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CHAPTER – 3

RESULTS AND DICUSSION 3.1 EXTRACTION AND PHYTOCHEMICAL SCREENING 3.1.1 Physical status and percentage yield

Double maceration method was used for extraction of phytoconstituents using

chloroform and methanol at room temperature. Intermittent Shaking of the flasks

results in uniform extraction of active constituents by the solvents. Aqueous extract was

prepared using soxhlet extractor.

The physical status and percentage yield of leaves, bark and root of Soymida febrifuga

are given in Table12.The maximum percentage of yield was obtained from bark

aqueous extract followed by methanol extract.

3.1.2. Preliminary Phytochemical Screening of various extracts of Soymida

febrifuga

The results of phytochemical screening of leaves, bark and root are given in Table13.

Test tube analysis of the extracts was carried out following the standard procedures

as described earlier. The methanol extract of leaves were found to contain Glycosides,

Tannins, Flavonoids, Reducing sugar.

The chloroform extract of bark of Soymida febrifuga was found to contain secondary

metabolites namely Glycosides, Steroids, Triterpenoids. Alkaloids, Steroids,

Triterpenoids, Tannins, Saponins, and Flavonoids are present in methanol extract.

Steroids, Triterpenoids and Tannins were found in aqueous extract.

138

The root methanol extract contains Glycosides, Triterpenoids, Tannins, Flavonoids and

Reducing sugars. Flavonoids were also detected in aqueous extract.The results reveal

that metanol extract of bark is rich in different classes of important chemical

constituents.

Phytochemical screening helps in identifying the chemical constituents of a plant drug.

This inturn helps in distinguishing from the adulterants. With the knowledge of the

chemical constituents, the pharmacological activities can also be predicted.

3.1.3. Treatment of bark powder with different chemical reagents

Treatment of bark powder with different chemical reagents is shown in Table 14.These

studies help in identification of authentic sample.

3.1.4. Fluorescence characteristics of Bark

Fluorescence characteristics of bark powder are depicted in Table 15.

The results of treatment of bark with chemical reagents and fluorescence

characteristics of bark can be used for identification of the authentic sample and

to distinguish from adulterants.

139

Table 12: Physical status and percentage yield of the extracts

Plant Part Solvent Color Yield in % w/w Code

Leaves Chloroform Dark green 2.01 SFLC

Leaves Methanol Dark 16.91 SFLM

Leaves Water Greenish brown 12.61 SFLA

Bark Chloroform Brownish 3.50 SFBC

Bark Methanol Reddish brown 23.84 SFBM

Bark Water Blackish brown 26.75 SFBA

Root Chloroform Pale yellow 2.96 SFRC

Root Methanol Light brown 19.21 SFRM

Root Water Pale brown 17.63 SFRA

Table 13: Preliminary Phytochemical Screening of various extracts of Soymida

febrifuga

S.No. Name of the

Compound

Name of the Extract

SFLC SFLM SFLA SFBC SFBM SFBA SFRC SFRM SFRA

1 Alkaloids --- --- --- --- +++ --- --- --- ---

2. Glycosides --- +++ ---- +++ +++ --- --- +++ ---

3. Triterpenoids --- --- --- +++ +++ +++ --- +++ ---

4. Steroids --- --- --- +++ +++ +++ --- --- ---

5. Tannins --- +++ --- --- +++ +++ --- +++ ---

6. Saponins --- --- --- --- +++ --- --- --- ---

7. Flavonoids --- +++ --- --- +++ --- --- +++ +++

8. Reducing

sugars --- +++ --- --- +++ --- --- +++ ---

140

Table 14: Treatment of bark powder with chemical reagents

Reagents used Observation

Powder Reddish brown

Powder + Aceticacid Cherry red

Powder + Conc. Sulphuric acid Dark reddish brown

Powder + Conc. Nitric acid Orange

Powder + Conc. Hydrochloric acid Light brown

Powder + Ferric Chloride solution Dark greenish brown

Powder + 5% Iodine solution Cinnamon brown Powder + Ammonia Solution Reddish brown

Powder + Picric acid solution. Yellowish orange

Table 15: Fluorescence characteristics of Bark

Reagents

UV light

Visible light Short (254

nm) Long (366nm)

Bark Powder Bark Powder Bark Powder

Powder Light yellow Black Coffee brown

Powder +Acetic acid No color No color Cherry red

Powder +I N sulphuric acid Brown Dark Borwn Dark reddish borwn

Powder+ Picric Acid solution No color Light yellow Yellowish orange

Powder + 1N Hydrochloric acid No color No color Light brown

Powder + 1N Nitric acid Orange Green Orange

Powder + 5% Iodine solution Yellow Light green Brown

Powder + 5% Ferric chloride Yellow Light green Greenish brown

141

3.2 IN VITRO STUDIES

3.2.1 Free radical scavenging activity

DPPH free radical scavenging activity

The results of DPPH free radical scavenging activity of chloroform, methanol and

aqueous extracts are shown in Table 16.

The role of free radicals in diabetes and hepatic problems has been confirmed by

several reports cited earlier.Therefore before planning for invivo experiments to

evaluate hypoglycemic activity and hepatoprotective activities, free radical scavenging

activity and anti oxidant potential of the selected fractions was investigated.

DPPH radical is a stable free radical, during reaction with antioxidants it is reduced

which can be visualized as a change in the color from purple to yellow. All the extracts

bleached DPPH indicating their free radical scavenging activity.

The concentration of the standard and extracts required to produce 50% scavenging

action on DPPH radical is given as IC 50 values. The standard showed a percentage

inhibition of 66.56 at a dose of 5µg/ml. The IC50 value of the standard was found to be

3.65µg/ml. All extracts of the bark at all tested concentrations showed antioxidant

activity. Further, the activity was found to be dose dependant. The percentage

inhibition at a dose of 5µg/ml with chloroform, methanol and aqueous extracts was

22.63, 67.17 and 68.94 respectively. The corresponding IC50 values of the extracts were

found to be 17.95, 3.18 and 3.58 µg/ml respectively.

Abnormally high levels of free radicals and decreased anti oxidant protection are the

main cause for Diabetes, diabetic complications and hepatic diseases. Methanol and

aqueous extracts were found to have significant DPPH free radical scavenging activity.

The scavenging ability of methanol and aqueous extracts was comparable to the

142

standard. SFBM has better antioxidant activity among the three extracts as indicated by

its IC50.

The increasing order of the IC50 values of the test compounds is as follows:

SFBM <SFBA<Vit C< SFBC

Alkaloids, Glycosides, Steroids, Triterpenoids, Tannins, Saponins, and Flavonoids are

the chief active principles identified in the bark extracts. All these compounds are

reported to possess free radical scavenging and anti oxidant activities. Tannins and

flavonoids have a strong free radical scavenging activity and anti inflammatory activity.

They regulate cellular activities of inflammation related cells, macrophages,

lymphocytes, Neutrophils. They also modulate metabolizing enzymes like,

Phospholipase A2, Cyclo Oxygenase (COX), Lipoxygenase (LOX) and Nitric oxide

synthase (NOS)

Superoxide free radical scavenging activity

The results of superoxide radical scavenging activity of chloroform, methanol and

aqueous extracts are shown in Table 17. Superoxide is a precursor for ROS like

hydrogen peroxide, hydroxyl radical, singlet oxygen which are the main culprits

reacting with biological membranes and resulting in tissue damage. Endogenously,

super oxide radical is scavenged by superoxide dismutase. So, compounds which

possess superoxide radical scavenging activity protect the biological membranes and

help in prevention and treatment of free radical mediated diseases.

All extracts of the bark at all tested concentrations showed free radical scavenging

activity. Further the activity was found to be dose dependant. The IC50 value of the

standard was found to be 0.72 µg/ml. The percentage inhibition of the standard was

found to be 61% at a conc. of 5µg/ml . The IC50 values of the extracts were found to be

11.78, 1.30 and 4.26 µg/ml .The percentage inhibition of methanol and aqueous

extracts at a dose of 5µg/ml was found to be 87.7 and 56. The aqueous and methanol

extracts of the bark showed significant antioxidant activity. Chloroform extract also

143

exhibited antioxidant activity but was less when compared to the remaining extracts.

SFBM has potent antioxidant activity among the three extracts as indicated by the IC50

value.

The increasing order of IC50 Values of the test compounds is gives as follows:

Gallicacid <SFBM<SFBA<SFBC

The results of DPPH and Superoxide free radical scavenging activity suggest that these

plant extracts protect the cells from oxidative damage by possessing significant

antioxidant and free radical scavenging activities. Oxidative stress is involved in

pathology of cancer, Diabetes, Liver diseases etc. The antioxidant activity may be

responsible for traditional uses of the bark of Soymida febrifuga.

3.2.2 5-Lipoxygenase inhibiting activity

The results of 5-Lipoxygenase inhibiting activity of chloroform, methanol and aqueous

extracts are shown in Table 18. The IC50 value of standard (Nor Dihydroguaritic acid)

was found to be 5.34µg/ml. The percentage inhibition was 57.79 at 5µg/ml

concentration.

