The Flavonoid Composition And Coloration Mechanism In Water Lily Petals

Water lily,an herb of genus Nymphaea,the family Nymphaeaceae,is a precious perennial aquatic flower plant and divides into two ecological groups,namely,Tropical and Hardy water lily.In this study,the petals of 35 and 119 cultivars of tropical and hardy groups were collected,respecitvely.After the color measurement,the composition and content of the flavonoids in the petals were investigated using high performance liquid chromatography with diode array detector(HPLC-DAD)and HPLC electrospray ionization multi-stage mass spectrometry(HPLC-ESI-MSn).The differences of the flavonoid composition among cultivars of tropical and hardy water lily were analyzed.The chemical coloration mechanism of water lily color was also discussed.The main research results were as follows:1.The distribution of water lily flower colorsIn the coordinates of chromatic components a*and b*,the distribution of hardy water lily flower colors starts from the second quadrant of yellow area,astride the first quadrant,most cultivars colors mainly distribute in the fourth quadrant of pink and red area,which closing to a*coordinate,and no colors distribute in the third quadrant.Tropic water lily has the special blue and bluish violet colors,while,hardy water lily lacks.The colors of tropic water lily are rich and distribute in all quadrants,namely,the second quadrant consisting of yellow and white cultivars,the first quadrant including compound color;most pink,red,blue and bluish violet cultivars concentrating in the fourth quadrant and the white cultivar'He Hua Water Lily' distribute in the third quadrant.2.Identification of flavonoids in water lily petalsThe HPLC methods for analyzing the flavonoids in water lily petals were developed by optimizing analytical conditions,such as the solvent system,elution gradient,temperature and flow rate etc.Thirty four flavonoid components,including eleven anthocyanins,twenty two flavonol glycosides and one chalcone galactoside,were characterized in tropic water lily according to the standards or tentatively identified by using HPLC-MSn.Seventeen flavonoids of them were firstly reported in flowers of tropic water lily,including four anthocyanins:3-rhamnosyl-5-galactoside(3Rh5Ga),3-(2"-galloyl-6"-oxalyl-rhamnoside)(3galloyl-oxalylRh)and 3-(6"-acetyl-glucopyranoside)(3acetylG)of delphinidin(Dp)and 3-2"-galloyl-galactopyranoside)-5-rhamnoside(3galloylGa5Rh)of cyanidin(Cy);twelve flavonol glycosides:7-rhamnosyl-(1?2)-rhamnoside(7RhRh),3-galactoside(3Ga),3-galloylrhamnoside(3galloylRh)of myricetin(My);7-galactosyl-(1?2)-rhamnoside(7GaRh),7-galactoside(7Ga),3-acetylgalactoside(3acetylGa)of quercetin(Qu);7-galactosyl-(1?2)-rhamnoside(7GaRh),7-galloylgalactosyl-(1?2)-rhamnoside(7galloylGaRh),3-galactoside(3Ga),3-(3"-acetylrhamnoside)(3-3"acetylRh)of kaempferol(Km);7-galactoside(7Ga),7-xyloside(7Xy)of isorhamnetin(Is);one chalcone glycosides:2'-galactoside(2'Ga)of chalcononaringenin(Chal).Glycosides of fourteen anthocyanins,five flavones,fourty-six flavonols,two flavanones and eight chalcones,seventy five components in all were characterized in 119 hardy water lily,petals,in which six anthocyanins:3-galactopyranoside(3Ga),3-2"-galloyl-galactopyranoside(3galloylGa),3-6"-acetyl-galactopyranoside(3acetylGa),3-2"-galloyl-6"-acetyl-galactopyranoside(3galloyl-acetylGa)of delphinidin(Dp);3-6"-acetyl-galactopyranoside(3acetylGa),3-2"-galloyl-6"-acetyl-?