抗耐甲氧西林金黄色葡萄球菌（MRSA）天然产物的筛选及研究
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摘要
金黄色葡萄球菌(Staphylococcus aureus)是引起化脓性感染和医院感染的常见病原菌,由于细菌本身的进化和药物的滥用导致其耐药性逐渐增强,特别是耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus, MRSA)严重影响人类健康,如何防治已成为全球关注的焦点。MRSA具有多重耐药的特性,对目前绝大多数的抗菌药耐药,甚至对目前临床最为有效的万古霉素也开始出现耐药,一旦MRSA发展到对万古霉素普遍耐药,MRSA感染患者将面临无药可治的危险境地,因此研究开发新的抗MRSA药物具有重大意义。
中药中蕴藏着多种有效成份,天然化合物极具结构复杂性和多样性,是开发新药的一个巨大宝藏,也是我国新药开发的一个优势所在。许多中药都具有抗菌活性,如板蓝根、穿心莲、溪黄草、黄柏和鱼腥草等,其单体成分如小檗碱、大黄素、穿心莲内酯和去氢贝母碱是其抗菌活性成分等。多种耐药菌对中药的抗菌物质仍然敏感,提示中药在耐药菌株的治疗方面也有明显优势。
本课题对几十种中草药提取物和中药单体进行了较为系统的研究,并对抗MRSA活性较强的中草药和中药单体进行了深入研究,旨在寻找到新的抗MRSA活性成分,为开发新的抗MRSA药物提供理论依据和实验基础。
课题分为三个部分：
一、对23种中草药提取物的抗MRSA活性进行了筛选
采用96孔板法初步测定了23种中草药水提物和醇提物的抗MRSA活性,从中筛选出8种具有较强抗MRSA活性的中草药,分别为仙鹤草、蒲公英、夏枯草、半枝莲、侧柏叶、桑叶、木瓜、大血藤,且抑菌效果与浓度呈正相关性。进一步测定了这8种中草药提取物对11株MRSA的最低抑菌浓度(MIC)和最低杀菌浓度(MBC),其中仙鹤草水提取物和醇提物抗MRSA活性最强,其MIC和MBC的生药浓度均为6.25mg.mL-1。
二、对33种中药单体的抗MRSA活性进行了研究
采用96孔板法测定了33种中药单体对MRSA的抗菌活性,从中发现10种中药单体对MRSA具有抑制活性,分别为花旗松素、黄芩素、二氢杨梅素、原花青素、没食子酸、姜黄素、熊果酸、白秋李醇、水飞蓟宾、芦竹碱、杨梅苷,其MIC分别为50μg.mL-1、50gg.mL-1、1O0μg.mL1、1OOgg.mL"1、200gg.mL-1、400μg.mL-1、400μg.mL-1、400μg·mL-1、400μg.mL-1、400μg.mL-1、400μg.mL-1、800μg.mL-1。同时我们进一步研究了花旗松素、二轻杨梅素、原花青素3种抑菌作用相对较强的中药单体对常见抗菌素的增敏作用,实验结果结果表明这3种中药单体对氧氟沙星、头孢西丁钠、氯霉素、头孢唑啉钠等4种常见抗菌素的抗MRSA活性无显著的增敏作用。
三、仙鹤草提取物抗MRSA有效成分的深入研究
采用溶剂分离和柱层析相结合的方法对仙鹤草醇提物进行了分离,采用96孔板法逐步验证分离物的活性,结果石油醚萃取部分经硅胶柱层析(乙酸乙酯：石油醚=7：3洗脱),分得5个有较强抗MRSA活性的成分。
通过实验确证了水提物中抗MRSA活性物质为鞣质,并对仙鹤草中鞣质的提取和纯化工艺进行了初步研究。
本课题从中草药及中药单体入手进行了较为系统的抗MRSA活性物质筛选,得到了一些有价值的结果,为后续进一步的研究奠定了基础。研究结果也表明,许多中草药中的确含有抗MRSA活性物,但如何分离纯化得到抗菌效果好的活性物质,是下一步研究的重点和难点。
MRSA (methicillin-resistant Staphylococcus aureus), is a common pathogens to cause pyogenic infection and nosocomial infection. Because of the bacterium evolution and drug abuse, the resistance of bacteria has increased gradually, especially MRSA. It has become a global focus. MRSA has the characteristic of antimicrobial drug resistance, even began to resist to vancomycin which is considered the present most effective medicine. Once the MRSA resists vancomycin universally, a dangerous situation is appearing:patients who have infected MRSA will not be cured by any drugs. So it has a great significance to research and develop new drug for treatment of MRSA.
