Hypoglycemic effects of ethanolic extract from mesona chinensis benth on diabetic mice

159 HNUE JOURNAL OF SCIENCE DOI: 10.18173/2354-1059.2018-0041 Natural Sciences 2018, Volume 63, Issue 6, pp. 159-166 This paper is available online at HYPOGLYCEMIC EFFECTS OF ETHANOLIC EXTRACT FROM Mesona chinensis BENTH. ON DIABETIC MICE Nguyen Thi Thu, Nguyen Thi Phuong Thao, Nguyen Quoc Dat, Dao Thi Sen and Nguyen Thi Hong Hanh Faculty of Biology, Hanoi National University of Education Abstract. Both diabetes mellitus and obesity lead to a variety of local and systemic detrimental effects. This study was conducted to investigate the hypoglycemic effects of ethanolic extract from Mesona chinensis Benth. on diabetic mice due to exogenous glucose, alloxan-induced diabetic and obese diabetic mice. Hyperglycemia was induced by taking glucose 3 g/kg body weight (bw) or injecting of alloxan monohydrate 100 mg/kg bw or feding high-fat diet. Blood glucose level, HbA1c, glucose tolerability was assessed before, during and after treatment with ethanolic extract of Mesona chinensis Benth. at dose 400 mg/kg bw. The finding of the present investigation showed that extract did not reduce blood glucose level immediately but was effective after 15 days of treatment. The using of ethanolic extract from Mesona chinensis Benth. effects to decrease glucose levels more than half at the 15 th day from 10.90 to 4.68 mmol/L in alloxan-induced diabetic group; from 13.20 to 5.26 in obese diabetic group. Blood glucose tolerance and HbA1c values were also significantly improved after 15 days of treatment. These findings suggest that ethanolic extract from Mesona chinensis Benth. had hypoglycemic effect on diabetic mice. Keywords: Mesona chinensis Benth., hypoglycemic, alloxan-induced, diabetes mellitus, mice, obesity. 1. Introduction The worldwide incidence of diabetes mellitus and obesity continues to escalate despite the increased awareness of its pathogenesis and treatment. Both of these metabolic disorders lead to a variety of local and systemic detrimental effects. Obesity and diabetes have the interdependence relationship. Both insulin resistance and defective insulin secretion appear very prematurely in obese patients [1]. Obesity is believed to account for 80-85% of the risk of developing type 2 diabetes. The statistics in 2000 show that the number of diabetic patients worldwide was 177 million people which increased to 284.8 million in 2010 [2]. Over the last few decades, the rate of obesity and diabetes in Vietnam has rapidly increased along with the development of the economy. According to World Health Organization (WHO), Vietnam is among the countries with the highest rate of diabetes mellitus in the world due to dangerous complications such as kidney failure, high blood pressure, necrosis of the foot, heart attack, retinal damage [3]. Received July 6, 2018. Revised August 13, 2018. Accepted August 20, 2018. Contact Nguyen Thi Hong Hanh, e-mail address: hanhphucmauhong111@yahoo.com Nguyen Thi Thu, Nguyen Thi Phuong Thao, Nguyen Quoc Dat, Dao Thi Sen and Nguyen Thi Hong Hanh 160 A number of oral medicines are currently used for treatment of diabetes. However, these chemical drugs often have high costs, cause unwanted side effects and have high rates of secondary failure [4]. Therefore, along with the development of the science, the tendency of use products derived from herbals combined with dietary changes and physical activity is increasingly used in prevention and treatment of this disorder. Numerous natural products such as crude extracts and isolated compounds from plants appear to be applicable as treatments of diabetes, including Spirulina platensis [5], Carum carvi L. [6], Eruca sativa [7], Dioscorea cayenensis [8], Nelumbo nuficera Gaertn [9], Andrographis paniculata (Burm. F.) [10], Pterocarpus marsupium [11], Cleome droserifolia [12]. Mesona chiensis Benth. is an ideal, natural and safe pharmaceutical ingredient with rich nutrients and special medical functions. It is an important agricultural and medicinal plant of high economic value in Southeast Asia and China, which has been intensively studied in recent years [13]. Some studies have shown that Mesona chiensis Benth. contains 17 amino acids (including 7 essential amino acids), carbohydrates, fats, fiber, polyphenols, and flavonoids [14, 15]. With the extremely beneficial effects of the compounds found in Mesona chiensis Benth., mouse testing was used to evaluate the effects of these compounds in the treatment of diabetes. Moreover, Mesona chiensis Benth. has also been shown to reduce glucose and triglyceride levels in humans in China [16]. However, in Vietnam, there is no study on hypoglycemic effects of Mesona chinensis Benth. in prevention and treatment of diabetes mellitus. This study was conducted to investigate the hypoglycemic effects of ethanolic extract from Mesona chinensis Benth. on diabetes mice, contribute to supplement new medicine sources for Vietnamese traditional medicine. 