An inducible hydrolase from Mortierella isabellina (basidiomycetes). The deacylation of (+)-usnic acid

An inducible enzyme catalysing the hydrolysis of (+)-usnic acid to (+)-2-desacetylusnic acid and acetic acid has been purified 150-fold from the mycelium of Mortierella isabellina grown in the presence of (+)-usnic acid. Purification was achieved by treatment with protamine sulfate, (NH₄)₂SO₄ fractionation, negative adsorption on alumina Cγ gel and hydroxylapatite followed by chromatography on DEAE-cellulose and Sephadex G-200. The elution pattern from a Sephadex G-200 column indicated a MW of ca 7.6 × 10⁴ for the enzyme. The apparent K_m value for (+)-usnic acid at the pH optimum (pH 7) was 4.0 × 10^?5 M. The enzyme was specific for (+)-usnic acid and inactive towards (?)-usnic acid, (+)-isousnic acid or certain phloracetophenone derivatives. Its activity was enhanced in the presence of divalent metal ions such as Co²⁺, Ni²⁺, Mn²⁺, Mg²⁺ and Zn²⁺.     

Minor phenolics from Angelica keiskei and their proliferative effects on Hep3B cells

A new coumarin, (?)-cis-(3′R,4′R)-4′-O-angeloylkhellactone-3′-O-β-d-glucopyranoside (1) and two new chalcones, 3′-[(2E)-5-carboxy-3-methyl-2-pentenyl]-4,2′,4′-trihydroxychalcone (4) and (±)-4,2′,4′-trihydroxy-3′-{2-hydroxy-2-[tetrahydro-2-methyl-5-(1-methylethenyl)-2-furanyl]ethyl}chalcone (5) were isolated from the aerial parts of Angelica keiskei (Umbelliferae), together with six known compounds: (R)-O-isobutyroyllomatin (2), 3′-O-methylvaginol (3), (?)-jejuchalcone F (6), isoliquiritigenin (7), davidigenin (8), and (±)-liquiritigenin (9). The structures of the new compounds were determined by interpretation of their spectroscopic data including 1D and 2D NMR data. All known compounds (2, 3, and 6–9) were isolated as constituents of A. keiskei for the first time. To identify novel hepatocyte proliferation inducer for liver regeneration, 1–9 were evaluated for their cell proliferative effects using a Hep3B human hepatoma cell line. All isolates exhibited cell proliferative effects compared to untreated control (DMSO). Cytoprotective effects against oxidative stress induced by glucose oxidase were also examined on Hep3B cells and mouse fibroblast NIH3T3 cells and all compounds showed significant dose-dependent protection against oxidative stress.     

Formation of (-⊃-1′,2′-epi-2-cis-xanthoxin acid from a precursor of abscisic acid

Tomato shoots and avocado mesocarp supplied with (±)-[2-¹⁴C]-5-(1,2-epoxy-2,6,6-trimethylcyclohexyl)-3-methylpenta-cis-2-trans-4-dienoic acid metabolize it into (+)-abscisic acid and a more polar material that was isolated and identified as (?)-epi-1′(R),2′(R)-4′(S)-2-cis-xanthoxin acid. The (+)-1′(S),2′(S)-4′(S)-2-cis-xanthoxin acid recently synthesized from natural violaxanthin, has the 1′,2′-epoxy group on the opposite side of the ring to that of the 4′(S)-hydroxyl group and the compound is rapidly converted into (+)-abscisic acid. The 1′,2′-epoxy group of (?)-1′,2′-epi-2-cis-xanthoxin acid is on the same side of the ring as the 4′(S) hydroxyl group: the compound is not metabolized into abscisic acid. The configuration of the 1′,2′-epoxy group probably controls whether or not the 4′(S) hydroxyl group can be oxidized. (+)-2-cis-Xanthoxin acid is probably not a naturally occurring intermediate because a ‘cold trap’, added to avocado fruit forming [¹⁴C]-labelled abscisic acid from [2-¹⁴C]mevalonate, failed to retain [¹⁴C] label.     

