report of the work done - v.g.vaze...
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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
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
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
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
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 < 0.01 highly significant as compared treatment with Disease Control.
* 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
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
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
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
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.
Exp. Grp. 1st Month 2nd Month 3rd Month
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
Data are expressed as Mean ± SD, n = 18. ANOVA followed by multiple comparison two tail “t” Test.
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
** P < 0.01 highly significant as compared treatment with Disease Control.
* P < 0.05 Significant as compared treatment with Disease Control
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..
Exp. Grp. 1st Month 2nd Month 3rd Month
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
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
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
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
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
Table 3.6: Serum levels of Lipid peroxide in terms of Malondialdehyde in Normal
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.
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 control (NC) animals. After treatment of
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 control group values of LPO were 7.5650 n/mol.
In DC and VC values were higher which is (11.8237 n/mol, 11.4728 n/mol) respectively.
DC TD1 TD2 SDExperimental group
LPO
Table 3.6: Serum levels of Lipid peroxide in terms of Malondialdehyde in Normal
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.
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
rol (NC) animals. After treatment of
stem extract the alteration of erythrocyte membrane lipid peroxides
were reversed back to near normal. However, there was no such alternations exist in the
ol group values of LPO were 7.5650 n/mol.
In DC and VC values were higher which is (11.8237 n/mol, 11.4728 n/mol) respectively.
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.
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- induced diabetic control rats.
Consequently, the levels of SOD were significantly (P < 0.01) improved after treatment
extracts in Alloxan-induced diabetic rats. The above observations
may clearly suggest that increased levels of SOD of Pongamia pinnata
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 significantly decrease and it was 4.79 SOD
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
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.
As it is evident from table no 3.8: as well as fig No 3.9: showed the SOD enzymes
induced diabetic control rats.
Consequently, the levels of SOD were significantly (P < 0.01) improved after treatment
induced diabetic rats. The above observations
Pongamia pinnata extract has free
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
ntly decrease and it was 4.79 SOD
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
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.