CN116640618A - 一种具有提升免疫力及健脑功效的牡丹肽、牡丹籽油肽及其制备方法、应用 - Google Patents
一种具有提升免疫力及健脑功效的牡丹肽、牡丹籽油肽及其制备方法、应用 Download PDFInfo
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- CN116640618A CN116640618A CN202310529500.3A CN202310529500A CN116640618A CN 116640618 A CN116640618 A CN 116640618A CN 202310529500 A CN202310529500 A CN 202310529500A CN 116640618 A CN116640618 A CN 116640618A
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Abstract
本发明公开了一种具有提升免疫力及健脑功效的牡丹肽、牡丹籽油肽及其制备方法、应用。该牡丹籽油提取自籽牡丹籽仁和牡丹籽壳,含各种成分不饱和脂肪酸,成分丰富。经动物实验证实,该牡丹籽油具有明显的增强免疫力和健脑功效。该牡丹籽油与胶原三肽一起复配制备得到一种牡丹籽油肽微胶囊,该牡丹籽油肽微胶囊能够有效释放游离脂肪酸和胶原三肽,易于在体内吸收,生物吸收效果佳。
Description
技术领域
本发明涉及牡丹籽油技术领域,具体涉及一种具有提升免疫力及健脑功效的牡丹肽、牡丹籽油肽及其制备方法、应用。
背景技术
牡丹(PaeoniasuffrutzcosaAndr.)是我国专有的宝贵花草。根据用途可将牡丹分为观赏牡丹和油用牡丹两类。油用牡丹产的牡丹籽油被誉为“液体黄金”,牡丹籽油不仅成分结构合理,而且还含有丰富的营养。牡丹籽油含有的a-亚麻酸是我们人体所必需的但需要通过摄取外来食物来获得的重要营养物质。运用气相色谱-质谱联用(GC-MS)分析方法的研究结果显示,牡丹籽油富含不饱和脂肪酸以及一部分不皂化物(甾醇类、脂溶性维生素E和角鳖烯等)。
翟文婷等研究发现牡丹籽油可以保护四氯化碳诱导的急性肝损伤的模型组小鼠。朱宗磊分别将低、中、高浓度的牡丹籽油喂给高血糖小鼠,发现小鼠低密度脂蛋白胆固醇(LDL-C)指标水平明显降低,HDL-C水平明显升高。给糖尿病小鼠喂一定浓度的牡丹籽油,经测定结果显示能够显著地降低其血糖水平,牡丹籽油对正常小鼠的葡萄糖耐量也有一定的改善。张萍将牡丹籽油和乙醇混合,当牡丹籽油所占比例低于一半时,牡丹籽油含量越高,清除DPPH自由基越多。由此可见,牡丹籽油具有十分广泛的应用价值,继续开发其更多的生物功能仍然具有十分重要的意义。
发明内容
有鉴于于此,本发明提供了一种具有提升免疫力及健脑功效的牡丹肽、牡丹籽油肽及其制备方法、应用。
本发明目的之一提供一种牡丹籽油的提取方法,包括:
将经酸热处理的牡丹籽仁和牡丹籽壳研磨成粉,混匀于去离子水中,加入胃蛋白酶和风味蛋白酶进行酶解;
将经灭酶处理的酶解液经离心,收集清油及乳状液;
将乳状液用木瓜蛋白酶破乳,得到的油脂与清油合并,得到粗牡丹籽油;
将所述粗牡丹籽油经超声和萃取,得到所述牡丹籽油。
进一步的,所述胃蛋白酶的用量为所述牡丹籽仁和牡丹籽壳粉末重量的0.5%,所述风味蛋白酶的用量为所述牡丹籽仁和牡丹籽壳粉末重量的0.25%。
进一步的,所述木瓜蛋白酶用量为所述乳状液的占比为0.15mg/mL。
进一步的,“萃取”采用了体积比为3:7的石油醚和乙酸乙酯的混合溶液,从中间乙酸乙酯相中收集所述牡丹籽油。
本发明目的之一提所述的提取方法得到的牡丹籽油,其中,包含:
3.055%的肉豆蔻酸;
4.261%的7-十六碳烯酸;
3.141%的9-十六碳烯酸;
11.716%的棕榈酸;
0.109%的2-己基-环丙烷辛酸;
0.052%的十七酸;
26.121%的亚油酸;
32.931%的亚麻酸;
6.177%的硬脂酸;
2.875%的8,11,14-二十碳三烯酸;
