A new acylated flavonol diglycoside from <Emphasis Type="Italic">Atriplex littoralis</Emphasis>

A new acetylated flavonol glycoside: patuletin 3-O-[5′″-O-feruloyl-β-D-apiofuransyl (1′″→2′′)-β-D-glucopyranoside] (2), together with a known patuletin 3-O-β-D-glucopyranoside (1) were isolated from the aerial part of Artiplex littoralis L. (Chenopodiacease). Their structures were elcidated by acid hydrolysis and spectroscopic methods including UV, ¹H, ¹³C NMR and ESI-MS for both compounds, additionally 2D-NMR, HSQC, HMBC experiments were performed for 2.     

Chemosystematic investigations on phenolics from flowerheads of Central European taxa of Hieracium sensu lato (Asteraceae)

 HPLC-UV and HPLC-MS investigations of phenolic acids and flavonoids in flowerheads of 84 samples of 76 taxa belonging to 66 species of Hieracium resulted in the identification of three phenolic acids (chlorogenic acid, 3,5-dicaffeoyl quinic acid, 4,5-dicaffeoyl quinic acid) and six flavonoids (apigenin 4^′-O-β-D-glucuronide, isoetin 4^′-O-β-D-glucuronide, luteolin, luteolin 7-O-β-D-glucoside, luteolin 7-O-β-D-glucuronide, luteolin 4^′-O-β-D-glucoside). The contents of these secondary metabolites were quantified by HPLC using quercetin and cynarin as internal standards. In contrast to the previously investigated genera Leontodon and Crepis, cichoric acid and caffeoyl tartaric acid were not found in any of the investigated Hieracium taxa. Results of HPLC analyses revealed only a limited degree of qualitative variation between the different taxa, and luteolin 7-O-β-D-glucuronide and isoetin 4^′-O-β-D-glucuronide were the only compounds, which were not detectable in some of the investigated taxa. Quantitative patterns of phenolics differed markedly between particular taxa and Principal Component Analysis of the quantification results yielded separate clusters for the members of the subgenera Hieracium and Pilosella. Received January 23, 2001 Accepted October 11, 2001     

A new isoflavone glycoside from <Emphasis Type="Italic">Trifolium pratense</Emphasis> L.

The isoflavonoid composition of red clover (Trifolium pratense L.) has been studied. The following compounds have been detected: cyclopolyol (+)-pinitol, not found in clover before; known isoflavones formononetin, prunetin, genistein, and prunetin-4′-O-β-D-glucopyranoside; isoflavone monogalactosides formononetin-7-O-β-D-galactopyranoside, inermin-3-O-β-D-galactopyranoside, and genistein-7-O-β-D-galactopyranoside; and a novel compound, prunetin-4′-α-D-glucopyranoside. The structures of these compounds have been proven by UV, IR, 1H NMR, 13C NMR, mass, and circular dichroism spectra. (+)-Pinitol is known to possess biological activity.     

Biotransformation of soybean isoflavones by a marine <Emphasis Type="Italic">Streptomyces</Emphasis> sp. 060524 and cytotoxicity of the products

A marine Streptomyces sp. 060524 capable of hydrolyzing the glycosidic bond of isoflavone glycosides, was isolated by detecting its β-glucosidase activity. 5 isoflavone aglycones were isolated from culture filtrates in soybean meal glucose medium. They were identified as genistein (1), glycitein (2), daidzein (3), 3′,4′,5,7-tetrahydroxyisoflavone (4), and 3′,4′,7-trihydroxyisoflavone (5), based on UV, NMR and mass spectral analysis. The Streptomyces can selectively hydroxylate at the 3′-position in the daidzein and genistein to generate 3′-hydroxydaidzein and 3′-hydroxygenistein, respectively. The Strain biotransformed more than 90% of soybean isoflavone glycosides into their aglycones within 108 h. 3′-hydroxydaidzein and 3′-hydroxygenistein exhibited stronger cytotoxicity against K562 human chronic leukemia than daidzein and genistein.     

