Phytochemical investigations and antiproliferative secondary metabolites from Thymus alternans Klokov growing in Slovakia

Context: Thymus alternans Klokov (Lamiaceae) is a neglected species of the genus Thymus (Sect. Serpyllum) endemic to Carpathian area, where it is used as a flavouring agent and for medicinal purposes. Objective: The aim of the work was to identify antiproliferative constituents from the flowering aerial parts of this plant. Materials and methods: Thymus alternans extracts were analyzed by HPLC-MSn and subjected to exten- sive chromatographic separations. The isolated compounds (phenolics and triterpenes) were structurally elucidated by MS and 1D and 2D NMR experiments. Essential oil (EO) composition was determined by GC-FID and GC-MS. Six purified triterpenes and EO were assayed for in vitro antiproliferative activity against a panel of human cancer cells, namely, breast (MDA-MB 231), colon (HCT-15 and HCT116), lung (U1810), pancreatic (BxPC3), melanoma (A375) and cervical carcinoma (A431) cells. Results: The structures of the isolated compounds were achieved on the basis of H-NMR and MS experiments. Luteolin-40-O-b-D-glucopyranoside (P1), chrysoeriol-7-O-b-D-glucopyranoside (P2), chrysoeriol- 5-O-b-D-glucopyranoside (P3), apigenin-7-O-b-D-glucopyranoside (P4), rosmarinic acid (P5), rosmarinic acid- 30-O-b-D-glucopyranoside (P6), caffeic acid-3-O-b-D-glucopyranoside (P7), 3a-hydroxy-urs-12,15-diene (T1), a-amyrin (T2), b-amyrin (T3), isoursenol (T4), epitaraxerol (T5), and oleanolic acid (T6). GC-MS analysis revealed that the EO of T. alternans was devoid of phenols and belonged to the nerolidol-chemotype, that is typical of the Sect. Serpyllum. The six purified triterpenes (T1-T6) were active with IC50 ranging from 0.5 to 5 lM being comparable or better than those of reference compounds betulinic acid and cisplatin. The EO exhibited significant effects on A375, MDA-MB 231 and HCT116 cell lines with IC50 in the range of 5–8 lg/mL. Conclusion: The reported results suggest that T. alternans can be considered as a good source of phyto- constituents with possible importance in the pharmaceutical field.