CN110563779A - 一种枣核提取物及其提取分离方法和应用 - Google Patents
一种枣核提取物及其提取分离方法和应用 Download PDFInfo
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Abstract
本发明公开了一种枣核提取物及其提取分离方法和应用。一种枣核提取物包括19种化合物,分别鉴定为:3,3'‑二甲氧基鞣花酸‑4‑O‑β‑D‑葡萄糖、5‑羟甲基糠酸、儿茶素、无刺枣苄苷Ⅱ、柚皮素、山奈酚、(E)‑3,3'‑二甲氧基‑4,4'‑二羟基二苯乙烯、异香草醛、棕榈油烯酸、3,3'‑O‑二甲基鞣花酸、胡萝卜苷、原儿茶酸、没食子酸、二氢查尔酮‑4'‑β‑D‑吡喃葡萄糖苷、3‑氧代齐墩果酸、东莨菪内酯、对羟基间甲氧基苯甲酸、α,β‑D‑吡喃葡萄糖、脱氢双没食子酸;枣核提取物可应用于制备治疗阿尔茨海默症和糖尿病药物以及美白化妆产品。
Description
技术领域
本发明涉及天然药物提取分离和生物医学领域,更具体的涉及一种枣核提取物及其提取分离方法和应用。
背景技术
枣为鼠李科枣属物,具有很高的食用价值和药用价值,在中国已有四千年的栽培历史,在中医学被认可为一味重要的中药。随着社会日益发展,人们经济水平的提高,对生活质量要求也有所提高,保健意识越来越强,这使得人们对具有药食同源作用的大枣展开更深入的研究并有了更深层次的认识,促使红枣系列产品获得人们的喜爱,提高了在国内外市场的需求量。
大枣是滋补保健佳品且其药理活性广泛,其中保肝抗癌功效尤为明显。其果实、叶片、果核、树皮以及根都可以入药。文献中对枣属植物果肉的化学成分研究较多,对枣核化学成分的研究报道甚少,调研发现大枣经过加工后大量枣核遭废弃填埋处理或作为燃料,造成资源浪费和环境污染。基于史料记载,枣核具有生津止渴、解毒敛疮、润肠道和促进消化的功效,本发明对枣核化学成分进行分析研究,并作定量分析和生物活性筛选实验,对充分利用枣核功能成分,帮助解决枣质资源开发的问题,同时对中药资源开发具有重要现实意义。
发明内容
为解决上述问题,本发明提供了一种枣核提取物及其提取分离方法和应用。
本发明的第一个目的是提供一种枣核提取物,包括以下组分:
3,3'-二甲氧基鞣花酸-4-O-β-D-葡萄糖(1)、5-羟甲基糠酸(2)、儿茶素(3)、无刺枣苄苷Ⅱ(4)、柚皮素(5)、山奈酚(6)、(E)-3,3'-二甲氧基-4,4'-二羟基二苯乙烯(7)、异香草醛(8)、棕榈油烯酸(9)、3,3'-O-二甲基鞣花酸(10)、胡萝卜苷(11)、原儿茶酸(12)、没食子酸(13)、二氢查尔酮-4'-β-D-吡喃葡萄糖苷(14)、3-氧代齐墩果酸(15)、东莨菪内酯(16)、对羟基间甲氧基苯甲酸(17)、α,β-D-吡喃葡萄糖(18)、脱氢双没食子酸(19),其结构如下:
本发明第二个目的是提供上述枣核提取物的提取分离方法,包括以下步骤:
步骤1:将枣核粉碎后用体积浓度为90%的乙醇-水溶液提取四次,每次浸泡48小时,合并提取液并减压浓缩,得到浸膏;
步骤2:加水分散浸膏,得浸膏溶液,用石油醚进行萃取,取出上层石油醚萃取液,对下层浸膏溶液反复萃取三次直到上层石油醚萃取液接近无色,合并多次石油醚萃取液,得石油醚萃取层浸膏,减压浓缩回收石油醚溶剂,石油醚萃取后得到的下层浸膏溶液按同样方法用饱和正丁醇作为萃取液依次萃取三次,得正丁醇萃取层浸膏,正丁醇萃取后得到的下层浸膏溶液按同样方法用乙酸乙酯作为萃取液依次萃取三次,得乙酸乙酯萃取层浸膏;
步骤3:将正丁醇萃取层浸膏采用湿法上样过反相聚酰胺柱,分别用纯水、体积浓度为20%乙醇-水溶液、40%乙醇-水溶液、60%乙醇-水溶液、80%乙醇-水溶液、纯乙醇梯度洗脱,结合TLC检测分析将同一梯度洗脱剂洗脱后的正丁醇萃取层浸膏溶液合并,最终将正丁醇萃取层浸膏按照洗脱剂体积浓度划分为D.1~D.6六段,各段洗脱剂分别为纯水、体积浓度为20%乙醇-水溶液、40%乙醇-水溶液、60%乙醇-水溶液、80%乙醇-水溶液、纯乙醇;将D.1~D.6六段分别通过柱层析,并洗脱,制备化合物(1)~(6);
步骤4:将乙酸乙酯萃取层浸膏采用干法上样过100-200目硅胶柱,用CH2Cl2-CH3OH溶液进行梯度洗脱,结合TLC检测分析将同一梯度洗脱剂洗脱后的乙酸乙酯萃取层浸膏溶液合并,将乙酸乙酯萃取层浸膏按照CH2Cl2-CH3OH溶液体积浓度比例范围划分为极性不同的Fr.1~Fr.8八段馏分,各段的CH2Cl2-CH3OH溶液体积浓度比例范围分别为130:1-120:1、110:1-100:1、95:1-75:1、70:1-60:1、55:1-40:1、35:1-25:1、20:1-10:1、8:1-3:1;将Fr.1~Fr.8八段分别通过柱层析,并洗脱,制备化合物(7)~(19)。
优选地,所述化合物(1)~(6)具体是通过以下步骤分离制得:将D.2过聚酰胺反相柱,分别用纯水、体积浓度为10%甲醇-水溶液、20%甲醇-水溶液、40%甲醇-水溶液梯度洗脱,用甲醇冲柱,结合TLC检测将同一梯度洗脱剂洗脱后的D.2合并,将D.2按照洗脱剂体积浓度划分为三段A2-1、A2-2、A2-3,各段洗脱剂分别为10%甲醇-水溶液、20%甲醇-水溶液、40%甲醇-水溶液,用Sephadex LH-20柱分离A2-2并反复用体积浓度为10%甲醇-水溶液分离最终得到化合物(1);
