CN113968869B - 愈创木烷倍半萜内酯类化合物Artemvulactone及其制备方法和应用 - Google Patents
愈创木烷倍半萜内酯类化合物Artemvulactone及其制备方法和应用 Download PDFInfo
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- CN113968869B CN113968869B CN202111315594.1A CN202111315594A CN113968869B CN 113968869 B CN113968869 B CN 113968869B CN 202111315594 A CN202111315594 A CN 202111315594A CN 113968869 B CN113968869 B CN 113968869B
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Abstract
本发明涉及一种愈创木烷倍半萜内酯类化合物Artemvulactone及其制备方法和应用，本发明首先将北艾Artemisia vulgaris L.的叶片粉末经浸提、浓缩后，通过萃取得到粗馏分，进一步采用小孔树脂、葡聚糖凝胶和正相、反相硅胶等柱层析法进行纯化，最后通过高效液相色谱法制备得到六个结构新颖的愈创木烷倍半萜内酯类化合物，本发明的化合物Artemvulactone对LPS诱导RAW264.7细胞一氧化氮的IC50为0.9±0.2μM，相比Dexamethasone的IC50值4.1±0.2μM更低，具有显著的抗炎活性，可作为活性成分应用于化妆品和医药领域。
Description
技术领域
本发明属于医药领域，具体涉及一种愈创木烷倍半萜内酯类化合物Artemvulactone及其制备方法和应用。
背景技术
北艾（Artemisia vulgaris L.）为菊科蒿属植物，广泛分布于中国和蒙古国的一种传统中草药。北艾叶具有广泛的医药用途，自古被用作消炎止痛，驱寒逐湿，温经安胎。研究表明北艾中含有多种化学成分，包括萜类、黄酮类、酚酸类、多糖类及香豆素类等。其中萜类化合物具有重要的生理活性和药用价值，如植物生理激素赤霉素和脱落酸，抗疟疾药物的青蒿素以及抗肿瘤药物的紫杉醇等。
萜类化合物在自然界中分布广泛，截至到2019年，从自然界中发现的萜类化合物已达80,000多个。萜类化合物是一类碳氢骨架以异戊烯为基本结构单元组成的天然产物，其中以三个异戊烯构成的为倍半萜，倍半萜内酯则是基本骨架为三个异戊烯单元构成的内酯化合物。目前，自然界中已分离鉴定的愈创木烷型倍半萜已有数百种，中国专利CN110305092B公开了一种从瑞香科瑞香属植物芫花的根中提取分离6个愈创木烷倍半萜，具有明显的对H2O2诱导的人SH-SY5Y细胞氧化损伤的神经保护活性；中国专利CN112159378A公开了一种从腺梗豨莶中制备的吉马烷型倍半萜内酯类化合物，对于RANKL诱导的破骨细胞具有明显的抑制分化效果，绝大多数化合物的IC50均在10 μM以下，且在10μM下不显示细胞毒性，可用于预防骨质疏松药物领域；中国专利CN11840275A公开了一种从白术中制备出的倍半萜二聚体类化合物，对CREB具有明显的激活作用，可用于抗焦虑或抗抑郁药物领域。中国专利CN109761994A公开了一种从千叶蓍提取分离的降碳类愈创木烷型倍半萜内酯类化合物，其能抑制小鼠巨噬细胞的NO释放量，50μM可将NO释放量降低50%左右，具有一定的抗炎活性；中国专利CN109761948A公开了了一种海绵来源杂萜类化合物dysiarenone，对COX-2酶的表达和炎症介质PGE2的释放显示出很强的抑制活性，其IC50值为6 .4 μM，约为avarol的10倍，为阳性药Dexamethasone的25倍，可用于制备抗炎和抗肿瘤药物。目前看来，对于天然产物中制备的愈创木烷型倍半萜内酯类化合物的药理活性，尤其是抗炎活性并不高，CN109761994A中制备的愈创木烷型倍半萜内酯类化合物将巨噬细胞内NO释放量降低50%需要50 μM，因此，对于挖掘一种具有显著抗炎活性的愈创木烷型倍半萜内酯类化合物可为研制抗炎药物提供新的先导化合物，进一步消除因炎症而引发的各种疾病，减轻因非甾体抗炎药引起的不良胃肠道反应和肝、肾毒性，另外，可以丰富萜类化合物数据库，对于揭示其药效作用提供科学依据。
