Phytochemical Characterization and Anticancer Activity of Clerodendrum chinense Leaf Extract Against Breast and Cervical Cancer Cells

Abstract

Cancer remains a significant global health challenge, necessitating novel therapeutic interventions. Clerodendrum chinense leaf extract (CCL) has gained interest for its potential anticancer properties due to its bioactive composition. This study aims to evaluate the cytotoxic effects of CCL against MCF-7 breast cancer and HeLa cervical cancer cells and elucidate its mechanisms of action. High-performance liquid chromatography identified verbascoside, isoverbascoside, and hispidulin as the major bioactive compounds. CCL exhibited time- and dose-dependent cytotoxicity, with MCF-7 cells showing greater sensitivity (IC₅₀ = 126.8 µg/mL, 72 h) than HeLa cells (216.1 µg/mL, 72 h). Flow cytometry confirmed apoptotic induction, with late apoptosis increasing at moderate concentrations (16.03-23.55%) and necrosis prevailing at higher doses (50.80-63.68%). Reactive oxygen species generation was significantly elevated in MCF-7 (70.2%) and HeLa (60.4%) cells at 250 µg/mL. CCL effectively suppressed colony formation and cell migration in a dose-dependent manner. Molecular docking studies demonstrated that apoptosis induction of CCL bioactive compounds may mediate through the pro-apoptotic BCL2 associated X, apoptosis regulator (BAX) regulator. These findings highlight the potential of CCL as a natural anticancer agent with multiple mechanisms, including reactive oxygen species (ROS)-induced apoptosis, BAX activation, and inhibition of proliferation and metastasis.

Keywords: Clerodendrum chinense; HELA; MCF-7; ROS; anticancer; apoptosis; verbascoside.

Figure 1

The HPLC chromatograms of (a) verbascoside (1), isoverbascoside (2), and hispidulin (3) standards; (b) CCL. All detected at a wavelength of 334 nm UV.

Figure 2

Effects of CCL and its bioactive compounds at various concentrations on the viability of MCF-7 cells. Data are expressed as Mean ± SD (n = 3).

Figure 3

Effects of CCL and its bioactive compounds at various concentrations on the viability of HeLa cells. Data are expressed as Mean ± SD (n = 3).

Figure 4

Effects of CCL and its bioactive compounds at various concentrations on the viability of RAW264.7 cells. Data are expressed as Mean ± SD (n = 3).

Figure 5

Apoptosis and necrosis induction by CCL in (A) MCF-7 and (B) HeLa cells at different concentrations. (n = 3) Data are expressed as Mean ± SD. All experiments were conducted in three independent replicates. **** p < 0.0001 indicates statistically significant differences compared with control (0 µg/mL).

Figure 6

Molecular docking simulation results indicate that binding of (A) BAX activator (BTC-8), (B) verbascoside, (C) isoverbascoside, and (D) hispidulin to BAX protein.

Figure 7

The results of the 3D molecular docking simulation highlighting (A) the hydrogen bond interactions and (B) the hydrophobic interactions observed in the superimposition of the BAX activator (BTC-8), verbascoside, isoverbascoside, and hispidulin with the BAX protein.

Figure 8

ROS formation induced by CCL in (A) MCF-7 and (B) HeLa cell lines, and (C) quantitative analysis of ROS formation. Data are expressed as Mean ±SD (n = 3). All experiments were conducted in three independent replicates. **** p < 0.0001 indicates statistically significant differences compared with control (0 µg/mL).

Figure 9

Effect of CCL on colony formation in (A) MCF-7 and (B) HeLa cells, and (C) quantitative analysis of colony formation. Data are expressed as Mean ± SD (n = 3). All experiments were conducted in three independent replicates. * p < 0.05 and **** p < 0.0001 indicate statistically significant differences compared with control (0 µg/mL).

Figure 10

Effect of CCL on the migration of (A) MCF-7 and (B) HeLa cells, and (C) quantitative analysis of cell migration after 48 h. Data are expressed as Mean ± SD (n = 3). All experiments were conducted in three independent replicates. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 indicate statistically significant differences compared with control (0 µg/mL).