RP-HPLC测定蕲龙胶囊中华蟾酥毒基和酯蟾毒配基的含量

目的建立蕲龙胶囊中华蟾酥毒基和酯蟾毒配基的含量测定方法。方法采用高效液相色谱法,Phenomenex lunaC18(250 mm×4.6 mm,5μm)色谱柱,流动相为乙腈-水(50:50),流速0.8 mL/min,检测波长296 nm。结果方法学考察结果表明,本研究建立的分析方法有较好的重现性和稳定性。华蟾酥毒基平均加样回收率为98.5%(RSD=0.55%,n=6);酯蟾毒配基平均加样回收率为101.0%(RSD=0.67%,n=6)。结论本方法定量准确、重复性好,可用该制剂的质量控制。     

Cinobufagin exerts anti-proliferative and pro-apoptotic effects through the modulation ROS-mediated MAPKs signaling pathway

Abstract

Cinobufagin (CBG) is a cardiotoxic bufanolide steroid secreted by the skin and parotid venom glands of the Asiatic toad Bufo bufo gargarizans (called Chan-Su). Although CBG is known to exhibit anti-cancer activities, very little is known about its potential mechanism(s) of action. In this study, we investigated whether CBG mediates its effect through the modulation of the mitogen-activated protein kinases (MAPKs) signaling pathway in human multiple myeloma (MM) U266 cells. We found that CBG caused the significant activation of ERK, JNK and p38 MAPK in U266 cells. CBG showed much higher cytotoxicity against U266 cells as compared to peripheral blood mononuclear cells (PBMC). Induction of CBG increased reactive oxygen species (ROS) generation from mitochondria, which is associated with the induction of apoptosis as characterized by increased sub-G1 DNA contents of cell cycle, positive Annexin V binding, activation of caspase-3 and cleavage of PARP. Inhibition of ROS generation by N-acetyl-l-cysteine (NAC) significantly prevented CBG-induced ERK, JNK and p38 MAPK activation and apoptosis. CBG also down-regulated the expression of various downstream gene products that mediate cell proliferation, survival, angiogenesis and metastasis. Interestingly, ERK, JNK and p38MAPK pharmacological inhibitors blocked CBG-induced MAPKs activation and ERK inhibitor (PD98059) also prevented the CBG-induced caspase-3 activation and PARP cleavage in U266 cells. Taken together, these findings suggest that CBG can act as a potent anticancer agent against MM and possibly exerts its effects through the ROS-mediated activation of ERK, JNK and p38 MAPK leading to the activation of caspase-3 in U266 cells.     

Immunomodulatory effects of cinobufagin isolated from Chan Su on activation and cytokines secretion of immunocyte in vitro

The objective of this study was to evaluate the immunomodulatory effects of cinobufagin (CBG) isolated from Chan Su (Venenum Bufonis) in vitro. In this paper, our results show that CBG signi?cantly stimulated cell proliferation of splenocytes and peritoneal macrophages (PMΦ) and markedly enhanced the phagocytic activation of PMΦ. CBG also significantly increased CD4⁺CD8⁺ double-positive T-cell populations and the percentage of S-phase cells of splenic lymphocytes. The levels of several Th1 cytokines, including interferon-γ and tumor necrosis factor-α, are signi?cantly increased after CBG treatment, whereas the levels of the Th2 cytokine interleukin-4 and interleukin-10 are signi?cantly decreased. As a result, the ratio of Th1/Th2 also increased. Taken together, these results indicated that CBG had potential immune system regulatory effects and suggested that this compound could be developed as a novel immunotherapeutic agent to treat immune-mediated diseases such as cancer.     

灵猫香解毒丸的质量标准研究

目的:制定灵猫香解毒丸的含量测定标准.方法:采用反相高效液相色谱法测定蟾酥中华蟾酥毒基和酯蟾毒配基.结果:华蟾酥毒基和酯蟾毒配基分别在48.84～784.44,50.28～804.48 ng与峰面积呈良好线性关系,平均回收率100.5%,100.3%.结论:本实验定量方法简便、实用,重复性好,能够较好控制灵猫香解毒丸的质量.     

华蟾素对SD大鼠膀胱肿瘤的抑制作用

目的研究华蟾素对SD大鼠膀胱肿瘤的治疗作用。方法 50只SD大鼠按随机分为对照组和实验组,实验组40只大鼠膀胱灌注N-甲基-N-亚硝基脲(N-methyl-N-nitrosourea,MUN),每3周1次,共4次;于第14周将实验组大鼠随机分为生理盐水组(膀胱灌注生理盐水)、丝裂霉素组(5 mg/kg)、低浓度华蟾素组(5 mg/kg)及高浓度华蟾素组(20 mg/kg),每周灌注1次,连续灌注5次,计算抑瘤率,TUNEL法检测肿瘤细胞的凋亡情况。结果 MNU膀胱灌注成功建立膀胱肿瘤动物模型,以膀胱肿瘤质量计算高浓度华蟾素组的抑瘤率为76.7%,高浓度华蟾素组抑瘤率较丝裂霉素组及低浓度华蟾素组高;TUNEL法检测高浓度华蟾素组AI值为(24.21±1.24)%,较其他各组AI值明显增高,提示高浓度华蟾素组凋亡指数高于丝裂霉素及低浓度华蟾素组。结论华蟾素对膀胱肿瘤有抑制生长、促进凋亡的作用,且高浓度时抑瘤作用及促凋亡作用较强。     

