Content uploaded by Patrick Nwabueze Okechukwu
Author content
All content in this area was uploaded by Patrick Nwabueze Okechukwu on May 21, 2016
Content may be subject to copyright.
ANTIDIABETIC ACTIVITY OF CRUDE STEM EXTRACTS OF COSCINIUM FENESTRATUM ON
STREPTOZOTOCIN-INDUCED TYPE-2 DIABETIC RATS
Research Article
MALARVILI A/P SELVARAJA1, ADOLPH WILLIAM NDYEABURA1, GABRIEL AKYIREM AKOWUAH2 AND PATRICK
N OKECHUKWU1*
1Biotechnology program, Faculty of Applied Sciences, UCSI University, 2
Received: 23 July 2011, Revised and Accepted: 11 Aug 2011
School of Pharmacy, Faculty of Pharmaceutical Sciences, UCSI
University, No. 1,Jalan Menara Gading, UCSI Heights, 56000 Kuala Lumpur, Malaysia. Email: patrickn@ucsi.edu.my
ABSTRACT
Coscinium fenestratum (CF) (Gaertn.) Colebr traditionally has been used for the treatment of many diseases including diabetes Mellitus. In the
present study, we investigated the effects of crude dichloromethane (DCM), Ethyl Acetate (EA), and Butanol (BuOH) extract of CF on streptozotocin-
induced diabetic rat’s models. The crude stem extracts at concentration of 250mg/kg were administered for 4 weeks and the effects of extracts on
blood glucose, body weight and carbohydrate metabolizing enzymes Hexokinase and Lactate Dehydrogenase (LDH) were determined. The total
phenolic content, anti-oxidant activity and phytochemical screening and HPLC profiling of extracts were also investigated. DCM and EA extracts
possesses a significant (p<0.001) hypoglycemic effect by lowering the blood glucose levels and increasing the body weight in STZ induced diabetic
rats. The activities of hexokinase and LDH increases in the diabetic group treated with DCM and EA extract compared to the diabetic rats group. The
total phenolic content (TPC) and antioxidant studies revealed the presence of phenolic and antioxidant activity in DCM and EA extracts. Phytochemical
screening, TPC of DCM and EA extract confirmed the presences of phenols, alkaloids, flavonoids, terpenes, saponins, tannins, steroids and possess strong
antioxidant properties. In conclusion, DCM and EA stem extract of CF showed strong plasma glucose lowering and antioxidant activity. These effects may
be as a result of the presence of phytochemicals alkaloids, flavonoids, terpenes, tannins, and steroids in DCM and EA stem extract.
Keywords: Coscinium fenestratum; Antidiabetic; Streptozotocin; Antioxidant; Diabetes.
INTRODUCTION
Diabetes and its complications have been a major cause of
premature death all over the world and most of these are avoidable
1. In 2006 Malaysian Third National Health and Morbidity Survey
showed prevalence of Type 2 Diabetes Mellitus for Adults aged 30
years and above to be 14.9% upped by almost 79.6% in the space of
10 years from 1996 to 2006. The prevalence of type 2 diabetes is the
highest among Indian ethnic at 19.9% for those aged 30 and above 2
The β-cells from the islet of Langerhans in the pancreas detect the
post meal rise in blood glucose and begin to stimulate insulin release
to enhance the diffusion of glucose into cells to produce energy to
fuel body functions or store as glycogen. The release of insulin from
the pancreas is not only stimulated by increase blood glucose, it is
also stimulated by incretins, vagal nerve stimulation and other
factors as well
.
3. Pancreatic β-cells of the islet of Langerhans are the
only cells that produce insulin, but they have a limited capacity for
regeneration, which is predisposing factor for the development of
diabetes mellitus 4. Intercellular signalling cascade results in storage
of glucose as glycogen, if the insulin receptors fail to initiate the
intercellular signalling the body compensate by secreting greater
amount of insulin hyperglycaemia occur. Also when the body cannot
produce enough insulin or is unable to effectively use the insulin it
makes (insulin resistance) glucose accumulate in the blood leading
to hyperglycaemia. Hyperglycaemia is the most consistent sign of
diabetes, but is not a sensitive indicator at the onset of the disease 5
In the past few years new evidence and studies has made a major
impact on diabetes management. From new targets for control,
emphasis and recognition of the cluster of cardiovascular risk
factors that make up the metabolic syndrome in which type 2
diabetes is a principal player and new classes of pharmacological
agents targeting novel pathways as well as major outcome studies,
these have changed algorithms for the management of diabetes care.
