Potentiation of GABAA receptors expressed in Xenopus oocytes by perfume and phytoncid.

To study the effects of perfume and phytoncid on GABAA receptors, ionotropic GABAA receptors were expressed in Xenopus oocytes by injecting mRNAs that had been prepared from rat whole brain. Essential oil, perfume and such phytoncid as leaf alcohol, hinokitiol, pinene, eugenol, citronellol and citronellal potentiated the response in the presence of GABA at low concentrations (10 and 30 microM), possibly because they bound to the potentiation-site in GABAA receptors and increased the affinity of GABA to the receptors. Since it is known that the potentiation of GABAA receptors by benzodiazepine, barbiturate, steroids and anesthetics induces the anxiolytic, anticonvulsant and sedative activity or anesthetic effect, these results suggest the possibility that the intake of perfume or phytoncid through the lungs, the skin or the intestines modulates the neural transmission in the brain through ionotropic GABAA receptors and changes the frame of the human mind, as alcohol or tobacco does.     

外用不同浓度茉莉酸甲酯诱导的茶树挥发物的种类和时序变化

【目的】明确MeJA对茶树挥发物的诱导作用。【方法】采用顶空活体取样法对不同浓度MeJA处理后的茶苗挥发物进行抽提,并利用GC-MS对挥发物进行鉴定。【结果】不同剂量MeJA显著地影响茶树挥发物的种类组成和释放量,50μL MeJA处理可显著诱导茶树释放香叶烯、萜品油烯、罗勒烯等10种单萜类化合物,法呢烯、橙花叔醇和红没药烯等7种倍半萜类化合物,苯甲醇、苯乙腈和吲哚等5种氨基酸衍生物,以及3种未知化合物;而100μL MeJA处理仅能诱导茶树释放7种化合物。不同挥发物对MeJA处理的响应时间不同,但其释放量都具有昼高夜低的趋势。并且,释放量的大小明显受到光照强度的影响。【结论】外用MeJA喷雾处理可诱导茶树挥发物的产生和释放。     

武夷岩茶炭焙工艺对肉桂乌龙茶挥发性组分的影响

以武夷岩茶当家茶树品种肉桂(Camellia sinensis ‘Rougui’)鲜叶制成的乌龙茶为试材,基于顶空固相微萃取法(HS-SPME)结合气相色谱-质谱联用技术(GC-MS),探讨武夷岩茶炭焙工艺对肉桂乌龙茶挥发性组分的影响。实验中共检测到样本挥发性成分443个,其中包括96个杂环化合物、81个酯类化合物、31个萜类化合物、42个芳香烃类化合物、55个酮类化合物、24个其他烃类化合物、35个醇类化合物、24个醛类化合物、15个酚类化合物、14个胺类化合物、10个酸类化合物、5个含氮化合物、3个含硫化合物等。经主成分分析及聚类分析显示,焙火工艺是影响乌龙茶挥发性成分含量的重要影响因子。随炭焙程度增加,不同类别物质中的多数挥发性成分含量随之显著升高,且以美拉德反应产物等具有茶叶烘炒香的吡嗪类、糠醛类衍生物、吡咯类等化合物最具代表性;同时,部分含量丰富且具有乌龙茶特殊花果香气的醇类、萜烯类物质如香叶醇、反式-橙花叔醇、植物醇、α-法尼烯等,及具清新花香的吲哚含量显著降低。然而,大多数挥发性差异代谢物随炭焙程度增加相对含量显著升高,并不代表茶叶中芳香物质总量增加。研究表明,乌龙茶精制烘焙过程中,香气物质的积累主要来自热作用下的美拉德反应及非酶促的降解和氧化,如黄酮苷类物质水解。     

Green, oolong and black tea extracts modulate lipid metabolism in hyperlipidemia rats fed high-sucrose diet

