CN111233757B - 一种夜交藤中极性化合物的纯化制备方法 - Google Patents

一种夜交藤中极性化合物的纯化制备方法 Download PDF

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CN111233757B
CN111233757B CN201811432508.3A CN201811432508A CN111233757B CN 111233757 B CN111233757 B CN 111233757B CN 201811432508 A CN201811432508 A CN 201811432508A CN 111233757 B CN111233757 B CN 111233757B
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梁鑫淼
刘典
金红利
刘艳芳
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Dalian Institute of Chemical Physics of CAS
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Abstract

本发明提供了一种夜交藤提取物中极性化合物的纯化制备方法。采用高正交性的反相/亲水二维制备液相色谱，实现夜交藤中极性化合物的分离纯化。第一维采用极性共聚反相XAqua C18色谱柱，对夜交藤的极性组分进行制备，降低样品复杂性。选取三个代表性馏分，采用两性离子键合的亲水色谱柱Click XIon对其进行第二维制备。该方法采用线性梯度的洗脱方式，流动相组成为乙腈、甲醇和水，甲酸作为添加剂，无缓冲盐添加，便于样品制备后处理。该方法解决了极性化合物在常规色谱填料上的弱保留问题，可以实现夜交藤中极性化合物的高效制备，为其他中药、生物样品以及药物制剂中极性化合物的分离纯化提供了良好的技术方案。

Description

一种夜交藤中极性化合物的纯化制备方法
技术领域
本发明属于分析化学领域，涉及一种夜交藤中极性化合物的纯化制备方法，具体地说是一种通过极性共聚反相色谱结合两性离子的亲水色谱的二维制备液相色谱方法，实现夜交藤中极性化合物的高效分离纯化。
背景技术
中药水煎液中含大量极性成分，包括极性生物碱、极性糖苷、有机酸以及多酚类等化合物。这些极性成分在中药中具有多种重要的生理活性，包括镇痛、抗菌、抗病毒等。目前，在新药发现过程中，天然极性成分越来越多的作为先导化合物被发现并发挥着非常重要的作用。(Niko,S.R.,et al.,The Last Decade of Antinociceptive Alkaloids:Structure,Synthesis,Mechanism of Action and Prospect.Current Topics inMedicinal Chemistry,2013.13(17):p.2134-2170；Teplova,V.V.,et al.,NaturalPolyphenols:Biological Activity,Pharmacological Potential,Means of MetabolicEngineering (Review).Applied Biochemistry and Microbiology,2018.54(3):p.221-237；Leeson,P.D.and B.Springthorpe,The influence of drug-like concepts ondecision-making in medicinal chemistry.Nat Rev Drug Discov,2007.6(11):p.881-890；Leeson,P.D.and S.A.St-Gallay,The influence of the 'organizational factor'on compound quality in drug discovery.Nat Rev Drug Discov,2011.10(10):p.749-765.)
