【作者】 支欢欢；

【导师】 魏永巨；

【作者基本信息】 河北师范大学 ， 化学生物学， 2014， 博士

【摘要】 根据香豆素类化合物在苯环和酯环上取代基的类型与位置等结构特点，分类研究比较了40多种香豆素类化合物的荧光性质，考察了环境因素（pH、溶剂极性、有序介质、共存离子、放置和光照时间等）对荧光波长和强度的影响，总结了荧光性质与分子结构之间的经验规律。提出了中成药复方胆通胶囊中羟甲香豆素的荧光分析方法，并研究了中药材羌活、宽叶羌活中紫花前胡苷的荧光分析方法和三维荧光-化学计量学方法。论文包括以下5部分。1.研究了2种苯环和酯环上均无取代基的香豆素类化合物的荧光性质，包括香豆素（苯并吡喃酮）和二氢香豆素。香豆素稀溶液在通常情况下没有荧光，其碱性溶液在紫外光照射时可产生香豆素二聚体的荧光，β-环糊精（β-CD）可使荧光稳定，最大激发和发射波长λex/λem=360/495nm，荧光量子产率Y=0.09。二氢香豆素的λex/λem=272/300nm，呈现取代苯的波长特征，Y=0.023。二者的光谱差异说明，苯环和酯环上的取代基以及酯环上的酯键、羰基和3、4位双键对香豆素类化合物的荧光都有重要影响。2.研究了8种仅酯环上有取代基的香豆素类化合物的荧光性质，包括香豆素-3-羧酸、香豆素-3-羧酸乙酯、3-乙酰基香豆素、3-丁酰基香豆素、4-羟基香豆素、4-甲氧基香豆素、华法林钠和双香豆素。此类香豆素的荧光较弱或没有荧光。香豆素-3-羧酸仅在pH2.0左右有荧光，λex/λem=295/418nm，Y=0.010；香豆素-3-羧酸乙酯在pH1.9~7.9有荧光，λex/λem=292/418nm，Y=0.009。4-羟基香豆素水溶液在pH5.3~13.5范围内有荧光，λex/λem=285/366nm，Y=0.013；4-甲氧基香豆素在pH1.20~12.05范围内有极弱荧光；华法林钠水溶液在pH6.2~13.3范围内有荧光，λex/λem=306/385nm，Y=0.020。3-乙酰基香豆素、3-丁酰基香豆素和双香豆素无荧光。3.研究了8种仅苯环上有取代基的香豆素类化合物的荧光性质，包括6-甲基香豆素、7-甲基香豆素、紫花前胡苷、花椒毒素、补骨脂素、异补骨脂素、7-甲氧基-8-羟基香豆素和7-羟基-8-乙酰基香豆素。综合比较了这些香豆素和本实验室曾研究过的10多种此类香豆素的荧光性质，得到如下经验规律：（1）苯环上弱给电子取代的香豆素类化合物自身荧光很弱，例如6-甲基香豆素和7-甲基香豆素的中性稀溶液基本没有荧光，但是，在pH大于12.20时，出现二聚体荧光，λex/λem分别为379/508nm和355/490nm，Y分别为0.090和0.17。（2）不饱和呋喃香豆素荧光较弱，饱和呋喃和吡喃香豆素荧光较强，且线型比角型香豆素的荧光强。例如补骨脂素为不饱和线型呋喃香豆素，Y=0.029；异补骨脂素为不饱和角型呋喃香豆素，Y=0.015；白花前胡甲素饱和角型吡喃香豆素，Y=0.11；紫花前胡苷为饱和线性呋喃香豆素，Y=0.81。（3）7-OH邻位有-OH或吸电子基团导致荧光减弱，7-OCH3邻位有-OH则导致荧光猝灭。（4）酯环在碱性条件下水解开环导致荧光猝灭。4研究了9种苯环和酯环均有取代基的香豆素类化合物的荧光性质，包括6-甲氧基-4-甲基香豆素、6-羟基-4-甲基香豆素、7-羟基-4-甲基香豆素、7-羟基-4-甲氧基甲基香豆素、7-乙酰氧基-4-甲基香豆素、7-甲氧基-4-甲基香豆素、7-乙氧基-4-甲基香豆素、7-甲氧基香豆素-3-羧酸和4-甲基-7-氧香豆素-β-D-吡喃半乳糖苷。通过综合比较此类香豆素的荧光性质，得到如下经验规律：（1）酯环上取代基为4-CH3的香豆素类化合物荧光性质取决于苯环的取代基。（2）苯环或酯环上多个取代基时-OH或-COOH（吸电子或给电子能力更强的取代基）对荧光性质起决定性作用。（3）当6位取代基为甲氧基而且其他位置没有-OH时，香豆素类化合物的荧光图谱中呈现3个激发峰。（4）荧光激发波长与整个分子的共轭体系有关，但发射荧光的基团为酯环。5.研究了中成药复方胆通胶囊中羟甲香豆素的溶液荧光分析方法，测定结果与药品标示量一致。研究了中药羌活与宽叶羌活中紫花前胡苷的溶液荧光分析方法，并用高效液相色谱法对测定结果的可靠性进行了验证，结果表明两种方法测定宽叶羌活中紫花前胡苷的含量基本一致，分别为2.94%和3.00%；而羌活中紫花前胡苷的测定结果有一定差别，分别为0.208%和0.187%，原因是羌活中的共存荧光成分对紫花前胡苷的测定有干扰。为了解决这一问题，探讨建立了用三维荧光-化学计量学方法测定中药羌活和宽叶羌活中紫花前胡苷含量的新方法，得到满意结果，证明三维荧光-化学计量学方法是解决复杂中药样品定量分析的一种有效方法。更多还原

【Abstract】 According to the structural features of coumarins with different substituent located atbenzene ring and/or lactone ring, more than40coumarin compounds are classified into4groups and their fluorescent properties are studied. Influences of environmental conditions(pH, solvent polarity, orderly medium, coexisting ions, placing or illumination time, etc.) onfluorescence wavelength and intensity are investigated. Empirical rules of fluorescenceproperty vs. molecular structure are summarized. A fluorimetric method for determination ofHymecromone in Chinese patent medicine Compound Dantong Capsule is proposed, and afluorimetric method as well as a3D fluorescence-chemometrics