Iranian Journal of Pharmaceutical Research (2008), 7 (1): 61-68 Copyright © 2008 by School of Pharmacy

Received: June 2006 Shaheed Beheshti University of Medical Sciences and Health Services
Accepted: September 2006

Original Article

Antidiabetic and In Vivo Antioxidant Activity of Ethanolic Extract of

Bacopa monnieri Linn. Aerial Parts: A Possible Mechanism of Action

Tirtha Ghosha*, Tapan Kumar Maityb, Pinaki Sengupta b,

Deepak Kumar Dash b and Anindya Bose a

Institute of Pharmacy and Technology, Salipur, Cuttack district, Orissa, India. bDepartment
a

of Pharmaceutical Technology, Jadavpur University, Kolkata, India.

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Abstract

Diabetes mellitus is a metabolic disorder affecting carbohydrate, fat and protein metabolism

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that affects nearly 10% of the population every year. The treatment of diabetes mellitus has been
confined to use of oral hypoglycemic agents and insulin, the former being reported to possess
serious side effects. This leads to increasing demand for herbal products with antidiabetic factor
with little side effects.
This article describes the antihyperglycaemic activity, in vivo antioxidant potential,
of
effect on glycosylation of hemoglobin and in-vitro peripheral utilisation of glucose of the
ethanolic extract of the aerial parts of Bacopa monnieri. The extract produced significant
decrease in the blood glucose level when compared with the controls in alloxan induced
hyperglycemic rats both in the single dose as well as multiple dose experiment at the tested
ive

dose level and is comparable with the standard drug glibenclamide. It was observed that
the ethanolic extract reversed the weight loss of the diabetic rats and they returned to near
normal. The extract prevented significant elevation of glycosylated hemoglobin in vitro,
with IC50 value being 11.25 µg/ml that is comparable with the reference drug α-tocopherol.
Administration of the exract and glibenclamide significantly decreased the levels of
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TBARS, increased the content of GSH and increased the activity of SOD and CAT in liver
of diabetic rats. The extract increased peripheral glucose utilisation in the diaphragm of
diabetic rats in vitro, which is comparable with the action of insulin. Thus, the extract might
have insulin like activity and the antihyperglycemic effect of the extract might be due to an
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increase in peripheral glucose consumption as well as protection against oxidative damage

in alloxanised diabetes.

Keywords: Bacopa monnieria; Diabetes; Antioxidant activity; Antihyperglycemic

activity.

Introduction diabetes mellitus is affecting nearly 10% of

the population every year (1). The treatment of
Diabetes mellitus is a metabolic disorder diabetes mellitus in clinical practice has been
affecting carbohydrate, fat and protein confined to use of oral hypoglycemic agents and
metabolism. A worldwide survey reported that insulin, the former being reported to be endowed
with characteristic profiles of serious side effects
* Corresponding author: (2). This leads to increasing demand for herbal
E-mail: tghosh75@yahoo.co.in products with antidiabetic factor with little side

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Ghosh T, Maity TK, Sengupta P, Dash DK and Bose A / IJPR (2008), 7 (1): 61-68

effects. A large number of plants have been were housed in acrylic cages in standard
recognized to be effective in the treatment of environmental conditions (25-30 °C). They were
diabetes mellitus (3). allowed free access to standard dry pellet diet
Bacopa monnieri L. (Fam. Scrophulariaceae) (Hindustan Lever, Kolkata, India) and water ad
is a creeping, glabrous, succulent herb, rooting libitum. All experiments were carried out as per
at nodes, distributed throughout India in all plain the guidelines of the Institutional Animal Ethical
districts, ascending to an altitude of 1,320 m. The Committee.
plant is reported to show sedative, antiepileptic,
vasoconstrictor and anti-inflammatory activity Drugs and chemicals used
(4). It has been reported that the plant contains Bovine serum albumin (Sigma chemical St.
tetracyclic triterpenoid saponins, bacosides A Louis, MO, USA), thiobarbituric acid, nitro blue
and B, hersaponin, alkaloids viz. herpestine tetrazolium chloride (NBT), hemoglobin (Loba
and brahmine and flavonoids (4, 5). In folklore Chemie, Mumbai, India), trichloro acetic acid
practice, some of the tribes of Orissa, India use (Merck Ltd, Mumbai, India), 5,5’-dithio bis-2-

