CN107964032A - 马尾松中一种三萜皂苷及其制备工艺和用途 - Google Patents
马尾松中一种三萜皂苷及其制备工艺和用途 Download PDFInfo
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Abstract
本发明属于植物次生代谢化学成分分析及天然产物应用领域，具体涉及一种文献中从未报道过的三萜皂苷即3‑O‑β‑D‑吡喃葡萄糖基(1″→6′)‑β‑D‑吡喃葡萄糖基乌苏酸28‑O‑β‑D‑吡喃葡糖酯苷的制备工艺及该三萜皂苷在制备抗补体产品中的应用。本发明提出的制备工艺具有路线简单、制备产物得率高且质量稳定、原料成本低、产物纯度高、适合工业化生产等特点。实验证实3‑O‑β‑D‑吡喃葡萄糖基(1″→6′)‑β‑D‑吡喃葡萄糖基乌苏酸28‑O‑β‑D‑吡喃葡糖酯苷具显著的补体抑制活性。
Description
技术领域
本发明属于植物次生代谢化学成分分析及天然产物应用领域，具体涉及一种文献中从未报道过的三萜皂苷即3-O-β-D-吡喃葡萄糖基(1″→6′)-β-D-吡喃葡萄糖基乌苏酸28-O-β-D-吡喃葡糖酯苷(对应英文名为3-O-β-D-glucopyranosyl-(1″→6′)-β-D-glucopyranosylursolic acid 28-O-β-D-glucopyranosyl ester)及其制备工艺和抗补体活性用途。
背景技术
马尾松(Pinus massoniana Lamb)为松科(Pinaceae)松属(Pinus L.)乔木，广泛分布于我国亚热带东部湿润地域，并向北延至北热带地区，是我国最重要的松科植物之一。其松树皮、松针等具有祛风活血、明目安神、活血止痛、舒筋、止血、解毒、止痒等功效，广泛用于食品、保健品、添加剂等方面。松树植物提取物具有镇痛、抗炎、祛痰、镇咳、抑菌、镇静、平喘、调血脂、抗衰老、抗氧化、抗肿瘤、抗突变等活性(王巍，等，中国医院药学杂志，2008，28(7)：549～552；肖云川，等，中草药，2015，46(23)：3460～3465)。
补体(complement)是存在于正常动物和人的组织液与血清中的一组经活化后具有酶活性的蛋白质，可辅助和补充特异性抗体，介导免疫溶血及溶菌作用，因此被称为补体(徐小娜，等，天然产物研究与开发，2015，27：355～359，327)。补体具有多种生物学活性，既执行非特异性免疫应答，同时也参与特异性免疫反应。然而，在有些条件下，补体过度激活所产生的活化片段可介导炎症的发生，引发各种病理反应，如老年性痴呆、类风湿性关节炎、急性心肌梗死、系统性红斑狼疮和急性呼吸窘迫综合征等。最新研究发现，重症非典型性肺炎(SARS)和禽流感亦与补体系统的过度激活密切相关，然而，到目前为止，临床上并无有效的补体抑制剂出现。自然界中存在大量的抗补体活性成分，而且从天然产物中提取抗补体功能成分具有易吸收、成本低和副作用小等优点，因此，从天然产物中寻找抗补体活性成分的研究一直受到国内外学者的高度关注(杨庆雄，等，安徽农业科学，2011，39(26)：15962～15964)。
本发明以提取萃取方法和柱色谱分离技术从马尾松植物原料中制备得到一种文献中从未报道过且抗补体活性显著的乌苏酸型三萜皂苷新化合物3-O-β-D-吡喃葡萄糖基(1″→6′)-β-D-吡喃葡萄糖基乌苏酸28-O-β-D-吡喃葡糖酯苷。
发明内容
本发明的目的是提供一种具有显著抗补体活性的三萜皂苷即3-O-β-D-吡喃葡萄糖基(1″→6′)-β-D-吡喃葡萄糖基乌苏酸28-O-β-D-吡喃葡糖酯苷(该化合物对应的英文名为3-O-β-D-glucopyranosyl-(1″→6′)-β-D-glucopyranosylursolic acid 28-O-β-D-glucopyranosyl ester)。
本发明的另一个目的是提供从松科松属乔木马尾松中制备3-O-β-D-吡喃葡萄糖基(1″→6′)-β-D-吡喃葡萄糖基乌苏酸28-O-β-D-吡喃葡糖酯苷的工艺。
本发明的第三个目的是提供3-O-β-D-吡喃葡萄糖基(1″→6′)-β-D-吡喃葡萄糖基乌苏酸28-O-β-D-吡喃葡糖酯苷的抗补体活性功效用途。
