Evaluation of Hydro-Alcoholic Root Extract of Nyctyanthes Arbortristis for Antidiabetic Activity on Wistar Albino Rats

Diabetes mellitus is associated with the dysfunction of β-cells of pancreas and liver metabolism with raised blood glucose level either due to insulin insufficiency or insulin resistance. ^[1] Abnormalities in secretion of insulin can lead to chronic hyperglycemia resulting in the damage or impaired function of various body organs such as eyes, kidney, nerve, heart and blood vessels. ^[2,3] India has second highest number of adults with diabetes (20-79 years) in the world. ^[4] Now a days herbal remedies are waged all over the world even in absence of proof of their therapeutic value as compared to the conventional medications. As the synthetic medicines used for the chronic diseases (diabetes) have unwanted side effects to the host. ^[5] Many studies revealed that the strong antidiabetic action of the plant derived oral antihyperglycemic medicines due to their secondary metabolites; flavonoids, phenolics and terpenoids. ^{[6, 7]} Synthetic oral hypoglycemic medications and insulin are the standard antidiabetic drugs have successfully reduced the blood glucose level, but possess side effects without changing the progress of diabetic problems. This is the major reason for the changing of alternate medication adapted by the diabetic patients towards the herbal drugs with potentially less or non-existent side effects. ^[8] According to the International Diabetes Federation (IDF), 8.8% of the adult population have diabetes, with the men having slightly higher rates (9.6%) than women (9.0%). Current global statistics shows that 537 million people have diabetes, and this number is projected to reach 643 million by 2030, and 783 million by 2045. ^[9]  Nyctanthes arbortristis is a value aided medicinal plant belongs to Oleaceae family. The plant generally grows in tropical and subtropical regions. It commonly known as Night jasmine, Harsinghar & Parijat. It is a common wild hardy large shrub or small tree. Different parts of this plant are used in Indian systems of medicine for various pharmacological actions like as anti-leishmaniasis ^[10], anti-viral ^[11], anti-fungal ^[12], anti-pyretic ^[13], anti-histaminic ^[14], anti-malarial ^[15], mast cell stabilizer ^[16], anti-oxidant ^[17], anti-inflammatory ^[18] and many more activities. In the present study, we designed a method to evaluate the antidiabetic activity hydro-alcoholic root extract of N. arbortristis using alloxan-induced diabetic in Wistar albino rats.

MATERIALS AND METHODS

Chemicals and Reagents

Alloxan monohydrate was purchased from S.D. Fine Chemicals Ltd., Boisar. Glibenclamide tablets and other chemicals of high analytical grade were purchased for the study.

Collection of Plant Material and Extraction: Fresh and mature plant roots of Nyctanthes arbortristis Linn. has been collected in bulk quantities from the medicinal garden at U.D.P.S and Chandaka Forest, Bhubaneswar, Odisha, India. The plant specimen was authenticated by IMMT, BBSR, Odisha, India. The voucher specimen of N. arbortristis Linn. (Voucher No. IMMT-134/22) have been preserved in the institution herbarium of U.D.P.S, Bhubaneswar for future reference.  The dried parts (root) were then grated to coarse powder by mechanical grinder and allowed to extract with hydro-alcohol (water 30: ethanol 70) in Soxhlet apparatus. The crude extracts were evaporated to dryness under vacuum and placed it in vacuum desiccator for further drying. Preliminary phytochemical investigation was performed and on the eve of the phytochemicals of hydro-alcoholic root extracts of N. arbortristis (HARNA) was selected for antidiabetic screening in Wistar albino rats.

Animals: Wistar albino rats weighing 150-200 g were used for the present study. All animals were maintained in the animal house of Dadhichi College of Pharmacy, Cuttack, Odisha, under controlled conditions of temperature 25±2? C, relative humidity 55-60 %, and 12-h light-dark cycles for experimental purposes. They are grouped into experimental and control groups, kept in polypropylene cages with sterile paddy husk as bedding and free access to standard pellets to feed and water ad libitum. All the studies conducted were approved by the Institutional Animal Ethical Committee (IAEC) of Dadhichi College of Pharmacy, Sundargram, Cuttack, Odisha (Approval No. -1200/PO/Re/S/08/CCSEA).

Acute toxicity studies

An acute oral toxicity study as per OECD-423 guidelines (acute toxic class method) was done by taking rats (n=6) of either sex. The animals were kept fasting for 12h (night), with only access to water ad libitum before administration of test extract dose (2 g/kg HARNA) body weight by intra-gastric tube and observed for 14 days. No mortality was seen and the same procedure was repeated for higher doses (2.5 g/kg and 3 g/kg HARNA) body weight. Mortality was not observed. ^[19] Repeat this procedure and at a dose of 5 g/kg HARNA, behavioral changes were observed in animals.

