Formation mechanism of p-methylacetophenone from citral via a tert-alkoxy radical intermediate

The aim of this study was to clarify the formation mechanism of a potent off-odorant, p-methylacetophenone, from citral under acidic aqueous conditions. An acidic aqueous solution (pH 3.0) containing 10 mg/L of citral was stored at 40 degrees C for 2 weeks. Among the compounds detected in the stored citral solution, 4-(2-hydroxy-2-propyl)benzaldehyde was identified for the first time as a degradation product from citral. The formation of p-methylacetophenone and 4-(2-hydroxy-2-propyl)benzaldehyde behaved the same when antioxidants were added to the citral solution. In addition, both compounds were formed by the Fe(2+)-induced decomposition of 8-hydroperoxy-p-cymene, another compound identified in the stored citral solution. These results suggested that both p-methylacetophenone and 4-(2-hydroxy-2-propyl)benzaldehyde can be formed via the same radical intermediate [p-CH3C6H4C(CH3)2O*] that can be derived from the O-O bond homolysis of 8-hydroperoxy-p-cymene. On the other hand, the degradation of 8-hydroperoxy-p-cymene without Fe2+ under acidic aqueous conditions did not yield p-methylacetophenone and 4-(2-hydroxy-2-propyl)benzaldehyde, but the degradation of citral without Fe2+ did. Therefore, other than the decomposition of 8-hydroperoxy-p-cymene, a mechanism to generate the tert-alkoxy radical intermediate was proposed for the formation of p-methylacetophenone and 4-(2-hydroxy-2-propyl)benzaldehyde in the citral solution.     

Inhibition of citral degradation by oil-in-water nanoemulsions combined with antioxidants

The aim of the present study was to investigate the effects of oil-in-water (O/W) nanoemulsions combined with six different natural antioxidants on the stability of citral. Acidic emulsions (lecithin-stabilized palm kernel lipid in pH 3 buffer) containing 1000 ppm citral and 1000 ppm antioxidants (black tea extract, ascorbic acid, naringenin, tangeretin, β-carotene, and tanshinone) were stored at 25 and 50 °C, respectively. The emulsions with and without antioxidants were analyzed by solid phase microextraction gas chromatography (SPME-GC) to monitor the degradation process of citral and the formation of different off-flavor compounds, such as α,p-dimethylstyrene and p-methylacetophenone. The results suggested that encapsulation of citral in emulsions and the addition of the appropriate antioxidants (β-carotene, tanshinone, and black tea extract) could greatly enhance citral's chemical stability during storage.     

Determination of theaflavins including methylated theaflavins in black tea leaves by solid-phase extraction and HPLC analysis

A quantitative method for four theaflavins and two methylated theaflavin derivatives in black tea leaves was developed by solid-phase extraction and a high-performance liquid chromatographic method with photodiode array detection. The theaflavins in black tea leaves were extracted three times with 40 vol 50% aqueous ethanol (mg dry tea powder/mL) containing 2% ascorbic acid. The ethanol extracts were diluted 4-fold with distilled water. All diluted extracts were directly applied to the solid-phase C18 cartridge column without concentration. The fraction of theaflavins was obtained by 40% ethanol extraction after rinsing with water followed with 15% ethanol extraction. An aliquot of theaflavins after concentration was injected onto an ODS C18 reversed-phase column, and four theaflavins and two methylated theaflavins were sufficiently separated by a linear gradient system using distilled water and acetonitrile with 0.5% acetic acid. This analytical method is sensitive for the determination of a small amount of methylated theaflavins, since various interfering substances produced during the fermentation process were eliminated in advance by solid-phase extraction. Using this analytical method, we also demonstrated that methylated theaflavins were easily produced during the manufacture of black tea.     

Role of polyphenol oxidase and peroxidase in the generation of black tea theaflavins.

