|Year : 2014 | Volume
| Issue : 1 | Page : 42-48
Anti-arthritic activity of ethanolic extract from the leaves of Commiphora caudata (Linn.) in complete Freund's adjuvant-induced arthritic rats
Girija Pashikanti1, Umasankar Kulundaivelu1, Venkateshwar Rao Jupalli2, Sharvana Bhava Bandaru Sheshagiri1, Venkateshwarlu Eggadi1
1 Department of Pharmacology, Vaagdevi College of Pharmacy, Hanamkonda, Warangal, Andhra Pradesh, India
2 Department of Chemistry, Talla Padmavathi College of Pharmcy, Ursu, Warangal, Andhra Pradesh, India
|Date of Web Publication||1-Jul-2014|
Departments of Pharmacology, Vaagdevi College of Pharmacy, Hanamkonda, Warangal, Andhra Pradesh
Source of Support: None, Conflict of Interest: None
Objective: Rheumatoid arthritis is a chronic multi-system disease of unknown cause. It affects people in their prime of life, predominantly between the ages of 20 and 50 years with an unpredictable course. The present study was to evaluate the anti-arthritic activity of ethanolic extract of Commiphora caudata leaves in complete Freund's adjuvant (CFA)-induced arthritic rats. Materials and Methods: Arthritis was induced by sub-cutaneous injection of 0.1 ml of CFA in rats. Arthritic arts were divided into different groups and ethanolic extract of C. caudata leaves (EECCL) was administered at doses of 200 and 400 mg/kg, p.o for 28 days. The control group received Tween 20 for 28 days. Dexamethasone (1 mg/kg, i.p) was used as a standard drug. Paw volumes were recorded on 7, 14, 21 and 28 day using a plethysmometer. Blood samples were collected at the end of the experiment from all the groups to analyze the serological rheumatic factors: C-reactive protein (CRP), serum rheumatic factor (SRF) and hematological parameters. EECCL was also tested for protein denaturation and membrane stabilization activities. Statistical Analysis: All data were expressed as mean ± SEM. One-way ANOVA followed by Dunnett's test was used to compare the mean values of test groups and control. Results: The EECCL (200 and 400 mg/kg, p.o) showed significant (P < 0.01, P < 0.001) reduction in paw volume, change in body weight in CFA rats at 28 day when compared with arthritic control rats. In addition, EECCL with dose 400 mg/kg, p.o significantly (P < 0.05, P < 0.001) increase the red blood cells, hemoglobin and above the 400 mg/kg dose significantly (P < 0.001) decrease the white blood cells, erythrocyte sedimentation rate, SRF and CRP when compared with arthritic control rats. Conclusion : The results obtained from the present study revealed the potential anti-arthritic activity of ethanolic extract from the leaves of C. caudata.
Keywords: Anti-arthritic activity, Commiphora caudata leaves, complete Freund′s adjuvant, dexamethasone
|How to cite this article:|
Pashikanti G, Kulundaivelu U, Jupalli VR, Sheshagiri SB, Eggadi V. Anti-arthritic activity of ethanolic extract from the leaves of Commiphora caudata (Linn.) in complete Freund's adjuvant-induced arthritic rats. Niger J Exp Clin Biosci 2014;2:42-8
|How to cite this URL:|
Pashikanti G, Kulundaivelu U, Jupalli VR, Sheshagiri SB, Eggadi V. Anti-arthritic activity of ethanolic extract from the leaves of Commiphora caudata (Linn.) in complete Freund's adjuvant-induced arthritic rats. Niger J Exp Clin Biosci [serial online] 2014 [cited 2019 Mar 23];2:42-8. Available from: http://www.njecbonline.org/text.asp?2014/2/1/42/135729
| Introduction|| |
Rheumatoid arthritis (RA) is a systemic autoimmune disease of unknown etiology, affecting 1-1.5% of the population worldwide.  The disease is characterized by articular inflammation and by the formation of an inflammatory and invasive tissue, rheumatoid pannus that eventually leads to the destruction of joints. Anti-inflammatory drugs and analgesics including steroids are used to suppress the symptoms, while disease-modifying antirheumatic drugs (DMARDs), newer therapies such as anti-CD20 therapy (rituximab) and abatacept, anti-tumor necrosis factor (TNF)-α therapy (etanercept, infliximab and adalimumab) are often required to inhibit or halt the underlying immune process. However, all of these agents are associated with numerous side effects. In recent days, researchers are directed toward traditional system of medicine for the discovery of drugs that are long acting anti-inflammatory with minimum side effects. Although there is no ideal animal model for RA at this time, rat adjuvant arthritis shares many features of human RA, and the sensitivity of this model to anti- arthritic agents support the view that the adjuvant arthritis is the best available model of RA.  Herbal medicines are being accepted and used increasingly by general populations in both eastern and western countries because of the ethnic acceptability and compatibility having fewer side effects. 
