A novel diarylheptanoid-bearing sesquiterpene moiety from the rhizomes of Alpinia officinarum

Abstract A new diarylheptanoid analogue-bearing sesquiterpene moiety, named Alpinisin A, was isolated from the rhizomes of Alpinia officinarum Hance. The new structure was determined by various spectroscopic techniques 1H-nuclear magnetic resonance (1H NMR), 13C-attached proton test (13C-APT), heteronuclear single quantum coherence (HSQC), heteronuclear multiple bond correlation (HMBC), 1H-1H correlation spectroscopy (1H–1HCOSY), nuclear overhauser effect spectroscopy (NOESY) and high resolution electrospray ionization mass spectrometry (HR–ESI–MS). The compound was tested for cytotoxic activity in vitro against human tumour cell lines (gastric carcinoma cell -7901 (SGC-7901), Michigan Cancer Foundation-7 (MCF-7) and Caski), which showed significant inhibitory effects with IC50 levels of 11.42, 15.14 and 14.78 μM, respectively. The novel chemical structure characterised with a diarylheptanoid linked to a chain-like sesquiterpenoid should be highlighted.


Introduction
Alpinia officinarum Hance is a member of the genus Alpinia in the Zingiberaceae family, which is a perennial plant that is mainly produced in Guangdong, Guangxi, Hainan, Yunnan and Taiwan provinces (Huang & Yang 2009). Currently, the rhizomes of A. officinarum have been used for stomach pain, cholera, dysentery, abdominal pain and vomiting (Shin et al. 2002;Huang et al. 2008). Therefore, the rhizomes of A. officinarum are a common medicine used for splenic and gastric illnesses in China. The present investigation suggested that a wide range of pharmacologic activities of A. officinarum have been developed, such as antiulcer, antidiarrhoeal, anti-inflammatory, analgesic and hypolipidemic effects (Matsuda et al. 2006;Lee et al. 2009;Xia et al. 2010). The chemical constituents that have been isolated from the rhizomes of A. officinarum are mainly flavonoids, monoterpenes, sesquiterpenes, diarylheptanoids, phenylpropanoids and the polymers of diarylheptanoids and flavonoids (Ly et al. 2003;An et al. 2006;Sun et al. 2008;Zou et al. 2016). Therefore, we initiated a chemical study using the rhizomes of A. officinarum with a focus on diarylheptanoids and completed the isolation of a new compound (Alpinisin A) from the ethyl acetate extract of the rhizomes of A. officinarum. The cytotoxicity of the compounds was tested by MTT against tumor cell lines (SGC-7901, MCF-7 and Caski), and it was shown that the compound possessed anti-tumour activity and had a valid inhibitory effect with IC 50 levels of 11.42, 15.14 and 14.78 μM, respectively.
The configuration of compound 1 was determined by NOESY analysis. The configuration of the two double bonds was determined as Z and E by NOESY correlation analysis and the chemical shift values at δ C 15.0 (C-14″′) and 21.4 (C-15″′). Chemical shift values of the methyl groups linked to the double bonds could be applied to determine the geometry of the double bonds. Chemical shift value of the methyl group is greater than 20.0 ppm, the configuration of the double bond is E. In contrast, chemical shift value of the methyl group is less than 20.0 ppm, the configuration of the double bond is Z. In addition, observed NOESY cross-peaks from H-2″″ to H-15″″ and H-6″″ to H-8″″ confirmed the configuration. The correlations between 2.30 (1H, m H-4″′) and 2.76 (1H, m H-4), and 2.00 (1H, m H-5) in the NOESY spectrum suggest that the three protons were in the same planer of the six-member ring, thus the relative configuration was determined according to the above information as shown in Figure S2.

General experimental procedures
Specific rotations were obtained on a Perkin-Elmer 241 MC polarmeter. IR spectra were obtained on a Nicolet iS-5 Infrared spectrophotometer. uV spectra were measured on a JASCO-uV-V650 spectrophotometer. NMR spectra were obtained with a Bruker AVIII-600 spectrometer (chemical shift values are presented as δ values with tetramethylsilane (TMS) as the internal standard). HR-ESI-MS spectrum was performed on a Waters LCT Premier XE time-of-flying spectrometer. Silica gel (200-300 mesh; Qingdao Marine Chemical Group, Co, Qingdao, China) and ODS (40-60 μm; Merck Germany). Sephadex LH-20 (Amersham Pharmacia Biotech AB) was used for column chromatography.

Plant material
The rhizomes of A. officinarum were collected in Haikou county of Hainan Province of China in July 2013. The plant was identified by Professor Niankai Zeng of Hainan Medical university. A voucher specimen (No. 20130716) has been deposited in the School of Pharmacy of Hainan Medical university.

Cytotoxicity bioassays
The cytotoxic activity of compound 1 was tested against human cancer cell lines (SGC-7901, MCF-7, and Caski) using the MTT method, as previously described (Zhang et al. 2012). A reference compound (5-fluorouracil, purity >98%; Sigma Chemical Co, St. Louis, MO, uSA) was used to assess the cytotoxicity of the tested compounds. The number of cells was counted on a haemocytometer using the trypan blue dye exclusion technique to assure cell viability >95%. Briefly, cells were plated in 96-well plates for 24 h before treatment and continuously exposed to different concentrations of tested and reference compounds. Then, the cells were cultured in triplicate for 48 h with the test compounds. Cells cultured with medium alone were used as controls. MTT (5 mg/mL) reagent was added 4 h before the end of culture, the plates were centrifuged (1400r/m for 5 min), and the untransformed MTT was carefully removed by pipetting. Dimethyl sulfoxide working solution (150 μL) was added to each well. The absorbance was measured on an ELISA reader at 490 nm and the inhibition rate and IC 50 value of the compounds were calculated. The percentage of cell death was calculated using the following formula: Compound 1 had cytotoxicity towards the three cancer cell lines with IC 50 values of 11.42, 15.14 and 14.78 μM, respectively, in comparison to the positive control (5-fluorouracil) with IC 50 values ranging from 5.31-8.27 μM.

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