化妆品天然乳化剂的研究
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摘要
本课题主要完成了大豆磷脂的分离提纯、酶水解改性及其改性产物性能测定以及使用大豆磷脂制备维生素C脂质体的研究。
采用浓缩大豆磷脂为原料,探索了以乙醇、乙酸乙酯、异丙醇作为溶剂以及使用无机盐沉淀方法提取大豆磷脂主要乳化成分磷脂酰胆碱的工艺条件。最终确定以乙醇作为提取溶剂,提取温度为45(±1)℃,料液比为1:4,提取次数为6次,磷脂酰胆碱在提取物中的百分含量达到45%以上,提取率在80%以上。
使用提纯后的产物进行酶水解改性的研究,其最优条件是:反应温度为38(±1)℃、反应时间为7h、酶的用量为100IU/mL、钙离子的浓度为0.025M/L、缓冲液的pH值为8.50、底物浓度为10.0%。在最佳水解反应条件下所测得的酸价为55.03mgKOH/g,水解率在87.6%-94.0%。
经硅胶柱层析分离可以得到不同极性的三类磷脂,担体与待分离物的比例为100:1。采用梯度洗脱方式进行分离,洗脱剂的条件是氯仿与甲醇的比例由1:1改变至1:2到1:4,直至用甲醇洗脱。分别得到未知组分磷脂、卵磷脂以及溶血磷脂的浓缩液。未知组分磷脂的高效液相色谱峰面积经归一化计算后达到96.32%,溶血磷脂的含量达到了67.52%。
采用R.K.Gupta法测定溶血磷脂的HLB值为9,与水解前的磷脂相比提高50%,乳化稳定性与市售非离子乳化剂乳化效果相似。
采用薄膜分散—超声乳化法制备,以包封率为考察指标,经正交优化选择,确定脂质体的最佳配方:卵磷脂/胆固醇/维生素E的摩尔比为4∶2∶0.34;维生素C的浓度为3.3mg/mL;PBS缓冲液的pH值为7.93;成膜时所用氯仿的量为25mL,此时包封率可达58.32%。用扫描电子显微镜观察脂质体的结构,平均粒径为310nm。经脂质体包裹以后,维生素C的稳定性增强。
The extracted phosphatidylcholine from soybean phospholipds and modified phosphatidylcholine with phospholipaseA2 .Tested modified products emulsifying properties and reseached on the preparation of vitminC liposome made by soybean phospholipids.
Phosphatidylcholine was extracted from soybean phospholipds and alcohol、ethyl actate、isopropanol as optimal sovent and inorganic salt precipitate method respectuvely. The optimal conditions of the extract were: alchohol as extract solvent and its valume was 4 times to the soybean phospholipds weight at 45(±1)℃for 6 times. The phosphatidylcholine concentration could reach above 45% and the yield could reach above 80%.
Phosphatidylcholine was modified by phospholipaseA2. The optimal conditions: the temperature was 38(±1)℃,the time of the hydrolysis was 7h, the concentration of PLA2 was 100IU/mL, the concentration of calcium was 0.025mol/mL,the value of tris-HCl buffers wais 8.50. The acidity of the hydrolysis in this situation was 55.03,the rate of hydrolysis was 87.6%-94.0%.
Gain the value of Rf of each ingredient by TLC, the Rf value of each ingredient was 0.90,0.56,0.32. Separated the products of the hydrolysis by silica gel columchromatography and used gradient elution for separation. Changed the proportion of chloroform / methanol 1∶1 to 1∶2, and then to 1∶4 ,untill only used the methanol as eluant.The content of lysophospholipid gained 67.52% .
Lysophospholipids HLB value sas tested by R.K.Gupta method was 9 and improved 50% than lecithin. The emulsifying stability of lysophospholipids was nearly the same as other emulsifier ,like AEO.
To optimize the preparation formulation of vitamin C liposomes. The liposomes were prepared by film dispersion-ultrasonic emulsify method. Taking entrapment efficiency as index, and designing an orthogonal experiment. The optimal formulation were: phosphatidylcholine / cholesterin / vitamin E (molar ratio)4:2:0.34; vitamin C concentration :3.3mg/mL;PBS buffer pH value:7.93;chloroform for film forming :25 mL. Under the formulation the entrapment efficiency was 58.32%. The structure of liposome was observed under the scan electronic microscope. The main diameter of the liposome particles was 310 nm. The stability of vitaminC was improved after entraped by phosphatidylcholine.