蟾毒灵、华蟾酥毒基及酯蟾毒配基大鼠体内药动学研究

目的 建立检测大鼠血浆中3种蟾蜍甾烯类化合物蟾毒灵、华蟾酥毒基及酯蟾毒配基的HPLC法,并用于蟾酥在大鼠体内的药动学研究。方法 分别于大鼠尾iv给予蟾酥提取物0.8 mg/kg后2、5、10、15、20、30、45、60、90 min,眼眶取血,采用乙腈沉淀蛋白与液液萃取相结合方法进行血浆样品预处理,以HPLC法测定大鼠血浆中蟾毒灵、华蟾酥毒基及酯蟾毒配基的质量浓度,以Kinetica软件拟合药动学参数。结果 蟾毒灵、华蟾酥毒基及酯蟾毒配基均得到很好的分离,重现性、精密度、线性关系良好,达到体内分析要求;经非房室模型拟合,得到蟾毒灵、华蟾酥毒基及酯蟾毒配基在大鼠体内主要药动学参数;iv给药30 min时,血药浓度均降至C_max的1/5以下。结论 建立的蟾毒灵、华蟾酥毒基及酯蟾毒配基血药浓度测定方法操作简单、结果准确可靠;蟾蜍甾烯类成分在大鼠体内代谢较为迅速,所得数据可为蟾酥提取物的药动学研究提供参考。     

HPLC法测定蟾皮中蟾毒配基类成分的含量

目的用HPLC法测定蟾皮中蟾蜍灵、脂蟾毒配基、华蟾蜍精等成分的含量。方法样品经乙醇室温浸渍提取,采用HPLC法测定。色谱柱为DiamonsilTMC18(250 mm×4.6 mm,5μm),流动相为甲醇-乙腈-水(体积比为4∶1∶5),流速为1.0 mL.min-1,检测波长为296 nm,柱温为40℃。结果蟾蜍灵、脂蟾毒配基和华蟾蜍精分别在0.0848~0.5088、0.0884~0.5304和0.2000~1.2000μg内与色谱峰面积线性关系良好,相关系数r分别为0.9999、0.9992和1.0000。蟾蜍灵、脂蟾毒配基和华蟾蜍精的平均回收率分别为99.3%、99.6%和99.0%,RSD分别为0.5%、0.2%和0.6%(n=9)。结论HPLC法可用于蟾皮中蟾蜍灵、脂蟾毒配基和华蟾蜍精的含量测定。     

基于大鼠胸主动脉血管环的蟾酥舒缩调控作用研究及效应成分发现

目的揭示蟾酥提取物对大鼠胸主动脉血管环舒缩的双向调控作用及潜在效应成分组成。方法利用离体生物能量信号采集系统,评价蟾酥提取物及其水洗脱部位和70%乙醇洗脱部位对大鼠胸主动脉血管环的舒缩调控作用;并在此基础上,建立UHPLC-Q TOF-MS/MS联用方法,对各样品进行成分解析;根据成分鉴定结果,逐一考察其中主要成分的血管调控效应。结果蟾酥提取物对静息状态下的大鼠胸主动脉血管环表现出先收缩后舒张的双向调控作用;而其水洗脱部位仅表现出血管收缩效应,70%乙醇洗脱部位仅表现出血管舒张效应。进一步研究发现,肾上腺素、5-羟色胺是蟾酥提取物水洗脱部位中发挥血管收缩效应的主要成分;而华蟾酥毒基、酯蟾毒配基、华蟾毒它灵、去乙酰华蟾毒精和脂蟾毒精是蟾酥提取物70%乙醇洗脱部位中发挥血管舒张效应的主要成分。结论蟾酥提取物对于血管舒缩的双向调控作用,与其中水溶性成分肾上腺素、5-羟色胺诱导血管收缩,脂溶性成分华蟾酥毒基、酯蟾毒配基、华蟾毒它灵、去乙酰华蟾毒精和脂蟾毒精诱导血管舒张密切相关,为中药单味药多成分作用于同一器官的多靶点协同拮抗调控效应研究提供了数据支撑。     

干蟾皮中蟾毒内酯类成分的提取工艺研究

目的 探讨干蟾皮中蟾毒内酯类成分的最佳提取工艺.方法 通过正交试验法考察乙醇体积分数、乙醇用量、提取时间、提取次数对干蟾皮中蟾毒内酯类成分提取效果的影响,并以酯蟾毒配基和华蟾酥毒基的总量-总毒基量为考察指标,采用超高效高压液相色谱-紫外检测器检测.结果 最佳提取工艺为以10倍量80%乙醇提取2次,每次2 h.结论 所优选出的提取工艺方法可行、稳定、合理.     

反相高效液相色谱法测定梅花点舌丸中酯蟾毒配基、华蟾毒配基的含量

目的 :建立测定梅花点舌丸中酯蟾毒配基、华蟾毒配基的反相高效液相色谱法。方法 :采用KromasilC18 柱 ,流动相为乙腈∶0 5 %KH2PO4 溶液 (50∶50) ,检测波长为296nm。结果 :该方法线形关系良好 ,酯蟾毒配基的平均加样回收率为97 40 % ,RSD为2 2 % ;华蟾毒配基的平均加样回收率为97 78 % ,RSD为2 6 %。结论 :方法简便可靠 ,分离度较好 ,可用于梅花点舌丸的质量控制。