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Anti-diabetic properties of Momordica charantia L. polysaccharide in alloxan-induced diabetic mice
International Journal of Biological Macromolecules 81 (2015) 538–543
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Anti-diabetic properties of Momordica charantia L. polysaccharide in alloxan-induced diabetic mice Xin Xu a,c , Bin Shan b , Cai-Hu Liao b , Jian-Hua Xie a,∗ , Ping-Wei Wen a , Jia-Yi Shi d a
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China College of Yingdong Food Science and Engineering, Shaoguan University, Shaoguan 512005, China College of Food Science and Engineering, Yangzhou University, Yangzhou 225127, China d School of Food Science and Engineering, Nanjing University of Finance and Economics, Nanjing 210023, China b c
a r t i c l e
i n f o
Article history: Received 6 May 2015 Received in revised form 18 August 2015 Accepted 24 August 2015 Keywords: Momordica charantia L. Polysaccharide Hypoglycemic effect Anti-diabetic Alloxan-induced
a b s t r a c t A water-soluble polysaccharide (MCP) was isolated from the fruits of Momordica charantia L., and the hypoglycemic effects of MCP were investigated in both normal healthy and alloxan-induced diabetic mice. MCP was orally administered once a day after 3 days of alloxan-induction at 100, 200 and 300 mg/kg body weight for 28 day. Results showed that fasting blood glucose level (BGL) was significantly decreased, whereas the glucose tolerance was marked improvement in alloxan-induced diabetic mice, and loss in body weight was also prevented in diabetic mice compared to the diabetic control group. The dosage of 300 mg/kg body weight exhibited the best effects. In addition, MCP did not exhibit any toxic symptoms in the limited toxicity evaluation in mice. The results suggest that MCP possess significantly dose-dependent anti-diabetic activity on alloxan-induced diabetic mice. Hence, MCP can be incorporated as a supplement in health-care food, drugs and/or combined with other hypoglycemic drugs. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Momordica charantia L. (M. charantia L.) commonly known as ‘bitter gourd’, ‘bitter melon’, or ‘karela’ is a multi purpose herb widely cultivated in many tropical and subtropical regions of the world. The fruit of M. charantia L. are used as vegetable in different parts of the world. Apart from their role in food consumption, a wide array of pharmacological activities of the fruits of M. charantia L., such as antihyperglycemic [1], antidiabetic [2–4], antifungal [5], antioxidant effects [6,7], cytotoxic activities [4], and inhibitory activity against protein tyrosine phosphatas 1B [8] have been reported. Yuan et al. reported that the aqueous extracts from M. charantia L. had significant hypoglycemic effect in alloxan-induced diabetic mice [9]. However, the studies on anti-diabetic effects of M. charantia L. were mainly focused on the activity of the extracts [3,10].
Abbreviations: MCP, polysaccharide from the fruits of Momordica charantia L; BGL, blood glucose level; HPLC, high performance liquid chromatography; PMP, 1phenyl-3-methyl-5-pyrazolone; Mw , molecular weight. ∗ Corresponding author at: State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, Jiangxi, China. E-mail address: [email protected] (J.-H. Xie). http://dx.doi.org/10.1016/j.ijbiomac.2015.08.049 0141-8130/© 2015 Elsevier B.V. All rights reserved.
Diabetes mellitus, one of the most serious diseases, has a significant impact on health, quality of life, and life expectancy of patients as well as on the health care system in the modern world. It is a chronic disease characterized by high BGL, which can be broadly categorized into type 1 and type 2, the former develops as a result of insulin deficiency and the latter is due to insulin resistance. The number of adults with diabetes in the world was 347 million [11], and this number is likely to more than double by 2030. In 2012 diabetes was the direct cause of 1.5 million deaths. More than 80% of diabetes deaths occur in low- and middle-income countries. Yang et al. [12] estimated that more than 92 million men and women are living with diabetes in China, or almost 10 percent of adults in the world’s largest population of 1.3 billion people. Although different types of synthetic oral hypoglycemic agents and insulin are available for the treatment of diabetes mellitus, insulin cannot be taken orally and the synthetic agents can produce serious side effects and toxicity. Therefore, search for safe and effective agents has continued to be an important area of active research. Thus, there is a growing interest to identify natural anti-diabetic agents from natural materials to treat diabetes. It has been reported that most polysaccharides derived from higher plants are relatively non-toxic and do not cause significant side effects, which is a major problem associated with synthetic compounds [13–16]. Polysaccharides were found to be the main active compounds in M. charantia L., which exhibited
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immunoenhancing and antioxidant properties [7], and could protect against cerebral ischemia/reperfusion injury [17]. However, the hypoglycemic activity of MCP was not frequently reported. In this study, the MCP was extracted from M. charantia L. and its hypolipidemic activity was determined in alloxan-induced diabetic mice. 2. Materials and methods
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were housed in air-conditioned animal house with standard environmental conditions of constant temperature of 20–25 ◦ C, relative humidity of 45–55%, 12 h light–dark cycle. Before and during the experiment mice were fed standard pellet diet and water ad libitum. After randomization into various groups according to the sex before the experiment, the mice were acclimatized for a period of 2–3 days. Prior to experimental treatments, animals were subjected to fasting for 18 h but were allowed free access to water. All the experiments were done during daytime.
