目的 對(duì)蟾酥甲醇提取物中化學(xué)成分進(jìn)行初步分析，并利用斑馬魚(yú)模型評(píng)價(jià)其抑制血管生成及抗炎活性。方法 采用液質(zhì)聯(lián)用（LC-MS）技術(shù)，用反相C₁₈色譜柱，以0.1%甲酸溶液-乙腈為流動(dòng)相進(jìn)行梯度洗脫，采用電噴霧-三重四級(jí)桿飛行時(shí)間質(zhì)譜正/負(fù)離子雙重掃描方式，對(duì)提取物中的樣品進(jìn)行分析；采用受精后24 h（24 hpf）綠色熒光標(biāo)記血管的轉(zhuǎn)基因斑馬魚(yú)作為實(shí)驗(yàn)動(dòng)物模型，熒光顯微鏡下觀察背部節(jié)間血管生成情況，計(jì)算節(jié)間血管長(zhǎng)度，研究PTK787（陽(yáng)性藥，0.2 μg/mL）和蟾酥甲醇提取物2、4、6 μg/mL濃度對(duì)斑馬魚(yú)血管生成的影響；采用受精后3 d（3 dpf）健康綠色熒光標(biāo)記炎癥細(xì)胞的轉(zhuǎn)基因斑馬魚(yú)作為實(shí)驗(yàn)動(dòng)物模型，設(shè)置對(duì)照組、模型（CuSO₄，20 μmol/L）組、布洛芬（陽(yáng)性藥，20 μmol/L）組和蟾酥甲醇提取物（0.4、0.8 μg/mL）組，計(jì)數(shù)神經(jīng)丘周?chē)装Y細(xì)胞個(gè)數(shù)。結(jié)果 蟾酥甲醇提取物中共檢測(cè)出33個(gè)色譜峰，并鑒定出其中7個(gè)為結(jié)合型蟾蜍二烯酸內(nèi)酯。與對(duì)照組比較，蟾酥甲醇提取物4、6 μg/mL濃度組斑馬魚(yú)血管生成受到抑制，血管長(zhǎng)度變短，具有顯著性差異（P<0.05、0.01）；與對(duì)照組比較，CuSO₄模型組斑馬魚(yú)遷移到神經(jīng)丘側(cè)線部位的炎癥細(xì)胞數(shù)量顯著增多（P<0.01）；與模型組比較，蟾酥甲醇提取物0.8 μg/mL組炎癥細(xì)胞遷移減少，具有顯著差異（P<0.01）。結(jié)論 通過(guò)LC-MS法從蟾酥甲醇提取物中鑒定出7個(gè)結(jié)合型蟾蜍二烯酸內(nèi)酯，蟾酥甲醇提取物具有抑制血管生成和抗炎活性。

Objective To preliminarily analyze the chemical constituents in methanol extract of Bufonis Venenum (MEBV), and to evaluate its anti-angiogenesis and anti-inflammatory activities in zebrafish. Methods A reversed-phase C₁₈ column with 0.1% formic acid solution-acetonitrile as mobile phase was used for gradient elution by liquid-mass spectrometry (LC-MS). The samples were analyzed by electrospray and triple quadrupole time-of flight mass spectrometry (TFMS) with positive/negative ion dual scanning. The transgenic zebrafish with green fluorescent protein (GFP) labeled blood vessels at 24 hpf (hours post fertilization) were used as the experimental animal model. The angiogenesis of back internode was observed under fluorescent microscope, the development of dorsal longitudinal anastomotic vessel (DLAV) and the length of inter-segmental blood vessels (ISV) were measured to study the effects of MEBV (2, 4, and 6 μg/mL) and PTK787 (positive medicine, 0.2 μg/mL) on zebrafish angiogenesis at different concentrations. The transgenic zebrafish with GFP labeled inflammatory cells on 3 dpf (days post fertilization) were used to study the effect of of MEBV on inflammatory response by counting the number of inflammatory cells around the neuromast. The control group, model group (CuSO₄,20 μmol/L), ibuprofen group (positive drug,20 μmol/L) and MEBV (0.4 and 0.8 μg/mL) groups were set. Results A total of 33 chromatographic peaks were detected in methanol extract of toad venom, and seven of them were conjugated bufadienolides. In the experiment of antiangiogenesis, the formation of new blood vessels were inhibited at the concentration of 4 and 6 μg/mL, and the vascular development was not complete and the length of ISV was significantly decreased (P < 0.05 and 0.01). The results of anti-inflammatory experiment showed that compared with model group, the migration of inflammatory cells in zebrafish at the concentration of 0.8 μg/mL was significantly decreased (P < 0.01). Conclusion Seven Bufonadienolides were identified by LC-MS from the MEBV. Bufonis Venenum has anti-angiogenesis and anti-inflammatory activities.

山東省優(yōu)秀青年人才基金項(xiàng)目（ZR2020YQ60）；齊魯工業(yè)大學(xué)（山東省科學(xué)院）科教產(chǎn)融合創(chuàng)新試點(diǎn)工程項(xiàng)目（2020KJC-ZD08）