Preparative separation of high-purity troxerutin and related substances from mother liquor of troxerutin by silica gel column chromatography and semi-preparative liquid chromatography    
Shaojing Liu1,2,3, Bei Qin1,2*, Hongfang Han1, Li Li1,2, Lili Yu1,2, Xiaojing Xu1,2       
1. College of pharmacy, Xi’an Medical University, Shaanxi 710021, China
2. Institute of Medicine, Xi’an Medical University, Shaanxi 710021, China
3. College of Chemical Engineering, Department of harmaceutical Engineering, Northwest University, Shaanxi 710069, China 
 
 
Abstract: Troxerutin (TRO) is a mixture of semi-synthetic flavonoids prepared by hydroxyethylation of rutin, and it is commonly used for the treatment of cerebrovascular diseases. The main active ingredient is trishydroxyethyl rutin. The mother liquor of TROcontains a lot of TRO and other derivatives of hydroxyethylated rutin. In order to make full use of the mother liquor of TRO, an efficient method was developed for recovering high-purity TRO from mother liquor of TRO by combining silica gel column chromatography with semi-preparative liquid chromatography. In the silica gel column chromatographic separation, the ratio of silica gel to sample and eluent composition were investigated to obtain optimum separation effect. The results showed that when the ratio of silica gel to sample was 50, and acetoneethyl acetatewaterglacial acetic acid (10:10:3:1, v/v/v/v) was used as the eluent, the separation effect of TRO and adjacent impurities was good. Moreover, 150 g ofTRO with a purity of 80% could be obtained from 1 kg of mother liquor of TRO by the silica gel column chromatographic separation, and the results were consistent with the quality standard of TRO raw material. Subsequently, the semi-preparative HPLC was performed, and 100 g TRO with a purity of up to 98% (w/w) was obtained. Meanwhile, tetrahydroxyethylrutin and tetrahydroxyethylquercetin with purity greater than 98% were obtained. This work proposed the separation and preparation of TRO with high-purity from the production waste of TRO for the first time, which had certain environmental benefits and economic benefits.                       
Keywords: Mother liquor of troxerutin; Troxerutin; Silica-gel column chromatography; Semi-preparative HPLC; NMR 
CLC number: R917                Document code: A                 Article ID: 10031057(2020)748707
 
 
1. Introduction
Troxerutin (TRO) is a commonly used medicine for the treatment of cerebrovascular diseases, also known as vitamin P4, and its trade name is venoruton. It is chemically designated as 2-[3,4-bis (2-hydroxyethoxy)phenyl]-3-[[6-O-(6-deoxy-α-L-mannopyra-nosyl)-β-D-glucopyranosyl]oxy]-5-hydroxy-7-(2-hydroxyethoxy)-4H-1-benzopyran-4-one (tris (hydroxyethyl)rutin)[1,2] (Fig. 1). TRO is well-known due to its diverse range of biologicalactivities, including anti-oxidant, anti-thrombotic, anti-inflammatory, anti-fibrinolytic, odema-protective and radioprotective properties. As an oral capillary preservatory drug, TRO is used for thrombophlebitis, capillary hemorrhage and in the treatment of chronic venous insufficiency (CVI) diseases. In addition to oral application, TRO as an antioxidant is used for topical treatment or as a protective agent on the vein wall to prevent the development of varicose veins[3–6]. The effectiveness and safety of TRO in various pathological conditions have been broadly discussed, and the good results are shown in elderly patients and pregnant women[7–10].
 
 
 
Figure 1. The molecular structures of rutin and TRO. 
 
