网络药理学及分子对接法探讨三仁汤主要成分对新型冠状病毒肺炎（COVID-19）的机制研究

目的通过网络药理学及分子对接法探讨三仁汤主要成分对新型冠状病毒肺炎(COVID-19)的作用机制。方法通过中药系统药理学分析平台(TCMSP)及中医整合药理学研究平台V2.0(TCMIP V2.0)获得三仁汤君药(苦杏仁、白蔻仁、薏苡仁)全部化学成分并筛选,结合相关文献筛得主要活性成分。借助Uni Port数据库查询活性成分对应靶标蛋白,Cytoscape3.7.2构建成分-靶点网络。通过STRING网站构建PPI蛋白互作网络,运用cytoHubba分析关键子网络。CTD数据库对三仁汤君药活性成分靶蛋白进行GO、KEGG富集分析。以三仁汤君药活性成分及洛匹那韦等相关化学药物为配体,通过CB-Dock网站分别与新型冠状病毒(SARS-CoV-2)3CL水解酶进行分子对接。结果三仁汤君药共有39个活性成分,对应有效靶标168个。GO功能富集分析筛得生物学过程(BP)、细胞组成(CC)、分子功能(MF)分别为25条、14条及2条。KEGG富集筛得先天免疫系统(innate immune system)、免疫系统中的细胞因子信号转导(cytokine signaling in immune system)及白介素信号通路(signaling by interleukins)等信号通路,共36条。分子对接提示三仁汤君药活性成分白桦脂酸、枣素及齐墩果酸与SARS-CoV-2 3CL水解酶结合性较好,三者的Vina得分与3CLpro抑制剂洛匹那韦及Rd Rp抑制剂瑞德西韦的得分相近。结论三仁汤君药可能通过调控炎症因子,参与炎症相关信号通路,以多成分、多靶点、多途径参与COVID-19的多种生理过程,对治疗COVID-19发挥一定的干预作用,且其活性成分具有潜在抗SARS-CoV-2的可能性。     

Inhibition of Organic Anion Transporting Polypeptide 1B1 and 1B3 by Betulinic Acid: Effects of Preincubation and Albumin in the Media

Betulinic acid (BA), a plant-derived pentacyclic triterpenoid, may interact with the members of the organic anion transporting polypeptide 1B subfamily. Here, we investigated the interactions of BA and its analogs with OATP1B1/3 and rat Oatp1b2 in vitro and in vivo. BA inhibited the activity of OATP1B1/3 and rat Oatp1b2 in vitro. Systemic exposure of atorvastatin was substantially altered with the intravenous co-administration of BA (20 mg/kg). Preincubation (incubation with inhibitors, followed by washout) with BA led to a sustained inhibition of OATP1B3, which recovered rapidly in the media containing 10% fetal bovine serum. The addition of albumin to the media decreased intracellular concentrations of BA and expedited the recovery of OATP1B3 activity following preincubation. For asunaprevir and cyclosporin A (previously known to inhibit OATP1B3 upon preincubation), the addition of albumin to the media shortened recovery time with asunaprevir, but not with cyclosporin A. Overall, our results showed that BA inhibits OATP1B transporters in vitro and may incur hepatic transporter-mediated drug interactions in vivo. Our results identify BA as another OATP1B3 inhibitor with preincubation effect and suggest that the preincubation effect and its duration is impacted by altered equilibrium of inhibitors between intracellular and extracellular space (e.g., albumin in the media).     

HPLC法快速测定8种中药中齐墩果酸和熊果酸

目的建立一种快速分析方法,同时测定中药材马鞭草Verbena officinalis、女贞子Ligustrum lucidum、夏枯草Prunella vulgaris、白花蛇舌草Hedyotis diffusa、败酱草Patrinia scabiosaefolia、枇杷叶Eriobotrya japonica、山楂Crataegus pinnatifida、木瓜Chaenomeles Fructus papaya中齐墩果酸和熊果酸的量。方法使用快速溶剂萃取法,采用甲醇作为提取溶剂,静态萃取时间6 min,制备样品溶液,液相色谱分析采用Acclaim C_(30)色谱柱,以乙腈-0.2%醋酸(85∶15)为流动相,体积流量为0.3 mL/min,紫外检测波长为205 nm。结果样品提取时间为10 min,待测物齐墩果酸和熊果酸达到基线分离,且无样品基质干扰,r大于0.999,平均回收率在95.8%～102.7%,本法测定结果与《中国药典》2015年版方法的平均质量分数差异不显著。结论本方法简便、快速,且一法多用,可用于8种中药中齐墩果酸和熊果酸的测定。     

