Flavonol glycosides and triterpenes from the leaves of Uncaria ...

Flavonol glycosides and triterpenes from the leaves of Uncaria rhynchophylla (Miq.) Jacks. / Asian Journal of TraditionalMedicines, 2009, 4 (3)Regular ArticlesFlavonol glycosides and triterpenes from the leaves ofUncaria rhynchophylla (Miq.) Jacks.Bin Ma a , Suiku Liu a , Yuanyuan Xie a , Yoshihiro Kano b , Dan Yuan a *a. School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang110016, China;b. Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, JapanAbstractTen compounds were isolated from the leaves of Uncaria rhynchophylla (Miq.) Jacks. by the chromatographic techniques. On thebasis of physico-chemical properties and spectral methods, they were identified as five flavonol glycosides, quercetin-3-O-α-Lrhamnopyranosyl-(1→6)-β-D-galactopyranoside(1), rutin (2), hyperoside (3), kaempferol-3-O-α-L-rhamnopyranosyl-(1→6)-β-Dgalactopyranoside(4) and kaempferol-3-O-β-D-galactopyranoside (5), three triterpenoids, 6β–hydroxyursolic acid (6), 3β, 6β,19α-trihydroxyurs-12-en-28-oic acid (7), and 3β, 6β, 23–trihydroxyurs-12-en-28-oic acid (8), and two phytosterols, β–sitosterol (9)and daucosterol (10) Compounds 1, 4 and 6 were obtained from the Uncaria sp. for the first time with compounds 2, 8 and 10 fromthis plant for the first time.Key words: Uncaria rhynchophylla; flavonol glycosides; triterpenes; chemical constituentIntroductionGambirplant (Gouteng) is a well-known herb,distributed in southern China. The hook and stem ofU. rhynchophylla, U. sinensis and U. macrophylla arerecorded in the Chinese and Japanese Pharmacopoeias[1, 2], which were often used to treat hypertension,epilepsia and vascular disease. Although oxindoleand indole alkaloids are responsible for its beneficialeffects on hypertension and some neurodegenerativediseases, recent pharmacological studies indicatedthat the phenol-containing fraction that was preparedfrom the methanol extract, and contained hyperin,* Author to whom correspondence should be addressed. Address:School of Traditional Chinese Materia Medica, ShenyangPharmaceutical University, Shenyang 110016, China. Tel & Fax:+86-24-2398-6502; E-mail: yuandan_kampo@163.comReceived: 2008-11-19 Accepted: 2009-04-01(-)epicatechin, caffeic acid and procyanidin B 2alsoshowed the protection against cerebral ischemia andweak antihypertensive activity [3] .In this paper, ten known compounds were isolatedfrom the leaves of U. rhynchophylla, and wereidentified as quercetin-3-O-α-L-rhamnopyranosyl-(1→6)-β-D-galactopyranoside (1), rutin (2), hyperoside(3), kaempferol-3-O-α-L-rhamnopyranosyl-(1→6)-β-D-galactopyranoside (4), kaempferol-3-O-β-Dgalactopyranoside(5), 6β-hydroxyursolic acid (6), 3β,6β, 19α-trihydroxyurs-12-en-28-oic acid (7), 3β, 6β,23-trihydroxyurs-12-en-28-oic acid (8), β-sitosterol(9) and daucosterol (10) Compounds 1, 4 and 6 wereobtained from the Uncaria sp. for the first time withcompounds 2, 8 and 10 from this plant for the firsttime.Experimental section85

