不同基原秦皮、香豆素单体抗菌作用对比研究
作者：刘丽梅,王瑞海,陈琳,杨庆,翁小刚,孙建辉
【摘要】 目的 研究4种基原秦皮提取物、5种香豆素单体、5种单体混合物、苦枥白蜡树树皮已知香豆素指纹区样品、未知成分指纹区样品的体外抑菌作用。方法 采用体外抑菌试验微量定量检测方法,测定最小抑菌浓度及最低杀菌浓度。结果 4种基原秦皮苦枥白腊树、尖叶白腊树、白腊树、宿柱白腊树树皮均有明显抑菌、杀菌作用,抑菌强度依次为：宿柱白腊树＞苦枥白腊树＞尖叶白腊树＞白腊树。5种香豆素单体秦皮素、秦皮乙素对9种细菌均有明显抑菌、杀菌作用,秦皮甲素、秦皮苷对金黄色葡萄球菌、表皮葡萄球菌有一定抑菌、杀菌作用,抑菌作用依次为：秦皮素＞秦皮乙素＞秦皮甲素＞秦皮苷＞6,7-二甲氧基-8-羟基香豆素。苦枥白腊树树皮提取物对所试9种细菌均有明显抑菌作用；单体混合物、已知香豆素指纹区样品对金黄色葡萄球菌、表皮葡萄球菌、乙型溶血性链球菌、绿脓杆菌、大肠杆菌、肺炎克雷伯氏菌、卡他菌有明显抑菌作用；未知成分指纹区样品对金黄色葡萄球菌有明显抑菌作用,对表皮葡萄球菌、甲型溶血性链球菌、乙型溶血性链球菌、大肠杆菌、沙门氏菌也有一定抑菌作用。结论 秦皮抑菌强弱与已知香豆素成分含量高低基本一致。秦皮中苷元的抑菌作用优于苷,5种香豆素类单体中秦皮素、秦皮乙素抑菌作用最强。
【关键词】 秦皮；香豆素；抑菌作用
Abstract：Objective To study bacteriostasis in vitro of 4 kinds of origins ash bark extract, 5 kinds of coumarin monomer, 5 kinds of monomer mixture, known coumarin fingerprint region sample and unknown component fingerprint region of Fraxinus rhynchophylla Hance. Methods Bacteriostasis experiment in vitro and microamount and quantitative detection methods were adopted to measure minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Result Four kinds of origins ash bark including Fraxinus rhynchophylla Hance, Fraxinus chinensis Roxb. var. acuminata Lingelsh., Fraxinus chinensis, Fraxinus stylosa Lingelsh. had obvious bacteriostasis and bactericidal action, bacteriostasis intensity order was Fraxinus stylosa Lingelsh. ＞Fraxinus rhynchophylla Hance＞Fraxinus chinensis Roxb. var. acuminata Lingelsh. ＞Fraxinus chinensis. Five kinds of coumarin monomer：fraxetin, aesculetin had obvious bacteriostasis and bactericidal action, aesculin and fraxin had certain bacteriostasis and bactericidal action to Staphylococcus aureus, Staphylococcus epidermidis. Bacteriostasis and bactericidal action order was fraxetin＞aesculetin＞aesculin＞fraxin＞6,7-dicho- methoxyl-8-Hydroxycoumarin. Fraxinus rhynchophylla Hance extract had obvious bacteriostasis to 9 kinds of bacterium；monomer mixture and coumarin fingerprint region sample had obvious bacteriostasis to Staphylococcus aureus, Staphylococcus epidermidis, beta streptococcus, aeruginosus Bacillus, Bacillus coli, Klebsiella pneumoniae, catarrh. Unknown component fingerprint region sample had obvious bacteriostasis to Staphylococcus aureus and certain bacteriostasis to Staphylococcus epidermidis, alpha streptococcus, beta streptococcus, Bacillus coli, salmonella. Conclusion The changes of ash bark bacteriostasis were basically identical with known coumarin component content. Aglycone bacteriostasis of ash bark was superior to glycoside, fraxetin and aesculetin bacteriostasis were most powerful in 5 kinds of coumarin monomer.
