Hamamelis japonica (Hamamelidaceae), widely known as Japanese witch hazel, is a deciduous flowering shrub that produces compact clumps of yellow or orange-red flowers with long and thin petals. As a part of our ongoing search for phenolic constituents from this plant, eleven phenolic constituents including six flavonol glycosides, a chalcone glycoside, two coumaroyl flavonol glycosides and two galloylated compounds were isolated from the flowers. Their structures were elucidated as methyl gallate ( 1 ), myricitrin ( 2 ), hyperoside ( 3 ), isoquercitrin ( 4 ), quercitrin ( 5 ), spiraeoside ( 6 ), kaempferol 4'- O -β-glucopyranoside (7), chalcononaringenin 2'- O -β-glucopyranoside ( 8 ), trans -tiliroside ( 9 ), cis -tiliroside ( 10 ), and pentagalloyl- O -β-D-glucose ( 11 ), respectively. These structures of the compounds were identified on the basis of spectroscopic studies including the on-line LCNMR-MS and conventional NMR techniques. Particularly, directly coupled LC-NMR-MS afforded sufficient structural information rapidly to identify three flavonol glycosides ( 2 - 4 ) with the same molecular weight in an extract of Hamamelis japonica flowers without laborious fractionation and purification step. Cytotoxic effects of all the isolated phenolic compounds were evaluated on HCT116 human colon cancer cells, and pentagalloyl- O -β-D-glucose ( 11 ) was found to be significantly potent in inhibiting cancer cell growth. Hamamelis , commonly known as witch hazel, is a genus of fragrant flowering plants belonging to the family Hamamelidaceae. Six major species including H. virginiana , H. ovalis , H. vernalis , H. mollis , H. mexicana and H. japonica are naturally distributed in the North America and eastern Asia. 1 , 2 Of them, H. virginiana L. (North American witch hazel) is the most well-known species which originally used by the Native Americans to treat burns and injuries. 3 Hamamelis preparations from leaves, twigs, and bark of this species have been widely used as commercial ingredients for cosmetic products and treatment of dermatological disorder, therefore, research studies of phytochemical composition and biological evaluation on this genus have been focused on the H. virginiana . 3 - 6 H. japonica Sieb. et Zucc. (Japanese witch hazel) is one of common Hamamelis species in eastern Asia that is native to Japan, 7 and popularly cultivated as an ornamental plant in Korea. It is a deciduous shrub about 2 - 3 m tall which typically flowers with curled and crimped yellow petals during the February to March. 8 The flowers of H. japonica has been traditionally used as antipyretic and antihemorrhagic agents, 9 however, only a few papers were previously published on the species of H. japonica . 