Acetic Acid Induce Vascular Permeability Biology Essay

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Body weight of the animals before and after termination was noted and no any significant change observed. Further skin and fur, eyes and mucous membranes and also respiratory, circulatory, autonomic and central nervous system and somatomotor activity and behavior pattern were also unchanged. Additionally, there was no any sign of tremors, convulsion, salivation, diarrhoea; lethargy, sleep and coma were noted. The onset of toxicity and signs of toxicity also noted but found negative.

The acute oral toxicity study was done according to the OECD guidelines 423 (Acute Toxic Method). A starting dose used was 2000 mg/kg, p. o. of TPF and TPR was administered to 3 male rats, observed for three days. There was no considerable change in body weight before and after treatment of the experiment and no signs of toxicity were observed. When the experiments were repeated again with the same dose level, 2000 mg/kg, p. o. of methanolic extracts of flower and root for 3 days more and observed for 14 days, no change were observed from the first set of experiment. LD50 cut off mg/kg body weight was observed as X-unclassified.

The TPF and TPR did not produce lethality up to the dose level of 2000 mg/kg.

5.2.2 Anti-inflammatory activity

5.2.2.1. Acetic acid induce vascular permeability (acute study)

Methanolic extracts of the plant were reduced the extent of peritoneal inflammation induced by injection of acetic acid. The absorbance was considerably (P<0.01) reduced in all doses of TPF and TPR. Standard drug treatment Indomethacin exhibited strongest activity with 65.31% inhibition in dye leakage as compared to control. (Table 6.34) TPF and TPR at the dose of 400 mg/kg were exhibits 61.58 % and 52.49 % inhibition respectively.

Similarly, FEA and REA were also reduced the amount of dye leakage and, the reduction in vascular permeability by FEA (31.94 %) and REA (22.65 %) was found to be 31.94 % (P< 0.05) and 22.65 % correspondingly. The effect demonstrated by ethyl acetate extracts was lesser as compared with the effect produced by the methanolic extracts. (Table 6.35)

Table 6.34: Effect of TPF and TPR on acetic acid induced vascular permeability in mice.

Groups

Dose (mg/kg)

Absorbance #

% inhibition

Control

CMC

1.848 ± 0.12

0

Indo

5

0.641 ± 0.10**

65.31

TPF

100

1.560 ± 0.15

15.58

200

1.141 ± 0.04*

38.26

400

0.710 ± 0.11**

61.58

TPR

100

1.564 ± 0.09

15.37

200

1.397 ± 0.10

24.40

400

0.878 ± 0.80**

52.49

#Values are expressed as mean ± SEM. n = 6. * P < 0.05, ** P < 0.001 when compared with control (ANOVA followed by Dunnett's t-test).

Table 6.35: Effect of FEA and REA on acetic acid induced vascular permeability in mice.

Groups

Dose (mg/kg)

Absorbance #

% Inhibition

Control

CMC

1.85 ± 0.12

0

Indo

5

0.64 ± 0.10**

65.36

FEA

100

1.82 ± 0.12

1.42

200

1.43 ± 0.21

22.71

400

1.26 ± 0.15*

31.94

REA

100

1.84 ± 0.12

0.58

200

1.55 ± 0.20

16.27

400

1.43 ± 0.23

22.65

#Values are expressed as mean ± SEM. n = 6. * P < 0.05, ** P < 0.001 when compared with control (ANOVA followed by Dunnett's t-test).

5.2.2.2. Cotton pellet induced granuloma in rats (chronic study)

Table 6.36 shows chronic anti-inflammatory effects of the TPF and TPR at the doses of 100, 200 and 400 mg/kg. Changes in the cotton pellets weights (wet weight-dry weight) of the test substances were compared with the controls. TPF and TPR showed significant (P< 0.01), dose dependant chronic anti-inflammatory effect. TPF and TPR (400 mg/kg) showed 37.06 % and 31.25 % inhibition respectively; whereas Indo treated group exhibited 46.52 %. Their effects are in comparable magnitude with the standard anti-inflammatory drug.

However, FEA and REA at the dose of 400 mg/kg were showed 28.03 % and 20.81 % inhibition in granuloma formation respectively and found to be statistically significant (P< 0.01) as compared with control group. At low dose (100 mg/kg) both FEA and REA does not inhibit the granuloma formation. Standard compound indomethacin was showed 42.20 % inhibition. The inhibition observed in middle group of FEA and REA also lesser but statistically significant (P< 0.05, P< 0.01) (Table 6.37)

Table 6.36: Effect of TPF and TPR on % inhibition on cotton pellet induced chronic inflammation in rats

Groups

Dose (mg/kg)

Wet weight (g)#

Dry weight (g)#

Difference (g)

% Inhibition

Control

CMC

154.62± 2.04

56.87± 3.70

97.75

0.00

Indo

10

73.98±2.03**

21.7±1.03**

52.28

46.52

TPF

100

138.57±2.40*

41.05±1.61**

97.52

0.24

200

108.87±4.37*

33.47±1.18**

75.4

22.86

400

84.12±2.25**

22.6± 1.24**

61.52

37.06

TPR

100

142.18±1.87*

44.62±1.87**

97.56

0.19

200

120.33±1.50*

38.83±1.82**

81.5

16.62

400

96.23±1.56**

29.03±2.96**

72.57

31.25

#Values are expressed as mean ± SEM. n = 6. * P < 0.05, ** P < 0.001 when compared with control (ANOVA followed by Dunnett's t-test).

