Research Article Genetic Variation of Flavonols Quercetin ...

Research ArticleGenetic Variation of Flavonols Quercetin Myricetin andKaempferol in the Sri Lankan Tea (Camellia sinensis L) andTheir Health-Promoting Aspects

Brasathe Jeganathan1 P A Nimal Punyasiri2 J Dananjaya Kottawa-Arachchi3

Mahasen A B Ranatunga3 I Sarath B Abeysinghe3

M T Kumudini Gunasekare4 and B M Ratnayake Bandara5

1Department of Food Science and Technology Faculty of Agriculture University of Peradeniya Peradeniya Sri Lanka2Institute of Biochemistry Molecular Biology and Biotechnology University of Colombo 00300 Colombo Sri Lanka3Tea Research Institute of Sri Lanka Talawakelle Sri Lanka4Coordinating Secretariat for Science Technology amp Innovation 3rd Floor Standard Charted BuildingJanadhipathi Mawatha 00100 Colombo Sri Lanka5Department of Chemistry Faculty of Science University of Peradeniya Peradeniya Sri Lanka

Correspondence should be addressed to P A Nimal Punyasiri nimalibmbbcmbaclk

Received 27 March 2016 Accepted 9 May 2016

Academic Editor Rong Di

Copyright copy 2016 Brasathe Jeganathan et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

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

1 Introduction

Tea is the second most popular beverage after water whichrepresents a major source of dietary polyphenols [1] Thusthe scientific community is interested in exploring the health-promoting constituents present in tea namely flavan-3-olsflavonols and their derivatives [2] Recent epidemiologicalstudies have demonstrated a protective effect of fruits andvegetables against the incidence of degenerative diseases [3]Several classes of compounds have been assumed as potentialprotective factors one of which is the flavonoids generally

considered as nonnutritive agents Flavonoids are known assecondary metabolites which are the largest class of polyphe-nols widely distributed in the plant kingdom It can be subdi-vided into six major subclasses based on the structural vari-ations flavones flavanones isoflavones flavonols flavanolsand anthocyanidins [4]

According to the study conducted byHollman et al [5] teais a good source of flavonols Flavonols have recently receivedmuch attention due to their antioxidant antimicrobial anti-cancer antiatherosclerotic and antiproliferative properties[6ndash8] The flavonols exist in tea plant (Camellia sinensis L)

Hindawi Publishing CorporationInternational Journal of Food ScienceVolume 2016 Article ID 6057434 9 pageshttpdxdoiorg10115520166057434

2 International Journal of Food Science

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140

120

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(mAU

)

5 10 15 20

(min)

(1) (2)

(3)4579

8490

16479

(1) Myricetin(2) Quercetin(3) Kaempferol

DAD1 A Sig = 3704 Ref = 450100 (015-0501D)

(a)

(min)


120

100

80

60

40

20

0

(mAU

)

(1) (2)

(3)

5 10 15 20

(KQM_NBD_230113DAD1 A Sig = 3704 Ref = 450100KQM_NBD_220113 2013-01-23 12-21-38021-1101D)

1463

2036

2229

2615

3020

3294

3709

4487 8259

15967

(b)

Figure 1 (a) HPLC chromatogram of mixed standard solution comprising kaempferol quercetin and myricetin (b) Representative teaaccession quantified and resolved by HPLC

as flavonol glycosides with a sugar residue and themost com-mon flavonol aglycones in tea are quercetin kaempferol andmyricetin [9]

Ceylon tea from Sri Lanka acclaimed as the best tea in theworld has its inherent unique characteristics and reputationrunning over more than a century [10] The objectives of thisstudy were to investigate the variation in flavonol aglyconesnamely quercetin kaempferol andmyricetin in tea (Camelliasinensis L) accessions including beverage and nonbeveragetypes Therefore we report here the first systematic study onthe flavonol content in representative accessions of the SriLankan tea germplasm

2 Materials and Methods

21 Collection and Preparation of Tea Samples for Analysis50 g of tender tea shoots (two apical leaves and the bud) of 87beverage type and 6 nonbeverage type germplasm accessionswas collected in triplicate from the ex situ field gene bankof Tea Research Institute of Sri Lanka (TRI) Talawakelle(latitude 6∘541015840N longitude 80∘421015840E) and brought to thelaboratory at 4∘C Selection criteria for germplasm accessionsin this study included the attributes such as morphologicalvariations origins made tea quality and sensitivity to bioticstresses [11ndash13]The tea samples were immediately stored in aminus80∘C freezer for 6 h to two weeks and freeze-dried for 24 h(Labconco Corporation MS USA) The freeze-dried leaveswere ground to a fine powder (passed through 500120583mmesh)sealed in triple laminated aluminium foil packages andstored at 20∘C until biochemical analysis [14]

22 Extraction of Flavonols 100mg of freeze-dried tea sam-ple was hydrolysed in duplicate with 05mL of 6M HCl and45mL of 60 methanol for 2 h in a water bath preheated to95∘C Hydrolysed sample extracts were topped up to 5mL

with 60methanol and filtered through 045120583mnylon filtersinto HPLC vials

23 Preparation of Standards and Calibration Curves Stan-dards of myricetin quercetin and kaempferol (Sigma StLouis USA) were prepared in 60 methanol Calibrationcurves were constructed for the individual compounds Peakidentity was also confirmed by the photodiode array UVspectra

24 Quantification of Flavonols Analysis of individualflavonols was carried out in the Agilent 1260 Infinity HPLCsystem comprising of solvent delivery system quaternarypump temperature-controlled column oven autosamplerdegasser unit and a photodiode array detector withUVdetec-tion at 370 nm and Open LabChemstation Software systemThe compounds were separated on a 46 times 250mm 5 120583mparticle and reversed phase ZORBAX Eclipse Plus C 18 col-umn (Agilent USA) (Phenomenex Inc USA) with a guardcolumn made of the same material (Security-Guard Phe-nomenex Inc USA) at 30∘C 30HPLC grade acetonitrile in0025M potassium dihydrogen orthophosphate buffer solu-tion adjusted to a pH of 25 with 6MHCl was used as mobilephase at a flow rate of 10mLmin The injection volumewas 10 120583L Quantification was carried out from the integratedpeak areas of the sample against the corresponding standardgraph and results were expressed on drymatter basis (Figures1(a) and 1(b)) The dry matter contents of the freeze-driedleaves were determined in accordance with ISO15731980

