【作者】 王进；

【导师】 岳永德；

【作者基本信息】 中国林业科学研究院 ， 生态学， 2012， 博士

【摘要】 我国竹资源丰富，竹叶被《中华本草》等药典收载，具有一定的药用价值。竹叶提取物有显著的抗氧化、抑菌等生物活性，成为开发植物源抗氧化剂、植物药和天然香料的重要来源。本研究以簕竹属（Bambusa）的小琴丝竹（B. multiplex cv. Alphonse-Karr）、青皮竹（B. textilis McClure）、银丝竹（B. multiplex cv. Silverstripe）、小佛肚竹（B. ventricosa McClure）、孝顺竹（B. multiplex var. multiplex）、凤尾竹（B. multiplex cv.Fernleaf）、观音竹（B. multiplex var. riviereorum R.Maire）等7种竹叶为研究对象，以清除DPPH自由基的方法筛选出高抗氧化活性的竹种、以家兔体内模型筛选出竹叶中的药效成分，利用高压制备液相色谱对所筛选出的抗氧化成分和药效成分进行分离制备，并应用液相色谱-飞行时间质谱（LC/Q-TOF-MS）、核磁共振等对活性组分开展了结构鉴定，分别建立了高效薄层色谱（HPTLC）和高效液相色谱（HPLC）测定竹叶提取物中4种黄酮碳苷的检测方法，并开展了顶空-固相微萃取方法（HS-SPME）和水蒸气蒸馏法（HD）两种前处理方法分析竹叶挥发性成分的比较。旨在为簕竹属竹叶资源应用于天然食品抗氧化剂、天然药物等提供理论基础，促进竹叶资源的高附加值利用。主要研究结果如下：1.建立了HPTLC同时测定市售竹叶黄酮中4种黄酮碳苷（异荭草苷、荭草苷、牡荆苷、异牡荆苷）的方法。市售竹叶黄酮样品用甲醇提取后，点样于高效硅胶板上，以四氢呋喃-甲苯-甲酸-水（16:8:2:1, v/v/v/v）为展开剂，经全自动展开仪（ADC2）展开，350nm下紫外扫描检测。结果表明，4种黄酮碳苷在薄层板上分离良好，在竹叶黄酮样品中的添加回收率94-100%，在30ng-1200ng/斑点的范围内（其中，异荭草苷200-1200ng/斑点，异牡荆苷100-600ng/斑点，荭草苷160-960ng/斑点，牡荆苷-360ng/斑点）相关系数r0.9995，该方法简便可靠，适合于市售竹叶黄酮中4种黄酮碳苷的同时测定，为竹叶黄酮产品的质量控制提供技术支撑。2.建立了HPLC同时测定不同竹种竹叶中4种黄酮碳苷（异荭草苷、荭草苷、牡荆苷、异牡荆苷）的方法。竹叶样品用60%甲醇提取，采用反相C₁₈色谱柱（250×4.6mmI.D.,5μm），以乙腈-水（含有0.5%乙酸）（15:85, v/v）为流动相，流速为1.0mL/min，柱温为30℃，检测波长为345nm。结果表明，4种黄酮碳苷在30min内达到基线分离，在0.2-100μg/mL的范围内线性关系良好，在竹叶提取物中的添加回收率88-106%，该方法简便、灵敏、准确，成功用于9种不同竹叶提取物中黄酮碳苷的测定。3.利用DPPH分光光度法测定了簕竹属7种竹叶提取物的抗氧化活性，不同竹种DPPH自由基清除率从大到小依次为：青皮竹（84.67%）>银丝竹（69.06%）>小佛肚竹（51.48%）>孝顺竹（43.88%）>凤尾竹（37.83%）>小琴丝竹（35.86%）>观音竹（30.52%）。利用优化后的薄层色谱生物自显影法（DPPH-TLC）对7种竹叶样品的抗氧化活性进行筛选，结果显示，不同竹叶的抗氧化活性的大小与DPPH分光光度法的测定结果一致。其中，青皮竹叶的抗氧化能力较强，其抗氧化活性IC50值为25.26mg/L。对青皮竹叶提取物进行抗肿瘤活性筛选，发现青皮竹叶提取物对白血病细胞（K562）、胃癌细胞（BGC823）、肺癌细胞（A549）等3种肿瘤细胞均无细胞毒性。4.青皮竹叶提取物具有较高的抗氧化活性，为明确青皮竹叶中抗氧化成分，取青皮竹叶，以60%甲醇超声波提取30min后，再振荡提取12h，重复提取2次，合并提取液浓缩、冻干，通过化学显色反应，发现青皮竹叶中抗氧化成分主要为黄酮类和酚酸类化合物，以DPPH-TLC活性追踪的方法，利用高压制备液相色谱从青皮竹叶中分离制备出3种主要抗氧化成分，通过紫外、质谱、核磁共振等数据综合分析，鉴定了3种抗氧化成分，分别为（1）木犀草素-6-C-葡萄糖木糖苷；（2）木犀草素-6-C-葡萄糖鼠李糖苷；（3）木犀草素-6-C-葡萄糖苷。其中，木犀草素-6-C-葡萄糖木糖苷和木犀草素-6-C-葡萄糖鼠李糖苷是首次从簕竹属竹种中发现。5．利用LC-Q-TOF-MS技术对青皮竹叶水煎液中的主要化学成分进行结构鉴定，共检出20种化合物，包括黄酮类、酚酸类、香豆素类。结合紫外光谱和核磁共振技术，鉴定了其中10种化合物，分别为（1）木犀草素-6-C-葡萄糖木糖苷、（2）木犀草素-6-C-葡萄糖鼠李糖苷、（3）异荭草苷、（4）荭草苷、（5）反式对香豆酸、（6）顺式对香豆酸、（7）香豆素、（8）牡荆苷、（9）芹菜素-8-C-葡萄糖鼠李糖苷、（10）异牡荆苷。6.以青皮竹叶水煎液对家兔灌胃后，采集不同时相血浆，利用液相色谱的指纹图谱比较，发现青皮竹叶水煎液中的3种化合物能够进入血液，分别用高分辨质谱、紫外光谱和核磁共振对3种化合物进行鉴定，分别为反式对香豆酸、顺式对香豆酸、芹菜素-8-C-葡萄糖鼠李糖苷，本研究首次阐明了青皮竹叶的药效物质基础。7.分别采用顶空-固相微萃取（HS-SPME）和水蒸气蒸馏法（HD）对簕竹属7种竹叶中挥发性成分进行富集，以气相色谱-质谱联用仪（GC-MS）进行定性检测，用面积归一化法确定各组分的相对含量。结果表明，两种方法共鉴定竹叶发性成分79种，包括酸类、酚类、酮类、酯类、醛类等化合物，其中HD-GC/MS鉴定出76种，HS-SPME-GC/MS鉴定出18种。HD-GC/MS测定竹叶挥发性成分的数量和相对含量上均高于HS-SPME-GC/MS方法，HS-SPME-GC/MS适合于快速测定竹叶挥发性成分，具有简单、快速等特点。两种方法在7种竹叶中均检出的化合物有8种，包括：α-紫罗兰酮，香叶基丙酮，β-紫罗兰酮，4-[2,2,6-三甲基-7-氧杂二环[4.1.0]庚-1-基]-3-丁烯-2-酮，抗氧剂264，二氢猕猴桃内酯，植酮，邻苯二甲酸二异丁酯，这些共同检出的化合物在食品香精、日用化工、食品防腐剂和医药中间体等领域广泛应用，为簕竹属竹叶挥发性成分的开发利用提供研究基础。综上所述，本研究建立了HPLC和HPTLC两种测定竹叶黄酮碳苷的方法，筛选出青皮竹叶提取物具有较高的抗氧化活性，并鉴定出3种抗氧化成分，从青皮竹叶水煎液中鉴定了10种化合物，包括3种药效成分，簕竹属竹叶的挥发性成分含有香精香料等组分，为竹叶资源应用于天然抗氧化剂、天然药物等提供研究基础。更多还原

