Mechanisms of imperatorin on regulating Pglycoprotein in blood-brain barrier based on network pharmacology and in vitro experiment

OBJECTIVE To explore mecha⁃nisms of imperatorin on regulating P-glycoprotein(P-gp)in blood-brain barrier(BBB)based on net⁃work pharmacology combined with in vitro experi⁃ment.METHODS Drug targets were predicted using the Pharmapper and Swiss targets data⁃bases;disease targets were obtained through the Genecards database;intersections between drugs and disease targets were screened by Cytoscape software;the obtained core targets were used to construct protein-protein interaction(PPI)network,gene ontology(GO)functions,and Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analysis.The effects of imperatorin(20,50,100μmol·L^(-1))on P-gp activity were monitored in hCMEC/D3 in vitro BBB model,and the effects of imperatorin on the expression of target proteins were verified using Western blot method.RESULTS 55 drug targets and 3102 disease targets were obtained from the network pharmacology screening,and 37 core targets were obtained after the combination.Enrichment analysis showed that core targets were closely related to chemical synaptic trans⁃mission regulation,neurotransmitter receptor activity,protein kinase regulation activity,G proteincoupled receptor signaling pathway,neural active ligand receptor interaction pathway,PI3K-Akt sig⁃naling pathway,VEGF signaling pathway,etc..In vitro experimental validation suggested that all tested concentration groups of imperatorin signifi⁃cantly reduced the activity and expression of P-gp,which were achieved by significantly downregu⁃lating the phosphorylation levels of PI3K and Akt,and repressing the expression of VEGFR2 pro⁃tein.CONCLUSION Network pharmacology was used to predict the core targets and signaling pathways of imperatorin on regulating P-gp in BBB and relevant validation was conducted through in vitro experiments,providing a refer⁃ence basis for further exploration of the mecha⁃nisms of imperatorin on regulating P-gp in BBB.

An LC-MS/MS Method for Determination of Imperatorin and Isoimperatorin in Rat Plasma and Application to a Pharmacokinetic Study

A highly sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for the de-termination of imperatorin and isoimperatorin in rat plasma using psoralen as internal standard. Analyses were performed on a Shimadzu HPLC system using a Shimadzu C18 column eluted with a mobile phase of methanol-water (25:75), containing 0.1% formic acid at a flow rate of 0.4 ml/min. The detection was performed by positive ion elec-trospray ionization in multiple reactions monitoring mode, monitoring the transitions m/z 203→147 and m/z 203→159 for imperatorin and isoimperatorin, respectively. The method was validated over the concentration range of 2.5 -1000.0 ng/mL for imperatorin and isoimperatorin. The limits of detection and quantification were 1.0 and 3.0 ng/mL, repectively for both analytes. The intra-day and inter-day accuracy and precision of the assay were less than 10.9%. This method has been applied successfully to a pharmacokinetic study involving the intragastric administration of extract of Angelicae Dahuricae Radix to rats.

Protective effects of imperatorin against cerebral ischemia/reperfusion-induced oxidative stress through Nrf2 signaling pathway in rats

OBJECTIVE To investigates the effects of imperatorin on the oxidative stress in the cerebral cortex and hippocampus after focal cerebral ischemia/reperfusion injury.METHODS Transient focal cerebral ischemia/reperfusion model in male Sprague-Dawley rats was induced by 2 h middle cerebral artery occlusion followed by 24 h reperfusion.Imperatorin(1.25 and 2.5 mg·kg-1)or vehicle were administered intraperitoneally at 1,5 and 9 h after the onset of ischemia.At 24 h after reperfusion,the biomarkers of oxidative stress such as the levels of reactive oxygen species(ROS),lipid peroxidation products malondialdehyde(MDA),nitric oxide(NO)and total antioxidant capacity(T-AOC),the activities of inducible nitric oxide synthase(iN OS),superoxide dismutase(SOD)and catalase(CAT)in the cerebral cortex and hippocampus were observed.We also assessed the nuclear factor erythroid 2-related factor 2(Nrf2),heme oxygenase-1(HO-1),and the NAD(P)H-quinone oxidoreductase 1(NQO-1)protein expression by Western blot.RESULTS As compared to vehicle-treated animals,imperatorin treatment significantly reduced the ROS,MDA,NO levels and i NOS activity,increased T-AOC and the activities of SOD and CAT.Furthermore,imperatorin treatment also significantly induced the nuclear translocation of Nrf2,enhanced the protein expression of HO-1 and NQO-1 in the cerebral cortex and hippocampus.CONCLUSION Our findings indicate that imperatorin can protect the brain against the excessive oxidative stress induced by cerebral ischemia/reperfusion through activation of Nrf2 signaling pathway.

