INTRODUCTION:

Arthritis is a chronic non-communicable autoimmune disorder affecting over 1% of the total populace, mainly between age groups of 30–55, even though it can occur at any age, with females more affecting than males^1-4. It is mainly characterized by non-specific peripheral joints inflammation, deformities of the joints, arthralgia, dyskinesia, and damage of articular tissues^5-8.

The exact etiology of arthritis is not known yet, but some inflammatory cytokines such as “interleukin-1 (IL-1), tumor necrosis factor-(TNF-α), interleukin-6 (IL-6), and C-reactive proteins” are considered the major markers involved in the progression of inflammation and destruction of joint during the development of arthritis^9-14.

Nowadays, the treatment of arthritis emphasizes the attenuation of inflammation in the joints of patients, but its complete treatment is not explored yet¹⁵. Conventionally available medicines such as glucocorticoids, methotrexate, NSAIDs, analgesics, disease-modifying anti-rheumatic drugs (DMARDs) like Janus Kinase inhibitors (Tofacitinib, Upadacitinib, Filgotinib), TNF alpha inhibitors (Infliximab, Adalimumab, Golimumab), Interleukin-6 inhibitors (Tocilizumabviz) have been reported to possess limited effectiveness in the arthritis treatment^16,17. Moreover, chronic use of these medicines produces various undesired effects such as gastric toxicity, cardiovascular complications, nephrotoxicity, and unable to prevent the damage of joints, which limit their use in the management of arthritis^18-21.

Therefore, exploring new drugs with more efficacy and less toxicity to treat arthritis has become an absolute necessity. Medicinal plants have always been an enormous source of treatment for various ailments since ancient times²²due to their easy affordability, less cost, with minimal side effects^23-26. Despite this, most of the traditionally used plant-based medicines possess significant potential against inflammation and oxidative stress in several animal experimental models without any incidence of severe side effects viz. Terminalia chebula, Piper nigrum, Zingiber officinale, Ammania baccifera, Neolamarckia cadamba, Akebia quinata, Andrographis paniculata, Curcuma longa, etc^{27- 31}.

Cordia myxa (or lasora), family Boraginaceae is a valuable, fast-growing medicinal plant^{32, 33} used traditionally for its various pharmacological activities such as immunomodulatory, analgesic, antimicrobial, anti-inflammatory, antihelminthic along with cardiovascular, respiratory, gastrointestinal protective effects^34-38. Various plant parts are reported to be used in different disorders due to their diverse constituents like flavonoids, saponins, alkaloids, glycosides, sterols, tannins, coumarins, resins, oil, mucilage, and gums, etc. As no scientific studies revealing the anti-arthritic potential of leaves extract of this plant are reported. Therefore, the present study is designed to investigate the anti-arthritic potential of leaves extracts of C. myxa in Freund’s complete adjuvant (FCA)-induced arthritis in rats.

MATERIALS AND METHODS:

Plant material:

Cordia myxa L. leaves were procured from local area of Yamuna Nagar, Haryana, were air-dried and authenticated by Dr. K Madhava Chetty, Assistant Professor, Department of Botany, Shri Venkateshwar University Tirupati. A voucher specimen of the plant (0620) has been retained there for future reference. All the chemicals, solvents, and reagents used in the study were of analytical grade.

Preparation of extracts:

The dried plant material was coarsely powdered, and the powdered material was first defatted using petroleum ether to remove fatty materials. It was air-dried and subjected to Soxhlet extraction using ethanol for 48 hours. The extract obtained was concentrated using the rotary evaporator (IKA Works INC., North America), dried, weighed subsequently, and then stored in a refrigerator to avoid microbial contamination. The extract obtained was further investigated for various bioactive compounds using phytochemical screening tests and procedures³⁹.

Animal Housing:

Wistar albino rats (160–170 g) were used for the study. Animals were kept in plastic rat cages with stainless steel coverlids and rice straw was used as a bedding material. The animals were facilitated with environmental conditions of photoperiod (12:12 h dark: light cycle) and temperature (25±2°C). They were kept on the commercial diet and water ad libitum. The protocol was approved by Institutional Animal Ethics Committee with protocol no. (MMCP/IAEC/94) and as per the guidelines of the CPCSEA.

