Phytochemical Constituents and Antiproliferative Activities of Essential Oils from Four Varieties of Malaysian Zingiber officinale Roscoe against Human Cervical Cancer Cell Line

This study evaluates the volatile metabolic constituents and anticancer potential of essential oils distilled from the rhizomes of four Malaysian Zingiber officinale Roscoe (Zingiberaceae family) varieties (Bentong (BE), Cameron Highlands (CH), Sabah (SA), and Bara (BA)). The ginger essential oils were analyzed by gas chromatography coupled with quadrupole mass spectrometry (GC qMS). A total of 58 secondary compounds were tentatively identified, representing 82.6–87.4% of the total ion count. These metabolites comprise mainly of monoterpene hydrocarbons (19.7–25.5%), oxygenated monoterpenes (23.6–33.7%), sesquiterpene hydrocarbons (21.3–35.6%), oxygenated sesquiterpenes (1.5–3.9%), and other minor classes of compounds (0.7–2.7%). Principal component analysis (PCA) enabled differentiation of the analyzed ginger essential oils according to their varieties, with respect to their metabolites and relative quantities. The antiproliferative activity against the HeLa cervical cancer cell line was investigated via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The oils were found to exhibit strong antiproliferative activities with IC50 values of 23.8, 35.3, 41.3, and 42.5 μg/mL for BA, BE, SA, and CH, respectively. These findings suggest that the differences among the secondary metabolites and their abundance in different varieties of Z. officinale essential oils appear to be related to their antiproliferative potential. The strong antiproliferative effects of these oils signified their potential in the prevention and chemotherapy of cervical carcinoma treatment.


Introduction
Zingiber officinale Roscoe is a well-known herb belonging to the Zingiberaceae family which consists of 53 genera and approximately 1300 species [1,2]. This perennial rhizomatous plant only propagates through its rhizome (colloquially referred to as ginger), with physical appearances as pale yellowish, thick-lobed, with tuberous joints [2]. This flowering plant is seasonally cultivated in many tropical and subtropical regions throughout the world, including Australia, China, Indonesia, India, Malaysia, Nigeria, Spain, and others [3,4]. Albeit both ginger flowers and bruised stems have a pleasant aroma, the rhizomes are most often used as a spice and condiment due to its spicy flavor and woody aroma. Apart from being one of the most internationally traded spices, the Z. officinale rhizome is also highly valued for its health-promoting properties. Ginger rhizomes have been extensively documented as folk and traditional medicines for the treatment of numerous ailments, such as asthma, dyspepsia, heart palpitation, rheumatism, and vomiting [1,5]. The sesquiterpenic profile revealed a major contribution of its sesquiterpenes analogues, with SA oil having the highest relative amount (35.6%). The major sesquiterpenes were α-curcumene (2.2-4.5%), zingiberene (7.9-14.0%), β-bisabolene (4.6-8.4%), and βsesquiphellandrene (3.2-5.2%) for all samples. The concentration of zingiberene was much lower in BE (7.9%) than the other varieties (12.1%, 14.0%, and 10.8% for CH, SA, and BA, respectively). It is noted that this compound constituted the highest in the total composition of CH, SA, and BA. δ-Elemene was found at the low concentrations of 0.3%   The sesquiterpenic profile revealed a major contribution of its sesquiterpenes analogues, with SA oil having the highest relative amount (35.6%). The major sesquiterpenes were α-curcumene (2.2-4.5%), zingiberene (7.9-14.0%), β-bisabolene (4.6-8.4%), and β-sesquiphellandrene (3.2-5.2%) for all samples. The concentration of zingiberene was much lower in BE (7.9%) than the other varieties (12.1%, 14.0%, and 10.8% for CH, SA, and BA, respectively). It is noted that this compound constituted the highest in the total composition of CH, SA, and BA. δ-Elemene was found at the low concentrations of 0.3% and 0.1%, in CH and SA, respectively, while it was not detected in BE and BA. Farnesol was present in CH at a low concentration of 0.1% and was absent in the other three varieties. Produced by dephosphorylation of farnesyl-PP, farnesol has been reported to play a critical role in the antiproliferation and apoptosis of various tumor cells [26]. Non-terpenic constituents only accounted for 0.7-2.7% of the total compounds, with the highest relative content present in BE. 2-Nonanol, an acyclic alcohol, was found in BA at only 0.6%; meanwhile, it was absent in the oils from all the other varieties.
Z. officinale essential oil was reportedly composed of a relatively high amount of sesquiterpenes and a low concentration of monoterpenes, and this is the case typically associated with Eastern Asian origin [27][28][29][30][31][32][33][34][35]. However, a similarly large number of studies have detailed the preponderance of monoterpenoids from various other locations [28,32,[35][36][37][38][39][40][41]. Most of the monoterpenoid-rich oils from previous studies were characterized by significantly high amounts of αand β-citral. For instance, Wohlmuth et al. reported the amount of citral from Australian-grown Z. officinale oils to be in the range of 28.1-70.8% [40]. In a study by Vairappan et al., the content of citral (25.2-28.3%) in the hydro-distilled oil varieties from Malaysia was significantly high [38]. The result did not agree well with the current finding, where the relative amount was comparatively lower (12.9-18.1%) [42]. There are also a few studies where the occurrence of citral is lower and other compounds predominate, similar to the current finding. Mollenbeck et al. found that the Z. officinale oil from Madagascar contained camphene (30.8%) as the major compound, while citral was present in a comparatively lower concentration (13.9%) [36]. Nandi et al. reported α-citral concentration to be 7.6-9.8% in the monoterpenoid-rich oils from China and Bangladesh, while β-citral was absent in both oils [28]. The low amounts of citral in Z. officinale essential oil have been associated with its loss during drying processes, immaturity of the harvested rhizome, the long storage period before analysis, and changes in climatic conditions during cultivation [37,40]. Other factors that may influence the chemical variation in Z. officinale oils include geographical origins, agricultural practices, soil qualities, methods of extraction, and genetics, to adapt to the diverse environmental surroundings [40,43]. These findings demonstrated that these variations may impart significant effects on the flavor and aroma qualities of different varieties of ginger.

