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.
Trang 1STUDY 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
increasing at an alarming rate globally
Ac-cording to a World Health Organization
re-port 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
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 adminis-tration 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 phy-tochemicals are being investigated
anti-di-Corresponding author: Ho My Dung, Hanoi Medical
University.
Email: homydung@hmu.edu.vn
Received: 15 June 2017
Accepted: 16 November 2017
Trang 2abetic potential for their [5] Using an herbal
remedy as an alternative therapy for
diabe-tes treatment would reduce individuals
de-pendence 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
tra-ditional therapy for pain, fever, digestive
disorders, heart diseases, diabetes,
can-cers 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
improv-ing the immune system [9; 10] However,
adequate characterization of CF2 effect is
yet to be done and no study has been
per-formed using a type 2 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
1 Materials
Experimental Medicine
CF2 powder extracted from leaves,
stems and shoots of Basket Plant was
sup-plied by Institute of Marine Biochemistry,
Viet Nam
Experimental Animals
Swiss male white mice, from 6 - 8 weeks
of age, and weighing between 23 - 27 grams, were used for this study The mice were obtained from National Institute of Hy-giene and Epidemiology, Viet Nam The ex-perimental animals were caged individually and acclimatised to laboratory conditions for 2 weeks prior to the experiment The study was carried out at the Pharmaceutical Department of Hanoi Medical University Machines and Chemicals
- Streptozotocin 1 g (Sigma-Aldrich, Sin-gapore), 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,
cholester-ol (Transasia, India)
2 Method
The study was divided into two stages [5]:
* The first stage:
Before ending the study, all mice base-line fasting glucose levels checked from pe-ripheral blood samples
+ Group 1: Control condition (n = 10) mice were randomized to one of two group: Normal fat diet regime (NFD) for 8 weeks + Group 2: Diabetic condition (n = 70): High fat diet regime (HFD) in 8 weeks fol-lowing Fabiola and Srinivasan method with 43% saturated fat combined siro fructose 55% [6]
After 8 weeks, the fasting glucose level
in all mice was checked Mice in group 2
Trang 3were injected intraperitoneally with STZ at
a dose of 100 mg/kg Mice in group 1 were
injected intraperitoneally with citrate pH 4.5
After 72 hours (t0), the blood of all mice
of group 2 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
ran-domized into four groups from, group 2 to
group 5
* The second stage: The study was
car-ried out in a continuous 14-day period Mice
were divided into five groups of ten animals:
- Group 1: NFD regime + drinking
dis-tilled 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 2 mg/kg/day
(low-dose group);
- Group 5: HFD regime + STZ injection
100 mg/kg + drinking CF2 4 mg/kg/day (high-dose group)
After 7 days (t1) and 14 days (tc) of treat-ment, the fasting glucose level of all mice were tested After 14 days of treatment, all animals blood lipid index (total
cholester-ol (TC), triglyceride (TG), HDL-Chcholester-olestercholester-ol, 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 Ex-cel software version 2010 The levels of sig-nificance between groups were determined using student's t-test and Avant-après test Data is shown as mean ± standard devia-tion All data was considered significant at
p < 0.05
III RESULTS
Figure 1 Wight of mice over time
Trang 4The 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
Time
Blood glucose level (mmol/l)
2-1 (t- test Student)
Group 1: Normal control Group 2: Diabetic 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
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 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
Group
Blood glucose level (mmol/l) (X ± SD) (n = 10)
t 0 (before treatment)
t 1 (after 7 days
of treatment)
t c (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*
% in change in comparison to t0 ↑ 21.3 ↑ 20.43
Group 3: HFD + STZ + gliclazide
80 mg/kg/day 16.45 ± 4.40* 15.49 ± 4.08** 12.74 ± 3.32***
% in change in comparison to t0 ↓ 4.07 ↓ 19.45
Trang 5Group 4: HFD + STZ + CF2 2 mg/
kg/day 16.64 ± 4.53* 15.09 ± 4.92** 13.76 ± 3.11***
% in change in comparison to t0 ↓ 6.97 ↓ 13.1
Group 5: HFD + STZ + CF2 4 mg/
kg/day 16.89 ± 5.87* 11.82 ± 3.76*** 10.73 ± 3.84***
% 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)
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 were in creased in group 2 (diabetic control) in comparison to group 1 (normal control) Blood
lip-id disorders at gliclazlip-ide 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)
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Group 1 Group 2 Group 3 Group 4 Group 5
Trang 6Histopathological 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
Trang 7IV DISCUSSION
After 8 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
con-trol mice After mice with HFD eating regime
were injected with STZ at a dose of 100 mg/
kg, the blood glucose level nearly 3 times
higher than before the injection This
mod-el was stably maintained during 2 weeks
of treatment and similar to the model of
ShaoYu [7] These results prove a rich
ener-gy eating regime combined with a low-dose
STZ injection has an effect on
hyperglyce-mia and successfully creates model of type
2 like diabetes
After 7 days and 14 days of treatment,
gli-clazide 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
glu-cose-lowering effect of CF2 at dose of 2 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 2 mg/kg and
gli-clazide 80 mg/kg in reducing blood glucose
level CF2 powder is there a contains
ec-dysteroid which was proved to reduce blood
glucose level in experimental animals In
Ki-zelsztein’s study (2009),
20-hydroxyecdys-on at dose of 10 mg/kg/day reduces blood
glucose level in type 2-like diabetic mice
after 13 weeks through increasing
circulat-ing 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
glucose, insulin, HbA1c, and key carbohy-drate an enzymes [9]
Besides reducing blood glucose, CF2 effected lipid disorder conditions CF2 at two doses of 2 mg/kg/day and 4 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 consis-tent with previous research showing 20-hy-droxyecdyson in Quinoa extract decreased total cholesterol and triglyceride in diet-in-duced obesity mice [10]
The result of histopathological examina-tion showed that there was positive change
in liver and pancreas structure of mice af-ter 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
liv-er 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 2 mg/kg and 4 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
Trang 8I would like to express my deepest
grat-itude to the Department of Pharmacology,
Hanoi Medical University for supporting us
in this study
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