In vitro antitumor capacity of extracts obtained from the plants Plukenetia volubilis (Sacha inchi) and Moringa oleifera in gastric cancer

Introduction

More than 19 million people had their first case of cancer, and nearly half of them died in 2022 (WHO, 2024). Gastric or stomach cancer is the fifth most common cancer worldwide, with more than one million cases annually, of which 70% of patients die (Ferlay et al., 2021), mainly because diagnoses occur at late stages and because of this, the probability of success of conventional therapies at these stages is low, making it the fourth most lethal neoplasm globally (Sung et al., 2021). Despite the efforts made in the last four decades, the cost-benefit and long-term survival picture for many cancer pathologies remains bleak. Between 1971 and 2007, an increase in survival of only 17% has been achieved in ovarian cancer (Lloyd, Cree, & Savage, 2015), while in breast cancer, the increase has been 38% in 10-year survival. Significant barriers to major advances include low rates of early detection, lack of effective prognostic and predictive strategies, and the emergence of chemoresistance, which ultimately leads to patient death.

Canonical chemotherapy for the treatment of gastric cancer is primarily based on combinations of cisplatin and 5-fluorouracil (5-FU) or its derivatives, such as oxaliplatin and capecitabine. The genotoxic effects of chemotherapy and radiotherapy are the same as those that lead to the initiation and maintenance of cancer, and it is puzzling that genotoxic agents are given preference in cancer treatment over other substances that may act more specifically. Approximately 25% of all new anticancer drugs approved in the last 30 years are related to natural products (Newman & Cragg, 2020). In addition, such natural compounds obtained through diet offer options for preventing and treating many diseases, including cancer (Cragg & Pezzuto, 2016). Natural products have been so successful that they have doubled human life expectancy in the 20th century. For more than five decades, they have been positioned as weapons in the battle against cancer, thanks to the presence of exotic structures rich in functional groups (Verdine, 1996). About 1 million natural products, of which more than half come from plants, are the most critical anticancer products (Demain & Vaishnav, 2011).

Given the characteristics of natural products, many studies have focused on uncovering their therapeutic potential in cancer research. An example of this is the study of extracts from the Moringa oleifera, known as ‘the tree of life,’ a tropical and subtropical plant with several recognized biological properties, mainly anti-inflammatory (Cheenpracha et al., 2010). Regarding its antitumor capacity, several studies have been carried out based mainly on extracts from the leaf in ovarian, prostate, and breast cancer tumor lines (Al-Asmari et al., 2015; Del Mar Zayas-Viera, Vivas-Mejia, & Reyes, 2016; Ghosh, 2013), hepatocarcinoma and leukemia (Khalafalla et al., 2010), multiple myeloma (Parvathy & Umamaheshwari, 2007), KB human tumor lines (Sreelatha, Jeyachitra, & Padma, 2011) and those derived from esophageal cancer (Tiloke, Phulukdaree, & Chuturgoon, 2016), colorectal cancer (Al-Asmari et al., 2015), as well as in animal approaches using the Ehrlich solid tumor model (W. K. Khalil, Ghaly, Diab, & ELmakawy, 2014), among other studies (Khor, Lim, Moses, & Abdul Samad, 2018).

Plukenetia Volubilis is another plant on which the study of its components in cancer has focused, although not as extensively as Moringa. P. volubilis is commonly known as Sancha inchi (SI). It is distributed along the western and northern edge of the Amazon basin, through Brazil, Bolivia, Peru, Ecuador, Colombia, Venezuela, and Suriname, and in the Lesser Antilles (del-Castillo, Gonzalez-Aspajo, de Fátima Sánchez-Márquez, & Kodahl, 2019). In recent years, P. volubilis has attracted attention because of the abundance and composition of its seed oil, which is now commercially available. Although the biological function of SI has not been fully delineated, its beneficial impact in modulating non-communicable diseases has gained popularity worldwide for its antioxidant, anti-inflammatory, and immunomodulatory properties, mainly from leaves and fruit hulls (Nascimento et al., 2013; Wuttisin, Nararatwanchai, & Sarikaphuti, 2021). It is also recognized because its consumption has been associated with the prevention of cardiovascular diseases, inflammatory diseases, dermatitis, and control of tumor proliferation, especially given its recognized high content of essential fatty acids, as well as the hypolipidemic (Cárdenas, Gómez Rave, & Soto, 2021) and antitumor activity in cervical and lung tumor lines (Nascimento et al., 2013). It is also recognized as a sustainable crop (Kodahl & Sørensen, 2021).

