CN110974834A - 黄褐毛忍冬酸水解产物在制药中的应用及其制备方法 - Google Patents
黄褐毛忍冬酸水解产物在制药中的应用及其制备方法 Download PDFInfo
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Abstract
本发明公开了黄褐毛忍冬酸水解产物在制药中的应用及其制备方法，上述化合物中常春藤皂苷元对丙型肝炎病毒蛋白酶NS3/4A具有一定的抑制作用，可单独用于制备预防和治疗丙型肝炎的药物，也可与其它组分复配用于预防和治疗丙型肝炎；咖啡酸、咖啡酸乙酯对艾滋病病毒蛋白酶具有较好的抑制作用，可单独用于制备预防和治疗艾滋病的药物，也可与其它组分复配用于预防和治疗艾滋病。黄褐毛忍冬酸水解产物具有较好抗艾滋病和丙型肝炎的作用，可用于艾滋病患者、丙型肝炎患者和艾滋病‑丙型肝炎联合感染病人的用药。
Description
技术领域
本发明涉及药物技术领域，特别是黄褐毛忍冬酸水解产物在制备治疗艾滋病和丙型肝炎药物中的应用及制备方法。
背景技术
HIV-1是造成艾滋病的病原体，其中HIV-1蛋白酶是艾滋病病毒圆熟和感染过程中所必须的天冬氨酸蛋白酶，由99个氨基酸单体形成的二聚体结构。HIV-1蛋白酶是寻找抗艾滋病病毒药物的重要靶点。
另一方面，单链RNA黄病毒丙型肝炎病毒(HCV)是造成丙型肝炎的重要原因，感染丙型肝炎病毒常导致肝硬化和肝癌，它由9.6kb的基因组编码形成结构蛋白C,E1,E2,和非结构蛋白NS2,NS3,NS4A,NS4B,NS5A and NS5B。由于NS3-NS4A蛋白酶在HCV病毒复制过程中扮演着重要的作用，该丝氨酸蛋白酶被公认为是筛选抗丙型肝炎药物的有效靶点.
当前公认的抗艾滋病药物包括高效抗逆转录病毒治疗法(HAART)，是通过艾滋病病毒蛋白酶抑制剂(HIV PR)和逆转录酶抑制剂联用,已经显著降低艾滋病人死亡率提高感染艾滋病病毒人员的生活质量.5,6然而由于艾滋病病毒容易突变并且很快产生耐药性，造成艾滋病病毒的防治和治疗不足，导致艾滋病比其他感染性疾病的死亡人数更多。
人体感染丙型肝炎病毒所致的丙型肝炎，已成为全球最棘手的健康问题,目前全世界已有1.7亿人感染。若无药物的治疗,长期感染丙型肝炎病毒将发展成为慢性肝炎、肝坏死、肝癌等疾病。当前最有效的治疗方法是聚乙二醇干扰素联合利津巴韦，该方法对于广大发展中国家普遍存在的I型丙型肝炎治疗有效率低于50％,长期使用干扰素存在许多副作用，如：白细胞、血小板、嗜中性白血球的减少和抑郁症等；长期使用利津巴韦，也将产生溶血性贫血等副作用，使得患者不能持续治疗，因此发展独特、有效、更安全的丙型肝炎治疗药物尤为紧迫。
丙型肝炎病毒蛋白酶NS3/4A在病毒圆熟过程中起着重要的作用，已被证实是发现治疗丙型肝炎药物的有效靶点之一。目前三种丙型肝炎NS3/4A蛋白酶抑制剂，BILN 2061,VX-950和SCH 503034，已被临床证实疗效显著，但多为肽类抑制剂，合成困难，并有一定的毒副作用。
中药黄褐毛忍冬系忍冬科植物(Lonicera fulvotomentosa Hsu et S.C.Cheng)的干燥花蕾或带初开的花，又名山银花，具有清热解毒，疏散风热的功效，主治温病发热、热毒血痢、痈肿疔疮、丹毒、热毒血痢、风热感冒、温病发热等多种感染性疾病。该植物主要分布于中国广西西北部、贵州西南部和云南。生长在海拔850-1300米的山坡岩旁灌木林或林中。
发明内容
本发明所要解决的技术问题是克服现有治疗艾滋病和丙型肝炎的药物合成困难、副作用多的缺陷，提供黄褐毛忍冬酸水解产物在治疗艾滋病和丙型肝炎药物中的应用，同时提供酸水解产物制备、分离和纯化的方法。
为了解决上述技术问题，本发明采用如下的技术方案：
