WO2021196505A1 - 青钱柳叶黄酮提取物在制备抗菌药物和/或抗菌剂中的用途 - Google Patents

青钱柳叶黄酮提取物在制备抗菌药物和/或抗菌剂中的用途 Download PDF

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WO2021196505A1
WO2021196505A1 PCT/CN2020/110253 CN2020110253W WO2021196505A1 WO 2021196505 A1 WO2021196505 A1 WO 2021196505A1 CN 2020110253 W CN2020110253 W CN 2020110253W WO 2021196505 A1 WO2021196505 A1 WO 2021196505A1
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antibacterial
drug
extract
cyclocarya
ethanol
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French (fr)
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孙美灵
刘杰
廖文
廖敏钧
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四川大学华西医院
四川万格农业科技有限公司
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Priority to US17/595,038 priority Critical patent/US20220305070A1/en
Priority to JP2021560359A priority patent/JP2023519453A/ja
Publication of WO2021196505A1 publication Critical patent/WO2021196505A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/52—Juglandaceae (Walnut family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9789—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/37—Extraction at elevated pressure or temperature, e.g. pressurized solvent extraction [PSE], supercritical carbon dioxide extraction or subcritical water extraction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization

Definitions

  • the invention belongs to the field of medicines, and specifically relates to the use of the flavonoid extract of Cyclocarya longifolia leaves in the preparation of antibacterial drugs and/or antibacterial agents against drug-resistant bacteria.
  • MRSA Methicillin-resistant Staphylococcus aureus
  • MRSA develops to the level of general resistance to vancomycin
  • the person infected with MRSA The patient will face a dangerous situation with no cure. Therefore, in view of the increasingly serious situation of bacterial resistance, it is urgent to develop drugs with anti-drug-resistant bacteria activity and sensitization to antibiotics.
  • Cyclocarya paliurus (Batal.) ljinskaja also known as green money plum, money tree, sweet tea tree, etc.
  • Green money plum also known as green money plum
  • money tree also known as sweet tea tree, etc.
  • Cyclocarya paliurus (Batal.) ljinskaja is a unique single genus of Cyclocarya paliurus (Batal.) ljinskaja in the Juglans family. It is endemic to my country and is an endangered tree species.
  • the green money willow is a kind of tall and fast-growing broad-leaved tree, because its tree shape resembles a willow, the fruit is round like a copper coin, and the color is green and drooping, hence the name "green money willow”.
  • the purpose of the present invention is to provide a new use of the flavonoid extract of Cymbidium aureum leaves in the preparation of antibacterial drugs and/or antibacterial agents against drug-resistant bacteria.
  • the present invention provides the use of the flavonoid extract of Cyclobalanopsis vulgaris leaves in the preparation of antibacterial drugs and/or antibacterial agents, and the bacteria are drug-resistant bacteria.
  • the drug-resistant bacteria are drug-resistant gram-positive bacteria, preferably drug-resistant Staphylococcus aureus, and more preferably methicillin-resistant Staphylococcus aureus.
  • the content of the flavonoids from the Cyclocarya leaf flavonoids is 50wt.% or more, preferably 67.3581wt.% or more.
  • the flavonoids extract of Cyclocarya longiflora leaves is prepared by the following method: taking Cyclocarya longiflora leaves, extracting with a solvent, retaining the extract, and concentrating or drying it to obtain.
  • the Cyclobalanopsis macrophylla leaves are dried Cyclobalanopsis macrocarpa leaf powder; and/or, the solvent is water, and the mass ratio of the solvent to the Cyclobalanopsis leaves is (5-15):1, preferably 10: 1; and/or, the extraction temperature is 70-110°C, the number of extractions is 1 to 5 times, and the extraction time is 30-120min each time. Preferably, the extraction temperature is 90°C and the number of extractions is 2 times, The extraction time is 90 minutes each time.
  • the extraction liquid before the extraction liquid is concentrated or dried, it further includes a purification process, and the purification process includes the following steps:
  • Ethanol is added to the extract to make the volume fraction of ethanol in the obtained system 50%-70%, and after mixing uniformly, centrifuge to retain the supernatant; preferably, the volume fraction of ethanol in the system is 60%.
