Unveiling the anticancer potential of Anamirta cocculus (L.) Wight& Arn.: Evidences from cytotoxicity studies, apoptosis analysis, and molecular docking

Abstract

Anamirta cocculus, a woody climber, is extensively utilised in traditional Asian medicine. This study investigates the cytotoxic effects of A. cocculus leaf extracts on various cancer cell lines as well as on a normal cell line. The ethyl acetate extract exhibited potent anticancer activity, with the highest cytotoxicity observed against ovarian cancer cell line (PA1) (IC₅₀ = 8.30 ± 0.38 µg/mL) and colorectal adenocarcinoma cell line (HT29) (IC₅₀ = 17.97 ± 0.63 µg/mL). Notably, the extract displayed low toxicity (18.72 ± 0.73%) on the normal human keratinocyte cell line (HaCaT) at a concentration of 100 µg/mL, indicating selective cytotoxicity towards cancer cells. The acetone extract also demonstrated significant cytotoxicity against various cancer cell lines, including A498, MG63, PA1, and UM-SCC-83B. The ethyl acetate extract of A. cocculus demonstrated potent inhibition of colony formation in HT29 and PA1 cancer cell lines while inducing apoptosis, as evidenced by membrane blebbing, chromatin condensation, and DNA fragmentation. The number of late apoptotic cells increased with an increase in concentrations of ACLE. Molecular docking studies of compounds identified through GC–MS analysis revealed strong interactions with key apoptotic proteins, including caspase-8, p53, caspase-3, and caspase-9. Compounds such as vitamin E, epoxylathyrol, squalene, and phytol showed high binding affinity to these proteins, suggesting their role in apoptosis induction. The possibility of induction of apoptotic proteins through indirect interaction by binding to other proteins or receptors cannot be ruled out. The cytotoxic effects may result from individual, combined, or synergistic actions of these compounds. Among these, epoxylathyrol emerged as a particularly promising anticancer drug candidate based on ADME analysis and binding affinity assessments, warranting further investigation.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-024-04096-2.

Introduction

Cancer represents a severe and life-threatening global health issue. Conventional treatment modalities, such as chemotherapy, radiation therapy, and surgery, are limited by issues including recurrence, severe side effects, and high financial costs. As a result, there is an urgent need for the development of novel, safe, and more effective therapeutic strategies (Choudhari et al. 2020). Consequently, there is a renewed interest in identifying novel anticancer molecules from plants, as plants offer a repository of bioactive molecules. This is supported by the fact that many drugs currently used for the treatment of cancer are isolated from plants. In addition, various medicinal plants have been directly utilised as a primary therapeutic option due to their perceived therapeutic values. Such plants serve as a valuable source of distinctive chemical compounds with the potential for therapeutic effects (Debela et al. 2021). Phytochemicals and their derivatives found in plants show promising efficacy in cancer treatment and reducing adverse reactions. Many of these natural compounds possess significant antitumor potential. The development of effective anticancer therapies based on phytochemicals involves initial testing of natural extracts from plants or plant materials, followed by active compound purification through bioassay-guided fractionation (Fridlender et al. 2015).

The increasing incidence of cancer, along with the significant limitations of conventional treatments, such as elevated costs and toxicity, presents a major obstacle in cancer therapy. In light of this, phytomolecules are expected to play a pivotal role in the future of cancer treatment. Their high biodegradability and biocompatibility make them promising candidates for integration into cancer therapeutic strategies (Yadav et al. 2020). Several phytochemicals with potential anticancer activity, including curcumin, epigallocatechin, isothiocyanates, gossypol, sulforaphane, and garcinol, are currently being investigated across various experimental models to evaluate their therapeutic efficacy and toxicological profiles (Ijaz et al. 2018).

This study explores the anticancer potential of the leaf extract of Anamirta cocculus (L.) Wight & Arn., a woody climber of the Menispermaceae family, widely recognized in Asia for its traditional medicinal applications. The stems and roots of A. cocculus are notably rich in alkaloids, which contribute to their antibacterial, antimicrobial, and sympatholytic properties. Furthermore, its seeds contain picrotoxin, a compound historically employed as an antidote for barbiturate and morphine poisoning, as well as a nerve tonic for the management of neurological disorders, including schizophrenia and epilepsy, despite its inherent toxicity.

To date, no studies have been reported in the literature regarding the anticancer effects of Anamirta cocculus extracts. In this study, we investigated the cytotoxic potential l of this plant against ten cancer cell lines and a normal cell line. The mechanism of cell death induced by the leaf extract was explored through in vitro assays on both cancerous and normal cell lines. To identify potential molecular targets, in silico prediction techniques were employed, providing insights into the interactions between the extract's bioactive compounds and key proteins involved in apoptotic pathways.

The findings from this study could pave the way for the isolation of novel anticancer compounds from A. cocculus, opening new avenues for further research and potential therapeutic applications in cancer treatment.

Materials and methods

Collection and sample preparation

A. cocculus was collected from Palakkad, Kerala, India (10.749448º N and 76.121046º E). Plant specimen was identified by Dr. A. K. Pradeep, Assistant Professor, Department of Botany, University of Calicut, and was deposited in the Calicut University Herbarium with the accession number 7190. The leaves were washed in running water, dried in an oven at 40ºC, and powdered. Extracts were prepared from the leaf powder by sequential extraction with solvents with increasing polarity in the order petroleum ether, chloroform, ethyl acetate, acetone, and water. Each of the solvent extracts was dried and dissolved in DMSO and used for further analyses.

Cell lines and cell culture

The cell lines used in the present study were human colorectal adenocarcinoma cell lines (HT29, HCT116), human epidermoid carcinoma cell line (A431), human renal carcinoma cell line (A498), human cervical cancer cell line (HeLa), human breast adenocarcinoma cell line (MCF7), human osteosarcoma cell line (MG63), human ovarian teratocarcinoma cell line (PA1), and human pancreatic epithelioid carcinoma cell line (PANC1) were purchased from National Centre for Cell Science (Pune), India, and human squamous cell carcinoma cell line (UM-SCC-83B), and human keratinocyte cell line (HaCaT) were obtained as a gift. HT29 was maintained and propagated in McCoy’s 5A medium, while HCT116, HeLa, MCF7, PANC1, and UM-SCC-83B were cultured in DMEM, and A431, A498, MG63, and PA1 were cultured in MEM medium. All media contained 10% (v/v) foetal bovine serum, along with streptomycin (100 µg/mL) and penicillin (100U/mL). All the cultures were incubated at 37 °C in a humidified environment with 5% CO₂. All assays were done after treating with the extracts for 48 h, except where otherwise mentioned.

Cytotoxicity assay

To assess cytotoxicity, all cancer and normal cell lines were treated with A. cocculus leaf extracts at a concentration of 100 µg/mL, prepared in DMSO, and incubated for 48 h at 37ºC under 5% carbon dioxide. The cytotoxicity was assessed by MTT assay (Mosmann 1983). For calculation of percentage mortality, the viability of cells grown in medium containing vehicle alone (0.1% DMSO) was considered to be 100%. IC₅₀ values of the extracts were determined for cell lines exhibiting more than 50% mortality by treating them with different concentrations of extracts. Probit analysis was carried out to calculate the IC₅₀ value using IBM SPSS statistics 24.

