Cytotoxic activity of Indigofera aspalathoides (Vahl.) extracts in cervical cancer (HeLa) cells: Ascorbic acid adjuvant treatment enhances the activity

Abstract

ABSTRACT: Background: The folkloric use of Indigofera aspalathoides (Vahl) in anti-inflammatory and anti-cancer treatment dates back to circa 300 BC. However, reports on scientific validation for folkloric usage are not available yet. Purpose: The objective of the study is to assess the cytotoxic potential of I. aspalathoides and to provide a rationale for its ethnopharmacological use with lime juice (ascorbic acid) as an adjuvant. Methods: Using Soxhlet, the active principles of I. aspalathoides were extracted using methanol and ethanol as solvent and studied using gas chromatography coupled with mass spectrometry (GC–MS). We assessed the in vitro antioxidant efficacy by studying the DPPH radical scavenging activity, ferrous reducing antioxidant capacity (FRAC), and ABTS cation radical scavenging activity. Using MTT assay, the cytotoxic activity of SV-ME and EE against human cervical cancer (HeLa) and normal murine fibroblast (L929) cells were evaluated. The cytotoxicity and apoptosis induction was studied by dual acridine orange/ethidium bromide (AO/EB) fluorescent probe and Hoechst dye. Mitochondria membrane potential (Δψm) was assessed using JC1. Ascorbic acid (AA) additive/synergism was studied by co-treating HeLa cells with AA along with SV-ME and SV-EE at IC50 concentration. Results: SV-ME and EE exert higher DPPH radical scavenging at 200 μg/mL. Both the extracts showed effective ABTS radical cation scavenging activity and ferrous reducing antioxidant capacity. Further, SV-ME and SV-EE treatment affected the cell viability and induced apoptosis, reduced cell viability with a concomitant increase in nuclear condensation and DNA fragmentation in a dose-dependent manner in cancer (HeLa) cells, but not in normal (L929) cells. Both the extracts increased the mitochondria membrane potential (Δψm) depolarization and altered the cellular redox state in HeLa cells. In addition, adjuvant ascorbic acid (AA) treatment increased the cytotoxic efficacy of the SV-ME and SV-EE in HeLa cells. Conclusion: I. aspalathoides used in the Siddha medicine for antitumor treatment have a very potent cytotoxic effect against cervical cancer cells (HeLa) in vitro. The cytotoxic and antitumor effect may have plausible involvement of Nrf2-ARE signaling pathway by restoration of cellular redox homeostasis.

Keywords

• Indigofera aspalathoides
• Sivanar Vembu
• Methanol extract
• Ethanol extract
• Anticancer
• Apoptosis