CN107439787A - 一种具有抗pm2.5颗粒物功效的核桃低聚肽 - Google Patents
一种具有抗pm2.5颗粒物功效的核桃低聚肽 Download PDFInfo
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Abstract
本发明涉及一种具有抗PM 2.5颗粒物功效的核桃低聚肽。所述低聚肽粉肽含量在80wt%以上,其中95%以上核桃肽的分子量小于1500Dalton。所述方法包括使用碱提酸沉法提取核桃蛋白,过滤、酶解,再将蛋白酶解液依次经过微滤膜与超滤膜高效分离纯化,最后浓缩喷雾,得到低聚肽粉。所述的核桃低聚肽粉可用于制备治疗或者预防PM 2.5颗粒物引起的症状的药物、食品或保健品。
Description
技术领域
本发明涉及到核桃低聚肽粉在抗PM 2.5颗粒物功效上的应用,属于生物技术领域。
背景技术
雾霾是特定气候条件与人类活动相互作用的结果,其主要成分包括烟雾、煤烟及粉尘等各种固态或液态颗粒或气溶胶。高密度人口的经济及社会活动必然会排放大量细颗粒物(PM 2.5),一旦排放超过大气循环能力和承载度,细颗粒物浓度将持续积聚,此时如果受静稳天气等影响,极易出现大范围产生。大量的细颗粒物既是污染物,又是重金属、多环芳烃及病毒等有害物质的载体,不仅对大气能见度和气候有巨大的影响,其中可吸入颗粒物能够进入人体呼吸系统,更能对人体健康造成直接的严重威胁,已经成为危害人类健康和生命的重要杀手。因此制备效果较为显著的用于预防或者治疗PM 2.5引起的相关症状的药物已经成为药物或者保健食品研究领域的热点之一
核桃(Juglans regia L)又名胡桃,属于四大坚果之一,具有很高的营养药用价值。在我国古医药书籍中,有明确记载,李时珍《本草纲目》记述“补气养血、润燥化痰、益命门、利三焦,温肺润肠。治肺润肠。治虚寒喘咳,腰脚重疼,心腹疝痛,血痢肠风,散肿毒,”宋刘翰《开宝本草》载“胡桃(即核桃)味甘、平、无毒。食之令人肥健,润肌黑发,取瓤烧令黑,未断烟,和松脂,研傅瘰疬疮”。唐化孟诜《食疗本草》中说,核桃仁能“通经脉、黑须发,常服骨肉细腻光润”。崔禹锡的《食经》说它“多食利小便,去五痔”。《医林纂要》一书的评价是,
可以“补肾,润命门,固精,润大肠,通热秘,止寒泻虚泻。”等等。
核桃中含有丰富的蛋白质,含量高达24%,其氨基酸比例合理,容易吸收,对人体有着重要的生理作用。为了获得比核桃原蛋白在营养、功能性及生物活性上更加优良的低聚肽粉,已尝试了多种制备方法并获得意想不到的生物活性,如免疫调节、抗疲劳等。考虑到核桃蛋白及低聚肽的特有性质,本发明进一步按照前期申请专利CN 105567774 A的制备方法所获得的核桃低聚肽粉以考察其抗PM 2.5颗粒物的生物活性。
发明内容
本发明公开了参照前期核桃低聚肽粉的制备工艺获得核桃低聚肽粉,进行体内改善PM 2.5颗粒物引起机体不适的功效研究。研究发现核桃低聚肽粉能明显改善PM 2.5引起的心脏扩大,促进肠道对PM 2.5颗粒物的分泌,增强巨噬细胞对PM 2.5颗粒物的吞噬功能,可用于制备治疗或者预防PM 2.5颗粒物引起的症状的药物、食品或保健品。
为达到上述目的,本发明采用的技术方案是:
一种核桃低聚肽粉,采用GB/T 22492-2008附录A与附录B的检测方法,测得肽含量在80wt%以上,其中95%以上核桃肽的分子量小于1500Dalton,其分子量分布如下:
分子量Dalton分布
所述核桃低聚肽粉是通过下述方法制备得到的:
(1)核桃粕的预处理:将核桃去壳、脱油,得到脱脂核桃粕。
(2)蛋白质提取:将一定量脱脂后的核桃粕,与水按重量比为1∶5~1∶15混合,调节pH至9~11于室温提取1~2h,提取完成后,过滤,滤液备用;使用同样的条件再次对滤渣进行提取,提取完成后,过滤,滤液与第一次蛋白提取液合并。调节滤液pH为3~5,静置0.5~2h,弃去上清液,最后往沉淀物中加入体积比为1∶10~1∶20的水,搅拌均匀,得到核桃蛋白液。