【Abstract】 China is rich in bamboo resource. Bamboo leaves have been used in traditional Chinesemedicine and collected by Chinese Materia Medica. It was found that extract of bamboo leaveshas multiple biological activities, such as antioxidation, antifungi, and can be used as apharmaceutical intermediate and food additive. To develop new plant drug, dietary supplementand value-added products from bamboo resources. The leaves of B. multiplex cv. Alphonse-Karr, B. textilis McClure, B. multiplex cv. Silverstripe, B. ventricosa McClure, B. multiplex var.multiplex, B. multiplex cv. Fernleaf and B. multiplex var. riviereorum R.Maire were collectedfrom Nanjing forestry university and Jiangxi academy of forestry in China. In this study, high-performance thin-layer chromatography （HPTLC） and high-performance liquidchromatography （HPLC） methods were optimized and validated for the simultaneousdetermination of flavone C-glycosides. DPPH-TLC and DPPH spectrometry were used toscreen the compounds with antioxidant activity from bamboo leaves. Compounds showedbiological active were isolated by preparative chromatography and identified by liquidchromatography/quadrupole time-of-flight mass spectrometry （LC-Q-TOF-MS） and nuclearmagnetic resonance （NMR）. Volatile compounds were enriched through head space solid phasemicroextractions （HS-SPME） and hydrodistillation （HD） technologies, and analyzedqualitatively with gas chromatography-mass spectrometry （GC-MS）.（1） A simple HPTLC method was developed for the simultaneous determination ofisoorientin, isovitexin, orientin and vitexin in the commercial samples of bamboo-leaf-flavonoids. The flavone C-glycosides were extracted from bamboo-leaf-flavonoids withmethanol and chromatographed on silica gel60plates in automatic developing chamber（ADC2） with tetrahydrofuran-toluene-formic acid–water （16+8+2+1,v/v/v/v） as mobile phase.Quantitation was obtained with UV detection at350nm. Polynomial calibration plots wereconstructed in the concentration range200-1200ng/zone for isoorientin and100-600ng/zonefor isovitexin and160-960ng/zone for orientin and30-360ng/zone for vitexin with good correlation coefficients （r≥0.9995）. The average recovery was found to be93.95%forisoorientin,95.35%for isovitexin,99.79%for orientin and100.46%for vitexin. The proposedHPTLC method was found to be simple, precise, specific and accurate and can be used formanufacturing quality control of bamboo-leaf-flavonoids.