Tea catechol oxidase: Isolation,purification and kinetic characterization

Cathechol oxidase extracted from tea leaves was purified over 200-fold, using isoelectric focusing. The purified catechol oxidase was free of peroxidase and flavanol gallate esterase activities. Further, this enzyme was shown to have optimum activity near pH 5·7 and a K_m of 2·3 × 10^?3 M (at 25°) for (?)-epigallocatechin gallate. The purified enzyme was found to be capable of epimerizing tea flavanols at their C-2 position whether oxidation of the flavanol occurs (aerobic conditions) or not (anaerobic conditions). When oxygen is present, gallic acid is formed as a result of oxidation of either (?)-epigallocatechin gallate or (?)-epicatechin gallate. Formation of gallic acid is a side reaction of the oxidation of the flavanol gallates and is named oxidative degallation; no esterase per se is involved in this reaction.     

Antioxidants from Steamed Used Tea Leaves and Their Reaction Behavior

The most efficient steaming conditions below 200 °C for extracting antioxidants from used tea leaves and their reaction behavior during the steaming treatment were investigated. The antioxidative activity of the steamed extracts increased with increasing steaming temperature, and the yield of the ethyl acetate extract fraction from each steamed extract showing the greatest antioxidative activity also increased. Caffeine, (?)-catechin, (?)-epicatechin, (?)-gallocatechin, (?)-epigallocatechin, (?)-catechin gallate, (?)-epicatechin gallate, (?)-gallocatechin gallate, (?)-epigallocatechin gallate and gallic acid were identified from the ethyl acetate extract fraction. Quantitative analyses demonstrated that the catechins with a 2,3-cis configuration decreased with increasing steaming temperature, whereas the corresponding epimers at the C-2 position increased. Each pair of epimers showed similar antioxidative activity to each other, indicating that the epimerization reaction did not contribute to the improved antioxidative activity. It is concluded from these results that the improvement in antioxidative activity at higher steaming temperatures was due to the increased yield of catechins and other antioxidants.     

Effect of cadmium on the phenolic compounds formation in the callus cultures derived from various organs of the tea plant

The effects of cadmium (6.3 × 10^?5 M or 10.6 × 10^?5 M) on the growth of tea plant (Camellia sinensis L.) callus cultures derived from leaves, stems, and roots and on the formation, in these cultures, of phenolic compounds, including flavans and lignin, which are characteristic of the tea plant, were investigated. In the calli derived from leaves and stems, cadmium treatment decreased the biomass increment, while in the calli derived from roots, growth characteristics remained at the control level. Under the effect of cadmium, the content of phenolic compounds, including flavans, in the leaf calli decreased, while in the stem and root calli, it either increased (at the cadmium concentration of 6.3 × 10^?5 M), or was close to a control one (at the cadmium concentration of 10.6 × 10^?5 M). The lignin content in the root and stem calli increased, but it did not change in the leaf calli. All this data demonstrate that the cadmium-induced changes in phenolic metabolism of the tea plant callus culture depended both on the cadmium concentration in the medium and on the origin of calli.     

Production of phenolic compounds in callus cultures of tea plant under the effect of 2,4-D and NAA

The effect of hormone-like compounds at different concentrations: 2,4-D (2 × 10^?6; 2 × 10^?5; and 2 × 10^?4M) and 1-NAA (2 × 10^?7; 2 × 10^?6; 2 × 10^?5; 4 × 10^?5, and 6 × 10^?5 M) on the growth and production of phenolic compounds, including flavans and lignin, was investigated in callus culture of tea plant (Camellia sinensis L., a highly productive strain IFR ChS-2). The growth of the culture was vigorous, and production of phenolic compounds therein was efficient in the medium containing 2 × 10^?5 M 2,4-D. Substitution of 1-NAA for 2,4-D in all the cases decelerated the growth of the culture. These changes were more pronounced when 2 × 10^?7 and 2 × 10^?6 M 1-NAA was used; in this case, biomass accumulation decreased by 1.5–2.0 times as compared with control material growing on the medium with 2 × 10^?5 M 2,4-D. In the presence of 1-NAA, the content of total soluble phenolic compounds and flavans in the calli rose by 30% on the average as compared with control material. Accumulation of lignin remained essentially the same. Therefore, the replacement of 2,4-D with 1-NAA in the nutrient medium used for the growing of highly productive strain of tea plant callus did not induce considerable changes in its ability to produce phenolic compounds.     