本发明提供了黄褐毛忍冬酸水解产物在治疗艾滋病和丙型肝炎药物的应用。
本发明还提供了从黄褐毛忍冬(Lonicera fulvotomentosa Hsu et S.C.Cheng)中提取、分离和纯化酸水解产物的方法：取黄褐毛忍冬干燥花蕾或带初开的花20Kg，粉碎，用2mol/L硫酸-乙醇溶液水解，每次3h。药渣用水清洗，2mol/LNaOH调pH中性,加等体积氯仿加热回流提取3次,每次1h。减压浓缩氯仿提取液，得
膏1528.07g。通过硅胶柱(7.8×114cm)层析分离，石油醚-乙酸乙酯(纯石油醚-80:1-乙酸乙酯)梯度洗脱得到308个流份，Fr.1～Fr.308。Fr.2(1.5g)经硅胶柱色谱(3×64cm)分离,石油醚-乙酸乙酯(100:1-20:1)梯度洗脱，得化合物β-谷甾醇(987mg)。Fr.11(1.8g)通过硅胶柱层析(3×64cm)，石油醚-乙酸乙酯(100:1-50:1)梯度洗脱，得Fr.11A22-25(195mg)，运用制备薄层色谱(石油醚：乙酸乙酯＝80:1)，得化合物5,5'-二丁氧基-2,2'-联呋喃(65mg)。Fr.14(850mg)通过硅胶柱层析(3.5×75cm)，石油醚-乙酸乙酯(100:1-40:1)梯度洗脱，获得38个流份,其中Fr.14A13混合溶剂重结晶得化合物二十九烷-10-醇(18mg)。Fr.38(10.5g)经过反复硅胶柱层析(3.5×75cm)，石油醚-乙酸乙酯(100:1-30:1)梯度洗脱，得化合物常春藤皂苷元乙酯(6.3g)。Fr.56经过硅胶柱层析(3×50cm)，二氯甲烷-甲醇(100:1-60:1)梯度洗脱得化合物齐墩果酸(25mg)。Fr.65-69(15.0g)经过硅胶柱层析(3.6×70cm)，二氯甲烷-甲醇(100:1-40:1)梯度洗脱，并用混合溶剂重结晶，在Fr.65-69B15-52和Fr.65-69E11-分别获得化合物咖啡酸乙酯(4.481g)和化合物咖啡酸(1.36g)。Fr.85(1.2g)经ODS硅胶柱(2.5×50cm)，甲醇-水(5:5-100:0)梯度洗脱，其中Fr.85A22(108mg)硅胶柱分离(2.0×30cm)，二氯甲烷-甲醇(50:1-15:1)梯度洗脱，得化合物异香草醛(15mg)。Fr.124-285(279g)经硅胶柱层析(4.0×100cm)，二氯甲烷-甲醇(50:1-15:1)梯度洗脱，得化合物常春藤皂苷元(24.5g)。
为了确认所提取化合物的结构及验证其抗艾滋病和丙型肝炎的作用，发明人做了相关的实验如下：
1、化合物的结构鉴定
采用上述技术方案从黄褐毛忍冬酸水解产物中所提取获得的化合物，经1H-NMR,13C-NMR,MS和与文献对照，被确定为：
化合物1白色针晶(氯仿)。EI-MS m/z:414[M+]。薄层对照Rf值与对照品与β-谷甾醇一致，故鉴定该化合物为β-谷甾醇。
化合物2白色针晶(石油醚-乙酸乙酯)。EI-MS m/z:162.1[M+].1H-NMR(CDCl3,400MHz)δ:0.87(3H,s,H-9),1.11(3H,s,H-9′),1.45(1H,m,H-8和H-8′),1.68(1H,m,H-4),1.74(1H,m,H-4′),4.32(1H,t,H-6和H-6′),7.53(1H,dd,J＝3.3,5.6Hz,H-7和H-7′),7.72(1H,dd,J＝3.3,5.6Hz,H-3和H-3′).13C-NMR(CDCl3,100MHz)δ:13.7(C-9和C-9′),19.4(C-8和C-8′),29.4((C-7),30.7(C-7′),65.6(C-6和C-6′),128.7(C-3和C-3′),131.1(C-4和C-4′),131.9(C-2和C-2′),167.5(C-5和C-5′)。以上氢谱、碳谱和质谱数据与文献[1]报道的5,5′-dibutoxy-2,2′-bifuran基本一致，故确定化合物2为5,5'-二丁氧基-2,2'-联呋喃。
