{"id":53299,"date":"2023-12-31T11:06:57","date_gmt":"2023-12-31T11:06:57","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=53299"},"modified":"2024-01-05T06:30:08","modified_gmt":"2024-01-05T06:30:08","slug":"investigation-of-antioxidant-and-anticancer-activity-againts-mcf-7-and-hela-cancer-cells-of-melinjo-gnetum-gnemon-l","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol16no4\/investigation-of-antioxidant-and-anticancer-activity-againts-mcf-7-and-hela-cancer-cells-of-melinjo-gnetum-gnemon-l\/","title":{"rendered":"Investigation of Antioxidant and Anticancer Activity againts MCF-7 and HeLa Cancer Cells of Melinjo (Gnetum gnemon L.)"},"content":{"rendered":"\n

In<\/strong>troduction<\/strong><\/p>\n\n\n\n

Cancer\nremains one of the leading causes of morbidity and mortality globally. There\nare many diseases caused\nby oxidative stress such as cancer 1<\/sup> .\nIt occurs from a multiple-step process which includes initiation, promotion,\nand progression stages, called carcinogenesis 2<\/sup>.<\/sup> The incidence rate of\ncancer rises to 18.1 million new cases and 9.6 million cancer deaths in 2018.\nFrom 36 different types of cancer, cancer mainly affects women in the form of\nbreast and cervical cancer 3<\/sup>.<\/sup>\nIn present, various treatments i.e chemotherapy, radiotherapy, surgery, and\nchemically derived drugs are used as cancer treatment 4<\/sup>. However, those modality treatments have\nsome side effects 5<\/sup>.\nTherefore, discovery and development studies were still required to find new\nand effective anticancer drugs 6<\/sup>.\n<\/p>\n\n\n\n

Generally, plants have been used\nwidely to prevent and treat cancer for a long period 7<\/sup>.\nPlants medicine has been recognized as a source of potential therapeutic due to\ntheir biologically active compounds. Thus, many anticancer drugs and\nantioxidants are derived from natural sources 8<\/sup>.\nOver 60% of chemotherapeutic drugs are identified and isolated from plants or\ntheir synthetic derivatives 6<\/sup>.\nPlant-derived agents have an important role in the treatment of cancer due to\nproducing a wide range of chemical constituents called a secondary metabolite (9). For\ninstance, among the many plants, Gnetum gnemon<\/em> L. (G. gnemon<\/em>)  local name: Melinjo has been identified with\nthe presence of secondary metabolite and polyphenols 10<\/sup>,11<\/sup>. Melinjo (G. gnemon<\/em>) is a\ngymnospermae plant native to Southeast Asia whose seeds and fruits are commonly\nused in Indonesian cuisine 11<\/sup>. The seed flour of melinjo was\nevaluated for nutritional composition, antioxidant activity and functional\nproperties. It consisted primary utilitarian groups such as: amines, amides, amino acids, polysaccharides,\ncarboxylic acids, esters and lipids 12<\/sup>.<\/p>\n\n\n\n

Data from preclinical studies show the potential of the melinjo\nseeds as antioxidants, decreasing uric acid, antimicrobial, anti-obesity, and\nanti-cancer  13<\/sup>,<\/sup>14<\/sup>,<\/sup>11<\/sup>,<\/sup>15<\/sup>. Therefore, extensive\ninvestigation of the potential of the seeds as anticancer agents is necessary.\nThus, thoroughly evaluating cytotoxic activity and screening raw extracts of\nthe seeds would confirm these effects. <\/p>\n\n\n\n

Ma<\/strong>terial and Methods<\/strong><\/p>\n\n\n\n

Chemical\nand Reagents<\/strong><\/p>\n\n\n\n

The\nantioxidant was performed by colorimetric through DPPH assay and MTT assay were\nconducted for cytotoxicity. The DPPH and MTT kit was purchased\nfrom Sigma-Aldrich. All chemicals and solvents were analytical grades and\nobtained from commercial sources.<\/p>\n\n\n\n

Materials Collection and Identification<\/strong><\/p>\n\n\n\n

The edible parts of G.gnemon <\/em>were selected because they are widely consumed. G.gnemon<\/em> seeds were harvested in March 2021 from the Pesawaran Regency of Lampung Province, Indonesia. The botanical identification of G.gnemon <\/em>was performed at the Botanical Laboratory, Faculty of Mathematics and Natural Sciences, University of Lampung, Lampung, Indonesia.<\/p>\n\n\n\n

