基于指纹图谱和UPLC-MS/MS定量测定对不同产地陈皮的质量评价研究

目的 采用HPLC指纹图谱与化学计量学相结合、UPLC-MS/MS测定多黄酮成分含量的方法,测定不同产地陈皮质量,为其鉴别和质量控制提供依据。方法 采用HPLC-DAD指纹图谱检测并结合“中药色谱指纹图谱相似度评价系统（2012A版）”建立6个产区,49个不同批次陈皮药材（S1～S49）指纹图谱,并进行相似度评价和共有峰确认,结合聚类分析（hierarchical clustering analysis,HCA）、主成分分析（principal component analysis,PCA）、正交偏最小二乘法-判别分析（orthogonal partial least-squares discrimination analysis,OPLS-DA）等化学计量学综合分析。采用UPLC-MS/MS法建立了8个黄酮类成分柚皮素、柚皮苷、木犀草苷、川陈皮素、橙皮素、橙皮苷、橘皮素和芸香柚皮苷的含量测定方法对60批7个产地陈皮药材进行评价。结果 建立了49批不同产地陈皮药材的指纹图谱,相似度为0.864～0.999,共标定了11个共有峰,HCA分析49批陈皮明显分为4类;PCA得到5个主成分的累积方差贡献率为92.748%;OPLS-DA表明8、7、10和11号峰可能是影响陈皮药材质量的差异标志物;含量测定结果表明不同产地陈皮中黄酮类含量存在显著差异,广东新会以川陈皮素和橘皮素为主要标志成分,四川荷花池以木犀草苷为差异性成分,江西樟树以柚皮素和柚皮苷为差异性成分,广西玉林则以橙皮素、橙皮苷和芸香柚皮苷为主,重庆云阳以川陈皮素和橙皮苷为主。结论 建立了稳定性强的不同产地陈皮HPLC指纹图谱和8个黄酮类UPLC-MS/MS定量测定方法,结合化学计量学可用于陈皮药材综合评价和质量控制。     

川陈皮素的全合成

 目的：研究川陈皮素的化学全合成路线。方法：从基本的化工原料间苯二酚开始，经6步合成此化合物，即间苯二酚溴代，醚化，苯环上亲核取代，付-科反应上乙酰基，缩合与关环。结果：得到的样品经，等波谱分析¹H-NMR，MS证明是川陈皮素。结论：此合成路线是完全可行的。     

Nobiletin,a Polymethoxylated Flavone,Inhibits Glioma Cell Growth and Migration via Arresting Cell Cycle and Suppressing MAPK and Akt Pathways

Nobiletin, a bioactive polymethoxylated flavone (5,6,7,8,3^',4^'‐hexamethoxyflavone), is abundant in citrus fruit peel. Although nobiletin exhibits antitumor activity against various cancer cells, the effect of nobiletin on glioma cells remains unclear. The aim of this study was to determine the effects of nobiletin on the human U87 and Hs683 glioma cell lines. Treating glioma cells with nobiletin (20–100 µm ) reduced cell viability and arrested the cell cycle in the G0/G1 phase, as detected using a 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay and propidium iodide (PI) staining, respectively; however, nobiletin did not induce cell apoptosis according to PI‐annexin V double staining. Data from western blotting showed that nobiletin significantly attenuated the expression of cyclin D1, cyclin‐dependent kinase 2, cyclin‐dependent kinase 4, and E2 promoter‐binding factor 1 (E2F1) and the phosphorylation of Akt/protein kinase B and mitogen‐activated protein kinases, including p38, extracellular signal‐regulated kinase, and c‐Jun N‐terminal kinase. Our data also showed that nobiletin inhibited glioma cell migration, as detected by both functional wound healing and transwell migration assays. Altogether, the present results suggest that nobiletin inhibits mitogen‐activated protein kinase and Akt/protein kinase B pathways and downregulates positive regulators of the cell cycle, leading to subsequent suppression of glioma cell proliferation and migration. Our findings evidence that nobiletin may have potential for treating glioblastoma multiforme. Copyright © 2015 John Wiley & Sons, Ltd.     

