WO2023088073A1

WO2023088073A1 - Multifunctional american ginseng hydrolyzed peptide, preparation method therefor, and application thereof

Info

Publication number: WO2023088073A1
Application number: PCT/CN2022/128319
Authority: WO
Inventors: 张轩铭; 刘可春; 靳梦; 李晓彬; 张云; 屠鹏飞; 张姗姗; 夏青; 王利振; 盛文龙; 李培海
Original assignee: 山东省科学院生物研究所
Priority date: 2021-11-17
Filing date: 2022-10-28
Publication date: 2023-05-25
Also published as: CA3203208A1; CN114032273B; CN114032273A

Abstract

The present invention relates to a multifunctional American ginseng hydrolyzed peptide, a preparation method therefor, and an application thereof. The content of active peptide in the American ginseng hydrolyzed peptide provided by the present application is up to 85% or above, and the molecular weight range of the active peptide is less than 10 kD. The present application also provides a preparation method for the American ginseng hydrolyzed peptide The American ginseng hydrolyzed peptide is extracted by using an ionic liquid solution and a pepsin-trypsin two-stage digestion process. The process is green and safe and simple in operations, and facilitates scale-up production. The American ginseng hydrolyzed peptide of the present application has significant functional effects, such as immunoregulation, blood pressure reduction, blood sugar reduction, inflammation resistance, oxidation resistance, etc., and pharmacodynamic experiments show that the use of a drug combination mode has a synergistic effect on partial symptoms.

Description

A kind of multifunctional American ginseng hydrolyzed peptide and its preparation method and application

technical field

The invention relates to a multifunctional American ginseng hydrolyzed peptide, a preparation method and application thereof, and belongs to the field of processing medical functional products.

Background technique

Organic solvents are often used in the extraction and separation of natural active ingredients. They not only have strong volatility or toxicity, but also cause environmental pollution during the production process. Ionic liquids are liquid substances composed of larger, asymmetrically structured organic cations and smaller inorganic/organic anions. Compared with traditional solvents, ionic liquids have the characteristics of good thermal stability, non-volatility, safety and environmental protection, and easy reuse. And because of its high selectivity to target substances, it can effectively extract active ingredients in natural products, and has become a new type of green solvent in the field of purification and separation.

Gutowski first proposed the concept of ionic _liquid two-phase water in 2003, _and the researches are _more on the ionic liquid/inorganic salt system. An aqueous two-phase system in which ionic liquid is collected and the lower phase is enriched in potassium phosphate. Compared with the traditional aqueous two-phase system, the ionic liquid two-phase aqueous system not only creates a mild environment for the extraction, but also avoids the use of organic solvents in the extraction process. Short, difficult to emulsify, high extraction rate, and easy recycling of ionic systems, it has broad application prospects in the separation of natural active substances.

American ginseng has the effects of invigorating qi and nourishing yin, clearing heat and promoting body fluid, and is a nourishing and health care product with extremely high medicinal value. American ginseng contains various active ingredients such as ginsenosides, polysaccharides, sterols, proteins and polypeptides. Proteins and peptides are the more important active ingredients in American ginseng except saponins; studies have shown that they have the functions of regulating immunity, lowering blood pressure, lowering blood sugar, lowering blood fat, anti-oxidation, and anti-radiation damage. As a biological macromolecule, protein is generally difficult to be directly absorbed by the human body, especially for patients and the elderly. Due to the decline in body function, the digestion and absorption capacity is relatively poor, and it is more difficult to absorb protein components in food. As a hydrolyzate of protein, peptide components have better physical and chemical properties than protein in many aspects, and can be directly absorbed in the gastrointestinal tract without digestion, and their absorption efficiency is even more significant than that of amino acids.

The preparation of American ginseng polypeptide in the prior art mainly uses organic solvents or expensive extraction equipment. The preparation process is complicated and the cost is high, which is not conducive to the promotion and application of the market. The purity of polypeptide products is low, how to simplify the extraction process and effectively improve the purity of American ginseng polypeptide is a technical problem faced by those skilled in the art.

Contents of the invention

Aiming at the deficiencies of the prior art, the application provides a multifunctional American ginseng hydrolyzed peptide and its preparation method and application.

The multifunctional American ginseng hydrolyzed peptide provided by this application has an active peptide content of over 85%.

Based on the advantages of human body absorption and utilization of peptide components, the invention provides a preparation method of multifunctional American ginseng hydrolyzed peptide; the extraction efficiency is excellent, the operation process is green and safe, and the active peptide content of the final product can reach more than 85%. This technology has obtained high-quality American ginseng active peptides to a great extent, and has broad application prospects in the development of functional products such as anti-oxidation, immune regulation, anti-inflammation, lowering blood pressure and lowering blood sugar.

Technical scheme of the present invention is as follows:

A multifunctional American ginseng hydrolyzed peptide. The hydrolyzed peptide is extracted from an ionic liquid solution, purified with an inorganic salt, and then enzymolyzed by a two-step pepsin-trypsin digestion process. The active peptide content in the hydrolyzed peptide is measured by mass The fraction is more than 85%, and the molecular weight range of the active peptide is <10kD.

Preferably according to the present invention, the active peptide content in the hydrolyzed peptide is above 90% by mass fraction, and the molecular weight range of the active peptide is <10kD.

Further preferably, the active peptide content in the hydrolyzed peptide is above 92% by mass fraction, and the molecular weight range of the active peptide is <3kD.

