CN106511409A - 一种复方番木瓜籽醇提物及其在增强免疫力中的应用 - Google Patents
一种复方番木瓜籽醇提物及其在增强免疫力中的应用 Download PDFInfo
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Abstract
本发明公开了复方番木瓜籽醇提物及其制备方法以及具有增强机体免疫力的功能。本发明以复方番木瓜籽醇提物和海南裂叶山龙眼叶为研究对象,根据中华人民共和国卫生部《免疫‑保健食品检验与评价技术规范》的药理方法,研究复方番木瓜籽醇提物对小鼠体液免疫、非特异性免疫和免疫脏器指数的影响。结果表明,单味番木瓜籽醇提物高、中剂量组及其复方组能显著提高小鼠的胸腺指数、促进血清溶血素的生成以及增强腹腔巨噬细胞吞噬功能,表明了单味番木瓜籽醇提物及其复方番木瓜籽醇提物具有增强机体免疫的功能。
Description
技术领域
本发明涉及番木瓜的新用途,具体涉及一种复方番木瓜籽醇提物及其在增强免疫力中的应用。
背景技术
番木瓜(Carica papaya L)又名万寿果,为番木瓜科植物的果实,在我国广东、广西、海南、云南、福建等省广泛种植。研究表明,番木瓜果实含有番木瓜碱、木瓜蛋白酶、凝乳酶、胡萝卜素等,同时还含有17种以上氨基酸及多种营养元素,具有护肝、抗炎抑菌、降低血脂等功效。番木瓜中含有丰富的苄基硫代葡萄糖苷(benzyl glucosinolate,BG),尤其番木瓜籽中含量最高,此物质在硫苷酶或者肠道菌的作用下水解成具芳香烷侧链的异硫氰酸酯—异硫氰酸苄酯(benzyl isothiocyanate,BITC)。已有研究表明,BITC对肝癌、前列腺癌、乳腺癌、宫颈癌、肺癌、胰腺癌和卵巢癌等肿瘤细胞均具有较好的抗肿瘤作用。
海南裂叶山龙眼Heliciopsislobata(Merr.)Sleum.,又名大叶萝卜树、那托、定朗,为山龙眼科假山龙眼属植物。海南裂叶山龙眼的根皮与叶入药,其药性凉,味淡、涩,有清热解毒之功效,民间主要用于治疗腮腺炎和皮炎;海南山区黎族群众则长期用来治疗肿瘤,目前无免疫活性报道,本发明以海南裂叶山龙眼叶入药。
发明内容
发明目的:本发明的目的是开发番木瓜籽新的药理活性,提供一种复方番木瓜籽醇提物及其在增强免疫力中的应用。
技术方案:为了实现以上目的,本发明所采取的技术方案为:
一种复方番木瓜籽醇提物,由下述原料药提取制备而成:番木瓜籽5-20份、海南裂叶山龙眼叶1-3份。
所述复方番木瓜籽醇提物,所述的制备方法如下:
称取粉碎的番木瓜籽,加入乙醇提取两次,合并滤液,减压浓缩,浓缩液依次用石油醚、乙酸乙酯、正丁醇分别萃取3次,最后水层减压浓缩,加入乙醇,抽滤,滤液浓缩至浸膏。另取海南裂叶山龙眼叶,晾干,粉碎,粗粉用乙醇回流提取两次,合并提取液,减压浓缩得浸膏,合并番木瓜籽醇提物和海南裂叶山龙眼叶醇提物,添加辅料,制备成口服液、片剂、胶囊剂、颗粒剂中的一种。
所述复方番木瓜籽醇提物,所述的制备方法如下:称取粉碎的番木瓜籽10份,加入5-15倍番木瓜籽重量的体积比为50%-95%的乙醇提取两次,合并滤液,减压浓缩,浓缩液依次用石油醚、乙酸乙酯、正丁醇分别萃取3次,最后水层减压浓缩成浸膏,加入番木瓜籽重量的体积比为50%-95%的乙醇,抽滤,滤液浓缩至浸膏;另取海南裂叶山龙眼叶2份,晾干,粉碎,粗粉用体积比为50%-95%的乙醇回流提取两次,合并提取液,减压浓缩得浸膏,合并番木瓜籽醇提物和海南裂叶山龙眼叶醇提物,添加辅料,制备成口服液、片剂、胶囊剂、颗粒剂中的一种。
