9. in vivo pharmacological studiesshodhganga.inflibnet.ac.in/bitstream/10603/62402/8/17. chapter ix...
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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.
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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
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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.
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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)
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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.
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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
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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,
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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.
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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
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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
Prophylactic Model Therapeutic Model
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
Prophylactic Model Therapeutic Model
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
compared to normal control.
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
Prophylactic Model Therapeutic Model
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
Prophylactic Model Therapeutic Model
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
compared to normal control.
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
number of cells thereby confirming the efficacy of the extract at the higher dose as
well as dose dependent effect showed in Figure 9.12 to 9.16.
146
Figure 9.12 Normal control Figure 9.13 CFA Induced
Figure 9.14 CFA+ Methothrexate
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
number of cells thereby confirming the efficacy of the extract at the higher dose as
well as dose dependent effect showed in Figure 9.17 to 9.21.
Figure 9.17 Normal control Figure 9.18 CFA Induced
Figure 9.19 CFA+ Methothrexate
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.
Figure 9.22 Normal control Figure 9.23 CFA Induced
149
Figure 9.24 CFA+ Methothrexate
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
Figure 9.31 Normal control Figure 9.32 CFA Induced
Figure 9.33 CFA+ Methothrexate
59
Figure 9.34 CFA+ETGP 200 mg Figure 9.35 CFA+ ETGP400 mg
Histopathology of paw tissues in vivo antiarthritic activity of GP
Figure 9.36 Normal control Figure 9.37 CFA Induced
60
Figure 9.38 CFA+ Methothrexate
Figure 9.39 CFA+ETGP 200 mg Figure 9.40 CFA+ ETGP400 mg
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.
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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