阅读说明：本技术 一种从短梗五加果中综合提取多种有效成分的方法及其应用 (Method for comprehensively extracting multiple effective components from acanthopanax sessiliflorus fruits and application thereof ) 是由 张宝堂 范斌 蒋乐文 于 2021-08-10 设计创作，主要内容包括：本发明公开了一种从短梗五加果中综合提取多种有效成分的方法及其应用,该方法包括：将短梗五加果清洗干燥后粉碎过筛,采用超临界CO-(2)萃取得到短梗五加果油和萃余物；将萃余物置于乙醇水溶液中加热回流后,固液分离得到滤液和滤渣；将滤渣加水超声提取后过滤并浓缩得到水提物,随后将水提物依次经盐析去蛋白、超滤除小分子和树脂脱色后浓缩干燥,即得短梗五加果多糖；进一步将滤液纯化后可得到短梗五加果多酚。本发明所提供的从短梗五加果中综合提取多种有效成分的方法,对短梗五加果起到了一个综合利用的效果,与一批原料提取一类成分相比,明显缩短了工艺所需时间、减少了植物原料的损耗,从而大大降低了生产成本。(The invention discloses a method for comprehensively extracting various effective components from acanthopanax sessiliflorus fruits and application thereof, wherein the method comprises the following steps: cleaning and drying Acanthopanax sessiliflorus fruit, pulverizing, sieving, and supercritical CO 2 Extracting to obtain acanthopanax sessiliflorus fruit oil and raffinate; placing the extraction residues in an ethanol water solution, heating and refluxing, and carrying out solid-liquid separation to obtain filtrate and filter residues; adding water into the filter residue, performing ultrasonic extraction, filtering and concentrating to obtain an aqueous extract, sequentially performing salting-out deproteinization, ultrafiltration to remove micromolecules, resin decoloration, concentration and drying on the aqueous extract to obtain acanthopanax sessiliflorus fruit polysaccharide; further purifying the filtrate to obtain the acanthopanax sessiliflorus fruit polyphenol. The method for comprehensively extracting various effective components from acanthopanax sessiliflorus fruits provided by the invention has a comprehensive utilization effect on acanthopanax sessiliflorus fruits, and has the advantages of simple process, low cost and high yieldCompared with the extraction of a class of components, the method obviously shortens the time required by the process, reduces the loss of plant raw materials and greatly reduces the production cost.)

1. A method for comprehensively extracting a plurality of effective components from acanthopanax sessiliflorus fruits is characterized in that,

the method comprises the following steps:

s1, cleaning, drying, crushing and sieving the acanthopanax sessiliflorus fruits by adopting supercritical CO₂Extracting to obtain acanthopanax sessiliflorus fruit oil and raffinate;

s2, placing the raffinate obtained in the step S1 in an ethanol water solution, heating and refluxing, and carrying out solid-liquid separation to obtain filtrate and filter residue;

s3, adding water into the filter residue obtained in the step S2, carrying out ultrasonic extraction, filtering and concentrating to obtain an aqueous extract, sequentially carrying out salting out and deproteinization, ultrafiltration to remove small molecules, resin decoloration, concentration and drying on the aqueous extract, and obtaining the acanthopanax sessiliflorus fruit polysaccharide.

2. The method for extracting multiple effective components from Acanthopanax sessiliflorus fruit in combination according to claim 1,

in step S3, the method further comprises,

concentrating the filtrate obtained in the step S2, recovering ethanol, diluting with water, filtering out insoluble substances, performing chromatographic separation with macroporous resin, washing with water, removing impurities, and eluting with ethanol water solution to obtain Acanthopanax sessiliflorus fruit polyphenol.

3. The method for extracting multiple effective components from Acanthopanax sessiliflorus fruit in combination according to claim 2,

in step S3, the concentration of the ethanol aqueous solution in the elution process is 60-70 v%.