Substrate specificity of a recombinant chicken <Emphasis Type="Italic">β</Emphasis>-carotene 15,15′-monooxygenase that converts <Emphasis Type="Italic">β</Emphasis>-carotene into retinal

The recombinant β-carotene 15,15′-monooxygenase from chicken liver was purified as a single 60 kDa band by His-Trap HP and Resource Q chromatography. It had a molecular mass of 240 kDa by gel filtration indicating the native form to be tetramer. The enzyme converted β-carotene under maximal conditions (pH 8.0 and 37°C) with a k _cat of 1.65 min⁻¹ and a K _m of 26 μM and its conversion yield of β-carotene to retinal was 120% (mol mol⁻¹). The enzyme displayed catalytic efficiency and conversion yield for β-carotene, β-cryptoxanthin, β-apo-8′-carotenal, β-apo-4′-carotenal, α-carotene and γ-carotene in decreasing order but not for zeaxanthin, lutein, β-apo-12′-carotenal and lycopene, suggesting that the presence of one unsubstituted β-ionone ring in a substrate with a molecular weight greater than C₃₀ seems to be essential for enzyme activity.     

A novel isoflavone glycoside fromCaragana alaica

3-O-Rhamnopyranosylisorhamnetin, 3-O-glucopyranosylisorhamnetin, 7-O-galactopyranosylluteolin, quercitrin, isoquercitrin, wistin, and a novel isoflavonoid, 3′-hydroxy-6,4′-dimethoxy-7-O-β-D-glucopyranosylisoflavone, were isolated from the aerial parts ofaragana alaica. Of these, the previously described compounds were identified on the basis of their physicochemical and spectral characteristics, whereas the spectral analysis and conversion to a known compound, cladrastin, allowed the structural elucidation of the novel isoflavone glycoside.     

The leaves of the common box,Buxus sempervirens (Buxaceae), become red as the level of a red carotenoid,anhydroeschscholtzxanthin, increases

Carotenoids from the leaves of the common box,Buxus sempervirens (Buxaceae), which turn red in late autumn to winter, were analyzed by reversed-phase HPLC. A novel carotenoid, monoanhydroeschscholtzxanthin (3), was isolated from the red-colored leaves. UV-VIS, MS,¹H-NMR and CD spectral data showed that the structure of 3 was (3S)-2′, 3′, 4′, 5′-tetradehydro-4, 5′-retro-β, β-caroten-3-ol. As well as anhydroeschscholtzxanthin (2), the major red carotenoid in the leaves, eschscholtzxanthin (4) was identified. Very small amounts of yellow carotenoids (neoxanthin, violaxanthin, lutein and β-carotene), which are major components of green leaves, were present in the red-colored leaves. The amounts of chlorophylla andb in the leaves decreased markedly during coloration, even at the early stages, whereas those of the yellow carotenoids decreased gradually. In contrast, the content of 2, a red carotenoid, increased steadily during coloration. The biosynthetic pathway of 2 inB. sempervirens was deduced tentatively on the basis of the individual carotenoid contents during autumnal coloration.     

Algal Carotenoids 51. Secondary carotenoids 2.Haematococcus pluvialis aplanospores as a source of (3S, 3′S)-astaxanthin esters

Aplanospores ofHaematococcus pluvialis MUR 145 contained 0.7% carotenoids (dry wt. basis) consisting of β,β-carotene (5% of total carotenoid), echinenone (4%), canthaxanthin (4%), (3S,3′S)-astaxanthin diester (34%), (3S,3′S)-astaxanthin monoester (46%), (3S,3′S)-astaxanthin (1%) and (3R,3′R,6′R)-lutein (6%). The astaxanthin esters were examined by TLC and HPLC and VIS,¹H NMR and mass spectra recorded. Their chirality was determined by the camphanate method (Vecchi & Müller, 1979) after anaerobic hydrolysis. The tough cell wall of the aplanospores required enzymatic treatment prior to pigment extraction. The potential use of this microalga as a feed ingredient in aquaculture is discussed briefly.     