TRO is a mixture of semi-synthetic flavonoids prepared by hydroxyethylation of rutin, and the main active ingredient is trishydroxyethyl rutin. Compared with rutin, TRO is soluble in water[11]. Moreover, it is easy to be absorbed by human body, and the curative effect is reliable. Because there are four dissociated hydroxyl groups in rutin, when the main component trishydroxyethyl rutin is formed during the production process of TRO, related substances, such as monohydroxyethyl rutin, dihydroxyethyl rutin, trishydroxyethyl rutin isomer and tetrahydroxyethyl rutin, are also formed[12,13]. The proportion of each component in hydroxyethyl rutin derivatives is related to the reaction conditions, and it is difficult to separate and purify each derivative due to their similar properties[14,15]. Based on the above analysis, the mother liquid of TRO also contains a large amount of TRO and other hydroxyethyl rutin derivatives. The contents of TRO and tetrahydroxyethyl rutin in the mother liquid of TRO are about 20% and 10%, respectively, as determined by HPLC. The direct discharge of mother liquid not only causes loss of active ingredients, but also leads to serious environmental pollution. Therefore, the recovery of TRO from the mother liquor is of great significance, and there is no relevant research report in the literature.
In the present study, we aimed to combine silica gel chromatography with semi-preparative liquid chromatography for the separation and purification of TRO from mother liquor of TRO. The results showed that 150 g of TRO with a purity of 80% could be obtained from 1 kg of mother liquor of TRO by the silica gel column chromatographic separation. Trihydroxyethylrutin, tetrahydroxyethylrutin and tetrahydroxyethylquercetin with the purity greater than 98% were obtained by combining silica gel column chromatography with semi-preparative liquid chromatography from the production waste of TRO for the first time. The study laid the foundation for re-use of mother liquor of TRO and reduced environmental pollution, which had certain environmental benefits and economic benefits.
2. Experimental
2.1. Reagents and materials
Acetonitrile and methanol used for HPLC analyses were of chromatographic grade and purchased from Sigma-Aldrich Co., Ltd. (Shanghai, China). Acetone, ethyl acetate, glacial acetic acid and methanol were of analytical grade and obtained from Guangdong Guanghua Sci-Tech Co., Ltd. (Guangdong, China). Distilled water was supplied by Hangzhou Wahaha Group Co., Ltd. Mother liquor of TRO was provided by Yabao Pharmaceutical Group Co., Ltd. (Shanxi, China). The silica gel (200–300 mesh) and thin-layer chromatography (TLC) plates (10 cm×20 cm) were purchased from Qingdao Ocean Chemical Company (Qingdao, China).
2.2. Apparatus
The semi-preparative HPLC system consisted of two Shimadzu LC-6AD pumps, a Shimadzu SPD-M10Avp UV detector (Shimadzu, Kyoto, Japan), a 1.0-mL sampleloop, and an ZORAX SB-C18 column (9.4 mm×250 mm,5 μm, Agilent Co. Ltd., USA). The analytical HPLC system used in this study consisted of an Aglient 1260 System, an Aglient DAD detector, a 20-μL sample loop, and an Aglient LC Solution Workstation (Agilent Co., Ltd., USA). The column applied in this work was an Aglient TC-C18 column (250 mm×4.6 mm, 5 μm, Agilent Co., Ltd., USA). The nuclear magnetic resonance (NMR) spectrometer used here was a Varian VNMRS 600 NMR system (Agilent Co., Ltd., USA).
2.3. Silica gel column chromatography
Mother liquid powder of TRO was dissolved in methanolwater (1:1, v/v) and applied to a silica gel chromatographic column (30 cm×3.5 cm). Isocratic solvent system consisting of acetoneethylacetatewaterglacial acetic acid (10:10:3:1, v/v/v/v) was employed in chromatographic process. The eluent was collected by fraction size of 10 mL, and each fraction was analyzed for its TRO content using TLC. The fractions containingTRO were pooled together, and the solvent was evaporated. Finally, the dried residue was obtained.
2.4. TLCanalysis
TLC was performed on silica gel plates (Qingdao Ocean Chemical Company, China). The plates were developed in acetoneethylacetatewaterglacial acetic acid (10:10:3:1, v/v/v/v) at room temperature, and then the plate was dried and dipped in a 5% sulfuric acid solution. Finally, the plates were heated with a hair dryer until the separated spots appeared.
2.5. Semi-preparative HPLC
The partially purified fractions obtained from the silica gel column chromatography were evaporated to dryness under reduced pressure and dissolved in the mobile phase. Subsequently, the sample solution was purified using semi-preparative HPLC (Shimadzu, Kyoto, Japan).
The separation was performed with an Agilent ZORAX SB-C18 column (9.4 mm×250 mm, 5 μm, Agilent Co., Ltd., USA) at room temperature. The mobile phase composed of acetonitrilewater (20:80, v/v) was isocratically eluted at a flow rate of 1.0 mL/min. The wavelength was set at 254 nm. The volume of injection sample was 250 μL. The effluent of fraction peaks was collected according to the elution profile.
The effluents with retention times of 12 to 14 min,24 to 30 min and 34 to 36 min were collected separately. The organic solvent was concentrated under reduced pressure, and then lyophilization was carried out to remove water. Finally, pale yellow amorphous powders a, b and c were obtained, and the chromatogram was shown in Figure 2.  
 