Coscinium fenestratum is a woody climber found in south East Asia
and has been widely used as a medicinal plant
.
6. The infusion and
tincture preparation of stem is widely used in the traditional
Ayurvedic system for the treatment of diabetes mellitus7. In the
siddah system of medicine, the powdered stem is dissolved in milk
and given to the diabetic patients 8. The rural people of
Kanyakumarl District, Tamilnadu, India use the decoction of the
stem for treatment of diabetes 9. Alcoholic extracts of CF has been
reported to possess anti-diabetic effect 10
MATERIALS AND METHODS
. In the present study, we
investigated the effects of crude dichloromethane (DCM), Ethyl
Acetate (EA), and Butanol (BuOH) extracts from the stem of CF on
blood glucose, body weight and carbohydrate metabolizing enzymes
Hexokinase (HK) and Lactate Dehydrogenase (LDH) in
streptozotocin-induced diabetic rat’s models. The total phenolic
content, anti-oxidant activity and phytochemical screening of
extracts were also investigated.
Collection of plant material
The stems of the plant CF (20kg) were collected from the jungles of
Pahang. Mr.Shamsul Khamis a plant taxonomist from the Laboratory of
Natural Products (NATPRO) in Institute of Bioscience, University Putra
Malaysia (UPM), Selangor DarulEhsan specifically identified the plant.
Preparation of Plant Extract
The stem of CF was washed, cut, dried and grinded into a powder
with a miller in the Institute of Bioscience, UPM. Approx. 2kg of stem
powder was macerated with 8L Methanol into four 5L conical flask
for two days at room temperature. The extractwas filtered using
Whatman Filter papers and the filtrate concentrated by evaporation
at 35oC to 37o
Animals
C using rotary evaporator to give a concentrated semi-
solid crude methanol extract. The dried methanol extract about 10g
was suspended in 300mL of water and then partitioned with DCM,
EA and BuOH respectively
Healthy adult Male Wistar albino rats weighing 170-230g were used
for the study. Rats were maintained under standard laboratory
conditions (12h light/12hr dark cycle; 25o
Acute toxicity studies
C; 35-60% humidity). The
animals were fed with standard rat pellet diet and water ad libitum.
Rats were allowed to acclimatize for 2 weeks prior to the experiment.
The acute toxicity study of the crude extracts from stems of C. F. was
performed in rats (n=10). In this assay, increasing doses of crude
extracts were orally administered to groups of animals for each dose
after a 12h fast. Animals receiving the vehicle (saline) served as
control. The signs and symptoms associated with the crude extracts
Asian Journal of Pharmaceutical and Clinical Research
Vol. 4, Issue 4, 2011 ISSN - 0974-2441
Academic Sciences
Okechukwu et al.
Asian J Pharm Clin Res, Vol 4, Issue 4, 2011, 47-51
48
administration (5g/kg, p.o. volume of 10mL/kg of body weight)
were observed at 0, 30, 60,120,180 and 240 min after
administration and then once a day for the next 14 days. At the end
of the period the number of survivor was recorded. The acute
toxicological effect was estimated by the method described by 11 and
the death, when occurred, was expressed as LD 50 according to 12
Induction of Diabetes
.
Diabetes was induced 13
Study Design
in overnight fasted wistar rats were induced
diabetes using Streptozotocin (STZ) (sigma, St Louis, MO). STZ was
dissolved in ice-cold citrate buffer (0.1M. pH 4.5). STZ and
administered in a dosage of 60mg/kg by intraperitoneal (i.p.)
injection. Blood glucose level and the bodyweight of the rats were
assessed 72 hours post STZ injection to confirm hyperglycaemia and
only rats with elevated blood glucose levels above 11 mmol/L or
200mg/dL were used for this study.
Total number of 30 rats were used and divided into 5 groups (n=6).
Group 1: Normal Treated with saline (1mL)
Group 2: Diabetic Positive control treated with Tolbutamide
(100mg/kg).
Group 3: Diabetic treated with DCM crude extract (250mg/kg).
Group 4: Diabetic treated with EA crude extract (250mg/kg).
Group 5: Diabetic treated with BuOH crude extract (250mg/kg).
