Geranylated flavanones from the secretion on the surface of the immature fruits of Paulownia tomentosa

Chemical investigation of the methanol extract of the viscous secretion on the surface of immature fruits of Paulownia tomentosa furnished nine geranylated flavanones, 6-geranyl-5,7-dihydroxy-3',4'-dimethoxyflavanone (1), 6-geranyl-3',5,7-trihydroxy-4'-methoxyflavanone (2), 6-geranyl-4',5,7-trihydroxy-3',5'-dimethoxyflavanone (3), 6-geranyl-4',5,5',7-tetrahydroxy-3'-methoxyflavanone (4), 6-geranyl-3,3',5,7-tetrahydroxy-4'-methoxyflavanone (5), 4',5,5',7-tetrahydroxy-6-[6-hydroxy-3,7-dimethyl-2(E),7-octadienyl]-3'-methoxyflavanone (6), 3,3',4',5,7-pentahydroxy-6-[6-hydroxy-3,7-dimethyl-2(E),7-octadienyl]flavanone (7), 3,3',4',5,7-pentahydroxy-6-[7-hydroxy-3,7-dimethyl-2(E)-octenyl]flavanone (8), and 3,4',5,5',7-pentahydroxy-3'-methoxy-6-(3-methyl-2-butenyl)flavanone (9), along with six known geranylated flavanones. Among these, compounds 4, 6-9 and the known 6-geranyl-3',4',5,7-tetraahydroxyflavanone (diplacone), 6-geranyl-3,3',4',5,7-pentahydroxyflavanone (diplacol) and 3',4',5,7-pentahydroxy-6-[7-hydroxy-3,7-dimethyl-2(E)-octenyl]flavanone showed potent radical scavenging effects towards DPPH radicals.     

Geranylated phenolic constituents from the fruits of Artocarpus nobilis

Chemical investigation of the combined dichloromethane and ethyl acetate extracts of the fruits of Artocarpus nobilis, furnished four new geranylated phenolic constituents, 2,4,4'-trihydroxy-3-[(2E)-5-methoxy-3,7-dimethylocta-2,6-dienyl]chalcone (4), 1-(3,4-dihydro-3,5-dihydroxy-2-methyl-2-(3-methyl-2-butenyl)-2H-1-benzopyran-6-yl-3-(4-hydroxyphenyl)-2(E)-propen-1-one (5), 8-geranyl-3',4',7-trihydroxyflavone (8), 3'-geranyl-4',5,7-trihydroxyflavanone (9), together with known related compounds, xanthoangelol (1), xanthoangelol B (2), 3-geranyl-2,3',4,4'-tetrahydroxychalcone (3), lespeol (6), 8-geranyl-4',7-dihydroxyflavanone (7), and isonymphaeol-B (10). Compounds 3, 8 and 10 showed strong antioxidant activity against DPPH radical by spectrophotometric method.     

Coumarin glucosides from Cruciata taurica

Two new coumarin glycosides (1 and 2) along with two known coumarin glucosides, daphnin (3) and daphnetin glucoside (4) were isolated from the aerial parts of Cruciata taurica. The structures of the new compounds were elucidated by spectral methods and chemical means as 7-O-(6' -acetoxy-beta-D-glucopyranosyl)-8-hydroxycoumarin (1) and 7-O-[6 '-O-(3',4'-dihydroxycinnamoyl)-beta-D-glucopyranosyl]-8-hydroxycoumarin (2). The phylogenetic significance of coumarins in C. taurica was discussed.     

Interaction of lipophilic quinones with membrane fragments of Paracoccus denitrificans and Staphylococcus epidermidis.

In quinone-depleted mitochondrial and Paracoccus denitrificans membranes the quantum yield of fluorescence of ostruthin (6-geranyl-7-hydroxycoumarin) was maintained, whereas an increase in the quantum yield took place after extraction of Staphylococcus epidermidis membrane. A marked quenching effect of ubiquinone and menaquinone each with two isoprene units in the side chain on the ostruthin fluorescence was found with all types of quinone-depleted particles. When the homogues of menaquinone and ubiquinone with six isoprene units in the side chain were re-incorporated, a quenching of the ostruthin fluorescence was observed in the S. epidermidis membranes but not in those of P. denitrificans. The different behaviour of both bacterial preparations is attributable to the more specific finding of ubiquinone in the particles of P. denitrificans.     

