CN110229205A - 酸枣仁单体成分斯皮诺素钠的制备方法和应用 - Google Patents
酸枣仁单体成分斯皮诺素钠的制备方法和应用 Download PDFInfo
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Abstract
本发明公开了酸枣仁单体成分斯皮诺素钠的制备方法和应用。将酸枣仁粉碎加入适量酸水解液消化，调节pH至中性，收集上清液，加入乙酸乙酯萃取数次，获得酸枣仁多酚粗提液；该粗提液经大孔树脂及高效液相色谱柱进行逐级分离，获得纯度大于98％的斯皮诺素钠单体成分，可作为对照品使用。该制备方法工艺简单，获得的斯皮诺素钠具有很好的水溶性，方便大规模生产。抗肿瘤活性实验显示：斯皮诺素钠能够显著抑制多种肿瘤细胞增殖，且对正常细胞无明显作用。酸枣仁单体成分斯皮诺素钠可用来制备预防和/或***疾病的功能食品和/或药品等相关产品。
Description
技术领域
本发明涉及酸枣仁药用有效成分的制备，具体属于酸枣仁单体成分斯皮诺素钠及其制备方法，以及该化合物在制备预防和/或***疾病的功能食品和/或药品等相关产品中的应用。
背景技术
酸枣仁，鼠李科植物酸枣(Ziziphus jujuba Mill.var.spinosa)的干燥成熟种子，是一种药食同源的传统中药，作为药用已有上千年的历史，功用以补肝、宁心、敛汗、生津为主，中医将其列为镇静催眠、养心安神的良药。酸枣仁主要产于山西、河北、陕西、辽宁、河南等地，其中含有多种发挥镇静催眠的活性成分，如脂肪酸、三萜类化合物、多酚类化合物、生物碱以及氨基酸等。研究显示：酸枣仁中多酚类物质的主要成分为斯皮诺素及其衍生物，该物质能够发挥镇静催眠、抗抑郁等功效。
目前关于酸枣仁中斯皮诺素的制备方法已有报道，但现有制备方法获得的斯皮诺素均难溶于水，生物利用率较低。本发明对现有制备技术进行了工艺优化改进，获得了水溶性好，纯度大于98％的斯皮诺素钠；同时通过抗肿瘤活性实验，首次发现斯皮诺素钠能够发挥显著的抗肿瘤效应，且其抗肿瘤活性显著高于斯皮诺素，而对正常细胞无明显作用。因此，本发明提供了斯皮诺素钠的制备方法及该化合物在制备预防和/或***疾病的功能食品和/或药品等相关产品中的应用。
发明内容
本发明的目的在于提供酸枣仁单体成分斯皮诺素钠的制备方法，所述制备方法工艺简单，耗时短，方便规模化生产；获得的斯皮诺素钠单体水溶性好、纯度高，可作为对照品使用；斯皮诺素钠抗肿瘤药效显著，可在制备预防和/或***疾病的功能食品和/或药品等相关产品中应用。
本发明的上述目的是通过以下技术方案实现的：
一种酸枣仁单体成分斯皮诺素钠的制备方法，包括如下步骤：
第一步：酸枣仁中多酚粗提液的制备，参照中国专利申请号CN201810038716.9公开的方法制备，具体如下：
将酸枣仁粉碎，加入酸解液水解消化，然后用NaOH溶液调pH至中性终止消化；离心，弃沉淀，在上清液中加入乙酸乙酯萃取，再离心，取下层水相溶液，除净有机溶剂，即得酸枣仁多酚粗提液，采用福林酚比色法测定多酚含量；
第二步：将上述获得的多酚粗提液经大孔树脂柱静态吸附，用超纯水和30％丙酮分别洗脱大孔树脂各3次；再用50％丙酮洗脱，并收集该洗脱液，于60℃下旋转蒸发体积至1/4以下，真空干燥成粉末即为酸枣仁活性多酚组分ZSSP；
所述的大孔树脂为SP207大孔树脂；静态吸附时间为：12h-16h；
第三步：取第二步获得的ZSSP粉末溶解在40％的甲醇中，采用高效液相色谱柱进行精细分离，收集出峰时间为22.444的峰5馏分，于60℃下旋转蒸发，真空冷冻干燥，即获得高纯度的斯皮诺素钠单体成分。
所述的高效液相色谱柱分离的色谱条件为：色谱柱Phenomenex Hydro-RP C18(250mm×10mm，4μm)，流动相为：40％甲醇溶液；流速1mL/min，检测波长330nm。
取适量的斯皮诺素钠溶解于超纯水中，采用MTT法检测其抗肿瘤活性，结果显示：斯皮诺素钠可显著抑制多种肿瘤细胞的增殖，诱导肿瘤细胞凋亡。
