大蒜活性蛋白提取、分离及制剂工艺的研究
|
摘要
论文主要分析大蒜冻干粉中活性蛋白的成分,并对冻干粉制剂工艺中辅料(赋形剂)的配比以及包衣增重量对片剂累积溶出度的影响进行了研究,从而确定优化后的冻干粉片剂的处方及工艺,并对优化后的处方和工艺进行了验证。
试验使用膜分离法从大蒜中提取分离超氧化歧化酶(superoxide dismutase, SOD)和水溶性多肽,再通过冷冻干燥法对大蒜纳滤浓缩液进行冻干处理得到大蒜粉。用邻苯三酚经典自氧化法测定大蒜粉中SOD的活力,SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)测定冻干粉中活性蛋白的种类及其相对分子质量。
将大蒜冻干粉与不同种类辅料按处方量混合,再通过湿法制粒压片法制成片剂,并用合适浓度的玉竹多糖溶液进行包衣。采用中心组合设计方法安排试验点,结合响应曲面优化法对制剂的处方及工艺进行优化。按照试验设计改变片剂辅料配方中微晶纤维素与乳糖的比例及玉竹多糖包衣液增重,用考马斯亮蓝法(Bradford法)测定蛋白质含量从而测定累积溶出度。实验数据通过SAS软件建立二元线性回归方程,利用绘制出的响应曲面图及等值线图确定优化的处方及工艺。
试验结果表明,辅料配比及包衣液的增重量对包衣片的累积溶出度均有明显影响,且相对于包衣液的增重量,辅料配比对累积溶出度的影响更为明显。优化后的辅料比例为微晶纤维素：乳糖=57：43、包衣液增重为10%,验证试验的数据表明包衣片剂累积溶出度的实测值与预测值十分接近,且片剂外观、硬度、片剂重量差异均符合药典规定。并且试验制得的大蒜包衣片剂能很好的掩盖大蒜辛辣味及刺激性气味,更适合服用。
The active component of garlic lyophilized powder which was obtained using membrane separation method, the excipient ratio in the preparation engineering, effects on the tablet dissolution rate of the coating material weight was studied in this research. And the formula of the lyophilized powder preparation and the engineering parameters were determined, and the quality assessment of the tablet was performed at last.
Superoxide dismutase (SOD) and soluble peptide was separated and extracted from garlic using membrane separation method. Next, garlic lyophilized powder got from the garlic concentrated solution using lyopyilization method. The SOD activity was tested using the classic adjacent pyrogallol since oxidation method. And the kinds and the relative molecular weight of the active protein in the garlic lyophilized powder were tested with SDS-PAGE method.
Mixing garlic lyophilized powder with different kinds of excipents, using Jade bamboo polysaccharide solution as coating material, garlic tablets were made with wet granulating method. In order to get better preparation parameters, central composite design method was used to design experiments. The preparation formulas and the engineering parameters were optimized combining with the Creating response-surface design method. According to the experiments which were designed above, the ratio of the Microcrystalline cellulose (MCC) and lactose, and the coating material weight was determined. Bradford method was used to test the protein content to determine the dissolution rate. Finally, the experimental results were put into the SAS software to build dimensional linear fit function, create response surface and contour maps to get the final optimization parameters of preparation and engineering.
The study suggests that the excipent ratio and coating material weight has an obvious effect on the tablet dissolution rate. And compared to the coating material weight, the excipent ratio plays a more important role. The optimized ratio of MCC and lactose is 57:43. To validate the experimental result, the coating material weight increased by 10%, and final result suggests that the experimental values are very close to the predictive ones. And the tablet appearance, hardness, weight variances all conforms to the Chinese Pharmacopieas.In addition, the garlic tablet made with above methods can well mask the pungent smell and be better for administration.