Synthesis and antimicrobial activities of N6-hydroxyagelasine analogs and revision of the structure of ageloximes

(+)-N⁶-Hydroxyagelasine D, the enantiomer of the proposed structure of (?)-ageloxime D, as well as N⁶-hydroxyagelasine analogs were synthesized by selective N-7 alkylation of N⁶-[tert-butyl(dimethyl)silyloxy]-9-methyl-9H-purin-6-amine in order to install the terpenoid side chain, followed by fluoride mediated removal of the TBDMS-protecting group. N⁶-Hydroxyagelasine D and the analog carrying a geranylgeranyl side chain displayed profound antimicrobial activities against several pathogenic bacteria and protozoa and inhibited bacterial biofilm formation. However these compounds were also toxic towards mammalian fibroblast cells (MRC-5). The spectral data of N⁶-hydroxyagelasine D did not match those reported for ageloxime D before. Hence, a revised structure of ageloxime D was proposed. Basic hydrolysis of agelasine D gave (+)-N-[4-amino-6-(methylamino)pyrimidin-5-yl]-N-copalylformamide, a compound with spectral data in full agreement with those reported for (?)-ageloxime D.     

Metabolism of nicotine in Nicotiana glauca

A mixture of (?)-nicotine-[2′-³H] and (±)-nicotine-[2′-¹⁴C] was administered to Nicotiana glauca plants for 3 days, resulting in the formation of radioactive nornicotine (49·5% incorporation) and myosmine (2·05% incorporation). Negligible activity was detected in anabasine, cotinine, or 3-acetylpyridine, the last two compounds being added as carriers to the harvested plants. The radioactive nornicotine consisted of 48% (?)-nornicotine-[2′-¹⁴C,³H] and 52% (+)-nornicotine-[2′-¹⁴C]. Thus if (+)-nornicotine is formed from (?)-nicotine the transformation must involve loss of the hydrogen from C-2′. Myosmine is presumably formed from nicotine via nornicotine. However by feeding myosmine-[2′-¹⁴C] to N. glauca it was shown that the dehydrogenation is not reversible, no activity being detected in nornicotine. Nicotinic acid (0·14% incorporation) was a metabolite of myosmine-[2′-¹⁴C]. Essentially all the activity of the nicotinic acid was located on its carboxyl group, indicating that myosmine was a direct precursor.     

Biosynthesis of the bisbenzylisoquinoline alkaloid,tetrandrine

The incorporation of (±)-coclaurine, (±)-norcoclaurine, (±)-N-methylcoclaurine and didehydro-N-methyleoclaurinium iodide into tetrandrine in Cocculus laurifolius has been studied and specific utilization of (±)-N-ethylcoclaurine demonstrated. The evidence indicates that tetrandrine is formed in the plants by oxidative dimerization of N-methylcoclaurine. Double labelling experiment with (±)-N- [¹⁴C]-methyl- [1-³H]-coclaurine demonstrated that the hydrogen atom at the asymmetric centre in the 1-benzylisoquinoline precursor is retained in the bioconversion into tetrandrine. Parallel feedings of (+)-(S)- and (?)-(R)-N-methylcoclaurines showed that the stereospecificity is maintained in the biosynthesis of tetrandrine from the 1-benzylisoquinoline precursor.     

Novel dammarane saponins from Gynostemma pentaphyllum and their cytotoxic activities against HepG2 cells

Two new dammarane saponins, 2α,3β,12β-trihydroxydammar-20(22),24-diene-3-O-[β-d-glucopyranoxyl(1→2)-β-d-6″-O-acetylglucopyranoside (1, namely damulin C) and 2α,3β,12β-trihydroxydammar-20(21),24-diene-3-O-[β-d-glucopyranoxyl(1→2)-β-d-6″-O-acetylglucopyranoside (2, namely damulin D), were isolated from the ethanol extract of Gynostemma pentaphyllum, which had been heat processed by steaming at 125 °C. The NMR spectroscopic data of the novel saponins were completely assigned by using a combination of 2D NMR experiments including ¹H–¹H COSY, HSQC, and HMBC. Their cytotoxic activities of human liver adenocarcinoma HepG2 cells were evaluated in vitro. They showed cytotoxicities against HepG2 cell line with IC₅₀ of 40 ± 0.7 and 38 ± 0.5 μg/ml, respectively.     

