Preparation method of Acanthopanax senticosus extract

Abstract

The invention discloses a preparation method of an Acanthopanax senticosus extract, comprising: step 1, preparing Acanthopanax senticosus leaves and auxiliary Chinese medicine into powder; step 2, adding a first low eutectic solvent to a mixture of Acanthopanax senticosus powder and auxiliary Chinese medicine powder, placing the mixture in a microwave extractor for extraction, and centrifuging to obtain a first clear liquid and a first filter residue; step 3, adding a second low eutectic solvent to the first filter residue obtained in step 2, placing the mixture in a microwave extractor for extraction and centrifugation to obtain a second clear liquid and a second filter residue; step 4, adding a third low eutectic solvent to the second filter residue obtained in step 3, placing the mixture in a microwave extractor for extraction and centrifugation to obtain a third clear liquid and a third filter residue; step 5, collecting the first clear liquid, the second clear liquid and the third clear liquid, decompressing and concentrating the mixture, and spray drying the mixture to obtain an Acanthopanax senticosus extract. The invention can simultaneously extract flavonoids, Acanthopanax senticosus glycoside B, Acanthopanax senticosus glycoside E and Isofraxidin.

Description

Technical Field

[0001] The present invention relates to the technical field of natural product extraction, and more specifically, to a method for preparing an acanthopanax senticosus extract. Background Art

[0002] Acanthopanax senticosus, commonly known as Siberian ginseng, belongs to the genus Acanthopanax of the Araliaceae family and is mainly distributed in Heilongjiang, Jilin, Liaoning, Hebei and Shanxi in my country. Acanthopanax senticosus has a long history of being widely used as a medicine in Chinese medicine and pharmacy, and has the effects of tonifying the middle and replenishing essence, replenishing qi and strengthening the spleen, strengthening the body, strengthening the tendons and bones, and strengthening the will. Acanthopanax senticosus contains a variety of active ingredients such as flavonoids (such as quercetin), polysaccharides, saponins, coumarins (such as isoflurane) and lignans (eleutheroside E, etc.). At present, the research on Acanthopanax senticosus mainly focuses on the extraction of Acanthopanax senticosus ingredients.

[0003] As a new type of green solvent, deep eutectic solvents have the characteristics of simple preparation, low price, biodegradability, and the ability to improve the efficiency of organic reactions. They have been used in the field of separating natural products. For example, deep eutectic solvents have shown good extraction effects on baicalin and phenolic compounds. However, due to the large difference in chemical polarity of acanthopanax senticosus glycosides, flavonoids and isoflurane in Acanthopanax senticosus, it is difficult to extract the above active ingredients simultaneously using traditional extraction methods.

[0004] The application number is 202110534593.X, and the name is a method for synthesizing a low eutectic solvent and a method for extracting and purifying acanthoside B. It uses a low eutectic solvent mixed with choline chloride, L-proline, and water to extract acanthoside B, and the content of the obtained acanthoside B is up to 1.4062±0.0213. The application number is 202110889827.2, and the name is a low eutectic solvent and its preparation method and its use in extracting isofraquinone from Acanthopanax senticosus. It uses a low eutectic solvent mixed with choline chloride, acid and water to extract isofraquinone, and the content of the obtained isofraquinone is up to 0.6394±0.0128. Summary of the invention

[0005] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0006] Another object of the present invention is to provide a method for preparing an Acanthopanax senticosus extract, which can simultaneously extract flavonoids, eleutheroside B, eleutheroside E and isofraquinone, and by adding auxiliary Chinese medicine to form a co-solvent system with the target compound, the extraction rate of the target compound total flavonoids, eleutheroside B, eleutheroside E and isofraquinone is improved.

[0007] In order to achieve these purposes and other advantages according to the present invention, a method for preparing an Acanthopanax senticosus extract is provided, comprising:

[0008] Step 1: Dry and crush the Acanthopanax senticosus leaves to a particle size of 40 meshes to obtain Acanthopanax senticosus powder; at the same time, dry and crush the auxiliary Chinese medicines to a particle size of 40 meshes to obtain auxiliary Chinese medicine powders; set aside

[0009] Step 2, adding a first low eutectic solvent to the mixture of Acanthopanax senticosus powder and auxiliary Chinese medicine powder, placing it in a microwave extractor and extracting it at 300-600W for 15-30min at a temperature of 60-80°C, and centrifuging to obtain a first clear liquid and a first filter residue, wherein the mass ratio of the Acanthopanax senticosus powder to the auxiliary Chinese medicine powder is 60-100:1, and the first low eutectic solvent is prepared from choline chloride, amino acid, and water in a molar ratio of 1:0.5-1.5:200-400;

[0010] Step 3, adding a second low eutectic solvent to the first filter residue obtained in step 2, placing in a microwave extractor and extracting at 300-600W for 15-30min at a temperature of 60-80°C, and centrifuging to obtain a second clear liquid and a second filter residue, wherein the second low eutectic solvent is prepared from choline chloride, a polyol, and water in a molar ratio of 1:5-10:50-100;

[0011] Step 4: adding a third low eutectic solvent to the second filter residue obtained in step 3, placing the mixture in a microwave extractor and extracting it at 300-600W for 15-30min at a temperature of 60-80°C, and centrifuging to obtain a third clear liquid and a third filter residue, wherein the third low eutectic solvent is prepared from choline chloride, sugar, and water in a molar ratio of 1:2-4:10-20;

[0012] Step 5: collect the first clear liquid, the second clear liquid and the third clear liquid, concentrate under reduced pressure and then spray dry to obtain the Acanthopanax senticosus extract. The vacuum degree of the reduced pressure concentration is -0.06MPa to -0.09MPa.

[0013] Preferably, the solid-liquid ratio (g / mL) of Acanthopanax senticosus powder and the first deep eutectic solvent in step 2 is 1:10-20, the volume of the second deep eutectic solvent in step 3 and the volume of the third deep eutectic solvent in step 4 are 1 / 4-1 / 2 and 1 / 4-1 / 3 of the volume of the first deep eutectic solvent, respectively.

