CN115067460A - 一种红枣色素纳米颗粒及其制备方法 - Google Patents
一种红枣色素纳米颗粒及其制备方法 Download PDFInfo
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- CN115067460A CN115067460A CN202210688199.6A CN202210688199A CN115067460A CN 115067460 A CN115067460 A CN 115067460A CN 202210688199 A CN202210688199 A CN 202210688199A CN 115067460 A CN115067460 A CN 115067460A
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Abstract
一种红枣色素纳米颗粒及其制备方法,将红枣色素提取物溶解于水中作为内水相,与磷脂、中性脂质等的混合油相剪切制备W/O初乳,初乳与外水相经机械方法复乳化,经优化后的冻干工艺加工,获得担载红枣色素的多囊脂质体。本发明制备方法针对红枣色素水溶性好的特点,结合多囊脂质体较一般脂质体包封体积大、水溶性成分载药量高等优点制备的红枣色素多囊脂质体,很大程度上提高了对红枣色素的生物利用率,能够有效减少突释现象以实现缓释放,且经本工艺制备的多囊脂质体具更好的稳定性,预期具有良好的抗氧化活性,可用于紫外线所致皮肤损伤的防护及修复,达到一定抗衰老功能,对于红枣色素综合开发利用前景的开拓有重要意义。
Description
技术领域
本发明属于生物工程领域,是一种红枣色素纳米颗粒及其制备方法。
背景技术
红枣,一种药食同源食物,有“活性维生素丸”之美称,在我国种植广泛。红枣枣皮中富含色素,色泽自然鲜艳、安全无毒、具有特定的药理药效功能,广泛用于食品、医药及化妆品着色。随着红枣类产品的日渐丰富,如何对枣皮加工副产物中色素开发也成为我国发展林业经济的契机之一。红枣色素属于类黄酮类物质,水溶性好,溶液澄清透明,色泽鲜艳自然,安全性高,实红枣色素对光照、碱、还原剂稳定性较好,但抗氧化能力较差,受Cu2+、Al3 +、Fe3+影响较大。天然红枣皮色素对金黄色葡萄球菌属,李斯特氏菌属,乳酸杆菌属,脂环酸芽孢杆菌属等革兰氏阳性菌和大肠杆菌属,变形杆菌属,痢疾杆菌属,肺炎杆菌属,不动杆菌属等革兰氏阴性菌显示抗菌活性。红枣色素对大豆油脂质过氧化、Fe2+诱导的卵黄脂蛋白过氧化、亚油酸脂质过氧化与Fe2+诱导的亚油酸脂质过氧化有较好的抑制作用,且能明显抑制小鼠肝组织自发性脂质或氧化保护肝脏,对过氧化造成的红胞损伤起到保护作用,抑制红细胞溶血的发生,避免氧化损伤。枣皮中总酚酸、类黄酮和花色苷含量丰富,实验表明,枣皮色素的抗氧化能力与黄酮含量相关,为开发枣皮色素药用价值提供了理论基础,可作为具备潜在保健功能活性成分及天然色素进行深度开发。
目前,红枣色素的提取、纯化工艺较为完备,但就如何提高其稳定性和生物利用率,更好的探究作为食品活性成分或天然色素添加方面的应用不多。作为一种常见的纳米运载体系,脂质体技术目前已在不同领域得到了较为广泛的应用,其具有低毒性、易于制备及工业化生产、生物相容性好等优点,在食品领域相关产品开发中,可通过脂质体技术提高活性成分稳定性、生物利用度及达到缓控释作用,普通脂质体由单个或多个同心脂质双分子层组成,分为单层脂质体和多层脂质体,其中单层脂质体又可以分为小单层脂质体(SUV)和大单层脂质体(LUV),其机械稳定强度不足,对水溶性成分包裹效率有限。多囊脂质体系统(multivesicular systems)为内部含有多个囊泡的脂质体,多囊系统可以很好地解决普通脂质体包封体积小、水溶性药物载药量低、脂质膜一旦破裂药物便立即释放等问题。采用多囊脂质体技术对红枣色素进行包封,探索制备工艺,极大的提高了其缓慢释放能力,为作为新型食品功能性色素、抗氧化类系列化妆品天然色素的应用提供了依据。
