Nature’s Sugar Destroyer: Phytochemical Diversity And Free Radical Scavenging Efficacy Of Gymnema Sylvestre Sourced From Valsad, Gujarat

Since the beginning of human history, Medicinal plants have been used for therapeutic purposes (James & alewo,2014). Gymnema sylvestre also referred to as “Meshasringi”, is one such medicinal plant (khan et al., 2020). About 40 species make up the genus with G. sylvestre, G.montanum, G.yunnanense, G.inodorum, being particularly valued for their medicinal properties (Tiwari et al., 2020). Gymnema sylvestre is commonly known as “Gurmar” in local language. This herb is significant in India. It is widely found in Konkan, The Western Ghats, the deccan peninsula, and in Western and Northern India (Neel et al., 2025).

Table 1: Taxonomical Classification (Srivastava et al., 2019)

Vernacular Names:

Sanskrit : Meshashringi, Madhunashini, Vishani-vishanika, Meshavalli, Tiktadugdha, Varttika, Cakshushya

Hindi : Medhaa singee OR Merasingi, Gudmaar (“gurmar” comes from 2 Hindi words- “gur”, meaning sugar, and “mar”, meaning to destroy. When combined “gurmar” literally means “the sugar killer”.)

English: Periploca of the woods, Australian cow plant, Cow plant.

Gujarati: Mardashingi, Dhuleti

Marathi: Vakundi, Kavali, Kalikardori

Telugu:  Podapatri

Tamil: Adigam, Cherukurinja

Kannada: Sannagerasehambu, Kadhasige

Malayalam: Cakkarakkolli, Madhunashini

Bengali: Mera-singi

Oriya: Gudmari  (Triveni et al., 2012); (Sagar et al., 2015).

The word Gymnema comes from Greek where “gymnos” means naked and “nema” means thread.  Then sylvestre is Latin means “of the forest” (Damor & Polaiah, 2022). Gymnema sylvestre is commonly known as the “destroyer of sugar” as a traditional name because by chewing it’s leaves it will destroy the flavour of sweetness (Chowdhary & Rasool, 2010).

Gymnema sylvestre was put in consideration as one of the most significant botanicals to cure diabetes in the Ayurvedic. Gymnema sylvestre has been used in India to treat diabetes for over 2,000 years (Padmapriya et al., 2010).  Also is a part of Indian Pharmacopoeia as well as a system of medicine as an anti-diabetic plant. One of the ancient books on medicine, Shushruta, mentions Gymnema sylvestre as a curative agent in glycosuria and urinary disorder. It is a curing herb possessing several possibilities as stated by folk medicine, Ayurveda and Homeopathic Systems of medicine (Das et al., 2022).

It is a natural plant that thrives in the open woodland of India, China, Indonesia, Japan, Malaysia, Sri Lanka, Vietnam, and South Africa (EI Shafey et al., 2013). It is found in the forests of Madhya Pradesh, Andhra Pradesh, Kerela, Tamil Nadu, Uttar Pradesh, West Bengal, Bihar, and Chhattisgarh in India. The plant’s high demand in Asia is causing it to become rare and be widely grown in southern Indian States, Particularly Tamil Nadu (Devi et al., 2021)

It is a large woody climber that can grow between 100 and 1000 meters. The simple, green, oval, or elliptical leaves measure 2 to 6 cm long and 1 to 4 cm wide. They usually have a rounded or heart- shaped base, are petiolate, have a smooth edge, and are hairless on both surfaces. The leaves are opposite and have a pointed tip, with a network of veins. They also feature an astringent, slightly bitter taste and a distinct smell (Srivastava et al., 2019).

The compound leaves are a distinctive light, bright green colour and have a feathery appearance. Each of their three- to five-centimetre-long leaves has two to four pairs of primary leaflets, or pinnae, which are further subdivided into one or two pairs of secondary leaflets, or pinnules. This makes them doubly pinnate (Hajare, 2018).

Flowers are greenish-yellow and arranged in spirals within lateral corymbose cymes. The calyx is covered in fine hair and is divided at the base. The corolla is bell-shaped. The corona has projections with a lying-down base. It is surrounded by two rows of short, stiff hairs, which are also fringed. The cymose gynostegium does not have a corona (Prakash, 2018)

The recent clinical trials have been performed in India and demonstrated that extract of Gymnema sylvestre can be used to control Blood Sugar. Gymnema sylvestre has found

popularity in the United States today as an adjunct therapy in diabetes (Srinivasan et al., 2019). Additionally, it is used to treat snakebite, family planning, inflammations, eye problems, asthma, and cough. Furthermore, it has hepatoprotective, stomachic, diuretic, antimicrobial, anti-hypercholesteraemic, and anti-saccharine properties (Chowdhary & Rasool, 2010). It can be found in Ayurvedic formulations like Mritasanjivani Sura, Nyagrodhadi Churna, Ayaskriti, and Mahavisagarbha Taila. Diabetes, inflammation, eye conditions, asthma, and dental cavities are all treated with these formulations.

