Article pubs.acs.org/IECR
Cite This: Ind. Eng. Chem. Res. 2018, 57, 15840−15845
Preparative Separation of Ginkgolic Acids from the Sarcotesta of Ginkgo biloba L. by β‑Cyclodextrin Clathration Coupled with pHZone-Refining and Recycling Countercurrent Chromatography Huijiao Yan,† Long Chen,† Aiying Bai,‡ Hengqiang Zhao,† Meng Kong,§ and Li Cui*,†
Downloaded via KAOHSIUNG MEDICAL UNIV on November 26, 2018 at 09:00:37 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
†
Key Laboratory of TCM Quality Control, Shandong Analysis and Test Center, Qilu University of Technology (Shandong Academy of Sciences), Jinan, Shandong 250014, People’s Republic of China ‡ Jinan Center for Disease Control And Prevention, Jinan, Shandong 250001, People’s Republic of China § College of Life Science, Shandong Normal University, Jinan, Shandong 250014, People’s Republic of China ABSTRACT: Ginkgolic acids (GAs), bioactive compounds found in Ginkgo biloba L., have received much attention in pharmacological research. Separation of individual GAs from their mixtures is, however, difficult. Herein, an efficient method for the preparative separation of five major GAs from the sarcotesta of Ginkgo biloba L. was developed using β-cyclodextrin (β-CD) clathration coupled with pH-zone-refining countercurrent chromatography (PZRCCC) and recycling CCC. Clathration by β-CD was employed to enhance the hydrophilicity of the GAs. Separation of β-CD-GA clathrates was achieved by PZRCCC using a two-phase solvent system composed of n-heptane−ethyl acetate− methanol−water (2:1:1.5:1, v/v). The upper phase was acidified with 40 mM HCl (stationary phase), and the lower phase was made alkaline with 5 mM triethylamine. Three GAs were obtained with purities over 98% together with two mixtures. The mixtures were separated in recycling CCC mode using n-heptane−ethyl acetate−methanol−acetic acid (5:4:1:1, v/v) as the solvent system. Five GAs, including C13:0, C15:0, C15:1, C17:1, and C17:2, were successfully separated with purities over 98%, and their structures were confirmed by comparison with a mixture of GA standard samples coupled with ESI-MS data.
1. INTRODUCTION Ginkgo biloba L., the last remaining member of the gymnosperm species, has been described as a living fossil.1 Pharmacological research has shown that extracts from the leaves and seeds have useful bioactivity in the treatment of cognitive diseases.2,3 Ginkgo biloba leaf extract is one of the most extensively used phytopharmaceutical drugs in the treatment of cardiovascular, cerebrovascular, and neurological diseases.4 Ginkgolic acids (GAs) are abundant in the leaves, seeds, and sarcotesta of G. biloba,5 which are considered to be toxic, mutagenic, and allergenic and are limited to less than 10 μg/g (w/w) in the products. On the other hand, GAs have beneficial effects, including antitumor, antidepressant, antiparasitic, antibacterial, and molluscicidal activities.6−10 In particular, GAs inhibit enzymes such as HIV protease, fatty acid synthase, and tyrosinase, among others.11−13 GAs are natural 6-alkylsalicylic acids containing a hydrophobic chain composed of 13−17 carbon atoms with 0−2 double bonds (Figure 1).14 Because of their particular structures, GAs are low-polarity organic acids with strong hydrophobicity. Phytochemistry studies have described separation of GAs using traditional methods, including silica gel chromatography and reverse phase C8 and C18 HPLC.15−18 Normal phase chromatography, such as traditional silica gel separation, is tedious, requiring a long time for separation and © 2018 American Chemical Society
Figure 1. Chemical structures and schematic diagram of the β-CD clathration with GAs.
