New Sesquiterpene Glycosides from the Flowers of Aster koraiensis and Their Inhibition Activities on EGF- and TPA-Induced Cell Transformation

In total, four new eudesmane-type sesquiterpene glycosides, askoseosides A–D (1–4), and 18 known compounds (5–22) were isolated from the flowers of Aster koraiensis via chromatographic techniques. Chemical structures of the isolated compounds were identified by spectroscopic/spectrometric methods, including NMR and HRESIMS, and the absolute configuration of the new compounds (1 and 2) was performed by electronic circular dichroism (ECD) studies. Further, the anticancer activities of the isolated compounds (1–22) were evaluated using the epidermal growth factor (EGF)-induced as well as the 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced cell transformation assay. Among the 22 compounds, compounds 4, 9, 11, 13–15, 17, 18, and 22 significantly inhibited both EGF- and TPA-induced colony growth. In particular, askoseoside D (4, EGF: 57.8%; TPA: 67.1%), apigenin (9, EGF: 88.6%; TPA: 80.2%), apigenin-7-O-β-d-glucuronopyranoside (14, EGF: 79.2%; TPA: 70.7%), and 1-(3′,4′-dihydroxycinnamoyl) cyclopentane-2,3-diol (22, EGF: 60.0%; TPA: 72.1%) showed higher potent activities.


Introduction
Aster koraiensis Nakai (syn. Asteromoea koraiensis and Gymnaster koraiensis), belonging to the family Compositae (Asteraceae), is an endemic plant in the Republic of Korea distributed throughout the Korean peninsula. The plant presents beautiful pale purple flowers, which are used as tea and the young shoots are used as a food ingredient. Traditionally, extracts of the aerial parts and roots of A. koraiensis are used to treat pertussis, pneumonia, and chronic bronchitis [1,2]. Further, the extracts have also been reported to be anti-diabetic [3,4], antinociceptive [5], liver protective [6], and anti-inflammatory [7] activities. Several phytochemical studies have reported the presence and biological effects of sesquiterpenoids, polyacetylenes, flavonoids, caffeoylquinic acids, and saponins in A. koraiensis [2,[8][9][10]. Recently, it has been reported that astersaponin I, saponin isolated from the aerial parts of this plant, inhibits the SARS-CoV-2 infection [11]. Most of these studies have been conducted on the aerial parts, roots, and leaves of this plant, and, currently, research on components and physiological activities specific to the flowers of this plant is relatively lacking compared to that of other parts of the plant. In the case of Compositae plants, since the flower parts are often used for edible or medicinal purposes, many studies on the components and efficacy specific to the flower part have been conducted [12][13][14][15][16][17][18][19], but studies on the flower of this plant have not been sufficiently conducted. Therefore, it is necessary to study the components of the flower of this herb and the efficacy of these components.
Cancer is a major cause of death in humans, worldwide, and approximately one in five people develop cancer during their lifetime [20]. As a result of continuous research over the past decades to discover new anticancer compounds from various natural products, such as plants, and to identify their anticancer properties, about 25% of all anticancer drugs approved in 1981 and 2019 were related to natural products. As such, natural products are a rich source of numerous bioactive compounds that have potential as therapeutic agents for various diseases, including cancer [21]. In addition, natural compounds are less toxic and effective in cancer prevention and treatment [22].
As part of the research to discover new bioactive substances of plants distributed in Korea, the flower extract of A. koraiensis, which showed a significant anti-carcinogenic effect through our preliminary screening, was selected as the target plant for this study. As mentioned above, this plant is endemic to Korea, but little is known about its cancer-preventive constituents, especially in its flowers. The anti-carcinogenic activity in this study was evaluated by the epidermal growth factor (EGF)-induced and 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced cell transformation assays. Neoplastic cell transformation is one of the basic mechanisms of tumorigenesis, and various cell transformations can be induced by carcinogens, such as EGF and TPA [23]. EGF signaling is known to stimulate cell proliferation and survival in several types of human cancer cells [24][25][26]. In addition, treatment with TPA, a skin tumor promoter, induces transformation and forms colonies in soft agar [27]. Therefore, substances that inhibit cell transformation by these carcinogens, such as EGF and TPA, can be considered to have anti-carcinogenic efficacy.
In this study, four new eudesmane-type sesquiterpenes (1−4), along with 18 compounds (5−22) were isolated from the flowers of A. koraiensis via chromatographic separation methods and their anti-carcinogenic activities were evaluated by EGF-and TPAinduced cell transformation assay.

