Purification of Four Flavonoid Glycosides from Lotus (Nelumbo nucifera Gaertn) plumule by Macroporous Resin Combined with HSCCC

Purification of Four Flavonoid Glycosides from Lotus (Nelumbo nucifera Gaertn) plumule by... Abstract High-speed counter-current chromatography (HSCCC) combined with macroporous resin (MR) column was successfully applied to the isolation and purification of four flavonoid glycosides from the medicinal herb Lotus plumule (LP). A polar two-phase solvent system composed of ethyl acetate–n-butanol–water (1:2:3, v/v/v) was selected by high-performance liquid chromatography (HPLC) and run on a preparative scale where the lower aqueous phase was used as the mobile phase with a head-to-tail elution mode. Quercetin-3-O-β-D-glucopyranoside (15 mg), isorhamnetin-3-O-β-D-glucopyranoside (13 mg), apigenin 6-C-β-D-glucopyranosyl-8-C-α-L-arabinopyranoside (18 mg) and apigenin 6,8-di-C-β-D-glucopyranoside (48 mg) were obtained in a one-step HSCCC separation from 240 mg of the sample. The purity of each compound was over 95% as determined by HPLC. Chemical structures of the isolated compounds were identified by electrospray ionization mass spectrometry (ESI–MS-MS) and nuclear magnetic resonance (NMR) methods. Moreover, the four compounds were isolated from LP for the first time. Introduction Lotus (Nelumbo nucifera Gaertn.) is an aquatic perennial plant of economic importance that is widely distributed in India, Japan and China (1). Almost all tissues of lotus, including leaves, leaf stalks, flower petals, flower stalks, flower stamens, flower pistils, seeds and rhizomes, are used as traditional Chinese medicines for treatment of various diseases, such as cancer, depression, diarrhea, heart problems, hypertension, insomnia, inflammation, skin diseases, neuronal disorders, poisoning and some other symptoms (2–4). LP is the germ inside the lotus seed with bitter taste and generally removed before lotus seeds are eaten. Previous phytochemical investigations on LP have found a variety of secondary metabolites such as flavonoids, alkaloids and polyphenols (5–8). Among them, flavonoids are the main active components. Our previous studies on LP also disclosed that the extract of this plant was rich in flavonoids, which possesses strong antioxidant activities (9). Column chromatography is commonly adopted in separation and purification of flavonoids from Lotus (10). But these traditional methods are time consuming, low efficiency and are complicated in operation. Compared to conventional repeated column chromatography, high-speed counter-current chromatography (HSCCC), invented by Ito, is a support-free liquid–liquid partition chromatography technique without solid support matrix, which eliminates the irreversible adsorptive loss of samples onto the solid support matrix (11). Furthermore, it has numerous advantages such as low solvent consuming, high sample recovery, crude samples injection permission and predictive scale-up from analytical to preparative scale and so on (12). This technique has been successfully used in the preparative separation and purification of natural products and other researches (13–16). Isolation and separation of flavonoids from petals, leaves of N. nucifera by HSCCC has been reported (17–19), however, no report has been published on the separation and purification of flavonoid glycosides from LP using HSCCC. Therefore, this study focused on the establishment of an efficient method for the isolation and purification of four flavonoid glycosides (Figure 1) with high purity from LP by HSCCC. The chemical structures of the flavonoids were elucidated by ESI–MS, 1H-NMR and 13C-NMR. Figure 1. View largeDownload slide Chemical structures of the four analyzed flavonoid glycosides from LP by HSCCC. Figure 1. View largeDownload slide Chemical structures of the four analyzed flavonoid glycosides from LP by HSCCC. Experimental Reagents and materials All analytical-grade reagents used for fraction and HSCCC separation were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Chromatographic grade acetonitrile and acetic acid used for high-performance liquid chromatography (HPLC) analysis was purchased from Jiangsu Hanbon Science & Technology Co., Ltd. (Jiangsu, China). All aqueous solutions were prepared with pure water produced by direct-Q3 (18.2 MΩ) system (Millipore, Billerica, MA, USA). LP were supplied by Guangchang white Lotus Research Institute and identified by professor Gang Xu (Institute of Applied Chemistry, Jiangxi Academy of Sciences, Nanchang, China). Apparatus The HSCCC instrument was performed in the present study using a model TBE-300B HSCCC (Shanghai Tauto Biotech Co., Ltd., Shanghai, China). The apparatus was equipped with three multilayer coil separation columns connected in series (total volume: 280 mL, the diameter of PTFE tube: 1.6 mm) and a 20 mL manual sample loop. The revolution radius (the distance between the holder axis and central axis of the centrifuge) was 5 cm, and the β-values of the multilayer coil varied from 0.5 at the internal layer to 0.8 at the external terminal layer (β = r/R, where r is the distance from the coil to the holder shaft, and R is the revolution radius or the distance between the holder axis and central axis of the centrifuge). The rotation speed of the apparatus could be regulated between 0 and 1,000 r.p.m. The HSCCC system was equipped with a HX-105 constant-temperature circulating regulator (Beijing Changliu Scientific Instrument, Beijing, China), which was used to control the separation temperature. The system was also equipped with a TBP-5002 pump, a model Quik Sep UV-50 UV detector (H&E Co., Ltd, Beijing, China). and a HIE-50A chromatography workstation (H&E Co., Ltd, Beijing, China). In the separation