Abstract Four flavonoids including apigenin-7,4′-dimethylether, genkwanin, quercetin, and kaempferol were isolated in a preparative or semi-preparative scale from the leaves of wild Aquilaria sinensis using an improved preparative high-speed counter-current chromatography apparatus. The separations were performed with a two-phase solvent system composed of hexane–ethyl acetate, methanol–water at suitable volume ratios. The obtained fractions were analyzed by HPLC, and the identification of each target compound was carried out by ESI-MS and NMR. The yields of the above four target flavonoids were 4.7, 10.0, 11.0 and 4.4%, respectively. All these four flavonoids exhibited nitrite scavenging activities with the clearance rate of 12.40 ± 0.20%, 5.84 ± 0.03%, 28.10 ± 0.17% and 5.19 ± 0.11%, respectively. Quercetin was originally isolated from the Thymelaeaceae family, while kaempferol was isolated from the Aquilaria genus for the first time. In cytotoxicity test these two flavonoids exhibited moderate inhibitory activities against HepG2 cells with the IC50 values of 12.54 ± 1.37 and 38.63 ± 4.05 μM, respectively. Introduction Aquilaria sinensis (Lour.) Gilg (Thymelaeaceae), a principal source of the expensive agilawood, is distributed in the south China such as Hainan, Guangxi, Guangdong, Fujian and Taiwan provinces. It is one of the most valuable forest products currently known and traded all over the world (1). Agilawood is of particular interest, but becoming scarce year by year. However, the resource of leaves of A. sinensis is abundant and available two quarters per year in southern China. Traditionally, these leaves are used in China for treatments for inflammation and anaphylaxis (2). They are also broadly used as a main component in several health foods including A. sinensis tea, honey and flavor. The ethanol extract from the leaves of A. sinensis was confirmed to have analgesic, anti-inflammatory, and nitrite scavenging activities (3, 4). Several previous studies have indicated that the main compounds from the leaves of A. sinensis are flavonoids, benzophenone glycoside and triterpenoids. These compounds exhibited notable antinociceptive, anti-inflammatory, antioxidative, α-glucosidase inhibitory and laxative activities (2, 5–10). Considering their various biological activities, a large quantity of pure bioactive compounds (with a focus on flavonoids) from the leaves of A. sinensis is needed for further pharmacological studies and industrial applications. Traditional separation and purification methods of flavonoids from the leaves of A. sinensis require multiple chromatographic steps using silica gel, polyamide column, sephadex LH-20, preparative HPLC, etc. These methods are more or less non-green, tedious and time consuming with a potential risk of loss of target compounds due to the highly irreversible adsorptive, contaminative and denaturing effects of the solid matrix. High-speed counter-current chromatography (HSCCC), a unique liquid–liquid partition chromatographic technique without solid matrix, can yield a highly efficient separation of a large amount of samples in several hours and also permits introduction of crude samples directly into the separation column without extensive preparation (11). HSCCC has been successfully applied to the isolation and purification of a number of natural products including flavonoids (12–14). It is an effective and economical separation technology especially for flavonoid-like compounds that can be adsorbed and lost in the solid-liquid chromatographic process. However, to our best knowledge, no report has been published on the use of HSCCC for the isolation and purification of compounds from wild A. sinensis leaves. Because of usually limited distribution space and relatively small amount of stationary phase, usually only milligram to hundred milligram amounts of purified compounds can be obtained by HSCCC apparatuses widely used at present. In order to increase the preparation quantity ranged from gram to ten gram by HSCCC, HSCCC apparatus with a high β values