In Vitro and In vivo approaches to evaluate Uncaria tomentosa bark extract loaded FDOFs on Osteoarthritis models

Abstract

Osteoarthritis is one the leading health concerns worldwide affecting two third million with no proper treatment ensured to restore the normal function and completely relieving the joint pain. Oral fast dissolving films have promising action and targeted delivery with high drug loading capacity. The present investigation involves the study the invitro and invivo activity of developed Oral fast dissolving films of U. tomentosa bark extract with optimised F5 and F13 formulations. For invitro evaluation a three dimensional OA model was prepared with first passage chrondrocytes grown on trypsin EDTA media in 1: 3 ratio. The OA agarose model was prepared with C20A4 chondrocytes on agarose gel (25 ± 5oC) in phospahate buffer solution. Cultivation of chrondrocytes was done with 1 mL of RPMI-1640 (10% FBS) which was added with 20% (IL-1β) solution on third day of incubation and media was replaced time to time. The incubated cell line with 20,000 cells/well in 96-well plates were treated with 5 µL of 0.5% MTT reagent on fifth day of incubation and absorbance was measured at 570 nm. The effects were studied for 7, 13, 27, 35 days for the study effects of FDOFs on the cell lines were (Control, IL-1β, F5, and F13 treated IL-1β injected types). The chondrocytes in agarose constructs cultured only in media (RPMI-FBS) without IL-1b, served as control. The GAG, HYP and DNA quantitation analyses along with DNA content assay were performed to study the arthritic effect of optimized FDOF’s i.e F5. For invivo studies Monoiodoacetate (MIA) induced arthritis models which is well established to understand weight bearing and response to tactile stimuli though the ongoing procedure is not known. The invivo protocol was performed in seven week old male wistar rats with negative control of MIA and positive control as Celecoxib. The assessment of pain and thickness of the knee were estimated to be indicators of osteoarthritic potential. The study results revealed the F5 formulation has efficacy on the OA models which need a clinical investigation in humans.