发明内容
针对上述现有的技术问题，本发明提供了一种愈创木烷倍半萜内酯类化合物Artemvulactone及其制备方法和应用，本发明首先将北艾叶粉末经有机溶剂浸提、浓缩后，通过机溶剂萃取得到粗馏分，进一步采用小孔树脂、葡聚糖凝胶和正相、反相硅胶等柱层析法进行纯化，最后通过高效液相色谱法制备得到六个结构新颖的愈创木烷倍半萜内酯类化合物；
所述愈创木烷倍半萜内酯类化合物Artemvulactone的结构式如1~6所示：
所述愈创木烷倍半萜内酯类化合物Artemvulactone的制备步骤如下：
（1）取干燥的北艾叶粉末，溶剂浸提，提取液浓缩得到粗提物；
（2）将粗提物悬浮于水中，有机溶剂萃取，浓缩得到粗馏分；
（3）粗馏分经柱层析、高效液相制备得到化合物。
所述步骤（1）中北艾叶粉末经过筛后粒径为20目。
所述步骤（1）溶剂中北艾叶粉末的含量为105~150g/L。
所述步骤（1）中溶剂为95%乙醇，冷浸提取3次，单次浸提时间为6~8h。
所述步骤（2）中有机溶剂萃取为石油醚，二氯甲烷，乙酸乙酯依次萃取。
所述步骤（3）中柱层析包括小孔树脂柱层析、正相硅胶柱层析、ODS反相硅胶柱层析、Sephadex LH-20凝胶柱层析中的一种或多种，洗脱体系为甲醇、甲醇-甲酸、甲醇-水、甲醇-水-甲酸、或石油醚-乙酸乙酯。
所述步骤（3）中高效液相采用反相硅胶柱进行制备，流动相为甲醇-水、甲醇-水-甲酸
或乙腈-水、乙腈-水-甲酸。
所述步骤（2）中石油醚萃取的粗馏分部分，经小孔树脂柱层析，采用梯度为60：40~100：0的甲醇-水体系梯度洗脱，进一步经Sephadex LH-20葡聚糖凝胶柱层析，采用甲醇洗脱，然后经ODS反相硅胶柱层析，甲醇-水洗脱体系梯度为40:60~100:0，接着经正相硅胶柱层析，以梯度为95:5~0:100的石油醚-乙酸乙酯进行洗脱，最后经高效液相制备，以甲醇-水洗脱，得到化合物1-6。
与现有技术相比，本发明的有益效果如下：
本发明提供的愈创木烷倍半萜内酯类化合物Artemvulactone具有显著的抗炎活性，对
LPS诱导的RAW264.7细胞一氧化氮的IC50为0.9 ± 0.2 μM，优于Dexamethasone的IC50值4.1 ± 0.2 μM，本发明为研究和开发新的抗炎、抗肿瘤药物提供了新的先导化合物，同时本发明所述的愈创木烷倍半萜内酯类化合物Artemvulactone与药用辅料可制成片剂、胶囊剂和注射剂，可应用于化妆品和医药领域中，具有较好的应用前景。
附图说明
图1为化合物1-6的分子结构式；
图2为化合物1-6的H-H COSY和HMBC主要相关；
图3为化合物1-6的NOESY相关；
图4为化合物1的X-射线单晶衍射晶体结构图；
图5为化合物2的ECD谱图；
图6为化合物3的ECD谱图；
图7为化合物4的X-射线单晶衍射晶体结构图；
图8为化合物5的X-射线单晶衍射晶体结构图；
图9为化合物6的ECD谱图。
具体实施方式
本发明下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。实施例中所用到的各种常用化学试剂，均为市售产品。
除非另有定义，本发明所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不用于限制本发明。
为使本发明的目的、技术方案和优点更加清楚明了，下面结合具体实施方式，对本发明进一步详细说明。应该理解，这些描述只是示例性的，而并非要限制本发明的范围。此外，在以下说明中，省略了对公知结构和技术的描述，以避免不必要地混淆本发明的概念。
以下实施例对本发明做进一步的描述，但该实施例并非用于限制本发明的保护范围。
实施例1化合物的制备
（1） 取干燥的北艾叶2kg粉碎、过20目筛，加入18 L 95%乙醇充分搅匀后冷浸提取3次，合并提取液浓缩得到粗提物；
（2） 将粗提物悬浮于水中，依次采用石油醚，二氯甲烷，乙酸乙酯萃取5次，分别合并萃取液，浓缩得到3个粗馏分；
