CN110279752B - 速生桉叶提取物及其制备方法和抗hiv应用 - Google Patents
速生桉叶提取物及其制备方法和抗hiv应用 Download PDFInfo
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- CN110279752B CN110279752B CN201910493456.9A CN201910493456A CN110279752B CN 110279752 B CN110279752 B CN 110279752B CN 201910493456 A CN201910493456 A CN 201910493456A CN 110279752 B CN110279752 B CN 110279752B
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Abstract
本发明公开了一种速生桉叶提取物,由速生桉叶经体积浓度90%的甲醇水溶液超声波提取后经制备型高效液相分离而得。发明人利用体外细胞TZM‑bl‑HIV‑IIIB感染系统进行抗HIV‑1活性的研究,发现速六号的细胞毒性很低,但对HIV‑1的复制具有明显抑制作用。采用TOA(Time of Addition)法就速六号对HIV病毒抑制机制进行研究,发现速六号对病毒抑制的作用方式与临床用药恩夫韦地非常相似,可能是病毒进入抑制剂。迄今为止,有关速生桉叶抑制HIV‑1复制成分的体内、外研究报道很少,本发明首次提出并证实速生桉叶提取物具有良好抗HIV‑1活性,为开发临床治疗艾滋病药物提供了新选择。
Description
技术领域
本发明属于速生桉应用和HIV治疗技术领域,尤其涉及一种速生桉叶提取物及其制备方法和抗HIV应用。
背景技术
人类免疫缺陷病毒(Human immunodeficiency virus,HIV)可引起艾滋病(Acquired immunode-ficiency syndrome,AIDS),该病疫情形势严峻,虽然高效抗逆转录病毒疗法(Highly Active Antiretroviral Therapy,HAART)能够有效地抑制HIV-1复制并降低艾滋病死亡率,但其无法完全清除患者体内的HIV-1病毒,寻找新的药物治疗艾滋病成为必然趋势。来自天然产物的活性成分具有低毒性、高效性以及成本低廉的优点,是抗HIV-1药物筛选的来源。我国有丰富的药用植物资源,许多天然植物提取物具有抗HIV-1活性,如蓼科大属植物、三宝木属植物、小叶买麻藤植物等。利用天然植物提取物抗HIV-1活性的研究呈增长的趋势。目前,已有从天然药物中分离出抗HIV-1活性的成分的报道,如生物碱类、黄酮类、萜类等化合物。
速生桉(Eucalyputs grandis E.urophylla)为桃金娘科(Myrtaceae)植物,自1890年引入后,广西开始大面积种植。现代药理学研究发现,速生桉叶含有大量有效成分,已被用于治疗感冒,流感,发烧,肌肉疼痛,溃疡,内部疼痛和炎症,也有多项研究将速生桉提取物与抗病毒及抗肿瘤特性联系起来。
发明内容
本发明要解决的技术问题是提供一种速生桉叶提取物及其制备方法和抗HIV应用,为开发植物来源的艾滋病治疗药物提供候选药物,为充分利用桉树资源开辟新途径。
为解决上述技术问题,本发明采用以下提取方案:
速生桉叶提取物,由速生桉叶经体积浓度90%的甲醇水溶液超声波提取后经制备型高效液相分离而得。
该提取物中含有槲皮苷(Quercetin)、常春藤苷(Hederacoside C)、当归(sweroside)苷、长寿花糖苷(roseoside)、马栗树皮苷(Esculin sesquihydrate)、葫芦素(Cucurbitacin I)、牛磺胆酸(Taurocholic acid)。
