Chapter 6 : Hypoglycemic test

Chapter 6 : Hypoglycemic test

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 Chapter 6 : Hypoglycemic test

6. Evaluation of hypoglycemic activity of Leaves of Grewia nervosa by

glucose tolerance test

6.1. Introduction

Diabetes mellitus is a multifactorial disease which is characterized by hyperglycemia

(Scoppola et al., 2001), lipoprotein abnormalities (Scoppola et al., 2001), raised basal
metabolic rate ( Nawata et al., 2001), defect in reactive oxygen species scavenging enzymes
(Kesavulu et al., 2000) and altered intermediary metabolism of major food substances.
Diabetes is a major degenerative disease in the world today (Ogbonnia et al., 2008), affecting
at least 150 million people and having complications which include hypertension,
atherosclerosis and microcirculatory disorders.

Yet with the tremendous scientific advances witnessed in this century medical science cannot
claim it knows all that needs to be known about this disease, including its management. At
present, the oral anti-diabetic agents belong to sulphonylureas, biguanides, α-glucosidase
inhibitors, thiazolidinediones and meglitinide derivatives.The major limitations of these drugs
are their side effects. The search for appropriate hypoglycemic agents has been focused on
plants used in traditional medicine and so far a number of hypoglycemic agents have been
derived from different plant origin.

The present study was conducted to screen the hypoglycemic effect of methanolic extract of
Grewia nervosa and its different fractions both in normal and glucose overloaded swiss
albino mice.

6.2. Principle

A glucose tolerance test (GTT) (Trinder, 1969) is one of the most acceptable methods to
evaluate the hypoglycemic activity. It is a medical test in which glucose is given and blood
samples taken afterward to determine how quickly it is cleared from the blood. The test is
usually used to test for diabetes, insulin resistance, and sometimes reactive hypoglycemia or
rarer disorders of carbohydrate metabolism. Many variations of the GTT have been devised
over the years for various purposes, with different standard doses of glucose, different routes
of administration, different intervals and durations of sampling, and various substances

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 Chapter 6 : Hypoglycemic test

measured in addition to blood glucose. At the present study, hypoglycemic effect of different
extractives of Grewia nervosa were examined & compared with relative to that of control and
standard group. Here glibenclamide was used as a standard drug.

6.3. Experimental Design

Twenty experimental animals were randomly selected and divided into four groups denoted
as group-I, group-II, group-III and group- IV consisting of 5 mice in each group. Each group
received a particular treatment i.e. control, standard and the dose of methanolic extract 400
mg/ml and 200 mg/ml respectively. Prior to any treatment, each mouse was weighed properly
and the doses of the test samples and control materials were adjusted accordingly.

Figure 6.1: Swiss Albino Mice

6.4. Preparation of Test Materials

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 Chapter 6 : Hypoglycemic test

6.4.1. Preparation of Test Samples

In order to administer the crude extract or portioning fractions at doses of 400 mg/kg body
weight 100 mg of the extract/fraction were measured and triturated unidirectional way by the
addition of small amount of Tween-80 (a suspending agent). After proper mixing of extract
and suspending agent, normal saline was slowly added. The final volume of the suspension
was made up to 3.0 ml and for 200 mg/kg body weight dose 100mg of extract/fraction were
measured and triturated unidirectional way by the addition of small amount of Tweeen-80.
After mixing the made up to 5ml by the addition of normal Saline

6.4.2. Preparation of Standard Sample

For the preparation of standard (Glibenclamide) at the dose of 10-mg/kg body weight, 10 mg
tablet was dissolved into 3.0 ml normal saline (0.9% NaCl).

6.4.3. Preparation of glucose solution

Glucose solution (10%) was collected from commercial market named 10% DA of Orion
Pharmaceutical.

Table 6.1: Test samples used in the evaluation of hypoglycemic effect of methanolic extract
of Grewia nervosa leaves

Test samples Group Purpose Dose (mg/kg) Route of

administration
1%Tween 80 & I Control 0.15 ml/10 gm Oral
DMSO in saline of body weight
Glibenclamide II Standard 10 Oral
MEL III Test Sample 400 Oral
MEL IV Test Sample 200 Oral
10% Glucose All Inducing 2 Oral
solution Glucose Level
MEL=Methanolic extract of leaves

6.5. Procedure

Twenty five fasted mice were divided into five groups of five mice each. Each groups
received treatment.

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 Chapter 6 : Hypoglycemic test

After 30 min of extract administration all groups were treated with 10% glucose solution
(2gm/kg body wt.)

After 30, 90 and 120 minutes of glucose loading, blood samples were collected from tail vein

By using glucometer blood glucose level is measured

Figure 6.3: Schematic representation for determining blood glucose level of mice after
administration of Grewia nervosa extract

0min 30 mins 1 hr 30 mins 90mins 120

mins

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 Chapter 6 : Hypoglycemic test

30 mins

Blood glucose Treatment Glucose load BG determination after glucose

load

at fasting period

Figure 6.4: Pictorial diagram of the procedure of evaluation of

hypoglycemic effect of methanolic extract of Grewia nervosa

6.6. Determination of Plasma Glucose Level

The glucose level is determined after the enzymatic oxidation in the presence of glucose
oxidase. The resultant Hydrogen peroxide (H2O2) is oxidatively coupled with 4-
aminophenazone and phenol in the presence of peroxidase (POD) to yield a red violet
quinoneimine dye as an indicator, the concentration of which is proportional to the
concentration of glucose (Trinder, 1969) and done by a glucometer named caresense from
korea)

6.7. Results and discussion

The methanolic extract of G.nervosa leaves at a dose of 200 mg/kg and 400 mg/ml body
weight were subjected to screening for hypoglycemic activity by oral glucose tolerance test.

