Cancer is a condition of abnormal cell proliferation of tissue cells in the body that becomes malignant. It can attack other parts of the body and affect the normal function of the body organs. The sample used in this study was tubers of paku atai merah (Angiopteris ferox Copel), then extracted using 96 ethanol eluent to obtain a thick extract. The ethanolic extract of A. ferox was fractionated using column chromatography to get the active fraction to characterize the compound using thin-layer chromatography and gas chromatography-mass spectroscopy (GC-MS) and tested its cytotoxic effectiveness on MCF-7 and HeLa cancer cells. The results of this study were obtained from fractionation using the column chromatography method to get sub-fraction C and the results of compound characterization using GC-MS and obtained variations in the class of compounds contained in the sample: amino acids, glucosinolates, alkaloids, flavonoids, and terpenoids. Based on the cytotoxic effect test of sub-fraction C on MCF-7 cells, the results obtained moderate cytotoxic effects with an IC50 value of 61.027 ug/mL, and HeLa cells had an IC50 value of 521.03 ug/mL, which was categorized as having a weak cytotoxic effect. Based on the results obtained from this study, it can be concluded that sub-fraction C of A. ferox tubers has a cytotoxic effect on MCF-7 cells to be used as a reference for tracing pure compounds from A. ferox tuber.

Cancer is acondition of abnormal cell proliferation of tissue cells in the body thatbecomes malignant. It can attack other parts of the body and affect the normalfunction of body organs1-3. Cancer is a severe problem. As many as 8.2 million cases of death arecaused by cancer. Breast cancer is the first most common sufferer in the Asianregion, with 23% of breast cancer2. Breast cancer can be characterized by a disruption in the proliferationof abnormal mammary cells that turn into malignant cells through variouspathways of cell mutagenesis. One of the mechanisms of breast cancer is signaltransduction of estrogen receptors (ERα and ERβ) which is a factor inactivating or suppressing the expression of target genes on ligand binding4,5. The ERα has a significant role of about 75% in the pathogenesis of breastcancer by promoting the growth of breast tumor cells. The ERα reacted withcyclin D1, which can activate cyclin-dependent kinases (CDKs) to change thetransition of cells from the G1 phase to the S phase into cancer cells5,6.

Varioustechnological and scientific developments for cancer treatment have beencarried out, starting from surgery, radiotherapy, chemotherapy, immunotherapy,hormone therapy, stem cell transplantation, and radiation therapy7,8. However, some of these therapies have various side effects: hair loss,decreased white blood cells, and decreased immune quality. The high cost ofcancer treatment is not proportional to the success rate of therapy in cancer7,9,10. Therefore, to minimize excessive side effects on cancer treatment,several natural ingredients have been developed by looking at the cytotoxiceffects of secondary metabolite compounds in plants that function as adjuvantanticancer therapy that have proliferative pro-apoptotic properties11-14.

One of theplants with anticancer activity is paku atai merah or Angiopteris feroxCopel from the Marratiaceae family. The community has widely used A. ferox,especially in the Dayak area, Kalimantan, as a medicinal plant to treat variousdiseases. It is because A. ferox tubers contain a variety of compounds asreported in several studies by Nur et al7,15,16. Based on the results of phytochemical screening, the ethanolic extract ofA. ferox tubers contains compounds such as flavonoids, tannins,saponins, steroids, terpenoids, phenolics, and angiopterosides. The variouscompounds in the A. ferox tubers also have antioxidant activity inreducing 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals with a strong categoryand iron ions with a potent category. It also has strong categories forantioxidant activity using the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid) (ABTS), nitric oxide (NO), and lipid peroxidase methods4. Extracts and fractions of A. ferox tubers have also been reportedto have anticancer activity on breast cancer cells (MCF-7 and T47D), coloncancer cells (WiDr), and epithelioid cancer cells (HeLa), indicating that theethyl fraction acetate has an effect on each cancer cell with a toxic category2,7. Based on the activity background as an anticancer from A. feroxtubers, this study focuses on tracing the active compounds with anticanceractivity by isolating the active compounds from A. ferox tubers and thencharacterizing the compounds using gas chromatography-mass spectroscopy (GC-MS)and testing the cytotoxic effect on MCF-7 and HeLa cells.

The materials usedwere ethanol 70% (OneMed, Indonesia), ethanol 96% (JT-Baker), silica gel 60 GF254 (Merck, Germany), thin-layer chromatography plate (TLC, Merck, Germany),acetonitrile (JT-Baker), methanol (Merck, Germany), ethyl acetate (Merck,Germany), FeCl3 (Sigma Aldrich, Germany), H2SO4(Merck, Germany), phosphate-buffered saline (PBS, Gibco), penicillin-streptomycin(Gibco), sodium dodecyl sulfate (SDS, Merck, Germany), trypsin EDTA 0.25%(Gibco), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),and A. ferox Copel tuber simplicia obtained from West Kutai, EastKalimantan, Indonesia, and has been identified at the Anatomy and ScienceLaboratory of Universitas Mulawarman, Samarinda, Indonesia.

