Antidiabetic Effect of
Luffa acutangula Fruits
and Histology of Organs in Streptozotocin Induced
Diabetic in Rats
S. Mohan
Raj*, Shahid Mohammed1, Vinoth Kumar S.2, Santhosh
Kumar C3. and Subal Debnath3,
1Jogaiah Institute of Technology and Sciences College of Pharmacy, Kalagampudi, W.G, Andhra Pradesh
2Gogulakrishna College of
Pharmacy, Sulurpet, Andhra Pradesh.
3Srikrupa Institute of
Pharmaceutical Sciences, Vil. Velkatta, Kondapak (Mdl), Dist. Medak, Siddipet, Andhra Pradesh –
502 277.
ABSTRACT:
The Antidiabetic activity of fruits and
seeds ethanolic extract of Luffa acutangula (Cucurbitaceae) was studied in a Streptozotocin
{STZ) induced diabetic in rats. The acute toxicity and lethality (LD50)
and the Phytochemicals analysis of the extract were
also evaluated. The results showed that the extract (200 and 400 mg/kg)
significantly (P<0.05) reduced fasting blood sugar of Streptozotocin
diabetic rats in a dose-related manner, with maximum hypoglycemic effect at
after 21 days. Acute toxicity and lethality test of the extract in rats gave an
oral LD50 greater than 5 g/kg. It is clearly evident from the study
that the streptozotocin administration caused the
significant increase in the blood glucose level at 0 day (p<0.001). The 50%
ethanolic extract of the fruits of Luffa acutangula showed the significant effect compared with
the respective diabetic control group, decrease the blood glucose level at a
dose of 200 mg/kg and 400 mg/kg (p<0.001), the standard drug glibenclamide also showed the significant decrease the
blood glucose level after 21 days (241.33-105.33, p<0.001). Finally the 400
mg/kg and the standard drug showed the significant decrease in the blood
glucose level after 21 days treatment (p<0.001). The findings indicate that
the leaves of Luffa Acutangula
may be beneficial as an Antidiabetic therapy.
KEYWORDS: Streptozotocin, Luffa Acutangula, Antidiabetic
INTRODUCTION:
Diabetes Mellitus is considered as one of the five leading causes
of death in the world. An epidemic by all standards, nearly 250 million people
suffer from diabetes across the world. Synthetic anti-diabetic drugs increase
the insulin secretion or decrease the blood glucose level but they are also
producing many harmful effects. Since, increase in the use of these drugs in
diabetes therapy leads to many side effects and undesirable hazards, there is
worldwide trend to go back to natural resources, i.e., traditional plant1.
In industrialized countries the people are seeking for safer alternative to
allopathic medicine because of the increasing realization on the adverse side
effects of many modern remedies. The current interest in and demand for herbs
is a worldwide phenomenon, WHO currently encourages, recommends and promotes
traditional/ herbal remedies in national healthcare programmes
because such drugs are easily available at low cost, are comparatively safe and
people have faith in such remedies2. Luffa
Acutangula wild (Cucurbitaceae),
commonly known as “Turai” (Hindi), “Peerkku” (Tamil), and Kritavedhana”
(Sanskrit), is an pale yellowish – brown and
cylindrical or club – shaped. The seeds are pale green and bitter in taste and
cultivated in wastelands especially along the coastal areas in throughout
India.
The plant is commonly used
all over the world for the treatment of diabetes, anti- inflammatory , bronchodialator , antimicrobial activity, nutritional
values, antiproliferative and anti- antiangiogenic activity and potent CNS depressant (1-4).
The ethno medicinal use of the plant in the treatment of diabetes in the all
over world, we evaluated the Antidiabetic potentials
of L.acutangula fruits extract in
diabetic rats.