[0014] Preferably, the amino acid in step 2 is a basic amino acid, including one or more of arginine, lysine and histidine.

[0015] Preferably, the polyol in step three includes one or more of ethylene glycol, propylene glycol, and 1,4-butanediol.

[0016] Preferably, the sugar in step 4 includes one or more of glucose, rhamnose and fructose.

[0017] Preferably, the auxiliary Chinese medicine is one or more of Phragmites australis, Angelica sinensis, Ophiopogon japonicus and Panax notoginseng.

[0018] The present invention further claims protection for the product obtained by the preparation method of the Acanthopanax senticosus extract, wherein the obtained product comprises Acanthopanax total flavonoids, isofraquinone, eleutheroside B and eleutheroside E, wherein the content of isofraquinone (mg / g) is ≥0.6829; the content of eleutheroside B (mg / g) is ≥1.4867; the content of eleutheroside E (mg / g) is ≥1.5923; and the total flavonoids of Acanthopanax senticosus (mg / g) is ≥41.236.

[0019] The present invention has at least the following beneficial effects:

[0020] First, the preparation method of the Acanthopanax senticosus extract provided by the present invention proposes for the first time to use different eutectic systems for extraction in a certain order, and can simultaneously extract the target products flavonoids, eleutheroside B, eleutheroside E and isoflurane;

[0021] Second, the preparation method of the Acanthopanax senticosus extract provided by the present invention screens out for the first time a Chinese medicinal agent that has a synergistic extraction effect on the target product in Acanthopanax senticosus; and discloses the optimal synergistic ratio of the auxiliary Chinese medicinal agent and Acanthopanax senticosus;

[0022] Thirdly, the preparation method of Acanthopanax senticosus extract provided by the present invention has improved extraction rates of various target products compared with existing extraction methods;

[0023] Fourthly, the preparation method of Acanthopanax senticosus extract provided by the present invention adopts a low eutectic solvent, which is economical, readily available, safe, and has strong compatibility with existing microwave extraction technology, and can simplify the subsequent purification process.

[0024] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can implement the invention with reference to the description.

[0026] It should be understood that terms such as “having”, “including” and “comprising” used herein do not specify the existence or addition of one or more other elements or combinations thereof.

[0027] The number of equipment and processing scale described here are used to simplify the description of the present invention. The application, modification and variation of the preparation method of Acanthopanax senticosus extract of the present invention will be obvious to those skilled in the art.

[0028] Example 1

[0029] Step 1: Dry the Acanthopanax senticosus leaves and crush them into a particle size of 40 meshes to obtain Acanthopanax senticosus powder; at the same time, dry the Phragmites australis and crush them into a particle size of 40 meshes to obtain auxiliary Chinese medicine powder; set aside

[0030] Step 2, adding a first low eutectic solvent to the mixture of Acanthopanax powder and Phragmites australis powder, placing it in a microwave extractor and extracting it at 300W for 15min at a temperature of 60°C, and centrifuging to obtain a first clear liquid and a first filter residue, wherein the mass ratio of the Acanthopanax powder to the Phragmites australis powder is 100:1, the first low eutectic solvent is prepared from choline chloride, arginine, and water in a molar ratio of 1:0.5:200, and the solid-liquid ratio of the Acanthopanax powder to the first low eutectic solvent is 1:10;

[0031] Step 3: adding a second low eutectic solvent to the first filter residue obtained in step 2, placing the mixture in a microwave extractor and extracting it at 300 W for 15 min at a temperature of 60° C., and centrifuging to obtain a second clear liquid and a second filter residue, wherein the second low eutectic solvent is prepared from choline chloride, ethylene glycol, and water in a molar ratio of 1:5:50, and the volume of the second low eutectic solvent is 1 / 4 of the volume of the first low eutectic solvent;

[0032] Step 4: adding a third low eutectic solvent to the second filter residue obtained in step 3, placing the mixture in a microwave extractor and extracting it at 300 W for 15 min at a temperature of 60° C., and centrifuging to obtain a third clear liquid and a third filter residue, wherein the third low eutectic solvent is prepared from choline chloride, rhamnose, and water in a molar ratio of 1:2:10, and the volume of the third low eutectic solvent is 1 / 4 of the volume of the first low eutectic solvent;

[0033] Step 5: collecting the first clear liquid, the second clear liquid and the third clear liquid, concentrating under reduced pressure and then spray drying to obtain the Acanthopanax senticosus extract.

[0034] Example 2

[0035] Step 1: Dry and crush the Acanthopanax senticosus leaves to a particle size of 40 meshes to obtain Acanthopanax senticosus powder; at the same time, dry and crush Angelica sinensis and Panax notoginseng to a particle size of 40 meshes to obtain Angelica sinensis powder and Panax notoginseng powder respectively; set aside

[0036] Step 2, adding a first low eutectic solvent to a mixture of Acanthopanax powder, Angelica powder and Panax notoginseng powder, placing the mixture in a microwave extractor at 600W for 30min at a temperature of 80°C, and centrifuging to obtain a first clear liquid and a first filter residue, wherein the mass ratio of the Acanthopanax powder to the total mass ratio of Angelica and Panax notoginseng powder is 100:1, wherein the Angelica powder and Panax notoginseng powder have no specific ratio relationship, and the first low eutectic solvent is prepared from choline chloride, amino acid and water in a molar ratio of 1:1.5:400, wherein the amino acid is a mixture of arginine and lysine in any proportion, and the solid-liquid ratio of the Acanthopanax powder to the first low eutectic solvent is 1:20;

[0037] Step 3, adding a second low eutectic solvent to the first filter residue obtained in step 2, placing the mixture in a microwave extractor and extracting it at 600W for 30min at a temperature of 80°C, and centrifuging to obtain a second clear liquid and a second filter residue, wherein the second low eutectic solvent is prepared from choline chloride, a polyol, and water in a molar ratio of 1:10:100, wherein the polyol is a mixture of propylene glycol and 1,4-butanediol in any proportion, and the volume of the second low eutectic solvent is 1 / 3 of the volume of the first low eutectic solvent;