夜交藤为植物何首乌的藤茎，是一种在临床上广泛使用以治疗睡眠障碍和糖尿病的中药。研究发现，夜交藤中含有芪类、酚酸、黄酮和蒽醌类等化合物，但关于其中极性成分的研究却鲜有报道。(Chen,Y.-L.,et al.,Prescription patterns of Chinese herbalproducts for patients with sleep disorder and major depressive disorder inTaiwan.Journal of Ethnopharmacology,2015.171:p.307-316；Feng,S.,et al.,Hypoglycemic Activities of Commonly-Used Traditional Chinese Herbs.TheAmerican Journal of Chinese Medicine,2013.41(04):p.849-864；Wang,G.-y.,et al.,Rapid Characterization of the major chemical constituents from PolygoniMultiflori Caulis by liquid chromatography tandem mass spectrometry andcomparative analysis with Polygoni Multiflori Radix.Journal of SeparationScience,2017.40(10):p.2107-2116.)。在我们的研究中发现，夜交藤的极性组分具有多巴胺受体-2激动活性。因此，开展夜交藤中极性成分的分离纯化工作对全面认识夜交藤药理活性具有非常重要的作用。
目前关于复杂样品中极性成分的分离纯化仍然面临非常大的挑战。传统的中、低压色谱柱，如凝胶柱、硅胶柱等无法实现极性成分的分离。高效逆流色谱分辨率较低，无法实现微量成分的制备，也难以获得纯度较高的单体化合物。(Ito,Y.,pH-zone-refiningcounter-current chromatography:Origin,mechanism,procedure andapplications.Journal of Chromatography A,2013.1271(1):p.71-85；Li,Y.,et al.,Two-stage fractionation of polar alkaloids from Rhizoma coptidis bycountercurrent chromatography considering the strategy of reactiveextraction.Journal of Chromatography A,2015.1378(Supplement C):p.58-64；Ma,F.,et al.,Preparative separation and purification of two highly polar alkaloidsderived from Semen Strychni extracted with dichloromethane by high-speedcountercurrent chromatography.Journal of Separation Science,2016.39(19):p.3709-3715；)。制备型高效液相色谱作为一种高效的分离纯化方法被广泛应用于中药等复杂样品，但常用的反相碳十八柱对极性化合物的几乎没有保留能力，且一维色谱的分离制备能力十分有限，无法适应复杂样品的纯化。因此需要筛选合适的色谱填料，对极性化合物的提供合适的保留，实现其高效分离纯化。
发明内容
针对上述问题，本发明的目的是解决极性化合物在常规色谱填料上的弱保留问题，实现夜交藤中极性化合物的高效制备
具体的技术方案为：
本发明涉及一种夜交藤中极性化合物的纯化制备方法，具体是一种通过极性共聚反相色谱结合两性离子的亲水色谱的二维制备液相色谱方法，流动相由水(A)、甲醇或乙腈(B)组成，采用甲酸作为流动相添加剂，无缓冲盐添加，实现夜交藤中极性化合物的高效分离纯化。
