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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

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


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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

effective diabetes treatment drug.
Chapter 2: MATERIALS AND METHODS
2.1. MATERIAL
2.1.1. Material: Andiabet is a hard capsule includes 200 mg of dried
leaves (Lagerstroemia speciosa (L.) Pers) 70% alcoholic extract with 200
mg of dried stems, roots, leaves Gynostemma pentaphyllum (Thunb.)
Makino water extract and 133 mg dried root Anemarrhenae Aspheloides
(Bunge) extracted 50% alcohol. Weight of one capsule is 590 mg. The
inoculants are manufactured according to basic standards and supplied by
Traphaco Joint Stock Company, Hanoi.
2.1.2. Animals: Normal Swiss mice (both sexes, weighing 18 - 22g


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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

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,


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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.


Blood glucose levels (mmol/l) (X ± SD)
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**

↓ 17,44 %

↓ 20,52 %

5,03 ± 0,25


respectively after 1 week (T1) and 2 week (T2), compared to the control


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group (p 0,05

29,60 ± 1,15***

37,17 ± 1,89***

< 0,001

p vs

% increased

< 0,001
< 0,001

***: 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

↑ 207,4

< 0,001


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***: 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


17,53 ± 3,61

11,83 ± 3,91**

↓ 4,6 %

↓ 35,6 %

11,69 ± 3,78**

14,84 ± 5,01*

↓ 36,4 %

↓ 19,3 %

% reduced vs nontreatmnet
G4: Andiabet 0,68g/kg

17,89 ± 6,3

% reduced vs nontreatment
G5: Andiabet 1g/kg

19,23 ± 6,3

% reduced vs nontreatment
G6: Andiabet 2g/kg
% reduced vs nontreatment



0,50 ± 0,17

1,40 ± 0,11

1,12 ± 0,40

3,85 ± 0,56∆∆∆

0,89 ± 0,28 ∆∆∆

1,79 ± 0,22 ∆∆∆

1,64 ± 0,47 ∆∆∆

3,86 ± 0,52

0,81 ± 0,18

1,89 ± 0,31

1,60 ± 0,63

3,70 ± 0,61

0,81 ± 0,22

2,29 ± 0,21***

1,05 ± 0,66***


Comment: All of Andiabet groups displayed significantly reducing
LDL-C levels and increasing HDL-C levels, compared to the
nontreament (p
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

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Table 3.7. Effect of Andiabet on PBG and blood glucose AUC after 2
hours orally sucrose administration in normal mice
Groups

PBG

%¯

AUC

%¯

(n =10)

(mmol/L)

PBG

(mmol/L)

AUC

Normal Mice
Gr 1: control

10,93 ± 1,29

16,82 ± 1,98

Gr 2: acarbose 14mg/kg


10,15 ± 1,29

2,9

15,05 ± 1,49*

10,52

p vs group control: * p < 0,05; ** p < 0,01: *** p < 0,001

Comment: acarbose at 14 mg/kg dose and metformin at 250 mg/kg
dose both decreased significantly PBG and AUC, compared to control
(p

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

STZ-induced diabetic mice
NT diabetic group

32,33 ± 1.01

58,71 ± 7,61

Gr acar 14 mg/kg

29,41 ± 5,50

9,03

50,06 ± 10,84

14,73

Gr met 250 mg/kg

18,43 ± 5,21**

42,99

21,69 ± 6,31***

63,05

Gr Andiabet 1 g/kg

26,10 ± 4,15*

reduced PBG and AUC (p > 0.05).
v Oral sucrose tolerance test
Table 3.10. Effect of Andiabet on PBG and blood glucose AUC after
2 hours orally sucrose administration in T2D mice
Groups

PBG

%¯

AUC

%¯

(n =10)

(mmol/L)

PBG

(mmol/L)

AUC

STZ-induced diabetic mice
NT diabetic group

32,89 ± 1,10

66,22 ± 7,89


Gr Andiabet 2g/kg

25,33 ± 4,97*

22,98

42,12 ± 8,92**

36,39

p vs nontreatment (NT) group: * p < 0,05; ** p < 0,01: *** p < 0,001

Comment: 3 groups were acarbose 14 mg/kg, metformin 250 mg/kg
and Andiabet 2g/kg displayed significantly inhibiting PBG compared
to the nontreatment group (p < 0,05). The Andiabet group at the dose
1g/kg had no inhibited the PBG. However, all of groups displayed
significantly reducing AUC by 47,32% (Acarbose 14mg/kg group);
50,22% (Metformin 250mg/kg group) and 39,43 % (Andiabet 1g/kg);
36,39 % (Andiabet 2g/kg), respectively.
v Oral starch tolerance test
Table 3.11. Effect of Andiabet on PBG and blood glucose AUC after 2
hours orally starch administration in T2D diabetic mice
Groups

PBG

%¯

AUC


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**

90

100

110

Times (min)
120

Figure 3.1. Blood glucose levels in Hyperinsulinemic - euglycemic clamp
test in T2D diabetic mice.


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Glucose ìnusion rate (ml/h)

2
Andiabet
2g/kg

1.6

Chứng trắng

1.2

Chứng bệnh

0.8

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


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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

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
administration similar to the effect of acarbose 14 mg/kg/day and
metformin 250 mg/kg/day and this effect depends on the dose of
Andiabet. The mechanism of hypoglycemic effect of Andiabet may be
due to the inhibition of α-glucosidase similar to acarbose’s mechanism
or increasing insulin sensitivity like that of metformin’s mechnism, or
there are may be any other mechanism besides these two mechanisms?
4.2.3.4. Inhbitivity on hyperglycemic postprandial blood glucose of
Andiabet in starch tolerance test.
Andiabet is able to improve oral starch tolerance test, depending
on the dose. The hypoglycemic effect of Andiabet at the dose of 2g/kg
in this test was better than that of acarbose 14mg/kg and metformin
250mg/kg may suggested Andiabet's ability to inhibit hyperglycemic
postprandial due to inhibition of α-glucosidase and/or α-amylase
enzymes. In addition, Andiabet may also have mechanisms that
increase insulin sensitivity, like metformin, increase the ability to
transport glucose into the target cells, reduce hyperglycemic
postprandial and AUC.
4.2.4. Antagonism with insulin resistance of Andiabet in type 2
diabetic mice.
In this study, the technique of "Hyperinsulinemic - euglycemic
clamp test" was used to directly assess insulin resistance in type 2
diabetes mice, which is the first model we have implemented in Vietnam.
We have also improved several the technical details: Instead of exposing
the carotid artery and neck veins on both sides to place a fixed catheter,
the catheter is inserted into vein’s tail to simultaneously infuse insulin
solution and glucose solution. Blood samples were given at the tip of
mice’s tail instead of carotid artery. The mice were fasted overnight


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weeks orally administration, showed the hypoglycemic effect in
normal mice. The percentage of reduction were 14.3% and 17.1%,
respectively.


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- Andiabet at all 3 doses levels: 0.68 g/kg/day; 1 g/kg/day and 2
g/kg/day orally for 2 weeks improved the glucose homeostasis in type
2 diabetic mice. The blood glucose levels significantly decreased by
9.1%; 35.6% and 19.3%, respectively, comparable to the nontreated
diabetic mice group (p