In Japan, Gymnema teas and Gymnema chewing gum are made from G. sylvestre leaves. They are promoted as a natural way to control obesity, boost insulin secretion through the regeneration of pancreatic beta cells, and reduce sweet cravings on the tongue. The juice from the root is used to treat vomiting and dysentery. A paste made from the plant, combined with mother’s milk, is applied to treat mouth ulcers. Gymnema preparations have shown anti-allergic activity (Chavda, 2015).

Many phytochemicals, including terpenoids, saponins, gymnemic acids, and gymnema saponins, are found in G. sylvestre leaves. Gymnemic acid, the active ingredient, was isolated from leaves and is frequently used as an anti-sweetener, anti-hypercholesterolemia, and antidiabetic (Entooru et al., 2021). sylvestre stems contain a number of chemical components with therapeutic significance, such as stigmasterol and triterpenoid saponin. Gymnema root powder has also been used to treat liver diseases, constipation, stomach issues, snake bites, and water retention. In traditional medical systems, it is also used to treat infections (Saumendu, 2010). Gymnema sylvestre is a promising candidate for evaluating its anti-oxidant and anti-microbial properties, which may benefit diabetes patients (Gunasekaran et al., 2019).

Also, there are various products, dietary supplements, and health food available in the market that are based on Gymnema in different parts of Asia, Europe and America (Damor & Polaiah, 2022).

MATERIALS AND METHODOLOGY

Plant Collection

The whole plant material (panchang) of Gymnema sylvestre (Asclepiadaceae) was collected from Village Marla of district Valsad, Gujarat, India. The collected material was washed thoroughly with tap water, after that the material was dried at room temperature for 6-7 days, after drying the dried plant material then mechanically ground into a fine powder using a mixer grinder. Approximately 150 gm of the homogeneous powder was packaged in an airtight zip-lock bag and stored under ambient condition to maintain stability until further use or analysis.

Plant Extract Preparation

The plant extract was prepared using the cold maceration method. Three different solvents were used for the extraction process which are Methanol, Ethanol and Water For each solvent, 10g of powdered plant material was soaked in 200 mL of the respective solvent. The mixture stood for 72 hours (3 days) at room temperature. During this time, the mixture was shaken gently 1 to 2 times a day to help with extraction. After 72 hours, the extract was filtered through Whatman filter paper. The liquid that passed through was collected into a petri dish and was allowed to evaporate completely under fan over a period of 2-3 days. Once fully dried, the petri plates were stored in laboratory refrigerator for the further process. The percentage yield was calculated by the standard formula-

Yield (%) = Weight of dry extractWeight  of plant powder

Qualitative Analysis (Phytochemical Screening)

• Test For Alkaloids

1) Dragendroff’s Test: Take 1ml extract and 2ml Dragendroff’s reagent, the formation of orange Precipitate indicates the presence of alkaloids.

• Test For Carbohydrate

1. Iodine Test: Take 1 ml extract and add 2-3 drops of iodine solution, The formation of Blue-Black coloured complex indicates the presence of Carbohydrate.

• Test For Glycosides

1)  Keller- Kelani Test: Take some amount of extract and add 1ml G.A.A., + 2% fecl3 solution (2drops) – pour into test tube with 1ml conc. H₂SO₄, 2 Layers formed Upper-red brown Lower-blue green indicates presence of Glycosides.

• Test For Proteins

1. Millon’s Test: Take 1ml extract and add 1ml millions reagent. The formation of White precipitate indicates the presence of Proteins.

• Test For Phenolics

1. Lead acetate Test: Take 1ml extract add 5ml dis. Water then add 3ml 10% lead acetate. The formation of White ppt indicates the presence of Phenol.

