CN114748532A - 一种从油橄榄果中制备的具有耐缺氧活性的组分及其应用 - Google Patents
一种从油橄榄果中制备的具有耐缺氧活性的组分及其应用 Download PDFInfo
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Abstract
本发明提供一种从油橄榄果中制备的具有耐缺氧活性的组分及其应用,所提供的提取物,是将油橄榄果的水提取煎液进行浓缩后,在浓缩液中加入乙醇;静置过滤后得到上清液;上清液浓缩后,进行干燥后获得。本发明所提供的提取物可用于制备预防治疗摄氧不足的制品。本发明所制备的橄榄果提取物具有耐缺氧活性的有效成分,可将所提供的提取物制成可随身携带的胶囊功能食品、保健制品,从而增加了油橄榄果加工的经济效益。
Description
技术领域
本发明属于活性物质筛选分离技术领域,具体涉及一种从油橄榄果中制备的具有耐缺氧活性的组分及其应用。
背景技术
氧气是一切需氧生物生存的基本条件,缺氧会引发一系列级联反应,导致自由基生成,线粒体功能破坏,严重时甚至导致神经细胞死亡。
儿童的生长发育不正常,成年人出现脑疲劳,老年人记忆力下降,均与人的大脑缺氧有一定的关系。而具有提高缺氧耐受力的营养物质可以在某种程度上消除大脑中的自由基和补充供氧量,调节脑神经细胞和脑部其他组织细胞的新陈代谢,促进大脑神经传导系统的正常运转,或提高肌体对氧的利用率,减少在缺氧情况下对人体功能的损伤,进而可加速对缺氧环境的适应过程。
目前,耐缺氧保健食品在保健食品市场中占据的份额越来越大,据中国保健协会保健品市场工作委员会的数据,截至目前,共批准耐缺氧功能保健食品二百余种,其主要成分多为鲨烯、红景天、西洋参等。在我国传统的养生保健文化中,耐缺氧保健食品有着独特的地位,可以开发利用的资源极为丰富,因此,研发耐缺氧保健食品具有广阔的市场发展前景。
发明内容
本发明的目的在于针对现有技术的不足,提供一种从油橄榄果中制备的具有耐缺氧活性的组分及其应用,所提供的组分具有显著的耐缺氧保健功能,从而弥补现有技术的不足。
本发明首先提供一种从油橄榄果中制备的具有耐缺氧活性的提取物,其制备方法如下:
将油橄榄果的水提取液进行浓缩后,在浓缩液中加入乙醇;静置过滤后得到上清液;上清液浓缩后,进行干燥获得粉末状提取物;
所述的进行浓缩,是使水提取液的相对密度为1.10-1.20;
所述的乙醇加入到浓缩液中的体积百分比终浓度为65-85%;
所述的获得上清液,是使用离心或过滤方式获得上清液;
更进一步的,所述的上清液进行浓缩前,先进行脱色处理;
所述的脱色处理,其中一个方式是将上清液通过色谱柱,色谱柱选用氧化铝色谱柱或聚酰胺色谱柱或凝胶色谱柱或大孔树脂色谱柱,或将上清液依次通过氧化铝色谱柱、聚酰胺色谱柱、凝胶色谱柱和大孔树脂色谱柱中的任意两种色谱柱。
本发明所提供的提取物可用于制备用于预防治疗摄氧不足的制品;
本发明另一个方面还提供过一种用于预防治疗摄氧不足的制品,所述的制品中包含有药理有效浓度的上述的油橄榄果提取物;
所述的制品,为食品或药品;
更具体的,所述的制品为胶囊。
本发明所制备的橄榄果提取物具有耐缺氧活性的有效成分,可将所提供的提取物制成可随身携带的胶囊功能食品、保健食品,从而增加了油橄榄果加工的经济效益。
附图说明
图1:D-1和D-2对PC12细胞毒性筛选检测数据图;
图2:D-1和D-2对PC12的低氧保护作用数据图;
图3:NO及Ca2+含量测定结果图。
具体实施方式
本发明提供一种从油橄榄果中制备的具有耐缺氧活性的组分及其应用,所提供的组分具有显著的耐缺氧保健功能。
本发明提供的从油橄榄果中制备的具有耐缺氧活性的提取物的方法,其一种具体的步骤如下:
将油橄榄果的水提取液进行浓缩后,在浓缩液中加入乙醇;静置过滤后得到上清液;上清液浓缩后,进行干燥获得粉末状提取物;
其中油橄榄果的水提取煎液,其一个实施例中的具体记载,是取油橄榄果,加水煎煮两次,合并水提液;第一次加水为总重量的8-12倍量,煎煮1-2h,第二次加水为总重量的6-10倍量,煎煮1-2h。
