Biotechnological approaches for the production of gymnemic acid from Gymnema sylvestre R. Br.

Diabetes is a chronic disease that affects several organs and can be treated using phytochemicals found in medicinal plants. Gymnema sylvestre (Asclepiadaceae) is one such medicinal plant rich in anti-diabetic properties. The plant is commonly known as madhunashini in Sanskrit because of its ability to cure diabetes (sugar). Gymnemic acid (GA) is a phytochemical (a triterpenoid saponin) responsible for the herb's main pharmacological activity. This secondary metabolite has a lot of potential as a phytochemical with pharmacological properties including nephroprotection, hypoglycemia, antioxidant, antimicrobial, and anti-inflammatory. Gymnema has acquired a lot of popularity in recent years due to its low side effects and high efficacy in healing diabetes, which has led to overexploitation by pharmaceutical enterprises for its biomass in the wild for the purification of gymnemic acid. Modern biotechnological techniques involving the establishment of cell and organ cultures from G. sylvestre will assist us in fulfilling the need for gymnemic acid production. The present review provides insights on the establishment of cell and organ cultures for the production of a potent antidiabetic molecule gymnemic acid. Further, the review also delves into the intricacies of the different strategies for improved production of gymnemic acid using various elicitors. There is huge potential for sustainable production of gymnemic acid which could be met by establishment of bioreactor scale production. Understanding and engineering the biosynthetic pathway could also lead to improved GA production. KEY POINTS: • Gymnemic acid is one of the potential anti-diabetic molecules from madhunashini • Cell and organ culture offers potential approach for gymnemic acid production • Elicitation strategies have improved the gymnemic acid production.

Anti-inflammatory and antioxidant activities of Gymnema Sylvestre extract rescue acute respiratory distress syndrome in rats via modulating the NF-κB/MAPK pathway.

Acute respiratory distress syndrome (ARDS) is one of the major causes of mortality in COVID-19 patients, due to limited therapeutic options. This prompted us to explore natural sources to mitigate this condition. Gymnema Sylvestre (GS) is an ancient medicinal plant known to have various therapeutic effects. This investigation examined the therapeutic effect of hydroalcoholic extract of Gymnema Sylvestre (HAEGS) against lipopolysaccharide (LPS)-induced lung injury and ARDS in in vitro and in vivo models. UHPLC-HRMS/GC-MS was employed for characterizing the HAEGS and identified several active derivatives including gymnemic acid, gymnemasaponins, gymnemoside, gymnemasin, quercetin, and long fatty acids. Gene expression by RT-qPCR and DCFDA analysis by flow cytometry revealed that several inflammatory cytokine/chemokine, cell injury markers, and reactive oxygen species (ROS) levels were highly upregulated in LPS control and were significantly reduced upon HAEGS treatment. Consistent with the in vitro studies, we found that in LPS-induced ARDS model, pre-treatment with HAEGS significantly suppressed the LPS-induced elevation of inflammatory cell infiltrations, cytokine/chemokine marker expression, ROS levels, and lung injury in a dose-dependent manner. Further mechanistic studies demonstrated that HAEGS suppressed oxidative stress by modulating the NRF2 pathway and ameliorated the ARDS through the NF-κB/MAPK signalling pathway. Additional fractionation results revealed that fraction 6 which has the exclusive composition of gymnemic acid derivatives showed better anti-inflammatory effects (inhibition of IL-6 and IL-1ß) at lower concentrations compared to HAEGS. Overall, HAEGS significantly mitigated LPS-induced lung injury and ARDS by targeting the NF-κB/MAPK signalling pathway. Thus, our work unravels the protective role of HAEGS for the first time in managing ARDS.

Radio-protective efficacy of Gymnema sylvestre on Pangasius sutchi against gamma (60Co) irradiation.

