Secondary Metabolites, Antioxidant, and Antiproliferative Activities of Dioscorea bulbifera Leaf Collected from Endau Rompin, Johor, Malaysia

Breast cancer is among the most commonly diagnosed cancer and the leading cause of cancer-related death among women globally. Malaysia is a country that is rich in medicinal plant species. Hence, this research aims to explore the secondary metabolites, antioxidant, and antiproliferative activities of Dioscorea bulbifera leaf collected from Endau Rompin, Johor, Malaysia. Antioxidant activity was assessed using 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), and 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) assays, while the cytotoxicity of D. bulbifera on MDA-MB-231 and MCF-7 breast cancer cell lines was tested using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cell cycle analysis and apoptosis were assessed using flow cytometry analysis. Phytochemical profiling was conducted using gas chromatography-mass spectrometry (GC-MS). Results showed that methanol extract had the highest antioxidant activity in DPPH, FRAP, and ABTS assays, followed by ethyl acetate and hexane extracts. D. bulbifera tested against MDA-MB-231 and MCF-7 cell lines showed a pronounced cytotoxic effect with IC50 values of 8.96 μg/mL, 6.88 μg/mL, and 3.27 μg/mL in MCF-7 and 14.29 μg/mL, 11.86 μg/mL, and 7.23 μg/mL in MDA-MB-231, respectively. Cell cycle analysis also indicated that D. bulbifera prompted apoptosis at various stages, and a significant decrease in viable cells was detected within 24 h and substantially improved after 48 h and 72 h of treatment. Phytochemical profiling of methanol extract revealed the presence of 39 metabolites such as acetic acid, n-hexadecanoic acid, acetin, hexadecanoate, 7-tetradecenal, phytol, octadecanoic acid, cholesterol, palmitic acid, and linolenate. Hence, these findings concluded that D. bulbifera extract has promising anticancer and natural antioxidant agents. However, further study is needed to isolate the bioactive compounds and validate the effectiveness of this extract in the In in vivo model.


Introduction
Millions of mortalities were reported globally as a result of cancer, and the numbers of new cases are expected to increase in the future [1]. Breast tumour is the second highest cancer recorded worldwide and the most common cause of cancer death among women. It has been ascertained that about 25% of all new identified cancer case is breast cancer, which accounts for 15% of deaths in women each year [2,3].
Breast cancer is a commonly diagnosed cancer in Malaysia, having a standardized age rate of 47.4 per 100,000 [4,5].
e National Cancer Registry of Malaysia (NCR) records that 21,773 Malaysians who are perceived with cancer and approximations show that unregistered cases have reached about 10,000 cases every year. It is likely that by 75 years old, ¼ of Malaysians will be diagnosed with cancer [6]. erefore, discovering new drugs with minimal toxicity is a broad scientific challenge, and there is an immediate need to search for a new agent to mitigate the menace by using alternative evidence-based herbal medicines.
Dioscorea bulbifera (Figure 1) is a member of the family Dioscoreaceae, categorised in the order Dioscoreales and genus Dioscorea. It is usually referred to as air potato, air yam, or bulbil-bearing yam. It is a prolific climbing plant indigenous to Southern Asia and West Africa, mostly found along forest edges. e direction of circular twinning had been reported in D. bulbifera and can grow to a height of 12 m. It has shiny green leaves that are alternate with a long leafstalk. Resembling true yam leaves, D. bulbifera has an annual vegetative cycle. Hence, this study was directed toward discovering secondary metabolites, antioxidant, and antiproliferative effects of D. bulbifera leaf extract from Kampung Peta, Endau Rompin, Mersing, Johor, Malaysia.  [7]. Identification was made, and the voucher specimen was deposited in the Herbarium of Centre of Research for Sustainable Uses of Natural Resources (CoR-SUNR), Faculty of Applied Sciences and Technology, Universiti Tun Hussein Onn Malaysia. Fresh sample was cleaned using tap water to eliminate impurities or soil debris. e sample was crushed to a fine powder with an electric blender after shade drying at room temperature and kept in a zip lock bag in a freezer (−20°C) for further analysis [8,9].

