CN113244289B - 香樟果花色苷的提取方法 - Google Patents
香樟果花色苷的提取方法 Download PDFInfo
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Abstract
本发明涉及化工领域，公开了一种香樟果花色苷的提取方法，采集的香樟果原料清洗，去核，冷冻干燥，粉碎，过筛处理得香樟果粉；采用乙酸乙酯按一定料液比对香樟果粉进行浸提脱脂处理，得脱脂香樟果粉；将脱脂香樟果粉与低共熔溶剂按照一定料液比进行混合，加水稀释，经磁力充分振荡提取；将所得溶液置于超高压条件下进行超高压处理；向所得溶液中加入一定比例的反萃取溶液，离心取上清液，得香樟果花色苷粗提液；将樟果花色苷粗提液经大孔树脂和葡聚糖凝胶洗脱，得香樟果花色苷精提液。经脱脂和低共熔溶剂反复萃取配合超高压处理带来的优越破壁效果，三者结合下大幅度提高了香樟果花色苷的提取效率。
Description
技术领域
本发明涉及植物花色苷的高效提取领域，特别涉及一种香樟果花色苷的提取方法。
背景技术
花色苷是广泛存在于自然界的天然水溶性色素，存在于植物的花、果实、茎、叶和根器官的细胞液中，使其呈现由黄、红、紫、蓝、黑等不同颜色，具有抗氧化、保护心血管、改善视力、维持肠道菌群多样性等功效，并可显著增强机体免疫力，提升机体应对病原菌和病毒侵袭时的抗病能力，保障身体健康。花色苷来源广泛且储量丰富，其中植物是天然花色苷的主要来源，我国植物种类数目位居世界第三，为花色苷的提取应用提供了多样且丰富的来源。香樟果是香樟树的果实，其中含有丰富的花色苷类物质，香樟树种植广泛、产果量高、结实年限长，香樟果可以进一步加以利用，使其成为可用资源。同时由于花色苷提取效率低及其自身性质不稳定等严重限制了香樟果花色苷在食品、医疗、保健等行业的开发和利用。若能对花色苷的提取制备达到工业化生产规模，必将推动其在人类健康事业中的广泛应用。当前，多种花色苷的提取方法已经较为成熟，已在不同花色苷的提取中得到一定的应用。因此，花色苷在生物、医药、食品等行业均具有广阔的发展应用前景。
花色苷是含有两个芳香环和一个杂环(C6-C3-C6)结构的花青素糖苷衍生物，基本结构为2-苯基苯并吡喃阳离子，母环上不同侧链结构会形成不同的花色苷，花色苷结构上的差异决定了其生物学性状和功能的多样性。花色苷最基础的生物活性为抗氧化性，在对人体有益的大多数生理功能中，强大的抗氧化能力是发挥其他生理功能，如清除自由基、抗癌、抗心血管疾病的基础。在体外实验中花色苷表现出能促使癌细胞凋亡和显著抑制前列腺癌细胞的增殖的生理作用，另有研究表明花色苷对食道癌、肺癌、皮肤癌、结肠癌等也有一定的预防和治疗效果；花色苷还在防治心血管疾病方面也有显著疗效。此外，花色苷在保护肝脏、保护神经、改善记忆力、抗辐射损伤、抗血栓等方面也有积极效果。
低共熔溶剂(Deep-eutectic solvent，DES)是通过氢键受体(Hydrogen-bondacceptor，HBA)和氢键供体(Hydrogen-bond donor，HBD)的氢键作用而形成的一种熔点比其任一组分的熔点都要低，在室温状态下常呈现液体状态的一种混合物，低共熔溶剂的合成原料均为常见化合物，来源丰富且价格低廉，制备过程仅需将几种化合物混合并于一定温度下加热搅拌直至形成均一的无色液体即可，易于制备及提取使用。
研究发现，低共熔溶剂挥发性低、化学性质稳定、热稳性高、溶解能力强，在天然产物提取中具有良好的实验效果。低共熔溶剂的原料有很多种，一般为胆碱类化合物作为氢键受体，金属盐及有机酸作为氢键供体，其组成成分有些为人体内的代谢中间体或产物，或者为人体所必须的维生素，而氢键供体大多为对人体无害的有机酸或者其他化合物，本身对人体无害且便于自然界微生物的降解。所以，低共熔溶剂还展现出了无毒、制备简单、价格低廉、可生物降解等突出特点，这使得低共熔溶剂在绿色化学领域备受青睐。
