Composite plant extract as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of composite plant extracts, and discloses a composite plant extract and a preparation method and application thereof.The composite plant extract comprises wheat germ peptide and swertiamarin, and the mass ratio of the wheat germ peptide to the swertiamarin is 1: (0.8-1.2); the types and quantity of amino acids in the wheat germ peptide meet the following conditions: (1) Glu + Lys + Pro + Val + His + Gly is greater than 32%, and (2) Gly + Ala + Val + Ile + Pro + Thr + Lys + His is greater than 62%. The stability of swertiamarin in the active component of the composite plant extract provided by the invention is high. In order to repair and relieve problem skin, the wheat germ peptide and the swertiamarin can increase target expression of different pathways and can also synergistically promote target expression on the same pathway, and the wheat germ peptide and the swertiamarin can also promote mutual expression of targets among the pathways, so that the effect of repairing and relieving the problem skin is achieved. And the soothing and repairing effects on the skin are improved through multi-dimensional synergy.

Description

Technical Field

[0001] The present invention relates to the technical field of composite plant extracts, and more specifically, to a composite plant extract and a preparation method and application thereof. Background Art

[0002] In recent years, with the changes in dietary structure and the increase in life and work pressure, people's skin barrier has been damaged to varying degrees, which can easily lead to various skin problems. For example: the skin's tolerance to stimulation is reduced, and symptoms such as stinging, burning, tightness, and itching are prone to occur; the skin's ability to resist bacterial invasion is reduced, and symptoms such as acne and acne dermatitis appear.

[0003] In order to solve the problem of damaged skin barrier, especially for those with fragile skin, such as infants, pregnant women, teenagers with acne-damaged skin, and those with sensitive skin, the market has launched products with soothing and repairing effects. However, the soothing and repairing products on the market currently have the following problems: (1) The stability of the active ingredients of the products is poor. The active ingredients of commonly used soothing products are mainly scutellaria baicalensis. Scutellaria baicalensis is mainly found in Gentianaceae plants (such as Gentiana, Swertia vulgaris, and Gentiana macrophylla), and Gentianaceae plants generally also contain gentiopicroside and polyphenols, which are more easily oxidized. These ingredients produce some oxidation products after oxidation, which accelerate the oxidation of scutellaria baicalensis. In addition, scutellaria baicalensis is a terpene lactone glycoside, which is inherently unstable in structure and is easily degraded by ring opening. (2) The product's repair and soothing effect on damaged skin is relatively simple, and usually only focuses on the inhibitory effect on anti-inflammatory and anti-allergic targets, without considering the comprehensive soothing of multiple pathways such as oxidative stress and neural hyperresponsiveness of damaged skin, resulting in poor repair and soothing effects on the skin. Summary of the invention

[0004] The invention provides a composite plant extract, wherein the active ingredient of the composite plant extract, swertiamarin, has high stability and is superior to the prior art in repairing and soothing damaged skin.

[0005] Another object of the present invention is to provide a method for preparing the composite plant extract.

[0006] Another object of the present invention is to provide application of the composite plant extract in repairing and soothing daily chemical products.

[0007] In order to solve the above technical problems, the present invention provides a composite plant extract, comprising wheat germ peptide and swertiamarin, wherein the mass ratio of wheat germ peptide to swertiamarin is 1:(0.8-1.2); the types and quantities of amino acids in the wheat germ peptide simultaneously meet the requirements of ① and ②: ①Glu+Lys+Pro+Val+His+Gly>32%; ②Gly+Ala+Val+Ile+Pro+Thr+Lys+His>62%.

[0008] The composite plant extract provided by the present invention has the following beneficial effects: 1. Swertiamarin has high stability The composite plant extract provided by the present invention contains wheat germ peptides with specific proportions and amino acid characteristics. When Glu+Lys+Pro+Val+His+Gly>32%, the above amino acids form covalent bonds with metal ions through their amino and carboxyl groups to chelate the metal ions in swertiamarin; when Gly+Ala+Val+Ile+Pro+Thr+Lys+His>62%, the isoelectric points of these amino acids are all pH=5.5~6.5, which can stabilize the pH value of swertiamarin, so it can effectively inhibit the degradation of swertiamarin and improve the stability of swertiamarin. Experiments have shown that after the composite plant extract provided by the present invention is left standing at 48°C for one month, the color of the swertiamarin contained therein does not change, the degradation rate is less than 5%, and the pH drop value of swertiamarin does not exceed 0.5, and it has excellent stability.