Key words：ash bark；coumarin monomer；bacteriostasis
秦皮为木犀科植物苦枥白蜡树Fraxinus rhynchophylla Hance、白蜡树Fraxinus chinensis Roxb.、尖叶白蜡树Fraxinus szaboana Lingelsh.、宿柱白蜡树Fraxinus stylosa Lingelsh.的干燥枝皮或干皮,具有清热燥湿、收涩、明目的功能,用于热痢、泄泻、赤白带下、目赤肿痛、目生翳膜等。秦皮主要含有香豆素类化学成分,如：秦皮甲素(aesculin)、秦皮乙素(aesculetion)、秦皮苷(fraxoside)、秦皮素(fraxetol)、6,7-二甲氧基-8-羟基香豆素[1-2]。本研究对4种基原秦皮提取物、5种香豆素单体、5种单体混合物和以苦枥白蜡树树皮为代表的秦皮提取物、已知香豆素指纹区样品、未知成分指纹区样品进行了体外抑菌试验,旨在阐明其抑菌、杀菌作用的强弱,为客观地评价秦皮质量、指导临床用药提供科学依据。
1 实验材料
1.1 药物
4种基原秦皮：苦枥白蜡树、尖叶白蜡树、白蜡树、宿柱白蜡树树皮提取物；5种香豆素单体：秦皮甲素、秦皮乙素、秦皮苷、秦皮素、6,7-二甲基-8-羟基香豆素(均为自制,纯度＞98%)；以苦枥白蜡树为代表的秦皮提取物,5种香豆素单体混合物(按苦枥白蜡树提取物中各单体含量：秦皮甲素2.08%、秦皮乙素0.14%、秦皮苷0.84%、秦皮素0.37%、6,7-二甲基-8-羟基香豆素0.03%混合而成)；苦枥白蜡树已知香豆素指纹区样品,未知成分指纹区样品。上述样品以DMSO溶解,实验前配制。
1.2 试剂及培养基
注射用硫酸链霉素(批号050312)、注射用青霉素钠(批号U204063),均为华北制药股份有限公司。营养肉汤(批号980110)、营养琼脂(批号990330),北京海淀区微生物培养基制品厂。
1.3 菌种
标准株：金黄色葡萄球菌(26112)、乙型(B)溶血性链球菌(32310)、绿脓杆菌(10211)、大肠杆菌(4413)、沙门氏杆菌(50094-14)，由北京生物制品检定所提供。临床分离株：表皮葡萄球菌、甲型(A)溶血性链球菌、肺炎克雷伯氏菌肺炎亚种、卡他菌由中国中医科学院中药所提供。
2 方法
2.1 菌液制备
每种菌以接种环挑取同类型菌落2～3个,接种于营养肉汤内,35 ℃孵育16～18 h后,以无菌营养肉汤校正浓度达3.0 亿/mL左右,再以营养肉汤稀释,使菌浓度为300～3 000个/mL。
2.2 最小抑菌浓度测定
参考文献[3]方法,受试样品加入96孔圆底培养板中,每孔0.1 mL。每个样品设8个浓度,苦枥白蜡树树皮、尖叶白蜡树树皮、白蜡树树皮、宿柱白蜡树树皮、苦枥白蜡树树皮提取物、5种香豆素单体混合物、苦枥白蜡树已知香豆素指纹区样品及未知成分指纹区样品终浓度分别为250、100、40、16、6.4、2.56、1.02、0.409 mg原药材/mL；秦皮中5种香豆素类单体终浓度分别为5 000、2 000、800、320、128、51.2、20.48、8.19 μg/mL,然后接种稀释菌液0.1 mL/孔。同时设定溶剂对照、细菌对照、培养基对照,设定青霉素或链霉素为阳性对照,以微量振荡器混匀后,置37 ℃温孵24 h,在黑色背景下观察判断结果。有细菌生长孔呈现混浊,未见混浊则为无细菌生长,以无细菌生长的药物最小浓度作为该药对该试验菌的最小抑菌浓度(MIC)。