The preparation method of the above-mentioned American ginseng hydrolyzed peptide comprises the following steps:

(1) After the American ginseng is crushed, add the ionic liquid solution according to the mass volume ratio g/mL 1: (10-35), extract at 40-70°C for 0.5-4 hours, and then separate the solid and liquid to obtain the American ginseng crude protein extract;

(2) Take the American ginseng crude protein extract prepared in step (1), add inorganic salts according to the volume-to-mass ratio mL/g (5-15): 0.8, ultrasonically vibrate for 0.5-2 hours, and centrifuge at 3000-5000 r/min for 5-5 hours. 15min to separate the liquid; remove the lower liquid, add polyethylene glycol to the upper liquid, let it stand for 12-48h, centrifuge at 3000-5000r/min for 15-30min, collect the precipitate to obtain the American ginseng protein extract;

(3) Dissolve the protein extract prepared in step (2) with 10 to 15 times the amount of water, and undergo a two-step pepsin-trypsin digestion process to obtain American ginseng protein hydrolyzate, then add sulfosalicylic acid solution and place it at room temperature for 30- After 90 minutes, the solid and liquid were separated, and the precipitate was redissolved, then subjected to ultrafiltration and freeze-drying to obtain the hydrolyzed peptide of American ginseng.

Preferably according to the present invention, in step (1), the ionic liquid is 1-butyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium hexafluorophosphate, 1-butyl -3-methylimidazolium bistrifluoromethanesulfonylimide, 1-octyl-3-methylimidazolium chloride or 1-hexyl-3-methylimidazolium hexafluorophosphate.

Further preferably, the selected ionic liquid is 1-butyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium hexafluorophosphate or 1-butyl-3-methylimidazolium Bistrifluoromethanesulfonylimide salt.

More preferably, the ionic liquid is 1-butyl-3-methylimidazolium tetrafluoroborate.

Preferably according to the present invention, the final concentration of the ionic liquid in step (1) is 0.001-0.01 mol/L.

Further preferably, the final concentration of the ionic liquid is 0.002-0.008 mol/L.

More preferably, the final concentration of the ionic liquid is 0.004mol/L.

Preferably according to the present invention, in step (2), the inorganic salt is dipotassium hydrogen phosphate, sodium citrate, potassium chloride, sodium dihydrogen phosphate or ammonium sulfate.

Further preferably, the inorganic salt is dipotassium hydrogen phosphate, sodium citrate or sodium dihydrogen phosphate.

More preferably, the inorganic salt is dipotassium hydrogen phosphate.

Preferably according to the present invention, in step (2), the type of polyethylene glycol added is polyethylene glycol 200, 400, 600, 800 or 1000.

Further preferably, the added type of polyethylene glycol is polyethylene glycol 400, 600 or 800.

More preferably, the polyethylene glycol added is polyethylene glycol 600.

Preferably according to the present invention, in step (2), the amount of polyethylene glycol added is 5%-20% by mass fraction.

Further preferably, the added amount of polyethylene glycol is 12%-18% by mass fraction.

More preferably, the added amount of polyethylene glycol is 15% by mass fraction.

Preferably according to the present invention, in step (2), the ultrasonic oscillation frequency is 40KHz-100KHz.

Further preferably, the ultrasonic oscillation frequency is 60KHz-80KHz.

More preferably, the ultrasonic vibration frequency is 70KHz.

Preferably according to the present invention, in step (3), the first digestion process is pepsin addition 10-100mg/mL, pH value 1.5-4, NaCl final concentration 0.01-0.08mol/L, enzymolysis time 0.5-4h, two The advanced digestion process is as follows: the amount of trypsin added is 4-20 mg/mL, the pH value is 7.5-9.5, the final concentration of KH ₂ PO ₄ is 0.05-0.12 mol/L, and the enzymatic hydrolysis time is 1-6 hours; the amount of sulfosalicylic acid added is mass volume Ratio g/mL 0.15: (8~25); Ultrafiltration molecular weight cut-off range is <10kDa.

Further preferably, the primary digestion process is pepsin addition 30-80 mg/mL, pH value 2-3, NaCl final concentration 0.025-0.06 mol/L, enzymolysis time 1-3 hours, and the secondary digestion process is trypsin addition Amount of 8~16mg/mL, pH value of 8~9, final concentration of KH ₂ PO ₄ 0.07~0.10mol/L, enzymatic hydrolysis time of 2~5h; the amount of sulfosalicylic acid added is mass volume ratio g/mL 0.15:( 10～15); ultrafiltration molecular weight cut-off range is <6kDa.

More preferably, the primary digestion process is pepsin addition 60 mg/mL, pH 2.5, NaCl final concentration 0.04 mol/L, enzymolysis time 2 h, and the secondary digestion process is trypsin addition 12 mg/mL, pH 8.5 , KH ₂ PO ₄ final concentration 0.08mol/L, enzymatic hydrolysis time 3h; the amount of sulfosalicylic acid added is the mass volume ratio g/mL 0.15:12; the ultrafiltration cut-off molecular weight range is <3kDa.

The application of the above-mentioned American ginseng hydrolyzed peptide as an active ingredient in the preparation of functional food, health food, medicine or daily chemical products.

Preferably according to the present invention, the above-mentioned American ginseng hydrolyzed peptide is used as an active ingredient in the preparation of immune regulation, blood pressure lowering, blood sugar lowering, anti-inflammation or anti-oxidation functional products.

A drug for lowering blood sugar, the active ingredient of which contains: acarbose and the above-mentioned American ginseng hydrolyzed peptide.

Preferably according to the present invention, the mass ratio of acarbose to American ginseng hydrolyzed peptide in the drug for lowering blood sugar is 1:1.

