Evaluation
of Ginkgo biloba in Diabetic Nephrotoxicity.
M.K. Kale1*, M.P. Patil2 and K.P. Bhusari1
1Sharad Pawar College
of Pharmacy, Wanadongri, Nagpur 441110
2Tapi Valley’s, College of Pharmacy, Faizpur, Ta. Yawal , Dist. Jalgaon (MS)
ABSTRACT:
KEYWORDS: Diabetes, Oxidative stress, Nephrotoxicity, Ginkgo
biloba.
INTRODUCTION:
Nephrotoxicity is the major cause of morbidity and
mortality in diabetes and it is the leading cause of end stage renal disease1,2.
Different studies were carried out in diabetes showed that it is the major
disorder responsible for nephrotoxicity. The major
key factor involved is the generation of free radicals along with depletion of
first line defense system of body3,4. Alloxan
has been widely used to induce diabetes in experimental models. It causes
necrosis of b- cells of islets of langerhans
of pancreas5. Hyperglycemia induced oxygen free radicals acts as a
mediator of diabetic complications. Several studies showed that the
overproduction of superoxide anion by the mitochondrial electron-transport
chain seems to be the first event in diabetes to produce free radicals6.
Other key events are increased polyol pathway7,
increased advanced glycosylation end product
formation8 and activation of protein kinase
C9. Alloxan induced diabetes is
responsible for generation of free radicals in the cells10. Although
the mechanism is not known for nephrotoxicity, but
may be due to oxidative stress and subsequent oxidative damages leading cell
and tissue injury. Ginkgo biloba is a flavonoid that
occurs in conjugated form11.
It has been
reported that the active constituents responsible for antioxidant activity are quercetin, isorhamnetin and
kaempherol12,13. Hence it was proposed to evaluate the antioxidant
activity of Ginkgo biloba
in long-term diabetic complications such as diabetic nephrotoxicity.
Experimental Material and
methods:
Animals – Sprague-Dawley rats
of both sex weighing between 150-200gms were used for experiment. They were
maintained in a clean polypropylene cage with food and water ad libitum.
Institutional Animal Ethical committee under guidelines of CPCSEA, New Delhi,
INDIA, approved the protocol.
Chemicals – Diacetyl monoxime, thiosemicarbazide, metaphosphoric acid, picric acid, uric acid, trichloroacetic acid, thiobarbituric
acid, pyrogallol, hydrogen peroxide, 5,5 dithio-bis-2
nitro benzoic acid (DTNB). Chemicals were purchased from LOBA Chemie, Burgoyne, Merck and Sigma Aldrich Co. U.S.A.
Methods- The rats were divided into four groups of six
animals in each group. Group I served as normal control and received normal
saline solution daily intraperitoneally. Group II
served as diabetic control and received alloxan
120mg/kg i.p, at the interval of 14 days14.
Group III received alloxan, i.p
plus Ginkgo biloba
300mg/kg orally, daily15. Group IV received alloxan,
i.p plus Vit E 150mg/kg
orally, daily16.
Sugar level of each rat was maintained and monitored
throughout the protocol by GOD-POD method. At the end of the protocol, blood
was withdrawn from retro-orbital plexus and levels of serum creatinine,
urea and uric acid17 were determined. Serum creatinine
was estimated by alkaline picrate method, serum urea
by diacetyl monoxime method
and serum uric acid by Henry-Caraway’s method. Along with this blood parameter,
the oxidative stress parameters like lipid peroxidation
in erythrocyte suspension was determined. The endogenous antioxidant enzymes,
superoxide dismutase, catalase and reduced
glutathione were determined in erythrocyte lysate and
blood respectively to find out the extent of oxidative stress.
Lipid peroxidation18 – It was determined by
in separated RBC suspension. To the suspension phosphate buffered saline, trichloroacetic acid was added and centrifuged. To the
supernatant, thiobarbituric acid was added and the
mixture was heated on boiling water bath for 1 hour and cooled immediately. The
absorbance was measured spectrophotometrically at 532 nm. The lipid peroxidation was calculated on the basis of the molar
extinction coefficient of malondialdehyde (1.56 ´ 105)
and expressed in terms of nanomoles of MDA/g Hb.
Superoxide dismutase19 – It was determined
in erythrocyte lysate prepared from RBC suspension.
To the lysate, Tris-HCl
buffer (pH 8.2), EDTA and pyrogallol were added. An
increase in absorbance was recorded at 420 nm for 3 minutes by
spectrophotometer. The activity of SOD is expressed in terms of units/mg
protein.
Catalase20 – Catalase
activity was determined in erythrocyte lysate. To the
lysate, phosphate buffer was added (pH 7.0). To this
hydrogen peroxide was added and increase in absorbance was recorded at 240 nm
for 1 minute. The molar extinction coefficient of hydrogen peroxide was used to
determine catalase activity and it is expressed in
terms of units/mg protein.
Reduced glutathione21 – It was determined in
whole blood. Blood was added in distilled water followed by addition of
precipitating mixture of metaphosphoric acid, EDTA
and NaCl. It was centrifugedand
clarified. To the filtrate, phosphate solution was added followed by addition
of DTNB reagent. Absorbance was measured at 412 nm and expressed in terms of mm DTNB
conjugated/g Hb.