10 , 11 In order to identify chemical constituents in crude extracts obtained from plant materials, we have performed the hyphenated LC-NMR-MS technique in parallel with conventional isolation and NMR study. On-line LC-NMR-MS is recently known as a powerful analytical technique with which one can elucidate structures of natural products without time consuming isolation, and it is being widely accepted as a powerful structure-guided screening tool. 12 - 14 As a part of our phytochemical investigation for the flowering plant H. japonica , eleven phenolic compounds including six flavonol glycosides ( 2 - 7 ), a chalcone glycoside ( 8 ), two coumaroyl flavonol glycosides ( 9 and 10 ) and two galloylated compounds ( 1 and 11 ) were identified from the ethyl acetate extract of H. japonica flowers with the aid of online LC-NMR-MS as well as conventional NMR techniques after isolation. Details of isolation and structure elucidation process for phenolic components from the flowers of H. ja ponica are presented in this report in addition to their cytotoxic effects against human colon cancer cells. General experimental procedures − Conventional NMR spectral data including ¹ H, ¹³ C NMR, HSQC, and HMBC spectra were recorded using a Varian Unity INOVA 500 spectrometer in MeOH- d ₄ , and DMSO- d ₆ and chemical shifts are expressed in δ (ppm) and the coupling constants are in Hz. LC-NMR-ESIMS was performed on a Varian VNMRS 600 MHz NMR spectrometer connected to a Varian Prostar HPLC system using a 150 μL triple-resonance microflow cryogenic probe and a Varian 320-MS TQ mass spectrometer (Palo Alto, CA). Column chromatography was carried out on silica gel 60 (70 - 230mesh) (Merck, Darmstadt, Germany) and Sephadex LH-20 (GE Healthcare, Uppsala, Sweden). HPLC separations were carried out with an Agilent HP1100 series system (Santa Clara, CA) which comprises a degasser, two binary mixing pumps, a column oven and a diode array detector. Waters SunFire ^TM ‚ C18 (5 μm, 4.6 × 150 mm) (Milford, MA) and SunFire ^TM ‚ Prep C18 (5 μm, 10 × 150 mm), SunFire ^TM ‚ Prep C18 OBD (5 μm, 19 × 150mm) HPLC columns were used for analytical and semi-preparative HPLC analysis. UV absorptions were monitored at the wavelengths of 254, 280 and 350 nm. Plant materials − The flowers of H. japonica were collected at Arboretum of Korea Expressway Corporation in Jeonju, Jeonbuk Province, Republic of Korea in March 2009. A voucher specimen was deposited at the herbarium in the College of Pharmacy, Mokpo National University (No. P2009HJ). Extraction and isolation − The dried flowers of H. japonica (338.5 g) were powdered and extracted with MeOH (1.5 L × 3) at room temperature. After filtration, the MeOH extract was evaporated under reduced pressure to obtain a dark brown residue (77.1 g). The concentrated MeOH extract was suspended in water and partitioned with n -hexane and EtOAc and n -BuOH. Isolation of phenolic compounds from EtOAc extract was performed as follows. The EtOAc extract (14.3 g) was subjected to Sephadex LH-20 chromatography on elution with MeOH, and silica gel column chromatography with CHCl ₃ /MeOH step-gradient system (91:9 → 25:75) to afford 12 fractions. Each fraction was screened by analytical HPLC mode in order to trace phenolic compounds. As a result, two major fractions 7 and 10 were selected, and both fractions were separated by HPLC using the gradient eluent system with acetonitrile and water containing 0.1% formic acid, i.e. 12% acetonitrile to 35% acetonitrile for 30 min. Compounds ( 1 - 10 ) were isolated from fraction 7, and compound 11 was isolated as a main phenolic compound from fraction 10. Amounts of the isolated compounds