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. 2024 Mar 21;16(1):16–23. doi: 10.4103/jpbs.jpbs_878_23

Melinjo Seeds (Gnetum gnemon L.) Antioxidant Activity and Cytotoxic Effects on MCF-7 Breast Cancer Cells: A Study Based on Tracing of Resveratrol Compound

Asep Sukohar 1, Muhammad Iqbal 2,✉, Ramadhan Triyandi 2, Sahidin 3
PMCID: PMC11060622  PMID: 38694961

ABSTRACT

Introduction:

Breast cancer is one of the deadliest cancers worldwide. One of the polyphenols, namely, resveratrol, has been proven to have anticancer activity. Melinjo seeds which contain resveratrol need to be tested for their potential as an anti-breast cancer agent. This study aims to determine the antioxidant activity and cytotoxic effect of melinjo seeds based on solvent variations and resveratrol tracing.

Methods:

Extraction of melinjo seeds was performed using the soxhletation method. Antioxidant test was performed using the 2,2-diphenyl-1-picrylhydrazil method. The in vitro cytotoxic test was carried out using the microtetrazolium method. Cytotoxic test was carried out on MCF-7 breast cancer cells using a concentration range of melinjo seeds between 31,25 and 1000 μg/mL. Antioxidant and anticancer potentials are expressed in inhibitory concentration (IC)50 values. Resveratrol was traced using preparative high-performance liquid chromatography (Prep-HPLC).

Results:

Melinjo seed ethanol extract provided the largest total phenolics (126,154 ± 0,865 mg GAE/g sample) and total flavonoids (44,576 ± 0,611 mg QE/g sample) among all solvent fractions. The antioxidant activity of melinjo seeds from ethanol extract, n-hexane fraction, ethyl acetate fraction, and ethanol fraction was 263,307 ppm, 317,595 ppm, 160,878 ppm, and 181,159 ppm, respectively. The ethyl acetate fraction of melinjo seeds showed the strongest cytotoxic effect (94.6 μg/mL) among all extracts and solvent fractions. Prep-HPLC showed that the ethanol extract of melinjo seeds contained resveratrol, while the ethanol and ethyl acetate fractions of melinjo seeds were thought to contain resveratrol derivatives.

Conclusion:

The antioxidant activity of melinjo seeds showed a cytotoxic effect on MCF-7 cells, which varied based on solvent polarity and total phenolic and total flavonoid. The ethyl acetate fraction which is thought to contain resveratrol derivatives provides the most potent antioxidant activity and cytotoxic effect. These results indicate that melinjo seeds containing resveratrol and its derivatives have the potential for anticancer of the breast. Further studies are still needed in determining the structure of resveratrol compounds and their derivatives to ensure their biological activity and mechanism of action.

KEYWORDS: Antioxidant, breast cancer, cytotoxic, melinjo seeds, resveratrol

INTRODUCTION

Breast cancer is the most common cancer among women worldwide.[1] There are around 2.1 million cases of breast cancer worldwide, and around 630,000 die from the disease.[2] Breast cancer is associated with oxidative stress conditions at the cellular and molecular levels.[3,4] Oxidative stress is a physiological state in which reactive oxygen species (ROS) or reactive nitrogen species (RNS) exceed antioxidant metabolites, resulting in high levels of free radicals. Increased ROS/RNS formation can be overcome with antioxidants.[4,5] Thus, the role of antioxidants is very important to inhibit ROS/RNS, which mediates oxidative stress in breast cancer cells.

Existing breast cancer treatments still cause drug resistance and attack normal cells.[6] Meanwhile, many anticancer studies have focused on the active ingredients of natural ingredients as chemopreventive agents to address drug resistance and safety problems.[7] Natural compounds are most preferred for the prevention and treatment of cancer because of their anticancer capabilities, easy availability, potential to overcome resistance, safety, and efficiency. Among natural compounds, polyphenols (flavonoid, catechin, hesperetin, flavone, quercetin, phenolic acid, ellagic acid, lignan, stilbene, etc.) represent a large and diverse group used in the prevention and treatment of cancer. Natural flavonoids have antioxidant, anti-inflammatory, and anticancer activities through various pathways. Flavonoids induce apoptosis in breast cancer.[8] In other studies, flavonoids (apigenin, luteolin, myricetin) selectively reduced cell viability of ovarian cancer cells (A2780, OVCAR-3, SKOV-3) by affecting intra-cellular ROS.[9] Furthermore, the cytotoxic potential of the Carlina acaulis L. plant in the ethyl acetate fraction (the most abundant polyphenolic compound and the strongest antioxidant potential) against human colorectal adenocarcinoma (HT29) and human cervical cancer (HeLa) cells shows the most promising anticancer activity.[10] Thus, polyphenols contained in the phenolic and flavonoid compound groups have a relationship with antioxidant and anticancer activity.

