Phytochemical Screening and Pharmacognostic studies and Antidiabetic activity of Meshashringi (Gymnema sylvestre (Retz.) R.Br. ex. Sm.) Leaf and Stem Components

Suchitra Boxi , Haleshi C

Department of Studies in Botany, Davangere University Davanagere – 577 007, Karnataka, India

Corresponding Author Email: haleshi@rediffmail.com

DOI : https://doi.org/10.51470/ABP.2026.05.01.04

Abstract

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Introduction

Phytochemicals are bioactive substances that are derived from plants.They are referred to as secondary metabolites.^[1] Bioactive components are naturally produced by all plant parts, including the stem and leaves.^{[2] [3]} Gymnema sylvestre, another names is Gurmar, it is a Hindu word that means ‘sugar destroyer’.^[4] Where the English term “Gymnema” is originates. Gymnema sylvestre have lot of medicinal value, which is decreases blood sugar, are becoming more and more in demand in thepharmaceutical industry.^[5]Gurmar leaves are used to treat ailments due to their unique ability to block the sensation of sweet foods on the tongue and inhibit the absorption of glucose in the intestinal tract.^[6] For this reason, it is known as the sugar destroyer.^[7]GC-MS analysis has made significant breakthroughs in identifying chemical components and elucidating their structures due to its high sensitivity. Plant extracts contain numerous potential components, but only small amounts of active chemicals. Sensitive bioassays are necessary to detect these small quantities and the wide range of chemicals present. The introduction of TLC and chromatographic techniques such as GC-MS and HPLC has made analyzing these substances easier and more affordable. Gymnema sylvestre leaves, a popular herb for treating diabetes, are increasingly sought after by the pharmaceutical industry.^[8] The leaves contain gymnemic acid IV, deacyl gymnemic acid, and gymnemagenin—the plant’s main active ingredient—commonly used to treat hypercholesterolemia, diabetes, and related conditions.^[9] Additionally, the leaves possess stomachic, cough-suppressing, and diuretic properties.^[10]Glucose levels in the blood are elevated by Diabetes Mellitus, which is a common disease nowadays.^[11]Diabetes has a negative impact on various organ systems in the human body, including the heart and kidneys, and is especially damaging for those with chronic diabetes.^[12] It is also utilized by pharmaceutical companies to produce herbal medications.^[13]Study on phytochemical analysis and chemical constituents of Gymnema sylvestre.

Materials and Methods

Plant material collection

The plant material was collected from Hassan, Karnataka. The plant samplewas identified with the help of floras ^{[14] [15]} and received authentication from Herbarium of GKVK, UAS, Bangalore.

Microscopic Studies

The transverse sections of leaf and stem were stained with alcoholic safranin, and mounted in Canada balsam using the microtechnique method of Johanson.^[16]

Histochemical Tests

The following reagents were used to confirm the alkaloids ^[17].

Wagner’s Reagent

40 ml of dist. H₂O, 0.5 gm of Iodine and 1 gm of Potassium Iodide were dissolved. Formation of a reddish-brown color indicates the presence of alkaloids.

Mayer’s Reagent

40 ml of dist. H₂O, 12.45 gm of Mercury Chloride and 40 gm of Potassium Iodide were dissolved. Formation of pale yellow or white color indicates the presence of alkaloids.

Plant Leaf Extract Preparation

The collected plant material was shade-dried, powdered, and stored in a closed container for further use. The dried powdered material was subjected to continuous hot extraction using a Soxhlet apparatus with 90% methanol as solvents.^[10] This methanol extract was stored in the refrigerator for further use.^[18]

Isolation of Gymnemic acid

1% aqueous KOH solution was mixed with a crude leaf and stem extract, and the mixture was continuously stirred using a magnetic stirrer for 40 to 60 mint. Filter the extract. Diluted HCl was then slowly added to the filter solution.After 50 mint, the gymnemic acids precipitated. The concentrated solution was filtered and then left to dry at room temperature. As a result, gymnemic acid was precipitated.

Various color tests to confirm the Gymnemic acid

Phenolic test

2ml of methanol, taken in a clean test tube, then a small quantity of gymnemic acid was diluted. Addition of few drops of FeCl₃, a dark blue color was appearing, its indicated the presence of Phenol.

Glycoside test

In a test tube a pinch of gymemic acid was dissolved in a 2ml of methanol and 1ml of alpha napthol solution was added from the side of the test tube. At the junction of the two layers, a bluish red ring appeared, signifying the presence of glycoside.