Experimental design:

Arthritis was induced in rats by administering intradermally single dose (0.1ml) of Freund’s Complete Adjuvant (FCA) containing 10mg/mL Mycobacterium tuberculosis H37Ra suspension in sterile paraffin oil (Sigma Aldrich, India) into their right hind foot paw (Freund’s complete adjuvant-induced arthritis)^40-42. About 9 days later, treatment with ethanolic extracts in different doses and methotrexate (0.3mg/Kg) was started and continued up to 21 days.

Animal Grouping:

Animals were divided into 6 groups of six rats each. The first group (Healthy Control, HC) received normal saline whereas the second group (Arthritic control, AC) was administered FCA (0.1ml, intradermally a single dose into right hind foot paw). The third group (MTX) was kept on methotrexate treatment at a dose of 0.3mg/Kg orally. However the fourth (ECME 100mg/Kg), fifth (ECME 200 mg/kg) and sixth group (ECME 400 mg/kg) received ethanolic extract orally at a dose of 100mg/Kg, 200mg/Kg and 400mg/Kg respectively.

The paw volume was measured of all the animal groups using plethysmograph and paw thickness using vernier caliper before FCA immunization and then at 0th, 7th, 14th, and 21st day after 9th day of FCA immunization⁴³ along with monitoring of body weights at regular intervals. On the last day of study, blood was withdrawn, and serum was collected by centrifugation to assess hematological parameters which includes RBC count, WBC count, platelet count, hemoglobin concentration, and ESR. Then animals were sacrificed for histopathological examination⁴⁴.

Statistical analysis:

All the results were expressed as mean ± standard error mean (S.E.M). The estimated data was analyzed statistically using one-way ANOVA followed by Tukey’s test.

RESULTS:

The phytochemical screening reveals the presence of flavonoids, glycosides, alkaloids, and tannins in the ethanolic extract of Cordia myxa leaves (Table 1).

Table 1: Phytochemical Screening

Plant Constituents	Test	EE
Alkaloids	Dragondroff’s test	++
	Hager’s test	++
	Mayer’s test	++
	Wagner test	++
Carbohydrates	Benedict’s test	+
Carbohydrates	Molisch’s test	+
Glycosides	Borntrager’s test	++
Glycosides	Keller killani	++
Proteins	Biuret’s test	-
Saponins	Foam’s test	+
Flavonoids	Lead acetate test	++
Flavonoids	Vanillin HCl test	++
Steroids	Libermann test	-
Steroids	Salkowaski test	-
Tannins	Gelatin test	-

(+ indicates the presence of constituents and ─ indicates the absence of constituents)

Effect on paw volume:

A significant (P < 0.001) increase in paw volume was observed in all the rats treated with FCA compared to healthy control. ECME (200 and 400mg/kg) considerably reduced paw volume in a dose dependent manner as compared to arthritic control starting from day 7(P < 0.01) to day 21(P < 0.001) measured after 9 days of FCA administration. However, ECME 100 mg/kg was less effective (P< 0.01) upto day 14, it significantly (P < 0.01) reduced paw volume on day 21. MTX i.e. group treated with methotrexate showed significant (P < 0.001) reduction in paw volume from day 14 onwards. The change in paw volume of ECME treated group (400mg/kg, 0.995±0.0207; 200mg/kg, 1.058±0.0897 and 100mg/Kg, 1.208±0.0552) was evident as compared to arthritic control (1.498±0.1202) on day 21(Figure 1).

Effect on paw thickness:

Treatment with FCA cause increased paw thickness in all the groups compared to healthy control. However, treatment with different ethanolic extracts (ECME 100, 200, and 400 mg/kg) ameliorates the effect and reduces paw thickness significantly in a dose-dependent manner (Figure 2). The potency of ECME 400mg/Kg (5.583 ± 0.664) was found comparable to MTX treated rats (5.5 ± 0.548).

