CN101744853A - 以三七二醇组皂苷为有效成分的抗肿瘤药物及其应用 - Google Patents
以三七二醇组皂苷为有效成分的抗肿瘤药物及其应用 Download PDFInfo
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Abstract
本发明涉及医药技术领域,提供一种以三七二醇组皂苷为有效成分的抗肿瘤药物,三七二醇组皂苷在制备抗肿瘤药物中的应用。本发明公开了三七二醇组皂苷具有抗肿瘤活性,苔盼蓝拒染法和四噻唑蓝(MTT)法均表明,三七二醇组皂苷能抑制肿瘤细胞的生长;蛋白质免疫印迹法表明,三七二醇组皂苷可以降低肿瘤细胞中硫氧还蛋白和细胞周期素D1的表达水平。
Description
技术领域:
本发明涉及医药技术领域,具体地,涉及以三七二醇组皂苷为有效成分的抗肿瘤药物,三七二醇组皂苷在制备抗肿瘤药物中的应用。
背景技术:
三七,又名田七、人参三七,是五加科人参属植物Panax notoginseng(Burk)F.H.Chen的根茎,是我国特产的珍贵药材,主要产于云南、广西等地。三七总皂苷是三七的主要活性成分,药理作用广泛,如具有抗疲劳、提高耐缺氧能力、降血糖、活血化瘀、提高机体免疫功能、清除自由基、抗炎、抗氧化等药理作用。近年来对三七的进一步研究表明:三七总皂苷中可分为三七二醇组皂苷(PanaxadialSaponins,PDS))和三七三醇组皂苷(Panaxtriol saponins,PTS)。到目前为止,有关三七二醇组皂苷(以三七二醇型皂苷Rb1为主的从三七中分离提取出的有效部分)具有抑制肿瘤细胞生长的作用未见报道。
发明内容:
本发明的目的在于提供一种以三七二醇组皂苷作为有效成分,并提供三七二醇组皂苷在抗肿瘤药物中的应用。
本发明的上述目的是通过下述的技术方案得以实现的:
三七二醇组皂苷作为有效成分抗肿瘤药物,其特征是含有三七二醇组皂苷作为有效成分,并含有常规药用载体。
三七二醇组皂苷有效成分在制备抗肿瘤药物中的应用。
三七二醇组皂苷在制备抗肿瘤药物中的应用。
本发明化合物用作药物时,可以直接使用,或者以药物组合物的形式使用。该药物组合物含有0.1-99%,优选为0.5-90%的本发明化合物,其余为药物学上可接受的,对人和动物无毒和惰性的可药用载体和/或赋形剂。
所述的药用载体或赋形剂是一种或多种固体、半固体和液体稀释剂、填料以及药物制品辅剂。将本发明的药物组合物以单位体重服用量的形式使用。本发明的药物可经注射(静注、肌注)和口服两种形式给药。
附图说明:
图1是苔盼蓝拒染法检测PDS对Hela细胞生长的影响;
图2是四噻唑蓝(MTT)法测定PDS对Hela细胞生长的影响;
图3是Western Blot检测PDS降低Hela细胞中Trx蛋白表达水平;
图4是Western Blot检测PDS降低Hela细胞中CyclinD1蛋白表达水平。
具体实施方式:
下面描述本发明的具体实施方式,但是本发明的内容不局限于此。本发明通过下列实施方式证明了三七二醇组皂苷能抑制肿瘤细胞的生长,但不仅限于这些实施例子。
实施例1:
三七二醇组皂苷,用少量的DMSO溶解后,按常规加注射用水,精滤,灌封灭菌制成注射液。
实施例2:
三七二醇组皂苷,用少量的DMSO溶解后,将其溶于无菌注射用水中,搅拌使溶解,用无菌抽滤漏斗过滤,再无菌精滤,分装于安瓿中,低温冷冻干燥后无菌熔封得粉针剂。
为了更好地理解本发明的实质,下面将用本发明的试验例来说明本发明三七二醇组皂苷的药理作用结果,但不以此试验例来限定本发明。
实验例1:
1、细胞
Hela细胞:人宫颈癌细胞。
2、药品及主要试剂
三七二醇组皂苷从昆明制药集团股份有限公司获得,一抗Anti-human Trx由日本京都大学淀井溶司教授提供;一抗Anti-human CyclinD1购自Santa cruz;Protein Marker,购自Fermentas公司;RPMI Medium 1640,购自Invitrogencorporation;蛋白质定量试剂盒(BCA法),购自Beyotime公司;Chemiluminesentsubstrate,购自生物晶美公司;PVDF膜,购自Millipore公司;TRIS Base,购自NOVON公司;Acrylamide,购自Solarbio公司;十二烷基硫酸钠,购自xiamen sanlandchemicals company limited;琼脂糖、考马斯亮蓝R-250、Aprotimin、PMSF和NP-40,均购自Sanland-chem