Nobiletin and its derivatives overcome multidrug resistance (MDR) in cancer: total synthesis and discovery of potent MDR reversal agents

Our recent studies demonstrated that the natural product nobiletin (NOB) served as a promising multidrug resistance (MDR) reversal agent and improved the effectiveness of cancer chemotherapy in vitro. However, low aqueous solubility and difficulty in total synthesis limited its application as a therapeutic agent. To tackle these challenges, NOB was synthesized in a high yield by a concise route of six steps and fourteen derivatives were synthesized with remarkable solubility and efficacy. All the compounds showed improved sensitivity to paclitaxel (PTX) in P-glycoprotein (P-gp) overexpressing MDR cancer cells. Among them, compound 29d exhibited water solubility 280-fold higher than NOB. A drug-resistance A549/T xenograft model showed that 29d, at a dose of 50 mg/kg co-administered with PTX (15 mg/kg), inhibited tumor growth more effective than NOB and remarkably increased PTX concentration in the tumors via P-gp inhibition. Moreover, Western blot experiments revealed that 29d inhibited expression of NRF2, phosphorylated ERK and AKT in MDR cancer cells, thus implying 29d of multiple mechanisms to reverse MDR in lung cancer.     

基于化学计量学方法结合正交偏最小二乘判别分析的陈皮饮片HPLC指纹图谱研究

目的建立陈皮饮片HPLC指纹图谱分析方法,为其质量控制提供技术参考。方法采用HPLC法建立17批陈皮饮片的指纹图谱,通过聚类分析(clusteranalysis,CA)、主成分分析(principalcomponentanalysis,PCA)、正交偏最小二乘判别分析(orthogonal partial least square discrimina te analysis,OPLS-DA)对指纹图谱进行评价。结果建立了陈皮饮片指纹图谱,相似度均0.9,确定了25个共有峰,其中14个峰的变量投影重要性(variableimportance projection,VIP)均1,通过与对照品谱图比对,确定了13号峰为芸香柚皮苷、14号峰为橙皮苷、23号峰为川陈皮素、24号峰为3,5,6,7,8,3′,4′-七甲氧基黄酮、25号峰为桔皮素。结论该方法简单可靠,可用于陈皮饮片鉴别和质量控制。     

基于网络药理学和设计空间的乳腺康提取工艺研究

目的通过网络药理学及设计空间法对乳腺康提取工艺参数进行研究。方法借助网络药理学对乳腺康潜在的活性成分进行筛选,并与酪氨酸激酶进行分子对接,结合《中国药典》2020年版确定指标性成分,采用HPLC法运用设计空间进行乳腺康提取工艺研究。结果筛选出乳腺康中甘草查尔酮A、川陈皮素、蒲公英甾醇等核心成分与酪氨酸激酶的亲和力与临床推荐用药相似;设计空间得到最佳提取工艺:浸泡时间为30 min、溶媒量为12倍、提取时间为45~75 min、乙醇体积分数为65%~80%、提取2~3次。结论实验所得到的工艺合理可行,验证实验与理论值预测值接近,具有一定的实用价值。该研究基于质量源于设计(QbD)理念的乳腺康提取工艺,稳定可靠,为其进一步的工艺开发及质量控制提供思路。     

