In dia n Jou rn al of Experimen tal Biology
Vo l. 4 1, June 2003, pp. 636-640
Lowering of blood sugar by water extract of Azadirachta indica and
Abroma augusta in diabetes rats
Eshrat Halim M
National C hemical Laboratory, Pune 411008, Indi a
Received 5 July 2002; revised 2 J March 2003
Combinatio n ( I: I) of water ex trac t of dri ed powder of roo t a nd leaves (200 mg/kg body wt) of A. augus/([ a nd A. indica
respec ti ve ly was ad mini stered ora ll y to a ll oxa n diabetic rats once a day for 8 weeks . Thi s treatm e nt ca used sig ni ficant
lowering of blood s uga r in fa sted as estim a ted by g lucose to le ran ce tes t. The treatme nt resu lted in a sig nifi ca nt red uc ti o n in
serum lipi ds . Aqueo us extract also decreased thc formatio n of lipid peroxides estimated as thiobarbituric ac id reac ti ve
substa nce, (TBARS) , a nd increased a nt iox id a nts (supe roxide di s mutase, catalase, g luta thi o ne peroxidase and glutathi o ne
tran sferase) in e ryt hrocy tes. There was red uction in LPO as TBARS in hea rt , li ver, kidney , and mu scles. It also prevented
decrease in body weight. Prese nt study showed that Abrollla augusta roots a nd A. il1dica leaves whe n give n toget he r as
wate r cxtrac t had hypogl ycae mi c action a nd had better effect than g ive n alonc.
Keywords : AbrOllla augusta, A zadiracllla indica, Blood suga r, Di abetes, Hy pog lycae mi a
M any herbal products have been desc ribed for the
care of diabetes mellitus in ancient literature of
Ayurveda in India. Hypoglycaemic action in animals
and humans by herbs have also been re viewed 1.2.
Artemisia herba alba is reported to have hypoglycemic effect3 . Extracts of ripe leaves, tender leaves,
fruits and fl owers of Azadirachta indica (neem) have
been reported to possess antidiabetic and antiviral
activit/·s. Effect of extract of leaves has been studied
on cardiov ascular system of anaesthetized monkey s
4
and rabbits . Neem has bee n show n to possess a
number of ph armacolog ical effects like cardiovascular, antimicrobial and immunomodulatory
action 6 . Isolated studies have reported antiinflammatory , immunostimulant a nd hypoglycaemic
effects of A. indica leaf extract. One well studied
property of neem is its hypoglycaemi c effece · ll . The
plant Abroma augusta is effective in the treatment of
di abetes and in amenorrhoea. Abromine, the active
I2
constituent, has been identified as betaine . 13 . The
leaves contain octacosanol, tarasxerol, ~-sitosterol
acetate and mixture of long chain fatty diols 13. 14.
Recently we have demonstrated the antidiabetic actilS
vity of A. augusta water e xtract . In Ayurveda extracts of more than one pl ant are also used in combination for treatment. Therefore, in the present study,
' Correspo nden t author: E- mail : prs@ems. ncl.res. in
we studied the combined effect of A. augusta and
A. indica in alloxan induced di abetic rats.
Materials and Methods
Plant material - The root bark of Abroma augusta
was obtained from Khasia Hills, Assam, India and
was identified by Nation al Botanical Research In stitute, Lucknow. Root bark (25 g) was air-dri ed a nd
gro und in an electrical mill. Freshly collected neem
leaves were air dried and powdered as menti oned
above. Equal qua ntiti es of root and leaves powder of
Abroma augusta and Azadirachta indica respectively
were mixed. Powder (25g) of individual plant or mixture of plants was well extracted by soaking in distilled water (lOOml) for two day s at 4 °C with freque nt
stirring. The extract was concentrated in a lyophilizer
and kept separately in airtight containers in a deep
freeze until use.
Animals- H ealthy adult male albino rats (25) of
Wi star strain (l00-200 g) originally obtained from
Center for Cellular and Molecular Biology, Hyderabad India, were fed on a pellet diet (Hindustan Lever
Ltd, Mumbai ) and water was provided ad libitum.
