Abstract
Water lily, the member of the Nymphaeaceae family, is the symbol of Buddhism and Brahmanism in India. Despite its limited researches on flower color variations and formation mechanism, water lily has background of blue flowers and displays an exceptionally wide diversity of flower colors from purple, red, blue to yellow, in nature. In this study, 34 flavonoids were identified among 35 tropical cultivars by high-performance liquid chromatography (HPLC) with photodiode array detection (DAD) and electrospray ionization mass spectrometry (ESI-MS). Among them, four anthocyanins: delphinidin 3-O-rhamnosyl-5-O-galactoside (Dp3Rh5Ga), delphinidin 3-O-(2″-O-galloyl-6″-O-oxalyl-rhamnoside) (Dp3galloyl-oxalylRh), delphinidin 3-O-(6″-O-acetyl-β-glucopyranoside) (Dp3acetylG) and cyanidin 3- O-(2″-O-galloyl-galactopyranoside)-5-O-rhamnoside (Cy3galloylGa5Rh), one chalcone: chalcononaringenin 2′-O-galactoside (Chal2′Ga) and twelve flavonols: myricetin 7-O-rhamnosyl-(1→2)-rhamnoside (My7RhRh), quercetin 7-O-galactosyl-(1→2)-rhamnoside (Qu7GaRh), quercetin 7-O-galactoside (Qu7Ga), kaempferol 7-O-galactosyl-(1→2)-rhamnoside (Km7GaRh), myricetin 3-O-galactoside (My3Ga), kaempferol 7-O-galloylgalactosyl-(1→2)-rhamnoside (Km7galloylGaRh), myricetin 3-O-galloylrhamnoside (My3galloylRh), kaempferol 3-O-galactoside (Km3Ga), isorhamnetin 7-O-galactoside (Is7Ga), isorhamnetin 7-O-xyloside (Is7Xy), kaempferol 3-O-(3″-acetylrhamnoside) (Km3-3″acetylRh) and quercetin 3-O-acetylgalactoside (Qu3acetylGa) were identified in the petals of tropic water lily for the first time. Meanwhile a multivariate analysis was used to explore the relationship between pigments and flower color. By comparing, the cultivars which were detected delphinidin 3-galactoside (Dp3Ga) presented amaranth, and detected delphinidin 3′-galactoside (Dp3′Ga) presented blue. However, the derivatives of delphinidin and cyanidin were more complicated in red group. No anthocyanins were detected within white and yellow group. At the same time a possible flavonoid biosynthesis pathway of tropical water lily was presumed putatively. These studies will help to elucidate the evolution mechanism on the formation of flower colors and provide theoretical basis for outcross breeding and developing health care products from this plant.
Figures
Citation: Zhu M, Zheng X, Shu Q, Li H, Zhong P, Zhang H, et al. (2012) Relationship between the Composition of Flavonoids and Flower Colors Variation in Tropical Water Lily (Nymphaea) Cultivars. PLoS ONE 7(4): e34335. https://doi.org/10.1371/journal.pone.0034335
Editor: Xiaoyu Zhang, University of Georgia, United States of America
Received: November 13, 2011; Accepted: February 26, 2012; Published: April 2, 2012
Copyright: © 2012 Zhu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors appreciate the support of the National Natural Science Foundation of China, No. 31071824, as well as International Science & Technology Cooperation Program of China, no. 2011DFA30560. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Water lily (an aquatic herb of genus Nymphaea, family Nymphaeaceae), a precious perennial aquatic flower plant, is divided into two ecological groups, namely Tropical and Hardy water lily [1]. There are about 50 species in the whole world, five of which originate from China: N. alba L., N. candida Presl., N. tetragona Georgi., N. lotus L.var. pubescens and N. atellata Willd [2]. It is called subaqueous nymph and symbolized as spotlessness, trueness and coquettishness. Like lotus, water lily is not only an ornamental plant but also an important water purification one. Because the roots of water lily can absorb the poisonous substances like mercury, lead, phenol, etc and filter the microorganism in water, it plays an important role in decontaminating water, afforesting and landscaping [3], [4]. In the meanwhile, flowers and roots of water lily can both be made into tea and liquor, and the whole plant has been useful in the therapies of nephritis and is reputedly a detoxicant and aphrodisiac along with astringent, diuretic properties [5]. Furthermore, as shown in Fig. 1, tropical water lily owns flowers with the special colors of blue, violet and bluish purple which hardy water lily lacks of, for this reason the former is more favorable by people. However, little is known about the formation and genetic mechanism of the flower colors on tropical water lily, the study on its pigments of flower petals will illuminate the formation of flower colors.