• Test For Flavonoids

1. Lead acetate Test: Take 1ml extract add few drops of 10% lead acetate Solution. Yellow precipitates indicate the presence of Flavonoids

• Test For Tannins

1. FeCl₃ Test: Take 1ml extract and add 5% ferric chloride solution. Dark green or Blackish-blue colouration indicates the presence of Tannins

• Test For Terpenoids

1. Salkowski’s Test: Take 1ml extract, add 2ml chloroform and 2ml con H₂SO₄, forms Reddish-brown colour ring indicates the presence of Terpenoids

• Test For Saponins

1) Foam Test: Take 1ml extract, add 3ml dist. Water & shake it vigorously the formation of Foam layers indicates the presence of Saponins

• Test For Steroids

1. Salkowski’s Test: Take 1ml filtrate and add 2ml chloroform plus 2ml con. H₂SO₄ Reddish-brown/ rusty color formation indicates the presence of Steroids. (Sayma et al., 2025) ;(Shaikh and Patil., 2020) ;(Dubale et al., 2023).     

Quantitative Analysis

The quantitative analysis of phytochemicals is done for the determination of exact amount of phytochemicals are present. For conducting quantitative analysis, the stock solution was prepared with a concentration of 1mg/ml in Methanol, Ethanol and Water solvent. 10mg dry extracts was dissolved in 10ml of Respective solvents to prepare the stock solution. Similarly, the standard solution was prepared with a concentration of 1 mg/ml.

DPPH ASSAY:

This is a popular and common method used to check the presence of free radical scavenging activity of a given plant material. 2, 2-diphenyl-1-picrylhydrazyl (DPPH) is a dark crystalline powder having stable free radical molecules. 4mg (0.004%) of DPPH was prepared by dissolving in 100 ml methanol. 3ml of prepared DPPH was added to various concentration of sample (200, 400, 600, 800, and 1000 µg/ml) and incubate for 30 minutes at room temperature in dark, the absorbance was read at 517 nm in triplicates for each concentration in spectrophotometer. The application of DPPH is important for antioxidant assay. The DPPH antioxidant assay is based on the ability of DPPH, a stable free radical, to decolorize in the presence of antioxidants, thus a low value corresponds to a good scavenging ability. Ascorbic acid solution of same concentration can be used as standard for the free radical scavenging assay. The result was expressed by IC₅₀ value. Radical scavenging activity was compared with inhibition concentration (IC₅₀ following formula was used for calculating DPPH scavenging activity: (Sayma et al., 2025).

DPPH scavenging activity (%) = [(absorbance of blank - absorbance of sample) / absorbance of blank)] × 100

Determination of Total Phenol Content:

The total phenolic content was determined by using Folin-Ciocalteau method with using gallic acid as a standard in different concentrations with modified series are formed. Standard solution is form with 10mg Galic acid powder with 10ml water as a solvent. Standard solution concentration series were 10mg/ml to 50mg/ml. Then add 0.5ml of 2N FC reagent and after that added 10ml of distil water and then added 1 ml 20% Sodium carbonate make volume up to 25ml. Put it for incubation for 30min. Than take OD at 765nm (Siddiqui et al.,2017). Similarly for plant extract, 1ml plant extract are taken with respective solvents in which add 0.5ml 2N FC reagents and then add 10ml water after that added 1ml 20% sodium carbonate than make volume up to 25ml for 30min keep it for incubation. Using UV Spectrophotometer, the absorbance is determined at 765nm. Quantification of total phenolic content was carried in micrograms of Galic acid equivalents per g of sample. Then determine the total phenolic content with using this formula (Siddiqui et al.,2017).

C = C1 × V/m

Where C = total phenolic content in mg/ml, in GAE (gallic acid equivalent),

C1 = concentration of gallic acid established from the calibration curve in mg/ml,

V = Volume of extract in ml, and m = the weight of the plant extract in g (Siddiqui et al.,2017)

Determination of Total Flavonoid Content:

The flavonoids content was determined in different extracts by modified colorimetric method. First made a standard series with slight modification and used quercetin as standard. Standard solution of quercetin is formed by dissolving 10mg with 10ml methanol to make 1mg/ml then the solution used to make different concentrations series of 50mg/ml to 500mg/ml. To the series add 4ml water and then add 5% sodium nitrite and then, 0.3ml 10% aluminium chloride and after that, 2ml 1M Sodium hydroxide are added. Then make volume up to 10ml. after that take OD at 510nm.

Same as standard for solvents also plant extract, 1ml plant extract are taken in which 4ml water and then 0.3ml 5% sodium nitrite and then, 0.3ml 10% aluminium chloride and then, 2ml 1M sodium hydroxide are added. In both standard and extract final up to volume is 10ml. Using UV Spectrophotometer, the absorbance is determined at 510nm. The flavonoid content was calculated from standard curve of quercetin is determined and expressed as mgQEg-¹ (quercetin equivalent) using the formula: QE = C × V/M (Kaviya et al.,2024). 

