兰州大学机构库 >药学院
植物多酚及其衍生物的抗菌活性评价与作用机制研究
Alternative TitleEvaluation of antimicrobial activity and mechanism of action of plant polyphenols and their derivatives
胡勇梅
Subtype硕士
Thesis Advisor刘映前
2023-05-29
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name药学硕士
Degree Discipline药学
Keyword植物多酚 plant polyphenols 抗菌活性 antimicrobial activity 紫檀芪 pterostilbene 黄曲霉 Aspergillus flavus 黄曲霉毒素 aflatoxin
Abstract

农业和食品行业中真菌、细菌及其毒素污染现状和传统化学杀菌剂频繁不当施用诱发的耐药菌株的大量出现和农药残毒超标等问题已经引起了公众对食品安全和生态环境的广泛担忧。因此,从自然资源中探索高效的抑菌物质来代替或减少传统化学农药用量是作物病害绿色防控的有效途径。多酚是植物自身通过多种生物合成途径形成的重要次生代谢产物,存在于多种可食用作物和中草药的根、皮、茎、叶和果实中,作为植物王国中的第二大化学物质群,其具有的抗菌和抗氧化特性使其成为抗菌剂开发和食品储藏等相关领域的研究热点之一。开展植物多酚抗菌活性评价工作,寻找高效低毒的植物多酚结构,阐明其抗菌作用机制对绿色抑菌剂的开发和多酚在食品储藏行业中的应用具有极其重要的意义。 
本研究评价了多种植物多酚及其衍生物的抗植物病原真菌和食源性细菌活性,并进一步探究了相应高活性多酚及其衍生物的抗菌作用机制。现将论文主要研究内容分述如下: 
1、植物多酚及其衍生物的抗菌活性评价 
本章活性筛选测定了包括酚酸、黄酮、苯丙素、木脂素、二苯乙烯和简单取代酚类等多种结构类型的酚类化合物对植物病原真菌和食源性细菌的抑菌活性。其中,没食子酸丁酯、没食子酸异戊酯、反式-阿魏酸、咖啡酸乙酯、水杨酸、肉桂酸、光甘草定、紫檀芪、3’-羟基紫檀芪、厚朴酚、和厚朴酚、丁香酚、异丁香酚甲醚、异丁香酚、α-细辛脑、橄榄醇、百里香酚、香芹酚等天然植物多酚和多种简单烷基取代苯酚具有良好的抗植物病原真菌活性。其中,光甘草定、紫檀芪、厚朴酚、和厚朴酚、百里香酚、香芹酚和 6-叔丁基间甲酚对黄曲霉、扩展青霉、匍枝根霉、核果褐腐病菌和灰葡萄孢菌等 5种植物病原真菌具有优异的广谱抗真菌活性,EC50均低于 50 μg/mL。黄酮类化合物川陈皮素对匍枝根霉具有特异性抑菌活性,EC50为14.03 μg/mL;黄腐酚、异黄腐酚对灰葡萄孢菌具有特异性抑菌活性;EC50分别为 2.51、4.29 μg/mL。食源性细菌活性筛选发现没食子酸异戊酯、厚朴酚、和厚朴酚和橄榄醇对蜡样芽孢杆菌、单核增生李斯特菌、大肠杆菌O157:H7和肠炎沙门氏菌具有优异的抗细菌活性,MIC90均小于 200 μg/mL。 
2、二苯乙烯类化合物的抗菌活性评价及紫檀芪对黄曲霉的抗菌机制与毒素代谢影响研究 
第二章活性筛选发现天然源二苯乙烯类骨架结构具有良好的抗植物病原真菌活性,其中紫檀芪、3’-羟基紫檀芪和苯烯莫德对黑曲霉、黄曲霉、扩展青霉、小麦赤霉、核果褐腐病菌、灰葡萄孢菌和胶孢炭疽菌等植物病原真菌具有广谱抗真菌活性。进一步体外活性评价表明,紫檀芪对黄曲霉的抑菌活性最佳,其对菌丝生长抑制的 EC50为 15.94 μg/mL,对黄曲霉的抑菌效果优于广泛用于食品防腐保鲜的天然产物纳他霉素(EC50=22.01 μg/mL)。紫檀芪对孢子萌发抑制的 EC50为 3.86 μg/mL。液体培养基中,紫檀芪对黄曲霉菌丝生物量的抑制呈浓度依赖性。体内防效评价发现,在紫檀芪浓度为 250和 500 μg/mL时,能够有效抑制黄曲霉对花生的侵染。毒素代谢影响研究发现,75 μg/mL紫檀芪能够显著抑制黄曲霉毒素代谢途径关键基因 afl A、afl B、afl C、afl R、afl S和真菌代谢全局调控基因Lea A和 V eA的表达,从而减少 37.62%黄曲霉毒素 B1的生物合成。机制探究结果表明紫檀芪能够诱导黄曲霉细胞内活性氧的大量生成,干扰细胞内氧化还原平衡,造成线粒体膜电位崩塌和细胞膜完整性丧失,通过多种作用方式最终导致黄曲霉细胞死亡。 
3、酰基化间苯三酚衍生物的合成、抗菌活性评价和作用机制浅析 
本章以间苯三酚为合成子,进行酰基化衍生合成、抗菌活性评价和作用机制研究。体外活性评价发现化合物 2b能够有效抑制灰葡萄孢菌、核果褐腐病菌和匍枝根霉的菌丝生长,其 EC50分别为 1.39,1.18和 7.76 μg/mL。体内抗菌实验中,化合物 2b在 200 μg/mL对灰霉病和褐腐病的治疗效率分别为 76.26%和83.35%,略低于商业化杀菌剂嘧霉胺和甲基硫菌灵的治疗效果。毒性评价发现化合物 2b具有较低的细胞毒性和植物毒性。初步机制探究表明,化合物 2b通过抑制孢子萌发、破坏菌丝细胞膜完整性、导致细胞内容物渗漏等途径引起灰葡萄孢菌丝细胞死亡,发挥抗菌作用。

Other Abstract

