INTRODUCTION:

Herbal drugs are use of therapeutic herbs to prevent and treat diseases and ailments or to support health and healing. These are drugs or preparations made from a plants and used for any of such purposes. Herbal drugs are the oldest form of health care known to mankind. However herbal medicines suffer from a range of short-comings. These include insufficient and unacceptable evidences of safety, efficacy, standardization and inconsistent production practices.^1-3A large number of Indian medicinal plants have been used in the treatment of antidiabetic and they were reported to have no side effects. Plants have always been a very good source of drugs, and many of the currently available drugs have been derived directly or indirectly from them.

Ethno botanical information suggests that about 800 plants possess anti-diabetic potential.^4,5

Diabetes mellitus refers to a group of diseases that affect how your body uses blood sugar (glucose). Glucose is vital to your health because it's an important source of energy for the cells that make up your muscles and tissues. It's also your brain's main source of fuel. The underlying cause of diabetes varies by type. However, regardless of the type of diabetes you have, it can result in an excess of sugar in your blood. Too much sugar in your blood can lead to serious health problems. Chronic diabetes conditions include type 1 diabetes and type 2 diabetes. Potentially reversible diabetes conditions include prediabetes and gestational diabetes. Prediabetes occurs when your blood sugar levels are higher than normal, but not high enough to be classified as diabetes. The prediabetes is often the precursor of diabetes unless appropriate measures are taken to prevent progression. Gestational diabetes occurs during pregnancy but may resolve after the baby is delivered.^6-8 According to literature survey different parts of Gymnema Sylvestre reported to possess beneficial effects as digestive, anti-inflammatory, diuretic and anthelmintic. It is believed to be used in dyspepsia, constipation, jaundice, haemorrhoids, cardiopathy, asthma, bronchitis and leucoderma. A scrutiny of literature revealed some notable pharmacological activities of the plant such as antidiabetic, antiobesity, hypolipidaemic, antimicrobial, free radical scavenging and anti-inflammatory. It is also reported to be bitter, astringent, acrid, thermogenic, anodyne, digestive, liver tonic emetic, stomachic, stimulant, laxative, cardio tonic, expectorant, antipyretic and uterine tonic. It is useful in dyspepsia, constipation and jaundice, haemorrhoids, renal and vesicle calculi, cardiopathy, asthma, bronchitis, amenorrhea, conjunctivitis and leucoderma.^9-12

MATERIALS AND METHODS:

Plant Material:

The leaves of Gymnema Sylvestre are collected form Asansol, West Bengal, India. A herbarium sheet was prepared and it was identified and authenticated (CNH/35/2011/TECH II/446) by the Botanical Survey of India, Howrah, West Bengal, India.

Preparation of extract:

The leaves were dried in shade to avoid too many chemical changes occurring and made into coarse powder. Ethanol was used as solvent for extraction and extraction was performed in soxhlet apparatus. The extraction vessel was made up of borosil glass which contains round bottom flask. The plant material to be extracted was packed in the soxhlet assembly and a condenser through which refluxing was done .Heat was supplied through a heating mantle .The extract was collected after evaporating the solvent using rota evaporator. The extract was kept in airtight container at room temperature until further use.

Physiochemical Studies:¹³

Determination of Ash value:

The total ash obtained was boiled with 25 ml of alcohol for few minute. The insoluble matter was collected on ash less filter paper, washed with hot water and ignited for 15 min at temperature not exceeding 450ºC. The difference in weight represents the alcohol-soluble ash. The percentage of alcohol - soluble ash was calculated with reference to the air dried drug.

Determination of extractive value:

About 5gms of air-dried, coarsely powdered roots of Gymnema Sylvestre were weighed accurately and separately macerated with 100ml of alcohol in stoppered flask for 24 hrs, shaking frequently during the first 6 hrs and was allowed to stand for 18 hrs. Then it was filtered and 25ml of the filtrate was evaporated to dryness in a tarred flat bottom shallow dish, dried at 105ºC and weighed. The percentage of alcohol-soluble extracts was calculated with reference to the air-dried drug.