Animals were treated respectively for 4 weeks orally. Glucose
measured by glucometer and body weight was measured using
weight machine on week 0, 2 and 4. Blood samples were collected
from the tail tip under mild ether anesthesia. After 4 weeks the rats
were sacrificed using chloroform and liver dissected out and 2g of
liver weighed and used for biochemical assay. CF DCM extract was
selected for further test (phytochemical analysis, Biochemical assay
and antioxidant properties).
Biochemical assay
Hexokinase (EC2.7.1.1) enzyme activity
To a 0.1mL of Liver tissue homogenate 2.28mL of tris(200mmol L-1)
/ MgCl2 buffer (20mol L-1), pH 8, 0.5mL of 0.67M glucose, 0.1mL of
16 mM ATP, 0.1 Ml OF 6.8 mM NAD and 0.01mL of 300 U mL-1
Lactate dehydrogenase enzyme activity
glucose-6-phosphate dehydrogenase were added. The solution was
mixed thoroughly and the absorbance was measured at 340 nm.
To 0.05mL of Liver tissue homogenate 2.5mL of Tris (81.3mmol, pH
7.2) NaCl (203.3 mmol) per NADH 0.244 mmol L-1) were added and
the solution mixed thoroughly. Then 0.5 mL of Tris (81.3mmol, pH
7.2) per NaCl (203.3mmol) per pyruvate (9.76mmol L-1
Antioxidant activity
) was added.
The solution was mixed well and absorbance read at 339nm.
Determination of Total Phenolic Content (TPC)
The amount of phenol in the DCM extract was determined with
Folin-Ciocalteu Reagent (FCR) using the method of 14as modified by
Crop Research Report. 2.5ml of 10% FCR and 2ml of Na2 CO3
(2%w/v) was added to 0.5ml of each sample (3 replicates) of DCM
extract solution (1mg/ml). The resulting mixture was incubated at
450
Ferrous ion chelating (FIC) assay
C with shaking for 15min. The absorbance of the samples was
measured at 765 nm using UV/visible light. Results were expressed as
milligrams of Gallic acid (0-0.5mg/ml) dissolved in distilled water.
Into a 1mL of extract sample 1 mL of 0.1mM FeSO4
Chelating Effect (%) = [1- {Absorbance
and 1mL of
0.25mM Ferrozine were added and the mixture left to stand for 10
minutes at room temperature. Absorbance reading versus blank
(mixture without extract) was taken at 562 nm. The capability of
extract to chelate ferrous ion was calculated relative to negative
control using formula
sample / Asborbancecontrol
DPPH free-radical scavenging activity
}] x 100
The method of 15
DPPH scavenging activity (%) = [(Abs
was used for the determination of scavenging
activity of DPPH free radical. One ml of 0.135 mM DPPH prepare in
methanol was mixed with 1.0ml of DCM extract ranging from 0.2-0.8
mg/ml. The reaction mixture was vortexed thoroughly and left in
dark at room temperature for 30min. The absorbance was measured
spectrophoto-metrically at 517nm. The scavenging ability of the
plant extract was calculated using this equation;
control - Abs sample )] / [(Abs
control
Where Abs
)] x 100
control is the absorbance of DPPH + methanol; Abs sample
Phytochemical analysis
is
the absorbance of DPPH Radical + sample (i.e. DCM extract or
standard).
A small portion of the dry DCM extract was used for the
phytochemical tests for compounds which include tannins,
flavonoids, alkaloids, saponins, and steroids in accordance with
methods of 16,17
RESULTS
, with little modifications. Exactly 1.0g of the DCM
extract was dissolved in 10ml of distilled water and filtered (using
Whatman No 1 filter paper) A blue colouration resulting from the
addition of ferric chloride reagent to the filtrate indicated the
presence of tannins in the extract. Exactly 0.5g of DCM extract was
dissolved in 5ml of 1 % HCl on steam bath. A militre of the filtrate
was treated with few drops of Dragendorff’s reagent. Turbidity or
precipitation was taken as indicative of the presence of alkaloids.
About 0.2g of DCM extract was dissolved in methanol and heated. A
chip of magnesium metal was added to the mixture followed by the
addition of few drops of concentrated HCl. The occurrence of a red
or orange colouration was indicative of flavonoids. One gram of each
extract was separately dissolved in 10 ml of chloroform and equal
volume of concentrated sulphuric acid was added by sides of the test
tube. The upper layer turn red and sulphuric acid layer showed
yellow with green fluorescence. This indicated the presence of
steroids.
