9. in vivo pharmacological studiesshodhganga.inflibnet.ac.in/bitstream/10603/62402/8/17. chapter ix...

126

9. IN VIVO PHARMACOLOGICAL STUDIES

9.1 Acute oral toxicity study

The purpose of acute toxicity testing are to obtain information on the

biologic activity of a chemical and gain insight into its mechanism of action. The

information on acute systemic toxicity generated by the test is used in hazard

identification and risk management in the context of production, handling, and use

of chemicals. The LD50 value, defined as the statistically derived dose that, when

administered in an acute toxicity test, is expected to cause death in 50% of the

treated animals in a given period, is currently the basis for toxicological

classification of chemicals.

9.1.1 Materials and methods

9.1.2 Experimental animals

Healthy adult female wistar Albino rat, weighing about 20-25 g were

procured from Biogen, Bangalore. The study was approved by the Institutional

Animal Ethical Committee, Sri Ramachandra University, Chennai (IAEC/XXXIV/

SRU/294/2013Version-I) animals were housed individually in a well ventilated

polypropylene cage. 12-hr light/12-hr dark artificial photo period was maintained.

Room temperature of 25°C (±3° C) and relative humidity of 50–70 % were

maintained in the room. Animals had free access to pelleted feed (Nutrilab rodent,

Tetragon Chemie Pvt Ltd., India) and reverse osmosis (Rios, USA) purified water ad

libitum. For experimental purpose the animals were kept fasting overnight but

allowed for access to water.

127

9.1.3 Acute toxic class method

Acute oral toxicity study was performed according to the OECD

(Organization for Economic Co-operation and Development) test guideline 423-

Acute toxic class method. It is a stepwise procedure with three animals of single sex

per step. Depending on the mortality and morbidity status of the animal, an average

of 2-4 steps may be necessary to allow judgment on the test substance. The

procedure is to fix a minimal number of animals, which allows acceptable database

scientific conclusion. The method uses different defined doses (5, 50, 500, 2000

mg/kg body weight) and the results allow a substance to be ranked and classified

according to the -Globally Harmonized System (GHS) for the classification of

extracts which cause acute toxicity.

Procedure

Three healthy, wistar albino rats weighing 20-25 g were selected for the

study. The rats were fasted over-night and provided with water ad libitum.

Following the period of fasting, a dose limit at 2000 mg/kg body weight of ethanol

extract dissolved in olive oil was administered orally to the animals of test group.

The animals of control group received olive oil. In each case the volume

administered was 10 ml/kg body weight. Following administration, the animals were

closely observed during the first 3 hr, and 48 hr, thereafter, 14 days, for toxic sign

and symptoms such as convulsions, salivation, diarrhoea, lethargy, sleep, coma and

death. The weight of the animals was monitored throughout the experimental period.

9.1.4 Results and discussion

During the acute toxicity study, the ethanol extract was administered

orally and animals were observed for mortality, changes in the autonomic nervous

128

system, central nervous system and behavioral responses. The observations were

tabulated in table 9.1.

Table 9.1 Observations of acute oral toxicity

Group Treatment Animal No. Organs Observations

I

GP (2000 mg/kg

body weight)

121 Skin, eyes, brain,

lung, heart, liver,

kidney, adrenals,

spleen and sex

glands

No abnormality

detected

122 No abnormality

detected

123

No abnormality

detected

There was no mortality observed even at 2000 mg/kg body weight for the

extract. No abnormality in the general behavior of the test animals either in the

short term or long term, was observed. There was no body weight loss during the

observation period. All the animals were found to be normal and there were no gross

behavioral changes till the end of the observation period. This observation revealed

that the ethanol extract of the plant was found to be very safe up to 2000 mg/kg of

body weight known as maximum tolerated dose (MTD) by acute toxicity model

study as per OECD guidelines 423. Hence from this, 1/10th and 1/5th of MTD was

selected and the effective doses were fixed as 200 and 400 mg/kg for further

pharmacological studies.

129

9.2 IN VIVO ANTI-ARTHRITIC ACTIVITY OF ETHANOL

EXTRACT OF Glycosmis pentaphylla

9.2.1 Animals

Male Wister rats (150 -250 g) were procured from Biogen Laboratory

Animal Facility Bangalore. The animals were maintained in animal house at a room

temperature of 19-230C, with relative humidity of 50 to 70 % and alternating 12 Hr

dark –light. The animals had free access to standard laboratory feed and water

adlibitum. The rats were acclimatized to the environment for 1 week prior to

experiment use. The study was approved by the institutional animal ethics

committee (IAEC/XXXIV/SRU/294/2013Version-I)