It has been reported earlier that when macerated tea leaf is fermented at lower pH, the resultant black tea contains increased levels of theaflavin, an important quality marker in black tea. In an attempt to investigate the biochemistry and chemistry underlying this observation, in vitro oxidation experiments using polyphenol oxidase (PPO) from fresh tea leaves, horseradish peroxidase (POD), and tea catechins, precursors for theaflavins, were carried out. In vitro oxidation experiments using crude tea PPO resulted in higher content of theaflavins at pH 4.5 in comparison with pH 5.5, the normal pH of the macerated tea leaf. When purified PPO was used in the in vitro system, surprisingly a reversal of this trend was observed, with more theaflavins being formed at the higher pH. A combination of pure tea PPO and POD led to an observation similar to that with the crude enzyme preparation, suggesting a possible role for POD in the formation or degradation of theaflavin. POD was observed to oxidize theaflavins in the presence of H(2)O(2), leading to the formation of thearubigin, another black tea pigment. This paper demonstrates that tea PPO, while oxidizing catechins, generates H(2)O(2). The amount of H(2)O(2) produced is greater at pH 5.5, the optimum pH for PPO activity, than at pH 4.5. Hence, an observed increase of theaflavins in black teas fermented at pH 4.5 appears to be due to lower turnover of formed theaflavins into thearubigins.     

Molecular binding of black tea theaflavins to biological membranes: relationship to bioactivities

Molecular dynamics simulations were used to study the interactions of three theaflavin compounds with lipid bilayers. Experimental studies have linked theaflavins to beneficial health effects, some of which are related to interactions with the cell membrane. The molecular interaction of theaflavins with membranes was explored by simulating the interactions of three theaflavin molecules (theaflavin, theaflavin-3-gallate, and theaflavin-3,3'-digallate) with a mixed bilayer composed of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE). The simulations show that the theaflavins evaluated have an affinity for the lipid bilayer surface via hydrogen bonding. The molecular structure of theaflavins influenced their configuration when binding to the bilayer surface, as well as their ability to form hydrogen bonds with the lipid headgroups. The theaflavin-bilayer interactions studied here help to define structure-function relationships of the theaflavins and provide a better understanding of the role of theaflavins in biological processes. The significance of the results are discussed in the context of black tea composition and bioactivity.     

alpha-Glucosidase inhibitory profile of catechins and theaflavins

To clarify the postprandial glucose suppression effect of flavonoids, the inhibitory effects of catechins and theaflavins against alpha-glucosidase (AGH) were examined in this study. It was initially demonstrated that theaflavins and catechins preferentially inhibited maltase rather than sucrase in an immobilized AGH inhibitory assay system. For the maltase inhibitory effects of theaflavins, the effects were observed in descending order of potency of theaflavin (TF)-3-O-gallate (Gal) > TF-3,3'-di-O-Gal > TF-3'-O-Gal > TF. This suggests that the AGH inhibition induced by theaflavins is closely associated with the presence of a free hydroxyl group at the 3'-position of TF as well as the esterification of TF with a mono-Gal group. In addition, the R-configuration at the 3'-position of TF-3-O-Gal showed a higher inhibitory activity than the S-configuration. As a result of a single oral administration of maltose (2 g/kg) in rats, a significant reduction in blood glucose level was observed at a dose of 10 mg/kg of TF-3-O-Gal, demonstrating for the first time that TF-3-O-Gal can suppress glucose production from maltose through inhibition of AGH in the gut.     

Identification of the astringent taste compounds in black tea infusions by combining instrumental analysis and human bioresponse

Application of taste dilution analyses on freshly prepared black tea infusions revealed neither the high molecular weight thearubigen-like polyphenols nor the catechins and theaflavins, but a series of 14 flavon-3-ol glycosides as the main contributors to the astringent taste perceived upon black tea consumption. Among these glycosides, the apigenin-8-C-[alpha-l-rhamnopyranosyl-(1-->2)-O-beta-d-glucopyranoside] was identified for the first time in tea infusions. Depending on the structure, the flavon-3-ol glycosides were found to induce a velvety and mouth-coating sensation at very low threshold concentrations, which were far below those of catechins or theaflavins; for example, the threshold of 0.001 micromol/L found for quercetin-3-O-[alpha-l-rhamnopyranosyl-(1-->6)-O-beta-d-glucopyranoside] is 190000, or 16000 times below the threshold determined for epigallocatechin gallate or theaflavin, respectively. Moreover, structure/activity considerations revealed that, besides the type of flavon-3-ol aglycon, the type and the sequence of the individual monosaccharides in the glycosidic chain are key drivers for astringency perception of flavon-3-ol glycosides.     