[0009] 2. The composite plant extract of the present invention repairs and soothes the skin through the following multiple pathways (1) Antioxidant stress pathway In the composite plant extract provided by the present invention, wheat germ peptide can increase the content of Nrf2 and keap1 in the body and improve the body's own anti-oxidative stress ability, while swertiamarin can synergistically enhance the increase of the content of Nrf2 and keap1 in the body and inhibit the anti-oxidative stress of the skin. Experiments have shown that the composite plant extract provided by the present invention can increase the expression of Nrf2 and keap1 by up to 130% and 175% respectively.

[0010] (2) Neural hyperreflexia pathway In the composite plant extract provided by the present invention, wheat germ peptide and swertiamarin synergistically increase the body's expression of micro RNA-145 and inhibit the body's expression of PAK1, thereby inhibiting the skin's neural hyperresponsiveness. Experiments have shown that the composite plant extract provided by the present invention can increase the body's expression of micro RNA-145 by up to 85%, and inhibit the expression of PAK1 by up to 33%.

[0011] (3) Anti-inflammatory factor pathway On inflammatory targets (such as TNFα, IL-1β), the scutellaria glycoside in the composite plant extract provided by the present invention can significantly inhibit the expression of inflammatory mediators TNFα and IL-1β, and wheat germ peptide can stabilize the presence of scutellaria glycoside and have a certain synergistic strengthening effect. The combination of the two has a good anti-inflammatory effect on the skin. Experiments have shown that the inhibition rate of the composite plant extract provided by the present invention on the expression of inflammatory factors TNFα and IL-1β is up to 27% and 19% respectively.

[0012] (4) Antioxidant pathway Experiments have shown that wheat germ peptides and swertiamarin in the composite plant extract provided by the present invention can significantly promote the expression of GCLC to eliminate excess free radicals, and experiments have shown that wheat germ peptides and swertiamarin have a synergistic effect on the expression of GCLC and have excellent antioxidant effects on the skin. Experiments have shown that the composite plant extract provided by the present invention can increase the GCLC of the body by up to 646%.

[0013] (5) Aquaporin AQP3 pathway Experiments have shown that the wheat germ peptide in the composite plant extract provided by the present invention can significantly increase the expression of the water channel protein AQP3, improve the problem of skin dehydration, dryness and itching, and thus play a role in moisturizing and soothing the skin. Experiments have shown that the AQP3 expression increase rate of the body after the composite plant extract provided by the present invention is up to 75%.

[0014] In addition, in the above (1) antioxidant stress pathway, wheat germ peptide can increase the content of Nrf2 and keap1 in the body. After the Nrf2 content is increased, it can induce the expression of anti-inflammatory proteins and related genes in downstream (4) antioxidant pathways and (3) anti-inflammatory factor pathways, such as heme oxygenase-1 (HO-1), phosphoamidite adenine dinucleotide quinone oxidoreductase-1 (NADPH quinone oxidoreductase 1, NQO1), glutamate-cysteine ​​ligase (glutamate-cysteine ​​ligase, GCL) catalytic subunit (GCL catalytic subunit, GCLC), and superoxide dismutase (SOD).

[0015] Preferably, the molecular weight of the wheat germ peptide described in the present invention is 150-2000 Daltons, and the purity is >90%; the gentiopicroside content in the swertiamarin is <1%, the total phenol content is <0.05%, and the purity is >90%.

[0016] The invention improves the purity of swetiamarin, controls the relative contents of gentiopicroside and total phenols in swetiamarin, and effectively reduces the oxidation acceleration effect of gentiopicroside and total phenol oxidation products on swetiamarin.

[0017] The method for preparing wheat germ peptide of the present invention comprises the following steps: S1. adding medium temperature amylase to the wheat germ granule slurry for enzymatic hydrolysis, and obtaining solid M1 by centrifugation; S2. Add papain and bromelain to the solid M1 solution for enzymatic hydrolysis, and then centrifuge and membrane treat in sequence to obtain wheat germ peptide. Preferably, the enzymatic hydrolysis conditions in S1 are: adjusting the pH of the wheat germ particle slurry to 5-7, and the enzymatic hydrolysis temperature to 50-60°C, The amount of medium-temperature amylase added is 0.3%~0.5% of the mass of wheat germ particles.

[0018] Preferably, the S2 enzymolysis conditions are: adjusting the solid M1 solution pH to 6-7, the enzymolysis temperature to 50-60°C, the papaya egg The amount of white enzyme added is 0.1%~0.2% of the mass of solid M1, and the amount of bromelain is 0.05%~0.1% of the mass of solid M1.

