CN104940211B - 白桦脂酸在制备抗真菌生物被膜药物中的应用 - Google Patents
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Abstract
本发明涉及白桦脂酸及其药学上可接受的盐在制备抗真菌生物被膜药物中的应用。本发明提供了一种有效的抗白念珠菌生物被膜的方法，本发明所用化合物白桦脂酸在较低的浓度下具有抑制白念珠菌生物被膜生长，或抑制已成熟的生物被膜的作用，其最低有效浓度仅为2.0μmol/L。本发明不仅为白桦脂酸提供了一种新的用途，也为临床上抑制真菌生物被膜的形成或破坏成熟的真菌生物被膜提供了有效的药物。
Description
技术领域
本发明涉及医药化学技术领域，具体地说，是白桦脂酸在制备抗真菌生物被膜药物中的应用。
背景技术
生物被膜是一个微生物聚集群体，是微生物不可逆的与无活力生物或活组织表面接触，由自身产生的细胞外基质包裹着活细菌细胞形成的微生态结构。生物被膜可形成于自然环境中，也可形成于人类宿主体内，它具有保护微生物细胞，抵抗各种理化刺激，调节基因表达等作用。在日常生活和工业生产中，细菌生物被膜普遍存在，绝大多数细菌为了生产，聚集形成生物被膜，从而可以生长在各种物体表面的保护膜中。早在1991年有研究表明，细菌生物被膜具有三维结构，细菌含量不到1/3，而绝大部分是细菌分泌的粘性物质。生长在细菌生物被膜上的细菌在其生物学特性上，与浮游菌有明显的不同。浮游菌易获得营养和氧气，也易于排出代谢物，故繁殖快；而被膜细菌由于胞外多糖基质的屏障难以获得营养供给而代谢缓慢。微生物产生生物被膜是一种重要的耐药机制，在植入性器材，比如各种人工假体、血管内插管等常常发生真菌生物被膜感染，微生物形成生物被膜之后，耐药性可发生极为显著的变化，据文献报道，白色念珠菌形成生物被膜之后，对唑类抗真菌药物的MIC大大增加，是游离细胞的128倍以上。如何防治和克服生物被膜耐药问题，已成为临床医学亟需客服的难题。
白桦脂酸(Betulinic acid，BA)是一种五环三萜类化合物，在自然界中广泛存在，在白桦树皮中含量尤为丰富，1995年Pisha发现体内外实验中白桦脂酸都能诱导黑色素瘤细胞的凋亡。此外，研究表明该类化合物具有显著的抗肿瘤和抗HIV活性。白桦脂酸结构如下所示。
中国期刊《华中科技大学学报(医学版)》，2010年10月第39卷第5期刊登的论文《白桦脂酸抑制鼻咽癌细胞CNE-1生长及其机制探讨》，公开了白桦脂酸对鼻咽癌细胞的抑制作用，MTT结果显示：小剂量的白桦脂酸即对鼻咽癌CNE-1细胞有抑制生长作用。中国期刊《中国药学杂志》，2012年5月第47卷第9期刊登的论文《白桦脂酸抑制食管鳞状细胞癌增殖及相关机制的实验研究》，研究抗肿瘤药物白桦脂酸对人食管鳞癌细胞的抑制作用及抗肿瘤作用机制，结果显示：白桦脂酸能抑制人食管鳞癌KYSE170细胞的生长，这一作用可能是通过诱导细胞凋亡、阻滞细胞停留在S期来实现的。中国期刊《吉林大学学报》，2009年5月第40卷第3期刊登的论文《白桦脂酸体外抗肿瘤的活性和机制》，公开了白桦脂酸对肿瘤细胞(人宫颈癌细胞HeLa，人乳腺癌细胞MCF-7，ZR)的生长具有抑制作用，细胞生长抑制率随剂量的增加而增加。黑龙江中医药大学2012年6月博士学位论文《白桦脂酸诱导人宫颈癌HeLa细胞凋亡及机制的研究》公开了白桦脂酸有明显的抑制宫颈癌HeLa细胞生长的作用，并诱导HeLa细胞凋亡，其作用机制可能与诱导caspase-3、CytoC蛋白的过度表达，开放线粒体凋亡通路有关。
但是，关于白桦脂酸的抗真菌生物被膜作用，目前还未见报道。
发明内容
本发明的主要目的是提供白桦脂酸的新的药理用途。
本发明的再一的目的是，提供一种抗真菌生物被膜的药物组合物。
为实现上述目的，本发明采取的技术方案是：
白桦脂酸及其药学上可接受的盐在制备抗真菌生物被膜药物中的应用。