 
Figure 2. Semi-preparative HPLC chromatogram.   
 
2.6. HPLC analysis and NMR identification of sample
Each purified fraction obtained by silica gel column chromatography and semi-preparative HPLC was analyzed by HPLC. The analyses were performed on an Aglient TC-C18 column (250 mm×4.6 mm, 5 μm, Agilent Co., Ltd., USA) at the column temperature at 30 ºC. The mobile phase, composed of acetonitrile0.1%glacial acetic acid (20:80, v/v), was isocratically eluted at a flow rate of 0.5 mL/min,and the effluent was monitored at 254 nm by an Aglient DAD detector. The purity of compounds a, b and c was determined with the area normalization method on HPLC with a purity of 98.94%, 98.14%and98.34%,respectively. The chromatogram was shown in Figure 3. 
 
 
Figure 3. Analytical HPLC chromatogram. Identification of semi-preparative HPLC peak fractions was carried out by 1H NMR and 13C NMR.   
3. Results and discussion
3.1. Optimization of silica gel column chromatographic conditions
In order to separate and prepare TRO from mother liquor of TRO, silica-gel column chromatography was first conducted. In the separation process, separation was achieved by utilizing different adsorption capacities of the components in the mixture to the solid adsorbent. In this work, two variables, ratio of silica gel to sample and composition of eluent, were investigated to obtain optimum separation efficiency. In general, the separation efficiency will be improved by increasing the ratio of adsorbent to sample. However, excessive adsorbent causes waste of resources. The ratio of silica gel to sample was determined to be 50 after inspection.
Binary, trivalent, and multi-component solvent systems are often used for elution in silica gel column chromatography. A large proportion of solvent plays a role in dissolving the sample and its separation, and a small proportion of the solvent plays a role in improving Rf. On the basis of the pre-experiment, the separation effect of three different solvent ratios (acetoneethyl acetatewaterglacial acetic acid, 10:9:4:1, 10:10:3:1, 10:11:2:1, v/v/v/v) were compared. Based on the separationeffect of the main component and the adjacent impurities, acetoneacetic acid ethyl esterwaterglacial acetic acid (10:10:3:1, v/v/v/v) was determined as eluent.
Mother liquor of TRO was processed according to the above-mentioned conditions. The eluent was collected by fraction size of 10 mL, and a total of 86 bottles (3BV) of eluent were collected. The results indicated that no TRO was detected in the first 30 bottles of eluent (the first column volume), and the amount of TRO in 31 to 70 bottles of eluent (2nd column volume to 3.4th column volume) was sharply increased and then significantly decreased from 71st bottle (3.4th column volume). Meanwhile, starting from the 71st bottle (3.4th column volume), impurities with less polarity were significantly increased. Therefore, samples of 31 to 70 bottles (2nd column volume to 3.4th column volume) were combined for further separation.
