Indian Journal of Clinical Biochemistry, 2002, 17 (2) 115-123

REVERSAL OF DIABETIC RETINOPATHY IN S T R E P T O Z O T O C I N I N D U C E D DIABETIC RATS

USING TRAD I T I ONA L INDIAN ANTI-DIABETIC PLANT, A Z A D / R A C H T A / N D / C A
(L.)
Halim Eshrat M. All Hussain
National Chemical Laborator~, Pune - 411008

ABSTRACT
This study was carried out to investigate the effect of oral feeding of water extract of fresh
leaves of Azadirachta ind/ca (Fam: Meliaceae) in streptozotocin induced diabetes and its
associated retinopathy in rats. Treatment of the diabetic rats with aqueous extract of leaves of
A. indica at a dose of 250 mg/kg body weight for 16 weeks resulted in gradual but significant
fall in blood glucose and improvement in serum total, LDL and HDL cholesterol and
triacylglycerol which increased it1 diabetic rats. It also showed improvement in body weight
and reversed retinopathy.

KEY WORDS
Azadirachtaindica, diabetes mellitus, diabetic retinopathy, hypoglycemic effect, hypolipidemic

effect

INTRODUCTION
Diabetes poses an increasing public health problem
across the world. In particular, the increasing
prevalence of type II diabetes affects life
expectancies and more so obese people. Long term
complications of diabetes mellitus include
retinopathy with potential loss of vision, nephropathy
leading to renal failure, peripheral neuropathy with
risk of foot ulcers, amputation and charcoal joints.
However, much of the clinical and economic toll of
diabetes arises from complications of the disease,
such as capillary basement membrane thickening,
retinopathy, nephropathy, neuropathy, and
accelerated arteriosclerosis (1,2).
Early development of cataract of lens is due to the
increased rate of sorbitol formation, caused by
hyperglycemia. Glycosylation of retinal proteins and
retinal micro vascular abnormalities lead to
retlnopathy and blindness (3). Glycosylation of
lys~ne residues of lens proteins also causes cataract
formation. A variety of plant preparations have been
mentioned in Ayurveda and other indigenous
Author for Correspondence :

Dr. Halim Eshrat M. All Hussain

C/o Dr. Paul Ratnasamy
Director, National Chemical Laboratory
Pune-411008
Indian Journal of Clinical Biochemmtc: 2002

systems of medicine used in India, which are

claimed to be useful in diabetes mellitus and their
complications (4,5). The present therapy is grossly
incomplete in wew of the poor modification of
incidence of certain complications such as cataract
and diabetic nephropathy (3). If a drug can reverse
these complications either partly out of these plants,
or completely, it will be a great advantage.
Azard/kachta #7dicaJuss, leaves (Fam: Meliaceae,
common name: neem) have been reputed to
possess
cardiovascular,
antimicrobial,
immunomodulatory, hypoglycemic and a number
of other effects (6,7). A bitter principle, nimbi din,
isolated from seeds of neem tree was effective in
reducing fasbng blood glucose at a dose of 200mg/
kg in alloxan diabetic rabbits (8). Aqueous extract
of tender neem leaves was reported to reduce blood
glucose and this effect was stated to be due to
blocking the action of epinephnne on glycogenolysis
and peripheral utilization of glucose (9).
Earlier our preliminary studies have shown moderate
hypoglycemic and anti diabetic activity and of neem
leaves in animal models of NIDDM. There are no
reports on the ability of neem leaves to reverse
changes in retinopathy. In this paper we report this
new observation that streptozotocin induced
diabetes and associated retinopathy treatment with
the water extract of the neem leaves not 0nly
controls fasting blood sugar but also improves lipid
profile and retinopathy.
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Indian Journal of Clinical Biochemistry, 2002, 17 (2) 115-123

MATERIALS AND METHODS

Preparation of aqueous extract
Freshly collected neem leaves (10g) were ground
and homogenized with 50 ml of distilled water in an
electric blender in the cold room at 4~ The
homogenate was filtered through sterilized cheese
cloth. The clear filtrate was concentrated by
lyophilization and stored in a deep freeze (-20~
At the time of use, it was dissolved in water ( 1gin
dry residue in 1 ml water) and administered orally
at a dose of 250 mg/kg b.w in the morning for 16
weeks by bulged steel tube.