4. The method for extracting multiple effective components from Acanthopanax sessiliflorus fruit in combination according to claim 1,

in the step S2, in the step S,

the ethanol content in the ethanol water solution is 75-88 v%;

and/or the temperature and the time of the heating reflux are 66-75 ℃ and 1-3h respectively.

5. The method for extracting multiple effective components from Acanthopanax sessiliflorus fruit in combination according to claim 1 or 4,

in the step S1, in the step S,

the supercritical CO₂The extraction process conditions are as follows: extracting at 30-60 deg.C under 15-50MPa for 1-6 h.

6. The method for extracting multiple effective components from Acanthopanax sessiliflorus fruit in combination according to any one of claims 1 to 5,

in the step S3, in the step S,

the salting-out is specifically that calcium chloride is added into the water extract according to the mass-volume ratio of 1g to 20-40mL, the pH is controlled to be 5-6, and precipitation is carried out for 30-60 min.

7. The method for extracting multiple effective components from Acanthopanax sessiliflorus fruit in combination according to any one of claims 1 to 5,

in the step S3, in the step S,

the ultrafiltration is specifically that an ultrafiltration membrane is adopted to intercept molecules with the molecular weight of more than 40000 daltons, and trapped fluid is collected.

8. The method for extracting multiple effective components from Acanthopanax sessiliflorus fruit in combination according to any one of claims 1 to 7,

in step S3, in the ultrasound extraction process,

the adding amount of water is 10-25mL corresponding to 1g of filter residue;

and/or the power and time of the ultrasonic extraction are respectively 100-600W and 10-30 min.

9. The method for extracting multiple effective components from Acanthopanax sessiliflorus fruit in combination according to claim 8,

in step S3, the concentration is specifically performed by controlling the volume of the water extract to be 0.1 to 0.2 times of the volume before concentration.

10. Use of the method according to any one of claims 1 to 9 for the integrated extraction of the various active ingredients from acanthopanax sessiliflorus fruits.

Technical Field

The invention belongs to the technical field of extraction of plant active ingredients, and particularly relates to a method for comprehensively extracting multiple active ingredients from acanthopanax sessiliflorus fruits and application thereof.

Background

The acanthopanax sessiliflorus is also called crow fruit and acanthopanax sessiliflorus, wild species are mainly produced in alpine dense forest in northeast China, and north China, Korean peninsula and Russia are also distributed. The acanthopanax sessiliflorus fruit has the effects of medicine and food, contains rich active ingredients, volatile oil, phenolic acid, flavone, polysaccharide, glycoside and other effective ingredients, has high content in the acanthopanax sessiliflorus fruit, has wide pharmacological action, and has the effects of resisting inflammation, easing pain, resisting aging, resisting tumors, resisting platelet aggregation, reducing blood pressure and regulating immunity.

In the prior art, the extraction technology of effective active ingredients in acanthopanax sessiliflorus fruits is only limited to extract one kind or one kind of ingredients, and other ingredients with higher values still exist in residues after extraction, so that the waste of resources is caused.

Specifically, for the development of the acanthopanax sessiliflorus fruit volatile oil, steam distillation and solvent methods are mainly reported at present. Wherein, the steam distillation method is simple to operate, but the active ingredients of the volatile oil can be damaged at high temperature, and the oil yield is not high, so that the steam distillation method is generally only suitable for laboratory research; in the solvent method, the extraction rate is higher than that of the squeezing method because the extraction is directly performed by using solvents such as ether and ethanol, but the quality is affected because volatile oil components obtained by extraction contain a large amount of impurities, and a large amount of solvents remain in the herb residue and are difficult to remove, so that the further utilization of the herb residue is affected.

Specifically, for the deproteinization purification of acanthopanax sessiliflorus fruit polysaccharide, sevag reagent method, trichloroacetic acid method and enzyme method are mainly reported at present, and the three methods have good removal effect on protein, but have respective limitations. Wherein, the sevag reagent consists of chloroform and n-butyl alcohol, the chloroform is a highly toxic solvent and has pungent smell, and the method can completely remove the protein by repeating for more than 5 times; trichloroacetic acid has a pungent odor although it is low in toxicity, and the acidity easily destroys part of the polysaccharide structure; the enzyme method is simple and convenient to operate, but new impurity proteins are introduced and specific action conditions are required.