Acute toxicity studies
The oral toxicity of crude extracts was evaluated with dose five –
fold higher than maximum tested antiulcer dose (1000mg/kg, p. o.).
At this dose, no signs and symptoms of acute toxicity were observed
in all treated rats. No significant difference was observed in the
weight of heart, liver, kidney, or lungs when they were compared
with those of control group (saline). None of the treated rats died
during the 14 days of observation after the administration of crude
extracts. The results obtained indicated the absence of acute toxic
effect of crude extract.
Antidiabetic activity
All the crude stem extracts of CF exhibited plasma glucose lowering
activity (hypoglycaemic) in STZ –induced diabetic rats compared to
the negative control as shown on Table 1. Group I did not show any
effect compared to treated group. Group II-IV, showed significant
reduction in sugar level when compared with negative control rats
(P<0.001) 35.5%, 57.8%, and 43.5% respectively. In contrast Group
V did not show significant effect (P>0.001) when compared to
control groups with percentage reduction of only 19%. Similarly,
the body weight of the negative control group was decreased
compared to the treated groups which continuously increase as
shown in Table 2. Group III showed a significant (p<0.001) increase
in weight 5.85%, Group IV 4.13%, Group II 1.81% while Group IV
0.9% when compared to control group.
Biochemical assay
Hexokinase
Hexokinase enzyme activity was increased in Groups II 0.893 u/g,
Group III 1.821 u/g, and Group IV 1.140 u/g compared to Group I
2.532 u/g Table 3
Okechukwu et al.
Asian J Pharm Clin Res, Vol 4, Issue 4, 2011, 47-51
49
Lactate Dehydrogenase
Similarly LDH enzyme activity was increased in Groups II 0.434u/g,
Group III 1.413 u/g, and Group IV 0.822u/g compared to Group I
2.351u/g Table 4.
Antioxidant activity
DCM crude extract contain a total phenol content of
1092.3310+34.8918 mg GAE/100g, 47.13+ 0.44 % Ferrous Ion
Chelating activity and 66.30+ 0.12 % DPPH free radical scavenging
activity. Table 5
Phytochemical analysis
DCM crude stem extract was proved to have flavonoids, saponins,
tannins, alkaloids, terpenes and steroid, cardiac glycosides were
absent.
Table 1: Effect of Coscinium Fenestratum crude DCM extract on blood glucose level in normal and diabetic rats
Groups
Treatment
Blood Glucose Concentration(mg/dl)
Reduction
(%)
Day 0
Day 3
Day 7
Day 10
I
Negative control
248.65+ 1.85
292.65 +1.93
300.13 + 1.83
306.28 + 1.87
-18.8
II
Positive Control (Tolbutamide
100mg/kg)
274.65 +1.48
221.75 + 1.46***
184.73 + 1.42***
177.05 + 1.60***
35.5
III
DCM (250mg/kg)
271.47 + 1.24
217.08 + 1.42***
180.10 + 1.63***
114.18 + 2.56***
57.8
IV
EA (250mg/kg)
275.47 + 1.45
224.90 + 1.27***
192.68 + 1.11***
155.67 + 1.06***
43.5
V
BuOH (250mg/Kg)
281.42 + 1.50
264.30 + 1.51***
245.48 + 1.26***
227.88 + 1.52***
19.0
Each value represents Mean + SEM; n = 6, analysis by one – way ANOVA followed by Dunnett’s test, significant at ***P<0.001, in comparison to
negative control.
Table 2: Effect of Coscinium Fenestratum Crude DCM Extract on Body Weight in Normal and Diabetic Rats
Groups Treatment Body weight (g) % of Body weight
Increase
Day 0
Day 3
Day 7
Day 10
I
Negative control
204.07 + 1.05
201.10 +1.31
197.33+ 0.98
189.02+ 1.53
- 7.38
II
Tolbutamide (100mg/kg)
199.53+ 1.76
200.50+ 1.80
202.03+ 1.85***
ns
203.22 + 1.80***
1.81
III
DCM (250mg/kg)
215.82+ 1.07
219.27+ 1.13***
222.30+ 1.16***
225.12+ 1.03***
5.85
IV
EA (250mg/kg)
222.30+ 0.88
224.60+ 0.94***
226.65+ 0.89***
228.15+ 0.89***
4.13
V
BuOH (250mg/Kg)
222.45+ 0.97
223.13+ 0.97***
222.80+ 0.96***
224.50+ 1.00***
0.9
Each value represents Mean + SEM; n = 6, analysis by one – way ANOVA followed by Dunnett’s test, significant at (***P<0.001, NS –Not Significant)
in comparison to negative control.
