Metabolism of administered (2RS)-naringenin in flavonoid-producing cultured cells of Sophora flavescens

Abstract

Cultured cells of Sophora flavescens produce (2S)-naringenin-derived prenylated flavanone sophoraflavanone G and liquiritigenin-derived trifolirhizin 6Ј-O-malonate. The regulation of flavonoid biosynthesis was examined by analyzing the metabolites produced in the cultured cells fed (2RS)-naringenin. The amount of sophoraflavanone G in cells fed 0.1 or 0.3 mM (2RS)-naringenin was two-fold that in control cells, although the conversion ratio was only 5 to 10% of the administered (2S)-naringenin. On the other hand, (2R)-naringenin, which does not occur naturally, was efficiently converted into its 4Ј,7-di-O-b-D-glucoside. (2S)-Naringenin prenylation activity was higher at the logarithmic growth stage. The cells fed (2RS)-naringenin at a lower concentration (below 0.1 mM), accumulated sophoraflavanone G as the main prenylated flavanone. In contrast, cells fed 0.3 mM (2RS)-naringenin accumulated 8-prenylnaringenin and leachianone G, intermediates of sophoraflavanone G in large amounts. Accumulation of trifolirhizin 6Ј-O-malonate was suppressed by the addition of naringenin.Key words: Naringenin; (2R)-naringenin 4Ј,7-di-O-b-D-glucoside; Sophora flavescens; sophoraflavanone G. Original PaperCopyright © 2004 The Japanese Society for Plant Cell and Molecular Biology precursor of SFG but not TM, to the culture medium, and examined its effect on the production of these secondary metabolites. Materials and methods General(2RS)-Naringenin was purchased from Nacalai Tesque (Kyoto, Japan). HPLC-photodiode array analysis was carried out using a Waters Alliance PDA system (Tokyo, Japan). 1 H and 13 C NMR spectra were recorded in DMSO-d 6 with spectrometers (Varian Unity plus 500 and Varian Gemini 300) operating at 500 MHz and 300 MHz for 1 H, and 125 MHz for 13 C, respectively. Mass spectra were recorded on a JEOL JMS DX-303 spectrometer. UV and CD spectra were measured with a UV-1600 visible spectrophotometer and a J-725N spectrometer, respectively. Plant materials and culture methodsThe origin and subculturing of callus cultures (Yamamoto et al. 1991a) and the establishment of cell suspension cultures (Yamamoto et al. 1996) of S. flavescens were described previously. Cell suspension cultures (0.5 g) were subcultured in 100 ml-flasks containing 20 ml of Murashige and Skoog (1962) medium with 1 mM 2,4-D and 1 mM kinetin at 14 days interval on a rotary shaker at a speed of 100 rpm in the dark at 25°C. For the experiment, filter-sterilized 300 mM (2RS)-naringenin solution in 20 ml of DMSO was aseptically added to the flask (final concentration 0.3 mM), and cultured for another one day. Twenty ml of DMSO was added to the control cells. Isolation and identification of the metabolitesThe naringenin-fed cells collected from 60 flasks (120 g) were extracted three times with MeOH by ultrasonication in ice-water bath for 30 min. The MeOH extract after concentration was resuspended in H 2 O, partitioned with CHCl 3 and n-BuOH, successively. The residual H 2 O layer was passed through Diaion HP-20 column (Mitsubishi Chem, Tokyo, J...

Simultaneous Determination of Twenty-Two Components in Asari Radix et Rhizoma by Ultra Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Mass Spectrometry

“…Thus, the compound corresponding to peak 5 was tentatively identified as the isomer of (2R)-naringenin 5,7-di-O-glucoside. According to the UV spectra of peak 5, the compound corresponding to peak 5 may be (2R)naringenin 4′,7-di-O-hexoside [30]. According to the fragmentation pathways in the study by Quang et al [8], the compound corresponding to peak 18 was tentatively identified as N-isobutyl-2E,4E,8Z-decatetraenamide (l " Fig.…”

Analysis of Active Metabolites of Sophora flavescens for Indoleamine 2,3-dioxygenase and Monoamine Oxidases using Ultra-Performance Liquid Chromatography-Quadrupole time-of-Flight Mass Spectrometry

“…), the main compounds 4, 5, and 10 included m/z 445 [M−H] − , 471 [M−H] − , and 455 [M−H] − , respectively.Comparison to the literature led to the assignments of trifolirhizin (4), kushenol K (5), and kurarinol (10), respectively[13,[21][22][23]29]. The four minor metabolites retained in the SFR Fr.3 were identified.…”

“…The mass (MS) spectra of all metabolites were carefully compared in the context of the previous Sophora genus (Fabaceae) literature, including the experimental t R , UV, m/z values, High-resolution electrospray ionization mass spectrometry (HRESIMS) data, error ppm, Tandem mass spectrometry (MS 2 ), and molecular formulae data (sub. 1) [6,15,16,[20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37]. In S. flavescens, these metabolites are structurally diverse, containing alkaloids, flavanone, flavanol, flavone, isoflavone, pterocarpan, chalcone, furocoumarin, and dibenzoyl derivatives as substituents, which are encountered with prenyl, lavandulyl or glycoside groups [17, 18, 21, 24-29, 35, 38-40].…”