[0019] The method for preparing swertiamarin of the present invention comprises the following steps: S1. The raw material containing swertiamarin was crushed, extracted by ultrasonic reflux, and concentrated to obtain an intermediate extract N1; S2. The flocculant is added to the intermediate extract N1 for flocculation, followed by refrigeration, centrifugation, and concentration to obtain the intermediate extract N2; S3. The activated carbon is added to the intermediate extract N2 for decolorization, and the intermediate extract N3 is obtained by filtering and concentrating; S4. The intermediate extract N3 is sequentially adsorbed and eluted by a macroporous resin to obtain an intermediate extract N4; S5. adding molecular imprinting polymer to the intermediate extract N4, performing molecular imprinting polymer adsorption separation, and obtaining swertiamarin after elution and concentration.

[0020] Preferably, the amount of flocculant added to S2 is 2% to 4% of the mass of the intermediate extract N1; the concentration method is reduced pressure concentration, and the concentration temperature is 65 to 75°C; the amount of activated carbon added to S3 is 0.5% to 1% of the mass of the intermediate extract N2.

[0021] Preferably, the ratio of the S4 intermediate extract N3 converted into the mass of the input medicinal materials to the dry weight of the macroporous resin is 1:(5-7); the ratio of the S5 intermediate extract N4 converted into the mass of the input medicinal materials to the mass of the molecular imprinting polymer is 1:(2-3).

[0022] The vacuum degree of the reduced pressure concentration in the present invention is ≥0.08.

[0023] The present invention also provides a method for preparing a composite plant extract, comprising the following steps: Wheat germ peptide and scutellaria baicalensis are added to butanediol in proportion for compounding, sterilized, cooled and filtered in sequence to obtain a composite plant extract; the wheat germ peptide is prepared according to any of the above-mentioned wheat germ peptide preparation methods; the scutellaria baicalensis is prepared according to any of the above-mentioned scutellaria baicalensis preparation methods.

[0024] The present invention also provides the use of the composite plant extract in preparing a repairing or soothing daily chemical product.

[0025] Preferably, the soothing daily chemical product is selected from toner, cream, lotion, essence, facial mask, shampoo and shower gel. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the calibration test spectrum of amino acid standards.

[0027] Figure 2 This is the amino acid test spectrum of the wheat germ peptide obtained in Example 1.

[0028] Figure 3 This is the amino acid test spectrum of the wheat germ peptide obtained in Example 2.

[0029] Figure 4 This is the amino acid test spectrum of the wheat germ peptide obtained in Example 3.

[0030] Figure 5 This is the amino acid test spectrum of the wheat germ peptide obtained in Comparative Example 1.

[0031] Figure 6 This is the amino acid test spectrum of the wheat germ peptide obtained in Comparative Example 2.

[0032] Figure 7 This is the amino acid test spectrum of the wheat germ peptide obtained in Comparative Example 3.

[0033] Figure 8 This is the amino acid test spectrum of the wheat germ peptide obtained in Comparative Example 4.

[0034] Fig. 9 This is a WB image of skin tissue of PAK1 mice in the DNCB-induced dermatitis model.

[0035] Fig.10 This is the WB image of skin tissue of Nrf2, Keap1, and GCLC mice in the UVB light damage model. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below in conjunction with specific implementation methods.

[0037] The medium-temperature amylase used in the examples of the present invention and the comparative examples was purchased from Shanghai Yuanye Biotechnology Co., Ltd. with an enzyme activity of 4000u / g; papain was purchased from Shanghai Yuanye Biotechnology Co., Ltd. with an enzyme activity of 600u / mg; bromelain was purchased from Shanghai Yuanye Biotechnology Co., Ltd. with an enzyme activity of 800u / mg; and fig enzyme was purchased from Shanghai Yuanye with an enzyme activity of 100u / mg.

[0038] The wheat germ peptides in all the examples and comparative examples were prepared at a concentration of 4000 ppm.

[0039] The flocculant composition in all the examples and comparative examples is 1% chitosan in 1% citric acid solution.

