CN117327039B - 一种可用于治疗急慢性鼻炎的木犀草素的提取方法 - Google Patents
一种可用于治疗急慢性鼻炎的木犀草素的提取方法 Download PDFInfo
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Abstract
本发明属于有效成分提取工艺领域,具体涉及一种可用于治疗急慢性鼻炎的木犀草素的提取方法及应用。方法包括先将野菊花中加入广藿香,混合,然后加溶剂浸泡,提取,得提取液,然后向提取液中加入酶1酶解,得酶解液;再将酶解液中过聚酰胺树脂柱纯化,用水洗涤,加乙酸乙酯‑丙酮溶液洗脱,收集洗脱液,旋干,得粗品;最后将粗品加碱性乙醇溶液,加酶2酶解,加盐酸析晶抽滤即得。本发明提取工艺温和,木犀草素的提取率和纯度均很高,其提取的木犀草素可用于治疗急慢性鼻炎的治疗。
Description
技术领域
本发明属于有效成分提取工艺领域,具体涉及一种可用于治疗急慢性鼻炎的木犀草素的提取方法。
背景技术
木犀草素是一种天然黄酮类化合物,其性质为黄色针状结晶,熔点228~230℃,化学名为3,4,5,7一四羟基黄酮,广泛存在于自然界多种植物当中,富含木犀草素的植物常被用于治疗疾病,具有多种药理作用。化学分析研究表明,等摩尔浓度条件下的木犀草素与槲皮素及辣椒素相比,木犀草素具有最强的抗氧化活性;药理及临床实验的结果还表明木犀草素具有明显的治疗慢性鼻炎的作用,其抗菌、抗病毒作用显著,此外还具有降低血脂和胆固醇等功效。
木犀草素是一种多酚羟基的化合物,由于羟基间存在分子间作用力,晶格能较高,其亲脂亲水性均较差;其特殊的结构导致其在多种溶剂中的溶解性低,对其制备提出了一定的挑战。目前木犀草素的半合成工艺、全合成工艺操作复杂,成本高;从天然植物中提取是木犀草素的最佳获取方式。研究创新有效的提取工艺成为本领域技术人员孜孜不懈的追求。
中国发明专利申请CN105566273A公开了一种玉米须中木犀草素的提取方法,以玉米须为原料,应用微波-超声辅助提取玉米须中木犀草素,该提取技术分为两个阶段:前期采用微波炉微波提取,后期采用超声波清洗器超声波提取。
另一中国发明专利申请CN107382937A公开了一种从木犀草中提取木犀草素的方法,其包括粉碎,提取、过滤,酸水解,离心分离,上中性氧化铝脱色纯化,活性炭脱色、浓缩结晶等步骤。该发明在提取过程中采用超声波结合高压脉冲电场处理和微波结合高压脉冲电场处理,得到的提取液采用酸配合高频电磁波处理进行水解,最后将得到的水解液先后经离心分离、中性氧化铝脱色纯化、活性炭脱色、浓缩结晶、离心、重结晶等工艺,来提高成品木犀草素的提取率和纯度。
现有技术已对木犀草素的提取进行了探索,但是要么提取率和纯度不高,要么工艺复杂,普适性较差,在大生产中较难推广,且木犀草植物在我国种植较少,供货较为有限;从含量较高的本土野菊花、大面积种植的广藿香中来提取木犀草素纯品,现有技术尚未有公开。
发明内容
针对上述不足,本发明提供了一种可用于治疗急慢性鼻炎的木犀草素的提取方法,提取率高,纯度高。
为解决上述技术问题,本发明采用的技术方案为:
一种可用于治疗急慢性鼻炎的木犀草素的提取方法,包括如下步骤:
(1)将野菊花中加入广藿香,混合,然后加溶剂浸泡,提取,得提取液;
(2)向提取液中加入酶1酶解,得酶解液;
(3)将酶解液中过聚酰胺树脂柱纯化,用水洗涤,加乙酸乙酯-丙酮溶液洗脱,收集洗脱液,旋干,得粗品;
