【作者】 娄在祥；

【导师】 王洪新；

【作者基本信息】 江南大学 ， 食品营养与安全， 2010， 博士

【摘要】 本论文以牛蒡为原料,对牛蒡叶有效成分与牛蒡根菊糖的提取、分离及其活性进行了相关研究。为牛蒡综合深度加工利用提供了科学依据。具体研究内容及结果如下:1.牛蒡叶、根有效成分提取分离研究采用超声微波协同萃取技术提取多酚成分,发现提取时间、微波功率、料液比、提取次数对多酚提取有显著影响,分析得出了较佳提取条件为:以质量分数为70%的乙醇溶液为溶剂,提取时间30s,微波功率500w,超声波功率50w,料液比1g:20mL,提取次数为2次。在此条件下,多酚平均得率为10.28 mg/g,总提取物平均得率为232.79mg/g (牛蒡叶,干重)。通过扫描电镜观察发现,在超声微波协同萃取过程中,样品的微观结构被明显破坏,从而大大提高了多酚得率,加快了提取速度,取得最高得率所需的提取时间比超声波辅助提取和加热搅拌提取分别缩短了29.5min和5h。因此超声微波协同萃取技术在工业化提取多酚方面具有很大潜力,超声微波协同萃取为植物中多酚化合物的分析与鉴定提供了一种新的样品制备技术。采用超声微波协同萃取技术从牛蒡根中提取菊糖。分析得出了较佳提取条件为:以水为溶剂,提取时间60s,微波功率400w,料液比1g:15mL,菊糖平均得率为99.03 mg/g。结果表明,超声微波协同萃取处理破坏了样品的微观结构,因此与超声波辅助提取、加热搅拌提取相比,取得最高得率的提取时间分别显著缩短了120s与240s,提高了提取效率。采用二级超滤对菊糖提取液进行纯化处理,确定了较佳操作条件为:一级超滤膜截留分子量为10KD,超滤压力为0.5MPa,二级超滤膜为0.5KD,超滤压力为1.0MPa。提取液经过两级超滤处理后,制备了高纯度菊糖产品,为白色粉末,纯度达91.3%。将牛蒡叶总提取物依次用石油醚、乙酸乙酯、正丁醇、水分别萃取,得到四个组分,分别命名为PF、EF、BF、WF。乙酸乙酯组分经柱层析预处理后,再通过高速逆流色谱制备了对香豆酸与绿原酸,纯度分别为98.8%与98.3%,并通过UV、ESI-MS、1H-NMR与13C-NMR等波谱分析方法进行了鉴定。2.牛蒡叶成分的分析鉴定及5种主要成分同时检测方法的建立利用UPLC-MS/MS技术,通过与标准品比较保留时间、紫外光谱、质谱等,并进行分析,鉴定了牛蒡叶的十一种成分。这些化合物是槲皮素、二咖啡酰奎尼酸、苯甲酸、槲皮苷、咖啡酸、木犀草素、绿原酸、水杨酸、对香豆酸、牛蒡子苷、芦丁等。其中,水杨酸、对香豆酸、苯甲酸是在牛蒡叶中首次发现。并建立了同时测定牛蒡叶中五种主要活性成分(绿原酸、苯甲酸、咖啡酸、对香豆酸、芦丁)的HPLC分析方法,色谱条件为:色谱柱Waters symmetry C18;流动相甲醇(A)+ 20mmol/L甲酸水溶液(B),梯度洗脱;流速1mL/min;柱温30℃;检测波长280nm。所建立的方法方便快速,检测限达0.005μg/mL,回收率为95.7-105.8%,精密度为1.58-2.86%。3.牛蒡叶功能性成分的抗氧化、抗菌活性牛蒡叶总提取物清除DPPH自由基的IC50值为0.9 mg/mL,抑制脂质过氧化的IC50值为0.8 mg/mL;在浓度为2.5 mg/mL时,该提取物的羟自由基清除活性为55.21%,超氧阴离子自由基清除活性为79.80%。在总提取物萃取分级组分中,乙酸乙酯组分(EF)具有最强的抗氧化活性,其清除DPPH自由基、抑制脂质过氧化的的IC50值分别为90.67与133.91μg/mL,EF的抗氧化活性与TBHQ相近,因此EF有望替代合成抗氧化剂。且EF与TBHQ表现为协同增效作用,这为组合抗氧化剂的使用提供了基础。牛蒡叶总提取物对六种食品相关细菌(大肠杆菌、痢疾志贺氏菌、鼠伤寒沙门氏菌、枯草芽孢杆菌、肺炎链球菌、金黄色葡萄球菌)具有抑制活性,最低抑菌浓度(MIC)为1.5-2.5mg/mL;对三种真菌(黑曲霉、酿酒酵母、扩展青霉)的抑制效果不明显。探讨了牛蒡叶总提取物的萃取分级组分对6种食品相关菌的抑制情况。结果表明,乙酸乙酯组分(EF)能够抑制全部受试菌,EF的抗菌活性最强,其MIC为88-352μg/mL。杀菌试验表明,经过12h,在浓度为1×MIC,3×MIC和5×MIC时,EF能够杀灭4/6,6/6和6/6的受试菌。所以,EF是一种有效的天然杀菌剂资源。从牛蒡叶中纯化得到的对香豆酸的抗菌活性比绿原酸强,对香豆酸对肺炎链球菌、金黄色葡萄球菌、枯草芽孢杆菌、痢疾志贺氏菌、鼠伤寒沙门氏菌、大肠杆菌的MIC分别为:20、10、20、20、20、80μg/mL。通过经口急性毒性初步试验测定得出EF的LD50>10000mg/kg,属于实际无毒级。对于各个组分,抗氧化、抗菌活性与其多酚含量密切相关(R=0.86 ~0.99)。因此组分的抗菌、抗氧化活性主要是绿原酸、对香豆酸、芦丁、咖啡酸、水杨酸等多酚化合物共同作用的结果。4.对香豆酸、绿原酸的抗菌机理在多酚化合物绿原酸、对香豆酸作用下,都引起了痢疾志贺氏菌细胞膜通透性增大,导致了明显的胞内离子泄露及电导率的增大。流式细胞仪的分析表明,经过绿原酸或对香豆酸作用后,都导致了细菌细胞膜破裂。透射电镜实验结果直观的表明,对香豆酸、绿原酸都能导致细胞膜破裂或孔洞形成,导致细菌细胞内容物外泄,最终导致痢疾志贺氏菌死亡。对香豆酸、绿原酸进入细菌细胞后,其作用靶标是DNA。琼脂糖凝胶电泳、荧光光谱分析表明,对香豆酸或绿原酸都不能断裂细菌基因组DNA,而是结合DNA。圆二色谱表明对香豆酸、绿原酸结合到DNA的链上以后,使其双螺旋结构变得松散,并造成了细菌DNA构象的改变。从而改变了细菌的各种生理功能,最终导致细菌死亡。更多还原

【Abstract】 Burdock (Arctium lappa L.) is a popular vegetable in China and Japan, exhibiting various biochemical activities. The extraction, separation, activities of the active components in burdock leaves and the inulin in burdock root were studied. The present study could provide help for the deep development of burdock and its application in food industry.1. The extraction and separation of active components from burdock leaves and burdock rootsThe simultaneous ultrasonic and microwave assisted extraction (UMAE) technique was employed to obtain phenolics. The effects of UMAE variables on the yield of phenolics were investigated. The optimized conditions were as follows: solvent to solid ratio was 20:1 (mL/g), extraction time was 30s, microwave power was 500 W and two times of extraction. Moreover, the phenolic yield of UMAE (10.28mg/g) was higher than that by maceration extraction (ME) and ultrasonic extraction, indicating a significant reduction of extraction time and an improvement of efficiency. The phenomenon is related to the strong disruption of leaf tissue structure by microwave induced expansion and ultrasonic shaking, which had been observed with the scanning electron microscopy. The results suggest that UMAE is a good alternative for the extraction of phenolics, with a great