Catechin and Catechin Fractions as Biochemical Markers to Study the Diversity of Indian Tea (Camellia sinensis (L.) O. Kuntze) Germplasm

The heterogeneous Indian tea germplasm includes ‘China’, ‘Assam’, ‘Cambod’, and their hybrids which were evaluated using biochemical markers viz., total catechin and their fractions, for varietal identification and characterization. Principal component analysis (PCA) of biochemical characters showed that the total catechin and trihydroxylated catechin has higher eigenvalues. The first two principal components (PCs) could differentiate more than 90% of the clones studied. This grouping based on first two principal component matrices differentiated ‘China’, and their hybrids with ‘Assam’ and ‘Cambod’ variety. Morphologically indistinct large‐leaved ‘Cambod’ variety and ‘Assam’ varieties could not be differentiated using biochemical markers, since both varietal types taxonomically belong to a single species. Clones of ‘China’ type showed low total catechin content and catechin ratio which are distinctly grouped. The ‘China–Assam’ and ‘China–Cambod’ hybrids formed intermediate groups between ‘China’ PC group and ‘Cambod’/‘Assam’ PC groups, providing evidence for genetic control of catechin ratio variation. Tea clones which are differentially positioned in the PC group could be explained based on the genetic contribution by other varietal type as parents. This biochemical characterization will be a useful tool in the development of quality‐tea clones with different proportion of total catechin and their fractions.     

High Level of Genetic Diversity Among the Selected Accessions of Tea (Camellia sinensis) from Abandoned Tea Gardens in Western Himalaya

To revive cultivation of the tea unique to the western Himalayan region, it is important to evaluate the seed-derived bushes available in the area's abandoned gardens. This study used quantitative leaf characters, catechin content, and AFLP markers to assess these China cultivar type bushes. Compared with other China cultivar germplasm, these accessions showed a higher level of diversity among themselves. Among the quantitative morphological characters, leaf length is important in distinguishing the accessions studied, with a high loading value in the principal component analysis. The catechins and AFLP markers displayed the genetic makeup of the accessions. Other than total catechins, the trihydroxylated catechins showed a high loading value in differentiating the accessions. The genetic control of the ratio of dihydroxylated and trihydroxylated catechins is found to be based on a correlation with AFLP markers. The genetic similarity between Kangra Asha and Kangra Jat suggests that Kangra Jat must be descended from Kangra Asha. Kangra Jat is well adapted to local environmental conditions, as is evident from its high catechin content.     

Growth and photosynthetic and biochemical responses of tea cultivars to blister blight infection

Growth characteristics such as leaf area, fresh and dry mass, and shoot length, and physiological parameters such as photosynthetic and transpiration rates, stomatal conductance, and water use efficiency were reduced by blister blight significantly more in a susceptible tea clone TES-34 than in a tolerant clone SA-6. Also the contents of total sugars, nitrogen, amino acids, proteins, polyphenols, and catechin were reduced more in diseased plant leaves. However, the reduction was more prominent in susceptible than in tolerant clone. Among the different hybrids of tea, Assam hybrid UPASI-3 was highly susceptible to blister blight followed by Cambod UPASI-27 and China UPASI-9. Similarly, tea seedling cv. Caline was highly susceptible to blister blight when compared to tea clone UPASI-3. Susceptibility of tea cultivars to blister blight infection is connected with many physical barriers including leaf area, shoot length, moisture contents, and other physiological and biochemical parameters.     

Effects of catechin concentration on the formation of black tea polyphenols during in vitro oxidation

An in vitro model fermentation system, containing purified catechins and partially purified polyphenol oxidase (EC 1.14.81.1) from green tea shoots, has been used to determine the efrect of catechin mixtures of different concentration and proportions on the formation of theaflavin and thearubigin. Increases in total catechin concentration, 25% above that typical in green tea shoots of Malawi-grown bushes, inhibited polyphenol oxidase activity and, consequently, depressed theaflavin levels. Individual or combined concentrations of epicatechin gallate and epigallocatechin gallate in excess of 110 mM were shown to be responsible for enzyme inhibition, whereas epicatechin and epigallocatechin had no effect. Fermentation of a catechin mixture, containing the four major catechins, epicatechin, epigallocatechin, epigallocatechin gallate and epicatechin gallate, at equal individual concentrations (55 mM), produced, after 3O min, total theaflavin levels 68% higher and thearubigin levels only 25% higher than those from a standard catechin mixture fermented under similar conditions. Continued fermentation of this mixture produced no further theaflavin, but the thearubigin fraction increased significantly, due to subsequent oxidation of the excess of simple catechins. A new catechin mixture was, therefore, calculated to give a similar level of theaflavin to that of the previous mixture without leaving an excess of unoxidized simple catechins. The catechin proportions and concentrations of the latter mixture agree well with those of the green shoots of quality Kenyan teas or similar quality Malawi teas grown during the dry cold season. The results indicate that a high ratio of simple to gallocatechins will facilitate a high theaflavin-thearubigin ratio in black tea.     

