tea is the second most popular beverage after water which represents a major source of dietary polyphenols .
thus the scientific community is interested in exploring the health - promoting constituents present in tea , namely , flavan-3-ols , flavonols , and their derivatives .
recent epidemiological studies have demonstrated a protective effect of fruits and vegetables against the incidence of degenerative diseases .
several classes of compounds have been assumed as potential protective factors , one of which is the flavonoids , generally considered as nonnutritive agents .
flavonoids are known as secondary metabolites which are the largest class of polyphenols widely distributed in the plant kingdom .
it can be subdivided into six major subclasses based on the structural variations : flavones , flavanones , isoflavones , flavonols , flavanols , and anthocyanidins . according to the study conducted by hollman et al .  
flavonols have recently received much attention due to their antioxidant , antimicrobial , anticancer , antiatherosclerotic , and antiproliferative properties [ 68 ] .
the flavonols exist in tea plant ( camellia sinensis l. ) as flavonol glycosides with a sugar residue and the most common flavonol aglycones in tea are quercetin , kaempferol , and myricetin .
ceylon tea from sri lanka , acclaimed as the best tea in the world , has its inherent unique characteristics and reputation running over more than a century .
the objectives of this study were to investigate the variation in flavonol aglycones , namely , quercetin , kaempferol , and myricetin in tea ( camellia sinensis l. ) accessions including beverage and nonbeverage types . therefore , we report here the first systematic study on the flavonol content in representative accessions of the sri lankan tea germplasm .
50  g of tender tea shoots ( two apical leaves and the bud ) of 87 beverage type and 6 nonbeverage type germplasm accessions was collected in triplicate from the ex situ field gene bank of tea research institute of sri lanka ( tri ) , talawakelle ( latitude 654n , longitude 8042e ) , and brought to the laboratory at 4c .
selection criteria for germplasm accessions in this study included the attributes such as morphological variations , origins , made tea quality , and sensitivity to biotic stresses [ 1113 ] .
the tea samples were immediately stored in a 80c freezer for 6  h to two weeks and freeze - dried for 24  h ( labconco corporation , ms , usa ) .
the freeze - dried leaves were ground to a fine powder ( passed through 500  m mesh ) , sealed in triple laminated aluminium foil packages , and stored at 20c until biochemical analysis .
100  mg of freeze - dried tea sample was hydrolysed in duplicate with 0.5  ml of 6  m hcl and 4.5  ml of 60% methanol for 2  h in a water bath preheated to 95c .
hydrolysed sample extracts were topped up to 5  ml with 60% methanol and filtered through 0.45  m nylon filters into hplc vials .
analysis of individual flavonols was carried out in the agilent 1260 infinity hplc system comprising of solvent delivery system , quaternary pump , temperature - controlled column oven , autosampler , degasser unit and a photodiode array detector with uv detection at 370  nm , and open lab / chemstation software system .
the compounds were separated on a 4.6  250  mm , 5  m particle , and reversed phase zorbax eclipse plus c 18 column ( agilent , usa ) ( phenomenex inc . , usa ) with a guard column made of the same material ( security - guard , phenomenex inc . , usa ) at 30c .
30% hplc grade acetonitrile in 0.025  m potassium dihydrogen orthophosphate buffer solution adjusted to a ph of 2.5 with 6  m hcl was used as mobile phase at a flow rate of 1.0  ml / min .
quantification was carried out from the integrated peak areas of the sample against the corresponding standard graph and results were expressed on dry matter basis ( figures 1(a ) and 1(b ) ) .
the dry matter contents of the freeze - dried leaves were determined in accordance with iso1573:1980 .
experimental data were analysed using sas system for windows v 9.1 ( sas institute inc .
absolute methanol ( analytical reagent grade ) , anhydrous sodium carbonate , and acetonitrile ( hplc gradient grade ) were supplied by fisher scientific uk ; quercetin , kaempferol , and myricetin were purchased from sigma chemicals ; and potassium dihydrogen orthophosphate and hydrochloric acid ( analytical grade ) were purchased from british drug house ltd . , england .
the major flavonols present in tea leaves make up 2 - 3% of the water - soluble solids in tea [ 15 , 16 ] .  
table 1 shows the amounts of myricetin , quercetin , and kaempferol quantified in the beverage and nonbeverage type accessions of the sri lankan tea germplasm .
