Qualitative analysis for Flavonol and Chalcone.
Using analysis by HPLC-DAD, 22 glycosides of flavonol (f1–f12, f14–f23) and one glycosides of chalcone (f13) (Fig. 4) have been detected by the characterization of UV-vis Absorption Spectroscopy for flavonol and chalcone. The data of HPLC-DAD and HPLC-ESI(+/−)-MS2 including retention time of HPLC, UV characteristic absorption wavelength, molecular ion, aglycone ion and some important fragment ions were summarized in Table S3.
Compared the references with characteristic of MS and UV Spectroscopy [30], 5 aglycones of flavonol and one chalcone have been found, containing 4 aglycones of flavonol: isorhamnetin (f16 and f20), kaempferol (f1, f7, f10, f15, f21 and f22), myricetin (f1, f2, f3, f8, f9, f12 and f14) and quercetin (f4, f5, f6, f11, f17, f18, f19 and f23); one aglycone of chalcone: chalcononaringenin (f13) (the chemical structure was illustrated in Fig. 3).
Through analysis by HPLC, 7 compounds were already known in comparison with standards and references: myricetin 3′-O-xyloside (My3′Xy) (f9); quercetin 3-O-rhamnoside (Qu3Rh) (f11); myricetin 3-O-(2″-acetylrhamnoside) (My3acetylRh) (f14); quercetin 3-O-(3″-acetylrhamnoside) (Qu3acetylRh) (f17); quercetin 3′-O-xyloside (Qu3′Xy) (f18); quercetin 3-O-(2″-acetylrhamnoside) (Qu3acetylRh) (f19) and kaempferol 3-O-(2″-acetylrhamnoside) (Km3-2″acetylRh) (f22). Except for f1, f3 and f4, which were not identified exactly, the rest of the 12 compounds were detected in the petals of water lily for the first time in this study.
Under the negative ion modes, the relative abundance about aglycone ion ([Y0−]) and aglycone ion free radical ([Y0-H]−.) of flavonoid 3-O-glycoside and 7-O-glycoside was different. When glycosylation took place in 3-position, the relative abundance of [Y0-H]−. was higher than that of [Y0−], the situation is reverse when glycosylation happened to 7-position [31]. This conclusion further verified the structure of the known flavonoids (f11, f14, f17 and f19), meanwhile we could suppose f6 and f23 to be quercetin 7-O-hexoside and quercetin 3-O-acetylhexoside. Only in one research, it had been reported that galactose glycosylated with flavonoid [21], so peak f6 and f23 were identified as quercetin 7-O-galactoside (Qu7Ga) and quercetin 3-O-acetylgalactoside (Qu3acetylGa) tentatively. As we all know, the characteristic of UV absorbance wavelength about the above two glycosides flavonoid was different, the band of flavonoid 7-O-glycoside caused bathochromic shifts compared with 3-O-glycoside [32]. As a result the structure of five compounds was identified as follows: f8: myricetin 3-O-galactoside (My3Ga), f15: kaempferol 3-O-galactoside (Km3Ga), f16: isorhamnetin 7-O-galactoside (Is7Ga), f20: isorhamnetin 7-O-xyloside (Is7Xy), f21: kaempferol 3-O-(3″-acetylrhamnoside) (Km3-3″acetylRh).
Although only one disaccharide has been isolated from leaves of the water lily Nymphaea×marliacea (white petals) [21], we could still not ensure the linking style between two monosaccharides, although the usual style was 1→2 and 1→6. Based on data of MS, the aglycone quercetin of f5 was judged to be connected by two sugars: one hexose and one rhamnose, and quercetin was certainly glycosided with disaccharide because m/z 463 [M-H-146]− was detected except for m/z 447 [M-H-162]−. The relative abundance of fragment ion m/z 303 [Y0+] was higher than that of m/z 463 [M+H-146]+, the connection type of glycosidic bond of disaccharide was 1→2 [33]. Compared the characteristic of UV absorbance of the known compounds (f11; f17 and f19), f5 was identified as quercetin 7-O-galactosyl-(1→2)-rhamnoside (Qu7GaRh) tentatively. As the same way, f2 and f7 were tentatively identified as myricetin 7-O-rhamnosyl-(1→2)-rhamnoside (My7RhRh) and kaempferol 7-O-galactosyl-(1→2)-rhamnoside (Km7GaRh) separately. Peak f10 had fragment ions at m/z 449 [M+H-152]+ and m/z 315 [galloylhexose+H]+ (86), it illustrated that f10 was acylated by gallic acid, and the galloyl connected with galactose. According to above information, the structure of f10 was identified as kaempferol 7-O-galloylgalactosyl-(1→2)-rhanmoside (Km7galloylGaRh) provisionally.