were as follows; 1 ( t _R 5.62 min; 276.0 mg), 2 ( t _R 14.26 min; 5.2 mg), 3 ( t _R 14.62 min; 16.7 mg), 4 ( t _R 14.93 min; 34.6 mg), 5 ( t _R 17.86 min; 33.9 mg), 6 ( t _R 18.91 min; 103.3 mg), 7 ( t _R 19.75 min; 19.1 mg), 8 ( t _R 22.74 min; 13.3 mg), 9 ( t _R 26.8 min; 141.0 mg), 10 ( t _R 28.00 min; 24.3 mg), and 11 ( t _R 16.33 min; 193.7 mg), respectively. Methyl gallate (1) − white amorphous powder; UV λ _max 277 nm (HPLC solvent); ¹ H NMR (CD ₃ OD, 500 MHz): δ 7.04 (2H, H-2,6, s), 3.81 (3H, O-CH ₃ , s); ¹³ C NMR (CD ₃ OD, 125 MHz): δ 169.2 (C=O), 146.6 (C-3,5), 139.9 (C-4), 121.5 (C-1), 110.1 (C-2,6), 52.4 (O-CH ₃ ); ESIMS m / z 183 [M−H] ⁻ . Quercitrin (5, quercetin 3-O-β-rhamnoside), Spiraeoside (6, quercetin 4'-O-β-glucoside) − UV, ¹ H NMR, ¹³ C NMR and ESIMS data were described in the previous results. 15 Kaempferol 4'-O-β-D-glucoside (7) − yellow amorphous powder; UV λ _max 266, 315, 363 nm (HPLC solvent); ¹ H NMR (CD ₃ OD, 500 MHz): δ 8.18 (2H, d, J = 8.5 Hz, H-2',6'), 7.22 (2H, d, J = 8.5 Hz, H-3',5'), 6.40 (1H, s, H-8), 6.19 (1H, s, H-6), 5.03 (1H, d, J = 7.0 Hz, H-1''), 3.93 (1H, brd, J = 11.0 Hz, H-6''), 3.72 (1H, dd, J = 11.0, 6.5 Hz, H-6''), 3.48~3.52 (3H, m), 3.42 (1H, m); ¹³ C NMR (CD ₃ OD, 125 MHz): δ 179.4 (C-4), 166.1 (C-7), 162.9 (C-5), 160.3 (C-4'), 158.5 (C-8a), 147.3 (C-2), 137.8 (C-3), 130.5 (C-2',6'), 126.7 (C-1'), 117.5 (C-3',5'), 104.6 (C-4a), 101.9 (C-1''), 99.5 (C-8), 94.7 (C-6), 78.4 (C-2''), 78.1 (C-5''), 75.0 (C-3''), 71.5 (C-4''), 62.6 (C-6''); ESIMS m / z 447 [M−H] ⁻ . Chalcononaringenin 2'-O-β-D-glucoside (8) − yellow amorphous powder; UV λ _max 371 nm (HPLC solvent); ¹ H NMR (CD ₃ OD, 500 MHz): δ 8.03 (1H, d, J = 16.0 Hz, H-α), 7.67 (1H, d, J = 16.0 Hz, H-β), 7.61 (2H, d, J = 8.5 Hz, H-2,6), 6.84 (2H, d, J = 8.5 Hz, H-3,5), 6.22 (1H, d, J = 2.0 Hz, H-3'), 5.99 (1H, d, J = 2.0 Hz, H-5'), 5.15 (1H, d, J = 7.5 Hz, H-1''), 3.92 (1H, dd, J = 12.0, 1.5 Hz, H-6''), 3.75 (1H, dd, J = 12.0, 5.5 Hz, H-6''), 3.56 (1H, t, J = 8.5 Hz, H-2''), 3.52 (1H, t, J = 9.0 Hz, H-3''), 3.48 (1H, m, H-5''), 3.45 (1H, t, J = 9.5 Hz, H-4''); ¹³ C NMR (CD ₃ OD, 125 MHz): δ 194.6 (C=O), 168.0 (C-6'), 166.1 (C-4'), 162.0 (C-2'), 161.2 (C-4), 144.3 (C-β), 131.9 (C-2,6), 128.6 (C-1), 126.1 (C-α), 117.0 (C-3,5), 107.6 (C-1'), 102.0 (C-1''), 98.5 (C-5'), 95.8 (C-3'), 78.6 (C-5''), 78.6 (C-3''), 75.1 (C-2''), 71.3 (C-4''), 62.5 (C-6''); ESIMS m / z 433 [M−H] ⁻ . trans-Tiliroside (9) − pale yellow amorphous powder; UV λ _max 267, 315 nm (HPLC solvent); ¹ H NMR (CD ₃ OD, 500 MHz): δ 7.97 (2H, d, J = 8.5 Hz, H-2',6'), 7.39 (1H, d, J = 16.0 Hz, H-7'''), 7.27 (2H, d, J = 8.5 Hz, H-2''',6'''), 6.79 (2H, d, J = 8.5 Hz, H-3',5'), 6.77 (2H, d, J = 8.5 Hz, H-3''',5'''), 6.27 (1H, s, H-8), 6.11 (1H, s, H-6), 6.06 (1H, d, J = 16.0 Hz, H-8'''), 5.24 (1H, d, J = 7.5 Hz, H-1''), 4.31 (1H, dd, J = 11.8, 2.0 Hz, H-6''), 4,19 (1H, dd, J = 11.8, 6.5 Hz, H-6''), 3.53~3.47 (3H, m), 3.35 (1H, m); ¹³ C NMR (CD ₃ OD, 125 MHz): δ 179.5 (C-4), 169.0 (C-9'''), 166.0 (C-7), 163.0 (C-5), 161.6 (C-4'), 161.3 (C-4'''), 159.4 (C-2), 158.4 (C-8a), 146.7 (C-7'''), 135.4 (C-3), 132.4 (C-2',6'), 131.3 (C-2''',6'''), 127.2 (C-1'''), 122.8 (C-1'), 116.9 (C-3''',5'''), 116.1 (C-3',5'), 