was designed and assembled under patents CN201310032823.8 and CN201320047321.8 by Prof Tian You Zhang’s group in Guangdong, China (15, 16). In the current study, four flavonoids (Figure 1) were successfully purified from wild A. sinensis leaves by this improved preparative HSCCC apparatus, and their anti-cancer activity was investigated. Figure 1. View largeDownload slide Structures of the four flavonoids from the wild Aquilaria sinensis leaves. Figure 1. View largeDownload slide Structures of the four flavonoids from the wild Aquilaria sinensis leaves. Experimental Chemicals and reagents Silica gel (100–200 mesh) was purchased from Qingdao Ocean Chemical Co. (Qing-dao, China); and pre-coated silica gel HSGF254 thin layer chromatography (TLC) plates were obtained from Jiangyou Silica Gel Development Co. (Yantai, China). High performance liquid chromatography (HPLC) grade methanol (MeOH) was from Merck Chemical Co. (Darmstadt, Germany). Aanalytical grade n-hexane, ethyl acetate (EtOAc), MeOH, n-butanol, petroleum ether (b.p. 60–90°C), acetone, sodium nitrite, sulfanilic acid, N-ethylenediamine, citrate sodium, monosodium phosphate and muriatic acid were purchased from Guangzhou Chemical Reagent Co. (Guangzhou, China). All cell culture reagents were obtained from Invitrogen Co. (Carlsbad, CA, USA). Cell culture dishes and plates were purchased from Corning Inc. (New York, USA). 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were from Sigma-Aldrich Co. (St. Louis, MO, USA). Human hepatocellular cancer cell lines (HepG2) were established and maintained in our laboratory. Instruments The present study utilized a GX-6L high-speed counter-current chromatograph equipped with a multilayer coiled separation column with a total capacity of 1,000 mL (if six units are connected in series, the total column capacity becomes 6.0 L); a manual sample injection valve with a 20-mL or 50-mL sample injection loop (An engineering HSCCC prototype, which designed and assembled under patents CN201310032823.8 and CN201320047321.8, Guangdong, China); and an HD-2000 ultraviolet detector (Jiapeng, Shanghai, China). An LC-10Avp liquid chromatography (HPLC) system used was equipped with a CTO-10ASvp column oven, a manual sample injection valve (model 7725) with a 20-μL loop, and an SPD-10Avp ultraviolet detector (Shimadzu, Kyoto, Japan); YMC-Pack ODS-A columns (5 μm, 250 × 4.6 mm2 I.D.) for analytic purposes (YMC, Kyoto, Japan). Identification of purified samples was carried out with an ESI-mass spectrometer a Finnigan LCQ Advantage MAX spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) and a nuclear magnetic resonance (NMR) instrument from a Bruker AV-300 or Bruker AV-400 (Bruker Biospin, Rheistetten, Baden-Württemberg, Germany). A UV–spectrophotometer (Beijing’s General Instrument Co., Ltd, China) and Microplate Reader (TECAN SpectraII Plate Reader, Research Triangle Park, NC, USA) were also used. Plant materials The leaves of wild A. sinensis were collected from Sanxiang Town of Zhongshan City, Guangdong Province, China in May, 2011. The plant material was botanically authenticated by Prof Zhijian Feng in College of Forestry, South China Agricultural University. A voucher specimen (No. JNU-2267) was deposited in the herbarium of South China Agricultural University. Preparation of crude sample Oven-dried leaves of wild A. sinensis (1.0 kg) were extracted thrice each with 10.0 L of 70% acetone for 1 h under ultrasonication. The combined extract was condensed under vacuum to give a syrupy extract, which was then diluted with water to a total volume of 5 L and then partitioned successively with petroleum ether (5.0 L × 4), EtOAc (5.0 L × 5), and n-butanol (5.0 L × 3). The combined layers of each organic solvent were evaporated in vacuo to yield a petroleum ether-soluble fraction (FP, 33.8 g), an EtOAc-soluble fraction (FE, 43.5 g), and a n-butanol-soluble fraction (FB, 21.7 g), respectively. The FP was further subjected to silica gel column chromatography using a gradient elution with petroleum ether/EtOAc (10:1–4:1, V/V) to give