2.1. Materials and chemicals 2.5. Induction of experimental diabetes The fruit of M. charantia L. were purchased from local markets in July 2007 at Shaoguan, Guangdong province, China. They were washed, dried, then cut into pieces, and seeds were removed from the pulps, then ground to homogeneous powder (40–60 mesh) and stored at a dry place for further use. Alloxan, bovine serum albumin and d-glucose were purchased from Sigma Chemical Co. (St. Louis, USA). Xiangke Pill was produced by Guangzhou Zhongyi Pharmaceutical Company Ltd., which is composed of Chinese herbal medicines (Kudzuvine root, Rehmannia, Astragalus mongholicus, etc.) and chemical composition (Glibenclamide), is an effective compound used for treating diabetes in China. All other reagents used in the experiment were of analytical grade. 2.2. Preparation of MCP The preparation of polysaccharide was performed as described by Yi et al. with minor modifications [18]. Briefly, the dried fruits of M. charantia L. powder were extracted with water at 80 ◦ C for 2 h. The extracts were filtered through glass wool and centrifuged (6000 × g, 10 min) to separate the supernatant and the residue. The associated proteins in the solution were removed using the Sevag method. After removing the Sevag reagent, the water phase were concentrated and precipitated with ethanol, and kept at 4 ◦ C overnight in refrigerator to precipitate polysaccharide. The precipitates were collected and then re-dissolved in water, then further dialyzed (MWCO 8000) in distilled water for 24 h before concentration [19]. Lastly, the precipitate was frozen, and then was submitted to lyophilized. The M. charantia L. polysaccharide was obtained. 2.3. Analysis of MCP The total carbohydrate content was examined using anthronesulfuric acid method with d-glucose as standard. Protein content was analyzed by the Folin-Ciocalteu reagent. Uronic acid contents were determined according to Blumenkrantz and Asboe-Hansen’s method [20] with d-glucuronic acid as the standard. The molecular weight of MCP was evaluated by high performance liquid chromatography (HPLC) equipped with UltrahydrogelTM Linear Column (7.8 mm × 300 mm). HPLC was performed on 0.5% MCP (20 L) dissolved in distilled water with 0.7% NaCl as the mobile phase at 0.5 mL/min and 35 ◦ C. The columns were calibrated with T-series Dextran as standards. Monosaccharide composition of the MCP was analyzed by HPLC after 2 M trifluoroacetic acid hydrolysis and labeling of the hydrolysate with 1-phenyl-3-methyl-5-pyrazolone (PMP), on a Shim-Pack C18 column (4.6 mm × 250 mm) using an HPLC system, eluted with 82.0% PBS (0.1 mol/L, pH 7.0) and 18.0% acetonitrile at a flow rate of 1 mL/min.
Animals were allowed to fast 18 h and were injected with ice cold alloxan monohydrate freshly prepared with water (2%) at a dose of 70 mg/kg body weight [21]. Fasting BGL were measured after a week when the condition of diabetes was stabilized. Animals with marked hyperglycemia (fasting blood glucose concentration level above 11.1 mmol/L) were considered to be diabetic and used for the experiments [21]. 2.6. Assessment of MCP on normal mice and alloxan-induced diabetic mice The normal mice were divided into three groups (Groups I–III). Group I was served as normal control and received appropriate volumes of vehicle (distilled water) orally only; Group II was served as positive control (Xiaoke pill a dose of 200 mg/kg); Groups III was given orally MCP in the form of mucilage at dose of 200 mg/kg body weight. Animals were treated once a day for 28 days. The mice with marked hyperglycemia were divided into three groups (Groups IV–VIII). Group IV was served as diabetic control and received appropriate volumes of vehicle (distilled water) orally only. Group V was served as positive control and received Xiaoke pill a dose of 200 mg/kg, orally. Groups VI, VII and VIII were given orally MCP in the form of mucilage at doses of 100, 200 and 300 mg/kg body weight, respectively. Animals were treated once a day for 28 days. 2.7. Assessment of MCP on glucose tolerance After overnight fasting, the hypoglycemic effects of MCP in normal healthy and alloxan-induced diabetic mice were assessed by the improvement of glucose tolerance. Fasting blood glucose was taken from over-night fasted mice. The mice (n = 10 per group) were divided into six groups. Group a (normal control), normal mice treated with vehicle (distilled water) alone; Group b (diabetic control), diabetic mice treated with vehicle alone; Group c (positive control), diabetic mice treated with Xiaoke pill (200 mg/kg body weight); Group d (MCP-L), diabetic mice treated with MCP (100 mg/kg body weight); Group e (MCP-M), diabetic mice treated with MCP (200 mg/kg body weight); Group f (MCP-H), diabetic mice treated with MCP (300 mg/kg body weight). The mice of all the groups were given glucose (as aqueous solution, 2 g/kg, body weight) after 90 min of MCP and vehicle administration. Blood samples were collected for glucose levels estimation just prior to the administration of the glucose (0 min) and 30, 60, and 120 min after the glucose loading. 2.8. Measurements of blood glucose, body weight changes and food intake
2.4. Animals Kunming mice (weighing 20 ± 2 g) of both sexes were purchased from the Sun Yat-sen University (Guangzhou, China). All animal protocols were approved by the institutional animal care and use committee of Sun Yat-sen University (Gangdong, China). All mice
Blood samples were centrifuged (4 ◦ C, 4200 × g, 15 min) and the serum obtained was used for blood glucose determination. Stored plasma samples were analyzed for glucose level by using the Trinder’s glucose-oxidase method [22]. After 0, 7, 14 and 28 days’ treatment with, serum from fasting mice were collected to detect
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Fig. 1. HPLC analysis for monosaccharide composition of MCP. Monosaccharides were identified and quantified by comparing to authentic ones used as standard. Rib, ribose; Man, mannose; Rha, rhamnose; Glu, glucose; Gal, galactose; Xyl, xylose; Ara, arabinose; Fuc, fucose.
blood glucose levels. Body weight was measured at 3:00 p.m. on day 0, 7, 14, and 28. Food intake was measured daily before injection.
Fig. 2. Effects of MCP on fasting blood glucose levels in alloxan-induced diabetic rats. Group IV (diabetic control), diabetic rats treated with vehicle (distilled water) alone; Group V (positive control), diabetic rats treated with xiaoke pill (XKP) (200 mg/kg body weight); Group VI (MCP-L), diabetic rats treated with MCP (100 mg/kg body weight); Group VII (MCP-M), diabetic rats treated with MCP (200 mg/kg body weight) and Group VIII (MCP-H), diabetic rats treated with MCP (300 mg/kg body weight); The values were expressed as mean ± S.D. (n = 10 for each group), *p < 0.05 significant from the normal control, # p < 0.05 significant from the diabetic control.
