CN115073295A - 马齿苋中不饱和脂肪酸及其提取分离方法和应用 - Google Patents
马齿苋中不饱和脂肪酸及其提取分离方法和应用 Download PDFInfo
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- CN115073295A CN115073295A CN202210860526.1A CN202210860526A CN115073295A CN 115073295 A CN115073295 A CN 115073295A CN 202210860526 A CN202210860526 A CN 202210860526A CN 115073295 A CN115073295 A CN 115073295A
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- unsaturated fatty
- fatty acid
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- column chromatography
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Abstract
本发明属于医药领域,提供了马齿苋中不饱和脂肪酸及其提取分离方法和应用,包括:一种新的不饱和脂肪酸11‑羰基‑十八碳‑(7E,9E)‑二烯酸甲酯和一种新的天然产物13‑羰基‑十八碳‑(9E,11E)‑二烯酸甲酯的化学结构。从马齿苋中提取分离不饱和脂肪酸单体及其组合物的新方法包括:通过60%乙醇超声提取、聚酰胺柱色谱、乙酸乙酯超声萃取、正相硅胶柱色谱、半制备HPLC分离纯化工艺,成功分离并鉴定出新的不饱和脂肪酸酯、以及亚麻酸、亚油酸、油酸等不饱和脂肪酸酯衍生物。本发明公开上述新型不饱和脂肪酸及已知化合物的抗炎活性,本发明中的不饱和脂肪酸单体及其组合物可应用于炎症相关药物、食品、保健品和化妆品等产品的开发。
Description
技术领域
本发明属于医药领域,具体涉及从药食两用马齿苋中分离鉴定新型不饱和脂肪酸、不饱和脂肪酸单体和组合物的提取分离方法及其抗炎活性的应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解,而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
马齿苋(Portulaca oleracea L.)为马齿苋科马齿苋属一年生肉质草本植物,为药食两用植物,又名长寿菜,是亚洲、非洲、欧洲、南美洲、北美洲等诸多地区的传统药物。《中国药典》记载马齿苋的地上部分药用,其性寒味酸,具有止痢、清热解毒、止血凉血等功效。现代医学研究表明马齿苋具有抗炎、抑菌、抗氧化、抗衰老、降血压、降血糖、降血脂、抗动脉粥样硬化、抗肿瘤、增强免疫、保肝、抗哮喘等诸多药理活性。马齿苋富含生物碱、有机酸、多糖、黄酮、木脂素、萜类等多种类型的化学成分。
GC-MS检测分析表明马齿苋是ω-3不饱和脂肪酸的丰富植物来源,其中ω-3不饱和脂肪酸亚麻酸的含量是菠菜的5倍;不同产地马齿苋药材中α亚麻酸和亚油酸总量为0.12-0.29%。ω-3不饱和脂肪酸(如亚麻酸)及ω-6不饱和脂肪酸(如亚油酸)是人体不能通过自身合成的,但对人体的生长发育、特别是预防各种心血管疾病和维持健康的免疫系统至关重要,具有抗炎、抗氧化、降血脂、降血压、软化血管、促进微循环、增强免疫力等诸多药理活性。不饱和脂肪酸作为马齿苋发挥药理功能和营养潜力的重要成分,其提取分离及其应用在食品工业、膳食补充剂、药物、化妆品和身体护理等产品开发中具有广阔前景。