Figure 1: Effect of C. myxa on paw volume in FCA-induced arthritic rats

Data are expressed as mean±S.E.M. (n = 6). Data was analyzed by one-way Analysis of Variance (ANOVA) followed by Tukey’s test for comparison. a^***P < 0.001 as compared to control. b^***P < 0.001 as compared to FCA. b^**P < 0.01 as compared to FCA. b^*P < 0.05 as compared to FCA.

Figure 2: Effect of C. myxa on paw thickness in FCA-induced arthritic rats

Data are expressed as mean ± S.E.M. (n = 6). Data was analyzed by one-way Analysis of Variance (ANOVA) followed by Tukey’s test for comparison. a^***P < 0.001 as compared to control. b^***P < 0.001 as compared to FCA. b^**P < 0.01 as compared to FCA. b^*P < 0.05 as compared to FCA.

Effect on body weight:

The body weight of rats after FCA administration showed a significant weight loss compared to healthy rats. ECME (400mg/kg) significantly (P<0.01) attenuated the body weight loss starting from day 7. ECME (100mg/kg and 200mg/Kg) however showed significant (P < 0.05) response from day 14 as compared to arthritic control (Figure 3). The body weight of ECME (400 mg/kg, 173.667±2.658; 200 mg/kg, 166.83 ±3.868 and 100mg/kg, 166.5±4.324) was evident as compared to arthritic control (152.33±1.505) which was comparable to methotrexate treated group, MTX (174.33±0.816) on day 21.

Figure 3: Effect on body weight

Effect on hematological parameters:

The decreased levels of hemoglobin concentration, RBC count, and increased levels of WBC count, platelet count and, ESR were observed in arthritic control group. Treatment with methotrexate (MTX) and ECME (100, 200, and 400mg/Kg) showed amelioration in these altered parameters viz. increase in hemoglobin concentration, RBC count, and decrease in the level of WBC count, platelet count and ESR in the arthritic rats. The results of these hematological parameters are presented in Table 2.

Table 2: Effect on Haematological parameters

	RBC Count (millions/ mm³)	Hb (gm/ dL)	WBC Count (/mm³)	ESR (mm/ hr)	Platelet count (x10³/mm³)
Healthy control	7.628	14.733	6878.33	11.45	3.968
Arthritic control	5.383	10.35	9412.33	14.32	5.65
MTX	7.218	14.483	7221.66	11.65	3.838
ECME 100 mg/kg	5.875	11.015	8449.16	12.34	4.551
ECME 200 mg/kg	6.16	12.583	7481.83	12.02	4.573
ECME 400 mg/kg	7.198	14.076	7223	11.96	3.967

Histopathological studies:

Histopathological examination of the paw tissue section of healthy control rats revealed visible articular cavity and smooth articular cartilage surface without pathological alteration or infiltration of inflammatory cells in the synovium (Figure 4A). However, histopathological changes such as infiltration of inflammatory cells, edema formation, changes in sub-synovial collagen fiber structure, and thickening of extracellular matrix were observed in the FCA-induced arthritic group (Figure 4B). These pathological changes induced by FCA were markedly mitigated on the treatment of the arthritic rats with methotrexate (MTX) (Figure 4C) and ECME (100, 200, and 400mg/kg orally) for 21 days (Figure 4D-4F).

Figure 4: Histopathological studies

(4A- HC; 4B- AC; 4C-MTX; 4D- ECME 100mg/kg; 4E-ECME 200mg/Kg; 4F- ECME 400mg/Kg)

DISCUSSION:

The present study investigated the protective role of ethanolic extract in FCA-induced arthritis. The arthritic condition may ensue as a result of increased levels of inflammatory cytokines due to dysregulation in cellular as well as humoral immunity. These circulatory cytokines in the synovium are considered one of the main parameters involved in the progression of arthritis^45,46. NSAIDs and other commercially available anti-inflammatory drugs are used for arthritis these days, but their long-term use causes various side effects⁴⁷. Within few years, the popularity of alternative medicine has increased considerably. WHO has suggested evaluating medicinal plants as effective therapeutic agents, especially in areas where modern drugs are not readily available⁴⁸. Therefore, the present study investigated the protective effect of ethanolic extract of C. myxa in FCA induced arthritis in rats. FCA is a well-established model used to induce arthritis in animals and shows several clinical and histological similarities to human arthritis⁴⁹. FCA treated rats mainly possess paw swelling, increased paw thickness, loss of body weight due to alterations in the metabolic activities of diseased rats⁵⁰. The determination of paw swelling and paw thickness is a simple, sensitive, and quick procedure for evaluating and assessing the degree of inflammation and the therapeutic and curative effects of drugs⁵¹. The results of the present study showed that there is a significant increase in swelling in paw along with paw thickness in arthritic control rats when compared with the normal control group. However, on treatment with extract at the dose 100, 200, and 400mg/kg/day, significant attenuation in paw swelling and paw thickness was observed in a dose-dependent manner.

Moreover, this study found that there is significant weight loss after the injection of the FCA. The changes in body weight of animals have been considered to investigate pharmacological responses against disease. The result of the present study showed that there is a relationship between weight loss and joint inflammation. However, treatment with ECME (100, 200, and 400 mg/kg, orally) significantly attenuated body weight loss induced by FCA, comparable to the positive control group.

With previous findings of the hematological parameters, the results showed a significant reduction in the hemoglobin (Hb) and red blood corpuscles (RBC). The significant decrease in these parameters in the arthritis-induced rats could be attributed to premature red blood cells destruction and abnormal iron storage in the synovial tissue and reticuloendothelial system. These reasons might have caused an iron deficiency in bone marrow due to reduced iron release through the mononuclear phagocyte system activation, which leads to anemia. Some inflammatory cytokines are also released during arthritis, causing the reduction in folic acid absorption responsible for decreased Hb concentration and RBC count⁵². Comparing the arthritic control group with the ethanolic extract (ECME at 100, 200, and 400mg/kg/day orally) treated group, the maximum increase in the RBCs and hemoglobin (Hb) was observed.

On the other hand, the WBC count, platelet count, ESR in the arthritic rats were increased moderately due to the activation of the immune system against the entering antigens of arthritis which leads to the development of unnecessary leucocytes⁵³. The amelioration in the level of WBC count, platelet count, and ESR was observed in all the groups treated with the ethanolic extract (ECME at 100, 200, and 400mg/kg/day orally). However, ECME 400mg/kg showed better results than other groups.

The histopathological examination provides the prominent morphologically pathological characterization of arthritis⁵⁴. FCA-induced arthritic rats (Group 2) revealed observable peri-articular and synovial inflammation and intense inflammatory cells infiltration and pannus formation. It is documented that irregulating pro- and anti-inflammatory agents promote the development and progression of arthritis^55,56. In the present study, histological analysis showed the protective effects of ethanolic extract (ECME 100, 200, and 400mg/kg/day) of C. myxa on synovitis within joints against arthritis. The best recovery and restoration of the ankle joint was seen with ECME 400mg/Kg compared to methotrexate control (MTX 0.3mg/Kg); however, a complete restoration was not observed in any case.

The presence of flavonoids, alkaloids, glycosides, and tannins in the leaves of C. myxa are thought to be responsible for their anti-arthritic effect due to their anti-inflammatory action and ability to scavenge free radicals to protect the body against free radicals and other pro-oxidative compounds.

CONCLUSION:

The results obtained from the current investigation contribute to exploring the anti-arthritic potential of leaves extracts of C. myxa in FCA-induced arthritis in rats. Chemical constituents present in the leaves extract may be responsible for protecting synovial membrane, vascular permeability, and preventing cartilage damage, which could finally improve the arthritic condition. Moreover, further evaluation of extracts is required to identify and isolate the potent bioactive compound responsible for its anti-arthritic activity, facilitating the utilization of C. myxa in arthritic patients clinically.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors are thankful to management of MM College of Pharmacy, MM (Deemed to be University, Mullana (Ambala) for providing support and necessary facilities.