Commiphora caudata (Burseraceae) is an ethnomedicinal plant found in India and Sri Lanka. The various parts of C. caudata, namely bark and leaves, have been used in the folk medicine for treating vata, pitta, diabetes, low back pain, sciatica, fever, arthritis and urinary retention. ,, From the above literature, we selected this plant to evaluate anti-arthritic activity.
| Materials and methods|| |
Drugs and Chemicals
Complete Freund's adjuvant (CFA) was procured from Sigma Aldrich chemicals Pvt. Ltd, Hyderabad, India, Dexamethasone was obtained from SD fine chemicals Pvt. Ltd, Mumbai, India and all other chemicals used in this study were obtained commercially and were of analytical grade.
Male Wistar rats (150-200 g) were obtained from Mahaveer Enterprises, Hyderabad, India. The rats were housed at a temperature of 25 ± 1°C and relative humidity of 45-55% under 12:12 light-dark cycle. The animals had free access to food pellets and water ad labitum. The experimental protocol was approved (IAEC NO:1047/ac/07/CPCSEA) by the Institutional Animal Ethics Committee (IAEC), Vaagdevi College of Pharmacy, Warangal, and performed in accordance with the guidelines of Committee for Control and Supervision of Experimentation on Animals (CPCSEA), Government of India on animal experimentation.
Fresh leaves of C. caudata were collected from the Kondapally hills, Krishna district, A.P, India, and authenticated by Dr. E. Narasimha Murthy, Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Central University, Hyderabad, Andhra Pradesh, India.
Preparation of Ethanolic Extract
Freshly collected plant material was dried under shade and then milled to obtain a coarse powder. The coarsely powdered material (1 kg) was extracted with petroleum ether to remove the fatty material and further, plant material was packed in Soxhlet apparatus and subjected to continuous extraction with ethanol. The liquid extract was collected and concentrated under reduced pressure until a waxy mass was obtained. The mass obtained was weighed in each case.
Acute Toxicity Studies
The acute toxicity study was carried out as per the procedure given in Organisation for Economic Co-operation and Development (OECD) Guideline No. 420.  The male Wistar albino rats (200-250 g) were used in the study. After the sighting study (allow selection of the appropriate starting concentration for the main study), C. caudata at the dose of 2 g/kg body weight was given to five animals. The animals were continuously observed for 14 days for mortality and general behavior.
Membrane Stabilization Method
The effect of EECCL on hemolysis of human red blood cells (HRBCs) induced by heat and distilled water was evaluated according to the method as described by Shinde et al. (1989).  Briefly, blood (2 ml) was collected from healthy volunteers and mixed with the equal volume of sterilized Alsever's solution containing dextrose, citrate and citric acid followed by centrifugation at 4000 rpm for 5 minutes. The packed cells were washed in normal saline at 4°C until use. The reaction mixture (4.5 ml) consisted of 2 ml hypotonic saline (0.25% w/v NaCl), 1 ml of phosphate buffer (pH 7.4), 1 ml of test solution (50,100, 250, 500, 1000 μg/ml) and 0.5 ml of 10% HRBC suspension. Positive and negative controls were simultaneously run with 1 ml of isotonic saline was used instead of test solution and without HRBC suspension, respectively. Two controls were prepared, one with 1.0 ml of isotonic saline without RBCs, (control 1 ) and the other one with 1 ml of extract solution without RBCs (control 2 ). Dexamethasone (50,100, 250, 500, 1000 μg/ml) was used as a standard. The mixtures were incubated at 37°C for 30 min and centrifuged at 3000 rpm. The supernatant was separated and hemoglobin content was measured spectrophotometrically at 560 nm.