Determination of Total Tannin Content:

This phytochemical is produced by the plant as a defence mechanism, offering protection through its strong taste. To determine the total tannin content, the potassium iodate method was employed. Tannic acid was used as the standard, and a concentration series ranging from 100mg/mL to 500mg/mL was prepared. The solution was then placed in a cold water bath for 5 minutes. Afterward, 5 mL of 2.5% potassium iodate (KIO₃) was added, and the mixture was incubated in a test tube for 25 to 30 minutes. The optical density was measured at 550 nm (Willis and Allen.,1998).

For the plant extract, 1 mL of the extract, along with the respective solvent, was used, and the same procedure was followed. The tannin content was calculated from the standard curve of tannic acid and expressed as milligrams of tannic acid equivalent per gram (mg TE/g). The calculation was done using the formula: 

TE = C × V/M.

RESULTS

Plant Extract Yield (%) value

Standard formula for Yield (%):

Yield (%) = (Weight of Dry Extract ÷ Weight of Plant Powder Taken) × 100

Calculations:

1. Yield (%) for Methanolic Extract   = (1.893 ÷10) ×100 = 18.93%
2. Yield (%) for Ethanolic Extract      = (0.872÷10) × 100 = 8.72%
3. Yield (%) for Water Extract            = (1.676 ÷10) ×100 = 16.76%

(Graph 1: Yield of Different Extracts)

Qualitative analysis

Table 1: Phytochemical Screening

(The table represents the results of the phytochemical tests performed. ‘++’ sign indicates sign indicates – Highly Present, ‘+’ sign indicates – Moderately present, the ‘-’ sign indicates absence of the respective phytochemicals)

Quantitative Analysis

Quantitative Estimation of Total Phenol Content (TPC)

The process of finding out sample’s overall phenolic content is known as the Total Phenolic content (TPC). The extraction solvent has a direct impact on the yields. Here, Gallic acid was used as a reference to measure TPC of the Whole plant of Gymnema sylvestre. The substances found in the plant sample also exhibit antioxidant qualities.

(Graph 2 : Gallic acid standard Curve)

Table 2: Standard reading of Total Phenol Content

Graph 3: The Phenol Content In different extracts of Gymnema sylvestre.

Table 3: Total Phenolic Content (TPC) In different extracts of Gymnema sylvestre.

Quantitative Estimation of Total Flavonoid Content (TFC)

The process of finding out sample’s overall Flavonoids content is known as the Total Flavonoid content (TFC). The extraction solvent has a direct impact on the yields. Here, Quercetin was used as a reference to measure TFC of the Whole plant of Gymnema sylvestre. The substances found in the plant sample also exhibit antioxidant qualities.

(Graph 4 : Quercetin Standard Curve)

Table 4: Standard Reading of Total Flavonoid Content

Graph 5 : Total Flavonoid Content In different extracts of Gymnema sylvestre 

Table 5: Total Flavonoid Content (TFC) In different extracts of Gymnema sylvestre.

Quantitative Estimation of Total Tannin Content (TTC)

The process of finding out sample’s overall Flavonoids content is known as the Total Tannin content (TTC). The extraction solvent has a direct impact on the yields. The tannin content in the whole plant of Gymnema sylvestre was quantified by using Tannin acid as a standard. The substances found in the plant sample also exhibit antioxidant qualities.

Graph 6 : Tanic acid Standard Curve

Table 6: Standard Reading of Total Tannin Content

Graph 7 : Total Tannin Content In different extracts of Gymnema sylvestre

Table 7: Total Tannin Content (TTC) In different extracts of Gymnema sylvestre

Antioxidant Activity

Estimation of DPPH Assay:

Free radicals are unpaired electrons in uncharged compounds. It is poisonous and can cause oxidative stress, which harms living cell or tissue. Here DPPH Assay was performed for the different solvents in the plant Gymnema sylvestre. Ascorbic acid used as the standard for DPPH Assay, and the inhibitory activity was computed using the OD of the sample extracts.

Table 8:  Inhibitory Activity of Ascorbic Acid

Graph 8 : Ascorbic Acid Standard

Table 9: Depicting for the results for DPPH Assay for Plant Gymnema sylvestre

Graph 9 : Various Solvent Extracts Inhibition Against Standard Inhibition