【作者】 张梅；

【导师】 潘大仁；

【作者基本信息】 福建农林大学 ， 生物化学与分子生物学， 2012， 博士

【摘要】 杜鹃花属是杜鹃花科中最大的一个属，而我国又是杜鹃花属植物种类最丰富的国家，是世界杜鹃花属植物的主要分布中心。该属植物除了具有极高的欣赏价值外，还具有较高的药用价值，在医药、食品等具有广阔的开发应用前景。黄酮类化合物是植物中重要的次生代谢产物，而杜鹃属植物是一种优良的类黄酮潜在资源。但是目前对杜鹃各方面的研究和开发主要是以野生品种为主，而由于对野生品种的盲目采集，导致杜鹃自然资源严重匮乏，可供利用的野生杜鹃资源越来越少，且目前对杜鹃黄酮类化合物也没有一个系统的研究，限制了人们对杜鹃属黄酮类化合物潜在资源的进一步开发和利用。本论文以目前人工栽培非常成功的锦绣杜鹃、白杜鹃等为材料，对杜鹃中黄酮类化合物进行了系统研究，为进一步利用栽培杜鹃的黄酮类化合物提供了重要的理论依据和技术支撑，同时也为今后黄酮类化合物扩大生产提供了资源保障。本文主要研究内容有：（1）将BP人工神经网络技术与传统的正交试验方法相结合对杜鹃黄酮类物质提取工艺进行优化，并对优化后杜鹃总黄酮提取液进行抑菌实验；（2）采用颜色反应、高效液相-质谱联用技术等分析方法分析杜鹃叶和花中黄酮类化合物的主要成分；（3）应用HPLC法，建立了同时测定杜鹃提取液中4种主要黄酮类成分的测定方法，同时，用建立的HPLC方法对锦绣杜鹃和白花杜鹃叶、枝的黄酮类主要成分含量的年动态变化进行分析研究；（4）研究不同类型不同浓度的外源诱导子对杜鹃黄酮类化合物含量的累积及叶片相关酶活性的影响，选择对黄酮类化合物含量提高最适的诱导子类型和诱导子浓度。主要的研究结果如下：1、在正交试验的基础上，应用BP人工神经网络模型确定锦绣杜鹃叶黄酮提取的最优化工艺：料液比1：20，超声波提取时间为40min，提取温度为50℃，乙醇质量分数为40%。应用优化工艺提取的总黄酮含量高于正交试验各结果，且节约了成本，降低了能耗。表明将BP人工神经网络技术与传统的正交试验方法相结合提出的试验设计和数据处理的方法是可行的，该原理和方法也可用于其他植物黄酮类物质提取工艺的优化。2、锦绣杜鹃和白杜鹃叶总黄酮提取液对青霉菌、黑曲霉等霉菌几乎无抑菌作用，对细菌则有不同程度的抑制作用，原液抑菌率都达到100%。其中对金黄色葡萄球菌抑菌效果最好，大肠杆菌和枯草芽孢杆菌次之，并且都随总黄酮提取液浓度的提高，抑菌效果增强。而白杜鹃叶总黄酮提取液的抑菌效果要好于锦绣杜鹃叶的，这可能是与其提取的总黄酮含量的高低有关。锦绣杜鹃叶总黄酮提取液的最低抑菌浓度（MIC）：金黄色葡萄球菌抑菌和大肠杆菌为0.03125g/ml，枯草芽孢杆菌为0.0625g/ml。白杜鹃叶总黄酮提取液的最低抑菌浓度（MIC）：金黄色葡萄球菌抑菌、大肠杆菌和枯草芽孢杆菌均为0.03125g/ml。3、本文用Waters C18(4.6mm×250mm，5μm)反相色谱柱，在甲醇和水的流动相梯度洗脱，洗脱程序为：0~24min，31%~42%B；24~30min，42%~50%B；30~35min，50%~60%B；35~40min，60%B；检测波长356nm，流速为0.7mL/min，柱温30℃条件下可以使锦绣杜鹃叶中的黄酮类化合物HPLC得到很好的分离效果。在HPLC分离的基础上，通过液质联用技术初步鉴定了杜鹃叶中的5个黄酮类化合物，分别是金丝桃苷、异槲皮苷、广寄生苷、槲皮苷、槲皮素。杜鹃花中的6个杜鹃花黄酮类化合物，分别为锦葵花素戊糖苷、杨梅黄素3-鼠李糖苷、槲皮素-3-半乳糖苷、槲皮素-3-O–阿拉伯糖苷、槲皮素-3-鼠李糖苷槲皮素。4、锦绣杜鹃叶总黄酮及黄酮主要成分含量的季节变化变化规律呈现出一定的相似性，黄酮含量随着春季气温回升，芽的发育，新枝叶的生长，黄酮的含量大幅度增加，在3~6月份杜鹃叶总黄酮含量均表现出较高水平，进入9月，总黄酮含量又有所回升，11月总黄酮大幅度下降。锦绣杜鹃叶四种主要黄酮类成分中槲皮苷含量最大，其次为槲皮素，最少为异槲皮苷。锦绣杜鹃枝总黄酮含量的季节性变化规律与叶有所不同，枝在进入9月份时，并没有象叶一样进入第二个黄酮的增长期，这可能与枝的含量太低，变化不明显有关，且杜鹃枝含量明显低于叶。白杜鹃枝叶总黄酮与其主要成分含量的季节性变化规律同锦绣杜鹃枝叶呈现出一定的相似性，但白杜鹃枝叶黄酮类化合物含量较高于锦绣杜鹃。5、不同浓度的SA处理，杜鹃总黄酮及主要成分含量均在处理第8天时，达到最高值。其中低浓度的SA (50μg/mL)对黄酮含量的影响最明显，高浓度的SA起抑制作用。PAL活性随着诱导子处理时间的增加而增加，且在第4天和第8天PAL活性均达到较高值，说明SA处理后，PAL活性维持较高水平的时间较长。100μg/mL浓度的SA处理，PAL活性增幅最大。50μg/mL浓度与100μg/mL浓度的SA处理后，PPO活性都有较大幅度的提高。POD活性在第4天就达到较高值，这可能与其对外界刺激作出的应激反应有关.研究结果说明不同浓度的SA处理对POD活性都有显著提高，是提高POD活性较好的诱导子。6、一定浓度的外源MeJA诱导子处理，杜鹃总黄酮及主要成分含量的影响显著，其中高浓度的MeJA(150μg/mL)对总黄酮及主要成分含量的影响最明显，但低浓度(50μg/mL)的MeJA对杜鹃黄酮含量基本没有影响。100~200μg/mL浓度的MeJA处理后，PAL活性都显著提高，说明对杜鹃喷施100μg/mL-200μg/mL浓度范围的MeJA后可显著提高PAL酶活性。100μg/mL浓度的MeJA处理后，PPO活性提高最明显。POD活性在第4天就达到最高值，且各处理POD活性均有大幅度的提高，这说明不同浓度的MeJA处理对POD活性都有显著提高，也是提高POD活性较好的诱导子。7、不同浓度的GA3处理杜鹃植株，浓度为100μg/mL的GA3处理对杜鹃总黄酮含量的影响最明显。而高浓度的GA3（200μg/mL）对杜鹃黄酮含量基起到抑制作用。不同浓度的MeJA处理对黄酮类4种成分含量的影响不同，金丝桃苷、槲皮苷2种黄酮类成分含量提高幅度最明显的处理浓度均为100μg/mL。而异槲皮苷和槲皮素2种黄酮类成分含量提高幅度最明显的处理浓度却为150μg/mL。50μg/mL、100μg/mL浓度的GA3处理后，对PAL活性的影响较为显著，PAL活性维持较高水平的时间较长。100μg/mL浓度的GA3处理后，PPO活性提高的幅度最显著。低浓度的GA3（50μg/mL）处理后，POD活性在处理第4天后有较大幅度的提高，其余浓度的GA3处理，反而降低了叶片POD的活性。、8、不同浓度的ABA处理杜鹃植株，结果表明，浓度为150μg/mL的ABA处理对杜鹃总黄酮及主要成分含量的影响较为显著。低浓度（50μg/mL）的ABA处理，PAL活性增加幅度最大。100μg/mL浓度的ABA处理后，PPO活性提高的幅度最明显，且在处理后第12天PPO活性还保持一定的水平，对PPO活性影响最少的浓度是50μg/mL。而对POD活性影响较大的浓度则是200μg/mL和150μg/mL。而50μg/mL和100μg/mL浓度的ABA处理，POD活性反而下降。结果表明，高浓度的ABA对POD活性起到提高作用，但低浓度的ABA对POD活性反而起到一定的抑制作用。更多还原

