www.mdpi.org/molecules Two New Triterpenoids from Photinia serrulata

Two new triterpenoids, 2alpha,3beta,11alpha,13beta-tetrahydroxy-12-ketooleanan-28-oic acid(1) and 3beta-hydroxy-12-keto-9(11)-ursen-28,13beta-olide (2) were isolated from the leaves ofPhotinia serrulata. Their structures were identified by spectral methods. Compounds 1 and2 were assessed for cytotoxic activity against three human tumor cell lines (A-549, HCT-8,and BEL-7402), and they showed no cytotoxic effects at concentrations up to 5microg/mL.


Introduction
Photinia serrulata Lindl.belongs to the Rosaceae family and is found throughout the East and South of Asia [1].Its tender leaves are used as edible vegetables in the south of China, and the matured leaves, known in China as "Shi-Nan", are used for the treatment of nephropathy, rheumatism, and spermatorrhea [2].Despite the wide use of the leaves of this plant in Traditional Chinese Medicine, reports on the chemical constituents of this plant are scarce.During our investigation, two new compounds including one oleanane-and one ursane-type triterpenoid 1 and 2 (Figure 1) were isolated, and their structures were characterized by means of spectroscopic methods.The cytotoxic activity of compounds 1 and 2 against three human tumor cell lines (A-549, HCT-8, and BEL-7402) were also assessed.In this paper, we report the isolation and structure determination of these new isolates.
The UV absorption at 259 nm and IR peak at 1632 cm -1 indicated the presence of an α,β-unsaturated ketone.In addition, a lactone ring between C-13 and C-28 inferred from the molecular mass, was also confirmed by the absorption peak at 1780 cm -1 in the IR spectrum.The hydroxyl at C-3 was assigned a β orientation by comparison of its 13 C-NMR data with literature data [6], and ROESY correlations of H-3 (δ 3.18) with Me-23 (δ 1.04).The cis-fusion of D/E rings could be judged from the large coupling constant of Hax-18 (J=12.0Hz).It was noted that the signal for C-9 appeared at a very abnormally down-field position, and the reasons for this phenomenon still remain unclear.Taken together, the structure of 2 was identified as 3β-hydroxy-12-keto-9(11)-ursen-28,13β-olide.The acetate of 2 has previously been described in the literature as a result of chemical transformation [7].However, as a naturally occurring compound, 2 was isolated for the first time, the 1 H-and 13 C-NMR data of 2 have now been unambiguously assigned on the basis of 2D NMR experiments for the first time.The cytotoxic activity of compounds 1 and 2 against three human tumor cell lines (A-549, HCT-8, and BEL-7402) were evaluated.However, they exhibited no cytotoxic effects at concentrations up to 5 μg/mL (data not shown).

General
Melting points were recroded on an XRC-1 micromelting apparatus.Optical rotations were determined on a JASCO-20C digital polarimeter.UV spectra were recorded on a Shimadzu UV-2401PC spectrophotometer.IR spectra were obtained with a Bruker Tensor 27 FT-IR spectrophotometer with KBr pellets. 1 H-NMR (400 MHz) and 13 C-NMR (100 MHz) spectra were recorded on a Bruker AM-400 spectrometer, with TMS as an internal reference.2D NMR spectra were measured with a DRX-500 spectrometer.FABMS were recorded on a VG Auto Spec-3000 spectrometer.ESIMS and HRESIMS were carried our with an API QSTAR Pulsar 1 spectrometer.Silica gel (200-300 mesh and 10-40 μm) for column chromatography and GF 254 for TLC were obtained from Qingdao Marine Chemical Factory (Qingdao, P. R. China).Sephadex LH-20 was obtained from Amersham Pharmacia Biotech (Sweden).

Plant Material
The dried and matured leaves of P. serrulata Lindl.were purchased from Nanjing Pharmaceutical Ltd.Corporation of Jiangsu Province (P.R. China) in March, 2006, and identified by Mrs. Xuedong Geng (Nanjing Pharmaceutical Ltd.Corporation of Jiangsu Province).A voucher specimen (CHYX0392) was deposited at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences.

Extraction and Isolation
The dried and powdered plant materials of P. serrulata (15 kg) were extracted three times with 80% EtOH under reflux.The extracts were concentrated and suspended in water followed by successive partition with petroleum ether, EtOAc and n-BuOH, respectively.The EtOAc extracts (866 g) were subjected to column chromatography (CC) over silica gel (200-300 mesh) and eluted with CHCl 3 -MeOH (15:1) to afford fractions 1-3.Fraction 2 (160 g) was submitted to CC over silica gel, eluted with CHCl 3 -MeOH-EtOAc (6:1:1) to give fractions

Table 1 .
1 H-and 13 C-NMR spectra data for compounds 1 and 2 a .