CN113563296B - 一种间苯三酚杂萜类化合物的抗新冠用途及制备方法 - Google Patents
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Abstract
本发明公开了一种间苯三酚杂萜类化合物的抗新冠用途及制备方法，该间苯三酚杂萜类化合物具有抗新冠病毒活性，所述新冠病毒包括：SARS‑CoV‑2。该间苯三酚杂萜类化合物为大羽鳞毛蕨的提取物。本发明首次发现大羽鳞毛蕨提取物具有抗新冠作用，提取物间苯三酚杂萜类化合物对新冠病毒感染的VeroE6及Calu‑3均具明显的抑制作用，同步的毒性测试表明显示出低毒性，可以用于作为制备治疗抗新冠病毒的药物。
Description
技术领域
本发明涉及一种大羽鳞毛蕨提取物，具体涉及一种间苯三酚杂萜类化合物的抗新冠用途及制备方法。
背景技术
大羽鳞毛蕨为鳞毛蕨科(Dryopteridaceae)鳞毛蕨属(Dryopteris)植物粗莖鳞毛蕨(Dryopteris wallichiana)的干燥根莖及叶柄残基。鳞毛蕨类植物通常具有味苦，微寒，有小毒，归肝、胃经，具有清热解毒、驱虫等功效。
近年来出现的由SARS-CoV-2病毒(Severe Acute Respiratory SyndromeCoronavirus 2，严重急性呼吸综合征冠状病毒2)引起的严重急性呼吸系统综合征，即COVID-19(CoronaVirus Disease 2019，新型冠状病毒肺炎)，给经济和社会带来损害，当前还没有效的抗病毒药物被批准用于预防或治疗高传染性的SARS-CoV-2和中东呼吸道冠状病毒综合征(MERS-CoV)。
一些前体和潜在的候选药物，如瑞德西韦和氯喹，已被认为是针对COVID-19的治疗方案。瑞德西韦是核苷类似物，是RNA依赖性RNA聚合酶(RdRP)抑制剂，通过靶向病毒RNA聚合酶来降低病毒复制(EC50＝0.77μM)有效抑制Vero E6细胞的SARS-CoV-2感染。一项试验发现，在同情给药中短期使用瑞得西韦治疗中没有观察到明显的治疗效果。氯喹被广泛用于治疗自身免疫性疾病和疟疾，并可应用于对抗某些病毒感染。该药物可以通过增加病毒与细胞融合所介导的pH或与病毒复制的后期相互作用来阻止病毒感染，最近指出它可以抑制病毒Vero E6细胞中的SARS-CoV-2(EC50＝1.13μM))。一些临床试验表明，采用氯喹治疗COVID-19患者显示出临床部分有效性，但大多数临床数据仍然是初步的。到目前为止，还没有开发出针对SARS-CoV-2的临床抗病毒药物。
发明内容
本发明的目的是提供一种间苯三酚杂萜类化合物的抗新冠用途及制备方法，解决了目前尚未有SARS-CoV-2的抗病毒药物，该间苯三酚杂萜类化合物对新冠病毒感染的VeroE6及Calu-3均具明显的抑制作用，同步的毒性测试表明显示出低毒性。
为了达到上述目的，本发明提供了一种间苯三酚杂萜类化合物的抗新冠用途，该间苯三酚杂萜类化合物的结构式如下所示：
该间苯三酚杂萜类化合物具有抗新冠病毒活性，所述新冠病毒包括：SARS-Cov-2。
优选地，所述间苯三酚杂萜类化合物为大羽鳞毛蕨的提取物。
优选地，所述间苯三酚杂萜类化合物用于作为制备治疗抗新冠病毒的药物。
优选地，所述间苯三酚杂萜类化合物与药学上可接受的辅料制成制剂用于作为制备治疗抗新冠病毒的药物。
优选地，所述制剂包含：胶囊剂、片剂、颗粒剂、凝胶剂、缓释剂、口服液、滴丸剂和纳米制剂中任意一种。
