Summary of new aspect of the thesis: Nghiên cứu độc tính và tác dụng hạ glucose máu của viên Andiabet trên thực nghiệm

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Several new models have been implemented: evaluate the effect inhbitivity on hyperglycemic postprandial blood glucose of reagent in glucose/sucrose/starch tolerance test in normal and diabetic mice and the effect antagonism with insulin resistance in type 2 diabetic mice (the technique of "Hyperinsulinemic - euglycemic clamp test")

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Nội dung Text: Summary of new aspect of the thesis: Nghiên cứu độc tính và tác dụng hạ glucose máu của viên Andiabet trên thực nghiệm

1 INTRODUCTION Type 2 diabetes mellitus (T2D) is a metabolic disorder with complex mechanisms and pathophysiological abnormalities. It is extremely hard to reverse T2D disease state with monotherapy. Thus combination therapy is becoming a promising alternative choice in clinical practice by designing drug combinations or compound drugs to interact with multiple targets and achive synergitic treatment effect. For example, traditional Vietnamese medicines are attracting more attentions for their efficacy and less frequent side effects for treating T2D. Lagerstroemia speciosa (L.) Pers; Gynostemma pentaphyllum (Thunb.) Makino; Anemarrhenae Aspheloides (Bunge) has been reported to alleviate hyperglycemia and hyperlipemia in many preclinical and clinical studies and have been combined into the soft form of Vinabetes. However, Vinabetes has only been extracted in laboratory and there are no adequate pharmacological studies. Andiabet is a compound of 3 Vietnamese herbal medicines composition above. The aim of this study was to investigate the toxicity and the hypoglycemic effect of Andiabet in experimental animals with the following objectives: 1. Determine of acute toxicity and longterm toxicity by Andiabet. 2. Evaluate the hypoglycemic effect and several hypoglycemic mechanisms of Andiabet tablets in experiment. Chapter 1. OVERVIEW 1.1. OVERVIEW ABOUT THE DIABETES MELLITUS 1.1.1. Definition, classification, diagnostic criteria for diabetes and pathogenetic mechanism of diabetes type 2 1.1.1.1. Definition “Diabetes is a metabolic disorder characterized by hyperglycemia, the result of a deficiency of insulin secretion; impaired functioning of insulin; or both. Chronic hyperglycemia is often
2 associated with damage, disorders and impaired function of many organs, especially the eyes, kidneys, nerves, heart and blood vessels.” 1.1.1.2. Classification: Diabetes is divided into 4 types: diabetes type 1, type 2, diabetes pregnancy and special types. In which, type 1 and type 2 diabetes is most common. 1.1.1.3. Diagnostic criteria for diabetes Four diagnostic tests for diabetes are currently recommended, including measurement of fasting plasma glucose; 2-hour (2-h) post- load plasma glucose after a 75 g oral glucose tolerance test (OGTT); HbA1c; and a random blood glucose in the presence of signs and symptoms of diabetes. People with fasting plasma glucose values of ≥ 7.0 mmol/L (126 mg/dl), 2-h post-load plasma glucose ≥ 11.1 mmol/L (200 mg/dl), HbA1c ≥ 6.5% (48 mmol/mol); or a random blood glucose ≥ 11.1 mmol/L (200 mg/dl) in the presence of signs and symptoms are considered to have diabetes 1.1.1.4. Pathogenetic mechanism of diabetes type 2 Insulin resistance in peripheral tissues and insulin secretion disorders are two important and related closely factors in the pathogenesis of type 2 diabetes, which usually occurs before the clinical manifestations of diabetes (from the pre-diabetes phase). However, in patients with type 2 diabetes who are not overweight, the manifestation of insulin secretion is the opposite, whereas in type 2 diabetes patients with obesity, insulin resistance is the main condition. Genetic and environmental factors play a role in promoting disease development and development. 1.2. GROUPS OF MEDICINE TREATMENT FOR DIABETES Currently, in addition to lifestyle adjustment (diet and exercise), drugs should be used to treat of type 2 diabetes. The drugs for treatment of type 2 diabetes focus on the following main groups: 1.2.1. Drugs stimulate insulin secretion include: KATP channel