25 Statistical Analysis The results were expressed asmean plusmnSD of triplicate data Experimental data were analysed usingSAS system forWindows V 91 (SAS Institute Inc NC USA)

International Journal of Food Science 3

26 Reagents Absolute methanol (analytical reagent grade)anhydrous sodium carbonate and acetonitrile (HPLC gradi-ent grade) were supplied by Fisher Scientific UK quercetinkaempferol and myricetin were purchased from SigmaChemicals and potassium dihydrogen orthophosphate andhydrochloric acid (analytical grade) were purchased fromBritish Drug House Ltd England

3 Results and Discussion

31 Flavonols Content of Beverage and Nonbeverage TypeAccessions The major flavonols present in tea leaves makeup 2-3 of the water-soluble solids in tea [15 16] Table 1shows the amounts of myricetin quercetin and kaempferolquantified in the beverage and nonbeverage type accessionsof the Sri Lankan tea germplasm These results are agreeableto a greater extend with the results reported for 3 commercialsamples of green tea and 30 commercial samples of black tea[17] and those reported for 4 commercial samples of greentea and 2 commercial samples of black tea [18] Accordingto the mean values of flavonols quercetin (150mg gminus1) isabundantly present in the beverage type accessions of the SriLankan tea germplasm followed by kaempferol (131mg gminus1)and myricetin (094mg gminus1) The mean value of total flavon-ols in 89 beverage type C sinensis was 375mg gminus1

Among beverage type accessions the highest quercetincontents were reported in the exotic accessions PBGT41(323mg gminus1) followed by PBGT73 China PBGT70and PBGT12 whereas TRI1114 showed the lowest content(036mg gminus1) The highest kaempferol contents wereobserved in the exotic accessions PBGT61 (221mg gminus1)followed by PBGT68 PBGT48 PBGT49 and PBGT70Among the 9 exotic accessions examined PBGT41 reportedthe highest myricetin content (163mg gminus1) followed byPBGT49 PBGT68 PBGT70 and PBGT61 and INTRI6recorded the lowest content (033mg gminus1)

Conversely myricetin was available in trace amountsrecorded in the nonbeverage type accessions except Camelliasasanqua (037mg gminus1) and Camellia rosaflora (020mg gminus1)Highest kaempferol contents were observed in Camelliajaponica donkelarri and Camellia japonica Red Camel-lia rosaflora has reported the highest quercetin content(344mg gminus1) when considering the entire germplasm whichis higher than that of the beverage type exotic cultivarPBGT41 This special characteristic can be utilized in futuregermplasm studies

The specific patterns of flavonol occurrence in plantspecies vary based on their synthesis and distribution in theplant organism [19] In addition to intrinsic factors flavonolcontent is strongly influenced by extrinsic factors such asvariations in plant type and growth season climate degree ofmaturity preparation and processing [20ndash24]

Several studies report varying content of flavonol in teas[1 25 26] This is possibly due to the difference in teasamples used (fresh tea leaves or processed tea) and analyticalmethods utilized resulting in different extraction efficienciesAccording to Finger et al [27] flavonol glycosides arenot affected by polyphenol oxidase and varying content in

different tea types can be due to difference in tea varietiesgeographic location or agricultural conditions

32 Health Aspects of Tea Flavonols The increasing knowl-edge on functional ingredients in food provides new oppor-tunities for plant sciences to produce crops with higheramounts of selected health-promoting phytochemicals usingcrop husbandry techniques plant breeding techniques andgenetic engineering [28] Recent studies suggest that higherintakes of flavonoids from food are associated with reducedrisk of degenerative diseases including cancer and heartdisease [29 30]

The three flavonols that is myricetin quercetin andkaempferol quantified in this study have also been exten-sively investigated for their health benefits Flavonols nat-urally occur as either glycosides (with attached glycosylgroups) or aglycones However dietary intake of the aboveflavonols is mainly in the glycoside form During digestionand absorption sugarmoieties can be released from the glyco-sides Digestive tractmicrobiota could also assist in the diges-tion and the absorption process of the flavonols Followingabsorption several organs contribute to flavonol metabolismincluding the liver giving rise to glucuronidated methylatedand sulfated forms of flavonolswhich are found in the plasmaFurther free flavonols (aglycone) are also found in theplasmaThese circulating flavonols and theirmetabolites havediverse biological activities [31]

Several mechanisms of actions such as antioxidant activ-ity anti-inflammatory activity and modulation of signalingpathways are responsible for the beneficial effects of flavonols[32]

Prolonged oxidative stress could lead to several degen-erative diseases including cardiovascular disease Ability ofhydrogen radical donation from the phenolic group and thepresence of an unpaired electron in the aromatic ring makethe flavonols strong antioxidants in vivo thus scavenging thereactive oxygen species and other free radicals Ability tochelate metal ions also reduces the production of harmfuloxidant species in vivo [33] Flavonols could contribute to theantioxidant properties through the protection or improve-ment of endogenous antioxidants especially by stimulatingglutathione-S-transferase (GST) an enzyme that protectscells from the damage caused by free radicals [34]

The inflammatory response is part of the innate immuneresponse which triggers the inflammatory cascade forminginflammatory mediators such as prostaglandin and throm-boxane Although acute inflammation is essential in dealingwith invading pathogens and injury chronic inflammationcan cause tissue destruction and is involved in the patho-genesis of degenerative diseases Commercially availableanti-inflammatory drugs are used to prevent the forma-tion of prostaglandins and thromboxanes by inhibiting thecyclooxygenase (COX) enzymes involved in the productionof the above during inflammation Flavonoids can suppressthe expression of the COX gene through interactionswith cellsignaling pathways such as the protein kinase C NF-120581B andtyrosine kinase pathways [35]

It has been reported that flavonols including myricetinquercetin and kaempferol could play an important role in


Table 1 Flavonol content of Sri Lankan tea accessions (all units are in mg gminus1 data represent the mean of six replicates plusmn standard error)

