Evaluation of antiarthritic activity of ethanolic extract of Boswellia serrata on CFA induced method in albino rats

Corresponding author: M Swaroopa Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. Evaluation of antiarthritic activity of ethanolic extract of Boswellia serrata on CFA induced method in albino rats M. Swaroopa *, P. Sailaja and Y. Prapurna Chandra Department of pharmacology, Ratnam Institute of pharmacy, Pidathapolur(V), Muthukur(M), SPSR Nellore Dt. 524346A.P. India. World Journal of Biology Pharmacy and Health Sciences, 2025, 22(3), 095–106 Publication history: Received on 31 March 2025; revised on 27 May 2025; accepted on 30 May 2025 Article DOI: https://doi.org/10.30574/wjbphs.2025.22.3.0478 Abstract Rheumatoid arthritis (RA) is an autoimmune, inflammatory condition mainly characterized by inflammation of the synovial membranes. It predominantly affects women ages 30 to 50, with a prevalence rate of 1in 150.The present study was to evaluate the anti-arthritic activity of ethanolic extract of Boswellia Serrata Bark in complete Freund’s adjuvant (CFA)-induced arthritic rats.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 Boswellia Serrata Bark (EEBSB) was administered at doses of 100 and 200 mg/kg, p.o for 15 days. The control group received Normal saline. Diclofenac (10 mg/kg, i.p) was used as a standard drug. Paw volumes were recorded on 0, 5and 15 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. EEBSB was also tested for protein denaturation and membrane stabilization activities The collected data were analyzed using paired t-tests and analysis of variance (ANOVA) with SPSS. The data were analyzed and presented as mean differences. A P-value of < 0.05 was considered statistically significant." The EEBSB (100 and 200 mg/kg, p.o) showed significant (P < 0.01, P < 0.05) reduction in paw volume, change in body weight in CFA rats at 15 day when compared with arthritic control rats. The results obtained from the present study revealed the potential anti-arthritic activity of ethanolic extract from the Barkof B. Serrat. Keywords: Anti-arthritic activity; Boswelia Serrata Bark; Complete Freund’s adjuvant; Diclofenac 1. Introduction The degradation of joint surfaces, which causes pain, stiffness, and swelling in the joints, is a hallmark of arthritis. Usually, degenerative changes brought on by overuse or injury, like in osteoarthritis (OA), or inflammatory mechanisms, like those found in rheumatoid arthritis (RA), are to blame. RA shares several characteristics with other, less common forms of arthritis, such as psoriatic arthritis, scleroderma, and systemic lupus erythematosus. 350 million individuals worldwide suffer from arthritis, making it one of the most deceptive illnesses. A recent study found that one in four American people suffers from arthritis, which is characterised by excruciating joint pain.1Arthritis causes the deterioration of cartilage that typically shields the joints. It triggers an inflammatory response along with an increase in the growth of synovial cells. As a result, excessive accumulation of synovial fluid in the joints forms layers in the synovial cells, leading to inflammation at the joint sites. The condition's histology frequently demonstrates that it also damages the auricular cartilage, leading to joint alkalosis2. It is not a single illness, but rather a collection of linked conditions that are all referred to as "Arthritis." About 300,000 children and 47 million adults are impacted in the US alone3. World Journal of Biology Pharmacy and Health Sciences, 2025, 22(3), 095–106 96 If proper treatment is not received in a timely manner, the problem may result in permanent disability. Due to lost income and the cost of prescription drugs, it causes severe financial hardship on a global scale4. 1.1. Rheumatoid arthritis (RA): Figure 1 Rheumatoid arthritis Figure 2 Difference between normal and Rheumatoid arthritis Rheumatoid arthritis (RA) is an autoimmune, inflammatory condition mainly characterized by inflammation of the synovial membranes. It predominantly affects women ages 30 to 50 years,with a prevalence rate of 1 in 150. Boswellia serrata (also known as Salai or SalaiGuggal) is a relatively large, branching tree that grows in the hilly regions of India, Northern Africa, and the Middle East. In India, states including Andhra Pradesh, Gujarat, Madhya Pradesh, Jharkhand, and Chhattisgarh are important commercial producers of Boswellia serrata. In different places, this tree is known by different names. Known as Indian olibanum, SalaiGuggal, loban, or kundur, it is one of the most valued plants in Ayurveda. Its resin has been utilized in religious