CN111840339A - 一种从路路通中提取混合果酸和抑菌精油的方法 - Google Patents
一种从路路通中提取混合果酸和抑菌精油的方法 Download PDFInfo
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Abstract
本发明属于中药提取技术领域,具体涉及一种从路路通中提取混合果酸和抑菌精油的方法;采用前处理、一次提取、梯度洗脱、二次提取、纯化的方法对路路通进行提取,可同时从中提取到熊果酸、齐墩果酸和具有抑菌效果的挥发油,提取方法简单,尽可能的减少了浪费,同时,所提取的抑菌精油对大肠杆菌和枯草芽孢杆菌的抑菌圈直径分别达到6.1mm和14.7mm,熊果酸和齐墩果酸的纯度分别达到95.3%和92.7%。
Description
技术领域
本发明属于中药提取技术领域,具体涉及一种从路路通中提取混合果酸和抑菌精油的方法。
背景技术
路路通,是金缕梅科植物枫香树的干燥成熟果序,是一种传统中药材,有祛风活络,利水,通经的作用,用于关节痹痛,麻木痉挛,水肿胀满,乳少,经闭。
熊果酸是一种五环三萜类化合物,具有镇静、抗炎、抗菌、抗糖尿病、降低血糖等多种生物学效应,同时还具有明显的抗氧化功能。齐墩果酸是从龙胆科植物獐牙菜属的青叶胆全草或女贞子的果实中分离提取而得的一种五环三萜类化合物。目前采用路路通进行熊果酸和齐墩果酸的提取还比较少见,对路路通的提取液多是提取路路通酸、鞣质等有效成分为主。
公开号为CN104370992A的专利文件公开了一种从女贞子中提取熊果酸和齐墩果酸混合物的方法,其是先采用酸水解技术将女贞子中以糖配体方式存在的熊果酸与齐墩果酸水解成游离态,优先分离出原料中富含熊果酸和齐墩果酸的提取物料,然后取出脂溶性有机化合物,再采用碱性乙醇混合溶剂,辅以超声提取,经洗涤、乙醇溶解等处理获得目标产物。但其仅是提取熊果酸和齐墩果酸,对于女贞子中的脂溶性有益成分却被损失掉了,并且整个提取过程较为繁琐。
发明内容
本发明为解决上述问题,提供了一种从路路通中提取混合果酸和抑菌精油的方法。
具体是通过以下技术方案来实现的:
一种从路路通中提取混合果酸和抑菌精油的方法,包括以下步骤:
1、前处理:将路路通干燥后碾压,过筛后取大颗粒物质,弃粉末及路路通梗杂质,将大颗粒物质采用碱浸0.5-1h后再用50-65℃热水浸泡2-3h,干燥后粉碎得到路路通粉末。
2、发酵:将微生物菌剂接种于培养液中培养2-3天后以微波辐射3-5s得到发酵液;将发酵液与路路通粉末按1-1.5:0.8-1.2的比例混合,在温度为12-18℃、湿度60-70%条件下发酵5-10天;
进一步,所述的微生物菌剂是将纤维素酶、果胶酶、酵母菌按6-10:2.5-4:2-4.5的比例混合而成。
3、一次提取:将发酵完毕的混合物以20-25kV/cm的电场强度,10-15μs的脉冲宽度,600-650Hz的脉冲频率进行PEF处理5-7s,过滤得发酵药液和药渣,烘干药渣。
4、梯度洗脱:对发酵液进行硅胶柱层析,以环己烷-乙酸乙酯(7-9:1-2.5) 进行梯度洗脱,乙酸乙酯以6-8%的比例递增,每份50ml,合并第2-4份,得化合物A,合并第18-24份,得化合物B。
5、二次提取:将步骤3中的药渣以水为溶剂进行亚临界萃取,取萃取相,减压蒸馏,取蒸馏液,浓缩后得到抑菌精油。