Table 3: Enzyme Activity of Hexokinase Enzyme
Groups
Enzyme Activity (U/g)
Group I-Non- Diabetic Control
2.603 + 0.052
Group II -Diabetic Control
0.867 + 0.031
Group III- DCM
***
1.854 + 0.019
Group IV- EA
***
1.153 + 0.007***
Value expressed as Mean + SEM, n = 5, analysis by One-way ANOVA followed by Dunnett’s test, significant at ***p<0.001, in comparing to Group I : U,
u mol reduction of NAD
Table 4: Enzyme Activity of Enzyme Lactate Dehydrogenase
+
Groups
Enzyme Activity (U/g)
Group I-Non- Diabetic Control
2.387 + 0.027
Group II -Diabetic Control
0.430 + 0.016
Group III- DCM
***
1.441 + 0.019
Group IV- EA
***
0.852 + 0.021***
Value expressed as Mean + SEM, n = 5, analysis by One-way ANOVA followed by Dunnett’s test, significant at ***
Table 5: Total Phenolic Content
p<0.001, in comparing to Group I: U,
u mol conversion of NAD to NADH
TPC ( mg GAE / 100g )
100% DCM C. fenestratum extract
1092.3310 +34.8918
Results are means +SEM ( n = 3)
Table 6: Ferrous ion Chelating (Fic) Activity
Chelating Effect (%)
100% DCM C. fenestratum extract
47.13 +0.44
Results are means + SEM (n = 3)
Table 6: DPPH Free Radical Scavenging Activity (%)
Scavenging Effect (%)
100% DCM C. fenestratum extract
66.30 + 0.12
Results are means + SEM (n = 3)
Okechukwu et al.
Asian J Pharm Clin Res, Vol 4, Issue 4, 2011, 47-51
50
DISCUSSION
Diabetes mellitus is a worldwide problem, and type 2 diabetes is
found to be more prevalent. Patients in this group range from insulin
deficiency and insulin resistance to a predominantly secretary defect
with some insulin resistance.
The oral toxicity of crude extracts was evaluated with dose five –fold
higher than maximum tested antidiabetic dose (1000mg/kg, p. o.).
At this dose, no signs and symptoms of acute toxicity were observed
in all treated rats. No significant difference was observed in the
weight of heart, liver, kidney, or lungs when they were compared
with those of control group (saline). None of the treated rats died
during the 14 days of observation after the administration of crude
extracts. The results obtained indicated the absence of acute toxic
effect of crude extracts.
18
STZ is well known for its selective pancreatic islet β-cells
cytotoxicity and has been extensively used to induce diabetes
mellitus in animals. It interferes with cellular metabolic oxidative
mechanisms 19. Intraperitoneal administration of STZ effectively
induced diabetes in normal rats, as observed by hyperglycemia,
when compared with normal rats. Persistent hyperglycemia, the
common characteristic of diabetes can cause most diabetic
complications and it is normalized by the action of insulin 20. In this
study significant hyperglycemia was achieved within 48 hours after
STZ (60mg/kg body wt. i.p.) injection. STZ induced diabetic rats with
more than levels above 11 mmol/L or 200mg/dL of blood glucose
were considered to be diabetic and used for the study. In this study
it was observe that the oral administration of the extract (250mg/kg
body wt) could reverse the above mentioned diabetic effect, possibly
due to an insulin-like effect of the extract on peripheral tissues,
either by promoting glucose uptake and metabolism, or by inhibiting
hepatic gluconeogenesis. This result was in-line with traditional
claim and the previous reports by 10 and 21. A number of compounds
have also been shown to exert hypoglycaemic activity through
stimulation of insulin release 22. The hypoglycaemic potency of the
extract was comparable with tolbutamide, a standard
hypoglycaemic drug. Tolbutamide has long been used to treat
diabetes and is known to act by stimulating insulin secretion
through action on the pancreatic β-cells 23
Hexakinase is an insulin – dependent and insulin- sensitive enzyme
and are almost completely inhibited or inactivated in diabetic rat
liver in the absence of insulin
.
