4.1 preliminary qualitative phytochemical study of the...

Results and Observations

128

Many plants have been used for the treatment of diabetes mellitus in Indian system of

medicine and in other ancient systems of the world, out of these only a few have been

evaluated as per modern system of medicine. From many such plants only extracts have been

prepared and their usefulness evaluated in experimental diabetes in animals. Most of them

seem to act directly on pancreas (pancreatic effect) and stimulate insulin level in blood. Some

have extra-pancreatic effect by acting directly on tissues like liver, muscle etc. and alter

favorably the activities of the regulatory enzymes of glycolysis, gluconeogenesis and other

pathways. Many of its products / chemical constituents are known to possess wide array of

medicinal properties.

This study demonstrated the antidiabetic, hypoglycemic effect and antioxidant

properties of Aqueous extract of Leaves of S. nigrum (ALSN) and Aqueous extract of Aerial

parts of M. pentaphylla (AAMP) on normal and alloxan induced diabetic rats.

4.1 Preliminary Qualitative Phytochemical study of the plant extracts

The data represented in Table- 4.01 depicted the preliminary phytochemical investigation

reports of the various extracts from S.nigrum and M.pentaphylla, indicates that the pet.ether extract

from the leaves of S.nigrum was found to contain tannins and fats and oils; where as chloroform

extract shown the presence of tannins, alkaloids, phytosterols and coumarins while the ethanol

extract was found to contain carbohydrates, poly peptides, tannins, alkaloids, phytosterols,

flavonoids, coumarin, terpenoids and steroids; and the aqueous extract of leaves of S.nigrum

(ALSN) showed the presence of carbohydrates, poly peptides, saponins, tannins, alkaloids,

flavonoids, coumarin, terpenoids, steroids as phytoconstituents but devoid of glycosides, fats and

oils.

At the same time, the pet.ether extract from the aerial parts of M.pentaphylla was found to

contain phytosterols, fats & oils; where as chloroform extract shown the presence of phytosterols,

flavonoids, coumarins and steroids; while the ethanol extract was found to contain carbohydrates,

proteins, tannins, alkaloids, flavonoids, coumarin, terpenoids and steroids; and the aqueous extract

of aerial parts of M.pentaphylla (AAMP) showed the presence of carbohydrates, glycosides,

polypeptides, saponins, tannins, alkaloids, flavonoids, terpenoids and steroids as phytoconstituents

but devoid of fats and oils.


129

TABLE – 4.01: Preliminary qualitative phytochemical analysis of the plant extracts

For various extracts of S. nigrum

Test for Carbohydrate Glycoside Polypeptides Saponins Tanins Fats and

Oils

Alkaloids Phytosterols Flavonoids Coumarin Terpenoids Steroids

Type of extract

Pet ether - - - - + + - - - - - -

Chloroform - - - - + - + + - + - -

Ethanol + - + - + - + + + + + +

Aqueous + - + + + - + - + + + +

For various extracts of M. pentaphylla

Test for Carbohydrate Glycoside Polypeptides Saponins Tanins Fats and

Oils

Alkaloids Phytosterols Flavonoids Coumarin Terpenoids Steroids

Type of extract

Pet ether - - - - - + - + - - - -

Chloroform - - - - - - - + + + - +

Ethanol + - + - + - + - + + + +

Aqueous + + + + + - + - + - + +

(+) shows the presence of constituents, (-) shows the absence of constituents.


130

SECTION - I

Hypoglycemic and Anti-Diabetic Study


131

4.2. Selection of potent extracts in reducing blood glucose level on alloxan induced

hyperglycemic acute model

4.2.1. Effects of various extracts of leaves of S. nigrum in Single dose treated alloxan

induced diabetic rats in oral route.

The perusal of Table– 4.02 shows that petroleum ether extract, chloroform extract,

ethanol extract and aqueous extract of leaves of S. nigrum registered 9.05%, 7.31%, 31.32%

and 55.13% decrease in fasting blood glucose levels respectively at the dose level of

100mg/kg b.w in oral route in alloxan induced diabetic rats at the end of 10 hours. However

at the same time solvent control (tween + water) showed almost no change in blood sugar

level. The standard drug glibenclamide (2.5mg/kg b.w.) posses 72.11 % decrease in blood

glucose level at the same time.

The alcoholic extract significantly decreases the blood sugar level at the end of 6hrs

and 8hrs in the level of p<0.01 and at the end of 10hrs in the level of p<0.001, while the

aqueous extract showed significant decrease of blood sugar level starting from 2hrs to the end

of 10hrs at the level of p<0.001, when compared with solvent control. However the standard

drug possess a significant decrease of blood sugar level beginning from 1hr (p<0.01) upto

10hrs @ p<0.001, when compared with solvent control.

The blood glucose lowering property of aqueous extract of S.nigrum is comparable

with standard drug glibenclamide (2.5mg/kg b.w.).Among the tested extracts the aq. ext. at

the dose level of 100mg/kg through oral route possess highest percentage of decrease in

blood glucose level. Therefore the aqueous extract of S.nigrum was selected for the

evaluation of hypoglycemic and anti-diabetic study.


132

4.2.2. Effects of various extracts of aerial parts of M. pentaphylla in Single dose treated

alloxan induced diabetic rats in oral route.

The data represented in Table– 4.03 shows that petroleum ether, chloroform, ethanol

and aqueous extract of aerial parts of M. pentaphylla registered 6.20%, 20.63%, 48.76% and

64.92% decrease in fasting blood glucose levels respectively at the dose level of 500mg/kg

b.w in oral route in alloxan induced diabetic rats at the end of 10 hours. However, at the same

time, solvent control (tween + water) group showed almost no change in blood sugar level.

The standard drug glibenclamide (2.5mg/kg b.w.) posses 72.11 % decrease in blood glucose

level at the same time.

The ethanolic extract significantly decreases the blood sugar level starting from 2hrs

up to the end of 10hrs in the level of p<0.001, while the aqueous extract showed significant

decrease of blood sugar level starting from 1hr to the end of 10hrs at the level of p<0.001,

when compared with solvent control. However the standard drug possess a significant

decrease of blood sugar level at 1hr with a level of p<0.01 and from 2 hrs up to the end of

10hrs @ p<0.001, when compared with solvent control.

The blood glucose lowering property of aqueous extract of aerial parts of M.

pentaphylla is comparable with standard drug glibenclamide (2.5mg/kg b.w).Among the

tested extracts the aq. ext. at the dose level of 500mg/kg through oral route possess highest

percentage of decrease in blood glucose level. Therefore the aqueous extract of M.

pentaphylla was selected for the evaluation of further hypoglycemic and anti-diabetic study.


133

TABLE – 4.02: Effects of various extracts of leaves of S. nigrum in Single dose treated alloxan induced diabetic rats in oral route.

Groups

&

Treatments

Blood Glucose Levels(mg/dl)

0 hr 1 hr 2 hrs 4 hrs 6 hrs 8 hrs 10 hrs

%age

decrease at

the end of

10hrs

I. Solvent Control

(Tween + Water) 271.66 ± 10.01 272.5 ± 5.03 282.5 ± 4.85 278.16 ± 9.16 287.33 ± 13.09 269.66 ± 14.14 268.83 ± 9.81 -

II. Glibencamide

(2.5mg/kg) 282.66 ± 2.62 225.16 ± 4.04b 176.16 ± 5.22c 114.33 ± 6.62c 105.83 ± 7.12c 88.33 ± 3.27c 78.83 ± 7.21c 72.11

III. Pet Ether Extract

(100 mg/kg) 285.16 ± 9.54 265.33 ± 11.67 275.33 ± 4.65 277.66 ± 9.79 270.83 ± 10.65 251.66 ± 11.06 259.33 ± 10.24 9.05

IV. Chloroform Extract

(100mg/kg) 273.5 ± 4.52 268.16 ± 7.21 277.66 ± 10.47 271.33 ± 8.61 276.66 ± 10.04 264.16 ± 7.54 253.5 ± 10.06 7.31

V. Ethanol Extract

(100mg/kg) 295.83 ± 6.25 274.66 ± 10.45 269.33 ± 9.40 245.33 ± 12.1a 226.33 ± 9.00b 217.66 ± 6.21b 203.16 ± 8.29c 31.32

VI. Aqueous Extract

(100mg/kg) 283.83 ± 10.08 254.33 ± 11.9 214.83 ± 8.51c 158.83 ± 7.76c 141.83 ± 10.25c 137.66 ± 10.83c 126.83 ± 7.91c 55.13

F (5,30) 1.28 4.26** 33.29** 58.95** 56.79** 61.87** 76.91** -

Values are expressed in MEAN ± S.E.M of six animals. One Way ANOVA followed by Dunnet’s t-test.

(F-value denotes statistical significance at *p<0.05, **p<0.01)

(t-value denotes statistical significance at ap<0.05,

bp<0.01 and

cp<0.001 respectively, in comparison to group-I.


134

TABLE – 4.03: Effects of various extracts of aerial parts of M. pentaphylla in Single dose treated alloxan induced diabetic rats in oral

route.

Groups

&

Treatments



%age

decrease at

the end of

10hrs

I. Solvent Control

(Tween + Water) 271.66 ± 10.01 272.5 ± 5.03 282.5 ± 4.85 278.16 ± 9.16 287.33 ± 13.09 269.66 ± 14.14 268.83 ± 9.81 -

II. Glibencamide

(2.5mg/kg) 282.66 ± 2.62 225.16 ± 4.04b 176.16 ± 5.22c 114.33 ± 6.62c 105.83 ± 7.12c 88.33 ± 3.27c 78.83 ± 7.21c 72.11

III. Pet Ether Extract

(500mg/kg) 274.16 ± 8.50 267.5 ± 6.92 268.33 ± 10.38 271.66 ± 11.87 261.66 ± 13.33 268.33 ± 12.82 257.16 ± 10.17 6.20

IV. Chloroform Extract

(500mg/kg) 268.16 ± 5.71 272.33 ± 6.68 265.16 ± 9.92 257.66 ± 11.91 246.16 ±13.16b 254.5 ± 10.01 212.83 ± 12.57 20.63

V. Ethanol Extract

(500mg/kg) 289.5 ± 5.79 256.66 ± 8.81 216.5 ± 12.94c 188.66 ± 7.30c 164.16 ± 6.26c 146.66 ±10.45c 148.33 ± 9.71c 48.76

VI. Aqueous Extract

(500mg/kg) 264.16 ± 6.74 226.83 ± 11.77c 174.16 ± 8.00c 136.66 ± 8.13c 121.16 ± 8.93c 109.83 ± 7.92c 92.66 ± 4.60c 64.92

F (5,30) 1.83 8.32** 28.62** 58.09** 51.79** 65.35** 85.11** -




bp<0.01 and



135

4.3. Acute toxicity study: (LD50 determination)

Vehicle control animals showed no mortality at any dose level, so the data associated

with vehicle treatment were not inserted in the tables.

4.3.1. LD50 determination of ALSN

Results of the ALSN treatments have been summarized in Table-4.04-a. From Fig.

4.01, LD50 was found to be log 2.966 and their antilog value is 926.12. Therefore, the LD50

value of ALSN is 926.12 mg/kg body weight. So basing upon the resulted LD50, the doses

selected for further study were 50 mg/kg b.w. and 100 mg/kg b.w.

Table- 4.04 –a:

Gr. Dose

(mg/kg)

Log dose

(x) Dead Total

Death

%

Corrected

%

Probit

(y) x

2 xy Y

I 500 2.699 0 10 0 2.5 3.04 7.284 8.204 8.477

II 750 2.875 2 10 20 20 4.16 8.265 11.96 9.608

III 1000 3 6 10 60 60 5.25 9 15.75 10.412

IV 1500 3.176 9 10 90 90 6.28 10.086 19.945 11.544

V 2000 3.301 10 10 100 100 6.96 10.896 22.974 12.348

Σx=15.051 Σy=25.69 Σ(x2)=45.533 Σ(xy)=78.835

Mean (x )

= 1.881

Mean (y)

= 3.211

The regression co-efficient,

So, putting the values in the equation, we get:

78.835 – (15.051 x 25.69)

5 78.835 – 77.33 1.505

b = = = = 6.46

(15.051)2 45.533 – 45.30 0.233

45.533 -

5

Linear regression equation: Y = y + b (x- x),

Where x and y are the mean values of x and y, ‘b’ is known as the regression co-efficient.