REFERENCES:

1. Singh VS, Dhawale SC, Shakeel F, Faiyazuddin M, Alshehri S. Anti-arthritic potential of Calotropis procera leaf fractions in FCA-induced arthritic rats: Involvement of cellular inflammatory mediators and other biomarkers. Agriculture. 2021; 11: 68. doi: https://dx.doi.org/10.3390/agriculture11010068

2. Xin C, Ruijing W, Peimin B, Qingke W, Seshadri VDD, Liu L. Evaluation of antiarthritic activity of nimbolide against Freund’s adjuvant induced arthritis in rats. Artificial Cells, Nanomedicine, and Biotechnology. 2019; 47(1): 3391-3398. doi: https://dx.doi.org/10.1080/21691401.2019.1649269

3. Bihani GV, Rojatkar SR, Bodhankar SL. Anti-arthritic activity of methanol extract of Cyathocline purpurea (whole plant) in Freund's complete adjuvant-induced arthritis in rats. Biomedicine and Aging Pathology. 2014; 4(3): 197-206. doi: https://dx.doi.org/10.1016/j.biomag.2014.04.007

4. Kilimozhi D, Parthasarathy V, Manavalan R. A review on arthritis. Research Journal of Pharmacy and Technology. 2011; 4(1): 29-36.

5. Tanaka T. Introduction for inflammation and cancer. Seminars in Immunopathology. 2013; 35: 121-122. doi: https://dx.doi.org/10.1007/s00281-012-0360-6

6. Putterman C. Introduction: New treatment paradigms in rheumatoid arthritis. The American Journal of Orthopedics (Belle Mead NJ). 2007; 36(3 Suppl): 2-3.

7. Khalimatun SS, Jais MR, Elysha NI, Reezal I. The effect of Nigella sativa and Eucheuma cottonii in collagen-induced arthritis mice. Research Journal of Pharmacy and Technology. 2020; 13(3): 1319-1323. doi: https://dx.doi.org/10.5958/0974-360X.2020.00243.7

8. Kilimozhi D, Parthasarathy V, Manavalan R. A review on arthritis. Research Journal of Pharmacy and Technology. 2011; 4(1): 29-36.

9. McInnes IB, Schett G. The pathogenesis of rheumatoid arthritis. The New England Journal of Medicine. 2011; 365(23): 2205-2219. doi: https://dx.doi.org/10.1056/NEJMra1004965

10. Reddy D, Trost LW, Lee T, Baluch AR, Kaye AD. Rheumatoid arthritis: Current pharmacological treatment and anesthetic considerations. Middle East Journal of Anesthesiology. 2007; 19(2): 311-313.

11. Bhardwaj LK, Chandrul KK, Sharma US. Evaluation of anti-arthritic activity of Ficus benghalensis Linn. root extracts on Freund’s adjuvant induced arthritis in rats. The Journal of Phytopharmacology. 2016; 5: 10-14. doi: https://dx.doi.org/10.31254/phyto.2016.5103

12. Khan MY, Aziz I, Ahmad I, Roy M, Verma VK. A Natural cure to arthritis by phytomedicines- A review. Asian Journal of Research in Pharmaceutical Sciences. 2015; 5(4): 216-220. doi: https://dx.doi.org/10.5958/2231-5659.2015.00031.4

13. Spandana K, Chary VT, Shivani M. Rheumatoid arthritis. Research Journal of Pharmaceutical Dosage Forms and Technology. 2019; 11(2): 131-133. doi: https://dx.doi.org/10.5958/0975-4377.2019.00021.1

14. Suneel KA, Venkatarathanamma V, Naga SV, Nageswara RT, Pavan KK, Vijayalakshmi. A anti-arthritic activity of Annona squamosa leaves methanolic extract on adjuvant induced arthritis in rats. Research Journal of Pharmacology & Pharmacodynamics. 2017; 9(2): 46-56. doi: https://dx.doi.org/10.5958/2321-5836.2017.00009.X

15. Bullock J, Rizvi SAA, Saleh AM, Ahmed SS, Do DP, Ansari RA, et al. Rheumatoid arthritis: a brief overview of the treatment. Medical Principles and Practice. 2019; 27(6): 501–507. doi: https://dx.doi.org/10.1159/000493390