A drug for lowering blood pressure, the active ingredient of which contains: captopril and the above-mentioned American ginseng hydrolyzed peptide.

Preferably according to the present invention, the mass ratio of captopril to American ginseng hydrolyzed peptide in the blood pressure lowering drug is 1:2.

Beneficial effect of the present invention

1. The present invention provides a high-purity American ginseng hydrolyzed peptide, the active peptide content in the hydrolyzed peptide is more than 85% by mass, and the molecular weight range of the active peptide is <10kD; the American ginseng hydrolyzed peptide provided by this application Significant antioxidant effect;

2. The method for preparing the hydrolyzed peptide of American ginseng in the present invention is simple to operate, does not require expensive equipment, and significantly reduces the amount of organic solvent used. The active peptide content of the prepared hydrolyzed peptide of American ginseng reaches more than 85%.

3. The present invention provides the combined administration of American ginseng hydrolyzed peptide and acarbose, the inhibition of α-glucosidase and α-amylase activity is better than that of the two alone, and has a synergistic effect; The invention provides that the combined administration of American ginseng hydrolyzed peptide and captopril has a better inhibitory effect on ACE than that of the two used alone, and has a synergistic effect; Play a stronger ability to regulate blood pressure and blood sugar; help to improve the scientific and technological content of my country's American ginseng resource processing and utilization technology, and have important economic and social benefits.

4. The hydrolyzed peptide of American ginseng provided by the present invention has significant anti-inflammatory effect, effectively inhibits NO release, and the inhibition rate can reach 33.5%; it can efficiently regulate the expression of related inflammatory mediators or inflammatory factors induced by LPS, and exert an effective anti-inflammatory effect.

5. The hydrolyzed peptide of American ginseng provided by the present invention has significant immunoregulatory effect, can effectively stimulate macrophages, significantly enhance the phagocytic function of macrophages, and enhance immunity.

6. The hydrolyzed peptide of American ginseng provided by the present invention can effectively hinder the generation of angiotensin II, which has the effect of raising blood pressure, so as to achieve the effect of treating hypertension symptoms, and the inhibition rate can reach 77.2%.

7. The hydrolyzed peptide of American ginseng provided by the present invention has inhibitory effects on both α-glucosidase and α-amylase activities, and the inhibition rates can reach 42.5% and 27.2% respectively; the inhibitory effect on α-glucosidase activity is even better than Acarbose positive group.

Description of drawings

Figure 1 Effect of hydrolyzed peptides of American ginseng on LPS-induced NO level in RAW 264.7 cells.

Fig. 2 The inhibitory effect of American ginseng hydrolyzed peptide on ACE enzyme.

Detailed ways

The present invention will be described in detail below in conjunction with specific embodiments, and the described examples are helpful for the understanding and implementation of the present invention, and are not intended to limit the present invention. The protection scope of the present invention is not limited by the specific embodiments, but by the claims.

The contents not described in detail in the embodiments are all according to the prior art in this field.

source of material

1-Butyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium hexafluorophosphate, 1-octyl-3-methylimidazolium chloride and other ionic liquid reagents were purchased from Shanghai Chengjie Chemical Co., Ltd.; inorganic salt reagents such as dipotassium hydrogen phosphate, sodium citrate, and ammonium sulfate were purchased from Sinopharm Chemical Reagent Co., Ltd.; polyethylene glycol was purchased from Beijing Suolaibao Technology Co., Ltd.; pepsin (1 :3000), trypsin (1:250), etc., were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

Example 1

A preparation method of multifunctional American ginseng hydrolyzed peptide, the specific steps are as follows:

(1) After crushing 1kg American ginseng, add 0.004mol/L 1-butyl-3-methylimidazolium tetrafluoroborate solution according to the mass volume ratio g/mL 1:20, and extract at 55°C for 2 hours to obtain American ginseng crude protein Extraction solution;

(2) Take American ginseng crude protein extract, add dipotassium hydrogen phosphate according to the volume to mass ratio of mL/g 12:0.8, 70KHz ultrasonic vibration for 1h, and centrifuge at 4000r/min for 10min; separate the liquid, remove the lower liquid, and supernatant Add polyethylene glycol 600 with a final concentration of 15% to the solution, let it stand at 4°C for 24 hours, centrifuge at 4000r/min for 20 minutes, and collect the precipitate to obtain the American ginseng protein extract.

(3) The protein extract is dissolved in 12 times the amount of water, the amount of pepsin added for primary digestion is 60mg/mL, the pH value is 2.5, the final concentration of NaCl is 0.04mol/L, the enzymatic hydrolysis time is 2h, and the amount of trypsin added for secondary digestion is 12mg/mL mL, pH value 8.5, KH ₂ PO ₄ to a final concentration of 0.08mol/L, enzymatic hydrolysis time 3h; then add sulfosalicylic acid at a mass volume ratio of g/mL 0.15:12 and place at room temperature for 60min, solid-liquid separation, precipitation complex After dissolution, ultrafiltration with a cut-off molecular weight range <3kDa, and freeze-drying to obtain 184g of the extract preserved in powder form.

Example 2

(1) After crushing 1kg American ginseng, add 0.01mol/L 1-butyl-3-methylimidazolium tetrafluoroborate solution according to the mass volume ratio g/mL 1:35, and extract at 70°C for 0.5h to obtain crude American ginseng protein extract;

(2) Take the American ginseng crude protein extract, add dipotassium hydrogen phosphate according to the volume to mass ratio of mL/g 15:0.8, oscillate ultrasonically at 100KHz for 0.5h, and centrifuge at 5000r/min for 5min; separate the liquid, remove the lower liquid, and top Add polyethylene glycol 200 with a final concentration of 20% to the supernatant, let it stand at 4°C for 48 hours, centrifuge at 5000r/min for 15 minutes, and collect the precipitate to obtain the American ginseng protein extract.