Sample Extraction.
Methanol, ethyl acetate, and hexane solvents were used for the extraction of plant samples by successive maceration methods as described by Bhunu et al. [10] with some modifications. e mixture was filtered using a vacuum filter and then evaporated using a rotary evaporator where the yield of extracts was found; hexane (6.22%), ethyl acetate (8.63%), and methanol (20.6%) were used. e extract was later used to identify its phytochemicals, antioxidant, and antiproliferative effects.

Determination of Antioxidant Activities
DPPH scavenging activity of the sample was determined according to Bakar et al. [11] and Hassan and Bakar [12]. Briefly, a methanol solution of DPPH (0.3 mM) was added into the samples (2.5 mL). e extract was kept in a light protected place at room temperature for 30 min after mixing vigorously. e absorbance was read at 518 nm. e following equation was used for calculating the scavenging or antioxidant activity (AA): For the suppression concentration (IC 50 ), the quality of sample that readily inhibits cell division by half was determined explicitly for every cell multiplying curve.

Cell Cycle Analysis by Propidium Iodide (PI) Staining.
Cell cycle analysis was carried out with some modifications as described by Queiroz et al. [16]. Cells (1 × 10 6 ) were nurtured for 24 h and subsequently treated for 24 h, 48 h, and 72 h using crude extracts at the IC 50 values. Altogether, the detached and adhering cells were reaped and reassigned in a disinfected centrifuge tube. e cells were then centrifuged (1,200 rpm) for 15 min at 4°C. Cold phosphate buffered saline (PBS) was used to wash the cells where the cells were resuspended with 0.5 mL of cold PBS, and ice-cold ethanol (70%) was then added to the cell suspension and incubated at −20°C for 2 h. en, the ethanol was expelled from the sample after centrifugation. e cells that have been rinsed twice using cool PBS previously were stained with propidium iodide (500 μL of 10 μg/mL) and RNase (100 μg/ mL) at room temperature for about 30 min. Dispersion of the cell cycle was analysed using flow cytometry.

Annexin V/PI Apoptosis Assay.
is method was performed according to Tor et al. [17]. Briefly, cells were plated in 6-well plates and treated with IC 50 values for 24 h, 48 h, and 72 h. e viability of the cells was measured using an annexin V-FITC/PI kit, following the manufacturer's procedure. Both treated and control cells were centrifuged and washed twice using PBS. At room temperature, cell staining was carried out using FITC annexin V (5 µL) and 10 µL PI for 15 min, and cell death was also analysed using flow cytometry.
e selected crude extract was analysed using gas chromatography (GC-MS-2010 Plus-Shimadzu) in order to determine the secondary metabolites present. e segment (30.0 m length, 0.25 mm ID, and 0.25 μm thickness) was set at 50°C for 4 min which was then expanded to 300°C at the rate of 3°C/min, and after that supported for 10 min. e temperature at the injector was set at 250°C, and the volume was set at 0.1 L. e present helium rate of the bearer gas was set at 1 mL/min with an aggregate run span of an hour. Electron ionization and mass spectra were performed at 70 eV and between the range of 40-700 m/z [18].

Statistical Analysis.
All the data were presented as mean ± standard error of mean (S.E.M). e data were statistically analysed by one-way ANOYA, followed by Dunnett's posthoc using Prism version 15.0. e level of statistical significance was set at p < 0.05.