There is a growing interest in different areas about the potential of promising plants, including Plukenetia volubilis and Moringa oleifera, but there remains a lack of focused studies evaluating their specific effects on gastric cancer, especially for P.volubilis, and particularly with regard to its unprocessed seed oil. For this plant there is research predominantly focused on its nutritional value and general antioxidant or anti-inflammatory properties, with only limited exploration into its potential to modulate cancer-related pathways such as apoptosis, cell cycle arrest or proliferation inhibition in gastric tumors, in fact, the direct impact of its components in this context is still unknown. This represents a critical gap, particularly given the increasing global burden of gastric cancer and the need for novel, plant-derived chemopreventive agents.

The selection of M. oleifera and P. volubilis for this study was based on their traditional medicinal use, promising bioactivity, and particularly their diverse phytochemical profiles, which support potential anticancer properties. M. oleifera leaves are rich in flavonoids (e.g., quercetin, kaempferol), glucosinolates, isothiocyanates (e.g., niazimycin, benzyl isothiocyanate), phenolic acids, and alkaloids, compounds that have demonstrated antiproliferative, antioxidant, and pro-apoptotic effects in various tumor models, including breast, colon, prostate, and leukemia cell lines (Chiș et al., 2023; Pop, Kerezsi, & Ciont, 2022). P. volubilis, although less extensively studied, has attracted attention for its seed oil’s high content of polyunsaturated fatty acids (especially α-linolenic and linoleic acid), as well as tocopherols, phytosterols (β-sitosterol, stigmasterol), terpenoids, and phenolic (Chirinos et al., 2013; Đurović, Radovanović, Tomić, Marjanović, & Mandić, 2025; Valencia, Romero-Orejon, Viñas-Ospino, & Barriga-Rodriguez, 2021).

Regarding M. oleifera previous studies have shown that its extracts, especially from leaves and seeds, induce apoptosis, inhibit cell proliferation and generate DNA damage in different tumor cell lines such as breast, colon and lung cancer (Al-Asmari et al., 2015; Kuete et al., 2011; Sreelatha et al., 2011; Tiloke, Phulukdaree, & Chuturgoon, 2013). Despite these findings, the effect of M. oleifera on gastric cancer remains poorly explored, despite the fact that this neoplasm represents one of the leading causes of cancer mortality worldwide. For such reason, it is a priority to investigate the antitumor potential of Moringa oleifera-derived compounds in in vitro models of gastric cancer. A preliminary study by Kuete et al. (2011) showed that methanolic extracts of M. oleifera exert cytotoxic activity against AGS (human gastric adenocarcinoma) cells, suggesting a possible direct effect on cell viability (Kuete et al., 2011). However, the molecular mechanisms involved in this cytotoxicity have not yet been thoroughly characterized.

The species tested in this study are adaptable to tropical climates and easy to cultivate, which allows large-scale production in regions of Latin America, Africa and Asia. This facilitates continuous access to plant biomass for extraction and analysis of bioactive compounds, reducing research and development costs. In addition, both plants have a history of traditional use as foods or supplements, suggesting a favorable safety profile. This makes their compounds good candidates for preclinical studies with lower risk of serious adverse effects compared to synthetic agents. The fact of considering promising plants such as these in this type of studies encourages ethnobotanical and biotechnological research in regions with high biodiversity, promoting local scientific development and the sustainable use of endemic natural resources.

Accordingly, this work aimed to study the effect of extracts obtained from these two plants on cytotoxicity, inhibition of cell proliferation and migration through in vitro assays in the AGS tumor line, as well as the study of the possible mechanism responsible for these effects, seeking to advance in the development of a phytotherapeutic approach for gastric carcinoma.

Methods

Results

Cytotoxic effect of Plukenetia volubilis and Moringa oleifera derivative extracts in AGS tumor cells

Cells were exposed to various concentrations of the extracts and oil for 72 hours. The effect on viability was evidenced by the MTT assay. The results revealed that the extracts of both plants affect the survival of the AGS gastric cancer cell line, with CC₅₀ values ranging from 94.5 mg/mL to 158.3 μg/mL ( Figure 1). The extracts showed a CC₅₀ above 100 μg/mL for AGS cells, except for the M. oleifera cyclohexane extract, which showed a CC₅₀ of 94.3 μg/mL. Given the CC₅₀ values obtained for the extracts on AGS cells, it can be deduced that, except for the cyclohexane extract of M. oleifera, which showed high cytotoxicity, the extracts present moderate toxicity to these cells ( Figure 1). Similarly, the oil obtained from P. volubilis seed also showed cytotoxic potential on AGS cells with a reduction in cell viability of 47% ( Figure 1).

