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. 2025 Jun 20;15(7):220. doi: 10.1007/s13205-025-04392-5

Dose-dependent apoptotic effect of Woodfordia fruticosa on hepatocellular carcinoma (HepG2) cells by upregulating bax and caspase-9

Byeong-Su Kang 1,#, Kyubae Lee 2,#, Hyeon-Ju Lee 1, Hyeon-Ji Han 1, Gyunam Kim 2, Dain Kim 2, Eon-Bee Lee 1,✉
PMCID: PMC12181531  PMID: 40546395

Abstract

This study investigated the dose-dependent apoptotic effect of Woodfordia fruticosa (WF) in HepG2 hepatocellular carcinoma cells, focusing on Bax and caspase-9 upregulation. The ethanol extracts, WF70 (70% ethanol) and WF30 (30% ethanol), were tested for cytotoxicity, pro-apoptotic properties, and cell cycle effects. MTT assays revealed a dose- and time-dependent reduction in cell viability, with IC50 values of 14.39 µg/mL and 15.96 µg/mL for WF70 and WF30, respectively. Flow cytometry analysis showed that WF70 induced significantly higher apoptosis rates (54.82% at 30 µg/mL) than WF30 (27.84%), confirming its stronger pro-apoptotic potential. Cell cycle analysis demonstrated a dose-dependent increase in G2/M phase arrest, reaching 30.69% and 24.53% with 30 µg/mL WF70 and WF30, respectively. Gene expression analysis revealed a significant upregulation of pro-apoptotic markers (Bax: 3.65-fold, Caspase-9: 4.32-fold, Caspase-3: 2.12-fold) and a marked downregulation of an anti-apoptotic marker (Bcl-2: 0.08-fold) after treatment with 30 µg/mL WF70. Thus, WF70 exerts a stronger apoptotic effect through both intrinsic (mitochondrial) and extrinsic apoptotic pathways, primarily driven by Bax-mediated mitochondrial membrane destabilization and Caspase-9 activation. This study highlights the potential of WF as an anticancer agent for hepatocellular carcinoma, although this warrants further in vivo validation and optimization for therapeutic applications.

Keyword: Woodfordia Fruticosa, Anticancer, HepG2 cell, Apoptosis

Introduction

Hepatocellular carcinoma (HCC) is a major global health concern characterized by the malignant transformation of liver cells (Llovet et al. 2021). There is an urgent need for novel therapeutic approaches for HCC due to its rising incidence and especially due to the complexities associated with its diverse etiological factors such as chronic hepatitis infection and alcohol abuse (Gromowski et al. 2023). Cases of HCC are further complicated by the generally late diagnosis and limited success of existing treatments (Blum 2005).

The concept of apoptosis or programmed cell death is particularly significant in HCC (Marquardt and Edlich 2019). Since cancer cells often evade apoptosis, leading to unchecked growth, it is crucial to understand and utilize this process in cancer therapy. The strategy of triggering apoptosis in cancer cells could potentially revolutionize treatment approaches for HCC, targeting cancer cells more effectively while minimizing harm to normal cells (Fabregat 2009). This has prompted an investigation into natural treatments, particularly plant extracts, as a method for combating cancer (Khan et al. 2019). Plant-derived compounds have demonstrated great potential due to their varied and powerful bioactive elements. These natural substances are adept at targeting multiple cancer-related pathways, including those responsible for apoptosis, thus offering a comprehensive approach to cancer treatment (Sun et al. 2019).

Recent studies have demonstrated the potential of natural plant extracts in cancer treatment. For example, in A-549 lung cancer cells, the methanolic extract of Ipomoea purpurea leaves was able to arrest the S phase in the cell cycle and trigger the intrinsic pathway of apoptosis (Beheshti et al. 2021). Moreover, the efficacy of Eclipta alba extract was demonstrated against HCT-116 cells, as evidenced by migration and clonogenic assays (Nelson and kumar et al. 2020). Notably, among these natural agents, Woodfordia fruticosa (WF), a plant renowned in traditional medicine, has shown significant promise. WF is widely recognized for its diverse medicinal properties, including strong antioxidant, anti-inflammatory, antimicrobial, and hepatoprotective effects (Chandan et al. 2008). These pharmacologic activities are largely attributed to its rich phytochemical profile, which includes flavonoids, tannins, and polyphenols (Das et al. 2007). These bioactive compounds in WF strongly warrant further investigation regarding their role in the induction of apoptosis, especially because oxidative stress and chronic inflammation are key contributors to the progression of HCC. The methanolic extract of WF has previously demonstrated antiproliferative potential in the human hepatoma PLC/PRF/5 cell line (Nitha et al. 2014), but the specific mechanisms underlying its anticancer effects, particularly its influence on apoptotic pathways, remain unclear.

Building on this knowledge, the current study aims to thoroughly investigate the impact of WF on HepG2 cells, which are commonly used as a representative model for studying HCC. We seek to determine the pro-apoptotic effects of various preparations of WF extract in these cells, with a particular focus on crucial apoptotic genes such as Bax and caspase-9. By elucidating the apoptotic pathway through which WF exerts its effects, this study aims to further uncover its potential as a natural therapeutic agent for HCC.

Materials and methods

Reagents and chemicals

HepG2 human liver cancer cells (HB-8065) were acquired from the American Type Culture Collection (Manassas, VA, United States). The Annexin V/fluorescein isothiocyanate (FITC) apoptosis detection kit was obtained from BD pharming (NJ, USA). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), streptomycin, and penicillin were procured from Thermo Fisher Scientific (Waltham, MA, USA). The 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT) assay kit and cell cycle analysis kit were provided by Sigma-Aldrich (St. Louis, MO, United States). WF flowers were sourced from Herbworld (Andong, Gyeongsangbuk-do, Korea).

Preparation of the WF extract

To produce the WF extract, 20 g of finely ground WF was placed into a 200-mL conical flask and 100 mL of either 30% ethanol (WF30) or 70% ethanol (WF70) was separately added. This study utilized 30 and 70% ethanol based on preliminary solubility tests, ensuring the extraction of both hydrophilic and moderately lipophilic bioactive compounds. The top of the flask was tightly sealed with aluminum foil, placed on a reciprocating shaker, and agitated continuously for 24 h at 150 rpm to maximize extraction efficiency. The resulting mixture was filtered sequentially through muslin cloth, Whatman No. 1 filter paper, and a vacuum-assisted pressure pump (AP-9925 Auto Science) to remove particulate matter. Afterward, the solvent was evaporated using a rotary vacuum evaporator (RE52) in a 50 °C water bath, and the concentrated residues were collected for experimental use.

A concentrated solution of 10,000 mg/L was prepared by dissolving 1 g of the dried plant residue in 5 mL of acetone, then diluting it to 100 mL with tap water. The final stock solution (100 mg/mL) was prepared by dissolving the extract in either 30% ethanol or 70% ethanol, depending on the respective extraction solvent. The final dried extracts were stored at − 20 °C until further use, and the extraction yields were recorded for standardization.

Cell culture and treatment

HepG2 cells were grown in DMEM, supplemented with 10% FBS, streptomycin (100 µg/mL), and penicillin (100 µg/mL), and maintained at 37 °C in a moisture-rich environment with 5% CO2. The untreated group was regarded as the control (CT), while the HepG2 cells in the test groups were subjected to various concentrations of WF (1, 5, 10, 20, and 30 µg/mL) for different durations (3, 6, 12, 24, 48 and 72 h).

Cell viability assay

The viability of the HepG2 cells was assessed using an MTT assay. Cells were seeded in 96-well culture plates at a density of 1x104 cells per well, then incubated at 37 °C with varying concentrations of WF70 and WF30 (1, 5, 10, 20 and 30 µg/mL) for 48 h. Meanwhile, a separate set of cells was treated with 10 µg/mL of WF30 or WF70 for various durations ranging from 0 to 48 h. After this, MTT solution (5 mg/mL) was placed into each well. The absorbance was then measured at 450 nm using an Epoch microplate reader (Winooski, VT, USA). The half-maximal inhibitory concentration (IC50) values were determined via nonlinear regression analysis using GraphPad Prism version 7.0 (GraphPad Software, Inc., La Jolla, CA, USA). All experiments were conducted in triplicate.

Evaluation of apoptosis by annexin V-FITC

The cells underwent treatment with varying doses (10 and 30 µg/mL) of WF70 and WF30. After a 48-h incubation period, they were collected and rinsed twice using cold PBS in preparation for the analysis of apoptosis. Next, the cells were treated with 5 µL of Annexin V/FITC and 5 µL of PI solution, followed by a 15-min incubation at room temperature in the dark. Subsequently, apoptosis was assessed using an Accuri C6 flow cytometer. Following the guidelines of the apoptosis kit, the number of cells in each phase of the cell cycle was determined using the Flowing Software 2.5.1 (Turku Bioscience Center, Turku, Finland). All experiments were conducted in triplicate. The apoptotic rate was calculated as the sum of the proportions of cells in the early (Annexin V/FITC + PI-) and late (Annexin V/FITC + PI +) phases of apoptosis.

Cell cycle analysis

Flow cytometric analysis was used to evaluate the distribution of cells in each phase of the cell cycle. Initially, cells were seeded in 10-cm plates and treated with different concentrations of WF70 (10, 20, and 30 µg/mL) for 48 h. After treatment, the cells were gathered, rinsed twice using cold PBS, fixed in 70% ethanol, then stained with propidium iodide (PI). A flow cytometer (Bio‑Rad Laboratories) was used for the cell analysis, and the results were processed using Flowing Software 2.5.1 (Turku Bioscience Center, Turku, Finland).