3.2. Effect of MCP on normal mice 2.9. Acute toxicity A separate experiment was performed to know whether any toxic effects are produced by MCP on mice. Four groups of mice were administered orally by the single dose of 2.5, 5 and 10 times of effective MCP dose. Then mice were observed with regard to gross behavioral, neurological, autonomic and toxic effects at the short time intervals within 24 h. Food consumption, feces and urine were also examined at 2 h and then at 6 h intervals within 24 h. 2.10. Data analysis All the data were expressed as mean ± standard deviation (S.D.) in each group. Statistical calculations by SPSS version 10.0 software (SPSS Inc., Chicago, USA) were carried out. One-way ANOVA was applied for determining differences between results of samples. Duncan test was taken to compare the data. The values were considered to be significantly different when the P-value was less than 0.05. 3. Results and discussion 3.1. Analysis of MCP After the precipitate was filtered, and lyophilized to produce a yellow powder, the polysaccharide was obtained, and the percentage yield was 2.3%. The total carbohydrate content and protein content of MCP was 72.6 ± 1.2% and 8.9 ± 0.2%, respectively. The uronic acid content in MCP determined according to Blumenkrantz and Asboe-Hansen’s method was 20.1 ± 0.4%. The molecular weight of polysaccharide was determined in a range of 85–100 kDa. To further investigate the composition of MCP, HPLC technique was used in this study. Specifically, different monosaccharide standards were run on HPLC and their retention time was recorded to identify the monosaccharide component of MCP. The monosaccharide composition analysis revealed it contained arabinose, xylose, galactose and rhamnose in a ratio of 1.00:1.12:4.07:1.79 according to the retention time and peak areas of HPLC (Fig. 1).
Results of the effect of oral administration of MCP on the BGL in normal mice are presented in Table 1. In normal control, positive control (Group II) and MCP groups (Group III), the BGL observed at the 7th day after treatment were 5.06 ± 0.25, 5.15 ± 0.27 and 5.17 ± 0.18 mmol/L, respectively. After 28 days of treatment, the BGL in normal control, positive control and MCP groups were 5.09 ± 0.16, 5.01 ± 0.33 and 5.03 ± 0.41 mmol/L, respectively (Table 1). Though the BGL in Group II and Group III decreased, the effect was significant. The results indicated that the MCP had significant hypoglycemic effect on normal healthy mice. 3.3. Effect of MCP on alloxan-induced diabetic mice Diabetes mellitus is a serious chronic disease, mostly accompanied by characteristic long-term complications. With the increasing of the incidence rate, the study is necessary to get more in depth about the polysaccharides for treatment of diabetes mellitus from natural materials. Therefore, this study was also designed to determine whether MCP had an anti-diabetic activity in alloxan-induced hyperglycemic mice. In a long-term study, alloxan-induced diabetic mice were treated with MCP and Xiaoke pill, respectively, once a day, for 28 days. The anti-hyperglycemic effect of repeated oral administration of MCP on the BGL in diabetic mice is shown in Fig. 2. The various BGL of severely diabetic mice were estimated before and after 7, 14 and 28 days of treatment. Seven days after alloxan administration, blood glucose values were 3–5 folds higher in all the groups in comparison to normal control (Group I) and were not statistically (P > 0.05) different from each other. The blood glucose concentration had been kept in high level among 16–22 mmol/L in diabetic control group (Group IV) throughout the whole experiment periods. But the blood glucose in the MCP groups lowered significantly, especially that in the MCP groups at 200 and 300 mg/kg. The BGL decreased by 55.76% and 57.28% in the animals treated with MCP-M and MCP-H respectively, after 28 days of treatment (Fig. 2). In the positive control (Group V), reduction of BGL was also significant (P < 0.05) from 28 days after administration.
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Table 1 The effect of MCP on fasting blood glucose levels in normal rats. Treatment groups
Normal control (Group I) Positive control (Group II) MCP (Group III) a *
Dose (mg/kg)
200 200
Blood glucose levels in rats at different days (mmol/L)a Day 0
Day 7
Day 14
Day 28
4.92 ± 0.44 5.21 ± 0.29 5.34 ± 0.13
5.06 ± 0.25 5.15 ± 0.27 5.17 ± 0.18
5.75 ± 0.57 5.09 ± 0.23* 5.12 ± 0.14*
5.24 ± 0.16 5.01 ± 0.33* 5.03 ± 0.41*
Values were expressed as mean ± S.D. (n = 10 for each group). p < 0.05 significant from the normal control.
12.5 mmol/L after 120 min. The glucose levels of these mice were statistically equal to that of Xiaoke pill (reduction of 31.26%). MCPH showed a higher hypoglycemic effect (Fig. 3). On the other hand, no significant difference in BGL was observed for the normal mice treated with the extract when compared to 0 min of this group. After 120 min, no significant difference was observed between the blood levels of the mice treated with the MCP and those treated with the standard drugs. These results clearly indicated that MCP could improve glucose tolerance of alloxan-induced diabetic mice. 3.5. Effect of MCP on body weight in alloxan-induced diabetic mice
Fig. 3. Effects of MCP on the oral glucose tolerance in normal and alloxan-induced diabetic rats. Group a (normal control), normal rats treated with vehicle (distilled water) alone; Group b (diabetic control), diabetic rats treated with vehicle alone; Group c (positive control), diabetic rats treated with xiaoke pill (XKP) (200 mg/kg body weight); Group d (MCP-L), diabetic rats treated with MCP (100 mg/kg body weight); Group e (MCP-M), diabetic rats treated with MCP (200 mg/kg body weight) and Group f (MCP-H), diabetic rats treated with MCP (300 mg/kg body weight). The values were expressed as mean ± S.D. (n = 10 for each group), *p < 0.05 significant from the normal control, # p < 0.05 significant from the diabetic control.
