Induction of Isoflavonoid and Retrochalcone Branches of the Flavonoid Pathway in Cultured Glycyrrhiza echinata Cells Treated with Yeast Extract

Yeast extract-treated suspension cultures of a new cell line, AK-1, of Glycyrrhiza echinata were induced to produce an isoflavonoid phytoalexin (medicarpin) and metabolites of retrochalcone/flavone pathway (echinatin, licodione, and 7,4′-dihydroxyflavone). From these cells, putative full-length cDNAs encoding cytochrome P450s,(2S)-flavanone 2-hydroxylase and isoflavone 2′-hydroxylase, were cloned.     

In Vitro Antioxidative Activity of (−)-Epicatechin Glucuronide Metabolites Present in Human and Rat Plasma

Recently we identified four conjugated glucuronide metabolites of epicatechin, (?)-epicatechin-3′-O-glucuronide (E3′G), 4′-O-methyl-(?)-epicatechin-3′-O-glucuronide (4′ME3′G), (?)-epicatechin-7-O-glucuronide (E7G) and 3′-O-methyl-(?)-epicatechin-7-O-glucuronide (3′ME7G) from plasma and urine. E3′G and 4′ME3′G were isolated from human urine, while E7G and 3′ME7G were isolated from rats that had received oral administration of (?)-epicatechin (Natsume et al. (2003), Free Radic. Biol. Med. 34, 840–849). It has been suggested that these metabolites possess considerable in vivo activity, and therefore we carried out a study to compare the antioxidant activities of the metabolites with that of the parent compound. This was achieved by measuring superoxide scavenging activity, reduction of plasma TBARS production and reduced susceptibility of low-density-lipoprotein (LDL) to oxidation. (?)-Epicatechin was found to have more potent antioxidant activity than the conjugated glucuronide metabolites. Both (?)-epicatechin and E7G had marked antioxidative properties with respect to superoxide radical scavenging activity, plasma oxidation induced by 2,2′-azobis-(2-aminopropane) dihydrochloride (AAPH) and LDL oxidation induced by copper ions or 2,2′-azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN). In contrast, the other metabolites had light antioxidative activities over the range of physiological concentrations found in plasma.     

Erymildbraedin A and B,two novel cytotoxic dimethylpyrano-isoflavones from the stem bark of Erythrina mildbraedii: evaluation of their activity toward endocrine cancer cells

Two new dimethylpyrano-isoflavones, named erymildbraedin A (4) and B (5), were isolated from the stem bark of the Cameroonian medicinal plant Erythrina mildbraedii, along with four known ones, the linear congeners, scandenone (1), erysenegalinsein M (2), 5,4′-dihydroxy-2′-methoxy-8-(3,3-dimethylallyl)-2″,2″-dimethylpyrano[5,6:6,7]isoflavone (3), and the angular isoflavone eryvarin B (6), and two other compounds, fraxidin and scoparone. Their structures were elucidated by the usual spectroscopic methods and isoflavone effects on the growth of human breast, prostate, and endometrial adenocarcinoma cells were determined. Isoflavones 1, 3, and 6 strongly inhibited the growth of all three cell lines, supporting the notion that a non-oxidized isoprenyl group at C-8 is requisite for cytotoxic activity.     

The plant antifungal isoflavone genistein is metabolized by Armillaria mellea Vahl to give non-fungitoxic products

Abstract

Armillaria mellea, the causal agent of root rot, is a fungal pathogen which proved able to convert the leguminous plant antifungal compound 4′,5,7-trihydroxyisoflavone (genistein) into intermediate metabolites. After suitable periods of incubation, the metabolites were extracted and concentrated from liquid culture media, containing both the isoflavone and the fungus. After purification by column chromatography, the molecular structure of the metabolites was determined by means of mass spectrometry and nuclear magnetic resonance analyses. Five different compounds were identified: 1,3,5-trihydroxybenzene, 4-hydroxyphenylacetic acid, 2,5-dihydroxyphenylacetic acid (homogentisic acid), its lactone 5-hydroxy-2(3H)-benzofuranone, and 1,4-benzoquinone. In vitro experiments showed that while the starting compound, i.e. genistein, has some activity in inhibiting the growth of the fungal pathogen, the degradation products are devoid of any appreciable fungitoxic activity. Moreover, results show that the isoflavone metabolites can be, at least partially, utilized by A. mellea as a carbon source.     