Leucoflavonine,a new bioactive racemic flavoalkaloid from the leaves of Leucosceptrum canum

A new flavoalkaloid racemate, leucoflavonine (1), together with its flavonoid precursor pectolinarigenin (2), was isolated from the leaves of Leucosceptrum canum collected from Tibet. Its structure was established by comprehensive spectroscopic analysis. Chrial separation of the enantiomers of 1 was achieved, and their absolute configurations were determined as S-(+)- and R-(?)-leucoflavonines ((+)-1a and (?)-1b) by comparison of their computational and experimental optical rotations. Biological assays indicated that both (+)-1a and (?)-1b exhibited inhibitory activity against acetylchlorinesterase (AChE) in vitro (IC₅₀?=?68.0?±?8.6 and 18.3?±?1.8?μM, respectively). Moreover, (?)-1b displayed cytotoxicity against human hepatoma cells HepG2 (IC₅₀?=?52.9?±?3.6?μM), and inhibited the production of interleukelin-2 (IL-2) in Jurkat cells (IC₅₀?=?16.5?±?0.9?μM), while (+)-1a showed no obvious activity in these assays.     

(−)-9′-O-(α-l-Rhamnopyranosyl)lyoniresinol from Lespedeza cuneata suppresses ovarian cancer cell proliferation through induction of apoptosis

Lespedeza cuneata (Dum. Cours.) G. Don. (Fabaceae), known as Chinese bushclover or sericea lespedeza, has been used in traditional medicine to treat diabetes, hematuria, and insomnia, and it has been reported that bioactive compounds from L. cuneata possess various pharmacological properties. However, there has been no study to determine the active compounds from L. cuneata with potential activity against ovarian cancer. This study aimed to isolate cytotoxic compounds from L. cuneata and identify the molecular mechanisms underlying the apoptosis pathway in ovarian cancer cells. Based on cytotoxic activity identified in the screening test, chemical investigation of the active fraction of L. cuneata led to the isolation of nine compounds including four lignanosides (1–4), three flavonoid glycosides (5–7), and two phenolics (8–9). Cytotoxicity and the molecular mechanism were examined by methyl thiazolyl tetrazolium (MTT) assay and Western blot analysis. Of the isolated compounds, (?)-9′-O-(α-l-rhamnopyranosyl)lyoniresinol (3) demonstrated the strongest effect in suppressing A2780 human ovarian carcinoma cell proliferation in a dose-dependent manner, with an IC₅₀ value of 35.40?±?2.78?μM. Control A2780 cells had normal morphology, whereas cell blebbing, shrinkage, and condensation were observed after treatment with compound 3. Western blotting analysis showed that compound 3 inhibited A2780 human ovarian cancer cell viability by activating caspase-8, caspase-3, and PARP, which contributed to apoptotic cell death. These results suggest that (?)-9′-O-(α-l-rhamnopyranosyl)lyoniresinol (3) has potent anticancer activities against A2780 human ovarian carcinoma cells through the extrinsic apoptotic pathway. Therefore, (?)-9′-O-(α-l-rhamnopyranosyl)lyoniresinol is an excellent candidate for the development of novel chemotherapeutics.     

Purification and characterization of a novel O-methyltransferase from Flammulina velutipes

An enzyme catalyzing the methylation of phenolic hydroxyl groups in polyphenols was identified from mycelial cultures of edible mushrooms to synthesize O-methylated polyphenols. Enzyme activity was measured to assess whether methyl groups were introduced into (?)-epigallocatechin-3-O-gallate (EGCG) using SAM as a methyl donor, and (?)-epigallocatechin-3-O-(3-O-methyl)-gallate (EGCG3″Me), (?)-epigallocatechin-3-O-(4-O-methyl)-gallate (EGCG4″Me), and (?)-epigallocatechin-3-O-(3,5-O-dimethyl)-gallate (EGCG3″,5″diMe) peaks were detected using crude enzyme preparations from mycelial cultures of Flammulina velutipes. The enzyme was purified using chromatographic and two-dimensional electrophoresis. The purified enzyme was subsequently analyzed on the basis of the partial amino acid sequence using LC–MS/MS. Partial amino acid sequencing identified the 17 and 12 amino acid sequences, VLEVGTLGGYSTTWLAR and TGGIIIVDNVVR. In database searches, these sequences showed high identity with O-methyltransferases from other mushroom species and completely matched 11 of 17 and 9 of 12 amino acids from five other mushroom O-methyltransferases.     

