CN110251499A - 蒲公英提取物及其在制备预防和治疗妇***道疾病药物中的用途 - Google Patents

蒲公英提取物及其在制备预防和治疗妇***道疾病药物中的用途 Download PDF

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CN110251499A
CN110251499A CN201910498473.1A CN201910498473A CN110251499A CN 110251499 A CN110251499 A CN 110251499A CN 201910498473 A CN201910498473 A CN 201910498473A CN 110251499 A CN110251499 A CN 110251499A
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梁引库
段红波
张萍
高飞雄
李云祥
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Shaanxi University of Technology
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Abstract

本发明公开了蒲公英提取物及其在制备预防和治疗妇***道疾病药物中的用途，所述蒲公英提取物主要由4‑香豆酸、阿魏酸、槲皮素戊糖苷、3,5‑二‑O‑咖啡酰奎尼酸、4,5‑二‑O‑咖啡酰奎宁酸和木犀草素组成。试验结果显示，蒲公英提取物对白假丝酵母菌和加德纳菌有良好的抑制作用，对白假丝酵母菌和加德纳菌的最低抑菌浓度分别为0.4mg/mL和0.2mg/mL，可用于制备预防和治疗由白假丝酵母菌和加德纳菌引起妇***道疾病药物。本发明所用蒲公英提取物的原材料取材方便，生产成本低，产品质量好，价格低廉，绿色环保，适合推广应用。因此，可成为一种新型的中药***病治疗药物。