9.2.2 Induction of arthritis

Animals were injected intraperitoneal with 0.1 ml of Complete Freunds

Adjuvant (CFA) into the right hind paw of each rat. Animals were assigned to

groups of 6 animals. Arthritic control group received only intraperitoneal injection

of CFA, but non-arthritic control (IFA group) received intraperitoneal injection of

0.1 ml Incomplete Freunds Adjuvant (IFA) (Sterile Paraffin Oil). ETGP (200 mg/ kg

and 400 mg /kg), Methotrexate (0.1 ml/ kg) were administered to rats in the various

groups respectively. Paw volume was measured by water displacement

plethysmograph (Fereidoni, et. al, 2000) for both the ipsilateral (injected paw) and

the contralateral paw (non- injected paw) before intraperitoneal injection of CFA

(day 0) and every week. The edema component of inflammation was quantified by

measuring the different in foot volume between day 0 and day 28. (Pearson et.al,

1956)

130

9.3 Two sets of experiments were carried out

9.3.1 The test drugs on established arthritis (Therapeutic protocol)

Intraperitoneal injection of 0.1 ml CFA was followed by drug

administration on day 9 with the onset of arthritis.

Group 1 : Non Control/ IFA (Intraperitoneal injection of 0.1 ml of IFA)

Group 2 : Arthritic control/ CFA (Intraperitoneal injection of 0.1 ml CFA)

Group 3 and 4 : ETGP 200 mg/ kg and 400 mg/ kg respectively from day 9

Group 5 : Methotrexate (0.1 ml/ kg)

9.3.2 Test drug when given before inducing arthritis (Prophylactic

protocol)

Drugs were administered on day 0 and CFA was injected intraperitoneal

24 hrs later.

Group 1 : Non Control/ IFA (Intraperitoneal injection of 0.1 ml of IFA)

Group 2 : Arthritic control/ CFA (Intraperitoneal injection of 0.1 ml CFA)

Group 3 and 4 : ETGP 200 mg/ kg and 400 mg/ kg respectively from day 0

Group 5 : Methotrexate (0.1 ml/ kg)

Scores for ipsilateral and contralateral paw volume were individually

normalized as percentage of change from their value at day 0 and then averaged for

each treatment group. The extract and standard was suspended in 0.5 % CMC. All

the drugs were freshly prepared.

131

9.3.3 Arthritis index

Rats were evaluated every week for arthritis. The physical symptoms of

arthritis were judged by the following grading system (Wooley PH, et al, 1981): 0 =

normal paws; 1 = erythema of toes; 2 = erythema and swelling of paws; 3 = swelling

of ankles; 4 = complete swelling of the whole leg and inability to bend it. The

maximum achievable score is thus 16. Arthritis index for each rat was calculated by

adding the four scores of individual paws. A sensitized animal was considered to

have arthritis when atleast one non-injected paw was inflammed (Philippe L, et al,

1997).

9.3.4 Measurement of body weight

Body weight for each rat was recorded before and every week after

adjuvant inoculation to assess food intake and weight gain throughout the period of

arthritis. The difference between body weight in each day and that of day 0 was

calculated to determine the change in body weight in arthritic rats. Percentage of

body weight changes was calculated.

9.3.5 Measurement of paw volume changes

Volumes of hind paws were measured before and daily after adjuvant

inoculation by using water displacement plethysmometry (David LB, et al, 2001).

The changes of volumes of hind paws, from those of day 0, were calculated.

9.3.6 Biochemical analysis

At the end of experiment, on the 28th day all animals were anaesthetized

and blood was withdrawn by retro-orbital puncture and collected in plain and EDTA

containing tubes, respectively for serum separation. The homogenized samples were

132

subjected to biochemical examination like BUN (Fischbach FT et al; 2004), CR, TP,

ALT, AST, ALP, and CRP in blood and in liver SOD, GSH, LPO, GPX and

PROTEIN.

9.3.7 Statistical analysis

The results are presented as the mean ± standard error. Clinical edema

volumes measured in different treatment groups were compared with adjuvant non-

treated control group (group II) and non-adjuvant control group (group I) by one

way ANOVA and Dunnett's Multiple Comparison Test. Also, significance tests were

calculated to determine the differences between the effects of different doses.