其中所述的反相色谱柱为极性共聚的C18色谱柱XAqua C18；亲水色谱柱为两性离子修饰的Click XIon色谱柱。色谱操作参数如下：色谱柱内径为4.6-50mm；样品浓度为1mg/mL-1g/mL；进样量为1μL-10mL；流速为0.7-80mL/min；柱温为25-40℃。
操作步骤为：
1)夜交藤极性组分先经第一维反相高效液相色谱分离，色谱柱为极性共聚的碳十八柱，洗脱方式为线性梯度、台阶梯度，收集一维制备的馏分；
2)将一维反相制备的馏分经过第二维亲水色谱进行分离，色谱柱两性离子键合的亲水色谱柱，选择权利要求1所述的流动相对馏分进行第二维制备，获得极性化合物单体。
所述夜交藤极性组分的制备方法为：称取0.5公斤～100公斤的夜交藤药材粉末，用5～30倍量的50％～95％乙醇溶液在50-100℃加热回流提取1-5次，每次回流提取的时间为1-3小时，合并提取液抽滤，用旋转蒸发仪40～80℃条件下减压回收乙醇，得到固体粗提物。将固体粗提物用水溶解后，采用氯仿、二氯甲烷、乙酸乙酯等有机溶剂进行萃取，除去弱极性成分，收集水层，40～80℃条件下减压浓缩得到固体，采用50％～95％乙醇对固体复溶，将复溶样品上固相萃取柱(30μm～60μm)进行前处理，用3-5倍柱体积0％-10％的0.1％～5％甲酸-水和甲醇洗脱，收集洗脱液，40～80℃减压浓缩，得到固体样品；之后，采用碳十八柱对该固体样品进行制备，采用5％-90％甲醇-水进行线性梯度或台阶梯度洗脱，添加剂甲酸的比例为0.05％～1％，流速为50-100mL/min，检测器波长选择为190-400nm；采用体积浓度为5％-40％二甲基亚砜-水混合溶剂对该固体样品进行溶解，得到浓度为100-1000mg/mL的样品，进样量为1-5mL，收集在“死时间”流出的馏分，40～80℃减压浓缩，得到固体样品，为夜交藤的极性成分。
步骤1)中夜交藤极性组分经第一维反相高效液相色谱分离，梯度洗脱参数为：梯度时间范围为30-100min，流动相如权利要求1中所述，采用线性梯度或台阶梯度进行洗脱，其中流动相(B)体积浓度变化为由0％～15％到60％～100％，添加剂甲酸的比例为0.05％～1％，流速为50-100mL/min，检测器波长选择为190-400nm；采用纯水对极性组分进行溶解，得到浓度为50-100mg/mL的样品，进样量为5-20mL，根据色谱峰进行馏分收集，将每个馏分减压浓缩至干后备用，以进行第二维亲水色谱分离。
步骤2)中选取一维反相制备馏分中的代表性馏分，采用亲水色谱进行第二维制备，得到极性化合物单体；洗脱参数为：梯度时间范围为20-50min，流动相为权利要求1中所述流动相，流动相(B)体积浓度变化为由100％～90％到40％～60％，添加剂甲酸的比例为0.05％～1％，流速为10-30mL/min，检测波长为190-400nm，柱温为30℃分别收集每个馏分中的色谱峰，并进行浓缩，经核磁实验确定，获得极性化合物单体。
所述化合物P1为烟酸，分子式为C6H5NO2，分子量为123；化合物P2为1-C-guaiacylglycerol 3-O-β-D-glucopyranoiside，分子式为C16H24O10，分子量为398；化合物P3为腺嘌呤，分子式为C5H5N5，分子量为135。化合物结构信息如下：
Figure BDA0001882949180000041
化合物P1为烟酸，分子式为C6H5NO2，分子量为123；化合物P2为1-C-邻甲氧苯基丙三醇-3-O-β-D-glucopyranoiside，分子式为C16H24O10，分子量为398；化合物P3为腺嘌呤，分子式为C5H5N5，分子量为135。
本发明的有益效果
该方法解决了极性化合物在常规色谱填料上的弱保留问题，可以实现夜交藤中极性化合物的高效制备，为其他中药、生物样品以及药物制剂中极性化合物的分离纯化提供了良好的技术方案。
附图说明
图1为夜交藤极性组分在XAqua C18色谱柱上的第一维制备结果。
图2为馏分6、7和8在Click XIon色谱柱上的第二维制备结果。
具体实施方式
现结合实例，对本发明做进一步说明。实例仅限于说明本发明，而非对本发明的限定。
实施例1：夜交藤极性成分的制备
