Report of the work done - V.G.Vaze...

Report of the work done

Adult male Wistar rats were used for the study. Diabetes was induced by alloxan

with a dose of 150mg/kg. The rats having fasting Blood sugar level 250mg/dl where

included in study. They were treated orally with Distilled water (NC), alloxan induced

(DC) Pongamia pinnata aquous extract (PPAqExt) (TD1) Pongamia pinnata alcoholic

extract PPAlcExt (TD2), Metformin (SD), DMSO (VC) over a period of three months.

The present study was undertaken to assess body weight, Sugar profile, Lipid profile,

antioxidant status and histopathology of diabetic animals.

The results are depicted in the following manner in present chapter.

1. Plant extraction:- The extraction procedure was repeated many times since

every time only 25 gms of the plant material could be extracted. Aqueous and

alcoholic extracts were obtained at multiple times. Every time the yield varies

slightly hence the total yield, average, and percentage yield are illustrated in

tabular form.

2. Body profile:- Three parameters were selected like Body weight, Food Intake

and water intake. All the parameters were documented before and after the study

and the profiles are presented.

3. Antioxidant Assay in vitro:- Assay was performed using a stable free radical 1,

1-diphenyl-2-picryl hydrazyl (DPPH) assay

4. Biochemical profile:-

a) Sugar profile includes three main parameters, Blood sugar fasting (BSF),

Glyco Hb (HbA1c) and Mean Blood Glucose (MBG).

b) Lipid Profile includes four parameters viz. total cholesterol (TC),

Triglycerides (TG), Low density lipoprotein (LDL), High Density

Lipoprotein (HDL).

5. Antioxidant Assay in vivo: - Assay was performed at the end of study. The

antioxidant enzymes assay like LPO, SOD and AR were performed form serum,

liver, nerve and lens homogenate.

6. Histopathological examination:- histopathology of the organs like pancreas,

liver was carried out in support of the biochemical parameters.

7.

Results:-

1. Plant extraction:-

The extraction procedure was repeated many times since every time only 25 gms

of the plant material could be extracted. Aqueous and alcoholic extracts were

obtained at multiple times. Every time the yield varies slightly hence the total

yield, average, and percentage yield are illustrated in tabular form.

2. In vitro antioxidant activity of plant extract by DPPH method:-

The extract was analyzed for their antioxidant activity so that it shouldn’t be toxic

to the experimental animals. The DPPH radical scavenging activity of Pongamia pinnata

stem extracts is shown in Figure 1. Among the extracts tested, ethanol extract had better

scavenging activity (EC50 value of 2.5 mg/ml) followed by Aqueous (EC50 value of 9

mg/ml). When compared to butylated hydroxytoluene (BHT) which had an EC50 value

of 2.0 µg/ml, the EC50 value of ethanol was quite high as compared to aqueous.

Sr. No Aqueous Extract

(gms)

Alcoholic extract

(gms)

1. 1.380 1.115

2. 0.758 1.545

3. 2.216 1.339

4. 1.400 1.291

5. 2.283 0.732

6. 0.913 0.864

7. 2.388 0.670

8. 0.922 0.636

9. 0.891 0.573

10. 1.071 0.669

Total 14.222 9.434

Average ± SD 1.4222 ± 0.6379 0.9434 ± 2.5814

% Yield 5.6 % 3.7%

Figure 1: DPPH radical scavenging activity of Pongamia pinnata stem extract at different

concentrations.

As with ethanol extract, even at 5 mg/ml concentration the percentage DPPH

scavenging was 83.6 %. Wheras same concentration at aqueous extract it showed 38.5%.

3.1.1 Sugar profile:-

Table 3.9: Blood sugar fasting values of normal and experimental groups at the end

of each month (of all three experiments) in mg/dl.

Exp.