Table 6.37: Effect of FEA and REA on % inhibition on cotton pellet induced chronic inflammation in rats

Groups

Dose (mg/kg)

Wet weight (g)#

Dry weight (g)#

Difference (g)

% inhibition

Control

CMC

150.62± 1.36

55.87± 3.17

94.75

0

Indo

10

79.45± 1.37**

24.68±1.64**

54.77

42.20

FEA

100

138.83±1.90**

44.71±1.64**

94.12

0.66

200

112.55±2.99**

37.67±1.81**

74.88

20.97

400

96.0± 3.21**

27.81±1.46**

68.19

28.03

REA

100

141.35± 1.05*

47± 2.87*

94.35

0.42

200

122.38±2.36**

40.67±1.81**

81.71

13.76

400

107.88±3.09**

32.85±0.99**

75.03

20.81

#Values are expressed as mean ± SEM. n = 6. * P < 0.05, ** P < 0.001 when compared with control (ANOVA followed by Dunnett's t-test).

5.2.3 Analgesic activity

5.2.3.1. Formalin induce nociception in mice (Chemical induction)

Table 6.38 depicts the effect of TPF and TPR on formalin induce nociceptive response in mice. As shown, all the three doses of the TPF and TPR were significantly (P< 0.01) impaired the time spent on licking of injected paw, both in the early (0- 5 min) and late phases (15- 30 min) of nociception. However, the effect in the early phase appears to be rather higher than in the late phase.

While, FEA (400 gm/kg) REA (400 mg/kg) treated group shows statistically significant (P< 0.01) reduction in nociceptive response in both early and late phases. REA treated groups produced somewhat lesser extent of effect in both phases as compared to FEA treatment. (Table 6.39)

Table 6.38: Effect of TPF and TPR on nociceptive response on formalin induced nociception in mice

Groups

Dose

(mg/kg)

Nociceptive response (sec)#

% inhibition

Early Phase

Late phase

Early phase

Late phase

Control

CMC

58.1± 1.09

39.28± 1.04

--

--

Penta

2

13.52 ± 1.36**

7.98± 1.06**

76.73

79.68

TPF

100

44.77± 1.70*

33.38± 1.70

22.94

15.02

200

35.1± 1.48*

20.57± 1.19*

39.59

47.63

400

18.3± 1.47**

13.7± 1.41**

68.50

65.12

TPR

100

44.63± 1.13*

36.37± 2.27

23.18

7.41

200

36.43± 1.51*

34.7± 1.16*

37.29

11.66

400

24.07± 1.03**

16.34± 1.08**

58.57

58.40

#Values are expressed as mean ± SEM. n = 6. * P < 0.05, ** P < 0.001 when compared with control (ANOVA followed by Dunnett's t-test).

Table 6.39: Effect of FEA and REA on nociceptive response on formalin induced nociception in mice

Groups

Dose

Nociceptive response (s)#

% inhibition

Early Phase

Late phase

Early phase

Late phase

Control

CMC

69.23± 2.07

47.62± 1.16

--

--

Penta

2

13.23± 1.18*

9.43± 1.10**

80.89

80.20

FEA

100

50.52± 1.23*

36.52± 1.52*

27.03

23.31

200

42.48± 0.91*

26.02± 2.03**

38.64

45.36

400

34.32± 1.28**

22.37± 1.03**

50.43

53.02

REA

100

57.45± 1.65*

45.63± 1.55

17.02

4.18

200

40.35± 0.68*

43.43± 1.89

41.72

8.80

400

34.85± 1.28*

30.32± 1.76**

49.66

36.33

#Values are expressed as mean ± SEM. n = 6. * P < 0.05, ** P < 0.001 when compared with control (ANOVA followed by Dunnett's t-test).

5.2.3.2. Acetic acid induce writhing in mice (Peripheral analgesia)

Among the extract treated groups the higher dose of TPF (400 mg/kg) was showed better (65.31 %) and statistically significant (P< 0.01) inhibit the number of writhings induced by acetic acid. Indo treated group shows grater inhibition (73.18 %) in writings, whereas, TPR treated animals were also exhibit statistically significant (P< 0.01) effect with 59.48 % inhibition. (Table 6.40) Hence, the protective effect shown by TPF and TPR is dose dependant.

Similarly, high dose (400 mg/kg) of FEA (48.34 %) and REA (36.66 %) shows better and statistically significant inhibition of number of writhings. (Table 6.41) Standard (Indo, 5 mg/kg) treated group demonstrate highest no. of reduction in writhing (16.83± 0.86) as compared to control (CMC suspension) group (50± 1.13) and with 66.34% inhibition. Nevertheless, FEA and REA at medium dose (200 gm/kg) level also produce statistically significant inhibition (P< 0.01 and P< 0.05).

Table 6.40: Effect of TPF and TPR on writhings induced by acetic acid in mice

Groups

Dose (mg/kg)

No. of writhing#

% Inhibition

Control

CMC

57.16 ± 1.50

--

Indo

5

15.33±1.16**

73.18

TPF

100

38±1.54**

33.53

200

32.5±0.51**

43.15

400

19.83±0.43**

65.31

TPR

100

41.83±0.43**

26.83

200

35.5±1.28**

37.9

400

23.16±1.03**

59.48

#Values are expressed as mean ± SEM. n = 6. * P < 0.05, ** P < 0.001 when compared with control (ANOVA followed by Dunnett's t-test).

Table 6.41: Effect of FEA and REA on writhings induced by acetic acid in mice

Groups

Dose (mg/kg)

No. of writhing#

% Inhibition

Control

CMC

50 ± 1.13

--

Indo

5

16.83± 0.86**

66.34

FEA

100

43.5± 1.21*

13

200

35.16± 1.06**

29.68

400

25.83± 1.40**

48.34

REA

100

47.16± 1.47

5.68

200

39.5± 0.51*

21

400

30.67± 1.74**

36.66

#Values are expressed as mean ± SEM. n = 6. * P < 0.05, ** P < 0.001 when compared with control (ANOVA followed by Dunnett's t-test).