Accessions Myricetin (mg gminus1) Quercetin (mg gminus1) Kaempferol (mg gminus1) Total flavonols (mg gminus1)Estate selections

1 B275 054 plusmn 008 085 plusmn 006 090 plusmn 006 229 plusmn 0192 CH13 092 plusmn 010 111 plusmn 011 219 plusmn 012 422 plusmn 0693 CV4B1 056 plusmn 006 091 plusmn 008 158 plusmn 023 305 plusmn 0524 CY9 168 plusmn 017 169 plusmn 011 170 plusmn 016 507 plusmn 0015 DEL40 046 plusmn 002 137 plusmn 001 148 plusmn 006 331 plusmn 0566 DG39 108 plusmn 012 209 plusmn 011 134 plusmn 018 451 plusmn 0537 DG7 069 plusmn 018 043 plusmn 004 105 plusmn 009 217 plusmn 0318 DK24 075 plusmn 001 242 plusmn 011 133 plusmn 009 450 plusmn 0859 DN 085 plusmn 032 117 plusmn 030 073 plusmn 011 275 plusmn 02310 DT1 109 plusmn 004 204 plusmn 018 060 plusmn 005 374 plusmn 07311 DT95 088 plusmn 011 092 plusmn 012 269 plusmn 009 449 plusmn 10312 DUN7 099 plusmn 006 190 plusmn 052 143 plusmn 008 433 plusmn 04613 H158 045 plusmn 004 071 plusmn 004 124 plusmn 015 241 plusmn 04014 HS10A 081 plusmn 011 107 plusmn 002 151 plusmn 029 339 plusmn 03515 K145 108 plusmn 007 129 plusmn 051 148 plusmn 079 386 plusmn 02016 KEN163 092 plusmn 008 078 plusmn 016 146 plusmn 015 316 plusmn 03617 KP204 122 plusmn 017 243 plusmn 007 084 plusmn 010 450 plusmn 08318 MO241 060 plusmn 003 102 plusmn 013 147 plusmn 010 308 plusmn 04419 MT18 185 plusmn 095 176 plusmn 004 119 plusmn 004 481 plusmn 03620 N2 112 plusmn 012 169 plusmn 009 106 plusmn 021 387 plusmn 03521 NAY3 152 plusmn 005 201 plusmn 005 122 plusmn 010 476 plusmn 04022 NL83 135 plusmn 008 171 plusmn 007 073 plusmn 028 379 plusmn 05023 PK2 131 plusmn 018 196 plusmn 017 098 plusmn 015 425 plusmn 04924 PLLG2 074 plusmn 011 106 plusmn 012 192 plusmn 003 372 plusmn 06125 PO26 050 plusmn 007 063 plusmn 002 136 plusmn 010 248 plusmn 04626 QT44 110 plusmn 003 114 plusmn 003 146 plusmn 002 369 plusmn 02027 S106 091 plusmn 001 073 plusmn 003 160 plusmn 002 324 plusmn 04628 TC10 086 plusmn 007 175 plusmn 009 173 plusmn 008 434 plusmn 05129 TC9 148 plusmn 014 168 plusmn 018 187 plusmn 002 503 plusmn 01930 TRI1114 064 plusmn 006 036 plusmn 010 045 plusmn 003 145 plusmn 01431 TRI1294 093 plusmn 007 140 plusmn 025 210 plusmn 044 443 plusmn 05932 TRI2142 078 plusmn 004 167 plusmn 022 134 plusmn 023 379 plusmn 04533 TRI26 106 plusmn 006 153 plusmn 015 214 plusmn 029 473 plusmn 05434 TRI3011 067 plusmn 013 125 plusmn 007 069 plusmn 013 260 plusmn 03335 W3 079 plusmn 010 098 plusmn 022 146 plusmn 021 323 plusmn 03536 WT26 070 plusmn 018 085 plusmn 002 126 plusmn 012 281 plusmn 02937 TRI2016 061 plusmn 008 055 plusmn 007 067 plusmn 009 184 plusmn 006

Introductions from Assam38 ASM410 076 plusmn 006 108 plusmn 013 101 plusmn 006 285 plusmn 01739 TRI2022 181 plusmn 010 212 plusmn 053 063 plusmn 010 456 plusmn 07940 TRI2023 135 plusmn 005 159 plusmn 026 079 plusmn 009 374 plusmn 04141 TRI2024 068 plusmn 008 063 plusmn 005 109 plusmn 020 240 plusmn 02542 TRI2025 040 plusmn 004 059 plusmn 015 099 plusmn 006 199 plusmn 03043 TRI2026 079 plusmn 017 094 plusmn 007 087 plusmn 011 260 plusmn 00844 TRI3047 059 plusmn 002 130 plusmn 059 083 plusmn 014 273 plusmn 03645 TRI3055 105 plusmn 006 242 plusmn 015 107 plusmn 006 454 plusmn 07946 TRI625 067 plusmn 006 103 plusmn 001 087 plusmn 016 257 plusmn 01847 TRI629 044 plusmn 004 040 plusmn 005 018 plusmn 003 102 plusmn 014

Introductions from Indo-China48 TRI777 065 plusmn 005 175 plusmn 013 134 plusmn 023 374 plusmn 05649 TRI2043 083 plusmn 005 150 plusmn 064 089 plusmn 002 321 plusmn 038


Table 1 Continued

Accessions Myricetin (mg gminus1) Quercetin (mg gminus1) Kaempferol (mg gminus1) Total flavonols (mg gminus1)Unknown introductions

50 INTRI6 033 plusmn 004 041 plusmn 007 114 plusmn 013 187 plusmn 04551 VHMOR 091 plusmn 015 152 plusmn 007 164 plusmn 021 407 plusmn 039