Grp Preinduction Induction 1st Month 2nd Month 3rd Month

NC 68.64 ± 2.71 85.07 ± 2.13 52.46 ± 12.16 75.54 ± 12.46 89.65 ± 9.22

DC 76.97 ± 2.54 506.4 ± 10.95 331.49 ± 30.76 348.66 ± 49.51 339.28 ± 112.24

TD1 71.15 ± 4.45 399.86 ± 2.3 240.08 ± 59.76* 223.14 ± 55.97* 191.13 ± 34.71**

TD2 76.35 ± 4.59 467.4 ± 52.44 96.2 ± 41.2** 113 ± 22.44** 89.03 ± 8.4**

SD 79.2 ± 5.23 506.37 ± 72.52 100.27 ±10.87** 85.53 ± 3.83** 73.32 ± 5.66**

VC 80.75 ± 1.9 458.1 ± 59.85 341.1 ± 58.4 330.88 ± 77.51 324.6 ± 65.53

Data are expressed as Mean ± SD, n = 18. ANOVA followed by multiple comparison two tail “t” Test.

** P < 0.01 highly significant as compared treatment with Disease Control.

* P < 0.05 Significant as compared treatment with Disease Control.

0

10

20

30

40

50

60

70

80

90

100

1 5 10 20 30

% D

PP

H S

cav

eng

ing

Concentration in mg/ml

DPPH AssayAqueous

Alcohol

50

55

60

65

70

75

80

85

90

95

1 5 10 20 30

% D

PP

H S

cav

eng

ing

Concentration in ug/ml

DPPH Assay BHT

Figure 3.10: Comparative Blood Sugar fasting activities of each group for three

Months.

Table No: 3.9 and fig 3.10 shows that before inducing diabetes all the values of

Blood sugar fasting (BSF) were in normal range between 68.6 ± 2.21 up to 80.75 ± 1.9.

After inducing diabetes with Alloxan the values reached their peaks except in Normal

Control (NC) and the values are between 399.86 ± 2.3 up to 506.4 ± 10.95. In Disease

Control rats (DC) BSF level remained on higher side more than 300 mg/dl. Treatment

with both the doses of Pongamia pinnata Aqueous extract (PPAqExt) and Pongamia

Pinnata Alcoholic extract (PPAlcExt) produced reduction in the blood glucose level and

the values were 240 ± 9.76, 223.13 ±5.97, 191.13 ± 4.71 for Aqueous extract and 96.2 ±

41.2, 113 ± 22.4, 89.03 ± 8.4 for Alcoholic extract respectively. Highly significant

reduction was achieved with the alcoholic extract (P< 0.005). Whereas in Standard drug

(SD) Metformin the BSF values are 100.27 ± 0.87, 82.52 ± 3.83, 73.31 ± 5.66. Which

shows that the PPExts treated group values are almost similar when compared to

Metformin and rats reached towards normalcy.

Table 3.10: Glyco Hb values of normal and experimental groups at the end of each

Batch (of all three experiments) in %.

Exp. Batch NC DC TD1 TD2 SD VC

1st Batch 7.3 ±

0.3

15.2 ±

1.37

9.9 ±

0.4**

7.8 ±

0.7**

7.8 ±

0.8**

13.6 ±

0.43

0

100

200

300

400

500

600

Preinduction Induction 1st Month 2nd Month 3rd Month

mg

/dl

BSFNC

DC

TD1

TD2

SD

VC

Fig No:- 3.10

2nd

Batch 7.9 ±

0.04

12.6 ±

0.37

9.3 ±

0.21**

9.1 ±

0.11**

6.2 ±

0.2**

13.7 ±

0.35

3rd

Batch 7.5 ±

0.30

13.1 ±

1.37

9.5 ±

0.4**

8.3 ±

0.76**

7.0 ±

0.8**

12.9 ±

0.43



* P < 0.05 Significant as compared treatment with Disease Control

Figure 3.11: Comparative Glyco Hb activities of each group for three Months.

Glycosylated hemoglobin (GHb) reflects the metabolic control of glucose level

over a period of time unaffected by diet, insulin, other drugs, or exercise on the day of

testing. The levels of HbA1c were significantly higher in DC and VC group. Table No:

3.10 and Fig No: 3.11 indicate that after the treatment with PPExts produces highly

significant reduction (P ≤ 0.001) in Blood GHb (TD1 9.9%, 9.3%, 9.5%) and (TD2 7.8%,

9.1%, 8.3%) as compared to DC (15.2%, 12.6%, 13.1%)

Table 3.11: Mean Blood Glucose values of normal and experimental groups at the

end of each Batch (of all three experiments) in mg/dl.

