木蝴蝶黄酮标准品的制备及总黄酮分离纯化工艺研究
|
摘要
木蝴蝶,别名千张纸、玉蝴蝶、云破纸等,是一种常见中药材,是紫葳科木蝴蝶属的成熟种子。具有润肺、止咳、舒肝、利咽、和胃、生肌等功效。木蝴蝶的有效成分主要为黄酮类化合物,以千层纸苷、木蝴蝶苷A、黄芩苷、木蝴蝶苷B等为主。目前,有关木蝴蝶的研究主要集中在有效成分的含量测定、分离鉴定及其药理作用方面,其中有效成分含量测定主要是利用HPLC或RP-HPLC对某一成分进行测定,仅有本研究室的胡殿丽采用黄芩苷为标准品,测定了木蝴蝶醇提物的总黄酮含量,而未见以木蝴蝶苷A为标准品测定木蝴蝶总黄酮的含量和采用大孔吸附树脂法分离纯化木蝴蝶醇提物的报道。随着人们对植物药物的研究开发,木蝴蝶醇提取物越来越多的应用于医药、食品等领域,对其质量也提出了更高的要求。所以本文以此为基础,选择木蝴蝶种子为原料,得到了木蝴蝶苷A,并进行了结构鉴定和纯度测定,并对大孔吸附树脂分离纯化木蝴蝶总黄酮的工艺进行系统研究。主要研究内容如下：
1.木蝴蝶种子经干燥、粉碎,用70%的乙醇提取,提取液经浓缩、醇沉、离心、上硅胶色谱柱,采用氯仿：甲醇系统梯度洗脱,分离出了木蝴蝶苷A。经13CNMR和'HNMR进行结构鉴定,通过熔点测定、紫外吸收、RP-HPLC法进行含量测定,最终确定其纯度为98%。
2.以自制的木蝴蝶苷A为标准品,采用紫外分光光度法,测得木蝴蝶苷A的标准工作曲线为C (μg/mL)=16.7491 A-0.2975, R2=0.9998,线性工作范围为1.70-17.0μg/mL。通过方法学考察得知该方法重复性、稳定性良好,精密度实验相对标准偏差较小,加标回收率较高,可以用来测定木蝴蝶总黄酮的含量。
3.采用静态吸附与动态吸附相结合的方法比较5种不同类型的大孔吸附树脂的吸附解吸性能,综合考虑发现：HPD100树脂的吸附与解吸效果优于其它树脂。因此,选择HPD100树脂分离纯化木蝴蝶总黄酮。
4.考察了HPD100树脂吸附木蝴蝶总黄酮的工艺条件,结果发现：当上样浓度为5.303mg/mL, pH值为2-3,流速为3.0mL/min时,HPD100树脂对木蝴蝶总黄酮的吸附率较大；水洗液用量为3BV时,水溶性杂质可以基本除尽；以90%乙醇为解吸剂,用量为8BV时,木蝴蝶总黄酮解吸效果较好。在此条件下,经一次分离纯化后木蝴蝶总黄酮纯度达到88.3%,且工艺稳定,重现性好。
5.对HPD100树脂吸附木蝴蝶总黄酮的动力学与热力学进行考察,结果发现：在150min时,HPD100树脂吸附木蝴蝶总黄酮的过程基本达到吸附平衡；HPD100树脂吸附木蝴蝶总黄酮的动力学符合拟2级动力学模型；Freundlich等温吸附方程更适合描述HPD100树脂吸附木蝴蝶总黄酮的过程,且吸附过程的焓变△H均为负值,绝对值小于4OKJ/moL,吸附过程是一个放热的物理吸附过程。吉布斯自由能变AG小于0,吸附过程能够自发进行。熵变AS小于0,吸附过程是一个熵减小的过程。
Semen Orxyli, which is the mature seed of Oroxylum indicum (L.) Vent, has been used a traditional Chinese medicine for its abilities of moistening lung, relieving cough, smoothing liver, eliminating phlegm, promoting new growth of tissues etc. A number of academic studies show that the main chemical components of Oroxylum indicum are Oroxin A, Oroxin B,Tetuin, Baicalin and so on. So far, the research of Oroxylum indicum has mainly focused on the determination, separation and pharmacological actions of the active components.The contents of the active components were determined by using HPLC or RP-HPLC method to test a single component. Only Dian-li Hu has determined the contents of total flavonoids with Baicalin as a chemical reference substance. But the determined of total flavonoids with Oroxin A as a standard and purification of total flavonoids from Oroxylum indicum by macroporous adsorption resins have not been reported. With the development of plant-based drugs, The ethanol extracts from Oroxylum indicum become used more and more in pharmaceutical industry, food industry and other fields, and their qualities are required of higher standard. Based on this, this paper selects Oroxylum indicum as the raw material, obtains Oroxin A and then carries out the structural identification and purity determination, makes the systematical research on the technical conditions of separation and purification total flavonoids from Oroxylum indicum. The main content includes:
1. The mature seeds of Oroxylum indicum were first dried,crushed and extracted by 70% ethanol, the extracts after being condensed, alcohol precipitation and centrifugation were separated by silica gel column chromatography. The eventual result is that the Oroxin A was found. Then its structure was identified by 13CNMR and 1HNMR, and the purity of Oroxin A was determined by melting point, UV absorption and RP-HPLC. The result showed that the purity of Oroxin A was 98%.
2. The standard working curve is C (μg/mL)=16.7491 A-0.2975, R2= 0.9998,which was obtained by ultraviolet spectrophotometry with home-made Oroxin A as a standard. At the same time, the studying of methodology showed the method was inaccurate, reliable and could be repeated. It could be used to determine total flavonoids of Oroxylum indicum.