(3) Dissolve the protein extract with 10 times the amount of water, add 20 mg/mL of pepsin for primary digestion, pH value 4, final concentration of NaCl 0.08mol/L, enzymatic hydrolysis time 4 hours, add 4 mg/mL of trypsin for secondary digestion mL, pH value 9, KH ₂ PO ₄ to final concentration 0.12mol/L, enzymatic hydrolysis time 6h; then add sulfosalicylic acid according to the mass volume ratio g/mL 0.15:25 and leave it at room temperature for 90min, separate solid and liquid, precipitate and recombine After dissolution, ultrafiltration with a molecular weight cut-off range <6kDa, and freeze-drying to obtain 171 g of the extract preserved in powder form.

Example 3

(1) After crushing 1kg American ginseng, add 0.001mol/L 1-butyl-3-methylimidazolium tetrafluoroborate solution at a mass-volume ratio of g/mL 1:10, and extract at 40°C for 4 hours to obtain American ginseng crude protein Extraction solution;

(2) Take American ginseng crude protein extract, add dipotassium hydrogen phosphate according to the volume to mass ratio of mL/g 5:0.8, oscillate ultrasonically at 40KHz for 2h, and centrifuge at 3000r/min for 15min; separate the liquid, remove the lower liquid, and supernatant Add polyethylene glycol 1000 with a final concentration of 5% to the solution, let it stand at 4°C for 12 hours, centrifuge at 3000r/min for 30 minutes, collect the precipitate to obtain the American ginseng protein extract.

(3) Dissolve the protein extract in 15 times the amount of water, add 100mg/mL pepsin for primary digestion, PH value 1.5, final NaCl concentration 0.01mol/L, enzymolysis time 0.5h, add 20mg trypsin for secondary digestion /mL, pH value 7.5, KH ₂ PO ₄ to a final concentration of 0.05mol/L, enzymatic hydrolysis time 1h; then add sulfosalicylic acid at a mass volume ratio of g/mL 0.15:8 and place at room temperature for 30min, solid-liquid separation, precipitation After reconstitution, ultrafiltration with a cut-off molecular weight range of <10kDa, and freeze-drying obtained 176 g of the extract preserved in powder form.

Example 4

A preparation method of multifunctional American ginseng hydrolyzed peptide, the difference from Example 1 is:

(1) The ionic liquid is 1-butyl-3-methylimidazolium hexafluorophosphate solution;

(2) inorganic salt is sodium citrate;

(3) Others were the same as in Example 1, and obtained 166 g of the extract preserved in powder form.

Example 5

(1) ionic liquid is 1-octyl-3-methylimidazolium chloride salt solution;

(2) inorganic salt is ammonium sulfate;

(3) Others were the same as in Example 1, and obtained 163 g of the extract preserved in powder form.

Comparative example 1

A preparation method of multifunctional American ginseng hydrolyzed peptide, the difference from Example 1 is that the inorganic salt is anhydrous sodium carbonate, and the others are the same, and 158 g of the extract preserved in powder form is obtained.

Comparative example 2

A method for preparing a multifunctional American ginseng hydrolyzed peptide. The difference from Example 1 is that the ionic liquid is 1-heptyl-3-methylimidazolium chloride, and the others are the same, and 160 g of the extract preserved in powder form is obtained.

Comparative example 3

After crushing 1kg American ginseng, add 15 times the amount of 10mmol/L Tris-HCl (pH 7.4), extract at 45°C for 2 hours, centrifuge at 5000r/min for 30 minutes, keep the supernatant, add 1.5 times the volume of acetone to the supernatant, and After standing at 4°C for 24 hours, the precipitate was collected, and the preparation steps of enzymatic digestion were the same as in Example 1 to obtain 155 g of the extract preserved in powder form.

Comparative example 4

After 1kg of American ginseng is crushed, add 20 times the amount of compound enzyme solution (trypsin:papain=1:1w/w, 500U/L), hydrolyze at 37°C for 4h, centrifuge at 5000r/min for 15min, and the supernatant is cut off in the molecular weight range < 10kDa ultrafiltration and freeze-drying to obtain 161 g of the extract preserved in powder form.

Application Test Example 1

Detection of active peptide content of American ginseng

(1) Preparation of biuret solution: Weigh 0.6g CuSO ₄ 5H ₂ O, dissolve in 100mL distilled water, add 1.8g potassium sodium tartrate, then add 1g KI, after the added reagent is completely dissolved, stir and add Dilute 20mL of 6mol/L NaOH solution to 200mL with distilled water, mix well and set aside.

(2) Draw a standard curve: take the concentration of reduced glutathione (GSH) as the abscissa, and the absorbance value as the ordinate, and the operation is as follows: add 1 mL of the concentration of 0.05, 0.1, 0.2, 0.3, 0.4, 0.5mg/mL GSH standard solution, add 4mL biuret solution, develop color at 50°C for 20min, measure and record the absorbance value at 540nm.