114.9 (C-8'''), 105.7 (C-4a), 104.2 (C-1''), 100.1 (C-8), 95.0 (C-6), 78.1 (C-2''), 75.9(C-5''), 75.9 (C-3''), 71.8 (C-4''), 64.5 (C-6''); ESIMS m / z 593 [M−H] ⁻ . cis-Tiliroside (10) − brown amorphous powder; UV (HPLC solvent) λ _max 267, 317 nm; ¹ H NMR (CD ₃ OD, 500 MHz): δ 7.96 (2H, d, J = 8.0 Hz, H-2',6'), 7.51 (2H, d, J = 8.0 Hz, H-2''',6'''), 6.82 (2H, d, J = 8.0 Hz, H-3',5'), 6.69 (1H, d, J = 12.8 Hz, H-7'''), 6.68 (2H, d, J = 8.0 Hz, H-3''',5'''), 6.31 (1H, s, H-8), 6.19 (1H, s, H-6), 5.51 (1H, d, J = 12.8 Hz, H-8'''), 5.20 (1H, d, J = 6.5 Hz, H-1''), 4.21 (1H, brd, J = 11.0 Hz, H-6''), 4,17 (1H, dd, J = 11.0, 6.0 Hz, H-6''), 3.50~3.40 (3H, m), 3.29 (1H, m); ¹³ C NMR (CD ₃ OD, 125 MHz): δ 179.5 (C-4), 167.9 (C-9'''), 166.1 (C-7), 163.1 (C-5), 161.6 (C-4'), 160.1 (C-4'''), 159.6 (C-2), 158.5 (C-8a), 145.6 (C-7'''), 135.3 (C-3), 133.9 (C-2',6'), 132.4 (C-2''',6'''), 127.6 (C-1'''), 122.8 (C-1'), 116.2 (C-8'''), 116.1 (C-3''',5'''), 115.8 (C-3',5'), 105.8 (C-4a), 104.2 (C-1''), 100.1 (C-8), 95.0 (C-6), 78.1 (C-2''), 75.8 (C-3''), 75.6 (C-5''), 71.7 (C-4''), 64.2 (C-6''); ESIMS m / z 593 [M−H] ⁻ . 1,2,3,4,6-Penta-O-galloyl-β-D-glucose (11) − brownish amorphous powder; UV (HPLC solvent) λ _max 281 nm; ¹ H NMR (CD ₃ OD, 500MHz): δ 7.14 (2H, s, galloyl protons), 7.08 (2H, s, galloyl protons), 7.00 (2H, s, galloyl protons), 6.98 (2H, s, galloyl protons), 6.93 (2H, s, galloyl protons), 6.26 (1H, d, J = 8.0 Hz, anomeric proton H-1), 5.93 (1H, t, J = 10.0 Hz, H-3), 5.65 (1H, t, J = 9.5 Hz, H-4), 5.61 (1H, dd, J = 10.0, 8.5 Hz, H-2), 4.54 (1H, d, J = 10.5 Hz, H-5), 4.45~4.39 (2H, m, H-6); ESIMS m / z 939 [M−H] ⁻ . LC-NMR-MS method − One dimensional ¹ H NMR spectra for the compounds 2 - 4 were obtained in the continuous-flow mode with isocratic HPLC condition (acetonitrile/D ₂ O containing 0.1% HCOOH = 14:86) under UV detection at 306 nm. 10 μl of the mother liquor sample (20 mg/mL in CH ₃ OH- d ₄ ) was injected on to an Ascentis Express ODS column (2.7 μm, 4.6 × 150 mm) (Sigma-Aldrich, St. Louis, MO) at a flow rate of 0.5 mL/min. The standard WET1D sequence was used for the pre-saturation of ¹ H frequency in the HOD, ACN, and HCOOH. NMR data were acquired with 9.058 kHz sweep width using 28K time domain points with an acquisition time of 1.60 second. ¹ H NMR spectra were collected by 32 scans each during the chromatographic elution. ¹ H NMR spectra were referenced to the acetonitrile resonance (1.96 ppm). For electrospray ionization (ESI) mass spectrometry, deuterium-hydrogen back-exchange was performed using a makeup pump (0.2 mL/min, H ₂ O with 0.1% HCOOH) to detect molecular ion peaks in the LC eluent, as partial hydrogen-deuterium exchange by D ₂ O often leads to erroneous molecular weight determination. Myricitrin (myricetin 3-O-α-L-rhamnoside) (2) − t _R 23.64 min; ¹ H NMR (recorded on-line in HPLC solvent ACN-D ₂ O, 600 MHz): δ 7.90 (2H, s, H-2',6'), 7.39 (1H, d, J = 2.7 Hz, H-8), 7.13 (1H, d, J = 2.7 Hz, H-6), 5.77 (1H, s, H-1''), 4.57 (1H, s, H-2''), 4.00 (1H, m, H-3''), 3.31 (1H, m, H-4''); ESIMS m / z 463 [M−H] ⁻ (C ₂₁ H ₂₀ O ₁₂ ). Hyperoside (quercetin 3-O-β-D-galactoside) (3) − t _R 25.98 min; ¹ H NMR (recorded on-line in HPLC solvent ACN-D ₂ O, 600 MHz): δ 8.74 (1H, d, J = 3.0 Hz, H-2'), 8.61 (1H, dd, J = 10.2, 3.0 Hz H-6'), 7.91 (1H, d, J = 10.2 Hz, H-5'), 7.40 (1H, d, J = 3.0 Hz, H-8), 7.12 (1H, d, J = 3.0 Hz, H-6), 5.38 (1H, d, J = 9.6 Hz, H-1''), 4.11 (1H, d, J = 4.2 Hz, H-4''), 3.82 (1H, dd, J = 12.0, 10.0 Hz, H-2''), 3.79 (2H, m, H-6'',3''), 3.70 (1H, dd, J = 13.2, 7.8 Hz, H-6''), 3.64 (1H, t, J = 7.2 Hz, H-5''); ESIMS m / z 463 [M−H] ⁻ (C ₂₁ H ₂₀ O ₁₂ ). Isoquercitrin (quercetin 3-O-β-D-glucoside) (4) − t _R 28.95 min; ¹ H NMR (recorded on-line in HPLC solvent ACN-D ₂ O, 600 MHz) δ 8.69 (1H, d, J = 2.4 Hz, H-2'), 8.60 (1H, dd, J = 10.2, 2.4 Hz, H-6'), 7.90 (1H, d, J = 10.2 Hz, H-5'), 7.39 (1H, d, J = 2.7 Hz, H-8), 7.12 (1H, d, J = 2.7 Hz, H-6), 5.49 (1H, d, J = 9.0 Hz, H-1''), 3.84 (1H, dd, J = 15.0, 2.7 Hz, H-6''), 3.73 (1H, dd, J = 15.0, 6.0 Hz, H-6''), 3.70 (1H, t, J = 10.8 Hz, H-2''), 3.61 (1H, t, J = 10.8 Hz, H-3''), 3.53 (1H, t, J = 10.8 Hz, H-4''), 3.44 (1H, m, H-5''); ESIMS m / z 463 [M−H] ⁻ (C ₂₁ H ₂₀ O ₁₂ ). Cytotoxicity test − HCT116 colon cancer cell line was a gift from Prof. Sun-Young Ra, Yonsei University College of Medicine, Republic of Korea. The cells were maintained in Dulbecco’s modified Eagles medium (DMEM) supplemented with 10% FBS and 1% penicillin/streptomycin, and cultured at 37 ℃ in an atmosphere containing 5% CO ₂ . HCT116 cells were seeded in 96 well plates at the density of 3 × 10 ⁴ cells/mL and incubated at 37 ℃ overnight. The cells were then treated with various concentrations (2.5, 5, 10, 20 and 40 μM) of compounds in serum-free media. DMSO served as a negative control. After 72 h incubation, media was removed, and cells were again incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) solution for 3 h. 50 μL of DMSO was then added to each well to dissolve the purple formazan produced by the MTT. Absorbance was measured using a spectrophotometer at a wavelength of 540 nm. The shade-dried and milled flowers of H. japonica were exhaustively extracted with MeOH, and then this extract was suspended in H ₂ O and partitioned with n -hexane, EtOAc, n -BuOH and H ₂ O, successively. The EtOAc extract was fractionated by column chromatography on Sephadex LH-20, silica gel and HPLC to afford nine flavonoid glycosides ( 2 - 10 ) and two galloylated compounds ( 1 and 11 ) ( Fig. 1 ). HPLC peak profile and UV spectra of these compounds were obtained by photodiode array detector (190 - 400 nm). Compounds 2 - 7 showed maximal absorption bands at around 260 and 360 nm which indicated they were flavone derivatives. Also, compounds 8 - 10 exhibited characteristic UV absorption bands indicating the presence of a chalcone (371 nm) and acylated flavone (267 and 315 nm) moieties, respectively ( Fig. 2 ). ESI-MS spectra of 2 - 4 exhibited the same pseudomolecular ion peaks [M−H] ⁻ at m / z 463 corresponding to the molecular formula C ₂₁ H ₂₀ O ₁₂ , and their chromatographic profile displayed that LC peaks of 2 - 4 appeared consecutively under optimized conditions. In order to identify each flavonoid quickly without subsequent isolation step, on-line LC-NMR-MS studies were performed in the continuous-flow mode with isocratic HPLC condition using acetonitrile/D ₂ O containing 0.1% HCOOH (14:86) mixture under a wavelength of 306 nm ( Fig. 3 ). Compound 2 showed three aromatic aglycone signals at δ 7.90 (2H, s, H-2',6'), 7.39 (1H, d, J = 2.7 Hz, H-8) and 7.13 (1H, d, J = 2.7 Hz, H-6), corresponding to myricetin (3,5,7, 3',4',5'-hexahydroxyflavone), in addition to several charac-teristic proton signals typical with a α-rhamnopyranose at δ 3.30~5.80 including δ 5.77 (1H, s, H-1'') and 4.57 (1H, s, H-2''). 