Determination of moisture content:

About 5gm of powdered leaves of Gymnema Sylvestre were weighed accurately and was taken separately in a china dish. It was kept at 105 – 110°C for 30min in a hot air oven; the residue was cooled and weighed. The percentage of moisture content was then calculated with reference to the air-dried drug.

Preliminary phytochemical Test:

Preliminary phytochemical studies of the leaf extract were conducted as per the standard procedure.^14-17

Experimental Design:

Anti-diabetic activity:

Animal selection:

Animals for the proposed activities are selected as per the experimental protocol approved by Institutional Animal Ethical Committee (Vide: JNMC/IAEC/Res- 2/9/2019 dated 19^th December 2019) in accordance with OECD – 423 guidelines.49

Determination of LD50:

The method of OECD Guidelines was adapted for the determination of LD50. Male albino rat of 20-25g weight were used. Acute toxicity studies were performed following OECD guideline no. 423 (Acute Toxic Class Method), in which Swiss albino rat of either sex (20-25g weight) were treated with ethanolic extracts of Gymnema Sylvestre. It was observed that the test extracts did not show any remarkable undesired signs, change in behavior; change in body weight, mortality and morbidity even at 2000mg/kg dose after completion of 14 days observation. Therefore, 1/10th (200mg/kg b. w.) and 1/5th (400mg/kg b. w.) of these doses were selected for further study.

Anti-diabetic activity:

Pharmacological screening for Anti-diabetic activity of was done by using male albino Wistar rats. The glucose tolerance test (GTT) study and evaluation of their effects (Single dose and Multidose treatment study) on blood glucose level and histopathology of liver in Alloxan induced diabetic rats was done.

Drugs and chemicals:

Alloxan was purchased from Sigma Aldrich, USA. Daonil tablets were purchased from local market.

Instruments used:

Micro pipettes, glucometer and Digital balance

Animal Experiment Model:

Six animals in each group. (n = 6)

Group-I: Control Oral-1mL of 1% gum acacia suspension.

Group -II: Diabetic Control Alloxan 120mg/kg

Group -III: Standard Drug Glibenclamide - 2.5mg/kg

Group-IV: Ethanolic extract of Gymnema Sylvestre (leaves) 100mg/kg

Group-V: Ethanolic extract of Gymnema Sylvestre (leaves) 200mg/kg

Oral glucose tolerance test (OGTT):¹⁸

Fasting blood glucose level of experimental animal was determined at zero time after overnight fasting with free access to water. Rats were divided into six groups each containing six rats. The first group of animals received 1 ml of 1% gum acacia suspension orally (Control animals). Remaining groups received Glibenclamide (2.5 mg/kg- standard) and ethanolic extracts of selected plants (100 and 200mg/kg), by oral route using an orogastric tube respectively. Glucose (2gm/kg) was orally administered 30 min. After the administration of extracts or Glibenclamide or gum acacia suspension. Blood samples were collected from the tail vein under ether anesthesia just prior to and 30, 60, 120 and 240 min after glucose loading. Glucose levels were estimated using glucose-oxidase-peroxidase reactive strips and a glucometer (Sugar-check, Wockhardt Ltd, Mumbai, India). Effect of ethanolic extracts on blood glucose levels in alloxan induced diabetic rats [Single dose (Acute) treatment].