3 inhibitors are: sulfonylurea and meglitinide. The incretin modulators are: GLP-1 analogues: Exenatid, Liraglutid, Lixisenatid and DPP-4 inhibitors: sitagliptin, vildagliptin, saxagliptin, linagliptin, alogliptin. 1.2.2. Drugs reduce insulin resistance include: metformin and thiazolidinedion: pioglitazon 1.2.3. Drugs reduce/slow absorption of glucid: drugs a-glucosidase inhibitors include: acarbose (Precose, Glucobay) and miglitol (Glyset). 1.2.4. Drugs increase renal glucose excretion: SGLT2 inhibitors are: Dapagliflozin, Canagliflozin and Empagliflozin 1.3. SEVERAL DIABETIC RESEARCH METHODS IN EXPERIMENTANT 1.3.1. The invivo research model. 1.3.1.1. Models of type 1 diabetes: Spontaneous type 1 diabetes (T1D) models and secondary T1D models induced by chemicals, removing pancreas or virus. 1.3.1.2. Models of type 2 diabetes: Obese and non-obese spontaneous T2D rodents models. Secondary T2D models: induced by chemicals or a high-fat diet combined with low-dose STZ and by genetic modification. 1.3.1.3. Several methods to evaluate hypoglycemic effect in invivo: Assessing the ability of the drug on glucose tolerance test, polysaccharide absorption. Assessing the drug’s effect of increasing insulin sensitivity to the target tissue via the technique "Hyperinsulinemic - euglycemic clamp test" 1.3.2. The invitro research model. The invitro research model can be divided into two categories: assessing the of drug’s effect on organs, isolated cells and on enzymes involved glucose homeostasis. 1.4. OVERVIEW COMPONENTS OF ANDIABET AND RESEARCHS RELATED TO ANDIABET
4 Lagerstroemia speciosa (L.) Pers; Gynostemma pentaphyllum (Thunb.)Makino; Anemarrhenae aspheloides (Bunge) was estimated for hypoglycemic virtue in many preclinical and clinical studies and have been combined into the soft form of Vinabetes. Vinabetes with a 1,5:1,5:1 weight ratio of the three herbal composition, similar to Andiabet was performed acute toxicity test and LD50 dose was 42.98 g/kg mice. Vinabetes has also been studied for long term toxicity test in rabbits for 4 weeks at 2 doses of 1.8 g/kg/day and 3.6 g/kg/day. Vinabetes at the doses for 4.5g/kg and 9g/kg for 4 consecutive weeks in normal mice displayed significantly reducing blood glucose levels (by 34 % and 44 %, respectively) compared to the control (p
5 each) and Newzealand White rabbits (both sexes, weighing 1,8 – 2,5 kg each) obtained from Central Hygienic and Epidemiologic Institude were housed at 24±20C and provided with food and water ad libitum. 2.1.3. Chemicals. Streptozotocin (Sigma-Aldrich, Singapore). Diamicron (gliclazid) 30mg (Servier, France). Blood glucose meter and glucose kit (On Call Plus, ACON Biotech, America). Triglycerid kit and total cholesterol kit (DIALAB GmbH, Austria). 2.2. EXPERIMENT DESIGN 2.2.1. Method of the acute and long term toxicity test 2.2.1.1. Acute toxicity test (following WHO guidelines) The mice were randomized into many groups of 10 animals in each. In all group, mice were given an equal volume (0.2ml/10g) of Andiabet with increasing doses up to a highest dose of 44.25g/kg mice. The mice were observed for 4 consecutive hours, the number of mice was died in the first 72 hours were counted and the whole body condition in 7 days after treatment were reported. LD50 dose was determined according to Litchfield - Wilcoxon method. 2.2.1.2. Long term toxicity test (following WHO guidelines) Rabbits are given two doses of Andiabet: 0.21 g/kg/day (equivalent to the expected dose in human) and 0.64 g/kg/day (3 times the expected dose in humans) for 90 consecutive days . Assess general condition and weight change. Evaluate hematopoietic function, liver, kidney function, liver and kidney histopathology of rabbits 2.2.2. Evaluate the hypoglycemic effect and several hypoglycemic mechanisms of Andiabet tablets in experiment. 2.2.2.1. Hypoglycemic effect of Andiabet in normal mice. The mice were randomized into 4 groups of 10 animals in each. - Group 1 (Control): received with distilled water - Group 2 (positive control): received with gliclazid 80 mg/kg. - Group 3 (Andiabet 0,68g/kg): received with Andiabet 0,68 g/kg/day
