Development of quinone-rich fraction of Ardisia crispa (Thunb.) A.DC roots mitigates rheumatoid arthritis by suppressing angiogenesis, in vitro and in vivo

Angiogenesis, a process of new blood vessel formation from pre-existing ones, can perpetuate synovial inflammation and joint destruction at its equivocal balance in rheumatoid arthritis (RA). Targeting excessive angiogenesis underlies the early development of RA, a chronic autoimmune joint condition, has therefore become a promising approach for the disease intervention. Limitations of current arthritis therapies have encouraged for rapid development of alternative adjuncts from natural sources. 2-methoxy-6-undecyl-1,4-benzoquinone (BQ) is a pbenzoquinone derivative of Ardisia crispa roots (Family: Primulaceae), with proven anti-angiogenic properties. However, it is still unclear how BQ is able to inhibit angiogenesis in RA. Hence, this present study investigated the anti-arthritic potential of quinone-rich fraction (QRF) (a rich fraction containing the BQ) from Ardisia crispa roots, targeting angiogenesis inhibition in vitro and in vivo. Preparation of test samples from Ardisia crispa roots using n-hexane, followed by fractionation and isolation via column chromatography, yielded Ardisia crispa roots hexane extract (ACRH) [13.64 g; 24.0%, weight per weight (w/w)], QRF (1.99 g; 14.6%, w/w), and BQ (0.04 g; 1.9%, w/w), respectively. Subsequently, gas chromatography-mass spectrometry (GC-MS) analyses have confirmed the benzoquinonoid content in each sample [ACRH (6.1%, peak-area-percent), QRF (30.5%, peak-area-percent), and BQ (81.4%, peak-area-percent)]. Antiangiogenic and anti-arthritic activities of ACRH, QRF, and BQ were tested via cell viability MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, tube formation, cell invasion, and cell apoptosis assays in vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cells (HUVECs) and interleukin-1β (IL-1β)-induced human fibroblast-like synoviocytes for rheumatoid arthritis (HFLS-RA), respectively. ACRH, QRF, and BQ exhibited a narrow therapeutic range in VEGF-induced HUVECs (IC50=1.09±0.18 μg/mL, 3.85±0.26 μg/mL, and 1.34±0.16 μg/mL) and IL-1β-induced HFLS-RA (IC50=3.60±1.38 μg/mL, 4.47±0.34 μg/mL, and 1.09±0.09 μg/mL), respectively. ACRH, QRF, and BQ at 0.05, 0.5, and 5 μg/mL, respectively, significantly inhibited/suppressed (P0.05) reduced the arthritic scores, ankle swelling, and paw edema in arthritic rats after 13 days treatment, respectively. Additionally, at all doses, QRF also showed no significant (P>0.05) modulation on the body weights and organ weights, i.e., liver, kidney, and spleen, respectively. Protein expression analyses using enzyme-linked immunosorbent assay (ELISA) and multiplex assay demonstrated that QRF at all doses (3, 10, and 30 mg/kg) significantly (P<0.05) attenuated VEGF-A, PI3K, AKT, NF-κB, p38, STAT3, and STAT5 proteins level in arthritic rats in a dose-independent manner. QRF at all doses also significantly (P