Gene expression using real-time polymerase chain reaction

HepG2 cells were exposed to different concentrations of WF70 (10, 20, and 30 µg/mL) for 48 h. Total RNA was extracted using the TRIzol reagent (Ambion, Life Technologies, United States). The Nanodrop device was used to assess the purity and quantity of RNA. To synthesize complementary DNA, a real-time polymerase chain reaction (RT-PCR) premix (Pioneer, Korea) was utilized, with the addition of random hexamers. RNA was quantified using the CFX96 Touch™ real-time PCR detection system (Bio-Rad, United States) along with IQTM SYBR Green Supermix for real-time PCR (Bio-Rad, Singapore). The products of the reaction were stored at − 20 °C. The SYBR Green technique was used to analyze the genes expression of Bax, Bid, Bcl-2, Caspase-3, Caspase-8, and Caspase-9 in HepG2 cells treated for 48 h, with GAPDH gene serving as the internal control. Each reaction mixture had a final volume of 25 mL, consisting of 12.5 mL of Master mix (Bioneer, Korea), 1 mL of synthesized cDNA (0.1–1 mg for GAPDH, Bax, Bid, Bcl-2, Caspase-3, Caspase-8, and Caspase-9), 1 mL (10 mM) of both forward and reverse primers for each gene (Takapuzist company, Tehran, Iran), and 9.5 mL of DEPC water. The primer sequences are listed in Table 1. PCR was conducted separately for each gene and repeated thrice. PCR involved an initial denaturation at 94 °C for 5 min then 40 cycles consisting of denaturation at 94 °C for 30 s, annealing at specific temperatures for 40 s (61.5 °C for Bax and Bid, 54 °C for Bcl-2, 63.5 °C for Caspase-3, 8, 9, and 57 °C for GAPDH), and extension at 72 °C for 45 s. This was concluded with a final extension at 72 °C for 5 min. After the reaction, data on the Ct (threshold cycle) was retrieved from the instrument. The gene expression levels were then quantified using the ΔΔCt approach (Pfaffl 2001). The outcomes were visualized using GraphPad Prism version 7.0 (GraphPad Software, Inc., La Jolla, CA, USA).

Table 1.

Primers used for the real-time polymerase chain reaction

Gene Primer sequence
Bax F: 5′-CCCGAGAGGTCTTTTTCCGAG-3′
R: 5′-CCAGCCCATGATGGTTCTGAT-3′
Bcl-2 F: 5′-CCTGTGGATGACTGAGTACC-3′
R: 5′-GAGACAGCCAGGAGAAATCA-3′
Bid F: 5′-CTTAGCCAGAAATGGGATG-3′
R: 5′-AGTCACAGCTATCTTCCAG-3′
Caspase-3 F: 5′-AAAGCACTGGAATGACATC-3′
R: 5′-CGCATCAATTCCACAATTTC-3′
Caspase-8 F: 5′-CTACAGGGTCATGCTCTATC-4′
R: 5′-ATTTGGAGATTTCCTCTTGC-4′
Caspase-9 F: 5′-CTGAGCCAGATGCTGTCCCAT-3′
R: 5′-CCAAGGTCTCGATGTACCAGGAA-3′
GAPDH F: 5′-GCAAGTTCAACGGCACGATCAAG-6′
R: 5′-CTACTCAGCACCAGCATCACC-6′
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Statistical analysis

Statistical evaluation of the data was conducted via GraphPad Prism (GraphPad Software, Inc., La Jolla, CA, USA), utilizing one-way analysis of variance followed by Tukey’s post hoc test to compare the gene expression levels between the treated samples and controls, ensuring statistical robustness and minimizing type I errors. The results are displayed as the mean ± standard deviation (SD), and p < 0.05 was considered statistically significant. To ensure reliability of the results, all experiments were conducted in triplicate, and appropriate negative controls were included.

Results

Cell proliferation using the MTT Assay

Cell viability was assessed after treatment with WF70 (Fig. 1a) and WF30 (Fig. 1b) extracts at a concentration of 10 µg/mL. At 6, 12, 24, and 48 h, decreasing cell viability was observed in cells treated with WF70 extract (79.02, 61.84, 53.36, and 45.69%, respectively) and those treated with WF30 extract (78.11, 64.00, 59.15, and 49.52%, respectively). These findings highlight the sustained, time-dependent cytotoxic nature of both extracts, with the WF70 extract demonstrating a consistently stronger effect at the same concentration.

Fig. 1.

Fig. 1

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Time- and dose-dependent cytotoxic effects of WF70 and WF30 on HepG2 cell viability. a, b HepG2 cell viability after treatment with (a) 10 µg/mL WF70 and (b) 10 µg/mL WF30 over time. Cells were incubated for 6, 12, 24, and 48 h, and viability was assessed via the MTT assay. Control (CT) cells were unaffected. Cell viability gradually decreased after treatment with both WF70 (which exhibited a significant reduction at 24 and 48 h) and WF30. Both extracts exhibited a time-dependent cytotoxic effect, which was stronger in WF70. c, d HepG2 cell viability after treatment with increasing concentrations (1, 5, 10, 20, and 30 µg/mL) of (c) WF70 and (d) WF30 for 24 h. Cell viability significantly decreased alongside increasing concentrations of WF70 (most pronounced at 30 µg/mL) and WF30. Both extracts demonstrated a concentration-dependent cytotoxic response, which was stronger in WF70. e, f Determining the half-maximal inhibitory concentration (IC50) of (e) WF70 and (f) WF30, calculated based on the dose–response curve. WF70 had an IC50 of 14.39 µg/mL, indicating its effectivity in reducing HepG2 cell viability at relatively low concentrations. WF30 had a slightly higher IC50 of 15.96 µg/mL, indicating slightly less potency than WF70. All data are presented as mean ± standard deviation (SD) from three independent experiments

The cell viability of HepG2 cells was also assessed after treatment with WF70 (Fig. 1c) and WF30 (Fig. 1d) extracts with various concentrations and compared to a non-treated control group (CT). For both extracts, a concentration of 5 µg/mL resulted in a marginal decline in cell viability (WF70: 99.071%, WF30: 99.471%), whereas a concentration of 10 µg/mL resulted a notable reduction in cell viability (WF70: 87.370%, WF30: 90.806%). The most substantial impact on cell viability was observed at 30 µg/mL, the highest concentration tested (WF70: 33.710%, WF30: 46.392%). These findings illustrate the clear concentration-dependent cytotoxic effect of WF extracts on HepG2 cells, which was consistently more pronounced with WF70. Additionally, WF70 and WF30 demonstrated IC50 values of 14.39 µg/mL and 15.96 µg/mL, respectively (Fig. 1e–f).

Flow cytometry analysis of apoptosis in HepG2 cells after exposure to varying concentrations of WF30.

Figure 2 illustrates the influence of WF30 extract on the induction of apoptosis in HepG2 cells as evaluated via flow cytometry analysis. The percentage of cells undergoing apoptosis after treatment with different concentrations of WF30 (control, 10 µg/mL, and 30 µg/mL) was determined through increased binding of Annexin V and uptake of PI (Fig. 2a–c). There was a significant dose-dependent increase in apoptosis in the cells treated with WF30, with apoptosis rates of 13.72% and 27.84% at concentrations of 10 and 30 µg/mL, respectively (p < 0.001 for both; Fig. 2d). Regarding the different stages of apoptosis, most cells in the control group were alive (Q4: 96.43%), with very few cells in early (Q3: 0.11%) or late apoptosis (Q2: 3.04%). Meanwhile, after treatment with WF30, there was a notable increase in early and late apoptotic cells and a corresponding decrease in live cells at a concentration of 10 µg/mL (Q3: 0.21%, Q2: 12.04%, Q4: 87.44%), and this effect was even more pronounced at a concentration of 30 µg/mL (Q3: 0.46%, Q2: 22.29%, Q4: 77%) (Fig. 2e). Thus, WF30 induced apoptosis in a dose-dependent manner, with higher concentrations leading to a greater proportion of cells undergoing both early and late apoptosis.

Fig. 2.

Fig. 2

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Flow cytometric analysis of apoptosis induced by WF30 extract in HepG2 cells. a–c Representative flow cytometry dot plots displaying Annexin V-FITC and PI staining used to assess apoptosis in HepG2 cells treated with WF30 at different concentrations: (a) Control (CT, untreated cells), (b) 10 µg/mL, and (c) 30 µg/mL. Various quadrants represent different cells, as follows: Q1, necrotic cells (Annexin V-/PI +); Q2, late apoptotic cells (Annexin V +/PI +); Q3, early apoptotic cells (Annexin V +/PI-); and Q4, viable cells (Annexin V-/PI-). d Quantifying the total apoptotic cell population (Q2 + Q3). Apoptotic cells increased significantly after WF30 treatment in a dose-dependent manner (CT: 3.15%, 10 µg/mL VF30: 13.72%, 30 µg/mL VF30: 27.84%). Statistical analysis revealed highly significant differences compared to the control at both concentrations (p < 0.001). e Stacked bar graph illustrating the distribution of HepG2 cells in different apoptotic stages (Q1–Q4) after treatment with WF30 at 10 and 30 µg/mL. In the CT group, few cells were in early or late apoptosis, and most were viable (Q3: 0.11%, Q2: 3.04%, Q4: 96.43%). After WF30 treatment, early and late apoptotic cells increased, while live cells decreased at a concentration of 10 µg/mL (Q3: 0.21%, Q2: 12.04%, Q4: 87.44%), and this effect was even more pronounced at 30 µg/mL (Q3: 0.46%, Q2: 22.29%, Q4: 77%) These results indicate a dose-dependent induction of apoptosis by WF30. Values are presented as mean ± SEM. ***p < 0.001 versus control

Flow cytometry analysis of apoptosis in HepG2 cells after exposure to varying concentrations of WF70.

