Glycosidic Flavonoids as Rat-Liver Injury Preventing Compounds from Green Tea

A glycosidic flavonoids-rich fraction from green tea leaves was purified to isolate five glycosidic flavonoids, guided by the detection of a preventive effect on D-galactosamine-induced liver injury in rats. These were identified as a flavone C-glycoside (1) and trisaccharide flavonols (2-5) based on the spectroscopic analyses. These compounds suppressed the D-galactosamine-induced increase of plasma alanine aminotransferase and asparatate aminotransferase activities in rats.     

A Primeverosidase as a Main Glycosidase Concerned with the Alcoholic Aroma Formation in Tea Leaves

We isolated and characterized a primeverosidase from fresh tea leaves (Camellia sinensis var. sinensis cv. Yabukita) as a main glycosidase involved in alcoholic aroma (geraniol, linalool, benzyl alcohol, 2-phenylethanol, linalool oxides etc.) formation from their aroma precursors (β-primeverosides: 6-O-β-D-xylopyranosyl-β-D-glucopyranosides) in tea leaves.     

The action of Bacillus subtilis liquefying amylase on 6-deoxy-6-iodoamylose

Benzyl 2-acetamido-2-deoxy-3-O-β-D-galactopyranosyl-α-D-glucopyranoside (1) was chosen as a model bioside to develop a standard procedure for the selective cleavage of glycosidic linkages in polysaccharides containing 2-amino-2-deoxyhexose residues. Treatment of 1 with hydrazine in the presence of hydrazine sulphate resulted in quantitative N-deacetylation with the formation of benzyl 2-amino-2-deoxy-3-O-β-D-galactopyranosyl-α-D-glucopyranoside (2). The galactosyl glycosidic linkage in 2 could be selectively cleaved by acid hydrolysis. Oxidation of 2 with periodate destroyed the galactose residue. Treatment of 2 with nitrous acid cleaved the 2-amino-2-deoxy-D-glucosyl linkage to give 2,5-anhydro-3-O-β-D-galactopyranosyl-D-mannose (3) and benzyl alcohol.     

trans- and cis-Linalool 3,6-Oxide 6-O-β-d-Xylopyranosyl-β-d-glucopyranosides Isolated as Aroma Precursors from Leaves for Oolong Tea

New glycosidic aroma precursors (1 and 2) of the main volatile constituents, trans- and cis-linalool 3,6-oxides (linalool oxides I and II), were isolated from oolong tea leaves (Camellia sinensis var. sinensis cv. Maoxie). The isolation was guided by an enzymatic hydrolysis with acetone powder prepared from fresh tea leaves (cv. Yabukita) followed by GC or GC-MS analyses. Chromatographic purification of hot water extracts of the tea leaves on active charcoal, Amberlite XAD-2, and Sephadex LH-20 columns as well as HPLC gave two new glycosides, trans- and cis-linalool 3,6-oxide 6-O-β-d-xylopyranosyl-β-d-glucopyra-nosides (1 and 2).     

Profiling the Lipophilic Fractions of Pithecellobium dulce Bark and Leaves Using GC/MS and Evaluation of Their Antioxidant,Antimicrobial and Cytotoxic Activities

Pithecellobium dulce has been used in traditional medicine to treat various ailments owing to its restorative properties. The biological activities and chemical profiles of the lipophilic fraction of P. dulce bark and leaves were assessed herein. Fatty acid methyl esters (FAME) and unsaponifiable matter (USM) were prepared and analyzed by GC/MS. A total of 40 compounds were identified in the bark saponifiable fraction, whereas 9 compounds were annotated in the leaves. Palmitic acid methyl ester was the major compound identified accounting for 41.48 % of the bark and 19.03 % of the leaves composition. Besides, linolenic acid methyl ester (22.40 %) and linoleic acid (12.69 %) were annotated in the leaves saponifiable fraction. A total of 63 compounds were detected in the bark USM and 4 compounds were identified in the leaves. Phytol represented the major component in the leaves (52.57 %) followed by lupeol (20.68 %) and lupenone (8.60 %). Meanwhile, n‐dodecane dominated in the bark USM accounting for 24.69 % of the total composition. The leaves and bark lipophilic fractions revealed moderate antioxidant and antibacterial activities. Both extracts showed no antifungal activity. No cytotoxicity was observed for both lipophilic fractions. P. dulce offers a good source of antioxidant compounds that can be introduced to food and pharmaceutical industry.     