Identification of Herbal Compound Imperatorin with Adverse Effects on ANO1 and CFTR Chloride Channels

Calcium-activated chloride channels（CaCCs） are the crucial regulators of transepithelial fluid secretion, smooth muscle contraction and sensory transduction. Recently, compelling evidence has indicated that TMEM16A（ANO1 or anoctamin-1） is a bona fide calcium-acvtivated chloride channel. A few small molecule CaCCs regulators are available for functional and therapeutic studies. We screened 126 natural compounds from Chinese herbs. Screening was performed with an iodide influx assay in Fischer rat thyroid epithelial cells to coexpress ANO1 and an iodide-sensitive fluorescent indicator（EYFP-H148Q/I152L）. Imperatorin, a coumarin compound, was identified to inhibit ANO1-mediated chloride transport activated by multiple calcium-elevating agonists. The inhibitory effect is dose-dependent with IC50～14.63 μmol/L. Interestingly, imperatorin activated CFTR chloride channel with EC50～35.52 μmol/L. The adverse effects of imperatorin on CaCC and CFTR chloride channels will make it useful in pharmacological dissection of chloride transport in airway and intestinal epithelium. Further studies are required to evaluate the therapeutic effects of imperatorin on hypertension, asthma and certain tumors.

Imperatorin alleviates Aβ-induced spatial learning memory impairment and neuroinflam⁃mation in model mice of Alzheimer disease

OBJECTIVE To investigate the effects of imperatorin on the spatial learning memory impairment and neuroinflammation in model mice of Alzheimer disease(AD)induced by intracerebroventricular injection of Aβ1-42.METHODS Mouse model of AD was established by injection of Aβ1-42 into the lateral ventricles.Im⁃peratorin(2.5 and 5.0 mg·kg-1,daily)was inject⁃ed by intraperitoneally 1 h after intracerebroven⁃tricular injection for 13 d.The effect of imperato⁃rin on the spatial learning and memory impair⁃ment was assessed by eight arm maze tests.The levels of cytokines TNF-α,IL-1β,IL-6,IL-18 and chemokines MCP-1 in mouse cortex and hip⁃pocampus were detected by ELISA.The protein expression of NF-κB P65,TLR4,MyD88,p-P38,p-ERK,and p-JNK were detected by Western blotting.RESULTS As compared with the AD model group,imperatorin treatment significantly attenuated Aβ1-42-induced spatial learning and memory impairment assessed by eight arm maze tests.In addition,imperatorin significantly reduced the levels of cytokines TNF-α,IL-1β,IL-6,IL-18 and chemokines MCP-1 in the cerebral cortex and hippocampus.Meanwhile,Western blotting results showed that imperatorin treat⁃ment significantly down-regulated the protein expression of NF-κB P65,TLR4,MyD88,p-P38,p-ERK,and p-JNK.CONCLUSION Imperatorin has neuroprotective effects in the Aβ1-42 induced AD model mice and its mechanism may be partially associated with the inhibition of inflam⁃matory response in the cortex and hippocampus.