Thin Layer Chromatography (TLC)

The number of chemical constituents present in the crude plant extract was determined using Thin Layer Chromatography (TLC). The analysis was carried out on pre-coated silica gel plates (grade F₂₅₄).^[19] Thin layer chromatographic examination revealed the presence of several prominent bioactive compounds in the plant extract. Separation of the chemical components of Gymnema sylvestre leaf extract was achieved using a solvent system consisting of dichloromethane, methanol, and formic acid in the ratio of 75:25:10.^[20]

GCMS Analysis

Various bioactive compounds present in the plant extract were identified using Gas Chromatography–Mass Spectrometry (GC–MS) analysis^[21]. The analysis was performed using a PerkinElmer Clarus 600 GC–MS system equipped with TurboMass™ version 5.2 software. Separation was achieved using a non-polar standard capillary column operated with a split injection mode at a ratio of 25:1. Helium was used as the carrier gas, and 1 µl of the methanolic extract was injected at an injector temperature of 220°C.The oven temperature program was initiated with a 3-mint delay at an initial temperature of 180°C, followed by a gradual increase at a rate of 7°C per mint up to 300°C, and maintained at this temperature for 15 mint. The mass spectrometer was operated in electron ionization (EI) mode at an ionization energy of 70 eV, with a scan range of 50–600 Da. Identification of the compounds present in the test sample was carried out based on their retention time, molecular weight, and mass spectral data.^[22]

HPLC Analysis of Leaf and stem Extract from Gymnema sylvestre

Quantitative Phytochemical analysis

The leaf extracts were filtered using a Sartorius RC membrane syringe filter, and an injection volume of 20 µl was used for analysis. High-performance liquid chromatography (HPLC) was performed using a Shimadzu HPLC system equipped with a Supelcosil LC-18 C18 column (250 × 4.6 mm). The mobile phase consisted of solvent A, comprising 1 mM KH₂PO₄ with 0.5 ml phosphoric acid in 1000 ml of distilled water, and solvent B, acetonitrile.The chromatographic system was operated at a back pressure of approximately 250 psi, with a flow rate of 1.0 ml/mint, and detection was carried out using a UV detector set at 210 nm. Although the total run time was 40 mint, a longer run time of 60 mintwould be preferable for improved separation of compounds. ^[23]

 Anti-Diabetic activity of Gymnema sylvestre leaf and stem extracts

α-Amylase Inhibition Assay By Starch-Iodide Method

The chosen extracts and compounds were screened for α-amylase inhibition using a slightly modified version of the Ononamadu et al. (2020) approach.The following were added in triplicate to 250 μl of each extract (62.5, 125, 250, 500, and 1000 μg/ml) in a test tube: Phosphate buffered α-amylase (250 μl, 0.05 mg/ml), starch (250 μl, 1% w/v), and 250 μl of phosphate buffer (200 mM, pH 6.9 and containing 6 mM sodium chloride) were added to the reaction mixture, which was then incubated for 15 mint at 37°C.After stopping the enzymatic reaction with one molar HCl (20 μl), 100 μl of iodine reagent (5 mM I₂ and 5 mM KI) was added.At 625 nm, the absorbance was measured and the color shift was reported. There was no test extract or chemical in the control reaction, which represented 100% enzyme activity. Acarbose was used as the benchmark.^[24]

α – Glucosidase inhibition assay

The method described by Shai et al. (2011) was slightly modified to evaluate the effect of the samples on α-glucosidase activity.Various concentrations of the sample were preincubated with 400 μl of α-glucosidase (0.067 U/ml) for 30 mint. The reaction was initiated by adding 200 μl of 3.0 mM p-nitrophenyl-α-D-glucopyranoside (pNPG) prepared in 0.1 M sodium phosphate buffer (pH 6.9).After incubation at 37°C for 30 mint, the reaction was terminated by adding 2 ml of 0.1 M Na₂CO₃.^[25]

Results

Morphology of dried plant material:

The powdered leaf and stem material of Gymnema sylvestre was yellowish green in color. The powder characteristics are shown in figure 1. Powder microscopy of leaves and stem revealed the presence of vessels, different types of stone cells, Starch cells, parenchymatous cells, palisade and spongy parenchymatous cells, fibers multicellular, unicellular trichome with upper epidermal cells.^[26]

A: Plant powder sample, B: Trichome, C: Epidermis with cortex, D: Stone cells,E: Pericycle,F:MassesofStarchgrain,G:Fibrous,H:Parenchymatous cells,I:Stone cells, J: Palisade and Spongy Parenchyma, K: Epidermis with cortex

Microscopic Studies

Transverse section of Stem:A circular outline is visible on the stem (Figure 2A), with a margin that is either entirely or partially wavy. Trichomes consist of groups of multicellular uniseriate cells. The outermost layer of the epidermis is composed of barrel-shaped or rectangular cells and is covered by a thick cuticle. The outermost portion of the cortex consists of 3–4 rows of polygonal or oval parenchymatous cells, with no distinct endodermis present. The pericycle is represented by scattered groups of stone cells with thick walls. Vascular tissues found in the stem anatomy include primary xylem, secondary xylem, interaxillary phloem, pith, cambium, and primary phloem.^[27]