发明内容
解决的技术问题:本发明提供一种红枣色素纳米颗粒及其制备方法,该方法极大的提高了提取所得的红枣色素的稳定性和生物利用率,为农产品加工剩余物高值化利用提供了技术方法,红枣色素发挥其功能特性提供了良好的途径。
技术方案:一种红枣色素纳米颗粒制备方法,将提取得到的枣皮色素溶解于超纯水中,将磷脂、胆固醇、三油酸甘油酯按质量比1:(0.1-1):(0.1-1)溶解于二氯甲烷中,将两相经高速剪切混合制得初乳;将初乳与外水相经涡旋混合后,旋蒸除去有机溶剂,制得红枣色素多囊脂质体,经冷冻干燥后,可以获得红枣色素纳米颗粒。
上述初乳制备条件为:以一定比例的水相与油相,剪切下形成初乳,内水相:油相体积比=1:(0.1-5),剪切速率为1000-30000rpm,初乳化时间为0-300s;所述初乳:外水相体积比=1: (0.1-3),复乳化时间为0-130s。
上述多囊脂质所使用的外水相为水溶性成分。
上述水溶性成分为氨基酸、盐或水溶性高分子中的至少一种。
上述氨基酸为L-赖氨酸、色氨酸、丝氨酸中的至少一种。
上述制备方法制得的红枣色素纳米颗粒。
有益效果:本发明提供的一种红枣色素纳米颗粒及其制备方法,存在于枣皮中的色素经复合酶法提取,纯化后主要功能成分包括对香豆酸、(-)-表儿茶素、槲皮素-3-O-刺槐苷、芦丁、山奈酚3-O-刺槐苷、槲皮素3-O-α-L-阿拉伯糖基-(1→2)-α-L-鼠李糖苷、槲皮素3-O-β-D-木糖基-(1→2)-α-L-鼠李糖苷、槲皮素。在该方法中,对红枣色素进行多囊脂质体的制备,经过优化后的制备条件制备得到的脂质体可以获得高的包封体积,有利于对水溶性色素的有效利用,实现更好的经济效益,能有效的解决应用过程中的突释现象,实现缓释效果,较普通脂质体相比获得相同体积下的良好稳定性和高的生物利用率,同时更高效的发挥色素所含的功能性成分的功能性。
附图说明
图1为各因素对MVLs包封率影响(a)磷脂浓度(b)磷脂与胆固醇之比(c)磷脂与三油酸甘油酯之比(d)L-赖氨酸浓度(e)初乳与外水相之比(f)油相与内水相之比(g)初乳化转速;
图2为不同温度下脂质体悬液的渗漏率;
图3为不同释放介质下脂质体的释放率;
图4为光学显微镜下的多囊脂质体(a)100×(b)400×;
图5为透射电镜下的多囊脂质体;
图6为含有不同冻干保护剂(分别设置5%、10%和15%(w/v)3个浓度)的枣皮色素多囊脂质体冻干粉末。(a)葡萄糖;(b)海藻糖;(c)乳糖;(d)甘露醇;
图7为DPPH清除率(a)红枣色素、Vc、色素MVLs、空白MVLs和普通脂质体;(b) MVLs不同释放时间;
图8为红枣色素、色素MVLs、空白MVLs和普通脂质体的ABTS+清除率比较;
图9为红枣色素、色素MVLs、空白MVLs对(a)L929细胞及(b)HUVEC细胞存活率影响。
具体实施方式
下面将结合具体实施例来详细说明本发明,在此本发明的示意性实施例及其说明用来解释本发明,但并不作为对本发明的限定。
为了进一步提高对水溶性色素的包封效率,本发明采用复乳法制备了红枣色素多囊脂质体,利用对膜材等处方条件及设备参数等工艺条件的优化,提高脂质体稳定性的同时,增加其生物利用度,且就该制备的多囊脂质体对色素发挥抗衰老作用及其机制进行探索,拓展了红枣色素的综合利用前景。同时脂质体冻干工艺进行了优化,制备的冻干粉,丰富红枣色素在不同类产品中的应用,为提高红枣生产中副产品加工的综合利用提供依据与方法
实施例1
枣皮色素的提取