2. Content 2.1. Materials and methods * Materials Animals: Male white mice (Mus musculus) weighing about 20 g (5-6 weeks of age) were purchased from the National Institute of Hygiene Epidemiology. Animals were maintained in a temperature (21 ± 2°C) and humidity (50 ± 20%) controlled room with a 12 h dark-light cycle at Human and Animal Physiology Department, Faculty of Biology. Mice were weighed every week and assessed physiological function every day. Physiological parameters include food intake, activity, urine, hairiness and cornea. Plant extract: Mesona chinensis Benth. was collected in 2016 in Dinh Hoa district of Thai Nguyen province. After harvesting, Mesona chinensis Benth. is dried, stored at 25-35 o C in a dry place. Ethanolic extract from Mesona chinensis Benth. was produced at the Biochemistry Department, Faculty of Biology according to the method described previously [17]. * Experimental design - Effect of ethanolic extract from Mesona chinensis Benth.on diabetic mice by exogenous glucose Animals were divided into three groups (six mice/group): (1) Normal control group (I) received 0.9% NaCl; (2) Standard group (II) received gliclazide (Stada Arzneimittel; 20 mg/kg bw); (3) Test group (III) received ethanolic extract of Mesona chinensis Benth. (400 mg/kg bw). All mice were fasted 12 hours before taking the drug/extracts. After 4 hours, all mice of three groups were measured the blood glucose level and then given oral glucose (3 g/kg bw). After that, blood glucose level was measured every 0.5 hours within 2.5 hours. Hypoglycemic effects of ethanolic extract from Mesona chinensis Benth. on diabetic mice 161 - Effect of ethanolic extract from Mesona chinensis Benth. on alloxan-induced and obese diabetic mouse model Animals were divided into four groups (six mice/group): (1) Normal control group (I) received 0.9% NaCl; (2) Standard group (II) received gliclazide (Stada Arzneimittel; 20 mg/kg bw); (3) Test alloxan-induced diabetic group (III) received ethanolic extract of Mesona chinensis Benth. (400 mg/kg bw); (4) Test obese diabetic group (IV) received ethanolic extract of Mesona chinensis Benth. (400 mg/kg bw). Group I, II, III were fed on standard pellets and water while group IV were fed on high-fat diet (65% standard pellets + 35% boiled lard in weight) during the experiment. * Alloxan induced diabetes 18-hour fasted mice were injected alloxan (Sigma Aldrich Chemicals Pvt Ltd, USA) dose of 100 mg/kg into the abdominal cavity [18]. After 1 hr of alloxan administration, the animals were fed on standard pellets and water. After 72 hour of injection, the animals having blood glucose level above 180 mg/dL (10 mmol/L) were selected for the investigation. * Obese diabetes The animals were fed on high-fat diet (65% standard pellets + 35% boiled lard in weight). After six weeks, the animals having blood glucose level above 180 mg/dL (10 mmol/L) were selected for the investigation. * Blood index measuring Blood sugar level was measured with an autoanalyzer by using Accu Check advantage II glucose kit. At the end of investigation, blood was collected from the caudal vena. Blood glucose levels and HbA1c were detected using an automated blood analyzer (Type Architect C8000; Abbott Ltd., United States of America) in Medlatec Hospital of Hanoi City. * Oral glucose tolerance test Oral glucose tolerance test was performed in the morning after an overnight fast. Mice were given glucose dose 3 g/kg bw. Blood samples were collected from the caudal vena at 0, 30, 60, 120 and 180 minutes for glucose to measure the blood glucose level. * Statistical analysis All values are expressed as the mean ± standard deviation. Statistical analyses were conducted using SPSS for Windows, version 16.0 (SPSS, Inc., Chicago, IL, USA). Multiple comparison tests for different groups were conducted. One way ANOVA with post-hoc test or independent-sample t test were used when appropriate. The criterion for statistical significance was set at P < 0.05. 