【作者】 袁高峰；

【导师】 李铎；

【作者基本信息】 浙江大学 ， 食品科学， 2008， 博士

【摘要】 近年来,研究报导共轭亚麻酸(CLNA)具有细胞毒性、抑制癌症形成和改变脂代谢等一系列功能,以及其在体内能转化为共轭亚油酸(CLA),这引起了人们对CLNA研究的极大兴趣。本学位论文对(CLNA)最主要的两种异构体：α-桐酸(c9,t11.t13-CLNA)和石榴酸(c9,t11.c13-CLNA)分析、代谢和功能进行了一系列研究。优化了CLNA甲酯化的程序,以此优化建立的程序对我国天然种质资源进行了分析；证实了CLNA能在大鼠、小鼠和人体内代谢转化为c9.tl1-CLA,在小鼠内α-桐酸的代谢转化率显著高于石榴酸；进一步发现CLNA能降低小鼠肝脏甘油三酯含量,影响小鼠各组织器官脂肪酸组成,显著升高人体8-异前列腺素F2α水平第一部分共轭亚麻酸的分析与资源研究通过对不同的催化方法进行比较,优化了CLNA甲酯化的程序。对不同的含有CLNA的脂类样本通过气相色谱进行定量分析时,应该根据CLNA的化学形式选择合适的甲酯化方法。对自由脂肪酸含量低的脂质甲酯化时直接采用碱性催化的方法(0.5N甲醇钠甲醇溶液55℃反应20min)进行,而对自由脂肪酸含量高的复杂脂质可以采用先皂化裂解(0.3N氢氧化钾90%乙醇溶液37℃皂化裂解2h),再采用酸性催化的方法(1N硫酸甲醇溶液50℃反应10min)进行甲酯化。以此优化建立的气相色谱分析方法,对我国天然种质资源进行分析,结果表明栝楼、石榴和苦瓜籽油等富含CLNA。栝楼籽油CLNA总含量达36.9%,其中最主要的异构体为石榴酸,含量达32.6%。对三个不同品种的石榴籽进行分析,结果表明石榴籽油中石榴酸含量达73.4%-77.5%,CLNA总含量达83.4%-87.9%。对六个不同品种的苦瓜籽进行分析,结果表明苦瓜籽油中主要含有α-桐酸,含量达56.0%-63.7%,CLNA总含量达65.2%-71.9%。以氯仿：甲醇(2∶1)和石油醚两种溶剂系统萃取栝楼籽油,对其脂质组成和脂质各组分脂肪酸组成等进行了分析。结果表明甘油三酯是栝楼籽油的最主要的脂质组分(95.8-98.4%),总脂中最主要的脂肪酸是亚油酸(32.7-32.7%)和石榴酸(32.6%-33.3%)。栝楼籽油甘油三酯组分的脂肪酸组成与总脂非常相似,而磷脂脂肪酸组成与总脂之间存在显著差异,磷脂组分石榴酸含量(2.4-9.4%)显著低于总脂(32.6%-33.3%)。不同提取方法磷脂脂肪酸组成也存在显著的不同。第二部分共轭亚麻酸在动物和人体内的代谢通过以2g含有石榴酸的栝楼籽油给SD大鼠灌胃,调查石榴酸在大鼠血浆和各组织器官中24h内的代谢和整合状况。代谢物通过HPLC、GC-MS鉴定和分析,结果表明石榴酸能整合到大鼠血浆和不同的组织器官中,部分石榴酸在血浆、肝脏、肾脏、脑、心脏和脂肪等组织器官中快速地转化为c9,t11-CLA；肝脏和血浆中石榴酸和CLA的浓度高于脑、心脏、肾脏和脂肪组织。以对照日粮、含有1%α-桐酸和1%石榴酸的日粮饲喂ICR小鼠6周(n=8),比较研究α-桐酸和石榴酸代谢为c9,t11-CLA的代谢转化效率。在α-桐酸和石榴酸饲喂的小鼠各组织器官中,通过GC-MS鉴定得到同一代谢产物c9,t11-CLA。α-桐酸的转化率在所有的小鼠组织器官中均显著高于石榴酸。α-桐酸和石榴酸代谢转化率也存在组织器官间的差异：α-桐酸在脂肪组织、肾脏和脾脏的转化率最大,而在心脏中最低；石榴酸在肝脏的转化率最大,在心脏中的转换率最低。这些结果表明,与大鼠一样在小鼠体内也存在α-桐酸和石榴酸代谢为c9,t11-CLA的△13-双键饱和反应：△13位双键的几何构型可能影响酶的活性,α-桐酸和石榴酸代谢转化率的差异可能是由于△13位双键饱和酶底物特异性选择的结果。采取平行对照实验,招募30个健康青年人(年龄21-35岁,男性24人,女性6人),以葵花籽油作为安慰剂,以我国特殊的天然资源栝楼籽研究石榴酸在人体内的代谢和整合状况。结果表明石榴酸能整合到人体血浆和红细胞膜中,部分石榴酸能在人体内代谢转化为c9,t11-CLA。石榴酸在人体血浆中代谢转化为c9,t11-CLA的效率为34%,在人体红细胞膜中代谢转化为c9,tl1-CLA的效率为28%。据我们所知,这是首个发现CLNA能在人体内整合和代谢为c9,t11-CLA的研究。这表明在大鼠、小鼠和人体内均存在CLNA转化为c9,t11-CLA的△13-双键饱和反应,但具体的代谢机制还需进一步研究。第三部分共轭亚麻酸功能研究以对照日粮,含1%CLA、1%α-桐酸、1%石榴酸和1%α-亚麻酸的日粮饲喂ICR小鼠6周,比较调查CLA、α-桐酸和石榴酸以及α-亚麻酸对动物体脂、血脂和肝脂等功能的影响(n=8)。α-桐酸、石榴酸和α-亚麻酸饲喂,小鼠体重和内脏脂肪量没有显著变化,而CLA饲喂小鼠内脏脂肪量显著降低。CLA、α-桐酸和石榴酸饲喂对小鼠血脂等也没有显著影响,但α-桐酸和石榴酸能显著降低肝脏甘油三酯的含量。这表明,α-桐酸和石榴酸可能对抑制甘油三酯在肝脏内集聚具有积极的作用。以对照日粮,含1%CLA、1%α-桐酸和1%石榴酸的日粮饲喂ICR小鼠6周,比较研究CLNA和CLA对小鼠各组织器官脂肪酸的影响(n=8)。在心脏和脂肪组织中,饲喂CLA、α-桐酸和石榴酸小鼠18:2n-6的含量均显著降低,相应地各组小鼠脂肪组织n-6多不饱和脂肪酸(n-6 PUFA)和PUFA含量显著降低。石榴酸饲喂,肝脏、肾脏和心脏中22:6n-3含量显著升高,相应地石榴酸饲喂肝脏、肾脏和心脏n-3 PUFA含量显著增加,石榴酸可能通过提高22:6n-3和n-3 PUFA含量而间接起有利的生理功能；而CLA饲喂后肝脏和肾脏中22:6n-3含量显著下降。饲喂CLA后,肝脏、肾脏20:4n-6含量显著下降,而饲喂CLNA后,各组织器官20:4n-6含量没有显著变化。这些结果表明虽然α-桐酸和石榴酸结构相似,但它们对小鼠组织器官脂肪酸组成的作用存在不同；CLNA在小鼠体内可能通过代谢物(CLA)或底物状态(CLNA)单独影响组织器官脂肪酸组成。采取平行对照实验,招募30个健康青年人(年龄21-35岁,男性24人,女性6人),以葵花籽油作为安慰剂,研究石榴酸对人体血脂、炎症因子和脂质过氧化等的影响。据我们所知,这是首个调查CLNA对人体功能的研究。结果表明,以含3g/d石榴酸的栝楼籽干预4周,对血脂、载脂蛋白和炎症因子等均没有显著影响,而显著升高尿液8-异前列腺素F2α水平。石榴酸有可能通过本身整合到人体内或通过代谢物c9,t11-CLA而起升高8-异前列腺素F2α水平的作用,但其具体的机制还不清楚。石榴酸诱导脂质过氧化水平升高的机制以及可能产生的影响还需要进一步研究。更多还原

