Flavonoids from Polygonum hydropiper L.(Polygonaceae)

Journal of Chemistry, Vol. 42 (4), P. 512 - 515, 2004<br /> <br /> <br /> Flavonoids from Polygonum hydropiper L.<br /> (Polygonaceae)<br /> Received 6th-Jan.-2004<br /> Nguyen Van Dau, Vu Nhuan Thang<br /> Faculty of chemistry, Hanoi University of Science, VNU<br /> <br /> <br /> summary<br /> Five flavonoids along with -sitosterol were isolated from the leaves of Polygonum<br /> hydropiper L. (Polygonaceae). Four flavonoids were identified as quercetin, quercetin-3-O- -L-<br /> rhamnoside (quercitrin), quercetin-3-sulphate and (+)-catechin. Their structures were<br /> established on the basis of chemical evidence and spectroscopic techniques, including 2D NMR<br /> methods.<br /> <br /> <br /> I - Introduction flavonoids in Polygonum hydropiper L., we now<br /> report the isolation and identification of four<br /> Many species of the genus Polygonum flavonoids, quercetin, quercetin-3- -L-rhamnoside<br /> (Polygonaceae) are used in traditional medicine (quercitrin), quercetin-3-sulphate and (+)-catechin<br /> against kidney stones and as antidiabetic, from the leaves of Polygonum hydropiper L.<br /> diuretic, and antidiarrhoeal agents [1]. The<br /> genus Polygonum produces a wide range of II - Experimental<br /> secondary metabolites including flavonoids,<br /> anthraquinones, naphthoquinones, sesquiterpen- 1. General experimental procedures<br /> oids, lignans, coumarins, stilbene glycosides,<br /> phenol carboxylic acids and so on [2]. UV spectra were determined in<br /> Polygonum hydropiper L. is a small herb, spectroscopic grade MeOH on a Shimadzu UV-<br /> 30 - 60 cm in height, which widely distributes VIS GCB-2855 spectrophotometer, and IR<br /> in Europe and Asia. It grows wild in wet spectra on a Impact- 410 Nicolet FT-IR<br /> lowland areas along canals, pools, fields, etc. spectrometer as pressed KBr disks. 1H, 13C<br /> There are few papers reported on Polygonum NMR, 1H-1H COSY, HMQC and HMBC NMR<br /> hydropiper L. showing the presence of spectra were recorded on a Bruker AV 500<br /> sesquiterpenes, dialdehydes, polygodial and instrument at 500 MHz for 1H and 125.7 MHz<br /> warburganal, which exhibit antifeedant, for 13C. TLC was carried out on precoated silica<br /> antimicrobial, plant growth inhibitory, cytotoxic gel 60F254 aluminum sheets (Merck); for column<br /> and pesticidal activities, together with several chromatography (CC), normal phase silica gel<br /> related drimane-type sesquiterpenes. In 60 (0.040 - 0.063 mm, Merck), and polyamide<br /> addition, other kinds of metabolites are also (6S, 0.160 mm, Riedel-Haen) were used.<br /> found, such as tri-p-coumaryl glucoside from Compounds were detected by UV fluorescence<br /> the roots, antioxidative sulphated flavonoids and/or spraying with vanillin-H2SO4 reagent<br /> from the leaves, etc [3, 4]. followed by heating them above 100oC for about<br /> In the course of a chemical examination of 5 min.<br /> <br /> 512<br /> 2. Plant material 0.68 (toluene-acetone, 4 : 6, v/v), turns bright-<br /> red with vanillin-H2SO4, and dark-blue with the<br /> P. hydropiper was collected from Truc<br /> FeCl3 solution. IR (KBr) max 3303, 1623, 1511,<br /> Chinh commune, Truc Ninh district, Nam Dinh<br /> 1465, 1034, 814 cm-1. 