3.2. Optimization of HPLC separation conditions
As a semi-synthetic flavonoid compound, the phenolic hydroxyl group in the molecular structure of TRO makes the compound acidic. In order to achieve a good resolution for the target compound and reduce the tailingof the chromatographic peak, glacial acetic acid, formic acid or phosphate buffer solution are often added to determine flavonoids. The effects of 0.1% glacial aceticacid, 0.1% formic acid, and 0.1 mol/L phosphate buffer solution on the chromatographic peaks were examined. The optimum HPLC mobile phase was found to be acetonitrilewater (0.1% CH3COOH) in an isocratic solvent system. The best separations of samples were achieved using the mobile phase of acetonitrilewater (0.1% CH3COOH, 20:80, v/v) at a flow rate of 0.5 mL/min, an wavelength of 254 nm and a column temperature of 30 ºC.
3.3. Optimization of prep-HPLC separation conditions
In order to obtain pure target compound, the partially purified fractions obtained from the silica gel column chromatography were subjected to semi-preparative HPLC for further purification. Selection of semi-preparative HPLC conditions was based on the analytical HPLC conditions. In order toobtain good chromatographic peak shape and resolution, acetic acid was added to the mobilephase in analytical HPLC.However, it is difficult to remove glacial acetic acid under the conditions of decompression due to its higher boiling point. In addition, glacial acetic acid may cause hydrolysisof the sample. Therefore, in the semi-preparative HPLC, 0.1% aqueous glacial acetic acid in the mobile phase was replaced with pure water.
Different injection volumes (100, 250, 500 and 1000 μL)and different flow rates (1, 2 and 3 mL/min) were investigated. The results indicated that the best separationconditions were achieved using acetonitrilewater (20:80, v/v) at a flow rate of 1 mL/min and a wavelength of 254 nm, and the optimum injection volume was 250 μL.
3.4. Structural identification 
The chemical structure of compound b was identifiedby1H NMR and 13C NMR. 1H NMR (D2O) δ: 7.48 (1H, 2′-H), 7.23 (1H, 6′-H), 6.68 (1H, 5′-H), 6.05(1H, 8-H; 1H, 6-H), 0.90, 0.91 (3H, 12″-H); 4.97, 4.97 (1H, 1″-H),4.40 (1H, 7″-H), 3.08–4.02 (-CH, -CH2 on glycosyl group).13C NMR (D2O) δ: 180.177 (C=O), 167.177 (C-7), 162.372 (C-9), 159.028 (C-5), 158.484 (C-3′), 153.142 (C-4′), 149.127 (C-2), 136.934 (C-3), 125.458 (C-1′), 124.510 (C-6′), 116.133 (C-5′), 114.481 (C-2′), 107.605(C-10), 105.132 (C-8), 103.672 (C-6), 100.846(C-7″), 96.344 (C-1″), 78.460 (3′, 4′-OCH2-), 77.875 (7-OCH2-), 76.863 (C-10″), 74.485 (C-2″), 73.023 (C-8″), 72.746 (C-5″), 72.691 (C-3″), 72.619 (C-9″), 72.564 (C-4″), 71.513 (C-11″), 70.454 (C-6″), 62.788, 62.670 (-CH2OH), 19.402 (C-12″). Based on the above spectrum data and with reference to the related literature[16,17], it was confirmed that the sample b was trishydroxyethyl rutin.