[0040] The specific preparation method of the molecular imprinted material described in all the embodiments and comparative examples comprises the following steps: 1.1 Preparation of dispersed phase: 4 mmol acrylamide and 20 mmol N, N'-methylenebisacrylamide were dissolved in 20 mL of water, and 1 mmol of template molecule 2-(hydroxymethyl)-6-((4-ethoxynaphthalen-1-yl)oxy)-tetrahydro-2H-pyran-3,4,5-triol was added, uniformly dispersed, and magnetically stirred for 0.5 h at room temperature to form a prepolymer to obtain solution A; the 2-(hydroxymethyl)-6-((4-ethoxynaphthalen-1-yl)oxy)-tetrahydro-2H-pyran-3,4,5-triol has the structural formula:

[0041] 1.2 Preparation of continuous phase: Dissolve 24 g of L4 polyoxyethylene lauryl ether (BRIJ 30) in 300 mL of n-hexane under nitrogen atmosphere to form a continuous phase as solution B; 1.3 Preparation of inverse dispersion system: Under magnetic stirring, slowly transfer solution A to solution B and stir at room temperature for 60 min under nitrogen atmosphere to obtain a white emulsion; 1.4 Polymerization reaction: 0.5 mL of 10% (w / v) potassium persulfate was added to the white emulsion to initiate the polymerization reaction, and the reaction was continued at 50°C and 300 r / min for 2 h to obtain a polymer microsphere mixture; 1.5 Post-treatment: Add ethanol to the polymer microsphere mixture to aggregate and precipitate the polymer, and filter to obtain the polymer. Transfer to a 240-mesh sieve, wash with water to remove fine powder, and then dry. Put the dried polymer into a Soxhlet extractor, extract the template molecules in the polymer with ethanol, and vacuum dry the polymer at 50-55°C to constant weight. After drying, classify the imprinted material with sieves of different mesh sizes to obtain molecular imprinted polymers of different sizes.

[0042] Wherein, the template molecule: 2-(hydroxymethyl)-6-((4-ethoxynaphthalen-1-yl)oxy)-tetrahydro-2H-pyran-3,4,5-triol, has the structural formula:

[0043] The preparation method is specific and comprises the following steps: (1) Add 10.0 mmol of D-glucose and 10 mL of acetic anhydride to a round-bottom flask to obtain a mixture, and cool the mixture to 0°C. Then, add 0.1 m perchloric acid (70 wt %) dropwise to the mixture while stirring. After the addition is completed, continue stirring at 0°C for 5 h to obtain a reaction solution. Dilute the reaction solution with dichloromethane (40 mL). Then wash the diluted reaction solution with water (20 mL), saturated NaHCO3 solution (20 mL) and brine (20 mL), respectively. Dry the organic layer with anhydrous sodium sulfate, and then remove the organic solvent in the organic layer by rotary evaporator to obtain acetyl-protected glucose with a yield of 95%.

[0044] (2) Add 2.0 mmol of acetyl-protected glucose and 20 mL of dichloromethane to a round-bottom flask to obtain a mixed solution. Add 0.83 mL of 48% mass fraction boron trifluoride ether solution (Cat. No.: Titan Technology 01375737) to the mixed solution at 0 °C. Continue to stir the mixed solution at 0 °C for 10 minutes, then add 2.6 mmol of 1-ethoxy-4-hydroxynaphthalene to the mixed solution, then warm it to room temperature and continue stirring for 24 hours. After the reaction is completed, cool to 0 °C, add 10 mL of saturated NaHCO3 aqueous solution to the mixed solution at 0 °C, and wash the organic layer with water (10 mL x 3). After separation, further extract the aqueous layer with 20 mL of dichloromethane. Combine the dichloromethane organic phases, dry them with anhydrous sodium sulfate, and concentrate them under reduced pressure. After column chromatography purification, the acetyl-protected template molecule is obtained with a yield of 88%.

[0045] (3) 1.5 mmol of the acetyl-protected template molecule was dissolved in a mixture of 10 mL of methanol and 1.5 mL of 25% by mass aqueous ammonia solution. After stirring at room temperature for 24 hours, the solvent was removed under reduced pressure. The template molecule was purified by column chromatography with a yield of 89%.

[0046] The spectral data of the template molecule are as follows:1H NMR (500 MHz, CD3OD) δ (ppm) 8.38–8.36 (m,1H), 7.79–7.77 (m, 1H), 7.50–7.44 (m, 3H), 7.40–7.37 (m, 1H), 7.24–7.22 (m,1H), 5.10 (d, J = 7.5 Hz, 1H), 4.54 (m, 2H), 4.39 (m, 1H), 4.21 (dd, J1 =11.0 Hz, J2 = 5.5 Hz, 1H), 3.77–3.67 (m, 2H), 3.59–3.50 (m, 2H), 161 (m, 3H).13C NMR (125 MHz, CD3OD) δ (ppm) 154.4, 135.8, 128.4, 127.3, 127.1, 127.0,126.3, 123.2, 123.0, 110.6, 102.6, 77.7, 75.9, 75.0, 71.2, 68.1, 60.9, 15.8.HRMS (ESI) m / z calcd. for C16H18O6 [M+H]+ 350.1366, found 350.1354. For details of the equipment and method for extracting scutellaria baicalensis glycosides from medicinal materials in the method for preparing scutellaria baicalensis glycosides described in all embodiments and comparative examples, please refer to the embodiment section of the Chinese patent application "Equipment for Ultrasonic Extraction of Medicinal Materials and Method for Ultrasonic Extraction of Medicinal Materials" with application number: 202410591953.3.