试验使用膜分离法从大蒜中提取分离超氧化歧化酶(superoxide dismutase, SOD)和水溶性多肽,再通过冷冻干燥法对大蒜纳滤浓缩液进行冻干处理得到大蒜粉。用邻苯三酚经典自氧化法测定大蒜粉中SOD的活力,SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)测定冻干粉中活性蛋白的种类及其相对分子质量。
将大蒜冻干粉与不同种类辅料按处方量混合,再通过湿法制粒压片法制成片剂,并用合适浓度的玉竹多糖溶液进行包衣。采用中心组合设计方法安排试验点,结合响应曲面优化法对制剂的处方及工艺进行优化。按照试验设计改变片剂辅料配方中微晶纤维素与乳糖的比例及玉竹多糖包衣液增重,用考马斯亮蓝法(Bradford法)测定蛋白质含量从而测定累积溶出度。实验数据通过SAS软件建立二元线性回归方程,利用绘制出的响应曲面图及等值线图确定优化的处方及工艺。
试验结果表明,辅料配比及包衣液的增重量对包衣片的累积溶出度均有明显影响,且相对于包衣液的增重量,辅料配比对累积溶出度的影响更为明显。优化后的辅料比例为微晶纤维素：乳糖=57：43、包衣液增重为10%,验证试验的数据表明包衣片剂累积溶出度的实测值与预测值十分接近,且片剂外观、硬度、片剂重量差异均符合药典规定。并且试验制得的大蒜包衣片剂能很好的掩盖大蒜辛辣味及刺激性气味,更适合服用。
The active component of garlic lyophilized powder which was obtained using membrane separation method, the excipient ratio in the preparation engineering, effects on the tablet dissolution rate of the coating material weight was studied in this research. And the formula of the lyophilized powder preparation and the engineering parameters were determined, and the quality assessment of the tablet was performed at last.
Superoxide dismutase (SOD) and soluble peptide was separated and extracted from garlic using membrane separation method. Next, garlic lyophilized powder got from the garlic concentrated solution using lyopyilization method. The SOD activity was tested using the classic adjacent pyrogallol since oxidation method. And the kinds and the relative molecular weight of the active protein in the garlic lyophilized powder were tested with SDS-PAGE method.
Mixing garlic lyophilized powder with different kinds of excipents, using Jade bamboo polysaccharide solution as coating material, garlic tablets were made with wet granulating method. In order to get better preparation parameters, central composite design method was used to design experiments. The preparation formulas and the engineering parameters were optimized combining with the Creating response-surface design method. According to the experiments which were designed above, the ratio of the Microcrystalline cellulose (MCC) and lactose, and the coating material weight was determined. Bradford method was used to test the protein content to determine the dissolution rate. Finally, the experimental results were put into the SAS software to build dimensional linear fit function, create response surface and contour maps to get the final optimization parameters of preparation and engineering.
The study suggests that the excipent ratio and coating material weight has an obvious effect on the tablet dissolution rate. And compared to the coating material weight, the excipent ratio plays a more important role. The optimized ratio of MCC and lactose is 57:43. To validate the experimental result, the coating material weight increased by 10%, and final result suggests that the experimental values are very close to the predictive ones. And the tablet appearance, hardness, weight variances all conforms to the Chinese Pharmacopieas.In addition, the garlic tablet made with above methods can well mask the pungent smell and be better for administration.