所述复方番木瓜籽醇提物,所述的制备方法如下:称取粉碎的番木瓜籽10份,加入10倍番木瓜籽重量的体积比为75%的乙醇提取两次,合并滤液,减压浓缩,浓缩液依次用石油醚、乙酸乙酯、正丁醇分别萃取3次,最后水层减压浓缩成浸膏,加入番木瓜籽重量的体积比为75%的乙醇,抽滤,滤液浓缩至浸膏;另取海南裂叶山龙眼叶2份,晾干,粉碎,粗粉用体积比为75%的乙醇回流提取两次,合并提取液,减压浓缩得浸膏,合并番木瓜籽醇提物和海南裂叶山龙眼叶醇提物,添加辅料,制备成口服液、片剂、胶囊剂、颗粒剂中的一种。
所述复方番木瓜籽醇提物在制备提高免疫力的保健品或药品的应用。
本发明以复方番木瓜籽醇提物为研究对象,根据卫生部《免疫-保健食品检验与评价技术规范》的药理方法,研究其对小鼠体液免疫、非特异性免疫和免疫脏器指数的影响。结果表明,复方番木瓜籽醇提物能显著提高小鼠的胸腺指数、促进血清溶血素的生成以及增强腹腔巨噬细胞吞噬功能,表明了复方番木瓜籽醇提物具有增强机体免疫的功能,而且复方番木瓜籽醇提物效果更明显,由于单味番木瓜籽,对进一步开发番木瓜籽和海南裂叶山龙眼叶有一定的参考价值。
具体实施方式
实施例1:称取粉碎的番木瓜籽1kg,加入9倍体积比为75%的乙醇,提取两次,合并滤液,减压浓缩,浓缩液分别用石油醚、乙酸乙酯、正丁醇萃取,最后水层减压浓缩至300mL,加入其2倍量乙醇,抽滤,滤液浓缩至浸膏,另取海南裂叶山龙眼叶200g,晾干,粉碎,粗粉用体积比为75%的乙醇回流提取两次,合并提取液,减压浓缩得浸膏,合并番木瓜籽醇提物和海南裂叶山龙眼叶醇提物,添加辅料,制备成口服液。
实施例2:称取粉碎的番木瓜籽0.5kg,加入9倍体积比为50%的乙醇,提取两次,合并滤液,减压浓缩,浓缩液分别用石油醚、乙酸乙酯、正丁醇萃取,最后水层减压浓缩至300mL,加入其2倍量乙醇,抽滤,滤液浓缩至浸膏,另取海南裂叶山龙眼叶0.3kg,晾干,粉碎,粗粉用体积比为95%的乙醇回流提取两次,合并提取液,减压浓缩得浸膏,合并番木瓜籽醇提物和海南裂叶山龙眼叶醇提物,添加辅料,制备成片剂。
实施例3:称取粉碎的番木瓜籽2kg,加入9倍体积比为95%的乙醇,提取两次,合并滤液,减压浓缩,浓缩液分别用石油醚、乙酸乙酯、正丁醇萃取,最后水层减压浓缩至300mL,加入其2倍量乙醇,抽滤,滤液浓缩至浸膏,另取海南裂叶山龙眼叶100g,晾干,粉碎,粗粉用体积比为55%的乙醇回流提取两次,合并提取液,减压浓缩得浸膏,合并番木瓜籽醇提物和海南裂叶山龙眼叶醇提物,添加辅料,制备成胶囊剂。
实施例4:称取粉碎的番木瓜籽1kg,加入9倍体积比为75%的乙醇,提取两次,合并滤液,减压浓缩,浓缩液分别用石油醚、乙酸乙酯、正丁醇萃取,最后水层减压浓缩至300mL,加入其2倍量乙醇,抽滤,滤液浓缩至浸膏,另取海南裂叶山龙眼叶200g,晾干,粉碎,粗粉用体积比为70%的乙醇回流提取两次,合并提取液,减压浓缩得浸膏,合并番木瓜籽醇提物和海南裂叶山龙眼叶醇提物,添加辅料,制备成颗粒剂。
实施例5:复方番木瓜籽醇提物的免疫活性研究
1.实验材料
1.1材料与试剂
番木瓜籽,从水果市场搜集所得,放入烘箱,于110℃灭酶活1h,水洗后40℃烘干,粉碎,过筛,放入密封袋储藏备用。
