【作者】 谌馥佳；

【导师】 刘兆普； 刘玲；

【作者基本信息】 南京农业大学 ， 应用海洋生物学， 2013， 博士

【摘要】 菊芋(Helianthus tuberosus L.)属于菊科(Compositae)向日葵属多年生草本植物,广泛的分布在我国大部分地区,是一种适应性极强,具有生态和经济双重价值的作物。菊芋广泛用于造纸业以及食品、药物、饲料、糖和酒精的工业生产。目前菊芋产品的开发多是利用块茎和茎秆,而在我国多数菊芋叶片未能得到开发和利用,同时也加重了环境污染和资源浪费。由于菊芋地上部分具有抗逆性强,极少病虫害的特点,引起了人们对其化学成分及生物活性的关注。本文对菊芋叶片中抑菌活性成分进行了系统性研究,并开发其防治植物病害的新应用。这对于有效利用和开发菊芋这一自然资源具有重要的意义。本文主要研究结果如下：1.菊芋叶片化学成分中,总糖和总蛋白含量比总酚和总黄酮要高的多。南芋叶片中各成分含量除总黄酮差异不明显外,总糖、总蛋白和总酚均显著高于野生型和青芋。花期是菊芋叶片各化学成分积累的重要时期,总酚、总黄酮、总糖和总蛋白含量均显著高于现蕾期和块茎膨大期。菊芋叶片中水解氨基酸的总含量,可达叶片干重的12.445%,其中脯氨酸、天门冬氨酸、谷氨酸和亮氨酸为菊芋叶片中氨基酸的主要组成成分。游离氨基酸中脯氨酸含量最高。2.本文分析并鉴定了菊芋叶片中的酚类物质,有10种属于绿原酸类(CGAs)化合物,包括3种绿原酸异构体、4种二咖啡酰奎尼酸异构体、咖啡酸、P-香豆酰奎尼酸、阿魏酰奎尼酸,还有4种其他化合物,分别是异鼠李素-O-葡萄糖苷、咖啡酰葡萄糖、山奈酚葡萄糖醛酸苷、山奈酚-3-O-葡萄糖苷。本文研究发现在所有菊芋品种中咖啡酸的含量最低,绿原酸与4,5-二咖啡酰奎宁酸的含量最高。南芋叶片各酚酸类物质的含量显著高于野生型和青芋,因此南芋品种可作为酚酸类物质开发和利用的重要资源。以南芋为例,花期的各种酚酸含量普遍高于现蕾期和块茎膨大期,且差异极显著。3.本文采取的乙醇减压回流法作为抑菌活性物质的提取方法,具有提取效率高、抑菌效果好以及对环境友好等的优点,适合大规模生产的需要。减压回流法所得提取物浸膏的总酚和总黄酮含量显著高于回流法和室温浸提法,绿原酸含量也显著高于其他两种方法。乙酸乙酯层浸膏总酚和总黄酮含量最高,分别可达306.38mg/g和97.33mg/g,这说明菊芋叶片中的黄酮类和酚类物质属于极性中等偏大的活性成分。正丁醇层浸膏的绿原酸含量最高可达76.09mg/g,占6种酚酸总含量的55.70%。乙酸乙酯层浸膏中除绿原酸外的其他酚酸含量均为最高。4.菊芋叶片乙醇粗提物(减压回流法)具有广泛的抑菌谱,可以用于多种植物病害的防治。尤其对半知菌亚门的番茄灰霉病菌、苹果炭疽病菌、小麦纹枯菌和鞭毛菌亚门的辣椒疫霉病菌的抑制效果较好,在供试浓度10g.L-1下,抑制率分别可达98.22%、89.77%、74.62%和87.85%。石油醚、氯仿、乙酸乙酯以及正丁醇浸膏对四种植物病原真菌均有一定的抑制效果。氯仿层对辣椒疫霉病菌的抑制效果最好,EC50值仅为0.875g.L-1；乙酸乙酯层对小麦纹枯病菌的抑制效果最好,EC50值仅为0.300g.L-1；正丁醇层对小麦纹枯病菌的抑制效果最好,EC50值仅为0.232g.L-1,对辣椒疫霉病菌的抑制效果次之,ECs0值为0.839g.L-1。菊芋叶片浸膏用于番茄灰霉病的防治,在有效浓度相同的情况下,与商品化的杀菌剂多菌灵的防效相当,且差异不显著,安全无公害。因此开发菊芋叶片作为果蔬保鲜剂,对于菊芋资源的综合利用具有重要意义。5.结合生物活性追踪试验,包括生长速率法和生物自显影法,筛选菊芋叶片中抑菌活性较好的正丁醇层中活性物质,通过柱层析(AB-8柱、聚酰胺柱、ODS柱、Toyopearl HW-40凝胶柱)和制备液相色谱分离得到10种化合物,通过HPLC-ESI-MS、1H-NMR结合化学性质和标准品对照,鉴定了6种化合物为已知酚酸类物质,分别是绿原酸、咖啡酸和4种二咖啡酰奎宁酸的同分异构体。6.咖啡酸、3,4-二咖啡酰奎宁酸和1,5-二咖啡酰奎宁酸能有效的抑制小麦赤霉病菌的生长,MIC值分别可达108、60和4.2μgmL-1。总酚含量与各粗提物对辣椒疫霉病菌和小麦纹枯病菌的EC50呈现明显的负相关性,咖啡酸的含量同各粗提物对小麦纹枯病菌的EC50值呈显著负相关性,咖啡酸和1,5-二咖啡酰奎宁酸含量同各粗提物对辣椒疫霉病菌的EC50值呈现显著的负相关性,推测菊芋叶片中这些酚酸类抑菌物质可能单独、也可能协同抑制植物病原真菌。氯仿层萃取物和正丁醇层萃取物处理的辣椒疫霉病菌菌丝形态和细胞超微结构发生一些变化：菌丝凹陷,局部断裂或者出现裂纹,生长点异常；菌丝体细胞壁显著增厚,出现质壁分离,氯仿层萃取物处理还能使菌丝细胞器大量降解。通过本文的研究将菊芋叶片提取物应用于防治植物病害,不仅从废物利用的角度,大大提高了菊芋产业的经济附加值,而且开拓了植物源农药的新用途及新思路,具有十分重要的研究价值。更多还原

【Abstract】 Helianthus tuberosus L.(Jerusalem artichoke), Asteraceae family, is a perennial herb distributed in most of china. Helianthus tuberosus has been used in food, pharmaceutical, feed, sugar, paper, and bioethanol industry, and in desert and tideland control.Nowadays, the products originating from tubers and stems in Helianthus tuberosus were fully utilized, whereas it aggravates environmental pollutions and wastes of resources that quite a lot of Helianthus tuberosus leaves have not been utilized. Given its wide adaptation (it is resistant to drought, cold, wind stress, diseases and pests), Helianthus tuberosus has both economic and ecological values. Therefore, more studies were focused on the chemicals and their bioactivities of aerial part Helianthus tuberosus. Chemical constituents of Helianthus tuberosus leaves and their antifungal activities were systematically investigated for making full use of Helianthus tuberosus and control plant diseases.The main results described are as following:1. The main components in Helianthus