Antihyperglycemic and hypolipidemic activity of methanolic extract of Amaranthus viridis leaves in experimental diabetes.

To investigate the antihyperglycemic and hypolipidemic effects of methanolic extract of leaves of Amaranthus viridis (MEAV) in normal and Streptozotocin (STZ) induced diabetic rats. The anti-hyperglycemic and hypolipidemic activity of methanolic extract of leaves of Amaranthus viridis was evaluated by using normal and STZ induced diabetic rats at dose of 200 mg/kg and 400 mg/kg by mouth per day for 21 days. Blood glucose levels and body weight was monitored at specific intervals, and different biochemical parameters, serum cholesterol, serum triglyceride, high density lipoprotein, low density lipoprotein, very low density lipoprotein were also assessed in the experimental animals. Histology of pancreas was performed. The statistical data indicated a significant increase in the body weight, decrease in the blood glucose, total cholesterol and serum triglycerides after treatment with MEAV. High density lipoprotein (HDL) cholesterol level was significantly increased when treated with extract. Histologically, focal necrosis was observed in the diabetic rat pancreas; however, was less obvious in treated groups. The MEAV has beneficial effects in reducing the elevated blood glucose level and body weight changes, and improves the lipid profile of STZ induced rats.

Introduction

According to the International Diabetes federation (IDF), Type-2 diabetes currently affects 246 million people worldwide and is expected to increase to 380 million by 2025. Metabolic syndrome is a combination of related disorders, including impaired glucose tolerance, abdominal obesity, high cholesterol and high blood pressure, which increase the risk of cardiovascular disease and diabetes. Insulin resistance is a key pathophysiologic feature of metabolic syndrome and type-2 diabetes, and is strongly associated with a co-existing cardiovascular risk factors and accelerated atherosclerosis.[1] Therefore, the search for more effective and safer hypoglycemic compounds has continued to be an important area of active research and after the recommendations made by World Helath Organization (WHO) on diabetes mellitus (WHO, 1980), research on hypoglycemic compounds from medicinal plants has become an important aspect of this project.

Amaranthus viridis grows annually as an erect, monoecious herb, and is around 100-300 cm tall. It probably originated in America, and is found throughout India in waste places. It is widespread in tropical and subtropical regions of the world. It is commonly known as slender amaranth or green amaranth (English), Tanduliya, Vishaghna (Sanskrit) and chilikiraesoppu (kannada).[3] The Amaranthus plants (Amaranthaceae) are spread throughout the world, growing under a wide range of climatic conditions and they are able to produce grains and leafy edible vegetables. Nutritionally, Amaranth grain has 2-3 times higher biological value than common cereal grains while containing substantially higher levels of protein with 2-3 times higher lysine content.[4] Current industrial and public interest in the use of Amaranth grain has not only been linked to its recognized nutritional properties, but also to its potential beneficial use as therapeutic adjunct in diets for hypercholesterolemia susceptible individuals.[5]

Recent studies have demonstrated a variety of important and unique nutraceutical type applications for the grains of the Amaranth plant,[5] including its use as an adjunct to lower blood glucose in non-insulin dependent diabetes and in other applications to lower blood serum cholesterol level.[67] Hence, an attempt was made to investigate the leaves of Amaranthus viridis for an antihyperglycemic and hypolipidemic potential.

However, literature indicates that there is no scientific evidence to support the antihyperglycemic and hypolipidemic effects of Amaranthus viridis. The present study investigates the action of methanolic extract of Amaranthus viridis (MEVA) in the different models of rats to ascertain the scientific basis for the use of this plant in the treatment of diabetes and hyperlipidemia.

Materials and Methods

Plant Material

Amaranthus viridis (Amaranthaceae) leaves, collected in May-June, 2008 from Gandhi Krishi Vignan Kendra (GKVK) University of Agricultural Sciences, Bangalore, were authenticated by Dr. Rajanna, the taxonomist of the university. The voucher specimen was then deposited in the herbarium of the Pharmacognosy department, PES College of Pharmacy, Bangalore, Karnataka, India, with reference no. MAV-26.

Extraction

The leaves (60 g) were shade dried and powdered and soxhlet-extracted with methanol (400 ml). The extract was concentrated using rotary evaporator under reduced pressure (yield-4.8% w/w), and was stored in a refrigerator at 4°C, until use for the biological testing and phytochemical screening.