MATERIALS AND METHODS:
Animals:
Sprague-Dawley rats (150-185g) and Swiss
albino mice (20-25 gm) of either sex and of approximately the same age were
selected for the study. The study was carried in accordance with the rules and
regulations laid by the Institutional Animal Ethics Committee. They were kept
in the departmental animal house at 26 ± 2°C and
relative humidity 44 – 56% in polypropylene cages. The animals were exposed to
alternate 12 hours of darkness and light each. Animals were provided with
standard rodent pellet diet (Dayal, India) and the
food was withdrawn 18-24 h before the experiment though water was allowed ad
libitum. All experiments were performed in the
morning according to current guidelines for investigation of experimental pain
in conscious animals64. The standard orogastric
cannula was used for oral drug administration in
experimental animals.
Plant
material:
The fruits of Luffa Acutangula (Family-Cucurbitaceae)
were collected from the local market in month of January 2011. The plant
material was authenticated by Dr. A.B.S Murthy, Botanist. A voucher specimen
has been deposited at the museum of our college.
Preparation of plant extract:
The powdered plant material (1000g) was macerated with petroleum
ether to remove fatty substances and filtered by using Whatman filter paper no.
1. The filtrate is dried under reduced pressure by using rotavapour
(Buchi, USA) and then dried in lyophilizer
(Labconco, USA). The residue was then stored in a
desiccator. The marc left was dried and then further exhaustively extracted
with of 50% ethanol for 3 days (3 X 5L). The extract was separated by
filtration and concentrated on rotavapour (Buchi, USA) and then dried in lyophilizer
(Labconco, USA) under reduced pressure and low
temperature to obtain 87.52 g of solid residue (yield 8.75 % w/w). The extract
obtained was further subjected to toxicological and pharmacological
investigations, which was subsequently subjected to phytochemical analysis
using standard procedures3.
Acute toxicity tests (OECD 423):
This test involves the administration of a simple bolus
dose of test substances to faster healthy young adult rodents by oral gavage, observation for up to 15days after dosing and
recording of body weight and the necropsy of all the animals. In this method
pre-specified fixed doses of the test substances were used i.e,
5mg/Kg, 50mg/Kg, 300mg/Kg, 2000mg/Kg and the mortality due to these doses were
observed. Generally female animals were used for this study and each dose group
should consist of 3 animals (Table no-3).
IN VIVO ANTIDIABETIC ACTIVITY:
Experimental induction of diabetes:
All animals were allowed to adapt to metabolic cages for 3 days,
after which they were fasted overnight and 65mg/kg of Streptozotocin
freshly dissolved in normal saline was injected intraperitoneally.
After Streptozotocin treatment, all animals were
returned to their cages and given free access to food and water. Blood glucose
levels were measured 3 days after Streptozotocin
injection and used as parameter to obtain matching pairs of rats with diabetes
of similar level of severity. Only rats with fasting blood glucose levels
greater than 200 mg/dl were considered diabetic and then included in this
study. The mean blood concentration of glucose in normoglycemic
rats was 95 mg/dl. Diabetic rats were randomly assigned to four different
groups (n = 6 animal/groups). All treatments started 3 days after Streptozotoicn injection.
Experimental Design:
Group I- Control rats received vehicle
solution (1% carboxy methyl cellulose)
Group II-Diabetic control rats received 30
mg/kg body weight in (1% CMC)
Group III-Diabetic rats treated with
extract 200 mg/kg body weight in (1% CMC)
Group IV- Diabetic rats treated with
extract 400 mg/kg body weight in (1% CMC)
Group V-Diabetic rats treated with Glibenclamide
10 mg/kg body Weight in aqueous solution
The vehicles and the drugs were administered orally using intra
gastric tube daily for three weeks. After three weeks of treatment the rats
were fasted overnight, the blood samples were analyzed for blood glucose
content. Then the animal was sacrificed by cervical decapitation. The liver,
kidney and pancreas was exposed and per fused with cold phosphate buffer saline
of pH 7.4. Blood free liver and kidney were taken out and homogenized in a
glass Teflon homogenizer separately (10% w/v). Incubations were done at 37ºC
under controlled conditions for biochemical estimations. Fresh blood drawn was
centrifuged for 10 min at 2000 rpm. Erythrocyte sediment is resuspended
twice in physiological NaCl solution (1:10) and
centrifuged again in same solution. 250 ml of washed erythrocytes are then resuspended
in 1000 ml
physiological NaCl solution and stored at 4°C in the dark until SOD
measurement4.