所述的进行浓缩,可以采用旋蒸方式,浓缩至65℃时相对密度为1.10-1.20;所述的液体相对密度,是指其密度与标准大气压下4℃纯水的密度的比值。
所述的乙醇加入到浓缩液中的体积百分比浓度为65-85%;
所述的步骤2)获得上清液,可以使用离心或过滤方式获得;
更进一步的,所述的上清液进行浓缩前,先进行脱色处理;
所述的脱色处理,其中一个方式是将上清液通过色谱柱,色谱柱选用氧化铝色谱柱或聚酰胺色谱柱或凝胶色谱柱或大孔树脂色谱柱,或将上清液依次通过氧化铝色谱柱、聚酰胺色谱柱、凝胶色谱柱和大孔树脂色谱柱中的任意两种色谱柱。
步骤2)中上清液浓缩后,进行干燥后获得粉末状提取物,其中一种干燥方式为喷雾干燥,条件为:进风温度为80-120℃,出风温度为80-90℃,物料温度为70-90℃,雾化压力为0.2-0.4兆帕,喷雾速度为5-10mL/s。
本发明所提供的提取物可用于制备用于预防摄氧不足活性成分的制品,其中一种产品是将提取物与辅料联合使用来制备胶囊。
所述的辅料可以是食品或药品领域中常用的辅料,例如淀粉、乳糖或糊精。
除非另有说明,本发明所采用的百分数均为重量百分数。
下面结合实施例和附图对本发明进行详细的描述。
实施例1:制备油橄榄果提取物
取油橄榄果1000g,加水煎煮两次,第一次加水为总重量的8倍量,煎煮1h,第二次加水为药材重量的6倍量,煎煮1h,合并煎液。
将煎液在0.07MPa、60-70℃下减压浓缩至65℃时相对密度为1.10,然后加入95%(v/v)乙醇使含醇量达65%(v/v),搅拌,静置24小时,过滤,得水提液。
将水提液滤过使澄清,通过氧化铝色谱柱(300g,柱子直径为4cm,柱高为100cm),收集流出上清液,上清液在80℃真空度为0.01MPa的条件下浓缩,再经喷雾干燥,条件为进风温度为100℃,出风温度为80℃,物料温度为80℃,雾化压力为0.2兆帕,喷雾速度为5mL/s;即得固体粉末47g油橄榄提取物D-1。
实施例2:制备油橄榄果提取物
取油橄榄果1000g,加水煎煮两次,第一次加水为总重量的10倍量,煎煮1.5h,第二次加水为总重量的8倍量,煎煮1.5h,合并水提液。将水提液在0.07MPa、60-70℃下减压浓缩至65℃时相对密度为1.15,然后加入95%(v/v)乙醇使含醇量达75%(v/v),搅拌,静置24小时,过滤,得上清液。
上清液离心使澄清,通过聚酰胺色谱柱(1000mL,柱子直径为5cm,柱高为120cm),收集流出液体,液体在80℃真空度为0.01MPa的条件下浓缩,再经喷雾干燥;条件为进风温度为120℃,出风温度为100℃,物料温度为90℃,雾化压力为0.4兆帕,喷雾速度为10mL/s;即得固体粉末52g油橄榄提取物D-2。
实施例3:耐缺氧活性实验
下面通过细胞活性试验对上述实施例1和实施例2所制备油橄榄提取物D-1、D-2的耐缺氧活性进行检测。
实验中使用的PC12细胞购自中国科学院上海细胞库;DMEM高糖培养基、马血清、青-链霉素、CoCl2和CCK-8均为Bioss公司产品;复方丹参滴丸(27mg*180s)购自天士力制药集团股份有限公司;NO和Ca2+检测试剂盒购自北京索莱宝科技有限公司。
所使用的仪器包含有超净工作台(SW-CJ-2FD)、细胞低氧研究装置(mic-101,Billups-Rothenberg);多功能酶标仪(Spectra Max i3,Molecular Devices)。
1、细胞毒性测定
PC12细胞培养于含有5%FBS、10%灭活马血清、100U/mL青霉素和100μg/mL链霉素的DMEM培养基中。将处于对数生长期的PC12细胞以1×105/mL接种于96孔板中,待细胞贴壁后,加入以完全培养基配制好的1、10、50、100、200和500μg/mL的样品溶液,将其置于5%CO2培养箱处理24h,处理结束后向96孔板中加入CCK-8试剂,10μL/孔,于37℃孵育1h后,测定450nm波长处的吸光值,计算细胞活力。
2、油橄榄提取物对低氧状态PC12细胞的保护作用