All the extracts exhibited dose dependant inhibition of 5-lipoxygenase enzyme. The

percentage inhibition of chloroform, methanol and aqueous (SFBC,SFBM&SFBA) was

found to be 2.87, 57.29 and 50.73 at 5µg/ml concentration. The IC50 values were found

to be 37.1, 4.05 and 4.75µg/ml respectively. The IC50 value of methanol extract was

less than the standard used. However, chloroform extract exhibited less activity when

compared to methanol and aqueous extracts. Methanol extract is confirmed to posses

significant 5-LOX inhibitory activity as indicated by the IC50 value. 5-Lipoxygenase

inhibitors have a very important role to play in treatment of asthma and as anti-

inflammatory agents. Antioxidants are known to inhibit lipoxygeneases. Therefore, 5-

lipoxygenase inhibiting activity of chloroform, methanol and aqueous extracts of bark

of Soymida febrifuga is also related to their anti oxidant activity.

144

3.2.3. Evaluation of Anti Cancer Activity on selected cell lines

The results of anti cancer activity of chloroform, methanol and aqueous extracts are

represented in Table 19. There was marked cell growth inhibition by chloroform extract

in human breast tumor, adenocarcinoma, mammary gland (negative) MCF-7, Human

epidermoid carcinoma 4-431, Fibrosarcoma HT-1080 cells. Methanol extract did not

exhibit cytotoxic activity on any of the cell lines upto 100 µg/ml. Chloroform and

aqueous extracts exhibited good cytotoxic activity. Aqueous extract was effective on

three cell lines tested i.e. MDA - MB - 231, MCF-7, A-431. The IC50 value was found

to be 77.2 µg/ml for MCF-7 cell lines, and a percentage inhibition of 26.3 and 48.8 at

100 µg/ml was observed with MDA-MB-231 and A-431 cell lines. It was not active

even upto 100 µg/ml on HT-1080 cell lines. Chloroform extract exhibited cytotoxic

activity on all the tested cell lines. This extract showed 37 percent inhibition on MDA-

MB-231 cell lines at 100 µg/ml. The IC50 values of SFBC were found to be 26.8µg/ml

for MCF-7, 77.88µg/ml for A-431 and 76.9 µg/ml for HT-1080 cell lines.

The bark extracts of Soymida febrifuga revealed the presence of steroids, triterpenoids,

alkaloids, tannins, glycosides& flavonoids, which are characterized for their anti cancer

andanti mutagenic activities. Constituents in combined form (or) independently are

responsible for the anti cancer activity of the bark extracts. The possible mechanism

of action may be expected to be radiomimetic, nucleotoxic, cytotoxic effect (or) by

acting as spindle poisons.

3.2.4. Evaluation of Antihelmintic activity

The results of Antihelmintic activity are shown in Table 20. Results are expressed as an

average of six observations. It was observed that methanol and aqueous extracts of

Soymida febrifuga bark showed significant Antihelmintic activity. Chloroform extract

did not possess the activity in the concentrations tested. Among the extracts, methanol

extract(50mg/ml) showed shortest time of paralysis and death. At the test dose of

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50mg/ml, the time required to paralyze the worms was 58+0.21, 30+0.25, 36+0.41 min

with chloroform, methanol and aqueous extracts. Death was recorded at 65.0+0.22,

40+0.23 and 45+0.28 min.with chloroform, methanol and aqueous extracts. Whereas

the time required to paralyze and kill the worms with standard (Albendazole) was

48.0+0.38and 60.0+0.33 min. respectively. The activity of methanol and aqueous

extracts was comparable to that of standard drug Albendazole with respect to time

taken to paralyze and to kill the worms. The potency of the extracts was inversely

proportional to the time taken for paralysis /death of the worms. The control group

animals were alive up to 24hrs. These results are in agreement with the traditional use

of Soymida febrifuga as Antihelmintic in folklore medicine.

Chemotherapeutic agents act as antihelmintics by disruption of neuromuscular

physiology, blockade of energy metabolism, disrupting reproductive system which is

highly efficient in these parasites. The tested extracts may act as antihelmintics by

anyone or a combination of these mechanisms. As the extracts caused paralysis as well

as death, they can be confirmed to possess more potent Antihelmintic activity.

146

Table 16: DPPH free radical scavenging activity

S.No Test

Substance (Extracts)

Dose Response Dose (µg/ml)

Percent inhibition

IC50 µg/ml

1 SFBC

5 22.63

17.95 10 38.37

25 62.56

2

SFBM

1 22.14

3.18 2.5 49.36

5 67.17

3

SFBA

1 20.83

3.58 2.5 32.78

5 68.94

4 VIT C

1 11.92

3.65 2.5 38.31

5 66.56

y = 1.923X +15.69 R2 = 0.981

y = 10.844X + 15.49 R2 = 0.9317

147

Table 17: Superoxide free radical scavenging activity

S.No Test Substance (extract) Dose Response Dose

(µg/ml) Percent inhibition IC50 µg/ml

1 SFBC

5 37.06

11.78 10 48.91

25 71.96

2 SFBM

0.5 19.86

1.30 1 45.85

2.5 87.70

3 SFBA

1 27.22

4.26 2.5 36.34

5 55.50

Standard

Gallic acid

0.25 24.13

0.72 0.5 44.79

1 61.46

y = 1.6967X + 30.02 R2 = 0.97

y = 32.62X + 7.046 R2 = 0.978

y = 7.285X + 19.38 R2 = 0.985

y = 47.283X + 15.789 R2 = 0.9379

148

Table 18: 5-Lipoxygenase inhibiting activity

S.

No

Test

Substance

(Solubility)

Dose response

Dose

(µg/

ml)

Percent inhibition IC50

µg/ml

1 SFBC

(DMSO)

2.5 0.37

37.1 5 2.87

10 11.0

2 SFBM

(DMSO)

1 20.67

4.05 2.5 39.41

5 57.29

3 SFBA

(DMSO)

1 11.02

4.75 2.5 32.43

5 50.73

4

Standard

(NDGA)

(DMSO)

1 29.18

5.34 2.5 41.73

5 57.19

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Table 19: Anti Cancer Activity

S.No. Extract

Name

Cytotoxic potential in cell lines

MDA-MB-231 MCF-7 A-431 HT-1080

1 SFBC 37% inhibition at

100 µg/ml

IC 50

26.8 µg/ml

IC 50

77.8 µg/ml

IC 50

76.9 µg/ml

2 SFBM Not active upto

100 µg/ml

Not active upto

100 µg/ml

Not active upto

100 µg/ml

Not active

Upto 100 µg/ml

3. SFBA 26.3 %inhibition

at 100 µg/ml

IC 50

77.2 µg/ml

48.8

%inhibition at

100µg/ml

Not active upto

100 µg/ml

Table 20: Evaluation of Antihelmintic activity

S.No Group Extract Dose

mg/ml

Response

Time taken for

paralysis (min)

Time taken for

death (min)

1 I Vehicle No Response No Response

3

4

5

II

III

IV

SFBC

10

25

50

No Response

No Response

58 + 0.21

No Response

No Response

65 + 0.22

6

7

8

V

VI

VII

SFBM

10

25

50

60 + 0.23

45 + 0.21

30 + 0.25

75 + 0.21

60 + 0.22

40 + 0.23

9

10

11

VIII

IX

X

SFBA

10

25

50

58 + 0.31

48 + 0.28

36 + 0.41

68 + 0.41

59 + 0.31

45 + 0.28

12 XI Albendazole

(Standard) 25 48 + 0.38 60 + 0.33

Values expressed as Mean + SD of six observations, n=6.

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3.3 FRACTIONATION AND IN VITRO STUDY OF METHANOL

EXTRACT OF BARK OF SOYMIDA FEBRIFUGA

3.3.1. Percentage yield of isolated fractions

The percentage yield of fractions isolated from methanol extract is given in Table

21.The yield of Acetone fraction was more than the remaining two fractions.

3.3.2. Phytochemical Screening of isolated fractions

The results of Phytochemical screening of the fractions are represented in Table 22.

TLC analysis and test tube reactions revealed the presence of alkaloids, glycosides,

triterpenoids and saponins in AFSF. Triterpenoids are present in MF1SF. Alkaloids,

triterpenoids and saponins were present in MF2SF.

3.3.3. Free radical scavenging activity of the selected fractions

DPPH free radical scavenging activity of fractions isolated is given in Table 23. The

standard Vit C showed a percent inhibition of 66.56 at a dose of 5 µg/ml. The IC50

value was found to be 3.65 µg/ml. IC50 of the fractions were found to be 2.04, 2.86 and

2.71 µg/ml respectively. They showed a percent inhibition of 89.14, 80.19 and 83.19

respectively. Further, the IC50 values of the fractions were less than that of crude

extract. Therefore, the fractions possessed better free radical scavenging property than

the crude extract. Fractionation has improved the antioxidant potential.

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Table 21: Comparative yields of isolated fractions

S.No Solvent used Code no Yield % w/w

1. Acetone:chloroform(80:20) AFSF 38.12

2. Methanol :Acetone(40:60) MF1SF 25.85

3. Methanol :Acetone(80:20) MF2SF 16.05

Table 22: Phytochemical Screening of fractions

S. No. Name of the Compound AFSF MF1SF MF2SF

1 Alkaloids +++ --- +++

2. Glycosides +++ --- ---

3. Triterpenoids +++ +++ +++

4. Steroids --- --- ---

5. Tannins --- --- ---

6. Saponins +++ --- +++

7. Flavonoids --- --- ---

152

Table 23: DPPH free radical scavenging activity of the isolated fractions

S.No

Test

Substance

(Extracts)

Dose Response Dose

(µg/ml)

Percent inhibition

IC50µg/ml

1 AFSF

1 65.11

2.038 2.5 80.04

5.0 89.14

2

MF1SF

1 64.11

2.86 2.5 72.81

5.0 80.19

3

MF2SF

1 65.12

2.71

2.5 74.80

5.0 83.19

4

VIT-C

1 11.92

3.65 2.5 38.31

5.0 66.56

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3.4. ACUTE TOXICITY STUDY RESULTS

All the fractions (AFSF, MF1SFand MF2SF) isolated were tested for their acute

toxicity. In this study, the rats did not produce behavioral, neurological and autonomic

profiles. The fractions did not produce lethality or death during 72h period of the

study. As the fractions were found to be non-toxic up to the highest dose tested (2g/kg,

b.w), they were selected for the studies in experimental animals.