Trypan blue dye exclusion

Cell viability assessment of ethyl acetate extract of A. cocculus leaf (ACLE) was conducted on PA1 and HT29 cell lines employing the trypan blue dye exclusion method. Cells displaying compromised cell membrane integrity, indicative of cell death, exhibited a distinct blue staining (Strober 2001).

Microscopy

Cytomorphological changes induced by the extracts were observed after 48 h using phase contrast microscopy and a field emission scanning electron microscope (FE-SEM). For light microscopy, both the control and treated cells were washed with ice-cold phosphate-buffered saline (pH 7.4) and visualised under an inverted microscope. For FE-SEM, the control and treated cells were harvested, rinsed with ice-cold PBS, and then fixed in 4% glutaraldehyde in PBS. The cells were washed twice with ice-cold PBS and later dehydrated by passing through a series of ascending concentrations of acetone (75–85-100%), and images were acquired with CARL-Zeiss Gemini 300 SEM (Fischer et al. 2012).

Colony forming assay

Around 500 cells were seeded in 12-wells plate, and incubated for 24 h in CO₂ incubator. Subsequently, the cells were subjected to treatment with varying concentrations of the extract and incubated for seven days. A vehicle control with 0.1% DMSO was also kept. Later, the cells were fixed in ice-cold methanol and stained with 0.25% Giemsa stain. Images were taken under a stereomicroscope, and colonies were counted manually (Franken et al. 2006).

Scratch wound healing assay to assess cell migration

Cells were seeded in a 12-wells plate to obtain a confluent monolayer and scratched in the middle to create an artificial gap. The dislodged and floating cells were removed by a gentle wash with a growth medium. Cells were then exposed to IC₅₀ concentrations of the extracts, and the cell migratory capacity in terms of gap closure was assessed at 0, 24, and 48 h by taking a series of photographs (Martinotti and Ranzato 2020).

Assays for apoptosis

DNA degradation assay

Both treated and untreated cells were re-suspended in lysis buffer (20 mM Tris; pH 8.0, 10 mM EDTA, and 0.2% Triton X-100) after a PBS wash, followed by incubation for 15 min at room temperature. DNA extraction from these cell lysates was carried out using conventional methods as previously described (Gong et al. 1994). The isolated DNA was separated on a 1.2% agarose gel and photographed using the gel documentation system.

Genotoxicity evaluation by alkaline comet assay

The comet assay was employed for the identification of DNA strand breaks in individual cells, with gel electrophoresis conducted under alkaline conditions following a protocol previously outlined (Pu et al. 2015).

Acridine orange/ethidium bromide dual staining

The control and treated cells were collected, rinsed with ice-cold PBS, and adjusted to a cell density of 1X10⁴ cells/mL using PBS. Subsequently, the cell suspension was supplemented with acridine orange (AO)-ethidium bromide (EB) solution (1:1) to achieve a final concentration of 100 µg/mL prior to observation under a fluorescence microscope (Liu et al. 2015). The percentage of apoptotic cells was calculated.

Assessment of mitochondrial membrane potential (δψm)

The mitochondrial membrane potential of both untreated and treated cells was assessed using a lipophilic cationic dye, rhodamine-123 (5 µg/mL) staining for 30 min at 37 °C, and analysed for rhodamine-123 fluorescence using fluorescent microscopy (Lu et al. 2018). Additionally, cells were counterstained with DAPI. The increased permeability of the apoptotic cell membrane allowed more DAPI to enter, resulting in more intense blue colouration (Wallberg et al. 2016).

Identification of compounds in extracts by GC–MS analysis

GC–MS analysis of the ethyl acetate extract and acetone extract was conducted using a Shimadzu gas chromatography system equipped with an ELITE-5MS column with 30 m length, 0.25 mm ID, and 0.25 µm thickness. Initially, the oven temperature was maintained at 80 °C for 1 min, then gradually increased to 260 °C at a rate of 5 °C/min. Helium was utilised as a carrier gas, adjusted to achieve a column velocity flow of 1 mL/min. A sample volume of 1 μL was injected into the system. Identification of components was performed by comparing mass spectral fragmentation patterns with those stored in the MS library [NIST11 (National Institute of Standards and Technology, USA) and amp; WILEY 8].

In vitro antioxidant activity measurement by DPPH scavenging assay

The DPPH (1,1-diphenyl-2-picryhydrazyl) radical scavenging activity of the extracts was evaluated using L-ascorbic acid as a positive control, as previously described (Baliyan et al. 2022). DMSO was used as the solvent control.

In silico docking studies

Molecular docking was conducted to explore the location where the ligand binds to the protein and to understand the mechanism of their interaction, using AutodockVina software (Trott and Olson 2010). Native 3D protein structures of target proteins were downloaded from the RCSB PDB database (https://www.rcsb.org/) followed by preparation for docking using Discovery Studio 2021 client software. Structure of the compounds identified in GC–MS (ligands) was downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/), prepared in Open Babel GUI (O'Boyle et al. 2011) software, and virtual molecular screening was done by multiple ligand docking using PyRx software. The resulting protein–ligand interaction model 3D interactions were visualised with the Discovery Studio 2021 version.

The three-dimensional conformer structures of nine compounds identified from the ACLE extract were retrieved from the PubChem compound repository (Kim et al. 2016) and converted into PDBQT files using the MGLTools 1.5.7 within AutoDock Vina. Additionally, the three-dimensional crystal structures of crucial proteins involved in apoptosis, including caspase-3 (PDB ID-3DEI), caspase-8 (PDB ID-3KJQ), caspase-9 (PDB ID-1JXQ), and TP53 (PDB ID-3DCY), were obtained from the RCSB PDB (https://www.rcsb.org) (Berman et al. 2000). These proteins underwent preparation using MGLTools 1.5.7 integrated into Autodock Vina, which involved removing water molecules, addition of Kollaman charges, and polar hydrogens, and then converting them into pdbqt format.

For generating receptor grids and conducting molecular docking experiments, PyRX (Dallakyan and Olson 2015) was utilized. Docking procedures were executed, and only the best docking score was obtained using Discovery Studio Visualiser.

n silico ADME analysis

The pharmacokinetic characteristics of the identified compounds were assessed based on Lipinski’s rule of five (Lipinski et al. 2001), taking into account factors such as water solubility and skin permeation values (Daina et al. 2017). The web tool Swiss ADME (http://www.swissadme.ch/index.php) was employed to analyse the ADME parameters of all the GC–MS-identified compounds.

Statistical analysis

Results were expressed as mean values with standard error of the mean (SEM), based on observations from four separate experiments with each experiment in triplicates. The IC₅₀ was determined through probit analysis using IBM SPSS Statistics 24. Data visualisation was carried out using Origin (Version 9) and Graphpad Prism 10.2.2 (397). Statistical significance of the differences between means was assessed using ANOVA with Dunnett’s multiple comparison test, performed with GraphPad Prism 10.2.2 (397), and significance was accepted at p < 0.05.

