Separation of Four Flavonol Glycosides from Solanum rostratum Dunal Using Solvent Sublation Followed by HSCCC and Low Column Temperature Preparative HPLC

Separation of Four Flavonol Glycosides from Solanum rostratum Dunal Using Solvent Sublation... Abstract Hyperoside, 3′-O-methylquercetin 3-O-β-D-galactopyranoside, astragalin and 3′-O-methylquercetin 3-O-β-D-glucopyranoside from an invasive weed Solanum rostratum Dunal were separated and purified successfully by high-speed counter-current chromatography (HSCCC) with a solvent system composed of n-hexane-ethyl acetate–methanol–water (1:7:1:7, v/v) and gradient elution mode preparative high-performance liquid chromatography (prep-HPLC) with low column temperature. In the sample pretreatment section, target compounds in aqueous extract of the weed were concentrated using solvent sublation. Two target fractions with purities of 93.75% and 93.68% were obtained from HSCCC. Their chemical structures were identified. The fraction 1 is a pure compound hyperoside and the fraction 2 is the mixture of astragalin, 3′-O-methylquercetin 3-O-β-D-galactopyranoside and 3′-O-methylquercetin 3-O-β-D-glucopyranoside by nuclear magnetic resonance and liquid chromatography-mass spectra. Then, the three flavonol glycosides in the fraction 2 were separated and purified successfully by prep-HPLC with low column temperature. Introduction High-speed counter-current chromatography (HSCCC), being a support-free liquid–liquid partition method (1), eliminates irreversible adsorption of sample onto the solid support (2), and has been widely used in preparative separation of active compounds from traditional Chinese herbs and other natural products (3–5). Solvent sublation (SS) is a kind of adsorptive bubble separation technique (6) in which many water-soluble active compounds with surface-activity can be adsorbed on the bubble surface and then collected in organic phase. Therefore, this technique can effectively separate and concentrate the water-soluble compounds. Preparative high-performance liquid chromatography (prep-HPLC) is easy to be used for separation of compounds because of its high separation efficiency and recovery (7). So, it can be used in the last procedure to separate the mixture which has not been separated completely (8, 9). Solanum rostratum Dunal, a newly invasive weed species in China, seriously breaks the ecological balance in the invasive areas because it inhibits and excludes native plants by releasing allelochemicals (10). In addition, this invasive weed also severely affects agricultural and animal husbandry production due to its extensive infestation in agricultural fields and grasslands (11, 12). It is worth mentioning that there are quantities of useful substances in this weed. For example, methylprotodioscin, a kind of cytotoxin found in this weed (13), was toxic to cancerous cells (14), so it can be used for the treatment of people with cancer. These researches provide a new way to develop and utilize the S. rostratum Dunal instead of destroy it. Therefore, it is significative to discover more active substances from the invasive weed for resource utilization. The objective of the present paper is to separate and purify flavonoids from S. rostratum Dunal by HSCCC combined with low column temperature prep-HPLC. The active compounds in aqueous S. rostratum Dunal extract were separated and concentrated by SS. Then, hyperoside, astragalin, 3′-O-methylquercetin 3-O-β-D-galactopyranoside and 3′-O-methylquercetin 3-O-β-D-glucopyranoside with high purity were obtained after further separation and purification using HSCCC and low column temperature prep-HPLC from flotation product. Experimental Chemicals and reagents Solanum rostratum Dunal plant was supplied by the Chinese Academy of Agricultural Sciences. Phosphoric acid was purchased from Yili Fine Chemicals Co., Ltd (Beijing, China). Methanol, acetonitrile and trifluoroacetic acid used as mobile phase for HPLC analysis and prep-HPLC purification were all of chromatography grade (J&K, China). Ammonium sulfate, isopropanol, hydrochloric acid and sodium hydroxide used for SS and n-hexane, ethyl acetate and methanol used for HSCCC were all of analytical grade and purchased from Beijing Chemical Reagent Factory (Beijing, China). Apparatus The SS apparatus is similar to the one mentioned in the previous papers (15). An AB204-N electronic balance (Mettler, Switzerland) and a Mettler Toledo 320-S pH meter (Mettler, Switzerland) were used. Semi-preparative HSCCC was performed using a Model GS10A multilayer coil planet centrifuge (Beijing Institute of New Technology Application, Beijing, China) equipped with a polytetrafluoroethylene multilayer coil of 110 m × 1.6 mm I.D. with a total capacity of 240 mL. The β value of the preparative column ranged from 0.5 to 0.8 (β = r/R, where r is the distance from the holder shaft to the coil and R is the distance between the holder axis and central axis of the planet centrifuge or the rotation radius). The solvent was pumped into the column with a Model NS-1007 constant-flow pump (Beijing Institute of New Technology Application, Beijing, China). Continuous monitoring of the effluent was achieved with a Model 8823A-UV Monitor (Beijing Institute of New Technology Application, Beijing, China) at 254 nm. A manual sample injection valve with a 10-mL loop (for the preparative HSCCC) was used to introduce the sample into the column. A portable recorder Yokogawa Model 3,057 (Sichuan Instrument Factory, Chongqing, China) was used to plot the chromatogram. A rotary evaporator was also used. Prep-HPLC was performed using a FLEXA Purification System (Agela, China) with an YMC-Pack ODS-A column (250 mm × 20 mm I.D., 5 μm, YMC, Japan) to separate and purify the mixture from HSCCC. And the products from prep-HPLC were analyzed by analytical HPLC with an Inertsil ODS-3 column (250 mm × 4.6 mm I.D., 5 μm). A Shimadzu LC-20AT chromatograph system (Shimadzu, Japan) was used to analyze the aqueous extract, the flotation products and the purification products from HSCCC. Identification of purified products was carried out by LCMS-IT-TOF and 1H NMR, 13C NMR spectra. LCMS-IT-TOF was performed using a Shimadzu (Kyoto, Japan) HPLC system consisting of a solvent delivery pump (LC-20AD), an auto-sampler (SIL-20AC), a DGU-20A3 degasser, a photodiode array detector (SPD-M20A), a communication base module (CBM-20A), a column oven (CTO-20A), an Inertsil ODS-SP column (150 × 4.6 mm I.D., 5 μm), and a hybrid ion trap/time-of-flight instrument (Shimadzu Corp., Kyoto, Japan) equipped with an ESI source. NMR spectra were performed in DMSO-d6 and CD3COCD3 using a Bruker high-resolution AV400NMR spectrometer (Bruker Biospin Corporation, USA). Preparation of extract of Solanum rostratum Dunal using SS for HSCCC Dried powder of S. rostratum Dunal (60.0 g) was extracted twice, each with 1,000 mL of pure water for 90 min by water reflux method. The residue was filtrated and washed with 200 mL of pure water. The HPLC chromatograms (Figure 1) of the S. rostratum Dunal extract showed that the retention time of fraction 1 was about 20.8 min and fraction 2 was 23.0 min. Subsequently, ammonium sulfate was added into the combined extracts, and the aqueous