3. Static adsorption and dynamic adsorption method were combined to study the five different types of macroporous adsorption and desorption properties The results showed that HPD100 resin have good adsorption capacity than other resins. As a result, HPD100 resin was selected to separate and purify the total flavonoids of Oroxylum indicum.
4. The purification of the process conditions total flavonoids of Oroxylum indicum attached by HPD100 resin found that when the concentration of the sample is 5.303mg/mL, pH value is 2-3, velocity of flow is 3.0 mL/min,the adsorption rate of the total flavonoids of Oroxylum indicum is relatively big,when the amount of water used is 3 BV,the water-soluble impurities can be basically divisible, using the 90% ethanol as the desorption agent,the amount used is 8BV, the desorption effect of the flavonoids is relatively good. Under such condition, the purity of the total flavonoids of Oroxylum indicum can reach to 88.3% after division and purification with stable process and good reproducibility.
5. The kinetic and thermodynamic experimental results of studying HPD100 resin's adsorption of Oroxylum total flavonoids show that the process basically reaches equilibrium at 150 min, and its kinetics follows the pseudo-second-order kinetic model. Besides, Freundlich isotherm adsorption equation is more suitable for the whole process, and the enthalpy change AH in adsorption process is negative and its modulus is less than 40KJ/mol. The adsorption process is an exothermic physical process. The Gibbs free energy AG is less than 0, so the adsorption process can occur spontaneously. the entropy change AS are less than 0, which indicates that the adsorption reduces the degrees of the entire system's freedom.
1.木蝴蝶种子经干燥、粉碎,用70%的乙醇提取,提取液经浓缩、醇沉、离心、上硅胶色谱柱,采用氯仿：甲醇系统梯度洗脱,分离出了木蝴蝶苷A。经13CNMR和'HNMR进行结构鉴定,通过熔点测定、紫外吸收、RP-HPLC法进行含量测定,最终确定其纯度为98%。
2.以自制的木蝴蝶苷A为标准品,采用紫外分光光度法,测得木蝴蝶苷A的标准工作曲线为C (μg/mL)=16.7491 A-0.2975, R2=0.9998,线性工作范围为1.70-17.0μg/mL。通过方法学考察得知该方法重复性、稳定性良好,精密度实验相对标准偏差较小,加标回收率较高,可以用来测定木蝴蝶总黄酮的含量。
3.采用静态吸附与动态吸附相结合的方法比较5种不同类型的大孔吸附树脂的吸附解吸性能,综合考虑发现：HPD100树脂的吸附与解吸效果优于其它树脂。因此,选择HPD100树脂分离纯化木蝴蝶总黄酮。
4.考察了HPD100树脂吸附木蝴蝶总黄酮的工艺条件,结果发现：当上样浓度为5.303mg/mL, pH值为2-3,流速为3.0mL/min时,HPD100树脂对木蝴蝶总黄酮的吸附率较大；水洗液用量为3BV时,水溶性杂质可以基本除尽；以90%乙醇为解吸剂,用量为8BV时,木蝴蝶总黄酮解吸效果较好。在此条件下,经一次分离纯化后木蝴蝶总黄酮纯度达到88.3%,且工艺稳定,重现性好。
5.对HPD100树脂吸附木蝴蝶总黄酮的动力学与热力学进行考察,结果发现：在150min时,HPD100树脂吸附木蝴蝶总黄酮的过程基本达到吸附平衡；HPD100树脂吸附木蝴蝶总黄酮的动力学符合拟2级动力学模型；Freundlich等温吸附方程更适合描述HPD100树脂吸附木蝴蝶总黄酮的过程,且吸附过程的焓变△H均为负值,绝对值小于4OKJ/moL,吸附过程是一个放热的物理吸附过程。吉布斯自由能变AG小于0,吸附过程能够自发进行。熵变AS小于0,吸附过程是一个熵减小的过程。
Semen Orxyli, which is the mature seed of Oroxylum indicum (L.) Vent, has been used a traditional Chinese medicine for its abilities of moistening lung, relieving cough, smoothing liver, eliminating phlegm, promoting new growth of tissues etc. A number of academic studies show that the main chemical components of Oroxylum indicum are Oroxin A, Oroxin B,Tetuin, Baicalin and so on. So far, the research of Oroxylum indicum has mainly focused on the determination, separation and pharmacological actions of the active components.The contents of the active components were determined by using HPLC or RP-HPLC method to test a single component. Only Dian-li Hu has determined the contents of total flavonoids with Baicalin as a chemical reference substance. But the determined of total flavonoids with Oroxin A as a standard and purification of total flavonoids from Oroxylum indicum by macroporous adsorption resins have not been reported. With the development of plant-based drugs, The ethanol extracts from Oroxylum indicum become used more and more in pharmaceutical industry, food industry and other fields, and their qualities are required of higher standard. Based on this, this paper selects Oroxylum indicum as the raw material, obtains Oroxin A and then carries out the structural identification and purity determination, makes the systematical research on the technical conditions of separation and purification total flavonoids from Oroxylum indicum. The main content includes:
1. The mature seeds of Oroxylum indicum were first dried,crushed and extracted by 70% ethanol, the extracts after being condensed, alcohol precipitation and centrifugation were separated by silica gel column chromatography. The eventual result is that the Oroxin A was found. Then its structure was identified by 13CNMR and 1HNMR, and the purity of Oroxin A was determined by melting point, UV absorption and RP-HPLC. The result showed that the purity of Oroxin A was 98%.
2. The standard working curve is C (μg/mL)=16.7491 A-0.2975, R2= 0.9998,which was obtained by ultraviolet spectrophotometry with home-made Oroxin A as a standard. At the same time, the studying of methodology showed the method was inaccurate, reliable and could be repeated. It could be used to determine total flavonoids of Oroxylum indicum.
