Ginseng glycopeptide and preparation process thereof

Abstract

The present application provides a kind of ginseng glycopeptide and its preparation process, the preparation process includes the following steps: (1) extracting ginseng glycopeptide, collecting extract, alcohol precipitation is carried out, and ginseng glycoprotein is obtained;(2) the ginseng glycoprotein is carried out enzymolysis, and enzyme solution is collected;The enzyme used in the enzymolysis is the combination of alpha-pancreatic trypsin, trypsin and pepsin;(3) the enzyme solution is carried out solid-liquid separation, and the ginseng glycopeptide is obtained;The solid-liquid separation includes centrifugation, primary ultrafiltration, secondary ultrafiltration.The present application is combined by extraction, alcohol precipitation, enzymolysis, ultrafiltration technology, greatly improves the extraction rate of ginseng glycopeptide, wherein the three enzymes of enzymolysis synergistic effect, ginseng glycoprotein is more fully hydrolyzed, effectively improves ginseng glycoprotein conversion utilization rate, and the purity of ginseng glycopeptide can reach 95%, which lays a good foundation for its application in medicine, cosmetics and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to a ginseng glycopeptide and a preparation process thereof. BACKGROUND

[0002] Panax ginseng C.A.Mey is a perennial herbaceous plant in the family Araliaceae of the order Apiales, with a history of about 600 million years. It is known as the "plant fossil". As one of the precious Chinese medicinal materials, Panax ginseng has been used by traditional Chinese medicine for more than 2,000 years. In recent years, more and more external preparations of Panax ginseng have emerged, such as compound Panax ginseng volatile oil aerosol, Panax ginseng liniment and compound Panax ginseng resorcinol liniment, which have broad application value. The material basis for multiple pharmacological effects in Panax ginseng mainly includes ginsenosides, polysaccharides, proteins, polypeptides, glycopeptides and volatile oils. Among them, ginsenosides, polysaccharides, polypeptides and glycopeptides have been proven to have many biological activities. Peptide components are widely used in the fields of drugs and cosmetics due to their small molecular weight, high bioavailability and unique biological activity.

[0003] Glycoprotein was proposed by Montreuil in 1973, which is a complex composed of a protein molecule and one or more substances containing carbohydrate groups, except nucleic acids. Glycopeptides refer to glycoproteins and proteoglycans, which are regions formed by covalent bonding of sugars and amino acids or polypeptide chains. Ginseng glycopeptides (GGP) have a relative molecular mass of 200-50,000 Da, and their sugar part is mainly composed of mannose, rhamnose, galactose, glucose, fucose, N-acetylglucosamine and N-acetylgalactosamine. The peptide part is mainly composed of 17 kinds of amino acids, including glycine, serine and threonine.

[0004] CN113151389A discloses a ginseng glycopeptide, a preparation method and medical use, and belongs to the field of medicine. The ginseng glycopeptide is obtained by decoction extraction, ethanol precipitation separation, double-enzyme hydrolysis, dialysis and freeze-drying, etc. from ginseng as raw material, with a yield of more than 14% and a purity of more than 90%. Animal model experiments show that the ginseng glycopeptide can enhance the cellular immune function, humoral immune function, monocyte-macrophage phagocytic function and NK cell activity of immunocompromised mice.

[0005] CN1931874A discloses a preparation method and pharmaceutical preparation of ginseng glycopeptide, and also discloses a new medical use of ginseng glycopeptide. The method uses ginseng as raw material, uses the principle that substances with different molecular weights have different solubilities in different concentrations of ethanol, uses 50% ethanol reflux extraction and 80% ethanol precipitation to obtain crude ginseng glycopeptide, controls the approximate range of the molecular weight of the crude ginseng glycopeptide, and then removes impurities through refrigeration, decolorization and ultrafiltration to obtain refined ginseng glycopeptide. The ginseng glycopeptide is subjected to liquid preparation, filling and sealing, sterilization and packaging to produce ginseng glycopeptide injection. The pharmaceutical preparation can be used for preventing and treating diabetes and its complications and the like.

[0006] CN114158725A discloses a preparation method of ginseng enzymatic hydrolysate containing polypeptide polysaccharide, comprising the following steps: 100 parts by weight of ginseng dry product is crushed, 500-2000 parts by weight of water is added, 1-8 parts by weight of saccharase is added at a temperature of 30-65℃, and reaction is carried out for 0.5-12 hours; 2) at the same temperature, 0.1-8 parts by weight of alkaline protease and 0.1-8 parts by weight of neutral protease are added at the same time, and reaction is carried out for 0.5-12 hours, or 0.1-8 parts by weight of alkaline protease is added first, reaction is carried out for 0.5-12 hours, then 0.1-8 parts by weight of neutral protease is added, and reaction is carried out for 0.5-12 hours; the pH value in step 2) is maintained at 6-9; 3) the enzyme reaction in step 2) is terminated.

