low temperature in comparison to 24 °C grown embryos. These data suggest that low and high temperature treatment provoked an oxidative stress in E. senticosus embryos, as shown by the increase in lipid peroxidation. The increase in lipid peroxidation was paralleled by a rise in lipoxygenase (LOX) activity and hydrogen peroxide (H₂O₂) content. However, this stress was more prominent at high temperature than low temperature grown embryos. This result suggests that the reduced growth of embryo at 30 °C was concomitant with reduced efficiency of these protective enzymes. On the other hand, increases in antioxidant activities at 12 and 18 °C could also be a response to the cellular damage; however, this increase could not stop the deleterious effects of low temperature, but reduced stress severity thus allowing embryo growth to occur.     

Influence of inoculum density and aeration volume on biomass and bioactive compound production in bulb-type bubble bioreactor cultures of Eleutherococcus koreanum Nakai

This study deals with the effects of initial inoculum density and aeration volume on biomass and bioactive compound production in adventitious roots of Eleutherococcus koreanum Nakai in bulb-type bubble bioreactors (3-L capacity). While the fresh and dry weights of the roots increased with increasing inoculum density, the highest percentage dry weight and accumulation of total target compounds (eleutheroside B and E, chlorogenic acid, total phenolics, and flavonoids) were noted at an inoculum density of 5.0 g L⁻¹. Poor aeration volume (0.05 vvm) stunted root growth, and high aeration volume (0.4 vvm) caused physiological disorders. Moreover, an inoculum density of 5.0 g L⁻¹ and an aeration volume of 0.1 vvm resulted in the highest concentration of total target compounds and least root death. Such optimization of culture conditions will be beneficial for the large-scale production of E. koreanum biomass and bioactive compounds.     

Immunologically active polysaccharides of acanthopanax senticosus

From the alkaline aqueous extract of Acanthopanax (Eleutherococcus) senticosus two homogeneous polysaccharides, a glucan with a mean M, of 150 000 and a heteroxylan with a mean M, of 30 000, have been isolated by DEAE-Sepharose CL-6B and Sephacryl S-400 column chromatography. Their structures were elucidated mainly by permethylation, periodate oxidation, Smith degradation, reduction experiments, partial hydrolysis and ¹³C NMR. The isolated crude polysaccharide mixture and the heteroxylan enhance phagocytosis in in vitro and in vivo immunological tests.     

Application of bioreactor system for large-scale production of Eleutherococcus sessiliflorus somatic embryos in an air-lift bioreactor and production of eleutherosides

Embryogenic callus was induced from leaf explants of Eleutherococcus sessiliflorus cultured on Murashige and Skoog (MS) basal medium supplemented with 1 mg l(-1) 2,4-dichlorophenoxyacetic acid (2,4-D), while no plant growth regulators were needed for embryo maturation. The addition of 1 mg l(-1) 2,4-D was needed to maintain the embryogenic culture by preventing embryo maturation. Optimal embryo germination and plantlet development was achieved on MS medium with 4 mg l(-1) gibberellic acid (GA(3)). Low-strength MS medium (1/2 and 1/3 strength) was more effective than full-strength MS for the production of normal plantlets with well-developed shoots and roots. The plants were successfully transferred to soil. Embryogenic callus was used to establish a suspension culture for subsequent production of somatic embryos in bioreactor. By inoculating 10 g of embryogenic cells (fresh weight) into a 3l balloon type bubble bioreactor (BTBB) containing 2l MS medium without plant growth regulators, 121.8 g mature somatic embryos at different developmental stages were harvested and could be separated by filtration. Cotyledonary somatic embryos were germinated, and these converted into plantlets following transfer to a 3l BTBB containing 2l MS medium with 4 mg l(-1) GA3. HPLC analysis revealed that the total eleutherosides were significantly higher in leaves of field grown plants as compared to different stages of somatic embryo. However, the content of eleutheroside B was highest in germinated embryos. Germinated embryos also had higher contents of eleutheroside E and eleutheroside E1 as compared to other developmental stages. This result indicates that an efficient protocol for the mass production of E. sessiliflorus biomass can be achieved by bioreactor culture of somatic embryos and can be used as a source of medicinal raw materials.     

