INTRODUCTION:

RA is considered as an autoimmune disorder with systemic, chronic inflammation affecting many tissues but principally attacking the joints to produce a nonsupportive synovitis that frequently progress to destroy cartilage and underlying bone results disability with progression and severity of disease in long term onset. RA has many factors which includes inflammatory (Neutrophil, macrophages, IL-6 and TNF-α), Immune responses (generation of self antibodies, anti citrulline production and metabolic factors) as well as environmental factors and aging. In clinical situations, the medication prescribed for the management is a lifelong process and according to FDA and ELUAR the therapies existing for disease prevention and management contains immunosuppressive agents, corticosteroids and NSAIDs which are known for their unavoidable side effects¹.

In the present investigation, Momordica charantia seed belonging to Cucurbitaceae family also known as bitter gourd is used as preventive treatment for management of RA which is reported to have anti inflammatory, wound healing and immunomodulatory actions^2,3. Seed of Momordica chirantia contains p-Insulin, α and β glycoproteins (Momorcharins), charantin, charine, cryptoxanthin, cucurbitins, cucurbitacines, cytokinines and lectins and many amino acids⁴ of medicinal importance. Reverse pharmacological actions of aqueous extracts of Momordica charantia (MCAE) in collagen induced RA model in rats was used here which is a well accepted model for induction of RA with immunological intervention in mice, rat and non human primates^5,6 used here to bridge this traditional knowledge with the emerging biomedical sciences which is proved by the positive responses on inflammatory markers, immunological markers and also in disease symptomatology in terms of histopathological and radiological changes in treatment group.

MATERIALS AND METHODS:

Animals:

All experiments were carried out on healthy male wistar rats weighing 150 - 180gms, obtained from registered breeders and study was approved by Institutional Animal Ethics Committee (IAEC), Pharmacy Dept. Faculty of Pharmacy, The M. S. University of Baroda, vide protocol number MSU/IAEC/2018-19/1802 dated 29/12/2018. Guidelines of The Committee for Control and Supervision of Experiments on Animals (CCSEA) and The Prevention of Cruelty to Animals act (PCA), 1960, Department of Animal Husbandry and Dairying, Ministry of Fisheries Animal Husbandry and Dairying Government of India (DAHDMoFAH&D) were followed for experimentations. Animals were housed in well-controlled conditions of temperature (22±2°C), humidity (55±5%) and 12hrs/12hrs light-dark cycle maintained according to the guidelines. The animals had free access to conventional laboratory diet (VRK nutritional solutions, Vadodara, Gujarat, India) with purified water ad libitum.

Chemicals and kits:

Bovine Collagen (Type II) was purchased from Chondrex Inc Redmond, WA supplied by Krishgen Biosystem Mumbai. Complete Freund’s Adjuvant (CFA- each ml contains 1mg heat killed and dried Mycobacterium tuberculosis in 0.85ml paraffin oil and 0.15 ml mannide monooleate) was purchased from Sigma-Aldrich, Bangalore, India. Methotrexate was obtained as a gift sample from Alembic pharmaceuticals, Vadodara, Gujarat.

Kits for estimation:

Estimation kits of C - reactive protein (C-RP) and Rheumatoid factor were obtained from ADI enterprises, Vadodara. ELISA kits for estimation of TNF-α, Interleukin-6 were purchased from Krishgen biosystems Mumbai. X-rays of rats were done at Dr. Angela Lobo's Veterinary Clinic, Kirti mandir compound, Tilak Road, opp. SSG Hospital Vadodara, Gujarat. Histopathological investigations were performed by Sakshi histopathology lab, Vadodara, Gujarat.

Plant extract:

Momordica charantia dry seeds were procured from vegetable field Khargone, M.P. were washed thoroughly and dried seeds were authenticated by Dr. Padmanabhi S. Nagar assistant professor department of Botany at The Maharaja Sayajirao University of Baroda, Vadodara with Ref: Bot/30317/aut/1/TD04. Simple maceration technique was used to extract out all the components of powdered seed in aqueous extract for 10-12 hrs at room temperature with occasional shaking and filtrate was taken out by muslin cloth (cheese cloth) after 12 hrs. The filtrate was evaporated to get dry extract and labeled as MCAE (Momordica charantia aqueous extract). It was stored at 2-8°C till further use. At the time of dosing the extract was taken out from the storage and brought to the room temperature for preparing the fresh dose to be given.

Methods:

1. Experimental design for induction of Rheumatoid Arthritis:

Animals were randomized according to body weight into six group (n=6) as follows-

Group I- Normal control group (animal of this group were not sensitized with any inducing agent throughout the study period). Here in this study collagen induced RA protocol was designed for 42 days and group II, III, IV and V and VI were sensitized with collagen 0.1ml (CFA1: CIA1). The induction was done by Bovine collagen type II which was premixed with Complete Freund’s Adjuvant before injecting in animals in 1:1 ratio to get a stable emulsion (tested by uniform droplet formation in cold water) and 0.1ml of this prepared emulsion was injected by sub planter route on day 0 for induction of RA in each animal once only in the study⁷.

