从羊毛醇中提取胆固醇的工艺研究
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摘要
胆固醇是具有重要生理作用和工业用途的天然物质,有广泛的市场需求。羊毛脂衍生物羊毛醇中含有大量的游离胆固醇,采用适当的方法将胆固醇从羊毛醇中提取出来具有重要的应用价值。为此,本文对萃取-层析-结晶为核心的高含量胆固醇制备工艺进行了系统研究。
针对羊毛醇成分复杂的特点,建立了紫外-示差检测器联用的反相高效液相色谱法对胆固醇和十八醇的含量进行定量分析,建立了气相色谱法对甾醇与脂肪醇等杂质进行定性分析,两种分析方法相结合可以对羊毛醇及分离产物的组成有更全面的了解。
开展了液-液萃取法脱除羊毛醇中极性杂质的研究。以十八醇与胆固醇二元混合物为羊毛醇的模拟研究对象,使用正己烷溶解样品,从多种极性萃取剂中初步筛选出选择性较高的甲醇/水混合溶剂为萃取剂；以羊毛醇为研究对象,通过考察水含量、原料浓度和温度等参数对分配系数和萃取选择性的影响,得到优化的工艺条件：萃取剂为甲醇/水(90：10,v/v),温度35～40℃,原料浓度80～100mg/mL,该条件下总杂质对胆固醇的选择性系数高于3。在优化的工艺条件下进行逆流萃取,可有效脱除羊毛醇中极性较强的杂质,将其酸值由3.16降为0.91,同时胆固醇含量提高5-6%。
对萃取得到的精制羊毛醇进行了层析分离研究,以胆固醇与羊毛甾醇的分离度为指标,筛选出200～300目硅胶为合适的吸附剂,考察了流动相组成、温度和上载量等因素对层析分离效果的影响,得到优化的工艺条件：流动相为丙酮/正己烷(4：96,v/v)混合溶剂,温度35℃,以胆固醇计适宜上载量为2.4%(wt%)。在优化的工艺条件下可得到含量为88.63%的胆固醇粗品,回收率为96.33%。筛选出丙酮/正己烷(50：50,v/v)混合溶剂为有效的再生溶剂,验证了萃取除杂有利于硅胶的再生,经过7次大上样量层析-再生循环使用后硅胶仍能保持其分离能力不变。
最后对溶剂结晶法精制胆固醇工艺进行了研究。考察了溶剂种类、固液比和析晶温度等因素的影响。以无水甲醇为溶剂,固液比为1：20,70℃回流溶解,3h内缓慢冷却至30℃,析晶0.5h,可得到纯度为98.7%,熔程为147.1-147.4℃的胆固醇产品,胆固醇收率为69.4%。
Cholesterol, which is a typical basic molecular in physiology, has been widely applied in various industry fields. The wool wax alcohol, which is one of the derivatives of lanolin, has plenty of cholesterol, so it is significant to isolate the cholesterol. This study was focus on the separation process of cholesterol, including the liquid-liquid extraction, column chromatography and crystallization.
In wool wax alcohol, the components were complex. In order to get comprehensive analysis result, quantitative detection of several components was carried out on HPLC while the qualitative detection of all components was carried out on GC. So the analysis conditions of HPLC and GC were determined at first.
The liquid-liquid extraction was used to remove the highly polar impurities in wool wax alcohol. A series of polar solvent was chosen to separate octadecanol from cholesterol in hexane and the mixture solvent of methanol/H2O was screened out as the proper extractant due to its high selectivity. Then the extraction of impurities in wool wax alcohol was studied. Several influencing factors were studied, such as the ratio of methanol to H2O, temperature and the concentration of wool wax alcohol. The optimized operation conditions were as follows:methanol/H2O (9:1,v/v) was used as extractant, the proper temperature was 35～40℃and the concentration of wool wax alcohol was 80-100mg/mL, then the value of selectivity coefficient was higher than 3. The optimum conditions above were used for countercurrent extraction. The acid value of wool wax alcohol was decreased from 3.16 to 0.91 after extraction and the purity of the cholesterol was evaluated by 5-6%.
Then the extracted wool wax alcohol was purified by column chromatography. According to the resolution of cholesterol and lanosterol, silica was proved to be the proper adsorbent and the process conditions were optimized, including composition of eluent, temperature and the charge of column. The optimum conditions were as follows:the mixture solvent of acetone/hexane (4:96, v/v) was used as eluent, the proper temperature was 35℃and the proper charge of column was 2.4%. After separated by column chromatography under the conditions above, the purity of cholesterol was 88.63% and the recovery was 96.33%. The silica was regenerated by the mixture solvent of acetone/ hexane (50:50, v/v). It was formed that the extraction process ensured complete regeneration. The separation ability of silica gel remained almost unchanged after 7 circulations of usage and regeneration.