Estrogenic activity of osthole and imperatorin in MCF-7 cells and their osteoblastic effects in Saos-2 cells

There is an increasing interest in phytoestrogens due to their potential medical usage in hormone replacement therapy(HRT). The present study was designed to investigate the in vitro effects of estrogen-like activities of two widespread coumarins, osthole and imperatorin, using the MCF-7 cell proliferation assay and their alkaline phosphatase(ALP) activities in osteoblasts Saos-2 cells. The two compounds were found to strongly stimulate the proliferation of MCF-7 cells. The estrogen receptor-regulated ERα, progesterone receptor(PR) and PS2 m RNA levels were increased by treatment with osthole and imperatorin. All these effects were significantly inhibited by the specific estrogen receptor antagonist ICI182, 780. Cell cycle analysis revealed that their proliferation stimulatory effect was associated with a marked increase in the number of MCF-7 cells in S phase, which was similar to that observed with estradiol. It was also observed that they significantly increased ALP activity, which was reversed by ICI182,780. These results suggested that osthole and imperatorin could stimulate osteoblastic activity by displaying estrogenic properties or through the ER pathway. In conclusion, osthole and imperatorin may represent new pharmacological tools for the treatment of osteoporosis.

欧前胡素调节ERK/MAPK信号通路对肺结核大鼠炎症反应的影响

目的 探讨欧前胡素(Imp)调节细胞外调节蛋白激酶(ERK)/有丝分裂原激活蛋白激酶(MAPK)信号通路对肺结核大鼠炎症反应的影响。方法 64只大鼠随机分为对照组12只及造模组52只,造模组大鼠通过尾部注射结核杆菌方法建立肺结核大鼠模型,之后随机分为模型组、Imp低(Imp-L,25 mg/kg)、中(Imp-M,50 mg/kg)、高剂量(Imp-H,100 mg/kg)组、Imp-H+ERK特异性激活剂(EGF,100 mg/kg Imp+25 mg/kg EGF)组。干预结束后,主动脉采血,ELISA检测各组大鼠血清中白细胞介素-6(IL-6)、γ干扰素(IFN-γ)、环氧化酶-2(COX-2)水平;分离肺组织,HE、Tunel分别检测大鼠肺组织病理学变化及细胞凋亡情况;统计肺组织中结核杆菌菌落数;Western blot检测p-ERK1/2/ERK1/2、p38 MAPK表达水平。结果 与对照组相比,模型组肺组织严重病变,IL-6、IFN-γ、COX-2、细胞凋亡率、结核杆菌菌落数、p-ERK1/2/ERK1/2、p38 MAPK表达均显著增加(P<0.05);与模型组相比,Imp-L组、Imp-M组、Imp-H组病理损伤得到缓解,IL-6、IFN-γ、COX-2、细胞凋亡率、结核杆菌菌落数、p-ERK1/2/ERK1/2、p38 MAPK表达显著降低,以Imp-H组变化最为显著(P<0.05);与Imp-H组相比,Imp-H+EGF组病理损伤进一步加重,IL-6、IFN-γ、COX-2、细胞凋亡率、结核杆菌菌落数、p-ERK1/2/ERK1/2、p38 MAPK表达显著增加(P<0.05)。结论 Imp可降低肺结核炎症反应,与抑制ERK/MAPK信号通路的激活有关。