PURPOSE: Freshwater fish Pangasius sutchi was used in this study as a vertebrate model. We evaluated the induction of certain antioxidant enzymes in various vital organs. The radioprotective efficacy of Gymnema sylvestre leaves extract (GS) [25 mg/kg Body Weight (B.W)] and its bioactive compound Gymnemagenin (GG) [0.3 mg/kg B.W] was compared with Amifostine (Ami), the only radioprotector clinically approved by the US-FDA [Ami- 83.3 mg/kg B.W] against different doses of gamma radiation - 60Co (Lethal Dose: LD30-9.2 Gy, LD50-10.2 Gy and LD70-11.4 Gy). MATERIALS AND METHODS: This study was done via stress marker enzymes, cell cycle analysis (CCA) and DNA damage assay prediction with molecular docking, which are reported here for the first time. The results indicate an elevated LPO level and decreased level of CAT, SOD and GSH due to oxidative stress initiation by 60Co Ionizing Radiation (IR) on 4th day and slightly reduced on 32nd day while the reverse observed when the fishes were pretreated with Ami, GS and GG. Similarly, CCA and dead/live cells counts were conducted with pretreatment of Ami, GS and GG against 60Co IR dose (LD50-10.2 Gy). RESULTS: In CCA, G0/G1 phase was observed to be the highest in Ami and lowest in GG, against 60Co IR doses 10.2 Gy which was 51.76 ± 7.55. The dead cells range observed in pretreated group of Ami, GS and GG was lowest in Ami and highest in GG and live cells (highest in Ami and lowest in GG) as compared to 60Co IR group (86.43 ± 3.42 and 8.77 ± 5.95). Thus, antioxidant profile improvement by oxidative stress reduction and gradual progression of different phases of cell cycle except the apoptotic phase along with the live cells counts indicates that the radio-protective efficacy of GS is similar to Ami. CONCLUSION: Predictive assessment was carried out by docking of Ami, various components of GS with p53, NF-κß cells and Rad51 proteins structures responsible for CCA, apoptosis and repair mechanism. These structural proteins were docked with other structural proteins like USP7, TNF-α and partner and localizer of BRCA2 associated (PALB2/BRCA2) complex which made us perform these systemic efforts to find the functional activity of these known radio-protectants.

Development of a Building Block Strategy to Target the Classification, Identification, and Metabolite Profiling of Oleanane Triterpenoids in Gymnema sylvestre Using UHPLC-qTOF/MS.

The major class of bioactive metabolites in Gymnema sylvestre, a popular Ayurvedic medicinal plant for the treatment of diabetes mellitus, is oleanane triterpenoids. In this study, a targeted, biosynthesis-inspired approach using UHPLC-qTOF/MS was implemented to elucidate the whole chemical profile of this plant for the standardization of the Vietnamese G. sylvestre variety. The known compounds were first determined to identify the building blocks of the biosynthetic intermediates and the construction rules for synthesizing oleanane triterpenoids in the plant. These blocks were recombined to build a virtual library of all reasonable compounds consistent with the deduced construction rules. Various techniques, including relative mass defect filtering, multiple key ion analysis, mass fragmentation analysis, and comparison with standard references, were applied to determine the presence of these predicted compounds. Conventional isolation and structure elucidation of six of the new compounds were carried out to identify the new building blocks and validate the assignments. Consequently, 119 peaks were quickly assigned to oleanane triterpenoids, and among them, 77 peaks were predicted to be new compounds based on their molecular formulas and mass fragmentation patterns. All the identified metabolites were then classified into different layers to analyze their logical relationships, and a multilayered chemical profile of the oleanane triterpenoids was constructed. This new approach is expected to be practical for characterizing structures of modular secondary metabolites, such as triterpenoid saponins, and for proposing biosynthetic relationships among compounds of the same class of metabolites in medicinal plants.

De novo transcriptome of Gymnema sylvestre identified putative lncRNA and genes regulating terpenoid biosynthesis pathway.