The cholesterol was further purified by crystallization. The process conditions were optimized as follows:methanol was used as the crystallizing solvent, ratio of solid to liquid was 1:20, the temperature was 70℃at beginning and cooled to 30℃slowly in 3 hours, and then kept for 30 minutes. The purity of cholesterol was 98.7% at last while the recovery was 69.4%, and the melting range of which was between 147.1-147.4℃.
针对羊毛醇成分复杂的特点,建立了紫外-示差检测器联用的反相高效液相色谱法对胆固醇和十八醇的含量进行定量分析,建立了气相色谱法对甾醇与脂肪醇等杂质进行定性分析,两种分析方法相结合可以对羊毛醇及分离产物的组成有更全面的了解。
开展了液-液萃取法脱除羊毛醇中极性杂质的研究。以十八醇与胆固醇二元混合物为羊毛醇的模拟研究对象,使用正己烷溶解样品,从多种极性萃取剂中初步筛选出选择性较高的甲醇/水混合溶剂为萃取剂；以羊毛醇为研究对象,通过考察水含量、原料浓度和温度等参数对分配系数和萃取选择性的影响,得到优化的工艺条件：萃取剂为甲醇/水(90：10,v/v),温度35～40℃,原料浓度80～100mg/mL,该条件下总杂质对胆固醇的选择性系数高于3。在优化的工艺条件下进行逆流萃取,可有效脱除羊毛醇中极性较强的杂质,将其酸值由3.16降为0.91,同时胆固醇含量提高5-6%。
对萃取得到的精制羊毛醇进行了层析分离研究,以胆固醇与羊毛甾醇的分离度为指标,筛选出200～300目硅胶为合适的吸附剂,考察了流动相组成、温度和上载量等因素对层析分离效果的影响,得到优化的工艺条件：流动相为丙酮/正己烷(4：96,v/v)混合溶剂,温度35℃,以胆固醇计适宜上载量为2.4%(wt%)。在优化的工艺条件下可得到含量为88.63%的胆固醇粗品,回收率为96.33%。筛选出丙酮/正己烷(50：50,v/v)混合溶剂为有效的再生溶剂,验证了萃取除杂有利于硅胶的再生,经过7次大上样量层析-再生循环使用后硅胶仍能保持其分离能力不变。
最后对溶剂结晶法精制胆固醇工艺进行了研究。考察了溶剂种类、固液比和析晶温度等因素的影响。以无水甲醇为溶剂,固液比为1：20,70℃回流溶解,3h内缓慢冷却至30℃,析晶0.5h,可得到纯度为98.7%,熔程为147.1-147.4℃的胆固醇产品,胆固醇收率为69.4%。
Cholesterol, which is a typical basic molecular in physiology, has been widely applied in various industry fields. The wool wax alcohol, which is one of the derivatives of lanolin, has plenty of cholesterol, so it is significant to isolate the cholesterol. This study was focus on the separation process of cholesterol, including the liquid-liquid extraction, column chromatography and crystallization.
In wool wax alcohol, the components were complex. In order to get comprehensive analysis result, quantitative detection of several components was carried out on HPLC while the qualitative detection of all components was carried out on GC. So the analysis conditions of HPLC and GC were determined at first.
The liquid-liquid extraction was used to remove the highly polar impurities in wool wax alcohol. A series of polar solvent was chosen to separate octadecanol from cholesterol in hexane and the mixture solvent of methanol/H2O was screened out as the proper extractant due to its high selectivity. Then the extraction of impurities in wool wax alcohol was studied. Several influencing factors were studied, such as the ratio of methanol to H2O, temperature and the concentration of wool wax alcohol. The optimized operation conditions were as follows:methanol/H2O (9:1,v/v) was used as extractant, the proper temperature was 35～40℃and the concentration of wool wax alcohol was 80-100mg/mL, then the value of selectivity coefficient was higher than 3. The optimum conditions above were used for countercurrent extraction. The acid value of wool wax alcohol was decreased from 3.16 to 0.91 after extraction and the purity of the cholesterol was evaluated by 5-6%.
