Anti-Diabetic effects of lotus (nelumbo nucifera) leaves in alloxan-induced diabetic mice

JOURNAL OF SCIENCE OF HNUE Chemical and Biological Sci., 2012, Vol. 57, No. 8, pp. 138-147 This paper is available online at ANTI-DIABETIC EFFECTS OF LOTUS (Nelumbo nucifera) LEAVES IN ALLOXAN-INDUCED DIABETIC MICE Nguyen Thi Hong Hanh, Ma Thi Thu Le and Le Thi Tuyet Faculty of Biology, Hanoi National University of Education Abstract. To evaluate the anti-diabetic property of lotus (Nelumbo nucifera) leaves, experimentation took place making use of an animal model before proceeding to clinical trials. Diabetes was induced by intraperitoneal injection of alloxan (75 mg/kg of body weight). Lotus leaves were orally administered once a day for 15 days in the amount of 600 mg/kg, followed by a daily injection of alloxan for three days. Blood glucose levels were measured on the 5th, 10th, 15th days of the experiment. Body weight (BW) and blood glucose levels were determined on the mice which were given alloxan. A comparison was made between normal mice, mice which were diabetic, and diabetic mice which were fed lotus leaves. The results indicated that the administration of lotus leaves tended to bring the parameters (BW, blood glucose levels) significantly closer to the norm. From the histopathological examination that was done, we found that the pancreas of mice treated with lotus leaves both increased the size and restored the normal endocrine cell population size of the islets of langerhans. A kidney histology showed that no difference was found in the structure of the glomerulus and vessels when comparing normal mice and diabetic mice which were given lotus leaves. We therefore conclude that lotus leaves have an anti-diabetic effect and should be the subject of future studies. Keywords: Hypoglycemic, diabetic, alloxan, mice, lotus leaf, Nelumbo nucifera. 1. Introduction Diabetes mellitus (DM) is a chronic disease caused by an inherited and/or acquired deficiency in production of insulin by the pancreas, or by an ineffectiveness of the insulin produced. DM is a common metabolic disorder which is accompanied by micro and macrovascular complications that result in significant morbidity and mortality [6]. DM Received September 27, 2011. Accepted February 7, 2012. Biology Subject Classification: 362 196. Contact Nguyen Thi Hong Hanh, e-mail address: honghanhnt111@gmail.com 138 Anti-diabetic effects of lotus (Nelumbo nucifera) leaves in alloxan-induced diabetic mice is considered to be one of the five leading causes of death in the world, and the disease is increasing rapidly in most parts of the world. In 1995, the World Health Organization reported that approximately 150 million people worldwide had diabetes mellitus, and this number may well double by 2025 [10]. In modern medicine, no satisfactory effective therapy has been available to date to cure diabetes mellitus [6]. Because synthetic drugs are expensive, of limited availability, have undesirable side effects and carry an uncertain risk when used during pregnancy, some medical practitioners prefer to make use of hypoglycemic drugs of plant origin to treat chronic conditions [2]. Plants which have been shown to have hypoglycemic action alter blood glucose levels through various mechanisms. More and more patients want to use natural products that have anti-diabetic properties. Among the traditional medicinal plants that are reported to have hypoglycemic properties are garlic, bitter melon, aloe, onion, guava and spirulina [2, 4]. Lotus leaf is particularly useful as a medicine to treat diabetes. The leaves have been known to contain 0.2 - 0.3% tannins and 0.77 - 0.84% alkaloids. Flavonoids are considered to be a main component of lotus leaf and flavonoids do have anti-diabetic properties [10]. In Vietnam, lotus plants are widely grown. Some parts of the lotus plant have been used as a tradition medicine. The lotus plant is easy to find, inexpensive, non-toxic and accepted by many Vietnamese people as a means of treating diabetes, and yet data on the mechanism or even effectiveness of lotus leaf on diabetes mellitus was not known. The present study was designed to evaluate the hypoglycemic effect of lotus leaf on mice which had alloxan induced diabetes. 2. Content 2.1. Time and place of study This study was done from November 2010 to July 2011 at the Faculty of Biology, Hanoi National University of Education. 2.2. Materials and methods 2.2.1. Materials Animals: 120 male white mice (Mus musculus), Swiss strain, 4 weeks old (16 -18 g), were obtained from the National Institute of Hygiene Epidemiology. Plant material: leaves of lotus plants (Nelumbo nucifera), of the Nelumbonaceae family, were collected at Gia Lam, Hanoi. 