Chemosystematics of tea trees based on tea leaf polyphenols as phenetic markers

This study examined the polyphenols of tea leaves as chemotaxonomic markers to investigate the phenetic relationship between 89 wild (the small-leaved C. sinensis var. sinensis and large-leaved C. sinensis var. assamica), hybrid, and cultivated tea trees from China and Japan. (?)-Epigallocatechin 3-O-gallate, EGCG (1); (?)-epigallocatechin, EGC (2); (?)-epicatechin 3-O-gallate, ECG (3); (?)-epicatechin, EC (4); (+)-catechin, CA (5); strictinin, STR (6); and gallic acid, GA (7) were used as polyphenolic markers. Of the 13 polyphenol patterns observed, Principal Component Analysis (PCA) indicated that the structure-types of the flavonoid B-rings, such as the pyrogallol-(EGCG (1) and EGC (2)) and catechol-(ECG (3) and EC (4)) types, greatly influenced the classification. Ward’s minimum-variance cluster analysis was used to produce a dendrogram that consisted of three sub-clusters. One sub-cluster (A) was composed of old tea trees ‘Gushu’ cha (C. sinensis var. assamica) and cv ‘Taidi’ cha, suggesting that relatively primitive tea trees contain greater amounts of compounds 3 and 4 and lower amounts of compounds 1 and 2. The other two sub-clusters B and C, made up of Chinese hybrids (sub-cluster B) and Japanese and Taiwanese tea trees (sub-cluster C), had lower contents of 3 and 4 than sub-cluster A. Therefore, PCA and cluster analysis indicated that the greater the amounts of 1 and 2 (and the lower of 3 and 4), the more recent the origin of the tea line. Based on morphological characteristics, geographical information, and the historical information on tea trees, these results show good agreement with the current theory of tea tree origins, and this suggests that the Xishuangbanna district and Puer City are among the original sites of the tea tree species.     

Interaction of Growth Retardants and Temperature in Growth, Flowering, Regeneration, and Auxin Activity of Begonia × cheimantha Everett

Soil application of CCC reduced stem and leaf growth in Begonia plants. This effect was evident with all concentrations tested at 18°C, whereas at 21 and 24°C no growth–retarding effect was observed with 2 × 10^?2 M CCC, and with 5 × 10^?3 M growth was even stimulated. Flowering was promoted by CCC in long day and neur–critical temperature, particularly under low light intensity in the winter. The formation of adventitious buds in leaves of plants grown at 21 and 24°C was stimulated when the plants received 5 × 10^?2 and 2 × 10^?2 M CCC, while 8 7times; 10^?2 M was inhibitory. In plants grown at 18°C bud formation was inhibited by all CCC concentrations. Root formation in the the leaves was usually stimulated by high CCC concentrations, while root elongation was reduced. The level of ether–extractable. acidic auxin (presumably IAA) in the leaves was lowered by CCC treatment of the plants, hut this required higher CCC concentrations at higt than at low temperature. When applied to detached leaves CCC stimulated bud formation at concentrations ranging from 10^?4 to 10^?2 M in leaves planted at 18 and 21°C. At 24°C budding was inhibited by 10^?2 M CCC, the lower concentrations being stimulatory also at this temperature. Root formation and growth were not much affected by CCC treatment of the leaves, but increased with the temperature. Soil application of Phosfon (4 × 10^?4 M) had no effect on growth and flowering, nov did it affect the subsequent regeneration of buds and roots in the leaves. In detached leaves Phosfon stimulated bud formation with au optimum at 10^?6 M. Root formation was stimulated by Phosfon at all temperatures, the optimal concentration being 10^?5 M, whereas root length was conversely affected. Foliar application of B-995 to intact plants and treatment of detached leaves greatly inhibited the formation of buds and had little effect on root formation. B-99D reduced the growth and delayed flowering in the plants.     

Drechslerol-A,a new phytotoxic metabolite produced by Drechslera maydis,a strain from costus speciosus

Drechslera maydis causes severe leaf blight disease of Costus speciosus. One compound, drechslerol-A, isolated from the culture filtrate of this fungus was elucidated as (cis) hentetracont-10-ene-12-hydroxymethyl-4-ol on the basis of spectral analysis. The production of necrotic lesion was observed on the leaves of Costus speciosus with 1.6 × 10⁻⁴ M concentration. In the most sensitive reaction drechslerol-A shows complete killing effect on the root growth of wheat seedlings at the concentration of 3.2 × 10⁻⁵ M.     

