In vitro cytotoxic activity, molecular docking study, and chemical composition of Zingiber cassumunar root oil

: The cytotoxic activities against cancer cell lines of eight known sesquiterpene and phenylbutenoids, namely, ( − )- β -sesquiphellandrene (1), ( E )-1-(3,4-dimethoxyphenyl)buta-1-ene (2), ( E )-1-(3,4-dimethoxyphenyl)buta-1,3-diene (3), ( E )-1-(2,4,5-trimethoxyphenyl)buta-1-ene (4), ( E )-1-(2,4,5-trimethoxyphenyl)buta-1,3-diene (5), ( E )- 4-(3,4-dimethoxyphenyl)buta-3-eneyl acetate (6), ( ± )- trans -3-(3,4-dimethoxyphenyl)-4- [ ( E )-3,4-dimethoxystyryl ] cyclohex-1-ene (7), and ( ± )- cis -3-(3,4-dimethoxyphenyl)-4- [ ( E )-3,4-dimethoxystyryl ] cyclohex-1-ene (8) were evalu-ated. All compounds were isolated from the rhizomes of Zingiber cassumunar Roxb. (Plai) using classical column chro- matography. Compounds 1, 7, and 8 exhibited good cytotoxic activity against acute lymphoblastic leukemia (MOLT-3) with half maximal inhibitory concentration (IC 50 ) values of 16.39 ± 1.22, 16.41 ± 3.68, and 14.38 ± 0.78 µ g / ml; promyelocytic leukemia (HL-60) with IC 50 values of 7.64 ± 0.33, 15.25 ± 0.88, and 13.02 ± 0.91 µ g / ml; and hormone-independent breast cancer (MDA-MB-231) with IC 50 values of 27.71 ± 1.41, 28.99 ± 2.30, and 27.94 ± 2.24 µ g / ml, respectively. Compounds 3, 7, and 8 displayed good anticancer activity against cervical carcinoma (HeLa) with IC 50 values of 18.68 ± 0.62, 20.86 ± 1.68, and 18.89 ± 1.26 µ g / ml, respectively. The results showed that two diastereomers (7 and 8) have good activity against the broad range of tested cancer cell lines. From molecular docking analysis, the binding energy and interaction between the isolated compounds and topoisomerase II (Top2) was calculated and could be used to evaluate cytotoxic activity. Molecular docking showed that 7 and 8 interacted with Top2 ( α and β types) using two or three hydrogen bonding, whereas the other compounds that also displayed this interaction had at least one hydrogen bonding. Additionally, only 7 exhibited non-toxic effect against normal embryonic lung cell line (MRC-5); therefore, the biological activity of 7 can serve as a basis for the study of anti-cancer agents in the near future.


INTRODUCTION
The Zingiberaceae plant family commonly found in Southeast Asia is known for its medicinal, pharmacological, and nutritional properties. Zingiber cassumunar Roxb., a plant in the Zingiberaceae family known as "Plai" or "Wan-fai", is used as traditional medicine to relieve pain, flatulence, and asthma [1][2][3][4]. Essential oil from the rhizomes of Z. cassumunar Roxb. has a wide variety of biological activities, such as antibacterial [5,6], antimicrobial [7], anti-inflammatory [8,9], antioxidant [10], anticoagulant [11], anticancer [12], and insecticidal effects [13]. Additionally, phytochemicals from Plai essential oil have been investigated as well. The major components that can be isolated from this plant, including terpenes, phenylbutenoids, and curcuminoids, depend on the crude preparation methods, such as solvent extraction and distillation [14]. Most of the isolated components also exhibit good biological activities. For example, curcuminoid groups have strong antioxidant activity [15], and phenylbutenoids exhibit good antiinflammatory activity [16]. Nowadays, cancer is a major cause of deaths around the world. Cancer treatment procedures include surgery, radiation therapy, and chemotherapy. In chemotherapy, patients receive chemotherapeutic drugs that target and eradicate cancer cell lines. Nevertheless, chemotherapeutic drugs also affect regular cells and cause many undesirable adverse effects, such as nausea, vomiting, and hair loss [17]. Currently available commercial chemotherapeutic drugs are slightly expensive and can be carcinogenic [18,19]. Therefore, the discovery of new anticancer drugs is very important to prevent the drug resistance of cancer cell lines and reduce the side effects of chemotherapeutic agents. Natural products have served as a productive source for novel drug discovery, particularly anticancer drugs [20]. Almost half of the drugs approved in the previous decade are based on natural products [21,22]. In addition, over one-third of therapeutic drugs derived from natural products in all pharmaceutical development stages are cancer treatment agents. Thus, the exploration of new natural products for drug development is highly recommended. In this work, we reported the isolation of eight known secondary metabolites from Plai oil via classical liquid column chromatography. The 1 H and 13 C nuclear magnetic resonance (NMR) spectroscopy and mass spectroscopy of all the isolated compounds from Plai oil were elucidated. The results corresponded with previous reports [23][24][25] in which these compounds were separated by high-performance liquid chromatography (HPLC). However, the spectroscopic data of some compounds are not clear, and only few studies have reported the cytotoxic activity of the sesquiterpene and phenylbutenoids isolated in the present study against cancer cell lines. We also used molecular docking to predict the binding sites, binding energy, and interaction of the assigned compounds and biological molecules.
Herein, the completed reports on the spectroscopic data along with the cytotoxic activity and molecular docking study of the isolated compounds were evaluated and accomplished. The phytochemicals were tested for cytotoxic activity against ten normal and drug-resistant cancer cell lines and normal embryonic lung cell line . The study could contribute to the discovery of effective anticancer drugs.