将D.3过聚酰胺反相柱,分别用纯水、体积浓度为10%甲醇-水溶液、20%甲醇-水溶液、40%甲醇-水溶液、50%乙醇-水溶液、60%乙醇-水溶液梯度洗脱,结合TLC检测和HPLC图谱分析将同一梯度洗脱剂洗脱后的D.3合并,将D.3按照洗脱剂体积浓度分为五段A3-1、A3-2、A3-3、A3-4、A3-5,各段洗脱剂分别为10%甲醇-水溶液、20%甲醇-水溶液、40%甲醇-水溶液、50%乙醇-水溶液、60%乙醇-水溶液,A3-1经CHP柱层析分离得到化合物(2)和化合物(3);A3-5经硅胶柱层析,用CH2Cl2-CH3OH溶液进行梯度洗脱,CH2Cl2与CH3OH体积浓度比例分别为18:1、15:1、12:1、8:1、4:1,结合TLC检测和HPLC图谱确定得到化合物(4);
将D.4过硅胶柱,用CH2Cl2-CH3OH溶液进行梯度洗脱,CH2Cl2与CH3OH体积浓度比例分别为15:1、12:1、10:1、8:1、5:1,结合TLC检测将同一梯度洗脱剂洗脱后的D.4合并,将D.4按照洗脱剂体积浓度分为四段A4-1、A4-2、A4-3、A4-4,各段CH2Cl2与CH3OH体积浓度比例分别为15:1-12:1、10:1、8:1、5:1,A4-4过LH-20柱,用体积浓度为20%甲醇-水溶液洗脱,得到化合物(5);
将D.5过硅胶柱,依次用CH2Cl2-CH3OH溶液进行梯度洗脱,CH2Cl2与CH3OH体积浓度比例分别为20:1、15:1、13:1、10:1、5:1,结合TLC检测合并相同馏分,经比例为10:1的CH2Cl2-CH3OH溶液洗脱后过Sephadex LH-20反相柱得到化合物(6);
优选地,所述化合物(7)~(19)具体是通过以下步骤分离制得:将Fr.2干法上样过硅胶柱层析,用体积浓度比例范围为40:1-5:1的石油醚-乙酸乙酯溶液梯度洗脱结合TLC检测和HPLC图谱合并相同馏分后分为三段B2-1、B2-2、B2-3,各段的石油醚-乙酸乙酯溶液体积浓度比例范围分别为70:1-40:1、35:1-20:1、15:1-5:1,将B2-2过硅胶柱层析,用石油醚-乙酸乙酯溶液进行梯度洗脱,石油醚与乙酸乙酯体积浓度比例分别为20:1、15:1、13:1、10:1、5:1,经比例为15:1的石油醚-乙酸乙酯溶液洗脱后结合TLC检测和HPLC图谱检测得到化合物(7);将B2-3过硅胶柱层析,用石油醚-乙酸乙酯溶液进行梯度洗脱,石油醚与乙酸乙酯体积浓度比例分别为18:1、15:1、12:1、10:1、8:1、5:1,经比例为8:1的石油醚-乙酸乙酯溶液洗脱后结合HPLC图谱检测得到化合物(8);
将Fr.3干法上样过硅胶柱层析,用体积浓度范围为70:1-5:1的CH2Cl2-CH3OH溶液梯度洗脱结合TLC跟踪监测和HPLC谱图分析划分为四段B3-1、B3-2、B3-3、B3-4,各段CH2Cl2-CH3OH溶液体积浓度范围分别为70:1-60:1、55:1-40:1、35:1-20:1、15:1-1:1,将B3-1干法上样硅胶柱层析,石油醚-乙酸乙酯溶液按25:1、20:1、18:1、15:1、12:1、8:1、4:1、1:1的体积浓度梯度洗脱,经比例为12:1石油醚-乙酸乙酯溶液洗脱后反复硅胶柱层析得到化合物(11);将B3-2过Sephadex LH-20柱凝胶分离,分别用纯水、10%甲醇-水溶液、20%甲醇-水溶液、30%甲醇-水溶液梯度洗脱,经30%甲醇-水溶液和10%甲醇-水溶液洗脱后结合TLC跟踪检测合并相同馏分分别得到化合物(9)、(10);
将Fr.4干法上样过硅胶柱,分别选择体积浓度比例为80:1、70:1、65:1、60:1、55:1、50:1、45:1、40:1、35:1、30:1、25:1、20:1、10:1、5:1的CH2Cl2-CH3OH溶液梯度洗脱,甲醇冲柱,结合TLC监测和HPLC图谱解析将Fr.4划分为三段B4-1、B4-2、B4-3,各段的CH2Cl2-CH3OH溶液体积浓度比例范围分别为60:1-50:1、35:1-25:1、20:1-10:1,将B4-1经CHP、LH-20柱反复分离得到化合物(14);将B4-2过CHP层析柱,分别用20%甲醇-水溶液、40%甲醇-水溶液、60%甲醇-水溶液、80%甲醇-水溶液梯度洗脱,甲醇冲柱,经80%甲醇-水溶液和40%甲醇-水溶液洗脱后结合TLC跟踪检测和HPLC图谱分析分别得到两个纯化合物(12)、(13);将B4-3进行HPLC分析得到化合物(15);
将Fr.5干法上样过硅胶柱,用体积浓度为65:1、60:1、55:1、50:1、45:1、40:1、35:1、30:1、25:1、20:1、10:1、5:1的CH2Cl2-CH3OH溶液梯度洗脱,结合TLC跟踪检测合并相似组分划分为五段B5-1、B5-2、B5-3、B5-4、B5-5,各段的CH2Cl2-CH3OH溶液体积浓度范围分别为65:1-55:1、50:1-40:1、35:1-30:1、25:1-20:1、10:1-5:1,对B5-3中出现无色结晶状物质,洗涤后得到化合物(16);对B5-4反复硅胶柱层析,用体积浓度比例范围为8:1-3:1的石油醚-乙酸乙酯溶液进行梯度洗脱,得到化合物(17);对B5-5反复硅胶柱层析,用体积浓度比例范围为8:1-3:1的CH2Cl2-CH3OH溶液进行梯度洗脱,得到化合物(18);