3.5. SCREENING OF THE FRACTIONS FOR HYPOGLYCEMIC AND

ANTI HYPERGLYCEMIC ACTIVITY IN RATS

3.5.1 Effect of fractions on blood glucose levels in euglycemic rats

The effect of fractions on blood glucose levels in euglycemic rats are shown in Table

24 and Fig 10. In euglycemic rats, the fraction AFSF at the test doses 200 and

400mg/kg produced a significant (P<0.05) hypoglycemic effect after 2h of the

treatment, while the extracts MF1SFand MF2SF exhibited the same at 200 and

400mg/kg doses after 4h of the treatment. The significant hypoglycemic effect of the

fraction, AFSF, was observed up to 12h. All the fractions at all the test doses exhibited

the hypoglycemic effect at 4, 6 and 8h of the study. AFSF (200 mg/kg b.w) and the

reference drug Glibenclamide (10mg/kg) exhibited the significant effect with p value as

<0.01 after 2h of the treatment. The percentage reduction in blood glucose levels

reached to maximum level ranging from 34.4 to 54.1 after 6h of administration with all

the fractions at all test doses. However, the maximum hypoglycemia of 54.1, 37.8 and

43.0% at 6h of the study was observed with the dose 200mg/kg, of AFSF, MF1SF and

MF2SF respectively. The % reduction in blood glucose of AFSF (200mg/kg; 54.1%)

was well comparable to that of the reference drug, Glibenclamide (10mg/kg; 54.8%).

154

3.5.2 Effect of fractions on oral glucose tolerance in normal rats

The effect of the fractions on oral glucose tolerance is shown in Table 25 and Fig

11.The fraction AFSF at the entire test doses (100, 200 and 400mg/kg, b.w.) showed

a significant reduction in blood glucose levels after 90 min of glucose loading. Whereas

MF1SF and MF2SF at 200 and 400mg/Kg, b.w showed a significant reduction in blood

glucose levels after 90 min of glucose loading. All the fractions exhibited improved

glucose tolerance up to 120 min of the study. In both the test and standard groups, the

blood glucose levels of the animals reached to less than normal values after 120 min of

the investigation. The significant effect of the extracts at all the test doses was

comparable to that of the reference drug, Glibenclamide (10mg/kg, b.w.).

3.5.3. Effect of fractions on fasting blood glucose levels in alloxan induced

Diabetic rats

The effect of fractions on blood glucose levels in alloxan induced diabetic rats are

shown in Table 26 and Fig 12. All the fractions AFSF, MF1SF and MF2SF at all the test

doses (100, 200 and 400mg/kg, b.w.) significantly (p<0.001) lowered the blood glucose

levels after 2h of the treatment and continued the effect up to 24h of the study. The

maximum reduction in blood glucose levels was observed after 6h with all the fractions

at all the test doses. The percentage reduction in blood glucose levels was high with

200mg/kg of the fractions, showing 33.0%, 32.0% and 32.5% for AFSF, MF1SF and

MF2SF respectively. Though the effect of fractions was significant with 400mg/kg, b.w

after 6h, the percentage reduction in blood glucose levels was less than that of the dose

200mg/kg. The significant (p<0.001) antihyperglycemic effect and percentage

reduction in blood glucose levels with 200mg/kg of AFSF was well comparable to that

of the reference drug, Glibenclamide ( 10mg/kg,b.w) at any time interval of the study

and the maximum % reduction in blood glucose levels of 33.0% and 33.46% was

observed with AFSF (200mg/kg b.w) and the reference standard Glibenclamide

(10mg/kg, b.w.)

155

Plants with proven hypoglycemic effects were found to contain compounds like

triterpenoids, glycosides, alkaloids, and saponins. All these compounds are present in

the fractions of methanol extract of Soymida febrifuga bark. The observed

hypoglycemic effect may be a result of individual compound (or) a combination of

these constituents. The possible hypoglycemic activity exhibited by these fractions may

be due to potentiation of pancreatic secrection of insulin from β–cell of Islets of

Langerhan’s (or) by Stimulation of uptake of glucose by peripheral tissues (or) by

inhibition of endogenous glucose production (or) stimulation of gluconeogenesis in

liver and muscles.

3.5.4. Effect of fractions on body weight and blood glucose levels in Alloxan

induced Type-II diabetes in rats (subacute study)

The results of the study are shown in Table 27 Fig. 13 and 14. The observations made

on the results of different parameters studied are as follows:

i. Body weight

There was a gradual decrease in body weight of animals in diabetic control group. The

animals treated with fractions and reference drug showed a gradual and significant

(p<0.01) increase in the body weight after 7 days of the treatment. The increase in the

body weight was observed till the end of the study (21 days). The significant (p<0.01)

effect of AFSF (200mg/kg), MF1SF (200mg/kg) and MF2SF (200mg/kg) on body

weight of the animals was comparable to that of the reference drug, Glibenclamide

(10mg/kg) at each time interval of the study. A significant reduction in body weight

observed in alloxan-induced diabetic rats was due to increased excretion of glucose and

reduced uptake of glucose by peripheral tissues and glycogen synthesis. The

improvement in body weight of the animals when treated with fractions (AFSF, MF1SF

and MF2SF) may be due to potentiation of insulin secretion which inturn reverses these

effects.

156

ii. Blood glucose level:

All the fractions, AFSF, MF1SF and MF2SF at 200mg/kg, b.w. exhibited a significant

(p<0.001) antihyperglycemic effect after 7 days of the treatment and continued the

effect up to the end of the study i.e. 21 days. The effect of the extracts in lowering the

blood glucose levels was gradually increased and was maximum after 21 days showing

30.2%, 29.7% and 29.1% for AFSF, MF1SF and MF2SF respectively. The significant

anti hyperglycemic effect of the fractions was well comparable to that of the reference

drug, Glibenclamide (10 mg/kg b.w. 30.81%) at all time intervals of the study. Further,

the percentage reduction in blood glucose levels with the fraction AFSF after 21 days

was very near to that of the reference drug.

3.5.5 Effect of fractions on Serum Biochemical parameters in Alloxan induced

diabetic rats (sub acute study)

Serum insulin

The results of the study are shown in Table 28, Fig. 15, 16,17,18,19 and 20. In AFSF,

MF1SF and MF2SF groups, a significant (p<0.001) increase in serum insulin level after

21 days was observed. The percent increase in serum insulin level was 68.1 and 64.6%

and 71.8% with AFSF, MF1SF and MF2SF respectively, which was very close to the

effect shown by the reference drug, Glibenclamide (10mg/kg, 68.75%).

Serum Glutamate Oxaloacetate Transaminase (SGOT) and Serum Glutamate

Pyruvate Transaminase (GPT) level

After 21 days of AFSF, MF1SF and MF2SF supplementation, there was a significant

decrease in serum GOT and GPT levels. The significant effect (p<0.001) of the extracts

was comparable to that of the reference drug. The percentage decrease in SGOT levels

with AFSF, MF1SF and MF2SF was 38.48, 37.06 and 36.05. The percentage decrease

in SGPT levels with AFSF, MF1SF and MF2SF was 42.27, 42.56 and 42.12

157

respectively. The percentage decrease in SGOT and SGPT levels of the fractions was

comparable to the standard.

There is a rise in SGOT and SGPT levels in Diabetes induced rats. This may be due to

increased accumulation of these amino acids (Glutamate and alanine) due to

mobilization from protein stores. GOT and GPT stimulates gluconeogenesis there by

increasing glucose production from amino acids. The fractions tested significantly

reduced their levels suggesting anti diabetogenic activity.

Serum triglyceride level

In groups treated with fractions and reference drug, a significant (p<0.001) decrease in

serum triglyceride level was recorded after 21 days of the study. The percent reduction

in serum triglyceride level in AFSF, MF1SF and MF2SF treated groups were 12.18,

12.55 and 12.26 respectively, while it was 11.7 in standard group (Glibenclamide,

10mg/kg, b.w.).

Serum cholesterol level

All the fractions AFSF, MF1SF and MF2SF at 200mg/kg b.w. significantly (p<0.001)

lowered serum cholesterol level after 21 days. The percent reduction in serum

cholesterol level in AFSF, MF1SF and MF2SF groups were 41.13, 40.16% and 39.96%

respectively. The percent reduction with standard was 44.3%. The statistically

significant (p<0.01) antihypercholesterolemic effect of the fractions was comparable to

that of the reference drug.

There is an association between Diabetes and hyperlipidemia. Insulin is responsible for

activation of a lipolytic enzyme lipoprotein lipase which hydrolyses triglyceride under

normal conditions. Destruction of β–cells results in decreased plasma Insulin and

ultimately hyperlipidemia. Significant control of plasma lipid levels by the fractions

suggests that they produce the action by improving secretion of insulin. The effect of

158

the fractions on cholesterol and triglyceride levels may be due to decrease in activity of

enzymes involved in cholesterol biosynthesis (or) low levels of lipolysis which are in

turn controlled by insulin.