16 Compounds 3 and 4 exhibited typical quercetin (3,5,7,3',4'-pentahydroxyflavone) signals at δ 8.74, 8.69 (1H, d, J = 2.4~3.0 Hz, H-2'), δ 8.61, 8.60 (1H, dd, J = 10.2, 2.4~3.0 Hz, H-6'), δ 7.91, 7.90 (1H, d, J = 10.2 Hz, H-5'), δ 7.40, 7.39 (1H, d, J = 2.7~3.0 Hz, H-8) and δ 7.12 (1H, d, J = 2.7~3.0 Hz, H-6). The sugars were assigned to be galactopyranose and glucopyranose by comparison of their NMR data with published data. 17 , 18 The large vicinal J values (9.0~9.6 Hz) of the anomeric proton (H-1'') indicated that these sugars had β-configurations. The site of attachment between each sugar and the aglycone in 2 - 4 was unambiguously determined to be at C-3 as O -glycoside from long-range correlations observed in the supplementary HMBC experiments. From these results, compounds 2 - 4 were identified as myricitrin (myricetin 3- O - α -L-rhamnoside), hyperoside (quercetin 3- O - β -D-galactoside) and isoquercitrin (quercetin 3- O - β -Dglucoside), respectively. This experiment showed that directly coupled LC-NMR-MS could be an effective and powerful tool to analyze the aglycone and glycone in flavonol glycosides with the same molecular weight without laborious purification step. The structures of compounds 1 and 5 - 11 were elucidated by interpretation of one dimensional (1D) and two dimensional (2D) NMR, UV and ESI-MS spectral analyses after semi-preparative HPLC purification. They were also confirmed by comparison of their chemical and spectral data with those previously reported in the literature. Compounds 5 and 6 showed the pseudomolecular ion peaks [M−H] ⁻ at m / z 447 and 463 corresponding to the molecular formula C ₂₁ H ₂₀ O ₁₁ and C ₂₁ H ₂₀ O ₁₂ , respectively. Aromatic proton signals for a quercetin moiety (H-6, 8 and H-2', 5', 6') were shown in both ¹ H NMR spectra. Sugar signals in the NMR spectra of 5 and 6 were assigned to be α -rhamnopyranose and β -glucopyranose, respectively, in comparison with the previously reported spectroscopic data, and their attached position were identified to be C-3 in 5 and C-4' in 6 based on HMBC correlation of H-1'' proton to C-3 and to C-4', respectively. 19 - 21 Compound 7 showed the [M−H] ⁻ peak at m / z 447, and a pair of proton NMR signals at δ 8.18 (2H, d, J = 8.5 Hz) and 7.22 (2H, d, J = 8.5 Hz) suggested that it is kaempferol- O -glycoside. The unambiguous structure of 7 was determined to be kaempferol 4'- O -β-D-glucopyranoside, which was confirmed by 2D NMR data and in comparison with the previously reported spectroscopic data. 19 , 20 , 22 , 23 Chalcononaringenin 2'- O - β -D-glucoside ( 8 ) displayed the molecular ion peak [M−H] ⁻ at m / z 433, corresponding to a molecular formula C ₂₁ H ₂₂ O ₁₀ , and also its aglycone exhibited proton NMR signals of an α , β -unsaturated ketone at δ 8.03 (1H, d, J = 16.0 Hz), 7.67 (1H, d, J = 16.0 Hz), a 1,4-disubstituted aromatic at δ 7.61 (2H, d, J = 8.5 Hz), 6.84 (2H, d, J = 8.5 Hz), a 1,3,4,5-tetrasubstituted aromatic moiety at δ 6.22 (1H, d, J = 2.0 Hz), 5.99 (1H, d, J = 2.0 Hz). The structure of sugar moiety and its position was identified to be a 2'- O -β-Dglucopyranoside from further HSQC and HMBC experiments. 