Phaeophytin Analogues from Ligularia knorringiana

A new phaeophytin, ligulariaphytin A, together with five known phaeophytins, were isolated from the aerial parts of Ligularia knorringiana. The structure of ligulariaphytin A was elucidated as 131-hydroxy-131,132-peroxyphaeophorbide A ethyl ester (1), and the five known compounds were identified as 132-hydroxyphaeophorbide A ethyl ester (2), 173-ethoxyphaeophorbide A (3), phaeophytin B (4), phaeophytin A (5), and phaeophorbide B ethyl ester (6), respectively, based on spectroscopic analysis and by comparison of their spectral data with those reported previously in the literature. All compounds were evaluated for their in vitro cytotoxic activities against cultured Hela cell, and were found to show only very weak cytotoxicity.


Introduction
The genus Ligularia Cass. is highly diversified in China, consisting of about 110 species [1]. More than 40 species of Ligularia have been studied chemically, and more than 300 compounds, including terpenoids, pyrrolizidine alkaloids, flavonoids and benzofuran derivatives, have been isolated, with a variety of biological activities [2][3][4]. One of us and co-workers have been studying the diversity of Ligularia by means of analyses of terpenoid composition and neutral DNA sequences and have found OPEN ACCESS that intra-specific diversity is present in many species [5,6]. Although root terpenoid composition has been successfully used as the chemical index, another class of chemicals would be useful to understand the complex diversity. In this study, we focused on phaeophytins, which are found commonly in aerial parts of higher plants. To the best of our knowledge, phaeophytins in Ligularia have not been investigated. In this report, we describe isolation of phaeophytins from L. knorringiana, whose chemical constituents have not been reported. We isolated a new phaeophytin (1) (Figure 1) along with five known phaeophytins, 13 2 -hydroxyphaeophorbide A ethyl ester (2) [7], 17 3 -ethoxyphaeophorbide A (3) [8], phaeophytin B (4) [9], phaeophytin A (5) [9], and phaeophorbide B ethyl ester (6) [7] from the aerial parts of L. knorringiana. The new compound 1 has a rare four-membered peroxide structure.
Previous works revealed that phaeophytins possess potent antioxidant activities [13] and cytotoxic activities [14]. All compounds isolated and prepared in this study were therefore assayed for in vitro cytotoxicity against the Hela cell line by the methylthiazolyltetrazolium (MTT) assay [15]. Compounds 1 and 2 showed only very weak cytotoxicity, with IC 50 values of 80, 90 µg/mL, respectively.
The results indicate that various phaeophytins are present in Ligularia, suggesting that the compounds may be a useful index in the study of chemical diversity. Compound 1 has a rare fourmembered peroxide-containing phaeophytin and such compound has only been isolated from the Asteraceae once before [11]. The analogues, bidenphytin A and B have been previously isolated from Biden pilosa (Asteraceae) [11]. These data suggest that phaeophytins are a useful index to compare various plants chemically.

General
Melting points were measured on an XT-4 micro-melting point apparatus and are uncorrected. Optical rotations were recorded on a JASCO P-1020 polarimeter at room temperature. UV spectra were measured on a Shimadzu UV-260 spectrophotometer in absolute MeOH. IR spectra were recorded on an Avatar 360 FT-IR ESP spectrometer in KBr. A Bruker Daltonics APEXШ 7.0 TESLA FTMS mass spectrometer for HRESIMS. NMR spectra were recorded on a Varian XTIPC-400 spectrometer (400 MHz for 1 H and 100 MHz for 13 C). Analytical and preparative TLC were run on silica gel plates (GF 254 , Yantai Institute of Chemical Technology, Yantai, China). Spots were observed under UV light and visualized by spraying with 10% H 2 SO 4 , followed by heating. Column chromatography (CC) was performed on silica gel (200-300 mesh and 300-400 mesh; Qingdao Marine Chemical Factory, Qingdao, China) and Lichroprep RP 18 gel (40-60 μm, Merck, Darmstadt, Germany).

Conclusions
As a part of our chemical investigation on the aerial parts of Ligularia knorringiana, a new phaeophytin, ligulariaphytin A, together with five known phaeophytins, were isolated and their structures elucidated by spectroscopic methods including 2D-NMR techniques. The results indicate that various phaeophytins are present in Ligularia, suggesting that the compounds may be a useful index in the study of chemical diversity. Compounds 1 and 2 showed very weak cytotoxicity against cultured HeLa cella.