16. Mysler E, Mariana C, Ana L. Current and emerging DMARDs for the treatment of rheumatoid arthritis. Open Access Rheumatol ogy: Research and Reviews. 2021; 13: 139-152. doi: https://dx.doi.org/10.2147/OARRR.S282627

17. Mohanty S, Pal A, Si SC. Flavonoid as nutraceuticals: A therapeutic approach to rheumatoid arthritis. Research Journal of Pharmacy and Techology. 2020; 13(2): 991-998. doi: https://dx.doi.org/10.5958/0974-360X.2020.00184.5

18. Smolen JS, Landewe R, Bijlsma J, Burmester G, Katerina C, Maxime D, et al. EULAR recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs: 2016 update. Annals of the Rheumatic Diseases. 2017; 76(6): 960-977. doi: https://dx.doi.org/10.1136/annrheumdis-2016-210715

19. Choudhary M, Kumar V, Gupta P, Singh S. Investigation of antiarthritic potential of Plumeria alba L. leaves in acute and chronic models of arthritis. BioMed Research International. 2014: 1-12. doi: https://dx.doi.org/10.1155/2014/474616

20. Elumalai A, Prakash Y G. Evaluation of anti-arthritic activity of ethanolic extract of Pisonia grandis R. Br. Asian Journal of Pharmaceutical Research. 2012; 2(3): 91-93.

21. Kayalvizhi MK, Samuel V, Nirmala P, Balamurugan K. Anti-arthritic and anti-inflammatory effect of Cetylmyristeolate against Freund’s adjuvant induced arthritis model. Research Journal of Pharmacology and Pharmacodynamics. 2012; 4(2): 91-93.

22. UmiKalsum, Khotimah H, Riawan W, Trisnaini HA, Zubaidah E. Sugar Palm Fruit (Arenga pinnata) diminish pain and inflammatory symptoms of osteoarthritis in male wistar rat (Rattus norvegicus). Research Journal of Pharmacy and Techology. 2018; 11(9): 3745-3751. doi: https://dx.doi.org/10.5958/0974-360X.2018.00686.8

23. Garg AK, Faheem M, Singh S. Role of medicinal plant in human health disease. Asian Journal of Plant Science & Research. 2021; 11(1): 19-21. doi: https://dx.doi.org/10.24941/ijcr.40170.11.2020

24. Polat S, Gurol A. Safety of herbal medicines in children. IntechOpen. 2020: 1-10. doi: https://dx.doi.org/10.1136/adc.88.12.1032

25. Azhar MF, Siddiqui MT, Ishaque M, Tanveer A. Study of ethnobotany and indigenous use of Calotropis procera (Ait.) in Cholistan desert, Punjab, Pakistan. Journal of Agricultural Research. 2014; 52(1): 117-126.

26. Rai R. Herbal folk remedies in cure of arthritis by ethnic communities in Madhya Pradesh, India. Asian Journal of Research in Pharmaceutical Sciences. 2016; 6(3): 177-184. doi: https://dx.doi.org/10.5958/2231-5659.2016.00024.2

27. Premkumar B, Murthy MS, Rajagopal K. Medicinal plants for inflammatory arthritis. Research Journal of Pharmacognosy & Phytochemistry. 2014; 6(2): 51-65.

28. Ali B, Mujeeb M, Aeri V, Mir SR, Faiyazuddin M, Shakeel F. Anti-inflammatory and antioxidant activity of Ficus carica Linn. leaves. Natural Product Research. 2012; 26(5): 460-465. doi: https://dx.doi.org/10.1080/14786419.2010.488236

29. Kumar SS, Bhosle D, Janghel A, Deo S, Raut P, Verma C, Agrawal M, Amit N, Sharma M, Giri T, Tripathi DK, Ajazuddin, Alexander A. Indian medicinal plants used for treatment of rheumatoid arthritis. Research Journal of Pharmacy and Technology. 2015; 8(5): 597-610. doi: https://dx.doi.org/10.5958/0974-360X.2015.00099.2