136

The Linear regression equation: Y = 6.6136x – 14.77, R2 = 0.9947

Corrected Formula: For the 0% dead: 100 (0.25/N)

For the 100% dead: 100/N (N-0.25), where N is the nos. of animals in

each group.

Fig. - 4.01. Graphical method of determination of LD50 value of ALSN in rats

y = 6.6136x - 14.77

R2 = 0.9947

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

6.5

7

7.5

0 0.5 1 1.5 2 2.5 3 3.5

Log dose

Prob

it

Probit (y)

Linear (Probit (y))

4.3.2. LD50 determination of AAMP

The test results presented in the Table-4.04-b denoted that, AAMP was found to

slightly toxic in the group treated with 5000 mg/kg registering the death (20%) of only two

out of ten animals. So the doses selected for further study were 250 mg/kg b.w. and 500

mg/kg b.w.

TABLE – 4.04 -b:

Group Dose (mg/kg) Route Dead/Total Death %

I 500 Oral 00/10 0

II 1000 Oral 00/10 0

III 2000 Oral 00/10 0

IV 3000 Oral 00/10 0

V 4000 Oral 00/10 0

VI 5000 Oral 02/10 20

Result: Mortalities after 48hrs were recorded as shown in the Table.


137

4.4. Effect of ALSN and AAMP on glucose loaded hyperglycemic rats (oral glucose

tolerance test)

The experimental results of the effect of aqueous extract of leaves of S. nigrum and

aqueous extract of aerial parts of M. pentaphylla in glucose loaded hyperglycemic rats are

depicted in Table 4.05. ALSN registered a reduction of 23.62% and 37.81% (p<0.01) in the

blood glucose level in dose levels of 50 mg/kg and 100 mg/kg respectively, while the

standard drug registered 47.83% with statistical significant reduction of p<0.001 at the end of

4h. However, AAMP significantly reduces the blood glucose level in both the dose levels of

250mg/kg and 500 mg/kg with a percentage reduction of 32.91% (p<0.01) and 46.43%

(p<0.001), respectively, at the end of 4 hrs. The statistical significance of one way analysis of

variance showed significant reduction of blood glucose with p<0.05 to p<0.01 starting from

1h to 4h.

4.5. Hypoglycemic activity study of ALSN and AAMP.

4.5.1. In single dose treated normoglycaemic rats

The experimental results of the effect of aqueous extract of leaves of S. nigrum and

aqueous extract of aerial parts of M. pentaphylla in normoglycemic rats are presented in

Table 4.06, shown that ALSN at both the tested dose levels of 50 mg/kg and 100 mg/kg,

reduces the blood glucose levels significantly with effect from 6h (p<0.01) till the end of 10h

(p<0.001) onwards, registering 21.13% and 39.69% reduction at the end of 10h, respectively.

While in the standard treated group, the % reduction of glucose levels calculated as 43.59%

with a statistical significance of p<0.001 at the same time. However, AAMP at dose levels of

250 mg/kg and 500 mg/kg, reduces the blood glucose levels significantly with effect from 2h

(p<0.05) till the end of 10h (p<0.001) onwards, registering 25.61% and 41.48% reduction at

the end of 10h, respectively. The results of the normoglycemic model showed that both the

test extracts have dose dependent hypoglycemic effect as supported by one way ANOVA

(p<0.05 to p<0.01).

4.5.2. In multi dose (30 days) treated normoglycaemic rats

The results of effects of ALSN and AAMP on blood sugar levels of normoglycemic

rats are illustrated in Table 4.07. The test result indicates that, in animals treated with 50 and

100 mg/kg of ALSN, there is a significant reduction (p<0.05 to p<0.01) in blood glucose level

from 15th

day onwards, and registered 21.7 and 33.9% reduction at the end of 30 days.

However the standard drug glibenclamide at the same day reduces the blood glucose 33.88%

with p<0.001, when compared with solvent control group. While in animals treated AAMP

with the dose levels of 250 and 500 mg/kg, there is a significant reduction (p<0.001) in blood

glucose level from 15th

day onwards, and registered 38.3 and 49.2 % fall, at the end of 30

days. The statistical significance of one way ANOVA showed significant reduction of blood

glucose with p<0.01 from 15th

day onwards within the groups.


138

TABLE – 4.05: Effects of Single dose treatment of ALSN and AAMP in Glucose loaded hyperglycemic rats in oral route.

Groups

&

Treatments


0 hr 0.5 hr 1 hr 2 hrs 4 hrs %age decrease at

the end of 10hrs

I. Solvent Control

(Tween + Water) 144.16 ± 5.06 135.16 ± 4.65 129.16 ± 2.88 121.5 ± 1.72 113.5 ± 2.47 21.26

II. Glibencamide

(2.5mg/kg) 157.83 ± 6.53 134.66 ± 5.25 117.00 ± 4.57 95.16 ± 2.03c 82.33 ± 2.31c 47.83

III. ALSN (50mg/kg) 151.66 ± 3.84 139.66 ± 3.11 122.33 ± 5.37 116.5 ± 5.03 115.83 ± 2.22 23.62

IV. ALSN (100mg/kg) 158.66 ± 5.73 136.5 ± 5.90 116.33 ± 5.25 105.5 ± 4.25b 98.66 ± 3.56b 37.81

V. AAMP (250mg/kg) 148.33 ± 9.71 131.33 ± 6.69 112.66 ± 3.30a 99.5 ± 1.54b 99.5 ± 1.54b 32.91

VI. AAMP (500mg/kg) 142.83 ± 2.65 128.16 ± 4.16 106.16 ± 5.74b 90.66 ± 3.62c 76.5 ± 4.42c 46.43

F (5,30) 1.24 0.62 2.89* 13.37** 29.76**




bp<0.01 and



139

TABLE – 4.06: Effect of ALSN and AAMP in Single dose treated on Normoglycemic rats in oral route.

Groups

&

Treatments



%age

decrease at

the end of

10hrs

I. Solvent Control

(Tween + Water) 103.5 ± 2.71 101.5 ± 3.88 104.16 ± 4.33 102.66 ± 4.26 102.83 ± 3.23 104.66 ± 3.04 103.16 ± 2.84 -

II. Glibencamide

(2.5mg/kg) 101.33 ± 6.45 99.5 ± 5.77 94.66 ± 4.53 89.33 ± 3.63a 79.16 ± 2.56b 67.33 ± 2.90c 57.16 ± 3.26c 43.59

III. ALSN (50mg/kg) 108.83 ± 4.03 106.16 ± 3.91 105. 83 ± 3.57 98.83 ± 4.05 95.16 ± 4.33 87.33 ± 3.02b 85.83 ± 3.04b 21.13

IV. ALSN (100mg/kg) 98.66 ± 3.56 96.83 ± 3.16 94.16 ± 2.61 91.16 ± 2.84 87.66 ± 3.01b 80.33 ± 3.63c 59.5 ± 3.21c 39.69

V. AAMP

(250mg/kg) 95.66 ± 2.52 92.83 ± 2.31 88.83 ± 2.86a 87.33 ± 5.10a 82.16 ± 5.44b 73.83 ± 2.18c 71.16 ± 2.78c 25.61

VI. AAMP (500mg/kg) 105.66 ± 7.23 102.16 ± 6.85 99.33 ± 5.70 85.83 ± 4.47a 79.66 ± 4.06b 64.50 ± 3.08c 61.83 ± 2.88c 41.48

F (5,30) 0.99 1.01 2.53* 2.60* 5.96** 24.30** 24.76**




bp<0.01 and



140

TABLE – 4.07: Effect of ALSN and AAMP in Multi- dose treated on Normoglycemic rats in oral route.

Groups

&

Treatments


0th

day 5th

day 10th

day 15th

day 20th

day 25th

day 30th

day

%age

decrease at

the end of 30th

day

I. Solvent Control

(Tween + Water) 99.5 ± 4.80 98.66 ± 4.66 96.5 ± 4.77 101.16 ± 4.04 96.83 ± 4.15 97.83 ± 5.60 102.16 ± 4.90

II. Glibencamide

(2.5mg/kg/day) 92.5 ± 2.68 86.83 ± 2.75 79.83 ± 2.95c 59.5 ± 2.77c 62.33 ± 2.33c 56.16 ± 2.68c 61.16 ± 3.29c 33.88

III. ALSN

(50mg/kg/day) 101.83 ± 4.20 98.66 ± 4.98 93.33 ± 5.37 89.5 ± 4.88 88.16 ± 4.53 84.66 ± 4.74 79.66 ± 6.50a 21.77

IV. ALSN

(100mg/kg/day) 87.5 ± 6.41 85.16 ± 5.22 81.5 ± 6.30 76.66 ± 5.45b 74.33 ± 3.26b 72.83 ± 6.26b 57.83 ± 6.60b 33.90

V. AAMP

(250mg/kg/day) 92.66 ± 4.13 89.83 ± 4.22 84.16 ± 4.71 71. 16 ± 3.29c 67.5 ± 4.51c 60.33 ± 3.44c 57.16 ± 4.64c 38.31

VI. AAMP

(500mg/kg/day) 91.5 ± 6.48 89.16 ± 6.80 82.16 ± 5.67 66.66 ± 3.62c 62.66 ± 4.32c 57.66 ± 5.15c 46.5 ± 4.46c 49.18

F (5,30) 1.16 1.42 1.86 14.00** 13.13** 12.27** 11.49**




bp<0.01 and



141

4.6. Anti-diabetic activity study of ALSN and AAMP.

4.6.1. In single dose treated alloxan induced hyperglycemic rats

The perusal of Table 4.08 concerning with the results of the antidiabetic activity in

alloxan induced diabetic rats, showed that ALSN in both dose levels (50 and 100 mg/kg),

reduces the blood glucose significantly, starting from 2h (p<0.01) to the end of 10h (p<0.001)

of the study in a dose dependent manner, while the standard drug, glibenclamide showed

similar effect during the course of the experiment. AAMP in both dose levels (250 and 500

mg/kg), significantly reduces the blood glucose levels, starting from 1h (p<0.01) to the end of

10h (p<0.001) of the study in dose dependent manner too. However, the percent decrease of

blood sugar at the end of 10h calculated as 46 to 55% for ALSN, 55 to 65% for AAMP, while

standard drug showed 72% at the same time. The statistical significance of one way ANOVA

showed significant reduction of blood glucose @ p<0.01 starting from 1 hr up to the end of

10 hr within the groups.

4.6.2. In multi dose treated alloxan induced hyperglycemic rats

The study results depicted in Table 4.09 reveals that, the extract ALSN reduces the

blood glucose level to an extent of 46.49% and 59.23% at 50mg/kg and 100mg/kg body

weight respectively at the end of the 30th

day of the study. However the individual data for

ALSN treated group shows a statistical significance ranges between p<0.05 to p<0.001,

throughout the experiment when compared with solvent control and analysis of variance

registered p<0.01 level of significance. In case of groups treated with AAMP, the extract

reduces the blood glucose level to an extent of 51.14% and 65.86% at 250mg/kg and

500mg/kg dose level respectively at the end of the 30 day of the study, where as the standard

drug glibenclamide registered 66.79% of reduction at the same day of the study. However the

individual data for AAMP treated group shows a statistical significance ranges between

p<0.01 to p<0.001 throughout the experimental result when compared with solvent control;

while analysis of variance registered p-value less than 0.01 from 5th

day onwards till 30 the

day of treatment.

So, in the above glucose lowering models, AAMP was found to be significantly more

potent in reducing the blood glucose levels than that of ALSN, in the acute and sub-acute

models of normal and alloxan induced diabetic as well as glucose loaded hyperglycemic

animals.


142

TABLE – 4.08: Anti-diabetic activity of ALSN and AAMP in Single dose treated on Alloxan induced diabetic rats in oral route.