International InC;TEMED,购自HUALVYUAN BIOTECHNOLOGY;X射线胶片,购自中国乐凯胶片集团公司;二抗affinity purified antibodyperoxidase labled goat anti-mouse lgG(H+L)HSA liquid conjugate和affinitypurified antibody peroxidase labled goat anti-rabbit lgG(H+L)HSA liquidconjugate,购自美国Kirkegaard & laboratories;苔盼蓝(Trypan Blue),购自MP Biomedicals生物医学公司;四噻唑蓝(MTT),购自美国Promega公司。
1、主要实验仪器
BHC-1300II A/B3生物安全柜,苏州安泰空气技术有限公司生产;TS100生物倒置相差显微镜,日本NIKON公司生产;CO2培养箱,美国NBS公司生产;TS-1脱色摇床,金纭市杰瑞尔电器有限公司生产;电子分析天枰,北京塞多利斯仪器系统有限公司生产;旋涡混合器,海门市其林贝尔仪器制造有限公司生产;BYY-6C双稳定时电泳仪,北京六一仪器有限公司生产;酶标仪,美国MD公司生产;J-25离心机,美国贝克曼公司生产;XJX-IIX射线摄影暗匣,上海跃进医用光学器械厂生产;血球计数板,上海市求精生化试剂仪器有限公司;96孔板细胞培养板,BiousingBiotechnology有限公司生产。
实验例2:
苔盼蓝拒染法测定Hela细胞的存活率:
(一)Hela细胞株的培养
Hela细胞用含10%胎牛血清、100U/mL青霉素和100μg/mL链霉素的RPMI1640培养基,于37℃、5%CO2和95%湿度下进行贴壁培养。每2-3天更换培养基一次,细胞达约70%~80%融合时,传代或用于实验。
(二)Hela细胞的排板
用0.25%的胰酶将贴壁的Hela细胞消化起来,1500rpm离心5min,除去胰酶。将细胞均匀的排在直径6cm的培养皿里,排板密度为1×105cell/皿,排4板,在含有10%胎牛血清、100U/mL青霉素和100μg/mL链霉素的RPMI1640培养基,于37℃、5%CO2和95%湿度下进行贴壁培养。
(三)梯度浓度三七二醇组皂苷刺激
细胞培养过夜,换新鲜RPMI1640培养基,将四板Hela细胞做好相应的标记,对照组不加三七二醇组皂苷,实验组分别用200、400、800μg/ml的浓度三七二醇组皂苷刺激,24h后收细胞。
(四)苔盼蓝拒染法的活细胞计数
取5μl的细胞悬液与5μl 0.4%的苔盼蓝均匀混合,小心加到血球计数板的小室里,在倒置显微镜下数活细胞的数目。
(五)细胞的存活率计算
存活率%=加药组细胞数/对照组细胞数×100%
试验例2:
四噻唑蓝(MTT)法测定Hela细胞的存活率
(一)Hela细胞株的培养
Hela细胞用含10%胎牛血清、100U/mL青霉素和100μg/mL链霉素的RPMI1640培养基,于37℃、5%CO2和95%湿度下进行贴壁培养。每2-3天更换培养基一次,细胞达约70%~80%融合时,传代或用于实验。
(二)Hela细胞的排板
用0.25%的胰酶将贴壁的Hela细胞消化起来,1500rpm离心5min,除去胰酶。将细胞均匀的排在96孔板中,排板密度为1×104cell/孔,在含有10%胎牛血清、100U/mL青霉素和100μg/mL链霉素的RPMI1640培养基,于37℃、5%CO2和95%湿度下进行贴壁培养。
(三)梯度浓度三七二醇组皂苷刺激
细胞培养过夜,换新鲜RPMI1640培养基,对照组不加三七二醇组皂苷,实验组分别用200、400、800μg/ml的浓度三七二醇组皂苷刺激。各组设5个平行实验。
(四)四噻唑蓝(MTT)试验
取出待测的96孔细胞培养板,每孔加MTT溶液(5mg/ml)10ul,继续孵育4小时后,3000rpm室温下离心15min,小心去掉上清,每孔加入150ul二甲基亚砜,小心混匀,用酶标仪在波长570nm处测定吸光度值。
(五)细胞的存活率计算
存活率%=加药组A值/对照组A值×100%
实验例3:
三七二醇组皂苷降低Hela细胞中Trx和CyclinD1蛋白表达水平
(一)细胞株培养和三七二醇组皂苷刺激处理