There are a variety of effective components in Chinese traditional medicine. For the structural complexity and diversity, natural compounds are great treasures to develop new drugs, which are also the advantages of our country.Many herbs have antimicrobial activities, such as Radix Isatidis, Andrographis paniculata(Burm.f.) Nees, Herba corydalis Bungeanae, Phellodendron amurense and Houttuynia cordata Thunb. Their components such as berberine, emodin, andrographolide and peiminine are the effective constituent. Chinese medicines remain to be sensitive to multiple drug resistance bacteria, which suggest the obvious advantages in treatment of resistant strains.
Traditional Chinese medicine monomer, which has strong activities against MRSA, has been made deep researches. The aim of our research is to find a new anti-MRSA active ingredients and provide a theoretical basis and experimental base for the development of new drug for treatment of MRSA.
There are three parts in the study:
(I)23kinds of Chinese herbs extracts against MRSA were screened
Anti-MRSA activities among23kinds of Chinese herbal medicine water and ethanol extracts were determinated by96well plates initially. Eight species which have strong anti-MRSA activities are obteined, they are:Grimonia pilosa Ledeb, Taraxacummongolicum Hand. Mazz,Prunella vulgaris L, Scutellaria barbata D. Don, Platycladu orientalis, Chaenomeles sinensis, Sargentodoxa cuneata, Morus alba L, and the inhibitory effect was positively correlated with concentration. The minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of8kinds of Chinese herbs extracts against11strains MRSA are determined. The water extract and alcohol extract of G rimonia pilosa Ledeb has the strongest activity in anti-MRSA, and the MIC and MBC of the drug concentration was6.24mg.ml-1.
(Ⅱ) The anti-MRSA activities of33kinds of traditional Chinese medicine monomer were studied
33kinds of Chinese herbal monomer were detected anti-MRSA activities, and10kinds of traditional Chinese medicine monomers which have inhibitory activity on MRSA were found. They are:Taxifolin, Noroxylin, Dihydromyricetin, proanthocyanidins, gallic acid, Curcumin, Vrsolic Acid, Patchouialcohol, Silymarin, Gramine, and Myricetrin. The MIC of these monomers is:50μg.mL-1,50μg.mL-1,100μg.mL-1,100μg.mL-1,200μg.mL-1,400μg.mL-1,400μg.mL-1,400μg.mL-1,400μg.mL-1,400μg.mL-1,400μg.mL-1,800μg.mL-1respectively. At the same time, the Taxifolin, Dihydromyricetin, proanthocyanidins, bacteriostasis, which had relatively strong ant-MRSA activities, were investigated. The results showed that with these3kinds of traditional Chinese medicine monomers, the anti-MRSA activities of ofloxacin, cefoxitin sodium, chloramphenicol and cefazolin sodium were not getting stronger.
(Ⅲ) Effective components of Grimonia pilosa Ledeb extracts on MRSA were researched
Solvent separation and column chromatography combination were used to separete acohol extracts from Grimonia pilosa Ledeb, and the activities of associative detachments were tested with96well plates.5strong anti-MRSA activity components from petroleum ether portion by silica gel column chromatography was obtained (ethyl acetate:petroleum ether=7:3elution).
It was confirmed that anti-MRSA activities in water extracts was Tanni. And tannin extraction and purification process were studied preliminary.