Glucose estimation:
The collected serum samples of different study group were
subjected to the serum glucose level estimation by enzymatic GOD-POD method by
using glucose diagnostic kit (Auto-span).We can also measure the change of
optical density directly from Bio Chemical analyzer at 505 nm5.
Total cholesterol estimation
The serum cholesterol level was estimated by wybenga
and pileggi method using cholesterol diagnostic
reagent kit (span). Reagents were Mixed well and kept in the boiling water bath
exactly for 90 seconds. Mixer was allowed to cool to room temperature under
running tap water. Absorbance was measured at 560 nm6.
Triglycerides estimation:
The triglycerides level was estimated by Glycerol phosphate oxidase (GPO) method. Mix and Incubate at
37oC for 10 minutes. Read the absorbance against reagent
blank on a spectrophotometer at 520 nm against reagent blank. The final colour stable for 30 minutes7.
Bio-chemical parameters:
Serum was analyzed for the following parameters Aspartate aminotransferase/Serum glutamic oxaloacetic transaminase (ASAT)/ (SGOT), Alanine
amino transferase/Serum glutamate pyruvate
transaminase (ALAT)/(SGPT),
Alkaline phosphates (ALP) and cholesterol9,8,10.
Determination of Serum glutamic oxaloacetic transaminase (SGOT):
The SGOT activity was determined according to the method of IFCC
modified method using SGOT (Liquizone diagnostic reagent
kit).
Determination of Serum glutamate pyruvate
transaminase (SGPT) or ALAT:
The SGPT activity was determined according to the method of IFCC
modified method using SGPT (Liquizone diagnostic
reagent kit).
Determination of serum alkaline phosphatase
(SALP):
The alkaline phosphates level was estimated by p- Nitro phenyl
phosphate (PNPP) method (Qualigens diagnostic reagent
kit).
STATISTICAL ANALYSIS:
All the values were expressed as mean ± SEM (standard error mean) for six
rats. Statistical analysis was carried out by using PRISM software package
(version 3.0). Statistical significance of differences between the control and
experimental groups was assessed by One-way ANOVA followed by Newman-Keuls Multiple Comparison Test. The value of probability
less than 5% (P < 0.05) was considered statistically significant.
RESULTS AND DISCUSSION
The main property in this regard is their Antidiabetic
activity which enables them to attenuate the problem of hypoglycemia. Antidiabetic plants an important role in inhibiting the
glucose level and, thus providing protection to human against hyperglycemia11.
Realizing the fact, this research was carried out to evaluate the Antidiabetic activity of 50% ethanolic extract of fruits of
Luffa Acutangula
in streptozotoicn induced diabetic rats. The phytoconstituents were extracted by using different solvent
of increasing polarity like Petroleum ether, Ethanol (50%).The extractive
values were presented in (Table no.1)
Phytochemical evaluation
The phytoconstituents were
identified by chemical tests which showed the various phytoconstituents
in the both extracts, the phytochemical evaluation shows the presence of flavonoids, phenolic compounds,
tannins, glycosides, saponins, phytosterols
and carbohydrate in the ethanolic extract.
LD50 value of 50 % Ethanolic extract of Luffa Acutangula.
There were no mortality is observed by using 2000mg/kg b.w to the mice after 48 hours observation. So 1/10 and 1/5
of the dose i.e., 200mg/kg and 400mg/kg dose were selected for the study
In vivo Anti diabetic studies
Effect of 50% ethanolic extract of fruits of Luffa Acutangula on
streptozotocin induced diabetic rats after 0 day and
21 days.
