【作者】 姚曦；

【导师】 岳永德；

【作者基本信息】 中国林业科学研究院 ， 生态学， 2014， 博士

【摘要】 中国竹类资源丰富，栽培利用历史悠久。本研究以牡竹属（Dendrocalamus）的梁山慈竹（Dendrocalamus farinosus）竹秆为研究对象，对竹秆中黄酮、酚酸等次生代谢组分进行了分离鉴定；分析了竹秆多糖的组成和分子量分布，利用红外、核磁和热重等手段对竹多糖结构进行表征；对比分析了经水蒸气蒸馏法（HD）提取得到的梁山慈竹竹秆和竹中各挥发性成分组成，并比较二者抑菌活性；分别研究了包括木质素、综纤维素、多戊糖、各种抽提物、灰分、无机元素等一般性化学组成；建立了竹秆中以异荭草苷为代表的黄酮类化合物离子液体-微波辅助萃取工艺；评价了包括梁山慈竹和其他3属共16种竹秆提取物的抑菌和抗氧化活性。旨在为梁山慈竹资源的高附加值利用提供理论基础。主要研究结果如下：1.采集梁山慈竹竹秆，用95%乙醇浸提，大孔树脂柱层析。分别用水、30%、60%和80%乙醇依次洗脱。经硅胶柱色谱、凝胶柱色谱、高压制备色谱及重结晶等步骤，从该植物中得到10种单体化合物。分别鉴定为：（1）异荭草苷、（2）对羟基苯甲醛、（3）苜蓿素、（4）苜蓿素-7-O-葡萄糖苷）、（5）阿魏酸、（6）对香豆酸、（7）对羟基苯甲酸、（8）异牡荆苷、（9）2,6-二甲氧基-1,4-对苯醌、（10）β-谷甾醇。2.对梁山慈竹竹秆水溶性多糖（BCP）进行了研究。利用高效离子色谱法（HPIC）分析了水解后的BCP的单糖组成，分别是葡萄糖、阿拉伯糖、木糖、半乳糖和甘露糖等五种，相对含量分别为40.57%、21.88%、19.89%、17.5%和0.15%。高效凝胶色谱法（HPGPC）测定出纯化后的BCP的重均相对分子量Mw为16900Da，多分散性系数D（Mw/Mn）为1.33。傅立叶红外光谱分析表明BCP具有多糖特有的吸收峰。核磁波谱分析结果也同样表明BCP符合吡喃型酸性多糖的特征。热稳定性（TGA）分析结果显示，梁山慈竹BCP在240℃时开始分解，到340℃时其质量损失大约为65%；当温度达到500℃时，BCP的质量缓慢地损失达73%；此后随着温度的升高，多糖样品最终成了碳化结构。3.经水蒸气蒸馏法（HD）提取，气相色谱-质谱联用法（GC/MS）分析了梁山慈竹竹秆及其竹叶中挥发性成分，检测出梁山慈竹竹秆挥发发性组分40种，占色谱总流出物相对含量的92.77%，主要为脂、酸、醇、醛、酮、酚、呋喃以及烯烃等类物质；从竹叶挥发性成分中初步鉴定出76个化合物，占色谱总流出物相对含量的70.91%，主要为脂、酮、醛、醇、烷烃、呋喃以及萘类等。竹秆和竹叶中共同检出的化合物有10种，分别为：正己醛，（E）-2-己烯醛、苯甲醛、苯乙醛、大马士酮、β-紫罗兰酮、月桂酸乙酯、法尼基丙酮、棕榈酸乙酯、叶绿醇。这些共有成分在日化、食品香精领域有着广泛的用途，为梁山慈竹挥发性成分的开发利用提供研究基础。将提取的梁山慈竹秆和叶的挥发油稀释成5个浓度梯度对4种测试菌种进行抗菌效果测定，处理24h后，高浓度（25mg mL-1）竹秆挥发油对枯草芽孢杆菌有较强抑制作用，对金黄色葡萄球菌和大肠杆菌有中度抑制作用，对白色假丝酵母菌抑制效果一般；高浓度（25mg mL-1）竹叶挥发油对除白色假丝酵母菌外的其余3种细菌均有良好的抑制作用。竹叶挥发性成分的抑菌活性优于竹秆挥发油成分，这和其中所含醇、醛、酚、酸类等物质含量有关。4.梁山慈竹竹秆基本化学组成的结果如下：灰分含量为1.24%，苯醇抽出物含量为3.27%，冷、热水抽提物分别为9.16%和11.58%；1%NaOH抽出物为30.71%，多戊糖含量为19.44%，木质素含量为22.18%，综纤维素含量为72.70%。采用微波消解-电感耦合等离子体质谱法（ICP/MS）同时测定了梁山慈竹竹秆中18种无机元素含量，其中较高元素含量的趋势为：Ca（587.13mg·kg-1）>Mg（448.75mg·kg-1）>Al（76.51mg·kg-1）>Fe（66.38mg·kg-1）>Na（51.4mg·kg-1）>Zn（16.47mg·kg-1）> Mn（15.52mg·kg-1）> Cr（5.26mg·kg-1），其中Ca和Mg的含量非常丰富， Ca元素的含量达到587.13mg·kg-1，Mg含量也在448mg·kg-1；含量最低的为Hg元素，仅0.07mg·kg-1。其余元素含量在0.09mg·kg-1~3.54mg·kg-1之间。5.以黄酮类物质为目标化合物，结合微波辅助萃取技术，建立了以[bmim][BF4]离子液体为提取溶剂的，梁山慈竹竹秆中异荭草苷的提取方法。考察了提取时间、提取温度、液固比等对提取率的影响。结合响应面优化设计实验，探索适合提取竹秆中异荭草苷的最佳提取工艺为：以1.5mol/L [bmim][BF4]的为溶剂，0.5g竹秆样品，提取温度60.2℃，提取时间12.36min，液固比16.74:1（mL/g），此参数条件下异荭草苷提取得率为1.693mg/g。经与热回流、超声波提取，以及传统溶剂微波萃取等常规方法比较显示出优越性，其提取率高出其他3种方法12.46~22.7%，提取时间上也由2h大大缩短至12min。6.评价了梁山慈竹、硬头黄竹和9种刚竹属（Phyllostachys）、5种大明竹属（Pleioblastus）共16种竹秆提取物的生物活性。包括以稻白叶枯病菌（Xanthomonas oryzaepv. Oryzae）、番茄青枯菌（Ralstonia solanacearum）、金黄色葡萄球菌（Staphylococcusarueu）和大肠杆菌（Escherichia coli）为靶标的抗细菌活性，以及稻瘟菌（Magnaporthegrisea）、苹果炭疽菌（Colletotrichum gloeosporioides）、苹果腐烂菌（Valsa mali Miyabe etYamada）、番茄灰霉菌（Botrytis cinerea）等为靶标的抗真菌活性，以及抗氧化活性。