Extraction method of pseudo-ginseng flower glycopeptide, obtained product and application

Abstract

The invention discloses an extraction method of sanchi flower glycopeptide, an obtained product and application, sanchi flower is taken as a main raw material, firstly, extraction is performed with ethanol or an ethanol aqueous solution, then, ionic liquid extraction and deep eutectic solvent extraction are performed on filter residues in sequence, and the sanchi flower glycopeptide is obtained through alcohol precipitation, pigment removal, free protein removal and enzymolysis of the obtained extracting solution. The extraction method of the sanchi flower glycopeptide is simple in process, high in safety, high in yield and purity and suitable for industrial production, and the obtained sanchi flower glycopeptide has antioxidant activity, can be prepared into drugs, health care products, food and cosmetics and has good application value.

Description

Technical Field

[0001] The invention relates to a method for extracting notoginseng flower glycopeptide, a product obtained therefrom and an application thereof, and belongs to the technical field of extraction and application of effective components of traditional Chinese medicines. Background Art

[0002] Notoginseng flower, also known as Tianqi flower, mountain lacquer flower, ginseng flower, and golden flower, is the dried inflorescence of Panax notoginseng (Burk.) FHChen, a plant of the genus Panax in the Araliaceae family. Notoginseng flower has the effects of clearing away heat and detoxifying, calming the liver and improving eyesight, promoting fluid production and quenching thirst, calming and anti-inflammatory, promoting blood circulation and removing blood stasis, clearing the throat and moistening the throat, and lowering blood pressure. Studies have shown that notoginseng flower is rich in saponins, proteins, polysaccharides, dietary fiber, and a large number of mineral elements, and has a good nutritional effect on the human body. At present, the development and application of notoginseng flower mainly focus on small molecule compounds such as saponins, while the research on glycopeptide compounds with significant biological activity is insufficient, and there is no relevant report yet.

[0003] Glycopeptides refer to regions in glycoproteins and proteoglycans where sugars are covalently linked to amino acids or polypeptide chains. Glycopeptides have been widely used in medicines, foods, health foods, and cosmetics due to their structural stability, potential health benefits, and pharmacological activities. Glycopeptides have some of the characteristics of both polypeptides and polysaccharides, so their extraction methods are usually optimized based on the properties of polypeptides and polysaccharides. In recent years, ionic liquids and low eutectic solvents have attracted much attention as new solvents due to their important role in the dissolution of bioactive polysaccharides, starches, and proteins. These solvents can significantly improve the solubility of macromolecular proteins, polysaccharides, and glycopeptides, providing a broad platform for the development of natural renewable resources, and are expected to replace traditional organic solvents and solve the problems faced in the extraction process of natural compounds. In addition, glycopeptide components have been shown to have a variety of biological activities, such as antioxidant properties, which make them potentially useful in the treatment of certain diseases.

[0004] At present, there are no reports on the extraction of glycopeptides from Panax notoginseng flowers. The extraction of glycopeptides from Panax notoginseng flowers is of great significance for the further development and utilization of Panax notoginseng flowers. Summary of the invention

[0005] The purpose of the present invention is to provide a method for extracting a Panax notoginseng flower extract, more specifically, a method for extracting a Panax notoginseng flower glycopeptide, wherein the Panax notoginseng flower is first preliminarily purified, and then extracted with an ionic liquid and a ternary low eutectic solvent as solvents to obtain a Panax notoginseng flower crude glycoprotein, and then further purified and enzymolyzed to obtain a final glycopeptide. The present invention is simple to operate, highly safe, and pollution-free. The obtained extract contains a Panax notoginseng flower glycopeptide component, enriches the active ingredients in the Panax notoginseng flower, and has a high extraction rate of the Panax notoginseng flower glycopeptide.

[0006] The specific technical solution adopted by the present invention is as follows:

[0007] A method for extracting a notoginseng flower extract, wherein the extract is mainly notoginseng flower glycopeptide, the method comprising the following steps:

[0008] (1) extracting Panax notoginseng flower powder with ethanol or ethanol aqueous solution to obtain an extract 1 and a filter residue 1;

[0009] (2) drying the filter residue 1 and extracting it with an ionic liquid to obtain an extract 2 and a filter residue 2;

[0010] (3) drying the filter residue 2, and extracting it with a low eutectic solvent under microwave to obtain an extract 3 and a filter residue 3;