将Fr.6与Fr.7段合并,干法上样硅胶柱层析,依次用体积浓度比例为45:1、40:1、35:1、30:1、25:1、20:1、10:1、5:1、1:1的CH2Cl2-CH3OH溶液梯度洗脱,甲醇冲柱,根据TLC薄层分析结果划分为五段B6-1、B6-2、B6-3、B6-4、B6-5,各段的CH2Cl2-CH3OH溶液体积浓度比例范围分别为45:1-40:1、35:1-30:1、25:1-20:1、10:1、5:1-1:1,结合HPLC分析对B6-2经体积浓度比例为25:1的CH2Cl2-CH3OH溶液反复洗脱后得到化合物(19)。
本发明的第三个目的是提供上述枣核提取物在制备治疗阿尔茨海默症药物方面的应用。
本发明的第四个目的是提供上述枣核提取物在制备治疗糖尿病药物方面的应用。
本发明的第五个目的是提供上述枣核提取物在制备化妆品美白产品方面的应用。
本发明的有益效果为:
本发明中对枣核中的化学成分进行提取、分离、定量分析,并且研究枣核分离得到的单体化合物在生物医药及化妆品领域的应用,充分利用枣核功能成分,帮助解决枣质资源开发的问题,同时对中药资源开发以及化妆品行业都具有重要现实意义。
附图说明
图1为本发明实施例提供的枣核90%乙醇提取液中部分化合物在高效液相色谱图中的出峰位置图;
图2为本发明实施例中正丁醇萃取液的高效液相色谱图;
图3为本发明实施例中乙酸乙酯萃取液的高效液相色谱图;
图4为本发明实施例中各标准品在高效液相色谱图中的出峰位置图;
图5为本发明实施例中各标准品的紫外光谱图。
具体实施方式
下面结合本发明实施例中的附图,对本发明技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
结合图1~3,图1分别与图2-3进行对照,表明枣核90%乙醇提取液正丁醇萃取层中含有极性偏大的目标活性化合物以及枣核90%乙醇提取液乙酸乙酯萃取层中含有极性中等的目标活性化合物,化合物种类较多,对分离起到指导作用,本实施例从枣核中提取分离到19种化合物的方法,包括以下步骤:
步骤1:将18kg粉碎好的枣核置于渗漉桶中,用60L体积浓度为90%乙醇-水溶液浸泡48h提取4次,过滤提取液,合并滤液并浓缩,得到浸膏1.1kg;
步骤2:加水分散浸膏,得浸膏溶液,用石油醚进行萃取,取出上层石油醚萃取液,对下层浸膏溶液反复萃取三次直到上层石油醚萃取液接近无色,合并多次石油醚萃取液,得石油醚萃取层浸膏38g,减压浓缩回收石油醚溶剂,石油醚萃取后得到的下层浸膏溶液按同样方法用饱和正丁醇作为萃取液依次萃取三次,得正丁醇萃取层浸膏89g,正丁醇萃取后得到的下层浸膏溶液按同样方法用乙酸乙酯作为萃取液依次萃取三次,得乙酸乙酯萃取层浸膏45g;
步骤3:将100-200目聚酰胺用甲醇浸泡24小时活化后倒入直径为15cm的柱层析玻璃仪器中,达到合适高度后用水反复冲洗直至洗脱液无甲醇味,将正丁醇萃取层浸膏加入少量水稀释并搅拌均匀,使粘稠度适中,加入聚酰胺填料中使其匀速沉降展开并吸附3小时。在样品层上方铺设脱脂棉后分别用纯水、体积浓度为20%乙醇-水溶液、40%乙醇-水溶液、60%乙醇-水溶液、80%乙醇-水溶液、纯乙醇梯度洗脱,结合薄层色谱TLC跟踪检测结果,同一梯度洗脱液合并浓缩后将正丁醇萃取层浸膏划分为六段D.1~D.6;
D.1未分离;
将D.2过聚酰胺反相柱,分别用纯水、体积浓度为10%甲醇-水溶液、20%甲醇-水溶液、40%甲醇-水溶液梯度洗脱,用甲醇冲柱,结合TLC检测将同一梯度洗脱剂洗脱后的D.2合并,将D.2按照洗脱剂体积浓度划分为三段A2-1、A2-2、A2-3,各段洗脱剂分别为10%甲醇-水溶液、20%甲醇-水溶液、40%甲醇-水溶液,用Sephadex LH-20柱分离A2-2并反复用体积浓度为10%甲醇-水溶液分离最终得到18mg化合物(1);
将D.3过聚酰胺反相柱,分别用纯水、体积浓度为10%甲醇-水溶液、20%甲醇-水溶液、40%甲醇-水溶液、50%乙醇-水溶液、60%乙醇-水溶液梯度洗脱,结合TLC检测和HPLC图谱分析将同一梯度洗脱剂洗脱后的D.3合并,将D.3按照洗脱剂体积浓度分为五段A3-1、A3-2、A3-3、A3-4、A3-5,各段洗脱剂分别为10%甲醇-水溶液、20%甲醇-水溶液、40%甲醇-水溶液、50%乙醇-水溶液、60%乙醇-水溶液,A3-1经CHP柱层析分离得到28mg化合物(2)、15mg化合物(3);A3-5经硅胶柱层析,用CH2Cl2-CH3OH溶液进行梯度洗脱,CH2Cl2与CH3OH体积浓度比例分别为18:1、15:1、12:1、8:1、4:1,洗脱的馏分经TLC检测呈一清晰圆点,合并浓缩后结合HPLC图谱显示为单一峰形可确定得到20mg化合物(4);
将D.4过硅胶柱,用CH2Cl2-CH3OH溶液进行梯度洗脱,CH2Cl2与CH3OH体积浓度分别为15:1、12:1、10:1、8:1、5:1,结合TLC检测将同一梯度洗脱剂洗脱后的D.4合并,将D.4按照洗脱剂体积浓度分为四段A4-1、A4-2、A4-3、A4-4,各段CH2Cl2与CH3OH体积浓度比例分别为15:1-12:1、10:1、8:1、5:1,A4-4过LH-20柱,用体积浓度为20%甲醇-水溶液洗脱,得到10mg化合物(5);