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.     

Glycosides from Breynia fruticosa and Breynia rostrata

Glycosides, 3-acetyl-(?)-epicatechin 7-O-β-glucopyranoside (1), 3-acetyl-(?)-epicatechin 7-O-(6-isobutanoyloxyl)-β-glucopyranoside (2), 3-acetyl-(?)-epicatechin 7-O-[6-(2-methyl-butanoyloxyl)]-β-glucopyranoside (3), (5Z)-6-[5-(2-hydroxypropan-2-yl)-2-methyl-tetrahydrofuran-2-yl]-3-methylhexa-1,5-dien-3-O-β-glucopyranoside (4), hydroquinone O-[6-(3-hydroxyisobutanoyl)]-β-galactopyranoside (5), 4-(4-O-β-glucopyranosyl-phenoxy)-1-O-β-glucopyranosyl-1,3-benzenediol (6), 7,8-erythro-dihydroxy-3,4,5-trimethoxy-phenyl-propane8-O-β-glucopyranoside (7), 6,7-dimethylbenzofuranol 5-O-β-xylopyranosyl-(1 → 6)-β-glucopyranoside (8), along with 30 known glycosides, were isolated from Breynia fruticosa and Breynia rostrata (Euphorbiaceae). Their structures were determined on the basis of spectroscopic analysis and chemical methods.     

The Inhibition of α-Amylase by Tea Polyphenols

The inhibition of α-amylase from human saliva by polyphenolic components of tea and its specificity was investigated in vitro. Four kinds of green tea catechins, and their isomers and four kinds of their dimeric compounds (theaflavins) produced oxidatively during black tea production were isolated. They were (?)-epicatechin (EC), (?)-epigallocatechin (EGC), (?)-epicatechin gallate (ECg), (?)-epigallocatechin gallate (EGCg), (?)-catechin (C), (?)-gallocatechin (GC), (?)-catechin gallate (Cg), (?)-gallocatechin gallate (GCg), theaflavin (TF1), theaflavin monogallates (TF2A and TF2B), and theaflavin digallate (TF3). Among the samples tested, EC, EGC, and their isomers did not have significant effects on the activity of α-amylase. All the other samples were potent inhibitors and the inhibitory effects were in the order of TF3>TF2A>TF2B>TFl>Cg> GCg > ECg > EGCg. The inhibitory patterns were noncompetitive except for TF3.     

In Vitro Antioxidative Activity of (−)-Epicatechin Glucuronide Metabolites Present in Human and Rat Plasma

Recently we identified four conjugated glucuronide metabolites of epicatechin, (?)-epicatechin-3′-O-glucuronide (E3′G), 4′-O-methyl-(?)-epicatechin-3′-O-glucuronide (4′ME3′G), (?)-epicatechin-7-O-glucuronide (E7G) and 3′-O-methyl-(?)-epicatechin-7-O-glucuronide (3′ME7G) from plasma and urine. E3′G and 4′ME3′G were isolated from human urine, while E7G and 3′ME7G were isolated from rats that had received oral administration of (?)-epicatechin (Natsume et al. (2003), Free Radic. Biol. Med. 34, 840–849). It has been suggested that these metabolites possess considerable in vivo activity, and therefore we carried out a study to compare the antioxidant activities of the metabolites with that of the parent compound. This was achieved by measuring superoxide scavenging activity, reduction of plasma TBARS production and reduced susceptibility of low-density-lipoprotein (LDL) to oxidation. (?)-Epicatechin was found to have more potent antioxidant activity than the conjugated glucuronide metabolites. Both (?)-epicatechin and E7G had marked antioxidative properties with respect to superoxide radical scavenging activity, plasma oxidation induced by 2,2′-azobis-(2-aminopropane) dihydrochloride (AAPH) and LDL oxidation induced by copper ions or 2,2′-azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN). In contrast, the other metabolites had light antioxidative activities over the range of physiological concentrations found in plasma.     