Hela细胞用含10%胎牛血清、100U/mL青霉素和100μg/mL链霉素的1640培养基,于37℃、5%CO2和95%湿度下进行贴壁培养。每2-3天更换培养基一次,细胞达约70%~80%融合时,传代或用于实验。
将细胞接种于平板中,培养过夜后,三七二醇组组皂苷处理细胞。分组处理如下:对照组不加药,实验组分别加200ug/ml,400ug/ml,800ug/ml的三七二醇组皂苷。上述处理后,置于37℃和5%CO2培养箱中培养24小时,然后收集细胞,以提取蛋白:1500rpm离心5min收获细胞,分别加入100ul细胞裂解液(150mM NaCl,0.5%NP-40,10mM Tris-HCl,pH 7.2,0.1mM PMSF,2ug/ml aprotinin,200ug/ml NaN3),充分混悬后于冰浴下放置裂解50min(中间振荡5~6次),4℃下15000rpm离心15min,转上清至新离心管中,-80℃保存。
(二)免疫印迹
1.蛋白含量的测定
用BCA试剂盒测定:将10ul标准品或待测样本与200ulWR工作溶液混合,37℃孵育30min,将反应管温度冷却至室温。测定562nm光密度值,绘制标准曲线,并计算蛋白浓度。
2.蛋白免疫印迹(Western Blot)
总蛋白加样量为6ug,与2×SDS凝胶加样缓冲液混合后,95℃热变性5min,用15%的SDS-PAGE胶电泳分离后,将蛋白条带用电转移到PVDF膜上,用5%脱脂牛奶4℃封闭过夜;取出PVDF膜,用含0.1%Tween-20的TPBS冲洗后,加入1000倍稀释的一抗室温孵育75min;用含0.1%Tween-20的TPBS冲洗,加入5000倍稀释的二抗室温孵育75min;用含0.1%Tween-20的TPBS冲洗,将PVDF膜浸入发光底物溶液中反应1min,X射线胶片曝光。
结果表明:三七二醇组皂苷能抑制Hela细胞的生长(图1,图2),同时可以降低Hela细胞中Trx和CyclinD1的蛋白表达水平(图3,图4),这说明三七二醇组皂苷可用于肿瘤的防治及治疗。
硫氧还蛋白(thioredoxin,Trx)是一种普遍存在于原核生物和真核生物中的小分子蛋白质,它与NAPDH和硫氧还蛋白还原酶组成细胞内主要的氧化还原系统。根据表达部位的不同,硫氧还蛋白分为3类:Trx-1、Trx-2和spTrx。到目前为止,人类疾病中有关Trx的研究大都集中在Trx-1。TRX-1是生物体所必须的基因,如果选择性地敲除小鼠的Trx-1基因,小鼠则早期胚胎致死,不能存活(Matsui M.,Oshima M.,Oshima H.,Takaku K.,Maruyama T.,Yodoi J.,and Taketo M.M.Early embryonic lethality caused by targeted disruption of the mousethioredoxin gene.Dev.Biol.1996,178:179-185)。Trx-1是细胞内一种有效的抗氧化剂,能保护细胞、减轻氧化应激所致的损伤。Trx-1能抑制p38MAPK和凋亡信号调节激酶1相关的凋亡信号传导途径,增强细胞的抗凋亡能力(Saito M.,Nishitoh,H.,Fujii,M.,Takeda,K.,Tobiume,K.,Sawada,Y.,Kawabata,M.,Miyazono,K.,and Ichijyo,H.Mammalian thioredoxin is a direct inhibitorof apoptosis signal-regulating kinase(ASK)1.EMBO,1998,17:2596-2606;Hashimoto S.,Matsumoto K.,Gon Y.,Furuichi S.,Maruoka S.,Takeshita I.,Hirota K.,Yodoi J.,and Horie T.Thioredoxin negatively regulates p38 MAPkinase activation and IL-6 production by tumor necrosis factor-alpha.Biochem.Biophys.Res.Commun.1999,258:443-447.)。与对照组相比,三七二醇组皂苷能随剂量的增加降低Hela细胞中Trx蛋白表达水平,由于Trx具有抗凋亡,保护细胞的作用,因此三七二醇组皂苷有可能是通过降低Trx的表达来抑制肿瘤细胞的生长。
