Sesquiterpenes from Curcuma wenyujin with their inhibitory activities on nitric oxide production in RAW 264.7 cells

Abstract One new sesquiterpene, 7α,11-epoxy-6α-hydroxy-carabrane-4,8-dione, along with 10 known ones were isolated from the essential oil of Curcuma wenyujin Y.H. Chen et C. Ling. Their structures were established based on extensive spectroscopic analysis. The absolute configuration of compound 1 was determined by the CD analysis of the insitu formed [Rh2(OCOCF3)4] complex, and the CD data analysis based on the octane rule of cyclohexanone. The inhibitory effects of these sesquiterpenes on nitric oxide production in lipopolysaccharide-activated macrophages were also evaluated. Furthermore, the biosynthesis pathway of the isolated compounds was proposed.


Introduction
Curcuma wenyujin Y.H. chen et C. Ling., a perennial herbaceous plant of Zingiberaceae, is distributed especially in Zhejiang Province, China. The rhizomes of this plant are a kind of important medicine officially documented in Chinese Pharmacopoeia (Committee of National Pharmacopoeia 2015) which has been used for the treatment of 'Oketsu' (various syndromes caused by the obstruction of blood circulation such as arthralgia, psychataxia and dysmenorrhoea) (Sasaki et al. 2003). The main active constituents are divided into phenolic pigments (Lin et al. 2012) and essential oils (Srinivasan 1953;Ishii et al. 2011;Rameshkumar et al. 2015). Previous studies indicated that the main bioactive constituents of rhizoma curcumae are sesquiterpenoids (Takano et al. 1995;Firman et al. 1988;Matsuda, Morikawa, Toguchida et al. 2001) and diarylheptanoids (Syu et al. 1998;Zeng et al. 2007), which possess anticancer (Lim et al. 2010), vasorelaxant , anti-inflammatory (Claeson et al. 1996;Sodsai et al. 2007), heptoprotective (Song et al. 2001) and immunological activities (Motterlini et al. 2000). Our research works have recently led to the isolation of several new sesquiterpenoids and diarylheptanoids from the dried rhizomes of C. kwangsiensis, C. phaeocaulis and C. wenyujin (Lou et al. 2009(Lou et al. , 2010Li et al. 2010Li et al. , 2011Liu et al. 2013Liu et al. , 2014. Most of the isolated compounds exhibited potent inhibitory activities against nitric oxide (NO) production in lipopolysaccharide (LPS)-activated macrophages. as part of our ongoing research, this paper describes the isolation and characterisation of 10 sesquiterpenoids as well as their inhibitory effects on NO production. Furthermore, the plausible biogenetic pathway for these compounds was proposed ( Figure S3).
The discovery of these biogenetically related compounds in C. wenyujin is highly meaningful from a chemotaxonomy perspective. as shown in Figure S3, compound 1 was supposed to be the precursor of compound i, which was isolated from C. wenyujin previously and its biogenetic pathway was also proposed (Parker & Roberts 1967;Xia et al. 2015). Curdione, a sesquiterpene isolated from C. wenyujin (Inayama et al.1985), was a key biosynthetic precursor of compounds 9 and 10. Compounds 2-7, 11 were also considered to be synthesised through the similar pathway.
The effects of compounds 1-11 with respect to the inhibition of nitric oxide (NO) production in LPS-activated RaW 264.7 in macrophage cells were evaluated. as shown in Table S2, hydrocortisone (IC 50 64.34 ± 7.49 μM) was used as a positive control. Compound 2 exhibited stronger inhibitory activity against NO production (IC 50 14.99 ± 1.21 μM) than other elemane-type sesquiterpenes in this paper. Comparison of the inhibitory effects of these compounds, with those of the previously isolated γ-elemenolides (Lou et al. 2010;Ma et al. 2015) revealed that the orientation of H-8 was of pivotal importance ( Figure S14). Furthermore, compound 9 (IC 50 23.28 ± 1.47 μM) showed stronger inhibitory activity than 10 (IC 50 45.49 ± 2.96 μM) probably due to the different orientation of the isopropyl at C-7.