Inhibitory effects of oolong tea polyphenols on pancreatic lipase in vitro

Fifty-four polyphenols isolated from tea leaves were evaluated for their inhibitory activities against pancreatic lipase, the key enzyme of lipid absorption in the gut. (-)-Epigallocatechin 3-O-gallate (EGCG), which is one of major polyphenols in green tea, showed lipase inhibition with an IC50 of 0.349 microM. Moreover, flavan-3-ol digallate esters, such as (-)-epigallocatechin-3,5-digallate, showed higher activities of inhibition on lipase with an IC50 of 0.098 microM. On the other hand, nonesterified flavan-3-ols, such as (+)-catechin, (-)-epicatechin, (+)-gallocatechin, and (-)-epigallocatechin, showed zero and/or the lowest activities against pancreatic lipase (IC50 > 20 microM). These data suggested that the presence of galloyl moieties within the structure was required for enhancement of pancreatic lipase inhibition. It is well-known that flavan-3-ols are polymerized by polyphenol oxidase and/or heating in a manufacturing process of oolong tea. Oolonghomobisflavans A and B and oolongtheanin 3'-O-gallate, which are typical in oolong tea leaves, showed strong inhibitory activities with IC50 values of 0.048, 0.108, and 0.068 microM, respectively, even higher than that of EGCG. The oolong tea polymerized polyphenols (OTPP) were prepared for the assay from oolong tea extract, from which the preparation effectively subtracted the zero and/or less-active monomeric flavan-3-ols by preparative high-performance liquid chromatography. The weight-average molecular weight (Mw) and number-average molecular-weight (Mn) values of OTPP were 2017 and 903, respectively, by using gel permeation choromatography. OTPP showed a 5-fold stronger inhibition against pancreatic lipase (IC50 = 0.28 microg/mL) by comparison with that of the tannase-treated OTPP (IC50 = 1.38 microg/mL). These data suggested that the presence of galloyl moieties within their chemical structures and/or the polymerization of flavan-3-ols were required for enhancement of pancreatic lipase inhibition.     

Induction of apoptosis by the oolong tea polyphenol theasinensin A through cytochrome c release and activation of caspase-9 and caspase-3 in human U937 cells

This study examined the growth inhibitory effects of theasinensin A (from oolong tea) and black tea polyphenols, including theaflavin (TF-1), a mixture (TF-2) of theaflavin-3-gallate (TF-2a) and theaflavin-3'-gallate (TF-2b), and theaflavin-3,3'-digallate (TF-3) in human cancer cells. Theasinensin A, TF-1, and TF-2 displayed strong growth inhibitory effects against human histolytic lymphoma U937, with estimated IC50 values of 12 microM, but were less effective against human acute T cell leukemia Jurkat, whereas TF-3 and (-)-epigallocatechin-3-gallate (EGCG) had lower activities. The molecular mechanisms of tea polyphenol-induced apoptosis as determined by annexin V apoptosis assay, DNA fragmentation, and caspase activation were further investigated. Loss of membrane potential and reactive oxygen species (ROS) generation were also detected by flow cytometry. Treatment with tea polyphenols caused rapid induction of caspase-3, but not caspase-1, activity and stimulated proteolytic cleavage of poly(ADP-ribose) polymerase (PARP). Pretreatment with a potent caspase-3 inhibitor, Z-Asp-Glu-Val-Asp-fluoromethyl ketone, inhibited theasinensin A induced DNA fragmentation. Furthermore, it was found that theasinensin A induced loss of mitochondrial transmembrane potential, elevation of ROS production, release of mitochondrial cytochrome c into the cytosol, and subsequent induction of caspase-9 activity. These results indicate that theasinensin A allows caspase-activated deoxyribonuclease to enter the nucleus and degrade chromosomal DNA and induces DFF-45 (DNA fragmentation factor) degradation. The results suggest that induction of apoptosis by theasinensin A may provide a pivotal mechanism for their cancer chemopreventive function.     