process, the temperature of separation columns was maintained at 30°C, and the effluents were achieved at 254 nm. The HPLC equipment used was an Agilent 1260 system (Agilent Technologies Co., Ltd., Santa Clara, CA, USA) including a quaternary pump (G1311 C), an auto-sampler (G1329B), a thermostated column compartment (G1316A), a ultraviolet absorption detector (G1314D), and an Agilent ChemStation workstation was used for data acquisition and processing. ESI–MS spectrums were obtained with a Thermo LCQ advantage ion-trap mass spectrometer (Thermo, San Jose, CA, USA). The nuclear magnetic resonance (NMR) spectrometer were Bruker Avance 400 and 600 NMR system (Bruker, Fallanden, Switzerland). Preparation of resin-purified sample The air-dried LP (100 g) were pulverized and extracted three times (each for 0.5 h) with 70% ethanol using ultrasonic-assisted. The resulting extracts were combined, filtered and concentrated under reduced pressure to remove ethanol to give aqueous fluid (crude extract). The fluid was loaded into a MR column (70 cm × 3.5 cm, containing 300 g D-101 MR), and eluted with 0, 30, 70 and 100% ethanol (~1500 mL for each gradient). The water–ethanol (70:30) fraction was concentrated to produce 3.12 g of resin-purified sample for subsequent HSCCC isolation and purification. Selection of two-phase solvent system The partition coefficient was the ratio of the solute distributed between the mutually equilibrate two solvent phases. The partition coefficients were determined by HPLC as follows: About 5 mg of resin-purified was added to each test tube, and then 2 mL of each phase of a pre-equilibrated two-phase solvent system was added. After thorough mixing and left to stand at room temperature until equilibrium was attained between the two phases, 1 mL of each phase was dried and re-dissolved in 1 mL acetonitrile, then analyzed by HPLC to obtain K values of all target compounds, respectively. The partition coefficient (K) is expressed as K = Aupper/Alower, where Aupper and Alower were the HPLC peak areas of objective compounds in the upper and lower phase, respectively. Preparation of solvents system and sample solution In this study, the HSCCC separation was carried out in two-phase solvent system composing of ethyl acetate–n-butanol–water (1:2:3, v/v/v). The mixed solvents were shaken vigorously in a separatory funnel and equilibrated at room temperature. The upper phase and lower phase were separated and degassed by sonication for 30 min prior to use. The sample solution for HSCCC separation was prepared by dissolving 240 mg of crude sample in 10 mL solvent mixture of upper phase and lower phase (1:1, v/v) of the solvent system. HSCCC separation procedure HSCCC was performed as follows: the column of HSCCC was first entirely filled with the upper phase (stationary phase) by constant flow pump at 15 mL/min and then the apparatus was run at 900 r.p.m. About 30 min later, the lower mobile phase was pumped into the inlet of the column at the flow rate of 2 mL/min in the head-to-tail elution mode. After a clear mobile phase eluted at the tail outlet and the hydrodynamic equilibrium was established, then ~10 mL sample solution was injected into the injection valve. Afterwards, he effluent from the outlet of the column was continuously monitored with a UV detector at 254 nm and the peak fractions were collected manually according to the chromatographic profile and evaporated under vacuum. After target compounds were eluted, the centrifuge was stopped and solution in column was pumped out with pressured nitrogen. HPLC analysis and identification of HSCCC peak fractions The crude extract, resin-purified sample and each peak fraction obtained by HSCCC were analyzed by HPLC. The HPLC analyses were carried out using Agilent Zorbax Eclipse XDB-C18 column (250 × 4.6 mm ID, 5 μm). The HPLC solvents were H2O with 0.4% acetic acid (v/v) as an aqueous solvent (A) and CH3CN as an organic solvent (B), respectively. The gradient condition was as follows: 0–5 min, 20–30% B; 5–6 min, 30% B; 6–28 min, 30–48% B, 28–42 min, 48% B. A flow rate was set at 1.0 mL/min, a column temperature was 30°C, sample injection volume was 10 μL and the detector wavelength was 254 nm. Their chemical structures were identified by electrospray ionization mass spectrometry (ESI–MS-MS) and NMR methods. Results Concentration of flavonoids by D-101 MR The crude extract sample extracted directly from LP contains various types of compounds, which can be seen from the HPLC chromatogram shown in Figure 2A. A D-101 macroporous absorption resin that was packed into a column was selected to concentrate flavonoids before HSCCC separation because of the relatively low amount of the target and the interference from the impurities in the crude sample. The results given in Figure 2B showed that the impurities were removed effectively by using D-101 macroporous resin (MR) after analysis of the elution of the target compounds by HPLC method. Figure 2. View largeDownload slide The HPLC chromatogram of the crude extract of LP (A) and resin-purified sample (B). HPLC conditions: column, Agilent Zorbax Eclipse XDB-C18 (250 mm∗4.6 mm i.d., 5 μm); mobile phase, consisted of A (0.4% acetic acid in water) and B (acetonitrile), which was programmed as follows: 0–5 min, 20–30% B; 5–6 min, 30% B; 6–28 min, 30–48% B, 28–42 min, 48% B; flow rate, 1 mL/min; UV wavelength, 254 nm; column temperature, 30°C. Figure 2. View largeDownload slide The HPLC chromatogram of the crude extract of LP (A) and resin-purified sample (B). HPLC conditions: column, Agilent Zorbax Eclipse XDB-C18 (250 mm∗4.6 mm i.d., 5 μm); mobile phase, consisted of A (0.4% acetic acid in water) and B (acetonitrile), which was programmed as follows: 0–5 min, 20–30% B; 