迷迭香酸对黄嘌呤氧化酶的抑制作用_尚雁君

第二军医大学学报Acad J Sec M il M ed Univ

　2006Feb ;27(2) 189　

论　著

迷迭香酸对黄嘌呤氧化酶的抑制作用

尚雁君1,黄才国1*,蒋三好2,朱大元2,魏善建1,焦炳华1

(1.第二军医大学基础医学部生物化学和分子生物学教研室,上海200433,2.中国科学院上海药物研究所,上海201203)[摘要]　目的:研究迷迭香酸对黄嘌呤氧化酶的抑制作用。方法:将20、40、60μg /ml 迷迭香酸或1μg /ml 阳性对照别嘌呤醇,分别加入黄嘌呤溶液(测尿酸生成量:1mmol /L ;测超氧离子:50μmol /L )和0.1U /ml 黄嘌呤氧化酶中,用生化仪测定5min 尿酸生成量和超氧离子生成(NBT 显色法)。在1ml 2×105/ml HL -60细胞悬液中加入100μl 6mo l /L 黄嘌呤、100μl 0.1U /ml 黄嘌呤氧化酶、500μg /ml 迷迭香酸,分别以Annexin Ⅴ-P I 双标试剂盒法(以1μg /ml 别嘌呤醇为阳性对照)或细胞周期法(以100U /ml SO D 为阳性对照)测定细胞凋亡率。结果:

迷迭香酸显著抑制尿酸生成和超氧离子引起的N BT 显色,两种方法测得其IC 50分别为56μg /ml 和21μg /ml ;对细胞凋亡的抑制率均在40%以上。

结论:

迷迭香酸是黄嘌呤氧化酶的竞争性抑制剂。

[关键词]　迷迭香酸;黄嘌呤氧化酶;尿酸;超氧离子;细胞凋亡

[中图分类号]　R 285.5 [文献标识码]　A [文章编号]　0258-879X (2006)02-0189-03

Inhibition of xathine oxidase by rosmarinic acid

S HA N G Y an -jun 1,HU A NG Cai -guo 1*,JI AN G San -hao 2,Z H U Da -y uan 2,W EI Shan -jian 1,JIAO Bin -hua 1(1.Depar tme nt of Bio chemistry and M o lecular Bio log y ,Co llege of Basic M edical Science s ,Second M ilitary M edical U niver sity ,Shanghai 200433,China ;2.Shanghai I nstitute of M ateria M edica ,Shanghai 201203)

[ABSTRACT ]　Objective :T o study the inhibito ry effect of rosmarinic acid on x anthine ox idase.Methods :Xanthine ox idase (0.1U /ml )w as incuba ted with xa nthine (1mmol /L for determining for ma tion of uric acid ;50μmo l/L fo r de te rmining super -o xide anions )in the presence of 20,40and 60μg /ml rosmarinic acid o r allo purino l as positiv e contro l.T he forma tion o f uric acid w as deter mined by automatic bio chemical analyzer 5min after reactio n and the production of supero xide anio ns w as meas -ured by N it ro Blue Btetr azo lium (NBT )reduction.HL -60cells (1ml ,2×105/ml )wer e pretrea ted w ith xanthine (100μl ,6mol /L )and xanthine o xidase (100μl ,0.1U /ml ),then ro smarinic acid (500μg /ml )o r allopurinol (1μg /ml ,a s positive con -trol )(A nnexin Ⅴ-P I kit )was added to de te rmine the cell a po pto sis rate.H L -60cells (1ml ,2×105/ml )w ere also pre treated with xanthine (100μl ,6mo l/L )and x anthine ox idase (100μl ,0.1U /m l ),then ro smarinic acid (500μg /ml )or SO D (100U /ml ,a s positive contro l )(cell cycle me tho d )was added to de te rmine the cell apopto sis rate.Results :Ro smarinic acid obvio usly inhibited the production of uric acid and supero xide anion -induced reaction in N BT assay ,with their IC 50being 56μg /ml and 21μg /ml ,respec tively.T he rates of apoptosis inhibitio n by ro sma rinic acid w ere bo th o ver 40%by Annexin Ⅴ-PI kit and cell cy -cle me tho d.C onclusion :Rosmarinic acid is a competitive inhibito r of x anthine o xidase.[KEY WORDS ]　rosmarinic acid ;xanthine oxidase ;uric acid ;super oxide anio ns ;apo pto sis

[A cad J Sec M il M ed U niv ,2006,27(2):189-191]

[基金项目]　国家自然科学基金(29632050).S upported b y National Natural Science Foundation of China (29632050).[作者简介]　尚雁君,硕士生.E -m ail :syjsmm u @

*Corres ponding

autho r.E -mail :hu angcaig @h

丹参是临床上常用的活血化瘀药,是唇形科植

物丹参Salvia miltiorrhiza Bung e 的根,常用于妇科病、冠心病、缺血性脑卒中、动脉粥样硬化等症的治疗。临床上丹参制剂对冠心病、脑血栓、肝炎、肝硬化等有显著的疗效。丹参的活性成分主要分为脂溶性和水溶性两类。中医传统用药方法是用其水煎剂,即丹参的水溶性部位,所以研究丹参的水溶性成分更有意义[1]

。研究表明其水溶性成分主要是丹参素、原儿茶醛、丹酚酸。丹酚酸是一类既有咖啡酰缩酚酸结构又有新木脂素骨架的水溶性成分。丹酚酸类化合物包括丹酚酸A 、B 、C 、D 、E 、F 、G 、H 、I ,迷迭香酸(ro smarini acid ),紫草酸(litho spermic acid )

等,其中迷迭香酸是由1分子丹参素和1分子咖啡

酸缩合而成。黄嘌呤氧化酶是人体内产生尿酸过程中的关键酶,同时也是治疗痛风时药物的作用靶点,本文研究其对黄嘌呤氧化酶的抑制作用。1　材料和方法

1.1　试剂　H L -60细胞株,购自上海中国科学院细胞所;Annexin Ⅴ-PI 双标试剂盒购自晶美公司;黄