Figure 1. Effect of derivatives obtained from each of the plants under study on cell viability.

Data were normalized with respect to the untreated control (100% survival) and are shown as the mean and standard deviation of at least three independent experiments performed in quadruplicate for each dose. The cytotoxic concentration 50 (CC₅₀) of each derivative is shown.

The CH extract from M. oleifera was 1.6 times more effective than its counterpart from P. volubilis, while the extract obtained with PE was the most cytotoxic. However, at the highest dose tested, the latter induced more cell death (88%) than CH extract (72%) despite having the lowest CC₅₀. It was also observed that the cytotoxic effect of the extracts and the oil is dose-dependent, as detailed in the viability curves ( Figure 1).

Antiproliferative effect of Plukenetia volubilis and Moringa oleifera derivative extracts in AGS tumor cells

The effect on cell proliferation was determined by cell viability assay with the CellTiter-Blue^® kit (Promega). All derivatives, extracts, and oil affected the proliferation of tumor cells at concentrations below the CC₅₀, and this effect is maintained over time.

Moringa extracts had better inhibitory dynamics for the doses required and the time employed compared with P.volubilis extracts. For example, the EtOH and PE extracts of M. oleifera exhibited a significant inhibitory effect from the second day of treatment, compared to the same extracts from P. volubilis ( Figure 2a,b,d,e). Moreover, among all extracts tested, the EtOH extract of M. oleifera showed almost complete inhibition at the evaluated concentrations ( Figure 2a). In contrast, the antiproliferative effect of the cyclohexane of M. oleifera is not as evident since it was found that the lowest dose of this extract showed an innocuous effect on the AGS line ( Figure 2c). This behavior was also observed in the EtOH extract from P.volubilis where the 10 ug/mL dose also had no effect on inhibition of cell proliferation ( Figure 2d). Despite this, it is this extract that exhibits the most significant antiproliferative potential among the P. volubilis group, first, because it achieves an evident antiproliferative effect up to a dose of 50 ug/mL, and second, the inhibition was observed from the second day of treatment, events that do not occur with the other two extracts ( Figure 2d,e,f).

Figure 2. Effect of M. oleifera and P. volubilis extracts on cell proliferation.

Data were normalized with respect to the untreated control (100% survival) and are shown as the mean and standard deviation of at least two independent experiments performed in triplicate for each dose.

On the other hand, the P. volubilis oil generated a varied inhibitory effect on proliferation at all concentrations tested, especially at the highest concentration ( Figure 3); moreover, the effect was maintained until the end of the treatment.

Figure 3. Effect of P. volubilis oil on cell proliferation of the AGS line.

Data were normalized with respect to the untreated control (100% survival) and are shown as the mean and standard deviation of at least two independent experiments performed in triplicate for each dose.

Death inducing effect of Plukenetia volubilis oil in AGS tumor cells

Fluorescence microscopy revealed that cells treated with the different derivatives and N-ethyl nitrosourea showed a generally lower proportion of dead cells (red fluorescence) relative to live cells (green fluorescence), as shown in Figure 4. The viability of AGS cells treated with the extracts and oil at the respective CC₅₀ ranged from 43% to 69%, with the CH and EtOH extracts of M. oleifera affecting cell viability the most, with viability percentages close to 40%. All extracts produced a low mortality of AGS cells, with the percentages obtained being less than 10% ( Table 1). On the other hand, P. volubilis oil at a concentration of 25% showed viability percentages of 77% and mortality of 23%, being the highest mortality of all the plant derivatives evaluated ( Table 1, Figure 4). N-ethyl nitrosurea, used to induce stomach cancer in C57Bl6 mice, produced 89% viability and 9% mortality. With doxorubicin, viability was observed in 38% of cells, and mortality was 66%. These results suggest that P. volubilis oil is cytotoxic to AGS cells at concentrations of 25% v/v or higher. As expected, most cells of the untreated group show fluoresce green (live), and only a small proportion of cells show red fluoresce ( Figure 4j).

Figure 4. Effect of extracts and oil on the viability/cytotoxicity of AGS cells.

