A new DNA marker CafLess-TCS1 for selection of caffeine-less tea plants.

A clonal line of Camellia taliensis, 'Taliensis-akeme' has a recessive caffeine-less gene. To accelerate breeding of caffeine-less tea cultivars using this gene, DNA markers are indispensable for selecting heterozygotes that do not show a caffeine-less phenotype as parental lines. Therefore, we tried to determine the sequence of the six tea caffeine synthase (TCS) genes to search for polymorphisms and to prepare one of the TCS genes as a selection marker. Six TCS genes and the caffeine-less trait were mapped on the reference linkage map of tea. Strong linkage between the caffeine-less phenotype and TCS1 indicate that it is a promising candidate as a causative gene of the caffeine-less trait. We decided to use a three-nucleotide insertion in TCS1 that can be distinguished by sequencing as a selection marker named 'CafLess-TCS1'. Caffeine-less individuals appeared in the progeny population of caffeine-less heterozygous individuals selected using 'CafLess-TCS1'. These results confirmed that the developed 'CafLess-TCS1' will be an effective selection marker for breeding of caffeine-less tea cultivars.

Low and medium but not high doses of green tea polyphenols ameliorated dextran sodium sulfate-induced hepatotoxicity and nephrotoxicity.

Our previous study indicated that a diet containing a high dose (1%) of green tea polyphenols (GTPs) disrupted liver and kidney function via a reduction in antioxidant enzyme and heat shock protein (HSP) levels in both colitis and non-treated ICR mice. In the present study, we assessed the effects of 0.01%, 0.1%, and 1% dietary GTPs on liver and kidney physiological functioning in dextran sulfate sodium (DSS)-exposed and normal mice. GTPs at 0.01% and 0.1% significantly suppressed DSS-increased serum aspartate 2-oxoglutarate aminotransferase (AST) and alanine aminotransferase (ALT) levels. In contrast, GTPs at 1% increased kidney weight, serum creatinine levels, and thiobarbituric acid-reactive substances (TBARs) in both the kidney and the liver in normal mice, as compared with DSS-exposed mice. GTPs at 0.01% and 0.1% remarkably upregulated the expression of heme oxygenase-1 (HO-1) and heat shock protein 70 (HSP70) mRNA in the liver and kidney of mice exposed to DSS, whereas GTPs at 1% abolished it. Our results indicate that low and medium doses of GTPs have beneficial effects on DSS-induced hepatotoxicity and nephrotoxicity via upregulation of self-protective enzymes, while these effects disappeared at a high dose.

Chemical analysis and acetylcholinesterase inhibitory effect of anthocyanin-rich red leaf tea (cv. Sunrouge).

BACKGROUND: The purpose of this study was to evaluate the effects of leaf order or crop season on anthocyanins and other chemicals in the anthocyanin-rich tea cultivar 'Sunrouge' (Camellia sinensis x C. taliensis) by using high-performance liquid chromatography, and to study the effect of 'Sunrouge' extract on acetylcholinesterase (AChE) activity in human neuroblastoma SK-N-SH cells. RESULTS: The total anthocyanin content was higher in the third (3.09 mg g⁻¹) than in the second (2.24 mg g⁻¹) or first crop season (1.79 mg g⁻¹). The amount of anthocyanins contained in the stem was high (1.61 mg g⁻¹). In the third crop season, the concentrations of delphinidin-3-O-ß-D-(6-(E)-p-coumaroyl)galactopyranoside (DCGa), cyanidin-3-O-ß-D-(6-(E)-p-coumaroyl)galactopyranoside, delphinidin-3-O-ß-D-galactopyranoside, delphinidin-3-O-ß-D-(6-O-(Z)-p-coumaroyl)galactopyranoside, cyanidin-3-O-ß-D-galactoside, and delphinidin-3-O-ß-D-glucoside were 1.57 mg g⁻¹, 0.52 mg g⁻¹, 0.40 mg g⁻¹, 0.22 mg g⁻¹, 0.14 mg g⁻¹, and 0.11 mg g⁻¹, respectively. DCGa accounted for about 50% of the anthocyanins present. The suppressive effect of 'Sunrouge' water extract on AChE activity in human neuroblastoma SK-N-SH cells was the strongest among the three tea cultivars ('Sunrouge', 'Yabukita' and 'Benifuuki'). CONCLUSION: These results suggested that 'Sunrouge' might protect humans from humans from AChE-related diseases by suppressing AChE activity. To obtain sufficient amounts of anthocyanins, catechins and/or caffeine for a functional food material, 'Sunrouge' from the third crop season should be used.