（2） An HPLC method was optimized and validated for the simultaneous determination offlavone C-glycosides, including isoorientin, isovitexin, orientin and vitexin in the leaves ofdifferent species of bamboo. Extraction was carried out with60%methanol. The separationwas performed using a C₁₈ column （4.6×250mm,5μm） at30℃. The solvent system consistedof a mixture of water with0.5%（v/v） glacial acetic acid and acetonitrile （85/15, v/v） at a flowrate of1.0mL/min. The detection wavelength was set at345nm. Satisfactory separation ofthese marker compounds was obtained in less than30min. The optimized HPLC methodproved to be linear in the concentration range tested （0.2-100μg/mL, r2≥0.9997）, accurate（88-106%）. The proposed method was validated to be simple and reliable and can be a tool forquality control of bamboo leaf extract.（3） The DPPH radical scavenging activity of the leaf extract from seven different speciesof bamboo were investigated by UV-Vis spectrometry. The radical scavenging rates of sevensamples ranged from30.52%to51.48%. The same results achieved by DPPH-TLC methodrevealed that the extract of B. textilis McClure showed high antioxidant activity with an IC50of25.26mg/L. A MTT method has been used for screening the anti-tumor activity of the extractof B. textilis McClure. The results showed no cytotoxic activity against the tumor cells testedK562, BGC823andA549).（4） A qualitative survey using a simple derivatization steps with the DPPH and NP reagentwas carried out. The flavone C-glycosides and phenolic acids found in bamboo leavesexamined could be the main antioxidant compounds. To characterize the main antioxidantcompounds, the leaf of B. textilis McClure was extracted with60%methanol by ultrasonicwave technology. The leaf extract of B. textilis McClure was further fractionated usingpreparative chromatography. Three antioxidant fractions were isolated by DPPH-TLCbioactive-guided fractionation. According to the data of UV, MS, and NMR spectra, three main antioxidant compounds were identified as5,7,3’,4’-tetrahydroxy-6-C-β-D-glu-xylflavonoside（1）,5,7,4’-trihydroxy-6-C-β-D-glu-rha flavonoside （2） and isoorientin（3）.Compound （1） and （2） were first isolated from bambusa.（5） LC-Q-TOF-MS method for analyzing the constituents in the water extract of theleaves of B. textilis McClure has been established. Of the20detected compounds includingflavonoids, phenolic acids, coumarins,10were identified from their MS, UV, NMR spectra, orby comparing the retention time and mass spectrometry data with that of reference compoundsand reference literatures. The identified compounds were as follows:5,7,3’,4’-tetrahydroxy-6-C-β-D-glu-xyl flavonoside（1）,5,7,4’-trihydroxy-6-C-β-D-glu-rha flavonoside（2）, isoorientin（3）,orientin（4）, trans-p-coumaric acid（5）, cis-p-coumaric acid（6）, coumarin（7）, vitexin（8）,5,7,4’-trihydroxy-8-C-β-D-glucose-α-L-rhamnosyl flavonoside（9）, isovitexin（10）.