抗氧化剂对辐照猪肉理化和感官品质的影响

为减少猪肉辐照灭菌后产生的异味和脂肪氧化,研究了叔丁基对苯二酚、虾青素、维生素E、茶多酚4种抗氧化剂对辐照猪肉理化和感官品质的影响。采用2 g/L抗氧化剂浸泡处理,透氧包装,辐照剂量2.6 kGy,冷藏10 d。分析测定辐照猪肉感官品质、过氧化值、硫代巴比妥酸反应物、挥发性物质、抗氧化剂对羟自由基清除能力,筛选出适合猪肉辐照的高效抗氧化剂。结果表明：叔丁基对苯二酚在储藏期可以很好地减轻辐照异味并抑制脂肪氧化,效果优于虾青素、维生素E、茶多酚。叔丁基对苯二酚和维生素E可以有效地降低脂肪辐照后产生的挥发性物质含量。叔丁基对苯二酚（0.5 g/L）对羟自由基的清除率为52.5%,高于其他抗氧化剂,可抑制猪肉辐照过程中羟自由基参与的反应。     

Fermentation characteristics of some assamica clones and process optimization of black tea manufacturing

Changes in the specific activities of polyphenol oxidase (PPO), peroxidase (POD), and protease and in the relative amounts of flavan-3-ols for eight genetically derived cultivated teas at various stages of leaf maturity and in four succescive seasons were examined. A series of investigations were carried out to study the cross-reactivity of complex polyphenols and PPO-generated orange-yellow theaflavins, as well as of POD oxidized substrates, producing brown so-called thearubigins during fermented tea processing. From the estimation of five major catechins, PPO activities in young shoots, and theaflavin and thearubigin contents of crushed, torn, and curled (CTC) black teas, the superior variety and flavorful flush characteristics were refined. Notable protein hydrolysis by endogenous protease as measured from free amino acids and formation of tannin-protein complex (browning products) was obtained for cultivar character and product quality. Results showed that process optimization with respect to time, temperature, moisture, and pH maximizes PPO-catalyzed desirable theaflavin pigments, whereas POD-mediated chemical reaction produces dull color.     

Antioxidative activities of volatile extracts from green tea, oolong tea, and black tea

Antioxidative activities of volatile extracts from six teas (one green tea, one oolong tea, one roasted green tea, and three black teas) were investigated using an aldehyde/carboxylic acid assay and a conjugated diene assay. The samples were tested at levels of 20, 50, 100, and 200 micrograms/mL of dichloromethane. The results obtained from the two assays were consistent. All extracts except roasted green tea exhibited dose-dependent inhibitory activity in the aldehyde/carboxylic acid assay. A volatile extract from green tea exhibited the most potent activity in both assays among the six extracts. It inhibited hexanal oxidation by almost 100% over 40 days at the level of 200 micrograms/mL. The extract from oolong tea inhibited hexanal oxidation by 50% in 15 days. In the case of the extract from roasted green tea, the lowest antioxidative activity was obtained at the level of 200 micrograms/mL, suggesting that the extract from roasted green tea contained some pro-oxidants. The extracts from the three black teas showed slight anti- or proactivities in both assays. The major volatile constituents of green tea and roasted green tea extracts, which exhibited significant antioxidative activities, were analyzed using gas chromatography-mass spectrometry. The major volatile chemicals with possible antioxidative activity identified were alkyl compounds with double bond(s), such as 3,7-dimethyl-1,6-octadien-3-ol (8.04 mg/kg), in the extract from green tea and heterocyclic compounds, such as furfural (7.67 mg/kg), in the extract from roasted green tea. Benzyl alcohol, which was proved to be an antioxidant, was identified both in a green tea extract (4.67 mg/kg) and in a roasted tea extract (1.35 mg/kg).     