The figure shows a representative photograph of AGS cells treated with cyclohexane (a), ethanol (b), and petroleum ether (c) extracts of P. volubilis. Photographs (d), (e) and (f) correspond to AGS cells treated with cyclohexane, ethyl and petroleum ether extract of M. oleifera, respectively. Cells treated with P. volubilis oil 25% v/v (g), N-ethyl Nitrosurea 25% v/v (h), Doxorubicin 5μg/mL (I) and untreated (J): fluorescence microscopy, magnification 10×.

Table 1. Effect of Plukenetia volubilis and Moringa oleifera extracts on the viability of AGS cells.

Treatment	% live cells	% dead cells
P. volubilis CH	68.5 ± 6.8	9.7 ± 1.2
P. volubilis EtOH	59.0 ± 2.5	8.6 ± 1.5
P. volubilis PE	58.9 ± 1.9	8.4 ± 2.3
M. olífera CH	43.5 ± 3.3	1.6 ± 0.3
M. olífera EtOH	47.7 ± 8.8	4.7 ± 1.8
M. olífera PE	69.7 ± 4.0	7.6 ± 2.4
P. volubilis oil (25% v/v)	77.1 ± 1.2	22.6 ± 0.5
N-ethyl nitrosurea (25% v/v)	88.8 ± 0.9	8.6 ± 0.6
Doxorrubicin (5 μg/mL)	37.8 ± 3.7	65.8 ± 8.2
DMSO 20%	52.5 ± 0.22	39.3 ± 10.6
Nontreatment	99.5	0.5

Effect of P. volubilis oil in the AGS cell cycle

The effect on the content of the DNA in AGS cells treated with the different extracts of M. oleifera and P. volubilis and the oil of P. volubilis was analyzed by flow cytometry with PI staining. The assays revealed statistically significant changes only in cells treated with P. volubilis oil compared to untreated cells. In cells treated with P. volubilis oil, the percentage of cells in the sub-G1 phase increased from 5.1 ± 2.2% in control cells to 28.2% ± 12.3 within 8 hours of treatment (p = 0.0027), and the percentage of cells in G1 phase decreased from 80% to 53% ± 12.8% (p = 0.0025). With leaf extracts, no statistically significant differences in arrest were observed in the sub-G1 phase cell population compared to untreated cells (p > 0.005) ( Figure 5).

Figure 5. Effect of extacts and oil on the cell cycle of AGS cells.

Data correspond to the X ± SD of the percentages of AGS cells treated for 8 hours with P. volubilis and M. oleifera leaf extracts and P. volubilis oil at the corresponding CC₅₀. Statistical test ANOVA two-way comparison multiple treatments, Dunnet statistical test p<0.0001 data analyzed in Graph Pad Prisma 80. A value of p>0.05 (NS), p<0.05 (*), p<0.001 (**), and p<0.001 (***) was considered statistically significant (ANOVA) to compare treatments after confirming the normal distribution of the data. Sub G1 cells: hypo diploid cell population (apoptosis); G1 cells: diploid cell population or in G0 - G1 phase; G2/M cells: tetraploid cell population; and S cells: cell population in synthesis phase.

Effect of M. oleifera petroleum ether and P. volubilis ethanolic extracts in the AGS cell membrane.

AGS cell death after treatment with M. oleifera and P. volubilis derivatives extracts was characterized by measuring phosphatidylserine translocation and cell membrane permeability, which occur sequentially during apoptosis. The PE extract of M. oleifera and the EtOH extract from P. volubilis induced phosphatidylserine externalization over time, with membrane permeability increasing between 48 and 72 hours of treatment ( Figure 6b, e). In contrast, the remaining extracts showed a reversal in the maintained curves over time, increasing progressively. The P. volubilis oil induced a membrane permeability greater than the phosphatidylserine translocation ( Figure 6d).

Figure 6. Assessment of apoptosis/necrosis induced by plants derivatives on AGS cells.

The figure shows the mean and standard deviation of luminescence and fluorescence of at least two independent experiments performed in sextuplicate for the corresponding CC₅₀ for each extract or oil compared to untreated cells as negative control (NC). DMSO was used as a positive control of the assay (h). In panels b and e, there is a significant delay between the appearance of PS: Annexin V binding and the loss of membrane integrity, suggesting an apoptotic phenotype leading to secondary necrosis.