[0007] At present, the extraction methods of ginseng glycopeptide mainly include water extraction, alcohol extraction, dilute salt solution or buffer solution extraction, acid and alkali solution extraction, ultrasonic wave assisted method and microwave assisted method, etc. The above extraction methods have advantages and disadvantages, and it is difficult to achieve efficient extraction of ginseng glycopeptide by a single method, and the effective utilization rate of ginseng is low in industrial production.

[0008] Therefore, developing a method for extracting ginseng glycopeptide with high efficiency, high yield and high purity is the research focus in the field. SUMMARY

[0009] In view of the deficiencies of the prior art, the purpose of the present application is to provide a ginseng glycopeptide and a preparation process thereof, which is efficient, has high yield, and the prepared ginseng glycopeptide has high purity.

[0010] To achieve the purpose of the present application, the following technical solutions are adopted:

[0011] In a first aspect, the present application provides a preparation process of ginseng glycopeptide, which comprises the following steps:

[0012] (1) extracting ginseng, collecting the extraction liquid, and performing alcohol precipitation on the extraction liquid to obtain ginseng glycoprotein;

[0013] (2) subjecting the ginseng glycoprotein to enzymolysis, and collecting the enzymolysis liquid;

[0014] The enzyme used in the enzymolysis is a combination of alpha-chymotrypsin, trypsin and pepsin;

[0015] (3) performing solid-liquid separation on the enzymolysis solution to obtain the ginseng glycopeptide;

[0016] The solid-liquid separation includes centrifugation, primary ultrafiltration and secondary ultrafiltration.

[0017] The ginseng glycopeptide is extracted in a combined manner, and the extraction, alcohol precipitation and enzymolysis technologies are combined to greatly improve the extraction rate of the ginseng glycopeptide while effectively controlling the industrialization cost. The enzymolysis uses a combination of alpha-chymotrypsin, trypsin and pepsin, and the three enzymes synergistically act on the ginseng glycoprotein to improve the conversion utilization rate of the ginseng glycoprotein, and the prepared ginseng glycopeptide has higher purity, which lays a good foundation for application in medicines, cosmetics and the like. The process of secondary ultrafiltration can effectively control the molecular weight of the ginseng glycopeptide to obtain ginseng glycopeptide with more uniform molecular weight distribution and easier absorption.

[0018] Preferably, the ratio of the extracted material to the liquid is 1: (10-15), for example, it can be 1:11, 1:12, 1:13 or 1:14, etc. For 1 g of ginseng, the extraction agent is 10-15 mL.

[0019] Preferably, the extraction agent used in the extraction is water.

[0020] Preferably, the extraction time is 1.5-2.5 h, for example, it can be 1.7 h, 2 h or 2.3 h, etc.

[0021] Preferably, the extraction is repeated 2-3 times.

[0022] Preferably, the extraction further includes a soaking step before the extraction, and the ratio of the soaked material to the liquid is 1: (5-15), for example, it can be 1:6, 1:8, 1:10, 1:12 or 1:14, etc.

[0023] Preferably, the extraction further includes a concentration step after the extraction.

[0024] Preferably, the relative density of the extracted liquid obtained by the concentration is 1.0-1.2 g / mL, for example, it can be 1.05 g / mL, 1.1 g / mL or 1.15 g / mL, etc.

[0025] Other specific point values in the above numerical ranges can be selected, which will not be described here one by one.

[0026] Preferably, the alcohol content in the system during the alcohol precipitation is 60-80%, for example, it can be 62%, 65%, 70%, 75% or 78%, etc.

[0027] Preferably, the reagent used in the alcohol precipitation is an ethanol aqueous solution.

[0028] Preferably, the mass percentage of ethanol in the ethanol aqueous solution is 80-90%, for example, it can be 82%, 84%, 86% or 88%, etc.

[0029] Preferably, the alcohol precipitation is followed by the steps of reduced pressure concentration and freeze-drying.

[0030] Preferably, the mass ratio of α-chymotrypsin, trypsin and pepsin is (3-5):(2-3):1, for example, it can be 3:2:1, 3:3:1, 4:2:1, 4:3:1, 5:2:1 or 5:3:1, etc.

[0031] Preferably, the total amount of enzyme in the enzymolysis accounts for 4-8% of the mass percentage of ginsenoside in the substrate, for example, it can be 4.5%, 5%, 5.5%, 6%, 7%, 7.2%, 7.4%, 7.6% or 7.8%, etc.