Exp. Batch NC DC TD1 TD2 SD VC

1st Batch

93 ±

8.50

313 ±

38.17

166 ±

11.13**

107 ±

21.38**

107 ±

22.4**

269 ±

10.78

2nd Batch 110 ± 241 ± 144 ± 149 ± 63 ± 272 ±

0

2

4

6

8

10

12

14

16

18

NC DC TD1 TD2 SD VC

Gly

co H

b i

n %

Experimental Group

Glyco Hb1ST Batch2ND Batch3RD Batch

Fig No:- 3.11

5.65 9.89 5.65** 19.79** 21.21** 14.14

3rd

Batch 102 ±

5.52

255 ±

20.41

152 ±

9.11**

121 ±

10.12**

93 ±

15.21**

252 ±

19.56




Figure 3.12: Comparative Mean Blood Glucose activities of each group for three

Months.

Mean blood glucose (MBG) is average blood Glucose concentration over a period

of time. GHb on the other hand reflects the mean Blood glucose level over an extended

period of time. Table No: 3.11 and Fig No: 3.12 exhibit that treated group with PPExts

(TD1, TD2) produces highly significant (P ≤ 0.01) reduction in MBG in TD1 (166, 144,

152 mg/dl) and TD2 (107, 149, 121 mg/dl) as compared to DC (313, 241, 255 mg/dl) and

VC (269, 272, 252 mg/dl). MBG is higher for DC and VC i.e. above 250mg/dl. Whereas

after treatment with PPExts it come down to 125.67 ± 21.38 mg/dl in TD2 and 87.67 ±

22.47 in SD which is as equal to NC. Where as in TD1 it is 154 ± 11.13 mg/dl. It was

evident that DC and VC had very high amount of glucose level throughout the study.

Whereas in case of treated groups it is decreased gradually and attain good control over

MBG.

0

50

100

150

200

250

300

350

400

NC DC TD1 TD2 SD VC

mg

/dl

Experimental group

MBG 1ST Batch

2ND Batch

3RD Batch

Fig No:- 3.12

Table 3.12: Average values of BSF, Glyco Hb and MBG of all the normal and

experimental groups at the end of three months.

Exp. Grp BSF (mg/dl) Glyco Hb (%) MBG (mg/dl)

NC 72.55 ± 18.77 7.56 ± 0.30 101.67 ± 8.50

DC 339.81 ± 8.59 13.63 ± 1.37 269.67 ± 38.17

TD1 218.11 ± 24.85* 9.56 ± 0.30** 154 ± 11.13**

TD2 99.41 ± 12.03** 8.4 ± 0.65** 125.67 ± 21.38**

SD 86.37 ± 13.49** 7.0 ± 0.80** 87.67 ± 22.47**

VC 332.19 ± 8.32 13.4 ± 0.43 264.33 ± 10.78

Data are expressed as Mean ± SEM, n=18. ANOVA followed by multiple comparison two tail “t” Test.


* P < 0.05 Significant as compared treatment with Disease Control.

Figure 3.13: Average Comparison of BSF, MBG, and Glyco Hb activities of each

group at the end of three months.

0

50

100

150

200

250

300

350

400

NC DC TD1 TD2 SD VC

mg

/dl

Experimental group

Sugar Profile BSF

MBG

Fig No:- 3.13

0

2

4

6

8

10

12

14

16

NC DC TD1 TD2 SD VC

Gly

co H

b i

n %

Experimental group

Glyco Hb AverageFig No:- 3.13

Table No 3.12 and fig No. 3.13 shows that the reduction in the BSF level by

PPExts and standard drug Metformin were 121.7, 240.4, 253.44 mg/dl for BSF, 4.13%,

5.23%, 6.63% for Glyco Hb and 114.68, 144.68, 184.68 mg/dl for MBG. The average

value of BSF in diabetic control group were 339.81 ± 8.59 which is reduced in PPExts

treated group to 218.11 ± 24.85 and 99.41 ± 12.03 mg/dl. Glyco Hb levels were reduced

from 13.63 (DC), 13.65 (VC) to 9.5% (TD1), 8.4% (TD2), 7.0% (SD). Similarly MBG

levels were reduced from 269.68 (DC), 264.33 (VC) to 154 (TD1), 125.67 (TD2), 87.67

(SD).

3.4.2: Lipid profile

Table 3.13: Total Cholesterol values of Normal and Experimental groups at the end

of each month (of all three experiments) in mg/dl.

