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4410 J. Med. Plants Res. ovarian cancer cell lines (SKOV3) and human cervical cancer cell lines (Hela) in vitro. B. yinchowense is abundantly distributed in the Northwest of China and widely used in Chinese folk medicine. No evidence is available in the literatures concerning its constituents and pharmacological activities. To systematically evaluate its potential anticancer activity, the constituents were studied by activity-guided fraction and result in the isolation of 13 saikosaponins. Their structures were identified on the basis of spectral data. The isolation, structure elucidation and evaluation for cytotoxic activities were described in this study. MATERIALS AND METHODS General experimental procedure Nuclear magnetic resonance (NMR) spectra were recorded on a Brucker DRX-500 spectrometer (Brucker Biosciences Corporation, Billerica, MA) with tetramethylsilane (TMS) as internal standard operating at 500 and 125 MHz for 1 H and 13 C, respectively. Fast atom bombardment-mass spectra (FAB-MS) and high resolutionfast atom bombardment-mass spectra (HR-FABMS) were recorded on a Micromass Autospec-Q instrument (Micromass Ltd., Manchester, UK). Infrared (IR) spectra were recorded in KBr discs using a Perkin-Elmer 983G spectrophotometer (Perkin-Elmer Ltd., USA). Gas chromatography (GC) analysis was carried out on an Agilent 6890N gas chromatography (Agilent Co., USA) using an HP-5 capillary column. Column chromatography was performed with silica gel (100 to 200 mesh, Qingdao Marine Chemical Co., Qingdao, P. R. China), Sephadex LH-20 (25 to 100 μm, GE Healthcare Bioscences AB, Uppsaki, Sweden), octadecyl silica (25 to 40 μm, Merck, USA), D101 macroporous resins (Tianjin Gujiao Factory, Tianjin, P. R. China), MCI Gel CHP20P (75 to 150 µm, Mitsubishi Chemical, Japan). Thin layer chromatography (TLC) was performed on precoated silica gel GF254 (0.2 mm thick, Qingdao Marine Chemical Co., Qingdao, P. R. China) and spots were detected by spraying with 10% ethanolic H2SO4 reagent. 3-(4, 5dimethylthiiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Plant collection The roots of B. yinchowense were collected from Dingxi County, Gansu province, China, in August, 2009 and identified by the author, Professor Ruile Pan of the Institute of Medicinal Plant, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China, where a voucher specimen (No. 20090815) was deposited. Extraction The dried and powdered roots (800 g) of B. yinchowense were extracted with 60% ethanol containing 0.5% ammoniae aqua (three times, 1 L each) at room temperature for 12 h. The ethanol extracts were combined and evaporated in vacuo, to yield a dark brown residue (120 g), which was dissolved in H2O-MeOH (5:95) solution (200 ml), and then portioned with n-hexane of 200 ml) to get the nhexane-soluble fraction. The H2O-MeOH (5:95) layer was evaporated to remove residual MeOH, and then distilled water (200 ml) was added. This aqueous solution was