【Abstract】 It was reported that conjugated linolenic acid (CLNA) have a cytotoxic effect on cultured human tumor cells, inhibit carcinogenesis and alter the lipid metabolism in animals. This has led to increased interest in CLNA. This thesis focuses on the main isomers of CLNA, a-eleostearic acid (a-ESA, c9, t11, t13-CLNA) and punicic acid (9c, t11, cl3-CLNA), and consists of three parts:part one investigated the analysis of CLNA; part two investigated the metabolism of CLNA in animals and humans, and part three investigated physiological roles in animals and humans.Part one:Analysis of conjugated linolenic acidDifferent methods for methylating CLNA were compared. The results showed that precise methylation methods should be chosen according to the chemical forms of CLNA. For lipids with negligible content of free fatty acid (FFA). it can be methylated by 0.5 N NaOMe/methanol (55℃,20 min). For lipids with considerable content of FFA, it can be methylated by two-step method:First, the lipids were hydrolyzed into FFA by 0.3 N KOH/90% ethanol (37℃,2 h). Second, the FFA was methylated by 1 N H2SO4/methanol (50℃,10 min).The lipid classes and CLNA content in seed oils of Trichosanthes kirilowii (TK), three pomegranate and six bitter melon cultivars were investigated. The predominant lipid was triacylglycerol (TAG) in the analyzed samples ranging from 92.6 to 97.0%. In Trichosanthes kirilowii seed oil, the major fatty acids were 18:1 (22.6%),18:2n-6 (32.6%) and PA (32.6%), and the total CLNA was 36.9%. In pomegranate cultivars seed oil, punicic acid was the most predominant fatty acid, ranging from 73.4 to 77.5%, and the total CLNA ranged from 83.4 to 87.9%. In bitter melon seed oils, the total CLNA ranged from 65.2 to 71.9%. and a-ESA was the predominant one, accounting for 56.0 to 63.7%. TK, pomegranate and bitter melon were rich in CLNA. However, the main CLNA in TK and pomegranate was PA, whereas in bitter melon it was a-ESA.The TK seed oil was extracted with two solvents, chloroform-methanol (C:M extract) and petroleum ether (PE extract). The oil content of TK seed was 33.3-34.6%. The predominant lipid component was TAG (95.8-98.4%) with smaller amounts of sterol ester, FFA, diacylglycerol, sterol and PL. The major fatty acids were linoleic acid (32.7-32.7%) and PA (32.6%-33.3%) followed by oleic acid (22.6-22.9%) in the total lipids (TL). The TAG fatty acid profile is very similar to TL, whereas there exit differences between PL and