所述的盐为盐酸盐、硫酸盐、磷酸盐、醋酸盐等；所述的真菌为白念珠菌。
所述的白桦脂酸结构如下所示：
白桦脂酸在制备抑制白念珠菌生物被膜形成的药物中的应用。
白桦脂酸在制备消除白念珠菌生物被膜的药物中的应用。
所述的药物为膏药、乳剂、喷雾剂或膜剂。
为实现上述第二个目的，本发明采取的技术方案是：
一种抗真菌被膜的药物组合物，包括有效量的白桦脂酸和药学上可接受的辅料或载体。
所述的真菌为白念珠菌。
所述的药物为膏药、乳剂、喷雾剂或膜剂。所述的药物用于抑制真菌生物被膜的生长增殖，或用于抑制已形成的真菌生物被膜。
本发明优点在于：
本发明提供了一种有效的抗白念珠菌生物被膜的方法，本发明所用化合物白桦脂酸在较低的浓度下具有抑制白念珠菌生物被膜生长，或抑制已成熟的生物被膜的作用，其最低有效浓度仅为2.0μmol/ml。本发明不仅为白桦脂酸提供了一种新的用途，也为临床上抑制真菌生物被膜的形成或破坏成熟的真菌生物被膜提供了有效的药物。
具体实施方式
下面结合具体实施方式对本发明作详细说明，但是应理解所述实施例仅是范例，不对本发明构成任何限制。应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改或替换均落入本发明的保护范围。
实施例1白桦脂酸对白念珠菌生物被膜的作用
1、药物
白桦脂酸：购自上海一研生物科技有限公司；氟康唑：购自Sigma公司；两性霉素B：购自Sigma公司；XTT购自Sigma公司；二甲亚砜(DMSO)购自Sigma公司；PBS溶液购自Sigma公司
白桦脂酸用DMSO溶解，使用前经0.22μm的过滤器过滤除菌，-20℃避光保存；两性霉素B与氟康唑分别溶解于DMSO溶液中。XTT用灭菌的PBS缓冲溶液配成.5mg/ml的饱和溶液，经0.22μm的过滤器过滤除菌；menadione用100％丙酮配成10M溶液，XTT、menadione于-80℃保存。实验前先将药液取出并于保温箱中梯度融化。
2、实验用菌株
白念珠菌SC5315，购自ATCC。实验菌株于沙堡葡萄糖琼脂培养基(SDA)划板活化，于30℃培养48h后，挑取单克隆再次划板活化，取第二次所得单克隆置于SDA平板，用上述方法培养后于4℃保存备用。
3、培养液的配置
RPMI 1640液体培养液：RPMI1640(Gibco BRL)10g、NaHCO32.0g、吗啡丙磺酸(MOPS，Sigma)34.5g，加三蒸水900ml溶解，NaOH调pH至7.0，三蒸水定容至1000ml，0.22μm微孔滤膜过滤除菌，分装后于4℃保存备用。
沙堡葡萄糖琼脂固体培养基(SDA):蛋白胨10g，葡萄糖40g，琼脂18g，加三蒸水900ml溶解，加入2mg/ml氯霉素水溶液50ml，调整pH至7.0，以三蒸水定容至1000ml，高压灭菌(121℃，15min)后子4℃保存备用。
YPD培养液：酵母浸膏10g，蛋白胨20g，葡萄糖20g，加三蒸水900ml溶解，定容至1000ml，高压灭菌(121℃，15min)后4℃保存备用。
PBS缓冲溶液(PH7.2)：氯化钠8.0g、氯化钾0.4g、磷酸氧二钠0.133g、磷酸二氧钾0.06g、碳酸氧钠0.35g，加三蒸水1000ml溶解后，高压灭菌(121℃，15min),4℃保存备用。
4、XTT法测定白桦脂酸体外抗白念珠菌生物被膜活性
XTT还原法的基本原理：四氮唑盐能够在电子耦合剂甲萘醌的作用下，通过具有线粒体活性的酵母细胞转化为具有特殊颜色的甲月替四氮唑盐。
4.1培养条件
实验前用接种圈从4℃保存的SDA培养基上挑取白念珠菌SC5314单克隆，接种至1ml的YPD培养液中，于30℃，200rpm震荡培养，活化16h，使真菌处于指数生长期后期。取该菌液至1.5ml玻璃管中，离心弃除上清液，用1ml PBS缓冲液吹打混匀，离心弃除上清液，重复用PBS缓冲液洗一次，用1ml RPMI 1640培养液吹打混匀，用血细胞计数板计数，以RPMI1640培养液调整菌液浓度至106cells/ml，将菌液接种到96孔板1-11号孔，37℃恒温箱中静置培养90min。