Isothermal DNA amplification facilitates the identification of a broad spectrum of bacteria,fungi and protozoa in <Emphasis Type="Italic">Eleutherococcus</Emphasis> sp. plant tissue cultures

In vitro cultures of Eleutherococcus sieboldianus originating from surface sterilized leaf explants were found to be associated with several microorganisms. The associations included bacteria, fungi and protozoa within the rhizosphere and inside root hairs. To determine if this phenomenon is unique to this species, plant tissue cultures of E. gracilistylus and E. senticosus were included in our studies for comparison. A methodology consisting of isothermal amplification, cloning and sequencing was established for analysing 16S ribosomal DNA of cultivated and non-cultivated bacteria from different tissue types. The same methodology was used to obtain internal transcribed spacer regions and 18S regions of fungal and protozoan rDNA. Comparative analyses of sequencing data resulted in the identification of various genera within the Firmicutes and γ-proteobacteria kingdoms and a broad spectrum of fungal genera related to several uncultured fungi. In addition, amoebal and chrysophyte species were detected. Most of the species were identified in different plant organs and in in vitro culture cell types indicating the microorganisms are systemically distributed. The presence of identical microorganisms in different plant species argues for an evolutionary long-lasting and stable association between the plant genus and the microinhabitants.     

A comparative study on root and bark extracts of Eleutherococcus senticosus and their effects on human macrophages

BackgroundEleutherococcus senticosus or Siberian ginseng is a medicinal plant containing adaptogenic substances believed to regulate immune responses. Both, the root and stem bark are commonly used in traditional medicines.PurposeThe purpose of the present study is to chemically characterize E. senticosus root and bark extracts and to compare their effects on functions of human primary macrophages.Study design and methodsHPLC-DAD-MS analysis was used to characterize chemical constituents of alcoholic extracts from E. senticosus root and bark. The data obtained and available databases were combined for network pharmacology analysis. Involvement of predicted pathways was further functionally confirmed by using monocyte-derived human macrophages and endotoxin-free E. senticosus root and bark extracts.ResultsChemical analysis showed that the root extract contained more syringin, caffeic acid, and isofraxidin than the bark extract. At variance, bark extract contained more sesamin and oleanolic acid. Coniferyl aldehyde and afzelin were below the limit of quantification in both extracts. Network pharmacology analysis indicated that constituents of E. senticosus might affect the immune cell phenotype and signaling pathways involved in cell metabolism and cytoskeleton regulation. Indeed, both extracts promoted actin polymerization, migration, and phagocytosis of E. coli by macrophages pointing to macrophage polarization towards the M2 phenotype. In addition, treatment with E. senticosus root and bark extracts decreased phosphorylation of Akt on Ser473 and significantly reduced expression of the hemoglobin scavenger receptor CD163 by macrophages. Neither extract affected expression of CD11b, CD80, or CD64 by macrophages. In addition, macrophages treated with the bark extract, but not with the root extract, exhibited activated p38 MAPK and NF-κB and released increased, but still moderate, amounts of proinflammatory TNF-α and IL-6, anti-inflammatory IL-10, and chemotactic CCL1, which all together point to a M2b-like macrophage polarization. Differently, the root extract increased the IL-4-induced expression of anti-inflammatory CD200R. These changes in monocytes are in agreement with an increased M2a macrophage polarization.ConclusionThe ability of E. senticosus root and bark extracts to promote polarization of human macrophages towards anti-inflammatory M2a and M2b phenotypes, respectively, might underlay the immunoregulatory activities and point to potential wound healing promoting effects of this medicinal plant.     