Transgenic aequorin monitors cytosolic calcium transients in soybean cells challenged with β-glucan or chitin elicitors

Transgenic soybean (Glycine max L.) cells expressing aequorin were used to monitor changes in cytosolic Ca²⁺ concentrations in response to treatment with fungal elicitors. After an apparent lag phase of about 60 s, both chitin fragments and β-glucan elicitors caused a rapid increase in cytosolic Ca²⁺ concentration, which peaked within 2–2.5 min of treatment. The Ca²⁺ concentration then decreased and reached the basal level after about 5 min in the case of the treatment with chitin fragments, while a second rise in the Ca²⁺ concentration with a maximum occurring after about 7–8 min was observed in the case of β-glucan treatment. Calibration of the signals showed that the elicitors enhanced the cytosolic Ca²⁺ concentration from resting concentrations as low as 0.1 lM to highest levels of about 2 lM. Dose-response experiments showed that the concentration of elicitors giving a Ca²⁺ response at the 50% level was 0.4 nM for the chitin fragment and 28 lM and 72 lM, respectively, for a synthetic hepta-β-glucoside and a fungal β-glucan fraction. The β-glucan- or N,N′,N′′,N′′′-tetraacetyl chitotetratose (CH4)-induced Ca²⁺ signals were inhibited by both the Ca²⁺ chelator 1,2-bis-(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and by the Ca²⁺-channel inhibitor La³⁺. Neomycin, whose target in plant cells has not yet been clearly identified, reduced predominantly the expression of the second peak of the biphasic Ca²⁺ curve following β-glucan treatment. Bacterial cyclic β-glucans known to suppress β-glucan-induced phytoalexin production were also found to function as a suppressor for the Ca²⁺ response that was elicited by the fungal β-glucans. The results clearly show that the increase in the cytosolic Ca²⁺ concentration is an early and rapid event in the elicitor-sensing mechanism of soybean cells, and is probably connected with the subsequent activation of defence responses. Received: 23 July 1998 / Accepted: 16 October 1998     

Purification and characterization of UDP-glucose: anthocyanin 3′,5′-<Emphasis Type="Italic">O</Emphasis>-glucosyltransferase from <Emphasis Type="Italic">Clitoria ternatea</Emphasis>

A UDP-glucose: anthocyanin 3′,5′-O-glucosyltransferase (UA3′5′GT) (EC 2.4.1.-) was purified from the petals of Clitoria ternatea L. (Phaseoleae), which accumulate polyacylated anthocyanins named ternatins. In the biosynthesis of ternatins, delphinidin 3-O-(6″-O-malonyl)-β-glucoside (1) is first converted to delphinidin 3-O-(6″-O-malonyl)-β-glucoside-3′-O-β-glucoside (2). Then 2 is converted to ternatin C5 (3), which is delphinidin 3-O-(6″-O-malonyl)-β-glucoside-3′,5′-di-O-β-glucoside. UA3′5′GT is responsible for these two steps by transferring two glucosyl groups in a stepwise manner. Its substrate specificity revealed the regioselectivity to the anthocyanin′s 3′- or 5′-OH groups. Its kinetic properties showed comparable k _cat values for 1 and 2, suggesting the subequality of these anthocyanins as substrates. However, the apparent K _m value for 1 (3.89 × 10⁻⁵ M), which is lower than that for 2 (1.38 × 10⁻⁴ M), renders the k _cat/K _m value for 1 smaller, making 1 catalytically more efficient than 2. Although the apparent K _m value for UDP-glucose (6.18 × 10⁻³ M) with saturated 2 is larger than that for UDP-glucose (1.49 × 10⁻³ M) with saturated 1, the k _cat values are almost the same, suggesting the UDP-glucose binding inhibition by 2 as a product. UA3′5′GT turns the product 2 into a substrate possibly by reversing the B-ring of 2 along the C2-C1′ single bond axis so that the 5′-OH group of 2 can point toward the catalytic center. K. Kogawa, N. Kato, K. Kazuma, and N. Noda contributed equally to this work.     

<Emphasis Type="Italic">Aspergillus niger</Emphasis> metabolism of citrus furanocoumarin inhibitors of human cytochrome P450 3A4

Fungi metabolize polycyclic aromatic hydrocarbons by a number of detoxification processes, including the formation of sulfated and glycosidated conjugates. A class of aromatic compounds in grapefruit is the furanocoumarins (FCs), and their metabolism in humans is centrally involved in the “grapefruit/drug interactions.” Thus far, the metabolism by fungi of the major FCs in grapefruit, including 6′, 7′-epoxybergamottin (EB), 6′, 7′-dihydroxybergamottin (DHB), and bergamottin (BM), has received little attention. In this study, Aspergillus niger was observed to convert EB into DHB and a novel water-soluble metabolite (WSM). Bergaptol (BT) and BM were also metabolized by A. niger to the WSM, which was identified as BT-5-sulfate using mass spectrometry, UV spectroscopy, chemical hydrolysis, and ¹H and ¹³C nuclear magnetic resonance spectroscopy. Similarly, the fungus had a capability of metabolizing xanthotoxol (XT), a structural isomer of BT, to a sulfated analog of BT-5-sulfate, presumably XT-8-sulfate. A possible enzyme-catalyzed pathway for the grapefruit FC metabolism involving the cleavage of the geranyl group and the addition of a sulfate group is proposed.     