5–6 min, 30% B; 6–28 min, 30–48% B, 28–42 min, 48% B; flow rate, 1 mL/min; UV wavelength, 254 nm; column temperature, 30°C. HSCCC separation and peak fraction analysis HSCCC was introduced in this study to purify each compound effectively. In the present study, nine solvent systems were tested for the selection of ideal K-value for four compounds (Table I). The separation time was ~350 min in each separation run, the four compounds were eluted in order of increasing K-value and good resolution could be obtained. A total of 15 mg of quercetin-3-O-β-D-glucopyranoside, 13 mg of isorhamnetin-3-O-β-D-glucopyranoside, 18 mg of apigenin 6-C-β-D-glucopyranosyl-8-C-α-L-arabinopyranoside and 48 mg of apigenin 6,8-di-C-β-D-glucopyranoside were separated from the above resin-purified sample (240 mg) within 350 min by HSCCC in only one-step. The HPLC analysis of each HSCCC fraction revealed that the purities of the four components (I–IV, Figure 1) were 95.7, 98.3, 96.6 and 96.8%, respectively (Figure 3), and their corresponding HSCCC chromatogram were shown in Figure 4. Table I. The Partition Coefficient (K-value) of Compounds I, II, III and IV in the Two-Phase Solvent Systems of Ethyl acetate–n-butanol–water by HPLC Analysis Solvent system  Ratio (v/v/v/v)  K values  I  II  III  IV  n-Hexane–ethyl acetate–methanol–water  1:1:1:1  0.09  0.12  0.22  0.14  n-Hexane–ethyl acetate–methanol–water  1:2:1:2  0.11  0.45  0.57  0.65  n-Hexane–ethyl acetate–methanol–water  1:5:1:2  0.05  0.14  0.23  0.73  Chloroform–methanol–water  3:1:3  16.56  18.35  17.86  16.46  Chloroform–methanol–water  4:1:2  14.56  19.35  14.78  13.65  Chloroform–methanol–water  5:1:3  18.53  17.65  14.56  16.86  Ethyl acetate–n-butanol–water  1:2:3  0.67  0.83  0.92  1.04  Ethyl acetate–n-butanol–water  2:3:5  0.42  1.11  2.21  3.34  Ethyl acetate–n-butanol–water  4:1:5  0.55  1.14  1.42  2.68  Solvent system  Ratio (v/v/v/v)  K values  I  II  III  IV  n-Hexane–ethyl acetate–methanol–water  1:1:1:1  0.09  0.12  0.22  0.14  n-Hexane–ethyl acetate–methanol–water  1:2:1:2  0.11  0.45  0.57  0.65  n-Hexane–ethyl acetate–methanol–water  1:5:1:2  0.05  0.14  0.23  0.73  Chloroform–methanol–water  3:1:3  16.56  18.35  17.86  16.46  Chloroform–methanol–water  4:1:2  14.56  19.35  14.78  13.65  Chloroform–methanol–water  5:1:3  18.53  17.65  14.56  16.86  Ethyl acetate–n-butanol–water  1:2:3  0.67  0.83  0.92  1.04  Ethyl acetate–n-butanol–water  2:3:5  0.42  1.11  2.21  3.34  Ethyl acetate–n-butanol–water  4:1:5  0.55  1.14  1.42  2.68  Ethyl acetate-n-butanol-water (1:2:3) gave the best performance in separating I, II, III and IV with K values all locating within the interval between 0.5 and 2. Figure 3. View largeDownload slide The HPLC chromatogram of the four target compounds (A–D) separated and purified by HSCCC. HPLC conditions are the same to the above Figure 2. Figure 3. View largeDownload slide The HPLC chromatogram of the four target compounds (A–D) separated and purified by HSCCC. HPLC conditions are the same to the above Figure 2. Figure 4. View largeDownload slide HSCCC chromatogram of the resin-purified sample from LP. Two-phase solvent system: ethyl acetate–n-butanol–water (1:2:3, v/v/v). The other conditions of HSCCC were as follows: flow rate: 2.0 mL/min; revolution speed: 900 rpm; detection wavelength: 254 nm; column temperature: 30°C. Figure 4. View largeDownload slide HSCCC chromatogram of the resin-purified sample from LP. Two-phase solvent system: ethyl acetate–n-butanol–water (1:2:3, v/v/v). The other conditions of HSCCC were as follows: flow rate: 2.0 mL/min; revolution speed: 900 rpm; detection wavelength: 254 nm; column temperature: 30°C. Structural identification The structural identification of isolated compounds was performed according to ESI–MS, 1H-NMR and 13C-NMR as follows: Compound I: yellow amorphous powder, C21H20O12; ESI–MS m/z: 487 [M + Na]+; 1H-NMR (600 MHz, DMSO-d6) δ 12.64 (s, 5-OH), 7.58 (1 H, d, J = 2.0 Hz, H-2’), 7.57 (1 H, dd, J = 8.0, 2.0 Hz, H-6’), 6.84 (1 H, d, J = 8.8 Hz, H-5’), 6.40 (d, J = 1.9 Hz, H-8), 6.20 (d, J = 2.0 Hz, H-6), 5.46 (d, J = 7.4 Hz, 1 H-1”). 13C-NMR (150 MHz, DMSO-d6) δ 177.46 (C-4), 164.15 (C-7), 161.25 (C-5), 156.34 (C-2), 156.19 (C-9), 148.47 (C-3´), 144.82 (C-4´), 133.33 (C-3), 121.18 (C-1´), 121.1 (C-6’), 116.1 (C-5’), 115.1 (C-2’), 103.99 (C-10), 100.8 (C-1”), 98.6 (C-6), 93.4 (C-8), 74.0 (C-2”), 76.4 (C-3”), 69.9 (C-4”), 77.5 (C-5”), 60.9 (C-6”). Comparing the above data with the literature data (20), compound I was identified as quercetin-3-O-β-D-glucopyranoside. Compound II: yellow amorphous powder, C22H22O12; ESI–MS m/z: 479 [M + H]+; 1H-NMR (600 MHz, DMSO-d6) δ 12.61 (1 H, s, 5-OH), 7.95 (1 H, d, J = 2.1 Hz, H-2′), 7.51 (1 H, dd, J = 8.5, 2.1 Hz, H-6′), 6.93 (1 H, d, J = 8.5 Hz, H-5′), 6.45 (1 H, d, J = 2.1 Hz, H-8), 6.22 (1 H, d, J = 2.1 Hz, H-6), 5.57 (1 H, d, J = 7.5 Hz, H-1″), 3.85 (3 H, s, OCH3-3′); 13C-NMR (150 MHz, DMSO-d6) δ 177.5 (C-4), 164.1 (C-7), 161.2 (C-5), 156.3 (C-2), 156.2 (C-9), 149.4 (C-4′), 146.9 (C-3′), 133.0 (C-3), 122.0 (C-1′), 121.1 (C-6′), 115.2 (C-5′), 113.5 (C-2′), 100.8 (C-1”), 104.0 (C-10), 98.7 (C-6), 93.5 (C-8), 74.1 (C-2”), 76.5 (C-3”), 69.8 (C-4”), 77.6 (C-5”), 60.1 (C-6”). These ESI–MS, 1H-NMR and 13C-NMR data were similar to those in previous report (21), and compound II was identified as isorhamnetin-3-O-β-D-glucopyranoside. Compound III: yellow amorphous powder, C26H28O14; ESI–MS m/z: 565 [M + H]+, 563 [M − H]-, 587 [M + Na]+; 1H-NMR (400 MHz, DMSO-d6) δ 6.81 (1 H, s, H-3), 8.09 (1 H, d, J = 8.9 Hz, H-2′), 8.11 (1 H, dd, J = 8.9, 2.1 Hz, H-6′), 6.93 (2 H, d, J = 8.9 Hz, H-3′, 5′), 4.67 (1 H, d, J = 9.6 Hz, H-1′′), 4.72 (1 H, d, J = 9.3 Hz, H-1′′′), 3.99 (1 H, s, H-2′′), 4.11 (1 H, s, H-2′′′), 3.44 (1 H, s, H-3′′), 