9.4 Immunohistochemistry

Immunohistochemistry (IHC) refers to the process of detecting antigens

in cells of a tissue section by exploiting the principle of antibodies binding

specifically to antigens in biological tissues. IHC takes its name from the roots

"immuno," in reference to antibodies used in the procedure, and "histo," meaning

tissue (compare to immunocytochemistry). The procedure was conceptualized and

first implemented [Albert Coons, 1941]

Immunohistochemical staining is widely used in the diagnosis of

abnormal cells such as those found in cancerous tumors. Specific molecular markers

are characteristic of particular cellular events such as proliferation or cell death. IHC

is also widely used in basic research to understand the distribution and localization

of biomarkers and differentially expressed proteins in different parts of a biological

tissue.Visualising an antibody-antigen interaction can be accomplished in a number

of ways. In the most common instance, an antibody is conjugated to an enzyme,

https://en.wikipedia.org/wiki/Antigen

https://en.wikipedia.org/wiki/Antibody

https://en.wikipedia.org/wiki/Biological_tissue

https://en.wikipedia.org/wiki/Immunocytochemistry

https://en.wikipedia.org/wiki/Albert_Coons

133

such as peroxidase, that can catalyse a colour-producing reaction. Alternatively, the

antibody can also be tagged to a fluorophore, such as fluorescein orrhodamine.

9.4.1 Materials and methods

CD4 (#SC7219, Santa Cruz, U.S.A.))

IL-2 (#SC7896 Santa Cruz, U.S.A.)

TGF-beta (#SC-146, Santa Cruz)

TNF-alpha 2 (#SC52746 Santa Cruz)

9.4.2 Protocol

1. Sections deparaffinized in xylene and hydrated through descending

grades of alcohol.

2. Heat-induced antigen retirival was retrieval was carried out by

microwaving at HI-90 for 20 minutes using citrate buffer (pH 6.8)

for TNF-alpha or Tris-EDTA buffer (pH 8.0) for CD4, IL-2 and

TGF-beta .

3. Each step herein is preceded by three washings in PBST (Phosphate

buffered saline with 0.1% tween 20).

4. Endogenous peroxidase quenching by incubation with 3%H2O2 for

30 minutes.

5. Blocking with 5% goat serum in 1%BSA for 30 minutes.

6. Primary antibody incubation at 4°C overnight.

7. Biotinylated secondary antibody incubation for 30minutes at room

temperature.

https://en.wikipedia.org/wiki/Peroxidase

https://en.wikipedia.org/wiki/Fluorophore

https://en.wikipedia.org/wiki/Fluorescein

https://en.wikipedia.org/wiki/Rhodamine

134

8. Avidin-Biotin peroxidase incubation for 30 minutes at room

temperature.

9. Staining with DAB (3,3’-diaminobenzidine)chromogen for 15-20

minutes.

10. Counterstaining with Harris hematoxylin for 30 seconds.

9.5 Histopathological examination

The rats from each group at the end of the experimental period were

euthanized using anaesthetic ether. The left hind paw from all the rats were

amputated proximal to the ankle joint and were fixed in 10% neutral buffered

formalin for 24 hours followed by decalcification in 10% formic acid for 4 days

approximately. After decalcification, the digits were trimmed off and the ankle joints

and paw tissue were transected in a mid-sagittal plane. The ankle joint and paw

tissues were passed through series of ascending grades of alcohol and embedded in

paraffin. 3-4 micron thickness of tissue section were sectioned and then stained with

Hematoxylin and Eosin (Bancroft and Gamble, 2008) for histopathological

evaluation of joints and paw tissues for arthritis and other associated lesions.

9.6 Results and discussion

Presence of various phytochemical constituents may be responsible for

many pharmacological actions like antiinflammatory, antioxidant and antiarthritic.

9.6.1 Effect of Drugs on Adjuvant Induced Arthritis on paw volumn

The models of the adjuvant arthritis investigated in the present study

were the acute and poly-arthritis/ chronic models corresponding to day 0-10 and

days 10-18 post adjuvant inoculation respectively. The one way ANOVA (treatment

135

X time) revealed a significant (p< 0.05) effect of drug treatment. Total edema

produced by each treatment is expressed in arbitrary units as AUC of the time course

curves.

All arthritis control animals showed acute inflammatory edema at the

ipsilateral (injected paw) around 4-6 days followed by subsequent chronic poly-

arthritis model which begins around day 10-12. The progress of inflammatory

edema in the contralateral (non-injected) paw was evident on day 12 with systemic

inflammation. Throughout the 28 day experiment, there was no significant change in

the paw volume of the non inflamed control group injected IFA. ETGP 200 mg/ kg

and 400 mg/ kg modified the time course significantly (p< 0.05) and reduced the

acute edema in the ipsilateral paw with the lowest dose table 9.5 used (200 mg/ kg)

significantly (p< 0.05) reducing the edema with percentage inhibition when

compared with standard Methotrexate. (Table 9.5 and Figure 9.3 to 9.6)

This Methotrexate and ETGP completely presented the spread of the

arthritic from the ipsilateral to the contralateral paws of the treatment animals. ETGP

400 mg/ kg did not exhibit significant anti-arthritic activity. ETGP 200 mg/ kg and

400 mg/ kg did not show any significant effect compared to standard.