24 Two acylated flavonoids 9 and 10 had the same molecular formula C ₃₀ H ₂₆ O ₁₃ as confirmed by the ion peak [M−H] ⁻ at m / z 593, as well as the same aglycone and sugar moieties of kaempferol 3- O - β -glucopyranoside as established by NMR data. The ¹ H NMR spectrum of 9 exhibited the signals for a trans -coumaroyl group at δ 7.27, 6.77 (each 2H, d, J = 8.5 Hz, H-2''', 6''' and H-3''', 5''') and δ 7.39, 6.06 (each 1H, d, J =16.0 Hz, H-7''', 8'''). In contrast, compound 10 showed the proton signals for a cis -coumaroyl moiety at δ 7.51, 6.68 (each 2H, d, J = 8.0 Hz, H-2''', 6''' and H-3''', 5''') and δ 6.69, 5.51 (each 1H, d, J = 12.8 Hz, H-7''', 8'''). Acylated positions in 9 and 10 were assigned to be C-6'' of glucopyranose, which was confirmed on the basis of HMBC correlations of two methylene protons H-6'' to C-9'''. 25 , 26 Thus, structures of these compounds were identified as trans -tiliroside ( 9 , kaempferol 3- O - β -D-(6- O - trans - p -coumaroyl)glucopyranoside) and cis -tiliroside ( 10 , kaempferol-3- β -D-(6- O - cis - p -coumaroyl) glucopyranoside). Phenolic compounds 1 and 11 showed maximal UV absorption bands at around 280 nm and aromatic protons (each 2H, s) indicating the presence of galloyl moieties. Based on the MS, ¹ H and ¹³ C NMR spectral data they were assigned as methyl gallate ( 1 ) and 1,2,3,4,6-penta- O -galloyl- β -D-glucose ( 11 ). 27 , 28 Anti-proliferative effects of compounds from H. japonica were assessed by viability percentage of cancer cells. The cytotoxicity was measured using MTT assay on HCT116 human colon cancer cells at the concentration of 2.5 - 40 μM. As shown in Fig. 4 , penta- O -galloyl- β -glucose ( 11 ) exhibited strong cytotoxicity in comparison to other compounds. Cytotoxic effects of an extract 29 and antitumor effects of quercetin glycosides ( 3 - 6 ) 15 from the identical plant materials have been recently described by our groups. The results showed that type or position of a glycone affected anti-tumor activity and toxicity, which were characterized using cell viability test, western blotting assay and zebrafish-based in vivo assay. 15 In summary, combination studies of on-line HPLC-hyphenated NMR spectroscopy and conventional NMR techniques provided valuable information for chemical composition of plant extract from the flowers of H. japonica . Secondary metabolites in this plant have not been investigated in depth except for the presence of spiraeoside ( 6 ). 10 Additionally, cell viability test on MTT assay afforded an information of anti-proliferation effects for the identified compounds 1 - 11 against human colon cancer cells. Considering our phytochemical and biological studies, it is assumed that tannins and flavonol glycosides may be major principles responsible for anti-tumor effects of the extract.