6 - Group 4 (Andiabet 2g/kg): received with Andiabet 2g/kg/day Mice were given distilled water or reagents continuously for 2 weeks in the mornings. Blood samples were collected at times: start the treatmnet (to); 1 week (t1) and 2 weeks after oral the reagent (t2). 2.2.2.2. Hypoglycemic effect of Andiabet in T2D mice. - Phase 1: High- fat diet combined with low dose STZ-induced diabetic mice model. Mice is divided into 2 groups: Group 1 (n = 10 animals) eating a normal fat diet (NFD). Group 2 (n = 100 animals): eating a high fat diet (HFD). After 8 weeks, diabetes was induced by a single i.p injection of STZ (100mg/kg) for all mice in group 2, and mice in group 1 were injected by solvent of STZ. Blood samples were taken at 3 points: start the study, before injection of STZ and 72 hours after injection of STZ. 72 hours after injection of STZ or solvent, animals which glucose level develop more than 10 mmol/L were selected. - Phase 2: Assess the hypoglycemic effect of Andiabet in type 2 diabetic mice.The animals were randomly assigned to one of six groups of 10 mice each. The first group were regarded as control group, eating a normal fat diet. The type 2 diabetic (T2D) mice divided into 5 groups from second to sixth groups were studied for the hypoglycemic effect of Andiabet. - Group 1 (control): NFD mice received with saline - Group 2 (nontreatment): T2D mice received with distilled water - Group 3 (positive control):T2D mice + gliclazid 80 mg/kg. - Group 4 (Andiabet 0,68 g/kg):T2D mice + Andiabet 0,68g/kg/d. - Group 5 (Andiabet 1 g/kg): T2D mice + Andiabet 1 g/kg/d. - Group 6 (Andiabet 2 g/kg): T2D mice + Andiabet 2 g/kg/d. After being orally administered with the reagent for 2 consecutive weeks, Evaluate: weight of mice after 2, 4, 6 and 8 weeks had a high fat diet. The average blood glucose levels measured at times: t0, t1, t2. Lipid profiles: TC, TG, HDL-C, LDL-C. Observe the histopathology,
7 weight of liver and pancreas. 2.2.2.3. Evaluate inhibitivity on hyperglycemic postprandial blood glucose of Andiabet in glucose/sucrose/starch tolerance test in normal and diabetic mice. s Evaluate inhibitivity on hyperglycemic postprandial blood glucose of Andiabet in glucose/sucrose/starch tolerance test in normal mice. The normal animals were classified into 5 groups (10 mice/groups): - Group 1 (Control): received with distilled water - Group 2 (positive control): received with Acarbose 14 mg/kg/d. - Group 3 (positive control): received with Metformin 250mg/kg/d - Group 4 (Andiabet 1g/kg): received with Andiabet 1 g/kg/d. - Group 5 (Andiabet 2g/kg): received with Andiabet 2 g/kg/day Mice were given distilled water or reagents continuously for 2 weeks in the mornings. The 15th day, glucose (2g/kg-oral) was administered to the mice in each group (glucose tolerance test) or sucrose at 4 g/kg was administered to the mice in each group (sucrose tolerance test) or potato starch at 6 g/kg was administered to the mice in each group (starch tolerance test). Blood samples were taken at 30, 60, 120 min after the glucose/sucrose/starch load for the assay of glucose/sucrose/starch. Evaluation: peak blood glucose (PBG) - The maximum blood glucose level of the whole group after loading glucose/sucrose/starch. The area under of curve (AUC) is calculated following formula: !"#!$" !$"#!&" AUC = ( % ) x (t30 – t0) + ( % ) x (t60 - t30) + !&"#!'%" ( ) x (t120 - t60) % In which, C0, C30, C60, C120 are measured blood glucose levels at the times: before use drugs (t0), 30 minutes (t30), 60 minutes (t60) and 120 minutes (t120). s Evaluate inhibitivity on hyperglycemic postprandial blood glucose of Andiabet in glucose/sucrose/starch tolerance test in T2D diabetic mice.
