IN-VITRO AND IN-VIVO ANTIDIABETIC ACTIVITY OF METHANOLIC EXTRACT OF AERIAL PARTS OF ALANGIUM SALVIFOLIUM SUB-SPECIES HEXAPETALUM (WANGERIN)

IN-VITRO AND IN-VIVO ANTIDIABETIC ACTIVITY OF METHANOLIC EXTRACT OF AERIAL PARTS OF ALANGIUM SALVIFOLIUM SUBSPECIE HEXAPETALUM (WANGERIN)

H. Seena ^{* 1}, N. Kannappan ¹ and P. Manoj Kumar ²

Department of Pharmacy ¹, Annamalai University, Annamalai Nagar, Chidambaram - 608002, Tamil Nadu, India.

Department of Chemistry ², The Dale View College of Pharmacy And Research Centre, Trivandrum, 695575, Kerala, India.

ABSTRACT: Alangium salvifolium is a plant that is traditionally used to treat many diseases like laxative, antiepileptic, jaundice, antiulcer agent, agent to alleviate spasms, anthelmintic, emetic, antiprotozoal agent and hypoglycemic agent. Its subspecies Alangium salvifolium subsp. Hexapetalum (Wangerin) is also known for a variety of traditional use like hemorrhoids, rheumatism, and antidote for snake bite. The present work aims for the evaluation of the in-vitro and in-vivo anti-diabetic activity of methanolic extract of aerial parts of Alangium salvifolium subsp. Hexapetalum (Wangerin). The in-vitro antidiabetic activity was evaluated by a starch-iodine color assay method. The in-vivo method was performed by administering orally the methanolic extract of Alangium salvifolium subsp. Hexapetalum in streptozotocin-induced male Albino Wistar rats weighing 200 g. The study was compared using standard metformin hydrochloride (10 mg/kg body weight). The in-vitro method showed a dose-dependent anti-diabetic activity that is as the dose increases the percentage inhibition of enzyme activity also increases. The in-vivo anti-diabetic activity was accessed by comparing the body weight and blood glucose level on 0^th, 5^th, 10^th and 15^th day using a glucometer. Thus, the present study reveals that the methanolic extract of aerial parts of Alangium salvifolium subsp. Hexapetalum (Wangerin) was efficient in lowering blood glucose levels.

Antidiabetic, Streptozotocin, Methanolic extract, Metformin hydrochloride, Histopathology

INTRODUCTION: Plants are widely used in Chinese medicine and Ayurveda-Indian traditional medicine. The traditional system of medicine is still continued to be widely practiced. They emphasize on the use of plants as a source of medicine for various ailments is increasing due to many reasons like an increase in population, side effects of several synthetic drugs, high treatment cost and resistance of many drugs ¹.

Diabetes mellitus is a complex disorder marked by hyperglycemia due to increased hepatic glucose production, decreased insulin secretion and reduced insulin action. Diabetes Mellitus is the leading cause of end-stage renal disease, non-traumatic lower extremity amputations and the affected individuals are susceptible to long term complications affecting the organs like skin, eye, nerves, blood vessels and kidney ². Management of diabetes with agents lacking side effects is still a challenge to the medical field. In spite of many advances in the management of this disease, the diabetes-related mortality graph is still increasing. Available synthetic drugs which are used for the treatment of this disease are allied with adverse effect and are unable to control metabolism adequately.

Because of this reason, there is a growing interest in herbal remedies ³. The literature surveys unveils that Alangium salvifolium is traditionally used as laxative, antiepileptic, astringent, antiulcer agent, pungent, purgative, agent to alleviates spasms, anthelmintic, emetic, antiprotozoal agent and hypoglycemic agent. Antidiabetic activity of A. salvifolium in aqueous extract of stem and leaves has been reported ⁴.

The aim of the present work is to scientifically reveal the hypoglycaemic activity of methanolic extract of aerial parts of its subspecie A. hexapetalum.

MATERIALS AND METHODS:

Plant Collection and Verification: The aerial parts of Alangium salvifolium subsp. Hexapetalum was collected from Tirunelveli district, Tamil Nadu. The plant was identified and authenticated by Mr. Chelladurai, Research officer- Botany, Central Council for Research in Ayurveda and Siddha, Government of India (Accession no. FTN/183/2018).

Preparation of Extract: The plant’s aerial parts were desiccated in air shade and ground to coarse powder through a mixer. Using the Soxhlet extraction method, the powdered drug was processed with solvents of increasing polarity. The extracts of petroleum ether, chloroform, methanol and water were subjected to preliminary phytochemical study.

Preliminary Phytochemical Study: The methanolic extract of aerial parts of Alangium salvifolium subsp. Hexapetalum was subjected to preliminary phytochemical screening to find the presence of phytoconstituents like alkaloids, carbohydrates, flavanoids, phenolic content and saponins.