large quantities of solvent, and separation of GAs is difficult. Moreover, the long hydrophobic chains of GAs result in strong absorption in reverse phase chromatography. Because of their important biological properties and difficult separation, an efficient method for isolation and purification of GAs is urgently required. High-speed countercurrent chromatography (HSCCC) could be a good choice for efficient separation. It is a Received: Revised: Accepted: Published: 15840
August 30, 2018 October 24, 2018 October 25, 2018 October 25, 2018 DOI: 10.1021/acs.iecr.8b04167 Ind. Eng. Chem. Res. 2018, 57, 15840−15845
Article
Industrial & Engineering Chemistry Research
2.3. Preparation of GA Extract. The fresh sarcotesta were dried in the dark at room temperature. The dry sarcotesta were extracted three times with petroleum ether and the combined extracts concentrated under reduced pressure at 40 °C. 2.4. Preparation of β-CD-GA Clathrates. β-CD (30 g) was added to 2 L of reverse osmosis Milli-Q water and stirred magnetically (1000 rpm) at 60 °C until complete dissolution. GA extract (10 g) was then added to the β-CD solution and incubated in the dark for 48 h at 60 °C. The solution was then concentrated under reduced pressure at 40 °C to obtain β-CDGA clathrates. 2.5. Selection of CCC Solvent System. The partition coefficients (KD values) of the target compounds were determined by HPLC to select a suitable solvent system as follows. Each phase of the solvent system and 10 mg of the βCD-GA clathrates were added to a tube and shaken vigorously. After complete separation of the two phases, 1 mL of each layer was removed and dried in a stream of nitrogen. The residues were dissolved in 1 mL of methanol, filtered through a 0.45 μm filter membrane, and analyzed by HPLC. The KD values of the target compounds were calculated according to the equation K = AU/AL, where AU and AL were the peak areas of the target compound in the upper and lower phases, respectively. 2.6. Preparation of Solvent Systems and Sample Solutions. For PZRCCC separation, a two-phase solvent system composed of n-heptane−ethyl acetate−methanol− water (2:1:1.5:1, v/v) was added to a separating funnel. After being shaken vigorously, the mixture was left to stand for 10 min and then separated into two phases for the experiment. The upper phase was acidified with 40 mM HCl (stationary phase), and the lower phase was made alkaline with 5 mM triethylamine (TEA). The β-CD-GA clathrates (2.0 g) were dissolved in 10 mL of acidified upper phase and 10 mL of lower phase before addition of TEA. For recycling CCC separation, a two-phase solvent system composed of n-heptane−ethyl acetate−methanol−acetic acid (5:4:1:1, v/v) was used. The upper phase was the stationary phase. The fraction obtained from PZRCCC separation was dissolved in 10 mL of isometric upper and lower phase. 2.7. Separation Procedure. PZRCCC Separation. The column of the CCC instrument was first entirely filled with the upper phase at 20 mL/min in head-to-tail mode. The sample solution of β-CD-GA clathrates was then injected with the manual sample loop. The lower phase was pumped into the head of the CCC column at 2.0 mL/min, while the apparatus was rotated at 800 rpm in a clockwise direction. The separation temperature was set at 25 °C. The effluents were continuously monitored at 254 nm with a portable recorder. The retention of the stationary phase was defined as the stationary phase relative to the total column capacity after separation. Each fraction was combined according to the HPLC results. Organic solvents in each fraction were removed under reduced pressure at 40 °C. Five GA fractions were obtained in the one-step separation. Recycling CCC Separation. The three-stage configuration of the recycling CCC is shown in Figure 2. In the first stage, the six-way valve was in the collection position (Figure 2A). The HSCCC column was entirely filled with the upper phase at 20 mL/min in head-to-tail mode. The apparatus was then rotated at 800 rpm in a clockwise mode, and the lower phase was pumped into the head of the CCC column at 2.0 mL/min. After hydrodynamic equilibrium had been established, the
liquid−liquid partition chromatographic technique, which eliminates the potential for irreversible adsorption of sample onto a solid support. Furthermore, HSCCC gives high recoveries, has good separation efficiency, is easily scaled-up and simple to operate, and has low solvent consumption.19,20 It has recently become a useful tool for preparative isolation and purification of various natural products.21−23 pH-zone-refining countercurrent chromatography (PZRCCC) is an excellent large-scale preparative mode for organic acids that can increase sample loading capacity, concentrate minor impurities, and provide a high concentration of target compound in the fractions.24,25 The long hydrophobic chains in the GAs, however, make solvent system selection for PZRCCC difficult, and the similar KD values of the GAs result in poor peak resolution during traditional HSCCC separation. In this study, β-cyclodextrin (βCD), which has a relatively hydrophobic central cavity and hydrophilic outer surface, was introduced as a molecular host to enhance the hydrophilicity of GAs for PZRCCC separation. Furthermore, recycling CCC was used to enhance the theoretical plate number of the traditional HSCCC separation. This combination of methods was developed for the preparative separation of GAs from the sarcotesta of G. biloba and is the first report of this approach to the best of our knowledge. The aim of this work was to develop preparative separation of the major GAs from the sarcotesta of G. biloba for use in complementary pharmacological investigations and quality control.