【Abstract】 China is the country with richest species of Rhododendron Linn., it is the main distributioncenter of the Rhododendron Linn.in the world. Ii is the largest genus of the Ericaceae, it is notonly with extremely high appreciation value, but also has good medicinal value, so that it hasbroad prospects of its application in the fields such as foods, medicine. Flavonoids is the mostimportant secondary metabolites in the plants. The flavonoids compounds widely exist in theRhododendron Linn., which is a broad class of substances with bioactivity, so it is a kind of thepotential natural resources. Nowadays the main ways to research the Rhododendron Linn.arecentered on the feral breed, but the blind use of resources lead to a seriously storage of availablenatural resources of the Rhododendron Linn.. And at present there is not a systematical researchwith flavonoids so that it restricts peoples to develop and utilize them further. This paper usedthe Rhododendron pulchrum Sweet. and the Rhododendron mucronatum (BL.) which weresuccessfully grown by artificial cultivation as the materials, to research the flavonoidscompounds scientifically. Consequently, it can provide the important theoretical and techniquefoundation for further utilizing the flavonoids compounds in Cultivated Rhododendron.Simultaneously, it also can provides a guarantee for enlarging the flavonoids production.The major research contents of this paper were:(1) By combining BP artificial neuralnetwork with traditional orthogonal tests, optimiz the extraction processes for flavonoids inRhododendron and then conducted the antibacterial experiments.(2) Analysed flavonoids in theleaves and flower of Rhododendron with color reaction and HPLC-MS.(3) Established a HPLCmethod for determination the major compounds of flavonoids and study the dynamic variation ofthe contents of flavonoids in the leaf and stem of Rhododendron.(4) By studying on theinfluences of the different exogenous elicitor on the content of flavonoids and related enzymeactivities of Rhododendron, selected the exogenous elicitor with fit types and concentration toincrease the content of flavonoids. The main results were as follows:1. On the base of the orthogonal tests, apply BP artificial neural network to determine theoptimal conditions for extraction of total flavonoids in Rhododendron pulchrum Sweet. was thatthe solid-liquid ratio of1:20,40min of extraction time,50℃of extraction temperature,40%ofethanol concentration. Thwas optimal process was better than traditional orthogonal tests, it canreduce the cost and the energy consumption. The results show artificial neural network combinedwith traditional orthogonal design constitute the baswas on which a new method of test data analyzing and processing was put forward and we also can use it to optimize the extractionprocess of flavonoids in other plants.2. The flavonoids of Rhododendron pulchrum Sweet. and Rhododendron mucronatum(BL.) had almost no bacteriostaswas to molds such as penicillin sp. and aspergillus niger, buthad different inhibition effects to bacteria. The antibacterial rate of the extraction reach100%.The inhibition effects on staphylococcus aureus was the best, and next