Flavonol glycosides and triterpenes from the leaves of Uncaria rhynchophylla (Miq.) Jacks. / Asian Journal of TraditionalMedicines, 2009, 4 (3)HO6758OH1O9 2 1'104O3OR 22' 3'R 1OH1 R 1 =-OH, R 2 =- β Gal 6 - α Rha2 R 1 =-OH, R 2 =- β Glc 6 - α Rha3 R 1 =-OH, R2 =- β Gal4 R 1 =-H, R 2 =- β Gal 6 - α Rha5 R 1 =-H, R 2 =- β Gal6'5'4'HO232414 5R 11125 2610236 R 1 =-H, R 2 =-H7 R 1 =-H, R 2 =-OH8 R 1 =-OH, R 2 =-H96OH128729R 21813 171427153020 211916COOH2822R 1 O231411199510H612H8721 2220131418H17H1615239 R 1 =-H10 R 1 =- β Glc282429252627Fig. 1. The structures of compounds 1–10Fig.1 The structures of compounds 1–10General experimental proceduresMelting points were determined on a Yanacomicro melting-point apparatus (uncorrected) (Japan).UV spectra was obtained using a Shimadzu UV-2201spectrophotometer. 1 H NMR and 13 C NMR spectrawere recorded on a Bruker ARX-300 spectrometerwith TMS as the internal standard. ESI-MS datawere acquired on a Shimadzu QP8000α HPLC-Mass spectrometer. Preparative HPLC was carriedout using a Shimadzu LC-8A solvent delivery pumpand a Shimadzu SPD-10AVP detector. Silica-gel forchromatography was purchased from Qindao OceanChemicals Co., China, and polyamide was purchasedfrom the Zhejiang Plastics Plant, China. SephadexLH-20 was purchased from GE Healthcare, Sweden.Plant materialsLeaves of U. rhynchophylla were collected inOctober 2005 in Fujian Province, China. A voucherspecimen is held in the Department of TraditionalChinese Medicines, Shenyang PharmaceuticalUniversity. Species identification was confirmed byProfessor Zerong Jiang, Shenyang PharmaceuticalUniversity.Extraction and isolationThe air-dried leaves (2.5 kg) of U. rhynchophyllawere percolated with 75 % aqueous EtOH. The EtOHextract (670 g) was suspended in water and partitionedsuccessively with n-hexane (3 × 4 L), CHCl 3(3 × 4 L)and n-BuOH (3 × 4 L) to obtain the n-hexane extract(25 g), CHCl 3extract (55 g) and n-BuOH extract (250g), respectively. The n-BuOH extract was subjectedto silica-gel column chromatography (10 × 150 cm,200-300 mesh, Qindao Ocean Chemicals Co., China)involving elution with a CHCl 3-MeOH gradient.Several fractions (150 ml each) were collected,analyzed by TLC and grouped, accordingly.Three grams of the fraction eluted with CHCl 3-MeOH (20:1-10:1) (total 10 g) was subjected torepeated silica-gel column chromatography using aCHCl 3-MeOH solvent system and the eluates weregrouped on the basis of TLC analysis into six majorfractions (F1-F6). Fraction F2 was further subjectedto ODS open column chromatography (3.0×30 cm, 50μm, YMC Co., Ltd, Japan) eluting with MeOH-H 2O(50:50-100:0). The fraction eluted with MeOH-H 2O(85:15) was purified a further two times using ODSopen column chromatography to give compounds86