Structure of a geranyl-alpha-pyrone from Mimulus aurantiacus leaf resin

A previously reported but misidentified geranyl-alpha-pyrone, in addition to six known compounds, was isolated from the leaf resin of Mimulus aurantiacus. HMBC NMR analyses of the geranyl-alpha-pyrone resolved uncertainties in the site of attachment of the two side chains and necessitated a revision of the previously reported structure. This compound is shown to be 3-geranyl-4-hydroxy-6-(2-hydroxypropyl)-2-pyrone.     

Constituents of two Flourensia species

The MeOH extract of aerial parts of Flourensia riparia Grisebach (Asteraceae) afforded a sesquiterpene lactone, 4beta-hydroxy-4,10alpha-dimethyl-7alphaH,8alphaH-eudesman-11-ene-8,12-olide, together with septuplinolide, its isomer at positions C-5 and C-10. In addition, known flavonoids, p-hydroxyacetophenone derivatives, carabrone and isoalantolactone were identified. Three known flavonoids and a benzofuran were isolated from Flourensia campestris Wedd.     

Mechanistic characterization of omega-3 desaturation in the green alga Chlorella vulgaris

alpha-Linolenic acid (ALA, 9(Z),12(Z),15(Z)-octadecatrienoic acid) derivatives are important plant lipids which play a critical key role in cold tolerance. The final steps of ALA biosynthesis feature a series of regio- and stereoselective dehydrogenation reactions which are catalyzed by a set of enzymes known as fatty acid desaturases. In conjunction with ongoing research into the structural biology of these remarkable catalysts, we have examined the mechanism of double bond introduction at C15,16 as it occurs in a model photosynthetic organism, Chlorella vulgaris. The individual deuterium kinetic isotope effects associated with the C-H bond cleavages at C-15 and C-16 of a thialinoleoyl analogue were measured via competition experiments using appropriately deuterium-labelled 7-thia substrates. A large kinetic isotope effect (KIE) (k(H)/k(D)=10.2+/-2.8) was observed for the C-H bond-breaking step at C-15 while the C-H bond cleavage at C-16 was found to be relatively insensitive to deuterium substitution (k(H)/k(D)=0.8+/-0.2). These results point to C-15 as the site of initial oxidation in omega-3 desaturation and imply that the Chlorella and corresponding plant systems share a common active site architecture.     

Biotransformation of ent-13-epi-manoyl oxides difunctionalized at C-3 and C-12 by filamentous fungi

Biotransformation of ent-3beta,12alpha-dihydroxy-13-epi-manoyl oxide with Fusarium moniliforme gave the regioselective oxidation of the hydroxyl group at C-3 and the ent-7beta-hydroxylation. The action of Gliocladium roseum in the 3,12-diketoderivative originated monohydroxylations at C-1 and C-7, both by the ent-beta face, while Rhizopus nigricans produced hydroxylation at C-7 or C-18, epoxidation of the double bond, reduction of the keto group at C-3, and combined actions as biohydroxylation at C-2/epoxidation of the double bond and hydroxylation at C-7/reduction of the keto group at C-3. In the ent-3-hydroxy-12-keto epimers, G. roseum originated monohydroxylations at C-1 and C-7 and R. nigricans originated the oxidation at C-3 as a major transformation, epoxidation of double bond and hydroxylation at C-2. Finally, in the ent-3beta-hydroxy epimer R. nigricans also originated minor hydroxylations at C-1, C-6, C-7 and C-20 and F. moniliforme produced an hydroxylation at C-7 and a dihydroxylation at C-7/C-11.     

