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In vivo Studies on Antiarthritic Activity of Cissus quadrangularis against

Adjuvant Induced Arthritis

Article  in  Journal of Clinical & Cellular Immunology · January 2015

DOI: 10.4172/2155-9899.1000327

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 Clinical & Cellular Immunology Bhujade et al., J Clin Cell Immunol 2015, 6:3
http://dx.doi.org/10.4172/2155-9899.1000327

Research Article Open Access

In vivo Studies on Antiarthritic Activity of Cissus quadrangularis against

Adjuvant Induced Arthritis
Arti Bhujade, Suhas Talmale and Patil MB*
University Department of Biochemistry, L.I.T. Premises, R.T.M. Nagpur University, Nagpur, India
*Corresponding author: Patil MB, PhD, University Department of Biochemistry, L.I.T. Premises, R.T.M. Nagpur University, Nagpur-440033, India, Tel.:

+91-7506035459/+ 9860018333; E-mail: mbpatil@hotmail.com

Received date: March 05, 2015, Accepted date: May 05, 2015, Published date: May 12, 2015
Copyright: © 2015 Bhujade A et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

Objective: Arthritis is a chronic crippling, skeletal and muscular disorder having quite similar symptoms as that of
rheumatoid arthritis (RA) for which currently there is no medicine available for permanent cure. Even modern drugs
used for the amelioration of the symptoms, offer only temporary relief but produce severe side effects. In the
indigenous system of medicine, Cissus quadrangularis (CQ) has been reported to be useful in the treatment of
arthritis. However, no systematic study had been reported regarding its anti-arthritic activity. Hence, this work has
been aimed at the scientific validation of its ethno-pharmacological claim about anti-arthritic efficacy.

Methods: In the present study, anti-arthritic activity of AFCQ (Active Fraction of Cissus quadrangularis) obtained
from acetone extract of Cissus quadrangularis has been reported by employing CFA (Complete Freund's Adjuvant)
induced arthritis model in Wistar rats as an in vivo experimental model. Rat paw edema was induced by
carrageenan and altered hematological and biochemical parameters were determined.

Results: AFCQ at the dose of 100 mg/kg body weight was found to be more efficient in inhibiting the rat paw
edema as comparable to standard drugs celecoxib and methotrexate. The results had indicated that AFCQ
possesses a significant anti-arthritic activity against CFA induced arthritis. Results were analyzed through
histopathology and radiography.

Conclusion: The results of above experiments revealed that AFCQ was more effective as anti-arthritic drug as
compared to celecoxib and methotrexate, in CFA induced arthritic rats.

Keywords: Cissus quadrangularis; Antiarthritic activity; CFA;
DMARDs; NSAIDs
Introduction
Arthritis is one of the most common medical problems in the
world. Many people throughout the world are affected with arthritis
and other rheumatic conditions. At present there exists only treatment
for arthritis, but no permanent cure. Osteoarthritis is a degenerative
form of arthritis which occurs when the cartilaginous lining that
cushions the ends of bones in joints get deteriorated, leaving bones to
rub against each other. RA on the other hand, is not associated with
wear and tear. It is an autoimmune disease in which an autoimmune
response gets stimulated against the joints [1,2]. The inflammatory
mediators digest the cartilages, bones, tendons and ligaments. These
mediators are inhibited by many or various families of anti-
inflammatory drugs, which include the non-steroidal anti-
inflammatory drugs (NSAIDs). This class of drug, of which aspirin is a
member, inhibits inflammation by interfering with an inflammatory
mediator enzyme known as cyclooxygenase (COX). Cyclooxygenases
are of two types; cyclooxygenase-1 (COX-1) and cyclooxygenase-2
(COX-2). Out of these, COX-2 has been considered to be more
dangerous than COX-1. Clinical trials demonstrate that although
NSAIDs are effective in decreasing the symptoms associated with
arthritis but they increase the gastrointestinal ulcerations [3]. Another
very common COX-2 inhibitor is Vioxx (Rofecoxib) which specifically
inhibits COX-2, but Vioxx was verified to increase the risk of heart
attack, stroke and serious cardiovascular problems [4]. The current
treatment of RA is intended to minimize the associated pain and
inflammation using non-steroidal anti-inflammatory drugs (NSAIDs)
as well as to decelerate the progress of the disease by using disease-
modifying anti rheumatic drugs (DMARDs). DMARDs suppress the
immunological reactions involved in the progression of RA. Drugs
that manifest the effects of both DMARDs and NSAIDs will be more
effective in the treatment of RA, but there is a scarcity of such drugs
acting through multiple mechanisms to bring about the cure of RA.
Hence, the treatment of RA involves the combined use of NSAIDs and
DMARDs [5]. RA is an autoimmune disease, which is chronic,
affecting the people of all ethnic groups worldwide. Even though
various categories like immunosuppressants, NSAIDs, steroidal anti-
inflammatory drugs are being used till now, but due to severe adverse
effects of these drugs, the development of new anti-arthritic drugs are
aimed towards the discovery of safe, potent drugs with minimal side
effects. Hence, complementary and alternative medicines are
commonly preferred in this context [6]. In Indian system of traditional
medicine, employment of medicinal plants, as a re-emerging health
aid, has been fuelled by the rising costs of prescription drugs in the
maintenance of personal health and well-being and the bio
prospecting of new plant derived drugs has been taken up seriously
[2]. This fact improves the thought that believes the emergence of
serious infectious diseases that might best be met with new anti-
infective agents from traditional plant remedies [7]. This has greatly

