本文主要研究内容
作者张瑜（2019）在《DHA和2-DHA-单甘酯的制备纯化及调节HepG2细胞脂质代谢的比较研究》一文中研究指出：二十二碳六烯酸(Docosahexaenoic acid,DHA)是一种n-3多不饱和脂肪酸,它在维护人体营养健康和预防治疗某些疾病方面发挥重要作用。目前,市场上高浓度DHA多为乙酯型,相比于乙酯型,游离型和甘油酯型DHA更易吸收。此外,即使同为甘油酯型,DHA在甘油骨架上的位置不同对其生理活性亦有重要的影响。根据甘三酯在体内的代谢特点,DHA位置分布不同的甘三酯会形成DHA和2-单甘酯型DHA(2-DHAMAG)。本论文制备了高浓度DHA和2-DHA-MAG,并对2-DHA-MAG酰基转移规律进行了研究,同时以DHA和2-DHA-MAG代表DHA位置分布不同的甘三酯的代谢产物,比较其对HepG2细胞脂质代谢的影响。主要研究内容如下:首先,以金枪鱼油水解产物脂肪酸为原料,通过低温溶剂结晶和尿素包合分别富集DHA。结果表明,以乙腈作为低温结晶溶剂,可显著缩短结晶所需时间,在脂肪酸与乙腈比为1:10(w/v)、结晶温度-60°C、结晶时间1 h的条件下,使DHA的含量从26.85%提高至56.31%,回收率为60%左右;采用尿素包合法对DHA富集时,在脂肪酸:尿素:溶剂为1:3:12(w/w/v)、包合温度-8°C、包合时间10 h的条件下,DHA含量从26.85%提高至77.21%,回收率为50%左右。而在相同条件下,可使裂殖壶菌藻油水解产物脂肪酸中DHA含量从48.20%分别提升至79.82%和83.12%。然后,通过柱直径为5 cm的制备型高效液相色谱将尿素包合后的DHA脂肪酸浓缩物进一步纯化,使DHA含量提高至95.23%,回收率为49.33%。其次,采用酶法醇解金枪鱼油和裂殖壶菌藻油制备富含DHA的2-MAG,探究脂肪酶种类、底物摩尔比、反应时间、反应温度和加酶量的影响。结果表明,在油脂与无水乙醇摩尔比为1:60、反应时间2 h、反应温度30°C、Lipozyme 435添加量为8%的条件下,金枪鱼油中2-MAG含量在22.10%~27.40%、藻油中2-MAG含量在26.50%~30.95%。经正己烷和85%乙醇-水萃取后,金枪鱼油和藻油2-MAG的含量提高至94%以上,回收率在70%左右,而DHA含量分别达到53.22%和74.76%。然后通过2-MAG与乙腈之比1:10(w/v)、-40°C下结晶10 h,使金枪鱼油2-MAG中DHA含量提高至67.63%,2-MAG含量保持在90%以上;通过分子蒸馏使金枪鱼油2-MAG中DHA含量提高至59.95%,但2-MAG含量下降至69.81%,说明在蒸馏过程中2-MAG发生了酰基转移。再次,研究了2-MAG在不同温度(25°C、40°C、50°C)和不同溶剂(正己烷、乙醇、叔丁醇、丙酮、乙腈、二氯甲烷)中的酰基转移规律,结果表明,2-MAG酰基转移符合一级可逆反应模型。在无溶剂体系中,温度提高25°C,酰基转移程度增加5.6倍;不同溶剂对酰基转移影响不同,酰基转移速率常数的大小为正己烷>无溶剂体系>二氯甲烷>乙醇≈丙酮≈乙腈>叔丁醇,与所选溶剂的log P值呈正相关(40°C R=0.932、50°C R=0.939,p<0.01)。酰基转移后2-MAG和1-MAG组分中脂肪酸分布结果表明,40°C下,乙醇、丙酮、乙腈和叔丁烷体系中酰基转移程度较小,DHA在2-MAG中的含量高于其在1-MAG中的含量,证明DHA酰基转移速率相对较慢;50°C相同体系中,DHA在两组分中分布差异较小,可能由于温度升高,其酰基转移速率提高。最后,探讨了DHA和2-DHA-MAG对HepG2细胞脂质代谢的影响。结果表明,浓度低于50μmol/L的DHA和2-DHA-MAG均对细胞无毒害作用,均能有效地被HepG2细胞吸收,两者对细胞中DHA含量的提高无显著性差异;相对于DHA,2-DHA-MAG更有效地降低油酸诱导的脂肪变性肝细胞中总甘油三酯和总胆固醇的含量,可使细胞内甘三酯含量多下降25.21%,胆固醇含量多下降32.64%,这是因为2-DHA-MAG更有效地促进脂肪氧化关键基因PPARα和CPT-1的表达、抑制胆固醇合成关键基因HMGCR的表达。综上所述,本论文建立了高浓度DHA的富集方法,将DHA含量提高至95.23%;建立了2-单甘酯型DHA的合成和纯化方法,制备了2-MAG含量94%以上、DHA含量为74%左右的产品,并发现温度和溶剂对其酰基转移具有重要影响。通过细胞实验证实相对于DHA,2-DHA-MAG具有更显著地降低肝细胞总甘三酯和总胆固醇水平的功效,表明sn-2位DHA能更好地调节脂质代谢,为开发新剂型DHA提供了理论支持。
Abstract
er shi er tan liu xi suan (Docosahexaenoic acid,DHA)shi yi chong n-3duo bu bao he zhi fang suan ,ta zai wei hu ren ti ying yang jian kang he yu fang zhi liao mou xie ji bing fang mian fa hui chong yao zuo yong 。mu qian ,shi chang shang gao nong du DHAduo wei yi zhi xing ,xiang bi yu yi zhi xing ,you li xing he gan you zhi xing DHAgeng yi xi shou 。ci wai ,ji shi tong wei gan you zhi xing ,DHAzai gan you gu jia shang de wei zhi bu tong dui ji sheng li huo xing yi you chong yao de ying xiang 。gen ju gan san zhi zai ti nei de dai xie te dian ,DHAwei zhi fen bu bu tong de gan san zhi hui xing cheng DHAhe 2-chan gan zhi xing DHA(2-DHAMAG)。