2. MATERIALS AND METHODS 2.1. Reagents and Materials. n-Heptane, n-hexane, ethyl acetate, methanol, acetic acid, petroleum ether (60−90 °C), hydrochloric acid (HCl), and triethylamine (TEA) used for the preparation of crude extract and CCC separations were of analytical grade (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China). HPLC-grade methanol and acetic acid were purchased from the Fisher Company (Fairlawn, NJ). Water was deionized using an osmosis Milli-Q system (18.2 MΩ) (Millipore, Bedford, MA). β-CD used for the PZRCCC separation was purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Fresh G. biloba sarcotesta were obtained from ginkgo trees in the city of Jinan (Shandong, China) and identified by Dr. Jia Li (College of Pharmacy, Shandong University of Traditional Chinese Medicine). A voucher specimen (2016100701) has been deposited at the Shandong Analysis and Test Center. 2.2. Apparatus. The HSCCC equipment was a TBE-300C (Tauto Biotechnique, Shanghai, China) with three multilayer coil separation columns of 300 mL (diameter of the PTFE tube was 2.6 mm) and a 20 mL manual sample loop. The HSCCC apparatus was equipped with four other instrument modules, including a TBP-5002 constant-flow pump (Tauto Biotechnique, Shanghai, China), an 8823A-UV monitor operated at 254 nm (Beijing Emilion Technology, Beijing, China), a model 3057 portable recorder (Yokogawa, Sichuan Instrument Factory, Sichuan, China), and a DC-0506 low constant temperature bath (Tauto Biotechnique) to maintain the temperature at 25 °C. HPLC separation was performed on a Waters 600 system (Bedford, MA) consisting of a Waters 600 pump, a photodiode array detector (DAD), and an automatic sample injector with Waters Symmetry C18 column (250 × 4.6 mm, i.d. 5 μm, USA). 15841
DOI: 10.1021/acs.iecr.8b04167 Ind. Eng. Chem. Res. 2018, 57, 15840−15845
Article
Industrial & Engineering Chemistry Research
with ESI-MS data (Agilent 6120 LC/MS system, Agilent Technologies, Palo Alto, CA). The MS conditions were as follows: mass range, 100−650 m/z; capillary temperature, 300 °C; spray voltage, 4 kV; skimmer voltage, 40 V; auxiliary gas (nitrogen) flow, 6 L/min; positive ionization mode.
