阅读说明：本技术 一种橡木中鞣花酸的制备方法 (Preparation method of ellagic acid in oak ) 是由 石万银 徐剑 杨跃军 李韦琴 王道清 于 2021-07-13 设计创作，主要内容包括：本发明涉及一种橡木中鞣花酸的制备方法,包括提取、干燥、纯化,馏分收集和浓缩步骤,使用液相色谱柱对橡木提取物直接进行分离纯化,操作简单,转移率高,只需在相应时间收集洗脱液即可获取高纯度鞣花酸,工艺简单、操作方便、周期短、效率高。(The invention relates to a preparation method of ellagic acid in oak, which comprises the steps of extraction, drying, purification, fraction collection and concentration, wherein the oak extract is directly separated and purified by using a liquid chromatographic column, the operation is simple, the transfer rate is high, the high-purity ellagic acid can be obtained by collecting eluent only in corresponding time, and the preparation method has the advantages of simple process, convenience in operation, short period and high efficiency.)

1. A method for preparing ellagic acid in oak is characterized by comprising the following steps: the method comprises the following operation steps:

extraction: pulverizing oak, extracting with ethanol under heating for 2-3 times, and filtering to obtain oak extractive solution;

and (3) drying: concentrating and drying the oak crude extract, and removing ethanol to obtain oak extract;

and (3) purification: dissolving oak extract with ethanol solution, filtering, and eluting with chromatographic column;

collecting eluent: collecting the eluent of corresponding time to obtain ellagic acid eluent;

concentration: and concentrating the ellagic acid eluent to obtain the ellagic acid.

2. The method of claim 1, wherein the ellagic acid is produced from oak wood by: in the extraction step, the concentration of the ethanol is 60-70%.

3. The method of claim 2, wherein the ellagic acid is produced from oak wood by: in the extraction step, the ratio of the mass (g) of the oak to the volume (ml) of the ethanol is as follows: the mass (g) of oak and the volume (ml) of ethanol are 1: 10-15.

4. The method of claim 1, wherein the ellagic acid is produced from oak wood by: the extraction operation steps in the extraction step are heating reflux extraction for 2-3 times at 80-90 ℃, and each time lasts for 1-2 hours.

5. The method of claim 1, wherein the ellagic acid is produced from oak wood by: in the purification step, 40-60 times of 60-70% ethanol solution is added into the oak extract for ultrasonic dissolution.

6. The method of claim 1, wherein the ellagic acid is produced from oak wood by: in the purification operation step, a C18 reversed phase chromatographic column is used, the mobile phase is acetonitrile-0.1% -0.2% formic acid, the flow rate is 25-35mL/min, and the elution gradient is as follows:

7. the method of claim 6, wherein the ellagic acid is produced from oak wood by: in the purification operation step, the temperature of a chromatographic column is 25-35 ℃.

8. The method of claim 1, wherein the ellagic acid is produced from oak wood by: the collecting time period of the ellagic acid eluate is 50-55 min.

9. The method of claim 1, wherein the ellagic acid is produced from oak wood by: in the concentration step, vacuum concentration and drying are carried out under reduced pressure at the temperature of 55-65 ℃.

Technical Field

The invention belongs to the technical field of plant extract preparation, and particularly relates to a preparation method of ellagic acid in oak.

Background

The main components of oak include cellulose, hemicellulose, lignin, tannin and trace components. In the long-term natural aging process, by virtue of the slow action of acid in alcohol, water and wine and oxygen in the air, macromolecular substances such as lignin, tannin, hemicellulose and the like are decomposed and oxidized to generate various fragrant components, and ellagic acid is a typical representative of a product obtained by decomposing oak tannin.

The research shows that the ellagic acid has strong capacity of eliminating free radicals and resisting oxidation. Ellagic acid has scavenging effect on oxygen free radical and hydroxyl free radical, and has higher capacity of scavenging free radical than sesamol, folium Canarii albi extract, xanthophyll, and proanthocyanidins. Ellagic acid shows obvious inhibiting effect on canceration induced by chemical substances and other multiple canceration, and especially has good inhibiting effect on colon cancer, esophageal cancer, liver cancer, lung cancer, tongue and skin tumor, etc.

