CN105663051A - 一种白藜芦醇丝素蛋白纳米粒的制备方法 - Google Patents
一种白藜芦醇丝素蛋白纳米粒的制备方法 Download PDFInfo
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Abstract
本发明公开了一种白藜芦醇丝素蛋白纳米粒的制备方法,属于白藜芦醇领域。本发明从花生根须中提取得白藜芦醇浓缩液,对蚕丝脱胶得丝素蛋白,再通过透析得丝素蛋白溶液,将白藜芦醇浓缩液和丝素蛋白溶液分别装入双轴静电喷射装置的内外通道中,经内外通道注射,最后在喷头处混合,喷头处的溶液在静电场的作用下发生破裂形成微液滴,经冷冻干燥后即得白藜芦醇丝素蛋白纳米粒,本发明制得的白藜芦醇丝素蛋白纳米粒颗粒均匀,而且具有良好的水溶性,不易被氧化,而且整个制备过程中未使用有机溶剂,无有机溶剂残留,无毒无污染。
Description
技术领域
本发明公开了一种白藜芦醇丝素蛋白纳米粒的制备方法,属于白藜芦醇领域。
背景技术
白藜芦醇是一种活性多酚物质,在虎杖、葡萄、花生、毛脉酸模等植物中存在。白藜芦醇具有广泛的生理药理活性,如抗氧化、抑制血小板聚集、抗真菌、抗肿瘤、降低血清和肝脏的脂质,保护肝脏等作用。但其对光敏感,易氧化,水溶性差,生物利用度低,影响其在医药领域的广泛应用。
白藜芦醇纳米脂质体制备过程中存在有机试剂残留和磷脂成分易氧化等问题,且现有实验室制备方法生产成本高、周期长,制备过程难以控制,放大为工业化生产存在难度;储存过程中易受到温度、pH等外界环境的影响发生泄露,稳定性有待进一步提高;白藜芦醇纳米脂质体进入体内与特定受体相结合的作用机理尚不明确,其安全性与毒性仍存在争议;其在血液循环过程中与体内微环境间作用途径与机制尚无定论,不同给药方式对其释放过程影响也尚无研究。
生物大分子是目前高分子学科很活跃的一个研究领域,其研究对象为糖类、蛋白质和核酸等。作为生物大分子的蚕丝蛋白,是人类利用很早的天然蛋白质之一,具有纯度高,来源广等优点,对其进行研究及有效利用具有重要的意义。蚕丝中,丝素蛋白占总质量的70~80%,丝胶蛋白占20~30%。丝素蛋白是一种纤维蛋白,其在生物整体、细胞和分子生物学3个水平的实验结果都表明丝素蛋白安全可靠,具有与胶原蛋白相同的生物、细胞亲和性,适合于开发生物功能材料。
发明内容
本发明主要解决的技术问题:针对目前白藜芦醇化学性质不稳定且水溶性差,因此将其制成脂质体以改善其不足,但是白藜芦醇纳米脂质体进入体内与特定受体相结合的作用机理尚不明确,其安全性与毒性仍存在争议,而且白藜芦醇纳米脂质体制备过程中存在有机试剂残留和磷脂成分易氧化等问题,提供了一种白藜芦醇丝素蛋白纳米粒的制备方法,本发明从花生根须中提取得白藜芦醇浓缩液,再对蚕丝脱胶得丝素蛋白,通过透析得丝素蛋白溶液,将白藜芦醇浓缩液和丝素蛋白溶液分别装入双轴静电喷射装置的内外通道中,经内外通道注射,最后在喷头处混合,喷头处的溶液在静电场的作用下发生破裂形成微液滴,经冷冻干燥后即得白藜芦醇丝素蛋白纳米粒,本发明制得的白藜芦醇丝素蛋白纳米粒颗粒均匀,而且具有良好的水溶性,不易被氧化,而且整个制备过程中未使用有机溶剂,无有机溶剂残留,无毒无污染。
为了解决上述技术问题,本发明所采用的技术方案是:
(1)称取1~2kg花生根须,放入超声清洗仪中清洗20~30min,将洗涤后的根须移入烘箱,在50~60℃下干燥10~12h,干燥结束后用气流粉碎机将根须粉碎并过100目标准筛,得到干燥花生根粉末;
(2)称取500~600g上述得到的花生根粉末装入陶瓷酶解罐中,加入3~4L蒸馏水和花生根粉末总质量1~2%复合酶剂,用搅拌棒搅拌均匀后,再用浓度为0.5mol/L的醋酸溶液调节pH至5~6,放入恒温箱中在35~45℃下酶解2~3h,酶解后过滤,得酶解液,其中所述的复合酶剂为木聚糖酶和果胶酶按质量比为1:2复配制得;
(3)量取400~500mL上述酶解液装入2L具塞三角瓶中,再加入300~400mL无水乙醇,放置在恒温水浴振荡器中,在75~85℃下振荡提取1~2h,提取结束后将提取液移入卧式离心机中,以4000~5000r/min转速离心15~20min,分离得上清液后,在40~45℃下旋蒸去除多余乙醇,即得白藜芦醇浓缩液,备用;(4)按蚕丝、蒸馏水和无水碳酸钠质量比为9:2:5称取总质量为700~800g混合物装入铁锅中,放置在电磁炉上,以1000~1200W功率加热煮制40~50min,去除蚕丝蛋白表面的丝胶蛋白,过滤后用去离子水冲洗滤渣,直至淋洗液澄清为止,将滤渣干燥,得丝素蛋白;
(5)称取50~60g丝素蛋白倒入1L烧杯中,加入10~15g无水氯化钙、700~800mL去离子水和200~300mL无水乙醇,用保鲜膜密封烧杯口,放置在摇床上,在60~70℃下振荡溶解30~40min,待溶液冷却至室温后将溶液装入透析袋中,以流动蒸馏水作为透析液透析1~2天后,收集透析袋中的溶液即为纯净的丝素蛋白溶液;
(6)将等体积的丝素蛋白溶液和备用的白藜芦醇浓缩液分别装入双轴静电喷射装置的内外通道中,在室温和45~55%相对湿度条件下,经内外通道注射,最后在喷头处混合,喷头处的溶液在静电场的作用下发生破裂形成微液滴,经冷冻干燥后即得白藜芦醇丝素蛋白纳米粒。
本发明的应用的方法:本发明制得的白藜芦醇丝素蛋白纳米粒,可通过适当工艺制成各种剂型,如粉剂、颗粒剂、片剂、胶囊剂、溶液剂、油膏剂等,可作成药品、保健品,也可作成添加剂加入食品、饮料、制成各种形式的保健食品、饮料、酒类,可以直接溶于水相中,方便其操作使用,可以起到抗氧化、抑制血小板聚集、抗真菌、抗肿瘤、降低血清和肝脏的脂质,保护肝脏等作用。
本发明的有益效果是:
(1)本发明制得的白藜芦醇丝素蛋白纳米粒颗粒均匀,而且具有良好的水溶性,不易被氧化;
(2)本发明整个制备过程中未使用有机溶剂,无有机溶剂残留,无毒无污染,而且工艺简单,成本低。
具体实施方式
称取1~2kg花生根须,放入超声清洗仪中清洗20~30min,将洗涤后的根须移入烘箱,在50~60℃下干燥10~12h,干燥结束后用气流粉碎机将根须粉碎并过100目标准筛,得到干燥花生根粉末;称取500~600g上述得到的花生根粉末装入陶瓷酶解罐中,加入3~4L蒸馏水和花生根粉末总质量1~2%复合酶剂,用搅拌棒搅拌均匀后,再用浓度为0.5mol/L的醋酸溶液调节pH至5~6,放入恒温箱中在35~45℃下酶解2~3h,酶解后过滤,得酶解液,其中所述的复合酶剂为木聚糖酶和果胶酶按质量比为1:2复配制得;量取400~500mL上述酶解液装入2L