CN106893758A - 拟海桑提取物的生物活性cpe+mtt测定方法 - Google Patents

拟海桑提取物的生物活性cpe+mtt测定方法 Download PDF

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CN106893758A
CN106893758A CN201510960617.2A CN201510960617A CN106893758A CN 106893758 A CN106893758 A CN 106893758A CN 201510960617 A CN201510960617 A CN 201510960617A CN 106893758 A CN106893758 A CN 106893758A
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cpe
cell
cultivate
per hole
mdck cell
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褚方强
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Qingdao Qingquan Biotechnology Co Ltd
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Qingdao Qingquan Biotechnology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

Abstract

本发明涉及拟海桑提取物的生物活性CPE+MTT测定方法：MTT法：MDCK细胞37ºC下在通入5%CO2和95%氧气，含有10%FBS的RPMI-1640细胞悬浮液中培养。每孔加入2uL，50ug/mL含药物的甲醇溶液的维持液继续培养72h。然后，每孔加入20uLMTT溶液，继续培养4h。以DMSO更换溶解MTT的介质，至Formazan结晶完全溶解。酶标仪测定每孔540nm处的OD值，计算IC50。CPE法：单层MDCK细胞首先与流感病毒于37ºC培养1h。弃去病毒稀释液，将细胞于37ºC保持在含有不同浓度测试药物的感染媒介中。37ºC培养48h后，用100uL 4%的多聚甲醛与MDCK细胞在室温下固定20min。除去甲醛后，MDCK细胞用0.1%的结晶紫染色30min。将板洗净干燥，酶标仪在570nm处测定各孔中结晶紫染色的强度，计算IC50值。本发明的方法相简单快速、而且检测结果可靠，适于推广应用。

Description

拟海桑提取物的生物活性CPE+MTT测定方法
技术领域
本发明属于海洋生物医药技术领域，具体涉及一种拟海桑提取物的生物活性CPE+MTT测定方法。
背景技术
拟海桑(Sonneratiaparacaseolaris)属于海桑科海桑属，为我国稀有濒危红树植物，仅天然分布于海南岛，分布范围狭窄，个体数量稀少。正处于濒危状态，被列入《中国生物多样性国情研究报告》中国被子植物濒危及稀有种名录。在前期的生物活性筛选实验中，拟海桑的甲醇提取物对小鼠白血病肿瘤细胞株P-388和人肝癌细胞株BEL-7402显示较强的细胞毒活性。因此，本项目选择拟海桑进行细致的化学成分研究，期望发现海洋来源活性先导化合物。
拟海桑化学成分的研究只有一篇报道，2012年郭跃伟课题组对采自广东湛江的拟海桑进行化学成分研究，从中分离得到一个新骨架化合物ParacaseolideA(69)是一个二聚-烷基丁内酯类化合物内酯甲素，体外药理活性筛选显示该化合物对细胞周期分裂蛋白25B具有显著的抑制活性。
发明内容
本发明旨在为拟海桑的提取物提供一种方便可靠的生物活性测定方法。
拟海桑提取物的生物活性CPE+MTT测定方法：包括MTT法和CPE法；
MTT法：MDCK细胞37ºC下在通入5%CO2和95%氧气，含有10%FBS的RPMI-1640细胞悬浮液中培养。将细胞密度调至5×104个/mL，在96孔细胞培养板中每孔加入200uL培养24h。每孔加入2uL，50ug/mL含药物的甲醇溶液的维持液继续培养72h。然后，每孔加入20uLMTT(5mg/mL的RPMI-1640)溶液，继续培养4h。以DMSO更换溶解MTT的介质，至Formazan结晶完全溶解。酶标仪测定每孔540nm处的吸光值(OD值)。获得剂量反应曲线，计算IC50。
CPE法：单层MDCK细胞首先与流感病毒(A/PuertoRico/8/34(H1N1),PR/8)于37ºC培养1h。弃去病毒稀释液，将细胞于37ºC保持在含有不同浓度测试药物的感染媒介中(RPMI1640,4ug/mL胰蛋白酶)。37ºC培养48h后，用100uL4%的多聚甲醛与MDCK细胞在室温下固定20min。除去甲醛后，MDCK细胞用0.1%的结晶紫染色30min。将板洗净干燥，酶标仪(Bio-Rad,USA)在570nm处测定各孔中结晶紫染色的强度。计算IC50值，即感染后48h抑制CPE为50%时的化合物浓度。
本发明的测定方法相对于现有技术简单快速、而且检测结果可靠，适于推广应用。
具体实施方式
下面结合具体实施例对本发明做进一步详细说明。
采用MTT+CPE法，对从拟海桑中提取的三萜类和木质素类化合物进行抗流感病毒H1N1活性筛选，具体方法如下：
MTT法：MDCK细胞37ºC下在通入5%CO2和95%氧气，含有10%FBS的RPMI-1640细胞悬浮液中培养。将细胞密度调至5×104个/mL，在96孔细胞培养板中每孔加入200uL培养24h。每孔加入2uL，50ug/mL含药物的甲醇溶液的维持液继续培养72h。然后，每孔加入20uLMTT(5mg/mL的RPMI-1640)溶液，继续培养4h。以DMSO更换溶解MTT的介质，至Formazan结晶完全溶解。酶标仪测定每孔540nm处的吸光值(OD值)。获得剂量反应曲线，计算IC50。
CPE法：单层MDCK细胞首先与流感病毒(A/PuertoRico/8/34(H1N1),PR/8)于37ºC培养1h。弃去病毒稀释液，将细胞于37ºC保持在含有不同浓度测试药物的感染媒介中(RPMI1640,4ug/mL胰蛋白酶)。37ºC培养48h后，用100uL4%的多聚甲醛与MDCK细胞在室温下固定20min。除去甲醛后，MDCK细胞用0.1%的结晶紫染色30min。将板洗净干燥，酶标仪(Bio-Rad,USA)在570nm处测定各孔中结晶紫染色的强度。计算IC50值，即感染后48h抑制CPE为50%时的化合物浓度。
结果见表1，测试结果表明，在50ug/mL浓度水平，化合物SP-1、4、5和17显示中等强度抗病毒活性，化合物SP-10、16和32显示弱的抗病毒活性。
表1部分单体化合物对H1N1流感病毒致细胞(MDCK)病变的抑制率
对以上有抗H1N1病毒活性的化合物(抑制率大于30%)进行了IC50测试，IC50结果显示只有化合物SP-1对H1N1病毒具有强的抑制活性，IC50值为28.4ug/mL。非常接近阳性药利巴韦林的活性。