A single intraperitonial injection of 120mg/kg of alloxan monohydrate was employed to induce diabetes in overnight fasted male Wistar albino rats weighing 170-200gm. After 72 hr, animals with blood glucose levels higher than 250mg/dl were considered diabetic and were included in the study. Animals were divided into five groups including six rats each. Group I: Normal control rats administered 1ml of 1% gum acacia suspension; Group II: Diabetic control rats administered 1ml of 1% gum acacia suspension; Group III: Diabetic rats administered Glibenclamide (2.5mg/kg), Group IV: Diabetic rats administered Gymnema Sylvestre ethanol extract (100mg/kg), Group V: Diabetic rats administered Gymnema Sylvestre ethanol extract (200mg/kg Group orally. Blood samples were collected from the tail vein prior to and at 30 min, 60 min, 2, 4, and 6 h intervals after the administration of the extract and blood glucose levels were estimated using glucometer

Effect of ethanol extract on blood glucose in alloxan induced diabetic rats [Multi dose (sub acute) treatment:¹⁹

Diabetes was induced in overnight fasted adult male Wistar albino rats weighing 170-200gm by a single intraperitoneal injection of 120mg/kg of alloxan monohydrate. After 72 hr, animals with blood glucose levels higher than 250mg/dl were considered diabetic and were included in the study. Animals were divided into five groups including six rats each. Group I: Normal control rats administered 1ml of 1% gum acacia suspension; Group II: Diabetic control rats administered 1ml of 1% gum acacia suspension; Group III: Diabetic rats administered Glibenclamide (2.5mg/kg) Group IV: Diabetic rats administered Gymnema Sylvestre ethanol extract (100mg/kg), Group V: Diabetic rats administered Gymnema Sylvestre ethanol extract (200mg/kg) orally. These rats were given the same doses of the extract once daily for 15 days in this study. Blood samples were collected from the tail vein of non-fasted rats on days 0, 5, 10 and 15 of extract administration and blood glucose levels were estimated using glucometer.

Histopathological studies:²⁰

Pancreatic tissues from rats of all groups of Multi dose (Sub acute) treatment were subjected to histopathological studies. The whole pancreas from each animal was removed after sacrificing the animal under anesthesia and was collected in 10% formalin solution and immediately processed by the paraffin technique. Sections of 5µm thickness were cut and stained by hematoxylin and eosin (H and E) for histological examination.

Statistical analysis:

Values are presented as mean ±S.E.M. Statistical difference between treatments and the controls were tested by one-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparison test using the “Stat” statistics computer program. A difference in the mean values of P< 0.05 was considered to be statistically significant.

RESULTS:

Physiochemical Studies:

The extract of leaves of Gymnema Sylvestre was subjected to evaluate its alcohol soluble ash, alcohol soluble extractive value and moisture content. Each determination was carried out and the values were taken.

The result was reported in table-1.

Table No.1 Percentage yield of values

Name of the Plant	VALUES (%w/w)
Name of the Plant	Alcohol Soluble ash	Alcohol soluble extractive	Moisture Content
Gymnema Sylvestre	2.84	6.4	0.8

Table No.2 Qualitative Chemical examination of extract of Gymnema Sylvestre leaves

S. No	Name of the Experiment	Gymnema Sylvestre
1	Detection of alkaloids Dragendroff’s/ Kraut’s test	Positive
2	Hager’s test	Positive
3	Mayer’s/ Bertrand’s/Valser’s test	Positive
4	Wagner’s test	Positive
5	Picric acid test	Positive
1	Detection of Carbohydrates Barfoed’s test	Positive
2	Molish’s test	Positive
3	Seliwanoff’s Test	Positive
6	Test for starch	Positive
1	Detection of Reducing sugars Benedict’s test	Positive
2	Fehling’s test	Positive
1	Detection of Glycosides Keller-Killani test	Positive
2	Kedee’s test	Positive
1	Detection of Proteins and Amino acids Biuret test	Positive
2	Millon’s test	Positive
3	Ninhydrin test	Positive
1	Detection of Flavonoids Alkaline reagent test (NaOH)	Positive
2	Shinoda’s test/ Mg- HCL reduction test	Positive
3	Ferric chloride test	Positive
1	Detection of Phenolic compounds Iodine test	Positive
2	Ferric chloride test	Positive
1	Detection of Tannins Gelatin test	Positive
2	Lead sub acetate test	Positive
3	Detection of Saponins Foam test: vigorously shaken	Positive
4	Haemolysis test	Positive
1	Detection of Phytosterols Salkowski’s test	Positive
2	Libermann-Burchard’s test	Positive
1	Detection of Cholesterol 2ml CHCl₃+ 5mL plant extract, (evaporated on H₂O bath) + 3mL conc. H₂SO₄ (boiled on H₂O bath)	Negative
1	Detection of Terpinoides 2ml CHCl₃+ 5mL plant extract, (evaporated on H₂O bath) + 3mL conc. H₂SO₄ (boiled on H₂O bath)	Positive
1	Detection of Triterpinoides Salkowski’s test	Positive
1	Detection of Diterpenes Copper acetate test	Positive
1	Detection of Lignins Labat test	Positive
2	Furfuraldehyde test	Positive
1	Detection of Carotenoids Carr-Price reaction	Positive
1	Detection of Quinones Alcoholic KOH test	Positive
2	Conc. HCl test	Positive
3	Sulphuric acid test	Positive
1	Detection of Anthraquinones Borntrager’s test	Positive
2	Ammonium hydroxide test	Positive
1	Detection of Anthocyanins HCl test	Negative
1	Detection of Emodins Plant extract + 2mL NH₄OH + 3mL benzene	Negative