5.2.3.3 Hot plate method in mice (Thermal induction)

TPF at the dose 400 mg/kg showed a significant analgesic effect (P<0.01) against thermally induced pain at 30, 60, 90 and 120 min of the study period (Table 6.42), with more than 50% protection was achieved at 60 min. TPR at the dose 400 mg/kg significantly increased the hot-plate latency time in mice starting from 30 min (P< 0.05), at 120 min it showed the maximum protection (~112%) which is fairly similar to TPF (400 mg/kg). By contrast, Pentazocine was shown to provide protection against thermally induced stimuli throughout the observation period (P< 0.01 at 30, 60, 90 and 120 min), with maximum protection (~157%) conferred after 120 min.

FEA and REA at the dose 400 mg/kg were showed a significant analgesic effect (P<0.01) against thermally induced pain at 60, 90 and 120 min of the study period (Table 10), with 90.06% and 80% protection was achieved at 60 min respectively. REA at the dose 400 mg/kg significantly increased the hot-plate latency time in mice starting from 60 min (P< 0.01), at 90 min it showed the maximum protection (~98%) which is to some extent lesser than FEA (400 mg/kg).

Ethyl acetate extracts were shown to have lesser extent of % protection against nociception in thermal induction than methanolic extracts. Additionally, ethyl acetate extracts were produced higher degree of activity at 90 min; after, the analgesic action gets decreased gradually. By contrast, methanolic extract may show similar degree of activity like standard drug Pentazocine.

Table 6.42: Effect of TPF and TPR on latency time and % protection against thermal induction on hot plate method in mice

Groups

Dose

(mg/kg)

Latency time/ reaction time# in sec (% PATI)

Pre drug

Latency

30 min

60 min

90 min

120 min

Control

CMC

4.34± 0.17

4.41± 0.18

4.8± 0.46

4.93± 0.44

6.14± 0.47

Penta

2

5.07± 0.21

6.60± 0.51**

(30.18)

10.25± 0.64**

(102.17)

13.18± 0.51**

(159.96)

13.06± 0.72**

(157.59)

TPF

100

4.78± 0.24

5.03± 0.15

(5.23)

7.55± 0.41**

(57.95)

7.12± 0.42*

(48.95)

8.39± 0.44*

(62.30)

200

5.12± 0.39

5.29± 0.35

(3.32)

6.73± 0.34*

(31.45)

8.31± 0.57**

(62.30)

9.92± 0.40**

(93.75)

400

5.37± 0.26

7.33± 0.49**

(36.50)

8.63± 0.52**

(60.71)

11.03± 0.75**

(105.40)

11.41± 0.41**

(112.48)

TPR

100

4.59± 0.31

4.78± 0.22

(4.14)

6.82± 0.41*

(48.58)

7.82± 0.54**

(70.37)

8.33± 0.35*

(81.48)

200

5.37± 0.26

4.77± 0.23

(-11.17)

7.04± 0.29*

(31.10)

8.06± 0.53**

(68.72)

8.8± 0.38**

(63.87)

400

4.49± 0.25

5.9± 0.51*

(31.40)

7.81± 0.73**

(73.94)

9.1± 0.47**

(102.67)

9.54± 0.53**

(112.47)

Values in parenthesis indicate the % protection against thermal induction (PATI). #Values are expressed as mean ± SEM. n = 6. * P < 0.05, ** P < 0.001 when compared with control (ANOVA followed by Dunnett's t-test).

Table 6.43: Effect of FEA and REA on latency time and % protection against thermal induction on hot plate method in mice

Groups

Dose (mg/kg)

Latency time/ reaction time# in sec (% PATI)

pre drug latency

30 min

60 min

90 min

120 min

Control

CMC

4.97± 0.38

5.07± 0.49

5± 0.38

5.55± 0.35

4.97± 0.18

Penta

2

5.18± 0.40

7.81±0.40** (50.77)

10.89±0.45** (110.23)

12.38±0.95** (139.0)

12.97±0.20** (150.39)

FEA

100

4.83± 0.35

6.01± 0.52 (24.43)

6.96± 0.30* (44.10)

8.53± 0.52* (76.60)

8.05± 0.26* (66.67)

200

5.28± 0.29

5.19± 0.49 (-1.70)

7.12± 0.31* (34.85)

9.88± 0.42* (87.12)

8.53± 0.31* (61.55)

400

5.03± 0.45

6.44± 0.51 (28.03)

9.56±0.41** (90.06)

10.38± 0.72** (106.36)

9.59±0.53** (90.66)

REA

100

5.77± 0.43

5.25± 0.50 (-9.01)

7.33± 0.55* (27.04)

8.6± 0.52* (49.05)

7.63± 0.19* (32.24)

200

5.68± 0.33

6.15± 0.59 (8.27)

7.43± 0.32* (30.81)

9.98± 0.71* (75.70)

8.78± 0.28* (54.58)

400

4.85± 0.53

6.37± 0.34 (31.34)

8.73±0.41** (80.0)

9.59± 0.44** (97.73)

8.44±0.29** (74.02)

Values in parenthesis indicate the % protection against thermal induction (PATI). #Values are expressed as mean ± SEM. n = 6. * P < 0.05, ** P < 0.001 when compared with control (ANOVA followed by Dunnett's t-test).