30. Raj A, Shakya MK, Singh T, Agrawal M, Katiyar NS. Evaluation of anti-arthritic activity of leaves extracts of Neolamarckia cadamba. Research Journal of Pharmacy and Technology. 2022; 15(2): 873-7. doi: https://dx.doi.org/10.52711/0974-360X.2022.00146

31. Samuel SM, Pramod K, NBijin E, Ajithkumar KC, Jijith US. Herbal remedies for rheumatoid arthritis. Research Journal of Pharmacognosy & Phytochemistry. 2016; 8(1): 32-36. doi: https://dx.doi.org/10.5958/0975-4385.2016.00007.8

32. Padhi M, Singh SP. Surface sterilization for reducing microbial contamination in in vitro propagation of lasora (Cordia myxa Roxb.) using nodal segments. International Journal of Current Microbiology and Applied Sciences. 2017; 6(8): 836-842. doi: https://dx.doi.org/10.20546/ijcmas.2017.608.106

33. Al-Snafi AE. The pharmacological and therapeutic importance of Cordia myxa- a review. IOSR Journal of Pharmacy. 2016; 6(6): 47-57.

34. Rashed K, Luo MT, Zhang LT, Zheng YT. Evaluation of anti-HIV-1 activity of Cordia myxa L. and phytochemical profile. Banat's Journal of Biotechnology. 2014; 5(9):51-56. doi: https://dx.doi.org/10.7904/2068–4738–V(9)–51

35. Pandey B, Deshpande BS, Sing S, Chandrakar V. Estimation of elemental contents of Cordia myxa and its antimicrobial activity against various pathogenic microorganisms. Indian Journal of Scientific Research. 2014; 4(1): 39-44.

36. Inas ZA, Hala AK, Gehan HH. Gastroprotective effect of Cordia myxa L. fruit extract against indomethacin-induced gastric ulceration in rats. Life Science Journal. 2011; 8(3): 433-445.

37. Aberoumand A. Preliminary evaluation of some phytochemical and nutrients constituents of Iranian Cordia myxa fruits. International Journal of Agriculture and Food Science. 2011; 1(2): 3033.

38. Ali KA. Study of effect of alcoholic extract of fruit of Cordia myxa in total count and differential count of white blood cells in rats. Quarterly Journal of the Royal Meteorological Society. 2008; 7(2): 21-24.

39. Khandelwal KR. Preliminary phytochemical screening in practical pharmacognosy. Nirali Parkashan, Pune. 2004: 449-1156.

40. Kamal RM, Sabry MM, Aly ZY, Hifnawy MS. Phytochemical and in-vivo anti-arthritic significance of Aloe thraskii Baker in combined therapy with methotrexate in adjuvant-induced arthritis in rats. Molecules. 2021; 26: 1-12. doi: https://dx.doi.org/10.3390/molecules26123660

41. Pearson CM, Wood FD. Studies of polyarthritis and other lesions in rats by injection of mycobacterial adjuvant. I. General Clinical and Pathological Characteristics and some Modifying Factors. Arthritis Rheumatology. 1959, 2: 440–45. doi: https://dx.doi.org/10.1084/jem.113.3.485

42. Bhujade A, Talmale S, Patil MB. In vivo studies on antiarthritic activity of Cissus quadrangularis against adjuvant induced arthritis. Journal of Clinical and Cellular Immunology. 2015; 6: 1-8. doi: https://dx.doi.org/10.4172/2155-9899.1000327

43. Wang S, Wang Y, Liu X, Guan L, Yu L, Zhang X. Anti-inflammatory and anti-arthritic effects of taraxasterol on adjuvant-induced arthritis in rats. Journal of Ethnopharmacology. 2016; 187: 42-48. doi: https://dx.doi.org/10.1016/j.jep.2016.04.031

