Patent Application
20070287674
The present invention deals with the characterization of a paniculoside IV (1) and ent-16,17-dihydroxy-(−)-kauran-19-oic acid (2) [X. Jiang, M. Yunbao, X. YunIong, Phytochemistry 1992, 31, 917]. Chemical structures of these two compounds are shown in
General Analytical Instrumentation: TLC: Kieselgel F254 (0.25 mm: Merck). Column chromatography (CC): silica gel (70-230 mesh; Merck), flash chromatography (FC): silica gel (230-400 mesh; Merck). Optical rotation: Jasco DIP-360 digital polarimeter. UV Spectra: Hitachi-UV-3200 spectrophotometer. IR spectra: Jasco-320-A spectrophotometer. 1H-NMR, 13C-NMR, COSY, HMQC and HMBC Spectra: Bruker spectrometer. EI-MS and FAB-MS spectra: JMS-HX-110 spectrometer.
The shade-dried ground plant material (whole plant) of Pulicaria undulata L. (Asteraceae) was exhaustively extracted with methanol at room temperature. The extract was evaporated and dissolved in water and partitioned with hexane, chloroform, ethyl acetate and n-butanol. The ethyl acetate soluble extract was subjected to column chromatography (silica gel, Hexane/CHCl3 mixtures of increasing polarity, CHCl3, CHCl3/MeOH mixtures of increasing polarity) and fifteen fractions (1-15) were collected. Pulicarside 1 was obtained from Fr. 8 when it was subjected to FC (silica gel, CHCl3/MeOH (10:90)). Compound 1 was obtained from Fr. 9 when it was subjected to FC (silica gel, CHCl3/MeOH (15:85). Compound 1 was also obtained when pulicarside 1 as subjected to acid hydrolysis: pulicarside 1 was refluxed with 0.5 N HCl for 2 h. After neutralization with NH4OH, it was extracted with n-butanol. The n-butanol fraction was evaporated under reduced pressure to give glucoside without acetonyl moiety, 1H-NMR data of which were identical with paniculoside IV (compound 1) (16, β-17-hydroxy-ent-1-α-auzan 19-O-D-glucopyranosyl ester [K. Yamasaki, H. Kohada, T. Kobayashi, N. Kaneda, R. Kasai, O. Tanaka, K. Nishi, Chem. Pharm. Bull. 1977, 25, 2895].
The chloroform soluble fraction was submitted to column chromatography (silica gel, Hexane/CHCl3 mixtures of increasing polarity) and twenty fractions (1-20) were collected. Compound 2 was obtained from Fr. 12 (EtOAc/hexane (45:55) and also from base hydrolysis of pulicarside 1 when it was refluxed with 5% aqueous KOH solution for 2 h. The mixture was then neutralized with a dilute HCl solution and extracted with n-butanol (3×6 ml). The combined n-butanol fractions were evaporated to gave aglycone having similar 1H-NMR data as that of already reported ent-16,17-acetonyl-(−)-kauran-19-oic acid [M. S. Correa, G. M. S. P. Guilhon, L. M. Conserva, Fitoterapia 1998, LXIX, 277].
α-Glucosidase (E.C.3.2.1.20) enzyme inhibition assay was performed according to the slightly modified method of Matsui et al. α-glucosidase (E.C.3.2.1.20) from Saccharomyces species, purchased from Wako Pure Chemical Industries Ltd. (Wako 076-02841). The enzyme inhibition was measured spectrophotometrically at pH 6.9 and at 37° C. using 0.7 mM p-nitrophenyl-α-D-glucopyranoside (PNP-G) as a substrate and 500 m units/mL enzyme, in 50 mM sodium phosphate buffer containing 100 mM NaCl. 1-Deoxynojirimycin (0.425 mM) and acarbose (0.78 mM) were used as positive control. The increment in absorption at 400 nm, due to the hydrolysis of PNP-G by α-glucosidase, was monitored continuously on microplate spectrophotometer (Spectra Max Molecular Devices, USA).) [T. Matsui, C. Yoshimoto, K. Osajima, T. Oki, and Y. Osajima. Biosci. Biotech. Biochem., 1996, 60, 2019].
Table 1 Result of In vitro quantitative studies on compounds 1 and 2 against known α-glucosidase inhibitors.
A critical analysis of the chemical structure shows that when the ring is cleaved in case of the compound, 1 (paniculoside IV) showed inhibitory effect on the enzyme (IC50 406.7±20) and when the sugar molecule is replaced by the COO— group from the molecule in case of the compound, 2 (Ent-16, 17-dihydroxy-(−)-kauran-19-oic acid) (IC50 62.2±0.008) the compound also showed a promising inhibitory activity against the enzymes compared to pulicarside 1. So the COO— group is playing a crucial role for the inhibitory effect on the enzyme.
