CN105949257B - 一种异黄酮苷类化合物的制备和用途 - Google Patents
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Abstract
本发明属医药及天然产物化学领域，具体涉及一种新的异黄酮苷类化合物及其制备方法和用途。所述的异黄酮苷类化合物结构如下，本发明首先采用正相（硅胶）柱色谱分离，再采用反相（开放ODS）柱色谱分离，最后经甲醇重结晶得到化合物2′‑羟基大豆苷。本发明的化合物能够用于预防和治疗由于体内自由基产生过多或清除自由基能力下降引起的各种疾病。
Description
技术领域
本发明属医药及天然产物化学领域，具体涉及一种新的异黄酮苷类化合物及其制备方法和用途。
背景技术
绿豆为豆科植物绿豆(Vigna radiate L.)的干燥成熟种子，具有清热解毒、燥湿的功效。可用于治疗吐泻、斑疹、疔疮、疥癣等病症。《本草汇言》中记载用绿豆治疔毒、癍疹，金、石、丹、火诸毒及霍乱吐下。目前，并未有绿豆植株的药理活性及活性物质的报道。
异黄酮苷类化合物在植物界普遍存在，目前从绿豆植株中发现的新的异黄酮苷化合物为2′-羟基大豆苷结构，是尚未发现的新化合物。
近年来，硅胶与ODS等填料在天然药物化学成分的提取、分离纯化、制剂工艺改革、制剂质量分析等方面有了较广泛的应用研究，并明确显示出其独特的作用。利用硅胶与ODS的相反的吸附功能和物质的相似相容原理可从中药提取液中分离精制有效成分或有效部位，最大限度地去粗取精，促进中药现代化研究的发展。
自由基在信号传导和免疫等方面具有重要的作用，但过多的活性氧自由基就会产生破坏行为，通过链式反应攻击细胞内或体液中的生物分子，如DNA、脂质、蛋白质等，导致人体正常细胞和组织的损坏，从而引起多种疾病，如癌症、衰老和各种心血管疾病。与此同时，具有自由基清除活性的合成抗氧化剂或天然抗氧化剂已成为国内外各学科领域的研究热点，其中对异黄酮类化合物的研究是自由基清除剂研究中的热门领域。
发明内容
本发明的一个目的在于提供一种新的异黄酮苷化合物及其化学结构和名称，即2′-羟基大豆苷，英文名为2'-hydroxydaidzin，其具体结构式如下：
该化合物具有如下光谱学特征：1H-NMR及13C-NMR及HMBC谱如表1所示。
表1.化合物2′-羟基大豆苷的1H-NMR及13C-NMR及HMBC谱数据
HR-ESIMS数据：对于该化合物来说，其高分辨质谱给出分子离子峰(negative ionmode):m/z＝455.0929[M+Na]+(calcd.for C21H20O10Na,455.0949)，分子式：C21H20O10。
本发明的另一个目的在于提供上述异黄酮苷化合物的制备方法。具体步骤如下：
(1)取绿豆植株的干燥茎枝，用乙醇或甲醇提取，过滤，合并滤液，得醇提液；将醇提液减压回收醇至无醇味，得粗提液；
(2)所获得的粗提液依次用不同极性有机溶剂萃取，弃去萃取液，再用正丁醇萃取，合并正丁醇萃取液，减压回收正丁醇，得绿豆植株提取物；
(3)所得的绿豆植株提取物，用适量甲醇分散，加入适量硅胶，搅拌均匀，装入预先处理好的硅胶柱中，以两种有机溶剂混合的混合有机溶剂(100:0～0:100)进行梯度洗脱，收集混合有机溶剂(50:50～30:70)洗脱液，浓缩至浓稠状，加适量甲醇分散；上开放ODS柱，以醇-水(0:100～100:0)，优选为醇-水(15:85～30:70)，进行梯度洗脱，收集醇-水(15:85～25:75)洗脱液，经浓缩得到异黄酮苷化合物；