5.2.4 Anti-arthritis activity

5.2.4.1. Freund's adjuvant induced arthritis in rat

Effect of on primary lesions

The observation made on different days of treatment period in freund's complete adjuvant induced arthritis showed that there was a less increased in paw swelling in DF and extracts treated animals as compared to control group. The assessment made on the 21st day showed that, treatment with all extracts as well as DF significantly (P< 0.01) reduce the injected paw swelling (primary lesions) as compared with control group. The TPF, TPR, FEA and REA at the doses of 400 mg/kg, p.o. showed percentage inhibition in mean paw volume by 70.37%, 62.96%, 58.02% and 49.38% respectively. While, DF treatment group showed 80.25 % percentage inhibition in mean paw volume on day 21. (Table 6.44)

Additionally, photographic images of injected paw of animals were also helping to reveals the protective effects of extract and DF treatment. (Fig. 6.66)

Effect of on body weight

Changes in body weight have also been used to assess the course of the disease and the response to therapy of anti-inflammatory drugs as the incidence and severity of arthritis increased, the changes in the body weight of the rat also occurred during the course of experimental period. The body weight reduction was less for groups which received DF (3.8 g) and TPF (4.6 g) (Table 6.45) when compared to control group (16.5 g). TPR, FEA and REA at dose of 400 mg/kg, p.o. showed statistically significant (P< 0. 01) reduction in weight as compared with control group.

Table 6.44: Effect of TPF, TPR, FEA and REA on Injected Paw Volume (Primary Lesions) in CFA induced arthritis in rat.

Groups

Dose (mg/kg)

Mean increase in paw volume# (mL)

Day 5

Day 10

Day 15

Day 20

Control

CMC

0.43 ± 0.03

0.66 ± 0.01

0.82 ± 0.01

0.81 ± 0.01

DF

5

0.27 ± 0.01** (37.21)

0.31± 0.02** (53.03)

0.31± 0.02** (62.20)

0.16± 0.02** (80.25)

TPF

400

0.29 ± 0.01** (32.56)

0.33± 0.02** (50.0)

0.34± 0.01** (58.54)

0.24± 0.02** (70.37)

TPR

0.31 ± 0.01** (27.91)

0.38± 0.01** (42.42)

0.43± 0.01** (47.56)

0.30± 0.02** (62.96)

FEA

0.37 ± 0.03 (13.95)

0.48± 0.03** (27.27)

0.58± 0.08** (29.27)

0.53± 0.07** (34.57)

REA

0.40 ± 0.05 (6.98)

0.51± 0.04** (22.73)

0.59± 0.08** (28.03)

0.57± 0.02** (29.63)

Values in parenthesis indicate the % inhibition. #Values are expressed as mean ± SEM. n=6 ** P < 0.01 when compared with control (ANOVA followed by Dunnett's t-test).

* P < 0.05, ** P < 0.01 when compared with control (ANOVA followed by Dunnett's t-test).

Fig 6.66: Showing effect of TPF, TPR, FEA and REA on % rise in ankle diameter of injected paw in FCA induced arthritis in rats.

control2

2 mg2

a. Control

b. DF (5 mg/ kg)

10 mg

5 mg2

c. REA (400 mg/kg)

d. FEA (400 mg/kg)

2 mg1

std2

e. TPR (400 mg/kg)

f. TPF (400 mg/kg)

Fig 6.67: Showing the photographs of injected paw in FCA induced arthritis in rats taken at 21st day of the study.

Table 6.45: Effect of TPF, TPR, FEA and REA on body weight in CFA induced arthritis in rat.

Group

Dose (mg/kg)

Mean body weight# (gm)

Reduction in body weight (gm)

0 day

21 day

Control

CMC

178.5 ± 1.088

162.0 ± 0.96

16.5

DF

5

188.5 ± 2.094

184.7 ± 1.78**

3.8

TPF

400

198.3 ± 4.645

193.7 ± 3.99**

4.6

TPR

201 ± 4.351

195.5 ± 2.43**

5.5

FEA

197.8 ± 4.498

188.3 ± 1.62**

9.5

REA

202 ± 3.512

190.2 ± 2.09**

11.8

#Values are expressed as mean ± SEM. n=6 * P < 0.05, ** P < 0.01 when compared with control (ANOVA followed by Dunnett's t-test).

Effect on scoring systems

Arthritis score is increased from day-7 to day-21, the maximum score was peaked at 21st day. (Table 6.46) All extracts were showed dose dependent reduction in arthritis score as compared to vehicle treated group. Among the extracts TPF showed maximum effect at dose of 400 mg/kg, p.o. and comparable to the group treated with standard drug DF.

Flexion pain test score was employed to assess effect of extract treatment on inhibition of arthritic pain. This test score increases as disease progress takes place. All extracts and DF decreases flexion pain test score on day-7 and day- 21 in a dose dependant manner. (Table 6.46)

Results of mobility score are shown in Table 6.46. All extracts at the dose of 400 mg/kg p.o. showed measurable reduction in mobility score (3-1) on 21st day. Reduction in mobility score on 14th day is somewhat less but in a dose dependent manner.

In case of stance scoring system the maximum score referred to the normal condition of animals while reduction in score indicates severity of disease progression. Effect of TPF, TPR, FEA and REA on stance score was observed and it found to be dose dependant and comparable with the effect of DF. (Table 6.46)

Table 6.46: Effect of TPF, TPR, FEA and REA on various pain test scores in FCA induced arthritis in rat.

Groups

Dose (mg/kg)

Arthritis score

Flexion pain

test Score

Mobility score

Stance score

Control

CMC

4 (4,3)

3 (3,3)

3 (3,3)

1 (1,1)

DF

5

2 (4,2)**

1 (2,1)**

1.5 (2,1)**

2 (2,1)**

REA

400

3.5 (4,3)

2.5 (3,2)*

2.5 (3,2)

3 (3,1)

FEA

4 (4,3)

2.5 (3,2)*

3 (3,2)

2.5 (3,1.5)

TPR

3.5 (4,3)

2 (3,2)*

2 (3,2)**

2 (3,2)**

TPF

3 (4,2)

2.5 (3,1)*

2 (3,1) **

2 (3,1) **

Values are expressed as median (maximum, minimum). n=6 * P < 0.05, ** P < 0.01 when compared with control (ANOVA followed by Dunnett's t-test).

