CN102225197A - 顶体蛋白的用途及提取方法 - Google Patents
顶体蛋白的用途及提取方法 Download PDFInfo
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- CN102225197A CN102225197A CN2011101577332A CN201110157733A CN102225197A CN 102225197 A CN102225197 A CN 102225197A CN 2011101577332 A CN2011101577332 A CN 2011101577332A CN 201110157733 A CN201110157733 A CN 201110157733A CN 102225197 A CN102225197 A CN 102225197A
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- acrosin
- cancer
- animal semen
- dried powder
- acid
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
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- C—CHEMISTRY; METALLURGY
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Abstract
本发明提供的顶体蛋白的用途及提取方法,主要解决了现有应用动物精液制备治疗肿瘤、抑郁症等多种疾病中的药物纯度低、需服用的剂量大及精液中的非特异性蛋白、脂肪和糖类化合物等杂质对治疗效果的干扰的问题;本发明提供的顶体蛋白的用途及提取方法,极大地提高了药物的纯度和药效,突破了现有技术的局限性。获取了顶体蛋白在治疗子宫肌瘤、乳房瘤、胃癌、肺癌、肝癌、白血病、大肠癌、卵巢癌、乳腺癌、宫颈癌、恶性淋巴瘤及治疗抑郁症等疾病的药物中的应用;极大地提高了该类药物的纯度,并降低了患者的服用剂量和动物精液冻干粉中含有的其他物质可能造成的影响。
Description
技术领域
本发明涉及顶体蛋白的用途及制备方法。
背景技术
众所周知,肿瘤是当前严重影响人类健康,威胁人类生命的主要疾病之一,已成为人类致死的首要原因。据统计,中国每年有160万人死于癌症,世界上每年有740万人死于癌症。虽然目前治疗癌症的药物和治疗手段不断在更新,但癌症发病率和死亡率仍在不断增加,预计2030年全世界将有超过1200万人死于癌症。
癌是机体正常细胞快速产生异常细胞,并发生转移,扩散到其他器官,同时异常细胞增生过程中,吸收营养,破坏人体免疫力和抵抗力,导致机体功能紊乱,失去平衡,最终导致人死亡。
目前国际上对癌症病因及发病机理尚未完全认识,对于癌症的治疗,常规疗法无外乎手术治疗、药物化疗、放射性治疗和以毒攻毒等。他们对癌症的治疗虽能起一定作用,使部分患者病情暂时好转,但容易再次复发和转移,同时正常的组织细胞及机体免疫力不可避免地在治疗过程中受损,给患者带来难以忍受的痛苦和食欲不振、呕吐、发烧、大小便失禁、抑郁、失眠等症状,患者生活质量严重降低。经过传统的化学治疗后,患者会头发脱落、骨瘦如柴,不能饮食吞咽。致使许多患者未死于癌症而死于放化疗和手术。当这些弊端日益显现时,一些人进而转向基因和免疫学等途径,但迄今未取得实质性的进展。
专利CN200710017785.3已描述了动物精液药材在治疗肿瘤、抑郁症等多种疾病中的药物应用,专利申请201010219062.3描述了动物精液冻干粉的制备方法。但精液的主要成分为水,约占90%以上,在有形成分中,除成熟精子外,尚有1%的其他细胞,如未成熟的精原细胞、精母细胞以及生殖管道脱落的上皮细胞和少量白细胞,除细胞外,精液内还含有脂肪及蛋白质颗粒、色素颗粒、前列腺液的磷脂小体,无机盐、酶类、乳酸及果糖等;现有技术并未确定动物精液中何种物质起到治疗肿瘤、抑郁症等疾病的作用,直接应用动物精液制备的药物纯度也较低,因此效用较差,患者的服用剂量大。
发明内容
本发明提供的顶体蛋白的用途及提取方法,主要解决了现有应用动物精液制备治疗肿瘤、抑郁症等多种疾病中的药物纯度低、需服用的剂量大及精液中的非特异性蛋白、脂肪和糖类化合物等杂质对治疗效果的干扰的问题;本发明提供的顶体蛋白的用途及提取方法,极大地提高了药物的纯度和药效,突破了现有技术的局限性。
本发明的具体技术解决方案如下:
顶体蛋白可以用于抑制癌细胞的扩散,尤其是用于抑制MCF-7、Bel7402、BGC823、A2780、A549或HCT-8细胞的扩散。
顶体蛋白在制备治疗癌症、抑郁症的药物中的应用。癌症包括妇科肿瘤、消化道肿瘤、肺癌、肝癌、白血病和恶性淋巴瘤;妇科肿瘤包括子宫肌瘤、卵巢癌、乳房瘤、乳腺癌和宫颈癌;消化道肿瘤包括食道癌、胃癌、大肠癌。
顶体蛋白可以用于制备胶囊、颗粒、片剂、滴丸、中西药复方制剂、口服液或注射液。
本发明顶体蛋白提纯通过下述方法得到:
1]取动物精液或复溶的动物精液冻干粉,动物精液一般选择健康驴、牛、羊或马的精液,其中优选为驴精液;向其中加入浓度为1%~5%的稀酸溶液,再加入无机酸将pH调节至2~5,以2000r/min~8000r/min的转速离心15~30min后弃去沉淀物,较佳范围为离心转速2500r/min~6000r/min,离心时间7~20min;然后将得到的上清液合并,浓缩;其中,1%~5%的稀酸溶液可选醋酸溶液、甲酸溶液、丙酸溶液、丁酸溶液、盐酸溶液、氢溴酸溶液、硫酸溶液、磷酸溶液等,其中优选为醋酸溶液,稀酸溶液浓度为1%~5%,较佳范围为1.7%~4.5%,优选为2%;调节pH的无机酸为1mol/L的硫酸、盐酸、氢溴酸或磷酸,其中优选为盐酸;无机酸调节pH范围为2~5,优选为2.8~4.6,最佳pH为3;其中动物精液与稀酸的质量比为1∶1~1∶20,较佳范围为1∶2~1∶15,最优比为1∶4;动物精液冻干粉与复溶溶剂水的质量比为1∶1~1∶20,较佳范围为1∶5~1∶15,最优比为1∶10,其中动物精液冻干粉复溶溶液与稀酸的质量比为1∶1~1∶16,较佳范围为1∶2~1∶12,最优比为1∶5.5;
2]向步骤1处理所得的上清液中加入无机铵盐,调节饱和度至0.2-0.5后在4℃环境中放置3-5小时,饱和度的调节的较佳范围为0.25-0.48,优选为0.3;再以2000r/min~8000r/min离心15~30min,较佳范围为离心转速2500r/min~6000r/min,离心时间7~20min;然后弃去上清液,沉淀用醋酸缓冲溶液溶解后,得到顶体蛋白粗提物;无机铵盐包括硫酸铵、硫酸氢铵、碳酸铵、碳酸氢铵、磷酸铵等,其中优选的为硫酸铵;醋酸缓冲溶液为0.1mol/L,pH为1.8~4,较佳范围为2~3.5,优选pH为2;
3]将经步骤3处理所得的蛋白粗提物进行柱层析,用醋酸缓冲溶液平衡洗脱,处理完成后收集洗脱液;一般是用葡聚糖凝胶柱、羟丙基葡聚糖凝胶柱、丙烯酰胺凝胶柱或琼脂糖凝胶柱进行柱层析,其中优选的为葡聚糖凝胶柱和羟丙基葡聚糖凝胶柱;醋酸缓冲溶液是pH=2,0.1mol/L的醋酸缓冲溶液,醋酸缓冲溶液平衡洗脱的流速为0.5ml/min,处理完成后收集在680nm处有最大吸收的洗脱液;
4]将洗脱液以2000r/min~8000r/min离心5~10min,弃去上清液,所得沉淀洗涤后以2000r/min~8000r/min继续离心1~3次,每次离心5~10min,收集沉淀,即得到顶体蛋白。
本发明的优点在于:
本发明通过大量的实验研究,突破现有技术应用上的局限性,获取了顶体蛋白在治疗子宫肌瘤、乳房瘤、胃癌、肺癌、肝癌、白血病、大肠癌、卵巢癌、乳腺癌、宫颈癌、恶性淋巴瘤及治疗抑郁症等疾病的药物中的应用;极大地提高了该类药物的纯度,并降低了患者的服用剂量和动物精液冻干粉中含有的其他物质可能造成的影响。
经检测,动物精液中顶体蛋白含量约为0.1%~0.4%。经提纯后,顶体蛋白含量大于90%,与动物精液或其冻干粉相比,药物浓度得到了很大提高,并且剔除了精液中的非特异性蛋白、脂肪和糖类化合物,从药效上提高抗肿瘤活性,并且降低了服用剂量,更方便临床应用。
可制成可制成胶囊、颗粒、片剂、滴丸、口服液、注射液等各种制剂应用于临床,优选为胶囊。本品也可与其他药物制成中西药复方制剂应用。
具体实施方式
顶体蛋白是卵子与精子受精过程中所不可缺少的生物活性物质。其化学成分为丝氨酸蛋白溶解类物质,可溶解卵膜。当卵子和精子都成功地被输送到受精部位时,精子的头部一旦接触到卵子表面时,位于其头部顶端的细胞器之一的顶体就随之发生顶体反应,由顶体部分伸长为顶体丝,并分泌出多种酶蛋白,即顶体蛋白,顶体蛋白溶解卵膜,使精子头部穿透卵的放射冠和透明带,而进入卵子,完成受精过程。因此,顶体蛋白活性临床上常成为精液分析评价精子功能的定量指标。
顶体蛋白可通过对冷冻保存的精子使用去垢剂,声处理和低pH等方法提取,并用凝胶过滤,离子交换层析,等电聚集,亲和层析和电泳等生化技术,可获得纯顶体蛋白。但不同哺乳动物和采用的不同分离方法所得到的顶体蛋白分子量差异较大,如人顶体素为30KD~70KD,而猪的约为41KD~56KD。
顶体素分子是由酶原区、催化区、尾区三部分组成,其中酶原区、催化区氨基酸序列无种属差异,尾区有种属差异。
经药效学实验证实,顶体蛋白对MCF-7、Bel7402、BGC823、A2780、A549及HCT-8六种人癌细胞株有抑制作用,对小鼠S180肿瘤抑制作用明显,与动物精液冻干粉相比,抑瘤作用明显,同时药用剂量也显著降低。同时本品与化疗药物联用能明显增加对化疗药物治疗肿瘤的治疗作用,提示本品有应用于治疗肿瘤的广泛前景。人体临床应用情况表明,本品治疗癌症有实质性的临床应用效果;将动物精液提纯得到顶体蛋白属于重大创新和发明,有利于降低患者服药剂量,为上述疾病的治疗提供了一种新的药用物质。
顶体蛋白可制成胶囊、颗粒、片剂、滴丸、口服液、注射液等各种制剂应用于临床,优选为胶囊,顶体蛋白具有较好的稳定性,优选的胶囊在遮光、密封条件下可保存24个月,可稳定有效的应用于临床。本品也可与其他药物制成中西药复方制剂应用。
以下结合实施例进一步说明本发明,而非限制本发明:
实施例1:
向1kg动物精液中加入4L的2%醋酸溶液,用1mol/L的盐酸调节pH为3,以5000r/min转速离心20min,弃去沉淀物,上清液合并,浓缩;
上清液加硫酸铵至饱和度为0.3,于4℃放置4小时,以3000r/min转速离心20min,弃去上清液。沉淀用0.1mol/L(pH 2)的醋酸溶液溶解即得顶体蛋白粗提物
柱层析:将粗提物用Sephadex G-25凝胶柱进行柱层析,以0.1mol/L(pH2)的醋酸缓冲溶液平衡洗脱,流速为0.5ml/min。用紫外分光光度法检测,收集按Lowry法在680nm处有最大吸收的洗脱液;
将洗脱液以3000r/min离心10min,弃去上清液,所得沉淀用纯蒸馏水洗涤后继续离心,收集沉淀,即得到顶体蛋白,收率为0.133%,用ELISA定量检测法测定顶体蛋白含量,含量92%。
实施例2:
将50g动物精液冻干粉用500ml蒸馏水溶化,加入3L的3%醋酸溶液,用1mol/L的盐酸调节pH为4,以6000r/min转速离心15min,弃去沉淀物,上清液合并,浓缩;
上清液加硫酸铵至饱和度为0.4,于4℃放置4小时,以3500r/min转速离心15min,弃去上清液,沉淀用0.1mol/L(pH2.5)的醋酸缓冲溶液溶解即得顶体蛋白粗提物;
柱层析:将粗提物用Sephadex LH-20凝胶柱进行柱层析,以0.1mol/L(pH2)的醋酸缓冲溶液平衡洗脱,流速为0.5ml/min;用紫外分光光度法检测,收集按Lowry法在680nm处有最大吸收的洗脱液;
将洗脱液以3000r/min离心10min,弃去上清液,所得沉淀用纯蒸馏水洗涤后继续离心,收集沉淀,即得到顶体蛋白,收率为2.8%,用ELISA定量检测法测定顶体蛋白含量,含量大于93.7%。
顶体蛋白的药效学实验如下:
MTT试验:用胰酶消化MCF-7、Bel7402、BGC823、A2780、A549及HCT-8六种人癌细胞株(中国科学院上海细胞所),以含10%小牛血清的RPMI1640培养液配制细胞悬液,浓度为104/ml,于96孔培养板内每孔接种100μl(1000个细胞/孔)。用培养基稀释药物,设6个浓度:10、20、40、80、160、320μg/ml,每组设三个平行孔,每孔加样0.1ml。置37℃、5%CO2孵箱中培养96小时后弃去培养液,每孔加入0.1ml 0.5%MTT溶液(RPMI1640配制)。37℃保温4小时,弃上清,每孔加入DMSO 0.1ml溶解Formazan颗粒,振荡混匀,用酶标仪检测(参比波长450nm、检测波长570nm),计算药物对细胞生长的抑制率。
表1.MTT法观察顶体蛋白对人癌细胞株的生长抑制作用结果
结果表明,在本实验条件下,320μg/ml顶体蛋白对MCF-7、Bel7402、BGC823、A2780有较强的抑制作用,对A549及HCT-8有一定的抑制作用。这为该药应用于肿瘤的治疗提供了临床前研究基础。
S180移植性肿瘤抑制实验:
选用昆明种小鼠,18~22g,雌,50只,分为空白对照组,动物精液冻干粉组、2.5mg·kg-1、5mg·kg-1、10mg·kg-1低中高剂量组,每组10只。将全身状况良好的荷瘤动物处死,无菌条件下取出腹水,以生理盐水稀释,于每鼠腋窝下接种0.2ml瘤液(S180细胞:1.0×107个/ml)。接种后24小时,将动物按体重随机分组。动物精液冻干粉64mg·kg-1剂量组、顶体蛋白2.5mg·kg-1、5mg·kg-1、10mg·kg-1低中高剂量组每天灌胃给药一次,连续给药10天,末次给药后24小时,颈椎脱臼处死动物,分别称体重及瘤重,计算肿瘤抑制率,进行疗效评价。并将各组结果进行统计学处理。
表2.顶体蛋白对小鼠肉瘤S180的生长抑制作用
表2显示,动物精液冻干粉的抑瘤率为29.1%(P<0.05),顶体蛋白2.5mg·kg-1组为44.8%(P<0.05),5mg·kg-1组为48.3%(P<0.05),10mg·kg-1组为56.2%(P<0.05)。这表明,在提纯后,与动物精液冻干粉相比,顶体蛋白给药剂量不但降低,而且对肿瘤的抑制率也明显增强。
与化疗药物联合用药的增效实验:
选用昆明种小鼠,18~22g,雌,50只,设空白对照组,环磷酰胺20mg·kg-1治疗组,环磷酰胺20mg·kg-1治疗组分别与顶体蛋白2.5mg·kg-1、5mg·kg-1、10mg·kg-1联合给药组。将肿瘤生长良好、全身状况较好的荷瘤动物处死,无菌条件下取出腹水,以生理盐水稀释,于每鼠腋窝下接种0.2ml瘤液(S180细胞:1.0×107个/ml)。接种后24小时,将动物按体重随机分组,每组10只。环磷酰胺腹腔注射给药20mg·kg-1,隔天一次,共计5次,顶体蛋白每天一次,连续给药10天。次给药后24小时,颈椎脱臼处死动物,分别称体重及瘤重,计算肿瘤抑制率,进行疗效评价。
由表3可见,环磷酰胺20mg·kg-1给药5次的抑瘤率为19.6%(P>0.05)。环磷酰胺20mg·kg-1与顶体蛋白2.5mg·kg-1合用后,抑瘤率为36.6%((P<0.05)。环磷酰胺20mg·kg-1与顶体蛋白5mg·kg-1合用后,抑瘤率为38.0%(P<0.05)。环磷酰胺20mg·kg-1与顶体蛋白10mg·kg-1合用后,抑瘤率为40.6%(P<0.05)。顶体蛋白与环磷酰胺合用后能明显增加对环磷酰胺治疗肿瘤的增效作用。
表3.顶体蛋白对S180肉瘤小鼠化疗增效作用
注:与空白组比,#P<0.05
临床药效验证:
将顶体蛋白试用于肿瘤门诊患者,共收集病例300例(主要为手术、放疗、化疗后病灶转移复发的患者),每例患者均口服20mg顶体蛋白,每日3次,服用3个月,查体检查,经统计有效率达到87%,显效率达到59%,治愈率达到8%。将顶体蛋白试用于226例抑郁症随机统计,每例患者均口服20mg顶体蛋白,每日3次,试用1个月,总有效率89%,治愈率14%。
由此可见,动物精液提纯物-顶体蛋白对子宫肌瘤、乳房瘤、胃癌、肺癌、肝癌、白血病、大肠癌、卵巢癌、乳腺癌、宫颈癌、恶性淋巴瘤及治疗抑郁症、癫痫等疾病有临床应用价值。
顶体蛋白稳定性实验:
取顶体蛋白20g,加入无水乳糖10g,混合均匀,测定顶体蛋白含量,计算粒重,无菌条件下灌装胶囊,制成1000粒。取灌装的3批胶囊,模拟市售包装,以顶体蛋白含量、水分、微生物限度(仅在0月和24个月测定)为检查指标在温度40℃±2℃、相对湿度75%±5%条件下进行0、1、2、3、6个月加速实验,在温度25℃±2℃、相对湿度60%±10%条件下进行0、3、6、9、12、18、24个月长期实验,并将结果与0月比较,可确定产品有效期为24个月。结果见表4。
表4.顶体蛋白稳定性实验结果
迄今为止,国内外尚未有顶体蛋白治疗各种肿瘤及疾病的药物文献报道,是医药技术领域有价值的新发明,为国内外首先发现和揭示。
Claims (10)
1.顶体蛋白用于抑制癌细胞的扩散。
2.顶体蛋白用于抑制MCF-7、Bel7402、BGC823、A2780、A549或HCT-8细胞的扩散。
3.顶体蛋白在制备治疗癌症的药物中的应用。
4.顶体蛋白在制备治疗抑郁症的药物中的应用。
5.根据权利要求3所述的顶体蛋白,其特征在于:所述癌症包括妇科肿瘤、消化道肿瘤、肺癌、肝癌、白血病和恶性淋巴瘤。
6.根据权利要求5所述的顶体蛋白,其特征在于:所述妇科肿瘤包括子宫肌瘤、卵巢癌、乳房瘤、乳腺癌和宫颈癌;所述消化道肿瘤包括食道癌、胃癌、大肠癌。
7.根据权利要求3至6任一所述的顶体蛋白,其特征在于:所述顶体蛋白用于制备胶囊、颗粒、片剂、滴丸、中西药复方制剂、口服液或注射液。
8.一种顶体蛋白的制备方法,其特殊之处在于,包括以下步骤:
1]取动物精液或复溶的动物精液冻干粉,向其中加入浓度为1%~5%的稀酸溶液,再加入无机酸将pH调节至2~5,以2000r/min~8000r/min的转速离心15~30min后弃去沉淀物,将得到的上清液合并,浓缩;选取动物精液时,动物精液与稀酸的质量比为1∶1~1∶20;选取动物精液冻干粉时,动物精液冻干粉与复溶溶剂水的质量比为1∶1~1∶20,动物精液冻干粉复溶溶液与稀酸的比例为质量比为1∶1~1∶16;
2]向步骤1处理所得的上清液中加入无机铵盐,调节饱和度至0.2-0.5后放置3-5小时,再以2000r/min~8000r/min离心15~30min,弃去上清液,沉淀用醋酸缓冲溶液溶解后,得到顶体蛋白粗提物;
3]将经步骤3处理所得的蛋白粗提物进行柱层析,用醋酸缓冲溶液平衡洗脱,处理完成后收集洗脱液;
4]将洗脱液以2000r/min~8000r/min离心5~10min,弃去上清液,所得沉淀洗涤后以2000r/min~8000r/min继续离心1~3次,每次离心5~10min,收集沉淀,即得到顶体蛋白。
9.根据权利要求8所述的顶体蛋白的制备方法,其特征在于:所述步骤1中,稀酸溶液是浓度为1.7%~4.5%的醋酸溶液、甲酸溶液、丙酸溶液、丁酸溶液、盐酸溶液、硫酸溶液或磷酸溶液,调节pH的无机酸为1mol/L的硫酸、盐酸、氢溴酸或磷酸,调节pH至2.8~4.6;动物精液与稀酸的质量比为1∶4;选取动物精液冻干粉时,动物精液冻干粉与复溶溶剂水的质量比为1∶10,动物精液冻干粉复溶溶液与稀酸的比例为质量比为1∶5.5;所述步骤2中无机铵盐是硫酸铵、硫酸氢铵、碳酸铵、碳酸氢铵或磷酸铵,调节饱和度至0.3后在4℃环境中放置3-5小时;所述醋酸缓冲溶液是pH=2,0.1mol/L的醋酸缓冲溶液;所述步骤3中,是用葡聚糖凝胶柱、羟丙基葡聚糖凝胶柱、丙烯酰胺凝胶柱或琼脂糖凝胶柱进行柱层析,所述醋酸缓冲溶液是pH=2,0.1mol/L的醋酸缓冲溶液,醋酸缓冲溶液平衡洗脱的流速为0.5ml/min,处理完成后收集在680nm处有最大吸收的洗脱液;所述步骤4中,洗涤沉淀使用纯蒸馏水;所述步骤1至4中的离心转速为2500r/min~6000r/min。
10.根据权利要求9所述的顶体蛋白,其特征在于:所述动物精液冻干粉为健康驴、牛、羊或马的精液冻干粉。
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