1.622%的11-二十碳烯酸;
3.057%的花生酸;
0.049%的山嵛酸;
0.05%的二十四碳酸;
0.0352%的豆蔻酸甲酯;
0.0269%的十五烷酸甲酯;
0.254%的棕榈油酸甲酯;
0.103%的棕榈酸甲酯;
0.727%的棕榈酸乙酯;
2.432%的2-已基环丙烷辛酸甲酯;
0.02%的十七烷酸甲酯;
0.061%的7,10-十八碳二烯酸甲酯;
0.099%的亚油酸甲酯;
0.053%的油酸甲酯;
0.211%的亚麻酸甲酯;
0.135%的硬脂酸甲酯;
0.085%的亚油酸乙酯;
0.152%的油酸乙酯;
0.127%的亚麻酸乙酯;
0.141%的2-辛基环丙烷辛酸甲酯;
0.063%的二十碳烯酸甲酯。
本发明目的之一提供一种牡丹籽油肽微胶囊的制备方法,包括:
采用所述的提取方法制得的牡丹籽油;
将乳清分离蛋白溶解于蒸馏水中,与玉米糖浆混合均匀后加入所述牡丹籽油和胶原三肽以及磷脂,采用高速剪切分散乳化机对混合液进行乳化,形成预乳状液,再经过高压均质(压强30MPa)均质2次后,对乳状液进行喷雾干燥,进料速率25rpm,进风温度180℃,出风温度90~95℃,得到牡丹籽油肽微胶囊。
进一步的,乳清分离蛋白、玉米糖浆、牡丹籽油、胶原三肽以及磷脂的质量配比为15:(45~50):(32~35):(15~18):2。
本发明目的之一提供所述的提取方法制得的牡丹籽油、或所述的牡丹籽油或所述的制备方法制得的牡丹籽油肽微胶囊在制备增强免疫力制品中的应用。
本发明目的之一提供所述的提取方法制得的牡丹籽油、或所述的牡丹籽油或所述的制备方法制得的牡丹籽油肽微胶囊在制备健脑制品中的应用。
与现有技术相比,本发明至少具有以下有益效果之一:
本发明利用牡丹籽仁和牡丹籽壳作为原材料,对其进行提取得到了一种特殊的牡丹籽油,该牡丹籽油含各种成分不饱和脂肪酸,成分丰富。经动物实验证实,这些牡丹籽油具有明显的增强免疫力和健脑功效。
本发明进一步将这些牡丹籽油与胶原三肽一起复配,制备得到一种牡丹籽油肽微胶囊,该牡丹籽油肽微胶囊能够有效释放游离脂肪酸和胶原三肽,易于在体内吸收,生物吸收效果佳。并且经动物实验证实,这些牡丹籽油肽微胶囊能提高小鼠碳廓清能力、能对ConA诱导的小鼠淋巴细胞转化能力、小鼠巨噬细胞吞噬鸡红细胞的能力、NK细胞活性、抗体生成细胞数产生显著影响;并且能够上调AD大鼠海马取SorLA、SNX27mRNA表达量,通过对APP贩运途径的影响减少Aβ的生成和沉积,从而抑制神经元细胞凋亡,提供大鼠空间探索能力,从而大大降低患AD的风险;具有明显的增加免疫力和健脑功能。
附图说明
图1为实施例2提供的牡丹籽油肽微胶囊模拟胃肠液体外释放曲线。
图2为实施例3提供的牡丹籽油肽微胶囊模拟胃肠液体外释放曲线。
图3为对比例2提供的牡丹籽油肽微胶囊模拟胃肠液体外释放曲线。
图4为对比例3提供的牡丹籽油微胶囊模拟胃肠液体外释放曲线。
图5为增强免疫力实验中各组小鼠的足跖肿胀度结果。
图6为增强免疫力实验中各组小鼠的HC50结果。
图7为增强免疫力实验中各组小鼠的吞噬指数结果。
图8为增强免疫力实验中各组小鼠的淋巴增殖(OD差值)结果。
图9为增强免疫力实验中各组小鼠的溶血空斑(×103/全脾)结果。
图10为增强免疫力实验中各组小鼠的吞噬率结果。
图11为增强免疫力实验中各组小鼠的NK细胞活性结果。
图12为健脑功能研究实验中各组大鼠的逃避潜伏期结果。
图13为健脑功能研究实验中各组大鼠的目标象限游泳时间百分比结果。
图14为健脑功能研究实验中各组大鼠的海马Aβ1-42含量结果。
图15为健脑功能研究实验中各组大鼠海马SorLA、SNX27mRNA表达量结果。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。本发明中未详细单独说明的试剂均为常规试剂,均可从商业途径获得;未详细特别说明的方法均为常规实验方法,可从现有技术中获知。
1、实验材料及预处理
