Abstract: This study was undertaken to investigate the antidiabetic effect of aqueous and hydroalcoholic extract of Phyllanthus amarus Schum and Thonn, a medicinal plant used in Togo for treatingdiabetes and many others diseases. Diabetes was induced in fasted rats (12 h) by a single intraperitoneal injection of 120 mg kg-1 of alloxan monohydrate. Two doses (500 and 1000 mg kg-1) of the both extracts of Phyllanthus amarus were administered orally to diabetic rats. The normal control group receives distilled water only. After 15 days treatment, body weight gain, blood glucose level, serum insulin, total cholesterol, triglycerides and malondialdehyde were evaluated. At the doses tested, aqueous and hydroalcoholic extract of P. amarus decrease significantly blood glucose level after 15 days of administration. Aqueous extract reduce body weight gain contrary to hydroalcoholic extract. Serum insulin increases in group treated with extracts in compared to diabetic control group. The hydroalcoholic extract reduce the malondialdehyde concentration in the serum. Values of total cholesterol and triglycerides are similar in all the groups. This study demonstrated the potential antidiabetic property of aqueous and hydroalcoholic extract of Phyllanyhus amarus thus justifying its traditional usage.
INTRODUCTION
Diabetes is the world’s largest endocrine disease involving metabolic disorders of carbohydrate, fat and protein. The number of diabetic patients is gradually increasing worldwide. Currently there are over 150 million diabetics worldwide and this is likely to increase to 300 million or more by the year 2025 (IDF, 2003).
Diabetes mellitus is characterized by high blood glucose levels due to absolute or relative deficiency of circulating insulin levels. The increased blood glucose level generates glucose auto oxidation and auto oxidative glycosylation of proteins which leads to oxidative stress by increasing the reactive oxygen species. Reactive oxygen species contribute to the development of diabetic complications (Wiernsperger, 2003).
Though different types of oral hypoglycemic agents are available along with insulin for the treatment of diabetes mellitus, there is a growing interest in herbal remedies, due to the side effects associated with these therapeutic agents (Proks et al., 2002). More than 400 species have been reported to present antidiabetic activity (Yeh et al., 2003).
Phyllanthus amarus Schum and Thonn is a small shrub which belongs to Euphorbiaceae family. The plant is distributed in tropical and subtropical countries and is used for the treatment of various diseases and disorders, such as jaundice, constipation, diarrhea, kidney ailments. Phytochemistry of this species is well known and reveals the presence of gallotannins, lignans like phyllanthin and hypophyllanthin and alkaloids such as isobubbialine and epibubbialine (Joseph and Raj, 2011; Annamalai and Lakshmi, 2009). Compounds isolated from Phyllanthus were found to possess anti-viral property against hepatitis B virus in vitro (Huang et al., 2003). Hydroalcoholic extract of Phyllanthus amarus induced liver regenerative effect against liver cell injury (Chattopadhyay et al., 2006). The extract also possessed anti-tumour, anti-carcinogenic and anti-inflammatory properties (Kassuya et al., 2006; Kiemer et al., 2003). Gallotannin and ellagitannins (geraniin and corilagin) were shown to be the most potent mediators of the antiviral HIV activity (Notka et al., 2003). Studies revealed the hypoglycemic activity of the plant (Raphael et al., 2002; Adeneye et al., 2006) but no studies have been conducted to show the effect of the plant on serum insulin level (hormone involved in regulating blood glucose). This study was undertaken to evaluate the antidiabetic effect (effect on blood glucose level, on serum insulin, serum triglyceride and cholesterol) of Phyllanthus amarus in alloxan induced diabetic rats.
MATERIALS AND METHODS
Chemicals: Alloxan monohydrate, DPPH (2, 2-diphenyl-1- picrylhydrazyl) and AAPH (2, 2’-azobis 2 amidinopropanedihydrochloride) were purchased from sigma France.