3.48 (1 H, s, H-3′′′), 3.82 (1 H, s, H-4′′), 3.80 (1 H, s, H-4′′′), 3.85 (1 H, s, H-5′′), 3.90 (1 H, s, H-5′′′), 3.63 (1 H, s, H-6′′); 13C-NMR (100 MHz, DMSO-d6) δ 182.36 (C-3), 164.18 (C-2), 161.24 (C-7), 161.28 (C-4′), 158.77 (C-5), 155.04 (C-9), 129.41 (C-2′), 128.76 (C-6′), 121.17 (C-1′), 116.27 (C-3′), 115.94 (C-5′), 108.42 (C-6), 104.52 (C-8), 103.04 (C-10), 102.29 (C-3), 70.2 (C-5”), 70.11 (C-5′′′), 74.82 (C-3”), 75.03 (C-3′′′), 74.28 (C-1′′), 73.72 (C-1”′), 68.77 (C-2”), 69.12 (C-2′′′), 69.12 (C-4”), 70.22 (C-4′′′), 61.67 (C-6”). Comparing the above data with the literature data (22), compound III was identified as apigenin 6-C-β-D-glucopyranosyl-8-C-α-L-arabinopyranoside. Compound IV: yellow amorphous powder, C16H18O9; ESI–MS m/z: 595 [M + H]+, 593 [M-H]-, 617 [M + Na]+; 1H-NMR (400 MHz, DMSO-d6) δ 13.67 (1 H, s, 5-OH), 8.00 (2 H, t, J = 20.1 Hz, H-2′, H-6′), 6.90 (2 H, d, J = 8.1 Hz, H-3′, H-5′), 4.77 (1 H, d, J = 9.6 Hz, H-1′′), 4.71 (1 H, d, J = 9.3 Hz, H-1′′′), 3.92 (1 H, s, H-2′′), 3.90 (1 H, s, H-2′′′), 3.87 (1 H, s, H-3′′), 3.85 (1 H, s, H-3′′′), 3.82 (1 H, s, H-4′′), 3.77 (1 H, s, H-4′′′), 3.75 (1 H, s, H-5′′), 3.66 (1 H, s, H-5′′′), 3.63 (1 H, s, H-6′′), 3.17 (1 H, s, H-6′′′); 13C-NMR (100 MHz, DMSO-d6) δ 182.66 (C-4), 164.48 (C-2), 161.64 (C-7), 161.28 (C-4′), 158.67 (C-5), 155.54 (C-9), 129.41 (C-2′), 128.76 (C-6′), 121.97 (C-1′), 116.27 (C-3′), 115.94 (C-5′), 108.52 (C-6), 105.52 (C-8), 104.04 (C-10), 102.99 (C-3), 82.28 (C-5”), 81.41 (C-5′′′), 79.30 (C-3”), 78.64 (C-3′′′), 74.28 (C-1′′′), 73.72 (C-1”), 71.40 (C-2”), 71.03 (C-2′′′), 70.54 (C-4”), 70.32 (C-4′′′), 61.67 (C-6”), 61.08 (C-6′′′). These ESI–MS, 1H-NMR and 13C-NMR data were similar to those in previous report (23), and compound IV was identified as apigenin 6,8-di-C-β-D -glucopyranoside. Discussion According to the literature (24), the partition coefficient (K) of the target compound in different two-phase solvent systems is critical for its successful isolation and separation by HSCCC. In an HSCCC separation, the selection of a suitable two-phase solvent system is the first and most important step, and a good solvent system can provide an ideal partition coefficient (K) for the target compounds in the range of 0.5–2.0 (25–27). Therefore, some two-phase solvent systems including n-hexane–ethyl acetate–methanol–water, chloroform–methanol–water and ethyl acetate–n-butanol–water each at different volume ratio, respectively, were tested in this article. The K-value of the target compounds were shown in Table I. K-value of the four target compounds were high when using the system of chloroform–methanol–water (3:1:3, 4:1:2, 5:1:3, v/v/v), which demonstrated that most target compounds were dissolved in the upper phase. However, the use of n-hexane–ethyl acetate–methanol–water (1:1:1:1, 1:2:1:2, 1:5:1:2, v/v/v/v) was not suitable in this step for K-value were too low. The results indicated that the solvent system acetate–n-butanol–water (4:1:5, v/v/v) provided proper K-value for separating compounds I (K-value: 0.55), II (K-value: 1.14) and III (K-value: 1.42) but was unsuitable for compound IV (K-value: 2.68). While, when acetate–n-butanol–water (1:2:3, v/v/v) was used as the two phase solvent system, the K-value of all the compounds were suitable for separation. Therefore, this two-phase solvent system was selected for the HSCCC separation in the present paper. Recently, interest in plant-derived food additives has grown, mainly because synthetic antioxidants suffer from several drawbacks. Flavonoids are well-known antioxidants which have many beneficial health effects such as antiviral, anti-inflammatory, antibacterial and muscle-relaxing properties (28). In DPPH, free radical scavenging assay, quercetin-3-O-β-D-glucopyranoside and isorhamnetin-3-O-β-D-glucopyranoside from aerial parts of Halostachys caspica were screened and exhibited weaker antioxidant activity with IC50 values of 82.55 and 165.62 μg/mL, respectively, comparing with positive control of BHT (IC50 18.8 μg/mL) (29). Schaftoside (compound III) and vicenin-2 (compound IV), the di-C-glycosyl flavonoids isolated from Centaurea calolepis Boiss. and Lychnophora ericoides Mart. respectively, showed significant antioxidant properties (30, 31). Conclusion In this work, the results of our study clearly demonstrated that HSCCC could provide highly efficient preparative separation of quercetin-3-O-β-D-glucopyranoside, isorhamnetin-3-O-β-D-glucopyranoside, apigenin 6-C-β-D-glucopyranosyl-8-C-α-L-arabinopyranoside and apigenin 6,8-di-C-β-D-glucopyranoside from LP. In combination with pre-separation of macroporous absorption resin column prior to HSCCC separation, four flavonoid glycosides with high purity were obtained, making them suitable for use in further chemical research and pharmacological studies. Funding This work was supported by the Key Project of Scientific and Technical supporting plan programs Foundation of Jiangxi (No. 20171BBH80017), Science Foundation for Young Doctors of Jiangxi Academy of Science (2016-YYB-07), National Natural Science Foundation of China (No. 31471629), and Introduction of Overseas Technical and Managerial Personnel Program of Tianjin City (20171200118) and State Administration of Foreign Experts Affairs, P.R. China (20173600003). References 1 Liao, C.H., Lin, J.Y.; Purification, partial characterization and anti-inflammatory characteristics of lotus (Nelumbo nucifera Gaertn) plumule polysaccharides; Food chemistry , ( 2012); 135: 1818– 1827. 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Purification of Four Flavonoid Glycosides from Lotus (Nelumbo nucifera Gaertn) plumule by Macroporous Resin Combined with HSCCC

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Oxford University Press