Antioxidant Activity of the Plant Extracts.
Since one antioxidant assay is not sufficient to provide an adequate picture of the scavenging activities, D. bulbifera leaf extracts were subjected to several antioxidant assays. Antioxidant Evidence-Based Complementary and Alternative Medicine potential of D. bulbifera leaf extracts was evaluated using DPPH, FRAP, and ABTS assays (Table 1). For the DPPH assay, the methanol extract of D. bulbifera had the highest scavenging activity with the value of 79.0 ± 0.31 %, followed by the ethyl acetate extract with 23.2 ± 0.05 % and hexane extract with 11.5 ± 0.31 %, respectively. e same observation was also found in the FRAP assay where the methanol extract demonstrated highest reducing capacity with a value of 65.6 ± 0.35 mM Fe 2+ /g, followed by ethyl acetate with 59.5 ± 0.10 mM Fe 2 + /g and hexane extract with 14.9 ± 0.05 mM Fe 2+ /g, respectively. Whereas for the ABTS assay, the methanol extract also had the highest scavenging activity with a value of 31.34 ± 2.06 mg AEAC/g, followed by the ethyl acetate extract with 10.98 ± 0.64 mg AEAC/g and hexane extract with a value of 9.50 ± 0.48 mg AEAC/g. Plant phenolics are a significant group of compounds that serve as major antioxidants or free radical scavengers. Hence, in this analysis, the observed high free radical scavenging behavior of the methanol extract may have accounted for its polarity for the radical scavenging ability of the plant extracts. e radical scavenging activities may be due to the presence of some flavonoids with a free hydroxyl group, capable of donating hydrogen and electron [12]. e findings showed that the plant extracts had substantial disparities in their ability to compare ascorbic acid, which may also be ascribed to various characteristics, reaction, and mechanisms. Organic media can be used in solubilising DPPH radical chromogens. A strong positive correlation (R 2 � 0.9399, p < 0.05) was observed between the activities.
ere was also a similar relationship in FRAP, where a strong positive linear correlation (R 2 � 0.8143, p < 0.05) was found. FRAP also correlates with ABTS• + (R 2 � 0.87), whereas ABTS• + showed a stiff positive association to VCEAC (R 2 � 0.6784). e practical difference in these comparisons was DPPH•, which also correlates poorly with ABTS (R 2 � 0.53).  Figure 2). In MCF-7, there was a momentous arrest at sub-G 1 , at 24 h-72 h, and the number of cells at S and G 2 /M phases and the number of cells at G 0 /G 1 were reduced substantially. In MDA-MB-231, there was a significant arrest at sub-G 1 and G 2 /M for 24 h, 48 h, and 72 h, and the number of cells in G 0 / G 1 and the number of cells in G 2 /M were markedly reduced. At 48 h, there was also considerable arrest at sub-G 1 and G 0 / G 1 , and the number of cells in S and the number of cells in G 2 / M phases were markedly reduced, and eventually at 72 h, there was a substantial arrest at sub-G 1 and S, and the number of cells in G 0 /G 1 and G 2 /M phases decreased significantly.  Figure 3 shows the percentages of cell viability after treatment of D. bulbifera methanol extract in MCF-7. At the end of treatment (after 72 h), the percentage of feasible cells decreased ominously by 7.78%, while the percentage of cells increased by 3.99% at early apoptosis. e cell proportion also rises by 1.06% in the late apoptotic period. Nevertheless, the percentage of cells in early apoptosis decreased by 3.8% for the control culture at 72 h; the late apoptotic stage also decreased by 0.02%, while the number of viable cells         However, for the control culture at 72 h, the proportion of cells with early apoptosis decreased by 4.14%, the late apoptotic stage also decreased by 14.62%, while the apoptotic period increased by 1.08%, and a number of viable cells also decreased by 11.92%.

