Ribes orientale: A novel therapeutic approach targeting rheumatoid arthritis with reference to pro-inflammatory cytokines, inflammatory enzymes and anti-inflammatory cytokines.

Abstract

ETHNOPHARMACOLOGICAL RELEVANCE\nThe roots of Ribes orientale (Family Grossulariaceae) have long been used as a folk remedy to treat rheumatism and joints pain in Northern Areas of Pakistan.\n\n\nAIM OF THE STUDY\nThe purpose of study was to observe the preventive efficacy of roots of Ribes orientale (RO) aqueous ethanolic extract (30:70) and its aqueous and n-butanol fractions in treating rheumatoid arthritis and to determine its possible mechanism of action.\n\n\nMATERIAL AND METHODS\nArthritis was evaluated in vitro using heat induced bovine serum albumin and egg albumin denaturation and membrane stabilizing assays at 50-6400\u202fμg/ml concentration of extract/fractions whereas, in vivo arthritis was evaluated at 50, 100, 200\u202fmg/kg doses of extract/fractions in formaldehyde model by measuring rat paw volume/diameter. Moreover, highest effective dose (200\u202fmg/kg) of extract/fractions was evaluated in Freünd complete adjuvant (FCA) model. Arthritis was induced in Sprague Dawley rats by immunization with 0.1\u202fml FCA in left footpad. RO extract/fractions at 200\u202fmg/kg were orally administered from day 0, 30\u202fmin prior to adjuvant injection and sustained for 28 days. Paw volume/diameter, arthritic score, body weight, and hematological (WBC, RBC, ESR, Hb and Platelet count) and biochemical (AST, ALT, ALP, urea, creatinine, CRP and RF) parameters were observed. The mRNA expression levels of COX-2, IL-1β, IL-6, NF-kB, TNF-α, IL-4 and IL-10 were measured by real time reverse transcription polymerase chain reaction (RT-PCR) whereas, PGE2 and TNF-α levels in serum samples were measured by Enzyme linked immunosorbent assay (ELISA). Furthermore, radiographs of hind paws and histological changes in ankle joint were analyzed in adjuvant injected rats. The anti-oxidant activity of plant extract and fractions was evaluated using DPPH and reducing power assays. In addition, phytochemistry, total phenolic and flavonoid contents, and HPLC analysis of most active fraction (aqueous fraction) were performed.\n\n\nRESULTS\nResults showed that RO extract and fractions (notably aqueous fraction) significantly reduced protein denaturation and protected erythrocyte membrane in concentration dependent manner. Similarly, extract/fractions induced dose-dependent decrease in paw volume/diameter in the formaldehyde model. Plant extract and fractions significantly suppressed paw swelling and arthritic score, prevented cachexia and remarkably ameliorated hematological and biochemical changes. Furthermore, RO extract/fractions downregulated gene expression levels of PGE2, COX-2, IL-1β, IL-6, NF-kB and TNF-α whereas, upregulated those of IL-4 and IL-10, compared with FCA control rats. The radiographic and histopathologic improvement in joint architecture was also observed in RO treated rats. Piroxicam, used as reference drug, also significantly suppressed arthritis. Additionally, plant exhibited notable anti-oxidant activity and phytochemical analysis revealed the presence of flavonoids and polyphenols.\n\n\nCONCLUSION\nResults indicated that suppression of pro-inflammatory enzymes/cytokines, inhibition of protein denaturation, lysosomal membrane stabilizing abilities, and redox/free radical scavenging properties of RO extract and fractions support anti-arthritic and immunomodulatory property of Ribes orientale that might be due to its polyphenolic and flavonoid constituents. This suggests that Ribes orientale roots may be used as a therapeutic agent for treating human arthritis.