In summary, there is a need to develop a method for extracting multiple effective components from acanthopanax sessiliflorus fruit, which can improve the utilization of acanthopanax sessiliflorus fruit raw material, obtain acanthopanax sessiliflorus fruit volatile oil with higher yield, and improve the purity of acanthopanax sessiliflorus fruit polysaccharide and acanthopanax sessiliflorus fruit polyphenol obtained.

Disclosure of Invention

In view of the above, the invention provides a method for comprehensively extracting various effective components from acanthopanax sessiliflorus fruits and application thereof.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a method for comprehensively extracting multiple effective components from acanthopanax sessiliflorus fruits comprises the following steps:

s1, cleaning, drying, crushing and sieving the acanthopanax sessiliflorus fruits by adopting supercritical CO₂Extracting to obtain acanthopanax sessiliflorus fruit oil and raffinate;

s2, placing the raffinate obtained in the step S1 in an ethanol water solution, heating and refluxing, and carrying out solid-liquid separation to obtain filtrate and filter residue;

s3, adding water into the filter residue obtained in the step S2, carrying out ultrasonic extraction, filtering and concentrating to obtain an aqueous extract, sequentially carrying out salting out and deproteinization, ultrafiltration to remove small molecules, resin decoloration, concentration and drying on the aqueous extract, and obtaining the acanthopanax sessiliflorus fruit polysaccharide.

In the above technical solution, step S3 further includes,

concentrating the filtrate obtained in the step S2, recovering ethanol, diluting with water, filtering out insoluble substances, performing chromatographic separation with macroporous resin, washing with water, removing impurities, and eluting with ethanol water solution to obtain Acanthopanax sessiliflorus fruit polyphenol.

Further, in the above technical solution, in step S3, the concentration of the ethanol aqueous solution in the elution process is 60-70 v%.

In the above technical solution, in step S2, the content of ethanol in the ethanol aqueous solution is 75-88 v%.

In the above technical solution, in step S2, the temperature and time of the heating reflux are 66-75 ℃ and 1-3h, respectively.

In a specific embodiment of the present invention, in step S2, the number of times of the heating reflux is 1 to 3, and the reflux solutions obtained by heating reflux for a plurality of times are combined and then filtered to obtain a filtrate.

Further, in the above technical solution, in step S1,

the supercritical CO₂The extraction process conditions are as follows: the extraction pressure is 15-50MPa, the extraction temperature is 30-60 ℃, and the extraction time is 1-6 h.

Specifically, in the above technical solution, in step S3,

the salting-out is specifically carried out in a way that the mass volume ratio of the salting-out to the water extract is 1 g: adding calcium chloride at a ratio of 20-40mL, controlling pH at 5-6, and precipitating for 30-60 min.

Specifically, in the above technical solution, in step S3, the ultrafiltration specifically includes using an ultrafiltration membrane to intercept molecules having a molecular weight greater than 40000 daltons, and collecting the intercepted solution.

Still further, in the above technical solution, in step S3, the amount of water added is 10-25mL corresponding to 1g of filter residue during the ultrasonic extraction.

Still further, in the above technical solution, in step S3, the power and time of the ultrasonic extraction are respectively 100-.

In a specific embodiment of the present invention, in step S3, the number of times of ultrasonic extraction is 1-3, ultrasonic post-filtration is performed, and the filtrates are combined and then concentrated to obtain an aqueous extract.

In detail, in step S3, the concentration is specifically performed by controlling the volume of the water extract to be 0.1 to 0.2 times of the volume before concentration.

The invention also provides the application of the method in comprehensively extracting various effective components in the acanthopanax sessiliflorus fruit.