Methyl Linoleate Dimer and Methyl 8-Phenyloctanoate in the Copper-catalyzed Decomposition Products of Methyl Linoleate Hydroperoxides

Cochliophilin A (5-hydroxy-6,7-methylenedioxyflavone, 1), known as a host-specific attractant towards the zoospores of Aphanomyces cochlioides, a cause of root rot and damping-off diseases of Chenopodiaceae, was found in the Amaranthaceae plant, Celosia cristata, that is susceptible to the pathogen. The content of 1 in Celosia seedlings was quantified as 1.4 μg/g fresh weight. A new isoflavone, cristatein (5-hydroxy-6-hydroxymethyl-7,2′-dimethoxyisoflavone, 2), and five known flavonoids were also identified.     

Metabolism of the Fungicide S-n-Butyl S′-p-tert-Butylbenzyl N-3-Pyridyldithiocarbonimidate (S-1358) in Rats

S-1358 was rapidly absorbed, metabolized and readily excreted via urine and feces from orally dosed rats. Excretion of radioactivity was almost complete within 4 days. The radioactivity was distributed mainly in stomach, intestines, liver and kidneys. It seems that S-1358 and its metabolites do not persist in organs and tissues following a single oral dosing.

Major urinary metabolites of the benzyl-labeled S-1358 were p-(1,1-dimethyl-2-hydroxyethyl)benzyl methyl sulfide [B], p-(1,1-dimethyl-2-hydroxyethyl)benzyl methyl sulfone [A], p-(1-methyl-1-carboxylethyl)benzyl methyl sulfide [D], p-(1-methyl-1-carboxylethyl)benzyl methyl sulfone [C] and their glucuronide conjugates. Fecal metabolites were S-n-butyl S′-(1, 1-dimethyl-2-hydroxyethyl)benzyl N-3-pyridyldithiocarbonimidate [MR], A, B, C and D. These metabolites were also found in the bile. The pyridine-labeled S-1358 gave rise to 2-(3′-pyridylimino)-4-carboxylthiazolidine [HM] and 3-aminopyridine [AP] in the urine, and MR and AP in the feces. Intact S-1358 was a major component of the fecal radioactivity.     

Multiplex CRISPR/Cas9‐mediated metabolic engineering increases soya bean isoflavone content and resistance to soya bean mosaic virus

Isoflavonoids, which include a variety of secondary metabolites, are derived from the phenylpropanoid pathway and are distributed predominantly in leguminous plants. These compounds play a critical role in plant–environment interactions and are beneficial to human health. Isoflavone synthase (IFS) is a key enzyme in isoflavonoid synthesis and shares a common substrate with flavanone‐3‐hydroxylase (F3H) and flavone synthase II (FNS II). In this study, CRISPR/Cas9‐mediated multiplex gene‐editing technology was employed to simultaneously target GmF3H1, GmF3H2 and GmFNSII‐1 in soya bean hairy roots and plants. Various mutation types and frequencies were observed in hairy roots. Higher mutation efficiencies were found in the T₀ transgenic plants, with a triple gene mutation efficiency of 44.44%, and these results of targeted mutagenesis were stably inherited in the progeny. Metabolomic analysis of T₀ triple‐mutants leaves revealed significant improvement in isoflavone content. Compared with the wild type, the T₃ generation homozygous triple mutants had approximately twice the leaf isoflavone content, and the soya bean mosaic virus (SMV) coat protein content was significantly reduced by one‐third after infection with strain SC7, suggesting that increased isoflavone content enhanced the leaf resistance to SMV. The isoflavone content in the seeds of T₂ triple mutants was also significantly increased. This study provides not only materials for the improvement of soya bean isoflavone content and resistance to SMV but also a simple system to generate multiplex mutations in soya bean, which may be beneficial for further breeding and metabolic engineering.     