Preparation and Extraction<\/strong><\/p>\n\n\n\n

Based on our previous study 16<\/sup>, seeds were air-dried in air shade at room temperature, then ground to a uniform powder. The powder of dry seeds G. gnemon<\/em> (300 g) was macerated in 1.2 L of ethanol for three days and then filtered. The thick extracts were obtained by concentrating with a rotary evaporator at 40\u00b0C and stored at 4\u00b0C until being used. For qualitative phytochemical analysis, the residue was reconstituted in a solvent before testing.<\/p>\n\n\n\n

Qualitative Phytochemical Analysis<\/strong><\/p>\n\n\n\n

The ethanol seed extract was subjected to the qualitative phytochemicals screening of some chemical compounds i.e. alkaloids, flavonoids, saponins, tannins, and triterpenoids. Mayer test for identifying alkaloids, Shinoda test, Foam test, Braemer\u2019s test, and Salkowski test for flavonoids, saponin, tannin and terpenoid respectively 17,18<\/sup>.<\/p>\n\n\n\n

DPPH Antioxidant Activity Determination<\/strong><\/p>\n\n\n\n

The DPPH was used to determine the free radical scavenging activity 19, 20.<\/sup> A total 2 mL of DPPH (50 ppm in ethanol) was mixed with 2 mL extract. This was incubated for 30 min at room temperature (23-25\u00b0C) and protected from light. The absorbance was measured with a spectrophotometer at 517 nm 16<\/sup> then the IC50<\/sub> value (\u00b5g\/mL) determination was followed. Experiments were conducted in triplicates.  <\/p>\n\n\n\n

Cell Culture<\/strong><\/p>\n\n\n\n

MCF-7 and HeLa cell lines were used during the experiment. Cells were cultured in a complete growth medium: Roswell Park Memorial Institute (RPMI 1640) media supplemented with 10% fetal bovine serum (FBS), antibiotics (100 I.U\/mL penicillin and 100 \u00b5g\/mL streptomycin) at 37\u00b0C, 5% CO2<\/sub> incubator humidity. Cells were cultured in healthy conditions and exponentially growing cells (-80% confluency) were used for experiments.<\/p>\n\n\n\n

Cytotoxicity Assay<\/strong><\/p>\n\n\n\n

The cytotoxicity of the ethanol extract on MCF-7 and HeLa cells was measured using an MTT assay. The cells were seeded in 96-microwell plates a density of the cell 2×104<\/sup> cells\/well then incubated for 24 hours (h). A total 100 \u00b5L amount of extract was added to test wells and the microplates were incubated for 24 h. In addition, removing the supernatant and add 20 \u03bcL of MTT solution to each well and incubated for 3\u2009h. The supernatant was added with 100\u2009\u03bcl DMSO and dissolved formazan crystals. The amount of formazan crystal was measured at wavelength 570 nm 21<\/sup>. The percentage of the cell viability of the cells treated with ethanol extract was calculated according to the formula:<\/p>\n\n\n\n

% Cell viability  = (Abssample<\/sub>– Abscontrol<\/sub>)\/Abscontrol<\/sub>x100%<\/p>\n\n\n\n

The inhibitory concentration (IC) 50 value was calculated from a graph plotting % cell viability against the concentration of the sample.<\/p>\n\n\n\n

Observation of Morphological Changes<\/strong><\/p>\n\n\n\n

MCF-7 and HeLa cells were seeded in 96-microwell plates at the density of the cell 2×104<\/sup> cells\/well then incubated for 24 h. They were then treated with the extract and cell morphology was observed and analyzed after 24 h incubation.<\/p>\n\n\n\n

Statisctic Analysis<\/strong><\/p>\n\n\n\n

All data analysis were performed using Microsoft Excel software 2016 and the result was presented as mean \u00b1 SD with triplicate (n=3).<\/p>\n\n\n\n

Re<\/strong>sults<\/strong><\/p>\n\n\n\n

Sample extraction<\/strong><\/p>\n\n\n\n

The G.gnemon<\/em> seeds extract\nwas obtained by evaporation of ethanol as a solvent and the\npercentage by weight yield was estimated at 0.55% based on the solvent used.\nThe qualitative phytochemical investigation result found flavonoid, tannin, and\nterpenoid (Table 1).<\/p>\n\n\n\n

Tabel 1: Qualitative Phytochemical screening of G.gnemon <\/em>L. seed extracts.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n
\n

Constituents<\/strong><\/p>\n<\/td>\n

\n

Test<\/strong><\/p>\n<\/td>\n

\n

Result<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Alkaloid<\/p>\n<\/td>\n