将D.5过硅胶柱,依次用CH2Cl2-CH3OH溶液进行梯度洗脱,CH2Cl2与CH3OH体积浓度分别为20:1、15:1、13:1、10:1、5:1,结合TLC检测合并相同馏分,经比例为10:1的CH2Cl2-CH3OH溶液洗脱后过Sephadex LH-20反相柱得到12mg化合物(6);
步骤4:称量乙酸乙酯萃取层浸膏共45g,加入适量甲醇将样品完全溶解后,取一定量的100-200目硅胶拌样,充分混合均匀后平铺在托盘中放置通风橱里风干,待溶剂完全挥发后将样品倒入研钵中研磨至细腻均匀的粉末,过80-100目筛后收集样品搁置待用。取适量100-200目硅胶柱层析填料放入大烧杯中并加入二氯甲烷搅拌均匀后放置在超声波清洗器中除去气泡后装柱,将研磨后的样品均匀的平铺在填料上层,并在样品层上方垫加棉花以防冲散样品层影响分离效果。用体积浓度比例范围为130:1-3:1的CH2Cl2-CH3OH溶液作为洗脱剂进行梯度洗脱,最后用甲醇冲柱,结合薄层硅胶色谱TLC检测最终将该层划分为极性由小到大的8段,并命名为Fr.1、Fr.2、Fr.3、Fr.4、Fr.5、Fr.6、Fr.7、Fr.8,各段的CH2Cl2-CH3OH溶液体积浓度比例范围分别为130:1-120:1、110:1-100:1、95:1-75:1、70:1-60:1、55:1-40:1、35:1-25:1、20:1-10:1、8:1-3:1;
Fr.1未分离;
将Fr.2采用硅胶柱层析干法上样,用体积浓度比例范围为40:1-5:1的石油醚-乙酸乙酯溶液梯度洗脱并结合TLC跟踪监测和HPLC谱图分析大致划分为B2-1、B2-2、B2-3三段,各段的石油醚-乙酸乙酯溶液体积浓度比例范围分别为70:1-40:1、35:1-20:1、15:1-5:1。对B2-2进行硅胶柱层析,石油醚-乙酸乙酯溶液按20:1、15:1、12:1、10:1、5:1、3:1的体积浓度比例进行配制,分离过程中发现比例为15:1的石油醚-乙酸乙酯溶液洗脱馏分呈粉红色,经旋转蒸发仪浓缩后用TLC和HPLC检测得到17mg纯化合物(7);将B2-3同样过硅胶柱,石油醚-乙酸乙酯溶液按18:1、15:1、12:1、10:1、8:1、5:1的体积浓度比例梯度洗脱,其中比例为8:1的石油醚-乙酸乙酯溶液洗脱馏分浓缩后经HPLC分析含有一个主要化合物,经硅胶柱反复洗脱后得到9mg化合物(8);
将Fr.3采用硅胶柱层析干法上样,用体积浓度比例范围为70:1-5:1的CH2Cl2-CH3OH溶液梯度洗脱,TLC跟踪监测和HPLC谱图分析大致划分为四段B3-1、B3-2、B3-3、B3-4,各段的CH2Cl2-CH3OH溶液体积浓度比例范围分别为70:1-60:1、55:1-40:1、35:1-20:1、15:1-1:1。将B3-1样品溶解完全后用100-200目硅胶拌样,采用干法上样进行硅胶柱层析,石油醚-乙酸乙酯溶液按25:1、20:1、18:1、15:1、12:1、8:1、4:1、1:1的体积浓度比例梯度洗脱,经比例为12:1石油醚-乙酸乙酯溶液洗脱后反复硅胶柱层析得到18mg化合物(11);将B3-2过Sephadex LH-20柱凝胶分离,分别用纯水、10%甲醇-水溶液、20%甲醇-水溶液、30%甲醇-水溶液梯度洗脱,在分离过程中结合薄层色谱TLC分析跟踪检测,经30%甲醇-水溶液和10%甲醇-水溶液洗脱后合并相同馏分并用旋转蒸发仪将溶剂蒸干,最终分离得到只有两个点以及少量杂质的样品,用填料CHP-20P继续分离,结合TLC跟踪检测合并相同馏分分别得到11mg化合物(9)、30mg化合物(10);
将Fr.4采用干法上样过硅胶柱,分别选择体积浓度比例为80:1、70:1、65:1、60:1、55:1、50:1、45:1、40:1、35:1、30:1、25:1、20:1、10:1、5:1的CH2Cl2-CH3OH溶液梯度洗脱后甲醇冲柱,结合TLC薄层色谱跟踪监测合并相似的馏分并进行HPLC图谱解析,根据分析结果再分离并分别命名为B4-1、B4-2、B4-3,各段的CH2Cl2-CH3OH溶液体积浓度比例范围分别为60:1-50:1、35:1-25:1、20:1-10:1,其余阶段的馏分蒸干后移至样品瓶中搁置待用。将B4-1经过CHP、LH-20柱反复分离最后得到12mg化合物(14);将B4-2过CHP层析柱,分别用20%甲醇-水溶液、40%甲醇-水溶液、60%甲醇-水溶液、80%甲醇-水溶液梯度洗脱,甲醇冲柱,经80%甲醇-水溶液和40%甲醇-水溶液洗脱后经TLC跟踪检测并结合HPLC图谱分析,得到纯化合物25mg(12)和13mg(13);B4-3中有结晶状物质出现,过滤晶体进行HPLC分析得到20mg化合物(15);
将Fr.5采用干法上样过硅胶柱,依次用体积浓度比例为65:1、60:1、55:1、50:1、45:1、40:1、35:1、30:1、25:1、20:1、10:1、5:1的CH2Cl2-CH3OH溶液梯度洗脱,结合TLC跟踪检测合并相似组分最终分为B5-1、B5-2、B5-3、B5-4、B5-5段,各段的CH2Cl2-CH3OH溶液体积浓度比例范围分别为65:1-55:1、50:1-40:1、35:1-30:1、25:1-20:1、10:1-5:1