枣皮色素的提取方法参照CN202011315667.2获得,使用了经优化后的复合酶法提取。首先将干燥的枣皮经复合酶(纤维素酶:果胶酶:蛋白酶=4000-6000U:3000-4000U:2000:3000U) 处理后,43℃孵育90min,90℃灭酶5min;加入等体积的0.01-0.08M NaOH后25℃恒温摇床160rpm处理90min后离心去除沉淀调pH至中性。提取所得的枣皮色素粗提物经大孔树脂超声纯化。
样品总黄酮的含量测定
a.总黄酮标准曲线的制作
准确称取吸取芦丁标准品用60%乙醇溶解,配制成0.2mg/mL母液,再用乙醇稀释成 0μg/mL、20μg/mL、40μg/mL、80μg/mL、160μg/mL、320μg/mL、640μg/mL,分别吸取1.5mL 不同浓度芦丁标品溶液,加入5wt.%NaNO2 0.5mL,避光6min,加入10wt.%AlCl3·6H2O0.5mL,避光静置6min,加入4wt.%NaOH溶液5mL,避光6min后用分光光度计在510nm 波长处测定吸光度。以芦丁质量浓度为横坐标,吸光度值为纵坐标,绘制标准曲线。
所得标准曲线的方程式为:y=0.0013x+0.0374,R2=0.998,符合标准曲线的要求。
b.样品总黄酮的测定
准确吸取待测色素溶液1.5mL置于25mL比色管中。然后按a步骤的方法测定吸光度值。再根据标准曲线算出总黄酮含量。
复乳法制备红枣色素多囊脂质体
a.初乳制备
准确称取卵磷脂、胆固醇、三油酸甘油酯,加入二氯甲烷充分溶解,加入0.5mg/mL的红枣皮色素,在16000rpm的剪切条件下乳化150s。
b.多囊脂质体制备
将制备好的初乳倒入40mmol/L L-赖氨酸水溶液中,2000rpm磁力搅拌1min。复乳于35℃中旋蒸7min除去有机溶剂,得多囊脂质体。
c.单因素优化制备条件
固定其他参数不变,依次以:①卵磷脂浓度(10mg/mL、20mg/mL、30mg/mL、40mg/mL);②磷脂与胆固醇质量之比(4:1、2:1、4:3、1:1);③磷脂与三油酸甘油酯质量之比(4:1、2:1、 4:3、1:1);④L-赖氨酸浓度(20mmol/L、30mmol/L、40mmol/L、50mmol/L、60mmol/L);⑤油相与内水相质量之比(1:2、1:1、2:1、3:1);⑥初乳与外水相质量之比(1:1、1:2、1:3、1:4);⑦初乳化转速(10000rpm、13000rpm、16000rpm、19000rpm)。
以包封率表征确定单因素条件,经优化后的多囊脂质体对枣皮色素的包封率可以达到88%以上(图1)。
样品包封率测定
将制备好的脂质体悬液于4℃、2500rpm离心15min后弃去上清液,使用外水相洗涤后再次离心,反复三次后将沉淀分散至1mL外水相中,加入4mL异丙醇溶液破囊,于510nm 处测吸光度值计算脂质体中包封的色素含量,计做W包。取脂质体悬液1mL加入4mL异丙醇破囊后计算悬液中色素含量,计做W总。包封率的计算方法如(1)。
样品渗漏率测定
将制备好的脂质体悬液测定包封率E0,于4℃和室温下保存48h后测定包封率E48h,渗漏率的计算如(2)。
如图2,制备所得的脂质体悬液于4℃和室温下保存时,360h的渗漏率分别为22.92%和 55.60%,脂质体悬液在两种条件下保存时,色素均会发生一定程度的渗漏,而在4℃环境保存时,脂质体的渗漏率较低,且随着时间增大的趋势较缓。
样品释放率测定
取适量制备好的脂质体悬液于离心管中,另加等体积的PBS或生理盐水作为释放介质,离心管置于37℃,100rpm的恒温摇床中,于特定时间后取出离心管2500rpm离心15min后弃去上清液,将沉淀重悬,异丙醇破囊后于510nm处测定脂质体中枣皮色素的含量,计算释放率,释放率计算公式如(3).其中,Q0为0时刻脂质体中枣皮色素的含量,Qt为t时刻脂质体中枣皮色素的含量.