Estrogenic and Acetylcholinesterase-Enhancement Activity of a New Isoflavone, 7,2′,4′-Trihydroxyisoflavone-4′-O-β-D-Glucopyranoside from Crotalaria Sessililflora

A new isoflavone, 7,2′,4′-trihydroxyisoflavone-4′-O-β-D-glucopyranoside has been isolated from the aerial part of Crotalaria sessiliflora. The isoflavone glucoside enhanced the proliferation of the MCF-7 human breast cancer cell line, which possesses estrogen receptor (ER) and responds to estrogen in culture. The estrogenic property of the isoflavone glucoside was blocked by the known ER antagonist tamoxifen, indicating the involvement of the ER. Furthermore, the isoflavone glucoside was found to enhance the acetylcholinesterase (AChE) activity of the rat neuronal cell line PC12 at low concentrations of nerve growth factor (NGF).     

Lasiolactols A and B Produced by the Grapevine Fungal Pathogen Lasiodiplodia mediterranea

A strain of Lasiodiplodia mediterranea, a fungus associated with grapevine decline in Sicily, produced several metabolites in liquid medium. Two new dimeric γ‐lactols, lasiolactols A and B ( 1 and 2 ), were characterized as (2S*,3S*,4R*,5R*,2′S*,3′S*,4′R*,5′R*)‐ and (2R*,3S*,4R*,5R*,2′R*,3′S*,4′R*,5′R*)‐(5‐(4‐hydroxymethyl‐3,5‐dimethyl‐tetrahydro‐furan‐2‐yloxy)‐2,4‐dimethyl‐tetrahydro‐furan‐3‐yl]‐methanols by IR, 1D‐ and 2D‐NMR, and HR‐ESI‐MS. Other four metabolites were identified as botryosphaeriodiplodin, (5R)‐5‐hydroxylasiodiplodin, (–)‐(1R,2R)‐jasmonic acid, and (–)‐(3S,4R,5R)‐4‐hydroxymethyl‐3,5‐dimethyldihydro‐2‐furanone ( 3  –  6 , resp.). The absolute configuration (R) at hydroxylated secondary C‐atom C(7) was also established for compound 3 . The compounds 1  –  3 , 5, and 6 , tested for their phytotoxic activities to grapevine cv. Inzolia leaves at different concentrations (0.125, 0.25, 0.5, and 1 mg/ml) were phytotoxic and compound 5 showed the highest toxicity. All metabolites did not show in vitro antifungal activity against four plant pathogens.     

Flemiphyllin,an isoflavone from stems of Flemingia macrophylla

A new isoflavone, designated flemiphyllin, was isolated from a petrol extract of the stems of Flemingia macrophylla. Its structure was established as 5,7,4′-trihydroxy-3′,5′,8-tri(3-methylbut-2-enyl) isoflavone on the basis of physical and chemical evidence.     

Chemical and Biological O-Demethylation of Rotenone Derivatives

3-O-Demethyl and 2,3-O,O-didemethyl derivatives of natural rotenone (5′β-rotenone), 5′α-rotenone, d-epirotenone (5′β-epirotenone) and 5′α-epirotenone are obtained upon reacting 5′β-rotenone or 5′β-epirotenone with two or three molar equivalents of boron tribromide followed by recyclization of the E-ring using sodium bicarbonate. 3-Methoxy-¹⁴C-5′β-rotenone is prepared in 16% yield by treating 3-O-demethyl-5′β-rotenone with methyl-¹⁴C iodide in the presence of alkali followed by epimerization of the ¹⁴C-5′β-epirotenone byproduct for increased yield of ¹⁴C-5′β-rotenone. 3-O-Demethylation is established as a detoxification mechanism for 5′β-rotenone or for one of its metabolites based on the expiration by mice and rats of 27% and 13%, respectively, of the administered radiocarbon as ¹⁴carbon dioxide.     

Quercetin metabolites and protection against peroxynitrite-induced oxidative hepatic injury in rats

Quercetin has strong antioxidant potency. Quercetin-3′-O-sulphate (Q3′S) and quercetin-3-O-glucuronide (Q3GA) are the main circulating metabolites after consumption of quercetin-O-glucoside-rich diets by humans. However, information about how these quercetin metabolites function in vivo is limited. Hence, this study evaluated the efficacy of Q3′S and Q3GA for the protection of oxidative injury using in vitro and in vivo experiments. Peroxynitrite-mediated hepatic injury in rats was induced by administration of galactosamine/lipopolysaccharide (GalN/LPS). Twenty-four hours after GalN/LPS treatment, plasma ALT and AST levels δ increased significantly. However, pretreatment with 4^G-α-D-glucopyranosyl rutin, a quercetin glycoside (30 mg/kg body weight), prevented these increases and reduced nitrotyrosine formation, indicating that consumption of quercetin glycosides prevent oxidative hepatotoxicity. Moreover, physiological levels of Q3′S and Q3GA (1 µM) effectively prevented peroxynitrite-induced nitrotyrosine formation in human serum albumin in in vitro experiments. These findings indicate peroxynitrite-induced oxidative hepatotoxicity is protected by the in vivo metabolites of quercetin, Q3′S and Q3GA.     

