用HPLC法建立SPT抑制剂筛选模型的研究
摘要
目的：丝氨酸棕榈酰转移酶(SPT)[EC2.3.1.50]是体内鞘脂合成的关键酶,在调节鞘脂代谢中起着枢纽作用,影响各种鞘脂类成分在体内的合成、分布及功能,蝉花(Cordyceps cicadae)是我国传统的一种名贵中药,与冬虫夏草同属虫菌复合体,其主要的活性成分多球壳菌素(myriocin, ISP-1)是SPT的天然抑制剂,具有较强的免疫抑制活性,还具有抗动脉粥样硬化、抗真菌等药理作用,这些活性可能通过调节同一靶点—SPT,寻找SPT更优的抑制剂具有广阔的临床价值,为此本文建立一种操作简便、精密度和准确性高的SPT抑制剂筛选平台,为抑制剂的筛选实验提供可能的技术手段,也为进一步的研究奠定基础。
方法：大鼠肺脏中SPT活性较高,本文以大鼠肺脏为酶源,超声粉碎,冷冻离心,制备含酶裂解液,与底物反应后,加入内标物,液液萃取酶反应产物,HPLC测定酶反应含量,从而建立抑制剂筛选平台,通过内标法计算抑制率,评价抑制效果。实验优化裂解,萃取,酶反应等各个步骤的实验条件,为此,还建立了邻苯二甲醛(OPA)柱前衍生-HPLC-UV的测定方法,考察了衍生化反应体系中缓冲盐浓度和pH、OPA量、2-巯基乙醇量、衍生反应时间、衍生反应温度等因素对二氢鞘鞍醇(Sa.酶产物)和植物鞘鞍醇(Phy.内标物)长链碱衍生物影响及稳定性。在酶反应体系中加入myriocin标准品,测试平台的可行性,而后通过加入几种不同的中药提取物,进行了初步的筛选实验。
结果：柱前衍生在25℃反应2h为优化的反应条件,检测波长为230nm,二氢鞘鞍醇和植物鞘鞍醇样品衍生产物在放置4℃冰箱7h内稳定,测定结果的RSD均小于4.0%,二氢鞘鞍醇线性范围为3.125—100.0μg／mL(r=0.9993)、植物鞘鞍醇衍生物线性范围为5.0—500.0μg／mL(r=0.9971)；检测限(S／N=3：1)：Phy为0.9μg／mL,Sa为0.5μg／mL。超声裂解大鼠肺脏制备的酶裂解液,裂解均匀,蛋白含量稳定,通过优化萃取过程,不同条件下的萃取率保持在70.0%到85.0%之间,上清液量为90μL,高液进样量40μL(底物浓度为：L-丝氨酸(200 mM),棕榈酰辅酶A(5 mM))为优化的酶反应条件,在抑制剂筛选试验中,除禅花菌丝体提取物抑制率为69.20%,其它中药提取物没有明显的抑制作用。
结论：实验建立了检测SPT反应产物的高效液相色谱法,在此基础上建立了SPT抑制剂体外筛选模型,应用本方法对4种中药提取物进行了检测,没有发现对SPT有明显抑制作用的提取物,今后可加大筛选力度,以期找到更优的抑制剂。本研究在参考国外文献的基础上,在国内首次建立一种灵敏度高、成本低、方便一般实验室操作的SPT抑制剂筛选模型,与通常的同位素标记法比较,具有设备简单,没有放射性危险等优点。大力开展筛选新的丝氨酸棕榈酰转移酶抑制剂具有广阔的前景,尤其是从中药中发现明显效果的抑制剂具有良好的理论价值和经济价值。
Purpose:Serine palmitoyl transferase (SPT) [EC2.3.1.50] is the key enzyme in sphingolipid synthesis. plays a pivotal role in the regulation of sphingolipid metabolism, impact Various components of sphingolipids in vivo synthesis, distribution and function. Cordyceps cicadae is a Chinese traditional medicinal mushroom, which belongs to the class Ascomycetes and DongChongXiaCao group in Chinese herbs, the main active ingredient myriocin is a natural inhibitor of SPT, has strong immunosuppressive activity, but also has anti-atherosclerotic, anti-fungal and other pharmacological effects, such activity may regulate the same target-SPT, SPT find better inhibitors have broad clinical value. this paper has developed a kind of SPT inhibitor screening platform with simple, high precision and accuracy, lay the foundation and technical means for further study.