Results

Anamirta cocculus extracts exhibit differential cytotoxicity: The MTT assay was employed to assess the cytotoxicity of A. cocculus leaf extracts prepared with petroleum ether (ACLP), chloroform (ACLC), ethyl acetate (ACLE), acetone (ACLA), methanol (ACLM), and water (ACLW). The cell lines employed for the cytotoxicity were HT29, HCT116, A431, A498, HeLa, MCF7, MG63, PA1, PANC1, UM-SCC-83B, and HaCaT. Among these, ACLE extract exhibited higher cytotoxicity across the cell lines tested, with PA1 showing the highest mortality (98.64 ± 0.40%), followed by HT29, MG63, PANC1, HCT116, A498, MCF7, UM-SCC-83B, and A431. Of the different extracts, ACLA also demonstrated significant cytotoxicity, specifically against PA1 and MG63. All six extracts showed minimal cytotoxicity towards the human keratinocyte cell line, HaCaT, indicating differential toxicity towards cancer cell lines (Table 1).

Table 1.

Differential cytotoxicity of various organic solvent extracts on different cancer cell lines, and non-cancerous human keratinocyte cell line. Mortality rate after 48 h of incubation is listed in the table as mean ± SEM

Cell line	Percentage mortality (48 hrs) mean±SEM
	Petroleum ether extract(ACLP)	Chloroform extract (ACLC)	Ethyl acetate extract (ACLE)	Acetone extract (ACLA)	Methanol extract (ACLM)	Water extract (ACLW)
A431	5.74±0.87	4.17±1.04	57.54±2.93	51.35±3.18	0.75±2.92	6.58±0.98
A498	15.06±2.55	1.09±1.72	68.54±0.69	66.54±1.70	13.00±0.92	12.71±1.58
HCT116	10.78±1.82	9.47±2.66	69.18±2.26	31.52±1.39	9.68±2.39	11.53±2.25
HeLa	18.21±3.47	9.18±1.71	60.11±1.24	37.85±2.31	15.91±3.40	6.78±3.07
HT29	0.63±1.03	16.75±1.34	91.16±0.24	25.01±1.83	15.33±2.37	12.29±1.78
MCF7	5.42±2.69	1.17±1.57	67.23±1.47	20.84±2.55	12.61±2.58	6.19±2.57
MG63	11.56±2.45	5.48±2.20	86.76±1.04	81.33±1.33	8.10±2.69	17.33±2.22
PA1	4.77±2.81	7.19±0.84	98.64±0.40	92.72±0.58	4.06±1.43	2.87±2.21
PANC1	24.73±2.31	24.35±1.08	79.16±1.32	33.46±1.75	28.13±3.91	26.79±1.65
UM-SCC-83B	7.51±2.61	1.61±1.60	62.44±1.07	61.59±0.77	43.93±0.10	24.42±3.29
HaCaT	5.43±1.90	17.55±2.64	18.72±0.73	30.23±0.10	10.57±1.88	29.54±3.29

Percentage mortalities above 90% are shown in bold

Values are represented as mean ± SEM (n = 6)

IC₅₀ of ACLE extract and ACLA extract on different cell lines: A concentration-dependent cytotoxicity was observed for both ACLE extract and ACLA extract when tested towards cell lines HT29, HCT116, A431, A498, HeLa, MCF7, MG63, PA1, PANC1, UM-SCC-83B, and HaCaT. The lowest IC₅₀ observed was for ACLE extract towards PA1 with an IC₅₀ value of 8.30 ± 0.38 µg/mL (Table 2). Thus, the extract may contain potent anticancer molecule(s). Therefore, it will be worth investigating to purify and identify the anticancer molecules from the ethyl acetate extract of A. cocculus. The other cell line highly sensitive to ACLE extract is HT29, with an IC₅₀ value of 17.97 ± 0.63 µg/mL. Both PA1 and HT29 cell lines exhibited an increase in mortality with an increase in incubation time (Fig. 1A).

Table 2.

IC₅₀ values of ethyl acetate, and acetone extracts of A. cocculus on different cell lines after 48 h incubation

Cell Line	IC50 (48 h) (µg/mL)
	ACLE extract	ACLA extract
PA1	8.30 ± 0.38	47.75 ± 0.61
HT29	17.97 ± 0.63	> 100
MG63	48.68 ± 3.83	51.98 ± 0.86
PANC1	63.45 ± 0.87	> 100
MCF7	85.60 ± 2.15	> 100
UM-SCC-83B	88.92 ± 2.89	> 100
A498	89.83 ± 0.40	92.16 ± 2.45
HCT116	90.7 ± 0.81	> 100
HeLa	90.17 ± 1.31	> 100
A431	95.33 ± 1.71	92.21 ± 1.45
HaCaT	> 100	> 100

Values are represented as mean ± SEM (n = 6)

Fig. 1.

(A) Evaluation of cell viability on treatment with extracts was assessed using MTT assay. The percentage mortality of cells was assessed for different solvent extracts of Anamirta cocculus at a concentration of 100 μg/mL on PA1 and HT29 cell lines following 24, 48, and 72 h of exposure. (B) The percentage of viable cells on treatment with different concentrations of ACLE extract on PA1, and HT29 cell lines after incubation for 48 h assessed by trypan blue assay. The cells grown in medium without DMSO (control) are also included to show that the concentration of DMSO used is not affecting the cells significantly. Data are expressed as mean ± SEM (n = 6). Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns – not significant

Trypan blue dye exclusion assay: In addition to the MTT assay, cell viability was further assessed using the trypan blue dye exclusion method. Two cell lines, PA1 and HT29, which were more sensitive to ACLE extract, were subjected to trypan blue staining followed by treatment with ACLE extract. In both cell lines, as the concentration of ACLE extract increased, cell viability decreased (Fig. 1B). Trypan blue staining for cell viability closely correlated with MTT assay results, confirming the cytotoxicity data obtained using the MTT assay.

ACLE inhibits colony formation in cancer cell lines: Exposure of PA1 and HT29 cells to ACLE extract demonstrated a concentration-dependent suppression of their colony-forming potential. ACLE extract at IC₅₀ concentration, the inhibition of colony formation in the PA1 cell line was to the extent of 85.2 ± 1.72%. At the same time, colony formation was inhibited up to 79.33 ± 1.70% when HT29 cells were treated with an IC₅₀ concentration ACLE extract (Fig. 2).

Fig. 2.