solution was used for the separation and concentration of SS. In order to prepare accurately the aqueous ammonium sulfate solution, the classical relationship equation (16) between the mass concentration of ammonium sulfate and the volume concentration was applied. The initial volume of aqueous solution in the flotation column was 400 mL. Figure 1. View largeDownload slide HPLC chromatogram of the Solanum rostratum Dunal extract and structural formula of target compounds. Chromatographic conditions: an Apollo C18 column (150 × 4.6 mm I.D., 5 μm), methanol (A) and 0.5% aqueous phosphoric acid (B) as mobile phase, the flow rate was 1.0 mL/min with a gradient elution of 20–100% A from 0 to 40 min, the detection wavelength was 254 nm and temperature of column oven was 30°C. Figure 1. View largeDownload slide HPLC chromatogram of the Solanum rostratum Dunal extract and structural formula of target compounds. Chromatographic conditions: an Apollo C18 column (150 × 4.6 mm I.D., 5 μm), methanol (A) and 0.5% aqueous phosphoric acid (B) as mobile phase, the flow rate was 1.0 mL/min with a gradient elution of 20–100% A from 0 to 40 min, the detection wavelength was 254 nm and temperature of column oven was 30°C. The parameters of SS, such as sublation solvent, solution pH, (NH4)2SO4 concentration in aqueous solution, nitrogen flow rate, flotation time, the volume of sublation solvent and repeat times can greatly influence the recovery of target compounds. In this paper, the parameters mentioned above were studied in this order by single factor experiment, and the specific process was the same as that reported in the previous study (15, 16). All the SS experiments were performed at room temperature. During the SS procedure, target compounds can be effectively separated and concentrated from the aqueous extract to the upper phase. Then the flotation product was concentrated to dryness under reduced pressure and then used for subsequent HSCCC isolation. Preparation of two-phase solvent system and sample solution The solvent system is essential to the separation process in HSCCC. An appropriate solvent system should satisfy the following requirements: it can dissolve the sample well and will not cause decomposition or denaturation of the sample; it can form stable two-phase solvent system and have a good retention of stationary phase; the partition coefficient (K) of the target components in it should be between 0.2 and 5 (17). The K value was determined by HPLC as follows: suitable amount of crude extract was dissolved in 2 mL of each phase of the pre-equilibrated two-phase solvent system with violent shaken in order to reach a thorough equilibrium at room temperature. After 3 h standing, 1 mL of each phase was taken out and evaporated to dryness. The residues were dissolved with 1 mL methanol and then analyzed by HPLC. The K value was calculated as the peak area of the target compound in the upper phase divided by the peak area of the target compound in the lower phase. The two-phase solvent system utilized in the present study was prepared by mixing n-hexane-ethyl acetate–methanol–water (1:5:1:5, 1:6:1:6, 1:7:1:7, v/v) in a separatory funnel. Then, the separatory funnel was shaked violently at room temperature to make the two-phase system thoroughly equilibrated. The upper phase and the lower phase were separated and degassed by ultrasonic for 30 min shortly before use. The sample solutions were prepared by dissolving the extract obtained in section 2.3 in 5.0 mL upper phase and 5.0 mL lower phase. Separation procedure for HSCCC In separation procedure, the multilayer coiled column was entirely filled with the upper phase as the stationary phase at first. Then the lower phase as mobile phase was pumped into the head end of the column at a flow rate of 2.0 mL/min, while the apparatus was rotated at 800 rpm. After a clear mobile phase eluted at the tail outlet, which means hydrodynamic equilibrium was established, the sample solution was injected through the sample port. A UV detector was used to monitor the effluent from the tail end of the column continuously at 254 nm. Each peak fraction was collected according to the chromatogram. The retention of the stationary phase was calculated from the volume of the stationary phase collected from the column before the sample was injected. Further separation for prep-HPLC The fraction 2 from HSCCC was dried with rotary evaporator with the temperature of 55°C, and it was dissolved by methanol and water (7:4, v/v). A YMC-Pack ODS-A column (250 mm × 20 mm I.D., 5 μm) was used for the separation procedure. The mobile phase was composed of acetonitrile (A) and water including 0.0125% trifluoroacetic acid (B), and a gradient elution of 14–21% A at 0–100 min was used with the detection wavelength of 254 nm. The flow rate was 8.0 mL/min and the injection volume was 0.5 mL. The prep-HPLC experiments were performed at 10°C temperature approximately. HPLC analysis The analytical HPLC was used to analyze the aqueous extract, the flotation product and the purification product from HSCCC with an Apollo C18 column (150 × 4.6 mm I.D., 5 μm). In the HPLC analysis, the mobile phase was composed of methanol (A) and 0.5% aqueous phosphoric acid (B). The flow rate was 1.0 mL/min with a gradient elution of 20–100% A at 0–40 min. The detection wavelength was 254 nm and the injection volume was 10 μL. It is important to note that the aqueous extracts and the flotation products should be diluted with methanol (1–2 mL), and desalted with centrifuge at 2,500 rpm for 10 min. The temperature of all the HPLC analytical experiments was 30°C. Each effluent from prep-HPLC was analyzed by HPLC with an Inertsil ODS-3 column (250 mm × 4.6 mm I.D., 5 μm), and the purity was obtained with the method of area normalization. It is noted that the constituent of the mobile phase used in this section is the same as that of the mobile phase used in prep-HPLC, while the gradient elution changed to 14% A at 0–10 min, 14–19% A at 10–40 min, 19–20.5% A at 40–90 min. The temperature of column oven is 10°C. Results The condition of SS was listed as follows: isopropanol as the sublation solvent, pH 3, 350 g/L of ammonium sulfate concentration in aqueous phase, 40 mL/min of nitrogen flow rate, 30 min of flotation time, and 10.0 mL of flotation solvent volume. After 30 min of flotation time, 10.0 mL of isopropanol was transferred from the top of the flotation column to a 50-mL conical flask, then additional 10.0 mL of isopropanol was added in the top of the flotation column, and the flotation procedure was kept 30 min again in the same condition. In this condition, the recovery of fraction 1 and fraction 2 was 90.51% and 92.07%, respectively. With a two-phase solvent system composed of n-hexane-ethyl acetate–methanol–water (1:7:1:7, v/v), the purity of fraction 1 and fraction 2 separated by HSCCC were 93.75% and 93.68%, respectively, according to the HPLC analysis. The structural identification of the two fractions obtained from HSCCC was confirmed by LCMS-IT-TOF, 13C NMR and 1H NMR analysis. The fraction 1 was a pure compound hyperoside. LCMS-IT-TOF: m/z 463.1 [M−H]−, m/z 465.1 [M+H]+, MS2 yielded ions m/z 301.1 ([M−H−-162], loss of glucose or galactose). 