Comparative Studies on a Standardized Subfraction of Red Onion Peel Ethanolic Extract (Plant Substance), Quercetin (Pure Compound), and Their Cell Mechanism and Metabolism on MDA-MB-231

This study indicates the presence of quercetin in subfraction F1 and the standardized value of F1 derived from research using ultra-performance liquid chromatography (UPLC) and AlCl3 colorimetric assays, which further proved that both F1 and quercetin are potential growth inhibitors in MDA-MB-231 cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In the process, staining of F1-treated cells with annexin/propidium iodide (PI) reduced cell proliferation and induced only S and G2 phases of cell cycle arrest in the treated cells by flow cytometry. Quercetin reduced cell proliferation by inducing apoptosis and S phase arrest. The 5′-bromo-2′-deoxyuridine (BrdU) incorporation of DNA synthesis in MDA-MB-231 cells was also inhibited after F1 and quercetin treatments. F1 and quercetin induced CYP1A1 and CYP1B1 gene expression, but only F1 induced CYP2S1 gene expression in the treated cells. Both F1 and quercetin inhibited the proliferation of MDA-MB-231 cells in different ways, but F1 is likely a better potential anticancer agent derived from the green approach towards breast cancer treatment.

erefore, more studies on natural products or compounds to explore the anticancer properties for alternative cancer therapeutics with reduced toxicity are warranted. Red onion is rich in bioactive compounds and displays numerous pharmacological properties, including antimicrobial, antioxidant, anti-inflammatory, antihypertensive, and immunoprotective effects. Considerable epidemiological research has also revealed that this vegetable exhibits anticancer therapeutic value [6][7][8][9], and dietary intake of onion has been shown to reduce the risk of developing breast cancer [10,11]. erefore, the potential of natural products derived from onions containing these highly bioactive compounds could be used as new anticancer agents. A closer inspection of several alternative medical treatments has also proven that onion-derived natural products carry anticarcinogenic properties with less toxicity.
is study aimed to indicate the presence of quercetin (the main flavonoid of red onion peel ethanolic extract) in a subfraction of red onion peel ethanolic extract (F1) and the standardized value of F1 derived from research using ultraperformance liquid chromatography (UPLC) and aluminium chloride (AlCl 3 ) colorimetric assays. e study then investigated the growth inhibitory effect of F1 and quercetin. e analysis used red onion peel instead of flesh because the peel contains higher concentrations of quercetin aglycon than the flesh [12,13]. e F1 produced in-house contains high quercetin levels that can be considered a potential anticancer agent. Preliminary results showed that F1 reduced the growth of breast cancer MCF-7 cells. e study also showed a better in vitro anticancer effect for F1 than for camptothecin (a commercially used anticancer drug) on oral cancer CAL-27 cells. Many in vitro and in vivo studies have also been conducted to confirm the anticancer properties of red onion peels. However, the cellular mechanism and cell metabolism induced by F1 have not been studied in detail.
is study first investigated the phytochemical properties of F1 and the cytotoxic effects of both F1 and quercetin in MDA-MB-231 cells. en, the study examined the cell cycle profile, apoptosis induction, and 5′-bromo-2′-deoxyuridine (BrdU) incorporation of DNA synthesis in MDA-MB-231 cells. e mRNA expression of CYP genes in F1-and quercetin-treated MDA-MB-231 cells was also investigated in this study. e findings will facilitate the discovery of a green approach to novel anticancer agents from natural products for breast cancer treatment with bioavailable, safe, cost-effective, and minimal side effect properties in the future.

