INTRODUCTION:

G. sylvestre is one of the important medicinal plant used from neolithic times. It's imperishable, woody rambler spread through in dry timber up to height of 600 m (1). It's generally known as Gurmar in Hindi. The Greek words “Gymnos” means naked and “nema” means thread while the specific description “sylvestre” is of Latin origin means “ of the timber” (2).G. sylvestre is generally circulated in India, Malaysia, Sri Lanka, Indonesia, Japan, Vietnam, tropical Africa, and South Western region of the people’s democracy of China. It's also plant in Banda, Konkan, Western Ghats, and Deccan extending to the corridor of Western and Northern India (Fig. 1).

The rubric Gymnema consists of 40 species distributed from Western Africa to Australia. Gymnema acuminatum (Roxb.) wall, Gymnema aurantiacum, Gymnema balsamicum, Gymnema elegans W and A, Gymnema lactiferum, Gymnema latifolium, Gymnema tingens W and A, Gymnema inodorum, Gymnema yunnanense, and Gymnema spartum are some of the important species of Gymnema (3,4). Ayurvedic and Homeopathic system of drug used G. sylvestre as a potent antidiabetic condiment. It also reported to be a bitter, tangy, diuretic, laxative, stomachic, liver alcohol, goad, expectorant, and antipyretic agent. The conversational names of G. sylvestre as depicted in Table 1.

Fig. 1: Leaves, flowers, and fruit of Gymnema sylvestre

Table 1: Vernacular names of Gymnema sylvestre (5,6)

Language	Common name
Hindi	Gudmar
Kannada	Kadhasige
Malayalam	Chakkarakolli, Madhunashini
Tamil	Sirukurunja/Sakkaraikkolli
Sanskrit	Jaboli
Telugu	Podapatri
English	Periploca of the wood
Marathi	Kavali, Kalikardori, Vakundi
Gujarati	Dhuleti, Mardashingi

DISTRIBUTION:

Gymnema sylvestre is distributed in whole Asia, tropical Africa, Malaysia and Srilanka. In India it grows in open wood lands and backcountry lands at an altitude of 100-1000m. It's plant in the Deccan peninsula, extending to corridor of northern and western India. It's abundantly plant in Kota quarter of Rajasthan. Some area like Mukundara Hills National Park and Garadiya Mahadeva serves as a bank for this factory.

MORPHOLOGY:

Gymnema sylvestre is a large woody twinning shrub growing hectically running over the covers of high trees in Timbers. Stem is upstanding, hard, twinning and fanned. The youthful stems, branches are smooth and spherical. Leaves are elliptic, base acute to acuminate, rough and contrary. The taste of splint is slightly bitter and tangy. It also possesses remarkable property of paralyzing the sense of the taste for sweet substances for many hours.

Flowering season:

April and November.

Fruiting season:

Downtime (December-March)

Taxonomic position:

Kingdom: Plantae

Subkingdom: Tracheobionta

Division: Magnoliophyta

Class: Magnoliopsida

Subclass: Asteridae

Order: Gentianales

Family: Asclepiadaceae

Genus: Gymnema

Species: sylvestre

Mechanism of action (Gymnemic Acid):

G. Sylvester leaves have been plant to cause hypoglycemia in laboratory creatures and shown a use in herbal drug to treat diabetes mellitus in grown-ups. When leaf extract of plant, administered to a diabetic case, there's stimulation of the pancreas by virtue of which there's an increase in insulin release. These composites have also been plant to increase fecal excretion of cholesterol (8,9). There are some possible mechanisms by which the leaves extract of G. Sylvester or (Gymnemic acid) retain its hypoglycemic acid effects are 1) It initiates regeneration of islet cells of pancreas, 2) It may increases the secretion of insulin, 3) It causes inhibition of glucose immersion from intestine, 4) It increases application of glucose as it increase the conditioning of enzymes responsible for application of glucose by insulin dependent pathways, an increase in Phosphorylase activity, drop in Gluconeogenic enzymes and Sorbitol dehydrogenase (7).