two sub-fractions (FP1 and FP2) on the basis of TLC tracing. Preparation of two-phase solvent system and sample solution The two-phase solvent systems composed of n-hexane–EtOAc–MeOH–water (HEMW) at various volume ratios were used for HSCCC separation. Each set of solvent system was added to a separatory funnel and thoroughly equilibrated at room temperature for 2 h. The upper phase and lower phase were separated and degassed by sonication for 30 min shortly before use. The sample solutions were prepared by dissolving FP1, FP2, FE or FB in the mobile phase of the selected solvent system. Selection of two-phase solvent systems Successful separation by HSCCC largely depends upon the selection of suitable two-phase solvent systems. In the previous research on separation of flavonoids, many different organic solvent systems were ever selected, among which HEMW was used most frequently, 60% of the reported solvent systems for the isolation of free flavonoids from plant extracts where most flavonoids had suitable partition coefficient in the above solvent system with different volume ratios (17, 18). According to the above procedure, several different volume ratios of HEMW solvent systems (Tables I and II) were made and tested in the present study. K-values of the target compounds were measured to predict the retention volume of the tested systems. Table I. The K-Values of Apigenin-7,4′-Dimethylether and Genkwanin Measured in Different Ratios of HEMW Solvent Systems Two-phase solvent system HEMW K-value Apigenin-7,4′-dimethylether Genkwanin 5:5: 5:5 0.10 0.44 5:2.5: 5:5 0.11 0.82 5:10: 5:5 0.03 0.12 5:5: 6:5 0.18 0.84 5:5: 7.5:5 0.36 1.65 5:5: 10:5 0.70 3.13 5:6: 6:5 0.19 0.74 5:7.5: 6:5 0.07 0.23 5:7.5: 7.5:5 0.33 1.05 Two-phase solvent system HEMW K-value Apigenin-7,4′-dimethylether Genkwanin 5:5: 5:5 0.10 0.44 5:2.5: 5:5 0.11 0.82 5:10: 5:5 0.03 0.12 5:5: 6:5 0.18 0.84 5:5: 7.5:5 0.36 1.65 5:5: 10:5 0.70 3.13 5:6: 6:5 0.19 0.74 5:7.5: 6:5 0.07 0.23 5:7.5: 7.5:5 0.33 1.05 Table II. The K-Values of Quercetin and Kaempferol Measured in Different Ratios HEMW Systems Two-phase solvent system HEMW K-value Quercetin Kaempferol 5:4: 5:4 0.04 0.24 7:4: 5:4 0.02 0.13 5:6: 5:4 0.14 0.44 5:7: 5:4 0.79 1.81 5:5: 5:5 0.18 0.63 Two-phase solvent system HEMW K-value Quercetin Kaempferol 5:4: 5:4 0.04 0.24 7:4: 5:4 0.02 0.13 5:6: 5:4 0.14 0.44 5:7: 5:4 0.79 1.81 5:5: 5:5 0.18 0.63 HSCCC separation procedure The stationary phase was pumped into the column from head to tail. After the column was totally filled, the rotor was rotated at 400 rpm. Then, the mobile phase was pumped into the column at a flow-rate of 10 mL/min until hydrodynamic equilibrium was reached, when no further stationary phase was displaced from the column. The volume of stationary phase displaced from the column was noted to calculate the retention of the stationary phase in the column. Then, 420 mg of FP1, 700 mg of FP2 and 780 mg of FE were pumped into a 20-mL (2.0% of coil volume) sample loop and injected into the column through the injection valve. The effluent from the tail end of the column was continuously monitored with a UV absorbance detector at 340 nm. The data were recorded immediately after sample injection. Fractions were collected manually when chromatographic peaks were detected. A 1-mL aliquot was taken from each fraction and analyzed for quantity and purity by HPLC. HPLC analysis and identification of HSCCC fractions Each peak fraction of HSCCC was analyzed by RP-HPLC where 360 nm was chosen as the UV detection wavelength. Flow rate was set at 1.0 mL/min. Identification of the HSCCC peak fractions was based on the data of ESI-MS, 1H and 13C NMR. Nitrite scavenging test Anti-cancer activities of the four flavonoids were evaluated by the nitrite scavenging activity assay using a UV spectrophotometer at a wavelength of 544 nm performed as described previously (19). The conditions were modified as follows: sodium nitrite (5 μg/mL), naphthyl ethylene diamine dihydrochloride (0.2% w/v), sulfanilic acid (0.4% w/v), reaction temperature 37°C, reaction