13 C NMR (DMSO-d6, 400 MHz): 177.4 (C-4), 164.4 (C-7), 161.1 (C-5), 156.3 (C-2), 156.2 (C-9), 133.4 (C-3), 103.8 (C-10), 98.7 (C-6), 93.5 (C-8), 148.5 (C-4′), 144.8 (C-3′), 121.9 (C-6′), 121.0 (C-1′), 115.9 (C-5′), 115.2 (C-2′), 101.8 (C-1″), 75.8 (C-5″), 73.2 (C-3″), 71.2 (C-2″), 67.9 (C-4″), 60.1 (C-6″). 1 H NMR (CD3COCD3, 400 MHz): δ ppm 12.39 (1 H, s, 5-OH), 8.04 (1 H, d, J = 2.1 Hz, H-2′), 7.65 (1 H, dd, J = 2.3 Hz, 8.6 Hz, H-6′), 6.95 (1 H, d, J = 8.4 Hz, H-5′), 6.55 (1 H, d, J = 1.9 Hz, H-8), 6.31 (1 H, d, J = 2.1 Hz, H-6), 5.19 (1 H, d, J = 1.9 Hz, H-1″), 3.93∼3.60 (6 H, H-2″∼H-6″, m). The peaks assigned in 1H NMR and 13C NMR corresponded to the reported literature (18). Fraction 2: LCMS-IT-TOF: m/z 447.1 and 477.1 [M−H],−MS2 yielded ions m/z 285.0 and 314.0 ([M−H]−-162, loss of glucose or galactose). Seen from the 1H NMR analysis of fraction 2, there were three single peaks near δppm 12.48. So, the fraction 2 was the mixture of three compounds. The proportion of compound 2–1, compound 2–2 and compound 2–3 was 1:1:0.5 in the fraction 2. Then the fraction 2 was further separated by prep-HPLC, astragalin, 3′-O-methylquercetin 3-O-β-D-galactopyranoside and 3′-O-methylquercetin 3-O-β-D-Glucopyranoside were collected and identified by 1H NMR and 13C NMR again. The three compounds were obtained with the purity of 96.7%, 95.3% and 99.9%, respectively, and their NMR data were as follows. Astragalin (compound 2–1): 13C NMR (DMSO-d6, 400 MHz): 177.3 (C-4), 164.8 (C-7), 161.1 (C-5), 156.1 (C-2), 156.1 (C-9), 130.8 (C-3), 103.7 (C-10), 98.9 (C-6), 93.8 (C-8), 159.9 (C-4′), 130.8 (C-2′), 130.8 (C-6′), 120.9 (C-1′), 115.1 (C-3′), 115.1 (C-5′), 101.6 (C-1″), 77.5 (C-5″), 76.4 (C-3″), 74.3 (C-2″), 69.8 (C-4″), 60.8 (C-6″). 1H NMR (CD3COCD3, 400 MHz): δ ppm 8.16 (1 H, d, J = 8.92 Hz, H-2′), 8.16 (1 H, d, J = 8.92 Hz, H-6′), 6.99 (1 H, d, J = 8.56 Hz, H-3′), 7.00 (1 H, d, J = 8.84 Hz, H-5′), 6.56 (1 H, d, J = 2.04 Hz, H-8), 6.31 (1 H, d, J = 2.20 Hz, H-6), 5.27 (1 H, d, J = 7.36 Hz, H-1″), 3.55∼3.27 (6 H, H-2″∼H-6″, m). The peaks assigned in 1H NMR and 13C NMR corresponded to the reported literature (19). 3′-O-Methylquercetin 3-O-β-D-galactopyranoside (compound 2–2): 13C NMR (DMSO-d6, 400 MHz): 177.3 (C-4), 164.8 (C-7), 161.1 (C-5), 156.4 (C-2), 156.4 (C-9), 133.1 (C-3), 103.7 (C-10), 100.9 (C-6), 93.8 (C-8), 149.4 (C-3′), 147.0 (C-4′), 121.8 (C-6′), 121.0 (C-1′), 115.1 (C-5′), 113.5 (C-2′), 101.6 (C-1″), 75.9 (C-5″), 73.0 (C-3″), 71.2 (C-2″), 67.9 (C-4″), 60.2 (C-6″), 55.9 (OCH3). 1H NMR (CD3COCD3, 400 MHz): δ ppm 8.23 (1 H, d, J = 2.04 Hz, H-2′), 7.63 (1 H, dd, J = 2.04 Hz, 8.44 Hz, H-6′), 6.98 (1 H, d, J = 8.44 Hz, H-5′), 6.31 (1 H, d, J = 2.20 Hz, H-8), 5.46 (1 H, d, J = 8.00 Hz, H-1′), 3.97 (1 H, s, OCH3), 3.96∼3.57 (6 H, H-2″∼H-6″, m). The peaks assigned in 1H NMR and 13C NMR corresponded to the reported literature (20). 3′-O-Methylquercetin 3-O-β-D-glucopyranoside (compound 2–3): 13C NMR (DMSO-d6, 400 MHz): 164.9 (C-7), 161.1 (C-5), 133.0 (C-3), 100.8 (C-6), 149.4 (C-3′), 146.9 (C-4′), 122.0 (C-6′), 121.0 (C-1′), 101.6 (C-1″), 77.4 (C-5″), 69.7 (C-4″), 55.6 (OCH3). 1H NMR (CD3COCD3, 400 MHz): δ ppm 8.08 (1 H, d, J = 1.92 Hz, H-2′), 7.69 (1 H, dd, J = 2.08 Hz, 8.48 Hz, H-6′), 6.98 (1 H, d, J = 8.44 Hz, H-5′), 6.31 (1 H, d, J = 2.20 Hz, H-8), 5.46 (1 H, d, J = 8.00 Hz, H-1′), 3.97 (1 H, s, OCH3), 3.55∼3.27 (6 H, H-2″∼H-6″, m). The peaks assigned in 13C NMR and 1H NMR corresponded to the reported literature (20). Discussion Selection of sublation solvent and other conditions of SS procedure Seen from the HPLC and UV result of the aqueous extract, the target compounds might be flavonol glycosides. According to our previous report (15), polyethylene glycol (PEG) was a better choice as the sublation solvent for flavonol glycosides, while the alcohols with low molecular weight such as isopropanol, n-propanol, n-butanol took the second place. However, the viscosity and boiling point of PEG were higher, so the PEG is difficult to move out from the target compounds. For this reason, the alcohols with low molecular weight, especially isopropanol was more suitable because of its lower viscosity and boiling point. Other conditions such as solution pH, (NH4)2SO4 concentration in aqueous solution, N2 flow rate and flotation time on the recovery were adjusted only a little on the original basis of our previous work (15). Partition coefficients and HSCCC purification For the selection of the phase system and the optimization of the HSCCC purification, the ternary solvent system ethyl acetate–n-butanol–water (5:2:5, 5:4:5, v/v) was firstly tried according to the selection principle expounded by Ito (21) for flavonol glycosides. However, the ternary system could not separate the target compounds well (shown in Supplementary Figures 1S and 2S), and there was not any fraction with high purity collected from HSCCC because the polarity of ethyl acetate–n-butanol–water might be a little high, so quaternary solvent system with weaker polarity such as n-hexane–ethyl acetate–methanol–water (1:5:1:5, 1:6:1:6 and 1:7:1:7, v/v) were selected subsequently. The measured K values were listed in Table I. The result showed that the three solvent systems were all suitable for the separation of target compounds. In order to choose an optimal system, target compounds were separated and purified by HSCCC using three solvent systems (n-hexane–ethyl acetate–methanol–water: 1:5:1:5, 1:6:1:6, and 1:7:1:7, v/v) successively, and the retention ratios of stationary phase in each system were reached 51.36%, 52.27% and 53.18%, respectively. The results are shown in Figure 2. Compared with these three solvent systems, the purities of final products (fraction 1 and fraction 2) were 96.42% and 88.37% using 1:6:1:6, and 93.75% and 93.68% using 1:7:1:7, respectively, which were better than that using 1:5:1:5. The purity of fraction 2 (88.37%) using 1:6:1:6 was lower than that using 1:7:1:7 (93.68%). So, solvent system 1:7:1:7 was the most suitable for the purification of the flavonol glycosides by HSCCC. Table I. The Partition Coefficient (K) of the Flavonol Glycosides Solvent system Fraction 1 Fraction 2 n-Hexane–ethyl acetate–methanol–water (1:5:1:5, v/v) 1.509 2.102 n-Hexane–ethyl acetate–methanol–water (1:6:1:6, v/v) 1.514 2.157 n-Hexane–ethyl acetate–methanol–water (1:7:1:7, v/v) 1.532 2.201 Solvent system Fraction 1 Fraction 2 n-Hexane–ethyl acetate–methanol–water (1:5:1:5, v/v) 1.509 2.102 n-Hexane–ethyl acetate–methanol–water (1:6:1:6, v/v) 1.514 2.157 n-Hexane–ethyl acetate–methanol–water (1:7:1:7, v/v) 1.532 2.201 Table I. The Partition Coefficient (K) of the Flavonol Glycosides Solvent system Fraction 1 Fraction 2 n-Hexane–ethyl acetate–methanol–water (1:5:1:5, v/v) 1.509 2.102 n-Hexane–ethyl acetate–methanol–water (1:6:1:6, v/v) 1.514 2.157 n-Hexane–ethyl acetate–methanol–water (1:7:1:7, v/v) 1.532 2.201 Solvent system Fraction 1 Fraction 2 n-Hexane–ethyl acetate–methanol–water (1:5:1:5, v/v) 1.509 2.102 n-Hexane–ethyl acetate–methanol–water (1:6:1:6, v/v) 1.514 2.157 n-Hexane–ethyl acetate–methanol–water (1:7:1:7, v/v) 1.532 2.201 Figure 2. View largeDownload slide HSCCC chromatograms of optimization suitable solvent systems for separation of the target compounds from