8 Performed on type 2 diabetes mice, similar to that in normal mice. 2.2.2.4. Antagonism with insulin resistance of Andiabet in type 2 diabetic mice model. The diabetic mice were divided into 4 groups (7 mice/group), given reagents or distilled water for 2 consecutive weeks. - Goup 1 (control): Normal mice received with distilled water - Group 2 (nontreatment):T2D mice received with distilled water - Group 3 (Andiabet 1g/kg): T2D mice + Andiabet 1g/kg/day - Group 4 (Andiabet 2g/kg): T2D mice + Andiabet 2g/kg/day On the 15th day, the technique "Hyperinsulinemic - euglycemic clamp test" was used to assess insulin resistance. A blood sample was collected from the mouse tail, at the time (t0). Insulin was infused continuously and maintained a constant speed at a rate of 4 mU/kg/min, throughout the experiment. Glucose (20%) was infused stimultaneously: the blood glucose concentration measured at the time (t0) was able to determined the rate infusion. Then, the blood samples were measured every 10 minutes, glucose infusion rate (GIR) was adjusted continuously (blood glucose levels were always ~ 7.5 - 8.3 mmol/L). Blood glucose concentration, glucose infusion rate at the “steady state” (from 80 - 120 minutes) were compared vs control group. 2.3. STATISTICS All data were expressed as mean ± SE. Student's t-test and one-way ANOVA was used for statistical analysis. Significantly different when the p < 0.05. Chapter 3: RESULT 3.1. ACUTE TOXICITY AND CHRONIC TOXICITY OF ANDIABET 3.1.1. Acute toxicity test: The mice in all groups were observed within the first 72 hours after taking the Andiabet, we noticed that in the all of doses groups: Mice displayed operation, eating and
9 drinking normally; feces and urine normally; pink lining, silky hairs and mice had good reflexes with the stimulations. In higheast dose group were 44,25 g/kg mice, none of the dead mice were observed within the first 72 hours. Such that, Andiabet has no acute toxicity at a dose of 44.25 g/kg and LD50 dose has not been determined. 3.1.2. Chronic toxicity test Andiabet at 2 dose levels: 0.21g/kg/day and 0.64g/kg/day orally for 90 consecutive days without any changes in hematological and blood biochemical indices and histopathology of liver and rabbit kidney. 3.2. HYPOGLYCEMIC ACTIVITY AND SEVERAL HYPOGLYCEMIC MECHANISMS OF ANDIABET IN EXPERIMENTAL. 3.2.1. Hypoglycemic effect of Andiabet in normal mice. Table 3.1.Blood glucose levels of normal mice after 2 weeks taking Andiabet Groups Blood glucose levels (mmol/l) (X ± SD) (n = 10) To T1 T2 Group 1: control 3,53 ± 0,72 4,99 ± 1,14 4,97 ± 0,92 Group 2: Gliclazid 80mg/kg 3,55 ± 0,51 4,12 ± 0,58* 3,95 ± 0,72** % reduced vs control ↓ 17,44 % ↓ 20,52 % Group 3: Andiabet 0,68g/kg 3,34 ± 0,21 5,03 ± 0,25 4,26 ± 0,44* % reduced vs control - ↓ 14,3 % Group 4: Andiabet 2 g/kg 3,40 ± 0,60 4,92 ± 0,79 4,12 ± 0,61* % reduced vs control ↓ 1,4 % ↓ 17,1 % p vs control: *: p < 0,05; **: p < 0,01. Comment: At the time T0, blood glucose levels in all mice groups was similar (p > 0.05). The positive control group was used gliclazide 80 mg/kg had a significant hypoglycemic effect 17.44% and 20.52%, respectively after 1 week (T1) and 2 week (T2), compared to the control
