Abstract
Context: In the Indian traditional system of medicine, Melothria heterophylla (Lour.) Cogn., (Cucurbitaceae) is prescribed for the treatment of diabetes mellitus.
Objective: In the present study, the antidiabetic effect of ethanol extract of Melothria heterophylla (EEMH), and its active isolated constituents were investigated in streptozotocin (STZ)-induced diabetic Swiss albino rats.
Method: Successive Soxhlet extraction of the dried total aerial parts with petroleum ether for defatting and then with ethanol (95%) to obtain ethanol extract, which was concentrated under reduced pressure. Hyperglycemia was induced in rats by STZ (50 mg/kg, body weight). Twenty-four hours after STZ induction, respective groups of diabetic rats received EEMH (200 and 400 mg/kg, body weight), gallic acid (GA) (2 and 4 mg/kg, body weight), and rutin (RU) (2 and 4 mg/kg, body weight), respectively, orally daily for 15 days. Glibenclamide (0.5 mg/kg, orally) served as reference. Blood glucose levels and change in body weight were measured on every 5th day during 15 days of treatment. Biochemical parameters, viz., serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), alkaline phosphatase (ALP) and serum insulin, were measured.
Results: EEMH and its active constituents significantly (p < 0.01) normalized blood glucose levels and serum biochemical parameters as compared to those of STZ controls. Both GA (4 mg/kg) and RU (4 mg/kg) exhibited maximum glucose lowering effect (69.1 and 66.7%, respectively) in diabetic rats compared to the other dose (2 mg/kg) at the end of the study. EEMH, gallic acid and RU also showed significant increase in serum insulin, and body weight of STZ-induced diabetic rats.
Conclusion: Therefore, ethanol extract of Melothria heterophylla, GA and RU demonstrated remarkable antidiabetic activity in STZ-induced diabetic rats.
Keywords::
Introduction
Diabetes mellitus, both insulin-dependent diabetes mellitus (IDDM) and nonIDDM type, is a common and serious disorder throughout the world (CitationKeen, 1986; CitationHarris et al., 1987). This metabolic disorder often leads to physical disability arising from the vascular complications of coronary artery disease and cerebrovascular disease, resulting in renal failure, blindness, and limb amputation in addition to neurological complications and premature death (CitationWeidmann et al., 1993; CitationCho et al., 2005). Treatment of diabetes mellitus by insulin and oral hypoglycemic drugs fails to prevent these complications in many patients, indicating that additional alternative treatment could be helpful. Plant derived drugs are gaining popularity in the treatment of diabetic mellitus (CitationPari, 1999). The major advantages of herbal medicine seem to be their efficacy, low incidence of side effects. Melothria heterophylla (Lour.) Cogn. (Cucurbitaceae), popularly known as kudari, is a scandent herb with tuberous roots found throughout India ascending up to 2100 m in the hills. It is reported to be useful for stimulant, invigorating, diabetes, cuts, fever, anti-inflammatory and purgative properties (CitationKirtikar & Basu, 2000; CitationAnonymous, 1962; CitationPant & Samant, 2010) and antioxidant (CitationMondal et al., 2009). However, there is no scientific evidence to support the antidiabetic effect of Melothria heterophylla. Hence, the objective of this study was to evaluate the antidiabetic activity of ethanol extracts of the aerial parts of M. heterophylla and its active isolated constituents in streptozotocin (STZ)-induced diabetic rats.
Material and methods
Plant material
The aerial parts of Melothria heterophylla were collected from young matured plants during August and September 2010, from the rural belt of Mayurbhanj district, Odisha, India and identified by Dr. P. Venu, taxonomist of Botanical Survey of India, Howrah, India. A voucher specimen (CNH/I-I (65)2006/Tech.II/1661) was deposited in the Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India. The collected plant material was washed, shade-dried, and then milled to course powder by a mechanical grinder for further studies.
Extraction and isolation
The powdered plant material (2 kg) was defatted with petroleum ether (60–80°C) and then extracted with 3L of ethanol (95%) in a Soxhlet apparatus. The solvent was then removed under reduced pressure, to obtain petroleum ether (Melothria heterophylla (PEMH), yield 4.38%) and ethanol (EEMH, yield 10.1%) extract, respectively. The ethanol extract was partitioned successively between ethyl acetate-water system and then between n-butanol-water system (3×1L). The respective solvents were removed similarly under reduced pressure, which produced ethyl acetate fraction (EAF) (120 g) and n-butanol fraction (NBF) (60 g). Both the fractions were evaluated for antidiabetic activity in STZ-induced diabetic rats. EAF was found to be more potent than NBF. Hence, EAF was further exploited for isolation, which led to the isolation of gallic acid (GA), a phenolic acid and rutin (RU), a flavonoid glycoside. GA was isolated as a white colored compound and RU as a yellowish amorphous powder, which were characterized as GA and RU based on their melting point and spectroscopic (IR, 1H, 13C Nuclear magnetic resonance (NMR) and Mass Spectrometry (MS)) data (CitationLatha & Daisy, 2011; CitationAbdullah et al., 2008).