Clonogenic survival assay was performed to assess the impact of ACLE extract on colony formation in PA1 and HT29 cell lines after 7 days of incubation with ACLE extract at IC₂₀ and IC₅₀ concentrations. A Representative images of colony formation: (A-I) PA1-DMSO Control, (A-II) PA1 treated with IC₂₀ concentration, (A-III) PA1 treated with IC₅₀ concentration, (A-IV) HT29-DMSO Control, (A-V) HT29 treated with IC₂₀ concentration, and (A-VI) HT29 treated with IC₅₀ concentration. B Quantitative analysis of colony formation: (B-I) Percentage of colony formation in PA1 cell line and (B-II) Percentage of colony formation in HT29 cell line following treatment with ACLE extract at IC₂₀ and IC₅₀ concentrations. Cells grown in standard growth medium served as the control, and cells grown in DMSO at a final concentration of 0.1% are shown as the DMSO control, demonstrating the no significant effect of DMSO on colony formation. Data are presented as mean ± SEM (n = 6). Statistical significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns–not significant

Treatment of PA1 cells with ACLE extract resulted in diminished wound closure at a concentration of IC₂₀ and complete cessation of wound closure at IC₅₀ concentration. In a similar fashion, exposure of HT29 cells to ACLE extract at concentrations of IC₂₀ leads to decreased wound closure, and at IC₅₀ concentration, complete cessation of wound closure. This result can be attributed to a concentration-dependent reduction in the migration ability of the treated cells (Fig. 3).

Fig. 3.

A Scratch wound healing assay in PA1 cells to assess the effect of ACLE extract on cell migration. Cells were treated with IC₂₀ and IC₅₀ concentrations of the extract, and wound closure, reflecting cell migratory capacity, was monitored at 0, 24, and 48 h. (A-I) Represents wound closure in 0.1% DMSO control cells, (A-II) in cells treated with IC₂₀ concentration, and (A-III) in cells treated with IC₅₀ concentration. The images indicate that ACLE extract reduces cell migration, as shown by the slower closure of the wound in treated cells compared to the control. B Scratch wound healing assay in HT29 cells following treatment with ACLE extract. Cells were exposed to IC₂₀ and IC₅₀ concentrations of the extract, and gap closure, indicating cell migration, was assessed at 0, 24, and 48 h. (B-I) Represents wound closure in 0.1% DMSO control cells, (B-II) in cells treated with IC₂₀ concentration, and (B-III) in cells treated with IC₅₀ concentration. The images demonstrate that ACLE extract impairs cell migration, evidenced by reduced wound closure in treated cells relative to controls

Morphological changes on treatment with ACLE extract

Both PA1 and HT29 cells manifested dose-dependent cytomorphological alterations indicative of apoptosis induction, as elucidated through observations via both phase contrast and scanning electron microscopy (Fig. 4). In the untreated control group, PA1 cells exhibited a polygonal shape, forming confluent aggregations. However, following 48 h of ACLE extract treatment; these cells underwent a transition to a rounded morphology, displayed shrinkage, and presented with dead floating cells and fragmented cellular particles. Similarly, untreated HT29 cells displayed colonies of polygonal cells, whereas the ACLE extract-treated HT29 cells revealed cells with a shrunken and rounded morphology, and many of the cells were floating.

Fig. 4.

A Phase contrast microscopic images depicting cytomorphological changes in PA1 and HT29 cancer cell lines after 48 h of treatment with ACLE extract. The images include: (a-i) PA1- DMSO Control; (a-ii) PA1 treated at IC₂₀ concentration; (a-iii) PA1 treated at IC₅₀ concentration; (b-i) HT29-Control; (b-ii) HT29 treated at IC₂₀ concentration; (b-iii) HT29 treated at IC₅₀ concentration. B Scanning electron microscopic images illustrating the cytomorphological changes in PA1 and HT29 cancer cell lines after 48 h of treatment with ACLE extract. The images include: (a-i) PA1- DMSO Control; (a-ii) PA1 treated at IC₂₀ concentration; (a-iii) PA1 treated at IC₅₀ concentration; (b-i) HT29-Control; (b-ii) HT29 treated at IC₂₀ concentration; (b-iii) HT29 treated at IC₅₀ concentration

Assays for apoptosis

DNA fragmentation assay

DNA strand breaks, a prominent characteristic of apoptotic cells, were identified in the DNA isolated from ACLE extract-treated cells when separated on 1.2% agarose gels compared to control (Supplementary data: Fig. 1).

Genotoxicity evaluation by alkaline single-cell electrophoresis (comet assay)

The presence of DNA strand breaks in cancer cells was discerned through comet assay (single-cell electrophoresis), a method for evaluating genotoxicity. On ACLE extract treatment, PA1 and HT29 cells exhibited longer comet tail lengths in a concentration-dependent manner, demonstrating the occurrence of DNA damage (Fig. 5).

Fig. 5.

Genotoxicity assessment in PA1 and HT29 cancer cell lines using the alkaline comet assay after 48 h of treatment with ACLE extract. The images illustrate the extent of DNA damage in: A PA1 cells treated with DMSO control, B PA1 cells treated with ACLE extract at IC₂₀ concentration, C PA1 cells treated with ACLE extract at IC₅₀ concentration, D HT29 cells treated with DMSO control, E HT29 cells treated with ACLE extract at IC₂₀ concentration, and F HT29 cells treated with ACLE extract at IC₅₀ concentration. The comet assay results demonstrate increased DNA damage in cells treated with ACLE extract, with more pronounced effects observed at the higher concentration (IC₅₀), indicating the genotoxic potential of the extract

Acridine orange/Ethidium bromide dual staining

Fluorescence microscopy of ACLE extract-treated PA1 and HT29 cells when subjected to dual staining with acridine orange (AO) and ethidium bromide (EB), a distinct staining pattern was observed, confirming the induction of apoptosis in a concentration-dependent manner (Fig. 6). Acridine orange, which is permeable across intact cell membranes, imparted a green stain, whereas EB, selectively entering cells with compromised membrane integrity to interact with DNA, emitted fluorescence in shades of yellow to orange, indicative of the stages of apoptosis. The quantitative analysis revealed a significant increase in the number of late apoptotic cells with higher concentrations of the ACLE extract. In the PA1 cell line, the percentage of late apoptotic cells reached 82.34 ± 1.09%, while in the HT29 cell line, it was 54.65 ± 1.76% at a concentration of 20 µg/mL of ACLE extract (Fig. 7).

Fig. 6.

Effect of ACLE extract on apoptosis in PA1 and HT29 cell lines following 48 h of treatment. Apoptotic cells were stained with acridine orange, which imparted a green colour, and ethidium bromide (EB), which emitted fluorescence in shades of yellow to orange. A PA1 cell line: (A-I) DMSO Control, (A-II) Cells treated with 10 µg/mL ACLE extract, (A-III) Cells treated with 20 µg/mL ACLE extract. B HT29 cell line: (B-I) DMSO Control, (B-II) Cells treated with 10 µg/mL ACLE extract, (B-III) Cells treated with 20 µg/mL ACLE extract. The images show that treatment with ACLE extract induces apoptosis in both PA1 and HT29 cell lines, as indicated by the increased presence of orange-stained cells, which correspond to late apoptotic or necrotic cells. The effect is more pronounced at higher concentrations

Fig. 7.