3. Static adsorption and dynamic adsorption method were combined to study the five different types of macroporous adsorption and desorption properties The results showed that HPD100 resin have good adsorption capacity than other resins. As a result, HPD100 resin was selected to separate and purify the total flavonoids of Oroxylum indicum.
4. The purification of the process conditions total flavonoids of Oroxylum indicum attached by HPD100 resin found that when the concentration of the sample is 5.303mg/mL, pH value is 2-3, velocity of flow is 3.0 mL/min,the adsorption rate of the total flavonoids of Oroxylum indicum is relatively big,when the amount of water used is 3 BV,the water-soluble impurities can be basically divisible, using the 90% ethanol as the desorption agent,the amount used is 8BV, the desorption effect of the flavonoids is relatively good. Under such condition, the purity of the total flavonoids of Oroxylum indicum can reach to 88.3% after division and purification with stable process and good reproducibility.
5. The kinetic and thermodynamic experimental results of studying HPD100 resin's adsorption of Oroxylum total flavonoids show that the process basically reaches equilibrium at 150 min, and its kinetics follows the pseudo-second-order kinetic model. Besides, Freundlich isotherm adsorption equation is more suitable for the whole process, and the enthalpy change AH in adsorption process is negative and its modulus is less than 40KJ/mol. The adsorption process is an exothermic physical process. The Gibbs free energy AG is less than 0, so the adsorption process can occur spontaneously. the entropy change AS are less than 0, which indicates that the adsorption reduces the degrees of the entire system's freedom.
引文
[1]国家药典委员会.中华人民共和国药典[M].北京：化学工业出版社.2005：43
[2]中国药典[S].一部.2005：46
[3]中药辞海[M].北京：中国医药科技出版社.1996：807
[4]Tomimori T, Imoto Y, Ishida M, et al. Studies on the Nepalesecrude drugs (VIII). On the flavonoid constituents of the seed of Oroxylum indicum Vent[J]. Shoyakugaku Zasshi,1988, 42 (1):98
[5]陈亮亮,宋晓凯,侯文彬,等.木蝴蝶化学成分的研究[J].中草药,2007,38(2)：186-187
[6]Li J Chen, David E G, Jonathan J. Isolation and identification four flavonoid constituents from the seeds of Oroxylum indicum by high-speed counter-current chromatography [J].chromatogr,2003,98 (8):95
[7]Subramanian, S Sankara, Nair A G R. Flavonoids of the leaves of Oroxylum indicum and Pajanelia longifolia[J]. Phytochemistry (Elsevier),1972,11(1):439～405
[8]Subramanian S S, Nair A G R. Flavonoids of the stem bark of Oroxylum indicum [J]. Current Science,1972,41(2):62
[9]陈仲夏,赵志远.木蝴蝶化学成分的研究-二种新黄酮苷木蝴蝶甲素和木蝴蝶乙素[J].药学学报,1964,11(11)：762-767
[10]Biswanath Dindal, Bikas Chandra Mohantal, Shio Arima, et al. Flavonoids from the Stembark of Oroxylum indicum[J]. Natural Product Sciences,2007,13(3):190～194
[11]T.Hari Babu, K.Manjulayha, G.Suresh Kumar, et al. Gastroprotective flavonoid constituents from Oroxylum indicum Vent[J]. Bioorganic&Medicinal Chemistry letters,2010,20: 117～120
[12]Mehta C R, Mehta T P. Tetuin.A glucoside from the seeds of Oroxylum indicum [J]. Current Science,1953,22:114
[13]Nair A G Ramachandran, Joshi B S. Oroxindin-a new flavone glucuronide from Oroxylum indicum Vent.[J].Proc Indian Acad Sci SectA,1979,88(A):323
[14]Dey A K, Mukherjee A, DasPC, et al. Occurrence of aloeemodin in the leaves of Oroxylum indicum Vent. [J].Indian J ChemSectB OrgChemMed Chem,1978,16(11):1042
[15]Teshima Ko Ichiro, Kaneko Tetsuo, Ohtani Kazuhiro, et al. Phe-nylethanoids and cyclo-hexylethanoids from Oroxylum indicum [J]. NatMed,1996,50(4):307
[16]胡殿丽.木蝴蝶总黄酮提取工艺和脂肪酸成分研究[D].[硕士学位论文].郑州：郑州大学,2010,5