基于网络药理学与分子对接的欧前胡素治疗宫颈癌的作用机制

目的探讨欧前胡素治疗宫颈癌的活性成分和作用机制。方法通过使用网络药理学筛选欧前胡素药用成分和疾病宫颈癌靶点并进行相关的分子对接技术验证。中草药系统药理平台查找欧前胡素的有效成分和作用靶点,采用OMIM和Gen⁃eCards数据库,对宫颈癌相关的目标基因进行交叉筛选,Cytoscape软件分析宫颈癌和欧前胡素的共同靶点,String数据库分析并建构PPI网络分析,Rstudo 4.0软件分析并绘图,DAVID生物信息数据库统计,GO富集、KEGG富集分析。分子对接使用AutoDock分析。结果PPI网络结果显示TERT、ESR1、PTGS2、PGR、PPARG、CA9、ESR2、AR、CASP9、ALPL、CLK1可能为欧前胡素和宫颈癌的核心靶点。GO富集得到26个生物过程,KEGG富集得到6条主要通路,包括Chemical cancer recep⁃tor activation、Estrogen、Breast cancer、VEGF、Prolactin等信号通路。分子对接结果显示LYS-243、ARG-240和GLU-272等化合物能与宫颈癌的核心靶点有良好的结合力。结论欧前胡素可能通过与ESR1、PGR、PTGS2、ESR2、CASP9等结合,在VEGE、雌激素、孕激素等信号通路发挥抗治疗宫颈癌的作用。

利鼻片HPLC指纹图谱及多组分含量测定

目的建立多组分含量测定结合化学计量学分析,评价利鼻片质量。方法采用高效液相色谱法,Waters SunFire C18色谱柱(4.6 mm×250 mm,5μm);乙腈(A)-0.55%磷酸水溶液(B)为流动相;梯度洗脱,流速:1.0 ml·min^(-1);切换检测波长:323、280、248 nm;柱温:35℃,进样量:10μl。采用中药色谱指纹图谱相似度评价系统结合聚类分析与正交偏最小二乘判别分析评价15批利鼻片质量。结果建立的利鼻片HPLC指纹图谱共确定了19个共有峰,并指认出其中7个色谱峰;利鼻片中菊苣酸、绿原酸、咖啡酸、黄芩苷、木兰脂素、欧前胡素、异欧前胡素的平均回收率分别为100.30%、99.87%、99.25%、99.45%、99.08%、98.75%和98.58%,RSD均<1.5%(n=6)。15批利鼻片中上述7个成分的含量测定结果分别为15.05~17.93、10.17~12.51、7.03~9.30、4.42~6.50、1.08~2.76、1.01~1.76、1.00~1.90 mg·g^(-1)。不同批次利鼻片存在差异,区分的特征峰为菊苣酸、绿原酸和木兰脂素。聚类分析结果表明3家企业的样品各自聚为一类,S1~S4(陕西康惠)聚为一类,S5~S10(通化民泰)聚为一类,S11~S15(长春银诺克)聚为一类。正交偏最小二乘法判别分析结果表明这4种化学成分〔10号峰菊苣酸(VIP值为2.216)、6号峰未知(VIP值为1.934)、4号峰绿原酸(VIP值为1.438)、13号峰木兰脂素(VIP值为1.013)〕显著影响利鼻片的分类,是引起该制剂质量差异的潜在标志性成分,可以考虑将这些差异标志性成分作为指标成分监控利鼻片的质量。结论建立的综合质量评价法简便、专属、准确,可客观评价利鼻片质量。

