目的 鉴定华蟾素片中的多肽类成分.方法 采用超声提取加超滤的方法提取华蟾素片中的多肽类成分,采用纳升液相-线型离子阱-静电轨道阱(Nano LC-LTQ-Orbitrap)质谱对华蟾素片多肽进行鉴定,通过数据库匹配以及生物信息学手段对华蟾素片多肽进行分析.结果 采用Nano LC-LTQ-Orbitrap对华蟾素片进行鉴定,共鉴定到163条序列不同的肽段,且均为多价态的肽段.此外,通过对氨基酸出现频次进行统计并对肽段进行亲水性评分发现,华蟾素片中的肽段主要为亲水性肽段.通过对其前体蛋白进行分析发现,华蟾素片多肽主要来源于角蛋白、胶原蛋白、转录翻译相关蛋白以及组蛋白等.结论 通过Nano LC-LTQ-Orbitrap可以快速对华蟾素片中的多肽进行鉴定,这种方法可为药物中多肽类成分的分析鉴别提供参考.

OBJECTIVE To proteins in toad venom.To investigate the by proteomic approach.METHODS The total proteins from the ear-side gland of toad were digested by trypsin,and the peptides were further analyzed by the NanoLC-linear trap quadropole (LTQ)-Orbitrap Velos Pro..The raw data acquired by mass spectrometer were imported into MaxQuant software for the identification of peptides and proteins.The proteins were categorized based on gene ontology annotation in biological process,cellular component and molecular function.RESULTS A total of 407 protein groups and 880 peptides were identified.There were 76 pathways associated with the identified proteins in toad venom,including the 5-HT receptor mediated signaling pathway,beta adrenergic receptor signaling pathway,blood coagulation,cadherin signaling pathway,cholesterol biosynthesis,etc..CONCLUSION This study lays the foundation for further exploration of the proteins in toad venom and their functions.

目的 评价蟾酥药材的刺激性.方法 大鼠足趾一次性sc给予蟾酥10和50 mg· mL-1,以肿胀度(炎症)和提足反应(疼痛)为指标,并结合UPLC-MS足部炎性介质的相对含量变化来反应刺激性.结果 蟾酥使大鼠出现明显的提足反应,低浓度(10 mg·mL-1)和高浓度(50 mg·mL-1)组10 min内提足次数分别为(14±5)和(99±8)次(P＜0.01),而生理盐水组未出现提足反应;蟾酥使大鼠足部出现明显肿胀,与生理盐水组肿胀度(0.071 ±0.009) mL相比,蟾酥低浓度和高浓度组30 min后的肿胀度分别为(0.702±0.093)和(1.027±0.11)mL(P ＜0.01).蟾酥也引起大鼠足部组织中促炎物质前列腺素E2和前列腺素F2α的相对含量明显增加,抗炎物质前列腺素E1和8,9-DiHETrE的相对含量降低,并且不同剂量给药组间也有显著性差异.结论 本实验发现蟾酥对大鼠足部具有一定的刺激性,并且刺激性存在明显的剂量依赖性.

目的:研究细胞上清中6种炎症介质花生四烯酸（AA）,白三烯B4（LTB4）,前列腺素G2（PGG2）,前列腺素F2α（PGF2α）,15-脱氧-δ-12,14-PGJ2（15d-PGJ2）,5,15-过氧化氢二十碳四烯酸（5,15-Di HETE）的变化,探讨芫花酯甲干预花生四烯酸代谢产生的致炎、诱导平滑肌收缩的作用机制。方法:选取RAW264.7作为体外实验模型,采用三重四极杆质谱,分析空白组及低、高质量浓度组芫花酯甲（0,1.62,6.48 mg·L-1）作用于RAW264.7细胞（5×104个/m L）48 h后细胞上清中6种炎症相关介质的相对含量变化,以探讨芫花酯甲的致炎机制。结果:与空白组相比,AA,PGF2α,PGG2,5,15-Di HETE的相对含量显著升高（P＜0.05）;与空白组相比较,LTB4在高浓度芫花酯甲刺激时相对含量显著升高,但对于15d-PGJ2,高浓度的芫花酯甲刺激时相对含量呈现下降的趋势（P＜0.01）。结论:芫花酯甲增加了巨噬细胞上清液中炎症相关介质AA,5,15-Di HETE及促炎介质LTB4,PGG2,PGF2α水平,减低了抗炎介质15d-PGJ2的水平,提示芫花酯甲通过影响促炎介质和抗炎介质的双重途径,发挥致炎和诱导平滑肌收缩作用。

Toad venom is the Bufo bufo gargarizans or B. melanostictus after the ears of the gland secretion, used in the treatment of various cancers in recent years. Research shows that the main anti-tumor components in bufadienolide. Bufadienolide have free type structure and conjunct type structure. To identify and clarify the difference between bufogenin and bufotoxin contained in Bufonis Venenum, which was from B. bufo gargarizans, an UPLC-TQ-MS method has been established. UPLC-TQ-MS method was used to identify and quantify the major bufadienolides in Bufonis Venenum. UPLC-TQ-MS assay with positive ion mode was performed on a Waters ACQUITY UPLC BEH C, (2.1 mm x 100 mm, 1.7 µm) with the mobile phase consisting of 0. 1% aqueous formic and acidacetonitrile in gradient elution at a flow rate of 0.4 mL · min⁻¹ and the column temperature was set at 35 °C. By comparing their retention time and high resolution mass data of Bufonis Venenum extracts, 37 effective components were primarily identified by MS/MS analysis in positive ion mode. Twenty-six of them were free-type bufadienolides (bufogenin), 11 of them were conjugated bufadienolides. There were significant differences in the main composition between fresh and processed Bufonis Venenum. The study found that the chemical composition of toad venom through great changes after processing, conjunct type content is much less, free type content as well change.