time 30 min, citric acid/sodium dihydrogenphosphate buffer solution at pH value of 3.0 or 7.0, and sample concentration of 3.0 mg/mL. The reaction mixture (3 mL) containing sodium nitrite (2 mL), sample solution (0.5 mL) and buffer solution (0.5 mL) (for adjusting the pH value of 3.0 or 7.0) was incubated at 37°C for 30 min. After incubation, 0.5 mL of the reaction mixture mixed with 2 mL of sulfanilic acid and allowed to stand for 5 min for completing diazotization. Then, 1 mL of naphthyl ethylene diamine dihydrochloride was added, mixed and allowed to stand for 30 min at 37°C. A pink colored chromophore was formed in diffused light. The absorbance of sample solutions was measured at 544 nm against the corresponding blank solution (distilled water), and the % scavenging value was computed according to the following formula: Nitritescavengingpercentage=Acontrol−AsampleAcontrol×100%where, the Acontrol is the absorbance of solution without the addition of sample solution. Cytotoxicity test Cytotoxic activities against cancer cells of the four flavonoids were evaluated using MTT assay, which was performed as described previously (20) with Doxorubin (Dox) served as the positive control. Briefly, cells were plated on 96-well plates at 3 × 103 cells per well for HepG2 cell lines. After 48 h of exposure, the cells were stained with MTT. Absorbance at 570 nm was used to measure with a multiplate reader. Statistical analysis All data were expressed as mean±SEM (standard error of mean). Results were analyzed by one-way analysis of variance (ANOVA), and significant differences were determined by post-hoc Tukey test using SPSS 11.0 software, where differences were statistically significant at P < 0.05. Results Preparative isolation of four flavonoids by HSCCC The current study performed with an optimized HEMW two-phase solvent systems at the volume ratio of 5:7.5: 6:5 has achieved the preparative separation of apigenin-7,4′- dimethylether and genkwanin from 420 mg of FP1 and 700 mg of FP2 (Figure 2A–D), where 20 mg (4.7%) of apigenin-7,4′-dimethylether with purity of 99.7%; and 70 mg (10%) of genkwanin with purity of 93.1% were obtained each in a single HSCCC run. The detailed chemical structures of apigenin-7, 4′-dimethylether and genkwanin (Figure 1) were confirmed by the comparison of their NMR and MS with the data from literature (21, 22). Figure 2. View largeDownload slide HSCCC separations of FP1 and FP2. HSCCC chromatograms of FP1 (A) and FP2 (B): Experimental conditions: column volume: 1000 mL; phase system: HEMW (5:7.5:6:5, V/V); stationary phase: lower aqueous; mobile phase: organic upper phase; rotational speed: 400 rpm; detection wavelength: 340 nm; retention of stationary phase: 77.6%. RP-HPLC profiles of apigenin-7,4′-dimethylether (C) and genkwanin (D). Column: Welch Ultimate C18 (250 × 4.6 mm2 i.d., 5 μm); isocratic, eluant (V/V): (C) AcN:H2O:AcOH = 60:20: 2; (D) MeOH: 2% AcOH in water = 70:30. Note: The optional detection wavelengths of the prepartive HSCCC apparatus matching UV absorbance detector are 220, 254, 280 and 340 nm, and 340 nm was chosed to HSCCC separations of the four flavonoids in this study. Figure 2. View largeDownload slide HSCCC separations of FP1 and FP2. HSCCC chromatograms of FP1 (A) and FP2 (B): Experimental conditions: column volume: 1000 mL; phase system: HEMW (5:7.5:6:5, V/V); stationary phase: lower aqueous; mobile phase: organic upper phase; rotational speed: 400 rpm; detection wavelength: 340 nm; retention of stationary phase: 77.6%. RP-HPLC profiles of apigenin-7,4′-dimethylether (C) and genkwanin (D). Column: Welch Ultimate C18 (250 × 4.6 mm2 i.d., 5 μm); isocratic, eluant (V/V): (C) AcN:H2O:AcOH = 60:20: 2; (D) MeOH: 2% AcOH in water = 70:30. Note: The optional detection wavelengths of the prepartive HSCCC apparatus matching UV absorbance detector are 220, 254, 280 and 340 nm, and 340 nm was chosed to HSCCC separations of the four flavonoids in this study. As shown in Figure 3A–C, quercetin (86 mg, 11.0%) with purity of 99.4% and kaempferol (34 mg, 4.4%) with purity