补肾中药有效成分对SAMP8小鼠氧化应激水平的影响

[目的]观察补肾中药有效成分补骨脂素、齐墩果酸、淫羊藿苷对快速老化(SAMP8)小鼠氧化应激水平的影响。[方法]选取9月龄雄性SMAP8小鼠,采用随机数字表法随机分为补骨脂素组、齐墩果酸组、淫羊藿苷组及模型组。同时另取同龄段的抗快速衰老亚系1(SAMR1)小鼠作为对照组。治疗组分别给予补骨脂素(50 mg/kg)、齐墩果酸(50 mg/kg)、淫羊藿苷(20 mg/kg)灌胃,对照组与模型组均给予等量的生理盐水灌胃,每次0.3 m L,每日1次,连续灌胃4周后断头取脑。采用酶联免疫吸附实验(ELISA)法检测SMAP8小鼠大脑皮质中谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)的含量。[结果]SMAP8小鼠大脑皮质中SOD、CAT和GSH-Px活性较同龄SAMR1小鼠明显降低,模型组大脑皮质SOD、CAT和GSH-Px活性均低于对照组,经4周治疗后,治疗组大脑皮质中SOD、CAT和GSH-Px活性均高于模型组,对于以上指标,补骨脂素、齐墩果酸、淫羊藿苷3种组间比较差异无统计学意义(P0.05)。[结论]补肾中药对SAMP8的氧化应激损伤具有一定保护作用,从而可以达到防治老年性痴呆的作用。     

红波罗花的化学成分研究

目的对红波罗花Incarvillea delavayi全草的化学成分进行研究。方法采用90%乙醇进行回流提取,通过各种色谱技术对各部位进行分离,以各种光谱分析技术对分离化合物的结构进行鉴定。结果从红波罗花90%乙醇提取物中分离得到14个化合物,结构类型包括单萜生物碱、环己乙醇、三萜类等,分别鉴定为5-羟乙基-6-羟基-3-甲基苯并呋喃(1)、cleroindicin B(2)、3,4,5-三甲氧基苯甲酸乙酯(3)、3,4,5-三甲氧基苯甲酸甲酯(4)、6-羟基苯并二氢呋喃(5)、2-(4'-乙氧基苯基)-乙醇(6)、tecomine(7)、(+)-epidihydrotecomanine(8)、5-hydroxy skytanthine(9)、δ-skytanthine(10)、isoincarvilline(11)、mairine B(12)、coelobillardierine(13)、3β-乙酰基齐墩果酸(14)。结论化合物1为新化合物,命名为波罗花醇A;化合物3~6、9~11、13为首次从该植物中分离得到。     

黄芪甲苷和齐墩果酸对体外神经干细胞增殖和Jagged1 mRNA表达的影响

目的 观察黄芪甲苷和齐墩果酸对体外神经干细胞增殖和相关基因Jagged1表达的影响。方法 采用机械吹打法从胎鼠脑内获得神经干细胞,随机分组,分别给予不同浓度(10^-6,5×10^-7,10^-7,5×10^-8,10^-8,5×10^-9,10^-9 mol·L^-1)的黄芪甲苷、齐墩果酸进行干预,72 h后用WST法检测细胞的增殖情况,PCR检测Jagged1表达水平。结果 黄芪甲苷对体外神经干细胞增殖影响不显著,高剂量尚存在细胞毒性。齐墩果酸能促进神经干细胞增殖,且呈一定的剂量效应关系,与对照组和黄芪甲苷组比较均有显著差异(P<0.05)。细胞未分化状态下Jagged1基因存在一定的表达,增殖率越高Jagged1表达水平越高;与同浓度黄芪甲苷组比较,齐墩果酸显著上调Jagged1表达(P<0.05)。结论 不同浓度的黄芪甲苷和齐墩果酸对体外培养神经干细胞增殖和Jagged1表达的影响不同;齐墩果酸作用优于黄芪甲苷。     

