Four organosulfur compounds from the seeds of Capsella bursa-pastoris and their anti-inflammatory activities

Abstract Phytochemical investigation of the seeds of Capsella bursa-pastoris led to the isolation of four organosulfur compounds. There were two new compounds, 10-methylsulfinyl-decanamide (1) and 11-methylsulfinyl-undecanamide (2), along with two known compounds (3 − 4), which all have a sulfoxide group and an amide or a nitrile group. Their chemical structures were elucidated by analysing UV, IR, ESI-MS and NMR spectroscopy. In addition, compounds 1 − 4 were evaluated for their anti-inflammatory activities by using LPS-induced RAW 264.7 cells. Compounds 1 − 4 exhibited potential anti-inflammatory activities on NO release characterised by decreasing the mRNA expression levels of inducible NO synthase (iNOS), cytokines cyclooxygenase-2 (COX-2) and interleukin 6 (IL-6). Graphical Abstract


Introduction
Capsella bursa-pastoris (Brassicaceae), widely distributed all over the world (Han et al. 2015), is a kind of annual herb enriching with proteins, dietary fibers, vitamins, minerals and amino acids (Iurisson 1976;Maillard et al. 1988). As a typical plant homology of medicine and food, it has been commonly known as shepherd's purse with the function of clearing heat and eliminating dampness, suppressing hyperactive liver for improving eyesight, cooling blood to stop bleeding, and resolving food stagnancy and the clinical application for the treatment of nephritis edema, hematuria, hematochezia, menorrhagia, eye swelling and pain, infantile indigestion, diarrhea, dysentery, chyluria, hypertension and other diseases (Choi et al. 2017;Xie et al. 2022). Phytochemical studies have led the isolation of flavonoids (Kub ınov a et al. 2013;Lu et al. 2021), polyphenols (Lu et al. 2021), lignin (Cha et al. 2017), organic acids (Kuroda et al. 1976) and alkaloids (El-Abyad et al. 1990) from the aerial parts of this plant. Pharmacologic investigations have found that it has the effects on hemostatic (Kuroda and Kaku 1969), lowering blood pressure (Kuroda and Kaku 1969), anti-inflammatory (Choi et al. 2014), antioxidant (Yildirim et al. 2020) and anti-cancer (Kuroda et al. 1976).
Previous chemical and pharmacological studies had focused on the aerial parts of C. bursa-pastoris, but its seed was rarely reported. 'Ben Cao Gang Mu' (Compendium of Materia Medica) records that shepherd's purse seed has the function of improving eyesight, relieving eye pain, supplementing the deficiency of entrails (Heart, liver, spleen, lung and kidney) to treat abdominal distension, cure chills, clear heat and remove toxic substance. In addition, shepherd's purse seed is widely used in the folk treatment of jaundice in children. By comparison, we could speculate that its seed may be different from the aerial parts in terms of chemical composition and biological activities. Consequently, it was necessary to dig out the chemical constituents and biological activities of the seed of C. bursa-pastoris.
The structures of two known OSC were identified as 10-methylsulfinyl-decanenitrile (3) and 11-methylsulfinyl-undecanenitrile (4) by analyses of their ESI-MS (Figures S15 and S20) and NMR data, and comparison with those reported in the literature (Cha et al. 2018). The two known OSC (3-4) bearing a sulfoxide and a nitrile moiety were isolated for the first time from the seeds of C. bursa-pastoris.
As nutraceutical agents, OSC are widely present in our bodies and the natural environment, and have various effects such as immunomodulatory, fibrinolytic, antioxidant, anti-inflammatory, antiplatelet and antiaging actions (Goncharov et al. 2016). All our obtained compounds belong to OSC in this study, and the structure of new compounds 1 À 2 are novel as bearing both a sulfoxide group and an amide group. The characterisation of 1 À 2 enriched not only the diversity of chemical constituents of the seeds of C. bursa-pastoris, but also the diversity of OSC.