Serum total proteins level

The fractions AFSF, MF1SF and MF2SF at 200mg/kg showed a significant increase in

serum total protein level with a percentage of 65.0, 43.18 and 41.14 respectively. In

Glibenclamide treated group, it was 48.7%. Though the statistically significant effect of

the fractions and reference drug were comparable to each other, the effect of AFSF was

greater than that of the other two fractions and Glibenclamide.

From the above findings it is confirmed that these fractions have antidiabetic activity

and are capable of correcting the altered biological parameters.

159

Table 24: Effect of fractions on blood glucose levels in euglycemic rats.

Group No.

Treatment Dose (mg/kg)

Blood glucose levels (mg/dl) at different hours 0h 2h 4h 6h 8h 12h 24h

I Control --- 80.5 + 9.51 80.5 + 7.62 (0.12%)

78.4 + 8.01 (2.6%)

78.3 + 9.10 (2.73%)

79.3 + 8.01 (1.49%)

77.5 + 8.29 (3.7%)

80.8 + 8.08 (0.37%)

II Glibenclamide 10 84.1 + 8.06 60.7 + 3.5 b (27.82%)

51.5 + 6.2 c (38.7%)

38.0 + 6.1c (54.8%)

48.5 + 8.4c (42.3%)

61.3 + 8.0 a (27.1%)

73.5 + 7.2 (12.6%)

III AFSF 100 90.2 + 20.1 67.9 + 10.1 (24.7%)

60.3 + 11.5 b (33.2%)

48.8 + 10.9 c (45.9%)

54.6 + 10.3c (39.47%)

63.6 + 10.1 a (29.5%)

74.6+ 13.52 (5.3%)

IV AFSF 200 85.6 + 7.9

61.7 + 5.9 b (27.9%)

53.9 + 5.6 c (37.0%)

39.3 + 7.1c (54.1%)

51.1 + 7.3c (39.1%)

62.8 + 7.3 a (26.6%)

73.7 + 6.9 (13.9%)

V AFSF 400 85.0 + 8.2 62.9 + 5.8 a (26%)

53.6 + 8.6 c (36.9%)

40.8 + 7.1c (52%)

50.8 + 6.0c (40.2%)

63.2 + 7.3 a (25.6%)

79.6 + 6.9 (6.35%)

VI MF1SF 100 85.5 + 13.9 71.6 + 12.3 (16.25%)

66.6 + 12.21 (22.1%)

55.6 + 12.2b (34.9%)

63.5 + 10.3a (25.7%)

70.5 + 9.1 (17.5%)

76.5 + 11.9 (10.5%)

VII MF1SF 200 85.0 + 8.39 66.8 + 11.3 (21.4%)

61.5 + 8.5 a (27.6%)

44.8 + 7.4 c (47.3%)

56.8 + 6.8 c (33.1%)

66.5 + 7.5 (21.7%)

74.1 + 6.5 (12.9%)

VIII MF1SF 400 86.5 + 10.53

67.9 + 8.1 (21.5%)

59.9 + 10.7 b (30.6%)

46.3 + 8.9 c (45.9%)

59.5 + 8.6 b (31.2%)

68 + 8.1 (21.3%)

81.6 + 8.5 (5.6%)

IX MF2SF 100 86.4 + 13.4 70.6 + 12.12 (18.2%)

67.3 + 10.1 (22.4%)

56.6 + 12 c (34.4%)

64.5 + 11.3 a (25.3%)

70.3 + 11.1 (18.9%)

77.3 + 12.1 (10.8%)

X MF2SF 200 85.3 + 10.2 65.5 + 11.2 (22.9%)

59.3 + 8.9 b (30.5%)

48.55 + 7.8 c (43%)

59.3 + 10.8 b (30.5%)

65 + 6.5 (23.5%)

79.8 + 19 (6.4%)

XI MF2SF 400 85.0 + 8.21 66.4 + 10.8 (21.6%)

60.4 + 6.31 b (28.9%)

50.67+10.1c (41%)

60.9 + 9.7 b (28.3%)

65.9 + 7.3 (22.4%)

80.2 + 9.5 (5.6%)

All Values are expressed as Mean + SD, n = 6; Figures in parenthesis indicate the percentage hypoglycemia when compared to ‘0’ h

values(s), P value- a <0.05; b <0.01; c <0.001when compared with control at the respective time interval.

160

Fig 10: Effect of fractions on blood glucose levels in euglycemic rats

161

Table 25: Effect of fractions on oral glucose tolerance in normal rats

Group No.

Treatment Dose (mg/kg)

Blood glucose levels (mg/dl) at different time intervals 0min 30min 60min 90min 120min

I Control --- 85.7 + 5.0 128.1 + 1.5 (49.4%)

119.7 + 1.1(39.67%)

103.0 +1.3(20.18%)

88.9 + 2.56(3.7%)

II Glibenclamide 10 84 +5.48 135.1 + 10.8 (61.9%)

111.7 + 11.5(32.9%)

80.9 + 2.5c

(3.6%) 73.0 + 0.9 c

(13.09%) III AFSF 100 83.1 + 8.45 136.1 + 9.1

(63.7%) 115.2 + 10.3

(38.7%)87.8 + 10.1a

(5.7%)72.5 + 10.6 b

(12.76%)IV AFSF 200 83.6 + 5.75 135.6 + 9

(61.4%) 112.8 + 9.6

(34.9%)82.0 + 2.9 c

(1.9%)73.2 + 0.08 c

(12.4%)V AFSF 400 84.51 + 5.21 135.0 + 8.5

(59.7%) 115.75 + 9.1

(36.98%) 86.15 + 8.5 a

(1.76%) 73.9 + 1.21 c

(12.5%) VI MF1SF 100 84.5 + 8.15 136.5 + 8.0

(61.5%) 120.7+9.0(42.8%)

90.0+ 10(15.6%)

72.9 + 8.9 a

(13.75%)VII MF1SF 200 84.3 + 6.79 135.81 + 1.1

(60.1%) 115.1 + 8.5

(36.4%)85.9 + 9.07 b

(1.89%)74 + 1.38 c

(12.2%)VIII MF1SF 400 83.6 + 7.5 139.1+ 10.6

(66.2%) 114.6 + 10.5

(37.08%)88.8 + 10.1 a

(6.2%)73.6 + 8.6 b

(13.16%)IX MF2SF 100 84.5 + 8.10 137.5 + 10.8

(62.7%) 120.0 + 10.5

(42.0%)91.9 + 11.1

(8.75%)74.8 + 8.19 a

(11.47%)X MF2SF 200 85.17 + 6.9 137.8 + 12.1

(60.85%) 118.5 + 8.5

(38.2%)84.13 + 8.01 b

(1.2%)75.5 + 2.5 b

(11.1%)XI MF2SF 400 83.28 + 7.31 134.0+ 10.0

(60.9%) 116.1 + 10.0

(39.4%)86.11 + 10.4 a

(3.4%)73.4 + 8.5 c

(11.86%)All Values are expressed as Mean + SD, n = 6; Figures in parenthesis are increase or decrease in blood glucose levels when compared to ‘0’ min value (s) P value- a <0.05; b <0.01; c <0.001 when compared with control at the respective time interval.

162

Fig 11: Effect of fractions on blood glucose levels on oral glucose tolerance in

normal rats

163

Table 26: Effect of fractions on blood glucose levels in Alloxan induced diabetic rats

Group No.

Treatment Dose

(mg/kg)

Blood glucose levels (mg/dl) at different hours

0h 2h 4h 6h 8h 12h 24h

I Control --- 279.4 + 2.5 280.4 + 2.3 (0.35%)

281.3 + 3.1 (0.68%)

281.4 +6.1 (0.72%)

281.8 + 3.1 (0.85%)

280.4 + 3.2 (0.35%)

280.6 + 2.8 (0.43%)

II Glibenclamide 10 280.3 + 1.6 235.0 + 4.8c (16.6%)

214.0 + 5.0c (23.65%)