Exp. Grp 1st Month 2nd Month 3rd Month

NC 58.39 ± 4.65 50.64 ± 1.3 53.42 ± 2.14

DC 74.44 ± 16.46 71.92 ± 8.17 98.63 ± 9.24

TD1 63.03 ± 2.01* 61.33 ± 1.67* 70.54 ± 3.45*

TD2 52.76 ± 4.91* 56.58 ± 3.96* 59.01 ± 2.62*

SD 61.38 ± 4.53* 57.05 ± 11.03* 62.92 ± 1.72*

VC 73.47 ± 5.26 81.37 ± 2.45 100.97 ± 14.21




0

20

40

60

80

100

120

140

NC DC TD1 TD2 SD VC

mg

/dl

Experimental group

TC1st Month

2nd Month

3rd Month

Fig No:- 3.14

Figure 3.14: Comparative Total cholesterol activities of each group for three

months.

Table No 3.13 and Fig No. 3.14 indicate that there is good control on serum TC

level in both the treatment groups (TD1, TD2) as well as in SD. Significant increases in

the level of cholesterol were observed in diabetic rats when compared to Normal control

group. Treatment with PPExts (TD1, TD2) and SD group showed highly significant (P ≤

0.05) reduction in TC. The TC values in the 1st month were 63.03 ± 2.01, 52.76 ± 4.91,

61.38 ± 4.53 when compared with DC (74.44 ± 16.46) and VC (73.47 ± 5.26). In the 2nd

month were 61.33 ± 1.67, 56.58 ± 3.96, 57.03 ± 11.03 when compared with DC (71.92 ±

8.17) and VC (81.37 ± 2.45). Whereas in 3rd

month they were 70.54 ± 3.45, 59.01 ± 2.62,

62.92 ± 1.72 when compared with DC (98.63 ± 9.24) and VC (100.97 ± 14.21).

Table 3.14: Triglyceride values of normal and experimental groups at the end of

each month (of all three experiments) in mg/dl..

Exp. Grp. 1st Month 2nd Month 3rd Month

NC 58.85 ± 15.84 60.24 ± 4.93 60.6 ± 16.53

DC 114.79 ± 27.75 165.88 ± 38.63 234.5 ± 36.67

TD1 76.33 ± 2.58** 75.62 ± 16.94** 157.76 ± 7.11**

TD2 57.63 ± 7.82** 66.15 ± 6.71** 82.01 ± 30.05**

SD 66.01 ± 9.28** 62.51 ± 7.16** 68.5 ± 9.93**

VC 89.63 ± 8.62 148.01 ± 16.6 183.92 ± 7.17




Figure 3.15: Comparative Triglyceride activities of each group for three months.

Table No 3.14 and Fig No. 3.15 indicate TG level in both the treatment groups

(TD1, TD2) as well as in SD were significantly reduced (P ≤ 0.01) over a period of three

months. Although significant increase in the level of TG were observed in diabetic rats

when compared to Normal control group. The values for TG for in 1st month were 76.33

± 2.58, 57.63 ± 7.82, 66.01 ± 9.28 when compared with DC (114.79 ± 0) and VC (89.63

± 8.62). In the 2nd

month were 75.62 ± 16.94, 66.15 ± 6.71, 62.51 ± 7.16 when compared

with DC (165.88 ± 38.63) and VC (148.01 ± 16.6). Whereas in 3rd

month they were

157.76 ± 7.11, 82.01 ± 30.05, 68.5 ± 9.93 when compared with DC (234.5 ± 36.67) and

VC (183.92 ± 7.17).

Table 3.15: Low density lipoprotein values of Normal and Experimental groups at

the end of each month (of all three experiments) in mg/dl.


NC 29.31 ± 3.3 29.67 ± 0.09 22.98 ± 3.16

DC 46.22 ± 4.34 45.18 ± 6.07 45.29 ± 6.43

TD1 32.86 ± 2.34* 33.24 ± 3.13* 26.07 ± 0.35*

TD2 26 ± 5.31* 28.91 ± 2.02* 30 ± 8.99*

SD 27.87 ± 12* 28.72 ± 6.81* 19.9 ± 8.85**

VC 31.23 ± 23.99 34.95 ± 1.01 51.95 ± 6.65


0

50

100

150

200

250

300

NC DC TD1 TD2 SD VC

mg

/dl

Experimental group

TG1st Month

2nd Month

3rd Month

Fig No:- 3.15



Figure 3.16: Comparative Low density lipoprotein activities of each group for three

months.