Gymnema sylvestre is a highly valuable medicinal plant in traditional Indian system of medicine and used in many polyherbal formulations especially in treating diabetes. However, the lack of genomic resources has impeded its research at molecular level. The present study investigated functional gene profile of G. sylvestre via RNA sequencing technology. The de novo assembly of 88.9 million high quality reads yielded 23,126 unigenes, of which 18116 were annotated against databases such as NCBI nr database, gene ontology (GO), KEGG, Pfam, CDD, PlantTFcat, UniProt & GreeNC. Total 808 unigenes mapped to 78 different Transcription Factor families, whereas 39 unigenes assigned to CYP450 and 111 unigenes coding for enzymes involved in the biosynthesis of terpenoids including transcripts for synthesis of important compounds like Vitamin E, beta-amyrin and squalene. Among them, presence of six important enzyme coding transcripts were validated using qRT-PCR, which showed high expression of enzymes involved in methyl-erythritol phosphate (MEP) pathway. This study also revealed 1428 simple sequence repeats (SSRs), which may aid in molecular breeding studies. Besides this, 8 putative long non-coding RNAs (lncRNAs) were predicted from un-annotated sequences, which may hold key role in regulation of essential biological processes in G. sylvestre. The study provides an opportunity for future functional genomic studies and to uncover functions of the lncRNAs in G. sylvestre.

In vivo pharmacokinetic interaction by ethanolic extract of Gymnema sylvestre with CYP2C9 (Tolbutamide), CYP3A4 (Amlodipine) and CYP1A2 (Phenacetin) in rats.

Gymnema sylvestre (GS) is a medicinal herb used for diabetes mellitus (DM). Herbs are gaining popularity as medicines in DM for its safety purpose. The aim of the present study was to evaluate in vivo pharmacokinetic (PK) interaction between allopathic drugs tolbutamide (TOLBU), amlodipine (AMLO), and phenacetin (PHENA) at low (L) and high (H) doses with ethanolic extract (EL) from GS. EL was extracted and subjected to TLC, total triterpenoid content (19.76 ± 0.02 W/W) and sterol content (0.1837 ± 0.0046 W/W) estimation followed by identification of phytoconstituents using HRLC-MS and GC-MS. PK interaction study with CYP2C9, CYP3A4 and CYP1A2 enzymes were assessed using TOLBU, AMLO and PHENA respectively to index cytochrome (CYP) mediated interaction in rats after concomitant administration of EL extract (400 mg/kg) from GS for 7 days. The rats were divided into four groups for each PK study where, group I and II were positive control for low and high dose of test drugs (CYP substrates) while group II and IV were orally administered EL. The PK study result of PHENA indicated that area under the plasma concentration-time curve (AUC0-24) was significantly (P < 0.0001) increased by 1.4 (L) and 1.3-fold (H), plasma concentration (Cmax) was significantly (P < 0.001) increased by 1.6 (L) and 1.4-fold (H). Whereas for TOLBU; clearance rate (CL) was significantly (P < 0.0001) decreased by 2.4 (L) and 2.3-fold (H), Cmax, was significantly (P < 0.001) decreased by 26.5% (L) and 50.4% (H) and AUC0-24 was significantly (P < 0.0001) decreased by 59.8% (L) and 57.5% (H). Thus, EL is seen to be interacting with CYP1A2 by inhibiting its metabolic activity. HRLC-MS and GC-MS helped identify the presence of gymnemic acid (GA), triterpenoids and steroids in EL which could be the reason for PK interaction of CYP1A2 and CYP2C9. Also, in silico structure based site of metabolism study showed Fe accessibility and intrinsic activity for GA-IV, GA-VI, GA-VII and GA-X with CYP2C9. PK parameters of AMLO were not significantly affected by pre-treatment of EL. Thereby our findings indicate that co-administration of GS with drugs that are metabolized by CYP2C9 and CYP1A2 could lead to potential HDI.

Production of Gymnemic Acid from Cell Suspension Cultures of Gymnema sylvestre.