10 group (p 0,05 After 4 weeks 29,60 ± 1,15*** 37,17 ± 1,89*** < 0,001 % increased ↑ 16,3 ↑ 42,5 After 6 weeks 33,60 ± 1,43*** 42,89 ± 1,93*** < 0,001 % increased ↑ 32,0 ↑ 64,4 After 8 weeks 35,90 ± 1,45*** 48,47 ± 2,27*** < 0,001 % increased ↑ 41,1 ↑ 85,8 ***: p < 0,001: vs before study time. Comment: The weight of mice in the HFD group increased respectively by 42.5 %; 64.4 % and 85.8 % after 4, 6 and 8 weeks, compared to the control group (p 0,05 After 8 weeks 5,77 ± 0,67 6,32 ± 0,93 > 0,05 % change ↑ 7,4 ↑ 13,7 72h after injection of STZ 5,98 ± 0,92 17,09 ± 6,33*** < 0,001 % change ↑ 11,4 ↑ 207,4
11 ***: p < 0,001: p vs before study; (∆∆∆): p < 0,001: p vs after 8 weeks Comment: 72h after injection of STZ, blood glucose levels in HFD group increased strongly (207,4%) compared with control group (increased 11,4%) (p < 0,001) and before injection of STZ (p < 0,001). 3.2.2.3. Hypoglycemic effect of Andiabet in T2D mice. Table 3.4. Effect of Andiabet on blood glucose levels of T2D diabetic mice Blood glucose levels (mmol/l) (X ± SD) Group (n=10) To T1 T2 G1: control 5,54 ± 0,86 5,51 ± 0,81 5,46 ± 0,46 G2: nontreatment 17,88 ± 6,23 18,38 ± 4,46 18,38 ± 4,39 G3: Gliclazid 80mg/kg 17,36 ± 5,26 14,18 ± 5,23* 13,83 ±3,45** % reduced vs nontreatmnet ↓ 22,9 % ↓ 24,8 % G4: Andiabet 0,68g/kg 17,89 ± 6,3 13,99 ± 3,61* 16,71 ± 4,46 % reduced vs nontreatment ↓ 23,9 % ↓ 9,1% G5: Andiabet 1g/kg 19,23 ± 6,3 17,53 ± 3,61 11,83 ± 3,91** % reduced vs nontreatment ↓ 4,6 % ↓ 35,6 % G6: Andiabet 2g/kg 18,04 ± 5,27 11,69 ± 3,78** 14,84 ± 5,01* % reduced vs nontreatment ↓ 36,4 % ↓ 19,3 % p vs nontreatment: *: p < 0,05; **: p < 0,01; ***: p < 0,001. Comment: At 1week oral administration, Andiabet at doses (0.68g/kg and 2g/kg) markedly reduced blood glucose levels (by 23,9% and 36,4%, respectively) compared to the nontreatment (p
12 Lipid indexes mmol/l (X ± SD) Groups (n=10) TC TG HDL-C LDL-C G1: control 2,75 ± 0,55 0,50 ± 0,17 1,40 ± 0,11 1,12 ± 0,40 G2: 3,85 ± 0,56∆∆∆ 0,89 ± 0,28 ∆∆∆ 1,79 ± 0,22 ∆∆∆ 1,64 ± 0,47 ∆∆∆ Nontreatment G3: Gliclazid 3,86 ± 0,52 0,81 ± 0,18 1,89 ± 0,31 1,60 ± 0,63 80mg/kg G4: Andiabet 3,70 ± 0,61 0,81 ± 0,22 2,29 ± 0,21*** 1,05 ± 0,66*** 0,68g/kg G5: Andiabet 3,63 ± 0,38 0,9 ± 0,13 2,07 ± 0,16** 1,15 ± 0,32** 1g/kg Group 6: 3,23 ± 0,50** 0,62 ± 0,16** 2,15 ± 0,21*** 0,81 ± 0,43*** Andiabet 2g/kg p vs control: ∆∆∆: p
13 injected STZ had a silver color, were less uniform, the tissue density were somewhat loosecompared to the control group. v Observe the microscopic liver: In nontreatment group, 100% of samples had severe fat degeneration. Andiabet at all of doses improved this situation: 2/3 of samples had mild fat degeneration and 1/3 of the sample had medium fat degeneration. v Observe the pancreas in general: Pancreas in all group were pale pink, the tissue’s density were tough and firm and didn’t have congestion or injury. v Observe the microscopic pancreas: In nontreatment group, 100% of samples were severe degenerated. Andiabet at all of doses improved this situation: the degeneration of pancreastic islet cells reduced, pancreastic islets decreased slightly in size, and the density of islets was smaller than normal. 