Aromatic hydroxylation and sulfation of 5-hydroxyflavone by Streptomyces fulvissimus.

The conversion of 5-hydroxyflavone by various microorganisms was studied. Among them, Streptomyces fulvissimus was the sole microbe which produced a new polar metabolite from 5-hydroxyflavone in addition to 5,4-dihydoxy- and 5,3,4-trihydroxyflavone. The structure of this polar metabolite was determined to be 5,4-dihydroxyflavone-4-sulfate on the basis of mass, infrared, and nuclear magnetic resonance spectroscopies. These results demonstrate that S. fulvissimus catalyzes sulfation at the 4 position of 4,5-dihydroxyflavone.     

Regioselective formation of quercetin 5-O-glucoside from orally administered quercetin in the silkworm, Bombyx mori

The cocoons of some races of the silkworm, Bombyx mori, have been shown to contain 5-O-glucosylated flavonoids, which do not occur naturally in the leaves of their host plant, mulberry (Morus alba). Thus, dietary flavonoids could be biotransformed in this insect. In this study, we found that after feeding silkworms a diet rich in the flavonol quercetin, quercetin 5-O-glucoside was the predominant metabolite in the midgut tissue, while quercetin 5,4'-di-O-glucoside was the major constituent in the hemolymph and silk glands. UDP-glucosyltransferase (UGT) in the midgut could transfer glucose to each of the hydroxyl groups of quercetin, with a preference for formation of 5-O-glucoside, while quercetin 5,4'-di-O-glucoside was predominantly produced if the enzyme extracts of either the fat body or silk glands were incubated with quercetin 5-O-glucoside and UDP-glucose. These results suggest that dietary quercetin was glucosylated at the 5-O position in the midgut as the first-pass metabolite of quercetin after oral absorption, then glucosylated at the 4'-O position in the fat body or silk glands. The 5-O-glucosylated flavonoids retained biological activity in the insect, since the total free radical scavenging capacity of several tissues increased after oral administration of quercetin.     

In vivo formation of omega-oxidized metabolites of leukotriene C4 in the rat

[3H8]Leukotriene C4 was administered to germfree rats and to conventional rats having a bile duct cannula. Several radioactive metabolites were isolated. Two polar biliary metabolites from conventional rats were identified as N-acetyl-omega-carboxy-leukotriene E4 and N-acetyl-omega-hydroxy-leukotriene E4. A polar fecal metabolite from germfree rats was found to be N-acetyl-omega-carboxy-leukotriene E4. Chemical identities were established using UV spectroscopy and cochromatographies with authentic compounds in several HPLC systems. The fecal metabolite was further characterized by reductive desulfurization followed by gas-liquid-radiochromatography. The yield of the two biliary metabolites was 5% of the administered tritium after three hours and the yield of fecal N-acetyl-omega-carboxy-leukotriene E4 was 3.5% after three days.     

Metabolism of vitamin D. A new cholecalciferol metabolite, involving loss of hydrogen at C-1, in chick intestinal nuclei

1. A comparison was made of the nature and intestinal intracellular distribution of the metabolites formed in vitamin D-deficient chicks from [4-(14)C]cholecalciferol and [1-(3)H]cholecalciferol. 2. The simultaneous administration of the two radioactive substances showed the presence in blood, liver, intestine, kidney and bone of cholecalciferol, its ester, 25-hydroxycholecalciferol and a further metabolite of cholecalciferol more polar than 25-hydroxycholecalciferol. The (3)H/(14)C ratios in these four radioactive components were the same as that of the dosed material (4.7:1) with the exception of the most polar material. The (3)H/(14)C ratio was lower in the fourth, most polar, metabolite (0.4:1-1.8:1) in all tissues examined, with the exception of blood. 3. In the chick intestine the polar metabolite accounted for almost 70% of the radioactivity in this tissue after a dose of 0.5mug. of [4-(14)C,1-(3)H]cholecalciferol. This polar metabolite from the intestine also had the lowest (3)H/(14)C ratio of all the tissues. It appears that in the chick intestine the polar metabolite reaches a maximum concentration of 1ng./g. of tissue, above which it cannot be increased irrespective of the dose of the vitamin. 4. The intestinal intracellular organelle with the highest concentration of (14)C radioactivity is the nucleus, and this radioactivity is almost entirely due to the polar metabolite with the lowered (3)H/(14)C ratio, in this case <0.2:1. It appears to be further localized in the chromatin of the nuclei. However, about half of the polar metabolite in the intestine is extranuclear. 5. Double-labelled 25-hydroxycholecalciferol was prepared and after its administration to vitamin D-deficient chicks the polar metabolite with the lowered (3)H/(14)C ratio was detected in liver, kidney, intestine, bone, muscle and heart. 6. None of the polar metabolite with the lowered (3)H/(14)C ratio was detected 16hr. after dosing with either the double-labelled vitamin or the double-labelled 25-hydroxycholecalciferol in blood and adipose tissue of vitamin D-deficient chicks, nor in the intestine, liver and kidney of supplemented birds. 7. The reasons for this loss of (3)H relative to (14)C are discussed in relation to possible chemical structures of this new polar metabolite.     