采用浓缩大豆磷脂为原料,探索了以乙醇、乙酸乙酯、异丙醇作为溶剂以及使用无机盐沉淀方法提取大豆磷脂主要乳化成分磷脂酰胆碱的工艺条件。最终确定以乙醇作为提取溶剂,提取温度为45(±1)℃,料液比为1:4,提取次数为6次,磷脂酰胆碱在提取物中的百分含量达到45%以上,提取率在80%以上。
使用提纯后的产物进行酶水解改性的研究,其最优条件是:反应温度为38(±1)℃、反应时间为7h、酶的用量为100IU/mL、钙离子的浓度为0.025M/L、缓冲液的pH值为8.50、底物浓度为10.0%。在最佳水解反应条件下所测得的酸价为55.03mgKOH/g,水解率在87.6%-94.0%。
经硅胶柱层析分离可以得到不同极性的三类磷脂,担体与待分离物的比例为100:1。采用梯度洗脱方式进行分离,洗脱剂的条件是氯仿与甲醇的比例由1:1改变至1:2到1:4,直至用甲醇洗脱。分别得到未知组分磷脂、卵磷脂以及溶血磷脂的浓缩液。未知组分磷脂的高效液相色谱峰面积经归一化计算后达到96.32%,溶血磷脂的含量达到了67.52%。
采用R.K.Gupta法测定溶血磷脂的HLB值为9,与水解前的磷脂相比提高50%,乳化稳定性与市售非离子乳化剂乳化效果相似。
采用薄膜分散—超声乳化法制备,以包封率为考察指标,经正交优化选择,确定脂质体的最佳配方:卵磷脂/胆固醇/维生素E的摩尔比为4∶2∶0.34;维生素C的浓度为3.3mg/mL;PBS缓冲液的pH值为7.93;成膜时所用氯仿的量为25mL,此时包封率可达58.32%。用扫描电子显微镜观察脂质体的结构,平均粒径为310nm。经脂质体包裹以后,维生素C的稳定性增强。
The extracted phosphatidylcholine from soybean phospholipds and modified phosphatidylcholine with phospholipaseA2 .Tested modified products emulsifying properties and reseached on the preparation of vitminC liposome made by soybean phospholipids.
Phosphatidylcholine was extracted from soybean phospholipds and alcohol、ethyl actate、isopropanol as optimal sovent and inorganic salt precipitate method respectuvely. The optimal conditions of the extract were: alchohol as extract solvent and its valume was 4 times to the soybean phospholipds weight at 45(±1)℃for 6 times. The phosphatidylcholine concentration could reach above 45% and the yield could reach above 80%.
Phosphatidylcholine was modified by phospholipaseA2. The optimal conditions: the temperature was 38(±1)℃,the time of the hydrolysis was 7h, the concentration of PLA2 was 100IU/mL, the concentration of calcium was 0.025mol/mL,the value of tris-HCl buffers wais 8.50. The acidity of the hydrolysis in this situation was 55.03,the rate of hydrolysis was 87.6%-94.0%.
Gain the value of Rf of each ingredient by TLC, the Rf value of each ingredient was 0.90,0.56,0.32. Separated the products of the hydrolysis by silica gel columchromatography and used gradient elution for separation. Changed the proportion of chloroform / methanol 1∶1 to 1∶2, and then to 1∶4 ,untill only used the methanol as eluant.The content of lysophospholipid gained 67.52% .
Lysophospholipids HLB value sas tested by R.K.Gupta method was 9 and improved 50% than lecithin. The emulsifying stability of lysophospholipids was nearly the same as other emulsifier ,like AEO.
To optimize the preparation formulation of vitamin C liposomes. The liposomes were prepared by film dispersion-ultrasonic emulsify method. Taking entrapment efficiency as index, and designing an orthogonal experiment. The optimal formulation were: phosphatidylcholine / cholesterin / vitamin E (molar ratio)4:2:0.34; vitamin C concentration :3.3mg/mL;PBS buffer pH value:7.93;chloroform for film forming :25 mL. Under the formulation the entrapment efficiency was 58.32%. The structure of liposome was observed under the scan electronic microscope. The main diameter of the liposome particles was 310 nm. The stability of vitaminC was improved after entraped by phosphatidylcholine.
引文
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[4] Yuri Wedmid,Craig Evans,Wolfgang J.Baumann. Synthesis of Cyclic Glycerol Acetal Phosphates: Proton and Carbon-13 NMr Characteristics of Isomeric 1,3-Dioxolane Phosphate Structures[J]. J.Org.Chem.,1980,45:1582-1586.
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[20] Ricardo Madoery,Clelia G.Gattone,Gerardo Fidelio. Bioconversion of phospholipids by immobilized phospholipase A2[J].Journal of Biotechnology ,1995,40:145-152.
[21] Carmen Virto,Patrick Adlercreutz. Lysophosphatidylcholine synthsis with Candida Antarctica lipase B(Novozym435)[J].Enzyme and Microbial Technology,2000,26:630-635.
[22] Josh P.Kupferberg,Shinji Yokoyama,Ferenc J.Kezdy. The Kinetics of the Phospholipase A2–catalyzed Hydrolysis of Egg Phosphatidylcholine in Unilamellar Vesicles[J]. The Journal of Biological Chemistry,1981,256(12):6274-6281.
[23]H.Kunze,E.Bohn.Separation andanalysis of amino alcohol-containing diacylglycero- -phspholipid and their hydrolytic[J]. Journal of Chromatography,1993,636:221-229.