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© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com
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0021-9665
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1945-239X
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10.1093/chromsci/bmx088
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Abstract

Abstract High-speed counter-current chromatography (HSCCC) combined with macroporous resin (MR) column was successfully applied to the isolation and purification of four flavonoid glycosides from the medicinal herb Lotus plumule (LP). A polar two-phase solvent system composed of ethyl acetate–n-butanol–water (1:2:3, v/v/v) was selected by high-performance liquid chromatography (HPLC) and run on a preparative scale where the lower aqueous phase was used as the mobile phase with a head-to-tail elution mode. Quercetin-3-O-β-D-glucopyranoside (15 mg), isorhamnetin-3-O-β-D-glucopyranoside (13 mg), apigenin 6-C-β-D-glucopyranosyl-8-C-α-L-arabinopyranoside (18 mg) and apigenin 6,8-di-C-β-D-glucopyranoside (48 mg) were obtained in a one-step HSCCC separation from 240 mg of the sample. The purity of each compound was over 95% as determined by HPLC. Chemical structures of the isolated compounds were identified by electrospray ionization mass spectrometry (ESI–MS-MS) and nuclear magnetic resonance (NMR) methods. Moreover, the four compounds were isolated from LP for the first time. Introduction Lotus (Nelumbo nucifera Gaertn.) is an aquatic perennial plant of economic importance that is widely distributed in India, Japan and China (1). Almost all tissues of lotus, including leaves, leaf stalks, flower petals, flower stalks, flower stamens, flower pistils, seeds and rhizomes, are used as traditional Chinese medicines for treatment of various diseases, such as cancer, depression, diarrhea, heart problems, hypertension, insomnia, inflammation, skin diseases, neuronal disorders, poisoning and some other symptoms (2–4). LP is the germ inside the lotus seed with bitter taste and generally removed before lotus seeds are eaten. Previous phytochemical investigations on LP have found a variety of secondary metabolites such as flavonoids, alkaloids and polyphenols (5–8). Among them, flavonoids are the main active components. Our previous studies on LP also disclosed that the extract of this plant was rich in flavonoids, which possesses strong antioxidant activities (9). Column chromatography is commonly adopted in separation and purification of flavonoids from Lotus (10). But these traditional methods are time consuming, low efficiency and are complicated in operation. Compared to conventional repeated column chromatography, high-speed counter-current chromatography (HSCCC), invented by Ito, is a support-free liquid–liquid partition chromatography technique without solid support matrix, which eliminates the irreversible adsorptive loss of samples onto the solid support matrix (11). Furthermore, it has numerous advantages such as low solvent consuming, high sample recovery, crude samples injection permission and predictive scale-up from analytical to preparative scale and so on (12). This technique has been successfully used in the preparative separation and purification of natural products and other researches (13–16). Isolation and separation of flavonoids from petals, leaves of N. nucifera by HSCCC has been reported (17–19), however, no report has been published on the separation and purification of flavonoid glycosides from LP using HSCCC. Therefore, this study focused on the establishment of an efficient method for the isolation and purification of four flavonoid glycosides (Figure 1) with high purity from LP by HSCCC. The chemical structures of the flavonoids were elucidated by ESI–MS, 1H-NMR and 13C-NMR. Figure 1. View largeDownload slide Chemical structures of the four analyzed flavonoid glycosides from LP by HSCCC. Figure 1. View largeDownload slide Chemical structures of the four analyzed flavonoid glycosides from LP by HSCCC. Experimental Reagents and materials All analytical-grade reagents used for fraction and HSCCC separation were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Chromatographic grade acetonitrile and acetic acid used for high-performance liquid chromatography (HPLC) analysis was purchased from Jiangsu Hanbon Science & Technology Co., Ltd. (Jiangsu, China). All aqueous solutions were prepared with pure water produced by direct-Q3 (18.2 MΩ) system (Millipore, Billerica, MA, USA). LP were supplied by Guangchang white Lotus Research Institute and identified by professor Gang Xu (Institute of Applied Chemistry, Jiangxi Academy of Sciences, Nanchang, China). Apparatus The HSCCC instrument was performed in the present study using a model TBE-300B HSCCC (Shanghai Tauto Biotech Co., Ltd., Shanghai, China). The apparatus was equipped with three multilayer coil separation columns connected in series (total volume: 280 mL, the diameter of PTFE tube: 1.6 mm) and a 20 mL manual sample loop. The revolution radius (the distance between the holder axis and central axis of the centrifuge) was 5 cm, and the β-values of the multilayer coil varied from 0.5 at the internal layer to 0.8 at the external terminal layer (β = r/R, where r is the distance from the coil to the holder shaft, and R is the revolution radius or the distance between the holder axis and central axis of the centrifuge). The rotation speed of the apparatus could be regulated between 0 and 1,000 r.p.m. The HSCCC system was equipped with a HX-105 constant-temperature circulating regulator (Beijing Changliu Scientific Instrument, Beijing, China), which was used to control the separation temperature. The system was also equipped with a TBP-5002 pump, a model Quik