(4)将粗品加碱性乙醇溶液,加酶2酶解,加盐酸析晶抽滤即得。
优选地,步骤(1)中所述野菊花和广藿香的质量比为1:1.5-2。
优选地,步骤(1)中所述溶剂为山梨醇、聚乙二醇600酒石酸和水的混合物,所述山梨醇、聚乙二醇600、酒石酸和水的质量比为0.01-0.05:0.01-0.05:0.01-0.03:1。
优选地,步骤(1)中所述提取的温度为60-70℃,提取的时间为0.5-2h,提取的次数为1-3次。
优选地,步骤(2)中所述酶包括质量比为1:1-5的纤维素酶和糖化酶的混合物,所述酶的用量为提取液质量的0.05-0.1%。
优选地,步骤(2)中所述酶解温度为55-70℃,酶解的pH为5.0-7.5,酶解的时间为2-4h。
优选地,步骤(3)中所述聚酰胺树脂的粒径为60-80目;所述乙酸乙酯和丙酮的体积比为1:0.2-0.5,用量为5-8倍柱体积,所述洗脱的流速为1-1.5mL/min。
优选地,步骤(4)中所述碱性乙醇溶液为1-5%的氨水乙醇溶液,所述粗品与碱性乙醇的质量体积比为1:1-2。
优选地,步骤(4)中所述酶2为质量比为1:0.5-1:0.5-1的角蛋白酶、丝氨酸蛋白酶和氨基肽蛋白酶的混合物,所述酶解的时间为2-4h,所述加盐酸后调pH至1.0-1.5。
本发明再一目的是提供上述提取方法提取的木犀草素在制备治疗急慢性鼻炎中的药物中的应用。
本发明还有一个目的是提供上述提取方法在提取木犀草素中的应用。
与现有技术相比,本发明的积极和有益效果在于:
(1)本发明经过特定的溶剂浸泡提取,促进了有效成分的溶出;
(2)进一步采用特定的酶进行酶解,不仅能将木犀草素更完全的提取出来,而纯化后加特定的酶2酶解,更能将木犀草素从与糖类、蛋白质等结合的复合物中分离出来,提高其提取率。
(3)本发明采用复合树脂进行纯化,以及采用特定的溶剂进行复溶和结晶,能明显协同提高木犀草素的提取纯度。
(4)本发明通过广泛的研究,发现广藿香和野菊花共同提取,还能促进木犀草素的提取。
具体实施方式
下面结合具体实施例,对本发明作进一步详细的阐述,下述实施例不用于限制本发明,仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明,实施例中未注明具体条件的实验方法,通常按照常规条件,下述实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到。
其中,纤维素酶供应商为夏盛酶制剂;糖化酶供应商为南宁东恒华道生物科技有限责任公司;
角蛋白酶供应商为山东丰泰科技生物有限公司,丝氨酸蛋白酶供应商为西安达尔闻生物科技有限公司,氨基肽蛋白酶、菠萝蛋白酶、木瓜蛋白酶的供应商为南宁东恒华道生物科技有限责任公司;无花果蛋白酶供应商为
实施例1
本实施例木犀草素的提取方法为:
(1)将野菊花中加入广藿香,二者质量比为1:1,混合,然后加溶剂浸泡30min,在65℃下提取1h,得提取液;
其中,溶剂为山梨醇、聚乙二醇600、酒石酸和水的混合物,所述山梨醇、聚乙二醇、酒石酸和水的质量比为0.02:0.02:0.02:1。
(2)向提取液中加入0.08%酶,在55℃、pH为7.5下酶解2h,得酶解液;
酶为质量比1:3的纤维素酶和糖化酶的混合物。
(3)将酶解液过聚酰胺树脂柱纯化,用水洗涤,然后用5BV体积比为1:0.3的乙酸乙酯-丙酮溶液洗脱,收集洗脱液,旋干,得粗品;
