Hypoglycemic activity of Erythrina variegata leaf in streptozotocin-induced diabetic rats

Abstract

Context: Erythrina variegata Linn. (Fabaceae), commonly known as Tiger’s Claw, is a thorny deciduous tree grown in tropical and subtropical regions of Eastern Africa, Southern Asia, and Northern Australia. In India, its leaves are traditionally used for diabetes mellitus.

Objective: To evaluate the hypoglycemic activity of methanol extract of E. variegata leaf (MEEV) in streptozotocin (STZ)-induced diabetic Wistar rats.

Materials and methods: Hyperglycemia was induced in rats by single intraperitoneal injection of STZ (55 mg/kg body weight). Three days after STZ induction, the hyperglycemic rats were treated with MEEV orally at the doses of 300, 600, and 900 mg/kg body weight daily for 21 days. Glibenclamide (1 mg/kg, orally) was used as reference drug. The fasting blood glucose levels were measured on every 7th day during the 21 days of treatment. Serum biochemical parameters including lipid content were estimated.

Results and discussion: MEEV at the doses of 300, 600, and 900 mg/kg orally significantly (P < 0.01) and dose-dependently reduced and normalized blood glucose levels as compared to that of STZ control group; the dose 900 mg/kg being the most potent showing complete normalization of blood glucose levels. Serum biochemical parameters including lipid profile were significantly (P < 0.01) restored toward normal levels in META-treated rats as compared to STZ control animals.

Conclusion: This study concludes that E. variegata leaf demonstrated promising hypoglycemic action in STZ-induced diabetic rats substantiating its ethnomedicinal use.

Keywords::

• Hypoglycemic
• glibenclamide
• streptozotocin
• diabetes

Introduction

Diabetes mellitus is a metabolic disorder with the highest rate of prevalence and mortality worldwide (Powers, 2008), affecting 25% of the population, afflicting 150 million people, predicted to rise to 300 million by 2025 (Vats et al., 2000). Plants have long been a source of traditional antidiabetic medications. Although different types of oral hypoglycemic agents are available along with insulin for the treatment of diabetes mellitus, certain adverse effects and weak effectiveness of them have led to the search for more effective hypoglycemic agents. Hence, there is an increased investigation toward the antidiabetic plant products that are frequently preferred by the patients. Herbal preparations alone or in combination with oral hypoglycemic agents sometimes produce a good therapeutic response in some resistant cases, where modern medicines alone do not work (Anturlikar et al., 1995). The major merits of herbal medicine seem to be its efficacy, low incidence of side effects, and low cost.

Erythrina variegata Linn. (Fabaceae), commonly known as Tiger’s Claw, is a thorny deciduous tree grown in tropical and subtropical regions of Eastern Africa, Southern Asia, and Northern Australia. Different parts of E. variegata have been used traditionally as nervine sedative, febrifuge, antiasthmatic, antiepileptic for treatment of convulsion, fever, inflammation, bacterial infection, insomnia, helminthiasis, cough, cuts, and wounds (Warrier et al., 1997; Anon., 2002; Rastogi & Mehrotra, 2006). Its leaf has been ethnomedicinally used in India for treatment of diabetes mellitus (Monique & Melanie, 2006). Previous workers reported antibacterial and analgesic activities of its leaf (Sato et al., 2003; Haque et al., 2006). However, there are no experimental reports demonstrating its antidiabetic potential. This study therefore investigated the hypoglycemic effect of the methanol extract of E. variegata leaf (MEEV) against streptozotocin (STZ)-induced diabetic Wistar rats to justify the traditional and folkloric attributes.

Materials and methods

Plant material

The leaves of E. variegata were collected during September 2009 from Majhitar region of Sikkim state, India. The plant species were identified with the help of available literature (Gurung, 2002) and authenticated by Dr. A. K. Panda, Taxonomist, Botanical Survey of India (BSI), Gangtok branch, Sikkim, India; a voucher specimen (No. HPI/AK-01) was deposited at the Department of Pharmacology, Himalayan Pharmacy Institute, India for future reference. The collected leaves were thoroughly washed with running tap water and shade dried (24–26°C) for 3–4 weeks and ground mechanically into a coarse powder.