  • the purification process further includes the following steps: after removing the ethanol in the above supernatant, eluting through a polyamide resin column, collecting the eluate; the eluent used for elution is water and 60% ethanol in sequence. , Among them, the amount of water is 5BV, and the amount of 60% ethanol is 5BV.
  • the present invention also provides a flavonoid extract of Cyclocarya serrata leaf, which is as described above.
  • the present invention also provides an antibacterial drug, which is prepared by using the above-mentioned Cyclocarya flavonoids extract as an active ingredient and pharmaceutically acceptable auxiliary materials.
  • the present invention also provides an antibacterial agent, which is prepared by using the above-mentioned Cyclobalanopsis flavonoids extract as the active ingredient, plus auxiliary materials commonly used in the field of preparations.
  • the experimental results show that the flavonoids extract of Cyclocarya longifolia has obvious killing effect on methicillin gold-resistant Staphylococcus aureus strains, and overcomes the resistance of methicillin gold-resistant Staphylococcus aureus strains to most antibacterial drugs. problem.
  • the flavonoid extract of Cyclobalanopsis vulgaris leaves can be prepared into antibacterial drugs or antibacterial agents against drug-resistant bacteria, and has a good application prospect in solving the worldwide problem of drug-resistant bacteria infection.
  • Antibacterial drugs refer to drugs with bactericidal or antibacterial activity.
  • Antibacterial agents refer to preparations with bactericidal or antibacterial activity, and generally cannot be used to treat diseases.
  • the flavonoid extract of Cyclocarya serrata leaf refers to an extract containing flavonoids extracted from Cyclocarya serrata leaves.
  • Flavonoids from Cyclocarya serrata leaf refer to flavonoids extracted from the leaves of Cyclocarya serrata.
  • Flavonoids are A general term for a class of compounds with a 2-phenyl chromone (flavone) structure.
  • Figure 1 shows the detection results of 16S RNA and mecA genes.
  • Figure 2 shows the bactericidal effect curve of Cyclocarya flavonoids on each strain.
  • the raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.
  • the collected eluate was recovered by ethanol to be in the form of a liquid extract, and dried under vacuum at 50° C. for 24 hours to obtain a powdered solid, namely the flavonoid extract of Cymbidium aureum leaves, which was weighed and stored for later use.
  • Drugs the flavonoid extract powder of Cymbidium acuminata prepared in Example 1, penicillin sodium and oxacillin sodium (purchased commercially available products).
  • Staphylococcus aureus (ATCC25923), MRSA-1, MRSA-2, MRSA-3, MRSA-4 and MRSA-5 are all MHB (Mueller-Hinton Broth) medium.
  • the mecA gene is a specific drug resistance gene of MRSA, which plays a decisive role in the resistance of MRSA.
  • the PCR method was used to verify the existence of mecA gene in each strain. The specific method is as follows:
  • the bacterial genomic DNA extraction kit was used to extract the genomic DNA of each strain. See the kit instructions for the extraction steps. Using the genomic DNA of each strain as a template, a multiplex PCR technique was used to amplify 449 bp fragments of 16S RNA and mecA genes.
  • the primer sequence used is: 16S RNA upstream primer 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.1) and downstream primer 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO.2); mecA upstream primer 5'-CTCAGGTACTGCTATCCACC- 3'(SEQ ID NO.3) and downstream primer 5'-CACTTGGTATATCTTCACC-3' (SEQ ID NO.4).
  • the PCR amplification procedure is: 94°C for 3min, 94°C for 45s, 50°C for 45s, 72°C for 1min and 30s, 30 cycles, 72°C for 5min.
  • the MHB medium used in this test contains 2% NaCl (w/v).
  • test bacteria respectively inoculate each bacteria in 5ml MHB medium, culture overnight at 37°C in a shaker, adjust the turbidity of the bacterial liquid to 0.5 McDaniel units, and then dilute the bacterial liquid 100 times for use.
  • the final bacterial concentration is 5 ⁇ 10 5 cfu/ml.
