A New Phenolic Glycoside from Chamaecyparis obtusa var. breviramea f. crippsii

A new phenolic glycoside, 3-methoxyphenol 1-O-α-L-rhamnopyranosyl-(1→6)-O-β-D-glucopyranoside (1), was isolated from the 90% acetone extract of the branches and leaves of Chamaecyparis obtusa var. breviramea f. crippsii along with another 10 known phenolics 2–11. Their structures were determined mainly by means of MS, 1D- and 2D-NMR data. Cytotoxicities of compounds 3 and 5–11 were tested on BGC-823, Hela and A549 cancer cell lines, the results showed that compound 8 was bioactive and its IC50 values were 6.9, 29.7 and 52.9 μM, respectively.


Introduction
There are six species in the genus Chamaecyparis, which are mainly distributed in North America, Japan, and Taiwan [1]. Chamaecyparis plants have been found to be rich sources of monoterpenes [2], sesquiterpenoids [3], diterpenes [2,4] and lignans [5][6][7], some of which have shown antitumor, antimalarial and antibacterial activities [8][9][10]. Chamaecyparis obtusa (Sieb. et Zucc.) Endl. var. breviramea f. crippsii is a cultivated variety of C. obtusa [11]. According to the literature, no chemical constituents of this plant has been reported until now. As part of serial investigations on the Cupressaceae and in order to seek more novel bioactive compounds, we carried out an extensive chemical study on C. obtusa var. breviramea f. crippsii. In this paper, we report the isolation and structure elucidation of a new phenolic glycoside 1, together with ten other known phenolics 2-11 from the branches and leaves of C. obtusa var. breviramea f. crippsii, in addition to a screening of their cytotoxicity.

Results and Discussion
The n-BuOH fraction of the branches and leaves of C. obtusa var. breviramea f. crippsii was subjected to column chromatographies on silica gel, Sephadex LH-20 and MCI, and preparative HPLC, to afford a new phenolic glycoside 1, together with the 10 known phenolics 2-11, which were identified by comparison of spectra data with the reported literature values (Figure 1).    13 C-NMR spectrum. The IR spectrum of 1 showed absorption bands for OH (3423 cm −1 ) and aromatic (Ph) groups (1596 cm −1 ). Its UV spectrum revealed the presence of aromatic (Ph) groups (200, 220, 273 nm). The 1 H and 13 C spectra (see Table 1) showed the presence of a m-substituted aromatic group (δ C 147.9 (C-1), 124. , and a methoxy (δ c 56.6), which suggested that compound 1 was a phenolic glycoside. The 1 H-and 13 C-NMR data of 1 (see Table 1) were a close match to those of itoside I [12]. In the HMBC spectrum of 1 (Figure 2), the correlation from the anomeric proton 4.72 (d, J = 1.2 Hz, H-1'') to the methine at 67.7 (C-6') indicated that the linkage between the α-L-rhamnopyranose and the β-D-glucopyranose was C-1''→C-6'. The cross peak between δ H 4.86 (d, J = 6.3 Hz, H-1') and δ C 147.9 (C-1) suggested that the substituted site of the β-D-glucopyranose on the phenolic aglycone was C-1'→C-1. The correlation between δ H 3.88 (s, OMe) and δ C 150.8 (C-3) indicated that the OMe was located at C-3. Thus, the structure of 1 was identified as 3-methoxyphenol 1-O-α-L-rhamnopyranosyl-(1→6)-O-β-D-glucopyranoside. In the primary bioactivity test, the methanol extract of this plant showed cytotoxicities on the cancer cell lines A549, BGC-823, Du145 and MDA-MB-231 with IC 50 values of 0.94, 1.07, 0.95 and 0.96 μg/mL, respectively. In order to find the cytotoxic constituents, among the 11 compounds obtained and identified from the n-BuOH fraction of this plant, compounds 3 and 5-11 were tested for cytotoxicity against the Hela, BGC-823 and A549 cancer cell lines, and only compound 8 showed cytotoxicity, with IC 50 values of 6.9, 29.7 and 52.9 μM, respectively.

General
Optical rotations were measured at 17 °C on a Horiba SEAP-300 polarimeter. IR spectra were obtained on a Bio-Red FTS-135 spectrophotometer. UV spectra were measured on a 2401PC spectrophotometer. NMR spectra were recorded on a Bruker AM-400 or DRX-500 spectrometer, using TMS as an internal standard. ESIMS spectra were obtained on a VG Autospec-3000 spectrometer. HPLC was performed using an Agilent 1100 autopurification system equipped with a DAD detector (190-950 nm). Precoated silica gel plates (Meijing, China) were used for TLC. Detection was done by spray plates with 8% anisaldehyde-sulfuric acid, followed by heating.

Plant Material
Branches and leaves of C. obtusa var. breviramea f. crippsii were collected from Kunming Botany Garden, Yunnan Province, People's Republic of China, in August 2010. It was identified by Associated Prof. Zhong Shu Yue from Kunming Institute of Botany, Chinese Academy of Sciences.

Cytotoxicity Activity Assay
The human tumor cell lines Du145 (prostate carcinoma) and MDA-MB-231 (breast carcinoma), BGC-823 (gastric carcinoma), Hela (cervical carcinoma) and A549 (non-small cell lung carcinoma) were bought from the Chinese Academy of Medical Sciences. The cytotoxicity assays were performed according to a published procedure [23].

Conclusions
This work was part of a series of investigations on antitumor compounds from Cupressaceae plants. Compound 1 was found to be a new phenolic glycoside, and the other ten compounds were found for the first time in C. obtusa var. breviramea f. crippsii. The random cytotoxic screening results showed that the significant cytotoxicity of the methanol extract was not caused by these simple phenolics isolated from the n-BuOH fraction, so the sesquiterpenes, diterpenes and podophyllotoxin derivatives isolated from the petroleum ether and EtOAc fraction might be responsible for the strong cytotoxicity of the crude methanol extract, which will be extensively investigated and reported in future work.