Groups

&

Treatments



%age

decrease at

the end of

10hrs

I. Solvent Control

(Tween + Water) 271.66 ± 10.01 272.5 ± 5.03 282.5 ± 4.85 278.16 ± 9.16 287.33 ± 13.09 269.66 ± 14.14 268.83 ± 9.81

II. Glibencamide

(2.5mg/kg) 282.66 ± 2.62 225.16 ± 4.04b 176.16 ± 5.22c 114.33 ± 6.62c 105.83 ± 7.12c 88.33 ± 3.27c 78.83 ± 7.21c 72.11

III. ALSN (50mg/kg) 266.26 ± 9.15 257.16 ± 7.65 243.83 ± 6.96b 178.66 ± 10.93c 159.33 ± 7.63c 146.33 ± 6.25c 141.5 ± 5.47c 46.85

IV. ALSN (100mg/kg) 283.83 ± 10.08 254.33 ± 11.9 214.83 ± 8.51c 158.83 ± 7.76c 141.83 ± 10.25c 137.66 ± 10.83c 126.83 ± 7.91c 55.31

V. AAMP (250mg/kg) 269.66 ±10.89 253.83 ± 12.53 229.66 ± 8.46c 169.66 ± 9.96c 152.16 ± 9.62c 141.66 ± 8.86c 119.83 ± 5.43c 55.56

VI. AAMP (500mg/kg) 264.16 ± 6.74 226.83 ±11.77b 174.16 ± 8.00c 136.66 ± 8.13c 121.16 ± 8.93c 109.83 ± 7.92c 92.66 ± 4.60c 64.92

F (5,30) 0.91 3.84** 33.61** 40.73** 45.22** 47.08** 95.18**




bp<0.01 and



143

TABLE – 4.09: Antidiabetic activity of ALSN and AAMP in Multi- dose treated in Alloxan induced diabetic rats in oral route.

Groups

&

Treatments


0th

day 5th

day 10th

day 15th

day 20th

day 25th

day 30th

day

%age

decrease at

the end of 30th

day

I. Solvent Control

(Tween + Water) 285.66 ± 12.71 279.83 ± 11.81 257.66 ±11.67 246.83 ±11.80 239.33 ±10.39 235.83 ± 9.76 231.83 ± 6.30 -

II. Glibencamide

(2.5mg/kg/day) 296.66 ± 13.07 199.66 ±10.21c 138.16 ±10.33c 116.83 ± 6.60c 115.16 ± 5.24c 103.83 ± 7.46c 98.5 ± 6.82c 66.79

III. ALSN

(50mg/kg/day) 278.16 ± 10.51 248.16 ±13.33 210.66 ±11.83a 178.16 ±12.95b 172.33 ±10.84c 157.5 ± 7.39c 148.83 ± 7.17c 46.49

IV. ALSN

(100mg/kg/day) 272.66 ± 13.92 208.16 ±13.53b 157.16 ±12.61c 138.33 ±10.09c 129.33 ± 9.18c 126.33 ± 9.61c 111.16 ± 7.35c 59.23

V. AAMP

(250mg/kg/day) 269.83 ± 10.70 221.66 ± 9.17b 173.83 ±12.17c 151.5 ±10.92c 142.16 ±12.83c 133.33 ±11.26c 131.83 ± 8.71c 51.14

VI. AAMP

(500mg/kg/day) 275.83 ± 9.95 192.83 ± 7.98c 149.5 ±10.25c 128.83 ±7.96c 116.83 ± 5.53c 106.66 ± 7.94c 94.16 ± 4.90c 65.86

F (5,30) 0.68 8.79** 15.38** 21.23** 25.31** 29.60** 52.30** -




bp<0.01 and



144

4.7. Effect of ALSN and AAMP on the Urine Sugar of Multi-dose treated diabetic rats

The perusal of Table-4.10 indicates that there was ameliorative changes occurred in

the urine sugar of the animals treated with the standard drug and the extracts when measured

on 0th

, 10th

, 20th

and 30th

day of treatment. All the groups showed the presence of 0.75% of

urine sugar on the 0th

day, however, the diabetic control group animals showed the presence

of more than 1% of sugar in urine starting from the 10th

day till the end of the 30th

day. ALSN

at the dose levels of 50mg/kg & 100mg/kg b.w could able to reduce the amount of sugar in

urine to 0.5% and 0.25% respectively at the end of 30th

day; and AAMP at both the dose

levels (250mg/kg and 500mg/kg b.w), registered a decrease in the urine sugar up to 0.25% in

the treated animals at the same time, while the standard drug glibenclamide at the tested dose

level decreased the urine sugar of the animals up to a level of zero % at the end of the

treatment day.

4.8. Effect of ALSN and AAMP on Peripheral Glucose-uptake by isolated rat hemi-

diaphragm

The results of study on glucose uptake by isolated rat hemidiaphragm are shown in

Table 4.11, which reveals that ALSN at 50 mg/ml and 100mg/ml concentration exhibited the

glucose uptake of 3.71 and 4.73 mg/g/30min (p<0.05) respectively, by the isolated rat hemi-

diaphragm, while the groups treated with AAMP at 250 mg/ml and 500mg/ml concentration

registered the uptake of 5.34 and 6.81 mg/g/30min, with statistical significance of p<0.05 and

p<0.001 respectively, when compared with diabetic control group. At the same time, the

group treated with only insulin, showed 6.36mg/g/30min of the glucose uptake with a

statistical significance of p<0.001. However, insulin and ALSN (50 mg/ml and 100mg/ml)

combination respond to significant (p<0.001) increase of 6.33 and 7.16mg/g uptake of

glucose at the same time when compared with diabetic control group; Insulin and AAMP (250

mg/ml and 500mg/ml) combination respond to significant (p<0.001) increase of 7.23 and

8.45 mg/g uptake of glucose at the same time. The extent of glucose uptake differ

significantly ranges from p<0.05 to p<0.001 when compared with diabetic control group,

which showed 3.21 mg/g glucose uptake within 30 min and the F-value showed the statistical

significance of p<0.01 among the groups.


145

TABLE – 4.10: Effect of ALSN and AAMP on Urine Sugar in Multi- dose treated in Alloxan induced diabetic rats.

Groups

&

Treatments

Urine Sugar

0th

day 10th

day 20th

day 30th

day

I. Normal Control _ _ _ _

II. Diabetic Control

(Tween + Water) + + + + + + + + + + + + + + +

III. Glibencamide (2.5mg/kg/day) + + + + + -

IV. ALSN (50mg/kg/day) + + + + + + + + + +

V. ALSN (100mg/kg/day) + + + + + + + +

VI. AAMP (250mg/kg/day) + + + + + + + +

VII. AAMP (500mg/kg/day) + + + + + + +

Urine Sugar: (+) indicates 0.25 %, (+ +) indicates 0.50 %, (+ + +) indicates 0.75 % and (+ + + +) indicates more than 1 % of Sugar.


146

TABLE – 4.11: Effect of ALSN and AAMP on Glucose-uptake by isolated rat hemi-diaphragm.

Groups and Treatments

(Incubation medium) Glucose uptake (mg/g/30 min)

I. Tyrode solution with Glucose (2 g%) – Diabetic Control 3.21 ± 0.18

II. Tyrode solution with Glucose (2 g%) + Insulin (0.25 IU/ml) 6.36± 0.62c

III. Tyrode solution with glucose (2 g%) + ALSN (50 mg/ml) 3.71 ± 0.55

IV. Tyrode solution with glucose (2 g%) + ALSN (100 mg/ml) 4.73 ± 0.26a

V. Tyrode solution with Glucose (2 g%) + AAMP (250 mg/ml) 5.34 ± 0.45a

VI. Tyrode solution with Glucose (2 g%) + AAMP (500 mg/ml) 6.81 ± 0.30c

VII. Tyrode solution with glucose (2 g%) + Insulin (0.25 IU/ml + ALSN (50 mg/ml) 6.33 ± 0.39c

VIII. Tyrode solution with Glucose (2 g%) + Insulin (0.25 IU/ml + ALSN (100 mg/ml) 7.16 ± 0.42c

IX. Tyrode solution with Glucose (2 g%) + Insulin (0.25 IU/ml + AAMP (250 mg/ml) 7.23 ± 0.39c

X. Tyrode solution with Glucose (2 g%) + Insulin (0.25 IU/ml + AAMP (500 mg/ml) 8.45 ± 0.45c

F (9, 50) 21.47**




bp<0.01 and



147

4.9. Effect of ALSN and AAMP Glycogen concentration in liver and kidney

Glycogen content in liver and kidney decreased to a greater extent in diabetic control

compared to normal control (Table 4.12). ALSN at the dose levels of 50mg/kg & 100mg/kg

b.w, at the end of the 30th

day of experiment, elevate the glycogen content of liver up to 23.96

and 31.23 mg/g (p<0.01) and kidney glycogen up to 12.16 and 16.41 mg/g (p<0.05) of the

tissue respectively, while the standard drug glibenclamide at the same time registered

glycogen content of 35.91 and 20.38 mg/g for liver and kidney with the statistical

significance of p<0.001 and p<0.01 respectively. However the statistical analysis shows

significant rise of glycogen content by ALSN while compared to diabetic control group in

dose levels of 100mg/kg body weight only. On the other hand, AAMP at tested dose levels

(250mg/kg and 500mg/kg b.w), at the end of the 30th

day of experiment, showed more

potency than ALSN by significantly elevating the glycogen content of liver up to 29.12 and

34.65 mg/g (p<0.05 and p<0.001) and kidney glycogen up to 16.64 and 19.43 mg/g (p<0.05

to p<0.01) of the tissue, respectively, while compared to diabetic control group. The one way

analysis of variance shows significance in both the test groups ranging from p<0.05 in kidney

glycogen to p<0.01 in liver glycogen.

4.10. Effect of ALSN and AAMP Plasma Insulin levels in multi-dose treated diabetic rats

The results of the test depicted in Table 4.13. Both the extracts, ALSN & AAMP, at the

tested dose levels could significantly (p<0.001) increase insulin concentrations in a

progressive manner in diabetic treated rats when compared with diabetic control. The ALSN

at the dose level of 50 and 100 mg/kg recorded a maximum increase in insulin concentration

of 103.5 and 117.66 µU/ml respectively on 30th

day; While the AAMP at 250 and 500 mg/kg

dose levels, recorded a maximum increase in insulin concentration of 126.5 and 156.16

µU/ml respectively on 20th

day. On the other hand, glibenclamide showed maximum plasma

insulin concentration of 181.83 µU/ml at the end of 20th

day, while on 30th

day it registered

119.66 µU/ml of plasma insulin with statistical significance of p<0.001. The one way

ANOVA showed significance of p<0.01 among the groups starting from 5th

day till the end of

30th

day of treatment.


148

TABLE – 4.12: Effect of ALSN and AAMP on the Glycogen concentration in liver and kidney (at the end of 30th

day of treatment)

Groups and Treatments Liver Glycogen (mg/gm tissue) Kidney Glycogen (mg/gm tissue)

I. Normal Control 37.41 ± 2.63 21.05 ± 2.37


(Tween + Water) 18.88 ± 1.84

c 9.10 ± 1.14

b

III. Glibencamide (2.5mg/kg/day) 35.91 ± 2.57c 20.38 ± 2.38

b

IV. ALSN (50mg/kg/day) 23.96 ± 2.70 12.16 ± 2.28

V. ALSN (100mg/kg/day) 31.23 ± 2.49b 16.41 ± 2.35

a

VI. AAMP (250mg/kg/day) 29.12 ± 1.98a 16.64 ± 2.91

a

VII. AAMP (500mg/kg/day) 34.65 ± 1.52c 19.43 ± 2.29

b

F (6, 35) 8.67** 3.70*


(F-value denotes statistical significance at *p<0.05, **p<0.01), (t-value denotes statistical significance at ap<0.05,

bp<0.01 and

cp<0.001

respectively, in comparison of Gr-II with Gr-I and Gr-III to Gr-VII with Gr-II.)


149

TABLE – 4.13: Effect of ALSN and AAMP on Plasma Insulin Levels of diabetic rats


Plasma Insulin (µU/ml)

0th

day 5th

day 10th

day 20th

day 30th

day

I. Diabetic Control 21.66 ± 3.75 23.33 ± 4.79 21.25 ± 2.80 26.66 ± 3.44 23.83 ± 2.73

II. Glibencamide (2.5mg/kg/day) 33.83 ± 3.87a 74.16 ± 7.00c 169.83 ± 11.71c 181.83 ± 9.28c 119.66 ± 7.64c

III. ALSN (50mg/kg/day) 23.18 ± 4.16 43.13 ± 2.41 78.83 ± 7.33 c 91.66 ± 8.25c 103.5 ± 8.31c

IV. ALSN (100mg/kg/day) 30. 83 ± 4.55 59.66 ± 5.13c 112.33 ± 8.99c 114.5 ± 7.92c 117.66 ± 7.2c

V. AAMP (250mg/kg/day) 24.16 ± 3.43 56.83 ± 4.97b 89.66 ± 7.56c 126.5 ± 9.12c 113.33 ± 9.42c

VI. AAMP (500mg/kg/day) 31.66 ± 2.45 79.83 ± 6.74c 144.5 ± 12.43c 156.16 ± 10.15c 117.83 ± 6.21c

F (5,30) 2.06 17.94** 44.26** 70.09** 51.49**



bp<0.01 and

cp<0.001 respectively, in comparison to Gr-I.)