186.5 + 3.2c

(33.5%) 198.3 + 4.2c

(29.3%) 216.0 + 9.3 c

(22.9%) 239.1 + 9.2 c

(14.7%) III AFSF 100 280.4 + 2.1 240 + 1.6 c

(14.4%) 219.0 + 2.1c

(21.8%) 190.8 + 2.2c

(31.9%) 200.0 + 2.3c

(28.6%) 221.5 + 3.2 c

(21.0%) 242.4 + 2.3c

(13.5%) IV AFSF 200 280.5 + 3.9

236.5 + 3.2c

(15.6%) 215.3 + 4.5c

(23.3%) 187.7 + 3.3c

(33.1%) 199.4 + 3.2c

(28.9%) 217.6 + 4.3 c

(22.4%) 239.9 + 5.8c

(14.4%) V AFSF 400 284.3 + 3.5 253.0 + 3.5c

(11%) 229.8 + 2.3c

(19.16%) 196.6 + 4.8c

(30.8%) 214.5 + 4.2c

(24.6%) 220.5 + 2.5 c

(22.4%) 253.5 + 6.3c

(10.8%) VI MF1SF 100 280.6 + 2.4 250.6 + 4.3c

(10.7%) 225.0 + 7.8c

(19.8%) 199.6 + 9.3c

(28.8%) 204.4 + 8.5c

(27.2%) 229.6+ 8.8 c

(18.1%) 246.6 + 8.8c

(12.1%) VII MF1SF 200 280.5 + 1.4 240.2 + 4.01c

(14.4%) 220.4 + 4.8c

(21.4%) 190.6 + 5.1c

(32%) 202.4 + 8.3c

(27.8%) 221.0 + 10.3 c

(21.2%) 243.3 + 10.3c

(13.3%) VIII MF1SF 400 282.3 + 1.6

258.1 + 3.6c

(8.6%) 214.9 + 2.1c

(23.8%) 199.9 + 5.6c

(29.1%) 215.3 + 4.6c

(23.8%) 223.3 + 4.6 c

(20.9%) 245.0 + 3.8c

(13.5%) IX MF2SF 100 280.3 + 1.5 255.4 + 4.1c

(8.7%) 227.1 + 6.8c

(18.9%) 198.3 + 3.6c

(29.2%) 205.3 + 5.9c

(26.7%) 227.5 + 8.3 c

(18.7%) 247.5 + 5.8c

(11.6%) X MF2SF 200 281.4 + 1.6 242.1 + 2.9c

(13.8%) 221.4 + 4.3c

(21.3%) 189.6 + 5.8c

(32.5%) 200.7 + 8.5c

(28.5%) 223.5 + 10.4 c

(20.4%) 244.4 + 10.2c

(13.1%) XI MF2SF 400 282.5 + 1.5 256.2 + 3.5c

(9.3%) 216.4 + 1.8c

(23.39%) 199.4 + 5.9c

(29.5%) 215.5 + 3.6c

(23.7%) 220.5 + 4.6 c

(21.94%) 249.5 + 2.8 c

(11.6%) All Values are expressed as Mean + SD, n = 6; Figures in parenthesis indicate the percentage hypoglycemia when compared to ‘0’ h values(s), P value- a <0.05; b <0.01; c <0.001 when compared with control at the respective time interval.

164

Fig 12: Effect of fractions on blood glucose levels in alloxan induced diabetic rats.

165

Table 27: Effect of fractions on body weight and blood glucose levels in alloxan induced diabetic rats in Sub acute study

(21 days)

Groups Dose

(mg/kg

b.w)

Body weight in grams Blood glucose levels (mg/dl)

1st day After 1st day After

7 days 14 days 21 days 7 days 14 days 21 days

Diabetic control-I - 220.6±6.2 207.5±9.4 190.4±8.5 179.0±5.9 359.8±2.6 360.1±3.5

(0.08%)

362.5±2.5

(0.75%)

361.5±2.04

(0.08%)

Glibenclamide-II 10 219.3±10.8 230.2±9.03 c 244.4±8.9c 256.3±10.8c 363.5±6.8 334.5±2.5c

(7.97%)

291.5±6.9c

(19.8%)

251.5±4.9c

(30.81%)

AFSF-III 200 219.9±10.6 229.0±8.9 c 242.4±5.3c 250.4±8.3c 364.3±4.8 334.4±5.2c

(8.2%)

304.4±1.8c

(16.44%)

254.2±2.6c

(30.2%)

MF1SF- IV 200 218.4±11.5 225.8±7.6 b 238.4±6.5c 246.5±5.6c 366.5±4.81 336.3±5.6c

(8.3%)

306.5±4.0c

(16.2%)

257.6±4.3c

(29.7%)

MF2SF -V 200 219.5±

10.6

227.6±1.8c 239.0±7.5c 249.5±5.8c 365.4±4.9 339.4±5.9c

(7.7%)

306.6±8.0c

(16%)

258.6±3.8c

(29.1%)

All values are expressed as Mean±SD, n=6; Values given in the parenthesis are percent blood glucose reduction when compared to 1st day value(s); P value- a <0.05; b <0.01; c <0.001 when compared with control at the respective time interval.

166

Fig 13: Effect of fractions on body weight in Alloxan induced diabetic rats (sub

acute study)

Fig 14: Effect of fractions on blood glucose levels in Alloxan induced diabetic rats (sub acute study)

167

Table 28: Effect of fractions on different serum biochemical parameters in Alloxan induced diabetic rats in Sub acute study

(21 days).

Groups Dose (mg/kg

b.w)

Serum Insulin levels in µIU/ml

SGOT (U/L) SGPT (U/L) Serum Triglyceride levels (mg/dl)

Serum Cholesterol (mg/dl)

Serum Total protein (g/dl)

1 st day

After 21days

1 st day

After 21days

1 st day

After 21days

1 st day

After 21days

1 st day

After 21days

1 st day

After 21days

Diabetic control- I

- 10.7±0.6 10.9±0.6 (1.86%)

111.6±3.0 112.5±4.2 (0.8%)

74.6±2.8 75.6±2.1 (1.3%)

180.8±1.5 181.2±1.8 (0.22%)

122.4±6.0 122.9±3.5 (0.4%)

4.2±0.5 4.3±0.4 (0.7%)

Glibenclamide - II

10 11.2±0.6 18.9±1.9 (68.75%)

112.6±2.5 71.7±6.3c (36.3%)

72.7±2.3 42.3±3.3c (41.8%)

172.5±2.9 152.3±3.0c (11.7%)

121.2±4.3 67.5±10.3c (44.3%)

3.9±0.2 5.8±0.7c (48.7%)

AFSF- III 200 11.3±0.3 19.0±2.2 c (68.1%)

112.5±2.1 69.2±2.9c (38.48%)

73.9±2.0 42.6±4.8c (42.27%)

171.5±2.6 150.6±2.6c (12.18%)

121.3±3.2 71.4±8.3c (41.13%)

4.1±0.2 6.0±0.7c (65.34%)

MF1SF - IV 200 11.4±0.4 19.1±2.8 c (64.6%)

112.5±3.6 70.8±4.6c (37.06%)

72.6±1.7 41.7±2.2c (42.56%)

170.5±4.8 149.1±3.9c (12.55%)

120.5±4.04 72.1±8.4c (40.16%)

4.4±0.3 5.7±0.5c (43.18%)

MF2SF - V 200 11.3±0.6 19.4±4.0 c (71.68%)

111.8±2.9 71.5±4.0c (36.05%)

73.6±1.8 42.6±1.9c (42.12%)

174.6±4.9 153.2±4.2c (12.26%)

120.1±3.61 72.1±7.4c (39.96%)

4.4±0.3 5.3±0.32a (41.14%)

All values are expressed as Mean±SD, n=6; Values given in the parenthesis are the percent increase or decrease in respective parameter level;

P value – a <0.05; b <0.01; c <0.001 when compared with control at the respective time interval.

168

Fig 15: Effect of fractions on Serum Insulin Levels in Alloxan induced diabetic rats

Fig 16: Effect of fractions on SGOT Levels in Alloxan induced diabetic rats

169

Fig 17: Effect of fractions on SGPT Levels in Alloxan induced diabetic rats

Fig 18: Effect of fractions on Serum Triglyceride Levels in Alloxan - induced diabetic rats

170

Fig 19: Effect of fractions on Serum Cholesterol Levels in Alloxan - induced diabetic rats

Fig 20: Effect of fractions on Serum Total Protein Levels in Alloxan - induced diabetic rats

171

3.6. SCREENING OF THE FRACTIONS FOR ALDOSE REDUCTASE

INHIBITORY ACTIVITY IN IN VITRO AND IN VIVO MODELS

3.6.1 Assessment of Aldose reductase inhibitory activity of fractions using rat

lens homogenate.

From Albumin standard graph (Fig 21), the protein concentration of rat lens

homogenate was found to be 20µg/ml and it was calculated with the dilution factor,

100. The final concentration obtained was 2mg/ml. The enzyme activity as well as the

specific activity of the lens homogenate was calculated and they were found to be

14.11U/ml and 7.06U/mg, respectively.

Enzyme inhibitory values (ARI %) and IC50 values (µg/ml) of the test compounds

against lens homogenate are given in Table 29. All the readings were expressed as

Mean±SD. IC50 value of quercetin, which is taken as standard, was found as 1.35±0.08

µg/ml. The IC50 values of AFSF, MF1SF MF2SF and Glibenclamide were found as 7.59

+0.02, 15.6+0.83, 29.75+0.71 and 57.11+0.75 µg/ml respectively. These values

indicate that the ARI activity of AFSF is better than that of MF1SF, MF2SF. The plant

has potential aldose reductase inhibiting activity. The ARI activity of all the samples

tested was dose-dependent. There is no significant ARI activity for Glibenclamide.

The results obtained clearly indicate that AFSF, MF1SF and MF2SF of Soymida

febrifuga showed ARI activity. ARI activity is more for AFSF when compared to

MF1SF, MF2SF and Glibenclamide employed in the present study.

3.6.2 Assessment of Aldose reductase inhibitory activity of fractions using rat

kidney homogenate

The protein concentration of kidney homogenate was obtained from albumin standard

graph (Fig-21) and it was found to be 2.2 mg/ml, after calculating with the dilution

172

factor, 100. Enzyme activity as well as the specific activity of the kidney homogenate

was found to be 9.68U/ml and 4.39U/mg, respectively.