Table No 3: OGTT (Oral glucose tolerance test)

Animal Group (n= 6)	Treatment	Blood glucose concentration (mg/dl) (Mean ± SEM)
Animal Group (n= 6)	Treatment	0 min	30 min	60 min	120 min	240 min
I	Normal control (1% gum acacia)	92.8± 2.9	161.6± 4.1	162.7±2.6	161.2± 3.5	132.3± 3.2
II	Glibenclamide(2.5mg/kg)	96.0±2.4	143.0±2.4 *	145.8±1.2 *	137.0±2.2 *	98.9±1.2 *
III	Ethanolic extract of Gymnema Sylvestre (100mg/kg)	98.2±4.2	130.0±2.7 *	133.2±3.5 *	124.7±2.6 *	109.0±1.4 *
IV	Ethanolic extract of Gymnema Sylvestre (200mg/kg)	97.6±3.9	131.0±3.0 *	132.7±3.2 *	125.7±2.9 *	114.0±1.4 *

*P< 0.01. Significant, compared to normal control

Fig. 1 Effect of Ethanolic extract of Gymnema Sylvestre on the blood glucose levels in glucose loaded rats (OGTT)

Table No 4: Blood glucose levels in alloxan-diabetic rats (Single dose treatment / acute study)

Animal Group (n= 6)	Treatment	Blood glucose concentration (mg/dl) (mean ± S.E.M)
Animal Group (n= 6)	Treatment	0min	30min	60min	120min	240min	360min
I	Normal control (1% gum acacia)	87.2±2.4	88.4±2.1^# ^#	90.50±2.3 ^# ^#	90.86±1.8 ^# ^#	94.4± 2.2 ^# ^#	95.76± 3.1^# ^#
II	Diabetic control	288.8±5.0	296.4±5.1*	297.1±5.2*	291.3±4.1*	293.5±3.5*	294.0± 4.1*
III	Glibenclamide (2.5mg/kg)	298.3±6.2	285.1±7.0*	271.3±9.3*	259.0±11.9* ^#	253.8±12.1*^# ^#	236.9 ±11.8*^# ^#
IV	Et. GS (100mg/kg)	278.1± 5.0	273.4± 5.5*^#	265.2 ±6.0*^# ^#	254.8±6.2*	248.1±6.6*^# ^#	234.7 ±6.3*^# ^#
V	Et. GS (200mg/kg)	290.5± 6.7	278.2 ±6.1* ^#	269.6 ±4.2*^# ^#	258.2±5.4*	250.8±5.1* ^# ^#	234.2 ±6.2*^# ^#

*P< 0.01. Significant, compared to normal control,

^#P< 0.05 &^# ^#P< 0.01 Significant, compared to diabetic control n = no of animals in each group.