[0011] (4) combining extract 2 and extract 3, adding ethanol to precipitate, and collecting the precipitate to obtain notoginseng flower crude saccharoprotein;

[0012] (5) adding water to redissolve the crude saccharide protein from Panax notoginseng flower, and then passing it through a DEAE-23 fiber chromatography column for adsorption, and then eluting it with water and a sodium chloride solution in sequence, ultrafiltering the sodium chloride eluate to remove sodium chloride, and then combining it with the water eluate to obtain a decolorized crude saccharide protein solution from Panax notoginseng flower;

[0013] (6) removing free protein from the decolorized crude notoginseng flower glycoprotein solution by the Sevag method to obtain a purified notoginseng flower glycoprotein solution;

[0014] (7) The purified notoginseng flower glycoprotein solution is first enzymatically hydrolyzed with trypsin and then with alkaline protease to obtain notoginseng flower glycopeptide.

[0015] Furthermore, in step (1), the notoginseng flower powder is extracted with ethanol or a high concentration of ethanol aqueous solution, and the concentration of the ethanol aqueous solution used is greater than or equal to 80wt%, such as 80%, 85%, 90%, 95%. The number of extractions can be once or multiple times, such as twice, three times, or four times, preferably three times. The temperature of each extraction is 20-85°C, the amount of ethanol or ethanol aqueous solution used each time is 1-2 times the mass of the notoginseng flower powder, the time of each extraction is 30-60min, and the conditions of each extraction can be the same or different.

[0016] Preferably, in step (1), 75-80wt% ethanol solution of 1-2 times the mass of Panax notoginseng flower powder is added during the first extraction, stirred and extracted for 30-60min at room temperature, and filtered to obtain a filtrate and a filter residue; during the second extraction, 85-90wt% ethanol solution of 1-2 times the mass of Panax notoginseng flower powder is added to the filter residue obtained by the first extraction, and stirred and extracted for 30-60min at 55-65°C to obtain a filtrate and a filter residue; during the third extraction, 1-2 times the mass of Panax notoginseng flower powder is added to the filter residue obtained by the second extraction, and stirred and extracted for 30-60min at 75-85°C to obtain a filtrate and a filter residue; all filtrates of the three times are combined to obtain an extract 1. By gradually increasing the ethanol concentration and combining temperature control, impurities of different solubility can be effectively removed step by step, while improving the extraction rate of the target component. This method not only improves the extraction efficiency, but also reduces the complexity and cost of subsequent purification steps.

[0017] Furthermore, in step (2), the filter residue 1 is placed in a ventilated place to dry in the shade to a constant weight, and then extracted with an ionic liquid. The ionic liquid is an aqueous solution of 1-ethyl-3-methylimidazolium acetate with a concentration of 0.05-0.1 mol / L. The mass ratio of the filter residue 1 to the ionic liquid is 1:1-4, for example, 1:1, 1:2, 1:3, 1:4.

[0018] Furthermore, in step (2), the mixture of the filter residue 1 and the ionic liquid is extracted on a shaker, the shaker speed is 300-400 rpm, the extraction temperature is room temperature, and the extraction time is 6-12 hours. The ionic liquid can effectively promote the dissolution of the active ingredient, can achieve the extraction of the active ingredient at room temperature, avoid protein denaturation or impurity interference caused by high temperature and complex operation, and at the same time improve the extraction efficiency and purity, simplify the experimental steps, and maintain the biological activity and structural integrity of the glycoprotein.

[0019] Further, in step (3), the filter residue 2 is dried to constant weight and then extracted with a low eutectic solvent. The low eutectic solvent is a ternary low eutectic solvent, which is obtained by mixing choline chloride, guaiacol and lactic acid in a molar ratio of 1-2:1:1-2. The present invention has obtained a low eutectic solvent combination suitable for Panax notoginseng flower through a large number of experimental explorations, which has high solubility and selectivity, can more efficiently extract the target substance, and optimize the extraction efficiency and purity.

[0020] Furthermore, in step (3), the mass ratio of the filter residue 2 to the low eutectic solvent is 1:14-18.

[0021] Preferably, in step (3), microwave extraction technology is used for extraction, microwave heating is used to quickly increase the reaction temperature and rate, and the high efficiency dissolving capacity of the low eutectic solvent is used to achieve a fast, efficient and environmentally friendly extraction effect, which can significantly shorten the extraction time and improve the extraction rate of the target component. The extraction temperature is 50-85°C, such as 50°C, 60°C, 70°C, 80°C, 85°C, the microwave power is 100-130W, such as 100W, 110W, 120W, 130W, and the extraction time is 15-30min.