Presynaptic Control of the Synthesis and Release of Dopamine from Striatal Synaptosomes: A Comparison Between the Effects of 5-Hydroxytryptamine, Acetylcholine, and Glutamate

The effects of 5-HT and glutamate on dopamine synthesis and release by striatal synaptosomes were investigated and compared with the action of acetylcholine, which acts presynaptically on this system. 5-HT inhibited (28%) synthesis of [¹⁴C]dopamine from L-[U-¹⁴C]tyrosine, at 10-⁵M and above. This contrasts with the action of acetylcholine, which stimulated [¹⁴C]-dopamine synthesis by 24% at 10-⁴ M. Tissue levels of GABA were unaffected by either 5-HT or acetylcholine up to concentrations of 10-⁴ M. The inhibitory action of 5-HT (5 × 10^?5 M and 2 × 10^?4 M) on [¹⁹C]dopamine synthesis was completely abolished by methysergide (2 × 10^?6 M). Higher concentrations of methysergide (10^?4 M) or cyproheptadine (10^?5 M) inhibited [¹⁴C]dopamine synthesis by 28% and 25%, respectively, when added alone to synaptosomes. However, only methysergide prevented the further inhibition of synthesis caused by 5-HT. At concentrations of 2 × 10^?5 M and above, 5-HT stimulated [¹⁴C]dopamine release. This releasing action differed from that of acetylcholine, which occurred at lower concentrations (e.g., 10^?6 M). Methysergide (up to 10^?4 M) or cyproheptadine (2 × 10^?4 M) did not reduce the 5-HT (5 × 10^?5 M)-induced release of [¹⁴C]dopamine, but methysergide (10^?4 M) showed a potentiation (49%) of this increased release. The stimulatory effects of 5-HT (2 × 10^?5 M) and K⁺ (56 mM) on [¹⁴C]dopamine release were additive, indicating that two separate mechanisms were involved. However, when both agents were present the stimulatory effect of K⁺ (56 mM) on [¹⁴C]dopamine synthesis was not seen above the inhibitory effect of 5-HT. Glutamate (0.1-5 mM) did not affect [⁴C]dopamine release or its synthesis from L-[U-¹⁴C]tyrosine. It is concluded that 5-HT modulates the synthesis of dopamine in striatal nerve terminals through a presynaptic receptor mechanism, an action antagonised by methysergide. The releasing action of 5-HT apparently occurs through a separate mechanism which is also distinct from that involved in the response to K⁺ depolarisation.     

Early Effects of Penconazole on H+ and K+ Transport,Electrolyte Leakage and Transmembrane Electrical Potential in Egeria densa Leaves

The early effects of penconazole (PCZ) at relatively high concentration (10^?4 to 5 × 10^?4 M) on changes in pH and in titratable acidity of the medium, transmembrane electrical potential difference (Em), electrolyte leakage and cell morphology were investigated in Egeria densa leaves. At the lowest (10^?4 M) concentration and in the presence of a very low (10 μM) K⁺ concentration, triazole induced an early, moderate hyperpolarization of Em, associated with a decrease of net K⁺ uptake, suggesting some increase in the passive permeability to K⁺. This Em hyperpolarization was no longer detectable at high (2 mM) K⁺_out concentration. At high PCZ concentrations (3 × 10^?4 M and 5 × 10^?4 M) the early hyperpolarization detectable in the presence of a low K⁺_out concentration became transient, and was followed by a marked depolarization. PCZ, at these concentrations, suppressed acidification of the medium, stimulated electrolyte leakage and, in the mesophyll cells, induced some shrinking of the cytoplasm and its disconnection from the cell walls. These results are interpreted as due to an early effect of this triazole leading to the disorganization of the plasma membrane.     

Flavan-3-ols from the leaves of Malaysian Uncaria longiflora var. pteropoda (Miq.) Ridsd

A novel flavonoid, (?)-2R,3R-3,5,4′-trihydoxyflavan-[6,7:5″,6″]-2″-pyranone, named uncariechin (1), was isolated from the methanolic extract of the leaves of Uncaria longiflora var. pteropoda (Miq.) Ridsd. along with the known (?)-epiafzelechin (2) and (?)-epicatechin (3), methyl 4-hydroxybenzoate and 4-hydroxybenzaldehyde, four pentacyclic oxindole alkaloids, isopteropodine, pteropodine, uncarine F and isopteropodic acid, previously found in the stems, and two coumarins, scopoletin and 3,4-dihydroxy-7-methoxycoumarin. Structures of the compounds were elucidated by 1D and 2D NMR, FTIR, UV, MS, and experimental as well as calculated electronic circular dichroism (ECD) data. Compounds 2 and 3 were evaluated for their neurotoxic and neuroprotective properties against differentiated SH-SY5Y neuroblastoma cell lines using the MTS assay. Compounds 2 and 3 did not show any neurotoxic effects but showed strong protective potential against hydrogen peroxide-induced neurotoxicity with maximum cell viability at a concentration of 1 μM.     