\n

Mayer test<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

Flavonoid<\/p>\n<\/td>\n

\n

Shinoda test<\/p>\n<\/td>\n

\n

+<\/p>\n<\/td>\n<\/tr>\n

\n

Saponin<\/p>\n<\/td>\n

\n

Frothing test \/ Foam test<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

Tanin<\/p>\n<\/td>\n

\n

Braemer\u2019s test<\/p>\n<\/td>\n

\n

+<\/p>\n<\/td>\n<\/tr>\n

\n

Terpenoid<\/p>\n<\/td>\n

\n

Salkowski test<\/p>\n<\/td>\n

\n

+<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Abbreviations: (+), present;(\u2212), absence; N, Not indicated.<\/p>\n\n\n

DPPH\nAntioxidant Activity Determination<\/strong><\/p>\n\n\n\n

The most common method to determine the antioxidant activity of\nvarious compounds is the DPPH assay 22<\/sup>.\nThis assay was measured by changing the purple ethanol solution of DPPH. The\nantioxidant agents can convert DPPH into 1-1 diphenyl-2-picryl hydrazine<\/em>\nin the yellow molecule, by transferring electrons or hydrogen 8<\/sup>.\nThe result of antioxidant activity as mean \u00b1 SD was 543.19\u00b111.43 \u00b5g\/mL in 3\nreplications (Figure 1). The ascorbic acid as a standard with a coefficient\ncorrelation (R2<\/sup>) of  0.9993\nhas IC50<\/sub> 5.42 \u00b1 0.009 \u00b5g\/mL.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 1:<\/strong> The IC50<\/sub> of ethanol G. gnemon<\/em> L. seeds extract in DPPH assay (mean \u00b1 SD).<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Cytotoxicity\nassay<\/strong><\/p>\n\n\n\n

The cell viability on MCF-7 and HeLa cells was assessed in vitro\nthrough MTT assay. The extract is revealed as moderate cytotoxicity (Figure.\n2). According to ISSO: 10993-5 standard, percentages of cell viability over 80%\nare considered as non-cytotoxicity; within 80%\u201360% weak; 60%\u201340% moderate and\nunderneath 40% strong cytotoxicity respectively following above 80% as\nnon-cytotoxicity, within 80-60% as a weak, 60-40% moderate and below 40% strong\ncytotoxicity 23<\/sup>.\nBase on Figure. 2 indicated that after being treated with the extract for 24 h,\nsignificantly decreased the % cell viability (p<0.05) at the final\nconcentration, up to 47.55% and 62% for MCF-7 and HeLa cells respectively. <\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 2: <\/strong>Decreased % cell viability of MCF-7 and HeLa cancer cell line by G. gnemon <\/em>extrats.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

The cytotoxicity of the extract (IC50<\/sub>)\nwas determined in comparison to the anticancer drug doxorubicin (Table.2). The\nIC50<\/sub> values obtained refer to 50% of cells inhibited by extracts.\nBased on Table 2. Shown that G.gnemon<\/em> extract has IC50<\/sub>\n300-400 \u00b5g\/mL for both cancer cells. Moreover, the extract indicates\nmoderate cytotoxic (100-1000 \u00b5g\/mL) 24<\/sup>.\n<\/p>\n\n\n\n

Table 2: Result of cytotoxicity against MCF-7 and HeLa cell lines<\/strong><\/p>\n\n\n\n\n\n\n\n\n
\n

 <\/strong><\/p>\n<\/td>\n

\n

Cytotoxic activity <\/strong><\/p>\n

(IC50<\/sub>, \u00b5g\/mL)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

MCF-7<\/strong><\/p>\n

(mean \u00b1 SD)<\/strong><\/p>\n<\/td>\n

\n

HeLa<\/strong><\/p>\n

(mean \u00b1 SD)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

G.gnemon <\/em>extract<\/p>\n<\/td>\n

\n

316.19 \u00b1 45.76<\/p>\n<\/td>\n

\n

489.57 \u00b1 4.03<\/p>\n<\/td>\n<\/tr>\n

\n

Doxorubicin<\/p>\n<\/td>\n

\n

0.14 \u00b1 0.12<\/p>\n<\/td>\n

\n

2.38 \u00b1 3.5<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

The\nIC50 <\/sub>in MTT assay (mean \u00b1 SD) of three independent experiments, n=3<\/p>\n\n\n\n