Quantitative analysis of late apoptotic cells in PA1 and HT29 cell lines following 48 h of treatment with ACLE extract. The bar graphs depict the proportion of late apoptotic cells, as determined by staining with acridine orange and ethidium bromide. Results are expressed as mean ± SEM (n = 6). Statistical significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns – not significant

Assessment of mitochondrial membrane potential (ΔΨM) and apoptosis

In the intrinsic apoptotic pathway, a critical event is the mitochondrial membrane depolarisation, which leads to augmented permeability of the outer membrane due to pore formation, accompanied by the release of proapoptotic molecules such as cytochrome c. The alteration in mitochondrial membrane potential (ΔΨM), a characteristic feature of the intrinsic apoptotic pathway, was evaluated using the mitochondria-specific, voltage-dependent fluorescent probe Rhodamine-123. ACLE extract induced a dose-dependent reduction in ΔΨM after 48 h, as evidenced by fluorescence microscopy (Fig. 8). Conversely, untreated cells exhibited relatively low disruption of ΔΨM. DAPI, a fluorescent stain, was employed to elucidate nuclear changes during apoptosis and quantify the percentage of apoptotic cells with condensed and fragmented chromatin. The figure (Fig. 8) illustrates the extent of nuclear changes during apoptosis, as determined by DAPI staining in both control and treated groups. Treatment with ACLE extract demonstrated apoptosis in both PA1 and HT29 cells, characterised by increased permeability to DAPI, the presence of nuclear apoptotic bodies, and chromatin condensation.

Fig. 8.

Effect of ACLE extract on mitochondrial membrane potential in PA1 and HT29 cell lines following 48 h of treatment. The images show changes in mitochondrial membrane potential. A PA1 cell line: (A-I) DMSO Control, (A-II) PA1 treated with 10 µg/mL ACLE extract, (A-III) PA1 treated with 20 µg/mL ACLE extract. B HT29 cell line: (B-I) DMSO Control, (B-II) HT29 treated with 10 µg/mL ACLE extract, (B-III) HT29 treated with 20 µg/mL ACLE extract. The images demonstrate the effect of ACLE extract on mitochondrial membrane potential, with alterations observed in treated cells compared to controls, indicating potential mitochondrial dysfunction

Identification of compounds in ACLE and ACLA extracts by GC–MS analysis

The two bioactive extracts of A. cocculus, ACLE and ACLA, were subjected to GC–MS analysis to delineate the organic constituents present. The ACLE extract was found to contain octadecane, neophytadiene, n-Hexadecanoic acid, heneicosane, phytol, epoxylathyrol, dotriacontane, squalene, and vitamin E (Supplementary data: Fig. 2, and Table 3), whereas in ACLA extract hexadecyl acrylate, neophytadiene, 3,7,11,15-tetramethyl-2-hexadecen-1-ol, 1-( +)-ascorbic acid 2,6_dihexadecanoate, cyclononasiloxane, octadecamethyl, pentacosane, ageratriol, bis(2-ethylhexyl)phthalate, pentacosane, tetracosamethyl-cyclododecasiloxane, dotriacontane, 1,3-benzenedicarboxylic acid, bis(2-ethylhex, dotriacontane, and heptacosane were present (Supplementary data: Fig. 3, and Table 4).

Table 3.

List of compounds identified in the ethyl acetate extract of A. cocculus via GC–MS

Peak	R.time	Area	Area%	Height	Height%	A/H	Mark	Name
1	26.123	45,226	1.11	19,367	1.33	2.34	MI	Octadecane
2	26.87	923,455	22.73	354,005	24.4	2.61	MI	Neophytadiene
3	27.376	150,210	3.7	62,074	4.28	2.42	MI	Neophytadiene
4	27.749	314,212	7.73	112,392	7.75	2.8	MI	Neophytadiene
5	29.324	253,168	6.23	86,632	5.97	2.92	MI	n-Hexadecanoic acid
6	30.152	51,859	1.28	21,530	1.48	2.41	MI	Heneicosane
7	32.19	116,160	2.86	29,342	2.02	3.96	MI	Phytol
8	38.674	131,562	3.24	38,203	2.63	3.44	MI	Epoxylathyrol
9	40.368	39,069	0.96	16,712	1.15	2.34	MI	Dotriacontane
10	43.44	1,840,935	45.3	669,714	46.16	2.75	MI	Squalene
11	48.731	197,723	4.87	40,767	2.81	4.85	MI	Vitamin E

Table 4.

List of compounds identified in the acetone extract of A. cocculus via GC–MS

Peak	R.Time	Area	Area%	Height	Height%	A/H	Name
1	23.75	22,630	1.79	11,118	2.99	2.04	hexadecyl acrylate
2	26.87	96,833	7.65	40,955	11.03	2.36	Neophytadiene
3	27.38	24,892	1.97	9319	2.51	2.67	Neophytadiene
4	27.758	24,503	1.93	10,797	2.91	2.27	3,7,11,15-Tetramethyl-2-hexadecen-1-ol
5	29.31	39,629	3.13	18,327	4.94	2.16	l-( +)-Ascorbic acid 2,6-dihexadecanoate
6	36.292	18,147	1.43	7711	2.08	2.35	Cyclononasiloxane, octadecamethyl-
7	37.227	50,801	4.01	15,290	4.12	3.32	Pentacosane
8	38.669	43,114	3.4	15,471	4.17	2.79	Ageratriol
9	38.83	74,154	5.86	27,129	7.31	2.73	Pentacosane
10	39.258	119,242	9.42	43,425	11.7	2.75	Bis(2-ethylhexyl) phthalate

Assessment of in vitro antioxidant activity of the extracts by DPPH scavenging assay

Of the six extracts tested, the DPPH radical scavenging activity results showed that ACLE extract, followed by ACLA extract, and ACLC extract having excellent scavenging effects on DPPH radicals than the other extracts (Fig. 9). Overall, ACLE extract revealed the best antioxidant properties (low IC₅₀ value = 0.246 ± 0.02 µg/mL) (Table 5).

Fig. 9.

Antioxidant activity of six different extracts of Anamirta cocculus at various concentrations, as determined by the DPPH assay. The bar graphs represent the mean percentage of DPPH radical scavenging activity ± SEM (n = 6) for each extract at different concentrations. The data illustrate the effectiveness of each extract in neutralizing DPPH radicals, reflecting their antioxidant capacity

Table 5.

IC₅₀ values of ethyl acetate, acetone, and chloroform extracts of A. cocculus determined with DPPH assay

	A. cocculus extract	IC50 (in µg/mL)
1	ACLE	0.246 ± 0.02 µg/mL
2	ACLA	1.271 ± 0.01 µg/mL
3	ACLC	1.326 ± 0.04 µg/mL
4	Ascorbic acid	4.452 ± 0.01 µg/mL

Values are represented as mean ± SEM (n = 6)

In silico analysis

For molecular docking studies, the ligands identified from ACLE extract in the GC–MS analysis were docked against apoptotic proteins, viz., caspase-3 (PDB ID-3DEI), caspase-8 (PDB ID-3KJQ), caspase-9 (PDB ID-1JXQ), and P53 (PDB ID-3DCY). The 3-D structure of these proteins was downloaded from the PDB database. The docking results were visualised and analysed using Discovery Studio client 2021.