Linear regression equation: Y＝0.617x+0.253, R ² ＝0.9961, linear range 0.05～0.5mg/mL

(3) Detection of active peptide content: Weigh 5 mg of extract powder, dilute to 10 mL, and set aside. Draw 1mL of sample solution into a graduated colorimetric test tube, add 4mL of biuret reagent, mix well, develop color according to the "standard curve", measure the absorbance value at 540nm, and compare the standard curve to obtain the concentration of active peptide in the sample solution. Mass (mg), calculate active peptide content (%) in the extract, see Table 1.

Table 1 Active peptide content determination result

It can be concluded from Table 1 that the preparation method involved in the present invention has the dual advantages of large amount of active peptide target components and high enrichment rate, and the content of active peptides in the hydrolyzed peptides of American ginseng provided by the present invention reaches more than 85%.

Compared with Example 1, Comparative Example 1 and Comparative Example 2 only changed the proportioning types of ionic liquid and inorganic salt, resulting in a significant decrease in the content of American ginseng peptide in the product; The effect is remarkable in the state of salt ratio.

In Comparative Example 3, the total protein was prepared by the traditional organic solvent precipitation method. Although the enzymatic digestion preparation process was the same as that of the present invention, the yield and the content of active peptide components were greatly reduced. In Comparative Example 4, only a single compound enzymatic hydrolysis process was used to prepare the active peptides of American ginseng, and the purity of the active peptides in the final product was also low. The results show that the ionic liquid-two-stage enzymatic hydrolysis technology established in the present invention has higher yield and higher purity of the hydrolyzed peptides prepared by the American ginseng compared with the traditional technology, and the content of active peptides in the hydrolyzed peptides of American ginseng reaches more than 85%.

Application test example 2

Antioxidant Experiment of American Ginseng Hydrolyzed Peptide

(1) DPPH·free radical scavenging experiment

Take American ginseng hydrolyzed peptide and dilute it into different concentrations for later use. Take 1mL of diluents of different concentrations in a test tube, add 1mL of 1mmol/mL DPPH solution, add absolute ethanol to 4mL, shake well and place in the dark for 30min, measure the absorbance at 517nm, and calculate the DPPH free radical scavenging rate.

DPPH·clearance rate (%)=(1-A _j /A ₀ )*100%

(A _j = sample + DPPH absorbance, A ₀ = DPPH + ethanol absorbance)

(2) OH radical scavenging experiment

Take American ginseng hydrolyzed peptide and dilute it into different concentrations for later use. Take 1mL of diluents of different concentrations in a test tube, add 2mL of 6mmol/L FeSO ₄ solution, 2mL of 6mmol/L salicylic acid, 2mL of 6mmol/L H ₂ O ₂ solution in turn, mix well and let stand for 30min to measure the absorbance at 510nm, calculate the OH free Base clearance.

OH scavenging rate (%)=[1-(A _j -A _i )/A ₀ ]*100%

(A _j = absorbance after adding sample, A ₀ = absorbance of blank control, A _i = absorbance of sample without adding H ₂ O ₂ )

(3) Experimental results, see Table 2

Table 2 Antioxidant activity of hydrolyzed peptides of American ginseng

As can be seen from Table 2, the _IC50s of the two groups of experiments in Example 1 were 0.3519 mg/mL and 0.4993 mg/mL respectively, which showed a more excellent free radical scavenging ability compared with Comparative Example 3 and Comparative Example 4, especially ·The difference of OH free radical experiment is significant. The above experimental results show that the ionic liquid-two-stage enzymatic hydrolysis process provided by the present invention can obtain American ginseng hydrolyzed peptides with more efficient antioxidant capacity than the traditional process, which may be related to the higher purity of the target components in the final product.

Application test example 3

Anti-inflammatory experiment of American ginseng hydrolyzed peptide

(1) RAW264.7 cells were inoculated in a 96-well plate (1×10 ⁵ cells/mL), and the experimental groups were as follows: blank control group (DMEM culture medium), model group (1 μg/mL LPS), low, medium, and high levels in Example 1. concentration administration group (25, 50, 100 μg/mL+1 μg/mL LPS), comparative example 3 administration group (100 μg/mL+1 μg/mL LPS) and comparative example 4 administration group (100 μg/mL+1 μg/mL LPS), with 3 replicate wells in each group.

(2) After culturing in a constant temperature incubator at 37°C and 5% _CO2 for 24 hours, pipette 100 μL of the culture supernatant and transfer it to a microplate plate, measure the OD value with a microplate reader at 540 nm according to the instructions of the NO kit, and calculate Inhibition rate of NO release by hydrolyzed peptides of American ginseng.

(3) The experimental results are shown in Figure 1. As an important intercellular communication substance with various biological activities, nitric oxide (NO) plays an important role in regulating other pathophysiological processes such as vasodilation and inflammatory immune response. Role. When inflammation occurs in the body, the protein expression of nitric oxide synthase (iNOS) can be significantly up-regulated, which in turn can induce the production of a large amount of NO, triggering subsequent related pathological reactions. Therefore, how to effectively block the pathway of NO synthesis is one of the important methods to regulate the inflammatory response

.

The high-concentration group (100 μg/mL) in Example 1 showed the strongest inhibitory effect on NO release, and its inhibition rate could reach 33.5%. The inhibition rates of NO in the administration groups of Comparative Example 3 and Comparative Example 4 at a concentration of 100 μg/mL were only 16.8% and 17.9%, respectively. Therefore, the American ginseng hydrolyzed peptide obtained by the present invention can efficiently regulate the expression of related inflammatory mediators or inflammatory factors induced by LPS, and then play an anti-inflammatory effect in mouse macrophage RAW 264.7.