Compared with the prior art, the invention has the following advantages:

(1) the method for comprehensively extracting various effective components from the acanthopanax sessiliflorus fruits fully exerts the characteristic of high efficiency of supercritical extraction, can efficiently extract volatile components and fat-soluble components from the acanthopanax sessiliflorus fruits, and has high yield of products; compared with a solvent method, the obtained product has high purity and good quality, and no organic solvent residue exists in the product;

(2) according to the method for comprehensively extracting various effective components from acanthopanax sessiliflorus fruits, any entrainer is not added in the supercritical extraction process, so that the extraction product does not contain any impurity;

(3) the method for comprehensively extracting multiple effective components from acanthopanax sessiliflorus fruits provided by the invention is to extract multiple effective components from supercritical CO₂Extracting polysaccharide components from the raffinate, and refluxing with high-concentration ethanol firstly, so that non-saccharide impurities in the crude polysaccharide obtained by water extraction are less, the difficulty in subsequent polysaccharide purification is reduced, and the high-concentration ethanol reflux is recovered from ethanol, diluted with water and purified by a macroporous resin column, so that the high-purity acanthopanax sessiliflorus fruit polyphenol is obtained;

(4) the method for comprehensively extracting various effective components from acanthopanax sessiliflorus fruits provided by the invention has the advantages that the polysaccharide is separated and purified without adopting the traditional alcohol precipitation method, the polysaccharide loss is low, proteins are removed by salting out, and impurities such as micromolecular substances, oligosaccharides and the like can be removed after ultrafiltration;

(5) the method for comprehensively extracting various effective components from acanthopanax sessiliflorus fruits provided by the invention has a comprehensive utilization effect on the acanthopanax sessiliflorus fruits, obviously shortens the time required by the process and reduces the loss of plant raw materials compared with the method for extracting one kind of components from a batch of raw materials, thereby greatly reducing the production cost.

Drawings

FIG. 1 is a process flow chart of the method for comprehensively extracting various effective components from Acanthopanax sessiliflorus fruit according to the embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments.

It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

In the examples, the means used are conventional in the art unless otherwise specified.

The terms "comprises," "comprising," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The dried acanthopanax sessiliflorus fruits used in the embodiment of the invention are commercially available products.

Example 1

The embodiment of the invention provides a method for comprehensively extracting multiple effective components from acanthopanax sessiliflorus fruits, which specifically comprises the following steps:

s1, cleaning the acanthopanax sessiliflorus fruits, drying the acanthopanax sessiliflorus fruits at 50 ℃ to constant weight, crushing the acanthopanax sessiliflorus fruits, sieving the crushed acanthopanax sessiliflorus fruits with a 20-mesh sieve, putting 1kg of the acanthopanax sessiliflorus fruits into a supercritical extraction kettle, extracting the acanthopanax sessiliflorus fruits at 50 ℃ under the extraction pressure of 40MPa for 120min by using carbon dioxide fluid, and extracting the acanthopanax sessiliflorus fruits at 55 ℃ under the analyzer pressure of 5MPa to obtain 42.55g of acanthopanax sessiliflorus fruit extract (acanthopanax sessiliflorus fruit oil) and extract residues;

s2, mixing supercritical CO₂Refluxing the extracted raffinate with 10L 80 v% ethanol water solution at 70 deg.C for 1.5h, repeating twice, mixing the two refluxed solutions, and performing solid-liquid separation to obtain filtrate and residue;

s3, adding 10L of water into the filter residue obtained in the step S2 according to the amount that 12.5mL of water is added into 1g of filter residue, carrying out ultrasonic extraction for 20min under the power of 300W, repeating ultrasonic extraction for 2 times according to the same process conditions, filtering, merging the filtrate, concentrating the filtrate to obtain 2L of water extract, adding 60g of calcium chloride into the water extract, adjusting the pH to 5.5, carrying out reaction precipitation for 60min, carrying out centrifugal filtration to obtain supernatant, carrying out salting-out on the supernatant again under the same process conditions to obtain deproteinized liquid, passing through an ultrafiltration membrane with the molecular weight cutoff of 4 ten thousand daltons, collecting the trapped liquid, passing through decolorizing resin, concentrating the eluent, and carrying out spray drying to obtain 38.62g of acanthopanax brachypus fruit polysaccharide.