本发明的另一目的是提供所述的间苯三酚杂萜类化合物的制备方法，该制备方法包含：将大羽鳞毛蕨以有机溶剂作为提取溶剂通过浸渍提取、回流提取、渗漉提取、微波提取和超声提取中任意一种方式提取，将提取液浓缩，得到浓缩液；将所述浓缩液采用硅胶柱层析，石油醚-乙酸乙酯梯度洗脱，经TLC检测合并组分，将以体积比为100:0～1:1的石油醚-乙酸乙酯洗脱得到的洗脱液作为组分一；将组分一去除杂质叶绿素和叶黄素，经过HPLC正相柱纯化获得化合物3和化合物4，HPLC正相柱采用的洗脱液为含0.5％乙酸的正己烷–乙酸乙酯或含0.5％乙酸的石油醚-乙酸乙酯。
优选地，所述大羽鳞毛蕨采用浸渍提取的方式提取。
优选地，所述有机溶剂选自乙醚、丙酮、乙酸乙酯、乙醇和甲醇中任意一种或两种以上。
优选地，采用体积比从100:0到0:100的石油醚-乙酸乙酯进行梯度洗脱。
优选地，所述含0.5％乙酸的正己烷–乙酸乙酯或含0.5％乙酸的石油醚-乙酸乙酯中，正己烷或石油醚与乙酸乙酯的体积比为95:5。
本发明的间苯三酚杂萜类化合物的抗新冠用途及制备方法，解决了目前尚未有SARS-CoV-2的抗病毒药物，具有以下优点：
本发明的间苯三酚杂萜类化合物，对新冠病毒感染的Vero E6及Calu-3均具明显的抑制作用，同步的毒性测试表明显示出低毒性，本发明的活性化合物3-4在抗新冠病毒感染Vero E6细胞实验中，化合物3的EC50值为4.5μM，化合物4的EC50值为12.1μM，化合物3和4的EC50值为6.8μM；在抗新冠病毒感染Calu-3细胞实验中，化合物3的EC50值为20.2μM，化合物4的EC50值为30.0μM，化合物3和4的EC50值为24.8μM，对氯喹没有活性的肺细胞也显示出明确的抑制活性。
本发明的间苯三酚杂萜类化合物通过对大羽鳞毛蕨提取获得，利用多种色谱方法(正相硅胶、反相硅胶、凝胶和高效液相色谱HPLC)分离纯化得到抗新冠活性化合物，本发明首次发现大羽鳞毛蕨提取物具有抗新冠作用，可以作为抗新冠药用植物进一步研究。
附图说明
图1为本发明化合物1-6在10μM终浓度下的抗新冠病毒感染Vero E6细胞活性抑制率。
图2为本发明活性化合物3-4在抗新冠病毒感染Vero E6细胞活性EC50值。
图3为本发明活性化合物3-4在抗新冠病毒感染Calu-3细胞活性EC50值。
图4为本发明活性化合物3-4在细胞毒性CC50值。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、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。
以下实验中使用的试剂及仪器等，如下：
1、试验材料：羽鳞毛蕨2019年12月产地云南，经中国科学院昆明植物研究所鉴定为大羽鳞毛蕨的干燥鳞茎。用粉碎机将大羽鳞毛蕨干燥鳞茎粉碎后过40目孔径筛，密封保存备用。
2、试剂：甲醇，乙醚、乙腈均为色谱分析纯，为国药集团化学试剂公司产品。
3、仪器：Agilent 1260HPLC高效液相；KERN电子分析天平(北京赛多利斯仪器系统有限公司)；Heidolp旋转蒸发仪(德国Hei-VAP公司)；色谱柱ZORBAX SB-C18(4.6×250mm，Agilent，美国)和HPLC正相柱。
实验例1大羽鳞毛蕨提取物
将大羽鳞毛蕨干燥块茎4.7kg粉碎后，用乙醚(或丙酮)室温浸泡3次，减压浓缩获得浸膏。该部分经硅胶柱层析石油醚-乙酸乙酯梯度洗脱(体积比100:0～0:100)，石油醚-乙酸乙酯的体积比100:0～1:1收集的组分为Fr.A，石油醚-乙酸乙酯体积比1:1～0:100收集的组分为Fr.B)。
Fr.A(60.0g)经过凝胶除去叶绿素和叶黄素等杂质。进一步经过HPLC正相柱(体积比为95:5的n-hexane–EtOAc，0.5％HOAc，2mL/min)组纯化获得化合物3-6，化合物3(黄色油状物)含量320mg，化合物4(黄色油状物)含量260mg，化合物5(黄色油状物)含量50mg，化合物6(黄色油状物)含量30mg；