  • Cyclobalanopsis macrophylla leaf flavonoids application solution accurately weighed 79.8mg of Cyclobalanopsis macrocarpa leaf flavonoids extract powder prepared in Example 1 (according to the Cyclobalanopsis macrophylla leaf flavonoids content of 67.3581wt.%, it is calculated that the powder contains the flavonoids Willow leaf flavonoids 53.75mg), dissolved in 0.32ml DMSO, added 3.68ml MHB medium to dilute, to obtain Cyclocarya serrata leaf flavonoids application solution with a concentration of 13.44mg/ml.
  • Preparation of the mother liquor of penicillin sodium and oxacillin sodium solution accurately weigh 100mg of penicillin sodium or oxacillin sodium and dissolve in 1ml deionized water, filter and sterilize through 0.22 ⁇ m to obtain the mother liquor of penicillin sodium or oxacillin sodium.
  • the concentration of the flavonoids in Cyclocarya longifolia leaves were 6720 ⁇ g/ml, 3360 ⁇ g/ml, 1680 ⁇ g/ml, 840 ⁇ g/ml, 420 ⁇ g/ml, 210 ⁇ g/ml, 105 ⁇ g/ml, 52.5 ⁇ g/ ml, 26.25 ⁇ g/ml;
  • the concentration of penicillin sodium solution or oxacillin sodium solution are: 128 ⁇ g/ml, 64 ⁇ g/ml, 32 ⁇ g/ml, 16 ⁇ g/ml, 8 ⁇ g/ml, 4 ⁇ g/ml, 2 ⁇ g/ml, 1 ⁇ g/ ml, 0.5 ⁇ g/ml.
  • Sensitivity test Add 100 ⁇ l of the above-prepared bacterial solution to the drug wells of each concentration and the medium control wells. Place the culture well plate in a humid box and incubate at 34°C for 24 hours. Take 20 ⁇ l of each bacterial solution, dilute 50 times with culture medium, take 100 ⁇ l and spread it on the culture plate, and incubate overnight at 37°C. The lowest drug concentration for colony growth was judged to be the minimal inhibitory concentration (MIC).
  • MIC minimal inhibitory concentration
  • Sterilization test add 100 ⁇ l of the above-prepared bacterial solution to the flavonoid holes of the Cyclocarya sylvestris leaves and the medium control wells of each concentration. After mixing thoroughly, incubate overnight at 37°C in a humidified box. Take 20 ⁇ l of bacterial solution from each well, dilute it with MHB medium 10 times, spread 100 ⁇ l on a culture plate, incubate at 37°C overnight, and count the number of colonies.
  • Sterilization rate (1-the number of colonies in the drug group / the number of colonies in the medium control group) ⁇ 100%
  • MBC 50 is the lowest concentration that reaches 50% of the bactericidal effect.
  • the flavonoids of Cyclocarya paliurus leaves prepared by the present invention can effectively kill MRSA.
  • the present invention provides a flavonoid extract of Cyclocarya longiflora leaves, which has an obvious killing effect on methicillin gold-resistant Staphylococcus aureus strains and overcomes methicillin.
  • Aureus-resistant Staphylococcus aureus strains are resistant to most antibacterial drugs.
  • the flavonoid extract of Cyclobalanopsis vulgaris leaves can be prepared into antibacterial drugs or antibacterial agents against drug-resistant bacteria, and has a good application prospect in solving the worldwide problem of drug-resistant bacteria infection.