Effect of Isolated Compounds on EGF-and TPA-Induced Cell Transformation
The cell transformation soft agar assay has been used in evaluating the efficacy of various compounds for development and screening of new anticancer agents by quantifying the proliferation of anchorage-independent cells [26]. To identify the biological effects of the isolated compounds (1-22), we performed the EGF-and TPA-induced cell transformation assay using the mouse epithelial cell line, JB6 Cl41 (Table 3 and Figure 6). JB6 CI41 cells are widely used to study the molecular mechanisms of tumor promotion and anti-tumor drugs. In addition, both TPA and EGF are well-known tumor promoters that are useful for studying malignant cell transformation [26]. Each compound was used at a concentration of 50 µM and the percentage growth inhibition of cells was determined by comparison with that of the controls (treatment with EGF or TPA alone). Among the 22 compounds, compounds 2, 4, 9, 10-15, 17, 18, and 22 inhibited EGF-induced colony growth, while compounds 1, 3-5, 6, 9, 11, 13, 14-18, and 22 inhibited TPA-induced colony growth by more than 50%, respectively (Table 3). Further, compounds 4, 9, 11, 13-15, 17,  18, and 22 displayed significant inhibition of both EGF-and TPA-induced colony growth (Figure 6), of which askoseoside D (4, EGF: 57.8%; TPA: 67.1%), apigenin (9, EGF: 88.6%; TPA: 80.2%), apigenin-7-O-β-D-glucuronopyranoside (14, EGF: 79.2%; TPA: 70.7%), and 1-(3 ,4 -dihydroxycinnamoyl) cyclopentane-2,3-diol (22, EGF: 60.0%; TPA: 72.1%) displayed higher potent activities than that of others. We demonstrated that the isolated compounds significantly inhibited EGF-and TPA-induced cell transformation. These results indicate that constituents of the flowers of A. koraiensis could exert anticancer effects by preventing EGF-and TPA-induced tumorigensesis. In previous studies, flavonoids, apigenin (9) and its derivatives, isolated from the flowers of A. koraiensis showed cytotoxicity to several cancer cells, A549, SK-OV-3, SK-MEL-2, and HCT15 [43]. Apigenin (9) and its derivatives are well-known natural bioactive substances obtained from many plant sources and have been reported to have anticancer, antidiabetic, antioxidant, and antiviral effects. In particular, these compounds have been reported to exert a broad-spectrum of anticancer effects against several types of cancer, such as liver, lung, breast, colorectal, and prostate cancers [44]. Polyacetylenes isolated from the roots of this plant, including gymnasterkoreayne B (19) and gymnasterkoreayne C (20), showed significant cytotoxicity to L1210 tumor cells [9]. On the other hand, gymnasterkoreayne B (19), a major component isolated from A. koraiensis, has been reported to have an antioxidant effect [45], and also gymnasterkoreayne E (18) and gymnasterkoreayne B (19) isolated from the aerial parts of this plant have been reported to have cholesterol modulatory activity [32]. Table 3. Inhibitory activities of compounds on EGF-and TPA-induced cell transformation.

Compounds
Inhibitory Activity % (EGF) a Inhibitory Activity % (TPA) b    Among the compounds tested in this study, apigenin (9) and apigenin-7-O-β-Dglucuronopyranoside (14) showed potent anti-carcinogenic effects, and other flavonoids (10-13 and 15) also showed significant effects. Further, polyacetylenes (16-18) showed significant anti-carcinogenic effects. These results suggest that various components of A. koraiensis may have both cytotoxic activities to cancer cells and anti-carcinogenic effects. Therefore, we suggest that the flower of this plant and its active compounds have potential as anticancer agents or chemo-preventive agents against cancer.

