INTRODUCTION:

Diabetes mellitus (DM) is a chronic complicated metabolic disorder characterized by increased blood glucose level resulting from the defects in insulin secretion, insulin action, or both. Hyperglycemia is suggested to be one of the major cause contributing to diabetic complication.^[1]The incidence of DM is rising at alarming rate all over the word in the coming years. According to the diabetic atlas of the International Diabetic Federation, 382 million people were affected by diabetes worldwide in the year 2013 and diabetes prevalence is expected to 592 million by the year 2035.^[2]The World Health Organization projects that diabetes will be the 7^th leading cause of death in 2030.^[3]

Management of diabetes is a global health problem and successful treatment is yet to be discovered.^[4]Currently available therapies for diabetes include insulin and various oral antidiabetic agents such as sulfonylureas, biguanides and glinides. Many of them have a number of serious adverse effects; therefore, the search for more effective and safer hypoglycemic agents is one of the important areas of investigation.^[5]

Medicinal plants are rich source of various bioactive phytochemicals. Many Indian medicinal plants have been found to be useful in the management of diabetes acting through variety of mechanisms. Medicinal plants provide better alternatives as they are less toxic, easily available and affordable and many of the currently available drugs have been derived directly or indirectly from them.^[6,
7]

MATERIALS AND METHODS:

Plant material collection, processing, preparation of extracts and isolation of fraction:

The leaves of Ammania baccifera Linn were collected December from the nearby regions of Bhopal in the month of December. The plant was identified by Department of Botany, Safia College, Bhopal M.P. Leaves were shade dried for a month,and powdered in mechanical grinder and stored in airtight container.

The air dried powdered leaves (1000g) were defatted with petroleum ether and remaining marc was extracted with methanol (70%v/v) and concentrated in rotary evaporator under reduced pressure to get methanol extract (100.5g). The methanol extract was chromatographed on silica gel column (70cmX15cm, 60-120mesh, 2kg) chromatography and preparative TLC.

Column was first eluted with chloroform, then polarity of mobile phase was gradually increased by adding methanol in different concentrations (100:0, 95:5, 90:10, 85: 15, 80:20, 70:30, 60:40v/v).

178 fractions each of 50mL were collected and TLC was performed of each fraction individually and eluates were monitored for the presence of various constituents. Fractions were pooled on the basis of their TLC profile, pooled fractions (40-65) were selected for the isolation of constituents. Further purification was performed by preparative TLC of isolated constituents to offered AB1.

Preliminary phytochemical test for phytoconstituents:

The freshly prepared extract was subjected for qualitative chemical tests for the presence various phytoconstituents viz. alkaloids, glycosides, tannins, flavonoids and phenolic compounds, fats, resins, steroids, proteins and amino acids etc. ^[8]

Chemicals:

STZ and glibenclamide was obtained from Sigma-Aldrich Company (Bangalore, India). The other experimental chemicals used were of analytical grade and were purchased from Hi Media (Mumbai, India).

Acute oral toxicity studies:

Healthy Male Albino mice 220-250 g (Wistar strain) were selected for acute oral toxicity. Animals were acclimatized with free access to food and water. The study group used 6 animals in each group.Animals were kept fasting for overnight providing only water, after that the extracts were administered orally at the dose level of 5 mg/kg bodyweight intragastric tube and observed for 14 days. If mortality was observed in 2-3 animals then the dose assigned was assigned as toxic dose. If mortality was observed in one animal, then the same dose was repeated again to confirm the toxic dose. If the mortality was not observed, the procedure was repeated for further higher dose such as 50, 250, 500 and 2000mg/kg body weight. ^{[8, 9]}

Experimental animals:

Adult male albino Wistar rats with body weight (b.w.) above 180g -200g were taken for the study. They have been housed at poly propylene confines and kept up in standard environment (12 h light and 12 h dark cycle, (25 ± 3) °C). The rats were fed with standard rat pellet diet and given water ad libitum and maintained at Central Animal House. All studies were conducted as per the guideline of CPCSEA, and the study was endorsed by the Institutional Animal Ethical Committee of Vedica College of B. Pharmacy, RKDF University, Bhopal. Animals were adapted for 3 days in the research laboratory before start of the experiments.