(S)-Linalyl, 2-Phenylethyl,and Benzyl Disaccharide Glycosides Isolated as Aroma Precursors from Oolong Tea Leaves

Three glycosides, 6-O-β-d-xylopyranosyl-β-d-glucopyranosides (β-primeverosides) of the aroma constituents, linalool, 2-phenylethanol, and benzyl alcohol, were isolated as aroma precursors from the tea leaves (Camellia sinensis var. sinensis cv. Shuixian and Maoxie, cultivars for oolong tea). The isolation was guided by acid or enzymatic hydrolysis, and subsequent GC and GC-MS analyses. The linalyl glycoside is the first example of naturally occurring (S)-linalyl β-primeveroside.     

Engineering of glucoside acceptors for the regioselective synthesis of beta-(1-->3)-disaccharides with glycosynthases

Glycosynthase mutants obtained from Thermotogamaritima were able to catalyze the regioselective synthesis of aryl β-d-Galp-(1→3)-β-d-Glcp and aryl β-d-Glcp-(1→3)-β-d-Glcp in high yields (up to 90 %) using aryl β-d-glucosides as acceptors. The need for an aglyconic aryl group was rationalized by molecular modeling calculations, which have emphasized a high stabilizing interaction of this group by stacking with W312 of the enzyme. Unfortunately, the deprotection of the aromatic group of the disaccharides was not possible without partial hydrolysis of the glycosidic bond. The replacement of aryl groups by benzyl ones could offer the opportunity to deprotect the anomeric position under very mild conditions. Assuming that benzyl acceptors could preserve the stabilizing stacking, benzyl β-d-glucoside firstly assayed as acceptor resulted in both poor yields and poor regioselectivity. Thus, we decided to undertake molecular modeling calculations in order to design which suitable substituted benzyl acceptors could be used. This study resulted in the choice of 2-biphenylmethyl β-d-glucopyranoside. This choice was validated experimentally, since the corresponding β-(1→3) disaccharide was obtained in good yields and with a high regioselectivity. At the same time, we have shown that phenyl 1-thio-β-d-glucopyranoside was also an excellent substrate leading to similar results as those obtained with the O-phenyl analogue. The NBS deprotection of the S-phenyl group afforded the corresponding disaccharide quantitatively.     

Production of Melilotic Acid by Action of Taphrina wiesneri

The in vitro action of Taphrina wiesneri on coumarin and its related compounds were examined. Melilotic acid, which accumulates in larger amounts in infected cherry leaves than in healthy leaves, was produced from coumarin, 3,4-dihydrocoumarin, o-coumaric acid or o-coumaryl glucoside by the action of acetone-dried cells of the fungus. From the results it is suggested that in cherry plants infected with the fungus melilotic acid may be formed from these precursors contained as ordinary components in cherry leaves. Possible mechanisms of the conversion of coumarin to melilotic acid are also discussed.     