9.6.2 Arthritic score

Results of arthritic score for the therapeutic and prophylactic model

showed significant (p< 0.05) score for the arthritic groups with wide spread

erythemia and increased hind paw swelling particularly for the ETGP 200 mg/ kg

body weight as group and lower scores for the anti-arthritic groups 400 mg/kg and

Methotrexate (0.1 ml/ kg). (Table 9.2 and Figure 9.1)

136

Table 9.2 Effect on arthritic score in adjuvant induced arthritis

Groups Arthritic Index Score

Prophylactic Model Therapeutic Model

Normal control 0 ± 0 0 ± 0

Arthritic control 4 ± 0.16 4 ± 0.16

ETGP 200 mg/ kg 2.4 ± 0.14 2.4 ± 0.14

ETGP 400 mg/ kg 3 ± 0.17* 2 ± 0.16*

Methotrexate 0.1 mg/ kg 1.6 ± 0.14** 1.4 ± 0.13**

Values are expressed as mean ± SEM (n= 6 ) *p< 0.05, **p< 0.01

compared with arthritic control.

Figure 9.1 Effect on arthritic score in adjuvant induced arthritis

AR

THR

ITIC

SC

OR

E

GROUPS

Prophylactic Model Therapeutic model

137

Table 9.3 Effect of root of ETGP on change in body weight

Groups % Body Weight Change


Days Day

7

Day

14

Day

21

Day

28

Day

7

Day

14

Day

21

Day

28

Normal control 3.2 3.8 4.87 5.28 3.2 3.4 4.17 4.88

Arthritic control 3 3.92 6.16 7.29 3 3.92 6.16 7.29

ETGP 200 mg/ kg 2.8 3.76 4.14 4.2 2.99 3.46 4.47 4.96

ETGP 400 mg/ kg 2.61 3.84 4.24 4.8 1.27 2.58 4.58 5.2

Methotrexate 0.1mg/ kg 1.95 2.51 2.89 3.22 2.2 2.84 3.2 3.1

Figure 9.2 Effect of ETGP root on change in body weight

9.6.3 Effect of Glycosmis pentaphylla treatment on Paw volume

Effect of vehicle, Glycosmis pentaphylla 200 mg/ kg and 400 mg/ kg

body weight, Methotrexate 0.1ml/kg body weight on paw volumes of CFA induced

arthritic animals with untreated pathogenic arthritic animals are tabulated in table

9.5 and graphically represented in Figure 9.3 to 9.6.

Normal control Arthritic control EEGP 200 mg/kg

138

Table 9.4 Effect of root of ETGP on paw volume on complete Freund’s adjuvant induced arthritic in rats

Groups Paw Volume


Days 0 7 14 21 28 0 7 14 21 28

Normal

control

1.22 ±

0.04

1.21±

0.04

1.32±

0.04

1.45±

0.03

1.24±

0.02

1.22

±0.03

1.2±

0.11

1.24±

0.01

1.24±

0.11

1.28±

0.12

Arthritic

control

1.37 ±

0.03

3.15±

0.06*

2.24±

0.06**

2.28±

0.02

2.34±

0.06**

1.37

±0.03

2.55±

0.15*

2.03±

0.08**

2.5±

0.18**

2.4±

0.02**

ETGP

200 mg/ kg

1.24 ±

0.02

3.39±

0.09*

2.18±

0.05**

2.63±

0.03**

2.02±

0.06**

1.24

±0.03

3.29±

0.19*

2.3±

0.33**

2.03±

0.26**

1.9±

0.03**

ETGP

400 mg/ kg

1.31 ±

0.03

3.52±

0.06*

1.77±

0.05**

2.02±

0.02**

1.85±

0.06**

1.31

±0.03

3.34±

0.19*

1.72±

0.24**

1.98±

0.02**

1.76±

0.03**

Methotrexate

0.1 mg/ kg

1.31±

0.03

2.94±

0.11*

1.52±

0.05**

1.77±

0.02**

1.66±

0.06**

1.47

±0.02

3.39±

0.03*

1.95±

0.02**

1.65±

0.33**

1.53±

0.03**

Values are expressed as mean ± SEM; n=6 rats in each group, *p< 0.05 compared with normal control, **p< 0.01 compared with arthritic

control.