[0022] Furthermore, in step (4), the mass ratio of the total mass of extract 2 and extract 3 to anhydrous ethanol is 1:4-5, and the mixture is allowed to stand at 0-4°C for 12-24 hours for alcohol precipitation.

[0023] Further, in step (5), DEAE-23 cellulose column chromatography is used for decolorization. The crude glycoprotein of Panax notoginseng flower is added to water 15-25 times its mass for redissolution to obtain a crude glycoprotein solution of Panax notoginseng flower, and the solution is added to a DEAE-23 fiber chromatography column (DEAE cellulose DE-23) for adsorption for 10-15min, and then eluted with water and 0.5-1mol / L NaCl aqueous solution in turn, with an elution rate of 2-3BV / h and an elution volume of 3-7BV. DEAE-23 cellulose column chromatography has efficient adsorption capacity, can effectively remove pigments and impurities in glycoprotein crude extracts, and will not cause a large amount of loss of Panax notoginseng flower glycoprotein. It is also suitable for the separation of acidic glycoproteins and neutral glycoproteins, and has high purification efficiency and ease of operation.

[0024] Further, in step (6), the Sevag reagent can be a mixture of chloroform and n-butanol, or a mixture of dichloromethane and n-butanol, and the volume ratio of chloroform or dichloromethane to n-butanol is 4: 1. Dichloromethane has lower toxicity and higher safety, and has good solubility and extraction efficiency, can effectively reduce occupational hazard risks and meet experimental requirements, so a mixture of dichloromethane and n-butanol is preferred.

[0025] Furthermore, in step (6), the amount of Sevag reagent used is 15-25% of the volume of the decolorized notoginseng flower crude saccharoprotein solution.

[0026] Furthermore, in step (7), trypsin and alkaline protease are used for enzymatic hydrolysis, which can effectively decompose glycoprotein into soluble polypeptides or amino acids, improve extraction efficiency and reduce impurities, and optimize enzymatic hydrolysis conditions to improve product yield and quality. The amount of trypsin used is 5500-6500u / g, indicating that 5500-6500 units of enzyme activity are added per gram of Panax notoginseng flower glycoprotein.

[0027] Furthermore, in step (7), the amount of alkaline protease used is 180,000-220,000 u / g, indicating that 180,000-220,000 units of enzyme activity are added per gram of Panax notoginseng flower glycoprotein.

[0028] Furthermore, in step (7), trypsin is added to the solution at 30°C, and then the temperature is raised at a rate of 10-15°C / 60min until it reaches 60°C, and then alkaline protease is added, and the enzymolysis is continued for 2-3 hours. By gradually raising the temperature, the enzymolysis efficiency and product quality can be improved, while avoiding the damage of enzyme activity caused by high temperature, thereby achieving efficient and stable glycoprotein decomposition.

[0029] The present invention also provides a product obtained according to the above method, which is a Panax notoginseng peptide. It has been verified that the Panax notoginseng peptide has a good antioxidant effect. Therefore, the present invention also provides the use of the Panax notoginseng peptide in the preparation of antioxidant products. The antioxidant product can be a medicine, a health product, a food or a cosmetic.

[0030] The beneficial effects of the present invention are:

[0031] 1. The present invention discovered the glycopeptide components in Panax notoginseng flower for the first time through extraction and separation, enriching the active ingredients in Panax notoginseng flower.

[0032] 2. The present invention can effectively remove impurities step by step and retain target components by gradually increasing the ethanol concentration and combining temperature control, thereby improving extraction efficiency and reducing purification costs.

[0033] 3. The present invention uses ionic liquids to promote the dissolution of active ingredients, avoid problems caused by high temperature and complex operations, and improve extraction efficiency and purity.

[0034] 4. The ternary low eutectic solvent used in the present invention has higher dissolving power and selectivity than the binary low eutectic solvent. Combined with microwave extraction technology, the target component can be extracted quickly, efficiently and environmentally friendly.

[0035] 5. The DEAE-23 cellulose column chromatography method adopted in the present invention can efficiently adsorb and remove pigments and impurities in the crude glycoprotein extract without causing a large loss of Panax notoginseng flower glycoprotein.

[0036] 6. The dichloromethane used in the present invention has low toxicity, high safety, high dissolution and extraction efficiency, and can reduce occupational hazards.