2.2. Results and discussion The acute oral toxicity study results of ethanolic extract of Mesona chinensis Benth. have been reported from previous studies showing no mortality up to 8,000 mg/kg [17]. The physiological status of mice was normal during the 72 h monitoring. This result demonstrates that an oral administration of concentrated Mesona chinensis Benth. is non-toxic to experimental mice. 2.2.1. Effect of Mesona chinensis Benth. extract on blood glucose level of diabetic mice by exogenous glucose For dose of 3 g/kg glucose, the results of the change in blood glucose level are shown in the Table 1. Table 1 shows that the blood glucose level drop sharply after 4 hours of using gliclazide. Blood glucose level in this group decreased from 7.65 mmol/L to 2.25 mmol/L, much lower than the other two groups (P = 0.039). Blood glucose levels remained low and continued to decrease Nguyen Thi Thu, Nguyen Thi Phuong Thao, Nguyen Quoc Dat, Dao Thi Sen and Nguyen Thi Hong Hanh 162 after mice were given glucose. Meanwhile, the glucose tolerability of the mice receiving the extract was almost no different from that of the normal control group. After 4 hours of taking the ethanolic extract of Mesona chinensis Benth., the blood glucose levels decreased slightly from 8.50 mmol/L to 7.85 mmol/L. Thus, the Mesona chinensis Benth. extract had mild responsibility for reducing blood glucose that secreted by exogenous glucose. Table 1. Blood glucose level of diabetic mice by exogenous glucose Animal group Blood glucose level (mmol/L) Initial After 4 hour 30 minutes 60 minutes 90 minutes 120 minutes 150 minutes Normal control 8.10±0.14a 8.11±0.28a 23.35±0.49a 22.50±0.71a 22.15±0.78a 21.95±0.78a 21.60±0.85a Standard treated Gliclazide 7.65±0.21a 2.25±0.35b 6.85±0.35b 4.80±1.14b 2.25±0.35b 1.90±0.14b 1.60±0.14b Ethanolic extract 8.50±0.57a 7.85±0.21a 20.75±0.35a 19.25±0.35a 18.60±0.57a 17.75±0.21a 17.15±0.35a Values are expressed in mean ± S.D; each group contains 6 animals (n = 6). Different letters represent significant difference statistically (P < 0.05, P-values obtained by ANOVA post-hoc test) Index of glucose tolerance is important to evaluate the effects of a drug that treats the diabetes. The ability to inhibit glucose uptake in the small intestine by ingestion and glucose intake has been widely considered as a hypothesis on the effects of medicinal products for diabetes mellitus [19-21]. However, natural products need a certain period to take effect. 2.2.2. Effect of Mesona chinensis Benth. extract on diabetic mice during 15 days treatment The target of treating is controlling glucose in blood stream and preventing the complication. To evaluate the effects of Mesona chinensis Benth. extract on treating diabetes mellitus, testing of blood glucose level, oral glucose tolerance, HbA1c level is required. * Effect of Mesona chinensis Benth. extract on blood glucose level of diabetic mice during 15 days treatment The results of changing in blood glucose level of diabetic mice during 15 days treatment are shown in the Figure 1. Figure 1 shows that the blood glucose level drop sharply after 15 days of using gliclazide. Blood glucose level in this group decreased from 10.80 mmol/L to 2.05 mmol/L, much lower than the other three groups. After 15 days of using the ethanolic extract of Mesona chinensis Benth., blood glucose levels of alloxan-induced diabetic group and obese diabetic group decreased significantly compared to normal control group (P = 0.001 and P = 0.003, respectively). Blood glucose level in alloxan-induced diabetic group decreased from 10.90 mmol/L to 4.68 mmol/L. Blood glucose levels in obese diabetic group decreased from to 13.20 mmol/L to 5.26 mmol/L. Meanwhile, blood glucose level in normal control group remained stable at about 11 mmol/L. Hypoglycemic effects of ethanolic extract from Mesona chinensis Benth. on diabetic mice 163 Figure 1. Effect of Mesona chinensis Benth. extract on blood glucose level of diabetic mice during 15 days treatment Values are expressed in mean; each group contains 6 animals (n = 6). Different letters