3.2.3. Effect inhbitivity on hyperglycemic postprandial blood glucose of Andiabet in glucose/sucrose/starch tolerance test in normal and diabetic mice. 3.2.3.1. Effect inhibitivity on hyperglycemic postprandial blood glucose of Andiabet in glucose/sucrose/starch tolerance test in normal mice. v Oral glucose tolerance test Table 3.6. Effect of Andiabet on PBG and blood glucose AUC after 2 hours orally glucose administration in normal mice Groups PBG %¯ AUC %¯ (n =10) (mmol/L) PBG (mmol/L) AUC Normal mice Gr1: control 9,28 ± 2,15 13,58 ± 3,05 Gr 2: acarbose 14mg/kg 7,86 ± 0,99 15,3 11,33 ± 1,58 16,57 Gr metformin 250mg/kg 7,37 ± 1,13* 20,58 11,95 ± 1,23 12
14 Gr 4 Andiabet 1g/kg 9,95 ± 1,87 - 13,88 ± 2,55 - Gr 5 Andiabet 2g/kg 8,22 ± 1,90 11,4 11,65 ± 1,99 14,2 p vs control: * p
15 (n =10) (mmol/L) PBG (mmol/L) AUC Normal mice Gr control 8,93 ± 1,52 13,99 ± 1,47 Gr acarbose 14mg/kg 7,51 ± 0,98* 15,9 13,32 ± 0,83 4,79 Gr metformin 250mg/kg 7,24 ± 0.37* 18,92 12,71 ± 1,39 9,15 Gr Andiabet 1g/kg 7,85 ± 1,12 12,09 12,82 ± 1,61 8,36 Gr Andiabet 2g/kg 7,29 ± 1,27* 18,36 12,88 ± 1,62 7,93 p vs group control: * p < 0,05; ** p < 0,01: *** p < 0,001 Comment: All 3 groups of acarbose, metformin and Andiabet 2g/kg inhibited PBG compared to the control group (p
16 of PBG was 19.27% and 22.89%, respectively (p
17 (n =10) (mmol/L) PBG (mmol/L) AUC STZ-induced diabetic mice NT diabetic group 29,24 ± 4,14 47,92 ± 6,16 Gr 2: Acar 14mg/kg 19,59 ± 8,49 33,03 29,86 ± 1,28* 37,69 Gr 3: Met 250mg/kg 18,16 ± 3,73** 37,89 22,90 ± 4,69** 52,21 Gr 4: Andiabet 1g/kg 19,26 ± 11,12 34,13 36,33 ± 2,27 24,18 Gr 5: Andiabet 2g/kg 15,18 ± 4,19*** 48,08 24,94 ± 6,81** 47,95 p vs nontreatment (NT) group: * p < 0,05; ** p < 0,01: *** p < 0,001 Comment: 2 groups are metformin and andiabet at 2g/kg dose significantly inhibited PBG, compared to the nontreatment group (p
18 2 Andiabet 2g/kg 1.6 Glucose ìnusion rate (ml/h) Chứng trắng 1.2 Chứng bệnh 0.8 Andiabet 1g/kg 0.4 0 80 90 100 110 120 Time (min) Figure 3.2. Glucose infusion rate in Hyperinsulinemic - euglycemic clamp test in T2D diabetic mice Comment: In the figure 3.3.The blood glucose level of all 4 groups was maintained steady state in the range of 7.5 - 8.3 mmol/L during the “clamping time” from 80-120 minutes. In the figure 3.2. Glucose infusion rate gradually decreased from the control group, to Andiabet 2g/kg, Andiabet 1g/kg and was lowest in the nontreatment group. Chapter 4. DISCUSSION Lagerstroemia speciosa (L.) Pers; Gynostemma pentaphyllum (Thunb.) Makino; Anemarrhenae Aspheloides (Bunge) are traditional Vietnamese herbal medicines that is widely used to treat type 2 diabetes. With the desire to create an effective diabetes treatment product, derived from herbs, Traphaco company had extracted and prepared Andiabet, a hard capsules. Andiabet is a compound of 3 Vietnamese herbal medicines composition above. To be able to use Andiabet to support treatment of type 2 diabetes, it is necessary to study the safety as well as pharmacological effects and study some mechanisms of action of Andiabet tablets. 4.1. ACUTE AND LONGTERM TOXICITY OF ANDIABET 4.1.1. Acute toxicity of Andiabet