Half-sib family52 TRI3013 048 plusmn 004 075 plusmn 011 081 plusmn 012 205 plusmn 01853 TRI3014 082 plusmn 004 222 plusmn 003 184 plusmn 004 488 plusmn 07254 TRI3015 116 plusmn 008 164 plusmn 005 139 plusmn 003 419 plusmn 02455 TRI3041 128 plusmn 006 233 plusmn 036 216 plusmn 053 576 plusmn 05656 TRI3050 134 plusmn 010 184 plusmn 012 144 plusmn 002 461 plusmn 02657 TRI3072 088 plusmn 009 151 plusmn 025 130 plusmn 007 369 plusmn 03258 TRI3073 079 plusmn 008 151 plusmn 021 192 plusmn 038 422 plusmn 05759 TRI3045 092 plusmn 018 084 plusmn 012 108 plusmn 019 284 plusmn 013

Full-sib family60 TRI3016 059 plusmn 006 215 plusmn 018 167 plusmn 025 441 plusmn 08061 TRI3017 096 plusmn 006 204 plusmn 014 247 plusmn 033 547 plusmn 07862 TRI3018 081 plusmn 009 171 plusmn 002 116 plusmn 010 368 plusmn 04563 TRI3019 109 plusmn 010 192 plusmn 028 113 plusmn 007 415 plusmn 04764 TRI3026 063 plusmn 002 081 plusmn 006 083 plusmn 007 227 plusmn 01165 TRI3036 040 plusmn 005 089 plusmn 021 120 plusmn 016 248 plusmn 04066 TRI4004 093 plusmn 010 200 plusmn 004 226 plusmn 003 518 plusmn 07167 TRI4049 084 plusmn 007 123 plusmn 027 139 plusmn 013 345 plusmn 02868 TRI4052 078 plusmn 012 114 plusmn 006 147 plusmn 020 339 plusmn 03569 TRI4053 045 plusmn 003 094 plusmn 011 093 plusmn 010 232 plusmn 02870 TRI4061 170 plusmn 005 227 plusmn 031 224 plusmn 012 621 plusmn 03271 TRI4063 063 plusmn 004 142 plusmn 004 127 plusmn 022 332 plusmn 04272 TRI4067 098 plusmn 019 102 plusmn 022 126 plusmn 007 326 plusmn 01573 TRI4068 078 plusmn 005 064 plusmn 018 113 plusmn 010 255 plusmn 02674 TRI4071 076 plusmn 012 110 plusmn 017 130 plusmn 013 316 plusmn 02875 TRI4076 162 plusmn 007 191 plusmn 012 074 plusmn 008 427 plusmn 06176 TRI4078 100 plusmn 008 133 plusmn 016 155 plusmn 004 388 plusmn 02877 TRI4079 091 plusmn 007 137 plusmn 008 116 plusmn 006 344 plusmn 02378 TRI4085 127 plusmn 009 115 plusmn 024 090 plusmn 006 331 plusmn 01979 TRI3022 148 plusmn 005 195 plusmn 006 219 plusmn 011 563 plusmn 036

Exotics types80 China 085 plusmn 010 298 plusmn 021 102 plusmn 012 485 plusmn 11981 PBGT61 134 plusmn 002 170 plusmn 003 221 plusmn 017 524 plusmn 04482 PBGT68 142 plusmn 008 239 plusmn 007 163 plusmn 006 544 plusmn 05183 PBGT48 131 plusmn 013 234 plusmn 010 141 plusmn 005 506 plusmn 05784 PBGT49 146 plusmn 014 241 plusmn 008 123 plusmn 004 510 plusmn 06385 PBGT12 115 plusmn 083 261 plusmn 028 105 plusmn 015 481 plusmn 08886 PBGT41 140 plusmn 056 323 plusmn 039 103 plusmn 008 566 plusmn 11787 PBGT70 139 plusmn 015 285 plusmn 015 121 plusmn 003 545 plusmn 09088 PBGT73 096 plusmn 007 305 plusmn 004 102 plusmn 009 503 plusmn 11989 Yabukita 111 plusmn 017 212 plusmn 003 194 plusmn 009 517 plusmn 054

Mean (119899 = 89) 094 150 131 375Nonbeverage types

90 C sasanqua 037 plusmn 005 159 plusmn 017 052 plusmn 003 248 plusmn 06791 C japonica donkelarri ND 113 plusmn 002 208 plusmn 006 321 plusmn 10492 C japonica herculea ND 196 plusmn 018 095 plusmn 009 291 plusmn 09893 C japonica Red ND 136 plusmn 007 202 plusmn 005 338 plusmn 10394 C japonicaWhite ND 170 plusmn 007 160 plusmn 006 331 plusmn 09695 C rosaflora 020 plusmn 000 344 plusmn 003 103 plusmn 008 467 plusmn 177

Mean (119899 = 6) 028 186 137 333ND not detected


Table 2 Flavonols profile of three varieties of C sinensis (all units are in mg gminus1 data represent the mean of six replicates plusmn standard error)

Compounds var sinensis (119899 = 10) var assamica (119899 = 19) ssp lasiocalyx (119899 = 60) Nonbeverage types (119899 = 6)Myricetin 107 plusmn 035 086 plusmn 026 096 plusmn 037 028 plusmn 002Quercetin 211 plusmn 072 144 plusmn 056 143 plusmn 064 186 plusmn 082Kaempferol 125 plusmn 025 143 plusmn 045 130 plusmn 052 137 plusmn 063Total flavonols 443 plusmn 097 373 plusmn 088 369 plusmn 118 329 plusmn 066

reducing the risk of cardiovascular disease cancer andneurodegradative and other degenerative diseases throughthe above-mentioned mechanisms and several other mech-anisms Several epidemiological studies also have found thebeneficial effects of flavonols [36 37] Therefore it could beargued that dietary items high in flavonols could be beneficialto human health

Although flavonol glycosides can be important contrib-utors to black tea flavour [38] these were hydrolysed byacid treatment to affordmyricetin quercetin and kaempferolfor HPLC analysis due to the fact that the dietary forms offlavonol glycosides are initially hydrolysed to aglycones atthe oral cavity [39ndash42] and continued in the digestive tract[5 43] Even during the fermentation processes the flavonoidglycosides undergo enzymatic hydrolysis and the level offlavonoid aglycones increase However the chemical form offlavonol prior to and after digestion is of great importance indetermining the bioavailability and bioactivity