24. Decrease enzymatic activity of
hexokinase has also been reported in diabetic animals, resulting in
depletion of liver and muscle glycogen 25. Administration of extract
and tolbutamide to STZ treated rats resulted in an increased activity
of hexakinase in liver. Increased hexokinase activity was observed in
the STZ-induced diabetic rats treated with extract which would have
resulted in the activation of glycolysis, which, in turn, increased the
utilization of glucose by restored insulin secretion in treated rats 26.
Lactate dehydrogenase (LDH) activity increase in extracts treated
groups compared to diabetic control group. The decreased activity
of this enzyme in the diabetic condition diminishes the reducing
equivalent of oxidative stress leading to diabetic complications. The
mechanism of action of this extracts is not yet known however from
its effect on LDH the extract seems to increase flux of glucose into
the glycolytic pathway and pentose monophosphate shunt in an
attempt to reduce high blood glucose levels and may have increased
the production of the reducing agent, NADPH with concomitant
decrease in oxidative stress 27
Accumulating evidence indicates that ROS not only are strongly
associated with lipid peroxidation resulting in deterioration of food
materials, but are also involved in the development of pathology of
many clinical disorders, such as cancer, gastric ulcers, Alzheimer,
diabetes, arthritis and ischaemic reperfusion
.
28,29. The removal of
free radicals and ROS is probably one of the most effective defences
of the body to maintain the oxidative –antioxidant balance. The
extract showed very high content of polyphenols and free radical
scavenging (DPPH) and chelating (FIC) effects (Antioxidant) and
phytochemical screening confirmed the presence of flavonoids,
saponins, tannins, alkaloids, terpenes. There is an increasing interest
in phytochemicals such as polyphenols, saponins, tannins, alkaloids
terpenes due to their potentially positive effect against certain
diseases including diabetes. They can act as free radical scavengers,
neutralising dangerous reactive oxygen species and metal ion
chelators, which are responsible for antioxidant properties30
CONCLUSION
.
In conclusion our result indicates that DCM crude extract from the
stem of CF possess antidiabetic action. The mechanism of action may
be by stimulation of the pancreatic β-cells and the enzymes that
regulate glycolysis. The effect maybe as a result of the presence
phytochemicals which provided antioxidant properties and have
been reported to possess antidiabetic effect.
ACKNOWLEDGEMENT
UCSI University for the research grant provided for this experiment.
REFERENCES
1. Erem C, Yildiz R, Kagvaci H, Karahan C, Deger O, Can G,
Prevalence of Diabetes, obesity and hypertension in a Turkish
population (Trabzon city). Diabetes Research and Clinical
Practice 2001; 54:203-208.
2. Letchuman GR, Wan Nazaimoon WM, Wan Mohamad WB,
Chandran LR, Tee GH, Jamaiyah, Prevalence of diabetes in the
Malaysia National Health Morbidity Survey III 2006, Med J
Malaysia 2010; 65:173-179.
3. Friedman MH, Principles and Models of Biological transport.
2nd
4. Okamoto H, In molecular biology of the islet of Langerhans
Bioessays 1985; 2:15-21.
ed. New York: Springer Publishers 2008.
5. Tierney LM, McPhee SJ, Papadakis MA, Current-medical
Diagnosis and Treatment. Prentice. 38. Prentice-Hall Int. Inc,
USA; 1999.
6. Pinho MMP, Pinto MMM, Kijjoa A, Pharadia K, Diaz JG and Herz
W, Protoberberine alkaloids from Coscinium fenestratum.
Phytochemistry 1992; 31:1403-1407.
7. Varier PS, Indian Medicinal plants compendium of 500 Species
vol.2. Orient Longman Ltd., Hyderabad, pp. 191-193.
8. Chinnaiah A, 1000 Siddha medicine notes (Tamil). Siva
Publishers, Sivakasi,p.33.
9. Kalavincela T, Studies on rural medicine of Kanyakumari
district with special reference to medicinal plants. M.Phil
Thesis, Manonmaniam Sundranar University, p.185.
10. Shirwaikar A, Rajendran K, and Punitha ISR, Antidiabetic
activity of alcoholic stem extract of Coscinium fenestratum in
streptozotocin-nicotinamide induced type 2 diabetic rats. J
Ethnopharmacology 2005; 97:369-374.