Table 6.2: Data representing concentration of glucose in plasma (mmol/l) of mice at different
time intervals after the administration of normal saline (control) at the dose of 0.15 ml/10 gm
of body weight body weight .

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 Chapter 6 : Hypoglycemic test

Mice no 0 minute 30 minute 90 minute 120 minute

Indv. Mean Indv. Mean Indv. Mean Indv. Mean

Data data data data

M1 7.7 16 10.2 7.2

M2 6.6 13.8 8.3 7.1

M3 7.2 6.64 16.7 14.08 11.4 8.98 7.4 6.76

M3 5.1 9.8 6.1 5.6

M4 6.6 14.1 8.9 6.5

Table 6.3: Data representing concentration of glucose in plasma (mmol/l) of mice at different
time intervals after the administration of glibenclamide (standard) at the dose of 2mg/kg body
weight.

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 Chapter 6 : Hypoglycemic test

Mice no 0 minute 30 minute 90 minute 120 minute

Indv. Mean Indv. Mean Indv. Mean Indv. Mean

data data data data

M1 5.8 10.1 5 4.7

M2 5.5 9.1 4.9 4.6

M3 6.1 5.82 11.3 10.06 4.8 4.6 4.4 4.18

M3 6.8 11.9 4.4 3.7

M4 4.9 7.9 3.9 3.5

Table 6.4: Data representing concentration of glucose in plasma (mmol/l) of mice at different
time intervals after the administration of methanolic extract at the dose of 200 mg/kg body
weight

Mice no 0 minute 30 minute 90 minute 120 minute

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 Chapter 6 : Hypoglycemic test

Indv. Mean Indv. Mean Indv. Mean Indv. Mean

Data Data data data

M1 3.7 9.9 4.1 5

M2
4.7 4.72 7 8.54 4.2 4.3 4.3 4.82

M3 5.8 8.7 4.6 5.2 .

M3 4.3 9 4.5 4.5

M4 5.1 8.1 4.1 5.1

Table 6.5: Data representing concentration of glucose in plasma (mmol/l) of mice at different
time intervals after the administration of methanolic extract at the dose of 400mg/kg body
weight.

Mice no 0 minute 30 minute 90 minute 120 minute

Indv. Mean Indv. Mean Indv. Mean Indv. Mean

data Data data data

M1 3.8 11.6 5.8 3.8

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 Chapter 6 : Hypoglycemic test

M2 3.76
3.6 13.8 11.9 6.6 6.22 4.4 4.14

M3 3.9 10.3 6.3 4.2

M3 4 12.1 6.5 4

M4 3.5 11.7 5.9 4.3

Table 6.6: Hypoglycemic activity of methanolic extract of G.nervosa

Animal Group Dose mg/kg Mean ± SEM

0 minute 30 minute (% 90 minute( % of 120 minute

of change) change) (% of
change)
Control 14.08 ± 1.203 8.98 ± 0.897 6.76 ± 0.326
6.64± 0.436 (+110.92)a (-36.49) (-51.19)b
Glibenclamid 10
10.06 ± 1.62 4.6 ± 0.202 4.18 ± 0.243
e 5.82± 0.315 (+72.21)a (- 53.56)b*** (-57.72)b***
MEL 200 4.72± 0.35 8.54± 0.482 4.3 ± 0.10 4.82 ±0.17

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 Chapter 6 : Hypoglycemic test

(+86.92)a* (- 49.01)b*** (-43.06)b***

MEL 400 11.9 ± 1.25 6.22 ± 0.15 4.14 ± 0.10
3.76± 0.09 (+217.89)a (-65.48)b (-64.95)***
Each value represent the mean ± SEM, N=5. *P< 0.05, **P< 0.01, ***P< 0.001 compared to control
Dunnett's t-test after analysis. Figures in parenthese are the increase (+) or decrease (-) of blood
glucose level; a= compared to the glucose level at zero time; b= Compared to the glucose level at 30
min after glucose load.

16
conc. of glucose in blood(mmol/L)

14
12
10
8 Control
Standard
6
Me200
4 Me400
2
0
0 min 30min 90min 120min
Time

Figure 3.5 : Curves of glucose concentration in blood in different concentration in different group of
mice receiving different dose of methanolic extract.

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 Chapter 6 : Hypoglycemic test

16

Conc. of glucose in blood (mmol/L)

14
12
10
8 Control
6 Standard
Me200
4
Me400
2
0
0 min 30 min 90 min 120 min
Time

Figure 6.6 : Concentration of blood glucose showed by methanolic extract of G.nervosa in different
times.

6.7.1. Discussion

After 30 minutes of glucose load, percent of glucose level change was positive and there was
no significant reduction in blood glucose level for the methanolic extract.(Table-3.6). But
after 90 minutes of glucose load, methanolic extract showed reduction in blood glucose level
49.01% (***P<0.001) and 65.48% for 200mg/kg and 400mg/kg dose compared to 30 minute
glucose level respectively while the control itself exhibited 36.49% reduction and for
standard it was 53.56%(***P<0.001). (Table-3.6).And after 120 minute of glucose load the
percent of glucose reduction were 43.06%(***P<0.001) and 64.95% (***p<0.01) for
methanolic extract at 200mg/kg and 400mg/kg respectively compared to 30 min blood
glucose level where control Glibenclamide shows 51.19% and 57.72% reduction in blood
glucose respectively.

After 90 min and 120 min of glucose load methanolic extract shows significant
antihyperglycemic effect.

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 Chapter 6 : Hypoglycemic test

In conclusion, we can say that the methanolic extract of Grewia nervosa showed potential
antihyperglycemic actions which sustained for longer duration.

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