were escherichia coli andbacillus subtilwas. The higher concentration of the flavonoids extraction, the more inhibitioneffects increased. The inhibition effects of the extracts in Rhododendron mucronatum (BL.) wasbetter than Rhododendron pulchrum Sweet.. Thwas could be due to the content of theflavonoids. The MIC of the flavonoid extracts from Rhododendron pulchrum Sweet. were asfollows:0.03125g/ml for staphylococcus aureus and escherichia coli;0.0625g/ml for bacillussubtilwas. The MIC of the flavonoid extracts from Rhododendron mucronatum (BL.) was0.03125g/ml for staphylococcus aureus, escherichia coli and bacillus subtilwas.3. In thwas paper, the separation was performed on Waters C18column (4.6mm×250mm，5μm) by gradient elution(0~24min,31%~42%B;24~30min,42%~50%B;30~35min,50%~60%B;35~40min,60%B) using methanol(B) and water(B) as the mobile phase. Thedetection wavelength was356nm and the flow rate was0.7mL/min with column temperature at30℃. By the HPLC-MS/MS with electrospray ionization, five constituents were identified asquercetin-3-galactoside, quercetin-3-glucoside, quercetin-3-0-arabinoside,quercetin-3-rhamnoside, quercetin in the leaf and six constituents were identified asmalvidin-pentoside, myricetin-rhamnoside, quercetin-3-galactoside, quercetin-3-0-arabinoside,quercitrin, quercetin in the flower of Rhododendron pulchrum Sweet..4. The seasonal dynamic variation of the contents of the total flavonoids and the maincomponent in the leaf of Rhododendron pulchrum Sweet.had some similarities. With the springtemperatures rebound and the development of the bud, the content of flavonoids increasedsignificantly. In3~6months, the content of flavonoids was in a high standard, and then it fellrapidly. In September, the contents started to climb back up. When it turned cold in November, itfell rapidly again. In the four components of the flavonoids, the most abundant content wasquercetin-3-glucoside, and then was quercetin, wasoquercitrin was lease. The seasonal dynamicvariation of the contents of the total flavonoids in the stem was different from leaf. It didn’t enterthe second growth in September. Thwas may be related to the low contents in stem which had noobvious change. The content in stem was lower than leaf dwastinctly. The seasonal dynamicvariation of the contents of the total flavonoids in the leaf of Rhododendron mucronatum (BL.)and Rhododendron pulchrum Sweet. was similar, but the content of the former was higher than the latter.5. Spraying the Rhododendron pulchrum Sweet with SA of different concentration, thecontents of the total flavonoids and the main component all reached the maximum value on theeighth day. The low concentration of SA (50μg/mL) had the most dwastinct influence on thecontent of flavonoids. The high concentration of SA had an inhibitory effect. The PAL activityincreased with the induction processing time increased. On the eighth day, it reached the highestvalue. On the4th and8th day, PAL activity was in a higher standard. So using SA can make thePAL preserve high activity in a longer period. With100μg/mL concentration of SA, the PALactivity had the biggest