1H- and 13C-NMR<br /> in September 2003. The species was identified<br /> (MeOD), see Table. Compound 3 was pale-<br /> by Tran Van On (Dept. of Herbarium, Hanoi<br /> Pharmacy Institute). A voucher specimen has yellow crystals (0.5 g), mp 315 - 316oC; Rf 0.72<br /> been deposited in the Laboratory of Natural (toluene-acetone, 4 : 6, v/v), turns bright-yellow<br /> Products Chemistry, Hanoi University of with vanillin-H2SO4, and dark-blue with the<br /> Science. FeCl3 solution. IR (KBr) max 3419, 1663, 1610,<br /> 1562, 1014, 822 cm-1. UV (MeOH) max, nm:<br /> 3. Extraction and isolation 256, 374; +AlCl3: 269, 433; + (AlCl3+HCl):<br /> The dried powdered aerial parts of P. 256, 369; +AcONa: 273, 433;<br /> hydropiper L. were extracted 5 times with 70% +(H3BO3+AcONa): 260, 388. 1H and 13C NMR<br /> methanol at room temperature. The aqueous (MeOD), see table.<br /> methanol extracts were combined and The n-butanol extract (5.0 g) was separated<br /> concentrated in vacuo to a suitable volume by column chromatography over polyamide (50<br /> (about 0.5 L), then partitioned with n-hexane, g), eluting with mixtures of methanol-water.<br /> dichloromethane, ethyl acetate and n-butanol, Compound 4 was obtained as pale-yellow<br /> respectively. needles by the fractional crystallization in<br /> The ethyl acetate extract (10.0 g) was aqueous methanol.<br /> chromatographed over silica gel (100 g), eluting Compound 4 was pale-yellow needles, mp<br /> with toluene (100 mL), followed by increasing 255oC (dec.); Rf 0.81 (ethyl acetate-methanol, 7<br /> ratio of ethyl acetate to toluene (10% 100% : 3, v/v), turns dark-yellow with vanillin-H2SO4.<br /> ethyl acetate) to yield 80 fractions which were UV (MeOH) max, nm: 255 and 372; +AlCl3:<br /> combined into 5 main fractions (EA1 EA5). 270, 442; +AlCl3+HCl: 264, 429; + MeONa:<br /> The fraction EA4 and EA5 were subjected to 244, 329; +AcONa: 272, 390; +AcONa+H3BO3:<br /> fractional crystallization from a mixture of 259, 386. 1H-and 13C-NMR (MeOD), see table.<br /> toluene and acetone to yield a pale-yellow<br /> amorphous powder signed as compound 1.<br /> III - Results and discussion<br /> Compound 1 was pale-yellow amorphous<br /> powder (0.92 g), mp 178 - 179oC; Rf 0.56 The aqueous ethanolic extract (MeOH : H2O<br /> (toluene-acetone, 4 : 6, v/v), turns dark-blue = 7 : 3) of the aerial parts of P. hydropiper was<br /> with the FeCl3 solution. IR (KBr) max 3267, concentrated in vacuo, suspended in water and<br /> 1664, 1598, 1501, 1449, 1158, 1065, 963, 814 then partitioned with solvents of increasing<br /> cm-1. 1H- and 13C-NMR (MeOD), see Table. polarity (n-hexane, dichloromethane, ethyl<br /> Acid hydrolysis of 1 was carried out in heating acetate, and n-butanol). The ethyl acetate<br /> with 10% HCl for 30 min. followed by residue was fractionated on silica gel column<br /> extraction with ethyl acetate. The concentrated chromatography to give compounds 1 - 3.<br /> organic layer was compared with quercetin by<br /> Compound 1 was obtained as a pale-yellow<br /> TLC, developing in a mixture of toluene-<br /> acetone-water (10 : 20 : 1, v/v/v). amorphous powder. The structure of 1 was<br /> identified on the basis of comparison of its<br /> The fractions EA2 and EA3 were further spectroscopic (IR, 1H- and 13C-NMR) data with<br /> purified by repeated open CC (silica gel) using literature values and physicochemical evidence.