The main goal of this study was to compare effects of ethanol-soluble fractions prepared from various types of teas on sucrose-induced hyperlipidemia in 5-week old male Sprague-Dawley rats. Rats (n = 6-8 per group) weighed approximately 200 g were randomly divided into control diet, sucrose-rich diet, green tea, oolong tea and black tea groups. Control-diet group was provided with modified AIN-93 diet while the others consumed sucrose-rich diet. Tea extracts (1% w/v) were supplied in the drink for green tea, oolong tea and black tea groups. Results indicated sucrose-rich diet induced hypertriglyceridemia and hypercholesterolemia. Food intake was reduced by oolong tea extract. Consuming oolong and black tea extracts also significantly decreased body weight gains and food efficiency. Hypertriglyceridemia was normalized by green and black tea drink on day 18 and by oolong tea extract on day 25, respectively. Hypercholesterolemia was normalized by green tea on day 18 and by oolong tea and black tea on day 25, respectively. Plasma HDL-cholesterol concentrations were not affected by any tea extract. The triglyeride content in the liver as well as the cholesterol content in the heart of rats fed sucrose-rich diet were elevated and were normalized by all types of tea drink tested. Although green and oolong tea extracts contained similar composition of catechin, our findings suggest green tea exerted greater antihyperlipidemic effect than oolong tea. Apparent fat absorption may be one of the mechanisms by which green tea reduced hyperlipidemia as well as fat storage in the liver and heart of rats consumed sucrose-rich diet.     

Effects of oolong tea on metabolism of plasma fat in mice under restraint stress

We investigated the effects of oolong tea on the basic metabolism of plasma lipids in mice under restraint stress. When a lipid emulsion (Intralipid 20%; a lipid emulsion containing 20% soybean oil) was injected intravenously into mice, the restraint stress prolonged the half-life (T 1/2) of elimination for plasma triglyceride (TG) from 28.7 to 55.5 min. The elimination rate per minute was 48.2% in stressed mice with the rate in starved control mice as 100%. Therefore, TG metabolism was disrupted by the stress, and the use of TG as an energy source decreased. We found that the metabolism of lipids significantly response to the restrained stress in the present study. Plasma TG was 515.9 +/- 29.9mg/dl 35min after Intralipid administration in control stressed mice, 478.7 +/- 26.7 mg/dl in the stressed group given caffeine 100 mg/kg of body weight, and 418.3 +/- 18.4 mg/dl in the stressed group given 1,000 mg/kg oolong tea, an improvement by 7.2% and 18.9%, respectively, with the value for the untreated control group. The intake of oolong tea alleviated the stress-induced decrease in the rate of blood lipid metabolism; this effect may have arisen from some non-specific stress-relieving property of the tea or from acceleration of lipid metabolism by properties of polyphenols, etc. in tea. Oolong tea had anti-stress effects on plasma TG metabolism, and the effects did not depend on caffeine.     

Melanogenesis inhibition by an oolong tea extract in b16 mouse melanoma cells and UV-induced skin pigmentation in brownish guinea pigs

To investigate the new physiological functions of oolong tea, the effects on melanogenesis were studied. An oolong tea extract inhibited melanogenesis without affecting cell growth in B16 mouse melanoma cells. However, the oolong tea extract hardly showed any inhibitory effect on mushroom tyrosinase in a cell-free system. The effects of an oolong tea extract on the intracellular tyrosinase level in B16 cells were therefore studied. All the levels of activity, protein and mRNA were decreased in the oolong tea extract-treated cells. We also investigated the inhibitory effects of oolong tea on the pigmentation induced by ultraviolet B (UVB) by using brownish guinea pigs in vivo. The number of 3,4-dihydroxyphenylalanine (DOPA)-positive melanocytes increased by UVB was repressed by an oral administration of oolong tea. These results imply that oolong tea might be effective in whitening and that its inhibitory effect on melanogenesis was involved in the decrease of intracellular tyrosinase at the mRNA level.     

γ-Aminobutyric AcidA Receptor α5-Subunit Creates Novel Type II Benzodiazepine Receptor Pharmacology

A cDNA encoding a protein with 70% amino acid identity to the previously characterized gamma-aminobutyric acidA (GABAA) receptor alpha-subunits was isolated from a rat brain cDNA library by homology screening. As observed for alpha 1-, alpha 2-, and alpha 3-subunits, coexpression of this new alpha-subunit (alpha 5) with a beta- and gamma 2-subunit in cultured cells produces receptors displaying high-affinity binding sites for both muscimol, a GABA agonist, and benzodiazepines. Characteristic of GABAA/benzodiazepine type II sites, receptors containing alpha 2-, alpha 3- or alpha 5-subunits have low affinities for several type I-selective compounds. However, alpha 5-subunit-containing receptors have lower affinities for zolpidem (30-fold) and Cl 218 872 (three-fold) than measured previously using recombinantly expressed type II receptors containing either alpha 2- or alpha 3-subunits. Based on these findings, a reclassification of the GABAA/benzodiazepine receptors is warranted.     