It is clearly evident from (Table no.2) that the streptozotocin administration caused the significant increase
in the blood glucose level at 0 day (71.83-250.66, p<0.001). The 50%
ethanolic extract of the fruits of Luffa Acutangula showed
the significant effect compared with the respective diabetic control group,
decrease the blood glucose level at a dose of 200 mg/kg and 400 mg/kg
(241.33-185.26, 241.33-118.16,p<0.001), the standard drug glibenclamide also showed the significant decrease the
blood glucose level after 21 days (241.33-105.33, p<0.001). Finally the 400
mg/kg and the standard drug showed the significant decrease in the blood
glucose level after 21 days treatment (p<0.001). Streptozotocin
diabetes causes an increase in blood glucose level in rats. Streptozotocin
exerts its diabetogenic action when it is
administered parenterally. The action of Streptozotocin in the pancreas is preceded by its rapid
uptake by the β cells. Rapid uptake by insulin-secreting cells has been
proposed to be one of the important features determining Streptozotocin
diabetogenicity. Our studies show that oral
administration of 50 % ethanolic extract of the fruits of Luffa
Acutangula decreases blood glucose level
in diabetic rats.
Table No. 1. Percentage yield of various extracts of Luffa Acutangula
|
Plant
used |
Part used |
Method |
Percentage
yield |
|
|
Luffa Acutangula
|
Fruits |
Cold
percolation |
Pet.
ether |
50%ethanol |
|
2.35% w/w |
8.75%w/w |
|||
Table No. 2. Anti-diabetic effect (Glucose level) of 50%
ethanolic extract of Luffa Acutangula (ELA) on streptozotocin
induced diabetic rats after 0 and 21 days.
|
Groups |
Treatment |
Dose |
0 day
(mg/dl) |
After 7 day (mg/dl) |
After 14
days (mg/dl) |
After 21
days (mg/dl) |
|
I |
Control |
V.1% CMC |
71.83
±7.12 |
72.33±6.26 |
73.16±5.72 |
72.16±6.57 |
|
II |
Dia.Control |
30 mg/kg |
250.66±23.59*** |
243.12±23.23*** |
246.32±24.72*** |
241.33±23.33*** |
|
III |
STZ +ELA
200 |
200mg/kg |
246.33±22.43* |
222.16±21.34* |
205.12±19.76* |
185.26±18.12* |
|
IV |
STZ +ELA
400 |
400mg/kg |
240.32±21.26* |
198.16±18.12** |
182.16±17.7** |
118.16±10.67*** |
|
V |
Gliben-Clamide |
10mg/kg |
235.84±20.12*** |
152.84±14.67*** |
132.26±12.27*** |
105.33±10.52*** |
Values are expressed as Mean ± SEM of 6 rats in each group and 4
rats in Diabetic control group. *P < 0.05, ** P <
0.01, ***P < 0.001 compared with diabetic control group, and
normal control group.
Table No. 3. Effect of 50% ethanolic extract of Luffa Acutangula (ELA) on the
glucose level, cholesterol, and triglyceride in blood serum
|
Groups |
Treatment |
Dose |
Glucose
level (mg/dl)
after 21 days |
Cholesterol (mg/dl) |
Triglycerides (mg/dl) |
|
I |
Control |
V.1%CMC |
72.16±6.57 |
73.52±6.27 |
80.16±7.57 |
|
II |
Diabetic |
30 mg/kg |
241.33.±23.33*** |
118.38±7.12*** |
130.23±12.27*** |
|
III |
STZ +ELA
200 |
200mg/kg |
185.26±18.12* |
98.27±7.62* |
98.27±9.16* |
|
IV |
STZ + ELA
400 |
400mg/kg |
118.16±10.67** |
88.16±6.67** |
88.16±6.67** |
|
V |
Glibenclamide |
10 mg/kg |
105.33±10.52*** |
83.12±7.31*** |
83.12±7.72*** |
Values are expressed as Mean ± SEM of 6 rats in each group and 4
rats in Diabetic control group. * P < 0.05, ** P <
0.01, ***P < 0.001 compared with diabetic control group, compared
and normal control group.