Application test example 4

Immunomodulation experiment of American ginseng hydrolyzed peptide

(1) 42 male and female Kunming mice, with a body weight of 18 to 22 g, were randomly divided into 7 groups, 6 in each group, and a blank control group, a model group, a low, middle and high dose group in Example 1, a drug administration group in Comparative Example 3 and Comparative example 4 administration group. The test animals in each group were intragastrically administered for 7 days, once a day, 0.2 mL/10 g each time. The blank control group and the model group were given equal volumes of normal saline, the doses of the low, middle and high dose groups in Example 1 were 50mg/10g, 100mg/10g, and 250mg/10g respectively, and the doses of the comparative example 3 administration group and the comparative example 4 administration group were 250mg /10g. On the 4th day, except the blank group, each mouse in the other groups was intraperitoneally injected with cyclophosphamide.

(2) On the 4th day of the experiment, 1 mL of starch broth was intraperitoneally injected into each mouse, and 1 mL of 1% chicken red blood cells were injected into the abdominal cavity of each mouse after the end of the administration period, and 1 mL of normal saline was injected into the abdominal cavity after 30 minutes. Then the mice were sacrificed by cervical dislocation, the peritoneal fluid was dropped on the glass slide, fixed with methanol:acetone (1:1, V:V) for 5 min, stained with Giemsa, and 50 macrophages were observed under the oil microscope, and the phagocytosis rate was calculated according to the following formula: Phagocytosis index (one macrophage can phagocytose several chicken red blood cells).

Phagocytosis rate/100%=(the number of macrophages that phagocytized chicken red blood cells/50 macrophages)×100%

Phagocytosis index/100%=(the total number of phagocytosed chicken red blood cells/50 macrophages)×100%

(3) Experimental results, see Table 3

Table 3 Effect of hydrolyzed peptides of American ginseng on peritoneal phagocytosis of chicken red blood cells in mice

Compared with the model group *(p<0.05), **(p<0.01)

The activation of macrophages after stimulation can significantly enhance their phagocytosis. Macrophages have active phagocytosis, can remove antigenic substances and denatured cells in the body, and play an important role in both specific and nonspecific immunity. Embodiment 1 group compares with normal control group, the phagocytosis index to mouse peritoneal macrophage is shown as: middle dose group, high dose group effect is extremely significant (P<0.01), has statistical difference; The phagocytosis rate of the cells showed a significant effect in the middle-dose group (P<0.05), and a very significant effect in the high-dose group (P<0.01), with statistical differences. Compared with the experimental results of Comparative Example 3 and Comparative Example 4, the continuous administration of Example 1 for 7 days can better strengthen the phagocytic function of mouse peritoneal macrophages.

Application Test Example 5

ACE Inhibition Experiment of American Ginseng Hydrolyzed Peptides

(1) Example 1 sample is diluted to 0.25mg/mL, 0.5mg/mL, 1.25mg/mL with 50mmol/L Tris-HCl buffer solution (containing 300mmol/L NaCl, pH value 7.5), comparative example 3 and The samples of Comparative Example 4 were diluted to 1.25 mg/mL respectively. 40 μL of angiotensin-converting enzyme (ACE solution, 0.25 units/mL) and 10 μL of sample solution were added to each well of a 96-well plate, and reacted at 37° C. for 10 min. Then 150 μL of N-[3-acryloyl]-L-phenylalanyl-glycyl-glycine solution (FAPGG-Tris-HCl, 0.88 mmol/L) was added for detection. For the blank control, 10 μL of Tris-HCl buffer was added instead of the sample solution, and the concentration of captopril in the positive control group was 50 μg/mL; in addition, the combined administration group of Example 1, the combined administration group of Comparative Example 3 and the combined administration of Comparative Example 4 were set. group (sample 50 μg/mL+captopril 25 μg/mL).

(2) Monitor the absorbance attenuation caused by the degradation of FAPGG by ACE at a wavelength of 340nm. The ACE activity is expressed as the slope of this absorbance decrease, and the inhibitory performance of the active peptide within 40min is recorded. The formula for calculating the inhibition rate is: ACE inhibition rate (%)=(1-ΔA sample/ΔA blank)×100%.

(3) The experimental results are shown in Figure 2. The ACE inhibitory effect of embodiment 1 administration group under the test concentration presents dose-effect relationship, shows the strongest ACE inhibitory effect when the reaction concentration is 250 μ g/mL, and the inhibitory rate can reach 77.2%, and the effect of captopril positive control group quite. However, at this concentration, the ACE inhibition indicators of Comparative Example 3 and Comparative Example 4 were weaker than that of Example 1, and the inhibition rates were only 60.3% and 55.6%. The combined administration of low-dose captopril and the sample of Example 1 will also bring excellent ACE inhibitory effect, and the inhibition rate is as high as 86.1%, which is significantly higher than the combined drug effect of the same dose of Comparative Example 3 and Comparative Example 4, even better than that of captopril. Topril or embodiment 1 high concentration administration result alone. Therefore, the ionic liquid-two-stage enzymatic hydrolysis preparation technology provided by the present invention can obtain American ginseng hydrolyzed peptides with more significant ACE inhibitory effect, and may have a synergistic effect with traditional ACE antihypertensive drugs. Vascular pressure is regulated by the renin-angiotensin system, and renin promotes the production of angiotensin I, which is converted to angiotensin II by angiotensin-converting enzyme (ACE). The American ginseng active peptide sample involved in the present invention can hinder the generation of angiotensin II, which has the effect of raising blood pressure, so as to achieve the effect of treating hypertension symptoms.