引文
[1]李时珍.本草纲目(下)[M].北京：华夏出版社,1998：1070-1076
[2]Bianchini F,Vainio H. Allium vegetables and organosulfur compounds:do they help prevent cancer? [J].Environ Health Perspectives,2001,109(9):893-902
[3]Ackermann RT,Mulrow CD,Ramirez G,et al. Garlic shows promise for improving some cardiovascular risk factors [J].Arch Intern Med,2001,161(6):813-824
[4]Harris JC,Cottrell SL,Plurnmer S,et al.Antimicrobial properties of Allium sativum(garlie)[J].Appl Microbiol Biotechnol,2001,57(3):282-286
[5]贾江滨,刘静,谭亚非.大蒜化学成分研究进展[J].广东药学,1999,9(1)：1-5
[6]O'Gala E A,Hill D J,Maslin D J. Activities of garlic oil, garlic powder, and their diallyl constituents against helicobacter pylori[J]. Appl Environ Microbiol,2000, 66(5):2269-2273
[7]Masakatsu H,Hiroko N,Yukio S. Kinetic assay of enzymes with an ammonia gas-sensing electrode:Part 2.In situ monitoring of serum leucine aminopeptidase [J].Analytica Chimica Acta,1983,149:203-208
[8]严常开,曾繁典.大蒜的主要化学成分及其药理作用研究进展[J].中国新药杂志,2004,13(8)：688-691
[9]贾江滨,许重远,罗景慧,等.大蒜中含硫氨基酸研究进展[J].中草药,2000,31(6)：468-470
[10]陈能煜,伍睿,陈丽,等.大蒜研究进展[J].天然产物研究与开发,2000,12(2)：67-74
[11]刘德军.大蒜[M].北京：中国中医药出版社,2001：76-80
[12]Auger J,Yang W, Arnault I,et al.High-performance liquid chromatographic-inductively coupled plasma mass spectrometric evidence for Se-"alliins" in garlic and onion grown in Se-rich soil [J]. Journal of Chromatography A,2004, 1032:103-107
[13]袁勤生.现代酶学[M].上海：华东理工大学出版社,2001：78-80
[14]段永新,傅庭治,傅家瑞.硒在大蒜体内的生物富集及其抗氧化作用[J].园艺学报,1997,24(4)：343-347
[15]Cai XJ,Block E,Uden P,et al. Allium chemistry:identification of selenoamino acids in ordinary and selenium-enriched garlic,onion and broccoli using gas chromatography with atomic emission detection[J].J.Agric Food Chem,1995,43: 1754-1757
[16]Takeshi T,Mizuno T,Komuto M. Isolation of an antitumor compound From Agaricus blazei Murill and its mechanism of action[J].J Nutr,2001,131(5): 1409-1413
[17]孟月生,陆道培.大蒜新素抗人巨细胞病毒作用及其在骨髓移植中的作用[J].中华血液学杂志,1992,13(12)：627-630
[18]Zakaria El Astal. The inhibitory action of aqueous garlic extract on the growth of certain pathogenic bacteria[J]. Eur Food Res Technol,2004,218:460-464
[19]Mousumi Banerjee,Prabir K,Sarkar.Inhibitory effect of garlic on bacterial pathogens from spices[J]. World Journal of Microbiology & Biotechnology,2003, 19:565-569
[20]叶景华.大蒜药用研究现状[J].山东中药学院学报,1996,20(5)：350-352
[21]Sheela C G. Antidiabetic effects of onion and garlic sulfoxide amino acids in rats [J].Planta Med,1995,61(4):356-357