6、纯化:对化合物A采用丙酮洗涤后回收丙酮,再用冰醋酸洗涤一遍,回收冰醋酸,干燥粉碎得到熊果酸;对化合物B采用乙醇萃取,取萃取相重结晶,干燥后粉碎得到齐墩果酸,将齐墩果酸和熊果酸混合即得到混合果酸。
7、应用:混合果酸和抑菌精油可应用于制备化妆品、医药领域;抑菌精油还可用于制备食品保鲜剂。
综上所述,本发明的有益效果在于:本发明采用前处理、一次提取、梯度洗脱、二次提取、纯化的方法对路路通进行提取,可同时从中提取到熊果酸、齐墩果酸和具有抑菌效果的挥发油,提取方法简单,尽可能的减少了浪费,同时,所提取的抑菌精油对大肠杆菌和枯草芽孢杆菌的抑菌圈直径分别达到6.1mm和 14.7mm,熊果酸和齐墩果酸的纯度分别达到95.3%和92.7%。
在制备方法中,我们对路路通进行干燥后先碾压再过筛去除梗、叶、粉末等杂质,这是由于路路通果序较硬,在进行碾压后可将较硬的部分破坏,同时路路通有许多粉末状杂质,直接提取会对产物的纯度造成影响;先碱浸再用热水浸泡,可软化果序,也避免了有益成分先被碱溶液浸泡出来,造成损失。对路路通采用微生物菌剂发酵,可使植物细胞壁被分解,大量的活性物质溶出,极大程度的提高了有效成分的含量,且在发酵过程中以路路通中的有效成分为前体,经微生物的代谢可以形成新的化合物,降低药物的毒副作用。对发酵物进行脉冲电场处理,电极中的金属物质会少量释放到发酵物的油溶性成分中,提高有效成分活性的同时也对微生物菌剂进行了灭活处理。采用硅胶柱层析的方式最大限度的提取目标,并且通过梯度洗脱的方式可同时将熊果酸和齐墩果酸洗脱出来,简化步骤,也保证了果酸的纯度。在二次提取时采用了亚临界萃取法,以水为溶剂减少了有机溶剂的使用,同时也避免了采用有机溶剂造成挥发油损失的问题,省去了后续对有机溶剂回收的步骤。纯化时采用不同的溶剂对化合物A进行洗涤,先采用丙酮尽可能的将熊果酸洗涤出来,再采用冰醋酸洗涤一遍则是去除上一步丙酮所带出的其他成分,进一步提高熊果酸的纯度。
具体实施方式
下面对本发明的具体实施方式作进一步详细的说明,但本发明并不局限于这些实施方式,任何在本实施例基本精神上的改进或代替,仍属于本发明权利要求所要求保护的范围。
实施例1
一种从路路通中提取混合果酸和抑菌精油的方法,包括以下步骤:
1、前处理:将路路通干燥后碾压,过筛后取大颗粒物质,弃粉末及路路通梗杂质,将大颗粒物质采用氢氧化钠碱溶液浸泡1h后,再用60℃热水浸泡2h,干燥后粉碎得到路路通粉末。
2、发酵:将微生物菌剂接种于培养液中培养3天后以功率为400W的微波辐射4s得到发酵液;将发酵液与路路通粉末按1.5:1的比例混合,在温度为 16℃、湿度65%条件下发酵7天;
进一步,所述的微生物菌剂是将纤维素酶、果胶酶、酵母菌按8:3:3.5的比例混合而成。
3、一次提取:将发酵完毕的混合物以23kV/cm的电场强度,12μs的脉冲宽度,625Hz的脉冲频率进行PEF处理6s,过滤得发酵药液和药渣,烘干药渣。
4、梯度洗脱:对发酵液进行硅胶柱层析,以环己烷-乙酸乙酯(8:2)进行梯度洗脱,乙酸乙酯以7%的比例递增,每份50ml,合并第2-4份,得化合物A,合并第18-24份,得化合物B。
5、二次提取:将步骤3中的药渣以水为溶剂进行亚临界萃取,取萃取相,减压蒸馏,取蒸馏液,浓缩后得到抑菌精油。