抑菌圈的结果表明，斑苦竹提取物对水稻白叶枯菌抑制效果最明显，24小时抑菌圈直径达18.33mm；长叶苦竹提取物对番茄青枯菌抑制效果最强，抑菌圈直径为19.33mm；硬头黄竹和红壳竹的竹秆提取物对金黄色葡萄球菌和大肠杆菌均表现出突出的作用，抑菌圈直径均超过18mm。生长速率法的结果显示，在0.04g（竹粉）L的浓度下，水竹提取物对番茄灰霉菌的抑制作用最强，48小时的抑制率为56.84%；毛金竹对苹果腐烂菌有很强的抑制作用，抑制率高达80.94%；水竹提取物对稻瘟菌以及毛金竹对苹果炭疽菌的各自抑制率均超过了40%。用DPPH法评价了竹秆提取物的抗氧化活性。供试竹秆提取物抗氧化活性强度排序为：TBHQ＞毛竹＞梁山慈竹＞毛金竹＞硬头黄竹＞BHT＞实心苦竹＞杭州苦竹＞雷竹＞早竹＞刚竹＞斑苦竹＞红壳竹＞水竹＞紫竹＞长叶苦竹＞丽水苦竹＞白哺鸡竹。16种竹秆提取物抗氧化活性差异较大，其中毛竹提取物清除DPPH自由基的能力最强，IC50值为136.5mg/L。梁山慈竹提取物次之，也达到了141.5mg/L，二者效果都优于人工合成抗氧化剂二丁基羟基甲苯（BHT）的抗氧化效果。活性最差的为白哺鸡竹提取物，1g TBHQ对DPPH自由基的清除效果相当于11.76g白哺鸡竹提取物。更多还原

【Abstract】 China is rich in bamboo resources and has a long history both in cultivation and utilization.To promote the basic research and comprehensive utilization of bamboo resources, chemicalcomponents of bamboo culms were studied. The compounds from Dendrocalamus farinosusculms were isolated and identified, and the composition and relative molecular mass（Mw） ofbamboo culm polysaccharides was analyzed by high performance ion chromatography（HPIC） and high performance gel penetration chromatography （HPGPC）, alone with thestructure characterized by IR and1HNMR&1CNMR. Volatile compounds both from D.farinosus culm and leaves were collected through hydrodistillation （HD） technologies andanalyzed qualitatively with chromatography-mass spectrometry （GC/MS）, and thenantibacterial effects of two samples were compared. The chemical compositions ofDendrocalamus farinosus culm were tested, including ash content,1%NaOH extractive, hotwater extractive, benzene-alcohol extractive, pentosan, klason lignin, holocellulose andelements. The Ionic liquid-based microwave-assisted was applied to the extraction of isorientinin Dendrocalamus farinosus culm.The antibacterial activity and antioxidant activity of16bamboo culms extract were evaluated. The main results were as following:1. Extracting compounds from Dendrocalamus farinosus culm with95%ethanol coldsoak extract method. Extraction was purified by AB-8macroporous absorption resin. Then30%ethanol,60%ethanol,80%ethanol and acetine eluted respectively. The elution wereisolated by Sephadex LH-20, GX-281High Voltage HPLC Separate system and Sepacorelow-pressure HPLC Separate system.9compounds were isolated from Dendrocalamusfarinosus. On the basis of physic-chemical characters and spectroscopic analysis such as UVIR, NMR and MS,10compounds were identified as:4-hydroxybenzaldehyde（1）, tricin（2）,p-hydroxybenzoic acid （3）, β-sitosterol （4）, isoorientin （5）, isovitexin （6）, tricin-7-O-glucopyranoside （7）,2,6-Dimethoxy-1,4-benzoqunone （8）, ferulic acid（9）,p-coumaric acid（10）.2. The results showed that the water-soluble polysaccharide of Dendrocalamus