The enzyme inhibitory values (ARI %) as well as the IC50 values (µg/ml) of the test

samples against kidney homogenate are given in Table-30. All the readings were

expressed as Mean±SD. IC50 value of quercetin, which is taken as standard, was found

to be 1.49±0.001 µg/ml. IC50 values of AFSF, MF1SF and MF2SF were found to be

12.5±0.07, 24.19+0.29 and 22.09 ±0.13 µg/ml. The IC50 value of AFSF is less than that

of MF1SF and MF2SF.The results confirm that AFSF possesses significant ARI

activity as indicated by its IC50 value. The plant possesses good ARI activity. However

IC50 value of Glibenclamide was found as 64.73±0.08 µg/ml. It has no significant ARI

activity against rat kidney homogenate.

3.6.3 Effect of fractions on body weight and blood glucose levels in experimental

Animals

Table 31 depicts the change in weight of all the animals during experimental period.

All the values were given in Mean±SD. All the test samples treated groups were

compared to that of control group, which is treated only with galactitol. There is a

significant increase in the body weight of animals treated with test compounds when

compared to control.

The blood glucose estimation was determined on day 1 and day 15 using blood serum

samples in normal rats fed with galactose. The changes in the blood glucose levels of

the experimental animals during the study period were recorded in Table 32. There is a

significant decrease in blood glucose level in rats treated with all test samples when

compared to control. The hypoglycemic activity of AFSF is similar to that of

Glibenclamide. The blood glucose level decreasing activity of the test groups was given

in their decreasing order of activity

173

3.6.4 Assessment of Aldose reducatase inhibitory activity of fractions in invivo

models by HPLC analysis

The derivatized lens homogenates were analyzed by HPLC (Fig 23-29) and the peak

heights of samples were noted. By using the standard graph of galactitol (Fig 22)

concentrations of the test samples were calculated and the values were given in

Mean±SD (Table-33). The galactitol content of the standard, test fractions and control

is shown in Fig 30. The galactitol concentration of quercetin (10mg/kg) was found to

be 10.66+0.82 µg/ml. In Glibenclamide treated animals galcatitol concentration was

found to be 20.86+0.34 µg/ml In the groups treated with AFSF, MF1SF and MF2SF

(200 mg/kg bw), galactitol concentration was 3.59+0.18, 11.37+0.99 and 13.08+0.61

µg/ml.

There is a significant decrease (P<0.001) in galactitol concentration with quercetin

treated group. Similarly there is a significant decrease (P<0.001) in galactitol

concentration with AFSF, MF1SF and MF2SF treated groups compared to control

group. However the galactitol concentration in Glibenclamide treated group was found

to be higher than that of control. So it has no significant ARI activity.

174

Fig 21 . Standard Graph of Albumin

175

Table 29: Effect of fractions on Aldose Reductase Inhibitory activity on Rat lens

homogenate

S.No Test sample Concentration

(µg/ml)

ARI (%)

IC50

(µg/ml)

1 Quercetin

0.5 35.76±0.47

1.35±0.08 1 57.43±0.65

5 70.60±0.47

10 91.38±0.57

2 AFSF

5 39.68±0.26

7.59±0.02 10 58.26±0.28

50 73.58±0.35

100 89.14±0.68

3 MF1SF

5 28.65+0.35

15.6+0.83 10 48.36+0.61

50 69.10+0.29

100 75.46+0.72

4 MF2SF

5 29.48±0.61

29.75±0.71 10 49.62±0.83

50 64.66±0.43

100 74.64 ±0.47

5 Glibenclamide

0.5 0.90 ±0.04

57.11±0.04 1 2.35 ±0.02

5 4.38 ±0.03

10 9.69 ±0.01

All values are expressed as Mean±S.D, n=3.

The test compounds were given in increasing order of IC50 values. Quercetin < AFSF< MF1SF < MF2SF < G

176

Table 30: Effect of fractions on Aldose Reductase Inhibitory activity on Rat

Kidney homogenate

S.No Test Sample Concentration

( µg/ml)

ARI (%)

IC50 (µg/ml)

1 Quercetin

0.5 35.65±0.44

1.49±0.001 1 55.90±0.30

5 70.54±0.42

10 92.38±0.50

2 AFSF

5 34.62±0.28

12.5±0.07 10 59.32±0.28

50 71.51±0.36

100 90.39±0.48

3 MF1SF

5 30.62+0.34

24.19+0.29 10 45.32+0.42

50 74.51+0.38

100 89.69+0.33

4 MF2SF

5 32.11±0.42

22.09±0.13 10 50.73±0. 61

50 68.51±0.48

100 90.11±0.26

5 Glibenclamide

0.5 0.63±0.01

64.73±0.08 1 1.93±0.04

5 4.12±0.02

10 8.36±0.03

All values are expressed as Mean±S.D, n=3.

The test compounds were given in increasing order of IC50 values:

Quercetin< AFSF < MF2SF < MF1SF < G

177

Table 31: Effect of fractions on Body weight of experimental animals

S.No Treating Groups Initial weights(g) Final weights(g)

1 Control 196.5±2.74 232.33±5.16

(18.23%)

2 Quercetin 196.5±2.74 245.67±5.2c

(25.02%)

3 AFSF 195.67±8.2 247.33±4.08c

(26.4%)

4. MF1SF 195.67+8.16 234.10+5.48

(19.60%)

5. MF2SF 196.07±5.02 249.03±6.31c

(27.26%)

6. Glibenclamide 196.5±2.74 227.33±4.08

(15.69%)

All values are expressed as Mean±S.D, n=6, P value- a <0.05; b <0.01; c <0.001 when

compared with Control.

178

Table 32: Effect of fractions on Blood Glucose Levels in experimental animals

S.No Group Initial

(On 1st day)

Final

(On 15th day)

1 Control 77.65+1.21 205.17+1.02

2 Quercetin 75.81 +1.32 43.08+1.17c

(43.17 %)

3 AFSF 73.56 +1.21 33.30+ 1.26c

(54.73%)

4

MF1SF 72.76+1.01

44.93+1.2c

(38.25%)

5 MF2SF 71.63 + 0.99 45.48+ 1.04c

(36.5%)

6 Glibenclamide 73.43 + 0.63 32.30+ 1.21c

(56.0%)

All values are expressed as Mean±SD, n=6, P value- a <0.05; b <0.01; c <0.001 when

compared with Control.

The blood glucose level decreasing activity of the test groups was given in their

decreasing order of activity:

Glibenclamide > AFSF > Q > MF1SF > MF2SF

179

Fig 22: Glactitol Standard Graph

0

2000000

4000000

6000000

0 20 40 60

y = 103148x + 186253R2 = 0.9951

180

Fig 23: HPLC of Standard Galactitol

Time (min)

Fig 24: HPLC of lens homogenate of Control group

Time (min)

mA

U

mA

U

181

Fig 25: HPLC of lens homogenate of Quercetin treated rats

Time (min)

Fig 26: HPLC of lens homogenate of AFSF treated rats

Time (min)

mA

U

mA

U

182

Fig 27: HPLC of lens homogenate of MF1SF treated rats

Time (min)

Fig 28: HPLC of lens homogenate of MF2SF treated rats

Time (min)

mA

U

mA

U

183

Fig 29: HPLC of lens homogenate of Glibenclamide treated rats

Time (min)

mA

U

184

Table 33: Estimation of lens Galactitol levels by HPLC

S.No Groups Peak height Galactitol Concentration

(µg/ml)

1 Control 1952362±60159.2 20.69±0.66

2 Quercetin 1043126±74744.31 10.66±0.82c

3 AFSF 402732.3+16507.73 3.59+0.18c

4. MF1SF 1107936±90293.38 11.37+0.99c

5 MF2SF 1262811±55329.84 13.08±0.61c

6 Glibenclamide 1705143±30775.6 20.86±0.34

All values are expressed as Mean±SD, n=6, P value- a <0.05; b <0.01; c <0.001 when

compared with Control.

Fig 30: Comparison of galactitol content of test groups with control

185

3.7 SCREENING OF THE FRACTIONS FOR HEPATOPROTECTIVE AND

ANTI HEPATOTOXIC ACTIVITY

3.7.1 Evaluation of hepatoprotective activity of fractions in CCl4 induced acute

hepatotoxicity in rats

CC14 induced acute hepatotoxicity in rats causes alterations in the serum biochemical

parameters as well as in the normal cellular architecture of liver. The results of the

study are shown in Tables 34, 35 and Figs 31,32,33,34 and 35.

Biochemical parameters: The evaluation of the preventive action in liver damage

induced by CC14 has been used widely as an indicator of the liver protective ability of

drugs in general, even if CCl4 liver injury resembles the damage due to acute viral

hepatitis.

In the present investigation, rats treated with CCl4 developed severe hepatic damage

that was observed as a significant increase (P<0.001) in the serum levels of GPT, GOT,

ALP and TB. This might be due to release of the enzymes (SGPT, SGOT and SALP)

from the cytoplasm of hepatic cells into the blood circulation rapidly after the rupture

of the plasma membrane and cellular damage resulting from the CC14 induced lipid

peroxidation . Treatment with fractions, AFSF, MF1SF and MF2SF at 50 and l00mg/kg

b.w p.o. statistically (P<0.001) reduced the levels of these marker enzymes in CC14

treated rats in hepatoprotective studies. CCl4 intoxication elevated the levels of

hepatospecific markers (SGPT, SGOT, and ALP). Silymarin (50mg/Kg b.w)

significantly (P<0.001) reduced the elevated levels of these parameters. The percentage

recovery was 92.79% in SGPT, 94.59% in SGOT and 70.87% in ALP levels. There

was a significant (P<0.001) reduction in biochemical parameters in groups treated with

AFSF, MF1SF and MF2SF. The percentage recovery observed with AFSF (100mg/kg)

was better than the remaining fractions. The % recovery values are 94.22% in SGPT,

97.12% in SGOT, 73.97 in ALP and further, AFSF (100mg/kg) offered better

protection when compared to Silymarin (50 mg/kg).

