A new patchoulane-type sesquiterpenoid glycoside from the roots of Croton crassifolius

Abstract A new patchoulane-type sesquiterpenoid glycoside (1), together with five known sesquiterpenoids (2–6), was isolated from the roots of Croton crassifolius. Their structures were elucidated on the basis of spectroscopic methods. This is the first report about the sesquiterpenoid glycoside from C. crassifolius. All the isolated compounds 1–6 were evaluated for their cytotoxic activities against human tumour cell lines HL-60 and A549, but none showed significant activity.


Introduction
The genus Croton belongs to the Euphorbiaceae family, comprising approximately 1300 species distributed throughout tropical and subtropical regions of the world (Salatino et al. 2007). The plants of Croton are generally known for containing diverse terpenoids with a good range of biological activities (Kuo et al. 2007;Pan et al. 2014;Sun et al. 2014;Shahwar et al. 2015). Croton crassifolius Geisel. is mainly distributed in southern China, Laos, Thailand, and Vietnam (Qiu et al. 1996). The plant of C. crassifolius, which is known as 'jiguxiang' in China, has been used for the treatment of cancer, stomachache, sore throat and rheumatism (Song 1999). Previous phytochemical investigations on C. crassifolius showed that the main components were clerodane diterpenoids with cytotoxic (Boonyarathanakornkit et al. 1988;Qiu et al. 2016), antiviral (Wang et al. 2012) and anti-angiogenic activities (Huang, Li et al. 2014;Wang et al. 2016). However, the sesquiterpenoids constituents of this plant species are rarely reported . As part of our ongoing search for new bioactive constituents from traditional plants with medicinal interest, we carried out the investigation of the roots of C. crassifolius. The present paper deals with the isolation and structural elucidation of a new patchoulane-type sesquiterpenoid glycoside (1), together with five known sesquiterpenoids (2-6) ( Figure 1). The cytotoxic activities of 1-6 were evaluated against human tumour cell lines HL-60 and A549.

Results and discussion
The air-dried and powdered roots of C. crassifolius were extracted with 70% acetone and partitioned with petroleum and CHCl 3 . The water-soluble and CHCl 3 fraction were subjected to MCI resin, HW-40F, reversed-phase C18, and repeated silica gel column chromatography (CC), followed by Sephadex LH20, to afford a new sesquiterpenoid glycoside (1), together with five known sesquiterpenoids (2-6). Their structures were elucidated on the basis of spectroscopic methods including HRESIMS, 1D and 2D nMR spectra and comparison with the data reported in the literature.
The six isolated compounds 1-6 were evaluated for their cytotoxic activities against two human tumour cell lines HL-60 and A549 using MTT and SRB assays. However, none showed significant activity (cellular proliferation inhibition rate < 50% at 20 μM), and the results were presented in Table S2.

General experimental procedures
Optical rotations were measured on a Perkin-Elmer 341 polarimeter. IR spectrum was recorded on a Perkin-Elmer 577 spectrometer using KBr discs. One-dimensional and two-dimensional nMR experiments were performed at room temperature using a Bruker Ascend 500 MHz ( 1 H) and 126 MHz ( 13 C) spectrometer. HRESIMS analyses were carried out on an Agilent 6224 TOF mass spectrometer with an ESI interface. Silica gel (200-300 mesh, Qingdao Haiyang Chemical Co Ltd), C 18 reversed-phase (RP-18) silica gel (20-45 μm; Fuji Silysia Chemical Ltd), Toyopearl HW-40F (32-63 μm; Tosoh) and Sephadex LH-20 gel (Amersham Biosciences) were used for CC. Pre-coated silica gel GF 254 plates (Qingdao Haiyang Chemical Co.Ltd) were used for TLC. All solvents used for CC were of analytical grade (Shanghai Chemical Reagents Co, Ltd).

Plant material
The dried roots of C. crassifolius were collected from Guangxi Province, People's Republic of China in December 2011, and authenticated by Prof. He-Ming Yang. A voucher specimen (SIMMXLJ-89) was deposited at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, People's Republic of China.

Cytotoxic activity
The cytotoxic activities against the cancer cell lines HL-60 and A549 were determined by the MTT (Mosmann 1983) and SRB (Vichai & Kirtikara 2006) assays, respectively. Briefly, 100 μL of each cell line (5 × 104 cells mL −1 ) was seeded in 96-well microplates and incubated at 37 °C for 24 h. Then, the test compound (20 μM) was added and the microplates were further incubated for 72 h. Compounds that inhibited 50% of the growth of the cancer cells were evaluated again at six concentrations; each concentration of the compound was tested in two parallel wells. Cisplatin was used as a positive control.

Conclusions
One new patchoulane-type sesquiterpenoid glycoside, named cyperenoic acid-9-O-β-dglucopyranoside (1), together with five known sesquiterpenoids (2-6), was isolated from the roots of C. crassifolius. All the isolated compounds showed no significant cytotoxic activity against human tumour cell lines HL-60 and A549. This is the first report about the sesquiterpenoid glycoside from C. crassifolius.

Disclosure statement
no potential conflict interest was reported by the authors.