[0047] In the wheat germ peptide preparation method described in all the embodiments and comparative examples, the total peptide testing method in S24 is the lowry method.

[0048] Example 1 A composite plant extract comprises wheat germ peptide and swertiamarin, wherein the mass ratio of wheat germ peptide to swertiamarin is 1:1.

[0049] Wherein, the preparation method of wheat germ peptide comprises the following steps: S1. First, the wheat germ particles were soaked in 10 times the mass of pure water for 2 hours at 200 rpm to obtain wheat germ particle slurry; then, the pH of the wheat germ particle slurry was adjusted to 6.5, the temperature was heated to 55°C, and 0.4% of the mass of the wheat germ particles was added with medium-temperature amylase, and enzymolysis was performed for 2 hours; finally, centrifuged at 7000 rpm, filtered, and the oil layer and the water layer were discarded to obtain solid M1; S2. Add papain and bromelain to the solid M1 solution for enzymatic hydrolysis, centrifugation and membrane separation in sequence to obtain wheat germ peptide, which specifically comprises the following steps: S21. Add 20 times the mass of pure water to solid M1 and stir to dissolve; S22. The solid M1 solution was adjusted to pH 6.5, heated to 55 ° C, 0.15% of the mass of solid M1 papain and 0.075% of the mass of solid M1 bromelain were added, enzymatic hydrolysis for 2h, centrifuged at 7000rpm, filtered to obtain the supernatant M2; S23. The supernatant M2 is ultrafiltered using a 2000 Dalton ultrafiltration membrane at a pressure of 5 bar. When the retentate remains below 30%, 30% pure water is added and ultrafiltration is continued until the mass of the permeate is the same as that of M2, and the permeate M3 is taken; S24. The permeate M3 was nanofiltered with a 150 Dalton nanofiltration membrane, and the retentate was taken. When the retentate remained below 30% by nanofiltration, 30% of pure water was added to the retentate mixture until the total peptide content was 2.1%, thereby obtaining a wheat germ peptide mother solution; Wherein, the preparation method of swertiamarin comprises the following steps: S1. The Swertia japonica was crushed and then subjected to reduced pressure ultrasonic reflux extraction for 1.2 h, with 50% by mass ethanol as the extraction solvent, until the mass of the concentrate was 125% of the crude drug amount, to obtain the intermediate extract N1; S2. First, add 3% flocculant of the intermediate extract N1, stir for 20 min, and refrigerate at 0~8℃ for 24 h; second, 7000rpm Centrifuge to obtain a clear liquid, filter and obtain the supernatant; finally, reduce the pressure and concentrate the obtained supernatant, extract until the mass of the concentrated liquid is 225% of the crude drug amount, and obtain the intermediate extract N2; the reduced pressure and concentration process is: vacuum degree 0.09, temperature 70°C; S3. Add 0.75% of the mass of the intermediate extract N2 to 250 mesh coconut shell activated carbon and stir at room temperature for 1h, filter through a 0.45μm membrane until clear, and concentrate the filtrate with a membrane until the mass of the concentrate is 350% of the crude drug amount to obtain the intermediate extract N3; wherein the membrane molecular weight cutoff is 150 Daltons and the pressure is 12bar; S4. The intermediate extract N3 was added to the D101 macroporous resin column, and firstly subjected to dynamic adsorption at a flow rate of 1BV / h, then washed with 2BV of pure water at a flow rate of 1BV / h, and finally eluted with 3BV of 10% ethanol at a flow rate of 1BV / h to obtain the intermediate extract N4; wherein, the intermediate extract N3 was converted into a ratio of the input medicinal material mass to the macroporous resin dry weight of 1:6; S5. Add molecular imprinting polymer to the intermediate extract N4; first, wash the adsorbed molecular imprinting polymer with pure water twice the mass of the molecular imprinting polymer; then, elute with 95% ethanol 4.5 times the mass of the molecular imprinting polymer; finally, perform nanofiltration in step S24 of the wheat germ peptide preparation method and concentrate to a content of 2.1% of scutellaria glycoside to obtain a scutellaria glycoside mother liquor; the mass ratio of the intermediate extract N4 converted into the mass of the input medicinal material to the molecular imprinting polymer is 1:2.5; the scutellaria glycoside content test refers to the determination method of scutellaria glycoside in the 2020 edition of the Chinese Pharmacopoeia P140; The method for preparing the composite plant extract comprises the following steps: mixing wheat germ peptide mother solution, swertiamarin mother solution, 50% butanediol and pure water in proportion, sterilizing at 93° C. for 30 minutes, cooling to 40° C., and filtering through a 0.22 μm filter membrane to obtain the wheat germ complex.