Biotechnological approaches to enhance the biosynthesis of ginkgolides and bilobalide in Ginkgo biloba

Ginkgo biloba is one of the oldest living tree species and its extracts or powdered leaves are one of the best selling herbal preparations. The main bioactive constituents are flavonoids and the terpene trilactones, ginkgolides and bilobalide, which are responsible for their pharmacological activity. However, there are many difficulties for ginkgo leaves supply and the chemical synthesis is far from of being applicable for commercial-scale production. G. biloba cell cultures have arisen as a useful alternative source of pharmacologically active terpene trilactones. This review sheds light on the chemistry and biosynthesis of terpene trilactones with the aim of increasing the production of these high value compounds by biotechnological approaches. Different biotechnological strategies to improve ginkgolides and bilobalide production will be discussed, including screening and selection of in vitro ginkgo cultures, cell differentiation levels of these cultures, optimization of culture conditions, feeding and elicitation strategies. Special attention will be paid in developing new methodologies to enhance ginkgo cell biomass and provide high amounts of these bioactive terpene trilactones using large-scale cell cultures.     

Effect of nitrogen source on biomass and bioactive compound production in submerged cultures of Eleutherococcus koreanum Nakai adventitious roots

Ammonium to nitrate ratios of 0:30, 5:25, 10:20, 15:15, 20:10, 25:5, and 30:0 mM were tested to determine the optimal NH(4)(+) :NO(3)(-) ratio for improving biomass and bioactive compound production in Eleutherococcus koreanum Nakai adventitious roots using 3-L bulb-type bubble bioreactors. A high ammonium nitrogen ratio had a negative effect on root growth, and the highest fresh and dry weights were obtained when NH(4)(+):NO(3)(-) ratios were 5:25 and 10:20 (mM) after 5 weeks of culture. Although the total production of eleutherosides B and E was slightly higher at the 10:20 ratio than at the 5:25 ratio (NH(4)(+):NO(3)(-)), we proposed that the optimal NH(4)(+):NO(3)(-) ratio was 5:25 mM. This ratio achieved both the highest total production of five target bioactive compounds (eleutherosides B and E, chlorogenic acid, total phenolics, and flavonoids) and the highest root biomass. Furthermore, increasing NH(4)(+):NO(3)(-) ratios to 10:20 decreased pH in the medium, interrupted the absorption of essential minerals from the culture medium, and resulted in low biomass and increased relative oxidative stress levels, which were evaluated by determining 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity. Therefore, nitrate rather than ammonium nitrogen was more essential not for only biomass production but also for bioactive compound production in E. koreanum adventitious root cultures. The optimal nitrogen source ratio produced 5.63 g L(-1) of biomass and 24.41 mg of the five total bioactive compounds per gram of biomass (dry weight basis). The development of such in vitro culture technology will benefit the pilot-scale production of E. koreanum-based bioactive compounds for commercialization.     

Quality control of roots of Eleutherococcus senticosus by HPLC

An HPLC method based on several known methods for the determination of eleutherosides B and E was developed, optimised and validated in terms of linearity, precision (repeatability and intermediate precision on different days and at different concentration levels) and accuracy (recovery). The extraction procedure, the extraction solvent and the extraction yield were evaluated and optimised. A reversed-phase RP-18 column gradient eluted with a two-phase system consisting of phosphoric acid:water (0.5:99.5) and acetonitrile was used to evaluate the samples; detection was at 220 nm. Although eleutherosides B and E are commercially available, they are very costly, and therefore ferulic acid was chosen as external standard. The correction factors for the response of ferulic acid against both eleutherosides were determined and validated. This method, accepted by the European Pharmacopoeia Commission, will be included in the monograph on Eleutherococcus senticosus roots to assay the content of eleutherosides B and E.     