3. RESULTS AND DISCUSSION 3.1. Selection of the PZRCCC Solvent System. For successful PZRCC separation, the solvent system requires an appropriate partition coefficient (Kacid ≫ 1 and Kbase ≪ 1) as well as good sample solubility.26 Since GAs are low-polarity organic acids with a long hydrophobic chain, traditional PZRCCC solvent systems do not easily separate them. β-CD is commonly used in the food industry to alter the chemical or physical characteristics of components. Cyclodextrins are cyclic (α-1,4)-linked oligosaccharides containing a relatively hydrophobic central cavity, which can form clathrates, and a hydrophilic outer surface. The long hydrophobic chains of GAs can insert into the hydrophobic central cavity, significantly improving the aqueous solubility of the compounds (schematic shown in Figure 1). As shown in Table 1, four solvent systems were tested for the separation of GA clathrates, including chloroform− Table 1. KD Values of GAs in PZRCCC Separation with Different Solvent Systems KD values of compounds 1−5 solvent system CHCl3/MeOH/H2O (4:3:3, v/v)
1
2
3
4
5
Kbase Kacid Kacid Kbase
0.04 0.31 5.66 0.01
0.03 0.25 6.16 0.02
0.11 0.30 8.44 0.03
0.04 0.15 11.45 0.03
0.06 0.36 12.68 0.05
Kacid Kbase
7.37 0.07
7.68 0.09
9.39 0.10
11.91 0.14
12.85 0.18
Kacid Kbase
8.60 0.12
8.87 0.15
9.56 0.18
13.43 0.20
13.62 0.29
Figure 2. Schematic diagrams and chromatograms of recycling CCC separation procedure. Solvent system: n-heptane−ethyl acetate− methanol−acetic acid (5:4:1:1, v/v); flow-rate: 2.0 mL/min; detection wavelength: 254 nm. (A) Collection; (B) recycling; (C) separation of Fr. I; (D) separation of Fr. IV.
n-heptane/EtOAc/ MeOH/H2O (2:1:3:1, v/v) n-heptane/EtOAc/ MeOH/H2O (2:1:2:1, v/v) n-heptane/EtOAc/ MeOH/H2O (2:1:1.5:1, v/v)
sample solution was injected into the sample loop. After the sample solution had been entered into the column, the separation mode was changed to the second stage and the sixway valve was in the recycling position (Figure 2B). In this stage, target compounds were separated after the appropriate number of recycling cycles. The separation then progressed to the third stage. The six-way valve was switched to the collection position (Figure 2A), and the eluent was manually collected into tubes. The five high-purity β-CD-GA clathrates were then decomposed by addition of 0.1 M NaCl solution and heating for 15 min at 55 °C. The GAs were obtained by extraction with n-hexane. 2.8. HPLC Analysis and Identification of CCC Fractions. HPLC analyses of the crude extract, mixed standards sample, and CCC fractions were performed on Waters 600 HPLC equipment with a C18 column (Waters Symmetry, 4.6 × 250 mm, 5 μm i.d.). The mobile phase was methanol and 0.5% aqueous acetic acid (90:10, v/v) at a flowrate of 1.0 mL/min. The eluate was monitored at a wavelength of 310 nm. Identification of the purified GAs was performed by comparison with a mixture of GA standard samples coupled
methanol−water (4:3:3, v/v) and n-heptane−ethyl acetate− methanol−water at different ratios (2:1:3:1, 2:1:2:1, and 2:1:1.5:1, v/v). When chloroform−methanol−water (4:3:3, v/v) was used, the Kbase and K acid values were less than 1. This indicated that the GA clathrates would be eluted rapidly with poor separation. When the n-heptane−ethyl acetate−methanol−water solvent system was used, suitable Kbase and K acid values were obtained. In n-heptane−ethyl acetate−methanol− water (2:1:3:1, v/v), the Kacid values were 5.66−12.68 while the Kbase values were less than 0.1. When the proportion of methanol was reduced in this solvent system, the hydrophobicity of the lower phase decreased, Kacid values increased, and Kbase values decreased. In n-heptane−ethyl acetate− methanol−water (2:1:1.5:1, v/v), the Kacid values were 8.60− 13.62 while the Kbase values were 0.12−0.29. Solvent systems containing n-heptane−ethyl acetate−methanol−water at different ratios (2:1:3:1, 2:1:2:1, and 2:1:1.5:1, v/v) were then tested in the PZRCCC separation. 3.2. PZRCCC Separation. Figure 3A shows the PZRCCC separation of GAs using n-heptane−ethyl acetate−methanol− water (2:1:3:1, v/v) containing 10 mM HCl in the upper phase and 10 mM TEA in the lower phase. The sample loading was 2.0 g, and the flow-rate was 2.0 mL/min. The retention of the 15842
DOI: 10.1021/acs.iecr.8b04167 Ind. Eng. Chem. Res. 2018, 57, 15840−15845
Article
Industrial & Engineering Chemistry Research
Figure 3. Chromatogram of the optimization procedure with different ratio of solvent in PZRCCC separation. Basic solvent system: n-heptane− ethyl acetate−methanol−water with HCl in upper phase as retention acid and TEA in lower as elution alkali. Flow-rate: 2.0 mL/min; detection wavelength: 254 nm. (A) 2:1:3:1, v/v, 10 mM HCl and 10 mM TEA; (B) 2:1:2:1, v/v, 10 mM HCl and 10 mM TEA; (C) 2:1:1.5:1, v/v, 10 mM HCl and 10 mM TEA; (D) 2:1:1.5:1, v/v, 40 mM HCl and 5 mM TEA.