Drugs and chemicals

STZ was from Sigma Chemical Co., USA. Glibenclamide was from Hoechst, India. All other reagents used were of analytical grade obtained commercially.

Preparation of extract

The powdered plant material (400 g) was extracted using Soxhlet extraction apparatus with 95% aqueous methanol. The extract was filtered and evaporated to dryness in vacuo at 40°C temperature to yield the dry extract (MEEV, 13.13%). The dry extract was kept in a vacuum desiccator until use. Preliminary phytochemical analysis (Harborne, 1998) revealed the presence of flavonoids, alkaloids, and steroids in MEEV.

Animals

Adult male Wistar albino rats weighing 170–200 g were used for the study. All the animals were under the age of 8–12 weeks. They were housed in a clean polypropylene cage and maintained under standard laboratory conditions (temperature 25 ± 2°C with dark/light cycle 12/12 h). They were fed with standard pellet diet (Hindustan Lever, Kolkata, India) and water ad libitum. The animals were acclimatized to laboratory conditions for 1 week prior to experiment. All experimental methods were reviewed and approved by Institutional Animal Ethics Committee, Himalayan Pharmacy Institute (Reg. no. HPI/09/60/IAEC/0074).

Acute toxicity

MEEV was administered orally to male Swiss albino mice to evaluate the acute toxicity as reported previously (Anon., 2008).

Oral glucose tolerance test (OGTT)

The OGTT was performed in overnight fasted normal rats. Rats were divided into five groups (n = 6). Group I served as normal control (NC) and received distilled water (5 ml/ kg b.w., p.o.) and groups II, III, and IV received MEEV at the doses of 300, 600, and 900 mg/kg b.w., p.o., respectively. Group V received glibenclamide 1 mg/kg b.w. p.o. 30 min after these treatments, all groups received glucose (4 g/ kg b.w.) orally. Blood was withdrawn from the tail vein just prior to and 30, 60, and 120 min after the oral glucose administration (Schoenfelder et al., 2006; Barik et al., 2008). Blood glucose levels were measured using glucose oxidase-peroxidase reactive strips and a portable glucometer (AccuSure blood glucose monitoring system).

Induction of experimental diabetes mellitus

The rats were rendered diabetic by a single intraperitoneal dose of 55 mg/kg b.w. STZ freshly dissolved in ice-cold 0.1 M citrate buffer (pH 4.5). After 72 h, fasting blood glucose (FBG) levels were measured and only those animals showing blood glucose level ≥ 225 mg/dl were used for the following investigation. The day on which hyperglycemia had been confirmed was designated as day 0 (Li et al., 2007; Schmatz et al., 2009).

Treatment schedule and estimation of FBG level

Normal and hyperglycemic rats were divided into seven groups (n = 6) receiving the following treatments (Mohammadi & Naik, 2008):

• Group I: Nondiabetic or normal control, received the vehicle, that is distilled water 5 ml/kg b.w., p.o. (NC).

• Group II: Nondiabetic control, received MEEV 600 mg/ kg b.w., p.o.

• Group III: Diabetic control, received the vehicle, that is distilled water 5 ml/kg b.w., p.o. (DC).

• Group IV: Diabetic treatment, received MEEV 300 mg/ kg b.w., p.o.

• Group V: Diabetic treatment, received MEEV 600 mg/kg b.w., p.o.

• Group VI: Diabetic treatment, received MEEV 900 mg/ kg b.w., p.o.

• Group VII: Diabetic treatment, received glibenclamide 1 mg/kg b.w., p.o.

The above treatment was continued daily for 21 days. Fasting blood glucose concentrations were measured with a portable glucometer (AccuSure blood glucose monitoring system) at days 0, 7, 14, and 21.

Body weight

The body weights of rats of each group were recorded on 1st, 7th, and 15th day of MEEV treatment.