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Abstract

提供了青钱柳叶黄酮提取物在制备抗菌药物和/或抗菌剂中的用途，所述菌为耐药细菌。实验证明，青钱柳叶黄酮提取物对耐甲氧西林金黄色葡萄球菌株具有明显的杀灭作用，克服了耐甲氧西林金黄色葡萄球菌株对绝大多数抗菌药物的耐药的问题。

Description

青钱柳叶黄酮提取物在制备抗菌药物和/或抗菌剂中的用途 技术领域
本发明属于药物领域，具体涉及青钱柳叶黄酮提取物在制备针对耐药细菌的抗菌药物和/或抗菌剂中的用途。
背景技术
众所周知，细菌耐药已成为全球范围的重大公共健康问题，抗生素的滥用是造成细菌耐药的重要原因。耐甲氧西林金黄色葡萄球菌(Methicillin-resistant Staphylococcus aureus，简称“MRSA”)具有多重耐药性并伴有高发病率和高死亡率，是导致坏死性肺炎、严重败血症、坏死性筋膜炎等疾病的重要致病菌，是医院感染和社区感染的重要病原菌之一，给临床治疗带来了极大的困难。MRSA对绝大多数的抗菌药物或制剂耐药，甚至对目前临床抗耐药菌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的程度，感染MRSA的患者将面临无药可治的危险境地。因此，针对细菌耐药日趋严重的情况，开发具有抗耐药菌活性、对抗生素具有增敏作用的药物，刻不容缓。
青钱柳，Cyclocarya paliurus(Batal.)ljinskaja，又名青钱李、摇钱树、甜茶树等，系胡桃科青钱柳属植物特有单种属，为我国所特有，属濒危树种。青钱柳是一种高大速生阔叶乔木，因其树形似柳，果实圆形似铜钱，色青而下垂，故名“青钱柳”。据《中国中药资源志要》记载，其青钱柳叶具有清热消肿、止痛的功能，可用于治疗顽癣。长期以来，民间用青钱柳叶片做茶，具有清热解暑、降血糖、降血压和延年益寿的作用。
目前还未见将青钱柳提取物作为抗耐药细菌的药物或制剂的报道，更未见将青钱柳提取物作为抗耐甲氧西林金黄色葡萄球菌的药物或制剂的报道。
发明内容
本发明的目的在于提供青钱柳叶黄酮提取物在制备针对耐药细菌的抗菌药物和/或抗菌剂中的新用途。
本发明提供了青钱柳叶黄酮提取物在制备抗菌药物和/或抗菌剂中的用途，所述菌为耐药细菌。
进一步地，所述耐药细菌为耐药性革兰氏阳性菌，优选为耐药性金黄色葡萄球菌，更优选为耐甲氧西林金黄色葡萄球菌。
进一步地，所述青钱柳叶黄酮提取物中，青钱柳叶黄酮的含量为50wt.％以上，优选为67.3581wt.％以上。
进一步地，所述青钱柳叶黄酮提取物是通过以下方法制得的：取青钱柳叶，加溶剂提取，保留提取液，浓缩或干燥，即得。
进一步地，所述青钱柳叶为干燥的青钱柳叶粉末；和/或，所述溶剂为水， 溶剂与青钱柳叶的质量比为(5～15)：1，优选为10：1；和/或，所述提取温度为70～110℃，提取次数为1～5次，提取时间为每次30～120min，优选的，所述提取温度为90℃，提取次数为2次，提取时间为每次90min。
进一步地，所述提取液在浓缩或干燥之前，还包括提纯工艺，所述提纯工艺包括以下步骤：
在提取液中加入乙醇，使所得体系中乙醇的体积分数为50％～70％，混合均匀后，离心，保留上清液；优选的，所述体系中乙醇的体积分数为60％。
进一步地，所述提纯工艺还包括以下步骤：将上述上清液中的乙醇除去后，通过聚酰胺树脂柱洗脱，收集洗脱液；洗脱采用的洗脱剂依次为水、60％乙醇，其中，水的用量为5BV，60％乙醇的用量为5BV。
本发明还提供了一种青钱柳叶黄酮提取物，所述青钱柳叶黄酮提取物如上所述。
本发明还提供了一种抗菌药物，所述抗菌药物是以上述的青钱柳叶黄酮提取物为活性成分，加上药学上可接受的辅料制得的。
本发明还提供了一种抗菌剂，所述抗菌剂是以上述的青钱柳叶黄酮提取物为活性成分，加上制剂领域常用的辅料制得的。
