【作者】 吕兆林；

【导师】 张柏林； 金幼菊；

【作者基本信息】 北京林业大学 ， 植物学， 2009， 博士

【摘要】 黄酮类化合物和挥发油是竹叶中的主要功能性成分,具有显著的生物学功效,应用前景广阔。本文以来自福建省南屏市的毛竹（Phyllostachys heterocycla（Carr.）Mitford Cv.Pubescens Mazel ex H.de leh.）、苦竹（Pleioblastus amarus（Keng）Keng f.）、绿竹（Dendrocalamopsis oldhami（Munro）Keng f.）、黄甜竹（Acidosasa edulis Wen）为实验材料,通过高效液相色谱仪/四级杆-飞行时间/串联质谱联用仪（HPLC-ESI-Q-TOF-MS/MS）手段鉴定了四种竹叶提取液中常量、微量及痕量的黄酮类化合物结构;建立了简便快捷的竹叶中黄酮类化合物质量监控的二极管阵列高效液相色谱（HPLC-DAD）方法,对四种竹叶中总黄酮化合物及主要黄酮化合物进行了定量测定,获得了竹叶黄酮类化合物随种类、季节变化的数据资料;优化了无污染溶剂提取竹叶黄酮的工艺,以大孔树脂吸附法对竹叶黄酮粗提物进行了纯化;采用体外化学方法及大鼠体内模型评价了不同竹叶提取物的抗氧化能力,并结合竹叶黄酮化合物指纹图谱,探讨了其抗氧化机理;用不同提取方法研究竹叶挥发油,以气质联用仪（GC/MS）为分析手段,鉴定了毛竹、苦竹、绿竹和黄甜竹叶挥发油组分结构;建立了竹叶挥发油组分快速、简便的气相色谱（GC）内标定量法,获得竹叶挥发油组分随种类变化的数据资料;研究了毛竹、苦竹、绿竹和黄甜竹叶的挥发油的抑菌效果,并结合竹叶挥发油指纹图谱,探讨了其挥发油的抑菌机理。1.竹叶黄酮化合物的结构鉴定首次使用HPLC-ESI-Q-TOF/MS/MS技术对毛竹、苦竹、绿竹及黄甜竹四种竹种竹叶黄酮化合物结构进行了鉴定,已鉴定16种毛竹叶黄酮化合物（其中Mono-C-glyeosylflavones 5种,O-glyeosylflavones 3种,Flavonoid aglyeones 1种,其他类黄酮化合物7种）、24种苦竹叶黄酮化合物（其中Di-C-glyeosylflavones 4种,Mono-C-glyeosylflavones 1种,O,C-Diglyeosylflavones 11种,O-glyeosylflavones 2种,Flavonoid aglycones 1种,其他类黄酮化合物5种）、绿竹叶黄酮化合物17种（其中Mono-C-glycosylflavones 5种,O,C-Diglycosylflavones 2种,O-glycosylflavones 3种,Flavonoid aglycones 2种,其他类黄酮化合物5种）、黄甜竹叶黄酮化合物25种（其中Di-C-glycosylflavones 2种,Mono-C-glyeosylflavones 1种,O,C-Diglycosylflavones 7种,O-glycosylflavones 1种,Flavonoid aglycones 1种,其他类黄酮化合物13种）。2.竹叶黄酮化合物的季节变化（1）以月为周期,收集2007年09月至2008年06月期间福建省南屏市毛竹、苦竹、绿竹及黄甜竹竹叶,对四种竹叶黄酮化合物的研究发现,从秋季、冬季、春季至夏季,四种竹叶中黄酮含量呈上升的趋势,即随着叶片的发育和成熟,总黄酮（TF）的含量也在增加。（2）从秋初到夏初,四种竹叶中黄酮化合物的变化趋势与其总黄酮的变化趋势相同,呈现上升的趋势,不同的是来自毛竹叶6种黄酮化合物、苦竹叶8种黄酮化合物、绿竹叶6种黄酮化合物以及黄甜竹叶8种黄酮化合物的变化幅度大小不一。3.竹叶黄酮提取工艺研究（1）以热回流提取为竹叶黄酮的提取手段,利用二次回归正交旋转组合设计试验,得到竹叶黄酮提取得率y与各影响因素（提取剂乙醇浓度X₁、提取时间X₂、液固比X₃）间的回归数学模型:y=-12.6432+0.3114X₊1.7292X₂+0.5018X₃-0.0106X₁X₂+0.1115X₂X₃-0.0023X₁²-0.2808X₂²-0.0459X₃²,对模型进行主效应、单因素和交互效应分析,竹叶黄酮提取得率因素的主次顺序为提取时间、液固比、提取剂乙醇浓度,提取剂乙醇浓度与提取时间之间、提取时间与液固比之间的交互作用显著,而提取剂乙醇浓度与液固比之间的交互作用不显著;利用理想点法,对数学模型求解,获得最优的竹叶黄酮提取参数（即乙醇浓度为59.86%,提取时间3.94小时,液固比为10:1）;试验验证,预测值和试验值的误差不超过3%。（2）采用最优化提取条件,提取毛竹、苦竹、绿竹及黄甜竹竹叶黄酮化合物,得到黄酮化合物粗提物,其中总黄酮含量3.74%-5.88%,总黄酮回收率58.6%-84.9%,粗提物实物得率6.5%-15.03%。利用选择性好、性价比高的HP-20树脂,对竹叶黄酮粗提物进行柱纯化,黄酮粗提取含量可由4.76%增至20.78%（HPLC测定）。4.竹叶黄酮抗氧化研究（1）以每克提取物相当的VC质量表示其总抗氧化能力测试表明,毛竹的总抗氧化能力是0.102 gVC/g粗提物,苦竹是0.088 gVC/g粗提物,绿竹是0.084 gVC/g粗提物,黄甜竹是0.068 gVC/g粗提物:毛竹柱纯化物的总抗氧化能力是0.101gVC/g毛竹柱纯化物。以每克提取物相当的槲皮素的质量表示其还原能力测试表明,毛竹的还原能力是0.069g槲皮素/g粗提物,苦竹是0.068g槲皮素/g粗提物,绿竹是0.074g槲皮素/g粗提物,黄甜竹是0.062g槲皮素/g粗提物;毛竹柱纯化物的还原能力是0.131g槲皮素/g毛竹柱纯化物。以IC50表示其清除DPPH自由基能力测试表明,毛竹清除DPPH自由基能力是2.26 mg/g,苦竹是2.46 mg/g,绿竹是2.22 mg/g,黄甜竹是2.67 mg/g;毛竹柱纯化物清除DPPH自由基能力是0.96 mg/g。以8mg/g含量的四种竹叶粗提物计算,黄甜竹的清除羟基自由基的能力最强,其次是绿竹、苦竹和毛竹。综合上述结果,可以看出四种竹叶提取物均具有良好的体外抗氧化作用,以绿竹的抗氧化能力尤为突出。