The hypoglycemic activity of MCP-H (300 mg/kg dose) in alloxandiabetic mice was similar to that of Xiaoke pill 200 mg/kg (Fig. 2). Normal control showed significant variation on the BGL throughout the experimental period. The results showed that MCP possessed remarkable hypoglycemic effect on hyperglycemia in diabetic rats. M. charantia have been reported to have significant hypoglycemic and antidiabetic effects [1–3]. Furthermore, some investigators have attempted to purify the active fractions from fruits of M. charantia and reported that saponins [23], charantin [3], and peptides [9] from it exhibited hypoglycemic effects. The ethnopharmacological studies of MCP confirmed the ethnobotanical data for the plant M. charantia L., which is traditionally used as an infusion of M. charantia L. by Chinese population for treatment diabetes. 3.4. Effect of MCP on glucose tolerance The effect of MCP on oral glucose tolerance in alloxan-induced diabetic mice was presented in Fig. 3. As shown in Fig. 3, the glucose tolerance of the diabetic mice control was severely worse than that of normal control (P < 0.05), and both the diabetic groups treated with MCP showed a significant improvement (P < 0.05) in the tolerance performance. Basal glycemia remained high in the diabetic control group, whereas MCP-L, MCP-M, and MCP-H groups caused a reduction of blood glucose (23.68, 29.02 and 31.27%, P < 0.05, respectively) at 120 min tested when compared to 30 min. In diabetic mice, the MCP-H showed a higher hypoglycemic effect throughout the period studied, reaching glycemic levels below
Compared to the normal control mice (Group I), all the alloxaninduced diabetic mice exhibited significant (P < 0.05) loss of body weight (Table 2), although there was no significant difference in their food intake. Before embarking on the experiment, all the groups had no significant difference in body weight (P > 0.05). A significant decrease in body weight was detected in the diabetic control, MCP-L, MCP-M, and MCP-H groups as compared to the normal control group from 7 days after treatment (P < 0.05). However, the body weights in the MCP-treated groups were significantly (P < 0.05) and dose-dependently increased as compared to those of the diabetic control from 14 days after administration, which is comparable to that of the positive control. To the end of trial, the mice in MCP-L, MCP-M, and MCP-H groups got an increase in body weigh of 31.19%, 36.25% and 38.05%, respectively (Table 2). The data indicated that MCP could significantly increase the body weight in alloxan-induced diabetic mice. The ability of the MCP to protect body weight loss seems to be as a result of its ability to reduce hyperglycemia. Alloxan is widely used in studies of experimental diabetes as this agent produce hyperglycemia by selective cytotoxic effect on pancreatic ˇ cells [24]. Alloxan-induced diabetes is widely used in the laboratory to mimic diabetic pathology and for screening anti-diabetic drugs. The generally accepted mechanism for alloxaninduced diabetes is that free radicals generated by alloxan initiates damage that ultimately leads to ˇ cells death and hypoinsulinemia [25]. The study of digestion and absorption property of polysaccharide extract in the animal is still not very clear. Hu et al. [26] investigated the gastric, intestinal digestion, and fermentation of polysaccharide by given oral administration polysaccharide from Plantago asiatica L. The effects of the polysaccharide on mouse colon, nutrient metabolism and colon microbiota were also studied. It was found that salivary amylase had no effect on the polysaccharide; however, the polysaccharide was influenced in later gastrointestinal digestion. A steady decrease in molecular weight (Mw ) of the polysaccharide may due to the breakdown of glycosidic bonds. In addition, there was no monosaccharide released throughout the whole digestion period, suggesting that the gastrointestinal digestion did not result in a production of free monosaccharide. In previous studies, it has been shown that polysaccharide have anti-hyperglycemic activity in streptozotocin- or alloxan-induced
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Table 2 The effects of MCP on body weight in alloxan-induced diabetic rats. Treatment groups
Dose (mg/kg)
Body weight in rats at different days after treatmenta (g) Day 0
Normal control Diabetic control Positive control MCP-L MCP-M MCP-H a * # a−d
200 100 200 300
18.62 18.87 18.89 18.85 18.51 18.45
Day 7 ± ± ± ± ± ±
0.72 0.72 0.93 0.59 0.89a 1.06a
23.18 21.53 23.58 20.35 21.32 21.41
Day 14 ± ± ± ± ± ±
1.93 1.03* 0.95# 1.46* 0.62b, * 1.83b, *
26.57 23.22 25.13 23.88 24.24 24.43
± ± ± ± ± ±
Day 28 1.99 1.64* 1.24* 2.07* 1.22c, * , # 0.73* , #
27.98 23.56 25.49 24.73 25.22 25.47
± ± ± ± ± ±
0.54 0.83* 0.63* 0.82*,# 1.16d, * , # 1.02* , #
Values expressed as mean ± S.D. (n = 10 for each group). p < 0.05 significant in comparison with normal control. p < 0.05 significant in comparison with diabetic control. Data within a column in groups of MCP without the same superscripts differ significantly in comparison with MCP-L (P < 0.05).
diabetic animals [27–31]. It has been shown in previous studies that the hypoglycemic effect of Lycium barbarum polysaccharide was possibly due to the intestinal absorption of glucose, or improving the metabolism of glucose, increasing insulin secretion from the ˇ-cells of the islets of langerhans or ameliorating insulin sensitivity, and preventing the destruction of ˇ cells [32]. The current study indicated that the dose of MCP played a vital role in fasting the blood glucose reduction and the improvement of glucose tolerance in alloxan-induced type 2 diabetic mice, thus proving the hypoglycemic effects of MCP. In the present study, the mechanism of the hypoglycemic effects of MCP was not investigated, it could be an improvement of insulin resistance of T2DM based on the lowered FINS and HOMA-IR levels of the diabetic mice [31], however, it remains to be further studied and to be proven.
3.6. Acute toxicity experiment The behavior of the treated mice appeared normal. No toxic effect was reported up to 5 and 10 times of effective dose of the MCP and there were no death in any of these groups, and there was no significant difference in the body weights and food consumption when compared to the vehicle treated group. Also, no gross pathological changes were seen. Only the consumption of food was increased by 20% in 10 and 15 time doses during 4 h but remaining normal afterwards. Thus, it was concluded that MCP was safe at a dose of 3000 mg/kg body weight. Furthermore, polysaccharides from M. charantia L. may make very good agent for the treatment of diabetes as it is often eaten in our diet and is therefore unlikely to be harmful to human health.
4. Conclusions Preliminary pharmacological assays suggested that MCP administered orally in alloxan-induced diabetic mice could significantly reduce BGL, increase the glucose tolerance and body weight of diabetic mice. Toxicity data proved that the MCP did not show any toxic reactions. The results give a scientific support for the proper use of M. charantia L. in folk medicine for the treatment of diabetes. In view of the protective property in the hypoglycemic and its relatively non-toxic nature, MCP would be a promising candidate for the development as a potential agent for the treatment of diabetes in human. Further studies need to carried out and undertaken to insight into the accuracy mechanisms of this action.