rituals and cultural celebrations for ages. In Ayurvedic medicine, frankincense (SalaiGuggal) is highly valued for its analgesic, anti-inflammatory, anti-arthritic, and anti-proliferative qualities. Additionally, it is frequently used to treat skin disorders like dermatitis and eczema. Furthermore, Boswelliacarterii frankincense is widely used in Traditional Chinese Medicine (TCM) to encourage vasodilation and alleviate pain in diseases like cancer, gonorrhoea, and leprosy. World Journal of Biology Pharmacy and Health Sciences, 2025, 22(3), 095–106 97 2. Materials and Methods 2.1. Drugs and Chemicals Complete Freund’s adjuvant (CFA) was procured from Sigma Aldrich chemicals Pvt. Ltd, Hyderabad, India, Diclofenac was obtained from SD fine chemicals Pvt. Ltd, Chennai, India and all other chemicals used in this study were obtained commercially and were of analytical grade. 2.2. Animals 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:RIPH/2023-24/CPCSEA) by the Institutional Animal Ethics Committee (IAEC), Ratnam Institute ofPharmacy, Nellore and performed in accordance with the guidelines of Committee for Control and Supervision of Experimentation on Animals (CPCSEA), Government of India on animal experimentation. 2.3. Plant Material Figure 3 Boswelliaserrata 2.4. Taxonomical Classification • Kingdom: Plantae • Order: Sapindales • Family: Burseraceae • Genius: Boswellia • Species: B. serrata • Family:: Burseraceae 2.5. Collection The Fresh Bark of B. Serrata were collected from the Tirumala hills, chittor district, A.P, India, and authenticated by Dr. MadhavaChetty from Sri Venkateswara University, Department of Botany, Tirupati, Andhra Pradesh, India. 2.6. 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. World Journal of Biology Pharmacy and Health Sciences, 2025, 22(3), 095–106 98 2.7. 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), B. Serrata 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. 2.8. Pharmacological activity 2.8.1. Adjuvant-Induced Arthritis 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 2.8.2. Experimental design The arthritic animals were divided into five groups each containing six animals, and one group of normal non-arthritic animals. Ethanolic extract of B. Serrata was given at doses of 100 and 200 mg/kg, for 15 days, as a suspension in CMCat a dose of 0.2 ml/kg to different groups of animals. • Group I: Normal control (Saline) • Group II:Positive control (0.1 ml of CFA (CMC)) • Group III: Arthritic animals received Diclofenac a dose of 10 mg/kg • Group IV: Arthritic animals received EEBSB at a dose of 100 mg/kg Group • V: Arthritic animals received EEBSB at a dose of 200 mg/kg "For the induction of arthritis, 0.1 ml CFA was administered subcutaneous injection into the foot pad of the left hind paw of rats, excluding the control group.All rats were monitored for an acute inflammatory response indicative of arthritis. The treatment regimen was carried out fromday5 to day15. Throughout the study, standard protocols were adhered to. Baseline measurements for parameters such as body weight, paw thickness, ankle circumference, and paw volume were recorded onday0. Subsequently, the aforementioned parameters, along with an arthritic score, were assessed on days 5 and 15. Onday15, all rats were euthanized using a high dose of pentobarbital (50 mg/kg i.p.). The inflamed limbs were excised above the ankle joints and analyzed for histopathological evidence of rheumatoid arthritis." Diclofenac 10 mg/kg was administered in distilled water for oral administration to each rat.The body mass (g) of all rats was recorded on days 0, 5, and 15 during the experiment. The percentage variation in body mass was determined using the formula: Percentage variation=(Wc −Wt)×100/Wc, Where Wc is the average change in body mass of the arthritic control group and Wt is the average change in body mass of the treated group. Ankle circumference was measured on days 0, 5, and 15 to evaluate inflammation as an acute lesion on the injected limb. The percentage reduction in ankle circumference was calculated using the formula: Percentage reduction= (Dc− Dt)×100/Dc Where Dc is the average change in ankle circumference of the arthritic control group and Dt is the average change in ankle circumference of the treated group." "Paw volume of the injected limb was measured on days 0, 5, and 15 by immersing it vertically to the level of the lateral malleolus in the plethysmometer. The average change in paw volume was calculated, and the percentage reduction in paw swelling was determined using the formula: Percentage reduction= (Vc− Vt)×100/Vc World Journal of Biology Pharmacy and Health Sciences, 