来自于市售的凤丹牡丹种子,脱壳机脱壳,收集牡丹籽仁和牡丹籽壳,加入2倍重量的0.1M的柠檬酸溶液浸泡12h,于65℃温度下烘干至水分于3.5%~4.0%之间。
2、提取牡丹籽油
实施例1:
将经酸热处理的牡丹籽仁和牡丹籽壳研磨成粉,以1:10的料液比加入至去离子水中混合,加入牡丹籽粉重量的0.5%的胃蛋白酶(CAS:9001-75-6,北京索莱宝)和0.25%的风味蛋白酶(CAS:9001-92-7,北京索莱宝),在pH4.5、60℃条件下酶解4h,100℃水浴处理10min后,5000rpm离心15min,收集清油及乳状液,同时分离渣相,将其与离心后得到的水相混合,于50℃、pH9条件下搅拌1h使其充分混合,5000rpm二次离心15min,合并两次离心得到的清油和乳状液,将乳状液用木瓜蛋白酶破乳,得到的油脂与清油合并,得到粗牡丹籽油。其中,木瓜蛋白酶用量为所述乳状液的占比为0.15mg/mL。
向粗牡丹籽油中加入1倍体积的去离子水,超声处理30min后(超声功率400W,超声温度61℃),取上层油相;再加入2倍体积的石油醚和乙酸乙酯的混合溶液(体积比为3:7),超声处理30min后(超声功率400W,超声温度20℃),取中间乙酸乙酯相,即得所述牡丹籽油。
对比例1:
将经酸热处理的牡丹籽仁和牡丹籽壳研磨成粉,加入80%的乙醇溶液中充分热回流处理8h,过滤得到乙醇浸膏。在乙醇浸膏中加入1倍体积的去离子水,超声处理30min后(超声功率400W,超声温度61℃),取上层油相;再加入2倍体积的石油醚和乙酸乙酯的混合溶液(体积比为3:7),超声处理30min后(超声功率400W,超声温度20℃),取中间乙酸乙酯相,即得所述牡丹籽油。
3、脂肪酸含量检测
气相色谱检测条件:色谱柱:HP-5MS(30m×0.25mm×0.25μm)弹性石英毛细管柱;柱流量:0.8m1/min;柱温:初温80℃以8℃/min速率升温到250℃;载气:高纯氦气;进样口温度:250℃;进样方式:分流20:1;GC/MS接口温度:250℃,EI源:(70eV),离子源:230℃,四极杆温度:150℃,EM电压:1294V,扫描范围:27~460amu。
液相色谱检测条件:AgilentPoroshell120EC-C18(3.0×50nm,2.7μm)色谱柱,DAD检测器;检测波长214nm;柱温为30℃;流速为0.6mL/min;进样体积10μL;流动相:乙腈(A)-0.1%磷酸(B);梯度洗脱顺序为0~10min,A8%~16%,B92%~84%;10~22min,A16%~80%,B84%~20%。
表1质量百分比
成分 | 实施例1 | 对比例1 |
肉豆蔻酸 | 3.055 | 0.081 |
7-十六碳烯酸 | 4.261 | 0.061 |
9-十六碳烯酸 | 3.141 | 2.675 |
棕榈酸 | 11.716 | 7.435 |
2-己基-环丙烷辛酸 | 0.109 | 0.203 |
十七酸 | 0.052 | 0.142 |
亚油酸 | 26.121 | 5.422 |
亚麻酸 | 32.931 | 6.537 |
硬脂酸 | 6.177 | 0.205 |
8,11,14-二十碳三烯酸 | 2.875 | 2.364 |
11-二十碳烯酸 | 1.622 | - |
花生酸 | 3.057 | 0.291 |
山嵛酸 | 0.049 | - |
二十四碳酸 | 0.05 | - |
豆蔻酸甲酯 | 0.035 | - |
十五烷酸甲酯 | 0.027 | - |
棕榈油酸甲酯 | 0.254 | 0.226 |
棕榈酸甲酯 | 0.103 | 0.068 |
棕榈酸乙酯 | 0.727 | 0.653 |
2-已基环丙烷辛酸甲酯 | 2.432 | 1.435 |
十七烷酸甲酯 | 0.020 | - |
7,10-十八碳二烯酸甲酯 | 0.061 | 0.363 |
亚油酸甲酯 | 0.099 | 0.499 |
油酸甲酯 | 0.053 | 0.043 |
亚麻酸甲酯 | 0.211 | 18.247 |
硬脂酸甲酯 | 0.135 | 22.86 |