（2）基于体外抗氧化试验结果,选取绿竹粗提物作为大鼠体内抗氧化测试的原料,将大鼠划分为:①空白组（喂养未加胆固醇饲料和每天灌喂生理盐水）;②负对照组（喂养加胆固醇饲料和每天灌生理盐水）;③正对照组（喂养加胆固醇饲料和每天灌VE,VE用量为0.0025g/kg体重）,④低剂量组（喂养加胆固醇饲料和每天灌100mg/kg体重竹叶提取物）,⑤高剂量组（喂养加胆固醇饲料和每天灌400mg/kg体重竹叶提取物）,饲养6周后,对肝脏组织检测表明,喂胆固醇饲料及大浓度的竹叶提取物高剂量组大鼠的超氧化物歧化酶（SOD）活力最小,与其他组的差异显著;低剂量组的过氧化氢酶（CAT）活力最大,与负对照组和高剂量组差异不显著,与空白组及正对照组差异显著;谷胱甘肽过氧化物酶（GSH-Px）活力高剂量组的值最低,与其它四组差异显著;对丙二醛（MDA）而言,高剂量组值最低,与正负对照组差异显著,与空白和低剂量组差异不显著。对肾脏组织检测而言,SOD活力同样是高剂量组最小,与其他组的差异显著;CAT的活力空白组最大,与其他组差异显著;GSH-Px在高剂量组偏低,但差异不显著;MDA在高、低剂量组的偏低,两组间差异不显著,与其它三组差异显著,负对照组的MDA值最大,其次是空白组,这两组间差异不显著,正对照组的MDA值居于中间,与其它四组差异显著。竹叶提取物显著影响大鼠的肝肾抗氧化酶活力及MDA水平,且提高竹叶提取物含量能明显提升大鼠的抗氧化能力。5.竹叶挥发油研究（1）采用同时水蒸气蒸馏萃取（SDE）、挥发油提取器（VOD）、超临界萃取（SFE）和索氏提取（SEM）等四种提取方法研究竹叶挥发油,采用SDE和VOD提取的化合物以醇、羧酸、烷烃类为主,SFE获得的化合物以烷烃、羧酸为主,SEM获得的化合物以烷烃类为主,SDE和VOD提取到的挥发油信息较多。（2）用SDE收集毛竹、苦竹、绿竹及黄甜竹竹叶挥发油,四种竹叶的挥发油实物得率在0.1%-0.3%之间,以苦竹为最大,其次为黄甜竹、绿竹和毛竹。毛竹叶挥发油以羧酸、烷烃和醇类化合物为主;苦竹叶挥发油成分与毛竹叶很相似;绿竹、黄甜竹叶挥发油成分以羧酸、醛和烷烃类化合物为主。（3）用内标法定量研究了竹叶挥发油中7种主要化合物（十二烷酸,十四酸,6,10,14-三甲基-2-十五酮,十六烷酸,二十三烷,二十七烷,9-十八烯醛）,十六烷酸在四种竹叶挥发油中的含量最高;毛竹和苦竹中居于第二位的是十二烷酸,而绿竹和黄甜竹居于第二位的是9-十八烯醛;毛竹、苦竹中居于第三位的是6,1014-三甲基-2-十五酮;绿竹和黄甜竹居于第三位的是十二烷酸。（4）研究了毛竹叶挥发油对大肠杆菌（Escherichia coil）、枯草杆菌（Escherichia coli）、荧光假单胞杆菌（Pseudomonas fluorescens）、黄杆菌（Flavobacterium）和酿酒酵母（Saccharomyces cerevisiae）等8种测试菌株的抑菌作用,结果显示毛竹叶挥发油均表现出较强的抑菌活性,且对大肠杆菌（革兰氏阳性菌）和枯草杆菌（革兰氏阴性菌）的最低抑菌浓度为2.25mg/mL。综上所述,竹叶中含有丰富的黄酮类及挥发油等活性物质,这两类化合物随竹种、季节的变化而变化,具有显著的抗氧化和抑菌活性,这些资料有利于竹类资源品质甄别及其在食品领域的深加工利用。更多还原

【Abstract】 Bamboo-leaf-flavonoids and bamboo-leaf-volatile oils are claimed to have bioactivities and be potential for use as functional components.The leaves of Phyllostachys heterocycla（Carr.） Mitford cv.Pubescens Mazel ex H.de leh., Pleioblastus amarus（Keng） Keng f.,Dendrocalamopsis oldhami（Munro） Keng f.and Acidosasa edulis Wen were collected monthly from the Nanping City of Fujian Province from Sep.2007 to Jun.2008.Electrospray ionization（ESI）-tandem quadrupole/orthogonal-acceleration time-of-flight（Q-TOF） mass spectrometer,in combination with high performance liquid chromatograph（HPLC） was used to identify the structures of trace,tiny or ordinary flavonoid compounds extracted from the four kinds of bamboo leaves.Total flavonoids’ level and the main flavonoid compounds from the four kinds of bamboo leaves were quantified with the help of diode array high performance liquid chromatography（HPLC-DAD）.GC/MS was used to confirm the volatile oil components of bamboo leaves collected from Phyllostachys heterocycla（Carr.） Mitford cv. Pubescens Mazel ex H.de leh.,Pleioblastus amarus（Keng） Keng f., Dendrocalamopsis oldhami（Munro） Keng f.and Acidosasa edulis Wen.The objectives of this study were to:（1） obtain the data of bamboo leaves flavonoids according to differences in growing seasons and bamboo’s species;（2） establish to an optimized and unpolluted process to extract and purify bamboo’s flavonoids;（3） evaluate the