上述速生桉叶提取物在制备治疗艾滋病的药物或辅助治疗艾滋病的保健品方面的应用。
治疗艾滋病的药物为抗HIV的药物。
抗HIV的药物为抑制HIV-1复制的药物。
药物为口服药物制剂。
口服药物制剂为胶囊剂、颗粒剂、片剂或丸剂。
上述速生桉叶提取物的制备方法,按以下步骤进行操作:
<1>超声波提取
新鲜速生桉叶片风干粉碎,按重量体积比50:1加入体积浓度为90%的甲醇水溶液,在80℃超声提取60分钟,放置2天后,在常温下再次超声提取120分钟,然后用真空泵抽滤收集滤液,滤液在冻干机中冷冻干燥为冻干粉末;
<2>制备型高效液相分离
将冻干粉末溶解于乙腈的水溶液中,采用制备型高效液相进行分离;制备型高效液相的分离条件为:
色谱柱为Waters XTerra-C18(150mm×3.9mm.I.D.5μm);流动相为:A液与B液的混合液,其中,A液为乙腈,B液为水;流速:1.0mL/min;洗脱条件:混合液中A液体积浓度由30%至80%,梯度洗脱;柱温:35℃;洗脱时长:40min;检测波长:260nm;收集保留时间在28-30min的洗脱液,该洗脱液冷经冷冻浓缩至干,去除其中的有机溶剂,即得。
针对速生桉资源丰富、来源广泛的特点,发明人研制了一种速生桉叶提取物(简称速六号),由速生桉叶经体积浓度90%的甲醇水溶液超声波提取后经制备型高效液相分离而得。发明人利用体外细胞TZM-bl-HIV-IIIB感染系统进行抗HIV-1活性的研究,发现速六号的细胞毒性很低,但对HIV-1的复制具有明显抑制作用。采用TOA(Time of Addition)法就速六号对HIV病毒抑制机制进行研究,发现速六号对病毒抑制的作用方式与临床用药恩夫韦地非常相似,可能是病毒进入抑制剂。迄今为止,有关速生桉叶抑制HIV-1复制成分的体内、外研究报道很少,本发明首次提出并证实速生桉叶提取物具有良好抗HIV-1活性,为开发临床治疗艾滋病药物提供了新选择。
附图说明
图1是速六号的制备工艺流程图。
图2是制备速生桉叶提取物的HPLC色谱图。
图3是速六号对TZM-b1细胞生长影响的体外实验结果图。
图4是速六号对HIV-1抑制作用的体外实验结果图。
图5是速六号与临床药物AZT、恩夫韦地对HIV-1活性影响比较实验结果图。
具体实施方式
一、速生桉叶提取物的制备
1、速生桉叶来源
速生桉叶采集于广西南宁市郊区的桉树林,采集时间为春季,选取位于速生桉树中部叶龄为3年生树叶,叶片大小控制为叶长8~17cm,叶宽3~7cm。
2、超声波提取
新鲜速生桉叶片风干粉碎,按重量体积比50:1加入体积浓度为90%的甲醇水溶液,在80℃超声提取60分钟,放置2天后,在常温下再次超声提取120分钟,然后用真空泵抽滤收集滤液,滤液在冻干机中冷冻干燥为冻干粉末。
3、制备型高效液相分离
按照10kg鲜叶对应1ml的量,将冻干粉末溶解于体积浓度10%的乙腈水溶液中,溶液经15000g离心15min,上清液采用制备型高效液相进行分离。
制备型高效液相的分离条件为:色谱柱为Waters XTerra-C18(150mm×3.9mm.I.D.5μm);流动相为:A液与B液的混合液,其中,A液为乙腈,B液为水;流速:1.0mL/min;洗脱条件:混合液中A液体积浓度由30%至80%,梯度洗脱;柱温:35℃;洗脱时长:40min;检测波长:260nm;收集保留时间在28-30min的洗脱液(对应A液体积浓度约为60%),该洗脱液冷经冷冻浓缩至干,去除其中的有机溶剂,即得。(如图2所示)
4、速六号的质谱法鉴定
采用线性离子阱质谱仪对速六号成分进行鉴定。
质谱条件为:ESI电喷雾离子源;正离子方式扫描;碰撞气为氩气,碰撞气压力为1.5mT;氮气为雾化器雾化气,雾化气压力为50psi;雾化室温度为50℃,雾化挡板电压为600V;喷雾针电压为5000V;检测电压为1000V;干燥气温度为250℃;干燥气压力为20psi。