Determination of Total Flavonoid and Quercetin Content by UPLC and AlCl3
Colorimetric Assays. Quercetin content in subfraction F1 was detected by an ACQUITY UPLC system (Waters Corporation, Milford, MA, USA) incorporated with a photodiode array as the detector in the School of Biological Sciences, USM. F1 was prepared as described in Chao's previous study [14]. A 2.0 μL aliquot of F1 or quercetin standard solution was injected into the UPLC BEH C18 column (100 mm × 2.1 mm i.d., 1.7 µm particle size, Waters Corporation, Milford, Massachusetts, USA). e standard and sample solutions were injected twice to ensure reproducibility. e temperature was set at room temperature, and the mobile phase used was isocratic mode, in which elution was performed with 95% acetonitrile. e flow rate of the analysis was set as 0.1 mL/min with a total runtime of 10 min (7 min for analysis and 3 min reequilibration). e detection of the elution peaks was set at λ � 360 nm. e retention time shown in the chromatograms by Empower 2 Software (Waters Corporation, Milford, MA, USA) indicated the presence and the level of quercetin in the F1 solution. e total flavonoid content in F1 was also determined by an AlCl 3 colorimetric assay adapted from Sembiring's study [15] with slight modification. First, 50 μL F1 (1.0 mg/ mL) and various concentrations of quercetin standard solution (25, 50, 100, and 200 μg/mL) were prepared. Absolute ethanol was used instead of quercetin or sample solution as the blank solution. Each solution was then added to the respective well in the 96-well plate. en, 150 μL of 99.7% ethanol and 10 μL of 10% (w/v) AlCl 3 solution were added to each well. Next, 10 μL of 1.0M sodium acetate solution was added to the solution mixture in the wells. All reagents in the wells were mixed and incubated for 40 min at room temperature in the dark. e absorbance of the standard versus blank solutions was then quantified at 415 nm using a Multiskan Spectrum Microplate Reader ( ermo Fisher Scientific, Waltham, MA, USA). e absorbance of the sample and standard solutions was also measured in a range of wavelengths from 350 to 600 nm using a microplate reader for purity analysis. All sample and standard solutions were prepared in triplicate, and the absorbance values were measured, recorded, and interpolated into the linear quercetin calibration curve. e linear regression equation of the calibration curve for a straight line is Y � mx + c, where Y � absorbance of extract, m � slope of the calibration curve, x � concentration of F1, and c � intercept. e total flavonoid content of F1 was expressed as mg quercetin equivalents (QEs) per gram of dry extract (mg/g) and calculated by using the following formula: C � cV/M, where C � total flavonoid content of F1 (mg QEs/g dry extract), c � concentration of quercetin obtained from the calibration curve in mg/mL, V � volume of extract in mL, and M � mass of extract in grams. e MDA-MB-231 cells were incubated at 37°C in a humidified atmosphere of 5% (v/v) carbon dioxide (CO 2 ) and grown with a doubling time of approximately 38 h. e MDA-MB-231 cells were trypsinized when the number of cells in the culture flasks had reached sufficient growth. e cells were seeded in 96-well plates at a cell density of 2 × 10 4 cells/mL for growth inhibitory effect analysis or in 6-well plates at a cell density of 1.2 × 10 6 cells/mL for cell cycle, apoptotic effect, DNA synthesis, and CYP mRNA expression analyses. e cells were then subjected to treatment with 0.1% dimethyl sulfoxide (DMSO) (control), 50 μg/mL F1 (determined in this study), or 60 μg/mL quercetin [16] in growth media that contained only 2% FBS. e treated cells were incubated at 37°C in a 5% (v/v) CO 2 incubator for 24, 48, and 72 h prior to further analysis.

Growth Inhibitory Effect Analysis of F1 and Quercetin by MTT Assay.
e cells treated with F1 and quercetin in 96well plates, as described above, at the end of each incubation period were added to 24 μL of 5 mg/mL MTTreagent (Sigma-Aldrich, St. Louis, MO, USA) in each well. e reaction was incubated for 4 h. e solution was carefully removed without disturbing the formazan crystals formed in each well. Next, 100 μL acidified isopropanol (Sigma-Aldrich, St. Louis, MO, USA) was added to each well and agitated to promote homogeneous colour development. Following colour development, the colour intensity in each well was read at 570 nm using an enzyme-linked immunoassay (ELISA) plate reader (Tecan, Männedorf, Switzerland). and quercetin in 6-well plates, as described above, were trypsinized at the end of each incubation period, and the cell suspension was transferred into Falcon tubes. e cell suspension with the old medium in the tube was centrifuged at 1,000 g for 5 min. e supernatant was then discarded, and the cell pellet was washed with phosphate-buffered saline (PBS). After washing, a 1 × 10 6 cell suspension was transferred into new Falcon tubes, and the cell suspension was then centrifuged as above.