(F-value denotes statistical significance at *p<0.05, **p<0.01).


150

4.11. Study of the effect of the extracts on Beta cell degranulation score in multi-dose

treated diabetic rat pancreas

The perusal of Table 4.14 reveals that both glibenclamide and the test extracts

produced degranulation and loss of immunostainable insulin content of islet of beta cells. The

degree of depletion was initiated in the standard and extract treated groups in 5th

day post

treatment with a depletion of 25%. The degranulation was increased progressively with time

showing a maximum of 50% and 75% depletion of the beta cells in the ALSN treated groups

at the end of 30th

day of study at dose levels of 50 mg/kg and 100mg/kg b.w, respectively,

where as no such degranulation was observed in the diabetic control group; while AAMP

showed a consistent increase in the degranulation profile exhibiting maximum of 50% and

75% degree of depletion of the beta cells at the dose levels of 250mg/kg and 500 mg/kg b.w

respectively at the end of the study, where as the standard treated groups showed a maximum

of 75% degranulation of the beta cells starting from 20th

day onwards.

TABLE – 4.14: Effect of ALSN and AAMP on Beta cell degranulation score.


Beta cell degranulation score

0th

day 5th

day 10th

day 20th

day 30th

day

I. Diabetic Control 0 0 0 0 0

II. Glibencamide (2.5mg/kg/day) 0 + ++ +++ +++

III. ALSN (50mg/kg/day) 0 0 + ++ ++

IV. ALSN (100mg/kg/day) 0 + ++ ++ +++

V. AAMP (250mg/kg/day) 0 + + ++ ++

VI. AAMP (500mg/kg/day) 0 + ++ ++ +++

0 = Normal granularity, + = About 25 % of cells are degranulated,

++ = About 50 % of cells are degranulated, +++ = About 75 % of cells are

degranulated, ++++ = Almost all the are cells degranulated.

At least 3 randomly selected sections from each of the treatment group were used for

scoring


151

4.12. Sub –acute study of ALSN and AAMP on the diabetic rats at the end of 30th

day of

treatment.

4.12.1. Study of the effect of ALSN and AAMP on Serum lipid profile.

The Table 4.15 illustrates the levels of lipid profile such as total lipids, phospholipids,

total cholesterol, triglycerides, HDL, LDL, VLDL and free fatty acids. The diabetic control

group rats registered significant (p<0.001) high levels of all the parameters with marked

decrease in the HDL levels (p<0.05) when compared with that of the normal control group.

Both the extracts at the tested dose levels showed a dose dependent and significant (P < 0.05

to p<0.001) reduction in total lipids, triglycerides, LDL, VLDL and free fatty acids, however

a marked decrease in the levels of total cholesterol and phospholipids were also recorded in

the extract treated groups when compared to diabetic control group. The elevations in HDL

levels were approaching almost normal values when compared to normal control group. The

one way analysis of variance showed a significancy of p<0.01 within the tested groups in all

experimental models.

4.12.2. Study of the effect of ALSN and AAMP on levels of Serum Enzymes, Total

Proteins, Total Bilirubin, Direct Bilirubin, Albumin and Globulin in multi-dose treated

diabetic rat.

From the Table-4.16, the data showed ASAT, ALAT and ALP enzyme activities of

alloxan induced diabetic rats showed significantly higher than normal rats. Both the extract

treated rats had significantly (p<0.05 to p<0.001) reduced enzymatic activities when

compared with that of the diabetic control rats especially at higher tested dose levels, while

the standard drug glibenclamide also registered a significant (p<0.001) decrease at the same

time.

The total protein, albumin and globulin contents in serum were also markedly lowered

in diabetic rats when compared with solvent control, but in the drug treated diabetic animals

it returned to nearly normal. The diabetic rats showed an increase in total and direct bilirubin

contents in serum than that of normal group, but in standard and extract treated diabetic

animals the level they approaches to almost normal. However, in all experiments, AAMP at

250mg/kg and 500 mg/kg b.w registered more potency in altering the activities of the tested

parameters than that of ALSN at 50 mg/kg and 100mg/kg b.w respectively. The one way

analysis of variance showed a significancy of p<0.01 within the tested groups of ASAT,

ALAT and ALP models and p<0.05 within the tested groups of direct bilirubin and globulin

models.


152

TABLE – 4.15: Effect of ALSN and AAMP on Serum Lipid profile.


Serum Lipid profile

Total

Lipids

(mg/dl)

Total

Cholesterol

(mg/dl)

Phospholipids

(mg/dl)

Triglycerides

(mg/dl)

HDL

(mg/dl)

LDL

(mg/dl)

VLDL

(mg/dl)

Free

Fatty

Acids

(mg/dl)

I. Normal Control 113.66

± 7.76

75.65

± 5.90

105.5

± 8.10

62.08

± 6.93 51.5 ± 4.68 11.73 ± 1.86

12.42

± 1.40

412.41

± 77.47


(Tween + Water) 393.16

± 23.54c

188.16

± 16.15c

212.91

± 15.42b

189.08

± 20.19c 32.66 ± 3.43a

117.68 ±

6.21b 37.82 ± 1.94c

1324.58

± 137.14c

III. Glibencamide

(2.5mg/kg/day) 141.83

± 17.37c

96.75

± 10.31b

127.75

± 13.40b

78.33

± 6.44c 54.11 ± 6.11b

26.98

± 3.35b

15.66

± 1.84c

675.41

± 80.69c

IV. ALSN (50mg/kg/day) 221.5

± 15.16c

166.41

± 15.99

189.5

± 18.83

113.83

± 10.25b 32.66 ± 4.49 110.98 ± 6.23

22.77

± 2.34b

917.08

± 70.60b

V. ALSN (100mg/kg/day) 216.83

± 28.62c

147.06

± 21.36

173.33

±15.37

97.83

± 13.67c 44.66 ± 3.36

82.83

± 2.84a

19.57

± 2.42c

796.33

± 56.85b

VI. AAMP

(250mg/kg/day) 186.33

± 24.93c

153.75

± 23.06

157.58

± 24.60a

95.5

± 10.99c 46.5 ± 5.35

88.15

± 5.87a 19.1 ± 1.80c

892.25

± 58.01b

VII. AAMP

(500mg/kg/day) 159.83

± 17.82c

117.33

± 13.85b

136.5

± 14.93b

67.58

± 7.64c 56.41 ± 4.81b

47.4

± 5.95b

13.52

± 2.50c

763.08

± 69.39c

F (6, 35) 20.28** 6.09** 5.13** 13.37** 4.23** 4.37** 22.47** 11.30**



bp<0.01 and

cp<0.001



153

TABLE–4.16: Effect of ALSN and AAMP on Serum Biochemical parameters


Serum Biochemical parameters

ASAT

(u/l)

ALAT

(u/l)

ALP

(u/l)

TB

(mg/dl)

DB

(mg/dl)

Albumin

(gm/dl)

Total

Protein

(gm/dl)

Globulin

(gm/dl)

I. Normal Control 22.83 ± 2.42

28.91 ± 2.41

105.38 ± 11.12

0.91 ± 0.14 0.25 ± 0.07 3.71 ± 1.00 6.61 ± 0.64 2.9 ± 0.34


(Tween + Water) 42.08

± 2.80c 57.16

± 4.14c 258.5

± 15.64c 1.55 ± 0.38a 0.31 ± 0.03 3.37 ± 1.20 4.38 ± 0.68 1.01 ± 0.25a

III. Glibencamide

(2.5mg/kg/day) 24.2

± 3.14c 29.41

± 3.58C 135.25

± 13.15c 0.74 ± 0.12a 0.22 ± 0.01 4.69 ± 1.01 5.95 ± 0.71 1.26 ± 0.26

IV. ALSN (50mg/kg/day) 39.75 ± 2.50

42.41 ± 4.08a

143.16 ± 10.84c

1.30 ± 0.11 0.41 ± 0.04 3.53 ± 1.15 4.26 ± 0.69 0.73 ± 0.13

V. ALSN (100mg/kg/day) 32.25

± 2.30a 40.78

± 4.12b 138.91

± 11.01c 1.22 ± 0.17 0.38 ± 0.04 3.70 ± 1.15 4.81 ± 0.93 1.11 ± 0.24

VI. AAMP (250mg/kg/day) 34.11 ± 2.44

32.91 ± 2.73c

148.16 ± 9.39c

1.20 ± 0.15 0.41 ± 0.04 4.43 ± 0.84 5.28 ± 0.78 0.85 ± 0.17

VII. AAMP (500mg/kg/day) 27.41

± 2.42b 32.41

± 1.46c 141.45 ± 5.98c

1.01 ± 0.08 0.21 ± 0.03 3.85 ± 0.58 5.43 ± 0.72 1.58 ± 0.24

F (6, 35) 8.22** 8.94** 18.27** 1.92 3.93* 0.33 1.29 3.08*



bp<0.01 and

cp<0.001



154

4.12.3. Study of the effect of ALSN and AAMP on the activities of Glucose-6-

Phosphatase, Hexokinase, HMG CoA reductase & Arginase in the Livers of

experimented rats.

The Table 4.17 presents the levels of Glucose-6-Phosphatase, Hexokinase, HMG

CoA reductase & Arginase in the livers of the normal & 30 days drug treated diabetic rats.

The diabetic control group rats registered significant (p<0.01 to p<0.001) increase in the

levels of all the parameters except Hexokinase which showed significant decrease (p<0.001)

when compared with that of the normal control group. The groups treated with Insulin,

glibenclamide, *ALSN and *AAMP at the tested dose levels, showed a *dose dependent and

significant (P < 0.05 to p<0.001) reduction in glucose-6-phosphatase, HMG CoA reductase

and arginase activities, however a significant (p<0.05 to p<0.01) increase in the levels of

hexokinase were recorded in the extract and other drug treated groups when compared to

diabetic control group. The one way analysis of variance showed a significancy of p<0.05 to

p<0.01 within the tested groups in the experimented models.

TABLE–4.17: Effect of ALSN and AAMP on the activities of Liver Glucose-6-

Phosphatase, Hexokinase, HMG CoA reductase & Arginase.

Groups and Treatments Glucose-6-

Phosphatase Hexokinase

HMG CoA

reductase Arginase

I. Normal Control 0.63 ± 0.13 0.176 ± 0.120 1.56 ± 0.17 2.3 ± 0.37

II. Diabetic Control 3.56 ± 0.44c 0.076 ± 0.009c 4.31 ± 0.41c 4.1 ± 0.28b

III. Glibencamide

(2.5mg/kg/day) 2.2 ± 0.48a 0.143 ± 0.014b 1.4 ± 0.13c 2.58 ± 0.42a

IV. ALSN (50mg/kg/day) 2.4 ± 0.39 0.098 ± 0.011 3.18 ± 0.29a 3.11 ± 0.35

V. ALSN (100mg/kg/day) 1.61 ± 0.41b 0.128 ± 0.016a 2.51 ± 0.31b 2.81 ± 0.41a

VI. AAMP (250mg/kg/day) 2.53 ± 0.45 0.116 ± 0.012 3.11 ± 0.15a 3.03 ± 0.33

VII. AAMP (500mg/kg/day) 1.42 ± 0.33b 0.136 ± 0.014a 2.33 ± 0.24c 2.23 ± 0.40 a

VIII. Insulin 1.1 ± 0.18c 0.163 ± 0.015b 1.68 ± 0.26c 2.73 ± 0.41a

F (7,40) 6.72** 6.23** 15.59** 2.68*

Glucose-6-Phosphatase activity is expressed in µmole of inorganic phosphate

liberated/15- min/mg protein at 370C; Arginase activity is expressed in enzyme units. 1 unit

of arginase is amount of enzyme that produces 1 µmole of Ornithine /min/mg of protein at

370C; HMG CoA reductase activity is inversely proportional to the ratio of HMG CoA /

mevalonate; Hexokinase activity of liver is expressed in µmole of glucose phosphorylated /

hr / mg protein.

Values are expressed in MEAN ± S.E.M of six animals. One Way ANOVA followed by

Dunnet’s t-test. (F-value denotes statistical significance at *p<0.05, **p<0.01) and (t-value

denotes statistical significance at ap<0.05,

bp<0.01 and

cp<0.001 respectively, in comparison

of Gr-II with Gr-I and Gr-III to Gr-VIII with Gr-II).