Estimation of serum biochemical parameters

After 21 days treatment, blood samples were drawn from overnight fasted rats by retroorbital venipuncture technique from light-ether anesthetized animals. The nonheparinized blood was allowed to coagulate before being centrifuged (4000 rpm for 20 min) and the serum separated. Serum levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDLC), high-density lipoprotein cholesterol (HDLC), glycosylated hemoglobin (HbA_1C), aspartate transaminase (AST), alanine transaminase (ALT), and alkaline phosphatase (ALP) were estimated enzymatically using commercially available reagent kits (Erba Diagnostics and Span diagnostics Ltd.).

Statistical analysis

The data were expressed as mean ± standard error of mean (SEM). Statistical significance was analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test of significance using Graph Pad (Instat) software version 4.0. P values of < 0.05 were considered as statistically significant.

Results

Acute toxicity

The MEEV did not show any toxic effect or death up to the dose of 5000 mg/kg, b.w., p.o. in mice.

Oral glucose tolerance (OGTT)

Effects of the MEEV on glucose-loaded rats are shown in Table 1. Results of the OGTT strongly supported the improved ability of glucose tolerance with treatment of MEEV and glibenclamide. Among the groups, the concentrations of blood glucose baseline (0 min) were not significantly different. Although plasma glucose levels were increased after loading with glucose, animals treated with MEEV at 600 and 900 mg/kg showed slight increase when compared with the NC group at 30, 60, and 120 min during OGTT. Glibenclamide significantly blocked (P < 0.01) the increase in blood glucose levels after glucose administration at 120 min.

Table 1.  Effect of methanol extract of Erythrina variegata leaf (MEEV) on oral glucose tolerance in normal rats.

Time (min)	Blood glucose level (mg/dl)
	Control glucose (4 g/kg)	MEEV 300 mg/kg + glucose (4 g/kg)	MEEV 600 mg/kg + glucose (4 g/kg)	MEEV 900 mg/kg + glucose (4 g/kg)	Glibenclamide (1 mg/kg) + glucose (4 g/kg)
0	85.83 ± 3.60	77.52 ± 4.25	91.33 ± 1.17	88.16 ± 2.53	78.16 ± 5.01
30	135.65 ± 3.94	132.16 ± 2.27	117.16 ± 2.28**	103.83 ± 1.91**	80.05 ± 3.31**
60	121.79 ± 4.42	114.50 ± 3.38*	102.13 ± 3.12**	91.50 ± 1.33**	70.50 ± 2.93**
120	115.66 ± 2.89	101.31 ± 3.05**	95.27 ± 1.43**	84.91 ± 2.56**	68.29 ± 4.05**

Values are mean ± SEM (n = 6).

*P < 0.05, **P < 0.01 when compared with control group at corresponding time.

FBG levels

Fasting blood sugar levels measured in normal and STZ-induced diabetic rats after a single day and at the end of 7, 14, and 21 days of treatment are given in Table 2. Here, diabetic rats had a significant effect on blood glucose response after treat for 21 days. NC rats did not show any significant variation in the blood glucose throughout the experimental period. Administration of STZ (55 mg/ kg, i.p.) led to several fold elevation of blood glucose levels relative to that of the NC group, indicating stable hyperglycemia during the experimental period. FBG level of normal animals treated with MEEV at 600 mg/ kg (Group II) did not vary significantly from that of the NC group. Although MEEV at 300 and 600 mg/ kg reduced hyperglycemia significantly (P < 0.01) as compared to the diabetic control (DC) group, it failed to restore the level to that of the NC group; while MEEV at 900 mg/ kg or glibenclamide (1 mg/kg) significantly (P < 0.01) reduced the blood glucose levels close to NC group level.

Table 2.  Effect of methanol extract of Erythrina variegata leaf (MEEV) on fasting blood glucose levels in normal and streptozotocin (STZ)-induced diabetic rats.