[17]赵丽娟,李铁纯,丁旭光,等.木蝴蝶挥发油成分的研究[J].化学工程师,2002,88(1)：47-48
[18]赵丽娟,张捷莉,李学成.木蝴蝶挥发性化学成分的气相色谱-质谱分析[J].食品科技,2006,8(2)：252-253
[19]Ali Mohd, Chaudhary Anurag,Ramachandram Ramid, et al. New pterocarpans from Oroxylum indicum stem bark[J]. Indian J Chem SectB OrgChemMed Chem,1999,38(8): 950
[20]Ali Rasadah Mat, Houghton, Peter J, et al. Antifungal activity of some Bignoniaceae found in Malaysia[J]. Phytotherapy Res,1998,12(5):331
[21]Joshi Krishna C, Prakash Lalit, Shah Rama Kant. Chemical examination of the roots of tabebuia rosea and heartwood of Oroxylum indicum[J]. Planta Medica,1977,31(3):257-258
[22]Vasanth Saradha, Natarajan Meenaksh, Sundaresan Rukman. Ellagic acid from Oroxylum indicum Vent[J]. Indian Drugs,1991,28(11):507
[23]刘凡,何美英.HPLC法测定木蝴蝶中黄芩苷的含量[J].湖南中医药导报,2002,8(6)：365-366
[24]仝会娟,张强,康琛,等.木蝴蝶药材中黄芩素和白杨素的含量测定[J].中国中医药信息杂志,2009,12：52-53
[25]李曼玲,康琛,张强,等.中药材木蝴蝶专属性成分木蝴蝶苷B的提取分离及质量标准研究[J].中华中医药学会第十届中药鉴定学术会议论文集,2010,491-507
[26]缪建荣,杨红美,曾建国,等.木蝴蝶中黄酮类化合物的HPLC-ESI-MS分析[J].中草药,2006,37(4)：505-507
[27]姚琰.HPLC法测定木蝴蝶提取物中木蝴蝶苷A的含量[J].中国药房,2010,13(12)：1751-1752
[28]蔡鹰,丁安伟.木蝴蝶药材HPLC指纹图谱研究[J].中国实验方剂学杂志,2007,13(12)：6-8
[29]孙楠,莫卫民,胡宝祥,等.白杨黄酮的单扫描示波极谱法测定及应用[J].理化检验化学,2005,41(9)：636-641
[30]Le Thi Diem, Hong N, guyen X. T. Influence of flavonoids from Oraxylum indicum Vent towards a-chymotrypsin in relation to inflammation[J]. Tap ChiDuocHoc,2005, (8):23
[31]胡庭俊,刘珊珊,赵灵颖,等.木蝴蝶提取物制备及其抗菌抗炎活性的研究[J].中国兽牧兽医,2010,37(3)：225-228
[32]Ali Rasadah Mat, Houghton P J Raman A, et al. Antinicrobial and anti-inflammatory activities of extracts and constituents of Oroxylim indicum[J]. Phytomedicine,1998,5(5): 375
[33]戴卉卿,陈承瑜,杨滨.22种黄酮、酚酸类化合物和9种中药清除AAPH活性考察[J].中国中药杂志,2010,35(17)：2296-2302
[34]吴立军主编.天然药物化学,第四版,北京：人民卫生出版社,2003,172
[35]吴剑锋主编.天然药物化学,北京：高等教育出版社,2005,232
[36]高荫榆,魏强,范青生,等.高速逆流色谱分离提取天然产物技术研究进展[J].食品科学,2005,29(02)：461-465
[37]甘春芳,韦万兴,周敏,等.高速逆流色谱技术在天然产物分离中的应用[J].应用化 工,2009,35(10)：1505-1506
[38]Ying-Hung Lee, Albert Linton Charlesb, Hsien-Feng Kungc, Chi-Tang Hod, Tzou-Chi Huanga. Extraction of nobiletin and tangeretin from Citrus depressa Hayata by supercritical carbon dioxide with ethanol as modifier[J]. Industrial Crops and Products,2003,14(3): 78～82
[39]杨宜婷,罗琥捷,叶树勇,等.不同储存年限广陈皮的多甲氧基黄酮提取研究[J].食品工业科技.2011,(5)：50-56
[40]刘国柱.中药中黄酮类化合物全定性分析策略[D].[博士学位论文].长沙：湖南师范大学,2010
[41]X.G.Chen, L.Kong, X.Y.Su,H.J.Fu, R.H.Zhao, H.F.Zou. Separation and identification of compounds in Rhizoma chuanxiong by comprehensive two-dimensional liquid chromatography coupled to mass spectrometry[J]. J.Chromatogr.A,2004,1040:169～178
[42]X.G.Chen, L.Kong, X.Y.Su, C.S.Pan, M.L.Ye, H.F.Zou. Integration of ion-exchange chromatography fractionation with reversed-phase liquid chromatography-atmospheric pressure chemical ionization mass spectrometer and matrix-assisted laser desorption/ionization time-of- flight mass spectrometry for isolation and identification of compounds in Psoralea corylifolia[J]. J.Chromatogr.A,2005,1089:87～100
[43]徐双双,孙瑜,井风,等.硅胶柱色谱结合高速逆流色谱法分离纯化荷花中黄酮类化合物[J].色谱,2011,(12)：1244-1248
[44]杜琪珍,赵永春,李博.常春油麻藤花黄酮类化合物结构鉴定及抗氧化活性分析[J].食品科学,2011,32(23)：111-115
[45]Rice Evans C, Miller N J. The relative anthioxidant activities of plant derived ployphenloic flavonoids[J]. free radicalRes,1995,22:375～383
[46]胡春,丁萧霖.黄酮类化合物在不同氧化体系中的抗氧化作用研究[J].食品与发酵工程,1996,(3)：46-53
[47]Terao J, Piskula M, Yao Q. Protective effect of epicatechin, epicatechin gallate,and quercetin on lipid peroindation in phospholipids bilayers[J]. Archives Biochem & Biophys,1994, 308(1):278～284
[48]陈丽娜,吴琼.沙棘籽及果皮渣黄酮粗提物抗氧化作用研究[J].食品研究与开发,2011,32(10)：51-53
[49]樊廷俊,赵君,李凌,等.人角膜内皮细胞体外氧化损伤及黄酮抗氧化保护作用的机制[J].国际眼科杂志,2008,8(8)：1536-1514