[0038] Step 4: adding a third low eutectic solvent to the second filter residue obtained in step 3, placing the mixture in a microwave extractor and extracting it at 600 W for 30 min at a temperature of 80° C., and centrifuging to obtain a third clear liquid and a third filter residue, wherein the third low eutectic solvent is prepared from choline chloride, sugar, and water in a molar ratio of 1:4:20, wherein the sugar is a mixture of glucose and fructose in any proportion, and the volume of the third low eutectic solvent is 1 / 4 of the volume of the first low eutectic solvent;

[0039] Step 5: collecting the first clear liquid, the second clear liquid and the third clear liquid, concentrating under reduced pressure and then spray drying to obtain the Acanthopanax senticosus extract.

[0040] Example 3

[0041] Step 1, dry the Acanthopanax senticosus leaves and crush them into a particle size of 40 mesh to obtain Acanthopanax senticosus powder; at the same time, dry the Phragmites australis, Chinese Angelica sinensis, Ophiopogon japonicus, and Panax notoginseng respectively and crush them into a particle size of 40 mesh to obtain Phragmites australis powder, Chinese Angelica sinensis powder, Ophiopogon japonicus powder, and Panax notoginseng powder; set aside

[0042] Step 2, adding a first low eutectic solvent to the mixture of Acanthopanax powder and auxiliary Chinese medicine powder, placing it in a microwave extractor and extracting it at 500W for 20min at a temperature of 70°C, and centrifuging to obtain a first clear liquid and a first filter residue, wherein the mass ratio of the Acanthopanax powder to the auxiliary Chinese medicine powder is 80:1, and the first low eutectic solvent is prepared from choline chloride, amino acid, and water in a molar ratio of 1:1:300, wherein the auxiliary Chinese medicine powder is a mixture of Phragmites australis powder, Angelica sinensis powder, Ophiopogon japonicus powder, and Panax notoginseng powder in any proportion, the amino acid is a mixture of arginine, lysine, and histidine in any proportion, and the solid-liquid ratio of Acanthopanax powder to the first low eutectic solvent is 1:15;

[0043] Step 3, adding a second low eutectic solvent to the first filter residue obtained in step 2, placing the mixture in a microwave extractor and extracting it at 400W for 25min at a temperature of 75°C, and centrifuging to obtain a second clear liquid and a second filter residue, wherein the second low eutectic solvent is prepared from choline chloride, a polyol, and water in a molar ratio of 1:8:80, wherein the polyol is a mixture of ethylene glycol, propylene glycol, and 1,4-butanediol in any proportion, and the volume of the second low eutectic solvent is 1 / 2 of the volume of the first low eutectic solvent;

[0044] Step 4: adding a third low eutectic solvent to the second filter residue obtained in step 3, placing the mixture in a microwave extractor and extracting it at 500 W for 20 min at a temperature of 70° C., and centrifuging to obtain a third clear liquid and a third filter residue, wherein the third low eutectic solvent is prepared from choline chloride, sugar, and water in a molar ratio of 1:3:15, wherein the sugar is a mixture of glucose, fructose, and rhamnose in any proportion, and the volume of the third low eutectic solvent is 1 / 3 of the volume of the first low eutectic solvent;

[0045] Step 5: collecting the first clear liquid, the second clear liquid and the third clear liquid, concentrating under reduced pressure and then spray drying to obtain the Acanthopanax senticosus extract.

[0046] Comparative Example 1

[0047] Step 1: Dry the Acanthopanax senticosus leaves and crush them into 40 mesh size to obtain Acanthopanax senticosus powder; set aside

[0048] Step 2, adding 75% ethanol solution to Acanthopanax senticosus powder, placing in a microwave extractor for extraction at 300W for 15min at a temperature of 60°C, and centrifuging to obtain a first clear liquid and a first filter residue, wherein the solid-liquid ratio of Acanthopanax senticosus powder to 75% ethanol solution is 1:10;

[0049] Step 3, add 75% ethanol solution to the first filter residue obtained in step 2, place in a microwave extractor and extract at 300W for 15min at a temperature of 60°C, and centrifuge to obtain a second clear liquid and a second filter residue, wherein the volume of the 75% ethanol solution in this step is 1 / 4 of the volume of the 75% ethanol solution in step 1;

[0050] Step 4: add 75% ethanol solution to the second filter residue obtained in step 3, place in a microwave extractor and extract at 300W for 15min at 60°C, and centrifuge to obtain a third clear liquid and a third filter residue. The volume of the 75% ethanol solution in this step is 1 / 4 of the volume of the 75% ethanol solution in step 1;

[0051] Step 5: collecting the first clear liquid, the second clear liquid and the third clear liquid, concentrating under reduced pressure and then spray drying to obtain the Acanthopanax senticosus extract.

[0052] Comparative Example 2

[0053] Step 1: Dry the Acanthopanax senticosus leaves and crush them into a particle size of 40 meshes to obtain Acanthopanax senticosus powder; at the same time, dry the Phragmites australis and crush them into a particle size of 40 meshes to obtain auxiliary Chinese medicine powder; set aside

[0054] Step 2: Add 75% ethanol solution to the mixture of Acanthopanax senticosus powder and Phragmites australis powder in a microwave extractor, extract at 300W for 15min at 60°C, and centrifuge to obtain a first clear liquid and a first filter residue, wherein the solid-liquid ratio of Acanthopanax senticosus powder to 75% ethanol solution is 1:10;

[0055] Step 3, add 75% ethanol solution to the first filter residue obtained in step 2, place in a microwave extractor and extract at 300W for 15min at a temperature of 60°C, and centrifuge to obtain a second clear liquid and a second filter residue, wherein the volume of the 75% ethanol solution in this step is 1 / 4 of the volume of the 75% ethanol solution in step 1;

[0056] Step 4: add 75% ethanol solution to the second filter residue obtained in step 3, place in a microwave extractor and extract at 300W for 15min at 60°C, and centrifuge to obtain a third clear liquid and a third filter residue. The volume of the 75% ethanol solution in this step is 1 / 4 of the volume of the 75% ethanol solution in step 1;

[0057] Step 5: collecting the first clear liquid, the second clear liquid and the third clear liquid, concentrating under reduced pressure and then spray drying to obtain the Acanthopanax senticosus extract.

