Flavonoids in translucent bracts of the Himalayan Rheum nobile (Polygonaceae) as ultraviolet shields
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- Iwashina, T., Omori, Y., Kitajima, J. et al. J Plant Res (2004) 117: 101. doi:10.1007/s10265-003-0134-2
UV-absorbing substances were isolated from the translucent bracts of Rheum nobile, which grows in the alpine zone of the eastern Himalayas. Nine kinds of the UV-absorbing substances were found by high performance liquid chromatography (HPLC) and paper chromatography (PC) surveys. All of the five major compounds are flavonoids, and were identified as quercetin 3-O-glucoside, quercetin 3-O-galactoside, quercetin 3-O-rutinoside, quercetin 3-O-arabinoside and quercetin 3-O-[6″-(3-hydroxy-3-methylglutaroyl)-glucoside] by UV, 1H and 13C NMR, mass spectra, and acid hydrolysis of the original glycosides, and direct PC and HPLC comparisons with authentic specimens. The four minor compounds were characterised as quercetin itself, quercetin 7-O-glycoside, kaempferol glycoside and feruloyl ester. Of those compounds, quercetin 3-O-[6″-(3-hydroxy-3-methylglutaroyl)-glucoside] was found in nature for the first time. The translucent bracts of R. nobile accumulate a substantial quantity of flavonoids (3.3–5 mg per g dry material for the major compounds). Moreover, it was clarified by quantitative HPLC survey that much more of the UV-absorbing substances is present in the bracts than in rosulate leaves. Although the flavonoid compounds have been presumed to be the important UV shields in higher plants, there has been little characterisation of these compounds. In this paper, the UV-absorbing substances of the Himalayan R. nobile were characterised as flavonol glycosides based on quercetin.
KeywordsFlavonoidsHimalayan alpine plantsQuercetin 3-O-[6″-(3-hydroxy-3-methylglutaroyl)-glucoside]Rheum nobileTranslucent bractsUV-absorbing substances
Rheum nobile Hook. f. & Thomson (Polygonaceae) is endemic to the alpine zone of the eastern Himalayas and grows to a height of about 1.5 m (Kikuchi et al. 1999; Omori and Ohba 1999). One of the outstanding features of the species is the large translucent cream-coloured bracts concealing the huge inflorescence (Ohba 1988). A series of biological studies of R. nobile revealed various peculiar structures and functions (Omori and Ohba 1996, 1999, 2003; Terashima et al. 1993, 1995; Kikuchi et al. 1999; Omori et al. 2000). One important function of the translucent bracts is to produce a greenhouse effect for the inflorescences against extreme cold in high mountains such as the Himalayas (Omori and Ohba 1999). Another function was presumed to be defence against noxious UV radiation, especially UV-B. Indeed, Omori et al. (2000) proved that the translucent bracts of R. nobile selectively absorb UV-radiation, though visible light was almost completely transmitted. Moreover, they presumed that the epidermis and hypodermal layers of R. nobile contain many flavonoids dissolved in the cells to screen UV radiation. The characterisation of the flavonoids, however, was not performed. It was expected that in many plant species such as Vaccinium reticulatum (Ericaceae), Dubautia menziesii (Compositae) and Rumex acetosella (Polygonaceae) the flavonoids are important UV-absorbing substances (Caldwell et al. 1983). Indeed, it was shown that two acylated flavonol glycosides, kaempferol 3-O-(3″,6″-di-p-coumaroylglucoside) and quercetin 3-O-(3″,6″-di-p-coumaroylglucoside) in Pinus sylvestris (Pinaceae) (Jungblut et al. 1995), quercetin 3-O-galactoside, myricetin 3-O-rutinoside and two p-coumaroyl kaempferol 3-O-glucosides in Quercus ilex (Fagaceae) (Skaltsa et al. 1994), and glycosides of quercetin, apigenin and luteolin in Olea europaea (Oleaceae) (Karabourniotis et al. 1992) act as UV-absorbing substances. However, the isolation and identification of the flavonoids as UV shields have been accomplished only to a limited extent in most plant species, especially alpine plants.
In this survey, the flavonoid compounds in the translucent bracts and leaves of the Himalayan Rheum nobile were isolated and identified as UV shields.
Materials and methods
Collection of material
Isolation of the flavonoids
Dry bracts and leaves (3 g) were extracted with methanol. After concentration in vacuo, the extracts were applied to preparative paper chromatography using BAW (n-BuOH/HOAc/H2O = 4:1:5, upper phase), 15% HOAc and then BEW (n-BuOH/EtOH/H2O = 4:1:2.2) as solvent systems. Each flavonoid was purified by Sephadex LH-20 column chromatography (with 70% MeOH as solvent) and finally obtained as pale yellow powders (major compounds) or pure solutions (minor ones).