化合物3白色粉末(氯仿)。碘显下显橙色，在硫酸显加热后显紫色斑点。可溶于石油醚、二氯甲烷等。mp.83-84.EI-MSm/z:424.1H-NMR(CDCl3,400MHz)δ:3.58(1H,br s,H-10),1.46(2H,br s,H-9),1.43(2H,br s,H-11),1.39,1.25(48H,s H-2～H-8,H-12～H-28),0.89(3H,t,J＝6.5Hz,H-1),0.88(3H,t,J＝6.5Hz,H-29).13C-NMR(CDCl3,100MHz)δ:72.0(C-10),37.5(C-9和C-11),31.9(C-7),31.9(C-13),29.7(C-5,C-6,C-14,C-15,C-16,C-17,C-18,C-19,C-20,C-21,C-22,C-23,C-24和C-25),29.6(C-4和C-26),25.7(C-8和C-12),14.1(C-1和C-29)。其Dept谱显示：结构中有一个甲基，5个亚甲基，2个次亚甲基，无季碳峰。以上氢谱、碳谱和质谱数据与文献报道的二十九烷-10-醇基本一致[2]，故确定化合物3为二十九烷-10-醇。
化合物4无色固体,[ɑ]D 25.9＝+60°(C＝0.05，CHCl3)，UVλmaxnm(logε):242(3.2)；IRυmax KBr cm-1:3425.8(C-OH),2927(C-H),1725.17(C＝O)；HREIMSm/z:523.3757[M+Na]+(calcd for C32H52O4,500.3866)；化合物4的1H和13C NMR光谱数据如表1所示，与常春藤皂苷元非常相近[3]主要不同之处在于δH4.10(H-1'),1.23(H-2')和δC60.2(C-1'),14.4(C-2'),提示在化合物4的结构中存在一个乙酰基。在HMBC光谱中,发现一个乙酰基信号在δH4.10(H-1')和δC177.5(C-28)和14.4(C-2')存在远程相关，质子δH2.86(H-18)和碳δC122.5(C-12),143.5(C-13),46.4(C-17)和177.5(C-28)之间的相关，以及质子δH 1.23(H-2')和碳δC60.2(C-1')之间存在相关。这些证据提示在C-28位链接了一个乙酰基。见图1-2。基于上述事实，化合物4被证明为ethyl(3β)-3,23-dihydroxyolean-12-en-28-oate。
表1化合物4的1H和13C NMR光谱数据
a-eSignals bearing the same superscript were overlapped。
化合物5白色针晶；易溶于氯仿-丙酮、氯仿-甲醇等混合有机溶剂，10％硫酸-甲醇TLC显紫红色斑点。EI-MS m/z：456.36；1H-NMR(CDCl3,400MHz)δ:0.76(3H,s),0.78(3H,s),0.91(3H,s),0.92(3H,s),0.93(3H,s),0.99(3H,s),1.14(3H,s),3.22(1H,dd,J＝4.2,10.2Hz),5.12(1H,t-like,H-12)；13C-NMR(CDCl3,100MHz)δ:15.4,16.6,17.2,18.2,22.9,23.4,23.5,23.6,25.9,27.7,28.0,30.7,32.4,32.5,33.0,33.8,37.0,37.7,38.1,39.3,40.9,41.6,45.8,46.5,47.6,55.3,80.9,122.6,143.6,183.7.上述光谱数据与文献报道基本一致[4]，故鉴定该化合物5为齐墩果酸。