represent significant difference statistically (P < 0.05, P-values obtained by ANOVA post-hoc test) These demonstrated that the use of Mesona chinensis Benth. extract has long-term effects to diabetic in mice caused both by alloxan and obesity. The ability of Mesona chinensis Benth. on reducing glucose and triglyceride levels were tested in human by Charoonsri Chusak et al. (2014) [16]. Alloxan is a factor that causes to increase glucose in blood stream through the glucokinase inhibition, which reduces glucose oxidation and inhibits insulin synthesis [22]. The hypoglycemic activity of the Mesona chinensis Benth. extract used in the present investigation may occur through the mechanism as reported by Noor et al, 2008 [23]. This is an interesting finding and suggests that flavonoids have the potential for antioxidant activity. Thus, this compound may help to improve the insulin secretion function of the pancreas as well as the insulin resistance of the cell. The results from our previous research have shown that the flavonoid and total phenol content of the ethanol extracts is 37.5% and 26.56% [17]. Besides, Mesona chinensis Benth. extract was also effective with mice diabetic mice due to obesity. This effect can be indirectly mediated obesity therapeutic effect of Mesona chinensis Benth. * Effect of Mesona chinensis Benth. extract on oral glucose tolerance of diabetic mice The results of oral glucose tolerance test of diabetic mice after 15 days treatment are shown in Figure 2. Figure 2. Effect of Mesona chinensis Benth. extract on oral glucose tolerance of diabetic mice after 15 days treatment Values are expressed in mean; each group contains 6 animals (n = 6). Different letters represent significant difference statistically (P < 0.05, P-values obtained by ANOVA post-hoc test) Nguyen Thi Thu, Nguyen Thi Phuong Thao, Nguyen Quoc Dat, Dao Thi Sen and Nguyen Thi Hong Hanh 164 The results show that after 15 days of treatment, the tolerability of the batches was markedly different (Figure 2). Figure 2 shows that the blood glucose levels of all groups increase after 30 minutes of using 3 g/kg glucose. This increase caused by assemble glucose in cells. As a result, glucose inhibits glucokinase to reduce the glucose oxidation and insulin synthesis. After 120 minutes, the blood glucose levels of the mices receiving the extract of Mesona chinensis Benth. decreased compared to normal control group (P < 0.0001). Blood glucose level in alloxan-induced diabetic group decreased from 19.58 mmol/L to 11.44 mmol/L. Blood glucose levels in obese diabetic group decreased from to 15.40 mmol/L to 9.00 mmol/L. Meanwhile, blood glucose level in normal control group decrease slightly from 30.10 mmol/L to 26.75 mmol/L. This indicates that Mesona chinensis Benth. extract not only effect to increase the ability of glucose tolerance in diabetes mellitus mice caused by alloxan, but also had effect in diabetes mellitus caused by obesity. The results was consistent with some earlier researches about effects on reducing glucose level such as Spirulina platensis, Carum carvi L., Eruca sativa and Nelumbo nuficera [5, 6, 7, 24]. * Effect of Mesona chinensis Benth. extract on HbA1c diabetic mice after 15 days treatment The results of HbA1c after 15 days treatment are shown in Figure 3. Figure 3. Effect of Mesona chinensis Benth. extract on HbA1c of diabetic mice after 15 days treatment Values are expressed in mean; each group contains 6 animals (n = 6). Different letters represent significant difference statistically (P < 0.05, P-values obtained by ANOVA post-hoc test) Figure 3 showed that treatment with 400 mg/kg/day of dose, HbA1c level tended to be lower when compared to the normal control and similar with group using gliclazide. This demonstrated that Mesona chinensis Benth. extract tended to reduce HbA1c level in diabetes mellitus mice. Our research had resemble results with other studies on some herbs. For example, HbA1c level decreases in diabetes mellitus mice though using mixture extracted from Syzygium nervosum, Psidium guajava, Nelumbo nucifera [24, 25], and effects of cinnamon after 12 weeks [26]. 3. Conclusion The finding of the present investigation showed that extract did not reduce blood glucose level immediately but was effective after 15 days of treatment. 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