J Clin Cell Immunol Volume 6 • Issue 3 • 1000327

ISSN:2155-9899 JCCI, an open access journal
Citation: Bhujade A, Talmale S, Patil MB (2015) In vivo Studies on Antiarthritic Activity of Cissus quadrangularis against Adjuvant Induced
Arthritis. J Clin Cell Immunol 6: 327. doi:10.4172/2155-9899.1000327
increased the use of traditional medicinal plants which has made
medicinal plants as an integral part of the health delivery system in
developing countries.
In Ayurveda, plant extracts (rasayanas) are considered to be rich in
phytochemicals. Cissus quadrangularis (CQ) is commonly known as
‘Bone Setter’ (Hadjod). The properties of CQ have already been
reported in literature. The stem extract contains high percentage of
calcium (4% by weight) and phosphorus [8], where both these
elements are essential for bone fracture healing. As reported earlier,
CQ has been recommended for treating painful and inflammatory
conditions like arthritis. Its use for wound healing, as antimicrobial
and in the treatment of skin diseases is very common [9]. Analysis of
CQ stem has shown that it has putative ability to promote mineral
growth in bones [8]. The plant has been reported to possess various
biological activities like antiosteoporotic, analgesic, anticancer and
antibacterial [10-13]. The use of CQ in arthritic disorders has not been
systematically investigated. Therefore, the present study was designed
to determine the anti-arthritic efficacy of CQ.
Methods
Plant material
Plant material was collected from Vidarbh region of Maharashtra
(India) in March 2008. The plant was authenticated at Department of
Botany, Rashtrasant Tukdoji Maharaj Nagpur University, Nagpur
(India) and voucher specimens were deposited in the herbarium. The
plant was identified to be Cissus quadrangularis Linn. (CQ) and the
relevant voucher specimen number was 9433.
Isolation of AFCQ
The dried powder of the stem of Cissus quadrangularis was
extracted in different solvents using soxhlet apparatus in the
increasing order of polarity. Acetone fraction was found to be more
effective in inhibiting inflammatory mediators like cycloxygenases and
lipoxygenases (COX and LOX). Hence, it was subjected to further
purification by adsorption column chromatography to isolate an active
fraction, which could prevent inflammation. This active fraction was
designated as AFCQ (Active Fraction of Cissus quadrangularis) [13].
Reagents and chemicals
Complete Freund Adjuant (CFA) and Carrageenan were purchased
from Sigma Aldrich chemical company [St. Louis, USA]. Celecoxib,
Methotrexate, other chemicals and solvents of analytical grade were
purchased from authorized and standard companies.
Animals and experimental design
Animals: Wistar albino rats weighing between 180 ± 20 g and age
2-3 months were selected for in vivo studies. During experiment
animals were acclimatized to the standard laboratory conditions
(Temp. 25 ± 2°C) and maintained at 12 h light, 12 h dark cycle. The
animals were fed with standard diet (Hindustan Lever) and water ad
libitum. The animals were maintained as per the norms of Animal
Ethics Committee. Experimental set ups were cleared by the
Institutional Animal Ethics Committee.
Acute toxicity study: A group of six rats was orally administered
with AFCQ in graded doses of 0.050, 0.075, 0.10, 0.20, 0.25, 0.5, 1.0
and 1.5 g/kg body weight (b. w.). Rats were continuously observed for
J Clin Cell Immunol
ISSN:2155-9899 JCCI, an open access journal
Page 2 of 8
mortality and behavioral responses initially for 48 h and once daily
thereafter till 14 days. LD50 (lethal dose 50), as obtained from this
experiment was 1000 mg/kg b. w. Dose selection was performed by
taking 1/10th of the LD50. Therefore, the dose of the AFCQ selected
for present experiments was 100 mg/kg b. w. Celecoxib (NSAIDs) 50
mg/kg b. w. [14] and Methotrexate (DMARDs) 0.3 mg/kg b. w. [15]
were used as standard drugs for comparing the efficacy of AFCQ in
experimental animals.
Carrageenan induced edema in rats: The rat paw edema was
induced by carrageenan as reported elsewhere [16]. The experimental
animals were divided into five group’s containing six rats in each
group. Group I received normal saline and served as control. Group II