Figure 3 details the effects of different concentrations of WF70 extract (control, 10 µg/mL, and 30 µg/mL) on the induction of apoptosis in HepG2 cells (Fig. 3a–c). There was a significant, dose-dependent increase apoptotic cells after treatment with WF70, with apoptosis rates of 28.18 and 54.82% at concentrations of 10 and 30 µg/mL, respectively (p < 0.001 for both; Fig. 3d). Regarding the different stages of apoptosis, most cells in the control group were alive (Q4: 91.61%), with a very small percentage in early (Q3: 3.21%) or late apoptosis (Q2: 4.38%). After treatment with WF70, there was a notable increase in early and late apoptotic cells and a corresponding decrease in live cells at a concentration of 10 µg/mL (Q3: 6.15%, Q2: 25.79%, Q4: 67.12%). This trend continued at a higher concentration of 30 µg/mL, wherein there was a moderate increase in early apoptotic cells, a significant increase in late apoptotic cells, and a further decrease in live cells (Q3: 7.93%, Q2: 49.01%, Q4: 41.94%) (Fig. 3e). Similar to WF30, treatment with WF70 also induced apoptosis in a dose-dependent manner, with higher concentrations leading to greater proportions of cells in both early and late apoptosis.

Fig. 3.

Fig. 3

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Flow cytometric analysis of apoptosis induced by WF70 extract in HepG2 cells. a–c Representative flow cytometry dot plots displaying Annexin V-FITC and PI staining used to assess apoptosis in HepG2 cells treated with WF70 at different concentrations: (a) Control (CT, untreated cells), (b) 10 µg/mL, and (c) 30 µg/mL. Various quadrants represent different cells, as follows: Q1, necrotic cells (Annexin V-/PI +); Q2, late apoptotic cells (Annexin V +/PI +); Q3, early apoptotic cells (Annexin V +/PI-); and Q4, viable cells (Annexin V-/PI-). d Quantifying the total apoptotic cell population (Q2 + Q3). Apoptotic cells increased significantly after WF70 treatment in a dose-dependent manner (CT: 7.59%, 10 µg/mL VF70: 28.18%, 30 µg/mL VF70: 54.82%). Statistical analysis revealed highly significant differences compared to the control at both concentrations (p < 0.001) e Stacked bar graph representing the distribution of HepG2 cells across different apoptotic stages (Q1–Q4) after treatment with WF70 at 10 and 30 µg/mL. In the CT group, few cells were in early or late apoptosis, and most were viable (Q3: 3.21%, Q2: 4.38%, Q4: 91.61%). After treatment with 10 µg/mL WF70, there was a notable increase in early and late apoptotic cells, while live cells decreased (Q3: 6.15%, Q2: 25.79%, Q4: 67.12%). After treatment with 30 µg/mL WF70, there was a surge in both early and late apoptotic cells, with a further decrease in live cells (Q3: 7.93%, Q2: 49.01%, Q4: 41.94%). Thus, WF70 induces apoptosis in a dose-dependent manner, with significantly higher rates of apoptosis at 30 µg/mL compared with WF30. Values are presented as mean ± SEM. ***p < 0.001 versus control

Progression of apoptosis in HepG2 cells at various time intervals after exposure to WF30

Figure 4a illustrates the progression of apoptosis in cells at various time intervals after treatment with WF30 at a concentration of 20 µg/mL. An increasing rate of apoptosis was first seen at 12 h (10.71%), which more than doubled at 24 h (24.34%), followed by a significant surge at 48 h (54.26%), then reaching a plateau at 72 h (55.79%). Thus, WF30 induced a time-dependent apoptotic response in the treated cells, with the most pronounced effect occurring from 24 to 48 h.

Fig. 4.

Fig. 4

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Time-dependent apoptosis in HepG2 cells treated with WF30. a Flow cytometry dot plots illustrating the progression of apoptosis in HepG2 cells treated with 20 µg/mL WF30 at various time points (Control [CT], 12 h, 24 h, 48 h, and 72 h). Annexin V-FITC and PI staining was done to differentiate between viable (Q4), early apoptotic (Q3), late apoptotic (Q2), and necrotic cells (Q1). A time-dependent increase in apoptotic cell populations (Q2 + Q3) was observed, with the highest percentage at 72 h. b Quantifying cell distribution across different apoptosis stages over time. At 12 h, both early and late apoptosis slightly increased (Q3: 6.32%, Q2: 0.09%), and at 24 h, these markedly increased (Q3: 23.05%, Q2: 6.07%). At 48 h, early apoptosis peaked, and late apoptosis significantly increased (Q3: 37.33%, Q2: 10.16%). At 72 h, early apoptosis slightly decreased, while late apoptosis continued to rise (Q3: 30.44%, Q2: 13.59%). Viable cells (Q4) declined over time, confirming the sustained apoptotic effect of WF30

Figure 4b shows the percentage of cells in different stages of apoptosis at various time points after treatment with WF30 as analyzed via flow cytometry. In the control group (CT), nearly all cells were alive (Q4: 99.10%), with negligible percentages in early (Q3) and late (Q2) apoptosis. Both early and late apoptosis slightly increased at 12 h (Q3: 6.32%, Q2: 0.09%) and markedly increased at 24 h (Q3: 23.05%, Q2: 6.07%). At 48 h, the trend became more pronounced, with both early and late apoptosis significantly increasing alongside a corresponding decrease in live cells (Q3: 37.33%, Q2: 10.16%, Q4: 50.41%). By 72 h, early apoptosis slightly decreased, late apoptosis continued to rise, and live cells moderately recovered (Q3: 30.44%, Q2: 13.59%, Q4: 53.19%). These results suggest a dynamic response to WF30 treatment, with a peak in early apoptosis at 48 h followed by a continued increase in late apoptosis, indicating a shift toward terminal cell death stages over time.

Progression of apoptosis in HepG2 cells at various time intervals after exposure to WF70

Figure 5a shows the percentage of apoptosis in HepG2 cells over time after treatment with WF70 at a concentration of 20 µg/mL. The apoptosis rate significantly increased at 12 h (15.73%), slightly decreased at 24 h (15.47%), then the upward trend continued again at 48 h (22.07%) and 72 h (27.47%). Thus, WF70 induced apoptosis in HepG2 cells in a time-dependent manner, with a notable increase in apoptotic cells over 72 h.

Fig. 5.

Fig. 5

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Time-dependent apoptosis in HepG2 cells treated with WF70. a Flow cytometry dot plots showing the progression of apoptosis in HepG2 cells treated with 20 µg/mL WF70 at different time points (Control [CT], 12 h, 24 h, 48 h, and 72 h). Annexin V-FITC and PI staining was done to differentiate between viable (Q4), early apoptotic (Q3), late apoptotic (Q2), and necrotic cells (Q1). A time-dependent increase in apoptotic cell populations (Q2 + Q3) was observed, with the highest percentage at 72 h. b Quantifying cell distribution across different apoptosis stages over time. At 12 h, there was minor increase in both early and late apoptosis (Q3: 1.39%, Q2: 4.08%). At 24 h, there was a smaller increase in early apoptosis and a significant rise in late apoptosis (Q3: 1.69%, Q2: 12.3%). At 48 h, early apoptosis remained relatively stable, whereas late apoptosis continued to rise, (Q3: 1.43%, Q2: 24.98%). At 72 h, there was only a slight increase in early apoptosis, while late apoptosis rose (Q3: 1.64%, Q2: 35.40%). Viable cells (Q4) declined over time, confirming the sustained apoptotic effect of WF70. c Comparison of apoptosis induction between WF70 and WF30 at 72 h. WF70-treated cells exhibited a significantly higher apoptotic response than WF30-treated cells (55.78% vs. 27.47%, p = 0.0007), suggesting that WF70 is more effective in inducing apoptosis in HepG2 cells. Values are expressed as mean ± SEM. ***p < 0.001

Figure 5b represents the cell distribution across the different stages of viability and apoptosis after treatment with WF70 as analyzed via flow cytometry. At 12 h, there was a slight increase in early apoptosis and a more notable increase in late apoptosis, while most cells remained live (Q3: 1.39%, Q2: 4.08%, Q4: 93.67%). At 24 h, there was a smaller increase in early apoptosis, a significant rise in late apoptosis, and a corresponding decrease in live cells (Q3: 1.69%, Q2: 12.3%, Q4: 85.27%). At 48 h, early apoptosis remained relatively stable, whereas late apoptosis continued to rise, and the live cells were further reduced (Q3: 1.43%, Q2: 24.98%, Q4: 72.37%). At 72 h, there was only a slight increase in early apoptosis, a pronounced elevation in late apoptosis, and a further reduction of live cells (Q3: 1.64%, Q2: 35.40%, Q4: 61.48%). This progression suggests a time-dependent response to WF70 treatment, with an increase in late apoptotic cells over time. Thus, the compound effectively induced apoptosis, progressing from early to late stages, alongside an increasing duration of exposure.

Figure 5c presents the differences in apoptosis induction between treatment with WF70 and WF30 for 72 h. A more substantial apoptotic response was seen in cells treated with WF70 versus WF30 (55.78% vs. 27.47%, p = 0.0007), indicating the higher efficacy of WF70 in triggering apoptosis after a 72-h exposure period.