Screening anti-MRSA substance from Chinese herbal medicine and traditional Chinese medicine monomer was the first step and some valuable results were obtained, which made further research foundation. The results also showed that, many Chinese herbal medicines contain antioxidant MRSA, but how to get good antibacterial substance, how to separate and purify, is emphasis and difficulty for the next step research.
中药中蕴藏着多种有效成份,天然化合物极具结构复杂性和多样性,是开发新药的一个巨大宝藏,也是我国新药开发的一个优势所在。许多中药都具有抗菌活性,如板蓝根、穿心莲、溪黄草、黄柏和鱼腥草等,其单体成分如小檗碱、大黄素、穿心莲内酯和去氢贝母碱是其抗菌活性成分等。多种耐药菌对中药的抗菌物质仍然敏感,提示中药在耐药菌株的治疗方面也有明显优势。
本课题对几十种中草药提取物和中药单体进行了较为系统的研究,并对抗MRSA活性较强的中草药和中药单体进行了深入研究,旨在寻找到新的抗MRSA活性成分,为开发新的抗MRSA药物提供理论依据和实验基础。
课题分为三个部分：
一、对23种中草药提取物的抗MRSA活性进行了筛选
采用96孔板法初步测定了23种中草药水提物和醇提物的抗MRSA活性,从中筛选出8种具有较强抗MRSA活性的中草药,分别为仙鹤草、蒲公英、夏枯草、半枝莲、侧柏叶、桑叶、木瓜、大血藤,且抑菌效果与浓度呈正相关性。进一步测定了这8种中草药提取物对11株MRSA的最低抑菌浓度(MIC)和最低杀菌浓度(MBC),其中仙鹤草水提取物和醇提物抗MRSA活性最强,其MIC和MBC的生药浓度均为6.25mg.mL-1。
二、对33种中药单体的抗MRSA活性进行了研究
采用96孔板法测定了33种中药单体对MRSA的抗菌活性,从中发现10种中药单体对MRSA具有抑制活性,分别为花旗松素、黄芩素、二氢杨梅素、原花青素、没食子酸、姜黄素、熊果酸、白秋李醇、水飞蓟宾、芦竹碱、杨梅苷,其MIC分别为50μg.mL-1、50gg.mL-1、1O0μg.mL1、1OOgg.mL"1、200gg.mL-1、400μg.mL-1、400μg.mL-1、400μg·mL-1、400μg.mL-1、400μg.mL-1、400μg.mL-1、800μg.mL-1。同时我们进一步研究了花旗松素、二轻杨梅素、原花青素3种抑菌作用相对较强的中药单体对常见抗菌素的增敏作用,实验结果结果表明这3种中药单体对氧氟沙星、头孢西丁钠、氯霉素、头孢唑啉钠等4种常见抗菌素的抗MRSA活性无显著的增敏作用。
三、仙鹤草提取物抗MRSA有效成分的深入研究
采用溶剂分离和柱层析相结合的方法对仙鹤草醇提物进行了分离,采用96孔板法逐步验证分离物的活性,结果石油醚萃取部分经硅胶柱层析(乙酸乙酯：石油醚=7：3洗脱),分得5个有较强抗MRSA活性的成分。
通过实验确证了水提物中抗MRSA活性物质为鞣质,并对仙鹤草中鞣质的提取和纯化工艺进行了初步研究。
本课题从中草药及中药单体入手进行了较为系统的抗MRSA活性物质筛选,得到了一些有价值的结果,为后续进一步的研究奠定了基础。研究结果也表明,许多中草药中的确含有抗MRSA活性物,但如何分离纯化得到抗菌效果好的活性物质,是下一步研究的重点和难点。
MRSA (methicillin-resistant Staphylococcus aureus), is a common pathogens to cause pyogenic infection and nosocomial infection. Because of the bacterium evolution and drug abuse, the resistance of bacteria has increased gradually, especially MRSA. It has become a global focus. MRSA has the characteristic of antimicrobial drug resistance, even began to resist to vancomycin which is considered the present most effective medicine. Once the MRSA resists vancomycin universally, a dangerous situation is appearing:patients who have infected MRSA will not be cured by any drugs. So it has a great significance to research and develop new drug for treatment of MRSA.