如图3,37℃时,0.5h内脂质体在PBS(pH=7.2)和0.9%NaCl中的释放率分别为7.06%和13.33%,释放率均未达到40%,认为没有发生突释现象。在24h后,释放率分别达到87.34%和82.75%,脂质体能够实现药物的完全释放。
样品的表征
a.光学显微镜
将制备所得的多囊脂质体分别于100×,400×光学显微镜下观察形态结构。(图4)
b.透射电镜
取一滴样品于铜网栅上,4min后用滤纸吸取多余液体。干燥1min后。将干燥后的样品放入透射电镜电镜中观察。(图5)
光学显微镜和透射电镜表明,经优化后的工艺可以制备出多囊脂质体,卵形或者圆形,粒径分布不均,内部有大大小小的囊泡将色素包裹于其中,各囊泡之间由脂质层分隔开。
冻干工艺方法
将制备好的多囊脂质体悬液(MVLs)2mL分别置于10mL的西林瓶中并加入冻干保护剂摇匀。本实验以葡萄糖、海藻糖、乳糖、甘露醇(分别设置5%、10%、15%(w/v)3个浓度)作为冻干保护剂。首先将样品放入-80℃冰箱中预冻24h,设置条件为-55℃和低于100 Pa的真空压强,最终可得到枣皮色素多囊脂质体冻干粉末。
如图6,从冻干样品的外貌观察,发现甘露醇的冻干效果最好,冻干粉末外观饱满,体积及高度基本无塌陷变化。葡萄糖、海藻糖和乳糖冻干样品塌陷及萎缩严重,且黏稠未能够形成细腻粉末。综上,甘露醇作为冻干保护剂形成的样品外观形态最佳。
抗氧化能力测定
a.DPPH清除能力
将色素(溶于水)、空白MVLs、色素MVLs、Vc和普通脂质体配制成5、10、15、20、 30、40μg/mL的浓度,然后将200μmol/L的DPPH·乙醇溶液2mL分别与等体积的色素水溶液、空白MVLs、色素MVLs、Vc溶液以及普通脂质体溶液混合。在室温下避光保存30 min,吸取200μL于96孔板中,利用酶标仪测量混合液在517nm处的吸光度。每组样品设置3 组平行,取平均值。利用公式(4)计算得出样品的DPPH自由基清除效率:
式中A0:蒸馏水、乙醇+DPPH溶液的吸光值
A1:样品溶液+DPPH溶液的吸光值
A2:样品溶液+乙醇溶液的吸光值
如图7:a所示,各样品均对DPPH自由基具有不同的清除效果,以Vc作为阳性对照,抗氧化能力最强。经纯化制备的枣皮色素,其DPPH清除能力呈现剂量依赖的增长趋势,且在40μg/mL时,清除率达到约70%。而普通脂质体及多囊脂质体未表现出明显的清除能力,如图7b显示,多囊脂质体在经24h释放后其清除能力与经同时间的枣皮色素的清除能力几乎一致,认为脂质体较为完好的包裹了枣皮色素,经释放后可以发挥其对DPPH自由基的清除作用。
a.ABTS清除能力
将色素(溶于水)、空白MVLs、色素MVLs和普通脂质体配置成5、10、20、30、40、 60μg/mL的浓度,然后将7mmol/L的ABTS水溶液与2.45mmol/L的K2S2O8等体积混合,避光放置24h后制得ABTS溶液。用去离子水将ABTS溶液稀释至A734nm=0.70±0.02然后将 4mLABTS溶液与100μL的样品混合。避光保存30min,吸取150μL于96孔板中,利用酶标仪测量混合液吸光度值。每组样品设置3组平行,取平均值。利用公式计算得出样品的ABTS 自由基清除效率,计算方法如(5):