已有学者报道了从马齿苋提取不饱和脂肪酸粗提取物的多种方法。如徐德钦等利用正己烷在60℃超声提取4h(正交实验法优化马齿苋中α-亚麻酸的提取工艺参数研究.食品工业科技,2010,31(2):215-217)、超临界CO2流体萃取(萃取压力10-50MPa,萃取温度30-60℃)获得含有α-亚麻酸的马齿苋粗提取物(徐德钦.一种采用超临界CO2流体萃取马齿苋亚麻酸的方法.中国发明专利CN102219669A,2010)。刘长君分别采用水蒸气蒸馏法、索氏提取法(提取溶剂包括环己烷、乙醚和石油醚,温度分别为80℃、45℃和60℃,提取时间1.5h)、以及超声提取法从马齿苋中提取不饱和脂肪油,通过GC-MS分析比较,发现石油醚超声提取法的提取效果最好(马齿苋中脂肪酸提取工艺比较.现代农业科技,2012,(14):281-289)。敬思群等将马齿苋籽采用超声辅助酶解预处理(55℃超声15分钟),再用石油醚进行索氏提取,GC-MS检测发现该方法制备的马齿苋籽油含有8种不饱和脂肪酸,其中亚麻酸含量高达40%,其次为亚油酸29.43%和油酸15.61%(超声辅助酶解-索氏提取联用法提取马齿苋籽油及其脂肪酸组成分析.食品与发酵工业,2014,40(7):218-222)。敬思群等将马齿苋全草(根茎叶)经亚临界异丁烷萃取(萃取压力0.8M Pa,萃取温度40℃,萃取时间150min)获得全草油浸膏,再经脱蜡获得含有亚油酸和亚麻酸的马齿苋全草油(马齿苋全草油的亚临界流体萃取、脂肪酸分析及抗氧化活性.食品与发酵工业,2015,41(9):203-208)。敬思群等进一步挖掘了马齿苋水煎煮后的剩余残渣,利用亚临界异丁烷萃取(萃取压力0.8MPa,萃取温度40℃,萃取150min)残渣获得马齿苋油粗浸膏,再利用皂化水解、尿素包合法富集获得多不饱和脂肪酸的粗提取物(一种从马齿苋渣制备马齿苋多不饱和脂肪酸的方法.中国发明专利CN104059774A,2014;尿素包合法富集马齿苋全草油多不饱和脂肪酸及脂肪酸分析.粮食与油脂,2015,28(2):33-36;皂化水解及尿素包合工艺对马齿苋油脂肪酸组成及抗氧化性影响.食品科技,2015,40(2):82-86)。李冠文等将新鲜马齿苋经石油醚索氏提取后所得的脂肪油,通过尿素包合方法富集纯化获得多不饱和脂肪酸粗提取物(尿素包合法富集纯化马齿苋中多不饱和脂肪酸的工艺优化.食品安全质量检测学报,2021,12(15):6186-6191)。
上述文献或专利多是对马齿苋中不饱和脂肪酸粗提物进行制备,并利用GC-MS或HPLC-MS对粗提取物中亚麻酸等不饱和脂肪酸的含量进行分析,尚未对粗提取物进行深入的分离和结构鉴定。不饱和脂肪酸在空气中不稳定,在高温条件下容易发生氧化,如何从马齿苋中分离纯化不饱和脂肪酸单体或制备纯度较高的组合物,其大规模制备方法及其应用有待深入挖掘。
发明内容
为了解决上述问题,本发明提供一种新的不饱和脂肪酸单体11-羰基-十八碳-(7E,9E)-二烯酸甲酯和一种新的天然产物13-羰基-十八碳-(9E,11E)-二烯酸甲酯的化学结构,同时提供一种从马齿苋药材中提取分离该不饱和脂肪酸单体和组合物方法,该方法具有操作简单、纯度较高、经济环保、适合大规模制备。本发明还提供了结构新颖不饱和脂肪酸单体和已知的不饱和脂肪酸在抗炎方面的应用。
为了实现上述目的,本发明采用如下技术方案:
本发明的第一个方面,提供了一种不饱和脂肪酸单体,所述不饱和脂肪酸单体为11-羰基-十八碳-(7E,9E)-二烯酸甲酯,分子式为C19H32O3,其化学结构如下:
本发明的第二个方面,提供了一种天然产物,所述天然产物为13-羰基-十八碳-(9E,11E)-二烯酸甲酯,分子式为C19H32O3,其化学结构如下:
本发明的第三个方面,提供了一种不饱和脂肪酸组合物,包括:上述的不饱和脂肪酸单体和/或上述的天然产物。
本发明的第四个方面,提供了一种不饱和脂肪酸的分离纯化方法,包括:
将马齿苋进行醇提,取提取液浓缩,过滤,收集滤液;
将所述滤液采用聚酰胺柱色谱洗脱,收集洗脱流分Fr.1-4;
将所述流分Fr.2用乙酸乙酯萃取,收集萃取液浓缩成浸膏;
将所述浸膏采用正相硅胶柱洗脱,收集多个流分;
将流分Fr.(9-15)过正相硅胶柱色谱洗脱,得到多个流分;
将流分Fr.(9-15)-(1-17)过正相硅胶柱色谱洗脱,得到多个流分;
将Fr.(9-15)-(1-17)-(12-14)用半制备型HPLC进行分离,得到亚麻酸甲酯、亚麻酸乙酯、亚油酸甲酯、亚油酸乙酯;
将流分Fr.(9-15)-(48-63)用半制备型HPLC进行分离,得到13-羰基-十八碳-(9E,11E)-二烯酸甲酯和11-羰基-十八碳-(7E,9E)-二烯酸甲酯。
现有文献所述方法多是对马齿苋不饱和脂肪酸粗提取物进行制备,对马齿苋不饱和脂肪酸单体的分离制备方法,特别是本发明所提供的新型不饱和脂肪酸的分离纯化方法及其抗炎活性尚未有报导。
本发明的第五个方面,提供了上述的不饱和脂肪酸单体、上述的天然产物、含上述不饱和脂肪酸、及其盐或衍生物的组合物中任意一项在制备抗炎药物中的应用。
本发明的有益效果
本发明提供的不饱和脂肪酸的提取分离纯化方法是依次采用60%乙醇超声提取、聚酰胺柱色谱、乙酸乙酯超声萃取、正相硅胶柱色谱、半制备HPLC进行分离纯化。该方法操作简便、成本较低、有机溶剂可回收使用、可大规模制备,得到的化合物纯度大于90%。上述不饱和脂肪酸单体或其组合物可以用于炎症相关药物、食品、保健品和化妆品的开发。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。
图1.本发明新不饱和脂肪酸的高分辨质谱图。
图2.本发明新不饱和脂肪酸的1H NMR谱图。
图3.本发明新不饱和脂肪酸的13C NMR谱图。
图4.本发明新不饱和脂肪酸的HSQC谱图。
图5.本发明新不饱和脂肪酸的HMBC谱图。
图6.本发明新不饱和脂肪酸的1H-1H COSY谱图。
图7.马齿苋中不饱和脂肪酸的抗炎活性(n=3)(注:与Control相比,####p<0.0001;与LPS相比,****p<0.0001,***p<0.001,**p<0.01,*p<0.05)。
具体实施方式
应该指出,以下详细说明都是例示性的,旨在对本发明提供进一步的说明。除非另有指明,本发明使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
一种新的不饱和脂肪酸11-羰基-十八碳-(7E,9E)-二烯酸甲酯[methyl(7Z,9E)-11-oxooctadeca-7,9-dienoate],分子式为C19H32O3,其化学结构如下所示:
一种新天然产物13-羰基-十八碳-(9E,11E)-二烯酸甲酯[methyl(9Z,11E)-13-oxooctadeca-9,11-dienoate],分子式为C19H32O3,其化学结构如下:
在一些实施例中,本发明所述不饱和脂肪酸组合物包括上述不饱和脂肪酸和已知不饱和脂肪酸如亚麻酸甲酯(methyl linolenate)、亚麻酸乙酯(ethyl linolenate)、亚油酸甲酯(methyl linoleate)、亚油酸乙酯(ethyl linoleate)或其衍生物形成的组合物,或其与任意来源不饱和脂肪酸形成的组合物。已知不饱和脂肪酸的化学结构式如下:
其次,本发明提供了上述不饱和脂肪酸单体和组合物的分离纯化方法。
在一些实施例中,所述方法分7步进行。在步骤1中将马齿苋药材用6倍量60%乙醇超声提取2次,每次30min。将提取液浓缩至浓度为0.5g生药/mL,提取液置于4℃冰箱静置,过滤,获得滤液。
在步骤2中将上述所得滤液上样于聚酰胺柱色谱,依次用水-乙醇(1:0→4:6→2:8→0.5:9.5)梯度洗脱,洗脱液浓缩,合并为4个流分Fr.1-4。
在步骤3中将上述所得流分Fr.2用乙酸乙酯进行萃取。萃取液减压浓缩成浸膏(不 饱和脂肪酸组合物I)。
在步骤4中将上述浸膏进一步过正相硅胶柱,依次用石油醚-乙酸乙酯(1:0→9:1→8:2→7:3→6:4→1:1→3:7→0.5:9.5)洗脱,得到20个流分。
在步骤5中,将上述所得流分Fr.(9-15)(不饱和脂肪酸组合物II)过正相硅胶柱色谱,用石油醚-乙酸乙酯(15:1)等度洗脱,得到10个流分。
在步骤6中,将步骤5所得流分Fr.(9-15)-(1-17)过正相硅胶柱色谱,用正己烷-乙酸乙酯(25:1→5:1→0:1)梯度洗脱,得到19个流分。其中Fr.(9-15)-(1-17)-(12-14)用半制备型HPLC进行分离,甲醇:水(95:5)为流动相,流速1.5mL/min,得到亚麻酸甲酯(tR1=33.2min)、亚麻酸乙酯(tR2=37.5min)、亚油酸甲酯(tR3=41.6min)、亚油酸乙酯(tR4=47.4min)。
在步骤7中,将步骤5中所得流分Fr.(9-15)-(48-63)用半制备型HPLC进行分离,甲醇:水(85:15)为流动相,流速1.5mL/min,得到13-羰基-十八碳-(9E,11E)-二烯酸甲酯(tR1=35.0min)和11-羰基-十八碳-(7E,9E)-二烯酸甲酯(tR2=36.9min)。
再次,本发明提供了上述不饱和脂肪酸的抗炎活性。
在一些实施例中,上述不饱和脂肪酸适用于马齿苋不饱和脂肪酸单体及其组合物,或含有马齿苋不饱和脂肪酸的其它混合物。
下面结合具体的实施例,对本发明做进一步的详细说明,应该指出,所述具体实施例是对本发明的解释而不是限定。
实施例1新颖不饱和脂肪酸的结构鉴定
所述新不饱和脂肪酸命名为11-羰基-十八碳-(7E,9E)-二烯酸甲酯[methyl(7Z,9E)-11-oxooctadeca-7,9-dienoate],结构如下,表1为该化合物(1)的1H NMR(600MHz,CDCl3)谱和13C NMR(150MHz,CDCl3)谱数据。
表1新不饱和脂肪酸1的1H NMR和13C NMR数据归属
化合物1为黄色油状固体,易溶于二氯甲烷、乙酸乙酯、甲醇。在GF254正相硅胶薄层板上以石油醚-乙酸乙酯(15:1)为展开剂时Rf=0.22,UV 254nm下有暗斑,碘熏显黄色。
Positive-HR-ESI-MS显示(图1)m/z 309.2427为[M+H]+峰(元素组成C19H33O3,计算值309.2424),m/z 331.2243为[M+Na]+峰(元素组成C19H32NaO3,计算值331.2249),不饱和度为4。1H NMR(600MHz,CDCl3)谱(图2)结合HSQC谱(图4)显示,δ6.07(1H,d,J=15.6Hz),7.13(1H,ddd,J=15.6,10.2,2.4Hz)为一对反式烯烃氢信号,δ6.18(1H,dm,J=15.0,6.6Hz),6.17(1H,dm,J=15.0,7.8Hz)为另一对反式烯氢信号;δ3.66(3H,s,-OCH3)为甲氧基信号;δ2.53(2H,t,J=7.2Hz),2.30(2H,t,J=7.2Hz),2.17(2H,dd,J=12.6,7.2Hz),1.61(4H,m),1.44(2H,m),1.31(10H,m)为11个亚甲基信号,δ0.89(3H,t,J=7.2Hz)为一个甲基信号。