Drugs and Chemicals: For in-vitro hypoglycaemic activity different concentrations of methanolic extract of aerial parts of Alangium salvifolium subsp. Hexapetalum prepared were 200,400,600 and 800 µg along with α- amylase solution (1 mg/ml), phosphate buffer and 1% starch solution. For the in-vivo study the methanolic extract of aerial parts of Alangium salvifolium subsp. hexapetalum of 200 mg and 400 mg/kg body weight was prepared in a 10% acacia solution and was administered orally. The study was compared with standard metformin hydrochloride (10 mg/kg body weight) prepared in a 10% acacia solution.

In-vitro Antidiabetic Activity:

Starch-Iodine Colour Assay: Mixed 20 µl of α-amylase solution (1 mg/ml) with 390 µL of phosphate buffer (0.02 M phosphate buffer pH 7.0 containing 0.006 M NaCl, pH 7.0) having different concentration of extracts. The above samples were incubated at 37 °C for 10 min. After incubation added 100 µl of starch solution (1%) and the mixture was re-incubated for another 1 h. To the resulting solution added 0.1 ml of 1% iodine solution and 5 ml distilled water. The absorbance was measured at 565 nm. Blank determinations were carried out under the same reaction conditions. Percentage inhibition of enzyme activity was calculated using the formula ⁶.

% Inhibition = (A-C) × 100 / (B-C)

Where, A = absorbance of the sample, B = absorbance of blank (without α-amylase) and C = absorbance of the control (without starch)

Acute Toxicity Study: Acute toxicity study of methanolic extract of the aerial parts of Alangium salvifolium subsp. Hexapetalum was determined in Wistar albino rats according to OECD guidelines no. 425 ⁵. The overnight fasted animals were orally administered with the methanolic extract 2000 mg/kg body weight. Animals were continuously monitored for the first 3 h and then for 14 days for death, signs of discomfort, general behaviour and nervous manifestations.

Experimental Animals: Healthy male Wistar albino rats (200 g) were used for the study. They were maintained under standard conditions as per CPCSEA guidelines. The experimental protocol was approved by the Institutional Animal Ethics Committee (Registration no. 1118/PO/Re/S/07/ CPCSEA) The Dale View college of pharmacy and Research center, Trivandrum (IAEC approval no. DVCP/2018/005). The animals were habituated for at least one week before use.

Streptozotocin-Induced Anti-diabetic Activity: Prior to the experimental study, animals were fasted by depriving them of food for overnight but allowing free access to water.

Induction of Experimental Diabetes: In experimental animals, diabetes was induced by intraperitoneal (i.p.) injection of nicotinamide (230 mg/kg) 15 min before streptozotocin (65 mg/kg i.p) administration. As STZ induces fatal hypoglycemia due to massive pancreatic insulin release, STZ-treated rats were provided with a 10% glucose solution after 3 h for the next 24 h to prevent fatal hypoglycemia. Different doses of the extracts were selected on the basis of acute toxicity study. Experimental design animals with blood glucose levels above 250 mg/dl were confirmed as diabetic and selected for the study. They were divided into five groups comprising six animals in each group.

Group I: Normal control administered with a 10% acacia solution.

Group II: Negative control, induced with diabetes and no treatment received.

Group III: Treated with reference drug Metformin hydrochloride at (10 mg/kg body weight).

Group IV: Treated with methanolic extract of aerial parts of A. salvifolium subsp. Hexapetalum at 200 mg/kg body weight orally for 15 days.

Group V: Treated with methanolic extract of aerial parts of A. salvifolium subsp. Hexapetalum at 400 mg/kg body weight orally for 15 days.

Blood samples were collected from the tail vein of the overnight fasted rats and blood glucose level was determined on 0^th, 5^th, 10^th and 15^th day along with body weight. On the 15^th day, all the animals were humanely sacrificed using CPCSEA recommended euthanasia procedure (Carbon dioxide inhalation method) and evaluated for the biochemical parameters along with histopathology of the pancreas tissue ⁷.

Statistical Analysis: All values were expressed as Mean ± SEM. The data was statistically analyzed by Bonferroni Multiple Comparison Test. A P-value of less than 0.05 was considered as statistically significant.

RESULTS AND DISCUSSION:

Preliminary Phytochemical Screening: Many phytoconstituents present in plants are used to screen and analyze biologically active compounds. Alkaloid-the secondary metabolite present in plants has been investigated for antidiabetic activity. Glycolysis is the center for carbohydrate metabolism where the sugar is converted to glucose by a series of reactions catalyzed by enzymes like hexokinase, pospho fructokinase and pyruvate kinase. Alkaloid amplifies the activity of these enzymes and leads to glucose transport, carbohydrate digestion and absorption. They also cause the regeneration of pancreatic beta cells and insulin secretion. Similarly, flavonoids are also reported to have anti-diabetic activity. They act by targeting glycogen synthesis, glycolysis and gluconeogenesis ⁸. The preliminary phytochemical screening of methanolic extract of aerial parts of A. salvifolium subsp. Hexapetalum confirms the presence of flavonoids, alkaloids and phenolic content.

