Cytotoxic Properties of C17 Polyacetylenes from the Fresh Roots of Panax ginseng on Human Epithelial Ovarian Cancer Cells

Although C17 polyacetylenes from Panax ginseng exhibit cytotoxic properties against various tumor cells, there have been few experiments on epithelial ovarian carcinoma cells. This study aimed to investigate the cytotoxic effects of C17 polyacetylenes from P. ginseng against ovarian cancer cell lines. Four unreported (1–4) and fifteen known (5–19) C17 polyacetylenes were obtained from the roots of P. ginseng using repeated chromatography (open column, MPLC, and preparative HPLC). The chemical structures of all the compounds were determined by analyzing their spectroscopic data (NMR, IR, and optical rotation) and HR-MS. The structures of new polyacetylenes were elucidated as (3S,8S,9R,10R)-(-)-heptadeca-9,10-epoxy-4,6-diyne-3,8-diyl diacetate (1), (3S,8S,9R,10R)-(−)-heptadeca-1-en-9,10-epoxy-4,6-diyne-3,8-diyl diacetate (2), (−)-haptadeca-9,10-epoxy-8-methoxy-4,6-diyne-3,11-diol (3), and (3R,9R,10R)-(+)-3-acetoxy-9,10-dihydroxyheptadeca-1-en-4,6-diyne (4), named ginsenoynes O, P, and Q, and 3-acetyl panaxytriol, respectively. Subsequently, in vitro experiments on A2780 and SKOV3 human epithelial ovarian carcinoma cells were performed to assess the cytotoxic properties of the isolates. Among the isolates, panaquinquecol 4 (15) exhibited the most remarkable cytotoxic effects on both human ovarian cancer cells A2780 (IC50 value of 7.60 μM) and SKOV3 (IC50 value of 27.53 μM). Therefore, C17 polyacetylenes derived from P. ginseng may warrant further investigation for their therapeutic potential in epithelial ovarian cancer.


Introduction
Ovarian cancer, identified as a group of malignant neoplasms, poses a lethal threat to women worldwide [1]. It is the third most common gynecological malignancy, with around 31 million officially recorded cases and 20 million deaths in 2020 [2]. Even the most frequent type of ovarian cancer, epithelial cancer, has a poor prognosis due to the asymptomatic characteristics of its early stages, with a 5-year relative survival rate of only 30% [3,4]. Currently, the common treatment procedure is to first have cytoreductive surgery and to then proceed with platinum-based chemotherapy [5]. However, platinum-based drugs damage normal cells substantially, which can result in organ dysfunction syndrome [6]. Platinum resistance in the treatment of ovarian cancer is also the main factor decreasing survival rate and increasing the risk of relapse [7]. Therefore, novel drugs are urgently required for the effective treatment of ovarian cancer.
Numerous anti-cancer agents derived from traditional medicines have been developed as alternatives to platinum-based chemotherapy with reduced toxic side effects and resistance [8]. Panax ginseng Meyer (Araliaceae) and its constituents have anti-tumor effects and resistance [8]. Panax ginseng Meyer (Araliaceae) and its constituents have anti-tumor effects in various cancer cell lines, including ovarian cancer [9]. The unique saponin, 20-(S)ginsenoside Rg3, exhibited anti-tumor effects in ovarian cancer cell lines (SKOV3 and A2780) and in vivo models by inhibiting the Warburg effect caused by reduced lactate synthesis or glucose metabolism [10]. In addition to ginsenosides, a recent review has indicated that C17 polyacetylenes, such as panaxydiol and panaxytriol in Panax species, effectively suppress cell proliferation in several cancer cell lines, inducing cell cycle arrest at multi-phases [11]. Although C17 polyacetylenes have potential cytotoxic properties and are abundant among the polyacetylenes found in P. ginseng, few investigations on their activity in human ovarian cancer have been conducted. In our preliminary study, we identified the cytotoxic property of polyacetylene-enriched extracts from the fresh roots of P. ginseng on epithelial ovarian cancer cells. Therefore, the present research aimed to identify C17 polyacetylenes in P. ginseng with cytotoxic effects on human ovarian cancer cell lines.

Structure Elucidation of Isolated Compounds
Compound 1 was isolated as yellow oil. The chemical formula was determined to be C21H30O5 by measuring HR-DART-MS (m/z 380.23495 [M + NH4] + ; calcd for C21H34NO5, 380.24370) (Supplementary Materials Figure S1). The IR spectroscopy exhibited absorption bands at 1380, 1746, and 2250 cm −1 , indicating that 1 has a conjugated triple bond and an
Compound 3 was isolated as yellow oil, and its chemical formula was identified as C 18 (Table 1; Figures S16 and S17). The connections from C-1 to C-3 and from C-8 to C-17 in 3 were confimed using an analysis of the COSY data ( Figure S18). The position of the methoxy group was determined to be C-8 using an analysis of the HMBC spectrum ( Figure 3 and Figure S19). The stereochemistry of the epoxide ring was determined to be cis configuration using the J value analysis ( 3 J H9-H10 = 4.5 Hz). Although the NOESY spectrum exhibited correlations between H-8/H-11 and H-9/H-10 ( Figure S20), the relative energies of all conformers exhibited approximate values in the conformer search for 3. As a highly oxygenated derivative with scarce amount, we could not determine the absolute configuration of 3. Thus, the structure of 3 was identified as (−)-haptadeca-9,10-epoxy-8methoxy-4,6-diyne-3,11-diol, named ginsenoyne Q.