<br /> mixtures of toluene-acetone and then by means On acid hydrolysis 1 gave quercetin which was<br /> of fractional crystallization compounds 2 and 3 confirmed by comparison with an authentic<br /> were successfully isolated. Compound 2 was sample. This result proved 1 to be a glycoside of<br /> white-off needles (0.55 g), mp 175 - 177oC; Rf quercetin. The IR spectrum of 1 indicated the<br /> 513<br /> presence of hydroxyl (3267 cm-1), conjugated and H6’; C3’ - H2’ and H5’; C4’-H2’, H5’ and H6’; On<br /> carbonyl (1666 cm-1), aromatic (1598, 1501 and the basis of spectroscopic analysis and<br /> 1449 cm-1), and ether (1157, 1065 cm-1) groups. comparison in the coupling constant of the<br /> 1<br /> H- and 13C-NMR data determined the quercetin methine protons H2 and H3 (J = 16.1 Hz)<br /> skeleton. For example, the 1H-NMR spectra compound 2 was identified as (+)-catechin.<br /> displayed the characteristic signals of 5 Compound 3 was obtained as pale-yellow<br /> aromatic protons, 6.21 (1H, d, J = 2.1 Hz, H-6), needles. Its IR spectrum showed absorption<br /> 6.37 (1H, d, J = 2.1 Hz, H-8), 7.35 (1H, d, J = bands due to hydroxyl (3419 cm-1), carbonyl<br /> 2.1 Hz, H-2’), 6.92 (1H, d, J = 8.3 Hz, H-5’), (1663 cm-1), aromatic (1610, 1562 cm-1), and<br /> 7.32 (1H, dd, J = 2.1 and 8.3 Hz, H-6’). The ether (1014 cm-1) groups. The UV maxima (257<br /> 13<br /> C-NMR (DEPT) spectra of 1 exhibited 21 nm, band II; 375 nm, band I) changed on<br /> carbon resonances: ten quaternary carbons (C), addition of aluminum chloride (269 and 433<br /> ten methines (CH), and one methyl (CH3) (see nm, respectively) and of sodium acetate (274<br /> table). The 1H- and 13C-NMR spectral data and 433 nm, respectively), indicating that 3 is a<br /> supported the presence of L-rhamnose, and from flavonoid and that hydroxyl groups are located<br /> the chemical shift values and the coupling at the 5 and 3’positions. The structure of 3 was<br /> constants of the anomeric proton, the made clearly by comparing its 1H- and 13C-<br /> glycosidation was found to be -linkage at C-3. NMR data with literature values reported for<br /> Consequently, the structure of 1 was established quercetin and further confirmed by the direct<br /> as quercetin-3-O- -L-rhamnoside. comparison with an authentic sample in the<br /> Compound 2 was isolated as white-off same TLC conditions.<br /> crystals. In its DEPT-13C-NMR spectrum fifteen n-butanol residue was separated on a<br /> carbons [comprising seven quaternary carbons polyamide column, eluting with mixtures of<br /> (C), seven methines (CH) and one methylene methanol-water. Several fractions were<br /> (CH2)] were observed. The 1H-NMR and subjected to fractional crystallization in aqueous<br /> COSY-spectrum showed the presence of methanol to afford compounds 4 as pale-yellow<br /> substituted benzene rings [ 5.80 (1H, d, J = 2.3 needles. The UV spectrum of 4 indicates the<br /> Hz, H-6), 5.90 (1H, d, J = 2.3 Hz, H-8), 6.86 maxima (255 nm, band II; 373 nm, band I)<br /> (1H, d, J = 1.8 Hz, H-2’), 6.79 (1H, d, J = 8.0 characterized flavonol-type compounds.