ben lun wen zhi bei le gao nong du DHAhe 2-DHA-MAG,bing dui 2-DHA-MAGxian ji zhuai yi gui lv jin hang le yan jiu ,tong shi yi DHAhe 2-DHA-MAGdai biao DHAwei zhi fen bu bu tong de gan san zhi de dai xie chan wu ,bi jiao ji dui HepG2xi bao zhi zhi dai xie de ying xiang 。zhu yao yan jiu nei rong ru xia :shou xian ,yi jin qiang yu you shui jie chan wu zhi fang suan wei yuan liao ,tong guo di wen rong ji jie jing he niao su bao ge fen bie fu ji DHA。jie guo biao ming ,yi yi jing zuo wei di wen jie jing rong ji ,ke xian zhe su duan jie jing suo xu shi jian ,zai zhi fang suan yu yi jing bi wei 1:10(w/v)、jie jing wen du -60°C、jie jing shi jian 1 hde tiao jian xia ,shi DHAde han liang cong 26.85%di gao zhi 56.31%,hui shou lv wei 60%zuo you ;cai yong niao su bao ge fa dui DHAfu ji shi ,zai zhi fang suan :niao su :rong ji wei 1:3:12(w/w/v)、bao ge wen du -8°C、bao ge shi jian 10 hde tiao jian xia ,DHAhan liang cong 26.85%di gao zhi 77.21%,hui shou lv wei 50%zuo you 。er zai xiang tong tiao jian xia ,ke shi lie shi hu jun zao you shui jie chan wu zhi fang suan zhong DHAhan liang cong 48.20%fen bie di sheng zhi 79.82%he 83.12%。ran hou ,tong guo zhu zhi jing wei 5 cmde zhi bei xing gao xiao ye xiang se pu jiang niao su bao ge hou de DHAzhi fang suan nong su wu jin yi bu chun hua ,shi DHAhan liang di gao zhi 95.23%,hui shou lv wei 49.33%。ji ci ,cai yong mei fa chun jie jin qiang yu you he lie shi hu jun zao you zhi bei fu han DHAde 2-MAG,tan jiu zhi fang mei chong lei 、de wu ma er bi 、fan ying shi jian 、fan ying wen du he jia mei liang de ying xiang 。jie guo biao ming ,zai you zhi yu mo shui yi chun ma er bi wei 1:60、fan ying shi jian 2 h、fan ying wen du 30°C、Lipozyme 435tian jia liang wei 8%de tiao jian xia ,jin qiang yu you zhong 2-MAGhan liang zai 22.10%~27.40%、zao you zhong 2-MAGhan liang zai 26.50%~30.95%。jing zheng ji wan he 85%yi chun -shui cui qu hou ,jin qiang yu you he zao you 2-MAGde han liang di gao zhi 94%yi shang ,hui shou lv zai 70%zuo you ,er DHAhan liang fen bie da dao 53.22%he 74.76%。ran hou tong guo 2-MAGyu yi jing zhi bi 1:10(w/v)、-40°Cxia jie jing 10 h,shi jin qiang yu you 2-MAGzhong DHAhan liang di gao zhi 67.63%,2-MAGhan liang bao chi zai 90%yi shang ;tong guo fen zi zheng liu shi jin qiang yu you 2-MAGzhong DHAhan liang di gao zhi 59.95%,dan 2-MAGhan liang xia jiang zhi 69.81%,shui ming zai zheng liu guo cheng zhong 2-MAGfa sheng le xian ji zhuai yi 。zai ci ,yan jiu le 2-MAGzai bu tong wen du (25°C、40°C、50°C)he bu tong rong ji (zheng ji wan 、yi chun 、shu ding chun 、bing tong 、yi jing 、er lv jia wan )zhong de xian ji zhuai yi gui lv ,jie guo biao ming ,2-MAGxian ji zhuai yi fu ge yi ji ke ni fan ying mo xing 。