（3） 取石油醚萃取后的粗馏分，采用小孔树脂柱层析，以甲醇-水系统进行梯度洗脱，洗脱梯度为60:40、70:30、80:20、90:10、100:0，经TLC薄层色谱分析，划分为7个馏分（A~G）；
选取馏分D进行纯化，步骤如下：
Step 1：经SephadexLH-20凝胶柱层析，以甲醇进行洗脱，得到5个馏分（D1~D5）；
Step 2：取馏分D3经ODS柱层析，以甲醇-水系统进行梯度洗脱，洗脱梯度为40:60、50:50、55:45、60:40、65:35、70:30、75:25、80:20、85:15、90:10、95:5和100:0，经TLC薄层色谱分析、合并，划分为7个馏分（D3-1~D3-7）；
Step 3：取馏分D3-5经正相硅胶柱层析，以石油醚-乙酸乙酯系统进行梯度洗脱，洗脱梯度为95:5、93:7、91:9、89:11、87:13、85:15、83:17、81:19、79:21、77:23、75:25、73:27、71:29、69:31、67:33、65:35，0:100，经TLC薄层色谱分析、合并，划分为5个馏分（D3-5-1~D3-5-5）；
（4） 取馏分D3-5-2通过HPLC进行制备和纯化，以63%的甲醇-水为流动相，经C18反相色谱柱纯化得到化合物1~5；
（5） 取馏分D3-5-3通过HPLC进行制备和纯化，以55%的甲醇-水为流动相，经C18反相色谱柱制备得到10个馏分（D3-5-3-1~ D3-5-3-10）；
（6） 取馏分D3-5-3-9通过HPLC进行制备和纯化，以53%乙腈-水洗脱，经C18反相色谱柱纯化得到化合物6。
实施例2化合物鉴定
所得的化合物经系统的波谱学方法鉴定，结果如下：
化合物1：白色粉末，[α] D25+ 83.8 (c 0.08, MeOH)，UV (MeOH)λmax(logε)212(2.69)nm；HRESIMS给出准分子离子峰[M+Na]+峰m/z 383.1462(calcd for C20H24O6Na,383.1462)，结合1H-NMR、13C-NMR确定其分子式为C20H24O6，计算不饱和度为9；通过铜靶单晶X-射线衍射的方法，确定该结构绝对构型为1R,3R,4S,5R,6S,7R,8S,10Z,2’Z（见附图3），命名为ArtemvulactoneA，结构式如下：
化合物2：白色粉末，[α] D25+ 112.5(c 0.04, MeOH)，UV (MeOH) λmax(logε)207(2.91)nm；HRESIMS给出准分子离子峰[M+Na]+峰m/z 385.1614(calcd for C20H26O6Na,385.1614)，结合1H-NMR、13C-NMR确定其分子式为C20H26O6，计算不饱和度为8；通过B3LYP/631G(d) PCM/MeOH相对热自由能(AG)对化合物2的几何构型进行了Boltzmann分布分析，在B3LYP/6-31+G条件下进行了几何优化和频率计算，计算了（CAM-B3LYP/TZVP）（-B3LYP/6-311G(d,p)）ECD谱（见附图4），确定化合物2的绝对构型为1R,3R,4S,5R,6S,7R,8S,10Z,2’R，命名为ArtemvulactoneB，结构式如下：
化合物3：白色粉末，[α] D25+110.0(c 0.04, MeOH)，UV(MeOH)λmax(logε)206(2.78)nm；HRESIMS给出准分子离子峰[M+Na]+峰m/z 385.1617(calcd for C20H26O6Na,385.1617)，结合1H-NMR、13C-NMR确定其分子式为C20H26O6，计算不饱和度为8；通过B3LYP/631G(d) PCM/MeOH相对热自由能(AG)对化合物3的几何构型进行了Boltzmann分布分析，在B3LYP/6-31+G条件下进行了几何优化和频率计算，计算了（CAM-B3LYP/TZVP）（-B3LYP/6-311G(d,p)）ECD谱（见附图5），确定化合物3的绝对构型为1R,3R,4S,5R,6S,7R,8S,10Z，命名为ArtemvulactoneC，结构式如下：