其中B5-3出现无色结晶状物质,洗涤后得到8mg化合物(16);B5-4反复硅胶柱层析进行分离,用体积浓度比例范围为8:1-3:1的石油醚-乙酸乙酯溶液梯度洗脱,分离得到12mg化合物(17);将B5-5反复硅胶柱层析,用体积浓度比例范围为8:1-3:1的CH2Cl2-CH3OH溶液梯度洗脱,分离得到28mg化合物(18);
将Fr.6与Fr.7合并,干法上样进行硅胶柱层析,依次用体积浓度比例为45:1、40:1、35:1、30:1、25:1、20:1、10:1、5:1、1:1的CH2Cl2-CH3OH溶液梯度洗脱后用甲醇冲柱,根据TLC薄层分析结果合并相似馏分分为B6-1、B6-2、B6-3、B6-4、B6-5五个不同极性段,各段的CH2Cl2-CH3OH溶液体积浓度比例范围分别为45:1-40:1、35:1-30:1、25:1-20:1、10:1、5:1-1:1,结合HPLC分析选取B6-1、B6-2、B6-3进行再分离,B6-2经体积浓度比例为25:1的CH2Cl2-CH3OH溶液反复洗脱后得到10mg化合物(19)。
对提取的19种化合物分别通过核磁共振技术(1HNMR、l3CNMR),紫外光谱技术(UV),质谱技术(MS)等现代波谱技术确定具体结构,谱图数据如下表1~16所示:
表1化合物(1)的波谱数据
表2化合物(2)的波谱数据
表3化合物(3)的波谱数据
表4化合物(4)的波谱数据
表5化合物(5)的波谱数据
表6化合物(6)的波谱数据
表7化合物(7)的波谱数据
表8化合物(8)的波谱数据
表9化合物(11)的波谱数据
表10化合物(12)的波谱数据
表11化合物(13)的波谱数据
表12化合物(14)的波谱数据
表13化合物(15)的波谱数据
表14化合物(16)的波谱数据
表15化合物(17)的波谱数据
表16化合物(19)的波谱数据
化合物(9):13C-NMR(101MHz,CDCl3):δ179.23(-COOH)、130.03(C-9)、129.75(C-10)、33.96(C-2)、31.93(C-14)、29.70~29.08(8C)、24.71(C-3)、22.69(C-15)、14.11(C-16);13H NMR(400MHz,CDCl3)δ:5.37(2H,m,H-9,H-10)、2.36(2H,t,H-2)、2.36(4H,d,H-8,H-11)、1.90(2H,t,H-3)、1.27~1.95(16H,H4-7,H12-15)、0.88(3H,m,-CH3);
化合物(18):13C NMR(101MHz,DMSO):δ97.37(Cβ-1),92.69(Cα-1),77.24(Cβ-5),77.21(Cβ-3),75.31(Cβ-2),73.56(Cα-3),72.84(Cα-2),72.42(Cα-5),71.08(Cα-4),70.78(Cα-4),61.70(Cβ-6),61.37(Cα-6)。
这19种化合物及其结构式如下表17所示:
表17枣核中各化合物及结构式
结合图4~5,本发明实施例提供上述枣核标准化提取物定量分析方法,包括以下步骤:
步骤1:称取10.0g粉碎后枣核放入100mL锥形瓶中,加入40mL体积浓度为75%甲醇-水溶液,在室温下超声提取90min,静置后离心10min倒出上清液,残渣再加入溶剂提取两次,合并提取液并浓缩定容至10mL;
步骤2:用电子天平精确称取1.0mg各标准品儿茶素、柚皮素、山奈酚、3,3'-O-二甲基鞣花酸、原儿茶、没食子酸、二氢查尔酮-4'-β-D-吡喃葡糖苷、3-氧代齐墩果酸、东莨菪内酯,用甲醇溶液完全溶解后在1mL容量瓶中定容,最终配制成1.0mg/mL的标准品储存液,取出0.5mL并定容至1mL容量瓶中配制成浓度为0.5mg/mL的溶液,然后逐级稀释成0.20mg/mL、0.10mg/mL、0.05mg/mL、0.025mg/mL的浓度梯度标准品溶液,用保鲜膜封口遮光处理后保存于4℃冰箱待用;
步骤3:用高效液相色谱仪对标准品溶液进行检测,不断调整色谱条件,确定最佳色谱条件和吸收波长,最佳色谱条件为:色谱柱LunaC-18柱(5um,250×4.60nm,Phenonemenx公司)流动相:乙腈(A)—0.2%磷酸水(B)系统,流速:1mL/min,柱温:30℃,进样量:20μL,检测波长:230nm、254nm、280nm、320nm,分析方法见下表18所示
表18 HPLC分析方法
步骤4:用高效液相色谱仪分析上述配制的各化合物的系列标准溶液,分别记录各化合物的峰面积,以标准溶液浓度为横坐标,峰面积为纵坐标,绘制标准曲线,各化合物标准曲线及其参数见下表19:
表19标准品标准曲线表
结果表明,各化合物浓度在25-200mg/L范围内时峰面积与浓度呈良好的线性关系,R2值均大于0.9997;
步骤5:精确吸取供试液用高效液相色谱仪进行检测分析,根据紫外图谱找出各对照品化合物对应的峰位置,记录其响应峰面积并用标准曲线法计算各化合物的含量,结果见表20;
表20枣核中各化合物含量
