从大豆卵磷脂中分离磷脂酰胆碱和脑苷脂的研究
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摘要
磷脂酰胆碱(PC)具有重要生理功能,高纯度磷脂酰胆碱现已被广泛用于营养、食品、医药、化妆品等领域,市场需求较大。脑苷脂(Ce)类化合物具有多样结构和较强生物活性。随着抗肿瘤、抗病毒、免疫调节等活性的发现,可望开发成为防治慢性疾病的新药。近年来国外对脑苷脂的提取纯化工艺、检测方法、生物活性等方面的研究有较大进展,但是我国对脑苷脂类化合物的研究较少,因此加强此方面的研究是必要的。主要结论如下：
(1)用无水乙醇搅拌提取磷脂酰胆碱的提取率和纯度都高于95%7,醇搅拌提取和无水乙醇摇床提取。使PC提取率达58.2%,得率36.3%,纯度达44.1%。
(2)应用50nm的无机陶瓷膜过滤粉末磷脂的乙醇提取液,可得到纯度55.6%的磷脂酰胆碱,PC得率66.1%。
(3)使用硅胶柱层析分离纯化磷脂酰胆碱,在梯度洗脱的情况下,比较了异丙醇甲醇和氯仿甲醇两种流动相对分离纯化磷脂酰胆碱的影响。应用氯仿甲醇洗脱体系,可得到的PC 97%以上,PE 96%以上,PC得率86.5%的产品。应用异丙醇甲醇体系,可得到PC 95%以上,得率83.0%的产品。
(4)用石油醚萃取大豆卵磷脂中的中性脂质,获得粗脑苷脂类物质。通过单因素试验和正交试验,确定了石油醚萃取分离粗脑苷脂类物质的最佳条件为：料液比1：1、提取温度50℃,提取时间30min,在此条件下得到6.13%的粗脑苷脂。
(5)经质谱检测纯化的组分中主要有两种分子类型的神经酰胺(Cer),分别是C18：0d18:1Cer和C25:0h d18:1Cer;主要有两种分子类型的脑苷脂,分别是C16:0h t18:1Ce和C17：0Ce。
(6)通过HPLC-ELSD分析,二次柱层析得到的神经酰胺含量99.43%回收率为98.23%,脑苷脂含量为98.86%,回收率为97.27%。
Phosphatidylcholine(PC) has many important physiological functions, being widely used in the filed of nutrition, food, medicine, cosmetics and so on. Cerebroside(Ce) was desirabled biological activity and diverse structure. Ce was developing to a new drug for prevention of chronic diseases with the discovery of activity such as the anti-tumor, anti-virus and immune regulation. Recently year, they were has great progress in extraction and purification, detection and biological activities of Ce in abroad. It was necessary to improve the research progress of Ce in China because we were research less on cerebroside.
(1) Enrichment PC by ethanol extract from soy powder lecithin. Compare with the influence of alcohol content and extraction methods for the extraction rate of PC. The results showed that the extraction rate of PC to 58.2% by stirring extract of ethanol.
(2) Applied inorganic ceramic membrane of 50nm ultrafiltration of ethanol extract of powder lecithin obtained purity of PC 55.6%, yield of PC 66.1%.
(3) In the gradient elution conditions, compared with isopropyl/alcohol and chloroform/ methanol system by silica gel column chromatography. Used chloroform/methanol elution can obtained purity of PC more than 97.0%, isopropanol/methanol elution can obtained purity of PC more than 95.0% by the silica gel column chromatography. The purity and recovery of PC haven't apparent discrimination that contrast two elution system.
(4) Cerebrosides were separated from neutral lipids in soybean lecithin by petroleum ether extraction to obtain the crude cerebrosides. The optimal conditions for extraction obtained by the single factor experiments and the orthogonal experiments were listed as follows:ratio of solvent to stuff of 1:1, extraction temperature of 50℃and extraction time of 30 min. Under these conditions, the highest yield of the crude cerebrosides was 6.73%.
(5) Ce and Cer by EIS/MS analysis that there were two molecular types of Cer(C18:0 d18:1Cer rand C25:0h d18:1Cer) and two molecular types of Ce(C16:0 t18:2Cerand C17:0Ce).