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the decoction of the aerial parts of the plant nitrobenzoic acid (DTNB) were used. All the
for the treatment of diabetes mellitus. In the solvents were of analytical grade and purchased
present paper we report the antihyperglycaemic from local market.
activity, in vivo antioxidant potential, effect
on glycosylation of hemoglobin and in vitro
peripheral utilisation of glucose of the ethanolic
extract of the aerial parts of B. monnieri using
standard laboratory procedures.
SI Screening for antidiabetic activity
The method of Joy and Kuttan was followed
(6). The acclimatized animals were kept fasting
for 24 h with water ad libitum and injected
of
intraperitoneally a dose of 150 mg/kg of alloxan
Experimental monohydrate in normal saline. After one hour,
the animals were provided feed ad libitum. The
Plant Material blood glucose level was checked before and
The plant was identified by the taxonomists 72 h after alloxan injection. The animals were
ive

of the Botanical Survey of India, Govt. of India, considered diabetic when the blood glucose
Shibpur, Howrah, India. After authentication, level was raised beyond 300 mg/dl of blood.
fresh aerial parts of the young and matured This condition was observed at the end of 72 h
plants were collected in bulk from the rural belt after alloxan injection.
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of Salipur, Orissa, India during early summer,

washed, shade dried and then milled in to coarse Effect on oral glucose tolerance in rats
powder by a mechanical grinder. After overnight fasting, a 0-min blood sample
was taken from the tip of the tail of each rat of
Preparation of extract
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different groups under mild ether anesthesia.

The powdered plant material (400 g) was Without delay a glucose solution (2 g/kg) was
defatted with petroleum ether (60-80 °C) and administered by a gavage. Four more samples
then extracted with 1.5 litre of ethanol (95%) in were taken at 30, 60, 90 and 120 min after glucose
a soxhlet apparatus. The solvent was removed administration (7). All blood samples were
under reduced pressure, which obtained a taken for the estimation of the blood glucose.
greenish-black sticky residue (yield: 11.6% w/w Estimation of blood glucose was carried out with
with respect to dried plant material). The dried the haemoglucostrips supplied by M/s Lifescan,
extract was stored in a desiccator till further Inc. USA with the help of a Johnson & Johnson
study. ONE TOUCH blood glucometer.

Animals Used Single dose study

Wistar albino rats of either sex, weighing The animals were segregated into five
180-250 g supplied by M/s Ghose enterprises, groups of six rats in each. Group I and II rats
Kolkata, India were used. The selected animals were randomly selected from normal rats that

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 Antidiabetic and Antioxidant Activity of Bacopa monnieri Linn.

received only distilled water and the extract (300 5gm/ml in 0.01 M phosphate buffer (pH 7.4)
mg/kg, p.o.) respectively. Group III to Group V was incubated for 72h in presence of 2 g/100ml
animals were selected from the alloxanised rats. concentration of glucose in order to find out the
Group III animals served as diabetic control. best condition for hemoglobin glycosylation.
Group IV animals received glibenclamide The assay was performed by adding 1 ml of
(600 µg/kg) and group V was treated with the glucose solution, 1ml of hemoglobin solution
extract (300 mg/kg) in a similar manner. Blood and 1ml of gentamicin (20 mg/100ml) in 0.01
samples were collected from the tip of tail of M phosphate buffer (pH 7.4). The mixture
each rat under mild ether anesthesia at 0 h, 1 was incubated in dark at room temperature.
h, 2 h and 4 h after the administration of test The degree of glycosylation of hemoglobin
samples and tested for glucose concentration in presence of different concentration of
as above. the extract and their absence was measured
colorimetrically at 440 nm. α-tocopherol was
Multidose study used as standard.

