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.
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 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 m/kg or CF2 mg/kg body weight daily by oral route of administration during 14 days Result: CF2 has blood glucose-lowering effect in type 2-like diabetic mice for 14 days treatment at doses of mg/kg and 4mg/kg daily (p < 0.05) The blood glucose- lowering effect of CF2 at dose of mg/kg daily is similar to gliclazide 80 mg/kg daily CF2 at dose of 4mg/kg is more effective than that of mg/kg and gliclazide 80 mg/kg in reducing blood glucose level Conclusion: CF2 has effect on lowering blood glucose level at doses of mg/kg and 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 increasing at an alarming rate globally According to a World Health Organization report an estimated 422 million adults globally were living with diabetes in 2014, compared to 108 million in 1980 [1] The number of people with diabetes aged 20 - 79 years was Corresponding author: Ho My Dung, Hanoi Medical University Email: homydung@hmu.edu.vn Received: 15 June 2017 Accepted: 16 November 2017 JMR 111 E2 (2) - 2018 predicted to rise to 642 million by 2040 [2] The health consequences of diabetes can overwhelm the health care systems due to the severity of the long term complications of diabetes [1] Several oral hypoglycemic agent are available to lower blood glucose levels in diabetics However, their administration may cause side effects in patients [3; 4] Therefore, there is on urgent need to find new prevention strategies and treatments for diabetes Recently, many plant-based natural products that contain certain phytochemicals are being investigated anti-di9 JOURNAL OF MEDICAL RESEARCH abetic potential for their [5] Using an herbal remedy as an alternative therapy for diabetes treatment would reduce individuals dependence on synthetic oral hypoglycemic agents [6] The plant kingdom offers a wide field of possible effective oral hypoglycemic agents Basket Plant (whose scientific name is Callisia fragrans) has been used as a traditional therapy for pain, fever, digestive disorders, heart diseases, diabetes, cancers and many other airments [7; 8] CF2 powder is extracted from the leaves, stems and shoots of Basket Plant CF2 powder contains the active component ecdysteroid which can be used in treating diabetes; preventing inflammation and osteomalacia; protecting the nervous system; and improving the immune system [9; 10] However, adequate characterization of CF2 effect is yet to be done and no study has been performed using a type diabetes model The objective of this study was to evaluate the hypoglycemic action of CF2 in a model of induced type 2-like diabetic mice Type-2 like diabetes was individual by high fat diet (HFD) combined with streptozotocin (STZ) injection II MATERIALS AND METHODS Materials Experimental Medicine CF2 powder extracted from leaves, stems and shoots of Basket Plant was supplied by Institute of Marine Biochemistry, Viet Nam Experimental Animals Swiss male white mice, from - weeks 10 of age, and weighing between 23 - 27 grams, were used for this study The mice were obtained from National Institute of Hygiene and Epidemiology, Viet Nam The experimental animals were caged individually and acclimatised to laboratory conditions for weeks prior to the experiment The study was carried out at the Pharmaceutical Department of Hanoi Medical University Machines and Chemicals - Streptozotocin g (Sigma-Aldrich, Singapore), Buffer solution Citrate pH 4.5 - Diamicron (gliclazide) tablets 30 mg (Servier ,France) - Blood glucose monitoring system On Call EZII (ACON Biotech, USA) - Animal blood counter Vet Exigo (Bonle Medical AB, Sweden) - Chemistry analyzer Erba and Test strips: blood triglyceride, HDL-C, cholesterol (Transasia, India) Method The study was divided into two stages [5]: * The first stage: Before ending the study, all mice baseline fasting glucose levels checked from peripheral blood samples + Group 1: Control condition (n = 10) mice were randomized to one of two group: Normal fat diet regime (NFD) for weeks + Group 2: Diabetic condition (n = 70): High fat diet regime (HFD) in weeks following Fabiola and Srinivasan method with 43% saturated fat combined siro fructose 55% [6] After weeks, the fasting glucose level in all mice was checked Mice in group JMR 111 E2 (2) - 2018 JOURNAL OF MEDICAL RESEARCH were injected intraperitoneally with STZ at a dose of 100 mg/kg Mice in group were injected intraperitoneally with citrate pH 4.5 After 72 hours (t0), the blood of all mice of group was checked The mice were considered diabetic if their blood glucose concentration was more than 10.0 mmol/L at that time Diabetic mice were then randomized into four groups from, group to group * The second stage: The study was carried out in a continuous 14-day period Mice were divided into five groups of ten animals: - Group 1: NFD regime + drinking distilled water; - Group 2: HFD regime + STZ injection 100mg/kg + drinking distilled water; - Group 3: HFD regime + STZ injection 100 mg/kg + drinking gliclazide 80 mg/kg/ day; - Group 4: HFD regime + STZ injection 100 mg/kg + drinking CF2 mg/kg/day (low-dose group); - Group 5: HFD regime + STZ injection 100 mg/kg + drinking CF2 mg/kg/day (high-dose group) After days (t1) and 14 days (tc) of treatment, the fasting glucose level of all mice were tested After 14 days of treatment, all animals blood lipid index (total cholesterol (TC), triglyceride (TG), HDL-Cholesterol, LDL-Cholesterol) were checked Animals were subjected to a full gross necrospy From 30% of each group, mices livers and pancreas were removed for histopathology examination Statistical analysis Data was analyzed using Microsoft Excel software version 2010 The levels of significance between groups were determined using student's t-test and Avant-après test Data is shown as mean ± standard deviation All data was considered significant at p < 0.05 III RESULTS Figure Wight of mice over time JMR 111 E2 (2) - 2018 11 JOURNAL OF MEDICAL RESEARCH The results in Figure showed that the weight of mice in the HFD eating regime was significantly 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 The change in blood glucose level of mice in type 2-like diabetes model Blood glucose level (mmol/l) ( X ± SD) (n = 10) Time p2-1 Group 1: Normal control Group 2: Diabetic control (t- test Student) Before studying 5.55 ± 0.74 5.79 ± 0.88 > 0.05 After weeks 5.45 ± 0.74 5.66 ± 1.04 > 0.05 After STZ injection 72 hours 5.36 ± 0.81 16.55 ± 5.48 * < 0.001 *: p < 0.05: In comparison to before studying Show in Table 1, the blood glucose level of mice in HFD regime group showed no differences compared to NFD regime group at the time before studying and after weeks (p > 0.05) However, after STZ injection 72 hours, the blood glucose level of mice in group was significantly increased compared to level in group (p < 0.001) Table Effect on blood glucose level of mice after weeks of treatment Blood glucose level (mmol/l) Group ( X ± SD) (n = 10) t0 (before treatment) t1 (after days of treatment) tc (after 14 days of treatment) Group 1: NFD + distilled water 5.22 ± 0.73 5.34 ± 0.68 5.35 ± 0.76 Group 2: HFD + STZ + distilled water 16.31 ± 3.32* 19.41 ± 3.77* 18.68 ± 1.26* ↑ 21.3 ↑ 20.43 15.49 ± 4.08** 12.74 ± 3.32*** ↓ 4.07 ↓ 19.45 % in change in comparison to t0 Group 3: HFD + STZ + gliclazide 80 mg/kg/day % in change in comparison to t0 12 16.45 ± 4.40* JMR 111 E2 (2) - 2018 JOURNAL OF MEDICAL RESEARCH Group 4: HFD + STZ + CF2 mg/ kg/day 16.64 ± 4.53* 15.09 ± 4.92** 13.76 ± 3.11*** ↓ 6.97 ↓ 13.1 11.82 ± 3.76*** 10.73 ± 3.84*** ↓ 27.02 ↓ 31.00 % in change in comparison to t0 Group 5: HFD + STZ + CF2 mg/ kg/day 16.89 ± 5.87* % in change