Sep UV-50 UV detector (H&E Co., Ltd, Beijing, China). and a HIE-50A chromatography workstation (H&E Co., Ltd, Beijing, China). In the separation process, the temperature of separation columns was maintained at 30°C, and the effluents were achieved at 254 nm. The HPLC equipment used was an Agilent 1260 system (Agilent Technologies Co., Ltd., Santa Clara, CA, USA) including a quaternary pump (G1311 C), an auto-sampler (G1329B), a thermostated column compartment (G1316A), a ultraviolet absorption detector (G1314D), and an Agilent ChemStation workstation was used for data acquisition and processing. ESI–MS spectrums were obtained with a Thermo LCQ advantage ion-trap mass spectrometer (Thermo, San Jose, CA, USA). The nuclear magnetic resonance (NMR) spectrometer were Bruker Avance 400 and 600 NMR system (Bruker, Fallanden, Switzerland). Preparation of resin-purified sample The air-dried LP (100 g) were pulverized and extracted three times (each for 0.5 h) with 70% ethanol using ultrasonic-assisted. The resulting extracts were combined, filtered and concentrated under reduced pressure to remove ethanol to give aqueous fluid (crude extract). The fluid was loaded into a MR column (70 cm × 3.5 cm, containing 300 g D-101 MR), and eluted with 0, 30, 70 and 100% ethanol (~1500 mL for each gradient). The water–ethanol (70:30) fraction was concentrated to produce 3.12 g of resin-purified sample for subsequent HSCCC isolation and purification. Selection of two-phase solvent system The partition coefficient was the ratio of the solute distributed between the mutually equilibrate two solvent phases. The partition coefficients were determined by HPLC as follows: About 5 mg of resin-purified was added to each test tube, and then 2 mL of each phase of a pre-equilibrated two-phase solvent system was added. After thorough mixing and left to stand at room temperature until equilibrium was attained between the two phases, 1 mL of each phase was dried and re-dissolved in 1 mL acetonitrile, then analyzed by HPLC to obtain K values of all target compounds, respectively. The partition coefficient (K) is expressed as K = Aupper/Alower, where Aupper and Alower were the HPLC peak areas of objective compounds in the upper and lower phase, respectively. Preparation of solvents system and sample solution In this study, the HSCCC separation was carried out in two-phase solvent system composing of ethyl acetate–n-butanol–water (1:2:3, v/v/v). The mixed solvents were shaken vigorously in a separatory funnel and equilibrated at room temperature. The upper phase and lower phase were separated and degassed by sonication for 30 min prior to use. The sample solution for HSCCC separation was prepared by dissolving 240 mg of crude sample in 10 mL solvent mixture of upper phase and lower phase (1:1, v/v) of the solvent system. HSCCC separation procedure HSCCC was performed as follows: the column of HSCCC was first entirely filled with the upper phase (stationary phase) by constant flow pump at 15 mL/min and then the apparatus was run at 900 r.p.m. About 30 min later, the lower mobile phase was pumped into the inlet of the column at the flow rate of 2 mL/min in the head-to-tail elution mode. After a clear mobile phase eluted at the tail outlet and the hydrodynamic equilibrium was established, then ~10 mL sample solution was injected into the injection valve. Afterwards, he effluent from the outlet of the column was continuously monitored with a UV detector at 254 nm and the peak fractions were collected manually according to the chromatographic profile and evaporated under vacuum. After target compounds were eluted, the centrifuge was stopped and solution in column was pumped out with pressured nitrogen. HPLC analysis and identification of HSCCC peak fractions The crude extract, resin-purified sample and each peak fraction obtained by HSCCC were analyzed by HPLC. The HPLC analyses were carried out using Agilent Zorbax Eclipse XDB-C18 column (250 × 4.6 mm ID, 5 μm). The HPLC solvents were H2O with 0.4% acetic acid (v/v) as an aqueous solvent (A) and CH3CN as an organic solvent (B), respectively. The gradient condition was as follows: 0–5 min, 20–30% B; 5–6 min, 30% B; 6–28 min, 30–48% B, 28–42 min, 48% B. A flow rate was set at 1.0 mL/min, a column temperature was 30°C, sample injection volume was 10 μL and the detector wavelength was 254 nm. Their chemical structures were identified by electrospray ionization mass spectrometry (ESI–MS-MS) and NMR methods. Results Concentration of flavonoids by D-101 MR The crude extract sample extracted directly from LP contains various types of compounds, which can be seen from the HPLC chromatogram shown in Figure 2A. A D-101 macroporous absorption resin that was packed into a column was selected to concentrate flavonoids before HSCCC separation because of the relatively low amount of the target and the interference from the impurities in the crude sample. The results given in Figure 2B showed that the impurities were removed effectively by using D-101 macroporous resin (MR) after analysis of the elution of the target compounds by HPLC method. Figure 2. View largeDownload slide The HPLC chromatogram of the crude extract of LP (A) and resin-purified sample (B). HPLC conditions: column, Agilent Zorbax Eclipse XDB-C18 (250 mm∗4.6 mm i.d., 5 μm); mobile phase, consisted of A (0.4% acetic acid in water) and B (acetonitrile), which was programmed as follows: 0–5 min, 20–30% B; 5–6 min, 30% B; 6–28 min, 30–48% B, 28–42 min, 48% B; flow rate, 1 mL/min; UV wavelength, 254 nm; column temperature, 30°C. Figure 2. View largeDownload slide The HPLC chromatogram of the crude extract of LP (A) and resin-purified sample (B). HPLC conditions: column, Agilent Zorbax Eclipse XDB-C18 (250 mm∗4.6 mm