Influence of WF70 on the cell cycle distribution in HepG2 Cells

Figure 6 displays the distribution of HepG2 cells across the cell cycle phases after treatment with WF70 extract at varying concentrations (control, 10 µg/mL, 20 µg/mL, and 30 µg/mL). In the control group, most cells were in the G0/1 phase (77.39%), with fewer cells in the S (8.65%) and G2/M (13.96%) phases. There was a trend of decreasing cells in the G0/1 phase and increasing cells in the G2/M phase seen with increasing treatment concentrations of 10 µg/mL (G0/1 phase: 68.96%, G2/M phase: 21.78%), 20 µg/mL (G0/1 phase: 67.22%, G2/M phase: 24.53%), and 30 µg/mL (G0/1 phase: 61.06%, G2/M phase: 30.69%). Meanwhile, cells in the S phase remained relatively stable across all concentrations. This indicated that treatment with increasing concentrations of WF70 induced a concentration-dependent increase in G2/M phase cell cycle arrest in HepG2 cells.

Fig. 6.

Fig. 6

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Effect of WF70 extract on cell cycle distribution in HepG2 cells. a–d Flow cytometry analysis showing the distribution of HepG2 cells across different cell cycle phases (G0/1, S, G2/M) after treatment with increasing concentrations of WF70 extract: (a) Control (CT), (b) 10 µg/mL, (c), 20 µg/mL, and (d) 30 µg/mL. In the control group, most cells were in the G0/1 phase (77.39%), with less in the S (8.65%) and G2/M (13.96%) phases. After treatment with WF70, there was a concentration-dependent decrease in G0/1 cells and a corresponding increase in G2/M phase cells seen at concentrations of 10 µg/mL (G0/1: 68.96%, G2/M: 21.78%), 20 µg/mL (G0/1: 67.22%, G2/M: 24.53%), and 30 µg/mL (G0/1: 61.06%, G2/M: 30.69%), indicating G2/M phase cell cycle arrest. The S phase remained relatively stable across all conditions. e Quantitative representation of the relative proportions of HepG2 cells in each cell cycle phase at different WF70 concentrations. The increasing accumulation of cells in the G2/M phase suggests that WF70 induces cell cycle arrest, which could contribute to its cytotoxic effects

Apoptosis pathway gene expression after treatment with WF70

Figure 7 displays the changes in the expression levels of various genes (Bax, Bid, Bcl-2, Caspase-3, Caspase-8, and Caspase-9) involved in the apoptotic pathway after treatment with various concentrations of WF70 extract (control, 10 µg/mL, 20 µg/mL, and 30 µg/mL). A concentration-dependent increase was seen in both Bax expression (from 1.61-fold at 10 µg/mL to 3.65-fold at 30 µg/mL, Fig. 7a). Compared to the control, significant upregulation was detected at 20 µg/mL (p = 0.002) and 30 µg/mL (p < 0.001), while the increase at 10 µg/mL was not statistically significant (p = 0.11). Bid expression (from 1.37-fold at 10 µg/mL to 2.67-fold at 30 µg/mL, Fig. 7b). Significant upregulation was observed at both 20 µg/mL and 30 µg/mL compared to the control group (p < 0.001 for both), while the change at 10 µg/mL was not statistically significant (p = 0.20). Meanwhile, expression of the anti-apoptotic gene Bcl-2 significantly decreased in response to increasing WF70 concentration (0.08-fold at 30 µg/mL, Fig. 7c), with statistically significant reductions observed at 20 µg/mL (p = 0.005) and 30 µg/mL (p < 0.001), while the change at 10 µg/mL was not significant (p = 0.85). In response to increasing WF70 concentration, gene expression was also upregulated for Caspase-3, Caspase-8, and Caspase-9 (Fig. 7d–f). Caspase-3 expression increased from 1.52-fold at 10 µg/mL to 2.12-fold at 30 µg/mL, with statistically significant differences observed at 20 µg/mL (p = 0.009) and 30 µg/mL (p = 0.001), while the increase at 10 µg/mL was not significant (p = 0.07) (Fig. 7d). Caspase-8 expression showed a robust and statistically significant increase at all tested concentrations (p = 0.009 at 30 µg/mL, p < 0.001 at 10 and 20 µg/mL) (Fig. 7e). Caspase-9, which exhibited the most pronounced response among the caspases, increased from 2.01-fold at 10 µg/mL to 4.32-fold at 30 µg/mL, with significance at all concentrations (p = 0.003 at 10 µg/mL, p < 0.001 at 20 and 30 µg/mL) (Fig. 7f). Figure 7g illustrates a heat map that compares the levels of gene expression. These results show that WF70 induced apoptosis in a dose-dependent manner by upregulating pro-apoptotic genes (Bax, Bid, Caspase-3, Caspase-8, and Caspase-9) and downregulating an anti-apoptotic gene (Bcl-2).

Fig. 7.

Fig. 7

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Dose-dependent modulation of apoptotic gene expression in HepG2 cells treated with WF70 extract. a–f Quantitative analysis of apoptotic gene expression following WF70 treatment at concentrations of 10, 20, and 30 µg/mL compared to controls (CT). Gene expression levels were analyzed via qRT-PCR and normalized to the control group. Several trends were noted with increasing WF70 concentrations. a Bax expression significantly increased (10 µg/mL: 1.61-fold, p = 0.11; 20 µg/mL: 2.48-fold, p = 0.002; 30 µg/mL: 3.65-fold, p < 0.001). b Bid expression followed a similar trend (10 µg/mL: 1.37-fold, p = 0.20; 20 µg/mL: 2.01-fold, p < 0.001; 30 µg/mL: 2.67-fold, p < 0.001). c The anti-apoptotic gene Bcl-2 was drastically downregulated (10 µg/mL: 0.92-fold, p = 0.85; 20 µg/mL: 0.46-fold, p = 0.005; 30 µg/mL: 0.08-fold, p < 0.001). d Caspase-3 expression increased (10 µg/mL: 1.52-fold, p = 0.07; 20 µg/mL: 1.80-fold, p = 0.009; 30 µg/mL: 2.12-fold, p = 0.001). e Caspase-8, a marker of the extrinsic apoptotic pathway, consistently increased (10 µg/mL: 1.72-fold, p < 0.001; 20 µg/mL: 2.18-fold, p < 0.001; 30 µg/mL: 2.74-fold, p < 0.001). f Caspase-9, associated with intrinsic apoptosis, showed the most significant increase (10 µg/mL: 2.01-fold, p = 0.003; 20 µg/mL: 3.29-fold, p < 0.001; 30 µg/mL: 4.32-fold, p < 0.001). g Heat map representation of gene expression levels, illustrating the dose-dependent upregulation of pro-apoptotic genes (Bax, Bid, Caspase-3, Caspase-8, and Caspase-9) and downregulation of the anti-apoptotic gene (Bcl-2). Thus, both intrinsic and extrinsic apoptotic pathways are activated in HepG2 cells after WF70 treatment. Values are expressed as mean ± SEM. Statistical significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001

Discussion

Liver cancer remains one of the most challenging malignancies to treat, and its pathogenesis typically involves disrupted apoptotic processes (Wong 2011). Apoptosis plays a key role in preventing cancer progression by regulating cell proliferation (Carneiro and El-Deiry 2020). WF is a medicinal plant with potential anticancer effects, and one of its constituents, ellagic acid, may reduce hepatocarcinogenesis by scavenging free radicals and protecting against oxidative stress, as well as regulating apoptosis and inhibiting angiogenesis (Hussein and Khalifa 2014; Das et al. 2017). WF is a medicinal plant with potential anticancer effects, but its precise mechanisms remain unclear (Nitha et al. 2014, 2013). Our results provide strong evidence that WF promotes apoptosis in HepG2 cells by modulating apoptotic gene expression and inducing cell cycle arrest. This study sheds light on the apoptotic mechanisms of WF, further supporting its potential as a therapeutic candidate for liver cancer treatment. Nevertheless, the mechanisms underlying WF-induced apoptosis need further exploration to fully understand its therapeutic potential.

Considering the importance of plant-derived compounds in modulating apoptotic pathways, phytochemical extraction is a key factor in evaluating bioactive components. The choice of extraction methods is critical, since different solvents can yield distinct phytochemical compositions. Ethanol is a commonly used solvent in phytochemical extractions because of its effectiveness across a wide range of plant compounds, particularly those with moderate polarity (Abubakar and Haque 2020). Previous research has demonstrated that WF can be extracted using various solvents, including methanol (Tayab et al. 2021), ethanol (Sujanamulk et al. 2020), and water (Chandan et al. 2008). Two different ethanol concentrations, 30 and 70%, were used in this study to optimize the extraction of both polar and nonpolar bioactive compounds, ensuring a comprehensive evaluation of the apoptotic effects of WF. In general, higher ethanol concentrations extract more nonpolar compounds, whereas lower concentrations favor polar compounds (Zhang et al. 2018). This variability in solubility can yield extracts with distinct phytochemical compositions and subsequently, various biological activities. Certain bioactive constituents such as flavonoids (Abotaleb et al. 2018) and tannins (Zhang et al. 2023) in WF have been previously implicated in the induction of apoptosis, thus highlighting the importance of investigating the fractionation and purification of WF extracts to pinpoint the active components responsible for its cytotoxic effects.

To investigate the apoptotic effects of WF extracts, this study examined the cytotoxicity of WF70 and WF30 at a consistent concentration of 10 µg/mL (Fig. 1a–b). WF70 had a greater cytotoxic effect, suggesting that the higher ethanol concentration may have facilitated the extraction of the key bioactive compounds involved in cell death or improved its ability to penetrate cellular membranes. In support of this inference, an increase in ethanol concentration has previously been correlated with higher phenolic content in plant extracts (Lohvina et al. 2022). The dose-dependent cytotoxicity was further analyzed by evaluating the effects of various extract concentrations (Fig. 1c–d). For both extracts, a low concentration of 5 µg/mL induced only a marginal decline in cell viability. However, a significant reduction in viability was observed at 10 µg/mL, with WF70- and WF30-treated cells exhibiting 87.37 and 90.81% viability, respectively. This trend was even more pronounced at higher concentrations, thereby highlighting the time- and concentration-dependent cytotoxic effect of WF extracts on HepG2 cells. The reduction in cell viability was more pronounced with WF70, suggesting potential differences in the phytochemical composition or mechanisms of action between the two extracts. Nevertheless, further investigation is needed to characterize the specific bioactive components of WF and their roles in the induction of apoptosis.