进一步Fr.B(5.3g)经Sephadex LH-20(体积比为1:1的氯仿–甲醇)洗脱，后经硅胶柱层析，石油醚–乙酸乙酯(体积比1:5–1:1)，再经过HPLC ZORBAX SB-C18柱子(体积比为30:35:35的H2O–MeOH–isoPropanol，0.5％HCOOH，2.0mL/min)，得到化合物1(黄色油状物)为45mg，化合物2(黄色油状物)为10mg。
通过核磁、质谱、紫外、圆二色、旋光多种波谱分析方法，确定化合物1-化合物6的结构如上所示。
化合物1的核磁表征数据如下所示：
1H NMR(600MHz,acetone-d6,25℃):δ19.02(s,1H)；5.48(t,6.4,1H)；4.82(m,1H)；4.52(m,1H)；2.51(s,3H)；2.39–2.36(m,1H)；2.37–2.29(m,1H)；2.34-2.45(m,2H)；2.16(ddd,16.2,10.8,6.4,1H)；2.06-2.00(m,1H)；2.02(d,3.3Hz,2H)；1.98-1.83(m,2H)；1.85-1.87(m,1H)；1.81(m,1H)；1.79-1.87(1H)；1.73(s,3H)；1.58-1.60(m,1H)；1.58(m,2H)；1.46(td,12.8,4.3,1H)；1.38(m,1H)；1.34(s,3H)；1.30(s,3H)；1.23(td,12.9,3.5,1H)；1.14(s,3H)；0.79(s,3H)。
13C NMR(150MHz,acetone-d6,25℃)δ200.6；196.8；189.1；180.0；174.9；148.9；133.2；129.4；83.8；47.8；108.358.0；50.4；47.8；39.639.1；38.4；37.8；27.8；27.3；25.8；25.2；24.9；23.1；19.2；17.9；17.1；15.0；12.4。
化合物1的质谱表征数据为：ESI+:m/z 511[M+H]+。
化合物2的核磁表征数据如下所示：
1H NMR(600MHz,acetone-d6,25℃):δ19.01(s,1H)；5.47(t,6.3,1H)；4.82(m,1H)；4.49(m,1H)；2.51(s,3H)；2.39–2.36(m,1H)；2.37–2.29(m,1H)；2.35-2.47(m,2H)；2.18(m,1H)；2.06-2.00(m,1H)；2.02(d,3.3Hz,2H)；1.98-1.83(m,2H)；1.86(m,1H)；1.81(m,1H)；1.79-1.87(1H)；1.73(s,3H)；1.58-1.60(m,1H)；1.58(m,2H)；1.46(td,12.5,3.8,1H)；1.38(m,1H)；1.34(s,3H)；1.30(s,3H)；1.24(td,12.8,3.9,1H)；1.14(s,3H)；0.79(s,3H)。
13C NMR(150MHz,acetone-d6,25℃)δ200.7；197.0；189.1；180.1；175.0；149.0；133.3；129.8；84.0；47.8；108.358.0；50.4；47.8；39.639.1；38.4；37.8；27.9；27.3；25.8；25.2；24.8；23.1；19.2；18.0；17.2；15.0；12.3。
化合物2的质谱表征数据为：ESI+:m/z 511[M+H]+。
化合物3的核磁表征数据如下所示：
