Compounds from Dryopteris Fragrans (L.) Schott with Cytotoxic Activity

One new coumarin, dryofracoumarin A (1), and eight known compounds 2–9 were isolated from Dryopteris fragrans (L.) Schott. Their structures were established on the basis of extensive spectroscopic data analyses and comparison with reported spectroscopic data. The new compound 1 was determined to be 8-hydroxyl-4-isopropyl-7-methyl-6-methyl-2H-benzopyran-2-one. Two dimers, trans- and cis-3-(3,4-dimethoxyphen-yl)-4-[(E)-3,4-dimethoxystyryl]cyclohex-1-ene (compounds 8 and 9), were isolated from the Dryopteris genus for the first time. The other six were esculetin (2), isoscopoletin (3), methylphlorbutyrophenone (4), aspidinol (5), albicanol (6) and (E)-4-(3,4-dimethoxyphen-yl)but-3-en-1-ol (7). All compounds were evaluated for their cytotoxic effects by the MTT assay. Compounds 2, 3, 8 and 9 showed significantly cytotoxic effects against three cell lines (A549, MCF7 and HepG2), 1 and 5 against two cell lines (A549 and MCF7), and 6 against one cell line (MCF7). Their IC50 values ranged between 2.73 ± 0.86 μM and 24.14 ± 3.12 μM. These active compounds might be promising lead compounds for the treatment of cancer.


Introduction
Traditional Chinese Medicines (TCMs), are commonly used as representative alternative and complementary medicines for the treatment of cancer in Asian countries [1]. The current treatment modality for patients with lung, breast or other cancer is chemotherapy based on taxane, a famous and efficient drug isolated from the bark of the Pacific yew tree [2][3][4]. However, its efficacy is frequently attenuated due to drug resistance and side effects [5], so it is urgent to identify new targets to treat cancer from TCMs.
Dryopteris fragrans (L.) Schott (Chinese name: Xiang-Lin-Mao-Jue) is a deciduous perennial herb of the Dryopteris genus, which grows mainly in small communities on the talc slopes of the alpine region and the lava cracks of volcanoes ( Figure 1). It is distributed in many countries, especially in Wu-Da-Lian-Chi, Heilongjiang Province, Northeast China [6]. D. fragrans was used for the treatment of skin diseases, especially on treatment of psoriasis, arthritis, rash, dermatitis and barbiers [7,8].
Previous investigations also showed that its extracts have antimicrobial properties and anticancer activity [9,10]. Many constituents from D. fragrans have been isolated, including sesquiterpenes, phloroglucins, phenolic glycosides, itosterols and essential oils. Among them, more than 10 compounds from have anticancer activities [11][12][13][14][15]. For example, aspidin BB and albicanol suppressed in vivo two-stage carcinogenesis on mouse skin [10]. However, there are no reports available on the cytotoxic activity of compounds from the ethanolic extract of D. fragrans. In this study, compounds with significant cytotoxic activity on A549, MCF7 and HepG2 cells (in vitro lung, breast and liver cancer models) were reported.

Effects of Compounds on Cytotoxic Activity
Lung cancer in males or breast cancer in females are the most frequently diagnosed cancers and the leading cause of cancer death in both developed and developing countries [17,18]. All compounds were investigated for their anticancer activity in vitro against A549 (human lung cancer), MCF7 (human breast cancer), along with HepG2 [19] (human liver cancer) cancer cell lines using pseudolaric acid B as standard [20]. Pseudolaric acid B was isolated from the ethanolic extract of the root bark of Pseudolarix kaempferi with the purity of 99.3% by Dr. Qinshi Zhao (Kunming Institute of Botany, Chinese Academy of Sciences). IC 50 values, the concentration of the test compounds inhibiting 50% of the cell growth at 48 h, was calculated by Reed and Muench's method [21]. Among all the compounds, 2, 3, 8 and 9 showed promising activity against three cancer cell lines at below 30 μM concentration, with 1 and 5 against two lines (A549 and MCF7) and 6 against one line (MCF7) ( Table 2).

Extraction and Isolation
Air-dried, powdered whole plants of Dryopteris fragrans (L.) Schott (3 kg) were extracted three times with 95% ethanol at room temperature. After removal of the solvent by evaporation, the residue (240 g) was suspended in H 2 O and partitioned with EtOAc. The EtOAc fraction (135 g) was subjected to silica gel column chromatography with a gradient elution system of petroleum ether-acetone
As great attentions have been paid to the development of novel anticancer molecules from TCMs, the anticancer activities of all isolated compounds were tested by the MTT assay. Compounds 2, 3, 8 and 9 showed significantly cytotoxic effects against three human cancer cell lines including A549, MCF7 and HepG2, compounds 1 and 5 against two cancer cell lines (A549 and MCF7), and compound 6 against one cancer cell line (MCF7). Consistent with previous studies, the cytotoxic effects of compounds 2, 3, 5, 6 were demonstrated [30][31][32][33][34] again. Importantly, cytotoxic effects of compounds 1, 8, 9 were provided for first time. All these active compounds might be promising lead compounds for the treatment of cancer.