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For multidose study, administration of
test samples was continued for 10 days, once Determination of peripheral consumption of
daily through oral route. Blood samples were glucose in vitro
collected from the tip of tail and the estimation
of blood glucose was carried out as above on the
1, 3, 7 and 10 day of the drug administration.
Body weights of all the animals were recorded
just prior to and on the 10th day of the study
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The method of Chattopadhyay et al. was
followed (13). Peripheral glucose consumption
was studied in rat diaphragm preparation from
animals fasted for 36 h prior to the experiment. The
animals were sacrificed by cervical dislocation
of
to determine the change in the body weight, if and the diaphragms were quickly taken out;
any. followed by dividing each diaphragm into four
pieces. The pieces of diaphragms were incubated
Determination of in vivo antioxidant activity in the nutrient solution with constant oxygenation
On the 10th day following study, the and shaking (90 cycles / min) at 37 °C for 90 min
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animals were deprived of food overnight in accordance with the procedure. The nutrient
and sacrificed by cervical dislocation. The solution with the diaphragms was aerated for 10
livers were dissected out, washed in ice-cold min and used immediately. Glucose was added
saline, patted dry and weighed. A 10 % w/v to a final concentration of 500 mg%. Each piece
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of homogenate was prepared in 0.15 M Tris- of diaphragm is incubated in 2.5 ml of glucose

HCl buffer and processed for the estimation of nutrient mixture. The results were expressed as
lipid peroxidation by the method of Fraga et al. glucose consumption per 10 mg of dry diaphragm
(8). A part of homogenate after precipitating (by subtracting glucose concentration after
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proteins with trichloro acetic acid (TCA) incubation from glucose concentration before
was used for estimation of glutathione by the incubation). The dry weight was determined
method of Ellman et al. (9). The rest of the after oven drying the diaphragm at 105 °C for
homogenate was centrifuged at 15000 rpm for 2 h.
15 min at 4 °C. The supernatant thus obtained
was used for the estimation of SOD by the Statistical analysis
method described by Kakkar et al. (10) and Statistical significance was determined
CAT activity was measured by the method of by one way analysis of variance (ANOVA)
Maehly et al. (11). followed by Dunnet’s t-test. P<0.05
indicates significant difference between
Determination of in vitro glycosylation of group means.
hemoglobin
The degree of glycosylation of hemoglobin Results and Discussion
in vitro was measured colorimetrically as
suggested by Fluckiger et al. (12). Hemoglobin, Table 1 shows the blood glucose level of

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Ghosh T, Maity TK, Sengupta P, Dash DK and Bose A / IJPR (2008), 7 (1): 61-68

Table 1. Effect of ethanolic extract of Bacopa monnieri aerial parts (300 mg/kg, p.o.) on oral glucose tolerance test (OGTT) in normal
and alloxan induced diabetic rats.
Blood sugar level (mg/dl)
Groups Treatment
Fasting 30 min 60 min 90 min 120 min
I Normal 75.00±0.77 149.83 ±2.32 176.83±2.09 125.17±2.83 80.16 ±1.83
II Normal + Extract 74.83±0.79 NS 146.50 ±1.78NS 181.17 ±2.39NS 124.17 ±3.78NS 85.67± 3.18NS

Diabetic control
III 250.33±3.10* 322.33± 4.16* 374.17±5.16* 319.33 ±3.29* 317.83 ±2.67*
(Alloxan only)

IV Diabetic+ Extract 77.50±1.50* 141.83± 2.91* 176.17 ±3.52* 127.50 ±2.80* 87.50 ±1.43*
V Diabetic+ Glibenclamide 76.50 ±2.02* 151.56± 3.45* 185.33±2.53* 126.83 ±2.46* 92.50 ±1.50*
Values are mean ± SEM for n=6; *P < 0.05 = significant; NS = Not significant; Group II and III are compared with group I while Group
IV and V are compared with group III.