化合物4：白色粉末，[α] D25+ 130.0 (c 0.04, MeOH)，UV (MeOH)λmax(logε)207(2.89)nm；HRESIMS给出准分子离子峰[M+Na]+峰m/z 369.1669(calcd for C20H26O5Na,369.1669)，结合1H-NMR、13C-NMR确定其分子式为C20H26O5，计算不饱和度为8；通过铜靶单晶X-射线衍射的方法，确定该结构绝对构型为3R,4S,5S,6S,7R,8S,10Z,2’R（见附图6），命名为ArtemvulactoneD，结构式为：
化合物5：无色胶状，[α] D25+27.5 (c 0.08, MeOH)，UV (MeOH)λmax(logε)206(2.50)nm；HRESIMS给出准分子离子峰[M+Na]+峰m/z 369.1671(calcd for C20H26O5Na,369.1671)，结合1H-NMR、13C-NMR确定其分子式为C20H26O5，计算不饱和度为8；通过铜靶单晶X-射线衍射的方法，确定该结构绝对构型为3R,4S,5S,6S,7R,8S,10Z（见附图7），命名为ArtemvulactoneE，结构式为：
化合物6：无色胶状，[α] D25+42.5 (c 0.04, MeOH)，UV (MeOH)λmax(logε)257(2.78)nm；HRESIMS给出准分子离子峰[M+Na]+峰m/z 367.1514(calcd for C20H26O5Na,367.1514)，结合1H-NMR、13C-NMR确定其分子式为C20H24O5，计算不饱和度为9；通过B3LYP/631G(d) PCM/MeOH相对热自由能(AG)对化合物6的几何构型进行了Boltzmann分布分析，在B3LYP/6-31+G条件下进行了几何优化和频率计算，计算了（CAM-B3LYP/TZVP）（-B3LYP/6-311G(d,p)）ECD谱（见附图8），确定化合物6的绝对构型为5R,6S,7S,8R,10Z，命名为ArtemvulactoneF，结构式为：
上述化合物1~6的H-H COSY和HMBC主要相关见附图1；化合物1-6的NOESY相关见附图2；化合物1~6的1H-NMR见表1、2，13C-NMR见表3；
表1：化合物1~3的1H-NMR（CDCl3）数据
| Position | 1 | 2 | 3 |
| 1 | 4.17,s(-OH) | 4.14,s(-OH) | 4.14,s(-OH) |
| 2α | 1.96,d | 1.95,d | 1.95,s |
| 2β | 2.50,d | 2.48,d | 2.48,d |
| 3 | 3.57,s | 3.56,s | 3.56,s |
| 5 | 2.59,d | 2.58,d | 2.57,d |
| 6 | 3.93,dd | 3.90,dd | 3.90,dd |
| 7 | 3.37,m | 3.33,m | 3.31,m |
| 8 | 5.41 – 5.45,m | 5.30 – 5.35,m | 5.30 – 5.34,m |
| 9 | 5.34,dd | 5.25,dd | 5.29,dd |
| 13α | 5.75,d | 5.81,d | 5.79,d |
| 13β | 6.31,d | 6.33,d | 6.33,d |
| 14 | 1.92 – 1.94,m | 1.87 – 1.93,m | 1.91 – 1.93,m |
| 15 | 1.73,s | 1.73,s | 1.73,s |
| 2’α | - | 2.43,dd | 2.24,dd |
| 2’β | - | - | 2.31,dd |
| 3’α | 6.22,dd | 1.47 – 1.52,m | 2.16,dt |
| 3’β | - | 1.73 – 1.78,m | - |
| 4’ | 1.92 – 1.94,m | 0.94,t | 1.00,dd |
| 5’ | 2.03,dd | 1.21,d | 1.00,dd |
表2：化合物4~6的1H-NMR（CDCl3）数据
| Position | 4 | 5 | 6 |