Flavonol glycosides and triterpenes from the leaves of Uncaria rhynchophylla (Miq.) Jacks. / Asian Journal of TraditionalMedicines, 2009, 4 (3)6 (40 mg) and 9 (60 mg). Fraction F4 was furthersubjected to ODS open column chromatographyeluting with MeOH-H 2O (50:50-100:0). The fractioneluted with MeOH-H 2O (75:25) was purified a furthertwo times using ODS open column chromatography togive compounds 7 (15 mg) and 8 (23 mg).Four grams of the fraction eluted with CHCl 3-MeOH (10:1-8:1) (total 13 g) was subjected to silicagelcolumn chromatography and eluted with a CHCl 3-MeOH gradient to obtain six fractions (F7-12).Fraction F8 (300 mg) was separated on a SephadexLH-20 (3.5×60 cm, GE Healthcare, Sweden) column,involving gradient elution with CHCl 3-MeOH(50:50) and was recrystallized in CHCl 3-MeOH toafford compound 10 (140 mg). Fraction F10 (450mg) was subjected to repeated polyamide columnchromatography (CHCl 3-MeOH; 20:1-1:1) , thenfurther purified by preparative HPLC (Shim-packPRC-ODS, 2.0×30 cm, Shimadzu Co. Ltd, Japan)using MeOH-H 2O (55:45) as an eluent at a flow rateof 12 ml/min to afford compounds 3 (18 mg) and 5 (11mg).Three grams of the fraction eluted with CHCl 3-MeOH (8:1–5:1) (total 17 g) was subjected to silicagelcolumn chromatography and eluted with a CHCl 3-MeOH gradient to obtain nine fractions (F13-21).Fraction F17 (200 mg) was separated on a polyamidecolumn, involving gradient elution with CHCl 3-MeOH(50:50), then further purified by preparative HPLCusing MeOH-H 2O (40:60) as an eluent at a flow rateof 12 ml/min to afford Compounds 1 (19 mg), 2 (25mg) and 4 (20 mg).Results and discussionCompound 1Amorphous yellow powder, m.p. 193-195 °C. HCl-Mg reaction and Molish reaction: positive. UV (MeOH)λ max: 255, 358 nm. ESI-MS (m/z): 633 [M+Na] + , 649[M+K] + , 609 [M-H] - . Molecular formula: C 27H 30O 16.1H NMR (300 MHz, DMSO-d 6): δ H12.60 (1H, s,-OH-5), 7.66 (1H, dd, J = 8.5, 2.0 Hz, H-6’), 7.52 (1H,d, J=2.0 Hz, H-2’), 6.82 (1H, d, J=8.5 Hz, H-5’), 6.40(1H, d, J=1.9 Hz, H-8), 6.20 (1H, d, J=1.9 Hz, H-6),5.33 (1H, d, J=7.5 Hz, gal-1’’), 4.42 (1H, s, rha-1’’’),1.06 (3H, d, J=6.0 Hz, rha-6’’’). 13 C NMR (75 MHz,DMSO-d 6) see Table 1. All data were identical tothose of quercetin-3-O-α-L-rhamnopyranosyl-(1→6)-β-D-galactopyranoside [4] .Compound 2Amorphous yellow powder, m.p. 174-177 °C. HCl-Mg reaction and Molish reaction: positive. UV (MeOH)λ max: 257, 358 nm. ESI-MS (m/z): 633 [M+Na] + , 649[M+K] + , 609 [M-H] - . Molecular formula: C 27H 30O 16. 