将处于对数生长期的PC12细胞以1×105/mL接种于96孔板中,待细胞贴壁后,加入以完全培养基配制好的1、10和100μg/mL的油橄榄提取物(浓度较高时,样品稀释比较低,培养基相对含量也降低,在后续不同处理可能影响细胞营养物质摄取,因此选择了1、10和100μg/mL浓度),将其置于5%CO2培养箱中预处理6h。预处理结束后,向96孔板中加入终浓度为600μM的CoCl2溶液进行造模处理。24h后,加入10μL/孔的CCK-8试剂,于37℃孵育1h后,测定450nm波长处的吸光值,计算细胞活力。
3、NO及Ca2+含量测定
将处于对数生长期的PC12接种于6孔板中,细胞密度为5×106/mL,待细胞贴壁后,加入终浓度为100μg/mL的样品溶液,预处理6h,向96孔板中加入终浓度为600μM的CoCl2溶液进行造模处理。处理结束后,吸取上清液,以NO检测试剂盒测定不同处理PC12细胞的NO释放量。
将接种于6孔板中处理后的PC12细胞,以PBS清洗一遍后,加入细胞裂解液,待细胞完全裂解后,转移裂解液至离心管中,以12000r/min离心5min,收集上清液,以Ca2+检测试剂盒测定Ca2+含量。
所有数据用均数±标准差(x±s)表示,数量资料采用方差分析,组间差异采用Student T检验。
4、实验结果
4.1油橄榄提取物的细胞毒性检测
油橄榄提取物处理PC12细胞24h,细胞活力检测结果发现,油橄榄提取物D-1和D-2与control组相比,没有显著性差异,表明两种油橄榄提取物均不具有细胞毒性(图1)。
4.2油橄榄提取物对低氧状态PC12细胞的保护作用
以复方丹参滴丸作为阳性对照,细胞活力测定结果发现,100μg/mL的复方丹参滴丸对CoCl2诱导的缺氧模型具有较好的保护作用,呈现极显著差异。油橄榄提取物D-1处理浓度达到100μg/mL时,对低氧状态PC12细胞具有显著的保护作用;油橄榄提取物D-2在处理浓度为50μg/mL时,即表现出对PC12细胞的低氧保护作用,浓度达到100μg/mL时,具有极显著差异(图2)。
4.3NO及Ca2+含量的检测
PC12细胞的神经元延伸与NO的产生有关,实验结果发现,经CoCl2诱导的PC12细胞NO含量显著降低,加入复方丹参滴丸及油橄榄提取物后,均可显著提高NO的含量。
Ca2+含量测定结果发现,低氧处理组的Ca2+含量较正常对照组显著升高,表明低氧导致细胞发生钙超载,经复方丹参滴丸和油橄榄提取物处理后,Ca2+含量较低氧处理组显著降低。实验结果表明,油橄榄提取物可以通过减轻低氧导致的钙超载来保护低氧对PC12细胞的损伤,发挥耐缺氧活性。
研究结果发现,油橄榄提取物D-1和D-2均可以保护低氧对PC12细胞的损伤作用,且当处理浓度为100μg/mL时,与CoCl2处理组相比具有极显著差异。
结果表明油橄榄提取物D-1和D-2在500μg/mL的浓度范围内,不具有细胞毒性;当处理浓度达到100μg/mL时,与CoCl2处理组相比,具有显著性差异,表明有很好的耐缺氧作用;具有开发为耐缺氧功能食品的潜力。
Claims (10)
1.一种从油橄榄果中制备的具有耐缺氧活性的提取物,其特征在于,所述的提取物的制备方法如下:
将油橄榄果的水提取液进行浓缩后,在浓缩液中加入乙醇;静置过滤后得上清液;上清液浓缩后,进行干燥后获得粉末状提取物。
2.如权利要求1所述的提取物,其特征在于,所述的将油橄榄果的水提液进行浓缩后,是使水提取煎液的相对密度为1.10-1.20。
3.如权利要求1所述的提取物,其特征在于,所述的乙醇加入到浓缩液中的体积百分比终浓度为65-85%。
4.如权利要求1所述的提取物,其特征在于,所述的获得上清液,是使用离心或过滤方式获得上清液。
5.如权利要求1所述的提取物,其特征在于,所述的上清液,其进行浓缩前,先进行脱色处理。
6.如权利要求5所述的提取物,其特征在于,所述的脱色处理,是上清液通过色谱柱。
7.权利要求1所述的提取物在制备用于预防治疗摄氧不足的制品中的应用。
8.一种用于预防治疗摄氧不足的制品,其特征在于,所述的制品中包含有药理有效浓度的权利要求1所述的提取物。
9.如权利要求8所述的制品,其特征在于,所述的制品为食品或药品。
10.如权利要求9所述的制品,其特征在于,所述的制品为胶囊。
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