farinosusculm were arabinose、galactose、glucose、xylose and mannose with the contents of21.88%,7.5%,40.57%,19.89%and0.15%respectively by HPIC. The average molecular weights（Mw） of BCP was16900Da and the polydispersity was1.33. Infrared spectrogram showedthat BCP had the characteristic absorption peak of polysaccharides.1HNMR and13CNMRindicated that its main chains were composed of β-pyranglycoside linkage. Thermal analysisshowed the degradation of BCP occurred in the second stage rapidly. The mass loss was about65%during the temperature240~340℃.3A total of40volatile compounds were extracted and separated in bamboo culm,including ester, acid, wine, aldehydes, ketone, phenol, etc., while76compounds wereseparated in bamboo leaves. Comparison analysis showed that there were10volatilecompounds both in Dendrocalamus farinosus culm&leaves, included caproaldehyde,（E）-2-Hexenal, benzaldehyde, phenylacetaldehyde, beta-Damascenone, beta-Ionol, ethyl laurate,Farnesyl acetone, ethyl palmitate, Phytol. These compounds have been applied in the field offragrance and flavor, daily-use chemical, and food preservative.The volatile compounds from Dendrocalamus farinosus culm&leaves were diluted into aseries of concentration of sample solutions:5.0mg mL-1,10.0mg mL-1,15.0mg mL-1,20.0mg mL-1,25.0mg mL-1, and the antibacterial effects of sample solutions on Bacillus subtili,Staphyloccocus aureus, Candida albicans and Escherichia coli were tested by using filterpaper method. After24hours’ culture, the volatile compounds from culm had a stronginhibition effect to B. subtili at high concentration （25.0mg mL-1）. The volatile compoundsfrom leaves had strong inhibition effect to B. subtili, S. aureus and E. coli. The volatilecompounds from leaves had better inhibition effect than that from culms, due to the content ofwine, aldehydes, phenol, acid. 4. The chemical compositions of Dendrocalamus farinosus culm were tested. Resultsshow that the chemical components of Dendrocalamus farinosus are determined as ash content1.24%,1%NaOH extractive30.71%, hot water extractive1.58%, cold water extractive9.16%,benzene-alcohol extractive3.27%, pentosan19.44%, klason lignin22.18%, holocellulose72.7%. The contents of18kinds of inorganic elements was investigated by inductively coupledplasma-mass spectrometry （ICP/MS）, combinning with mircowave sample digestion, whichwere Ca（587.13mg·kg-1）, Mg（448.75mg·kg-1）,Al（76.51mg·kg-1）, Fe（66.38mg·kg-1）,N（a51.4mg·kg-1）, Zn（16.47mg·kg-1）, Mn（15.52mg·kg-1）, C（r5.26mg·kg-1）, N（i3.54mg·kg-1）,M（o3.03mg·kg-1）, C（u2.48mg·kg-1）, P（b2.46mg·kg-1）, C（d30.39mg·kg-1）, S（e0.35mg·kg-1）,Ag（0.26mg·kg-1）,As（0.23mg·kg-1）, Co（0.09mg·kg-1）, Hg（0.07mg·kg-1） respectively.Ca, Mg, Al, Fe, Na