化合物6白色结晶(甲醇)EI-MS m/z:208，180，163.分子式：C11H12O4。1H-NMR(CD3OD，400MHz)δ:7.51(1H,d，J＝16.0Hz,H-7),7.01(1H,d,J＝2.0Hz,H-2),6.94(1H，dd,J＝2.4Hz，8.0Hz,H-6),6.75(1H,d,J＝8.0Hz,H-5),6.22(1H,d,J＝16.0Hz,H-8),4.18(2H,q,J＝7.2Hz,H-10),1.28(3H,t,J＝7.6Hz,H-11)；13C-NMR(CD3OD,100MHz)δ:168.8(C-9),149.0(C-4)，146.3(C-7)，146.2(C-3)，127.2(C-1)，122.4(C-6)，115.9(C-5)，114.7(C-2)，114.5(C-8)，60.9(C-10)，14.1(C-11)。以上氢谱、碳谱数据与文献报道的咖啡酸乙酯基本一致[5]，故确定化合物6为咖啡酸乙酯。
化合物7黄色结晶(甲醇)ESI-MS m/z:203(M+23)+。分子式：C9H8O4。1H-NMR(CD3OD，600MHz)δ:7.56(1H,d，J＝12.0Hz,H-7),7.07(1H,d,J＝2.0Hz,H-2),6.96(1H，dd,J＝2.4Hz，12.0Hz,H-6),6.81(1H,d,J＝6.0Hz,H-5),6.25(1H,d,J＝18.0Hz,H-8)；13C-NMR(CD3OD，150MHz)δ:113.8(C-2),114.2(C-8),115.1(C-5),121.5(C-6)，126.4(C-1),145.4(C-7),145.7(C-3),148.0(C-4),169.7(C-9)。以上氢谱与碳谱数据与文献报道的咖啡酸本一致[6]，故确定化合物7为咖啡酸。
化合物8淡黄色油状液体(甲醇)，TCL在254nm紫外下显紫色荧光。EI-MS m/z:152.2；1H-NMR(CDCl3,400MHz)δ:3.35(3H,s,-OCH3),6.60(1H,d,J＝8.0Hz,ArH),7.41(1H,d,J＝2.0Hz,ArH),7.44(1H,m,ArH),9.56(1H,s，ArCHO)。13C-NMR(CDCl3,100MHz)δ:55.9,109.7,123.5,135.5,148.6,152.6,161.9,178.3。以上氢谱、碳谱数据与文献报道的异香草醛基本一致[7]，故确定化合物8为异香草醛。
化合物9白色粉末(甲醇重结晶)。ESI-MS：m/z 495.4[M+23]+；1H-NMR(CDCl3，400MHz)δ:0.52(3H,s),0.70(3H,s),0.86(9H,s),1.08(3H,s),2.73(1H,d,J＝8Hz),3.05(1H,m),3.42(1H,m),4.14(1H,m),4.38(1H,t-like),5.15(1H,s),12.0(1H,s)；13C-NMR(CDCl3,100MHz)δ:178.6(C-28),143.9(C-13),121.7(C-12)，69.7(C-3),64.6(C-23)，47.4(C-5),46.4(C-9)，45.9(C-17)，46.4(C-19)，41.2(C-4)，42.5(C-14)，41.5(C-18)，39.5(C-8)，38.6(C-1),37.0(C-10)，32.3(C-21)，32.7(C-7)，33.5(C-29)，32.8(C-22)，31.0(C-20)，23.3(C-16)，22.8(C-11)，24.01(C-30)，19.1(C-6)，17.6(C-26)，16.6(C-25)，15.9(C-25)，12.9(C-24)化合物的氢谱、碳谱和质谱数据与文献报道一致[3],故确定化合物9为常春藤皂苷元。
2、纯度检查：