1H NMR(600MHz,acetone-d6,25℃):δ18.58(s)；16.49(s)；11.28(s)；10.17(s)；5.57(t,6.6,1H)；4.53(d,2.1,1H)；4.83(s,1H)；4.43(dd,10.3,2.3,1H)；3.52(s,2H)；2.70(s,3H)；3.03(ddd,15.1,8.3,6.4,1H)；2.94(ddd,15.6,8.3,6.6,1H)；2.74(ddd,16.64,5.57,2.65,1H)；2.56(m,1H)；2.37(m,1H)；2.02(m,1H)；1.96-2.03(m,1H)；2.37(m,1H)；2.17(ddd,16.3,10.7,6.7,1H)；1.91-1.84(overlapping signals),1.89-1.96(overlapping signals)；1.88(m,1H)；1.81(s,3H)；1.77-1.83(d,3.24,1H)；1.64(m,2H)；1.63-1.70(m,13.01,3.38,1H)；1.58-1.69a(m,2H)；1.48(s,3H)；s 1.48(s,3H)；1.45(m,12.8,4.3,1H)；1.37(m,1H)；1.23(td,12.8,4.0,2H)；1.14(s,3H)；0.96(t,7.23,3H)；0.79(s,3H)。
13C NMR(150MHz,acetone-d6,25℃)δ207.3；204.3；199.9；188.3；179.7；172.6；162.6；161.7；158.5；148.9；133.9；130.2；112.1；109.2；108.3；106.1；104.8；103.9；83.4；58.1；50.4；47.8；46.5；45.0；39.6；39.1；38.4；37.8；29.2；27.3；25.9；25.0；24.9；23.2；20.2；19.2；17.2；17.1；15.0；14.4；12.6。
化合物3的质谱表征数据为：ESI-:m/z 717[M-H]-.
化合物4的核磁表征数据如下所示：
1H NMR(600MHz,acetone-d6,25℃):δ18.58(s)；16.49(s)；11.28(s)；10.17(s)；5.55(t,6.41,1H)；4.54(s,1H)；4.83(d,2.2,1H)；4.44(dd,10.5,2.2,1H)；3.53(s,2H)；2.70(s,3H)；3.03(ddd,16.11,7.26,1H)；2.96(dt,16.11,7.26,1H)；2.74(ddd,16.52,5.54,2.57,1H)；2.57(m,1H)；2.35(m,1H)；2.03(m,1H)；1.96-2.03(m,1H)；2.35(m,1H)；2.20(ddd,16.3,10.85,6.93,1H)；1.91-1.84(overlapping signals),1.89-1.96(overlapping signals)；1.89(m,1H)；1.81(s,3H)；1.76-1.83(m,3.24,1H)；1.66(m,2H)；1.57-1.60(dd,13.01,3.38,1H)；1.58-1.69(m,2H)；1.48(s,3H)；1.48(s,3H)；1.45(m,12.8,4.1,1H)；1.38(m,1H)；1.23(td,12.9,3.80,2H)；1.15(s,3H)；0.95(t,3H)；0.80(s,3H)。
13C NMR(150MHz,acetone-d6,25℃)δ207.3；204.3；199.9；188.3；179.7；172.6；162.6；161.6；158.5；148.9；133.9；130.4；112.1；109.2；108.4；106.1；104.8；103.9；83.4；58.1；50.4；47.8；46.4；45.0；39.6；39.1；38.4；37.8；29.2；27.3；26.0；25.0；24.9；23.2；20.2；19.0；17.2；17.1；15.0；14.3；12.6。
化合物4的质谱表征数据为：ESI-:m/z 717[M-H]-.