Cyclin是20世纪80年代由Hunt等从研究卵母细胞首先发现的,之后在其它一些物种中也发现和证实了cyclin的存在,现在已发现的哺乳动物cyclin有14大类,它们可分为G1期cyclin和M期cyclin。Cyclin的表达具有典型的周期性和时相特异性,CyclinD1在细胞的增殖过程中起着重要作用,是G1期细胞增殖信号(Tashiro E,Tsuchiya A,Imoto M.Functions of cyclin D1as an oncogene and regulationof cyclin D1 expression.Cancer Sci,2007,98:629-635.)。很多肿瘤的发生与cyclinD1的过表达有关Chen CH,Shen J,Lee WJ,Chow SN.overexpression ofcyclin D1 and c-Myc gene products in human primary epithelial ovarian cancer.Int J Gynecol Cancer,2005,15:878-883;Lantsov D,Meirmanov S,NakashimaM,Kondo H,Saenko V,Naruke Y,Namba H,Ito M,Abrosimov A,Lushnikov E,Sekine I,Yamashita Sh.Cyclin D1 over-expression in thyroid papillarymicrocarcinoma:its association with tumour size and aberrant beta-cateninexpression.Histo-pathology,2005,47:248-256;Mega S,Miyamoto M,EbiharaY,Takahashi R,Hase R,Li L,Shichinohe T,Kawarada Y,Uehara H,KanekoH,Hashimoto H,Murakami Y,Itoh T,Morikawa T,Kondo S.Cyclin D1,E2F1expression levels are associated with characteristics and prognosis ofesophageal squamous cell carcinoma.Dis Esophagus,2005,18:109-113.),它还调节视网膜母细胞瘤蛋白(Rb)的磷酸化状态(Diehl JA,Zindy F,Sherr CJ.Inhibition of cyclin D1 phosphorylation on threonine-286 prevents its rapiddegradation via the ubiquitin-proteasome pathway.Genes Dev,1997,11:957-972.)。与对照组相比,三七二醇组皂苷能随剂量的增加降低Hela细胞中CyclinD1蛋白表达水平,由于CyclinD1是细胞周期G1的调节蛋白,因此三七二醇组皂苷有可能是通过降低CyclinD1的表达来抑制肿瘤细胞的生长。
Claims (2)
1.三七二醇组皂苷作为有效成分抗肿瘤药物,其特征是含有三七二醇组皂苷作为有效成分,并含有常规药用载体。
2.三七二醇组皂苷有效成分在制备抗肿瘤药物中的应用。
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CN102323423A (zh) * | 2011-05-31 | 2012-01-18 | 昆明理工大学 | 检测曲妥珠影响MCF-7细胞中Trx-1蛋白表达的方法 |
CN104165850A (zh) * | 2014-08-13 | 2014-11-26 | 四川省人民医院 | 一种羟氯喹亚麻酸酯治疗肿瘤的有效性体外评价方法 |
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CN102323423A (zh) * | 2011-05-31 | 2012-01-18 | 昆明理工大学 | 检测曲妥珠影响MCF-7细胞中Trx-1蛋白表达的方法 |
CN104165850A (zh) * | 2014-08-13 | 2014-11-26 | 四川省人民医院 | 一种羟氯喹亚麻酸酯治疗肿瘤的有效性体外评价方法 |
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