Part 3 Relationship between the Composition of Flavonoids and Flower Colors Variation in Tropical Water Lily (Nymphaea) Cultivars

Preparation of standard solutions

The standards of Mv3G5G and rutin weighted accurately were dissolved in 0.1% (V/V) HCl-methanol and methanol, respectively, and then diluted to a series of concentrations (mg/mL): 0.01, 0.025, 0.05, 0.1, 0.2, 0.4, 0.6 and 0.8.

Extraction and Preparation of the Flavonoids

The extraction method of flavonoids was modified from that of Yang et al. [36]. Approximately 0.6 g of frozen petal was powdered in liquid nitrogen with mortars and pestles and extracted for the first time with 3 mL 70% (V/V) methanol aqueous solution containing 0.1% HCl shaken in a QL-861 vortex (Kylinbell Lab Instruments, Jiangsu, China), sonicated in KQ-500DE ultrasonic cleaner (Ultrasonic instruments, Jiangsu Kunshan, China) at 20°C for 20 min, centrifuged in SIGMA 3K30 (SIGMA centrifugers, Germany) (12000 rpm, 10 min), and the supernatant was collected. Additional 2 mL and 1 mL extraction solution was supplemented to the residue, and repeated aforesaid operation for second and third times. All extract was pooled and filtrated through 0.22 µm reinforced nylon membrane filters (Shanghai ANPEL, Shanghai, China) before the HPLC-DAD and HPLC-MS analyses. Three replicates were performed for each sample.

HPLC-DAD Systems and Conditions

HPLC analysis was performed on a Dionex (Sunnyvale, CA, USA) system including a P680 HPLC pump, an UltiMate 3000 autosampler, a TCC-100 thermostated column compartment and a PDA100 photodiode array detector. The liquid chromatograph was equipped with an ODS-80Ts QA C18 column (250 mm×4.6 mm i.d., Tosoh, Tokyo, Japan), which was protected with a C18 guard cartridge (Shanghai ANPEL Scientific Instrument, Shanghai, China). Eluent A was 10% formic acid aqueous solution; eluent B was 0.1% formic acid in acetonitrile [44]. A gradient elution as follows was used: 8% B at 0 min, 18% B at 15 min, 23% B at 25 min, 40% B at 45 min, 8% B at 50 min. The flow rate was 0.8 mL·min−1 and aliquots of 10 µL of analytes were injected. Column temperature was maintained at 35°C for all analyses. Chromatograms were acquired at 520 and 350 nm for anthocyanins and other flavonoids, respectively, and DAD data were recorded from 200 to 800 nm.

HPLC-MS System and Conditions

HPLC-ESI-MSn analysis for anthocyanins and other flavonoids were carried out in an Agilent-1100 HPLC system equipped with a UV detector and a LC-MSD Trap VL ion-trap mass spectrometer via an ESI source (Agilent Technologies, Palo Alto, CA, USA). The HPLC separation conditions were the same as mentioned above. The MS conditions were as follows: anthocyanins were adopted in positive-ion (PI) mode and other flavonoids were employed in negative-ion (NI) mode. ESI was performed by using the following conditions: capillary voltage, 4.0 kV; a nebulization pressure, 241.3 kPa; and a gas (N2) temperature, 350°C; flow rate, 8.0 L·min−1. Capillary offset and exit voltage were 77.2 V and 127.3 V, respectively for PI, and −77.2 V and −127.3 V separately for NI. MS spectrum was recorded over the range from m/z 100 to 1000.

Statistical Analysis

Correlations between petal color parameters and flavonoid compositions of individual varieties were analyzed by a multiple linear regression (MLR) (SPSS17.0 Inc., CHI, IL, USA) with stepwise method.

Supporting Information

Relationship between the Composition of Flavonoids and Flower Colors Variation in Tropical Water Lily (Nymphaea) Cultivars

Showing 1/5: Figure_S1.tif

https://ndownloader.figstatic.com/files/338382/preview/338382/preview.jpg

Figure S1.

The graphs of glycosides of flavonoids (350 nm) separated between longer column (250 mm) (a) and shorter column (150 mm) (b).

https://doi.org/10.1371/journal.pone.0034335.s001

(TIF)

Table S1.

Linearity of response for Mv3G5G and rutin using the optimized method. Calibration fitting: y = kx+m1. 1 In the regression equation y = kx+m, y refers to the peak area, x is concentration of the standard substances (µg/mL), r2 is the correlation coefficient of the equation.

https://doi.org/10.1371/journal.pone.0034335.s002

(DOC)

Table S2.

Intra- and inter-day precision of 31 main flavonoids in the extract of water lily petals by HPLC-DAD.

https://doi.org/10.1371/journal.pone.0034335.s003

(DOC)

Table S3.

HPLC-DAD and HPLC-ESI-MSn analysis as well as the structure characterization and tentative identification of glycosides of flavonol and chalcone in petals of water lily.

https://doi.org/10.1371/journal.pone.0034335.s004

(DOC)

Table S4.