Effect of M. oleifera and P. volubilis derivatives in the activation of the caspases in AGS cells

To complement the study of the effector mechanisms of cell death, the enzymatic activity of some apoptotic markers, such as caspases 3, 8, and 9, was evaluated. Overall, all extracts increased the enzymatic activity of the caspases studied in treated AGS cells compared to the untreated control. This activity increased gradually in all treated groups and remained consistent for 72 hours. Both extracts and oil induced higher caspase eight activity compared to caspases 3 and 9 ( Figure 7a). The CH extract from M. oleifera and P. volubilis showed the most significant effect on caspase 8 induction, but it was the oil the derivative that induced the highest caspase 8 activity in AGS cells at 72 hours ( Figure 7b). On the contrary, caspase 9 showed the lowest activity in the different treatments, exhibiting almost identical fluorescence in all derivatives, except for the M. oleifera CH extract ( Figure 7a), which induced a discrete increase in the expression of caspase 9 compared to the other treatments.

Figure 7. Enzymatic activity of caspases 3, 8, and 9 in tumor cells treated withplants derivatives.

The figure shows the mean and standard deviation of the fluorescence of at least two independent experiments performed in sextuplicate for the corresponding CC₅₀ of each plant derivative, a) extract or b) oil. Panel c shows the caspase activity on positive (DMSO) and negative (NC, Vehicle) controls. A higher activity is observed in the group treated with the apoptosis inducer regarding the three caspases in a gradual manner over time compared to the negative control and the vehicle. MOEtOH: M.oleifera ethanol extract; PVEtOH; P. volubilis etanol extract; MOPE: M.oleifera petroleum ether extract; PVPE; P. volubilis petroleum ether extract; MOCH: M.oleifera ciclohexane extract; PVCH; P. volubilis ciclohexane extract; NC: negative control (untreated cells).

Inhibition of cell migration

The potential of biological derivatives to inhibit the migration ability of AGS cells was studied by analyzing the closure between the edges of the deliberately provoked wound in the cell monolayer. This analysis was performed over time and evaluated qualitatively. Of all the derivatives studied, the ethanolic extract at a concentration of 100 μg/mL revealed through microphotographs a difference with respect to its untreated counterpart in terms of wound closure at the end of treatment after 48 hours. Comparative analysis of the two groups over time allows us to identify a clear difference in the progress of wound closure in the untreated cells, which is synonymous of the maintenance of their migratory capacity, unlike what was observed in the treated tumor cells ( Figure 8).

Figure 8. Inhibition of migration of AGS cells.

The figure shows a photomicrograph of the scraping at 0 (a,b), 24 (c,d), and 48 (e,f ) hours: a,b: Wound in the monolayer of AGS cells at 0 hours c: Wound area in control (untreated) cells shows no significant wound closure at 24 hours. e: At 48 hours, the wound has almost healed in control cells. d,f: Wounds in treated cells (CC₅₀ dose) healed at a slower rate, with an almost negligible decrease in the area studied; wounds in treated cells remained with few cells within the area studied at 24 and 48 hours, magnification 10×.

Discussion

The present study provides new insights into the in vitro antitumor potential of Moringa oleifera and Plukenetia volubilis extracts against gastric cancer cells. Our results showed significant cytotoxic and antiproliferative effects across all tested derivatives, supporting the role of these plant species as promising sources of bioactive compounds with therapeutic potential. Notably, the unprocessed seed oil of P. volubilis exhibited a pronounced cytotoxic effect, inducing DNA fragmentation and apoptosis, and representing, to our knowledge, the first report of its antitumor activity in gastric cancer.

Rather than isolating specific compounds, this study focused on evaluating the biological activity of crude extracts, aligning with a phytotherapeutic approach that considers the synergistic interactions among multiple natural constituents. Crude extracts are widely used in traditional medicine and modern herbal-based formulations, and their activity may not be attributable to a single compound but to the complex chemical interplay within the matrix. This strategy allows the identification of promising plant-based extracts for further development, whether as standardized phytoproducts or as the basis for compound isolation and drug design.

Therefore, this work focused on demonstrating in vitro the biological activity on stomach cancer cells of derivatives obtained from two promising plants, P. volubilis, and M. oleifera, which have been attributed several benefits for human health, exploring different biological approaches to demonstrate their antitumor capacity.