Allanxanthone B, a polyisoprenylated xanthone from the stem bark of Allanblackia monticola Staner L.C

In addition to five known compounds including three xanthones, tovophyllin A, rubraxanthone and garciniafuran, one pentacyclic triterpene, lupeol and one phytosterol, stigmasterol, a polyisoprenylated xanthone named allanxanthone B was isolated from the stem bark of Allanblackia monticola. The structure of the new compound was assigned as 2-geranyl-1,3,6-trihydroxy-2',2'-dimethyl[5',6':7,8]xanthone by means of spectroscopic analysis. The antimicrobial activities of some of these compounds against a range of micro-organisms are also reported.     

Studies on the boronation of methyl-β-d-cellobioside—a cellulose model

The conversion of phenylboronic acid (PBA) with methyl-β-d-cellobioside (Me-β-d-clb) and cellodextrins (DP_w 12) was investigated to gain a basic understanding of the interactions of boric acid derivatives with oligo- and polyglucans. By means of MS and NMR experiments, it was possible to show a first stage formation of a six-membered ring at C-4 and C-6 of the non-reducing glucose occurs as in the case of monosaccharides. If the amount of reagent is increased the formation of seven-membered rings at the secondary OH moieties is observed. Even the existence of two of these large ring-systems in the direct neighborhood was found. Application of an excess of boronation reagent led to dimerization reactions of Me-β-d-clb via the primary reducing glucose residue as confirmed by DOSY NMR studies. Preliminary ¹³C NMR studies for the interaction of cellodextrins with PBA in DMSO solution confirmed a functionalization at the trans-1,2-diol moieties of these oligomers. The amount of reagent applied may either was shown to lead to soluble products or to insoluble cross-linked material.     

Hydrophobic interaction of isoprenic coumarin ostruthin with nonphosphorylating mitochondrial fragments

New emission maximum at 395 nm appeared in the fluorescence spectrum of ostruthin (6-geranyl-7-hydroxycoumarin) when allowed to interact with Keilin-Hartree preparation. Incubation of Keilin-Hartree preparation with umbelliferone (7-hy-droxycoumarin) did not result in any changes in its fluorescence spectrum. Different changes were observed in the fluorescence spectrum of ostruthin at incubation with bovine serum albumin, which suggest a different nature of interaction. Changes in pH or viscosity influenced the intensity, but the emission maximum of the ostruthin fluorescence spectrum was either not or very little shifted. Increase in concentration of aliphatic alcohols induced similar changes in the fluorescence spectra of both coumarins as Keilin-Hartree preparation did in the case of ostruthin. This indicated the hydrophobic nature of interaction between ostruthin and Keilin-Hartree preparation.Preincubation of Keilin-Hartree preparation with sulfhydryl reagents did not alter the fluorescence response of ostruthin. Removing lipids from Keilin-Hartree preparation resulted in a decrease in the quantum yield of ostruthin fluorescence at 395 nm and in the maximum number of binding sites. On the other hand, mild extraction of neutral lipids with pentane retained the quantum yield unaltered. At least two types of binding sites are present in Keilin-Hartree preparation from which one includes phospholipids, the other probably proteins. The maximum number of binding sites (39–50 nmoles/mg protein) corresponds to the amount of ostruthin needed for uncoupling of rat liver mitochondria.     

Effect of natural and synthetic benzyl benzoates on calmodulin

The present investigation describes the effect of the spasmolytic benzylbenzoates 1-9 from Brickellia veronicifolia on CaM using a functional in vitro enzymatic assay. Bovine brain PDE1 was used as a monitoring enzyme. The most active natural inhibitors of the system CaM-PDE1 were benzyl benzoates 3-5, which inhibited the activity of PDE1 in a concentration-dependent manner. In addition, three series of analogs of compound 4, compounds 10a-32a, were prepared and assayed. The benzyl benzoates from the first series, namely 10a-24a, possess no substituents on ring B but different number and position of hydroxyl or methoxy groups in ring A. The second group (25-32a), on the other hand, possesses an A ring identical to that on compound 4, but different substituents in Ring B. The most active compounds were 14a, 15a and 30a. These compounds were two to six times more potent than chlorpromazine, a well known CaM inhibitor. Benzyl benzoates 14a and 15a have methoxyl groups at C-2/C-4 and C-3/C-4 in ring A, respectively; while 30a, in addition to the methoxyl groups at C-2/C-6 of ring A, hold a benzoyloxy moiety at C-3' of ring B. Kinetic studies revealed that compounds 3, 4, 14a, 15a and 30a behave as competitive CaM antagonists.     

