摘要
目的从黄芩中提取具有拮抗内毒素(LPS)活性的物质。方法采用大孔吸附树脂和高效液相色谱(HPLC)等技术将黄芩水提物分离成若干组分,应用生物传感器技术检测各组分与LPS的活性中心LipidA的结合活性,动态比浊法鲎试验检测各组分(2.0mg/L)对LPS(0.2μg/L)的中和作用,籍此筛选出活性组分;逆转录聚合酶链式反应(RT-PCR)检测所筛组分对LPS诱导的RAW264.7细胞的Toll样受体4(TLR4)mRNA表达的影响,ELISA法检测所筛组分对LPS(100μg/L)刺激的RAW264.7细胞释放肿瘤坏死因子α(TNF-α)的影响。结果获得5种HPLC产物S3K1-5,其中S3K1与LipidA结合活性最高,S3K1(2.0mg/L)可显著抑制LPS(0.2μg/L)介导的鲎试剂的凝结反应(P〈0.05);以LPS(100μg/L)刺激RAW264.7细胞,其TLR4mRNA表达增强,给予40、80、160mg/L的S3K1预处理后能减弱LPS诱导RAW264.7细胞的TLR4mRNA表达(P〈0.05);单纯LPS刺激RAW264.7细胞释放的TNF-α为(1757.61±207.25)ng/L(对照组),给予40、80、160mg/L的S3K1预处理后TNF-α浓度依次为(1324.53±149.73)、(893.87±140.77)、(799.97±124.21)ng/L,随S3K1剂量递增而减低,均显著低于对照组(P〈0.05)。结论从黄芩中提取到具有拮抗LPS活性的组分S3K1,S3K1在体外对LPS具有一定的中和作用,可抑制LPS诱导的RAW264.7细胞的TLR4mRNA表达,进而抑制LPS诱导的RAW264.7细胞活化。
Objective To isolate and extract the anti-lipopolysaccharide (LPS) active compounds from Scutellaria baicalensis Georgi. Methods A series of fractions was prepared by macroporous adsorptive resins and HPLC from the aqueous extract of the herb. Their binding activity to lipid A and the neutralization to LPS were detected by the biosensor technology and kinetic turbidimetric limulus test. The active compounds against LPS were screened. RT-PCR and ELISA was used respectively to detect the expression of TLR4 mRNA and measure the release of TNF-α in RAW264.7 ceils exposure to LPS ( 100 μg/L) with or without the screened compound pretreatment. Results Five HPLC products were isolated from the herb and named from S3K1 to 5. Among them, S3K1 had the highest bind activity to lipid A and significantly inhibited the coagulation reaction induced by LPS (0.2 μg/L) at the dose of 2.0 mg/L. The expression of TLR4 mRNA was increased after the ceils exposure to LPS, however, this effect was attenuated by the pretreatment with S3K1 in doses of 40, 80, 160 mg/L. The supernatant TNF-α level was significant decreased in a negatively dose-dependent manner when S3K1 was used to treat the ceils before LPS stimulation. Conclusion An active anti-LPS compound, S3K1 is attained from Scutellaria baicalensis Georgi, which neutralizes LPS, inhibits the expression of TLR4 mRNA and suppresses the activity of RAW264.7 ceils after exposure to LPS.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2008年第5期382-385,共4页
Journal of Third Military Medical University
基金
国家重点基础研究发展规划项目("973"项目)(2005CB522600)
重庆市自然科学基金(2005BB5290)~~
关键词
黄芩
内毒素
生物传感器
大孔吸附树脂
Scutellaria baicalensis Georgi
lipopolysaccharide
biosensor
macroporous adsorptive resins