CN112048028B - 一种光稳定的改性可得然胶抗氧化剂及其制备方法和应用 - Google Patents
一种光稳定的改性可得然胶抗氧化剂及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种光稳定的改性可得然胶抗氧化剂及其制备方法和应用,属于生物质材料技术领域。本发明考虑到了羧甲基可得然胶本身物理特性—反应的空间位阻和水溶性问题,采用EDC/DMAP酯化反应制备改性产品。本发明方法的改性过程中,极大程度地保留了羧甲基可得然胶的羧基,选择先将EGCG琥珀酰化,再与羧甲基可得然胶剩余的羟基反应,制得EGCG改性可得然胶抗氧化剂Na‑CMCUD‑EGCG。本发明方法不仅有利于提高EGCG的接枝率,接枝率达70‑150mg EGCG/g Na‑CMCUD‑EGCG),还有助于产物的水溶性;所得产物具有较强的抗氧化活性和光稳定,可应用于食品工业、医药工业、日化领域中。
Description
技术领域
本发明涉及一种光稳定的改性可得然胶抗氧化剂及其制备方法和应用,属于生物质材料技术领域。
背景技术
可得然胶,又称热凝胶,凝结多糖,是由微生物发酵生产的,以β-1,3-糖苷键构成的水不溶性葡聚糖,相对分子量约为50~200万,来源广泛。可得然胶生物降解性好,具有生物相容性,且无毒无副作用。但可得然胶本生并无明显的生物活性,也不溶于水,这大大限制了它的广泛应用。羧甲基可得然胶是通过对可得然胶进行羧基化改性而得的衍生物,不仅改善了可得然胶水不溶性,还赋予了其更多的生物活性,包括抗氧化活性、抗肿瘤活性、免疫特性等。但这些生物活性提高有限,尤其是抗氧化活性。所以为了能大幅度提升抗氧化活性,扩大它在日用品、医药等方面的应用,开发可得然胶新型抗氧化剂聚合物的工作至关重要。
Cai等人(Cai W D,Zhu J,Wu L X,et al.Preparation,characterization,rheological and antioxidant properties of ferulic acid-grafted curdlanconjugates[J].Food chemistry,2019,300:125221.)制备的阿魏酸接枝可得然胶衍生物采用的是自由基介导的方式,主要研究了其流动性和抗氧化活性(DPPH自由基清除活性和Trolox等效抗氧化能力),未提及铁螯合能力。
发明内容
技术问题:
本发明的目的是:1)通过EGCG的接枝,可得然胶可获得更多方面良好的生物活性,例如强抗氧化活性、抗菌活性、抗癌活性、抑酶作用等,从而可以应用于更多领域;2)增强可得然胶的铁螯合能力,减少活性羟基自由基的堆积(可造成氧化损伤);3)具备较好的光稳定性,能够用于日化、食品和医药领域中。
技术方案:
提供一种制备具有光稳定的改性可得然胶抗氧化剂的方法,所述方法包括如下步骤:
(1)将羧甲基可得然胶钠盐(Na-CMCUD)分散于溶剂中,获得羧甲基可得然胶钠盐溶液;将表没食子儿茶素没食子酸酯衍生物(EGCG)、二酸酐、缩合助剂分散于有机溶剂中,发生缩合反应,反应结束后获得混合液;
(2)在步骤(1)所得的混合液中加入助剂,并将步骤(1)所得的羧甲基可得然胶钠盐溶液滴加到混合液中,进行缩合反应,反应完全后,即得改性可得然胶抗氧化剂(记作Na-CMCUD-EGCG)。
在本发明的一种实施方式中,步骤(1)所述溶剂为DMSO与水的混合溶液。其中,DMSO与水的体积比为(40-80):100(v/v)。进一步优选(40-50):100。
在本发明的一种实施方式中,步骤(1)羧甲基可得然胶钠盐溶液的浓度为8-18mg/mL。进一步优选8-9mg/mL。