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normal and experimental animals after oral of the body proteins (14). In the present study, it
administration of glucose (2 g/kg). Extract as was observed that the ethanolic extract reversed
well as standard drug treated animals showed
more significant decrease in peak blood glucose
level after 1 h. After 2 h, the extract treated
animals tended to bring the values near normal.
The results of Table 2 reveals that the extract
SI the weight loss of the diabetic rats and they
returned to near normal.
During diabetes the excess glucose
present in the blood reacts with hemoglobin
to form glycosylated hemoglobin. The rate
of
produced significant decrease in the blood of glycosylation is directly proportional to
glucose level when compared with the controls concentration of blood glucose and with
in alloxan induced hyperglycaemic rats in improvement of glycemic control glycosylated
the single dose experiment at the tested dose hemoglobin also decreases (15). Hence the
level and is comparable with the standard drug estimation of glycosylation of hemoglobin
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glibenclamide. is a well established parameter useful in the

In the multi dose study (Table 3), the test management and prognosis of the disease (16).
extract constantly maintained significant Our study gave a clear view that the ethanolic
reduction of the glucose level in diabetic rats extract prevented significant elevation of
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throughout the experimental period suggesting glycosylated hemoglobin in vitro, with IC50
the antihyperglycaemic property of the extract. value being 11.25 µg/ml that is comparable
Diabetes mellitus causes failure to use of glucose with the reference drug α- tocopherol (Table 4).
for energy that leads to increased utilization and Further, since the non-enzymatic glycosylation
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decreased storage of protein responsible for of hemoglobin is an oxidative reaction (17),

reduction of body weight essentially by depletion an antioxidant is expected to inhibit the

Table 2. Effect of single dose treatment of ethanolic extract of Bacopa monnieri aerial parts (300 mg/kg, p.o.) on blood glucose level in
normal and alloxan induced diabetic rats.
Blood glucose level (mg/dl)
Group Treatment
Basal value 1h 2h 4h
I Normal 76.33±0.71 76.17±0.65 75.83±0.95 76.17± 0.79
II Normal + Extract 76.17 ±0.87NS 75.83 ±0.70NS 75.17 ±0.75NS 74.33±0.71NS
III Diabetic control (Alloxan only) 349.67±2.95* 350.17±2.71* 349.83±2.62* 350.17± 2.79*
IV Diabetic+ Glibenclamide 343.17 ±5.12 NS
319.50±5.35* 298.83±3.91* 284.83± 3.65*
V Diabetic+ Extract 338.50 ±3.19NS 289.33±4.89* 266.83±3.37* 246.83± 3.20*
Values are mean ± SEM for n=6; *P < 0.05 = significant; NS = Not significant; Group II and III are compared with group I while Group
IV and V are compared with group III.

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 Antidiabetic and Antioxidant Activity of Bacopa monnieri Linn.

Table 3. Effect of multiple dose treatment of ethanolic extract of Bacopa monnieri aerial parts (300 mg/kg, p.o., once daily) on blood
glucose level and change in body weight after 15 days in normal and alloxan induced diabetic rats.
Blood glucose level (mg/dl) Change in body
Group Treatment
Basal value Day 1 Day 3 Day 7 Day 10 weight (g)

I Normal 76.33 ± 0.71 76.17 ± 0.48 75.83 ± 0.40 76.17 ± 0.65 76.50 ± 0.56 (+) 9.83 ± 1.47

Normal +
II 76.17 ± 0.87NS 75.00 ± 0.73NS 75.17 ± 0.54NS 74.83 ± 0.60NS 73.33 ± 0.67* (+) 10.00 ± 0.97NS
Extract

Diabetic control
III 349.67 ± 2.95* 356.83 ± 2.83* 353.83 ± 3.39* 354.33 ± 3.90* 354.17 ± 3.83* (-) 8.83 ± 0.87*
(Alloxan only)