the Solanum rostratum Dunal extract. Solvent system: A. n-hexane–ethyl acetate–methanol–water (1:5:1:5, v/v); B. n-hexane–ethyl acetate–methanol–water (1:6:1:6, v/v); C. n-hexane–ethyl acetate–methanol–water (1:7:1:7, v/v). Figure 2. View largeDownload slide HSCCC chromatograms of optimization suitable solvent systems for separation of the target compounds from the Solanum rostratum Dunal extract. Solvent system: A. n-hexane–ethyl acetate–methanol–water (1:5:1:5, v/v); B. n-hexane–ethyl acetate–methanol–water (1:6:1:6, v/v); C. n-hexane–ethyl acetate–methanol–water (1:7:1:7, v/v). Further separation of target compounds by prep-HPLC In order to separate the three compounds from the fraction 2, different mobile phases, such as methanol water with 0.5% aqueous phosphoric acid, acetonitrile water with 0.05% trifluoroacetic acid were used with different gradient. However, the three compounds still could not be separated. Considering the temperature is also an significant parameter for enhancing resolution in HPLC systems especially for separating solutes with subtle differences in their morphology (22, 23), we tried to change column temperature to make the three compounds in fraction 2 separate well. The separation procedure is shown in Figure 3. When the temperature was reduced to 10°C, there was a great tendency to be separated of the three compounds in fraction 2 (seen from Figure 3A and B). The reason might be the low temperature affected the K values of these compounds differently and sharply. And then they were finally separated well by the adjustment of the gradient (seen from Figure 3C), furthermore, it could be used to analyze the subsequent elute from prep-HPLC qualitatively. As shown in Figure 4, fraction 2–1, 2–2 and 2–3, which represent astragalin, 3′-O-Methylquercetin 3-O-β-D-galactopyranoside and 3′-O-methylquercetin 3-O-β-D-glucopyranoside, respectively, were separated well, and three individual peaks were collected and identified by 1H NMR and 13C NMR. According to the HPLC analysis (shown in Figure 4), the purities of astragalin, 3′-O-methylquercetin 3-O-β-D-galactopyranoside and 3′-O-methylquercetin 3-O-β-D-glucopyranoside were reached to 96.7, 95.3 and 99.9%, respectively. Figure 3. View largeDownload slide Preparative HPLC chromatogram of three compounds (2, 3) in fraction 2. Chromatographic conditions: an Inertsil ODS-3 column (250 mm × 4.6 mm I.D., 5 μm), acetonitrile and water including 0.0125% trifluoroacetic acid as mobile phase, the flow rate was 1.0 mL/min, the detection wavelength was 254 nm and temperature of column oven was 10°C. The gradient condition: A(a), 15–19% acetonitrile at 0–40 min, 19–23% acetonitrile at 40–80 min; B(a), 15–19% acetonitrile at 0–40 min, 19–21.5% acetonitrile at 40–80 min; C(a), 14% acetonitrile at 0–10 min, 14–19% acetonitrile at 10–40 min, 19–20.5% acetonitrile at 40–90 min. A(b), B(b) and C(b) were the partial enlarged view of A(a), B(a) and C(a), respectively. Figure 3. View largeDownload slide Preparative HPLC chromatogram of three compounds (2, 3) in fraction 2. Chromatographic conditions: an Inertsil ODS-3 column (250 mm × 4.6 mm I.D., 5 μm), acetonitrile and water including 0.0125% trifluoroacetic acid as mobile phase, the flow rate was 1.0 mL/min, the detection wavelength was 254 nm and temperature of column oven was 10°C. The gradient condition: A(a), 15–19% acetonitrile at 0–40 min, 19–23% acetonitrile at 40–80 min; B(a), 15–19% acetonitrile at 0–40 min, 19–21.5% acetonitrile at 40–80 min; C(a), 14% acetonitrile at 0–10 min, 14–19% acetonitrile at 10–40 min, 19–20.5% acetonitrile at 40–90 min. A(b), B(b) and C(b) were the partial enlarged view of A(a), B(a) and C(a), respectively. Figure 4. View largeDownload slide Prep-HPLC chromatograms of astragalin (2–1), 3′-O-methylquercetin 3-O-β-D-galactopyranoside (2) and 3′-O-methylquercetin 3-O-β-D-glucopyranoside (2, 3). Chromatographic conditions: a YMC-Pack ODS-A column (250 mm × 20 mm I.D.,5 μm), acetonitrile (A) and water including 0.0125% trifluoroacetic acid (B) as mobile phase, the flow rate was 8.0 mL/min with a gradient elution of 14–21% A at 0–100 min, the detection wavelength was 254 nm and temperature was 10°C, the analytical HPLC conditions were same as those in Figure 3. Insets: The HPLC chromatogram of fraction 2–1, 2–2 and 2–3 from prep-HPLC, respectively. Figure 4. View largeDownload slide Prep-HPLC chromatograms of astragalin (2–1), 3′-O-methylquercetin 3-O-β-D-galactopyranoside (2) and 3′-O-methylquercetin 3-O-β-D-glucopyranoside (2, 3). Chromatographic conditions: a YMC-Pack ODS-A column (250 mm × 20 mm I.D.,5 μm), acetonitrile (A) and water including 0.0125% trifluoroacetic acid (B) as mobile phase, the flow rate was 8.0 mL/min with a gradient elution of 14–21% A at 0–100 min, the detection wavelength was 254 nm and temperature was 10°C, the analytical HPLC conditions were same as those in Figure 3. Insets: The HPLC chromatogram of fraction 2–1, 2–2 and 2–3 from prep-HPLC, respectively. Conclusions In this paper, hyperoside, astragalin, 3′-O-methylquercetin 3-O-β-D-galactopyranoside and 3′-O-methylquercetin 3-O-β-D-glucopyranoside were separated from S. rostratum Dunal extract by HSCCC with solvent system composed of n-hexane–ethyl acetate–methanol–water (1:7:1:7, v/v) and gradient elution mode prep-HPLC with low column temperature for the first time. Our study demonstrates that HSCCC combined with low column temperature prep-HPLC is a very efficient method for the preparative separation of flavonoids and bioactive compounds from S. rostratum Dunal. The identification and characterization of four flavonol glycosides were useful in the resource utilization of invasive plants. The SS-HSCCC-low column temperature prep-HPLC method for separating and concentrating active compounds from the aqueous extract of natural product provides a new way and understanding of SS-HSCCC-prep-HPLC technique. Future studies of SS-HSCCC-prep-HPLC technique will likely expand the application in separation fields of other alien plant. Supplementary data Supplementary material is available at Journal of Chromatographic Science online. Funding Financial support from the National Key Research and Development Program of China (2017YFF0207800), the National Natural Science Foundation of China (NSFC, Grant No. 81671411 and 21075007), Program for New Century Excellent Talents in University (NCET-11-0563), Beijing Nova program interdisciplinary (Z161100004916045), Special Fund for Agro-scientific Research in the Public Interest (project 200803022 and 201103027) and the Fundamental Research Funds for the Central Universities (YS1406) is gratefully acknowledged. References 1 Gutzeit , D. , Winterhalter , P. , Jerz , G. ; Application of preparative high-speed counter-current chromatography/electrospray ionization mass spectrometry for a fast screening and fractionation of polyphenols ; Journal of Chromatography. A , ( 2007 ); 1172 ( 1 ): 40 – 46 . Google Scholar CrossRef Search ADS PubMed 2 Ito , Y. ; Recent advances in counter-current chromatography ; Journal of Chromatography. A , ( 1991 ); 538 ( 1 ): 3 – 25 . 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For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Chromatographic Science Oxford University Press

