Ⅳ型胶原酶模型的建立及其在中药活性成分筛选中的应用
|
摘要
目的建立Ⅳ型胶原酶筛选模型,并运用此模型对中药活性成分进行评价。方法利用Ⅳ型胶原酶特异性水解明胶释放的末端氨基与2,4,6-三硝基苯磺酸(TNBS)发生显色,吸光度值可以衡量Ⅳ型胶原酶含量,优化Ⅳ型胶原酶浓度、琥珀酰凝胶浓度、显色剂用量及时间参数。结果确定了Ⅳ型胶原酶筛选模型的条件:5.0×10~(-5) g/L酶溶液、20 g/L琥珀酰凝胶溶液、60μl 0.05%TNBS、显色30 min,阳性药盐酸四环素在1.0 g/L下对该酶的抑制率是19.39%,并呈现一定的量效关系,表明成功建立该筛选模型。应用该模型筛选发现,阿魏酸和川芎嗪具有明显的抑制Ⅳ型胶原酶活性,而水杨酸和小檗碱则具有促进作用。结论Ⅳ型胶原酶的筛选模型成功建立并应用到中药活性成分筛选中,为快速评价中药活性成分及其作用机制提供了有益参考。
Objective To establish a type Ⅳ collagenase screening model and use it to evaluate the active ingredients of TCM. Methods As type Ⅳ collagenase hydrolyze gelatin specifically, the released terminal amino group react with 2,4,6-trinitrobenzene sulfonic acid(TNBS) with color; the absorbance can be measured by UV for type Ⅳ collagenase content. The screening model is established by optimizing the concentration of type Ⅳ collagenase, succinyl gel concentration, the amount of color reagent and time. Results The conditions of the type Ⅳ collagenase screening model are finally determined: 0.05 mg/L type Ⅳ collagenase solution, 20 g/L succinyl gel solution, 0.05 % 60 μl TNBS, coloration for 30 min, the inhibitory rate of positive drug tetracycline hydrochloride at 1.0 g/L is 19.39 %, and showed a certain dose-effect relationship, indicating that the screening model of type Ⅳ collagenase is established successfully. Using the model, we find that ferulic acid and ligustrazine show inhibitory activity, while salicylic acid and berberine promote inversely. Conclusion The screening model for type Ⅳ collagenase is successfully established and applied to screen the active ingredients of TCM, which provide a useful reference for rapid evaluation of active ingredients and the mechanism of action.
Objective To establish a type Ⅳ collagenase screening model and use it to evaluate the active ingredients of TCM. Methods As type Ⅳ collagenase hydrolyze gelatin specifically, the released terminal amino group react with 2,4,6-trinitrobenzene sulfonic acid(TNBS) with color; the absorbance can be measured by UV for type Ⅳ collagenase content. The screening model is established by optimizing the concentration of type Ⅳ collagenase, succinyl gel concentration, the amount of color reagent and time. Results The conditions of the type Ⅳ collagenase screening model are finally determined: 0.05 mg/L type Ⅳ collagenase solution, 20 g/L succinyl gel solution, 0.05 % 60 μl TNBS, coloration for 30 min, the inhibitory rate of positive drug tetracycline hydrochloride at 1.0 g/L is 19.39 %, and showed a certain dose-effect relationship, indicating that the screening model of type Ⅳ collagenase is established successfully. Using the model, we find that ferulic acid and ligustrazine show inhibitory activity, while salicylic acid and berberine promote inversely. Conclusion The screening model for type Ⅳ collagenase is successfully established and applied to screen the active ingredients of TCM, which provide a useful reference for rapid evaluation of active ingredients and the mechanism of action.
引文
[1]Nd S J, Tebben A, Gilmore J L, et al. A molecular modeling analysis of novel non-hydroxamate inhibitors of TACE[J]. Bioorganic&Medicinal Chemistry Letters, 2007, 17(5):1408.
[2]Maskos K. Crystal structures of MMPs in complex with physiological and pharmacological inhibitors[J]. Biochimie, 2005, 87(4):249-263.
[3]薛建元,林言箴,尹浩然,等. IV型胶原纤维及其酶在胃癌浸润转移中的作用[J].中国肿瘤临床, 1995(4):241-243.
[4]潘玉夏.急性髓系白血病患者基质金属蛋白酶-2、9(MMP-2、MMP-9)的表达及其意义[D].石家庄:河北医科大学, 2003.
[5]朱艳虹,陈真,钱之玉,等.西红花酸对乙醛诱导的肝星状细胞增殖和胶原合成的影响[J].中国临床药理学与治疗学, 2013, 18(8):841-847.
[6]李晓龙.槲皮素对高糖环境下人脐静脉内皮细胞活性以及MMP-2与MMP-9表达的影响[D].石家庄:河北医科大学, 2014.
[7]林洪生,李树奇.川芎嗪,苦参碱对癌细胞与内皮细胞粘附及粘附因子表达的影响[J].中国新药杂志, 1999, 8(6):384-386.
[8]Liu L, Hugins W R, Shack S, et al. Cinnamic acid:Auatural product with potential use in cancer intervention[J]. Int J Cancer, 1995, 62(3):345.
[9]高冬,陈文元,郑良朴,等.血府逐瘀汤诱导内皮细胞迁移中Ⅳ型胶原酶的作用研究[J].中国中医基础医学杂志, 2011,17(12):1341-1342+1347.
