ANABOLIC EFFECT ON BONE FORMATION OF NANO ENCAPSULATED VOLATILE OIL OF RUTA GRAVEOLENS LEAVES IN LARVAL ZEBRAFISH MODELAbstract
Aim: The present research aims to screen anabolic activity on bone formation and bone density in normal and Teriparatide (Rh PTH biosynthetic human parathyroid hormone 1-34) induced inhibition of osteogenesis in zebrafish larval model to alleviate osteoporosis and other bone and joint disease or disorders of the Nano encapsulated essential oil isolated from the Ruta graveolens (NELVORG) Family: Rutaceae using a novel, fast, economical and genetically tractable method for screening developmental aspects of bone formation in a high throughput fashion through the visualization of embryonic and larval skeleton of zebrafish (Danio rerio) model. Method: The volatile oil (VO) was isolated from the leaves of R. graveolens and extracted VO was subjected to GC-MS Analysis. The nano-encapsulated volatile oil of R. graveolens leaves (NELVORG) was prepared by solvent displacement method, and particle size was standardized by scanning electron microscope (SEM). We implemented 3Rs (Reduction, Replacement, Refinement) ethical principle to minimize harm to the vertebrate animals. Preliminary toxicological studies were evaluated on whole embryo and larvae, showed no mortality up to 1µl/ml. Zebrafish larvae (n=6) at 3dpf were taken in 24 well plates containing embryo medium. 30ng/ml teriparatide was made in 20mM sodium dihydrogen phosphate, 0.9% sodium chloride, and 2.13% g/l mannitol were added to the well from 3 d.p.f to 6 d.p.f to induce catabolic effects in bones. At 6 d.p.f embryo medium containing 30ng/ml Teriparatide were removed and replaced with the combination of 30ng/ml teriparatide + NELVORG in triplicates till 9 d.p.f.10µg/ml of Alendronate used as a standard drug and 0.1% DMSO was a vehicle control. Zebrafish larvae were collected from the well at 9dpf and subjected to alizarin red staining for quantification and area of stained portion were analysed using image pro plus to calculate the density of staining. Results: The result showed NELVORG significantly protected the catabolic effect of teriparatide and rescue bone loss and density and comparable to the standard drug alendronate in zebrafish larval model in-vivo. So, Nano encapsulated volatile oil of the leaf of R. graveolens may be developed as a novel nontoxic potential candidate for the prevention or treatment of osteoporosis.