tuberosus leaves are sugar and protein. The contents of total sugars, proteins and phenolics in cultivar Nan Yu were significantly higher than the wild accession and cultivar QingYu, while the contents of total flavonoids between the three cultivars were not significantly different.The contents of total phenolics, flavonoids, sugars and proteins were highest at flowering stage. Thus, the flowering stage is an important period for chemicals accumulation.Content of hydrolysis amino acids was up to12.445%for dried weight leaves, while the main amino acids in Helianthus tuberosus leaves were proline, aspartic acid, glutamic acid and leucine. Proline was the main free amino acid in Helianthus tuberosus leaves.2. Ten chlorogenic acids (CGAs) were identified (chlorogenic acid, two isomers of chlorogenic acid, caffeic acid, p-coumaroyl-quinic acid, feruloyl-quinic acid,3,4-dicaffeoyquinic acid,3,5-dicaffeoylquinic acid,1,5-dicaffeoylquinic acid and4,5-dicaffeoylquinic acid) by comparing their retention times, UV-Vis absorption spectra and MS/MS spectra with standards. In addition, four other phenolic compounds, including caffeoyl glucopyranose, Isorhamnetin glucoside, kaempferol glucuronide and kaempferol-3-o-glucoside. The variations in phenolic concentrations and proportions in Helianthus tuberosus leaves were influenced by genotype and plant growth stage. Cultivar NanYu had the highest content of phenolic acids, in particular3-o-caffeoylquinic acid and4,5-dicaffeoylquinic acid compared with the other genotypes (wild accession and QingYu),while caffeic acid was detected in low concentration in all genotypes of all the phenolic acids. Cultivar NanYu of Helianthus tuberosus is a potential source of natural phenolics. Considering various growth stages, the contents of all phenolic acids in cultivar NanYu was significantly higher at flowering stage than at budding and tuber swelling stages.3. The antifungal components in Helianthus tuberosus leaves were screened by different solvents, different extracting methods combined with bioassay.The result showed that ethanol-reluxing under vacuum suited for manufacture on a large scale, with the characteristics of high extraction efficiency, more antifungal effect and good environmental compatibility. The contents of total phenolics and total flavonoids in refluxed extract under vacuum were significantly higher than the refluxed and macerated extracts. The total phenolics and flavonoids mainly presented in ethyl acetate fraction, with the contents of306.38mg/g and97.33mg/g in dired extracts. The content of chlorogenic acid in crude refluxed extract under vacuum was significantly higher than other methods, so was the n-butanol fractions. The content of chlorogenic acid was up to76.09mg/g in dired extracts, which was significantly higher than other fractions, accounting for55.70%of the total amount of the six main phenolic acids. Ethyl acetate fraction had the great amount of all the phenolic acids except chlorogenic acid, which further demonstrate that phenlics might be concentrated in polar fractions.4. The ethanol crude extracts from Helianthus tuberosus leaves were