Identification of the flavonoids
The flavonoids were identified by UV spectra according to Mabry et al. (1970), 1H- and 13C-nuclear magnetic resonance (1H- and 13C-NMR) spectra, liquid chromatography-mass spectrometry (LC-MS), acid hydrolysis and the characterisation of their products according to Iwashina et al. (2001), and direct PC and HPLC comparisons with authentic specimens.
UV, MS, 1H and 13C NMR, HPLC, and LC-MS data for the compounds isolated are given below.
Quercetin 3-O-rutinoside (rutin, D1)
PC Rf: 0.42 (BAW), 0.49 (BEW), 0.46 (15% HOAc), 0.32 (5% HOAc); UV-dark purple, UV/NH3-yellow. UV λmax (nm): MeOH 257, 266sh, 359; +NaOMe 273, 327, 413 (inc.); +AlCl3 274, 431; +AlCl3/HCl 269, 300, 365, 398; +NaOAc 273, 326, 399; +NaOAc/H3BO3 262, 379. HPLC retention time 5.63 min. LC-MS: ESI+ 633 [M+Na]+, ESI− 609 [M-H]−, calculated for C27H30O16.
Quercetin 3-O-arabinopyranoside (guaijaverin, D2)
PC Rf: 0.62 (BAW), 0.63 (BEW), 0.25 (15% HOAc), 0.10 (5% HOAc); UV-dark purple, UV/NH3-yellow. UV λmax (nm): MeOH 257, 266sh, 357; +NaOMe 273, 329, 409 (inc.); +AlCl3 275, 435; +AlCl3/HCl 269, 300, 363, 402; +NaOAc 273, 326, 394; +NaOAc/H3BO3 261, 378. 1H NMR (270 MHz, pyridine-d5): δ 8.40 (1H, d, J = 1.2 Hz, H-2′), 8.12 (1H, dd, J = 1.2 and 9.8 Hz, H-6′), 7.28 (1H, dd, J = 0.6 and 9.1 Hz, H-5′), 6.72 (1H, d, J = 1.8 Hz, H-8), 6.68 (1H, d, J = 0.9 Hz, H-6), 6.09 (1H, d, J = 5.2 Hz, arabinosyl H-1), 3.8–5.0 (m, sugar protons). 13C NMR (67 MHz, pyridine-d5): (quercetin) δ 157.6 (C-2), 135.5 (C-3), 178.9 (C-4), 162.8 (C-5), 99.8 (C-6), 165.9 (C-7), 94.5 (C-8), 157.7 (C-9), 105.2 (C-10), 122.8 (C-1′), 116.4 (C-2′), 147.0 (C-3′), 150.8 (C-4′), 117.5 (C-5′), 122.3 (C-6′); (arabinose) δ 104.5 (C-1), 72.8 (C-2), 74.1 (C-3), 68.2 (C-4), 66.4 (C-5). HPLC retention time 7.71 min. LC-MS: ESI+ 457 [M+Na]+, ESI− 433 [M-H]−, calculated for C20H18O11.
Quercetin 3-O-galactoside (hyperin, D3a) and quercetin 3-O-glucoside (isoquercitrin, D3b)
PC Rf: 0.56 (BAW), 0.62 (BEW), 0.32 (15% HOAc), 0.17 (5% HOAc); UV-dark purple, UV/NH3-yellow. UV λmax (nm): MeOH 257, 266sh, 360; +NaOMe 273, 330, 410 (inc.); +AlCl3 275, 433; +AlCl3/HCl 268, 299, 363, 401; +NaOAc 273, 325, 395; +NaOAc/H3BO3 261, 378. HPLC retention time 6.30 min (3-O-glucoside), 6.12 min (3-O-galactoside). LC-MS: ESI+ 487 [M+Na]+, ESI− 463 [M-H]−, calculated for C21H20O12.