Four new immunomodulating steroidal glycosides from the stems of Stephanotis mucronata

Four new C-21 steroidal glycosides, mucronatosides E (1), F (2), G (3), and H (4), were isolated from the stems of Stephanotis mucronata. Two of them had the rare aglycone with a double bond between C-6 and C-7. Their structures were elucidated on the basis of spectroscopic data and chemical evidence. These isolated compounds were assayed for their immunological activities in vitro against concanavalin A (Con A)- and lipopolysaccharide (LPS)-induced proliferation of mice splenocytes. Compounds 2 and 4 showed immunosuppressive activities in a dose-dependent manner, while compounds 1 and 3 possessed immunoenhancing activities.     

The microbiological transformation of 7alpha-hydroxy-ent-kaur-16-ene derivatives by Gibberella fujikuroi

The biotransformation of 7alpha-hydroxy-ent-kaur-16-ene (epi-candol A) by the fungus Gibberella fujikuroi gave 7alpha,16alpha,17-trihydroxy-ent-kaur-16-ene and a seco-ring B derivative, fujenoic acid, whilst the incubation of candicandiol (7alpha,18-dihydroxy-ent-kaur-16-ene) and canditriol (7alpha,15alpha,18-trihydroxy-ent-kaur-16-ene) afforded 7alpha,18,19-trihydroxy-ent-kaur-16-ene and 7alpha,11beta,15alpha,18-tetrahydroxy-ent-kaur-16-ene, respectively. The presence of a 7alpha-hydroxyl group in epi-candol A avoids its biotransformation along the biosynthetic pathway of gibberellins, and directs it to the seco-ring B acids route. The 15alpha-hydroxyl group in canditriol inhibits oxidation at C-19 and direct hydroxylation at C-11(beta). The formation of fujenoic acid, from 7alpha-hydroxy-ent-kaur-16-ene, probably occurs via 7alpha-hydroxykaurenoic acid and 7-oxokaurenoic acid, with subsequent hydroxylation at the C-6(beta) position.     

Simultaneous determination of coumarin, 7-hydroxycoumarin and 7-hydroxycoumarin glucuronide in human serum and plasma by high-performance liquid chromatography

A HPLC method was developed for the determination of the metabolites of coumarin and 7-hydroxycoumarin in plasma and serum. Separation was based on gradient elution of 7-hydroxycoumarin glucuronide, 7-hydroxycoumarin, coumarin and finally 4-hydroxycoumarin (which is used as an internal standard). Standards, prepared in plasma or serum, and samples were treated with trichloroacetic acid, mixed and centrifuged. The supernatant was removed and analyzed by reversed-phase high-performance liquid chromatography on a C₁₈ column. The limit of detection was 50 ng/ml for 7-hydroxycoumarin and 200 ng/ml for coumarin and 7-hydroxycoumarin glucuronide. The linear range was 0.5–100 μg/ml for each of the analytes. The percentage relative standard deviation about the mean measured concentrations were all below 10%. There was no statistical difference between the standard curves prepared in plasma or serum. The method developed was applied to the determination of each of the three compounds in serum, after the administration of 7-hydroxycoumarin, and in plasma after the administration of coumarin. The concentrations of total 7-hydroxycoumarin in the serum samples were also determined by another HPLC method and the results were compared. There was no statistical difference between the results determined.     