川陈皮素防治肝缺血再灌注损伤的作用机制研究

目的探究川陈皮素防治肝缺血再灌注损伤(hepatic ischemia reperfusion injury,HIRI)的作用及机制。方法 SD大鼠随机分为对照组、模型组和川陈皮素(10 mg/kg)组,采用ELISA法检测各组大鼠血清中丙氨酸转氨酶(alanine aminotransferase, ALT)和天冬氨酸转氨酶(aspartate aminotransferase, AST)活性;采用苏木素-伊红(HE)染色法考察各组大鼠肝组织病理变化;采用透射电子显微镜(TEM)观察各组大鼠肝细胞线粒体结构;采用Western blotting和qRT-PCR考察各组大鼠肝组织自噬相关蛋白Beclin1、微管相关蛋白1轻链3-Ⅱ(microtubule associated protein 1 light chain 3-Ⅱ,LC3-Ⅱ)、自噬相关基因-7 (autophagy-related gene-7,ATG-7)、p62蛋白和mRNA表达情况。采用H2O2诱导大鼠肝细胞BRL建立氧化损伤细胞模型,设置对照组、模型组和川陈皮素(10μmol/L)组,采用Western blotting和qRT-PCR考察各组细胞Beclinl、LC3-Ⅱ、ATG-7、p62蛋白和mRNA表达情况;采用MitoTracker染色和免疫荧光法考察各组细胞自噬蛋白与线粒体的共定位情况;考察自噬抑制剂和诱导剂对川陈皮素细胞保护作用的影响。结果与对照组比较,模型组大鼠血清ALT和AST活性均显著升高(P0.05),肝组织病变,肝细胞线粒体数目增加,结构错乱,受损的线粒体被吞噬,肝组织中LC3-Ⅱ、Beclin1、ATG-7蛋白和mRNA表达水平显著升高(P0.05),p62蛋白和mRNA表达水平显著降低(P0.05);与模型组比较,川陈皮素组大鼠血清ALT和AST活性明显降低(P0.05),肝组织病变程度减轻,线粒体形态变化得到明显改善,肝组织中LC3-Ⅱ、Beclinl、ATG-7蛋白和mRNA表达水平显著降低(P0.05),p62蛋白和mRNA表达水平显著升高(P0.05)。氧化损伤细胞模型中BRL细胞LC3-Ⅱ、Beclin1、ATG-7蛋白和mRNA表达水平显著升高(P0.05),p62蛋白和mRNA表达水平显著降低(P0.05),Beclin1和MitoTracker的共定位数目增加;与模型组比较,川陈皮素组BRL细胞中LC3-Ⅱ、Beclin1、ATG-7蛋白和mRNA表达水平显著降低(P0.05),p62蛋白和mRNA表达水平显著升高(P0.05),Beclin1和MitoTracker的共定位数目减少;自噬抑制剂3-MA (1、5 mmol/L)显著增强川陈皮素对BRL细胞的保护作用(P0.05),自噬诱导剂雷帕霉素显著抑制川陈皮素对BRL细胞Beclin1蛋白和mRNA表达水平的下调作用(P0.05)。结论川陈皮素对HIRI具有较好的防治作用,其机制可能与抑制线粒体自噬有关。     

基于UPLC特征图谱和Q-Marker量值传递评价经典名方半夏白术天麻汤颗粒剂的关键生产工艺

目的 基于特征图谱相关性和质量标志物（quality markers,Q-Marker）转移率评价半夏白术天麻汤（Banxia Baizhu Tianma Decoction,BBTD）颗粒剂的生产环节,明确关键控制点。方法 采用Acclaim^TM RSLC Lot Validation-120 C₁₈色谱柱（100 mm×2.1 mm,2.2 μm）进行分离,流动相为乙腈-水溶液,梯度洗脱,体积流量0.3 mL/min,检测波长235 nm,柱温30℃。以BBTD基准样品为参比,建立3批中试样品的UPLC指纹图谱,并进行相似度评价,分析Q-Marker在各生产环节的损失,评价生产工艺的合理性。结果 与BBTD基准样品相比,提取液、浓缩液、干膏粉、颗粒剂中均存在对应特征峰,以10种指标性成分及其总量表征的3批BBTD中试样品关键质量属性与其基准样品基本保持一致。结论 基于特征图谱和Q-Marker在各环节之间的量值传递,为经典名方BBTD制备工艺评价和质量控制提供了的理论依据。     

RP-HPLC法同时测定橘叶中3个黄酮类成分的含量

目的:建立同时测定橘叶中川陈皮素、橘红素、5-去甲川陈皮素含量的反相高效液相色谱法。方法:采用Kromasil C18(250 mm×4.6 mm,5μm)色谱柱,流动相为甲醇-乙腈-水(体积比30∶30∶40),流速1.0 mL.min-1,检测波长342 nm;柱温为室温(25℃)。结果:川陈皮素、橘红素、5-去甲川陈皮素线性范围分别为0.239～2.870μg(r=0.9995),0.102～1.224μg(r=0.9995),0.054～0.643μg(r=0.9995);平均回收率(n=6)分别为98.06%,98.02%,97.61%,RSD分别为2.6%,1.9%,2.0%。结论:本法快速、简便、准确,重复性好,可用于橘叶川陈皮素、橘红素、5-去甲川陈皮素的定量分析和质量控制。     

RP—HPLC法测定陈皮药材中川陈皮素的含量

目的：测定中药陈皮中川陈皮素的含量。方法：采用ＲＰ－ＨＰＬＣ法、色谱条件为μ－ＢｏｎｄａｐａｋＣ１８柱，四氢呋喃－乙腈－甲醇－水为流动相，检测波长３２８ｎｍ，结果：不同批号陈皮药材中川陈皮素的含量为０．０５１－０．５０９％，平均加样回收率为９９．２％，ＲＳＤ为１．９％，结论：含量测定方法的为控制陈皮药材的质量标准提供了依据。