Treatment	Fasting blood sugar level (mg/dl)
	0 day	7th day	14th day	21st day
Normal control	82.43 ± 4.30**	86.66 ± 4.37**	81.39 ± 5.28**	83.20 ± 6.69**
NC + MEEV 600 mg/kg	95.16 ± 5.12**	85.50 ± 5.10**	80.60 ± 5.62**	76.61 ± 5.00**
STZ control (55 mg/kg)	367.80 ± 8.36^¶	535.50 ± 21.26^¶	438.76 ± 25.29^¶	310.87 ± 22.94^¶
STZ + MEEV 300 mg/kg	312.23 ± 22.84^¶	280.49 ± 19.69**^¶	176.77 ± 16.66**^¶	117.16 ± 9.38**
STZ + MEEV 600 mg/kg	425.16 ± 22.36^¶	284.65 ± 20.29**^¶	165.66 ± 15.63**^¶	99.36 ± 9.60**
STZ + MEEV 900 mg/kg	428.66 ± 22.75^¶	199.35 ± 18.20**^¶	103.28 ± 8.55**	62.53 ± 3.10**
STZ + Glibenclamide 1 mg/kg	378.18 ± 16.12^¶	175.95 ± 11.36**^¶	70.90 ± 4.94**	55.15 ± 3.73**

Values are mean ± SEM (n = 6).

**P < 0.01 when compared with STZ control group; ^¶P < 0.01 when compared with normal control group.

Effect on body weight

The effect of MEEV on body weight of normal and diabetic animals is presented in Table 3. NC animals were found to be stable in their body weight but diabetic rats showed significant reduction in body weight during 21 days. STZ caused body weight reduction, which was significantly (P < 0.01) reversed by MEEV treatment.

Table 3.  Effect of methanol extract of Erythrina variegata leaf (MEEV) on body weight in normal and streptozotocin (STZ)-induced diabetic rats.

Treatment	Body weight (g)
	0 day	7th day	14th day	21st day
Normal control	165.16 ± 1.66	178.66 ± 3.57**	188.00 ± 2.30**	195.17 ± 2.98**
NC + MEEV 600 mg/kg	157.83 ± 2.89	181.00 ± 2.85**	191.83 ± 1.30**	198.16 ± 2.54**
STZ control (55 mg/kg)	162.33 ± 1.77	152.50 ± 1.68^¶	139.87 ± 2.08^¶	123.00 ± 1.71^¶
STZ + MEEV 300 mg/kg	154.82 ± 3.15	162.16 ± 1.40^§	169.12 ± 2.67**^§	181.33 ± 1.94**^¶
STZ + MEEV 600 mg/kg	161.87 ± 2.90	169.33 ± 1.87**	178.16 ± 2.15**	188.71 ± 2.67**
STZ + MEEV 900 mg/kg	159.28 ± 3.20	171.29 ± 1.85**	182.27 ± 1.56**	193.56 ± 2.58**
STZ + Glibenclamide 1 mg/kg	162.50 ± 3.01	177.50 ± 61**	185.61 ± 2.78**	194.16 ± 1.13**

Values are mean ± SEM (n = 6).

**P < 0.01 when compared with STZ control group; ^§P < 0.05 and ^¶P < 0.01 when compared with normal control group.

Serum biochemical parameters

Results of biochemical parameters are represented in Table 4. MEEV had a significant (P < 0.01) effect in lowering HbA_1C. After 21 days, the effect of MEEV on groups II, VI, and VII was not significant as compared to the NC group. Treatment with MEEV at 600 and 900 mg/kg and glibenclamide (1 mg/kg) decreased HbA_1C significantly (P < 0.01) in the diabetic rats. There was a significant (P < 0.01) decrease in the level of serum HDL-cholesterol and significant (P < 0.01) increase in the levels of TC, LDLC, and TGs in diabetic rats when compared to NC rats. Administration of MEEV at 600 and 900 mg/kg and glibenclamide (1 mg/ kg) significantly (P < 0.01) brought their levels toward normal. The activities of serum enzymes AST, ALT, and ALP were found to be significantly (P < 0.01) increased in diabetic rats compared to normal rats. Oral administration of MEEV at 600 and 900 mg/kg and glibenclamide at 1 mg/ kg for 21 days significantly (P < 0.01) normalized the enzymatic activities in diabetic rats.

Table 4.  Effect of methanol extract of Erythrina variegata leaf (MEEV) on serum biochemical parameters in normal and streptozotocin (STZ)-induced diabetic rats.