Protein Denaturation Method
Protein denaturation ability of EECCL was evaluated by the method of Biswakanth et al., 2012.  Briefly, aqueous solution of bovine serum albumin (BSA) and test or standard drug solutions were adjusted to pH 6.3 and were incubated at 37°C for 20 min followed by heating at 57°C for 3 minutes. The resulting solution was added with 2.5 ml of phosphate buffer (pH 6.3) and the absorbance was measured at 416 nm. Positive and negative controls were simultaneously run without test or standard drug and BSA, respectively.
Induction of arthritis
Arthritis was induced in rats by the intraplantar injection of 0.1 ml of CFA in the left hind paw. The adjuvant contained heat killed Mycobacterium tuberculosis in sterile paraffin oil (10 mg/ml). The paw volume of all the animal groups was measured by a plethysmometer (UgoBasile, 7140) at 0, 7, 14, 21 and 28 days after the injection of CFA. 
The arthritic animals were divided into four groups each containing six animals, and one group of normal non-arthritic animals. Ethanolic extract of C. caudata was given at doses of 200 and 400 mg/kg, for 28 days, as a suspension in Tween 20 at a dose of 2 ml/kg to different groups of animals.
Group I: Normal animals received Tween 20 at a dose of 2 ml/kg as a suspension in distilled water (Normal control)
Group II: Arthritic animals received Tween 20 at a dose of 2 ml/kg as a suspension in distilled water (Disease control)
Group III: Arthritic animals received Dexamethasone a dose of 1 mg/kg
Group IV: Arthritic animals received EECCL at a dose of 200 mg/kg
Group V: Arthritic animals received EECCL at a dose of 400 mg/kg
The blood samples were collected by retro-orbital puncture of anaesthetized rats.  Hematological parameters were determined using cell counter (ABX Micros ESV 60 -HORIBA). Blood was centrifuged at 3500 rpm for 20 min and serum was separated for estimation of serological parameters like serum rheumatoid factor (SRF) and C-reactive protein (CRP) estimated.
Body weight, paw volume
Body weights and paw volumes of all the animals were measured on day 0, 7, 14, 21 and 28. The paw volume of all the rats was recorded by means of a plethysmometer (UgoBasile, 7140).
All the data were statistically evaluated with ANOVA and the differences among groups were determined by Dunnett's multiple comparison tests using Graph pad prism 5.0. Values were considered to be significant when P < 0.05. All the results were presented as mean ± SEM for six rats in each group.
| Results|| |
In the acute oral toxicity study, at a dose of 2 g/kg body weight of C. caudata, mortality was not observed. From the results, test drug doses of 0.2 and 0.4 g/kg body weight  were chosen for the efficacy studies.
Membrane Stabilization and Protein Denaturation
In the in vitro method of membrane stabilization, it was observed that EECCL showed good activity compared to positive control. Percentage stabilization was found to be 75.98% and 94.35% at a concentration of 1000 μg/ml of EECCL and standard Dexamethasone, respectively [Table 1]. The percentage inhibition of protein denaturation by Dexamethasone and EECCL at different doses is shown in [Table 2]. EECCL exhibited maximum inhibition of protein denaturation of 85.56% at 1000 μg/ml, which was comparable with that of the standard Dexamethasone that showed the maximum inhibition of 92.56% at the same concentration.
|Table 1: Effect of Commiphora caudata ethanolic extract on Membrane stabilization|
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|Table 2: Effect of Commiphora caudata ethanolic extract on % inhibition of protein denaturation|
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Effect of EECCL on Body Weight, Paw Volume
Body weight of all the animals in the control group increased till day 28, whereas body weight of all the animals in the arthritic control group was significantly decreased till day 28 as compared to the control group. Further, body weight of all the animals in EECCL- and Dexamethasone-treated groups increased significantly (P < 0.001) as compared to the arthritic control group [Table 3]. Intraplantar administration of CFA in rats induced severe inflammation and redness over a period of 24 hours. Oral administration of EECCL (200 and 400 mg/kg) ameliorated the alteration in paw volume significantly (P < 0.01, P < 0.001), respectively, on day 28 when compared with arthritic control [Table 4].