resolution. If the α value is close to 1, adjacent chromatographic peaks may not be completely separated, while they may be completely separated when the α value is above 1.5.27 A number of two-phase solvent systems were tested for their separative capabilities (Table 2). When ethyl acetate−n-
stationary phase was 75%. Unfortunately, the five compounds were only separated into two mixtures. Figure 3B shows the separation using n-heptane−ethyl acetate−methanol−water (2:1:2:1, v/v) containing 10 mM HCl in the upper phase and 10 mM TEA in the lower phase. The sample loading was 2.0 g, and the flow-rate was 2.0 mL/min. The retention of the stationary phase was 66.7%. The PZRCCC chromatogram was superior to that obtained with n-heptane−ethyl acetate− methanol−water (2:1:3:1, v/v). Compounds 2 and 3 were partially separated. The solvent system was then changed to nheptane−ethyl acetate−methanol−water (2:1:1.5:1, v/v) containing 10 mM HCl in the upper phase and 10 mM TEA in the lower phase (Figure 3C). Relatively good separation was achieved, providing three purified compounds (2, 3, and 5) and two mixtures. The flow rate was 2.0 mL/min, and the sample loading was 2.0 g. The retention of the stationary phase was 83.3%. Further modification to 40 mM HCl in the upper phase and 5 mM TEA in the lower phase gave satisfactory separation (Figure 3D). Five fractions were obtained, including Fr. I (compounds 1 and 2, 188.3 mg), Fr. II (compound 2, 120.3 mg), Fr. III (compound 3, 49.8 mg), Fr. IV (compounds 4 and 5, 147.3 mg), and Fr. V (compound 5, 148.9 mg). Fr. I and IV were further separated by recycling CCC. 3.3. Selection of the Recycling CCC Solvent Systems. In traditional HSCCC separation, a higher KD value might produce excessively broad peaks and extend the elution time, while a lower KD value might lead to poor peak resolution. The separation factor (α = K2/K1, K2 > K1) is a measure of
Table 2. KD Values of GAs in Recycling CCC Separation with Different Solvent Systems KD values of compounds 1−5 solvent system EtOAc/n-BuOH/H2O (4:1:5, v/v) n-hexane/EtOAc/MeOH/ H2O (5:2:5:2, v/v) CHCl3/MeOH/H2O (4:3:2, v/v) n-heptane/EtOAc/MeOH/ HOAc (5:4:1:1, v/v)
1
2
3
4
5
≫1
≫1
≫1
≫1
≫1
15.93
18.22
27.67
38.47
49.69
≪1
≪1
≪1
≪1
≪1
0.80
0.73
0.66
1.22
1.09
butanol−water (4:1:5, v/v) and n-hexane−ethyl acetate− methanol−water (5:2:5:2, v/v) were used, the target compounds were mainly distributed in the upper phase, with KD values far greater than 1. When chloroform−methanol− water (4:3:2, v/v) was used, the KD values were much lower than 1. The KD values obtained with n-heptane−ethyl acetate− methanol−acetic acid (5:4:1:1, v/v) were in the appropriate range of 0.66−1.22. However, the separation factors of the compounds in Fr. I and IV (Figure 3D) were too close to 1 15843
DOI: 10.1021/acs.iecr.8b04167 Ind. Eng. Chem. Res. 2018, 57, 15840−15845
Article
Industrial & Engineering Chemistry Research
Figure 4. HPLC chromatograms of the crude extract and the isolated GAs. (A) Crude extract, (B) mixture of standard samples, (C) GA1 (C13:0), (D) GA2 (C15:1), (E) GA3 (C17:2), (F) GA4 (C15:0), (G) GA5 (C17:1). Experimental conditions: Waters Symmetry C18 column (5 μm, 4.6 mm × 250 mm, i.d.,); mobile phase: methanol/0.5% aqueous solution of acetic acid (90:10, v/v). Flow rate: 1.0 mL/min; detection: 310 nm.