Cell Cycle Analysis of F1-and
e cells were then fixed and permeabilized by adding 70% ice-cold absolute ethanol dropwise with gentle mixing. e cell suspension was then stored at 4°C overnight to allow fixation. e ethanol-fixed cells were centrifuged at 1,000 g for  e cells treated with F1 and quercetin in 6-well plates, as described above, were also subjected to apoptotic effect analysis using the annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit ( ermo Fisher Scientific, MA, USA). Briefly, the trypsinized cells collected in 15 mL Falcon tubes were centrifuged at 400 g for 4 min at 4°C to remove the old medium. en, the cells were washed twice with 3 mL cold PBS. e cell suspension was counted, and 5 × 10 5 cells/mL were transferred into new Falcon tubes. e cells were centrifuged to remove the supernatant, and then the cell pellet was resuspended in 200 μL of 1x binding buffer. A volume of 5 μL annexin V-FITC was then added to 195 μL of the cell suspension, mixed well, and incubated in the dark at room temperature for 10 min. e cells were then washed with 200 μL of 1x binding buffer. e cells were resuspended in 190 μL of 1x binding buffer prior to the addition of 10 µL of 20 μg/mL PI. e mixture was transferred to a new sterile tube for flow cytometry analysis using a BD FACSCalibur (BD Biosciences, NJ, USA) with 488 nm excitation, 655-730 nm emission for PI, and 525 nm emission for FITC.

DNA Synthesis Analysis of F1-and Quercetin-Treated MDA-MB-231 Cells by BrdU.
e cells treated with F1 in 6well plates were also subjected to incorporation of 1.0 mM BrdU labelling solution (BrdU; Invitrogen, ermo Fisher Scientific, Waltham, MA, USA) into newly synthesized DNA (surviving mitosis ability) in the dark. e cells were then washed, trypsinized, and counted, whereby 1.0 × 10 6 cells of cell suspension from each treatment were pipetted into a 15 mL Falcon tube. e BrdU-labelled cells were incubated before the cells were spun down at 1,000 g for 5 min. e BrdU-labelled cells were then fixed and permeabilized with 5 mL fixation dropwise. e cell suspension was then stored at −20°C overnight to allow fixation, and then the cells were subjected to washing steps. e steps of DNA denaturation, neutralization of the acidic treatment, and finally resuspension of the cell pellets with the antibody staining solution were performed. e cell suspensions were then added to the anti-BrdU monoclonal antibody conjugated with FITC. e stained cells were then incubated and washed again, as described above, and resuspended in 500 μL PBS. e cell suspensions were then stained with 500 μL FxCycle ™ PI/ RNase Staining Solution. e stained cells were collected, as described above, and then transferred into a 5 mL round bottom polystyrene test tube. e tube was placed on ice prior to analysis with flow cytometer analysis using Cell-Quest software version 3.3 (BD Biosciences, Franklin Lakes, NJ, USA) from a total of 15,000 events. e percentage of BrdU-positive cells was analyzed using Summit software version 1.4 (Beckman Coulter, Brea, CA, USA).