Treatment	Glycosylated hemoglobin (% total Hb)	Triglycerides (mg/dl)	Total cholesterol (mg/dl)	HDL cholesterol (mg/dl)	LDL cholesterol (mg/dl)	AST (U/L)	ALT (U/L)	ALP (U/L)
Normal control	5.42 ± 0.29**	51.98 ± 2.83**	76.49 ± 2.86**	30.92 ± 1.65**	36.32 ± 2.40**	55.54 ± 2.36**	26.75 ± 1.57**	53.53 ± 2.33**
NC + MEEV 600 mg/kg	4.75 ± 0.12**	42.36 ± 1.29**	71.6 ± 3.72**	33.98 ± 1.39**	30.29 ± 1.77**	51.31 ± 1.88**	23.53 ± 1.23**	57.63 ± 2.06**
STZ control (55 mg/kg)	12.34 ± 0.47^¶	97.22 ± 3.18^¶	180.17 ± 3.81^¶	19.85 ± 1.04^¶	140.91 ± 2.02^¶	88.81 ± 2.26^¶	63.24 ± 2.56^¶	87.63 ± 2.64^¶
STZ + MEEV 300 mg/kg	10.96 ± 0.49*^¶	88.07 ± 2.57^¶	122.06 ± 4.37**^¶	24.21 ± 2.11	80.79 ± 1.77**^¶	81.46 ± 1.95^¶	52.44 ± 1.94**^¶	81.87 ± 1.09^¶
STZ + MEEV 600 mg/kg	7.66 ± 0.35**^¶	65.15 ± 4.03**^¶	106.59 ± 3.51**^¶	28.48 ± 2.14*	66.20 ± 2.07**^¶	68.63 ± 2.32**^¶	44.20 ± 1.88**^¶	73.07 ± 1.74**^¶
STZ + MEEV 900 mg/kg	5.77 ± 0.42**	50.72 ± 1.55**	92.94 ± 3.06**	31.33 ± 2.46**	50.74 ± 2.80**^¶	58.09 ± 2.75**	32.03 ± 2.25**	65.48 ± 2.23**^¶
STZ + Glibenclamide 1 mg/kg	4.42 ± 0.17**	40.95 ± 1.33**^§	87.48 ± 2.45**	32.14 ± 2.95**	47.82 ± 2.77**^¶	60.12 ± 2.33**	29.27 ± 1.73**	63.38 ± 1.44**^¶

Values are mean ± SEM (n = 6).

*P < 0.05 and **P < 0.01 when compared with STZ control group; ^§P < 0.05 and ^¶P < 0.01 when compared with normal control group.

Discussion

Diabetes mellitus is a chronic metabolic disorder caused by partial or complete insulin deficiency, which causes a disturbance in the uptake of glucose as well as glucose metabolism and leads to several acute and chronic complications. Phytotherapy has been highly accepted worldwide in the health care system for diabetes mellitus. In this study, the hypoglycemic activity of MEEV was evaluated in STZ-induced diabetic rats.

Here, STZ at the dose of 55 mg/kg, i.p., was used to induce hyperglycemia after performing a pilot study for optimized dose to elevate blood glucose >250 mg/dl. The use of lower dose of STZ (55 mg/kg) produced an incomplete destruction of pancreatic β cells even though the rats become permanently diabetic (Aybar et al., 2001). MEEV exhibited significant reduction in blood glucose in diabetic rats at the doses of 300, 600, and 900 mg/ kg. Although MEEV at 300 and 600 mg/kg reduced the hyperglycemia significantly as compared to the DC group, it failed to restore the FBG level to that of the NC group, while with MEEV at 900 mg/kg the blood glucose levels of diabetic rats were reduced to NC group level. Blood glucose levels in normal rats treated with MEEV 600 at mg/kg were insignificant from that of the NC group, indicating that MEEV maintained glucose homeostasis. The hypoglycemic action of MEEV may be due to promotion of insulin release from existing β cells of the islets of Langerhans. The plasma glucose lowering activity was compared with that of glibenclamide, the reference oral hypoglycemic which has been used for many years to treat diabetes mellitus, to stimulate pancreatic β cells (Stephen, 2001). From the results of this study, it appears that still insulin producing cells are functioning and the stimulation of insulin release could be responsible for most of the metabolic effects. It may be suggested that the mechanism of hypoglycemic action of MEEV is similar to glibenclamide.