Plant material: Whole plant of P. amarus was collected in July 2007 from Tsevie (at 36 km North-East of Lome, Togo). A voucher specimen was identified and kept in the herbarium of the Laboratory of Botany and Plant Ecology (Faculty of Science/University of Lome). Plant was cleaned out with water and cut into small pieces, dried and extracted with water or ethanol/water (1:1 v/v) as following. Plant material (200 g) was soaked in water (2 L) and heated in a water bath at 80°C for 1 h for the aqueous extract. The same quantity was macerated 72 h in ethanol/water solution (2 L) for hydroalcoholic extract. The crude extracts were filtered with Whatman paper and evaporated in vacuo at 40°C using a rotary evaporator (Buchi, France). The yield of the preparation was 16% (w/w) for Aqueous Extract (AE) and 13% (w/w) for Hydroalcoholic Extract (HAE).
Animals: Male Wistar rats (200-250 g) were obtained from DEPRE animal center (France) and were fed with conventional chow (toxins-free according to EU regulation) and tap water ad libitum. Rats were acclimatized for 7 days under standard environmental conditions of temperature, relative humidity and dark/light cycle. The animals were divided randomly into different groups for the experiment and were deprived of food except water 16 h prior to the experiments. All animal experiments were conducted according to the law set by European and national authorities and following the agreement of the ethical working group at the University Bordeaux 2.
Induction of diabetes in rats: Diabetes was induced in fasted rats (12 h) by a single intraperitoneal injection of 120 mg kg-1 of alloxan mono hydrate. Alloxan was freshly dissolved in distilled water and the injection volume was 20 mL kg-1. The diabetic state was assessed by measuring the non-fasting blood glucose level 10 days after alloxan injection. Rats with blood glucose level in a range of 200-500 mg dL-1 with polyuria and glucosuria were selected for the experiment. Blood glucose levels were measured with a glucometer (Optium Sense, France) on the tail vein.
Experimental design: Rats were divided into seven groups of seven rats:
| • | Group 1: Normal control rats received distilled water alone |
| • | Group 2: Diabetic control rats received distilled water alone |
| • | Group 3: Diabetic rats, treated with 500 mg kg-1 AE |
| • | Group 4: Diabetic rats, treated with 1000 mg kg-1 AE |
| • | Group 5: Diabetic rats, treated with 500 mg kg-1 HAE |
| • | Group 6: Diabetic rats, treated with 1000 mg kg-1 HAE |
| • | Group 7: Diabetic rats, treated with 100 mg kg-1 metformin (standard oral hypoglycemic agent) |
The plant extract dissolved in distilled water was fed orally by gastric intubation during 15 days. All the drugs were given as a single dose in the morning. Body weight and blood glucose levels were estimated on days 1, 7 and 15. After the last dose, animals were anaesthetized with ether. Blood samples collected by the orbital sinus puncture were centrifuged at 500x g and serums were kept at -20°C. Total Cholesterol (TC) and Triglyceride (TG) were estimated by enzymatic method using reagent kit (BioMérieux, France); insulin concentration was determined using commercially available enzyme-linked immunoassay kit (ELISA kit, USA). Malondialdehyde concentration was measured spectrophotometrically.
Antioxidant activities: The free radical scavenging activity of the extracts was determined by DPPH (2, 2-diphenyl-1- picrylhydrazyl) test and AAPH (2, 2’-azobis 2 amidinopropanedihydrochloride) test.
The free radical scavenging activity of the extracts was measured in vitro by the stable 2, 2-diphenyl-1- picrylhydrazyl free radical assay. Briefly, plant extract (0.25 mL) was added to 1.5 mL of methanolic solution of DPPH. Absorbance was determined at 517 nm after 30 min of incubation at room temperature. Quercetin was used as standard controls.
The percentage inhibition activity was calculated from:
where, Ac is the absorbance of the control and Ae is the absorbance of the extract/standard.
IC50 value denotes the concentration of sample, which is required to scavenge 50% of DPPH free radicals.
AAPH is a free radical generator inducing hemolysis in RBC (red blood count). The free radical scavenging activity of the extracts was determined by the method described by Sekiya et al. (2002).
Statistical analysis: All data were expressed as Mean±SEM. Statistical differences between treated groups and controls were determined by Analysis of Variance (ANOVA) followed by Fisher LSD test using Systat 10.0. Differences between groups were considered significant for p<0.05. Graphs were performed using Graph Pad Prism 4.
