2. We lead
BACKGROUND OF RESEARCH
Duration of research : January 2013 – April 2014
Place of research : Universiti Sains Malaysia,
Kampus Kesihatan, Kubang Kerian,
Kelantan.
General objective:
The general objective of this study is to evaluate
the antidiabetic activity in vivo and in vitro, and
also the effectiveness of the herbal alternatives,
Esulin ® in the management of diabetes.
Antidiabetic activity in vitro and in vivo in
herbal alternatives, Esulin® (AIFA Health
Sdn.bhd).
3. We lead
BACKGROUND OF RESEARCH
The specific objectives of this study are:
1) To identify phytochemicals and measure its contents in Esulin®
2) To evaluate toxicological test through brine shrimp lethality test, cell toxicity,
level of heavy metals determination and microbial contamination test from
Esulin®.
3) To evaluate in vitro antidiabetic effect of Esulin® extraction.
4) To investigate the efficacy of Esulin® aqueous extraction in management of
diabetes on biochemical parameters:
i. fasting blood glucose level
ii. fasting serum insulin level
iii. serum fructosamine
iv. the total cholesterol, triglycerides, high-density lipoprotein (HDL),
v. renal function
vi. liver function
4. We lead
METHOD: in vitro
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Phytochemicals Identification of Esulin®
• Qualitative phytochemical
screening test
• Quantitative phytochemical test
Toxicity Evaluation of Esulin®
• Brine Shrimp Lethality Bioassay
• Cytotoxicity Test
• Microbial Contamination Test
• Heavy Metal Detection
In Vitro Anti Diabetic Activity of Esulin®
• α-amylase Inhibitory Assay
• α-glucosidase Inhibitory Assay
5. We lead
METHOD: in vivo
• Research in diabetic rats conducted at Animal Research and Service Center
(ARASC), USM, and the study received approval from the Animal Ethics
Committee of USM (USM / Animal Ethics Approval / 2012 / (75) (347).
• Sprague-Dawley rats aged 2-3 months and weighing between 250-300g were
used as normal and diabetic rats induced 60mg / kg streptozotocin (STZ).
• This in vivo study using a total of 36 diabetic rats and 6 normal rats as a
positive control, and divided into seven groups, each group containing 6 rats.
• Each group received a different treatment as follows:
i. Non-diabetic rats (normal control)
ii. Diabetic rats without treatment (control diabetes)
iii. Diabetic rats treated with Esulin® (dose: 75 mg / day)
iv. Diabetic rats treated with Esulin® (dose: 150 mg / day)
v. Diabetic rats treated with Esulin® (dose: 300 mg / day)
vi. Diabetic rats treated with metformin (dose: 150 mg / day)
vii. Diabetic rats treated with insulin Humulin R (dose: 5U / day)
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RESULTS:
1) Phytochemicals in Esulin® and its percentages
2) Toxicity test of Esulin ®
Alkaloids Saponins Tannins Flavonoids
Analisis
kualitatif
present present present presen
Analisis
kuantitatif
0.44 21.38 17.07 12.00
Toxic Non toxic
brine shrimp lethality √
cytotoxicity test √
heavy metal detection √
microbial
contamination test
√
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TOXICITY TEST OF ESULIN ®
TEST RESULTS
1) Brine Shrimp Lethality Study
Brine shrimp lethality bioassay:
i. widely used in the bioassay for the bioactive
compounds.
ii. Artemia salina is a simple zoological
organism was used as a convenient monitor
for the screening.
Brine shrimp lethality test (BST) showed:
i. Good activity in Esulin® with concentration
lower than 40 000 µg/ml.
ii. The more higher concentration of Esulin®
extract treated on hatched Artemia salina,
the more higher percentage of viability after
24 hours.
2) Cytotoxicity Test
Effect of Esulin® on:
i. Human Bladder Smooth Muscle Cell
(HBdSMC),
ii. Vero cell
iii. Madin Darby Canine Kidney (MDCK) cell
Based on results:
i. Esulin® showed proliferation activity against
all normal cells tested.
ii. They were non cytotoxic on all three studied
cell lines at different concentrations.
iii. Esulin® was highly promoted growth of
HBdSMC and Vero cell however not active
against MDCK cell line after 72 hour of
treatment. 7
8. We lead
TOXICITY TEST OF ESULIN ®
TEST RESULTS
3) Microbial Contamination Test
Microbial contamination test was performed on:
i. Potato Dextrose Agar (PDA) for fungal
ii. Nutrient Agar (NA) for bacteria.
The results for bacteria contamination of Esulin® extract
showed:
i. There was no visible colony on the agar plate from
2 x 104 to 1 x 105 µg/ml extract dilution.
ii. Only at concentrations 101 to 104, the colony
counting was ranging from 3.33 x 104 to 6.6 x
101CFU/mL.
iii. The subsurface colonies are very small, which look
like little dots on the agar medium.
4) Heavy Metal Detection
Samples were determined by :
i. Flame atomic absorption spectrometer (FAAS).