increase. The activity of PPO could reach a high level on the8th day.When the concentration of SA was50μg/mL or100μg/mL, the activity had a higher increase.The was phenomenon explained that the concentration ranged50μg/mL to100μg/mL of SAcould increase the activity of PAL and PPO. The PPO activity could reach a higher value on the4th day. It may be correlated with the respond to external stimuli.Thwas results indicateddifferent concentration of SA was a better elicitor for improving POD activity.6. Spraying the Rhododendron pulchrum Sweet with MeJA of different concentration, thecontents of the total flavonoids and the main component were affected observably. The highconcentration of MeJA (150μg/mL) had the most dwastinct influence on them, while the lowconcentration (50μg/mL) of MeJA had almost no effect. The was phenomenon indicated that thehigher concentration of MeJA has an stronger effect on the contents of flavonoids. When theconcentration of MeJA ranged from100μg/mL to200μg/mL, the activity of PAL improvedmarkedly, so spraying rhododendron with the range of the concentration of MeJA could improvethe activity of PAL observably. Using100μg/mL concentration of MeJA, the activity of PPOimproved most obviously. The POD activity could reach maximum value on the4th day, thenthey came down rapidly. The activity of POD increased substantially with differentconcentration of MeJA, so it was also a better elicitor for improving POD activity.7. Using GA3of different concentration dwasposed Rhododendron pulchrum Sweet.,when the concentration was100μg/mL, the influence on the contents of the total flavonoids wasmost obvious. The high concentration of GA3（200μg/mL）had an inhibitory effect. Theinfluence on the contents of the4main components was different with different concentration ofMeJA. The best concentration for quercetin3-galactoside and quercetin-3-rhamnoside was100μg/mL. The best concentration for isoquercitrin and quercetin was150μg/mL. Using MeJA of50μg/mL or100μg/mL concentration, the activity of PAL changed much obviously The activityof PAL sustained at a high level for a long time. The was phenomenon indicated that theconcentration ranged from50μg/mL to100μg/mL of GA3had a greater impact on the activity of PAL. with the100μg/mL concentration of GA3, the activity of PPO improved most obviously.With the lower concentration of GA3(50μg/mL）, the activity of POD increased by a widermargin after the4th day, and other concentrations actually reduced the activity.8、Using different concentration of ABA dwasposed Rhododendron pulchrum Sweet.,when the concentration was150μg/mL, the influence on the contents of the total flavonoids andthe main components was obvious. When the concentration was low (50μg/mL), the activity ofthe PAL had a biggest augment. With the100μg/mL concentration of ABA, the activity of PPOimproved most obviously. When the concentration of ABA was200μg/mL and150μg/mL, theinfluence on the activity of POD was bigger. With50μg/mL and100μg/mL, the activity ofPOD was reduced. So the high concentration of ABA could increase the activity of POD, and thelower had an inhibitory action.更多还原