139

Figure 9.3 Effect on paw volume changes in adjuvant induced arthritis

(Prophylactic model)

Figure 9.4 Effect on paw volume changes in adjuvant induced arthritis

(Therapeutic model )

0 7 14 21 28

0

1

2

3

4

Control

CFA+ Veh

CFA+ Methotrexate

CFA+ Low Dose

CFA+ High Dose

*

** ** ****

*****

**

*** **

***

### ###

###

###

$$$$

###p<0.001 vs Control

*p<0.05, **p<0.01vs CFA + Vehicle

$$p<0.01 vs Std

Days

Pa

w V

olu

me

(ml)

140

Figure 9.5 Percentage reduction on anti-arthritic inflammation (prophylactic

model)

Figure 9.6 Percentage reduction on anti-arthritic inflammation (Therapeutic

model)

0 7 14 21 28

-20

0

20

40

60 CFA + Methotrexate

CFA + Low Dose

CFA + High Dose

Days

% r

edu

ctio

n

0 7 14 21 28-20

0

20

40

60

CFA + Methotrexate

CFA + Low Dose

CFA + High Dose

Days

% r

educ

tion

141

9.6.4 Biochemical parameters

Results for BUN showed significant (*p< 0.05) increase in 400 mg/ kg

dose than 200 mg/ kg dose and standard in prophylactic model when compared to

normal control.

The biochemical parameter of CR showed significant (*p< 0.05) increase

in 400 mg/ kg dose and standard than 200 mg/ kg dose in prophylactic model. In the

therapeutic model there is a significant (*p< 0.05) increase in both the dose and

standard when compared to normal control.

The total protein showed significant (*p< 0.05) decrease in 200 mg/ kg

and 400 mg/ kg dose than standard in prophylactic model and therapeutic model

when compared to normal control.

Results for ALT, AST, ALP showed significant (*p< 0.05) increase in

400 mg/ kg dose than standard in prophylactic model and therapeutic model when

compared to normal control.

The CRP showed significant (*p< 0.05) increase in 200 mg/ kg dose and

400 mg/kg than 400 mg/ kg dose and standard in prophylactic model and in

therapeutic model significant (*p< 0.05) increase in 200 mg/kg dose only when


Results for SOD showed significant (*p< 0.05) increase in 200 mg/ kg

dose and 400 mg/ kg than standard in prophylactic model and in therapeutic model

significant (*p< 0.05) increase in 400 mg/ kg dose and standard when compared to

normal control. Results for GSH showed significant (*p< 0.05) increase in 200 mg/

kg, 400 mg/ kg and standard in prophylactic model and in therapeutic model

significant (*p< 0.05) increase standard when compared to normal control.

142

Table 9.5 Effect on biochemical parameters in blood

Parameters Normal

control

Arthritic

control


ETGP

200 mg/

Kg

ETGP

400 mg/

Kg

Methothrexate

0.1mg/ kg

ETGP

200 mg/

Kg

ETGP

400

mg/Kg

Methothrexate

0.1mg/ kg

BUN 37.75±

1.18

45.50±

5.17**

40.50±

0.50** 39.00±

1.15*

41.75±

1.31**

35.50±

3.20**

34.00±

2.38**

39.25±

2.06**

CR(mg/dl) 0.61±

0.05

0.48±

0.11**

0.50±

0.014** 0.65±

0.03*

0.57±

0.06*

0.45±

0.04*

0.45±

0.09*

0.51±

0.05*

TP (g/dl) 7.21±

0.22

7.25±

0.38* 6.48±

0.08*

7.15±

0.10*

6.85±

0.19 5.98±

0.34*

6.38±

0.44*

6.53±

0.19

ALT(U/L) 96.50±

5.72

99.75±

6.16** 85.75±

9.75*

92.00±

3.32

71.75±

13.26 83.25±

4.57*

79.50±

7.46

71.25±

11.33

AST(U/L) 142.50±

3.57

186.25±

30.92*

146.50±

7.50**

127.00±

6.04**

115.00±

10.68*

139.0±

4.08**

115.00±

5.46**

107.50±

12.6*

ALP(U/L) 607.25±

7.98

685.00±

52.42**

823.50±

27.50*

518.00±

85.78

567.75±

13.14

813.50±

27.50**

498.00±

85.78

560.25±

13.99

CRP-hs

(mg/L)

4.23±

0.49

3.74±

0.09** 3.64±

0.49*

3.48±

0.53*

3.74±

0.37 3.11±

0.42*

3.33±

0.54

2.99±

0.35

LDH(U/L) 278.00±

32.50

232.00±

32.26*

250.00±

24.00

265.00±

35.69

274.00±

20.31

248.50±

19.97

260.00±

36.29

266.50±

17.85

Values are mean ± SEM, n=6 rats per group. One way ANOVA followed by Tukey’s multiple comparison test. when compared with

normal control group, *P<0.05, **P<0.01

ALP- Alkaline phosphatase, ALT- Alanine aminotransaminase, AST- Aspartate aminotransaminase,

CR-Creatinine, LDH- Lactate dehydrogenase, TP- Total protein, CRP- hs- High sensitive C- reactive Protein