Chemical constituents of the root bark of Erythrina droogmansiana

Two new compounds namely 7,4′-dihydroxy-2′-methoxy-3′-(3-methylbut-2-enyl)isoflavanone (1) and 5,7,3′-trihydroxy-8-(3-methylbut-2-enyl)-[6′′,6′′-dimethylpyrano(2′′,3′′:4′,5′)]isoflavone (4) were isolated from the root bark of Erythrina droogmansiana together with eight known compounds. Their structures were elucidated on the basis of spectroscopic analyses (1D- and 2D-NMR and ESI-HRMS) and by comparison with literature data. In addition, the correct ¹³C NMR of 5,7,2′,4′-tertahydroxy-8,5′-di-(3-metylbut-2-enyl)isoflavone (6) was assigned. The DPPH free radical scavenging properties of the isolated compounds were evaluated. Compounds 4–7 showed weak to moderate DPPH free radical scavenging activities.     

Isopropyl m-Chlorocarbanilate and Its Hydroxylated Metabolites: Their Effects on Cell Suspensions and Cell Divison in Soybean and Carrot

Hydroxylated metabolites of isopropyl m-chlorocarbanilate (chlorpropham) are found in intact soybean plants (Glycine max Merr.). The metabolites are isopropyl 2-hydroxy-5-chloro-carbanilate (2OH) and isopropyl 3-chloro-4-hydroxycarbanilate (4OH). The phytotoxicity of these metabolites and chlorpropham was tested in cell suspensions and roots of intact soybean seedlings and cell cultures of carrot (Daucus carota L.). The growth of soybean cell suspensions was inhibited with 50 μM chlorpropham. Ten μM chlorpropham usually slowed initial growth of the cultures while 5 μM and 0.1 μM chlorpropham had no effect. The 2OH and 4OH metabolites had no significant effect on dry weight over the same concentration range. Some metabolism of chlorpropham, 2OH and 4OH occurred during 6 and 48 h of incubation with soybean cells. The results are interpreted to mean that all three analogs penetrated into the cells, were metabolized, and some of the metabolites excreted back into the medium. Mitotic index studies of intact 3-day-old soybean roots showed that 2OH inhibited mitosis to a greater extent than chlorpropham, whereas 4OH produced only a slight and insignificant reduction compared to controls. Chlorpropham, 2OH and 4OH (at 50 μM) all reduced the growth of wild carrot cultures grown in the presence or absence of 2,4-D. Therefore, hydroxylation of chlorpropham at the 2′ or 4′ positions of the 5′ chlorinated benzene ring is not sufficient to render the compound nonphytotoxic in all plant systems.     

A New Isoflavone with Antifungal Activity from Immature Fruits of Lupinus luteus

A new isoflavone having antifungal activity was isolated from immature fruits of Lupinus luteus (Leguminosae), and named luteone. The structure was shown to be 5,7,2′,4′-tetrahy-droxy-6-(3,3-dimethylallyl)-isoflavone by degradative and spectroscopic studies.     

Fungitoxic dihydrofuranoisoflavones and related compounds in white lupin,Lupinus albus

Chromatographic investigation of a methanolic extract of white lupin roots has revealed the presence of six new dihydrofuranoisoflavones (lupinisoflavones A-F). Three monoprenylated (3,3-dimethylallyl-substituted) isoflavones (wighteone, luteone and licoisoflavone A), two diprenylated isoflavones [6,3′-di(3,3-dimethylallyl)genistein (lupalbigenin) and 6,3′-di(3,3-dimethylallyl)-2′-hydroxygenistein (2′-hydroxylupalbigenin)] and two pyranoisoflavones (parvisoflavone B and licoisoflavone B) have also been isolated from the same source. In addition to genistein, leaf extracts of L. italbus contain 3′-O-methylorobol which is presumed to be the precursor of lupisoflavone [5,7,4′-trihydroxy-3′-methoxy-6-(3,3-dimethylallyl)isoflavone]. Probable biogenetic relationships between the prenylated, and dihydrofurano-and pyrano-substituted isoflavones in roots and leaves of L. albus are briefly discussed.     