[0032] Preferably, the mass percentage of ginsenoside in the substrate in the enzymolysis is 5-8%, for example, it can be 5.5%, 6%, 6.5%, 7% or 7.5%, etc.

[0033] Preferably, the temperature of the enzymolysis is 40-50℃, for example, it can be 42℃, 44℃, 46℃ or 48℃, etc., and the time is 5-7 h, for example, it can be 5.5 h, 6 h or 6.5 h, etc.

[0034] Preferably, before the enzymolysis, the pH value of the ginsenoside is adjusted to 8-9, for example, it can be 8.3, 8.5 or 8.8, etc.

[0035] Other specific point values in the above numerical ranges can be selected, which will not be repeated here.

[0036] Preferably, the enzymolysis is followed by the step of inactivation.

[0037] Preferably, the inactivation is carried out by heating, and the temperature of the inactivation is 95-100℃, for example, it can be 96℃, 97℃, 98℃ or 99℃, etc., and the time is 8-12 min, for example, it can be 9 min, 10 min or 11 min, etc.

[0038] Preferably, the solid-liquid separation is carried out by centrifugation.

[0039] Preferably, the solid-liquid separation is followed by the step of freeze-drying.

[0040] Preferably, the molecular weight cut-off of the first ultrafiltration is 1.5-2.5 KDa, for example, it can be 1.6 KDa, 1.8 KDa, 2 KDa, 2.2 KDa, 2.4 KDa, etc., and the filtrate is collected.

[0041] Preferably, the molecular weight cut-off of the second ultrafiltration is 400-600 Da, for example, it can be 450 Da, 500 Da, 550 Da, etc., and the cut-off liquid is collected.

[0042] Preferably, the preparation process comprises the following steps:

[0043] (1) Extracting ginseng in water for 1.5-2.5 h, the ratio of material to liquid is 1: (10-15), repeating 2-3 times, collecting the extract, concentrating to a relative density of 1.0-1.2 g / mL, then using 80-90% ethanol aqueous solution for alcohol precipitation, the alcohol content in the system during alcohol precipitation is 60-80%, collecting the solid, reducing pressure to concentrate, and freeze-drying to obtain ginseng glycoprotein;

[0044] (2) Adjusting the pH value of the ginseng glycoprotein to 8-9, then performing enzymolysis at 40-50℃ for 5-7 h, adjusting the pH value to 8-9, and collecting the enzymolysis liquid;

[0045] The enzyme used in the enzymolysis is a combination of α-chymotrypsin, trypsin and pepsin, and the mass ratio is (3-5) :(2-3) :1;

[0046] The total amount of enzyme used in the enzymolysis accounts for 4-8% of the mass percentage of ginseng glycoprotein;

[0047] (3) After centrifugation of the enzymolysis liquid, performing first ultrafiltration using a 1.5-2.5 KDa ultrafiltration membrane, collecting the filtrate, performing second ultrafiltration using a 400-600 Da ultrafiltration membrane, and collecting the cut-off liquid to obtain the ginseng glycopeptide.

[0048] In the second aspect, the present application provides a ginseng glycopeptide prepared by the preparation process of the first aspect.

[0049] Preferably, the weight average molecular weight of the ginseng glycopeptide is 700-850 Da, for example, it can be 720 Da, 740 Da, 760 Da, 780 Da, 800 Da, 820 Da, 840 Da, etc.

[0050] Preferably, the purity of the ginseng glycopeptide is ≥90%, for example, it can be 91%, 92%, 93%, 94% or 95%, etc.

[0051] Other specific point values in the above numerical ranges can be selected, which will not be described here one by one.

[0052] In a third aspect, the present application provides a ginseng glycopeptide for use in a pharmaceutical product or a cosmetic product.

[0053] Compared with the prior art, the present application has the following beneficial effects:

[0054] The ginseng glycopeptide is extracted by combination, and the extraction, alcohol precipitation and enzymatic hydrolysis technologies are combined to greatly improve the extraction rate of the ginseng glycopeptide while effectively controlling the industrialization cost. The three enzymes are combined to more fully hydrolyze the ginseng glycopeptide, effectively improve the conversion utilization rate of the ginseng glycopeptide, and the yield of the ginseng glycopeptide reaches more than 17%. The prepared ginseng glycopeptide has higher purity, and the highest purity can reach 95%, which lays a good foundation for the application in pharmaceutical products, cosmetics and the like. The process of twice ultrafiltration can effectively control the molecular weight of the ginseng glycopeptide, and the ginseng glycopeptide with smaller molecular weight, more uniform molecular weight distribution and easier absorption is obtained. DETAILED DESCRIPTION

[0055] The technical solutions of the present application are further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only used to help understand the present application, and should not be regarded as specific limitations on the present application.