The in silico docking study revealed the interactions between ligands and Caspase-3 (Fig. 10), Caspase-8 (Fig. 10), Caspase-9 (Fig. 11), and P53 (Fig. 11), and the binding scores obtained were given in Table 6.

Fig. 10.

Three-dimensional representations of the best fit ligand-receptor interactions for various compounds with caspase-3 and caspase-8. Interaction of each compound with caspase-3 and caspase-8: A Neophytadiene, B Dotriacontane, C Octadecane, D N-Hexadecanoic Acid, E Heneicosane, F Phytol, G Epoxylathyrol, H Squalene, and I Tocopherol. The images show the molecular docking results, illustrating the binding interactions of each ligand with caspase-3 and caspase-8, highlighting their potential roles in modulating apoptotic pathways

Fig. 11.

Three-dimensional representations of the best fit ligand-receptor interactions for various compounds with caspase-9 and p53. Interaction of each compound with caspase-9 and p53: A Neophytadiene, B Dotriacontane, C Octadecane, D N-Hexadecanoic Acid, E Heneicosane, F Phytol, G Epoxylathyrol, H Squalene, and (I) Tocopherol. The images show the molecular docking results, illustrating the binding interactions of each ligand with caspase-3 and caspase-8, highlighting their potential roles in modulating apoptotic pathways

Table 6.

Docking scores of the GC–MS identified compounds of ACLE extract

Ligand	Docking score (Binding affinity, kcal/mol)
	Caspase-3	Caspase-8	Caspase-9	P53
Tocopherol	−9.5	−8.8	−11.1	−8.2
Epoxylathyrol	−7.8	−6.8	−8	−8.5
Squalene	−6.9	−4.9	−6.2	−5.9
Phytol	−5.6	−5.2	−5.7	−5.7
Octadecane	−5.9	−3.6	−4.7	−4.5
Neophytadiene	−5.5	−5	−4.9	−5.3
N-Hexadecanoic_acid	−5.1	−5.1	−4.5	−5.3
Heneicosane	−5.1	−4.2	−3.9	−4.7
Dotriacontane	−4.9	−3.8	−4.4	−4.2

In our investigation, it was observed that tocopherol displayed the highest binding energy to caspase-3, caspase-8, and caspase-9 among the ligands tested. But epoxylathyrol showed the highest binding affinity to P53, the prominent tumour suppressor protein. Squalene and phytol exhibited less binding affinity to Csapase-3, Caspase-8, Caspase-9, and P53 compared to epoxylathyrol. These results indicate the potential for synergistic effects of these compounds in augmenting apoptosis in cancer cells, thereby prompting cellular demise.

ADME Analysis

In this study, Lipinski’s rule of five was applied to assess the pharmacokinetic and physiochemical parameters of compounds, focussing particularly on their drug-likeness as potential anticancer agents. The compounds under investigation, as indicated (Table 7), exhibited favourable drug-like characteristics. Notably, the logKp (cm/s) value, indicative of skin permeation, is significant; compounds with more negative values are less inclined to penetrate the skin. Additionally, lower logS values suggest greater water solubility, facilitating easier absorption in the intestine. Analysis of the ACLE extract revealed the presence of compounds with negative logKp and lower logS values. Of the different parameters considered, the maximum number of parameters is favourable to epoxylathyrol, making it a promising drug candidate.

Table 7.

ADME property prediction for the compounds identified in ACLE extract from Swiss ADME

Compound	Formula	aMW (g/mol)	bHB acceptor	cHB donor	dLog Po/w	eMolar reactivity	fESOL LogS	gBioavailability Score	hRule of five	iLogKp (cm/s)
Octadecane	C18H38	254.49	0	0	7.18	88.64	−6.33	0.55	1	−1.2
Neophytadiene	C20H38	278.52	0	0	7.07	97.31	−6.77	0.55	1	−1.17
N-hexadecanoic acid	C16H32O2	256.42	2	1	5.2	80.8	−5.02	0.85	1	−2.77
Heneicosane	C21H44	296.57	0	0	8.26	103.06	−7.41	0.55	1	−0.31
Phytol	C20H40O	296.53	1	1	6.22	98.94	−5.98	0.55	1	−2.29
Epoxylathyrol	C20H30O5	350.45	5	3	1.78	93.91	−2.88	0.55	0	−7.47
Dotriacontane	C32H66	450.87	0	0	12.24	155.94	−11.4	0.55	1	2.98
Squalene	C30H50	410.72	0	0	9.38	143.48	−8.69	0.55	1	−0.58
Vitamin E	C29H50O2	430.71	2	1	8.27	139.27	−8.6	0.55	1	−1.33

^aMW – Molecular weight (acceptable range: < 500)

^bHB – Hydrogen bond acceptor (acceptable range: ≤ 10)

^cHB – Hydrogen bond donor (acceptable range: ≤ 5)

^dLog P_o/w—Lipophilicity (acceptable range: < 5)

^eMolar refractivity should be between 40, and 130

^fESOL LogS-—solubility in water (acceptable range: < 10)

^gBioavailability score – four classes of probabilities (11%, 17%, 56% or 85%)

^hRule of five: Number of violations of Lipinski’s rule of five; recommended range: 0–4

ⁱNegative values of logK_p indicate low skin permeation

Discussion

Colon cancer is a highly aggressive malignancy associated with elevated morbidity and mortality rates, poor prognosis, and limited treatment options (Hossain et al. 2022). Ovarian cancer similarly presents a significant clinical challenge. While chemotherapy remains a key therapeutic strategy, its effectiveness is compromised by substantial toxic side effects, limiting patient tolerance (Yang et al. 2020). Additionally, the emergence of resistance to many chemotherapeutic agents further reduces their clinical efficacy. Thus, there is an urgent need for novel therapeutic agents that offer improved efficacy with reduced adverse effects. Natural products, particularly those derived from plants, have garnered attention as promising candidates for anticancer drug development. Medicinal and aromatic plants, in particular, provide a rich source of compounds with established anticancer activity while demonstrating lower toxicity to normal cells (Greenwell and Rahman 2015). This has led to an increased focus on the discovery of novel anticancer compounds from natural sources as potential drug candidates.

In this study, we screened ten cancer cell lines with six different solvent extracts from A. cocculus leaves. The cell lines with the highest cytotoxicity, PA1 and HT29, were chosen for further studies. Also, we examined the effects of ethyl acetate extract on colony formation, cell migration, mitochondrial permeability, and genotoxicity in PA1 and HT29 cell lines.

The results of this study highlight the significant anti-proliferative effects of Anamirta cocculus leaf extracts, specifically those prepared with ethyl acetate (ACLE) and acetone (ACLA). The ethyl acetate extract demonstrated a notably low IC₅₀ value against cancer cell lines PA1 (IC₅₀ = 8.30 ± 0.38 µg/mL) and HT29 (IC₅₀ = 17.97 ± 0.63 µg/mL). Particularly noteworthy is the enhanced potency of ACLE against the PA1 cell line, where it exhibited a six-fold lower IC₅₀ value compared to ACLA. Furthermore, ACLE was twice as effective on PA1 cells compared to HT29, suggesting potential selectivity. Both extracts (ACLE and ACLA) exhibited no significant cytotoxicity toward the non-cancerous human keratinocyte cell line (HaCaT), underscoring their potential as selective anticancer agents with minimal adverse effects on normal cells.