Separation of Four Flavonol Glycosides from Solanum rostratum Dunal Using Solvent Sublation Followed by HSCCC and Low Column Temperature Preparative HPLC

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Abstract

Abstract Hyperoside, 3′-O-methylquercetin 3-O-β-D-galactopyranoside, astragalin and 3′-O-methylquercetin 3-O-β-D-glucopyranoside from an invasive weed Solanum rostratum Dunal were separated and purified successfully by high-speed counter-current chromatography (HSCCC) with a solvent system composed of n-hexane-ethyl acetate–methanol–water (1:7:1:7, v/v) and gradient elution mode preparative high-performance liquid chromatography (prep-HPLC) with low column temperature. In the sample pretreatment section, target compounds in aqueous extract of the weed were concentrated using solvent sublation. Two target fractions with purities of 93.75% and 93.68% were obtained from HSCCC. Their chemical structures were identified. The fraction 1 is a pure compound hyperoside and the fraction 2 is the mixture of astragalin, 3′-O-methylquercetin 3-O-β-D-galactopyranoside and 3′-O-methylquercetin 3-O-β-D-glucopyranoside by nuclear magnetic resonance and liquid chromatography-mass spectra. Then, the three flavonol glycosides in the fraction 2 were separated and purified successfully by prep-HPLC with low column temperature. Introduction High-speed counter-current chromatography (HSCCC), being a support-free liquid–liquid partition method (1), eliminates irreversible adsorption of sample onto the solid support (2), and has been widely used in preparative separation of active compounds from traditional Chinese herbs and other natural products (3–5). Solvent sublation (SS) is a kind of adsorptive bubble separation technique (6) in which many water-soluble active compounds with surface-activity can be adsorbed on the bubble surface and then collected in organic phase. Therefore, this technique can effectively separate and concentrate the water-soluble compounds. Preparative high-performance liquid chromatography (prep-HPLC) is easy to be used for separation of compounds because of its high separation efficiency and recovery (7). So, it can be used in the last procedure to separate the mixture which has not been separated completely (8, 9). Solanum rostratum Dunal, a newly invasive weed species in China, seriously breaks the ecological balance in the invasive areas because it inhibits and excludes native plants by releasing allelochemicals (10). In addition, this invasive weed also severely affects agricultural and animal husbandry production due to its extensive infestation in agricultural fields and grasslands (11, 12). It is worth mentioning that there are quantities of useful substances in this weed. For example, methylprotodioscin, a kind of cytotoxin found in this weed (13), was toxic to cancerous cells (14), so it can be used for the treatment of people with cancer. These researches provide a new way to develop and utilize the S. rostratum Dunal instead of destroy it. Therefore, it is significative to discover more active substances from the invasive weed for resource utilization. The objective of the present paper is to separate and purify flavonoids from S. rostratum Dunal by HSCCC combined with low column temperature prep-HPLC. The active compounds in aqueous S. rostratum Dunal extract were separated and concentrated by SS. Then, hyperoside, astragalin, 3′-O-methylquercetin 3-O-β-D-galactopyranoside and 3′-O-methylquercetin 3-O-β-D-glucopyranoside with high purity were obtained after further separation and purification using HSCCC and low column temperature prep-HPLC from flotation product. Experimental Chemicals and reagents Solanum rostratum Dunal plant was supplied by the Chinese Academy of Agricultural Sciences. Phosphoric acid was purchased from Yili Fine Chemicals Co., Ltd (Beijing, China). Methanol, acetonitrile and trifluoroacetic acid used as mobile phase for HPLC analysis and prep-HPLC purification were all of chromatography grade (J&K, China). Ammonium sulfate, isopropanol, hydrochloric acid and sodium hydroxide used for SS and n-hexane, ethyl acetate and methanol used for HSCCC were all of analytical grade and purchased from Beijing Chemical Reagent Factory (Beijing, China). Apparatus The SS apparatus is similar to the one mentioned in the previous papers (15). An AB204-N electronic balance (Mettler, Switzerland) and a Mettler Toledo 320-S pH meter (Mettler, Switzerland) were used. Semi-preparative HSCCC was performed using a Model GS10A multilayer coil planet centrifuge (Beijing Institute of New Technology Application, Beijing, China) equipped with a polytetrafluoroethylene multilayer coil of 110 m × 1.6 mm I.D. with a total capacity of 240 mL. The β value of the preparative column ranged from 0.5 to 0.8 (β = r/R, where r is the distance from the holder shaft to the coil and R is the distance between the holder axis and central axis of the planet centrifuge or the rotation radius). The solvent was pumped into the column with a Model NS-1007 constant-flow pump (Beijing Institute of New Technology Application, Beijing, China). Continuous monitoring of the effluent was achieved with a Model 8823A-UV Monitor (Beijing Institute of New Technology Application, Beijing, China) at 254 nm. A manual sample injection valve with a 10-mL loop (for the preparative HSCCC) was used to introduce the sample into the column. A portable recorder Yokogawa Model 3,057 (Sichuan Instrument Factory, Chongqing, China) was used to plot the chromatogram. A rotary evaporator was also used. Prep-HPLC was performed using a FLEXA Purification System (Agela, China) with an YMC-Pack ODS-A column (250 mm × 20 mm I.D., 5 μm, YMC, Japan) to separate and purify the mixture from HSCCC. And the products from prep-HPLC were analyzed by analytical HPLC with an Inertsil ODS-3 column (250 mm × 4.6 mm I.D., 5 μm). A Shimadzu LC-20AT chromatograph system (Shimadzu, Japan) was used to analyze the aqueous extract, the flotation products and the purification products from HSCCC. Identification of purified products was carried out by LCMS-IT-TOF and 1H NMR, 13C NMR spectra. LCMS-IT-TOF was performed using a Shimadzu (Kyoto, Japan) HPLC system consisting of a solvent delivery pump (LC-20AD), an auto-sampler (SIL-20AC), a DGU-20A3 degasser, a photodiode array detector (SPD-M20A), a communication base module (CBM-20A), a column oven (CTO-20A), an Inertsil ODS-SP column (150 × 4.6 mm I.D., 5 μm), and a hybrid ion trap/time-of-flight instrument (Shimadzu Corp., Kyoto, Japan) equipped with an ESI source. NMR spectra were performed in DMSO-d6 and CD3COCD3 using a Bruker high-resolution AV400NMR spectrometer (Bruker Biospin Corporation, USA). Preparation of extract of Solanum rostratum Dunal using SS for HSCCC Dried powder of S. rostratum Dunal (60.0 g) was extracted twice, each with 1,000 mL of pure water for 90 min by water reflux method. The residue was filtrated and washed with 200 mL of pure water. The HPLC chromatograms (Figure 1) of the S. rostratum Dunal extract showed that the retention time of fraction 1 was about 20.8 min and fraction 2 was 23.0 min. Subsequently, ammonium sulfate was added into the combined extracts, and the aqueous