33 Utilization of Flavonol Diversity in Tea Breeding In asingle plant very large number of individual glycosides mayresult based on the type and number of sugar residuestogether with chain branching Some of these flavonols werequite useful for discriminating genotypes in different speciesVrhovsek et al [44] reported that myricetin glycosides hadsome significant level of variability among the blueberry cul-tivars This means that both the biosynthesis of the differentaglycones and their conjugation with different sugars arecontrolled by the genotype Another comprehensive survey offlavonol content in 100 vegetables and fruits grown in Chinareported that quercetin was widely distributed in edibleplants species [45]

Flavonol quercetin was the main compound found inmany fruits and vegetables [46] de Vries et al [47] exploredthe bioavailability of quercetin from red wine yellowonions and black tea and came to the conclusion that teais a rich source of flavonols Quercetin and kaempferolrhamnodiglucosides are characteristic compounds of Camel-lia sinensis According to Finger et al [27] black teacontains 0ndash095mg gminus1 quercetin rhamnodiglucoside and005ndash125mg gminus1 kaempferol rhamnodiglucoside Howevermyricetin and kaempferol were also detected in many foodsincluding tea [1 46 48 49]

Based on the records available less than 6 of the totalaccessions preserved in Sri Lankan tea germplasm havebeen utilized in the tea breeding programmes till 1998 andutilization of tea accessions has increased up to 136 by

the end of 2009 [50] The lack of information regardingthe genetic variation mainly biochemical and moleculardiversity of the existing population could be one reason forunderutilization of the germplasm [12]

According to present study the lowest mean value ofkaempferol content is observed for Assam introductions(Figures 2(a)ndash2(d))The ranges andmean values of quercetinand myricetin for full-sib and half-sib families are muchsimilar to the Assam introductions Therefore it is indicatedthat the TRI developed accessions have direct or indirectrelationship with Assam introductions Among the selectedaccessions all exotic accessions recorded higher contentsof quercetin than rest Besides the highest mean values oftotal flavonol contents were reported for exotic accessionsThese results revealed that those exotic accessions are havingdifferent geographic origin than other accessions present ingermplasm in Sri Lanka

34 Variations in Flavonols among Varieties of C sinensisAverage values of the flavonols of China type (var sinensis)Assam type (var assamica) and Cambod type (ssp lasio-calyx) of C sinensis (L) are given in Table 2 Flavonolsmyricetin and quercetin contents of the var sinensis werehigher than that of the var assamica and ssp lasiocalyxBeside var assamica had the highest kaempferol content thanother varieties and nonbeverage types Among all three vari-eties and nonbeverage types var sinensis showed the highesttotal flavonols Sri Lankan germplasm collection was pre-dominantly represented by ssp lasiocalyx followed by assam-ica and sinensis varieties Around 83 of the introductionsacquired prior to the 1960s mainly from India and Indo-China exhibit predominant Cambod type (ssp lasiocalyx)characters and introductions made in 21st century fromKorea reminiscent of more affinity to China type (var sinen-sis) However China type accessions are inadequately repre-sented in the collection [51] Therefore flavonols and theirglycosides can be useful to chemotaxonomic studies of teagermplasm

As evident from the present work the 9 exotic accessionswith different geographic origin selected for this study alongwith the nonbeverage type accessions especially Camelliarosaflora and Camellia japonica Red are worthwhile andthus merit focus in future germplasm studies The contentsof myricetin and myricetin glycosides are less in black teaunlikely in fresh tea they flush since they can still be affectedby tea processing Myricetin and myricetin glycosides are themost likely of the three flavonols to be oxidised [52] Howeverthe presence of myricetin is higher in tea compared to other


ES IA IC IU HS FS EX NB

MyricetinC

once

ntra

tion

(mg

g)

00

04

02

06

08

10

12

14

16

18

20

(a)ES IA IC IU HS FS EX NB

Con

cent

ratio

n (m

gg)

Kaempferol

00

06

03

09

12

15

18

21

24

27

30

(b)


Con

cent

ratio

n (m

gg)

Quercetin40

36

32

28

24

20

16

12

08

04

00

(c)

Con

cent

ratio

n (m

gg)

Total flavonols

ES IA IC IU HS FS EX NB12

24

18

30

36

42

48

54

60

66

(d)

Figure 2 Distribution of total and individual flavonols in cultivated Camellia sinensis varieties and nonbeverage types (ES estate selectionsIA introductions from Assam IC introductions from Indo-China IU unknown introductions HS half-sib families FS full-sib familiesEX exotic varieties NB nonbeverage types and e indicates outliers)

herbal plants [18]Therefore it is crucial to focus onmyricetinwhile discussing the flavonol content of tea

4 Conclusions

Significant amounts of myricetin quercetin and kaempferolhave been quantified in the beverage type tea accessionsof the Sri Lankan tea germplasm The var sinensis showedhigher content of myricetin quercetin and total flavonolthan var assamica and ssp Lasiocalyx therefore flavonolsand their glycosides can be useful to chemotaxonomic studiesof tea germplasm The nonbeverage type cultivars especiallyCamellia rosaflora and Camellia japonica Red along withthe exotic accessions resembling China type are worthwhilein future germplasm studies because they are rich sourcesof flavonols namely quercetin and kaempferol which arepotent antioxidants and health-promoting aspects Addition-ally the flavonol profiles can be effectively used in choosing

accessions in tea breeding programmes to generate progenieswith wide variations

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the National Research Councilof Sri Lanka (Grant no NRC11023)

References

[1] M G L Hertog P C H Hollman and B Van de PutteldquoContent of potentially anticareinogenic flavonoids of tea infu-sions wines and fruit juicesrdquo Journal of Agricultural and FoodChemistry vol 41 no 8 pp 1242ndash1246 1993


[2] H M Merken and G R Beecher ldquoLiquid chromatographicmethod for the separation and quantification of prominentflavonoid aglyconesrdquo Journal of Chromatography A vol 897 no1-2 pp 177ndash184 2000

[3] N J Temple and K K Gladwin ldquoFruit vegetables and the pre-vention of cancer research challengesrdquo Nutrition vol 19 no 5pp 467ndash470 2003