2.2.2. Methods * Experimental design Stage 1. The mice were fed a diet that was provided by the National Institute of Hygiene Epidemiology. Mice were kept in clean, dry cages 30 × 50 × 20 cm in size, 4 139 Nguyen Thi Hong Hanh, Ma Thi Thu Le and Le Thi Tuyet mice to a cage at 25 - 300C and 45 - 55% relative humidity. The mice were divided into 2 groups (n = 60 mice per group): - Group I: basal diet: 65 g feed/kg BW/day - Group II: rich lipid diet: basal diet + 13 g rich lipid food/kg BW/day After 6 weeks, we obtained Group 1: normal mice (NM) and Group 2: obese mice (OM). Stage 2. Induction of diabetes in experimental animals by a single intraperitonial injection. The dose administered was as follows (n = 20 mice per group): - NM + 75 mg alloxan/kg BW - NM + 100 mg alloxan/kg BW - NM + 125 mg alloxan/kg BW - OM + 75 mg alloxan/kg BW - OM + 100 mg alloxan/kg BW - OM + 125 mg alloxan/kg BW After injection, the mice were monitored for expression of diabetes. Diabetes was confirmed by measuring fasting blood glucose levels (FBG) on the third day after administration of alloxan. Mice with an FBG greater than 11.1 mmol/l were considered to be diabetic [10]. Stage 3. Study of anti-diabetic effects of lotus leaf The mice were divided into four groups (n = 16 mice per group) as given below: - Group I: normal control group (NC); - Group II: diabetes control group (DC); - Group III: diabetic mice were given 25 mL lotus leaf tea/kg BW/day (25 mL lotus leaf tea was made using 600 mg powdered lotus leaf) (DLL). During the 15 days that lotus leaf tea was administered to the mice, and the FBG level was measured once every 5 days. * Preparation of lotus leaf tea: Lotus leaves were oven dried at 45oC. The dried lotus leaves were pulverized to a coarse powder consistency and kept in a cool and dry place. A filter bag containing 600 mg of lotus leaves powder was allowed to steep in 25 mL boiling water for 5 to 7 minutes to obtain the lotus leaf tea. * Testing of fasting blood glucose: FBG levels were measured using the automatic analyzer Olympus AU 400 (made in Japan). * Acute toxicity evaluation in mice: Lotus leaf tea was tested for possible acute toxicity in male mice. Single oral administration of lotus leaf tea, at doses of 5000 mg, 6000 mg, 7000 mg and 8000 mg/kg BW/day (in 25 mL water) were given to the different groups of mice (10 mice in each group). General behavior was observed continuously for 1 hour, then after 4 hours, intermittently over the next 6 hours and then again at 24, 140 Anti-diabetic effects of lotus (Nelumbo nucifera) leaves in alloxan-induced diabetic mice 48 and 72 hours. Observers were alert for gross behavioral changes, grooming, alertness, sedation and loss of righting reflex [5, 10]. * The Histological study: Small slices obtained from the pancreas and kidneys of the animals were fixed in a 10% formalin solution and processed routinely. Sections 5 µ thick were cut and stained by Hematoxylin and Eosin (H&E) for histological examination. * Chemicals: Alloxan was purchased from the Sigma Chemical Co. * Statistical analysis: All of the data were expressed as a mean ± S.D. Statistical significance was calculated using the one-way analysis of MS Excel. 2.3. Results and discussion 2.3.1. Acute toxicity studies Mice were fed lotus leaf tea at an increasing dosage from 5000 to 8000 mg/kg BW. The result was expressed in Table 1. Table 1. Acute toxicity test results Dose (mg/kg BW) Total mice The number of dead mice after 72 h Mortality(%) 5000 10 0 0 6000 10 0 0 7000 10 0 0 8000 10 0 0 The acute oral toxicity study of lotus leaves showed no mortality up to 8000 mg/kg. The main purpose of this test is to get some idea of conspicuous behavioral changes and death, if any, and lotus leaf ingestion did not cause any toxic symptoms in the limited toxicity evaluation in male mice. The physiological status of mice was normal during the 72 h monitoring. This result demonstrates that an oral administration of concentrated lotus leaf tea is non-toxic to experimental mice. 2.3.2. Alloxan-induced diabetic mice Mice of equal weight were selected (16 - 18 g), divided into 2 groups and fed 2 different diets: a basal diet and a lipid rich diet. After 6 weeks, changes in body weight are as shown in Figure 1. After being fed for 6 weeks, the body weight of mice that were fed the basal diet increased 14.89 g, from 18.15 to 33.04 g (82.03%), while the body weight of mice that were fed a lipid rich diet increased 33.98 g, from 17.25 to 51.23g (1.97 times their original weight). When the body weight of mice increased more than 1.8 times their initial body weight, they are considered to be obese [3]. This is consistent with other studies of scientists using mice in Vietnam and abroad such as Srinivasan et al. [9]. 