_葡萄糖苷酶水解牛蒡子苷制备牛蒡子苷元_欧志敏

药　物　生　物　技　术

Pharm aceutical Biotechno logy 　2009, 16(5) :443～446443

β-葡萄糖苷酶水解牛蒡子苷制备牛蒡子苷元

欧志敏, 杨根生

＊

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, 冯　海

(浙江工业大学药学院, 杭州　310014)

摘　要　采用β-葡萄糖苷酶水解牛蒡子苷制备牛蒡子苷元。产率和反应初速度随β-葡萄糖苷酶用量的增多而提高。最佳水解反应条件为35℃,pH 5. 0, 40h 。产率随着牛蒡子苷初始浓度的提高而降低。在最佳反应V C

条件下,β-葡萄糖苷酶水解牛蒡子苷的过程符合单底物M ichaelis M enten 方程, v m s , 其中:

K m +C s

V m =2. 5×10-2mmol /L ·h , K m =3. 8×10-1mmol /L 。关键词　β-葡萄糖苷酶; 牛蒡子苷; 牛蒡子苷元

中图分类号:Q 503　　　文献标识码:A 　　　文章编号:1005-8915(2009) 05-0443-04

　　牛蒡子苷元是中药牛蒡子的有效成分之一, 具

有治疗或预防慢性肾功能衰竭及肾纤维化、抗病毒、抗肿瘤等功效[1～7]。从天然牛蒡子中可以分离提取牛蒡子苷元, 由于牛蒡子苷元在牛蒡子中含量低于1%,所以该方法收率低。牛蒡子苷的糖苷键断裂可以生成牛蒡子苷元, β-葡萄糖苷酶可以水解糖苷键, 因此采用β-葡萄糖苷酶水解牛蒡子苷可以生成牛蒡子苷元(图1) 。作者对β-葡萄糖苷酶水解牛蒡子苷制备牛蒡子苷元的工艺过程进行研究, 为牛蒡子苷元的制备提供了可靠方法

。

[8～12]

和上海融禾医药科技发展有限公司。β-葡萄糖苷酶(≥6u /mg ) 购自上海楷洋生物技术销售公司。其他试剂为分析纯。安捷伦高效液相色谱1200, 泵的型号为G1311A , 检测器的型号为G1365B 。

1. 2　β-葡萄糖苷酶水解牛蒡子苷制备牛蒡子苷元

分别用pH 3. 0、4. 0、5. 0、6. 0和7. 0的磷酸缓冲液配制浓度为0. 6、1. 2、1. 8、2. 4、3. 0m mol /L 的牛蒡子苷水溶液, 取20m l 牛蒡子苷溶液加入一定量的β-葡萄糖苷酶, 于一定温度的150r /min 摇床中转化一定时间。反应结束后, 精密加入8倍体积的甲醇终止反应, 放置过夜, 过滤取滤液, 经过0. 45μm 微孔滤膜过滤后得到的溶液用液相色谱分析牛蒡子苷和牛蒡子苷元的含量(方法1. 3) 。1. 3　牛蒡子苷和牛蒡子苷元的分析检测

色谱柱为Aglient Eclipse XDB -C8柱

Fig 1　P reparation o f ar ctigenin by hy drolyzatio n of arctii with β-g lucosidase

(4. 6mm ×150mm , 5μm ) 。检测条件:甲醇∶水=1∶1. 1, 体积流量∶1. 0ml /min , 进样量∶1μl , 紫外检测波长∶280nm , 柱温:30℃。标准品色谱图如图2。牛蒡子苷的标准曲线方程为Y =-0. 00459+9. 27356×10-4X , R =0. 99983。牛蒡子苷元的标准曲线方程为Y =0. 002+0. 00183X , R =0. 99987。

1　实验材料与方法1. 1　仪器与试剂

牛蒡子苷(纯度>98%) 和牛蒡子苷元(纯度>98%) 分别购自中国药品生物制品检定所

＊收稿日期:2009-01-07　　　修回日期:2009-03-02

基金项目:浙江省中医药科技计划项目(No . 2007CA100) 。作者简介:欧志敏, 女, 1973年生, 博士, 副教授。

　, T :(0571(; E ooz zmm . cn 。

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药物生物技术第16卷第5期　

明40h 后酶的活力降低不利于反应进行。40h 内β-葡萄糖苷酶没有发生明显的失活现象, 因此最佳反应时间确定为40h 。25m g β-葡萄糖苷酶用于转化, 40h 产率达到79. 8%。2. 2　反应液的pH 值对产率的影响