[0050] The calculation method of the crude drug amount is: crude drug amount = the mass of the raw material input into the process ÷ the mass of the material obtained in this step × 100%.

[0051] Example 2 This embodiment is the second embodiment of the present invention, which is different from the first embodiment in that: A composite plant extract comprises wheat germ peptide and swertiamarin, wherein the mass ratio of wheat germ peptide to swertiamarin is 1:1.2.

[0052] Wherein, the preparation method of wheat germ peptide comprises the following steps: S1. First, the wheat germ particles were soaked in 10 times the mass of pure water for 2 hours at 200 rpm to obtain wheat germ particle slurry; then, the pH of the wheat germ particle slurry was adjusted to 7, the temperature was heated to 50°C, and 0.5% of the mass of the wheat germ particles was added with medium-temperature amylase, and enzymolysis was performed for 2 hours; finally, centrifuged at 8000 rpm, filtered, and the oil layer and the water layer were discarded to obtain solid M1; S2. Add papain and bromelain to the solid M1 solution for enzymatic hydrolysis, centrifugation and ultrafiltration in sequence to obtain wheat germ peptide, which specifically comprises the following steps: S21. Add 20 times the mass of pure water to solid M1 and stir to dissolve; S22. The solid M1 solution was adjusted to pH 7, heated to 60 ° C, 0.2% of the solid M1 mass of papain and 0.05% of the solid M1 mass of bromelain were added, enzymatic hydrolysis for 2h, centrifuged at 8000rpm, filtered to obtain the supernatant M2; S23. The supernatant M2 is ultrafiltered using a 2000 Dalton ultrafiltration membrane at a pressure of 6 bar. When the retentate remains below 30%, 30% pure water is added and ultrafiltration is continued until the mass of the permeate is the same as that of M2, and the permeate M3 is taken; S24. The permeate M3 was nanofiltered using a 150 Dalton nanofiltration membrane, and the retentate was taken. When the retentate remained below 30% by nanofiltration, 30% of pure water was added to the retentate mixture until the total peptide content reached 3.7%, thereby obtaining a wheat germ peptide mother solution; Wherein, the preparation method of swertiamarin comprises the following steps: S1. The gentiana medicinal material was crushed and then subjected to reduced pressure ultrasonic reflux extraction for 1.5 hours, with 55% by mass ethanol as the extraction solvent, until the mass of the concentrated liquid was 150% of the crude drug amount, to obtain the intermediate extract N1; S2. First, add 4% flocculant of the intermediate extract N1, stir for 20 min, and refrigerate at 0~8℃ for 13 h; second, 8000 rpm Centrifuge to obtain a clear liquid, filter and obtain the supernatant; finally, concentrate the obtained supernatant under reduced pressure, extract until the mass of the concentrated liquid is 250% of the crude drug amount, and obtain the intermediate extract N2; the reduced pressure concentration process is: vacuum degree 0.09, temperature 65°C; S3. Add 1% of the intermediate extract N2 by mass and 200 mesh coconut shell activated carbon, stir evenly, and filter through a 0.45 μm membrane until clear. The filtrate was concentrated by membrane until the mass of the concentrate was 400% of the crude drug amount, and the intermediate extract N3 was obtained; wherein the membrane cut-off molecular weight was 150 Daltons and the pressure was 15 bar; S4. The intermediate extract N3 was added to the AB-8 macroporous resin column, firstly subjected to dynamic adsorption at a flow rate of 1BV / h, then washed with 2BV of pure water at a flow rate of 1BV / h, and finally eluted with 3BV of 10% ethanol at a flow rate of 1 BV / h to obtain the intermediate extract N4; wherein, the intermediate extract N3 was converted into a ratio of the input medicinal material mass to the macroporous resin dry weight of 1:7; S5. Add molecular imprinting polymer to the intermediate extract N4; first, wash the adsorbed molecular imprinting polymer with pure water twice the mass of the molecular imprinting polymer; then, elute with 95% ethanol five times the mass of the molecular imprinting polymer; finally, perform nanofiltration according to step S24 in the wheat germ peptide preparation method and concentrate to a content of 3.4% of scutellaria glycoside to obtain a scutellaria glycoside mother liquor; wherein the mass ratio of the intermediate extract N4 converted into the mass of the input medicinal materials to the molecular imprinting polymer is 1:2.