One of the polyphenol contents in melinjo seeds, namely, resveratrol, has been proven to have antioxidant activity.[11] Resveratrol is considered as one of the main compounds for developing new effective agents for cancer prevention or therapy.[12,13] Resveratrol also has broad protective activity against several types of cancer through inhibiting oxidative stress by influencing ROS.[14] Resveratrol has low cytotoxicity to normal cells and is considered safe for public use. Meanwhile, melinjo seeds also have high nutritional value and are consumed as the main food of the Indonesian population.[15] Research on resveratrol obtained from melinjo seeds for anti-breast cancer is still very minimal.[16] Therefore, this study emphasizes exploring the potential of melinjo seeds in Lampung Province as a chemoprevention agent for breast cancer by examining the antioxidant activity, total phenolic compound, and total flavonoid compound of melinjo seeds. Then, this exploration is directed at tracing the resveratrol compound contained in melinjo seeds.

SUBJECTS AND METHODS

Plant material

Melinjo seeds come from the Natar plantation area, South Lampung, Indonesia. Determination of melinjo plant was carried out at the Biology Laboratory, Faculty of Mathematics and Natural Sciences, University of Lampung.

Extract preparation

Melinjo seeds are dried in the sun covered with a transparent black cloth and then peeled and ground into a fine powder in a mechanical blender. Melinjo seed powder (50 g) was packed into a Soxhlet apparatus and extracted with 300 mL 96% ethanol (Merck) until circulation ended. The filtrate was then filtered using Whatman No. filter paper. 1, and the filtrate was concentrated using a rotary evaporator (Buchi R100) at 60°C. The extracts were dried, weighted, and stored at 4°C in storage vials for experimental use.

Total phenolic content

The total phenolic content of the extract was determined by the Folin-Ciocalteu method. Briefly, 200 μL crude extract (1 mg/mL) was made up to 3 mL with distilled water and mixed thoroughly with 0.5 mL Folin-Ciocalteu (Merck) reagent for 3 minutes, followed by the addition of 2 mL of 20% (w/v) sodium carbonate (Merck). The mixture was left for a further 60 minutes in the dark, and the absorbance was measured at 650 nm. The total phenolic content was calculated from the calibration curve, and the results are expressed as mg gallic acid equivalent per g dry weight.[17]

Total flavonoid content

The total flavonoid content of the crude extract was determined by the aluminum chloride colorimetric method. Briefly, 50 μL crude extract (1 mg/mL ethanol) was made up to 1 mL with methanol (Merck) and mixed with 4 mL distilled water and then 0.3 mL 5% sodium nitrite (NaNO2) (Merck) solution; 0.3 mL of 10% (w/v) aluminum chloride (AlCl3) (Merck) solution was added after 5 minutes of incubation, and the mixture was allowed to stand for 6 minutes. Then, 2 mL of 1 M sodium hydroxide (NaOH) (Merck) solution was added, and the final volume of the mixture was adjusted to 10 mL with distilled water. The mixture was left for 15 min, and the absorbance was measured at 510 nm. The total flavonoid content was calculated from the calibration curve, and the results were expressed as mg quercetin equivalent per g dry weight.[17]

Antioxidant test

The antioxidant activity of the extract was determined by the 2,2-diphenyl-1-picrylhydrazil (DPPH) test, as previously described with some modifications. Briefly, 200 μL of each extract (100–500 μg/mL) was mixed with 3.8 mL of DPPH (Sigma-Aldrich) solution and incubated in the dark at room temperature for 1 hour. The absorbance of the mixture was then measured at 517 nm. Data from absorbance measurements were analyzed for the percentage of antioxidant activity. The IC50 value was determined by probit analysis from concentration log data with free-radical binding percentage probit.[17]