Discussion
Medicinal plants have been of great interest as a source of natural antioxidants used for health promotion such as anticancer properties. In this present study, the radical scavenging activities of D. bulbifera leaf extracts were quantified using DPPH, FRAP, and ABTS assays. All the extracts demonstrated scavenging of stable DPPH and ABTS as well as reducing activity in FRAP assay. e secondary metabolites, namely, phenolics and flavonoids, present in this species make it potential for antioxidant activities by functioning as reducing agents [15,[19][20][21][22]. Numerous antioxidants that are described to have therapeutic potential such as vanillic acid, isovanillic acid, epicatechin, and myricetin are essential bioactive compounds in D. bulbifera. Tubers of D. bulbifera have displayed higher scavenging activity, reducing power, and ferrous ion chelating due to its high content of polyphenols such as oxalic acid, citric acid, malic acid, and succinic acid.
us, the secondary metabolites might be the key role players behind its biological activities [23]. e methanolic extract of D. bulbifera has been reported to demonstrate DPPH radical-scavenging activity [24]. Studies of antioxidants from different platinum-palladium bimetallic nanoparticles (PtNPs) made from D. bulbifera indicates that PtNPs could inhibit radical DPPH by up to 30.16%, while palladium (PdNPs) showed up to 28.97% of the activity. Despite this, Ptfor-PdNPs reveal 38.49% scavenging when compared separately with PtNPs and PdNPs. In addition, for Pt-PdNPs (56.71%), a synergistic enhancement activity against radical superoxide was also observed as contrasted and only PtNPs (31.87%) or PdNPs (27.1%).
Another study which was conducted by Li et al. [25] revealed that D. bulbifera and other Dioscorea species possessed high antioxidant activity as in the following order D. bulbifera, followed by D. collettii, D. nipponica, and D. opposita.
is indicates that members of the genus Dioscorea have therapeutic potential due to high antioxidants contents. Comparable study was carried out by Ghosh et al. [26]; copper nanoparticles (CuNPs) synthesised by D. bulbifera showed high radical scavenging activities that are slightly lesser when compared with ascorbic acid. Vitamin C also showed 14.11% of superoxide scavenging activity while CuNPs showed 48.39% of the activity.
e results of the present study indicated that the methanolic extract of D. bulbifera was found to be the most cytotoxic extract in both cell lines due to the presence of plentiful active compounds, namely, diosgenin in yam, the edible tubers of Dioscorea spp. ese results are compatible with the studies conducted by Li et al. [27] and Lee et al. [28]. Diosgenin has been shown to enhance metabolism of lipids, improve antioxidant activities, reduce glucose levels, and suppress inflammation [29]. Previous research presented that foods rich in diosgenin such as yam species (known as air potatoes) found to have a protective effect on myocardial I/R wound in rats due to apoptosis and necrosis and showed a cytotoxic effect on cancer cell lines [29][30][31][32][33][34].
It was reported by Nur and Nugroho [32] that D. bulbifera extracts demonstrated a cytotoxic effect on T47D cell lines.
is research result indicated that the chloroform extract of the D. bulbifera leaves was stronger in inhibiting the growth of the cells than the methanol extract. Similarly, Ghosh and his colleagues [26] revealed that different nanoparticles Pton-PdNPs, PdNPs, and PtNPs made from D. bulbifera tuber extract exhibited the antiproliferative effect where Pt-PdNPs had been the most cytotoxic nanoparticles at concentration 10 μg/mL. In vivo studies conducted on cytotoxic activities of the water extract, nonethyl acetate extract, ethyl acetate extract, ethanol extract, and diosbulbin B isolated from D. bulbifera showed that ethanol and ethyl acetate extracts reduced the lump weight in S180 and H22 tumour cells bearing mice while no such effect was observed in water and nonethyl acetate extracts [35].
In e result of the present study was similar with Srinivasan et al. [36] where diosgenin led to cell growth in the G 1 phase, consisting of 72% of cells in G 1 at 24 h and 82% and 76% at 48 h and 72 h posttreatment, respectively, compared to untreated cells. Similar observation was found in diosgenin-treated MDA-MB-231 cells where there was aggregation of cells in the G 1 process occurred (82% at 24 h, 81% at 48 h, and 71% at 72 h). In addition, Moalic et al. [37] found that cells treated with diosgenin suppressed 1547 cell proliferation rate after 12 h along with a significant accumulation of cells in the G 1 phase which was increased at 24 h. Consequently, the fraction of S phase cells decreased at 12 h. A sub-G 1 population, typically associated with apoptotic cells, appeared at 48 h when compared to controls. According to a similar study conducted by Wang and Weller [38], HepG2 cells treated with different concentrations of protodioscin (bioactive component in D. collettii) for up to 24 h, accumulated mainly in the G 2 /M phase in a dosedependent and time-dependent manner with consequent increase in the sub-G 1 phase of cell cycle. Moreover, diosgenin isolated from D. bulbifera was reported to induce S-phase arrest at a concentration of 13 µM in DU-145 prostate cancer cells. At 24 h of incubation phase, S arrest was relatively substantial, but at 48 h of incubation, there was no such effect, and arrest was not found to be important in MCF-7 compared to DU-145 [39].