Diabetic +
IV 343.17 ±5.12NS 264.33 ± 4.07* 235.83 ± 3.57* 219.33 ± 4.28* 205.33 ± 3.65* (+) 8.83 ± 0.98*
Glibenclamide

Diabetic +
V 338.50 ± 3.19NS 219.67 ± 3.58* 209.33 ± 3.96* 200.33 ± 2.68* 186.50 ± 1.80* (+) 9.16 ± 1.08*
Extract

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Values are mean ± SEM for n=6; *P < 0.05 = significant; NS = Not significant; Group II and III are compared with Group I while Group
IV and V are compared with Group III.

reaction. The extract is thus expected to possess

antioxidant activity.
Lipid peroixidation is one of the characteristic
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oxidative stress (21). GSH also functions as
free radical scavenger in the repair of radical
caused biological damage (22). A decrease
of
features of chronic diabetes. Alloxan gives rise was observed in GSH in liver during diabetes.
to dialuric acid, which undergoes oxidation and Administration of the extract and glibenclamide
leads to generation of O2ֹ, H2O2 and OHֹ (18). increased the content of GSH in liver of diabetic
Dialuric acid has been observed to stimulate rats (Table 5).
lipid peroxidation in vitro. In this context, a The cellular radical scavenging systems
ive

marked increase in the concentration of TBARS include the enzymes such as superoxide
was observed in liver of diabetic rats. Increased dismutase (SOD), which scavenges the
lipid peroxide concentration in the liver of superoxide ions by catalysing its dismutation
diabetic animals has already been reported (19). and catalase (CAT), a haeme enzyme which
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Administration of the exract and glibenclamide removes hydrogen peroxide (23). Therefore,
significantly decreased the levels of TBARS in reduction in the activity of these enzymes (SOD,
diabetic rats (Table 5). CAT) results in a number of deleterious effects
Glutathione (GSH), a tripeptide present in due to the accumulation of superoxide anion
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all the cells is an important antioxidant (20). radicals and hydrogen peroxide. Administration
Decreased glutathione levels in diabetes have of ethanolic extract and glibenclamide increased
been considered to be an indicator of increased the activity of SOD and catalase in diabetic rats

Table 4. Effect of ethanolic extract of Bacopa monnieri on percent inhibition of hemoglobin glycosylation in vitro.

Blood glucose level (mg/dl)

Group Treatment
Basal value 1h 2h 4h
I Normal 76.33 ± 0.71 76.17 ± 0.65 75.83 ± 0.95 76.17 ± 0.79
II Normal + Extract 76.17 ± 0.87NS 75.83 ± 0.70NS 75.17 ± 0.75NS 74.33 ± 0.71NS
III Diabetic control (Alloxan only) 349.67 ± 2.95* 350.17 ± 2.71* 349.83 ± 2.62* 350.17 ± 2.79*
IV Diabetic + Glibenclamide 343.17 ± 5.12 NS
319.50 ± 5.35* 298.83 ± 3.91* 284.83 ± 3.65*
V Diabetic + Extract 338.50 ± 3.19NS 289.33 ± 4.89* 266.83 ± 3.37* 246.83 ± 3.20*
Values are Mean ± S.D. for n=3; r = regression co-efficient.

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Ghosh T, Maity TK, Sengupta P, Dash DK and Bose A / IJPR (2008), 7 (1): 61-68

Table 5. Changes in the levels of TBARS and Reduced glutathione in liver of normal and diabetic rats.
Group Treatment TBARSa Reduced glutathioneb
I Normal 2.32 ± 0.06 58.07 ± 1.27
II Normal + Extract 2.07 ± 0.08NS 54.49 ± 1.39NS
III Diabetic 5.27 ± 0.10* 27.30 ± 1.57*
IV Diabetic + Extract 3.53 ± 0.11* 52.28 ± 0.88*
V Diabetic + Glibenclamide 4.20 ± 0.17* 48.47 ± 1.07*
a = nmole of MDA/mg of protein; b = µg/ mg of protein; values are Mean ± S.D. for n=6; Group II and III are compared with group I
while Group IV and V are compared with group III. *P < 0.05 = significant; NS = not significant.