The viability and proliferation of AGC tumor cells were affected by all biological derivatives, and inhibition of proliferation was maintained over time. M. oleifera and P. volubilis derivatives have cytotoxic activity for AGS tumor cells and can inhibit the proliferation of these cells during the first 96 hours after treatment. This effect may be due to a wide range of factors related to the preservation of phytochemicals, the percentage of water in the leaves, and the dissolution process to which the biological material is subjected. For example, solvents with more apolar and hydrophobic structures have a more significant effect at lower concentrations because of their ability to potentiate anticancer metabolites such as kaempferol, niazimycin, β-sitosterol-3-O-β-D-glucopyranoside and benzyl isothiocyanate present in M. oleifera leaf (Padayachee & Baijnath, 2020). Solvent polarity plays a critical role in the solubility of compounds, especially phenolics, as less polar solutes solubilize better in less polar solvents and more polar solutes in more polar solvents (Chahardehi, Arsad, Ismail, & Lim, 2021). Moreover, many biomolecules with cytotoxic and antiproliferative activity are inefficiently obtained with polar or hydrophilic solvents due to the presence of phenolic metabolites or hydrophobic residues. For this reason, organic solvents are often preferred in proliferation approaches (Lezoul, Belkadi, Habibi, & Guillén, 2020). However, there is evidence of significant anti-inflammatory and antioxidant activity of ethanolic extracts obtained from plants compared to organic solvents (Truong et al., 2019), as well as high in vitro antiproliferative capacity towards breast cancer cells (M. Khalil et al., 2021) and colorectal cancer lines (Mesas et al., 2021). In the specific case of Moringa, the ethanolic extract obtained from its seeds exhibited greater antiproliferative power than other extracts toward colon tumor lines, as demonstrated by Fuel et al. in 2021 (Fuel et al., 2021). These findings and those obtained in our study reinforce the fact that ethanolic extracts from different parts of plants promote biological effects of interest due to the different concentrations of flavonoids found in them (Xu, Chen, & Guo, 2019).

In the case of P. volubilis oil, which did not require chemical processing to obtain it, absolute cell culture death was observed at the highest dose tested. This event did not occur with the extracts in the viability assays. There is little evidence on the antitumor role of P. volubilis derivatives. An anti-hepatoma effect has been reported with peptides derived from the plant’s seed (He et al., 2023) and inhibition of cell growth in A459 and Hela lines due to methanolic and hexane extracts of its leaves, respectively, almost halving the cell population at the end of the assays (Nascimento et al., 2013). At the time of writing, there is no information on the in vitro antitumor potential of P. volubilis oil seeds.

The seed of P. volubilis has a particular chemical composition with a high amount of polyunsaturated fatty acids (PUFAs), the most important of which are linolenic acid (LA) and α-linolenic acid (ALA), which make up around 80% of these PUFAs (Cárdenas et al., 2021). Other vital metabolites in the oil include tocopherols, phytosterols, and terpenoids. The latter is responsible for vegetable oils’ physical characteristics; some have demonstrated antitumor effects, such as aristolene (da Anunciaçao et al., 2020) and cycloartenol (Niu et al., 2018). On the other hand, phytosterols and anticancer evidence exist for β-sitosterol (Bin Sayeed & Ameen, 2015), phytol (Alencar et al., 2018), and stigmasterol, which are some of the most abundant sterols in plant oil (Ramos-Escudero et al., 2019).

Li et al. identified the anticancer effects of stigmasterol on cell viability and proliferation in gastric cancer cells through mechanisms associated with the disruption of apoptosis (Li et al., 2018). Our findings show similarities with these results. Nonetheless, in Li’s study, they observed a G2/M phase arrest (Li et al., 2018). We observed that P. volubilis oil is a cell death-inducing agent favoring an increase of cells in the sub-G1 phase in a statistically significant way. In addition, all derivatives of M. oleifera and P. volubilis leaves, but also P. volubilis oil, induced caspase activity in AGS tumor cells, suggesting that apoptosis is the cytotoxic mechanism of cell death. The sub-G1 DNA content is characteristic of apoptotic cells, which further reinforces the cytotoxic and antiproliferative effect of the oil on the cells tested.

Studies of apoptotic markers such as phosphatidylserine externalization and caspase activity were performed to decipher the mechanisms responsible for cell death. In the early stages of cell death, translocation of phosphatidylserine to the outer layer of the cell membrane is observed, an event identified in our study by forming a luminescent complex with annexin V through luciferase conjugation. At later stages, destabilization of the cell membrane occurs, allowing entry of a fluorescent probe that binds to nucleic acids, indicating post-apoptotic necrosis, an apoptotic phenotype that results from a kinetic mismatch between an initial luminescent signal and a subsequent fluorescent signal.