Table No. 4. Effect of 50% ethanolic extract of Luffa Acutangula (ELA) SGOT,
SGPT,
SALP.
|
Groups |
Treatment |
Dose |
SGOT
(U/l) |
SGPT
(U/l) |
SALP
(U/l) |
|
I |
Control |
V.1%CMC |
64.73±6.23 |
22.16±2.62 |
231.62±22.12 |
|
II |
Diabetic |
30 mg/kg |
105.45±8.79*** |
48.42±5.12*** |
283.23±27.16*** |
|
III |
STZ+ELA
200 |
200 mg/kg |
71.84±7.12* |
32.17±2.82* |
239.57±21.96* |
|
IV |
STZ + ELA
400 |
400mg/kg |
65.38±6.25*** |
25.17±22.2*** |
235.91±22.79*** |
|
V |
Glibenclamide |
10 mg/kg |
66.34±6.56*** |
23.29±2.16*** |
233.23±22.77*** |
Values are expressed as Mean ± SEM of 6 rats in each group and 4
rats in Diabetic control group. * P < 0.05, **P <
0.01, ***P < 0.001 compared with diabetic control group and
normal control group
Table No. 5. Effect 50% ethanolic extract
of fruits of Luffa Acutangula
(ELA) on body weight of rats during anti-diabetic study
|
Group |
Treatment |
Dose |
0 week
(gm) |
1st
week (gm) |
2ndweek
(gm) |
3rd
week (gm) |
|
I |
Control |
V.1%CMC |
178.83±1.03 |
180.67±.86 |
182.17±.86 |
183.50±1.21 |
|
II |
Diabetic |
30 mg/kg |
180.83±1.33x |
180.66±1.11x |
178.66±1.09x |
164.50±1.06x |
|
III |
STZ +ELA
200 |
200 mg/kg |
154.83±1.35c |
155.33±1.66c |
157.16±1.23c |
161.16±1.06c |
|
IV |
STZ + ELA
400 |
400mg/kg |
153.83±1.35c |
156.33±1.28c |
158.67±0.95c |
162.67±0.86c |
|
V |
Glibenclamide |
10 mg/kg |
170.17±1.13c |
173.17±1.09c |
175.5±1.06c |
178.83±0.97c |
a P <
0.05, b P < 0.01, cP <
0.001 compared with diabetic control
group. x P < 0.05, y P <
0.01, zP < 0.001 compared with normal
control group.
Effect of 50% ethanolic extract of Luffa
Acutangula on streptozotocin
on cholesterol and triglyceride, in streptozotocin
induced diabetic rat’s serum.
It is clearly evident that Streptozotocin
caused significant elevation of serum markers. The Streptozotocin
treated group, the level of cholesterol (73.52-118.38, p<0.001),
triglyceride (80.16-130.23, p<0.001). In contrast, the groups treated with
50 % ethanolic extract of the fruits of Luffa
Acutangula at dose 200 and 400mg/kg once daily
for 21 days prevented the diabetes condition in a dose related manner. The
range of protection were found to be, cholesterol (118.38-98.27, 118.38-88.16
p<0.05, p<0.001), triglyceride (130.23 - 98.27, 130.23- 88.16 p<0.05,
p<0.001) and glibenclamide (10mg/kg) also showed
the significantly decrease with respect to diabetic control group
(118.38-83.12, p<0.001, p<0.01, 130.23-83.12,p<0.001,p<0.05)
in( Table no.3). It has been demonstrated that insulin deficiency in
diabetes mellitus leads to a variety of derangements in metabolic and
regulatory process, which in turn leads to accumulation of lipids such as
cholesterol and triglyceride in diabetic patients. The abnormal high
concentration of serum lipids in the diabetic subject is due mainly to increase
in the mobilization of free fatty acids from the peripheral fat depots. In
present study the 50 % ethanolic extract of the fruits of Luffa
Acutangula decreases the cholesterol level in a
significant manner. The 50 % ethanolic extract of the fruits of Luffa Acutangula also
decreases the triglycerides and lipid levels in diabetic rats.