[10] Nagata, Shigekazu. Apoptosis by Death Factor[J]. Cell, 1997, 88(3):355.
[11] Moller M N, Werther K A, Stangerup S E, et al. Angiogenesis in vestibular schwannomas:expression of extracellular matrix factors MMP-2, MMP-9,and TIMP-1[J]. Laryngoscope, 2010, 120(4):657-662.
[12] Choi W S, Jeon O H, Kim D S. CD40 ligand shedding is regulated by interaction between matrix metalloproteinase-2 and platelet integrin alpha(IIb)beta(3)[J]. Journal of Thrombosis&Haemostasis Jth, 2010, 8(6):1364-1371.
[13] Fang L, Teuchert M, Huber-Abel F, et al. MMP-2 and MMP-9 are elevated in spinal cord and skin in a mouse model of ALS[J]. Journal of the Neurological Sciences, 2010, 294(1-2):51-56.
[14] Brakus S M, Govorko D K, Vukojevic K, et al. Apoptotic and anti-apoptotic factors in early human mandible development[J]. European Journal of Oral Sciences, 2010, 118(6):537-546.
[15]华中科技大学.一种Ⅳ型胶原酶含量测定方法:CN101846632A[P].2010-09-29.
[16]李碧,方雄,龚晏,等.γ-氨基丁酸转氨酶抑制剂筛选方法的建立及其在天麻成分筛选中的应用[J].中草药, 2016, 47(3):454-458.
[17]李碧,方雄,褚福浩,等.琥珀酸半醛脱氢酶抑制剂筛选方法的建立及其在天麻成分筛选中的应用[J].北京中医药大学学报, 2016, 39(8):664-669.
[18] Burns F R, Stack M S, Gray R D, et al. Inhibition of purified collagenase from alkali-burned rabbit corneas[J]. Investigative Ophthalmology&Visual Science, 1989, 30(7):1569.
[2]Maskos K. Crystal structures of MMPs in complex with physiological and pharmacological inhibitors[J]. Biochimie, 2005, 87(4):249-263.
[3]薛建元,林言箴,尹浩然,等. IV型胶原纤维及其酶在胃癌浸润转移中的作用[J].中国肿瘤临床, 1995(4):241-243.
[4]潘玉夏.急性髓系白血病患者基质金属蛋白酶-2、9(MMP-2、MMP-9)的表达及其意义[D].石家庄:河北医科大学, 2003.
[5]朱艳虹,陈真,钱之玉,等.西红花酸对乙醛诱导的肝星状细胞增殖和胶原合成的影响[J].中国临床药理学与治疗学, 2013, 18(8):841-847.
[6]李晓龙.槲皮素对高糖环境下人脐静脉内皮细胞活性以及MMP-2与MMP-9表达的影响[D].石家庄:河北医科大学, 2014.
[7]林洪生,李树奇.川芎嗪,苦参碱对癌细胞与内皮细胞粘附及粘附因子表达的影响[J].中国新药杂志, 1999, 8(6):384-386.
[8]Liu L, Hugins W R, Shack S, et al. Cinnamic acid:Auatural product with potential use in cancer intervention[J]. Int J Cancer, 1995, 62(3):345.
[9]高冬,陈文元,郑良朴,等.血府逐瘀汤诱导内皮细胞迁移中Ⅳ型胶原酶的作用研究[J].中国中医基础医学杂志, 2011,17(12):1341-1342+1347.
[10] Nagata, Shigekazu. Apoptosis by Death Factor[J]. Cell, 1997, 88(3):355.
[11] Moller M N, Werther K A, Stangerup S E, et al. Angiogenesis in vestibular schwannomas:expression of extracellular matrix factors MMP-2, MMP-9,and TIMP-1[J]. Laryngoscope, 2010, 120(4):657-662.
[12] Choi W S, Jeon O H, Kim D S. CD40 ligand shedding is regulated by interaction between matrix metalloproteinase-2 and platelet integrin alpha(IIb)beta(3)[J]. Journal of Thrombosis&Haemostasis Jth, 2010, 8(6):1364-1371.
[13] Fang L, Teuchert M, Huber-Abel F, et al. MMP-2 and MMP-9 are elevated in spinal cord and skin in a mouse model of ALS[J]. Journal of the Neurological Sciences, 2010, 294(1-2):51-56.
[14] Brakus S M, Govorko D K, Vukojevic K, et al. Apoptotic and anti-apoptotic factors in early human mandible development[J]. European Journal of Oral Sciences, 2010, 118(6):537-546.
[15]华中科技大学.一种Ⅳ型胶原酶含量测定方法:CN101846632A[P].2010-09-29.
[16]李碧,方雄,龚晏,等.γ-氨基丁酸转氨酶抑制剂筛选方法的建立及其在天麻成分筛选中的应用[J].中草药, 2016, 47(3):454-458.
[17]李碧,方雄,褚福浩,等.琥珀酸半醛脱氢酶抑制剂筛选方法的建立及其在天麻成分筛选中的应用[J].北京中医药大学学报, 2016, 39(8):664-669.
[18] Burns F R, Stack M S, Gray R D, et al. Inhibition of purified collagenase from alkali-burned rabbit corneas[J]. Investigative Ophthalmology&Visual Science, 1989, 30(7):1569.