Gymnema sylvestre R. Br. is a popular herbal medicine. It has been used in ayurvedic system of medicine for thousands of years. It is popularly called as "Gur-mar" for its distinctive property of temporarily destroying the taste of sweetness and is used in the treatment of diabetes. The leaves of gymnema possess antidiabetic, antimicrobial, anti-hypercholesterolemic, anti-sweetener, anti-inflammatory, and hepatoprotective properties and have traditional uses in the treatment of asthma, eye complaints, and snake bite. The leaves contain triterpene saponins such as gymnemic acid which is an active ingredient of Gymnema. Since the cultivation of G. sylvestre is a very slow process and the content of gymnemic acid depends on the environmental factors, cell suspension culture is sought as an alternative means for the production of Gymnema biomass and to enhance the gymnemic acid content. In this chapter, the methods employed for the induction of callus and subsequent establishment of cell suspension cultures for the production of biomass and analysis of gymnemic acid using high performance liquid chromatography are described.

Anti-obesity effect of milk fermented by Lactobacillus plantarum NCDC 625 alone and in combination with herbs on high fat diet fed C57BL/6J mice.

The effect of dietary supplementation of milk fermented with indigenous probiotic Lactobacillus plantarum (LP625) alone and in combination with herbs (Aloe vera and Gymnema sylvestre) was investigated on high fat diet (HFD, 60 kcal% fat) fed mice for 12 weeks. Administration of LP625 alone or in combination with both herbs lowered the final body weight, however, a significant difference was observed with LP625 supplemented Gymnema sylvestre only as compared to the HFD fed group (25.06±0.18 vs 27.29±0.72 g, P<0.05). Similarly, the epididymal fat mass, fasting blood glucose and serum insulin levels were significantly (P<0.05) decreased by all treatment groups. In addition, a protective effect against the rise in serum and liver triglycerides, and in liver total cholesterol levels was found with the consumption of LP625 alone or in combination with herbs. Furthermore, the HFD fed mice showed a remarkable increase in the epididymal fat cell size, whereas administration of LP625 alone or in combination with herbs exhibited a significant decrease in the size. Finally, a significant increase in the relative mRNA expression of thermogenic proteins, i.e. uncoupling protein-2 (UCP-2, 1.16±0.25 fold change, P<0.05) and a decrease in pro-inflammatory markers, such as tumour necrosis factor-α and interleukin-6 (1.55±0.18 and 3.10±0.58 fold change, respectively, P<0.05) were due to LP625 supplementation in the HFD fed group. This shows that LP625 alone or supplemented with herbs seems to protect against diet induced obesity by decreasing the body and epididymal fat weight through upregulation of UCP-2 expression and reduced expression of pro-inflammatory cytokines.

Influence of milk fermented with Lactobacillus rhamnosus NCDC 17 alone and in combination with herbal ingredients on diet induced adiposity and related gene expression in C57BL/6J mice.

Obesity has become a major health problem in developed countries and is rapidly catching up in the developing world due to changes in their life style. Dietary incorporation of functional foods, including probiotic fermented milk and herbal ingredients, is being tried to ameliorate metabolic disorders. In the present study, the effect of dietary supplementation of a probiotic (Lactobacillus rhamnosus NCDC 17) fermented milk alone or either of the herbal preparations (Aloe vera/Gymnema sylvestre powders, 1% w/w) on the progression of obesity has been studied in C57BL/6J mice fed with a high fat diet for 12 weeks. At the end of the experimental period, oral administration of L. rhamnosus and herbs resulted in a significant decrease in the body weight, epididymal fat mass, fasting blood glucose and serum insulin levels. Supplementation of the probiotic L. rhamnosus alone and in combination with herbs showed a significant decrease in the adipocyte cell size and an increase in the number. Finally, obesity related adipokines levels were maintained at normal by the treatment groups. Thus, dietary intervention of milk fermented with probiotic L. rhamnosus alone or in combination with any of the herbal preparations seems to show anti-obesity and anti-inflammatory properties.

Triterpenoids from Gymnema sylvestre and their pharmacological activities.