CYP Expression Analysis in F1-and Quercetin-Treated MDA-MB-231 Cells by Real-Time PCR.
e cells treated with F1 and quercetin in 6-well plates, as described above, were also subjected to total RNA extraction using TRIzol Total RNA Isolation Reagent (Life Technologies Corporation, Carlsbad, CA, USA). e extracted total RNA pellet was then air-dried for 15 min and resuspended in 25 μL RNase-free water. Only the extracted total RNA with an absorbance A260/A230 ratio within 1.8-2.0 and an A60/280 ratio within 2.0-2.2, assessed using a NanoDrop ™ 2000C spectrophotometer ( ermo Fisher Scientific, USA), was used for cDNA conversion. e bands that indicated a good quality of RNA, assessed by 1% (w/v) agarose gel electrophoresis, were visualized and captured under UV light by an image analyzer (Syngene, Cambridge, UK). e extracted total RNA was then reverse-transcribed to cDNA using a Tetro cDNA Synthesis Kit (Bioline, London, UK). e reverse transcription products (cDNA) were kept at −20°C until the products were used for real-time polymerase chain reaction (PCR) gene expression analysis. All primers were designed using Primer Express software v3.0.1 ( ermo Fisher Scientific, Massachusetts. USA; Table 1). All oligonucleotide primers ordered from Integrated DNA Technologies (Coralville, USA) were packed in desalted lyophilized form. e primer stocks were dissolved in RNase-free water (Sigma-Aldrich, St. Louis, MO, USA) to generate a final 100 µM concentration of each primer solution. e primer solutions were stored at −20°C until use. Real-time (RT) PCR was performed using an Agilent AriaMx Real-Time PCR System (Agilent, California, USA). e PCR cocktail was prepared by adding 10 µL iTaq ™ Universal SYBR Green Supermix (Bio-Rad, CA, USA), 0.8 μL of 10 μM forwards, and reverse primers, each for the gene of interest (GOI), and 50 ng cDNA diluted in nuclease-free water to a total volume of 20 μL in each well of 0.1 mL, 8-tube quantitative (q) PCR Evidence-Based Complementary and Alternative Medicine strips (AITbiotech, Singapore). e reactions were initiated with a hot start at 95°C for 30 sec, followed by 40 cycles of amplification at 95°C for 15 sec as the denaturation step and 60°C for 1 min as the annealing and extension step. e reactions were continued at 1 cycle of thermal profile consisting of 95°C for 15 sec, followed by 60°C for 1 min, 95°C for 30 sec, and 60°C for 15 sec to generate a melting curve. e expression value of GOI was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), whereby the relation between the normalized GOI in treated and vehicle control samples was determined by the fold change using the 2 −ΔΔCt method [17]. e fold change value for the vehicle control was set as 1, whereas an expression change value >1 represented upregulation, and a value of <1 represented downregulation of the gene.

Statistical
Analysis. GraphPad Prism version 8.2.1 for Windows (GraphPad Software Inc., La Jolla, CA, USA) was used to generate the dose-response growth curves. e statistical analysis is presented as the mean ± standard deviation (SD) of triplicate determinations. One-way analysis of variance (ANOVA) was used to analyze the mRNA gene expression with an additional Dunnett's multiple comparisons test to compare 2 among 3 or more datasets. Data analysis was also performed for photographs using ImageJ scientific program (National Institutes of Health). e level of significance was set at α � 0.05 (95% confidence interval), where the confidence levels were indicated as statistically significant by asterisks * for p values <0.05, * * for p values <0.01, and * * * for p values <0.001.

Total Flavonoid Content of F1.
e analysis of total flavonoid and quercetin content in subfraction F1 using UPLC shows a narrow, sharp peak at approximately 2.5 min with a slight shoulder peak towards the right side of the chromatogram. e retention time of F1 (1,000 μg/mL) falls at approximately 2.549 to 2.553 min (Figures 1(a) and 1(b)), whereas the analysis detected 200 µg/mL quercetin at a retention time of 2.566 min (Figure 1(c)). UPLC analysis  showed that the retention time of F1 was similar to the retention time of quercetin. e analysis predicted the quercetin content of F1 to be approximately 135.085 μg/mL. e AlCl 3 colorimetric assay further revealed that the total flavonoid content in F1 was approximately 193.07 to 200.63 mg of quercetin equivalent (QE) per gram of dry extract (mg/g) using a standard curve (Figure 1(d)). F1 contains a high concentration of flavonol compounds, such as quercetin and its derivatives. e purity analysis showed a similar absorbance pattern at an absorbance peak of 420 nm, identical to the absorbance peak recorded for quercetin (data not shown). is result supports the presence of quercetin in F1 and the standardized value of F1 derived from our research.

Cytotoxic Effects of F1-and Quercetin-Treated MDA-MB-231 Cells.
e cytotoxic effect of F1 in MDA-MB-231 cells resulted in the ideal dose-response of the test compound on the cancer cells at 24 h (hillslope � −0.49; maximal response ≤25%) (Figure 2(a)) and 48 h (hillslope � −0.76; maximal response ≤25%) of treatment (Figure 2(b)). However, the ideal dose-response of F1 on the cancer cells did not persist until 72 h of treatment (Figure 2(c)). e ideal dose-response of quercetin was not observed on the cancer cells at 24 h of treatment (Figure 2(d)). e ideal dose-response of quercetin was observed on the cancer cells only at 48 h (hillslope � −2.247; maximal response ≤75%) (Figure 2(e)) and 72 h (hillslope � −2.785; maximal response ≤50%) of treatment (Figure 2(f )). Although quercetin showed a good later growth inhibitory effect on the cancer cells following 72 h of treatment, F1 showed a good early growth inhibitory effect on MDA-MB-231 cells. For the half-maximal effective concentration (EC 50 ) value, only MDA-MB-231 cells after F1 treatment for 48 h showed an EC 50 value ≤50 μg/mL (10.22 μg/mL); compounds that exhibited an EC 50 value ≥50 μg/mL were considered inactive. e differences in growth inhibitory activity of F1 and quercetin indicate that F1 is better suited for treatments that require immediate effect, while quercetin can be used for long-term treatment.