[0037] 7. The present invention improves the enzymatic hydrolysis efficiency and product quality by gradually increasing the temperature of the enzymatic hydrolysis conditions. Trypsin and alkaline protease work synergistically to efficiently decompose glycoproteins, improve extraction efficiency and optimize product yield and quality.

[0038] 8. The present invention is the first to separate notoginseng flower glycopeptide from notoginseng flower. The notoginseng flower glycopeptide has good antioxidant activity and is of great significance for the further development and utilization of notoginseng flower. DETAILED DESCRIPTION

[0039] The technical solution of the present invention is clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0040] Unless otherwise specified, the following concentrations are all mass percentages.

[0041] In the following examples and comparative examples, the trypsin used was purchased from Nanning Pangbo Bioengineering Co., Ltd. with an enzyme activity of 4000 u / g, and the alkaline protease used was purchased from Henan Wanbang Industrial Co., Ltd. with an enzyme activity of 200000 u / g.

[0042] Example 1

[0043] S1. Dry the Panax notoginseng flower under cool and ventilated conditions, and then crush it into powder. Take the Panax notoginseng flower powder, add 1.5 times the mass of the powder to an 80wt% ethanol solution, place it in a magnetic stirrer, stir at room temperature for 60 minutes at a stirring speed of 1500rpm, filter and take the residue, add 1.5 times the mass of the Panax notoginseng flower powder to the residue, stir at 60℃ for 60min, filter and take the residue, add 1.5 times the mass of the Panax notoginseng flower powder to the residue, stir at 80℃ for 60min, filter and take the residue, record it as residue 1.

[0044] S2. Place the filter residue 1 of S1 in a ventilated place and dry it in the shade to constant weight. Mix the filter residue 1 with 3 times the mass of 0.1 mol / L 1-ethyl-3-methylimidazolium acetate ionic liquid, place it on a shaker at a speed of 300-400 rpm, shake at room temperature for 12 hours, and finally centrifuge to obtain extract 2 and filter residue 2.

[0045] S3. The residue 2 of S2 was placed in an oven at 60°C and dried to constant weight. The residue 2 was mixed with 16 times by mass of a ternary low eutectic solvent (choline chloride, guaiacol and lactic acid mixed in a molar ratio of 1.5:1:1.5), placed in a microwave synthesizer, extracted at 80°C and 120W for 25 min, and finally centrifuged to obtain filtrate 3 and residue 3.

[0046] S4. Combine the extract 2 and extract 3 obtained in S2 and S3, add 4-5 times the mass of anhydrous ethanol after the combined extract, let stand at 4°C for 12h, filter out the precipitate, and redissolve the precipitate in water to obtain a 50mg / ml Panax notoginseng flower crude saccharide protein solution.

[0047] S5. Add the crude saccharide protein solution of Panax notoginseng flower in S4 to a DEAE-23 fiber chromatography column, adsorb for 10 minutes, and then elute with water and 0.6 mol / L NaCl aqueous solution in sequence, with an elution rate of 3 BV / h, and the elution volumes of water and sodium chloride solution are 5 BV respectively. The eluate of the sodium chloride aqueous solution is desalted by an ultrafiltration tube, and then all the eluates are combined to obtain a depigmented crude saccharide protein solution of Panax notoginseng flower.

[0048] S6. Remove free protein from the depigmented Panax notoginseng flower crude saccharoprotein solution of S5 by Sevag method. Add Sevag reagent (mixed by dichloromethane and n-butanol in a volume ratio of 4:1) to the depigmented Panax notoginseng flower crude saccharoprotein solution at a volume fraction of 20%, stir on a shaker at 400 rpm for 10 minutes at room temperature, centrifuge, and retain the upper aqueous solution as the Panax notoginseng flower saccharoprotein solution from which free protein has been removed.

[0049] S7. The notoginseng glycoprotein solution of S6 was heated to 30°C, and then 1.5 g of trypsin was added per g of notoginseng glycoprotein, and then the temperature was raised by 10°C every 60 minutes for enzymatic hydrolysis. When the temperature reached 60°C, alkaline protease (trypsin: alkaline protease = 3:2, m / m) was added, and the enzymatic hydrolysis was continued at 60°C for 2 hours. After the enzymatic hydrolysis, the temperature was raised to inactivate the enzyme, and then centrifuged, and the obtained filtrate was freeze-dried to obtain notoginseng glycopeptide.