O-Prenylated flavonoids from Dalbergia sissoo

A chalcone, 2,3-dimethoxy-4′-γ,γ-dimethylallyloxy-2′-hydroxychalcone (1) and an isoflavone, 7-γ,γ-dimethylallyloxy-5-hydroxy-4′-methoxyisoflavone (2) together with a known flavone, 7-hydroxy-6-methoxyflavone (3), a known isoflavone, biochanin A (4) and a known rotenoid, dehydroamorphigenin (5) were isolated from the root bark of Dalbergia sissoo. The structures of compounds 1–5 were elucidated on the basis of spectral and chemical studies.     

Four isoflavanones from the stem bark of Platycelphium voënse

From the stem bark of Platycelphium voënse (Leguminosae) four new isoflavanones were isolated and characterized as (S)-5,7-dihydroxy-2′,4′-dimethoxy-3′-(3″-methylbut-2″-enyl)-isoflavanone (trivial name platyisoflavanone A), (±)-5,7,2′-trihydroxy-4′-methoxy-3′-(3″-methylbut-2″-enyl)-isoflavanone (platyisoflavanone B), 5,7-dihydroxy-4′-methoxy-2″-(2?-hydroxyisopropyl)-dihydrofurano-[4″,5″:3′,2′]-isoflavanone (platyisoflavanone C) and 5,7,2′,3″-tetrahydroxy-2″,2″-dimethyldihydropyrano-[5″,6″:3′,4′]-isoflavanone (platyisoflavanone D). In addition, the known isoflavanones, sophoraisoflavanone A and glyasperin F; the isoflavone, formononetin; two flavones, kumatakenin and isokaempferide; as well as two triterpenes, betulin and β-amyrin were identified. The structures were elucidated on the basis of spectroscopic evidence. Platyisoflavanone A showed antibacterial activity against Mycobacterium tuberculosis in the microplate alamar blue assay (MABA) with MIC = 23.7 μM, but also showed cytotoxicity (IC₅₀ = 21.1 μM) in the vero cell test.     

New Alkaloids and α‐Glucosidase Inhibitory Flavonoids from Ficus hispida

Two new pyrrolidine alkaloids, ficushispimines A ( 1 ) and B ( 2 ), a new ω‐(dimethylamino)caprophenone alkaloid, ficushispimine C ( 3 ), and a new indolizidine alkaloid, ficushispidine ( 4 ), together with the known alkaloid 5 and 11 known isoprenylated flavonoids 6  –  16 , were isolated from the twigs of Ficus hispida. Their structures were elucidated by spectroscopic methods. Isoderrone ( 8 ), 3′‐(3‐methylbut‐2‐en‐1‐yl)biochanin A ( 11 ), myrsininone A ( 12 ), ficusin A ( 13 ), and 4′,5,7‐trihydroxy‐6‐[(1R*,6R*)‐3‐methyl‐6‐(1‐methylethenyl)cyclohex‐2‐en‐1‐yl]isoflavone ( 14 ) showed inhibitory effects on α‐glucosidase in vitro.     

Utilization of Bacterial Xanthine Oxidase for Inorganic Phosphorus Determination

Two host-specific phytotoxic metabolites, AK-toxin I and II, were isolated from a culture broth of Alternaria alternata Japanese pear pathotype, the fungus causing black spot disease of susceptible Japanese pear cultivars. From chemical, spectral and X-ray crystallographic data, AK-toxin I was characterized as 8-(2′S, 3′S)-2′-acetylamino-3′-methyl-3′-phenyl-propionyloxy]-(8R,9S)-9,10-epoxy-9-methyl-deca-(2E,4Z,6E)-trienoic acid. The structure of AK-toxin II was also assigned to be 3′-demethyl derivative of AK-toxin I by comparing the spectral data with those of AK-toxin I.