| 2α | 2.40 – 2.48,m | 2.49,dd | - |
| 2β | 2.70,d | 2.72,d | - |
| 3 | 3.39,s | 3.41,s | 6.19 – 6.21,m |
| 5 | 3.08,d | 3.09,d | 3.50,d |
| 6 | 3.69,t | 3.71,t | 3.71,t |
| 7 | 3.13,m | 3.13,m | 3.26,tt |
| 8 | 4.79,td | 4.80,td | 4.93,td |
| 9α | 2.14,dd | 2.21,dd | 2.43 – 2.48,m |
| 9β | 2.40 – 2.48,m | 2.43 – 2.47,m | 2.71,dd |
| 13α | 5.55,d | 5.56,d | 5.65,d |
| 13β | 6.13,d | 6.16,d | 6.22,d |
| 14 | 1.71,d | 1.74,d | 2.43 – 2.48,m |
| 15 | 1.66,s | 1.68,s | 2.33,s |
| 2’α | 2.37,m | 2.22 – 2.28,m | 2.25 – 2.27,m |
| 2’β | - | - | 2.28 – 2.31,m |
| 3’α | 1.42-1.49,m | 2.18 – 2.10,m | 2.13-2.19,m |
| 3’β | 1.69-1.74,m | - | - |
| 4’ | 0.92,t | 0.99,d | 1.01,d |
| 5’ | 1.17,d | 0.99,d | 1.01,d |
表3：化合物1~6的13C-NMR（CDCl3）数据
| Position | 1 | 2 | 3 | 4 | 5 | 6 |
| 1 | 80.7 | 80.6 | 80.7 | 136.6 | 136.6 | 133.7 |
| 2 | 42.2 | 42.2 | 42.3 | 33.3 | 33.3 | 195.1 |
| 3 | 63.2 | 63.1 | 63.1 | 63.6 | 63.6 | 136.2 |
| 4 | 67.4 | 67.4 | 67.4 | 67.0 | 66.9 | 169.4 |
| 5 | 60.4 | 60.4 | 60.4 | 51.5 | 51.5 | 51.7 |
| 6 | 75.6 | 75.5 | 75.6 | 78.1 | 78.0 | 81.6 |
| 7 | 48.3 | 48.3 | 48.2 | 56.6 | 56.5 | 55.2 |
| 8 | 71.5 | 71.9 | 72.0 | 70.0 | 70.1 | 69.2 |
| 9 | 122.6 | 122.2 | 122.3 | 41.3 | 41.5 | 44.6 |
| 10 | 138.8 | 138.9 | 139.0 | 128.6 | 128.6 | 144.8 |
| 11 | 135.6 | 135.7 | 135.5 | 137.1 | 137.0 | 136.3 |
| 12 | 168.8 | 168.7 | 168.7 | 168.9 | 168.8 | 168.5 |
| 13 | 124.8 | 124.8 | 124.9 | 120.7 | 120.8 | 122.0 |
| 14 | 24.9 | 25.0 | 25.0 | 22.2 | 22.2 | 21.4 |
| 15 | 19.8 | 19.8 | 19.8 | 19.1 | 19.0 | 20.1 |
| 1’ | 166.9 | 175.4 | 172.4 | 175.8 | 172.2 | 172.0 |
| 2’ | 127.0 | 41.6 | 43.5 | 41.5 | 43.5 | 43.4 |
| 3’ | 140.8 | 26.5 | 25.8 | 26.5 | 25.7 | 25.7 |
| 4’ | 20.7 | 12.0 | 22.6 | 12.0 | 22.6 | 22.6 |
| 5’ | 16.2 | 17.0 | 22.6 | 16.9 | 22.5 | 22.6 |