步骤6:精密吸取1000mg/L、500mg/L、200mg/L、100mg/L、50mg/L、25mg/L六种不同浓度的原儿茶酸标准溶液10μL,每天进样三次,并接连三天在同一时间进样测试,记录峰面积响应值,计算相对标准偏差RSD值,实验数据见表21:
表21日内、日间精密度实验
由表看出各化合物含量日内精密度RSD值低于2.35%,日间精密度RSD值低于1.99%,说明仪器的精密度良好;
步骤7:称取10.0g枣核样品共6份,按上述方法处理制备供试液,并按设定好的高效液相分析方法进行HPLC分析,进样量为20μL,准确记录每份供试品溶液中原儿茶酸的峰面积,并计算峰面积的平均值,然后计算RSD值,实验结果见表22:
表22重复性实验
所测原儿茶酸的平均含量为33.32μg/g,RSD值为0.57%,说明本实验建立的分析方法可重复性良好;
步骤8:精密称取六份10.0g枣核粉末药材,以原儿茶酸为例,已知样品中含量为33.32μg/g,向其中加入1300μg原儿茶酸对照品,然后进行HPLC分析,记录化合物峰面积响应值,计算化合物的平均回收率和RSD值,实验结果见表23,由表中数据显示RSD值为0.96%,平均回收率为92.98%。
表23回收率实验
本发明实施例提供枣核提取物中部分化合物的酶抑制活性,为其在制备治疗糖尿病药物和延缓阿尔茨海默症以及化妆品行业美白产品的应用提供依据,包括以下步骤:
步骤1:将原儿茶酸、柚皮素、5-羟甲基糠酸、山奈酚、儿茶素、α,β-D-吡喃葡萄糖用甲醇溶解配成1.0mg/mL的溶液,在96孔板中,1、2、3列孔中加入20μL 0.1mol/L磷酸缓冲液、20μL待测样品和20μLα-葡萄糖苷酶,作为样品组;4、5、6列孔中加入40μL 0.1mol/L磷酸缓冲液和20μL待测样品,作为控制组;7、8、9列孔中加入40μL 0.1mol/L磷酸缓冲液和20μLα-葡萄糖苷酶作为空白组;
在37℃下培养15min后,每孔加入20μL 2.5mmol/L PNPG糖苷,再在37℃下培养15min后,每孔加入80μL 0.2mol/L Na2CO3溶液终止反应。用酶标仪测定在405nm波长处的吸光值大小。用阿卡波糖作为阳性对照。抑制率%=[1-(OD样品-OD控制)/OD空白]×100%;
对各化合物按上述方法进行α-葡萄糖苷酶抑制活性测定结果见下表24,由表24可以看出柚皮素、山奈酚、儿茶素这三种黄酮类化合物的α-葡萄糖苷酶抑制率相对较高,这与黄酮类化合物B环上的羟基数目有关,其中儿茶素最高可达116.48%,与阳性对照物阿卡波糖抑制活性相当。黄酮类化合物属于天然药用组分,具有容易得到、毒副作用小等特点,基于此,本实验研究对α-葡萄糖苷酶抑制机制可以为临床开发新药提供理论基础和有利的作用。
表24α-葡萄糖苷酶活性抑制率
步骤2:将原儿茶酸、柚皮素、5-羟甲基糠酸、山奈酚、儿茶素、α,β-D-吡喃葡萄糖溶于甲醇溶液配成浓度为1.0mg/mL的溶液,在96孔板中,1、2、3列孔中均加入140μL 0.1mol/L磷酸缓冲液、20μL待测样品和20μL乙酰胆碱酯酶,作为样品组;4、5、6列孔中均加入160μL0.1mol/L磷酸缓冲液和20μL待测样品,作为控制组;7、8、9列孔中均加入160μL 0.1mol/L磷酸缓冲液(pH为7.4)和20μL乙酰胆碱酯酶,作为空白组;
在4℃下培养20min后,每孔加入10μL 15mM碘化乙酰硫代胆碱和10μL 2mM二硫代二硝基苯甲酸,再在37℃下培养20min,用酶标仪测定在412nm波长处的吸光值大小。用石杉碱甲作为阳性对照。实验结果如下表25所示:
表25乙酰胆碱酯酶活性抑制率
乙酰胆碱是脑内与学习、记忆密切相关的神经递质,生物化学研究发现阿尔茨海默症病人脑内乙酰胆碱能神经元缺少,乙酰胆碱含量不足,胆碱乙酰转移酶活性降低。胆碱酶抑制剂可抑制中枢突触间隙的乙酰胆碱酯酶,阻止乙酰胆碱的分解,提高脑内乙酰胆碱的含量,修复阿尔茨海默病中已丧失的胆碱能功能,阻止神经元间乙酰胆碱的代谢。近年来开发、寻找乙酰胆碱酯酶抑制剂受到人们的广泛关注。由本次实验结果可以看出原儿茶酸、5-羟甲基糠酸、山奈酚、儿茶素对乙酰胆碱酯酶抑制率比阳性对照物石杉碱甲高,可将其作为乙酰胆碱酯酶抑制剂应用于延缓阿尔茨海默症药物做进一步研究。
步骤3:准确称量并配制1mg/mL的L-酪氨酸标准溶液。取1mg酪氨酸酶加入5mLPBS缓冲液配成100U/mL酪氨酸酶溶液,4℃下保存,备用;
将原儿茶酸、柚皮素、5-羟甲基糠酸、山奈酚、儿茶素、α,β-D-吡喃葡萄糖用甲醇或DMSO溶解并配成1.0mg/mL的待测溶液,在96孔板中,1、2列孔中均加入80μL 0.1mol/L磷酸缓冲液、50μL溶剂和50μL酪氨酸酶,作为空白组;3、4列孔中均加入130μL 0.1mol/L磷酸缓冲液和50μL溶剂,作为空白背景组;5、6列孔中均加入80μL 0.1mol/L磷酸缓冲液、50μL待测样品和50μL酪氨酸酶,作为实验组;7、8列孔中均加入130μL 0.1mol/L磷酸缓冲液和50μL待测样品,作为实验背景组;最后向每个孔中加入20μL底物(L-酪氨酸)触发反应,96孔板在37℃下继续孵育30min,然后用酶标仪测定在475nm波长处的吸光值大小。用曲酸作为阳性对照。抑制率%=[1-(C实验-D实验背景)/(A空白-B空白背景)]×100%。实验结果如下表26所示:
表26络氨酸酶活性抑制率
酪氨酸酶抑制剂通过抑制酪氨酸酶的活性以阻断黑色素的合成反应链,减少其在皮肤内的生成,从而达到祛斑增白的效果,在化妆品领域的应用广泛。天然组分具有性能温和、高效祛斑、安全无毒的特点。为了使更有效的酪氨酸酶抑制剂得到研究并开发,本次实验考察了原儿茶酸、柚皮素、5-羟甲基糠酸、山奈酚、儿茶素、α,β-D-吡喃葡萄糖对络氨酸酶的抑制活性,实验结果表明以上化合物络氨酸酶抑制率均比阳性对照物曲酸的抑制率高,可将以上化合物作为开发应用于皮肤美白的药理学试剂的潜在成分,为深入开发枣核药材中的美白成分提供依据。
尽管已描述了本发明的优选实施例,但本领域内的技术人员一旦得知了基本创造性概念,则可对这些实施例作出另外的变更和修改。所以,所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
Claims (7)
1.一种枣核提取物,其特征在于:包括如下化合物:
3,3'-二甲氧基鞣花酸-4-O-β-D-葡萄糖(1)、5-羟甲基糠酸(2)、儿茶素(3)、无刺枣苄苷Ⅱ(4)、柚皮素(5)、山奈酚(6)、(E)-3,3'-二甲氧基-4,4'-二羟基二苯乙烯(7)、异香草醛(8)、棕榈油烯酸(9)、3,3'-O-二甲基鞣花酸(10)、胡萝卜苷(11)、原儿茶酸(12)、没食子酸(13)、二氢查尔酮-4'-β-D-吡喃葡萄糖苷(14)、3-氧代齐墩果酸(15)、东莨菪内酯(16)、对羟基间甲氧基苯甲酸(17)、α,β-D-吡喃葡萄糖(18)、脱氢双没食子酸(19);
其结构如下:
2.根据权利要求1所述的枣核提取物的提取分离方法,其特征在于,包括以下步骤:
步骤1:将枣核粉碎后用体积浓度为90%的乙醇-水溶液浸提四次,每次浸提48小时,合并提取液并减压浓缩,得到浸膏;
步骤2:加水分散浸膏,得浸膏溶液,浸膏溶液用石油醚反复萃取,直到上层石油醚萃取液为无色,合并多次石油醚萃取液,得石油醚萃取层浸膏;石油醚萃取后得到的下层浸膏溶液用饱和正丁醇萃取三次,收集正丁醇相,得正丁醇萃取层浸膏;正丁醇萃取后得到的下层浸膏溶液用乙酸乙酯萃取三次,收集乙酸乙酯相,得乙酸乙酯萃取层浸膏;
步骤3:将正丁醇萃取层浸膏采用湿法上样过反相聚酰胺柱,分别用纯水、体积浓度为20%乙醇-水溶液、40%乙醇-水溶液、60%乙醇-水溶液、80%乙醇-水溶液、纯乙醇梯度洗脱,结合TLC检测分析,按照上述洗脱顺序将同一梯度洗脱剂洗脱后的正丁醇萃取层浸膏溶液单独收集,最终将正丁醇萃取层浸膏按照洗脱剂体积浓度划分为D.1~D.6六段;将D.1~D.6六段分别通过柱层析,并洗脱,制备化合物(1)~(6);
步骤4:将乙酸乙酯萃取层浸膏采用干法上样过100-200目硅胶柱,用CH2Cl2-CH3OH溶液进行梯度洗脱,各梯度段的CH2Cl2-CH3OH溶液体积浓度范围分别为130:1-120:1、110:1-100:1、95:1-75:1、70:1-60:1、55:1-40:1、35:1-25:1、20:1-10:1、8:1-3:1;结合TLC检测分析,按照上述洗脱顺序将同一梯度洗脱剂洗脱后的乙酸乙酯萃取层浸膏溶液单独收集,最终收集到极性不同的Fr.1~Fr.8八段馏分,将Fr.1~Fr.8八段分别通过柱层析,并洗脱,制备化合物(7)~(19)。
3.根据权利要求2所述的枣核提取物的提取分离方法,其特征在于,所述化合物(1)~(6)具体是通过以下步骤分离制得:
将D.2过聚酰胺反相柱,分别用纯水、体积浓度为10%甲醇-水溶液、20%甲醇-水溶液、40%甲醇-水溶液梯度洗脱,用甲醇冲柱,结合TLC检测,将同一梯度洗脱剂洗脱后的D.2单独收集,并将D.2按照洗脱剂体积浓度划分为三段A2-1、A2-2、A2-3,各段洗脱剂分别对应10%甲醇-水溶液、20%甲醇-水溶液和40%甲醇-水溶液,用Sephadex LH-20柱分离A2-2并反复用体积浓度为10%甲醇-水溶液分离最终得到化合物(1);
将D.3过聚酰胺反相柱,分别用纯水、体积浓度为10%甲醇-水溶液、20%甲醇-水溶液、40%甲醇-水溶液、50%乙醇-水溶液、60%乙醇-水溶液梯度洗脱,结合TLC检测和HPLC图谱分析将同一梯度洗脱剂洗脱后的D.3单独收集,将D.3按照洗脱剂体积浓度分为五段A3-1、A3-2、A3-3、A3-4、A3-5,各段洗脱剂分别对应10%甲醇-水溶液、20%甲醇-水溶液、40%甲醇-水溶液、50%乙醇-水溶液、60%乙醇-水溶液;A3-1经CHP柱层析分离得到化合物(2)和化合物(3);A3-5经硅胶柱层析,用CH2Cl2-CH3OH溶液进行梯度洗脱,CH2Cl2与CH3OH体积浓度比例分别为18:1、15:1、12:1、8:1、4:1,结合TLC检测和HPLC图谱确定得到化合物(4);
将D.4过硅胶柱,用CH2Cl2-CH3OH溶液进行梯度洗脱,CH2Cl2与CH3OH体积浓度比例分别为15:1、12:1、10:1、8:1、5:1,结合TLC检测将同一梯度洗脱剂洗脱后的D.4合并,将D.4按照洗脱剂体积浓度分为四段A4-1、A4-2、A4-3、A4-4,各段CH2Cl2与CH3OH体积浓度比例分别对应15:1-12:1、10:1、8:1、5:1;A4-4过LH-20柱,用体积浓度为20%甲醇-水溶液洗脱,得到化合物(5);