本发明的技术方案概述如下：
下述结构式的化合物：
从马尾松中制备3-O-β-D-吡喃葡萄糖基(1″→6′)-β-D-吡喃葡萄糖基乌苏酸28-O-β-D-吡喃葡糖酯苷的工艺，包括如下步骤：
(1)取粉碎的马尾松植物原料，按质量比1∶2～1∶10加入体积百分浓度为60％～95％的丙酮水溶液，常温或加热或超声波或微波提取1～5次，提取液过滤、减压浓缩得粗提物；
(2)加入粗提物质量1～6倍的水，搅拌，加入粗提物质量1～6倍的正丁醇萃取1～6次，分离出正丁醇层，将正丁醇层冷却并除去沉淀，然后常温过滤并减压浓缩得正丁醇萃取物；
(3)正丁醇萃取物经大孔树脂D101和ODS真空柱层析之至少一种并结合SephadexLH-20柱层析制备得到3-O-β-D-吡喃葡萄糖基(1″→6′)-β-D-吡喃葡萄糖基乌苏酸28-O-β-D-吡喃葡糖酯苷。
在上述3-O-β-D-吡喃葡萄糖基(1″→6′)-β-D-吡喃葡萄糖基乌苏酸28-O-β-D-吡喃葡糖酯苷的制备工艺步骤中，所述马尾松植物原料包括马尾松的树枝、松针、树根、树干皮中的一种或几种。
3-O-β-D-吡喃葡萄糖基(1″→6′)-β-D-吡喃葡萄糖基乌苏酸28-O-β-D-吡喃葡糖酯苷在制备抗补体产品中的用途。
试验证实本发明的三萜皂苷化合物分子式为C48H78O18，化学名为3-O-β-D-吡喃葡萄糖基(1″→6′)-β-D-吡喃葡萄糖基乌苏酸28-O-β-D-吡喃葡糖酯苷，该化合物对应的英文名为3-O-β-D-glucopyranosyl-(1″→6′)-β-D-glucopyranosylursolic acid 28-O-β-D-glucopyranosyl ester。该化合物为白色无定型粉末，旋光度为(c，0.5，MeOH)。解析其电喷雾离子化质谱(ESI-MS)数据表明该三萜皂苷化合物分子量为942。
本发明提出的制备方法具有工艺路线简单、制备产物得率高且质量稳定、原料成本低、产物纯度高、适合工业化生产等特点。
通过经典途径的抗补体活性测定试验发现，3-O-β-D-吡喃葡萄糖基(1″→6′)-β-D-吡喃葡萄糖基乌苏酸28-O-β-D-吡喃葡糖酯苷比正对比组化合物银椴苷(tiliroside)和迷迭香酸(rosmarinic acid)的抗补体活性更强。因此，3-O-β-D-吡喃葡萄糖基(1″→6′)-β-D-吡喃葡萄糖基乌苏酸28-O-β-D-吡喃葡糖酯苷可广泛应用在抗补体药品、食品和保健品等产品制备中。
具体实施方式
参考下列实施例将更全面、更容易地理解本发明，给出实施例是为了更清楚地阐明本发明，而不是以任何方式限制本发明。
实施例1：
3-O-β-D-吡喃葡萄糖基(1″→6′)-β-D-吡喃葡萄糖基乌苏酸28-O-β-D-吡喃葡糖酯苷的制备
(1)取粉碎的马尾松树根为原料，按质量比1∶3加入体积百分浓度为65％的丙酮水溶液，常温提取4次，提取液过滤、减压浓缩得粗提物；
(2)加入粗提物质量5倍的水，搅拌，加入粗提物质量5倍的正丁醇萃取3次，分离出正丁醇层，将正丁醇层冷却并除去沉淀，然后常温过滤并减压浓缩得正丁醇萃取物；
(3)正丁醇萃取物经大孔树脂D101真空柱层析和Sephadex LH-20柱层析制备得到3-O-β-D-吡喃葡萄糖基(1″→6′)-β-D-吡喃葡萄糖基乌苏酸28-O-β-D-吡喃葡糖酯苷。
该制备所得化合物的结构鉴定：
制备所得的化合物为白色无定型粉末，其旋光度为(c，0.5，MeOH)。在薄层色谱展开试验中，以冰水-乙酸体积比94∶6的溶剂系统展开时该化合物的Rf值约为0.55。该制备的化合物电喷雾离子化质谱中的特征离子峰显示其m/z[M-H]-、[M]+和[2M]+分别为941、942和1884，表明该化合物分子量为942。结合该制备所得化合物的1H和13C核磁共振图谱(NMR)数据，确定其化学分子式为C48H78O18。在以1％的三氯化铁乙醇溶液(质量百分比)进行TLC薄层色谱喷雾显色反应时，该化合物呈深绿色，表明其分子中含有酚羟基团(Si，etal，Wood Science and Technology，2016，50(3)，645～659)。在红外光(IR)谱图中，该化合物在3395、1930、1720、1065和1030cm-1处有强吸收峰。