The current situation of fungal, bacterial, and toxin contamination in agriculture and food industries, as well as the emergence of resistant strains of fungi and bacteria caused by frequent and improper application of traditional antimicrobial agents and excessive pesticide residues, has sparked widespread public concern about food safety and environmental risks, prompting researchers to look for efficient antimicrobial substances derived from natural resources to replace or reduce the amount of traditional antimicrobial agents. Polyphenols are a type of secondary metabolite found primarily in the roots, bark, stems, leaves, and fruits of many edible crops and herbs. As the second largest group of chemicals in the plant kingdom, their antimicrobial and  antioxidant properties make them a hot research topic in the fields of antimicrobial agent development and food storage. It is critical to evaluate the antimicrobial activity and study the mechanism of action of plant polyphenols in order to promote the development of phenolic compounds for crop disease prevention and control and food preservation industries. In this study, we studied the anti-phytopathogenic fungal, bacterial, and food-borne bacterial activities of various plant polyphenols and their derivatives, as well as the mechanisms of action those polyphenols and their derivatives. 
The following are the key findings. 
1. Antimicrobial activity screening of plant polyphenols and their derivatives 
In this chapter, the antimicrobial activity of phenolic compounds, including flavonoids, phenolic acids, lignans, stilbenes, and simple phenolics, was determined through activity screening. Among them, butyl gallate, isoamyl gallate, trans-ferulic acid, ethyl caffeate, salicylic acid, cinnamic acid, glabridin, pterostilbene, 3'-hydroxypterostilbene, magnolol, honokiol, eugenol, isoeugenol methyl ether, isoeugenol, α-asarone, olivetol, thymol, carvacrol, and a variety of simple alkyl substituted phenols had good anti-phytopathogenic fungal activity. Among them, glabridin, pterostilbene, magnolol, honokiol, thymol, carvacrol and 6-tert-Butyl-m-
cresol had excellent and broad-spectrum antifungal activity against Aspergillus flavus, Penicillium expansum, Rhizopus stolonifer, Monilinia fructicola, and Botrytis cinerea, the EC50 values of five phytopathogenic fungi were all less than 50 μg/mL. Nobiletin had specific antifungal activity against Rhizopus stolonifer, with an EC50 of 14.03 μg/mL. Xanthohumol and isoxanthohumol showed specific antifungal activity against Botrytis cinerea with EC50 of 2.51 and 4.29 μg/mL, respectively. Following that, it was discovered that gallic acid isoamyl ester, magnolol, honokiol and olivetol exerted the best antibacterial activity against four food-borne bacteria, with MIC90 less than 200 μg/mL. 