[0053] The method for preparing the composite plant extract comprises the following steps: mixing wheat germ peptide mother solution, swertiamarin mother solution, 45% butanediol and pure water in proportion, sterilizing at 90° C. for 30 minutes, cooling to 43° C., and filtering through a 0.22 μm filter membrane to obtain the wheat germ complex.

[0054] Example 3 This embodiment is the third embodiment of the present invention, which is different from the first embodiment in that: A composite plant extract comprises wheat germ peptide and swertiamarin, wherein the mass ratio of wheat germ peptide to swertiamarin is 1:0.8.

[0055] Wherein, the preparation method of wheat germ peptide comprises the following steps: S1. First, the wheat germ particles were soaked in 10 times the mass of pure water for 2 hours at 200 rpm to obtain wheat germ particle slurry; then, the pH of the wheat germ particle slurry was adjusted to 5, the temperature was heated to 60°C, and 0.3% of the mass of the wheat germ particles was added. Medium-temperature amylase was enzymatically hydrolyzed for 2 hours; finally, centrifuged at 6000 rpm, filtered, and the oil layer and the water layer were discarded to obtain solid M1; S2. Add papain and bromelain to the solid M1 solution for enzymatic hydrolysis, centrifugation and ultrafiltration in sequence to obtain wheat germ peptide, which specifically comprises the following steps: S21. Add 20 times the mass of pure water to solid M1 and stir to dissolve; S22. The solid M1 solution was adjusted to pH 6, heated to 50 ° C, 0.1% of papain and 0.1% of bromelain by mass of solid M1 were added, enzymatic hydrolysis for 2h, centrifuged at 6000rpm, filtered to obtain a supernatant M2; S23. The supernatant M2 is ultrafiltered using a 2000 Dalton ultrafiltration membrane at a pressure of 4 bar. When the retentate remains below 30%, 30% pure water is added and ultrafiltration is continued until the mass of the permeate is the same as that of M2, and the permeate M3 is taken; S24. The permeate M3 was nanofiltered using a 150 Dalton nanofiltration membrane, and the retentate was taken. When the retentate remained below 30% after nanofiltration, 30% pure water was added to the retentate mixture until the total peptide content reached 3.2%, to obtain a wheat germ peptide mother solution; Wherein, the preparation method of swertiamarin comprises the following steps: S1. The Radix Gentianae was crushed and then subjected to reduced pressure ultrasonic reflux extraction for 1 h, with 45% by mass ethanol as the extraction solvent, until the mass of the concentrate was 100% of the crude drug amount, to obtain the intermediate extract N1; S2. First, add 2% flocculant of the intermediate extract N1, stir for 20 min, and refrigerate at 0~8℃ for 18 h; second, 6000 rpm Centrifuge to obtain a clear liquid, filter and obtain the supernatant; finally, concentrate the obtained supernatant under reduced pressure, extract until the mass of the concentrated liquid is 200% of the crude drug amount, and obtain the intermediate extract N2; the reduced pressure concentration process is: vacuum degree 0.08, temperature 75°C; S3. Add 0.5% of the intermediate extract N2 by mass and 300 mesh coconut shell activated carbon, stir evenly, and filter through a 0.45 μm membrane until clear. The filtrate was concentrated by membrane until the mass of the concentrate was 300% of the crude drug amount, and the intermediate extract N3 was obtained; wherein the membrane cut-off molecular weight was 150 Daltons and the pressure was 10 bar; S4. The intermediate extract N3 was added to the D101 macroporous resin column, firstly subjected to dynamic adsorption at a flow rate of 1BV / h, then washed with 2BV of pure water at a flow rate of 1BV / h, and finally eluted with 3BV of 10% ethanol at a flow rate of 1BV / h to obtain the intermediate extract N4; wherein, the intermediate extract N3 was converted into the input medicinal material mass and the macroporous resin dry weight of 1:5; S5. Add molecular imprinting polymer to the intermediate extract N4; first, wash the adsorbed molecular imprinting polymer with pure water of 2 times the mass of the molecular imprinting polymer; then, elute with 95% ethanol of 4 times the mass of the molecular imprinting polymer; finally, perform nanofiltration according to step S24 in the wheat germ peptide preparation method and concentrate to a content of scutellaria baicalensis glycoside of 2.8% to obtain a mother liquor of scutellaria baicalensis glycoside; wherein the mass ratio of the intermediate extract N4 converted into the mass of the input medicinal materials and the molecular imprinting polymer is 1:3.