1 HNMR (300 MHz, DMSO-d 6): δ H12.60 (1H, s, -OH-5),10.83 (1H, s, -OH-7), 9.68 (1H, s, -OH-4’), 9.19 (1H,s, -OH-3’), 7.54 (2H, m, H-2’, 6’), 6.84 (1H, d, J = 8.8Hz, H-5’), 6.38 (1H, d, J = 2.0 Hz, H-8), 6.19 (1H, d,J = 2.0 Hz, H-6), 5.35(1H, d, J = 7.3 Hz, glc-1’’), 4.38(1H, s, rha-1’’’), 0.99 (3H, d, J = 6.3 Hz, rha-6’’’). 13 CNMR (75 MHz, DMSO-d 6) see Table 1. All data wereidentical to those of rutin [5] .Compound 3Amorphous yellow powder, m.p. 221-224 °C. HCl-Mg reaction and Molish reaction: positive. UV (MeOH)λ max: 256, 354 nm. ESI-MS (m/z): 487 [M+Na] + , 503[M+K] + , 463 [M-H] - . Molecular formula: C 21H 20O 12.1H NMR (300 MHz, DMSO-d 6): δ H12.6 (1H, s,-OH-5), 7.67 (1H, dd, J = 8.5, 1.2 Hz, H-6’), 7.53 (1H,d, J = 1.6 Hz, H-2’), 6.82 (1H, d, J = 8.5 Hz, H-5’),6.41 (1H, d, J = 1.2 Hz, H-8), 6.21 (1H, d, J = 1.2 Hz,H-6), 5.38 (1H, d, J = 7.7 Hz, gal-1’’). 13 C NMR (75MHz, DMSO-d 6) see Table 1. All data were identicalto those of hyperoside [6] .Compound 4Amorphous yellow powder, m.p. 187-190 °C. HCl-Mg reaction and Molish reaction: positive. UV (MeOH)λ max: 265, 354 nm. ESI-MS (m/z): 617 [M+Na] + , 633[M+K] + , 593 [M-H] - . Molecular formula: C 27H 30O 15.1H NMR (300 MHz, DMSO-d 6): δ H12.60 (1H, s,-OH-5), 8.05 (2H, d, J = 8.6 Hz, H-2’, 6’), 6.8687

Flavonol glycosides and triterpenes from the leaves of Uncaria rhynchophylla (Miq.) Jacks. / Asian Journal of TraditionalMedicines, 2009, 4 (3)(2H, d, J = 8.6 Hz, H-3’, 5’), 6.41 (1H, s, H-8), 6.19(1H, s, H-6), 5.32 (1H, d, J = 7.6 Hz, gal-1’’), 4.40(1H, s, rha-1’’’), 1.06 (3H, d, J = 6.0 Hz, rha-6’’’). 13 C NMR (75 MHz, DMSO-d 6) see Table 1. Alldata were identical to those of kaempferol-3-O-α-Lrhamnopyranosyl-(1→6)-β-D-galactopyranoside[7] .Compound 5Amorphous yellow powder, m.p. 238-240 °C. HCl-Mg reaction and Molish reaction: positive. UV (MeOH)λ max: 266, 353 nm. ESI-MS (m/z): 471 [M+Na] + , 487[M+K] + , 447 [M-H] - . Molecular formula: C 21H 20O 11.1H NMR (300 MHz, DMSO-d 6): δ H12.60 (1H, s,-OH-5), 10.31 (1H, s, -OH-7), 8.07 (2H, d, J = 8.7 Hz,H-2’, 6’), 6.86 (2H, d, J = 8.7 Hz, H-3’, 5’), 6.43 (1H,s, H-8), 6.21 (1H, s, H-6), 5.41 (1H, d, J = 7.5 Hz,gal-1’’). 13 C NMR (75 MHz, DMSO-d 6) see Table 1.All data were identical to those of kaempferol-3-O-β-D-galactopyranoside [8] .Compound 6Table 1. 