of 98.7% were simultaneously obtained from 780 mg of FE at one HSCCC run by modifying the HEMW two-phase solvent system at the volumn ratio of 5:5: 5:5. The elucidation of their structures (Figure 1) was based on the NMR and MS analysis combined with the data comparison to the literature (23, 24). Figure 3. View largeDownload slide HSCCC separation of FE. (A) HSCCC chromatogram. Experimental conditions: column volume: 1,000 mL; two-phase solvent system: HEMW (5:5:5:5, V/V); stationary phase: lower aqueous; mobile phase: organic upper phase; rotational speed: 400 rpm; detection wavelength: 340 nm; retention of stationary phase: 82.4%. (B and C) RP-HPLC profiles of quercetin (B) and kaempferol (C). Analytical conditions: column: Welch Ultimate C18 (250 × 4.6 mm2 i.d., 5 μm); elution mode: isocratic; eluant (V/V): (B) MeOH: 0.4% phosphoric acid in water = 55:45; (C) MeOH: 0.05% phosphoric acid in water = 65:35. Figure 3. View largeDownload slide HSCCC separation of FE. (A) HSCCC chromatogram. Experimental conditions: column volume: 1,000 mL; two-phase solvent system: HEMW (5:5:5:5, V/V); stationary phase: lower aqueous; mobile phase: organic upper phase; rotational speed: 400 rpm; detection wavelength: 340 nm; retention of stationary phase: 82.4%. (B and C) RP-HPLC profiles of quercetin (B) and kaempferol (C). Analytical conditions: column: Welch Ultimate C18 (250 × 4.6 mm2 i.d., 5 μm); elution mode: isocratic; eluant (V/V): (B) MeOH: 0.4% phosphoric acid in water = 55:45; (C) MeOH: 0.05% phosphoric acid in water = 65:35. The K-value test of the target compounds indicated that the HEMW two-phase system at a volume ratio of = 5:7.5: 6:5 was suitable for the separation of apigenin-7,4′-dimethylether and genkwanin (K values of 0.07 and 0.23, Table I); and that at 5:5: 5:5 was suitable for the separation of quercetin and kaempferol (K values of 0.18 and 0.63, Table II). In vitro cancer-preventing activity As shown in Tables III, all the four flavonoids exhibited nitrite scavenging activities, in which quercetin is the most active compound at the scavenging rate of 28.10 ± 0.17%. In contrast, kaempferol is the poorest scavenger with scavenging rate of 5.19 ± 0.11%. The current determination was set in a condition with temperature at 37°C, pH value of 3.0 and reaction time of 30 min. Table III. Nitrite Scavenging Activities and Growth Inhibitory Activities to HepG2 Cells of the Four Flavonoilsa Compounds Nitrite scavenging rate (%) HepG2 IC50 (μM) Apigenin-7,4′-dimethylether 12.40 ± 0.20 >50 Genkwanin 5.84 ± 0.03 >50 Quercetin 28.10 ± 0.17 12.54 ± 1.37 Kaempferol 5.19 ± 0.11 38.63 ± 4.05 DOX – 0.17 ± 0.03 Compounds Nitrite scavenging rate (%) HepG2 IC50 (μM) Apigenin-7,4′-dimethylether 12.40 ± 0.20 >50 Genkwanin 5.84 ± 0.03 >50 Quercetin 28.10 ± 0.17 12.54 ± 1.37 Kaempferol 5.19 ± 0.11 38.63 ± 4.05 DOX – 0.17 ± 0.03 aData expressed as mean±SEM (standard error of mean) of three observation per sample. – Means no data. In vitro cytotoxicity Among the four flavonoids, Kaempferol and quercetin exhibited moderate inhibitory activities against human hepatocarcinoma cells (HepG2) with IC50 values of 12.54 ± 1.37, and 38.63 ± 4.05 μM (Table I), respectively. However, 4′-dimethylether and genkwanin showed only weak inhibitory activities against the growth of HepG2 cells, where their IC50 values were larger than 50 μM. Discussion Preparative isolation of four flavonoids by HSCCC Although HSCCC has been successfully applied to the isolation and purification of many natural products including flavonoids (13, 14, 22), so far it has not been applied to the leaves of wild A. sinensiss. In the present study separation conditions such as two-phase solvent systems and operating procedures applied to the wild A. sinensis leaves were successfully set up using an improved preparative HSCCC apparatus. This prototype HSCCC apparatus can produce excellent retention of stationary phase with a large sample loading capacity and high chromatographic resolution. The compounds with high purity can be obtained in hundred milligrams to grams from