Fig. 2 Ethanolic extract of Gymnema Sylvestre on blood glucose levels (BGL) in alloxan- diabetic rats (Single dose treatment/Acute study)

Table No 5: Change in body weight study

Animal Group (n= 6)	Treatment	Body weight of the animal (gm)				% Change in body Weight
Animal Group (n= 6)	Treatment	0^th Day	5^th Day	10^th Day	*15^thDay	% Change in body Weight
I	Normal control (1% gum acacia)	202.5	207.3	212.3	217.3	7.4 %
II	Diabetic control	203.7	191.2	183	179.7	11.8 %
III	Glibenclamide (2.5 mg/kg)	203	206.8	209.5	213.7	4.9 %
IV	Et.GS(100 mg/kg)	206.5	200.3	194.5	189.5	− 8.2 %
V	Et. GS(200 mg/kg)	216	210.7	205.3	202.2	− 6.5 %

*Compared to day 0 (Zero) n= no of animals in each group

A. Normal control rats showed normal pancreatic tissue.

B. Alloxan-induced diabetic rats had severe decrease in number of islets of Langerhans cells and β cells.

C. Pancreatic tissue of diabetic rats treated with Glibenclamide (2.5mg/kg).

D. Ethanol extract of Gymnema Sylvestre leave (100 mg/kg) showed partial restoration of normal cellular population and size of islet cells.

E. Ethanol extract of Gymnema Sylvestre leave (200 mg/kg) showed partial restoration of normal cellular population and size of islet cells.

DISCUSSION:

The present study aimed at evaluation of Gymnema Sylvestre leaves on pharmacological and phytochemical parameters. Gymnema Sylvestre leaves were subjected for various standardization parameters. The present study was attempted to evaluate the anti-diabetic activity of the leaves of Gymnema Sylvestre. The powder of Gymnema Sylvestre leaves was subjected to evaluate their alcohol soluble ash, alcohol soluble extractive value, moisture content and phytochemical evaluation. Ash values are helpful in determining the quality and purity of crude drugs. Extractive values are useful for evaluation of crude drugs and gives idea about the nature of chemical constituents present in them. In some cases the amount of drug soluble in a given solvent is an index of purity. The moisture content of the drug should be controlled and minimized in order to prevent decomposition of crude drugs either due to chemical change or microbial contamination. Phytochemical screening of methanolic extract of Gymnema Sylvestre leaves reveals the presence of alkaloids, glycosides, phenolic compounds and tannins, phytosterols, fixed oils and fats. The methanolic extract at a dose of 200mg/kg showed most effective as compared to control. Hence it can be exploited as an anti- diabetic therapeutic agent or adjuvant in existing therapy for the treatment of diabetic.

SUMMARY AND CONCLUSION:

In conclusion, the presented data indicate that administration of the 100mg/kg and 200mg/kg ethanolic extract of Gymnema Sylvestre leaves to rats with Alloxan induced diabetic reduced and prevented the blood sugar level, which supporting folk information regarding anti-diabetic activity of the plant Gymnema Sylvestre leaves.

ACKNOWLEDGEMENT:

We take pleasure to express our sincere thanks to our beloved Dr. D. Vikshapathi. M.Pharma, Ph.D Principal, Teegala Ram Reddy college of Pharmacy for his undivided attention, valuable suggestion, and endeavor throughout the during project work. Special thanks to all faculty members of Teegala ram reddy college of pharmacy for their help, support and encouragement during project work.

REFERENCES:

1. Bhaskar Ananda Raj V, Janet Beula S, Suresh KumarS. Ethanobotanical and pharmacognostical study of Givotia moluccana bark. International Journal of Pharmaceutical research and Development, 2012, 4(07), pg 148- 154.

2. Raina M.K. quality control of herbal and herbo- mineral formulations. Indian Journal of natural products, 2003, 19, pg 11-15

3. Mukesh S Sikarwar, Patil M.B. Anti- hyperlipidemic activity of Salacia chinensis root extract in triton induced and atherogenic diet induced hyperlipidemic rats. Indian Journal of Pharmacology, 2012, 44(1), pg 88-92.

