李宗云;高慧敏;王金华;曲婷;陈两绵;王智民;张启伟;
LI Zongyun1,2,3,GAO Huimin1,2,WANG Jinhua1,QU Ting1,2,CHEN Liangmian1,2,WANG Zhimin1,2,ZHANG Qiwei1,2 (1.Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China; 2.National Engineering Laboratory for Quality Control Technology of Chinese Herbal Medicine,Beijing 100700,China; 3.Jiangxi University of Traditional Chinese Medicine,Nanchang 330004,China)

• 1:中国中医科学院中药研究所
• 2:中药质量控制技术国家工程实验室
• 3:江西中医学院

摘要(Abstract)：

目的:评价蟾酥总蟾蜍甾烯(total bufadienolides from toad venom,TBFs)对H22荷瘤小鼠的肿瘤抑制作用,同步对体内组织中的代谢物进行初步分析。方法:采用HPLC和LC-MS对制备的受试药TBFs的化学组成进行表征;采用灌胃和腹腔注射2种方式给予H22荷瘤小鼠高、中、低3个剂量的受试药,给药后第13天处死,剥取瘤组织,称重,计算抑瘤率;同时取心、肝、脾、肺、肾各脏器组织,采用HPLC和LC-MS对其中的药物进行初步分析。结果:在对照品对照,在线紫外吸收光谱和质谱数据的基础上,明确了受试药TBFs的化学组成,以日蟾毒它灵、沙蟾毒精、蟾毒它灵、酯蟾毒精、华蟾毒它灵、蟾毒灵、华蟾酥毒基和酯蟾毒配基为主。与空白对照组比,灌胃给予TBFs对H22荷瘤小鼠的体重无影响,低(5 mg.kg-1),中(10 mg.kg-1),高剂量组(20 mg.kg-1)的抑瘤率分别为14.76%,16.38%,10.32%;腹腔注射给予TBFs,与空白对照组比,中、高剂量组在给药第5天时体重增加较慢,第13天时恢复正常;低(1.5 mg.kg-1),中(3 mg.kg-1),高剂量组(6 mg.kg-1)的抑瘤率分别为17.30%,19.80%,40.95%,呈一定的剂量依赖性。小鼠心、肝、脾、肺、肾脏器组织HPLC分析表明,肝组织中存在蟾蜍甾烯类成分,LC-DAD-MS初步推测其中的11个化合物。结论:灌胃给予TBFs对H22荷瘤小鼠肿瘤生长有一定的抑制趋势,但作用不明显;腹腔注射则展示了显著性抑瘤作用,同时给药初期也表现了一定的毒性反应;肝组织中推测的蟾蜍甾烯类化合物为进一步研究其体内代谢途径奠定基础。
Objective: To evaluate the inhibitory effect of total bufadienolides from toad venom against H22 tumor in mice and preliminarily analyze the structures of the metabolites in tissues.Method: HPLC and LC-MS were used for analysis of the chemical composition of TBFs.High,middle and low dosages of TBFs were orally administered or intra-peritoneally injected to H22 tumor-bearing mice for thirteen days.The animals were killed and the tumors were stripped and weighed.The metabolites in the tissues such as heart,liver,spleen,lung and kidney,were analyzed by HPLC and LC-MS.Result: The chemical composition of TBFs were identified by comparison of the retention times with those of reference substances,on-line UV spectra and MS data.Its main components are concerned with gamabufotalin,arenobufagin,bufotalin,resibufagin,cinobufotalin,bufalin,cinobufagin and resibufogenin.TBFs had no obvious influence on body weight of H22 tumor-bearing mice orally administered and the inhibition rate against tumor were 14.76%,16.38% and 10.32% for low(5 mg·kg-1),middle(10 mg·kg-1) and high dosage(20 mg·kg-1),respectively.The mice intra-peritoneally injected with middle and high-dose of TBFs gained body weight slower than the control mice on the 5th day and recovered on the 13th day.The inhibition rate against tumor were 17.30 %,19.80 % and 40.95 % for low(1.5 mg·kg-1),middle(3 mg·kg-1) and high dose(6 mg·kg-1),respectively.The inhibitory effect took on dose-dependent manner.Based on the HPLC analyses on heart,liver,spleen,lung and kidney,bufadienolides were found in the liver tissue and 11 compounds of them were tentatively identified by LC-DAD-MS.Conclusion: TBFs by oral administration had no inhibitory effect against H22 tumor in mice,however,TBFs by intra-peritoneal injection displayed the significantly inhibitory effect,accompanying some toxicity for early duration of the study.The identification of bufadienolides in the liver provides a good basis for the further investigation of the metabolic pathways of TBFs in vivo.