Experimental induction of diabetes:

Diabetes was prompted through single intraperitoneal injection of freshly prepared streptozotocin (STZ) (50 mg/ kg b.w.) in 0.1 M citrate buffer (pH = 4.5) to overnight starved rats^[10]. Diabetic rats were permitted to drink 20% glucose solution overnight to overcome the initial drug induced hypoglycemic death. The blood glucose level was measured after three days, and rats with glucose levels >250 mg/dL were considered as diabetic. At the time of induction, control rats were injected with 0.2 mL of vehicle (0.1Mcitrate buffer, pH 4.5) alone.

Experimental design:

In this experiment 42 rats (6 normal and 36 STZ diabetic existing rats) were used. They were separated into seven groups of 6 rats each. The methanolic extracts of Ammania baccifera Linn leaf, AB1 and glibenclamide 0.5 mL of 0.9% saline and administered orally (45 days).^[11]

Group I. Control rats (were given 0.5 mL of 0.9% saline orally for 45 days).

Group II. Diabetic group (STZ 50 mg/kg b.w.).

Group III. Diabetic rats were given methanolic extracts of Ammania baccifera Linn leaf (250 mg/kg b.w. dissolved in 0.5 mL of 0.9% saline) orally for 45 days.

Group IV. Diabetic rats were given methanolic extracts of Ammania baccifera Linn leaf (500 mg/kg b.w. dissolved in 0.5 mL of 0.9% saline) orally for 45 days.

Group V. Diabetic rats were given isolated fraction (AB1) from methanolic extracts of Ammania baccifera Linn leaf (25 mg/kg b.w. dissolved in 0.5 mL of 0.9% saline) orally for 45 days.

Group VI. Diabetic rats were given isolated fraction (AB1) from methanolic extracts of Ammania baccifera Linn leaf (50 mg/kg b.w. dissolved in 0.5 mL of 0.9% saline) orally for 45 days.

Group VII. Diabetic rats were given Glibenclamide (600 μg/ kg b.w. dissolved in 0.5 mL of 0.9% of saline) for 45 days ^[12]

Toward the study’s end (45 days), the animals were euthanized by ketamine (24 mg/kg/body) intramuscular injection and sacrificed by cervical dislocation between 9:00 and 11:00 am to minimize diurnal variation. The blood was gathered. The liver was dissected and washed with ice-cold saline immediately to remove blood. Fresh/frozen liver (1 g) was slashed and homogenized in ice-cold sucrose (15 mL, 250 mM) with a Potter-Elvehjem homogenizer for 2 minutes, centrifuged at 10,000 rpm for 30 minutes, and the supernatant was gathered and utilized as the source for different biochemical estimation. The body weight of all the animals was recorded proceeding to the treatment and sacrifice.

Biochemical analysis:

Estimation of blood glucose^[13]

Glucose level in plasma was determined by glucose oxidase/ peroxidase method using areagent kit. In brief, to 0.01 mL of plasma, standard and distilled water (blank) into 3 test tubes, 1.0 mL of the enzyme was added, mixed and kept at 37 °C for 15 minutes. The color developed was read at 505 nm against reagent blank.

Qualitative determination of plasma insulin^[14]

The plasma insulin was assayed by Enzyme Linked Immunosorbent Assay (ELISA) method using Boehringer- Mannheim kit. In brief, 0.1 mL of plasma was injected into the plastic tubes coated with monoclonal antiinsulinantibodies. Phosphate buffer and anti-insulin POD conjugate was added to form anti-insulin antibody–POD conjugate. Substrate chromogen solution was then added to form indicators reaction. A set of standards were also treated in a similar manner. After the development of color the absorbance was read at 420 nm.