Propagation:

G. sylvestre is propagated naturally by seed germination. But at the time of release of seeds from their pods, seeds have low humidity content together with dry environment and lower endosperm affect in veritably low germination, therefore the natural product of this factory species is veritably poor. Thus, it's veritably essential to make an artificial propagation protocol to maintain the actuality of this valuable plant. To help its defunct plant tissue culture fashion is exercising at large scale.

Traditional uses:

G. Sylvestre is used in folk, Ayurvedic system to treat type 1 and 2 diabetes. It's also used in the treatment of urinary complaints, stomach problems, piles, habitual cough, breathing troubles, asthma, eye complaints, cardiopathy, constipation, jaundice, and bronchitis (10,11,12). It's also used by trials to treat to neutralize the poison of snake bite (13).

Pharmacological uses:

G. sylvestre is one of the pivotal medicinal plants, which is well known for its anti-sweetening exertion. It used by Ayurveda for the treatment of Diabetes and as well as for various conditions described below

Fig 2: Pharmacological properties of G. sylvestre

Phytochemistry screening:

A number of phytochemical ingredients have been reported by several investigators. Plant comprises two resins one answerable in alcohol, saponins, gymnemic acid, stigmasterol, quercitol and amino acid derivatives betaine, choline, and trimethylamine (14). The leaves of G. sylvestre contains resins, albumin, chlorophyll, carbohydrates, tartaric acid, formic acid, butyric acid, anthraquinone derivatives, inositol alkaloids, organic acid (5.5), paraben, calcium oxalate (7.3), lignin (4.8), and 22 of cellulose (15). The leaves also enclose triterpene classes of oleanane saponins similar as gymnemic acids, gymnemasaponins, and dammarane saponins similar as gymnemasides and terpenoids as 6-octen1-ol,-dimethyl, isophytol, squalene, nerolidol, and β-amyrin. Leaves of G. sylvestre have acidic glycosides and anthraquinones and their derivatives (16). Gymnemic acids A2 and A3 held both glucuronic acid and galactose in their molecular structures while glucuronic acid was plant to be the only half in gymnemic acid A1 (17,18). Further, a gymnemic acids series (gymnemic acid I, II, III, IV, V, VI, and VII) were insulated and characterized from the hot water extract of dry leaves of G. sylvestre (19,20). An important 35 amino acid peptide gurmarin having a molecular weight of 4209 was insulated from G. sylvestre (21).

The qualitative phytochemical analyses were carried out following the Indian pharmacopoeia and the methods described by Harborne (1973). The four different solvent extracts obtained by successive solvent extraction were tested separately for the presence of various phytoconstituents namely alkaloids, amino acids, carbohydrates, fats and fixed oils, flavanoids, glycosides, saponins, gums, lignins, proteins, steroids, triterpenoids, tannins and phenolic compounds.

The leaves of Gymnema sylvestre subjected to solvent extraction exhibited high extractive value of 3.324% in petroleum ether followed by methanol (3.048%), chloroform (2.136%) and ethyl acetate (1.648%). There was only less than 1% of extractive value (0.74%) while using water. Among various solvent used, positive response for alkaloids, triterpenoids, phenolic compounds, tannins, proteins and aminoacids, steroids and sterols, fixed oil and fat and glycosides were observed in chloroform extract. Presence of alkaloids, carbohydrate, saponin, flavanoids and glycosides were detected in methanolic extract. Similarly, alkaloid, carbohydrate, saponin and phenolic compounds were confirmed in aqueous extract. The leaf extract of ethyl acetate exhibited the presence of alkaloids, phenolic compounds, tannins, flavanoids and glycosides. Moreover, gums and mucilage were absent in all the solvent extracts. The petroleum ether extract had only tri terpenoids and steroids only. In general, alkaloids were found to be present in all the extracts except petroleum ether. The chloroform extract had majority of phytochemical constituents followed by methanolic extract. The phytocompounds alkaloids, flavanoids and glycosides were at moderate level in the leaf extracts prepared with ethyl acetate and methanol (Table 2).