Indicanines B and C, two isoflavonoid derivatives from the root bark of Erythrina indica

In addition to two known compounds, 5,4'-di-O-methylalpinumisoflavone and cajanin, a new 3-phenylcoumarin metabolite, named indicanine B, and a new isoflavone derivative, named indicanine C, were isolated from the root bark of Erythrina indica. By means of spectroscopic analysis, the structures of the new compounds were characterized as 4-hydroxy-3-(4'-hydroxyphenyl)-5-methoxy-2",2"-dimethylpyrano [5",6":6,7] coumarin and 4'-hydroxy-5-methoxy-2",2"-dimethylpyrano [5",6":6,7] isoflavone, respectively. The 13C-NMR data of cajanin and the in vitro antimicrobial spectrum and potencies of the isolated compounds are also reported.     

Phytoestrogens as inhibitors of fungal 17beta-hydroxysteroid dehydrogenase

Different phytoestrogens were tested as inhibitors of 17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcl), a member of the short-chain dehydrogenase/reductase superfamily. Phytoestrogens inhibited the oxidation of 100 microM 17beta-hydroxyestra-4-en-3-one and the reduction of 100 microM estra-4-en-3,17-dione, the best substrate pair known. The best inhibitors of oxidation, with IC(50) below 1 microM, were flavones hydroxylated at positions 3, 5 and 7: 3-hydroxyflavone, 3,7-dihydroxyflavone, 5,7-dihydroxyflavone (chrysin) and 5-hydroxyflavone, together with 5-methoxyflavone. The best inhibitors of reduction were less potent; 3-hydroxyflavone, 5-methoxyflavone, coumestrol, 3,5,7,4'-tetrahydroxyflavone (kaempferol) and 5-hydroxyflavone all had IC(50) values between 1 and 5 microM. Docking the representative inhibitors chrysin and kaempferol into the active site of 17beta-HSDcl revealed the possible binding mode, in which they are sandwiched between the nicotinamide moiety and Tyr212. The structural features of phytoestrogens, inhibitors of both oxidation and reduction catalyzed by the fungal 17beta-HSD, are similar to the reported structural features of phytoestrogen inhibitors of human 17beta-HSD types 1 and 2.     

Phytoestrogens as inhibitors of fungal 17beta-hydroxysteroid dehydrogenase

Different phytoestrogens were tested as inhibitors of 17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcl), a member of the short-chain dehydrogenase/reductase superfamily. Phytoestrogens inhibited the oxidation of 100microM 17beta-hydroxyestra-4-en-3-one and the reduction of 100microM estra-4-en-3,17-dione, the best substrate pair known. The best inhibitors of oxidation, with IC(50) below 1microM, were flavones hydroxylated at positions 3, 5 and 7: 3-hydroxyflavone, 3,7-dihydroxyflavone, 5,7-dihydroxyflavone (chrysin) and 5-hydroxyflavone, together with 5-methoxyflavone. The best inhibitors of reduction were less potent; 3-hydroxyflavone, 5-methoxyflavone, coumestrol, 3,5,7,4'-tetrahydroxyflavone (kaempferol) and 5-hydroxyflavone, all had IC(50) values between 1 and 5microM. Docking the representative inhibitors chrysin and kaempferol into the active site of 17beta-HSDcl revealed the possible binding mode, in which they are sandwiched between the nicotinamide moiety and Tyr212. The structural features of phytoestrogens, inhibitors of both oxidation and reduction catalyzed by the fungal 17beta-HSD, are similar to the reported structural features of phytoestrogen inhibitors of human 17beta-HSD types 1 and 2.     