[22]杨丽君,周宏灏.大蒜对药物代谢酶及转运体活性影响研究进展[J].中国临床药理学杂志,2007,23(6)：453-456
[23]张桂梅,冯作华,郝天玲.大蒜素对T细胞激活的影响[J].中药药理与临床,1995,11(1)：26-28
[24]于新蕊.大蒜的化学成分及其药理作用研究进展[J].中草药,1994,25(3)：158-160
[25]McCord J M,Fridovich I. The utility of superoxide dismutase in studying free radical reactions[J]. Biol Chem,1969,245(6):1374-1377
[26]齐继成.超氧化岐化酶(SOD)的开发应用概括[J].中国制药信息,2003,19(11)：15-20
[27]李益新.超氧化岐化酶的结构和功能[J].生物化学与生物物理学进展,1985,7(3)：15-21
[28]Richardson JS,Thomas KA.Rubin BH.,et al.Crystal Structure of Bovine Cu,Zn Superoxide Dismutase at 3A Resolution:Chain Tracing and Metal Ligands [J] Proc Nat Acad Sci USA,1975,72(4):1349-1353
[29]Tainer JA,Getzoff ED,Richardson JS,et al. Structure and mechanism of copper, zinc superoxide dismutase[J] Nature,1983,306 (5940):284-287
[30]Borgstahl GE,Parge HE,Hickey MJ,et al. The structure of human mitochondrial manganese superoxide dismutase reveals a novel tetrameric interface of two 4-helix bundles[J] Cell,1992,71 (1):107-118
[31]Barondeau DP,Kassmann CJ,Bruns CK,et al. Nickel superoxide dismutase structure and mechanism[J]. Biochemistry,2004,43 (25):8038-8047
[32]迟乃玉,张庆芳,刘长江.SOD的化学特性及其应用[J].沈阳农业大学学报,1999,30(2)：171-175
[33]袁勤生.现代酶学[M].上海：华东理工大学出版社,2001,78-80
[34]张瑚,王军志,吴勇杰.超氧化岐化酶药用价值研究的进展[J].药学学报,2003,38(1)：71-74
[35]Harunobu A. Garlic Bioactive Constituents[J]. Nutrition,2007,9(19),715-716
[36]Jack P,Louise C,France G.Red blood cell Cu/Zn superoxide dismutase activity in sporadic amyotrophic lateral sclerosis[J]. Journal of the Neurological Sicences, 1994,127(1):121-123
[37]邓旭,李清彪,孙道华,等.从大蒜细胞中分离纯化出超氧化物歧化酶[J].食品科学,2001,(22)：47-49
[38]徐滢波,赵树进,郭勇.超氧化岐化酶检测方法评述[J].广东药学,2002,12(1)：9-12
[39]许申鸿,杭朗,李运平.超氧化岐化酶邻苯三酚测活法的研究及改进[J].化学通报,2001,8：516-519
[40]国占生.超氧化岐化酶活性的测定方法[J].衡水师专学报,2001,3(2)：39-41
[41]McCord J M,Fridovich I.The utility of superoxide dismutase in studying free radical reaction[J].Biol Chem,1969,244:6049-6055
[42]李海玲,彭书明,李凛,等.4种常用蛋白质浓度测定方法的比较[J].中国生化药物杂志,2008,4：277-282
[43]王孝平,形礼树.考马斯亮蓝法测定蛋白质的研究[J].天津化工,2009,3：40-42
[44]崔福德.药剂学[M].第五版.北京：人民卫生出版社,2004：685-704
[45]徐莲英.中药药剂学[M].北京：人民卫生出版社,1998：235-241
[46]江苏新医学院.中药大辞典[M].上海：上海人民出版社,1977：1156-1162
[47]路洪顺,刘鑫军,刘建敏.玉竹的开发利用价值与栽培技术[J].中国林副特产,2002,3(8)：16-17
[48]彭秧锡,刘士军,郭军,等.玉竹的研究开发现状与展望[J].食品研究与开发,2005,26(6)：120-123
[49]Qin HL,Li ZH,Wang P. A new furostanol glyeoside from Polygonatum Odoratum[J].Chin Chem Le-tt,2003,14(12):1259-1262
[50]李振,秦四海.皂贰的免疫调节作用机理与应用[J].动物保健,2005,9：24-25