RESULTS
Effect of the extracts of P. amarus on body weight in diabetic rats: Forthteen days after the treatment, diabetic control showed significant reduction in their body weight compared to normal control (Table 1). The body weight gain was 15% in the diabetic control vs 40.8 % in the normal control. The aqueous extract (500 and 1000 mg kg-1) reduced body weight while the hydroalcoholic extract caused body weight gain in diabetic rats.
Effect of the extracts of P. amarus on blood glucose level in diabetic rats: A marked rise in blood glucose level was observed in diabetic control compared to normal control rats (Table 2). At 500 and 1000 mg kg-1, the aqueous extract causes a significant decrease (p<0.05) in blood glucose level on 15th day compared to diabetic control. The hydroalcoholic extract decrease significantly (p<0.001) blood glucose level at 1000 mg kg-1.
| Table 1: | Effect of aqueous extracts (AE) and hydroalcoholic extract (HAE) on body weight gain (BWG) |
| C: Normal control; DC: Diabetic control; AE 500, AE 1000 mg kg-1: Diabetic rats fed with aqueous extract, respectively at 500 and 1000 mg kg-1; HAE 500, HAE 1000 mg kg-1: diabetic rats fed with hydroalcoholic extract, respectively at 500 and 1000 mg kg-1; Met 100 mg kg-1: Diabetic rats fed with metformin at 100 mg kg-1. The body weight gain (BWG) % represent (D15-D1)D1x100. The data were expressed as Mean±SEM (n = 7) and evaluated by ANOVA followed by Fisher LSD test at 5% | |
Effect of the extracts of P. amarus on biochemical parameters: Table 3 shows the levels of serum insulin, total cholesterol, triglycerides and malondialdehyde concentration in normal and diabetic rats. In diabetic control, serum insulin level was significantly decreased (p<0.001) whereas serum malondialdehyde was significantly (p<0.05) increased in compared with the normal rats. Diabetic group treated with the extracts (500 and 1000 mg kg-1) showed significant reduction in serum insulin level compared to control. Only the hydroalcoholic extract reduced significantly the concentration of serum malondialdehyde compared to control. Values of total cholesterol and triglycerides are similar in all the groups.
Antioxidant activities: Table 4 shows the free radical scavenging activity of the extracts determined by DPPH. Aqueous and hydroalcoholic extracts exhibited mild significant free radical scavenging activity compared to quercetin (standard control).
| Table 2: | Effect of different doses of aqueous extracts (AE) and hydroalcoholic extract (HAE) on blood glucose level |
| C: Normal control; DC: Diabetic control; AE 500 mg kg-1, AE 1000 mg kg-1: Diabetic rats fed with aqueous extract, respectively at 500 and 1000 mg kg-1; HAE 500, HAE 1000 mg kg-1: Diabetic rats fed with hydroalcoholic extract respectively at 500 and 1000 mg kg-1; Met 100 mg kg-1: Diabetic rats fed with metformin at 100 mg kg-1. The data were expressed as Mean±SEM (n = 7) and evaluated by ANOVA followed by Fisher LSD test at 5%. aaap<0.001 vs Normal Control; bp<0.05, bbp<0.01 and bbbp< 0.001vs Diabetic control | |
| Table 3: | Effect of aqueous extracts (AE) and hydroalcoholic extract (HAE) on serum insulin, MDA, triglyceride and total cholesterol |
| C: Normal control; DC: Diabetic control; AE 500 mg kg-1, AE 1000 mg kg-1: Diabetic rats fed with aqueous extract, respectively at 500 and 1000 mg kg-1; HAE 500, HAE 1000 mg kg-1: diabetic rats fed with hydroalcoholic extract, respectively at 500 and 1000 mg kg-1; Met 100 mg kg-1: Diabetic rats fed with metformin at 100 mg kg-1. The data were expressed as Mean±SEM (n = 7) and evaluated by ANOVA followed by Fisher LSD test at 5%. ap<0.01, aaap<0.001 vs Normal control; bp<0.05, bbp< 0.01 vs Diabetic control | |
| Table 4: | Antioxidant activities of aqueous and hydroalcoholic extracts (AE and HAE) in vitro |
| IC50: Concentration which scavenge (in vitro) 50% of DPPH and AAPH free radicals. Quercetin (QR) and Ascorbic Acid (AA) were used as standard controls | |
| Fig. 1: | Peroxyl radical scavenging effect of P. amarus extracts (AE and HAE) in vitro. Ascorbic Acid (AA) was used as positive control. Each point represent Mean±SEM (n = 4) |
IC50 was respectively 514.58 and 447.92 μg mL-1 for aqueous and hydroalcoholic extract.