Contrasting genetic patterns between two coexisting Eleutherococcus species in northern China

Climate oscillations are the key factors to understand the patterns in modern biodiversity. East Asia harbors the most diverse temperate flora, largely because an extensive terrestrial ice cap was absent during repeated Pleistocene glaciation–interglacial cycles. Comparing the demographic histories of species that are codistributed and are close relatives may provide insight into how the process of climate change influences species ranges. In this study, we compared the spatial genetic structure and demographic histories of two coexisting Eleutherococcus species, Eleutherococcus senticosus and E. sessiliflorus. Both species are distributed in northern China, regions that are generally considered to be sensitive to climatic fluctuations. These regions once hosted temperate forest, but this temperate forest was replaced by tundra and taiga forest during the Last Glacial Maximum (LGM), according to pollen records. Using three chloroplast DNA fragments, we assessed the genetic structure of 20 and 9 natural populations of E. senticosus and E. sessiliflorus, respectively. Extremely contrasting genetic patterns were found between the two species; E. sessiliflorus had little genetic variation, whereas E. senticosus had considerably higher levels of genetic variation (15 haplotypes). We speculated that a recent severe bottleneck may have resulted in the extremely low genetic diversity in E. sessiliflorus. In E. senticosus, populations in Northeast China (NEC) harbored all of the haplotypes found in this species and included private haplotypes. The populations in NEC had higher levels of genetic diversity than did those from North China (NC). Therefore, we suggest that both the NC and NEC regions can sustain LGM refugia and that lineage admixture from multiple refugia took place after the LGM elevated the local genetic diversity in NEC. In NEC, multiple genetic hot spots were found in the Changbai Mountains and the Xiaoxing'an Range, which implied that multiple locations in NEC may sustain LGM refugia, even in the Xiaoxing'an Range.     

Protective Effects of 3,4-Seco-Lupane Triterpenes from Food Raw Materials of the Leaves of Eleutherococcus Senticosus and Eleutherococcus Sessiliflorus on Arrhythmia Induced by Barium Chloride

As a traditional wild vegetable and food raw material, the leaves of Eleutherococcus senticosus and Eleutherococcus sessiliflorus are rich in 3,4-seco-lupane triterpenes, including chiisanoside (CSS), divaroside (DVS), sessiloside-A (SSA), and chiisanogenin (CSG). This study was conducted to evaluate the anti-arrhythmic effects of these 3,4-seco-lupane triterpenes. Evaluation of the cytotoxicity of compounds was performed by measuring cell viability and apoptosis with the CCK-8 assay. In vivo, arrhythmia was induced by rapid injection of BaCl₂ via rat caudal vein. The occurrence time and duration of arrhythmias in rats were studied. The levels of SOD and MDA in serum, and Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase in myocardial homogenate were detected by ELISA. The histopathological changes of rats myocardial were observed by HE staining. Changes in the expression of PKA and related proteins were detected by Western blot. The 3,4-seco-lupane triterpenes interactions with protein kinase A were analyzed by molecular docking. In the present study, we found that 3,4-seco-lupane triterpenes exhibited powerful anti-arrhythmic activity, especially DVS completely relieved the ventricular arrhythmia induced by BaCl₂. This study suggests that the leaves of E. senticosus and E. sessiliflorus might be used as functional food materials to prevent arrhythmia, and DVS can potentially be further developed as an anti-arrhythmic drug.     

<Emphasis Type="Italic">Aconitum</Emphasis> biotechnology: recent trends and emerging perspectives

The genus Aconitum (consists more than 250 species) is one of the most important clades of highly valued medicinal plants. Aconitum species are very essential in the traditional device of medication and feature excessive business demand in the herbal marketplace. Some of biologically energetic compounds, e.g., aconitine, indaconitine, pseudoacontine, and so on, had been recognized, and new formulations primarily based on those compounds are being produced as rapid rate. This has led to extensive and rather unregulated exploitation of the species in the wild making the genus a threatened group. Conventional breeding and propagation methods have contributed significantly, but these could not meet up with the ever increasing demands of herbal drug industry globally. Biotechnological interventions, therefore, emerge as an alternative approach in terms of higher production and conservation as well. In recent years, several reports have been published on in vitro propagation of various important Aconitum species. However, advanced biotechnological approaches, such as synthetic seed production and hairy root cultures, are still lacking with only a few reports available. The current review presents an updated overview and critical assessment of secondary data concerning the past and recent biotechnological approaches and interventions in genus Aconitum. This review also attempts to provide a detailed account of work explored so far in micropropagation and emphasizes over the areas not attempted yet, which will act as a baseline data as well as valuable information for different stakeholders and researchers working on various aspects of Aconitum biotechnology.     