-galactopyranoside(3galloyl-acetylGa)of cyanidin(Cy)and five flavonols:3-galactoside(3Ga),3-rhamnosyl-(1?6)-galactoside(3Rh(1?6)Ga),3-rhamnoside(3Rh)of myricetin(My);3-rhamnoside(3Rh)of quercetin(Qu);3-rhamnoside(3Rh)of kaempferol(Km)were aready reported.The rest sixty-four components were reported for the first time in hardy water lily.3.The composition and content differences of flavonoids between tropic and hardy water lily cultivars petalsFirst of all,the two groups of water lilies had the same anthocyanidins Dp and Cy with similar kinds of glycosylation and acylation.There were eleven anthocyanins detected in tropic water lily,including seven delphinidin derivants,three cyanidin derivants and one unknow components.However,in tropic water lily fourteen anthocyanins were detected,besides eight delphinidin derivants and six cyanidin derivants.In view of TA content,the highest one in hardy water lily was 5.22 mg/g FW and the mean TA contents of rest cultivars was about 0.88 mg/g FW.However,in deep red color of tropic water lily cultivars the highest TA content was only 1.43 mg/g FW,and the mean of rest tropic cultivars was about 0.39 mg/g FW.TA value was higher in hardy water lily than that of tropic water lily.Secondly,from the composition perspective of other flavonoids(flavone,flavonol,flavanone and chalcone),twenty two flavonol glycosides and one chalcone galactoside were characterized in tropic water lily petals.However,in hardy water lily petals glycosides of five flavones,fourty-six flavonols,two flavanones and eight chalcones were detected.Moreover,except the usual glycosides of My,Km,Qu,Is and Chalcone,glycosides of Pa,Lu,Ch and Na were also detected in hardy water lily petals.Quantity and variety of compounds in hardy water lily were much more than that of tropic water lily.From the perspective of TF content,the highest one in hardy water lily was 16.78 mg/g FW,but in tropic water lily was only 7.93mg/g FW which was less than half of hardy water lily.The main reason for the petal color differences between tropic and hardy water lily cultivars was due to the composition and content of flavonoids.4.Relationships between color parameters and flavonoid compositionsThere are many factors affecting the color of tropic water lily.Dp3galloylGa,Dp3'galloyl-acetylGa,Dp3galloyl-acetylGa,Cy3galloyl-acetylGa and Km3-2"acetylGaRh had positive effects on the value of a*,whereas Dp3galloylGa and Dp3'galloyl-acetylGa had negative effects on the value of b*.Qu3Rh and Qu3'Xy had positive effects on the value of L*,but Dp3'galloylGa had negative effects.However,Chalcone2'Ga had negative effects on the value of a*,but positive effects on the value of b*and L*.To obtain more vivid flower color,the contents of Dp3galloylGa,Dp3'galloyl-acetylGa,Dp3galloyl-acetylGa,Cy3galloyl-acetylGa and Km3-2"acetylGaRh could be increased.Increasing the contents of Chalcone2'Ga,the yellow color would become more deeper,and at the same time the lightness would be increased at a certain degree.A significant correlation existed between color parameters and flavonoid compositions of hardy water lily.Dp3galloylGa and Cy3acetylGa had negative effects on the value of L*,whereas Km3Ga and Qu7Ga had positive effects.Qu3Rh,Qu7Ga,Qu7A and THC2'digalloylG had positive effects of b*value,but Cy3acetylGa,Pa3Xy and Chalcone2'Ga had negative effects.The regression equation showed that there were mang factors affecting the value of a*,among which the contents of Dp3galloylGa,Cy3Ga(1?2)Ga and Cy3acetylGa had larger positive effects,but Qu7Ga and THC2'digalloylG had larger negative effects.So,increasing the contents of Dp3galloylGa,Cy3Ga(1?2)Ga and Cy3acetylGa would improve the red degree of petals,and at the same time decrease the lightness and yellow degree in some extent.The effective accumulation of THC2'digalloylG could decrease the red color and build more vivid yellow color in petals.5.Putative flavonoid biosynthesis pathway of water lily petalsThe flavonoid biosynthesis pathway in water lily petals was deduced according to the final products detected.The pathway began with the synthesis of 4,2',4',6'-tetrahydroxychalcone from 4-coumaroyl-CoA and malonyl-CoA.On one hand 4,2',4',6'-tetrahydroxychalcone was galactosylated to Chalcone2'Ga,on the other hand it was isomerized to naringenin,which was catalyzed by CHI.Then by the function of a series of enzymes,nine flavonoid aglycones were synthesized and formed final flavonoid glycosides by focusing on naringenin.