|Table 3: Effect of EECCL on rat body weight in CFA-induced arthritic rats|
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|Table 4: Effect of EECCL on rat paw edema in Freund's adjuvant-induced arthritic rats|
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The changes in hematological parameters in adjuvant-induced arthritic rats are shown in [Table 5]. The EECCL (400 mg/kg)-treated group reduces the WBC, ESR and increases the Hb (P < 0.001) and RBC (P < 0.05) significantly when compared with arthritic control. In addition, EECCL (200 mg/kg)-treated group reduces the WBC (P < 0.05), ESR (P < 0.01) and increases the Hb (P < 0.01) significantly and no effect on RBC when compared with arthritic control.
|Table 5: Effect of EECCL on rat hematological parameters in CFA-induced arthritic rats|
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The changes in serological parameters in adjuvant-induced arthritic rats are shown in [Table 6]. The results indicated that the treatment with EECCL (400 mg/kg) and Dexamethasone showed statistically significant (P < 0.001) reduction in levels of CRP, and SRF. The EECCL (200 mg/kg) showed significant CRP (P < 0.05) and SRF (P < 0.01) reduction when compared with arthritic control.
|Table 6: Effect of EECCL on rat serological parameters in CFA-induced arthritic rats|
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Histopathology of ankle joints in the normal control group showed normal joint structure, no cartilage destruction and no signs of inflammation or other distortion. However, arthritic animals showed focal cartilage destruction, mild to moderate hyperplasia of synovium, presence of pannus formation with destruction of joint space. Treatment with EECCL and dexamethasone group showed significant improvement in hyperplasia of synovium as compared to the disease control group [Figure 1]. The present study demonstrated that EECCL is able to suppress the swelling of the paws.
|Figure 1: Effect of EECCL on histopathological joints; (a) ankle joints in normal control group showed normal joint structure, no cartilage destruction and no signs of inflammation or other distortion, (b) arthritic animals showed focal cartilage destruction, mild to moderate hyperplasia of synovium, presence of pannus formation with the destruction of joint space, (c), (d) and (e) treatment with dexamethasone, EECCL (200 mg/kg) and EECCL (400 mg/kg) group showed significant improvement in hyperplasia of synovium as compared to the disease control group, respectively|
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Effect of EECCL on Histopathology of Arthritic Rats
GI: Vehicle-treated group,
GII: Disease control group
GIII: Dexamethasone-treated group (1 mg/kg),
GIV: EECCL (200 mg/kg)
GV: EECCL (400 mg/kg),
| Discussion|| |
RA is a chronic inflammatory disease characterized by fibroblastic proliferation, infiltration of the synovial lining by inflammatory cells that leads to expression of pro-inflammatory cytokines and a paucity of apoptosis resulting in bone and joint destruction.  Despite enormous research being carried out for allergic and immune disease, it still remains a disorder which can be ''controlled'' and not ''treated'', since no satisfactory treatment is available in allopathic system. Hence, there is a tremendous interest worldwide for the use of an alternative system of medicine. The research has been focused on formulations used in traditional medicine for the treatment of RA. 
CFA is used to initiate induction of arthritis. This model is the original model of RA, has been extensively used in pre-clinical screening of new anti-arthritis compounds and has successfully predicted activity in multiple new therapeutics. After a single injection of the adjuvant, rapid, reliable, robust, and easily measurable poly arthritis develops. The joint pathology seen in this animal model shares the cartilage degradation, bone resorption, and cellular influx seen in human RA. ,
Paw volumes and paw thickness are physical indicators of the inflammation in early as well as chronic phase of the disease. The progression of arthritis is characterized by an increase of the paw footpad and tibiotarsal joint diameters after day 14, which can be attributed to the delayed immunological flare in the disease.  The determination of paw swelling is an apparently simple, sensitive, and quick procedure for evaluating the degree of inflammation and assessing therapeutic effects of drugs. The EECCL-treated group showed significant reduction in paw volume suggesting the anti-inflammatory activity of the leaves. T-cell proliferation is an important mechanism of adjuvant diseases, specifically their differentiation into Th-1 helper cells.  Therefore, a possible mechanism for reduction in paw edema might be either a suppressive effect on Th-1 helper cells.
The increase in total WBC count in auto-immune arthritic rats was similar to the earlier reports. , This increase in WBC and platelet counts might be due to the stimulation of the immune system against the invading microorganism (CFA consists of M. tuberculosis, which might be responsible for the inflammation). This is evident by the infiltration of inflammatory mononuclear cells in the joints of adjuvant-induced arthritic rat.