Transverse section of Midrib: The midrib (Figure 2B) is oval-shaped on the abaxial side and features a small protrusion on the adaxial side. Single rows of cells on both sides of the midrib form multicellular, unbranched trichomes. The ground tissue consists of compact parenchyma. On the abaxial side, discontinuous wide strands form the vascular strand of the midrib. Radial rows of xylem elements are accompanied by numerous phloem patches located on both the upper and lower sides of the xylem strands.^[28]

Histochemical Tests

The present study only uses histochemical tests for alkaloids from different plant parts because Gymnena sylvestre is a rich source of alkaloids.Histochemistry analysis was carried out on the free-hand sections of G. Sylvestre leaf and stem using different reagents. Alkaloids were detected using a variety of reagents, includingwagner and mayer reagent. Alkaloids were recognized by using mayer reagent formation oflight yellow or cream in color. The presence of alkaloids was shown by the reddish-brownprecipitate produced by Wagner’s reagent.^[29]

Biochemical test

The outcomes of performing the phenolic test, which is the +ve test that indicates there is a -OH group in the molecule, resulted in the formation of a dark blue color. To confirm theglycosidic nature a blue-red ring was made.

Thin Layer Chromatography

Thin layer Chromatographic analysis demonstrated that the plant extract included a variety of strong biomolecules. The methanolic extract showed several spots of Deacyl Gymnemic acid, Gymnemagenin, and Gymnemic acid IV with Rf values of 0.51, 0.9, and 0.79 shown in Figure 3A. In a less polar system, the polarity of compounds with a high Rf value is low, and vice versa, according to TLC analysis, which gives an indication of the polarity of different chemical ingredients.^[30] Using a Dichloromethane: Methanol: Formic acid ratio of 75:25:10, An Rf value of 0.79 was discovered by Balamurali Krishna et al. (2012) in their TLC analysis of gymnemic acid from Gymnema sylvestre.^[31] The chromatogram was visualized under UV light (Normal light,366 nm, 254 nm, whight light) Figure 3B.

GC-MS Study

The methanolic extracts of Gymnema sylvestre obtained from both leaf and stem explants were analyzed for the presence of bioactive compounds using Gas Chromatography–Mass Spectrometry (GC–MS) in Figure 4. The identified active principles, along with their retention time (RT), molecular weight (MW), molecular structure, and percentage peak area, are Table 1.GC–MS analysis revealed that the methanolic extract of G. sylvestre contained a total of eleven distinct compounds, predominantly belonging to the classes of terpenes and both saturated and unsaturated fatty acids. Among the identified compounds, 9-octadecenoic acid (Z)- and its methyl ester were detected as major constituents, consistent with the findings reported by Parimala Devi.^[32]

HPLC Study:

The active principles extracted from the dried leaves were subjected to HPLC analysis and separated into three distinct fractions based on their retention times, in comparison with standard compounds, as described by Shimizu et al. (1997). Methanol was used as the extraction solvent for isolating the chemical constituents from Gymnema sylvestre.The results presented in Table 2 and Figure 5 illustrate the percentage composition of deacyl gymnemic acid, gymnemagenin, and gymnemic acid IV identified in the methanolic leaf extract.^[33]

Anti-Diabetic  activity Gymnema sylvestre leaf and stem extract

α-Amylase Inhibition Assay By Starch-Iodide Method

The anti-diabetic activity of the standard drug acarbose and the test sample was evaluated using the starch–iodide method, and the results are presented in Table 3 and Figure 6. Acarbose exhibited a concentration-dependent increase in inhibitory activity, with percentage inhibition rising from 21.92 ± 0.0882% at 62.5 µg/ml to 80.36 ± 0.0493% at 1000 µg/ml. Correspondingly, the optical density values decreased with increasing concentration, indicating effective inhibition of enzymatic activity.Similarly, the test sample demonstrated significant anti-diabetic activity in a dose-dependent manner. The percentage inhibition increased from 42.35 ± 0.0467% at 62.5 µg/ml to 77.94 ± 0.0874% at 1000 µg/ml, with a concomitant reduction in optical density values. Notably, the sample showed higher inhibitory activity than acarbose at lower concentrations (62.5–250 µg/ml). However, at higher concentrations (500 and 1000 µg/ml), acarbose exhibited slightly greater inhibition compared to the sample.^[34]