Determination of hemoglobin^[15]

Hemoglobin content in blood was determined by the cyanmethemoglobin method. In brief, the reaction mixture in a volume of 5.02 mL contained 5 mL of Drabkin’s reagent and 0.02 mL of blood. The reaction mixture was kept at room temperature for 5 min to ensure the completion of the reaction. The solution was read at 540 nm together with the standard solution of cyanmethemoglobin.

Estimation of glycosylated hemoglobin (HbA1c)^[16]

The saline washed erythrocytes (0.5 mL) were lysed with 5.5 mL of water, mixed and incubated at 37°C for 15 minutes. The contents were centrifuged and the supernatant was discarded, then 0.5 mL of saline was added, mixed and processed for estimation. To 0.02 mL of aliquot, 4 mL of oxalate hydrochloric solution was added and mixed. The contents were heated at 100 °C for 4 hours, cooled and precipitated with 2 mL of 40% TCA. The mixture was centrifuged and to 0.5 mL of supernatant, 0.05 mL of 80% phenol and 3.0 mL of concentrated H₂SO₄were added. The color developed was read at 480 nm after 30 minutes.

Estimation of liver glycogen^[17,18]

A known amount of the tissue was subjected to alkali digestion in a boiling water bath for 20 minutes after addition of 5 mL of 30% potassium hydroxide. The tubes were cooled and 3.0 mL of absolute ethanol and a drop of ammonium acetate were added. The tubes were then placed in a freezer overnight to precipitate the glycogen. The precipitated glycogen was collected after centrifugation at 3000× g for 10 minutes. The precipitate was washed thrice with alcohol and dissolved in 3 mL of water. Aliquots were taken and 4 mL of anthrone reagent was added to the tubes kept in an ice bath, mixed and heated in a boiling water bath for 20 minutes. The green color developed was read at 640 nm. Working standard glucose and a blank were treated similarly.

Statistical analysis:

All the results are expressed as mean ± SD, analysed by one-way ANOVA followed by Tukey’s multiple comparison analysis as post-hoc test. The P<0.05 was considered to be statistically significant.

RESULTS:

Phytochemical Screening:

The phytochemical screening of methanolic extract of Ammania baccifera revealed the presence of alkaloids, terpenoids, sterols, glycosides, phenolic compounds, tannins and flavonoids, carbohydrates and proteins and amino acids.

Acute Toxicity studies:

Acute oral toxicity studies showed no mortality up to the dose of 2000 mg/kg body weight. So, the selected extract is safe for long term use.

Hypoglycemic effect of Ammania baccifera leaves methanolic extract (250 and 500 mg/kg) in oral glucose tolerance test (OGTT):

Figure 1.1, shows the changes in the blood glucose levels in normal control and different experimental groups after oral administration of glucose (2g/kg b.w.). In all experimental groups oral feeding of glucose induced a significant elevation in blood glucose after 30 minutes as compared with corresponding values on 0 min. After 120 min the blood glucose level in normal control rats tend to return near normal level. The untreated diabetic rats showed maximum increase in blood glucose after 60 min and mild decline after 90 min. In diabetic rats treated with 500 mg/kg b.w. of methanolic extract of Ammania baccifera Linn leaf and Glibenclimide (group, the blood glucose level showed continues decline after 60 min and after 120 min the level reached to near initial value at 0 min.

Effects of methanolic extract and its isolated fraction of Ammania baccifera leaves on body weight (g) in STZ-induced diabetic rats:

Changes in body weight of the normal control, diabetic control and experimental rats are depicted in figure 1.2. The mean body weight of the rats of normal control group was significantly elevated after the 15, 30, and 45 days of treatment period when compared to their initial body weight. In contrast to this, the mean body weight of untreated diabetic control rats (group II) was significantly decreased (P≤0.05) after the 15, 30, and 45 days of experimental period when compared with their initial body weight (0 day).

Diabetic rats treated with methanolic extract of Ammania baccifera Linn leaf extract at different doses (250 and 500 mg/kg b.w./day). The dose at 500mg/kg b.w. showed stastically significant dose dependent decrease in mean body weight (P≤0.001).