Adjuvant disease is associated with an increase in the plasma levels of CRP.  CRP levels are closely linked to the rate of disease progression.  CRP is released from liver in response to the action of IL-6 in inflammatory condition and improves antigen presentation. The treatment with EECCL significantly reduced the levels of CRP. The effect may be due to suppression of all stages of disease progression like inhibition of synthesis of chemical mediators because EECCL is having proved anti-inflammatory activities.  This plant has been found to contain a substance like guggulsterone, which has been proved to be anti-inflammatory by the mechanisms may be associated with the inhibition of inflammatory mediator overproduction. ,,
Rheumatoid factor is the true marker of clinical presentation of RA.  Adjuvant disease shows elevated blood levels of rheumatoid factor. Rheumatoid factor generation in arthritis involves B-cell activation via toll-like receptors and several genetic predispositions to arthritic diseases.  The diseased control group showed significantly elevated levels of serum rheumatoid factor compared to the normal control group. The treatment with EECCL significantly reduced the levels of these biomarkers of inflammation and autoimmune stimulation in the treated rats. The results of the present study indicate that the anti-inflammatory effects of EECCL may be due to the inhibition of activation of B cell by medicinal constituents, because EECCL contains substantial amounts of plant steroids, which are reported to produce an anti-inflammatory action.
ESR is the indicator of the chronic inflammatory disease state.  Several factors which are increased in acute tissue damage, chronic inflammation and chronic infection could have a relevant role in increased erythrocyte aggregation.  RA accompanied by raised CRP levels and CRP play an important role in the induction and maintenance of increased erythrocyte aggregation in the blood of RA patients.  Treatment with EECCL showed reduction in ESR, thus reduction in the ESR brought about by treatment supports its anti-arthritic effect.
It is proposed that reduction in the Hb count during arthritis results from reduced erythropoietin levels, a decreased response of the bone marrow erythropoietin and premature destruction of RBCs. Thus, an increase in the Hb count brought about by EECCL further supports its anti-arthritic effect.
Histopathological evaluations of ankle joints in the normal control group showed normal joint structure, no cartilage destruction, and no signs of inflammation or other distortion. However, in arthritic animals showed mild to moderate hyperplasia of synovium; focal cartilage destruction; presence of pannus formation with destruction of joint space. It also showed the marked damage of articular structure indicating joint damage and inflammation. The pannus formation and bone erosion associated with the inhibition of neutrophil infiltration.  Hyperplastic synovial tissue (pannus) may erode cartilage, subchondral bone, articular capsule, and ligaments.  Treatment with EECCL and dexamethasone group showed significant improvement in hyperplasia of synovium as compared to the disease control group, which showed the protective effect of EECCL on hyperplasia of synovium which supports its anti-arthritic effect. The inhibition of pannus formation and bone erosion may be associated with the inhibition of neutrophil infiltration. Alterations observed in the above parameters during arthritic conditions were normalized to a greater extent in C. caudata-treated animals.
The anti-arthritic effect of EECCL was studied significantly by using in vitro inhibition of the protein denaturation model. The effect of EECCL on inhibition of protein denaturation of EECCL at different concentrations (dose levels) provided significant protection against denaturation of proteins.
Most of the investigators have reported that denaturation of protein is one of the causes of RA. Production of auto-antigens in certain rheumatic diseases may be due to in vivo denaturation of proteins. The mechanism of denaturation probably involves alteration in electrostatic, hydrogen, hydrophobic and disulphide bonding. 
The anti-arthritic effect of EECCL was studied significantly by using in vitro inhibition of the membrane stabilization model. The effect of EECCL on inhibition of membrane stabilization is shown in [Table 6]. EECCL at different concentrations (dose levels) provided significant protection against stabilization of the HRBCs membrane by hypotonicity-induced membrane lysis studied to establish the mechanism of anti-inflammatory action of C. caudata. The membrane stabilization action was studied to establish the mechanism of the anti-arthritic effect of C. caudata.
| Conclusion|| |
From the results obtained in the present study, it may be concluded that C. caudata possesses significant anti-arthritic activity. Further, research may be planned as an extension of this work which could prove C. caudata as a potent anti-arthritic agent.
| Acknowledgement|| |
The authors are grateful to Secretary, Viswabhara Educational society, Warangal for providing the necessary facilities to carry out the study.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]