The chemical structure of compound a was identified by1H NMR and 13C NMR. 1H NMR (D2O)  δ:7.67 (1H, 2′-H), 7.43 (1H, 6′-H), 6.90, 6.89 (1H, 5′-H), 6.37 (1H, 6-H), 6.33 (1H, 8-H), 0.95 (3H, 12″-H), 5.05 (1H, 1″-H), 4.49 (1H, 7″-H), 3.16–4.16 (-CH, -CH2 on glycosyl group).13C NMR (D2O) δ: 177.482 (C=O), 166.427 (C-7), 162.048 (C-9), 160.822 (C-5), 157.157 (C-3′), 152.921 (C-4′), 149.183 (C-2), 138.957 (C-3), 125.395 (C-1′), 124.826 (C-6′), 116.386 (C-5′), 114.655 (C-2′), 110.830 (C-10), 105.298 (C-8), 103.759 (C-6), 100.106 (C-7″), 96.921 (C-1″), 78.641 (3′, 4′-OCH2-), 78.072 (5, 7-OCH2-), 77.021 (C-10″), 74.445 (C-2″), 73.868 (C-8″), 72.951 (C-5″), 72.793 (C-3″), 72.651 (C-9″), 72.580 (C-4″), 71.521 (C-11″), 70.912 (C-6″), 62.844, 62.780, 62.741, 62.670 (-CH2OH), 19.307 (C-12″).By comparing the hydrogen spectrum and carbon spectrum of sample a and sample b, it was confirmed that the sample a was tetrahydroxyethyl rutin.
The chemical structure of compound c was identified by1H NMR and 13C NMR. 1H NMR (DMSO) δ: 12.62 (1H, 3-OH), 7.84, 7.84, 7.82 (1H, 2′-H), 7.82, 7.81, 7.80 (1H, 6′-H), 7.17, 7.16 (1H, 5′-H), 6.80, 6.80 (1H, 6-H); 6.38, 6.38 (1H, 8-H). 13C NMR (DMSO) δ: 177.970 (C=O), 164.466 (C-7), 160.672 (C-90), 156.136 (C-5), 155.132 (C-3′), 150.936 (C-4′), 147.757 (C-2), 137.209 (C-30), 122.225 (C-1′), 122.099 (C-6′), 113.658 (C-5′), 112.813 (C-2′), 104.965 (C-10), 98.019 (C-8), 92.682 (C-6), 73.631 (3′-OCH2-), 70.503 (4′-OCH2-), 70.343 (5-OCH2-), 70.153 (7-OCH2-), 60.061, 59.951, 59.406, 59.263, 59.097 (-CH2OH). By comparing the hydrogen spectrum and carbon spectrum of sample c and sample a, it was confirmed that the sample c was tetrahydroxyethylquercetin.
Acknowledgements
This work was supported by Shaanxi science and technology hall project (Grant No. 2018JM7060); Provincial Key Discipline Construction Project of pharmacy of Xi’an Medical University (Grant No. 2016YXXK08).
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硅胶柱层析和半制备液相色谱法从曲克芦丁母液中分离制备高纯度曲克芦丁及有关物质
刘少静1,2,3, 秦蓓1,2*, 韩红芳1, 李立1,2, 余丽丽1,2, 徐小静1,2
1. 西安医学院 药学院, 陕西 西安 710021
2. 西安医学院 药物研究所, 陕西 西安 710021
3. 西北大学 化工学院制药工程专业,  陕西 西安 710069      
摘要: 曲克芦丁(Troxerutin, TRO)是由芦丁羟乙基化制备的半合成黄酮类化合物的混合物,常用于治疗脑血管疾病,主要活性成分为三羟乙基芦丁(亦称曲克芦丁)。曲克芦丁生产母液中含有大量的三羟乙基芦丁及其它羟乙基芦丁衍生物,该研究采用硅胶柱层析和半制备液相色谱相结合的方法,TRO母液中回收制备高纯度的TRO。在硅胶柱色谱分离中:考察了硅胶与样品比例及洗脱液组成,以获得最佳的分离效果。结果表明,当硅胶与样品的比为50:1,丙酮乙酸乙酯冰醋酸(10:10:3:1, v/v/v/v)为洗脱剂时, TRO与相邻杂质的分离效果较好。用硅胶柱层析法, 1 kg TRO母液中制备得150 g (纯度80%) TRO。进一步采用半制备HPLC纯化,制备得纯度高达98% (w/w)TRO 100 g,同时得到纯度大于98%的四羟乙基芦丁和四羟乙基槲皮素。本文首次提出从TRO生产废液中分离制备高纯度TRO,具有一定的环境效益和经济效益。 
关键词: 曲克芦丁母液; 曲克芦丁; 硅胶柱层析; 半制备液相色谱法; NMR
 
   
 
Received: 2020-01-02; Revised: 2020-02-15; Accepted: 2020-04-12.
Foundation items: Shaanxi science and technology hall project (Grant No. 2018JM7060); Provincial Key Discipline Construction Project of pharmacy of Xi’an Medical University (Grant No. 2016YXXK08).
*Corresponding author. Tel.: +86-29-86177536, E-mail: liushaojingbmgw@163.com          
 
 
        
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