Specific Hydrolysis and Accumulation of Antiproliferative Lignans in the Fruit of Leuzea carthamoides (Willd.) DC
Anna Sólyomvárya, Zsolt Mervaib, Ibolya Molnár-Perlc, and Imre Boldizsára*
aDepartment of Plant Anatomy, Institute of Biology, Eötvös Loránd University, Pázmány Péter sétány 1/C, Budapest 1117, Hungary; bFirst Department of Pathology and Experimental Cancer Research, Semmelweis University, Üllői út 26, Budapest 1085, Hungary; cDepartment of Analytical Chemistry, Institute of Chemistry, Eötvös Loránd University, Pázmány Péter sétány 1/C, Budapest 1117, Hungary
*Corresponding author. phone: +36 1 381 21 65; fax: +36 1 381 21 66; e-mail: firstname.lastname@example.org
Dibenzylbutyrolactone-type lignan glycosides (tracheloside and carthamoside), their aglycones (trachelogenin and carthamogenin) and feruloyl-serotonin isomers were determined by LC-UV, LC-MS/MS and GC-MS techniques in the fruits of Leuzea carthamoides. The composition of the embryo and wall parts of fruits was analyzed before and after their hydrolysis. As a result of these studies, fruit part-specific accumulation of lignan glycosides and feruloyl-serotonins were confirmed, demonstrating that the embryo contains a high amount of lignan glycosides (tracheloside 32.9 mg/g, carthamoside 45.3 mg/g), while the wall part of the fruit accumulates feruloyl-serotonins (63.0 mg/g). Enzymatic hydrolysis of the embryo resulted in the quantitative transformation of lignan glycosides into their corresponding aglycones, allowing selective isolation of trachelogenin and carthamogenin. These aglycones were subjected to an antiproliferative study against the SW480 colon adenocarcinoma cell line. In this test, moderate activity of carthamogenin and a significant effect of trachelogenin were shown in a concentration range of 22-185 μM.
Fig. S1 HPLC-UV (λ=280 nm) elution profile of the unhydrolyzed fruit samples (A: whole fruit, B: embryo, C: fruit wall) completed by their corresponding enzymatic (D, E, F) and acidic (G, H, I) hydrolyzed versions. Peaks: 1, tracheloside; 2, carthamoside; 3 and 4 feruloyl-serotonin isomers; 5, trachelogenin; 6, carthamogenin; x, impurity. Data in parentheses represent the amount of individual compounds (normal printed), and the total amount of feruloyl-serotonin isomers (italic printed), given in mmol/100g. These data are the average values obtained from three parallel measurements; differences were characterized with the RSD percentages, varying from 2.0 RSD% (tracheloside in B sample) to 3.8 RSD% (carthamoside in C sample).
Fig. S2 Mass spectra of the isolated tracheloside (1A – 1D), carthamoside (2A – 2D), trachelogenin (5A – 5D) and carthamogenin (6A – 6D) obtained by LC-ESI-MS/(MS). Spectra 1A – 6A (first column) represent the ESI-MS spectra, while spectra 1B – 6D (second, third and forth columns) represent the ESI-MS/MS spectra obtained by three different collision induced dissociation (CID) energies [given in electron voltages (eV)]. Structures of fragment ions signed Fr a – Fr g are shown in Supplementary Fig. S3 with the same notations.
Fig. S3 Fragmentation patterns of lignans and feruloyl-serotonin. Masses of fragment ions (Fr a – Fr g) formed under different collision induced dissociation (CID) energies are shown in Supplementary Fig. S2.
Fig. S4 UV spectra (A-D) of lignan glycosides [tracheloside (A), carthamoside (B)] and those of their aglycones [trachelogenin (C), carthamogenin (D)] obtained from on-line LC separation (Supplementary Fig. S1A) of whole fruit extract. Numbers on the spectra indicate the absorption peaks (nm).
Fig. S5 GC-MS chromatograms of the unhydrolyzed intact whole fruit extract (trace A) and its enzymatic hydrolyzed version (trace B) completed with the MS spectra of characteristic compounds (trace C) and the fragmentation patterns of lignans (trace D). Fragment ions (Fr a – Fr c) found in trace C corresponds to those shown in trace D. Peaks: 1a, 1b, fructose; 2a, 2b, glucose; 3, inozitol; 4, linoleic acid; 5, stearic acid; 6, sucrose; 7, 9, 10 and 12 chlorogenic acid isomers; 8, sterol derivative; 11, trachelogenin; 13, carthamogenin; 14, 16, trisaccharides; 15, tracheloside; 17, carthamoside.
Fig. S6 Comparison of the lignan and feruloyl-serotonin composition in unhydrolyzed (Intact) and in enzymatic (Enz-30, Enz-60 and Enz-120) furthermore in acidic (Ac-30, Ac-60 and Ac-120), for 30, 60, and 120 min hydrolyzed whole fruit samples. Data are the average values obtained from three parallel measurements; differences were characterized with the RSD percentages, varying from 2.1 RSD% (trachelogenin in Ac-60 sample) to 5.3 RSD% (carthamoside in Enz-30 sample).