HPLC-MSn analysis of phenolic compounds and purine alkaloids in green and black tea

Tea is a complex mixture containing a range of compounds from simple phenolics to complex thearubigins, many of which have well-recognized antioxidant properties. This paper describes the application of high-performance liquid chromatography-mass spectrometry (HPLC-MS(n)) methods for the rapid and routine analysis of more than 30 phenolics in tea. Green and black tea infusions were injected directly onto a reversed phase HPLC column, and the phenolics eluted using two different mobile phase gradients, one optimized to resolve catechin derivatives and the other, flavonols and theaflavins. Compounds, identified on the basis of their retention time, absorbance spectrum, and MS fragmentation pattern, included (+)-catechin, (-)-epicatechin, theaflavin and their various gallate derivatives, quercetin and kaempferol mono-, di-, and triglycosides, quinic acid esters of gallic acid and hydroxycinnamates, and the purine alkaloids, caffeine and theobromine.     

Tyrosinase inhibitory activity of citrus essential oils

Thirteen kinds of citrus essential oils and their volatile flavor constituents were investigated for tyrosinase inhibitory activity. Eureka lemon, Lisbon lemon, Keraji, and Kiyookadaidai significantly inhibited the oxidation of L-dihydroxy phenylalanine (L-DOPA) by mushroom tyrosinase. Citral and myrcene among volatile flavor constituents of citrus essential oils exhibited tyrosinase inhibitory activities with Ki values of 0.318 and 2.38 mM, respectively. The inhibition kinetics analyzed by a Lineweaver-Burk plot indicated that citral is a noncompetitive inhibitor and myrcene is a competitive inhibitor. These results indicated that citral and myrcene are responsible for the tyrosinase inhibitory activity of citrus essential oils.     

Characterization of the key aroma compounds in the beverage prepared from Darjeeling black tea: quantitative differences between tea leaves and infusion

By application of the aroma extract dilution analysis on the volatile fraction isolated from a black tea infusion (Darjeeling Gold Selection), vanillin (vanilla-like), 4-hydroxy-2,5-dimethyl-3(2H)-furanone (caramel), 2-phenylethanol (flowery), and (E,E,Z)-2,4,6-nonatrienal (oat-flake-like) were identified with the highest flavor dilution (FD) factors among the 24 odor-active compounds detected in the FD factor range of 4-128. Quantitative measurements performed by means of stable isotope dilution assays and a calculation of odor activity values (OAVs; ratio of concentration to odor threshold in water) revealed, in particular, the previously unknown tea constituent (E,E,Z)-2,4,6-nonatrienal as a key odorant in the infusion and confirmed the important role of linalool and geraniol for the tea aroma. An aroma recombinate performed by the 18 odorants for which OAVs > 1 were determined in their "natural" concentrations matched the overall aroma of the tea beverage. In the black tea leaves, a total of 42 odorants were identified, most of which were identical with those in the beverage prepared thereof. However, quantitative measurements indicated that, in particular, geraniol, but also eight further odorants were significantly increased in the infusion as compared to their concentration in the leaves.     

Effects of tea polyphenols on the invasion and matrix metalloproteinases activities of human fibrosarcoma HT1080 cells

The effects of tea polyphenols on the invasion of highly metastatic human fibrosarcoma HT1080 cells through a monolayer of human umbilical vein endothelial cells (HUVECs) and the accompanying basal membrane were investigated. Among the tea polyphenols tested, epicatechin gallate (ECg), epigallocatechin gallate (EGCg), and theaflavin strongly suppressed the invasion of HT1080 cells into the monolayer of HUVECs/gelatin membrane, whereas epicatechin, epigallocatechin, tea flavonols, tea flavones, and gallate derivatives had no effect. Both theaflavin-digallate and theasinensin D showed a weak invasion inhibitory effect. ECg significantly inhibited the invasion without cytotoxicity against cancer cells and HUVECs. Ester-type catechins (ECg and EGCg) and theaflavin strongly suppressed the gelatin degradation mediated by matrix metalloproteinase (MMP) 2 and MMP-9, which were secreted into the conditioned medium of HT1080 cells. In conclusion, among the tea polyphenols tested, ECg was considered to be the agent with the most potential antimetastasis activity because it inhibited invasion in the absence of cytotoxicity.     