11. Souza Brito, A.R.M., Manual de Ensaios Toxicologicos in
vivo.Editora da Unicamp, Campinas,Sao Paulo 1995 pp. 15-22.
12. Litchfield,J.T., Wilcoxon, F., A simplified method of evaluation
dose-dependent experiments. Journal Pharmacology
Experimental Therapeutics 1949; 95, 99-113.
13. Kaleem M, Asif M, Ahmed Q, and Bano B, Antidiabetic and
Antioxidant activity of Annonna squamosa extract in
streptozotocin induced rats. Singapore Medical Journal 2006:
47(8), 670-675.
14. Spanos GA, Wrolstad RE: influence of processing and storage
on the phenolic composition of Thompson seedless grape juice.
JAgric food Chem 1990: 38, 1565-1571.
15. Kikuzaki H,Nakatani N, Antioxidant effect of some ginger
constitutents. J food Sc 1993:58, 1407-1410.
16. Aiyegoro,O.A., Afolayan,A.J., Okon,A.I., In vitro antibacterial
activity of crude extracts of the leaves of Helichrysum
longifolium in combination with selected antibiotics. Afr
J.Pharm Pharmacol 3(6): 293-300.
17. Trease,G.E., Evans, W.C., Textbook of pharmacognosy 12th
18. Home PD, Towards a new classification for diabetes: American
view,Diabetes News 1998; 29,1-3.
edition. BalliereTindiall: London; 1998.
19. Papaccio G, Pisanti FA, Latronico MV, Ammendola E and
Galdieri M, Multiple low dose and single high dose treatments
with streptozotocin do not generate nitric oxide. Journal Cell
Biochemistry 2000: 77, 82-91.
20. Gayathri M, and Kannabiran K Antidiabetic and ameliorative
potential of ficus bengalensis bark extract in streptozotocin
Okechukwu et al.
Asian J Pharm Clin Res, Vol 4, Issue 4, 2011, 47-51
51
induced diabetic rats. India Journal of Clinical Biochemistry
2008: 24(4) 758-763.
21. Mahapatra B Hypoglycemic activity of Cosinium fenestratum
Jorunal of research in Ayurveda and Siddha 1997:18, 89.
22. Palsamy P, Subramanian S, Resveratrol, a natural phytoalexin,
normalizes hyperglycemia in streptozotocin – nicotinamide
induced experimental diabetic rats. Biomedicne and
Pharmacotherapy, 2008: 77, 82-91.
23. Fisher,J.,1985.Drugs and chemicals that produce diabetes.
Trends in Pharmacological Science, 6, 72-75.
24. Gupta,B.L., Nehal,M., and Baquer,N.Z., Effect of experimental
diabetes on the activities of hexokinase,glucose – 6-phosphate
dehydrogenase and catecholamine in rat erythrocytes of
different ages. Indian Journal of experimental Biology 1997: 35,
792-795.
25. Murray,R.K., Granner,D.K, Mayes,P.A., Rodwell, V.W., Harper’s
Biochemistry (25th
26. Saravanan, G., Ponmurugan,P., Senthilkumar, G.P., Rajarajan, T.,
Modulatory effet of S-allylcysteine on glucose metabolism in
streptozocin induced diabetic rats. Journal of functional foods
2009; 336-340.
ed). Standford, Connecticut: Appleton &
Lange. Pp. 610-617.
27. Ugochukwu,N.H., and Babady, N.E., Antihyperglycemic effect of
aquous and ethanolic extracts of Gongronema lanfolium leaves
on glucose and glycogen metabolism in livers of normal and
streptozotocin-induced diabetic rats. Journal of life science
2003;73(15) 1925-1938.
28. Dalle-Donne,I., Giustarini,D., Colombo,R., Rossi,R., & Milzani,A.
Protein carbonylation in human diseases. Trend in Molcular
Medicine 2003;9,169-176.
29. Telci,A., Cakatay,U., Salman, S., Satman, I & Sivas, A. Oxidative
protein damage in early stage of type 1 diabetic patients.
Diabetic Research and Clinical Practice 2000; 50, 213-223.
30. Li, W.L., Zheng,H.C., Bukuru,J., De Kimpe,N., Natural Medicine
used in the traditional Chinese medical system for theraphy of
diabetes mellitus. Journal of ethnophamarmacology 2004; 1-21.