海南裂叶山龙眼叶由中国医学科学院药用植物研究所海南分所提供。
苄基硫代葡萄糖苷(BG)对照品(批号:12030606,购买于ITW Company);抗凝羊血(鸿泉生物),豚鼠血清(自制),SA缓冲液(biohao),都氏试剂(美国Sigma公司),Giemsa染液(上海源叶生物科技有限公司),鸡红细胞(自制),生理盐水(山东鲁抗辰欣),水为超纯水;甲酸、乙腈为色谱纯;其余试剂均为分析纯。
1.2仪器与设备
UV-2401PC(日本岛津),HW-3型恒温水浴(天津天大天发),DT5-2型低速台式离心机(北京时代北利离心机有限公司),3k18型高速冷冻离心机(美国Sigma公司),50i尼康光电显微镜,AUW220D型分析天平(日本岛津),UltiMate 3000 UHPLC(Thermo Fisher);HC-150T2型粉碎机(永康市绿可食品机械有限公司);GZX-9240ME型数显鼓风干燥箱(上海博迅实业有限公司);SY-180型超声仪(上海宁商超声仪器有限公司);SHZ-D(III)型循环水式真空泵(河南省予华仪器有限公司);AL104型电子天平(梅特勒-托利多仪器公司);RE-52AA旋转蒸发器(上海亚荣生化仪器厂);KDM型调温电热套(山东鄄城华鲁电热仪器有限公司)
1.3动物
SPF级昆明种小鼠,单一性别,体重18-22g,由海南省药物研究所提供,许可证号:SCXK(琼)2015-0007;豚鼠:健康豚鼠;鸡:健康家鸡。
2.实验方法
以复方番木瓜籽醇提物为研究对象,根据卫生部《免疫-保健食品检验与评价技术规范》的药理方法,研究其对小鼠体液免疫和非特异性免疫的影响考察免疫活性。
2.1复方番木瓜籽醇提物的制备:按上述实施例1方法制备。单味番木瓜籽醇提物的制备也是按上述实施例1前半部分方法。
2.2分组及给药方式
SPF级昆明种小鼠100只,18-22g,单一性别。每个实验随机分为5组(空白对照组、单味番木瓜籽醇提物低剂量组、单味番木瓜籽醇提物中剂量组、单味番木瓜籽醇提物高剂量组和复方番木瓜籽醇提物组),每组10只,实验前,将所有小鼠适应性饲养2天,自由饮水和取食。单味番木瓜籽醇提物低剂量组,中剂量组,高剂量组给药量分别为52.84、158.53、317.06mg/kg,复方番木瓜籽醇提物组给予复方番木瓜籽醇提物,空白对照组给予超纯水,每日1次,按小鼠体重给药,连续灌胃30天。
2.3复方番木瓜籽醇提物对小鼠体液免疫的影响
采集5只豚鼠血,分离出血清,将1mL压积SRBC加入到5mL豚鼠血清中,放4℃冰箱30min,经常震荡,离心取上清,制备补体,用时以SA液按1:8稀释。按照2.2项下分组、给药,制备2%的SRBC细胞悬液,每只鼠腹腔注射0.2mL进行免疫。4天后,摘除眼球取血、离心,用SA缓冲液稀释200倍。将稀释后的血清1mL置试管内,依次加入10%(v/v)SRBC 0.5mL,补体1mL。另设不加血清的对照管。置37℃恒温水浴中保温20min后,冰浴终止反应,离心。取上清液1mL,加都氏试剂3mL,同时取10%(v/v)SRBC 0.25mL加都氏试剂至4mL,充分混匀,放置10min后,于540nm处以对照管作空白,分别测定各管光密度值。血清溶血素的量以半数溶血值(HC50)表示,按下式公式计算:
2.4复方番木瓜籽醇提物对小鼠非特异性免疫的影响
2.4.1吞噬功能测定
