New Benzoyl Glucosides and Cytotoxic Pterosin Sesquiterpenes from Pteris ensiformis Burm.

Three new compounds: 2R,3R-pterosin L 3-O-β-D-glucopyranoside (1), β-D-xylopyranosyl(1→2)-7-O-benzoyl-β-D-glucopyranoside (2) and 4-O-benzoyl-β-D-xylo-pyranosyl(1→2)-7-O-benzoyl-β-D-glucopyranoside (3), together with nine known compounds, were isolated from the ethyl acetate extract of Pteris ensiformis. 5-[2-Hydroxyethylidene]-2(5H)-furanone (4), which had been synthesized, was isolated from natural sources for the first time. The structures of all isolated compounds were determined on the basis of mass and spectroscopic evidence. Compound 1 and pterosin B (5) show cytotoxicity against HL 60 cells (human leukemia) with the IC50 values of 3.7 and 8.7 μg/mL, respectively.


Introduction
For hundreds of years Pteris ensiformis Burm. (Pteridaceae) has been one of the most popular constituents of herbal beverages in Taiwan [1]. Some medium polarity extracts of different Pteris species show effective antitumor and antibacterial activity [2]. Chemical investigation of more than 30 species of the Pteridaceae has been reported [3]. Phytochemical investigations on the Pteris genus led to isolation of various phenolic compounds [4], flavonol glycosides [5][6], kauranes [7], and pterosinsesquiterpenes [8][9]. In bioactivity reports, for example, pterosin B, one of the main pterosins found in the genus Pteris, exhibits potent cytotoxic activity against HL 60 (human leukemia) cells [10][11].
The assignments of 13 C-NMR were confirmed by 2D NMR techniques. The 1 H-detected heteronuclear multiple bond connectivity (HMBC) spectrum showed the correlations of H-12/C-6, H-12/C-4, H-15/C-6, H-15/C-8, H-14/C-13, H-13/C-6, H-4/C-9, H-3/C-9, H-3/C-2, H-10/C-1, H-10/C-2, and H-10/C-11, which confirmed 1 as a pterosin-type sesquiterpene. In addition, the HMBC correlation of H-3/C-1' corresponds to the linkage between the pterosin moiety and the βglucopyranose (d, J = 7.6 Hz) (Fig. 2). The acid hydrolysis of 1 gave the aglycone and D-glucopyranose, which was confirmed by comparison of the 13 C-NMR spectra. The absolute configuration at C-2 and C-3 was deduced from the similarity of the CD spectrum {[θ] 20 330 +19800 (MeOH)} of 2R,3R-pterosin L [13]. Additionally, this configuration (2R,3R) was confirmed by NOESY correlations of H-10/H-3 and H-11/H-1' (Figure 2). Thus, the structure of compound 1, 2R,3R-pterosin L 3-O-β-D-glucopyranoside, was determined.  (Table 1) revealed the presence of glucopyranosyl and xylopyranosyl moieties [15]. The β-anomeric configuration for both glucopyranose and xlyopyranose were determined from a large 3 J H1'H2' coupling constant (7.6 Hz). Besides the signals of sugars, the 1 H-NMR spectrum of 2 showed a set of proton signals due to a monosubstituted aromatic ring at δ H   8.  Table 1). The β-D-glucopyranose was found to be connected to C-7 of the benzoyl moiety by observation of the HMBC correlation between  δ H 5.84 (Glc-H-1) and  δ C 166.3 (benzoyl-C-7). The structure of the sugar chain was assigned by a combination of COSY, HMQC, and HMBC experiments. Starting from the anomeric protons of each sugar unit, all of the proton signals could be identified using a COSY experiment. On the basis of assignments of all proton resonances, the linked carbon atoms were confirmed by HMQC experiment. In 1 H-NMR, the anomeric proton of xylose appears at ca. δ 5.0. The high-field anomeric proton signal of the xylose unit at δ 4.50 indicated a sugar-sugar linkage. The down-field shift of C-2' (δ 83.7) compared to the C-2' signal of 6 (δ 74.0) indicated that the xylosyl residue was connected to C-2' of the glucose ring. This linkage was confirmed by the correlation at δ  4.50/83.7 between H-1'' and C-2' in the HMBC spectrum. Thus, the structure of 2 was determined to be β-  (Table 1) were comparable to those of 2. Extra 13 C-NMR signals at 129.6 (2C), 130.6 (2C), 130.9, 134.7, 167.3 suggested an additional benzoyl group. The connection to C-4'' of the β-D-xylopyranosyl moiety was deduced from the HMBC correlation between δ H 4.29 (xyl-H-4'') and δ C 167.8 (benzoyl-C-7'''). Based on the above results, the structure of 3 was established as Glycosides with β-D-xylopyranosyl(1→2)-O-β-D-glucopyranoside moieties were isolated from this family plant, Pteridium esculentum [16]. This represents the second time these special disaccharide analogues are found in this family.
In this study 5-[2-hydroxyethylidene]-2(5H)-furanone (4) was isolated from a natural source for the first time, although it had been already synthesized by Siegel and Bruckner [17]. The structure of 4 was readily identified by comparison of its spectroscopic data ( 1 H-NMR, IR, and mass spectrometry data) [15]. We further confirmed its 13 C-NMR assignments by 1 H-1 H COSY, HMQC, and HMBC experiments ( Figure 3).