antioxidant activities of extracted bamboo-leaf flavonoids in vitro and in vivo according to obtained flavonoid’s fingerprint data;（4） set up a quick GC internal standard quantitative method to accumulate the data of bamboo-leaf volatile oil according to the varying in growing seasons and species;and（5） deal with the antimicrobial activities of volatile oil from Phyllostachys heterocycla（Can’.） Mitford cv.Pubescens Mazel ex H.de leh., Pleioblastus amarus（Keng） Keng f.,Dendrocalamopsis oldhami（Munro） Keng f.and Acidosasa edulis Wen bamboo leaves in combination with volatile oils’ fingerprint data.1.The structures identification of bamboo-leaf-fiavonoids 16 flavonoid’s compounds were identified from the bamboo-leaves of Phyllostachys heterocycla（Carr.） Mitford cv.Pubescens Mazel ex H.de leh.,including 5 mono-C-glycosylflavones,3 O-glycosylflavones,1 flavonoid aglycones and 7 other flavonoids;24 from Pleioblastus amarus（Keng） Keng f.leaves,including 4 Di-C-glycosylflavones,1 mono-C-glycosylflavones,11 O,C-Diglycosylflavones,2 O-glycosylflavones,1 flavonoid aglycones and 5 other flavonoids;17 from Dendrocalamopsis oldhami（Munro） Keng f.leaves,including mono-C-glycosylflavones,2 O,C-Diglycosylflavones,3 O-glycosylflavones,2 flavonoid aglycones and 5 other flavonoids;and 25 from Acidosasa edulis Wen leaves,including 2 Di-C-glycosylflavones, 1 mono-C-glycosylflavones,7 O,C-Diglycosylflavones,1 O-glycosylflavones,1 flavonoid aglycones and 13 other flavonoids,respectively.2.Seasonal diversification of bamboo-leaf flavonoids（1） The contents of total flavonoids（TF）,independent of bamboo’s species,increased with the bamboo-leaf development and maturation throughout this study period,and showed an upward trending from the fall,winter,and spring to summer.（2） Like TF from autumn to summer,the levels of individual flavonoid compounds in leaves of the four bamboo species showed an increasement during the whole growing seasons,but variation in the contents of individual flavonoid compounds depended on the bomboo’s species.3.Estiblishment of Bamboo-leaf flavonoids extraction technology（1） The yields of bamboo-leaf flavonoids（y） were greatly affected by ethanol concentration（X₁）,extraction time（X₂）,and liquid-solid ratio（X₃）.A return mathematical model,i.e.,y=-12.6432 + 0.3114X₁+ 1.7292X₂+ 0.5018X₃- 0.0106X₁X₂+ 0.1115X₂X₃-0.0023X₁² -0.2808X₂²-0.0459X₃²,was very good in describing the relationships between the extracted yields of bamboo-leaf flavonoids and the 3 parameters.Extraction time, liquid-solid ratio,and ethanol concentration in important order were interactive in influencing the yields of flavonoids extracted from bamboo leaves.The concentration of extracting agent and the extraction time,like the