[0058] Comparative Example 3

[0059] Step 1: Dry the Acanthopanax senticosus leaves and crush them into 40 mesh size to obtain Acanthopanax senticosus powder for later use.

[0060] Step 2, adding a first low eutectic solvent to the Acanthopanax powder, placing it in a microwave extractor and extracting it at 300W for 15 minutes at a temperature of 60°C, and centrifuging to obtain a first clear liquid and a first filter residue, wherein the first low eutectic solvent is prepared from choline chloride, arginine, and water in a molar ratio of 1:0.5:200, and the solid-liquid ratio of the Acanthopanax powder to the first low eutectic solvent is 1:10;

[0061] Step 3: Add the first low eutectic solvent to the first filter residue obtained in step 2, place in a microwave extractor and extract at 300W for 15 minutes at a temperature of 60°C, and centrifuge to obtain a second clear liquid and a second filter residue. The first low eutectic solvent in this step is 1 / 4 of the volume of the first low eutectic solvent in step 1;

[0062] Step 4: add the first deep eutectic solvent to the second filter residue obtained in step 3, place in a microwave extractor and extract at 300W for 15min at a temperature of 60°C, and centrifuge to obtain a third clear liquid and a third filter residue. The first deep eutectic solvent in this step is 1 / 4 of the volume of the first deep eutectic solvent in step 1;

[0063] Step 5: collecting the first clear liquid, the second clear liquid and the third clear liquid, concentrating under reduced pressure and then spray drying to obtain the Acanthopanax senticosus extract.

[0064] Comparative Example 4

[0065] Step 1: Dry the Acanthopanax senticosus leaves and crush them into a particle size of 40 meshes to obtain Acanthopanax senticosus powder; at the same time, dry the Phragmites australis and crush them into a particle size of 40 meshes to obtain auxiliary Chinese medicine powder; set aside

[0066] Step 2, adding a first low eutectic solvent to the mixture of Acanthopanax powder and Phragmites australis powder, placing it in a microwave extractor and extracting it at 300W for 15min at a temperature of 60°C, and centrifuging to obtain a first clear liquid and a first filter residue, wherein the mass ratio of the Acanthopanax powder to the Phragmites australis powder is 100:1, the first low eutectic solvent is prepared from choline chloride, arginine, and water in a molar ratio of 1:0.5:200, and the solid-liquid ratio of the Acanthopanax powder to the first low eutectic solvent is 1:10;

[0067] Step 3: Add the first low eutectic solvent to the first filter residue obtained in step 2, place in a microwave extractor and extract at 300W for 15 minutes at a temperature of 60°C, and centrifuge to obtain a second clear liquid and a second filter residue. The first low eutectic solvent in this step is 1 / 4 of the volume of the first low eutectic solvent in step 1;

[0068] Step 4: add the first deep eutectic solvent to the second filter residue obtained in step 3, place in a microwave extractor and extract at 300W for 15min at a temperature of 60°C, and centrifuge to obtain a third clear liquid and a third filter residue. The first deep eutectic solvent in this step is 1 / 4 of the volume of the first deep eutectic solvent in step 1;

[0069] Step 5: collecting the first clear liquid, the second clear liquid and the third clear liquid, concentrating under reduced pressure and then spray drying to obtain the Acanthopanax senticosus extract.

[0070] Comparative Example 5

[0071] Step 1: Dry the Acanthopanax senticosus leaves and crush them into a particle size of 40 meshes to obtain Acanthopanax senticosus powder; at the same time, dry the Phragmites australis and crush them into a particle size of 40 meshes to obtain auxiliary Chinese medicine powder; set aside

[0072] Step 2, adding a second low eutectic solvent to the mixture of Acanthopanax powder and Phragmites australis powder, placing the mixture in a microwave extractor at 300W for 15min at a temperature of 60°C, and centrifuging to obtain a first clear liquid and a first filter residue, wherein the mass ratio of the Acanthopanax powder to the Phragmites australis powder is 100:1, the second low eutectic solvent is prepared from choline chloride, polyol, and water in a molar ratio of 1:8:80, and the solid-liquid ratio of the Acanthopanax powder to the second low eutectic solvent is 1:10;

[0073] Step 3: Add a second low eutectic solvent to the first filter residue obtained in step 2, place in a microwave extractor and extract at 300W for 15 minutes at a temperature of 60°C, and centrifuge to obtain a second clear liquid and a second filter residue. The second low eutectic solvent in this step is 1 / 4 of the volume of the second low eutectic solvent in step 1;

[0074] Step 4: add a second low eutectic solvent to the second filter residue obtained in step 3, place in a microwave extractor and extract at 300W for 15 minutes at a temperature of 60°C, and centrifuge to obtain a third clear liquid and a third filter residue. The second low eutectic solvent in this step is 1 / 4 of the volume of the second low eutectic solvent in step 1;

[0075] Step 5: collecting the first clear liquid, the second clear liquid and the third clear liquid, concentrating under reduced pressure and then spray drying to obtain the Acanthopanax senticosus extract.

