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

Abstract

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.

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. 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]. The 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]. Therefore, 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. The 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.

[figure omitted; refer to PDF]

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. The 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 increased by 8.02%. Although the proportion of cells in the group of live cells decreased significantly by 28.43% in MDA-MB-231 after 72 h of treatment, the percentage of cells in early apoptosis increased by 29.37%. Similarly, at the necrotic level, the proportion of cells increased by 41.46% but decreased by 0.74% in the late apoptotic period. 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%.

3.3. Gas Chromatography-Mass Spectroscopy (GC-MS) Analysis

GC/MS investigation was conducted on D. bulbifera methanol extract. The peaks in the chromatogram were integrated and matched with the database spectra of recognised compounds stored in the GC-MS libraries of Pfleger–Maurer–Weber-drug, National Institute of Standard and Technology (NIST), WILEY229.LIB, Flavour, Fragrance, Natural and Synthetic Compounds (FFNSC1.3.lib), and Pesticides Library for toxicology (PMW_tox2).

The result of D. bulbifera methanol extract revealed 50 peaks, with 45 compounds identified, representing 98.49% of the entire extract (Figure 4). The major among them were acetic acid (34.68%), n-hexadecanoic acid (14.89%), 1,2,3-propanetriol, 1-acetate (acetin) (7.28%), hexadecanoate (4.01%), 7-tetradecenal (Z)- (2.92%), glycerol alpha-monoacetate (2.80%), phytol (2.46%), octadecanoic acid (2.26%), cholesterol (2.10%), palmitic acid (1.35%), linolenate (1.32%), megastigmatrienone and 8-oxabicyclo-oct-5-en-2-ol, 1,4,4-trimethyl (1.28%) each, 1,2,3-propanetriol (1.23%), and 4,4,5,8-tetramethylchroman-2-ol (1.22%).

[figure omitted; refer to PDF]

4. 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. The secondary metabolites, namely, phenolics and flavonoids, present in this species make it potential for antioxidant activities by functioning as reducing agents [15, 19–22]. 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. Thus, the secondary metabolites might be the key role players behind its biological activities [23]. The 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. This 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.

The 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. These 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 [29–34].

It was reported by Nur and Nugroho [32] that D. bulbifera extracts demonstrated a cytotoxic effect on T47D cell lines. This 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 this study, cell cycle analysis was conducted in MCF-7 and MDA-MB-231 cell lines after treated with D. bulbifera methanol extract at IC₅₀ concentration for 24 h, 48 h, and 72 h. At 24 h, the extract induced S and G₂/M phases arrest in MCF-7 and G₀/G₁ and G₂/M phases arrest in MDA-MB-231. At 48 h, growth in a number of the treated cells was observed in the sub-G₁ phase in MCF-7 and sub-G₁ and G₀/G₁ in MDA-MB-231, while significant decreases in the number of cells were observed in G₀/G₁, S, and G₂/M in MCF-7 and S and G₂/M in MDA-MB-231. At 72 h, D. bulbifera methanol extracts arrested MCF-7 at the sub-G₁ phase and MDA-MB-231 at sub-G₁ and S phases.

The result of the present study was similar with Srinivasan et al. [36] where diosgenin led to cell growth in the G₁ phase, consisting of 72% of cells in G₁ 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 G₁ 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 G₁ phase which was increased at 24 h. Consequently, the fraction of S phase cells decreased at 12 h. A sub-G₁ 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 G₂/M phase in a dose-dependent and time-dependent manner with consequent increase in the sub-G₁ 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 G₂/M. Diosgenin increased the p53 levels in NB4 cells, indicating its importance in controlling cell cycle arrest. Flow cytometric sub-G₁ 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. This 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-G₁ arrest. Demonstrating that dioscin caused cell cycle capture at the S stage, G₀/G₁ 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 G₀/G₁ 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 G₂/M stage had small changes. These 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 G₂/M stage cell population in Bel-7721, SMMC7721, and HepG2 HCC cells, suggesting that diosgenin could arrest the cell cycle in the G₂/M stage because of the way that extent of G₂/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. The outcome demonstrated that diosgenin from D. pseudojaponica hindered MCF-7 cells multiplication through G₀/G₁ arrest.

The 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 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. The 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 [46–49].

5. Conclusion

In conclusion, the effort presented herein has proved that D. bulbifera methanol extract had resilient antiproliferative activity when compared with a standard drug. The 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.

Acknowledgments

This research was financially supported by the Ministry of Higher Education of Malaysia (MOHE) under Fundamental Research Grant Scheme, FRGS, Vot No. 1560 (FRGS/1/2015/WAB01/UTHM/02/1) and also GPPS grant by UTHM (Vot No. U608). The authors would also like to thank Universiti Putra Malaysia (UPM) and Universiti Tun Hussein Onn Malaysia (UTHM) for providing infrastructural facilities to carry out this study.

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