186

The decrease in the levels of these enzymes may be a consequence of the stabilization

of plasma membrane as well as the repair of hepatic tissue damage caused by CC14. In

CC14 induced hepatotoxicity, elevation in serum TB level is due to defective excretion

of bile by the liver, indicating the loss of integrity of liver and necrosis. AFSF, MF1SF

and MF2SF at 50 and 100 mg/kg showed a significant depletion in serum TB levels

suggesting the possibility of the fractions, ability to persist or repair the damage of the

hepatocytes caused by CC14 in prophylactic and curative studies.

The maximal percentage reduction in the biochemical parameters was observed with all

the fractions at 100mg/kg b.w.p.o. Further, when compared to standard, AFSF at

1oomg/Kg offered better protection.

Histopathology: The photomiographs (Motic, Germany) of sections revealed

histopathological profile of CC14 intoxicated rats. They showed drastic alterations in

the histoarchitecture, i.e, fatty changes, necrosis, lymphocyte infiltration, dilatation of

sinusoidal spaces, ballooning degeneration and bleeding area in hepatic lobes. In

hepatoprotective studies of fractions, AFSF, MF1SF and MF2SF at l00mg/kg b.w.p.o

showed definite signs of protection and recovery against CC14 injury. Of the three test

fractions, AFSF at l00mg/kg b.w.p.o exhibited a remarkable recovery, towards

normalization of histological architecture of liver of the rats, which was almost similar

to that of Silymarin (50 mg/kg).

It is evident from the results of the study that the improvement in hepatospecific

biochemical parameters caused by AFSF at l00mg/kg b.w.p.o was well supported by

the improvement in histopathological findings, indicating recovery of the functional

status of the liver by the fractions.

187

3.7.2 Evaluation of antihepatotoxic activity of the fractions in CC14 induced acute

hepatotoxicity in rats.

In this study, prior intoxication of rats with CC14 caused hepatotoxicity, which was

evident from the elevated serum hepatospecific enzymes and bilirubin levels and

changes in the histology of liver of the rats. The results of the study are shown in

Tables 36 and 37, Figs 36,37,38,39 and 40.

Biochemical parameters: Both the standard drug (50mg/kg) and all the test fractions

(50 and 100 mg/kg) showed a significant (P<0.001) curative effect as evident from the

reduction in elevated SGPT, SGOT, SALP and serum TB levels. Standard drug

significantly reduced (P<0.001) the elevated levels of hepatospecific enzymes. It also

reduced the TB levels. The corresponding recovery values of standard are 94% in

SGPT, 94.33% in SGOT, 65.8% in ALP and 86.07% in TB levels. The groups treated

with AFSF, MF1SF and MF2SF also showed significant (P<0.001) reduction in the

observed biochemical parameters. Among them AFSF (100 mg/kg) offered better

protection even when compared to standard and the % recovery values are 97.13 in

SGPT, 97.38 in SGOT, 67.47 in ALP and 89.35 in TB levels. Among these bioactive

fractions, AFSF at 100 mg/kg exhibited a marked recovery by bringing SGPT, SGOT,

SALP, and Serum TB levels to near normal. It was even better than that of the

reference drug, Silymarin.

Histopathological studies: The same dose of CC14 intoxication of rats on alternate

days caused a severe liver damage involving disarrangement and degeneration of

hepatic cells with an intense centrilobular necrosis and fatty degeneration in other

areas. In this study, the test fractions at both the test doses showed a different degree of

curative effect, with a remarkable recovery from centrilobular necrosis and varying

degree of fatty degeneration. It is also evident from the study that AFSF (100mg/kg)

was the only test fraction, which restored the cellular architecture of the liver to near

normal and its effect was similar to that of the standard, Silymarin.

188

3.7.3 Effect of fractions on Thiopentone induced sleeping time of rats in CCl4

Induced hepatotoxicity

The hepatoprotective effect of the fractions AFSF, MF1SF and MF2SF was

substantiated in thiopentone sodium sleeping time experiment in rats. It has been

established that since the barbiturates are metabolized almost exclusively in the liver,

the sleeping time after a given dose is a measure of hepatic metabolism. When

thiopentone sodium, a barbiturate, is given to rats having hepatotoxicity induced by

CCl4, it causes an enhancement of sleeping time due to the delay in barbiturate

metabolism as a result of hepatic injury and resultant decrease in cytochromeP450-

mediated metabolic functional activity of hepatocytes. The results of this study are

given in Table 38 and Fig 41.

Thiopentone sodium at a dose of 25mg/kg (i.p.) caused sedation in rats of control group

for a period of 28.50±2.30 min. In toxic group, CC14 induced liver injury led to a

significant (P<0.001) increase in the duration of Thiopentone sodium induced sleeping

time(120.3+3.99 min). Pretreatment of rats with the fractions AFSF,MF1SF and MF2SF

at a dose of l00mg/kg significantly (P<0.001) shortened the Thiopentone sodium

induced sleeping time of rats in CC14 induced liver damage to 31.22+1.83,36.39+3.11

and 33.11+2.31 respectively as compared to the toxic group. Based on the percentage

reduction in sleeping time of the rats, the effect of AFSF at l00mg/kg was greater than

that of the reference drug, Silymarin(50mg/kg). As the fractions, AFSF, MF1SF and

MF2SF at l00mg/kg b.w.p.o were able to reduce the thiopentone sodium-induced

sleeping time of rats in CCl4 induced hepatotoxicity, it indicates that the fractions

possess hepatoprotective activity. The effect of the fractions may be due to their ability

to protect cytochrome P450, a xenobiotic metabolizing enzyme system in liver. The

protective effect of AFSF was found to be better than that of Silymarin(50mg/kg), a

hepatoprotectant.

189

3.7.4. Evaluation of hepatoprotective activity of fractions in Paracetamol induced

hepatotoxicity in rats

The fractions AFSF, MF1SF and MF2SF were evaluated for their hepatoprotective

effect in drug induced (paracetamol) hepatotoxicity in rats. The results are depicted in

Table 39 and 40, Fig 42,43,44,45 and 46.

Paracetamol is a common analgesic and antipyretic agent, safe in therapeutic doses that

can produce fatal hepatic, renal tubular necrosis in humans and experimental animals at

toxic doses. Hepatotoxicity of paracetmol has been attributed to the formation of toxic

metabolite, N-acetyl-p-benzoquinoneimine. Due to the liver injury the transport

function of the hepatocytes gets disturbed, resulting in leakage of the plasma

membrane, thereby increasing hepatospecific enzymes (SGPT, SGOT, SALP) and TB

in serum. Protection against Paracetamol induced toxicity has been used as a test for

potential of hepatoprotective activity.

In this study, the hepatic damage produced byParacetamol is evident from the

significantly (P<0.001) increased enzymes (GPT, GOT, ALP) and total bilirubin levels

in the serum of rats of toxic group. Pretreatment of rats with AFSF (50 and 100mg/kg

b.w), MF1SF (50 and 100mg/kg b.w) MF2SF (50 and 100mg/kg b.w) and standard

(50mg/kg b.w) for 7 days before Paracetamol administration, resulted in a significant

(P<0.01 to 0.001) protection against Paracetamol induced elevation in the levels of

serum hepato specific parameters mentioned above. The maximal protective effect with

respect to the four biochemical parameters was observed with AFSF 100mg/kg b.w,

which is greater than that of the reference drug, Silymarin (50mg/kg b.w).

Histopathological studies: The histological photographs of livers of the rats used in

the study are shown in Fig.44. Histological profile of the control rats showed normal

hepatocytes. Rats treated with Paracetamol exhibited intense centrilobular necrosis,

macro vesicular fatty changes. The liver sections of the animals treated with the

fractions AFSF, MF1SF and MF2SF as well as standard drug exhibited a remarkable

190

recovery from centrilobular necrosis with moderate accumulation of fatty lobules.

However, the protective effect offered by AFSF (100mg/kg) was relatively more than

that of the other test fractions and Silymarin (50mg/kg).

191

Table 34: Effect of Pre-treatment with fractions on different biochemical parameters in CCl4 induced liver damage in rats

GROUPS DOSE (mg/Kg) SGPT(U/I) SGOT(U/I) ALP (U/I) TB(mg/dl)

CONTROL - 29.01+6.56 30.18 + 2.52 184.9 + 3.30 0.31 + 0.19

TOXIC - 120.6+3.12d 148.8 + 6.85d 510.6 + 4.65d 2.11 + 0.15d

STANDARD 50 35.6 + 2.7c (92.79)

36.6 + 3.18c (94.59)

279.6 + 3.67c (70.87)

0.53 + 0.05c (87.78)

AFSF 50 40.36 + 2.7c (87.59)

40.31 + 1.35c (91.47)

300.62 +2.40c (64.59)

1.39 + 0.07c (40.0)

AFSF 100 34.3 + 2.1c (94.22)

33.6 + 2.57c (97.12)

269.5 + 3.91c (73.97)

0.42 + 0.14c (93.89)

MF1SF 50 43.63 + 1.06c (84.03)

42.58 + 1.49c (89.62)

334.4 + 3.67c (54.00)

1.21 + 0.07c (50.0)

MF1SF 100 38.61 + 1.60c (90.0)

38.43 + 1.98c (93.56)

280.6 + 2.69c (70.56)

0.59 + 0.07c (84.45)

MF2SF 50 41.39 + 1.01c (86.58)

46.38 + 2.84c (86.35)

306.0 + 2.45b (62.74)

1.00 + 0.02c (61.67)

MF2SF 100 36.1 + 5.71c (92.62)

37.18 + 1.67c (94.09)

275.1 + 2.69 c (72.28)

0.51 + 0.09c (89.45)

Data expressed as mean + SD, n = 6, values in parenthesis indicate percentage recovery.