According to the results obtained, the assays that faithfully represented the above described were the treatment of tumor cells with MOPE and PVEtOH extracts. For the rest of the extracts and the oil, an atypical behavior was observed in the curves, in which the first signal detected was fluorescence followed by luminescence. When comparing the signals of all derivatives with respect to the controls, especially the negative, it is inferred that in the former, other plausible mechanisms of cell death are occurring in these cells despite not exhibiting the orthodox apoptotic phenotype of this approach.

A possible cause of this atypia could be related to the time used for the analysis of the signal emission. Several assays of this type are based on the kinetic analysis of the signals in a time span of no more than 24, with short time ranges between detections (30 mins). It is very likely that these short kinetics provide a “zoom in” to events occurring in the earliest phase of treatment, thus increasing the likelihood of identifying the expected apoptotic phenotype. In any case, it should be taken into account that in some cases, as in ROS-mediated cell damage, some phytoelements can lead to the concurrence of two death mechanisms (apoptosis and necrosis) simultaneously. Among these phytoelements, vanicosides have been found, among other polyphenols, which can provoke oxidative stress in tumor cells and thus lead to cell death through various mechanisms (Bian, Wei, Zhao, & Li, 2020).

Regarding the caspase study, a higher activity of caspase 8 was observed in comparison with the rest of the enzymes, a sign indicative of the involvement of the extrinsic pathway of apoptosis, especially in the cells treated with the oil, since in these cells the enzyme was activated to a greater extent than in the other groups. Similarly, discrete involvement of the intrinsic pathway was identified through the activation of caspase 9 and the activation of caspase 3 confirmed apoptotic death in all groups, especially in the group treated with PVEtOH.

The cascades involved in the two pathways are different but converge in the activation of caspases. Initiation of the extrinsic pathway requires a trigger, which could be a phytochemical acting as a ligand, which subsequently binds to the death receptor (FAS) on the cell surface to form a complex involving intracellular activation of initiator caspase 8 with subsequent activation of executioner caspase 3 (Lavrik, Golks, & Krammer, 2005). According to the findings of this study, both extracts and oil function as inducers of cell death through the extrinsic pathway.

Several elements or signals can exert an activating role on proapoptotic events, among them ROS and NO. We decided to explore the induction of this type of molecules in tumor cells around Plukenetia Volubilis oil, since it was this derivative that had been exhibiting interesting characteristics in terms of toxicity, antiproliferation, caspases activation and cell cycle arrest, adding to this decision the fact that there are no reported antecedents on the induction role of metabolic stress in cancer for this derivative.

Treatment with P. volubilis oil leads to oxidative stress in AGS cells. The data obtained showed an evident increase in ROS and NO production in the treated tumor cells with the oil compared to the untreated group to a relatively similar degree to that induced by the positive control, especially for reactive nitrogen species. The role of NO as a tumor inductor molecule is ambiguous in comparison with ROS, as its effects depend on its concentrations and origin. At low concentrations it may exert a cytoprotective effect but at high levels it has been shown to act as a propapoptotic agent (Korde Choudhari, Chaudhary, Bagde, Gadbail, & Joshi, 2013). All these nitrosative alterations in DNA force cells to activate their repair mechanisms either by entering senescence pathways, or if the damage is very deep, apoptosis (Nakanishi, Shimada, & Niida, 2006), as observed in AGS cells according to the results obtained in cytotoxicity analysis, proliferation and apoptosis studies developed in our work.