Plant materials
The rhizomes of Z. cassumunar Roxb. were purchased from a local market in Bangkok, Thailand in September 2020. The voucher specimen (TTM-1000644) was deposited to Thai Traditional Medicine Herbarium, Department of Thai Traditional and Alternative Medicine, Ministry of Public Health, Bangkok, Thailand. The fresh rhizomes of Z. cassumunar Roxb. were washed and cut into small pieces before steam distillation using Clevenger apparatus. The Plai essential oil was kept in a dark bottle with light protection under N 2 atmosphere at 4°C.

Structure characterization
The melting points were measured using an SMP3 Stuart™ digital melting point apparatus from Bibby Sterlin, Ltd (Staffordshire, UK). To confirm the structure, the products were analyzed by nuclear magnetic resonance (NMR) and mass spectrometry. Proton and carbon NMR spectra were obtained using the Bruker AvanceIII-HD-400 spectrometer at 400 and 100 MHz, respectively. High-resolution mass spectra were measured with an ESI-TOF, i.e., MicroTOF mass spectrometer (Bruker Daltonics, Germany). The optical rotation was measured using a Jasco P-1020 Polarimeter (Tokyo, Japan). The spectroscopic data were also compared with previous reports, and the valid results  (

Cytotoxic activity
All materials were used as received. Among the 10 cancerous and 1 normal cell lines used for cytotoxicity screening of compounds, 9 cell lines were adherent to the culture wells, whereas only HL-60 and MOLT-3 grew in suspension. Each cell line was maintained in an appropriate culture medium supplemented with essential nutrients and maintained using standard procedures at 37°C with 95% humidity and 5% CO 2 . All the test compounds and positive controls, including doxorubicin and etoposide, were prepared as 10 mg/ml stock solutions in DMSO and freshly diluted with the corresponding cell culture medium for each cell line on the day of analysis.
Prior to the assay, the cells were inoculated as a suspension in the corresponding cell culture medium (100 ml for adherent cells and 75 ml for suspended cells) into 96-well microtiter plates (Costar No. 3599, Corning Incorporated, Corning, NY, USA) at a density of 5000-20 000 cells per well, depending on their growth rates. Adherent and suspended cells were then allowed to grow at 37°C with 95% humidity and 5% CO 2 for 24 h and 30 min, respectively. The cytotoxicity assay was initiated by adding an equal volume of cell culture medium containing either each test compound, positive control, or DMSO, at predetermined concentrations. Following 48 h of exposure to various treatments, cell viability was determined using MTT assay for adherent cells or XTT assay for suspended cells, as described below.
For adherent cells, 100 ml of the MTT reagent (0.5 mg/ml in serum-free cell culture medium) was added to each well, and the microtiter plates were further incubated for 2.5-4 h at 37°C with 95% humidity and 5% CO 2 [26,27]. The medium was subsequently replaced with 100 ml of DMSO to dissolve the purple formazan before the absorbance at 550 nm was measured using a Spectra-Max Plus 384 microplate reader (Molecular Devices, Sunnyvale, CA, USA) with a reference wavelength of 650 nm.
For suspended cells, 75 ml of the XTT reagent (prepared from 5 ml of 1 mg/ml XTT sodium in water and 100 ml of 0.383 mg/ml PMS in water) was added to each well, and the cells were further incubated for 4 h at 37°C with 95% humidity and 5% CO 2 [28]. Afterwards, the absorbance of orange formazan at 492 nm was measured with a reference wavelength of 690 nm using a SpectraMax Plus 384 microplate reader. For each well, the background absorbance (averaged from the wells containing the same volume of complete culture medium) was subtracted from either A550 or A492 to get the absolute absorbance. The average value from the duplicate wells, which had been treated with each concentration of the test compounds, was then compared with that of the untreated wells to yield the percentage of surviving cells.
The half maximal inhibitory concentrations (IC 50 value) was finally calculated from the dose-response curve as the concentration that inhibits the cell growth by 50% in comparison with the negative control following 48 h of exposure to each test compound.