6、纯化:对化合物A采用丙酮洗涤后回收丙酮,再用冰醋酸洗涤一遍,回收冰醋酸,干燥粉碎得到熊果酸;对化合物B采用乙醇萃取,取萃取相重结晶,干燥后粉碎得到齐墩果酸,将齐墩果酸和熊果酸混合即得到混合果酸。
实施例2
一种从路路通中提取混合果酸和抑菌精油的方法,包括以下步骤:
1、前处理:将路路通干燥后碾压,过筛后取大颗粒物质,弃粉末及路路通梗杂质,将大颗粒物质采用氢氧化钠碱溶液浸泡0.5h后,再用50℃热水浸泡2h,干燥后粉碎得到路路通粉末。
2、发酵:将微生物菌剂接种于培养液中培养2天后以功率为300W的微波辐射3s得到发酵液;将发酵液与路路通粉末按1:0.8的比例混合,在温度为12℃、湿度60%条件下发酵5天;
进一步,所述的微生物菌剂是将纤维素酶、果胶酶、酵母菌按6:2.5:2的比例混合而成。
3、一次提取:将发酵完毕的混合物以20kV/cm的电场强度,10μs的脉冲宽度,600Hz的脉冲频率进行PEF处理5s,过滤得发酵药液和药渣,烘干药渣。
4、梯度洗脱:对发酵液进行硅胶柱层析,以环己烷-乙酸乙酯(7:1)进行梯度洗脱,乙酸乙酯以6%的比例递增,每份50ml,合并第2-4份,得化合物A,合并第18-24份,得化合物B。
5、二次提取:将步骤3中的药渣以水为溶剂进行亚临界萃取,取萃取相,减压蒸馏,取蒸馏液,浓缩后得到抑菌精油。
6、纯化:对化合物A采用丙酮洗涤后回收丙酮,再用冰醋酸洗涤一遍,回收冰醋酸,干燥粉碎得到熊果酸;对化合物B采用乙醇萃取,取萃取相重结晶,干燥后粉碎得到齐墩果酸,将齐墩果酸和熊果酸混合即得到混合果酸。
实施例3
一种从路路通中提取混合果酸和抑菌精油的方法,包括以下步骤:
1、前处理:将路路通干燥后碾压,过筛后取大颗粒物质,弃粉末及路路通梗杂质,将大颗粒物质采用氢氧化钠碱溶液浸泡1h后再用65℃热水浸泡3h,干燥后粉碎得到路路通粉末。
2、发酵:将微生物菌剂接种于培养液中培养3天后以功率为500W的微波辐射5s得到发酵液;将发酵液与路路通粉末按1.5:1.2的比例混合,在温度为 18℃、湿度70%条件下发酵10天;
进一步,所述的微生物菌剂是将纤维素酶、果胶酶、酵母菌按10:4:4.5的比例混合而成。
3、一次提取:将发酵完毕的混合物以25kV/cm的电场强度,15μs的脉冲宽度,650Hz的脉冲频率进行PEF处理7s,过滤得发酵药液和药渣,烘干药渣。
4、梯度洗脱:对发酵液进行硅胶柱层析,以环己烷-乙酸乙酯(9:2.5)进行梯度洗脱,乙酸乙酯以8%的比例递增,每份50ml,合并第2-4份,得化合物A,合并第18-24份,得化合物B。
5、二次提取:将步骤3中的药渣以水为溶剂进行亚临界萃取,取萃取相,减压蒸馏,取蒸馏液,浓缩后得到抑菌精油。
6、纯化:对化合物A采用丙酮洗涤后回收丙酮,再用冰醋酸洗涤一遍,回收冰醋酸,干燥粉碎得到熊果酸;对化合物B采用乙醇萃取,取萃取相重结晶,干燥后粉碎得到齐墩果酸,将齐墩果酸和熊果酸混合即得到混合果酸。
实施例4
一种从路路通中提取混合果酸和抑菌精油的方法,包括以下步骤:
1、前处理:将路路通干燥后碾压,过筛后取大颗粒物质,弃粉末及路路通梗杂质,将大颗粒物质采用氢氧化钠碱溶液浸泡1h后再用65℃热水浸泡3h,干燥后粉碎得到路路通粉末。
2、发酵:将微生物菌剂接种于培养液中培养3天后以功率为450W的微波辐射5s得到发酵液;将发酵液与路路通粉末按1.5:1.2的比例混合,在温度为 15℃、湿度63%条件下发酵8天;