Flavanol glucosides from rhubarb and Rhaphiolepis umbellata

Investigations of rhubarb and the bark of Rhaphiolepis umbellata led to the isolation of new flavan-3-ol glucosides. Their structures were elucidated on the basis of ¹H and ¹³C NMR analysis hydrolytic studies as (+)-catechin 5-O-β-d-glucopyranoside and (?)-catechin 7-O-β-d-glucopyranoside.     

Dimeric phenolic constituents from the roots of Tamarix nilotica

The debarked roots of Tamarix nilotica contain the furanofuran lignan (±)-syringaresinol so far not reported from the Tamaricaceae, and the new natural product ellagic acid 3,3′-dimethyl ether 4-O-β-d-glucopyranoside. Further constituents were isoferulic acid, gallic acid, dehydrodigallic acid and ellagic acid. The structure of the isolated compounds was determined mostly by ¹H and ¹³C NMR spectroscopy.     

Metabolism of benzo[a]pyrene VI. Stereoselective metabolism of benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol to diol epoxides

(±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-1) and (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-2) are highly mutagenic diol epoxide diastereomers that are formed during metabolism of the carcinogen (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene. Remarkable stereoselectivity has been observed on metabolism of the optically pure (+)- and (?)-enantiomers of the dihydrodiol which are obtained by separation of the diastereomeric diesters with (?)-α-methoxy-α-trifluoromethylphenylacetic acid. The high stereoselectivity in the formation of diol epoxide-1 relative to diol epoxide-2 was observed with liver microsomes from 3-methylcholanthrene-treated rats and with a purified cytochrome P-448-containing monoxygenase system where the (?)-enantiomer produced a diol epoxide-2 to diol epoxide-1 ratio of 6 : 1 and the (+)-enantiomer produced a ratio of 1 : 22. Microsomes from control and phenobarbital-treated rats were less stereospecific in the metabolism of enantiomers of BP 7,8-dihydrodiol. The ratio of diol epoxide-2 to diol epoxide-1 formed from the (?)- and (+)-enantiomers with microsomes from control rats was 2 : 1 and 1 : 6, respectively. Both enantiomers of BP 7,8-dihydrodiol were also metabolized to a phenolic derivative, tentatively identified as 6,7,8-trihydroxy-7,8-dihydrobenzo[a]pyrene, which accounted for ～30% of the total metabolites formed by microsomes from control and phenobarbital-pretreated rats whereas this metabolite represents ～5% of the total metabolites with microsomes from 3-methylcholanthrene-treated rats. With benzo[a]pyrene as substrate, liver microsomes produced the 4,5-, 7,8- and 9,10-dihydrodiol with high optical purity (>85%), and diol epoxides were also formed. Most of the optical activity in the BP 7,8-dihydrodiol was due to metabolism by the monoxygenase system rather than by epoxide hydrase, since hydration of (±)-benzo[a]pyrene 7,8-oxide by liver microsomes produced dihydrodiol which was only 8% optically pure. Thus, the stereospecificity of both the monoxygenase system and, to a lesser extent, epoxide hydrase plays important roles in the metabolic activation of benzo[a]pyrene to carcinogens and mutagens.     

Completing the series of BVMOs involved in camphor metabolism of Pseudomonas putida NCIMB 10007 by identification of the two missing genes, their functional expression in E. coli, and biochemical characterization

The camphor-degrading Baeyer?CVilliger monooxygenases (BVMOs) from Pseudomonas putida NCIMB 10007 have been of interest for over 40?years. So far the FMN- and NADH-dependent type II BVMO 3,6-diketocamphane 1,6-monooxygenase (3,6-DKCMO) and the FAD- and NADPH-dependent type I BVMO 2-oxo-?³-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) have not been entirely studied, since it was not possible to produce those enzymes in satisfactory amounts and purity. In this study, we were able to clone and recombinantly express both enzymes and subsequently use them as biocatalysts for various mono- and bicyclic ketones. Full conversion could be reached with both enzymes towards (±)-cis-bicyclo[3.2.0]hept-2-en-6-one and with 3,6-DKCMO towards (?)-camphor. Further OTEMO gave full conversion with norcamphor. OTEMO was found to have a pH optimum of 9 and a temperature optimum of 20?°C and converted (±)-cis-bicyclo[3.2.0]hept-2-en-6-one with a k _cat/K _M value of 49.3?mM^?1?s^?1.     