Preliminary Phytochemical Screening

In order to determine the presence of phytoconstituents, a preliminary phytochemical study (colour reactions) with MEAV was performed.[8]

Animals

Throughout the experiment, experimental rats were processed in accordance with the instructions given by our institutional committee for the purpose of control and supervision on experiments on animals (CPCSEA).[9] Healthy Wister rats between 2-3 months of age and weighing 180-200 g were used for the study. Rats were kept in standard polypropylene cage and maintained under standard laboratory conditions of temperature (25±1°C), relative humidity (50±15%), 12 hour light-dark cycles, standard diet and water ad libitum.

Acute Toxicity Studies

The acute oral toxicity study was carried out according to the guidelines set by Organization for Economic co-operation and development (OECD) guidelines. Healthy Wistar rats (150-180 g) were used for this study. The two doses of 2000 mg/kg (by mouth) and 5000 mg/kg (by mouth) of the test samples were given to two groups containing 5 animals in each group. The treated groups were monitored for 14 days, for mortality and general behavior.

Effects of Methanolic Extract of Amaranthus viridis in Normal Rats

The MEAV was subjected to hypoglycemic studies at two dose levels: 200 mg/kg and 400 mg/kg (by mouth) for 21 days. Animals were grouped into 5 groups (n=6): Group 1 served as normal control, received normal saline 10 ml/kg orally;, Group 2 served as vehicle control, and received 3% v/v tween 80 in water 10 ml/kg orally; Group 3 served as standard, and received Glibenclamide at 10mg/kg orally. Group 4 and Group 5 received MEAV at doses 200 mg/kg and 400 mg/kg (by mouth). The treatments were given for 21 days. The blood samples were withdrawn from retro orbital plexus at 1st, 7th, 14th, and 21st day and blood glucose levels was estimated using Glucose oxidase and peroxidase (GOD-POD) kit (Acuurex, India).

Oral Glucose Tolerance Test (OGTT):

Rats were divided into five groups (n=6) and were administered normal saline, diabetic control (STZ, 70 mg/kg), Glibenclamide (10 mg/kg) and dose of 200 mg/kg and 400 mg/kg per oral of MEAV. Glucose solution 2g/kg was administered 30 minutes after the administration of the extract. Blood samples were withdrawn from the retro-orbital region at intervals of 60, 120 and180 minutes of glucose administration. Blood glucose levels’ were estimated using GOD-POD kit (Acuurex, India) (OECD 2001-guideline on acute oral toxicity (AOT) No.425)

Induction of Diabetes

The streptozotocin diabetic rat model was performed as per the method described by Kadnur and Goyal, 2005.[11] Rats were injected intraperitoneally (i.p.) with 70 mg/kg body weight, STZ (sigma chemical Co. U.S.A) dissolved in 0.1 M cold citrate buffer (PH 4.5). Forty eight hours after STZ administration, blood samples were drawn by retro orbital puncture and glucose levels determined to confirm diabetes. The diabetic rats exhibiting blood glucose levels in the range of 275 and 300 mg/100ml were selected for the studies.

Experimental Design

Rats were divided into five groups of six rats (n=6) each. Group 1 and 2 served as control and diabetic untreated control respectively. Group 3 served as standard and was treated with 10 mg/kg/day Glibenclamide (Ranbaxy, India). Group 4 and 5 were treated with methanolic extract of Amaranthus viridis at dose of 200 mg/kg and 400 mg/kg per oral for 21 days. Blood glucose levels and body weights were measured on day 1,7,14 and 21 of the study. Finally on day 21, blood was drawn by retro- orbital puncture technique. Blood samples’ were collected, allowed to clot and then centrifuged at 2500 rpm for 10minutes to obtain serum. Blood glucose was estimated by GOD-POD kit (Accuurex, India). All the lipid profile parameters were determined. Total cholesterol, HDL, low density lipoprotein (LDL), very low density lipoprotein (VLDL) were analyzed from serum (Harold varley). Triglycerides were determined using Hantzsch condensation method (Mac Donald).

Blood samples were drawn by retro-orbital puncture and fasting blood glucose levels were estimated on days 1, 7, 14 and 21 with the help of GOD-POD kit (Acuurex, India). Blood lipid profiles and body weights were determined.