At present, the ellagic acid is mainly prepared from pomegranate rind, blueberries, myrobalan and the like serving as raw materials by means of heating extraction, membrane separation, resin purification, biotransformation, organic solvent extraction and other methods, and the methods have the defects of complex process, inconvenient operation, long period, low efficiency and the like.

Disclosure of Invention

The invention aims to provide a purification method which is convenient and direct, has simple process, low production cost, easy popularization and high ellagic acid purity.

The technical scheme adopted by the invention for solving the technical problems is as follows: a preparation method of ellagic acid in oak comprises the following operation steps:

extraction: pulverizing oak, extracting with ethanol under heating for 2-3 times, and filtering to obtain oak extractive solution;

and (3) drying: concentrating and drying the oak crude extract, and removing ethanol to obtain oak extract;

and (3) purification: dissolving oak extract with ethanol solution, filtering, and eluting with chromatographic column;

collecting eluent: collecting the eluent of corresponding time to obtain ellagic acid eluent;

concentration: and concentrating the ellagic acid eluent to obtain the ellagic acid.

Preferably, in the extraction step, the concentration of ethanol is 60% -70%.

Preferably, in the extraction step, the ratio of oak mass (g) to ethanol volume (ml) is: the mass (g) of oak and the volume (ml) of ethanol are 1: 10-15.

Preferably, the extraction operation in the extraction step is heating reflux extraction at 80-90 ℃ for 2-3 times, each time for 1-2 hours.

Preferably, in the purification step, 40-60 times of 60% -70% ethanol solution is added into the oak extract for ultrasonic dissolution.

Preferably, in the purification operation step, a C18 reversed phase chromatographic column is used, the mobile phase is acetonitrile-0.1% -0.2% formic acid, the flow rate is 25-35mL/min, and the elution gradient is as follows:

time (min)	Acetonitrile (%)	0.1% -0.2% formic acid (%)
			0	3-5	97-95
5	3-5	97-96
			18	7-10	93-90
30	12-15	88-85
			60	27-30	73-70

Preferably, in the purification operation step, the column temperature of the chromatographic column is 25-35 ℃.

Preferably, the collection period of the ellagic acid eluate is 50-55 min.

Preferably, in the concentration step, reduced pressure vacuum concentration and drying are adopted, and the temperature is 55-65 ℃.

The invention has the following beneficial effects:

the method has the advantages of simple process and short production period: in the conventional process for preparing ellagic acid, methods such as resin purification or organic solvent extraction and the like are required to be firstly carried out after extraction. The invention directly concentrates and dries the oak chip extract, and then separates and purifies the oak chip extract by a preparation liquid phase, thereby obtaining high-purity ellagic acid and simultaneously achieving the purposes of simplifying the process and shortening the period.

Secondly, high transfer rate: the conventional process for preparing the ellagic acid needs to concentrate the oak chip extracting solution and then carry out resin purification, organic solvent extraction and other operations, the loss of the ellagic acid is large, and the invention directly prepares a liquid phase at high efficiency for separation and purification after concentrating and drying the oak chip extracting solution, thereby improving the transfer rate of the ellagic acid.

Thirdly, the product purity is high: the concentrated solution extracted from the oak chips is subjected to high-efficiency preparation and liquid phase separation and purification to obtain the high-purity ellagic acid monomer.

Detailed Description

Example 1

Extraction: pulverizing oak chips, extracting with 60% ethanol solution for 3 times (1000 mL each time), reflux extracting at 80 deg.C for 1 hr, collecting and mixing filtrates, coarse-filtering with filter screen, and coarse-filtering to obtain oak chip crude extract.

And (3) drying: concentrating the oak extractive solution at 55 deg.C under reduced pressure, and drying to obtain oak extract 12 g.

