Journal club 5 17 feb

JournAl club 5
17/02/2020
Dr Ashishkumar Baheti
JR 1, MD Pharmacology
MGIMS, Sevagram

Antidiabetic Activity of Vinca rosea Extracts in Alloxan-Induced
Diabetic Rats
Mohammed Fazil Ahmed, Syed Mohammed Kazim,
Syed Saﬁullah Ghori, Syeda Sughra Mehjabeen, Shaik Rasheed Ahmed,
Shaik Mehboob Ali, and Mohammed Ibrahim
Department of Pharmacology and Biotechnology, Nizam Institute of
Pharmacy, Nalgonda, Andhra Pradesh.
Center for Liver Research and Diagnostics, Deccan College of Medical
Sciences and Allied Hospitals Kanchanbagh, Hyderabad, Andhra Pradesh.
Department of Microbiology, Moana Marine Biotech Ltd.,
Vishakapatnam, Andhra Pradesh.
International Journal of Endocrinology Volume 2010.

1. Introduction
Diabetes mellitus is one of the common metabolic disorders with micro and
macrovascular complications that results in significant morbidity and mortality.
In modern medicine no satisfactory effective therapy is still available to cure
diabetes mellitus.
There is increasing demand by patients to use natural products with antidiabetic
activity due to side effects associated with the use of insulin and oral hypoglycemic
agents.
Vinca rosea (C. roseus) Linn. (Apocynaceae) is an herbaceous
subshrub also known as Madagascar periwinkle, Vinca rosea,
or Lchnera rosea worldwide.
It is cultivated mainly for its alkaloids, which are having
anticancer activities.
The two classes of active compounds in
Vinca are alkaloids and tannins.

The leaves and stems are the sources of dimeric alkaloids, vincristine and
vinblastine that are indispensable cancer drugs, while roots have antihypertensive,
ajmalicine and serpentine.
The leaves are used traditionally in various regions of the world including India,
West Indies as well as Nigeria to control diabetes.
The leaves have been known to contain 150 useful alkaloids among other
pharmacologically active compounds.
Signiﬁcant antihyperglycemic and hypotensive activity of the leaf extracts
(hydroalcoholic or dichloromethane-methanol) have been reported in laboratory
animals.
Fresh leaf juice of C. roseus has been reported to reduce blood glucose in normal
and alloxan diabetic rabbits.

Leaves and twigs of Catharanthus roseus have been reported to have
hypoglycaemic activity in streptozotocin induced diabetic rats.
In this study the prolonged eﬀect (up to 14 day) of the methanolic extracts of
whole plant of Vinca rosea in fasting blood glucose(FBG) and biochemical
parameters such as serum total cholesterol (TC), LDL, HDL, creatinine, urea, and
alkaline phosphatase were studied in alloxan induced diabetic rats.
Hence on the above fact no study has been carried out on methanolic extracts of
whole plant of Vinca rosea in alloxan induced diabetic rats.
Thus the present study is an attempt to test the antidiabetic activity of whole plant
of the Vinca rosea.

2. MaterialsandMethods
2.1. Plant Material.
The basic plant material of Vinca rosea Linn whole plant used for the investigation
2.2. Alcoholic Extraction.
The whole plants were collected and shadow dried.
The shade-dried whole plants were subjected to pulverization to get coarse
powder.
The coarsely powder whole plant (1kg) of Vinca rosea Linn was used for extraction
with methanol in soxhlet apparatus.
The extract was evaporated to dryness under vacuum and dried in vacuum
desiccator (15.5% w/w).

2.3. Animals.
Wistar albino rats (8–10 weeks) of both sexes were used.
Before and during the experiment, rats were fed with standard diet (Gold Moher,
Lipton India Ltd).
After randomization into various groups and before initiation of experiment, the
rats were acclimatized for a period of 7 days under standard environmental
conditions of temperature, relative humidity, and dark/light cycle.
Animals described as fasting were deprived of food and water for 16 hours ad
libitum.