Effect on haematological parameters

A) Total leukocyte (WBC) count

In arthritis condition there is a mild to moderate rise in the WBC count due to release of IL-1ß inflammatory response. Total leukocyte count was found to be decreased in extract and DF treated groups as compared to control group. The WBC count in TPF treated group at dose of 400 mg/kg, p.o. has count of 9.59 - 103 /mm3, while DF treated group showed WBC count of 9.53 -103/mm3 (Fig 6.67)

B) Haemoglobin

The results indicated that haemoglobin count was normalized in TPF and DF treated group as compared to control group (Fig 6.68). This indicates protective effect of TPF against anemia that occurs due to arthritic condition. However, ethyl acetate extract (400 mg/kg, p.o.) treated groups did not showed significant level of haemoglobin as compared to the control group.

* P < 0.05, ** P < 0.01 when compared with control (ANOVA followed by Dunnett's t-test).

Fig 6.67: Showing effect of TPF, TPR, FEA and REA on WBC count in FCA induced arthritis in rats.

** P < 0.01 when compared with control (ANOVA followed by Dunnett's t-test).

Fig 6.68: Showing effect of TPF, TPR, FEA and REA on haemoglobin count in FCA induced arthritis in rats.

C) Erythrocyte sedimentation rate (ESR)

The result indicated that, ESR was similarly lowered in TPF (3.54 mm/ hr) and DF (3.42 mm/ hr) treated group as compared to control group (Fig 6.69). Also, TPR at 400 mg/kg p.o. treated group showed significantly (P< 0.01) lowered values for ESR as compared to the control group.

** P < 0.01 when compared with control (ANOVA followed by Dunnett's t-test).

Fig 6.69: Showing effect of TPF, TPR, FEA and REA on Erythrocyte (RBC) sedimentation rate (ESR) in FCA induced arthritis in rats.

D) Concentration of C- reactive protein (CRP)

The rate of synthesis and secretion of CRP is increases within hours of an acute injury or the onset of inflammation. So, if CRP concentration is greater than 0.6 mg/dL a visible agglutination is observed in the presence of CRP latex reagent. The levels of CRP in serum were measured by semi-quantitative method. Methanolic extracts (400 mg/kg, p.o.) and DF significantly (P< 0.01) lowered the level of CRP as compared to control. (Fig 6.70). CRP level in TPF (400 mg/kg, p.o.) treated group (2 mg/dL) was lower than that of the DF treated group (1.8 mg/dL).

* P < 0.05, ** P < 0.01 when compared with control (ANOVA followed by Dunnett's t-test).

Fig 6.70: Showing effect of TPF, TPR, FEA and REA on concentration of CRP in FCA induced arthritis in rats.

E) Concentration of rheumatoid factor (RF)

Any serum sample containing 10 IU/mL or more of rheumatoid factor will show a clear agglutination. The levels of RF in serum were measured semi-quantitatively. In this study, serum sample of control showed 73.33 IU/mL of RF. TPF, TPR, FEA and REA 400 mg/kg, p.o. showed significantly lowered concentration of RF at concentration of 30, 33.33, 46.67 and 53.33 IU/mL respectively. The concentrations of RF in TPF (400 mg/kg, p.o.) treated group was fairly similar to that of the positive control group treated with DF (Fig 6.71).

* P < 0.05, ** P < 0.01 when compared with control (ANOVA followed by Dunnett's t-test).

Fig 6.71: Showing effect of TPF, TPR, FEA and REA on concentration of RF in FCA induced arthritis in rats.

5.2.5 Anti- hyperlipidemic activity

5.2.5.1 in- vivo study

5.2.5.1.1. High fat diet (HFD) induce hyperlipidemia (hyper-cholesterolemia) in rats (Chronic study)

High fat diet administration in animals was showed increased serum levels of cholesterol (236.8 mg/dl), triglyceride (229 mg/dl) and lipoproteins [LDL-c (164.3 mg/dl), VLDL-c (45.80 mg/dl)] however, the HDL-c level get decreased to 26.63 mg/dl. Administration of TPF (400 mg/kg/p.o.) to HFD rats results in significant (P< 0.01) decreased in the level of serum cholesterol (160.3 mg/dl) (Fig 6.72), triglyceride (138.7 mg/dl) (Fig 6.73), LDL-c (91.34 mg/dl) (Fig 6.74) and VLDL-c (27.73 mg/dl) (Fig 6.75), with significant (P< 0.01) increase in HDL-c level by 41.95 mg/dl (Fig 6.76)

All extract treated groups were showed the significant (P< 0.05, P< 0.01) reduction in serum cholesterol level. TPF and TPR at higher dose (400 mg/kg) were found to be more significant (P< 0.01) among the test groups as they showed reduced serum level of triglyceride, LDL-c, VLDL-c and increased serum level of HDL-c. Standard drug Simvastatin (Sim, 4 mg/kg) exhibited statistically significant (P< 0.01) and highest degree of protective effect throughout the study.

*P < 0.05, **P < 0.001, when compared with HFD (ANOVA followed by Dunnett's t-test)

Fig 6.72: Effect of TPF and TPR on serum cholesterol level in HFD induced hyperlipidemia in rats.

