基于UHPLC-Q-Orbitrap HRMS和HPLC-DAD法研究不同干燥方式对马蓝叶化学成分的影响

目的 研究阴干、晒干、真空冷冻干燥、热风干燥4种干燥方式对马蓝Baphicacanthus cusia叶化学成分的影响,寻找差异成分,并建立HPLC-DAD同时定量分析5种吲哚类生物碱成分的方法,以期为建立规范的马蓝叶干燥方式提供理论依据。方法 利用UHPLC-Q-Orbitrap HRMS对4种干燥处理的马蓝叶进行定性分析,并结合热图聚类分析（heat map clustering analysis,HCA）、主成分分析（principal component analysis,PCA）和正交偏最小二乘-判别分析（orthogonal partial least squares-discriminant analysis,OPLS-DA）筛选差异成分。结果 共鉴定出67个共有化合物。HCA和PCA将阴干和热风干燥归为一类。OPLS-DA筛选出14个差异成分,多为生物碱类,且呈现出不同的变化规律。HPLC-DAD结果表明,不同干燥方式马蓝叶中的5种吲哚类生物碱含量存在明显差异。其中,热风干燥样品的靛蓝含量最高,靛玉红含量最低;阴干样品的靛红、色胺酮、靛玉红含量最高。结论 不同的干燥方式对马蓝叶的品质有明显影响。热风干燥和晒干有利于吲哚苷水解合成靛蓝,但靛红和靛玉红的含量较低。而阴干和真空冷冻干燥样品的靛蓝含量较低,而靛玉红含量较高。阴干和热风干燥能显著提高马蓝叶药效成分的总含量。建议在初加工马蓝叶时使用阴干或热风干燥,为进一步探究马蓝叶的产地加工方式提供了数据支持,同时也为评估马蓝叶的质量提供了技术支持。     

大青叶化学成分研究

目的:研究大青叶乙醇总提取物中的化学成分。方法:应用各种柱色谱方法对其提取物进行化学成分分离,并应用化学方法和现代波谱分析方法鉴定所分离得到的化学成分。结果:从大青叶乙醇总提取物中分离得到3个化合物,经鉴定为吲哚类化合物靛玉红(indirubin)、喹唑酮类化合物色胺酮(tryptanthrin)及焦谷氨酸(L-py-roglutamic acid)。结论:焦谷氨酸为首次从菘蓝属植物中分离得到,色胺酮系从该植物叶中首次分离得到。     

Modulatory Effects and Action Mechanisms of Tryptanthrin on Murine Myeloid Leukemia Cells

Leukemia is the disorder of hematopoietic cell development and is characterized by an uncoupling of cell proliferation and differentiation. There is a pressing need for the development of novel tactics for leukemia therapy as conventional treatments often have severe adverse side effects. Tryptanthrin （6,12-dihydro-6,12-dioxoindolo- （2,1-b）-quinazoline） is a naturally-occurring, weakly basic alkaloid isolated from the dried roots of medicinal indigo plants （Ban-Lan-Gen）. It has been reported to have various biological and pharmacological activities, including anti-microbial, anti-inflammatory, immunomodulatory and anti-tumor effects. However, its modulatory effects and action mechanisms on myeloid cells remain poorly understood. In this study, tryptanthrin was shown to suppress the proliferation of the murine myeloid leukemia WEHI-3B JCS cells in a dose- and time-dependent manner. It also significantly reduced the growth of WEHI-3B JCS cells in vivo in syngeneic BALB/c mice. However, it exhibited no significant direct cytotoxicity on normal murine peritoneal macrophages. Flow cytometric analysis showed an obvious cell cycle arrest of the tryptanthrin-treated WEHI-3B JCS cells at the G0/G1 phase. The expression of cyclin D2, D3, Cdk 2, 4 and 6 genes in WEHI-3B JCS cells was found to be down-regulated at 24 h as measured by RT-PCR. Morphological and functional studies revealed that tryptanthrin could induce differentiation in WEHI-3B JCS cells, as shown by the increases in vacuolation, cellular granularity and NBT-reducing activity in tryptanthrin-treated cells. Collectively, our findings suggest that tryptanthrin might exert its anti-tumor effect on the murine myelomonocytic leukemia WEHI-3B JCS cells by causing cell cycle arrest and by triggering cell differentiation.     