具塞三角瓶中,再加入300~400mL无水乙醇,放置在恒温水浴振荡器中,在75~85℃下振荡提取1~2h,提取结束后将提取液移入卧式离心机中,以4000~5000r/min转速离心15~20min,分离得上清液后,在40~45℃下旋蒸去除多余乙醇,即得白藜芦醇浓缩液,备用;按蚕丝、蒸馏水和无水碳酸钠质量比为9:2:5称取总质量为700~800g混合物装入铁锅中,放置在电磁炉上,以1000~1200W功率加热煮制40~50min,去除蚕丝蛋白表面的丝胶蛋白,过滤后用去离子水冲洗滤渣,直至淋洗液澄清为止,将滤渣干燥,得丝素蛋白;称取50~60g丝素蛋白倒入1L烧杯中,加入10~15g无水氯化钙、700~800mL去离子水和200~300mL无水乙醇,用保鲜膜密封烧杯口,放置在摇床上,在60~70℃下振荡溶解30~40min,待溶液冷却至室温后将溶液装入透析袋中,以流动蒸馏水作为透析液透析1~2天后,收集透析袋中的溶液即为纯净的丝素蛋白溶液;将等体积的丝素蛋白溶液和备用的白藜芦醇浓缩液分别装入双轴静电喷射装置的内外通道中,在室温和45~55%相对湿度条件下,经内外通道注射,最后在喷头处混合,喷头处的溶液在静电场的作用下发生破裂形成微液滴,经冷冻干燥后即得白藜芦醇丝素蛋白纳米粒。
实例1
首先称取1kg花生根须,放入超声清洗仪中清洗20min,将洗涤后的根须移入烘箱,在50℃下干燥10h,干燥结束后用气流粉碎机将根须粉碎并过100目标准筛,得到干燥花生根粉末;称取500g得到的花生根粉末装入陶瓷酶解罐中,加入3L蒸馏水和酶解底物总质量1%复合酶剂,用搅拌棒搅拌均匀后,再用浓度为0.5mol/L的醋酸溶液调节pH至5,放入恒温箱中在35℃下酶解2h,酶解后过滤,得酶解液,其中所述的复合酶剂为木聚糖酶和果胶酶按质量比为1:2复配制得;量取400mL酶解液装入2L具塞三角瓶中,再加入300mL无水乙醇,放置在恒温水浴振荡器中,在75℃下振荡提取1h,提取结束后将提取液移入卧式离心机中,以4000r/min转速离心15min,分离得上清液后,在40℃下旋蒸去除多余乙醇,即得白藜芦醇浓缩液,备用;按蚕丝、蒸馏水和无水碳酸钠质量比为9:2:5称取总质量为700g混合物装入铁锅中,放置在电磁炉上,以1000W功率加热煮制40min,去除蚕丝蛋白表面的丝胶蛋白,过滤后用去离子水冲洗滤渣,直至淋洗液澄清为止,将滤渣干燥,得丝素蛋白;称取50g丝素蛋白倒入1L烧杯中,加入10g无水氯化钙、700mL去离子水和200mL无水乙醇,用保鲜膜密封烧杯口,放置在摇床上,在60℃下振荡溶解30min,待溶液冷却至室温后将溶液装入透析袋中,以流动蒸馏水作为透析液透析1天后,收集透析袋中的溶液即为纯净的丝素蛋白溶液;将等体积的丝素蛋白溶液和备用的白藜芦醇浓缩液分别装入双轴静电喷射装置的内外通道中,在室温和45%相对湿度条件下,经内外通道注射,最后在喷头处混合,喷头处的溶液在静电场的作用下发生破裂形成微液滴,经冷冻干燥后即得白藜芦醇丝素蛋白纳米粒。
本发明制得的白藜芦醇丝素蛋白纳米粒,可通过适当工艺制成各种剂型,如粉剂、颗粒剂、片剂、胶囊剂、溶液剂、油膏剂等,可作成药品、保健品,也可作成添加剂加入食品、饮料、制成各种形式的保健食品、饮料、酒类,可以直接溶于水相中,方便其操作使用,可以起到抗氧化、抑制血小板聚集、抗真菌、抗肿瘤、降低血清和肝脏的脂质,保护肝脏等作用。