(4)将粗品加1倍的1-5%的氨水乙醇溶液,加质量比为1:0.5:0.5的角蛋白酶、丝氨酸蛋白酶和氨基肽蛋白酶的混合物在30℃、pH为7.0下酶解2h,加盐酸调pH至1.0,搅拌析晶抽滤即得。
实施例2
本实施例木犀草素的提取方法为:
(1)将野菊花中加入广藿香,二者质量比为1:2,混合,然后加溶剂浸泡30min,在70℃下提取2h,得提取液;
其中,溶剂为山梨醇、聚乙二醇600、酒石酸和水的混合物,所述山梨醇、聚乙二醇、酒石酸和水的质量比为0.01:0.01:0.01:1。
(2)向提取液中加入0.05%酶,在55℃、pH为7.5下酶解4h,得酶解液;
酶为质量比1:5的纤维素酶和糖化酶的混合物。
(3)将酶解液过聚酰胺树脂柱纯化,用水洗涤,然后用5BV体积比为1:0.2的乙酸乙酯-丙酮溶液洗脱,收集洗脱液,旋干,得粗品;
(4)将粗品加2倍的5%的氨水乙醇溶液,加质量比为1:0.5:0.5的角蛋白酶、丝氨酸蛋白酶和氨基肽蛋白酶的混合物在30℃、pH7.0下酶解3h,加盐酸调pH至1.0,搅拌析晶抽滤即得。
实施例3
(1)本实施例木犀草素的提取方法为:将野菊花中加入广藿香,二者质量比为1:2,混合,然后加溶剂浸泡30min,在60℃下提取0.5h,提取3次,得提取液;
其中,溶剂为山梨醇、聚乙二醇600、酒石酸和水的混合物,所述山梨醇、聚乙二醇、酒石酸和水的质量比为0.05:0.05:0.03:1。
(2)向提取液中加入0.1%酶,70℃、pH为7.5下酶解2h,得酶解液;
酶为质量比1:1的纤维素酶和糖化酶的混合物。
(3)将酶解液过聚酰胺树脂柱纯化,用水洗涤,然后用5BV体积比为1:0.5的乙酸乙酯-丙酮溶液洗脱,收集洗脱液,旋干,得粗品;
(4)将粗品加1倍的1%的氨水乙醇溶液,加质量比为1:1:1的角蛋白酶、丝氨酸蛋白酶和氨基肽蛋白酶的混合物在30℃下酶解4h,加盐酸调pH至1.5,搅拌析晶抽滤即得。其中角蛋白酶供应商为山东丰泰科技生物有限公司,丝氨酸蛋白酶供应商为西安达尔闻生物科技有限公司,氨基肽蛋白酶的供应商为南宁东恒华道生物科技有限责任公司。
对比例1
本对比例与实施例1的区别在于步骤(1)溶剂不同;其余与实施例1保持一致。
(1)将野菊花中加入广藿香,二者质量比为1:1,混合,然后加溶剂浸泡30min,在65℃下提取1h,得提取液;
其中,溶剂为山梨醇、聚乙二醇600、酒石酸和水的混合物,所述山梨醇、聚乙二醇600、柠檬酸和水的质量比为0.02:0.02:0.02:1。
(2)向提取液中加入0.08%酶,在55℃、pH为7.5下酶解2h,得酶解液;
酶为质量比1:3的纤维素酶和糖化酶的混合物。
(3)将酶解液过聚酰胺树脂柱纯化,用水洗涤,然后用5BV体积比为1:0.3的乙酸乙酯-丙酮溶液洗脱,收集洗脱液,旋干,得粗品;
(4)将粗品加1倍的1-5%的氨水乙醇溶液,加质量比为1:0.5:0.5的角蛋白酶、丝氨酸蛋白酶和氨基肽蛋白酶的混合物在30℃下酶解2h,加盐酸调pH至1.0,搅拌析晶抽滤即得。
对比例2
本对比例与实施例1的区别在于步骤(2)酶不同;其余与实施例1保持一致。
(1)将野菊花中加入广藿香,二者质量比为1:1,混合,然后加溶剂浸泡30min,在65℃下提取1h,得提取液;
其中,溶剂为山梨醇、聚乙二醇600、酒石酸和水的混合物,所述山梨醇、聚乙二醇、酒石酸和水的质量比为0.02:0.02:0.02:1。
(2)向提取液中加入0.08%酶,在55℃、pH为7.5下酶解2h,得酶解液;