化合物5的核磁表征数据如下所示：
1H NMR(600MHz,acetone-d6,25℃):δ18.58(s)；14.17(s)；11.41(s)；9.11(s)；5.67(m,6.3)；4.63(dd,10.1,2.0)；4.86(d,1.2)；4.62(d,1.2)；3.48(d,16.4)；3.46(d,16.4)；3.16(dt,16.6,7.3)；3.11(dt,16.6,7.3)；2.72(ddd,16.7,5.7,3.1)；2.71(s)；2.51(ddd,16.7,11.3,5.7)；2.41(dd,15.6,6.3)；2.36(m,12.8,4.3,2.5)；2.25(ddd,15.6,11.0,6.3)；2.07–2.00(overlapping signals)；2.07–1.96(overlapping signals)；2.03(dd,2.5)；1.91–1.83(overlapping signals)；1.90(d,10.1)；1.85(s)；1.80(td,12.4,4.3)；1.70(m,7.3)；1.70–1.58(overlapping signals)；1.64–1.58(overlappingsignals)；1.52(s)；1.50(s)；1.47(td,12.8,4.3)；1.39(m,12.8,5.3,2.5)；1.15(s)；1.23(td,12.8,3.9)；0.98(t,7.3)；0.81(s)。
13C NMR(150MHz,acetone-d6,25℃)δ207.7,204.4,199.7,188.4,179.7,172.2,163.2,158.7,158.2,148.7,132.8,132.8,112.1,109.2,108.6,106.7,105.0,102.7,85.0,57.9,50.4,47.7,46.9,44.9,39.5,39.1,38.4,37.8,29.3,25.1,27.3,26.0,24.9,23.3,9.1,18.8,17.6,15.0,14.5。
化合物5的质谱表征数据为：ESI-:m/z 717[M-H]-。
化合物6的核磁表征数据如下所示：
1H NMR(600MHz,acetone-d6,25℃):δ18.55(s)；14.14(s)；11.46(s)；9.07(s)；5.72(m,6.3)；4.86(d,1.3)；4.62(dd,10.1,3.0)；4.59(d,1.4)；3.48(d,16.0)；3.46(d,16.0)；3.17(dt,16.6,7.3)；3.14(dt,16.6,7.3)；2.75(ddd,16.7,5.1,3.1)；2.70(s)；2.56-2.47(overlapping signals),2.39(m,12.8,4.3,2.8)；2.17(ddd,15.2,11.0,6.0)；210–2.00(overlapping signals)；2.04–1.96(overlapping signals)；2.07(dd,12.9,2.7)；1.93(overlapping signals)；2.04-1.96(overlapping signals)；1.85(s)；1.81(m,12.6,4.1)；1.70(m,7.3)；1.70–1.59(overlapping signals)；1.71–1.59(overlappingsignals)；1.55(s)；1.51(s)；1.47(td,12.8,4.3)；1.39(m,12.8,5.3,2.7)；1.15(s)；1.27(td,12.8,4.0)；0.98(t,7.3)；0.81(s)。
13C NMR(150MHz,acetone-d6,25℃)δ207.8,204.4,199.7,188.4,179.6,172.1,163.2,158.7,158.3,148.8,132.8,132.7,112.1,109.2,108.3,106.7,105.1,102.7,85.3,58.2,50.5,47.7,46.9,44.9,39.5,39.3,38.4,37.8,29.3,25.1,27.3,25.7,24.9,23.2,19.1,18.8,17.6,15.0,14.5。
化合物6的质谱表征数据为：ESI-:m/z 717[M-H]-。
实验例1药物活性实验
(1)将Vero E6细胞(或Calu-3细胞)接种到48孔板中，每孔约5×104个细胞，待第二天实验，然后放入37℃、5％CO2培养过夜。
(2)药物与细胞孵育：用含2％胎牛血清的DMEM培养基稀释化合物1-6。三倍倍比稀释药物，每个浓度设置3个复孔，共6个药物梯度；DMSO作为对照组，对照组采用含总体积2％胎牛血清的DMEM培养基稀释，给予药物同体积的二甲基亚砜。去除细胞上清后，在1.2的48孔板中加入100μL稀释后的化合物，对照组加入同等体积稀释后的DMSO，置37℃孵育1h。