Description

蒲公英提取物及其在制备预防和治疗妇***道疾病药物中的 用途
技术领域
本发明属于药物技术领域，具体涉及一种蒲公英提取物，以及该提取物在制备预防和治疗由白假丝酵母菌和加德纳菌引起的妇***道疾病药物中的用途。
背景技术
***病又名***炎症，是妇科门诊多见和频繁发生的病症。妇女的健康***对细菌、真菌等病原微生物的入侵有天然防御能力，而当健康***的天然防御***被破坏时，病原体趁虚而入，打破了菌群与***之间原有的生态平衡，某些致病菌在此适宜的生长条件下迅速生长，进而引发***炎症。
由于临床工作中诊断不正确、对治疗规范理解不透彻和对一些新的***感染、混合感染认识不足，以及日常生活中妇女们对妇科疾病缺乏全面正确的认识，缺乏相关的自身保健，加之多种不良生活作息等，使生理健康每况愈下，导致一些女性常年病魔缠身，且反复发作或久治不愈，给个人的正常工作和学习、家庭生活带来诸多不便。
临床上常见的***病有：细菌性***病、假丝酵母菌性***病、滴虫性***病、老年性***病及幼女性***病。其中细菌性***病和假丝酵母菌性***病是两种常见性***病，它们分别主要是由加德纳菌和白假丝酵母菌引起的。近年来，抗生素的滥用引起了细菌和真菌耐药等许多生物安全问题，所以寻找一种抑菌性能良好的天然药物成分作为***病治疗药物是极为重要的。
发明内容
本发明的目的是提供一种蒲公英提取物，并为该蒲公英提取物提供一种新的用途。
针对上述目的，本发明的蒲公英提取物包含下述相对质量百分含量的成分：4-香豆酸11％～12％、阿魏酸3.5％～4.5％、槲皮素戊糖苷10％～11％、3,5-二-O-咖啡酰奎尼酸34％～35％、4,5-二-O-咖啡酰奎宁酸3.5％～4.5％、木犀草素11％～12％。
上述蒲公英提取物由下述方法获得：
(1)水提
取干燥的蒲公英全草粉碎并过60～80目筛，按照料液比1g:25～35mL加入蒸馏水，于50～70℃下浸提30～40min，浸提2次，过滤，合并滤液，滤液浓缩2～3倍，离心分离，收集上清液。
(2)初步纯化
将上清液用0.45μm孔径的滤膜抽滤后超声5～10min，然后上预处理好的D101大孔吸附树脂柱，水洗2个柱体积后，用体积浓度为50％～70％的乙醇水溶液洗脱，收集洗脱液，将洗脱液浓缩4～6倍。
(3)再次纯化
将浓缩后的洗脱液用等体积的乙酸乙酯萃取，收集萃取液并浓缩5～6倍后，真空干燥，得到蒲公英提取物。
本发明蒲公英提取物在制备预防和/或治疗由白假丝酵母菌和加德纳菌引起的妇***道疾病药物中的用途。试验结果显示，本发明蒲公英提取物对白假丝酵母菌和加德纳菌有良好的抑制作用，对白假丝酵母菌和加德纳菌的最低抑菌浓度分别为0.4mg/mL和0.2mg/mL，可用于制备预防和治疗由白假丝酵母菌和加德纳菌引起妇***道疾病药物。
本发明所用蒲公英提取物的原材料取材方便，生产成本低，产品质量好，价格低廉，绿色环保，适合推广应用。因此，可成为一种新型的纯中药的***病治疗药物。
附图说明
图1是实施例1得到的蒲公英提取物的HPLC分析图，其中1是4-香豆酸；2是阿魏酸；3是槲皮素戊糖苷；4是3,5-二-O-咖啡酰奎尼酸；5是4,5-二-O-咖啡酰奎宁酸；6是木犀草素。
图2是实施例1得到的蒲公英提取物对白假丝酵母菌的抑菌试验。
图3是实施例1得到的蒲公英提取物对加德纳菌的抑菌试验。
具体实施方式
下面结合附图和实施例对本发明作进一步详细说明，但本发明的保护范围不仅限于这些实施例。
实施例1
制备蒲公英提取物
(1)水提
取新鲜蒲公英全草于60℃真空烘至恒重，烘干后粉碎并过80目筛，然后按照料液比1g:30mL加入蒸馏水，于60℃下浸提30min，浸提2次，过滤，合并滤液，滤液浓缩至2～3倍，在8000r/min离心10min，收集上清液。
(2)初步纯化
将步骤(1)收集的上清液用0.45μm孔径的滤膜抽滤器抽滤后，超声10min，然后上预处理好的D101大孔吸附树脂柱，水洗2个柱体积后，用体积浓度为60％的乙醇水溶液进行洗脱，收集洗脱液，将洗脱液浓缩4～6倍。
(3)再次纯化
将步骤(2)浓缩后的洗脱液用等体积的乙酸乙酯萃取，收集萃取液并浓缩5～6倍后，在60℃真空干燥箱中烘干，得到蒲公英提取物。
采用高效液相色谱(HPLC)测定所得蒲公英提取物的组成，HPLC***由一个配备有DiKMADiamonsil C18柱(250mm×4.6mm，5μm)的Agilent1100HPLC***和二极管阵列检测器(DAD)组成。样品用体积浓度为80％的甲醇水溶液溶解，柱温保持在40℃，流动相由乙腈(洗脱液A)和体积浓度为0.1％的磷酸水溶液(洗脱液B)组成，等度洗脱液的流速为1.0mL/min，按照20％A、80％B洗脱47min，进样量为10μL。所有多酚化合物的峰面积在280nm波长处测定。如图1所示，HPLC检测出6种化合物，分别是4-香豆酸、阿魏酸、槲皮素戊糖苷、3,5-二-O-咖啡酰奎尼酸、4,5-二-O-咖啡酰奎宁酸和木犀草素，这6种化合物在此提取物中相对百分含量分别为11.45％、3.96％、10.48％、34.24％、3.91％和11.80％。
实施例2
实施例1的蒲公英提取物对白假丝酵母菌和加德纳菌的抑制性能
(1)抑菌活性试验
通过纸片法测定蒲公英提取物对白假丝酵母菌(Candida albicans，CMCC85021)和加德纳菌(Gardnerella vaginalis，ATCC14018)的抑菌作用。将实验菌种复苏，活化2次。白假丝酵母菌传代于沙式液体培养基，在37℃、190r/min培养12h；加德纳菌使用HTM琼脂培养基平板在37℃5％CO2条件下培养12h，用液体培养基将菌落洗涤下来制备成菌悬液。用分光光度计测定各自菌体OD600nm值。当OD600nm＝0.6～0.7时，为最佳涂布浓度；若OD600nm>0.7时，用液体培养基进行适当稀释，再进行涂布。配制沙式琼脂培养基和HTM琼脂培养基，分别于115℃和121.3℃下灭菌20min，无菌辅酶Ⅰ(NAD)溶液在培养基冷却至50℃加入HTM琼脂培养基，混匀，分别制备平板。用移液枪吸取100μL经活化且浓度适宜的菌悬液于平板表面，用无菌L形玻棒将菌液涂匀。实验组：吸取10μL用40％二甲基亚砜溶解的样品溶液滴加到直径6mm无菌滤纸片上，适度烘烤，贴于平板表面。空白组：将10μL无菌40％二甲基亚砜(DMSO)滴加到直径6mm无菌滤纸片上，适度烘烤，贴于平板表面。分别于37℃、5％CO2条件下倒置培养18～36h，观察实验结果，测定抑菌圈直径大小。
试验结果显示，空白组40％DMSO纸片周围基本无抑菌圈，表明二甲基亚砜对真菌和细菌生长无影响；实验组蒲公英提取物周围有抑菌圈，表明蒲公英提取物对白假丝酵母菌和加德纳菌具有明显抑菌作用(见图2)。当蒲公英提取物的浓度为0.40～2.28mg/mL，白假丝酵母菌的抑菌圈直径为6.00～10.53mm，其中蒲公英提取物浓度为2.28mg/mL时，对白假丝酵母菌的平均抑菌圈直径为10.38±0.15mm；当蒲公英提取物的浓度为0.20～3.80mg/mL，加德纳菌的抑菌圈直径为6.00～11.85mm，其中蒲公英提取物浓度为3.80mg/mL时，对白假丝酵母菌的平均抑菌圈直径为11.86±0.17mm。说明蒲公英提取物对白假丝酵母菌和加德纳菌具有良好的抑制效果。
(2)最低抑菌浓度
按照方法(1)培养白假丝酵母菌和加德纳菌，用液体培养基将菌液浓度调到107CFU/mL，且配制4mg/mL蒲公英提取物母液。分别向装有2mL液体培养基的试管中加入蒲公英提取物母液，使培养基中蒲公英提取物最终浓度为0、0.05、0.10、0.20、0.40、0.60、0.80mg/mL，按4％(v/v)接入浓度为107CFU/mL的菌液，加德纳菌试管使用石蜡凡士林(V石蜡油:V凡士林＝1:1混合)封住液面。白假丝酵母菌和加德纳菌分别于37℃、190r/min和37℃培养24h，当样品OD600nm变化首次小于5％的蒲公英提取物的浓度为最低抑菌浓度，即为MIC。经分析发现，当蒲公英提取物的浓度大于0.4mg/mL时，白假丝酵母菌OD600nm值变化首次小于5％，当蒲公英提取物的浓度大于0.2mg/mL时，加德纳菌OD600nm值变化首次小于5％，可见，蒲公英提取物对白假丝酵母菌和加德纳菌的MIC值分别为0.4mg/mL和0.2mg/mL。
由上述可见，本发明蒲公英提取物对白假丝酵母菌和加德纳菌具有良好的抑制效果，对白假丝酵母菌和加德纳菌的MIC值分别为0.4mg/mL和0.2mg/mL，可用于制备预防和/或治疗由白假丝酵母菌和加德纳菌引起的妇***道疾病药物。