active against various phytopathogenic fungi.They were active in bioassays against Botrytis cinerea, Colletotrichum gloeosporioides and Rhizoctonia cerealis belonging to deuteromycete, Phytophthora capsici Leonian belonging to Mastigomycotin, with the inhibition rates of98.22%,89.77%,74.62%and87.85%(10g L-1).Chloroform fraction was more active against Phytophthora capsici Leonian, with the EC50value of0.875g L-1. Ethyl acetate and n-butanol fractions were more active against Rhizoctonia cerealis, with the EC50value of0.300g L-1and0.232g L-1, respectively. The control efficiencies of extract from Helianthus tuberosus leaves on grey mould caused by B. cinerea was equivalent to commercial preparation Carbendazim, but the natural extracts was safer with lower costs. The extracts of antifungal compounds from Helianthus tuberosus leaves were investigated for potential use in enhancing preservation of fruits and vegetables in storage, which is significant to comprehensive utilization of Helianthus tuberosus.5. The antifungal compounds in Helianthus tuberosus leaves were investigated by the activity-guided method. The antifungal activies were evaluated by the growth inhibition bioassay and TLC bioautography.The antifungal compounds in n-butanol fraction were isolated and purified by AB-8macroporous resin, polyamide, ODS, TSKgel Toyopearl HW-40column and PHPLC.There were ten compounds obtained from n-butanol fraction.Six of these compounds were identified as known phenolic acids by HPLC-ESI-MS,]H-NMR and compared with standards, which were Chlorogenic acid, Caffeic acid and four isomers of dicaffeoylquinic acid.6. Purified caffeic acid,3,4-DiCQA and1,5-DiCQA were active in bioassays against Gibberella zeae.There was a significant negative correlation between the contents of total phenolics and EC50Values against Phytophthora capsici Leonian and Rhizoctonia cerealis. It indicated that the phenolics might be major antifungal compounds. There was a significant negative correlation between the content of caffeic acid and EC50Values against Rhizoctonia cerealis,so was the content of caffeic acid,1,5-DiCQA and EC50Values against Phytophthora capsici Leonian. It was supposed that the phenolic acids might have independent and combined effects against phytopathogenic fungi. Scanning electron micrographs (SEM) of hyphae from P. capsici treated with chloroform and n-butanol fractions had been used to observe a series of marked structural and morphological alterations of hyphae. The changes included irregular hyphae in diameter, local denting, excessive branching, some of broken mycelium, or cracks on the surface of hyphae, which indicated the damage of cell wall. The micrographs of Transmission electron microscopy (TEM) indicated that the cellular ultrastructre of treated hyphae was damaged. The cell walls were thickened and plasmolysis was appeared. Organelles were degraded in chloroform fraction treatment.The extracts of antifungal compounds from Helianthus tuberosus leaves were investigated for potential use in improving industrial economic added value of Helianthus tuberosus largely from a view of waste utilization.更多还原