143

Table 9.6 Effect on antioxidant parameters in liver

Parameter Normal

control

Arthritic

control


ETGP

200 mg/ Kg

ETGP

400 mg/ Kg

Methothrexate

0.1mg/ kg

ETGP

200 mg/ Kg

ETGP

400 mg/ Kg

Methothrexate

0.1mg/ kg

SOD 0.35±

0.02

0.11±

0.006**

0.26±

0.005*

0.25±

0.005*

0.24±

0.006

0.26±

0.006

0.26±

0.005*

0.25±

0.02*

GSH 3.13±

0.008

1.03±

0.15**

2.41±

0.29*

2.4±

0.07*

2.4±

0.09* 2.49 ± 0.13 2.48 ± 0.04 2.46 ± 0.05*

LPO 0.11±

0.008

0.24±

0.04**

0.14±

0.02**

0.12±

0.02*

0.12±

0.03*

0.16±

0.02*

0.132±

0.01*

0.11±

0.005*

GPX 8.55±

0.32

17.93±

0.65**

11.28±

0.66*

10.12±

0.06

10.02±

0.39

10.66±

0.59*

10.51±

0.32

10.26±

0.53

Values are mean ± SEM, n=6 rats per group. One way ANOVA followed by Tukey’s multiple comparison test. when compared with

vehicle control group, *P<0.05, **P<0.01

SOD- Superoxide dismutase, GSH- Glutathione, LPO- Lipid peroxidase, GPX- Glutamate peroxidase.

144

Results for protein showed significant (*p< 0.05) increase in 200 and 400

mg/ kg than standard in prophylactic model and 200 mg/ kg therapeutic model when


The biochemical parameter of LPO showed significant (*p< 0.05)

increase in 400 mg/ kg and standard than 200 mg/ kg dose in prophylactic model and

in therapeutic model significant (*p< 0.05) increase in 200 mg/ kg, 400 mg/ kg and

standard when compared to normal control.

The GPX showed significant (*p< 0.05) increase in 200 mg/ kg than

standard and 200 mg/ kg dose in therapeutic model when compared to normal

control. All the bio chemical parameters and antioxidant parameter in liver value

showed in table 9.6 and 9.7.

9.6.5 Immunohistochemistry - IHC Inflammatory markers - CD4

The number of cells of CD4 in case of the group induced with CFA was

found to be high compared with the groups treated with the standard drug and the

extract treated groups. When compared with in groups treated with the extract of

400 mg and 200 mg. the group which received 400mg of the extract showed less

number of cells thereby confirming the efficacy of the extract at the higher dose as

well as dose dependent effect showed in Figure 9.7 to 9.11.

Figure 9.7 Normal control Figure 9.8 CFA Induced

145

Figure 9.9 CFA+ Methothrexate

Figure 9.10 CFA+ ETGP 200 mg Figure 9.11 CFA+ ETGP400 mg

IHC Inflammatory markers – IL 2

The number of cells of IL 2 in case of the group induced with CFA was

found to be more when compared with the groups treated with the standard drug and

the extract treated groups. The comparison with in groups treated with the extract

of 400 mg and 200 mg. the group which received 400mg of the extract showed less



146



Figure 9.15 CFA+ETGP 200 mg Figure 9.16 CFA+ ETGP400 mg

147

IHC (Tissue) Inflammatory markers – TGFβ1

The number of cells of TGFβ1 in case of the group induced with CFA

was found to be high compared with the groups treated with the standard drug and

the extract treated groups. When compared with in groups treated with the extract of

400 mg and 200 mg. the group which received 400mg of the extract showed less





148

Figure 9.20 CFA+ ETGP200 mg Figure 9.21 CFA+ETGP 400 mg

IHC Inflammatory markers – TNF-α

The number of cells of TNF-α in case of the group induced with CFA

was found to be more when compared with the groups treated with the standard drug

and the extract treated groups. The comparison with in groups treated with the

extract of 400 mg and 200 mg. the group which received 400mg of the extract

showed less number of cells thereby confirming the efficacy of the extract at the

higher dose as well as dose dependent effect showed in Figure 9.22 to 9.26.


149


Figure 9.25 CFA+ ETGP 200 mg Figure 9.26 CFA+ETGP 400 mg

9.6.6 Histopathology

The H&E section of the left ankle joints and paw tissue from

normal control group revealed normal histology of joint capsule,

synovium, periarticular tissues and paw tissue structure.

The H&E section of left ankle joints and paw tissue of the CFA

induced group revealed severe degree of dermal infiltration by

polymorphonuclear cells and panniculitis with mononuclear cells

infiltration. Skeletal muscular necrosis infiltrated by mononuclear

cells. Chronic proliferative synovitis characterized by hyperplasia

of synovivum, proliferation of connective tissues,

150

lymphoplasmocytic infiltration around the synctical blood vessels

and fibrosis. Moderate degrees of multinucleated giant cells

infiltration were also observed in panniculitis and synovitis.