zai mo rong ji ti ji zhong ,wen du di gao 25°C,xian ji zhuai yi cheng du zeng jia 5.6bei ;bu tong rong ji dui xian ji zhuai yi ying xiang bu tong ,xian ji zhuai yi su lv chang shu de da xiao wei zheng ji wan >mo rong ji ti ji >er lv jia wan >yi chun ≈bing tong ≈yi jing >shu ding chun ,yu suo shua rong ji de log Pzhi cheng zheng xiang guan (40°C R=0.932、50°C R=0.939,p<0.01)。xian ji zhuai yi hou 2-MAGhe 1-MAGzu fen zhong zhi fang suan fen bu jie guo biao ming ,40°Cxia ,yi chun 、bing tong 、yi jing he shu ding wan ti ji zhong xian ji zhuai yi cheng du jiao xiao ,DHAzai 2-MAGzhong de han liang gao yu ji zai 1-MAGzhong de han liang ,zheng ming DHAxian ji zhuai yi su lv xiang dui jiao man ;50°Cxiang tong ti ji zhong ,DHAzai liang zu fen zhong fen bu cha yi jiao xiao ,ke neng you yu wen du sheng gao ,ji xian ji zhuai yi su lv di gao 。zui hou ,tan tao le DHAhe 2-DHA-MAGdui HepG2xi bao zhi zhi dai xie de ying xiang 。jie guo biao ming ,nong du di yu 50μmol/Lde DHAhe 2-DHA-MAGjun dui xi bao mo du hai zuo yong ,jun neng you xiao de bei HepG2xi bao xi shou ,liang zhe dui xi bao zhong DHAhan liang de di gao mo xian zhe xing cha yi ;xiang dui yu DHA,2-DHA-MAGgeng you xiao de jiang di you suan you dao de zhi fang bian xing gan xi bao zhong zong gan you san zhi he zong dan gu chun de han liang ,ke shi xi bao nei gan san zhi han liang duo xia jiang 25.21%,dan gu chun han liang duo xia jiang 32.64%,zhe shi yin wei 2-DHA-MAGgeng you xiao de cu jin zhi fang yang hua guan jian ji yin PPARαhe CPT-1de biao da 、yi zhi dan gu chun ge cheng guan jian ji yin HMGCRde biao da 。zeng shang suo shu ,ben lun wen jian li le gao nong du DHAde fu ji fang fa ,jiang DHAhan liang di gao zhi 95.23%;jian li le 2-chan gan zhi xing DHAde ge cheng he chun hua fang fa ,zhi bei le 2-MAGhan liang 94%yi shang 、DHAhan liang wei 74%zuo you de chan pin ,bing fa xian wen du he rong ji dui ji xian ji zhuai yi ju you chong yao ying xiang 。tong guo xi bao shi yan zheng shi xiang dui yu DHA,2-DHA-MAGju you geng xian zhe de jiang di gan xi bao zong gan san zhi he zong dan gu chun shui ping de gong xiao ,biao ming sn-2wei DHAneng geng hao de diao jie zhi zhi dai xie ,wei kai fa xin ji xing DHAdi gong le li lun zhi chi 。
论文参考文献
论文详细介绍
论文作者分别是来自江南大学的张瑜，发表于刊物江南大学2019-10-21论文，是一篇关于单甘酯论文,纯化论文,酶法合成论文,酰基转移论文,脂质代谢论文,位置分布论文,江南大学2019-10-21论文的文章。本文可供学术参考使用，各位学者可以免费参考阅读下载，文章观点不代表本站观点，资料来自江南大学2019-10-21论文网站，若本站收录的文献无意侵犯了您的著作版权，请联系我们删除。
标签：单甘酯论文; 纯化论文; 酶法合成论文; 酰基转移论文; 脂质代谢论文; 位置分布论文; 江南大学2019-10-21论文;