Bixasteroid, a new compound from the fruits of Bixa orellana and its anti-inflammatory activity

Abstract Bixasteroid (1), one new steroid together with five known compounds (2–6), were isolated from the ethyl acetate fraction of ethanol extract of Bixa orellana fruits. All of these known compounds were isolated from the plant for the first time. Their structures were elucidated on the basis of spectroscopic analysis, and the absolute configuration of compound 1 was determined by X-ray crystallographic data analysis as well as by the quantum chemical ECD calculations. All the isolated compounds were tested for their anti-inflammatory activities. Compounds 1 and 2 showed inhibiting NO release activities in LPS-induced RAW 264.7 macrophages with the IC50 values of 4.72 ± 0.28 and 5.48 ± 1.48 μM, respectively. Graphical Abstract


Introduction
Bixa orellana L. (Bixaceae) is one of the oldest known plants yielding natural dye (Silva et al. 2010). It is native to Brazil and mainly distributes in Central and South America. As a famous dye yielding plant, it has been used earlier for body painting, and now the natural dyes extracted from the seeds of Bixa orellana are widely used in food, textiles, printing, cosmetics, pharmaceuticals and other industrial fields, which indicated its commercial potential. Bixa orellana has been used for centuries for the prevention and treatment of a number of heath disorders such as constipation, fevers, skin diseases, allergy, leprosy, infectious diseases, gonorrhea, diarrhea and asthma (Villar et al. 1997). Previous chemical investigations on Bixa orellana showed that there are many chemical constituents including carotenoids (Mercadante et al. 1997), apocarotenoids (Preston and Rickard 1980), sterols, aliphatic compounds, monoterpenes (Galindo-Cuspinera et al. 2002) and sesquiterpenes (Frega et al. 1998), triterpenoids (Beretta et al. 2018), and other miscellaneous compounds (Harborne 1975) that have been isolated and identified mostly from the seeds, seed coats and leaves of this plant. Many pharmacological investigations through various in vivo and in vitro experiments have demonstrated that the plant exhibited various pharmacological activities, such as antibacterial and antifungal (Irobi et al. 1996), antioxidant and free radical scavenging (Cardarelli et al. 2008), anti-inflammatory (Keong et al. 2011), anticarcinogenic (Tibodeau et al. 2010;Basting et al. 2020), enhanced gastrointestinal motility (Zhai et al. 2014), and neuropharmacological (Quanico et al. 2008) and anticonvulsant activities. However, the researches on the active chemical components of Bixa orellana are not enough. Therefore, a systematic chemical study was carried out on the EtOAc-soluble fraction of an EtOH extract of Bixa orellana fruits in this paper. As a result, one new compound and five known compounds were isolated and identified based on various chromatographic and spectral methods. We also tested the anti-inflammatory activity of these compounds in vitro.
The HMBC ( Figure S10, Supplementary materials) correlations from H-6 (d H 5.74) to C-4 (d C 38.8), C-8 (d C 44.3) and C-10 (d C 38.9), from H-19 (d H 1.24) to C-1 (d C 36.1), C-5 (d C 166.2), C-9 (d C 49.7) and C-10 (d C 38.9), and from H-1 (d H 1.19, 1.98) and H-4 (d H 2.33, 2.62) to C-3 (d C 79.0) and C-5 (d C 166.2), as well as the successive 1 H-1 H COSY correlations of H-1/H-2/H-3/H-4 indicated the connection of A and B rings. Meanwhile, the methoxyl group was proved to locate at C-3 by HMBC correlation from H-22 (d H 3.38) to C-3 (d C 79.0). Similarly, the connection of B and C rings can be deduced from the 1 H-1 H COSY correlations of H-12/H-11/H-9/H-8/H-14/H-15, along with the HMBC correlations from H-14 (d H 2.43) to C-9 (d C 49.7), C-8 (d C 44.3) and C-7 (d C 200.6). Finally, the HMBC correlations from H-18 (d H 1.04) to C-12 (d C 35.4), C-13 (d C 43.8) and C-17 (d C 146.3), and from H-15 (d H 2.25, 3.11) to C-13 (d C 43.8), C-14 (d C 44.0) and C-16 (d C 206.9) can determine the connection of C and D rings. In addition, the location of C-20/C-21 double bond and methyl group at C-21 were further confirmed by HMBC correlations from H-20 (d H 6.52) to C-13 (d C 43.8), C-16 (d C 206.9) and C-21 (d C 13.3), and from H-21 (d H 1.86) to C-17 (d C 146.3) and C-20 (d C 129.0) together with COSY correlation of H-20/H-21. Thus, the planar structure of compound 1 was deduced. The absolute configuration of compound 1 was confirmed by single crystal X-ray diffraction with Cu plank (Figure S15, Supplementary materials). From the data of X-ray diffraction, the absolute structure of compound 1 was elucidated as 3S, 8 R, 9S, 10 R, 13S and 14S [Flack parameter: 0.3(2)]. As the specific optical rotation of compound 1 wasn't zero, we believed that the data of X-ray diffraction is reliable. To further determine the absolute configuration of compound 1, its ECD was calculated by using the TDDFT method ( Figure S14, Supplementary materials). By comparing the theoretical calculation CD curves of 1 with the experimental CD spectrum, we found that the experimental CD spectrum agreed with the calculated CD curve of 3S,8R,9S,10R,13S,14S-enantiomer. Thus, the structure of compound 1 was elucidated as shown in Figure 1 and it is a new steroid, named bixasteroid, chemically 8 R,9S,10R,13S,14S-pregna-3S-methoxy-5,17(20)-diene-7,16-dione.