*P < 0.05, **P < 0.001, when compared with HFD (ANOVA followed by Dunnett's t-test)

Fig 6.73: Showing effect of TPF and TPR on serum triglyceride level in HFD induced hyperlipidemia in rats.

*P < 0.05, **P < 0.001, when compared with HFD (ANOVA followed by Dunnett's t-test)

Fig 6.74: Showing effect of TPF and TPR on serum LDL-c level in HFD induced hyperlipidemia in rats.

*P < 0.05, **P < 0.001, when compared with HFD (ANOVA followed by Dunnett's t-test)

Fig 6.75: Showing effect of TPF and TPR on serum VLDL-c level in HFD induced hyperlipidemia in rats.

*P < 0.05, **P < 0.001, when compared with HFD (ANOVA followed by Dunnett's t-test)

Fig 6.76: Showing effect of TPF and TPR on serum HDL-c level in HFD induced hyperlipidemia in rats.

While, the animals treated with Simvastatin (Sim, 4 mg/kg) and TPF (400 mg/kg) were showed significant (P< 0.01) decrease in body weight as compared to HFD group when measured on day 20 and day 30. Finally, the average body weight of animals of HFD, Sim and TPF (400 mg/kg) treated group was found to be 267.3 g, 207.0 g and 221 g respectively (Table 6.47). At the final day, HFD showed 50.23 % raise in initial body weight, as compared to this the TPR and TPF (400 mg/kg) were demonstrated 32.98 % and 25.17 % rise, and for Sim (4 mg/kg) it was found to be 17.13 %.

Table 6.47: Effect of TPF and TPR on body weight in HFD induced hyperlipidemia in rats.

Groups

Dose (mg/kg)

Mean body weight# (g)

Initial

Day 10

Day 20

Day 30

Control

CMC

174.8±7.43

183.0±10.2

193.8±5.17

195.2±3.41

HFD

--

179.0±5.03

214.2±4.93 (19.54)

231.2±7.28 (30.62)

267.3±8.10 (50. 23)

HFD + Sim

4

177.3±3.19

187.2±3.70 (5.56)

197.0±6.80** (11.14)

207.0±5.97** (17.13)

HFD + TPR

100

171.3±6.67

196.5±6.00 (14.92)

215.0 ±6.43 (25.51)

243.0±9.37* (43.04)

HFD + TPR

200

174.0±4.67

195.7±3.57 (12.59)

212.2±3.71 (19.79)

240.5±3.79* (38.69)

HFD + TPR

400

176.7±4.30

195.7±5.53 (10.73)

202.8±5.52** (15.39)

234.0±2.41** (32.98)

HFD + TPF

100

170.3±5.57

197.2±4.92 (16.01)

213.3±5.91 (26.62)

240.2±9.04* (42.0)

HFD + TPF

200

177.8±4.07

197.2±3.80 (11.01)

207.7±3.71* (19.45)

237.7±3.82** (33.91)

HFD + TPF

400

177.5±3.45

185.0±5.47 (4.46)

203.2±4.76** (14.58)

221.0±3.89** (25.17)

Values in parenthesis indicates % rise in initial body weight, #Values are expressed as mean ± SEM. n = 6. *P < 0.05, **P < 0.001, when compared with HFD group (ANOVA followed by Dunnett's t-test)

Fig 6.77 showed the effect of TPF and TPR on atherogenic index. However, only Simvastatin (Sim, 4 mg/kg) and the TPF (400 mg/kg) were showed significant (P< 0.01) atherogenic index as compared to HDF treated group.

Fig 6.77: Showing effect of TPF and TPR on atherogemnic index in HFD induced hyperlipidemia in rats.

5.2.5.1.2. Triton WR 1339-induced hyperlipidemia in rats (acute study)

Triton WR-1339 has causes a marked increase in cholesterol and triglyceride serum concentrations after intraperitoneal injection (200 mg/kg) as compared to normal group. Simultaneous administration of TPF and TPR at the dose of (100 and 200 mg/kg) does not shows significant reduction in total cholesterol, triglyceride, VLDL, LDL when compared with triton treated group. Whereas standard drug Gemfibrozil (250 mg/kg) and TPF (400 mg/kg) shows significant (P< 0.01) reduction in lipid profile as compared to triton treated group.

Fig 6.78 depicts the effect of methanolic extracts on cholesterol level. TPF (400 mg/kg) showed significant reduction in serum cholesterol level in 24 and 48 hrs after triton injection. Gemfibrozil (Gem, 250 mg/kg) displayed fairly greater effect with serum cholesterol level 127.1 mg/dl (24 hrs, P< 0.01) and 86.45 mg/dl (48 hrs, P< 0.01)

*P < 0.05, **P < 0.001, when compared with HCG (ANOVA followed by Dunnett's t-test)

Fig 6.78: Showing effect of TPF and TPR on serum cholesterol level in triton induced hyperlipidemia in rats.

Effect of TPF and TPR on serum triglyceride level was summarized in Fig 6.79 Methanolic extracts did not showed significant (P> 0.05) reduction in serum triglyceride level, excluding TPF (400 mg/kg) which was showed statistically significant (P< 0.05) reduction after 24 hrs of the triton injection. On the other hand, Gemfibrozil (Gem, 250 mg/kg) showed significant reduction in triglyceride level at 24 hrs (P< 0.01) and 48 hrs (P< 0.05) as compared to hyperlipidemic control group treated group.

*P < 0.05, **P < 0.001, when compared with HCG (ANOVA followed by Dunnett's t-test)

Fig 6.79: Showing effect of TPF and TPR on serum triglyceride level in triton induced hyperlipidemia in rats.