称取5kg的夜交藤药材粉末，用10倍量的70％乙醇溶液在80℃加热回流提取3次，每次回流提取的时间为2小时，合并提取液抽滤，用旋转蒸发仪50℃条件下减压回收乙醇，得到固体粗提物。将固体粗提物用水溶解后，采用乙酸乙酯进行分液，除去弱极性成分，将水层旋蒸浓缩拿到固体，采用70％乙醇对固体复溶，将复溶样品上XAqua C18SPE柱进行固相萃取，用3倍柱体积5％的0.1％甲酸-水和甲醇洗脱，收集洗脱液，50℃减压浓缩，得固体样品。之后，采用SunFire C18色谱柱对该固体样品进行制备，采用5％-90％甲醇-水进行线性梯度洗脱，添加剂甲酸的比例为0.1％，流速为80mL/min，检测器波长选择为254nm；采用体积浓度为30％二甲基亚砜-水混合溶剂对该固体样品进行溶解，得到浓度为600mg/mL的样品，进样量为2mL，收集在“死时间”流出的馏分，50℃减压浓缩，得到固体样品，为夜交藤的极性成分。
实施例2：夜交藤极性组分的一维制备
制备得到的夜交藤极性组分先经第一维反相高效液相色谱分离，色谱条件：色谱柱为XAqua C18柱；流动相组成为：0.1％甲酸-水(v/v)(A)和甲醇(B)；洗脱梯度为0-20min，体积浓度0％B；20-30min，体积浓度0-70％B；30-40min，体积浓度70％B；流速为80mL/min；检测波长为254nm；样品溶液浓度为51mg/mL，进样体积为10mL；按色谱峰进行馏分接取，共收集11个馏分，每个馏分50℃减压浓缩至干后备用；
实施例3：化合物P1的制备
选取一维制备馏分中的馏分6进行第二维的亲水高效液相色谱制备。色谱条件：色谱柱为Click XIon色谱柱；流动相组成为：0.1％甲酸-水(v/v)(A)和0.1％甲酸-乙腈(v/v)(B)；洗脱梯度为0-5min，体积浓度90％B；5-35min，体积浓度90-45％B；进样体积为400μL；流速为19mL/min；检测波长为254nm；柱温为30℃；收集色谱峰，50℃减压浓缩至干，得到化合物P1。高效液相色谱检测纯度大于95％，经理化测定，数据如下：P1，ESI-MS m/z:124.04[M+H]+.13C-NMR:δC(151MHz,D2O)168.68(C-α),145.04(C-4),143.64(C-6),143.06(C-2),135.28(C-3),126.59(C-5)；1H-NMR:δH(600MHz,D2O)9.02(1H,s,H-2),8.76(1H,dt,H-5),8.73(1H,dd,H-6),7.95(1H,dd,H-4).
实施例4：化合物P2的制备
选取一维制备馏分中的馏分7进行第二维的亲水高效液相色谱制备。色谱条件：色谱柱为Click XIon色谱柱；流动相组成为：0.1％甲酸-水(v/v)(A)和0.1％甲酸-乙腈(v/v)(B)；洗脱梯度为0-5min，体积浓度90％B；5-35min，体积浓度90-45％B；进样体积为500μL；流速为19mL/min；检测波长为254nm；柱温为30℃；收集色谱峰，50℃减压浓缩至干，得到化合物P2。高效液相色谱检测纯度大于95％，经理化测定，数据如下：P2，ESI-MS m/z:399.13[M+Na]+；13C-NMR:δC(151MHz,D2O)148.52(C-3),144.98(C-4),135.90(C-1),120.05(C-6),115.68(C-5),111.36(C-2),100.31(C-1,Glc),76.02(C-3,Glc),75.41(C-7),74.50(C-8),73.46(C-2,Glc),72.75(C-4,Glc),69.18(C-9),62.53(C-5,Glc),60.33(C-6,Glc),55.70(C-3-OCH3)；1H-NMR:δH(600MHz,D2O)7.04(1H,d,H-5),6.99(1H,d,H-2),6.86(1H,dd,H-6),5.01(1H,m,H-7),4.48(1H,d,H-1),3.79(1H,d,H-7),3.76(3H,s,H-3-OCH3),3.65(1H,d,H-),3.63(1H,d,H-),3.61(1H,d,H-),3.47(4H,m,H-2,3,4,Glc,H-8),3.39(1H,d,H-5,Glc)。
实施例5：化合物P3的制备