试验测定并解析的该制备所得化合物的核磁共振谱数据：1H(400MHz，δ，DMSO-d6)：3.19(1H，dd，J＝12.1&4.0Hz，H-3)，5.34(1H，t-like，H-12)，2.50(1H，d，J＝11.2Hz，H-18)，1.27(3H，s，H-23)，1.08(3H，s，H-24)，0.83(3H，s，H-25)，0.98(3H，s，H-26)，1.23(3H，s，H-27)，0.96(3H，d，J＝6.1Hz，H-29)，0.85(3H，d，J＝6.1Hz，H-30)，5.74(1H，s，H-1′)，5.47(1H，d，J＝8.0Hz，H-1″)，5.16(1H，s，H-1″′)；13C(100MHz，δ，DMSO-d6)：39.06(C-1)，26.86(C-2)，88.67(C-3)，39.96(C-4)，55.65(C-5)，18.36(C-6)，33.54(C-7)，40.17(C-8)，47.21(C-9)，36.85(C-10)，23.66(C-11)，127.56(C-12)，138.66(C-13)，41.59(C-14)，28.14(C-15)，24.25(C-16)，47.87(C-17)，53.65(C-18)，39.75(C-19)，39.06(C-20)，30.84(C-21)，36.72(C-22)，27.82(C-23)，16.43(C-24)，15.63(C-25)，16.72(C-26)，23.78(C-27)，176.07(C-28)，16.80(C-29)，21.26(C-30)，105.09(C-1′)，78.86(C-2′)，82.44(C-3′)，72.05(C-4′)，77.40(C-5′)，67.44(C-6′)，104.17(C-1″)，75.21(C-2″)，78.04(C-3″)，71.90(C-4″)，78.03(C-5″)，62.60(C-6″)，94.54(C-1″′)，72.43(C-2″′)，78.01(C-3″′)，69.87(C-4″′)，79.16(C-5″′)，61.06(C-6″′)。由以上1H及13C NMR数据，结合已知文献，表明该化合物的苷元为乌苏酸(蔡凡，等，广东药学院学报，2016，32(4)：428～430；夏文绮，等，中草药，47(8)：1272～1277)。1H核磁共振图谱中，该化合物的三个β-D-吡喃葡萄糖苷的各端基质子峰分别为δH 5.74(1H，s，H-1′)、5.47(1H，d，J＝8.0Hz，H-1″)和5.16(1H，s，H-1″′)。在该化合物的HMBC谱中显示其中的两个葡萄糖端基质子H-1′(δ5.14，1H，s)和H-1″′(δ5.16，1H，s)分别与乌苏酸苷元的C-3(δ88.67)和C-28(δ176.07)相关，而另外一个葡萄糖端基质子H-1″(δ5.47，1H，d，J＝8.0Hz)与葡萄糖基中C-6′(δ67.44)相关，因而确定其中的两个葡萄糖基分别连接在乌苏酸的C-3和C-28位，而另一个葡萄糖基则结合在与C-3相连葡萄糖基的C-6′处。
综合以上信息，该化合物被确定为3-O-β-D-吡喃葡萄糖基(1″→6′)-β-D-吡喃葡萄糖基乌苏酸28-O-β-D-吡喃葡糖酯苷，即3-O-β-D-glucopyranosyl-(1″→6′)-β-D-glucopyranosylursolic acid 28-O-β-D-glucopyranosyl ester。经检索，该化合物为文献中从未制备和报道过的新的乌苏酸型三萜皂苷类化合物。
实施例2：
3-O-β-D-吡喃葡萄糖基(1″→6′)-β-D-吡喃葡萄糖基乌苏酸28-O-β-D-吡喃葡糖酯苷的制备
(1)取粉碎的马尾松树干皮为原料，按质量比1∶4加入体积百分浓度为75％的丙酮水溶液，加热提取3次，提取液过滤、减压浓缩得粗提物；
(2)加入粗提物质量4倍的水，搅拌，加入粗提物质量4倍的正丁醇萃取4次，分离出正丁醇层，将正丁醇层冷却并除去沉淀，然后常温过滤并减压浓缩得正丁醇萃取物；
(3)正丁醇萃取物经ODS真空柱层析和Sephadex LH-20柱层析制备得到3-O-β-D-吡喃葡萄糖基(1″→6′)-β-D-吡喃葡萄糖基乌苏酸28-O-β-D-吡喃葡糖酯苷。