Flavonoid is the decisive pigment presented in most flower colors, among which anthocyanin is the key component. Flower petals with anthocyanin present red, pink, purple and blue. Except contribution of flower color, it also has important biological activities, such as antioxidative, antiinflammatory, antimicrobial, anti-platelet aggregation and antitumor activity, etc. [6]–[9]. At present, TLC (Thin-Layer Chromatography), HPLC-DAD, HPLC-MSn (multi-stage tandem Mass Spectrometry), UV-vis (Ultraviolet visible), HSCCC (High Speed Countercurrent Chromatography) and NMR (Nuclear Magnetic Resonance) are the important techniques in characterizing the distribution and identifying the structure of anthocyanins and have been used in many plants, like tree peony [10]–[12], purple corn [13], Ficus carica L. [14] and Vaccinium myrtillus [15].
Till now, there is no systematic study on the pigment constitutes of water lily petals except several limited reports by Fossen et al.. Among those reports, 9 glycosides of anthocyanidin were isolated from the red flowers and leaves of Nymphaba×marliacea var. Escarboucle, reddish leaves of N. alba and blue flowers of N. caerulea [16]–[19], and 11 glycosides of flavonol from the red petals and leaves of Nymphaba×marliacea var. Escarboucle and blue flowers of N. caerulea by a combination of chromatography, homo- and heteronuclear two-dimensional NMR techniques and electrospray MS [20], [21]. Apart from those literatures, other articles only concentrated on one species or one cultivar [22]–[26]. Due to the limitation of techniques and the limited number of accession, the real quantity and type of flavonoids presented in the flower petals of water lily remains unclear. It is necessary to use as many as possible accessions to characterize the distribution and identify the structure of anthocyanins in water lily plants, in order to provide a global knowledge on the flower color formation. Meantime, it will provide a theoretical basis for selection parents of breeding novel cultivars with optimal flower colors by outcrossing. In this study, chromatographic conditions were optimized in order to obtain higher separation efficiency and peak resolution of target compounds, and at the same time a rapid method by HPLC-DAD coupled with ESI-MS was established to detect anthocyanins and other flavonoids simultaneously and to analysis those compounds qualitatively and semi-quantitatively. The relationship between flower color and pigment composition was also discussed. The established technique will be helpful to obtain fingerprints for these plants, and isolate the important components for medical therapy or study on its anti-oxidant ability, parental selection for outcrossing and breeding. It is also important to explore the rare blue coloration of this plant, which will be a base for the breeding new cultivars with special colors within Nymphaeaceae family. The components of flower pigments will also be an important data for classification of cultivars.
Results
Identification of Flavonoids
In order to obtain higher separation efficiency and peak resolution of target compounds, chromatographic conditions were optimized (Figure S1). The developed method provided satisfactory precision and accuracy with over-all intra-day and inter-day variations of 0.03%–0.75% and 0.03%–3.5%, respectively (Table S1). All calibration curves showed good linear regression (r2≥0.9986) within test ranges. The limit of detection (LOD) of optimized method was 0.4537 and 0.7193 µg/mL for MV3G5G and rutin, respectively, while the limit of quantification (LOQ) was 1.5124 and 2.3977 µg/mL (Table S2).
Generally, glycosylation sites usually occurs at the 7-hydroxyl group for flavones and flavanones, the 3- and 7-hydroxyl for flavonols and flavan-3-ols, and the 3- and 5-hydroxyl for anthocyanidins [27]. Sugars combined with the aglycone are always hexose and pentose. Glucose is the most commonly encountered sugar, galactose, rhamnose, xylose and arabinose are not uncommon. But in the cultivars of water lily, galactose is more common one instead of glucose [16]–[21]. Acylated glycosides, in which one or more of the sugar hydroxyls are esterified with an acid, also occur. There are many kinds of acids which usually participate in acylation like acetic, oxalic, gallic, cinnamic, ferulic acid and so on [18], [19]. In this research the structure of flavonoids were deduced mostly through retention time of the HPLC analysis, elution order, UV-vis spectroscopy and MSn, and by comparing with the standard and the known structures published in other researchers, 11 anthocyanins and 22 glycosides of flavonol as well as one chalcone were detected and in the meanwhile the structure of them were identified or identified tentatively (Table 1 and Table S3).
Qualitative analysis for anthocyanins.