Effect of the 50% ethanolic
extract of fruits of Luffa Acutangula on SGOT, SGPT and SALP levels in blood
serum.
It is clearly evident that Streptozotocin
caused significant increase in SGOT (64.73 105.45, p<0.05, p<0.001),
SGPT(22.16-48.42, p<0.05, p<0.001) and SALP (231.62-283.23,
p<0.0001).The 50% ethanolic extracts of 200, and 400 mg/kg of fruits of Luffa Acutangula decreases the SGOT (105.45-71.84, 105.45 65.38,
p<0.001, p<0.01, p<0.05), SGPT (48.42-32.17, 48.42-25.17, p<0.001,
p<0.05) and SALP (283.23-239.57, 283.23-235.91, p<0.05, p<0.001)
levels with respect to diabetic control group and glibenclamide
(10mg/kg) also showed the significantly decrease with respect to diabetic
control group(105.45-66.34, 48.42-23.29, 283.23-233.23, p<0.0001, p<0.05)
as shown in table no. 5 and in fig. no. 4. A marked hike observed
in the activities of SGOT, SGPT and ALP in serum of diabetic rats indicates
injury caused to tissue due to STZ. When cell membrane gets damaged, the enzyme
which is normally located in the cytosol leaked into
the blood stream thus manifesting damage affected the liver and other tissues.
Thus the 50% ethanolic extract of fruits of Luffa
acutangula
200, and 400 mg/kg of decreases
the SGOT, SGPT and SALP levels with respect to diabetic control group and glibenclamide (10mg/kg) also showed the significantly
decrease up to normal level. The results were showed in (Table no 4)
Effect of 50% ethanolic extract of Luffa
Acutangula on body weight and food intake of
rats
The 50% ethanolic extract of fruits of Luffa
Acutangula at
a dose of 200 and 400 mg per kg of body weight, there is no significant
difference on body weight (183.5-178.83) and food intake (16.16-16.17) in the
end of the study (Table.no.5).
Histopathological studies on
Pancreas:
In the pancreas of normal control group rats, many round and
elongated islets were evenly distributed throughout the cytoplasm, with their
nucleus lightly stained than the surrounding cells. In Diabetic Control group
rats, the islets were damaged, shrunken in size and infiltration of lymphocytes
was observed. The treatment with 400 mg of Luffa Acutangulat
extract significantly reduced
pancreatic β-cells damage compare to 200mg plant extract.
The histological sections of the pancreas, kidney, and tissues
were observed to know the effect of Luffa Acutangula fed in non-diabetic and diabetic
rats. This was done to observe any protective or harmful effect of Luffa Acutangula extract
on non-diabetic and diabetic rats.
.
CONCLUSION:
In conclusion, the present study clearly demonstrates that the
ethanol fruits and seeds extract of Luffa acutangula is an effective antidiabetic
agent with multiple therapeutic effects mediated by a combination of preventing
the β-cell destruction, histological architecture of the pancreatic
islets, improving glucose disposa12. As a result indicates that Luffa acutangula extracts
were more useful effective in comparison with glibenclamide
in attenuating the increased serum parameters resulting from damage of
STZ-induced diabetic rats and that the Luffa acutangula extract
treatment may be of use as an antidiabetic supplements.
Overall, our report from the present analysis should be ground data for
undertaking further study, and it is useful information for investigating new
sorghum materials for food additives and human health.
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Received on 06.12.2011
Modified on 24.12.2011
Accepted on 12.02.2012
© A&V Publication all right reserved
Research Journal of Pharmacognosy and Phytochemistry.
4(2): March-April 2012,
64-69