[0056] The terms "comprising", "including", "containing", "have" or "including" or any other variant thereof used in this document are intended to cover non-exclusive inclusion. For example, a composition, step, method, article or device that comprises a listed element does not necessarily include only those elements, but can also include other elements not explicitly listed or inherent to such composition, step, method, article or device.

[0057] "Optional" or "any of" means that the matter or event described thereafter can occur or not occur, and the description includes the case where the event occurs and the case where the event does not occur.

[0058] The indefinite articles "a" and "an" before an element or component of the application are not intended to be limiting, unless otherwise indicated, as to the number of such elements or components. Thus, "a" or "an" should be interpreted to mean "one or at least one" and the singular form of an element or component of the application includes the plural unless the number is clearly specified as being singular.

[0059] The description of the terms "one embodiment", "some embodiments", "exemplarily", "specific examples" or "some examples" and the like described in the present application means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this document, the illustrative description of the above terms is not necessarily directed to the same embodiment or example.

[0060] The reagents or instrument sources in the following examples are as follows:

[0061] Alpha-chymotrypsin: Shanghai Yuan Ye Biotechnology Co., Ltd.;

[0062] Trypsin: Shanghai Yuan Ye Biotechnology Co., Ltd.;

[0063] Pepsin: Shanghai Yuan Ye Biotechnology Co., Ltd.;

[0064] Ethanol: National Pharmaceutical Group Chemical Reagent Co., Ltd.;

[0065] Pure water: Hangzhou Wahaha Group Co., Ltd.;

[0066] Sodium hydroxide: Beijing Chemical Company.

[0067] Example 1

[0068] This example provides a ginseng glycopeptide, and the preparation method of the ginseng glycopeptide is as follows:

[0069] (1) The ginseng was crushed into coarse particles, soaked with 10 times water until there was no white core, then taken out, and boiled with 12 times water twice, each time for 2 hours. The extract was filtered, the filtrate was combined, concentrated to a relative density of 1.10 g / mL (50°C), cooled, and then 85% ethanol aqueous solution was added to adjust the alcohol content in the system to 70%. After standing for 48 h, the precipitate was taken out, stirred with water, and then the ethanol was recovered under reduced pressure. The freeze-dried product was ginseng glycoprotein;

[0070] (2) The ginseng glycoprotein was dissolved in deionized water, and 0.5 mol / L NaOH aqueous solution was added to adjust the pH to 8.5. At this time, the substrate concentration in the system was 7%, and 6% protease (E / S, the concentration ratio of protease to substrate) was added, including 3% alpha-chymotrypsin, 2.25% trypsin, and 0.75% pepsin. The reaction was carried out at 45°C and 180 rpm in a shaking bed for 6 h. After the reaction was completed, the inactivation was carried out in a water bath at 100°C for 10 min;

[0071] (3) The enzyme hydrolysate after inactivation was centrifuged at 4800 rpm for 10 min, and the supernatant was collected. The supernatant was first filtered through a 2kDa ultrafiltration membrane, and the filtrate was collected. The filtrate was filtered through a 500 Da ultrafiltration membrane, and the cut-off liquid was collected. The ultrafiltration was stopped when there was no filtrate dropping within 10 min. The freeze-dried product was ginseng glycopeptide.

[0072] Example 2

[0073] This example provides a ginseng glycopeptide, and the preparation method of the ginseng glycopeptide is as follows:

[0074] (1) Take ginseng powder into coarse particles, soak in 15 times water until there is no white core, take out, add 10 times water, decoct twice, every 2.5 hours, filter the extract, combine the filtrate, concentrate to the relative density of 1.0 g / mL (50℃), cool down, add 90% ethanol aqueous solution, adjust the alcohol content in the system to 60%, stand for 48 h, take the precipitate, add water, stir evenly, recover ethanol under reduced pressure, freeze-dry to obtain ginseng glycoprotein;

[0075] (2) Take ginseng glycoprotein, dissolve in deionized water, add 0.5 mol / L NaOH aqueous solution to adjust the pH to 8, at this time the substrate concentration in the system is 5%, add protease (E / S, the concentration ratio of protease to substrate) 8%, of which α-chymotrypsin 4%, trypsin 3% and pepsin 1%, react at 45℃, 180 rpm in a shaking bed for 5 h, after the reaction is completed, inactivate in a water bath at 100℃ for 10 min;

[0076] (3) Centrifuge the inactivated enzymatic product at 4800 rpm for 10 min, take the supernatant, first filter the supernatant through a 1.6 kDa ultrafiltration membrane, collect the filtrate, filter the filtrate through a 600 Da ultrafiltration membrane, collect the cut-off liquid, ultrafiltrate to the end point when there is no filtrate dropping within 10 min, freeze-dry to obtain ginseng glycopeptide.