[51]刘欣,杨凌,崔星.人参皂贰的活性综述[J].中国微生态学杂志,2005,17(1)：76-79
[52]侯如燕,宛晓春,文汉.茶皂贰的化学结构及生物活性研究进展[J].安徽农业大学学报,2005,32(3)：369-372
[53]胡青平,徐建国,朱志敏,等.山茱英总皂贰的抑菌作用研究[J].食品科学,2006,27(10)：162-164
[54]李会娟,樊均明.人参皂试Rgl的药理作用及机制[J].四川医学,2006,27(10)：1007-1009
[55]张勇慧,阮汉利,皮慧芳,等.湖北贝母生物碱单体的镇咳、祛痰和平喘作用[J].中草药,2005,36(8)：1205-1207
[56]聂珍贵,梁翠茵,高春艳,等.骆驼蓬生物碱平喘作用的研究[J].中国药理通讯,2003,20(3)：4-6
[57]樊晓霞,赵学增.天然生物碱抗肿瘤药理作用研究进展[J].中国现代医药杂志,2006,8(9)：133-135
[58]韩进,郭莹,万海同.中药生物碱类的药理作用及药代动力学研究[J].中医药学刊,2006,24(12)：2326—2328
[59]刘素彦.中药中生物碱类化合物的药代动力学研究进展[J].河北医药,2006,28(2)：124-126
[60]吴伟,催光华.星点设计-效应面优化法及其在药学中的应用[J].国外医学药学分册,2000,27(5)：292-298
[61]Iskandarani B,Clair JH,Patel P,et al.Simultaneous optimization of capsule and tablet fromulation using response surface methology[J].Drug Dev Ind Pharm, 1993,19(16):2089-2101
[62]Abu-lzza KA,Garcia-Contreas L,Lu DR.Preparation and evaluation of sustained release AZT-loaded microspheres:optimization of the release characteristics using response surface methodology[J].J Pharm Sci,1996,85(2):144-149
[63]Mcleod AD,Lam FC,Gupta PK,et al.Optimized synthesis of polyglutaraldehyde nanoparticles using central composite design[J].J Pharm Sci,1988,77(8):704-710
[64]Hassan EE,Parish RC,Gallo JM.Optimized formulation of magnetic chitosan microspheres containing the anticancer agent,oxantrazole[J].Pharm Res,1992, 9(3):390-397
[65]谌威,杨栋梁,刘佳佳,等.膜分离提取大蒜SOD及其水溶性多肽研究[J].化学工程与装备,2008,10：14-18
[66]杨明琰,张晓琦,沈俭,等.超氧化物歧化酶两种邻苯三酚自氧化测定活力方法的比较[J].微生物学杂志,2006,26(3)：40-42
[67]李智,韩静,李成顺.甘草浸膏吸湿性的研究[J].中药材,2007,30(2)：223-225
[68]罗晓健,辛洪亮,饶小勇,等.板蓝根泡腾片干法制粒工艺研究[J].中国中药杂 志,2008,33(12)：1402-1406
[69]Schagger H,Jagow G.Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100kDa[J]. Anal Biochem,1987,166(2):368-379
[70]Kyte J,Rodriguez H. A discontinuous electrophoretic system for separating peptides on polyacrylamide gels [J]. Anal Biochem,1983,133(2):515-522
[71]张晓楠,张延凤,曹云新,等.尿素-SDS-PAGE快速测定多肽的相对分子质量[J].细胞与分子免疫学杂志,2001,17(1)：87-97
[72]Anderson BL,Berry RW,Telser A.A sodium dodecyl sulfate-polyacrylamide gel electrophoresis system that separates peptides and proteins in the molecular weight range of 2500 to 90,000[J]. Anal Biochem,1983,132(2):365-375
[73]郭尧君.蛋白质电泳试验技术[M].第二版.北京：科学出版社,2005：92-118
[74]何忠效.生物化学试验技术[M].北京：化学工业出版社,2004：356-360
[75]回瑞华,侯冬岩,刘晓媛,等.不同产地大蒜抗氧化性能的研究[J].鞍山师范学院学报,2008,10(2)：15-18
[76]陈亚飞,孙宇,高丰衣,等.SDS-聚丙烯酰胺凝胶电泳法测定蛇毒抗瘤蛋白的相对分子质量[J].中国生化药物杂志,2004,25(5)：300-303
[77]中国药典2010年版第二部附录XC溶出度测定法[M].北京：化学工业出版社,2010