Induction of diabetes with STZ is associated with a characteristic loss of body weight, during the observation period of 21 days even though the food intake was more in diabetic rats than NC animals. It was due to increased muscle wasting and loss of tissue proteins (Swanston-Flat, 1990). STZ-induced insulin deficiency may lead to protein content decrease in muscular tissue by proteolysis (Shirwaikar et al., 2004). Diabetic rats treated with the MEEV showed significant improvement in body weight as compared to the STZ control animals; hence, MEEV exhibited marked effect in controlling the loss of body weights of diabetic rats.

Oral administration of MEEV decreased the level of HbA_1C (53.24%). Lower levels of total hemoglobin observed in diabetic rats might be due to the increased formation of HbA_1C. Glycohemoglobin is formed throughout the circulatory life of red blood cells (RBCs) by the addition of glucose to the N-terminal of the hemoglobin β chain. This process, which is nonenzymatic, reflects the average exposure of hemoglobin to glucose over an extended period (Mohammadi & Naik, 2008).

Lipids play a vital role in the pathogenesis of diabetes mellitus. It is well known that in uncontrolled diabetes mellitus, there is an increase in TC in blood, which may contribute to coronary artery diseases (Arvind et al., 2002). The most common lipid abnormalities in diabetes are hypertriglyceridemia and hypercholesterolemia. In this study, elevated levels of serum lipids such as TC, LDLC, and TGs were found in diabetic rats. STZ produced various cardinal symptoms of diabetes mellitus including hypoinsulinemia, a condition that is probably responsible for the elevation of serum cholesterol levels because the insulin has an inhibitory action on HMG-CoA reductase, a key enzyme that acts as rate limiting in the metabolism of cholesterol rich LDL particles (Murali & Goyal, 2002). In insulin-deficient diabetes, the concentration of serum fatty acids is elevated as a result of free fatty acid outflow from fat depots, where the balance of the free fatty acid esterification-TG lipolysis cycle is displaced in favor of lipolysis (Shirwaikar et al., 2004). High-density lipoprotein (HDL) is an antiatherogenic lipoprotein. It transports cholesterol from peripheral tissues into the liver and thereby acts as a protective factor against coronary heart disease. The level of HDLC, which increased after MEEV administration, might be due to the increase in the activity of lecithin cholesterol acyl transferase (LCAT), which may contribute to the regulation of blood lipids (Patil et al., 2004). Oral administration of MEEV reduced the elevated serum lipids such as TC, LDLC, and TGs toward normal in diabetic rats.

Elevation of serum biomarker enzymes such as SGOT, SGPT, and SALP was observed in diabetic rats indicating impaired liver function, which was obviously due to hepatocellular necrosis. It has been reported that liver necrosis occurred in STZ-induced diabetic rats (Ohaeri, 2001). Therefore, increase in the activities of AST, ALT, and ALP gives an indication on the hepatotoxic effect of STZ. Twenty-one days of treatment with MEEV restored all the above-mentioned serum hepatic biochemical parameters toward the normal values in a dose-dependent manner, thereby alleviating liver damage caused by STZ-induced diabetes.

In this study, administration of MEEV to STZ-induced hyperglycemic rats demonstrated prominent reduction in blood sugar level, normalization of serum biochemical profiles including lipid contents, comparing to STZ control rats. Therefore, it can be concluded that the MEEV is remarkably effective against STZ-induced diabetes in Wistar rats thereby validating its ethnomedicinal usage. From the observed oral hypoglycemic activity of E. variegata leaf extract in STZ-induced diabetic rats, it can be further inferred that E. variegata leaf can serve as an interesting candidate in complementary and alternative medicine for the effective management of diabetes mellitus.

Acknowledgement

The authors are grateful to Dr. H.P. Chhetri, Director of Himalayan Pharmacy Institute, Sikkim, India, for providing necessary facilities for this work.

Declaration of interest

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.