in comparison to t0 *: p < 0.001: In comparison to group **: p < 0.05: In comparison to group CF2 powder at two oral doses (2 mg/kg/day and mg/kg/day) used continuously after 14 days had effect to reduce blood glucose level in comparison to diabetic control group (p < 0.05 and p < 0.001) Glucose reducing effect of CF2 at dose of mg/kg/day was similar to reducing effect of gliclazide dose 80 mg/kg/day seen in group (p > 0.05) CF2 at dose of mg/kg/day was more effective than CF2 at dose of mg/kg/day and gliclazide 80 mg/kg/day (p < 0.05) 4.5 Concentration (mmol/l) 3.5 Group 2.5 Group 2 Group Group 1.5 Group 0.5 TC TG HDL-C LDL-C Figure Effect on blood lipid content of mice after weeks of treatment The result of figure shows lipid disorder conditions TC, TG, HDL-C, LDL-C were in creased in group (diabetic control) in comparison to group (normal control) Blood lipid 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 JOURNAL OF MEDICAL RESEARCH Histopathological examination of liver and pancreas shows that all mice in group (diabetic control) were in degenerate condition when compared to group (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 The results are shown in Figure and Figure Figure Histopathological image of mice liver after weeks of treatment Figure Histopathological image of mice pancreas after weeks of treatment 14 JMR 111 E2 (2) - 2018 JOURNAL OF MEDICAL RESEARCH IV DISCUSSION After weeks of consuming a high fat diet regime, the blood glucose level in mice was increased but there was no significant difference when compared to normal control mice After mice with HFD eating regime were injected with STZ at a dose of 100 mg/ kg, the blood glucose level nearly times higher than before the injection This model was stably maintained during weeks of treatment and similar to the model of ShaoYu [7] These results prove a rich energy eating regime combined with a low-dose STZ injection has an effect on hyperglycemia and successfully creates model of type like diabetes After days and 14 days of treatment, gliclazide 80 mg/kg/day, CF2 at a low dose (2 mg/kg/day), and a high dose (4 mg/kg/day) effectively in reduced blood glucose levels in type 2-like diabetic mice when compared to diabetic control group The blood glucose-lowering effect of CF2 at dose of mg/ kg daily was similar to gliclazide 80 mg/kg daily CF2 at dose of 4mg/kg daily was more effective than the dose of mg/kg and gliclazide 80 mg/kg in reducing blood glucose level CF2 powder is there a contains ecdysteroid which was proved to reduce blood glucose level in experimental animals In Kizelsztein’s study (2009), 20-hydroxyecdyson at dose of 10 mg/kg/day reduces blood glucose level in type 2-like diabetic mice after 13 weeks through increasing circulating adiponectin levels [8] Another study by Sumdaram showed that the administration of 20-OH-ecdysone results in a significant restoration towards normal levels of plasma JMR 111 E2 (2) - 2018 glucose, insulin, HbA1c, and key carbohydrate an enzymes [9] Besides reducing blood glucose, CF2 effected lipid disorder conditions CF2 at two doses of mg/kg/day and mg/kg/ day reduced total cholesterol, triglyceride and LDL-C and increased HDL-C, however, differences were not significant This was similar to gliclazides 80 mg/kg/day effect on regulating lipid disorders This is consistent with previous research showing 20-hydroxyecdyson in Quinoa extract decreased total cholesterol and triglyceride in diet-induced obesity mice [10] The result of histopathological examination showed that there was positive change in liver and pancreas structure of mice after 2-week of treatment with either dose of CF2 The experimental medicine improved lipid disorders which is possibly low, it also reduced the degenerate condition of the liver Another possible explanation for CF2 is regenerative effect on reason for recreating pancreas