化合物的鉴定使用仪器所带的工作软件Mass Hunter自动将谱图中所有峰的质谱图与NIST 2.0数据库进行比对,据此对谱图中的所有峰进行定性。鉴定到速六号中的主要化合物有七种:槲皮苷(Quercetin)、常春藤苷(Hederacoside C)、当归苷(sweroside)、长寿花糖苷(roseoside)、马栗树皮苷(Esculin sesquihydrate)、葫芦素(Cucurbitacin I)、牛磺胆酸(Taurocholic acid)。
化合物的相对含量测定利用C6-26的正构烷烃混标对谱图中所有峰的保留指数(Retetion index,RI)进行计算,再与相同或相似极性色谱柱下各物质的保留指数进行比对,用内标物(苯乙酮、正十四烷)的峰面积与相应定量物质的峰面积相比较对其进行相对含量计算。
结果显示,七种化合物的含量分别为:槲皮苷含量12.54%、常春藤苷含量3.81%、当归苷含量2.30%、长寿花糖苷含量1.55%、马栗树皮苷含量1.07%、葫芦素含量1.54%、牛磺胆酸含量3.85%。
二、利用TZM-bl-HIV-IIIB细胞株对速六号进行细胞毒性检测
1、实验试剂:无酚红DMEM细胞培养液购自GibcoTM公司(产品编号为21063-029);胎牛血清(fetal bovine serum,FBS)、青霉素、链霉素均购自美国Gibco公司;二甲基亚矾(DMSO)购自Sigma公司;荧光素酶检测试剂(Bright-GloTM Luciferase Assay System,产品编号为E2650)购自Promega公司;ATP检测试剂(CellTiter-Glo@Luminescent CellViability Assay,产品编号为G7571)购自Promega公司。
2、实验仪器:生物安全柜为苏州安泰空气技术有限公司产品;二氧化碳培养箱为Thermo公司产品;全自动荧光倒置显微镜(型号:Evos FL Auto2)为美国ThermoScientific公司产品;多功能酶标仪为BioTek公司产品;Microfuge18低速离心机为美国Beckman Coulter有限公司产品。
3、细胞来源:TZM-bl-HIV-IIIB细胞株由中国人民解放军军事医学科学院惠赠。
细胞特点:TZM-bl-HIV-IIIB又称为JC57BL-13细胞,来源于HeLa细胞系,TZM-bl-HIV-IIIB细胞稳定表达HIV-1受体CD4,以及共受体CXCR4和CCR5,并且在基因组中整合有受HIV-1启动子长末端重复序列(Long Terminal Repeat,LTR)控制的报告基因萤火虫荧光素酶。病毒感染后报告基因的表达水平可以指示病毒的复制水平。细胞株由中国人民解放军军事医学科学院惠赠。
4、细胞培养与传代:TZM-bl-HIV-IIIB细胞用含10%灭活的FBS、100U/mL青霉素和100μg/mL链霉素的DMEM培养基于37℃、5%CO2饱和湿度培养箱中培养,用0.25%胰酶37℃消化细胞5至10min后,加入5倍培养基将细胞充分吹打混匀,按1:3比例进行传代培养。
5、细胞毒性的检测实验步骤
取对数生长的TZM-bl-HIV-IIIB细胞,用0.25%胰酶37℃消化细胞制备成单细胞悬液,以5×105个/孔的密度接种于96孔板,每孔100μl,37℃的CO2培养箱培养,当细胞生长至融合度约80%时,加入5倍梯度稀释的药物40μl,阴性对照组为加入等体积的PBS处理TZM-bl-HIV-IIIB细胞,空白对照组加入等体积的培养液,培养箱中继续培养48小时后,加入细胞活性检测试剂Cell TiterGlo 20μl,水平震荡3min,37℃、5%CO2培养箱孵育10min后,用多功能酶标仪检测化学发光信号。每次每组设3个重复孔。
6、相对细胞活力计算方法为:细胞活力(%)=(A处理组-A空白组)/(A阴性对照组-A空白组)×100%。
7、实验结果(见表1)