Moderate degree of articular cartilage erosion and synovial

invasion with mononuclear cells infiltration in the eroded lesions

along with moderate degree of oestoclasts cells infiltration in the

bone components. Paw tissues revealed edema and severe

infiltration of mixed population of inflammatory cells (neutrophils

and mononuclear cells) in the subcutaneous tissues

The H&E section of the left ankle joints and paw tissues of the

reference control (methotrexate) has reduced the severity of

synovitis and inflammatory cells infiltration, muscular necrosis,

panniculitis and articular cartilage erosions and paw tissue

inflammatory cells infiltration to mild to moderate degree when

compared to that of the CFA induced arthritic group.

The H&E section of left ankle joint and paw tissue of arthritic

group treated with Glycosmsis pentaphylla at the dose level of 400

mg/kg b.wt (Both prophylactic and therapeutic dose) has revealed

mild to moderate degree of the panniculitis, moderate dermis and

subcutaneous infiltration of polymorphonuclear cell in the skin,

minimal to mild degree of synovitis , no articular erosions and

decreased proliferation of connective tissues, lymphocytic

infiltration around the blood vessels, decreased inflammatory

cellsinfiltrationin the subcutaneous paw tissues when compared to

the arthritic rats treated with Glycosmsis pentaphylla at the dose of

200mg/kg b.wt (both prophylactic and therapeutic dose)

151

Figure 9.27 Water Displacement plethysmograph Figure 9.29 Induction of arthritis

Figure 9.28 Measurement of Paw volume Figure 9.30 Paw after ETGP treatment

58

Histopathology of synovial joint in in-vivo anti-arthritic activity of GP



59


Histopathology of paw tissues in vivo antiarthritic activity of GP


60



9.6.6.2 Result and Discussion

The in vivo anti arthritic activity of the ETGP was evaluated in the

Complete Freund’s Adjuvant (CFA) induced arthritis model in rats. The Complete

61

Freund’s Adjuvant (CFA) induced arthritis model is considered as a chronic,

immunological, cellular and proliferative arthritis similar to clinical RA. In adjuvant

induced arthritis model, rats develop a chronic swelling in multiple joints with

influence of inflammatory cells, erosion of joint cartilage and bone destruction and

remodeling. These inflammatory changes ultimately result in the complete

destruction of joint integrity and function in affected animals. Complete Freund’s

Adjuvant (CFA) induced is considered as cell mediated autoimmunity due to the

structural mimicry between mycobacteria and cartilage proteoglycans in rats. This is

a most frequently used model for screening the anti–arthritic agents, especially

NSAIDs, as the inflammation associated with CFA is highly dependent on

Prostaglandin E2 (PGE2) generated by Cycloygenase (COX-2) [Anderson GD, et.

al, 1996].

The arthritis observed in rats is associated with a hyperalgesia

phenomenon and spontaneous behaviors, such as protection of the affected paw,

evidenced by curving and/ or elevation of the paw, as well as a voidance of

supporting the body on the paw (Claworthy et al, 1995). The hyperalgesia is more

evident during the acute inflammatory model, when spontaneous behaviors,

indicative of painful response are more pronounced. Increased paws diameter

(ipsilateral and contralateral paw), due to inflammation and edema is also observed

(Cain et al, 1997). The initial inflammatory response is developed within hours, but

more critical signals emerged from the 10th post inoculation day and thereafter and

the alteration remain detectable for several weeks (Cloparet et al, 1992). The results

of the present study indicate that ETGP exhibits anti-arthritic effects in rat with

Freund’s Adjuvant- Induced arthritis, either on its acute as well as its chronic model.

Pathogenesis of rheumatoid arthritis is multifactorial and recent research

has implicated oxygen free radicals as mediators of cartilage damage. Oxygen free

62

radicals such as superoxide and hydrogen peroxide are produced by

polymorphonuclear leukocytes when they ingest bacteria or immune complexes

(Gutteridge, 1986). In rheumatoid arthritis, it has been suggested that OH radical or

a similar oxidizing species, contribute to membrane damage, alteration in the protein

structure, conformation and antigenicity, production of auto-antibodies, hyaluronic

degradation and destruction of antioxidants within the synovial joints (Gutteridge,

1986). Many cellular defense mechanisms are afforded against the toxic effect of

these radicals in inflammation including serum sulfydryl groups, cerulopasmin, and

albumin and blood glutathione (Fahim et al, 1995).