155

4.12.3. Effect of ALSN and AAMP on Haematological parameters

The haematological parameters exhibited in Table–4.18, showed that the animals

treated with standard drug Glibenclamide and test extracts in the tested dose levels registered

normal values in RBC count, WBC count, Hb count, and clotting time. AAMP at 500 mg/kg

b.w showed a significant (p<0.05) increase in haemoglobin levels. However the diabetic rats

treated with solvent showed a decrease value of RBC, Hb content and an increased value of

WBC count and clotting time when compared with normal

The Neutrophil count appears to nearly equal with that of normal value in all the

tested groups treated with both the extracts at all dose levels, except AAMP at 500 mg/kg,

which showed a significancy of p<0.01 in increasing the neutrophil level.. In case of the

standard drug and test extract treated animals showed almost normal values in the other

haematological parameters like eosinophil, basophil, lymphocyte and monocytes. Therefore,

it might be suggested that the test ext. has no significant effect on the haematological

parameters and is evident for the safety use for a longer duration of time.

4.12.4. Effect of ALSN and AAMP on Body weight

The percentage losses in the body weight of the animals were recorded on 0th

, 10th

,

20th

& 30th

day of the experiment by using standard animal weighing procedure and the data

depicted in Table-4.19. The % loss of body weight during 30-days study in diabetic rats

under treatment of test and standard drug, showed that, there are significant (p<0.05 to

p<0.001) recovery of body weight when compared with solvent treated diabetic rats. On 0th

day, the recorded loss in body weight in all the groups was about 23 to 29%. In the solvent

control group, the loss in b.w had been increased progressively through out the experiment

recording a maximum value of 37.83 % at 30th

day of treatment, where as standard drug

glibenclamide registered a consistent & significant (p<0.001) reduction in the % age loss in

b.w starting from 10th

day onwards up to 30th

day recording a lowest value of 5.5% at the end

of the treatment. ALSN at the tested dose levels significantly (p<0.05 to p<0.001) decreased

the %age loss in body weight recording the lowest value of 8.83% at the 30th

day, while

AAMP shown more potency than that of ALSN, in recovering the loss in b.w., recording a

significant (p<0.001) lowest value of 5.33% at the same time when compared with that of the

solvent control.


156

TABLE–4.18: Effect of ALSN and AAMP on Serum Haematological parameters

Groups and

Treatments

Serum Haematological parameters

RBC

(millions/ml)

WBC

(1000/ml)

Hb

(g/dl)

Clotting

time

(min.)

Neutrophil

(%)

Eosinophil

(%)

Basophil

(%)

Lymphocyte

(%)

Monocyte

(%)

I. Normal Control 4.19 ± 1.35 6.65 ± 1.2 10.86 ± 1.05

1.08 ± 0.18

28.5 ± 2.40

3.5 ± 0.84 00 68.16 ± 4.39 1.8 ± 0.32


(Tween+Water) 2.41 ± 1.15 7.38 ± 1.14

7.75 ± 1.71a

1.61 ± 0.19

20.5 ± 2.45a

6.5 ± 1.25a 00 72.33 ± 5.13 4.1 ± 0.77a

III. Glibencamide

(2.5mg/kg/day) 4.05 ± 1.07 6.88 ± 1.37

10.41 ± 0.96

1.01 ± 0.14

26.91 ± 1.44

4.66 ± 0.76 00 67.33 ± 7.12 2.0 ± 0.82a

IV. ALSN

(50mg/kg/day) 3.15 ± 0.91 5.35 ± 1.06

8.16 ± 1.19

1.21 ± 0.22

20.75 ± 2.63

2.83 ± 0.90a 00 72.33 ± 9.17 2.3 ± 0.74

V. ALSN

(100mg/kg/day) 3.45 ± 0.87 6.58 ± 1.53

9.04 ± 1.72

1.18 ± 0.22

26.08 ± 2.15

3.33 ± 0.76a 00 69.16 ± 8.52 2.2 ± 0.64a

VI. AAMP

(250mg/kg/day) 3.27 ± 1.03 5.43 ± 1.11

9.01 ± 1.44

1.28 ± 0.23

25.83 ± 3.36

3.83 ± 1.07 00 71.66 ± 6.63 1.5 ± 0.23a

VII. AAMP

(500mg/kg/day) 3.65 ± 1.07 6.29 ± 1.53

10.63 ± 2.11a

1.08 ± 0.26

32.63 ± 2.99b

3.16 ± 1.08a 00 70.83 ± 7.12 1.7 ± 0.21a

F (6, 35) 0.30 0.33 1.53 0.86 2.77* 1.67 -- 0.08 2.18



bp<0.01 and

cp<0.001



157

TABLE – 4.19: Percentage loss in body weight in ALSN and AAMP in Multi- dose treated in Alloxan induced diabetic rats.

Groups

&

Treatments

Percentage loss in Body weight loss

0th

day 10th

day 20th

day 30th

day

I. Diabetic Control

(Tween + Water) 27.16 ± 2.25 31.5 ± 2.12 34.66 ± 2.27 37.83 ± 3.15

II. Glibencamide (2.5mg/kg/day) 26.16 ± 3.75 19.5 ± 2.36c 9.66 ± 1.05

c 5.5 ± 0.71

c

III. ALSN (50mg/kg/day) 29.33 ± 2.76 24.33 ± 1.94a 14.5 ± 1.23

c 11.5 ± 1.23

c

IV. ALSN (100mg/kg/day) 23.16 ± 1.40 20.33 ±0.91c 12.16 ± 1.01

c 8.83 ± 1.07

c

V. AAMP (250mg/kg/day) 25.16 ± 1.86 21.33 ± 1.64c 11.33 ± 0.84

c 7.16 ± 0.90

c

VI. AAMP (500mg/kg/day) 27.5 ± 3.01 19.16 ± 2.35c 9.33 ± 1.11

c 5.33 ± 0.92

c

F (5,30) 0.65 5.75** 52.11** 63.51**




bp<0.01 and



158

4.12.5. Histopathological studies (at the end of 30th

day of treatment)

Group-1: Served as normal saline control given orally.

Group-2: Served as diabetic control (solvent control) and received only vehicle

(tween + water) at a dose of 5 ml/kg orally.

Group-3: Received Glibenclamide at a daily dose of 2.5 mg/kg b.w orally.

Group-4: Received ALSN at a daily dose of 50 mg/kg b.w orally.

Group-5: Received ALSN at a daily dose of 100 mg/kg b.w orally.

Group-6: Received AAMP at a daily dose of 250 mg/kg b.w orally.

Group-7: Received AAMP at a daily dose of 500 mg/kg b.w orally.

4.12.5.1. Histopathology of Liver of the experimented animals

Group-1

The microscopic section of liver of rat of group-1, kept as normal saline control

resembled almost normal histology and no significant pathological change could be observed

(Fig. 4.02).

Group-2

The microscopic section of liver of rat of group-2, kept as diabetic control, received

only vehicle (tween + water), showed consistent findings of degenerative and fatty changes in

hepatocytes, characterized by presence of small to large fat vacuoles in the cytoplasm. In

some cases the fat droplets were so large that it pushed the nuclei in periphery giving signet

ring appearance. Centrilobular necrosis of hepatocytes was also observed as revealed by

individualization, disintegration and denucleation of hepatocytes. Very high mononuclear cell

infiltration, congestion in portal tract and proliferation of the bile duct was seen. There was

heavy congestion of portal blood vessels as well as central vein (Fig. 4.03).

Group-3

The microscopic section of liver of rat of group-3, received glibenclamide at a dose

level of 2.5 mg/kg b.w., prevailed very mild congestion of sinusoids and portal as well as

hepatic veins. The hepatocytes were showing vacoular changes mostly suggestive of hydropic

degeneration. There has been moderate degree of increase in the number of kupffer cells.

Very mild mononuclear cell infiltration in portal tract was also evident. A most interesting

finding was that mild degenerative and necrotic changes were found around central vein

while infiltrative and proliferative changes were seen in portal areas (Fig. 4.04 and 4.05).


159

Group-4

The microscopic section of liver of rat of group-4, treated with ALSN at a dose level

of 50 mg/kg b.w. daily showed degenerative and necrotic changes, which were more marked

in this group as compared to group-3. Congestion of blood vessels was also significant.

Similarly, mononuclear cell infiltration and proliferation of fibroblast cells were also more

marked than that of group-3. Proliferation of bile duct was also a significant finding (Fig.

4.06 and 4.07).

Group-5

The microscopic section of liver of rat of group-5, treated with ALSN at a dose level

of 100 mg/kg b.w. daily showed mild vacuolar degeneration, lesser centrilobular necrosis of

hepatocytes, disintegration and denucleation of hepatocytes than that of group-4. Very mild

mononuclear cell infiltration in portal tract was also seen. However, infiltrative and

proliferative changes resembled to that of group-4 (Fig. 4.08 and 4.09).

Group-6

The microscopic section of liver of rat of group-6, treated with AAMP at a dose level

of 250 mg/kg b.w. showed mild congestion of the central vein with vacuolar degeneration of

hepatocytes around the central vein; and mild necrotic changes, however, mononuclear cell

infiltrative and proliferative changes in the portal area resembled to that of groups treated

with ALSN. Proliferation of bile duct was n (Fig. 4.10 and 4.11).

Group-7

The microscopic section of liver of rat of group-7, treated with AAMP at a dose level

of 500 mg/kg b.w. showed very milder vacuolar degenerative and necrotic changes in the

hepatocytes as compared to group-4, 5 & 6. The mononuclear cell infiltration and

proliferation of fibroblast cells were rarely marked. Very mild infiltrative and proliferative

changes were also evident (Fig. 4.12 and 4.13).

4.12.5.1. Histopathology of Kidney of the experimented animals

Group-1

The microscopic section of kidney of rat of group-1, kept as normal saline control

resembled almost normal histological picture and no significant pathological change could be

observed (Fig. 4.14).

Group-2

The microscopic section of kidney of rat of group-2, kept as diabetic control, received

only vehicle (tween + water), showed severe degenerative and desquamative granular as well


160

as vacuolar changes in the epithelial cells of the proximal convoluted tubules. Heavy

congestion was observed in blood vessels of glomeruli and interstitial spaces. Severe amyloid

depositions were observed in glomeruli, which resembled an eosinophilic acellular hyaline

mass. Mononuclear infiltration was also found in interstitial spaces as well as in

periglomerular spaces (Fig. 4.15).

Group-3

The microscopic section of kidney of rat of group-3, received glibenclamide at a dose

level of 2.5 mg/kg b.w. prevailed very mild marked changes like amyloidosis in glomeruli,

individualization, mild necrosis and desquamation of the tubular epithelial cell. The

glomeruli were showing presence of acellular hyaline eosinophilic deposits and the lumen

was showing the deposition of proteinaceous substances. Some infiltrative changes were also

observed in the interstitial spaces (Fig. 4.16 & Fig. 4.17).

Group-4

The microscopic section of kidney of rat of group-4, treated with ALSN at a dose level

of 50 mg/kg b.w. daily showed dilatation & congestion of blood vessels, and marked focal

mononuclear cell infiltration in the interstitial spaces. The lining tubular epithelial cells were

showing degenerative and desquamative changes at places (Fig. 4.18 & Fig. 4.19).

Group-5

The microscopic section of kidney of rat of group-5, treated with ALSN at a dose level

of 100 mg/kg b.w. daily showed marked changes like amyloidosis in glomeruli; congestion of

glomerular as well as inter-tubular blood vessels. The presence of eosinophilic hyaline

deposits in the lumen of proximal and distal convoluted tubules was found. (Fig. 4.20 & Fig.

4.21).

Group-6

The microscopic section of kidney of rat of group-6, treated with AAMP at a dose

level of 250 mg/kg b.w. showed mild congestion of the blood vessels with vacuolar

degeneration in the epithelial cells of the proximal tubules, but the presence of eosinophilic

hyaline casts in the lumen of proximal and distal convoluted tubules along with mild

mononuclear cell infiltration in the interstitial spaces was also evident (Fig. 4.22 & Fig. 4.23).

Group-7

The microscopic section of kidney of rat of group-7, treated with AAMP at a dose

level of 500 mg/kg b.w. showed mild granular & vacuolar degeneration of the tubular

epithelial cells; almost nil necrosis and a very little desquamation of the tubular epithelial

cells. Some tubules showed the presence of cellular debris in the lumen (Fig. 4.24 & Fig.