are relative abundant in the bamboo culm among the elements above.5. Ionic liquid-based microwave-assisted extraction coupled with high performance liquidchromatography was first applied to isolate and determinate isorientin in Dendrocalamusfarinosus culm. Response surface experimental design was used to explore for the bestextraction conditions of isorientin: The amount of culm0.5g, in the1.5mol/L1-Butyl-3-methylimidazolium tetrafluoroborate （[bmim][BF4]） system, the extractiontemperature60.2℃, extraction time12.36min, and liquid-to-solid ratio of16.74:1（mL/g）.Under the condition, the extraction yield was1.693mg/g. Furthermore, the comparison of theproposed ILMAE approach with the conventional method was also studied to demostrate themethod feasibility. It is worth mentioning that the proposed approach could dramaticallyimprove both the extraction efficiency of the menthol and the total extraction time.6. The antimicrobial activity of the extracts from4genuses,16species bamboo culmsagainst X.oryzae pv.oryzae, S.arueus, R.solanacearum, E.coli, Botrytis cinerea, Valsa maliMiyabe et Yamada, M.grisea and C.gloeosporioides were evaluated.Filter paper in each extract to add a dose of0.02g. The results indicated that P.maculatusmaculatus showed the most obvious antibacterial activeites to X.oryzae pv.oryzae. Thediameter of antibacterial ring after24h was18.33mm. P. china f.hisauchii showed the most obvious antibacterial activeites to S.arueus, with the diameter of antibacterial ringwas19.33mm. Bambusa rigida Keng and P. iridescens extract showed most obviousantibacterial activeites to R.solanacearum and E.coli. The diameter of antibacterial ring wereboth exceed18mm. At the concentration of0.04g/mL, P.heteroclada showed most obviousantifungal activities to Valsa mali Miyabe et Yamada, the inhibition rates after48h was above56.84%. P.nigra var.henonis showed most obvious antifungal activities to Valsa mali Miyabeet Yamada, with the inhibition rates was80.94%. P.heteroclada and P.nigra var.henonisshowed most obvious antifungal activities to M.grisea and C.gloeosporioides, the inhibitionrates were both above40%.The antioxidant capacities of bamboo culms extracts were investigated using DPPH test.In the DPPH assays, the antioxidant activities were evident in all the extracts, and theP.heterocycla cv.Pubesceus extract indicated the most effective scavenging effect on theDPPH·. Its scavenging effect of DPPH·（IC50=136.5mg/L）.was even better than that of BHT.更多还原