Antitumor Constituents from Anthriscus Sylvestris ( L . ) Hoffm

Anthriscus sylvestris (L.) Hoffm. (Umbelliferae) is a perennial herb that grows in Europe and in parts of North America, Africa, Asia and New Zealand (Jeong, 2007; Oktavia, 2011). The roots of A. sylvestris have been used as antitussive, antipyretic, analgesic, diuretic, and cough remedy in Chinese traditional medicine, and the young aerial part of this plant is used for food (Kozawa et al., 1978a; Wang et al., 1982; Yang et al., 2010). This plant has a lignan named deoxypodophyllotoxin (Noguchi et al., 1940), which is known to have many bioactivities such as antitumor activity (Kozawa et al., 1978b; Ayres et al., 1990; Lim et al., 1999), anti-platelet aggregation activity, antiviral activity, antiproliferative activity, broad insecticidal activity, inhibition of passive cutaneous anaphylaxis reactions (Lin et al., 2004), liver protective action (Kiso et al., 1982), and anti-inflammatory activity (Lee et al., 2004). A. sylvestris contains highly lignans, phenylpropanoids, flavonoids, coumarins, organic acids, and so on (Tozaburo et al., 1979; Liang et al., 1990; Milovanovic et al., 1996; Ikeda et al., 1998; Bos et al., 2002; Koulman et al., 2007). Umbelliferae has high research value of anti-tumor activity. There were many reports. For example, the alcoholic extracts and seed oil of Petroselinum sativum (Umbelliferae) induced cell death in MCF-7 cells (Farshori et al., 2013). In course of searching for anti-tumor agents from medicinal herbs, the MeOH extract of the roots of A. sylvestris was found to be active against human chronic

In course of searching for anti-tumor agents from medicinal herbs, the MeOH extract of the roots of A. sylvestris was found to be active against human chronic School of Life Science and Engineering, Southwest Jiaotong University, Chengdu, China *For correspondence: jianghz10@sina.com, mcy195888@126.com

Antitumor Constituents from Anthriscus Sylvestris (L.) Hoffm
Hui Chen, He-Zhong Jiang*, Yong-Chao Li, Guo-Qing Wei, Yun Geng, Chao-Ying Ma* myelogenous leukemia cell K562 (Lim et al., 1999).In recent years, there is a trend to explore antitumor activities of some plants like A. Sylvestris, which are known for its food and medicinal properties, expected to find some drugs with relatively low toxicity and high activity.Oroxylum indicum (L.) can effectively target ER-negative breast cancer cells to induce apoptosis, without harming normal cells by cancer-specific cytotoxicity (Kumar et al., 2012).Syzygium aromaticum L showed inhibition activity of some cancer cell lines like HeLa (Dwivedi et al., 2011).In addition, some compounds like oleanolic acid and ursolic acid from food and medicinal herbs had relatively low toxicity and antitumor activities (Gayathri et al., 2009;George et al., 2012).In this study, the bioassayguided chemical investigation of the roots of A. sylvestris resulted in the isolation of nine known compounds, and their antitumor activities were tested.

Materials
The roots of A. sylvestris were purchased at Emei Mountain, Sichuan Province, China in October, 2012, and identified by Prof. Liang-Ke Song, Southwest Jiaotong University.The specimen (CE20121210) was deposited at the Specimens laboratory of Southwest Jiaotong University.