Cell culture conditions

MCF-7 cells were obtained from ATCC (American Type Cell Cancer), which is a collection from the cell culture laboratory of the Faculty of Medicine and Health Sciences, Muhammadiyah University of Yogyakarta (UMY), Indonesia. MCF-7 breast cancer cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich) and penicillin–streptomycin (1%) (Gibco) at 37°C in a 5% CO2-containing humidifier.[18]

Cytotoxic test

The cytotoxic test was carried out with the reagent [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide] (MTT). MCF-7 cells at a density of 5000 cells/well were distributed into 96-well plates and incubated for 24 hours in DMEM. Cells were treated with extract concentrations of 31.25, 62.5, 125, 250, 500, and 1000 μg/mL and incubated for 24 hours. For screening purposes, concentration determination is carried out by trial and error over various concentration variations. At the end of incubation, 100 μL of DMEM containing 5 mg/mL MTT was added to each well and incubated for 3 hours at 37°C. Living cells will react with MTT to form purple formazan crystals. The crystals were dissolved by adding 10% sodium dodecyl sulfate (SDS) blocking reagent in 0.01N HCl (Merck) and left in a dark place overnight; then the absorbance was read with an enzyme-linked immunosorbent assay (ELISA) reader (Tecan Infinite F50 GP) at a wavelength of 595 nm.[18] The cytotoxic test was carried out according to the Health Research Ethics Committee, Faculty of Medicine, University of Lampung, Indonesia (2548/UN26.18/PP.05.02.00/2022).

Cytotoxic test analysis

The data obtained in the form of the absorbance of each well were converted into the percentage of living cells and analyzed statistically using the correlation test method, followed by a significance test to determine the significance between the levels of the test extract and the percentage of living cells. Then IC50 is calculated using the linear regression method. IC50 is the concentration that causes the death of 50% of the cell population so that its cytotoxic potential can be identified.[18]

Resveratrol tracing

Resveratrol tracing using Prep-HPLC was done. The HPLC system used was HPLC preparative infinity II 1260 (Agilent). Preparation of standard resveratrol (Sigma-Aldrich) compounds with a concentration of 100 μg/mL was done as a comparison to pure resveratrol and to find the maximum wavelength from 200 nm to 400 nm. The mobile phase was used through a comparison of the mobile phase of distilled water: methanol 55: 45 using a non-polar C18 column measuring 150 mm in diameter of 30 mm; the volume rate of the mobile phase was 30 mL/min.[19] The wavelength results are obtained based on retention time (RT), sharp peaks, and percent area. Separation of compounds was done using wavelengths of 215 nm, 230 nm, 290 nm, and 309 nm. The reading of the chromatogram results can be seen from the peak (peak) and narrower (sharp). The test sample was made at 100 ppm.

RESULTS

Total phenolic content, total flavonoid content, and antioxidant activity

The search for resveratrol was preceded by extraction and fractionation of melinjo seeds. In sequence, the largest total phenolics of melinjo seeds are ethanol extract, ethanol fraction, ethyl acetate fraction, and n-hexane fraction. Meanwhile, the highest order of total melinjo seed flavonoids is ethanol extract, ethyl acetate fraction, ethanol fraction, and n-hexane fraction. The strongest antioxidant activity of melinjo seeds was demonstrated by the ethyl acetate fraction, ethanol fraction, ethanol extract, and n-hexane fraction. The ethyl acetate fraction showed the strongest antioxidant (160,878 ppm) and anticancer potential (IC50 = 94.6 μg/ml) [Table 1]. The active compounds contained in extracts and fractions are not known with certainty. Furthermore, it is necessary to trace the chemical compounds contained in the ethanol extract, n-hexane fraction, ethyl acetate fraction, and ethanol fraction to obtain resveratrol.

Table 1.

Testing melinjo seeds in various indicators

Test Ethanol extract n-Hexane fraction Ethyl acetate fraction Ethanol fraction
Total Fenolic (mg GAE/g sample) 126.154±0.865** 18.287±0.416** 47.680±0.635** 100.643±0.416**
Total Flavonoid (mg QE/g sample) 44.576±0.611* 16.667±0.258* 34.746±0.508* 23.559±0.224*
Antioxidant (ppm) 263.307* 317.595* 160.878* 181.159*
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***The phenolic, flavonoid, and antioxidant activities of melinjo seeds were investigated using solvents of varying polarity. Extraction was carried out using the soxhletation method. GAE: gallic acid equivalent; QE: quercetin equivalent. *p < 0.05 indicates a significant difference between samples in one test row. **p >0.05 indicates there is no significant difference between samples in one test row