低共熔溶剂的组成成分决定了其性能，通过选择合适的氢键给体和氢键供体以及不同的配比结合，可以实现较大范围内调节低共熔溶剂的物化性质。低共熔溶剂在天然产物提取领域相比常见的溶剂提取法在提取效率和提取效果方面展现出明显优势，然而比提高提取效率更重要的是，获得的提取物可以保持其生物活性并且对人类无害，成为食品、生物、催化等领域开发和利用天然产物的又一发展方向。
超高压辅助提取技术(Ultra-high pressure-assisted extraction，UHP)是利用水或其它流体为媒介，将样品在100MPa以上压力下保持一定时间，促使细胞壁结构破坏、目标产物充分释放的一种新型提取技术。与其它提取技术相比，超高压处理时提供给物料的能量相对较低，一般只破坏对生物大分子立体结构有贡献的氢键、离子键、疏水键等非共价键，而对共价键没有影响，具有能耗低、效率高、对生物活性物质影响小等优点，目前已被应用于多酚、多糖、黄酮等物质的提取，但是超高压辅助提取香樟果花色苷的研究仍鲜见报道。
发明内容
发明目的：针对现有技术中存在的问题，本发明提供一种香樟果花色苷的提取方法，经脱脂和低共熔溶剂反复萃取配合超高压处理带来的优越破壁效果，三者结合下大幅度提高了香樟果花色苷的提取效率。
技术方案：本发明提供了一种香樟果花色苷的提取方法，包括以下步骤：
S1：采集的香樟果原料清洗，去核，冷冻干燥，粉碎，过筛处理得香樟果粉；
S2：采用乙酸乙酯按1:1-1:3的料液比对香樟果粉进行浸提脱脂处理，得脱脂香樟果粉；
S3：将所述脱脂香樟果粉与低共熔溶剂按照1:20～30的料液比进行混合，加水稀释，经磁力充分振荡，在30-40℃下提取30-50min；
S4：将S3所得溶液置于压力为100-500MPa的超高压条件下进行超高压处理1-10min；
S5：向S4所得溶液中加入一定比例的反萃取溶液，离心取上清液，得香樟果花色苷粗提液；
S6：将樟果花色苷粗提液经大孔树脂和葡聚糖凝胶洗脱，得香樟果花色苷精提液。
优选地，在所述S3中，所述低共熔溶剂为摩尔比为1:1-1:3的氢给体和氢受体的混合物；
所述氢供体为氯化胆碱，所述氢受体为乳酸、尿素、丙二醇、丁二醇或乙酸。
优选地，在所述S3中，所述加水稀释是采用去离子水按照10％-40％的比例添加。
优选地，在所述S4中，超高压处理的具体工艺为：从大气压经2～5min升高至100-500MPa，保压1～3min，然后经1～3S泄压至大气压。
优选地，在所述S5中，所述反萃取溶液的添加比例为1:0.5-1:3。
优选地，所述S5中，反萃取溶液为酸性无水乙醇；
优选地，在所述的S1中，冷冻干燥的具体步骤为：先将香樟果原料在-80℃下冷冻24h，再用冷冻干燥机干燥24-36h。
优选地，在所述的S1中，过筛过程采用60-100目筛进行。
优选地，在所述的S6中，所述大孔树脂为D4020，大孔树脂洗脱时的洗脱剂为pH3.0的80％酸性乙醇。
优选地，在所述的S6中，所述葡聚糖凝胶为LH-20。
有益效果：香樟果花色苷为一类存在于细胞内的天然水溶性色素，在物料中加入脱脂试剂，由于花色苷不溶于脂类物质，因此可以将富含花色苷物料中的脂类和易溶于脂类中的黄酮类化合物分离脱除，本身花色苷属于类黄酮化合物，有着较相似的理化性质，通过脱脂能去除这一类杂质，有利于提取的花色苷具有较高纯度，而且脱脂试剂能使得富含花色苷的物料变得松散，有利于花色苷类物质溶出。在提取过程中采用优选的低共熔溶剂对香樟果花色苷进行提取，可以利用优选的低共熔溶剂一方面对物料细胞中的纤维基质进行一定程度的溶解，使更多的花色苷类物质与细胞基质分离并溶解于低共熔溶剂中，另一方面，低共熔溶剂通过分子之间强作用力直接萃取物料中的花色苷类物质。通过低共熔溶剂的提取可以将大部分花色苷类成分萃取出来。但是基质中的花色苷不能完全萃取出来，采用低共熔溶剂反复萃取，可以得到较高的萃取得率，在此基础上，通过利用超高压设备对提取溶液进行处理，在100-500MPa的超高压处理下，经脱脂和低共熔溶剂反复萃取之后的物料性质会发生进一步变化，物料结构进一步皱缩塌陷，花色苷穿出细胞壁或细胞中的其他结构，溶剂成分可以更好地与花色苷类物质接触，从而使物料中的花色苷能最大限度的溶出，最终达到较高的花色苷提取率，提取后物料基本已无颜色。