[50]Yerra Koteswara Rao, Shih-HuaFang, Yew-MinTzeng. Anti-inflammatory activities of flavonoids isolated from Caesalpinia pulcherrima[J]. Jurnal of Ethnopharmacology 2005, 249～253
[51]Sadzuka Y, yugiyama T, Hirota S, Modulation of cancer chemotherapy by green tea[J]. Chin Cancer Res,1998,4(1):153
[52]Guseppe G, Peter J. Potential toxicity of flavonoids and other dietary phenolics significance for their chemopreventive and anticancer properties [J]. Free Radic Biol Med, 2004,37(3):287～303
[53]Eleni Psarra, Dimitris P Makris, Stamatina Kallithraka, et al. Evaluation of the antiradical and reducing properties of selected Greek white wines:correlation with polyphenolic composition[J]. Journal of the Scicence of Food and Agriculture,2002,82:1014～1020
[54]Pan H, Lai S, Hsu C,et al. Acacetin induces apoptosis in human gastric carcinoma cells accompanied by activation of caspase cascades and production of reactive oxygen species[J]. J Agric Food Chem,2005,53(3):620～630
[55]ZHEN YS, CAOSS,XUEYC, et al. Green tea extract inhibits nueleceide transportsnd potentiontes the antitumor effect of antimetabolites[J]. Chin Med Sci,1991,6(1):1
[56]Marion Foti, Mario Plattelli, Maria Tiziana Baratta, et al. Flavonoids,coumarins,and cinnamic acids as antioxidants in a micellar system structure-activity relationship[J]. Journal of Agricultural and Food Chemistry,1996,44(2):497～501
[57]Sergeev, I.N. Genistein induces Ca2+-mediated, calpain/caspase-12 dependent apoptosis in breast cancer cells[J]. Biochemical and Biophysical research Communications,2004,321: 462～467
[58]Herzog A, Kindermann B, Doring F, Daniel H, Wenzel U. Pleiotropic molecular effects of the proapoptotic dietary constituent flavone in human colon cancer cells identified by protein and mRNA expression profiling[J]. Proteomics,2004:55～64
[59]Veronika Ullmannova PhD, Nicholas C, Popescu PhD. Inhibition of cell proliferation, induction of apoptosis, reactivationof DLC-1, and modulation of other gene expression by dietary flavone in breast cancer cell lines[J]. Cancer Detection and Prevention,2007,31, 110～118
[60]Dajas F, Rivera F, Blasina F, et al. Cell culture protection and in vivo neuroprotective capacity of flavonoids[J]. Neurotox Res,2003,5 (6):425～432
[61]Chen J, Xu J.Li J. Effect of puerarin on fibrinolytic activity and lipid peroxide in patients with coronary heart disease[J]. Zhong guo Zhong Xi Yi Jie He Za Zhi,1999,19(11): 649～650
[62]Weber C, Negrescu E, ErL W, et al. Inhibitors of protein tyrosine kinase suppress TNF-stimulated induction of endothelial cell adhesion molecules[J]. Jimmunol,1995,155: 445～461
[63]郭雪微,卢挺,等.天然黄酮类化合物在心脑血管疾病中应用进展[J].中国老年学杂志,2000,26(4)：1001-1005
[64]Nicholas K.H. Khoo, C.Roger White, Lucas Pozzo-Miller, et al. Dietary flavonoid quercetin stimulates vasorelaxation in aortic vessels[J]. Free RadicalBiology & Medicine,2010,49: 339～347
[65]郭超.毛蕊异黄酮的提取工艺优化及其对大鼠脑缺血损伤的保护作用[D].[硕十学位论文].西安：陕西师范大学,2011
[66]万巧凤,吴莉,等.槲皮素对甲型H1N1流感病毒诱导的A549细胞凋亡效应酶Caspase-3的影响[J].中国中医药信息杂志,2011,18(10)：42-44
[67]F. Cottiglial, G Loyl, D Garau1, C Floris2, et al. Antimicrobial evaluation of coumarins and flavonoids from the stems of Daphne gnidium [J]. Phytomedicine,2001,8(4):302～305
[68]J L S Goncalvesl, S G Leitao, et al. In vitro antiviral effect of flavonoid rich extracts of Vitex polygama (Verbenaceae) against acyclovir-resistant herpes simplex virus type [J].2001, 8(6):477～480
[69]HENDRICH S, LEE K W, XU X, et al. Defining food components as new nutrients[J]. Journal of Nutrion,1994,124:1789～1792
[70]韩正康.异黄酮植物雌激素-大豆黄酮对雄性动物生长及其有关内分泌的研究[J].畜牧与兽医,1999,31(1)：1-2
[71]胥正敏,陈建业,等.黄芪总黄酮对人正常骨髓间充质细胞的抗辐射损伤作用[J].中药药理与临床,2011,27(3)：39-42
[72]陆绮,赵晖,穆阳.复方当归提取物对皮肤抗氧化作用实验研究[J].中国美容医学,2007,16(11)：1574-1576