Conflicts of interest The authors report no conflicts of interest.
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Anti-diabetic properties of Momordica charantia L. polysaccharide in alloxan-induced diabetic mice Xin Xu a,c , Bin Shan b , Cai-Hu Liao b , Jian-Hua Xie a,∗ , Ping-Wei Wen a , Jia-Yi Shi d a
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China College of Yingdong Food Science and Engineering, Shaoguan University, Shaoguan 512005, China College of Food Science and Engineering, Yangzhou University, Yangzhou 225127, China d School of Food Science and Engineering, Nanjing University of Finance and Economics, Nanjing 210023, China b c
a r t i c l e
i n f o
Article history: Received 6 May 2015 Received in revised form 18 August 2015 Accepted 24 August 2015 Keywords: Momordica charantia L. Polysaccharide Hypoglycemic effect Anti-diabetic Alloxan-induced
a b s t r a c t A water-soluble polysaccharide (MCP) was isolated from the fruits of Momordica charantia L., and the hypoglycemic effects of MCP were investigated in both normal healthy and alloxan-induced diabetic mice. MCP was orally administered once a day after 3 days of alloxan-induction at 100, 200 and 300 mg/kg body weight for 28 day. Results showed that fasting blood glucose level (BGL) was significantly decreased, whereas the glucose tolerance was marked improvement in alloxan-induced diabetic mice, and loss in body weight was also prevented in diabetic mice compared to the diabetic control group. The dosage of 300 mg/kg body weight exhibited the best effects. In addition, MCP did not exhibit any toxic symptoms in the limited toxicity evaluation in mice. The results suggest that MCP possess significantly dose-dependent anti-diabetic activity on alloxan-induced diabetic mice. Hence, MCP can be incorporated as a supplement in health-care food, drugs and/or combined with other hypoglycemic drugs. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Momordica charantia L. (M. charantia L.) commonly known as ‘bitter gourd’, ‘bitter melon’, or ‘karela’ is a multi purpose herb widely cultivated in many tropical and subtropical regions of the world. The fruit of M. charantia L. are used as vegetable in different parts of the world. Apart from their role in food consumption, a wide array of pharmacological activities of the fruits of M. charantia L., such as antihyperglycemic [1], antidiabetic [2–4], antifungal [5], antioxidant effects [6,7], cytotoxic activities [4], and inhibitory activity against protein tyrosine phosphatas 1B [8] have been reported. Yuan et al. reported that the aqueous extracts from M. charantia L. had significant hypoglycemic effect in alloxan-induced diabetic mice [9]. However, the studies on anti-diabetic effects of M. charantia L. were mainly focused on the activity of the extracts [3,10].
Abbreviations: MCP, polysaccharide from the fruits of Momordica charantia L; BGL, blood glucose level; HPLC, high performance liquid chromatography; PMP, 1phenyl-3-methyl-5-pyrazolone; Mw , molecular weight. ∗ Corresponding author at: State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, Jiangxi, China. E-mail address: [email protected] (J.-H. Xie). http://dx.doi.org/10.1016/j.ijbiomac.2015.08.049 0141-8130/© 2015 Elsevier B.V. All rights reserved.
Diabetes mellitus, one of the most serious diseases, has a significant impact on health, quality of life, and life expectancy of patients as well as on the health care system in the modern world. It is a chronic disease characterized by high BGL, which can be broadly categorized into type 1 and type 2, the former develops as a result of insulin deficiency and the latter is due to insulin resistance. The number of adults with diabetes in the world was 347 million [11], and this number is likely to more than double by 2030. In 2012 diabetes was the direct cause of 1.5 million deaths. More than 80% of diabetes deaths occur in low- and middle-income countries. Yang et al. [12] estimated that more than 92 million men and women are living with diabetes in China, or almost 10 percent of adults in the world’s largest population of 1.3 billion people. Although different types of synthetic oral hypoglycemic agents and insulin are available for the treatment of diabetes mellitus, insulin cannot be taken orally and the synthetic agents can produce serious side effects and toxicity. Therefore, search for safe and effective agents has continued to be an important area of active research. Thus, there is a growing interest to identify natural anti-diabetic agents from natural materials to treat diabetes. It has been reported that most polysaccharides derived from higher plants are relatively non-toxic and do not cause significant side effects, which is a major problem associated with synthetic compounds [13–16]. Polysaccharides were found to be the main active compounds in M. charantia L., which exhibited
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immunoenhancing and antioxidant properties [7], and could protect against cerebral ischemia/reperfusion injury [17]. However, the hypoglycemic activity of MCP was not frequently reported. In this study, the MCP was extracted from M. charantia L. and its hypolipidemic activity was determined in alloxan-induced diabetic mice. 2. Materials and methods
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were housed in air-conditioned animal house with standard environmental conditions of constant temperature of 20–25 ◦ C, relative humidity of 45–55%, 12 h light–dark cycle. Before and during the experiment mice were fed standard pellet diet and water ad libitum. After randomization into various groups according to the sex before the experiment, the mice were acclimatized for a period of 2–3 days. Prior to experimental treatments, animals were subjected to fasting for 18 h but were allowed free access to water. All the experiments were done during daytime.