The HPLC chromatogram detected the extract aqua in the visible region at 525 nm, showed 11 anthocyanins, a1–a11 (Fig. 2) (the chemical structure demonstrated in Fig. 3). It showed that there were only two aglycones, delphinidin and cyanidin in the petals of water lily. Among those anthocyanins, 5 compounds (a2, a3, a7, a9 and a11) were already reported in the past researches. By the retention time of the HPLC analysis, elution order and UV-vis spectroscopy (Table 1), they were identified to be delphinidin 3′-O-(2″-O-galloyl-β-galactopyranoside) (Dp3′galloylGa) (a2), delphinidin 3-O-(2″-O-galloyl-β-galactopyranoside) (Dp3galloylGa) (a3), delphinidin 3′-O-(2″-O-galloyl-6″-O-acetyl-β-galactopyranoside) (Dp3′galloy-acetylGa) (a7), delphinidin 3-O-(2″-O-galloyl-6″-O-acetyl-β-galactopyranoside) (Dp3galloyl-acetylGa) (a9) and cyanidin 3-O-(2″-O-galloyl-6″-O-acetyl-β-galactopyranoside) (Cy3galloyl-acetylGa) (a11), and were verified by electrospray MS. In the rest of the compounds, a1, a4 and a8 have fragment ions at m/z 303 which corresponding to delphinidin aglycone, so these two components are presumed to be delphinidin derivatives. a5 and a6 have fragment ions at m/z 287 which corresponding to cyanidin aglycone, so they are presumed to be cyanidin derivatives. Owing to the low amount in samples and little information of MS, a10 could not been identified.
From Table 1, the value of E440/Evis-max (the ratio of absorbance value at 440 nm and that at visible maximum absorption wavelength) [28] about 11 anthocyanins is between 23%–47%. Glycosylation at different position discriminated the value of E440/Evis-max. We can see that when the glycosylation is at 3′-OH, the value is about 39% higher than that of at 3-OH, meanwhile the two delphinidin 3′-glycosides, a2 and a7, showed a hypsochromic shift at 12 nm in the UV-vis spectrum compared with the analogous of delphinidin 3-glycosides, a3 and a9. Fossen et al. have already reported the identification of some anthocyanins which acylated with acetic and (or) gallic acid, glycosylated with monosaccharide galactose instead of glucose, and the acetyl and galloyl group was determined to be situated in the 6″-position and the 2″-position on the sugar respectively [18], [19].
The MS data of a1, fragment ion at m/z 465 ([M+H-146u]+) and 449 ([M+H-162u]+), exhibited two sugars link to delphinidin aglycone, and the relative abundance of m/z 449 was higher than that of m/z 465 which demonstrated the molecular ion 611 ([M+H]+) loses m/z 162u easier. Because glycosidic bond at 5-position is cracked easily [29], we identified a1 as delphinidin 3-O-rhamonsyl-5-O-galactoside (Dp3Rh5Rh) tentatively. Peak a4 was deduced as delphinidin glycoside based on the following information: the protonated molecule ion at m/z 673 ([M+H]+), the Y0+ at m/z 303, and other fragment ions at m/z 601 ([M+H-72u]+) and 449 ([M+H-(72+152)u]+). So the structure of a4 was assigned as delphinidin 3-O-(2″-O-galloyl-6″-O-oxalyl-rhamnoside) (Dp3galloyl-oxalylRh). Peak a8 had the molecule ion at m/z 507 ([M+H]+), fragment ions at m/z 465 [M+H-42]+ and the Y0+ at m/z 303. It was in line with delphinidin 3-O-(6″-O-acetyl-β-galactopyranoside) (Dp3acetylGa) which had been reported in the paper of Fossen et al. [17]. So it was likely to be Dp3acetylGa. However, two similar compounds of a8 had been found in petals of Hardy water lily and the retention time of one compound was close to that of a8, the other was a little earlier than it (unpublished data). Because the retention time of glucose was later than that of galactose in HPLC, we tentatively identified a8 as 3-O-(6″-O-acetyl-β-glucopyranoside) (Dp3acetylG). The spectra of peak a6 detected in positive full scan mode showed the sodium adduct at m/z 769 ([M+Na]+) and the molecule ion at m/z 747 ([M+H]+) corresponding to the successive losses of sugar units and acyl group, and finally gave the protonated aglycone Y0+ (m/z 287), fragment ions at m/z 601 (M+H-146u]+), 449 ([M+H-(146+152)]+) and 439 ([M+H-(146+162)]+). As a result, the relative abundance about m/z 449 was higher than that of m/z 439. We could conclude that rhamnose was linked at 5-position and the galloyl is happen to be galactose. Finally, the peak a6 was tentatively identified as cyanidin 3- O-(2″-O-galloyl-galactopyranoside)-5-O-rhamnoside (Cy3galloylGa5Rh). We only got the information about aglycone (m/z 287) for a5 in MS, and judged it as cyanidin aglycone by UV absorption spectroscopy. Then a5 was tentatively identified as cyanidin derivative. Owing to the low content in samples and little information of MS, a10 could not identified exactly except for one anthocyanin.
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.