subjected to a column contained 1 kg D101 macroporous resin and was eluted successively with water (2 L), 90% ethanol (2 L), respectively. Evaporation of the respective solvents gave n-hexane (12 g), water (42 g) and 90% ethanol (32 g) fractions. The 90% ethanol fraction is saponin-enriched part. Each fraction was evaluated for the cytotoxic activity on the tumor cell lines (Table 1). It was shown that the activity resided in the saponin-enriched part. Isolation Saponin-enriched part (32 g) was subjected to MCI column, eluting with a gradient of water-methanol (from 100:0 to 5:95), to yield 5 fractions. Fraction 2 (8 g) was chromatographed repeatedly on silica gel using chloroform-methanol (8:2) and octadecylsilane (ODS)-C18 with the elution of methanol-water (7:3) to afford 1 (80 mg),2 (55 mg),3 (75 mg), 11 (5 mg) and 9 (17 mg). Fraction 3 (10 g) was separated into three sub-fractions by ODS column using methanol-water (7:3) as elution and the second sub-fraction was subjected to repeated column chromatography, first on silica gel, chloroform:methanol (8:2) and purified on pharmadex LH-20 (methanol) to obtain 4 (51 mg), 5 (38 mg), 6 (20 mg) and 12 (8 mg). Compound 7 (35 mg), 8 (37 mg), 13 (6 mg) and compound 10 (22 mg) were purified from Fraction 4 (5 g) by repeated semipreparative high performance liquid chromatography (HPLC) using methanolwater (60:40) as fluent. Characterization of isolation Saikosaponin a (1): White amorphous powder, mp 229 to 230°C, 1 H-NMR (C5D5N, 500 MHz): � 0.91, 0.92, 0.99, 1.12, 1.31, 1.37 (each 3H, s, tert-Me×6), 1.47 (3H, d, J=6.6 Hz, Fuc-CH3), 4.94 (1H, d, J=7.8 Hz, Fuc-1′-H), 5.35 (1H, d, J=8.4 Hz, Glc-1″-H), 5.66 (1H, dd, J=10.2, 2.4 Hz, 11-H), 6.00 (1H, d, J=10.2 Hz, 12-H). 13 C-NMR data (Table 1). Saikosaponin c (2): White amorphous powder, mp 207 to 209°C, 1 H-NMR (C5D5N, 500 MHz): � 0.86, 0.91, 0.96, 0.97, 1.14, 1.28, 1.35 (each 3H, s, tert-Me × 7), 1.66 (3H, d, J=6.0 Hz, Rha-CH3), 4.94 (1H, d, J=7.8 Hz, Glc-1′-H), 4.52 (1H, d, J=9.0 Hz, Rha-1″-H), 4.79 (1H, d, J =7.8 Hz, Glc-1′″-H), 5.64 (1H, dd, J=10.2, 2.4 Hz, 11- H), 5.90 (1H, d, J=10.2 Hz, 12-H). 13 C-NMR data (Table 1). Saikosaponin d (3): White amorphous powder, mp 227 to 228°C, 1 H-NMR (C5D5N, 500 MHz): � 0.91, 0.92, 0.99, 1.12, 1.31, 1.37 (each 3H, s, tert-Me×6), 1.47 (3H, d, J=6.6 Hz, Fuc-CH3), 4.94 (1H, d, J=7.8 Hz, Fuc-1′-H), 5.35 (1H, d, J=8.4 Hz, Glc-1″-H), 5.66 (1H, dd, J=10.2, 3.0 Hz, 11-H), 6.02 (1H, d, J=10.2 Hz, 12-H). 13 C-NMR data (Table 1). Saikosaponin b2 (4): White amorphous powder, mp 201 to 203°C, 1 H-NMR (C5D5N, 500 MHz): � 0.89, 0.92, 0.99, 1.01, 1.04, 1.68 (each 3H, s, tert-Me×6), 1.45 (3H, d, J=6.0 Hz, Fuc-CH3), 4.99 (1H, d, J=7.8 Hz, Fuc-1′-H), 5.40 (1H, d, J=7.8 Hz, Glc-1″-H), 6.70 (1H, dd, J=10.2, 1.8 Hz, 11-H), 5.72 (1H, d, J=10.2 Hz, 12-H). 13 C-NMR data (Table 1). Saikosaponin f (5): White amorphous powder, mp198 to 200°C 1 H-NMR (C5D5N, 500 MHz): � 0.82, 0.95, 0.99, 1.00, 1.01, 1.29, 1.35 (each 3H, s, tert-Me×7), 1.65 (3H, d, J=6.0 Hz, Rha-CH3), 4.94 (1H, d, J=7.8 Hz, Glc-1′-H), 4.52 (1H, d, J=9.0 Hz, Rha-1″-H), 4.79 (1H, d, J=7.8 Hz, Glc-1′″-H), 5.86 (1H, br, 12-H). 