Quercetin 3-O-[6″-(3-hydroxy-3-methylglutaroyl)-glucoside] (D4)
PC Rf: 0.61 (BAW), 0.45 (BEW), 0.44 (15% HOAc), 0.30 (5% HOAc); UV-dark purple, UV/NH3-yellow. UV λmax (nm): MeOH 258, 265sh, 358; +NaOMe 272, 330, 409 (inc.); +AlCl3 276, 434; +AlCl3/HCl 270, 297sh, 364, 400; +NaOAc 273, 325, 390; +NaOAc/H3BO3 262, 379. 1H NMR (270 MHz, pyridine-d5): δ 8.34 (1H, d, H-2′), 8.10 (1H, dd, J = 1.2 and 6.7 Hz, H-6′), 7.34 (1H, d, J = 8.2 Hz, H-5′), 6.71 (1H, d, H-8), 6.67 (1H, d, H-6), 6.12 (1H, d, J = 6.7 Hz, glucosyl H-1), 4.87 (1H, d, J = 11.5 Hz, H-6″A), 4.6 (1H, dd, H-6″B), 4.1–4.4 (m, sugar protons), 3.13 (1H, d, J = 10.4 Hz, H-8″A or H-8″B), 3.08 (1H, d, J = 9.5 Hz, H-8″B or H-8″A), 3.03 (1H, d, J = 5.5 Hz, H-10″A or H-10″B), 2.97 (1H, d, J = 4.9 Hz, H-10″B or H-10″A), 1.66 (3H, s, H-12″). 13C NMR (67 MHz, pyridine-d5): (quercetin) δ 157.6 (C-2), 135.2 (C-3), 178.7 (C-4), 162.7 (C-5), 99.8 (C-6), 166.0 (C-7), 94.6 (C-8), 158.1 (C-9), 105.2 (C-10), 122.3 (C-1′), 116.2 (C-2′), 146.9 (C-3′), 150.8 (C-4′), 117.6 (C-5′), 122.9 (C-6′); [6-(3-hydroxy-3-methylglutaroyl)-glucose] δ 104.3 (C-1), 75.8 (C-2), 78.4 (C-3), 71.1 (C-4), 75.9 (C-5), 64.4 (C-6), 171.7 (C-7), 46.5 (C-8), 70.0 (C-9), 46.3 (C-10), 175.2 (C-11), 28.1 (C-12). HPLC retention time 7.20 min. LC-MS: ESI+ 609 [M+H]+, ESI− 607 [M−H]−, calculated for C27H28O16.
Kaempferol glycoside (D5)
PC Rf: 0.71 (BAW), 0.51 (BEW), 0.51 (15% HOAc), 0.33 (5% HOAc); UV-dark purple, UV/NH3-dark greenish yellow. UV λmax (nm): MeOH 267, 293sh, 346.
Quercetin 7-O-glycoside (Y1)
PC Rf: 0.42 (BAW), 0.48 (BEW), 0.07 (15% HOAc), 0.01 (5% HOAc); UV-yellow, UV/NH3-bright yellow. UV λmax (nm): MeOH 250, 266sh, 366.
PC Rf: 0.79 (BAW), 0.70 (BEW), 0.40 (Forestal, HOAc/HCl/H2O = 30:3:10); UV and UV/NH3-yellow. UV λmax (nm): MeOH 255, 371; +NaOMe decomposition; +AlCl3 272, 456; +AlCl3/HCl 266, 303, 361, 426; +NaOAc 274, 324, 401; +NaOAc/H3BO3 260, 388.
Feruloyl ester (B1)
PC Rf: 0.49 (BAW), 0.52 (BEW), 0.69 (15% HOAc), 0.63 (5% HOAc); UV-bright blue, UV/NH3-bright green. UV λmax (nm): MeOH 244sh, 327; +NaOMe 262sh, 303sh, 391 (inc.); +AlCl3 244sh, 266sh, 323; +AlCl3/HCl 244sh, 323; +NaOAc 267sh, 330, 400; +NaOAc/H3BO3 287sh, 330. HPLC retention time 4.12 min.
Qualitative and quantitative HPLC analysis of isolated compounds and crude extracts
Qualitative HPLC separation of the isolated compounds was performed with Shimadzu HPLC systems using Shim-pack CLC-ODS [I.D. 6.0 × 150 mm (Shimadzu)], at a flow-rate of 1.0 ml/min; detection wavelength was 190–360 nm and eluent was MeCN/H2O/H3PO3 (22:78:0.2) according to Iwashina et al. (1996). In quantitative HPLC analysis, each 0.1 g of dry bract or leaf was extracted with 3 ml MeOH, and the extracts were directly analysed with the above HPLC systems, after filtration with Maishori-disk H-13-5 (Tosoh).
1H and 13C NMR spectra
1H and 13C NMR spectra (270 and 67 MHz) were recorded in pyridine-d5 using JEOL A-500 spectrometer with TMS as an internal standard. 13C-1H correlation spectroscopy (COSY) was obtained with the usual pulse sequence and data processing was performed with standard JEOL software.