<br /> Hz, H-5’), 6.75 (1H, dd J = 1.8 and 8.1 Hz, H- HO O<br /> 6’). In addition, two methylene protons [ 2.86 OH<br /> (1H, dd, J = 5.4 and 16.1 Hz, H-4a) and 2.53 OH<br /> (1H, dd, J = 8.1 and 16.1 Hz, H-4b)] and two OR<br /> methine protons [ 4.59 (1H, d, J = 7.5 Hz, H-2) HO O<br /> and 4.0 (1H, m, H-3)] were successively Quercetin, R = H; Quercitrin, R = L-rhamnose<br /> coupled. The 13C-NMR signals of 2 were Quercetin-3-sulfate, R = HSO3<br /> assigned with the help of an HMQC experiment,<br /> establishing direct following C-H bonds: H2 HO O<br /> OH<br /> (4.59) at C2 (82.8); H3 (4.0) at C3 (68.7); H4a<br /> (2.86) and H4 b (2.53) at C4 (28.4); H6 (5.8) at C6 OH<br /> OH<br /> (96.3); H8 (5.9) at C8 (95.5); H2’ (6.86) at C2’<br /> (115.2); H5’ (6.79) at C5’ (116.1) and H6’ (6.74) HO<br /> at C6’(120.0) (see table). The placement of the<br /> all substituents on the A and C rings was (+)-Catechin<br /> established using 2D 13C-1H long-range Bathochromic shifts were observed in<br /> correlation (HMBC) experiment. The long- addition of both AlCl3/ or AlCl3+HCl (the band<br /> range correlations were observed between C5- I: 442/429 nm, respectively) indicating the<br /> H6; C7-H6 and H8; C9 - H2 and H8; C1’-H2’, H3, H5’, presence of a hydroxyl group at C-5 coupled<br /> 514<br />  Table: 13C-NMR and 1H- data ( , ppm; J, Hz) for compounds 1- 4<br /> <br /> Post. 1 2 3 4<br /> c H ,J c H, J c H ,J c H ,J<br /> 2 159.3 82.8 4.59 d (7.5) 146.0 148.7<br /> 3 136.2 68.7 4.0 m 135.6 146.6<br /> 4 179.6 28.4 Ha 2.86 dd (5.4; 176.5 177.0<br /> 16.1) Hb 2.53 dd<br /> (8.1; 16.1)<br /> 5 163.1 157.8 161.0 162.5<br /> 6 99.8 6.21 d (2.1) 96.3 5.90 d (2.3) 98.2 6.20 d (2.0) 99.2 6.20 d (2.0)<br /> 7 165.7 157.5 165.0 165.6<br /> 8 94.7 6.38 d (2.1) 95.5 5.80 d (2.3) 93.5 6.50 d (2.0) 94.4 6.40 d (2.0)<br /> 9 158.4 156.8 152.6 158.2<br /> 10 103.5 100.8 103.0 104.5<br /> 1’ 122.8 132.2 122.0 124.0<br /> 2’ 116.9 7.35 d(2.1) 115.2 6.86 d (1.8) 117.1 7.80 d (2.0) 116.2 7.75 d (2.0)<br /> 3’ 146.3 146.1 140.0 146.0<br /> 4’ 149.7 146.2 146.2 148.0<br /> 5’ 116.3 6.92 d (8.3) 116.1 6.79 d (8.0) 115.6 7.00 d (2.0) 116.0 6.93 d (2.0)<br /> 7.70; dd 7.65 dd<br /> 6’ 122.9 7.32 dd (2.1; 8.3) 120.0 6.74; dd (1.8; 8.1) 120.0 121.7<br /> (2.0; 8.3) (2.0; 9.0)<br /> Rha.<br /> 1” 103.5 5.34 d (1.3)<br /> 2” 72.0 3.8 m<br /> 3” 71.9 3.46 dd<br /> (3.6; 6.1)<br /> 4” 73.3 3.36 m<br /> 5” 71.8 3.33 m<br /> 6” 17.6 0.99 d (6.2)<br /> <br /> with >C4=O, and AcONa+H3BO3 (the band I: to Asia Research Center for the financial<br /> 386 nm) or MeONa (423 and 329 nm) proving support for this work.<br /> the presence of two octo-hydroxyl groups at C-<br /> 3’and C-4’. The 1H- and 13C-NMR signals of 4 References<br /> and those of quercetin were almost<br /> superimposable on each other with the 1. Do Tat Loi. Medic. Plants and Medic.<br /> exception of the signal at C-3 (see experimental Ingredi. of Vietnam, Sci&Tech. Publisher,<br /> part). On acid hydrolysis with 10% HCl 4 gave Hanoi, 1995.<br /> quercetin which was identified by direct 2. Calis, A. Kurüzüm, L. O. Demirezer, .<br /> comparison with an authentic sample, and the Sticher, W. Ganci, P. and Rüedi. Nat. Prod.<br /> anion O-SO32-, which was confirmed by 62, 1101- 1105 (1999).<br /> precipitation with BaCl2. The group O-SO32- was 3. Y. Fukuyama, T. Sato, I. Miura, and Y.<br /> assigned to C-3 due to the unusual downfield Asakawa. Phytochem., Vol. 24, No. 7, P.<br /> shift of the C-3 signal (146.6 ppm). 1521 - 1524 (1985).<br /> Consequently, 4 was identified as quercetin-3- 4. A. Yagi, T. Uemura, N. Okamura, H.<br /> sulphate. Haraguchi, T. Imoto, and K. Hashimoto.<br /> Acknowledgements: The authors are grateful Phytochem., Vol. 35, No. 4, P. 885 - 887 (1994).<br /> <br /> 515<br /> 516<br />