Journal of Chromatographic Science, 2017, 1–7 doi: 10.1093/chromsci/bmx076 Article Article Isolation of Flavonoids From Wild Aquilaria sinensis Leaves by an Improved Preparative High-Speed Counter-Current Chromatography Apparatus Mao-Xun Yang1,2,†, Yao-Guang Liang3,†, He-Ru Chen2,4,*, Yong-Fang Huang5, Hai-Guang Gong5, Tian-You Zhang3, and Yoichiro Ito6 1 Department of Biomedicine, Zhongshan Torch Polytechnic, 60 ZhongShan Port Avenue, Zhongshan 528436, PR China, 2Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, PR China, 3Guangdong Bless Biotechnical Development Co., Ltd, Area A 4th Floor, Building 4, Science and Technology Garden, South China Modern Chinese Medicine Park, Zhongshan 528400, PR China, 4Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, 601 Huangpu Avenue West, Guangzhou 510632, PR China, 5College of Forestry, South China Agricultural University, 483 Wushan Avenue, Guangzhou 510642, PR China, and 6Laboratory of Bioseparation Technology, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 10, Room 8N230, 10 Center Drive, Bethesda, MD 20892-1762, USA * Author to whom correspondence should be addressed. Email: firstname.lastname@example.org † These two authors contribute equally. Received 11 March 2015; Revised 8 May 2017; Editorial Decision 19 July 2017 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 © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: email@example.com 1 2 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. Yang et al. 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 ESImass 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 Figure 1. Structures of the four ﬂavonoids from the wild Aquilaria sinensis leaves. 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), Isolation of Flavonoids From Wild Aquilaria sinensis Leaves by an Improved Preparative HSCCC Apparatus 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 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 5:2.5: 5:5 5:10: 5:5 5:5: 6:5 5:5: 7.5:5 5:5: 10:5 5:6: 6:5 5:7.5: 6:5 5:7.5: 7.5:5 0.10 0.11 0.03 0.18 0.36 0.70 0.19 0.07 0.33 0.44 0.82 0.12 0.84 1.65 3.13 0.74 0.23 1.05 3 tested in the present study. K-values of the target compounds were measured to predict the retention volume of the tested systems. 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: Nitrite scavenging percentage = Acontrol − Asample Acontrol × 100% Table II. The K-Values of Quercetin and Kaempferol Measured in Different Ratios HEMW Systems Two-phase solvent system HEMW 5:4: 5:4 7:4: 5:4 5:6: 5:4 5:7: 5:4 5:5: 5:5 where, the Acontrol is the absorbance of solution without the addition of sample solution. K-value Quercetin Kaempferol 0.04 0.02 0.14 0.79 0.18 0.24 0.13 0.44 1.81 0.63 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 4 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). 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). Yang et al. 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. 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), Figure 2. 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. Isolation of Flavonoids From Wild Aquilaria sinensis Leaves by an Improved Preparative HSCCC Apparatus 5 Figure 3. 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. 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 Genkwanin Quercetin Kaempferol DOX 12.40 ± 0.20 5.84 ± 0.03 28.10 ± 0.17 5.19 ± 0.11 – >50 >50 12.54 ± 1.37 38.63 ± 4.05 0.17 ± 0.03 a Data expressed as mean±SEM (standard error of mean) of three observation per sample. – Means no data. 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 apigenin7,4′-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). 6 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 antiangiogenic 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. 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