Effect of varying the 4'-position of arbekacin derivatives on antibacterial activity against MRSA and Pseudomonas aeruginosa

4'-Deoxy-4'-episubstituted arbekacin derivatives and 4'-epi-5-deoxy-5-episubstituted arbekacin derivatives were designed and synthesized. Arbekacin and 4'-epiarbekacin both displayed the same antibacterial activity against Staphylococcus aureus (including methicillin-resistant S. aureus (MRSA)) and Pseudomonas aeruginosa. The 4'-epi-5-deoxy-5-episubstituted arbekacin derivatives showed potent antibacterial activity. Among them, the antibacterial activity of 5,4'-diepiarbekacin was superior to that of arbekacin or 5-episubstituted arbekacin against Gram-positive and Gram-negative bacteria. The 6'-N-methyl derivative of the 5,4'-diepiarbekacin was effective against P. aeruginosa expressing an aminoglycoside-modifying enzyme AAC(6')-Ib.     

Amino acid sequence of pilin isolated from pseudomonas aeruginosa PAK

A polar metabolite of leukotriene C4 was formed by sequential conversions with soybean lipoxygenase I and liver peroxidase. The structure of this product was found to be 5(S), 15(S)-dihydroxy-6(R)-S-glutathionyl-7,9,13-trans-11-cis-eicosatetraenoic acid (15-hydroxy-delta 13-trans-leukotriene C3. The HPLC behaviour, the molar extinction coefficient and the biological activity of the metabolite are reported. Preliminary evidence suggests that this product is formed by mammalian tissues.     

Metabolism of [14C] amitraz in larvae of Boophilus microplus

Amitraz, 1, 5-di(2, 4-dimethylphenyl)-3-methyl-1, 3, 5-triazapenta-1, 4-diene, labelled with 14C in the 2-methyl groups was applied to B. microplus larvae by an immersion technique. The chemical penetrated readily but never appeared in large amounts internally due to rapid cleavage to N-2, 4-dimethylphenyl-N'-methylformamidine. The expected complementary cleavage product 2, 4-dimethylformanilide was not produced in equivalent quantity. However, large amounts of polar metabolite(s) were produced. Small quantities of 2, 4-dimethylaniline and an unidentified non-polar metabolite were also produced. Of the identified chemicals only amitraz and N-2, 4-dimethylphenyl-N'-methylformamidine were toxic to larvae. Piperonyl butoxide applied simultaneously with amitraz had only a slight effect on metabolism but had a three-fold synergistic effect. SKF 525-A similarly applied had a negligible effect on both metabolism and toxicity.     

Metabolism and binding in vitro of 5 alpha-androstane-3 beta, 17 beta-diol and of 5 alpha-androstane-3 alpha, 17 beta-diol in cell fractions of rat ventral prostate and liver.

Rat ventral prostate and liver were investigated for the binding in vitro to particulate fractions and for the metabolism of 5 alpha-androstane-3 beta, 17 beta-diol. Comparative investigations were carried out on the metabolism of 5 alpha-androstane-3 alpha, 17 beta-diol. Preparations of the liver were investigated in order to establish the organ specificity of the method. In the prostate, the bulk of the metabolites of 5 alpha-androstane-3 beta, 17 beta-diol was present as steroids of high polarity. Of the less polar metabolites, 17 beta-hydroxy-5 alpha-androstan-3-one, 3 beta-hydroxy-5 alpha-androstan, 17-one and 5 alpha-androstane-3 alpha, 17 beta-diol were detectable. The binding of a 5 alpha-androstane-3 beta, 17 beta-diol to mitochondria and microsomes was unspecific. In the liver, among the less polar metabolites, 3 beta-hydroxy-5 alpha-androstan-17-one was the main metabolite, and the binding was unspecific. The main metabolite in the prostate homogenate of 5 alpha-androstane-3 alpha, 17 beta-diol was 17 beta-hydroxy-5 alpha-androstan-3-one. The portion of highly polar steroids was very low. The portion of unmetabolized hormone was distributed almost equally among the different cell preparations except the nuclei, in which 17 beta-hydroxy-5 alpha-androstan-3-one was higher and 5 alpha-androstane-3 alpha, 17 beta-diol was lower than in the remaining cell fractions.     