欧前胡素通过抑制NLRP3炎性通路改善大鼠慢性子宫内膜炎症

目的探讨欧前胡素(imperatorin,Imp)对慢性子宫内膜炎(chronic endometritis,CE)的治疗及可能的作用机制。方法采用卵巢处注射苯酚胶浆法建立CE大鼠模型,分为对照组(CT组)、CE组、Imp低剂量组(ImpL组,5 mg∙kg^(-1)∙d^(-1))、Imp高剂量组(ImpH组,10 mg∙kg^(-1)∙d^(-1))、ImpH+脂多糖(LPS,10 mg/kg)组,称量质量检测子宫脏器指数;苏木精-伊红(HE)染色观察子宫组织病理变化;免疫组化检测子宫内膜血管内皮生长因子(VEGF)表达水平;ELISA测定血清白介素(IL)-6、肿瘤坏死因子(TNF-α)的含量;RT-qPCR和Western blot验证子宫组织IL-1β、核因子κB(NF-κB)、NOD样受体热蛋白结构域相关蛋白3(NLRP3)、基质金属蛋白酶(MMP)-9的表达。结果与CT组相比,CE组大鼠子宫和阴道内肿胀、充血,子宫内膜VEGF的表达降低(P<0.05),血清IL-6、TNF-α含量和子宫组织IL-1β、p-NF-κB/NF-κB、NLRP3、MMP-9表达增加(P<0.05);与CE组比较,ImpL、ImpH组大鼠子宫和阴道内结节肿胀、充血均有所缓解,子宫内膜VEGF的表达增加,血清IL-6、TNF-α含量和子宫组织IL-1β、p-NF-κB/NF-κB、NLRP3、MMP-9表达减少(P<0.05);LPS可部分逆转ImpH对CE大鼠的改善作用。结论Imp可能通过抑制NLRP3炎症通路的激活对CE有一定治疗作用。

欧前胡素抑制人肾癌786-O细胞增殖并促进其凋亡

目的探究欧前胡素(imperatorin)对人肾癌细胞786-O增殖及凋亡的影响,初步研究其作用机制。方法分别使用0,20,40,60,80,100,120,140,160,180,200μmol/L的欧前胡素处理786-O细胞24,48,72 h后,CCK-8法检测786-O细胞增殖,并计算IC 50,筛选最佳干预浓度进行后续实验。生物显微镜观察细胞形态变化;CCK-8实验和克隆形成实验检测各组细胞增殖能力的变化;流式细胞术检测各组细胞凋亡的情况;RT-qPCR和Western blot方法检测各组Fas、Caspase-8、Caspase-3、Bcl-2的表达情况。从TCMSP和HERB数据库收集欧前胡素作用靶点。由GeneCards数据库获取肾癌疾病靶点,使用STRING数据库构建蛋白互作网络,并筛选欧前胡素治疗肾癌的核心靶点。结果欧前胡素作用786-O细胞24 h和48 h的IC 50分别为94.36μmol/和65.66μmol/L。与0μmol/L组相比,40μmol/L组786-O细胞体积减小,细胞密度略减少,增殖能力受到抑制(P<0.01),细胞凋亡率增加(P<0.01);80μmol/L组细胞壁皱缩明显,细胞密度明显减少,增殖能力明显被抑制(P<0.001),细胞凋亡率明显增加(P<0.001)。RT-qPCR和Western blot实验结果显示,与0μmol/L组相比,40μmol/L组Fas、Caspase-8、Caspase-3的表达上调(P<0.05),80μmol/L组上述3个分子表达明显上调(P<0.01),Bcl-2则无明显变化。通过网络药理学方法筛选出7个欧前胡素治疗肾癌的核心靶点,分别为GSK3β、CDK2、PPARG、ESR1、MAPK14、AR、PTGS2。结论欧前胡素可能是通过靶向GSK3β调控死亡受体途径,进而抑制肾癌786-O细胞的增殖,并促进其凋亡。