2.1. Materials and chemicals 2.5. Induction of experimental diabetes The fruit of M. charantia L. were purchased from local markets in July 2007 at Shaoguan, Guangdong province, China. They were washed, dried, then cut into pieces, and seeds were removed from the pulps, then ground to homogeneous powder (40–60 mesh) and stored at a dry place for further use. Alloxan, bovine serum albumin and d-glucose were purchased from Sigma Chemical Co. (St. Louis, USA). Xiangke Pill was produced by Guangzhou Zhongyi Pharmaceutical Company Ltd., which is composed of Chinese herbal medicines (Kudzuvine root, Rehmannia, Astragalus mongholicus, etc.) and chemical composition (Glibenclamide), is an effective compound used for treating diabetes in China. All other reagents used in the experiment were of analytical grade. 2.2. Preparation of MCP The preparation of polysaccharide was performed as described by Yi et al. with minor modifications [18]. Briefly, the dried fruits of M. charantia L. powder were extracted with water at 80 ◦ C for 2 h. The extracts were filtered through glass wool and centrifuged (6000 × g, 10 min) to separate the supernatant and the residue. The associated proteins in the solution were removed using the Sevag method. After removing the Sevag reagent, the water phase were concentrated and precipitated with ethanol, and kept at 4 ◦ C overnight in refrigerator to precipitate polysaccharide. The precipitates were collected and then re-dissolved in water, then further dialyzed (MWCO 8000) in distilled water for 24 h before concentration [19]. Lastly, the precipitate was frozen, and then was submitted to lyophilized. The M. charantia L. polysaccharide was obtained. 2.3. Analysis of MCP The total carbohydrate content was examined using anthronesulfuric acid method with d-glucose as standard. Protein content was analyzed by the Folin-Ciocalteu reagent. Uronic acid contents were determined according to Blumenkrantz and Asboe-Hansen’s method [20] with d-glucuronic acid as the standard. The molecular weight of MCP was evaluated by high performance liquid chromatography (HPLC) equipped with UltrahydrogelTM Linear Column (7.8 mm × 300 mm). HPLC was performed on 0.5% MCP (20 L) dissolved in distilled water with 0.7% NaCl as the mobile phase at 0.5 mL/min and 35 ◦ C. The columns were calibrated with T-series Dextran as standards. Monosaccharide composition of the MCP was analyzed by HPLC after 2 M trifluoroacetic acid hydrolysis and labeling of the hydrolysate with 1-phenyl-3-methyl-5-pyrazolone (PMP), on a Shim-Pack C18 column (4.6 mm × 250 mm) using an HPLC system, eluted with 82.0% PBS (0.1 mol/L, pH 7.0) and 18.0% acetonitrile at a flow rate of 1 mL/min.
Animals were allowed to fast 18 h and were injected with ice cold alloxan monohydrate freshly prepared with water (2%) at a dose of 70 mg/kg body weight [21]. Fasting BGL were measured after a week when the condition of diabetes was stabilized. Animals with marked hyperglycemia (fasting blood glucose concentration level above 11.1 mmol/L) were considered to be diabetic and used for the experiments [21]. 2.6. Assessment of MCP on normal mice and alloxan-induced diabetic mice The normal mice were divided into three groups (Groups I–III). Group I was served as normal control and received appropriate volumes of vehicle (distilled water) orally only; Group II was served as positive control (Xiaoke pill a dose of 200 mg/kg); Groups III was given orally MCP in the form of mucilage at dose of 200 mg/kg body weight. Animals were treated once a day for 28 days. The mice with marked hyperglycemia were divided into three groups (Groups IV–VIII). Group IV was served as diabetic control and received appropriate volumes of vehicle (distilled water) orally only. Group V was served as positive control and received Xiaoke pill a dose of 200 mg/kg, orally. Groups VI, VII and VIII were given orally MCP in the form of mucilage at doses of 100, 200 and 300 mg/kg body weight, respectively. Animals were treated once a day for 28 days. 2.7. Assessment of MCP on glucose tolerance After overnight fasting, the hypoglycemic effects of MCP in normal healthy and alloxan-induced diabetic mice were assessed by the improvement of glucose tolerance. Fasting blood glucose was taken from over-night fasted mice. The mice (n = 10 per group) were divided into six groups. Group a (normal control), normal mice treated with vehicle (distilled water) alone; Group b (diabetic control), diabetic mice treated with vehicle alone; Group c (positive control), diabetic mice treated with Xiaoke pill (200 mg/kg body weight); Group d (MCP-L), diabetic mice treated with MCP (100 mg/kg body weight); Group e (MCP-M), diabetic mice treated with MCP (200 mg/kg body weight); Group f (MCP-H), diabetic mice treated with MCP (300 mg/kg body weight). The mice of all the groups were given glucose (as aqueous solution, 2 g/kg, body weight) after 90 min of MCP and vehicle administration. Blood samples were collected for glucose levels estimation just prior to the administration of the glucose (0 min) and 30, 60, and 120 min after the glucose loading. 2.8. Measurements of blood glucose, body weight changes and food intake
2.4. Animals Kunming mice (weighing 20 ± 2 g) of both sexes were purchased from the Sun Yat-sen University (Guangzhou, China). All animal protocols were approved by the institutional animal care and use committee of Sun Yat-sen University (Gangdong, China). All mice
Blood samples were centrifuged (4 ◦ C, 4200 × g, 15 min) and the serum obtained was used for blood glucose determination. Stored plasma samples were analyzed for glucose level by using the Trinder’s glucose-oxidase method [22]. After 0, 7, 14 and 28 days’ treatment with, serum from fasting mice were collected to detect
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Fig. 1. HPLC analysis for monosaccharide composition of MCP. Monosaccharides were identified and quantified by comparing to authentic ones used as standard. Rib, ribose; Man, mannose; Rha, rhamnose; Glu, glucose; Gal, galactose; Xyl, xylose; Ara, arabinose; Fuc, fucose.
blood glucose levels. Body weight was measured at 3:00 p.m. on day 0, 7, 14, and 28. Food intake was measured daily before injection.
Fig. 2. Effects of MCP on fasting blood glucose levels in alloxan-induced diabetic rats. Group IV (diabetic control), diabetic rats treated with vehicle (distilled water) alone; Group V (positive control), diabetic rats treated with xiaoke pill (XKP) (200 mg/kg body weight); Group VI (MCP-L), diabetic rats treated with MCP (100 mg/kg body weight); Group VII (MCP-M), diabetic rats treated with MCP (200 mg/kg body weight) and Group VIII (MCP-H), diabetic rats treated with MCP (300 mg/kg body weight); The values were expressed as mean ± S.D. (n = 10 for each group), *p < 0.05 significant from the normal control, # p < 0.05 significant from the diabetic control.