Discrimination of Z. officinale Oil Varieties via Principal Component Analysis (PCA)
Principal component analysis (PCA) was applied to selected secondary compounds to identify differences and inter-relationships between metabolic profiles of the analyzed Z. officinale varieties. The score and loading plots in Figure 2 represented 82% of the total data variance (PC −1, 47%, and PC −2, 35%). The score plot clearly indicated that all four Z. officinale varieties were well-segregated from each other (Figure 2a), which can be explained in terms of the loading plots of the components (Figure 2b). PC −1 separated BA from SA, CH, and BE. Positioned at the farthest upper left quadrant with the lowest PC −1 score, the segregation of BA can be characterized by the high amounts of sulcatone (0.5%), p-cymene (0.2%), linalool (1.2%), borneol (2.7%), 4-terpineol (0.3%), bornyl acetate (0.6%), and α-curcumene (4.5%). PC −2 separated CH and SA from BE. CH and SA were located near to each other in the same quadrant (positive PC −2 axis), suggesting similarity in terms of secondary compounds and their relative quantity. CH and SA can be segregated from the other varieties through the presence of δ-elemene (0.3%), copaene (0.3%), β-bisabolene (7.4-8.4%), γ-bisabolene (0.2%), and farnesol (0.1%) as the primary loading metabolites. BE, situated near the negative PC −2 axis, contained eucalyptol (4.1%), 2-nonanol (0.6%), 2-undecanol (0.2%), farnesal (0.2%), rosefuran (0.3%), and citronellal (0.8%) as the main discriminatory components separating that variety from the others. Even though most of the principal explanatory variables in the four Z. officinale oils contribute to a minor degree to each of their total relative contents, they were proven to have a noticeable influence in distinguishing between different varieties. However, the need for more studies incorporating larger sets of samples for each variety is preferred to confirm their class attributes and chemical resemblance.
(0.6%), and α-curcumene (4.5%). PC −2 separated CH and SA from BE. CH and SA were located near to each other in the same quadrant (positive PC −2 axis), suggesting similarity in terms of secondary compounds and their relative quantity. CH and SA can be segregated from the other varieties through the presence of δ-elemene (0.3%), copaene (0.3%), β-bisabolene (7.4-8.4%), γ-bisabolene (0.2%), and farnesol (0.1%) as the primary loading metabolites. BE, situated near the negative PC −2 axis, contained eucalyptol (4.1%), 2-nonanol (0.6%), 2-undecanol (0.2%), farnesal (0.2%), rosefuran (0.3%), and citronellal (0.8%) as the main discriminatory components separating that variety from the others. Even though most of the principal explanatory variables in the four Z. officinale oils contribute to a minor degree to each of their total relative contents, they were proven to have a noticeable influence in distinguishing between different varieties. However, the need for more studies incorporating larger sets of samples for each variety is preferred to confirm their class attributes and chemical resemblance.   Table S1.