4.25).


161

Fig. 4.02. Microscopic section of Liver of normal rat treated orally with normal saline,

showing normal histology. (100X, H&E)

Fig. 4.03. Microscopic section of Liver of diabetic rat treated orally with vehicle (tween +

water) at a daily dose of 5 ml/kg b.w, showing severe degenerative & fatty changes in

hepatocytes; centrilobular hepatic necrosis; individualization of hepatocytes, and congestion

of portal vein. (100X, H&E)


162

Fig. 4.04. Microscopic section of Liver of diabetic rat treated orally with Glibenclamide at a

daily dose of 2.5 mg/kg b.w, showing mild mononuclear cell infiltration and fibroblastic

proliferation around the portal tract. (100X, H&E)

Fig. 4.05. Microscopic section of Liver of diabetic rat treated orally with Glibenclamide at a

daily dose of 2.5 mg/kg b.w showing mild congestion of sinusoids and portal vein, vacoular

changes mostly suggestive of hydropic degeneration and very mild mononuclear cell

infiltration. (100X, H&E)


163

Fig. 4.06. Microscopic section of Liver of diabetic rat treated orally with ALSN at a daily

dose of 50mg/kg b.w showing congestion of blood vessels with single cell necrosis;

mononuclear cell infiltration and proliferation of fibroblast cells. (100X, H&E)


dose of 50mg/kg b.w showing marked centrilobular necrosis of hepatocytes around the

congested central vein. (100X, H&E)


164


dose of 100mg/kg b.w showing mild vacuolar degeneration; centrilobular necrosis of

hepatocytes; disintegration and denucleation of hepatocytes, and mononuclear cell infiltration

in portal tract. (100X, H&E)


dose of 100mg/kg b.w showing mild fatty change and mild congestion in the central vein.

(100X, H&E)


165

Fig. 4.10. Microscopic section of Liver of diabetic rat treated orally with AAMP at a daily

dose of 250mg/kg b.w showing mild congestion of the central vein with vacuolar

degeneration of hepatocytes around the central vein. (100X, H&E)


dose of 250mg/kg b.w showing mononuclear cell infiltration and proliferation of fibroblast

cells in the portal area; and proliferation of bile duct. (100X, H&E)


166


dose of 500mg/kg b.w showing very milder vacuolar degenerative & necrotic changes in the

hepatocytes. (100X, H&E)


dose of 500mg/kg b.w showing very mild infiltrative and proliferative, and mild vacuolar

degenerative changes. (100X, H&E)


167

Fig. 4.14. Microscopic section of Kidney of normal rat treated orally with normal saline,

showing normal histology. (100X, H&E)

Fig. 4.15. Microscopic section of Kidney of diabetic rat treated orally with vehicle (tween +

water) at a daily dose of 5 ml/kg b.w, showing severe congestion in blood vessels of

glomeruli & infiltrative changes in interstitial spaces. Severe eosinophilic acellular hyaline

mass depositions were observed in glomeruli. (100X, H&E)


168

Fig. 4.16. Microscopic section of Kidney of diabetic rat treated orally with Glibenclamide at

a daily dose of 2.5 mg/kg b.w, showing mild individualization, mild necrosis and

desquamation of the tubular epithelial cell. (100X, H&E)

Fig. 4.17. Microscopic section of Kidney of diabetic rat treated orally with Glibenclamide at

a daily dose of 2.5 mg/kg b.w showing mononuclear cell infiltration in interstitial spaces

along with amyloid changes in the glomeruli and proteinaceous deposits in the lumen of the

tubules. (100X, H&E)


169

Fig. 4.18. Microscopic section of Kidney of diabetic rat treated orally with ALSN at a daily

dose of 50mg/kg b.w showing dilatation of blood vessels, marked focal mononuclear cell

infiltration in the interstitial tissues. (100X, H&E)


dose of 50mg/kg b.w showing congestion of the interstitial blood vessels and focal

mononuclear cell infiltration. The lining tubular epithelial cells are showing degenerative

and desquamative changes at places. (100X, H&E)


170


dose of 100mg/kg b.w showing presence of eosinophilic hyaline deposits in the lumen of

proximal and distal convoluted tubules. (100X, H&E)


dose of 100mg/kg b.w showing marked changes like amyloidosis in glomeruli; congestion of

glomerular as well as inter-tubular blood vessels. The glomeruli are showing presence of

eosinophilic hyaline deposits in the lumen. (100X, H&E)


171

Fig. 4.22. Microscopic section of Kidney of diabetic rat treated orally with AAMP at a daily

dose of 250mg/kg b.w showing presence of eosinophilic hyaline casts in the lumen of

proximal and distal convoluted tubules along with mild mononuclear cell infiltration in the

interstitial spaces. (100X, H&E)


dose of 250mg/kg b.w showing mild congestion of the blood vessels with vacuolar

degeneration and mononuclear cell infiltration in the epithelial cells of the proximal tubules.

(100X, H&E)


172


dose of 500mg/kg b.w showing very mild granular & vacuolar degeneration of the tubular

epithelial cells. (100X, H&E)


dose of 500mg/kg b.w showing almost nil necrosis & degeneration of the epithelial cells; and

a very little desquamation of the tubular epithelial cells. (100X, H&E)


173

SECTION - II

Anti-oxidant activity study


174

4.13. Anti-oxidant activity study of ALSN and AAMP

4.13.1. Anti-oxidant activity study in-vitro

4.13.1.1. Determination of total phenolic content, total flavonoid content, total

antioxidant activity & Ferric Reducing Power of ALSN and AAMP.

The perusal of Table-4.20 depicted that the total phenolic contents of aqueous extract

of leaves of S.nigrum (ALSN) and aqueous extract of aerial parts of M. pentaphylla (AAMP)

are found to 33.83 µg & 75.16 µg of pyrocatechol equivalent/500mg respectively and the

total flavonoids contents of ALSN and AAMP were found to be 5.86 mg & 9.58 mg equivalent

of quercetin /gm, which is quantitatively a greater value.

The total antioxidant activity and the ferric reducing power of test extracts and

standard drug ascorbic acid were investigated by using different in vitro methods and are

presented in Table-4.20, Fig. 4.26 and Table-4.21, Fig. 4.27 respectively.

Fig. 4.26 shows that ALSN and AAMP were found to have total antioxidant activity of

54.16 mg and 98.66mg ascorbic acid equivalent/gm, respectively, in comparison to that of the

reference standard ascorbic acid which registered 117.83 mg ascorbic acid equivalent/gm.

Similarly, Fig. 4.27 represented the reductive capabilities of both the extracts (ALSN and

AAMP) compared to that of ascorbic acid. Both the extracts found to potentiate in reducing

the Fe3+ / ferricyanide complex to the ferrous form (Fe2+) which was monitored by measuring

the formation of Perl’s Prussian blue at 700nm, but in comparison, AAMP was found to have

more potent ferric reducing capacity than that of ALSN. The reducing power of the extracts

were found to be significant and in a concentration dependent manner.


175

TABLE – 4.20: Determination of Total Phenolic content, Total Flavonoid content, and

Total Antioxidant activity of ALSN and AAMP

Groups

Total Phenolic

content

(µg of pyrocatechol

equivalent /500mg)

Total Flavonoid

content

(mg equivalent of

quercetin /gm)

Total Antioxidant

activity

(mg equivalent of

ascorbic acid/gm)

I. Ascorbic acid - - 117.83 ± 6.82

II. ALSN 33.83 ± 3.11 5.86 ± 0.29 54.16 ± 3.24

III. AAMP 75.16 ± 3.62 9.58 ± 0.82 98.66 ± 3.81

Values are expressed in MEAN ± S.E.M (n =3).

Fig. 4.26. Graphical presentation of Total Antioxidant activity

Determination of total antioxidant activity

0

20

40

60

80

100

120

Ascorbic acid ALSN AAMP

Ab

sorb

an

ce


176

TABLE – 4.21: Determination of Ferric reducing power of ALSN and AAMP (FRAP

assay)

Concentration

(µg/ml)

Ferric Reducing Power

(Absorbance at 700 nm)

Ascorbic acid ALSN AAMP

100 0.15 ± 0.01 0.10 ± 0.01 0.10 ± 0.01

200 0.23 ± 0.02 0.20 ± 0.01 0.21 ± 0.02

300 0.26 ± 0.01 0.22 ± 0.02 0.25 ± 0.01

400 0.35 ± 0.02 0.29 ± 0.01 0.31 ± 0.01

500 0.58 ± 0.01 0.39 ± 0.01 0.46 ± 0.01


Fig. 4.27. Graphical presentation of Ferric reducing power of ALSN and AAMP

Ferric reducing power of ALSN & AAMP

0

0.1

0.2

0.3

0.4

0.5

0.6

0 100 200 300 400 500

Concentration (µg/ml)

Ab

so

rb

an

ce

Absorbance of Ascorbic acid

Absorbance of AAMP

Absorbance of ALSN



177

4.13.1.2. Effect of ALSN and AAMP on 1, 1-diphenyl-2-picrylhydrazyl (DPPH),

superoxide (O2•−), hydrogen peroxide (H2O2) and nitric oxide (NO) scavenging activity

The capacity of aqueous extract of leaves of S. nigrum (ALSN) and aqueous extract of

aerial parts of M. pentaphylla (AAMP) to scavenge DPPH, O2•−, H2O2 and NO were

measured in-vitro; the related IC50 values and the % scavenging results are mentioned in

Table-4.22 and Table-4.23 respectively. The graphical presentations of DPPH, O2•−, H2O2

and NO scavenging activities are presented in Fig. 4.28, Fig. 4.29, Fig. 4.30 and Fig. 4.31

respectively.

Both the extracts, ALSN and AAMP scavenges DPPH radical in a concentration

dependent manner. The antioxidants react with DPPH, a purple colored stable free radical

and convert it into a colorless α-α-diphenyl-β-picryl hydrazine. The amount of DPPH reduced

could be quantified by measuring a decrease in absorbance at 517 nm. Both ALSN and AAMP

significantly and concentration dependently reduced DPPH radicals. However at a

concentration of 500µg/ml, ALSN significantly (p< 0.001) scavenged 94.0 % of DPPH

radicals as compared to that of control and had an calculated IC50 value of 165µg/ml; while

AAMP at a concentration of 500µg/ml, significantly (p< 0.001) scavenged 98.0 % of DPPH

radicals as compared to that of control and registered an calculated IC50 value of 96.5µg/ml;

however, standard ascorbic acid registered 100% scavenging activity at 400 µg/ml onwards

with a significance of p<0.001 as compared to that of control.

From the investigations, it was found that ALSN and AAMP scavenged O2•−

significantly and in a concentration dependent manner. The O2•− scavenging activity was

determined by Phenazine methosulphate/NADH-NBT system wherein O2•− derived from

dissolved oxygen by Phenazine methosulphate/NADH coupling reaction reduces NBT. The

decrease of absorbance at 560 nm with antioxidants thus indicates the consumption of

superoxide anions in the reaction mixture. ALSN exhibited a maximum of 56.4% superoxide

scavenging activity with a significant extent (p<0.01) at a concentration of 500 µg/ml, when

compared to that of control, with the IC50 value of 417µg/ml; while AAMP exhibited a

maximum of 68.8% superoxide scavenging activity with a significancy of p<0.001 at a

concentration of 500 µg/ml, as compared to that of control, with the calculated IC50 value of

381.4 µg/ml; however, standard ascorbic acid registered 81.3% scavenging activity at 500

µg/ml with a significance of p<0.001 when compared to that of control group.

The spontaneous or catalytic dismutation of O2•− leads to the formation of H2O2,

which in the presence of a transition metal ion like Fe3+, decomposes into •OH radicals, a

highly damaging species in free radical pathology. The extract ALSN was found to scavenge


178

57.6 % of H2O2 in a significant extent of p<0.001 at 500 µg/ml with a calculated IC50 value

of 472µg/ml, while, AAMP was found to scavenge 66.8 % of H2O2 with a significancy of

p<0.001 at 500 µg/ml with a calculated IC50 value of 432.7 µg/ml; where as the standard

ascorbic acid registered 78.5% scavenging activity at 500 µg/ml with a significance of

p<0.001 when compared to that of control groups.