Effects of a Tea Cultivar "MK5601" on Behaviors and Hippocampal Neurotrophin-3 Levels in Middle-Aged Mice.

Dietary factors are thought to play an important role in the prevention of cognition diseases and depression in late life. In the present study, we compared the effects between the theogallin-rich tea cultivar, "MK5601" and a common Japanese tea cultivar, "Yabukita" on behaviors and hippocampal neurotrophin levels in experimental animals. Middle-aged mice (aged 8 mo) were given either of the tea infusions or water ad libitum for 4 mo. In the novel object location test, the middle-aged mice drinking water or "Yabukita" performed worse than young mice (aged 2-3 mo) although the middle-aged mice drinking "MK5601" retained spatial memory at the same level as the young mice. We also found that the middle-aged mice drinking "MK5601" showed high levels of neurotrophin-3 in the hippocampus. In conclusion, the "MK5601" tea infusion appears to be effective in preventing age-related changes in cognitive function, as compared with a common Japanese tea cultivar.

A Randomized, Placebo-Controlled Study on the Safety and Efficacy of Daily Ingestion of Green Tea (Camellia sinensis L.) cv. "Yabukita" and "Sunrouge" on Eyestrain and Blood Pressure in Healthy Adults.

The green tea (Camellia sinensis L.) cultivar “Sunrouge” contains anthocyanins, catechins and flavonols. To determine whether ingesting green tea containing anthocyanins improves visual function and blood pressure (BP) in healthy adults, a randomized, double-blind, placebo-controlled study was performed. A total of 120 healthy subjects, aged between 20 and 60 years and with a systolic BP (SBP) value of ≤125 and <155 and a diastolic BP (DBP) value <95, or a DBP of ≤75 mmHg and <95 mmHg and a SBP <155 mmHg, were randomly assigned to one of three groups. For 12 weeks, the placebo group received barley extract without catechin; another group received “Sunrouge” extract containing 11.2 mg anthocyanin and 323.6 mg epigallocatechin-3-O-gallate (EGCG); and a third group received “Yabukita” extract containing 322.2 mg EGCG. Home BP, accommodation ability, visual analog scale questionnaires for eyestrain, and metabolic-associated markers were analyzed at weeks 0, 4, 8, and 12 of the intake period. The ingestion of “Sunrouge” tea significantly improved accommodation ability and eyestrain in subjects younger than 45 years and in subjects who operated visual display terminals every day. It also elevated BP. “Yabukita” tea ingestion significantly increased serum adiponectin levels. No adverse effects were observed. We conclude that long-term intake of “Sunrouge” tea containing anthocyanins and flavonols might improve visual function.

Comparison of the Effects of Three Tea Cultivars (Camellia sinensis L.) on Nitric Oxide Production and Aortic Soluble Guanylate Cyclase Expression in High-Salt Diet-Fed Spontaneously Hypertensive Rats.

Studies have suggested that the consumption of green tea reduces the risk of cardiovascular diseases. Although epigallocatechin gallate (EGCG) is the best studied active substance characteristic of green tea, previous results on EGCG do not appear sufficient to explain completely the mechanism of cardiovascular protection by green tea. Therefore, we investigated the effect of three different tea cultivars, "Yabukita," "Sofu," and "Sunrouge," which have characteristic flavonoid compositions, on the nitric oxide (NO) production and the related protein expression in the aorta of spontaneously hypertensive rats (SHRs) fed a high-salt diet. As a result, the reduction of urinary NO metabolite (NOx) levels, which reflect whole-body NO production, caused by the high-salt diet were significantly prevented by all three tea infusions. The improvement of NOx reduction in the tea-intake groups was unlikely to be caused by the changes in oxidative damage. On the other hand, as a partial effect, only "Yabukita" or "Sofu" increased the expression of the soluble guanylate cyclase, a receptor for NO, in the thoracic aorta. In the present study, the differences in the composition of these three cultivars led to partially different effects on NO signaling in SHRs, suggesting the physiological significance of subdominant ingredients besides EGCG.

Anthocyanin-rich tea Sunrouge upregulates expressions of heat shock proteins in the gastrointestinal tract of ICR mice: A comparison with the conventional tea cultivar Yabukita.