[0077] Example 3

[0078] This example provides a ginseng glycopeptide, and the preparation method thereof is as follows:

[0079] (1) Take ginseng powder into coarse particles, soak in 15 times water until there is no white core, take out, add 15 times water, decoct twice, every 1.5 hours, filter the extract, combine the filtrate, concentrate to the relative density of 1.2 g / mL (50℃), cool down, add 80% ethanol aqueous solution, adjust the alcohol content in the system to 80%, stand for 48 h, take the precipitate, add water, stir evenly, recover ethanol under reduced pressure, freeze-dry to obtain ginseng glycoprotein;

[0080] (2) Take ginseng glycoprotein, dissolve in deionized water, add 0.5 mol / L NaOH aqueous solution to adjust the pH to 9, at this time the substrate concentration in the system is 8%, add protease (E / S, the concentration ratio of protease to substrate) 4%, of which α-chymotrypsin 2%, trypsin 1.5% and pepsin 0.5%, react at 45℃, 180 rpm in a shaking bed for 7 h, after the reaction is completed, inactivate in a water bath at 100℃ for 10 min;

[0081] (3) The inactivated enzymatic product was centrifuged at 4800 rpm for 10 min, and the supernatant was collected. The supernatant was first filtered through a 2.5 kDa ultrafiltration membrane, and the filtrate was collected. The filtrate was filtered through a 400 Da ultrafiltration membrane, and the retentate was collected. The ultrafiltration was stopped when no filtrate dropped within 10 min. The product was freeze-dried to obtain ginseng glycopeptide.

[0082] Example 4

[0083] This example provides a ginseng glycopeptide, and the preparation method thereof is as follows:

[0084] The difference from Example 1 is that in step (2), 6% of protease (E / S) is added, in which α-chymotrypsin is 2.25%, trypsin is 3%, and pepsin is 0.75%. The other raw materials, dosages, and preparation methods are the same as those in Example 1.

[0085] Example 5

[0086] This example provides a ginseng glycopeptide, and the preparation method thereof is as follows:

[0087] The difference from Example 1 is that in step (2), 6% of protease (E / S) is added, in which α-chymotrypsin is 2.25%, trypsin is 3%, and pepsin is 0.75%. The other raw materials, dosages, and preparation methods are the same as those in Example 1.

[0088] Example 6

[0089] This example provides a ginseng glycopeptide, and the preparation method thereof is as follows:

[0090] The difference from Example 1 is that in step (2), 6% of protease (E / S) is added, in which α-chymotrypsin is 2.25%, trypsin is 3%, and pepsin is 0.75%. The other raw materials, dosages, and preparation methods are the same as those in Example 1.

[0091] Example 7

[0092] This example provides a ginseng glycopeptide, and the preparation method thereof is as follows:

[0093] The difference from Example 1 is that in step (2), 10% of protease (E / S) is added, in which α-chymotrypsin is 5%, trypsin is 3.75%, and pepsin is 1.25%. The other raw materials, dosages, and preparation methods are the same as those in Example 1.

[0094] Example 8

[0095] This example provides a ginseng glycopeptide, and the preparation method thereof is as follows:

[0096] The difference from Example 1 is only that in step (2) protease (E / S) 3% is added, in which α-chymotrypsin 1.5%, trypsin 1.125% and pepsin 0.375%. Other raw materials, amounts and preparation methods are the same as in Example 1.

[0097] Comparative Example 1

[0098] This comparative example provides a ginseng glycopeptide, the preparation method of which is as follows:

[0099] The difference from Example 1 is only that in step (2) protease (E / S) 6% is added, in which α-chymotrypsin 4%, trypsin 2%. Other raw materials, amounts and preparation methods are the same as in Example 1.

[0100] Comparative Example 2

[0101] This comparative example provides a ginseng glycopeptide, the preparation method of which is as follows:

[0102] The difference from Example 1 is only that in step (2) protease (E / S) 6% is added, in which α-chymotrypsin 4.8%, pepsin 1.2%. Other raw materials, amounts and preparation methods are the same as in Example 1.

[0103] Comparative Example 3

[0104] This comparative example provides a ginseng glycopeptide, the preparation method of which is as follows:

[0105] The difference from Example 1 is only that in step (2) protease (E / S) 6% is added, in which trypsin 4.5% and pepsin 1.5%. Other raw materials, amounts and preparation methods are the same as in Example 1.