(Table 6). consumption.

Alloxan has been found to induce free radical The ethanolic extract of B. monnieri is

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generation and cause tissue injury (24). Since reported to be rich in saponins (26). Saponins
the extract showed in vivo antioxidative activity are reported to possess antidiabetic (27) and
in normal and diabetic rats, improvement of the antioxidant activity (28). Presence of saponins in
liver tissues and the subsequent increase in the
uptake and utilisation of blood glucose might
be the mechanism of action of this extract as
antidiabetic agent.
Alloxan causes irreversible destruction
SI the ethanolic extract was confirmed through our
preliminary phytochemical screening also. Thus,
the saponins in the extract may be suspected
to possess the activity that may be attributed
to their protective action on lipid peroxidation
of
of pancreas β-cells (25). Thus, the and at the same time the enhancing effects on
antihyperglycemic activity might be due to cellular antioxidant defense contributing to
extra pancreatic mechanism. Hence, the effect the protection against oxidative damage in
of ethanolic extract of B. monnieri aerial parts alloxanised diabetes.
on peripheral consumption of glucose was
ive

investigated. The result suggests that the extract Acknowledgement

produces an antidiabetic action mediated by an
increase in peripheral glucose consumption in The authors are thankful to the management
the rat diaphragm of diabetic rats, especially at of Institute of Pharmacy and Technology, Salipur,
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a concentration of 600 µg/ml (Table 7). Insulin India and the authorities of Jadavpur University,
increased the peripheral glucose consumption Kolkata, India for providing necessary facilities
in normal and diabetic rats. Thus, the extract to carry out the research work. The authors are
might have an insulin like activity and the also thankful to the taxonomists of Botanical
Survey of India, Shibpur, Howrah, India for
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antihyperglycemic effect of the extract might

be due to an increase in peripheral glucose proper identification of the plant.

Table 6. Changes in the activity of Catalase and Superoxide dismutase in liver of normal and diabetic rats.
Groups Treatment Catalasec Superoxide dismutased
I Normal 68.71 ± 4.72 5.93 ± 0.70
II Normal + Extract 66.52 ± 4.85 NS
6.25 ± 0.79NS
III Diabetic 40.78 ± 2.4* 4.22 ± 0.55*
IV Diabetic + Extract 52.26 ± 3.51* 5.74 ± 0.57*
V Diabetic + Glibenclamide 48.65 ± 4.85* 5.31 ± 0.57*
c = µ mole of H2O2 consumed/ min / mg of protein; d = Units/ mg of protein; One unit of activity means enzyme reaction responsible for
50% inhibition of NBT per min. Values are Mean ± S.D. for n=6; Group II and III are compared with group I while Group IV and V are
compared with group III. *P < 0.05 = significant; NS = not significant

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 Antidiabetic and Antioxidant Activity of Bacopa monnieri Linn.

Table 7. Effect of ethanolic extract of Bacopa monnieri on in vitro peripheral glucose consumption in diaphragm of normal and diabetic
rats.
Glucose consumption (mg/10 mg of diaphragm dry weight)
Ethanolic extract (µg/ml) Insulin
Control
300 (µg/ml) 600 (µg/ml) 5 U/ml
Normal rats 0.47 ± 0.03 0.59 ± 0.05NS 0.68 ± 0.05* 0.82 ± 0.06*
Diabetic rats 0.49 ± 0.03 0.64 ± 0.04* 0.78 ± 0.06* 0.90 ± 0.06*
Values were expressed as Mean ± SEM for n=6. *P < 0.05 = Significant; NS = not significant when compared to control

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