141 Nguyen Thi Hong Hanh, Ma Thi Thu Le and Le Thi Tuyet Figure 1. Average body weight of mice before and after 6 weeks After the obese mice were selected, alloxan at 3 dose levels: 75, 100, 125 mg/kg BW, was injected into the 2 groups of mice (NM and OM). The results are as shown in Table 2. Table 2. FBG before and 72 hours after injection of alloxan Dose (mg/kg) FBG (mmol/l) (x± SD) NM OM Before injection After injection 72 h Before injection After injection 72 h 75 6.99±0.05 7.64±0.08 6.14±0.18 12.31±0.67 100 7.27±0.24 8.22±0.30 6.32±0.20 13.21±0.46 125 7.15±0.09 Die 7.29±0.20 Die The results show the differences in FBG between NM and OM at different alloxan injection doses. At the dose of 125 mg alloxan/kg BW, all mice died within 72 hours. In group NM, FBG increased at doses of 75 and 100 mg alloxan/kg BW, from 6.99 to 7.64 mmol/L and 7.27 to 8.22 respectively. In the OM group, the FBG increased significantly. At the dose of 75 mg/kg BW, FBG increased from 6.14 to 12.31 mmol/L. At the dose of 100 mg/kg BW, blood glucose levels increased from 6.32 to 13.21. However, at the dose level of 100 mg alloxan/kg BW, we observed in the mice symptoms of tremors, fuzziness, cramping, thirst, slower reflexes, less flexibility and death from days 4 to 6. Thus doses at 100 mg alloxan/kg BW should not be used in future studies. There was a significant elevation in FBG after a single dose of alloxan at 75 mg/kg BW. Results from Taoying Zhou et al. concluded that mice with an FBG greater than 11.1 mmol/L were diabetic [10]. Thus, the above results indicate that alloxan may be used to successfully cause induced diabetes in mice at a dose of 75 mg alloxan/kg BW. In particular, the incidence of diabetes in obese mice (90%) is much higher than in normal mice (5%). This is because obesity increases the concentration of free fatty acids. Many studies show that 142 Anti-diabetic effects of lotus (Nelumbo nucifera) leaves in alloxan-induced diabetic mice the free fatty acid excess in the blood inhibits the synthesis of glucose transport proteins which changes insulin receptors on target cell membranes and causes insulin resistance effects in muscle cells and reduces the likelihood of the capture of glucose from the blood as it circulates in the liver and muscle cells and this leads to type 2 diabetes [3]. 2.3.3. Anti-diabetic effects of lotus leaf tea * Effect of lotus leaf tea on body weigh in alloxan-induced diabetic mice Body weight was significantly lower in the alloxan-induced diabetic mice compared to that of mice in the control group. The average weight of animals in the NC and the DC group before this study began was 33.04 g and 50.87 g while in the 15th day of the study the weights were found to be 38.78 g and 45.57 g respectively. Accordingly, significant differences in weight reduction were observed comparing the NC and DC groups. Normal control animals were found to have slightly increased body weight but diabetic mice showed a significant reduction in body weight after 15 days (Figure 2). Figure 2. Average body weight of mice Alloxan caused a reduction in body weight which is reversed by lotus leaf tea after 15 days of treatment. Lotus leaf tea brought the body weight significantly closer to the control level. After the treatment period the average of body weight in DLL group was 58.45 g while that of the DC group was 45.57 g. The average body weight of animals before the study was begun, after the induction of diabetes and also after treatment with lotus leaf tea for 15 consecutive days is shown in Figure 2. Alloxan-induced diabetes is characterized by severe loss in body weight, and this reduction is due to a loss or degradation of structural proteins, such structural proteins being known to contribute to body weight [6]. * Effect of lotus leaf tea on blood glucose level in alloxan-induced diabetic mice Three days after alloxan injection, the diabetic mice with an FBG higher than 11.1 mmol/L were selected for further studies. The effect of lotus leaf tea on the FBG of the diabetic mice is shown in Figure 3. 143 Nguyen Thi Hong Hanh, Ma Thi Thu Le and Le Thi Tuyet Figure 3. FBG before and after 15 days of treatment The FBG of the DLL group decreased significantly after 15 days of treatment. This FBG decrease was 8.66 mmol/L, approximately that of the NC group (an FBG of 7.23 mmol/L). Fifteen days of daily treatment with lotus leaf tea led to a 29.31% drop in blood glucose level. A maximum decrease in FBG was observed in the 15th day of treatment administering a dose of 600 mg/kg of lotus leaves with a decrease in FBG to 8.66 from an initial level of 12.05 mmol/L. In addition, there was a significant elevation in FBS levels in the DLL and the DC group (FBG of 14.21 mmol/L) at the end of the study. The average fasting blood glucose levels of animals after the