与现有技术相比，本发明的有益效果：本发明从酸枣仁中获得了易溶于水的斯皮诺素钠单体成分，提取工艺简单，易于规模化生产；本发明获得的水溶性斯皮诺素钠单体纯度大于98％，可作为对照品使用；本发明首次发现斯皮诺素钠可显著抑制多种肿瘤细胞的增殖，诱导肿瘤细胞凋亡，可在制备预防和/或***疾病的功能食品和/或药品等相关产品中应用。
附图说明：
图1酸枣仁活性多酚组分(ZSSP)的HPLC分离图谱峰；
图2斯皮诺素钠及斯皮诺素的液相色谱图；
图3斯皮诺素钠的一级质谱及二级质谱图；
图4斯皮诺素钠的分子结构图；
图5斯皮诺素钠及斯皮诺素对肿瘤细胞增殖的影响；
图6斯皮诺素钠对正常细胞增殖的影响；
图7斯皮诺素钠对肿瘤细胞凋亡的影响。
具体实施方式
实施例1：酸枣仁中斯皮诺素钠的制备方法
(1)酸枣仁多酚粗提液的制备
1)将酸枣仁粉碎，取酸枣仁粉末20g，按重量体积比(W/V＝1g:10mL)加入预先配置好的酸解液(浓硫酸和甲醇的体积比为1:9)200mL，用摇床在37℃下水解消化2h。然后加入适量10mol/L NaOH使消化溶液pH至中性终止消化；消化完成后，11,000rpm离心10min，弃沉淀，在上清液中加入等体积的乙酸乙酯萃取，后11,000rpm离心10min，溶液分为三层，取下层水相，重复萃取3次；将上述水相组分在60℃旋转蒸发，除净有机溶剂，得到酸枣仁多酚粗提液；
2)用福林酚比色法测定酸枣仁多酚粗提液中多酚的含量。以没食子酸为标准品，配制浓度梯度分别为：0，0.02，0.06，0.1，0.15，0.2，0.3，0.4，0.5，0.6mg/mL的标准液。加入100μL不同浓度的标准液或多酚样品，400μL去离子水到试管中，再加入100μL福林酚试剂混匀静置反应6min，随后各加入1mL 7％Na2CO3混匀静置90min，于760nm波长处测定吸光度。以吸光值为横坐标，没食子酸量(mg)为纵坐标做标准曲线，通过标准曲线计算出酸枣仁多酚粗提液中多酚的含量约为508.25mg。
(2)酸枣仁多酚粗提液中斯皮诺素钠的分离纯化
1)将上述获得的酸枣仁多酚粗提液上样于湿重约为30g的SP207大孔吸附树脂柱，静态吸附12h-16h，用超纯水和30％丙酮分别洗脱大孔树脂各3次；再用50％丙酮溶液洗脱，并收集该洗脱馏分，旋转蒸发有机溶剂，真空冷冻干燥成粉末，即为酸枣仁活性多酚组分，命名为ZSSP，于4℃保存备用；
2)称取ZSSP粉末，充分溶解于40％的甲醇溶液中，配制成浓度为5mg/ml的ZSSP甲醇溶液，用1ml针管吸取该溶液并通过0.22μm孔径的滤器滤于安捷伦液相小瓶中备用；
3)将Phenomenex Hydro-RP C18(250mm×10mm，4μm)柱按正确流速装于安捷伦1260infinityⅡ高效液相色谱仪上；用100％的甲醇和40％的甲醇以1ml/min的流速分别平衡色谱柱30min；
4)取步骤2)中准备好的ZSSP溶液100μl上样于上述3)平衡好的色谱柱中，在330nm的波长下用40％的甲醇溶液等度洗脱ZSSP，得到不同保留时间的六个峰(见图1)，收集出峰时间为22.444的峰5馏分，用质谱技术结合斯皮诺素标品鉴定峰5馏分为斯皮诺素钠(见图3和图4)，且其纯度为98.225％(见图2)。
制备大量样品：取步骤2)中备好的ZSSP溶液5ml上样于已平衡好的制备型色谱柱Phenomenex Hydro-RP C18(250mm×21.2mm，4μm)，流速10mL/min在岛津制备液相色谱仪上用上述4)同样的方法，收集峰5馏分；旋转蒸发有机溶剂，真空冷冻干燥成粉末。结果表明：20g酸枣仁原料中提取的斯皮诺素钠单体含量为43.2mg。
实施例2：酸枣仁斯皮诺素钠对肿瘤细胞增殖的影响