As was earlier noted that both LDL (Bad Cholesterol) and HDL is associated with

coronary heart disease (CHD) table No: 3.15 & Fig No: 3.16 respectively shows that

LDL level was increased in DC which is in the range of 45.18 ± 6.07 up to 46.22 ± 4.34

mg/dl) and in VC (31.23 ± 23.99 up to 51.95 ± 6.65) with respect to untreated control

group NC values were 22.98 ± 3.16 up to 29.67 ± 0.09. After administration of PPAqExt

as well as PPAlcExt, the level of these biosensors were corrected and significantly ( P ≤

0.05) resettled near to the control level. In TD1 they were 26.07 ± 0.35 up to 33.24 ± 3.13

and in TD2 they were 26 ± 5.31 up to 30 ± 8.99.

Table 3.16: High density lipoprotein values of Normal and Experimental groups at

the end of each month (of all three experiments) in mg/dl..


NC 14.73 ± 5.08 16.53 ± 7.98 16.86 ± 4.41

DC 12.88 ± 1.95 13.75 ± 4.7 13.1 ± 3.84

TD1 21.46 ± 8.59* 21.81 ± 8.45* 24.7 ± 5.48**

TD2 20.96 ± 2.92* 19.41 ± 6.59* 23 ± 6.37**

0

10

20

30

40

50

60

70

NC DC TD1 TD2 SD VC

mg

/dl

Experimental group

LDL1st Month

2nd Month

3rd Month

Fig No:- 3.16

SD 22.19 ± 2.89** 20.19 ± 0.77* 23.02 ± 3.32**

VC 14.36 ± 1.5 12.65 ± 0.43 13.61 ± 4.87




Figure 3.17: Comparative High density lipoprotein activities of each group for three

months.

Table No: 3.16 & Fig No: 3.17 respectively shows that HDL (Good Cholesterol)

level was significantly decreased in DC which is in the range of (12.88 ± 1.95 up to 13.75

± 4.7mg/dl) and VC (12.65 ± 0.43 up to 14.36 ± 1.5mg/dl) when compared to Normal

Control values were 14.73 ± 5.08 up to 16.86 ± 4.41 . In extract treated diabetic group

(TD1, TD2) it was observed that the serum level of these parameters were significantly

(P ≤ 0.05) re-established towards the Normal control level. In TD1 the values were 21.46

± 8.59 up to 21.81 ± 8.45 and in TD2 19.41 ± 6.59 up to 23 ± 6.37mg/dl.

Table 3.17: Average values of TC, TG, LDL and HDL of all the normal and

experimental groups at the end of three months.

0

5

10

15

20

25

30

35

NC DC TD1 TD2 SD VC

mg

/dl

Experimental group

HDL1st Month

2nd Month

3rd Month

Fig No:- 3.17

Exp.

Grp. TC (mg/dl) TG (mg/dl) LDL (mg/dl) HDL (mg/dl)

NC 54.15 ± 3.92 59.89 ± 0.92 27.32 ± 3.76 16.04 ± 1.14




Figure 3.18: Average Comparison of TC, TG, LDL and HDL activities of each

group at the end of three months.

Table No 3.17 and fig No. 3.18 indicate that the significant reduction was

achieved by PPExts and standard drug Metformin were 16.7, 25.52, 21.21 mg/dl for TC.

68.48, 103.12, 106.12 mg/dl for TG. 14.84, 17.26, 20.07 mg/dl for LDL whereas

significant recoveries like 9.42, 7.88, 8.56 mg/dl for HDL. The average value of TC in

diabetic control group were 81.66 ± 14.74 which is reduced in PPExts treated group to

64.96 ± 4.89 and 56.12 ± 3.15 mg/dl. TG levels were reduced from 171.72 ± 60.06 to

103.24 ± 47.21, 68.6 ± 12.37. LDL levels were reduced from 45.56 ± 0.57 to 30.72 ±

4.03, 28.30 ± 2.06. But significance recovery was observed in HDL values from 13.24 ±

0.45 in DC to 22.66 ± 1.77, 21.12 ± 1.8 In TD1, TD2 respectively.