Suppression of postprandial hypertriglyceridemia in rats and mice by oolong tea polymerized polyphenols

Oolong tea-polymerized polyphenols (OTPP) are characterized polyphenols produced from semi-fermented tea (oolong tea). In the present study, we evaluated the suppressive effects of oolong tea extract and OTPP on postprandial hypertriglyceridemia in rats and mice. Lymphatic recovery of triglycerides in rats cannulated in the thoracic duct was delayed by the administration of oolong tea extract at 100 and 200 mg per head, and more effectively than with green tea extract. OTPP delayed lymphatic triglyceride absorption at 20 mg/head, though (-)-epigallocatechin gallate (EGCG) did not do so at the same dose. OTPP also suppressed postprandial hypertriglyceridemia after administration of olive oil in mice. The area under the curve (AUC) of plasma triglycerides was significantly decreased, by 53% and 76%, in the 500 and 1,000 mg/kg OTPP groups respectively, as compared with the control group. These results suggest that OTPP is responsible for the suppression of hypertriglyceridemia by ingestion of oolong tea.     

Flavor Constituents of Pouchong Tea and a Comparison of the Aroma Pattern with Jasmine Tea

The aroma constituents of the highest quality pouchong tea were characterized by GC–MS. Forty-eight components, including five newly identified compounds were characterized. GC peak area percentages of the main components in pouchong tea were compared with those of jasmine tea to differentiate compounds contributing to the aroma characteristics of pouchong tea with superior floral elegant flavor. Nerolidol, jasmine lactone, methyl jasmonate, indole, benzyl cyanide and linalool oxides were found in much higher concentration in pouchong tea than in jasmine tea. These compounds seemed to contribute to the aroma characteristics of the highest quality pouchong tea.     

Inhibitory effect of methyl jasmonate and its related compounds on kinetin-induced retardation of oat leaf senescence

The senescence-promoting activities of methyl jasmonate and its related compounds were compared with respect to structure-activity relationships. The activities were assayed by using oat ( A vena saliva L. cv. Victor) leaf segments in the presence of 2 μg/ml kinetin. Dextrorotatory methyl jasmonate prepared from an authentic sample of the racemate mixture was less active than the naturally occurring levorotatory form especially at its low concentrations (0.1 to 2.5 μg/ml). The activity of jasmonic acid, the free acid form of methyl jasmonate, was much less than the methyl ester, and this relationship was true for the other compounds tested. The reduction of the unsaturated bond in the substituent at the C-2 position and the keto group at the C-3 position greatly reduced the activity. The length of the n -alkyl substituents at the C-2 position had also a significant effect on the activity. From these results, it is concluded that the important functional groups for the high senescence-promoting activity of the methyl jasmonate related compounds are the methyl acetate substituent at the C-l position, the 2' cis -pentenyl or n -pentyl group at the C-2, position and the keto group at the C-3 position in methyl jasmonate.     

GABAB Receptor-Mediated Enhancement of Vasoactive Intestinal Peptide-Stimulated Cyclic AMP Production in Slices of Rat Cerebral Cortex

Basal and vasoactive intestinal peptide (VIP)-stimulated accumulations of cyclic AMP were measured in slices of rat cerebral cortex. Neither gamma-aminobutyric acid (GABA) nor the selective GABAB receptor agonist (-)-baclofen stimulated basal cyclic AMP accumulation, whereas VIP caused a large dose-dependent increase in cyclic AMP levels. However, in the presence of 100 microM (-)-baclofen, the effects of VIP on cyclic AMP accumulation were significantly enhanced, with the responses to 1 microM and 10 microM VIP being approximately doubled. The enhancing effects of (-)-baclofen was dose related (1-1,000 microM), but an enhancing effect was not observed with 100 microM (+)-baclofen. In the presence of the GABA uptake inhibitor nipecotic acid (1 mM), GABA caused a similar dose-related enhancement of the VIP response. The ability of either GABA or (-)-baclofen to augment VIP-stimulated production of cyclic AMP was not mimicked by the GABAA, agonists isoguvacine and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) and was not antagonized by the GABAA antagonist bicuculline. The putative GABAB antagonist 5-aminovaleric acid (1 mM) significantly reduced the effect of (-)-baclofen. The ability of (-)-baclofen to enhance VIP-stimulated accumulation of cyclic AMP was observed in slices of rat cerebral cortex, hippocampus, and hypothalamus. These results indicate that GABA and (-)-baclofen can enhance VIP-stimulated accumulation of cyclic AMP in rat brain slices via an interaction with specific GABAB receptors.     

