CN113101308B - 一种土当归提取物及其制备方法和应用 - Google Patents
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Abstract
本发明属于中药技术领域，具体涉及一种预防和/或治疗前列腺癌的土当归（Angelica gigas Nakai）提取物及其制备方法和应用。本发明提供的制备方法得到的土当归提取物，主要成分为香豆素类化合物，少量黄酮类和酚酸类化合物，相较于现有土当归提取物的制备大都要用到多种有机溶剂萃取、大孔树脂洗脱以及后处理等，工艺简单，制造成本低，更适于土当归提取物的大规模工业化生产。用本发明的制备方法得到土当归提取物通过实验证明其对前列腺癌转基因小鼠模型肿瘤的发生和生长具有显著预防和治疗作用，对人前列腺癌异种移植瘤小鼠模型肿瘤生长具有显著抑制作用，为其在制备抗前列腺癌产品中的应用提供了广阔的前景。
Description
技术领域
本发明属于中药技术领域，具体涉及一种预防和/或治疗前列腺癌的土当归（Angelica gigas Nakai）提取物KO及其制备方法和应用。
背景技术
土当归，又名朝鲜当归，大独活，为伞形科当归属多年生草本植物Angelica gigasNakai（AGN）的干燥根，在我国东北，朝医在民间将“土当归”作为正品当归使用，认为其不仅具有我国中药当归（Angelicae sinensis）活血止痛的功效，更兼具清热解毒、祛风通络等作用，既可单独使用，也可复方配伍，是吉林朝鲜族自治州民间用于解毒的主药之一。
前列腺癌是全世界男性泌尿生殖系统最常见的肿瘤之一。在美国男性人群中，前列腺癌是发病率最高的恶性肿瘤，是第二位的癌症死亡原因。在我国，前列腺癌从30年前的罕见肿瘤、20年前的少见肿瘤、到如今已跃居我国男性恶性肿瘤发病率第6位，是我国成年男性近十年来发病率和死亡率上升最快的癌症。随着我国人口老龄化程度的加剧，男性前列腺癌的发病率将会越来越高。前列腺癌发病机制与雄激素受体密切相关，临床多采用雄激素去势疗法进行治疗，尽管对于多数患者在一定时间内疾病进展可被控制，但经过12-24个月治疗后，最终会发展为去势抵抗性前列腺癌，预后极差，因此仍迫切需要积极研究和开发一种新的前列腺癌治疗药物。
发明内容
鉴于现有技术存在的问题，本发明的目的在于提供一种土当归提取物及其制备方法和在预防和/或治疗前列腺癌中的应用。用本发明的制备方法得到土当归提取物通过实验证明其对前列腺癌转基因小鼠模型肿瘤的发生和生长具有显著预防和治疗作用，对人前列腺癌异种移植瘤小鼠模型肿瘤生长具有显著抑制作用，为其在制备抗前列腺癌产品中的应用提供了广阔的前景。
为实现上述发明目的，本发明采用以下技术方案。
一种土当归提取物的制备方法，具体包括以下步骤。
步骤1、取土当归药材，加入药材重量8倍量的95% v/v乙醇回流提取2小时，过滤，滤渣再加加入相当于生药材重量5倍量的95%乙醇回流提取2小时，过滤，合并滤液，减压浓缩至浸膏状，得到提取物1。
步骤2、步骤1中得到的提取物1，加入3倍量水混悬，过滤去除树脂、叶绿素等水不溶性杂质后，依次用石油醚、乙酸乙酯、正丁醇有机溶剂进行等体积萃取，各两次，经减压浓缩后得到各萃取物浸膏，将乙酸乙酯部位提取物中紫花前胡素和紫花前胡醇当归酯成分去除干净后，与正丁醇萃取物合并，得到土当归提取物KO，并经喷雾干燥后得干粉。
一种土当归提取物由上述制备方法制得。
一种药物组合物，包含所述述的土当归提取物和药学上可接受的载体。
所述土当归提取物和所述药物组合物在制备预防和/或治疗前列腺癌药物中的应用。