齐墩果酸诱导人肝癌Bel-7402细胞G2/M期阻滞及凋亡的机制研究

该研究旨在探讨齐墩果酸在诱导Bel-7402肿瘤细胞凋亡过程中,细胞周期分布的变化及其相关的分子机制。采用MTT法、台盼蓝拒染试验检测齐墩果酸对肝癌细胞增殖的抑制作用;PI单染检测细胞周期;Annexin V-FITC双染法观察细胞凋亡;Western blot法检测周期调控蛋白以及凋亡相关蛋白表达的变化。结果显示齐墩果酸能显著抑制Bel-7402肝癌细胞株增殖,G₂/M期的细胞比例显著升高,而G₁期细胞明显减少,明显增加细胞凋亡率;同时下调细胞周期蛋白cyclin B1,使蛋白磷酸酶Cdc25C(Ser216)磷酸化而失去活性、引起Cdk1(Tyr15)以及蛋白激酶Chk1的磷酸化水平升高。OA也可上调p21水平导致细胞周期阻滞在G₂/M期;此外Bcl-2表达减少,Bax,Cyt c表达增加,cleaved-caspase-9和cleaved-caspase-3蛋白表达增加。以上结果提示齐墩果酸能明显抑制肝癌细胞的生长,诱导细胞凋亡,可能是通过激活凋亡线粒体信号通路而实现的。     

Oleanolic acid isolated from ethanolic extract of Phytolacca decandra induces apoptosis in A375 skin melanoma cells: drug-DNA interaction and signaling cascade

OBJECTIVE: Oleanolic acid(OA) has been reported to have anticancer effects, but the extent of its cytotoxicity, its ability to interact with nuclear DNA, its action against skin melanoma, as well as the molecular mechanism of its action against cell proliferation and in support of cell death are still unexplored. This led us to examine the efficacy of OA, a bioactive compound isolated from Phytolacca decandra, on these issues in the present investigation.METHODS: Studies related to analyses of cell viability, drug-DNA interaction, cell proliferation, cell cycle and epidermal growth factor receptor(EGFR) activity were performed. To investigate whether cells undergo apoptosis, studies like fl uorescence microscopy, poly(ADP-ribose) polymerase(PARP) degradation, annexin V-fl uorescein isothiocyanate/propidium iodide assay, alteration in mitochondrial membrane potential and activity of some relevant signaling proteins were performed.RESULTS: OA displayed a minimal and negligible cytotoxic effect on normal HaCaT cells(skin keratinocytes) and peripheral blood mononuclear cells but by contrast it reduced A375 cell viability significantly. OA interacted with nuclear DNA quickly after exposure. It acted as an antiproliferative agent. It suppressed EGFR activity. OA administration led the cells to mitochondriadependent caspase 3-mediated apoptosis.CONCLUSION: OA interacts with cellular DNA, inhibits proliferation possibly through modulating EGFR activity and induces mitochondria-dependent caspase 3-mediated apoptosis in A375 cells which would qualify it as a potent anticancer agent.     

Interactions of the active components of Punica granatum (pomegranate) with the essential renal and hepatic human Solute Carrier transporters

Context: Solute carrier transporters (SLCs) are membrane proteins responsible for cellular influx of various substances including many pharmaceutical agents; therefore, they largely impact on drug disposition and elimination in body. Punica granatum Linnaeus (Lythraceae), pomegranate, is a fruit with antidiabetic potential. Oleanolic acid (OA), ursolic acid (UA), and gallic acid (GA) are the major bioactive components of pomegranate. Co-administration of these compounds with other drugs could result in altered drug pharmacokinetics, possibly due to competing for transporter proteins.

Objective: We investigated the interactions of these three compounds with the essential hepatic and renal SLC transporters.

Materials and methods: Uptake of radiolabeled transporter model substrates was assessed in HEK293 cells over-expressing SLC transporters including the organic anion transporters (OATs), organic anion transporting polypeptides (OATPs) and organic cation transporters (OCTs), in the presence or absence of 10.0?µM UA, OA, or GA. Their IC₅₀ values on specific SLC transporters were also evaluated using varying concentrations of the particular compound (ranging from 0.10?nM to 80.0?µM).

Results: Our results demonstrated UA could significantly inhibit OAT3 and OATP2B1 uptake (IC₅₀: 18.9?±?8.20?µM and 11.0?±?5.00?µM, respectively) and GA has a pronounced inhibitory effect on OATP1B3 uptake (IC₅₀: 1.60?±?0.60?μM).

Discussion and conclusion: Our study reports the interactions of OA, UA, and GA with the essential SLC transporters. This information may contribute to elucidating the drug–drug/herb interactions involved with these three compounds and form the basis of therapeutic optimization when drugs are co-administered.