(3)蛋白酶解:将上述核桃蛋白液加热至40~55℃,加入核桃粕重量0.5~2%的生物酶,搅拌酶解3~6h后,煮沸灭活30min,离心,上清液即为蛋白酶解液。
(4)分离纯化:将蛋白酶解液使用孔径为0.1~0.5μm的微滤膜进行过滤,透过液再经2000~20000Dalton超滤膜处理后,透过液在50~80℃下浓缩至固含为3~5wt%时,经干燥得到核桃低聚肽粉,产率为20~30wt%。
(5)肽含量与分子量分布测定:采用GB/T 22492-2008附录A与附录B的检测方法,测得肽含量在80wt%以上,其中95%以上的核桃肽的分子量小于1500Dalton。
所述的核桃仁的脱脂方法可以为冷榨、热榨或者有机溶剂脱脂法,优选地使用冷榨脱脂法。
所述的碱提酸沉法可包括连续逆流提取法和普通提取法。
所述生物酶选自食品级的中性蛋白酶(酶活力≥30万u/g)、木瓜蛋白酶(酶活力≥40万u/g)、菠萝蛋白酶(酶活力≥30万u/g)、碱性蛋白酶(酶活力≥20万u/g)、胃蛋白酶(酶活力≥50万u/g)、胰酶(酶活力≥3000u/g)中的一种或者它们的混合物,优选地使用中性蛋白酶。
所述的干燥方法可以为喷雾干燥、真空干燥、加热干燥、冷冻干燥。
一种组合物,其含有权利要求1-3任一所述的核桃低聚肽粉,和可药用或食用辅料;可为任意剂型,优选其是片剂、分散片、胶囊剂、软胶囊剂、微囊剂、颗粒剂、注射剂、粉针剂、冻干粉针剂、微丸剂、滴丸剂、糖浆剂、散剂、浸膏剂、煎膏剂、口服液体制剂及其他药学上可以接受的剂型。
本发明所述的核桃低聚肽粉可改善PM 2.5引起的心脏扩大,促进肠道对PM 2.5颗粒物的分泌,增强巨噬细胞对PM 2.5颗粒物的吞噬功能,可用于制备治疗或者预防PM 2.5颗粒物引起的症状的药物、食品或保健品。
附图说明
图1:核桃低聚肽粉对于纳米活性炭(PM 2.5)引起斑马鱼心脏扩大改善作用的表型图;
图2:核桃低聚肽粉对斑马鱼肠道分泌纳米活性炭(PM 2.5)促进作用的表型图;
图3:核桃低聚肽粉对斑马鱼体内吞噬纳米活性炭(PM 2.5)的巨噬细胞吞噬功能促进作用的表型图。
实施例
下面通过实施例对本发明作进一步说明。应该理解的是,本发明实施例所述方法仅仅是用于说明本发明,而不是对本发明的限制,在本发明的构思前提下对本发明制备方法的简单改进都属于本发明要求保护的范围。如无特别说明,实施例中用到的所有原料和溶剂均为市售产品。
制备实施例1:
将100kg冷榨脱脂后的核桃粕,与水按重量比为1∶10混合,调节pH至10于室温提取2h;提取完成后,过滤,滤渣按照第一次提取条件再次提取,提取完成后,过滤,滤液与第一次过滤液合并,调节pH为5,静置2h,弃去上清液,最后往沉淀物中加入体积比为1∶10的水,搅拌均匀。将上述核桃蛋白液加热至45℃,将pH值调节至中性,加入1kg中性蛋白酶(酶活力为30万u/g),搅拌酶解6h后,煮沸灭活30min,离心,上清液即为蛋白酶解液。将蛋白酶解液使用孔径为0.1μm的微滤膜进行过滤,透过液再经5000Dalton超滤膜处理后,透过液在80℃下浓缩至固含为3.8%时,进行喷雾干燥,进口温度为140℃,出口温度为55~65℃,得到高纯度、低分子量的淡黄色核桃肽粉(批号为Z-1),产率为20.8wt%。采用GB/T 22492-2008附录A与附录B的检测方法,测得肽含量为80.6wt%,小于1500Dalton分子量占97%。
生物活性实施例:
1评价核桃低聚肽粉对PM 2.5引起心脏扩大的改善作用
实验动物:野生型AB品系斑马鱼,以自然成对交配繁殖方式进行。年龄为受精后2天,共90尾,每实验组为30尾。
实验模型的建立:用PM 2.5处理2dpf野生型AB品系斑马鱼24h,建立PM 2.5诱发的心血管毒性模型。