1H-1H COSY谱(图6)显示烯氢δ7.13与δ6.07和δ6.18相关,烯氢δ6.18又与δ2.17相关,δ2.17与δ1.44相关、δ1.44与δ1.31相关、δ1.31与δ1.61相关、δ1.61与δ2.30(2H,t,J=7.2Hz)相关,说明化合物存在一个-CH=CH-CH=CH-(CH2)n-片段;此外,δ2.53(2H,t,J=7.2Hz)与δ1.61相关、δ1.61与δ1.31相关、δ1.31与δ0.89(3H,t,J=7.2Hz)相关,说明该化合物还存在一个-(CH2)n-CH3片段。
13C NMR(150MHz,CDCl3)谱(图3)结合HSQC谱(图4)显示19个碳信号,其中δ201.2和δ174.4为羰基碳信号;δ145.9,143.1,129.0,128.0为烯碳信号;δ51.6(-OCH3)为甲氧基信号;δ40.6,34.2,33.3,31.5,29.3,29.2,29.1,28.5,25.0,24.5,22.6为11个亚甲基碳信号,δ14.1为甲基碳信号。
HMBC谱(图5)显示烯氢H-9与C-7,C-8,C-11远程相关,烯氢H-10与C-8,C-11,C-12远程相关,说明-CH=CH-CH=CH-片段一端与羰基相连;H-12与C-11,C-13,C-14远程相关,H-13与C-11,C-12,C-14,C-15远程相关,H-14与C-12远程相关,H-15与C-17远程相关,H-16与C-14,C-15远程相关,H-18与C-16,C-17远程相关,说明羰基的另一端连接-(CH2)6-CH3片段。烯氢H-7与C-6,C-9远程相关,H-8与C-10远程相关,H-6与C-5,C-7,C-8远程相关,H-5与C-4,C-6,C-7远程相关,H-4与C-2远程相关,H-3与C-1,C-2,C-4远程相关,H-2与C-1,C-3,C-4远程相关,-OCH3与C-1相关,说明-CH=CH-CH=CH-片段的另一端与-(CH2)5-COOCH3相连。综上所述,确定化合物1为11-羰基-十八碳-(7E,9E)-二烯酸甲酯。
本发明所述一种不饱和脂肪酸新天然产物命名为13-羰基-十八碳-(9E,11E)-二烯酸甲酯[methyl(9Z,11E)-13-oxooctadeca-9,11-dienoate](Gas chromatographicstudy on high-temperature thermal degradation products of methyl linoleatehydroperoxides.Journal of the American Oil Chemists Society,1997,74(4):387-391),该化合物的分子式为C19H32O3,结构如下,为首次从马齿苋中分离得到。
实施例2不饱和脂肪酸单体化合物的分离纯化
步骤1.将4.5kg的马齿苋药材用6倍量60%乙醇在室温下超声提取2次,每次30min。将提取液浓缩至9L(浓度为0.5g生药/mL),提取液置于4℃冰箱静置,过滤。
步骤2.将步骤1中的6L滤液上样于聚酰胺柱色谱(2.4kg,100-200mesh,10×71cm×2),依次用水-乙醇(1:0→4:6→2:8→0.5:9.5)洗脱,洗脱液浓缩,合并为4个流分Fr.1-4。
