Research Paper
Natural Product Radiance, Vol. 8(1), 2009, pp.32-36
Effect of ethanolic extract of Feronia elephantum Correa
fruits on blood glucose levels in normal and
streptozotocin-induced diabetic rats
Rahul Gupta1, Samta Johri2 and A M Saxena1*
1
Department of Zoology, University of Lucknow, Lucknow-226 007, Uttar Pradesh, India
2
Department of Zoology, Mahila Girls P.G. College, Lucknow
*Correspondent author, E-mail: [email protected]
Phone: 09415028759 (Mob.), 0522-2357057 (R)
Received 2 January 2008; Accepted 20 August 2008
Abstract
Feronia elephantum Correa (Family — Rutaceae) commonly known as Wood
apple has been widely used in Indian folk medicine for treatment of blood impurities, leucorrhoea,
and as diuretic and liver tonic. It has not been studied experimentally for its blood glucose
lowering potential earlier hence present study was conducted. Oral administration of 250mg/kg
body weight of 95% ethanolic extract of unripe fruits significantly lowered the blood glucose levels
of fasted, fed and streptozotocin-induced diabetic male albino rats. It also depressed the peak
value in glucose loaded model. Further, study on histology of pancreatic β-cells granularity of
normal rats was also done. Marked degranulation in β-cells of extract treated rats, associated with
the blood glucose lowering was observed. Extract probably lowered the blood glucose concentrations
by stimulating insulin secretogogue activity.
Keywords: Blood glucose lowering, β-cells, Ethanolic extract, Feronia elephantum,
Streptozotocin-diabetes, Wood apple.
Materials and Methods
IPC code, Int. cl.8 — A61K 36/75, A61P 3/10
Introduction
In India, plant and herbs have
been widely used by Ayurveda for the
treatment of various ailments since vedic
glory. Indian traditional medicine is
indeed rich in a vast variety of flora,
having ample scope in search of effective
herbal remedy for diabetes1. Feronia
elephantum Correa [syn. F. limonia
(Linn.) Swingle]2, 3 is a moderate size slow
growing tree, with strait sharp strong
spines. It is commonly known as Wood
apple or Kath-Bel and belongs to
family—Rutaceae. Wood apple is
indigenous in South India, Ceylon and Java
and cultivated in many parts of India. The
tree grows up to an elevation of 450 m in
the Western Himalayas. Traditionally, it
32
produced hepatoprotective activity at
400mg/kg and 800mg/kg in albino rats8.
It has not been studied experimentally for
its blood glucose lowering potential, thus
in the present study we investigated the
blood glucose lowering activity of 95%
ethanolic extract of unripe fruits of
F. elephantum in normal and
streptozotocin-induced diabetic rats,
along with its effect on histology of insulin
producing pancreatic β-cells granularity.
is consumed as fruit and vegetable in Sri
Lanka4.
The fruits are sour and sweet, and
used in India as liver and cardiac tonic. It
is effective in treatment of diarrhoea,
dysentery, hiccough, sore throat and gum
diseases. In Ayurveda, it has been used for
leucorrhoea and removing blood
impurities and in Yunani medicine as
diuretic 2 . The unripe fruits contain
0.015% stigmasterol and seeds contain oil
high in saturated fatty acids5. An acidic
heteropolysaccharide isolated from the
F. elephantum was reported to inhibit
carcinoma cell growth6. Active compounds
from stem bark were found to possess
antimicrobial
activity 7 .
Oral
administration of aqueous extract of leaves
Unripe fruits of F. elephantum
(Fig. 1) were collected fresh in month of
November from a village Bakshi Ka
Taalab (latitude 26°59′ 46″ N and
longitude 80°54′ 9″ E) of Lucknow
following standard guideline given by
Harnischfeger9. The plant was botanically
identified and authenticated by Dr.
Nirmala Upadhyay and a voucher
specimen (LU 2/23560/06) was deposited
in the Botany Department of Lucknow
University. Unripe fruits were sliced, air
dried in shade and powdered
mechanically. Coarse powder of fruits was
extracted by using 95% ethanol in Soxhlet
extractor10. The extracted material was
concentrated and an alcohol free
semi-solid crude extract was obtained
(wet fruits collected-5kg; yield of dry
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Research Paper
weight/kg wet fruits-274.0g; crude extract (c) Glucose loaded: Animals were
yield/100g dried fruits weight-23.20g).
fasted for 18 h and blood was
Extract was orally fed to experimental
collected (at 0h) for glucose
group by metal canula at a single dose of
estimation. Now extract was fed and
250mg/kg body weight as prepared in 2%
after half an hour glucose (1.5g/kg
gum acacia suspension and control was
b.w. oral) solution was administered
fed only 2% gum acacia11, 12 . All the
and blood samples were collected for
chemicals used were of analytical grades
glucose estimation at 1/2, 1 and 3
from E. Merk Ltd., West Germany and
hour interval.