Induction of diabetes - The method of Sochor
et al. 16 was followed . Rats were starved overnight and
each rat received a s in gle subcutaneous injection o f
freshly prepared so lution of alloxan monohydrate
(20 mg/l 00 g body wt) in sodium-acetate buffer
(0.15 M ; pH 4.5). The volume to be injected was kept
HALlM : LOWERING OF BLOOD SUGAR BY EXTRACT OF AZADIRACHTA IN DICA AND ABROMA AUGUSTA
between 100-150 Ill. The same volume of acetate
buffer was given to each contro l rat. Next day a single
injection of 2 units of protamine-Zn insulin prepared
in normal saline was given to each alloxan treated rat.
Thi s procedure was continued for 6 days. It decreased
the mortality of the animals. Controls were given the
same volume of normal saline instead of insulin . Fasting blood sugar was checked every week from 7th
day . Anima ls became diabetic after one month. Animal s with fasting blood sugar> 150 mg/d l were used
as diabetic rats with or without treatment. Treatment
was started after one month when the diabetes was
well established .
Blood colleclion - The blood was collected retro
orbitally from the inner canth us of the eye using micro hematocrit capillaries, Mucaps. The blood was
collected in oxalate- sodium fluoride in Eppendorf
tubes.
Estimation (~f plasma glucose - It was estimated
by gl ucose oxidase method using the Kit from Ranbaxy Labs, New Delhi, India.
Glucose tolerance test - The standard oral glucose
tolerance test was performed on all animals before
and after 8 weeks of experiment.
Lipid peroxidation products and antioxidantsLipid peroxidation (LPO) products were estimated as
thiobarbituric acid reactive substance (TBARS) in
plasma and ti ssues 17. Among the antioxida nts, reduced glutathione was determined by the method of
Poliodoro et a1 18 . Superoxide d ismutase and catalase
was estimated by the method of Wendel 19 . 17.
Serum lipid proJile- Total cho lestero l (TC), HDL
cholesterol (HDLC) and triacy lglycerols (TG) were
estimated using standard kits of Randox, Mumbai .
LDL cholesterol (LDLC) was calculated from the
2o
above measurement by using Friedwalds form ul a .
LDLC = TC - (HDLC + TG).
Experimental design - Animals were divided into
5 groups of 5 each. Group 1 served as healthy
Tab le 2 -
controls. Group 2 were untreated diabetic rats. Group
3 diabetic rats were given water extract of A. augusta,
Group 4 diabetic rats received A. indica and Group 5
diabetic rats were treated with combination of
A. augusta and A. indica. The dose of the extract was
200mg in ml/day for each animal. Rats of Groups J
and 2 were given 4 ml of saline daily in place of pl ant
ex tracts.
Results and Discussion
It can be seen from Tab le 1 that treatment for 8
weeks with the extracts of the plants brought down
fasting plasma glucose (FPG). to norma l range. The
reduction was more (25%) when treated with combination of two plants than A. indica (43%) or A. augusta (37%) alone. Similar improvement was seen in
glucose to lerance studies also (Table2). In A. augusta
plus A. indica treated animals, the peak values (0.5 hr)
were in the normal range and by 2 hr the plasma glucose values were not on ly normal, but even lower
than the initial value. The values in the animals
treated with only one plant were in between diabetic
untreated and diabetic animals treated with both the
plants.
Results in Table 3 show that the treatment with
water extract of A. augusta and A.indica for 8 weeks
Table I - Effect of water ex tract of (200 mg I kg body wt) AbrOl/la augusta and Awdirachta indica alone or in combination on
the fasting plasma glucose level in rats*
[Values arc mean ± SD of 5 anim als]
Plasma glucose (mg/dl )
8 weeks
89.8 ± 5.2
99.4 ± 19.5
172.2 ± 15.4 b
285.6 ± 42 .6"
165.6 ± 26.0c
105.4 ± 26.6
95.8 ± 3.7
168.9 ± 28.2'
167 .9± 98.5"
80.1± 2.0
o weeks
Treatment
Normal
Di abetic Untreated
A. augusta
A. indica
A.augusta + A. illdica
*Initi al values were not show n.
