Antiangiogenic and Anticancer Potential of Supercritical Fluid Extracts from Nutmeg Seeds; In vitro, Ex vivo and In silico studies

Sawsan S. Al-Rawi^1*, Ahmad Hamdy Ibrahim², Marthad A. Hamde³, Dinesh Babu⁴, Mansoureh Nazari⁵, Mohd Omar Ab Kadir⁶, Aman Shah Abdul Majid⁷, Amin Malik Shah⁸

Abstract

Background: Angiogenesis is a vital process of forming new blood vessels that occurs during several normal physiological processes. It plays a substantial role in tumor growth and cancer by supplying oxygen and nutrients for the proliferating tumors. Nutmeg, the dried seed of Myristica fragrans, is known for its therapeutic properties. This study aimed to investigate the antiangiogenic and cytotoxic properties of nutmeg extracts derived by Soxhlet and supercritical fluid extraction (SFE) using in-vitro, ex-vivo, and in-silico studies. Method: Nutmeg anticancer property was evaluated against breast cancer cell MCF7 and colon cancer cell HCT116 using MTT in-vitro assay. The antiangiogenic property was investigated using 3D ex-vivo rat aorta assays. The chemical composition of nutmeg extract was characterized using GC/TOF-MS. Subsequently, the main compounds in the nutmeg extract were analyzed against the angiogenesis-associated molecules (COX-1, VEGFA, HIF, and EGF) by molecular docking and were compared with tamoxifen and 5-fluorouracil. Results: The SFE extracts exhibited higher antiangiogenic properties than the Soxhlet (IC50 31µg/mL). Nutmeg SFE extract exhibited higher cytotoxicity towards HCT116 than MCF7 cells. Several active compounds, including myristicin, eugenol, safrole, and α-asarone were identified in the nutmeg SFE extracts using GC/TOF-MS. Molecular docking revealed strong interactions between these compounds and angiogenesis molecular mediators. Particularly, myristicin blocked COX-1, VEGFA, HIF, and EGF enzymes, indicating possible binding interactions. Conclusion: This study highlights the limitless possibilities of nature's offerings in advancing human health. Nutmeg's potential compounds lie in their binding models, which have proven to be powerful tools against cancer and angiogenesis. Myristicin, α-asarone, safrole, and eugenol work synergistically to induce antiangiogenic effects, making nutmeg a promising natural source of angiogenesis inhibitors for future anticancer therapies. The Molecular docking result confirmed that the inhibition of COX-1, VEGF-A, HIF, and EGF by nutmeg compounds offers great potential in the treatment and prevention of various angiogenesis diseases.

Keywords: Angiogenesis; COX-1; VEGF; HIF; EGF; Cytotoxicity; Cancer; MCF7; HCT116; GC-TOF/ MS, and Nutmeg.

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