Antioxidant, Antiproliferative, Pro-apoptotic and cell cycle arrest properties of crude extract and biofractions of Hybanthus enneaspermus Linn. to combat breast cancer

Abstract

Objective: According to the World Health Organisation, breast cancer is presently the most common cancer diagnosed in women globally. Polyphenolic compounds act as antioxidants, improve health, and reduce risk and proliferation of various types of cancers. Hybanthus enneaspermus Linn. is a beneficial medicinal plant, reported to possess antimicrobial, antioxidant, anti-inflammatory, neuroprotective, cardioprotective, and nephroprotective properties etc. Methods: The current study involved the evaluation the antioxidant, antiproliferative, apoptotic and cell cycle arrest potential of the ethanolic leaf extract of Hybathus enneaspermus Linn. (EEHE), its toluene soluble, toluene insoluble, ethyl acetate and methanol soluble biofractions viz. TFHE, ITHE, EFHE, and MFHE to combat breast cancer. In vitro antioxidant activities were evaluated using DPPH, Hydrogen peroxide, Nitric oxide and ABTS free radical scavenging assays. In vitro antiproliferative activity against MCF-7 cells was assayed using the Sulforhodamine method, while apoptosis and cell cycle assays were analysed by flow cytometry. Results: MFHE exhibited significant in vitro antioxidant activity with IC50 values of 21.10±0.39 μg/mL and 25.99±4.66μg/mL, when compared against standard ascorbic acid with IC50 values of 11.19±1.09 μg/mL and 9.30±0.26μg/mL in DPPH and nitric oxide assays respectively. EFHE displayed substantial antioxidant potential in ABTS and hydrogen peroxide assays with IC50 values of 40.38±0.88μg/mL and 99.11± 13.59μg/mL, while ITHE showed considerable activity with IC50 < 100μg/mL in DPPH, nitric oxide and ABTS assays. TFHE demonstrated significant antiproliferative activity by sulforhodamine assay, with GI50 value of 10.22 6.72µg/mL, while EEHE and ITHE showed substantial activity with GI50 values of 41.42±3.74µg/mL and 64.37±7.07µg/mL respectively, as against the standard drug Adriamycin (GI50 < 10µg/mL) used. In the apoptosis assay, ITHE showed 11.31±0.82% cells in late apoptosis and 34.48±1.57 % cells in necrosis as compared to standard Adriamycin indicated 13.67±1.02 % cells in late apoptosis and 8.58±0.65 % cells in necrosis. In cell cycle analysis, ITHE displayed significant apoptotic activity with 20.15±1.37 % cells in SubG1 phase and 13.99±1.65 % cells arrested in G2-M phase as compared to the control. Conclusion: The study thus revealed that MFHE, EFHE and ITHE biofractions showed significant antioxidant activities, while EEHE, TFHE and ITHE exhibited substantial antiproliferative activity against mammary cancer cells. Additionally, ITHE induced remarkable apoptotic activity and cell cycle arrest in the MCF-7 cells. The therapeutic benefits may be credited to the bioactive constituents present in the ITHE fraction viz. polyphenolics, flavonoids etc.; however, the molecular mechanisms may need to be evaluated further.