13 C NMR (75 MHz) data of compounds 1–5 (DMSO-d 6)Position 1 2 3 4 52 156.4 156.5 156.4 156.6 156.43 133.6 133.4 133.5 133.4 133.34 177.5 177.5 177.5 177.5 177.65 161.3 161.3 161.3 161.3 161.36 98.8 98.8 98.7 99.0 98.87 164.3 164.2 164.2 164.0 164.38 93.7 93.7 93.6 93.9 93.89 156.4 156.7 156.3 156.6 156.410 104.0 104.0 104.0 103.8 104.01' 121.1 121.3 121.1 120.9 121.02' 115.3 115.3 115.2 131.0 131.13' 144.9 144.8 144.9 115.1 115.24' 148.6 148.5 148.5 160.1 160.15' 116.0 116.3 116.0 115.1 115.26' 122.0 121.7 122.0 131.0 131.11'' 102.1 101.3 101.8 102.2 101.72'' 71.2 74.2 71.3 71.2 71.33'' 73.1 76.5 73.2 73.1 73.24'' 68.4 70.1 68.0 68.1 68.05'' 73.6 76.5 75.9 73.6 75.96'' 65.2 67.1 60.2 65.4 60.31''' 100.1 100.8 100.12''' 70.5 70.5 70.53''' 70.7 70.6 70.74''' 72.0 71.9 72.05''' 68.1 68.3 68.46''' 18.0 17.8 18.088

Flavonol glycosides and triterpenes from the leaves of Uncaria rhynchophylla (Miq.) Jacks. / Asian Journal of TraditionalMedicines, 2009, 4 (3)Table 2. 13 C NMR (75 MHz) data of compounds 6-10Position 6 a 7 a 8 a 9 b 10 a1 39.4 41.7 39.7 37.2 37.02 24.1 26.5 24.8 31.6 29.83 78.6 78.8 71.8 71.8 78.14 39.3 39.8 42.6 42.3 38.95 56.4 56.5 47.7 140.7 140.46 67.6 67.8 66.1 121.7 121.57 33.3 41.3 39.9 31.9 31.68 41.6 40.5 38.0 31.9 31.79 48.1 48.4 47.2 50.1 50.010 37.1 37.2 36.1 36.5 36.511 23.7 24.1 22.3 21.1 20.812 126.1 128.5 124.7 28.3 28.113 138.6 139.4 137.2 42.3 42.014 43.0 42.7 41.6 56.8 56.415 31.0 29.3 27.3 24.3 24.116 25.0 28.4 26.6 39.8 39.517 48.6 48.4 46.7 56.0 55.818 53.6 54.7 52.2 11.9 11.519 39.6 72.8 38.1 19.1 18.820 41.2 42.4 40.7 36.1 35.921 31.1 27.0 29.7 18.8 18.622 37.5 38.6 35.5 33.9 33.723 28.7 28.6 65.7 26.0 25.924 18.8 18.1 17.4 45.8 45.625 17.2 17.2 13.3 29.1 29.026 17.6 18.3 17.2 19.4 19.027 23.8 24.8 23.6 19.8 19.528 179.9 180.7 178.5 23.0 22.929 18.1 27.2 16.2 12.0 11.730 21.4 16.8 20.0Glc-1 102.12 74.93 78.24 71.25 77.66 62.4a, bMeasured separately in C 5D 5N and CDCl 389

Flavonol glycosides and triterpenes from the leaves of Uncaria rhynchophylla (Miq.) Jacks. / Asian Journal of TraditionalMedicines, 2009, 4 (3)Amorphous colorless powder. Liebermann-Burchard reaction: positive. ESI-MS (m/z): 495[M+Na] + , 471 [M-H] - . Molecular formula: C 30H 48O 4.1H NMR (300 MHz, C 5D 5N): δ H0.95 (3H, d, J = 6.3Hz, H-29), 1.00 (3H, d, J = 6.0 Hz, H-30), 1.25 (3H,s, H-23), 1.43 (3H, s, H-26), 1.61 (3H, s, H-24), 1.62(3H, s, H-25), 1.72 (3H, s, H-27), 3.11 (1H, m, 4.2 Hz,H-3), 4.86 (1H, brs, H-6), 5.44 (1H, brs, H-12). 13 CNMR (75 MHz, C 5D 5N) see Table 2. All data wereidentical to those of 6β-hydroxyursolic acid [9] .Compound 7Amorphous colorless powder. Liebermann-Burchard reaction: positive. ESI-MS (m/z): 511[M+Na] + , 487 [M-H] - . Molecular formula: C 30H 48O 5.1H NMR (300 MHz, C 5D 5N): δ H1.12 (3H, d, J = 6.3Hz, H-30), 1.42 (3H, s, H-23), 1.48 (3H, s, H-26), 1.63(3H, s, H-29), 1.69 (3H, s, H-24), 1.73 (3H, s, H-25),1.77 (3H, s, H-27), 2.51 (1H, m, H-16α), 3.46 (1H, dd,J = 11.4, 4.1 Hz, H-3), 5.44 (1H, brs, H-12). 