紫色土中氮肥、磷肥、钾肥配施对白芷产量及有效成分的影响

在国家防止耕地“非粮化”政策下,为合理利用四川地区紫色土(坡地、荒地等)进行白芷种植,本研究采用3因素5水平二次正交回归旋转组合设计,探究紫色土中氮肥、磷肥、钾肥配施对川芷2号白芷品种产量和有效成分含量的影响。结果表明,紫色土中氮肥、磷肥、钾肥配施对白芷产量的影响大小为钾肥>磷肥>氮肥,钾肥和磷肥对产量的影响都达到显著或极显著水平,氮肥对白芷产量没有显著影响;氮肥、磷肥、钾肥配施对欧前胡素含量的影响为钾肥>氮肥>磷肥,对异欧前胡素含量的影响为磷肥>氮肥>钾肥,可见钾肥显著影响欧前胡素含量,磷肥显著影响异欧前胡素含量。通过统计频数法优化出施肥配方,在氮肥施用量为233.73~283.36 kg/hm^(2)、磷肥施用量为1380.66~1522.04 kg/hm^(2)、钾肥施用量为215.14~240.03 kg/hm^(2)时川芷2号能实现高产,在氮肥施用量为240.39~276.58 kg/hm^(2)、磷肥施用量为1097.69~1197.45 kg/hm^(2)、钾肥施用量为163.12~185.91 kg/hm^(2)时可以均衡地提高川芷2号根中欧前胡素和异欧前胡素含量。最终利用灰色关联度和逼近理想解排序法(DTOPSIS法)综合获得在紫色土中川芷2号的最优施肥方案:氮肥施用量为193.40 kg/hm^(2)、磷肥施用量为1449.26 kg/hm^(2)、钾肥施用量为239.88 kg/hm^(2)。

欧前胡素对恶病质模型小鼠的改善作用及机制研究

目的探讨欧前胡素对恶病质模型小鼠的改善作用及机制。方法将15只雄性C57BL/6J小鼠随机分为空白对照组、模型组和欧前胡素组,每组5只。除空白对照组外,余下小鼠均于背部皮下接种小鼠肺癌LLC细胞混悬液,于接种的第7天开始每日灌胃给药,欧前胡素组按60 mg/kg灌胃欧前胡素混悬液(溶剂为0.5%羧甲基纤维素钠溶液),空白对照组和模型组给予等体积0.5%羧甲基纤维素钠溶液,连续给药13 d。给药期间,每日定时记录小鼠的摄食量和体质量,每两日测量一次肿瘤的长径和短径,计算肿瘤体积,取材当日称量各组小鼠骨骼肌质量和肿瘤质量,并计算去瘤体质量;观察小鼠骨骼肌病理学变化,计算骨骼肌纤维横截面积;检测小鼠骨骼肌中信号转导和转录因子3(STAT3)磷酸化水平(以p-STAT3/STAT3比值计),肌肉萎缩盒F蛋白(MAFbx)、肌细胞生成素(Myog)、B细胞淋巴瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)、胱天蛋白酶3(Caspase3)蛋白和mRNA表达水平。结果与空白对照组比较,模型组小鼠体质量和骨骼肌质量均显著降低(P<0.05),摄食量有所下降;骨骼肌排列疏松,细胞间隙较大,骨骼肌纤维横截面积显著缩小;p-STAT3/STAT3比值和MAFbx、Bax、Caspase3蛋白表达量以及mRNA表达水平均显著升高(P<0.05);Myog、Bcl-2蛋白表达量以及mRNA表达水平均显著降低(P<0.05)。与模型组比较,欧前胡素组小鼠体质量、去瘤体质量、骨骼肌质量均显著升高(P<0.05),摄食量有所提高,同时肿瘤质量和肿瘤体积均显著降低(P<0.05);上述蛋白以及基因的表达均有显著改善(P<0.05)。结论欧前胡素能改善小鼠肿瘤恶病质状态,其作用机制可能与调节泛素-蛋白酶体系统、抗细胞凋亡相关。

当归头基于BP-ANN结合信息熵赋权法提取工艺参数优化

目的:通过反向传播神经网络(BP-ANN)结合熵权法多指标优化当归头的提取工艺。方法:以提取次数、加液量、提取时间为考察因素,用熵权法计算阿魏酸、绿原酸、欧前胡素、藁本内酯、干浸膏得率含量的多指标综合评分作为评价指标,先采用正交设计,再建立反向传播神经网络模型,通过网络训练,预测当归头的最优提取工艺。结果:优化得到当归头最优提取工艺为加9.6倍量水,提取时间67 min,提取3次,检测样本的网络预测值和实际测量值的相对误差小于1%。结论:通过相关数学模型分析和预测所得的当归头提取工艺稳定可行,可有效提高当归头中有效成分的提取效率。