Group III -Standard treatment group (MTX was administered in 0.6mg/kg/ week dose⁸ by i. p. route from day 1 after receiving collagen 0.1ml on day 0 and continued till day 42). Group IV- Test I (was treated with MCAE 100mg/kg dose by oral route as preventive treatment from day 1 to 42). Group V- Test II (was treated with preventive doses of MCAE 200mg/kg p.o. as preventive treatment from day 1 to 42).Group VI - Test III (was treated with preventive doses of MCAE 400mg/kg p.o. as preventive treatment from day 1 to 42).

2. Estimation of inflammatory and immune components:

a) Paw volume⁹

Paw volume measurement was done on day 1, 3, 5, 7, 11, 14, 17, 21, 28, 35 and 42 for estimation of the inflammation and disease progression with the help of Digital Plethysmometer purchased from Laboratory Enterprises Nashik, Maharashtra.

b) Arthritic Score⁹ and Arthritic index:

Arthritic scoring and Arthritic index were done on day 5, 21 on 0-4 scale and 0-3 scales of scoring given by blind observers who were unaware about the group’s identity. Methods were adopted by H. Gerhard (drug discovery and evaluation pharmacological assay) and procedures given by by Singh JA et. al., and Mondal P. et al.

3. Estimation of CRP, ESR, IL-6, TNF-α, Anti-CCP:

Estimation of biochemical parameters was done in blood samples collected from animals by retro orbital plexus with mild anaesthesia using pentobarbital 60mg/kg⁹ and the samples were centrifuged (CPR 30, REMI Centrifuge, India) at 4,500 RPM at 4⁰C for 10 min. these samples were utilized to estimate CRP (1, 7, 14, 21, 28, 35 and 42days) IL-, TNF-α and Anti-CCP (day 42). ESR was measured using western green tubes on day 1, 7, 14, 21, 28, 35, 42 using 2% sodium citrate solution as anticoagulant in collection tubes.

4. Estimation of Disease progression index (Disability, Radiological and histopathological examinations):

X-ray of left hind paw and fore paw/ right hind paw progressed the symmetric disease were taken with mild anaesthesia for assessment and confirmation of edema and bone disruption. After study period completion on day 42, animals were euthanized humanly with overdose of anaesthetic agent and left hind paw was taken out as a specimen sample and immediately bone and secondary lesion portions were fixed in 10% buffered neutral formalin solution and sent for histopathological examination.

Statistical analysis:

All the values are expressed as Mean ± SEM and statistically compared using GraphPad Prism 5 (San Diego, CA, USA) by applying repeated measure ANOVA for weakly analyzed data and one way ANOVA analysis was also done for final estimation. Significant difference between groups was analyzed using multiple comparisons via post hoc test (Bonferroni’s, Dunnett’s and Tukey’s). In tests *P<0.05, **p<0.01, *** P<0.001 *, **, *** were taken significant for comparison between normal and all groups. #, ##, ### denotes comparison between the std. and treatment groups. &, &&, &&& is considered for comparison between model control and other groups. A non-significant difference between normal and other groups was marked as ns.

RESULTS:

Results of inflammatory components (Paw volume measurement, Arthritic score (Scoring) and Arthritic index):

Effects of MCAE on Paw volume in collagen induced RA in rats:

Paw volume in all groups except normal control group was increased gradually. Model control group showed the increased inflammation and primary secondary lesions which is indicator of disease severity and inflammation. After day 7 the treatment control groups showed the significant decrease in paw volume where MCAE in 400mg/kg dose showed maximum effect on edema and inflammation as compared to model and std. control (figure1).

Figure 1: Effects of MCAE on Paw volume in collagen induced RA in rats

Values are expressed as Mean ± SEM. Statistically evaluated using one way ANOVA analysis. Significant values were compared with (*P<0.05, **p<0.01, *** P<0.001) *, **, *** (comparison between normal and all groups), #, ##, ### (comparison between the std. and treatment groups), &, &&, &&& (comparison between model control and other groups) ns is non-significant differences between normal and other groups.

Effects of MCAE on Arthritic Score in collagen induced RA in rats:

Preventive treatment of MCAE against collagen induced RA showed the significant difference from day 5 and 21. On day 5 the model control group showed the primary and secondary lesions, wounds and severe inflammation. Primary lesions of standard control were disappeared on day 21 but the inflammation was significantly high. In treatment control groups primary lesions were seen which were less severe than model control groups and these lesions were healed on day 21. When comparison was done between three doses; MCAE 100mg/kg, 200mg/kg and 400mg/kg, MCAE 400mg/kg dose showed significant decrease in arthritic score (figure 2).