实施例3：
(1)取粉碎的马尾松树枝为原料，按质量比1∶5加入体积百分浓度为85％的丙酮水溶液，超声波提取2次，提取液过滤、减压浓缩得粗提物；
(2)加入粗提物质量3倍的水，搅拌，加入粗提物质量3倍的正丁醇萃取5次，分离出正丁醇层，将正丁醇层冷却并除去沉淀，然后常温过滤并减压浓缩得正丁醇萃取物；
(3)正丁醇萃取物经大孔树脂D101真空柱层析和Sephadex LH-20柱层析制备得到3-O-β-D-吡喃葡萄糖基(1″→6′)-β-D-吡喃葡萄糖基乌苏酸28-O-β-D-吡喃葡糖酯苷。
实施例4：
3-O-β-D-吡喃葡萄糖基(1″→6′)-β-D-吡喃葡萄糖基乌苏酸28-O-β-D-吡喃葡糖酯苷的抗补体活性评价
1.待评价样品：上述制备的3-O-β-D-吡喃葡萄糖基(1″→6′)-β-D-吡喃葡萄糖基乌苏酸28-O-β-D-吡喃葡糖酯苷，经HPLC测定后纯度为98.8％。
2.正对比组：购自Sigma公司的具有显著抗补体活性的银椴苷(tiliroside)和迷迭香酸(rosmarinic acid)标准品，经HPLC测定后，二者纯度均≥98.8％。
3.抗补体活性实验方法：待测样品3-O-β-D-吡喃葡萄糖基(1″→6′)-β-D-吡喃葡萄糖基乌苏酸28-O-β-D-吡喃葡糖酯苷的抗补体活性评价试验操作严格按照文献(Si，etal，Journal of Asian Natural Product Research，2008，10(11)：1003～1008)中的抗补体经典途径测定方法及规程执行。
4.试验结果：
(1)以抗补体经典途径测定法评价3-O-β-D-吡喃葡萄糖基(1″→6′)-β-D-吡喃葡萄糖基乌苏酸28-O-β-D-吡喃葡糖酯苷的抗补体活性，试验结果如表1所示。
表1.基于抗补体经典途径测定方法，3-O-β-D-吡喃葡萄糖基(1″→6′)-β-D-吡喃葡萄糖基乌苏酸28-O-β-D-吡喃葡糖酯苷的补体抑制活性(IC50值)
a三次独立试验的平均值
通过抗补体经典途径评价方法，测得3-O-β-D-吡喃葡萄糖基(1″→6′)-β-D-吡喃葡萄糖基乌苏酸28-O-β-D-吡喃葡糖酯苷的50％抑制浓度(IC50)为78μM，而平行试验所测的正对比组化合物银椴苷和迷迭香酸的IC50值分别为103和180μM，即说明制备所得的乌苏酸型三萜皂苷3-O-β-D-吡喃葡萄糖基(1″→6′)-β-D-吡喃葡萄糖基乌苏酸28-O-β-D-吡喃葡糖酯苷的抗补体活性优于正对比组银椴苷和迷迭香酸。试验结果充分证实3-O-β-D-吡喃葡萄糖基(1″→6′)-β-D-吡喃葡萄糖基乌苏酸28-O-β-D-吡喃葡糖酯苷具有显著的抗补体作用，可广泛应用在抗补体药品、食品和保健品等产品制备中。
Claims (4)
1.下述结构式的化合物：
化学名为3-O-β-D-吡喃葡萄糖基(1″→6′)-β-D-吡喃葡萄糖基乌苏酸28-O-β-D-吡喃葡糖酯苷，其对应英文名为3-O-β-D-glucopyranosyl-(1″→6′)-β-D-glucopyranosylursolic acid28-O-β-D-glucopyranosyl ester。
2.权利要求1化合物的制备工艺，其特征是包括如下步骤：
(1)取粉碎的马尾松植物原料，按质量比1∶2～1∶10加入体积百分浓度为60％～95％的丙酮水溶液，常温或加热或超声波或微波提取1～5次，提取液过滤、减压浓缩得粗提物；
(2)加入粗提物质量1～6倍的水，搅拌，加入粗提物质量1～6倍的正丁醇萃取1～6次，分离出正丁醇层，将正丁醇层冷却并除去沉淀，然后常温过滤并减压浓缩得正丁醇萃取物；
(3)正丁醇萃取物经大孔树脂D101和ODS真空柱层析之至少一种并结合Sephadex LH-20柱层析制备得到3-O-β-D-吡喃葡萄糖基(1″→6′)-β-D-吡喃葡萄糖基乌苏酸28-O-β-D-吡喃葡糖酯苷。
3.根据权利要求2所述的工艺，其特征是所述马尾松植物原料包括马尾松的树枝、松针、树根、树干皮中的一种或几种。
4.权利要求1化合物在制备抗补体产品中的应用。
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