亚油酸乙酯 | 0.085 | 17.72 |
油酸乙酯 | 0.152 | 8.212 |
亚麻酸乙酯 | 0.127 | 1.277 |
2-辛基环丙烷辛酸甲酯 | 0.141 | 1.068 |
二十碳烯酸甲酯 | 0.063 | 0.078 |
花生酸甲酯 | - | 0.083 |
山嵛酸甲酯 | - | 0.525 |
二十三烷酸甲酯 | - | 0.033 |
木焦油酸甲酯 | - | 0.062 |
表1示出了实施例1和对比例1分别制得的牡丹籽油中各脂肪酸类物质的含量,其中,“-”表示未检出;其中,花生酸和山嵛酸的含量检测采用上述液相色谱条件,其他成分含量检测采用上述气象色谱条件(均采用标准品及外标法检测)。由表1可知,实施例1制得的牡丹籽油的成分主要集中为脂肪酸,而对比例1制得的牡丹籽油的成分主要集中为脂肪酸酯。
4、牡丹籽油肽微胶囊的制备
实施例2:
将15g乳清分离蛋白(河南郑州明瑞化工产品有限公司)溶解于60℃蒸馏水中,与48g玉米糖浆混合均匀后加入35g实施例1提供的牡丹籽油、15g胶原三肽(氨基酸序列CTP,西安天广源生物科技有限公司)以及2.0g乳化剂磷脂,采用高速剪切分散乳化机对混合液进行乳化,形成预乳状液,再经过高压均质(压强30MPa)均质2次后,对乳状液进行喷雾干燥,进料速率25rpm,进风温度180℃,出风温度90~95℃,得到牡丹籽油肽微胶囊。
实施例3:
将15g乳清分离蛋白溶解于60℃蒸馏水中,与48g玉米糖浆混合均匀后加入32g实施例1提供的牡丹籽油、18g胶原三肽(氨基酸序列CTP,西安天广源生物科技有限公司)以及2.0g乳化剂磷脂,采用高速剪切分散乳化机对混合液进行乳化,形成预乳状液,再经过高压均质(压强30MPa)均质2次后,对乳状液进行喷雾干燥,进料速率25rpm,进风温度180℃,出风温度90~95℃,得到牡丹籽油肽微胶囊。
对比例2:
将15g乳清分离蛋白溶解于60℃蒸馏水中,与48g玉米糖浆混合均匀后加入35g对比例1提供的牡丹籽油、15g胶原三肽(氨基酸序列CTP,西安天广源生物科技有限公司)以及2.0g乳化剂磷脂,采用高速剪切分散乳化机对混合液进行乳化,形成预乳状液,再经过高压均质(压强30MPa)均质2次后,对乳状液进行喷雾干燥,进料速率25rpm,进风温度180℃,出风温度90~95℃,得到牡丹籽油微胶囊。
对比例3:
将15g乳清分离蛋白溶解于60℃蒸馏水中,与48g玉米糖浆混合均匀后加入35g实施例1提供的牡丹籽油以及2.0g乳化剂磷脂,采用高速剪切分散乳化机对混合液进行乳化,形成预乳状液,再经过高压均质(压强30MPa)均质2次后,对乳状液进行喷雾干燥,进料速率25rpm,进风温度180℃,出风温度90~95℃,得到牡丹籽油微胶囊。
5、牡丹籽油肽微胶囊的体外释放实验
整个模拟消化过程所需要的温度为37±0.5℃。胃液消化:取3g实施例2~3和对比例2~3分别提供的微胶囊,加入25mL模拟胃液(迈基生物),用1mol/L的NaOH溶液将反应液的pH调成2.5,在摇床中以130r/min的速率消化2h。肠液消化:将上述步骤中所有的胃液内容物,加入1mol/L的NaOH溶液,将pH调至7.0,使在模拟胃液的反应停止,向反应液中加入2.5mL的模拟肠液(迈基生物),将pH调节至7.0,模拟肠道反应3h。
在消化0.5h、1.0h、1.5h、2.0h、2.5h、3.0h、3.5h、4.0h、4.5h和5.0h分别取消化液检测其中游离脂肪酸释放量以及胶原蛋白肽的释放量。用0.2mol/LNaOH滴定,以酚酞为指示剂,游离脂肪酸释放量=V×C×M/m,式中,V为氢氧化钠体积,C为氢氧化钠浓度,M为牡丹籽油中脂肪酸平均分子量,m为样品中油重量。