i.d., 5 μm); mobile phase, consisted of A (0.4% acetic acid in water) and B (acetonitrile), which was programmed as follows: 0–5 min, 20–30% B; 5–6 min, 30% B; 6–28 min, 30–48% B, 28–42 min, 48% B; flow rate, 1 mL/min; UV wavelength, 254 nm; column temperature, 30°C. HSCCC separation and peak fraction analysis HSCCC was introduced in this study to purify each compound effectively. In the present study, nine solvent systems were tested for the selection of ideal K-value for four compounds (Table I). The separation time was ~350 min in each separation run, the four compounds were eluted in order of increasing K-value and good resolution could be obtained. A total of 15 mg of quercetin-3-O-β-D-glucopyranoside, 13 mg of isorhamnetin-3-O-β-D-glucopyranoside, 18 mg of apigenin 6-C-β-D-glucopyranosyl-8-C-α-L-arabinopyranoside and 48 mg of apigenin 6,8-di-C-β-D-glucopyranoside were separated from the above resin-purified sample (240 mg) within 350 min by HSCCC in only one-step. The HPLC analysis of each HSCCC fraction revealed that the purities of the four components (I–IV, Figure 1) were 95.7, 98.3, 96.6 and 96.8%, respectively (Figure 3), and their corresponding HSCCC chromatogram were shown in Figure 4. Table I. The Partition Coefficient (K-value) of Compounds I, II, III and IV in the Two-Phase Solvent Systems of Ethyl acetate–n-butanol–water by HPLC Analysis Solvent system  Ratio (v/v/v/v)  K values  I  II  III  IV  n-Hexane–ethyl acetate–methanol–water  1:1:1:1  0.09  0.12  0.22  0.14  n-Hexane–ethyl acetate–methanol–water  1:2:1:2  0.11  0.45  0.57  0.65  n-Hexane–ethyl acetate–methanol–water  1:5:1:2  0.05  0.14  0.23  0.73  Chloroform–methanol–water  3:1:3  16.56  18.35  17.86  16.46  Chloroform–methanol–water  4:1:2  14.56  19.35  14.78  13.65  Chloroform–methanol–water  5:1:3  18.53  17.65  14.56  16.86  Ethyl acetate–n-butanol–water  1:2:3  0.67  0.83  0.92  1.04  Ethyl acetate–n-butanol–water  2:3:5  0.42  1.11  2.21  3.34  Ethyl acetate–n-butanol–water  4:1:5  0.55  1.14  1.42  2.68  Solvent system  Ratio (v/v/v/v)  K values  I  II  III  IV  n-Hexane–ethyl acetate–methanol–water  1:1:1:1  0.09  0.12  0.22  0.14  n-Hexane–ethyl acetate–methanol–water  1:2:1:2  0.11  0.45  0.57  0.65  n-Hexane–ethyl acetate–methanol–water  1:5:1:2  0.05  0.14  0.23  0.73  Chloroform–methanol–water  3:1:3  16.56  18.35  17.86  16.46  Chloroform–methanol–water  4:1:2  14.56  19.35  14.78  13.65  Chloroform–methanol–water  5:1:3  18.53  17.65  14.56  16.86  Ethyl acetate–n-butanol–water  1:2:3  0.67  0.83  0.92  1.04  Ethyl acetate–n-butanol–water  2:3:5  0.42  1.11  2.21  3.34  Ethyl acetate–n-butanol–water  4:1:5  0.55  1.14  1.42  2.68  Ethyl acetate-n-butanol-water (1:2:3) gave the best performance in separating I, II, III and IV with K values all locating within the interval between 0.5 and 2. Figure 3. View largeDownload slide The HPLC chromatogram of the four target compounds (A–D) separated and purified by HSCCC. HPLC conditions are the same to the above Figure 2. Figure 3. View largeDownload slide The HPLC chromatogram of the four target compounds (A–D) separated and purified by HSCCC. HPLC conditions are the same to the above Figure 2. Figure 4. View largeDownload slide HSCCC chromatogram of the resin-purified sample from LP. Two-phase solvent system: ethyl acetate–n-butanol–water (1:2:3, v/v/v). The other conditions of HSCCC were as follows: flow rate: 2.0 mL/min; revolution speed: 900 rpm; detection wavelength: 254 nm; column temperature: 30°C. Figure 4. View largeDownload slide HSCCC chromatogram of the resin-purified sample from LP. Two-phase solvent system: ethyl acetate–n-butanol–water (1:2:3, v/v/v). The other conditions of HSCCC were as follows: flow rate: 2.0 mL/min; revolution speed: 900 rpm; detection wavelength: 254 nm; column temperature: 30°C. Structural identification The structural identification of isolated compounds was performed according to ESI–MS, 1H-NMR and 13C-NMR as follows: Compound I: yellow amorphous powder, C21H20O12; ESI–MS m/z: 487 [M + Na]+; 1H-NMR (600 MHz, DMSO-d6) δ 12.64 (s, 5-OH), 7.58 (1 H, d, J = 2.0 Hz, H-2’), 7.57 (1 H, dd, J = 8.0, 2.0 Hz, H-6’), 6.84 (1 H, d, J = 8.8 Hz, H-5’), 6.40 (d, J = 1.9 Hz, H-8), 6.20 (d, J = 2.0 Hz, H-6), 5.46 (d, J = 7.4 Hz, 1 H-1”). 13C-NMR (150 MHz, DMSO-d6) δ 177.46 (C-4), 164.15 (C-7), 161.25 (C-5), 156.34 (C-2), 156.19 (C-9), 148.47 (C-3´), 144.82 (C-4´), 133.33 (C-3), 121.18 (C-1´), 121.1 (C-6’), 116.1 (C-5’), 115.1 (C-2’), 103.99 (C-10), 100.8 (C-1”), 98.6 (C-6), 93.4 (C-8), 74.0 (C-2”), 76.4 (C-3”), 69.9 (C-4”), 77.5 (C-5”), 60.9 (C-6”). Comparing the above data with the literature data (20), compound I was identified as quercetin-3-O-β-D-glucopyranoside. Compound II: yellow amorphous powder, C22H22O12; ESI–MS m/z: 479 [M + H]+; 1H-NMR (600 MHz, DMSO-d6) δ 12.61 (1 H, s, 5-OH), 7.95 (1 H, d, J = 2.1 Hz, H-2′), 7.51 (1 H, dd, J = 8.5, 2.1 Hz, H-6′), 6.93 (1 H, d, J = 8.5 Hz, H-5′), 6.45 (1 H, d, J = 2.1 Hz, H-8), 6.22 (1 H, d, J = 2.1 Hz, H-6), 5.57 (1 H, d, J = 7.5 Hz, H-1″), 3.85 (3 H, s, OCH3-3′); 13C-NMR (150 MHz, DMSO-d6) δ 177.5 (C-4), 164.1 (C-7), 161.2 (C-5), 156.3 (C-2), 156.2 (C-9), 149.4 (C-4′), 146.9 (C-3′), 133.0 (C-3), 122.0 (C-1′), 121.1 (C-6′), 115.2 (C-5′), 113.5 (C-2′), 100.8 (C-1”), 104.0 (C-10), 98.7 (C-6), 93.5 (C-8), 74.1 (C-2”), 76.5 (C-3”), 69.8 (C-4”), 77.6 (C-5”), 60.1 (C-6”). These ESI–MS, 1H-NMR and 13C-NMR data were similar to those in previous report (21), and compound II was identified as isorhamnetin-3-O-β-D-glucopyranoside. Compound III: yellow amorphous powder, C26H28O14; ESI–MS m/z: 565 [M + H]+, 563 [M − H]-, 587 [M + Na]+; 1H-NMR (400 MHz, DMSO-d6) δ 6.81 (1 H, s, H-3), 8.09 (1 H, d, J = 8.9 Hz, H-2′), 8.11 (1 H, dd, J = 8.9, 2.1 Hz, H-6′), 6.93 (2 H, d, J = 8.9 Hz, H-3′, 