potential for industrial application. Also, UMAE provides a new sample preparation technique for characterization of the phenolic compounds from plants. The inulin from burdock root was extracted by simultaneous ultrasonic/ microwave assisted extraction (UMAE). It was found that UMAE required a much shorter extraction time than conventional stirring extraction. The suitable condition for UMAE of inulin was under a simultaneous ultrasonic power of 50w and a microwave power of 400w, at a ratio of 1g of solid material to 15mL of water for an extraction time of 60s. A comparison of scanning electron microscopy images of raw and simultaneous ultrasonic/microwave treated burdock roots indicated microfractures and disruption of cell walls in burdock root flakes.After ultrafiltration, the purified inulin product was obtained, and the purity of inulin product was 91.3%. The burdock leaves extract was fractioned with petroleum ether, ethyl acetate, n-butanol and water, named as PF, EF, BF and WF. After activity-guide fractionation, separation by column chromatogram and high-speed counter-current chromatography, two purified active compounds (chlorogenic acid, p-coumaric acid) were obtained from EF. The compounds were then identified by UV、ESI-MS、1HNMR and 13CNMR.2. Identification of the compounds and development of a method for the determination of five compounds in burdock leavesThe compositions of burdock leaves were then identified by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The composition was identified based on retention time, UV and MS spectra compared with those of authentic compounds or literature data, and 11 compounds were characterized, providing a more complete identification of phenolic compounds in burdock leaves than previously reported. The compounds were quercetin, cynarin, o-hydrobenzoic acid, benzoic acid, quercitrin, caffeic acid, luteolin, chlorogenic acid, p-coumaric acid, arctiin and rutin. The occurrence of benzoic acid, o-hydrobenzoic acid and p-coumaric acid is reported for the first time. By optimizing the extraction, separation and analytical conditions, a sensitive and accurate high performance liquid chromatographic method has been developed for the simultaneous determination of five active compounds (benzoic acid, caffeic acid, chlorogenic acid, p-coumaric acid and rutin) in burdock leaves. The analysis was performed on a Waters symmetry C18 column at 30℃using 20mmol/L aqueous formic acid solution/methanol gradient system at a flow rate of 1.0mL·min-1 and photodiode array detection (PDA) at wavelength of 280 nm. The method showed good linearity and satisfactory accuracy and recoveries.3. The antimicrobial and antioxidant activities of active components from burdock leavesThe burdock leaf extract exhibited high lipid peroxidation inhibition activity, DPPH radical, hydroxyl and superoxide anion radicals scavenging ability. The antioxidant activities of each burdock leaves fraction were investigated alone and in combination with tertiary butylhydroquinone (TBHQ). The EF exhibited the highest antioxidant activity. Although TBHQ exhibited higher lipid peroxidation inhibitory activity than EF, the reducing power, superoxide anion scavenging capability, DPPH and hydroxyl radicals scavenging ability of EF were higher than those of synthetic antioxidant (TBHQ). Moreover, a synergistic antioxidant effect between EF and TBHQ was first demonstrated by isobolographic analysis, indicating that EF dramatically enhances the antioxidant efficiency of TBHQ. The results indicate that the EF could be used as sources of nature antioxidant in food industry, and allows a decrease of about 4 folds in the amounts of the synthetic compounds used. The