[0076] Comparative Example 6

[0077] Step 1: Dry the Acanthopanax senticosus leaves and crush them into a particle size of 40 meshes to obtain Acanthopanax senticosus powder; at the same time, dry the Phragmites australis and crush them into a particle size of 40 meshes to obtain auxiliary Chinese medicine powder; set aside

[0078] Step 2, adding a third low eutectic solvent to the mixture of Acanthopanax powder and Phragmites australis powder, placing in a microwave extractor for extraction at 300W for 15min at a temperature of 60°C, and centrifuging to obtain a first clear liquid and a first filter residue, wherein the mass ratio of the Acanthopanax powder to the Phragmites australis powder is 100:1, the third low eutectic solvent is prepared from choline chloride, sugars, and water in a molar ratio of 1:3:15, and the solid-liquid ratio of the Acanthopanax powder to the third low eutectic solvent is 1:10;

[0079] Step 3: Add the third low eutectic solvent to the first filter residue obtained in step 2, place in a microwave extractor and extract at 300W for 15 minutes at a temperature of 60°C, and centrifuge to obtain a second clear liquid and a second filter residue. The volume of the third low eutectic solvent in this step is 1 / 4 of the volume of the third low eutectic solvent in step 1;

[0080] Step 4: add a third low eutectic solvent to the second filter residue obtained in step 3, place in a microwave extractor and extract at 300W for 15min at a temperature of 60°C, and centrifuge to obtain a third clear liquid and a third filter residue. The volume of the third low eutectic solvent in this step is 1 / 4 of the volume of the third low eutectic solvent in step 1;

[0081] Step 5: collecting the first clear liquid, the second clear liquid and the third clear liquid, concentrating under reduced pressure and then spray drying to obtain the Acanthopanax senticosus extract.

[0082] The contents of eleutheroside B, eleutheroside E, isofraquinone and total flavonoids (recorded as hyperoside) were measured for the extracts of Acanthopanax senticosus obtained in Examples 1 to 3 and Comparative Examples 1 to 6. The present application adopts HPLC method to measure the contents of eleutheroside B, eleutheroside E and isofraquinone, and the specific operation is as follows: firstly accurately weigh 10 mg of eleutheroside B, eleutheroside E and isofraquinone in a 10 mL volumetric flask, add methanol to the volume, mix well, and prepare a 1 g / L standard solution for standby use, then take different volumes of standard solution to prepare standard working solutions of different concentrations, use a disposable syringe (5 mL) to filter the standard working solutions of each concentration through a 0.22 μm filter membrane, store them in a 1.5 mL brown glass injection bottle, and perform HPLC determination, wherein the detection wavelengths of eleutheroside B, eleutheroside E and isofraquinone are 220 nm, 206 nm and 344 nm, respectively, and draw a standard curve. The test method of the Acanthopanax senticosus extract samples obtained in Examples 1 to 3 and Comparative Examples 1 to 6 was the same as the standard test method. The standard working solution of each concentration was filtered through a 0.22 μm filter membrane using a disposable syringe (5 mL), stored in a 1.5 mL brown glass injection bottle, and HPLC was performed to record the peak areas corresponding to Acanthopanax senticosus glycoside B, Acanthopanax senticosus glycoside E, and Isofraxidin, and substituted into the mass concentration of the sample in the standard curve. The total flavonoid content was determined by spectrophotometry, specifically: accurately weigh an appropriate amount of hyperoside reference substance (dried to constant weight under reduced pressure at 120°C), and prepared into a 1 mL standard solution containing 0.06 mg with methanol. Different volumes of standard solutions were taken to prepare standard working solutions of different concentrations. After color development with AlCl3 and sodium acetate, the absorbance was measured at 400nm by spectrophotometry, and a standard curve was drawn. 10 mg of the Acanthopanax senticosus extract obtained in Examples 1 to 3 and Comparative Examples 1 to 6 was accurately weighed in a 50mL volumetric flask, and 80% methanol was added to dissolve and set to volume for later use. After color development with AlCl3 and sodium acetate, the absorbance was measured at 400nm by spectrophotometry, and the mass fraction of total flavonoids was calculated according to the standard curve. The results are shown in Table 1.

[0083] Table 1 Contents of eleutheroside B, eleutheroside E, isofraxidin and total flavonoids (expressed as hyperoside)

[0084]

[0085]

[0086] As shown in Table 1, the contents of eleutheroside B, eleutheroside E, isofraquinone and total flavonoids in the Acanthopanax extracts obtained in Examples 1 to 3 were not much different, among which the minimum content of eleutheroside B was 1.4867 mg / g, the minimum content of eleutheroside E was 1.5923 mg / g, the minimum content of isofraquinone was 0.6829 mg / g, and the minimum content of total flavonoids was 41.236 mg / g. Relative to the preparation method of Example 1, the three-step extraction of Comparative Example 1 adopts conventional 75% ethanol extraction, and no synergistic auxiliary Chinese medicine is added; the three-step extraction of Comparative Example 2 adopts conventional 75% ethanol extraction, but adds synergistic auxiliary Chinese medicine, the three-step extraction of Comparative Example 3 adopts the first low eutectic solvent extraction, and no synergistic auxiliary Chinese medicine is added, the three-step extraction of Comparative Example 4 adopts the first low eutectic solvent extraction, but adds synergistic auxiliary Chinese medicine, the three-step extraction of Comparative Example 5 adopts the second low eutectic solvent, but adds synergistic auxiliary Chinese medicine, and the three-step extraction of Comparative Example 6 adopts the third low eutectic solvent, but adds synergistic auxiliary Chinese medicine. The results in Table 1 show that the contents of Acanthopanax Senticosides B, Acanthopanax Senticosides E, Isofraxin and total flavonoids obtained in Example 1 are much higher than the contents of Acanthopanax Senticosides B, Acanthopanax Senticosides E, Isofraxin and total flavonoids obtained in Comparative Examples 1 to 6. Comparing the data of Comparative Example 1 and Comparative Example 2, Comparative Example 3 and Comparative Example 4, respectively, it can be seen that the addition of synergistic auxiliary Chinese medicine can significantly increase the content of eleutheroside B, eleutheroside E, isofraquinone and total flavonoids, but in the low eutectic system, the increase in the content of eleutheroside B, eleutheroside E, isofraquinone and total flavonoids is more obvious. This is because the synergistic auxiliary Chinese medicine can provide hydrogen bond donors, which is beneficial to the release of the target compound, and the amino acids and polysaccharides contained in the synergistic auxiliary Chinese medicine in the low eutectic system can be used as a supplement to the low eutectic system. This can be seen from the data of Comparative Examples 4 to 6. Comparative Example 5 uses the second low eutectic solvent, and its system does not contain amino acids and polysaccharides. The obtained eleutheroside B, eleutheroside E, isofraquinone and total flavonoids content are lower than those of Comparative Examples 4 and 6. From the data of Comparative Examples 2 to 6, it can be seen that the eutectic system has significantly improved the contents of eleutheroside B, eleutheroside E, isofraquinone and total flavonoids compared with the traditional ethanol extraction. From the data of Example 1 and Comparative Examples 4 to 6, it can be seen that the three groups of eutectic systems are extracted in a certain order relative to one group of eutectic systems extracted 3 times, and the contents of eleutheroside B, eleutheroside E, isofraquinone and total flavonoids are qualitatively improved.