2025, 22(3), 095–106 99 Where Vc is the average change in paw volume of the arthritic control group and Vt is the average change in paw volume of the treated group. Rats were monitored and periodically evaluated for arthritis on days 0, 5, and 15.Theseverityof arthritis was assessed using a grading scale: • Normal Paw =0 • Mild swelling and redness of digits=1 • Swelling and redness of digits = 2 • Severe swelling and redness = 3 • Visible deformity and inability to move the limb=4. The arthritic score for each rat was calculated by summing the four scores for individual paws.If the adjuvant-injected limb (left hind) showed inflammation, the experimental animal was considered to have developed acute arthritis." "For histopathological assessments, rats were euthanized on day 15 using a high dose of pentobarbital (50 mg/kg i.p.), and the inflamed limbs were removed above the ankle joints and examined for histopathological signs of rheumatoid arthritis. The excised left hind limb tissue from both control and treated rats was fixed in a 10% buffered formalin solution and sent to a pathologist for evaluation, who was unaware of the group assignments. 2.9. Statistical analysis The collected data were analyzed using paired t-tests and analysis of variance (ANOVA) with SPSS. The data were analyzed and presented as mean differences. A P-value of < 0.05 was considered statistically significant." 2.10. StatisticalAnalysis The collected data were analyzed using paired t-tests and analysis of variance (ANOVA) with SPSS. The data were analyzed and presented as mean differences. A P-value of < 0.05 was considered statistically significant." 3. Results and Discussion 3.1. "Impact of Boswellia serrata extract on body mass compared to DICLOFENAC On day 0, body mass was recorded for each rat in all groups and taken as baseline values. Throughout the study, the average %increase in mass in the control group was 2.21 (P = 0.07) from day 0 to day 5, and the average % increase in mass was 0.43 (P = 0.78) from day 5 to day 15. On day 5, a reduction in body mass was observed in all CFA-induced arthritic groups (Groups 2–6). By day 15, body mass had increased in both diclofenac and BSE-treated groups compared to day 5. A significant gain in body mass was observed in the diclofenac group (P < 0.05), while the change was not significant in any of the BSE-treated groups [Table 1a]. Upon intergroup comparison, body mass was significantly higher in the diclofenac-treated group (P < 0.01) and the BSE 180 mg/kg-treated group (P < 0.05) compared to the arthritic control group [Table 1b]." Table 1a Variation in bodymass (measured in grams) of different animal groups on days 0, 5,and 15 (all values presented as mean ± standard deviation; values on days 0 and 5 represent pre-treatment values, while values on day 15 represent post-treatment values; n=6/group; all treatments administered orally) GROUPS Days of study (mean±SD) % change DAY 0 DAY 5 DAY 15 DAY 0-5 DAY 5-15 I 188.5±5.677 192.67±4.73 193.5±5.42 −2.31 −0.53 II 186.83±6.213 176.83±10.74 169.83±10.404 5.45 3.99 III 185.83±8.185 176.33±9.788 189.5±6.84 5.21# −7.57# IV 185±8.538 177.17±9.12 180±8.977 4.33 −1.70 V 184.17±7.648 177.67±9.60 183.33±7.105 3.63 −3.29 World Journal of Biology Pharmacy and Health Sciences, 2025, 22(3), 095–106 100 Table 1 b Change in bodyweight of treated groups ascompared with arthritic control group GROUPS MEAN DIFFERENCE DAY 0 DAY 5 DAY 15 II VERSUS III 1.03 0.52 -19.69* IV 1.85 -0.34 -10.19 V 2.69 -0.85 -13.60 3.2. Impact ofBoswellia serrata extract on ankle circumference compared to diclofenac Prior to CFA injection, ankle circumference of both hind limbs of each rat in every group was measured on day 0 and used as baseline values. Throughout the study, ankle circumference remained nearly constant in the normal control group. In CFA-induced arthritic groups (2–6), ankle circumference significantly increased on day 5 compared to day 0 (P < 0.01). In Group 2 (arthritic control), ankle circumference increased significantly on day 15 compared to day 5. After drug treatment, ankle circumference values on day 15 were observed to decrease from the day 5 values, with significant reductions in Groups 3, 5, and 6 [Table 2a]. Upon intergroup comparison, ankle circumference was significantly reduced in theindomethacin 3 mg/kg-treated group (P < 0.05) and the BSE 180 mg/kg-treated group (P < 0.05) compared tothearthriticcontrolgroup[Table2b]. Table 2 a Change in ankle diameter (measured in cm) of different groups of animals on day 0, 5, and 15 (all values represented here are in mean ± standard deviation; values on day 0 and 5 represent the value before treatment and