extract of burdock leaves was tested for its antimicrobial potential against three Gram-positive (streptococcus pneumoniae, bacillus subtilis, Staphylococcus aureus), three Gram-negative bacteria (Shigella dysenteriae, Escherichia coli, salmonella typhimurium) and three kinds of fungi (Aspergillus niger, saccharomyces cerevisiae, penicillium). The extract could not significantly inhibit the growth of fungi. But, the extract was effective against both Gram-positive and Gram-negative bacteria. The MIC values ranged from 1.5 to 2.5 mg/mL. The antibacterial activities of burdock leaves fractions against six food-related bacteria were first investigated. The data from minimum inhibitory concentration (MIC) values showed that EF and other fractions effectively inhibited the growth of all test bacterial pathogens, the antibacterial activity of EF being much greater than BF and WF. The time-kill assay indicated that EF exhibited significant bactericidal activity against all the six pathogens. At 12 h, EF was bactericidal for 4/6, 6/6 and 6/6 strains at 1×MIC, 3×MIC and 5×MIC, respectively. The ethyl acetate fraction based on its lower MIC values, concentration and time-dependent antibacterial ?and bactericidal activities could be useful in control of bacterial pathogens. The antibacterial activities of chlorogenic acid, p-coumaric acid, rutin and benzoic acid against six food-related bacteria were investigated. The antibacterial activity of p-coumaric acid was higher than other conpounds. The MIC values of p-coumaric acid against streptococcus pneumoniae, Staphylococcus aureus, bacillus subtilis, Shigella dysenteriae, Escherichia coli and salmonella typhimurium were 20, 10, 20, 20, 20 and 80μg/mL, respectively. The oral acute toxicity test indicated that the LD50 of EF was beyond 10000mg/kg, and EF was actually non-toxic. For all the fractions, the antioxidant and capacity had a significant correlation with total phenolic content. The antibacterial ability also had a significant correlation with total phenolic content, suggesting that the activities were probably due to the combined action of phenolic compounds. The phenolic compounds of the fractions were then identified. Among them the contents of chlorogenic acid, o-hydrobenzoic acid, p-coumaric acid and rutin were high.4. The antibacterial mechanism of chlorogenic acid and p-coumaric acidChlorogenic acid and p-coumaric acid could increase the membrane permeabilization of G- bacteria, causing the ion leakage. The treatment of Shigella dysenteriae cells with phenolic compounds induced a major influx of PI, demonstrating that the phenolic compounds executed their antibacterial effects via binding to the surface of the cells, followed by the formation of membrane pores. Also, it was supported by the observations made under transmission electron microscopy. Taken together, the antibacterial activity of chlorogenic acid and p-coumaric acid was concluded to result from the disintegration of the cell membrane.The nucleic acid was the action target of two phenolic compounds after penetrating the Shigella dysenteriae cells. They didn’t breakdown the genomic DNA, but strongly binding to DNA. The results of fluorescence quenching experiments and circular dichroism indicated that chlorogenic acid and p-coumaric acid could bind to bacterial genomic DNA, change its conformation, and loosen the double helix of DNA. Both phenolic compounds binding to DNA influenced the function of gene. The results demonstrated that chlorogenic acid and p-coumaric acid have dual bactericidal mechanisms: disrupting bacterial cell membranes and binding to bacterial genomic DNA to inhibit cellular functions, ultimately leading to cell death.更多还原