[0087] In order to compare the effects of the extraction order of the three groups of deep eutectic solvents on the contents of eleutheroside B, eleutheroside E, isoflurane and total flavonoids, the present application conducted corresponding comparative experiments, which are comparative examples 7 to 12 respectively.

[0088] Comparative Example 7

[0089] Step 1: Dry the Acanthopanax senticosus leaves and crush them into a particle size of 40 meshes to obtain Acanthopanax senticosus powder; at the same time, dry the Phragmites australis and crush them into a particle size of 40 meshes to obtain auxiliary Chinese medicine powder; set aside

[0090] Step 2, adding a second low eutectic solvent to the mixture of Acanthopanax senticosus powder and Phragmites australis powder, placing the mixture in a microwave extractor at 300W for 15min at a temperature of 60°C, and centrifuging to obtain a second clear liquid and a second filter residue, wherein the mass ratio of the Acanthopanax senticosus powder to the Phragmites australis powder is 100:1, and the second low eutectic solvent is prepared from choline chloride, ethylene glycol, and water in a molar ratio of 1:5:50, and the volume of the second low eutectic solvent is 1 / 4 of the volume of the first low eutectic solvent;

[0091] Step 3, adding a first low eutectic solvent to the first filter residue obtained in step 2, placing in a microwave extractor and extracting at 300W for 15min at a temperature of 60°C, and centrifuging to obtain a first clear liquid and a first filter residue, wherein the first low eutectic solvent is prepared from choline chloride, arginine, and water in a molar ratio of 1:0.5:200, and the solid-liquid ratio of Acanthopanax senticosus powder to the first low eutectic solvent is 1:10;

[0092] Step 4: adding a third low eutectic solvent to the second filter residue obtained in step 3, placing the mixture in a microwave extractor and extracting it at 300 W for 15 min at a temperature of 60° C., and centrifuging to obtain a third clear liquid and a third filter residue, wherein the third low eutectic solvent is prepared from choline chloride, rhamnose, and water in a molar ratio of 1:2:10, and the volume of the third low eutectic solvent is 1 / 4 of the volume of the first low eutectic solvent;

[0093] Step 5: collecting the first clear liquid, the second clear liquid and the third clear liquid, concentrating under reduced pressure and then spray drying to obtain the Acanthopanax senticosus extract.

[0094] Comparative Example 8

[0095] Step 1: Dry the Acanthopanax senticosus leaves and crush them into a particle size of 40 meshes to obtain Acanthopanax senticosus powder; at the same time, dry the Phragmites australis and crush them into a particle size of 40 meshes to obtain auxiliary Chinese medicine powder; set aside

[0096] Step 2, adding a first low eutectic solvent to the mixture of Acanthopanax powder and Phragmites australis powder, placing it in a microwave extractor and extracting it at 300W for 15min at a temperature of 60°C, and centrifuging to obtain a first clear liquid and a first filter residue, wherein the mass ratio of the Acanthopanax powder to the Phragmites australis powder is 100:1, the first low eutectic solvent is prepared from choline chloride, arginine, and water in a molar ratio of 1:0.5:200, and the solid-liquid ratio of the Acanthopanax powder to the first low eutectic solvent is 1:10;

[0097] Step 3: adding a third low eutectic solvent to the first filter residue obtained in step 2, placing the mixture in a microwave extractor and extracting it at 300 W for 15 min at a temperature of 60° C., and centrifuging to obtain a third clear liquid and a third filter residue, wherein the third low eutectic solvent is prepared from choline chloride, rhamnose, and water in a molar ratio of 1:2:10, and the volume of the third low eutectic solvent is 1 / 4 of the volume of the first low eutectic solvent;

[0098] Step 4: adding a second low eutectic solvent to the second filter residue obtained in step 3, placing the mixture in a microwave extractor and extracting it at 300 W for 15 min at a temperature of 60° C., and centrifuging to obtain a second clear liquid and a second filter residue, wherein the second low eutectic solvent is prepared from choline chloride, ethylene glycol, and water in a molar ratio of 1:5:50, and the volume of the second low eutectic solvent is 1 / 4 of the volume of the first low eutectic solvent;

[0099] Step 5: collecting the first clear liquid, the second clear liquid and the third clear liquid, concentrating under reduced pressure and then spray drying to obtain the Acanthopanax senticosus extract.