Cytotoxic activity of Isolated Compounds
In previous studies on plants of this genus, illudin-series compounds were reported as precursors of pterosins. In a recent report [24], a cytokinetic experiment with HL 60 cells indicated that illudin S exerts a primary effect on DNA synthesis. Illudin S could cause a complete block at the G 1 -S phase interface of the cell cycle. However, a series of illudin compounds were regarded as anti-cancer and/or carcinogenic substances [11], and the conflict in these biological results suggested that the safety issue needs to be concerned when using extracts from ferns from the family Pteridaceae. The pterosin metabolites were studied for their bioactivity, e.g. antimicrobial and antitumor activity, and displayed less toxicity than illudins [11,25]. So far, we haven't yet found any illudin-series compounds in this plant by using a cytotoxicity-guided fractionation method. This might explain why Taiwanese people use this plant extract as one ingredient of combination herbal beverages without serious toxic effects. However, it is an important issue to be clarified. Some pterosin glycosides have been identified [13,25], but 3-O-β-D-glucose substituted pterosins are rare and their activity has not yet been reported. All purified compounds were evaluated for the cytotoxicity toward Hep G2 (human liver cancer), A549 (human lung carcinoma), MDA-MB-231 (breast carcinoma), MCF-7 (breast carcinoma), Ca9-22 (human oral squamous carcinoma), and HL 60 (human leukemia) cell lines. Among them, compound 1 and pterosin B (5) showed selective activity against HL 60 human leukemia cancer cells with the IC 50 values of 3.7 and 8.7 μg/mL, respectively. Pterosins are inactive to antioxidant assays, but play an important role to action of cytotoxicity to cancer cell lines.

General
Optical rotations were measured on a JASCO DIP-370 digital polarimeter. UV spectra were obtained on a Hitachi 220-20 spectrophotometer. IR spectra were measured on a Hitachi 260-30 spectrophotometer. 1 H-NMR and 13 C-NMR spectra were recorded on a Varian Inova 500, Varian Unity Plus 400, or Varian Gemini 200 spectrometers using TMS as internal standard. Chemical shifts were reported in parts per million (δ), and coupling constants (J) were expressed in Hertz. LR EI MS were collected on a Bruker APEX II mass or a Quattro GC-MS spectrometer having a direct inlet system. LR ESI-MS and HR ESI-MS were measured on a Bruker APEX II mass spectrometer.

Cytotoxicity Assays
Compounds were assayed for cytotoxicity against Hep G2, A549, MCF-7, MDA-MB-231, Ca9-22 and HL 60 cell lines using the MTT method [26]. Freshly trypsinized cell suspensions were seeded in 96-well microtitre plates at densities of 5,000-10,000 cells per well and tested compounds were added from a DMSO stock solution. After 3 days in culture, attached cells were incubated with MTT (0.5 μg/ml, 1 h) and subsequently dissoluble in DMSO. The absorbance was measured at 550 nm using a microplate reader. The IC 50 is the concentration of agent reduced cell growth by 50% under the experimental conditions. Results represent the mean two to three separate experiments, each performed in triplicate.

Acid hydrolysis of compound 1
A solution of compound 1 (5.0 mg) in 6% aqueous HCl (3.5 mL) was refluxed for 2 h. The reaction mixture was diluted with water and then extracted with ethyl acetate. The resulting glucose (in water) and 2R,3R pterosin L (in ethyl acetate) were identified by their corresponding 13 C-NMR spectra.