Methods:Rat lung activity of SPT was higher in body, so enzyme source from the rat lungs. to prepare lysates containing enzyme by sonication and centrifuged, substrate reaction, adding the internal standard, liquid-liquid extraction of enzyme reaction products, HPLC determination of content of enzyme reaction in order to establish inhibitor screening platform, Through the inhibition rate of the internal standard method to evaluate inhibitory effect. experimental optimization of the experimental conditions of cracking, extraction, enzymatic reaction steps. we developed a method of determination for Sa (product), Phy (internal standard) with HPLC,based on the pre-column derivatization with OPA.Several factors influencing the buffer salt concentration and pH, OPA volume, ME volume, derivative reaction time, reaction temperature were investigated and optimized. enzyme reaction system of Adding myriocin standard to test the feasibility of the platform, and then by adding several different herbal extracts to a preliminary screening test.
Results:The formed derivative was stable for more than 7h at 4℃in refrigerator, The detection wavelength was 230 nm, The der ivatizat ion react ion was performed at 25℃with 2h, The system offered the following analytical parameters: detection limi ts of 0.5μg/ml for Sa (signal-to-noise ratio S/N=3:1), the linear range response was from 3.125—100.0μg ml-1,0.9μg/mL for Phy (S/N=3:1), the linear range response was from 5.0—500. 0μg ml-1. rat lung lysates with Cracking uniform, protein stability, by optimizing the extraction process, different conditions of extraction rate remained at 70.0% to 85.0%, and the supernatant volume of 90μL, The HPLC injection volume 40μL (concentration of substrate: L-serine (200 mM), palmitoyl coenzyme A (5 mM)) for the optimized reaction conditions, in the inhibitor screening test, in addition to Cordyceps cicadae mycelium extracts inhibition rate of 69.20%, other herbal extracts no significant inhibition.
conclusion: Experiment established the SPT activity detected by HPLC, On this basis, establishment of the SPT inhibitors screening system in vitro. application of the method were tested 4 of herbal extracts and found that all extracts no significant inhibition. in the future can increase screening efforts in order to find better inhibitors. In this study, based on the reference to foreign literature, for the first time to establish a SPT inhibitor screening system in China for general laboratory, features with high sensitivity, low cost, easy operation, and comparison with isotope labeling method, has advantages of no radiation risk and simple equipment. Carrying out Vigorously screening of new SPT inhibitor has broad prospects, especially, inhibitor from traditional Chinese medicine of obvious effect has a good of the theoretical value and economic value.
方法：大鼠肺脏中SPT活性较高,本文以大鼠肺脏为酶源,超声粉碎,冷冻离心,制备含酶裂解液,与底物反应后,加入内标物,液液萃取酶反应产物,HPLC测定酶反应含量,从而建立抑制剂筛选平台,通过内标法计算抑制率,评价抑制效果。实验优化裂解,萃取,酶反应等各个步骤的实验条件,为此,还建立了邻苯二甲醛(OPA)柱前衍生-HPLC-UV的测定方法,考察了衍生化反应体系中缓冲盐浓度和pH、OPA量、2-巯基乙醇量、衍生反应时间、衍生反应温度等因素对二氢鞘鞍醇(Sa.酶产物)和植物鞘鞍醇(Phy.内标物)长链碱衍生物影响及稳定性。在酶反应体系中加入myriocin标准品,测试平台的可行性,而后通过加入几种不同的中药提取物,进行了初步的筛选实验。
结果：柱前衍生在25℃反应2h为优化的反应条件,检测波长为230nm,二氢鞘鞍醇和植物鞘鞍醇样品衍生产物在放置4℃冰箱7h内稳定,测定结果的RSD均小于4.0%,二氢鞘鞍醇线性范围为3.125—100.0μg／mL(r=0.9993)、植物鞘鞍醇衍生物线性范围为5.0—500.0μg／mL(r=0.9971)；检测限(S／N=3：1)：Phy为0.9μg／mL,Sa为0.5μg／mL。超声裂解大鼠肺脏制备的酶裂解液,裂解均匀,蛋白含量稳定,通过优化萃取过程,不同条件下的萃取率保持在70.0%到85.0%之间,上清液量为90μL,高液进样量40μL(底物浓度为：L-丝氨酸(200 mM),棕榈酰辅酶A(5 mM))为优化的酶反应条件,在抑制剂筛选试验中,除禅花菌丝体提取物抑制率为69.20%,其它中药提取物没有明显的抑制作用。
结论：实验建立了检测SPT反应产物的高效液相色谱法,在此基础上建立了SPT抑制剂体外筛选模型,应用本方法对4种中药提取物进行了检测,没有发现对SPT有明显抑制作用的提取物,今后可加大筛选力度,以期找到更优的抑制剂。本研究在参考国外文献的基础上,在国内首次建立一种灵敏度高、成本低、方便一般实验室操作的SPT抑制剂筛选模型,与通常的同位素标记法比较,具有设备简单,没有放射性危险等优点。大力开展筛选新的丝氨酸棕榈酰转移酶抑制剂具有广阔的前景,尤其是从中药中发现明显效果的抑制剂具有良好的理论价值和经济价值。
Purpose:Serine palmitoyl transferase (SPT) [EC2.3.1.50] is the key enzyme in sphingolipid synthesis. plays a pivotal role in the regulation of sphingolipid metabolism, impact Various components of sphingolipids in vivo synthesis, distribution and function. Cordyceps cicadae is a Chinese traditional medicinal mushroom, which belongs to the class Ascomycetes and DongChongXiaCao group in Chinese herbs, the main active ingredient myriocin is a natural inhibitor of SPT, has strong immunosuppressive activity, but also has anti-atherosclerotic, anti-fungal and other pharmacological effects, such activity may regulate the same target-SPT, SPT find better inhibitors have broad clinical value. this paper has developed a kind of SPT inhibitor screening platform with simple, high precision and accuracy, lay the foundation and technical means for further study.