按照2.2项下分组、给药,制备20%的鸡红细胞悬液,每只鼠腹腔注射该悬液1mL。间隔30min,颈椎脱臼处死小鼠,将其仰位固定于鼠板上,正中剪开腹壁皮肤,经腹腔注入生理盐水2mL,转动鼠板1min。然后吸出腹腔洗液1mL,平均分滴于2片载玻片上,放入垫有湿纱布的搪瓷盒内,移至37℃孵箱温育30min。孵毕,于生理盐水中漂洗,以除去未贴片细胞。晾干,以1:1丙酮甲醇溶液固定,用瑞氏染液染色1min,再用蒸馏水漂洗晾干。
用显微镜计数巨噬细胞,每张片计数100个,按下式计算吞噬百分率和吞噬指数。
2.4.2脏器指数测定
解剖取脾脏、胸腺称重,计算脏器指数。
脏器指数=脏器重量(mg)/小鼠体重(g)。
2.5数据处理
采用spss19.0软件进行数据处理、统计和分析,分析结果以表示,组与组间比较采用方差分析和t检验。
3.实验结果
3.1复方番木瓜籽醇提物对小鼠体液免疫的影响
表1复方番木瓜籽醇提物对小鼠血清溶血功能的影响(n=10,)
注:与空白对照组比较,**P<0.01;与低剂量组比较,#P<0.05。
由表1可知,单味番木瓜籽醇提物及复方番木瓜籽醇提物可不同程度的促进小鼠的血清溶血素生成,与空白对照组相比,单味番木瓜籽醇提物高、中剂量组和复方番木瓜籽醇提物组的半数溶血值有极显著性差异(P<0.01),而低剂量组则无显著性差异;与低剂量组比较,复方组的半数溶血值有显著性差异(P<0.05),表明单味番木瓜籽醇提物及其复方番木瓜籽醇提物能显著增强小鼠的体液免疫功能,而且复方番木瓜籽醇提物效果更明显,番木瓜籽和海南裂叶山龙眼叶具有协同作用。
3.2复方番木瓜籽醇提物对小鼠非特异性免疫的影响
表2复方番木瓜籽醇提物对小鼠吞噬率和吞噬指数的影响(n=10,)
组别 | 剂量(mg/kg) | 吞噬率(%) | 吞噬指数 |
空白对照组 | — | 52.50±5.88 | 1.07±0.26 |
单味番木瓜籽醇提物低剂量组 | 52.84 | 52.65±6.11 | 1.08±0.09 |
单味番木瓜籽醇提物中剂量组 | 158.53 | 64.84±8.02** | 1.71±0.41* |
单味番木瓜籽醇提物高剂量组 | 317.06 | 66.51±6.41** | 1.74±0.33* |
复方番木瓜籽醇提物组 | 193.50 | 69.89±5.22**# | 1.79±0.08**# |
注:与空白对照组比较,*P<0.05,**P<0.01;与低剂量组比较,#P<0.05。
由表2可知,与空白对照组相比,单味番木瓜籽醇提物高剂量组、中剂量组和复方番木瓜籽醇提物组的吞噬率有极显著性差异(P<0.01),单味番木瓜籽醇提物高、中剂量组的吞噬指数有显著性差异(P<0.05),复方番木瓜籽醇提物组的吞噬指数有极显著性差异(P<0.01);与低剂量组比较,复方番木瓜籽醇提物组的吞噬率和吞噬指数具有显著性差异(P<0.05),表明单味番木瓜籽醇提物高、中剂量组及其复方组可明显提高小鼠腹腔巨噬细胞的吞噬百分率和吞噬指数,可增强小鼠的非特异性免疫,而且复方番木瓜籽醇提物效果更明显,番木瓜籽和海南裂叶山龙眼叶具有协同作用。
3.3复方番木瓜籽醇提物对小鼠免疫器官指数的影响
表3复方番木瓜籽醇提物对小鼠体重和免疫器官指数的影响(n=10,)
注:与空白对照组比较,*P<0.05,**P<0.01;与低剂量组比较,#P<0.05。