[0106] Comparative Example 4

[0107] This comparative example provides a ginseng glycopeptide, the preparation method of which is as follows:

[0108] The difference from Example 1 is only that in step (2) α-chymotrypsin (E / S) 6% is added. Other raw materials, amounts and preparation methods are the same as in Example 1.

[0109] Comparative Example 5

[0110] This comparative example provides a ginseng glycopeptide, the preparation method of which is as follows:

[0111] The difference from Example 1 is only that in step (2) trypsin (E / S) 6% is added. Other raw materials, amounts and preparation methods are the same as in Example 1.

[0112] Comparative Example 6

[0113] The present comparative example provides a ginseng glycopeptide, and the preparation method thereof is as follows:

[0114] The difference between it and Example 1 is only that pepsin (E / S) 6% is added in step (2). The other raw materials, dosages and preparation methods are the same as those in Example 1.

[0115] Comparative Example 7

[0116] The present comparative example provides a ginseng glycopeptide, and the preparation method thereof is as follows:

[0117] The difference between it and Example 1 is only that step (3) is as follows:

[0118] After inactivation, the enzymatic product is centrifuged at 4800 rpm for 10 min, the supernatant is filtered through a 2 kDa ultrafiltration membrane, the filtrate is collected, and the ultrafiltration is stopped when no filtrate drops within 10 min. The freeze-drying gives a ginseng glycopeptide.

[0119] Comparative Example 8

[0120] The present comparative example provides a ginseng glycopeptide, and the preparation method thereof is as follows:

[0121] The difference between it and Example 1 is only that step (3) is as follows:

[0122] After inactivation, the enzymatic product is centrifuged at 4800 rpm for 10 min, the supernatant is filtered through a 500 Da ultrafiltration membrane, the retentate is collected, and the ultrafiltration is stopped when no filtrate drops within 10 min. The freeze-drying gives a ginseng glycopeptide.

[0123] Comparative Example 9

[0124] The present comparative example provides a ginseng glycopeptide, and the preparation method thereof is as follows:

[0125] The difference between it and Example 1 is only that step (3) is as follows:

[0126] After inactivation, the enzymatic product is centrifuged at 4800 rpm for 10 min, the supernatant is collected, and the freeze-drying gives a ginseng glycopeptide.

[0127] Test Example 1

[0128] The degree of hydrolysis of ginseng glycopeptide was determined by indantrione colorimetry. A glycine solution with a concentration of 20 μg / mL was prepared, and 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, and 1.0 mL of the glycine solution were respectively taken into test tubes, and distilled water was added to make up to 1.0 mL. Then, 1 mL of indantrione (0.5 g of indantrione, 0.3 g of fructose, 10.0 g of Na2HPO4·10H2O, and 6.0 g of KH2PO4) was added as a color developing agent. After mixing, the mixture was placed in a boiling water bath for 15 min. After cooling, 5 mL of ethanol solution with a volume fraction of 40% was added, and the mixture was mixed uniformly and then placed at room temperature for 15 min. Then, 200 μL of the mixture was precisely taken and placed in a 96-well plate, and ultrapure water was used as a blank. The absorbance at 570 nm was measured. The standard curve was drawn with the glycine concentration as the abscissa and the absorbance as the ordinate, and the regression equation was calculated as y = 0.0121x + 0.0657, R 2 = 0.999.

[0129] The content of polysaccharide was determined by the phenol-sulfuric acid method, and the total protein concentration was detected according to the BCA assay kit instructions. The purity (mass fraction % of glycoprotein) = mass fraction of polysaccharide + mass fraction of protein.

[0130] The ginseng glycopeptides prepared in Examples 1-8 and Comparative Examples 1-6 were detected, and the degree of hydrolysis, yield, and purity are shown in Table 1.

[0131] Table 1

[0132] hydrolysis degree yield (%) purity (%) Example 1 29.98 22.3 95 Example 2 27.47 21.8 94 Example 3 26.30 20.9 93.5 Example 4 23.5 19.1 90.2 Example 5 22.3 18.3 90.9 Example 6 22.0 19.0 91.0 Example 7 22.1 18.9 86.2 Example 8 18.4 17.2 83.4 Comparative Example 1 16.1 14.3 80.1 Comparative Example 2 15.3 13.7 79.6 Comparative Example 3 15.8 14.1 78.5 Comparative Example 4 14.0 12.3 65.2 Comparative Example 5 13.5 11.9 64.1 Comparative Example 6 13.8 12.1 63.9

[0133] According to the data in the table, in the preparation process, when the total amount of enzymes is constant, the excessive proportion of one of the enzymes α-chymotrypsin, trypsin, or pepsin will cause the decrease of the degree of hydrolysis; when the total amount of enzymes is too high, the degree of hydrolysis is almost unaffected, but the yield and purity decrease; when the total amount of enzymes is too low, the degree of hydrolysis limit decreases; when two enzymes are used or a single enzyme is used, the degree of hydrolysis, yield, and purity all decrease significantly, indicating that the three enzymes work synergistically to improve the hydrolysis of ginseng and ensure the yield and purity of ginseng glycopeptide.