The increased lipid peroxide level noticed in arthritic rats in our study

(group 2 in both therapeutic and prophylactic model) may be due to its release from

neutrophils and monocytes during inflammation (Greenwald, et al; 1980). At the

onset of inflammation, there is a rapid fall in the total iron content of blood plasma

followed by an increased deposition of iron protein in the synovial fluid. The drop in

plasma iron correlates closely with the activity of the inflammatory process. In the

synovial fluid of inflamed joints, the iron released during necrosis, might catalyze

the formation of OH (hydroxyl) radicals from H2O2, thus contributing increased

lipid peroxidation in arthritis. From the literature review, it is apparent that RA is

exposed to oxidative stress and is prone to lipid peroxidation (Heliovaara et al,

1994). In the present study, the concentration of lipid peroxidation was significantly

altered in arthritic rat after the administration of ETGP, when compared with

arthritic controlled rats.

Many cellular defense mechanisms are directed against the toxic effects

of these radicals in inflammatory process. SOD which converts superoxide radicals

to H2O2 is widely distributed in cells having oxidative metabolism and it is believed

to protect such cells against the toxic effects of superoxide anion. Increase delivery

63

of NADPH from stimulated HMB shunt during inflammation is proposed to lead to

the activation of SOD in arthritic rat. Increase production of NADPH from HMB

shunt during arthritis may cause and increase in SOD activity (Marklund, et al,

1987). This increase in enzyme activity appears to be protective against the

intracellular oxygen free radical (Kasama et al, 1988). Administration of ETGP to

arthritic rat caused a significant decrease in elevated SOD activity.

Superoxide anion is thought to be involved in inflammatory reactions as

they are produced by phagocytic cells (Babior et al, 1973). These cells are reported

to produce hydroxyl radical (Salin, et al; 1975) and singlet oxygen (Allen et al,

1972). Glutathione peroxidase is localized in cytoplasm and mitochondria, which

catalyses the degradation of various peroxides by oxidizing glutathione with the

formation of its conjugates. The increased activity of glutathione peroxides in liver

of arthritic rat, are shown by the present results, indicate the free radical defense

system against oxidative stress and health to explain the pathogenesis associated

with arthritis. After ETGP treatment, GPX level were significantly decreased.

After ETGP treatment, the alterations produced in arthritic rats with

respect to lipid peroxidation and antioxidant concentrations were modulated nearly

to normal levels. This antiperoxidative action observed in ETGP treated arthritic rats

might be due the presence of compounds like flavonoids and phenol in ETGP. These

compound shave been shown to scavenge free radicals, including hydroxyl and

superoxide anions and reduce the level of lipid peroxidation in stress induced

animals (Jovanovic, et al; 2000).

In the present investigation, reduction of paw swelling in the drug treated

rats from the third week onwards may be due to immunological protection rendered

by the plant extract. Adjuvant inoculation triggers the production of activated

64

macrophages and lymphocytes or their products like monokines, cytokines and

chemokines. These in turn produce lipid peroxides due to abnormal lipid

peroxidation leading to increased inflammation. In the current study, a significant

reduction in the inflammation was shown in Group IV and Group V of both

therapeutic and prophylactic treated animals when compared to that of Group II both

therapeutic and prophylactic treated animal’s toxin intoxicated animals indicating

the antiperoxidative nature of the plant extract. Complex network of cytokines and

growth factors with overlapping biological effects are observed in the perpetuation

of RA.

As a consequence of the inflammatory processes, several immunological

mediators such as CD4, TGFβ1,TNF–α and IL–2 have been implicated in the

pathological mechanism of synovial tissue proliferation, joint destruction and

programmed cell death in rheumatoid joint. It was reported that the expression of

inflammatory cytokines such as TNF–α and IL–2 and the tissue enzymes such as

metalloproteinase were observed to be increased in the sub–chondral bone region of

the knee joint samples from human osteoarthritis or rheumatoid arthritis patients.

Biological agents that specifically inhibit the effects of TNF–α or IL–2 or leukocyte

migration and accumulation in arthritis may have beneficial effects for joint

preservation. Thus, in this study, observing the invitro experiments such as

inhibition of protein denaturation, by the plant extracts and in vivo anti arthritic

effect analysis indicates proof towards the above theory of joint preservation. Plant

phenolic compounds have been found to possess potent anti inflammatory activity

(G Rao, et al, 2009]. It has also been reported that the flavonoids significantly

inhibit the leukocyte migration in a dose dependant manner

[Tian et al.1995 ] The presence of high content of flavonoids and phenol

in ethyl acetate extract could be responsible for the antiarthrtic activity.

http://www.researchgate.net/researcher/39265654_Tian-Shung_Wu

65

The pathological findings showed Figure 9.31 to 9.40 and suggest the

treatment of CFA induced arthritic group with Glycosmsis pentaphylla at the dose

level of 400 mg/kg b.wt (both at therapeutic and prophylactic level) have reduced

the severity of the arthritis and other associated tissue lesions in ankle joints and paw

tissues than the arthritic group treated with Glycosmsis pentaphylla at the dose level

of 200 mg/kg body weight.

66

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