Sunrouge is an anthocyanin-rich, new tea cultivar that contains similar levels of catechins as Yabukita, the most popular tea cultivar consumed in Japan. Interestingly, Sunrouge preparations have previously been shown to have more pronounced acetylcholinesterase inhibitory and anticolitis activities than those of Yabukita. In this study, we examined their effects on expressions of self-defensive molecules, including heat shock proteins (HSPs), which are molecular chaperones involved in homeostasis and longevity. Hot water extract from freeze-dried Sunrouge significantly upregulated messenger RNA (mRNA) expressions of HSP40, HSP70, and HSP32 (heme oxygenase-1), with grades greater than those shown by Yabukita. Oral administration of freeze-dried preparation of Sunrouge to male ICR mice at a dose of 1% in the basal diet for 1 month resulted in marked upregulations of several HSP mRNA expressions in mucosa from the gastrointestinal tract, especially the upper small intestine. Again, its efficacy was remarkably higher than that of Yabukita. Moreover, exposure of Caenorhabditis elegans to Sunrouge conferred thermoresistant phenotype, and also resulted in a significant life-span elongation. Taken together, our results suggest that Sunrouge is a unique and promising tea cultivar for regulating self-defense systems.

Rapid determination of caffeine in tea leaves.

For the purpose of efficient screening of low-caffeine tea shoots, a method for the rapid determination of caffeine was developed using high-performance liquid chromatography. Polyvinylpolypyrrolidone was packed in a pre-column and used to remove polyphenols from tea extracts on-line. The concentrations of caffeine extracted from powdered tea leaves at 50 degrees C during 1 day could be analyzed in 2-5 min intervals. The pre-column and the analytical column could be used for the analysis of more than 2000 samples.

Delphinidin-3-O-galactoside protects mouse hepatocytes from (-)-epigallocatechin-3-gallate-induced cytotoxicity via up-regulation of heme oxygenase-1 and heat shock protein 70.

Delphinidin-3-O-galactoside (D3G) is a water-soluble anthocyanin with antioxidant activity. (-)-Epigallocatechin-3-gallate (EGCG) is also known as a powerful antioxidant but concomitantly possesses a prooxidative property. We hypothesized that D3G is capable of protecting the EGCG-induced cytotoxicity and endoplasmic reticulum (ER) stress via inducing self-protective proteins and antioxidant enzymes. (-)-Epigallocatechin-3-gallate (200-500 µM) dose dependently decreased the viability of hepa1c1c-7 mouse hepatocytes, whereas D3G (50-500 µM) did not change it. Pretreatment with D3G significantly suppressed EGCG-induced cytotoxicity in a time-dependent manner (0, 6, and 24 hours). (-)-Epigallocatechin-3-gallate drastically decreased heme oxygenase-1 and heat shock protein 70 messenger RNA (mRNA) levels, whereas, pretreatment with D3G markedly attenuated their down-regulations. Delphinidin-3-O-galactoside remarkably decreased EGCG-induced ER stress responses such as C/EBP-homologus protein mRNA expression and X-box-binding protein-1 mRNA splicing. Taken together, our data suggest that D3G is capable of masking the EGCG-induced cytotoxicity and ER stress, presumably through up-regulation of antioxidant enzymes and heat shock proteins.

Effects of anthocyanin-rich tea "Sunrouge" on dextran sodium sulfate-induced colitis in mice.

Sunrouge, an anthocyanin-rich tea, has similar levels of catechins as "Yabukita," the most popular green tea cultivar consumed in Japan. Green tea polyphenols (GTPs) have attracted interest due to their potent antioxidative activities combined with a lack of side effects in humans at normal consumption levels. However, we previously reported that high doses (0.5 and 1%) of dietary GTPs can result in deterioration of colitis and failed to prevent colon carcinogenesis in inflamed colons. In the present study, we determined the inhibitory effects of Sunrouge on colitis in dextran sodium sulfate (DSS)-treated and untreated control mice. Five-week-old female ICR mice were administered a single dose of Yabukita or Sunrouge (extracts in 1 mL distilled water) via a stomach tube for 3 weeks. After 1 week of treatment, the mice were divided into four groups (two Yabukita and two Sunrouge groups) and given drinking water with or without 3% DSS for 2 weeks, then they were euthanized. Those treated with DSS developed watery diarrhea and bloody stools, and showed body weight loss, spleen hypertrophy, and shortening of the colon, as well as deteriorations in survival rate, liver function, colon mucosal interleukin-1ß level and expression of phase II detoxification enzyme mRNA. Sunrouge improved these DSS-induced symptoms, at least in part, whereas Yabukita showed either no effect or adverse effects in regard to some those parameters. It is suggested that the differences between Yabukita and Sunrouge on DSS-induced colitis might be due to the high levels of anthocyanins found in Sunrouge tea.

Anthocyanins from new red leaf tea 'Sunrouge'.