The result showed:
i. Six different types of heavy metals were found in
Esulin extraction which is copper (Cu), zinc (Zn),
cadmium (Cd), ferum (Fe), lead (Pb) and nickel (Ni).
ii. Amount of heavy metals that found in tested
samples below the maximum permissible level
fixed by Akta Makanan Malaysia 1983 & Undang-
undang Makanan Malaysia 1985 and World Health
Organisation (WHO) 1989. 8
9. We lead
Activity Percentage (%)
α-amylase inhibitor
Acarbose
63%
68%
•results showed
inhibition of α-
amylase activity and
α-glucosidase is the
same as the
percentage inhibition
of acarbose (standard
drug, oral
hypoglycaemic) in
delaying carbohydrate
digestion and
absorption of glucose
after consuming food
by patients
α-glucosidase inhibitor
Acarbose
91%
88.4%
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3) in Vitro antidiabetic activity of Esulin®
RESULTS:
10. We lead
RESULTS:
10
0
10
20
30
40
50
60
70
80
0.125 0.25 0.5 1 2
Inhibitionofα-amylase
activity(%)
Concentration (mg/ml)
Esulin
Acarbose
0
10
20
30
40
50
60
70
80
90
100
0.125 0.25 0.5 1 2
Inhibitionofα-glucosidase
activity(%)
concentration (mg/ml)
Esulin
Acarbose
Inhibitory effects of aqueous extract of Esulin ®
and acarbose on α-mylase activity.
Inhibitory effects of aqueous extract of Esulin ®
and acarbose on α- glucosidase activity.
11. We lead
RESULTS:
• The results for antidiabetic activity in vivo showed significantly (p <0.05)
decrease in blood sugar levels, serum fructosamine, total cholesterol and
triglycerides for each group of diabetic rats that received treatment of
Esulin® when compared with diabetic rats.
• Besides, serum levels of HDL cholesterol in treated with all doses of
Esulin® showed significantly increased compared with diabetic control
rats.
• Treatment of diabetic rats with 300 mg/day Esulin® produced a
significant increase in insulin levels compared to diabetic control
• Kidney and liver showed no toxicological effects of each rat despite
receiving high doses of Esulin® (300mg) per day.
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4) Antidiabetic activity in vivo in Esulin®
12. We lead
TOXICITY TEST OF ESULIN ®
TEST RESULTS
1) Renal function test
Effect of Esulin® extraction on:
i. Sodium level
ii. Potassium level
iii. Chloride level
iv. Uric acid level
v. Creatinine level
vi. Urea level
Based on results:
i. Diabetic rats treated with all doses of Esulin® extraction showed
significantly (p<0.05) decreased in serum creatinine and serum urea
level compared to diabetic control rats.
ii. However, there was no significant difference in serum sodium,
potassium, chloride and uric acid level in each group of diabetic rats.
2) Liver function test
Effect of Esulin® extraction on:
i. Total protein
ii. Albumin
iii. Total bilirubin
iv. Globulin
v. Alkaline phosphate
The results were showed:
i. All groups of diabetic rats that received extract of Esulin® showed
significantly increased (p<0.05) total protein level and albumin level
compared with diabetic control rats.
ii. Results also showed significant decreased of serum total bilirubin in
diabetic rats that received treatment of Esulin® extraction compared
with diabetic control rats.
iii. There was no significant difference in serum globulin and alkaline
phosphate level in each group of diabetic rats.
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13. We lead
RESULTS:
• In summary, the results for antidiabetic activity in vivo (animal study) in
the table below
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No. Parameters Result
Decrease Increase
1 Fasting blood glucose (FBG) √
2 Serum fructosamine level √
3 Serum insulin level √
4 Total cholesterol √
5 Triglycerides √
6 HDL- Cholesterol √
toxic Non-toxic
7 Renal function √
8 Liver function √
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CONCLUSION:
• The present study has shown the phytochemical and elemental
compositions , cytotoxicity, in vitro and in vivo antidiabetic activity of the
Esulin®.
• Phytochemical screening indicates a basis of the therapeutic application
of these herbs in traditional medical practice.
• Aqueous extract of this herbs exhibited significant cytotoxicity and
antidiabetic activity in both in vitro and in vivo models.
• So, this formulation herbal (Esulin®) can be used as alternative herbal
medicine in the treatment of diabetes and diabetic complication.
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15. We lead
BACKGROUND OF RESEARCHER:
• Prof. Ab. Aziz Al Safi Hj. Ismail
• aiaziz@usm.my
• Qualification:
• MD (UKM)(1983), MPH
(Mal)(1991),Ph.D(Liverpool)(1998)
• Position:
• Researcher
• Senior lecturer Jabatan Perubatan
Masyarakat, PPSP, USM.
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16. We lead
BACKGROUND OF RESEARCHER:
• Dr. Wan Ezumi Mohd Puad @
Mohd Fuad
• wanezumi@usm.my
• Qualification:
• B.Sc. (Hons.) (UKM) Genetics;
Ph.D. (USM) Pharmacology
• Position:
• Reseacher
• Senior lecturer Biomedics
(Bioperubatan), PPSK, USM.
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17. We lead
BACKGROUND OF RESEARCHER:
• Dr. Mohd Dasuki Sul'ain
• drdasuki@usm.my
• Qualification:
• B.Appl.Sc. (Hons.) (USM)
Biotechnology; M.Sc. (USM)
Biotechnology; Ph.D. (USM)
Pharmacology/Toxicology, Cert. of
WHO Collaborating Centre
(Germany) Reproductive
Toxicology
• Position:
• Researcher
• Senior lecturer Biomedics
(Bioperubatan), PPSK, USM.
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18. We lead
BACKGROUND OF RESEARCHER:
• Cik Farah ‘Atiqah binti Ab Rahman
• farah_tiqq@yahoo.com
• Qualification:
• BSc (Hons) Biology
• Position:
• Researcher
• Master student of Biomedics
(Bioperubatan), PPSK, USM.
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• Cik Tuan Noorfatiehah binti Tuan Kub
• tuannoorfatiehah@yahoo.com
• Qualification:
• BSc (Hons) Agricultural biotechnology
• Position:
• Researcher
• Master student of Biomedics
(Bioperubatan), PPSK, USM.