There are a variety of effective components in Chinese traditional medicine. For the structural complexity and diversity, natural compounds are great treasures to develop new drugs, which are also the advantages of our country.Many herbs have antimicrobial activities, such as Radix Isatidis, Andrographis paniculata(Burm.f.) Nees, Herba corydalis Bungeanae, Phellodendron amurense and Houttuynia cordata Thunb. Their components such as berberine, emodin, andrographolide and peiminine are the effective constituent. Chinese medicines remain to be sensitive to multiple drug resistance bacteria, which suggest the obvious advantages in treatment of resistant strains.
Traditional Chinese medicine monomer, which has strong activities against MRSA, has been made deep researches. The aim of our research is to find a new anti-MRSA active ingredients and provide a theoretical basis and experimental base for the development of new drug for treatment of MRSA.
There are three parts in the study:
(I)23kinds of Chinese herbs extracts against MRSA were screened
Anti-MRSA activities among23kinds of Chinese herbal medicine water and ethanol extracts were determinated by96well plates initially. Eight species which have strong anti-MRSA activities are obteined, they are:Grimonia pilosa Ledeb, Taraxacummongolicum Hand. Mazz,Prunella vulgaris L, Scutellaria barbata D. Don, Platycladu orientalis, Chaenomeles sinensis, Sargentodoxa cuneata, Morus alba L, and the inhibitory effect was positively correlated with concentration. The minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of8kinds of Chinese herbs extracts against11strains MRSA are determined. The water extract and alcohol extract of G rimonia pilosa Ledeb has the strongest activity in anti-MRSA, and the MIC and MBC of the drug concentration was6.24mg.ml-1.
(Ⅱ) The anti-MRSA activities of33kinds of traditional Chinese medicine monomer were studied
33kinds of Chinese herbal monomer were detected anti-MRSA activities, and10kinds of traditional Chinese medicine monomers which have inhibitory activity on MRSA were found. They are:Taxifolin, Noroxylin, Dihydromyricetin, proanthocyanidins, gallic acid, Curcumin, Vrsolic Acid, Patchouialcohol, Silymarin, Gramine, and Myricetrin. The MIC of these monomers is:50μg.mL-1,50μg.mL-1,100μg.mL-1,100μg.mL-1,200μg.mL-1,400μg.mL-1,400μg.mL-1,400μg.mL-1,400μg.mL-1,400μg.mL-1,400μg.mL-1,800μg.mL-1respectively. At the same time, the Taxifolin, Dihydromyricetin, proanthocyanidins, bacteriostasis, which had relatively strong ant-MRSA activities, were investigated. The results showed that with these3kinds of traditional Chinese medicine monomers, the anti-MRSA activities of ofloxacin, cefoxitin sodium, chloramphenicol and cefazolin sodium were not getting stronger.
(Ⅲ) Effective components of Grimonia pilosa Ledeb extracts on MRSA were researched
Solvent separation and column chromatography combination were used to separete acohol extracts from Grimonia pilosa Ledeb, and the activities of associative detachments were tested with96well plates.5strong anti-MRSA activity components from petroleum ether portion by silica gel column chromatography was obtained (ethyl acetate:petroleum ether=7:3elution).
It was confirmed that anti-MRSA activities in water extracts was Tanni. And tannin extraction and purification process were studied preliminary.
Screening anti-MRSA substance from Chinese herbal medicine and traditional Chinese medicine monomer was the first step and some valuable results were obtained, which made further research foundation. The results also showed that, many Chinese herbal medicines contain antioxidant MRSA, but how to get good antibacterial substance, how to separate and purify, is emphasis and difficulty for the next step research.
引文
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