实例2
首先称取1.5kg花生根须,放入超声清洗仪中清洗25min,将洗涤后的根须移入烘箱,在55℃下干燥11h,干燥结束后用气流粉碎机将根须粉碎并过100目标准筛,得到干燥花生根粉末;称取550g得到的花生根粉末装入陶瓷酶解罐中,加入3.5L蒸馏水和酶解底物总质量1.5%复合酶剂,用搅拌棒搅拌均匀后,再用浓度为0.5mol/L的醋酸溶液调节pH至5.5,放入恒温箱中在40℃下酶解2.5h,酶解后过滤,得酶解液,其中所述的复合酶剂为木聚糖酶和果胶酶按质量比为1:2复配制得;量取450mL酶解液装入2L具塞三角瓶中,再加入350mL无水乙醇,放置在恒温水浴振荡器中,在80℃下振荡提取1.5h,提取结束后将提取液移入卧式离心机中,以4500r/min转速离心17min,分离得上清液后,在43℃下旋蒸去除多余乙醇,即得白藜芦醇浓缩液,备用;按蚕丝、蒸馏水和无水碳酸钠质量比为9:2:5称取总质量为750g混合物装入铁锅中,放置在电磁炉上,以1100W功率加热煮制45min,去除蚕丝蛋白表面的丝胶蛋白,过滤后用去离子水冲洗滤渣,直至淋洗液澄清为止,将滤渣干燥,得丝素蛋白;称取55g丝素蛋白倒入1L烧杯中,加入13g无水氯化钙、750mL去离子水和250mL无水乙醇,用保鲜膜密封烧杯口,放置在摇床上,在65℃下振荡溶解35min,待溶液冷却至室温后将溶液装入透析袋中,以流动蒸馏水作为透析液透析1.5天后,收集透析袋中的溶液即为纯净的丝素蛋白溶液;将等体积的丝素蛋白溶液和备用的白藜芦醇浓缩液分别装入双轴静电喷射装置的内外通道中,在室温和50%相对湿度条件下,经内外通道注射,最后在喷头处混合,喷头处的溶液在静电场的作用下发生破裂形成微液滴,经冷冻干燥后即得白藜芦醇丝素蛋白纳米粒。
本发明制得的白藜芦醇丝素蛋白纳米粒,可通过适当工艺制成各种剂型,如粉剂、颗粒剂、片剂、胶囊剂、溶液剂、油膏剂等,可作成药品、保健品,也可作成添加剂加入食品、饮料、制成各种形式的保健食品、饮料、酒类,可以直接溶于水相中,方便其操作使用,可以起到抗氧化、抑制血小板聚集、抗真菌、抗肿瘤、降低血清和肝脏的脂质,保护肝脏等作用。
实例3
首先称取2kg花生根须,放入超声清洗仪中清洗30min,将洗涤后的根须移入烘箱,在60℃下干燥12h,干燥结束后用气流粉碎机将根须粉碎并过100目标准筛,得到干燥花生根粉末;称取600g得到的花生根粉末装入陶瓷酶解罐中,加入4L蒸馏水和酶解底物总质量2%复合酶剂,用搅拌棒搅拌均匀后,再用浓度为0.5mol/L的醋酸溶液调节pH至6,放入恒温箱中在45℃下酶解3h,酶解后过滤,得酶解液,其中所述的复合酶剂为木聚糖酶和果胶酶按质量比为1:2复配制得;量取500mL酶解液装入2L具塞三角瓶中,再加入400mL无水乙醇,放置在恒温水浴振荡器中,在85℃下振荡提取2h,提取结束后将提取液移入卧式离心机中,以5000r/min转速离心20min,分离得上清液后,在45℃下旋蒸去除多余乙醇,即得白藜芦醇浓缩液,备用;按蚕丝、蒸馏水和无水碳酸钠质量比为9:2:5称取总质量为800g混合物装入铁锅中,放置在电磁炉上,以1200W功率加热煮制50min,去除蚕丝蛋白表面的丝胶蛋白