Physiological and biochemical responses of micropropagated tea plants

Summary Physiological and biochemical responses of micropropagated tea plants grown under field conditions were investigated in comparison to vegetatively propagated (VP) plants. No significant variation was observed between tissue culture raised (TC) and VP plants in terms of photosynthetic carbon assimilation rate. However, clones showed significant variation among themselves. Carbon assimilation studies carried out with a radiotracer technique revealed that ‘Assam’ cultivar UPASI-27 assimilated a higher amount of labeled carbon dioxide followed by UPASI-3. However, UPASI-27 was marginally better than UPASI-3 in terms of mobilization of assimilates to the growing sinks. Both, UPASI-3 and UPASI-27 reassimilated higher quantities of photosynthates followed by BSB-1 and UPASI-26. Though there was a marginal variation in photosynthetic pigments of TC and VP plants, it was not statistically significant. Similarly, no significant variations were observed in certain substrates (polyphenols, catechins and amino acids) and enzymes (polyphenol oxidase, peroxidase and phenylalanine ammonia-lyase) except protease involved in the formation of quality constituents of made tea. However, clonal variation was evident with respect to photosynthetic pigments, substrates/enzymes. Under soil moisture stress, no significant variation was observed between VP and TC plants in terms of proline accumulation.     

Genetic base of tea (<Emphasis Type="Italic">Camellia sinensis</Emphasis> L.) cultivars in Sri Lanka as revealed by pedigree analysis

An understanding of genetic diversity and relationships among breeding materials is a prerequisite for crop improvement. Coefficient of parentage (COP) can be used to measure the genetic diversity among genotypes on the basis of pedigree information. In the present study, COP was estimated for 56 cultivars, including commercial tea cultivars developed by the Tea Research Institute of Sri Lanka and their parental lines. Mean COP of the 56 accessions studied was 0.097 and the value was raised up to 0.272 when non-related pair-wise comparisons were excluded. A single cultivar (Assam/Cambod introduction) was the nucleus of the commercial cultivars. Group mean COP of the cultivars derived from Assam/Cambod parentage was 0.17. Thirty-three percent of the pair-wise comparisons had 0.00 COP, highlighting that many cultivars were unrelated. Within the pedigree, 2 major COP clusters were identified: Assam/Cambod open-pollinated half-sib progenies, and full-sib progenies derived from crosses between Assam/Cambod and other parental lines. The elite groups within the pedigree, where Assam/Cambod parentage was concentrated, were also identified. Information generated in this study should be useful for effective utilization of available diversity in future breeding programmes as well as for proper conservation of genetic diversity in the adapted germplasm. This is the first report on estimates of genetic diversity based on COP in a woody perennial crop, such as tea.     

Dietary gallate esters of tea catechins reduce deposition of visceral fat, hepatic triacylglycerol, and activities of hepatic enzymes related to fatty acid synthesis in rats

Tea catechins, rich in (-)-epigallocatechin gallate and (-)-epicatechin gallate, or heat-treated tea catechins in which about 50% of the (-)-epigallocatechin gallate and (-)-epicatechin gallate in tea catechins was epimerized to (-)-gallocatechin gallate and (-)-catechin gallate, were fed to rats at 1% level for 23 d. Visceral fat deposition and the concentration of hepatic triacylglycerol were significantly lower in the tea catechin and heat-treated tea catechin groups than in the control group. The activities of fatty acid synthase and the malic enzyme in the liver cytosol were significantly lower in the two catechin groups than in the control group. In contrast, the activities of carnitine palmitoyltransferase and acyl-CoA oxidase in the liver homogenate were not significantly different among the three groups. These results suggest that the reduction in activities of enzymes related to hepatic fatty acid synthesis by the feeding of tea catechins or heat-treated tea catechins can cause reductions of hepatic triacylglycerol and possibly of visceral fat deposition.     