Selecting an appropriate solvent is critical for the optimal extraction of bioactive compounds, which directly relates to the cytotoxic potential of plant-based extracts. Ethanol is widely used for phytochemical extractions because of its ability to dissolve both polar and nonpolar compounds (Chemat et al. 2019). In this study, 30 and 70% ethanol were used to examine how solvent concentration affects the extraction efficiency of key bioactive components in WF. Since higher ethanol concentrations are known to enhance the extraction of pro-apoptotic phenolic compounds, flavonoids, and tannins (Do et al. 2014; Shofian et al. 2011), the observed difference in cytotoxicity between WF70 and WF30 can be attributed to the higher solubility of these bioactive compounds in 70% ethanol, leading to a more potent apoptotic response in HepG2 cells (Stanciauskaite et al. 2021). Moreover, previous research has shown that ethanol concentration significantly alters the phytochemical profile of plant extracts, ultimately influencing their biological activity (Gao et al. 2018). In this study, WF70 exhibited stronger pro-apoptotic effects compared to WF30, specifically in terms of greater cell cycle arrest at the G2/M phase, higher expression of pro-apoptotic genes (Bax, Bid, Caspase-3, Caspase-8, Caspase-9), and stronger suppression of Bcl-2. In line with this, previous studies have demonstrated that plant-derived bioactive compounds can effectively induce apoptosis and cell cycle arrest, reinforcing their potential as anticancer agents (Vieira et al. 2022; Thumpati et al. 2025).

The differences between WF70 and WF30 can be attributed to variations in their bioactive compound composition. WF contains multiple anticancer constituents, including β-sitosterol, kaempferol, ellagic acid, quercetin, and woodfordin A–D, among which many have been linked to the induction of apoptosis (Das et al. 2007). In particular, ellagic acid inhibits DNA topoisomerase, thereby impairing cell cycle progression (Constantinou et al. 1995), while quercetin acts as an antioxidant and pro-apoptotic agent (Granado-Serrano et al. 2012). Meanwhile, woodfordin C exhibits potent topoisomerase II inhibition and antitumor activity that is comparable to etoposide and adriamycin (Kuramochi-Motegi et al. 1992). Additionally, hydrolyzable tannins such as woodfordin D and oenothein A have demonstrated pro-apoptotic effects (Yoshida et al. 1991). Since ethanol concentration affects extraction efficiency, WF70 may contain higher levels of these potent apoptotic inducers, resulting in stronger cytotoxic effects.

Despite the strong pro-apoptotic effects observed, one notable limitation of our study is that the specific bioactive compounds responsible for these effects were not identified nor quantified. Although previous research on WF has isolated hydrolyzable tannins, flavonoids, polyphenols, and anthraquinone glycosides, their individual contributions to the induction of apoptosis have not been clearly elucidated (Das et al. 2007; Thakur et al. 2021; Giri et al. 2023). In particular, since woodfordin C has been identified as a topoisomerase II inhibitor, this may be involved in the anticancer properties of WF (Kadota et al. 1990). The isolation and structural elucidation of plant-derived compounds have been greatly advanced by the introduction of modern analytical technologies such as high-performance liquid chromatography (HPLC), gas chromatography–mass spectrometry (GC–MS), nuclear magnetic resonance (NMR), optical rotation (OR), and circular dichroism (CD) spectroscopy. These techniques offer rapid and precise results in the separation and purification of key constituents in natural products, structural verification, and stereochemical characterization (Giri et al. 2023). The unique chemical profile of WF can be explored using HPTLC. The application of high-performance thin-layer chromatography (HPTLC) at a short wavelength of 254 nm has been demonstrated to be a highly effective method for the identification of non-polar and aromatic compounds, including terpenoids, alkaloids and flavonoids. In contrast, long-wavelength analysis at 366 nm helps distinguish polar compounds with conjugated double bonds or ultraviolet-absorbing properties, including phenolics and flavonoids (Prajapati et al. 2025). Analytical methods such as metabolomics are employed to investigate and compare the chemical characteristics of biological substances. Liquid chromatography–high-resolution mass spectrometry (LC–HRMS) is a primary analytical tool in large-scale studies of biological samples containing a broad range of metabolites due to its high throughput, sensitivity and selectivity. To address this knowledge gap, advanced analytical techniques such as liquid chromatography-mass spectrometry (LC–MS) and high-performance liquid chromatography (HPLC) can be used to precisely identify and quantify these key apoptotic compounds in future research in order to enhance our understanding of the therapeutic potential of WF against HCC.

The concentrations of WF70 used in this study (10, 20, and 30 µg/mL) are similar to those used in previous research on plant-derived anticancer compounds, commonly ranging from 5 to 100 µg/mL (Cragg and Newman 2005). These concentrations effectively induced apoptosis in HepG2 cells while maintaining cell viability at lower doses (Fulda and Debatin 2006). However, although these in vitro findings provide mechanistic insights, their physiological relevance in vivo remains uncertain due to other factors such as absorption, metabolism, and systemic distribution (Liu 2003). Since many plant-based compounds exhibit low bioavailability due to rapid metabolism and poor solubility, advanced drug delivery strategies (e.g., nanoparticle formulations or structural modifications) are needed to enhance their therapeutic potential (Brigger et al. 2002). AgNPs synthesized from WF extract (WF-AgNPs) induce cell death via the apoptosis pathway, which is mediated by the generation of reactive oxygen species (ROS), DNA fragmentation, and disruption of mitochondrial membrane potential. This nanoparticle strategy, owing to its small size, facilitates easy penetration into cancer cells and accumulation within cellular organelles (Dubey et al. 2024). By selectively inducing cytotoxicity in cancer cells over normal cells, these nanoparticles represent a promising drug delivery strategy for cancer therapy (Sriram et al. 2010). Further pharmacokinetic studies can determine whether these in vitro doses maintain their effectivity in vivo, with the goal of translating into these results into meaningful clinical strategies for the treatment of liver cancer (Neagu et al. 2017).

Another topic for further investigation is whether the apoptotic effects of WF extracts seen in HepG2 cells are broadly applicable to other HCC cell lines. Previous research has reported the cytotoxic effects of WF in PLC/PRF/5 cells, suggesting its potential anticancer activity beyond HepG2 (Nitha et al. 2013). However, comparative studies under identical experimental conditions are lacking. In particular, other liver cancer cell lines such as Huh7 and SNU-449 can be the focus of future studies to determine whether WF70-induced apoptosis and cell cycle arrest are consistent across different genetic backgrounds and drug resistance profiles.

The more potent apoptotic effects of WF70 compared to WF30 suggest higher concentrations of key bioactive compounds that enable the more efficient activation of both intrinsic and extrinsic apoptotic pathways. In line with this, previous studies have demonstrated the ability of phenolic compounds to destabilize the mitochondrial membrane (via Bax/Bid) and induce death receptor-mediated apoptosis (via Caspase-8) (Etxebarria et al. 2008). This supports our hypothesis that differences in extraction efficiency significantly impact the anticancer potential of plant-derived compounds. Nevertheless, the specific active compounds in WF need to be isolated and characterized to further elucidate their mechanistic role in the induction of apoptosis. Advanced analytical techniques such as LC–MS/MS, HPLC, and bioactivity-guided fractionation can help identify these key apoptotic inducers and optimize WF extracts for enhanced selectivity against HCC.

Cell cycle progression is tightly regulated by checkpoints that ensure that cells do not proceed to the next phase until after DNA replication and repair are complete (Pucci et al. 2000). Disruptions in these checkpoints lead to uncontrolled proliferation, a hallmark of cancer (Matthews et al. 2022). Flow cytometry analysis of WF70-treated HepG2 cells revealed that WF70 induces G2/M phase arrest, as evidenced by the dose-dependent accumulation of cells in the G2/M phase alongside a decrease in cells in the G0/G1 phase (Fig. 6). A potential mechanism behind this is interference with mitotic checkpoint regulators such as cyclin-dependent kinases and cyclins. Similarly, many plant-derived compounds are known to exert anticancer effects by modulating CDK1, cyclin B1, and other key regulators of mitotic entry (Jung et al. 2021; Otto and Sicinski 2017). G2/M phase arrest is a critical checkpoint that prevents mitosis in the presence of DNA damage or replication stress. The observed increase in G2/M-arrested cells suggests that WF70 activates a DNA damage response, leading to the upregulation of checkpoint kinases such as Chk1 and Chk2, which prohibit mitosis under compromised genomic conditions (Kastan and Bartek 2004). Moreover, since prolonged G2/M arrest often results in apoptosis or senescence, these findings further support the pro-apoptotic effects of WF70. In addition, G2/M arrest has also been found to sensitize cancer cells to DNA-damaging agents, including chemotherapy and radiation therapy (Zhou and Giannakakou 2005). Thus, WF70 could potentially enhance the efficacy of conventional anticancer treatments by inducing a checkpoint-dependent apoptotic response. The use of WF70 should be further studied in combination therapies in order to determine if it has synergy with standard liver cancer treatments, with the goal of improving therapeutic outcomes and reducing the required chemotherapy doses.