Table 2: Phytochemical screening of crude leaf extracts of Gymnema sylvestre

Phytochemical Constituents
Solvent Used	Alkaloid	Carbohydrate	Saponin	Tri Terpenoid	Phenolic Compound and tannin	Protein and amino acid	Steroid and sterol	Fixed oil and fat	Flavone and flavanoid	Glycoside	Gum and Mucilage
Petroleum ether	-	-	-	+	-	-	+	+	-	-	-
Water	+	+	+	-	+	-	-	-	-	-	-
Methanol	++	+	+	-	-	-	-	-	++	++	-
Ethyl acetate	++	-	-	-	+	-	-	-	++	++	-
Chloroform	+	-	-	+	-	-	-	-	-	+	-

+++ = maximum; ++ = moderate; + = minimum; - = absent

Preparations of Gymnema sylvestre (gudmar) leaf powder:

Gymnema sylvestre (gudmar) leaf powder was prepared as per the method given by Farzana and Muhammad [2010] (22).

Fig 3: Flowchart for the preparation of Gymnema sylvestre (gudmar) leaf powder

Preparations of Gymnema sylvestre (gudmar) leaf extract:

Gymnema sylvestre leaf extract was prepared as per the method adopted by Killedar et al., [2012] (23). Leaf powder (500g) was extracted by 70 per cent ethanol extraction method with boiling on water bath for 1 hour and cooled extract was filtered through vacuum filtrations unit, evaporated to dryness on rotary film vacuum evaporator. The dried extract was kept in refrigerator for future use.

Fig 4: Flowchart for the preparation of Gymnema sylvestre (gudmar) leaf extract

Gravimetric Estimation of Gymnemic acid:

The air- dried leaf of Gymnema sylvestreR.Br variant was coarsely cut into pieces to aid the extraction and soaked in distilled water and kept for 48 hours. The obtained extract was decanted and filtered. So, latterly the extracts were concentrated over a scorching water bath on china- dishes by free evaporation. 25g of this water extract was dissolved in 10ml of 1N NaOH result. The result was acidified with needed volume of 1N HCl so as to get the precipitate. It was filtered and washed with water fully free from acid. The precipitate was dissolved in methanol and filtered. The filtrate was faded to remove the methanol and the residue was counted.

HPLC determination:

Chromatographic equipment and conditions A Waters (Milford, USA) gradient HPLC instrument equipped with two 515 pumps and controlled by an interface module PC2, manual injector valve (Rheodyne), reverse phase C18 (100 × 4.6mm i.d.) X bridge HPLC column (Waters, Milford, USA) and Waters 2996 PDA (Photo Diode Array) detector was used for HPLC analysis. Waters Empower software was used to control the equipments and for the analysis of data. The solvents were prefiltered by a Millipore filtration unit (Millipore, Billerica, MA).

The other operating conditions were as follows:

Eluent: Acetonitrile – water (80:20)

Flow-rate: 1.0ml/minute

Detector: PDA

Wavelength: 210nm

Amount injection: 20µl

Chemicals and standard solutions:

Standard gymnemagenin (98% pure) was obtained from M/s Natural Remedies, Bangalore, India. All chemicals and reagents used were of HPLC grade. The reference solution of gymnemagenin was prepared by dissolving 10 mg of standard in 5mL of 50% (v/v) aqueous methanol.

Sample preparation:

Leaves of G. sylvestre were collected from the nursery for estimation of GA. The leaf samples were prepared and anatomized according to the system of Toshihiro etal. 1994 (24). One gram of powdered leaves of G. sylvestre was counted directly into a round- bottom beaker, 50mL of 95 (v/ v) aqueous methanol was added, the admixture was refluxed on a water bath for 30 min, and also filtered. Another 50mL of 95 methanol was added; the sample was refluxed for a farther 30 min and filtered. The alcoholic extracts were combined and faded on a rotary evaporator. To the performing semi-solid, 5mL of 50 (v/ v) methanol and 1mL of potassium hydroxide result (containing 11 g/ 100 mL water) were added and the admixture was refluxed on a water bath for 1 h. The admixture was cooled and the whole result was again refluxed for 1 h after adding 0.9mL of concentrated hydrochloric acid. The performing result was cooled, acclimated to pH7.5 –8.5 with potassium hydroxide result, made up to a volume of 50mL with 50 (v/ v) methanol and filtered through Whatmann 1 sludge paper. The filtrate was subordinated to HPLC analysis. The quantum of gymnemagenin was determined from the estimation curve attained by plotting the concentration of standard against the peak area on the scrutinized chromatogram. HPLC chromatogram of standard gymnemic acid.