Cytotoxic test

The profile of the percentage of cell life to the ethyl acetate fraction of melinjo seeds showed a decrease in the percentage of cell life of MCF-7, which was greater with increasing levels of the fraction (dose-dependent phenomenon). The ethanol extract, ethanol fraction, and n-hexane fraction of melinjo seeds showed a weak cytotoxic effect on MCF-7 cells [Table 2]. In contrast, the ethyl acetate fraction showed a strong cytotoxic effect against MCF-7 cells (IC50 = 94.6 μg/ml) [Table 2]. A comparison of cell morphology showed that the ethyl acetate fraction of melinjo seeds had cytotoxic activity on MCF-7 cells. The higher the content of the ethyl acetate fraction of melinjo seeds, the stronger the cytotoxic potential [Figure 1 and 2].

Table 2.

Cytotoxic test of melinjo seeds in various solvents

Cytotoxic test Ethanol extract n-Hexane fraction Ethyl acetate fraction Ethanol fraction
IC50 (µg/mL) 535.09* 223.8* 94.6* 446.3*
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*p < 0.05 indicates a significant difference between samples in one test row.

Figure 1.

Figure 1

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MCF-7 cell morphology in treatment with ethyl acetate fraction. Control cell (a) and treatment cells of 31.25 μg/mL (b), 62.5 μg/mL (c), 125 μg/mL (d), 250 μg/mL (e), and 500 μg/mL (f). Notes: (1) Dead cell and (2) live cell. (40x magnification)

Figure 2.

Figure 2

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Effect of melinjo seeds on the viability of MCF-7 cell. (a) Ethanol extract; (b) ethanol fraction; (c) ethyl acetate fraction; (d) n-hexane fraction. The ethyl acetate fraction showed a 50% decrease in cell viability at a concentration of 94.6 m g/mL, while other melinjo seed extracts and fractions showed a weak cytotoxic effect

Resveratrol tracing

The results of separating resveratrol compounds in melinjo ethanol extract, melinjo ethanol fraction, melinjo ethyl acetate fraction, and n-hexane fraction were obtained at wavelengths of 215 nm, 230 nm, 290 nm, and 309 nm, respectively [Figures 3 and 4]. There are four wavelengths indicating the presence of resveratrol: at a wavelength of 215 nm (RT = 3.707 minutes), at a wavelength of 230 nm (RT = 3.707 minutes), at a wavelength of 290 nm (RT = 3.707 minutes), and at a wavelength of 309 nm (RT = of 3.707 minutes) [Figures 3 and 4]. Estimates of the presence of resveratrol derivatives in the ethanol extract of melinjo seeds were obtained at a wavelength of 215 nm (RT = 3.617 minutes and 4.129 minutes), as well as the ethanol extract at a wavelength of 230 nm (RT = 3.616 minutes and 4.129 minutes), at a wavelength of 290 nm (RT = 3.615 minutes and 4.127 minutes), and at a wavelength of 309 nm (RT = 3.614 minutes and 4.128 minutes) [Figures 3 and 4]. Thus, it can be said that the ethanol extract of melinjo seeds contains resveratrol. The resveratrol content in melinjo seed ethanol extract is estimated to be much smaller than standard resveratrol. This is indicated by the wide area and peak sharpness in the chromatography results [Figure 3b].

Figure 3.

Figure 3

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HPLC chromatogram of resveratrol tracing on melinjo seeds. (A) Elution at 215 nm wavelength. (B) Elution at 230 nm wavelength. (a) Resveratrol, (b) ethanol extract, (c) ethanol fraction, (d) ethyl acetate fraction, and (e) n-hexane fraction

Figure 4.

Figure 4

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HPLC chromatogram of resveratrol tracing on melinjo seeds. (A) Elution at 290 nm wavelength. (B) Elution at 309 nm wavelength. (a) Resveratrol, (b) ethanol extract, (c) ethanol fraction, (d) ethyl acetate fraction, and (e) n-hexane fraction