2.4. Oral Glucose Tolerance Test.
The rats of group III and IV were loaded with glucose (3g/kg, p.o.) 30 minutes after
drug administration.
Blood samples were collected from puncturing the retro orbital sinus just prior to
drug administration and 30, 90, 150 minutes after loading glucose. Serum glucose
level was measured immediately by using glucose estimation kit.
Group I (n=6) Vehicle control 5% Tween 80 p.o.
Group II (n=6) Normal glucose only
Group III (n=6) Test methanolic extract 300mg/kg + glucose (3g/kg, p.o.)
Group IV (n=6) Test methanolic extract 500 mg/kg + glucose (3g/kg, p.o.)
Group V (n=6) Test methanolic extract 300mg/kg Only in a vehicle
Group VI (n=6) Test methanolic extract 500 mg/kg only in a vehicle

2.5. Acute Oral Toxicity Studies.
Vinca rosea at the dose range of 100mg–2000mg/kg were administered orally to
diﬀerent group of rats comprised of ten rats in each group.
Mortality was observed after 72 hours.

2.6. Experimental Design.
Glibenclamide (5mg/kg) and saline were administered with the help of feeding
cannula for 14 days.
Group I (n=6) Normal control Saline
Group II (n=6) Diabetic control Alloxan (150mg/kg. ip).
Group III (n=6) Test group Whole plants extract (300mg/kg, p.o),
Group IV (n=6) Test group Whole plants extract (500mg/kg, p.o),
Group V (n=6) Standard control Standard drug, Glibenclamide (5mg/kg, p.o).

2.7.Induction of Diabetes in Experimental Animals.
Rats were made diabetic by a single intraperitoneal injection of alloxan
monohydrate (150mg/kg).
Two days after alloxan injection, rats with plasma glucose levels of >140mg/dl were
included in the study.
Treatment with plant extracts was started 48 h after alloxan injection.
2.8. Collection of Blood Sample and Blood Glucose Determination.
Blood samples were drawn from tail tip of rat at weekly intervals till the end of
study (i.e., 2 weeks).
Fasting blood glucose estimation and body weight measurement were done on day
1,7,and 14 of the study.
Blood glucose estimation can be done by one touch electronic glucometer using
glucose test strips.
On day 14, blood was collected from retro-orbital plexus under mild ether
anesthesia from overnight fasted rats and fasting blood sugar was estimated.

Serum was separated and analyzed for serum cholesterol, serum triglycerides by
enzymatic DHBS colorimetric method, serum HDL, serum LDL, serum creatinine,
serum urea and serum alkaline phosphatase hydrolyzed phenol amino antipyrine
method was estimated.
The whole pancreas from each animal was removed after sacrificing the animal
and was collected in 10% formaline solution, and immediately processed by the
paraffin technique. Sections of 5μ thickness were cut and stained by haematoxylin
and eosin (H & E) for histological examination.
2.9. Statistical Analysis.
All the values of body weight, fasting blood sugar, and biochemical estimations
were expressed as mean±standard error of mean (S.E.M.) and analyzed for ANOVA
and post hoc Dunnet’s t-test. Differences between groups were considered
significant at P < .01 levels.

3. Results
3.1. Glucose Tolerance
The supplementation of Vinca rosea improved the glucose tolerance in the fasted
normal rats. After that serum glucose level was lowered significantly (P <0.05) at
90 minutes and varied significantly (P < .01) lowered at 150 minutes.
Extract also showed significant hypoglycemic effect after 90 minutes of treatment.

3.2. Experimental Results.
The acute oral toxicity study of Vinca rosea showed no mortality upto 2000mg/kg.
Administration of alloxan (150mg/kg, i.p.) lead to 1.5 fold elevation of fasting
blood glucose levels (group 2), which was maintained over a period of 2 weeks.
Two weeks of daily treatment of various extract of Vinca rosea (group 3 & 4) lead
to a dose dependent fall in blood sugar levels by 25%–50%.

Vehicle control animals (group 1 ) were found to be slightly increased in their body
weight but diabetic rats (group 2) showed signiﬁcant reduction in body weight
during 14 days (Figure 4). Alloxan caused body weight reduction, which is reversed
by whole plant extract at high dose (500mg/kg) is more eﬀectively than whole
plant extract at low dose (300mg/kg) after 14 days of treatment (Figure 4).

Glibenclamide (5mg/kg) and extracts reversed the alloxan induce changes(Table 1 )

Histopathological studies showed normal acini and normal cellular population in
the islets of Langerhans in pancreas of control rats (Group I).
Extensive damage to the islets of Langerhans and reduced dimensions of islets
(Group II)

Restoration of normal cellular population size of islets with hyperplasia by
Glibenclamide (Group V) were also shown.
The partial restoration of normal cellular population and enlarged size of β-cells
with hyperplasia were shown by methanolic extracts (Group III & Group IV).