With regard to acylated compounds, there were four components (f14, f17, f19 and f22) isolated by Fossen et al., and the common acyl was acetyl and galloyl in water lily, so except for f10, f21 and f23, the rest acylated peak f12 was temporary identified as myricetin 3-O-galloylrhamnoside (My3galloylRh).
One glycoside of chalcone had been detected by the chromatogram monitored at 350 nm, depending on the characteristic of UV-vis spectrum, f13 presented an intense absorption at 366 nm and a weak absorption at 250 nm [32]. The result of MS (m/z: 457[M+Na]+, 273[Y0+], 433[M-H]−, and 271[Y0−]) complyed with previous reports [34]. Therefore f13 was identified as chalcononaringenin 2′-O-galactoside (Chal2′Ga), this compound was first reported in petals of water lily in this study and only existed in yellow flowers. Meanwhile it was also a principal component.
Relationships between color parameters and flavonoid compositions
The flower color is associated with type, content and physicochemical property of pigment, the pH value of vacuole, the shape of epidermis cell and organization structure in petals. However the composition of pigments is the most important one. So choosing L*, a*, b*, C* and h as dependent variable, 37 indexes included 34 contents of pigment components, TA (total anthocyanins content), TF (total flavonols and chalcones content) and CI (co-pigment index = TF/TA) as independent variable. The regression equations about the relationships between color parameters and flavonoids components were established to study the interactions between pigment composition and color formation by multiple linear regression (MLR) analysis [35]. Statistical results indicated as follows (n = 35, p<0.05):
L* = 61.074+0.032Qu3Rh+0.054Km7galloylGaRh - 0.031Dp3′galloylGa (R2 = 0.531)
a* = −3.113+0.040TA+0.065Qu3acetylGa (R2 = 0.850)
b* = −20.226−0.202Qu3acetylGa+0.007Chal2′Ga+0.016Qu3acetylRh (R2 = 0.673)
C* = 10.750+0.032TA+0.167Qu3acetylGa (R2 = 0.688)
h = −1.322+0.005Dp3galloylGa−0.001Dp3′galloylGa (R2 = 0.953)
From this MLR analysis, it can be seen that there are many factors affecting the color: TA; Dp3galloylGa; Dp3′galloylGa; Qu3Rh; Qu3acetylGa; Qu3acetylRh; Km7galloylGaRh and Chal2′Ga. TA and Qu3acetylGa had positive effects on the value of a* and C*, whereas Qu3acetylGa and Qu3acetylRh had negative effects on the value of b*, meanwhile Chal2′Ga had positive effects on the value of b*. Km7galloylGaRh and Qu3Rh had positive effects on the value of L*, but Dp3′galloylGa had negative effects on the value of L* and h. In addition Dp3galloylGa had a positive effect on the value of h. Based on those conclusions we can see increasing TA, Dp3′galloylGa and Qu3acetylGa, the value of a* and C* increased, but L* and b* decreased that means the flower colors change to be red and blue and much vivid. The compounds Chal2′Gal only exist in yellow water lily cultivars. And in the equation of b* the same result is gained: the higher contents of Chal2′Gal, the deeper of the yellow flower color. From the equations, we could see that increasing TA and the content of Qu3acetylGa the flower color would be more vivid.