胶原三肽含量检测:色谱柱为ThermoHypersilGOLDTMC18(2.1mm×100mm,1.9μm);流动相组成:A为0.1%甲酸水溶液,B为0.1%甲酸乙腈;梯度洗脱条件为:0~1.0min,5%流动相B;1.0~2.0min,5%~15%流动相B;2.0~12.0min,15%~40%流动相B;12.0~14.0min,40%~95%流动相B;14.0~16.5min,95%流动相B;16.5~17.5min,95%~5%流动相B;17.5~20.0min,5%流动相B。流速为0.30mL/min;进样量为5.0μL;柱温为40℃。
如图1~4分别示出了实施例2、3以及对比例2和3的体外释放曲线图。由图可知,在2h内,游离脂肪酸和胶原三肽释放量较少,而在2~5h内,游离脂肪酸和胶原三肽的释放量急剧增加,并且实施例2和3分别提供的牡丹籽油肽微胶囊的释放量均显著高于对比例2,说明实施例2和3提供的牡丹籽油肽微胶囊易于在体内吸收,生物吸收效果更佳。
6、牡丹籽油及牡丹籽油肽微胶囊的增强免疫力功能的研究
(1)受试物
上述实施例1和对比例1分别提供的牡丹籽油。实施例2~3和对比例2分别提供的牡丹籽油肽微胶囊。对比例3提供的牡丹籽油微胶囊。
(2)实验动物
SPF级ICR雄性小鼠,济南朋悦实验动物繁育有限公司提供。
(3)实验分组和剂量选择
将实验动物分为7个组,阴性组、实施例1~3和对比例1~3,每组12只。以植物油为稀释溶剂和阴性对照,采取灌胃方式给予受试物,灌胃量为0.2ml/10gBW,连续灌胃30d。
(4)实验方法
参照《保健食品检验与评价技术规范》(2003版)中增强免疫力功能检验方法。
(5)迟发型变态反应
10%绵羊红细胞(货号RCB001,北京博尔西科技有限公司)进行腹腔免疫,0.25ml/只。免疫后4d,测量足跖部厚度,测量部位皮下注射20%SRBC,25μl/只,24h后测量左后足跖部厚度3次,取平均值。
(6)ConA诱导的小鼠淋巴细胞转化实验
制单细胞悬液,细胞浓度至4×106个/ml。加入24孔培养板,每孔1.25ml,每孔加70μlConA液(刀豆蛋白A,用双蒸水配制成100μg/mL,过滤除菌,-20℃保存),培养72h。68h后,MTT法测定OD570nm。
(7)抗体生成细胞检测
腹腔注射10%绵羊红细胞,0.25ml/只。免疫4d后取脾,制成细胞悬液,将脾细胞悬液加入RPMI1640培养液中,浓度调至4×106个/ml。制备琼脂糖玻片,温育1.5h,加入补体,温育1.5h,计数溶血空斑数。
(8)半数溶血值(HC50)的测定
腹腔注射10%绵羊红细胞,0.25ml/只。免疫5d后,离心取血清,稀释,后依次加入10%绵羊红细胞,补体。温育20min,冰浴终止反应。离心取上清加都氏试剂,混匀,静置10min,测定OD540nm,溶血素量以HC50表示。
(9)小鼠碳廓清实验
小鼠尾静脉注射印度墨汁,注入墨汁后2、10min,分别取血25μl加到1ml0.2%Na2CO3溶液中,测定OD600nm,计算吞噬指数。
(10)小鼠腹腔巨噬细胞吞噬鸡红细胞实验
小鼠腹腔注射15%鸡红细胞悬液1.5ml,30min后脱臼处死,取腹腔巨噬细胞洗液2ml,滴于载玻片上,温育30min,甲醇固定,5%Giemsa-磷酸缓冲液染色,漂洗晾干。镜下计数,计算吞噬率和吞噬指数。
(11)NK细胞活性测定(乳酸脱氢酶LDH测定法)
YAC-1细胞(作为NK靶细胞,BJ-010245,上海邦景实业有限公司)调整浓度至5×105个/ml,RPMI1640完全培养液重悬小鼠脾脏制成的悬液至2×107个/ml。取YAC-1细胞和小鼠脾脏细胞各100μl,靶细胞自然释放孔加靶细胞和培养液各100μl,靶细胞最大释放孔加靶细胞和1%NP40各100μl,温育4h,每孔吸取上清100μl测定OD490nm。
(12)结果
如图5~7所示,经口给予受试物30d,迟发型变态反应和HC50测定中,与阴性对照组比较,对比例1提供的牡丹籽油作为受试物后,小鼠的足跖肿胀度差异无统计学意义(P>0.05),而实施例1提供的牡丹籽油、实施例2和3分别提供的牡丹籽肽微胶囊具有统计学意义(P<0.05)。在小鼠碳廓清功能实验中,与阴性对照组比较,对比例1提供的牡丹籽油作为受试物后无统计学差异(P>0.05),而实施例1提供的牡丹籽油、实施例2和3分别提供的牡丹籽肽微胶囊差异具有统计学意义(P<0.05),即受试物均能提高小鼠碳廓清能力。