[0100] Comparative Example 9

[0101] Step 1: Dry the Acanthopanax senticosus leaves and crush them into a particle size of 40 meshes to obtain Acanthopanax senticosus powder; at the same time, dry the Phragmites australis and crush them into a particle size of 40 meshes to obtain auxiliary Chinese medicine powder; set aside

[0102] Step 2, adding a third low eutectic solvent to the mixture of Acanthopanax senticosus powder and Phragmites australis powder, placing the mixture in a microwave extractor at 300W for 15min at a temperature of 60°C, and centrifuging to obtain a third clear liquid and a third filter residue, wherein the mass ratio of the Acanthopanax senticosus powder to the Phragmites australis powder is 100:1, the third low eutectic solvent is prepared from choline chloride, rhamnose, and water in a molar ratio of 1:2:10, and the volume of the third low eutectic solvent is 1 / 4 of the volume of the first low eutectic solvent;

[0103] Step 3: adding a second low eutectic solvent to the first filter residue obtained in step 2, placing the mixture in a microwave extractor and extracting it at 300 W for 15 min at a temperature of 60° C., and centrifuging to obtain a second clear liquid and a second filter residue, wherein the second low eutectic solvent is prepared from choline chloride, ethylene glycol, and water in a molar ratio of 1:5:50, and the volume of the second low eutectic solvent is 1 / 4 of the volume of the first low eutectic solvent;

[0104] Step 4: add the first low eutectic solvent to the second filter residue obtained in step 3, place it in a microwave extractor and extract it at 300W for 15 minutes at a temperature of 60°C, and centrifuge to obtain a first clear liquid and a first filter residue, wherein the first low eutectic solvent is prepared from choline chloride, arginine, and water in a molar ratio of 1:0.5:200, and the solid-liquid ratio of Acanthopanax powder to the first low eutectic solvent is 1:10; Step 5: collect the first clear liquid, the second clear liquid, and the third clear liquid, concentrate them under reduced pressure, and then spray dry them to obtain an Acanthopanax extract.

[0105] Comparative Example 10

[0106] Step 1: Dry the Acanthopanax senticosus leaves and crush them into a particle size of 40 meshes to obtain Acanthopanax senticosus powder; at the same time, dry the Phragmites australis and crush them into a particle size of 40 meshes to obtain auxiliary Chinese medicine powder; set aside

[0107] Step 2, adding a second low eutectic solvent to the mixture of Acanthopanax senticosus powder and Phragmites australis powder, placing the mixture in a microwave extractor at 300W for 15min at a temperature of 60°C, and centrifuging to obtain a second clear liquid and a second filter residue, wherein the mass ratio of the Acanthopanax senticosus powder to the Phragmites australis powder is 100:1, the second low eutectic solvent is prepared from choline chloride, ethylene glycol, and water in a molar ratio of 1:5:50, and the volume of the second low eutectic solvent is 1 / 4 of the volume of the first low eutectic solvent;

[0108] Step 3: adding a third low eutectic solvent to the first filter residue obtained in step 2, placing the mixture in a microwave extractor and extracting it at 300 W for 15 min at a temperature of 60° C., and centrifuging to obtain a third clear liquid and a third filter residue, wherein the third low eutectic solvent is prepared from choline chloride, rhamnose, and water in a molar ratio of 1:2:10, and the volume of the third low eutectic solvent is 1 / 4 of the volume of the first low eutectic solvent;

[0109] Step 4: adding a first low eutectic solvent to the second filter residue obtained in step 3, placing the mixture in a microwave extractor and extracting it at 300W for 15 minutes at a temperature of 60°C, and centrifuging to obtain a first clear liquid and a first filter residue, wherein the first low eutectic solvent is prepared from choline chloride, arginine, and water in a molar ratio of 1:0.5:200, and the solid-liquid ratio of Acanthopanax powder to the first low eutectic solvent is 1:10;

[0110] Step 5: collecting the first clear liquid, the second clear liquid and the third clear liquid, concentrating under reduced pressure and then spray drying to obtain the Acanthopanax senticosus extract.

[0111] Comparative Example 11

[0112] Step 1: Dry the Acanthopanax senticosus leaves and crush them into a particle size of 40 meshes to obtain Acanthopanax senticosus powder; at the same time, dry the Phragmites australis and crush them into a particle size of 40 meshes to obtain auxiliary Chinese medicine powder; set aside

[0113] Step 2, adding a third low eutectic solvent to the mixture of Acanthopanax senticosus powder and Phragmites australis powder, placing the mixture in a microwave extractor at 300W for 15min at a temperature of 60°C, and centrifuging to obtain a third clear liquid and a third filter residue, wherein the mass ratio of the Acanthopanax senticosus powder to the Phragmites australis powder is 100:1, the third low eutectic solvent is prepared from choline chloride, rhamnose, and water in a molar ratio of 1:2:10, and the volume of the third low eutectic solvent is 1 / 4 of the volume of the first low eutectic solvent;

[0114] Step 3, adding a first low eutectic solvent to the first filter residue obtained in step 2, placing in a microwave extractor and extracting at 300W for 15min at a temperature of 60°C, and centrifuging to obtain a first clear liquid and a first filter residue, wherein the first low eutectic solvent is prepared from choline chloride, arginine, and water in a molar ratio of 1:0.5:200, and the solid-liquid ratio of Acanthopanax senticosus powder to the first low eutectic solvent is 1:10;

[0115] Step 4: adding a second low eutectic solvent to the second filter residue obtained in step 3, placing the mixture in a microwave extractor and extracting it at 300 W for 15 min at a temperature of 60° C., and centrifuging to obtain a second clear liquid and a second filter residue, wherein the second low eutectic solvent is prepared from choline chloride, ethylene glycol, and water in a molar ratio of 1:5:50, and the volume of the second low eutectic solvent is 1 / 4 of the volume of the first low eutectic solvent;

[0116] Step 5: collecting the first clear liquid, the second clear liquid and the third clear liquid, concentrating under reduced pressure and then spray drying to obtain the Acanthopanax senticosus extract.

[0117] Example 1, Comparative Examples 7 to 11 The contents of eleutheroside B, eleutheroside E, isofraxidin and total flavonoids (expressed as hyperoside) were determined in the Acanthopanax senticosus extracts obtained. The results are shown in Table 2.