Claims (1)

1.拟海桑提取物的生物活性CPE+MTT测定方法，其特征在于，包括MTT法和CPE法；
MTT法：MDCK细胞37ºC下在通入5%CO2和95%氧气，含有10%FBS的RPMI-1640细胞悬浮液中培养；将细胞密度调至5×104个/mL，在96孔细胞培养板中每孔加入200uL培养24h；每孔加入2uL，50ug/mL含药物的甲醇溶液的维持液继续培养72h；然后，每孔加入20uLMTT(5mg/mL的RPMI-1640)溶液，继续培养4h；以DMSO更换溶解MTT的介质，至Formazan结晶完全溶解；酶标仪测定每孔540nm处的吸光值(OD值)；获得剂量反应曲线，计算IC50；
CPE法：单层MDCK细胞首先与流感病毒(A/PuertoRico/8/34(H1N1),PR/8)于37ºC培养1h；弃去病毒稀释液，将细胞于37ºC保持在含有不同浓度测试药物的感染媒介中(RPMI1640,4ug/mL胰蛋白酶)；37ºC培养48h后，用100uL4%的多聚甲醛与MDCK细胞在室温下固定20min；除去甲醛后，MDCK细胞用0.1%的结晶紫染色30min；将板洗净干燥，酶标仪(Bio-Rad,USA)在570nm处测定各孔中结晶紫染色的强度；计算IC50值，即感染后48h抑制CPE为50%时的化合物浓度。
CN201510960617.2A 2015-12-21 2015-12-21 拟海桑提取物的生物活性cpe+mtt测定方法 Pending CN106893758A (zh)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111676185A (zh) * 2020-06-29 2020-09-18 肇庆大华农生物药品有限公司 一种全悬浮培养型mdck细胞系的驯化方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111676185A (zh) * 2020-06-29 2020-09-18 肇庆大华农生物药品有限公司 一种全悬浮培养型mdck细胞系的驯化方法

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