(4)所得的异黄酮苷化合物，用甲醇重结晶，得到2′-羟基大豆苷。
本发明中，步骤(1)中所述的乙醇或甲醇的浓度为30％～100％。
本发明中，步骤(1)中所述的提取是用5～20倍量的30％～100％乙醇或甲醇闪式提取1～3次，并合并提取液。
本发明中，步骤(2)中所述的不同极性有机溶剂包括，但不仅限于：石油醚、环己烷、二氯甲烷、氯仿、醋酸乙酯。
本发明中，步骤(2)中所述的不同极性有机溶剂、正丁醇的用量为0.5～2倍体积，萃取次数为1～5次。
本发明中，步骤(3)中所述的混合有机溶剂为二氯甲烷-甲醇，或氯仿-甲醇，或二氯甲烷-乙醇，或氯仿-乙醇。
本发明中，步骤(3)中所述的醇-水为甲醇-水或乙醇-水。
本发明中，首先采用正相(硅胶)柱色谱分离，再采用反相(开放ODS)柱色谱分离，最后经甲醇重结晶得到化合物2′-羟基大豆苷。
本发明中，采用硅胶柱色谱分离时，采用干法上样；采用开放ODS柱色谱分离时，采用湿法上样。
本发明的再一个目的在于提供该化合物在制备用于预防和治疗由于体内自由基产生过多或清除自由基能力下降引起的各种疾病，比如心脏病、老年痴呆症、肿瘤和衰老等药物中的用途。
附图说明
图1为2′-羟基大豆苷的氢谱图。
图2为2′-羟基大豆苷的碳谱图。
图3为2′-羟基大豆苷的HMBC谱图。
图4为本发明实施例2′-羟基大豆苷的提取分离流程图。
具体实施方式
实施例一、2′-羟基大豆苷的分离制备
1.植物材料：豆科植物绿豆(Vigna radiate L.)的干燥茎枝。
2.分离制备方法：绿豆植株干燥茎枝25kg，用10、10倍量的95％乙醇闪式提取2次，过滤，合并滤液，减压回收乙醇至无醇味，得浓缩液约5L；分别用石油醚5L、二氯甲烷5L、正丁醇5L各萃取3次，合并正丁醇萃取液，减压回收正丁醇，得绿豆植株提取物；将绿豆植株提取物，用适量甲醇分散，并取适量硅胶拌样；将拌好的样品装入硅胶柱中，用二氯甲烷-甲醇(100:0～0:100)梯度洗脱，得到6个流分，流分6〔二氯甲烷-甲醇(20:80～0:100)〕经开放ODS柱，甲醇-水(15:85～30:70)梯度洗脱得4个流分，流分2〔甲醇-水(20:80)〕在甲醇中析出结晶，得到2′-羟基大豆苷。
实施例二、2′-羟基大豆苷的体外抗氧化活性测试
1.仪器：U-1900型分光光度计(Hitachi)，AB135-S型电子天平(Mettler Toledo)，酶标仪(BIO-RAD)，pH-2TC(0.01级)精密数显酸度计(上海之信仪器有限公司)。
2.试剂：氯化钠(NaCl)、氯化钾(KCl)、磷酸二氢钾(KH2PO4)、磷酸氢二钾(K2HPO4)、过硫酸钾(K2S2O8)、2,2′-连氮基-双-(3-乙基苯并二氢噻唑啉-6-磺酸)二铵盐(ABTS)，抗坏血酸。
实验所用的2′-羟基大豆苷为发明人自制，其化学结构经氢谱、碳谱、质谱确定无误，HPLC纯度在98％以上。
3.实验方法：用pH7.4的磷酸盐缓冲溶液(PBS)配制7mmol/L的ABTS溶液和2.45mmol/L的过硫酸钾溶液，将两种溶液等体积混合，并在室温下、于黑暗处反应12h，得自由基正离子(ABTS·+·)溶液。然后用pH7.4的PBS溶液稀释40倍，稀释后避光放置30min，在734nm处测定吸光度为0.71(可用于抗氧化活性的测试)。用甲醇配制2′-羟基大豆苷和抗坏血酸的系列浓度溶液，浓度分别为6mmol/L、3mmol/L、1.5mmol/L、0.3mmol/L和0.15mmol/L。将各浓度供试品100μL与3mL的ABTS·+·在室温下避光放置6min，用酶标仪在734nm下测定吸光度值AS。阴性对照用甲醇代替，用酶标仪在734nm下测定吸光度值AC。ABTS·+清除率(％)计算公式如下：