Sigma-Aldrich Chemicals, USA.
(d) Diabetic: Diabetes was induced in
Male albino rats (120-140g) of
rats by injecting streptozotocin
Charles Foster strain were used and
(35mg/kg b.w.) dissolved in the
maintained on Hind Lever pellets diet
chilled citrate-phosphate buffer
(Mumbai, India), housed in polypropylene
(pH 4.5) through tail by the method
cages at a temperature of 23 ± 2°C and
of Theodorou et al16. At the moment
relative humidity 60 ± 5 with 12 h each
of injection the rats had fasted for 18
of dark and light cycle. Water was allowed
hours. After the streptozotocin
ad libitum. The rats were divided in to
injection, all rats were returned to
experimental and control groups of six
their cages and given free access to
rats each. Acute toxicity study13 of extract
food and water. Two to three days after
on behavioral aspect was carried out up
injection, diabetes was confirmed by
to dose of 2500mg/kg body weight and
checking urine glucose by Diastex17.
found to be safe. The blood glucose
Then the experiments were performed
lowering effect of the extract was examined
using identical procedure as in the
in following four different experimental
fasted model (a).
models14:
Statistical analysis of data
Mean and standard error were
(a) Fasted: Blood was collected (at 0 h)
from the tail vein of the overnight determined and students't' test was
(18h) fasted rats and extract was fed. performed between experimental and
Again blood samples were collected control group. The results were
at 1, 3 and 4 hour interval after considered statistically significant, if the
feeding extract. Blood glucose p values were 0.05 or less.
concentrations were estimated by
Histology of Pancreas
Nelson's Somogyi method15.
Pancreas of anesthetized animals
(b) Fed: Excess amount of pellets were of extract treated and control group were
put in the cages on previous evening, taken out at the most effective hours
so that some pellets were found left associated with blood glucose lowering
over in the next morning. Blood was and fixed in alcoholic Bouin's fluid.
collected before (at 0h) and after Paraffin sections of pancreas were double
administration of the extract at 1, 3 stained with haematoxylin and eosine,
and 4 hour interval for glucose and granularity of insulin producing
estimation.
Vol 8(1) January-February 2009
β-cells was
microscope18, 19.
compared
under
Results
In fasted group of rats ethanolic
extract of unripe fruits produced
significant (P<0.05) blood glucose
lowering at 3h as compared to control
(Table 1). In extract treated group,
maximum percentage of blood glucose
lowering (16.29% at 3h) from the initial
value (0h) was higher as compared to
control (5.42% at 4h). Ethanolic extract
produced significant blood glucose
lowering in fed rats at 3h (P<0.01) as
compared to corresponding control.
Maximum percentage lowering of blood
glucose from the initial value (0h) was
higher in extract treated group (13.98%
at 3h) than control (7.08% at 4h).
In diabetic group of albino rats
extract of unripe fruits produced
significant (P<0.05) lowering of blood
glucose at 3h and 4h after the extract
administration. Maximum percentage of
blood glucose lowering in extract treated
group from the initial value (0h) was
11.26% at 3h, but no lowering recorded
in control.
Ethanolic extract of unripe fruits
produced significant (P<0.05)
suppression of blood glucose level at
1 and 3h in glucose loaded model as
compared to control (Table 2). In terms
of maximum percentage of blood glucose
rise from the initial value (0h), the extract
was exhibited more blood glucose
lowering effect (18.05% rise at 1h) than
2% gum acacia in control group (33.98%
rise at 1h).
During the histological
examination of pancreas of extract treated
fasted (Fig. 2B) and fed (Fig. 2D) rats,
33
Research Paper
degranulated β-cells were observed at
3 h post-treatment as compared to their
respective controls (Figs 2A and 2C),
associated with the blood glucose
lowering. In glucose loaded rats also, the
marked degranulation in β-cells
was observed at 3 h post-treatment
(Fig. 2F) as compared to corresponding
control (Fig. 2E).