Antiproliferative Evaluation
The cytotoxicity of the four varieties of Z. officinale essential oils was evaluated against the human adenocarcinoma cervical cancer (HeLa) cell line using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. All essential oil samples displayed significant cell proliferation activity, in which the growth of the HeLa cells declined in a dose-dependent manner (Figure 3). This result is in agreement with a study reported by Panyajai et al., where the oil of Zingiber ottensii also showed dose-dependent anticancer activity against the HeLa cell line [44]. The highest antiproliferative activity was shown by BA oil (IC 50 = 23.8 µg/mL), followed by BE (IC 50 [45]. Lee reported the IC 50 value of Korean ginger oil to be 60.6 µg/mL [46]. Interestingly, these data demonstrate that phenolic compounds (e.g., 6-gingerol, shogaol, etc.) in ginger are not the only bioactive molecules responsible for conferring the antiproliferative effects of cancer cells (typically with IC 50 values in the range of 16.0-253.4 µg/mL) [47][48][49][50][51]. It is likely that the encouraging antiproliferative activity of ginger is also attributable to the terpenic components (or synergistic effects of the components) present in the essential oils. For instance, α-zingiberene (or zingiberene) was found to induce DNA fragmentation, increase the sub-diploid cell population, and activate caspase-3, leading to the pathway for cancer apoptosis [46]. Likewise, the inhibition of Ishikawa and ECC-1 endometrial cell proliferation were lower when treated with citral (α-and β-) as opposed to treatment with Z. officinale oil [15]. The study found that the terpenoids that are present in the oil served as potent anticancer agents that activated the p53 tumor suppressor, which then triggered the apoptotic pathways in endometrial cancer cells. Hachlafi et al. reported that camphene (a major compound in BA) can trigger apoptosis by decreasing pro-caspases 9 and induce the increase of caspases 3 through the activation of poly (ADP-ribose) polymerase cleavage [52]. These findings indicated that the synergism of various secondary compounds plays an important role in inducing cell cycle arrest and apoptosis in different cancer cell lines.
thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. All essential oil samples displayed significant cell proliferation activity, in which the growth of the HeLa cells declined in a dose-dependent manner (Figure 3). This result is in agreement with a study reported by Panyajai et al., where the oil of Zingiber ottensii also showed dose-dependent anticancer activity against the HeLa cell line [44]. The highest antiproliferative activity was shown by BA oil (IC50 = 23.8 μg/mL), followed by BE (IC50 = 35.3 μg/mL), SA (IC50 = 41.3 μg/mL), and CH (IC50 = 42.5 μg/mL). These results were significantly higher as compared to the Z. officinale oil from other locations. For example, Santos et al. evaluated the antiproliferative activity of the oil from Southern Brazil against the HeLa human cervical cancer cell line with the IC50 value of 141.4 μg/mL [45]. Lee reported the IC50 value of Korean ginger oil to be 60.6 μg/mL [46]. Interestingly, these data demonstrate that phenolic compounds (e.g., 6-gingerol, shogaol, etc.) in ginger are not the only bioactive molecules responsible for conferring the antiproliferative effects of cancer cells (typically with IC50 values in the range of 16.0-253.4 μg/mL) [47][48][49][50][51]. It is likely that the encouraging antiproliferative activity of ginger is also attributable to the terpenic components (or synergistic effects of the components) present in the essential oils. For instance, α-zingiberene (or zingiberene) was found to induce DNA fragmentation, increase the sub-diploid cell population, and activate caspase-3, leading to the pathway for cancer apoptosis [46]. Likewise, the inhibition of Ishikawa and ECC-1 endometrial cell proliferation were lower when treated with citral (α-and β-) as opposed to treatment with Z. officinale oil [15]. The study found that the terpenoids that are present in the oil served as potent anticancer agents that activated the p53 tumor suppressor, which then triggered the apoptotic pathways in endometrial cancer cells. Hachlafi et al. reported that camphene (a major compound in BA) can trigger apoptosis by decreasing pro-caspases 9 and induce the increase of caspases 3 through the activation of poly (ADP-ribose) polymerase cleavage [52]. These findings indicated that the synergism

Preparation and Isolation of Essential Oil
Four different varieties of mature Zingiber officinale rhizomes (Bentong (BE), Bara (BA), Cameron Highlands (CH), and Sabah (SA)) were collected from local producers in Malaysia. The samples were rinsed with ultra-pure water to remove particulates, air-dried (12 h), and grated into small pieces. Approximately 800 g of the grated samples was subjected to steam distillation for 5 h. The oils were isolated and stored at 4 • C when not in use. Prior to analysis, oil samples were diluted to 3% v/v with acetone.