served as carrageenan control (positive control), Group III received
the doses of standard drug Celecoxib 50 mg/kg b. w. Group IV
received the doses of standard drug Methotrexate 0.3 mg/kg b. w.
Group V received the doses of AFCQ at 100 mg/kg b. w. Edema was
induced by injecting 0.1 ml of carrageenan (1% w/v; Sigma, USA) in
normal saline into the sub plantar region of the right hind paw of all
the experimental animals [16]. After 1 h of carrageenan administration
the paw volume was measured by plethismograph method with the
help of Mercury Replacement Plethysmometer starting from zero h
followed by 1 h and 6 h interval after administration of carrageenan
[17].
Induction of arthritis: Arthritis was induced in rats by the
intraperitoneal injection of 0.1 ml of Complete Freund’s Adjuvant
(CFA) in the left hind paw [18]. The CFA contains heat killed
Mycobacterium tuberculosis in sterile paraffin oil (10 mg/ml). The
arthritis develops within 14 days from the day of administration of
CFA i.e. from day of induction of arthritis [19]. Oral feeding of rats
with that of AFCQ and standard drugs (Celecoxib and Methotrexate)
was therefore started from the 14th day to 28th day of initiation of
arthritic condition. The paw volume of all the animal groups was
measured by using a plethysmometer at 0 day and then at 4, 7, 14, 21,
25 and 28 days after the injection of CFA [17]. On 29th day all the
experimental animals were sacrificed and different tissues were
collected for heamatological, biochemical and histopathological
analysis. Radiographical analysis (X-ray) of all the experimental
animals was also undertaken before they were subjected to sacrifice
[20].
Experimental set up for antiarthritic activity of AFCQ
Wistar albino rats of either sex weighing 180 ± 20 g and age 2-3
months were used for this study. Animals were divided into five
groups with six animals in each group. The standard drug Celecoxib,
Methotrexate and AFCQ were dissolved in distilled water and
administered immediately. Group I served as control (without
treatment i.e. negative control), Group II served as arthritic control
(positive control), Group III were treated with Celecoxib (NSAID),
Group IV treated with standard anti-rheumatic drug Methotrexate
(DMARD) and Group V was treated with AFCQ (test compound).
On 29th day, at the end of experiment, all animals were sacrificed by
cervical decapitation and blood was collected in plain and EDTA
containing tubes, respectively, for plasma/serum separation. The
plasma/serum samples were subjected to haematological and
biochemical examination by using Autoanalyser [Merck Microlab 200
model]. For histopathology, the right legs were removed, washed with
saline and stored in 10% formalin. The right ankle joint sections were
obtained, stained with eosin-haematoxylin stain [21] and examined
under microscope.
Volume 6 • Issue 3 • 1000327
Citation: Bhujade A, Talmale S, Patil MB (2015) In vivo Studies on Antiarthritic Activity of Cissus quadrangularis against Adjuvant Induced
Arthritis. J Clin Cell Immunol 6: 327. doi:10.4172/2155-9899.1000327

Page 3 of 8

The hind legs of all the experimental rats were radio photographed
(X-ray) and also examined for the soft tissue swelling and bony
erosions, if any.
Statistical analysis
All experimental results were expressed as mean ± SD. The
statistical significance of the difference between control, standard and
CQ extract treated groups was determined by two way ANOVA
followed by Bonferroni post-hoc test for multiple comparisons to
determine the significant levels. The results were considered
significant at *P value<0.05 and ×p<0.01.
Effect of standard drugs and AFCQ on CFA induced arthritis
in rats
There is a significant increase in rat paw volume in CFA injected
arthritic positive control rats (group II) when compared to the normal
control rats (group I). Oral AFCQ treatment at the dose of 100 mg/kg
b. w. (group V) and that of standard drugs Celecoxib at 50 mg/kg b. w.
(group III) and Methotrexate at 0.3 mg/kg b. w. (group IV) showed
significant reduction in rat paw edema volume when compared with
the arthritic group II as shown in Figure 2.