Comparable studies have demonstrated similar results with other plant-derived ethyl acetate extracts. For example, the ethyl acetate extract of Acalypha wilkesiana displayed substantial anti-proliferative activity against human glioblastoma (U87MG) and lung carcinoma (A549) cell lines, with GI₅₀ values of 28.03 ± 6.44 µg/mL and 89.63 ± 2.12 µg/mL, respectively, after 72 h of exposure (Lim et al. 2011). The ethyl acetate extract from Euphorbia platyphyllos leaves also showed notable anticancer activity against human breast cancer (MCF7) cells, with an IC₅₀ value of 46.24 ± 0.057 µg/mL (Aslanturk and Celik 2013). Additionally, the ethyl acetate extract of Derris indica demonstrated efficacy against MCF7 and the small-cell lung cancer line NCI-H187 (Vittaya et al. 2023), and the chloroform extract of Marantodes pumilum was cytotoxic to prostate cancer cell lines (PC3, LNCaP, and DU145) with IC₅₀ values below 15 µg/mL (Hanafi et al. 2023).

Other studies have reported the sensitivity of HT29 to various plant extracts. For instance, methanol extracts of Chromolaena odorata (IC₅₀ = 200 µg/mL, 72 h) (Adedapo et al. 2016) and Stachys pilifera (IC₅₀ = 612 µg/mL, 24 h) (Kokhdan et al. 2018), as well as ethanol extracts of Ficus hispida (IC₅₀ = 125 µg/mL, 24 h) (Sathiyamoorthy and Sudhakar 2018), Cnidium officinale Makino (IC₅₀ = 305.02 µg/mL, 24 h) (Cruz et al. 2014), and Cynodon dactylon (IC₅₀ = 625 µg/mL, 24 h) (Kanimozhi and Bai 2013), have all shown varying degrees of anticancer activity. In comparison, the ethyl acetate extract of A. cocculus exhibited a much lower IC₅₀ value of 8.30 ± 0.38 µg/mL for PA1 and 17.97 ± 0.63 µg/mL for HT29, suggesting the presence of highly potent anticancer compounds.

The PA1 cell line has also demonstrated sensitivity to various plant extracts, such as ethanol extract of Adhatoda vasica (IC₅₀ = 107.339 µg/mL) (Nikhitha et al. 2021) and methanol extract from Pergularia daemia (IC₅₀ = 30 µg/mL) (Martin 2011). Screening of other plant extracts, including Eucalyptus sp., Clausen sp., Trichopus zeylanicus, Begonia floccifera, Gynura pseudochina, Schefflera racemosa, and Decalepis arayalpathra, revealed IC₅₀ values between 40 and 100 µg/mL for PA1, while Garcinia morella (IC₅₀ = 0.9 µg/mL), Plectranthus urticoides (IC₅₀ = 1.0 µg/mL), Aristolochia tagala (IC₅₀ = 13 µg/mL), and Acronychia pedunculata (IC₅₀ = 20 µg/mL) displayed more promising results with IC₅₀ values under 30 µg/mL (Garg et al. 2007).

In this context, the ethyl acetate extract of A. cocculus, with an IC₅₀ value of 8.30 ± 0.38 µg/mL for PA1 and 17.97 ± 0.63 µg/mL for HT29, indicates the presence of highly potent anticancer compounds. Thus, ACLE extract merits further exploration as a promising source of novel anticancer agents.

The mechanisms of anticancer action of components from plant extracts are multiple and complex. Some molecules may disrupt the cell cycle by blocking cell proliferation and inducing apoptosis. Others may interfere with cellular signalling pathways and impede the formation of new blood vessels (angiogenesis), a process crucial for tumour growth (Rajabi et al. 2021). On treatment with ACLE extract, the colony-forming ability of PA1 and HT29 cells decreased significantly in a concentration-dependent manner compared to untreated cells. Treated cells exhibited 17-fold decreases in colony formation for both PA1 and HT29. The scratch wound healing assay demonstrated ACLE extract-induced inhibition of cell migration in PA1 and HT29 cell lines. The extract efficiently blocked the ability of cancer cell lines to grow and proliferate in forming colonies or migrate in the case of PA1 and HT29 cell lines, thereby showing the potential to prevent invasion and migration of cancer cells. The methanol extract from Eclipta alba could prevent or reduce colony formation and migration of the human colon cancer cell line, HCT116 (Nelson et al. 2020).

PA1 and HT29 cell lines exposed to ACLE extract exhibited various apoptotic characteristics, including membrane blebbing, nuclear chromatin condensation, cellular/nuclear fragmentation, loss of mitochondrial membrane potential, genotoxicity, and DNA laddering, indicating apoptosis-mediated cell death. Quercetin, a flavonoid derived from Acalypha indica L., induced DNA ladder formation in two breast cancer cells, MDA-MB-231 and MCF7, suggesting its involvement in DNA damage leading to apoptosis (Chekuri et al. 2023). The Cm epoxide, isolated from Curcuma mutabilis, induced apoptotic DNA fragmentation as demonstrated through agarose gel electrophoresis and comet assay in cancer cell lines HCT116, MDA-MB-231, and K562 (Soumya et al. 2021). The aqueous extracts from Aronia melanocarpa, Cornus mas, and Chaenomeles superba also caused DNA damage visualised by comet assay in the colon cancer cell line Caco-2 when treated at or below IC₅₀ concentration (Efenberger-Szmechtyk et al. 2020). Additionally, the aqueous extract of Rubus coreanum degraded or fragmented DNA in the HT29 cell line at its IC₅₀ concentration (Kim et al. 2005).

In the acridine orange/ethidium bromide (AO/EB) staining assay, ACLE extract-treated PA1 and HT29 cells exhibited fluorescence ranging from green to red, indicating different stages of apoptosis. Green fluorescence signifies early apoptotic cells, while red fluorescence marks late apoptotic or necrotic cells. These observations suggest that the ACLE extract effectively induces apoptosis in these cancer cell lines. Similar apoptotic effects have been reported in HT29 cells treated with methanolic extracts of Brucea javanica (Bagheri et al. 2018) and Primula auriculata (Behzad et al. 2016), supporting the potential of plant-based extracts in triggering apoptosis in cancer cells.

Within living organisms, a moderate rise in reactive oxygen species (ROS) can prompt cell proliferation and differentiation. Nonetheless, excessive ROS levels can induce oxidative damage in cells, a phenomenon also detrimental to cancer cells (Boonstra and Post 2004). Mitochondria stands out as a notable ROS source and are pivotal in apoptotic signalling pathways. Elevated ROS production has been linked to mitochondrial injury, accompanied by a significant reduction in mitochondrial membrane potential (MMP) (Wang et al. 2003). High ROS levels within cells can induce peroxidative damage to the cell membrane (Shamsi et al. 2008), resulting in changes in membrane permeability (Kim et al. 2004), and a subsequent decrease of MMP. In this study, the ACLE extract caused a dose-dependent decrease in membrane permeability and MMP in both PA1 and HT29 cell lines. This decrease in MMP during apoptosis may contribute to cell death by impairing mitochondrial functions. Methanol extracts of Luffa echinata Roxb. (Shang et al. 2012), Teucrium polium, and Prosopis farcta (Khodaei et al. 2018) increased ROS generation when applied to the colon cancer cell line (HT29), leading to MMP loss in the treated cells.

