Study on the hypoglycemic action of cf2 in induced type 2 - like diabetic mice model

Nội dung text: Study on the hypoglycemic action of cf2 in induced type 2 - like diabetic mice model

1. JOURNAL OF MEDICAL RESEARCH STUDY ON THE HYPOGLYCEMIC ACTION OF CF2 IN INDUCED TYPE 2-LIKE DIABETIC MICE MODEL Ho My Dung¹, Vu Thi Ngoc Thanh¹, Pham Thi Van Anh¹, Nguyen Thi Thanh Ha¹, Nguyen Thi Minh Hang² ¹Hanoi Medical University ²Institute of Marine Biochemistry - Vietnam Academy of Science and Technology To investigate the hypoglycemic action of CF2 in a type 2-like diabetic mice model induced by high fat diet (HFD) combined with streptozotocin (STZ) injection. Method: Model: mice were fed with HFD for 8 weeks, then injected with a dose of STZ (100 mg/kg body weight, intraperitoneal injection). Animal used: Swiss male mice. Drugs: type 2-like diabetic mice with HFD and STZ treated with either gliclazide 80 mg/kg body weight, CF2 2 m/kg or CF2 4 mg/kg body weight daily by oral route of ad- ministration during 14 days. Result: CF2 has blood glucose-lowering effect in type 2-like diabetic mice for 14 days treatment at doses of 2 mg/kg and 4mg/kg daily (p < 0.05). The blood glucose- lowering effect of CF2 at dose of 2 mg/kg daily is similar to gliclazide 80 mg/kg daily. CF2 at dose of 4mg/kg is more effective than that of 2 mg/kg and gliclazide 80 mg/kg in reducing blood glucose level. Con- clusion: CF2 has effect on lowering blood glucose level at doses of 2 mg/kg and 4 mg/kg daily for 14 days in type 2-like diabetic mice, induced by HFD and STZ 100 mg/kg intraperitoneal injection. Keywords: CF2, Callisia fragrans, ecdysteroid, type 2-like diabetic mice, STZ, HFD, blood glucose I. INTRODUCTION The prevalence of diabetes mellitus is predicted to rise to 642 million by 2040 [2]. increasing at an alarming rate globally. Ac- The health consequences of diabetes can cording to a World Health Organization re- overwhelm the health care systems due to port an estimated 422 million adults globally the severity of the long term complications were living with diabetes in 2014, compared of diabetes [1]. Several oral hypoglycemic to 108 million in 1980 [1]. The number of agent are available to lower blood glucose people with diabetes aged 20 - 79 years was levels in diabetics. However, their adminis- tration may cause side effects in patients [3; Corresponding author: Ho My Dung, Hanoi Medical 4]. Therefore, there is on urgent need to find University. new prevention strategies and treatments Email: homydung@hmu.edu.vn for diabetes. Recently, many plant-based Received: 15 June 2017 natural products that contain certain phy- Accepted: 16 November 2017 tochemicals are being investigated anti-di- JMR 111 E2 (2) - 2018 9
2. JOURNAL OF MEDICAL RESEARCH abetic potential for their [5]. Using an herbal of age, and weighing between 23 - 27 remedy as an alternative therapy for diabe- grams, were used for this study. The mice tes treatment would reduce individuals de- were obtained from National Institute of Hy- pendence on synthetic oral hypoglycemic giene and Epidemiology, Viet Nam. The ex- agents [6]. The plant kingdom offers a wide perimental animals were caged individually field of possible effective oral hypoglycemic and acclimatised to laboratory conditions agents. for 2 weeks prior to the experiment. The Basket Plant (whose scientific name study was carried out at the Pharmaceutical is Callisia fragrans) has been used as a tra- Department of Hanoi Medical University. ditional therapy for pain, fever, digestive Machines and Chemicals disorders, heart diseases, diabetes, can- - Streptozotocin 1 g (Sigma-Aldrich, Sin- cers and many other airments [7; 8]. CF2 gapore), Buffer solution Citrate pH 4.5 powder is extracted from the leaves, stems - Diamicron (gliclazide) tablets 30 mg and shoots of Basket Plant. CF2 powder (Servier ,France). contains the active component ecdysteroid - Blood glucose monitoring system On which can be used in treating diabetes; Call EZII (ACON Biotech, USA) preventing inflammation and osteomalacia; - Animal blood counter Vet Exigo (Bonle protecting the nervous system; and improv- Medical AB, Sweden) ing the immune system [9; 10]. However, - Chemistry analyzer Erba and Test adequate characterization of CF2 effect is strips: blood triglyceride, HDL-C, cholester- yet to be done and no study has been per- ol (Transasia, India). formed using a type 2 diabetes model. The 2. Method objective of this study was to evaluate the The study was divided into two stages hypoglycemic action of CF2 in a model of [5]: induced type 2-like diabetic mice. Type-2 * The first stage: like diabetes was individual by