Cytotoxicity of Compounds on NHDF Cell
We determined the cytotoxicity of the representative compounds (9, 14, and 22) in different concentrations, ranging from 0 to 50 µM in a normal cell line NHDF, using the WST-8 assay. The new compound 4 could not be tested due to an insufficient amount available to be evaluated. As shown in Figure 7, after 48 h of treatment with 50 µM of compounds 9, 14, and 22, NHDF cell viabilities were 90.4%, 99.8%, and 95.4%, respectively. The results indicated that the cell viability of the NHDF was not significantly inhibited, and was maintained above 90% at a concentration of 50 µM for each compound during the 48 h. Compared to 5-Fu, an approved chemotherapeutic agent used as a positive control, it was confirmed that the compounds had no effect on the cell viability of NHDF. compounds 9, 14, and 22, NHDF cell viabilities were 90.4%, 99.8%, and 95.4%, respectively. The results indicated that the cell viability of the NHDF was not significantly inhibited, and was maintained above 90% at a concentration of 50 µM for each compound during the 48 h. Compared to 5-Fu, an approved chemotherapeutic agent used as a positive control, it was confirmed that the compounds had no effect on the cell viability of NHDF.

Plant Material
Dried flowers of A. koraiensis were obtained and identified from the National Institute of Forest Science in 2019. A voucher specimen (No. Gyko- ) was deposited at the Herbal Medicine Resources Research Center, KIOM, Republic of Korea.

Extraction and Isolation
The dried flowers of A. koraiensis (129.0 g) were ground and extracted with 70% EtOH (3 L × 3 times), and then evaporated to obtain the total extract (47.2 g, 36.6%). A total of 45 g of the extract was used for isolation without partitioning.

Acid Hydrolysis and Sugar Identification
Sugar units of new compounds 1−4 (each 0.5 mg) were confirmed according to the method of Tanaka et al. [46,47]. The standards (L-glucose, D-glucose, L-rhamnose, and D-rhamnose; each 1.0 mg, Merck) and hydrolyzed compounds 1−4 were reacted and then analyzed using a UPLC system. A Waters CSH C 18 analytical column with 30% acetonitrile isocratic elution (0.05% formic acid in D.W./acetonitrile, 0.3 mL/min, 10 min, 250 nm) was carried out for analysis. Standard derivatives were recorded at t R 4.056 min (L-glucose derivative), t R 4.427 min (D-glucose derivative), t R 3.950 min (D-rhamnose derivative), and t R 7.307 min (L-rhamnose derivative).

Computational Methods
Conformer distribution, optimization, and ECD analysis were performed, as described previously [48]. All conformers proposed in the study were found using the Spartan'14 (Wave-function, Inc., Irvine, CA, USA). The conformers were subjected to geometry optimization using the Gaussian'09 (Gaussian, Inc., Wallingford, CT, USA) in the DFT [B3LYP functional/6-31+G(d,p) basis set] level, and ECD calculations were performed at the TDDFT(CAM-B3LYP/SVP basis set) level with a CPCM solvent model in MeOH.

WST-8 Assay
To estimate cytotoxicity of compounds, normal human dermal fibroblast (NHDF; ATCC) cells (5000 cells/well) were seeded into each well of 96-well plates. After 24 h incubation, the cells were treated with various concentration of the representative compounds 9, 14, 22, and 5-fluorouracil (5-Fu; Sigma Aldrich, St. Louis, MO, USA) for 48 h. The cytotoxicity of each compound was measured using Quanti-MAX WST-8 Cell Viability Assay Kit reagent (Biomax, Seoul, Republic of Korea). According to the instructions, the absorbance was measured at 450 nm using the Multiskan SkyHigh Spectrophotometer (Thermo Scientific, Vantaa, Finland).

Conclusions
In the current study, four new compounds (1)(2)(3)(4) were isolated from the flowers of A. koraiensis and their structures were identified through spectroscopic studies. The anticancer activity of a total of 22 compounds  isolated in this study was evaluated by cell transformation assay, and most of them showed significant anti-carcinogenic activity. In particular, four compounds, askoseoside D (4), apigenin (9), apigenin-7-O-β-Dglucuronopyranoside (14), and 1-(3 ,4 -dihydroxycinnamoyl) cyclopentane-2,3-diol (22), including new compound (4), showed higher activity than other compounds. In addition, compounds 9, 14, and 22 did not exhibit any toxic effects in the cell viability assay for NHDF, which is a normal skin cell line. This study not only expands the chemical composition of A. koraiensis, but also provides new information on their physiological activities. In addition, this study suggests the potential value of this plant and its active compounds as natural anticancer agents.