[2]Bianchini F,Vainio H. Allium vegetables and organosulfur compounds:do they help prevent cancer? [J].Environ Health Perspectives,2001,109(9):893-902
[3]Ackermann RT,Mulrow CD,Ramirez G,et al. Garlic shows promise for improving some cardiovascular risk factors [J].Arch Intern Med,2001,161(6):813-824
[4]Harris JC,Cottrell SL,Plurnmer S,et al.Antimicrobial properties of Allium sativum(garlie)[J].Appl Microbiol Biotechnol,2001,57(3):282-286
[5]贾江滨,刘静,谭亚非.大蒜化学成分研究进展[J].广东药学,1999,9(1)：1-5
[6]O'Gala E A,Hill D J,Maslin D J. Activities of garlic oil, garlic powder, and their diallyl constituents against helicobacter pylori[J]. Appl Environ Microbiol,2000, 66(5):2269-2273
[7]Masakatsu H,Hiroko N,Yukio S. Kinetic assay of enzymes with an ammonia gas-sensing electrode:Part 2.In situ monitoring of serum leucine aminopeptidase [J].Analytica Chimica Acta,1983,149:203-208
[8]严常开,曾繁典.大蒜的主要化学成分及其药理作用研究进展[J].中国新药杂志,2004,13(8)：688-691
[9]贾江滨,许重远,罗景慧,等.大蒜中含硫氨基酸研究进展[J].中草药,2000,31(6)：468-470
[10]陈能煜,伍睿,陈丽,等.大蒜研究进展[J].天然产物研究与开发,2000,12(2)：67-74
[11]刘德军.大蒜[M].北京：中国中医药出版社,2001：76-80
[12]Auger J,Yang W, Arnault I,et al.High-performance liquid chromatographic-inductively coupled plasma mass spectrometric evidence for Se-"alliins" in garlic and onion grown in Se-rich soil [J]. Journal of Chromatography A,2004, 1032:103-107
[13]袁勤生.现代酶学[M].上海：华东理工大学出版社,2001：78-80
[14]段永新,傅庭治,傅家瑞.硒在大蒜体内的生物富集及其抗氧化作用[J].园艺学报,1997,24(4)：343-347
[15]Cai XJ,Block E,Uden P,et al. Allium chemistry:identification of selenoamino acids in ordinary and selenium-enriched garlic,onion and broccoli using gas chromatography with atomic emission detection[J].J.Agric Food Chem,1995,43: 1754-1757
[16]Takeshi T,Mizuno T,Komuto M. Isolation of an antitumor compound From Agaricus blazei Murill and its mechanism of action[J].J Nutr,2001,131(5): 1409-1413
[17]孟月生,陆道培.大蒜新素抗人巨细胞病毒作用及其在骨髓移植中的作用[J].中华血液学杂志,1992,13(12)：627-630
[18]Zakaria El Astal. The inhibitory action of aqueous garlic extract on the growth of certain pathogenic bacteria[J]. Eur Food Res Technol,2004,218:460-464
[19]Mousumi Banerjee,Prabir K,Sarkar.Inhibitory effect of garlic on bacterial pathogens from spices[J]. World Journal of Microbiology & Biotechnology,2003, 19:565-569
[20]叶景华.大蒜药用研究现状[J].山东中药学院学报,1996,20(5)：350-352
[21]Sheela C G. Antidiabetic effects of onion and garlic sulfoxide amino acids in rats [J].Planta Med,1995,61(4):356-357