表1速六号对TZM-bl-HIV-IIIB细胞生长的影响
速六号浓度(ng/μl) | 52 | 10.4 | 2 | 0.4 | 0.08 |
相对细胞活力(%) | 83.11±6.73 | 93.76±2.96 | 96.33±7.49 | 87.44±5.84 | 89.85±9.59 |
如表1所示:速生桉叶的速六号加入TZM-bl-HIV-IIIB细胞的浓度分别为0.08ng/μl、0.4ng/μl、2ng/μl、10.4ng/μl、52ng/μl,48小时后相对细胞活力分别为(89.85±9.59)%、(87.44±5.84)%、(96.33±7.49)%、(93.76±2.96)%、(83.11±6.73)%。
8、结论:速六号在52ng/μl浓度以下对TZM-bl-HIV-IIIB细胞无毒性,其相对细胞活力为80%~90%,可在此浓度下可进行抗病毒活性筛选实验。
三、采用TZM-bl-HIV-IIIB病毒感染系统进行速六号对HIV病毒的抑制实验
1、实验试剂:无酚红DMEM细胞培养液购自GibcoTM公司(产品编号为21063-029);胎牛血清(fetal bovine serum,FBS)、青霉素、链霉素均购自美国Gibco公司;二甲基亚矾(DMSO)购自Sigma公司;荧光素酶检测试剂(Bright-GloTM Luciferase Assay System,产品编号为E2650)购自Promega公司;ATP检测试剂(CellTiter-Glo@Luminescent CellViability Assay,产品编号为G7571)购自Promega公司。
2、实验仪器:生物安全柜为苏州安泰空气技术有限公司产品;二氧化碳培养箱为Thermo公司产品;全自动荧光倒置显微镜(型号:Evos FL Auto2)为美国ThermoScientific公司产品;多功能酶标仪为BioTek公司产品;Microfuge18低速离心机为美国Beckman Coulter有限公司产品。
3、细胞来源:TZM-bl-HIV-IIIB细胞株由中国人民解放军军事医学科学院惠赠。
细胞特点:TZM-bl-HIV-IIIB又称为JC57BL-13细胞,来源于HeLa细胞系,TZM-bl-HIV-IIIB细胞稳定表达HIV-1受体CD4,以及共受体CXCR4和CCR5,并且在基因组中整合有受HIV-1启动子长末端重复序列(Long Terminal Repeat,LTR)控制的报告基因萤火虫荧光素酶。病毒感染后报告基因的表达水平可以指示病毒的复制水平。细胞株由中国人民解放军军事医学科学院惠赠。
4、HIV-1病毒株:毒株是野生型参考病毒株,该毒株属于类似泰国B’基因亚型T嗜性毒株。HIV-1IIIB病毒储存液来自中国疾控中心馈赠。
5、细胞培养与传代:TZM-bl-HIV-IIIB细胞用含10%灭活的FBS、100U/mL青霉素和100μg/mL链霉素的DMEM培养基于37℃、5%CO2饱和湿度培养箱中培养,用0.25%胰酶37℃消化细胞5至10min后,加入5倍培养基将细胞充分吹打混匀,按1:3比例进行传代培养。
6、抗病毒活性的检测
阳性对照组:阳性对照组仅仅加入病毒,但不加速六号。
阴性对照组:阴性对照组既不病毒也不加速六号。
抗病毒活性的检测实验步骤如下:
取对数生长的TZM-bl-HIV-IIIB细胞,用0.25%胰酶37℃消化细胞制备成单细胞悬液,以5×105个/孔的密度接种于96孔板,每孔100μl,37℃的CO2培养箱培养,当细胞生长至融合度约80%时,加入5倍梯度稀释的药物40μl,加入适当稀释的HIV-1IIIB病毒储存液60μl,保证感染复数为1。培养箱中继续培养48小时后,加入荧光素酶活性检测试剂Bright-Glo(Promega公司产品)20μl,水平震荡3min后,BioTek(Synergy H1)多功能酶标仪检测化学发光信号。