Example 2

The embodiment of the invention provides a method for comprehensively extracting multiple effective components from acanthopanax sessiliflorus fruits, which specifically comprises the following steps:

s1, cleaning the acanthopanax sessiliflorus fruits, drying the acanthopanax sessiliflorus fruits at 50 ℃ to constant weight, crushing the acanthopanax sessiliflorus fruits, sieving the crushed acanthopanax sessiliflorus fruits by a 30-mesh sieve, putting 2kg of the acanthopanax sessiliflorus fruits into a supercritical extraction kettle, extracting the acanthopanax sessiliflorus fruits at 45 ℃ under 35MPa for 90min by using carbon dioxide fluid, and extracting the acanthopanax sessiliflorus fruits at 50 ℃ under 5MPa by using an analyzer to obtain 73.55g of acanthopanax sessiliflorus fruit extract (acanthopanax sessiliflorus fruit oil) and extract residues;

s2, mixing supercritical CO₂Refluxing the extracted raffinate with 10L 85 v% ethanol water solution at 70 deg.C for 1.5h, repeating twice, mixing the two refluxed solutions, and performing solid-liquid separation to obtain filtrate and residue;

s3, adding 20L of water into the filter residue obtained in the step S2 according to the amount that 12mL of water is added into 1g of filter residue, carrying out ultrasonic extraction for 30min under the power of 400W, repeating ultrasonic extraction for 2 times according to the same process conditions, filtering, merging the filtrate, concentrating the filtrate to obtain 4L of water extract, adding 130g of calcium chloride into the water extract, adjusting the pH to 5.0, carrying out reaction precipitation for 60min, carrying out centrifugal filtration to obtain supernatant, carrying out salting-out on the supernatant again under the same process conditions to obtain deproteinized liquid, passing through an ultrafiltration membrane with the molecular weight of 4 ten thousand daltons, collecting trapped liquid, passing through decolorizing resin, concentrating the eluent, and carrying out spray drying to obtain 82.93g of acanthopanax sessiliflorus fruit polysaccharide.

Example 3

The embodiment of the invention provides a method for comprehensively extracting multiple effective components from acanthopanax sessiliflorus fruits, which specifically comprises the following steps:

s1, cleaning the acanthopanax sessiliflorus fruits, drying the acanthopanax sessiliflorus fruits at 50 ℃ to constant weight, crushing the acanthopanax sessiliflorus fruits, sieving the crushed acanthopanax sessiliflorus fruits with a 20-mesh sieve, putting 10kg of the acanthopanax sessiliflorus fruits into a supercritical extraction kettle, extracting the acanthopanax sessiliflorus fruits at 50 ℃ under the extraction pressure of 45MPa for 120min by using carbon dioxide fluid, and extracting the acanthopanax sessiliflorus fruits at 55 ℃ under the analyzer pressure of 5MPa to obtain 435.67g of acanthopanax sessiliflorus fruit extract (acanthopanax sessiliflorus fruit oil) and extract residues;

s2, mixing supercritical CO₂Refluxing the extracted raffinate with 120L 80 v% ethanol water solution at 80 deg.C for 1.0h, repeating twice, mixing the two refluxed solutions, and performing solid-liquid separation to obtain filtrate and residue;