[4] C A Rice-Evans and N J Miller ldquoStructure-antioxidant activ-ity relationships of flavonoids and isoflavonoidsrdquo in Flavonoidsin Health and Disease C A Rice-Evans and L Parker Eds pp199ndash219 Marcel Dekker New York NY USA 1998

[5] P C H Hollman J H M de Vries S D van Leeuwen M J BMengelers and M B Katan ldquoAbsorption of dietary quercetinglycosides and quercetin in healthy ileostomy volunteersrdquo TheAmerican Journal of Clinical Nutrition vol 62 no 6 pp 1276ndash1282 1995

[6] I E Dreosti M J Wargovich and C S Yang ldquoInhibition ofcarcinogenesis by tea the evidence from experimental studiesrdquoCritical Reviews in Food Science and Nutrition vol 37 no 8 pp761ndash770 1997

[7] L Jie D Lingliang W Chunyan and X Hairong ldquoFlavonolsin tea (Camellia sinensis) and its impact on sensory qualityrdquoJournal of Tea vol 36 no 1 pp 14ndash18 2010

[8] S A Wiseman D A Balentine and B Frei ldquoAntioxidants inteardquoCritical Reviews in Food Science andNutrition vol 37 no 8pp 705ndash718 1997

[9] L H Yao Y M Jiang J Shi et al ldquoFlavonoids in food and theirhealth benefitsrdquo Plant Foods for Human Nutrition vol 59 no 3pp 113ndash122 2004

[10] WW DModder and AM T Amarakoon Tea and Health TeaResearch Institute Talawakelle Sri Lanka 2002

[11] J D Kottawa-Arachchi M T K Gunasekare M A B Rana-tunga P A N Punyasiri L Jayasinghe and R P KarunagodaldquoBiochemical characteristics of tea (Camellia L spp) germ-plasm accessions in Sri Lanka correlation between black teaquality parameters and organoleptic evaluationrdquo InternationalJournal of Tea Science vol 10 no 1 pp 3ndash13 2014

[12] J D Kottawa-Arachchi M T K Gunasekare M A BRanatunga P A N Punyasiri and L Jayasinghe ldquoUse of bio-chemical compounds in tea germplasm characterization and itsimplications in tea breeding in Sri Lankardquo Journal of theNational Science Foundation of Sri Lanka vol 41 no 4 pp 309ndash318 2013

[13] J H N Piyasundara M T K Gunasekare and I PWickramas-inghe ldquoCharacterization of tea (Camellia sinensisL) germplasmin Sri Lanka using morphological descriptorsrdquo Sri Lanka Jour-nal of Tea Science vol 74 no 1 pp 31ndash39 2009

[14] P A N Punyasiri B Jeganathan J D Kottawa-Arachchi et alldquoNew sample preparationmethod for quantification of phenoliccompounds of tea (Camellia sinensis L Kuntze) a polyphenolrich plantrdquo Journal of AnalyticalMethods inChemistry vol 2015Article ID 964341 6 pages 2015

[15] Q K Cheng and Z M Chen Tea and Health Press of ChineseAgricultural Sciences Beijing China 1994

[16] D A Balentine S A Wiseman and L C M Bouwens ldquoThechemistry of tea flavonoidsrdquo Critical Reviews in Food Scienceand Nutrition vol 37 no 8 pp 693ndash704 1997

[17] J Peterson J Dwyer S Bhagwat et al ldquoMajor flavonoids in dryteardquo Journal of Food Composition and Analysis vol 18 no 6 pp487ndash501 2005

[18] H Wang and K Helliwell ldquoDetermination of flavonols ingreen and black tea leaves and green tea infusions by high-performance liquid chromatographyrdquo Food Research Interna-tional vol 34 no 2-3 pp 223ndash227 2001

[19] J Kuhnau ldquoThe flavonoids A class of semi-essential foodcomponents their role in human nutritionrdquo World Review ofNutrition And Dietetics vol 24 pp 117ndash191 1976

[20] M S McDonald M Hughes J Burns M E J Lean DMatthews and A Crozier ldquoSurvey of the free and conjugatedmyricetin and quercetin content of red wines of different geo-graphical originsrdquo Journal of Agricultural and Food Chemistryvol 46 no 2 pp 368ndash375 1998

[21] A Trichopoulou E Vasilopoulou P Hollman et al ldquoNutri-tional composition and flavonoid content of edible wild greensand green pies a potential rich source of antioxidant nutrientsin the Mediterranean dietrdquo Food Chemistry vol 70 no 3 pp319ndash323 2000

[22] C Lakenbrink S Lapczynski B Maiwald and U H Engel-hardt ldquoFlavonoids and other polyphenols in consumer brewsof tea and other caffeinated beveragesrdquo Journal of Agriculturaland Food Chemistry vol 48 no 7 pp 2848ndash2852 2000

[23] A J Stewart S Bozonnet W Mullen G I Jenkins M E JLean and A Crozier ldquoOccurrence of flavonols in tomatoesand tomato-based productsrdquo Journal of Agricultural and FoodChemistry vol 48 no 7 pp 2663ndash2669 2000

[24] C Ewald S Fjelkner-Modig K Johansson I Sjoholm and BAkesson ldquoEffect of processing onmajor flavonoids in processedonions green beans and peasrdquo Food Chemistry vol 64 no 2pp 231ndash235 1999

[25] U Justesen P Knuthsen and T Leth ldquoQuantitative analysis offlavonols flavones and flavanones in fruits vegetables andbeverages by high-performance liquid chromatography withphoto-diode array and mass spectrometric detectionrdquo Journalof Chromatography A vol 799 no 1-2 pp 101ndash110 1998

[26] P Mattila J Astola and J Kumpulainen ldquoDetermination offlavonoids in plant material by HPLC with diode-array andelectro-array detectionsrdquo Journal of Agricultural and FoodChemistry vol 48 no 12 pp 5834ndash5841 2000

[27] A Finger U H Engelhardt and V Wray ldquoFlavonol glycosidesin teamdashkaempferol and quercetin rhamnodiglucosidesrdquo Journalof the Science of Food and Agriculture vol 55 no 2 pp 313ndash3211991

