CN116919946A - 槲皮素在制备预防前列腺癌药物中的应用 - Google Patents
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Abstract
本发明公开了槲皮素在制备预防前列腺癌药物中的应用。所述前列腺癌由人源前列腺癌22RV1细胞系或人源前列腺癌PC3细胞系引起的前列腺癌。药效学实验表明,槲皮素动物实验表明槲皮素具有预防前列腺癌作用。
Description
技术领域
本发明属于医药领域,具体涉及槲皮素在制备预防前列腺癌药物中的应用。
背景技术
许多天然化合物能够抑制和调节肿瘤细胞的增殖,槲皮素(Quercetin,QCT,分子式:C15H10O7,分子量:302.24)就是其中之一。槲皮素是植物来源的天然黄酮类化合物,广泛存在于我们常见食物中,包括苹果,绿茶,洋葱,坚果,萝卜、香菜、花菜,卷心菜和许多其他食物槲皮素肠道吸收差,血中暴露量低、生物利用度低,导致槲皮素的临床应用都处于探索阶段。
槲皮素通过即通过多种代谢产物、肠道菌群及代谢物、炎症和氧化产生的全身介质以及多种机制实现多种生物学效应。目前已经发现槲皮素具有多种生物学效应,如抗肿瘤、抗氧化、抗炎作用、抗糖尿病、抗肥胖、关节炎、哮喘、抗衰老和抗情绪障碍、关节炎、哮喘、抗衰老和抗情绪障碍等。因槲皮素具有广泛的生物学效应,一直成为研究的热点之一。我们团队在2001年在国际上报道发现一种天然物质槲皮素可抑制前列腺癌雄激素受体的表达与功能,槲皮素为治疗前列腺癌开辟了新篇章。然而,槲皮素预防前列腺癌的目前没有相关研究。
发明内容
本发明首次提出了槲皮素在预防前列腺癌的应用。槲皮素可有效预防前列腺癌,应用前景广泛。
本发明的目的是提供槲皮素在制备预防前列腺癌药物中的应用。
本发明所提供的槲皮素的结构式如式I:
本发明所述前列腺癌由人源前列腺癌22RV1细胞系或人源前列腺癌PC3细胞系引起的前列腺癌。
本发明所述药物产品除含有上述槲皮素肠道菌群代谢物外,还可含有适宜的载体或赋形剂,以及包含其他起配伍协同作用的有效成分。所述载体包括药学领域常规的稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、吸附载体、润滑剂等。上述药物可以制成注射液、片剂、粉剂、颗粒剂、胶囊、口服液、膏剂、霜剂等多种形式;上述各种剂型的药物均可以按照药学领域的常规方法制备。
上述药物可通过注射、喷射、滴鼻、滴眼、渗透、吸收、物理或化学介导的方法导入机体如肌肉、皮内、皮下、静脉、粘膜组织;或是被其他物质混合或包裹后导入机体。
本发明具有如下有益技术效果:
本发明首次提出了槲皮素在预防前列腺癌的应用。槲皮素可有效预防前列腺癌的发生与发展,应用前景广泛。
附图说明
图1为22RV1细胞系前列腺癌裸鼠肿瘤生长曲线;
图2为22RV1细胞系前列腺癌裸鼠肿瘤体积统计图;
图3为22RV1细胞系前列腺癌裸鼠肿瘤重量统计图;
图4为PC3细胞系前列腺癌裸鼠肿瘤生长曲线;
图5为PC3细胞系前列腺癌裸鼠肿瘤体积统计图;
图6为PC3细胞系前列腺癌裸鼠肿瘤重量统计图;
具体实施方式
下面结合具体实施例对本发明作进一步阐述,但本发明并不限于以下实施例。所述方法如无特别说明均为常规方法。所述原材料如无特别说明均能从公开商业途径获得。
实施例1、槲皮素预防前列腺癌的作用
1.实验目的
通过动物实验评价槲皮素是否具有预防前列腺癌的作用
2.实验细胞及裸鼠来源
本实验采用人源前列腺癌22RV1细胞系和人源前列腺癌PC-3细胞系,购自美国典型培养物保藏中心,常规培养、传代、冻存。
雄性BALB/c裸鼠,SPF级别,6周龄。购于北京维通利华实验动物公司,许可证号为:SCXK(京)2016-0011,体重为22-24g。
3.裸鼠饲养
6周龄的雄性BALB/c裸鼠,SPF级别,体重22-24g。裸鼠饲养和注意事项同槲皮素治疗前列腺癌章节。本研究是在中国医学科学院实验动物伦理委员会和北京协和医学院的许可和指导下进行的。所有步骤均参照《实验动物伦理委员会的组织指南和伦理指南》进行。
4.主要实验仪器和设备
表1.仪器及设备
表2.主要实验试剂
表3.主要耗材
5、实验试剂
槲皮素:分子式C15H10O7,CAS:117-39-5分子量302.04,含量:HPLC≥98%,购自美国Sigma公司。
6、实验方法
6.1.细胞培养
本实验采用人源前列腺癌22RV1细胞系和人源前列腺癌PC-3细胞系,常规培养、传代、冻存。
6.2.人前列腺癌裸鼠移植瘤模型的建立
(1)SPF级动物房中饲养BALB/c裸鼠,并让BALB/c裸鼠适应新环境1周后方可进行实验;
(2)人源前列腺癌22RV1细胞系和人源前列腺癌PC-3细胞系常规培养、传代、冻存;