(6) Detected by HPLC-ELSD were known the sample 3 was ceramide (Cer), the content of Ce to 99.43% and retention time 18.20min, the sample 4 was Ce, retention time 25.13min and the content of Ce to 98.86%.
(1)用无水乙醇搅拌提取磷脂酰胆碱的提取率和纯度都高于95%7,醇搅拌提取和无水乙醇摇床提取。使PC提取率达58.2%,得率36.3%,纯度达44.1%。
(2)应用50nm的无机陶瓷膜过滤粉末磷脂的乙醇提取液,可得到纯度55.6%的磷脂酰胆碱,PC得率66.1%。
(3)使用硅胶柱层析分离纯化磷脂酰胆碱,在梯度洗脱的情况下,比较了异丙醇甲醇和氯仿甲醇两种流动相对分离纯化磷脂酰胆碱的影响。应用氯仿甲醇洗脱体系,可得到的PC 97%以上,PE 96%以上,PC得率86.5%的产品。应用异丙醇甲醇体系,可得到PC 95%以上,得率83.0%的产品。
(4)用石油醚萃取大豆卵磷脂中的中性脂质,获得粗脑苷脂类物质。通过单因素试验和正交试验,确定了石油醚萃取分离粗脑苷脂类物质的最佳条件为：料液比1：1、提取温度50℃,提取时间30min,在此条件下得到6.13%的粗脑苷脂。
(5)经质谱检测纯化的组分中主要有两种分子类型的神经酰胺(Cer),分别是C18：0d18:1Cer和C25:0h d18:1Cer;主要有两种分子类型的脑苷脂,分别是C16:0h t18:1Ce和C17：0Ce。
(6)通过HPLC-ELSD分析,二次柱层析得到的神经酰胺含量99.43%回收率为98.23%,脑苷脂含量为98.86%,回收率为97.27%。
Phosphatidylcholine(PC) has many important physiological functions, being widely used in the filed of nutrition, food, medicine, cosmetics and so on. Cerebroside(Ce) was desirabled biological activity and diverse structure. Ce was developing to a new drug for prevention of chronic diseases with the discovery of activity such as the anti-tumor, anti-virus and immune regulation. Recently year, they were has great progress in extraction and purification, detection and biological activities of Ce in abroad. It was necessary to improve the research progress of Ce in China because we were research less on cerebroside.
(1) Enrichment PC by ethanol extract from soy powder lecithin. Compare with the influence of alcohol content and extraction methods for the extraction rate of PC. The results showed that the extraction rate of PC to 58.2% by stirring extract of ethanol.
(2) Applied inorganic ceramic membrane of 50nm ultrafiltration of ethanol extract of powder lecithin obtained purity of PC 55.6%, yield of PC 66.1%.
(3) In the gradient elution conditions, compared with isopropyl/alcohol and chloroform/ methanol system by silica gel column chromatography. Used chloroform/methanol elution can obtained purity of PC more than 97.0%, isopropanol/methanol elution can obtained purity of PC more than 95.0% by the silica gel column chromatography. The purity and recovery of PC haven't apparent discrimination that contrast two elution system.
(4) Cerebrosides were separated from neutral lipids in soybean lecithin by petroleum ether extraction to obtain the crude cerebrosides. The optimal conditions for extraction obtained by the single factor experiments and the orthogonal experiments were listed as follows:ratio of solvent to stuff of 1:1, extraction temperature of 50℃and extraction time of 30 min. Under these conditions, the highest yield of the crude cerebrosides was 6.73%.
(5) Ce and Cer by EIS/MS analysis that there were two molecular types of Cer(C18:0 d18:1Cer rand C25:0h d18:1Cer) and two molecular types of Ce(C16:0 t18:2Cerand C17:0Ce).
(6) Detected by HPLC-ELSD were known the sample 3 was ceramide (Cer), the content of Ce to 99.43% and retention time 18.20min, the sample 4 was Ce, retention time 25.13min and the content of Ce to 98.86%.
引文
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