穿山甲高温砂炒炮制增效机制研究
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摘要
目的穿山甲传统生品不入药,需炮制后方可入药,因此对穿山甲高温砂炒的炮制机制进行研究。方法采用TLC、Nano LC-Q Exactive Orbitrap MS对穿山甲炮制前后的脂溶性成分及蛋白成分的变化进行分析,同时,对炮制过程中显著增加的环二肽成分的形成进行模拟炮制。并对L-丝-L-酪环二肽的活性进行筛选。结果穿山甲经砂炒炮制后,脂溶性成分无显著变化、蛋白成分显著减少,环二肽显著增加。环二肽的形成主要与炮制过程中的受热有关,在低温条件下,直链肽N端可环化生成L构型的环二肽,在高温环境下,直链肽N端及C端均可快速环化生成环二肽。L-丝-L-酪环二肽可延长凝血时间并增加奶牛乳腺上皮细胞增殖率和乳蛋白合成相关基因表达,同时还具有显著的镇痛活性,这与穿山甲传统功效相一致。结论穿山甲经炮制后产生的L-丝-L-酪环二肽可能是穿山甲炮制增效的物质基础。这对揭示穿山甲药效物质基础、寻找穿山甲替代资源、保护穿山甲具有重要意义。
Objective Traditional raw pangolin products are not used as medicine, which can only be used as medicine after processing. Therefore, the processing mechanism of high temperature sand-fried pangolin was studied. Methods The changes of liposolubility and protein composition of pangolin before and after processing were analyzed by TLC and Nano LC-Q Exactive Orbitrap MS. Meanwhile, the simulation processing of cyclic dipeptides, which were significantly increased during processing, was performed. The activity of L-serine-L-tyrosine cyclic dipeptide was screened. Results The results showed that there was no significant change in fat-soluble components, significant decrease in polypeptides and significant increase in cyclic dipeptides after the sand-fried processing of pangolin. The formation of cyclic dipeptides was mainly related to the heating of the processing. At low temperature, the N-terminal of the linear peptide could be cycled to form L-shaped cyclic dipeptides. At high temperature, the N-terminal and C-terminal of the linear peptide could be rapidly cycled to form cyclic dipeptides. L-serine-L-tyrosine cyclic dipeptide could prolong coagulation time and increase the proliferation rate of mammary epithelial cells and the expression of genes related to milk protein synthesis in dairy cows. It also had significant analgesic activity, which was consistent with the traditional efficacy of pangolin. Conclusion These results suggested that large amounts of L-serine-L-tyrosine cyclic dipeptide produced by the processing of pangolin may be one of the material bases for enhancing the processing efficiency of pangolin. It was of great significance for revealing the material basis of pharmacodynamics of pangolin, searching for alternative resources and protecting pangolin.
Objective Traditional raw pangolin products are not used as medicine, which can only be used as medicine after processing. Therefore, the processing mechanism of high temperature sand-fried pangolin was studied. Methods The changes of liposolubility and protein composition of pangolin before and after processing were analyzed by TLC and Nano LC-Q Exactive Orbitrap MS. Meanwhile, the simulation processing of cyclic dipeptides, which were significantly increased during processing, was performed. The activity of L-serine-L-tyrosine cyclic dipeptide was screened. Results The results showed that there was no significant change in fat-soluble components, significant decrease in polypeptides and significant increase in cyclic dipeptides after the sand-fried processing of pangolin. The formation of cyclic dipeptides was mainly related to the heating of the processing. At low temperature, the N-terminal of the linear peptide could be cycled to form L-shaped cyclic dipeptides. At high temperature, the N-terminal and C-terminal of the linear peptide could be rapidly cycled to form cyclic dipeptides. L-serine-L-tyrosine cyclic dipeptide could prolong coagulation time and increase the proliferation rate of mammary epithelial cells and the expression of genes related to milk protein synthesis in dairy cows. It also had significant analgesic activity, which was consistent with the traditional efficacy of pangolin. Conclusion These results suggested that large amounts of L-serine-L-tyrosine cyclic dipeptide produced by the processing of pangolin may be one of the material bases for enhancing the processing efficiency of pangolin. It was of great significance for revealing the material basis of pharmacodynamics of pangolin, searching for alternative resources and protecting pangolin.
引文
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[13]刘睿,朱振华,吴佳,等.羚羊角与山羊角蛋白质类成分比较研究[J].中国中药杂志,2018,43(16):3329-3334.
[14]Park H J,Cha D S,Jeon H.Antinociceptive and hypnotic properties of Celastrus orbiculatus[J].JEthnopharmacol,2011,137(3):1240-1244.
[15]刘贝,王淑美,王佰灵,等.三棱的环二肽类成分抗凝活性[J].中成药,2015,37(1):34-39.
[16]陈璐,常晨城,史彬林,等.含蛋氨酸二肽影响奶牛乳腺上皮细胞乳蛋白合成相关基因表达[J].动物营养学报,2016,28(12):4036-4043.
[17]Borthwick A D,Costa N C D.2,5-diketopiperazines in food and beverages:Taste and bioactivity[J].Crit Rev Food Sci Nutr,2017,57(4):718-742.
[18]Li Y,Li F,Zhu Y,et al.DFT study on reaction mechanisms of cyclic dipeptide generation[J].Struct Chem,2016,27(4):1165-1173.
[19]文秀英,徐保国,刘浩.穿山甲对心脏收缩功能和血小板聚集性的影响[J].中国中药杂志,1999,24(1):52-53.