Results

Effect of standard drugs and AFCQ on carrageenan induced

rat paw edema
There is a significant increase in rat paw volume in carrageenan
injected positive control animals as compared to the paw volumes of
normal control rats. Oral feeding of experimental animals with AFCQ
at the dose of 100 mg/kg body weight and that of standard drugs
(Celecoxib 50 mg/kg b. w. and Methotrexate 0.3 mg/kg b. w.) showed
significant reduction in paw edema volume when compared with the
arthritic group, as shown in Figure 1.

Figure 2: Effect of standard drugs and AFCQ on CFA induced

arthritis in rats. Data are mean ± S.D. of three independent
experiments. P value of less than 0.05 (*P<0.05) and less than 0.01
(×P<0.01) was considered as statistically significant.

Figure 1: Effect of standard drugs and AFCQ on carrageenan
induced inflammation in rats. Data are mean ± S.D. of three
independent experiments. P value of less than 0.05 (*P<0.05) and
less than 0.01 (×P<0.01) was considered as statistically significant.
Haematological investigations
Effect of standard drugs and AFCQ on haematological parameters:
Changes in haematological parameters in CFA induced arthritic rats
have been presented in Table 1. There appear to be a significant
decrease in RBC count and haemoglobin, rise in WBC count and ESR
of arthritic rats, when compared with that of control rats. Oral
treatment with standard drugs and AFCQ has significantly brought
back the altered haematological changes in CFA induced arthritic rats
when compared with the normal control group.

Parameters Group I Group II Group III Group IV Group V

Hb(g/dl) 13.43 ± 0.291 9.387 ± 0.193 9.97 ± 0.102 10.82 ± 0.133 13.10 ± 0.158

RBC count (× 106/mm3) 5.81 ± 0.059 4.45 ± 0.1794 5.30 ± 0.153 5.42 ± 0.344 5.58 ± 0.09

WBC count (× 103/mm3) 7.46 ± 0.132 15.36 ± 0.125 10.95 ± 0.218 12.81 ± 0.075 7.817 ± 0.131

ESR (mm/h) 1.267 ± 0.176 18.50 ± 0.289 3.00 ± 0.116 2.47 ± 0.240 1.307 ± 0.145

Table 1: Effect of standard drugs and AFCQ on blood parameters.

Biochemical investigations glucose content, total and individual protein levels have been
presented in Table 2. In CFA injected arthritic rats, there was a
Effect of standard drugs and AFCQ on serum proteins (total significant decrease in total protein and albumin levels and in A/G
protein, albumin, globulin and A/G ratio) and glucose: The changes in ratio, but significant increase in globulin and glucose levels on

J Clin Cell Immunol Volume 6 • Issue 3 • 1000327

ISSN:2155-9899 JCCI, an open access journal
Citation: Bhujade A, Talmale S, Patil MB (2015) In vivo Studies on Antiarthritic Activity of Cissus quadrangularis against Adjuvant Induced
Arthritis. J Clin Cell Immunol 6: 327. doi:10.4172/2155-9899.1000327

Page 4 of 8

comparison with the control group. On oral treatment with AFCQ,
Celecoxib and Methotrexate all these changes were restored back or
compensated to the normal level to certain extent.
Effect of standard drugs and AFCQ on acute phase proteins: The
level of C-reactive protein rises dramatically during inflammatory
processes. In arthritis-induced rats, this acute phase marker was
significantly increased when compared with the control group (Table
2). Treatment with AFCQ, Celecoxib and Methotrexate has
significantly decreased the levels of acute phase proteins in treated
arthritic rats.