results are shown in Figure 4. Five GAs, including compound 1 (peak 1 in Figure 4A, 19.2 mg), compound 2 (peak 2 in Figure 4A, 53.9 mg), compound 3 (peak 3 in Figure 4A, 11.2 mg), compound 4 (peak 4 in Figure 4A, 16.2 mg), and compound 5 (peak 5 in Figure 4A, 57.3 mg), were detected at retention times consistent with the standard samples. The isolated compounds were identified by comparison with the retention times from a mixture of standard GA samples. The molecular weights of GA1 (C13:0, 319.2285 [M−H]−), GA2 (C15:1, 347.2585 [M−H]−), GA3 (C17:2, 345.2664 [M−H]−), GA4 (C15:0, 373.2820 [M−H]−), and GA5 (C17:1, 371.2659 [M− H]−) were also consistent with those in the literature.16,28
(1.10 and 1.12). This indicated that those peaks would be difficult to separate in a traditional one-step separation procedure. Recycling CCC separation mode was therefore used for the separation of these two mixtures. 3.4. Recycling CCC Separation. Conventional CCC separation is a one-step separation procedure. Because of the insufficient number of theoretical plates, a one-step process may be insufficient to separate target compounds having similar structures and KD values. According to basic chromatographic theory, a longer CCC column would result in a higher number of theoretical plates. Recycling CCC strategies have, therefore, been used to increase the effective column length. Since GAs have similar structures, the separation factors for Fr. I (compounds 1 and 2) and Fr. IV (compounds 4 and 5) were 1.10 and 1.12. In a single HSCCC separation run, Fr. I and IV in Figure 3D would be coeluted because of the small differences in KD values. Figure 2C shows the recycling CCC separation mode for separation of Fr. I using n-heptane−ethyl acetate−methanol−acetic acid (5:4:1:1, v/v) as the solvent system. After 11 cycles, purified compounds 1 and 2 were obtained. Fr. IV was separated in eight recycling CCC cycles (Figure 2D). After sufficient separation, the effluent collected in the tubes was switched using the six-port valve. The purities of the separated compounds were >98%, as determined by HPLC. 3.5. Identification of the Isolated Compounds. The crude extract, a mixture of standard samples, and the CCC fractions were investigated by HPLC coupled with MS. The
4. CONCLUSIONS In conclusion, this study presents a combination of β-CD clathration coupled with PZRCCC and recycling CCC to separate five major GAs from the sarcotesta of G. biloba. The β-CD clathration increases the hydrophilicity of the GAs and improves the PZRCCC separation. It was also found that recycling CCC mode could enhance the resolution of these compounds having similar structures. The results demonstrated that HSCCC could be a powerful technique for separation of GAs from G. biloba. The purified individual GAs may be used in further pharmacological and toxicological investigations. The established strategy could also be used to separate other low-polarity organic acids containing a long hydrophobic chain from natural and synthetic sources on a preparative or semipreparative scale. 15844
DOI: 10.1021/acs.iecr.8b04167 Ind. Eng. Chem. Res. 2018, 57, 15840−15845
Article
Industrial & Engineering Chemistry Research
■