进一步,所述的微生物菌剂是将纤维素酶、果胶酶、酵母菌按7:2.5:4.5的比例混合而成。
3、一次提取:将发酵完毕的混合物以20kV/cm的电场强度,14μs的脉冲宽度,600Hz的脉冲频率进行PEF处理7s,过滤得发酵药液和药渣,烘干药渣。
4、梯度洗脱:对发酵液进行硅胶柱层析,以环己烷-乙酸乙酯(7:2.5)进行梯度洗脱,乙酸乙酯以6%的比例递增,每份50ml,合并第2-4份,得化合物A,合并第18-24份,得化合物B。
5、二次提取:将步骤3中的药渣以水为溶剂进行亚临界萃取,取萃取相,减压蒸馏,取蒸馏液,浓缩后得到抑菌精油。
6、纯化:对化合物A采用丙酮洗涤后回收丙酮,再用冰醋酸洗涤一遍,回收冰醋酸,干燥粉碎得到熊果酸;对化合物B采用乙醇萃取,取萃取相重结晶,干燥后粉碎得到齐墩果酸,将齐墩果酸和熊果酸混合即得到混合果酸。
一、药材筛选实验
1.1实验材料
样品1:本实验在与实施例1的同等条件下,在前处理时仅对路路通进行碾压过筛;
样品2:本实验在与实施例1的同等条件下,在前处理时直接将路路通粉碎,不进行碱浸和热水浸泡;
样品3:本实验在与实施例1的同等条件下,将微生物菌剂更换为等量的纤维素酶;
样品4:本实验在与实施例1的同等条件下,将微生物菌剂更换为等量的果胶酶;
样品5:本实验在与实施例1的同等条件下,不添加任何微生物菌剂进行发酵;:
样品6:本实验在与实施例1的同等条件下,在纯化时仅采用丙酮对化合物 A进行洗涤,回收丙酮后干燥粉碎得到;
样品7:本实验在与实施例1的同等条件下,在纯化时不对化合物B采用以乙醇进行萃取、重结晶;
样品8:本实验在与实施例1的同等条件下,不进行PEF处理。
1.2实验方法
取KM种雄小鼠,按体重随机分为空白组、给药组1-6和阿司匹林组,给各组均灌胃给药,每日一次,连续三天,给药量为10mg/kg,空白组灌胃等容量蒸馏水。末次给药后1h,于大鼠左后足跖足皮下注射1%交角菜胶生理盐水溶液 0.05ml致炎,用容积法分别测定各组致炎前后1、3、5、7h的足跖容积,并计算致炎前后足跖容积差值,结果如表1所示。
其中,给药组1为实施例1制备的抑菌精油,给药组2-6分别为样品1-5制备的抑菌精油;
足跖容积差值=致炎后足跖容积差值-致炎前足跖容积差值。
1.3实验结果
表1(单位:ml)
由实验结果可知,路路通抑菌精油能对抗角叉菜胶致小鼠足跖肿胀,表明其具有一定的抗炎作用,而综合各组数据结果,实施例1为最优选。
二、纯度测定实验
对实施例1和样品6、样品7中果酸的纯度进行测定,结果如表2所示。
表2
三、抑菌实验
3.1实验材料
3.1.1对样品3-5和样品8所制备的抑菌精油测定各自的抑菌效果,并与实施例1进行比较,结果如表3所示。
3.1.2培养菌种
配置50ml的LB培养基,无菌条件下,分别加入150L大肠杆菌、金黄色葡萄球菌和枯草芽孢杆菌,放置28℃摇床下培养48h。
3.2实验方法
在10ml离心管中用蒸馏水稀释培养好的菌液,使其在波长600nm时的吸光度为1.2左右。配置适量的固体LB培养基(在LB液体培养基的基础上加入 1.5%-2.5%的琼脂),倒入适量在培养皿中,待冷却凝固,加入200μl稀释好的菌液,采用涂布法均匀的涂布于培养皿上,最后将薄膜用蓝枪头压得圆形小薄膜,用牙签将其挑起放置在已涂有菌的培养皿中,注入抑菌精油5ml,用密封条密封,于37℃下倒置培养一至两天,观察结果。