Induction of diabetes and associated

retinopathy.
Healthy adult albino Wistar rats of both sexes
weighing between 150-200 gm were obtained from
the Centre for Cellular and Molecular Biology
(CCMB), Hyderabad and used in this study. The
animals were fed on a pellet diet (Hindustan Lever,
India) and water provided adlibitum. Diabetes was
induced by the method for experimental diabetic
retinopathy / nephropathy (10).
Twelve overnight fasted rats were injected with STZ
(60mg/kg dissolved in 3 mM citrate buffer pH 4.5)
intraperitoneally. After 10 days, ten rats showed
plasma glucose level > 300 mg /dl. They were
classified as diabetic and were included in the study
as described earlier by our laboratory (11). After
two weeks diabetic retinopathy developed in the rats.
Five of them were used as untreated diabetic
animals and the remaining five were treated with
water extract of neem (diabetic treated group at a
dose of 250mg/kg) daily once for 16 weeks. Normal
rats and diabetic untreated rats were given
equivalent volume of saline for 16 weeks.

Method for observing of retina.

Fluoresce in angiography's is a test used for
abnormal blood vessels. To do the test, a fluorescent
dye was injected into a vein in the leg (10). The dye
travels through the body including the eyes. With
special camera meant for this purpose photography
of the retina is taken by ophthalmology expert at
Indian Institute of Chemical Technology, Hyderabad
as the dye passes through it. The photographs show
what changes occurred in the retina and where those
changes are located.

Estimations
Indian Journal of Clinical Biochemisto/ 2002

Glucose tolerance test, plasma glucose , total

cholesterol, LDL, VLDL-and HDL-cholesterol and
triglycerides were estimated as described earlier
by us (12) using kits from Ranbaxy Laboratories,
New Delhi. Procedure given in the kits was followed.

RESULTS AND DISCUSSION

Streptozotocin (STZ) is known to induce not only
diabetes but also develop diabetic retinopathy
similar to early stage retinopathy of humans. For
the study of antidiabetic agents, STZ induced
hyperglycemia in rodents is considered to be a good
perliminiary screening model. It is a potent
methylating agent for DNA and acts as nitric oxide
donor in pancreatic cells. [3 cells are particularly
sensitive to damage by nitric oxide and free radicals
because of their low levels of free radical scavenging
enzymes. Major microvascular complications of DM
include retinopathy, most common cause of adult
blindness in developed countries (3).
The Streptozotocin injected rats developed not only
diabetes as indicated by increased fasting blood
glucose values (Table1), but also showed external
signs of retinopathy by 10 days. The eyes of diabetic
rats looked opaque even from outside (Fig1).
Therefore the retina was observed as mentioned
under methods with a special camera and the
photographs showed clear signs of retinopathy as
indicated by the presence of dilated vessels and
laser spots (Fig 2). After treatment for 16 weeks,
the eyes of the treated group of rats appeared normal
from outside (Fig 3). Photograph of the retina
indicated that the laser spots, disappeared (Fig 4),
pointing out that the treatment reversed the changes
in the eye, that is, abnormal changes of retinopathy
almost disappeared. In the untreated STZ diabetic
rats, there was injury to legs and edema of paws.
Treatment with water extract of neem leaves was
so effective that these complications also
disappeared in the diabetic rats after treatment. The
behavior of the animals was normal. This is a great
advantage, which is not seen with the existing drugs.
The biochemical parameters were also analyzed in
the normal, diabetic untreated and diabetic treated
rats and shown in tables (1,3). Since the untreated
diabetic rats had a very high FPG 335.6 + 13.25
mg/dl (Table 1), it is quite possible that their
pancreas is damaged. Treatment of the diabetic rats
with water extract of A. indica leaves brought down
the FPG to normal value, 100.8 +26.2mg/dl.
Glucose tolerance test was performed in another
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Indian Journal of Clinical Biochemistr~A, 2002, 17 (2) 115.123

group of rats with FPG of 170.6 +43 mg/dl. The

reason is that if GTT is performed with rats with
very high FPG of more than 300 mg/dl, some of the
animals may die due to glucose over load during
GI-F.
Treatment of diabetic rats with water extract of
Azadirachta ind/ca (neem) showed considerable