(3)将状态良好,无污染的人源前列腺癌22RV1细胞和PC-3细胞传代至第3-4代,细胞长满90%左右;
(4)将对数生长期的细胞按前述方法消化,完全培养基终止消化后,800rpm离心5min;
(5)使用移液枪弃去上清液,用PBS液重悬沉淀细胞,吹打混匀,800rpm离心5min;
(6)重复步骤3,彻底去除胎牛血清成分,PBS重悬细胞按细胞计数法进行计数;
(7)根据计数结果,选取2×106数量的22RV1细胞和5×105数量PC-3细胞均匀混悬于100μL PBS和基质胶的混悬液(2:1)中;
(8)将上述准备好的22RV1细胞和PC-3细胞置于4℃冰水混合液中运送至无菌动物房;
(9)将上述数量的人源前列腺癌22RV1细胞和PC-3细胞于无菌环境下分别接种于裸鼠右侧背部靠前腿皮下缓慢注射,每次接种前务必充分混匀细胞基质胶混悬液,保证每只裸鼠接种细胞数量一致。接种时注意防止细胞悬液沿注射器针口流出;
(10)每日观察裸鼠成瘤情况,4周后麻醉处死,观测肿瘤体积、质量等指标。
6.3.药物溶液的配制
槲皮素药物溶液:取适量羧甲基纤维素钠(CMC-Na)溶于0.9%氯化钠注射液(500mg:4.5g),使终浓度成含0.3%CMC-Na的生理盐水,保存于4℃供备用。根据实验用药剂量每次给药前称量各组裸鼠体重,根据给药剂量将槲皮素溶于0.3%CMC-Na的生理盐水的溶液予以灌胃。同时模型组、空白组等给予溶剂0.3%CMC-Na的生理盐水。
6.4.实验动物分组及给药方案
6.4.1分组
2种细胞系(22RV1、PC3),n=6
具体分组
(1)模型组(0.3%CMC-Na)
(2)提前给药组(200mg/kg,造模前2周起灌药)
(3)持续给药组(200mg/kg,造模前2周起灌药,种瘤后继续给药4周)
6.4.2给药方案
(1)模型组:造模前2周起予0.3%CMC-Na灌胃,灌胃2周后分别用人源前列腺癌22RV1细胞和PC-3细胞建立人前列腺癌裸鼠移植瘤模型,种瘤后继续给予0.3%CMC-Na灌胃至4周。
(2)提前治疗组:造模前2周起予槲皮素溶液以灌胃,槲皮素的剂量根据我们的槲皮素治疗实验选择高剂量200mg/kg/d,溶于0.3%CMC-Na的生理盐水。槲皮素灌胃2周后分别用人源前列腺癌22RV1细胞和PC-3细胞建立人前列腺癌裸鼠移植瘤模型,然后停止灌胃。
(3)持续给药组:造模前2周起予槲皮素溶液以灌胃,槲皮素的剂量根据我们的槲皮素治疗实验选择高剂量200mg/kg/d,溶于0.3%CMC-Na的生理盐水。槲皮素灌胃2周后分别用人源前列腺癌22RV1细胞和PC-3细胞建立人前列腺癌裸鼠移植瘤模型,种瘤后继续给药至4周。
治疗过程中注意观察裸鼠精神状态、进食量等变化,每两天记录移植瘤长短径。每三天记录裸鼠体重,根据体重随时调整药物剂量。6周疗程结束时,给予水合氯醛麻醉裸鼠后脱颈处死,迅速取出移植瘤并称重。比较各组移植瘤体积和重量抑制情况,比较各组裸鼠体重变化,评价药物对各组裸鼠毒副作用。实验结束同时收集干净粪便,每只小鼠无菌收集粪便量3g以上,放入EP管后于-80℃保存待用。粪便样品送16S rRNA检测。
6.5.移植瘤体积和抑瘤率计算
用Excel for Mac软件记录数据,移植瘤体积(V)=长径×短径2×0.5;各个治疗组抑瘤率(%)=(1-治疗组平均移植瘤体积/对照组平均移植瘤体积)×100%或(1-治疗组平均移植瘤重量/对照组平均移植瘤重量)×100%。用GraphPad PrismVersion 9for macOS(GraphPad Software,San Diego,CA,USA)的统计分析。数据表示为平均值±标准偏差(SD),小于0.05的p值被认为具有统计学意义。
7、实验结果
实验开始前一周让BALB/c裸鼠适应环境,实验开始后对BALB/c裸鼠进行随机分组并按照上述实验方案进行对应治疗。对模型组和提前给药组灌胃2周后,予以种瘤造模。之后每2天测量移植瘤长短径,计算移植瘤体积,观察各组变化情况。每3天测量裸鼠体重,根据体重变化随时调整药物剂量。用药治疗10-12天后各组出现差别,持续给药组移植瘤体积要小于模型组和提前给药组。治疗6周后,可以看到槲皮素在两种细胞系建立的前列腺癌裸鼠模型中提前给药组和持续给药组对比模型组有明显的统计学差异,具体见图1-6。
由图可知,槲皮素具有预防前列腺癌的作用,并且能够达到预防目的。
Claims (2)
1.槲皮素在制备预防前列腺癌药物中的应用;
所述槲皮素的结构式如式I所示:
2.根据权利要求1所述的应用,其特征在于:所述前列腺癌由人源前列腺癌22RV1细胞系或人源前列腺癌PC3细胞系引起的前列腺癌。
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