[0050] Example 2

[0051] The method of Example 1 was used to prepare Panax notoginseng flower glycopeptide, except that in step S1, Panax notoginseng flower powder was taken, 1.5 times the mass of 80wt% ethanol solution was added, placed in a magnetic stirrer, stirred at 1500 rpm for 60 min at room temperature, and then filtered to obtain the residue. The above operation was repeated twice to finally obtain residue 1.

[0052] Example 3

[0053] The notoginseng flower glycopeptide was prepared according to the method of Example 1, except that: in step S1, notoginseng flower powder was taken, 1.5 times by mass of 80wt% ethanol solution was added, the mixture was placed in a magnetic stirrer, and stirred at room temperature for 30 min at 1500 rpm, the residue was filtered out, 1.5 times by mass of 90wt% ethanol solution of notoginseng flower powder was added to the residue, the mixture was stirred at 60°C for 30 min, the residue was filtered out, 1.5 times by mass of anhydrous ethanol was added to the residue, the mixture was stirred at 80°C for 30 min, and the residue 1 was obtained by filtering.

[0054] Example 4

[0055] The notoginseng flower peptide was prepared according to the method of Example 1, except that in step S2, the filter residue 1 was mixed with an equal mass of 0.05 mol / L 1-ethyl-3-methylimidazolium acetate ionic liquid.

[0056] Example 5

[0057] The notoginseng glycoside peptide was prepared according to the method of Example 1, except that in step S3, extraction was performed at 50° C. and 100 W for 15 min.

[0058] Example 6

[0059] The Panax notoginseng peptide was prepared according to the method of Example 1, except that in step S7, the enzymatic hydrolysis was performed by increasing the temperature by 15° C. every 60 min.

[0060] Comparative Example 1

[0061] The notoginseng flower glycopeptide was prepared according to the method of Example 1, except that step S1 was omitted and the notoginseng flower powder was directly subjected to ionic liquid extraction.

[0062] Comparative Example 2

[0063] The Panax notoginseng peptide was prepared according to the method of Example 1, except that the order of steps S2 and S3 was exchanged, and step S3 was performed after step S1, and then step S2 was performed.

[0064] Comparative Example 3

[0065] The Panax notoginseng peptide was prepared according to the method of Example 1, except that in step S2, the ionic liquid was replaced with water.

[0066] Comparative Example 4

[0067] The Panax notoginseng peptide was prepared according to the method of Example 1, except that in step S2, the ionic liquid was replaced with 1-butyl-3-methylimidazolium chloride.

[0068] Comparative Example 5

[0069] The Panax notoginseng peptide was prepared according to the method of Example 1, except that in step S3, the ternary deep eutectic solvent was replaced with an equal mass of a binary deep eutectic solvent (choline chloride and lactic acid were mixed in a molar ratio of 2.5:1.5).

[0070] Comparative Example 6

[0071] The Panax notoginseng peptide was prepared according to the method of Example 1, except that in step S3, the ternary deep eutectic solvent was replaced with another ternary deep eutectic solvent of equal mass (betaine, propylene glycol and acetic acid were mixed in a molar ratio of 1.5:1:1.5).

[0072] Comparative Example 7

[0073] The notoginseng flower glycopeptide was prepared according to the method of Example 1, except that: in step S5, the DEAE-23 cellulose column chromatography method was replaced with the activated carbon adsorption method, and 3% of the volume of activated carbon was added to the notoginseng flower crude glycoprotein solution of S4, and the mixture was fully stirred and decolorized at 60°C for 40 minutes. After the decolorization was completed, the mixture was filtered, and the filtered liquid was the depigmented notoginseng flower crude glycoprotein solution.

[0074] Comparative Example 8

[0075] The notoginseng glycopeptide was prepared according to the method of Example 1, except that: in step S7, the notoginseng glycoprotein solution from which the free protein was removed in S6 was heated to 60°C, and then trypsin was added at a ratio of 1.5 g of trypsin per g of notoginseng glycoprotein, and enzymolysis was performed for 5 hours. After enzymolysis, the temperature was raised to inactivate the enzyme, and then centrifuged, and the obtained filtrate was freeze-dried to obtain notoginseng glycopeptide.