基于色泽与多指标成分分析的川明参质量快速评价研究

[目的]探索川明参外观色泽与多个指标成分间的关系,为快速、高效筛选优质川明参药材提供参考。[方法]以不同商品规格川明参药材为研究对象,首先利用分光测色仪测定川明参粉末色度值,然后采用热浸法、硫酸-苯酚法以及超高效液相色谱法分别测定水溶性浸出物、醇溶性浸出物、多糖、欧前胡素等指标成分质量分数,最后使用IBM SPSS Statistics 26.0软件分析色度值与各指标成分间的相关性。[结果]川明参粉末色度值L*与醇溶性浸出物、水溶性浸出物以及欧前胡素质量分数呈极显著负相关(P<0.01),与多糖质量分数呈显著负相关(P<0.05);ΔE*与水溶性浸出物及欧前胡素质量分数呈极显著负相关(P<0.01),与醇溶性浸出物、多糖质量分数呈显著负相关(P<0.05);a*、b*与醇溶性浸出物、水溶性浸出物、欧前胡素质量分数呈极显著正相关(P<0.01),与多糖质量分数呈显著正相关。[结论]色度值可作为快速判断评价川明参药材质量的指标,为全面评价川明参药材品质提供科学依据。

欧前胡素酯类衍生物的合成及抗肿瘤活性研究

天然产物欧前胡素具有广泛的抗肿瘤活性,应用前景巨大。本论文以欧前胡素为先导化合物,通过氧化、还原、酯化等反应设计合成了13个欧前胡素酯类衍生物,并经^(1)H NMR、^(13)C NMR、HRMS进行结构确证。通过MTT法测定了目标化合物在200μmol·L^(-1)下对宫颈癌细胞HeLa、结肠癌细胞HCT116和肝癌细胞HePG2三种肿瘤细胞的体外抗肿瘤活性。结果显示目标化合物3g对肝癌细胞HePG2表现出显著的抑制活性,超过了原料欧前胡素,与阳性对照药索拉非尼和5-氟尿嘧啶相当。分子对接结果显示3g可以和抗凋亡蛋白Mcl-1通过多种作用力稳定结合。

禹白芷苯丙氨酸解氨酶基因AdPAL1的克隆与表达分析

【目的】探究禹白芷中欧前胡素含量与苯丙氨酸解氨酶基因AdPAL1表达的相关性,并对AdPAL1进行克隆和原核表达。【方法】使用高效液相色谱法测定禹白芷快速生长期和收获期根和叶中欧前胡素的含量,并使用实时荧光定量PCR(qRT-PCR)方法测定AdPAL1基因在同时期根和叶中的表达情况,以观察欧前胡素含量与AdPAL1基因表达是否具有相关性。此外,以禹白芷转录组为基础,采用逆转录PCR(RT-PCR)技术从禹白芷根中克隆AdPAL1基因,并在大肠杆菌中进行表达。【结果】禹白芷快速生长期根中的欧前胡素含量远高于叶,而AdPAL1基因在根中的表达量也远高于叶。与快速生长期相比,收获期根中欧前胡素含量略有下降,AdPAL1基因在根中的表达量也同时下降。这些结果表明AdPAL1基因表达与欧前胡素含量具有相关性。此外,生物信息学分析显示,禹白芷AdPAL1基因(GenBank登录号:OQ822236)开放阅读框长1764 bp,编码557个氨基酸,其蛋白相对分子质量为61.10 kD,等电点为5.92,且具有苯丙氨酸解氨酶保守结构域(IPR005922,1~547),属于PAL蛋白家族成员。AdPAL1蛋白定位于细胞质,主要由α-螺旋和无规则卷曲构成,与石防风属植物KpPAL2蛋白亲缘关系最近,序列相似性高达99.28%。AdPAL1基因在大肠杆菌中表达的重组蛋白相对分子质量约为84.00 kD,与预期蛋白大小一致。【结论】初步确定禹白芷根和叶中欧前胡素含量与AdPAL1基因表达相关,并成功克隆AdPAL1基因,在大肠杆菌中成功表达。上述结果为进一步研究AdPAL1基因在欧前胡素生物合成中的作用机制提供参考。