However, as compared to DPPH; O2•−, H2O2 and NO were weakly scavenged by both

the extracts. S. nigrum extract at a concentration of 500 µg/ml also quenched 51.7% NO

released by a NO donor, SNP in a significant manner (p<0.01) showing the IC50 value is

483µg/ml, while M. pentaphylla extract at a concentration of 500 µg/ml scavenged 68.3%

NO significantly (p<0.001) showing the IC50 value is 247.5 µg/ml; where as the standard

ascorbic acid registered 66.5% scavenging activity at 500 µg/ml with a significance of

p<0.001 when compared to that of control groups. Incubation of SNP solution in PBS at 25

0C for 150 min resulted in the release of NO. Both the extracts effectively and dose

dependently decreased the release of NO. Control experiments showed that, even at high

concentrations, the extracts did not interfere with the reaction between nitrite and Griess

reagent.

However, both the extracts are having significant potential in scavenging the free

radicals in the above experiments, but AAMP registered more significant potency in free

radical scavenging activity than compared to that of ALSN.

TABLE – 4.22: Determination of IC50 value of ALSN and AAMP for 1,1-diphenyl-2-

picrylhydrazyl (DPPH), superoxide (O2•−), hydrogen peroxide (H2O2) and nitric oxide

(NO)

Extracts

IC50 values (µg/ml)

DPPH O2•− H2O2 NO

I. ALSN 165 417 472 483

II. AAMP 96.5 381.4 432.7 247.5


179

TABLE–4.23: Determination of 1,1-diphenyl-2-picrylhydrazyl (DPPH), superoxide

(O2•−), hydrogen peroxide (H2O2) and nitric oxide (NO) scavenging activity of ALSN

and AAMP.

Groups &

Treatments

DPPH O2•− H2O2 NO

% of

control

%

scavenging

activity

% of

control

%

scavenging

activity

% of

control

%

scavenging

activity

% of

control

%

scavenging

activity

Control 100.0 ± 5.4 00 100.0 ± 4.2 00 100.0 ± 3.7 00 100.0 ±2.7 00

Ascorbic acid

100 µg/ml 29.6 ± 2.1c 70.4 43.3 ± 3.7b 56.7 61.4 ± 4.8 38.6 77.4 ± 3.6 22.6

200 µg/ml 13.2 ± 1.5c 86.8 37.1 ± 4.8b 62.9 45.7 ± 3.5 54.3 68.3 ± 3.9a 31.7

300 µg/ml 3.4 ± 0.41c 96.6 29.4 ± 2.6c 70.6 38.6 ± 2.4b 61.4 59.1 ± 2.4b 40.9

400 µg/ml 0.00 100 23.9 ± 3.1c 76.1 27.2 ± 1.7c 72.8 56.7 ± 3.8b 43.3

500 µg/ml 0.00 100 18.7 ± 2.3c 81.3 21.5 ± 1.8c 78.5 33.5 ± 4.9c 66.5

ALSN

100 µg/ml 57.4 ± 4.8a 42.6 92.5 ± 7.4 7.5 97.3 ± 3.1 2.7 91.2 ± 5.7 8.8

200 µg/ml 46.8 ± 4.1a 53.2 83.7 ± 5.9 16.3 91.5 ± 4.8 8.5 88.3 ± 6.1 11.7

300 µg/ml 31.5 ± 4.7a 68.5 68.9 ± 4.2a 31.1 82.7 ± 5.0 17.3 73.5 ± 5.3 26.5

400 µg/ml 17.3 ± 3.2b 82.7 51.3 ± 3.2a 48.7 67.9 ± 4.6a 32.1 67.9 ± 3.5a 32.1

500 µg/ml 6.0 ± 1.1c 94.0 43.6 ± 2.7b 56.4 42.4 ± 4.7c 57.6 48.3 ± 3.9b 51.7

AAMP

100 µg/ml 48.3 ± 5.3a 51.7 87.4 ± 5.3 12.6 97.6 ± 4.9 2.4 59.3 ± 2.8b 40.7

200 µg/ml 33.5 ± 4.7a 66.5 63.8 ± 4.8 36.2 89.8 ± 4.3 10.2 57.6 ± 3.1b 42.4

300 µg/ml 13.3 ± 2.7b 86.7 56.9 ± 3.9a 43.1 74.7 ± 3.8 25.3 41.8 ± 4.5c 58.2

400 µg/ml 4.1 ±1.2c 95.9 48.7 ± 2.9c 51.3 58.7 ± 2.3b 41.3 35.6 ± 2.3c 64.4

500 µg/ml 2.0 ± 0.3c 98.0 31.2 ± 2.2c 68.8 33.2 ± 2.0c 66.8 31.7 ± 1.6c 68.3



bp<0.01 and

cp<0.001 respectively, in

comparison to control group)


180

Fig. 4.28. Graphical presentation of DPPH scavenging activity of ALSN and AAMP

DPPH Scavenging Activity

0

20

40

60

80

100

0 100 200 300 400 500


% D

PP

H S

ca

ven

ged

Ascorbic acid

AAMP

ALSN

Fig. 4.29. Graphical presentation of Superoxide scavenging activity of ALSN and AAMP

Superoxide Scavenging Activity

0

20

40

60

80

100

0 100 200 300 400 500


% S

up

eroxid

e s

caven

ged

Ascorbic acid

AAMP

ALSN


181

Fig. 4.30. Graphical presentation of Hydrogen peroxide scavenging activity of ALSN

and AAMP

Hydrogen peroxide scavenging activity

0

20

40

60

80

100

0 100 200 300 400 500


% H

yd

rogn

peroxid

e

scaven

ged

Ascorbic acid

AAMP

ALSN

Fig. 4.31. Graphical presentation of Nitric oxide scavenging activity of ALSN and AAMP

Nitric oxide scavenging activity

0

20

40

60

80

100

0 100 200 300 400 500


% N

itric

ox

ide s

ca

ven

ged

AAMP

Ascorbic acid

ALSN


182

4.13.2. Anti-oxidant activity study in-vivo

(On 30 days treated alloxan induced diabetic rat liver)

4.13.2.1. Effect of ALSN and AAMP on Thiobarbituric acid reactive substances

(TBARS), Hydroperoxides (HP), Malondialdehyde (MDA) and Conjugated dienes (CD)

activities on the multi-dose (30 days) treated diabetic rat liver.

Table-4.24 presents the estimated concentrations of liver Thiobarbituric acid reactive

substances (TBARS), Hydroperoxides (HP), Malondialdehyde (MDA) and Conjugated

dienes (CD) on 30th

day of the study of both test extracts (ALSN and AAMP) and standard

drug. The diabetic animals showed a significant (p<0.001) increase in the lipid peroxidation

products such as TBARS, HP, MDA and CD levels as compared with the normal control

group. In the ALSN treated alloxan induced diabetic animals, the lipid peroxidation products

such as TBARS, HP, MDA and CD levels are declined with an extent of 46.71, 20.85, 27.19,

23.72% respectively in case of 50mg/kg dose, while in 100mg/kg body weight dose level, the

% decrease became 57.88, 25.36,32.62, 31.81 respectively, with statistical significance of

p<0.001, as compared to that of the diabetic control group; where as in the AAMP treated

groups, the TBARS, HP, MDA and CD levels are declined with an extent of 53.47, 39.79,

29.78, 26.38% respectively, in case of 250mg/kg dose, while in 500mg/kg body weight dose

level, the % decrease became 69.20, 49.02, 38.29, 38.57 respectively, with statistical

significance of p<0.001, as compared to that of the diabetic control group. However, in a

similar way, the standard drug also showed a decreased value of 64.49, 34.15, 35.46, and

37.69 in percentage wise, respectively, with statistical significance of p<0.001. The data

clearly indicates that AAMP is significantly more potent in reducing the lipid peroxidation

products in the diabetic animals when compared with that of ALSN, in the tested dose levels.


183

4.13.2.2. Effect of ALSN and AAMP on Reduced glutathione (GSH), Glutathione

peroxidase (GSH-Px), Glutathione reductase (GR), Superoxide dismutase (SOD) and

Catalase (CAT) activities on the multi-dose (30 days) treated diabetic rat liver.

The perusal of the results are depicted in Table-4.25 showed that the antioxidant

enzymes like Reduced glutathione (GSH), Glutathione peroxidase (GSH-Px), Glutathione

reductase (GR), Superoxide dismutase (SOD) and Catalase (CAT) values are lowered

significantly (p<0.001) in diabetic rats as compared with normal control rats. The ALSN

showed an elevated value of an extent of 13.27, 6.89, 11.37, 15.25, 29.62% in case of

50mg/kg dose, while in 100mg/kg body weight dose level, it showed an increase of 28.45,

27.58, 20.40, 43.69, 50.21%, respectively as per the above mentioned order of enzymes, with

a statistical significance (p<0.001); where as the test extract AAMP showed an elevated value

of these enzymes to an extent of 24.24, 31.03, 15.45, 23.91, 14.91 % in 250mg/kg dose level,

while in 500mg/kg dose level, the respective enzyme percentage became 57.23, 55.17, 36.44,

54.04 and 44.11% with statistical significance of p<0.001. However the standard drug

glibenclamide, at the same time, registered an increased % of 47.69, 44.82, 48.10, 64.03, and

64.07 with respect to the above enzymes with statistical significance (p<0.001). So, the

resulted data clearly shows that AAMP is more potent in elevating the level of the antioxidant

enzyme system in the diabetic animals when compared with that of ALSN, in the tested dose

levels.


184

TABLE – 4.24: Determination of concentration of TBARS, HP, MDA and CD in the ALSN and AAMP treated rat liver.


Thiobarbituric acid

reactive substances

(TBARS)

(µM/100g wet tissue)

Hydroperoxides

(HP)


Malondialdehyde

(MDA)


Conjugated dienes

(CD)


I. Normal control 4.41 ± 0.71 15.66 ± 0.84 0.84 ± 0.05 55.41 ± 2.55


(Tween + Water) 26.5 ± 2.06

c 29.25 ± 1.04

c 1.41 ± 0.05

c 94.83 ± 3.01

c

III. Glibencamide

(2.5 mg/kg/day) 9.41 ± 1.16

c 19.26 ± 0.94

c 0.91 ± 0.04

c 59.08 ± 2.54

c

IV. ALSN (50mg/kg/day) 14.12 ± 0.41c 23.15 ± 0.97

c 1.10 ± 0.06

c 72.33 ± 3.63

a

V. ALSN (100mg/kg/day) 11.16 ± 0.83c 21.83 ± 1.13

c 0.95 ± 0.03

c 64.66 ± 2.60

c

VI. AAMP (250mg/kg/day) 12.33 ± 0.55c 17.61 ± 0.73

c 0.99 ± 0.04

c 69.81 ± 3.03

b


c 0.87 ± 0.10

c 58.25 ± 3.43

c

F (6, 35) 48.45** 38.39** 13.35** 32.32**



bp<0.01 and

cp<0.001

respectively, in comparison of Gr-II with Gr-I and Gr-III to Gr-VII with Gr-II).


185

TABLE – 4.25: Determination of concentrations of GSH, GSH-Px, GR, SOD and CAT in ALSN and AAMP treated rat liver.


Reduced

glutathione (GSH)

(µM/g wet tissue)

Glutathione

peroxidase

(GSH-Px)

(µM/g wet tissue)

Glutathione

reductase

(GR)

(µM/g wet tissue)

Superoxide

dismutase

(SOD)

(Units/mg protein)

Catalase

(CAT)

(Units/mg protein)

I. Normal control 21.66 ± 1.13 0.47 ± 0.04 5.41 ± 0.10 9.11 ± 0.13 8.38 ± 0.23


(Tween+Water) 12.58 ± 0.89

c 0.29 ± 0.02

c 3.43 ± 0.09

c 5.31 ± 0.17

c 4.76 ± 0.18

c

III. Glibencamide

(2.5 mg/kg/day) 18.58 ± 0.86

c 0.42 ± 0.01

b 5.08 ± 0.20

c 8.71 ± 0.34

c 7.81 ± 0.36

c

IV. ALSN (50mg/kg/day) 14.25 ± 0.91a 0.31 ± 0.01 3.82 ± 0.16

a 6.12 ± 0.09

b 6.17 ± 0.42

c

V. ALSN (100mg/kg/day) 16.16 ± 0.94a 0.37 ± 0.02 4.13 ± 0.18

a 7.63 ± 0.12

c 7.15 ± 0.53

c

VI. AAMP (250mg/kg/day) 15.63 ± 0.77b 0.38 ± 0.01

b 3.96 ± 0.19

b 6.58 ± 0.19

b 5.47 ± 0.37

a


b 4.68 ± 0.16

c 8.18 ± 0.24

c 6.86 ± 0.30

b

F (6, 35) 14.99** 6.91** 23.67** 46.82** 15.92**



bp<0.01 and

cp<0.001

respectively, in comparison of Gr-II with Gr-I and Gr-III to Gr-VII with Gr-II).