On the inhibition of 5-lipoxygenase product formation by tryptanthrin: mechanistic studies and efficacy in vivo

BACKGROUND AND PURPOSE

Leukotrienes (LTs) are pro-inflammatory mediators produced by 5-lipoxygenase (5-LO). Currently available 5-LO inhibitors either lack efficacy or are toxic and novel approaches are required to establish a successful anti-LT therapy. Here we provide a detailed evaluation of the effectiveness of the plant-derived alkaloid tryptanthrin as an inhibitor of LT biosynthesis.

EXPERIMENTAL APPROACH

We analysed LT formation and performed mechanistic studies in human neutrophils stimulated with pathophysiologically relevant stimuli (LPS and formyl peptide), as well as in cell-free assays (neutrophil homogenates or recombinant human 5-LO) and in human whole blood. The in vivo effectiveness of tryptanthrin was evaluated in the rat model of carrageenan-induced pleurisy.

KEY RESULTS

Tryptanthrin potently reduced LT-formation in human neutrophils (IC₅₀ = 0.6 µM). However, tryptanthrin is not a redox-active compound and did not directly interfere with 5-LO activity in cell-free assays. Similarly, tryptanthrin did not inhibit the release of arachidonic acid, the activation of MAPKs, or the increase in [Ca²⁺]_i, but it modified the subcellular localization of 5-LO. Moreover, tryptanthrin potently suppressed LT formation in human whole blood (IC₅₀ = 10 µM) and reduced LTB₄ levels in the rat pleurisy model after a single oral dose of 10 mg·kg⁻¹.

CONCLUSIONS AND IMPLICATIONS

Our data reveal that tryptanthrin is a potent natural inhibitor of cellular LT biosynthesis with proven efficacy in whole blood and is effective in vivo after oral administration. Its unique pharmacological profile supports further analysis to exploit its pharmacological potential.     

色胺酮纳米胶束的制备及其性质研究

目的制备色胺酮纳米胶束,改善色胺酮的水溶性,并进行体外性质考察。方法以二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000)为载体,用溶剂挥发法制备色胺酮纳米胶束,通过正交实验筛选制备胶束的最佳条件,核磁共振氢谱(1 HNMR)验证色胺酮包载于纳米胶束,用芘荧光探针法测定其临界胶束浓度(CMC),用紫外分光光度计测定其包封率和载药率,动态光散射法测定胶束的粒径,以粒径、外观形态和包封率为指标考察胶束的稳定性。结果色胺酮纳米胶束的CMC为8.93×10-6mol·L~(-1),色胺酮与聚合物投药比为0.442 9∶1(mol∶mol),真空干燥1h,水化5min时,胶束的包封率为32.24%±1.37%,载药率为5.468%±0.39%。色胺酮纳米胶束平均粒径为112.5nm,平均分散系数为0.208,4℃条件下胶束可稳定15d以上。结论制备色胺酮纳米胶束,将色胺酮的溶解度提高至1.625mmol·L~(-1),为改善色胺酮生物利用度的研究奠定了基础。     

基于蛋白质组学分析色胺酮抑制小鼠乳腺癌的作用机制

目的:采用非标记定量(Label-free)蛋白质组学技术研究色胺酮抗小鼠体内乳腺癌的作用机制。方法:采用超高效液相色谱-质谱联用技术检测色胺酮抗小鼠乳腺癌的表达蛋白,选择Ionoptics nano UPLC C18色谱柱(0.075 mm×250 mm,1.6μm),流动相0.1%甲酸水溶液-0.1%甲酸乙腈溶液梯度洗脱,正离子模式,扫描范围m/z 100～1 700,使用MaxQuant 1.6.5.0进行数据库检索。采用Label-free高分辨质谱的蛋白质组学技术筛选4T1乳腺癌小鼠模型组与色胺酮(100 mg·kg~(-1))口服给药组之间的差异表达蛋白,进行色胺酮抗乳腺癌的蛋白质组学研究。结果:共鉴定出3 997个蛋白质,其中有2 911个蛋白可定量。模型组与色胺酮组共750个差异表达蛋白,其中286个蛋白上调,464个蛋白下调。基因本体分析表明,这些差异表达蛋白主要参与增殖、细胞迁移、凋亡、免疫、血管生成和炎症调节等生物学过程。京都基因与基因组百科全书通路分析进一步表明,这些蛋白主要集中于T细胞受体,B细胞受体,Toll样受体,核转录因子-κB(NF-κB),Ras蛋白,白细胞介素-17,肿瘤坏死因子,磷脂酰肌醇3-激酶/蛋白激酶B(PI3K-Akt)和丝裂原活化蛋白激酶(MAPK)等信号通路。结论:与色胺酮抗4T1乳腺癌作用密切相关的差异表达蛋白包括上调蛋白白细胞分化抗原14(CD14),前列腺素G/H合酶2(PTGS2),泛素蛋白连接酶E3和下调蛋白CD44,70 kDa热休克蛋白1A(HSPA1A),巨噬细胞移动抑制因子(MIF),NF-κB,核糖体蛋白S6激酶α-4(RPS6KA4)和高迁移率族蛋白B1(HMGB1),提示色胺酮主要通过调节肿瘤炎症微环境来达到抑制小鼠乳腺癌的作用。     