2. Evaluation of antimicrobial activity of stilbenes, study on the mechanism of action and toxin metabolism of Aspergillus flavus 
In this chapter, seven stilbene compounds, namely resveratrol, oxyresveratrol, piceatannol, pterostilbene, 3'-hydroxypterostilbene, polydatin and tapinarof, were selected for antimicrobial activity assay and it was found that pterostilbene, 3-hydroxypterostilbene and tapinarof exhibited broad-spectrum antifungal activity against plant pathogenic fungi such as Aspergillus niger, Aspergillus flavus, Penicillium 
expansum, Fusarium graminearum, Monilinia fructicola, Botrytis cinerea and Colletotrichum gloeosporioides. Further in vitro activity evaluation showed that pterostilbene exerted the best inhibitory activity against Aspergillus flavus with an EC50 
of 15.94 μg/mL for mycelial growth inhibition and 3.86 μg/mL for spore germination inhibition, and concentration-dependent inhibition of Aspergillus flavus mycelial biomass production under liquid culture conditions. In vivo efficacy evaluation revealed that treatment with pterostilbene at 250 and 500 μg/mL could effectively inhibit Aspergillus flavus infestation on peanut. Toxin metabolism studies revealed that 75 μg/mL of pterostilbene significantly inhibited the expression of afl A, afl B, afl C, afl R, afl S and the global regulatory genes of fungal metabolism Lea A and V eA in the toxin metabolism pathway, thereby reducing the biosynthesis of aflatoxin B1 by 37.62%. The results of the mechanism investigation indicated that pterostilbene can induce a large 
production of reactive oxygen species in Aspergillus flavus cells, interfere with the intracellular redox balance, cause the collapse of mitochondrial membrane potential, and loss of cell membrane integrity, and eventually lead to the death of Aspergillus flavus cells through various modes of action. 
3. Synthesis, evaluation of antimicrobial activity and mechanism of action of acylated phloroglucinol derivatives 
In this chapter, the synthesis, antimicrobial activity evaluation and mechanism of action of acylated phloroglucinol derivatives were investigated, and it was found that compound 2b could effectively inhibit the mycelial growth of Botrytis cinerea, Monilinia fructicola and Rhizopus stolonifer with EC50 of 1.39, 1.18 and 7.76 μg/mL, respectively, and had low cytotoxicity and phytotoxicity. In an in vivo antimicrobial 
assay, the therapeutic efficiency of compound 2b at 200 μg/mL against gray mold and brown rot was 76.26% and 83.35%, respectively, which was slightly lower than that of the commercial fungicides pyrimethanil and thiophanate-methyl. Preliminary mechanistic studies suggest that compound 2b may exert its antifungal inhibitory effects by inhibiting spore germination, disrupting mycelial cell membrane integrity, 
and causing leakage of cell contents. 

MOST Discipline Catalogue医学 - 药学(可授医学、理学学位) - 药学
URL查看原文
Language中文
Other Code262010_220200941820
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/536464
Collection药学院
Affiliation
兰州大学药学院
Recommended Citation
GB/T 7714
胡勇梅. 植物多酚及其衍生物的抗菌活性评价与作用机制研究[D]. 兰州. 兰州大学,2023.
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