Bile acids. XXXVII. Identification of the 3 beta isomers of allocholic and allochenodeoxycholic acids as metabolites of 5 -cholestanol in the rat

Bile was collected for 18-24 days from adult male rats with cannulated bile ducts that had received intraperitoneally 0.8 mg of 5alpha-[4-(14)C, 3alpha-(3)H]cholestan-3beta-ol. Bile from the first 2 days containing 14.2% of the administered (14)C and 3.3% of the (3)H was hydrolyzed, and the bile acids were separated by acetic acid partition chromatography. The previously unidentified metabolite more polar than cholic and allocholic acids was identified by isotopic dilution as 3beta,7alpha,12alpha-trihydroxy-5alpha-cholanic acid and represented 3% of the biliary (14)C and 15% of the (3)H. Similarly, 3beta,7alpha-dihydroxy-5alpha-cholanic acid was identified in fractions more polar than allochenodeoxycholic acid and represented 0.6% of the biliary (14)C and 8% of the (3)H. More polar fractions contained 4% of the (14)C and 31% of the (3)H in unidentified metabolites.     

Metabolism of 3 H-digitoxigenin by rat liver, adrenal, and ovary homogenates

The metabolism of ³H-digitoxigenin was studied in rat liver, adrenal, and ovary homogenates under identical conditions. The major metabolite formed by liver and ovarian preparations was 3-epidigitoxigenin. Male liver homogenates showed higher epimerizing activity than female liver or ovary homogenates. In the adrenal preparations, the major metabolite formed was 3-digitoxigenone, and no sex difference was observed in its rate of formation. Adrenal and liver homogenates produced small amounts of digitoxigenin polar metabolites. The polar metabolites formed by the adrenal preparations were tentatively identified as 5-hydroxydigitoxigenin and 16β-hydroxydigitoxigenin.     

Microbial transformation of zearalenone to a zearalenone sulfate.

The conversion of zearalenone by various microorganisms was studied. A new polar metabolite was formed in addition to alpha- and beta-zearalenols. The structure of the new metabolite was determined as zearalenone-4-O-sulfate conjugate on the basis of enzymatic and acid hydrolysis, followed by mass spectrometry, nuclear magnetic resonance, and infrared spectroscopic analysis. The results obtained demonstrate that Rhizopus arrhizus catalyzes sulfation of zearalenone at the C-4 hydroxyl group.     

艾纳香化学成分的分离与鉴定

采用色谱技术对艾纳香的化学成分进行分离,根据理化性质和波谱学技术确定化合物的结构。结果从艾纳香的乙酸乙酯萃取液中分离得到7个已知成分,分别鉴定为木犀草素(1);木犀草素-7-甲醚(2);鼠李素(3);5,4′-二羟基-7-甲氧基-黄酮(4);5,4′-二羟基3,7,3′-三甲氧基-黄酮(5);北美圣草素(6);二氢斛皮素-4′-甲醚(7)。其中化合物4、5为首次从该植物中分离得到。     

Microbial transformation of zearalenone to a zearalenone sulfate.

The conversion of zearalenone by various microorganisms was studied. A new polar metabolite was formed in addition to alpha- and beta-zearalenols. The structure of the new metabolite was determined as zearalenone-4-O-sulfate conjugate on the basis of enzymatic and acid hydrolysis, followed by mass spectrometry, nuclear magnetic resonance, and infrared spectroscopic analysis. The results obtained demonstrate that Rhizopus arrhizus catalyzes sulfation of zearalenone at the C-4 hydroxyl group.     

Metabolism of testosterone sulfate in the rat: analysis of biliary metabolites.

Following intraperitoneal injection of a mixture of testosterone-7-3-H-17-sulfate and testosterone-4-14-C into male and female rats with bile fistulas, biliary metabolites were separated and purified by a combination of column chromatography, enzymic hydrolysis or solvolysis of the conjugate fractions and identification of the liberated aglycones. The injected steroids were extensively metabolized and excreted predominantly in the bile. The major portion of the 3H was excreted in the disulfate fraction in both sexes. Solvolysis of the disulfate revealed the sex-specific aglycone pattern: 5alpha-Androstane-3beta,17beta-diol was the major metabolite in the male rat, whereas 5alpha-androstane-3alpha,17beta-diol and polar steroids were found in the female. In marked contrast, testosterone was metabolized in a different way than testosterone sulfate. 14-C radioactivity was distributed in monoglucosiduronate, monosulfate, and diconjugate fractions. Analysis of the aglycones showed that polar steroids were the main metabolites in the male. In the female, testosterone was metabolized to polar steroids, androsterone, and 5alpha-androstane-3alpha,17beta-diol.     