method for analysis ofNodakenin in Chinese herbal medicine Qianghuo and Kuanyeqianghuo are investigated.Five parts are included in the dissertation.1. Fluorescent properties of coumarins with no substituent on benzene and lactone ringincluding coumarin (benzopyrone) and dihydrocoumarin were investigated. Dilute solution ofcoumarin was no fluorescence under ordinary condition, whereas its alkaline solution canproduce coumarin dimer’s fluorescence with maximum excitation and emission wavelengthsλex/λemat360/495nm and quantum yield (Y)0.09when irradiated by UV light. Addition ofβ-CD into the solution can stabilize the fluorescence. Three dimensional (3D) fluorescencespectra of dihydrocoumarin with λex/λem=272/300nm and Y=0.023were measured, and thespectra was recognized to be similar to substituted benzene. The spectral difference betweencoumarin and dihydrocoumarin illustrate that the presence of substituent on benzene andlactone ring and the cyclic ether oxygen, carbonyl group and ethylenic bond in lactone ringare important to the fluorescence of coumarins.2. Fluorescent properties of8kinds of coumarin with substituent only on lactone ring,including Coumarin-3-carboxylic acid, Ethyl coumarin-3-carboxylate,3-Acetylcoumarin,3-Butyrylcoumarin,4-Hydroxylcoumarin,4-Methoxycoumarin, Warfarin and Dicoumarin,were investigated. The fluorescence of these coumarins is weaker or no fluorescence.Coumarin-3-carboxylic acid produce fluorescence with λex/λem=295/418nm and Y=0.010 only under about pH2.0. Ethylcoumarin-3-carboxylate shows fluorescence with λex/λem=292/418nm and Y=0.009in pH1.9-7.9.4-Hydroxylcoumarin shows fluorescence withλex/λem=285/366nm and Y=0.013in pH5.3-13.5.4-Methoxycoumarin shows very weakfluorescence in pH1.2-12.05. Warfarin shows fluorescence with λex/λem=306/385andY=0.020in pH6.2-13.3.3-Acetylcoumarin,3-Butyrylcoumarin, and Dicoumarin have nofluorescence.3. Fluorescence properties of8kinds of coumarin with substituent only on benzene ring,including6-Methylcoumarin,7-Methylcoumarin, Nodaenin, Xanthotoxin, Psoralen,Isopsoralen,8-Hydroxy-7-methoxycoumarin and8-Acetyl-7-hydroxycoumarin wereinvestigated. The fluorescence properties of these coumarins and more than10kinds of thissort coumarins studied previously in our lab were comprehensively compared, and someempirical rules were obtained as follows:(1) Coumarins with weak electron-donating groupon the benzene ring showed weak fluorescence. For example,6-Methylcoumarin and7-Methylcoumarin have no fluorescence in neutral dilute solution, but give their dimerfluorescence with λex/λem=379/508nm, Y=0.090and λex/λem=355/490nm, Y=0.17,respectively, when pH≥12.20.