Animals
Male Swiss albino rat weighing 150–250 g were used for the present investigation. They were housed in clean polypropylene cages and were fed with standard pellet diet (Hindustan Lever, Kolkata, India) and water ad libitum. The animals were acclimatized to laboratory condition (temperature 25 ± 2°C) with dark/light cycle (14/10 h) for one week before the start of an experiment. All procedures were approved by Jadavpur University Animal Ethical Committee (CPCSEA/ORG/CH/2006/Reg.No.95), Kolkata, India.
Drugs and chemicals
STZ was obtained from Spectrochem Pvt. Ltd, Mumbai, India; glibenclamide was obtained from Hoechst, India. All other reagents used were of analytical grade and were obtained commercially.
Acute toxicity test
The animals were divided into five groups (n = 6). The EEMH suspension was administrated orally in increasing dose up to 2000 mg/kg, b.w (CitationLitchfield & Wilcoxon, 1949). The rats were observed continuously for 2 h for behavioral, neurological and autonomic profiles and after 24 and 72 h for any lethality (CitationTurner, 1965).
Induction of experimental diabetes
Diabetes was induced by a single intraperitoneal injection of freshly prepared STZ (50 mg/kg, b.w) in ice cold citrate buffer (0.1 M, pH 4.5) to overnight-fasted rats (CitationYoruk et al., 2003; CitationKanter et al., 2003). Diabetes was identified by measuring glucose levels 48 h after injection of STZ. Only rats with glucose levels greater than 300 mg/dL were used in experiments.
Oral Glucose Tolerance Test in normal and STZ-diabetic rats
The oral glucose tolerance test (OGTT) was performed for two different doses of the EEMH (at predetermined therapeutic doses), and its isolated compounds (GA and RU) in normal and STZ-induced diabetic rat model. Four days after diabetes induction, the OGTT was performed by feeding glucose in the form of a solution through orally to STZ-induced diabetic rats fasted for 18 h. The animals were divided into nine groups, each having six animals. Group-I served normal control and received normal saline only; the animals of group II were administered STZ only; group III-IV were treated with EEMH 200 and 400 mg/kg, b.w., p.o; group V-VI with GA 2 and 4 mg/kg, b.w., p.o; group VII-VIII with RU 2 and 4 mg/kg, b.w., p.o and group IX was treated with glibenclamide (a known hypoglycemic agent) 0.5 mg/kg (CitationDas et al., 2011), respectively. One hour later, glucose (2 g/kg) was administrated. EEMH and its isolated compounds were administered throughout 14 days consecutively. The blood glucose level of each animal was monitored on 0, 5th, 10th and 15th days after the administration of the test samples.
Estimation of blood glucose
Blood glucose levels from the tail vein were measured after glucose administration using one touch system Accu-Check Active Glucometer (Roche Diagnostics India Pvt. Ltd, India).
Biochemical estimations
Different biochemical parameters such as serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT) and alkaline phosphatase (ALP) were determined by using commercially available kits (Span Diagnostic Limited, Surat, India) (CitationReitman & Frankel, 1957). Serum insulin estimation was done spectrophotometrically using standard kits (Span Diagnostic Limited, Surat, India).
Statistical analysis
Data were statistically evaluated using one-way analysis of variance (ANOVA), followed by post hoc Dunnett’s test using version 10 of SPSS computer software. The values are expressed as mean ± SD and p values less than 0.01 was considered as significant.
Results
Acute toxicity studies
Acute toxicity studies revealed the non-toxic nature of the EEMH. There was no lethality or toxic reaction found at any doses selected until the end of the study period.
Acute administration of GA even at a dose as high as 5 g/kg body weight did not produce any signs of toxicity or mortality (CitationRajalakshmi et al., 2001). RU is also reported to be safe as well (CitationPalmer et al., 2002).
Effect of EEMH, GA and RU on fasting blood glucose and body weight in STZ-induced diabetic rats
After 24 h of treatment with STZ, the fasting blood-glucose level was significantly changed in the range of 300–350 mg/dL (). Treatment of EEMH led to a dose-dependent fall in blood sugar levels by 4.9–70.1%. The initial antidiabetic activity was observed on the 5th day. The observed effect with isolated compounds was more significant (p < 0.01) pronounced than that of the EEMH. Both GA and RU (4 mg/kg) exhibited maximum glucose lowering effect (69.1 and 66.7%, respectively) in diabetic rats compared to the other dose (2 mg/kg) at the end of the study. Glibenclamide significantly (p < 0.01) exhibited a 61.70% reduction in blood-glucose level at the end of the study when compared to diabetic control.
STZ produced significant (p < 0.01) loss in body weight as compared to normal animals during the study. Diabetic control continued to loose weight till the end of the study while EEMH, GA and RU showed significant improvement (p < 0.01) in body weight compared to diabetic control ().
Table 1. Effect of EEMH and GA and RU on the glucose level in STZ-induced diabetic rats.
Table 2. The effect on body weight changes during 14 days treatment of EEMH, GA and RU.