选取一维制备馏分中的馏分8进行第二维的亲水高效液相色谱制备。色谱条件：色谱柱为Click XIon色谱柱；流动相组成为：0.1％甲酸-水(v/v)(A)和0.1％甲酸-乙腈(v/v)(B)；洗脱梯度为0-5min，体积浓度90％B；5-35min，体积浓度90-45％B；进样体积为500μL；流速为19mL/min；检测波长为254nm；柱温为30℃；收集色谱峰，50℃减压浓缩至干，得到化合物P3。高效液相色谱检测纯度大于95％，经理化测定，数据如下：P3，ESI-MS m/z:136.05[M+H]+；13C-NMR:δC(151MHz,D2O)152.12(C-6),149.51(C-4),147.92(C-2),142.34(C-8),115.76(C-5)；1H-NMR:δH(600MHz,D2O)8.31(1H,s,H-7-NH),8.13(1H,s,H-2),8.09(1H,s,H-8).HMBC correlations:H-8(δH,8.09)with C-5(δC,115.76)and C-4(δC,149.51),H-2(δH,8.13)with C-4(δC,149.51)and C-6(δC,152.12)；HSQCcorrelations:H-2(δH,8.13)withC-2(δC,147.92)；H-8(δH,8.09)with C-8(δC,142.34)。

Claims (3)

1.一种夜交藤中极性化合物的纯化制备方法，其特征在于：采用反相/亲水二维制备色谱从夜交藤极性组分中高效分离纯化极性化合物，第一维反相色谱采用极性共聚的碳十八柱XAqua C18，第二维亲水色谱采用两性离子键合的亲水色谱柱Click XIon，流动相由水A、甲醇B或乙腈B组成，采用甲酸作为流动相添加剂，添加剂甲酸的体积浓度为0.05%~1%；
操作步骤为：
1）夜交藤极性组分先经第一维反相高效液相色谱分离，色谱柱为极性共聚的碳十八柱，洗脱方式为线性梯度、台阶梯度，收集一维制备的馏分；
2）将一维反相制备的馏分经过第二维亲水色谱进行分离，色谱柱两性离子键合的亲水色谱柱，选择流动相对馏分进行第二维制备，获得极性化合物单体；
步骤1）中夜交藤极性组分经第一维反相高效液相色谱分离，梯度洗脱参数为：梯度时间范围为30-100min，流动相如上所述，采用线性梯度或台阶梯度进行洗脱，其中流动相B体积浓度变化为由0%~15% 到60%~100%，添加剂甲酸的比例为0.05%~1%，流速为50-100mL/min，检测器波长选择为190-400nm；采用纯水对极性组分进行溶解，得到浓度为50-100mg/mL的样品，进样量为5-20mL，根据色谱峰进行馏分收集，将每个馏分减压浓缩至干后备用，以进行第二维亲水色谱分离；
步骤2）中选取一维反相制备馏分中的代表性馏分，采用亲水色谱进行第二维制备，得到极性化合物单体；洗脱参数为：梯度时间范围为20-50min，流动相如上所述，流动相B体积浓度变化为由100%~90% 到40%~60%，添加剂甲酸的比例为0.05%~1%，流速为10-30 mL/min，检测波长为190-400nm，柱温为30℃分别收集每个馏分中的色谱峰，并进行浓缩，经核磁实验确定，获得极性化合物单体。
2.按照权利要求1所述的分离纯化方法，其特征在于：色谱操作参数如下：色谱柱内径为4.6-50 mm。
3.按照权利要求1所述的分离纯化方法，其特征在于：所述夜交藤极性组分的制备方法为：称取0.5公斤~100公斤的夜交藤药材粉末，用5~30倍量的50%~95%乙醇溶液在50-100℃加热回流提取1-5次，每次回流提取的时间为1-3小时，合并提取液抽滤，用旋转蒸发仪40~80℃条件下减压回收乙醇，得到固体粗提物；将固体粗提物用水溶解后，采用氯仿、二氯甲烷、乙酸乙酯进行萃取，除去弱极性成分，收集水层，40~80℃条件下减压浓缩得到固体，采用50%~95%乙醇对固体复溶，将复溶样品上固相萃取柱：30μm~60μm，进行前处理，用3-5倍柱体积0%-10%的0.1%~5%甲酸-水和甲醇洗脱，收集洗脱液，40~80℃减压浓缩，得到固体样品；之后，采用碳十八柱对该固体样品进行制备，采用5%-90%甲醇-水进行线性梯度或台阶梯度洗脱，添加剂甲酸的比例为0.05%~1%，流速为50-100mL/min，检测器波长选择为190-400nm；采用体积浓度为5%-40%二甲基亚砜-水混合溶剂对该固体样品进行溶解，得到浓度为100-1000mg/mL的样品，进样量为1-5mL，收集在“死时间”流出的馏分，40~80℃减压浓缩，得到固体样品，为夜交藤的极性成分。
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