Discussion
In the present study, four test
models having different background of
blood glucose levels, that resemble some
patho-physiological condition of the body
have been used to increase the sensitivity
of experimental procedure and to spot
blood glucose lowering activity of
F. elephantum with certainty14. Male
albino rats were chosen as experimental
animal because, their blood glucose levels
are fairly stable and have been most widely
used in such studies, leading to generation
of adequate baseline data11. The ethanolic
extract was orally administered as this
route was thought to be most
preferable and in tune with the route of
administration of 250mg/kg b.w. dose as
reported earlier by various workers11, 12.
In the present study, 95% ethanolic
extract of unripe fruits was found to
produce significant lowering of
blood glucose levels in fasted, fed
and streptozotocin-induced diabetic
male albino rats. Extract was also
improved the
oral glucose
tolerance. In
the present
experiments,
b l o o d
glucose
lowering
e f f e c t
induced by
fruits extract,
persisted for
3 to 4 h after
feeding,
h e n c e
F. elephantum definitely lowers the
blood glucose in rats. In extract treated
normal rats, reduction in blood glucose
levels is associated with the degranulation
in pancreatic β-cells. It suggests that the
rate of extract induced insulin release
from β-cells is probably greater than the
rate at which β-cells replenish their
insulin stores.
Fig. 1: Feronia elephantum fruits
Fig. 2 (A-F): Showing, rich granulation in β-cells of islets of Langerhans of control group (A) Fasted, (C) Fed,
(E) Glucose loaded, and marked degranulation in β-cells of the extract treated group (B) Fasted, (D) Fed,
(F) Glucose loaded at 3 h post-treatment (Magnification-63X)
34
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Research Paper
Table 1 : Effect of 95% ethanolic extract of Feronia elephantum fruits at a single oral dose of 250mg/kg body
weight, on blood glucose levels of fasted, fed and streptozotocin-induced diabetic male albino rats
Group
Treatment
Blood glucose level mg/100ml (Mean ±S.E.) at time (hours)
0h
1h
3h
4h
Maximum % of
blood glucose
lowering from
initial value
Fasted
Control
EG
83.38±4.78 (6)
85.08±3.18 (6)
82.80±2.94 (6)
84.27±3.19 (6)
81.24±2.55 (6)
71.22±2.78a (6)
78.86±2.47 (6)
74.75±2.17 (6)
5.42% at 4h
16.29% at 3 h
Fed
Control
EG
103.65±4.23 (6)
103.36±2.16 (6)
100.14±2.10 (6)
94.94±3.08 (6)
102.97±1.90 (6)
88.90±2.52b (6)
96.31±4.26 (6)
92.05±2.34 (6)
7.08% at 4h
13.98% at 3h
Diabetic
Control
EG
189.55±6.91 (6)
195.34±5.14 (6)
198.64±7.24 (6)
181.52±8.93 (6)
211.33±8.40 (6)
173.35±10.24a (6)
209.62±7.16 (6)
180.31±7.92a (6)
No lowering.
11.26% at 3h
Significance between the control and EG (experimental group): aP<0.05, bP<0.01; In parenthesis the number of rats used is given.
Table 2 : Effect of 95% ethanolic extract of Feronia elephantum fruits at a single oral dose of 250mg/kg body
weight, on glucose tolerance (1.5g/kg b. w. orally) of male albino rats
Group
Glucose
Loaded
Treatment
Control
EG
Blood glucose level mg/100ml (Mean ±S.E.) at time (hours)
0h
1/2h
1h
3h
Maximum % of
blood glucose rise
from initial value
74.71±2.07 (6)
73.63±2.27 (6)
94.55±4.63 (6)
84.74±3.07 (6)
100.10±3.27 (6)
86.92±1.90b (6)
95.97±3.10 (6)
80.71±1.80b (6)
33.98% at 1h
18.05% at 1h
Significance between the control and EG (experimental group): aP<0.05, bP<0.01; In parenthesis the number of rats used is given.
Department of Botany, University of 3. Hanelt P, Buttner R, Mansfeld R and Kilian R,
Type-Feronia elephantum Correa (Syn.
Thus, we can conclude that Lucknow, Lucknow for her active help in
F. limonia (L.) Swingle) In: Mansfeld's
ethanolic extract of F. elephantum identification of the plant.
Encyclo-pedia of Agricultural and Horticultural
unripe fruits probably have insulin
Crops, 2nd Edn, Springer, Germany, 2001,
p. 1037.
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