s3, adding 120L of water into the filter residue obtained in the step S2 according to the amount that 12.8mL of water is added into 1g of filter residue, carrying out ultrasonic extraction for 30min under the power of 400W, repeating ultrasonic extraction for 2 times according to the same process conditions, filtering, merging the filtrate, concentrating the filtrate to obtain 24L of water extract, adding 700g of calcium chloride into the water extract, adjusting the pH to 5.0, carrying out reaction precipitation for 50min, carrying out centrifugal filtration to obtain supernatant, carrying out salting-out on the supernatant again under the same process conditions to obtain deproteinized liquid, passing through an ultrafiltration membrane with the molecular weight cutoff of 4 ten thousand daltons, collecting trapped liquid, passing through decolorizing resin, concentrating the eluent, and carrying out spray drying to obtain 429.93g of acanthopanax brachypus fruit polysaccharide; heating and concentrating the filtrate obtained in the step S2 to recover ethanol, adding water to dilute the filtrate by 2-4 times, performing chromatographic separation by using macroporous resin, washing the filtrate by using water to remove impurities, and eluting the filtrate by using an ethanol aqueous solution with the concentration of 60-70 v% to obtain 384.54g of acanthopanax sessiliflorus fruit polyphenol.

Comparative example 1

The invention provides a method for comprehensively extracting various effective components from acanthopanax sessiliflorus fruits, which comprises the following steps:

s1, cleaning the acanthopanax sessiliflorus fruits, drying the acanthopanax sessiliflorus fruits at 50 ℃ to constant weight, crushing the acanthopanax sessiliflorus fruits, sieving the crushed acanthopanax sessiliflorus fruits with a 20-mesh sieve, putting 1kg of the acanthopanax sessiliflorus fruits into a supercritical extraction kettle, extracting the acanthopanax sessiliflorus fruits at 50 ℃ under the extraction pressure of 40MPa for 120min by using carbon dioxide fluid, and extracting the acanthopanax sessiliflorus fruits at 55 ℃ under the analyzer pressure of 5MPa to obtain 42.55g of acanthopanax sessiliflorus fruit extract (acanthopanax sessiliflorus fruit oil) and extract residues;

s2, mixing supercritical CO₂Refluxing the extracted raffinate with 10L 92.5 v% ethanol water solution at 75 deg.C for 1.5h, repeating twice, mixing the two refluxed solutions, and performing solid-liquid separation to obtain filtrate and residue; s3, adding 10L of water into the filter residue obtained in the step S2 according to the amount that 13.0mL of water is added into 1g of filter residue, carrying out ultrasonic extraction for 20min under the power of 300W, and repeating the ultrasonic extraction according to the same technological conditionsPerforming sound extraction for 2 times, filtering, combining filtrates, concentrating the filtrate to obtain 2L of water extract, adding 60g of calcium chloride into the water extract, adjusting pH to 5.5, performing reaction precipitation for 60min, performing centrifugal filtration to obtain supernatant, performing salting-out on the supernatant again under the same process conditions to obtain deproteinized liquid, passing through an ultrafiltration membrane with the molecular weight cutoff of 4 ten thousand daltons, collecting the retentate, passing through decolorizing resin, concentrating the eluate, and spray-drying to obtain 37.86g of acanthopanax brachypus fruit polysaccharide.

Comparative example 2

The invention provides a method for comprehensively extracting various effective components from acanthopanax sessiliflorus fruits, which comprises the following steps:

s1, cleaning the acanthopanax sessiliflorus fruits, drying the acanthopanax sessiliflorus fruits at 50 ℃ to constant weight, crushing the acanthopanax sessiliflorus fruits, sieving the crushed acanthopanax sessiliflorus fruits by a 30-mesh sieve, putting 2kg of the acanthopanax sessiliflorus fruits into a supercritical extraction kettle, extracting the acanthopanax sessiliflorus fruits at 75MPa by using carbon dioxide fluid for 90min, and extracting the acanthopanax sessiliflorus fruits at 50 ℃ by using a resolver at 5MPa to obtain 79.38g of acanthopanax sessiliflorus fruit extract (acanthopanax sessiliflorus fruit oil) and extract residues;

s2, mixing supercritical CO₂Refluxing the extracted raffinate with 10L 85 v% ethanol water solution at 70 deg.C for 1.5h, repeating twice, mixing the two refluxed solutions, and performing solid-liquid separation to obtain filtrate and residue;