The results of separating the resveratrol compound from the ethanol fraction of melinjo against the resveratrol comparator were also adjusted for the retention time at each wavelength. The ethanol fraction of melinjo seeds is thought to contain a resveratrol derivative compound with a retention time of 2.669 minutes (215 nm) and 2.669 minutes (230 nm) [Figure 3]. Meanwhile, the ethanol fraction of melinjo seeds did not contain resveratrol or its alleged derivatives (290 nm) and was suspected to contain resveratrol derivatives (309 nm; RT = 2.255 minutes) [Figure 4]. The ethyl acetate fraction of melinjo seeds is also thought to contain a resveratrol derivative compound as indicated by a retention time of 2.601 minutes (215 nm) and 2.669 minutes (230 nm) [Figure 3]. The content of resveratrol derivatives in the ethyl acetate fraction is estimated to be quite high as indicated by the chromatographic peak character [Figure 3]. The ethyl acetate fraction of melinjo seeds did not show the presence of resveratrol or its derivatives at 290 nm and 309 nm [Figure 4]. The results of the elution of compound separation showed that the n-hexane melinjo fraction did not contain resveratrol or its derivatives [Figures 3 and 4].

DISCUSSION

Currently, there is increasing attention toward the application of natural compounds as preventive and therapeutic agents in the management of cancer.[14] The mechanism of action of these natural compounds is emphasized as antioxidants.[5] In this research, exploration of the antioxidant activity of melinjo seeds as a breast cancer chemoprevention agent with the target of tracing for resveratrol compounds was carried out through the extraction and fractionation stages. Apparently, the results showed that total phenolics and total flavonoids of melinjo seeds did not correlate with antioxidant activity. However, based on biological action, antioxidant activity is usually related to total phenolics.[20,21]

The ethyl acetate fraction of melinjo seeds actually shows the most potent antioxidant activity, even though the total phenolics and total flavonoids are lower than the ethanol extract of melinjo seeds [Table 1]. Research showed that the more non-polar the organic solvent used for sample extraction, the lower the total phenolic content and antioxidant activity.[21] In this study, the total phenolics actually decreased along with the non-polarity of the organic solvent. However, it is suspected that the antioxidant potential is also influenced by the type and total of flavonoids present. Interestingly, other studies actually show the opposite by showing that antioxidant activity values vary significantly according to the type of solvent and do not depend on the degree of polarity of the organic solvent.[21,22,23] It is known that various extraction factors, including method, temperature, time, and solvent system, significantly influence the antioxidant quality of plant-derived products.[21] It can be said that the two extraction stages, namely, the initial extraction stage (crude ethanol extract) and fractionation, are thought to influence the characteristics of the compounds (total phenolics and total flavonoids) on the antioxidant potential of melinjo seeds.

Cytotoxic test showed that the ethyl acetate fraction of melinjo seeds exerted a cytotoxic effect on MCF-7 breast cancer cells (IC50 = 94.6 μg/ml) with more potent antioxidant activity than other extracts and fractions [Tables 1 and 2]. This is in line with other research that the antioxidant potential of a plant is synergistic with its anticancer effect. Artemisia absinthium L. extract showed cytotoxic activity on DLD-1 and ECC-1 cancer cells through its antioxidant activity.[20] In other studies, the ethyl acetate fraction of Carlina acaulis L. also tended to show the strongest cytotoxic effect against human colorectal adenocarcinoma cancer cells (HT29) and human cervical cancer cells (HeLa) compared to other organic solvent fractions.[10] This indicates that ethyl acetate as a semipolar solvent dissolves more semipolar active substances (e.g., flavonoids, triterpenoids, and alkaloids), which are proven to have anticancer effects.[24,25,26]

In contrast to the antioxidant activity of the ethanol extract, the ethanol fraction and the n-hexane fraction are not in line with their cytotoxic effects. The ethanol extract and ethanol fraction of melinjo seeds containing resveratrol and suspected resveratrol derivatives did not show a strong cytotoxic effect [Table 2]. This is thought to be because the total content of resveratrol or its derivatives is relatively small due to the low content of resveratrol and its derivatives in plants.[12] Another factor that causes high antioxidant activity but does not cause a commensurate cytotoxic effect is the decomposition of antioxidants during action.[3] The n-hexane fraction actually provided a more potent cytotoxic effect than the ethanol extract and ethanol fraction. This is because organic solvents dissolve a variety of compounds with different polarities.[23] Differences in the types of compounds contained in different solvents will affect the type of compound binding to cancer cell receptors.[27] It is suspected that the n-hexane fraction contains non-polar phenolic compounds and other compounds that also have anticancer potential (alkaloids, terpenoids).[28,29] This shows the need for synergistic interactions between several compounds in melinjo seeds to have a cytotoxic effect on MCF-7 cells.