these results are agreeable to a greater extend with the results reported for 3 commercial samples of green tea and 30 commercial samples of black tea   and those reported for 4 commercial samples of green tea and 2 commercial samples of black tea .
according to the mean values of flavonols , quercetin ( 1.50  mg  g ) is abundantly present in the beverage type accessions of the sri lankan tea germplasm followed by kaempferol ( 1.31  mg  g ) and myricetin ( 0.94  mg  g ) . the mean value of total flavonols in 89 beverage type c. sinensis was 3.75  mg  g. among beverage type accessions , the highest quercetin contents were reported in the exotic accessions : pbgt41 ( 3.23  mg  g ) , followed by pbgt73 , china , pbgt70 , and pbgt12 whereas tri1114 showed the lowest content ( 0.36  mg  g ) .
the highest kaempferol contents were observed in the exotic accessions pbgt61 ( 2.21  mg  g ) followed by pbgt68 , pbgt48 , pbgt49 , and pbgt70 . among the 9 exotic accessions examined , pbgt41 reported the highest myricetin content ( 1.63  mg  g ) followed by pbgt49 , pbgt68 , pbgt70 , and pbgt61 and intri6 recorded the lowest content ( 0.33  mg  g ) .
conversely , myricetin was available in trace amounts recorded in the nonbeverage type accessions except camellia sasanqua ( 0.37  mg  g ) and camellia rosaflora ( 0.20  mg  g ) .
camellia rosaflora has reported the highest quercetin content ( 3.44  mg  g ) when considering the entire germplasm , which is higher than that of the beverage type exotic cultivar pbgt41 .
the specific patterns of flavonol occurrence in plant species vary based on their synthesis and distribution in the plant organism . in addition to intrinsic factors , flavonol content is strongly influenced by extrinsic factors such as variations in plant type and growth , season , climate , degree of maturity , preparation , and processing [ 2024 ] . several studies report varying content of flavonol in teas [ 1 , 25 , 26 ] .
this is possibly due to the difference in tea samples used ( fresh tea leaves or processed tea ) and analytical methods utilized , resulting in different extraction efficiencies . according to finger et al .
, flavonol glycosides are not affected by polyphenol oxidase and varying content in different tea types can be due to difference in tea varieties , geographic location , or agricultural conditions .
the increasing knowledge on functional ingredients in food provides new opportunities for plant sciences to produce crops with higher amounts of selected health - promoting phytochemicals using crop husbandry techniques , plant breeding techniques , and genetic engineering .
recent studies suggest that higher intakes of flavonoids from food are associated with reduced risk of degenerative diseases including cancer and heart disease [ 29 , 30 ] .
the three flavonols , that is , myricetin , quercetin , and kaempferol , quantified in this study have also been extensively investigated for their health benefits . flavonols naturally occur as either glycosides ( with attached glycosyl groups ) or aglycones . however , dietary intake of the above flavonols is mainly in the glycoside form . during digestion and absorption sugar moieties
digestive tract microbiota could also assist in the digestion and the absorption process of the flavonols .
following absorption several organs contribute to flavonol metabolism including the liver , giving rise to glucuronidated , methylated , and sulfated forms of flavonols which are found in the plasma .
. several mechanisms of actions such as antioxidant activity , anti - inflammatory activity , and modulation of signaling pathways are responsible for the beneficial effects of flavonols .
ability of hydrogen radical donation from the phenolic group and the presence of an unpaired electron in the aromatic ring make the flavonols strong antioxidants in vivo , thus scavenging the reactive oxygen species and other free radicals .
ability to chelate metal ions also reduces the production of harmful oxidant species in vivo .
flavonols could contribute to the antioxidant properties through the protection or improvement of endogenous antioxidants especially by stimulating glutathione - s - transferase ( gst ) , an enzyme that protects cells from the damage caused by free radicals .
the inflammatory response is part of the innate immune response which triggers the inflammatory cascade forming inflammatory mediators such as prostaglandin and thromboxane .
although acute inflammation is essential in dealing with invading pathogens and injury , chronic inflammation can cause tissue destruction and is involved in the pathogenesis of degenerative diseases . commercially available anti - inflammatory drugs
are used to prevent the formation of prostaglandins and thromboxanes by inhibiting the cyclooxygenase ( cox ) enzymes involved in the production of the above during inflammation .