超高压提取过程压力对提取效率的影响可以从以下不同阶段加以说明。升压阶段：压力在几分钟内(一般小于5min)迅速由常压升为几百兆帕，固体组织细胞内外形成了超高的压力差，提取溶剂在超高压力推动下迅速渗透到植物内部维管束和腺细胞内。随着压力迅速升高，细胞体积被压缩，如果超过其形变极限，会导致细胞破裂，细胞内的物质与溶剂接触被溶解；如果没有超过细胞的形变极限，提取溶剂在高压作用下，进入植物细胞内，有效成分溶解在提取溶剂中。保压阶段：超高压力引起体系的体积变化，推动了化学平衡的移动，溶剂的渗透、溶质的溶解快速达到平衡。因此保压阶段时间很短，一般几分钟之内即可完成。卸压阶段：卸压一般在几秒钟之内即可完成(一般卸压时间＜2s)，组织细胞的压力从几百兆帕的超高压迅速减小为常压，在反方向压力作用下，发生流体以及药物基质体积的爆破膨胀，对细胞壁、细胞膜、质膜、核膜、液泡、微管等形成强烈的冲击致使发生变形。如果变形超过了其变形极限，导致细胞结构出现松散、孔洞、破裂等结构变化，有效成分和溶剂充分接触，溶解了有效成分的溶液会向细胞外迅速扩散；如果在反方向压力作用下细胞壁的变形没有超过其变形极限(在高压作用下通透性增大)，细胞内部已经溶解了有效成分的溶剂在高渗透压差下快速转移到细胞外，达到提取的目的。在流体吸收外界施加的压缩能一定(压力一定)的情况下，卸压时间越短，细胞内流体在向外扩散的同时产生的冲击力越强，引起的湍动效应越强烈，形成的孔洞、碎片越多，一定质量的目的产物基质的有效比表面会越大，有效成分扩散的传质阻力就会越小、与溶剂接触也就会更充分，提取效率会更高。
因此，经脱脂和低共熔溶剂反复萃取配合超高压处理带来的优越破壁效果，三者结合下大幅度提高了香樟果花色苷的提取效率。
具体实施方式
下面结合具体实施例对本发明进行详细的介绍。
实施方式1：
采集的香樟果原料清洗，去核，-80℃下冷冻24h，再用冷冻干燥机干燥25h，机械粉碎，过60目筛得香樟果粉；采用乙酸乙酯按1:2的料液比对香樟果粉进行浸提脱脂处理，得脱脂香樟果粉；阴干后准确称取脱脂香樟果粉末1.00g与30mL摩尔比为1:2的氯化胆碱-乙酸型低共熔溶剂进行混合，按照20％的比例加水稀释后，磁力充分震荡，按提取温度40℃、提取40min。再将所得溶液在密闭容器中从大气压经3min升压至300Mpa，然后在此超高压条件下保压10min后，经2s泄压至大气压。超高压处理结束后加入2倍体积的反萃取溶液酸性无水乙醇，离心取上清液，即得到香樟果花色苷的粗提液；最后经D4020大孔树脂和LH-20葡聚糖凝胶洗脱，洗脱剂为pH 3.0的80％酸性乙醇，即可得到香樟花色苷精提液。
实施方式2：
本实施方式与实施方式1大致相同，不同点仅在于，采用乙酸乙酯按1:3的料液比对香樟果粉进行浸提脱脂处理。除此之外，本实施方式与实施方式1完全相同，此处不做赘述。
实施方式3：
本实施方式与实施方式2大致相同，不同点仅在于，阴干后准确称取脱脂香樟果粉末1.00g与20mL摩尔比为1:2的氯化胆碱-乙酸型低共熔溶剂进行混合。除此之外，本实施方式与实施方式2完全相同，此处不做赘述。
实施方式4：
本实施方式与实施方式3大致相同，不同点仅在于，阴干后准确称取脱脂香樟果粉末1.00g与20mL摩尔比为1:3的氯化胆碱-乙酸型低共熔溶剂进行混合。除此之外，本实施方式与实施方式3完全相同，此处不做赘述。
实施方式5：
本实施方式与实施方式4大致相同，不同点仅在于，将所得溶液在密闭容器中从大气压经3min升压至400Mpa，然后在此超高压条件下保压10min后，经2s泄压至大气压。除此之外，本实施方式与实施方式4完全相同，此处不做赘述。
实施方式6：
本实施方式与实施方式5大致相同，不同点仅在于，将所得溶液在密闭容器中从大气压经3min升压至300Mpa，然后在此超高压条件下保压5min后，经2s泄压至大气压。除此之外，本实施方式与实施方式5完全相同，此处不做赘述。