Metabolism of capsaicinoids: Evidence for aliphatic hydroxylation and its pharmacological implications

A new metabolic oxidation pathway of capsaicin (N-[(4-hydroxy-3-methoxyphenyl)-methyl]-8-methyl-(E)-6-nonenamide), a major pungent and pharmacologically active principle of hot peppers, was investigated. Incubation of capsaicin with phenobarbital-induced rat liver postmitochondrial supernatant enriched with NADPH-generating system produced N-(4, 5-dihydroxy-3-methoxybenzyl)-(E)-6-nonenylamide and a more polar metabolite. The latter metabolite was spectrophotometrically and chromatographically identical to authentic ω-hydroxycapsaicin. This new metabolite was also detected in the urine of rabbits given capsaicin by gastric intubation. Other analogs of capsaicin, such as dihydrocapsaicin and nonivamide, also formed similar metabolites via aliphatic hydroxylation. When tested for antinociceptive activity as well as pungency, the above polar metabolites were found to be inactive while their parent compounds exhibited strong sensory effects. Capsaicin interacted irreversibly with hepatic drug metabolizing enzymes, thereby inhibiting their activity as indicated by prolongation of pentobarbital sleeping time in rats. Such inhibition of drug metabolism was not observed with ω-hydroxycapsaicin. These findings suggest that metabolism of capsaicinoids via hydroxylation of their side chains plays an important role in the detoxification of these pharmacologically active substances.     

Translocation of the 5-alkoxy substituent of 2,5-dialkoxyarylalkylamines to the 6-position: effects on 5-HT(2A/2C) receptor affinity

Positional modification of 2,5-dimethoxyamphetamine analogues has been studied. Specifically, the 5-alkoxy substituent was translocated to the 6-position of the phenyl nucleus. Methoxy groups were also constrained by incorporation into appended dihydrofuran and furan rings. 2,6-Dimethoxy-4-methylamphetamine had an approximately 3-fold lower affinity for the 5-HT(2A) receptor compared to the parent 2,5-dimethoxy-4-methylamphetamine (DOM). The rigid compound based on the 2,3,5,6-tetrahydrobenzo[1,2-b;5,4-b']difuran nucleus and the aromatic analogue containing the benzo[1,2-b;5,4-b']difuran nucleus possessed an approximate 7- and 27-fold increase in affinity, respectively, compared to 2,6-dimethoxy-4-methylamphetamine, the non-rigid, positional isomer.     

Some high-performance liquid-chromatographic studies of the metabolism of aflatoxins by rat liver microsomal preparations.

The metabolism of aflatoxin B1 in vitro was examined in rat liver microsomal preparations. 2. H.p.l.c. (high-performance liquid-chromatographic) systems were used. A silica column was used to separate non-polar metabolites. A system utilizing a reversed-phase column which separates both poar and non-polar metabolites was also developed. 3. The principal metabolites of aflatoxin B1 found were aflatoxin M1, aflatoxin Q1 and a compound which co-chromatographed with a degradation product of aflatoxin B1 2,3-dihydrodiol. 4. The time course of metabolism of aflatoxin B1 by microsomal preparations isolated from control and phenobarbitone-pretreated rats was examined. The rate and extent of metabolism was greater with microsomal preparations from the latter. The formation of aflatoxin Q1 was enhanced 4--5-fold by phenobarbitone pretreatment, whereas the production of aflatoxin M1 was only increased 1--2-fold. The formation of the degradation product of aflatoxin B1 2,3-dihydrodiol was increased 4--5-fold by the pretreatment with phenobarbitone. 5. The microsomal metabolism of aflatoxins M1, P1 and Q1 was examined. Aflatoxin M1 apparently underwent very limited microsomal metabolism to more polar compounds. Aflatoxin P1 was not metabolized. The situation with aflatoxin Q1 was complicated in that it was metabolized in the absence of NADPH to an unidentified metabolite. Aflatoxin B1 appeared as a metabolite of aflatoxin Q1 only when NADPH was present, and the formation of more polar metabolites was also then observed.