The standard drug Gemfibrozil (250 mg/kg) significantly (P< 0.01) increase the serum level of HDL-c at 24 and 48 hrs as compared to hyperlipidemic control group. TPF and TPR also showed increase in serum HDL-c level; however, it was not found to be significant statistically. For, hyperlipidemic control group the HDL-c level was 37.45 mg/dl (24 hrs) and 38.20 mg/dl (48 hrs) and for TPF (400 mg/kg) it was 37.36 mg/dl (24 hrs) and 40.84 mg/dl (48 hrs). Hence, the methanolic extracts did not exhibit any considerable increment in the level of serum HDL-c. TPR (400 mg/kg) (Fig 6.80)

*P < 0.05, **P < 0.001, when compared with HCG (ANOVA followed by Dunnett's t-test)

Fig 6.80: Showing effect of TPF and TPR on serum HDL-c level in triton induced hyperlipidemia in rats.

Similar effect was observed with TPF and TPR on serum LDL-c level. Only TPF at higher dose (400 mg/kg) showed statistically significant (P< 0.01) reduction in LDL-c level after 48 hrs. While, gemfibrozil demonstrated the significant (P< 0.01) effect at 24 hrs (14.22 mg/dl) and 48 hrs (22.19 mg/dl) as compared to hyperlipidemic control group which showed LDL-c level 45.86 mg/dl at 24 hrs and 50.79 mg/dl at 48 hrs respectively. Although remaining test extracts were showed reduction in serum LDL-c level but it was non- significant (P> 0.05). (Fig 6.81)

*P < 0.05, **P < 0.001, when compared with HCG (ANOVA followed by Dunnett's t-test)

Fig 6.81: Showing effect of TPF and TPR on serum LDL-c level in triton induced hyperlipidemia in rats.

*P < 0.05, **P < 0.001, when compared with HCG (ANOVA followed by Dunnett's t-test)

Fig 6.82: Showing effect of TPF and TPR on serum VLDL-c level in triton induced hyperlipidemia in rats.

5.2.5.1.3. Hypolipidemic activity in rats

Methanolic extracts were tested for hypolipidemic activity in normal rats kept on normal diet. Simvastatin (4 mg/kg) was used as reference standard for the study which shows significant decrease in Serum cholesterol (P< 0.05), triglyceride (P< 0.01) and VLDL-c level (P< 0.05) however HDL-c level was significantly (P< 0.01) increased as compared to control group.

TPF and TPR did not produce any significant changes in serum cholesterol level in normal rats (Fig 6.83). But at the higher dose (400 mg/kg) TPF and TPR fairly reduced the cholesterol level but it wasn't found to be significant. Cholesterol level for control group was nearly 91.92 mg/dl and for Simvastatin it was 78.84 gm/dl.

*P < 0.05, when compared with control (ANOVA followed by Dunnett's t-test)

Fig 6.83: Showing effect of TPF and TPR on serum Cholesterol level in hypolipidemic in rats.

Serum triglyceride level was significantly reduced by TPF (400 mg/kg) (P< 0.05) and Simvastatin (Sim, 4 mg/kg) (P< 0.01) as compared to control. Remaining doses of extracts were showed any measurable change in triglyceride level. For Simvastatin, TPF (400 mg/kg) and control; triglyceride level was 106.2 gm/dl, 110.2 mg/dl and 128.6 mg/dl respectively.

*P < 0.05, **P < 0.001, when compared with control (ANOVA followed by Dunnett's t-test)

Fig 6.84: Showing effect of TPF and TPR on serum triglyceride level in hypolipidemic in rats.

Concurrently, serum HDL-c level did not found to be statistically significant in TPF and TPR treated group. Practically, TPF (400 mg/kg) was proved to increase the level (46.29 gm/dl) of HDL-c in normal rats, but it was not statistically significant (P> 0.05). Simvastatin (Sim, 4 mg/kg) treated group showed statistically significant (P< 0.01) rise in protective HDL-c level nearly to 55 mg/dl as compared to control group (41.19 mg/dl)

**P < 0.001, when compared with control (ANOVA followed by Dunnett's t-test)

Fig 6.85: Showing effect of TPF and TPR on serum HDL-c level in hypolipidemic in rats.

Similarly, all the extract and Simvastatin treated groups did not exhibits the statistically significant reduction in serum LDL-c level. Although, there was found to be somewhat reduction in Sim (4 mg/kg) (24.80 gm/dl) and TPF (400 mg/kg) (27.69 gm/dl) group but not significant (P> 0.05) as compared to control group (31.54 gm/dl).

Fig 6.86: Showing effect of TPF and TPR on serum LDL-c level in hypolipidemic in rats.

Again, in case of serum VLDL-c level, only Simvastatin (4 mg/kg) treated group showed significant (P< 0.05) reduction. Remaining extract treated groups did not alter the VLDL-c level in the serum to a significant (P> 0.05) level. TPF at higher the dose of 400 mg/kg was showed slight reduction (27.69 gm/dl) in VLDL-c level as compared to control group level (31.54 gm/dl).

*P < 0.05, when compared with control (ANOVA followed by Dunnett's t-test)

Fig 6.87: Showing effect of TPF and TPR on serum triglyceride level in hypolipidemic in rats.

5.2.5.1.4. HMG-CoA Reductase enzyme activity

The ratio of HMG-CoA / Mevalonate ratio was significantly (P< 0.01) increased by the standard drug Simvastatin (Sim, 4 mg/kg) (3.29) whereas, TPF and TPR at a dose of 400 mg/kg were also exhibited significant increase in the ratio as compared to control group. The middle dose (200 mg/kg) of both the extracts did not showed statistically significant effect as compared to control group. TPF 400 mg/kg showed higher effect (2.71), followed by Simvastatin; among the test extracts. (Fig 6.88)

**P < 0.001, when compared with control (ANOVA followed by Dunnett's t-test)

Fig 6.88: Showing effect of TPF and TPR on HMG- CoA/ mevalonate ratio in rats.