Parameters Group I Group II Group III Group IV Group V

Glucose(mg/dl) 81.28 ± 0.43 105.3 ± 0.47 88.2 ± 0.58 98.3 ± 0.55 90.3 ± 0.49

Total Protein(mg/dl) 8.01 ± 0.047 6.21 ± 0.055 7.04 ± 0.079 7.29 ± 0.018 7.87 ± 0.03

Albumin(mg/dl) 3.38 ± 0.034 2.11 ± 0.07 2.24 ± 0.058 3.210 ± 0.06 2.95 ± 0.027

Globulin(mg/dl) 3.21 ± 0.06 4.58 ± 0.027 4.12 ± 0.066 4.2 ± 0.016 3.51 ± 0.066

A/G ratio 0.76 ± 0.02 0.547 ± 0.02 0.56 ± 0.011 0.74 ± 0.012 0.59 ± 0.012

CRP(mg/dl) 1.23 ± 0.03 4.897 ± 0.02 2.55 ± 0.032 3.68 ± 0.031 1.59 ± 0.021

Table 2: Effect of standard drugs and AFCQ on glucose, minerals (copper, iron), acute phase protein and serum proteins of control and CFA
induced arthritis rats.

Effect of standard drugs and AFCQ on copper and iron levels in oxygen from the lungs to the tissues. Deficiency of iron leads to
blood serum: Ceruloplasmin and fibrinogen are synthesized in the anemia whereas large amount of free iron in the circulation causes
liver. Ceruloplasmin is a major copper carrying protein in the blood damage to the liver; heart and other metabolically active organs [23].
and contains 8 atoms of copper in its structure and it plays a major
In CFA injected arthritic rats, there was significant increase in
role in iron metabolism also [22]. Free copper ions are powerful
copper and iron levels as compared to the control group. It was
catalysts of free radical damage. By binding with copper,
observed that treatment of arthritic rats with AFCQ, Methotrexate and
ceruloplasmin prevents free copper ions from catalyzing oxidative
Celecoxib has brought back the changes to normal or appear to be
damage to liver. Increased copper ion concentration indicates the
compensated to some extent as presented in Table 3.
extent of an inflammatory condition of liver. Iron is essential for
formation of haemoglobin in red blood cells in order to transport

Parameters Group I Group II Group III Group IV Group V

Copper (ppm) 1.88 ± 0.012 2.16 ± 0.017 1.83 ± 0.015 2.20 ± 0.019 1.79 ± 0.011

Iron (ppm) 6.43 ± 0.058 11.63 ± 0.035 5.28 ± 0.038 10.05 ± 0.069 5.09 ± 0.0203

Table 3: Effect of standard drugs and AFCQ on minerals copper and iron of control and CFA induced arthritis rats.

Parameters Group I Group II Group III Group IV Group V

SGOT (U/L) 118.3 ± 0.35 138.3 ± 0.391 102.2 ± 0.58 106.1 ± 0.55 114.3 ± 0.54

SGPT (U/L) 46.3 ± 0.51 46.15 ± 0.53 50.89 ± 0.34 41.44 ± 0.32 45.86 ± 0.21

Alk. Phostphatase (U/L) 87.07 ± 0.69 478.3 ± 0.58 448.2 ± 0.44 221.0 ± 0.32 223.0 ± 0.61

Total Bilirubin (mg/dl) 0.53 ± 0.01 0.47 ± 0.016 0.53 ± 0.013 0.63 ± 0.016 0.63 ± 0.015

Table 4: Effect of standard drugs and AFCQ on liver function test (LFT) in control and CFA induced arthritis rats.

Parameters Group I Group II Group III Group IV Group V

Urea (mg/dl) 38.76 ± 0.32 54.15 ± 0.52 44.23 ± 0.42 42.2 ± 0.45 40.1 ± 0.55

Creatinine (mg/dl) 0.61 ± 0.013 0.697 ± 0.02 0.59 ± 0.019 0.67 ± 0.01 0.60 ± 0.015

Table 5: Effect of standard drugs and AFCQ on kidney function test (KFT) in control and CFA induced arthritis.