high fat diet Before ending the study, all mice base- (HFD) combined with streptozotocin (STZ) line fasting glucose levels checked from pe- injection. ripheral blood samples. II. MATERIALS AND METHODS + Group 1: Control condition (n = 10) mice were randomized to one of two group: 1. Materials Normal fat diet regime (NFD) for 8 weeks. Experimental Medicine + Group 2: Diabetic condition (n = 70): CF2 powder extracted from leaves, High fat diet regime (HFD) in 8 weeks fol- stems and shoots of Basket Plant was sup- lowing Fabiola and Srinivasan method with plied by Institute of Marine Biochemistry, 43% saturated fat combined siro fructose Viet Nam. 55% [6]. Experimental Animals After 8 weeks, the fasting glucose level Swiss male white mice, from 6 - 8 weeks in all mice was checked. Mice in group 2 10 JMR 111 E2 (2) - 2018
3. JOURNAL OF MEDICAL RESEARCH were injected intraperitoneally with STZ at (low-dose group); a dose of 100 mg/kg. Mice in group 1 were - Group 5: HFD regime + STZ injection injected intraperitoneally with citrate pH 4.5. 100 mg/kg + drinking CF2 4 mg/kg/day After 72 hours (t0), the blood of all mice (high-dose group). of group 2 was checked. The mice were After 7 days (t1) and 14 days (tc) of treat- considered diabetic if their blood glucose ment, the fasting glucose level of all mice concentration was more than 10.0 mmol/L were tested. After 14 days of treatment, all at that time. Diabetic mice were then ran- animals blood lipid index (total cholester- domized into four groups from, group 2 to ol (TC), triglyceride (TG), HDL-Cholesterol, group 5. LDL-Cholesterol) were checked. Animals * The second stage: The study was car- were subjected to a full gross necrospy. ried out in a continuous 14-day period. Mice From 30% of each group, mices livers and were divided into five groups of ten animals: pancreas were removed for histopathology - Group 1: NFD regime + drinking dis- examination. tilled water; Statistical analysis - Group 2: HFD regime + STZ injection Data was analyzed using Microsoft Ex- 100mg/kg + drinking distilled water; cel software version 2010. The levels of sig- - Group 3: HFD regime + STZ injection nificance between groups were determined 100 mg/kg + drinking gliclazide 80 mg/kg/ using student's t-test and Avant-après test. day; Data is shown as mean ± standard devia- - Group 4: HFD regime + STZ injection tion. All data was considered significant at 100 mg/kg + drinking CF2 2 mg/kg/day p < 0.05. III. RESULTS Figure 1. Wight of mice over time JMR 111 E2 (2) - 2018 11
4. JOURNAL OF MEDICAL RESEARCH The results in Figure 1 showed that the weight of mice in the HFD eating regime was sig- nificantly higher than weight of mice in the NFD eating regime at the sixth week (p < 0.001). Hower, 72 hours after STZ injection in the NFD eating group, the weight of the mice in the HFD group was slightly reduced. Table 1. The change in blood glucose level of mice in type 2-like diabetes model Blood glucose level (mmol/l) ( X ± SD) (n = 10) p2-1 Time Group 1: Normal Group 2: Diabetic (t- test Student) control control Before studying 5.55 ± 0.74 5.79 ± 0.88 > 0.05 After 8 weeks 5.45 ± 0.74 5.66 ± 1.04 > 0.05 After STZ injection 72 5.36 ± 0.81 16.55 ± 5.48 * < 0.001 hours *: p < 0.05: In comparison to before studying Show in Table 1, the blood glucose level of mice in HFD regime group showed no differ- ences compared to NFD regime group at the time before studying and after 8 weeks (p > 0.05). However, after STZ injection 72 hours, the blood glucose level of mice in group 2 was significantly increased compared to level in group 1 (p < 0.001). Table 2. Effect on blood glucose level of mice after 2 weeks of treatment Blood glucose level (mmol/l) Group ( X ± SD) (n = 10) t0 (before t1 (after 7 days tc (after 14 days treatment) of treatment) of treatment) Group 1: NFD + distilled water 5.22 ± 0.73 5.34 ± 0.68 5.35 ± 0.76 Group 2: HFD + STZ + distilled 16.31 ± 3.32* 19.41 ± 3.77* 18.68 ± 1.26* water % in change in comparison to t0 ↑ 21.3 ↑ 20.43 Group 3: HFD + STZ + gliclazide 16.45 ± 4.40* 15.49 ± 4.08** 12.74 ± 3.32*** 80 mg/kg/day % in change in comparison to t0 ↓ 4.07 ↓ 19.45 12 JMR 111 E2 (2) - 2018
