Investigating the impact of Terminalia chebula, an underutilized functional fruit, on oral squamous cell carcinoma: Exploring cell death mechanisms

Abstract

Ethnopharmalogical relevance: Terminalia chebula, known for its extensive use in traditional medicinal practices among indigenous cultures, is recognized for its effectiveness in treating various oral disorders. Healers in India and China utilize the ripe fruits of T. chebula to prevent and manage conditions such as dental cavities, gingivitis, bleeding gums and stomatitis. The fruits have also been traditionally used in Ayurvedic and Siddha medicines for treatment of various diseases including anticancer properties. It is also an important component of Tibetan traditional medicine used for the treatment of cancer. Studies have demonstrated the efficacy of T. chebula against lung and colon carcinoma.

Aim of the study: Despite its historical significance in oral health, the potential of T. chebula against oral cancer has not been explored, warranting further investigation into its bioactive properties. This study aims to explore the therapeutic potential of the hydroalcoholic extract of Terminalia chebula fruits and its fractions against oral squamous cell carcinoma (OSCC) using SCC9 cells focusing on their cytotoxicity, anti-proliferative effect and the synergistic action of its ethyl-acetate fraction with cisplatin (CP). Additionally it seeks to identify the bioactive phytoconstituents in EAF were identified using LC-ESI-QTOF-MS.

Materials and methods: Antioxidant activity of TYH and its fraction were assessed using DPPH and ABTS assays. Total phenolic (TPC) and total flavonoid content (TFC) were quantified via Folin-ciocalteau and alluminium chloride assays respectively. Cytotoxic and antiproliferative effects were assessed using MTT assay, clonogenic assay and cell migration assay. Apoptosis in EAF treated SCC9 cells was analysed by using DAPI, Giemsa staining and flow cytometry using Annexin V-FITC/PI apoptosis detection kit. Intracellular reactive oxygen species (ROS) was assessed using H₂DCFDA, western blotting examined expression of apoptosis related proteins in SCC9 cells. Combinational effect of EAF with cisplatin (CP) was also assessed and phytochemical constituents of EAF were analysed using LC-ESI-QTOF-MS.

Results: The ethyl acetate fraction (EAF) showed the highest antioxidant activity (IC50 value of 8.16 ± 0.59 μg/mL and 4.99 ± 0.82 μg/mL in DPPH and ABTS assays respectively) which reciprocated with a high TPC and TFC (528.46 ± 2.59 mgGAE/g and 49.10 ± 1.61 mgQE/g dry weight of the extract respectively) content. EAF significantly reduced cell viability with an IC₅₀ value of 86.73 ± 0.55 μg/mL, resulted in dose dependent cell death, and prevented the proliferation and migration in SCC9 cells. Further Annexin V-PI based flow cytometric analysis and caspase-3/7 enzyme activity assay confirmed the apoptotic effect of EAF in SCC9 cells. Intrinsic pathway of apoptosis post treatment with EAF was confirmed by western blotting with marker proteins, Bax, Bcl-2, Mcl-1, cleaved caspase, procaspase and PARP. A combinatorial study of EAF with the standard drug cisplatin also indicated a synergistic effect of the fraction in cisplatin treated cells with a CI value of 0.67571. LC-ESI-QTOF-MS led to identification of the presence of phenolics and gallotannins with anticancer properties in EAF.

Conclusion: This study demonstrates the potential of the hydroalcoholic extract of Terminalia chebula fruits (TYH), especially its ethyl acetate fraction (EAF), as a therapeutic agent against oral squamous cell carcinoma (OSCC).

Keywords: Anti-cancer; Anti-oxidant; Apoptosis; Ethyl acetate fraction (EAF); Oral squamous cell carcinoma (OSCC); Terminalia chebula.