induction of diabetes and also after treatment with lotus leaf tea for 15 consecutive days is shown in Figure 3. This indicates that lotus leaf tea has a significant effect on reducing blood glucose levels in diabetic mice. * Histological results Pancreas Histopathological studies (Figure 4) showed normal acini and normal cellular population in the islets of Langerhans in the pancreas of control mice (a). DC group: Extensive damage to the islets of Langerhans and reduced size of islets (b). DLL group: A partial restoration of normal cellular population and enlarged ß cells with hyperplasia were observed after administration of lotus leaf tea (c).The renewal of ß cells in diabetics has been studied using several animal models. The total ß cell mass reflects a balance between a renewal and loss of these cells. Lotus leaf tea has been shown to influence ß cell regeneration. Similar effects in alloxan-treated diabetic animals were reported when using Vinca rosea whole plant alcohol-based extracts [6]. In this study, pancreatic ß cells were destroyed using alloxan. Alloxan is well known for its selective pancreatic islet cell toxicity and has been extensively used to induce diabetes mellitus in animals (Prince and Menon) [8]. In our studies, damage to the pancreas in alloxan-treated diabetic control mice (b) and a regeneration of ß cells by lotus leaf tea after 15 days of treatment was observed. This could have occurred because those ß-cells which survived are induced by the lotus leaf tea to exert an insulin releasing effect. Also flavonoids from the lotus leaf tea might have a regenerative effects on ß cells, stimulate these cells to produce more insulin 144 Anti-diabetic effects of lotus (Nelumbo nucifera) leaves in alloxan-induced diabetic mice (pancreatotrophic action) or it might itself contain some insulin-like substances. The induction of a regenerative stimulus in diabetic mice triggers a pancreatic regenerative processes, thereby restoring functional activities of the pancreas [1]. Results from the histopathological studies indicate a healing of the pancreas by lotus leaf tea, and this is a possible mechanism of the anti-diabetic activity. Figure 4. Histopathological studies of the pancreas: NC group (a), DC group (b), DLL group (c) Kidney The histological examination of alloxan-induced diabetic mice renal tissue, when compared to the controls groups, revealed a mild increase in mesangial cells and matrix of glumeroli. A hyaline thickening of some arteriole wall was also noted. Through the use of lotus leaf tea, these pathologic changes improved with measurements more like that of the normal control groups (Figure 5) Figure 5. Histopathological studies of renal medulla: NC group (a), DC group (b), DLL group (c) Observed findings and a comparison between normal, diabetic and lotus leaf supplemented diabetic mice were: - NC group: The histological examination of the kidney from normal control mice showed that the various segments of kidney tubules were well preserved. Abundant glomeruli, the portion of the nephron segment with interspersed blood capillaries, were also clearly seen. Medulla endothelial cells were small and even sized and the Bowmann 145 Nguyen Thi Hong Hanh, Ma Thi Thu Le and Le Thi Tuyet cavity was large. Interstitial tissue and blood vessels were normal and there was an absence of congestion (Figures 5a, 6a). - DC group: The endothelial cells showed irregular swelling and the Bowmann cavity was narrow. Interstitial tissue and blood vessels were congested and some inflamed cells were seen (Figures 5b, 6b). - DLL group: Endothelial cells were smaller than the endothelial cells of mice in DC group but cell size was not as even as in the NC group. Interstitial tissue and blood vessels did not show swelling or congestion (Figures 5c, 6c). Figure 6. Histopathological studies of renal cortex: NC group (a), DC group (b), DLL group (c) With observations similar to our own, Noor et al. reported the anti-diabetic activity of Aloe vera in alloxan-induced diabetic mice [7]. Noor et al. suggested two possible explanations for this finding: A. vera may exert its effect by preventing the death of ß cells and/or it may permit recovery of partially destroyed ß cells. The anti-diabetic activity of the lotus leaf tea used in the present investigation may occur through the mechanism as reported by Noor et al. This is an interesting finding and suggests that lotus leaves tea may have antioxidant (flavonoid) properties to prevent these changes. In light of the results, the study indicates that lotus leaf tea has good anti-diabetic activity. Lotus leaf tea exhibited significant anti-hyperglycemic activities in alloxan-induced hyperglycemic mice. 3. 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