one unit of HSCCC columns, and ten to hundred gram grade samples (50 times injection volume of this study) can be separated if six units are connected in series. Four flavonoids including apigenin-7,4′-dimethylether, genkwanin, quercetin and kaempferol were isolated in a preparative or semi-preparative scale with excellent stationary phase retention for all the tested solvent systems by this improved preparative HSCCC apparatus used in this study, which lay the foundation for the industrial production of these compounds. Here, it is necessary to point out that quercetin was originally isolated from the Thymelaeaceae family to which A. sinensis belongs, and kaempferol was isolated for the first time from the Aquilaria genus to which A. sinensis belongs (25). In vitro anti-cancer activity Sodium nitrite (SNT) is ubiquitous in the environment and can also be formed from nitrogenous compounds by microorganisms present in the soil, water, saliva and the gastrointestinal tract. SNT is widely used in food and drug industries as a preservative. About 40% of absorbed nitrite is excreted unchanged in the urine while the metabolism of the rest is not accurately known (26). When we ingest nitrite, endogenous nitrosation may form N-nitrosocompounds (NOCs) that have been observed to induce tumors of the kidney in animals (27, 28). In the human body, primarily in the stomach, nitrite can react with amines, amides or amino acids to produce NOCs, most of which are potent animal carcinogens. Therefore, scavenging ingested nitrite is probably one way to prevent carcinogenesis. As an example, vitamin C is traditionally used as a drug for cancer prevention partially because it is an effective inhibitor of NOCs formation (29, 30). In our previous work, nitrite scavenging activities of the ethanol extract from the leaves of A. sinensis were demonstrated (4), although the bioactive constituents are unknown. Based on the full understanding of flavonoids, we believe that some of those flavonoids should play a role as a nitrite scavenger. According to the result of our study, all four flavonoids exhibited nitrite scavenging activities. However, it should be noted that the current determination was set in a mimetic gastric environment. The mechanism of nitrite scavenge by flavonoids is now under investigation in our laboratory. In vitro cytotoxicity Kaempferol and quercetin have been reported to inhibit cancer development through an anti-angiogenic mechanism (31, 32), in which human ovarian cancer cells and hamster buccal pouch cells were used. The present investigation indicated that both compounds also exhibited moderate inhibitory activities against human hepatocarcinoma cells (HepG2). However, apigenin-7,4′-dimethylether and genkwanin showed only weak inhibitory activities against the growth of HepG2 cells. From the structural differences among the four flavonoids (Figure 1), it is suggested that the methylation of phenolic hydroxyl group(s) in the molecules might decrease their anti-cancer activities. The aforementioned cancer inhibition of kaempferol and quercetin may be explained on the basis of the anti-angiogenic mechanism. Conclusion In summary, four bioactive flavonoids, namely apigenin-7,4′-dimethylether genkwanin, quercetin, and kaempferol were successfully purified in a preparative or semi-preparative scale from the leaves of wild A. sinesis under environment-friendly conditions by an improved preparative HSCCC apparatus, with an HEMW solvent system at different volume ratios. Among those, quercetin was originally isolated from the Thymelaeaceae family, while kaempferol was isolated from the Aquilaria genus for the first time. All these four flavonoids exhibited nitrite scavenging activities in a mimetic gastric environment, in which quercetin was the best scavenger. Among those flavonoids, quercetin and kaempferol exhibited moderate cytotoxic activities against HepG2. The current results partially disclose the bioactive constituents of the wild A. sinesis leaves. Supplementary Data Supplementary data are available at Journal of Chromatographic Science online. Funding This research was financially supported by the National Natural Science Foundation of China (No. 81172982), the Foundation of Science and Technology Planning Project of Guangdong Province (Nos. 2010A030100006 and 2013B020411003), and the Bureau of Science and Technology of Zhongshan City (No. 2016B2166). References 1 Editorial Committee of Chinese Pharmacopeia.; Chinese Pharmacopoeia 2010 ed . China Medical Science Press, Beijing, ( 2010); p. 128. 