Aside from its effects on apoptosis and cell cycle regulation, WF70 can also influence broader oncogenic pathways associated with the progression of liver cancer. In addition to interfering with mitotic checkpoint regulators (i.e., CDK1 and Chk1/Chk2), WF70 may also modulate oxidative stress-related signaling pathways, including the Nrf2/Keap1 and MAPK pathways, which are crucial in tumor cell survival and regulating apoptosis (Kansanen et al. 2013). Oxidative stress and mitochondrial dysfunction are key regulators of apoptosis in HCC. The pro-apoptotic effects of many plant-derived bioactive compounds involve increasing reactive oxygen species (ROS) levels, resulting in mitochondrial membrane permeabilization, cytochrome c release, and caspase activation (Asma et al. 2022). Future studies should investigate whether the pro-apoptotic mechanism of WF70 involves oxidative stress-induced apoptosis, which can be elucidated through experimental approaches such as mitochondrial membrane potential assays, ROS quantification, and mitochondrial-specific assessments (MitoSOX, mtDNA damage analysis) (Deng et al. 2018; O’Malley et al. 2020).

Many plant-derived compounds exert cytotoxic effects by inducing oxidative stress, resulting in mitochondrial dysfunction and apoptotic activation (Lee et al. 2012). Since WF70 can activate both intrinsic and extrinsic apoptotic pathways, it may synergize with targeted therapies such as tyrosine kinase inhibitors to amplify apoptotic signaling. Over the past decade, various structure-based in silico approaches such as homology modeling, molecular docking and molecular dynamics (MD) simulation have been established as accurate and efficient tools for screening compounds against receptor proteins. In particular, serine/threonine kinases, which are key regulators of the mitotic checkpoint controlling cell division, have emerged as notable targets in cancer therapy, and the development of their inhibitors has remained a central focus of ongoing research. Therefore, an insilico screening strategy to identify potential checkpoint inhibitors from WF-derived compounds may lead to the discovery of novel therapeutic candidates for HCC) and serve as an important foundation for future drug development (Mishra et al. 2023a). Future investigations should explore combination strategies with existing chemotherapeutics and include transcriptomic, proteomic, and metabolomic analyses to better clarify the molecular targets of WF70 beyond the apoptotic pathway. This broader understanding could refine the therapeutic applications of WF70 and optimize its clinical translation.

The apoptotic effects of WF70 are strongly associated with the upregulation of pro-apoptotic genes such as Bax, Bid, Caspase-3, Caspase-8, and Caspase-9, as well as the downregulation of the anti-apoptotic gene Bcl-2 (Fig. 7). A schematic overview of these apoptotic pathways is illustrated in Fig. 8, which visually summarizes the dose-dependent effects of WF70 and WF30 on intrinsic and extrinsic apoptotic mechanisms in HepG2 cells. Therefore, WF70 modulates both intrinsic and extrinsic apoptotic pathways, which aligns with previous studies demonstrating the pro-apoptotic effects of plant-derived compounds in liver cancer models (Alarifi et al. 2017; Kaiser et al. 2008). In this study, Annexin V/PI staining was used to confirm the induction of apoptosis, revealing a dose-dependent increase in apoptotic cell populations. The TUNEL assay can also be used to assess apoptosis in HCC models, thereby providing insights into DNA fragmentation as a hallmark of apoptosis (Darzynkiewicz et al. 2008). However, Annexin V/PI staining is more commonly utilized because of its ability to differentiate between early and late apoptotic cells, as well as its compatibility with flow cytometry for quantitative analysis (Vermes et al. 1995), thereby providing a robust assessment of apoptosis.

Fig. 8.

Fig. 8

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Schematic representation of the dose-dependent apoptotic mechanism of the Woodfordia fruticosa (WF) extract on HepG2 cells. The diagram illustrates the extraction process of WF from flowers, yielding two ethanol extracts: WF30 (using 30% ethanol) and WF70 (using 70% ethanol). Treatment with WF70 and WF30 induces apoptosis in HepG2 cells via the mitochondrial pathway. The pro-apoptotic protein Bax is upregulated, whereas the anti-apoptotic protein Bcl-2 is downregulated, resulting in mitochondrial outer membrane permeabilization (MOMP). This disruption activates Bid cleavage and activates caspase-8, subsequently enhancing the mitochondrial apoptotic pathway. Caspase-9 activation further promotes caspase-3 cleavage, ultimately triggering apoptotic cell death. The schematic highlights the interplay between intrinsic and extrinsic apoptotic signaling cascades in WF-induced apoptosis, with WF70 exhibiting a stronger apoptotic effect than WF30

RT-PCR analysis confirmed that WF70 significantly upregulated Bax and Bid, which are key regulators of mitochondrial membrane permeability, leading to the release of cytochrome c and activation of downstream caspases. Meanwhile, the decrease in Bcl-2 expression suggests that WF70 disrupts survival signaling in a manner that favors apoptosis. Moreover, the concurrent increase in Caspase-3, Caspase-8, and Caspase-9 expression further suggests the involvement of mitochondrial-dependent and death receptor-mediated mechanisms (Kuwana et al. 2020; Wang et al. 2023).

These findings highlight the potential of WF70 as a dual-pathway apoptosis inducer, supporting its therapeutic relevance for HCC. The upregulation of Bax and Bid correlates with mitochondrial membrane permeabilization, leading to apoptotic execution via Caspase-3 activation, which is a hallmark of apoptosis (Korsmeyer et al. 2000; Heimlich et al. 2004). Meanwhile, the simultaneous activation of Caspase-8 and Caspase-9 suggests that the extrinsic and intrinsic apoptotic pathways are triggered in parallel (Singh et al. 2019).

Despite these promising in vitro findings, further in vivo studies need to confirm the efficacy and pharmacokinetic profile of WF70 in a physiological system. Understanding its absorption, metabolism, and systemic distribution will be crucial to optimize its therapeutic potential. Additionally, the safety profile of WF70, specifically regarding its selectivity for cancer cells over normal liver cells, needs to be confirmed before its clinical application.

One of the main limitations of this study was its focus on mRNA expression without protein level validation. Although qRT–PCRR provided transcriptional insights, gene expression does not always correlate with protein activity due to factors such as posttranscriptional modifications and mRNA stability (Heimlich et al. 2004). Promoter methylation validation may indicate a potential cause of tumor initiation and progression in HCC. To investigate the potential mechanisms of WF and identify novel therapeutic targets for HCC, differentially expressed genes (DEGs) and biomarkers are analyzed by through transcriptomic approaches. Several studies have demonstrated that identifying differentially expressed genes is a promising strategy for discovering potential therapeutic interventions (Mishra et al. 2023b; Lin et al. 2014). Furthermore, gene alteration analysis, which provides information on changes in prognostic biomarkers, can offer insights into how such alterations contribute to cancer progression and metastasis, as well as to detection, diagnosis, and prognosis. Ultimately, apoptosis is regulated at the protein level, and thus the functional impact of gene expression changes should be confirmed via Western blot or ELISA assays for Bax, Bcl-2, and Caspase proteins. Additionally, proteomic analyses are also needed to examine posttranslational modifications, which are crucial for protein activity.

Another limitation was that only GAPDH was used as a housekeeping gene for normalization. Although negative controls ensured specificity, relying on a single reference gene may introduce variability. Hub genes such as BUB1, BUB1B, CCNA2, CCNB2, KIF2C, and NCAPH are considered potential biomarkers, as their overexpression can lead to abnormal cell division, thereby promoting tumor formation and metastasis in HCC (Mishra et al. 2023c). Transcriptomic technologies such as RNA sequencing (RNA-seq), single-cell RNA sequencing (scRNA-seq), DropSeq, and inDrop not only elucidate the molecular mechanisms of disease and regulatory pathways influenced by gene transcription levels, regulatory properties, and drug interventions, but also enable the prediction of potential therapeutic benefits or adverse effects. These approaches support the transcriptomic quantification of WF’s mechanism of action and its potential conversion into responsive biomarkers (Yang et al. 2020). Multiple reference genes should be used in future studies to improve the reliability of gene expression analysis and confirm apoptotic marker regulation using alternative normalization strategies.

Despite the extensive in vitro experiments using HepG2 cells in this study, these cannot fully replicate the tumor microenvironment. HepG2 cells also have disadvantages regarding their lack of metastatic potential, deficient drug metabolism, and simplified genetic background. In addition, the traditional 2D monolayer cultures used in this study do not mimic the 3D architecture and hypoxic conditions of solid tumors. To address these limitations, future studies should include additional HCC cell lines, patient-derived organoids, and in vivo xenograft models. Moreover, incorporating 3D culture systems, coculture models with stromal and immune components, and advanced bioengineered tumor models can provide a more physiologically relevant assessment of the anticancer effects of WF70.

The limited bioavailability of WF70 represents a major challenge in translating our findings into therapeutic applications. Similarly, many plant-derived compounds exhibit poor solubility, rapid metabolism, and low systemic distribution, thereby reducing their clinical efficacy. To enhance the stability and targeted delivery of WF70, future studies should explore advanced drug delivery strategies, such as nanoparticle formulations or liposomal encapsulation.

From a safety perspective, systemic toxicity and off-target effects remain critical concerns, especially because the selectivity of WF70 for cancer cells over normal liver cells has not been confirmed. This study did not assess the cytotoxic effects of WF70 on noncancerous hepatocytes, which is crucial in anticancer drug development. The toxicity profile of WF70 needs to be further characterized by evaluating normal liver cell lines and hepatotoxicity markers. In vivo toxicity assessments and dose optimization studies will be necessary before the clinical translation of our findings.

Despite these limitations, this study provides valuable insights into the pro-apoptotic mechanisms of WF70, specifically by demonstrating its ability to regulate apoptotic gene expression and induce cell cycle arrest. This research lays the groundwork for further therapeutic investigation, and further validation in other liver cancer models is necessary to fully elucidate the potential of WF70 as a novel anticancer agent.