与现有技术比，本发明的有益效果如下。
本发明提供的制备方法得到的土当归提取物，主要成分为香豆素类化合物，少量黄酮类和酚酸类化合物，工艺简单，制造成本低，相较于现有土当归提取物的制备大都要用到多种有机溶剂萃取、大孔树脂洗脱以及后处理等，步骤繁琐、工艺复杂，本发明制备方法更简单方便、制造成本更低，更适于土当归提取物的大规模工业化生产。
本发明提供的制备方法得到的土当归提取药理活性得到显著提高，使当归提取物具有了更高、更好的药用价值，具有显著预防和治疗前列腺癌作用。经药理实验证明，本发明提供的土当归提取物对前列腺癌转基因小鼠模型肿瘤的发生和生长具有显著预防和治疗作用。本发明土当归提取物KO对人前列腺癌异种移植瘤小鼠模型肿瘤生长具有显著抑制作用。为其在制备抗前列腺癌产品中的应用提供了广阔的前景。
附图说明
图1是各组小鼠体重情况比较结果。
图2是各组小鼠前列腺癌上皮瘤诱发前列腺癌肥大情况比较。
图3是两组小鼠肿瘤体积、重量及肿瘤肺转移情况差异。
图4是两组小鼠体重和肝肾重量变化情况。
具体实施方式
下面通过具体实施例对本发明进行说明，下述实施例中所述实验方法，如无特殊说明，均为常规方法；所述试剂和生物材料，如无特殊说明，均为市售。下述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
实施例1 土当归提取物KO的制备方法。
一种土当归提取物的制备方法，具体包括以下步骤。
步骤1、取土当归药材，加入药材重量8倍量的95% v/v乙醇回流提取2小时，过滤，滤渣再加5倍量95%乙醇回流提取2小时，过滤，合并滤液，减压浓缩得到提取物1。
步骤2、步骤1中得到的提取物1加适量水混悬，去除树脂、叶绿素等水不溶性杂质后，依次用石油醚、乙酸乙酯、正丁醇有机溶剂进行等体积萃取，各两次，经减压浓缩后得到各萃取物浸膏，将乙酸乙酯部位提取物中紫花前胡素和紫花前胡醇当归酯成分去除干净后，与正丁醇萃取物合并，得到土当归提取物KO，并经喷雾干燥后得干粉。
实施例2 本发明提供的土当归提取物对前列腺癌转基因小鼠模型肿瘤的发生和生长具有显著预防和治疗作用。
1实验材料。
1.1实验动物及饲料。
前列腺癌转基因小鼠（TRAMP）母代购于Jackson实验室，子代小鼠出生后3周尾部取样进行基因检测。所有小鼠使用AIN-93G饲料喂养，自由饮食和饮水。
1.2实验药物。
实施例1制备的土当归提取物KO，以80 mg/kg体重（2mg/鼠）的给药剂量用乙醇-PEG400-吐温80-5%葡萄糖溶液（EPTG比例为3:6:1:20）配制成药液，灌胃给药。-20℃保存，灌胃给药前预温。
1.3实验方法。
携带目标基因的雄性鼠为实验所需小鼠，小鼠7周年龄时，随机分为实验组（土当归提取物组分KO给药组，2mg/鼠/天，每组20只）和安慰剂组（EPTG喂养组，每组20只），并加设非携带前列腺癌基因的正常雄性小鼠对照组，EPTG喂养组（每组6只），给药及分组信息见表1。8周开始灌胃给药，每天一次，连续给药20周。所有小鼠AIN-93G饲料喂养，自由进食、饮水，采用12h-12h明暗交替照明，每周换3次水，换2次垫料。每天给药期间检查并记录小鼠健康状况和存活情况，如活动性、皮毛状态、呼吸状态、弯腰驼背姿势等。每周测定小鼠体重一次。小鼠19周龄开始检查其前列腺肿瘤发生情况，22周开始测试肿瘤体积，每周一次，并用公式：肿瘤体积=（长×宽2）/2计算出肿瘤体积的大小。
表1. 给药及分组信息。
1.4标本采集和数据分析。