Enantiospecific enzymatic preparation of (2S,3S)-3-alkylaspartic acids of current interest in the synthesis of stereoregular poly[β-(2S,3S)-3-alkylmalic acids] as new optically active functional polyesters

(2S,3S)-3-methyl- and 3-isopropylaspartic acids were synthesized by bioconversion of the corresponding alkylfumarates (mesaconate and 3-isopropylfumarate) using β-methylaspartase from cell-free extracts of Clostridium tetanomorphum. Optically pure (2S,3S)-3-alkylaspartic acids were transformed in several steps to benzyl (3S,4R)-3-alkylmalolactonates without any racemization of the two chiral centers. These optically active α,β-substituted-β-lactones were polymerized by anionic ring opening polymerization yielding optically active semi-crystalline polyesters. ¹³C NMR analysis of poly[benzyl β-3-isopropylmalate] in CDCl₃ has shown that only the iso-type stereosequence is present in the polymer, indicating that the macromolecular chain is constituted by the only units of benzyl β-(2S,3S)-3-isopropylmalate monomer. The polymerization reaction was done without any racemization of the two stereogenic centers as in the case of benzyl (3S,4R)-3-methylmalolactonate. © 1996 Wiley-Liss, Inc.     

Preparative separation of chlorogenic acid by centrifugal partition chromatography from highbush blueberry leaves (Vaccinium corymbosum L.)

Introduction – Blueberries (genus Vaccinium) have gained worldwide focus because of the high anthocyanin content of their fruits. In contrast, the leaves of blueberry have not attracted any attention, even though they contain large quantities of chlorogenic acid, a strong antioxidant compound. Objective – The aim of this investigation was the quantification and preparative isolation of chlorogenic acid (5‐caffeoylquinic acid, 5‐CQA) from blueberry leaves using a new separation scheme, centrifugal partition chromatography (CPC). Methodology – A water fraction containing a high concentration of 5‐CQA (14.5% of dry weight extract) was obtained by defatting a crude methanol extract from blueberry leaves. CPC was applied to isolate 5‐CQA from this water fraction using a two‐phase solvent system of ethyl acetate–ethanol–water at a volume ratio 4:1:5 (v/v/v). The flow‐rate of mobile phase was 2 mL/min with the ascending mode while rotating at 1200 rpm. The eluate was monitored at 330 nm. The structure of chlorogenic acid in the CPC fraction was confirmed with HPLC, UV, ESI/MS and NMR spectra. Results – The HPLC chromatogram showed that the fractions collected by CPC contained chlorogenic acid with 96% purity based on peak area percentage. The total amount of chlorogenic acid isolated from 0.5 g of a water fraction was 52.9 mg, corresponding to 10.6% of the water fraction. The isolated compound was identified successively as 5‐CQA with MS (parent ion at m/z 355.1 [M + H]⁺) and ¹H NMR spectra [caffeoyl moiety in the down field (δ 6.0–8.0 ppm) and quinic acid moiety in the up field (δ 2.0–5.5 ppm)]. Conclusion – 5‐CQA was successfully isolated from blueberry leaves by the CPC method in a one‐step procedure, indicating a further potential use for blueberry leaves. Copyright © 2010 John Wiley & Sons, Ltd.     

Potent Lipolytic Activity of Lactoferrin in Mature Adipocytes

Momilactone A, a major rice diterpene phytoalexin, could be synthesized by dehydrogenation at the 3-position of 3β-hydroxy-9β-pimara-7,15-dien-19,6β-olide in rice leaves. The presence of 3β-hydroxy-9β-pimara-7,15-dien-19,6β-olide in UV-irradiated rice leaves was confirmed by comparing the mass spectra and retention times after a GC/MS analysis of the natural and synthetic compounds. The soluble protein fraction from UV-irradiated rice leaves showed dehydrogenase activity to convert 3β-hydroxy-9β-pimara-7,15-dien-19,6β-olide into momilactone A. The enzyme required NAD⁺ or NADP⁺ as a hydrogen acceptor. The optimum pH for the reaction was 8. The K _m value to 3β-hydroxy-9β-pimara-7,15-dien-19,6β-olide was 36 μM when NAD⁺ was supplied as a cofactor at a concentration of 1 mM. 3β-Hydroxy-9β-pimara-7,15-dien-19,6β-olide and its dehydrogenase activity were induced in a time-dependent manner by UV irradiation.     