The Production of Phenylacetaldehyde from l-Phenylalanine in Tea Fermentation

Tea leaves macerated with l-phenylalanine generated rose like aroma. The gas chromatogram of the essential oil obtained from these leaves showed extremely large peak of phenylacetaldehyde. The evidence for degradation of l-phenylalanine to phenylacetaldehyde and carbon dioxide was given by the radioactive tracer experiment using l-phenylalanine-U-¹⁴C. The phenylacetaldehyde was presumed to be an intermediate product in tea fermentation from the data on the changes of the compound in tea fermentation process.     

Cytokinin activity of N-phenyl-N′-(4-pyridyl)urea derivatives

We have synthesized 35 N-phenyl-N′-(4-pyridyl)urea derivatives and tested their cytokinin activity in the tobacco callus bioassay. Among them, N-phenyl-N′- (2-chloro-4-pyridyl)urea is highly active, the optimum concentration of which is lower than 4 × 10^?9 M (0.001 ppm), 3 compounds, i.e. N-(2-methylphenyl)-N′-(2-chloro-4-pyridyl)urea, N-(3-methylphenyl)-N′-(2-chloro-4-pyridyl)urea and N-(3-chlorophenyl)-N′-(2-chloro-4-pyridyl) urea are as active as N⁶-benzyladenine (concentration for optimum yield: 4.4 × 10^?8 M or 0.01 ppm), and N-phenyl-N′-(2-methyl-4-pyridyl)urea and N-(2-chlorophenyl)-N′-(2-chloro-4-pyridyl)urea are as active as N-phenyl-N′-(4-pyridyl)urea (concentration for optimum yield: 4.7 × 10^?7 M or 0.1 ppm), while the activity of the other 29 compounds are not so remarkable and 11 of them are almost or completely inactive.     

Kinetics of phosphate uptake in excised maize root

The short term uptake of phosphate involving 10 min absorption followed by 5 min desorption, both at 30 °C, in the concentration range 1.0×10^?9 to 7.5×10^?2 M KH₂PO₄ by fresh and washed maize (Zea mays L. cv. Ganga Safed-2) roots can be described by a single isotherm having five phases (0 and I–IV) with regularly spaced kinetic constants. Almost identical kinetics were observed in both fresh and washed maize roots. The kinetics of phase 0 in the concentration range 1.0×10^?9–3.0×10^?5 M. was sigmoidal in fresh maize roots, however, in washed tissue exhibited 2 phases termed here as 0a and 0b. 0a covered the concentration range 1.0×10^?9–5.0×10^?6 M and 0b 6.0×10^?6–3.0×10^?5 M. In the concentration range 1.0×10^?4–7.5×10^?2 M four distinct phases, termed as I, II, III and IV were evident in both fresh and washed maize roots. Each phase obeyed Michaelis—Menten kinetics. The values of K_m and V_max have been estimated for each phase. The uptake isotherm was accompanied by discontinuous transitions.     

Nitrate availability and calcite production in Emiliania huxleyi Lohmann

High-calcifying cells of Emiliania huxleyi were grown on a synthetic seawater medium and the effect of nitrate (NO^- ₃) concentration on growth, calcite accumulation, calcification rate and DIC (dissolved inorganic carbon) utilisation determined. The stoichiometry between NO^- ₃ utilisation and calcite production was 1:6·5 (mol/mol). Calcification and growth were tightly coupled: calcite production ceased when cultures entered the stationary phase due to NO^- ₃ depletion, but by adding a pulse of NO^- ₃ growth and calcification were restored. The initial C/N ratio in the medium was important in relation to calcification rate. At 20 µM NO^- ₃ the total DIC (2 mM) was rapidly depleted, the calcification rate subsequently declining, whereas at 5 and 10 µM NO^- ₃ rates of calcification were constant at 20 g carbon cell^-1 × 10¹⁴·h^-1 throughout culture growth, excess DIC being present relative to the available NO^- ₃. Calcite production per unit NO^- ₃ was similar for isolates of E. huxleyi from neritic, oligotrophic and nitrate-rich waters. In laboratory cultures, where the photon flux density is optimised for growth, the initial NO^- ₃ concentration is a reliable indicator of final calcite yield.