Conclusions

Liver cancer remains a challenging disease to treat, partly due to abnormalities in cellular death processes. This study elucidates the influence of WF70 extract in specific apoptotic pathways in HepG2 cells. In particular, pro-apoptotic genes such as Bax, Bid, Caspase-3, Caspase-8, and Caspase-9 were upregulated, whereas the anti-apoptotic gene Bcl-2 was downregulated. These findings suggest the potential of WF as a novel therapeutic agent in liver cancer, although these promising observations from laboratory studies represent only the initial steps. To move toward clinical application, the safety and efficacy of WF need to be further confirmed in animal studies and human trials.

Acknowledgements

This work was supported by a Research Grant from Pukyong National University (2024)

Author contributions

Byeong-Su Kang†: Methodology (equal), Formal analysis (equal), Investigation (equal), Data curation (equal), Writing—original draft (equal), Visualization (equal). Kyubae Lee†: Writing—review and editing (equal), Data curation (equal). Hyeon-Ju Lee: Investigation (equal), Validation (lead), Formal analysis (equal). Hyeon-Ji Han: Data curation (equal), Visualization (equal). Gyunam Kim: Methodology (equal), Writing—review and editing (equal). Dain Kim: Writing—review and editing (equal). Eon-Bee Lee: Conceptualization (lead), Methodology (equal), Investigation (equal), Writing—original draft (equal), Writing—review and editing (lead), Supervision (lead), Project administration (lead), Funding acquisition (lead). †These authors contributed equally to this work.

Data availability

The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Conflict of interest

The authors have no conflict of interest to declare.

Footnotes

Byeong-Su Kang and Kyubae Lee have contributed equally to this work.

