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  • The anticholinesterase activities of all subfractions derive

    2020-05-22

    The anticholinesterase activities of all subfractions derived from EtOAc extract were detected and summarized in . Fraction 5 and its resulting subfractions (5B, 5C, 5D, 5C3, 5C4, 5D3, and 5D4) were found to show attractive activities. Subsequently, ten compounds (–) were obtained from these active subfractions by repeated silica gel column chromatography and preparative TLC. Among them, Uridine was determined to be a novel oxidative ring-opening rotenoid (). Known compounds – were characterized as 4-methoxylonchocarpin (), dorspoinsettifolin (), isobavachromene (), deguelin (), tephrosin (), barbigerone (),′, 5′-dimethoxy-6,6-dimethylpyrano isoflavone (),′-hydroxyisolonchocarpin (), and 12a-hydroxyrotenone () by direct comparison of their physical and spectroscopic characteristics with literature data. Of which, compound was reported from this species for the first time. Compound was isolated as pale amorphous solid and determined as CHO by negative HR-ESI-MS (/ 457.1506 [M−H], calcd. for 457.1499), indicating twelve degrees of unsaturation. The six CC double bonds and two carbonyl groups were confirmed from H and C NMR data (). The extra four degrees of unsaturation, after counting CC and CO bonds were ascribed to tetracyclic skeleton of . The combined analysis of the C NMR and HSQC spectra confirmed the presence of 24 carbon signals comprising 2 carbonyl, 6 aromatic, 1 oxygenated methylene, 5 methane, one of which was oxygenated, 5 quaternary carbon, four of them was oxygenated, 2 methyl, and 3 methoxyl carbons. Analysis of the H NMR spectrum indicated the presence of a 1,2,3,4-tetrasubstitued aromatic ring with two proton system forming two doublets at 7.72 and 6.54. A -dimethyl pyran ring was conducted by H NMR (two olefinic doublets at 6.61 and 5.62, a -dimethyl resonance at 1.44 and 1.48) and C NMR (an oxygenated quaternary carbon at 77.6). Its location on aromatic ring was determined on the basis of the HMBC correlations shown in . Additionally, the H NMR spectrum exhibited the typical ABC spin system for a propanol moiety with a pair of nonequivalent methylene proton signals at 4.01 and 4.14 and a methine proton multiplet at 4. 49. The C NMR and HSQC spectra showed both oxygenated methylene ( 84.2) and quaternary carbon ( 75.3) and a carbonyl resonance at 194.2. All the information was indicative of a 3-hydroxyisoflavanone skeleton for Uridine . Thus the propanol moiety must be located in ring C, which was confirmed by the HMBC correlations between the H-2 ( 4.49) with C-3 ( 75.3), C-4 ( 190.2), C-1′ ( 136.4) and C-9 ( 156.4). Apparently, the signals of the B-ring in were quite different with common benzene ring in normal isoflavanone. The placement of three methoxy substituents and their substitution pattern in B-ring were further determined from the HMBC correlations between one methoxyl proton ( 3.79) with C-2′ ( 168.8), the other two methoxyl protons ( 3.09, 3.26) with C-5′ ( 94.3) and NOESY correlations between 2′-methoxyl ( 3.79) with H-3′ ( 5.59), 5′-methoxyls ( 3.09, 3.26) with H-6′ ( 6.76). Finally, one relatively rare 2,2-dimethoxy-5-methoxycyclohexa-3,5-dienone moiety was deduced from detailed 1D and 2D NMR spectrum analysis. Furthermore, its attachment site in C-3 was supported by the HMBC cross peaks from H-6′ ( 6.76) to C-3 ( 75.3) and C-4 ( 190.2) (). Thereby, the planar structure of compound was established as shown in . It possesses chiral centers at C2 and C3, and therefore has four possible stereoisomers. Since it is quite difficult to find a reference compound with similar 2-sbustituted 3-hydroxyisofalvonone skeleton, the absolute configuration at C-3 of compound was determined by comparing its CD data with 3-hydroxyflavanones (trivial name kenusanone F 7-methyl ether). For (3)-isoflavanones with the B ring in the favoured equatorial position, a positive Cotton effect in the range (ca. 320–350 nm) for the n → π carbonyl transition can be predicted from the octant rule modified for the cyclic arylketone., , A positive Cotton effect at 347 nm indicated a same 3 configuration of compound . However, according to the Cahn–Ingold–Prelog rules, the priority order changes when hydrogen at C-3 in isoflavanones is replaced with hydroxyl group in 3-hydroxyisoflavanones. So the designation at C-3 in will be for this reason.