Comparison of anthocyanin components among cultivars
The main anthocyanins in the petals of tropic water lily were delphinidin glycosides, followed by cyanidin glycosides. Dp3′Ga was presented in all cultivars with blue colors, the highest amounts was detected in cultivar ‘Tai Guo Wang’ which the flower demonstrated blue purple (similar to Fig. 1E). Dp3Ga was only detected in ‘Huang Guan Zi', ‘34’ and ‘Fo Shou Lian’ which ranked the first three cultivars with the highest delphinidin derivates (Table S4, marked by boldface). It can be concluded that at 3-position of the B ring galactose was preferred to be linked, however, in tree peony [10], [11], Lycoris longituba [35] and lotus [36], glucose was abundant at the same position. It would be possibly unique for water lily plants, since flavonoids were thought to be evolutionarily adaptive for plants, the enzyme function for glycosylation of flavonoids might be also evolved in water lily. Within the blue group, Cy3Ga was the only one detected in the petal of ‘Huang Guan Zi’ with the highest TA value. The type of anthocyanins within flowers of ‘18’ (Fig. 1B), ‘27’ (Fig. 1C) and ‘Tai Guo Wang’ (Fig. 1I), the colors of which presented thin-blue, dark-blue and blue purple respectively, maybe due to the increased content of Dp3′Ga or TA value. It could be deduced that Dp3′Ga contributes most of the blue color formation in tropic water lily.
In contrast, in the amaranth group (represented by Fig. 1G), Dp3Ga was the only detected delphinidin derivatives with the highest amount. Compared with blue group, cultivars which were detected Dp3Ga presented amaranth, or detected Dp3′Ga for blue colors. In red group, the derivatives of Dp and Cy were more complicated, Dp3RhGa, Cy3Ga5Rh were also detected. ‘Albert Greenberg’ (Fig. 1D) and ‘Roxburgh’ (Fig. 1H) were illustrated with different colors, which can be deduced by the type of Dp and Cy derivatives and the amount of Cy derivative, the former contained Dp3RhGa, Dp3Ga, the latter with Dp3Ga, Dp3Rh, Dp3′Ga and Cy3Ga, although the content of Dp derivatives was close, the latter had the maximal amount of Cy derivatives.
To the best of our knowledge, besides glycosylation and hydroxylation, acylation also happened to tropic water lily. From Table S4 we can see 2″-galloyl-6″-acetyl presented in most cultivars of tropic water lily, but the highest amount existed in red group. It was obvious to know that 6″-acetyl and 2″-galloyl-6″-oxalyl were only detected in amaranth and red group respectively, but the contents were not high. 2″-galloyl was detected in amaranth, red group and some cultivars of blue group. No anthocyanins were detected within white and yellow group. These two groups might be useful breeding materials when crossing with the other groups to produce novel colors of cultivars.
Putative flavonoid biosynthesis pathway of tropical water lily
Flavonoid especially anthocyanin biosynthesis pathway is one of the most extensively studied pathway of plant secondary products [37]. With the detected component of flavonoids in tropical water lily, we deduced the putative flavonoid biosynthesis pathway relevant to flower color (Fig. 5). It rooted from coumaroyl-CoA and malonyl-CoA, with the enzymes of CHS, CHI and F3H, finally, synthesized dihydrokaempferol. Then it was divided into five sub-pathway for synthesis of anthocyanins and flavonols. With the function of F3′H, F3′5′H, DFR and ANS or FLS, anthocyanidins (cyanidin and delpinidin) and aglycone of flavonols were obtained, which finished the first important modification of hydroxylation of flavonoids. The obtained secondary metabolites were glycosylated by glycosyltransferase at different position in tropical water lily. Flavonoids including anthocyanins exist in glycosylated form in vivo, although most anthocyanins are glycosylated at 3-O-position and often at 5-O-position, the former is a perquisite for further modifications including second glycosylation, acylation and methylation [37]. In tropical water lily, glycosylation at 3-O-position was most often, and the galactose was preferred to be added to the anthocyanidins. The selection of different substrates or different position for glycosylation in different plants may shed light on the related function of glycosyltransferase during the evolution of plants or the enzymes themselves. It is still need to be further studied for the function of this kind of enzymes. Modification of flavonoids by hydroxylation, glycosylation, methylation and acylation played an important role for flower color formation. In tropical water lily, the glycosylated flavonoids were further modified by acyltransferase. It is presumed that acylation with aromatic organic acid to contribute for stabilization of flavonoids due to intra- and/or inter-molecular stacking as co-pigmentation. Most acylated flavonoids in the petals of tropical water lily may intensify blue color as a bathochromic effect. The exact flavonoid biosynthesis pathway relevant to flower color of tropical water lily was still need to be confirmed by more molecular biology or biochemical evidences.