4. Wealth of India. A Dictionary of Indian Raw materials and Industrial Products, National Institute of Science Communication, Council of Scientific and Industrial Research. New Delhi, Raw material. 10: 100-107. 2002.

5. Ivan AR. Medicinal plant of World: Chemical Constituents, Traditional Uses and Modern Medicinal Uses, Human Press Totowa, New Jersey. 2006, pg 283-289.

6. Jain SR, Sharma SN. Hypoglycemic drugs of Indian indigenous origin. Plant Medica. 1967 Nov, 15(04), pg 439-42.

7. Singh N, Gupta M. Effects of ethanolic extract of Syzygium cumini (Linn) seed powder on pancreatic islets of alloxan diabetic rats. Indian Journal of Experimental Biology. 2007, 45, pg 861-867.

8. Teixeira CC, Weinert LS, Barbosa DC, Ricken C, Esteves JF, Fuchs FD. Syzygium cumini (L.) Skeels in the treatment of type 2 diabetes. Diabetes Care. 2004 Dec 1, 27(12), pg 3019-20.

9. Manohar SH, Naik PM, Praveen N, Murthy HN. Distribution of gymnemic acid in various organs of Gymnema sylvestre. Journal of Forestry Research. 2009, 20(3):pg68–270.

10. Singh VK, Umar S, Ansari SA, Iqbal M. Gymnema sylvestre for diabetics. Journal of Herbs, Spices and Medicinal Plants. 2008, 14(1-2) pg 88–106.

11. Kanetkar P, Singhal R, Kamat M. Gymnema sylvestre. A memoir. Journal of Clinical Biochemistry and Nutrition. 2007, 41(2), pg 77–81.

12. Saneja A, Sharma C, Aneja KR, Pahwa R. Gymnema Sylvestre (Gurmar). A review. Der Pharmacia Lettre. 2010, 2(1), pg 275–284.

13. Bhaskar Ananda Raj V, Murugamani V, Anusha CH, Indraja N. Preliminary pharmacognostical evaluation of Givotia Moluccana leaves. International Journal of Pharmacognosy and Phytochemical Research. 2012, 4(2), p g 74-78.

14. Madhava Chetty K, Sivaji K, Tulasi Rao K. Flowering Plants of Chitoor District, A.P.India, 3rd ed; 2008(1),pg 185, 317.

15. Mukherjee P.K. Quality Control of Herbal Drugs. Business Horizons, New Delhi 200,:pg 187-191.

16. Raman N. Phytochemical Techniques.1sted; New India Publications, New Delhi, 2006, pg 19-25.

17. Kokate CK, Purohit AP, Gokhale S.B .Methods of crude drug evaluation Pharmacognosy, Nirali Prakashan, Pune, 1995, pg 88-99.

18. Rafiullah M.R.M, Siddiqui A.W, Ali M. Antidiabetic Activity of Some Indian Medicinal Plants. Pharmaceutical Biology.2006, 44(6), pg 95-99.

19. Kifle Z.D, Yesuf J.S, Atnafie S.A. Evaluation of in vitro and in vivo anti-diabetic, anti-hyperlipidemic and anti-oxidant activity of flower crude extract and solvent fractions of hagenia abyssinica (rosaceae).Journal of Experimental Pharmacology, 2020(12), pg. 151.

20. Belayneh Y.M, Birru E.M, Ambikar D. Evaluation of hypoglycemic, antihyperglycemic and antihyperlipidemic activities of 80% methanolic seed extract of Calpurnia aurea Benth in mice. Journal of Experimental Pharmacology, 2019(11), pg 73.

Received on 22.09.2022 Modified on 10.10.2022

Asian J. Pharm. Tech. 2023; 13(1):34-40.

DOI: 10.52711/2231-5713.2023.00007