[0134] Test Example 2

[0135] Molecular weight test

[0136] (1) Preparation of the control solution

[0137] The known molecular weight of dextran molecular weight standard was used, according to the size of the sample molecular weight, the weight average molecular weight (Mw) of 180, 1000, 5000, 12000, 50000 Da different molecular weight standard was selected to prepare 10 mg / mL solution as the control solution. Agilent GPC software version A.02.01 was used to analyze the data.

[0138] (2) Preparation of test solution

[0139] The ginseng glycopeptide prepared in examples 1-3 and comparative examples 7-9 was taken and dissolved in mobile phase to prepare a 10 mg / mL solution, which was filtered through a 0.45 μm microporous filter, and the filtrate was used as the test solution.

[0140] (3) Chromatographic conditions

[0141] Chromatographic column: Sepex SRT SEC-100 (7.8 mm x 300 mm, 5 μm); mobile phase: 0.7% sodium sulfate solution; flow rate: 0.5 mL / min; column temperature: 35°C; injection volume: 10 μL; liquid phase: LC-2030C3D high performance liquid chromatograph; detector: differential refractive index detector; theoretical plate number calculated by glucose peak should not be less than 5000 Da. The results obtained by detection are shown in Table 2.

[0142] Table 2

[0143] Sample weight average molecular weight (Da) Example 1 786 Example 2 713 Example 3 842 Comparative Example 7 695 Comparative Example 8 1365 Comparative Example 9 1152

[0144] According to the table data, the secondary ultrafiltration process is important for preparing the ginseng glycopeptide required by the present application. When only one ultrafiltration is performed, the molecular weight of the obtained ginseng glycopeptide is reduced; when only the second ultrafiltration process is performed, the molecular weight of the obtained ginseng glycopeptide is significantly increased; when the secondary ultrafiltration process is not used, the molecular weight of the obtained ginseng glycopeptide is also significantly increased.

[0145] Test example 3

[0146] Hydroxyl radical scavenging experiment

[0147] Main equipment: multifunctional enzyme label instrument (Spectra Max M5 Molecular Device)

[0148] Experimental method: the ginseng glycopeptide prepared in sample groups examples 1-8 and comparative examples 1-9 were diluted to 1 / 10, and the negative control group, 0.1 parts of ferrous sulfate, 0.1 parts of ethanol, 0.1 parts of salicylic acid, 0.1 parts of sample or negative control, 1 parts of ionized water, 0.1 parts of hydrogen peroxide were added into 1.5 mL centrifuge tube in turn, tightly cover the tube cap, shake up and down, keep in 37℃ water bath for 15 min, move the solution to 96-well plate, three replicates for each group. The absorbance at 510 nm was measured by microplate reader.

[0149] The hydroxyl radical scavenging rate was calculated by the following formula:

[0150] The hydroxyl radical scavenging rate % = {[A0-(AX-AX0)] / A0}x100%, wherein A0 is the absorbance of the negative control group, Ax is the absorbance of the sample group, and Axo is the absorbance of the sample background. The test results are shown in Table 3.

[0151] Table 3

[0152] Sample hydroxyl radical scavenging rate (%) Example 1 71.8 Example 2 70.2 Example 3 70.8 Example 4 69.1 Example 5 66.3 Example 6 66.9 Example 7 67.0 Example 8 65.3 Comparative Example 1 63.1 Comparative Example 2 61.2 Comparative Example 3 64.0 Comparative Example 4 59.4 Comparative Example 5 60.2 Comparative Example 6 57.6 Comparative Example 7 66.9 Comparative Example 8 61.3 Comparative Example 9 63.4

[0153] According to the table data, in the preparation process, when the total amount of complex enzymes is constant, the excessive proportion of the amount of one of the enzymes α-chymotrypsin, trypsin or pepsin will cause the decrease of the hydroxyl radical scavenging rate; when the total amount of enzymes is too high, the scavenging rate decreases slightly, and when the total amount of enzymes is too low, the scavenging rate decreases significantly; when the combination of two of the enzymes or the use of a single enzyme is used under the condition that the total amount of enzymes is constant, the hydroxyl radical scavenging rate decreases significantly, indicating that the three enzymes synergistically enhance the effect of ginseng glycopeptide in scavenging hydroxyl radicals, and further ensure the effect of ginseng glycopeptide in skin care. When the weight average molecular weight of ginseng glycopeptide is low, the scavenging rate decreases slightly, and when the molecular weight is large, the scavenging rate decreases significantly.