In Vivo antioxidant activity by Lipid Peroxide assay:

0

50

100

150

200

250

NC DC TD1 TD2 SD VC

mg

/dl

Experimental group

Lipid Profile

TC

TG

LDL

HDL

Fig No:- 3.18

DC 81.66 ± 14.74 171.72 ± 60.06 45.56 ± 0.57 13.24 ± 0.45

TD1 64.96 ± 4.89** 103.24 ± 7.21** 30.72 ± 4.03* 22.66 ± 1.77*

TD2 56.12 ± 3.15** 68.6 ± 12.37** 28.30 ± 2.06* 21.12 ± 1.8*

SD 60.45 ± 3.04** 65.67 ± 3.00** 25.49 ± 4.86* 21.80 ± 1.45*

VC 85.27 ± 14.15 140.52 ± 47.58 39.38 ± 11.04 13.54 ± 0.86

In order to establish a scientific basis for the utility of Pongamia pinnata in the

treatment of diabetes, it was decided to evaluate in vivo free radical scavenging activity

in Alloxan-induced diabetic rats. Earlier reports reveal that Alloxan-induced diabetic

animals may exhibit most of the diabetic complication mediated through oxidative stress.

Table 3.5: Estimation of Lipid Peroxidation levels in terms of MDA from serum.

Test

No.

Concentration

(nmol/ml)

Std

MDA

Sol (ml)

D/W

(ml)

30%

TCA

(ml)

Vortex

For

1

min

1%

TBA

(ml)

OD at

532 nm

1. 0 0 0.5 0.5 0.5 0

2. 5 0.1 0.4 0.5 0.5 0.3534

3. 10 0.15 0.35 0.5 0.5 0.6188

4. 15 0.2 0.3 0.5 0.5 0.9552

5. 20 0.25 0.25 0.5 0.5 1.2664

6. 25 0.3 0.2 0.5 0.5 1.5216

7. 30 0.35 0.15 0.5 0.5 1.9409

Figure 3.6: Standard graph for LPO Estimation

0

0.5

1

1.5

2

2.5

0 5 10 15 20 25 30 35

Ab

sorb

an

ce 532n

m

Lipid hydroperoxide (n/mol)

Lipid Peroxides Std GraphFig No:- 3.6

Table 3.6: Serum levels of Lipid peroxide in terms of Malondialdehyde in Normal

and experimental animals.

Exp.

Grp

Serum

(ml)

30%

TCA(ml)

NC 0.5 0.5

DC 0.5 0.5

TD1 0.5 0.5

TD2 0.5 0.5

SD 0.5 0.5

VC 0.5 0.5

Figure 3.7: Comparison of LPO

The blood serum of experimental rats was used to estimate the LPO values as

illustrated in Table 3.6 and fig, No. 3.7. The LPO values of DC and VC were

significantly increased as compared to normal cont

Pongamia pinnata stem extract the alteration of erythrocyte membrane lipid peroxides

were reversed back to near normal. However, there was no such alternations exist in the

erythrocytes of control group. In normal contr

In DC and VC values were higher which is (11.8237 n/mol, 11.4728 n/mol) respectively.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

NC

Ab

sorb

an

ce a

t 5

32

nm

Fig No:- 3.7


and experimental animals.

TCA(ml)

Vortex

for

1

min

1%TBA

(ml)

Incuba

te

in

water

bath

for

30

min

Cool &

Centrif

uge at

2000

Rpm

For

10

min

OD at

532nm

0.5 0.5347

0.5 0.8357

0.5 0.6317

0.5 0.6595

0.5 0.5716

0.5 0.8109

Figure 3.7: Comparison of LPO activities of each group at the end of three Months.



significantly increased as compared to normal control (NC) animals. After treatment of

stem extract the alteration of erythrocyte membrane lipid peroxides


erythrocytes of control group. In normal control group values of LPO were 7.5650 n/mol.


DC TD1 TD2 SDExperimental group

LPO


OD at

532nm

LPO

(n/mol)

0.5347 7.5650

0.8357 11.8237

0.6317 8.9374

0.6595 9.3307

0.5716 8.0871

0.8109 11.4728

activities of each group at the end of three Months.



rol (NC) animals. After treatment of

stem extract the alteration of erythrocyte membrane lipid peroxides


ol group values of LPO were 7.5650 n/mol.