s3, adding 20L of water into the filter residue obtained in the step S2 according to the amount that 12.5mL of water is added into 1g of filter residue, carrying out ultrasonic extraction for 30min under the power of 400W, repeating ultrasonic extraction for 2 times according to the same process conditions, filtering, merging the filtrate, concentrating the filtrate to obtain 4L of water extract, adding 130g of calcium chloride into the water extract, adjusting the pH to 6.5, carrying out reaction precipitation for 60min, carrying out centrifugal filtration to obtain supernatant, carrying out salting-out on the supernatant again under the same process conditions to obtain deproteinized liquid, passing through an ultrafiltration membrane with the molecular weight cutoff of 4 ten thousand daltons, collecting trapped liquid, passing through decolorizing resin, concentrating the eluent, and carrying out spray drying to obtain 80.58g of acanthopanax brachypus fruit polysaccharide.

The protein residue and polysaccharide content in the obtained acanthopanax sessiliflorus fruit polysaccharide are measured by the following methods respectively:

(1) protein content determination method (Coomassie brilliant blue method)

Drawing a standard curve: adding 0, 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL, 1.2mL, 1.4mL and 1.6mL of 1mg/mL standard protein solution into 9 test tubes respectively, and adding water to 2.0 mL; adding more than 0.1mL of solution into 9 test tubes, respectively, adding 5mL of Coomassie brilliant blue reagent, mixing under shaking, standing at 595nm for 10min, and measuring absorbance of each tube with tube 0 as blank; and drawing a standard curve by taking the concentration of the standard protein as an abscissa and the absorbance value as an ordinate.

And (3) sample content determination: sampling 0.1mL of the solution, adding 5mL of Coomassie brilliant blue reagent into each test tube, mixing thoroughly, standing for 10min, taking reagent blank as control, carrying out color comparison at 595nm, and determining absorbance value.

(2) Method for measuring polysaccharide content (phenol-sulfuric acid method)

Drawing a standard curve: weighing 50mg of glucose in a 100ml volumetric flask, adding water to dilute to a scale mark to obtain a 0.5mg/ml standard solution, respectively sucking 2ml, 4ml, 6ml, 8ml, 10ml and 12ml of the standard solution into a 50ml volumetric flask, adding water to dilute to the scale mark, respectively sucking more than 2ml of standard reference substances into 10ml test tubes with stoppers, adding 1ml of 5% phenol test solution, shaking uniformly, quickly adding 5ml of concentrated sulfuric acid, shaking for 2 minutes, heating in a boiling water bath for 15 minutes, taking out, cooling, adding water to the scale mark, and cooling. Adding 5% phenol and concentrated sulfuric acid at a ratio of 1: 5 into a 10ml volumetric flask, and adding water to the scale to obtain a control solution; the absorbance was measured at 585nm, and a standard curve was plotted with the absorbance A as the abscissa and the concentration C as the ordinate.

And (3) polysaccharide content determination: taking 2ml of dissolved polysaccharide solution to a 10ml test tube with a plug, adding 1ml of 5% phenol test solution, shaking up, quickly adding 5ml of concentrated sulfuric acid, shaking up for 2 minutes, placing in a boiling water bath for heating for 15 minutes, taking out, cooling, adding water to scale, cooling again, measuring absorbance at 585nm, and calculating the polysaccharide content.

The protein residue and polysaccharide content in the acanthopanax sessiliflorus fruit polysaccharides of examples 1-3 and comparative examples 1-2 were measured by the above method, respectively, and the results are shown in table 1 below.

TABLE 1 detection results of protein residue and polysaccharide content in Acanthopanax sessiliflorus fruit polysaccharide

	Protein residues	Polysaccharide content
			Example 1	1.23wt％	84.29wt％
Example 2	1.07wt％	81.89wt％
			Example 3	1.12wt％	82.34wt％
Comparative example 1	1.28wt％	75.84wt％
			Comparative example 2	2.57wt％	77.57wt％

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention.

It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.