The isolated fraction i.e. AB at dose 25mg/kg b.w. showed most significant decrease in mean body weight at 45^th day (P≤0.01) of treatment compared with their initial body weight (0 day).

In diabetic rats treated with glibenclamide, the body weight gain was significantly (P≤0.001) decreased after 15, 30 and 45 days of treatment period as compared with their initial body weight (0 day).

DISCUSSION:

Preliminary phytochemical screening revealed the presence of alkaloids, terpenoids, sterols, glycosides, phenolic compounds, tannins and flavonoids, carbohydrates and proteins and amino acids in methanolic extract of Ammania baccifera leaves.

In the present study, the continuous treatment with the methanolic extract showed significant hypoglycemic activity in OGTT and normoglycemic rats.

The increase in level of glycosylated hemoglobin is directly proportional to the decreased level of total hemoglobin in diabetic control rats. Glycosylated hemoglobin is used as one of the standard diagnosis method for estimating the extent of protein glycation in diabetes mellitus. The glycosylated hemoglobin level tells information about status and complications of diabetes mellitus. On oral administration of methanolic extract and its isolated fraction significantly decreased the glycosylated hemoglobin level possibly due to normoglycemic control mechanisms in experimental rats.

A marked increase in serum concentration of TC, TG, LDL and decreased HDL was observed with diabetic rats than normal control group which is often linked with hyperlipidaemia. Our result showed significantly (P<0.05) fall in TC, TG, and LDL levels as well at the same time raised HDL level near to control on oral administration of methanolic extract (500 mg/kg) and its isolated fraction (50mg/kg) after 45 days repeatedly. This implies that plant may possess insulin-like activity which would be helpful to reduce the lipid related complications. The significant control on serum lipids may prevent hypercholesterolemia and hypertriglyceridemia and also reduce the cardiovascular risk factors.

Increased serum concentration of qualitative diagnostic enzymes such as SGPT and SGOT were observed in diabetic rats indicating an altered liver function or liver mitochondrial injury in comparison to normal control rats. Insulin deficiency contributes to increased serum level of transaminase enzymes due to easily availability of amino acids which leads to enhanced occurrence of gluconeogensis and ketogenesis processes during diabetes. On treatment with methanolic extract and its isolated fraction significantly reversed the elevated marker enzymes i.e. SGOT and SGPT and restored to normal values indicates are vital of insulin secretion into circulations and also its hepatoprotective effect.

Reports of earlier studies suggested that various plants was proved to possessing wide variety of natural antioxidant constituents such as tannins, saponins, alkaloids, flavonoids, phenolic acids and poly phenols etc. which enhances free radical scavenging activities and responsible to change in antioxidant enzymes which may be helpful for treatment of diabetic related complications. The methanolic extract and its isolated fraction found to be potential anti-diabetic extract in STZ-induced diabetic model through reducing oxidative damage and modulating antioxidant enzymes by dose dependent manner.

CONCLUSION:

The Results of the study scientifically validates the traditional use of Ammania baccifera leaves. It may be concluded that methanolic extract of the plant at 500mg/ kg b.w. and its isolated fraction AB possess hypoglycemic activities but the isolated fraction exhibited the most significant hypoglycemic effect as compare to the methanolic extract of Ammania baccifera leaves and the isolated fraction may be used as hypoglycemic agent.

According to our findings, the methanolic extracts of Ammania baccifera Linn leaves have proved to possess antidiabetic activity in STZ induced diabetic rats, which might be due to increase in plasma insulin levels, and this would be responsible for the restoration of carbohydrate metabolizing enzymes and liver glycogen. Results of methanolic extracts of Ammania baccifera Linn at the dose of 250 and 500mg/kg b.w. showed significant anti-diabetic activity in dose dependent manner as compared to the standard drug glibenclamide.

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Received on 24.05.2018 Modified on 30.06.2018

Research J. Pharm. and Tech 2018; 11(11): 4773-4780.

DOI: 10.5958/0974-360X.2018.00869.7