Simple and efficient enzymatic transglycosylation of stevioside by β-cyclodextrin glucanotransferase from <Emphasis Type="Italic">Bacillus firmus</Emphasis>

Stevioside was subjected to 1,4-intermolecular transglycosylation using β-cyclodextrin glucanotransferase (β-CGtase) produced from an alkalophilic strain of Bacillus firmus. The reaction was carried out by traditional, ultrasound-assisted and microwave-assisted techniques. Reaction under microwave conditions was faster and was completed in 1 min yielding two 1,4 transglycosylated products, 4′-O-alpha-d-glycosyl stevioside (I) and 4′′-O-alpha-d-maltosyl stevioside (II) in 66% and 24%, respectively. The optimum transglycosylation occurred by using stevioside (1.24 mmol), β-CD (1.76 mmol) and β-CGtase (2 U/g) under microwave assisted reaction (MAR) in 5 ml sodium phosphate buffer (pH 7) at 50°C and 80 W power. MAR is therefore potentially a useful and economical method for faster transglycosylation of stevioside. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Phosphorylation is Involved in the Regulation of the Taurine Influx Via the β-system in Ehrlich Ascites Tumor Cells

The role of 3′,5′-cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), protein kinase C (PKC) and phosphatases in the regulation of the taurine influx via the β-system in Ehrlich ascites tumor cells has been investigated. The taurine uptake by the β-system in Ehrlich cells is inhibited when PKC is activated by phorbol 12-myristate 13-acetate (PMA) and when protein phosphatases are inhibited by calyculin A (CLA). On the other hand, taurine uptake by the β-system is stimulated by an increased level of cAMP or following addition of N⁶,2′-O-dibutyryl-3′,5′-cyclic adenosine monophosphate (dbcAMP). The effect of dbcAMP is partially blocked by addition of the protein kinase inhibitor H-89, and suppressed in the presence of CLA. It is proposed that the β-system in the Ehrlich cells exists in three states of activity: State I, where a PKC phosphorylation site on the transporter or on a regulator is phosphorylated and transport activity is low. State II, where the PKC phosphorylation site is dephosphorylated and transport activity is normal. State III, representing a state with high transport activity, induced by an elevated cellular cAMP level. Apparently, cAMP preferentially stimulates taurine transport when the β-system is in State II. Received: 8 September/Revised: 9 November 1995     

Structure identification and fermentation characteristics of pinoresinol diglucoside produced by <Emphasis Type="Italic">Phomopsis</Emphasis> sp. isolated from <Emphasis Type="Italic">Eucommia ulmoides</Emphasis> Oliv

Pinoresinol diglucoside (PDG) is the important antihypertensive compound in Eucommia ulmoides Oliv., a traditional Chinese herb medicine. The research objective was to certify the possibility of producing PDG through fermentation. PDG-producing endophytic fungi were isolated from E. ulmoides Oliv., and the highest PDG-yielding (11.65 mg/L) isolate, XP-8, was identified as Phomopsis sp. according to the morphological characteristics and the phylogenetic tree constructed on the basis of the gene sequence in the internal transcribed spacers district. The microbial PDG was isolated by using S-8 resin and purified to a purity of 98.7% using preparative high-performance liquid chromatography (HPLC). Information obtained from the UV spectrum (277 and 227 nm, in water solution), infra-red spectrum (3,428; 2,930; 2,877; 1,637; 1,600; and 1,513; 1,460; 1,421; 1,269; 1,223; 1,075; 658 cm⁻¹, in powder), molecular weight (682 Da, measured using HPLC-electrospray ionization mass spectrometry (ESI/MS) and tandem mass spectrometry), and nuclear magnetic resonance analysis show the microbial PDG is (+)-1-pinoresinol 4,4′-di-O-β-d-glucopyranoside, same as the plant-derived PDG. The microbial PDG is stable in pH range from 3 to 11 but less stable at temperature higher than 90 °C and in light exposure. During the fermentation, PDG production outside cells starts at the later stage of cell growth when the residual sugar in the medium was low. The study reveals the possibility for production of PDG by fermentation.     