Observation\nof Morphological Changes<\/strong><\/p>\n\n\n\n

We evaluated the morphology changes of both breast cancer and HeLa\ncells with microscopically examined after 24 hours given extract treatment. A\nmajor change in cell morphology was observed in cell shrinkage, cell wall\nblebbing (Figure.3)<\/p>\n\n\n\n\n\n
\"\"<\/td>\nFigure 3: <\/strong>Microscopic observations. G.gnemon<\/em> seeds extract effect on
MCF-7 and HeLa cells.<\/strong>

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

(A) MCF-7 untreated cells, (B) MCF-7 treated cells, (C) HeLa untreated cells, and (D) HeLa treated cells. Observation of cells using an inverted microscope (200x magnification). The arrow indicates that the cell is apoptosis.<\/p>\n\n\n\n

Morphological changes were revealed after MCF-7 and HeLa were\ntreated with G.gnemon<\/em> seeds extract compared to untreated cells. Figure\n(A) shows that untreated MCF-7 cells appear healthy, normal, and confluent\ncells while treated cell (B) it appeared that cell vacuolization was formed,\nand the cells appeared to be smaller and rounded. Crude extract of G.gnemon<\/em>\nalso induced cell death in HeLa cells. HeLa cells without treatment showed a\nflat polygonal shape with good permeability, complete nucleoli, clear and\nadherence (C) while in treated cells (D), the cell was observed shrinkage, the\nshape became fusiform cells or long circles, and some cells had started to\nundergo apoptosis.<\/p>\n\n\n\n

Discussion<\/strong><\/p>\n\n\n\n

There are many anticancer drugs that have been developed over time.\nHowever due to side effects of cancer treatments, a new anti-cancer substance\nthat is more effective and less side effects is needed. A large number of\nplants are well known as alternative medicine that considered less side effects. Our previous study has been\nconducted several plant medicines including robusta coffee 25<\/sup>, <\/strong>Polygonum pulchrum <\/em>26<\/sup>, <\/strong>Annona muricata<\/em> Linn 27<\/sup>, <\/strong>Jatropha gossypifolia <\/em>28<\/sup>.<\/strong> In the recent study, a part of species G.gnemon<\/em>\nL was used to evaluate for its antioxidant and anticancer activity. These\nplants had been intensively studied for their therapeutic properties. We\nobserved the presence of various phytochemicals (Table 1) and these\nphytochemicals showed antioxidant and anticancer activities. Phytochemical\ncompounds of natural products as antioxidants have a main role as radical\nscavenging that produce certain diseases. <\/strong>The reactive oxygen species\n(ROS) is a chemically reactive molecule in cells and associated with a variety\nof biological processes, such as cell proliferation, differentiation and\nprogrammed cell death ROS has a relationship with the oncogene function and\nsuppression function 29<\/sup>. Concisely, ROS will results in G1 phase inhibition\nleading to a significant influence on cell proliferation 16<\/sup>. Antioxidant presence deactivates free\nradicals by donating hydrogen atoms to free radicals that are dominant to\nscavenge radicals 30<\/sup>. Those are explanations of how antioxidant prosperity\ntoward on underlying cancer mechanism. An uncontrolled increase in cell\nproliferation and decreased cellular apoptosis are characteristics of cancer.\nApproaches for treating cancer should be apoptosis induction and growth\ninhibition of tumor cells 31<\/sup>.<\/p>\n\n\n\n

Some parts of G. gnemon<\/em> plant\nare known to have anticancer activity, especially in leaves and seeds. Recent\nstudies there were several reports the extract of G. gnemon<\/em> leaves was\nindicated have high IC50<\/sub> values. The ethanol extract of G. gnemon\n<\/em>leaves on previous studied showing the IC50<\/sub> values was at high\npotential toxicity 32<\/sup>. Moreover, Bioactivity assay of gnetumal <\/em>and p-coumaric\nacid<\/em> as a bioactive compound of this plant indicated possessed more potent\ntyrosinase inhibitory activity, with IC50<\/sub> values of 31.6 and 2.3 mM, respectively, than that of a positive\ncontrol kojic acid 33<\/sup>. In our study presented G.gnemon<\/em> seed extract have\nanticancer activity as moderate IC50<\/sub> values. A different result was shown\nby Narayan et al. which presented IC50<\/sub> value as potential toxicity 34<\/sup>. We presumably it could cause the different type and specification\nof sample testing, in which the amount of compound was known. The preparation\nsample testing would expressed a different result. The specific isolation\ncompound of plants can understand clearly the mechanism and further\ninvestigation on anticancer candidates. Such as like our studied that performed\nthe bioactive from Robusta Coffee such as caffeine and chlorogenic acid against\non cell line Hep-G2 35,36<\/sup>. <\/p>\n\n\n\n