ConA诱导淋巴细胞转化实验、抗体生成数、巨噬细胞吞噬能力、NK细胞活性试验结果如图8~11所示。经口给予小鼠对比例1~3分别提供的受试物30d,各组淋巴细胞的增殖能力下降,而吞噬率、NK细胞活性、抗体生成数与阴性对照组比较差异无统计学意义(均P>0.05)。而给予小鼠实施例1~3分别提供的受试物30d后,各组小鼠淋巴细胞增殖能力增强,溶血空斑、吞噬率和NK细胞活性均呈现不同程度的增强。由此说明,实施例1~3提供的受试物能对ConA诱导的小鼠淋巴细胞转化能力、小鼠巨噬细胞吞噬鸡红细胞的能力、NK细胞活性、抗体生成细胞数产生显著影响,从而说明本实施例提供的牡丹籽油以及牡丹籽油肽微胶囊具有明显的免疫力增强功能。
7、牡丹籽油及牡丹籽油肽微胶囊的健脑功能的研究
(1)受试物
上述实施例1和对比例1分别提供的牡丹籽油。实施例2~3和对比例2分别提供的牡丹籽油肽微胶囊。对比例3提供的牡丹籽油微胶囊。将这些牡丹籽油或者牡丹籽油肽微胶囊分别用植物油配制成浓度为5μg/μL的溶液。
盐酸多奈哌齐片,规格:5mg/片,由卫材(中国)药业有限公司提供,批号:H20050978。药片碾成粉末状后溶于灭菌超纯水中,磁力搅拌器搅拌,配制成浓度0.045mg/mL的混悬液。封口保存于4℃冰箱,备用。
1mgAβ1-42(淀粉样蛋白,美国Sigma公司)溶于200μL0.9%氯化钠溶液中后混匀,配制成浓度为5μg/μL的溶液,3000r/min离心1min,封口膜封好,锡纸包裹避光放置于数显恒温水浴锅中,37℃孵育3d。青霉素(规格:0.96g,批号:170401,华北制药),水合氯醛(规格:AR,批号:20161217,上海沪试实验室器材有限公司。
(2)实验动物
SPF级Wistar雄性大鼠,济南朋悦实验动物繁育有限公司提供。
(3)分组及造模
通过随机数表法将大鼠分为:正常组、空白组、模型组、实施例1、实施例2、实施例3、对比例1、对比例2、对比例3组和阳性组。
造模:大鼠适应性喂养1周后,除正常组外,其余各组行脑立体定位手术:大鼠称重后,使用6%水合氯醛,按100g/0.6mL剂量进行腹腔注射麻醉。待大鼠麻醉后(全身肌肉松弛),头部备皮,将其固定于脑立体定位仪上,常规消毒手术区皮肤,无菌条件下沿矢状线做约1cm切口,棉签沾取少量3%过氧化氢,擦拭创口直至骨膜分离,充分暴露出前囟,“人”字缝、“十”字缝。参照脑立体定位仪,以前囟“十”字缝中心为零点,沿正中线向后4mm,沿矢状线左右各3mm,标记位置,使用牙科钻钻孔,微量进样器吸取2μLAβ1-42溶液,沿钻孔处进针至硬膜下4mm深度,5min注射完毕,留针5min后轻缓出针,对侧行同样操作。大鼠注射结束后,缝合头皮,创口处用碘伏消毒,棉签涂抹马应龙痔疮膏,每只肌注10万单位青霉素用以预防感染。将大鼠撤下手术台,以取暖器保温,待苏醒后放回笼中,标注造模日期,即为模型组。
空白组相同位置注射0.9%氯化钠溶液,余相同。
给药:术后3天,取上述模型组中部分大鼠,分别给予实施例1、实施例2、实施例3、对比例1、对比例2、对比例3组和阳性组(盐酸多奈哌齐溶液)分别提供的药物溶液10mL·kg-1·d-1给予灌胃,连续灌胃4周。
(4)行为学检测
给药结束后对各组大鼠进行行为学检测。采用Morris水迷宫实验评价固本健脑液对模型动物空间学习记忆的影响。将水迷宫水池分为4个象限,平台放置于第3象限。往水迷宫中注水直至水面高出平台约2cm后,池中加入适量墨汁。提捏大鼠尾部将其面向池壁分别从4个象限的固定位置进入水中。记录其90s内寻找到平台所花费的时长(又称逃避潜伏期)和目标象限游泳时间,计算目标象限游泳时间百分比。该实验共计5d。
(5)取材
行为学检测结束后,所有大鼠腹腔注射6%水合氯醛麻醉(0.6mL/100g)后断头,于冰盘上快速开颅取出海马,放置于冷冻管中标记后放入液氮备用,防止蛋白、RNA降解。
(6)Aβ1-42含量测定