（6） A valid chromatographic fingerprint method using LC-Q-TOF-MS is proposed forstudying the constituents in rabbit plasma after oral administration of the water extract of theleaves of B. textilis McClure. Three compounds, including cis-p-coumaric acid, trans-p-coumaric acid and5,7,4’-trihydroxy-8-C-β-D-glucose-α-L-rhamnosyl flavonoside, weredetected in both the water extract of the leaves of B. textilis McClure and rabbit plasma.The results prove that three compounds could be the bioactive compounds of bambooleaves.（7） To conduct comparative study on the volatile compounds from bamboo leaves.Volatile compounds were enriched through head space solid phase microextractions （HS-SPME） method, and analyzed qualitatively with gas chromatography-mass spectrometry （GC-MS）, which were comparative by analyzed with the method of direct injection GC-MS basedon hydrodistillation extraction （HD）. Area normalization method was employed for thesubsequent quantification. The results showed that a total of79volatile compounds wereseparated and checked out with the two methods, including acids, phenols, ketones, esters,aldehydes, etc., in which76compounds were checked out with HD-GC/MS, while18compounds with HS-SPME-GC/MS. Comparison analysis showed that there were significantsimilarity in type of volatile compounds from different bamboo leaves between the two methods. The main volatile compounds from seven different bamboo leaves by the twomethods included3-buten-2-one,4-（2,6,6-trimethyl-2-cyclohexen-1-yl）-,（E）-,5,9-undecadien-2-one,6,10-dimethyl-,（E）-,3-buten-2-one,4-（2,6,6-trimethyl-1-cyclohexen-1-yl）-,（E）-,3-buten-2-one,4-（2,2,6-trimethyl-7-oxabicyclo[4.1.0] hept-1-yl）-,264butylated hydroxytoluene,2（4H）-benzofuranone,5,6,7,7a-tetrahydro-4,4,7a-trimethyl-,（R）-,2-pentadecanone,6,10,14-trimethyl-,1,2-benzenedicarboxylic acid, bis（2-methylpropyl） ester. These compounds havebeen applied in the field of flavor and fragrance, food preservative, and daily-use chemical.In conclusion, validated HPLC and HPTLC methods for determination of flavone C-glycosides in bamboo leaves were developed. The bamboo leaves of B. textilis McClureshowed high antioxidant activity and three antioxidant compounds were identified. Threebioactive compounds from the water extract of the leaves of B. textilis McClure were identified.Flavor components were found in the bamboo leaves of the Genus Bambusa. These resultsshould very useful in promoting the fine production of bamboo leaves as natural antioxidantand natural medicines.更多还原