Antimicrobial Activities of Tocklai Vegetative Tea Clones

Thirty-one Tocklai vegetative (TV) tea clones contained caffeine and total catechin 44.39 and 227.55 mg/g dry weight of leaves, respectively. The (−)-epigallocatechin gallate (EGCG) was the most abundant (109.60 mg/g) followed by -(−)-epigallocatechin (EGC, 44.54 mg/g), (−)-epicatechin gallate (ECG, 41.74 mg/g), (−)-epicatechin (EC, 27.42 mg/g) and +catechin (4.25 mg/g). Total catechins were highest in TV 20 (509.7 mg/g) and lowest in TV 6 (71.7 mg/g). The tea clones that contain high level of total catechin exhibited the strongest antimicrobial activity. Among caffeine and flavanol compounds, theaflavins (TF) present in black tea possess a similar antimicrobial potency as EC present in fresh leaves, and that the conversion of catechins to TF during fermentation in making black tea tends to alter their antimicrobial activities. The bioactive molecules other than catechins present in tea leaves may also contribute towards antimicrobial activity.     

Metabolite profiling and characterization of somaclonal variants in tea (Camellia spp.) for identifying productive and quality accession

A study has been undertaken to characterize 15 field grown somaclonal variants derived from cotyledonary tissues of UPASI-10 using morphological, physiological and biochemical characters. Although variants were derived from UPASI-10, a very few variants possessed unique "Chinery" characters while others exhibited "Assam" characters. However, no variant showed identical morphological characters aligning with the parent. Somaclonal variants showed distinct variation in terms of photosynthetic carbon assimilation, stomatal conductance and diffusion resistance. Proline accumulation and water use efficiency showed marginal variations among the variants. SE 8 and SE 10 recorded higher values of membrane stability index denoting their tolerant nature against stress. Class interval analysis based on physiological parameters grouped these plants into three clusters. Three variants grouped under good category representing higher values of productivity index followed by five variants under moderate category. Green leaf constituents and quality profile of made tea produced with crop shoots of variants exhibited wide variation. Center point radar graph analysis of quality constituents grouped these plants into three clusters. Variants SE 2 and SE 13 were segregated distinctly representing their black tea characters. When considering both the quality and productivity indices, SE 3 and SE 7 fall under moderate category and in future these two variants may be subjected to further quality tests for commercial exploitation.     

Complex effects of different green tea catechins on human platelets

Epigallocatechin gallate (EGCG), a major component of green tea, has been previously shown to inhibit platelet aggregation. The effects of other green tea catechins on platelet function are not known. Pre-incubation with EGCG concentration-dependently inhibited thrombin-induced aggregation and phosphorylation of p38 mitogen-activated protein kinase and extracellular signal-regulated kinases-1/2. In contrast EGCG stimulated tyrosine phosphorylation of platelet proteins, including Syk and SLP-76 but inhibited phosphorylation of focal adhesion kinase. Other catechins did not inhibit platelet aggregation. Interestingly, when EGCG was added to stirred platelets, a tyrosine kinase-dependent stimulation of platelet aggregation was observed. The two other catechins containing a galloyl group in the 3' position (catechin gallate, epicatechin gallate) also stimulated platelet aggregation, while catechins without a galloyl group (catechin, epicatechin) or the catechin with a galloyl group in the 2' position (epigallocatechin) did not.     

Standardized biosynthesis of flavan-3-ols with effects on pancreatic beta-cell insulin secretion

Flavan-3-ols, such as green tea catechins represent a major group of phenolic compounds with significant medicinal properties. We describe the construction and optimization of Escherichia coli recombinant strains for the production of mono- and dihydroxylated catechins from their flavanone and phenylpropanoid acid precursors. Use of glucose minimal medium, Fe(II), and control of oxygen availability during shake-flask experiments resulted in production yield increases. Additional production improvement resulted from the use of medium rather than high-copy number plasmids and, in the case of mono-hydroxylated compounds, the addition of extracellular cofactors in the culture medium. The established metabolic engineering approach allowed the biosynthesis of natural catechins at high purity for assessing their possible insulinotropic effects in pancreatic beta-cell cultures. We demonstrated that (+)-afzelechin and (+)-catechin modulated the secretion of insulin by pancreatic beta-cells. These results indicate the potential of applying metabolic engineering approaches for the synthesis of natural and non-natural catechin analogues as drug candidates in diabetes treatments.     

白化和黄化茶树品种绿茶游离氨基酸、儿茶素类及咖啡碱差异分析

为探究以白化和黄化茶树品种鲜叶为原料制成的绿茶滋味品质和代谢物差异,对广德市6个白化品种绿茶(奶白茶)和14个黄化品种绿茶(黄金芽茶)进行感官审评和代谢物分析。结果表明,奶白茶滋味鲜爽而收敛性略弱;黄金芽茶滋味收敛性强而鲜度低于奶白茶。游离氨基酸总量以及呈现鲜味、甜味的游离氨基酸在奶白茶中的含量显著高于黄金芽茶,而贡献收敛性的儿茶素类化合物和没食子酸含量以及呈现苦味的咖啡碱含量在奶白茶中显著低于黄金芽茶。偏最小二乘法判别分析(PLS-DA)表明导致两种绿茶滋味差异的标志性化合物有7种,分别是茶氨酸、精氨酸、谷氨酸、天冬氨酸、表没食子儿茶素没食子酸酯(EGCG)、表儿茶素没食子酸酯和咖啡碱。味觉活性值(Dot)最高的EGCG在黄金芽茶中的呈味贡献显著高于奶白茶。因此,游离氨基酸、儿茶素类化合物、没食子酸和咖啡碱含量差异导致白化和黄化茶树品种绿茶滋味不同。     

Inheritance and quantitative trait loci analyses of the anthocyanins and catechins of Camellia sinensis cultivar ‘Ziyan’ with dark-purple leaves

Owing to the potential health benefits, anthocyanin-rich teas (Camellia sinensis) have attracted interest over the past decade. Previously, we developed the cultivar ‘Ziyan,’ which has dark-purple leaves because of the accumulation of a high amount of anthocyanins. In this study, we performed a genetic analysis of this anthocyanin-rich tea cultivar and 176 of its naturally pollinated offspring. For two consecutive years, we quantified the anthocyanins and catechins of ‘Ziyan’ and the offspring population. While >60% of the offspring accumulated less than half of the amount of anthocyanins of ‘Ziyan,’ 17 (2018) and 15 (2019) individuals exceeded ‘Ziyan’ in anthocyanin content. A negative correlation between anthocyanin and total catechin content (r = −0.59, P < 0.001) was observed. The population was genotyped with 131 SSR markers spanning all linkage groups of the C. sinensis genome. Kruskal-Wallis tests identified 10 markers significantly associated with anthocyanins, catechins and their ratios in both years. Quantitative trait locus (QTL) analyses using the interval mapping method detected 13 QTLs, suggesting the dark-purple trait of ‘Ziyan’ is because of the pyramiding of anthocyanin-promoting alleles on at least five linkage groups. Two genetic loci reversely related to anthocyanin and total catechin contents were identified. This study provides valuable information for genetic improvement of purple tea cultivars and for fine-mapping related genes.     

Clonal variation of tea [Camellia sinensis (L.) O. Kuntze] in countering water deficiency

Various clones of tea [Camellia sinensis (L.) O. Kuntze] such as TTL-1, TTL-2, TTL-4, TTL-5, TTL-6, UPASI-2 and UPASI-3 planted in the field were subjected to soil moisture stress conditions by withholding irrigation. A control set of the same clones were maintained by watering regularly. The soil water content of the irrigated and non irrigated plants was monitored through the soil moisture status. The extent of effect of drought on tea plants were monitored through various physiological parameters such as shoot weight, leaf water potential, chlorophyll and carotenoid content, chlorophyll fluorescence (Fv/Fm), net photosynthetic rate, transpiration rate, stomatal conductance and biochemical parameters such as extent of proline accumulation and free radical generation. These parameters were studied on the 30 d of non irrigation and on the 5 d during recovery from drought. The plants recovered when re-irrigated after 30 d of non-irrigation, which suggests that permanent wilting did not occur due to non-irrigation up to 30 d. On the 30 d of non-irrigation the clones TTL-1, TTL-6 and UPASI-2 showed lesser reduction of shoot weight, leaf water potential, chlorophyll fluorescence, photosynthetic rate, transpiration rate and stomatal conductance and increased proline and lesser lipid peroxidation as compared to the other clones. From these results it can be concluded that the clones TTL-1, TTL-6 and UPASI-2 are comparatively more drought tolerant than the clones TTL-2, TTL-4, TTL-5 and UPASI-3.     

Improving the Polyphenol Content of Tea

Tea, prepared from the leaves of Camellia species, has one of the highest contents of flavonoids among common food and beverage products. Tea consumption has moved beyond its pleasant flavor and cultural significance since a number of health promoting properties have been ascribed to this widespread beverage (e.g., anticancer, antiobesity and hypotensive effects). The major bioactive compounds in tea are catechins (flavan-3-ols), a group of flavonoids that include, among others, (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin-3-gallate (ECG), and (-)-epigallocatechin-3-gallate (EGCG). These compounds are also the precursors of theaflavins and thearubigins, oxidation products responsible for the taste and colour of certain tea types such as black tea. The composition of the tea leaf, and thus tea quality, is influenced by many pre-harvest factors such as the genetic make-up of the plant, region of production, horticultural and harvesting practices, and environmental conditions. Once harvested, processing, brewing, and storage conditions influence the phenolic composition and quality of tea infusions as well. In the present review we aim at outlining our current knowledge about means to increase the catechin content of teas, a cornerstone for improving the health-promoting properties of this beverage.     

Structural characteristics of green tea catechins for formation of protein carbonyl in human serum albumin

Catechins are polyphenolic antioxidants found in green tea leaves. Recent studies have reported that various polyphenolic compounds, including catechins, cause protein carbonyl formation in proteins via their pro-oxidant actions. In this study, we evaluate the formation of protein carbonyl in human serum albumin (HSA) by tea catechins and investigate the relationship between catechin chemical structure and its pro-oxidant property. To assess the formation of protein carbonyl in HSA, HSA was incubated with four individual catechins under physiological conditions to generate biotin-LC-hydrazide labeled protein carbonyls. Comparison of catechins using Western blotting revealed that the formation of protein carbonyl in HSA was higher for pyrogallol-type catechins than the corresponding catechol-type catechins. In addition, the formation of protein carbonyl was also found to be higher for the catechins having a galloyl group than the corresponding catechins lacking a galloyl group. The importance of the pyrogallol structural motif in the B-ring and the galloyl group was confirmed using methylated catechins and phenolic acids. These results indicate that the most important structural element contributing to the formation of protein carbonyl in HSA by tea catechins is the pyrogallol structural motif in the B-ring, followed by the galloyl group. The oxidation stability and binding affinity of tea catechins with proteins are responsible for the formation of protein carbonyl, and consequently the difference in these properties of each catechin may contribute to the magnitude of their biological activities.     

Reduction of catechin astringency by the complexation of gallate-type catechins with pectin

The reductive effect of pectin on tea catechin astringency was investigated by using a taste sensor system and (1)H-NMR spectroscopy. The sensor analysis revealed that the astringency of gallate-type catechins (EGCg and ECg) was reduced by the addition of pectin, whereas that of non-gallate-type catechins (EGC and EC) hardly changed. Changes in the (1)H-NMR chemical shifts of the catechins and pectin in mixed solutions showed that the gallate-type catechins formed complexes with pectin more closely than the non-gallate-type catechins. These results demonstrate that complexation between the gallate-type catechins and pectin is a factor for reducing catechin astringency.     

Ex vivo modulation of chemical-induced mutagenesis by subcellular liver fractions of rats treated with rooibos (Aspalathus linearis) tea, honeybush (Cyclopia intermedia) tea, as well as green and black (Camellia sinensis) teas

Male Fischer rats were given unprocessed (not oxidized) and processed (oxidized) rooibos and honeybush teas as well as green and black teas as a sole source of drinking fluid for 10 weeks, and sub cellular liver fractions were prepared. Cytosolic fractions of rats consuming the unprocessed herbal teas, green and black teas significantly (P < 0.05) protected against 2-acetylaminofluorene (2-AAF)-induced mutagenesis in the Salmonella mutagenicity test with strain TA 98, using Aroclor 1254-induced microsomes. A marginal or no protection was obtained with the processed herbal teas. The mutagenic response of aflatoxin B1 (AFB1) against Salmonella strain TA 100 was significantly (P < 0.05) inhibited by cytosolic fractions from rats treated with processed and unprocessed herbal teas, while no effect was obtained with the green and black teas. Microsomal fractions prepared from livers of rats treated with both the processed and unprocessed rooibos teas and the unprocessed honeybush tea, significantly (P < 0.05) reduced the activation of AFB1 while no protection was observed against 2-AAF-induced mutagenesis. In contrast, microsomal fractions from rats treated with the green, black and unprocessed honeybush teas significantly (P < 0.05) enhanced the mutagenic response of 2-AAF. None of the tea treatments significantly affected the concentration of the microsomal liver cytochrome P450.