Trials were laid out to regularize non-destructive harvesting system of Gudmar in NWFP Nursery of the institute during 2003 – 05. In the trials, picky harvesting styles were used. All the leaves were not gathered at a time. Only mature leaves (60) were hand plucked in the month of October and youthful immature leaves were left on the plant. Alternate crop was done in the month of June. It was observed that the leaves left at the time of first crop during October progressed in June at the time of alternate crop. This system didn't affect the growth of the plant.

Thin layer chromatographic analysis:

TLC studies on alcohol, water and 50 alcohol extracts of the powdered medicines of all three samples were carried out. TLC plates were prepared by using Silica Gel-G as adsorbent. 100g Silica Gel-G was mixed with sufficient volume of distilled water to make slurry. The slurry was incontinently poured into a spreader and plates were prepared by spreading the slurry on glass plates of needed size. The consistence of the subcaste was fixed1.5 mm. Plates were allowed to state dry for one hour and subcaste was fixed by drying at 110’C for two hours. Using a micropipette, about 10µml of 1 w/ v result of extracts were loaded gradationally over the plate. The loaded plated was eluted by suitable mobile phase like TBA (t-BuOH-AcOH-H2O – 311 rate), BAW (n-BuOH – AcOH – H2O – 415 rate-Upper Phase), Forestal (AcOH – Con. HCl – H2O – 30310 rate), 60 AcOH and Water. Before elution, the tank was allowed 30 min for saturation with mobile phase. The extracts showed separation into bands. The chromatograms were observed under visible light and were mugged. The Rf value of the band was calculated by the rate between the distance traveled by the substance (cm) and the distance traveled by the mobile phase (cm).

Gas Chromatographic-Mass Spectroscopic Analysis:

The 50 ethanolic extract was examined in GC-MS for its chemical composition by GC-MS machine model, GC – Clarus 500; Perkin Elmer and Computer Mass Library (Wiley 138L) of composites with a GC column Elite – 1 (100 Methyl Poly Siloxane). The other conditions were as follows Injector GC-Clarus – 500; Perkin Elmer; Carrier gas inflow Helium 1ml/ min; Split rate – 125; Sample injected 1μl; Oven temperature – 110^oC – 2 min hold; Up to 270^oC at the rate of 5^oC/min – 4 min hold; Injector temperature 250^oC; Total GC- time 38 min; MS inlet line temperature 200^oC; Source temperature 200^oC; Electron energy 70eV; Mass Overlook 25-400; MS time 39 min.

FTIR analysis:

Dried powder of methanolic extract was used after performing KBr pelleting. The sample was loaded onto FTIR spectroscope (Shimadzu, IR Affinity Japan) in a scan range of 400 -4000 cm-1.

HPTLC Analysis:

Standard Preparation for HPTLC analysis:

A stock result of reference standard (1mg/ml) was prepared by directly importing 10mg Gymnemic acid as Gymnemagenin in 5ml of methanol, followed by vortexing and making up the total volume of result to 10ml with methanol. The above stock result was further diluted with methanol to give a working standard result of applicable concentrations ranging from0.3-0.7µg/ band.