[20]吴珊,农彩丽,何显科,等.穿山甲水提物镇痛作用的实验研究[J].广西医学,2012,34(1):7-9.
[21]侯士良,赵晶,董秀华,等.比较猪蹄甲、穿山甲泌乳作用实验研究[J].中国中药杂志,2000,25(1):46-48.
[22]刘鑫,赵龙,王媛,等.一类全新阿片样二肽的设计合成及镇痛活性研究[J].化学学报,2016,74(1):44-48.
[23]Takagi H,Shiomi H,Ueda H,et al.A novel analgesic dipeptide from bovine brain is a possible Met-enkephalin releaser[J].Nature,1979,282(5737):410-412.
[24]Sato T,Sakurada S,Sakurada T,et al.Comparison of the antinociceptive effect between the cyclic dipeptide cyclo[Tyr(Et)-homoarginine]and the linear dipeptide Boc-Tyr(Et)-homoarginine-OMe in rats[J].Jpn J Pharmacol,1984,34(1):1-8.
[25]Perazzo J,Castanho M A,Sa Santos S.Pharmacological potential of the endogenous dipeptide kyotorphin and selected derivatives[J].Front Pharmacol,2017,doi:10.3389/fphar.2016.00530.
[2]王妍,张国民,哈伟.穿山甲应用及炮制研究进展[J].中国现代中药,2015,17(3):280-284.
[3]姜南,王邱文,晁明娣.区域合作治理跨境穿山甲犯罪对策研究[J].野生动物学报,2018,39(4):978-984.
[4]Gaubert P,Antunes A,Meng H,et al.The complete phylogeny of pangolins:Scaling up resources for the molecular tracing of the most trafficked mammals on Earth[J].J Hered,2018,109(4):347-359.
[5]郭珊珊,彭建军,刘双,等.中国境内野生穿山甲现状及相关非法贸易概况[J].重庆师范大学学报:自然科学版,2019,36(1):48-54.
[6]贾斌,马养民,陈镝,等.天然产物吲哚二酮哌嗪生物碱的结构及生物活性[J].化学进展,2018,30(8):1067-1081.
[7]李文利,夏娟.二酮哌嗪类化合物生物合成研究进展[J].微生物学通报,2014,41(1):111-121.
[8]Martins M B,Carvalho I.Diketopiperazines:Biological activity and synthesis[J].Tetrahedron,2007,63(40):9923-9932.
[9]马雪梅,秦永祺.穿山甲化学成分的研究[J].药学学报,1988,23(8):588-592.
[10]马雪梅,秦永祺,郭启栋.穿山甲活性成分丝-酪环二肽的合成[J].中草药,1993,24(3):162.
[11]Dalai P,Pleyer H L,Strasdeit H,et al.The influence of mineral matrices on the thermal behavior of glycine[J].Orig Life Evol Biosph,2017,47(4):427-452.
[12]Hayasaka F,Yamamoto S,Sakai Y.Production method for cyclic dipeptide derived from native collagen[J].FoodSci Technol Res,2016,22(4):477-483.
[13]刘睿,朱振华,吴佳,等.羚羊角与山羊角蛋白质类成分比较研究[J].中国中药杂志,2018,43(16):3329-3334.
[14]Park H J,Cha D S,Jeon H.Antinociceptive and hypnotic properties of Celastrus orbiculatus[J].JEthnopharmacol,2011,137(3):1240-1244.
[15]刘贝,王淑美,王佰灵,等.三棱的环二肽类成分抗凝活性[J].中成药,2015,37(1):34-39.
[16]陈璐,常晨城,史彬林,等.含蛋氨酸二肽影响奶牛乳腺上皮细胞乳蛋白合成相关基因表达[J].动物营养学报,2016,28(12):4036-4043.
[17]Borthwick A D,Costa N C D.2,5-diketopiperazines in food and beverages:Taste and bioactivity[J].Crit Rev Food Sci Nutr,2017,57(4):718-742.
[18]Li Y,Li F,Zhu Y,et al.DFT study on reaction mechanisms of cyclic dipeptide generation[J].Struct Chem,2016,27(4):1165-1173.
[19]文秀英,徐保国,刘浩.穿山甲对心脏收缩功能和血小板聚集性的影响[J].中国中药杂志,1999,24(1):52-53.
[20]吴珊,农彩丽,何显科,等.穿山甲水提物镇痛作用的实验研究[J].广西医学,2012,34(1):7-9.
[21]侯士良,赵晶,董秀华,等.比较猪蹄甲、穿山甲泌乳作用实验研究[J].中国中药杂志,2000,25(1):46-48.
[22]刘鑫,赵龙,王媛,等.一类全新阿片样二肽的设计合成及镇痛活性研究[J].化学学报,2016,74(1):44-48.
[23]Takagi H,Shiomi H,Ueda H,et al.A novel analgesic dipeptide from bovine brain is a possible Met-enkephalin releaser[J].Nature,1979,282(5737):410-412.
[24]Sato T,Sakurada S,Sakurada T,et al.Comparison of the antinociceptive effect between the cyclic dipeptide cyclo[Tyr(Et)-homoarginine]and the linear dipeptide Boc-Tyr(Et)-homoarginine-OMe in rats[J].Jpn J Pharmacol,1984,34(1):1-8.
[25]Perazzo J,Castanho M A,Sa Santos S.Pharmacological potential of the endogenous dipeptide kyotorphin and selected derivatives[J].Front Pharmacol,2017,doi:10.3389/fphar.2016.00530.
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