Molecular docking
Molecular docking can be used to study the binding energy of small ligand on the enzyme. The geometry of ligands was fully optimized using the density functional theory (DFT) at B3LYP/6-31G (d, p) level implemented in Gaussian 09. The crystal structures of topoisomerase IIα (Top2α) (PDB Id: 4FM9) and (PDB Id: 3QX3) for Top2β are obtained from the Protein data bank. The binding interactions of ligands with Top2 are simulated via molecular docking using Autodock 4.2 [29]. The grid box size of 60, 60, and 60 along the X, Y and Z axes with a grid-point spacing of 0.375 Å was applied for molecular docking. The center of grid for Top2 was set to 50.093, 40

RESULTS AND DISCUSSION
The isolated metabolites consisted of one sesquiterpene and seven phenylbutenoids from Z. cassumunar Roxb., namely,  [25,34]. Nevertheless, 7 and 8 were easily isolated in the present study by normal column chromatography using silica gel 60 as the stationary phase.
The structures of the isolated compounds were displayed and confirmed by 1 H and 13 C NMR spectroscopy and mass spectroscopy (Fig. 1,  Supplementary data: Fig. S3-S10 and Table S1-S4). The isolated compounds (1-8) were tested for cytotoxic activity against four normal cancer cell lines (MOLT-3, HuCCA-1, A549, and HepG2) and normal embryonic lung cell line (MRC-5). The results are tabulated in Table 1. Compounds 1-8 exhibited good cytotoxic activity against acute lymphoblastic leukemia (MOLT-3). Particularly, compounds 1, 7, and 8 had the IC 50 values of 16.39 ± 1.22, 16.41 ± 3.68, and 14.38 ± 0.78 µg/ml, respectively. The other compounds displayed moderate anticancer activity against MOLT-3 with IC 50 values in the range of 30-36 µg/ml. Additionally, 1-8 showed poor cytotoxic activity against cholangiocarcinoma (HuCCA-1), lung carcinoma (A549), and hepatocarcinoma cell lines (HepG2) except for compound 5, which illustrated strong activity against HepG2 with an IC 50 value of 17.83 ± 4.14 µg/ml. More than half of the tested compounds exhibited no harmful effect against normal embryonic lung cells (MRC-5) except compounds 1, 3, and 8, which had IC 50 values of 30-31 µg/ml. Compounds 1-8 were also tested for cytotoxic activity against human malignant and drug-resistant cancer cell lines (HeLa, T47-D, H69AR, S102, MDA-MB-231, and HL-60), and the results (     The result of the cytotoxicity assay revealed that 1, 7, and 8 had potential cytotoxicity against MOLT-3 and HL-60 with low IC 50 values. Both cell lines are part of leukemia, which is a type of cancer that occurs in the bone marrow caused by the abnormal growth of white www.scienceasia.org Table 2 In vitro cytotoxic activity of compounds 1-8 against human malignant and drug resistance human cancer cell lines. HeLa (cervical carcinoma), T47-D (hormone-dependent breast cancer), H69AR (lung cancer, multidrug resistance), S102 (Thai liver cancer), MDA-MB-231 (hormone-independent breast cancer), HL-60 (promyelocytic leukemia). Results are expressed as mean ± standard error of inhibition perceptual for all cell lines. Doxorubicin and etoposide were used as positive control. Experiments were performed in triplicate. IC 50 values were obtained from 10 µg/ml concentration of substance; %C = % inhibition at the 50 µg/ml concentration of substance; inactive (IC 50 > 50 µg/ml; %C = 0). blood cells. Leukemia can be treated with radiation therapy, chemotherapy, and bone marrow transplant. Chemotherapy is the primary method of treatment for leukemia. The chemotherapy drugs currently used to treat leukemia are the anthracycline class, such as doxorubicin and daunorubicin [35,36]. Doxorubicin and etoposide are powerful anticancer drugs that block DNA replication by inhibiting the mechanism of action of DNA topoisomerase II (Top2) [37,38]. Top2 is an enzyme that controls the supercoiling of DNA; plays a key role in replication, translation, recombination, and segregation in the cell cycle; and is used as a marker for cancer cells. In mammals, Top2 can be divided into two isoforms, namely, Top2α and Top2β. Top2α is involved in DNA replication whereas Top2β plays an important role