5. JOURNAL OF MEDICAL RESEARCH Group 4: HFD + STZ + CF2 2 mg/ 16.64 ± 4.53* 15.09 ± 4.92** 13.76 ± 3.11*** kg/day % in change in comparison to t0 ↓ 6.97 ↓ 13.1 Group 5: HFD + STZ + CF2 4 mg/ 16.89 ± 5.87* 11.82 ± 3.76*** 10.73 ± 3.84*** kg/day % in change in comparison to t0 ↓ 27.02 ↓ 31.00 *: p < 0.001: In comparison to group 2 **: p < 0.05: In comparison to group 2 CF2 powder at two oral doses (2 mg/kg/day and 4 mg/kg/day) used continuously after 14 days had effect to reduce blood glucose level in comparison to diabetic control group 2 (p < 0.05 and p < 0.001). Glucose reducing effect of CF2 at dose of 2 mg/kg/day was similar to reducing effect of gliclazide dose 80 mg/kg/day seen in group 3 (p > 0.05). CF2 at dose of 4 mg/kg/day was more effective than CF2 at dose of 2 mg/kg/day and gliclazide 80 mg/kg/day (p < 0.05). 4.5 4 3.5 3 Group 1 2.5 Group 2 2 Group 3 Group 4 1.5 Group 5 Concentration (mmol/l) 1 0.5 0 TC TG HDL-C LDL-C Figure 2. Effect on blood lipid content of mice after 2 weeks of treatment The result of figure 2 shows lipid disorder conditions TC, TG, HDL-C, LDL-C werein creased in group 2 (diabetic control) in comparison to group 1 (normal control). Blood lip- id disorders at gliclazide group and CF2 groups were improved, with reduction in TC, TG, LDL-C and increase of HDL-C content in comparison to diabetic control group. However, none of these were significant differences (p > 0.05). JMR 111 E2 (2) - 2018 13
6. JOURNAL OF MEDICAL RESEARCH Histopathological examination of liver and pancreas shows that all mice in group 2 (di- abetic control) were in degenerate condition when compared to group 1 (normal control). Histopathological examination of liver and pancreas of mice treated with gliclazide and two doses of CF2 were improved significantly in comparison to samples from group 2. The results are shown in Figure 3 and Figure 4. Figure 3. Histopathological image of mice liver after 2 weeks of treatment Figure 4. Histopathological image of mice pancreas after 2 weeks of treatment 14 JMR 111 E2 (2) - 2018
7. JOURNAL OF MEDICAL RESEARCH IV. DISCUSSION glucose, insulin, HbA1c, and key carbohy- After 8 weeks of consuming a high fat drate an enzymes [9]. diet regime, the blood glucose level in mice Besides reducing blood glucose, CF2 was increased but there was no significant effected lipid disorder conditions. CF2 at difference when compared to normal con- two doses of 2 mg/kg/day and 4 mg/kg/ trol mice. After mice with HFD eating regime day reduced total cholesterol, triglyceride were injected with STZ at a dose of 100 mg/ and LDL-C and increased HDL-C, however, kg, the blood glucose level nearly 3 times differences were not significant. This was higher than before the injection. This mod- similar to gliclazides 80 mg/kg/day effect el was stably maintained during 2 weeks on regulating lipid disorders. This is consis- of treatment and similar to the model of tent with previous research showing 20-hy- ShaoYu [7]. These results prove a rich ener- droxyecdyson in Quinoa extract decreased gy eating regime combined with a low-dose total cholesterol and triglyceride in diet-in- STZ injection has an effect on hyperglyce- duced obesity mice [10]. mia and successfully creates model of type The result of histopathological examina- 2 like diabetes. tion showed that there was positive change After 7 days and 14 days of treatment, gli- in liver and pancreas structure of mice af- clazide 80 mg/kg/day, CF2 at a low dose (2 ter 2-week of treatment with either dose of mg/kg/day), and a high dose (4 mg/kg/day) CF2. The experimental medicine improved effectively in reduced blood glucose levels lipid disorders which is possibly low, it also in type 2-like diabetic mice when compared reduced the degenerate condition of the liv- to diabetic control group. The blood glu- er. Another possible explanation for CF2 is cose-lowering effect of CF2 at dose of 2 mg/ regenerative effect on reason for recreating kg daily was similar to gliclazide 80 mg/kg pancreas structure is the antioxidant activity daily. CF2 at dose of 4mg/kg daily was more of Basket Plant [11]. effective than the dose of 2 mg/kg and gli- V. CONCLUSION clazide 80 mg/kg in reducing blood glucose CF2 lower blood glucose level at doses level. CF2 powder is there a contains ec- of 2 mg/kg and 4 mg/kg daily over 14 days dysteroid which was proved to reduce blood in type 2-like diabetic mice when compared glucose level in experimental animals. In Ki- to a control group. zelsztein’s study (2009), 20-hydroxyecdys- The high dose was more effective than on at dose of 10 mg/kg/day reduces blood both the low dose and the glyceride 80mg/ glucose level in type 2-like diabetic mice kg dose. after 13 weeks through increasing circulat- Besides reducing blood glucose effect, ing adiponectin levels [8]. Another study by CF2 improved lipid disorder conditions. It Sumdaram showed that the administration lowered total cholesterol, triglyceride and of 20-OH-ecdysone results in a significant LDL-C and increased HDL-C. restoration towards normal levels of plasma JMR 111 E2 (2) - 2018 15
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