Sample preparation

Thesamples of A. ferox tubers collected were sorted by wet sorting, thenwashed under water to remove impurities still attached to the samples of A.ferox after the wet sorting was carried out. Then, the sample was choppedand dried by placing it in a simplicia oven at 40-60°C. Furthermore, afterdrying, the sample was done dry sorting and then pollinated for the extractionprocess.

Extraction

Theextraction procedure was adopted from our previous research2 under the sameconditions. Dry simplicia as much as 1.5 kg was made into coarse powder bypounding. The coarse powder obtained was reduced in size by blending so that a slightlycoarse powder was obtained, as much as 1.2 kg. In general, simplicia powderwith a larger surface area will improve the filtration because the surface ofthe simplicia powder in contact with the liquid filter is wider and breaks downthe cell wall so that the filtered liquid can enter the cell. Simplicia powderas much as 1.2 kg was put into a tightly closed container and soaked with 96%ethanol. The simplicia was then allowed to stand for 24 x 3 hours, stirringoccasionally for the first six hours, then allowed to stand for 24 x 3 hours.The filtrate was taken, the residue was re-macerated with 96%w/w ethanol. Thefiltrate was collected and evaporated. The viscous extract obtained wasweighed, and the yield was calculated.

Thin-layerchromatography

Theseparation was carried out by TLC of the extract obtained to determine theeluent used in column chromatography. The extract was dissolved with theinitial solvent and then spotted in the TLC and eluted with the appropriateeluent, after which it was put in a bucket and allowed to elude to the elutionlimit. The orientation of the eluent was carried out before separation by TLCusing the ratio of methanol : ethyl acetate (9 : 1) and (8 : 2), and then oneeluent was selected, which produced an excellent stain appearance with theratio (8 : 2). Observations of the appearance of the stains were performedunder UV lamps at λ of 254 and 366 nm17.

Columnchromatography

A set ofcolumn chromatography tools was prepared, then silica gel was inserted wet intothe column tube. A total of 10 g of the extract was mixed using silica powderto obtain a dry powder extract. The mixture was then put into a column thatalready contained silica gel 60 and eluted using an eluent from non-polar topolar (n-hexane, ethyl acetate, ethanol with gradient concentration),starting from 100 mL n-hexane eluent, then further elution using ethylacetate, and ethanol 96% with gradient concentration. The results of theobtained fractions were accommodated in a glass container. The incorporation ofthe fractions was carried out based on the color appearance of the solution andthe stains on the TLC plate. Based on the similarity of the TLC profile, thecombined fraction was then TLC to observe the spots at UV 254 nm and 366 nm.Eight fractions were obtained in the fractionation I process. The fractionswere grouped according to their color and TLC profile. Fraction III (3.092 g)was then separated by column chromatography (polyamide, 60 cm x 5 cm column)using an eluent ratio of methanol : ethyl acetate (80 : 20 and 20 : 80) toobtain a sub-fraction of 7 (A-G). Sub-fraction C was characterized by compoundprofiles using GC-MS.

Fractioncharacterization

Thecharacterization of the isolated fraction was carried out using a GC-MS toobtain the profile of the components in the fraction.

Cytotoxic assay ofMCF-7 and HeLa cells

The active isolatefraction obtained was then subjected to cytotoxic testing to see the toxiceffect of sub-fraction C on MCF-7 and HeLa cells using the MTT assay methodfollowing the test procedure from our previous research4 with a slightlymodified on serial concentration of sample test. The absorbance measurement ofthe sample using a microplate reader at a wavelength of 595 nm and theabsorbance data obtained were then analyzed by looking at the percentage ofcell viability and determining the IC50 value.

In this study, thesample used was A. ferox and then extracted using the maceration method.The maceration method was chosen because the extraction process is simple andavoids compound damage18. The extractionprocess using the maceration method uses 96% ethanol solvent to dissolve bothnon-polar and polar compounds so that the extraction process occurs entirely.Besides that, it avoids compound damage due to the growth of microorganismsduring the process of making thick extracts of A. ferox tubers. Theethanol extract obtained was then fractionated by a silica chromatographiccolumn eluted using several solvents based on a concentration gradient. Theresults of column chromatography show that from the results of columnchromatography, 42 fractions were obtained. The obtained fractions werecombined based on the TLC color and stain profile eluted using methanol : ethylacetate (8 : 2) in 10 mL. The merger results obtained eight fractions giveneach code (Figure 1). Fractions III were columnchromatographed again with methanol : ethyl acetate (80 : 20 and 20 : 80) in100 mL. The chromatography results obtained 35 fractions, which were thencombined based on spot color and stain profile using an eluent ratio ofmethanol : acetone (8 : 2) in 10 mL to obtain seven fractions from the combinedresults. Sub-fraction C (7-11) was characterized using GC-MS to determine theprofile of the compounds contained in the sub-fraction. Sub-fraction C waschosen for further characterization because the resulting spot pattern showedthe presence of phenolic compounds after being sprayed using the FeCl3reagent, which formed a blue spot (Figure 2A).