[0076] The Panax notoginseng peptides obtained in the above examples and comparative examples were evaluated in the following manner:

[0077] 1. Determination of Panax notoginseng flower glycopeptide content

[0078] 1.1 Determination of polysaccharide extraction rate from Panax notoginseng flower

[0079] 1.1.1 Drawing of standard curve

[0080] Accurately pipette 0μL, 10μL, 20μL, 30μL, 40μL, 50μL, 60μL, 70μL, 80μL, 90μL, and 100μL of glucose standard solution with a mass concentration of 0.1mg / mL into a centrifuge tube, add ultrapure water to make up to 100μL, mix well, and obtain standard solutions with mass concentrations of 0.00mg / mL, 0.01mg / mL, 0.02mg / mL, 0.03mg / mL, 0.04mg / mL, 0.05mg / mL, 0.06mg / mL, 0.07mg / mL, 0.08mg / mL, 0.09mg / mL, and 0.10mg / mL, respectively. Add 100 μL of 6% phenol solution, slowly add 500 μL of concentrated sulfuric acid, mix well, and place in a 100°C water bath for 5 min. When the temperature in the water bath drops to room temperature, take out the solution, measure the absorbance at 490 nm, and draw a standard curve of concentration and absorbance to obtain the AA regression equation (1).

[0081] Y = 4.2116x + 0.143, R 2 =0.9977 (1)

[0082] 1.1.2 Sample preparation

[0083] 1000 mg of Panax notoginseng flower powder was taken respectively, and Panax notoginseng flower glycopeptide powder was prepared by the methods of the above embodiments and comparative examples.

[0084] The Notoginseng Flower Glycine Peptide powders prepared in the examples and comparative examples were respectively diluted to 10 ml volumetric flasks to obtain sample solutions.

[0085] 1.1.3 Determination of sample solution

[0086] Accurately pipette 100 μL of sample solution into a centrifuge tube, add 100 μL of 6% phenol solution, slowly add 500 μL of concentrated sulfuric acid, mix well, and place in a 100°C water bath for 5 min. When the temperature in the water bath drops to room temperature, take out the sample and measure the absorbance at 490 nm. Substitute it into the regression equation (1) to calculate the mass concentration of polysaccharides in the sample solution, and calculate the polysaccharide extraction rate (W1) in Panax notoginseng flower according to equation (2).

[0087] W1 = polysaccharide mass concentration in sample solution * total volume of sample solution / mass of Panax notoginseng flower powder * 100% (2) 1.2 Determination of protein extraction rate in Panax notoginseng flower

[0088] 1.2.1 Drawing of standard curve

[0089] Accurately pipette 0 μL, 1 μL, 2 μL, 4 μL, 8 μL, 12 μL, 16 μL, and 20 μL of 0.5 mg / mL BSA standard solution into a 96-well plate, add distilled water to make up to 20 μL, add 200 μL of BCA working solution to each well, place at 37°C for 30 minutes, cool to room temperature, measure the absorbance at 562 nm, draw a standard curve of absorbance and concentration, and obtain regression equation (3).

[0090] Y = 1.0118x + 0.1438, R 2 = 0.999 (3)

[0091] 1.2.2 Sample preparation

[0092] The sample solution prepared in 1.1.2 above was used as the experimental sample.

[0093] 1.2.3 Determination of sample solution

[0094] Accurately pipette 20 μL of sample solution into a 96-well plate, add 200 μL of BCA working solution to each well, place at 37°C for 30 minutes, cool to room temperature, measure the absorbance at 562 nm, substitute into regression equation (3) to calculate the protein mass concentration in the sample solution, and calculate the protein extraction rate (W2) in Panax notoginseng flower according to equation (4).

[0095] W2 = protein concentration in sample solution * total volume of sample solution / mass of Panax notoginseng flower powder * 100% (4)

[0097] 1.3 Calculation of extraction rate of Panax notoginseng flower glycopeptide

[0098] The glycopeptide extraction rate (W) from Panax notoginseng flower was calculated according to formula (5).

[0099] W = W1 + W2 (5)

[0100] 1.4 Results

[0101] The results of the determination of the extraction rate of notoginseng flower glycosides are shown in Table 1. As can be seen from the table below, the extraction rates of notoginseng flower glycosides in Examples 1-6 are better than those in Comparative Examples 1-8, among which the extraction rate of Example 1 is the best. It can be seen that after being treated by the method of the present invention, the obtained notoginseng flower glycosides are extracted to the maximum extent.

[0102] Table 1 Results of determination of extraction rate of Panax notoginseng flower glycopeptide (n=3)

[0103]

[0104]

[0105] 2. In vitro antioxidant assay

[0106] 2.1DPPH free radical scavenging ability

[0107] Prepare a 0.1 mmol / L DPPH alcohol solution in anhydrous ethanol for immediate use and store in a refrigerator away from light.