flavonoids can suppress the expression of the cox gene through interactions with cell signaling pathways , such as the protein kinase c , nf-b , and tyrosine kinase pathways .
it has been reported that flavonols including myricetin , quercetin , and kaempferol could play an important role in reducing the risk of cardiovascular disease , cancer , and neurodegradative and other degenerative diseases through the above - mentioned mechanisms and several other mechanisms .
several epidemiological studies also have found the beneficial effects of flavonols [ 36 , 37 ] .
therefore , it could be argued that dietary items high in flavonols could be beneficial to human health .
although flavonol glycosides can be important contributors to black tea flavour , these were hydrolysed by acid treatment to afford myricetin , quercetin , and kaempferol for hplc analysis due to the fact that the dietary forms of flavonol glycosides are initially hydrolysed to aglycones at the oral cavity [ 3942 ] and continued in the digestive tract [ 5 , 43 ] . even during the fermentation processes
however the chemical form of flavonol prior to and after digestion is of great importance in determining the bioavailability and bioactivity . in a single plant very large number of individual glycosides may result based on the type and number of sugar residues , together with chain branching .
this means that both the biosynthesis of the different aglycones and their conjugation with different sugars are controlled by the genotype .
another comprehensive survey of flavonol content in 100 vegetables and fruits grown in china reported that quercetin was widely distributed in edible plants species .
explored the bioavailability of quercetin from red wine , yellow onions , and black tea and came to the conclusion that tea is a rich source of flavonols .
quercetin and kaempferol rhamnodiglucosides are characteristic compounds of camellia sinensis . according to finger et al .
, black tea contains 00.95  mg  g quercetin rhamnodiglucoside and 0.051.25  mg  g kaempferol rhamnodiglucoside .
however myricetin and kaempferol were also detected in many foods including tea [ 1 , 46 , 48 , 49 ] .
based on the records available , less than 6% of the total accessions preserved in sri lankan tea germplasm have been utilized in the tea breeding programmes till 1998 , and utilization of tea accessions has increased up to 13.6% by the end of 2009 .
the lack of information regarding the genetic variation , mainly biochemical and molecular diversity of the existing population , could be one reason for underutilization of the germplasm . according to present study ,
the lowest mean value of kaempferol content is observed for assam introductions ( figures 2(a)2(d ) ) .
the ranges and mean values of quercetin and myricetin for full - sib and half - sib families are much similar to the assam introductions .
therefore , it is indicated that the tri developed accessions have direct or indirect relationship with assam introductions . among the selected accessions , all exotic accessions recorded higher contents of quercetin than rest .
besides , the highest mean values of total flavonol contents were reported for exotic accessions .
these results revealed that those exotic accessions are having different geographic origin than other accessions present in germplasm in sri lanka .
lasiocalyx followed by assamica and sinensis varieties . around 83% of the introductions acquired prior to the 1960s , mainly from india and indo - china , exhibit predominant cambod type ( ssp .
lasiocalyx ) characters and introductions made in 21st century from korea reminiscent of more affinity to china type ( var .
therefore , flavonols and their glycosides can be useful to chemotaxonomic studies of tea germplasm .
as evident from the present work , the 9 exotic accessions with different geographic origin selected for this study along with the nonbeverage type accessions , especially camellia rosaflora and camellia japonica red , are worthwhile and thus merit focus in future germplasm studies .
the contents of myricetin and myricetin glycosides are less in black tea ; unlikely , in fresh tea , they flush since they can still be affected by tea processing .
myricetin and myricetin glycosides are the most likely of the three flavonols to be oxidised . however
therefore it is crucial to focus on myricetin while discussing the flavonol content of tea .
significant amounts of myricetin , quercetin , and kaempferol have been quantified in the beverage type tea accessions of the sri lankan tea germplasm . the var .
lasiocalyx ; therefore flavonols and their glycosides can be useful to chemotaxonomic studies of tea germplasm .
the nonbeverage type cultivars , especially camellia rosaflora and camellia japonica red along with the exotic accessions resembling china type , are worthwhile in future germplasm studies because they are rich sources of flavonols , namely , quercetin and kaempferol , which are potent antioxidants and health - promoting aspects .
additionally , the flavonol profiles can be effectively used in choosing accessions in tea breeding programmes to generate progenies with wide variations .
 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 . 
1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusions