Genetic Variation of Flavonols Quercetin, Myricetin, and Kaempferol in the Sri Lankan Tea (Camellia sinensis L.) and Their Health-Promoting Aspects.

Flavonol glycosides in tea leaves have been quantified as aglycones, quercetin, myricetin, and kaempferol. Occurrence of the said compounds was reported in fruits and vegetable for a long time in association with the antioxidant potential. However, data on flavonols in tea were scanty and, hence, this study aims to envisage the flavonol content in a representative pool of accessions present in the Sri Lankan tea germplasm. Significant amounts of myricetin, quercetin, and kaempferol have been detected in the beverage type tea accessions of the Sri Lankan tea germplasm. This study also revealed that tea is a good source of flavonol glycosides. The Camellia sinensis var. sinensis showed higher content of myricetin, quercetin, and total flavonols than var. assamica and ssp. lasiocalyx. Therefore flavonols and their glycosides can potentially be used in chemotaxonomic studies of tea germplasm. The nonbeverage type cultivars, especially Camellia rosaflora and Camellia japonica Red along with the exotic accessions resembling China type, could be useful in future germplasm studies because they are rich sources of flavonols, namely, quercetin and kaempferol, which are potent antioxidants. The flavonol profiles can be effectively used in choosing parents in tea breeding programmes to generate progenies with a wide range of flavonol glycosides.

New Sample Preparation Method for Quantification of Phenolic Compounds of Tea (Camellia sinensis L. Kuntze): A Polyphenol Rich Plant.

Chemical analysis of the Sri Lankan tea (Camellia sinensis, L.) germplasm would immensely contribute to the success of the tea breeding programme. However, the polyphenols, particularly catechins (flavan-3-ols), are readily prone to oxidation in the conventional method of sample preparation. Therefore, optimization of the present sample preparation methodology for the profiling of metabolites is much important. Two sample preparation methodologies were compared, fresh leaves (as in the conventional procedures) and freeze-dried leaves (a new procedure), for quantification of major metabolites by employing two cultivars, one is known to be high quality black tea and the other low quality black tea. The amounts of major metabolites such as catechins, caffeine, gallic acid, and theobromine, recorded in the new sampling procedure via freeze-dried leaves, were significantly higher than those recorded in the conventional sample preparation procedure. Additionally new method required less amount of leaf sample for analysis of major metabolites and facilitates storage of samples until analysis. The freeze-dried method would be useful for high throughput analysis of large number of samples in shorter period without chemical deterioration starting from the point of harvest until usage. Hence, this method is more suitable for metabolite profiling of tea as well as other phenol rich plants.

Quantification of gamma-aminobutyric acid in Sri Lankan tea by means of ultra performance tandem mass spectrometry.

Gamma-Aminobutyric acid (GABA), an important bioactive component of tea, acts as a major inhibitory neurotransmitter and is considered to influence other physiological processes in human as well as in planta. In the hereby presented study, the content of this valuable metabolite was investigated in two novel types of Ceylon Tea, explicitly "Silver Tips" and "White Tea", originating from minimally processed buds of the unique cultivar, "TRI 2043". The samples were subjected to hot water infusion, equivalent to the traditional beverage preparation procedure, and analyzed by means of hydrophilic interaction ultra performance liquid chromatography coupled to tandem mass spectrometry (HILIC LC-MS/MS). The registered GABA levels were compared with those obtained for the classic "Black Tea" and "Green Tea" samples from Sri Lanka. A high variation of GABA content was observed among the different tea types, especially in the case of "Silver Tips" and "White Tea", indicating the crucial influence of the manufacturing procedure (processing extent) on the final abundance of the bioactive component of interest. Furthermore, "White Tea" samples boasted the highest GABA concentration reported for this type of tea so far, reaching up to 50% of that characteristic of the high-priced "GABA Tea". Therefore, "White Tea" and "Silver Tips" were proved to be high quality tea with amounts of gamma-aminobutyric acid comparable with those described for similar types before. To our knowledge, this is the first report on HILIC LC-MS/MS application for the quantification of GABA and for in-depth characterization of teas from Sri Lanka.

Functional characterization of proanthocyanidin pathway enzymes from tea and their application for metabolic engineering.

Tea (Camellia sinensis) is rich in specialized metabolites, especially polyphenolic proanthocyanidins (PAs) and their precursors. To better understand the PA pathway in tea, we generated a complementary DNA library from leaf tissue of the blister blight-resistant tea cultivar TRI2043 and functionally characterized key enzymes responsible for the biosynthesis of PA precursors. Structural genes encoding enzymes involved in the general phenylpropanoid/flavonoid pathway and the PA-specific branch pathway were well represented in the library. Recombinant tea leucoanthocyanidin reductase (CsLAR) expressed in Escherichia coli was active with leucocyanidin as substrate to produce the 2R,3S-trans-flavan-ol (+)-catechin in vitro. Two genes encoding anthocyanidin reductase, CsANR1 and CsANR2, were also expressed in E. coli, and the recombinant proteins exhibited similar kinetic properties. Both converted cyanidin to a mixture of (+)-epicatechin and (-)-catechin, although in different proportions, indicating that both enzymes possess epimerase activity. These epimers were unexpected based on the belief that tea PAs are made from (-)-epicatechin and (+)-catechin. Ectopic expression of CsANR2 or CsLAR led to the accumulation of low levels of PA precursors and their conjugates in Medicago truncatula hairy roots and anthocyanin-overproducing tobacco (Nicotiana tabacum), but levels of oligomeric PAs were very low. Surprisingly, the expression of CsLAR in tobacco overproducing anthocyanin led to the accumulation of higher levels of epicatechin and its glucoside than of catechin, again highlighting the potential importance of epimerization in flavan-3-ol biosynthesis. These data provide a resource for understanding tea PA biosynthesis and tools for the bioengineering of flavanols.

Exobasidium vexans infection of Camellia sinensis increased 2,3-cis isomerisation and gallate esterification of proanthocyanidins.

Infection of leaves of tea (Camellia sinensis (Kuntze) L, cv TRI 2025) which was susceptible to blister blight (Exobasidium vexans Massee), resulted in a shift of the proanthocyanidin stereochemistry away from 2,3-trans (e.g. catechin and gallocatechin) and towards 2,3-cis (e.g. epicatechin and epigallocatechin). Infection also resulted in increased gallic acid esterification of the initiating subunits of proanthocyanidins. This was shown by both mass spectroscopy and phloroglucinolysis. Proanthocyanidins isolated from healthy tissue had a predominantly 2,3-trans stereochemistry which accounted for 53% and 61% of the total initiating and extension units of proanthocyanidin, respectively. Conversely in infected tissue, proanthocyanidin subunits with a 2,3-trans stereochemistry accounted for 26% and 40% of the total initiating and extension units, respectively. Infection had little impact on the hydroxylation state of the B-rings of proanthocyanidins. The products of acid hydrolysis under oxidative conditions had a slight excess of di-hydroxylated B-rings with cyanidin accounting for 58.3+/-0.05% and 60.4+/-0.2% of the total anthocyanidin recovered following hydrolysis of proanthocyanidin isolated from infected and healthy leaves, respectively. Similar results were obtained by phloroglucinolysis.