The antioxidant activity were\ncarried out to assess the capacity of plant extracts to scavenge free radicals.\nCompare with the previous study regarding the antioxidant activity of melinjo\nseed have been reported by Supriyadi et al<\/em>. tested with the 2,2\u2032-azino-bis(3-ethylbenzothiazoline-6-sulfonic\nacid<\/em> (ABTS) assay,  this study shown\nthat the melinjo seed has very weak category for antioxidant activity 37<\/sup>. This may be due to differences in the\nmethod of the assay. Extracts showing low antioxidant that measured use one\nmethod should not be discarded as poor sources of antioxidant without having\nbeen compared with other methods 38<\/sup>. Phenol compounds in the melinjo seed is\nthe main component to have the potential for antioxidant activity 39<\/sup>. High levels of phenol could be\nconsidered as a good source of antioxidants that will act in the prevention of\nmany diseases e.g carcinomas 40<\/sup>.<\/p>\n\n\n\n

According to the literature, mechanism of action flavonoids are anti-cell\nproliferation, induced apoptosis and cell cycle arrest, inhibition angiogenesis\nand suppression of metastasis 41, <\/sup>42<\/sup>.\nTerpenoid through induced apoptosis intrinsic pathway 43<\/sup>.\nTannins induced G1 arrest, phosphorylation of the tumor suppressor protein p5344<\/sup>. As known G. gnemon<\/em> has several\nanticancer activities with a certain mechanism such as inhibits endothelial\nsenescence 45<\/sup>. In our report studied that fraction of G.\ngnemon<\/em> seed showed antioxidant and cytotoxic activity against HeLa cell\nlines 16<\/sup>. In vivo studies exhibited Gnetin C<\/em> as\nanother bioactive compound that has lead activity over the stilbenoid. Gnetin\nC<\/em>-treated tumors showed reduced mitotic activity and angiogenesis and a\nsignificant increase in apoptosis compared to all the other groups. The data\nsuggest that Gnetin C<\/em> is more vigorous in declining tumor progression in\nprostate cancer xenografts than Res or Pter 46<\/sup>. Beside phytochemical compounds,\nthese plants are rich in resveratrol and gnetin C content 10<\/sup>.\nResveratrol is a natural polyphenolic phytoalexin that has been shown to have\nantioxidant activity and anticancer properties. Resveratrol suppresses cell\nproliferation and induced apoptosis through mitochondrial and p53 signaling\npathways in human cervical carcinoma 47<\/sup>.\nIn a previous study, gnetin C<\/em> induced apoptosis through inhibits the\nmTOR and MAPK pathway in acute myeloid leukemia (AML) 48<\/sup>.\nMorphological changes of the cancer cell are beginning event during the\ninduction of apoptosis i.e. cell blebbing, shrinkage, nuclear fragmentation,\nchromatin condensation, and so on 21, 49,<\/sup> in this study presented in Figure 3.\n<\/p>\n\n\n\n

Recently, some\nphytochemical compounds were identified by qualitative methods and the\nanticancer activity of the extract was classified as moderate cytotoxicity.\nThese studies exhibit that G.\ngnemon<\/em> is a natural source with potent\nactivity as anticancer candidates. Many bioactivities of this plant need to be investigated\nand developed with in vitro and in vivo assay. Further investigation needs to\nbe performed quantitative analysis and mechanism of action as anti-cancer of\nthese plant compounds. <\/p>\n\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

In the light of these findings, it is\napparent that G. gnemon <\/em>or melinjo seeds could be considered as a vital\nsource of antioxidants. It is observed that extract of G. gnemon<\/em> had\nsignificant anticancer activity in different cancer cell lines using in vitro\nmodels. We investigated the phytochemical, antioxidant and cytotoxicity of the G.\ngnemon<\/em> seed extract against the MCF-7 and HeLa cells line. Our findings\nrevealed that the extract was of moderate cytotoxicity. The % cell viability of\nMCF-7 and HeLa cells decreased as the concentration of the extracts increased.<\/p>\n\n\n\n

Conflict of Interest<\/strong><\/p>\n\n\n\n

There is no conflict of interest.<\/p>\n\n\n\n

Funding Source<\/strong><\/p>\n\n\n\n

There is no funding Sources.<\/p>\n\n\n\n

References<\/strong><\/p>\n\n\n\n

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