| 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. |
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