13 C-NMR data (Table 1). Saikosaponin b4 (6): White amorphous powder, mp 206 to 208°C, 1 H-NMR (C5D5N, 500 MHz): � 0.97, 1.01, 1.01, 1.12, 1.14, 1.88 (each 3H, s, tert-Me×6), 3.26 (3H, s, OCH3), 1.45 (3H, d, J=6.0 Hz, Fuc-CH3), 4.96 (1H, d, J=7.2 Hz, Fuc-1′-H), 5.33 (1H, d, J=7.8 Hz, Glc-1″-H), 5.60 (1H, d, J=3.0 Hz, 12-H). 13 C-NMR data (Table 1). 6″-O-acetylsaikosaponin a (7): White amorphous powder, mp 204 to 205°C, 1 H-NMR (C5D5N, 500 MHz): � 0.89, 0.92, 0.92, 0.98,
Table 1. 13 C-NMR data for compounds 1 to 12 (C5D5N, 125 MHz). Li et al. 4411 1 2 3 4 5 6 7 8 9 10 11 12 1 38.6 38.5 38.6 38.4 38.8 40.1 38.6 38.4 38.6 38.4 39.8 38.6 2 26.6 26.5 26.2 26.1 26.6 26.4 25.8 26.1 26.6 26.1 26.3 25.7 3 88.9 89.0 81.6 81.7 89.1 81.9 81.6 81.7 88.6 81.7 88.8 81.6 4 39.8 39.7 43.7 43.7 39.5 43.8 43.7 43.7 39.8 43.6 39.8 43.7 5 55.4 55.3 47.3 47.4 55.7 48.4 47.3 47.3 55.4 47.4 55.7 47.4 6 18.2 18.1 17.5 18.9 18.5 18.3 17.3 18.2 18.2 17.3 18.3 17.6 7 31.9 31.9 31.5 32.3 33.4 33.4 31.6 32.3 31.9 31.9 33.3 31.6 8 42.2 42.2 41.9 41.1 40.2 40.7 42.2 41.1 42.2 41.9 41.0 42.2 9 53.0 52.8 53.0 54.0 47.1 51.7 53.1 54.1 53.0 53.0 52.0 53.2 10 36.4 36.3 36.8 35.5 36.8 38.2 36.3 36.5 36.4 36.3 38.1 36.3 11 132.0 132.1 132.0 126.2 24.0 76.0 132.2 126.1 132.0 132.0 76.0 132.2 12 131.2 131.2 131.9 126.3 122.7 122.5 131.2 126.8 131.2 131.9 122.4 131.2 13 84.0 84.0 84.8 136.1 144.0 149.8 83.9 137.4 84.0 84.9 148.1 84.0 14 45.7 45.7 45.3 41.9 43.8 41.9 45.6 42.1 45.7 43.6 43.9 45.7 15 36.2 36.2 36.3 32.6 36.8 37.2 36.2 31.9 36.2 35.4 36.8 36.1 16 64.0 64.0 77.2 67.7 66.6 74.2 64.0 67.7 64.0 77.1 66.2 64.1 17 47.0 47.0 54.4 45.3 41.1 43.4 47.0 45.5 47.0 45.3 43.6 46.9 18 52.2 52.2 51.4 133.0 44.5 41.9 52.1 131.4 52.2 51.4 43.9 52.1 19 37.8 38.5 37.8 39.0 47.1 47.8 37.7 34.0 37.8 38.4 47.0 37.7 20 31.6 31.6 31.9 31.9 31.1 31.3 31.6 44.0 31.6 31.9 31.1 31.6 21 34.7 34.7 36.8 36.5 34.3 35.0 34.7 31.1 34.7 36.8 33.4 34.7 22 25.7 25.7 31.3 24.4 26.6 30.9 25.7 24.2 25.7 31.3 26.3 25.8 23 64.1 28.0 64.1 64.7 28.2 64.3 64.0 64.1 27.8 64.1 28.2 64.2 24 13.3 16.3 13.1 13.1 17.0 13.6 13.0 13.1 16.3 13.1 17.0 13.0 25 18.0 18.1 18.8 18.3 15.7 17.9 18.7 18.9 18.0 18.9 17.3 18.7 26 20.0 19.9 19.5 17.2 17.0 18.4 20.0 17.3 20.0 19.6 18.5 20.1 27 20.9 20.9 20.6 21.9 27.1 26.4 20.8 21.9 20.9 18.1 26.3 20.8 28 73.1 72.7 77.6 64.1 69.1 70.0 73.0 65.1 73.0 78.1 69.1 73.0 29 33.7 33.7 33.7 25.1 33.4 33.4 33.6 181.2 33.7 33.8 33.3 33.6 30 23.8 23.8 24.4 32.6 24.1 24.6 23.8 21.5 23.8 24.4 24.0 23.8 OCH3 - - - - - 53.8 - - - - 54.2 - 1′ 106.8 106.7 106.0 106.0 106.7 106.0 106.1 106.0 106.8 106.1 106.7 105.1 2′ 71.6 75.2 71.0 71.8 75.2 71.5 71.4 71.6 71.6 71.5 75.1 71.3 3′ 85.2 76.8 85.3 85.3 76.8 85.5 85.4 85.3 85.2 84.9 76.8 85.9 4′ 71.8 80.0 72.2 72.2 80.0 72.2 71.4 72.2 71.8 72.1 79.9 71.7 5′ 71.0 75.2 71.5 71.0 75.6 71.0 71.0 71.1 71.0 71.0 75.5 70.9 6′ 17.3 69.2 17.3 17.2 