Effect of EEMH, GA and RU on serum insulin in STZ-induced diabetic rats
STZ caused a significant (p < 0.01) decrease in serum insulin. Administration of EEMH and its isolated compounds caused significant (p < 0.01) increase in insulin levels at the end of the study period. Amongst the drug treated groups, GA (4 mg/kg, b.w.) showed maximum increase which was comparable to glibenclamide ().
Table 3. Effect of EEMH, GA and RU on serum insulin of STZ-treated diabetic rats.
Biochemical enzymes
shows the effect of the EEMH on the serum glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT) and ALP levels in normal and diabetic rats. The results showed that serum GOT, GPT and ALP levels in diabetic groups increased, when compared with normal rats. The administration of the GA and rutin significantly (p < 0.01) decreased the serum GOT, GPT and ALP levels, when compared with control diabetic rats.
Table 4. Effect of ethanol extract of M. heterophylla, GA and RU on serum biomarkers in STZ-induced diabetic rats after 14 days treatment.
Discussion
STZ is broad-spectrum antibiotic from Streptomyces achromogenes. Since the finding, that STZ possesses diabetogenic properties mediated by pancreatic β-cell destruction; this compound has been widely used to induce diabetes in experimental animals. Evidence has accumulated suggesting that STZ induces oxidative stress, which is caused by a relative overload of reactive oxygen species (ROS). Once, STZ enters inside the cell, it is spontaneously decomposed to form an isocyanate compound and a methyldiazohydroxide. Isocyanate compound and methyldiazohydroxide undergo intra molecular carboxylation and alkylation of cellular components respectively. The DNA damage of β cells of the pancreas, mainly by alkylation with carbonium ion produced by methyldiazohydroxide (CitationWeiss, 1982; CitationWilson & Leiter, 1990). It is well known that diabetes mellitus causes a disturbance in the uptake of glucose as well as glucose metabolism. Even the low dose of STZ produced an incomplete destruction of pancreatic β-cells even though the rats become permanently diabetic and induce disturbance in glucose metabolism (CitationAybar et al., 2001). The induction of diabetes was confirmed as reflected by the hyperglycemia, and body weight loss (CitationMi-Kyung et al., 2005; CitationCho et al., 2006a,b). The single high-dose STZ-induced diabetic rat is one of the animal models of human IDDM or type I diabetes mellitus. In this model, diabetes arises from irreversible destruction of the islet cells of the pancreas, causing degranulation or reduction of insulin secretion and leads to hyperglycemia. In this type I model of diabetes, insulin is markedly depleted, but not absent (CitationPushparaj et al., 2001). Although insulin has become one of the most important therapeutic agents known to medicine, there is a continuing effort to find insulin substitutes, secretagogues, or sensitizers from synthetic or plant sources for the treatment of diabetes. Phyto-constituents, such as saponins and flavonoids, play important roles as the hypoglycemic agents (CitationPushparaj et al., 2001; CitationHosseinzadeh et al., 2002). The isolated plant compounds such as GA and rutin from M. heterophylla are phenolic acid and flavonoid glycoside respectively, and the plants were used by traditional people for the treatment of diabetes. In the present study, the high dose of EEMH and its secondary metabolites had significantly reduced hyperglycemia in STZ diabetic rats.
Hyperglycemia is associated with the generation of ROS causing oxidative damage, particularly to the heart, kidney, eyes, nerves, liver, small and large vessels and gastrointestinal system. The increased levels of plasma glucose in STZ-induced diabetic rats were lowered by the administration of EEMH, GA and rutin. The plasma glucose lowering activity was compared with glibenclamide, a standard hypoglycemic drug. The possible mechanism by which EEMH and its isolated compounds mediated their antidiabetic effect could be by potentiation of pancreatic secretion of insulin from existing β-cells of islets (CitationTian et al., 1998), as was evident by the significant increase in the level of insulin in the extract and isolated compounds treated animals. The hypoglycemic activity of EEMH, GA and RU was compared with glibenclamide. From the results of the present study, it may be suggested that the mechanism of action of FRAE may be similar to glibenclamide action.
The increase in the activities of serum GOT, GPT and ALP indicated that diabetes may be induced due to liver dysfunction in STZ-induced diabetic rats. Therefore, increase in the activities of GOT, GPT and ALP in plasma may be mainly due to the leakage of these enzymes from the liver cytosol into the blood stream (CitationFortson et al., 1985), which gives an indication on the hepatotoxic effect of STZ. On the other hand, independent treatment of the diabetic rats with GA and rutin caused the reduction in the activity of these enzymes in plasma compared to the mean values of the diabetic group and consequently, may alleviate liver damage caused by STZ-induced diabetes. These results are in agreement with those obtained by CitationEl-Demerdash et al. (2005) in rats. This investigation reveals the potential effect of GA and rutin as the hypoglycemic agent.
Conclusion
In conclusion, it can be stated, that EEMH and its isolated compounds, GA and rutin, have marked beneficial effects, in reducing the elevated blood-glucose level of STZ-induced diabetic rats, justifying the folklore claim.
Acknowledgement
The authors would like to thank University Grants commission (UGC), New Delhi, India, for providing financial assistance and Jadavpur University, Kolkata, India, for providing research facilities to the corresponding author.
Declaration of interest
The authors report no declarations of interest.
Related Research Data
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