Chemical Composition and Antimicrobial Activity of the Volatile Fractions from Leaves and Flowers of the Wild Iraqi Kurdish Plant Prangos peucedanifolia Fenzl

The volatile fractions isolated from Prangos peucedanifolia Fenzl leaves and flowers were investigated for their phytochemical composition and biological properties. Flower and leaf hydrodistillation afforded 3.14 and 0.49 g of yellowish oils in 1.25 and 0.41% yields, respectively, from dry vegetable materials. According to the GC‐FID and GC/MS analyses, 36 (99.35% of the total oil composition) and 26 compounds (89.12%) were identified in the two oils, respectively. The major constituents in the flower volatile fraction were β‐pinene (35.58%), α‐pinene (22.13%), and β‐phellandrene (12.54%), while m‐cresol (50.38%) was the main constituent of the leaf volatile fraction. The antimicrobial activity was evaluated against several bacterial and fungal strains, on the basis of the minimum inhibitory concentration (MIC) by the micro‐ and macrodilution methods. The two volatile fractions showed moderate antifungal and antibacterial activities, especially against Trichophyton rubrum (MIC of 2×10³ μg/ml), Streptococcus mutans, Streptococcus pyogenes, and Staphylococcus aureus (MIC≤1.9×10³ μg/ml for all).     

Bacterial Canker of Sweet Cherry (Prunus avium) in Poland

In Polish climatic conditions cherry cankers resulting from infection by Pseudomonas morsprunorum continue development in summer, but the rate may be slower than in the period from April to June. However, from the well established, active canker developing under the dwarf shoot of the susceptible variety‘Hedelfińska’in July no P. morsprunorum were isolated. On the other hand, numerous strains of the genus Erwinia were found there which caused a hyper sensitivity reaction (HR) on tobacco leaves. From the cracks on the current-year cherry shoots, due to fresh infection and from symptomless leaves P. morsprunorum strains were isolated, always accompanied by those of the Erwinia genus in the approximate proportion 1: 1 or 1:2. The strains of Erwinia genus seemed similar to the DC and YC strains isolated by BILLING and BAKER from pear and apple trees infected by fireblight and also to the strains in group III of the bacteria isolated by CROSSE from cherry leaves. In two tests (immediately after isolation and after 4 months of storage) the strains of the Erwinia genus and 3 nonidentified isolates induced HR. At a third test, after 10 months of preservation these strains were HR-negative in contrast to the P. morsprunorum isolates. The fact that strains of the Erwinia genus inducing HR were isolated in large numbers from active cherry canker where pathogenic bacteria were not detected, may indicate that they play some role in the development of cherry tree canker.     

Studies on water transport through the sweet cherry fruit surface: IX. Comparing permeability in water uptake and transpiration

Water uptake and transpiration were studied through the surface of intact sweet cherry (Prunus avium L.) fruit, exocarp segments (ES) and cuticular membranes (CM) excised from the cheek of sweet cherry fruit and astomatous CM isolated from Schefflera arboricola (Hayata) Hayata, Citrus aurantium L., and Stephanotis floribunda Brongn. leaves or from Lycopersicon esculentum Mill. and Capsicum annuum L. var. annuum Fasciculatum Group fruit. ES and CM were mounted in diffusion cells. Water (deionized) uptake into intact sweet cherry fruit, through ES or CM interfacing water as a donor and a polyethyleneglycol (PEG 6000, osmotic pressure 2.83 MPa)-containing receiver was determined gravimetrically. Transpiration was quantified by monitoring weight loss of a PEG 6000-containing donor (2.83 MPa) against dry silica as a receiver. The permeability coefficients for osmotic water uptake and transpiration were calculated from the amount of water taken up or transpired per unit surface area and time, and the driving force for transport. Permeability during osmotic water uptake was markedly higher than during transpiration in intact sweet cherry fruit (40.2-fold), excised ES of sweet cherry fruit (12.5- to 53.7-fold) and isolated astomatous fruit and leaf CM of a range of species (on average 23.0-fold). Partitioning water transport into stomatal and cuticular components revealed that permeability of the sweet cherry fruit cuticle for water uptake was 11.9-fold higher and that of stomata 56.8-fold higher than the respective permeability during transpiration. Increasing water vapor activity in the receiver from 0 to 1 increased permeability during transpiration across isolated sweet cherry fruit CM about 2.1-fold. Permeability for vapor uptake from saturated water vapor into a PEG 6000 receiver solution was markedly lower than from liquid water, but of similar magnitude to the permeability during self-diffusion of ³H₂O in the absence of osmotica. The energy of activation for self-diffusion of water across ES or CM was higher than for osmotic water uptake and decreased with increasing stomatal density. The data indicate that viscous flow along an aqueous continuum across the sweet cherry fruit exocarp and across the astomatous CM of selected species accounted for the higher permeability during water uptake as compared to self-diffusion or transpiration.     