Chromatographic Condition
Gas chromatography−mass spectrometry (GC-MS) analyses were conducted on an Agilent Technologies 7890B GC system equipped with a 5977B GC/MSD quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA), a 7693A autosampler, and a split/split-less inlet. The chromatographic separation was performed using a HP-5ms (5% phenyl-methylpolysiloxane) capillary column of dimensions 30 m × 0.25 mm I.D. × 0.25 µm film thickness (Agilent Technologies, Santa Clara, CA, USA). The chromatographic con-ditions were: oven temperature program, 40 • C (hold 2 min), then heated at 3 • C min −1 to 270 • C; injector temperature, 300 • C; carrier gas, helium (purity of 99.999%) at a flow rate of 1.0 mL min −1 (24.02 cm s −1 ); injection volume, 1 µL, and using a split ratio of 20:1. The MS was operated in electron ionization (EI) mode at 70 eV, transfer line temperature of 300 • C, ion source temperature of 230 • C, mass scan range of 45-500 Da, and solvent delay time of 2.6 min.

Data Handling and Statistical Analysis
Data acquisition and processing were performed using Agilent MassHunter Qualitative Analysis 10.0 (Agilent Technologies, Santa Clara, CA, USA). The National Institute of Standards and Technology (NIST) 14 MS spectrum library was used for spectrum searching and identification. Retention indices (RI) values were determined with respect to a C 7 -C 30 series of n-alkane standards analyzed under the same GC conditions as above, using the Van den Dool and Kratz equation and compared with reported RI values [23,24,53]. The relative concentrations of tentatively identified components were calculated based on the acquired total ion chromatograms and presented as the mean ± standard deviation from three repeated independent experiments. The data were statistically analyzed using PCA (Unscrambler X 10.3; CAMO Software AS, Oslo, Norway) to identify differences for the obtained chemical profiles. All data were presented using Origin 8 (OriginLab Corporation, Northampton, MA, USA) and Excel software (Microsoft Corporation, Washington, DC, USA).

Cell Culture
The human cervical adenocarcinoma (HeLa) cell line was acquired from the American Type Culture Collection (Manassas, VA, USA). The cell was cultured in Dulbecco's modified Eagle's medium-high glucose (DMEM, Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin or streptomycin at 37 • C and 5% CO 2 .

Cell Viability Assay
A total of 3 × 10 3 cells/well were seeded into a 96-well plate in supplemented DMEM and incubated (5% CO 2 at 37 • C) for 24 h. The medium was removed and the essential oils, re-suspended in ethanol (Merck, Darmstadt, Germany) at a final concentration of 50 mg/mL, and diluted with supplemented DMEM to afford concentration ranges from 4.69 to 75.00 µg/mL, were added to each well. The final concentration of ethanol in each well was not more than 0.3%. On completion of the 24 h incubation period, the MTT assay, as described by Mossman [25], was performed to assess the cell viability. The optical density (OD) was recorded at 540 nm by using a SkanIT absorbance reader (Thermo Scientific, St. Peters, MO, USA). All experiments were performed in triplicate using media with cells only as a positive control, and only media with no cells as a negative control. The vehicle control was cells and media with 0.3% ethanol.

Conclusions
This study reported a detailed untargeted characterization of the phytoconstituents of essential oils derived from the rhizomes of four varieties of Z. officinale (BE, CH, SA, and BA) sourced in Malaysia, using GC-qMS. Interestingly, the metabolic profiles of the oils revealed differences in terms of their major classes of compounds according to varieties. SA was dominated by sesquiterpenic hydrocarbons, whilst BE, CH, and BA were dominated by oxygenated monoterpenes. α-Citral (9.8%) was identified as the most abundant metabolite in BE, whilst zingiberene (10.8-12.1%) in CH, SA, and BA. PCA successfully classified different Z. officinale oils into their corresponding varieties, characterized according to their metabolic compositions. The oils displayed strong antiproliferative activities against the HeLa cell line, in which BA exerted the most significant inhibition (IC 50 value of 23.8 µg/mL). This study indicated that essential oils extracted from different Z. officinale cultivars constitute different phytochemical diversity, and thus confer distinctive flavor characteristics and antiproliferative effects. Moreover, these data also demonstrated the strong antiproliferation potential of ginger essential oils, which can be further explored as a potent alternative for the prevention and treatment of cervical carcinoma.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants11101280/s1, Table S1: Secondary compounds analyzed and identified in different ginger oil varieties using GC−MS. Repeated entries for a given compound correspond to different ginger varieties, Figure S1