In order to further explore the other capabilities of these plant derivatives, we sought to explore their ability to block the migration of AGS cells by studying changes in their motility. Of all the products studied, only MOEtOH had a notable effect in inhibiting the motility of these cells. According to the data obtained for each group in relation to the distance between the edges of the wounds between 0 and 48 hours, it could be established that the recovery of the wound in the untreated cells was 66% while in those treated with the extract it was 48%. Although without statistical significance, these findings indicate that at least this extract contributes to the change of phenotype in stomach tumor cells with the consequent affectation in the migration capacity. Cell motility is a hallmark of tumor progression and loss of this property has been associated with improved prognosis. However, further studies are needed to establish whether the cells are incited by the action of the extracts to follow an apoptotic or antimetastatic pathway, such as a reversal of the epithelial-mesenchymal transition. However, further studies are needed to establish whether the cells are incited by the action of the extracts to follow an apoptotic or antimetastatic pathway, such as a reversal of the epithelial-mesenchymal transition. In an approach similar to that performed in our study, Shu X et al. (2018) were able to demonstrate at the cellular and molecular level the hypothesis related to the reversal of epithelial-mesenchymal transition in gastric cancer cells. In that work, extracts from M. oleifera seed were evaluated, and through migration and invasion assays the anti-metastatic potential in gastric cancer cells was evidenced, also revealing the possible cellular target of the extract, since a positive regulation of NDRG1 expression was observed in the treated cells (Shu et al., 2018), which is a gene with invasion and metastasis suppressor activity. Based on this evidence, gene expression assays or protein analysis could be performed in the cells treated with our extract, contemplating not only the evaluation of NDRG1 but also other reversion markers such as ZO-1 and vimentin, among others.

The observed biological activity may result from the synergistic interplay of various phytochemicals naturally co-occurring in the extracts, such as flavonoids, polyunsaturated fatty acids, and phytosterols. Synergy can arise when compounds act on multiple molecular targets within the same pathway or on different but converging pathways. Flavonoids may induce mitochondria-dependent apoptosis, and sterols can modulate membrane integrity as well as the sensitivity of death receptors. Fatty acids, on the other hand, can increase ROS-mediated stress. Together, these interactions can amplify cytotoxic signals and circumvent resistance mechanisms. Although not directly tested in this study, such multi-target synergy has been proposed as a key feature of crude plant extracts in cancer therapeutics. A proposed model of these interactions is presented in Figure 9.

Figure 9. Proposed mechanism of synergistic antitumor activity of crude plant extracts.

The diagram illustrates how key phytochemicals present in Moringa oleifera and Plukenetia volubilis extracts, such as flavonoids, polyunsaturated fatty acids, and phytosterols, may act on complementary cellular targets. These include induction of oxidative and nitrosative stress (ROS/NO), activation of death receptors (extrinsic apoptosis), mitochondrial dysfunction (intrinsic apoptosis), and caspase cascade activation. The convergence of these mechanisms enhances cytotoxicity in gastric cancer cells, supporting the hypothesis of synergistic activity in crude extracts.

To our knowledge, this is the first report demonstrating the in vitro antitumor activity of P. volubilis seed oil against gastric cancer cells, including its effects on proliferation, apoptosis induction, cell cycle arrest, and oxidative stress. While previous studies have explored the bioactivity of P. volubilis seed peptides and leaf extracts in other cancer models, such as liver, lung, or cervical carcinoma, no studies have addressed the direct cytotoxic and pro-apoptotic effects of the unprocessed seed oil in gastric cancer. In addition, this study is also one of the few that comparatively evaluates ethanolic and oil extracts from two different plants (P. volubilis and M. oleifera) in a unified experimental model, offering a broader understanding of their mechanisms of action. Our findings provide new evidence supporting the role of these natural derivatives as potential therapeutic agents for gastric cancer and offer a starting point for further investigation into their molecular targets and in vivo efficacy.

Overall, this study makes original contributions to the field of natural product research in oncology. This work can be considered as one of the first to report the antitumor activity of Plukenetia volubilis seed oil in gastric cancer cells, and one of the few that directly compares ethanolic and oil-based extracts from two botanically unrelated species in a unified experimental model. Identifying extrinsic apoptosis activation, induction of oxidative stress, and cell cycle arrest highlights key mechanistic insights. These findings support the continued investigation of these derivatives as phytotherapeutic agents and as lead candidates for developing novel, low-toxicity treatments against gastric cancer.

Nonetheless, using crude extracts limits the ability to attribute the observed biological effects to specific active compounds, and the potential contribution of individual constituents was not experimentally determined. Second, all experiments were conducted in a single gastric cancer cell line (AGS), and therefore, the findings may not fully represent other tumor types or gastric cancer subtypes. Although evidence of apoptosis and oxidative stress was identified, more in-depth molecular validation (e.g., western blotting, gene expression profiling) was not performed. Lastly, in vivo studies are needed to confirm the extracts safety, efficacy, and pharmacokinetics under physiological conditions. Despite these limitations, the present findings provide a valuable foundation for further research on the anticancer potential of M. oleifera and P. volubilis.

Ethics and consent

Ethical approval and consent were not required.