The red arrows indicate the biosynthesis pathway of anthocyanins, the dotted arrows indicate uncertain reaction. Cy: cyanidin; Dp: delphinidin; Qu: quercetin; Km: kaempferol; My: myricetin; Is: isorhamnetin; Ga: glactoside; G: glucoside; Rh: rhamnoside; CHS: chalcone synthase; CHI : chalcone isomerase, F3H: flavonoid 3-hydroxylase; F3′H: flavonoid 3′-hydroxylase; F3′5′H: flavonoid 3′,5′-hydroxylase; DFR: dihydroflavonol reductase; FLS: flavonol synthase; ANS: anthocyanidin synthase; GT: flavonoid glycosyltransferase; AT: acyltransferase; MT: methyltransferase; CoA: acetyl coenzyme A.
Discussion
As a perennial plant, tropical water lily is characteristic of some unique biological features such as the special flower colors like blue, violet and bluish purple and some cultivars blooming at night. In general, tropical water lily spreads all over the world and its flower colors are diverse. In this study, using an HPLC-DAD/MS analytical method to characterize flavonoids of water lily petals within 50 min, finally 34 flavonoids have been identified at 525 nm and 350 nm, respectively, in which three anthocyanins, twelve flavonols and one chalcone were discovered for the first time in petals of water lily.
Compared the graphs of HPLC with the results of MLR analysis, we could see that Dp3′galloyl-acetylGa (a7) and Dp3galloyl-acetylGa (a9) were the main anthocyanins in those 35 cultivars, but they did not take part in the MLR analysis and in reverse Dp3′galloylGa (a2) and Dp3galloylGa (a3) replaced them. Meanwhile compared with a2 compound, a7 owned an acetyl group at 6″ position, so did a9. However, there were no similar regularities in flavonols, so the results of MLR analysis were synthetic with anthocyanins, flavonols and chalcones. A correlation analysis indicated that the L* value increased with increasing proportions of Km7galloylGaRh (f10) and Qu3Rh (f11) and the reduction of the contents of a2. At the same time we could see that increasing the contents of TA, Qu3acetylGa (f23) and a2, the value of a* and C* increased while L* and b* decreased, which means that the flower colors changed to be red, blue and much vivid. The compound Chal2′Ga (f13) only existed in yellow water lily cultivars, and in the equation of b*, the same result was gained that the higher contents of f13, the deeper of the yellow flower color. So it was the indispensable compound in formation of the yellow flower in water lily.
The relationships between color parameters and flavonoid compositions showed that many kinds of compounds played an important role on color formation. To characterize the mechanism on flower color formation of special blue and bluish violet which hardy water lily lacks was necessary because it could provide powerful evidences for the ornamental breeding of hardy water lily with blue colors and help to classify cultivars of Nymphaea through phytochemical analysis. At the same time, a HPLC fingerprinting database of water lily cultivars could be established with the flavonoids composition data for discriminating cultivars.
Anthocyanins not only contribute to flower colors, but also play a vital role on bioactivity including anti-oxidant, anti-cancer, anti-allergic and anti-ulcer, therefore they were used as diets in cancer therapy and prevention [38]. Among five anthocyanidins, delphinidin and cyanidin inhibited LPS-induced COX-2 expression, but pelargonidin, peonidin and malvidin did not, the structure-activity relationship suggested that the ortho-dihydroxyphenyl structure of anthocyanidins on the B-ring appears to be related to the inhibitory actions, especially delphinidin as the most potent inhibitor [39]. Delphinidin also protects human HaCaT keratinocytes and mouse skin against UVB-mediated oxidative stress and apoptosis [40]. In tropical water lily, cyanidin and delphinidin are the main anthocyanidins, which may be judged for the high bioactivity for developing functional food or medicine materials.