实验步骤:随机选取90尾受精后2天(2dpf)野生型AB品系斑马鱼于六孔板中,每孔(实验组)均处理30尾斑马鱼,水溶给予PM 2.5诱发斑马鱼心血管毒性评价模型。水溶给予核桃低聚肽粉(Z-1)100μg/mL浓度,同时设置正常对照组(养鱼用水处理斑马鱼)和模型对照组,每孔(实验组)容量为3mL。与PM 2.5共同处理24h后,每个实验组随机选取10尾斑马鱼在解剖显微镜下拍照并采集数据,用NIS-Elements D 3.10高级图像处理软件进行图像分析统计斑马鱼心脏面积(A),根据心脏面积的统计学意义评价供试品对PM 2.5引起的心脏扩大改善作用。统计学处理结果用mean±SE表示,供试品对PM 2.5引起的心脏扩大改善作用计算公式为:心脏扩大改善作用(%)=(A模型对照组-A供试品组)/(A模型对照组-A正常对照组)×100%。实验结果如表1所示。
表1核桃低聚肽粉对PM 2.5引起心脏扩大改善作用
与模型对照组比较,***p<0.001
由表1可知,模型对照组斑马鱼心脏面积(13121像素)与正常对照组(10526像素)比较p<0.001,表明模型建立成功;核桃低聚肽在浓度为100μg/mL时,斑马鱼心脏面积为10849像素,心脏扩大改善作用为88%,可知核桃低聚肽对PM 2.5引起心脏扩大具有明显的改善作用。
2评价核桃低聚肽粉对斑马鱼纳米活性炭分泌进入肠道的促进作用
实验动物:黑色素等位基因突变型半透明Albino品系斑马鱼,以自然成对交配繁殖方式进行。年龄为受精后2天,共150尾,每实验组为30尾。
实验模型的建立:将62.5mg/mL纳米活性炭(PM 2.5)作为纳米颗粒,注射到2dpf斑马鱼卵黄囊中(相当于人肌肉注射),每尾斑马鱼注射10nL,即以625ng/尾剂量建立斑马鱼PM 2.5分泌模型。
实验步骤:随机选取150尾受精后2天(2dpf)黑色素等位基因突变型半透明Albino品系斑马鱼于六孔板中,每孔(实验组)均处理30尾斑马鱼,肌肉注射给予纳米活性炭建立斑马鱼纳米活性炭分泌进入肠道功能评价模型。水溶给予核桃低聚肽(Z-1)250μg/mL浓度,同时设置正常对照组(养鱼用水处理斑马鱼)和模型对照组,每孔(实验组)容量为3mL。供试品每天换液,处理6d后,统计体内纳米活性炭分泌进入肠道的斑马鱼数量(N),根据纳米活性炭分泌发生率的统计学意义来评价供试品对纳米活性炭分泌进入肠道的影响。统计学处理结果用mean±SE表示,纳米活性炭分泌发生率计算公式为:分泌发生率(%)=(N纳米活性炭分泌进入肠道/N总数)×100%。实验结果如表2所示。
表2核桃低聚肽粉对斑马鱼肠道分泌的促进作用
与模型对照组比较,*p<0.05
由表2可知,模型对照组斑马鱼纳米活性炭分泌尾数为15/30,纳米活性炭分泌发生率为50%。而核桃低聚肽在浓度为250μg/mL时,斑马鱼纳米活性炭分泌尾数为23/30尾,纳米活性炭分泌发生率为76.7%,表明核桃低聚肽具有明显促进斑马鱼纳米活性炭分泌进入肠道的作用。
3评价核桃低聚肽粉对吞噬纳米活性炭(PM 2.5)的巨噬细胞吞噬功能的促进作用
实验动物:黑色素等位基因突变型半透明Albino品系斑马鱼,以自然成对交配繁殖方式进行。年龄为受精后2天,共150尾,每实验组为30尾。
实验模型的建立:将62.5mg/mL纳米活性炭(PM 2.5)作为纳米颗粒,注射到2dpf斑马鱼血液循环中(相当于人静脉注射),每尾斑马鱼注射10nL,即以625ng/尾剂量建立斑马鱼PM 2.5吞噬模型。
实验步骤:随机选取150尾受精后2天(2dpf)黑色素等位基因突变型半透明Albino品系斑马鱼于六孔板中,每孔(实验组)均处理30尾斑马鱼,静脉注射给予纳米活性炭建立斑马鱼纳米活性炭吞噬模型。水溶给予核桃低聚肽粉(Z-1)浓度为250μg/mL,同时设置正常对照组(养鱼用水处理斑马鱼)和模型对照组,每孔(实验组)容量为3mL。供试品每天换液,处理2d后,加入中性红溶液对斑马鱼进行活体染色16h,染色结束,在解剖显微镜下统计吞噬了纳米活性炭的巨噬细胞数量(N),根据吞噬纳米活性炭的巨噬细胞数量的统计学意义来评价供试品对巨噬细胞吞噬作用的影响。统计学处理结果用mean±SE表示,巨噬细胞吞噬促进作用计算公式为:巨噬细胞吞噬促进作用(%)=(N供试品-N模型对照组)/N模型对照组×100%。结果如表3所示。