3.2. Effect of MCP on normal mice 2.9. Acute toxicity A separate experiment was performed to know whether any toxic effects are produced by MCP on mice. Four groups of mice were administered orally by the single dose of 2.5, 5 and 10 times of effective MCP dose. Then mice were observed with regard to gross behavioral, neurological, autonomic and toxic effects at the short time intervals within 24 h. Food consumption, feces and urine were also examined at 2 h and then at 6 h intervals within 24 h. 2.10. Data analysis All the data were expressed as mean ± standard deviation (S.D.) in each group. Statistical calculations by SPSS version 10.0 software (SPSS Inc., Chicago, USA) were carried out. One-way ANOVA was applied for determining differences between results of samples. Duncan test was taken to compare the data. The values were considered to be significantly different when the P-value was less than 0.05. 3. Results and discussion 3.1. Analysis of MCP After the precipitate was filtered, and lyophilized to produce a yellow powder, the polysaccharide was obtained, and the percentage yield was 2.3%. The total carbohydrate content and protein content of MCP was 72.6 ± 1.2% and 8.9 ± 0.2%, respectively. The uronic acid content in MCP determined according to Blumenkrantz and Asboe-Hansen’s method was 20.1 ± 0.4%. The molecular weight of polysaccharide was determined in a range of 85–100 kDa. To further investigate the composition of MCP, HPLC technique was used in this study. Specifically, different monosaccharide standards were run on HPLC and their retention time was recorded to identify the monosaccharide component of MCP. The monosaccharide composition analysis revealed it contained arabinose, xylose, galactose and rhamnose in a ratio of 1.00:1.12:4.07:1.79 according to the retention time and peak areas of HPLC (Fig. 1).
Results of the effect of oral administration of MCP on the BGL in normal mice are presented in Table 1. In normal control, positive control (Group II) and MCP groups (Group III), the BGL observed at the 7th day after treatment were 5.06 ± 0.25, 5.15 ± 0.27 and 5.17 ± 0.18 mmol/L, respectively. After 28 days of treatment, the BGL in normal control, positive control and MCP groups were 5.09 ± 0.16, 5.01 ± 0.33 and 5.03 ± 0.41 mmol/L, respectively (Table 1). Though the BGL in Group II and Group III decreased, the effect was significant. The results indicated that the MCP had significant hypoglycemic effect on normal healthy mice. 3.3. Effect of MCP on alloxan-induced diabetic mice Diabetes mellitus is a serious chronic disease, mostly accompanied by characteristic long-term complications. With the increasing of the incidence rate, the study is necessary to get more in depth about the polysaccharides for treatment of diabetes mellitus from natural materials. Therefore, this study was also designed to determine whether MCP had an anti-diabetic activity in alloxan-induced hyperglycemic mice. In a long-term study, alloxan-induced diabetic mice were treated with MCP and Xiaoke pill, respectively, once a day, for 28 days. The anti-hyperglycemic effect of repeated oral administration of MCP on the BGL in diabetic mice is shown in Fig. 2. The various BGL of severely diabetic mice were estimated before and after 7, 14 and 28 days of treatment. Seven days after alloxan administration, blood glucose values were 3–5 folds higher in all the groups in comparison to normal control (Group I) and were not statistically (P > 0.05) different from each other. The blood glucose concentration had been kept in high level among 16–22 mmol/L in diabetic control group (Group IV) throughout the whole experiment periods. But the blood glucose in the MCP groups lowered significantly, especially that in the MCP groups at 200 and 300 mg/kg. The BGL decreased by 55.76% and 57.28% in the animals treated with MCP-M and MCP-H respectively, after 28 days of treatment (Fig. 2). In the positive control (Group V), reduction of BGL was also significant (P < 0.05) from 28 days after administration.
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Table 1 The effect of MCP on fasting blood glucose levels in normal rats. Treatment groups
Normal control (Group I) Positive control (Group II) MCP (Group III) a *
Dose (mg/kg)
200 200
Blood glucose levels in rats at different days (mmol/L)a Day 0
Day 7
Day 14
Day 28
4.92 ± 0.44 5.21 ± 0.29 5.34 ± 0.13
5.06 ± 0.25 5.15 ± 0.27 5.17 ± 0.18
5.75 ± 0.57 5.09 ± 0.23* 5.12 ± 0.14*
5.24 ± 0.16 5.01 ± 0.33* 5.03 ± 0.41*
Values were expressed as mean ± S.D. (n = 10 for each group). p < 0.05 significant from the normal control.
12.5 mmol/L after 120 min. The glucose levels of these mice were statistically equal to that of Xiaoke pill (reduction of 31.26%). MCPH showed a higher hypoglycemic effect (Fig. 3). On the other hand, no significant difference in BGL was observed for the normal mice treated with the extract when compared to 0 min of this group. After 120 min, no significant difference was observed between the blood levels of the mice treated with the MCP and those treated with the standard drugs. These results clearly indicated that MCP could improve glucose tolerance of alloxan-induced diabetic mice. 3.5. Effect of MCP on body weight in alloxan-induced diabetic mice
Fig. 3. Effects of MCP on the oral glucose tolerance in normal and alloxan-induced diabetic rats. Group a (normal control), normal rats treated with vehicle (distilled water) alone; Group b (diabetic control), diabetic rats treated with vehicle alone; Group c (positive control), diabetic rats treated with xiaoke pill (XKP) (200 mg/kg body weight); Group d (MCP-L), diabetic rats treated with MCP (100 mg/kg body weight); Group e (MCP-M), diabetic rats treated with MCP (200 mg/kg body weight) and Group f (MCP-H), diabetic rats treated with MCP (300 mg/kg body weight). The values were expressed as mean ± S.D. (n = 10 for each group), *p < 0.05 significant from the normal control, # p < 0.05 significant from the diabetic control.