And (3) purification: adding 40 times of 60% ethanol into the dried extract, ultrasonic dissolving, filtering with microporous membrane, pouring into C18 reversed phase chromatographic column, gradient eluting with acetonitrile-0.1% formic acid according to the following table, flow rate of 25mL/min, detection wavelength of 265nm, and column temperature of 25 deg.C.

Collecting eluent: collecting ellagic acid eluate (collection time period is 50-55 min);

concentration: vacuum concentrating and drying the eluate at 55 deg.C under reduced pressure to obtain ellagic acid product.

Example 2

Extraction: pulverizing 100g oak chips, extracting with 65% ethanol solution for 2 times (1200 mL each time), reflux extracting at 85 deg.C for 1.5 hr, collecting and mixing filtrates, coarse filtering with filter screen, and coarse filtering to obtain oak crude extract.

And (3) drying: concentrating the crude extract of oak at 60 deg.C under reduced pressure, and drying to obtain oak extract 11.8 g.

And (3) purification: adding 50 times of 65% ethanol into the dried extract, ultrasonic dissolving, filtering with microporous membrane, pouring into C18 reversed phase chromatographic column, gradient eluting with acetonitrile-0.15% formic acid according to the following table, flow rate of 30mL/min, detection wavelength of 270nm, column temperature of 30 deg.C,

collecting eluent: collecting ellagic acid eluate (collection time period is 50-55 min);

concentration: vacuum concentrating and drying the eluate at 55 deg.C under reduced pressure to obtain ellagic acid product.

Example 3

Extraction: pulverizing 100g oak chips, extracting with 70% ethanol solution for 3 times (each time adding 1500 mL), reflux extracting at 90 deg.C for 2 hr, collecting and mixing filtrates, coarse filtering with filter screen, and coarse filtering to obtain oak chip coarse extractive solution.

And (3) drying: concentrating the oak extractive solution at 65 deg.C under reduced pressure, and drying to obtain oak extract 12.3 g.

And (3) purification: adding 60 times of 60% ethanol into appropriate amount of dried extract, ultrasonic dissolving, filtering with microporous membrane, pouring into C18 reversed phase chromatographic column, gradient eluting with acetonitrile-0.2% formic acid according to the following table with flow rate of 35mL/min, detection wavelength of 275nm, column temperature of 35 deg.C,

collecting eluent: collecting ellagic acid eluate (collection time period is 50-55 min);

concentration: vacuum concentrating and drying the eluate at 55 deg.C under reduced pressure to obtain ellagic acid product.

Comparative example 1

Selecting HPD-600 macroporous resin column to perform primary separation and purification on the ellagic acid crude extract, wherein the static adsorption time is 6h, 50% ethanol solution is used as eluent, and the desorption time is 3 h; controlling the sample concentration at 2.5mg/mL, the flow rate at 3mL/min, the sample injection volume at 50mL, eluting with 60% ethanol solution, and performing secondary separation and purification with macroporous resin CG-161 at a static adsorption temperature of 25 deg.C for 3.5h, and using 13% sodium hydroxide-60% ethanol solution as eluent; the sample loading volume is 40mL, the elution flow rate is 3mL/min, and the eluent is subjected to vacuum concentration and drying to obtain the ellagic acid.

The products of each example were weighed, purity determined and yield calculated, with the final results shown in Table 1

TABLE 1

Sample (I)	Finished product quality (mg)	Yield (%)	Purity (%)
				Example 1	450	0.45	98.05
Example 2	490	0.49	96.59
				Example 3	420	0.42	97.02
Comparative example 1	530	0.53	80.18

As can be seen from the purity data of the final product of each example, the ellagic acid extracted from oak by the method of the present invention is extremely high in purity and almost free of other impurities; compared with the common macroporous resin purification method, the method has the advantages that the purity of the ellagic acid prepared by the method is improved by nearly 20%, the operation is simpler and more convenient, multiple purification and elution are not needed, and the ellagic acid finished product with extremely high purity can be prepared only by collecting eluent of a chromatographic column.