19 At the highest dose of 44,25 g/kg, Andiabet has no acute toxicity for mice. Andiabet showed that it to be relatively safe because the ratio between maximum tolerance dose (44.25 g/kg) to therapeutic dose (the lowest dose which has hypoglycemic effect (0.68 g/kg) is 66: 1. Therefore, high recommended a trial dose of Andiabet on the human from 0.45 g/kg - 4.4 g/kg/day (in the range of 1/100 - 1/10 maximum tolerance dose). Andiabet hard capsule includes 200 mg of dried leaves (Lagerstroemia speciosa (L.) Pers) 70% with 200 mg of dried stems, roots, leaves Gynostemma pentaphyllum (Thunb.) Makino and 133 mg dried root Anemarrhenae Aspheloides (Bunge). The content of the active ingredient which contained in a hard Andiabet capsule is 533 mg. So the maximum dose in humans is 8 capsules/day, extrapolating 1 g/kg/day in mice (extrapolation coefficient in mice are 12 and adults’s weight is about 50 kg). Therefore, our next study used 3 doses of Andiabet: 0.68g/kg (equivalent to clinical dose); 1 g/kg (1.5 times the clinical dose) and 2 g/kg (3 times the clinical dose). 4.1.2. Longterm toxicity of Andiabet Vinabetes is a soft form and has components the same at Andiabet, that has been studied for longterm toxicity in rabbits for 4 weeks with 2 doses of 1.8 g/kg/day and 3.6 g/kg/day. The results showed that Vinabetes caused the damage on hepatocellular. Therefore, in the present study, the longterm toxicity test of Andiabet was conducted on rabbits for 90 consecutive days with 2 doses: 0.21 and 0.64 g/kg/day. The result showed that, Andiabet has no noticeable sign of long term toxicity after 90 days oral consecutive administration. 4.2. HYPOGLYCEMIC ACTIVITY AND SEVERAL HYPOGLYCEMIC MECHANISMS OF ANDIABET IN EXPERIMENTAL. 4.2.1. Hypoglycemic effect of Andiabet in normal mice. Results from table 3.7 showed that Andiabet has hypoglycemic effect on normal white mice. Andiabet at 0.68 g/kg/day and 2 g/kg/day doses orally for 2 weeks reduced the blood glucose levels by 14.3% and 17.1% compared to the control group (p
20 of 80mg/kg reduced better than Andiabet (20.52% vs 17.1%) in normal mice, because all three herbs contain many active ingredients with different hypoglycemic mechanisms, not only stimulate the pancreas to increase insulin secretion such as gliclazide, but it can also have many other mechanisms such as promotion the insulin sensitivity, inhibition of glycogen metabolism in the liver and other tissues in the body, and or inhibition of hepatic gluconeogenesis. .. and the effects were not shown in normal mice. 4.2.2. Hypoglycemic effect of Andiabet in type 2 diabetic mice. 4.2.2.1. Type 2 diabetes model. Recently, scientists have developed a new type 2 diabetes model by combining a high-fat diet (fat ~ 40-60% of calories) for a long time (1- 2 months) to cause insulin resistance. Then, pancreastitis were induced by low-dose STZ injection. In this way, we have type 2 diabetes mice. Mice had obesity characteristic: after 4, 6 and 8 weeks, the weight of mice in the HFD groups were by 42.5%; 64.4% and 85.8%, respectively. The difference compared to the NFD group was statistically significant (p