extraction time and liquid-solid ratio, showed an significant interaction to the yields of extracted flavonoids,whereas no significant interaction between the concentration of extracting agent and liquid-solid ratio was found in extracting bamboo-leaf flavonoids.The optimized procedure to extract bamboo-leaf flavonoids was 59.86%of ethanol concentration,3.94 hrs of extraction time, and the liquid-solid ratio of 10:1.The range in error differences between the predicted yields of flavonoids in mathematical model and the real yields of flavonids obtained from experiment did not exceed 3%.（2） The crude extractions of flavonoids from the leaves of four bamboo species were collected using above-given optimized extraction parameters.TF contents ranged from 3.74%to 5.88%in crude extractions.Recoveries in TF were from 58.6%to 84.9%,and yields of the crude extractions in four bamboo’s species were from 6.5%to 15.03%.The crude extractions of bamboo-leaf flavonoids were purified by column chromatography filled with HP-20 macroporons resin.HPLC determination showed that the contents of TF were increased from 4.76%to 20.78%after purfication.4.The antioxidant activities of bamboo-leaf flavonoids confirmed in vtiro and in vivo（1） Total antioxidant capacity in vitro,expressed as the equivalent quality of VC（g） per bamboo-leaf flavonoids extractions（g） was 0.102gVC/g for Phyllostaehys heterocycla（Corr.） Mitford cv.Pubescens Mazel ex H.de leh.,0.088gVC/g for Pleioblastus amarus（Keng） Keng f.,0.084gVC/g for Dendrocalamopsis oldhami（Munro） Keng f.and 0.068gVC/g for Acidosasa edulis Wen. Antioxidant capacity of flavonoid crude extracts obtained by purified-column in Phyllostachysheterocycla（Carr.） Mifford cv.PubescensMazel ex H.de leh.was 0.101gVC/g.Reduction ability,defined as the equivalent quality of quercetin（g） per bamboo-leaf flavonoids extractions（g）,was 0.069g quercetin/g for Phyllostachys heterocycla（Carr.） Mitfordcv.PubescensMazel ex H.de leh.,0.068g quercetin/g for Pleioblastus amarus（Keng） Keng f.,0.074g quercetin/g for Dendrocalamopsis oldhami（Munro） Keng f.,and 0.062g quercetin/g for Acidosasa edulis Wen.Reduction ability of flavonoid crude extract by purified-column in Phyllostachys heterocycla（Carr.） Mifford cv.Pubescens Mazel ex H.de leh.was 0.131g quercetin/g.The ability of bamboo-leaf flavonoids to scavenge DPPH,defined by their IC50,was 2.26 mg/g for Phyllostachys heterocycla（Carr.） Mifford cv.Pubescens Mazel ex H.de leh.,2.46 mg/g for Pleioblastus amarus（Keng） Keng f.,2.22 mg/g for Dendrocalamopsis oldhami（Munro） Keng f.,and 2.67 mg/g for Acidosasa edulis Wen. DPPH scavenged by column purified flavonoids crude extracts from Phyllostachys heterocycla（Carr.） Mifford cv.Pubescens Mazel ex H.de leh.was 0.96 mg/g. Among four bamboo species,the flavonoids from Acidosasa edulis Wen leaves（calculated at 8mg/g） had the best performance in hydroxyl radical scavenging ability in vitro, followed by Dendrocalamopsis oldhami（Munro） Keng f.,Pleioblastus amarus（Keng） Keng f.and Phyllostachys heterocycla（Carr.） Mifford cv.Pubescens Mazel ex H.de leh.