VC

After treatment with plant extracts (TD1, TD2) and standard drug (SD) values were come

down to 8.9374 n/mol, 9.3307 n/mol, and 8.0871 n/mol respectively.

3.3.3: In Vivo antioxidant activity by Superoxide Dismutase Assay

Oxidative stress in diabetes is coupled to a decrease in the antioxidant status,

which can increase the deleterious effects of free radicals. The SOD is one of the major

scavenging enzymes that remove free radicals in vivo. The SOD plays a prominent role

in scavenging free radical and restoring antioxidant activities in the tissue of diabetic

animals. A decreased activity of these antioxidants can lead to an excess availability of

superoxide anion (O2- ) and hydrogen peroxide (H2O2), which in turn generate hydroxyl

radicals (OH), resulting in initiation and propagation of LPO. The SOD can catalyze

dismutation of (O2) into H2O2, which is then deactivated to H2O by catalase or SOD

works in parallel with selenium-dependent glutathione peroxidase, which plays an

important role in the reduction of H2O2 in the presence of reduced glutathione forming

oxidized glutathione, and it protects cell protein and cell membranes against oxidative

stress. Therefore the activities of SOD enzyme were measured in control and

experimental rats.

Table 3.7: The OD of standard sample at 440nm of at varying concentration along

with their respective Linerised rate for plotting standard graph.

Tube

No.

SOD Stock

(µl)

Sample

Buffer (µl)

Final SOD

Activity (µ/ml)

Std Sample

OD at 440nm

Linerised

rate value

A 0 1000 0 0.722 1

B 20 980 0.025 0.576 1.2534

C 40 960 0.05 0.446 1.6188

D 80 920 0.1 0.335 2.1552

E 120 880 0.15 0.251 2.8764

F 160 840 0.2 0.194 3.7216

G 200 800 0.25 0.193 3.7409

Figure 3.8: Superoxide Dismutase standard curve.

Table 3.8: Serum levels of SOD Activity of normal and experimental group.

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 0.05 0.1 0.15 0.2 0.25 0.3

Lin

eari

zed

rate

SOD Activity (U/ml)

SOD Standard GraphFig No:- 3.8

Exp.

Grp

Radical

detector

(µl)

Sample

(µl)

Xanthine

Oxidase

(µl)

Incubate

96 well

Plate on

a

Shaker

for 20

Minutes

OD at

440nm

SOD

activity

(U/ml)

NC 200 10 20 0.105 12.55

DC 200 10 20 0.222 4.79

TD1 200 10 20 0.166 7.13

TD2 200 10 20 0.120 10.70

SD 200 10 20 0.108 12.15

VC 200 10 20 0.244 4.15

Figure 3.9: Comparison of SOD activities of each group at the end of three Months.

As it is evident from table no 3.8: as well as fig No 3.9: showed the SOD enzymes

were significantly (P < 0.01) decreased in Alloxan

Consequently, the levels of SOD were significantly (P < 0.01) improved after treatment

with Pongamia pinnata extracts in Alloxan

may clearly suggest that increased levels of SOD

radical scavenging activity, which may exert a beneficial effect against pathological

alterations caused by reactive oxygen species. In TD1, TD2 it was 7.13 SOD U/ml and

10.7 SOD U/ml where as in DC and VC it significa

U/ml, 4.15 SOD U/ml respectively. It was observed that the values obtained clearly

reveal that, SOD values were significantly (P < 0.01) reversed by administration of

Pongamia pinnata extracts in Alloxan induced diabetic r

0

2

4

6

8

10

12

14

NC

SO

D (

U/m

l)Fig No:- 3.9

Comparison of SOD activities of each group at the end of three Months.


were significantly (P < 0.01) decreased in Alloxan- induced diabetic control rats.


extracts in Alloxan-induced diabetic rats. The above observations

may clearly suggest that increased levels of SOD of Pongamia pinnata



10.7 SOD U/ml where as in DC and VC it significantly decrease and it was 4.79 SOD



extracts in Alloxan induced diabetic rats after 90 days.