在pH3. 0、4. 0、5. 0、6. 0和7. 0的磷酸缓冲液

A :Arctiin (2. 048min ) ; B :Arctigenin (3. 591min )

配制的20m l 0. 6mm ol /L 牛蒡子苷溶液中加入25mg β-葡萄糖苷酶, 于35℃、150r /min 摇床中反应40h (图5) 。β-葡萄糖苷酶在pH4. 0～6. 0范围内产率可以达到70%以上, 反应最佳pH 5. 0

。

Fig 2　Chroma to g raphic profile of the standards o f a rctiin and arctig enin

1. 4　反应初速度

反应初速度定义为摩尔产率在5%以内时, 单位时间内产物生成量。2　结果

2. 1　酶的用量对水解产率的影响

在pH 5. 0的20ml 0. 6mm ol /L 牛蒡子苷溶

液中分别加入5、10、15、20和25m g β-葡萄糖苷酶, 于35℃、150r /min 摇床中反应(图3、4) 。当底物浓度足够使酶饱和后, 反应速度将与酶量成正比。随着酶量的提高, 产率和反应初速度逐渐提高。应该加入多少酶量进行反应要根据生产成本进行选择。反应时间小于40h , 产率随着时间的延长而升高。40h 后, 产率提高幅度很小。表　　

Fig 5　Effect of pH value o n pro duct yie ld

2. 3　反应温度对产率的影响

在pH5. 0的20m l 0. 6m mol /L 牛蒡子苷溶液中加入25mg β-葡萄糖苷酶, 分别于20℃、25℃、30℃、35℃、40℃、45℃和50℃的摇床中(转速150r /m in ) 反应40h (图6) 。β-葡萄糖苷酶水解牛蒡子苷的最佳温度为35

℃。

Fig 6　Effect of tempe rature on hy drolysis of ar ctii by β-g luco sida se

2. 4　底物初始浓度不同对产率的影响

在pH 5. 0的初始浓度分别为0. 6、1. 2、1. 8、2. 4、3. 0mmol /L 的20ml 牛蒡子苷溶液中加入25mg β-葡萄糖苷酶, 于35℃、150r /min 摇床中反应(图7) 。随着牛蒡子苷初始浓度的提高产率逐渐降

欧志敏等:β-葡萄糖苷酶水解牛蒡子苷制备牛蒡子苷元

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在原药材中的含量, 有助于药物摄入人体快速起效, 为酶法炮制中药牛蒡子提供可借鉴的思路。

参考文献

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[6]Kang H S , Lee J Y , Kim C J . Anti -inflam matory activity of

arctigenin from Forsy thiae Fructu s [J ]. Jour nal o f E thno -pha rmacolog y , 2008, 116(2) :305.

[7]S w aru p V , Ghosh J , M ish ra M K , et al . Novel strategy for

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[8]Choi Y H , Kim J W , Yoo K P . High performance l iquid ch ro -matography -electrospray ionization MS -M S analys is of Forsythia koreana fruits , leaves , and stems . Enhancement of the effic iency of extraction of arctigenin by use of supercritical -fluid extraction [J ]. Chr omatog raphia , 2003, 57(1～2) :73.

[9]Huang W Y , Sh eu S J . S eparation and identification of th e

fifteen con stituen ts in forsy thiae fructus [J ]. Jour nal Food and Dr ug Analysism , 2007, 15(1) :33.

[10]Kim C Y , Ahn M J , Kim J . A p reparative isolation and puri -fication of arctigenin and matairesin ol from Forsythia koreana by cen trifugal partition ch rom atography [J ]. Journa l Sepa -ration Science , 2006, 29(5) :656.

[11]Liu S M , Chen K S , S chliemann W , et al . Isolation and i -dentification of arctiin and arctigenin in leaves of bu rdock (Arctium lappla L . ) by polyamide column ch romatography in combination w ith HP LC -ESI /M S [J ]. Ph ytochemica l Ana ly -, , .

