Supplementary Materialsmolecules-18-07448-s001. initiatives. Concerning triterpenoids from L, Appendino reported 3-cv cv
Supplementary Materialsmolecules-18-07448-s001. initiatives. Concerning triterpenoids from L, Appendino reported 3-cv cv Cucurbita pepo with a homology-based PCR technique . Careful study of the seed products of L. provides resulted in the isolation of three book multiflorane triterpenoids 1C3 combined with the known substances 4 and 5. The buildings of 1C3 had been determined based on NMR spectroscopy, including 1D and 2D (1H, 1H-COSY, NOESY, HSQC, HMBC) NMR, and EIMS. 2. Dialogue and Outcomes The seed products of 693.4024) predicated on HREIMS. The UV range demonstrated a heteroannular diene moiety (utmost 230, 237, 248 nm, log 3.85, 3.80, 3.63). The IR range showed rings assignable to ester groupings (utmost 1713, 1287 cm?1) and a nitro group (utmost 1527, 1341 cm-1). The 1H- and 13C-NMR spectra (Desk 1) exhibited indicators assignable to seven tertiary methyls, ten CH2 groupings including an oxymethylene [H 4.11 and 4.17 (each 1H, d)], three 526 [MCin Hz)in KLF1 Hz)in Hz)586.4016) predicated on HREIMS. The UV absorption music group demonstrated a heteroannular diene (utmost 222, 237 nm, log 3.93, 3.95). The IR range showed the current presence of ester groupings (utmost 1743, 1718, 1271 cm-1). The 1H- and 13C-NMR spectra (Desk 1) exhibited indicators assignable to seven tertiary methyls, ten CH2 groupings including an oxymethylene [H 4.10, 4.15 (each 1H, d)], three 584.3864) predicated on HREIMS. The UV range demonstrated a 5(6),7,9(11)-triene moiety (utmost 227, 304, 315, 334 nm, log 4.19, 3.98, 4.00, 3.72). The IR Kaempferol cost range showed rings assignable to ester groupings (utmost 1725, 1239 cm-1). The 1H- and 13C-NMR spectra (Desk 1) exhibited signals due to seven tertiary methyls, nine CH2 groups including an oxymethylene [H 4.08, 4.16 (each 1H, d)], three 524 [MCAcOH]+ as a base ion peak. Based on the spectral data, the structure of 3 was Kaempferol cost established as 3-acetoxymultiflora-5(6):7:9(11)-trien-29-benzoate. Open in a separate windows Physique 5 Selected 1H-1H COSY and HMBC correlations for 3. Compounds 1C5 were evaluated for cytotoxic activity against HL-60 and P388 cells using MTT methods (Table 2) . Although, 2 exhibited poor cytotoxic activity against HL-60 (IC50 25.7 M) and P388 (IC50 75.1 M), 1 and 3C5 showed no activity against either cell line. Compound 3 showed melanogenesis inhibitory activity with low cytotoxicity at 100 M (melanin content 66.9%, cell viability 92.5%) (Table 3). Compound 2 exhibited strong melanogenesis inhibitory activity, although probably due to its cytotoxic action (cell viability 32.8%, 69.3%, and 87.6% at 100, 30, and 10 M, respectively). Table 2 Cytotoxic activity of multiflorane-type triterpenes from seeds. L. produced in USA (California), were purchased from JA (Japan Agricultural Co-opwration)-Takatsuki in May, 2011. 3.3. Isolation Process Air-dried seeds (10 kg) were ground and extracted 3 for 3 days each with MeOH (10 L) employing an automatic percolator. Removal of the MeOH under reduced pressure left a greenish residue which was partitioned between Et2O and H2O. Evaporation of the Et2O phase gave a yellowish residue (216.1 g) which was subjected to silica gel (3.5 kg) column chromatography. Elution of the column with CHCl3 gave residue A (Fr. No. 1C18, 39.5 g), B (Fr. No. 19C25, 14.9 g) and C (Fr. No. 26C30, 10.6 g). Elution of the column with CHCl3/EtOAc (10:1) afforded residues D (Fr. No. 31C33, 21.5 g) and E (Fr. No. 34C57, 13.4 g) and subsequent column chromatography with CHCl3/EtOAc (2:1) to give residues F (Fr. (Fr. No. 58C68, 2.0 g). Elution was continued with EtOAc and MeOH to give residues G (Fr. No. 69C74, 2.0 g) and H (Fr. No. 75C77, 4.5 g). Residue B was rechromatographed on a silica gel (230C400 mesh, 500 g) column using 0.048, CHCl3); HREIMS m/z 0.11, CHCl3); HREIMS m/z 0.255, CHCl3); HREIMS m/z /em (rel. int.): 584 (33) [M]+, 524 (100) [MCHOAc]+, 509 (52), 457 (11), Kaempferol cost 387 (35), 295 (23), 285 (36), 251 (30), 225 (51). 3.7. Cytotoxicity Assay The cytotoxicity assay was decided previously . Briefly, the HL-60 and P388 cell lines (each 1 104 cells in 100 L) were treated with test compounds for 72 h, and MTT answer was added to the wells. The produced cells were labeled with 5 mg/mL MTT in phosphate-buffered saline (PBS), and the absorbance of formazan dissolved with 20% sodium dodecyl sulfate (SDS) in 0.1 N HCl was measured at.