solution was used for the separation and concentration of SS. In order to prepare accurately the aqueous ammonium sulfate solution, the classical relationship equation (16) between the mass concentration of ammonium sulfate and the volume concentration was applied. The initial volume of aqueous solution in the flotation column was 400 mL. Figure 1. View largeDownload slide HPLC chromatogram of the Solanum rostratum Dunal extract and structural formula of target compounds. Chromatographic conditions: an Apollo C18 column (150 × 4.6 mm I.D., 5 μm), methanol (A) and 0.5% aqueous phosphoric acid (B) as mobile phase, the flow rate was 1.0 mL/min with a gradient elution of 20–100% A from 0 to 40 min, the detection wavelength was 254 nm and temperature of column oven was 30°C. Figure 1. View largeDownload slide HPLC chromatogram of the Solanum rostratum Dunal extract and structural formula of target compounds. Chromatographic conditions: an Apollo C18 column (150 × 4.6 mm I.D., 5 μm), methanol (A) and 0.5% aqueous phosphoric acid (B) as mobile phase, the flow rate was 1.0 mL/min with a gradient elution of 20–100% A from 0 to 40 min, the detection wavelength was 254 nm and temperature of column oven was 30°C. The parameters of SS, such as sublation solvent, solution pH, (NH4)2SO4 concentration in aqueous solution, nitrogen flow rate, flotation time, the volume of sublation solvent and repeat times can greatly influence the recovery of target compounds. In this paper, the parameters mentioned above were studied in this order by single factor experiment, and the specific process was the same as that reported in the previous study (15, 16). All the SS experiments were performed at room temperature. During the SS procedure, target compounds can be effectively separated and concentrated from the aqueous extract to the upper phase. Then the flotation product was concentrated to dryness under reduced pressure and then used for subsequent HSCCC isolation. Preparation of two-phase solvent system and sample solution The solvent system is essential to the separation process in HSCCC. An appropriate solvent system should satisfy the following requirements: it can dissolve the sample well and will not cause decomposition or denaturation of the sample; it can form stable two-phase solvent system and have a good retention of stationary phase; the partition coefficient (K) of the target components in it should be between 0.2 and 5 (17). The K value was determined by HPLC as follows: suitable amount of crude extract was dissolved in 2 mL of each phase of the pre-equilibrated two-phase solvent system with violent shaken in order to reach a thorough equilibrium at room temperature. After 3 h standing, 1 mL of each phase was taken out and evaporated to dryness. The residues were dissolved with 1 mL methanol and then analyzed by HPLC. The K value was calculated as the peak area of the target compound in the upper phase divided by the peak area of the target compound in the lower phase. The two-phase solvent system utilized in the present study was prepared by mixing n-hexane-ethyl acetate–methanol–water (1:5:1:5, 1:6:1:6, 1:7:1:7, v/v) in a separatory funnel. Then, the separatory funnel was shaked violently at room temperature to make the two-phase system thoroughly equilibrated. The upper phase and the lower phase were separated and degassed by ultrasonic for 30 min shortly before use. The sample solutions were prepared by dissolving the extract obtained in section 2.3 in 5.0 mL upper phase and 5.0 mL lower phase. Separation procedure for HSCCC In separation procedure, the multilayer coiled column was entirely filled with the upper phase as the stationary phase at first. Then the lower phase as mobile phase was pumped into the head end of the column at a flow rate of 2.0 mL/min, while the apparatus was rotated at 800 rpm. After a clear mobile phase eluted at the tail outlet, which means hydrodynamic equilibrium was established, the sample solution was injected through the sample port. A UV detector was used to monitor the effluent from the tail end of the column continuously at 254 nm. Each peak fraction was collected according to the chromatogram. The retention of the stationary phase was calculated from the volume of the stationary phase collected from the column before the sample was injected. Further separation for prep-HPLC The fraction 2 from HSCCC was dried with rotary evaporator with the temperature of 55°C, and it was dissolved by methanol and water (7:4, v/v). A YMC-Pack ODS-A column (250 mm × 20 mm I.D., 5 μm) was used for the separation procedure. The mobile phase was composed of acetonitrile (A) and water including 0.0125% trifluoroacetic acid (B), and a gradient elution of 14–21% A at 0–100 min was used with the detection wavelength of 254 nm. The flow rate was 8.0 mL/min and the injection volume was 0.5 mL. The prep-HPLC experiments were performed at 10°C temperature approximately. HPLC analysis The analytical HPLC was used to analyze the aqueous extract, the flotation product and the purification product from HSCCC with an Apollo C18 column (150 × 4.6 mm I.D., 5 μm). In the HPLC analysis, the mobile phase was composed of methanol (A) and 0.5% aqueous phosphoric acid (B). The flow rate was 1.0 mL/min with a gradient elution of 20–100% A at 0–40 min. The detection wavelength was 254 nm and the injection volume was 10 μL. It is important to note that the aqueous extracts and the flotation products should be diluted with methanol (1–2 mL), and desalted with centrifuge at 2,500 rpm for 10 min. The temperature of all the HPLC analytical experiments was 30°C. Each effluent from prep-HPLC was analyzed by HPLC with an Inertsil ODS-3 column (250 mm × 4.6 mm I.D., 5 μm), and the purity was obtained with the method of area normalization. It is noted that the constituent of the mobile phase used in this section is the same as that of the mobile phase used in prep-HPLC, while the gradient elution changed to 14% A at 0–10 min, 14–19% A at 10–40 min, 19–20.5% A at 40–90 min. The temperature of column oven is 10°C. Results The condition of SS was listed as follows: isopropanol as the sublation solvent, pH 3, 350 g/L of ammonium sulfate concentration in aqueous phase, 40 mL/min of nitrogen flow rate, 30 min of flotation time, and 10.0 mL of flotation solvent volume. After 30 min of flotation time, 10.0 mL of isopropanol was transferred from the top of the flotation column to a 50-mL conical flask, then additional 10.0 mL of isopropanol was added in the top of the flotation column, and the flotation procedure was kept 30 min again in the same condition. In this condition, the recovery of fraction 1 and fraction 2 was 90.51% and 92.07%, respectively. With a two-phase solvent system composed of n-hexane-ethyl acetate–methanol–water (1:7:1:7, v/v), the purity of fraction 1 and fraction 2 separated by HSCCC were 93.75% and 93.68%, respectively, according to the HPLC analysis. The structural identification of the two fractions obtained from HSCCC was confirmed by LCMS-IT-TOF, 13C NMR and 1H NMR analysis. The fraction 1 was a pure compound hyperoside. LCMS-IT-TOF: m/z 463.1 [M−H]−, m/z 465.1 [M+H]+, MS2 yielded ions m/z 301.1 ([M−H−-162], loss of glucose or galactose). 