values on day 15 are after treatment n=6/group; all treatment administered orally) Groups Day 0 Day 5 Day 15 % change Day 0-5 Day 5-15 I 0.33±0.04 0.33±0.07 0.34±0.02 0.02 −1.09 II 0.34±0.03 0.61±0.04 0.65±0.05 −87.25* −6.91# III 0.33±0.03 0.63±0.05 0.41±0.04 −94.42* 36.82* IV 0.32±0.23 0.57±0.56 0.59±0.04 −82.44* −4.87 V 0.31±0.14 0.67±0.07 0.56±0.5 −117.82* 17.05# Table 2b Change in ankle diameter of treated groups as compared with arthritic control group variables groups Mean difference Day 0 Day 5 Day 15 Ankle diameter II VERSUS III -0.006 -0.024 0.343# IV 0.0118 0.046 0.160 V 0.002 -0.054 0.236# 3.3. "Impact of Boswellia serrata extract on paw swelling compared to diclofenac: Before CFA injection, paw swelling of both hind limbs of each rat in every group was measured on day 0 and used as baseline values. A slight increase in paw swelling was observed in the normal control group on day 15 compared to day 0. Paw swelling values were significantly (P < 0.01) higher in all CFA-induced arthritic groups (2–6) on day 5 compared to day 0. After drug treatment, paw swelling values on day 15 showed a decrease from the day 5 values, but this World Journal of Biology Pharmacy and Health Sciences, 2025, 22(3), 095–106 101 reduction was significant only in the diclofenac treated group table 3a Intergroup comparison revealed a reduction in paw swelling on day 15 in all groups when compared to the arthritic control group, but the decrease was significant in Groups 3 and 6 [Table 3b]." Table 3a Change in paw volume (measured in ml) of different groups of animals on day 0, 5, and 15 (all values represented here are in mean ± standard deviation values on day 0 and 5 represent the value before treatment and values on day 15 are after treatment; n=6/group; all treatment administered orally) Groups Day 0 Day 5 Day 15 % change Days 0-5 Days 0-5 I 0.74±0.01 0.75±0.013 0.78±0.01 0.01 −0.26 II 0.73±0.02 0.99±0.29 1.31±0.14 −53.1# −22.73 III 0.74±0.01 1.22±0.25 0.80±0.03 −74.27* 38.39# IV 0.76±0.02 1.33±0.41 0.99±0.08 −85.99# 28.16 V 0.74±0.02 0.99±0.20 0.96±0.08 −55.95* 13.44 Table 3b Effects of Boswellia serrata extract on paw volume in control and treatment group-on day 0, 5, and 15 Variables Groups Mean difference Day 0 Day 5 Day 15 Paw volume II versus III −0.020 −0.136 0.610# IV −0.040 −0.246 0.415 V −0.010 0.008 0.365 3.4. Effects of Boswellia serrata extract on arthritis index as compared to diclofenac: Complete Freund's Adjuvant-induced arthritic rats produced statistically significant (P < 0.01) increase in the arthritic index in all arthritic group. Following the drug treatment, arthritic index values on day 15 were found to have decreased from day 5 values, but it was significant only in diclofenac-treated group [Table4a]. Arthritis index kept on increasing from day 5 to 15 in all the groups, whether control or treated with diclofenac/BSE [Table 4a].Intergroup comparative analysis showed that there was reduction in the arthritic index on day 15 in all the groups when compared to the arthritic control group, but it was significantly reduced in Group 3, 5, and 6 [Table 4b] Table 4a Arthritis index of different groups of animals on day 0, 5 and 15 (all values represented hereare in mean±standard deviation;valuesonday0and5representthevalue before treatmentand values on day 15 are after treatment; n=6/group; all treatment administered orally Groups Day 0 Day 5 Day 15 % change (days 5-15) I 0 0±0 0±0 0.00 II 0 5.76±0.51 19.7±1.22 −250.12* III 0 5.76±0.51 10.36±0.98 −80.41* IV 0 5.43±0.51 16.36±2.04 −206.13* V 0 5.6±0.54 15.8±2.66 −188.82* World Journal of Biology Pharmacy and Health Sciences, 2025, 22(3), 095–106 102 Table 4b Effects of Boswellia serrata extract on Arthritis index in control and treatment group-on day 5 and 15 Variables Groups Mean difference Days 5 Days 15 II versus III 0.001 9.43* Arthritic index IV 0.343 3.53 V 0.177 4.02# 3.5. Histopathological studies Figure 4 Histopathology of ankle joint cartilage in arthritis control group showing keratinized epidermis with underlying loose subepidermal zone infiltrated by chronic inflammatory cells entrapping fat cells Figure 5 Histopathology of ankle joint bone in arthritis control group showing dense inflammation and pannus World Journal of Biology Pharmacy and Health Sciences, 2025, 22(3), 095–106 103 Figure 6 Histopathology of ankle joint in diclofenac treated group. Response to drug seen in the form of normal thin epidermis and skin appendages Figure 7 Histopathology of ankle joint bone in diclofenac treated group showing bony spicules with regenerating marrow tissue Figure 8 Histopathology of ankle joint cartilage in Boswelliaserrata extract treated group