Compounds from Kadsura angustifolia with anti-HIV activity

Four new cycloartane triterpenoids, angustific acid A (1), angustific acid B (2), angustifodilactone A (3) and angustifodilactone B (4) were isolated from the branches of Kadsura angustifolia together with six known compounds, micranoic acid B (5), nigranoic acid (6), schisandrin (7), schisantherin D (8), interiotherin B (9), schisantherin B (10). Their structures were established on the basis of extensive spectroscopic data analyses and comparison with spectroscopic data reported. Compound 1, characterized by the presence of a C-16/C-17, C-20/C-21 conjugated diene and a C-1/C-7 ester bridge formed in rings A and B, provided a novel structural skeleton for 3,4-secocycloartane triterpenoid derivatives. In addition, the anti-HIV activities of these compounds were determined in infected C8166 cells, and it was found that angustific acid A (1) exhibited the most potent anti-HIV activity with an EC₅₀ value of 6.1 μg/mL and a therapeutic index of more than 32.8.     

Prenylated coumarins and sesquiterpenoids from Ferula communis

From the latex of Ferula communis, two 4-hydroxycoumarin derivatives were isolated bearing a farnesylic and a 12-hydroxyfarnesylic residue, respectively, at C-3. Prenylated coumarins, which represent toxic principles of the plant, were absent in other samples, which gave, besides known compounds, a germacrane alcohol (hallohedycariol) and a daucane ester (siol p-hydroxybenzoate).     

Cloning and characterization of a glucosyltransferase that reacts on 7-hydroxyl group of flavonol and 3-hydroxyl group of coumarin from tobacco cells

In higher plants, secondary metabolites are often converted to their glycoconjugates by glycosyltransferases (GTases). We cloned a cDNA encoding GTase (NtGT2) from tobacco (Nicotiana tabacum L.). The recombinant enzyme expressed in Escherichia coli (rNTGT2) showed glucosylation activity against several kinds of phenolic compounds, particularly the 7-hydroxyl group of flavonoids and 3-hydroxycoumarin. The K(m) values of kaempferol and 3-hydroxycoumarin with rNTGT2 are 6.5 microM and 23.6 microM, respectively. The deduced amino acid sequence of NTGT2 shows 60-70% identity to that of anthocyanin 5-O-glucosyltransferase (A5GT); rNTGT2 did not show activity against the anthocyanins tested. NtGT2 gene expression was induced by treating tobacco cells with plant hormones such as salicylic acid. We consider that NtGT2 gene might have evolved from the same ancestral gene as the A5GT genes to the stress-inducible GTases that react on several phenolic compounds.     

Triterpenoid saponins from the fruits of Aesculus pavia

The compounds, named aesculiosides Ia-Ie, IIa-IId, and IVa-IVc, were isolated from an ethanol extract of the fruits of North American Aesculus pavia, along with two known compounds. Their structures were characterized as polyhydroxyoleanene pentacyclic triterpenoid saponins by spectroscopic and chemical analyses. These saponins were divided into three elution zones by chromatography according to the polarity because of the acyl substitution at C-21 and C-22 of the aglycone saponins moiety. These are structurally different from those isolated from Eurasian Aesculus hippocastanum and Aesculus chinensis in their oligosaccharide moieties.     

A continuous fluorometric assay for cytochrome P-450-dependent mixed function oxidases using 3-cyano-7-ethoxycoumarin

A direct fluorometric procedure for the continuous determination of cytochrome P-450-dependent mixed function oxidases, using 3-cyano-7-ethoxycoumarin substrate, is described. The reaction product, 3-cyano-7-hydroxycoumarin, is fluorescent at neutral pH values (excitation and emission wavelength maxima: 408 and 450 nm, respectively). Using hepatic microsomal preparations from control rats, the enzyme(s) had an apparent Km of 16 microM. Vmax values (0.5 nmol/min/mg protein) were induced 6- and 21-fold by pretreatment of rats with phenobarbitone and about 50- to 100-fold more sensitive than the ethoxyresorufin deethylase assay. Reaction rates using 3-cyano-7-pentoxycoumarin as substrate were generally much lower than with the ethoxy analog. 3-Cyano-7-ethoxycoumarin can also be used as a substrate to measure mixed function oxidases in isolated hepatocytes. However, 3-cyano-7-hydroxycoumarin shows a time- and concentration-dependent loss of fluorescence when incubated with such cells. This causes an approximately 5% underestimate of the true reaction rates.