LC-MS was surveyed with Symmetry C18 column [I.D. 2.1×150 mm (Waters)], at a flow-rate of 0.18 ml/min, eluting with 15% MeCN rising to 45% MeCN (30 min), ESI+ 3.0 kV, cone voltage 30 V, ESI− 3.0 kV, cone voltage 30 V, 400°C, ion energy 1.0 V.
Results and discussion
Identification of UV-absorbing substances
Five major compounds (D1, D2, D3a, D3b and D4) and four minor ones (D5, Y1, Y2 and B1) were characterised as UV-absorbing substances. The major compounds were isolated as pale yellow powders.
UV spectral properties of D1 were those of typical flavonol glycoside, i.e., the presence of absorption maxima at 359 (band I) and 257 nm (band II). On addition of shift reagents (NaOMe, AlCl3, AlCl3/HCl, NaOAc, NaOAc/H3BO3), the presence of free 5-, 7-, 3′- and 4′-hydroxyl groups and a substituted 3-hydroxyl group was shown. Quercetin, glucose and rhamnose were liberated by acid hydrolysis. Since 611 [M+H]+ appeared by LC-MS, the attachment of one molecule each of glucose and rhamnose to quercetin was shown. Finally, D1 was identified as quercetin 3-O-rutinoside, i.e., rutin, by direct HPLC and PC comparison with an authentic specimen. Rutin is widespread in higher plants and has been reported from the leaves and petioles of some Rheum species, R. palmatum var. tanguticum, R. undulatum and R. rhaponticum, as a major flavonoid (Blundstone 1967; Kawasaki et al. 1986).
UV spectral properties of D2 were essentially the same as those of D1, showing it to be flavonol glycoside with free 5-, 7-, 3′- and 4′-hydroxyl groups and a substituted 3-hydroxyl group. Quercetin and arabinose were obtained by acid hydrolysis. In addition, the molecular ion, 457 [M+Na]+, was shown by LC-MS, showing the attachment of one molecule of arabinose to quercetin. From the results described above, the compound was characterised as quercetin 3-O-monoarabinoside. Quercetin 3-O-arabinosides have been reported as 3-O-α-l-arabinofuranoside, 3-O-α-l-arabinopyranoside (Geiger and Groot-Pfleidere 1979) and 3-O-β-l-arabinopyranoside (Servettaz et al. 1984). In the 1H NMR spectra, the arabinosyl anomeric proton of the isolated flavonoid exhibited the coupling constant, J = 5.2 Hz, showing it to be α-l-arabinopyranoside (Markham and Geiger 1994). Finally, the flavonoid was identified as quercetin 3-O-α-l-arabinopyranoside, i.e., quaijaverin, by the comparison of 13C NMR spectral data with the literature (Markham et al. 1978; Matsuura et al. 1978; Pachaly and Klein 1987). Quercetin 3-O-arabinoside has been found in the Rheum species for the first time.
Flavonoid D3 appeared as a single spot on PC, but divided into two peaks on HPLC. Quercetin, glucose and galactose were liberated by acid hydrolysis of the original glycoside. UV spectral data of D3 showed the presence of free 5-, 7-, 3′- and 4′-hydroxyl groups and a substituted 3-hydroxyl group, showing that the sugar is attached to the 3-hydroxyl group of quercetin. The direct HPLC and PC comparisons with authentic hyperin and isoquercitrin showed that flavonoid D3 is a mixture of quercetin 3-O-galactoside (D3a) and quercetin 3-O-glucoside (D3b). Hyperin and isoquercitrin are also widespread in plants, and have been reported from the leaves and petioles of R. rhaponticum (Blundstone 1967; Kawasaki et al. 1986).
UV spectral data of flavonoid D4 showed the presence of free 5-, 7-, 3′- and 4′-hydroxyl groups and a substituted 3-hydroxyl group. Though quercetin and glucose, which were characterised by co-PC with authentic specimens, were liberated by usual acid hydrolysis, LC-MS data, 609 [M+H]+, exhibited the attachment of an unknown substance of molecular weight 144 on addition of one molecule of glucose to the 3-hydroxyl group of quercetin. 1H NMR data of the original glycoside indicated four methylene protons (δ 2.97, 3.03, 3.08 and 3.13) and a C-methyl proton (δ 1.66), which originate from the unknown substance, in addition to five aromatic protons (6, 8, 2′, 5′, 6′-positions) and a glucosyl anomeric proton (δ 6.12, J = 6.7 Hz).