Induction of diabetes

Alloxan monohydrate (120 mg/kg body weight) dissolved in normal saline and administered intraperitoneally to overnight fast (12 hours), Wistar albino rats (only access to water ad libitum) for developing diabetes. Then animals were accessed to a standard diet and water ad libitum. After 3 days, the blood glucose levels were measured by using glucometer, and the range above 200 mg/dl was kept for this study. ^[20]

Antidiabetic Study

Design of model

The animals were divided into six groups and six animals constituted a group.

Group-I: Normal control administered with 0.9% normal saline.

Group II: Diabetic control administered with Alloxan monohydrate (120 mg/kg) i.p, in 0.9% normal saline.

Group III: Diabetic control was administered with the standard drug glibenclamide (10 mg/kg), served as standard.

Group IV: Diabetic control administered with test drug HARNA (200 mg/kg)

Group-V: Diabetic control administered with test drug HARNA (250 mg/kg)

Group-VI: Diabetic control administered with test drug HARNA (300 mg/kg)

Estimation of blood glucose

The treatment was started after induction of diabetes except for, the normal control group and diabetic control groups for 15 days, orally. During this period, the animals in all groups had free access to diet and water ad libitum. Blood glucose levels were measured on 1^st, 5^th, 10^th and 15^th days of treatment. Blood was collected from the tail vein of the rats and glucose level was measured by using glucometer and strips, Sugar Check (Mumbai).

Statistical Analysis

All data were expressed as mean ± SD (n=6). Statistical analysis was performed at p< 0.05 to p< 0.01 between the groups by one-way analysis of variance (one-way ANOVA) followed by Tukey’s multiple comparison test using Graph Pad prism software (version 10).

RESULTS AND DISCUSSIONS

Acute toxicity study

In an acute toxicity study, the hydroalcoholic extract of the root of Nyctanthes arbortristis did not show any mortality up to a dose of 3 g/kg body weight. After all, there were no behavioral changes seen at this dose. So, 300 mg/kg was taken as a therapeutic dose.

Preliminary phytochemical test

Identification of phytoconstituents of hydroalcoholic root extract of Nyctanthes arbortristis was carried out by using different screening methods. Hydro-alcoholic root extract of Nyctanthes arbortristis showed the presence of different primary and secondary metabolites; alkaloids, carbohydrates, flavonoids, phenols, coumarins, steroids, resins, and phenol etc. ^[21] The findings of phytochemical constituents are summarized in Table 1.   

Table 1: Summary of Phytochemicals in Nyctanthes arbortristis Root.

(+) - present; (-) – negative

Antidiabetic Activity

The alloxan-induced hyperglycaemia was significantly (p <0.05 to 0.01) corrected by the hydro-alcoholic root extract of N. arbortristis at the end of the treatment (15 days). The effects of hydro-alcoholic root extract on fasting BGL on multi-dose treated alloxan induced diabetic rats are depicted in Table 2. The HARNA at dose of 200 mg/kg body weight showed 123.2 ± 5.87 mg/dl, 250 mg/kg body weight showed 95.0 ± 8.32 mg/dl and 300 mg/kg body weight showed 73.5 ± 5.38 mg/dl reduced glucose level at the end of 15 days of the study. However, at the same day the standard drug Glibenclamide at 10mg/kg body weight showed 69.2 ± 9.79 mg/dl of reduced BGL. Study results reveals that on treatment with crude hydro-alcoholic extract at dose levels of 200, 250 and 300 mg/kg body weight reduced the BGL to an extent of 51.23, 63.2 and 71.31% respectively. On the same day, however, compared to diabetic control (positive control) rats, the fasting mean BGL of diabetic rats given the conventional medication Glibenclamide at a dose of 10 mg/kg body weight decreased by 72.32%. However, when compared to the solvent control, the individual data for the test plant treated groups demonstrated a statistical significance p<0.05 to p<0.01 throughout the experimental outcome. When compared to the standard, the hydro-alcoholic extract of 300 mg/kg body weight generally shown a closer BGL reduction towards blood glucose level.  Numerous plant species are known in traditional medicine used for their hypoglycaemic properties and consequently used for treatment of DM ^{[22, 23]} although few traditionally used antidiabetic plants have received appropriate scientific screening. ^[24] The free radicals break down DNA strands in β cells, which reduces insulin release and utilization of glucose in tissue level. ^[25] Glibenclamide is reported to enhance the activity of β cells of islet of Langerhans of pancreas resulting in secretion of larger amounts of insulin which in turn brings down blood glucose level. ^[26] This study showed that hydro-alcoholic root extract of N. arbortristis reduced blood glucose levels in alloxan-induced diabetic rats in a dose-dependent manner shown in Figure 1.

Table 2: Effect of Hydro-alcoholic Root Extract of N. arbortristis on Fasting Blood Glucose Level in Alloxan induced Diabetic Rats.

n=6, values are expressed in Mean±S.D of six animals. One Way ANOVA followed by Tukey's multiple comparison test. Statistical significance at * p <0.05, ** p <0.01 respectively, in comparison diabetic control group).

Figure 1: Effect of HARNA on Fasting Blood Glucose Level in Alloxan Induced Diabetic Rats.