海马组织匀浆后,4℃,3000r/min离心15min,取上清液,参照大鼠Aβ1-42ELISA试剂盒(北京博奥森,批号:RA20216)说明书进行检测。依据酶标仪450nm处吸光度(A)及标准曲线,计算Aβ1-42蛋白含量。
(7)SorLA、SNX27mRNA检测
用TRIZOL法提取海马组织总RNA,使用紫外吸收测定法、变性琼脂糖凝胶电泳法测定总RNA浓度和纯度。浓度和纯度测定结束后,将RNA逆转录合成cDNA,并将cDNA配置于Real-timequantitativePCR反应体系中。具体如下:①取0.2mLPCR管,配制如下反应体系:2×qPCRMix12.5μL,cDNA2.5μL,ddH2O8.0μL,体积23μL,充分混匀后短暂低速离心,放置于冰盒中。②将23μL混合液加入PCR八连管中,加入特异性引物(遵循三重复原则),各组全部加样完毕后盖好,充分混匀后短暂离心防止挂壁;设置PCR程序期间将装有样本的PCR管避光放置于冰盒中。③PCR扩增:预变性,95℃,10min;4次扩增循环95℃,15s;退火,58℃,15s;复性,72℃,60s。为了建立PCR产物溶解曲线,按95℃,10s;60℃,60s;95℃,15s;并从60℃缓慢加热到99℃(仪器自动进行)。进行RT-PCR反应,反应引物分别为:β-actinS:tgctatgttgccctagacttcg,SEQ ID NO.1所示;β-actinA:gttggcatagaggtctttacgg,SEQ IDNO.2所示;SorLAS:tgctgctgtatgacgaactgag,SEQ ID NO.3所示;SorLAA:gcaggatcaggaacaagatagg,SEQ ID NO.4所示;SNX27S:gacgactccttgggacaatcat,SEQ IDNO.5所示;SNX27A:cgggcatctaactgttgctctg,SEQ ID NO.6所示。mRNA相对表达量以2-ΔΔCt表示,ΔCt=(CtER-Ctβ-actin);以模型组作为基线,采用以下公式计算各组ER mRNA表达差异倍数(foldschange)=2-ΔΔCt,其中ΔΔCt=(CtER-Ctβ-actin)实验组-(CtER-Ctβ-actin)空白组。
(8)结果
图12示出了各组大鼠的逃避潜伏期结果。图13示出了各组大鼠的目标象限游泳时间百分比。其中,与正常组和空白组相比,模型组的逃避潜伏期显著升高,目标象限游泳时间百分比显著降低。而与模型组相比,实施例1~3分别提供的牡丹籽油或牡丹籽油肽微胶囊给药模型AD大鼠后,其逃避潜伏期显著降低,而目标象限游泳时间百分比显著升高至与正常组以及空白组接近。由此说明,本发明提供的牡丹籽油或牡丹籽油肽微胶囊能够改善老年痴呆模型AD大鼠的逃避潜伏期和目标象限游泳时间。
图14示出了各组大鼠的海马Aβ1-42含量。其中,与正常组和空白组相比,模型组的AD大鼠海马Aβ1-42含量显著升高。而与模型组相比,实施例1~3分别提供的牡丹籽油或牡丹籽油肽微胶囊给药模型AD大鼠后,其AD大鼠海马Aβ1-42含量显著降低至与正常组以及空白组接近。由此说明,本发明提供的牡丹籽油或牡丹籽油肽微胶囊能够改善老年痴呆模型AD大鼠海马Aβ1-42含量。
图15示出了各组大鼠海马SorLA、SNX27mRNA表达量。其中,与正常组和空白组相比,模型组的AD大鼠海马Aβ1-42含量显著降低。而与模型组相比,实施例1~3分别提供的牡丹籽油或牡丹籽油肽微胶囊给药模型AD大鼠后,其AD大鼠海马SorLA、SNX27mRNA含量显著升高至与正常组以及空白组接近。由此说明,本发明提供的牡丹籽油或牡丹籽油肽微胶囊能够改善老年痴呆模型AD大鼠海马SorLA、SNX27表达。