[0154] The applicant declares that the process of the present application is illustrated by the above examples, but the present application is not limited to the above examples, that is, it does not mean that the present application must rely on the above examples to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific methods, etc. fall within the scope of protection and disclosure of the present application.

Claims

1. A preparation process for ginseng glycopeptides, characterized in that, The preparation process includes the following steps: (1) Extract ginseng, collect the extract, and precipitate the extract with alcohol to obtain ginseng glycoprotein; (2) The ginseng glycoprotein is enzymatically hydrolyzed, and the hydrolysate is collected; The enzymes used in the enzymatic hydrolysis are a combination of α-coagulase, trypsin, and pepsin. The enzymatic hydrolysis is performed at a temperature of 40-50℃ for 5-7 hours. Before the enzymatic hydrolysis, the pH value of the ginseng glycoprotein is adjusted to 8-9; The enzymes used in the enzymatic hydrolysis are a combination of α-coagulase, trypsin and pepsin, and the mass ratio of α-coagulase, trypsin and pepsin is (3-5):(2-3):

1. The total amount of enzymes during enzymatic hydrolysis accounts for 4%-8% of the mass percentage of ginseng glycoproteins. (3) The enzymatic hydrolysate is subjected to solid-liquid separation to obtain the ginseng glycopeptide; The solid-liquid separation includes centrifugation, primary ultrafiltration, and secondary ultrafiltration; The molecular weight cutoff for the first ultrafiltration is 1.5-2.5 kDa, and the filtrate is collected. The secondary ultrafiltration has a molecular weight cutoff of 400-600 Da, and the retentate is collected.

2. The preparation process according to claim 1, characterized in that, The extraction material-to-liquid ratio is 1:(10-15).

3. The preparation process according to claim 1, characterized in that, The extraction agent used is water.

4. The preparation process according to claim 1, characterized in that, The extraction time is 1.5-2.5 h.

5. The preparation process according to claim 1, characterized in that, The extraction process is repeated 2-3 times.

6. The preparation process according to claim 2, characterized in that, The extraction process is followed by a concentration step.

7. The preparation process according to claim 6, characterized in that, The relative density of the concentrated extract is 1.0-1.2 g / mL.

8. The preparation process according to claim 1, characterized in that, The alcohol content in the system during alcohol precipitation is 60%-80%.

9. The preparation process according to claim 1, characterized in that, The reagent used for alcohol precipitation is an aqueous solution of ethanol.

10. The preparation process according to claim 9, characterized in that, The ethanol aqueous solution contains 80%-90% ethanol by mass.

11. The preparation process according to claim 8, characterized in that, The alcohol precipitation process also includes steps of vacuum concentration and freeze drying.

12. The preparation process according to any one of claims 1-11, characterized in that, The preparation process includes the following steps: (1) Extract ginseng with water for 1.5-2.5 h, with a material-to-liquid ratio of 1:(10-15), repeat 2-3 times, collect the extract, concentrate it to a relative density of 1.0-1.2 g / mL, and then precipitate it with 80%-90% ethanol aqueous solution. The alcohol content in the system during the precipitation is 60%-80%. Collect the solid, concentrate it under reduced pressure, and freeze dry it to obtain ginseng glycoprotein. (2) Adjust the pH of the ginseng glycoprotein to 8-9, and then enzymatically hydrolyze it at 40-50℃ for 5-7 h, and collect the hydrolysate; The enzymes used in the enzymatic hydrolysis are a combination of α-coagulase, trypsin and pepsin, and the mass ratio of α-coagulase, trypsin and pepsin is (3-5):(2-3):

1. The total amount of enzymes during enzymatic hydrolysis accounts for 4%-8% of the mass percentage of ginseng glycoproteins. (3) After centrifuging the enzymatic hydrolysate, perform a first ultrafiltration using a 1.5-2.5 kDa ultrafiltration membrane, collect the filtrate, perform a second ultrafiltration using a 400-600 Da ultrafiltration membrane, collect the retentate, and obtain the ginseng glycopeptide.

13. A ginseng glycopeptide, characterized in that, The ginseng glycopeptide is prepared by the preparation process described in any one of claims 1-12.

14. The ginseng glycopeptide according to claim 13, characterized in that, The weight-average molecular weight of the ginseng glycopeptide is 700-850 Da.