The mean content (µg/g) of petal anthocyanin in 35 tropic water lily varieties.

https://doi.org/10.1371/journal.pone.0034335.s005

(DOC)

Acknowledgments

We would like to thank Chonghui Li, Hui Du, Jie Wu, Xia Jiang and Shanshan Li for their help in collecting plant materials and amending Fig. 6 and Fig. 5.

Author Contributions

Conceived and designed the experiments: L-SW Q-YS. Performed the experiments: M-LZ Y-JX HL X-CZ L-JW. Analyzed the data: M-LZ X-CZ. Contributed reagents/materials/analysis tools: M-LZ H-JZ HL P-XZ. Wrote the paper: M-LZ QY-S.

References

  1. 1.Huang GZ, Deng HQ, Li ZX, Li G (2009) Water lily. Beijing: China Forestry Publishing House. 3 p.
    • 2.The Chinese Academy of Sciences Flora Reipublicae Popularis Sinicae Editorial Board (1979) Flora Reipublicae Popularis Sinicae. Science Press, Beijing Vol. 27: 8–12. (In Chinese).
    • 3.Li QQ, Huang JY, Ji JB, Meng QG, Yang CJ, et al. (2005) The goddess of aquatic flowers-water lily. Practical Forestry Technology 10: 45–46. (In Chinese).
    • 4.Shi YF (2009) Excellent aquatic flowers-Lotus and Water lily. China Fruit & Vegetable 8: 40–41. (In Chinese).
    • 5.Devi Bown (1995) Encyclopedia of Herbs and their uses. London, New York, Stuttgart, Moscow: Dorling Kindersley Limited. 317 p.
      • 6.Bomser J, Madhavi DL, Singletary K, Smith MAL (1996) In Vitro Anticancer Activity of Fruit Extracts from Vaccinium Species. Planta Med 62: 212–216.
      • 7.Bravo L (1998) Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance. Nutr Rev 56: 317–333.
      • 8.Juranic Z, Zizak Z (2005) Biological activities of berries: From antioxidant capacity to anti-cancer effects. BioFactors 23: 207–211.
      • 9.Stone SZ, Yasmin T, Bagchi M, Chatterjee A, Vinson JA, et al. (2007) Berry anthocyanins as novel antioxidants in human health and disease prevention. Mol Nutr Food Res 51: 675–683.
      • 10.Wang LS, Shiraishi A, Hashimoto F, Aoki N, Shimizu K, et al. (2001) Analysis of Petal Anthocyanins to Investigate Flower Coloration of Zhongyuan (Chinese) and Daikon Island (Japanese) Tree Peony Cultivars. J Plant Res 114: 33–43.
      • 11.Li CH, Du H, Wang LS, Shu QY, Zheng YR, et al. (2009) Flavonoid composition and antioxidant activity of tree peony (Paeonia section Moutan) yellow flowers. J Agr Food Chem 57: 8496–8503.
      • 12.Zhang JJ, Wang LS, Shu QY, Liu ZA, Li CH, et al. (2007) Comparison of anthocyanins in non-blotches and blotches of the petals of Xibei tree peony. Sci Hortic (Amsterdam) 114: 104–111.
      • 13.Pedreschi R, Cisneros-Zevallos L (2007) Phenolic profiles of Andean purple corn (Zea mays L.). Food Chem 100: 956–963.
      • 14.Solomon A, Golubowicz S, Yablowicz Z, Grossman S, Bergman M, et al. (2006) Antioxidant Activities and Anthocyanin Content of Fresh Fruits of Common Fig (Ficus carica L.). J Agr Food Chem 54: 7717–7723.
      • 15.Dua QZ, Jerz G, Winterhalter P (2004) Isolation of two anthocyanin sambubiosides from bilberry (Vaccinium myrtillus) by high-speed counter-current chromatography. J Chromatogr A 1045: 59–63.
      • 16.Fossen T, Andersenet ØM (1999) Delphinidin 3′-galloylactosides from blue flowers of Nymphaea caerulea. Phytochemistry 50: 1185–1188.
      • 17.Fossen T, Larsen Å, Andersenet ØM (1998) Anthocyanins from flowers and leaves of Nymphaea×Marliacea cultivars. Phytochemistry 48: 823–827.
      • 18.Fossen T, Andersenet ØM (1997) Acylated anthocyanins from leaves of the water lily, Nymphaea×Marliacea. Phytochemistry 46(2): 353–357.
      • 19.Fossen T, Andersenet ØM (2001) Cyanidin 3-(6″-acetyl) and other anthocyanins from reddish leaves of the water lily Nymphaea alba. J Hortic Sci Biotech 76: 213–215.
      • 20.Fossen T, Larsen Å, Kiremire BT, Andersenet ØM (1999) Flavonoids from blue flowers of Nymphaea caerulea. Phytochemistry 51: 1133–1137.
      • 21.Fossen T, Froystein NA, Andersen ØM (1998) Myricetin 3-rhamnosyl (1→6) galactoside from Nymphaea×Marliacea. Phytochemistry 49: 1997–2000.
      • 22.Elegami AA, Bates C, Gray AI, Mackay SP, Skellern GG, et al. (2003) Two very unusual macrocyclic flavonoids from the water lily Nymphaea lotus. Phytochemistry 63: 727–731.