Figure1. The process of compoundfractions from the ethanol extract of A. ferox

Compoundcharacterization of sub-fraction C was carried out by looking at the profilesof the compounds found from sub-fraction using the GC-MS. The GC-MS datafragmentation (m/z) was processed using the ReSpect for phytochemical (http://spectra.psc.riken.jp/menta.cgi/respect/search/fragment) to see fragmentsthat indicate the intensity of secondary metabolites contained in the isolatesof A. ferox tubers (Figure 2B). Based on the GC–MS data obtainedfrom identifying the A. ferox tuber isolates, it was shown that thesub-fraction C tested contained secondary metabolites, such as alkaloids,flavonoids, and terpenoids (Table I). Alkaloid compoundsin the chromatograms obtained were indicated by peak numbers 4, 10, 14, 17, 18and 19 in fragments 41, 42, 55, 57, 58, 68, 69, 73, 82, 84, 96, 97, 98, 101,110, 113, 114, 129, 131 and 146. At the same time, the flavonoid compounds wereshown by peaks 5, 6, 11, 22, 24 and 25 in fragments 41, 42, 57, 69, 70, 71, 73,81, 84, 85 97, 103, 111, 129, 167, 199, 213, 256, 279 and 390. While theterpenoid compounds were shown by peaks 8, 12, 13, and 15 in fragments 55, 69,97, and 115. The data show terpenoid compounds at peak 12-15, alkaloids at peak17-19, and flavonoids at peak 22-25.

Figure2. Blue spot profile in TLCafter spraying FeCl3 reagent (a) and chromatogram of thesub-fraction C using the GC-MS (b)

TableI. Results ofidentification of compound groups using the GC-MS

ND: compound notdetermined

Evaluation of thecytotoxic effects of the A. ferox tuber fraction using the MTT assay method onMCF-7 and HeLa cells was performed to evaluate the potential of the A. feroxtuber fraction in inhibiting cell proliferation with percent cell viability andtoxic effect based on IC50 value. The IC50 value is aconcentration value required for a sample to give a toxic effect of 50% oncells categorized as strong cytotoxic effect <50 µg/mL, moderate cytotoxiceffect 50-200 µg/mL, weak cytotoxic effect 200-1,000 µg/mL and no cytotoxiceffect >1,000 µg/mL. The cytotoxic effectiveness test of the A. feroxtuber fraction (Figure 3) showed that the A. feroxtuber fraction had a moderate cytotoxic effect on MFC-7 cells with an IC50value of 61.027 µg/mL. Meanwhile, the A. ferox tuber fraction had a weakcytotoxic effect in HeLa cells with an IC50 value of >500 µg/mL.

Figure3. The graph of cytotoxicactivity of sub-fraction C of A. ferox tuber toward MCF-7 (A) andHeLa cells (B) and doxorubicin as positive control toward MCF-7 (C)and HeLa cells (D). The data were observed in triplicate (n=3)

Meanwhile, the IC50value of doxorubicin positive control against MCF-7 and HeLa cells obtained anIC50 value of 2.62 and 3.276 µg/mL, respectively, and included inthe strong cytotoxic category. This study showed that sub-fraction C of A.ferox extract had a toxic effect on MCF-7 but not on HeLa cells. Thismechanism is influenced by compounds' content in the sub-fraction of A.ferox, which could not cause apoptosis in HeLa cells. The sub-fraction C ofA. ferox tubers has activity on MCF-7 cells based on the analysis ofcompound groups using GC-MS containing several compounds (Table I). According toprevious research13,19, phenolic compoundscan inhibit the formation and growth of tumors by inducing cell cycle arrestand undergoing cell apoptosis. Phenolic compounds can induce cell cycle arrestwith multiple cell cycles from G1-S-G2 so that they can downregulate cyclinsand CDKs, and directly induce gene expression in p21, p27, and p53. Accordingto other studies20-22, flavonoidcompounds have the potential as pro-oxidants so that they can suppress theproliferation of cancer cells by inhibiting the epidermal growth factorreceptor or mitogen active protein kinase (EGFR/MAPK), phosphatidylinositide3-kinases (PI3K), protein kinase B (Akt), and nuclear factor-kappa-β (NF-kB)23.

Basedon the results obtained from this study, it can be concluded that sub-fractionC of A. ferox tubers has anticancer activity, which was tested using anMTT assay on MCF-7 cells with an IC50 value of 61.027 µg/mL in themoderate toxic effect category. This result occurs because the sub-fraction Cresults from the compound groups' characterization using GC-MS. Severalcompounds are obtained, i.e., amino acids, glucosinolates, alkaloids,flavonoids, and terpenoids, to have a toxic effect on cancer cells.

We want to thank theIndonesian Government, the Ministry of Education, Culture, Research, andTechnology, for the funding provided through a higher education cooperationgrant with contract No. 01/A/BAST/2021 and 0397/E.E4/PT.01.02/2021.

All authors have anequal contribution in carrying out this study.

None.

Theauthor declares there is no conflict of interest.

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