Fig 7　Effect of initial concentration of arctii on product yield

2. 5　水解反应动力学模型

在最佳反应条件下, 对牛蒡子苷初始浓度不同的水解过程进行研究, 发现β-葡萄糖苷酶催化牛蒡子苷水解的过程符合单底物Michaelis M enten 方

m s

程, v , 其中:V m =2. 5×10-2mmo l /L ·

K m +C s h , K m =3. 8×10-1mmo l /L 。实验数据与模拟曲线能够很好地吻合(图8)

。

Fig 8　Effect of initial co ncentra tion o f a rctii on intial ve -locity of reaction

3　讨论

近年来, 人们对酶法转化制备药物的过程进行过大量研究

[13]

。本文即采用β-葡萄糖苷酶水解牛

蒡子苷成功地制备了牛蒡子苷元。为了提高牛蒡子苷元的收率可以进一步筛选催化效率更高的β-葡萄糖苷酶、优化催化反应条件并选择更好的反应溶剂体系。牛蒡子苷和牛蒡子苷元在牛蒡子原药材中含量约为10∶1, 具体含量根据牛蒡子原药材的产地不同而异。牛蒡子苷元是牛蒡子摄入体内迅速起效的有效成分, 而原药材中的牛蒡子苷进入人体后需要经过体内酶的催化作用转化为牛蒡子苷元才能起效。可以进一步对β-葡萄糖苷酶炮制

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[12]Lu e W J , Chen Y L , Zhang Y X , et al . M icroemuls ion elec -trokinetic chromatog raphy for th e separation of arctiin and arctigenin in Fru ctus Arctii and its herbal p reparations [J ]. J ourna l Ch roma togr aph y B -Ana lytical Technolog ies in the

B iomed ica l and Li fe S ciences , 2007, 860(1) :127. [13]吴晓, 倪孟祥, 朱晓丽, 等. 利用嘧啶核苷磷酸化酶基因工程

菌酶法合成5-氟尿苷的转化率影响因素的研究[J ]. 药物生物技术, 2008, 15(4) :282.

Preparation of Arctigenin by Hydrolysis of Arctii with β-Glucosidase

O U Zhi -min , YANG Gen -sheng , FENG Hai

(Pharmaceutical school , Zhejiang University o f Technology , H ang Zhou , 310014, China )

A bstract 　Arctigenin w as synthesized by hy dro lysis o f arctii w ith β-g luco sidase as cataly st . Effect o f many factors o n the A rctigenin y ield w as investig ated . The results show ed that on mo re additio n of β-gluco sidase in reactio n , a hig her yield and initial velocity can be achieved . The optimal hydroly sis condi -tion is 35℃,pH 5. 0, 40h , 150r /min . The yield %decreased w ith initial concentration of arctii increase . The inhibition of arctigenin and arctii on hydroly sis reaction w as no t discovered w hen the substrate co n -centratio n is in the rang e of 0. 6～3. 0m mol /L . The yield reached 79. 8%w hen 25m g β-gluco sidase w as used in 20ml 0. 6mmo l /L arctigenin so lution fo r 40h . The process o f hydroly sis of arcii is in accordance with equation of Michaelis Menten equation w hen the reaction w as carried o ut under optim al hydroly sis condition . The kinetic constant is V m =2. 5×10-2m mol /L ·h , K m =3. 8×10-1mmo l /L . The m odel simulating curves w ere in go od agreem ent w ith the ex perimental data . It w as indicated tha t the reactio n mechanism assumptio n w as reasonable . Key words 　β-Glucosidase , A rctii , A rctigenin

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Studies on the Anti -influenza Virus Activity of Jinchan Oral Solution in vitro and in vivo

ZHANG Lei 1, LI M eng -qiu 1, DO U Jie 1, WU Yu -lin 2, ZHOU Chang -lin 1＊

12(School of Li f e S cience and Technology ; S chool of Pharmacy , China Pharmaceutical University ,

N anjing 210009, China )

A bstract 　T his research intended to study the antiviral activity of Jinchan o ral solution against influenza virus in vitro and in v ivo . Toxicity concentration of Jinchan oral solution to MDCK cells w as assessed by observatio n of cy topathic effect (CPE ) . Its inhibito ry activity ag ainst infiuenza A /FM 1/1/47/(H 1N 1) virus in vitro w as determined by hemagg lutination assay , and the antiviral activity w as compared by u -sing different methods . Viral pneum onia model in IC R mice w as used in vivo . The death rate and death pro tection rate w ere de tected to evalua te the anti -influenza viral effect of Jinchan oral so lution . The re -sults showed that Jinchan oral solutio n had no to xicity to M DCK cells in 1∶128dilution . The 50%in -hibito ry dilution o f 1∶686and 1∶740w as calculated w hen the cells w ere treated in direct deactivatio n delivery Ⅰand direct deactivation delivery II respectirely , w hile no inhibito ry activity w as fo und w hen the cells w ere treated in precaution delivery . Jinchan oral solutio n displayed the protective effect ag ainst influenza virus in infected mice at the do se o f 2. 0m l /kg . , ,