Cell Cycle Profile of F1-and Quercetin-Treated MDA-MB-231 Cells.
e histograms of the cell cycle profile of MDA-MB-231 cells treated with 0.1% DMSO (control), 50 μg/mL F1, and 60 μg/mL quercetin for 24, 48, and 72 h showed changes in the phases of the cell cycle (Figure 3(a)). e cell population in S phase for F1-treated MDA-MB-231 cells increased significantly to 53.13% (p < 0.001), accompanied by a decrease in the cell population in the G0/G1 phase to 39.68% (p < 0.001), compared to the respective controls (DMSO-treated MDA-MB-231), but with no significant effect on the cell population in G2/M phase at 24 h of treatment (Figure 3(b) (A)). Conversely, the distribution of cell cycle phases in MDA-MB-231 cells treated with quercetin for 24 h showed no significant difference compared to the controls. At 48 h of treatment, a significant increase in the cell population in both the S phase (39.34%; p < 0.001) and G2/M phase (39.70%; p < 0.001) was observed in F1-treated MDA-MB-231 cells, where a drop in the cell population to only 20.96% (p < 0.001) was observed in the G0/G1 phase compared with the controls (Figure 3(b) (B)). Quercetin only caused a reduction in the cell population in G0/G1 phase to 70.53% (p < 0.001) and an increase in the cell population in S phase to 25.72% (p < 0.001), but it had no significant effect on the cell population in G2/M phase. At 72 h of treatment, F1-treated MDA-MB-231 cells showed a further reduced cell distribution in the G0/G1 phase (27.09%, p < 0.001) with increased distribution in both the S (31.52%, p < 0.001) and G2/M (41.39%, p < 0.001) phases at 72 h of treatment (Figure 3(b) (C)). Similarly, quercetin-treated MDA-MB-231 constantly underwent cell cycle progression, shifting only from G0/G1 phase (61.02%, p < 0.001) to S phase (36.88%, p < 0.001) with no effect on the G2/M phase.
is phenomenon implies that F1 induced cell cycle arrest at both the S and G2/M phases, but quercetin induced cell cycle arrest at the S phase only in MDA-MB-231 cells. When quercetin treatment was prolonged to 48 h, 7.71% (p < 0.001) and 5.26% (p < 0.001) of cells were observed in the early and late apoptosis stages, respectively, compared to 2.43% of the control in the early apoptosis stage and 2.42% of the control in the later apoptosis stage (Figure 4(b) (B)). e apoptotic effect induced by quercetin at 72 h of treatment was most significant, as 18.48% (p < 0.001) was found in the early apoptosis stage and 8.13% (p < 0.001) in the late apoptosis stage (Figure 4(b) (C)). A significant reduction in cell viability from 90.96% in the control to 70.95% (p < 0.05) in the quercetin-treated MDA-MB-231 cells was also observed after 72 h of treatment, suggesting that quercetin could induce apoptosis in MDA-MB-231 cells but F1 did not (data not shown).