酶为质量比1:3的纤维素酶和糖化酶的混合物。
(3)将酶解液过聚酰胺树脂柱纯化,用水洗涤,然后用5BV体积比为1:0.3的乙酸乙酯-丙酮溶液洗脱,收集洗脱液,旋干,得粗品;
(4)将粗品加1倍的1-5%的氨水乙醇溶液,加质量比为1:0.5:0.5的菠萝蛋白酶、木瓜蛋白酶和氨基肽蛋白酶的混合物在30℃下酶解2h,加盐酸调pH至1.0,搅拌析晶抽滤即得。
对比例3
本对比例与实施例1的区别在于步骤(3)纯化步骤不同;其余与实施例1保持一致。
(1)将野菊花中加入广藿香,二者质量比为1:1,混合,然后加溶剂浸泡30min,在65℃下提取1h,得提取液;
其中,溶剂为山梨醇、聚乙二醇600、酒石酸和水的混合物,所述山梨醇、聚乙二醇、酒石酸和水的质量比为0.02:0.02:0.02:1。
(2)向提取液中加入0.08%酶,在55℃、pH为7.5下酶解2h,得酶解液;
酶为质量比1:3的纤维素酶和糖化酶的混合物。
(3)将酶解液过聚酰胺树脂柱纯化,用水洗涤,然后用5BV乙酸乙酯溶液洗脱,收集洗脱液,旋干,得粗品;
(4)将粗品加1倍的1-5%的氨水乙醇溶液,加质量比为1:0.5:0.5的角蛋白酶、丝氨酸蛋白酶和氨基肽蛋白酶的混合物在30℃下酶解2h,加盐酸调pH至1.0,搅拌析晶抽滤即得。
对比例4
本对比例为广藿香的提取,其广藿香的用量与实施例1相同。
(1)将广藿香加溶剂浸泡30min,在65℃下提取1h,得提取液;
其中,溶剂为山梨醇、聚乙二醇600、酒石酸和水的混合物,所述山梨醇、聚乙二醇、酒石酸和水的质量比为0.02:0.02:0.02:1。
(2)向提取液中加入0.08%酶,在55℃、pH为7.5下酶解2h,得酶解液;
酶为质量比1:3的纤维素酶和糖化酶的混合物。
(3)将酶解液过聚酰胺树脂柱纯化,用水洗涤,然后用5BV体积比为1:0.3的乙酸乙酯-丙酮溶液洗脱,收集洗脱液,旋干,得粗品;
(4)将粗品加1倍的1-5%的氨水乙醇溶液,加质量比为1:0.5:0.5的角蛋白酶、丝氨酸蛋白酶和氨基肽蛋白酶的混合物在30℃下酶解2h,加盐酸调pH至1.0,搅拌析晶抽滤即得。
对比例5
本对比例为野菊花的提取,其野菊花的用量与实施例1相同。
(1)将野菊花加溶剂浸泡30min,在65℃下提取1h,得提取液;
其中,溶剂为山梨醇、聚乙二醇600、酒石酸和水的混合物,所述山梨醇、聚乙二醇、酒石酸和水的质量比为0.02:0.02:0.02:1。
(2)向提取液中加入0.08%酶,在55℃、pH为7.5下酶解2h,得酶解液;
酶为质量比1:3的纤维素酶和糖化酶的混合物。
(3)将酶解液过聚酰胺树脂柱纯化,用水洗涤,然后用5BV体积比为1:0.3的乙酸乙酯-丙酮溶液洗脱,收集洗脱液,旋干,得粗品;
(4)将粗品加1倍的1-5%的氨水乙醇溶液,加质量比为1:0.5:0.5的角蛋白酶、丝氨酸蛋白酶和氨基肽蛋白酶的混合物在30℃下酶解2h,加盐酸调pH至1.0,搅拌析晶抽滤即得。
实验一、对本发明提取的木犀草素的提取率和纯度进行检测
采用HPLC测定木犀草素的含量:
供试品溶液的制备:精密称取样品1g,加25mL甲醇超声溶解,过滤、浓缩,定容至5ml的容量瓶中,0.45μm滤膜过滤,得供试品溶液。
对照品溶液的配制:精密称取木犀草素对照品适量,用甲醇溶解,制成木犀草素1μg/mL的对照品溶液,备用。
(1)色谱条件为:
十八烷基硅烷键合硅胶柱为色谱柱;