Yeast extract and methyl jasmonate-induced silymarin production in cell cultures of Silybum marianum (L.) Gaertn

The biosynthesis of the flavonolignan silymarin, a constitutive compound of the fruits of Silybum marianum with strong antihepatotoxic and hepatoprotective activities, is severely reduced in cell cultures of this species. It is well known that elicitation is one of the strategies employed to increase accumulation of secondary metabolites. Our study here reports on the effect of several compounds on the production of silymarin in S. marianum cultures. Yeast extract (YE), chitin and chitosan were compared with respect to their effects on silymarin accumulation in S. marianum suspensions and only yeast extract stimulated production. Jasmonic acid (JA) potentiated the yeast extract effect. One of the jasmonic acid derivatives, methyl jasmonate (MeJA), strongly promoted the accumulation of silymarin. Methyl jasmonate acted in a number of steps of the metabolic pathway of flavonolignans and its stimulating effect was totally dependent of "de novo" protein synthesis. Chalcone synthase (CHS) activity was enhanced by methyl jasmonate; however there did not appear to be a temporal relationship between silymarin accumulation and increase in enzyme activity. Also, this increase was not blocked by the protein synthesis inhibitor cycloheximide (CH). This study indicates that elicitor treatment promotes secondary metabolite production in S. marianum cultures and that jasmonic acid and its functional analogue plays a critical role in elicitation.     

GABAA and GABAB receptors modulate the K(+)-evoked release of sensory CGRP from the guinea pig urinary bladder

Superfusion of mucosa-free muscle slices of guinea-pig urinary bladder with 40 mM K+ produced a remarkable increase in calcitonin gene-related peptide-like immunoreactivity (CGRP-LI), that in this organ is entirely contained in capsaicin-sensitive nerves. GABA (1 mM) did neither affect the basal nor the 40 mM K+ evoked CGRP-LI release. Baclofen (0.1 mM) or muscimol (1 mM) did not affect the basal CGRP-LI outflow. However, baclofen (0.1 mM) significantly reduced by 32% and muscimol (0.1-1 mM) significantly increased by 60% and 70%, respectively the K(+)-evoked CGRP-LI release. These findings add neurochemical evidence to the functional data suggesting the existence of GABAA and GABAB receptors which modulate the efferent function of capsaicin-sensitive afferents.     

The Effects of Caffeine and Caffeine-containing Beverages on the Disposition of Brain Serotonin in Rats

Caffeine and caffeine-containing beverages (instant coffee, black tea, green tea, or oolong tea) caused a significant decrease in serum tryptophan, and significant increases in brain tryptophan, serotonin, and 5-hydroxyindole acetic acid over those in rats fed a control diet. Adenosine supplementation partially counteracted the increase of brain serotonin caused by caffeine. These results are interpreted as indicating that caffeine-containing beverages may have some nutritional and behavioral effects.     

Rapid identification of acylated flavonol tetraglycosides in oolong teas using HPLC-MSn

A method was developed to separate and identify acylated flavonol tetraglycosides (AFTGs) by combining isocratic HPLC with electrospray ionisation tandem mass spectrometry. Better separation was obtained for oolong tea infusion using a manually packed Sephadex LH-20 mini-column than with an ACCUBOND ODS solid-phase column. Seven unknown and one known AFTGs were found in oolong teas prepared by various semi-fermentation processes and their structures were identified by mass spectrometry. According to the analyses of diverse oolong teas including Dongding Oolong, Tieguanyin, Wuyi Oolong, Fenghuang Oolong, Gaoshan Shibi, Laocong Shuixian and Baihao Oolong, AFTGs seemed to be universally present, and each oolong tea could be classified into one of three groups (Dongding Oolong, Tieguanyin and Wuyi Oolong) on the basis of its AFTGs profile. The results suggest that the developed method is rapid and sensitive for identifying natural compounds.     