J Clin Cell Immunol Volume 6 • Issue 3 • 1000327

ISSN:2155-9899 JCCI, an open access journal
Citation: Bhujade A, Talmale S, Patil MB (2015) In vivo Studies on Antiarthritic Activity of Cissus quadrangularis against Adjuvant Induced
Arthritis. J Clin Cell Immunol 6: 327. doi:10.4172/2155-9899.1000327
Effect of standard drugs and AFCQ on Liver Function Test and
Kidney Function Test: Changes in biochemical parameters in CFA
induced arthritic rats are presented in Table 4. Serum glutamic
oxaloacetic transaminase (SGOT) and serum glutamic pyruvate
transaminase (SGPT) are not liver function tests, but are biomarkers
of liver injury [24]. Alkaline phosphatase (ALP), an enzyme present in
the cell linings of the biliary ducts of liver gets elevated in case of
inflammation, liver and bone disorders [25]. Bilirubin, a potent
antioxidant, is the yellow breakdown product of normal heam
catabolism. Here, it seems that there is not much change occurring in
SGOT and SGPT levels in control and arthritic groups, but ALP level
Parameters Group I Group II
Cholestrol (mg/dl) 89.38 ± 0.59 38.85 ± 0.28
Triglyceride (mg/dl) 57.05 ± 0.58 52.57 ± 0.26
Uric acid (mg/dl) 3.40 ± 0.06 5.47 ± 0.19
RA Factor (IU/L) 9.193 ± 0.09 15.0 ± 0.06
Table 6: Effect of standard drugs and AFCQ on cholesterol, triglycerides, uric acid and RA factor.
Figure 3: Histopathology of gastric mucosa of A-DEMARD, B-
AFCQ, C-NSAID, D-arthritic positive, E-Normal. The gastric
mucosal lining is seen to be disturbed due to synthetic drugs like
DEMARD, NSAID, but in AFCQ treated group, it was found to be
safe.
Effect of standard drugs and AFCQ on cholesterol, triglyceride, uric
acid and RA factor: Changes in cholesterol, triglyceride, uric acid and
RA factor levels are presented in Table 6. Cholesterol and triglycerides
are the biomarkers of cardiac health which get elevated in cardiac
J Clin Cell Immunol
ISSN:2155-9899 JCCI, an open access journal
Page 5 of 8
appears to be dramatically increased in arthritic groups. These
alterations were significantly reduced by treatment with AFCQ,
celecoxib and methotrexate, as shown in Table 4. Total bilirubin level
was decreased in arthritic control group as compared to normal and in
treated groups this level was restored back to normal level to some
extent, as presented in Table 4. Urea and serum creatinine are the
biomarkers of kidney injury [26]. Significant increase in the values of
blood urea and serum creatinine in CFA induced arthritic rats was
found when compared with the normal control group. These
alterations were significantly reduced by treatment with AFCQ,
Celecoxib and methotrexate (Table 5).
Group III Group IV Group V
56.47 ± 0.47 53.3 ± 0.86 48.75 ± 0.20
63.85 ± 0.22 60.28 ± 0.64 53.32 ± 0.185
2.21 ± 0.22 2.24 ± 0.13 3.13 ± 0.091
10.03 ± 0.09 9.83 ± 0.12 9.03 ± 0.22
diseases [27]. Uric acid and RA factor are found elevated in arthritis
[28]. In CFA induced arthritic rats, there is a significant decrease in
cholesterol and triglyceride level, but significant rise in uric acid and
RA factor level was noted when compared with the control group. On
treatment with AFCQ, Celecoxib and methotrexate, the altered
parameters were brought back to normal or compensated to some
extent (Table 6).
Histopathology studies
Histopathology study in CFA induced arthritic rats, standards and
controls shows the potent effect of AFCQ than standard drugs
(Celecoxib and methotrexate). It is clear from the histopathology
studies that AFCQ reduced the rate of destruction of bone and
cartilage. AFCQ and the standard drugs were found to increase the
bone life with prominent recovery after induction with CFA induced
arthritis. AFCQ treatment shows prominent recovery in gastric
mucosa when compared with the gastric mucosa of CFA induced
arthritic rats as shown in Figure 3 and Figure 4. Generally, in the
arthritic medication the most commonly observed side effects of
treatment are gastric problems. In the present investigation AFCQ
appears to show the remarkable improvement in gastric mucosal
linings, than the standard drugs.
Radiographic (x ray) investigations
The radiographic studies of CFA induced arthritic rats,
experimental, standard and normal control groups are shown in
Figure 5. The lateral X-ray radiograph of the arthritic control group
rats shows bony dissolution with thinning of the distal tibial extremity.
The tibio tarsal joint shows acute arthritis with fragmentation of tarsal
as well as proximal phalangeal joint. The surrounding soft tissue
swellings were not grossly prominent. In Celecoxib, Methotrexate and
AFCQ treated group lateral X-ray shows the periferal periosteitis
which was not as prominent as that in arthritic control, the periferal
inflammation or the edema was found to be insignificant (Fairly
cured). Analysis of results has revealed that the AFCQ drastically
reduced the inflammation as compared with arthritic control. There is
Volume 6 • Issue 3 • 1000327