[22]杨丽君,周宏灏.大蒜对药物代谢酶及转运体活性影响研究进展[J].中国临床药理学杂志,2007,23(6)：453-456
[23]张桂梅,冯作华,郝天玲.大蒜素对T细胞激活的影响[J].中药药理与临床,1995,11(1)：26-28
[24]于新蕊.大蒜的化学成分及其药理作用研究进展[J].中草药,1994,25(3)：158-160
[25]McCord J M,Fridovich I. The utility of superoxide dismutase in studying free radical reactions[J]. Biol Chem,1969,245(6):1374-1377
[26]齐继成.超氧化岐化酶(SOD)的开发应用概括[J].中国制药信息,2003,19(11)：15-20
[27]李益新.超氧化岐化酶的结构和功能[J].生物化学与生物物理学进展,1985,7(3)：15-21
[28]Richardson JS,Thomas KA.Rubin BH.,et al.Crystal Structure of Bovine Cu,Zn Superoxide Dismutase at 3A Resolution:Chain Tracing and Metal Ligands [J] Proc Nat Acad Sci USA,1975,72(4):1349-1353
[29]Tainer JA,Getzoff ED,Richardson JS,et al. Structure and mechanism of copper, zinc superoxide dismutase[J] Nature,1983,306 (5940):284-287
[30]Borgstahl GE,Parge HE,Hickey MJ,et al. The structure of human mitochondrial manganese superoxide dismutase reveals a novel tetrameric interface of two 4-helix bundles[J] Cell,1992,71 (1):107-118
[31]Barondeau DP,Kassmann CJ,Bruns CK,et al. Nickel superoxide dismutase structure and mechanism[J]. Biochemistry,2004,43 (25):8038-8047
[32]迟乃玉,张庆芳,刘长江.SOD的化学特性及其应用[J].沈阳农业大学学报,1999,30(2)：171-175
[33]袁勤生.现代酶学[M].上海：华东理工大学出版社,2001,78-80
[34]张瑚,王军志,吴勇杰.超氧化岐化酶药用价值研究的进展[J].药学学报,2003,38(1)：71-74
[35]Harunobu A. Garlic Bioactive Constituents[J]. Nutrition,2007,9(19),715-716
[36]Jack P,Louise C,France G.Red blood cell Cu/Zn superoxide dismutase activity in sporadic amyotrophic lateral sclerosis[J]. Journal of the Neurological Sicences, 1994,127(1):121-123
[37]邓旭,李清彪,孙道华,等.从大蒜细胞中分离纯化出超氧化物歧化酶[J].食品科学,2001,(22)：47-49
[38]徐滢波,赵树进,郭勇.超氧化岐化酶检测方法评述[J].广东药学,2002,12(1)：9-12
[39]许申鸿,杭朗,李运平.超氧化岐化酶邻苯三酚测活法的研究及改进[J].化学通报,2001,8：516-519
[40]国占生.超氧化岐化酶活性的测定方法[J].衡水师专学报,2001,3(2)：39-41
[41]McCord J M,Fridovich I.The utility of superoxide dismutase in studying free radical reaction[J].Biol Chem,1969,244:6049-6055
[42]李海玲,彭书明,李凛,等.4种常用蛋白质浓度测定方法的比较[J].中国生化药物杂志,2008,4：277-282
[43]王孝平,形礼树.考马斯亮蓝法测定蛋白质的研究[J].天津化工,2009,3：40-42
[44]崔福德.药剂学[M].第五版.北京：人民卫生出版社,2004：685-704
[45]徐莲英.中药药剂学[M].北京：人民卫生出版社,1998：235-241
[46]江苏新医学院.中药大辞典[M].上海：上海人民出版社,1977：1156-1162
[47]路洪顺,刘鑫军,刘建敏.玉竹的开发利用价值与栽培技术[J].中国林副特产,2002,3(8)：16-17
[48]彭秧锡,刘士军,郭军,等.玉竹的研究开发现状与展望[J].食品研究与开发,2005,26(6)：120-123
[49]Qin HL,Li ZH,Wang P. A new furostanol glyeoside from Polygonatum Odoratum[J].Chin Chem Le-tt,2003,14(12):1259-1262
[50]李振,秦四海.皂贰的免疫调节作用机理与应用[J].动物保健,2005,9：24-25