Effect of chloride and bicarbonate ions on Mg2+ -ATPase of plasma membranes of bream (Abramis brama L.) brain sensitive to GABAA-ergic compounds

Effect of Cl and HCO3- ions on the Mg2+ -ATPase activity of the plasma membrane of bream brain was investigated. Cl- (5 or 10 mM) and HCO3- (1-5 mM) individually have low effect on the "basal" Mg2+ -ATPase. Simultaneous presence of Cl- and HCO3- in the incubation medium significantly increased the enzyme activity. Maximum effect of anions on the enzyme is observed in the presence of Cl- (approximately 7 mM) and HCO3- (approximately 3 mM). Br- can replace Cl- under joint effect with HCO3-, while I- has half maximum activity compared with Cl-. Bicuculline (7 microM) inhibits completely the joint effect of Cl- and HCO3- on the enzyme, while it has no effect on the "basal" Mg2+ -ATPase activity. SH-reagents (5, 5-dithiobis-2-nitrobenzoic acid, N-ethylmaleimide), oligomycine and orthovanadate inhibited the Cl-, HCO3- -activated Mg2+ -ATPase. The obtained results demonstrated that Mg2+ -ATPase of the bream brain sensitive to GABAergic ligands at a fixed concentrations of Cl- and HCO3- ions in the incubation medium is Cl-, HCO3- -activated by Mg2+ -ATPase, whose activity meets a number of requirements to the system which may be involved by GABAA receptors in the Cl-/HCO3- -exchange processes.     

Enzymic Studies of the Distribution Pattern of 4-O-Methylglucuronic Acid Residues in Glucuronoxylans from Sunflower Hulls

Two epimers of methyl jasmonate were optically resolved by capillary gas chromatography, using heptakis (2,3,6-tri-O-methyl)-β-cyclodextrin as the chiral stationary phase. In the tea volatile concentrates, both of these epimers were present as only one enantiomer, their absolute configurations being ascertained as (–)-(1R,2R)-methyl jasmonate and (+)-(1R,2S)-methyl epijasmonate.

The thermal isomerization of methyl epijamonate to methyl jasmonate was also clarified by optically resolved gas chromatography to have occurred at the asymmetric carbon of the C-2 position that is connected to the carbonyl group.     

Methyl Jasmonate and Lactones Including Jasmine Lactone in Ceylon Tea

The ester and lactone fraction possessing the most attractive aroma was separated from the aroma concentrate of Ceylon flavory tea by silica-gel column chromatography and analyzed by GC-MS.

Methyl 2-(cis-2′-pentenyl)-cyclopentanone-3-acetate(methyl jasmonate), 5-(cis-2′-pentenyl)-5-pentanolide (jasmine lactone), 2,3-dimethyl-2-nonen-4-olide, 4-octanolide, 4-nonanolide and 5-decanolide were newly identified as the constituents of tes aroma. Former two compounds seemed to carry a major share of aroma character of Ceylon flavory tea.     

Effect of delta-opioid receptor over-expression on cortical expression of GABAA receptor alpha1-subunit in hypoxia

Recent studies show that both delta-opioid receptors (DOR) and GABA receptors play a neuroprotective role in the mature cortex. Since we have observed that DOR over-expression renders the cortex more tolerant to hypoxic stress, we asked whether DOR over-expression affects GABA receptors expression in the cortex under hypoxia. As the first step, we investigated the expression of GABAA receptor alpha1-subunit (GABAA Ralpha1, the most abundant alpha-subunit of GABA receptors in the adult brain) in the mouse cortex with transgenic DOR over-expression after hypoxia. The results showed that GABAA Ralpha1 expression was lower in the transgenic than wild-type cortex, suggesting that DOR overexpression induces an inhibitory effect on GABAA receptor expression. Hypoxia for 1-3 days significantly increased GABAA Ralpha1 expression in the wild-type cortex, which may be an adaptive strategy for protecting the cortex against hypoxic stress. Interestingly, such increase was not found in the transgenic cortex with DOR over-expression. This may represent an interactive regulation in the transgenic cortex to efficiently balance energy production and consumption for better adaptation to hypoxic environment. Since DOR over-expression increases cortical tolerance to hypoxia, an increase in GABA receptors expression (an energy-costing process) may not be necessary in the cortex with DOR over-expression.