3.3实验结果
表3
由实验结果可知,不进行发酵所提取到的抑菌精油其抑菌效果远低于经过发酵的提取方法;不采用PEF进行处理所提取的抑菌精油虽然对金黄色葡萄球菌的抑制效果好于实施例1,但对大肠杆菌和枯草芽孢杆菌的抑制效果较差,综合来看,实施例1为最优选。
Claims (10)
1.一种从路路通中提取混合果酸和抑菌精油的方法,其特征在于,包括以下步骤:前处理、一次提取、梯度洗脱、二次提取、纯化。
2.如权利要求1所述的一种从路路通中提取混合果酸和抑菌精油的方法,其特征在于,所述的前处理,是将路路通干燥后碾压,过筛后取大颗粒物质,弃粉末及路路通梗杂质,将大颗粒物质采用碱浸0.5-1h后再用50-65℃热水浸泡2-3h,干燥后粉碎得到路路通粉末。
3.如权利要求1所述的一种从路路通中提取混合果酸和抑菌精油的方法,其特征在于,所述的一次提取,将路路通粉末发酵5-10天后进行PEF处理,过滤得发酵药液和药渣,烘干药渣。
4.如权利要求3所述的一种从路路通中提取混合果酸和抑菌精油的方法,其特征在于,所述的PEF处理,是以20-25kV/cm的电场强度,10-15μs的脉冲宽度,600-650Hz的脉冲频率处理5-7s。
5.如权利要求3所述的一种从路路通中提取混合果酸和抑菌精油的方法,其特征在于,所述的发酵,包括以下步骤:
a.将微生物菌剂接种于培养液中培养2-3天后以微波辐射3-5s得到发酵液;
b.将发酵液与路路通粉末按1-1.5:0.8-1.2的比例混合,在温度为12-18℃、湿度60-70%条件下发酵后烘干得到。
6.如权利要求1所述的一种从路路通中提取混合果酸和抑菌精油的方法,其特征在于,所述的梯度洗脱,对发酵液进行硅胶柱层析,进行梯度洗脱,每份50ml,合并第2-4份,得化合物A,合并第18-24份,得化合物B。
7.如权利要求6所述的一种从路路通中提取混合果酸和抑菌精油的方法,其特征在于,梯度洗脱的条件为:以环己烷-乙酸乙酯(7-9:1-2.5)进行梯度洗脱,乙酸乙酯以6-8%的比例递增。
8.如权利要求1所述的一种从路路通中提取混合果酸和抑菌精油的方法,其特征在于,所述的二次提取,是将药渣以水为溶剂进行亚临界萃取,取萃取相,减压蒸馏,取蒸馏液,浓缩后得到抑菌精油。
9.如权利要求1所述的一种从路路通中提取混合果酸和抑菌精油的方法,其特征在于,所述的纯化,是对化合物A采用丙酮洗涤后回收丙酮,再用冰醋酸洗涤一遍,回收冰醋酸,干燥粉碎得到熊果酸;对化合物B采用乙醇萃取,取萃取相重结晶,干燥后粉碎得到齐墩果酸,将齐墩果酸和熊果酸混合即得到混合果酸。
10.一种从路路通中提取的混合果酸和抑菌精油的应用,其特征在于,所述的混合果酸和抑菌精油可应用于制备化妆品、医药领域;抑菌精油还可用于制备食品保鲜剂。
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CN113499298B (zh) * | 2021-06-24 | 2023-01-24 | 青岛黛优佳生物科技有限公司 | 一种具有美白祛斑协同增效作用的化妆品和应用 |
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