Discussion
Polyacetylenes, commonly found as secondary metabolites in plants, have one or more carbon-carbon triple bonds in their structure. However, because there are various secondary metabolic pathways involved in the biosynthesis of polyacetylene, these compounds are sometimes highly oxidized and may contain a variety of substituents. Ginseng (Panax spp.) is one of the most well-known plants that contains a significant amount of polyacetylene in addition to ginsenosides [28]. For many years, researchers have become more interested in discovering novel ginsenosides as well as polyacetylenes [29]. In this study, four newly reported C17 polyacetylenes-ginsenoynes O, P, and Q (1-3), and 3-acetyl panaxytriol (4)-were isolated from the fresh roots of P. ginseng. Although the presence of panaquinquecol 4 (15) and (3R,8E,10S)-8-heptadecene-4,6-diyne-3,10-diol (18) was reported in Panax quinquefolium and Oplopanax horridus, it was reported for the first time in P. ginseng in this study.

Discussion
Polyacetylenes, commonly found as secondary metabolites in plants, have one or more carbon-carbon triple bonds in their structure. However, because there are various secondary metabolic pathways involved in the biosynthesis of polyacetylene, these compounds are sometimes highly oxidized and may contain a variety of substituents. Ginseng (Panax spp.) is one of the most well-known plants that contains a significant amount of polyacetylene in addition to ginsenosides [28]. For many years, researchers have become more interested in discovering novel ginsenosides as well as polyacetylenes [29]. In this study, four newly reported C17 polyacetylenes-ginsenoynes O, P, and Q (1-3), and 3-acetyl panaxytriol (4)-were isolated from the fresh roots of P. ginseng. Although the presence of panaquinquecol 4 (15) and (3R,8E,10S)-8-heptadecene-4,6-diyne-3,10-diol (18) was reported in Panax quinquefolium and Oplopanax horridus, it was reported for the first time in P. ginseng in this study.
In this study, the polyacetylenes were more effective against the A2780 cells compared to SKOV3 cells. These results indicated that the p53 pathway is likely associated with compound-induced cell death, and SKOV3 cells without p53 activity seem to be less responsive against the compounds. Nevertheless, panaquinquecol 4 (15) exhibited significant cytotoxicity in both A2780 and SKOV3 cells (7.60 ± 1.33 and 27.53 ± 1.22 µM, respectively). It has been reported that panaquinquecol 4 (15), which was first isolated from P. quinquefolium, has strong cytotoxicity against human leukemia cells (L1210), with an IC 50 value 20 times lower than panaquinquecol 5, the C 14 polyacetylene [25]. However, the cytotoxic effects of panaquinquecol 4 (15) against other carcinoma cell lines and their mechanisms are not yet fully understood. Therefore, further investigation into this compound is required.

Plant Materials
The fresh roots of Panax ginseng C.A. Meyer (Araliaceae) were purchased from a local market (Chungcheongnam-do, Korea) and authenticated by Prof. Dae Sik Jang. A voucher specimen (PAGI-2019) of the raw material was deposited in the Laboratory of Natural Product Medicine, College of Pharmacy, Kyung Hee University, Seoul, Republic of Korea.

Computational Chemistry
At the Hartree-Fock level of theory, ab initio calculations for 1 were performed. Using Gaussian 16 software (version G16 C.01; Wallingford, CT, USA) and modified versions of the reported methods, all geometries and conformers of 1 were optimized using an HF/3-21* level of theory [15]. The cumulative Boltzmann distribution (up to 75%, Table  S1) was used among generated conformers to select the conformers of 1 for calculating the specific rotation. Specific rotations of single conformers at 589 nm were calculated using a CPCM/HF/3-21* level of theory (chloroform). The final specific rotation value of 1 was calculated using the relative Boltzmann weighting of conformers.

Deacetylation of Compound 4
Acid hydrolysis was performed to confirm the absolute configuration of 4 using the modified procedures previously described [34]. Compound 4 (1.0 mg) and 1N HCl (0.01 mL) in MeOH (1.0 mL) were stirred at room temperature for 24 h. After the reaction, it was partitioned with EtOAc and water to obtain a product. The EtOAc fraction was purified directly using HPLC under a gradient system [A: water, B: methanol, 70% B, 35 min].

Cell Culture and MTT Assay
A2780 and SKOV3 human ovarian cancer cell lines and RAW264.7 macrophages were obtained from American Type Culture Collection (ATCC). The cell culture and MTT assay were performed using the method described previously [35,36].
Experimental and calculated specific rotation data of 1 and 2; Table S2. The cytotoxicity of compounds 6, 11, and 15 isolated from P. ginseng in RAW264.7 macrophages.

Conflicts of Interest:
The authors declare no conflict of interest.