In 2005, Liu et al. [40] reported that NB4 cells treated with diosgenin resulted in the enlargement of cell size, and arrest was made at G 2 /M. Diosgenin increased the p53 levels in NB4 cells, indicating its importance in controlling cell cycle arrest. Flow cytometric sub-G 1 analysis showed that a dramatic hypodiploid number of K562 cells appeared after treatment with diosgenin for 48 h along with DNA fragmentation. Another research carried out by Hsu et al. [41] presented that treatment of squamous cell carcinoma-25 (SCC-25) with red mold Dioscorea cell cycle for 24 h caused arrest at the G2/M phase. is effect was also connected to the repression of CDK1 and cyclin B1 mRNA levels, ensuring cell proliferation inhibition.
On comparative research by Liu and his colleagues [40], it was found that dioscin fundamentally hinders multiplication of C6 glioma cells at the S stage because of an expansion in a few cells in the stage following expanding dosages of dioscin increment in sub-G 1 arrest. Demonstrating that dioscin caused cell cycle capture at the S stage, G 0 /G 1 stage decline in a few cells was observed. It has been recently detailed that dioscin restrained ROS creation, brought about DNA harm, and made cell cycle captured at the S stage, when contrasted and untreated, gathering the extent of the G 0 /G 1 stage decreased from 61.38% to 35.43%, and S stage increased from 30.62% to 59.77% by 5.0 mg/ml of dioscin while the extents of G 2 /M stage had small changes. ese findings showed that dioscin was able to block human HEp-2 and TU212 cells at the S stage [42].
A study performed by Li et al. [27] showed that diosgenin treatment in focus subordinate caused increment of G 2 /M stage cell population in Bel-7721, SMMC7721, and HepG2 HCC cells, suggesting that diosgenin could arrest the cell cycle in the G 2 /M stage because of the way that extent of G 2 /M stage cells with expanded in centralization of diosgenin. Lee and his colleagues [28] presented the antiproliferative impacts of diosgenin from yam (D. pseudojaponica) on malignant growth (MCF-7, A 549, and Hep G2) and typical (HS68 and clone 9) cells. e outcome demonstrated that diosgenin from D. pseudojaponica hindered MCF-7 cells multiplication through G 0 /G 1 arrest. e importance of the apoptosis idea for oncology lies in its being a directed marvel subject to incitement and hindrance. Even though little was thought about how settled helpful operators for disease influence its introduction, it appears to be sensible to propose that more prominent comprehension of the procedures included may prompt the advancement of enhanced treatment regimens [43]. Apoptosis is a physiological process of cell death that is in charge of the destruction of cells in normal tissues; it likewise occurs in different pathologic settings. Morphologically, it involves quick build up and sprouting of the cells with the arrangement of membrane-enclosed apoptotic bodies containing well-preserved organelles which are phagocytosed Evidence-Based Complementary and Alternative Medicine and digested by nearby resident cells [44]. Generally, apoptosis involved two central pathways; the first one is the stimulation of death receptors (DRs) in the tumour necrosis factor (TNF) superfamily, and the second one is the mitochondrial pathway initiated by Bcl-2 family proteins [45].
Several drugs that are used for cancer treatment have been appeared to cause apoptosis in quickly dividing normal cell numbers and tumours. Consequently, enhanced apoptosis is liable for a significant number of the antagonistic impacts of chemotherapy and tumour regression. Clear understanding of the procedures involved might lead to the improved treatment regimen. Hence, the realization of anticancer drugs that mediated the therapeutic impact by activating apoptosis is an additional necessary outcome [44].
Annexin-V-FITC/PI-flow cytometry analysis ascertained the induction of apoptosis by plant crude in both MCF-7 cells and MDA-MB-231. Distinctive biochemical features characterise apoptosis in the removal of damaged cells or tumour cells without causing irritation. e commencement of enzymatic and catabolic procedures in apoptosis in this manner empower cell morphological changes, for example, externalization of the plasma layer, phosphatidylserine (PS), shrinking of the cells, blebbing in cell membrane, condensation of chromatin, nuclear fragmentation, and apoptotic bodies formation [46][47][48][49].

Conclusion
In conclusion, the effort presented herein has proved that D. bulbifera methanol extract had resilient antiproliferative activity when compared with a standard drug. e antioxidant activities shown by methanol, ethyl acetate, and hexane extracts of the plant were significant compared with ascorbic acid, indicating the potential of the leaves of this species as natural antioxidants. Hence, antiproliferative activities demonstrated by leaf extracts authenticate the old style uses of this plant against various diseases of breast cancer inclusive.
Data Availability e datasets generated and/or analysed during the current study are available from the first author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.