Methods:Rat lung activity of SPT was higher in body, so enzyme source from the rat lungs. to prepare lysates containing enzyme by sonication and centrifuged, substrate reaction, adding the internal standard, liquid-liquid extraction of enzyme reaction products, HPLC determination of content of enzyme reaction in order to establish inhibitor screening platform, Through the inhibition rate of the internal standard method to evaluate inhibitory effect. experimental optimization of the experimental conditions of cracking, extraction, enzymatic reaction steps. we developed a method of determination for Sa (product), Phy (internal standard) with HPLC,based on the pre-column derivatization with OPA.Several factors influencing the buffer salt concentration and pH, OPA volume, ME volume, derivative reaction time, reaction temperature were investigated and optimized. enzyme reaction system of Adding myriocin standard to test the feasibility of the platform, and then by adding several different herbal extracts to a preliminary screening test.
Results:The formed derivative was stable for more than 7h at 4℃in refrigerator, The detection wavelength was 230 nm, The der ivatizat ion react ion was performed at 25℃with 2h, The system offered the following analytical parameters: detection limi ts of 0.5μg/ml for Sa (signal-to-noise ratio S/N=3:1), the linear range response was from 3.125—100.0μg ml-1,0.9μg/mL for Phy (S/N=3:1), the linear range response was from 5.0—500. 0μg ml-1. rat lung lysates with Cracking uniform, protein stability, by optimizing the extraction process, different conditions of extraction rate remained at 70.0% to 85.0%, and the supernatant volume of 90μL, The HPLC injection volume 40μL (concentration of substrate: L-serine (200 mM), palmitoyl coenzyme A (5 mM)) for the optimized reaction conditions, in the inhibitor screening test, in addition to Cordyceps cicadae mycelium extracts inhibition rate of 69.20%, other herbal extracts no significant inhibition.
conclusion: Experiment established the SPT activity detected by HPLC, On this basis, establishment of the SPT inhibitors screening system in vitro. application of the method were tested 4 of herbal extracts and found that all extracts no significant inhibition. in the future can increase screening efforts in order to find better inhibitors. In this study, based on the reference to foreign literature, for the first time to establish a SPT inhibitor screening system in China for general laboratory, features with high sensitivity, low cost, easy operation, and comparison with isotope labeling method, has advantages of no radiation risk and simple equipment. Carrying out Vigorously screening of new SPT inhibitor has broad prospects, especially, inhibitor from traditional Chinese medicine of obvious effect has a good of the theoretical value and economic value.
引文
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[65]肖朝华,周建华,吴衡生.多球壳菌素对高糖诱导肾小球系膜细胞肥大及细胞外基质合成的影响[J] 实用儿科临床杂志2006,21：268-270
[66]吴琼英, 马海乐, 骆琳, 吴守一, 高效液相色谱法测定血管紧张素转化 酶抑制剂的活性色谱2005.23(1)：79-81.
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[69]李长勇姜向明俞豪李丽英高效液相色谱法测定生物样品中磷酸鞘氨醇的含量首都医科大学学报2008.29(4)：459-461.
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[77]李冰岚,蝉药的本草学考证[J] 现代应用药学 1993.10：22-23.
[78]幸兴球,大蝉草和小蝉草的分类[J] 微生物学报1975.15：21-26.
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