General experimental procedures
Optical rotations were measured with an anton paar MCP 200 polarimeter. uV spectra were recorded on a Shimadzu uV 2201 spectrophotometer. IR spectra were conducted on a Bruker IFS 55 spectrometer. NMR experiments were performed on Bruker aRX-300 and aV-600 spectrometers. The chemical shifts were stated relative to TMS and expressed in δ values (ppm), with coupling constants reported in Hz. HR-MS (eSI) were obtained on an agilent 6210 TOF mass spectrometer. CD spectra were recorded on a Bio-Logic Science MOS-450 spectrometer. Silica gel GF 254 prepared for TLC and silica gel (200-300 mesh) for column chromatography (CC) were obtained from Qingdao Marine Chemical Factory (Qingdao, People's Republic of China). Sephadex LH-20 was a product of Pharmacia. Octadecyl silica gel was purchased from Merck Chemical Company Ltd. RP-HPLC separations were conducted using a LC-6aD liquid chromatograph with a YMC Pack ODS-a column (250 × 20 mm, 5 μm, 120 Å) and SPD-10a VP uV/VIS detector. all reagents were HPLC or analytical grade and were purchased from Tianjin Damao Chemical Company. Spots were detected on TLC plates under uV light or by heating after spraying with anisaldehyde H 2 SO 4 reagent.

Plant material
Rhizomes of C. wenyujin were collected from Wenzhou City, Zhejiang Province, China, and identified by Professor Qishi Sun, Department of Pharmaceutical Botany, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical university. a voucher specimen (CP-20090625) has been deposited in the herbarium of the Department of Natural Products Chemistry, Shenyang Pharmaceutical university.

Nitric oxide production bioassay
The nitrite concentration in the medium was measured as an indicator of NO production in accordance with the Griess reaction. Briefly, RaW 264.7 cells were seeded into 96-well tissue culture plates at a density of 1 × 10 5 cells/well, and stimulated with 1 μg/mL of LPS in the presence or absence of test compounds. after incubation at 37 °C for 24 h, cell-free supernatant (100 μL) was mixed with Griess reagent [100 μL; mixture of equal volumes of reagent a and reagent B; a:1% (w/v) sulphanilamide in 5% (w/v) phosphoric acid; B:0.1% (w/v) of N-(1-naphthyl) ethylenediamine]. absorbance was measured in a microplate reader at 540 nm. Nitrite concentrations and the inhibitory rates were calculated by a calibration curve prepared with sodium nitrite standards (Dirsch et al. 1998;Lou et al. 2009). experiments were performed in triplicate, and data are expressed as the mean ± SD of three independent experiments.

Absolute configuration of the secondary alcohol in 1
according to a published procedure (Frelek & Szczepek 1999), a sample of 1 (1.2 mg) was dissolved in a dry solution of the stock [Rh 2 (OCOCF 3 ) 4 ] complex (1.5 mg) in CH 2 Cl 2 (0.5 mL) and was subjected to CD measurements at a concentration of 2.5 mg/mL. The first CD spectrum was recorded immediately after mixing, and its time evolution was monitored until stationary (ca. 10 min after mixing). The inherent CD was subtracted. The observed sign of the e band at ca. 350 nm in the induced CD spectrum was correlated to the absolute configuration of the secondary alcohol moiety.

Conclusion
In summary, one previously unreported sesquiterpene together with 10 elemane-type sesquiterpenes were isolated from the rhizomes of C. wenyujin. Some of the isolated compounds had made a supplement of the previously proposed biosynthesis pathway. Furthermore, compounds 1-11 were evaluated for their effects on nitric oxide production in LPS-activated macrophages. Most of them exhibited stronger inhibitory effects than the positive control, hydrocortisone, and these results may substantiate the traditional therapeutic effects of C. wenyujin and its essential oil.

Supplementary material
Supplementary material relating to this article is available online, alongside Tables S1-S2 and Figures S1-S15.

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