,过滤后用去离子水冲洗滤渣,直至淋洗液澄清为止,将滤渣干燥,得丝素蛋白;称取60g丝素蛋白倒入1L烧杯中,加入15g无水氯化钙、800mL去离子水和300mL无水乙醇,用保鲜膜密封烧杯口,放置在摇床上,在70℃下振荡溶解40min,待溶液冷却至室温后将溶液装入透析袋中,以流动蒸馏水作为透析液透析2天后,收集透析袋中的溶液即为纯净的丝素蛋白溶液;将等体积的丝素蛋白溶液和备用的白藜芦醇浓缩液分别装入双轴静电喷射装置的内外通道中,在室温和55%相对湿度条件下,经内外通道注射,最后在喷头处混合,喷头处的溶液在静电场的作用下发生破裂形成微液滴,经冷冻干燥后即得白藜芦醇丝素蛋白纳米粒。
本发明制得的白藜芦醇丝素蛋白纳米粒,可通过适当工艺制成各种剂型,如粉剂、颗粒剂、片剂、胶囊剂、溶液剂、油膏剂等,可作成药品、保健品,也可作成添加剂加入食品、饮料、制成各种形式的保健食品、饮料、酒类,可以直接溶于水相中,方便其操作使用,可以起到抗氧化、抑制血小板聚集、抗真菌、抗肿瘤、降低血清和肝脏的脂质,保护肝脏等作用。
Claims (1)
1.一种白藜芦醇丝素蛋白纳米粒的制备方法,其特征在于具体制备步骤为:
(1)称取1~2kg花生根须,放入超声清洗仪中清洗20~30min,将洗涤后的根须移入烘箱,在50~60℃下干燥10~12h,干燥结束后用气流粉碎机将根须粉碎并过100目标准筛,得到干燥花生根粉末;
(2)称取500~600g上述得到的花生根粉末装入陶瓷酶解罐中,加入3~4L蒸馏水和花生根粉末总质量1~2%复合酶剂,用搅拌棒搅拌均匀后,再用浓度为0.5mol/L的醋酸溶液调节pH至5~6,放入恒温箱中在35~45℃下酶解2~3h,酶解后过滤,得酶解液,其中所述的复合酶剂为木聚糖酶和果胶酶按质量比为1:2复配制得;
(3)量取400~500mL上述酶解液装入2L具塞三角瓶中,再加入300~400mL无水乙醇,放置在恒温水浴振荡器中,在75~85℃下振荡提取1~2h,提取结束后将提取液移入卧式离心机中,以4000~5000r/min转速离心15~20min,分离得上清液后,在40~45℃下旋蒸去除多余乙醇,即得白藜芦醇浓缩液,备用;
(4)按蚕丝、蒸馏水和无水碳酸钠质量比为9:2:5称取总质量为700~800g混合物装入铁锅中,放置在电磁炉上,以1000~1200W功率加热煮制40~50min,去除蚕丝蛋白表面的丝胶蛋白,过滤后用去离子水冲洗滤渣,直至淋洗液澄清为止,将滤渣干燥,得丝素蛋白;
(5)称取50~60g丝素蛋白倒入1L烧杯中,加入10~15g无水氯化钙、700~800mL去离子水和200~300mL无水乙醇,用保鲜膜密封烧杯口,放置在摇床上,在60~70℃下振荡溶解30~40min,待溶液冷却至室温后将溶液装入透析袋中,以流动蒸馏水作为透析液透析1~2天后,收集透析袋中的溶液即为纯净的丝素蛋白溶液;
(6)将等体积的丝素蛋白溶液和备用的白藜芦醇浓缩液分别装入双轴静电喷射装置的内外通道中,在室温和45~55%相对湿度条件下,经内外通道注射,最后在喷头处混合,喷头处的溶液在静电场的作用下发生破裂形成微液滴,经冷冻干燥后即得白藜芦醇丝素蛋白纳米粒。
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