DCTD1

TD2SD

VC

Experimental Group

SOD

Comparison of SOD activities of each group at the end of three Months.


induced diabetic control rats.


induced diabetic rats. The above observations

Pongamia pinnata extract has free



ntly decrease and it was 4.79 SOD



ats after 90 days.

SOD (U/ml)

5 Histopathological examination of Alloxan induced diabetic rats.

3.5.1 Histopathology of the pancreas:

NC: Normal histological appearance of

pancreas with islet of langerhans.(Arrow)

DC: Multifocal mild degeneration in islet

of langerhans (Arrow)

TD1: Focal mild Degeneration in Islet of

langerhans , reduced islet of langerhans.

(Arrow)

TD2: Focal minimal Degeneration in Islet

of langerhans (Arrow)

SD: Focal Minimal Degeneration in Islet of

langerhans (Arrow)

VC: Multifocal degeneration in islet of

langerhans (Arrow)

Figure 3.32: Comparative histopathology of pancreas of experimental animals

Results of the cellular architecture and integrity of the pancreatic cells was

examined of treated non diabetic and diabetic rats [ with various treatments TD1, TD2,

SD, VC respectively] as a possible site of antidiabetic agents. Untreated Alloxan diabetic

rats DC group presented with damaged islets markedly reduced [shrunken in mass along

with multifocal degeneration of islet of langerhans. Whereas the non diabetic rats showed

preserved numerous, undamaged islets widely distributed throughout the exocrine

panaceas and demonstrated well stained nuclei [NC group]. Treatment with PPAqExt

caused a partial recovery in damage to islet cell – mild degenerated changes to cells of

islet of langerhans. [TD1] whereas more prominent recovery was produced by treatment

with PPAlcExt – islet cell of langerhans was preserved. [TD2]. There was not

significantly different from plate [NC]. Treatment with extract reversed lesions produced

by Alloxan administration, possible islet cell regeneration. Treatment with standard drug

Metformin [SD] group rats showed focal minimal degeneration in islet of langerhans rest of

the cellular architecture and integrity of the cell was normal along with structure, which

is similar to normal control group rats. Where as in case of vehicle control multifocal

degeneration in the islet of langerhans was observed [VC]. The extent of reversal and

recovery was partial with aqueous extract and was distinct with Alcoholic extract.

3.5.2 Histopathology of the Liver:

NC: Normal histological appearance of

central vein and hepatic cords.(Arrow)

DC: Multifocal mild MNC infiltration (black

arrow). Diffuse mild SCN (Arrow)

TD1: Multifocal mild fatty changes in

hepatocytes (Arrow)

TD2: Focal minimal dilated sinusoids with

normal central vein. (Arrow)

SD: Focal dialated sinusoids with normal

liver histology.(Arrow)

VC: Multifocal increased SCN with MNC

infiltration in portal triad.(Arrow)

Figure 3.33: Comparative histopathology of Liver of experimental animals

Investigation of the histology of the hepatocytes as examined in this study

revealed multifocal mononuclear cell (MNC) infiltration, diffuse moderate, mild single

cell necrosis (SCN) moreover the sinusoids were non radiating tend to be wider and

interrupted in the interrupted Alloxan diabetic rats. Also the hepatocytes were

degenerated and number of nuclei reduced. [DC] on the other hand NC group liver

histology showed normal histology of portal triad, sinusoids spaces and distinct

lobulation with a central vein. [NC]. Sinusoids radiate out from the central vain,

hepatocytes were distinct single or polynuclei. Treatment with PPAqExt caused partial

reversal in the lesions observed with Alloxan treatment where multifocal mild central

vein congestion with mild fatty changes in hepatocyte was observed. Also mild focal

sinusoidal congestion with multifocal Mono nuclear cells infiltration was observed.

[TD1] There was better improvement with PPAlcExt. The extract showed normal

hepatocytes with central vein similar to normal control group histology. Few areas

showing mild mononuclear cell infiltration was observed. Treatment with standard drug

metformin [SD] group rats showed normal liver histology were cellular architecture and

integrity of the hepatocytes was normal alonwith distinct cell nuclei and sinusoids were

non radiating, spaces between the sinusoids were normal, wider as similar as normal

control. Where as in case of vehicle control degenerative changes of hepatocytes was

observed along with multifocal single cell necrosis.[VC] The extent of reversal and

recovery was partial with aqueous extract and was distinct with Alcoholic extract.

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