灌胃给药20周后，统一处死，心脏取血，分离血清，-80°C冰箱保存；解剖小鼠，取出肿瘤组织，称重；如小鼠前列腺处未发生肿瘤，取出前列腺，称重；肿瘤和前列腺组织均分为两份，一份-80°C冻存，另一份福尔马林缓冲液固定，并用石蜡密封用作组织学分析。通过计算和评价土当归提取物组分KO给药组抑制携带前列腺癌基因小鼠肿瘤发生率、自发瘤的体积和重量、未发生肿瘤小鼠前列腺肥大程度来衡量其对TRAMP小鼠的抗前列腺癌功效。解剖小鼠时，同时收集小鼠的其它脏器组织，如肺、心脏、肾脏、脾脏等，用于考量药物安全性。
1.5实验结果。
（1）在小鼠状态方面：土当归提取物KO给药组TRAMP小鼠精神状态良好，与健康小鼠（未携带前列腺癌基因WildType小鼠）相似，反应机敏、活动自如，毛发有光泽，随着周龄的增加，饮水量和摄食量均逐步增加，体重稳步增长，无死亡；TRAMP小鼠安慰剂给药组与WildType小鼠相比，后期表现出肿瘤带来的食欲减退、疲劳、皮毛干燥无光泽，反应迟钝等现象（各组小鼠体重变化见图1）。本发明土当归提取物KO体内给药安全性良好。
（2）从小鼠8周龄开始灌胃给药，连续给药20周后，能够显著降低携带前列腺癌基因的小鼠肿瘤发病几率，见表2，减缓前列腺上皮肿瘤生长，表现为前列腺肥大，速度如图2所示。
表2. 各组小鼠前列腺癌发病率。
实施例3 本发明土当归提取物KO对人前列腺癌异种移植瘤小鼠模型肿瘤生长具有显著抑制作用。
1实验材料。
1.1实验动物及饲料。
免疫缺陷小鼠购于Jackson实验室，所有小鼠使用AIN-93G饲料喂养，自由饮食和饮水。
1.2实验药物。
实施例1制备的土当归提取物KO，以160mg/kg体重（4mg/鼠）的给药剂量用乙醇-PEG400-吐温80-5%葡萄糖溶液（EPTG比例为3:6:1:20）配制成药液，灌胃给药。-20℃保存，灌胃给药前预温。
1.3实验方法。
将LNAR-luc细胞注射于双免疫缺陷小鼠躯体后侧方皮下，接种2天后，将小鼠随机分为空白对照组、土当归提取物组分KO给药组，每组20只。每天给药一次，连续给药10周，每周称量一次体重，测量两次小鼠皮下成瘤的最长径和最宽径，并用公式：肿瘤体积=（长径×宽径2）/2计算出肿瘤体积的大小。10周后检测小鼠肺转移情况，处死小鼠，心脏取血，离心得血浆；解剖取出肿瘤组织，称重，计算抑瘤率，绘制皮下成瘤的生长曲线。
1.4实验结果。
随着给药时间进行，土当归提取物KO给药组小鼠肿瘤生长体积和重量、及肺转移情况明显低于安慰剂给药组，土当归提取物KO给药组小鼠肿瘤重量与安慰剂给药组相比降低了43%，结果如图3所示。两组小鼠体重及肝肾重量并未呈现显著性差异，说明土当归提取物组分KO安全性达到治疗要求，结果如图4所示。
Claims (3)
1.一种治疗前列腺癌的土当归提取物，其特征在于，所述土当归提取物的制备方法具体包括以下步骤：
步骤1、取土当归药材，加入药材重量8倍量的95% v/v乙醇回流提取2小时，过滤，滤渣再加入相当于生药材重量5倍量的95% v/v乙醇回流提取2小时，过滤，合并滤液，减压浓缩至浸膏状，得到提取物1；
步骤2、步骤1中得到的提取物1，加入3倍量水混悬，过滤去除树脂、叶绿素水不溶性杂质后，依次用石油醚、乙酸乙酯、正丁醇有机溶剂进行等体积萃取，各两次，经减压浓缩后得到各萃取物浸膏，将乙酸乙酯部位提取物中紫花前胡素和紫花前胡醇当归酯成分去除干净后，与正丁醇萃取物合并，得到土当归提取物，并经喷雾干燥后得干粉。
2.一种药物组合物，其特征在于，包含权利要求1所述的土当归提取物和药学上可接受的载体。
3.权利要求2所述的药物组合物在制备治疗前列腺癌药物中的应用。
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