将D.5过硅胶柱,依次用CH2Cl2-CH3OH溶液进行梯度洗脱,CH2Cl2与CH3OH体积浓度比例分别为20:1、15:1、13:1、10:1、5:1,结合TLC检测合并相同馏分,经比例为10:1的CH2Cl2-CH3OH溶液洗脱后过Sephadex LH-20反相柱得到化合物(6)。
4.根据权利要求2所述的枣核提取物的提取分离方法,其特征在于,所述化合物(7)~(19)具体是通过以下步骤分离制得:
将Fr.2干法上样过硅胶柱层析,用体积浓度范围为40:1-5:1的石油醚-乙酸乙酯溶液梯度洗脱,结合TLC检测和HPLC图谱合并相同馏分后分为三段B2-1、B2-2、B2-3,各段的石油醚-乙酸乙酯溶液体积浓度比例范围分别为70:1-40:1、35:1-20:1、15:1-5:1;将B2-2过硅胶柱层析,用石油醚-乙酸乙酯溶液进行梯度洗脱,石油醚与乙酸乙酯体积浓度比例分别为20:1、15:1、13:1、10:1、5:1,经比例为15:1的石油醚-乙酸乙酯溶液洗脱后结合TLC检测和HPLC图谱检测得到化合物(7);将B2-3过硅胶柱层析,用石油醚-乙酸乙酯溶液进行梯度洗脱,石油醚与乙酸乙酯体积浓度比例分别为18:1、15:1、12:1、10:1、8:1、5:1,经比例为8:1的石油醚-乙酸乙酯溶液洗脱后结合HPLC图谱检测得到化合物(8);
将Fr.3干法上样过硅胶柱层析,用体积浓度范围为70:1-5:1的CH2Cl2-CH3OH溶液梯度洗脱结合TLC跟踪监测和HPLC谱图分析划分为四段B3-1、B3-2、B3-3、B3-4,各段的CH2Cl2-CH3OH溶液体积浓度比例范围分别为70:1-60:1、55:1-40:1、35:1-20:1、15:1-1:1,将B3-1干法上样硅胶柱层析,石油醚-乙酸乙酯溶液按25:1、20:1、18:1、15:1、12:1、8:1、4:1、1:1的体积浓度比例梯度洗脱,经比例为12:1石油醚-乙酸乙酯溶液洗脱后反复硅胶柱层析得到化合物(11);将B3-2过Sephadex LH-20柱凝胶分离,分别用纯水、10%甲醇-水溶液、20%甲醇-水溶液、30%甲醇-水溶液梯度洗脱,经30%甲醇-水溶液和10%甲醇-水溶液洗脱后结合TLC跟踪检测合并相同馏分分别得到化合物(9)、(10);
将Fr.4干法上样过硅胶柱,分别选择体积浓度比例为80:1、70:1、65:1、60:1、55:1、50:1、45:1、40:1、35:1、30:1、25:1、20:1、10:1、5:1的CH2Cl2-CH3OH溶液梯度洗脱,甲醇冲柱,结合TLC监测和HPLC图谱解析将Fr.4划分为三段B4-1、B4-2、B4-3,各段的CH2Cl2-CH3OH溶液体积浓度比例范围分别为60:1-50:1、35:1-25:1、20:1-10:1,将B4-1经CHP、LH-20柱反复分离得到化合物(14);将B4-2过CHP层析柱,分别用20%甲醇-水溶液、40%甲醇-水溶液、60%甲醇-水溶液、80%甲醇-水溶液梯度洗脱,甲醇冲柱,经80%甲醇-水溶液和40%甲醇-水溶液洗脱后结合TLC跟踪检测和HPLC图谱分析分别得到两个纯化合物(12)、(13);将B4-3进行HPLC分析得到化合物(15);
将Fr.5干法上样过硅胶柱,依次用体积浓度比例为65:1、60:1、55:1、50:1、45:1、40:1、35:1、30:1、25:1、20:1、10:1、5:1的CH2Cl2-CH3OH溶液梯度洗脱,结合TLC跟踪检测合并相似组分划分为五段B5-1、B5-2、B5-3、B5-4、B5-5,各段的CH2Cl2-CH3OH溶液体积浓度比例范围分别为65:1-55:1、50:1-40:1、35:1-30:1、25:1-20:1、10:1-5:1,对B5-3中出现无色结晶状物质,洗涤后得到化合物(16);对B5-4反复硅胶柱层析,用体积浓度比例范围为8:1-3:1的石油醚-乙酸乙酯溶液进行梯度洗脱,得到化合物(17);对B5-5反复硅胶柱层析,用体积浓度比例范围为8:1-3:1的CH2Cl2-CH3OH溶液进行梯度洗脱,得到化合物(18);
将Fr.6与Fr.7段合并,干法上样硅胶柱层析,依次用体积浓度比例为45:1、40:1、35:1、30:1、25:1、20:1、10:1、5:1、1:1的CH2Cl2-CH3OH溶液梯度洗脱,甲醇冲柱,根据TLC薄层分析结果划分为五段B6-1、B6-2、B6-3、B6-4、B6-5,各段的CH2Cl2-CH3OH溶液体积浓度比例范围分别为45:1-40:1、35:1-30:1、25:1-20:1、10:1、5:1-1:1,结合HPLC分析对B6-2经体积浓度比例为25:1的CH2Cl2-CH3OH溶液反复洗脱后得到化合物(19)。
5.根据权利要求1所述的枣核提取物在制备治疗阿尔茨海默症药物方面的应用。
6.根据权利要求1所述的枣核提取物在制备治疗糖尿病药物方面的应用。
7.根据权利要求1所述的枣核提取物在制备化妆品美白产品方面的应用。
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