"p < 0 .001, bp < 0.01, cp < 0.05
Plasma g lucose leve l in g lucose toleran ce test in normal, diabetic untreated and diabetic treated after 8 weeks
[Values are mean ± SD of 5 animal s]
o hr
Treatment
Norm al
Di abetic untrea ted
A . augusta
A. indica
A.augusta + A indica
83 .0 ± 6 .5
160.7 ± 14.3"
89.8 ± 25 .2
87.8 ± 24.5
80. 1 ± 2.0
"P<O .OO I, hp<O.OI, ' P<0.02
0.5 hr
160.5 ± 4.2
240.5 ± 77.2 b
115 .0±2 I.1 b
111.2 ± 19.6 b
92.0 ± 2.5"
637
Pl as ma glu cose (mg/dl)
I hr
145.2 ± 5.3
270.0 ± 90.0 b
116.4 ± 16.0
114.3 ± 15 .2 b
86.3 ± 3.0"
1.5 hr
110.5 ± 6.7
282 .5 ± 8 1.4"
104.0 ± 11.4
102.0 ± 10.8
84 .7 ± 2.3"
2 hr
86.3 ± 5.2
273. 1 ± 83.2"
90.5 ± 25.0
82 .3 ± 2.5
72.5 ± 3.6c
INDIAN J EXP BIOL, JUNE 2003
638
weight, reduction in total hemoglobin and sugar in
uri ne (Tab le 6). After treatment wi th water extract of
combination of two plants, total hemog lobin was
nearly normal. The body weight also increased by
15-20% in the treated animals. Experiments will be
carried out in future by treating the animals for a
longer time to find out obesity in treated an imals. The
diabetic untreated rats showed retinopathy damage,
muscles and ski n injury on tail and feet, and paw
edema (Fig. 1). All these symptoms recovered after
treatment for 8 weeks with the water extract of
combination of two plants, A. augusta and A. indica.
These observations on reversal of ret inopathy and
tissue injury effects by t~e extract needs experimenta l
evidence and diagnostic documentation. From the
improvement in FPG and glucose tolerance in the
improved serum lipid profile, whi le HOLC was
unaffected. However, after treatment with the extract
of the two plants, the values of TC and LOLC/HDLC
returned to even below the values of normal group
and HOLC values were co nsiderably higher on ly in
the group treated with both the plants. This showed
that treatment with A.augusta and A. indica improved
lipid profile better than with either plant alo ne in
diabetic animals (Tables 4 and 5). There was increase
in LPO and decrease in antioxidant enzymes in
diabetic untreated an imals. After treatme nt with plant
extracts there was an improvement in treated animals
i.e. reduction in LPO and increase antioxidant
enzymes. All the three treatments (in combination or
individual plant) were more or less equall y effective.
The diabetic untreated animals showed loss in body
Table 3 - Effect of treatment for 8 weeks with water extract (200 mg/kg b.w ) of A. augusta and A. indica a lone or in
combinat ion on plasma lipid profi le of norma l, diabetic untreated and d iabetic treated rats
[Values are mean ± SD of 5 an ima ls]
Treatment
TC (mg/d l)
LDLC (mg/dl )
Normal
Diabetic untreated
A. augusta
A. il/dica
A.augusta + A. indica
160.0 ± 14A
235.0 ± 16.0"
185 .0± 14.8
1750 ±L2
138. 1 ± 35.8
82.2 ± 8.79
165.8 ± 6.7"
99.8 ± 16.6
95 .6 ± 16.2
91.1± 13.2
HDLC (mg/dl)
46.87
48.3
51.3
48 .2
64.6
± 8.6
± 2.5
± 8A
± 7.3
± 2.9
LDLC/HDLC
!.77±0.1 3
3.83±0.19"
1.9 ± OA
2.0 ± OA
1.56 ± 0.03
TG (mg/d l)
110.6 ± 5.2
188.5 ± 55.30e
175.0 ± 15.7 b
135.0 ± 13 .2e
125.2.± 22.1 b
"p < 0.001 , hp < O.O I , "P < 0.02
Table 4 - Effect of treatment with water extract (200mg/kg b.w) of A. at1fw,ta and A. indica alone or in combination
for 8 weeks on li pid peroxidation product as TBARS (LPO) . SOD, CAT glutathione peroxidase ( GPX) and