186

SECTION - III

Isolation and Characterization


187

4.14. Spectroscopic Characterization of the isolated compound from the aqueous extract

of aerial parts of M. pentaphylla (AAMP).

The isolated compound was found to be an white crystalline compound with melting

point of 322-3240C. The various absorption spectrums of the isolated compound from AAMP

showed peaks as follows:

FTIR (KBr) cm-1

: 3275.95 (OH Str.), 2944.78 (CH2 Str.), 1564.36 (CH=CH Str.),

1391.31 (CH Str.), 1062.73 (C-O-C Str.); and shown in Fig. 4.35.

1H-NMR (MeOD, 500 MHz): δ 5.41: CH (d, 1H, CH, H-2′′), 5.37: CH=C (s, 1H,

CH=C, H-14), 5.23: COOH (s, 1H, COOH, H-4a), 5.14: CH (d, 1H, CH, H-6′), 5.11: OH (s,

1H, OH, H-6′′), 5.09: OH (s, 1H, OH, H-5′′), 5.08: OH (s, 1H, OH, H-4′′), 5.04: CH (s, 1H,

CH, H-2′, 6′, 3′′), 5.01: OH (s, 1H, OH, H-4′, 5′), 4.88: CH (d, 1H, CH, H-6′′), 4.85: CH (t,

1H, CH, H-5′′), 4.02: CH (d, 1H, CH, H-3′), 3.91: CH (t, 1H, CH, H-4′, 5′), 3.74: CH (t, 1H,

CH, H-4′′), 3.38: CH (t, 1H, CH, H-10), 3.07: CH (t, 1H, CH, H-14b), 2.34: CH (t, 1H, CH,

H-5), 2.30: CH (t, 1H, CH, H-6), 1.98: CH (s, 2H, CH, H-1), 1.91: CH (d, 2H, CH, H-13),

1.77: CH (q, 2H, CH, H-11), 1.63: CH (t, 2H, CH, H-4), 1.52: CH (t, 2H, CH, H-3), 1.43: CH

(t, 1H, CH, H-12b), 1.40: CH (t, 2H, CH, H-8), 1.36: CH (t, 2H, CH, H-7,12), 1.22: CH (s,

3H, CH3, H-6a), 1.20: CH (s, 3H, CH3, H-9), 6b, 12a, 0.91: CH (s, 3H, CH3, H-2), and 0.77 :

CH (t, 1H, CH, H-8a) ppm; and depicted in Fig. 4.36 & Table 4.26.

.

13

C-NMR (CDCl3, 125 MHz): δ 37.7 (C-1), 24.7 (C-2), 35.2 (C-3), 23.1 (C-4), 180.3

(C-4a), 24.0 (C-5), 26.1 (C-6), 42.4 (C-6a), 39.6 (C-6b), 29.8 (C-7), 18.3 (C-8), 49.5 (C-8a),

33.0 (C-9), 77.5 (C-10), 23.7 (C-11), 28.3 (C-12), 30.2 (C-12a), 48.5 (C-12b), 21.1 (C-13),

23.9 (C-14), 37.8 (C-14a), 30.1 (C-14b), 90.6 (C-2′), 70.0 (C-3′), 63.1 (C-4′), 74.3 (C-5′),

90.9 (C-6′), 92.5 (C-2′′), 72.1 (C-3′′), 65.0 (C-4′′), 74.0 (C-5′′), 90.9 (C-6′′) ppm; and

illustrated in Fig. 4.37 & Table 4.26.

LCMS m/z: 752.38 [M]+, 734, 692, 604, 602, 586, 456, 296, 148, 132, 131, 115, 60.

The Isolated compound is designated as ‘M’. Mass values are expressed in m/z values.

LC‐MS spectroscopy showed the molecular ion peaks at 752.38 m/z value that correspond to

a molecular formula, C40H64O13 which was consistent with the theoretical value of 752.43.

Fragmentation ion peaks at m/z 734 correspond to the loss of hydroxyl unit, and other

fragmentation ion peaks were also observed at m/z 692, 604, 602, 586, 456, 296, 148, 132,

131, 115, 60; and the spectral data depicted in Fig. 4.34, Fig. 4.38 & Table 4.27.


188

TABLE – 4.26: 1H-NMR and

13C-NMR spectral data of the isolated compound

Sl.

No.

Position of the

Carbon atom δH

δC

1. 1 1.98: CH (s, 2H, CH) 37.7

2. 2 0.91: CH (s, 3H, CH3) 24.7

3. 3 1.52: CH (t, 2H, CH) 35.2

4. 4 1.63: CH (t, 2H, CH) 23.1

5. 4a 5.23: COOH (s, 1H, COOH) 180.3

6. 5 2.34: CH (t, 1H, CH) 24.0

7. 6 2.30: CH (t, 1H, CH) 26.1

8. 6a 1.22: CH (s, 3H, CH3) 42.4

9. 6b 0.91: CH (s, 3H, CH3) 39.6

10. 7 1.36: CH (t, 2H, CH) 29.8

11. 8 1.40: CH (t, 2H, CH) 18.3

12. 8a 0.77 : CH (t, 1H, CH) 49.5

13. 9 1.20: CH (s, 3H, CH3) 33.0

14. 10 3.38: CH (t, 1H, CH) 77.5

15. 11 1.77: CH (q, 2H, CH) 23.7

16. 12 1.36: CH (t, 2H, CH) 28.3

17. 12a 0.91: CH (s, 3H, CH3) 30.2

18. 12b 1.43: CH (t, 1H, CH) 48.5

19. 13 1.91: CH (d, 2H, CH) 21.1

20. 14 5.37: CH=C (s, 1H, CH=C) 23.9

21. 14a - 37.8

22. 14b 3.07: CH (t, 1H, CH) 30.1

23. 1′ - -

24. 2′ 5.04: CH (s, 1H, CH) 90.6

25. 3′ 4.02: CH (d, 1H, CH) 70.0

26. 4′

3.91: CH (t, 1H, CH) 63.1

27. 5.01: OH (s, 1H, OH)

28. 5′

3.91: CH (t, 1H, CH) 74.3

29. 5.01: OH (s, 1H, OH)

30. 6′

5.14: CH (d, 1H, CH) 90.9

31. 5.04: OH (s, 1H, OH)

32. 1′′ - -

33. 2′′ 5.41: CH (d, 1H, CH) 92.5

34. 3′′ 5.04: OH (s, 1H, OH) 72.1

35. 4′′

3.74: CH (t, 1H, CH) 65.0

36. 5.08: OH (s, 1H, OH)

37. 5′′

4.85: CH (t, 1H, CH) 74.0

38. 5.09: OH (s, 1H, OH)

39. 6′′

4.88: CH (d, 1H, CH) 90.9

40. 5.11: OH (s, 1H, OH)


189

Fig. 4.32: Formulated structure and IUPAC nomenclature of the isolated compound

H3C CH3

CH3

CH3

CH3

H3C CH3

O

OH

O

O

O OOH

OH

OH

HO

HO

OH

OH

2,2,6a,6b,9,9,12a-Heptamethyl-10-[4,5,6-trihydroxy-3-(3,4,5,6-tetrahydroxy-tet

rahydro-pyran-2-yloxy)

-tetrahydro-pyran-2-yloxy]-1,3,4,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-oc

tadecahydro-2H-picen

e-4a-carboxylic acid

2,2,6a,6b,9,9,12a-Heptamethyl-10-[4′,5′,6′-trihydroxy-3′-(3′′,4′′,5′′,6′′-tetrahydroxy-tetrahydro-pyran-

2-yloxy)-tetrahydro-pyran-2-yloxy]-1,3,4,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydro-

2H-picene-4a-carboxylic acid.

(An Oleanolic acid glycoside derivative)

Fig. 4.33: Numbering of the compound


190

Fig. 4.34: Flow Chart of Major Fragmentation Peaks as per the LC-MS Spectral data of

the isolated compound:

Cleavage of Pyran ring - H2O Cleavage of Picene ring

– 2H – C2H2O – C5H8O5

– O – O

– C5H6O4 – H

– O

– C3H3O

A′ = (M – C5H8O5)

= C35H56O8

Peak at m/z: 604

A = (M - H2O)

= C40H62O12

Peak at m/z: 734

A′′ = (M – C30H48O3)

= C10H16O10

Peak at m/z: 296

B′ = (M – C5H10O5)

= C35H54O8

Peak at m/z: 602

B = (M – C2H4O2)

= C38H60O11

Peak at m/z: 692

B′′ = (M – C35H56O8)

= C5H8O5

Peak at m/z: 148

C′ = (M – C5H10O6)

= C35H54O7

Peak at m/z: 586

C′′ = (M – C35H56O9)

= C5H8O4

Peak at m/z: 132

D′ = (M – C10H16O10)

= C30H48O3

Peak at m/z: 456

M = C40H64O13

Mol. Peak at m/z: 752.38

D′′ = (M – C35H57O9)

= C5H7O4

Peak at m/z: 131

E′′ = (M – C35H57O10)

= C5H7O3

Peak at m/z: 115

F′′ = (M – C38H60O11)

= C2H4O2

Peak at m/z: 60


191

TABLE- 4.27: Details of the Sequence of Fragmentations in LC-MS

Sl.

No. Isolated compound (M)

M – (part of the

molecule

fragmented)

Molecule shown the fragmentation peak

Fragmentation

occurred at

(m/z) value.

1.

O

O

O

O

OH

OH

OH

OH

OH OH

OH

CH3

CH3

CH3

CH3

CH3

CH3 CH

3

O

OH

M = C40H64O13

M - Nil

O

O

O

O

OH

OH

OH

OH

OH OH

OH

CH3

CH3

CH3

CH3

CH3

CH3 CH

3

O

OH

Mol. Formula = C40H64O13 (M)

752.38

2. -DO- M – H2O

O

O

O

O

OH

OH

OH

OH OH

OH

CH3

CH3

CH3

CH3

CH3

CH3 CH

3

O

OH

Mol. Formula = C40H62O12 (A)

734


192

3. -DO- M – C2H4O2

O

OH

O

O

OH

OH

OH OH

OH

CH3

CH3

CH3

CH3

CH3

CH3 CH

3

O

OH

Mol. Formula = C38H60O11 (B)

692

4. -DO- M – C5H8O5

O

O

OH

OH

OH

OH CH3CH3

CH3

CH3

CH3

CH3 CH3

O

OH

Mol. Formula = C35H56O8 (A′)

604


193

5. -DO- M – C5H10O5

O

O

O

OH

OH

OH CH3CH3

CH3

CH3

CH3

CH3 CH3

O

OH

Mol. Formula = C35H54O8 (B′)

602

6. -DO- M – C5H10O6

O

O

OH

OH

OH CH3

CH3

CH3

CH3

CH3

CH3 CH

3

O

OH

Mol. Formula = C35H54O7 (C′)

586

7. -DO- M – C10H16O10

OH

CH3

CH3

CH3

CH3

CH3

CH3 CH

3

O

OH

Mol. Formula = C30H48O3 (D′)

456


194

8. -DO- M – C30H48O3

O

O

O

OH

OH

OH

OH

OH OH

OH

Mol. Formula = C10H16O10 (A′′)

296

9. -DO- M – C35H56O8 O

OH

OH O

OH

Mol. Formula = C5H8O5 (B′′)

148

10. -DO- M – C35H56O9 CH+

CHO

OH

OH OH

Mol. Formula = C5H8O4 (C′′)

132


195

11. -DO- M – C35H57O9 O+

OH OH

OH

Mol. Formula = C5H7O4 (D′′)

131

12. -DO- M – C35H57O10 O+

OH OH

Mol. Formula = C5H7O3 (E′′)

115

13. -DO- M – C38H60O11

CH+

CH

OH

OH

Mol. Formula = C2H4O2 (F′′)

60


196

Fig. 4.35: FTIR Spectra of the isolated compound from AAMP


197

Fig. 4.36: 1H-NMR Spectra of the isolated compound from AAMP


198

Fig. 4.37: 13

C-NMR Spectra of the isolated compound from AAMP


199

Fig. 4.38: LC-MS Spectra of the isolated compound from AAMP

4.1 preliminary qualitative phytochemical study of the...

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