Hexane Extract of Echinops spinosissimus Turra subsp. spinosus from Tunisia: A Potential Source of Acetylated Sterols – Investigation of its Biological Activities

The hexane extract of Echinops spinosissimus Turra subsp. spinosus flower heads was analyzed for its fatty acid and sterol composition. Its physicochemical characteristics were also studied. The saponification, iodine and peroxide values were determined as 255 mg KOH/g, 42.57 g I₂/100 g and 110 m equiv. O₂/kg of oil, respectively. The oleic (C18:1; 61.14%), palmitic (C16:0; 21.36%) and linoleic (C18:2; 10.45%) acids were the dominant fatty acids. This extract was also found to contain high levels of β‐sitosterol and stigmasterol (44.97% and 34.95% of total sterols, respectively). On the other hand, the identification of terpenoid compounds was investigated by using GC/MS, which revealed fourteen major terpenoids mainly taraxasterol, lupeol, pseudotaraxasterol, lup‐22(29)‐en‐3‐yl acetate, taraxasteryl acetate, α‐amyrin, β‐amyrin, pseudotaraxasteryl acetate, hop‐20(29)‐en3‐β‐ol, α‐amirenone, along with β‐sitosterol and stigmasterol. Moreover, we have evaluated the in vitro antibacterial and antifungal activities of the unsaponifiable matter and a fraction isolated from this extract. These activities were conducted using the diffusion disc methods and broth microdilution assay. The resulted fraction from this extract showed the highest antibacterial activity with significant minimum inhibitory concentrations (MIC) values 125.0 μg/ml against Staphylococcus aureus, Micrococcus luteus and Bacillus cereus. However, it did exhibit no substantial antifungal activity.     

Chemical aspects of host‐acceptance behaviour in the bird cherry‐oat aphid Rhopalosiphum padi: host‐acceptance signals used by different morphs with the same genotype

Host acceptance by gynoparae and winged virginoparae of the bird cherry‐oat aphid Rhopalosiphum padi (L.) is investigated utilizing leaves and aqueous extracts of the primary and secondary hosts, as well as nonhost plants. Gynoparae are specialized to reproduce on bird cherry Prunus padus L., whereas virginoparae reproduce and feed on various grasses. Host acceptance is assessed using levels of reproduction and survival for adults, as well as survival for nymphs. Little is known of host acceptance by nymphs. The data show that gynoparae and winged virginoparae produce nymphs almost exclusively on their host plants, bird cherry and barley leaves, respectively, over 72 h. When tested with aqueous plant extracts, however, gynoparae produce nymphs almost exclusively on bird cherry extract and progeny numbers are found to be similar to those on plant leaves. Few nymphs are produced on artificial diet. By contrast, winged virginoparae produce nymphs on aqueous extracts of barley, bird cherry and bean, as well as on artificial diet. The numbers of nymphs deposited by gynoparae are similar on aqueous extracts of bird cherry leaves collected at different times during the growing season. When extracts from leaves of various Prunus species are tested, only leaves of P. padus and Prunus virginiana stimulate parturition. Oviparae, the sexual female nymphs of gynoparae, survive well for 96 h on both bird cherry and barley leaves but not on bean seedlings, whereas nymphs of winged virginoparae survive well only on barley leaves. They do not survive for 96 h on any plant‐leaf extracts, although they do survive on artificial diet.     