[0108] The notoginseng flower glycosides extract prepared in each example and comparative example was taken and water was added to prepare sample solutions with different concentrations (10.00, 5.00, 2.50, 1.25, 0.63) mg / ml.

[0109] Take 200μL of sample solutions of different concentrations and add 200μL of DPPH alcohol solution as the sample group. Take a blank tube, add 200μL of sample solvent, and then add 200μL of anhydrous ethanol as the blank control group. Take a blank tube, add 200μL of sample solvent, and then add 200μL of DPPH alcohol solution as the blank group. Take a blank tube, add 200μL of sample solution, and then add 200μL of anhydrous ethanol as the sample control group. After each group is fully mixed, stand at room temperature in the dark for 30 minutes, and immediately detect the absorbance at 517nm. Measure three times in parallel, calculate the DPPH clearance rate according to the following formula, and use Graphpad prism9.0 to process the data and calculate the clearance rate IC50.

[0110] DPPH clearance rate (%) = [(A1-A2)-(A3-A4)] / (A1-A2)*100%

[0111] Where:

[0112] A1: absorbance value of blank group;

[0113] A2: absorption value of blank control group;

[0114] A3: absorbance value of sample group;

[0115] A4: Absorption value of sample control group.

[0116] 2.2 ABTS free radical scavenging ability

[0117] Preparation of ABTS working solution: prepare 7.0mmol / L ABTS solution and 2.45mmol / L K2S2O8 solution respectively; mix equal volumes of the prepared ABTS solution and K2S2O8 solution, and place them at room temperature and away from light for 12 to 16 hours to obtain ABTS working solution. Dilute it with anhydrous ethanol before use until the absorbance at 734nm is 0.70±0.02, and store it away from light until tested.

[0118] The notoginseng flower glycosides extract prepared in each example and comparative example was taken and water was added to prepare sample solutions with different concentrations (10.00, 5.00, 2.50, 1.25, 0.63) mg / ml.

[0119] Take 50μL of sample solutions of different concentrations and add 80μL of ABTS working solution as the sample group. Take a blank tube, add 50μL of sample solvent, and then add 80μL of anhydrous ethanol as the blank control group. Take a blank tube, add 50μL of sample solvent, and then add 80μL of ABTS working solution as the blank group. Take a blank tube, add 50μL of sample solution, and then add 80μL of anhydrous ethanol as the sample control group. After each group is fully mixed, stand at room temperature in the dark for 6 minutes, and immediately detect the absorbance at 734nm. The measurement is repeated three times, and the ABTS clearance rate is calculated according to the following formula. Graphpad prism 9.0 is used to process the data and calculate the clearance rate IC50 (the sample concentration when the clearance rate is 50%).

[0120] ABTS clearance rate (%) = [(A1-A2)-(A3-A4)] / (A1-A2)*100%

[0121] Where:

[0122] A1: absorbance value of blank group;

[0123] A2: absorption value of blank control group;

[0124] A3: absorbance value of sample group;

[0125] A4: Absorption value of sample control group.

[0126] 2.3 Results

[0127] The results of the DPPH and ABTS free radical scavenging ability test of the notoginseng glycosides are shown in Table 2. As can be seen from the table below, the antioxidant activity of the notoginseng glycosides of Examples 1-6 is better than that of Comparative Examples 1-8. It can be seen that after being treated by the method of the present invention, the obtained notoginseng glycosides retain the biological activity of the extract to the maximum extent.

[0128] Table 2 Results of DPPH and ABTS free radical scavenging ability determination in Panax notoginseng flower peptides (n=3)

[0129]

[0130]

Claims

1. A method for extracting Panax notoginseng flower glycopeptide, characterized in that The following steps are involved: (1) extracting Panax notoginseng flower powder with ethanol or ethanol aqueous solution to obtain an extract 1 and a filter residue 1; (2) drying the filter residue 1 and extracting it with an ionic liquid to obtain an extract 2 and a filter residue 2; (3) drying the filter residue 2, and extracting it with a low eutectic solvent under microwave to obtain an extract 3 and a filter residue 3; (4) combining extract 2 and extract 3, adding ethanol to precipitate, and collecting the precipitate to obtain notoginseng flower crude saccharoprotein; (5) adding water to redissolve the crude saccharide protein from Panax notoginseng flower, and then passing it through a DEAE-23 fiber chromatography column for adsorption, and then eluting it with water and a sodium chloride solution in sequence, ultrafiltering the sodium chloride eluate to remove sodium chloride, and then combining it with the water eluate to obtain a decolorized crude saccharide protein solution from Panax notoginseng flower; (6) removing free protein from the decolorized crude notoginseng flower glycoprotein solution by the Sevag method to obtain a purified notoginseng flower glycoprotein solution; (7) The purified notoginseng flower glycoprotein solution is first enzymatically hydrolyzed with trypsin and then with alkaline protease to obtain notoginseng flower glycopeptide.