13 C NMR (DMSO-d6, 400 MHz): 177.4 (C-4), 164.4 (C-7), 161.1 (C-5), 156.3 (C-2), 156.2 (C-9), 133.4 (C-3), 103.8 (C-10), 98.7 (C-6), 93.5 (C-8), 148.5 (C-4′), 144.8 (C-3′), 121.9 (C-6′), 121.0 (C-1′), 115.9 (C-5′), 115.2 (C-2′), 101.8 (C-1″), 75.8 (C-5″), 73.2 (C-3″), 71.2 (C-2″), 67.9 (C-4″), 60.1 (C-6″). 1 H NMR (CD3COCD3, 400 MHz): δ ppm 12.39 (1 H, s, 5-OH), 8.04 (1 H, d, J = 2.1 Hz, H-2′), 7.65 (1 H, dd, J = 2.3 Hz, 8.6 Hz, H-6′), 6.95 (1 H, d, J = 8.4 Hz, H-5′), 6.55 (1 H, d, J = 1.9 Hz, H-8), 6.31 (1 H, d, J = 2.1 Hz, H-6), 5.19 (1 H, d, J = 1.9 Hz, H-1″), 3.93∼3.60 (6 H, H-2″∼H-6″, m). The peaks assigned in 1H NMR and 13C NMR corresponded to the reported literature (18). Fraction 2: LCMS-IT-TOF: m/z 447.1 and 477.1 [M−H],−MS2 yielded ions m/z 285.0 and 314.0 ([M−H]−-162, loss of glucose or galactose). Seen from the 1H NMR analysis of fraction 2, there were three single peaks near δppm 12.48. So, the fraction 2 was the mixture of three compounds. The proportion of compound 2–1, compound 2–2 and compound 2–3 was 1:1:0.5 in the fraction 2. Then the fraction 2 was further separated by prep-HPLC, astragalin, 3′-O-methylquercetin 3-O-β-D-galactopyranoside and 3′-O-methylquercetin 3-O-β-D-Glucopyranoside were collected and identified by 1H NMR and 13C NMR again. The three compounds were obtained with the purity of 96.7%, 95.3% and 99.9%, respectively, and their NMR data were as follows. Astragalin (compound 2–1): 13C NMR (DMSO-d6, 400 MHz): 177.3 (C-4), 164.8 (C-7), 161.1 (C-5), 156.1 (C-2), 156.1 (C-9), 130.8 (C-3), 103.7 (C-10), 98.9 (C-6), 93.8 (C-8), 159.9 (C-4′), 130.8 (C-2′), 130.8 (C-6′), 120.9 (C-1′), 115.1 (C-3′), 115.1 (C-5′), 101.6 (C-1″), 77.5 (C-5″), 76.4 (C-3″), 74.3 (C-2″), 69.8 (C-4″), 60.8 (C-6″). 1H NMR (CD3COCD3, 400 MHz): δ ppm 8.16 (1 H, d, J = 8.92 Hz, H-2′), 8.16 (1 H, d, J = 8.92 Hz, H-6′), 6.99 (1 H, d, J = 8.56 Hz, H-3′), 7.00 (1 H, d, J = 8.84 Hz, H-5′), 6.56 (1 H, d, J = 2.04 Hz, H-8), 6.31 (1 H, d, J = 2.20 Hz, H-6), 5.27 (1 H, d, J = 7.36 Hz, H-1″), 3.55∼3.27 (6 H, H-2″∼H-6″, m). The peaks assigned in 1H NMR and 13C NMR corresponded to the reported literature (19). 3′-O-Methylquercetin 3-O-β-D-galactopyranoside (compound 2–2): 13C NMR (DMSO-d6, 400 MHz): 177.3 (C-4), 164.8 (C-7), 161.1 (C-5), 156.4 (C-2), 156.4 (C-9), 133.1 (C-3), 103.7 (C-10), 100.9 (C-6), 93.8 (C-8), 149.4 (C-3′), 147.0 (C-4′), 121.8 (C-6′), 121.0 (C-1′), 115.1 (C-5′), 113.5 (C-2′), 101.6 (C-1″), 75.9 (C-5″), 73.0 (C-3″), 71.2 (C-2″), 67.9 (C-4″), 60.2 (C-6″), 55.9 (OCH3). 1H NMR (CD3COCD3, 400 MHz): δ ppm 8.23 (1 H, d, J = 2.04 Hz, H-2′), 7.63 (1 H, dd, J = 2.04 Hz, 8.44 Hz, H-6′), 6.98 (1 H, d, J = 8.44 Hz, H-5′), 6.31 (1 H, d, J = 2.20 Hz, H-8), 5.46 (1 H, d, J = 8.00 Hz, H-1′), 3.97 (1 H, s, OCH3), 3.96∼3.57 (6 H, H-2″∼H-6″, m). The peaks assigned in 1H NMR and 13C NMR corresponded to the reported literature (20). 3′-O-Methylquercetin 3-O-β-D-glucopyranoside (compound 2–3): 13C NMR (DMSO-d6, 400 MHz): 164.9 (C-7), 161.1 (C-5), 133.0 (C-3), 100.8 (C-6), 149.4 (C-3′), 146.9 (C-4′), 122.0 (C-6′), 121.0 (C-1′), 101.6 (C-1″), 77.4 (C-5″), 69.7 (C-4″), 55.6 (OCH3). 1H NMR (CD3COCD3, 400 MHz): δ ppm 8.08 (1 H, d, J = 1.92 Hz, H-2′), 7.69 (1 H, dd, J = 2.08 Hz, 8.48 Hz, H-6′), 6.98 (1 H, d, J = 8.44 Hz, H-5′), 6.31 (1 H, d, J = 2.20 Hz, H-8), 5.46 (1 H, d, J = 8.00 Hz, H-1′), 3.97 (1 H, s, OCH3), 3.55∼3.27 (6 H, H-2″∼H-6″, m). The peaks assigned in 13C NMR and 1H NMR corresponded to the reported literature (20). Discussion Selection of sublation solvent and other conditions of SS procedure Seen from the HPLC and UV result of the aqueous extract, the target compounds might be flavonol glycosides. According to our previous report (15), polyethylene glycol (PEG) was a better choice as the sublation solvent for flavonol glycosides, while the alcohols with low molecular weight such as isopropanol, n-propanol, n-butanol took the second place. However, the viscosity and boiling point of PEG were higher, so the PEG is difficult to move out from the target compounds. For this reason, the alcohols with low molecular weight, especially isopropanol was more suitable because of its lower viscosity and boiling point. Other conditions such as solution pH, (NH4)2SO4 concentration in aqueous solution, N2 flow rate and flotation time on the recovery were adjusted only a little on the original basis of our previous work (15). Partition coefficients and HSCCC purification For the selection of the phase system and the optimization of the HSCCC purification, the ternary solvent system ethyl acetate–n-butanol–water (5:2:5, 5:4:5, v/v) was firstly tried according to the selection principle expounded by Ito (21) for flavonol glycosides. However, the ternary system could not separate the target compounds well (shown in Supplementary Figures 1S and 2S), and there was not any fraction with high purity collected from HSCCC because the polarity of ethyl acetate–n-butanol–water might be a little high, so quaternary solvent system with weaker polarity such as n-hexane–ethyl acetate–methanol–water (1:5:1:5, 1:6:1:6 and 1:7:1:7, v/v) were selected subsequently. The measured K values were listed in Table I. The result showed that the three solvent systems were all suitable for the separation of target compounds. In order to choose an optimal system, target compounds were separated and purified by HSCCC using three solvent systems (n-hexane–ethyl acetate–methanol–water: 1:5:1:5, 1:6:1:6, and 1:7:1:7, v/v) successively, and the retention ratios of stationary phase in each system were reached 51.36%, 52.27% and 53.18%, respectively. The results are shown in Figure 2. Compared with these three solvent systems, the purities of final products (fraction 1 and fraction 2) were 96.42% and 88.37% using 1:6:1:6, and 93.75% and 93.68% using 1:7:1:7, respectively, which were better than that using 1:5:1:5. The purity of fraction 2 (88.37%) using 1:6:1:6 was lower than that using 1:7:1:7 (93.68%). So, solvent system 1:7:1:7 was the most suitable for the purification of the flavonol glycosides by HSCCC. Table I. The Partition Coefficient (K) of the Flavonol Glycosides Solvent system Fraction 1 Fraction 2 n-Hexane–ethyl acetate–methanol–water (1:5:1:5, v/v) 1.509 2.102 n-Hexane–ethyl acetate–methanol–water (1:6:1:6, v/v) 1.514 2.157 n-Hexane–ethyl acetate–methanol–water (1:7:1:7, v/v) 1.532 2.201 Solvent system Fraction 1 Fraction 2 n-Hexane–ethyl acetate–methanol–water (1:5:1:5, v/v) 1.509 2.102 n-Hexane–ethyl acetate–methanol–water (1:6:1:6, v/v) 1.514 2.157 n-Hexane–ethyl acetate–methanol–water (1:7:1:7, v/v) 1.532 2.201 Table I. The Partition Coefficient (K) of the Flavonol Glycosides Solvent system Fraction 1 Fraction 2 n-Hexane–ethyl acetate–methanol–water (1:5:1:5, v/v) 1.509 2.102 n-Hexane–ethyl acetate–methanol–water (1:6:1:6, v/v) 1.514 2.157 n-Hexane–ethyl acetate–methanol–water (1:7:1:7, v/v) 1.532 2.201 Solvent system Fraction 1 Fraction 2 n-Hexane–ethyl acetate–methanol–water (1:5:1:5, v/v) 1.509 2.102 n-Hexane–ethyl acetate–methanol–water (1:6:1:6, v/v) 1.514 2.157 n-Hexane–ethyl acetate–methanol–water (1:7:1:7, v/v) 1.532 2.201 Figure 2. View largeDownload slide HSCCC chromatograms of optimization suitable solvent systems for separation of the target compounds from