Chemical examination of the roots of Terminalia arjuna—the structures of arjunoside III and arjunoside IV,two new triterpenoid glycosides

The non-phenolic fraction of the alcoholic extract of the root bark of Terminalia arjuna yielded two new triterpenoid glycosides, arjunoside III and arjunoside IV in addition to arjunglucoside I and arjunetin. The structure of arjunoside III was established as the 28-β-d(+)-glucuronopyranoside of arjunic acid by a study of its chemical and spectroscopic (¹H and ¹³C NMR) data. Arjunoside IV was shown to be the 3-O-α- l(?)-rhamnoside of arjunic acid. Leucocyanidin, ellagic acid and gallic acid have been isolated from the phenolic part of the root extract.     

Furanocoumarin biosynthesis in Ruta graveolens cell cultures

The biosynthetic routes to four linear furanocoumarins—psoralen, xanthotoxin, bergapten. isopimpinellin-co-occurring in Ruta graveolens cell cultures have been investigated with six ¹⁴C-labelled compounds. Mevalonic acid was only poorly incorporated, in contrast to umbelliferone. In support of previous suggestions, 7-demethylsuberosin and (±)-marmesin were very good precursors of the linear furanocoumarins. 7-O-Prenylumbelliferone also was fairly well utilized, but this was probably owing to a prior ether cleavage yielding umbelliferone. Psoralen was well incorporated into bergapten and xanthotoxin, but not into the dimethoxylated isopimpinellin. Differences exist between the organized plant and its cell culture in terms of metabolic products and, by implication, precursor utilization. S(+)-Marmesin was obtained in small quantity from an acid-hydrolysable conjugate present in the culture medium. Syntheses of [2-¹⁴C]7-demethylsuberosin, [2-¹⁴C]osthenol, [2-¹⁴C]7-O-prenylumbelliferone, [3-¹⁴C] (±)-marmesin, and [3-¹⁴C]psoralen are described, as well as an improved method for separation of furanocoumarin mixtures by TLC and GLC.     

Seven new triterpenoids from the aerial parts of Ilex cornuta and protective effects against H2O2-induced myocardial cell injury

Seven new triterpenoids (1–7), together with two known ones (8–9), were isolated from the aerial parts ofIlex cornuta. The leaves of I. cornuta are the major source of “Kudingcha”, a popular herbal tea consumed in China and other countries. The structures of compounds 1–7 were determined as 20-epi-urs-12,18-dien-28-oic acid 3β-O-α-l-arabinopyranoside (1), 20-epi-urs-12,18-dien-28-oic acid 2′-O-acetyl-3β-O-α-l-arabinopyranoside (2), 20-epi-urs-12,18-dien-28-oic acid 3β-O-β-d-glucuronopyranoside-6-O-methyl ester (3), 3β,23-dihydroxy-20-epi-urs-12,18-dien-28-oic acid (4), 23-hydroxy-20-epi-urs-12,18-dien-28-oic acid 3β-O-α-l-arabinopyranoside (5), 23-hydroxy-20-epi-urs-12,18-dien-28-oic acid 3β-O-β-d-glucuronic acid (6), 23-hydroxy-20-epi-urs-12,18-dien-28-oic acid 3β-O-β-d-glucuronopyranoside-6-O-methyl ester (7), on the basis of spectroscopic analyses (IR, ESI–MS, HR-ESI–MS, 1D and 2D NMR) and chemical reactions. Protective effects against H₂O₂-induced H9c2 cardiomyocyte injury were tested in vitro for compounds 1–9, and the data showed that compound 4 had significant cell-protective effect. Compounds 1-9 did not show significant DPPH radical scavenging activity.     