P value-CCl4 vs. vehicle d <0.001; P value CCl4 vs. treatments a < 0.05; b<0.01; c<0.001

192

Fig 31: Effect of pre treatment with fractions on SGPT Levels in CCl4 induced Liver damage in rats.

Fig 32: Effect of pre treatment with fractions on SGOT Levels in CCl4 induced Liver damage in rats.

193

Fig 33: Effect of pre treatment with fractions on ALP Levels in CCl4 induced Liver damage in rats.

Fig 34: Effect of pre treatment with fractions on TB Levels in CCl4 induced Liver damage in rats.

194

Fig 35: Histopathalogical changes in hepatoprotective study

195

Table 35: Histopathological changes in liver of rats in hepatoprotective study (in CCl4 induced hepatotoxicity)

Microscopic

observation

Groups

Normal Toxic StandardAFSF

50

AFSF

100

MF1SF

50

MF1SF

100

MF2SF

50

MF2SF

100

Fatty changes -- ++++ -- + -- ++ + ++ +

Necrosis -- ++++ + + + ++ + ++ +

Lymphocytes

infiltration -- ++++ -- + -- ++ + + --

Dilatation of

sinusoidal

space

-- ++++ -- + -- ++ + + --

Bleeding area -- ++++ -- + -- ++ + + --

Ballooning

degeneration -- ++++ -- + -- + -- + --

Histopathological damage was expressed using the following score system:

-- absent; + few; ++ mild; +++ moderate; ++++ severe.

196

Table 36: Effect of Post treatment with fractions on different biochemical parameters in CCl4 induced liver damage in rats

GROUPS DOSE (mg/Kg) SGPT(U/I) SGOT(U/I) ALP (U/I) TB (mg/dl)

CONTROL - 20.93+1.98 19.01 + 1.54 127.6 + 4.88 0.25 + 0.27

TOXIC - 117.2+2.96d 130.1 + 1.86 d 430.1 + 6.27d 1.47 + 0.07 d

STANDARD 50 26.71 + 1.25 c

(94.0) 25.33 + 1.64 c

(94.33) 231.6 + 2.47 c

(65.8) 0.42 + 0.04c

(86.07)

AFSF 50 32.31 + 3.21c (88.19)

36.13 + 2.96c

(84.6) 249.1 + 4.63c

(59.86) 0.75 + 0.22c

(59.0)

AFSF 100 23.71 + 2.93c

(97.13) 21.91 + 3.08c

(97.38) 226.1 + 6.39c

(67.47) 0.38 + 0.12c

(89.35)

MF1SF 50 34.01 + 3.99c

(86.43) 35.12 + 5.16c

(85.48) 280.1 + 5.22c

(49.6) 0.81 + 0.22c

(54.1)

MF1SF 100 29.63 + 0.27c

(90.63) 28.71 + 2.03c

(91.26) 243.6 + 3.99c

(61.65) 0.52 + 0.17c

(77.87)

MF2SF 50 31.06 + 1.74c

(89.48) 32.63 + 2.47c

(87.75) 273.1 + 5.16c

(51.92) 0.73 + 0.09c

(60.66)

MF2SF 100 27.21 + 0.96c

(93.48) 26.31 + 1.29c

(93.43) 235.2 + 6.39c

(64.85) 0.46 + 0.12c

(82.79)

Data expressed as mean + SD, n = 6, values in parenthesis indicate percentage recovery.

P value-CCl4 vs. vehicle d<0.001; P value CCl4 vs. treatments a< 0.05; b<0.01; c<0.001

197

Fig 36: Effect of post treatment with fractions on SGPT Levels in CCl4 induced Liver damage in rats.

Fig 37: Effect of post treatment with fractions on SGOT Levels in CCl4 induced Liver damage in rats.

198

Fig 38: Effect of post treatment with fractions on ALP Levels in CCl4 induced Liver damaged in rats.

Fig 39: Effect of post treatment with fractions on TB Levels in CCl4 induced Liver

damage in rats.

199

Fig 40: Histopathological changes in liver of rats in Antihepatotoxic study

200

Table 37: Histopathological changes in liver of rats in Antihepatotoxic study

Microscopic

observation

Groups

Normal Toxic StandardAFSF

50

AFSF

100

MF1SF

50

MF1SF

100

MF2SF

50

MF2SF

100

Fatty changes -- ++++ -- + -- ++ + ++ +

Necrosis -- ++++ -- ++ -- ++ + ++ --

Lymphocytes

infiltration -- ++++ -- + -- + -- + --

Dilatation of

sinusoidal

space

-- ++++ + ++ + ++ + ++ +

Bleeding area -- ++++ -- + -- ++ + + +

Ballooning

degeneration -- ++++ -- + -- ++ + + --

Histopathological damage was expressed using the following score system:

-- absent; + few; ++mild; +++ moderate; ++++ severe.

201

Table 38: Effect of Pre-treatment with fractions on Thiopentone induced sleeping

time in rats.

Data expressed as Mean±SD, n=6, values in parenthesis indicate percentage recovery.

P value-CCl4 vs. vehicle d<0.001; p value CC14 vs.treatments- a <0.05; b <0.01; c <0.001

Fig 41: Effect of pre treatment of fractions on Thiopentone induced sleeping time

in rats

GROUPS DOSE (mg/kg) SLEEPING TIME (min)

Control - 28.50+5.63

Toxic - 120.3+9.77d

Standard 50 33.58+3.99 c

(92.27)

AFSF 100 31.22+4.48 c

(97.03)

MF1SF 100 36.39+7.61c

(91.38)

MF2SF 100 33.11+5.65c

(94.97)

202

Table 39: Effect of fractions on different serum biochemical parameters in Paracetamol induced liver damage in rats

GROUPS DOSE

(mg/Kg) SGPT(U/I) SGOT(U/I) ALP (U/I) TB(mg/dl)

CONTROL - 19.55+1.25 16.93 + 0.69 131.2 + 3.26 0.71 + 0.09

TOXIC - 51.21+3.40d 60.01 + 5.66 d 491.4 + 3.50 d 1.85 + 0.04d

STANDARD 50 21.13 + 3.74c

(95.0) 27.58 + 3.21c

(75.22) 231.7 + 3.21c

(72.1) 0.91 + 0.07c

(82.46)

AFSF 50 28.71+5.16c

(71.1) 31.71+2.96c

(65.74) 239.1+1.91c

(70.08) 1.28+0.12c

(50.0)

AFSF 100 20.71+2.03c

(96.34) 25.71+2.77c

(79.66) 226.1+4.19c

(73.69) 0.81+0.17c

(91.23)

MF1SF 50 24.39 + 1.49c

(84.71) 31.55 + 1.98c

(66.08) 241.2 + 3.69c

(69.52) 1.32 + 0.19c

(46.5)

MF1SF 100 22.18 + 1.49c

(91.7) 29.01 + 3.69c

(72.0) 234.1 + 5.17c

(71.47) 0.99 + 0.09c

(75.44)

MF2SF 50 28.88 + 1.84c

(70.53) 31.58 + 3.72c

(66.0) 240.3 + 4.53c

(69.8) 1.30 + 0.07c

(48.25)

MF2SF 100 22.0 + 2.08c

(92.26) 28.02 + 1.98c

(74.32) 232.0 + 3.94c

(72.02) 0.98 + 0.15c

(77.12)

Data expressed as mean + SD, n = 6, values in parenthesis indicate percentage recovery.

P value-Paracetamol vs. vehicle d <0.001; P value Paracetamol vs. treatments a < 0.05; b <0.01; c <0.001

203

Fig 42: Effect of pre treatment with fractions on SGPT Levels in Paracetamol induced Liver damage in Rats.

Fig 43: Effect of pre treatment with fractions on SGOT Levels in Paracetamol induced Liver damage in Rats.

204

Fig 44: Effect of pre treatment with fractions on ALP Levels in Paracetamol induced Liver damage in Rats.

Fig 45: Effect of pre treatment with fractions on TB Levels inParacetamol induced

Liver damage in Rats.

205

Fig 46: Histopathological changes in liver of rats in hepatoprotective study:

(drug induced hepatotoxicity)

206

Table 40: Histopathological changes in liver of rats in hepatoprotective study (drug induced hepatotoxicity)

Microscopic observation

Groups

Normal Toxic StandardAFSF

50

AFSF

100

MF1SF

50

MF1SF

100

MF2SF

50

MF2SF

100

Fatty changes -- ++++ -- + -- ++ + + --

Necrosis -- ++++ -- + -- ++ + + --

Lymphocytes

infiltration -- ++++ + + + + + + +

Dilatation of

sinusoidal space -- ++++ -- + + + + + +

Bleeding area -- ++++ -- + -- + + + --

Ballooning

degeneration -- ++++ -- + -- ++ + + --

Histopathological damage was expressed using the following score system:

-- absent; + few; ++ mid; +++ moderate; ++++ severe.