Sample Preparation:

500mg of leaf and callus samples were extracted using 10ml of methanol water (11) and 2ml of 11 potassium hydroxide (KOH) result by refluxing for 1 hour. 1.8ml of HCl was added to extract and refluxed again for an hour. The admixture attained was also cooled to room temperature and the pH was acclimated in between 7.5 to 8.5 with 1 NaOH result. Further, the admixture was diluted to 50ml with 50 of methanol in volumetric beaker and filtered through 0.45μm nylon sludge (Millipore). The volume was made up to 50 ml with methanol and the clear supernatant was used for HPTLC analysis (25, 26).

Chromatographic conditions:

Chromatographic separation were performed on TLC aluminium precoated silica gel 60 F254 Plate (Merck). 10µl of extracts (leaf and calli) and norms were applied in triplet to the plate as 8mm band length using CAMAG Linomat 5TLC applicator equipped with 100μL syringe (Hamilton, Bonaduz, Switzerland). After the application, the linear ascending development of plate was carried out in a glass binary trough chamber (CAMAG, Switzerland) pre-saturated using ethyl acetate and methanol as mobile phase (10ml) in the rate of 56 (v/ v) for 20 minutes at room temperature. The length of chromatogram run was 8cm from the lower edge of the plate. After development, the plate was dried with the aid of hair dryer for complete junking of mobile phase. The plate was farther derivatized by dipping in Anisaldehyde-sulphuric acid reagent for 2 sec. and dried at room temperature. After drying, the plate was hotted on HPTLC Plate heater at 110⁰C for 10 sec. and print proved using print- attestation chamber (CAMAG REPROSTAR 3).

Quantitative estimation was performed using CAMAG Scanner 3 in confluence with winCATS software Interpretation 1.4.6. The plate was scrutinized under UV 366nm and visible light after derivatization. The slit dimension used was 6.0 ×0.45mm with surveying speed of 20mm/sec., throughout the analysis. Precision of retention factor (Rf) values was grounded on 10 consecutive measures. The developed and derivatized HPTLC plate and HPTLC densitogram (at 540nm) for standard and sample tracks of leaf and callus extracts are shown in Plate 1 A and B and Fig. 2 A, B and C independently.

Future aspect:

Phytochemicals account for numerous pharmacological properties. They are observed to have anti-metastatic, anti-diabetic, hypoglycemic, anti-oxidant, hepatoprotective, anti-inflammation, anti-bacterial, anti-fungal, anti-viral etc. activities. Plants contain compounds such as favonoids, alkaloids, and tannins that render these life-saving therapeutic activities. It has been reported that about 80% of people from developing countries rely on natural medicines for the treatment of diseases and their primary health concerns. However, despite having great demand and therapeutic uses only 10% of the plants have been investigated for their therapeutic potential. Furthermore, some of these plants which could be a great source of biologically important novel phytoconstituents are on the verge of extinction due to unsustainable use, destruction of forests, and habitat destruction. One of these therapeutically important plants that contain significant biologically important phytochemicals is Gymnema sylvestre. It constitutes saponins, flavonol, glycosides, gymnemanol, gurmarin etc. These phytochemicals isolated from Gymnema sylvestre can provide pharmacological activities such as anti-diabetic, anti-oxidative, anti-metastatic, anti-inflammatory, lipid-lowering and several other properties. However, this plant is also subject to unsustainable use. It is disappearing very fast due to overexploitation and extensive collection to meet the demand. Many unauthorized preparations of this plant are found in the local market. People are using this plant as a cheap substitution for their anti-diabetic medicine without any knowledge of what part of the plant to be used which results in unnecessary destruction the whole plant. Thus, this plant is being wasted without being used up to their maximum potential. In order to prevent the waste of this plant, legal production of medicinal preparation from the plant should be ensured and sustainable use of this plant should be closely monitored. In addition to these, people should be also made aware of the proper use of the plant so that they can get maximum benefit from this plant. Furthermore, in future study, the isolated principles from Gurmar needs to be evaluated in scientific manner using various innovative experimental models and clinical trials to understand its mechanism of action, in search of other active constituents, so that its other therapeutic uses can be widely explore.

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Received on 02.06.2022 Modified on 22.06.2022

Research J. Science and Tech. 2022; 14(4):261-268.

DOI: 10.52711/2349-2988.2022.00042