structure is the antioxidant activity of Basket Plant [11] V CONCLUSION CF2 lower blood glucose level at doses of mg/kg and mg/kg daily over 14 days in type 2-like diabetic mice when compared to a control group The high dose was more effective than both the low dose and the glyceride 80mg/ kg dose Besides reducing blood glucose effect, CF2 improved lipid disorder conditions It lowered total cholesterol, triglyceride and LDL-C and increased HDL-C 15 JOURNAL OF MEDICAL RESEARCH Acknowledgements I would like to express my deepest gratitude to the Department of Pharmacology, Hanoi Medical University for supporting us in this study REFERENCES World Health Organization (2016) Global report on diabetes 2016 WHO K Ogurtsova, J D da Rocha Fernandes, Y Huang et al (2017) IDF Diabetes Atlas: Global estimates for the prevalence of diabetes for 2015 and 2040 Diabetes Res Clin Pract, 128, 40 - 50 Katzung B.G (2012), "Chapter 41: Pancreatic Hormones & Antidiabetic Drugs" Basic and clinical Pharmacology, The Mc GrawHill company, 743 - 765 Standards of Medical Care in Diabetes (2017) Summary of Revisions Diabetes Care, 40(Suppl 1), S4 - S5 Malviya N Jain S and Malviya S (2010) Antidiabetic potential of medicinal plants Acta Poloniae Pharmaceutica, Drug Research, 67 (2), 113 - 118 Suchý V., Zemlicka M., Svajdlenka E et al (2008) Medicinal plants and diabetes mellitus Ceska Slov Farm, 57(2), 78 84 Joash Ban Lee Tan, Wei Jin Yap, Shen Yeng Tan et al (2014) Antioxidant Content, Antioxidant Activity, and Antibacterial Activity of Five Plants from the Commelinaceae Family Antioxidants, 3(4), 758 - 769 Zilfikarov I.N Olennikov D.N., Toropova A.A et al (2008) Chemical composition of Callisia fragrans Wood Juice and its antioxidant activity (in vitro) Chemistry of plant raw material, 4, 95 - 100 L Dinan (2009) The Karlson Lecture Phytoecdysteroids: what use are they? 16 Arch Insect Biochem Physiol, 72(3), 126 41 10 Lafont R and Dinan L (2009) Innovative and future applications for ecdysteroids Ecdysone: structures and functions Springer Science, New York, 551 – 578 11 K Srinivasan, B Viswanad, L Asrat et al (2005) Combination of high-fat diet-fed and low-dose streptozotocin-treated rat: a model for type diabetes and pharmacological screening Pharmacol Res, 52(4), 313 - 320 12 Escalona C.N., Rivera R.F., Garduno S.L et al (2011) Antiobesity and hypoglycaemic effects of aqueous extract of Ibervillea sonorae in mice fed a high fat diet with fructose Journal of Biomedicine and Biotechnology, 12, - 10 13 Shao Yu WU, Guang fa WANG, Zhong LIU et al (2009) Effect of geniposide, a hypoglycemic glucoside, on hepatic regulating enzymes in diabetic mice induced by a high-fat diet and streptozotocin Acta Pharmacol Sin, 30(12), 202 - 208 14 Kizelsztein P., Govorko D., Komarnytsky S et al (2009) 20-Hydroxyecdysone decreases weight and hyperglycemia in a diet-induced obesity mice model Am J Physiol Endocrinol Metab, 296, 433 – 439 15 R Sundaram, R Naresh, P Shanthi et al (2012) Efficacy of 20-OH-ecdysone on hepatic key enzymes of carbohydrate metabolism in streptozotocin induced diabetic rats Phytomedicine, 19(8-9), 725 - 16 A S Foucault, V Mathe, R Lafont et al (2012) Quinoa extract enriched in 20-hydroxyecdysone protects mice from diet-induced obesity and modulates adipokines expression Obesity (Silver Spring), 20(2), 270 - 277 JMR 111 E2 (2) - 2018 ... performed using a type diabetes model The objective of this study was to evaluate the hypoglycemic action of CF2 in a model of induced type 2-like diabetic mice Type- 2 like diabetes was individual... weight of mice in the HFD eating regime was significantly 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... Effect on blood lipid content of mice after weeks of treatment The result of figure shows lipid disorder conditions TC, TG, HDL-C, LDL-C were in creased in group (diabetic control) in comparison