2. The extraction method according to claim 1, characterized in that: In step (1), the Panax notoginseng flower powder is extracted three times with ethanol or ethanol aqueous solution, the concentration of the ethanol aqueous solution is greater than or equal to 80wt%, the temperature of each extraction is 20-85°C, the solid-liquid ratio of each extraction is 1:1-2, and the extraction time is 30-60min; preferably, during the first extraction, a 75-80wt% ethanol solution of 1-2 times the mass of the Panax notoginseng flower powder is added, and the mixture is stirred and extracted for 30-60min at room temperature, and filtered to obtain a filtrate and a filter residue; during the second extraction, an 85-90wt% ethanol solution of 1-2 times the mass of the Panax notoginseng flower powder is added to the filter residue obtained from the first extraction, and the mixture is stirred and extracted at 55-65°C for 30-60min to obtain a filtrate and a filter residue; during the third extraction, anhydrous ethanol of 1-2 times the mass of the Panax notoginseng flower powder is added to the filter residue obtained from the second extraction, and the mixture is stirred and extracted at 75-85°C for 30-60min to obtain a filtrate and a filter residue; all the filtrates are combined to obtain an extract 1.

3. The extraction method according to claim 1, characterized in that: In step (2), the ionic liquid is a 0.05-0.1 mol / L 1-ethyl-3-methylimidazolium acetate aqueous solution.

4. The extraction method according to claim 1 or 3, characterized in that: In step (2), the mass ratio of the filter residue 1 to the ionic liquid is 1:1-4; preferably, the mixture of the filter residue 1 and the ionic liquid is extracted on a shaker with a shaker speed of 300-400 rpm, an extraction temperature of room temperature, and an extraction time of 6-12 h.

5. The extraction method according to claim 1, characterized in that: In step (3), the low eutectic solvent is obtained by mixing choline chloride, guaiacol and lactic acid in a molar ratio of 1-2:1:1-2; preferably, in step (3), the mass ratio of the filter residue 2 to the low eutectic solvent is 1:14-18; the extraction temperature is 50-85°C, the microwave power is 100-130W, and the extraction time is 15-30min.

6. The extraction method according to claim 1, characterized in that: In step (4), the mass ratio of the total mass of extract 2 and extract 3 to anhydrous ethanol is 1:4-5, and the mixture is allowed to stand at 0-4°C for 12-24 hours for alcohol precipitation.

7. The extraction method according to claim 1, characterized in that: In step (5), the crude saccharide protein from Panax notoginseng flower is added to water with a volume of 15-25 times its mass for redissolution to obtain a crude saccharide protein solution from Panax notoginseng flower, which is then added to a DEAE-23 fiber chromatography column for adsorption for 10-15 min, and then eluted with water and a 0.5-1 mol / L NaCl aqueous solution in sequence.

8. The extraction method according to claim 1, characterized in that: In step (6), the Sevag reagent used is a mixture of dichloromethane and n-butanol in a volume ratio of 4:1, and the amount of Sevag reagent used is 15-25% of the volume of the decolorized Panax notoginseng flower crude saccharoprotein solution.

9. The extraction method according to claim 1, characterized in that: In step (7), 5500-6500u of trypsin and 180000-220000u of alkaline protease are added to each gram of Panax notoginseng flower glycoprotein; preferably, the enzymatic hydrolysis conditions are: trypsin is added at 30°C, and then the temperature is increased at a rate of 10-15°C / 60min until it reaches 60°C, and then alkaline protease is added, and the enzymatic hydrolysis is continued for 2-3h.

10. Notoginseng flower glycosides prepared by the extraction method according to any one of claims 1 to 9 and use of notoginseng flower glycosides in the preparation of antioxidant products, preferably, the antioxidant products include medicines, health products, cosmetics, and foods.

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