Figure 2: Effects of MCAE on Arthritic Score in collagen induced RA in rats

Effects of MCAE on Arthritic Index in collagen induced RA in rats:

Arthritic index is a cumulative data for involvement of joints participating in disease progression and the other body parts expressing the symptoms as nodule formation and inflammation. In this study the animals treated with MCAE 400mg/kg showed the maximum effects on these symptoms and give the protective effects as compared to model control group (figure 3).

Figure 3: Effects of MCAE on Arthritic Index in collagen induced RA in rats

Effects of MCAE on biochemical indicators:

Markers of inflammation and immunological components like CRP, ESR, IL-6, TNF-α and Anti-CCP were also significantly increased in Model control group (CFA1:collagen1) animals. This rise was significantly prevented by Momordica charantia aqueous extract and highest effect was observed in MCAE 400mg/kg treatment control group as described in Table 1.

Table 1: Values expressed for inflammatory markers as Mean± SEM

Group

Inflammatory markers

Immunological markers

CRP

ESR

Anti-CCP

TNF-α

IL-6

Normal control

2.2±

0.34

4.67±

0.005

2.3±

0.15

175±

28.8

2000±

590

Model control

5.2±

0.53***

12±

1.4***

9±

0.54***

217±

125.8***

6000±

732***

Std. control

4.3±

0.34***

8.3±

0.73***

6.5±

0.72***

459±

43.15***

4250±

832***

Test I

3.1±

0.36

5.4±

0.30

2.9±

0.23

328±

74.2

916±

46.3

Test II

3.8±

0.34

6.5±

0.80

3.4±

0.37

363±

19.4

2458±

181

Test III

2.11±

0.08

4.7±

0.33

2.3±

0.12

178±

29.05

3125±

544

Effects of MCAE on disease progression index (Disability, Radiological and histopathological examinations):

Disability was checked by walking ability of animals and the wounds and inflammation as depicted in figure 4. photographic assessment, bone erosion and deformities were also seen in radiography and histopathology suggests that model control groups were highly affected with inducing agents and disease severity was clarified in tissues, bone and cellular components of animal which was prevented in MCAE treated groups and MCAE 400mg/kg dose was significantly prevented all these parameters of disease progression.

DISCUSSION:

In preclinical as well as clinical scenario RA is a disease which has the immunological and metabolic components associated with chronic inflammatory factors involved in pathophysiology¹⁰.

Complete Freud’s Adjuvant (CFA) is used for induction of RA, it follows the inflammatory pathway activation due to involvement of Mycobacterium tuberculosis responsible cytokine release and this event is further maintained by HSP-65 (Heat Shock Protein 65) activation^11,
12.

On the other hand when Bovine collagen type II is incorporated with CFA it follows the immunological cross linkage with the inflammatory responses in the form of anti collagen antibodies, severe inflammation in the form of secondary lesions and disability of walking.

Edema is an abnormal accumulation of fluid in the interstitial, located beneath the skin and in the cavities of the body, which can cause severe pain; which leads to swelling of joints. Paw volume, arthritic index and arthritic score are the major criteria in experimental investigations performed in preclinical studies of RA, which can give an insight about symptoms generating as a visual events due to edema and inflammation at the site of induction, primary and secondary lesions, involvement of other parts of body made up with cartilaginous substances¹³.

CRP and ESR are biomarkers selected for hematological estimations which can correlates the symptomatology with disease pathophysiology due to internal changes in body.

Anti-CCP (ACCP/ACPA) is one of the important criteria’s in the RA, as this is the major factor to identify that whether the progressed disease is osteoarthritis or RA. In this study model control group showed the higher levels of ACCP which proved that collagen has induced RA in animals. As ACCP is also associated with the metabolic dysbiosis, environmental factors and auto antibody generates due to immunological disbalance¹⁴. In clinical investigations also ACCP works as a self generated auto antibodies generated due to peptide citrullination.

IL-6 and TNF-α also shows the immunological intervention in disease generation and these are also confirmatory markers for initiation of inflammasomes in RA which confirms the cell infiltration and synovial damage via activation during Pannus formation^{15, 16}.

Photography of paws of animals was done to check the progression of disease and for comparison with other groups for primary and secondary lesions. X-ray (Radiography) in this study was taken to confirm inflammation, bone erosion and destruction. The x- rays of tarsotibial joints of collagen induced paw were taken and there was bone erosion, edema and soft tissue inflammation was clearly seen in model control group which suggests the pathogenesis of RA and disease severity which leads deformity and disability in animals.

CONCLUSIONS:

Aqueous extract of Momordica charantia seed in 400mg/kg accorded significant protection against both immunological and inflammatory components of RA and the results were proven better than golden standard treatment of RA Methotrexate.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENT:

The research reported in this manuscript has been funded by Government of India, Ministry of Science and Technology, Department of Science and Technology (WISE KIRAN DIVISION) with sanction order no. SR/WOS-A/LS-195/2018.

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Received on 18.07.2023 Modified on 16.11.2023

Research J. Pharm. and Tech. 2024; 17(3):1364-1369.

DOI: 10.52711/0974-360X.2024.00215