（2） Thus the flavonoid extracts from Dendrocalamopsis oldhami（Munro） Keng f.leaves was further selected to investigate their antioxidant activity in rat’s model.These rats were divided randomizely into five groups,named as blank control,negative control,positive control,low dosage bamboo-leaf extracts（LDBE） and high level dosage bamboo-leaf extracts groups（HLBE）,respectively.Statistically,each group had seven individuals.Rats in the blank control were fed with no cholesterol-based diet which was not supplemented with any bamboo-leaf extracts or Vit.E.Rats in the negative control were fed with cholesterol-based diet without any supplementation of bamboo-leaf extracts or Vit.E. 0.0025%Vit.E was added to the cholesterol-based diet of rats in positive control. 100mg/kg or 400mg/kg of freeze-dried bamboo-leaf extracts in rat’s body weight was added to the diets of LDBE or HLBE group,espectively.After feeding rats for six weeks, tissues collection from liver and kidney showed that bamboo-leaf extracts significantly influenced enzyme’s activities of superoxide dismutase（SOD）,catalase（CAT）, glutathione peroxidase（GSH-Px） and concentation malonaldehyde（MDA） levels in all tesed rats well-controlled of above mentioned activities.The higher antioxidant activity proportional to the dose of bamboo leaf extracts was observed in tested rats.5.The characteristics and activity of volatile oil compounds from bamboo leaves（1） The bamboos leaves collected from Phyllostachys heterocycla（Carr.） Mitford cv. Pubescens Mazel ex H.de leh.were pre-treated with four techniques,i.e.simultaneous distillation extraction（SDE）,volatile oil distillation（VOD）,supercritical fluid extraction （SFE） and Soxhlet’s extraction method（SEM）) to extract volatile oils.The major composition of volatile oils when SDE or VOD was used were alcohols,carboxylic acids, and alkane hydrocarbons.If SFE technique was chosen to treat bamboo leaves,the main compounds of the volatile oils were alkane hydrocarbons and carboxylic acids.Use of SEM technique to treat bamboo leaves produced more alkane hydrocarbons from volatile oil.It was clear that differences in the profiles of the volatile oil of bamboo leaves existed due to the application of different extraction techniques.The present study indicated that the use of SDE or VOD to extract bamboo leaves would give more information on volatile oils composition than the other techniques.