44. Clayden EC. Practical section cutting and staining. 5th Ed. Edinburgh: Churchill Livingstone 1971: 239-250.

45. Boutet MA, Courties G, Nerviani A, Le GB, Apparailly F, Pitzalis C, Blanchard F. Novel insights into macrophage diversity in rheumatoid arthritis synovium. Autoimmunity Reviews. 2021: 102758. doi: https://dx.doi.org/10.1016/j.autrev.2021.102758

46. Cheng Q, Wu H, Du Y. The roles of small‐molecule inflammatory mediators in rheumatoid arthritis. Scandinavian Journal of Immunology. 2021; 93(3): e12982. doi: https://dx.doi.org/10.1111/sji.12982

47. Wong RSY. Disease-Modifying Effects of Long-Term and Continuous Use of Nonsteroidal Anti-Inflammatory Drugs (NSAIDs) in Spondyloarthritis. Advances in Pharmacological Sciences. 2019: 1-6. doi: https://dx.doi.org/10.1155/2019/5324170

48. Almalag HM, Almuhareb AM, Alsharfi A, Alhawassi TM, Alghamdi AA, Alarfaj H, Omair MA, Alomari BA, Alblowi MS, Abouzaid HH, Alarfaj AS. Relationship between different anti-rheumatic drug therapies and complementary and alternative medicine in patients with rheumatoid arthritis: an interview based cross-sectional study. Saudi Pharmaceutical Journal. 2021; 29(5): 462-466. doi: https://dx.doi.org/10.1016/j.jsps.2021.04.009

49. Sivakumar G, Sivakumar GA, Rebecca. Evaluation of anti-arthritic activity of methanolic extract of Barleria prionitis on CFA induced rats. Asian Journal of Pharmacy and Technology. 2019; 9(3): 159-164. doi: https://dx.doi.org/10.5958/2231-5713.2019.00027.8

50. Banji D, Pinnapureddy J, Banji OJ, Kumar AR, Reddy KN. Evaluation of the concomitant use of methotrexate and curcumin on Freund's complete adjuvant-induced arthritis and hematological indices in rats. Indian Journal of Pharmacology. 2011; 43(5): 546. doi: https://dx.doi.org/10.4103/0253-7613.84970

51. Kshirsagar AD, Panchal PV, Harle UN, Nanda RK, Shaikh HM. Anti-inflammatory and antiarthritic activity of anthraquinone derivatives in rodents. International Journal of Inflammation. 2014: 1-12. doi: https://dx.doi.org/10.1155/2014/690596

52. Alghadir A, Miraj M, Ali S. Efficacy of curcumin with iontophoretic application on paw edema and hematological responses in collagen-induced arthritis rat models. Evidence Based Complementary and Alternative Medicine. 2020: 1-11. doi: https://dx.doi.org/10.1155/2020/4606520

53. Obiri DD, Osafo N, Ayande PG, Antwi AO. Xylopia aethiopica (Annonaceae) fruit extract suppresses Freund׳ s adjuvant-induced arthritis in Sprague-Dawley rats. Journal of Ethnopharmacology. 2014; 152(3): 522-531. doi: https://dx.doi.org/10.1016/j.jep.2014.01.035

54. Alavala S, Nalban N, Sangaraju R, Kuncha M, Jerald MK, Kilari EK, et al. Anti-inflammatory effect of stevioside abates Freund’s complete adjuvant (FCA)-induced adjuvant arthritis in rats. Inflammopharmacology. 2020; 28(6): 1579-1597. doi: https://dx.doi.org/10.1007/s10787-020-00736-0.

55. Premrajan P, Jayananadan A, Suresh SC, Variyar EJ. Protective effects of phytocompounds from Alpinia calcarata (Haw.) Roscoe in Freund’s adjuvant induced arthritis in rats. Indian Journal of Expiremental Biology. 2021; 58: 186-193.

56. Patel D, Kaur G, Sawant MG, Deshmukh P. Herbal Medicine− A natural cure to arthritis. Indian Journal of Natural Products and Resources. 2013; 4(1): 27-33.

Received on 29.05.2022 Modified on 17.10.2022

Research J. Pharm. and Tech 2023; 16(9):4025-4031.

DOI: 10.52711/0974-360X.2023.00660