ABTS·+清除率(％)＝(1-As/Ac)×100％
用SPSS 17.0软件计算半数抑制浓度(IC50)。
4.实验结果：见表2。
表2.化合物2′-羟基大豆苷体外ABTS·+清除结果
该发明测试结果表明，化合物2′-羟基大豆苷具有ABTS·+抑制活性，且活性强于维生素C，故该化合物具有较强的体外抗氧化活性。尤其适用于预防和治疗心血管系统疾病，且其制备原料来源广泛，成本低廉，具有良好的应用、开发前景。
Claims (9)
1.具有如下结构的2′-羟基大豆苷：
2.权利要求1所述化合物的制备方法，包括以下步骤：
(1)绿豆植株粗提液的制备：取绿豆植株适量，用乙醇或甲醇提取，过滤，合并滤液，得醇提液，将醇提液减压回收醇至无醇味，得粗提液；
(2)绿豆植株提取物的制备：取绿豆植株粗提液，依次用不同极性有机溶剂萃取，弃去萃取液，再用正丁醇萃取，合并正丁醇萃取液，减压回收正丁醇，得绿豆植株提取物；
(3)2′-羟基大豆苷化合物的制备：所得的绿豆植株提取物，用适量甲醇分散，加入适量硅胶，搅拌均匀，装入预先处理好的硅胶柱中，以两种有机溶剂混合的混合有机溶剂100:0～0:100进行梯度洗脱，收集混合有机溶剂50:50～30:70洗脱液，浓缩至浓稠状，加适量甲醇分散；上开放ODS柱，以醇-水0:100～100:0，进行梯度洗脱，收集醇-水15:85～25:75洗脱液，经浓缩得到2′-羟基大豆苷化合物；
(4)2′-羟基大豆苷化合物的纯化：取得到的2′-羟基大豆苷化合物，经甲醇重结晶，得到高纯度的2′-羟基大豆苷；
步骤(2)中，先用0.5～2倍体积石油醚或环己烷萃取1～5次后，再用0.5～2倍体积二氯甲烷或氯仿或醋酸乙酯萃取1～5次，最后用0.5～2倍体积的正丁醇萃取1～5次；
步骤(3)中所述的混合有机溶剂为二氯甲烷-甲醇，或氯仿-甲醇，或二氯甲烷-乙醇，或氯仿-乙醇。
3.如权利要求2所述的制备方法，其特征在于，步骤(1)中采用5～20倍量的30％～100％乙醇或甲醇提取1～3次，并合并提取液。
4.如权利要求2所述的制备方法，其特征在于，步骤(3)中以醇-水15:85～30:70洗脱液进行洗脱。
5.如权利要求2或4所述的制备方法，其特征在于，步骤(3)中所述的醇-水梯度洗脱，其醇-水为甲醇-水或乙醇-水。
6.一种药物组合物，包含权利要求1所述的2′-羟基大豆苷化合物和药学上可接受的载体。
7.一种药物制剂，包含权利要求1所述的2′-羟基大豆苷化合物或权利要求6所述的药物组合物，所述的药物制剂为片剂、胶囊、颗粒剂、口服溶液、注射剂。
8.权利要求1所述的2′-羟基大豆苷化合物或权利要求6所述的药物组合物在制备用于预防和治疗由于体内自由基产生过多或清除自由基能力下降引起的各种疾病的药物中的应用。
9.如权利要求8所述的应用，其特征在于，所述的由于体内自由基产生过多或清除自由基能力下降引起的各种疾病包括心脏病、老年痴呆症、肿瘤或衰老症。
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