[28] T P Mikkonen K R Maatta A T Hukkanen et al ldquoFlavonolcontent varies among black currant cultivarsrdquo Journal of Agri-cultural and Food Chemistry vol 49 no 7 pp 3274ndash3277 2001

[29] A Rodriguez-Mateos D Vauzour C G Krueger et alldquoBioavailability bioactivity and impact on health of dietaryflavonoids and related compounds an updaterdquo Archives ofToxicology vol 88 no 10 pp 1803ndash1853 2014

[30] A Kozłowska and D Szostak-Wegierek ldquoFlavonoidsmdashfoodsources and health benefitsrdquo Roczniki Panstwowego ZakładuHigieny vol 65 no 2 pp 79ndash85 2014

[31] L Marın E M Miguelez C J Villar and F Lombo ldquoBioavail-ability of dietary polyphenols and gut microbiota metabolismantimicrobial propertiesrdquo BioMed Research International vol2015 Article ID 905215 18 pages 2015

[32] J HG Lago A C Toledo-ArrudaMMernak et al ldquoStructure-activity association of flavonoids in lung diseasesrdquo Moleculesvol 19 no 3 pp 3570ndash3595 2014

[33] M M Silva M R Santos G Caroco R Rocha G Justinoand L Mira ldquoStructure-antioxidant activity relationships of


flavonoids a re-examinationrdquo Free Radical Research vol 36 no11 pp 1219ndash1227 2002

[34] H Fiander and H Schneider ldquoDietary ortho phenols thatinduce glutathione S-transferase and increase the resistance ofcells to hydrogen peroxide are potential cancer chemopreven-tives that act by two mechanisms the alleviation of oxidativestress and the detoxification of mutagenic xenobioticsrdquo CancerLetters vol 156 no 2 pp 117ndash124 2000

[35] M L Ferrandiz and M J Alcaraz ldquoAnti-inflammatory activityand inhibition of arachidonic acid metabolism by flavonoidsrdquoAgents and Actions vol 32 no 3-4 pp 283ndash288 1991

[36] J J Peterson J T Dwyer P F Jacques and M L McCulloughldquoAssociations between flavonoids and cardiovascular diseaseincidence or mortality in European and US populationsrdquoNutrition Reviews vol 70 no 9 pp 491ndash508 2012

[37] D F Romagnolo and O I Selmin ldquoFlavonoids and cancerprevention a review of the evidencerdquo Journal of Nutrition inGerontology and Geriatrics vol 31 no 3 pp 206ndash238 2012

[38] S Scharbert and T Hofmann ldquoMolecular definition of blacktea taste by means of quantitative studies taste reconstitutionand omission experimentsrdquo Journal of Agricultural and FoodChemistry vol 53 no 13 pp 5377ndash5384 2005

[39] I A Macdonald J A Mader and R G Bussard ldquoThe role ofrutin and quercitrin in stimulating flavonol glycosidase activityby cultured cell-free microbial preparations of human feces andsalivardquo Mutation Research Letters vol 122 no 2 pp 95ndash1021983

[40] A Laires P Pacheco and J Rueff ldquoMutagenicity of rutin and theglycosidic activity of cultured cell-freemicrobial preparations ofhuman faeces and salivardquo Food and Chemical Toxicology vol 27no 7 pp 437ndash443 1989

[41] S Hirota T Nishioka T Shimoda K Miura T Ansai and UTakahama ldquoQuercetin glucosides are hydrolyzed to quercetinin human oral cavity to participate in peroxidase-dependentscavenging of hydrogen peroxiderdquo Food Science and TechnologyResearch vol 7 no 3 pp 239ndash245 2001

[42] T Walle A M Browning L L Steed S G Reed and U KWalle ldquoFlavonoid glucosides are hydrolyzed and thus activatedin the oral cavity in humansrdquoThe Journal of Nutrition vol 135no 1 pp 48ndash52 2005

[43] VD Bokkenheuser CH L Shackleton and JWinter ldquoHydrol-ysis of dietary flavonoid glycosides by strains of intestinalBacteroides from humansrdquo Biochemical Journal vol 248 no 3pp 953ndash956 1987

[44] U Vrhovsek D Masuero L Palmieri and F Mattivi ldquoIdenti-fication and quantification of flavonol glycosides in cultivatedblueberry cultivarsrdquo Journal of Food Composition and Analysisvol 25 no 1 pp 9ndash16 2012

[45] J Cao W Chen Y Zhang Y Zhang and X Zhao ldquoContentof selected flavonoids in 100 edible vegetables and fruitsrdquo FoodScience and Technology Research vol 16 no 5 pp 395ndash4022010

[46] A Crozier M E J Lean M S McDonald and C BlackldquoQuantitative analysis of the flavonoid content of commercialtomatoes onions lettuce and celeryrdquo Journal of Agricultural andFood Chemistry vol 45 no 3 pp 590ndash595 1997

[47] J H M de Vries P C H Hollman I van Amersfoort M ROlthof andM B Katan ldquoRed wine is a poor source of bioavail-able flavonols in menrdquo Journal of Nutrition vol 131 no 3 pp745ndash748 2001

[48] M G L Hertog P C H Hollman and D P Venema ldquoOpti-mization of a quantitative HPLC determination of potentially

anticarcinogenic flavonoids in vegetables and fruitsrdquo Journal ofAgricultural and Food Chemistry vol 40 no 9 pp 1591ndash15981992

[49] M G L Hertog P C H Hollman and M B Katan ldquoContentof potentially anticarcinogenic flavonoids of 28 vegetables and9 fruits commonly consumed in the Netherlandsrdquo Journal ofAgricultural and Food Chemistry vol 40 no 12 pp 2379ndash23831992

[50] M T K Gunasekare M A B Ranatunga J H N Piyasundaraand J D Kottawa-Arachchi ldquoTea genetic resources in Sri Lankacollection conservation and appraisalrdquo International Journal ofTea Science vol 8 no 3 pp 51ndash60 2012

[51] M A B Ranatunga H G J Kumudumali J D KottawaArachchi M T K Gunasekere and D M D YakandawalaldquoGenetic structure of tea (Camellia sinensis (L) O Kuntze)germplasm in Sri Lanka implication on germplasm manage-ment and tea breedingrdquo in Proceedings of the Peradeniya Uni-versity International Research Sessions vol 18 Peradeniya SriLanka July 2014