Histopathological study22- Kidneys from the four
groups were isolated and fixed in 10% formalin solution. These were processed.
Sections of 4-6 mm were cut on microtome machine and were stained with haematoxylin and eosin. Finally they were examined under
light microscope and photographed. The changes evaluated were denoted as absent
(-), negligible (±), mild (+), moderate (++) and severe (+++).
In alloxan induced diabetic
group, there was rise in the levels of serum creatinine,
urea and uric acid. Co-administration of Ginkgo
biloba showed decrease in levels of these
parameters. And these results were comparable with alloxan
+ Vit E treated group, which was taken as a standard
antioxidant (Table 1).
Table 1: Effect of Ginkgo biloba on serum creatinine, urea and uric acid in rats (mean ± S.D, n=6).
|
Group |
Serum creatinine (mg/dl) |
Serum urea (mg/dl) |
Serum uric acid (mg/dl) |
Control
|
0.42 ± 0.25 |
21.6 ± 2.9 |
3.82 ± 0.39 |
|
Diabetic
control |
1.5 ± 0.31a |
34 ± 2.7a |
4.86 ± 0.26a |
|
Diabetic +Ginkgo biloba |
1.16 ± 0.16b |
28.6 ± 2.0b |
4.28 ± 0.21b |
|
Diabetic + Vit E |
0.7 ± 0.29b |
25 ± 3.0b |
4.04 ± 0.16 b |
ap<0.05 when
compared to control; bp<0.05 when
compared to diabetic
Plasma malondialdehyde level
was increased and levels of antioxidant enzyme system were decreased in
diabetic group as compared to control. Concurrent administration of Ginkgo biloba reduced
the plasma malondialdehyde and increased the levels
of antioxidant enzyme (Table 2).
In diabetic group, hemorrhage in medullary
area, infiltration of leukocytes around the glomerulus,
cloudy and sloughy swelling of tubular epithelial
cells and necrosis of tubular epithelium was observed. Co-administration of Ginkgo biloba showed
mild hemorrhage and infiltration of leukocytes around glomerulus
(Table 3).
Table 2: Effect of Ginkgo biloba on
oxidative stress in rats (mean ± S.D, n = 6)
Groups
|
Parameters
|
|||
|
Lipid peroxidation (nm
MDA/g Hb) |
Superoxide dismutase (units/mg protein) |
Catalase
(units/mg protein) |
Reduced glutathione (mm DTNB conjugated/g Hb) |
|
Control
|
260.4 ± 20.33 |
32.26 ± 2.73 |
290.4 ± 20.55 |
4.78 ± 0.29 |
|
Diabetic control |
515.4 ± 21.81a |
24.76 ± 3.78a |
220.6 ± 18.36a |
2.72 ± 0.22a |
|
Diabetic + Ginkgo
biloba |
390.4 ± 11.54b |
27.2 ± 2.56b |
232 ± 18.86b |
3.75 ± 0.17b |
|
Diabetic + Vit E |
325 ± 11.18b |
30.06 ± 2.46b |
250 ± 19.82b |
4.2 ± 0.22b |
ap<0.05
when compared to control; bp<0.05 when
compared to diabetic
Table 3: Effect of Ginkgo biloba on
kidneys of diabetic rats (histopathological study)
(mean ±
S.D, n=6)
|
Lesions. |
Groups. |
||||
|
Control |
Alloxan
treated (diabetic) |
Alloxan + GB-I |
Alloxan + GB-II |
Alloxan
+ Vit. E |
|
|
Hemorrhage. |
++ |
+++ |
+ |
++ |
++ |
|
Cloudy swelling
of tubules. |
± |
++ |
++ |
+ |
+ |
|
Necrosis and
sloughing of tubular epithelium. |
± |
+ |
+ |
+ |
± |
|
Infiltration of
leukocytes around glomerulus and interstitial
spaces. |
- |
++ |
+ |
+ |
- |
Absent -;
Negligible ±; Mild +; Moderate
++; Severe +++
DISCUSSION:
Results of this study confirmed that, prolonged
duration of diabetes produced nephrotoxicity as
evident by increased levels of serum creatinine, urea
and uric acid. In the present study, we investigated the potent antioxidant
effect of Ginkgo biloba
on diabetes induced nephrotoxicity. Concurrent
treatment with Ginkgo biloba
provided the functional and histological protection against renal damage.
Increase in serum creatinine, urea and uric acid was
lowered by Ginkgo biloba.
Co-administration of Ginkgo biloba effectively reduced increase in plasma MDA. Also
showed increased level of antioxidant enzymes (superoxide dismutase, catalase and reduced glutathione).
In summary, the present study provided evidence that coadministration of Ginkgo
biloba in diabetes reduces both functional and
histological renal damages induced by diabetes. This protection may be due to
antioxidant property of Ginkgo biloba.
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Received on 08.08.2011
Accepted on 11.09.2011
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Research Journal of Pharmacognosy and
Phytochemistry. 3(6): Nov. - Dec. 2011, 286-288