The peroxyl radical scavenging effects were also examined. Both extracts showed dose-dependent antioxidant activity. The aqueous extract presented the strongest effect but this effect was lower than that of ascorbic acid used as standard control (Fig. 1).
DISCUSSION
In the present study, alloxan-induced diabetes in rat approximates non-insulin-dependent diabetic state with non-fasting blood glucose ranging between 200 and 500 mg dL-1. Alloxan was described for the first time by Brugnalli in 1818. Several studies were conducted in vitro and in vivo to understand the cytotoxic effect of this drug, however, its precise mode of action remains a controversial subject (Janjic et al., 1999). Alloxan specifically interacts with beta cells pancreatic generating oxygen free Radicals (ROS) responsible for its toxic effects (Weaver et al., 1978; Boquist et al., 1983). It is well reported that, partial destruction of beta cells causes a decrease in insulin secretion which is materialized by a chronic hyperglycemia (Ene et al., 2008).
The effect of hydroalcoholic and aqueous extracts in diabetic rats compared to controls shows a significant decrease in diabetic blood glucose at 15th day of treatment. The hydroalcoholic extract was more effective at the end of the treatment. Alloxan caused body weight reduction, which was reversed by the hydroalcoholic extract at 500 mg kg-1. Diabetic group treated with aqueous extract showed a severe body weight reduction compared to diabetic controls. The values found are similar to those of animals treated with metformin (standard reference).The effect of aqueous extract on body weight could be attributed to tannins which are more abundant in this extract than hydroalcoholic. Indeed, several studies have shown that tannins have antinutritional properties that occur in rodents by weight loss due to complexing proteins in the intestinal lumen (Butler, 1992; Carbonaro et al., 2001) or by reduction in food intake (Oliveira et al., 2005).
After 15 days of daily administration, insulin levels in the groups treated with aqueous extract and hydroalcoholic extract was significantly increased compared to untreated controls group. Our previous study reveals that aqueous and hydroalcoholic extract of P. amarus reduced significantly blood glucose level in glucose tolerance test but had no effect on fasted blood glucose level suggesting an extra pancreatic mechanism of the extract (Lawson-Evi et al., 1997). Then, increased insulin levels can be attributed to glucose utilization in peripheral tissues. But it is possible that increased insulin concentration was due to beta cell regeneration as shown by Takasawa and Okamoto (2002). In the same way, increased insulin activity on liver cell regeneration by Phyllanthus amarus was reported by Chattopadhyay et al. (2007). At this time, no study has been conducted to show the action of the plant extract on beta cell regeneration but previous study in our laboratory had shown that low concentrations (<80 μg mL-1) of aqueous and hydroalcoholic extract of Phyllanthus amarus led to 10-15% Caco-cell proliferation (Lawson-Evi et al., 2008).
Abnormally high levels of free radicals in diabetic animals, cause membrane and tissue damage due to lipid peroxidation. Present result showed that the rate of malondialdehyde, a marker of lipid peroxidation, decreased significantly in the groups treated with hydroalcoholic extract compared to untreated controls. This confirms the antioxidant activity of extracts evaluated in vitro by induction of hemolysis rat erythrocytes and scavenging of free radical DPPH (2, 2-diphenyl-1- picrylhydrazyl). Antioxidant activity of P. amarus was confirmed by Shokunbi and Odetola (2008) and Kumaran and Karunakaran (2007). As a new strategy for alleviating the oxidative damage who leads to diabetes complications, interest has grown in the usage of natural antioxidants.
CONCLUSION
From the present study, it is concluded that Phyllanthus amarus extract exhibited antidiabetic activity by increasing serum insulin level in alloxanised rats. According to the antidiabetic and antioxidant activities, Phyllanthus amarus can help in the treatment of diabetes and in the prevention of its complications.
ACKNOWLEDGMENTS
Authors thanks French Cooperation Service (SCAC) for a fellowship for PhD studies in the University Bordeaux 2, Toxicology Department (France).