g lutathione transferase (GST) activities in erythrocytes in no rmal. diabetic untreated and diabetic treated rats
[Values are mean ± SD of animals]
Treatment
LPO
Control
Diabetic untreated
A. augusta
A. indica
A. augusta + A. illdica
286.9 ± 4.5
406.8 ± 6.2"
173A ± 6.5"
130.1 ±40 .1 "
200A± 10.11"
SOD
CAT
380A ± 44.3
271.8 ± A. I
205.8 ± 3.0"
2 10.6± 12S
218.25 ± 45.2"
268.3 ± 11.0a
298.2 ± 6.3"
332.6 ± 2A
323 .5 ± 1.8
325.6 ± 1.5
GPX
27.7 ± 2.0
20.2.± 1.7"
30.6 ± 12.2"
27.08 ± 8.2"
32.2 ± 3.5"
GST
27.7 ± 2.0
15.0 ± 1.09"
35.2 ± 146"
33 .2 ± 5A"
26.6 ± 1.8"
Values are expressed as-SOD - Units /mg protein; CAT-nmole of H2 0 2/min/mg protein ; GPx-nmole of
glutathione oxidizedlmin/mg protein; LPO - nmole of malondialdehyde/mg protein; and GST - ~mole of H20 2
utilizedlmin/mg protein.
"p < 0.00 1.
Table 5 -
Effect of treatment with water extract of A. augusta and A. indica for 8 weeks on lipid peroxidation product
as TBARS in normal, diabetic and diabetic treated rats
[Values are mean ± SD of 5 an imals]
Treatment
Control
Diabetic untreated
A. augusta + A. indica
"p *<0.05; bp<O.OO I
Heart
OA5 ± 0.004
2. 12 ± 0.D2
0.38 ± 0.07"
Concentration of TBARS (mM/ IOOg wet tissue)
Liver
Kidney
0.90 ± 0.006
IA6 ± 0.05
0.93 ± 0.04
1.25 ± 0.06
1.78 ± 0.05
0.81 ±0. 14b
Muscle
124.30 ± 4.2
200.2 ± 6
125.3 ± 13.2 b
HALlM : LOW ER ING OF BLOOD SUGAR BY EXTRACT OF AZADIRACHTA INDICA AND ABROMA AUGUSTA
Table 6 - Effect of treatment with water ex trac t of A. augusta
and A. indica alo ne o r in co mbination on total hae mog lob in, body
wei ght and urin e sugar of normal , di abetic untreated and diabetic
treated rats
[Values are mean ± SD of 5 anim alsl
Treatment
Haemoglobin
(gIlOOml )
Body
weight (g)
A.augusta
16.0 ± 4 .2
10.5 ± 1.2
13. 6 ± 0.2
A. indica
12.9 ± 1.3
A. augusta + A. indica
14.9 ± 0.3
132.9± 15 .8
120. 1 ± 2 .1
155.0 ± 3. 1"
(+ 16%)
15 3.2 ± 4.2"
(+ 15 %)
160.6 ± 20.5 b
(+ 20%)
Norm al
Diabetic untreated
Urine sugar
+++
"p *< 0.05; bp<O.OO I
639
of retinopathy, damage to skin of tail , injury to legs
and edema of paws that recovered after treatment of
water extract of two plants in combination. In
conclusion it can be stated that the water extract of the
two plants A. augusta and A. indica, has a synergistic
effect when given in combination. They have a strong
antihyperglycaemic, anti hyperlipidemic effect and are
capable of reversing the free radical and lipid
peroxidation mediated ti ssue damage and other
complications like retinopathy , swelling of feet and
damage to skin of tail.
Acknowledgement
The author is grateful to Dr Paul Ratnasa my,
Director Nation al Chemical Laboratory , Pune, for
providing facility and constant encouragement.
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I
2
3
4
5
6
Fig. 1- Di abet ic rat suffering from re tin o pathy da mage, mu sc le
and sk in injury on tail a nd feet, and paw edema.
present study, one can presume th at either blood
insulin levels are elev ated or sensitivity to insulin
increased during glucose tolerance test. It is also quite
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tissues. Since the treatment with water extract of the
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untreated di abetic animals (Tables 4, 5) that was
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