吲哚胺2,3-双加氧酶(IDO)的色胺酮类抑制剂筛选及其体外抗肿瘤作用

 目的  评价色胺酮衍生物吲哚胺2,3-双加氧酶 (indoleamine 2,3-dioxygenase,IDO)的抑制活性,并研究其作为 IDO 抑制剂的抗肿瘤作用。方法  采用基因工程手段表达、纯化重组人 IDO (rhIDO),建立 IDO 活性检测体系;以色胺酮衍生物作为对象,进行 IDO 抑制活性初筛,对抑制类型、半数抑制浓度以及抑制常数进行测定;构建高表达人 IDO 的 pcDNA3.1(+) hIDO 转染 HEK 293 细胞,评价色胺酮衍生物在细胞水平上的 IDO 抑制活性;采用 MTT 比色法考察色胺酮衍生物3对人非小细胞肺癌A549细胞的生长抑制作用。结果  6个被测色胺酮衍生物均具有IDO抑制活性,且细胞水平上的抑制效力高于酶活水平,抑制效力均优于目前通用的IDO抑制剂1 甲基色氨酸(1-methyl-tryptophan,1-MT)。色胺酮衍生物 3 作为最强的 IDO 抑制剂,其Ki值为 0.161 μmol/L。MTT 实验结果显示,色胺酮衍生物3 显著抑制 A549 细胞生长,IC50 为 8.77 μmol/L。结论  色胺酮衍生物是一类新型高效的 IDO 抑制剂,在体外对 A549 细胞具有较强的抗肿瘤活性。     

HPLC法测定大鼠血浆中色胺酮的浓度及药代动力学参数

目的建立测定血浆中色胺酮含量的HPLC法,并用于色胺酮在大鼠体内的药代动力学研究。方法采用ODSC18色谱柱(4.6 mm×250 mm),流动相为乙腈∶水(47∶53),流速为1.0 ml.min-1,检测波长为251 nm,柱温30℃,进样量20μl,内标物为桂皮醛。按56 mg.kg-1剂量给大鼠灌胃后,用HPLC法测定给药后不同时刻大鼠血浆中色胺酮的浓度。采用DAS 2.1.1软件分析,计算药动学参数。结果色胺酮在0.0183～1.1712 mg.L-1范围内线性关系良好(r2=0.999),最低定量限为0.02 mg.L-1。回收率大于95.0%,其日内、日间RSD均小于8%。大鼠灌胃色胺酮56mg.kg-1后,♀和♂T12分别为4.314和5.597 h,Tmax分别为5.2和4.6 h,Cmax分别为1.887和2.620 mg.L-1,AUC0→t分别为12.1和14.70 mg.h.L-1。结论本方法操作简便,准确,灵敏度高、重复性好,可用于色胺酮血药浓度检测及药代动力学研究。     

色胺酮热特性分析

目的:研究色胺酮的热特性。方法:用差示扫描量热法观察了色胺酮的晶型情况;用热重法测量了色胺酮的热失重曲线,并用Kissinger法计算了其表观活化能;同时应用红外吸收光谱和X-射线衍射图谱进行了佐证。结果:色胺酮可能存在有多晶型现象,利用热失重法测得的表观活化能为83.28KJ.mol-1。结论:获得了色胺酮的热力学特征,为该化合物的应用进行了基础研究。     

青黛治疗溃疡性结肠炎的机制研究进展

溃疡性结肠炎(ulcerative colitis,UC)是一种与自身免疫相关的慢性非特异性肠道炎症性疾病,中医认为UC的病理性质为本虚标实,病理因素主要包括湿、热、瘀、毒等,青黛有清热解毒、凉血消斑之效,作为复方制剂的重要成分之一,被长期用于UC的口服或局部治疗中。近年来研究者采用青黛单药及其主要成分口服或纳肛治疗UC亦取得良好效果,并针对其作用机制开展了大量研究。本文主要归纳总结了近年来青黛及其有效成分提取物(靛玉红、靛蓝、色胺酮)等治疗UC的机制研究。