实验结果表明，青钱柳叶黄酮提取物对甲氧西林金耐药黄色葡萄球菌株具有明显的杀灭作用，克服了甲氧西林金耐药黄色葡萄球菌株对绝大多数抗菌药物耐药的问题。青钱柳叶黄酮提取物可以制备成针对耐药细菌的抗菌药物或抗菌剂，在解决耐药细菌感染的世界性难题上应用前景优良。
抗菌药物是指具有杀菌或抑菌活性的药物。
抗菌剂是指具有杀菌或抑菌活性的制剂，一般不能用于治疗疾病。
青钱柳叶黄酮提取物是指从青钱柳叶中提取出来的含黄酮类化合物的提取物，青钱柳叶黄酮是指从青钱柳叶中提取出来的黄酮类化合物，黄酮类化合物是一类具有2-苯基色原酮(flavone)结构的化合物的总称。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1为16S RNA和mecA基因的检测结果。
图2为青钱柳叶黄酮对各菌株的杀菌作用曲线。
具体实施方式
本发明所用原料与设备均为已知产品，通过购买市售产品所得。
实施例1、本发明青钱柳叶黄酮提取物的制备
称取室温阴干的青钱柳叶500g，粉碎后加10倍(g/g)量水，90℃下提取2次，每次提取90min，经浓缩、离心后，取上清液，再浓缩。然后 加入乙醇至体系中的乙醇终浓度为65％(v/v)，混合均匀后，4800r/min离心，取上清液，除去乙醇直至液体无醇味。然后将液体利用聚酰胺树脂柱(填料为100g聚酰胺树脂)洗脱，先经5BV的水洗，然后用5BV的60％乙醇洗脱，收集洗脱液。将收集的洗脱液进行乙醇回收至呈流浸膏状，于50℃真空干燥24小时，得到粉末状固体，即青钱柳叶黄酮提取物，称量，保存备用。
采用紫外-可见分光光度法(《中国药典》2015年版通则0401)，以芦丁为对照，测得上述青钱柳叶黄酮提取物中青钱柳叶黄酮的含量为67.3581wt.％。
以下通过实验例证明本发明的有益效果。
实验例1、青钱柳叶黄酮对耐甲氧西林金黄色葡萄球菌的杀菌作用测试
1、菌株和药物
菌株：(1)普通金黄色葡萄球菌(ATCC25923)购自美国模式培养物研究所(American Type Culture Collection,ATCC)；(2)甲氧西林金耐药黄色葡萄球菌株(Methicillin-Resistant Staphylococcus Aureus，MRSA)均分离自四川大学华西医院，由临床微生物室提供：MRSA-1(临床编号：1911101191)、MRSA-2(临床编号：1911081137)、MRSA-3(临床编号：1911051296)、MRSA-4(临床编号：1911051125)、MRSA-5(临床编号：1911081165)。
药物：实施例1制得的青钱柳叶黄酮提取物粉末，青霉素钠和苯唑西林钠(购买市售产品)。
2、细菌的培养
培养基：普通金黄色葡萄球菌(ATCC25923)、MRSA-1、MRSA-2、MRSA-3、MRSA-4和MRSA-5的培养基均为MHB(Mueller-Hinton Broth)培养基。
培养条件：37℃，150rpm摇床培养。
3、实验方法
3.1 mecA基因的鉴定
mecA基因是MRSA特有的耐药基因，对MRSA的耐药性起决定性的作用。本实验采用PCR法验证各菌株中是否存在mecA基因，具体方法如下：
采用细菌基因组DNA提取试剂盒提取各菌株的基因组DNA。提取步骤详见试剂盒说明。分别以各菌株的基因组DNA为模板，采用多重PCR技术扩增16S RNA和mecA基因449bp片段。采用的引物序列为：16S RNA上游引物5'-AGAGTTTGATCCTGGCTCAG-3'(SEQ ID NO.1)和下游引物5'-GGTTACCTTGTTACGACTT-3'(SEQ ID NO.2)；mecA上游引物5'-CTCAGGTACTGCTATCCACC-3'(SEQ ID NO.3)和下游引物5'- CACTTGGTATATCTTCACC-3'(SEQ ID NO.4)。PCR扩增程序为：94℃ 3min，94℃ 45s，50℃ 45s，72℃ 1min30s，30循环，72℃ 5min。