[73]王灿,左艇,王琳琳.桑叶黄酮抗皮肤衰老实验研究[J].中国医药导报,2011,8(3)：30-32
[74]李秀丽,邹继红,刘青妍,等.槲皮素改善肝细胞脂质堆积作用的实验研究[J].时珍国医国药,2011,22(10)：2384-2386
[75]石荣,马越鸣,方圆圆,等.槲皮素在糖尿病模型大鼠肝脏甲基化代谢的研究[J].中药药理与临床杂志,2011,27(5)：34-37
[76]陆晓和,金念祖.类黄酮抗癌作用机制的研究进展[J].医学研究生学报,2009,22(5)：536-539
[77]袁黎明.制备色谱技术及应用[M].北京：北京工业出版社,2005
[78]张昌壮.木蝴蝶化学成分研究[D].[硕士学位论文].吉林,吉林大学,2011
[79]田厚伦.有机化学[M].北京：北京工业出版社,2004
[80]马陶陶,张群林,李俊.中药总黄酮的含量测定方法[J].安徽医药,2007,11(1)：1030-1032
[81]龙海燕,刘雁鸣,黄晓燕,等.紫外分光光度法测定灯盏细辛胶囊中总黄酮含量[J].中南药学,2011,9(10)：766-769
[82]胡殿丽,吴鸣建.木蝴蝶总黄酮超声提取工艺研究[J].时珍国医国药,2010,21(8)：35-37
[83]李坤平.大孔树脂吸附黄芩黄酮和布渣叶黄酮的应用基础研究[D].[硕士学位论文].广州中医药大学,2010
[84]王冬梅,尉芹,马希汉.大孔吸附树脂在药用植物有效成分分离中的应用[J].西北林学院学报,2002,17(1)：60-65
[85]Garcia-delgado R.A, Cotoruelo L.M, Separ.Sci.Tec.[J].1992,27 (7):975
[86]刘晓庚,贾红圣,曹崇江.大孔吸附树脂分离纯化女贞子原花青素的动力学和热力学研究[J].中国粮油学报,2009,24(1)：124-127
[87]Bell J.P, Tsezos M, J.Water Pollut Control Fed [J].1987,59(4):191～198
[2]中国药典[S].一部.2005：46
[3]中药辞海[M].北京：中国医药科技出版社.1996：807
[4]Tomimori T, Imoto Y, Ishida M, et al. Studies on the Nepalesecrude drugs (VIII). On the flavonoid constituents of the seed of Oroxylum indicum Vent[J]. Shoyakugaku Zasshi,1988, 42 (1):98
[5]陈亮亮,宋晓凯,侯文彬,等.木蝴蝶化学成分的研究[J].中草药,2007,38(2)：186-187
[6]Li J Chen, David E G, Jonathan J. Isolation and identification four flavonoid constituents from the seeds of Oroxylum indicum by high-speed counter-current chromatography [J].chromatogr,2003,98 (8):95
[7]Subramanian, S Sankara, Nair A G R. Flavonoids of the leaves of Oroxylum indicum and Pajanelia longifolia[J]. Phytochemistry (Elsevier),1972,11(1):439～405
[8]Subramanian S S, Nair A G R. Flavonoids of the stem bark of Oroxylum indicum [J]. Current Science,1972,41(2):62
[9]陈仲夏,赵志远.木蝴蝶化学成分的研究-二种新黄酮苷木蝴蝶甲素和木蝴蝶乙素[J].药学学报,1964,11(11)：762-767
[10]Biswanath Dindal, Bikas Chandra Mohantal, Shio Arima, et al. Flavonoids from the Stembark of Oroxylum indicum[J]. Natural Product Sciences,2007,13(3):190～194
[11]T.Hari Babu, K.Manjulayha, G.Suresh Kumar, et al. Gastroprotective flavonoid constituents from Oroxylum indicum Vent[J]. Bioorganic&Medicinal Chemistry letters,2010,20: 117～120
[12]Mehta C R, Mehta T P. Tetuin.A glucoside from the seeds of Oroxylum indicum [J]. Current Science,1953,22:114
[13]Nair A G Ramachandran, Joshi B S. Oroxindin-a new flavone glucuronide from Oroxylum indicum Vent.[J].Proc Indian Acad Sci SectA,1979,88(A):323
[14]Dey A K, Mukherjee A, DasPC, et al. Occurrence of aloeemodin in the leaves of Oroxylum indicum Vent. [J].Indian J ChemSectB OrgChemMed Chem,1978,16(11):1042
[15]Teshima Ko Ichiro, Kaneko Tetsuo, Ohtani Kazuhiro, et al. Phe-nylethanoids and cyclo-hexylethanoids from Oroxylum indicum [J]. NatMed,1996,50(4):307
[16]胡殿丽.木蝴蝶总黄酮提取工艺和脂肪酸成分研究[D].[硕士学位论文].郑州：郑州大学,2010,5
[17]赵丽娟,李铁纯,丁旭光,等.木蝴蝶挥发油成分的研究[J].化学工程师,2002,88(1)：47-48
[18]赵丽娟,张捷莉,李学成.木蝴蝶挥发性化学成分的气相色谱-质谱分析[J].食品科技,2006,8(2)：252-253
[19]Ali Mohd, Chaudhary Anurag,Ramachandram Ramid, et al. New pterocarpans from Oroxylum indicum stem bark[J]. Indian J Chem SectB OrgChemMed Chem,1999,38(8): 950
[20]Ali Rasadah Mat, Houghton, Peter J, et al. Antifungal activity of some Bignoniaceae found in Malaysia[J]. Phytotherapy Res,1998,12(5):331
[21]Joshi Krishna C, Prakash Lalit, Shah Rama Kant. Chemical examination of the roots of tabebuia rosea and heartwood of Oroxylum indicum[J]. Planta Medica,1977,31(3):257-258
[22]Vasanth Saradha, Natarajan Meenaksh, Sundaresan Rukman. Ellagic acid from Oroxylum indicum Vent[J]. Indian Drugs,1991,28(11):507
[23]刘凡,何美英.HPLC法测定木蝴蝶中黄芩苷的含量[J].湖南中医药导报,2002,8(6)：365-366
[24]仝会娟,张强,康琛,等.木蝴蝶药材中黄芩素和白杨素的含量测定[J].中国中医药信息杂志,2009,12：52-53
[25]李曼玲,康琛,张强,等.中药材木蝴蝶专属性成分木蝴蝶苷B的提取分离及质量标准研究[J].中华中医药学会第十届中药鉴定学术会议论文集,2010,491-507