69.1 17.2 17.3 17.3 17.3 17.3 68.5 17.2 1″ 106.8 102.9 106.8 106.7 102.9 106.7 106.4 106.7 106.8 106.5 105.3 104.5 2″ 75.9 72.6 75.9 75.8 72.7 75.8 75.3 75.8 75.9 75.3 74.8 86.8 3″ 78.5 72.5 78.8 78.8 72.6 78.4 78.1 78.3 78.5 77.8 78.3 77.6 4″ 71.8 73.8 71.8 71.6 73.8 72.2 71.6 71.8 71.8 72.1 71.4 70.5 5″ 78.4 70.5 78.9 78.5 70.5 78.4 75.3 78.7 78.4 75.6 78.3 78.1 6″ 62.7 18.1 62.7 62.7 18.4 62.7 64.8 62.7 62.7 64.8 62.5 62.2 1″′ - 105.2 - - 105.1 - - - - - 103.0 107.8 2″′ - 74.8 - - 74.8 - - - - - 72.5 76.1 3″′ - 78.4 - - 78.5 - - - - - 72.7 77.9 4″′ - 71.4 - - 71.4 - - - - - 73.8 71.0 5″′ - 78.4 - - 78.4 - - - - - 70.5 67.6 6″′ - 62.6 - - 62.6 - - - - - 18.3 - COCH3 - - - - - - 170.8 - - 170.8 - - COCH3 - - - - - - 20.8 - - 20.8 - -
Page 1 and 2: B Journal of Medicinal Plants Resea
Page 3 and 4: Editors Prof. Akah Peter Achunike E
Page 5 and 6: Electronic submission of manuscript
Page 7 and 8: Fees and Charges: Authors are requi
Page 9 and 10: ences Table of Contents: Volume 6 N
Page 11 and 12: supplement of dietary minerals. The
Page 13 and 14: several studies. MO leaves ethanoli
Page 15 and 16: (Nandave et al., 2009). In ocular d
Page 17 and 18: Journal of Medicinal Plants Researc
Page 19 and 20: Table 1. Pa and Pi of PTK inhibitor
Page 23 and 24: Table 1. Plant collection and stora
Page 25 and 26: Table 2. Count’d. Psydrax livida
Page 27 and 28: Mukandiwa et al. 4385 Figure 3. Chr
Page 29 and 30: Euphorbiaceae family to which S. af
Page 33 and 34: Table 1. Number of axillary shoots
Page 35 and 36: REFERENCES Hui et al. 4393 Figure 1
Page 37 and 38: en-indicycloethers have an antispas
Page 39 and 40: Table 2. Analysis of variance of yi
Page 41 and 42: Pirzad 4399 Table 3. Correlation co
Page 45 and 46: Table 1. Analysis of variance (ANOV
Page 47 and 48: A Figure 2. Shoot multiplication fr
Page 49 and 50: capacity could be a model for micro
Page 51: Journal of Medicinal Plants Researc
Page 55 and 56: Figure 1. Chemical structures of co
Page 57 and 58: potent activity against the HepG2 c
Page 59 and 60: Lippia spp. using gas chromatograph
Page 61 and 62: Table 1. Contd. Singulani et al. 44
Page 63 and 64: Previously, β-caryophyllene was id
Page 67 and 68: consumers' individual characteristi
Page 69 and 70: Table 3. Contd. Hong et al. 4427 C2
Page 73 and 74: RESULTS Buncharoen et al. 4431 Tabl
Page 75 and 76: Buncharoen et al. 4433 Figure 2. Ph
Page 77 and 78: Stemona curtisii Hkf. Proceeding of
Page 79 and 80: Table 1. Herbal tea samples. Aboule
Page 81 and 82: Figure 2. Chloride levels in herbal
Page 83 and 84: Table 3. Herbal tea ingredients and
Page 87 and 88: Mean feed intake (g) 6 5 4 3 2 1 0
Page 89 and 90: Percentage mortality Percentage mor
Page 91 and 92: involvement according to Okoye et a
Page 93 and 94: properties of green leafy vegetable
Page 95 and 96: Table 1. Percentage yield extracts
Page 97 and 98: application of betalian pigment of
Page 99 and 100: Conferences and Advert September 20

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