5′), 4.67 (1 H, d, J = 9.6 Hz, H-1′′), 4.72 (1 H, d, J = 9.3 Hz, H-1′′′), 3.99 (1 H, s, H-2′′), 4.11 (1 H, s, H-2′′′), 3.44 (1 H, s, H-3′′), 3.48 (1 H, s, H-3′′′), 3.82 (1 H, s, H-4′′), 3.80 (1 H, s, H-4′′′), 3.85 (1 H, s, H-5′′), 3.90 (1 H, s, H-5′′′), 3.63 (1 H, s, H-6′′); 13C-NMR (100 MHz, DMSO-d6) δ 182.36 (C-3), 164.18 (C-2), 161.24 (C-7), 161.28 (C-4′), 158.77 (C-5), 155.04 (C-9), 129.41 (C-2′), 128.76 (C-6′), 121.17 (C-1′), 116.27 (C-3′), 115.94 (C-5′), 108.42 (C-6), 104.52 (C-8), 103.04 (C-10), 102.29 (C-3), 70.2 (C-5”), 70.11 (C-5′′′), 74.82 (C-3”), 75.03 (C-3′′′), 74.28 (C-1′′), 73.72 (C-1”′), 68.77 (C-2”), 69.12 (C-2′′′), 69.12 (C-4”), 70.22 (C-4′′′), 61.67 (C-6”). Comparing the above data with the literature data (22), compound III was identified as apigenin 6-C-β-D-glucopyranosyl-8-C-α-L-arabinopyranoside. Compound IV: yellow amorphous powder, C16H18O9; ESI–MS m/z: 595 [M + H]+, 593 [M-H]-, 617 [M + Na]+; 1H-NMR (400 MHz, DMSO-d6) δ 13.67 (1 H, s, 5-OH), 8.00 (2 H, t, J = 20.1 Hz, H-2′, H-6′), 6.90 (2 H, d, J = 8.1 Hz, H-3′, H-5′), 4.77 (1 H, d, J = 9.6 Hz, H-1′′), 4.71 (1 H, d, J = 9.3 Hz, H-1′′′), 3.92 (1 H, s, H-2′′), 3.90 (1 H, s, H-2′′′), 3.87 (1 H, s, H-3′′), 3.85 (1 H, s, H-3′′′), 3.82 (1 H, s, H-4′′), 3.77 (1 H, s, H-4′′′), 3.75 (1 H, s, H-5′′), 3.66 (1 H, s, H-5′′′), 3.63 (1 H, s, H-6′′), 3.17 (1 H, s, H-6′′′); 13C-NMR (100 MHz, DMSO-d6) δ 182.66 (C-4), 164.48 (C-2), 161.64 (C-7), 161.28 (C-4′), 158.67 (C-5), 155.54 (C-9), 129.41 (C-2′), 128.76 (C-6′), 121.97 (C-1′), 116.27 (C-3′), 115.94 (C-5′), 108.52 (C-6), 105.52 (C-8), 104.04 (C-10), 102.99 (C-3), 82.28 (C-5”), 81.41 (C-5′′′), 79.30 (C-3”), 78.64 (C-3′′′), 74.28 (C-1′′′), 73.72 (C-1”), 71.40 (C-2”), 71.03 (C-2′′′), 70.54 (C-4”), 70.32 (C-4′′′), 61.67 (C-6”), 61.08 (C-6′′′). These ESI–MS, 1H-NMR and 13C-NMR data were similar to those in previous report (23), and compound IV was identified as apigenin 6,8-di-C-β-D -glucopyranoside. Discussion According to the literature (24), the partition coefficient (K) of the target compound in different two-phase solvent systems is critical for its successful isolation and separation by HSCCC. In an HSCCC separation, the selection of a suitable two-phase solvent system is the first and most important step, and a good solvent system can provide an ideal partition coefficient (K) for the target compounds in the range of 0.5–2.0 (25–27). Therefore, some two-phase solvent systems including n-hexane–ethyl acetate–methanol–water, chloroform–methanol–water and ethyl acetate–n-butanol–water each at different volume ratio, respectively, were tested in this article. The K-value of the target compounds were shown in Table I. K-value of the four target compounds were high when using the system of chloroform–methanol–water (3:1:3, 4:1:2, 5:1:3, v/v/v), which demonstrated that most target compounds were dissolved in the upper phase. However, the use of n-hexane–ethyl acetate–methanol–water (1:1:1:1, 1:2:1:2, 1:5:1:2, v/v/v/v) was not suitable in this step for K-value were too low. The results indicated that the solvent system acetate–n-butanol–water (4:1:5, v/v/v) provided proper K-value for separating compounds I (K-value: 0.55), II (K-value: 1.14) and III (K-value: 1.42) but was unsuitable for compound IV (K-value: 2.68). While, when acetate–n-butanol–water (1:2:3, v/v/v) was used as the two phase solvent system, the K-value of all the compounds were suitable for separation. Therefore, this two-phase solvent system was selected for the HSCCC separation in the present paper. Recently, interest in plant-derived food additives has grown, mainly because synthetic antioxidants suffer from several drawbacks. Flavonoids are well-known antioxidants which have many beneficial health effects such as antiviral, anti-inflammatory, antibacterial and muscle-relaxing properties (28). In DPPH, free radical scavenging assay, quercetin-3-O-β-D-glucopyranoside and isorhamnetin-3-O-β-D-glucopyranoside from aerial parts of Halostachys caspica were screened and exhibited weaker antioxidant activity with IC50 values of 82.55 and 165.62 μg/mL, respectively, comparing with positive control of BHT (IC50 18.8 μg/mL) (29). Schaftoside (compound III) and vicenin-2 (compound IV), the di-C-glycosyl flavonoids isolated from Centaurea calolepis Boiss. and Lychnophora ericoides Mart. respectively, showed significant antioxidant properties (30, 31). Conclusion In this work, the results of our study clearly demonstrated that HSCCC could provide highly efficient preparative separation of quercetin-3-O-β-D-glucopyranoside, isorhamnetin-3-O-β-D-glucopyranoside, apigenin 6-C-β-D-glucopyranosyl-8-C-α-L-arabinopyranoside and apigenin 6,8-di-C-β-D-glucopyranoside from LP. In combination with pre-separation of macroporous absorption resin column prior to HSCCC separation, four flavonoid glycosides with high purity were obtained, making them suitable for use in further chemical research and pharmacological studies. Funding This work was supported by the Key Project of Scientific and Technical supporting plan programs Foundation of Jiangxi (No. 20171BBH80017), Science Foundation for Young Doctors of Jiangxi Academy of Science (2016-YYB-07), National Natural Science Foundation of China (No. 31471629), and Introduction of Overseas Technical and Managerial Personnel Program of Tianjin City (20171200118) and State Administration of Foreign Experts Affairs, P.R. China (20173600003). References 1 Liao, C.H., Lin, J.Y.; Purification, partial characterization and anti-inflammatory characteristics of lotus (Nelumbo nucifera Gaertn) plumule polysaccharides; Food chemistry , ( 2012); 135: 1818– 1827. 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Published: Feb 1, 2018

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