Evaluation of in-vitro Anti-diabetic Activity: Alpha-amylase is an enzyme present in saliva and pancreatic juice. It breaks down insoluble starch molecules into absorbable molecules. Alpha-amylase inhibitors slow down the breakdown of carbohydrates and thus results in diminishing blood glucose. The methanolic extract of A. salvifolium subsp. Hexapetalum shows a dose-dependent action. As the concentration increases the % inhibition also increases.

TABLE 1: INHIBITION OF α-AMYLASE BY METHANOLIC EXTRACT OF A. SALVIFOLIUM SUBSP. HEXAPETALUM

FIG. 1: RELATIONSHIP BETWEEN DIFFERENT CONCENTRATIONS OF METHANOLIC EXTRACT OF A. SALVIFOLIUM SUBSP. HEXAPETALUM AND PERCENTAGE INHIBITION

Evaluation of in-vivo Anti-diabetic Activity: In this study streptozotocin along with nicotinamide is used to induce diabetes in animals. Streptozotocin enters the B cells through glucose transporter (GLUT2) and causes DNA alkylation.

Damage of DNA leads to activation of poly ADP-ribosylation which causes depletion of cellular NAD+ and ATP. Increased ATP dephosphorylation leads to the formation of superoxide radicals along with hydrogen peroxide and hydroxyl radicals. Streptozotocin release nitric oxide which cause damage of DNA thus results in the destruction of B cells by necrosis ⁹.

Effect of Extract on Body Weight: Streptozotocin (65 mg/kg) induced experimental animals showed a remarkable (P<0.0001) decrease in body weight compared to the control group.

Standard metformin hydrochloride and the extract-treated animals showed significant (P<0.0001) increase in body weight when compared to vehicle-treated streptozotocin rats.

FIG. 2: EFFECT OF METHANOLIC EXTRACT OF A. SALVIFOLIUM SUBSP. HEXAPETALUM ON BODY WEIGHT OF STREPTOZOTOCIN INDUCED RATS

Effect of Extract on Blood Glucose: The blood glucose levels were measured in experimental animals on 0^th, 5^th, 10^th and 15^th days of treatment. Streptozotocin administered diabetic rats showed a significant (P<0.0001) increase in blood sugar when compared to control group. A significant decrease (P<0.05) (P<0.001) in blood sugar levels was seen in experimental rats orally administered with methanolic extract of A. salvifolium subsp. Hexapetalum (200 mg/kg & 400 mg/kg body weight) when compared to negative group.

TABLE 2: EFFECT OF METHANOLIC EXTRACT OF A. SALVIFOLIUM SUBSP. HEXAPETALUM ON BLOOD GLUCOSE LEVEL OF STZ-INDUCED RATS

FIG. 3: HISTOPATHOLOGICAL EVALUATION OF PANCREAS

A) Pancreas of control group, B) Streptozotocin induced pancreas, C) Streptozotocin (65 mg/kg) + metformin HCl (10 mg/kg), D) Streptozotocin (65 mg/kg) + Extract (200 mg/kg), E) Streptozotocin (65 mg/kg) + Extract (400 mg/kg).

CONCLUSION: The present study deals with the pharmacological and preliminary phytochemical analysis of a methanolic extract of aerial parts of Alangium salvifolium subspecie Hexapetalum (Wangrein). According to the phytochemical evaluation, it shows the presence of phyto-constituents like alkaloids, phenolic contents and flavanoids. Studies have reported that alkaloids ¹⁰ and flavanoids ¹¹ play a key role in alleviating diabetes mellitus. Both in-vitro and in-vivo method of anti-diabetic activities were evaluated. The methanolic extract of Alangium salvifolium subspecie Hexapetalum showed a significant in-vivo anti-diabetic activity which was supported by the in-vitro study. This may be due to the presence of alkaloids and flavonoids present in the methanolic extract.

ACKNOWLEDGEMENT: For the excellent and helpful support to enable the successful completion of this work, the authors are thankful to Athmic Biotec Solutions Pvt. Limited, present at kalliyoor, Kerala, India. For presenting the opportunity to carry out the anti-diabetic in-vivo study the authors are indebted to The Dale View College of Pharmacy & Research Centre, Trivandrum, India.

CONFLICTS OF INTEREST: We declare no conflicts of interest.

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    How to cite this article:

    Seena H, Kannappan N and Kumar PM: In-vitro and in-vivo anti diabetic activity of methanolic extract of aerial parts of Alangium salvifolium subspecie Hexapetalum (Wangerin). Int J Pharm Sci & Res 2020; 11(2): 700-04. doi: 10.13040/IJPSR.0975-8232.11(2).700-04.

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