Pigments in green tea leaves (Camellia sinensis) suppress transformation of the aryl hydrocarbon receptor induced by dioxin

Environmental contaminants such as dioxins enter the body mainly through diet and cause various toxicities through transformation of the aryl hydrocarbon receptor (AhR). We previously reported that certain natural flavonoids at the dietary level suppress the AhR transformation induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In this study, we identified lutein and chlorophyll a and b from green tea leaves as the novel antagonists for AhR. These active compounds suppressed AhR transformation dose-dependently with the 50% inhibitory concentration (IC(50)) values against 0.1 nM TCDD-induced AhR transformation at 3.2, 5.0, and 5.9 microM, respectively. (-)-Epigallocatechin gallate, which is the most abundant flavonoid in green tea leaves, also showed stronger suppressive effects than did other major tea components, with the IC(50) value of 1.7 microM. Thus, these pigments of green tea leaves have the potential to protect from dioxin toxicity through the suppression of AhR transformation.     

Two types of oxidative dimerization of the black tea polyphenol theaflavin.

Theaflavin and its galloyl esters are polyphenolic pigments of black tea. In the course of studies on the oxidation mechanism of tea polyphenols, two theaflavin oxidation products named bistheaflavins A and B were isolated, and their structures were elucidated on the basis of MS and NMR spectroscopic analyses. Treatment of a mixture of (-)-epicatechin and (-)-epigallocatechin with banana fruit homogenate yielded bistheaflavin A together with theaflavin and theanaphthoquinone. The symmetrical structure of bistheaflavin A suggested that this compound was formed by oxidative C [bond] C coupling of two theaflavin molecules. In contrast, theaflavin in phosphate buffer (pH 7.3) was gradually oxidized to give bistheaflavin B and theanaphthoquinone. Bistheaflavin B possesses a bicyclooctane skeleton probably formed by intermolecular cyclization between dehydrotheaflavin and dihydrotheanaphthoquinone.     

Angiotensin I converting enzyme inhibitory peptides from in vitro pepsin-pancreatin digestion of soy protein

Angiotensin I converting enzyme (ACE) inhibitory activity was determined in the soy protein isolate (SPI) digest produced by in vitro pepsin-pancreatin sequential digestion. The inhibitory activity was highest within the first 20 min of pepsin digestion and decreased upon subsequent digestion with pancreatin. An IC(50) value of 0.28 +/- 0.04 mg/mL was determined after 180 min of digestion, while no ACE inhibitory activity was measured for the undigested SPI at 0.73 mg/mL. Chromatographic fractionation of the SPI digest resulted in IC(50) values of active fractions ranging from 0.13 +/- 0.03 to 0.93 +/- 0.08 mg/mL. Although many of the fractions showed ACE inhibition, peptides with lower molecular masses and higher hydrophobicities were most active. The findings show that many different peptides with ACE inhibitory activities were produced after in vitro pepsin-pancreatin digestion of SPI and lead to the speculation that physiological gastrointestinal digestion could also yield ACE inhibitory peptides from SPI.     

Protective effects of dietary chamomile tea on diabetic complications

Matricaria chamomilla L., known as "chamomile", has been used as an herbal tea or supplementary food all over the world. We investigated the effects of chamomile hot water extract and its major components on the prevention of hyperglycemia and the protection or improvement of diabetic complications in diabetes mellitus. Hot water extract, esculetin (3) and quercetin (7) have been found to show moderate inhibition of sucrase with IC50 values of 0.9 mg/mL and 72 and 71 microM, respectively. In a sucrose-loading test, the administration of esculetin (50 mg/kg body weight) fully suppressed hyperglycemia after 15 and 30 min, but the extract (500 mg/kg body weight) and quercetin (50 mg/kg body weight) were less effective. On the other hand, a long-term feed test (21 days) using a streptozotocin-induced rat diabetes model revealed that the same doses of extract and quercetin showed significant suppression of blood glucose levels. It was also found that these samples increased the liver glycogen levels. Moreover, chamomile extract showed potent inhibition against aldose reductase (ALR2), with an IC50 value of 16.9 microg/mL, and its components, umbelliferone (1), esculetin (3), luteolin (6), and quercetin (7), could significantly inhibit the accumulation of sorbitol in human erythrocytes. These results clearly suggested that daily consumption of chamomile tea with meals could contribute to the prevention of the progress of hyperglycemia and diabetic complications.