Quercetin 3-O-glycoside corresponding to molecular weight 608 has been isolated from blackberries, i.e., the fruits of Rubus sp. (Rosaceae) and identified as quercetin 3-O-[6″-(3-hydroxy-3-methylglutaroyl)-β-galactoside] (Wald et al. 1986). 13C NMR data of flavonoid D4 was compared with that of the glycoside described above and the attachment of a 3-hydroxy-3-methylglutaric acid function to the 6-position of the 3-O-glucosyl group of quercetin was recognised. Thus, flavonoid D4 was identified as quercetin 3-O-[6″-(3-hydroxy-3-methylglutaroyl)-β-d-glucopyranoside], which was found for the first time in nature. The acylated flavonoid glycoside having a 3-hydroxy-3-methyglutaroyl group has only been reported from Rubus sp. (Rosaceae) (Wald et al. 1986). This paper is the second report of an acylated flavonoid having a 3-hydroxy-3-methylglutaroyl group.
Minor compound Y1 showed the presence of free 3-, 5-, 3′- and 4′-hydroxyl groups and a substituted 7-hydroxyl group. Though quercetin was liberated by acid hydrolysis, glycosidic sugar was not detectable on account of the minute amount of the original glycoside. Thus, flavonoid Y1 was characterised as quercetin 7-O-glycoside.
Flavonoid Y2 appeared as a yellow spot on the paper chromatogram by UV light, and is soluble in diethyl ether, showing it to be flavonol aglycone. UV spectral data of the flavonoid showed the presence of free 3-, 5-, 7-, 3′- and 4′-hydroxyl groups. Finally, Y2 was identified as quercetin itself by direct PC and HPLC comparison with an authentic specimen.
Compounds D5 and B1
Minor compounds D5 and B1 were presumed to be flavonol, kaempferol glycoside, and aromatic acid, feruloyl ester following paper chromatographic and UV spectral properties, and acid hydrolysis.
Qualitative and quantitative HPLC analyses of UV-absorbing substances in bracts and leaves
Quantitative HPLC analyses of bracts and rosulate leaves of Rheum nobile
Dry weight (mg/g)c
Dry weight (mg/g)
Half-green (ca. 50% green area) (C)
Dry weight (mg/g)
Dry weight (mg/g)
Deep green (rosulate leaf) (E)
Dry weight (mg/g)
It has been reported that flavonoid compounds have many biological functions in plants. The well-known function is the attraction of pollinators to flowers as floral pigments, anthocyanins, flavones and flavonols (Harborne and Grayer 1994). The flavonoids, especially isoflavonoids in roots and seeds, as defensive agents such as allelopathy and phytoalexin are also frequently reported (Bohm 1998). Nevertheless, the most frequently cited function of flavonoids may be to serve as UV filters, especially in leaves of vascular plants. However, the characterisation of UV shields in plants has been only little carried out. Though the protection of plants against UV radiation has been studied in vitro in horticultural plants such as cucumber (Cucumis sativus) (Kondo and Kawashima 2000), maize (Zea mays) (Stapleton and Walbot 1994), Arabidopsis thaliana (Li et al. 1993), UV shields, especially for UV-B, which cause damage to DNA, of alpine plants such as Rheum nobile growing in the high elevations of the Himalayan region, have not been surveyed. It was shown in this survey that the flavonoid contents of the translucent bracts of R. nobile are extremely high and the inflorescences of the plants are, therefore, protected from strong UV radiation. The major UV shields of R. nobile were five flavonoid glycosides, quercetin 3-O-glucoside, 3-O-galactoside, 3-O-rutinoside, 3-O-arabinoside and a new glycoside, 3-O-(3-hydroxy-3-methylglutaroylglucoside). Since their UV absorption properties are similar, all these compounds act as UV shields. Anthraquinones such as emodin and aloe-emodin, and proanthocyanidins have been isolated from the roots, seedlings, and callus culture of the Rheum species as the substances having absorption maxima in the UV range (Tutin and Clewer 1911; Rai 1978; Kashiwada et al. 1986). However, anthraquinones and proanthocyanidins were not found from the translucent bracts and leaves of R. nobile, showing that flavonol glycosides only act as UV shields in this species. The bracts of R. nobile are highly adapted to the environmental conditions of the eastern Himalayan alpine region because they do not transmit UV radiation, which is harmful to the developing flowers and apical meristem. It is remarkable that the effect of the bracts are probably produced by flavonol glycosides only. Rheum alexandrae, which is native to southwestern China, and Saussurea obvallata (Compositae) distributed from Pakistan to southwestern China, and S. involucrata in the Tien Sian Mountains are also glasshouse plants and have the translucent bracts. Recently, it was reported that the bracts of R. alexandrae are also specialised for protecting flowers from UV radiation (Tsukaya 2002). It is very interesting to know what UV-absorbing substances such glasshouse plants retain in the translucent bracts and leaves.