[26]缪建荣,杨红美,曾建国,等.木蝴蝶中黄酮类化合物的HPLC-ESI-MS分析[J].中草药,2006,37(4)：505-507
[27]姚琰.HPLC法测定木蝴蝶提取物中木蝴蝶苷A的含量[J].中国药房,2010,13(12)：1751-1752
[28]蔡鹰,丁安伟.木蝴蝶药材HPLC指纹图谱研究[J].中国实验方剂学杂志,2007,13(12)：6-8
[29]孙楠,莫卫民,胡宝祥,等.白杨黄酮的单扫描示波极谱法测定及应用[J].理化检验化学,2005,41(9)：636-641
[30]Le Thi Diem, Hong N, guyen X. T. Influence of flavonoids from Oraxylum indicum Vent towards a-chymotrypsin in relation to inflammation[J]. Tap ChiDuocHoc,2005, (8):23
[31]胡庭俊,刘珊珊,赵灵颖,等.木蝴蝶提取物制备及其抗菌抗炎活性的研究[J].中国兽牧兽医,2010,37(3)：225-228
[32]Ali Rasadah Mat, Houghton P J Raman A, et al. Antinicrobial and anti-inflammatory activities of extracts and constituents of Oroxylim indicum[J]. Phytomedicine,1998,5(5): 375
[33]戴卉卿,陈承瑜,杨滨.22种黄酮、酚酸类化合物和9种中药清除AAPH活性考察[J].中国中药杂志,2010,35(17)：2296-2302
[34]吴立军主编.天然药物化学,第四版,北京：人民卫生出版社,2003,172
[35]吴剑锋主编.天然药物化学,北京：高等教育出版社,2005,232
[36]高荫榆,魏强,范青生,等.高速逆流色谱分离提取天然产物技术研究进展[J].食品科学,2005,29(02)：461-465
[37]甘春芳,韦万兴,周敏,等.高速逆流色谱技术在天然产物分离中的应用[J].应用化 工,2009,35(10)：1505-1506
[38]Ying-Hung Lee, Albert Linton Charlesb, Hsien-Feng Kungc, Chi-Tang Hod, Tzou-Chi Huanga. Extraction of nobiletin and tangeretin from Citrus depressa Hayata by supercritical carbon dioxide with ethanol as modifier[J]. Industrial Crops and Products,2003,14(3): 78～82
[39]杨宜婷,罗琥捷,叶树勇,等.不同储存年限广陈皮的多甲氧基黄酮提取研究[J].食品工业科技.2011,(5)：50-56
[40]刘国柱.中药中黄酮类化合物全定性分析策略[D].[博士学位论文].长沙：湖南师范大学,2010
[41]X.G.Chen, L.Kong, X.Y.Su,H.J.Fu, R.H.Zhao, H.F.Zou. Separation and identification of compounds in Rhizoma chuanxiong by comprehensive two-dimensional liquid chromatography coupled to mass spectrometry[J]. J.Chromatogr.A,2004,1040:169～178
[42]X.G.Chen, L.Kong, X.Y.Su, C.S.Pan, M.L.Ye, H.F.Zou. Integration of ion-exchange chromatography fractionation with reversed-phase liquid chromatography-atmospheric pressure chemical ionization mass spectrometer and matrix-assisted laser desorption/ionization time-of- flight mass spectrometry for isolation and identification of compounds in Psoralea corylifolia[J]. J.Chromatogr.A,2005,1089:87～100
[43]徐双双,孙瑜,井风,等.硅胶柱色谱结合高速逆流色谱法分离纯化荷花中黄酮类化合物[J].色谱,2011,(12)：1244-1248
[44]杜琪珍,赵永春,李博.常春油麻藤花黄酮类化合物结构鉴定及抗氧化活性分析[J].食品科学,2011,32(23)：111-115
[45]Rice Evans C, Miller N J. The relative anthioxidant activities of plant derived ployphenloic flavonoids[J]. free radicalRes,1995,22:375～383
[46]胡春,丁萧霖.黄酮类化合物在不同氧化体系中的抗氧化作用研究[J].食品与发酵工程,1996,(3)：46-53
[47]Terao J, Piskula M, Yao Q. Protective effect of epicatechin, epicatechin gallate,and quercetin on lipid peroindation in phospholipids bilayers[J]. Archives Biochem & Biophys,1994, 308(1):278～284
[48]陈丽娜,吴琼.沙棘籽及果皮渣黄酮粗提物抗氧化作用研究[J].食品研究与开发,2011,32(10)：51-53
[49]樊廷俊,赵君,李凌,等.人角膜内皮细胞体外氧化损伤及黄酮抗氧化保护作用的机制[J].国际眼科杂志,2008,8(8)：1536-1514
[50]Yerra Koteswara Rao, Shih-HuaFang, Yew-MinTzeng. Anti-inflammatory activities of flavonoids isolated from Caesalpinia pulcherrima[J]. Jurnal of Ethnopharmacology 2005, 249～253
[51]Sadzuka Y, yugiyama T, Hirota S, Modulation of cancer chemotherapy by green tea[J]. Chin Cancer Res,1998,4(1):153
[52]Guseppe G, Peter J. Potential toxicity of flavonoids and other dietary phenolics significance for their chemopreventive and anticancer properties [J]. Free Radic Biol Med, 2004,37(3):287～303
[53]Eleni Psarra, Dimitris P Makris, Stamatina Kallithraka, et al. Evaluation of the antiradical and reducing properties of selected Greek white wines:correlation with polyphenolic composition[J]. Journal of the Scicence of Food and Agriculture,2002,82:1014～1020
[54]Pan H, Lai S, Hsu C,et al. Acacetin induces apoptosis in human gastric carcinoma cells accompanied by activation of caspase cascades and production of reactive oxygen species[J]. J Agric Food Chem,2005,53(3):620～630