2.1薄层色谱法：照《中国药典》2005年版一部附录VI B薄层色谱法测定。
取提纯得到的咖啡酸、咖啡酸乙酯、异香草醛、常春藤皂苷元和常春藤皂苷元乙酯加甲醇制成1.0mg/ml的溶液，作为供试品溶液。
分别取上述供试液5μL，点于硅胶GF254薄层板上，采用上行展开方式，以石油醚-丙酮(3:1)；氯仿-丙酮(10:1)、氯仿-甲醇(25:1)为展开剂展开，随后硅胶GF254薄层板在紫外灯下(254nm)检识后，碘显后，置于日光下检查。结果表明：经三种展开系统多种显色方式检识，均呈单一斑点。点样量为所适用检测方法点样量的10倍量时，色谱中未见明显杂质斑点。故经薄层色谱法检识咖啡酸、咖啡酸乙酯、异香草醛、常春藤皂苷元和常春藤皂苷元乙酯为纯品。
2.2NMR检测
3、化合物抗艾滋病病毒蛋白酶和丙型肝炎病毒NS3/4A蛋白酶作用
测试原理：荧光法
检测仪器：Synergy II，电子分析天平METTLERAE-240瑞士。
试剂盒:SensoLyte 520HCV蛋白酶试剂盒(批号：AS-71145-1035，美国圣何塞AnaSpec)；SensoLyte 520HIV蛋白酶试剂盒(批号：S-71147-1026，美国圣何塞AnaSpec)。
蛋白酶：HCVNS3/4A蛋白酶(批号：AS-61017-10，美国圣何塞AnaSpec)。
HIV-1蛋白酶recombinant(批号：155-144，美国圣何塞AnaSpec)
受试药物:咖啡酸、咖啡酸乙酯、异香草醛、常春藤皂苷元和常春藤皂苷元乙酯。
阳性对照：Embelin(Sigma-Aldrich,Lot#:022M4726V)
Pepstatin(批号：Lot#1022，美国圣何塞AnaSpec)
数据处理与分析：数据以均数标准差表示(X±S)，通过微软Windows 2003Excel软件完成统计学处理，计量资料采用STDEV-t检验计算抑制率。
抑制率计算公式：
抑制率％＝100×(Fvehicle-Fsample)/Fvehicle；式中的F为对照组和样品组荧光值。
实验方法及结果：
测试溶液的配制(按试剂盒说明手册操作)：
缓冲溶液：将2X实验缓冲液(Component D)、1M DDT(Component F)和去离子水按1：0.06：1进行混合，冰浴备用。
HCVNS3/4A蛋白酶底物溶解：加0.12ml的DMSO(Component C)到HCV NS3/4A蛋白酶底物[Ac-Asp-Glu-Dap(QXLTM520)-Glu-Glu-Abu-COO-Ala-Ser-Cys(5-FAMsp)-NH2](ComponentA),作为储备液-20℃冻存。每次实验前取储备液，用缓冲溶液按1：100比例稀释，现配现用。
HIV蛋白酶和HCVNS3/4A蛋白酶溶液：用缓冲溶液将蛋白酶溶液稀释20或60倍。
测定方法(按试剂盒说明手册操作)：在384孔黑色平板(美国BD Falcon公司)中，先分别加入底物空白溶液(Substrate Control，SC)、溶剂空白溶液(Vehicle Control，VC)、阳性对照(Positive Control,PC)、抑制剂对照(Inhibitor Control,IC)、测试化合物对照(Test Compound Control,TCC),再加入HCVNS3/4A蛋白酶和底物。每个浓度均为3个复孔，震荡混匀后，37℃下孵育30分钟,经Synergy II在激发波长485nm和发射波长535nm下测试荧光强度。实验结果见表2。
表2黄褐毛忍冬酸水解产物抗艾滋病病毒蛋白酶和丙型肝炎病毒蛋白酶作用(n＝3)
-：未测定。