关键词(KeyWords)： 蟾酥总蟾蜍甾烯;H22肝癌;代谢产物;抗肿瘤
total bufadienolides from toad venom;H22 liver tumor;metabolites;anti-tumor

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(30801512);; 国家“重大新药创制”科技重大项目(2009ZX09301-005,2009ZX09308-003);; 中国中医科学院自选课题项目(Z02073)

作者(Authors): 李宗云;高慧敏;王金华;曲婷;陈两绵;王智民;张启伟;
LI Zongyun1,2,3,GAO Huimin1,2,WANG Jinhua1,QU Ting1,2,CHEN Liangmian1,2,WANG Zhimin1,2,ZHANG Qiwei1,2 (1.Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China; 2.National Engineering Laboratory for Quality Control Technology of Chinese Herbal Medicine,Beijing 100700,China; 3.Jiangxi University of Traditional Chinese Medicine,Nanchang 330004,China)

参考文献(References)：

• [1]中国药典.一部[S].2010:360.
• [2]程国华.蟾酥质量研究及其药理临床应用进展[J].中草药,2001,32(2):184.
• [3]李强.蟾酥中蟾毒配基类化合物的抗肿瘤研究进展[J].实用药物与临床,2009,12(2):132.
• [4]刘俊珊,张冬梅,栗原博.蟾酥及其活性成分抗肿瘤作用研究进展[J].国际药学研究杂志,2009,36(2):115
• [5]Gao H M,Zehl M,Leitner A,et al.Comparison of toad venomsfrom different Bufo species by HPLC and LC-DAD-MS/MS[J].JEnthnopharmacol,2010,131(2):368.
• [6]Ye M,Qu G Q,Guo H Z,et al.Novel cytotoxic bufadienolidesderived from bufalin by microbial hydroxylation and their struc-ture-activity relationships[J].J Steriod Biochem Mol Biol,2004,91(1/2):87.
• [7]Ye M,Ning L L,Zhan J X,et al.Biotransformation of cinobufa-gin by cell suspension cultures of Catharanthus roseus and Platyc-odon grandiflorum[J].J Mol Caral B,Enzym,2003,22(1/2):89.
• [8]Ye M,Han J,An D G,et al.New cytotoxic bufadienolides fromthe biotransformation of resibufogenin by Mycor polymorphosporus[J].Tetrahedron,2005,61(37):8947.
• [9]Xin X L,Zhan L B,Li F Y,et al.Microbial transformation ofbufotalin by Alternaria alternata AS 3.4578[J].J Asian NatProd Res,2009,11(1):7.
• [10]杨秀伟,邢增涛,崔景荣,等.华蟾毒精和羟基华蟾毒精的人肠内细菌代谢研究[J].北京大学学报:医学版,2001,33(3):199.
• [11]Ning J,Wu T H,Tian Y,et al.Identification of cinobufagin me-tabolites in the bile of rats[J].Xenobiotica,2010,40(1):48.
• [12]He X J,Tang J S,Qiao A M,et al.Cytotoxic biotransformedproducts from cinobufagin by Mucor spinosus and Aspergillus niger[J].Steriods,2006,71(5):392.
• [13]Qiao L,Zhou Y Z,Chen H,et al.One new bufadienolide bio-transformed from cinobufagin by Cunninghamella elegans[J].Chin Chem Lett,2008,19(3):299.
• [14]Ma X C,Xin X L,Liu K X,et al.Microbial transformation ofcinobufagin by Syncephalastrum racemosum[J].J Nat Prod,2008,71(7):1268.
• [15]Ma X C,Cui J,Zheng J,et al.Microbial transformation of threebufadienolides by Penicillium aurantigriseum and its applicationfor metabolite identification in rat[J].J Mol Catal B,Enzym,2007,48(1/2):42.
• [16]梁研,李萍,余伯阳,等.HLPC-MS对蟾蜍甾烯类化合物在大鼠胃肠道中吸收和转化的初步研究[J].中国天然药物,2007,5(4):285.