[55]ZHEN YS, CAOSS,XUEYC, et al. Green tea extract inhibits nueleceide transportsnd potentiontes the antitumor effect of antimetabolites[J]. Chin Med Sci,1991,6(1):1
[56]Marion Foti, Mario Plattelli, Maria Tiziana Baratta, et al. Flavonoids,coumarins,and cinnamic acids as antioxidants in a micellar system structure-activity relationship[J]. Journal of Agricultural and Food Chemistry,1996,44(2):497～501
[57]Sergeev, I.N. Genistein induces Ca2+-mediated, calpain/caspase-12 dependent apoptosis in breast cancer cells[J]. Biochemical and Biophysical research Communications,2004,321: 462～467
[58]Herzog A, Kindermann B, Doring F, Daniel H, Wenzel U. Pleiotropic molecular effects of the proapoptotic dietary constituent flavone in human colon cancer cells identified by protein and mRNA expression profiling[J]. Proteomics,2004:55～64
[59]Veronika Ullmannova PhD, Nicholas C, Popescu PhD. Inhibition of cell proliferation, induction of apoptosis, reactivationof DLC-1, and modulation of other gene expression by dietary flavone in breast cancer cell lines[J]. Cancer Detection and Prevention,2007,31, 110～118
[60]Dajas F, Rivera F, Blasina F, et al. Cell culture protection and in vivo neuroprotective capacity of flavonoids[J]. Neurotox Res,2003,5 (6):425～432
[61]Chen J, Xu J.Li J. Effect of puerarin on fibrinolytic activity and lipid peroxide in patients with coronary heart disease[J]. Zhong guo Zhong Xi Yi Jie He Za Zhi,1999,19(11): 649～650
[62]Weber C, Negrescu E, ErL W, et al. Inhibitors of protein tyrosine kinase suppress TNF-stimulated induction of endothelial cell adhesion molecules[J]. Jimmunol,1995,155: 445～461
[63]郭雪微,卢挺,等.天然黄酮类化合物在心脑血管疾病中应用进展[J].中国老年学杂志,2000,26(4)：1001-1005
[64]Nicholas K.H. Khoo, C.Roger White, Lucas Pozzo-Miller, et al. Dietary flavonoid quercetin stimulates vasorelaxation in aortic vessels[J]. Free RadicalBiology & Medicine,2010,49: 339～347
[65]郭超.毛蕊异黄酮的提取工艺优化及其对大鼠脑缺血损伤的保护作用[D].[硕十学位论文].西安：陕西师范大学,2011
[66]万巧凤,吴莉,等.槲皮素对甲型H1N1流感病毒诱导的A549细胞凋亡效应酶Caspase-3的影响[J].中国中医药信息杂志,2011,18(10)：42-44
[67]F. Cottiglial, G Loyl, D Garau1, C Floris2, et al. Antimicrobial evaluation of coumarins and flavonoids from the stems of Daphne gnidium [J]. Phytomedicine,2001,8(4):302～305
[68]J L S Goncalvesl, S G Leitao, et al. In vitro antiviral effect of flavonoid rich extracts of Vitex polygama (Verbenaceae) against acyclovir-resistant herpes simplex virus type [J].2001, 8(6):477～480
[69]HENDRICH S, LEE K W, XU X, et al. Defining food components as new nutrients[J]. Journal of Nutrion,1994,124:1789～1792
[70]韩正康.异黄酮植物雌激素-大豆黄酮对雄性动物生长及其有关内分泌的研究[J].畜牧与兽医,1999,31(1)：1-2
[71]胥正敏,陈建业,等.黄芪总黄酮对人正常骨髓间充质细胞的抗辐射损伤作用[J].中药药理与临床,2011,27(3)：39-42
[72]陆绮,赵晖,穆阳.复方当归提取物对皮肤抗氧化作用实验研究[J].中国美容医学,2007,16(11)：1574-1576
[73]王灿,左艇,王琳琳.桑叶黄酮抗皮肤衰老实验研究[J].中国医药导报,2011,8(3)：30-32
[74]李秀丽,邹继红,刘青妍,等.槲皮素改善肝细胞脂质堆积作用的实验研究[J].时珍国医国药,2011,22(10)：2384-2386
[75]石荣,马越鸣,方圆圆,等.槲皮素在糖尿病模型大鼠肝脏甲基化代谢的研究[J].中药药理与临床杂志,2011,27(5)：34-37
[76]陆晓和,金念祖.类黄酮抗癌作用机制的研究进展[J].医学研究生学报,2009,22(5)：536-539
[77]袁黎明.制备色谱技术及应用[M].北京：北京工业出版社,2005
[78]张昌壮.木蝴蝶化学成分研究[D].[硕士学位论文].吉林,吉林大学,2011
[79]田厚伦.有机化学[M].北京：北京工业出版社,2004
[80]马陶陶,张群林,李俊.中药总黄酮的含量测定方法[J].安徽医药,2007,11(1)：1030-1032
[81]龙海燕,刘雁鸣,黄晓燕,等.紫外分光光度法测定灯盏细辛胶囊中总黄酮含量[J].中南药学,2011,9(10)：766-769
[82]胡殿丽,吴鸣建.木蝴蝶总黄酮超声提取工艺研究[J].时珍国医国药,2010,21(8)：35-37
[83]李坤平.大孔树脂吸附黄芩黄酮和布渣叶黄酮的应用基础研究[D].[硕士学位论文].广州中医药大学,2010
[84]王冬梅,尉芹,马希汉.大孔吸附树脂在药用植物有效成分分离中的应用[J].西北林学院学报,2002,17(1)：60-65
[85]Garcia-delgado R.A, Cotoruelo L.M, Separ.Sci.Tec.[J].1992,27 (7):975
[86]刘晓庚,贾红圣,曹崇江.大孔吸附树脂分离纯化女贞子原花青素的动力学和热力学研究[J].中国粮油学报,2009,24(1)：124-127
[87]Bell J.P, Tsezos M, J.Water Pollut Control Fed [J].1987,59(4):191～198