（2） The yields of volatile oils of four bamboo species collected by SDE were between 0.1% and 0.3%,with Pleioblastus amarus（Keng） Keng f.the largest,followed by Acidosasa edulis Wen,Dendrocalamopsis oldhami（Munro） Keng f.and Phyllostachys heterocycla（Carr.） Mitford cv.Pubescens Mazel ex H.de leh..The volatile oil compounds of Phyllostachys heterocycla（Carr.） Mifford cv.Pubescens Mazel ex H.de leh.were mainly alcohols,carboxySc acids and alkane hydrocarbons.Volatile oil composition of Acidosasa edulis Wen was very similar to that of Phyllostachys heterocycla（Carr.） Mitford cv.Pubescens Mazel ex H.de leh.The volatile oil compounds of Acidosasa edulis Wen and Dendrocalamopsis oldhami（Munro） Keng f. mainly composed of carboxylic acids,aldehydes and alkane hydrocarbon.（3） Internal standard quantitative method confirmed 7 volatile oil compounds,including Dodecanoic acid,6,10,14-trimethyl-2-Pentadecanone,tetradecanoic acid,tricosane, heptacosane,hexadecanoic acid,and 9-octadecanal.The content of hexadecanoic acid was the highest in all of bamboo-leaf volatile oil.The next was dodecanoic acid in Pleioblastus amarus（Keng） Keng f.and Phyllostachys heterocycla（Carr.） Mitford cv. Pubescens Mazel ex H.de leh.leaves.9-octadecanal ranked in the second position in Acidosasa edulis Wen or Dendrocalamopsis oldhami（Munro） Keng f.leaves. 6,10,14-trimethyl-2-Pentadecanone ranked as the third position in Pleioblastus amarus（Keng） Keng f.and Phyllostachys heterocycla（Carr.） Mitford cv. Pubescens Mazel ex H.de leh.leaves.Among volatile oil compounds determined, dodecanoic acid ranked in the third positon in Acidosasa edulis Wen and Dendrocalamopsis oldhami（Munro） Keng f.（4） Bacillus subtilis,Escherichia coli,2 Pseudomonas fluorescens strains SHL5 and SHL7, Flavobacterium SHL45 and three Saccharomyces cerevisiae strains Ja64,Tokay and Y8 were tested to evaluate growth inhibition of tesed organims bamboo-leaf volatile oils. Among those four bamboo species,the volatile oil from the leaves of Phyllostachys heterocycla（Carr.） Mitford cv.Pubescens Mazel ex H.de leh.showed strongest antimicrobial activity.The minimal inhibition concentration（MIC） toward Escherichia coli and Bacillus subtilis were 2.25mg/mL.In collusions,the present study proved that bamboo leaves contained large amount of flavonoids and volatile oils,with diversity in their composition.The levels of bamboo-leaf flavonoids or volatile oil depended on the species of bamboo and growing seasons, exhibiting good performances in antioxidant and antimicrobial activity.The collection of these data should be very useful in identifying the species of bamboo,and promoting fine production of bamboo leaves as food additives.更多还原