Application test example 6

Hypoglycemic experiment of American ginseng hydrolyzed peptide

(1) Take 10 μL of American ginseng hydrolyzed peptide sample solution with different concentrations and 45 μL 5U/mL α-glucosidase phosphate solution (0.2mol/L, pH 5.0), shake and mix well, place in a 55°C incubator, and let it react for 10 minutes. Then, 35 μL of 5 mmol/L 4-nitrophenyl-α-D-glucopyranoside (PNPG) solution was added to start the reaction, and placed in an incubator at 55° C. for 30 min. Add 100 μL of 0.2 mol/L Na ₂ CO ₃ solution to terminate the reaction, and measure its absorbance at 405 nm. PBS solution was used instead of α-glucosidase solution to measure sample blank, and PBS solution was used instead of sample to measure enzyme solution blank. And calculate the inhibition rate according to the following formula:

α-glucosidase inhibition rate (%)=[1–(c-d)/(a-b)]×100%

Where: a, absorbance of α-glucosidase solution; b, absorbance of blank; c: absorbance of reaction mixture solution; d: absorbance of sample.

(2) Premix 100 μL American ginseng hydrolyzed peptide sample solutions with different concentrations with 200 μL α-amylase solution (16.67nkat/mL) at 37°C for 10 minutes, add 400 μL 1g/mL soluble starch solution at 37°C for 15 minutes, after the reaction Add 400 μL of DNS reagent to stop, the mixture was heated in a boiling water bath for 5 min, cooled in an ice water bath for 30 min, and its absorbance was measured at 540 nm. An equal volume of phosphate buffer (0.1mol/L, pH 6.8) was used as a control group instead of α-amylase solution, and the inhibition rate was calculated according to the following formula:

α-amylase inhibition rate (%)=(1–A ₀ /A)×100%

In the formula: A ₀ is the absorbance of the control group, and A is the absorbance of the sample group.

(3) Experimental results, see Table 4

Table 4 Inhibitory effect of American ginseng hydrolyzed peptide on α-glucosidase and α-amylase activity

Compared with acarbose group*(p<0.05)

α-glucosidase and α-amylase are key enzymes of digestion in the body and play a very important role in the catabolism of carbohydrates. Under the catalysis of enzymes, glucose enters the blood through the small intestine, which has an important relationship with the postprandial blood sugar level of diabetic patients. As can be seen from Table 4, the inhibitory rates of the α-glucosidase and α-amylase activities of the Example 1 group increased significantly with the increase of the concentration, and the inhibitory rates were 42.5% and 27.2% respectively when the concentration was 150 μg/mL. The comparison of experimental results shows that the inhibitory effect of Example 1 on the two glucosidases is significantly better than that of Comparative Example 3 and Comparative Example 4, and the inhibitory effect of Example 1 on the activity of α-glucosidase at high concentrations is even better than that of A Carbose positive group. The combined administration of Example 1 and acarbose inhibited 63.2% of α-glucosidase, which was significantly better than the inhibitory effects of 38.6% and 36.1% of the combined administration test group of the comparative example. Example 1 combined with acarbose The inhibitory effect of 55.7% on α-amylase by administration is significantly better than the inhibitory effect of 35.3% and 38.4% in the combined administration test group of the comparative example, and presents a significant difference compared with the administration of acarbose alone (p<0.05) , showing a synergistic effect. The American ginseng active peptide involved in the present invention can exert the effect of lowering blood sugar through highly efficient α-glucosidase and α-amylase inhibitory activity, and because it has the characteristics of natural source of active ingredients, it can be used as an auxiliary dietary supplement in clinical practice, with Broad development prospects.

In summary, the present invention establishes the preparation technology of ionic liquid enrichment-two-stage biomimetic enzymatic hydrolysis of American ginseng hydrolyzed peptide. This technology has the dual advantages of large acquisition of target components and high enrichment rate, and the content of active peptides reaches more than 85%. The process is green and safe, and the operation is simple, which is convenient for large-scale production. In addition, the obtained American ginseng hydrolyzed peptide product has many functional activities superior to the traditional preparation process, such as immune regulation, lowering blood pressure, lowering blood sugar, anti-inflammation, anti-oxidation, etc. Symptoms have a synergistic effect, so they can be used as active ingredients in functional foods, health products and even medicines. They are suitable for various conventional dosage forms and are easily accepted by consumers.

The embodiments described above are some, not all, embodiments of the present invention. The detailed description of the embodiments of the invention is not intended to limit the scope of the claimed invention but to represent only selected embodiments of the invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

Claims

A multifunctional American ginseng hydrolyzed peptide, characterized in that the hydrolyzed peptide is extracted from an ionic liquid solution, purified with inorganic salts, and then enzymolyzed by a pepsin-trypsin two-step digestion process, and the activity of the hydrolyzed peptide The peptide content is more than 85% by mass fraction, and the molecular weight range of the active peptide is <10kD.
The hydrolyzed peptide of American ginseng according to claim 1, wherein the active peptide content in the hydrolyzed peptide is more than 90% by mass fraction, and the molecular weight range of the active peptide is <10kD;

Preferably, the active peptide content in the hydrolyzed peptide is above 92% by mass fraction, and the molecular weight range of the active peptide is <3kD.
A preparation method of American ginseng hydrolyzed peptide, characterized in that it comprises the following steps:

(1) After the American ginseng is crushed, add the ionic liquid solution according to the mass volume ratio g/mL 1: (10-35), extract at 40-70°C for 0.5-4 hours, and then separate the solid and liquid to obtain the American ginseng crude protein extract;