Discussion
e preliminary results of this study indicate the presence of quercetin in subfraction F1 and the standardized value of F1 derived from our research. e results then demonstrated the growth inhibitory activity of F1 (plant substance) and quercetin (pure compound), whereby F1 is better suited for treatments that require immediate effect, while quercetin could be used for long-term treatment. showed lower mRNA expression levels of CYP1A1 and CYP1B1 (procarcinogenic compounds metabolism) than quercetin, but a higher mRNA expression level of CYP2S1 (cytotoxicity) was found in F1-treated MDA-MB-231 cells than in quercetin-treated MDA-MB-231 cells for 72 h of treatment.
F1 was revealed to be a potential agent to exhibit an antiproliferative effect and cell cycle arrest in MDA-MB-231 breast cancer because F1 contains a significant amount of total flavonoid content and quercetin. F1 has a consistent absorption pattern, which directly proves the total flavonoid content and quercetin content in F1. is phenomenon also clearly indicates that the method used to obtain F1 is standardized, whereby F1 always shows a maximum absorption peak at 420 nm at a high concentration. e method used to obtain F1 also shows that most of the flavonols are retained during the dialysis fractionation process. Ethanol was more suitable for extracting polyphenolic compounds found in onion than methanol, ethyl acetate, chloroform, and acetone because ethanol is the most common solvent in laboratory settings, relatively safer, and less costly than other extraction solvents [18,19]. is phenomenon strengthens the usage of ethanol as the extraction solvent to separate bioactive compounds in onion peels, such as anthocyanins, quercetin, and other flavonols, from the crude ethanolic extract of red onion peels (E1) [20,21].
MDA-MB-231, a poorly differentiated, highly aggressive, and invasive breast cancer cell line, is a model representing triple-negative breast cancer, characterized by the lack of oestrogen receptor, progesterone receptor, E-cadherin, and HER2 growth factor receptor but presenting with mutated p53 gene expression [22,23]. erefore, MDA-MB-231 cells are the ideal cell model for investigating the effectiveness of newly developed chemotherapeutic agents. In this study, F1 and quercetin were found to reduce the proliferation of MDA-MB-231 cells by inducing different cell cycle arrest profiles. Regulation of the cell cycle is crucial for the development of healthy cells. Nevertheless, cancerous cells exhibit uncontrolled cell proliferation and evasion of apoptosis resulting from dysfunction of the cell cycle checkpoint and destruction [24]. Uncontrolled cell growth and apoptosis resistance are the major defects in cancer cells, thus, discovering potential compounds targeting cell cycle mechanisms and apoptotic machinery could be effective against uncontrolled cell proliferation.  Figure 6: Effect of F1 (50 μg/mL) and quercetin (60 μg/mL) on the mRNA gene expression of (a) CYP1A1, (b) CYP1B1, and (c) CYP2S1 in MDA-MB-231 cells. e mRNA expression was normalized to the expression of GAPDH in each sample. e bar chart represents the mean ± SD of two independent experiments with significant differences indicated as * P < 0.05, * * P<0.01, and * * * P<0.001, compared to the controls. One-way ANOVA and Dunnett's multiple comparison test were used for the statistical analysis.
independent experiments with significant differences indicated as * P < 0.05, * * P<0.01, and * * * P<0.001 compared to the controls. One-way ANOVA and Dunnett's multiple comparison test were used for the statistical analysis. different molecular mechanisms of cell cycle regulation induced by F1. A study reported that 20 µM quercetin induced cell cycle arrest at the S and G2/M phases in MDA-MB-231 cells after 48 h of treatment [25]. is finding was linked to the increased signalling activities of p21 and GADD45, which contributed to G1/S phase and G2/M phase arrest, respectively, regulated by p53. Another study indicated that cell cycle arrest was observed at the G2/M phase after treatment of MDA-MB-231 cells with 100 µM quercetin for 24 and 48 h [16]. Nevertheless, Rivera's study showed cell cycle arrest at the G2/M phase in MDA-MB-231 cells after 48 h of treatment with 15 µM quercetin [26]. In this study, cell cycle arrest was observed at the S phase only after treatment with 60 µg/mL quercetin for 48 h, which is slightly in contrast to previously published findings, whereas F1 is able to obstruct cell cycle progression in both the S and G2/ M phases. is difference may be due to variation in the cell treatment concentration, where a higher concentration of quercetin was utilized for the experiments. Apoptosis analysis showed that an apoptotic effect was observed in MDA-MB-231 cells treated with quercetin, but F1 did not induce apoptosis in MDA-MB-231 cells. e induction of apoptotic effects in MBA-MD-231 cells in this study was consistent with other findings using the same cell line [16,[25][26][27]. is result suggests that cell proliferation inhibition by F1 in MDA-MB-231 cells occurred through mechanisms other than the apoptosis pathway. e F1 effect was further validated by detecting the proliferative cells incorporated with DNA synthesis using BrdU staining, where the dye acts as a thymidine analogue to replace thymidine nucleotides in the newly synthesized DNA [28]. e analysis found that a low percentage of BrdU-positive cells was detected in MDA-MB-231 cells treated with 50 µg/mL F1 and 60 µg/mL quercetin for 72 h.