Discovery of potent α-glucosidase inhibitor flavonols: Insights into mechanism of action through inhibition kinetics and docking simulations

Beside other pharmaceutical benefits, flavonoids are known for their potent α-glucosidase inhibition. In the present study, we investigated α-glucosidase inhibitory effects of structurally related 11 flavonols, among which quercetin-3-O-(3″-O-galloyl)-β-galactopyranoside (8) and quercetin 3-O-(6″-O-galloyl)-β-glucopyranoside (9) showed significant inhibition compared to the positive control, acarbose, with IC₅₀ values of 0.97 ± 0.02 and 1.35 ± 0.06 µM, respectively. It was found that while sugar substitution to C3-OH of C ring reduced the α-glucosidase inhibitory effect, galloyl substitution to these sugar units increased it. An enzyme kinetics analysis revealed that 7 was competitive, whereas 1, 2, 8, and 9 were uncompetitive inhibitors. In the light of these findings, we performed molecular docking studies to predict their inhibition mechanisms at atomic level.     

Alkenylresorcinols and cytotoxic activity of the constituents isolated from Labisia pumila

Phytochemical investigation on the leaves of Labisia pumila (Myrsinaceae), an important medicinal herb in Malaysia, has led to the isolation of 1-O-methyl-6-acetoxy-5-(pentadec-10Z-enyl)resorcinol (1), labisiaquinone A (2) and labisiaquinone B (3). Along with these, 16 known compounds including 1-O-methyl-6-acetoxy-5-pentadecylresorcinol (4), 5-(pentadec-10Z-enyl)resorcinol (5), 5-(pentadecyl)resorcinol (6), (−)-loliolide (7), stigmasterol (8), 4-hydroxyphenylethylamine (9), 3,4,5-trihydroxybenzoic acid (10), 3,4-dihydroxybenzoic acid (11), (+)-catechin (12), (−)-epicatechin (13), kaempferol-3-O-α-rhamnopyranosyl-7-O-β-glycopyranoside (14), kaempferol-4′-O-β-glycopyranoside (15), quercetin-3-O-α-rhamnopyranoside (16), kaempferol-3-O-α-rhamnopyranoside (17), (9Z,12Z)-octadeca-9,12-dienoic acid (18) and stigmasterol-3-O-β-glycopyranoside (19) were also isolated. The structures of these compounds were established on the basis of 1D and 2D NMR spectroscopy techniques (¹H, ¹³C, COSY, HSQC, NOESY and HMBC experiments), mass spectrometry and chemical derivatization. Among the constituents tested 1 and 4 exhibited strongest cytotoxic activity against the PC3, HCT116 and MCF-7 cell lines (IC₅₀ values ⩽10 μM), and they showed selectivity towards the first two-cell lines relative to the last one.     

Metabolism of 13-cis-retinoic acid: Identification of 13-cis-retinoyl-and 13-cis-4-oxoretinoyl-β-glucuronides in the bile of vitamin A-normal rats

The metabolism of 13-cis-[11-³H]retinoic acid has been examined in vitamin A-normal rats. Within 24 h after intravenous administration of the parent retinoid (15 μg/kg) to animals with biliary fistulas, 69 ± 9% of the dose was detected in the bile with 9 ± 6% being found in the urine. Analysis of the bile by reverse-phase high-pressure liquid chromatography demonstrated that the retinoic acid was being metabolized to several more polar compounds. A number of these compounds were sensitive to incubation with β-glucuronidase as evidenced by a change in their chromatographic behavior after treatment with the enzyme. Two of the metabolites have been identified as 13-cis-4-oxoretinoyl-β-glucuronide (8.1 ± 1.0% of the dose during the first 4 h after administration of the parent compound) and 13-cis-retinoyl-β-glucuronide (7.0 ± 4.4% of the dose). A comparison of the chromatographic profiles of bile from 13-cis- versus all-trans-retinoic acid-treated rats indicated a difference in their metabolism, with a greater proportion of the all-trans-retinoic acid being converted to compounds that eluted in the more polar regions of the column effluent.