13 C NMR(75 MHz, C 5D 5N) see Table 2. All data were identicalto those of 3β, 6β, 19α-trihydroxyurs-12-en-28-oicacid [10] .Compound 8Amorphous colorless powder. Liebermann-Burchard reaction: positive. ESI-MS (m/z): 511[M+Na] + , 487 [M-H] - . Molecular formula: C 30H 48O 5.1H NMR (300 MHz, C 5D 5N): δ H0.92 (3H, d, J = 4.2Hz, H-30), 1.01 (3H, d, J = 7.5 Hz, H-29), 1.20 (3H,s, H-26), 1.64 (3H, s, H-24), 1.67 (3H, s, H-27), 1.72(3H, s, H-25), 4.27 (1H, dd, J = 11.4, 4.1 Hz, H-3),4.42, 4.00 (2H, each 1H, d, J = 10.3 Hz, H-23), 5.45(1H, brs, H-12). 13 C NMR (75 MHz, C 5D 5N) seeTable 2. All data were identical to those of 3β, 6β,23-trihydroxyurs-12-en-28-oic acid [10, 11] .Compound 9Amorphous colorless powder. Liebermann-Burchard reaction: positive. Molecular formula:C 29H 50O. 1 H NMR (300 MHz, CDCl 3): δ H0.68 (3H,s, H-18), 0.80-0.93 (15H, each 3H, m), 3.53 (1H, m,H-3), 5.35 (1H, m, H-6). 13 C NMR (75 MHz, CDCl 3)see Table 2. All data were identical to those ofβ-sitosterol [12] .Compound 10Amorphous colorless powder. Liebermann-Burchard reaction and Molish reaction: positive.Molecular formula: C 35H 60O 6. 1 H NMR (300 MHz,C 5D 5N): δ H0.65 (3H, s, H-18), 0.84-1.01 (15H, each3H, m), 5.06 (1H, d, J = 7.8 Hz, glc-1′), 5.35 (1H, brs,H-6). 13 C NMR (75 MHz, C 5D 5N) see Table 2. Alldata were identical to those of daucosterol [9] .AcknowledgementThis study was supported by research fundsfrom the 2005 Scientific Technology Plan Project ofLiaoning Province Government, China.References[1] The State Pharmacopoeia Committee of China.Pharmacopoeia of the People’s Republic of China, 2005Edition, Vol. 1. Beijing: Chemical Industry Press, 2005,180.[2] Japanese Pharmacopoeia Editorial Committee. TheJapanese Pharmacopoeia, 15th Ed. Tokyo: HirohawaPress, 2006, 462-5.[3] Zhao Q, Watanabe Y, Sakakibara I, Zhang SH, MurakamiT, Higashida M, Matsumoto S. Antihypertensive activityand protection against cerebral ischemia of phenolcontainingfraction of Uncaria hooks and its components.Medical and Pharmaceutical Society for WAKAN-YAKU,2002, 8, Suppl 152.[4] Liang QL, Ding LS. Chemical study on the Leaves ofRaisin Tree (Hovenia acerba) (I). Zhongcaoyao, 1996,27(10): 581-3.[5] Jiang SJ, Zhu B, Zhang NP, Lin RC. Chemical study on theGaleobdolon chinense. Journal of China PharmaceuticalUniversity, 2002, 33(9): 487-8.[6] Zhang W, Zhao DB, Li MJ, Liu XH, Wang HQ. Studies onflavonoid constituents from herbs of Artemisia ordosicaII. China Journal of Chinese Materia Medica, 2006,31(23): 1959-61.[7] Brasseur T, Angenot L. Flavonol glycosides from leaves ofStrychnos variabilis. Phytochemistry, 1986, 25(2): 563-4.[8] Huang KY, Zhang DS, Gao HY, Wu LJ. Chemicalconstituents of Dioscorea bulbifera L. Journal of90

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