improvement in glucose tolerance also (Table2). The

results in diabetic rats showed abnormal glucose
tolerance during GTT. After treatment with water
extract of A. indica the glucose tolerance pattern
was normal. Nowadays the level of glycosylated
hemoglobin (HbA~c) is considered as a more reliable
index of glycemic control than fasting blood values.
We therefore estimated HbAlcvalues also (Table3).
In the untreated diabetic animals, the initial HbAlc
value increased to 9.4+ 2.4 % and to 6.4+0.9 in the
neem treated group, when compared with the normal
value of 3.4 +0.2%. After treatment the HbA!r level
did not come down but remained more or less the
same. In the neem, treated group the HbA~cvalue
came clown to 2.8+0.1% which is in the lower range
of normal value. Thus, water extract of neem leaves
brought about very good glycemic control.
In the diabetic rats there was an increase in total
cholesterol, LDL- cholesterol, LDL/HDL ratio and
triacylglycerol (table 4 ). But there was no significant
change in HDL cholesterol. After treatment with
water extract of leaves of Azad/rachta indica, there
was significant lowering of the serum lipids which
were closer to normal values. Perhaps still longer
treatment would have brought the lipid profile to

normal. There was slight increase in HDL cholesterol. This indicates that the water extract of
Azadirachta indica has favorable effect on the lipid
metabolism of diabetic rats also. There are reports
that some plants with hypoglycemic constituents
have hypolipidemic effect also (13).
The gain in body weight in the untreated diabetic
rats (Table 5), from 150 + 6 g to 190+6.09 (26.6 %)
was much less than that in normal rats from 160 +_
9.0 to 230+9 (43.7 %). But after treatment for 4
weeks with water extract of neem/eaves, gain in
weight in the diabetic treated animals was almost
equal to that in normal (41.8%). At the same time
the increase in weight of the treated animals is not
such as to lead to obesity.
Thus, the present study indicates that treatment
with water extract of neem leaves has favorable effect
not only on blood glucose and glucose tolerance
but also on lipid profile and body weight. Another
interesting feature of this plant product is that it
completely reversed the abnormal change in retina
and inflammation of paws. This points out the
promising effect of A. ind/ca being a useful antidiabetic agent and its ability to reverse complications
of retinopathy and cardiovascular changes in
diabetes. This suggests that further detailed studies
are desirable.
ACKNOWLEDGEMENT
The author is gratefu I to Dr. Paul Ratnasamy,
Director, National Chemical Laboratory, Pune, for
his interest and encouragement to this work.

Table 1. Effect of water extract of Azadirachta indica leaves on the fasting plasma glucose level in
STZ induced diabetic rats.
Group

Plasma glucose mg/dl, mean _+ S.D.

0 weeks

16 weeks

Normal

94.4+25.4

850

+ 28.2

Diabetic untreated

258.8 + 9.0

335.6 + 13.2

Diabetic + Azadirachta
indica

237.9+19.16

100.8+26.2"

*p< 0.005 when compared with untreated diabetic control.

Indian dournal of Clinical Biochemistry, 2002

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/ndian Journal of Clinical Biochemistry, 2002, 17 (2) 115-123

Table 2. Effect of treatment with water extract of Azadirachta indica leaves on glucose tolerance in
diabetic rats after 16 weeks treatment.
Plasma glucose mg/dl, mean
0hr.

0.5hr.

lhr.

1.5hr.

2hr.

Normal

95.5

143.5

137.2+320

118.4+1t.3

106.0

Diabetic
Untreated

170.6

250.6

271.0

285.6

274.0_+ 84.8

Diabetic
Treated

88.2

114.0

117.5

102.7

94.9 26.0"

n= 5 for each group

*p< 0.05 when compared with untreated diabetic group.

Table 3. Effect of treatment with water extract of leaves of A, indica on glycoslated

(HbA,~)
Group

Initial

After 4 weeks treatment

HbAlc(%)

Normal

3.4

4.0

Diabetic
Untreated

9.5

93

6.4

2_8

Diabetic
neem

Indian Journal of Clinical Biochemist~ 2002

hemoglobin

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Indian Journal o f Clinical Biochemistry, 2002, 17 (2) 115.123

Table 4. Effect of treatment with water extract of Azadirachta indica leaves on the plasma lipid
profile in diabetic rats.
Group

TC (mgldl)

Normal

167.6+ 15.8

Diabetic untreated
Diabetic treated

LDLC(mgldl)

HDLC(mg/dl)

LDLC/HDLC

TAG(mgldl)

76.0

44.9+13.2

1.7+0.3

116.0 +43.0

247.0

155.2

46.0+_120

3.2+0.4

183.9+19.0

176.0+13.9"

97.7

52.0+8.5

2.0+0.4

178.0+16.0

*p< O.005when compared with untreated control

Change in TAG = (triacylglycerol) is insignificant

Table 5. Effect of water extract of Azadirachta indica on the body weight of rats.
Group

