Nano-Encapsulated Volatile Oil of Ruta Graveolens Leaves Inhibited Glucocorticoid-Induced Osteoporosis in Zebrafish Larval Model

Abstract

Background: The present research aims to screen anti-resorptive activity to alleviate osteoporosis and other bone and joint disease of the Nano encapsulated essential oil isolated from the Ruta graveolens (NELVORG), Family: Rutaceae using a novel, fast, economical and genetically tractable method to evaluate 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. Nano-encapsulated volatile oil of R. graveolens leaves (NELVORG) was prepared by solvent displacement method. The Nanoparticle size was standardized by scanning electron microscope. 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. At 3 days post fertilization (3 d.p.f) zebrafish larvae (n=6 / group) were taken in 96 well plates containing embryo medium. Osteoporosis (OP) was induced by immersing zebrafish larvae from 3dpf to 6dpf in embryo medium containing 10µg/ml prednisolone. At 6 d.p.f, the E3 medium containing prednisolone was removed and replaced with the combination of 10µg/ml prednisolone and NELVORG in triplicate till 9 d.p.f was taken as a test group. Alendronate (10µg/ml) and (0.1%) DMSO was taken as a standard and control group respectively. At 9 d.p.f zebrafish larvae were collected and subjected to alizarin red staining for the labelling of the skeleton and the area of stained portion were analyzed using image pro plus analysis to calculate the density of staining. Results: The result showed NELVORG significantly prevented Glucocorticoid-induced bone loss and density and comparable to the standard drug Alendronate in zebrafish larval (ZF) 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.