Citation: Bhujade A, Talmale S, Patil MB (2015) In vivo Studies on Antiarthritic Activity of Cissus quadrangularis against Adjuvant Induced
Arthritis. J Clin Cell Immunol 6: 327. doi:10.4172/2155-9899.1000327
no visible gap formation in the tibiotarsal joint in AFCQ treated rats.
Standard drugs such as Celecoxib and Methotrexate also reduced
inflammation but comparatively to a lesser extent than AFCQ. For
coming to a fair conclusion, a detailed and long term protocol is
required with respect to AFCQ treatment of arthritic rats. But from
the present experiments, it can be inferred that although for a short
treatment, AFCQ has shown the encouraging results, as shown in
Figure 5.
Figure 4: Histopathology of ankle joint of A-DEMARD, B-AFCQ,
C-NSAID, D-arthritic positive, E-Normal. The condition of AFCQ
treated group is found to be much more improved than that in
DEMARD and NSAID treated groups where still osteoporotic
condition are seen.
Discussion
Perhaps the most important inflammatory conditions to affect
humanity are the varieties of arthritis and rheumatism diseases.
Throughout the world herbal medicines appear to be widely accepted
for treatment of these and many other diseases. Arthritis is a general
term used for many diseases that produce either inflammation of
connective tissues, particularly in joints or non-inflammatory
degeneration of these tissues.
RA is an autoimmune inflammatory disease that causes pain,
swelling, stiffness and loss of function in the joints. RA affects
approximately 2.1 million people worldwide (and nearly about 1
percent of the United States adult population) [29]. Current therapies
have various degrees of efficacy, but toxicity frequently limits their
J Clin Cell Immunol
ISSN:2155-9899 JCCI, an open access journal
Page 6 of 8
long term use. Animal models of arthritis are normally used to help
provide basic insights into autoimmune diseases and to test novel
experimental approaches for the treatment of the diseases with
potential anti-arthritic drugs. Some animal models of RA with a
proven track record of predictability for efficacy in humans include
CFA and collagen-induced arthritis in rats. These experimental
models of RA induce inflammation, bone resorption, cartilage damage
and pannus that resemble the human disease [30]. Clinical or live
phase parameters of body weight and paw swelling are used along with
the histopathology of the joint sections to predict the efficacy of
potential anti-arthritic drugs.
Figure 5: Radiography of ankle joint of A-DEMARD, B-AFCQ, C-
NSAID, D-arthritic positive, E-Normal. In AFCQ treated group,
the ankle joint had almost return to normal, but still some
inflammation is seen in DEMARD and NSAID treated groups.
CFA induced arthritis is the most extensively used chronic test
model in which the clinical and pathological changes are similar with
those seen in human RA [31,32]. Chronic inflammation in the CFA
model is manifested as a progressive increase in the volume of the
injected paw. It is notable that the inhibitory effect of AFCQ (100
mg/kg) on the volume of the injected paw was quite better than that of
Celecoxib (50 mg/kg) and Methotrexate (0.3 mg/kg). CFA-induced
polyarthritis is associated with an immune-mediated inflammatory
reaction and the rats are unique in developing polyarthritis after CFA
treatment [33]. The initial reaction of edema and soft tissue thickening
at the depot site in this model is caused by the irritant effect of the
adjuvant, whereas the late-phase arthritis and flare in the injected foot
are presumed to be immunological events [34]. The suppression of
such paw edema by a drug shows its immunosuppressive activity [35].
In this respect also, AFCQ was found to be more effective than that of
Methotrexate and Celecoxib. This reveals potent suppression of cell
mediated immunity in arthritic rats by AFCQ. The experimental rats
Volume 6 • Issue 3 • 1000327
Citation: Bhujade A, Talmale S, Patil MB (2015) In vivo Studies on Antiarthritic Activity of Cissus quadrangularis against Adjuvant Induced
Arthritis. J Clin Cell Immunol 6: 327. doi:10.4172/2155-9899.1000327
show that the treatment with AFCQ, Methotrexate and Celecoxib
inhibited the arthritis associated joint changes. In addition to this,
characteristic haematological alterations such as the decreased Hb
level and increased erythrocyte sedimentation rate were also
significantly restored by AFCQ, Methotrexate and Celecoxib
treatments. It is proposed that the reduction in the Hb level during
arthritis probably results either due to reduced erythropoietin level or
a decreased response of the bone marrow erythropoietin and
premature destruction of red blood cells. Similarly, an increase in the
ESR is attributed to the accelerated formation of endogenous proteins
such as fibrinogen and globulin and such a rise in ESR indicates an
active but obscure disease process [36]. Thus, the reduction in ESR
and increase in Hb content brought about by AFCQ treatment further
supports antiarthritic effect of AFCQ.