(2) Take the American ginseng crude protein extract prepared in step (1), add inorganic salts according to the volume-to-mass ratio mL/g (5-15): 0.8, ultrasonically vibrate for 0.5-2 hours, and centrifuge at 3000-5000 r/min for 5-5 hours. 15min to separate the liquid; remove the lower liquid, add polyethylene glycol to the upper liquid, let it stand for 12-48h, centrifuge at 3000-5000r/min for 15-30min, collect the precipitate to obtain the American ginseng protein extract;

(3) Dissolve the protein extract prepared in step (2) with 10-15 times the amount of water, and obtain American ginseng protein hydrolyzate through the two-step pepsin-trypsin digestion process, then add sulfosalicylic acid solution and place it at room temperature for 30- After 90 minutes, the solid and liquid were separated, and the precipitate was redissolved, then subjected to ultrafiltration and freeze-drying to obtain the hydrolyzed peptide of American ginseng.
The method according to claim 3, characterized in that, in step (1), the ionic liquid is 1-butyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium hexafluorophosphate salt, 1-butyl-3-methylimidazolium bistrifluoromethanesulfonimide salt, 1-octyl-3-methylimidazolium chloride or 1-hexyl-3-methylimidazolium hexafluorophosphate;

Preferably, the selected ionic liquid is 1-butyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium hexafluorophosphate or 1-butyl-3-methylimidazolium bis Trifluoromethanesulfonylimide salt;

Preferably, the ionic liquid is 1-butyl-3-methylimidazolium tetrafluoroborate.
The method according to claim 3, characterized in that the final concentration of the ionic liquid in step (1) is 0.001 to 0.01mol/L;

Preferably, the final concentration of the ionic liquid is 0.002-0.008mol/L;

Preferably, the final concentration of the ionic liquid is 0.004mol/L.
method as claimed in claim 3, is characterized in that, in step (2), inorganic salt is dipotassium hydrogen phosphate, sodium citrate, potassium chloride, sodium dihydrogen phosphate or ammonium sulfate;

Preferably, the inorganic salt is dipotassium hydrogen phosphate, sodium citrate or sodium dihydrogen phosphate;

Preferably, the inorganic salt is dipotassium hydrogen phosphate;

In step (2), the type of polyethylene glycol added is polyethylene glycol 200, 400, 600, 800 or 1000;

Preferably, the added type of polyethylene glycol is polyethylene glycol 400, 600 or 800;

Preferably, the added type of polyethylene glycol is polyethylene glycol 600;

Preferably, in step (2), the amount of polyethylene glycol added is 5% to 20% by mass fraction;

Preferably, the amount of polyethylene glycol added is 12% to 18% by mass fraction;

Preferably, polyethylene glycol is added in an amount of 15% by mass fraction;

Preferably, in step (2), the ultrasonic oscillation frequency is 40KHz～100KHz;

Preferably, the ultrasonic oscillation frequency is 60KHz-80KHz;

Preferably, the ultrasonic oscillation frequency is 70KHz.
The method according to claim 3, characterized in that, in step (3), a digestion process is pepsin addition 10-100 mg/mL, pH 1.5-4, NaCl final concentration 0.01-0.08 mol/L, enzymatic hydrolysis The time is 0.5-4h, the secondary digestion process is trypsin addition 4-20mg/mL, pH value 7.5-9.5, KH 2 PO 4 final concentration 0.05-0.12mol/L, enzymolysis time 1-6h; sulfosalicin The amount of acid added is mass-volume ratio g/mL 0.15: (8-25); the ultrafiltration molecular weight cut-off range is <10kDa;

Preferably, the primary digestion process is pepsin addition of 30-80 mg/mL, pH value 2-3, NaCl final concentration 0.025-0.06 mol/L, enzymolysis time 1-3 hours, and the secondary digestion process is trypsin addition 8～16mg/mL, pH value 8～9, KH 2 PO 4 final concentration 0.07～0.10mol/L, enzymatic hydrolysis time 2～5h; the amount of sulfosalicylic acid added is mass volume ratio g/mL 0.15: (10 ~15); ultrafiltration molecular weight cut-off range is <6kDa;

Preferably, the primary digestion process is pepsin addition 60mg/mL, pH value 2.5, NaCl final concentration 0.04mol/L, enzymolysis time 2h, and the secondary digestion process is trypsin addition 12mg/mL, pH value 8.5, The final concentration of KH 2 PO 4 is 0.08mol/L, and the enzymatic hydrolysis time is 3h; the amount of sulfosalicylic acid added is the mass volume ratio g/mL 0.15:12; the ultrafiltration cut-off molecular weight range is <3kDa.
The application of the hydrolyzed peptide of American ginseng as an active ingredient in the preparation of functional food, health food, medicine or daily chemical products according to claim 1;

Preferably, the above-mentioned American ginseng hydrolyzed peptide is used as an active ingredient in the preparation of products with immune regulation, blood pressure lowering, blood sugar lowering, anti-inflammation or anti-oxidation functions.
A drug for lowering blood sugar, characterized in that the active ingredient contains acarbose and the hydrolyzed peptide of American ginseng according to any one of claims 1-2;

Preferably, the mass ratio of acarbose to American ginseng hydrolyzed peptide in the drug for lowering blood sugar is 1:1.
A drug for lowering blood pressure, characterized in that the active ingredient contains captopril and the hydrolyzed peptide of American ginseng according to any one of claims 1-2;

Preferably, the mass ratio of captopril to American ginseng hydrolyzed peptide in the drug for lowering blood pressure is 1:2.