步骤3.将步骤2中Fr.2(15.3g)浸膏研磨成粉末,用乙酸乙酯进行超声萃取,共4次。提取液抽滤,合并滤液,减压浓缩成浸膏(为不饱和脂肪酸组合物I)。
步骤4.将步骤3中的浸膏进一步过正相硅胶柱(420g,200-300mesh,6×46cm),依次用石油醚-乙酸乙酯(1:0→9:1→8:2→7:3→6:4→1:1→3:7→0.5:9.5)洗脱,得到多个流分,依次进行编号,待用。
步骤5.将步骤4中编号为9-15的洗脱流分Fr.(9-15)(577mg,为不饱和脂肪酸组合 物II)过正相硅胶柱(86g,200-300mesh,2×59cm),用石油醚-乙酸乙酯(15:1)洗脱,得到多个流分,依次进行编号,待用。
步骤6.将步骤5中编号为1-17的洗脱流分Fr.(9-15)-(1-17)(300mg)过正相硅胶柱(60.6g,200-300mesh,50×2cm),依次用正己烷-乙酸乙酯(25:1→5:1→0:1)梯度洗脱,得到多个流分,依次进行编号,待用。其中,将步骤6中编号为12-14的洗脱流份Fr.(9-15)-(1-17)-(12-14)(143.9mg)用半制备型HPLC进行分离,甲醇:水(95:5)为流动相,流速1.5mL/min,得到亚麻酸甲酯(8.2mg,tR1=33.2min)、亚麻酸乙酯(4.4mg,tR2=37.5min)、亚油酸甲酯(16.9mg,tR3=41.6min)、亚油酸乙酯(4.4mg,tR4=47.4min)。
步骤7.将步骤5中编号为48-63的洗脱流分Fr.(9-15)-(48-63)(15.8mg)用半制备型HPLC进行分离,甲醇:水(85:15)为流动相,流速1.5mL/min,得到13-羰基-十八碳-(9E,11E)-二烯酸甲酯(1.7mg,tR1=35.0min)和11-羰基-十八碳-(7E,9E)-二烯酸甲酯(1.6mg,tR2=36.9min)。
实施例3不饱和脂肪酸单体化合物的抗炎作用:
(1)主要材料
实验所用药品为上述分离步骤制备所得,纯度大于90%,精密称定后用DMSO稀释至待测浓度。RAW 264.7小鼠巨噬细胞(美国ATCC公司);DMSO、LPS(美国Sigma-Alidrich公司);对氨基苯磺酰胺和萘乙二胺(国药集团化学试剂有限公司);3,4-二羟基-苯甲羟肟酸(美国MedChem Express公司);胎牛血清(美国Gemini Bioproduct公司);DMEM培养基(美国Gibco公司)。
(2)实验方法及步骤
细胞培养:将RAW 264.7小鼠巨噬细胞培养于包含10%FBS、0.29mg/L L-谷氨酰胺、100U/mL青霉素、100mg/mL链霉素的DMEM完全培养基中,将培养基放置在温度37℃、含5%CO2的培养箱中培养。
Griess试剂的配制:精密称取0.025g的萘乙二胺和0.25g的对氨基苯磺酰胺,加入5%H3PO4溶解,定容至25mL,摇匀即得。
NO生成抑制实验:在96孔板中接种RAW 264.7小鼠巨噬细胞(8.0×104cells/well),待细胞贴壁后,吸去原培养基。在空白组加200μL DMEM培养基;在模型组加200μL含LPS(1μg/mL)的DMEM培养基;在实验组加含待测样品浓度为3.125、6.25、12.5、25、50、100μM和LPS(1μg/mL)的200μL DMEM培养基;阳性对照组加入含100μM 3,4-二羟基-苯甲羟肟酸(Didox)和LPS(1μg/mL)的200μL DMEM培养基。孵育24h,每孔取100μL上清液加入另一个96孔板中,各加入100μL的Griess试剂,振摇15分钟,在570nm波长下测定溶液吸光度,根据NaNO2标准曲线计算NO生成抑制率,实验重复3次。