Anaemia is commonly noted in patients with chronic arthritis [37].
The most common explanations for this condition are gastrointestinal
blood loss due to arthritis medications and bone marrow changes in
patients with inflammatory arthritis, which prevent the release of iron
for incorporation into the red blood cells [38]. In the present study,
arthritic rats (group II) showed a reduced RBC count, reduced Hb
levels, and an increased erythrocyte sedimentation rate (ESR). All
these symptoms indicate an anaemic condition, which is observed
severely in the arthritic group (group II). The AFCQ treated group
(group V) and standard drugs treated group III and IV (Celecoxib and
Methotrexate) showed a significant recovery from the induced
anaemia as indicated by the RBC count and Hb level. WBC count is an
indicator of infectious and inflammatory diseases [39], the WBC count
was increased in arthritic rats, which was significantly suppressed by
the AFCQ treated group (group V) and standard drugs treated group
III and IV (Celecoxib and Methotrexate), as indicated by the
significant decrease in the WBC count.
C-reactive protein (CRP) is a member of the class of acute phase
reactants. It rises dramatically during inflammatory processes [40].
The level of C-reactive protein was found to be significantly reduced in
the AFCQ treated group (group V) and standard drugs treated group
III and IV (Celecoxib and Methotrexate). Ceruloplasmin, a protein
synthesized in the liver, contains 8 atoms of copper in its structure.
Free copper ions are powerful catalysts of free radical damage. By
binding with copper, ceruloplasmin prevents free copper ions from
catalyzing oxidative damage to liver. The arthritic rats and
Methotrexate treated rats exhibited significantly elevated copper level,
which was found significantly decreased in AFCQ and Celecoxib
treated rats.
The observed histopathological changes of proximal tibiotarsal
joints of all groups are shown in Figure 4. Group I showed the
histopathology of normal ankle joint. In Group II the arthritic rat joint
showed prominent abnormalities from the normal joint like edema
formation, degeneration with partial erosion of the cartilage,
destruction of bone marrow and extensive infiltration of inflammatory
exudates in the articular surface. The standard drug treated rat joints
showed normal bone marrow with less cellular infiltrates. AFCQ
treatment for 14 days showed less inflammatory signs like absence of
edema formation along with normal bone marrow status and less
cellular infiltrates on the articular surface with less cartilage
destruction. The overall prevention of the inflammatory signs of the
rat ankle joints was significant in 14 days AFCQ treated group as
compared with 14 days drug treated group. Degeneration of the ankle
joint was not observed in any of the drug treated groups when
compared with the arthritic control.
J Clin Cell Immunol
ISSN:2155-9899 JCCI, an open access journal
Page 7 of 8
Radiographic changes in RA conditions are useful diagnostic
measures which indicate the severity of the disease. Soft tissue swelling
is the earlier radiographic sign, whereas prominent radiographic
changes like bony erosions and narrowing of joint spaces can be
observed only in arthritis [41]. The radiographic features of the rat
joints in CFA induced arthritic model are shown in Figure 5. In CFA
induced arthritic rat (group II), soft tissue swelling along with
narrowing of the joint spaces were observed which implies the bony
destruction in arthritic condition. In standard drug Celecoxib and
Methotrexate treated groups the bony destruction was found to be
prevented and also there was a negligible swelling of the joints. Similar
to the histopathological studies, AFCQ treatment for 14 days has
shown significant prevention against bony destruction by showing less
soft tissue swelling and preventing the dissolution of bone when
compared with arthritic groups.
Treatment of a disease like arthritis is expected to address the
alterations in the multiple mediators and/or their effects to derive
clinical benefits due to medication. As evident from the results of
experiments presented in this paper, Cissus quadrangularis possesses
anti-inflammatory and anti arthritic activity and the effects of AFCQ
may be due to active constituents like phenolics present in it.
Conclusion
In conclusion, AFCQ appears to exert beneficial effects on multiple
pathological manifestations of carrageenan induced inflammation and
CFA induced arthritis in rats. But, further study is needed to prove
clinical effects of this plant. However, the present study validates the
ethnomedicinal claim of CQ in the treatment of arthritic conditions as
reported in literature.
Conflict of Interest
The authors declare no conflict of interest.
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Arthritis. J Clin Cell Immunol 6: 327. doi:10.4172/2155-9899.1000327
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