It has been shown that geraniol induces apoptosis in prostate cancer by disrupting mitochondrial membrane polarization in cancer cells (Kim et al. 2012). Similarly, α-bisabolol and thymol have demonstrated efficacy against acute lymphoid and myeloid leukemias through modifications of mitochondrial membrane polarization (Rigo et al. 2019). Additionally, β-elemene and other terpenes have been reported to affect cancer cell membranes. Terpenoids can facilitate the release of mitochondrial cofactors, such as cytochrome C, in cancer cells, thereby triggering caspase-dependent apoptotic pathways (Omari et al. 2021).

Among the six extracts prepared from A. cocculus, the antioxidant activity was highest for the ACLE, which was followed by ACLA and ACLC extracts, and is correlated to the anticancer activity of these extracts. The antioxidant activity of ACLE, ACLA, and ACLC was higher than that of ascorbic acid. A similar observation was made from the leaf extract of Crinum asiaticum L., pointing out that the petroleum ether extract of the leaves of C. asiaticu exhibited much higher (10.20 times) antioxidant activity than the ascorbic acid (Goswami et al. 2020). The aqueous extract and acetone extract of Baccharis dracunculifolia showed greater antioxidant activity (Gazim et al. 2022).

The GC–MS analysis of ACLE extract revealed the presence of many bioactive components in it, like vitamin E, squalene, epoxy lathyrol, neophytadiene, phytol, etc. Vitamin E has antioxidant activity, protects cell membranes (Traber and Atkinson 2007), and has anticancer effects like delays or blocks tumour development (Lu et al. 2010), reducing tumour size, proliferation, viability, and enhancing apoptosis. Studies have shown that squalene can inhibit aberrant hyperproliferation in human mammary epithelial cells, MCF10A (Katdare et al. 1997). Epoxylathyrol is reported to have anti-inflammatory and anticancer properties. A lathyrol derivative, lathyrol-3-phenyl-acetate-5,15-diacetate, isolated from Euphorbia genus (Caper Euphorbia) seed, showed potent cytotoxicity against lung cancer A549 cells and induced apoptosis with elevation of reactive oxygen species (ROS), reduction in mitochondrial membrane potential, cytochrome C release, and an increase in activity of capase-9 and caspase 3 (Zhang et al. 2017). Phytol, a constituent of chlorophyll, has been shown to possess anticancer properties as well as to enhance immune function. As some of the compounds identified in the extract are known to possess anticancer activity, it is possible that the observed growth inhibitory effect on cancer cell lines is due to the additive or synergistic effect of these compounds.

To elucidate the mechanism of cytotoxicity mediated through apoptosis, in silico docking studies were conducted on pivotal apoptosis-related proteins, including caspase-3, caspase-8, caspase-9, and P53. The compounds identified in GC–MS exhibited affinity to the above apoptosis targets, with the highest affinity of tocopherol towards caspase-3, caspase-8, and caspase-9, followed by epoxylathyrol, squalene, and phytol. Epoxylathyrol showed the highest affinity towards P53, followed by tocopherol, squalene, and phytol. This suggests their potential involvement in inducing apoptosis. The tumour suppressor protein P53 also plays a prominent role in apoptosis. The highest affinity observed for P53 was for epoxylathyrol (8.5 kcal/mol), which was followed by tocopherol (-8.2 kcal/mol). As epoxylathyrol does not violate Lipinski’s rule of five, and its higher water solubility (ESOL logS = -2.88) favours its high likeness—the more the water solubility, the more will be gastrointestinal absorption (Daina et al. 2017). As the compounds in the extract bind with high affinity to caspases 3, 8, and 9, and p53, the observed apoptosis on treatment may be the result of direct activation of these pro-apoptotic proteins. The possibility of induction of apoptosis, mediated through other proteins or receptors with which the compounds in the extract interacts, cannot be ruled out. The predicted higher affinity of epoxylathyrol to P53 may contribute to the P53-mediated apoptosis in the ACLE extract-treated cell lines. The natural compound rutin gave a docking score of -7.3 kcal/mol to P53 (Malla et al. 2023), while quercetin had a binding affinity of −6.72 kcal/mol with P53. These docking results on apoptosis targets support our experimental results.

Thus, in the present study, we showed that ACLE extract exerts potent growth inhibition on ovarian cancer cell line PA1 and colorectal carcinoma cell line HT29 in vitro. The mechanism of cytotoxicity is apoptosis, as revealed by in vitro and in silico studies. Though studies using cell lines clearly indicate the anticancer potential of extracts from A. cocculus, further animal studies are required to confirm its anticancer potential in in vivo models.

Conclusions

In conclusion, the present study revealed that ACLE extract produced potent growth inhibition of the ovarian cancer cell line, PA1, and colorectal carcinoma cell line, HT29, in vitro, and this effect is mediated through the induction of apoptosis as evidenced by membrane blebbing, nuclear chromatin condensation, cellular/nuclear fragmentation, loss of mitochondrial membrane potential, and DNA laddering. The docking studies conducted on apoptosis target proteins such as caspase-3, caspase-8, csapase-9, and P53 revealed possible mechanisms of action of the components present in the extract leading to apoptosis. The observed cytotoxicity may be the result of the activity of components in the extract, such as epoxylathyrol, vitamin E, squalene, or phytol, individually or in combination or synergy. The ADME analysis and binding affinity indicate the potential of epoxylathyrol as a possible anticancer molecule, which needs to be further explored. Thus, further study is required to delineate the contribution of the components in inducing cytotoxicity for possible application in cancer chemotherapy.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

A. cocculus

: Anamirta cocculus

MTT

: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

ADME

: Absorption, distribution, metabolism, and excretion analysis

FESEM

: Field Emission Scanning Electron Microscopy

NIST

: National Institute of Standards, and Technology

RCSB PDB

: Research Collaboratory for Structural Bioinformatics Protein Data Bank

ANOVA

: Analysis of Variance

ACLP

: Anamirta cocculus Leaf petroleum ether extract

ACLC

Anamirta cocculus Leaf chloroform extract

ACLE

: Anamirta cocculus Leaf ethyl acetate extract

ACLA

: Anamirta cocculus Leaf acetone extract

ACLM

: Anamirta cocculus Leaf methanol extract

ACLW

: Anamirta cocculus Leaf water extract

MMP

: Mitochondrial membrane potential

Data availability

The data analysed during the present investigation are available from the corresponding author upon reasonable request.

Declarations

Conflict of interests

The authors declare that they have no conflict of interests.

Ethics approval

This article does not contain any studies with human participants or animals performed by any of the authors.