2 Qi, J., Lu, J.J., Liu, J.H., Yu, B.Y.; Flavonoid and a rare benzophenone glycoside from the leaves of Aquilaria sinensis; Chemical Pharmaceutical Bulletin , ( 2009); 57: 134– 137. Google Scholar CrossRef Search ADS PubMed 3 Zhou, M.H., Wang, H.G., Suolangjiba, Kou, J.P., Yu, B.Y.; Antinociceptive and anti-inflammatory activities of Aquilaria sinensis (Lour.) Gilg. leaves extract; Journal of Ethnopharmacology , ( 2008); 117: 345– 350. Google Scholar CrossRef Search ADS PubMed 4 Yang, M.X., Mao, S.S., Chen, H.R.; Scavenging effect of different fractions extracted from leaves of Aquilaria Sinensis on NO2-; Journal of Beihua University (Natural Science Ed.) , ( 2012); 13: 406– 409. 5 Feng, J., Yang, X.W., Wang, R.F.; Bio-assay guided isolation and identication of ɑ-glucosidase inhibitors from the leaves of Aquilaria sinensis; Phytochemistry , ( 2011); 72: 242– 247. Google Scholar CrossRef Search ADS PubMed 6 Nie, C., Song, Y., Chen, D., Xue, P., Tu, P., Wang, K., et al. .; Studies on chemical constituents of leaves of Aquilaria sinensis; Zhongguo Zhongyao Zazhi , ( 2009); 34: 858– 860. Google Scholar PubMed 7 Wang, H.G., Zhou, M.H., Lu, J.J., Yu, B.Y.; Antitumor constituents from the leaves of Aquilaria sinensis (Lour.) Gilg; Linchan Huaxue Yu Gongye , ( 2008); 28: 1– 5. 8 Cheng, J.T., Han, Y.Q., He, J., Wu, X.D., Dong, L.B., Peng, L.Y., et al. .; Two new tirucallane triterpenoids from the leaves of Aquilaria sinensis; Archives of Pharmacal Research , ( 2013); 36: 1084– 1089. Google Scholar CrossRef Search ADS PubMed 9 Hara, H., Ise, Y., Morimoto, N., Shimazawa, M., Ichihashi, M., Iinuma, M.; Laxative effect of agawood leaves and its mechanism; Bioscience, Biotechnology, and Biochemistry , ( 2008); 72: 335– 345. Google Scholar CrossRef Search ADS PubMed 10 Lu, J.J, Qi, J, Zhu, D.N, Yu, B.Y.; Antioxidant activity and structure-activity relationship of the flavones from the leaves of Aquilaria sinensis; Chinese Journal of Nature Medicines , ( 2008); 6: 456– 460. Google Scholar CrossRef Search ADS 11 Zhang, T.Y, Wang, X.; High-Speed Counter-Current Chromatography Technology . Chemical Industry Press, Beijing, ( 2011); pp. 93– 94. 12 Ito, Y.; Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography; Journal of Chromatography A , ( 2005); 1065: 145– 168. Google Scholar CrossRef Search ADS PubMed 13 Wu, S.J., Sun, A.L., Liu, R.M.; Separation and purification of baicalin and wogonoside from the Chinese medicinal plant Scutellaria baicalensis Georgi by high-speed counter-current chromatography; Journal of Chromatography A , ( 2005); 1066: 243– 247. Google Scholar CrossRef Search ADS PubMed 14 Li, H.B., Chen, F.; Isolation and purification of baicalein, wogonin and oroxylin A from the medicinal plant Scutellaria baicalensis by high-speed counter-current chromatography; Journal of Chromatography A , ( 2005); 1074: 107– 110. Google Scholar CrossRef Search ADS PubMed 15 Zhang, T.Y., Li, N., Wu, J.; An engineering high performance countercurrent chromatography apparatus, ( 2013), CN 2013-10032823.8, 2013-01-28. 16 Zhang, T.Y., Li, N., Wu, J.; An engineering countercurrent chromatography apparatus, ( 2013), CN 201320047321.8, 2013-01-28. 