实施方式7：
本实施方式与实施方式6大致相同，不同点仅在于，提取温度为30℃。除此之外，本实施方式与实施方式6完全相同，此处不做赘述。
实施方式8：
本实施方式与实施方式7大致相同，不同点仅在于，提取时间为30min。除此之外，本实施方式与实施方式7完全相同，此处不做赘述。
实施方式1-8的实验结果
上述实施方式只为说明本发明的技术构思及特点，其目的在于让熟悉此项技术的人能够了解本发明的内容并据以实施，并不能以此限制本发明的保护范围。凡根据本发明精神实质所做的等效变换或修饰，都应涵盖在本发明的保护范围之内。
Claims (8)
1.一种香樟果花色苷的提取方法，其特征在于包括以下步骤：
S1：采集的香樟果原料清洗，去核，冷冻干燥，粉碎，过筛处理得香樟果粉；
S2：采用乙酸乙酯按1:1-1:3的料液比对香樟果粉进行浸提脱脂处理，得脱脂香樟果粉；
S3：将所述脱脂香樟果粉与低共熔溶剂按照1:20~30的料液比进行混合，加水稀释，经磁力充分振荡，在30-40 ℃下提取30-50 min；所述低共熔溶剂为氢受体氯化胆碱与氢供体为乙酸的混合物；所述氯化胆碱与所述乙酸的摩尔比为1:2-1:3；
S4：将S3所得溶液置于压力为100-500 MPa的超高压条件下进行超高压处理1-10 min；
S5：向S4所得溶液中加入一定比例的反萃取溶液酸性无水乙醇，离心取上清液，得香樟果花色苷粗提液；
S6：将香樟果花色苷粗提液经大孔树脂和葡聚糖凝胶洗脱，得香樟果花色苷精提液。
2.根据权利要求1所述的香樟果花色苷的提取方法，其特征在于，在所述S3中，所述加水稀释是采用去离子水按照10%-40%的比例添加。
3.根据权利要求1所述的香樟果花色苷的提取方法，其特征在于，在所述S4中，超高压处理的具体工艺为：
从大气压经2~5min升高至100-500 MPa，保压1~10 min，然后经1~3 S泄压至大气压。
4.根据权利要求1所述的香樟果花色苷的提取方法，其特征在于，在所述S5中，所述反萃取溶液的添加比例为1:0.5-1:3。
5.根据权利要求1至4中任一项所述的香樟果花色苷的提取方法，其特征在于，在所述的S1中，冷冻干燥的具体步骤为：先将香樟果原料在-80 ℃下冷冻24 h，再用冷冻干燥机干燥24-36 h。
6.根据权利要求1至4中任一项所述的香樟果花色苷的提取方法，其特征在于，在所述的S1中，过筛过程采用60-100目筛进行。
7.根据权利要求1至4中任一项所述的香樟果花色苷的提取方法，其特征在于，在所述的S6中，所述大孔树脂为D4020，大孔树脂洗脱时的洗脱剂为pH为3.0的80%酸性乙醇。
8.根据权利要求1至4中任一项所述的香樟果花色苷的提取方法，其特征在于，在所述的S6中，所述葡聚糖凝胶为LH-20。
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| 昝丽霞.香樟果皮花色苷提取工艺优化及抗氧化研究.《广西植物》.2015,第35卷(第4期),第603-608页. * |
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Application publication date: 20210813 Assignee: Earl Food (Guangzhou) Co.,Ltd. Assignor: HUAIYIN INSTITUTE OF TECHNOLOGY Contract record no.: X2023980052900 Denomination of invention: Extraction method of anthocyanins from camphor fruit Granted publication date: 20220517 License type: Common License Record date: 20231219 |