在本发明的一种实施方式中,步骤(1)所述缩合助剂为4-二甲氨基吡啶(DMAP)、N,N'-二环己基碳酰亚胺(DCC)、1-羟基苯并三唑(HOBT)。
在本发明的一种实施方式中,步骤(1)所述二酸酐为丁二酸酐。
在本发明的一种实施方式中,步骤(1)所述EGCG、丁二酸酐、缩合助剂的摩尔比为1:2~4:4~6。
在本发明的一种实施方式中,步骤(1)所述EGCG相对有机溶剂的浓度为50-85mg/mL。
在本发明的一种实施方式中,步骤(1)所述有机溶剂为DMSO、吡啶、甲苯。
在本发明的一种实施方式中,步骤(2)所述助剂为1-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC)和4-二甲氨基吡啶(DMAP)。
在本发明的一种实施方式中,步骤(2)所述Na-CMCUD重复单元、EGCG、EDC、DMAP的摩尔比为1:0.8~1.2:1~1.5:0.1~0.3。
在本发明的一种实施方式中,所述Na-CMCUD重复单元的结构如下:
在本发明的一种实施方式中,步骤(2)中缩合反应结束后,利用乙醇进行萃取,并用甲醇洗涤,此过程重复三次,过滤得到的沉淀、干燥,得到粉色固体,即为Na-CMCUD-EGCG。
在本发明的一种实施方式中,干燥是60℃真空干燥箱24h。
在本发明的一种实施方式中,具体的制备过程如下:
(1)提前将一定量的羧甲基可得然胶钠盐(Na-CMCUD)溶于二甲亚砜(DMSO)-水溶液,获得羧甲基可得然胶钠盐溶液,静置备用;取EGCG、丁二酸酐、DMAP混合溶于DMSO纯溶液,在30℃下搅拌24h,获得反应混合液;
(2)向反应混合液中加入1-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC)和4-二甲氨基吡啶(DMAP),搅拌10min;然后将准备好的羧甲基可得然胶钠盐溶液逐滴加入反应混合液中,在30℃下搅拌48h,反应结束;
(3)利用乙醇进行萃取,并用甲醇洗涤,此过程重复三次,过滤得到的沉淀置于60℃真空干燥箱中,干燥24h,得到粉色固体,即为羧甲基可得然胶钠盐-表没食子儿茶素没食子酸酯衍生物,记为Na-CMCUD-EGCG。
本发明利用上述制备方法提供改性可得然胶抗氧化剂。
在本发明的一种实施方式中,所述改性可得然胶抗氧化剂的结构如下所示:
其中,n为300-500。
在本发明的一种实施方式中,所述改性可得然胶抗氧化剂的接枝率为70-150mgEGCG/g。进一步优选90-150mg EGCG/g。
本发明还提供一种改善可得然胶抗氧化剂的抗氧化性的方法,所述方法包括:
(1)将羧甲基可得然胶钠盐(Na-CMCUD)分散于溶剂中,获得羧甲基可得然胶钠盐溶液;将表没食子儿茶素没食子酸酯衍生物(EGCG)、二酸酐、缩合助剂分散于有机溶剂中,发生缩合反应,反应结束后获得混合液;
(2)在步骤(1)所得的混合液中加入助剂,并将步骤(1)所得的羧甲基可得然胶钠盐溶液滴加到混合液中,进行缩合反应。
本发明还将上述改性可得然胶抗氧化剂应用于日化用品制备、食品工业、非疾病诊断和治疗的医药工业领域中。
有益效果:
在本发明中,改性可得然胶抗氧化剂(Na-CMCUD-EGCG)有更强的抗氧化性和光稳定性,Na-CMCUD-EGCG对DPPH自由基的抑制率在浓度为0.2mg/mL时,它的抑制率为91.45%,接近表没食子儿茶素没食子酸酯对DPPH自由基的抑制率;且在浓度为1.0mg/ml时,Na-CMCUD-EGCG的铁螯合能力达到了40%;在平均气温为34℃的正夏光照8小时后,0.2mg/mlNa-CMCUD-EGCG水溶液未发生明显的颜色变化,在化妆品中将会有较好的应用前景。