References

  1. Abotaleb M, Samuel SM, Varghese E et al (2018) Flavonoids in cancer and apoptosis. Cancers (Basel). 10.3390/cancers11010028 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Abubakar AR, Haque M (2020) Preparation of medicinal plants: basic extraction and fractionation procedures for experimental purposes. J Pharm Bioallied Sci 12:1–10. 10.4103/jpbs.JPBS_175_19 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Alarifi S, Ali H, Alkahtani S, Alessia MS (2017) Regulation of apoptosis through bcl-2/bax proteins expression and DNA damage by nano-sized gadolinium oxide. Int J Nanomedicine 12:4541–4551. 10.2147/IJN.S139326 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Asma ST, Acaroz U, Imre K et al (2022) Natural products/bioactive compounds as a source of anticancer drugs. Cancers (Basel) 14:6203 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Beheshti F, Shabani AA, Akbari Eidgahi MR et al (2021) Anticancer Activity of Ipomoea purpurea leaves extracts in monolayer and three-dimensional cell culture. Evid Based Complement Altern Med 2021:6666567. 10.1155/2021/6666567 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blum HE (2005) Hepatocellular carcinoma: therapy and prevention. World J Gastroenterol 11:7391–7400. 10.3748/wjg.v11.i47.7391 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brigger I, Dubernet C, Couvreur P (2002) Nanoparticles in cancer therapy and diagnosis. Adv Drug Deliv Rev 54:631–651. 10.1016/s0169-409x(02)00044-3 [DOI] [PubMed] [Google Scholar]
  8. Carneiro BA, El-Deiry WS (2020) Targeting apoptosis in cancer therapy. Nat Rev Clin Oncol 17:395–417. 10.1038/s41571-020-0341-y [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chandan BK, Saxena AK, Shukla S et al (2008) Hepatoprotective activity of Woodfordia fruticosa Kurz flowers against carbon tetrachloride induced hepatotoxicity. J Ethnopharmacol 119:218–224. 10.1016/j.jep.2008.06.020 [DOI] [PubMed] [Google Scholar]
  10. Chemat F, Abert Vian M, Ravi HK et al (2019) Review of alternative solvents for green extraction of food and natural products: panorama, principles, applications and prospects. Molecules. 10.3390/molecules24163007 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Constantinou A, Stoner GD, Mehta R et al (1995) The dietary anticancer agent ellagic acid is a potent inhibitor of DNA topoisomerases in vitro. Nutr Cancer 23:121–130. 10.1080/01635589509514368 [DOI] [PubMed] [Google Scholar]
  12. Cragg GM, Newman DJ (2005) Plants as a source of anti-cancer agents. J Ethnopharmacol 100:72–79. 10.1016/j.jep.2005.05.011 [DOI] [PubMed] [Google Scholar]
  13. Darzynkiewicz Z, Galkowski D, Zhao H (2008) Analysis of apoptosis by cytometry using TUNEL assay. Methods 44:250–254. 10.1016/j.ymeth.2007.11.008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Das PK, Goswami S, Chinniah A et al (2007) Woodfordia fruticosa: traditional uses and recent findings. J Ethnopharmacol 110:189–199 [DOI] [PubMed] [Google Scholar]
  15. Das U, Biswas S, Chattopadhyay S et al (2017) Radiosensitizing effect of ellagic acid on growth of Hepatocellular carcinoma cells: an in vitro study. Sci Rep 7:14043. 10.1038/s41598-017-14211-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Deng Y, Li X, Li X et al (2018) Corilagin induces the apoptosis of hepatocellular carcinoma cells through the mitochondrial apoptotic and death receptor pathways. Oncol Rep 39:2545–2552. 10.3892/or.2018.6396 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Do QD, Angkawijaya AE, Tran-Nguyen PL et al (2014) Effect of extraction solvent on total phenol content, total flavonoid content, and antioxidant activity of Limnophila aromatica. J Food Drug Anal 22:296–302. 10.1016/j.jfda.2013.11.001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Dubey D, Meher RK, Pradhan B et al (2024) Green synthesis of silver nanoparticles using leaf extract of Woodfordia fruticosa (L.) for potential therapeutic application. J Chem Technol Biotechnol 99:1743–1753. 10.1002/jctb.7681 [Google Scholar]
  19. Etxebarria A, Landeta O, Antonsson B, Basañez G (2008) Regulation of antiapoptotic MCL-1 function by gossypol: mechanistic insights from in vitro reconstituted systems. Biochem Pharmacol 76:1563–1576. 10.1016/j.bcp.2008.08.003 [DOI] [PubMed] [Google Scholar]
  20. Fabregat I (2009) Dysregulation of apoptosis in hepatocellular carcinoma cells. World J Gastroenterol 15:513–520 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Fulda S, Debatin K-M (2006) Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene 25:4798–4811. 10.1038/sj.onc.1209608 [DOI] [PubMed] [Google Scholar]
  22. Gao Z, Gao W, Zeng S-L et al (2018) Chemical structures, bioactivities and molecular mechanisms of citrus polymethoxyflavones. J Funct Foods 40:498–509. 10.1016/j.jff.2017.11.036 [Google Scholar]
  23. Giri S, Dey G, Sahu R et al (2023) Traditional uses, phytochemistry and pharmacological activities of Woodfordia fruticosa (L) Kurz: a comprehensive review. Indian J Pharm Sci. 10.36468/pharmaceutical-sciences.1062 [Google Scholar]
  24. Granado-Serrano AB, Martín MÁ, Bravo L et al (2012) Quercetin attenuates TNF-induced inflammation in hepatic cells by inhibiting the NF-κB pathway. Nutr Cancer 64:588–598. 10.1080/01635581.2012.661513 [DOI] [PubMed] [Google Scholar]
  25. Gromowski T, Lukacs-Kornek V, Cisowski J (2023) Current view of liver cancer cell-of-origin and proposed mechanisms precluding its proper determination. Cancer Cell Int 23:3. 10.1186/s12935-022-02843-0 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Heimlich G, McKinnon AD, Bernardo K et al (2004) Bax-induced cytochrome c release from mitochondria depends on alpha-helices-5 and -6. Biochem J 378:247–255. 10.1042/BJ20031152 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hussein RH, Khalifa FK (2014) The protective role of ellagitannins flavonoids pretreatment against N-nitrosodiethylamine induced-hepatocellular carcinoma. Saudi J Biol Sci 21:589–596. 10.1016/j.sjbs.2014.03.004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Jung Y, Kraikivski P, Shafiekhani S et al (2021) Crosstalk between Plk1, p53, cell cycle, and G2/M DNA damage checkpoint regulation in cancer: computational modeling and analysis. NPJ Syst Biol Appl 7:46. 10.1038/s41540-021-00203-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kadota S, Takamori Y, Nyein KN et al (1990) Constituents of the leaves of Woodfordia fruticosa Kurz. I. Isolation, structure, and proton and carbon-13 nuclear magnetic resonance signal assignments of woodfruticosin (woodfordin C), an inhibitor of deoxyribonucleic acid topoisomerase II. Chem Pharm Bull (Tokyo) 38:2687–2697. 10.1248/cpb.38.2687 [DOI] [PubMed] [Google Scholar]
  30. Kaiser CL, Chapman BJ, Guidi JL et al (2008) Comparison of activated caspase detection methods in the gentamicin-treated chick cochlea. Hear Res 240:1–11. 10.1016/j.heares.2008.03.003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kansanen E, Kuosmanen SM, Leinonen H, Levonen A-L (2013) The Keap1-Nrf2 pathway: mechanisms of activation and dysregulation in cancer. Redox Biol 1:45–49. 10.1016/j.redox.2012.10.001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kastan MB, Bartek J (2004) Cell-cycle checkpoints and cancer. Nature 432:316–323. 10.1038/nature03097 [DOI] [PubMed] [Google Scholar]
  33. Khan T, Ali M, Khan A et al (2019) Anticancer plants: a review of the active phytochemicals, applications in animal models, and regulatory aspects. Biomolecules. 10.3390/biom10010047 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Korsmeyer SJ, Wei MC, Saito M et al (2000) Pro-apoptotic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c. Cell Death Differ 7:1166–1173. 10.1038/sj.cdd.4400783 [DOI] [PubMed] [Google Scholar]
  35. Kuramochi-Motegi A, Kuramochi H, Kobayashi F et al (1992) Woodfruticosin (woodfordin C), a new inhibitor of DNA topoisomerase II. Experimental Antitumor Activity. Biochem Pharmacol 44:1961–1965. 10.1016/0006-2952(92)90098-4 [DOI] [PubMed] [Google Scholar]
  36. Kuwana T, King LE, Cosentino K et al (2020) Mitochondrial residence of the apoptosis inducer BAX is more important than BAX oligomerization in promoting membrane permeabilization. J Biol Chem 295:1623–1636. 10.1074/jbc.RA119.011635 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Lee J, Giordano S, Zhang J (2012) Autophagy, mitochondria and oxidative stress: cross-talk and redox signalling. Biochem J 441:523–540. 10.1042/BJ20111451 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Lin C-C, Chao P-Y, Shen C-Y et al (2014) Novel target genes responsive to apoptotic activity by Ocimum gratissimum in human osteosarcoma cells. Am J Chin Med 42:743–767. 10.1142/S0192415X14500487 [DOI] [PubMed] [Google Scholar]
  39. Liu RH (2003) Health benefits of fruit and vegetables are from additive and synergistic combinations of phytochemicals. Am J Clin Nutr 78:517S-520S. 10.1093/ajcn/78.3.517S [DOI] [PubMed] [Google Scholar]
  40. Llovet JM, Kelley RK, Villanueva A et al (2021) Hepatocellular carcinoma. Nat Rev Dis Prim 7:6. 10.1038/s41572-020-00240-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Lohvina H, Sándor M, Wink M (2022) Effect of Ethanol Solvents on Total Phenolic Content and Antioxidant Properties of Seed Extracts of Fenugreek (Trigonella foenum-graecum L.) Varieties and Determination of Phenolic Composition by HPLC-ESI-MS. Diversity 14:7 [Google Scholar]
  42. Marquardt JU, Edlich F (2019) Predisposition to apoptosis in hepatocellular carcinoma: from mechanistic insights to therapeutic strategies. Front Oncol 9:1421. 10.3389/fonc.2019.01421 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Matthews HK, Bertoli C, de Bruin RAM (2022) Cell cycle control in cancer. Nat Rev Mol Cell Biol 23:74–88. 10.1038/s41580-021-00404-3 [DOI] [PubMed] [Google Scholar]
  44. Mishra D, Mishra A, Rai SN et al (2023a) In silico insight to identify potential inhibitors of BUB1B from mushroom bioactive compounds to prevent breast cancer metastasis. Front Biosci 28:151. 10.31083/j.fbl2807151 [DOI] [PubMed] [Google Scholar]
  45. Mishra D, Mishra A, Nand Rai S et al (2023b) Demystifying the role of prognostic biomarkers in breast cancer through integrated transcriptome and pathway enrichment analyses. Diagnostics (Basel, Switzerland). 10.3390/diagnostics13061142 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Mishra D, Mishra A, Rai SN et al (2023c) Identification of prognostic biomarkers for suppressing tumorigenesis and metastasis of hepatocellular carcinoma through transcriptome analysis. Diagnostics (Basel, Switzerland). 10.3390/diagnostics13050965 [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Neagu M, Piperigkou Z, Karamanou K et al (2017) Protein bio-corona: critical issue in immune nanotoxicology. Arch Toxicol 91:1031–1048. 10.1007/s00204-016-1797-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Nelson VK, Sahoo NK, Sahu M et al (2020) In vitro anticancer activity of Eclipta alba whole plant extract on colon cancer cell HCT-116. BMC Complement Med Ther 20:355. 10.1186/s12906-020-03118-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Nitha A, Prabha SP, Ansil PN, Latha MS (2013) Antiproliferative effect of Woodfordia fruticosa Kurz flowers on experimentally induced hepatocellular carcinoma in rats and in human hepatoma cell line. J Pharm Res 6:239–248. 10.1016/j.jopr.2013.02.003 [Google Scholar]
  50. Nitha A, Prabha SP, Ansil PN, Latha M (2014) Curative effect of woodfordia fruticosa kurz flowers on n-nitrosodiethylamine induced hepatocellular carcinoma in rats. Int J Pharm Pharm Sci 6:150–155 [Google Scholar]
  51. O’Malley J, Kumar R, Inigo J et al (2020) Mitochondrial stress response and cancer. Trends in Cancer 6:688–701. 10.1016/j.trecan.2020.04.009 [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Otto T, Sicinski P (2017) Cell cycle proteins as promising targets in cancer therapy. Nat Rev Cancer 17:93–115. 10.1038/nrc.2016.138 [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45. 10.1093/nar/29.9.e45 [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Prajapati C, Rai SN, Singh AK et al (2025) Untargeted metabolomics and bioactivities assessment of xylaria ellisii, an endophytic fungus isolated from the leaf of the plant Acorus calamus Linn. Curr Pharm des. 10.2174/0113816128337697250106001808 [DOI] [PubMed] [Google Scholar]
  55. Pucci B, Kasten M, Giordano A (2000) Cell cycle and apoptosis. Neoplasia 2:291–299. 10.1038/sj.neo.7900101 [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Shofian NM, Hamid AA, Osman A et al (2011) Effect of freeze-drying on the antioxidant compounds and antioxidant activity of selected tropical fruits. Int J Mol Sci 12:4678–4692. 10.3390/ijms12074678 [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Singh R, Letai A, Sarosiek K (2019) Regulation of apoptosis in health and disease: the balancing act of BCL-2 family proteins. Nat Rev Mol Cell Biol 20:175–193. 10.1038/s41580-018-0089-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Sriram MI, Kanth SBM, Kalishwaralal K, Gurunathan S (2010) Antitumor activity of silver nanoparticles in Dalton’s lymphoma ascites tumor model. Int J Nanomedicine 5:753–762. 10.2147/IJN.S11727 [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Stanciauskaite M, Marksa M, Babickaite L et al (2021) Comparison of Ethanolic and Aqueous Populus balsamifera L. Bud Extracts by Different Extraction Methods: chemical Composition, Antioxidant and Antibacterial Activities. Pharmaceuticals (Basel). 10.3390/ph14101018 [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Sujanamulk B, Shyam Sunder S, Ratnakar Pawar B et al (2020) Comparison of antifungal efficacy of ethanolic extracts of Woodfordia fruticosa leaf and Punica granatum peel in uncontrolled diabetic patients wearing removable dentures: a randomized controlled clinical trial. Curr Med Mycol 6:15–20. 10.18502/cmm.6.3.3983 [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Sun L, Zhou W, Zhang H et al (2019) Modulation of multiple signaling pathways of the plant-derived natural products in cancer. Front Oncol 9:1153 [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Tayab MA, Chowdhury KAA, Jabed M et al (2021) Antioxidant-rich woodfordia fruticosa leaf extract alleviates depressive-like behaviors and impede hyperglycemia. Plants (Basel, Switzerland). 10.3390/plants10020287 [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Thakur S, Kaurav H, Chaudhary G (2021) A review on woodfordia fruticosa kurz (Dhatki): ayurvedic, folk and modern uses. J Drug Deliv Ther 11:126–131. 10.22270/jddt.v11i3.4839 [Google Scholar]
  64. Thumpati P, Rai SN, Prajapati C et al (2025) Targeting c-MYC G-Quadruplexes for cancer treatment with small molecules. Sci Pharm 93:6 [Google Scholar]
  65. Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C (1995) A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods 184:39–51. 10.1016/0022-1759(95)00072-i [DOI] [PubMed] [Google Scholar]
  66. Vieira C, Rebocho S, Craveiro R et al (2022) Selective extraction and stabilization of bioactive compounds from rosemary leaves using a biphasic NADES. Front Chem 10:954835. 10.3389/fchem.2022.954835 [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Wang Y, Li J, Xia L (2023) Plant-derived natural products and combination therapy in liver cancer. Front Oncol 13:1116532. 10.3389/fonc.2023.1116532 [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Wong RSY (2011) Apoptosis in cancer: from pathogenesis to treatment. J Exp Clin Cancer Res 30:87. 10.1186/1756-9966-30-87 [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Yang X, Kui L, Tang M et al (2020) High-throughput transcriptome profiling in drug and biomarker discovery. Front Genet 11:19. 10.3389/fgene.2020.00019 [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Yoshida T, Chou T, Matsuda M et al (1991) Woodfordin D and oenothein A, trimeric hydrolyzable tannins of macro-ring structure with antitumor activity. Chem Pharm Bull (Tokyo) 39:1157–1162. 10.1248/cpb.39.1157 [DOI] [PubMed] [Google Scholar]
  71. Zhang Q-W, Lin L-G, Ye W-C (2018) Techniques for extraction and isolation of natural products: a comprehensive review. Chin Med 13:20. 10.1186/s13020-018-0177-x [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Zhang L, Chen L, Tao D et al (2023) Tannin alleviates glyphosate exposure-induced apoptosis, necrosis and immune dysfunction in hepatic L8824 cell line by inhibiting ROS/PTEN/PI3K/AKT pathway. Comp Biochem Physiol C Toxicol Pharmacol 266:109551. 10.1016/j.cbpc.2023.109551 [DOI] [PubMed] [Google Scholar]
  73. Zhou J, Giannakakou P (2005) Targeting microtubules for cancer chemotherapy. Curr Med Chem Anticancer Agents 5:65–71. 10.2174/1568011053352569 [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.

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