由表3可知,与空白对照组相比,单味番木瓜籽醇提物高、中剂量组能明显提高小鼠的胸腺指数,具有显著性差异(P<0.05),复方番木瓜籽醇提物组具有极显著性差异(P<0.01),而体重和脾脏指数则没有明显差异;与单味番木瓜籽醇提物低剂量组比较,复方番木瓜籽醇提物组的胸腺指数也具有显著性差异(P<0.05)。表明该单味番木瓜籽醇提物高、中剂量组和复方番木瓜籽醇提物组使胸腺指数明显增加,有增强小鼠免疫的功能,而且复方番木瓜籽醇提物效果更明显,番木瓜籽和海南裂叶山龙眼叶具有协同作用。
Claims (5)
1.一种复方番木瓜籽醇提物,其特征在于,由下述原料药提取制备而成:番木瓜籽5-20份、海南裂叶山龙眼叶1-3份。
2.如权利要求1所述复方番木瓜籽醇提物,其特征在于,所述的制备方法如下:
称取粉碎的番木瓜籽,加入乙醇提取两次,合并滤液,减压浓缩,浓缩液依次用石油醚、乙酸乙酯、正丁醇分别萃取3次,最后水层减压浓缩,加入乙醇,抽滤,滤液浓缩至浸膏。另取海南裂叶山龙眼叶,晾干,粉碎,粗粉用乙醇回流提取两次,合并提取液,减压浓缩得浸膏,合并番木瓜籽醇提物和海南裂叶山龙眼醇提取物,添加辅料,制备成口服液、片剂、胶囊剂、颗粒剂中的一种。
3.如权利要求2所述复方番木瓜籽醇提物,其特征在于,所述的制备方法如下:称取粉碎的番木瓜籽10份,加入5-15倍番木瓜籽重量的体积比为50%-95%的乙醇提取两次,合并滤液,减压浓缩,浓缩液依次用石油醚、乙酸乙酯、正丁醇分别萃取3次,最后水层减压浓缩成浸膏,加入番木瓜籽重量的体积比为50%-95%的乙醇,抽滤,滤液浓缩至浸膏;另取海南裂叶山龙眼叶2份,晾干,粉碎,粗粉用体积比为50%-95%的乙醇回流提取两次,合并提取液,减压浓缩得浸膏,合并番木瓜籽醇提物和海南裂叶山龙眼醇提取物,添加辅料,制备成口服液、片剂、胶囊剂、颗粒剂中的一种。
4.如权利要求3所述复方番木瓜籽醇提物,其特征在于,所述的制备方法如下:称取粉碎的番木瓜籽10份,加入10倍番木瓜籽重量的体积比为75%的乙醇提取两次,合并滤液,减压浓缩,浓缩液依次用石油醚、乙酸乙酯、正丁醇分别萃取3次,最后水层减压浓缩成浸膏,加入番木瓜籽重量的体积比为75%的乙醇,抽滤,滤液浓缩至浸膏;另取海南裂叶山龙眼叶2份,晾干,粉碎,粗粉用体积比为75%的乙醇回流提取两次,合并提取液,减压浓缩得浸膏,合并番木瓜籽醇提物和海南裂叶山龙眼醇提取物,添加辅料,制备成口服液、片剂、胶囊剂、颗粒剂中的一种。
5.如权利要求1所述复方番木瓜籽醇提物在制备提高免疫力的保健品或药品的应用。
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CN109856265A (zh) * | 2019-01-15 | 2019-06-07 | 广西中医药大学 | 基于抗氧化和降血糖谱效关系的龙眼叶化学成分评价方法 |
CN109856265B (zh) * | 2019-01-15 | 2022-01-25 | 广西中医药大学 | 基于抗氧化和降血糖谱效关系的龙眼叶化学成分评价方法 |
CN110423792A (zh) * | 2019-08-28 | 2019-11-08 | 浙江海洋大学 | 一种牛磺酸修饰的新型抗氧化肽的制备方法 |
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