7、相对病毒复制水平计算方法为:
病毒复制(%)=(处理组-阳性对照组)/(阳性对照组-阴性对照组)×100%
8、实验结果(见表2)
表2速六号对HIV-1活性的影响
速六号浓度(ng/μl) | 52 | 10.4 | 2 | 0.4 | 0.08 |
相对病毒量(%) | 3.24±0.06 | 5.18±2.36 | 17.00±13.11 | 16.27±5.77 | 36.04±10.10 |
实验结果:如表2所示,分别用0.08ng/μl、0.4ng/μl、2ng/μl、10.4ng/μl、52ng/μl五个不同浓度作用于TZM-bl-HIV-IIIB感染系统,其相对病毒量分别为(36.04±10.10)%、(16.27±5.77)%、(17.00±13.11)%、(5.18±2.36)%、(3.24±0.06)%,随着速六号浓度的增加,TZM-bl-HIV-IIIB在TZM-b1中的相对病毒量降低,即抑制率增加;速六号对HIV-1病毒的半数抑制浓度(IC50)为0.66ng/μl,表现良好的抗病毒活性。
9、结论:在52ng/μl浓度以下,速六号对HIV-1病毒感染具有抑制的作用。
四、采用TOA(Time of Addition)法进行速六号抗HIV-1病毒机制研究
1、实验试剂:无酚红DMEM细胞培养液购自GibcoTM公司(产品编号为21063-029);胎牛血清(fetal bovine serum,FBS)、青霉素、链霉素均购自美国Gibco公司;二甲基亚矾(DMSO)购自Sigma公司;荧光素酶检测试剂(Bright-GloTM Luciferase Assay System,产品编号为E2650)购自Promega公司;ATP检测试剂(CellTiter-Glo@Luminescent CellViability Assay,产品编号为G7571)购自Promega公司。
2、实验仪器:生物安全柜为苏州安泰空气技术有限公司产品;二氧化碳培养箱为Thermo公司产品;全自动荧光倒置显微镜(型号:Evos FL Auto2)为美国ThermoScientific公司产品;多功能酶标仪为BioTek公司产品;Microfuge18低速离心机为美国Beckman Coulter有限公司产品。
3、细胞来源:TZM-bl-HIV-IIIB细胞株由中国人民解放军军事医学科学院惠赠。
细胞特点:TZM-bl-HIV-IIIB又称为JC57BL-13细胞,来源于HeLa细胞系,TZM-bl-HIV-IIIB细胞稳定表达HIV-1受体CD4,以及共受体CXCR4和CCR5,并且在基因组中整合有受HIV-1启动子长末端重复序列(Long Terminal Repeat,LTR)控制的报告基因萤火虫荧光素酶。病毒感染后报告基因的表达水平可以指示病毒的复制水平。细胞株由中国人民解放军军事医学科学院惠赠。
4、HIV-1病毒:HIV-1IIIB病毒储存液来自中国疾控中心馈赠。毒株是野生型参考病毒株,该毒株属于类似泰国B’基因亚型T嗜性毒株。
5、细胞培养与传代:TZM-bl-HIV-IIIB细胞用含10%灭活的FBS、100U/mL青霉素和100μg/mL链霉素的DMEM培养基于37℃、5%CO2饱和湿度培养箱中培养,用0.25%胰酶37℃消化细胞5至10min后,加入5倍培养基将细胞充分吹打混匀,按1:3比例进行传代培养。
6、抗病毒活性的检测
阳性对照组:阳性对照组仅仅加入病毒,但不加速六号。
阴性对照组:阴性对照组既不病毒也不加速六号。
7、TOA实验步骤
取对数生长的TZM-bl-HIV-IIIB细胞,用0.25%胰酶37℃消化细胞制备成单细胞悬液,以5×105个/孔的密度接种于96孔板,每孔100μl,37℃的CO2培养箱培养,当细胞生长至融合度约80%时,加入5倍梯度稀释的药物40μl,加入适当稀释的HIV-1IIIB病毒储存液60μl,保证感染复数为1。分别在病毒感染细胞的0小时以及每隔2小时加入对照药物和速六号,培养箱中继续培养48小时后,加入荧光素酶活性检测试剂Bright-Glo(Promega公司产品)20μl,水平震荡3min后,BioTek(Synergy H1)多功能酶标仪检测化学发光信号。
8、实验采用临床药物做对照,对照药物两种,一种为齐多夫定(AZT),是一种核苷类的逆转录酶抑制剂,另一种是恩夫韦地,是病毒进入抑制剂。
9、相对病毒复制水平计算方法为:
病毒复制(%)=(处理组-阳性对照组)/(阳性对照组-阴性对照组)×100%
10、速六号与临床药物对照药物对HIV-1活性影响的体外实验结果见图5。
如图5所示,速六号对HIV-1的活性确实是有抑制作用,在感染病毒时加药时间越近,病毒抑制效果越好,两小时后加药只能抑制30%的病毒,而在四小时时加药对病毒就没有抑制作用了。速六号对病毒抑制的作用方式与病毒进入抑制剂恩夫韦地非常相似,与核苷类的逆转录酶抑制剂AZT的抑制时效和抑制率基本不同。因此,推测速六号可能是一种HIV-1的进入抑制剂。
Claims (8)
1.一种速生桉叶提取物,其特征在于由速生桉叶经体积浓度90%的甲醇水溶液超声波提取后经制备型高效液相分离而得,具体按以下操作制备:
<1>超声波提取
新鲜速生桉叶片风干粉碎,按重量体积比50:1加入体积浓度为90%的甲醇水溶液,在80℃超声提取60分钟,放置2天后,在常温下再次超声提取120分钟,然后用真空泵抽滤收集滤液,滤液在冻干机中冷冻干燥为冻干粉末;
<2>制备型高效液相分离
将冻干粉末溶解于乙腈的水溶液中,采用制备型高效液相进行分离;制备型高效液相的分离条件为:
色谱柱为Waters XTerra-C18 150mm×3.9mm.I.D.5μm;流动相为:A液与B液的混合液,其中,A液为乙腈,B液为水;流速:1.0mL/min;洗脱条件:混合液中A液体积浓度由30%至80%,梯度洗脱;柱温:35℃;洗脱时长:40min;检测波长:260nm;收集保留时间在28-30min的洗脱液,该洗脱液冷经冷冻浓缩至干,去除其中的有机溶剂,即得。
2.根据权利要求1所述的速生桉叶提取物,其特征在于该提取物中含有槲皮苷、常春藤苷、当归苷、长寿花糖苷、马栗树皮苷、葫芦素、牛磺胆酸。
3.权利要求1所述速生桉叶提取物在制备治疗艾滋病的药物或辅助改善艾滋病的保健品方面的应用。
4.根据权利要求3所述的应用,其特征在于:所述治疗艾滋病的药物为抗HIV的药物。
5.根据权利要求4所述的应用,其特征在于:所述抗HIV的药物为抑制HIV-1复制的药物。
6.根据权利要求5所述的应用,其特征在于:所述药物为口服药物制剂。
7.根据权利要求6所述的应用,其特征在于:所述口服药物制剂为胶囊剂、颗粒剂、片剂或丸剂。
8.权利要求1所述速生桉叶提取物的制备方法,其特征在于按以下步骤进行操作:
<1>超声波提取
新鲜速生桉叶片风干粉碎,按重量体积比50:1加入体积浓度为90%的甲醇水溶液,在80℃超声提取60分钟,放置2天后,在常温下再次超声提取120分钟,然后用真空泵抽滤收集滤液,滤液在冻干机中冷冻干燥为冻干粉末;
<2>制备型高效液相分离
将冻干粉末溶解于乙腈的水溶液中,采用制备型高效液相进行分离;制备型高效液相的分离条件为:
色谱柱为Waters XTerra-C18 150mm×3.9mm.I.D.5μm;流动相为:A液与B液的混合液,其中,A液为乙腈,B液为水;流速:1.0mL/min;洗脱条件:混合液中A液体积浓度由30%至80%,梯度洗脱;柱温:35℃;洗脱时长:40min;检测波长:260nm;收集保留时间在28-30min的洗脱液,该洗脱液冷经冷冻浓缩至干,去除其中的有机溶剂,即得。
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