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<em>In vivo</em> Studies on Antiarthritic Activity of <em>Cissus quadrangularis</em> against Adjuvant Induced Arthritis | OMICS International
ISSN: 2155-9899
Journal of Clinical & Cellular Immunology

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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: [email protected]
Received March 05, 2015; Accepted May 05, 2015; Published May 12, 2015
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
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.
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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.

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 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 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.

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.
 
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.
 
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.
 
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.
 
Biochemical investigations

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

Effect of standard drugs and AFCQ on copper and iron levels in blood serum: Ceruloplasmin and fibrinogen are synthesized in the liver. Ceruloplasmin is a major copper carrying protein in the blood and contains 8 atoms of copper in its structure and it plays a major role in iron metabolism also [22]. 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. Increased copper ion concentration indicates the extent of an inflammatory condition of liver. Iron is essential for formation of haemoglobin in red blood cells in order to transport oxygen from the lungs to the tissues. Deficiency of iron leads to anemia whereas large amount of free iron in the circulation causes damage to the liver; heart and other metabolically active organs [23].

In CFA injected arthritic rats, there was significant increase in copper and iron levels as compared to the control group. It was observed that treatment of arthritic rats with AFCQ, Methotrexate and Celecoxib has brought back the changes to normal or appear to be compensated to some extent as presented in Table 3.

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 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).

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 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 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.
 
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 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.

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 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.

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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