(14) Beek, T. A. V.; Montoro, P.; Xie, P. S.; Van Beek, T. A. Chemical analysis and quality control of Ginkgo biloba leaves, extracts, and phytopharmaceuticals. J. Chromatogr. A 2009, 1216, 2002−2032. (15) Itokawa, H.; Totsuka, N.; Nakahara, K.; Takeya, K.; Lepoittevin, J.; Asakawa, Y. Antitumor principles from Ginkgo biloba L. Chem. Pharm. Bull. 1987, 35, 3016−3020. (16) Nagabhushana, K. S.; Ravindranath, B. Efficient medium-scale chromatographic group separation of anacardic acids from solventextracted cashew nut (Anacardiumoccidentale) shell liquid. J. Agric. Food Chem. 1995, 43, 2381−2383. (17) Van Beek, T. A.; Wintermans, M. S. Preparative isolation and dual column high-performance liquid chromatography of ginkgolic acids from Ginkgo biloba. J. Chromatogr. A 2001, 930, 109−117. (18) Li, R.; Shen, Y.; Zhang, X.; Ma, M.; Chen, B.; Beek, T. A. V. Efficient purification of ginkgolic acids from Ginkgo biloba leaves by selective adsorption on Fe3O4 magnetic nanoparticles. J. Nat. Prod. 2014, 77, 571−575. (19) Gao, Q.; Ma, R.; Chen, L.; Shi, S.; Cai, P.; Zhang, S.; Xiang, H. Y. Antioxidant profiling of vine tea (Ampelopsis grossedentata): Offline coupling heart-cutting HSCCC with HPLC-DAD-QTOF-MS/ MS. Food Chem. 2017, 225, 55−61. (20) Zhao, H.; Hu, X.; Chen, X.; Shi, S.; Jiang, X.; Liang, X.; Chen, W.; Zhang, S. Analysis and improved characterization of minor antioxidants from leaves of Malus doumeri using a combination of major constituents’ knockout with high-performance liquid chromatography- diode array detector-quadrupole time-of-flight tandem mass spectrometry. J. Chromatogr. A 2015, 1398, 57−65. (21) Liu, H.; Gong, C.; Liao, Z.; Chen, T.; Li, Y. Isolation and purification of three analogues from Clematis akebioides by molecularly imprinted Solid-Phase extraction and HSCCC. Chromatographia 2017, 80, 1651−1658. (22) Luan, G.; Wang, H.; Lv, H.; Hu, N.; Suo, Y.; Wang, X. Separation and purification of five flavone glucosides and one lignan from Caraganakorshinskii kom. by the combination of HSCCC and semi-preparative RPLC. Chromatographia 2016, 79, 823−831. (23) Chen, T.; Li, H.; Zou, D.; Liu, Y.; Chen, C.; Zhou, G.; Li, Y. Separation of three anthraquinone glycosides including two isomers by preparative high-performance liquid chromatography and highspeed countercurrent chromatography from Rheum tanguticum Maxim. ex Balf. J. Sep. Sci. 2016, 39, 3105−3112. (24) Zhang, L. J.; Xiao, W.; Liu, J. H.; Duan, W. J.; Wang, D. J.; Geng, Y. L. Use of pH-zone refining countercurrent chromatography for preparative separation of fangchinoline and tetrandrine from Stephania tetrandra. Chromatographia 2009, 69, 959−962. (25) Ito, Y.; Ma, Y. pH-zone-refining Countercurrent Chromatography. J. Chromatogr. A 1996, 753, 1−36. (26) Ito, Y. Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography. J. Chromatogr. A 2005, 1065, 145−168. (27) Wang, D.; Geng, Y.; Fang, L.; Shu, X.; Liu, J.; Wang, X.; Huang, L. An efficient combination of supercritical fluid extraction and highspeed counter-current chromatography to extract and purify (E)- and (Z)-diastereomers of α-asarone and β-asarone from Acorus tatarinowii Schott. J. Sep. Sci. 2011, 34, 3339−3341. (28) van Beek, T. A.; Wintermans, M. S. Preparative isolation and dual column high-performance liquid chromatography of ginkgolic acids from Ginkgo biloba. J. Chromatogr. A 2001, 930, 109−117.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Tel.:86-531-6860-6191. ORCID
Li Cui: 0000-0002-1901-7545 Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This work was supported by the following funding: Shandong Province Taishan Scholar Program (Lanping Guo), National Natural Science Foundation of China (21506119), Shandong Province Major Scientific and Technological Innovation Project (2017CXGC1301, 2017CXGC1308), and the Natural Science Foundation of Shandong Province (ZR2016YL006).