脑组织中淀粉样斑块沉积是导致AD中神经元丢失乃至痴呆的早期因素和重要原因。Aβ的生成与β-淀粉样前体蛋白(β-amyloidprecursorprotein,APP)各分泌酶(α-分泌酶、β-分泌酶、γ-分泌酶)密切相关,故关注APP转运途径,有效调节Aβ的产生和沉积过程是关键环节,也是AD治疗和研究的重要方向。近年来研究证实,SorLA对于Aβ的生成和沉积过程具有显著调控作用。在脑内,SorLA、SNX27能够通过多条途径影响Aβ水平,避免Aβ沉积引起神经元凋亡,从而大大降低患AD的风险。而上述实验结果表明,本发明提供的牡丹籽油或牡丹籽油肽微胶囊能够上调AD大鼠海马取SorLA、SNX27mRNA表达量,通过对APP贩运途径的影响减少Aβ的生成和沉积,从而抑制神经元细胞凋亡,提供大鼠空间探索能力,从而大大降低患AD的风险,为其可能作用机制之一。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到的变化或替换,都应涵盖在本发明的保护范围之内。
Claims (9)
1.一种牡丹籽油的提取方法,其特征在于,包括:
将经酸热处理的牡丹籽仁和牡丹籽壳研磨成粉,混匀于去离子水中,加入胃蛋白酶和风味蛋白酶进行酶解;
将经灭酶处理的酶解液经离心,收集清油及乳状液;
将乳状液用木瓜蛋白酶破乳,得到的油脂与清油合并,得到粗牡丹籽油;
将所述粗牡丹籽油经超声和萃取,得到所述牡丹籽油。
2.根据权利要求1所述的提取方法,其特征在于,所述胃蛋白酶的用量为所述牡丹籽仁和牡丹籽壳粉末重量的0.5%,所述风味蛋白酶的用量为所述牡丹籽仁和牡丹籽壳粉末重量的0.25%。
3.根据权利要求1所述的提取方法,其特征在于,所述木瓜蛋白酶用量为所述乳状液的占比为0.15mg/mL。
4.根据权利要求1所述的提取方法,其特征在于,“萃取”采用了体积比为3:7的石油醚和乙酸乙酯的混合溶液,从中间乙酸乙酯相中收集所述牡丹籽油。
5.权利要求1~4任一项所述的提取方法得到的牡丹籽油,其中,包含:
3.055%的肉豆蔻酸;
4.261%的7-十六碳烯酸;
3.141%的9-十六碳烯酸;
11.716%的棕榈酸;
0.109%的2-己基-环丙烷辛酸;
0.052%的十七酸;
26.121%的亚油酸;
32.931%的亚麻酸;
6.177%的硬脂酸;
2.875%的8,11,14-二十碳三烯酸;
1.622%的11-二十碳烯酸;
3.057%的花生酸;
0.049%的山嵛酸;
0.05%的二十四碳酸;
0.0352%的豆蔻酸甲酯;
0.0269%的十五烷酸甲酯;
0.254%的棕榈油酸甲酯;
0.103%的棕榈酸甲酯;
0.727%的棕榈酸乙酯;
2.432%的2-已基环丙烷辛酸甲酯;
0.02%的十七烷酸甲酯;
0.061%的7,10-十八碳二烯酸甲酯;
0.099%的亚油酸甲酯;
0.053%的油酸甲酯;
0.211%的亚麻酸甲酯;
0.135%的硬脂酸甲酯;
0.085%的亚油酸乙酯;
0.152%的油酸乙酯;
0.127%的亚麻酸乙酯;
0.141%的2-辛基环丙烷辛酸甲酯;
0.063%的二十碳烯酸甲酯。
6.一种牡丹籽油肽微胶囊的制备方法,其特征在于,包括:
采用如权利要求1~4任一所述的提取方法制得的牡丹籽油;
将乳清分离蛋白溶解于蒸馏水中,与玉米糖浆混合均匀后加入所述牡丹籽油和胶原三肽以及磷脂,采用高速剪切分散乳化机对混合液进行乳化,形成预乳状液,再经过高压均质(压强30MPa)均质2次后,对乳状液进行喷雾干燥,进料速率25rpm,进风温度180℃,出风温度90~95℃,得到牡丹籽油肽微胶囊。
7.根据权利要求6所述的制备方法,其特征在于,乳清分离蛋白、玉米糖浆、牡丹籽油、胶原三肽以及磷脂的质量配比为15:(45~50):(32~35):(15~18):2。
8.权利要求1~4任一所述的提取方法制得的牡丹籽油、或权利要求5所述的牡丹籽油或权利要求6或7所述的制备方法制得的牡丹籽油肽微胶囊在制备增强免疫力制品中的应用。
9.权利要求1~4任一所述的提取方法制得的牡丹籽油、或权利要求5所述的牡丹籽油或权利要求6或7所述的制备方法制得的牡丹籽油肽微胶囊在制备健脑制品中的应用。
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