(3)SARS-Cov-2感染细胞：在48孔板每孔加入5μL的SARS-CoV-2病毒稀释液(MOI＝0.01Vero E6与MOI＝0.05Calu-3)37℃继续孵育lh，去除感染物上清，用200μLPBS(磷酸盐缓冲液)洗一遍。重新在孔中加入200μL对应浓度含药物的培养基，继续培养24h，收取150μL上清待测。
(4)病毒RNA提取采用试剂盒Takara MiniBEST Viral RNA/DNA Extraction Kit：病毒裂解，在150μL细胞培养上清中加入50μL PBS溶液使总体积为200μL。依次加入200μL的Buffer VGB缓冲液、20μL的Proteinase K、1μL的Carrier RNA，振荡混匀，置于56℃水浴10min充分裂解。再加入200μL无水乙醇，振荡混匀。再经过过柱、洗涤、洗脱获得病毒RNA。
(5)病毒RNA逆转录：操作方法参见TakaraPrimeScriptTM RT reagent Kit withgDNA Eraser试剂盒。去除洗脱液中的DNA，再进行逆转录反应备用。
(6)采用标准曲线法获得病毒拷贝数：参照试剂盒Takara TB
Premix ExTaqTMII，用已知拷贝数的RBD质粒做标准品，特异性引物靶向RBD，计算出每个样品的拷贝数。以空白DMSO组拷贝数作为参照，获得化合物处理组抑制率。根据不同浓度化合物处理组抑制率，运用Prism6.0.1软件，拟合出抑制率曲线，并计算出半数有效浓度EC50。
如图1所示，为本发明的化合物1-6在10μM终浓度下的抗新冠病毒感染Vero E6细胞活性抑制率，可以看出化合物3和4(天然含量约为1.2:1比例)能够抑制新冠病毒感染Vero E6细胞。
如图2所示，为本发明的活性化合物3-4在抗新冠病毒感染Vero E6细胞活性EC50值，可以看出，化合物3的EC50值为4.5μM，化合物4的EC50值为12.1μM，化合物3和4的EC50值为6.8μM。
如图3所示，为本发明的活性化合物3-4在抗新冠病毒感染Calu-3细胞活性EC50值，可以看出，化合物3的EC50值为20.2μM，化合物4的EC50值为30.0μM，化合物3和4的EC50值为24.8μM，对氯喹没有活性的肺细胞(Calu-3)也显示出明确的抑制活性。
实验例2药物毒性实验
(1)获得处于对数生长期的Vero E6细胞，调整细胞密度至5×104个/孔，分别以100μL/孔接种于96孔板，培养过夜。
(2)给药前采用含总体积2％胎牛血清的DMEM培养基倍比稀释8个浓度梯度，药物梯度浓度设置为2000μM、1000μM、500μM、125μM、62.5μM、31.25μM、15.625μM、7.8125μM)，取每孔100μL此稀释后的药物分别加入1.2.1中96孔板中的Vero E6细胞中，终体积200μL。设置3个复孔，DMSO溶剂处理组为空白对照。
(3)在孵箱中孵育48h后，每孔加入10μL CCK-8工作液，继续孵育3h，测定450nm处吸光度。
(4)根据OD450值，分别计算与对照组相比，在各个浓度的药物的作用下Vero E6细胞的存活率。
如图4所示，为本发明的活性化合物3-4在细胞毒性CC50值，可以看出，化合物3的CC50值为160.0μM，化合物4的CC50值为140.2μM，化合物3和4的CC50值为163.2μM。
尽管本发明的内容已经通过上述优选实施例作了详细介绍，但应当认识到上述的描述不应被认为是对本发明的限制。在本领域技术人员阅读了上述内容后，对于本发明的多种修改和替代都将是显而易见的。因此，本发明的保护范围应由所附的权利要求来限定。
Claims (5)
1.一种间苯三酚杂萜类化合物在制备抗新冠药物中的用途，其特征在于，该间苯三酚杂萜类化合物的结构式如下所示：
该间苯三酚杂萜类化合物具有抗新冠病毒活性，所述新冠病毒为SARS-Cov-2。
2.根据权利要求1所述的用途，其特征在于，所述间苯三酚杂萜类化合物为大羽鳞毛蕨的提取物。
3.根据权利要求1所述的用途，其特征在于，所述间苯三酚杂萜类化合物用于作为制备抗新冠病毒的药物。
4.根据权利要求3所述的用途，其特征在于，所述间苯三酚杂萜类化合物与药学上可接受的辅料制成制剂用于作为制备抗新冠病毒的药物。
5.根据权利要求4所述的用途，其特征在于，所述制剂选自胶囊剂、片剂、颗粒剂、凝胶剂、缓释剂、口服液、滴丸剂和纳米制剂中任意一种。
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| Unusual terpenylated acylphloroglucinols from Dryopteris wallichiana;Cecilia Socolsky等;《Phytochemistry》;20120601;第80卷;第121页4.3节 * |
| 治疗新冠肺炎小分子药物研发国际现状和思考;李治非等;《国际药学研究杂志》;20200731;第47卷(第7期);第522-528页 * |
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