Other flavonoids were also important components for antioxidant activity, among which quercetin and kaempferol have high antioxidant activity; apigenin and chalcononaringenin or its derivates demonstrated high antioxidant ability due to hydroxylation of B-ring at 4-position [11]. In yellow group of tropical water lily, the content of Chal2′Ga accounted for 80% of the total amount of other flavonoids (TF). However, in blue group the amount of qucercetin was about 87.7% of TF, in amaranth group the amount of kaempferol reached 92.8% of TF. These abundant flavonoids will be good resource for bioactivity for future utilization.
Genes for enzymes involved in the synthesis of flavonoid for flower colors were cloned in model plants such as petunia, arabidopsis, rice etc. People tried to change or create novel flower colors through over-expressed or knocked down some genes related to this pathway [41]. The deduced pathway in this study will help to understand its synthesis of flavonoids and to clone the related genes for molecular breeding of tropical water lily with novel flower colors or studies on molecular mechanism for the formation of flower colors.
Materials and Methods
Standards and solvents
Malvidin 3,5-di-O-glucoside chloride (Mv3G5G) was purchased from Extrasynthese (Genay, France). quercetin 3-O-rutinoside (rutin) was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Acetonitrile and methanol used for high-performance liquid chromatography-photodiode array detection (HPLC-DAD) and electrospray ionization multistage mass spectrometry (ESI-MSn) analysis were of chromatographic grade and obtained from Alltech Scientific (Beijing, China). Trifluoroacetic acid (TFA; ≥99%) was purchased from Merck (Darmstadt, Germany). Methanol, formic acid and hydrochloric acid of analytical grade were obtained from Beijing Chemical Works (Beijing, China). HPLC-grade water was purchased from a Milli-Q System (Millipore, Billerica, MA, USA).
Plant Material and Petal Color Measurement
Petals of 35 tropical water lily cultivars were all collected in 2010 at Beijing Botanical Garden, Institute of Botany, the Chinese Academy of Sciences (Long. 39°48′N, Lat. 116°28′E, Alt. 76 m), Beijing, China. These cultivars introduced from all around the world have been planted in the same-sized containers (diameter, 40 cm; height, 30 cm) with two cultivars in each small pond (length×width×depth: 140 cm×140 cm×70 cm) in Beijing Botanical Garden for more than 3 years under the same cultivated conditions like fertilization, irrigation, disease and insect prevention and so on. Petals were harvested at the first blooming day.
The fresh petals were compared to Royal Horticultural Society Colour Chart (RHSCC) and the color parameters were measured with a spectrophotometer NF333 (Nippon Denshoku Industries Co. Ltd., Japan). Then petals from 35 cultivars were stored in refrigerator at −40°C for future analysis. In this study, 35 cultivars were classified into four groups roughly in terms of floral colors (Table 2). CIE 1976 L*a*b* (CIELAB), a software package, is used to measure different aspects of a flower color and takes into account all aspects of the color described by L*, a*, and b* parameters [42]. The L* describes the lightness of the color, ranging from black (L* = 0) to white (L* = 100). The a* negative and positive are for green and red, and the b* negative and positive are for blue and yellow, respectively [35]. The color coordinates measured were shown in Fig. 6: the L* values ranged from 44.36 to 99.92, a* values from −13.40 to 62.91 and b* values from −60.86 to 15.24. Two new parameters, Chroma [C* = (a*2+b*2)1/2] and hue angle [h = arctan b*/a* ], were derived from a* and b*. The Chroma parameter describes the saturation of the color. The C* value is higher, the color is more saturated. The hue angle parameter describes the hue of the color. Hue angle values are stepped counterclockwise across a continuously fading hue circle, some special colors' values of which are remarkable: magenta (0°/360°), yellow (90°), bluish-green (180°) and blue (270°) [42], [43].
The flower colors were identified by the RHSCC value.