Then the extracted wool wax alcohol was purified by column chromatography. According to the resolution of cholesterol and lanosterol, silica was proved to be the proper adsorbent and the process conditions were optimized, including composition of eluent, temperature and the charge of column. The optimum conditions were as follows:the mixture solvent of acetone/hexane (4:96, v/v) was used as eluent, the proper temperature was 35℃and the proper charge of column was 2.4%. After separated by column chromatography under the conditions above, the purity of cholesterol was 88.63% and the recovery was 96.33%. The silica was regenerated by the mixture solvent of acetone/ hexane (50:50, v/v). It was formed that the extraction process ensured complete regeneration. The separation ability of silica gel remained almost unchanged after 7 circulations of usage and regeneration.
The cholesterol was further purified by crystallization. The process conditions were optimized as follows:methanol was used as the crystallizing solvent, ratio of solid to liquid was 1:20, the temperature was 70℃at beginning and cooled to 30℃slowly in 3 hours, and then kept for 30 minutes. The purity of cholesterol was 98.7% at last while the recovery was 69.4%, and the melting range of which was between 147.1-147.4℃.
引文
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[2]Lower E S. Cholesterol in cosmetic formulation-Review[J]. Drug & Cosmetic Industry.1975,116: 54-57.
[3]陈玉昆.甾族天然药物的提取及生产工艺[M].北京：科学出版社,2009.
[4]谭仁祥.甾体化学[M].北京：化学工业出版社,2009.
[5]Cashin M F, Moravek V. The physiological action of chloesterol[J]. American Journal of Physiology.1927,82(2):294-298.
[6]Bandsma R, Stellaard F, Vonk R J, et al. Contribution of newly synthesized cholesterol to rat plasma and bile determined by mass isotopomer distribution analysis:bile-salt flux promotes secretion of newly synthesized cholesterol into bile[J]. Biochemical Journal.1998,329(Part 3): 699-703.
[7]Cuixart C B, Sanchez R G, Garcia J M, et al. Total cholesterol and HDL cholesterol distribution in Spanish children and adolescents:Ricardin Study[J]. Medicina Clinica.2000,115(17):644-649.
[8]谭兰兰.羊毛脂皂化及其胆甾醇提取的工艺研究[D].成都：四川大学,2002.
[9]Urata K, Takaishi N. Cholesterol as synthetic building blocks for artificial lipids with characteristic physical, chemical and biological properties [J]. European Journal of Lopid Science and Technology.2001,103(1):29-39.
[10]Druml W, Fischer M. Cholesterol improves the utilization of parenteral lipid emulsions[J]. Wiener Klinische Wochenschrift.2003,115(21-22):767-774.
[11]徐仁生.天然产物化学[M].北京：科学出版社,1997.
[12]Capponi A M. Regulation of cholesterol supply for mineralocorticoid biosynthesis[J]. Trends in Endocrinology and Metabolism.2002,13(3):118-121.
[13]Opitz J M, Gilbert-Barness E, Ackerman J, et al. Cholesterol and development:The RSH ("Smith-Lemli-Opitz") syndrome and related conditions[J]. Pediatric Pathology and Molecular Medicine.2002,21(2):153-181.
[14]林吉文.甾体化学基础[M].北京：化学工业出版社,1989.
[15]周学良.精细化工产品手册：生物化学品[M].北京：化学工业出版社,2002.
[16]Milewski S. Health-promoting properties of sheep products[J]. Medycyna Weterynaryjna.2006, 62(5):516-519.
[17]王贵珍.羊毛脂皂化产物分离及其胆甾醇提取的工艺研究[D].成都：四川大学,2004.
[18]徐生庚,裘爱泳,译.贝雷：油脂化学与工艺学(第五版)[M].北京：中国轻工业出版社,2001.
[19]Kligman A M. Lanolin allergy-crisis or comedy[J]. Contact Dermatitis.1983,9(2):99-107.
[20]Nguyen J C, Chesnut G, James W D, et al. Allergic contact dermatitis caused by lanolin (wool) alcohol contained in an emollient in three postsurgical patients[J]. Journal of the American Academy of Dermatology.2010,62(6):1064-1065.
[21]Reiterreimers M, Baltes W. The analysisi of wool wax in cosmetics[J]. Zeitschrift Fur Lebensmittel-Untersuchung Und-Forschung.1986,183(3):186-190.
[22]Motiuk K. Wool wax alcohols:A review[J]. Journal of American Oil Chemists'Society.1978,56: 658-951.
[23]Lamparczyk H, Miszkiel M. Gas-chromatographic evaluation of wool wax alcohols supported by pricipal component analysis[J]. Chromatographia.1991,31(5-6):243-246.
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