4. Discussion
 In light of the results, this study indicates that methanolic extracts of Vinca
rosea have good antidiabetic activity.
 Alcoholic extracts of Vinca rosea exhibited significant antihyperglycemic
activities in alloxan-induced hyperglycemic rats without significant change in
body weight; they can also improve the condition of Diabetic mellitus as
indicated by parameters like body weight & lipid profile along with serum
creatinine, serum urea and serum alkaline phosphatase.
 The total β cell mass reflects the balance between the renewal and loss of these
cells. It was also suggested that regeneration of islet ß cells following
destruction by alloxan may be the primary cause of the recovery of alloxan-
injected guinea pigs from the effects of the drug.

 Vinca rosea whole plant alcoholic extracts has been shown to act by β cell
regeneration.
 Similar eﬀects in streptozotocin-treated diabetic animals were reported by
pancreas tonic, ephedrine, and Gymnema sylvestre leaf extracts.
 It is found that methanolic whole plant extract at high dose (500mg/kg) is more
eﬀective than whole plant extract at low dose (300mg/kg) after 14 days of
treatment.

 Hence the above discussion revels that methanolic whole plant extract at high
dose (500mg/kg) is more effective and shows similar curative effect as standard
that is, Glibenclamide (5mg/kg).
 This could be due to the possibility that some β-cells are still surviving to act
upon by Vinca rosea extract to exert its insulin releasing effect.
 Histopathological studies reinforce the healing of pancreas, by Vinca rosea
extracts, as a possible mechanism of their antidiabetic activity.

5. Conclusions :
 The whole plant extracts did not show a consistent effect on normal blood
sugar levels but it effectively reversed the alloxan-induced changes in the blood
sugar level and the beta-cell population in the pancreas.
 It also showed a protective effect when it was given prior to alloxan
administration. The action of whole plant extracts on the pancreatic beta-cells
and absence of acute toxicity may offer a new hope to the diabetics in future.
 From the above discussion it conclude that alcoholic whole plant extracts of
Vinca rosea at high dose (500mg/kg) exhibited significant antihyperglycemic
activity than whole plant extract at low dose (300mg/kg) in alloxan-induced
diabetic rats.

 These extracts also showed improvement in parameters like body weight and
lipid proﬁle as well as regeneration of β cells of pancreas and so might be of
value in diabetes treatment.
 Further investigation is in necessary to determine the exact phytoconstituents
(s) responsible for antidiabetic eﬀect.

Criticism
1)Title – Clear, brief, Extraxt type, Study design was not mentioned
2) Abstract - Provided an accurate summary of the background, principal
findings and conclusions of the study
Introduction
3) Background- Included sufficient scientific background
4) Objectives - Yes, objectives were Clearly described.
24

Methods
5) Ethical statement – No, It was not mentioned
6) Study design - The number of experimental and control groups was mentioned
for each experiment; sampling method was mentioned.
7) Experimental procedures – were explained in detail, method of euthanasia was
not mentioned.
8) Experimental animals - Provided details of the animals used, including species,
strain, sex, developmental stage, weight, source of animal.
25

10) Sample size - the number of animals in each group was mentioned in each
experiment. Total number of animals used were not given.
11) Allocating animals to experimental groups – full details of how animals were
allocated, treated and assessed in different experimental groups given.
12)Experimental outcomes -Yes, they were Clearly defined
13) Statistical methods – Yes, details of the statistical method used for each
analysis was mentioned
26

Results
14) Baseline data- Yes, it was mentioned.
15) Numbers analysed - the mean of all the animals present in each group
was used in analysis
16) Outcomes and estimation – Yes, it was mentioned. P < 0.01 was
considered statistically significant, all the outcomes were mentioned
graphically and in tabular form.
17) Adverse events- No, there were no adverse events
27

Discussion
18) Interpretation/ scientific implications - Yes, interpretations of the
study were mentioned
19) Generalisability - Yes, the findings of the study were relevant to
human biology
20 ) Funding – NO, source of funding was not mentioned.
21) Conflicts of interest : No, conflicts of interest were not mentioned
28

Journal club 5 17 feb

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