3.2各菌株对青霉素钠、苯唑西林钠和青钱柳叶黄酮的敏感性测试
参照美国临床和实验室标准协会(Clinical and Laboratory Standards Institute，CLSI)有关抗菌药物敏感性试验的方法和标准，验证各菌株对药物的耐药性。本测试所采用的MHB培养基均含2％NaCl(w/v)。
试验菌的制备：分别接种各菌于5ml MHB培养基中，37℃摇床培养过夜，调整菌液麦氏比浊至0.5个麦氏单位后，再将菌液稀释100倍备用。最终细菌浓度为5×10 5cfu/ml。
青钱柳叶黄酮应用液的配制：精确称取实施例1制得的青钱柳叶黄酮提取物粉末79.8mg(根据青钱柳叶黄酮含量为67.3581wt.％，计算出粉末中含有青钱柳叶黄酮53.75mg)，溶解于0.32ml DMSO后，加入3.68ml MHB培养基稀释，得到青钱柳叶黄酮浓度为13.44mg/ml的青钱柳叶黄酮应用液。
青霉素钠和苯唑西林钠溶液母液的配制：精确称取100mg青霉素钠或苯唑西林钠溶于1ml去离子水中，经0.22μm滤过除菌，得青霉素钠或苯唑西林母液。吸取10.24μl青霉素钠或苯唑西林母液加入3.98976ml MHB培养基中，得256μg/ml的青霉素钠或苯唑西林钠应用液。
青钱柳叶黄酮，青霉素钠和苯唑西林钠药物浓度梯度的配制：取药物原浓度应用液100μl加入96孔板中第1和2孔。从第2孔起，加入培养基100μl连续对倍稀释至第9孔。设第10孔为培养基对照。从第1到第9孔，青钱柳叶黄酮类化合物的浓度分别为6720μg/ml，3360μg/ml，1680μg/ml，840μg/ml，420μg/ml，210μg/ml，105μg/ml，52.5μg/ml，26.25μg/ml；青霉素钠溶液或苯唑西林钠溶液浓度分别为：128μg/ml，64μg/ml，32μg/ml，16μg/ml，8μg/ml，4μg/ml，2μg/ml，1μg/ml，0.5μg/ml。
敏感性测试：在各浓度的药物孔和培养基对照孔中分别加入上述制备的菌液100μl。置培养孔板于湿盒中34℃孵育24h。取各菌液20μl，用培养基做50倍稀释后，取100μl涂布于培养平板，置37℃孵育过夜。将无菌落生长的最低药物浓度判定为最小抑菌浓度(minimal inhibit concentration，MIC)。
3.3青钱柳叶黄酮的杀菌作用测试
本测试所用MHB培养基均无NaCl添加。
试验菌的制备：同3.2所述。
青钱柳叶黄酮应用液的配制同3.2所述。
青钱柳叶黄酮浓度梯度的配制：同3.2所述。
杀菌作用的测试：在各浓度青钱柳叶黄酮孔和培养基对照孔中加入上述制备的菌液100μl。经充分混匀后，置湿盒中37℃孵育过夜。取各孔中菌液 20μl，用MHB培养基10倍稀释，100μl涂布于培养平板，置37℃孵育过夜，计数菌落数。
杀菌率＝(1-药物组菌落数/培养基对照组菌落数)×100％
药物最小杀菌浓度(minimum bactericidal concentration，MBC)由GraphPad Prism(Version7.04)采用非线性回归分析方式计算。MBC 50为到达50％杀菌效果的最低浓度。
4、实验结果
4.1 mecA基因的检测
从图1可以看出，在各MRSA菌株(MRSA-1、MRSA-2、MRSA-3、MRSA-4和MRSA-5)中均能检测到16S rRNA和mecA的表达；而在普通金黄色葡萄球菌(ATCC25923)中仅能检测到16S rRNA的表达。上述结果证实本实验所用MRSA临床分离株均为甲氧西林耐药金黄色葡萄球菌株，而普通金黄色葡萄球菌(ATCC25923)为β-内酰胺类药物敏感株。