本发明研究的EGCG接枝羧甲基可得然胶衍生物采用的是化学偶联法,对过程进行了优化,考虑到了羧甲基可得然胶本身物理特性—反应的空间位阻和水溶性问题,采用EDC/DMAP酯化反应。本发明研究的EGCG接枝羧甲基可得然胶衍生物在反应过程中,极大程度地保留了羧甲基可得然胶的羧基,选择先将EGCG琥珀酰化,再与羧甲基可得然胶剩余的羟基反应,这样不仅有利于提高EGCG的接枝率(接枝率:70-150mg EGCG/g Na-CMCUD-EGCG),还有助于产物的水溶性。产物有较强的抗氧化活性,在浓度为0.2mg/mL时对DPPH自由基的抑制率已达91.45%;在浓度为1.0mg/ml时,Na-CMCUD-EGCG的铁螯合能力达到了40%。产物有较强的光稳定性,在平均气温为34℃的正夏光照8小时后,0.2mg/ml Na-CMCUD-EGCG水溶液未发生明显的颜色变化。
附图说明
图1为Na-CMCUD-EGCG抗氧化剂的分子结构式。
图2为实施例1所得Na-CMCUD-EGCG、Na-CMCUD、EGCG的红外光谱图;Na-CMCUD(a)、Na-CMCUD-EGCG(b)、EGCG(c)的红外光谱图。
图3为实施例1所得Na-CMCUD-EGCG、EGCG的DPPH自由基清除率图。
图4为实施例1所得Na-CMCUD-EGCG的铁螯合能力。
图5为0.2mg/ml EGCG(A)、实施例1所得Na-CMCUD-EGCG(B)水溶液的颜色变化。
具体实施方式
DPPH自由基的抑制率测定:配置0.05mmol/L DPPH甲醇溶液,0.2-1.0mg/ml浓度梯度(0.2mg/ml、0.4mg/ml、0.6mg/ml、0.8mg/ml、1.0mg/ml)的样品水溶液;取3ml DPPH溶液和200μl各样品溶液充分混合2min,避光保存10min后,在518nm处测定吸光度。计算公式:DPPH自由基清除率(%)=(Abs0-Abs1)/Abs0×100;
其中,Abs1、Abs0分别代表含样品时DPPH溶液的吸光度和空白的DPPH溶液的吸光度。
金属螯合作用测定:配置0.2-1.0mg/ml梯度浓度(0.2mg/ml、0.4mg/ml、0.6mg/ml、0.8mg/ml、1.0mg/ml)的样品水溶液,取1ml上清液,依次加入1.35ml去离子水,50μL 2mmol/L FeCl2,200μL 5mmol/L菲啰嗪,混匀,在室温下放置10min后,在562nm处测试吸光度,铁螯合能力的计算公式:螯合能力(%)=(Abs0-Abs1)/Abs0×100;
其中,Abs1、Abs0分别指代含样品时混合溶液的吸光度和空白溶液的吸光度。
实施例1:
提前将0.43g Na-CMCUD(DS=0.99,以重复单元结构计,为1.78mmol)溶于26ml50%DMSO溶液,静置备用;取0.815g EGCG、0.53g丁二酸酐、0.98g DMAP混合溶于10ml DMSO纯溶液,在30℃下搅拌24h;再向反应体系中加入0.41g EDC(3ml 50%DMSO)和0.044g DMAP(3ml 50%DMSO),等待10min后,将提前准备的Na-CMCUD溶液逐滴加入其中,在30℃下搅拌48h,反应结束。将得到的反应溶液倒入乙醇中萃取,并用甲醇洗涤,此过程重复三次,过滤得到的沉淀置于60℃真空干燥箱中,干燥24h,得到粉色固体,即为EGCG-羧甲基可得然胶钠盐,记为Na-CMCUD-EGCG。
图1为Na-CMCUD-EGCG衍生物的分子结构式。各衍生物中EGCG接枝率的测定采用Folin-Ciocalteu法(Turkmen N,Sari F,Velioglu Y S.Effects of extractionsolvents on concentration and antioxidant activity of black and black matetea polyphenols determined by ferrous tartrate and Folin–Ciocalteu methods[J].Food chemistry,2006,99(4):835-841.)。具体操作如下:将1ml样品水溶液稀释到适当浓度,加入1ml稀释3倍之后的Folin-Ciocalteu试剂,加入2ml 28%(w/v)碳酸钠溶液,充分混匀2min,再加入2ml去离子水,测试总体积为6ml,静置30min后,在760nm处测试吸光度。本发明以EGCG水溶液(浓度范围是0.005至0.05mg/ml)为标样,得到接枝率的计算公式:y=17.05x+0.01583(R2=0.9922),其中,x代表了接枝的EGCG浓度(mg/mL),y代表了混合溶液在760nm处的吸光度,聚合物的接枝率由每克样品中所含EGCG毫克数当量表示。本发明的接枝率为130~140mg EGCG/g Na-CMCUD-EGCG。
图2为Na-CMCUD、Na-CMCUD-EGCG、EGCG的红外光谱图。由图可知,Na-CMCUD的红外谱图中有很明显的-COOH的不对称伸缩振动峰和对称伸缩振动峰,分别位于1587cm-1、1419cm-1处。与Na-CMCUD相比,Na-CMCUD-EGCG的谱图中出现了几处明显的EGCG特征峰,包括位于1650cm-1、1592cm-1、1214cm-1和1145cm-1处的吸收峰,分别对应于酯C=O的伸缩振动、芳香族C=C的伸缩振动、酚C-OH的伸缩振动和醇C-OH的伸缩振动。另外,从Na-CMCUD-EGCG的红外谱图中还发现羧基COO-的非对称伸缩振动峰受芳香族C=C的影响,从原来的1587cm-1(Na-CMCUD)处红移至1562cm-1(Na-CMCUD-EGCG)处。由此,可说明Na-CMCUD-EGCG已接枝成功。
图3显示了EGCG、Na-CMCUD-EGCG对DPPH自由基抑制趋势。由图可知,EGCG表现出最好的DPPH自由基抑制效果,在浓度为0.06mg/mL时,就已达到94.34%的抑制率。Na-CMCUD-EGCG对DPPH自由基的抑制率与浓度相关,浓度越高,抑制效果就越好,且Na-CMCUD-EGCG在浓度为0.2mg/mL时,对DPPH自由基的抑制率已经与EGCG相似,达到了91.45%。
图4显示了Na-CMCUD-EGCG的铁螯合能力情况。由图4可知,Na-CMCUD-EGCG的铁螯合能力与测试浓度相关,浓度越高,它的铁螯合能力越好,在浓度为1.0mg/ml时,铁螯合能力显示为40%。
图5显示了0.2mg/ml EGCG(A)、Na-CMCUD-EGCG(B)水溶液经光照8小时后的颜色变化。该实验的实验参数如下:夏天,平均气温为34℃。由图可知,EGCG水溶液已经由无色变为黄色,而Na-CMCUD-EGCG水溶液的颜色无明显变化。可见,所得Na-CMCUD-EGCG具有较好的光稳定性。
实施例2:
提前将0.43g Na-CMCUD(DS=0.99,以重复单元结构计,为1.78mmol)溶于26ml50%DMSO溶液,静置备用;取0.815g EGCG、0.53g丁二酸酐、0.98g DMAP混合溶于10ml DMSO纯溶液,在30℃下搅拌24h;再向反应体系中加入0.41g EDC(3ml 50%DMSO)和0.044g DMAP(3ml 50%DMSO),等待10min后,将提前准备的Na-CMCUD溶液逐滴加入其中,在30℃下搅拌24h,反应结束。将得到的反应溶液倒入乙醇中萃取,并用甲醇洗涤,此过程重复三次,过滤得到的沉淀置于60℃真空干燥箱中,干燥24h,得到粉色固体,即为EGCG-羧甲基可得然胶钠盐,该衍生物的接枝率为95~110mg EGCG/g Na-CMCUD-EGCG。