以乙腈-0.5%磷酸溶液(体积比为50:50)为流动相;
柱温30℃;检测波长285nm;
流速1.0mL/min;进样量20μL;
按外标法计算产品中木犀草素的含量。
提取率%=结晶干燥的粉末重量/广藿香和野菊花的总质量*100%;
纯度%=木犀草素的含量/结晶干燥的粉末重量*100%。
测定本发明实施例1-3、对比例1-5制备的样品中木犀草素的含量,其提取率和纯度结果如下表1所示。
表1
提取率% | 纯度% | |
实施例1 | 0.61 | 98.6 |
实施例2 | 0.63 | 97.2 |
实施例3 | 0.59 | 98.0 |
对比例1 | 0.48 | 90.1 |
对比例2 | 0.38 | 94.5 |
对比例3 | 0.55 | 83.2 |
对比例4 | 0.01 | 97.4 |
对比例5 | 0.52 | 98.5 |
结果表明,本发明对广藿香和野菊花进行共同提取,其提取方法制备的木犀草素的提取率高于单独提取广藿香和野菊花的木犀草素之和,说明二者药材所含的化学成分相互促进,提高了木犀草素的提取率。同时本发明采用二次酶解及特定的溶剂对药材进行提取,将木犀草素从与糖类、蛋白质等结合的复合物中分离出来,进一步提高了木犀草素的提取率及纯度。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (7)
1.一种可用于治疗急慢性鼻炎的木犀草素的提取方法,其特征在于,包括如下步骤:
(1)将野菊花中加入广藿香,混合,然后加溶剂浸泡,提取,得提取液;
(2)向提取液中加入酶1酶解,得酶解液;
(3)将酶解液中过聚酰胺树脂柱纯化,用水洗涤,加乙酸乙酯-丙酮溶液洗脱,收集洗脱液,旋干,得粗品;
(4)将粗品加碱性乙醇溶液,加酶2酶解,加盐酸析晶抽滤即得;
步骤(1)中所述溶剂为山梨醇、聚乙二醇600酒石酸和水的混合物,所述山梨醇、聚乙二醇600、酒石酸和水的质量比为0.01-0.05:0.01-0.05:0.01-0.03:1;
步骤(2)中所述酶为质量比为1:1-5的纤维素酶和糖化酶的混合物;
步骤(4)中所述酶2为质量比为1:0.5-1:0.5-1的角蛋白酶、丝氨酸蛋白酶和氨基肽蛋白酶的混合物;
步骤(1)中所述野菊花和广藿香的质量比为1:1.5-2。
2.根据权利要求1所述的提取方法,其特征在于,步骤(1)中所述提取的温度为60-70℃,提取的时间为0.5-2h,提取的次数为1-3次。
3.根据权利要求1所述的提取方法,其特征在于,步骤(2)中所述酶的用量为提取液质量的0.05-0.1%。
4.根据权利要求1所述的提取方法,其特征在于,步骤(2)中所述酶解温度为55-70℃,酶解的pH为5.0-7.5,酶解的时间为2-4h。
5.根据权利要求1所述的提取方法,其特征在于,步骤(3)中所述聚酰胺树脂的粒径为60-80目;所述乙酸乙酯和丙酮的体积比为1:0.2-0.5,用量为5-8倍柱体积,所述洗脱的流速为1-1.5mL/min。
6.根据权利要求1所述的提取方法,其特征在于,步骤(4)中所述碱性乙醇溶液为1-5%的氨水乙醇溶液,所述粗品与碱性乙醇的质量体积比为1:1-2。
7.根据权利要求1所述的提取方法,其特征在于,步骤(4)中所述酶解的时间为2-4h,所述加盐酸后调pH至1.0-1.5。
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