[0118] Table 2 Contents of eleutheroside B, eleutheroside E, isofraxidin and total flavonoids (expressed as hyperoside)

[0119]

[0120] Comparing the preparation methods of Example 1 and Comparative Examples 7 to 11, the difference lies in that the extraction order of the first deep eutectic solvent, the second deep eutectic solvent and the third deep eutectic solvent is different. The extraction order of Example 1 is the first deep eutectic solvent-the second deep eutectic solvent-the third deep eutectic solvent, the extraction order of Comparative Example 7 is the second deep eutectic solvent-the first deep eutectic solvent-the third deep eutectic solvent, and the extraction order of Comparative Example 8 is the first deep eutectic solvent-the third deep eutectic solvent-the second deep eutectic solvent; the extraction order of Comparative Example 9 is the third deep eutectic solvent-the second deep eutectic solvent-the first deep eutectic solvent; the extraction order of Comparative Example 10 is the second deep eutectic solvent-the third deep eutectic solvent-the first deep eutectic solvent, and the extraction order of Comparative Example 11 is the third deep eutectic solvent-the first deep eutectic solvent-the second deep eutectic solvent. The contents of Acanthopanax glycoside B and Isofraxidin obtained in Comparative Example 7 are significantly lower than those obtained in Example 1, the contents of Acanthopanax glycoside E and total flavonoids obtained in Comparative Example 8 are significantly lower than those obtained in Example 1, the contents of Acanthopanax E and Isofraxidin obtained in Comparative Example 9 are significantly lower than those obtained in Example 1, the data of Acanthopanax glycoside B and total flavonoids obtained in Comparative Example 10 are significantly lower than those obtained in Example 1, and the contents of Isofraxidin and total flavonoids obtained in Comparative Example 11 are significantly lower than those obtained in Example 1, because the three groups of low eutectic systems correspond to the difficulty of releasing the target compound. The subsequent purification of the Acanthopanax extract obtained in this application only needs to be adsorbed by a macroporous resin to meet the purification requirements.

[0121] As described above, according to the present invention, the present invention at least includes the following beneficial effects:

[0122] First, the preparation method of the Acanthopanax senticosus extract provided by the present invention proposes for the first time to use different eutectic systems for extraction in a certain order, and can simultaneously extract the target products flavonoids, eleutheroside B, eleutheroside E and isoflurane;

[0123] Second, the preparation method of the Acanthopanax senticosus extract provided by the present invention screens out for the first time a Chinese medicinal agent that has a synergistic extraction effect on the target product in Acanthopanax senticosus; and discloses the optimal synergistic ratio of the auxiliary Chinese medicinal agent and Acanthopanax senticosus;

[0124] Thirdly, the preparation method of Acanthopanax senticosus extract provided by the present invention has improved extraction rates of various target products compared with existing extraction methods;

[0125] Fourthly, the preparation method of Acanthopanax senticosus extract provided by the present invention adopts a low eutectic solvent, which is economical, readily available, safe, and has strong compatibility with existing microwave extraction technology, and can simplify the subsequent purification process.

[0126] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the specific embodiments shown and described herein.

Claims

1. A method for preparing an Acanthopanax senticosus extract, characterized in that: include: Step 1, drying and crushing the Acanthopanax senticosus leaves to a particle size of 40 meshes to obtain Acanthopanax senticosus powder; at the same time, drying and crushing the auxiliary Chinese medicines to a particle size of 40 meshes to obtain auxiliary Chinese medicine powders for standby use, wherein the auxiliary Chinese medicines are one or more of Phragmites australis, Angelica sinensis, Ophiopogon japonicus and Panax notoginseng; Step 2, adding a first low eutectic solvent to the mixture of Acanthopanax senticosus powder and auxiliary Chinese medicine powder, placing the mixture in a microwave extractor and extracting at 300-600W for 15-30min at a temperature of 60-80°C, and centrifuging to obtain a first clear liquid and a first filter residue, wherein the mass ratio of the Acanthopanax senticosus powder to the auxiliary Chinese medicine powder is 60-100:1, and the first low eutectic solvent is prepared from choline chloride, amino acid, and water in a molar ratio of 1:0.5-1.5:200-400, and the amino acid is a basic amino acid, and the basic amino acid is one or more of arginine, lysine, and histidine; Step 3: adding a second low eutectic solvent to the first filter residue obtained in step 2, placing the mixture in a microwave extractor and extracting it at 300-600W for 15-30min at a temperature of 60-80°C, and centrifuging to obtain a second clear liquid and a second filter residue, wherein the second low eutectic solvent is prepared from choline chloride, a polyol, and water in a molar ratio of 1:5-10:50-100, and the polyol is one or more of ethylene glycol, propylene glycol, and 1,4-butanediol; Step 4: adding a third low eutectic solvent to the second filter residue obtained in step 3, placing the mixture in a microwave extractor and extracting it at 300-600W for 15-30min at a temperature of 60-80°C, and centrifuging to obtain a third clear liquid and a third filter residue, wherein the third low eutectic solvent is prepared from choline chloride, sugar, and water in a molar ratio of 1:2-4:10-20, and the sugar is one or more of glucose, rhamnose, and fructose; Step 5, collecting the first clear liquid, the second clear liquid and the third clear liquid, concentrating under reduced pressure and then spray drying to obtain the Acanthopanax senticosus extract; Among them, the solid-liquid ratio of Acanthopanax senticosus powder and the first deep eutectic solvent in step 2 is 1:10~20g / mL, the volume of the second deep eutectic solvent in step 3 and the volume of the third deep eutectic solvent in step 4 are 1 / 4~1 / 2 and 1 / 4~1 / 3 of the volume of the first deep eutectic solvent, respectively.

2. The product obtained by the preparation method of Acanthopanax senticosus extract according to claim 1, characterized in that: The obtained product includes total flavonoids of Acanthopanax senticosus, isofraquinone, eleutheroside B and eleutheroside E, wherein the content of isofraquinone is ≥0.6829 mg / g; the content of eleutheroside B is ≥1.4867 mg / g; the content of eleutheroside E is ≥1.5923 mg / g; and the total flavonoids of Acanthopanax senticosus is ≥41.236 mg / g.

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