式中A0:蒸馏水+ABTS溶液的吸光值
A1:样品溶液+ABTS溶液的吸光值
A2:样品溶液+蒸馏水的吸光值
如图8所示,各样品对ABTS+自由基清除能力具有剂量依赖型增长的趋势,经制备的多囊脂质体清除能力明显高于枣皮色素及普通脂质体,从图中可以看到空白的多囊脂质体仅略低于在色素的多囊脂质体,认为磷脂及中性脂质三油酸甘油酯具有一定的ABTS+自由基清除能力,且发挥了较为关键的协同促进作用。
体外安全性分析
a.细胞毒性试验
本实验通过CCK-8法考察红枣色素、色素MVLs、空白MVLs对L929、HUVEC细胞的毒性。取对数生长期细胞于离心管离心(1000rpm离心5min)后弃去原培养基,再加入适量的新鲜培养基,混匀,计数后将细胞稀释为8×104个/mL,接种于96孔板(80μL/孔)。加药时,分别精密称取不同制剂,设置多个浓度梯度,分别加不同组的红枣色素。实验均对照组、空白组,将96孔板放置在培养箱中培养24小时。随后每孔加10μL(注意避光)CCK-8 溶液,充分振荡,再继续将96孔板放置在培养箱中培养2小时,于酶标仪中在450nm处测定吸光度(A),取平均值后根据公式算出对L929细胞的存活率影响,计算方法如(6)。
式中AS:实验孔(含细胞培养基、cck-8、待测样品)的吸光度;
AC:对照孔(含细胞培养基、cck-8、无待测样品)的吸光度;
AB:空白孔(不含细胞培养基和待测样品、cck-8)的吸光度。
如图9所示,所有浓度的红枣皮色素处理对L929和HUVEC细胞的细胞存活率均高于80%,表明红枣皮色素对细胞没有显著的影响。另外,多囊脂质体在浓度低于500μg/mL时两种细胞的存活率均高于80%,但是当大于500μg/mL时,L929细胞的存活率出现明显降低,而HUVEC细胞则细胞存活率有所上升,结合空白多囊脂质体分析说明膜材对L929细胞产生毒性而对HUVEC则有一定的促进作用。
Claims (6)
1.一种红枣色素纳米颗粒制备方法,其特征在于,将提取得到的枣皮色素溶解于超纯水中,将磷脂、胆固醇、三油酸甘油酯按质量比1:(0.1-1):(0.1-1)溶解于二氯甲烷中,将两相经高速剪切混合制得初乳;将初乳与外水相经涡旋混合后,旋蒸除去有机溶剂,制得红枣色素多囊脂质体,经冷冻干燥后,可以获得红枣色素纳米颗粒。
2.根据权利要求1所述一种红枣色素纳米颗粒制备方法,其特征在于,所述初乳制备条件为:以一定比例的水相与油相,剪切下形成初乳,内水相:油相体积比= 1:(0.1-5),剪切速率为1000-30000rpm,初乳化时间为0-300s;所述初乳:外水相体积比= 1:(0.1-3),复乳化时间为0-130s。
3.根据权利要求2所述一种红枣色素纳米颗粒制备方法,其特征在于,所述多囊脂质所使用的外水相为水溶性成分。
4.根据权利要求3所述一种红枣色素纳米颗粒制备方法,其特征在于,所述水溶性成分为氨基酸、盐或水溶性高分子中的至少一种。
5.根据权利要求4所述一种红枣色素纳米颗粒制备方法,其特征在于,所述氨基酸为L-赖氨酸、色氨酸、丝氨酸中的至少一种。
6.权利要求1-5任一所述制备方法制得的红枣色素纳米颗粒。
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