17 Costa, F.D.N., Leitao, G.G.; Strategies of solvent system selection for the isolation of flavonoids by countercurrent chromatography; Journal of Separation Science , ( 2010); 33: 336– 347. Google Scholar CrossRef Search ADS PubMed 18 Yuan, E.D., Liu, B.G., Ning, Z.X., Chen, C.G.; Preparative separation of flavonoids in Adinandra nitidaleaves by high-speed counter-current chromatography and their effects on human epidermal carcinoma cancer cells; Food Chemistry , ( 2009); 115: 1158– 1163. Google Scholar CrossRef Search ADS 19 Sfahlana, A.J., Mahmoodzadeh, A., Hasanzadeh, A., Heidari, R., Jamei, R.; Antioxidants and antiradicals in almond hull and shell (Amygdalus communis L.) as a function of genotype; Food Chemistry , ( 2009); 115: 529– 533. Google Scholar CrossRef Search ADS 20 Mosmann, T.; Rapid colorimetric assay for cellular growth and survival:application to proliferation and cytoxiclity assay; Journal of Immunological Methods , ( 1983); 65: 55– 63. Google Scholar CrossRef Search ADS PubMed 21 Chen, D., Bi, D., Song, Y.L., Tu, P.F.; Flavanoids from the stems of Aquilaria sinensis; Chinese Journal of Nature Medicines , ( 2012); 10: 287– 291. 22 Wei, Z.F., Luo, M., Zhao, C.J., Wang, W., Zhang, L., Zu, Y., et al. .; An efficient preparative procedure for main flavone aglycones from Equisetum palustre L. using macroporous resin followed by gel resin flash chromatography; Separation and Purification Technology , ( 2013); 118: 680– 689. Google Scholar CrossRef Search ADS 23 Wu, T., Lin, J.B., Yang, Y., Abdulla, R., Chen, J., Aisa, H.A.; Preparative isolation of three flavonoids from Flos Gossypii by high-speed counter-current chromatography; Separation and Purification Technology , ( 2009); 66: 295– 298. Google Scholar CrossRef Search ADS 24 Xiao, Z.P., Wu, H.K., Wu, T., Shi, H., Hang, B., Aisa, H.A.; Kaempferol and quercetin flavonoids from Rosa rugosa; Chemistry of Natural Compounds , ( 2006); 6: 736– 737. Google Scholar CrossRef Search ADS 25 Yang, M.X., Chen, H.R., Liang, Y.G., Zhang, T.Y., Xie, C.L., Jiang, J.P., et al. .; Method for separating active ingredient in leaves of Aquilaria sinensis by high-speed countercurrent chromatography, ( 2013), CN 2013-10062910.8, 2013-02-27. 26 Chow, C.K., Hong, C.B.; Dietary vitamin E and selenium and toxicity of nitrite and nitrate; ToxicologyToxicology , ( 2002); 80: 195– 207. Google Scholar CrossRef Search ADS 27 Grosse, Y., Baan, R., straif, K., Secretan, B., Ghissassi, F.E., Cogliano, V.; Carcinogenicity of nitrate, nitrite, and cyanobacterial peptide toxins; The Lancet Oncology , ( 2006); 7: 628– 629. Google Scholar CrossRef Search ADS PubMed 28 IARC.; Monographs on the Evaluation of Carcinogenesis Risks to Humans: Some N-nitrosocompounds , Vol. 17. International Agency for Research on Cancer, Lyon, ( 1998). 29 Mirvish, S.S.; Role of N-nitroso compounds (NOC) and N-nitrosation in etiology of gastric, esophageal, nasopharyngeal, and bladder cancer and contribution to cancer of known exposures to NOC; Cancer letters , ( 1995); 93: 17– 48. Google Scholar CrossRef Search ADS PubMed 30 Mirvish, S.S.; Inhibition by vitamins C and E in vivo nitrosation and vitamin C occurrence in the stomach; European Journal of Cancer Prevention : The Official Journal of the European Cancer Prevention Organisation (ECP) , ( 1996); 1: 131– 136. Google Scholar CrossRef Search ADS 31 Luo, H., Rankin, G.O., Liu, L., Daddysman, M.K., Jiang, B.H., Chen, Y.C.; Kaempferol inhibits angiogenesis and VEGF expression through both HIF dependent and independent pathways in human ovarian cancer cells; Nutrition and Cancer , ( 2009); 61: 554– 563. Google Scholar CrossRef Search ADS PubMed 32 Priyadarsini, R.V., Vinothini, G., Murugan, R.S., Manikandan, P., Nagini, S.; The flavonoid quercetin modulates the hallmark capabilities of hamster buccal pouch tumors; Nutrition and Cancer , ( 2011); 63: 218– 226. Google Scholar CrossRef Search ADS PubMed © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: email@example.com
Journal of Chromatographic Science – Oxford University Press
Published: Jan 1, 2018
It’s your single place to instantly
discover and read the research
that matters to you.
Enjoy affordable access to
over 12 million articles from more than
10,000 peer-reviewed journals.
All for just $49/month
It’s easy to organize your research with our built-in tools.
All the latest content is available, no embargo periods.
“Whoa! It’s like Spotify but for academic articles.”@Phil_Robichaud