the Solanum rostratum Dunal extract. Solvent system: A. n-hexane–ethyl acetate–methanol–water (1:5:1:5, v/v); B. n-hexane–ethyl acetate–methanol–water (1:6:1:6, v/v); C. n-hexane–ethyl acetate–methanol–water (1:7:1:7, v/v). Figure 2. View largeDownload slide HSCCC chromatograms of optimization suitable solvent systems for separation of the target compounds from the Solanum rostratum Dunal extract. Solvent system: A. n-hexane–ethyl acetate–methanol–water (1:5:1:5, v/v); B. n-hexane–ethyl acetate–methanol–water (1:6:1:6, v/v); C. n-hexane–ethyl acetate–methanol–water (1:7:1:7, v/v). Further separation of target compounds by prep-HPLC In order to separate the three compounds from the fraction 2, different mobile phases, such as methanol water with 0.5% aqueous phosphoric acid, acetonitrile water with 0.05% trifluoroacetic acid were used with different gradient. However, the three compounds still could not be separated. Considering the temperature is also an significant parameter for enhancing resolution in HPLC systems especially for separating solutes with subtle differences in their morphology (22, 23), we tried to change column temperature to make the three compounds in fraction 2 separate well. The separation procedure is shown in Figure 3. When the temperature was reduced to 10°C, there was a great tendency to be separated of the three compounds in fraction 2 (seen from Figure 3A and B). The reason might be the low temperature affected the K values of these compounds differently and sharply. And then they were finally separated well by the adjustment of the gradient (seen from Figure 3C), furthermore, it could be used to analyze the subsequent elute from prep-HPLC qualitatively. As shown in Figure 4, fraction 2–1, 2–2 and 2–3, which represent astragalin, 3′-O-Methylquercetin 3-O-β-D-galactopyranoside and 3′-O-methylquercetin 3-O-β-D-glucopyranoside, respectively, were separated well, and three individual peaks were collected and identified by 1H NMR and 13C NMR. According to the HPLC analysis (shown in Figure 4), the purities of astragalin, 3′-O-methylquercetin 3-O-β-D-galactopyranoside and 3′-O-methylquercetin 3-O-β-D-glucopyranoside were reached to 96.7, 95.3 and 99.9%, respectively. Figure 3. View largeDownload slide Preparative HPLC chromatogram of three compounds (2, 3) in fraction 2. Chromatographic conditions: an Inertsil ODS-3 column (250 mm × 4.6 mm I.D., 5 μm), acetonitrile and water including 0.0125% trifluoroacetic acid as mobile phase, the flow rate was 1.0 mL/min, the detection wavelength was 254 nm and temperature of column oven was 10°C. The gradient condition: A(a), 15–19% acetonitrile at 0–40 min, 19–23% acetonitrile at 40–80 min; B(a), 15–19% acetonitrile at 0–40 min, 19–21.5% acetonitrile at 40–80 min; C(a), 14% acetonitrile at 0–10 min, 14–19% acetonitrile at 10–40 min, 19–20.5% acetonitrile at 40–90 min. A(b), B(b) and C(b) were the partial enlarged view of A(a), B(a) and C(a), respectively. Figure 3. View largeDownload slide Preparative HPLC chromatogram of three compounds (2, 3) in fraction 2. Chromatographic conditions: an Inertsil ODS-3 column (250 mm × 4.6 mm I.D., 5 μm), acetonitrile and water including 0.0125% trifluoroacetic acid as mobile phase, the flow rate was 1.0 mL/min, the detection wavelength was 254 nm and temperature of column oven was 10°C. The gradient condition: A(a), 15–19% acetonitrile at 0–40 min, 19–23% acetonitrile at 40–80 min; B(a), 15–19% acetonitrile at 0–40 min, 19–21.5% acetonitrile at 40–80 min; C(a), 14% acetonitrile at 0–10 min, 14–19% acetonitrile at 10–40 min, 19–20.5% acetonitrile at 40–90 min. A(b), B(b) and C(b) were the partial enlarged view of A(a), B(a) and C(a), respectively. Figure 4. View largeDownload slide Prep-HPLC chromatograms of astragalin (2–1), 3′-O-methylquercetin 3-O-β-D-galactopyranoside (2) and 3′-O-methylquercetin 3-O-β-D-glucopyranoside (2, 3). Chromatographic conditions: a YMC-Pack ODS-A column (250 mm × 20 mm I.D.,5 μm), acetonitrile (A) and water including 0.0125% trifluoroacetic acid (B) as mobile phase, the flow rate was 8.0 mL/min with a gradient elution of 14–21% A at 0–100 min, the detection wavelength was 254 nm and temperature was 10°C, the analytical HPLC conditions were same as those in Figure 3. Insets: The HPLC chromatogram of fraction 2–1, 2–2 and 2–3 from prep-HPLC, respectively. Figure 4. View largeDownload slide Prep-HPLC chromatograms of astragalin (2–1), 3′-O-methylquercetin 3-O-β-D-galactopyranoside (2) and 3′-O-methylquercetin 3-O-β-D-glucopyranoside (2, 3). Chromatographic conditions: a YMC-Pack ODS-A column (250 mm × 20 mm I.D.,5 μm), acetonitrile (A) and water including 0.0125% trifluoroacetic acid (B) as mobile phase, the flow rate was 8.0 mL/min with a gradient elution of 14–21% A at 0–100 min, the detection wavelength was 254 nm and temperature was 10°C, the analytical HPLC conditions were same as those in Figure 3. Insets: The HPLC chromatogram of fraction 2–1, 2–2 and 2–3 from prep-HPLC, respectively. Conclusions In this paper, hyperoside, astragalin, 3′-O-methylquercetin 3-O-β-D-galactopyranoside and 3′-O-methylquercetin 3-O-β-D-glucopyranoside were separated from S. rostratum Dunal extract by HSCCC with solvent system composed of n-hexane–ethyl acetate–methanol–water (1:7:1:7, v/v) and gradient elution mode prep-HPLC with low column temperature for the first time. Our study demonstrates that HSCCC combined with low column temperature prep-HPLC is a very efficient method for the preparative separation of flavonoids and bioactive compounds from S. rostratum Dunal. The identification and characterization of four flavonol glycosides were useful in the resource utilization of invasive plants. The SS-HSCCC-low column temperature prep-HPLC method for separating and concentrating active compounds from the aqueous extract of natural product provides a new way and understanding of SS-HSCCC-prep-HPLC technique. Future studies of SS-HSCCC-prep-HPLC technique will likely expand the application in separation fields of other alien plant. Supplementary data Supplementary material is available at Journal of Chromatographic Science online. Funding Financial support from the National Key Research and Development Program of China (2017YFF0207800), the National Natural Science Foundation of China (NSFC, Grant No. 81671411 and 21075007), Program for New Century Excellent Talents in University (NCET-11-0563), Beijing Nova program interdisciplinary (Z161100004916045), Special Fund for Agro-scientific Research in the Public Interest (project 200803022 and 201103027) and the Fundamental Research Funds for the Central Universities (YS1406) is gratefully acknowledged. References 1 Gutzeit , D. , Winterhalter , P. , Jerz , G. ; Application of preparative high-speed counter-current chromatography/electrospray ionization mass spectrometry for a fast screening and fractionation of polyphenols ; Journal of Chromatography. A , ( 2007 ); 1172 ( 1 ): 40 – 46 . Google Scholar CrossRef Search ADS PubMed 2 Ito , Y. ; Recent advances in counter-current chromatography ; Journal of Chromatography. A , ( 1991 ); 538 ( 1 ): 3 – 25 . 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Published: Sep 1, 2018

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