Antitussive effect of a fixed combination of Justicia adhatoda,Echinacea purpurea and Eleutherococcus senticosus extracts in patients with acute upper respiratory tract infection: A comparative,randomized, double-blind,placebo-controlled study

BackgroundKan Jang® oral solution (KJ) is a fixed combination of aqueous ethanolic extracts of Justicia adhatoda L. leaf, Echinacea purpurea (L.) Moench root, and Eleutherococcus senticosus (Rupr. & Maxim.) Harms root. It is approved in Scandinavia as an herbal medicinal product for respiratory tract infection treatment.PurposeThe present clinical trial aimed to compare the antitussive effect of KJ with placebo (PL) and bromhexine (BH) among patients of 18–65 years old with non-complicated upper respiratory infections (URI; i.e., common cold).Study designWe performed a parallel-group, randomized, double-blinded, placebo-controlled trial in in 177 patients with acute URI over a 5 day period.MethodsWe investigated the antitussive effects of a KJ (30 ml/day; 762 mg genuine extracts with standardized contents of 0.2 mg/ml vasicine, 0.8 mg/ml chicoric acid, and 0.03 mg/ml eleutherosides B and E), bromhexine hydrochloride (24 mg/30 ml/day) and PL on cough and blood markers. The primary outcome was cough relief, which was assessed as the change of cough frequency from baseline (cough index). Secondary outcomes were safety with regards to reported adverse events (AEs) and hematological data.ResultsBoth KJ and BH relieved cough more effectively than placebo. On the third and fourth days of treatment, we observed faster improvement in the group receiving KJ compared to in the groups receiving BH (100%) or PL (100%), indicating a slightly shorter recovery time in the KJ group. KJ showed a good tolerability and safety profile.ConclusionKJ exerted significant antitussive effects in URI. The present data further support the therapeutic use of KJ in upper respiratory tract infections.     

Genetic Relationships among Far Eastern Species of the Family Araliaceae Inferred by RAPD Analysis

A molecular genetic study of Far Eastern species of the family Araliaceae by means of RAPD analysis was conducted. Using 21 primers we assessed variability at 595 loci. Based on matrices of genetic distances D, dendrograms of genetic relationships among eleven species of this family were constructed. Our results suggest that Acanthopanax sessiliflorus and Eleutherococcus senticosus belong to different genera, Aralia cordata andA. continentalis are different species, and A. elata and A. mandshurica probably cannot be regarded as distinct species. Genetic similarity of Far Eastern A. cordata and American A. hispida is shown.     

Improved gymnemic acid production in the suspension cultures of Gymnema sylvestre through biotic elicitation

Enhancement of secondary metabolite accumulation in cultured plant cells through biotic and abiotic elicitation has been recognised as an important biotechnological strategy. Gymnema sylvestre is a rich source of triterpenoid saponins—gymnemic acids used mainly in the treatment of diabetes I and II. The cell suspension cultures initiated from the leaves and stalks of in vitro-grown plantlets have shown to accumulate large amounts of gymnemic acid. The cell-free extracts of Aspergillus niger, Saccharomyces cerevisiae, Agrobacterium rhizogenes, Bacillus subtilis and Escherichia coli were employed as sources of biotic elicitors to study the effect on secondary metabolite production. All the elicitors have shown a positive response in terms of gymnemic acid, with the highest response induced by A. niger [98.65 ± 0.93 mg/gram dry cell weight (gDCW)], 11.2-fold, and the lowest by E. coli (33.25 ± 1.38 mg/gDCW), 3.8-fold, in comparison to the untreated cultures (8.79 ± 0.82 mg/gDCW). The suspension cultures of G. sylvestre can serve as a continuous source of gymnemic acids throughout the year, irrespective of the climatic and geographical barriers.