4.2对生物被膜的影响
(1)对白念珠菌生物被膜增殖的抑制效果
取无菌96孔板，于每排11号孔加RPMI 1640培养液150μl作为阳性对照；2-10号孔各加RPMI 1640培养液150μl；1号孔分别加RPMI 1640培养液297μl和白桦脂酸3μl或RPMI培养液297μl和氟康唑药液3μl或RPMI 1640培养液298.8和AmB药液1.2μl。1-10号孔10级倍比稀释，使各孔的白桦脂酸最终药物浓度分别为64、32、16、8、4、2、1、0.5、0.25和0.125μmol/L，FLC最终药物浓度分别为1024、512、256、128、64、32、16、8、4、和2μmol/L，AmB最终药物浓度分别为16、8、4、2、1、0.5、0.25、0.0625和0.03125μmol/L，各 孔中DMSO含量均低于1％。取出培养90min的生物被膜板，用PBS换成溶液洗3次，取配好的含药物培养液100μl加入到被模板对应的孔中，12号孔不培养生物被膜作为阴性对照。各药敏板于37℃继续静置培养24h
(2)对白念珠菌成熟生物被膜的抑制作用
培养24h之后，取无菌96孔板，于每排11号孔加RPMI培养液150ml作为阳性对照；2-10号孔各加RPMI 1640培养液150μl；1号孔分别加RPMI 1640培养液297μl和白桦脂酸3μl或RPMI培养液297μl和氟康唑药液3μl或RPMI1640培养液298.8和AmB药液1.2μl。1-10号孔10级倍比稀释，使各孔的白桦脂酸最终药物浓度分别为64、32、16、8、4、2、1、0.5、0.25和0.125μmol/L，FLC最终药物浓度分别为1024、512、256、128、64、32、16、8、4、和2μmol/L，AmB最终药物浓度分别为16、8、4、2、1、0.5、0.25、0.0625和0.03125μmol/L，各孔中DMSO含量均低于1％。取出培养24h的生物被膜板，用PBS清洗3次，取配好的含药物培养液100ml加入到被模板对应的孔中，12号孔作为阴性对照不培养生物被膜。各药敏板于37℃培养。
5、SMIC值测定
白念珠菌生物被膜于37℃培养24h后，取出生物被膜板，用PBS缓冲液轻轻洗3次，每孔加入200μl的XTT/menadione溶液，于37℃静置避光孵育2-3h，取出生物被膜板，吸取100μl的XTT/menadione溶液至无菌96孔板中，用Infinite M200多功能酶标仪于490nm测定各孔OD值。与阳性对照空相比，以OD值下降80％以上的最低浓度孔中的药物浓度为SMIC80。
上述实验均平行操作5到6次，当SMIC80值能准确重复或只差一个浓度时才被接受，并以较高浓度作为SMIC80值；当SMIC80值相差两个浓度以上时，则需重新实验，直到符合要求为止。
表1
表1的结果显示：白桦脂酸对白念珠菌生物被膜具有较强的抑制作用，当 白桦脂酸的浓度为64、32、16、8、4、2μmol/L时，均具有较强的抑制白念珠菌生物被膜增殖的作用，与两性霉素效果相当；当白桦脂酸的浓度为64、32、16、8、4μmol/L时，均具有较强的抗白念珠菌成熟生物被膜作用。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还可以做出若干改进和补充，这些改进和补充也应视为本发明的保护 。
Claims (1)
1.白桦脂酸在制备抑制真菌生物被膜形成的药物中的应用；所述的真菌为白念珠菌；所述抑制为抑制白念珠菌生物被膜的增殖，所述白桦脂酸最低有效浓度为2.0μmol/ml。
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