Effects of compounds 1-4 on cell viability of RAW 264.7 macrophages
The RAW 264.7 cells were treated with compounds 1 À 4 at a concentration of 100 lM and incubated for 24 h. The cell counting kit-8 (CCK-8) assay showed that cell viabilities of RAW 264.7 cells treated by 1 À 4 were over 85%, suggesting that 1 À 4 were not cytotoxic to RAW 264.7 cells (Table S1 in the supplementary material).

Effects of compounds 1-4 on NO production in LPS stimulated RAW 264.7 macrophages
Based on the cell viabilities in RAW 264.7 cells treated by compounds 1 À 4, the nontoxic concentration range (25 À 100 lM) was chosen for evaluating the inhibitory effects on LPSinduced NO production in RAW264.7 cells. The results (Table S1 and Figure S25 in the supplementary material) showed that 1-4 at a concentration ranging from 25 lM to 100lM exhibited potential anti-inflammatory activities by suppressing NO production.

Effects of compounds 1-4 on cytokine mRNA expression in LPS-stimulated RAW 264.7 macrophages
To further probe the inflammatory-relating action of mechanisms, qRT-PCR assay was applied to determine the inflammatory cytokines iNOS, COX-2 and IL-6 mRNA expression levels after treatment with compounds 1 À 4 at a concentration of 100 lM in LPS-stimulated RAW 264.7 cells. As shown in Figure S26 of the supplementary material, LPS stimulation led to higher mRNA expression levels of iNOS, COX-2 and IL-6, compared with the control group. Whereas compounds 1 À 4 significantly downregulated the mRNA expression levels of iNOS, COX-2 and IL-6 in LPS-induced RAW 264.7 cells, suggesting that the inhibitory effects of 1-4 on NO production might closely link to the downregulation of iNOS, COX-2 and IL-6.

General experimental procedures
A surveyor high performance liquid chromatography (Thermo, US) were used to record the UV spectra. Preparative HPLC separation was carried out on an LC-3000 liquid chromatography system, equipped with an UV detector (Beijing Chuangxin Hengtong Technology Co., Ltd., Beijing, China). The column applied in this work was an Innoval C18 column (21.2 Â 250 nm, 5 lm, Tianjin Bona Aijieer Technology Co., Ltd., Tianjin, China). The HR-ESI-MS was recorded on an Agilent G6230A TOF LC/MS (Agilent Technologies Co., Ltd., CA, US). IR spectra were recorded on a Frontier FT-IR with KBr pellets. 1 D and 2 D NMR experiments were recorded on 600 MHz for 1 H and 150 MHz for 13 C on an AVANCE NEO 600 (Bruker BioSpin, MA, US). The chemical shifts were given in ppm (d), relative to TMS as internal standard, and coupling constants are in Hz. Column chromatography (CC) was carried out by using silica gel (200-300 mesh, Marine Chemical Factory, Qingdao, China), Sephadex LH-20 (Pharmacia, Sweden) and macroporous resin AB-8 (Nan Kai College Chemical Inc., Tianjin, China), respectively. TLC was performed on GF254 plates pre-coated with silica gel 60 (5 À 20 lm, Yantai Huayang New Material Technology Co., Ltd., China).

Plant material
The seeds of Capsella bursa-pastoris were purchased from Bozhou Chinese medicine exchange center (the geographical coordinate of the producing area is 114 52 0 -115 31 0 E, 32 35 0 -33 08 0 N with an altitude of about 40 meters), Anhui province China in October 2021 and identified by Professor Bin Li, Department of Pharmaceutical Chemistry, Beijing Institute of Radiation Medicine. The seeds were identified through checking specimens by character identification method and microscopic identification method, and observing their aboveground parts after planting. A voucher Specimen (No. -1006 was deposited in specimen cabinet of Beijing Institute of Radiation Medicine.