4.2各菌株对青霉素钠、苯唑西林钠和青钱柳叶黄酮的敏感性比较
从表1可以看出，各MRSA菌株对青霉素钠和苯唑西林钠的敏感性均较普通金黄色葡萄球菌标准株(ATCC25923)低，MRSA菌株的MIC是普通金黄色葡萄球菌标准株(ATCC25923)的4～32倍，甚至更高。而各MRSA菌株对青钱柳叶黄酮的敏感性与普通金黄色葡萄球菌标准株(ATCC25923)一致或更敏感。上述结果说明MRSA对青钱柳叶黄酮的抗菌作用无耐药性。
表1 青霉素钠、苯唑西林钠和青钱柳叶黄酮对各菌株的MIC
Figure PCTCN2020110253-appb-000001
4.3青钱柳叶黄酮的杀菌作用
从图2和表2可以看出，青钱柳叶黄酮不仅对普通金黄色葡萄球菌标准株(ATCC25923)有杀伤作用，对各种MRSA也有明显的杀伤作用，且杀伤能力的大到小依次为：MRSA-4＞MRSA-5＞金黄色葡萄球菌ATCC25923＞MRSA-2＞MRSA-1＞MRSA-3。
所以，本发明制得的青钱柳叶黄酮能够有效杀灭MRSA。
表2 青钱柳叶黄酮对各菌株的MBC 50
Figure PCTCN2020110253-appb-000002
Figure PCTCN2020110253-appb-000003
综上所述，本发明提供了一种青钱柳叶黄酮提取物，该青钱柳叶黄酮提取物对甲氧西林金耐药黄色葡萄球菌株具有明显的杀灭作用，克服了甲氧西林金耐药黄色葡萄球菌株对绝大多数抗菌药物耐药的问题。青钱柳叶黄酮提取物可以制备成针对耐药细菌的抗菌药物或抗菌剂，在解决耐药细菌感染的世界性难题上应用前景优良。

Claims (10)

  1. 青钱柳叶黄酮提取物在制备抗菌药物和/或抗菌剂中的用途，所述菌为耐药细菌。
  2. 根据权利要求1所述的用途，其特征在于：所述耐药细菌为耐药性革兰氏阳性菌，优选为耐药性金黄色葡萄球菌，更优选为耐甲氧西林金黄色葡萄球菌。
  3. 根据权利要求2所述的用途，其特征在于：所述青钱柳叶黄酮提取物中，青钱柳叶黄酮的含量为50wt.％以上，优选为67.3581wt.％以上。
  4. 根据权利要求3所述的用途，其特征在于：所述青钱柳叶黄酮提取物是通过以下方法制得的：取青钱柳叶，加溶剂提取，保留提取液，浓缩或干燥，即得。
  5. 根据权利要求4所述的用途，其特征在于：所述青钱柳叶为干燥的青钱柳叶粉末；和/或，所述溶剂为水，溶剂与青钱柳叶的质量比为(5～15)：1，优选为10：1；和/或，所述提取温度为70～110℃，提取次数为1～5次，提取时间为每次30～120min，优选的，所述提取温度为90℃，提取次数为2次，提取时间为每次90min。
  6. 根据权利要求4或5所述的用途，其特征在于：所述提取液在浓缩或干燥之前，还包括提纯工艺，所述提纯工艺包括以下步骤：
    在提取液中加入乙醇，使所得体系中乙醇的体积分数为50％～70％，混合均匀后，离心，保留上清液；优选的，所述体系中乙醇的体积分数为60％。
  7. 根据权利要求6所述的用途，其特征在于：所述提纯工艺还包括以下步骤：将权利要求6所述上清液中的乙醇除去后，通过聚酰胺树脂柱洗脱，收集洗脱液；洗脱采用的洗脱剂依次为水、60％乙醇，其中，水的用量为5BV，60％乙醇的用量为5BV。
  8. 一种青钱柳叶黄酮提取物，其特征在于：所述青钱柳叶黄酮提取物如权利要求3～7任一项所述。
  9. 一种抗菌药物，其特征在于：所述抗菌药物是以权利要求8所述的青钱柳叶黄酮提取物为活性成分，加上药学上可接受的辅料制得的。
  10. 一种抗菌剂，其特征在于：所述抗菌剂是以权利要求8所述的青钱柳叶黄酮提取物为活性成分，加上制剂领域常用的辅料制得的。
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