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[25]吕洛,魏少敏,等.脂质体载药系统经皮给药的研究进展[J] .日用化学工业,2002,32(5) :51-54
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[2]赵国华,陈宗道.磷脂改性研究(J).粮食与油脂,1999,3:8-12.
[3]陈石良,许正宏,孙微,谷文英,陶文忻.磷脂酶D的研究进展(J).工业微生物,1999,29(12):47-50.
[4] Yuri Wedmid,Craig Evans,Wolfgang J.Baumann. Synthesis of Cyclic Glycerol Acetal Phosphates: Proton and Carbon-13 NMr Characteristics of Isomeric 1,3-Dioxolane Phosphate Structures[J]. J.Org.Chem.,1980,45:1582-1586.
[5]龚盛昭.天然活性化妆品的概况和发展前景(J).香精香料化妆品,2002,2:16-19.
[6]柏薇薇,卢晓泉.化妆品绿色原料--大豆(J).牙膏工业,2001,2:44-45.
[7]崔小明.大豆磷脂的开发与应用前景(J).甘肃化工,2001,1:10-12,43.
[8]兰云军,谷雪贤,银德海,罗卫平.磷脂的化学改性(J).西部皮革,2003,8: 31-36.
[9]刘琳琳,张辉,强西怀.大豆磷脂的开发与利用(J).陕西科技大学学报:自然科学版,2004,4:50-54.
[10]夏海涛,刘玉芬.大豆磷脂中磷脂酰胆碱的脂肪酸组成分析(J).分析化学,2003,1:126-126
[11]富校铁,孙树坤,朱秀清.磷脂酶A2水解浓缩大豆磷脂最佳技术条件的研究(J).大豆通报.2004,3:16-17.
[12]汪勇,王兴国,欧仕益,唐书泽,付亮,李爱军.磷脂酶A2水解大豆浓缩磷脂的研究(II)贫水相的水解及产品性能的测定[J].中国粮油学报.2003,1(5):49-52.
[13]姜宝奎.乙酰化大豆改性磷脂的制取工艺[J].粮食与食品工业.2005,1:26-27.
[14]李桂华,赵斌.羟基化改性磷脂的研制[J].郑州工程学院学报.2002,2:69-71.
[15]姜涌明,戴祝英,陈俊刚,胡健.分子酶学导论(M).北京:中国农业大学出版社,2000:12.
[16]梁歧,王利,陶红.大豆磷脂酶水解技术研究(J).中国油脂,2003,28(7):58-60.
[17] Evelyn L.Ridgley,Larry Ruben. Phospholipase from Trypanosoma Brucei releases arachidonic acid by sequential sn-1,sn-2 deacylation of phospholipids[J].Molecular & Biochemistry Parasitology, 2001,114:29-40.
[18] YESAIR;David,W.;et al. Methods for Making Lysophospholatidylcholine (P). WO : 97/28270 ,1997–08–07.
[19] Elyssa D.Bent,John D.Bell.Quatification of the interactions among fatty acid, lysophosphatidycholine, calcium, dimyristalphosphatidycholine vescles, and phospholipase A2(J). Biochimica et Biophysica Acta,1995,1254:349-360.
[20] Ricardo Madoery,Clelia G.Gattone,Gerardo Fidelio. Bioconversion of phospholipids by immobilized phospholipase A2[J].Journal of Biotechnology ,1995,40:145-152.
[21] Carmen Virto,Patrick Adlercreutz. Lysophosphatidylcholine synthsis with Candida Antarctica lipase B(Novozym435)[J].Enzyme and Microbial Technology,2000,26:630-635.
[22] Josh P.Kupferberg,Shinji Yokoyama,Ferenc J.Kezdy. The Kinetics of the Phospholipase A2–catalyzed Hydrolysis of Egg Phosphatidylcholine in Unilamellar Vesicles[J]. The Journal of Biological Chemistry,1981,256(12):6274-6281.
[23]H.Kunze,E.Bohn.Separation andanalysis of amino alcohol-containing diacylglycero- -phspholipid and their hydrolytic[J]. Journal of Chromatography,1993,636:221-229.
[24]周家春.溶血磷脂的开发应用(J).粮食与油脂,2002,3:33-34.
[25]吕洛,魏少敏,等.脂质体载药系统经皮给药的研究进展[J] .日用化学工业,2002,32(5) :51-54
[26]穆筱梅.脂质体在化妆品中的应用[J].广东化工,2006, 33(2):20-21.
[27]张根旺,刘晓见.脂质体化妆品及其应用[J] .郑州工程学院院报,2000,21(4):1-4.
[28] A. Go′mez-Hens, J.M. Ferna′ndez-Romero. Analytical methods for the control of liposomal delivery systems[J]. Trends in Analytical Chemistry,2006,25(2): 167-178.
[29]汪多仁.卵磷脂脂质体开发与应用进展[J] .化工中间体,2003,18:23-26.
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