The hypoglycemic activity of MCP-H (300 mg/kg dose) in alloxandiabetic mice was similar to that of Xiaoke pill 200 mg/kg (Fig. 2). Normal control showed significant variation on the BGL throughout the experimental period. The results showed that MCP possessed remarkable hypoglycemic effect on hyperglycemia in diabetic rats. M. charantia have been reported to have significant hypoglycemic and antidiabetic effects [1–3]. Furthermore, some investigators have attempted to purify the active fractions from fruits of M. charantia and reported that saponins [23], charantin [3], and peptides [9] from it exhibited hypoglycemic effects. The ethnopharmacological studies of MCP confirmed the ethnobotanical data for the plant M. charantia L., which is traditionally used as an infusion of M. charantia L. by Chinese population for treatment diabetes. 3.4. Effect of MCP on glucose tolerance The effect of MCP on oral glucose tolerance in alloxan-induced diabetic mice was presented in Fig. 3. As shown in Fig. 3, the glucose tolerance of the diabetic mice control was severely worse than that of normal control (P < 0.05), and both the diabetic groups treated with MCP showed a significant improvement (P < 0.05) in the tolerance performance. Basal glycemia remained high in the diabetic control group, whereas MCP-L, MCP-M, and MCP-H groups caused a reduction of blood glucose (23.68, 29.02 and 31.27%, P < 0.05, respectively) at 120 min tested when compared to 30 min. In diabetic mice, the MCP-H showed a higher hypoglycemic effect throughout the period studied, reaching glycemic levels below
Compared to the normal control mice (Group I), all the alloxaninduced diabetic mice exhibited significant (P < 0.05) loss of body weight (Table 2), although there was no significant difference in their food intake. Before embarking on the experiment, all the groups had no significant difference in body weight (P > 0.05). A significant decrease in body weight was detected in the diabetic control, MCP-L, MCP-M, and MCP-H groups as compared to the normal control group from 7 days after treatment (P < 0.05). However, the body weights in the MCP-treated groups were significantly (P < 0.05) and dose-dependently increased as compared to those of the diabetic control from 14 days after administration, which is comparable to that of the positive control. To the end of trial, the mice in MCP-L, MCP-M, and MCP-H groups got an increase in body weigh of 31.19%, 36.25% and 38.05%, respectively (Table 2). The data indicated that MCP could significantly increase the body weight in alloxan-induced diabetic mice. The ability of the MCP to protect body weight loss seems to be as a result of its ability to reduce hyperglycemia. Alloxan is widely used in studies of experimental diabetes as this agent produce hyperglycemia by selective cytotoxic effect on pancreatic ˇ cells [24]. Alloxan-induced diabetes is widely used in the laboratory to mimic diabetic pathology and for screening anti-diabetic drugs. The generally accepted mechanism for alloxaninduced diabetes is that free radicals generated by alloxan initiates damage that ultimately leads to ˇ cells death and hypoinsulinemia [25]. The study of digestion and absorption property of polysaccharide extract in the animal is still not very clear. Hu et al. [26] investigated the gastric, intestinal digestion, and fermentation of polysaccharide by given oral administration polysaccharide from Plantago asiatica L. The effects of the polysaccharide on mouse colon, nutrient metabolism and colon microbiota were also studied. It was found that salivary amylase had no effect on the polysaccharide; however, the polysaccharide was influenced in later gastrointestinal digestion. A steady decrease in molecular weight (Mw ) of the polysaccharide may due to the breakdown of glycosidic bonds. In addition, there was no monosaccharide released throughout the whole digestion period, suggesting that the gastrointestinal digestion did not result in a production of free monosaccharide. In previous studies, it has been shown that polysaccharide have anti-hyperglycemic activity in streptozotocin- or alloxan-induced
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Table 2 The effects of MCP on body weight in alloxan-induced diabetic rats. Treatment groups
Dose (mg/kg)
Body weight in rats at different days after treatmenta (g) Day 0
Normal control Diabetic control Positive control MCP-L MCP-M MCP-H a * # a−d
200 100 200 300
18.62 18.87 18.89 18.85 18.51 18.45
Day 7 ± ± ± ± ± ±
0.72 0.72 0.93 0.59 0.89a 1.06a
23.18 21.53 23.58 20.35 21.32 21.41
Day 14 ± ± ± ± ± ±
1.93 1.03* 0.95# 1.46* 0.62b, * 1.83b, *
26.57 23.22 25.13 23.88 24.24 24.43
± ± ± ± ± ±
Day 28 1.99 1.64* 1.24* 2.07* 1.22c, * , # 0.73* , #
27.98 23.56 25.49 24.73 25.22 25.47
± ± ± ± ± ±
0.54 0.83* 0.63* 0.82*,# 1.16d, * , # 1.02* , #
Values expressed as mean ± S.D. (n = 10 for each group). p < 0.05 significant in comparison with normal control. p < 0.05 significant in comparison with diabetic control. Data within a column in groups of MCP without the same superscripts differ significantly in comparison with MCP-L (P < 0.05).
diabetic animals [27–31]. It has been shown in previous studies that the hypoglycemic effect of Lycium barbarum polysaccharide was possibly due to the intestinal absorption of glucose, or improving the metabolism of glucose, increasing insulin secretion from the ˇ-cells of the islets of langerhans or ameliorating insulin sensitivity, and preventing the destruction of ˇ cells [32]. The current study indicated that the dose of MCP played a vital role in fasting the blood glucose reduction and the improvement of glucose tolerance in alloxan-induced type 2 diabetic mice, thus proving the hypoglycemic effects of MCP. In the present study, the mechanism of the hypoglycemic effects of MCP was not investigated, it could be an improvement of insulin resistance of T2DM based on the lowered FINS and HOMA-IR levels of the diabetic mice [31], however, it remains to be further studied and to be proven.
3.6. Acute toxicity experiment The behavior of the treated mice appeared normal. No toxic effect was reported up to 5 and 10 times of effective dose of the MCP and there were no death in any of these groups, and there was no significant difference in the body weights and food consumption when compared to the vehicle treated group. Also, no gross pathological changes were seen. Only the consumption of food was increased by 20% in 10 and 15 time doses during 4 h but remaining normal afterwards. Thus, it was concluded that MCP was safe at a dose of 3000 mg/kg body weight. Furthermore, polysaccharides from M. charantia L. may make very good agent for the treatment of diabetes as it is often eaten in our diet and is therefore unlikely to be harmful to human health.
4. Conclusions Preliminary pharmacological assays suggested that MCP administered orally in alloxan-induced diabetic mice could significantly reduce BGL, increase the glucose tolerance and body weight of diabetic mice. Toxicity data proved that the MCP did not show any toxic reactions. The results give a scientific support for the proper use of M. charantia L. in folk medicine for the treatment of diabetes. In view of the protective property in the hypoglycemic and its relatively non-toxic nature, MCP would be a promising candidate for the development as a potential agent for the treatment of diabetes in human. Further studies need to carried out and undertaken to insight into the accuracy mechanisms of this action.
Conflicts of interest The authors report no conflicts of interest.
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