A new phenolic glycoside from Saprosma merrillii

Abstract A new phenolic glycoside derivative, saproglucoside (1), along with five known phenolic glycoside derivatives (2–6) were isolated from the stems of Saprosma merrillii. The structure of the new compound 1 was determined by 1D and 2D NMR as well as by HRESIMS and hydrolysis. The inhibitory activities of all compounds against seven pathogenic bacteria and two cancer cell lines were evaluated.


Introduction
The genus Saprosma (Rubiaceae) consists of about 30 species, which are mainly distributed in the tropical Asian region, and has been used as a traditional medicine for the treatment of fever (Ling et al. 2002;Lu et al. 2010;Quang et al. 2002;Singh et al. 2006;Wang et al. 2011Wang et al. , 2014. Saprosma merrillii is an endemic medicinal plant in Hainan, P.R. China. Our group recently reported the isolation of three new ursane-type triterpenoids and many anthraquinone derivatives, which showed antibacterial and cytotoxic activity (Li et al. 2015;Zhang et al. 2014. In our ongoing investigation of natural antibacterial products from the S. merrillii, a new phenolic glycoside derivative, saproglucoside (1), along with five known phenolic glycoside derivatives (Figure 1), isotachioside (2) (Inoshiri et al. 1987), tachioside (3) (Inoshiri et al. 1987), canthoside D (4) (Kanchanapoom et al. 2002), canthoside C (5) (Kanchanapoom et al. 2002) and koaburaside (6) (Ogawa et al. 1973;Kosuge et al. 1994) were isolated from the stems of S. merrillii. Known compounds 2-6 were isolated from Saprosma for the first time. The isolation and structure elucidation of 1 and the inhibitory activities of all compounds against six pathogenic bacteria and two cancer cell lines are reported herein.
The structures of known compounds 2-6 were identified by comparison with their 1 H/ 13 C NMR spectra and [α] 25 D with those in the literature. The isolated compounds were evaluated for cytotoxic activities against the mouse melanoma cell line (B16F10) and the human lung adenocarcinoma cell lines (A549). However, the IC 50 values of all compounds were higher than 10 μM in the assay The antibacterial activities of all compounds were determined against seven terrestrial pathogenic bacteria Micrococcus tetragenus, Escherichia coli, Staphylococcus albus, Bacillus cereus, Saphylococcus. aureus, Micrococcus luteus and Bacillus subtilis. However, the MIC values of all compounds were higher than 20 μg/mL in the assay.

General
IR spectra were recorded on a Nicolet 6700 spectrophotometer. uV spectra were recorded on a Beckman Du 640 spectrophotometer. Optical rotations were measured on a JASCO P-1020 digital polarimeter. NMR spectra were recorded on a Bruker AV spectrometer (400 MHz for 1 H and 100 MHz for 13 C). TMS was used as an internal standard. HRESIMS spectra were measured on a Q-TOF ultima Global GAA076 LC mass spectrometer. Silica gel (Qing Dao Hai Yang Chemical Group Co.; 200-300 mesh) and octadecylsilyl silica gel (YMC; 12 nm-50 μM) were used for column chromatography (CC). Precoated silica gel plates (Yan Tai Zi Fu Chemical Group Co.; G60, F-254) were used for TLC. Semi-preparative HPLC was performed on an Agilent 1260 LC series with a DAD detector using an Agilent Eclipse XDB-C18 column (9.4 × 250 mm, 5 μM).

Plant material
The stems of S. merrillii were collected from Sanya, Hainan Province, China, in June 2014 and identified by Prof. Qiong-Xin Zhong, College of Life Science, Hainan Normal university. A voucher specimen (No. GFM20140615) has been deposited at the Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, Hainan Normal university.

Hydrolysis of Saproglucoside (1)
Acid hydrolysis of Saproglucoside (1): a solution of 1 (20 mg) in 5% H 2 SO 4 (2 mL) was heated under reflux for 3 h and then evaporated to dryness. The mixture was separated by the semi-preparative HPLC to obtain a d-(-)-pantolactone (4 mg) and the remainder (13 mg). The remainder further basic hydrolysis in 20% KOH (1.5 mL) was heated under reflux for 2 h. The mixture was neutralised with 1 N HCl and then evaporated to dryness. The mixture was extracted with ethyl acetate. After evaporation, the residue was purificated by the semi-preparative HPLC to give 6-methoxyl-7-hydroxytropic acid (1a) (7 mg). The water soluble residue evaporated to dryness (contained glucose). The contained glucose was purified by the semi-preparative HPLC.

Biological assays
Cytotoxic activity was evaluated by the MTT method as described previously (Scudiero et al. 1988). Two cancer cell lines, ECA-109 and A549 cell lines, were used. Epirubicin was used as a positive control. Antibacterial activity was determined by the conventional broth dilution assay (Pierce et al. 2008). Seven terrestrial pathogenic bacteria M. tetragenus, E. coli, S. albus, B. cereus, S. aureus, M. luteus and B. subtilis were used, and ciprofloxacin was used as a positive control.

Disclosure statement
No potential conflict of interest was reported by the authors.