Multiplicity of Cell Wall Degrading Glycosidic Hydrolases Produced by Apple Infecting Botrytis cinerea

It was shown that Botrytis cinerea, an isolate infecting apples, secreted in vivo and in culture a variety of glycosidic hydrolases with substrate specificity towards the polysaccharides of cell walls. The following enzymes were partially separated by column chromatography on DEAE-Sepharose CL-6B: two cellulases, three xylanases, one arabinanase, polygalacturonase, β-glucosidase, β-xylosidase, β-galactosidase, β-mannosidase and α-galactosidase. The activity of glycosidic hydrolases tested was strongly inactivated by NBS and weaker by PCMB, tetranitromethan, dibromoacetophenon and Fe³+, The results indicate synergistic action of the obtained cellulase, xylanase, polygalacturonase and arabinanase in apple cell wall degradation.     

Chemical Variability of the Leaf Essential Oil of Xylopia aethiopica (Dunal) A.Rich. from Côte d'Ivoire

The chemical composition of 48 essential‐oil samples isolated from the leaves of Xylopia aethiopica harvested in six Ivoirian forests was investigated by GC‐FID and ¹³C‐NMR analyses. In total, 23 components accounting for 82.5–96.1% of the oil composition were identified. The composition was dominated by the monoterpene hydrocarbons β‐pinene (up to 61.1%) and α‐pinene (up to 18.6%) and the sesquiterpene hydrocarbon germacrene D (up to 28.7%). Hierarchical cluster and principal component analyses allowed the distinction of two groups on the basis of the β‐pinene and germacrene D contents. The chemical composition of the oils of Group I (38 oil samples) was clearly dominated by β‐pinene, while those of Group II (10 samples) were characterized by the association of β‐pinene and germacrene D. The leaves collected in the four inland forests produced β‐pinene‐rich oils (Group I), while the oil samples belonging to Group II were isolated from leaves harvested in forests located near the littoral.     

A new flavanone and other flavonoids from green perilla leaf extract inhibit nitric oxide production in interleukin 1β-treated hepatocytes

A new flavanone, shisoflavanone A (1), and several flavonoids were purified from the ethyl acetate-soluble fraction of green perilla leaves (Perilla frutescens Britton var. crispa form viridis), and their structures were identified. Shisoflavanone A was elucidated as 8-hydroxy-6,7-dimethoxyflavanone based on its spectral data. Other constituents of the ethyl acetate-soluble fraction, i.e. 5,8-dihydroxy-7-methoxyflavanone (2), negletein (5,6-dihydroxy-7-methoxyflavone) (3), luteolin (4), apigenin (5), esculetin (6), and protocatechuic acid (7), were identified. This is the first time that constituents 2, 3, and 6 have been found in green perilla. Shisoflavanone A and the other constituents (except 7) significantly inhibited nitric oxide production in interleukin 1β-stimulated rat hepatocytes, which have been used to monitor the anti-inflammatory effects of herbal constituents. The present findings suggest that these constituents, including shisoflavanone A, may be involved in the anti-inflammatory effects of green perilla leaves.     

(Z)-3-Hexenyl-β-d-glucopyranoside in Fresh Tea Leaves as a Precursor of Green Odor

The glycoside fraction from fresh tea leaves was acetylated and separated by silica gel column chromatography.

A crystalline product was identified as (Z)-3-hexenyl-(tetra-O-acetyl)-β-d-glucopyranoside from spectrometric data which were identical with those of an authentic synthesized sample in all respects.

There are two possible processes for the formation of the greenish odor of plant materials, these being a biosynthetic process from the lipid and enzymatic hydrolysis of (Z)-3-hexenyl-β-d-glucoside.