离体实验认为，单体化合物的IC50在10g/mL以下就具有较强的抑制作用。常春藤皂苷元对丙型肝炎HCV NS3/4A蛋白酶具有一定的抑制作用，IC50＝19μg/mL。咖啡酸乙酯、咖啡酸和异香草醛对HIV-1蛋白酶的抑制性较强，IC50分别为1.0,1.5，3.5μg/mL。因此，黄褐毛忍冬酸水解产物具有抗艾滋病和丙型肝炎的作用。
本发明发现常春藤皂苷元对丙型肝炎病毒蛋白酶NS3/4A有一定的抑制作用，可单独用于制备丙型肝炎病毒蛋白酶NS3/4A抑制剂和预防治疗丙型肝炎的作用，也可与其它组分复配用于预防和治疗丙型肝炎；咖啡酸、咖啡酸乙酯和异香草醛可单独用于制备艾滋病病毒蛋白酶抑制剂和预防治疗艾滋病的作用，也可与其它组分复配用于预防和治疗艾滋病。发明人采用现代植物化学分离纯化技术从黄褐毛忍冬(Lonicera fulvotomentosa Hsuet S.C.Cheng)酸水解产物中提取分离得到化合物纯度在98％，可作为药品检验中的化学标准品应用。
附图说明
图1是化合物4的HMBC光谱图；
图2是化合物4的NOESY光谱图。
具体实施方式
下面结合实施例对本发明作进一步说明，但不作为对本发明的任何限制。
实施例1：化合物的提取、分离和纯化
黄褐毛忍冬药材于2016年3月采自贵州贵阳，由贵阳中医学院陈德媛教授(生药学专家)鉴定为黄褐毛忍冬(Lonicera fulvotomentosa Hsu et S.C.Cheng)，植物标本存放在贵阳中医学院药学系中药化学实验室(标本号LF2016)。
实施例2：经试验验证，常春藤皂苷元对丙型肝炎病毒蛋白酶NS3/4A的抑制率为IC50＝19.0μg/mL，可用于制备治疗丙型肝炎的药物。
实施例3：取常春藤皂苷元，加入适当的辅料，制成颗粒剂、胶囊剂、片剂、口服液、软胶囊剂、滴丸剂、注射剂等药剂上可以接受的剂型。
实施例4：经试验验证，咖啡酸对艾滋病病毒蛋白酶的抑制率为IC50＝1.5μg/mL，可用于制备艾滋病蛋白酶抑制剂，用于治疗艾滋病的药物。
实施例5：取咖啡酸加入适当的辅料，制成颗粒剂、胶囊剂、片剂、口服液、软胶囊剂、滴丸剂、注射剂等药剂上可以接受的剂型。
实施例6：经试验验证，咖啡酸乙酯对艾滋病病毒蛋白酶的抑制率为IC50＝1.0μg/mL，可用于制备艾滋病蛋白酶抑制剂，用于治疗艾滋病的药物。
实施例7：取咖啡酸乙酯加入适当的辅料，制成颗粒剂、胶囊剂、片剂、口服液、软胶囊剂、滴丸剂、注射剂等药剂上可以接受的剂型。
实施例8：经试验验证，异香草醛对艾滋病病毒蛋白酶的抑制率为IC50＝3.5μg/mL，可用于制备艾滋病蛋白酶抑制剂，用于治疗艾滋病的药物。
实施例9：取异香草醛加入适当的辅料，制成颗粒剂、胶囊剂、片剂、口服液、软胶囊剂、滴丸剂、注射剂等药剂上可以接受的剂型。
以上只是本发明的具体应用范例，本发明还有其他的实施方式，凡采用等同替换或等效变换形成的技术方案，均落在本发明所要求的保护范围之内。
Claims (4)
1.黄褐毛忍冬酸水解产物在制备治疗艾滋病和丙型肝炎药物中的应用。
2.一种权利要求1所述的黄褐毛忍冬酸水解产物的制备方法，其特征在于：黄褐毛忍冬干燥花蕾或带初开的花，粉碎，用2mol/L硫酸-乙醇溶液水解后，药渣用包括氯仿加热回流提取，浸膏用硅胶在内的多种色谱法分离，确定结构得化合物β-谷甾醇、5,5'-二丁氧基-2,2'-联呋喃、二十九烷-10-醇、常春藤皂苷元乙酯、齐墩果酸、咖啡酸乙酯、咖啡酸、异香草醛和常春藤皂苷元。
3.根据权利要求2所述的黄褐毛忍冬酸水解产物的制备方法，其特征在于：采用艾滋病病毒蛋白酶筛选，黄褐毛忍冬酸水解产物咖啡酸乙酯、咖啡酸、异香草醛显示抗艾滋病病毒蛋白酶作用。
4.根据权利要求2所述的黄褐毛忍冬酸水解产物的制备方法，其特征在于：采用丙型肝炎NS3/4A蛋白酶筛选，黄褐毛忍冬主要酸水解产物常春藤皂苷元显示抗丙型肝炎NS3/4蛋白酶作用。
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