NaNO2标准曲线:在96孔板中加入100μL的NaNO2标准溶液,浓度依次为1.5625、3.125、6.25、12.5、25、50μM和100μL的Griess试剂,在570nm波长处测定溶液吸光度值,并绘制NaNO2浓度-吸光度的标准曲线。
MTT实验:在上述96孔板中,每孔加入20μL浓度为2mg/mL的MTT溶液,培养3小时后吸去上清液,每孔加入100μL二甲基亚砜(DMSO)使沉淀溶解,在570nm下测定吸光度值。细胞存活率(%)=实验组吸光度/空白组吸光度×100%,实验重复3次。
(3)实验结果
实验结果表明从马齿苋中分离得到的不饱和脂肪酸(包括新化合物、新天然产物,以及已知不饱和脂肪酸酯)均可剂量依赖性地显著降低脂多糖诱导的RAW264.7小鼠巨噬细胞中一氧化氮的产生(见图7),EC50值在7.80~40.85μM之间。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
3.一种不饱和脂肪酸组合物,其特征在于,包括:权利要求1所述的不饱和脂肪酸单体和/或权利要求2所述的天然产物。
4.如权利要求3所述的不饱和脂肪酸组合物,其特征在于,还包括:亚麻酸甲酯、亚麻酸乙酯、亚油酸甲酯、亚油酸乙酯及其衍生物中的至少一种。
5.如权利要求3所述的不饱和脂肪酸组合物,其特征在于,还包括:其他活性成分或辅料。
6.一种不饱和脂肪酸的分离纯化方法,其特征在于,包括:
将马齿苋进行醇提,取提取液浓缩,过滤,收集滤液;
将所述滤液采用聚酰胺柱色谱洗脱,收集洗脱流分Fr.1-4;
将所述流分Fr.2用乙酸乙酯萃取,收集萃取液浓缩成浸膏;
将所述浸膏采用正相硅胶柱洗脱,收集多个流分;
将流分Fr.9-15过正相硅胶柱色谱洗脱,得到多个流分;
将流分Fr.9-15-1-17过正相硅胶柱色谱洗脱,得到多个流分;
将Fr.9-15-1-17-12-14用半制备型HPLC进行分离,得到亚麻酸甲酯、亚麻酸乙酯、亚油酸甲酯、亚油酸乙酯;
将流分Fr.9-15-48-63用半制备型HPLC进行分离,得到13-羰基-十八碳-(9E,11E)-二烯酸甲酯和11-羰基-十八碳-(7E,9E)-二烯酸甲酯。
7.如权利要求6所述的不饱和脂肪酸的分离纯化方法,其特征在于,醇提的具体条件为:6~8倍量60~70%乙醇超声提取2~3次,每次30~40min。
8.如权利要求6所述的不饱和脂肪酸的分离纯化方法,其特征在于,所述聚酰胺柱色谱洗脱的具体条件为依次用水-乙醇1:0→4:6→2:8→0.5:9.5进行梯度洗脱;
或,浸膏采用正相硅胶柱色谱洗脱的具体条件为:依次用石油醚-乙酸乙酯1:0→9:1→8:2→7:3→6:4→1:1→3:7→0.5:9.5洗脱;
或,流分Fr.9-15过正相硅胶柱色谱洗脱的具体条件为:用石油醚-乙酸乙酯15~16:1等度洗脱;
或,流分Fr.9-15-1-17正相硅胶柱色谱洗脱的具体条件为用正己烷-乙酸乙酯25:1→5:1→0:1梯度洗脱。
9.如权利要求6所述的不饱和脂肪酸的分离纯化方法,其特征在于,流分Fr.9-15-1-17-12-14用半制备型HPLC进行分离的条件为:甲醇:水95:5为流动相,流速1.5~2mL/min;
流分Fr.9-15-48-63用半制备型HPLC进行分离的条件为:甲醇:水85:15为流动相,流速1.5~2mL/min。
10.权利要求1所述的不饱和脂肪酸单体、权利要求2所述的天然产物、权利要求3-5所述的不饱和脂肪酸组合物中任意一项在制备抗炎药物中的应用。
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