CN107243011B - 松香烷型二萜糖苷在制备抗炎药物中的应用 - Google Patents
松香烷型二萜糖苷在制备抗炎药物中的应用 Download PDFInfo
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Abstract
本发明涉及医药技术领域，公开了从雷公藤(Tripteryginum Wilfordii Hook.F)中分得的松香烷型二萜糖苷化合物Wilfordoside A的提取分离和结构鉴定以及在制备抗炎药物中的应用。
Description
技术领域
本发明涉及医药技术领域，尤其涉及从雷公藤中分离鉴定的松香烷型二萜糖苷化合物wilfordoside A在制备抗炎药物中的应用。
背景技术
雷公藤( Tripteryginum Wilfordii Hook. F) 为卫矛科雷公藤属植物，又名黄檀根和断肠草，其药用部位为根。据《全国中草药汇编》记载，雷公藤味苦、性辛、凉，有大毒。具有祛风，解毒，杀虫作用。外用治风湿性关节炎，皮肤发痒，杀蛆虫、孑孓，灭钉螺，毒鼠。雷公藤主要分布于福建、湖南和江西等地区。临床常用于治疗类风湿性关节炎、慢性肾炎、红斑狼疮等难治性免疫功能亢进疾病。目前有雷公藤片、雷公藤多苷片、雷公藤内酯、雷公藤内酯软膏、雷公藤总萜片等国产药品广泛应用于临床，疗效显著。
雷公藤的抗炎有效成分主要有松香烷型二萜，目前报道的雷公藤中具有抗炎活性的松香烷型二萜基本为苷元化合物，如雷公藤甲素、雷公藤乙素、雷公藤内酯酮和雷公藤氯内酯醇等松香烷型二萜苷元。雷公藤中关于松香烷型二萜糖苷类化合物报道极少，而具有抗炎活性的松香烷型二萜糖苷还未见报道。为了进一步从雷公藤中发现新的抗炎活性成分，为新药开发奠定基础，发明人对雷公藤进行了化学成分的深入研究。结合抗炎药效活性筛选，从雷公藤中分离鉴定了具有抗炎活性的松香烷型二萜糖苷wilfordoside A。本发明公开了松香烷型二萜糖苷wilfordoside A的提取分离、结构鉴定以及在制备抗炎药物中的应用。
发明内容
本发明所涉及的从雷公藤中提取分离鉴定的具有抗炎活性松香烷型二萜糖苷为wilfordoside A（结构如下式Ⅰ）。
式Ⅰ：wilfordoside A结构式。
本发明的目的是提供化合物wilfordoside A在制备抗炎药物中的应用。本发明是通过下述技术方案来实现的。
一、提取分离
1．将雷公藤根切片，加入95%乙醇水溶液，加热回流提取3次，合并提取液，减压浓缩回收乙醇得到总浸膏。将总浸膏用乙酸乙酯溶解数次至不再溶解，合并乙酸乙酯溶液，减压浓缩回收乙酸乙酯得乙酸乙酯部位。
2．将乙酸乙酯部位经中性氧化铝色谱柱层析，用石油醚-乙酸乙酯（1:0，4:1，3:2，2:3，V/V）及乙酸乙酯-甲醇（1:0，3:1，1:1，0:1，V/V） 依次梯度洗脱，依次得到8个洗脱流份，将乙酸乙酯-甲醇（1:1，V/V）洗脱流份减压浓缩得中性氧化铝色谱柱洗脱部位。
3．将中性氧化铝色谱柱洗脱部位经MCI GEL色谱柱层析，用甲醇-水（0:1，1:9，3:7，1:1，7:3，9:1，1:0，V/V）依次梯度洗脱，依次得到7个洗脱流份，将甲醇-水（7:3，V/V）洗脱流份减压浓缩得MCI GEL色谱柱洗脱部位。
4．将MCI GEL色谱柱洗脱部位经Sephadex LH-20凝胶柱分离，甲醇洗脱，TLC检测合并，减压浓缩依次得到9个流份，将第6个流份减压浓缩得Sephadex LH-20凝胶柱洗脱部位。
5．将Sephadex LH-20凝胶柱洗脱部位经制备HPLC分离 (YMC-Actus ODS-AC18色谱柱，流动相：甲醇-水 38:62，V/V，检测波长210nm)，得化合物wilfordoside A。
二、结构鉴定
wilfordoside A：棕黄色粉末（甲醇），熔点212-213℃。用盐酸加热水解后薄层色谱检测有葡萄糖。根据ESI-MS（[M-H]-，m/z 521）结合氢核磁共振谱（1H-NMR）和碳核磁共振谱（13C-NMR）确定分子式为C27H38O10。紫外光谱图（224nm，287nm）和NMR谱显示结构中含有苯环。氢核磁共振谱（1H-NMR）和碳核磁共振谱（13C-NMR）谱中显示3个特征甲基(δ H 1.18，d，7.0Hz，H-17；δ H 2.10，d，1.4Hz，H-19；δ H 1.16，s，H-20) (δ C 18.7，C-17；δ C 19.0，C-19；δ C 18.4，C-20)和1个特征亚甲基(δ H 3.85，dd，12.1，2.0Hz，H-16；3.69，dd，12.1，4.9Hz，H-16)(δ C 68.8，C-16)，结合存在苯环结构说明该化合物为松香烷型二萜类化合物。