General
NMR spectra were recorded on a Bruker AV-400 MHz or an Avance III 600 spectrometer with TMS as an internal standard using CDCl 3 as solvent.
β-Sitosterol: C 29 H 50 O, white needle ccvrystal, was elucidated by comparing with authentic compound by TLC means.
These compounds were elucidated on the basis of spectral data and comparison with published literatures.The structures of compounds 1-9 were showed in Figure 1.DOI:http://dx.doi.org/10.7314/APJCP.2014.15.6.2803Antitumor Constituents from Anthriscus Sylvestris (L.) Hoffm Antitumor activities assay in vitro: The MTT bioassay determines the ability of viable cells to reduce the yellow tetrazolium salt [3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide] (MTT) to blue formazan crystals by mitochondrial enzymes.The in vitro antitumor activities or cytotoxicity of the samples were tested on HepG2 (human hepatocellular carcinoma), MG-63 (human osteosarcoma cells), B16 (melanoma cells) and HeLa (human cervical carcinoma cells) that were cultured on RPMI-1640 medium supplemented with 10% foetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin in 25 cm2 culture flasks at 37°C in humidified atmosphere with 5% CO 2 (Alabsi et al., 2013).The cells were harvested from the culture flasks at the exponential growth phase and resuspended in fresh medium at a cell density of 1×10 5 cells ml−1.The cell suspension was dispensed into a 96-well microplate at 100 μl per well and incubated in humidified atmosphere with 5% CO 2 at 37°C for 48 h, and then treated with the drugs (different polar parts of A. sylvestris and compounds 1, 6-8 and 9) at selected doses.Cell proliferation in the microplate was determined at various treatment intervals with the MTT assay.MTT colorimetric method is a kind of method of detecting the growth and survival of cells.
The antitumor activities of A. sylvestris extract and compounds were evaluated against HepG2 cell line, MG-63 cell line, B16 cell line and HeLa cell line.The results are presented in Table 1.Petroleum ether and chloroform fractions exhibited excellent inhibitory activities with the IC 50 value in the range of 18.25-45.66μg/ml.Ethyl acetate fraction had a weaker activity on B16, while n-butyl alcohol fraction had no inhibition.Among the isolations from the petroleum ether fraction, compound 7 exhibited significant inhibitory activities against the growth of the four tumor cells with the IC 50 value ranging from 12.24-43.25μg/ml.And compound 8 also showed strong inhibitory activities against MG-63, B16 cells.Compound 6 and 9 showed weaker inhibition for HepG2, HeLa.Other compounds were weak cytotoxic agents or inactive.Figure 2-5 shows the relationship with the cell proliferation and different fractions of A. sylvestris extract  Petroleum ether fraction, chloroform fraction, compounds 6, 7, and 9 on the proliferation of HeLa cells in culture (48 h treatment), in which the petroleum ether fraction and compound 7 showed the strongest inhibitory effect (see Figure 2).For HepG2 cells, only the inhibition of compound 9 is not dosage dependent among petroleum ether fraction, chloroform fraction, compounds 6, 7 and 9 (see Figure 3), And control tests were conducted by using different fractions and compounds to detect its inhibition at different concentration for B16 and MG-63 cells.It was found that only compounds 7 and 8 showed remarkable inhibitory activity, and the inhibition is dosage dependent (see Figure 4 and 5).Cell proliferation (% of control) equals the MTT value of extract-treated culture divided by that of control.

Discussion
Among all compounds, only compounds 7, 8 and 9 showed relatively potent antineoplastic activity.Compounds 7, 8 and 9 are all arylnaphthalenes, so it could be supposed that arylnaphthalenes are the key compounds in A. sylvestris against the four cancer cells.The series of these compounds are valuable for further investigation.The structures of compounds 7, 8 and 9 were similar, but the antitumor activities of them differ widely.Compare compound 7 with compound 8, it could be supposed that 7-benzene were very important to the antitumor activities and seemed to be active groups.For compound 7 and 9, different conformations of 8, 8'-H showed different antitumor activities.This information could offer some clues for chemical modification and structure transformation of compounds 7, 8 and 9.The fractions of petroleum ether and chloroform small-polar show strong antitumor activities, but ethyl acetate and n-butyl alcohol fractions are mostly inactive.These results illustrate that petroleum ether fraction of A. sylvestris is the activity component and of which lignans especially arylnaphthalenes could be the most important compounds.
In conclusion, petroleum ether and chloroform fractions of the crude extracts exhibited remarkable inhibitory activities against HepG2 and HeLa cells with the IC 50 value in the range of 18.25-45.66μg/ml.Furthermore, with the guide of antitumor inhibition, nine pure compounds with different antitumor inhibitory activity were isolated and identified.Especially, compound 7 showed remarkable inhibitory activity against tumor cells with the IC 50 value ranging from 12.24-43.25μg/ ml.It is first reported that control tests were conducted by compound 9 to detect its antitumor inhibition.We believe that our results have shown A. sylvestris to be a new source of antitumor.The isolated compounds could be used as lead compounds for drug screening.

Figure
Figure 1.The Structures of Compound 1-9

Figure
Figure 4. Cell Proliferation of Compound 7, 8 with Different Doses Against B16 Cells