General experimental procedures
UV spectra were taken on a JASCO V-550 UV/VIS spectrometer (JASCO Corporation, Tokyo, Japan). The optical rotations were obtained on a JASCO digital polarimeter (JASCO Corporation, Tokyo, Japan). IR spectra were carried out with a Nicolet Impact 410-FTIR spectrometer (JASCO Corporation, Tokyo, Japan). The NMR spectra were recorded on Bruker AV-400 and AV-600 spectrometers (Bruker Instrument, Inc., Zurich, Switzerland) used TMS as an internal reference. HPLC was performed on an Agilent 1200 HPLC system equipped with a diode array detector, using a column A (Ultimate XB-C18, 5 lm, 4.6 Â 250 mm, Welch, Potamac, MA, USA) with the flow rate of 1 mL/min for analysis, a column B (Ultimate XB-C18, 5 lm, 10 Â 250 mm, Welch, Potamac, MA, USA) with the flow rate of 3 mL/min for semi-preparative purification. CD spectra were measured with a J-810 spectrometer using CH 3 CN as solvent. HR-TOF-MS data were achieved on a Waters Synapt G2TOF mass spec-trometer (Waters Technologies, Massachusetts, USA). Open column chromatography (CC) was performed on silica gel (200-300 mesh, Haiyang Chemical Group Corporation, Qingdao, China), ODS (50 lm, YMC, Tokyo, Japan) and Sephadex LH-20 (25-100 mm, Pharmacia, Uppsala, Sweden). HSGF 254 silica gel TLC plates (0.2 mm thickness, 200 Â 200 mm, Qingdao Marine Chemical, Qingdao, China) were used for routine TLC analysis. The spraying reagent used for TLC detection was 10% H 2 SO 4 in ethanol.

Plant material
The experimental material, that is the fruits of Bixa orellana, was collected in The Republic of Ecuador, which is located in northwestern South America. The plant sample was authenticated by Dr. GX Zhou. A dry voucher specimen (EP-201701) was deposited in the Teaching & Research Office of Pharmacognosy, Jinan University.

Computational details for ECD of compound 1
Monte Carlo conformational searches were carried out by means of the Spartan's 10 software using Merck Molecular Force Field (MMFF). The conformers with Boltzmannpopulation of over 5% were chosen for ECD calculations, and then the conformers were initially optimized at B3LYP/6-31 þ g (d, p) level in CH 3 CN using the CPCM polarizable conductor calculation model. The theoretical calculation of ECD was conducted in CH 3 CN using Time-dependent Density functional theory (TDDFT) at the B3LYP/6-311 þ g (d, p) level for all conformers of compound 1. Rotatory strengths for a total of 50 excited states were calculated. ECD spectra were generated using the program SpecDis1.6 (University of W€ urzburg, W€ urzburg, Germany) and GraphPad Prism 5 (University of California San Diego, USA) from dipole length rotational strengths by applying Gaussian band shapes with sigma ¼ 0.3 eV.

Anti-inflammatory activity
NO release inhibition assay: The anti-inflammatory activity was investigated based on the inhibition of NO generation. The NO concentration was detected by the Griess reagent. Briefly, the murine macrophage RAW 264.7 cell line was cultured in DMEM with 10% FBS. The RAW 264.7 macrophages were seeded at the density of 4 Â 10 4 cells per well in 96-well culture plate (100 lL per well), then with LPS (0.1 lg/mL) in the presence or absence of test compounds. After 24 h, culture supernatant was reacted with Griess reagent for 10 min at room temperature in the dark. The optical density at 540 nm was monitored by using microplate reader. Inhibition (%) ¼ 100 Â (A LPS treated À A LPS ＋ sample treated )/(A LPS treated À A untreated ). Dexamethasone was used as the positive control.

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

Funding
This work was supported by the National Key R&D Program of China (No.2017YFC1703800).