13C-NMR谱中6个碳信号（δ C 95.7，C-1’；δ C 74.2，C-2’；δ C 79.0，C-3’；δ C 71.2，C-4’；δ C 78.4，C-5’；δ C 62.5，C-6’)结合1H-NMR谱中糖端基氢信号耦合常数（δ H 5.56，d，8.0Hz，H-1’）说明结构中含有β-葡萄糖。1H NMR (600MHz, CD3OD) δ H: 3.22 (1H, m, H-1), 2.67 (1H, m, H-1),2.55(1H, m, H-2) , 2.34(1H, m, H-2) , 2.23(1H, m, H-5), 2.40 (1H, m, H-6),1.55 (1H, m, H-6), 3.38(1H, m, H-7) , 3.03(1H, dd, J =16.1,3.8Hz, H-7) ,6.44(1H, s, H-12), 3.24(1H, m, H-15) , 3.85(1H, dd, J=12.1, 2.0Hz, H-16), 3.69(1H, dd, J=12.1, 4.9Hz, H-16), 1.18 (3H, d, J=7Hz, H-17), 2.10(3H, d, J =1.4Hz, H-19) , 1.16(3H, s, H-20) , 5.56 (1H, d, J=8 Hz, H-1’), 3.49(1H, dd, J=10.7, 8.0 Hz, H-2’) , 3.63(1H, dd, J=10.7, 6.4Hz, H-3’), 3.44 (1H, dd, J=8.7, 6.4 Hz, H-4’), 3.40(1H, m, H-5’) , 3.37(2H, m, H-6’), 3.66(3H, s, OCH3)。13C NMR (150MHz, CD3OD) δC: 33.5 (C-1), 26 (C-2), 125.8(C-3), 150 (C-4), 50.4(C-5), 21.1 (C-6), 27.6 (C-7), 131.8 (C-8), 132.8(C-9), 38.4(C-10), 153.9(C-11), 113 (C-12), 136(C-13), 150.3 (C-14), 35.8(C-15), 68.8 (C-16), 18.7 (C-17),169.4 (C-18), 19(C-19), 18.4(C-20), 95.7(C-1’), 74.2 (C-2’), 79(C-3’),71.2 (C-4’), 78.4 (C-5’), 62.5 (C-6’),61.4 (OCH3)。经查文献比较，以上波谱数据与文献(LI Hong-mei, WAN Da-wu, LI Rong-tao. New abietane-type diterpeneglycosides from the roots of Tripterygium wilfordii [J]. Journal of AsianNatural Products Research, 2015, 17(7):761-766.)报道的wilfordoside A一致，因此确定该化合物为wilfordoside A(结构见式Ⅰ)。
三、抗炎药效学评价
本发明对wilfordoside A进行了抗炎药效学评价。
1. wilfordoside A对脂多糖（LPS）诱导的大鼠膝关节滑膜原代成纤维细胞炎症因子白介素1β（IL-1β）过量表达的影响实验。
1.1 大鼠膝关节滑膜原代成纤维细胞的培养
雄性SD大鼠（180-220g）乙醚麻醉后处死，75% 酒精浸泡15-20分钟后，取出膝关节滑膜组织，剪碎，利用II型胶原酶（4mg/ml）消化2h，加入含10% 胎牛血清的RPMI Medium1640培养，3天左右细胞慢慢爬出，待细胞长至融合状态后，用0.25%胰酶（含0.1%的EDTA）消化2分钟，细胞传代继续培养，原代细胞3-6代用于下面的实验。
1.2 测定wilfordoside A对LPS诱导的膝关节滑膜原代成纤维细胞IL-1β表达的影响
wilfordoside A用DMSO溶解，溶解后母液浓度为10mM，临用前用磷酸盐缓冲液（PBS）稀释到1mM，实验终浓度为10μM。将细胞以2×106个/ml的浓度接种于48孔板，37℃，5%CO2条件下培养至80% 的融合状态，PBS洗涤两次。换成无血清培养基，加入wilfordoside A溶液，使最终浓度为10μM，0.5h后，加入刺激剂LPS（10ng/ml）。实验分为空白组，模型组，wilfordoside A给药组，阳性对照药泼尼松龙给药组（实验终浓度为10μM）。模型组用刺激剂LPS（10ng/ml）造模，给药组加入对应化合物，每组6个复孔，37℃，5% CO2条件下孵育24小时后，按大鼠IL-1β ELISA试剂盒说明要求进行测定OD值，检测波长450nm。