New red leaf tea cultivar 'Sunrouge' (Camellia taliensis × Camellia sinensis), for which an application for registration was made in 2009, is an anthocyanin-rich tea. The anthocyanin content of 'Sunrouge' was the highest among 4 tea cultivars, and was 8.4 times higher than that of 'Yabukita'. We purified and isolated 6 anthocyanins from 'Sunrouge' by chromatography, and identified them by LC/MS/MS and NMR analysis. As a result, the four anthocyanins were identified as delphinidin-3-O-ß-D-(6-(E)-p-coumaroyl)galactopyranoside (2), delphinidin-3-O-ß-D-(6-(E)-p-coumaroyl)glucopyranoside (3), cyanidin-3-O-ß-D-(6-(E)-p-coumaroyl)galactopyranoside (4), and cyanidin-3-O-ß-D-(6-(E)-p-coumaroyl)glucopyranoside (5), and the other two were estimated as delphinidin-(Z)-p-coumaroylgalactopyranoside (1), petunidin-(E)-p-coumaroylgalactopyranoside (6). Compound 3 was found in tea for the first time. In general, anthocyanins have various bioactivities, including relieving eyestrain and antioxidative effects, so it is expected that drinking 'Sunrouge' tea brings in similar bioactivities.

High-dose green tea polyphenols induce nephrotoxicity in dextran sulfate sodium-induced colitis mice by down-regulation of antioxidant enzymes and heat-shock protein expressions.

Previously, we reported that oral feeding of 1% green tea polyphenols (GTPs) aggravated the dextran sulfate sodium (DSS)-induced colitis in mice. In the present study, we assessed the toxicity of 1% GTPs in several organs from normal and DSS-exposed mice. Sixty-two male ICR mice were initially divided into four groups. Non-treated group (group 1, n = 15) was given standard diet and water, GTPs (group 2, n = 15) received 1% GTPs in diet and water, DSS (group 3, n = 15) received diet and 5% DSS in water, and GTPs + DSS group (group 4, n = 17) received 1% GTPs in diet and 5% DSS in water. We found that group 4 significantly increased (P < 0.05) kidney weight, the levels of serum creatinine and thiobarbituric acid-reactive substances in both kidney and liver, as compared with those in group 3. The mRNA expression levels of antioxidant enzymes and heat-shock proteins (HSPs) in group 4 were lower than those of group 3. For instance, heme oxygenase-1 (HO-1), HSP27, and 90 mRNA in the kidney of group 4 were dramatically down-regulated as compared with those of group 3. Furthermore, 1% GTPs diet decreased the expression of HO-1, NAD(P)H:quinone oxidoreductase 1 (NQO1) and HSP90 in kidney and liver of non-treated mice. Taken together, our results indicate that high-dose GTPs diet disrupts kidney functions through the reduction of antioxidant enzymes and heat-shock protein expressions in not only colitis but also non-treated ICR mice.

Metabolomics-driven nutraceutical evaluation of diverse green tea cultivars.

BACKGROUND: Green tea has various health promotion effects. Although there are numerous tea cultivars, little is known about the differences in their nutraceutical properties. Metabolic profiling techniques can provide information on the relationship between the metabolome and factors such as phenotype or quality. Here, we performed metabolomic analyses to explore the relationship between the metabolome and health-promoting attributes (bioactivity) of diverse Japanese green tea cultivars. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the ability of leaf extracts from 43 Japanese green tea cultivars to inhibit thrombin-induced phosphorylation of myosin regulatory light chain (MRLC) in human umbilical vein endothelial cells (HUVECs). This thrombin-induced phosphorylation is a potential hallmark of vascular endothelial dysfunction. Among the tested cultivars, Cha Chuukanbohon Nou-6 (Nou-6) and Sunrouge (SR) strongly inhibited MRLC phosphorylation. To evaluate the bioactivity of green tea cultivars using a metabolomics approach, the metabolite profiles of all tea extracts were determined by high-performance liquid chromatography-mass spectrometry (LC-MS). Multivariate statistical analyses, principal component analysis (PCA) and orthogonal partial least-squares-discriminant analysis (OPLS-DA), revealed differences among green tea cultivars with respect to their ability to inhibit MRLC phosphorylation. In the SR cultivar, polyphenols were associated with its unique metabolic profile and its bioactivity. In addition, using partial least-squares (PLS) regression analysis, we succeeded in constructing a reliable bioactivity-prediction model to predict the inhibitory effect of tea cultivars based on their metabolome. This model was based on certain identified metabolites that were associated with bioactivity. When added to an extract from the non-bioactive cultivar Yabukita, several metabolites enriched in SR were able to transform the extract into a bioactive extract. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that metabolic profiling is a useful approach for nutraceutical evaluation of the health promotion effects of diverse tea cultivars. This may propose a novel strategy for functional food design.

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.