取对数生长期的结肠癌细胞株HCT116、***细胞Hela、乳腺癌细胞MCF-7、肝癌细胞HepG2和正常结肠上皮细胞株FHC，将细胞制成悬液后，通过细胞计数，调整细胞密度为每孔6000个/100μL，接种到96孔板中，在37℃、5％CO2的培养箱培养过夜后，弃掉旧培养基，加入含不同浓度斯皮诺素钠的培养液，其终浓度分别为50、100、150、200、250μg/mL，设5个平行孔，继续孵育48h；弃去旧培养液，PBS洗2次，每孔加入20μL 5.0mg/mL MTT，继续孵育4h。弃去板中培养液，每孔加入150μL的DMSO溶解活细胞产生的甲瓒结晶，置摇床上振荡10min，在波长570nm处的酶标仪上测定各孔吸光值。
用上述同样的方法测定不同浓度(100、200、300、400、500μg/mL)斯皮诺素的抗肿瘤活性。
本发明获得的酸枣仁斯皮诺素钠，经上述体外抗肿瘤活性检测，结果显示：斯皮诺素钠可显著抑制肿瘤细胞HCT116、Hela、MCF-7和HepG2细胞的增殖(见图5)，而对正常细胞FHC无明显作用(见图6)；且斯皮诺素钠对肿瘤细胞的抑制能力显著高于斯皮诺素的(见图5和表1)。
表1：斯皮诺素和斯皮诺素钠对不同肿瘤细胞系的IC50值
实施例3：酸枣仁斯皮诺素钠对肿瘤细胞凋亡的影响
取对数生长期的HCT116和Hela细胞，用0.25％胰酶消化，按1×105个/每个皿的细胞密度接种于6孔细胞培养板中，放在37℃，5％CO2的培养箱中培养过夜，待细胞贴壁后，取出培养皿，换新鲜培养基，加入不同浓度的斯皮诺素钠溶液，同时设空白对照，每个样品设3个重复，在相同条件下继续孵育48h。取出处理后的细胞，用0.25％胰酶消化贴壁细胞，1000rpm离心5min，倒掉上清液，加入PBS洗2次；用200μL AnnexinV/Binding buffer的混合液悬浮各样品中的细胞，4℃避光反应30min；上机测试前5min，向每个样品中加入300μL的PI/Binding buffer混合液。将上述双染后的细胞用流式细胞仪检测，区分正常细胞、早期凋亡、晚期凋亡和坏死细胞，统计其凋亡百分率。注：正常细胞标记为AnnexinV-/PI-；早期凋亡细胞标记为AnnexinV+/PI-；晚期凋亡细胞标记为：AnnexinV+/PI+；坏死细胞标记为AnnexinV-/PI+。
流式细胞仪检测结果显示：斯皮诺素钠能够显著诱导***细胞Hela和结肠癌细胞HCT116发生凋亡(见图7)。
Claims (7)
1.一种酸枣仁单体成分斯皮诺素钠的制备方法，其特征在于，包括如下步骤：
第一步：将酸枣仁粉碎，加入酸解液水解消化，然后用NaOH溶液调pH至中性终止消化；离心，弃沉淀，在上清液中加入乙酸乙酯萃取，再离心，取下层水相溶液，除净有机溶剂，即得酸枣仁多酚粗提液，采用福林酚比色法测定多酚含量；
第二步：将上述获得的多酚粗提液经大孔树脂柱静态吸附，用超纯水和30％丙酮分别洗脱大孔树脂各3次；再用50％丙酮洗脱，并收集该洗脱液，于60℃下旋转蒸发体积至1/4以下，真空干燥成粉末即为酸枣仁活性多酚组分ZSSP；
第三步：取第二步获得的ZSSP粉末溶解在40％的甲醇中，采用高效液相色谱柱进行精细分离，收集出峰时间为22.444的峰5馏分，于60℃下旋转蒸发，真空冷冻干燥，即获得高纯度的斯皮诺素钠单体成分。
2.如权利要求1所述的酸枣仁单体成分斯皮诺素钠的制备方法，其特征在于，第二步中所述的大孔树脂为SP207大孔树脂，静态吸附时间为12h-16h。
3.如权利要求1所述的酸枣仁单体成分斯皮诺素钠的制备方法，其特征在于，第三步中所述的高效液相色谱柱分离的色谱条件：色谱柱Phenomenex Hydro-RP C18(250mm×10mm，4μm)，流动相为：40％甲醇溶液；流速1mL/min，检测波长330nm。
4.如权利要求1-3所述的任一方法制备的具有抗肿瘤活性的酸枣仁斯皮诺素钠。
5.如权利要求4所述的酸枣仁斯皮诺素钠在制备预防和/或***药物中的应用。
6.如权利要求4所述的酸枣仁斯皮诺素钠在制备预防和/或***的功能食品中的应用。
7.如权利要求4所述的酸枣仁斯皮诺素钠作为对照品使用。
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