[51]刘欣,杨凌,崔星.人参皂贰的活性综述[J].中国微生态学杂志,2005,17(1)：76-79
[52]侯如燕,宛晓春,文汉.茶皂贰的化学结构及生物活性研究进展[J].安徽农业大学学报,2005,32(3)：369-372
[53]胡青平,徐建国,朱志敏,等.山茱英总皂贰的抑菌作用研究[J].食品科学,2006,27(10)：162-164
[54]李会娟,樊均明.人参皂试Rgl的药理作用及机制[J].四川医学,2006,27(10)：1007-1009
[55]张勇慧,阮汉利,皮慧芳,等.湖北贝母生物碱单体的镇咳、祛痰和平喘作用[J].中草药,2005,36(8)：1205-1207
[56]聂珍贵,梁翠茵,高春艳,等.骆驼蓬生物碱平喘作用的研究[J].中国药理通讯,2003,20(3)：4-6
[57]樊晓霞,赵学增.天然生物碱抗肿瘤药理作用研究进展[J].中国现代医药杂志,2006,8(9)：133-135
[58]韩进,郭莹,万海同.中药生物碱类的药理作用及药代动力学研究[J].中医药学刊,2006,24(12)：2326—2328
[59]刘素彦.中药中生物碱类化合物的药代动力学研究进展[J].河北医药,2006,28(2)：124-126
[60]吴伟,催光华.星点设计-效应面优化法及其在药学中的应用[J].国外医学药学分册,2000,27(5)：292-298
[61]Iskandarani B,Clair JH,Patel P,et al.Simultaneous optimization of capsule and tablet fromulation using response surface methology[J].Drug Dev Ind Pharm, 1993,19(16):2089-2101
[62]Abu-lzza KA,Garcia-Contreas L,Lu DR.Preparation and evaluation of sustained release AZT-loaded microspheres:optimization of the release characteristics using response surface methodology[J].J Pharm Sci,1996,85(2):144-149
[63]Mcleod AD,Lam FC,Gupta PK,et al.Optimized synthesis of polyglutaraldehyde nanoparticles using central composite design[J].J Pharm Sci,1988,77(8):704-710
[64]Hassan EE,Parish RC,Gallo JM.Optimized formulation of magnetic chitosan microspheres containing the anticancer agent,oxantrazole[J].Pharm Res,1992, 9(3):390-397
[65]谌威,杨栋梁,刘佳佳,等.膜分离提取大蒜SOD及其水溶性多肽研究[J].化学工程与装备,2008,10：14-18
[66]杨明琰,张晓琦,沈俭,等.超氧化物歧化酶两种邻苯三酚自氧化测定活力方法的比较[J].微生物学杂志,2006,26(3)：40-42
[67]李智,韩静,李成顺.甘草浸膏吸湿性的研究[J].中药材,2007,30(2)：223-225
[68]罗晓健,辛洪亮,饶小勇,等.板蓝根泡腾片干法制粒工艺研究[J].中国中药杂 志,2008,33(12)：1402-1406
[69]Schagger H,Jagow G.Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100kDa[J]. Anal Biochem,1987,166(2):368-379
[70]Kyte J,Rodriguez H. A discontinuous electrophoretic system for separating peptides on polyacrylamide gels [J]. Anal Biochem,1983,133(2):515-522
[71]张晓楠,张延凤,曹云新,等.尿素-SDS-PAGE快速测定多肽的相对分子质量[J].细胞与分子免疫学杂志,2001,17(1)：87-97
[72]Anderson BL,Berry RW,Telser A.A sodium dodecyl sulfate-polyacrylamide gel electrophoresis system that separates peptides and proteins in the molecular weight range of 2500 to 90,000[J]. Anal Biochem,1983,132(2):365-375
[73]郭尧君.蛋白质电泳试验技术[M].第二版.北京：科学出版社,2005：92-118
[74]何忠效.生物化学试验技术[M].北京：化学工业出版社,2004：356-360
[75]回瑞华,侯冬岩,刘晓媛,等.不同产地大蒜抗氧化性能的研究[J].鞍山师范学院学报,2008,10(2)：15-18
[76]陈亚飞,孙宇,高丰衣,等.SDS-聚丙烯酰胺凝胶电泳法测定蛇毒抗瘤蛋白的相对分子质量[J].中国生化药物杂志,2004,25(5)：300-303
[77]中国药典2010年版第二部附录XC溶出度测定法[M].北京：化学工业出版社,2010