表3核桃低聚肽粉对斑马鱼巨噬细胞吞噬功能的促进作用
与模型对照组比较,***p<0.001
由表3可知,模型对照组吞噬纳米活性炭的巨噬细胞数量为12个,而核桃低聚肽在250μg/mL时,吞噬纳米活性炭的巨噬细胞数量为20个,巨噬细胞吞噬促进作用分别为61%,表明核桃低聚肽对斑马鱼巨噬细胞吞噬功能具有明显的促进作用。
Claims (9)
1.一种核桃低聚肽粉,其特征在于:采用GB/T 22492-2008附录A与附录B的检测方法,测得肽含量在80wt%以上,其中95%以上核桃肽的分子量小于1500 Dalton,其分子量分布如下:
分子量Dalton分布
优选地,肽含量为80.6wt%,小于1500Dalton分子量占97%。
2.权利要求1所述核桃低聚肽粉的制备方法,其特征包括下述步骤:将脱脂后的核桃粕,使用碱提酸沉法提取蛋白质,过滤、酶解,再将蛋白酶解液依次经过微滤膜与超滤膜高效分离纯化,最后浓缩干燥,得到核桃低聚肽粉。
3.根据权利要求2所述的制备方法,其特征在于包括下述步骤:
核桃粕的预处理:将核桃去壳、脱油,得到脱脂核桃粕。
蛋白质提取:将一定量脱脂后的核桃粕,与水按重量比为1∶5~1∶15混合,调节pH至9~11于室温提取1~2h,提取完成后,过滤,滤液备用;使用同样的条件再次对滤渣进行提取,提取完成后,过滤,滤液与第一次蛋白提取液合并。调节滤液pH为3~5,静置0.5~2h,弃去上清液,最后往沉淀物中加入体积比为1∶10~1∶20的水,搅拌均匀,得到核桃蛋白液。
蛋白酶解:将上述核桃蛋白液加热至40~55℃,加入核桃粕重量0.5~2%的生物酶,搅拌酶解3~6h后,煮沸灭活30min,离心,上清液即为蛋白酶解液。
分离纯化:将蛋白酶解液使用孔径为0.1~0.5μm的微滤膜进行过滤,透过液再经2000~20000 Dalton超滤膜处理后,透过液在50~80℃下浓缩至固含为3~5wt%时,经干燥得到核桃低聚肽粉,产率为20~30wt%。
4.根据权利要求2或3所述核桃低聚肽粉的制备方法,其特征在于所述的核桃仁的脱脂方法可以为冷榨、热榨或者有机溶剂脱脂法,优选地使用冷榨脱脂法;所述的碱提酸沉法可包括连续逆流提取法和普通提取法;所述的干燥方法可以为喷雾干燥、真空干燥、加热干燥、冷冻干燥。
5.根据权利要求2或3所述的制备方法,其特征在于:所述生物酶选自食品级的中性蛋白酶(酶活力≥30万u/g)、木瓜蛋白酶(酶活力≥40万u/g)、菠萝蛋白酶(酶活力≥30万u/g)、碱性蛋白酶(酶活力≥20万u/g)、胃蛋白酶(酶活力≥50万u/g)、胰酶(酶活力≥3000u/g)中的一种或者它们的混合物,优选地使用中性蛋白酶。
6.一种核桃低聚肽粉,其特征在于其利用权利要求2-5任一所述的方法进行制备得到。
7.一种组合物,其含有权利要求1或6所述的核桃低聚肽粉,和可药用或食用辅料;可为任意剂型,优选其是片剂、分散片、胶囊剂、软胶囊剂、微囊剂、颗粒剂、注射剂、粉针剂、冻干粉针剂、微丸剂、滴丸剂、糖浆剂、散剂、浸膏剂、煎膏剂、口服液体制剂及其他药学上可以接受的剂型。
8.权利要求1或6所述的核桃低聚肽粉在改善PM 2.5引起的心脏扩大;促进肠道对PM2.5颗粒物的分泌;增强巨噬细胞对PM 2.5颗粒物的吞噬功能方面的用途。
9.权利要求1或6所述的桃低聚肽粉用于制备治疗或者预防PM 2.5颗粒物引起的症状的药物、食品或保健品的应用。
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