Extraction and isolation
The dried seeds of C. bursa-pastoris (80 kg) were extracted with 70% EtOH. The pooled extracts were concentrated to yield a residue (6.69 kg) that was then extracted with petroleum ether (PE), dichloromethane (DCM), EtOAc and n-BuOH successively, yielding DCM extract (220 g) and n-BuOH extract (1000 g). A gray sludge precipitated from the DCM extract, and the precipitation was further separated by column chromatography over silica gel and preparative HPLC to afford compound 2 (840 mg). The remainder of DCM extract was further separated by column chromatography over silica gel, Sephadex LH-20 gel and preparative HPLC to afford compound 3 (6045 mg) and compound 4 (1967 mg).

Cell viability assay
The cell viability was evaluated using the CCK-8 assay (Xing et al. 2020). RAW 264.7 cells were seeded in 96-well plates at a density of 1 Â 10 4 cells per well and incubated overnight at 37 C under 5% CO 2 . Then, the cells were treated with compounds 1-4 at a concentration of 100 lM. After incubating under the same conditions for 24 h, 10 lL CCK-8 solution was added to each well. After 1-2 h incubation, the absorbance of each well was measured at 450 nm using a microplated reader (Multiskan MK-3, Thermo, USA).
3.6. Determination of NO content RAW 264.7 cells were plated in 96-well plates at a density of 1 Â 10 4 cells per well and incubated overnight at 37 C under 5% CO 2 . Then, the cells were treated with compounds 1 À 4 at a concentration of 25, 50 and 100 lM, respectively for 1 h, and then stimulated with LPS (1 lg/mL). Dexamethasone (Solarbio, D8040, Beijing, China) was used as the positive control. Twenty-four hours later, NO production was determined by measuring the nitrite content using Griess reagents (Hou et al. 2021). Fifty microliters of culture supernatants were transferred to 96-well plates and mixed with 50 lL Griess reagent A. After incubation at room temperature for 5 min, the samples were mixed with an equal volume Griess reagent B for another 5 min. The absorbance of each mixture at 540 nm was measured using a microplate reader.

Real-time polymerase chain reaction
RAW 264.7 cells were plated in 12-well plates at a density of 1 Â 10 5 cells per well and incubated overnight at 37 C under 5% CO 2 . Then, the cells were treated with compounds 1 À 4 at a concentration of 100 lM for 1 h, and then stimulated with LPS (1 lg/mL). Twenty-four hours later, the total RNAs were isolated using RNAprep pure Cell/Bacteria Kit (Tiangen, DP430, Beijing, China) according to the manufacturer's instructions. The total RNA was reversely transcripted into cDNA by FastKing RT Kit (Tiangen, KR116-02, Beijing, China). The primer sequences were as follows: All of the primers were provided by Tsingke (Tsingke Biotechnology Co., Ltd., Beijing, China). PCR amplification was carried out with a SuperReal PreMix Plus (SYBR Green) (Tiangen, FP205-02, Beijing, China). Under the following conditions: 5 C for 15 min followed by 40 cycles at 95 C for 10 s and 60 C for 32 s. b-actin was used as an internal control, and all reactions were performed three times. The relative gene expression was quantified using the 2-DDCt method (Gao et al. 2014).

Conclusion
The present paper has obtained four compounds from the seeds of C. bursa-pastoris., including two new compounds. Their chemical structures were characterised by spectroscopic methods, mainly ESI-MS and NMR data ( 1 H, 13 C and DEPT), and comparison with data reported in the literature. All these four compounds belong to OSC, and the structure of new compounds 1 À 2 are novel as bearing both a sulfoxide group and an amide group, so this study had enriched the species of OSC in the natural products. In addition, compounds 1 À 4 were screened for anti-inflammatory activities by using LPS-induced RAW 264.7 cells as a model. Compounds 1 À 4 exhibited potential anti-inflammatory activities on NO release characterised by decreasing the mRNA expression levels of inducible iNOS, COX-2 and IL-6, and the results presented herein agree with previous reports for the relation between organosulfur structural requirements and the inhibition of cytokine secretion.

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

Funding
The author(s) reported there is no funding associated with the work featured in this article.