Purification and characterization of thermostable β-1,3-1,4 glucanase from<Emphasis Type="Italic">Bacillus</Emphasis> sp. A8-8

In this study, the extracellular enzyme activity ofBacillus sp. A8-8 was detected on LB agar plates containing 0.5% of the following substrates: carboxymethylcellulose (CMC), xylan, cellulose, and casein, respectively. The β-1,3-1,4 glucanase produced fromBacillus sp. A8-8 was purified by ammonium sulfate and hydrophobic chromatography. The molecular size of the protein was estimated by SDS-PAGE as approximately 33 kDa. The optimum pH and temperature for the enzyme activity were 6.0 and 60°C, respectiveley. However, enzyme activity was shown over a broad range of pH values and temperatures. The purified β-1,3-1,4 glucanase retained over 70% of its original activity after incubation at 80°C for 2 h, and showed over 40% of its original activity within the pH range of 9 to 12. This suggests that β-1,3-1,4 glucanase fromBacillus sp. A8-8 is thermostable and alkalistable. In addition, β-1,3-1,4 glucanase had higher substrate specificity to lichenan than to CMC. Finally the activity of the endoglucanase was inhibited by Fe³⁺, Mg²⁺, and Mn²⁺ ions. However Co²⁺ and Ca²⁺ ions were increased its activity. These authors contributed equally to this work.     

Enzyme analyses demonstrate that β-methylbutyric acid is converted to β-hydroxy-β-methylbutyric acid via the leucine catabolic pathway by Galactomyces reessii

Galactomyces reessii accomplishes the enzymatic transformation of β-methylbutyric acid (isovaleric acid) to β-hydroxy-β-methylbutyric acid. The enzymatic basis for this bioconversion was evaluated by analyzing cell-free extracts of G. reessii for enzyme activities commonly associated with leucine catabolism. G. reessii extracts contained activities for acyl-CoA synthetase, acyl-CoA dehydrogenase, and enoyl-CoA hydratase, whereas β-methylbutyric acid hydroxylase, α-ketoisocaproate oxygenase, and acyl-CoA oxidase (with isovaleryl-CoA as substrate) were not observed. Furthermore, β-methylbutyric acid is initially activated to isovaleryl-CoA by acyl-CoA synthetase, dehydrogenated to methylcrotonyl-CoA by acyl-CoA dehydrogenase, hydrated to β-hydroxy-β-methylbutyric acid-CoA by enoyl-CoA hydratase, and hydrolyzed to β-hydroxy-β-methylbutyric acid in G. reessii extracts. Cell-free extracts converted both isovaleryl-CoA and methylcrotonyl-CoA into β-hydroxy-β-methylbutyric acid, thus demonstrating that β-methylbutyric acid is part of the leucine catabolic pathway. The rate of β-methylbutyric acid conversion to β-hydroxy-β-methylbutyric acid with cell-free extract was 0.013 μmol β-hydroxy-β-methylbutyric acid (mg protein)^–1 h^–1, while the conversion rate of leucine was fivefold lower. With whole cells, the highest production rate [0.042 μmol β-hydroxy-β-methylbutyric acid (g cells)^–1 h^–1] was also observed with β-methylbutyric acid. The results indicate that β-methylbutyric acid is transformed to β-hydroxy-β-methylbutyric acid through the leucine catabolic pathway. Received: 18 July 1997 / Accepted: 12 November 1997     

A new petunidin tetraglycoside and tulipanin fromOphiopogon seeds

Blue seed-coats ofOphiopogon jaburan have been found to contain two kinds of anthocyanins. By means of paper chromatographic and spectral analyses, one present as a minor component was determined to be delphinidin 3-rutinoside, tulipanin, and the major component, a new anthocyanin, was identified as petunidin 3-O-β-(2^G-glucosylrutinoside)-5′-glucoside, which the authors have named “ophionin”. Both anthocyanins were also present in the blue seed-coasts ofO. japonicus andO. planiscapus.