实施例3药理活性实验
实验方法：将实施例2中的化合物ArtemvulactoneA~F（化合物1~6）作为测试样品进行体外抑制脂多糖诱导的RAW264.7小鼠巨噬细胞一氧化氮释放实验，以磷酸钠地塞米松注射液（Dexamethasone，人工合成抗炎药）作为阳性药，不加样品处理为空白组，样品用DMSO溶解，DMSO在体系中的浓度控制在不影响实验检测浓度范围内，采用MTT法测试样品对RAW264.7小鼠巨噬细胞的细胞毒性，采用Griess法测定样品对脂多糖诱导RAW264.7小鼠巨噬细胞一氧化氮的抑制效果，每个样品浓度在测试中均设置3个复孔。
具体实验步骤：将RAW264.7小鼠巨噬细胞在DMEM培养基（包含10%FBS，1%青霉素，1%链霉素）中培养，放置于37℃、5% CO2培养箱中培养，将培养至对数生长期细胞按6×104cells/孔铺至96孔板中贴壁培养24 h后，用含1 μg/ml脂多糖和0-2μM浓度范围样品的DMEM培养18 h，取50 μl培养液上清及Griess试剂A和B各50μl添加至96孔板中，在540nm波长测量吸光值。
按上述方法得到的结果如表4所示；
表4为化合物ArtemvulactoneA~F（化合物1~6）对LPS诱导的RAW264.7细胞中NO抑制活性，及对RAW264.7细胞毒性测试，数据表示为平均值± SD, n = 3；IC50表示NO半数抑制浓度；CC50表示细胞活力半数抑制浓度：
| Compound | IC50/μM | CC50/μM |
| 1 | 1.4± 0.2 | >10 |
| 2 | 1.9± 0.1 | >10 |
| 3 | 2.3± 0.2 | >10 |
| 4 | 1.3± 0.2 | >6 |
| 5 | 0.9± 0.2 | >6 |
| 6 | 1.2 ± 0.2 | >6 |
| Dexamethasone | 4.1± 0.2 | - |
由药理活性研究结果显示，本发明的化合物ArtemvulactoneA~F（化合物1~6）具有明显的抑制脂多糖诱导的RAW264.7小鼠巨噬细胞一氧化氮的产生，相比Dexamethasone的IC50值4.1 ± 0.2 μM，6个化合物的IC50均低于Dexamethasone，具有抑制活性，其中化合物ArtemvulactoneE（化合物5）具有更显著的抑制效果，对LPS诱导的RAW264.7细胞一氧化氮的IC50为0.9 ± 0.2 μM，可作为抗炎活性原料应用于化妆品和医药产品。
需要说明的是，本说明书中描述的具体特征、结构、材料或者特点可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例以及不同实施例的特征进行结合和组合。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
Claims (1)
1.一种愈创木烷倍半萜内酯类化合物Artemvulactone的制备方法，其特征在于，包括以下步骤：
（1）取干燥的北艾叶粉碎后经20目过筛得到北艾叶粉末，将艾叶粉末与95%乙醇混合，
混合液中艾叶粉末含量为105~150 g/L，经冷浸提取3次，单次浸提时间为6~8 h，提取液浓缩得到粗提物；
（2）将粗提物悬浮于水中，依次用石油醚，二氯甲烷，乙酸乙酯进行萃取，浓缩得到粗馏分；
（3）将步骤（2）中石油醚萃取的粗馏分部分，经小孔树脂柱层析，采用梯度为60:40~100:0的甲醇-水体系梯度洗脱，得到7个馏分A~G；将所述馏分D进一步经Sephadex LH-20凝胶柱层析，采用甲醇洗脱，得到的5个馏分D1~D5；将所述馏分D3进一步ODS反相硅胶柱层析，采用梯度为40:60~100:0的甲醇-水洗脱体系，得到7个馏分D3-1~D3-7；将所述馏分D3-5经正相硅胶柱层析，以梯度为95:5~0:100的石油醚-乙酸乙酯进行洗脱，得到5个馏分D3-5-1~D3-5-5，所述馏分D3-5-2经高效液相制备，采用C18反相硅胶柱，以50~80%甲醇-水洗脱，得到化合物1-5结构式为：
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