■
REFERENCES
(1) Boonkaew, T.; Camper, N. D. Biological activities of Ginkgo extracts. Phytomedicine 2005, 12, 318−323. (2) Mazza, M.; Capuano, A.; Bria, P.; Mazza, S. Ginkgo biloba and donepezil: a comparison in the treatment of Alzheimer’s dementia in a randomized placebo-controlled double-blind study. Eur. J. Neurol. 2006, 13, 981−985. (3) Bastianetto, S.; Ramassamy, C.; Dore, S.; Christen, Y.; Poirier, J.; Quirion, R. The Ginkgo biloba extract (EGb 761) protects hippocampal neurons against cell death induced by β-amyloid. Eur. J. Neurosci. 2000, 12, 1882−1890. (4) Grollino, M. G.; Raschellà, G.; Cordelli, E.; Villani, P.; Pieraccioli, M.; Paximadas, I.; Malandrino, S.; Bonassi, S.; Pacchierotti, F. Cytotoxicity, genotoxicity and gene expression changes elicited by exposure of human hepatic cells to Ginkgo biloba leaf extract. Food Chem. Toxicol. 2017, 109, 486−496. (5) Sun, Y.; Tang, C.; Wu, X.; Pan, Z.; Wang, L. Characterization of alkylphenol components in Ginkgo biloba Sarcotesta by thermochemolysis-gas chromatography/mass spectrometry in the presence of trimethylsulfonium hydroxide. Chromatographia 2012, 75, 387−395. (6) Yang, X. M.; Wang, Y. F.; Li, Y. Y.; Ma, H. L. Thermal stability of ginkgolic acids from Ginkgo biloba and the effects of ginkgol C17:1 on the apoptosis and migration of SMMC7721 cells. Fitoterapia 2014, 98, 66−76. (7) Wu, L.; Jiang, X. G.; Shen, Y. J.; Lu, Z. X.; Tu, G. H.; Fu, X. L.; Chen, S. X.; Cao, J. P. Efficacy of ginkgolic acids against Cryptosporidium andersoni in cell culture. Parasitol. Res. 2011, 109, 1475−1479. (8) Kubo, I.; Muroi, H.; Himejima, M.; Yamagiwa, Y.; Mera, H.; Tokushima, K.; Ohta, S.; Kamikawa, T. Structure-antibacterial activity relationships of anacardic acids. J. Agric. Food Chem. 1993, 41, 1016− 1019. (9) Lee, J. H.; Kim, Y. G.; Ryu, S. Y.; Cho, M. H.; Lee, J. Ginkgolic acids and Ginkgo biloba extract inhibit Escherichia coli O157:H7 and Staphylococcus aureus biofilm formation. Int. J. Food Microbiol. 2014, 174, 47−55. (10) Li, X.; Deng, F.; Shan, X.; Pan, J.; Yu, P.; Mao, Z. Effects of the molluscicidal agent GA-C13:0, a natural occurring ginkgolic acid, on snail mitochondria. Pestic. Biochem. Physiol. 2012, 103, 115−120. (11) Oh, J.; Hwang, I. H.; Hong, C. E.; Lyu, S. Y.; Na, M. Inhibition of fatty acid synthase by ginkgolic acids from the leaves of Ginkgo biloba and their cytotoxic activity. J. Enzyme Inhib. Med. Chem. 2013, 28, 565−568. (12) Fu, Y.; Hong, S.; Li, D.; Liu, S. Novel chemical synthesis of ginkgolic acid (13:0) and evaluation of its tyrosinase inhibitory activity. J. Agric. Food Chem. 2013, 61, 5347−5352. (13) Fukuda, I.; Ito, A.; Hirai, G.; Nishimura, S.; Kawasaki, H.; Saitoh, H.; Kimura, K.; Sodeoka, M.; Yoshida, M. Ginkgolic acid inhibits protein SUMOylation by blocking formation of the E1SUMO intermediate. Chem. Biol. 2009, 16, 133−140. 15845
DOI: 10.1021/acs.iecr.8b04167 Ind. Eng. Chem. Res. 2018, 57, 15840−15845
