Integrated in Vitro, in Silico, and GC MS Evaluation of Annona Muricata Leaf Extract for Osteoporosis Therapy

Abstract

Background: Osteoporosis is a progressive skeletal disorder characterized by reduced bone mass and microarchitectural deterioration, leading to fragility fractures. Current pharmacological interventions are limited by adverse effects and incomplete modulation of bone remodeling. Plant derived bioactives offer a promising alternative due to their multitargeted mechanisms and favorable safety profiles.
Methods: The ethanolic leaf extract of Annona muricata Linn. (AMLE) was subjected to GC MS analysis to identify phytoconstituents. In silico docking and ADMET profiling were performed to evaluate binding affinities and pharmacokinetic properties of identified compounds against bone related proteins, including RANKL. In vitro assays employed MC3T3 E1 osteoblasts and RAW 264.7 osteoclasts. Cell viability was assessed by MTT assay, osteoblast differentiation by alkaline phosphatase (ALP) activity, and osteoclastogenesis by tartrate resistant acid phosphatase (TRAP) staining.
Results: GC MS profiling revealed bioactive molecules such as neophytadiene, tocopherol acetate, and ergostadienol. Docking studies demonstrated strong interactions with RANKL and other bone remodeling proteins, with binding scores comparable to reference ligands. In vitro, AMLE maintained >90% viability up to 50 μg/mL. ALP activity was enhanced two fold, indicating osteoblast stimulation, while TRAP positive osteoclasts were reduced by ~60%, confirming anti resorptive activity.
Conclusion: The integrated GC MS, in silico, and in vitro evidence highlights AMLE as a dual modulator of bone remodeling, capable of promoting osteogenesis while inhibiting resorption. These findings position Annona muricata as a promising candidate for plant based osteoporosis therapy, warranting further preclinical and clinical validation.