Initial Body weight (g)

After 4 weeks treatment

Normal

160 +90

230 +90

Diabetic untreated

150 6.0

190

Diabetic +neem

158+5.0

224+9.0*

* p< 0.005 when compared with untreated diabetic group

Indian Journal of Clinical Biochemistry, 2002

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Indian Journal of Clinical Biochemistry, 2002, 17 (2) 115.123

Fig. 1. Untreated Diabetic rat showing Opaque eyes with retinopathy

Indian Journal of Cfinical Biochemist~ 2002

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Indian Journal of Clinical Biochemistry, 2002, 17 (2) 115-123

Fig. 2. Untreated Diabetic rat with retinopathy showing dilated vessels and laser spots

Indian Journal of Cfinical Biochemistry, 2002

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Indian Journal of Clinical Biochemistry, 2002, 17 (2) 115-123

Fig. 3. Diabetic rat after treatment with A. indica leaves extract for 16 weeks showing normal
appearance

Fig. 4. Diabetic rats after treatment with A. indica leaves extracts

showing reversal of retinopathy
Indian Journal of Clinical Biochemistry, 2002

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Indian Journal of Clinical Biochemistr~, 2002, 17 (2) 115-123

REFERENCES
1.

Ziyadeh,F. N. (2000) Long - term prevention of renal insufficiency, excess matrix Gene expression, and
glomerular mesangial matrix expansion by treatment with monoclonal antitransforming growth factor-b
antibody in rib~rib, diabetic mice. Proc. Natl. Acad Sci. USA 97 (6), 8015-8020

2.

Wan, P.H., Lao, L. and Chalmoro,T.C. (1993) Meta- analysis of effects of Intensive blood glucose
control on the diabetic complication of type 1 diabetes. Lancet, 341, 877- 890.

3.

Kelvin, R. and Moss, S (1992) Visual impairment and diabetes, In Albert K. R.De Fronzo Keen, H P,
Zimmet Eds. International Textbook of Diabetes Mellitus. Chi Chester; Wiley. 1373-1384.

4.

Shukla, R., Sharma, S.B., Puri, D., Prabhu, K.M. and Murthy, P.S. (2000) Medicinal Plants for treatment
of diabetes mellitus. Ind. J Clin. Biochem 15 (August, suppl.), 169-177.

5.

Satyarati,G.V., Raina, M K and Sharma, M. (1976) Medicinal plants of India, Indian Council of Medical
Research, New Delhi p. 65-68.

Pillai, NR. and Santha Kumare.G. ( 1981 ) Hypoglycemic activity of Me/ia azasirachtalnd. J. Med. Res.
74,931-933.

7.

Ghattopadhyay, R.R. Possible mechanism of antihyperglycemic effect of Azad/rachta indicaleaf extract

part 1V. Gen. Pharmacal Col. Letrs. 27 (3) (1996) 431-434

Sonia, B. and Srinivasan, B.P. (1999) Investigations in to the Anti-diabetic activity of Azadirachta
ind/ca. Indian Journal of Pharmacology, 31, 138-141
.

Chattopadhyay,.R.R., Chattopadhyay, R.N. and Maitra, S.K. Effect of A. indica on hepatic glycogen in
rats. Ind. J. Pharmacol. 25, 174-175.

10.

Yotsumoto, T., Naitoh, T., Sakuya sT. and Tanaka,T.(1997) Effects of specific antagonists of angiotensin
II receptors and captopril on diabetic nephropathy in mice. J.Pharmacol.75, 59-64.

11.

Hussain,H.E.MA., Jamil, K. and Mala Rao. (2001) Hypoglycemic, hypolipidemic and antioxidant
properties of Tulsi (Oc/murn sanctumLinn) on strteptozotocin induced diabetes rats.lnd J. Clin. Biochem. 16
(2), 190-194.

12.

Hussain, H.EMA., Jamil, K. and Mala Rao. (2001) Preliminary studies on the hypoglycemic effect of
Abroma auEusta in alloxan diabetic rats. Ind.J.Clin.Biochem 16 (1), 77-80.

13.

Shukla,R., Anand, K., Prabhu, K.M and Murthy, PS. (1995)1 Hypolipidemic effect of water extract of
F/cusbenga/ensisin alloxan induced diabetes mellitus in rabbits. Ind. J. Clin. Biochem 14 (2), 122-124.

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