4-Methylthio-butanyl derivatives from the seeds of Raphanus sativus and their biological evaluation on anti-inflammatory and antitumor activities

Abstract

Ethnopharmacological relevance: Raphanus sativus seeds (Brassicaceae) known as Raphani Semen have long been used as anti-cancer and/or anti-inflammatory agents in Korean traditional medicine. This study was designed to isolate the bioactive constituents from the seed extracts of Raphanus sativus and evaluate their anti-inflammatory and antitumor activities.

Material and methods: Bioassay-guided fractionation and chemical investigation of a methanolic extract of the seeds of Raphanus sativus led to the isolation and identification of seven 4-methylthio-butanyl derivatives. Structural elucidation of the isolated compounds was carried out using 1D and 2D nuclear magnetic resonance (NMR) spectroscopy techniques ((1)H, (13)C, COSY, HMQC and HMBC experiments) and mass spectrometry.

Results: The isolated compounds were characterized as in the following: three new 4-methylthio-butanyl derivatives, sinapoyl desulfoglucoraphenin (1), (E)-5-(methylsulfinyl)pent-4-enoxylimidic acid methyl ester (2), and (S)-5-((methylsulfinyl)methyl)pyrrolidine-2-thione (3), together with four known compounds, 5-(methylsulfinyl)-4-pentenenitrile (4), 5-(methylsulfinyl)-pentanenitrile (5), sulforaphene (6), and sulforaphane (7). Full NMR data assignments of the three known compounds 4-6 were also reported for the first time. We evaluated the anti-neuroinflammatory effect of 1-7 in lipopolysaccharide-stimulated murine microglia BV2 cells. Compound 1 significantly inhibited nitrite oxide production with IC50 values of 45.36 μM. Moreover, it also reduced the protein expression of inducible nitric oxide synthase. All isolates were also evaluated for their antiproliferative activities against four human tumor cell lines (A549, SK-OV-3, SK-MEL-2, and HCT-15), and all of them showed antiproliferative activity against the HCT-15 cell, with IC50 values of 8.49-23.97 μM.

Conclusions: 4-Methylthio-butanyl derivatives were one of the main compositions of Raphanus sativus seeds, and activities demonstrated by the isolated compounds support the ethnopharmacological use of Raphanus sativus seeds (Brassicaceae) as anti-cancer and/or anti-inflammatory agents.

Keywords: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide; 4-Methylthio-butanyl derivatives; Anti-inflammation; Brassicaceae; CC; COSY; Cytotoxicity; DMEM; Doxorubicin (PubChem CID: 31703); Dulbecco's modified Eagle medium; ECL; ESI; FBS; HMBC; HMQC; HPLC; HR; HRP; IR; L-NMMA; LPLC; LPS; MS; MTT; N(G)-monomethyl-L-arginine; N(G)-monomethyl-L-arginine (PubChem CID: 135242); NCI; NMR; NO; NOS; Nitric oxide; RP; Raphanus sativus; SDS-PAGE; SRB; Sulforaphane (PubChem CID: 5350); Sulforaphene (PubChem CID: 6433206); TLC; UV; column chromatography; correlation spectroscopy; electrospray ionization; enhanced chemiluminescence; fetal bovine serum; heteronuclear multiple bond correlation; heteronuclear multiple quantum coherence; high resolution; horseradish peroxidase; iNOS; inducible nitric oxide synthase; infrared; lipopolysaccharide; low-pressure liquid chromatography; mass spectrometry; national cancer institute; nitric oxide; nitric oxide synthase; nuclear magnetic resonance; preparative high performance liquid chromatography; reversed-phase; sodium dodecyl sulfate polyacrylamide gel electrophoresis; sulforhodamine B; thin-layer chromatography; ultraviolet.