is phenomenon suggests that F1 inhibits DNA synthesis during cell proliferation only after 72 h of treatment. New cellular DNA content was still synthesized in MDA-MB-231 cells even after treatment with 50 µg/mL F1 and 60 µg/mL quercetin for 24 and 48 h. e decrease in cell viability observed in the cells that were treated with this F1 concentration and these treatment durations may be due to the toxicity derived from other bioactive compounds in F1, rather than the decrease in cellular DNA content.
CYP genes have been extensively confirmed to be involved in the metabolism of procarcinogenic compounds to highly carcinogenic metabolites [29,30]. Other studies have shown that the genes encoding these proteins are linked to cell signalling pathways critical for cell cycle regulation [31]. For instance, the aryl hydrocarbon receptor (AHR) responsible for activating CYP gene transcription is a protein that affects cell cycle regulation [32]. Several findings have shown that dietary flavonoids play a role as AHR ligands with either antagonist or agonist activity to inhibit cancer cell growth [33][34][35]. Additionally, flavonoids may also undergo CYP1-mediated oxidative metabolism to become antiproliferative products [33]. A study demonstrated the antiproliferative and cytostatic effects of a flavonoid lipid molecule, eupatorine, in breast cancer cells due to the involvement of CYP1-mediated metabolism [36]. e studies showed that cell cycle arrest at the G2/M phase induced by eupatorine could be reversed when MDA-MB-468 cells were coincubated with the CYP1 inhibitor acacetin. Another finding by Atherton confirmed that metabolites produced from the isoflavones daidzein and genistein via CYP1A1, CYP1A2, and CYP1B1 metabolism induced an antiproliferative response in MCF-7 cells [37].
In this study, the analysis of CYP genes showed that F1 and quercetin induced mRNA expression of CYP1A1 and CYP1B1, with the highest level observed at 48 h of treatment.
is phenomenon corresponded to the initiation of cell cycle arrest at the S phase by F1 and quercetin in MDA-MB-231 cells and the profound changes in cell cycle progression, which was also observed at 48 h of treatment with further induction of cell cycle arrest at the G2/M phase by F1. is finding is supported by numerous studies that revealed the role of quercetin in cancer proliferation in relation to its interaction with CYP family enzymes. For instance, quercetin was shown to be an agonist of CYP1A1 in breast cancer cells [38]. A study by Ciolino also showed that quercetin increases CYP1A1 mRNA level through mediation by the AHR receptor [39]. Furthermore, the metabolism of quercetin by the CYP1 enzyme, particularly CYP1A1 and CYP1B1, intensifies their antiproliferative effects in breast cancer cells [38]. Although F1 may not be as effective as quercetin in promoting metabolic activity, the results suggested that the antiproliferative effect of F1 and quercetin on MDA-MB-231 cells might be due to the metabolic activity of these CYPs, resulting in the production of active metabolites, which indirectly modulate the cell cycle progression and survival of MDA-MB-231 cells. For CYP2S1 gene expression analysis, F1 (but not quercetin) induced significantly high gene expression levels in MDA-MB-231 cells at 24 and 48 h of treatment. e selective expression of CYP2S1 in MDA-MB-231 cells treated with F1 suggested that CYP2S1 likely plays a role in the regulation of F1 anticancer activity, which is likely regulated by AHR [40].
In conclusion, the results provide more detailed information on the composition of the standardized subfraction F1, including its growth inhibitory activity, cell cycle arrest profile, and apoptosis induction in MDA-MB-231 cells in different ways. Moreover, inhibition of new cellular DNA synthesis and CYP gene expression may imply that F1 is a potential anticancer agent derived from the green approach towards breast cancer treatment.

Data Availability
No specific datasets were used and/or analyzed during this study. e datasets are available from the corresponding author and can be presented at reasonable request.

Disclosure
is manuscript is part of a preprint previously published as doi: 10.21203/rs.3.rs-341312/v2. A reference to this preprint has been included in the reference list [41].