Masanori Kuroyanagi

Flavonoid glycosides and limonoids from Citrus molasses

Molasses of tangerine orange (Citrus unshiu Markovich) is obtained as a waste product in the course of tangerine orange juice production. This molasses is expected to be a useful source of organic compounds such as flavonoids and limonoids. To elucidate a use for this molasses waste, we isolated and identified its organic constituents. Two new flavanonol glycosides were isolated from tangerine orange molasses, along with several flavonoids such as hesperidine, narirutin, eriodictyol, 3′,4′,5,6,7,8-hexamethoxy-3-O-β-d-glucopyranosyloxyflavone, and 3′,4′,5,6,7,8-hexamethoxy- 3-β-d-[4-O-(3-hydroxy-3-methylglutaloyl)]-glucopyranosyloxyflavone, and limonoids such as limonin, nomilin, and cyclic peptide, citrusin III. The structures of the new flavanonol glycosides were determined as (2R,3R)-7-O-(6-O-α-l-rahmnopyranosyl-β-d-glucopyranosyl)-aromadendrin and 7-O-(6-O-α- l-rahmnopyranosyl-β-d-glucopyranosyl)-3,3′,5,7-tetrahydroxy-4′-methoxyflavanone by means of spectral analyses using 1H-NMR, 13C-NMR, and 2D-NMR. Of these compounds, flavanone glycoside, hesperidin and narirutin were isolated as the main constituents. Thus, molasses is a promising source of flavonoid glycosides.

Preparative separation of the saponin lancemaside a from Codonopsis lanceolata by centrifugal partition chromatography

INTRODUCTION Lancemaside A is a saponin that inhibits decreases in blood testosterone level and thus prevents or ameliorates symptoms associated with male climacteric disorder. Our initial attempt to preparative isolation of lancemaside A from the saponin fraction of Codonopsis lanceolata roots by a preparative HPLC did not give a clear result. OBJECTIVE To develop a simple and efficient method for the preparative isolation of lancemaside A from the hot water extract of C. lanceolata roots using centrifugal partition chromatography (CPC). METHODOLOGY The saponin fraction obtained from the hot water extract of C. lanceolata roots was used as the sample for preparative-scale separation of lancemasides by CPC using n-hexane:n-butanol:methanol:0.1% aqueous formic acid (3:4:1:6, v/v) as the two-phase solvent system. The upper phase (organic phase) of the two-phase solvent system was used as the mobile phase, and 0.5 g of saponin fraction was applied for separation by CPC. Each fraction that was separated by CPC was analysed by HPLC, and the fractions containing each of the separated compounds were pooled together, and then were purified by simple preparative HPLC. RESULTS The demonstrated separation sequence, hot water extraction, DIAION HP-20 column chromatography, CPC and preparative HPLC, yielded lancemaside A, foetidissimoside A and astersaponin Hb in their pure forms. CONCLUSION The simple and efficient method for the preparative isolation of lancemaside A along with two other saponins, foetidissimoside A and astersaponin Hb, from the saponin fraction of C. lanceolata was established using CPC.

Rapid identification of triterpenoid saponins in the roots of Codonopsis lanceolata by liquid chromatography–mass spectrometry

Liquid chromatography coupled with sequential mass spectrometry (LC–MSn) has been used to identify 3,28-bidesmosidic triterpenoid saponins, lancemaside A (1), foetidissimoside A (2), aster saponin Hb (3), lancemaside E (4), lancemaside B (5), lancemaside F (6), lancemaside G (7), lancemaside C (8), and lancemaside D (9) in the roots of Codonopsis lanceolata. Structural information about both the aglycone and the sugar moiety at the C-3 position of saponins was obtained in the negative-ion mode. On the other hand, positive-ion spectra mainly provide structural information about the sugar chains of saponins, especially the oligosaccharide moiety at the C-28 position. During subsequent fragmentation of the product ions derived from the oligosaccharide moiety at the C-28 position, fragments produced by sequential loss of a monosaccharide unit were observed. Furthermore, the structural features of two unknown saponins in the roots of C. lanceolata were assigned on the basis of the fragmentation patterns of the known saponins. These studies demonstrate that LC–MSn analysis has great potential for the identification and characterization of triterpenoid saponins in plant extracts.

Investigation of UV-A light irradiation on tomato fruit injury during storage

We investigated the effect of UV-A light (wavelength 315 to 400 nm) irradiation during storage on tomato fruit injury. Mature green tomato fruit (cv. House Momotaro) were exposed to UV-A at doses of 0.02, 0.5, and 2 mW x cm(-2) throughout storage at 25 degrees C. The physiological disorders, fruit ripening, superoxide dismutase (SOD) activity, and increases in fruit temperature were evaluated. All UV-A-irradiated and nonirradiated tomatoes developed a full red color at the same time (2 weeks). Irradiated fruit ripened normally, and exposure of tomato fruits to UV-A did not lead to the discoloration of ripe tomato fruit at any dosage. The fruit temperature did not increase in response to various UV-A light doses and exposure times, and none of the UV-irradiated fruits showed physiological disorders (dull skin blemish, pitting). The SOD activity of UV-A-irradiated fruit exposed to the various UV-A doses did not significantly (P = 0.05) differ from that of fruit stored in dark conditions. The SOD results imply that UV-A light might not induce reactive oxygen species in UV-A-irradiated fruit.

2,5-Dihydroxy-4-methoxybenzophenone: a novel major in vitro metabolite of benzophenone-3 formed by rat and human liver microsomes.

1. When benzophenone-3 (2-hydroxy-4-methoxybenzophenone; BP-3) was incubated with liver microsomes of untreated rats in the presence of NADPH, the 5-hydroxylated metabolite, 2,5-dihydroxy-4-methoxybenzophenone (5-OH-BP-3), was formed as a major novel metabolite of BP-3. The 4-desmethylated metabolite, 2,4-dihydroxybenzophenone (2,4-diOH-BP), previously reported as the major in vivo metabolite of BP-3, was also detected. However, the amount of 5-OH-BP-3 formed in vitro was about the same as that of 2,4-diOH-BP. 2. The oxidase activity affording 5-OH-BP-3 was inhibited by SKF 525-A and ketoconazole, and partly by quinidine and sulfaphenazole. The oxidase activity affording 2,4-diOH-BP was inhibited by SKF 525-A, ketoconazole and α-naphthoflavone, and partly by sulfaphenazole. 3. The oxidase activity affording 5-OH-BP-3 was enhanced in liver microsomes of dexamethasone-, phenobarbital- and 3-methylcholanthrene-treated rats. The activity affording 2,4-diOH-BP was enhanced in liver microsomes of 3-methylcholanthrene- and phenobarbital-treated rats. 4. When examined recombinant rat cytochrome P450 isoforms catalyzing the metabolism of BP-3, 5-hydroxylation was catalyzed by P450 3A2, 1A1, 2B1, 2C6 and 2D1, while 4-desmethylation was catalyzed by P450 2C6 and 1A1.