Histopathology of The Pancreas of Streptozotocin Induced Diabetic Rats

On the last day of the study, the animals were sacrificed; quickly dissected and small slices of pancreas samples were fixed in 10% formalin. Thin sections of the tissue, 5-7 μm, were cut and stained with haematoxylin-eosin. The tissue sections were subjected to rehydration by exposing them to decreasing concentrations of alcohol, 100-30% and then stained with haematoxylin. The sections were dehydrated by using increasing concentrations’ of alcohol and then stained with eosin. They were then treated with diphenyxylene (DPX) and examined under the microscope.[12]

Statistical Analysis

The results are expressed as mean ± standard error of the mean (SEM). Statistical difference was tested by using one-way analysis of variance (ANOVA) followed by Dunnetts test. A difference in the mean P value < 0.05 was considered as significant.

Results

The percentage yield of MEAV was found to be 4.8% w/w respectively. The methanol extract contained glycosides, saponins, flavonoids, proteins, amino acids and carbohydrates.

Acute Toxicity Study

The various observations showed the normal behavior of the treated rats. No toxic effects were observed at a higher dose of 5 g/kg body weight. Hence, there were no lethal effects in any of the groups till the end of the study.

Effect of Methanolic Extract of Amaranthus viridis on Blood Glucose in Normal Rats

At dose 200 mg/kg and 400 mg/kg of MEAV, fasting blood glucose levels were assessed in normal rats at various time intervals [Figure 1].

Figure 1

Effect of Methanolic extract of leaves of Amaranthus viridis (MEAV) on blood glucose in normoglycemic rats during the study

Effect of Methanolic Extract of Amaranthus viridis on Blood Glucose in Glucose Fed Hyperglycemic Rats

At dose 200 mg/kg and 400 mg/kg of MEAV, blood glucose levels were assessed in glucose fed rat at various intervals [Figure 2].

Figure 2

Effect of Methanolic extract of leaves of Amaranthus viridis (MEAV) on blood glucose in glucose fed hyperglycemic rats

Anti-diabetic activity of methanolic extract of Amaranthus viridis in STZ induced diabetic rats

The results from the study clearly indicated that the methanolic extract exhibited significant hypoglycemic activity in STZ induced diabetic rats. At the end of 21 days of treatment, there was a 57.64%, 65.06% decrease (P < 0.01) of blood glucose levels with the methanolic extract (200 mg/kg and 400 mg/kg) [Figure 3]. The standard drug glibenclamide also indicated a significant decrease (70.80%) of blood glucose levels.

Figure 3

Anti-diabetic activity of Methanolic extract of leaves of Amaranthus viridis(MEAV) in Streptozotocin induced diabetic rats

Changes in Body Weight

At the end of 21 day treatment, the body weight of methanolic extract at 200 mg/kg and 400 mg/kg and standard drug treated group, increased significantly (P < 0.01) by 45.33%, 84.59%, 58.19%.

Changes of Histopathology of the Pancreas

After 21 days treatment period, the histopathological examination of the pancreas of the non diabetic rats showed round and elongated islets which were evenly distributed throughout the cytoplasm. In diabetic control animals, the cells were irregular, not well defined and necrosis of the cells could be clearly appreciated. The standard group showed a mild protection from STZ induced changes in the pancreatic islets. Methanolic extract at 200 mg/kg showed no significant changes in the cells when compared with the diabetic control. Methanolic extract at 400 mg/kg showed slight regeneration of beta cells when compared with the diabetic control [Figure 4].

Figure 4

Histological changes of rat pancreas of islets of langerhans a) Non diabetic normal histological structure of rat pancreas showing normal islet b) Diabetic control rat showing irregular cells, not well defined necrosis of cells is very clear c) Glibenclamide (10 mg/kg. p.o) treated rat pancreas showing mild protection from STZ-induced changes in the pancreatic islets d) MEAV extract (200mg/kg) showed no significant changes in cells were seen when compared with diabetic control e) MEAV (400 mg/kg) showed slight regeneration of β-cells were seen when compared with diabetic control

Lipid Profile

When compared to the diabetic control rats, significant (P < 0.01) reductions of 18.69%, 28.72% CHL (Cholesterol), 36.70%, 51.35% LDL, 31.28%, 39.87% VLDL and 27.34%, 39.48% TGL (Triglyceride) were found after treatment with methanolic extract of Amaranthus viridis leaves at doses of 200 mg/kg and 400 mg/kg. Also, there was a significant (P < 0.05) increase of (10.2%, 18.36%) HDL cholesterol in treated diabetic rats. In case of untreated diabetic rats, there was a fall in HDL level [Figure 5].