DPPH自由基抑制清除率:在浓度为0.2mg/mL时,对DPPH自由基的抑制率达到了80.23%。
铁螯合能力:在浓度为1.0mg/mL时,铁螯合能力达到了27.38%。
光稳定性:光照8小时后,Na-CMCUD-EGCG水溶液开始出现变色趋势的最低浓度为0.4mg/mL。
实施例3:
提前将0.40g Na-CMCUD(DS=0.78,以重复单元结构计,为1.65mmol)溶于26ml50%DMSO溶液,静置备用;取0.815g EGCG、0.53g丁二酸酐、0.98g DMAP混合溶于10ml DMSO纯溶液,在30℃下搅拌24h;再向反应体系中加入0.41g EDC(3ml 50%DMSO)和0.044g DMAP(3ml 50%DMSO),等待10min后,将提前准备的Na-CMCUD溶液逐滴加入其中,在30℃下搅拌48h,反应结束。将得到的反应溶液倒入乙醇中萃取,并用甲醇洗涤,此过程重复三次,过滤得到的沉淀置于60℃真空干燥箱中,干燥24h,得到棕粉色固体,即为EGCG-羧甲基可得然胶钠盐,该衍生物的接枝率为90~110mg EGCG/g Na-CMCUD-EGCG。
DPPH自由基抑制清除率:在浓度为0.2mg/mL时,对DPPH自由基的抑制率达到了71.47%。
铁螯合能力:在浓度为1.0mg/mL时,铁螯合能力达到了24.60%。
光稳定性:光照8小时后,Na-CMCUD-EGCG水溶液开始出现变色趋势的最低浓度为0.4mg/mL。
实施例4:
提前将0.43g Na-CMCUD(DS=0.52,以重复单元结构计,为1.78mmol)溶于26ml70%DMSO溶液,静置备用;取0.815g EGCG、0.53g丁二酸酐、0.98g DMAP混合溶于10ml DMSO纯溶液,在30℃下搅拌24h;再向反应体系中加入0.41g EDC(3ml 50%DMSO)和0.044g DMAP(3ml 50%DMSO),等待10min后,将提前准备的Na-CMCUD溶液逐滴加入其中,在30℃下搅拌48h,反应结束。将得到的反应溶液倒入乙醇中萃取,并用甲醇洗涤,此过程重复三次,过滤得到的沉淀置于60℃真空干燥箱中,干燥24h,得到棕色固体,即为EGCG-羧甲基可得然胶钠盐,该衍生物的接枝率为70~80mg EGCG/g Na-CMCUD-EGCG。
DPPH自由基抑制清除率:在浓度为0.2mg/mL时,对DPPH自由基的抑制率达到了28.25%。
铁螯合能力:在浓度为1.0mg/mL时,铁螯合能力达到了15.63%。
光稳定性:光照8小时后,Na-CMCUD-EGCG水溶液开始出现变色趋势的最低浓度为0.6mg/mL。
实施例5:
参照实施例1的方法,仅仅是改变羧甲基可得然胶钠盐溶液的浓度:
提前将0.43g Na-CMCUD(DS=0.99,以重复单元结构计,为1.78mmol)溶于50ml50%DMSO溶液,静置备用;
取0.815g EGCG、0.53g丁二酸酐、0.98g DMAP混合溶于10ml DMSO纯溶液,在30℃下搅拌24h;再向反应体系中加入0.41g EDC(3ml 50%DMSO)和0.044g DMAP(3ml 50%DMSO),等待10min后,将提前准备的Na-CMCUD溶液逐滴加入其中,在30℃下搅拌48h,反应结束。将得到的反应溶液倒入乙醇中萃取,并用甲醇洗涤,此过程重复三次,过滤得到的沉淀置于60℃真空干燥箱中,干燥24h,得到粉色固体,即为EGCG-羧甲基可得然胶钠盐,该衍生物的接枝率为110~120mg EGCG/g Na-CMCUD-EGCG。