Table 3: α-Amylase  Inhibition Assay

α – Glucosidase inhibition assay

The α-glucosidase inhibitory activity of the standard drug acarbose and the test sample was evaluated, and the results are presented in Table 4 and Figure 7. Acarbose exhibited a strongconcentration-dependent inhibitory effect, with percentage inhibition increasing from 20.39 ± 0.07% at 62.5 µg/ml to 96.69 ± 0.073% at 1000 µg/ml. This increase in inhibition was accompanied by a corresponding decrease in optical density values, indicating effective suppression of α-glucosidase activity.The test sample also demonstrated significant α-glucosidase inhibition in a dose-dependent manner. The percentage inhibition increased from 58.68 ± 0.0665% at 62.5 µg/ml to 95.59 ± 0.056% at 1000 µg/ml, with a marked reduction in optical density values as the concentration increased. Notably, the sample showed substantially higher inhibitory activity than acarbose at lower concentrations (62.5 and 125 µg/ml). At higher concentrations (500 and 1000 µg/ml), the inhibitory activity of the sample was comparable to that of the standard drug. ^[35]

Discussion

The present study histochemical tests are restricted to only histochemistry of alkaloids in different plant parts. Gymnema sylvestre leaf and stem free-hand sections were subjected to various reagent treatments in order to perform histochemical analysis. Several reagents were used to detect alkaloids, including Mayer’s, which produced a cream or pale yellow color indicating the presence of alkaloids, and Wagner’s, which produced reddish-brown precipitate indicating the presence of alkaloids. The outcomes of the phenolic test, which showed that the molecule had an OH group. At the intersection of the two layers, a bluish red ring formed, signifying the presence of glycoside.Powder anatomy of Gymnema sylvestre  revealed epidermal fragments with multicellular hairs. A few mesophyll and palisade parenchyma cells were observed, with the hairs being uniseriate and smooth. Glandular hairs were also present within the epidermal cells. The powder anatomy showed abundant starch grains, simple to compound round-shaped stone cells, non-lignified fibers, and vessels with cortical walls.Examine the TLC plate under UV light at 254 nm and 366 nm. Then, observe the plate under white light after dipping it in vanillin–H₂SO₄ reagent and heating it at 105°C ± 5°C for approximately 10 mint. The methanolic extract exhibited several spots corresponding to Deacyl Gymnemic acid, Gymnemagenin, and Gymnemic acid IV, with Rf values of 0.51, 0.90, and 0.79, respectively.A total of 11 compounds had beenidentified from the methanolic extract of G.sylvestrethrough GC-MS analysis. G. sylvestre methanolic extract included different compounds, the main constituents of Gymnema sylvestre are  Propanoic acid,4-(1-Aminoethyl)phenol,2-Methoxy-4-vinylphenol.Among these, deacyl gymnemic acid was found to be the predominant compound, accounting for 4.45% (w/w) of the extract. The gymnemagenin content was comparatively lower at 0.32% (w/w). gymnemagenin, an aglycone moiety of gymnemic acids IV, plays a crucial role in the biological activity of these compounds, particularly in modulating glucose uptake and inhibiting intestinal glucose absorption. The Gymnemic acid IV content was found to be the lowest among the three quantified compounds, at 0.17% (w/w). Alpha-amylase and alpha-glucosidase these enzymes are capable of breaking down carbohydrate into glucose, which can then be further absorbed by the body. This suggests that the bioactive constituents in the sample effectively inhibit α-amylase, there by potentially reducing the breakdown of starch into glucose.^[32]In the α-glucosidase inhibition assay, a similar concentration-dependent trend was observed, with significantly higher inhibition values. The greater inhibition observed in this assay compared to α-amylase suggests that the sample is more effective against α-glucosidase, which plays a key role in the final step of carbohydrate digestion.

Conclusion

According to the current study, using 90% methanol in a continuous hot extraction process with a Soxhlet apparatus yielded significant results, produced the maximum yield of extraction. The identification and quantification of phytoconstituents present in plant materials require analysis of Thin-Layer Chromatography (TLC).The resulting plant extract can be subjected to comprehensive analysis using gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC) to accurately identify, quantify, and characterize the diverse chemical constituents present. The identification of these 11 compounds through GC-MS with significant components. Chemical constituents can be analyzed by using HPLC techniques since they are proven to be accurate, exact, and time-efficient. The starch-iodide method confirmed that the test sample effectively inhibits both α-amylase and α-glucosidase activities in a concentration-dependent manner. The findings support the use of this sample as a natural therapeutic agent for diabetes management, especially in controlling postprandial blood glucose levels.

Limitation of study

The study has been carried out in the species of Gymnema sylvestre derived from the habitat of Karnataka, India.

Acknowledgement

The Himalaya Wellness Company in Bangalore is acknowledged by the authors for providing the facilities needed to conduct the research. Auther also thankful to Davangere University, Davangere, for provided all facility.

Financial support and sponsor

Nil

Conflicts of interest

There are no conflicts of interest.

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