      • 23.Zhang ZZ, ElSohly HN, Li XC, Khan SI, Broedel SE, et al. (2003) Phenolic Compounds from Nymphaea odorata. J Nat Prod 66: 548–550.
      • 24.Marquina S, Bonilla-Barbosa J, Alvarez L (2005) Comparative phytochemical analysis of four Mexican Nymphaea species. Phytochemistry 66: 921–927.
      • 25.Liu RN, Wang W, Ding Y (2007) A new flavonol glycoside and activity of compounds from the flower of Nymphaea candida. J Asian Nat Prod Res 9: 333–338.
      • 26.Jambor J, Skrzypczak L (1991, 1992) Flavonols from the flowers of Nymphaea alba L.. Acta Soc Bot Pol 60(1–2): 119–125.
      • 27.Abad-Garcia B, Berrueta LA, Garmon-Lobato S, Gallo B, Vicente F (2009) A general analytical strategy for the characterization of phenolic compounds in fruit juices by high-performance liquid chromatography with diode array detection coupled to electrospray ionization and triple quadrupole mass spectrometry. J Chromatogr A 1216: 5398–5415.
      • 28.Harborne JB (1958) Spectral methods of characterizing anthocyanins. J Biochem 70: 22–28.
      • 29.Cuyckens F, Claeys M (2004) Mass spectrometry in the structural analysis of flavonoids. J Mass spectrom 39: 1–15.
      • 30.Harborne JB (1962) Plant polyphenols 6. The flavonol glycosides of wild and cultivated potatoes. Biochem J 84: 100–106.
      • 31.Abulajiang · Ke yimu (2006) Study on the mass spectrometric analytical method of flavonoid glycosides. Beijing: Peking Union Medical College. (In Chinese).
        • 32.Markham KR (1982) The flavonoids advances in research. London, Britain. Academic Press 5–6: 37–51.
        • 33.Ma Y, Cuyckens F, Van den Heuvel H, Claeys M (2001) Mass spectrometric methods for the characterisation and differentiation of isomeric O-diglycosyl flavonoids. Phytochem Anal 12: 159–165.
        • 34.Yoshida H, Itoh Y, Ozeki Y, Iwashina T, Yamaguchi M (2004) Variation in chalcononaringenin 2′-O-glucoside content in the petals of carnations (Dianthus caryophyllus) bearing yellow flowers. Sci Hortic 99: 175–186.
        • 35.He QL, Shen Y, Wang MX, Huang MR, Yang RZ, et al. (2011) Natural variation in petal color in Lycoris longituba revealed by anthocyanin components. Plos One 6: 1–8.
        • 36.Yang RZ, Wei XL, Gao FF, Wang LS, Zhang HJ, et al. (2009) Simultaneous analysis of anthocyanins and favonols in petals of lotus (Nelumbo) cultivars by high-performance liquid chromatography-photodiode array detection /electrospray ionization masss pectrometry. J Chromatogr A 1216: 106–112.
        • 37.Springob K, Nakajima JI, Yamazaki M, Saito K (2003) Recent advances in the biosynthesis and accumulation of anthocyanins. Nat Prod Rep 20: 288–303.
        • 38.Androutsopoulos VP, Papakyriakoub A, Vourloumisb D, Tsatsakisa AM, Spandidosc DA (2010) Dietary flavonoids in cancer therapy and prevention: Substrates and inhibitors of cytochrome P450 CYP1 enzymes. Pharmacol Therapeut 126: 9–20.
        • 39.Hou DX, Yanagita T, Uto T, Masuzaki S, Fujii M (2005) Anthocyanidins inhibit cyclooxygenase-2 expression in LPS-evoked macrophages: structure-activity relationship and molecular mechanisms involved. Biochem Pharmacol 70: 417–425.
        • 40.Afaq F, Syed DN, Malik A, Hadi N, Sarfaraz S, et al. (2007) Delphinidin, an anthocyanidin in pigmented fruits and vegetables, protects human HaCaT keratinocytes and mouse skin against UVB-mediated oxidative stress and apoptosis. J Invest Dermatol 127: 222–232.
        • 41.Tanaka Y, Katsumoto Y, Brugliera F, John M (2005) Genetic engineering in floriculture. Plant Cell Tiss Org 80: 1–24.
        • 42.Gonnet JF (1998) Colour effects of co-pigmentation of anthocyanins revisted-1. A colorimetric denition using the CIELAB scale. Food Chem 63: 409–415.
        • 43.Gonnet JF (1999) Colour effects of co-pigmentation of anthocyanins revisted-2. A colorimetric look at the solutions of cyanin co-pigmented by rutin using the CIELAB scale. Food Chem 66: 387–394.
        • 44.Lin LZ, Harnly JM (2007) A screening method for the identification of glycosylated flavonoids and other phenolic compounds using a standard analytical approach for all plant materials. J Agric Food Chem 55: 1084–1096.
        Back to blog