5.2.5.2 in- vitro study

5.2.5.2.1. Platelet anti-aggregation activity

The percentage inhibition of platelet aggregation was significantly increased with the treatment of high concentration (1000 μg/ml) of TPF (41.40) and TPR (33.68). Whereas, treatment with standard drug, Heparin (Hep 20 μg/ml) was showed highest percentage (79.57) of platelet inhibition. The lower concentrations of TPF and TPR were also demonstrated anti-aggregation effect to a certain extent. (Table 6.48)

Table 6.48: Effect of TPF and TPR on platelet aggregation induced by ADP

Groups

Concentration (μg/ml)

Absorbance#

% Inhibition

ADP

10

2.23± 0.01

--

Hep

20

0.56± 0.17**

79.57

TPR

250

1.97± 0.01

16.65

500

1.82± 0.06**

23.35

1000

1.58± 0.04**

33.68

TPF

250

1.84± 0.09**

22.17

500

1.72± 0.13**

27.44

1000

1.41± 0.10**

41.40

#Values are expressed as mean ± SEM. n = 6. * P < 0.05, ** P < 0.001 when compared with control (ANOVA followed by Dunnett's t-test)

5.2.5.2.2. Anti-inflammatory activity

The inhibitory effect of different concentrations of TPF and TPR on protein denaturation was found to be concentration dependent are shown in Table 11. TPF and TPR at different dose levels (50 - 250 μg/ml) showed considerable ability to inhibit denaturation of egg albumin.

Table 6.49: Effect (% inhibition) of TPF and TPR on protein denaturation

Drug concentration

(µg/ml)

Inhibition of protein denaturation (%)

TPF

TPR

50

34.67 ± 0.79

24.78 ± 0.82

100

39.54 ± 0.84

30.72 ± 0.27

150

46.34 ± 0.89

39.91 ± 0.42

200

67.83 ± 0.45

52.23 ± 0.91

250

72.76 ± 0.39

64.71 ± 0.82

#Values are expressed as mean ± SEM. n = 3.

5.2.6 Antioxidant activity

5.2.6.1 DPPH radical scavenging activity

The results of the DPPH radical scavenging activity of Ascorbic acid and all samples were shown in Fig 6.89. All the samples were produced concentration dependant reduction in absorbance and rise in percentage inhibition. The scavenging ability of the TPF (IC50 44.62) was found to be greater among the all tested samples; however ascorbic acid was showed DPPH radical scavenging efficacy with IC50 37.79 (Table 6.50). Ethyl acetate extracts also exhibits similar antiradical effect as that of AA.

Fig 6.89: Showing effect of TPF, TPR, FEA and REA on the DPPH radical scavenging activity

5.2.7.2 Nitric oxide scavenging activity

TPF showed considerable scavenging of nitric oxide radical as compared to standard drug. The % scavenging and IC50 vales for ascorbic acid and TPF were 41.32 and 43.02 respectively (Table 6.50). However, all the samples were exhibited concentration dependant rise in percentage inhibition. The absorbance of the samples was reduced as increase in concentration (Fig 6 .90). The Higher concentration of each sample was showed percentage inhibition almost > 50%.

Fig 6.90: Showing effect of TPF, TPR, FEA and REA on the Nitric oxide radical scavenging activity

5.2.7.3 Anti-Lipid Peroxidation (ALP) By Using Liver Homogenate

Again, higher concentration of TPF shows grater anti- lipid peroxidation activity, as indicated by their % inhibition values (Table 6.50); but less as compared to ascorbic acid which shows nearly 73.80 % inhibition. The level of lipid peroxidation was suppressed concentration dependently by all the tested extracts. FEA (65.21 %) and REA (63.94 %) showed less inhibition as compared to ascorbic acid. As concentration of sample increase the absorbance of reaction mixture was decreased consequently. (Fig 6.91)

Fig 6.91: Showing effect of TPF, TPR, FEA and REA on the lipid peroxidation in rat liver homogenate

Table 6.49: Percentage inhibition by AA, TPF, TPR, FEA and REA in different in-vitro antioxidant models

Sr. no

Conc. (µg/mL)

DPPH Scavenging activity

Nitric Oxide Scavenging activity

Lipid Peroxidation inhibition

AA

20

27.94

36.05

24.94

40

45.16

50.89

38.23

60

63.84

58.21

51.01

80

74.02

69.73

55.57

100

85.67

82.81

73.80

TPF

20

38.09

49.97

45.71

40

43.67

54.57

51.24

60

48.81

60.48

56.58

80

63.63

65.04

62.61

100

72.92

70.77

69.20

TPR

20

39.22

37.67

38.67

40

43.41

42.72

45.13

60

47.38

52.14

49.94

80

62.30

56.66

55.27

100

70.30

63.29

60.25

FEA

20

43.79

43.95

39.57

40

48.58

48.50

45.03

60

52.52

55.91

49.89

80

55.57

61.66

57.61

100

59.49

65.20

63.94

REA

20

30.98

42.30

33.57

40

37.89

46.34

37.07

60

42.29

50.98

45.12

80

44.78

57.04

51.36

100

49.93

64.23

57.61

5.2.7.4 Reducing Power Assay

Accordingly, reducing power of all samples was found to be concentration dependant. The sample with higher absorbance has greater reducing capability. Among the extract the TPF showed higher reducing power (0.6794±0.004), and it was comparatively higher than standard drug ascorbic acid (0.5493±0.001). (Fig 6.92)

Fig 6.92: Showing effect of TPF, TPR, FEA and REA on the Reducing power capacity

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