Guaianolide Sesquiterpenes With Significant Antiproliferative Activities From the Leaves of Artemisia argyi

Four new guaiane-type sesquiterpenes, argyin H–K (1–4), and two known analogues (5 and 6) were isolated from the leaves of Artemisia argyi Lévl et Vant. The new compounds were characterized by the basic analysis of the spectroscopic data obtained (1H NMR, 13C NMR, HMBC, and NOESY experiments), and their absolute configurations were determined by empirical approaches, combined with the exciton chirality method and electronic circular dichroism calculations. To further understand the antitumor effects of A. argyi, the antiproliferative activities of these compounds against A549, MCF-7, and HepG2 cell lines were tested in vitro using CCK-8 assays. The results showed that these compounds had significant antiproliferative effects on MCF-7, with IC50 values of 15.13–18.63 μM, which were superior to that of oxaliplatin (i.e., IC50 22.20 μM).


INTRODUCTION
Artemisia argyi Levl. et Vant, an important species in the genus of Compositae, is distributed in China, Japan, Korea, Far East of Russia, etc. (Doh et al., 2016;Ozek et al., 2014). A. argyi often appears in people's life in various forms. As a common medicinal resource for curing eczema, diarrhea, hemostasis, and irregular menstruation in Chinese history, it has advantages of low price, easy access, wide application, and low toxicity (Li et al., 2018;Li et al., 2018;Wang et al., 2013). Pharmacological studies have shown that A. argyi is rich in terpenoids, flavonoids, and tannins (Zan et al., 2012;Tan and Jia, 1992;Dahae et al., 2018). In vitro experiments indicated that terpenoids exhibited various promising biological activities, including antibacterial, antivirus, and antitumor activities (Merfort, 2011;Zhang et al., 2005). In an effort to explore the structural diversity and biological activities of sesquiterpenes in A. argyi, a comprehensive phytochemical investigation was carried out, and all the compounds isolated were evaluated for their antiproliferative activities in A549, MCF-7, and HepG2 cell lines.

Chemistry
Compound 1 gave a molecular formula of C 20 H 28 O 7 (HR-ESI-MS m/z 403.1728 [M + Na] + , calcd for 403.1733), which suggested seven unsaturation degrees. The 1 H NMR signals at δ 6.04 (1H, d, J 3.3 Hz) and 5.68 (1H, d, J 3.3 Hz) indicated an exocyclic methylene group. In the 13 C NMR spectrum, apart from five characteristic carbon signals for a 3methylbutyryl group, 15 carbon resonances were observed and indicated a sesquiterpene structure.
The trans disposition of H-5 and H-6 was deduced from the large vicinal coupling constants (J 5,6 11.6 Hz) (Wang et al., 2014). The strong NOE correlation between H-6/H 3 -15 and H-8, H 3 -15/H-3 revealed their cis relationship; H-5/H-7, C 1 -OH, and   C 4 -OH indicated that they have co-facial orientations and assigned as α-oriented ( Figure 1B). The overall pattern of the experimentally CD of one well matched the calculated ECD curve, which elucidated the absolute configuration of one was 1S, 3R, 4S, 5R, 6S, 7R, 8S, and named argyin H ( Figure 3A). The exciton chirality method in CD spectra is a very useful method to determine the absolute configuration of organic molecules. It can be used to determine not only the spatial relationship between two identical chromophores but also their absolute configuration according to the interaction of two different conjugated systems (Ying et al., 1988;Luo et al., 2011). Due to the existences of two conjugate systems in the isolated compounds, the absolute configurations could be further studied by the exciton chirality method. The ECD spectrum of one showed negative exciton chirality around the UV maximum of 234 nm; the anticlockwise array of two coupling chromophores in space ( Figure 4A) and absolute configurations of two bridgehead stereogenic centers (6S, 8S) were thus determined. This result was confirmed by the unambiguous match of its experimental and calculated ECD curves.

Cytotoxic Activity
In order to test the cytotoxic activities of compounds 1-6 isolated from A. argyi, CCK-8 assay was used to evaluate the inhibitory effects against HepG2, A549, and MCF-7 cell lines ( Table 2). These six guaianolide sesquiterpenes showed  Each value represents the mean ± SD of three independent experiments.
Frontiers in Chemistry | www.frontiersin.org June 2021 | Volume 9 | Article 698700 5 strong inhibitory effect in a dose-dependent manner against three cell lines, and the most sensitive cell line is MCF-7, in which the IC 50 of 1-6 (15.13-21.62 μM) was lower than that of the positive control, oxaliplatin (22.20 μM). It can be seen that the angeloyloxy substitution could strengthen the inhibitory effects compared with others.

CONCLUSION
In our continuing investigation on A. argyi, four undescribed guaiane-type sesquiterpenes were isolated from the Artemisia argyi Lévl. et Van. The antiproliferative activities of these compounds against A549, MCF-7, and HepG2 cell lines were tested in vitro using CCK-8 assays. Notably, these compounds could induce more cell death than positive control (oxaliplatin). Among them, compounds 3 and 4 with angeloyloxy substitution displayed the most potent antiproliferative effects, which could offer a promising lead structure with anticancer activity.
The CH 2 Cl 2 extract was chromatographed on silica gel, eluting with CH 2 Cl 2 -CH 3 OH to obtain Fr. A 1 -Fr. A 6 (100:2 to 1:1). Combinations (Fr. A 2 and Fr. A 3 ) were then applied on reversed-phase ODS washing with aqueous MeOH the statistical analysis. WM and YS wrote the first draft of the manuscript. WM, DM, YZ, and GB wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.

FUNDING
This work was supported by the National Natural Science Foundation of China (Grant No. 81573694), and the National Drug Standard Improvement Project in 2021 (General Technology) (Grant No. 2021Z01). The project was also sponsored by "Liaoning BaiQianWan Talents Program in 2018."