1.3 实验结果
结果（见表1）表明，模型组IL-1β表达与空白组比较有极显著性升高（P<0.001），说明造模成功。wilfordoside A、泼尼松龙给药组与模型组比较均有极显著性差异（P<0.001），说明wilfordoside A能显著抑制LPS诱导的膝关节滑膜原代成纤维细胞炎症因子IL-1β的过量表达，抑制效果与阳性药泼尼松龙相当，说明wilfordoside A具有明显的抗炎活性。
表1. wilfordoside A对LPS诱导的滑膜原代成纤维细胞IL-1β表达的影响(
±S，n=6)。
*** 与模型组比较P<0.001
2. wilfordoside A对二甲苯引起的小鼠耳肿胀模型影响实验
wilfordoside A用2 %丙二醇水溶液溶解。取昆明种小鼠，雌雄各半，体重18-22g，随机均分成模型组，wilfordoside A给药组，阳性对照药吲哚美辛给药组，每组10只。各给药组按10mg/kg剂量灌胃给药，模型组给等体积2%丙二醇水溶液。连续给药3天，末次给药后1小时，将20μl二甲苯涂于小鼠右耳廓两面致炎, 左耳不涂为对照。1小时后将小鼠处死，剪下双耳, 用直径6mm 的打孔器分别在同一部位打下圆耳片, 称重。以左右耳片重量差作为肿胀度, 计算抑制率。实验结果见表2，各给药组的平均耳肿胀度与模型组相比均有显著性( P < 0.01) 差异，wilfordoside A抑制率为61.5%，其抑制率高于阳性药吲哚美辛。实验结果表明wilfordoside A具有显著的抗炎活性，优于阳性药吲哚美辛。
表2. wilfordoside A对二甲苯引起的小鼠耳肿胀模型影响实验结果(
±S，n=10)。
* 与模型组比较P < 0.01。
四、抗炎药物制剂制备
wilfordoside A或以其为基本活性的药物组合物可以与药学上合适的辅料或载体结合，采用公知方法制成注射剂、片剂、胶囊剂、滴丸剂、控释制剂、缓释制剂、纳米制剂等制剂抗炎药物。
具体实施方式
下面通过实施例作进一步描述，但本发明并不限于实施例所述范围。
实施例1：
将雷公藤干燥根119千克切片，加入95%乙醇400升，于70℃加热回流提取3次（1.5×1.5×1小时）。合并提取液，减压浓缩回收溶剂得浸膏12Kg。浸膏用乙酸乙酯溶解多次，合并乙酸乙酯溶液，减压浓缩回收溶剂得乙酸乙酯部位1.87Kg。
将乙酸乙酯部位浸膏1.87kg，经中性氧化铝柱色谱层析（200-300目，22.5Kg），石油醚-乙酸乙酯（1:0，4:1，3:2，2:3，V/V）及乙酸乙酯-甲醇（1:0，3:1，1:1，0:1，V/V）梯度洗脱，每个流份收集50L，依次得到8个洗脱流份(Fr.1~Fr.8)。
将Fr.7流份（即：上述乙酸乙酯-甲醇 1:1洗脱流份）（15.6g）经MCI GEL-CHP 20P色谱柱分离，甲醇-水（0:1，1:9，3:7，1:1，7:3，9:1，1:0，V/V）梯度洗脱，依次得7个洗脱流份（Fr.7.1~Fr.7.7）。
将Fr.7.5（即：上述甲醇-水7:3洗脱流份）经Sephadex LH-20凝胶柱分离，甲醇洗脱，TLC检测合并依次得到9个流份（Fr.7.5.1~Fr.7.5.9）。
取Fr.7.5.6（300mg）经制备HPLC分离 (YMC-Actus ODS-AC18色谱柱，流动相：甲醇-水 38:62，检测波长210nm)，得wilfordoside A（保留时间49.31min）（12mg）。
实施例2：
wilfordoside A的片剂制备：取50mg相应化合物与淀粉25mg，糊精25mg混合，用适量30%乙醇湿润，常规制粒，加入适量硬脂酸镁混合，压片，即得。
实施例3：
wilfordoside A的胶囊剂制备：取50mg相应化合物与淀粉60mg，糊精10mg，糖粉10mg混合，用适量30%乙醇湿润，常规制粒，装入硬胶囊中，即得。
Claims (2)
1. 松香烷型二萜糖苷化合物Wilfordoside A在制备抗炎药物中的应用。
2.如权利要求1所述的松香烷型二萜糖苷化合物与制剂允许的赋形剂或药用辅料组合在制备抗炎药物中的应用。
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