On the other hand, there are contradictions in preventing or treating cancer with antioxidants.[3] Increased ROS are oncogenic, causing damage to DNA, proteins, and lipids, increasing genetic instability and tumorigenesis. On the contrary, toxic levels of ROS production in cancer are anti-tumorigenic, resulting in increased oxidative stress and induction of tumor cell death.[30] This raises suspicions that the antioxidant activity of the extracts and test fractions in this study is not comparable to their cytotoxic effects [Tables 1 and 2]. The role of active compounds which should act as antioxidants to inhibit ROS is precisely in the balance of redox reactions needed to induce ROS. Thus, patients with cancer who take large amounts of antioxidants during the chemotherapy may actually block ROS-induced apoptosis of cancer cells.[3] This is thought to have contributed to the variation in cytotoxic effects based on antioxidant activity in this study.

Meanwhile, it is strongly suspected that the resveratrol derivative that was traced is present in the ethyl acetate fraction in large quantities [Figure 3d]. However, the chromatogram showed that the ethyl acetate fraction did not have the same compound elution as its comparator resveratrol [Figure 3a and d]. This suggests the presence of resveratrol derivatives or other active compounds in the fraction. Resveratrol as a natural compound is reported to inhibit the growth of breast cancer cells in ER-positive (example: MCF-7) and negative (example: MDA-MB-231, MDA-MB-435) breast cancer cell lines, resulting in cell-specific regulation of G1 Stage/S and G2/M of the cell cycle. Resveratrol can also inhibit the migration and invasion of breast cancer cells and induce their apoptosis so as to achieve the goal of breast cancer treatment.[12] Resveratrol can also cancel CYP1A activity induced by the aryl hydrocarbon benzo[a] pyrene (B[a]P) environment and is catalyzed by directly suppressing CYP1A1/1A2 enzyme activity and signal transduction pathways that increase the expression of carcinogen-activating enzymes in human breast cancer MCF-7.[31] The ethyl acetate fraction in this study, which is thought to contain a resveratrol derivative such as resveratrol oligomer polyphenol, is thought to have an anticancer effect on MCF-7 cells with this mechanism.

Previous studies have shown that melinjo seed extract also has a cytotoxic effect on various in vitro exposures to cancer cells, namely, human pancreatic cancer cells (PANC-1), human prostate cancer cells (PC-3), human colon cancer cells (HT-29), and human embryonic kidney epithelial cells (HEK-293T), with an overall IC50 of 38–90 μg/mL.[15] In another study, it was shown that resveratrol had a cytotoxic effect on breast cancer (4t1) and liver cancer (HepG2) cells (IC50 = ± 200 μM) and became more potent with incubation time.[32] In addition, resveratrol showed cytotoxic effects on lung cancer cells (A549), breast cancer cells (HCC-1806), and liver cancer cells (HepG2) with an average IC50 of 50 μg/mL in all these cells.[33] Furthermore, research on grape seed containing resveratrol showed that grape seed extract also exerts a cytotoxic effect on MCF-7 breast cancer cells (IC50 = 0.5–100 μg/mL).[34] In this study, the ethyl acetate fraction of melinjo seeds had a cytotoxic effect on MCF-7 cells (IC50 = 94.6 μg/mL). Meanwhile, in vitro cytotoxicity studies of medicinal plant extracts collected from various countries in Africa against various cancer cells showed IC50 in the range of 20–300 μg/mL.[35,36] Thus, the ethyl acetate fraction of melinjo seeds in this study has anticancer potential that is close to that of resveratrol and is not inferior to other plants (grapes) and also not much different from the potential of Africa medicinal plants.

Thus, the exploration of the antioxidant activity of melinjo seeds using the soxhletation method in this study varied according to their cytotoxic effects. Extraction factors, including the method and polarity of the solvent, as well as the exposure characteristics of the cancer cells tested can influence the antioxidant potential and cytotoxic effect.[35] The estimated mechanism of action of melinjo seeds which are thought to contain resveratrol and its derivatives also needs to be explored further using computational chemistry–molecular docking. In addition, tracing of anticancer activity needs to be carried out on various other types of cancer cells which are then linked to the immunodulatory characteristics of resveratrol and its derivatives from melinjo seeds.

Financial support and sponsorship

Higher Education for Technology and Innovation (HETI) Project University of Lampung.

Conflicts of interest

There are no conflicts of interest.

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