in the survival of some neurons [39,40]. The binding interactions among Top2α/β, the tested compounds, and commercial chemotherapy drugs (doxorubicin and etoposide) were studied by molecular docking approach [29,30]. In this work, molecular docking may be used to explain the binding mechanisms of isolated compounds with Top2α/β and their cytotoxicity against cancer cell lines. The binding energy values and binding interaction between the ligands and Top2α/β were calculated and are presented in Fig. 2, Fig. 3 and Table 3. The lowest binding energy value was found in doxorubicin with values of −8.81 ± 0.19 kcal/mol for Top2α and etoposide with values of −9.74 ± 0.18 kcal/mol for Top2β. In the case of Top2α, phenylbutenoids 7 and 8 also demonstrated low binding energy values similar to that of doxorubicin and lower than that of etoposide with value of −8.46 ± 0.17 and −8.12 ± 0.26 kcal/mol, respectively. Compounds 1-6 displayed high binding energy values and did not have H-bonding interaction with Top2α/β possibly because of the small molecular size and characteristics of hydrocarbons. The molecular docking results showed that the binding interactions between all analyzed compounds and Top2α/β in the binding pocket were within the radius of 5.0 Å (Fig. 2, 3, Supplementary data: Fig. S1 and S2). The hydrogen bonding interactions between phenylbutenoids (7 and 8) and Top2α/β were found in the amino acid residues GLN773 for Top2α and ASP479 for Top2β as demonstrated in Fig. 2 and Fig. 3. GLN773 and ASP479 also interacted with doxorubicin and etoposide via hydrogen bonding interactions with Top2α and Top2β, respectively. In addition, the other interactions occurred between ligands and amino acid residues such as carbon hydrogen bond, pi-alkyl, and pi-sigma. Docking study suggested that the compounds (7,8), doxorubicin, and etoposide bound in a binding pocket with H-bonding interactions through the methoxy group, carbonyl group, and hydroxyl group in the side chain of ligands. The results displayed that phenylbutenoids 7 and 8 bound in the pockets of Top2α and Top2β to a similar area as those of doxorubicin and etoposide. The crucial pharmacophores in the compounds 7 and 8 that interacted to Top2α and Top2β are dimethoxyphenyl groups. The O atoms in dimethoxyphenyl side chain can form H-bonding interaction with amino acid residues on Top2α and Top2β. These are significant groups that related to core skeleton of doxorubicin and etoposide which displayed H-bonding interaction. The cytotoxicity against cancer cell lines of doxorubicin demonstrated lower IC 50 value than that of compound 7 whereas the binding energy of doxorubicin and compound 7 with Top2 showed similar value. The result implied that doxorubicin may be involved in other enzymes or process in the inhibition of cancer cell lines. However, Top2 is one of several enzymes involved in the functioning of cancer cells. In previous report using molecular docking analysis, the binding interaction between anticancer drugs and Top2 revealed that GLN726 and GLN773 in Top2α and ASP479 and ARG503 in Top2β are important key points for anticancer drugs [33]. Thus, our results from molecular docking study corresponded to the cytotoxic activity against cancer cell lines, particularly MOLT-3 and HL-60. Therefore, 7 and 8 showed potential as Top2 inhibitors for the development of anticancer drugs in the near future.

CONCLUSION
The essential oil obtained from Z. cassumar contained potential constituents with strong cytotoxic activity against leukemia cancer cell lines, namely, acute lymphoblastic leukemia (MOLT-3) and promyelocytic leukemia (HL-60) cell lines. The sesquiterpene (1) and two cyclohexene diasteromers (7 and 8) showed high cytotoxicity against MOLT-3 and HL-60. However, only compound 7 exhibited no harmful effect against normal embryonic lung cells (MRC-5) with IC 50 value of > 50 µg/ml; thus, 7 may be a potent novel anticancer drug. Molecular docking demonstrated that the O atoms in dimethoxyphenyl side chain of 7 and 8 interacted with amino acid residues of Top2 (α and β types) through two or three hydrogen bonds.