Anticancer properties and metabolomic profiling of Shorea roxburghii extracts toward gastrointestinal cancer cell lines

Abstract

Background: Gastrointestinal cancer (GIC) ranks as the highest cause of cancer-related deaths globally. GIC patients are often diagnosed at advanced stages, limiting effective treatment options. Chemotherapy, the common GIC recommendation, has significant disadvantages such as toxicity and adverse effects. Natural products contain substances with diverse pharmacological characteristics that promise for use in cancer therapeutics. In this study, the flower of renowned Asian medicinal plant, Shorea roxburghii was collected and extracted to investigate its phytochemical contents, antioxidant, and anticancer properties on GIC cells.

Methods: The phytochemical contents of Shorea roxburghii extract were assessed using suitable methods. Phenolic content was determined through the Folin-Ciocalteu method, while flavonoids were quantified using the aluminum chloride (AlCl₃) method. Antioxidant activity was evaluated using the FRAP and DPPH assays. Cytotoxicity was assessed in GIC cell lines via the MTT assay. Additionally, intracellular ROS levels and apoptosis were examined through flow cytometry techniques. The correlation between GIC cell viability and phytochemicals, ¹H-NMR analysis was conducted.

Results: Among the four different solvent extracts, ethyl acetate extract had the highest phenolic and flavonoid contents. Water extract exhibited the strongest reducing power and DPPH scavenging activity following by ethyl acetate. Interestingly, ethyl acetate extract demonstrated the highest inhibitory activity against three GIC cell lines (KKU-213B, HepG2, AGS) with IC₅₀ values of 91.60 µg/ml, 39.38 µg/ml, and 35.59 µg/ml, while showing less toxicity to normal fibroblast cells. Ethyl acetate extract induced reactive oxygen species and apoptosis in GIC cell lines by downregulating anti-apoptotic protein Bcl-2. Metabolic profiling-based screening revealed a positive association between reduced GIC cell viability and phytochemicals like cinnamic acid and its derivatives, ferulic acid and coumaric acid.

Conclusions: This study highlights the potential of natural compounds in Shorea roxburghii in the development of more effective and safer anticancer agents as options for GIC as well as shedding light on new avenues for cancer treatment.

Keywords: Anticancer; Antioxidant; Gastrointestinal cancer; Medicinal plant; Metabolic profiling-based screening.

Fig. 1

DPPH radical scavenging activity of crude Shorea roxburghii extracts. (A) DPPH radical scavenging activity of crude Shorea roxburghii extracts with 500 μg/ml (^aP < 0.05 compared with ethanol, ^bP < 0.05 compared with hexane) (B) EC₅₀ values of DPPH scavenging effect in crude Shorea roxburghii extracts and the standard compound. (HEX; Hexane, EtAc; Ethyl acetate, EtOH: Ethanol, Water: Distill water, AAE: Ascorbic acid)

Fig. 2

Cytotoxicity of Shorea roxburghii extracts on gastrointestinal cancer cell lines. Gastrointestinal cancer cell lines: (A) KKU-213B, (B) HepG2, (C) AGS treated with four crude Shorea roxburghii extracts with a series concentration 0–200 µg/ml in 48 and 72 h (HEX; Hexane, EtAc; Ethyl acetate, EtOH: Ethanol, Water: Distill water)

Fig. 3

Cytotoxicity of ethyl acetate extract of Shorea roxburghii on gastrointestinal cancer cell lines and normal fibroblast. Gastrointestinal cancer cell lines: (A) KKU-213B, (B) HepG2, (C) AGS treated with crude ethyl acetated of Shorea roxburghii extracts with a series concentration 0–200 µg/ml in 48 and 72 h compared with (D) normal fibroblast cell. (EtAc; Ethyl acetate)

Fig. 4

Crude ethyl acetate of Shorea roxburghii extract induce intracellular ROS. KKU-213B and AGS cells were treated with crude ethyl acetate of Shorea roxburghii extract for 48 h, and intracellular ROS level was determined by flow cytometry. (EtAc; Ethyl acetate)

Fig. 5

Crude ethyl acetate of Shorea roxburghii extract induce apoptosis. (A) KKU-213B and AGS cells were treated with crude ethyl acetate of Shorea roxburghii extract for 48 h, and apoptosis was determined by flow cytometry using Annexin V/PI double staining. (B) Quantitative analysis of apoptosis is shown. (*P < 0.05, **P < 0.001 versus control.) (C) Western blot analysis was performed to assess the expression levels of Bcl-2 after treatment in AGS and (D) Quantitative analysis of Bcl-2 expression is shown. Original images of blots are shown in Fig. S1. (EtAc; Ethyl acetate)

Fig. 6

Gastrointestinal cancer cell viability-related bioactive compounds analyze by ¹H-NMR. OPLS scores and corresponding coefficient loading plots displaying significant metabolites with cell viability on (A) KKU-213B, (B) HepG2 and (C) AGS. (HEX; Hexane, EtAc; Ethyl acetate, EtOH: Ethanol, Water: Distill water, AAE: Ascorbic acid)