Two new chlorinated sesquiterpenes from Sigesbeckia orientalis

Abstract Two new chlorinated guaianane-type sesquiterpenes (named chlosigesolides A and B) together with eight known compounds were isolated from the leaves and twigs of Sigesbeckia orientalis. Their structures were determined by analysis of HR-ESI-MS, 1D- and 2D-NMR spectral data as well as comparison with the literature. Absolute configurations of new compounds were elucidated by NOESY and ECD methods. Chlosigesolide A inhibited NO production in LPS-stimulated RAW264.7 macrophages with IC50 value of 10.9 ± 0.8 μM. Other compounds exhibited inhibitory activity at IC50 in range of 26.5 to 49.7 μM. Graphical Abstract


Introduction
Herba Siegesbeckiae is an oriental medicine which typically refers for the dried aerial parts of Sigesbeckia orientalis L., S. pubescens (Makino) Makino, or S. glabrescens (Makino) Makino.It is being used in the traditional medicinal remedies to treat inflammation, especially rheumatism (Tao et al. 2021;Wang et al. 2021b).Chemical constituents from Sigesbeckia species and their anti-inflammatory activity, therefore, have been attracted a lot of studies in recent years.Sesquiterpenoids (Hang et al. 2021;Sun et al. 2021;Gao et al. 2021b), diterpenoids (Engels et al. 2020;Trang et al. 2021;Wang et al. 2021a;Hu et al. 2022), and flavonoids (Gao et al. 2021a) were reported to be major compositions of Sigesbeckia species.Hundreds of compounds have been isolated from the Herba Siegesbeckiae which showed anti-inflammatory activity, anti-bacterial activity, and cytotoxic activity (Tao et al. 2021;Wang et al. 2021b).In present study, chemical constituents from S. orientalis were attempted to investigate and led to the identification of two new chlorinated sesquiterpenes together with other eight known compounds.Anti-inflammatory activity of the isolated compounds was evaluated by inhibition of NO production in LPS-stimulated RAW264.7 macrophages.

Results and discussion
Chlosigesolide A (1) was isolated as a white amorphous powder.A molecular formula of 1 was determined to be C 20 H 25 O 6 Cl by a cluster of quasi-molecular ion peaks at m/ z 397.1416 demonstrated for (Z)-2 0 -methylbut-2 0 -enoyl group (angeoyl group).Finally, analysis ECD of 1 showed similar Cotton effects (negative Cotton effects at wavelengths 200-220 nm) with that of siegesorienolide C (negative Cotton effects at wavelengths 200-220 nm) indicating the same absolute configurations (Hang et al. 2021).According to the CIP priority rules, absolute configurations of 1 were determined to be 3S, 4S, 5S, 6S, 7 R, 8 R, and 9 R (Figure 1).Chlosigesolide B (2) was isolated as a white amorphous powder.A molecular formula of 2 was determined to be C 22 H 27 O 7 Cl by ammonium adductive molecular ion peaks at m/z 456.1800 with that of 1 and siegesorienolide C indicating the same absolute configurations (Hang et al. 2021).According to the CIP priority rules, absolute configurations of 2 were determined to be 3S, 4S, 5S, 6S, 7S, 8 R, and 9 R (Figure 1).
Compounds 1-10 were evaluated for their anti-inflammatory activity by inhibition of the NO production in LPS-stimulated RAW264.7 cells (Tham et al. 2021).At concentration as high as 50 mM, none of significant cytotoxic on the cells were found.Effect of compounds on NO production were then examined at concentrations below 50 mM.As shown in the Table S2, compounds 1-10 exhibited NO inhibitory activity with IC 50 values in the range of 10.9 to 49.7 lM.Compound 1 potentially inhibited NO production (IC 50 10.9 ± 0.8 lM) in comparison with positive control L-NMMA (IC 50 35.6± 3.0 lM).Our results suggest that the NO inhibition of compounds 1-10 could partly explain for the anti-inflammatory property of S. orientalis which has been used in the folk medicinal remedies for the treatment of inflammation.

General
Optical rotations were recorded using a Jasco P2000 polarimeter.ECD spectra were acquired on a Chirascan spectrometer.NMR spectra were measured on a Bruker AVANCE III spectrometer.HR-ESI-MS was obtained on an Agilent 6530 Accurate-Mass Q-TOF LC/MS system.Column chromatography was carried out using silica gel (40-63 mm) or ODS (150 mm) as stationary phase.Thin layer chromatography was performed on pre-coated plates.Preparative HPLC was acquired on an Agilent 1100 HPLC system (including quaternary pump, autosampler, DAD detector) equipped with YMC J'sphere ODS-H80 column (20 Â 250 mm, 4 mm).Flow rate was set at 3 mL/min.

Plant material
Samples of Sigesbeckia orientalis L. were collected at Tam Dao National Park (21 27 0 08.9 00 N, 105 38 0 30.6 00 E, 809 m), Vinh Phuc province, Vietnam in January 2021 and taxonomically identified by Dr. Nguyen The Cuong, Institute of Ecology and Biological Resources, VAST.Voucher specimen (No.BKHT21) was kept at the Hanoi University of Science and Technology.

Conclusions
Phytochemical study on the leaves and twigs of S. orientalis obtained two new chlorinated guaianane-type sesquiterpenes, named chlosigesolides A and B. Their structures were determined by analysis of HR-ESI-MS, 1 D-and 2 D-NMR spectral data.Absolute configurations of them were confirmed by ECD spectra.Chlosigesolide A inhibited NO production in LPS-stimulated RAW264.7 macrophages with IC 50 value of 10.9 ± 0.8 lM.