欧前胡素调节MAPK/mTOR/p70S6K信号通路对急性髓系白血病细胞恶性生物学行为的影响

目的探讨欧前胡素(IMP)对急性髓系白血病(AML)细胞恶性生物学行为的影响以及对MAPK/mTOR/p70S6K信号通路的调节机制。方法将人AML细胞HL-60进行培养传代,并分为对照组(未处理组),ERK抑制剂组(PD98059组),IMP低、中、高剂量组,IMP高剂量+ERK激活剂组(IMP高+Cearoin组),每组均设置6个重复。MTT法检测IMP对HL-60细胞的毒性;MTT法、软琼脂克隆形成实验检测HL-60细胞的增殖活性;流式细胞仪术检测HL-60细胞凋亡;Transwell小室检测HL-60细胞迁移和侵袭能力;ELISA检测HL-60细胞培养液中TNF-α、IL-1β、IL-6的水平;Western blot检测通路相关蛋白及Bcl-2、Beclin-1、LC3Ⅱ/Ⅰ蛋白的表达。结果与未处理组比较,PD98059组和IMP低、中、高剂量组HL-60细胞的存活率、克隆形成数量、迁移和侵袭数量、促炎因子TNF-α、IL-1β、IL-6水平以及ERK1/2、mTOR、p70S6K磷酸化水平、Bcl-2表达显著降低(P<0.05),细胞凋亡率、自噬相关蛋白Beclin-1、LC3Ⅱ/Ⅰ显著升高(P<0.05);与IMP高剂量组比较,IMP高+Cearoin组中ERK激活剂明显消除了IMP对上述指标的影响(P<0.05)。结论IMP可能通过抑制MAPK/mTOR/p70S6K信号通路,抑制AML细胞增殖、迁移和侵袭,促进细胞自噬和凋亡。

LC-MS/MS测定精油和面膜中禁用白芷组分欧前胡素和异欧前胡素

建立一种三重四极杆液相色谱-串联质谱法(LC-MS/MS),对精油和面膜中禁用白芷组分欧前胡素和异欧前胡素进行定性定量分析。试验前处理采用QuEChERS方法,制备样以乙腈作为萃取溶剂,以QuEChERS法进行提取,再经LC-MS/MS分析,以基质空白配标,外标法定量。结果显示欧前胡素和异欧前胡素在0.5~5.0 ng/mL范围内具有较好的线性关系,相关系数均大于0.99。加标水平在10、20、50 ng/g时,欧前胡素和异欧前胡素的回收率范围在84.3%~98.3%,目标物相对标准偏差<8.0%。此方法适用于精油和面膜化妆品中禁用白芷组分欧前胡素和异欧前胡素的分析检测。

HPLC法同时测定生骨补肾胶囊中两种活性成分含量研究

目的:同时建立生骨补肾胶囊中两种活性成分的测定方法,对该制剂的质量进行评价和控制。方法:采用HPLC法,Shim-pack VP-ODS色谱柱(4.6 mm×150 mm, 5μm),梯度洗脱:甲醇(A)-水(B)为流动相,流速0.8 mL/min,检测波长丹参酮IIA 270 nm、欧前胡素320 nm;柱温25℃,进样量20μL,制备3批供试品对丹参酮IIA、欧前胡素进行指标成分检测。结果:丹参酮IIA在3.75~200μg/mL线性关系良好(r=0.999 7),欧前胡素在3~120μg/mL线性关系良好(r=0.999 8)。结论:本研究所用方法简便快速、准确可靠,可作为生骨补肾胶囊的质量控制参考依据。