Figure 5

Anti-hyperlipidemic effect of Methanolic extract of leaves of Amaranthus viridis (MEAV) in streptozotocin induced diabetic rats

Discussion

The present manuscript discusses about the antihyperglycemic and hypolipidemic effects of the methanolic leaf extract of Amaranthus viridis on normal and STZ-induced diabetic rats. Acute toxicity studies revealed the non-toxic nature of the methanolic leaf extract of Amaranthus viridis. There was no lethality or any toxic reactions found with the selected dose until the end of the study period. The basal food intake of normal group rats were found to be 14.3 ± 0.2 g/rat/day whereas the food intakes were significantly (18.4 ± 0.2) increased in the diabetic group of rats (compared with normal), but no change in food intake was observed (14.5 ± 0.2) in the standard and sample treated rats.

The fall in blood glucose levels in normal rats were found to be significant (P < 0.05) at 400 mg/kg per oral on 14th day of the study, which was in accordance with the standard drug glibenclamide. However, MEAV 200 mg/kg doses have also shown significant fall in blood glucose level (P < 0.01) on 21st day of the study. The fundamental mechanism in diabetes mellitus involves the over production (excessive hepatic glycogenolysis and gluconeogenensis) and decreased utilization of glucose by the tissues.[16] STZ, is a slightly cytotoxic agent of pancreatic β-cells[13] and selectively destroys the pancreatic insulin secreting beta cells, leaving less active cells and resulting in a diabetic state.[14] Hence, in the present study, we observed an increased level of blood glucose in STZ induced rats. The hyperglycemic activity of MEAV has shown a significant (P < 0.05) fall in blood glucose level from 7th day onwards in both the doses of 200 mg/kg and 400 mg/kg per oral. STZ - induced diabetes is characterized by severe loss in body weight,[15] and this reduction is due to loss or degradation of structural proteins, as the structural proteins are known to contribute to body weight. In our study, a significant weight loss was observed in the diabetic group and significant improvement in weight was observed in the groups treated with MEAV. This may be due to the presence of 20% protein, all 8 essential amino acids (high in lysine, threonine and tryptophan), vitamins, calcium, minerals in MEAV to reduce hyperglycemia.

Flavonoids are one of the most numerous and wide spread groups of phenolic compounds in higher plants.[17] Some of them, due to their phenolic structure are known to be involved in the healing process of free radical mediated diseases including diabetes.[18] The MEAV possesses flavonoids as the anti-diabetic principle agent. Hyperglycemia is accompanied with dyslipidemia[19] and represents a risk factor for coronary heart diseases.This abnormally high level of serum lipids is mainly due to the uninhibited actions of lipolytic hormones on the fat depots, mainly due to the action of insulin. Under normal circumstances, insulin activates the enzyme lipoprotein lipase, which hydrolyses triglycerides. However, in diabetic state lipoprotein lipase is not activated due to insulin deficiency, resulting in hyper triglyceridemia,[20] and insulin deficiency is also associated with hyper cholesterolemia due to metabolic abnormalities.[21] The dyslipidemia is characterized by increase in TC, LDL, VLDL, TG and fall in HDL. This altered serum lipid profile was reversed towards normal after treatment with methanolic extract of Amaranthus viridis. The hypothesis is further supported by the pancreatic histology which showed protection of pancreatic β-cells from toxic effects of STZ and focal necrosis was observed in the diabetic rat pancreas; however, was less obvious in treated groups. From the above result, we can confirm that the methanolic extract of leaves of Amaranthus viridis at doses of 200 mg/kg and 400 mg/kg possesses significant antihyperglycaemic activity with long-term (21 day) treatment in rats. The sample drug showed optimum activity at 400 mg/kg.

On the basis of the current investigation, it was noted that the methanolic extract of Amaranthus viridis acted in a similar fashion to the glibenclamide (standard drug) in reducing the elevated blood glucose level and lipid profile of STZ induced diabetic rats (thus, justifying the claim made in Ayurvedic classics); and it can be suggested that these results provide pharmacological evidence for its folklore claim as an antidiabetic and hypolipidemic agent. Further studies to isolate, identify and characterize the active principle(s) are in the progress. The antihyperglycemia activity may be attributed to some of its active principles.