Because plants are estimated to produce over 200,000 metabolites, research into new natural substances that can be used in the pharmaceutical, agrochemical and agro-industrial production of drugs, biopesticides and food additives has grown in recent years. The global market for plant-derived drugs over the last decade has been estimated to be approximately 30.69 billion USD. A relevant specific example of a plant that is very interesting for its numerous pharmacological properties, which include antidiabetic, anticarcinogenic, and neuroprotective effects is Gymnema sylvestre, used as a medicinal plant in Asia for thousands of years. Its properties are attributed to triterpenoidic saponins. In light of the considerable interest generated in the chemistry and pharmacological properties of G. sylvestre triterpenes and their analogues, we have undertaken this review in an effort to summarise the available literature on these promising bioactive natural products. The review will detail studies on the isolation, chemistry and bioactivity of the triterpenoids, which are presented in the tables. In particular the triterpenoids oxidised at C-23; their isolation, distribution in different parts of the plant, and their NMR spectral data; their names and physico-chemical characterisation; and the biological properties associated with these compounds, with a focus on their potential chemotherapeutic applications.

In vivo and in vitro evaluation of sterols from Gymnema sylvestrte R. Br.

Gymnema sylvestre R. Br. is an important medicinal plant known for its antidiabetic potential. In the present study, phytosterols from G. sylvestre was identified and quantified in vivo and in vitro. Maximum callus induction was observed in MS medium supplemented with 0.5 mg L(-1) of 2, 4-D. The protein content was significantly high both in aerial plant parts and callus tissue. Phytosterols were identified using chromatographic and spectral studies. beta-sitosterol, campesterol and stigmasterol were identified both in vivo and in vitro. Lanosterol was identified only in callus culture. Phytosterols have reported for the first time in callus culture of G. sylvestre.

Abiotic elicitation of gymnemic acid in the suspension cultures of Gymnema sylvestre.

Elicitation is one of the few strategies that find commercial application in the enhancement of secondary metabolite production from plants as well as cell culture systems. Due to their immense medicinal value, production of saponins in suspension cultures has been attempted by many researchers. Gymnema sylvestre is a rich source of gymnemic acids (saponins) that find application in the treatment of diabetes. The present study is an attempt to evaluate the effect of various metal salts (cadmium chloride, mercuric chloride, silver nitrate, cupric chloride, cobaltous chloride and calcium chloride) in eliciting the response from G. sylvestre suspension cultures. The maximum gymnemic acid production in the suspensions was achieved on day 12 of culture, though the maximum biomass was obtained on day 16. Among the different salts, CdCl(2) gave maximum response (59.97 mg/gDCW) at 2 mM concentration after a 24 h time period, while, AgNO(3) gave the least response (18.35 mg/gDCW) on incubation of 48 h at 1 mM concentration, in terms of gymnemic acid accumulation. The accumulation of gymnemic acid was found to be dependent on treatment time and concentration of the elicitor. The enhanced gymnemic acid production shown by the suspensions in response to the metal salts indicates their role in evoking the plant defense mechanisms. These elicitation studies help in providing a platform for improved commercial supply of bioactive gymnemic acids.

In vitro production of gymnemic acid from Gymnema sylvestre (Retz) R. Br. ex roemer and schultes through callus culture under abiotic stress conditions.

Plant secondary metabolites have enormous potential for research and new drug development. Many secondary metabolites have a complex and unique structure and their production is often enhanced by biotic and abiotic stress conditions. Gymnemic acid (C(43)H(68)O(14)), a pentacyclic triterpenoid isolated from the leaves of Gymnema sylvestre, exhibits potent inhibitory effect on diabetes. The gymnemic acid content is determined by chromatographic methods: Camag HPTLC system equipped with a sample applicator Linomat IV and TLC scanner and integration software CAT 4.0. In HPLC C(18) (ODS) reverse phase column; water 486 UV detector; mobile phase, water/methanol (35:65, HPLC grade) + 0.1% acetic acid are used. Sample (20 microL) is applied with a flow rate of 1 mL/min and read at 230 nm with UV detector. The production of gymnemic acid is significantly higher in callus treated with 2,4-dichloro phenoxy acetic acid (2,4-D) and kinetin (KN). The blue light increases gymnemic acid accumulation upto 4.4-fold as compared with fluorescent light treatment and out of which 2.8 is found in leaves. Gymnemic acid is isolated from callus, grown under stress conditions followed by preparative TLC, simple and reproducible character based on HPTLC and high performance liquid chromatography.