[52] I McDowell R G Bailey and G Howard ldquoFlavonol glycosidesin black teardquo Journal of the Science of Food and Agriculture vol53 no 3 pp 411ndash414 1990

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


160

140

120

100

80

60

40

20

0

(mAU

)

5 10 15 20

(min)

(1) (2)

(3)4579

8490

16479


DAD1 A Sig = 3704 Ref = 450100 (015-0501D)

(a)

(min)


120

100

80

60

40

20

0

(mAU

)

(1) (2)

(3)

5 10 15 20

(KQM_NBD_230113DAD1 A Sig = 3704 Ref = 450100KQM_NBD_220113 2013-01-23 12-21-38021-1101D)

1463

2036

2229

2615

3020

3294

3709

4487 8259

15967

(b)

Figure 1 (a) HPLC chromatogram of mixed standard solution comprising kaempferol quercetin and myricetin (b) Representative teaaccession quantified and resolved by HPLC

as flavonol glycosides with a sugar residue and themost com-mon flavonol aglycones in tea are quercetin kaempferol andmyricetin [9]

Ceylon tea from Sri Lanka acclaimed as the best tea in theworld has its inherent unique characteristics and reputationrunning over more than a century [10] The objectives of thisstudy were to investigate the variation in flavonol aglyconesnamely quercetin kaempferol andmyricetin in tea (Camelliasinensis L) accessions including beverage and nonbeveragetypes Therefore we report here the first systematic study onthe flavonol content in representative accessions of the SriLankan tea germplasm

2 Materials and Methods

21 Collection and Preparation of Tea Samples for Analysis50 g of tender tea shoots (two apical leaves and the bud) of 87beverage type and 6 nonbeverage type germplasm accessionswas collected in triplicate from the ex situ field gene bankof Tea Research Institute of Sri Lanka (TRI) Talawakelle(latitude 6∘541015840N longitude 80∘421015840E) and brought to thelaboratory at 4∘C Selection criteria for germplasm accessionsin this study included the attributes such as morphologicalvariations origins made tea quality and sensitivity to bioticstresses [11ndash13]The tea samples were immediately stored in aminus80∘C freezer for 6 h to two weeks and freeze-dried for 24 h(Labconco Corporation MS USA) The freeze-dried leaveswere ground to a fine powder (passed through 500120583mmesh)sealed in triple laminated aluminium foil packages andstored at 20∘C until biochemical analysis [14]

22 Extraction of Flavonols 100mg of freeze-dried tea sam-ple was hydrolysed in duplicate with 05mL of 6M HCl and45mL of 60 methanol for 2 h in a water bath preheated to95∘C Hydrolysed sample extracts were topped up to 5mL

with 60methanol and filtered through 045120583mnylon filtersinto HPLC vials

23 Preparation of Standards and Calibration Curves Stan-dards of myricetin quercetin and kaempferol (Sigma StLouis USA) were prepared in 60 methanol Calibrationcurves were constructed for the individual compounds Peakidentity was also confirmed by the photodiode array UVspectra

24 Quantification of Flavonols Analysis of individualflavonols was carried out in the Agilent 1260 Infinity HPLCsystem comprising of solvent delivery system quaternarypump temperature-controlled column oven autosamplerdegasser unit and a photodiode array detector withUVdetec-tion at 370 nm and Open LabChemstation Software systemThe compounds were separated on a 46 times 250mm 5 120583mparticle and reversed phase ZORBAX Eclipse Plus C 18 col-umn (Agilent USA) (Phenomenex Inc USA) with a guardcolumn made of the same material (Security-Guard Phe-nomenex Inc USA) at 30∘C 30HPLC grade acetonitrile in0025M potassium dihydrogen orthophosphate buffer solu-tion adjusted to a pH of 25 with 6MHCl was used as mobilephase at a flow rate of 10mLmin The injection volumewas 10 120583L Quantification was carried out from the integratedpeak areas of the sample against the corresponding standardgraph and results were expressed on drymatter basis (Figures1(a) and 1(b)) The dry matter contents of the freeze-driedleaves were determined in accordance with ISO15731980

25 Statistical Analysis The results were expressed asmean plusmnSD of triplicate data Experimental data were analysed usingSAS system forWindows V 91 (SAS Institute Inc NC USA)























Table 1 Continued























MyricetinC

once

ntra

tion

(mg

g)

00

04

02

06

08

10

12

14

16

18

20


Con

cent

ratio

n (m

gg)

Kaempferol

00

06

03

09

12

15

18

21

24

27

30

(b)


Con

cent

ratio

n (m

gg)

Quercetin40

36

32

28

24

20

16

12

08

04

00

(c)

Con

cent

ratio

n (m

gg)

Total flavonols


24

18

30

36

42

48

54

60

66

(d)



4 Conclusions



Competing Interests


Acknowledgments


References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology























Table 1 Continued























MyricetinC

once

ntra

tion

(mg

g)

00

04

02

06

08

10

12

14

16

18

20


Con

cent

ratio

n (m

gg)

Kaempferol

00

06

03

09

12

15

18

21

24

27

30

(b)


Con

cent

ratio

n (m

gg)

Quercetin40

36

32

28

24

20

16

12

08

04

00

(c)

Con

cent

ratio

n (m

gg)

Total flavonols


24

18

30

36

42

48

54

60

66

(d)



4 Conclusions



Competing Interests


Acknowledgments


References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















MyricetinC

once

ntra

tion

(mg

g)

00

04

02

06

08

10

12

14

16

18

20


Con

cent

ratio

n (m

gg)

Kaempferol

00

06

03

09

12

15

18

21

24

27

30

(b)


Con

cent

ratio

n (m

gg)

Quercetin40

36

32

28

24

20

16

12

08

04

00

(c)

Con

cent

ratio

n (m

gg)

Total flavonols


24

18

30

36

42

48

54

60

66

(d)



4 Conclusions



Competing Interests


Acknowledgments


References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Research Article Genetic Variation of Flavonols Quercetin ...

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Transcript of Research Article Genetic Variation of Flavonols Quercetin ...