Claims (3)

1.一种蒲公英提取物，其特征在于：所述的蒲公英提取物包含下述相对质量百分含量的成分：4-香豆酸11％～12％、阿魏酸3.5％～4.5％、槲皮素戊糖苷10％～11％、3,5-二-O-咖啡酰奎尼酸34％～35％、4,5-二-O-咖啡酰奎宁酸3.5％～4.5％、木犀草素11％～12％。
2.根据权利要求1所述的蒲公英提取物，其特征在于所述的蒲公英提取物由下述方法获得：
(1)水提
取干燥的蒲公英全草粉碎并过60～80目筛，按照料液比1g:25～35mL加入蒸馏水，于50～70℃下浸提30～40min，浸提2次，过滤，合并滤液，滤液浓缩2～3倍，离心分离，收集上清液；
(2)初步纯化
将上清液用0.45μm孔径的滤膜抽滤后超声5～10min，然后上预处理好的D101大孔吸附树脂柱，水洗2个柱体积后，用体积浓度为50％～70％的乙醇水溶液洗脱，收集洗脱液，将洗脱液浓缩4～6倍；
(3)再次纯化
将浓缩后的洗脱液用等体积的乙酸乙酯萃取，收集萃取液并浓缩5～6倍后，真空干燥，得到蒲公英提取物。
3.权利要求1所述的蒲公英提取物在制备预防和/或治疗由白假丝酵母菌和加德纳菌引起的妇***道疾病药物中的用途。
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