TL in fatty acid profile and C:M extract and PE extract in fatty acid profile of PL fraction. The PL fraction (2.4-9.4%) contained much less amounts of PA than TL (32.6%-33.3%).Part two:metabolism of conjugated linolenic acid in animals and humansThe incorporation and metabolism of PA in rat tissues and plasma, one isomer of CLNA was studied by oral administration of TK seed oil, a unique PA-containing material, to rats and which were monitored for 24 h. The metabolite was identified and analyzed by HPLC and GC-MS. The results show that PA was incorporated and metabolized to c9,t11-CLA in rat plasma, liver, kidney, heart, brain and adipose tissue. The level of PA and CLA in liver and plasma was higher than in brain, heart, kidney and adipose tissue, and the lowest accumulation occurred in the brain.The conversion rate into c9,t11-CLA of a-ESA and PA was investigated in the second part of this thesis. Six-week-old male ICR mice (n=8) were fed either a control diet or diets supplemented with 1%α-ESA or PA mixtures in the form of TAG for 6 wk. The results showed thatα-ESA and PA converted into the same metabolite, c9,t11-CLA in mice. The relative conversion rate in all mice tissues in theα-ESA group was higher than in the PA group. There were significant tissue differences in relative conversion rate in both a-ESA and PA groups. The highest conversion rate in the a-ESA group was found in the adipose tissue, kidney and spleen, and the lowest was in heart. However the highest conversion rate in the PA group was the liver and the lowest was in the heart. It is possible that geometrical structure of△13-double bond affected the△13-double saturation reaction activity of enzyme and the difference in conversion rates of a-ESA and PA in vivo was due to substrate-specific effect of this enzyme.The metabolism of PA was investigated in healthy young human subjects. The study was a randomized, placebo-controlled parallel trial. Thirty subjects received TK seeds containing 3g PA in the form of TAG or control for 4 wks. The results showed that PA can be incorporated into the human plasma and red cell membrane. Partial of PA can also be metabolized into c9, t11-CLA. The conversion rate of PA into CLA was 34% in human plasma whereas 28% in red cell membrane. To our knowledge, this is the first study demonstrating the incorporation and metabolism of CLNA in humans. It suggests that the△l3-double bond saturation reaction by which CLNA converted into c9,t11-CLA can also be occurred in humans as in rats and mice. However, further studies are warranted to provide a clearer understanding of this mechanism of CLNAPart three:physiological roles