DPPH自由基抑制清除率:在浓度为0.2mg/mL时,对DPPH自由基的抑制率达到了84.25%。铁螯合能力:在浓度为1.0mg/mL时,铁螯合能力达到了33.47%。光稳定性:光照8小时后,Na-CMCUD-EGCG水溶液开始出现变色趋势的最低浓度为0.3mg/mL。若将酸酐化的EGCG替换为阿魏酸,制得阿魏酸-羧甲基可得然胶钠盐,该抗氧化剂对DPPH自由基的抑制率约为67%。
Claims (10)
1.一种制备具有光稳定的改性可得然胶抗氧化剂的方法,其特征在于,所述方法包括如下步骤:
(1)将羧甲基可得然胶钠盐分散于溶剂中,获得羧甲基可得然胶钠盐溶液;将表没食子儿茶素没食子酸酯衍生物、二酸酐、缩合助剂分散于有机溶剂中,发生缩合反应,反应结束后获得混合液;
(2)在步骤(1)所得的混合液中加入助剂,并将步骤(1)所得的羧甲基可得然胶钠盐溶液滴加到混合液中,进行缩合反应,反应完全后,即得改性可得然胶抗氧化剂。
2.根据权利要求1所述的方法,其特征在于,步骤(1)所述表没食子儿茶素没食子酸酯衍生物、二酸酐、缩合助剂的摩尔比为1:2~4:4~6。
3.根据权利要求1所述的方法,其特征在于,步骤(2)所述助剂为1-(3-二甲基氨基丙基)碳二亚胺盐酸盐和4-二甲氨基吡啶。
4.根据权利要求3所述的方法,其特征在于,羧甲基可得然胶钠盐的单元分子、表没食子儿茶素没食子酸酯衍生物、1-(3-二甲基氨基丙基)碳二亚胺盐酸盐和4-二甲氨基吡啶的摩尔比为1:0.8~1.2:1~1.5:0.1~0.3。
5.根据权利要求1所述的方法,其特征在于,步骤(1)所述溶剂为DMSO与水的混合溶液;其中,DMSO与水的体积比为(40-80):100。
6.根据权利要求1-5任一项所述的方法,其特征在于,步骤(2)中缩合反应结束后,利用乙醇进行萃取,并用甲醇洗涤,此过程重复三次,过滤得到的沉淀、干燥,得到粉色固体。
7.权利要求1-6任一项所述的方法制得的改性可得然胶抗氧化剂。
9.一种改善可得然胶抗氧化剂的抗氧化性的方法,其特征在于,所述方法包括:
(1)将羧甲基可得然胶钠盐分散于溶剂中,获得羧甲基可得然胶钠盐溶液;将表没食子儿茶素没食子酸酯衍生物、二酸酐、缩合助剂分散于有机溶剂中,发生缩合反应,反应结束后获得混合液;
(2)在步骤(1)所得的混合液中加入助剂,并将步骤(1)所得的羧甲基可得然胶钠盐溶液滴加到混合液中,进行缩合反应。
10.权利要求7-8任一项所述的改性可得然胶抗氧化剂在食品工业、医药工业、日化领域中的应用。
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CN104784197A (zh) * | 2015-03-30 | 2015-07-22 | 福州乾正药业有限公司 | EGCG与β-葡聚糖的组合物及其制备方法和药物应用 |
CN106188574B (zh) * | 2016-07-18 | 2018-11-20 | 上海交通大学 | 一种羧甲基可得然胶水溶液或水凝胶及其制备方法和应用 |
CN106519060A (zh) * | 2016-09-22 | 2017-03-22 | 江南大学 | 一种羧甲基可得然胶的制备 |
US10993917B2 (en) * | 2016-12-30 | 2021-05-04 | Agency For Science, Technology And Research | Nanocomplex |
CN107987181A (zh) * | 2017-11-03 | 2018-05-04 | 江南大学 | 一种羟丙基可得然胶的制备方法及应用 |
CN108498486B (zh) * | 2018-07-02 | 2020-04-28 | 天津大学 | 一种bsa-egcg-tps纳米颗粒的制备方法 |
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