(2) Unsaturated furan coumarins give weaker fluorescence,while saturated furan and pyran coumarins give stronger fluorescence; fluorescence of linearcoumarins is stronger than that of angle coumarins. For examples, Psoralen is an linerunsaturated furan coumarin, Y=0.029; Isopsoralen is an unsaturated angle furan coumarin, Y=0.015; Praeruptorin is a saturated angle pyran coumarin, Y=0.11; Nodakenin is a saturatedliner furan coumarins, Y=0.81.(3) A-OH or an electron-withdrawing group at ortho-positionof7-OH induce fluorescence decrease, and a-OH at ortho-position of7-OCH3lead tofluorescence quench.(4) Hydrolization of lactone bond and ring-opening under alkalinecondition result in fluorescence quench.4. Fluorescence properties of9kinds of coumarin with substituent both on benzene andlactone ring, including6-Methoxy-4-methylcoumarin,6-Hydroxy-4-methyl coumarin,7-Hydroxy-4-Methylcoumarin,7-Hydroxy-4-(meithoxymethyl) coumarin,7-Acetoxy-4-methylcoumarin,7-methoxy-4-methylcoumarin and4-Methyl-7-ethoxycoumarin, wereinvestigated. Comparison of fluorescent properties of above coumarins leads to some empirical rules as follows:(1) If the substituent of C4position on lactone ring is-CH3,fluorescent property depend on the substituent on benzene ring.(2) If there are multiplesubstituent on both benzene and lactone ring,-OH or-COOH, or stronger electron-donatinggroup or electron-withdrawing group play a decisive role to fluorescence property.(3) Whensubstituent at C6-position is methoxyl and without-OH at any other position, there will be3excitation peaks emerged in fluorescence spectrum.(4) The fluorescence excitationwavelength is associated with the conjugate system of the whole molecule; but thefluorescence emission group is lactone ring.5. A fluorimetric method was proposed for detecting Hymecromone in Chinese patentdrug Compound Dantong Capsule, and the obtained result was consistent with the drug label.A fluorimetric method was also studied for analysis of Nodakenin in Chinese herbal medicineQianghuo and Kuanyeqianghuo, respectively, and HPLC method was used to verify thereliability of the fluorimetric method. The content of Nodakenin in Kuanyeqianghuodetermined by two methods was almost the same,2.94%and3.00%, respectively. However,the results of Nodakenin in Qianghuo were different,0.208%and0.187%, respectively. Thedifference came from the interference of coexistent compounds. For solving the problem, a3D fluorescence-chemometrics method was investigated to determine Nodakenin in Chineseherbal medicine Qianghuo and Kuanyeqianghuo, a satisfied result was obtained, and the resultdemonstrated that3D fluorescence-chemometrics method is an effective method for analysisof complex traditional Chinese medicine.更多还原