of conjugated linolenic acid in animals and humansThe effects of CLA, a-linolenic acid (ALA), a-ESA and PA on body fat, plasma lipids and liver lipids were investigated in mice. Six-week-old male ICR mice (n=8) were fed either a control diet or one of four experimental diets supplemented with 1% ALA,1% a-ESA,1% PA and 1% CLA mixture, respectively, in the form of TAG for 6 wks. Body weight and adipose tissue was significantly reduced by CLA feeding, whereas those were not modified by ALA, a-ESA or PA. There was no significant effect of CLA, ALA, a-ESA and PA on plasma lipids. However, the TAG content in the liver was significantly reduced by a-ESA and PA feeding. It suggests that there exits differences in the physiological roles among CLNA and CLA although the CLNA can be metabolized into CLA in vivo.The influence of a-ESA and PA on fatty acid profiles in liver, kidney, heart, spleen and adipose tissue was investigated in mice, comparing with CLA. Six-week-old male ICR mice (n=8) were fed either a control diet or diets supplemented with 1% a-ESA, PA or CLA mixtures in the form of TAG for 6 wks. The content of 18:2n-6 was significantly reduced in heart and adipose tissue, correspondingly that of total n-6 PUFA and PUFA were significantly reduced in adipose tissue by CLA and CLNA feeding. The content of 18:2n-6 in kidney was also reduced by PA feeding. The content of 22:6n-3 was significantly increased in liver, kidney and heart by PA feeding, leading the increasing total n-3 PUFA content whereas those were not modified by a-ESA feeding in all tissues examined. In contrast to PA. supplementation of CLA significantly decreased the 22:6n-3 content in liver, heart. Feeding CLA also decreased the level of 20:4n-6 in liver, kidney and heart, whereas CLNA have no effect on that of 20:4n-6 in all tissues examined. CLNA incorporation and conversion to CLA could also exert beneficial activities in different tissues through the effect of the metabolite CLA or CLNA solely.The effect of PA on plasma lipids. inflammatary parameters and lipid peroxidation was investigated in healthy young humans. The study was a randomized, placebo-controlled parallel trial. Thirty subjects received TK seeds containing 3g PA in the form of TAG or control for 4 wks. The results showed that plamsa lipids and inflammatary parameters were not significantly affected, whereas the level of 8-iso-prostaglandinF2a in the urine, a lipid oxidation indicator in vivo was significantly elevated by PA supplemenation. Further studies of the mechanism behind, and the possible consequences of, the increased lipid peroxidation after PA supplementation are needed.更多还原