[0056] The method for preparing the composite plant extract comprises the following steps: mixing wheat germ peptide mother solution, swertiamarin mother solution, 50% butanediol and pure water in proportion, sterilizing at 95° C. for 30 minutes, cooling to 38° C., and filtering through a 0.22 μm filter membrane to obtain the wheat germ complex.

[0057] Comparative Example 1 This comparative example is the first comparative example of the present invention, and is different from Example 1 in that: A composite plant extract comprises wheat germ peptide and swertiamarin, wherein the mass ratio of wheat germ peptide to swertiamarin is 1:1.3.

[0058] Wherein, the preparation method of wheat germ peptide comprises the following steps: S1. First, the wheat germ particles were soaked in 10 times the mass of pure water for 2 hours at 200 rpm to obtain wheat germ particle slurry; then, the pH of the wheat germ particle slurry was adjusted to 7.5, the temperature was heated to 65°C, and 0.6% of the mass of the wheat germ particles was added with medium-temperature amylase, and enzymolysis was performed for 2 hours; finally, centrifuged at 5000 rpm, filtered, and the oil layer and the water layer were discarded to obtain solid M1; S2. Add papain and bromelain to the solid M1 solution for enzymatic hydrolysis, centrifugation and ultrafiltration in sequence to obtain wheat germ peptide, which specifically comprises the following steps: S21. Add 20 times the mass of pure water to solid M1 and stir to dissolve; S22. The solid M1 solution was adjusted to pH 5.5, heated to 65 ° C, 0.25% of the mass of solid M1 papain and 0.04% of the mass of solid M1 bromelain were added, enzymatic hydrolysis for 2h, centrifuged at 5000rpm, filtered to obtain the supernatant M2; S23. The supernatant M2 is ultrafiltered using a 2000 Dalton ultrafiltration membrane at a pressure of 3.5 bar. When the retentate remains below 30%, 30% pure water is added and ultrafiltration is continued until the mass of the permeate is the same as that of M2, and the permeate M3 is taken; S24. The permeate M3 was nanofiltered using a 150 Dalton nanofiltration membrane, and the retentate was taken to a total peptide content of 1.8% to obtain a wheat germ peptide mother solution; Wherein, the preparation method of swertiamarin comprises the following steps: S1. The Swertia japonica was crushed and then subjected to reduced pressure ultrasonic reflux extraction for 45 min, with 40% ethanol as the extraction solvent, until the mass of the concentrate was 90% of the crude drug amount, to obtain the intermediate extract N1; S2. First, add 1.5% of the mass of the intermediate extract N1 into a flocculant, stir for 20 minutes, and refrigerate at 0-8℃ for 12 hours; secondly, The mixture was centrifuged at 5000 rpm to obtain a clear liquid, and the supernatant was filtered to obtain the supernatant; finally, the obtained supernatant was concentrated under reduced pressure, and the concentrate was extracted until the mass of the concentrate was 180% of the crude drug amount, and the intermediate extract N2 was obtained; the reduced pressure concentration process was: vacuum degree 0.07, temperature 80°C; S3. Add 0.4% of the intermediate extract N2 by mass and 350 mesh coconut shell activated carbon, stir evenly, and filter through a 0.45 μm membrane until clear. The filtrate was concentrated by membrane until the mass of the concentrate was 250% of the crude drug amount, and the intermediate extract N3 was obtained; wherein the membrane cut-off molecular weight was 150 Daltons and the pressure was 8 bar; S4. The intermediate extract N3 was added to the D101 macroporous resin column, firstly subjected to dynamic adsorption at a flow rate of 1BV / h, then washed with 2BV of pure water at a flow rate of 1BV / h, and finally eluted with 3BV of 10% ethanol at a flow rate of 1BV / h to obtain the intermediate extract N4; wherein, the intermediate extract N3 was converted into a ratio of the input medicinal material mass to the macroporous resin dry weight of 1:4; S5. Add molecular imprinting polymer to the intermediate extract N4; first, wash the adsorbed molecular imprinting polymer with pure water twice the mass of the molecular imprinting polymer; then, elute with 95% ethanol 5.5 times the mass of the molecular imprinting polymer; finally, perform nanofiltration according to step S24 in the wheat germ peptide preparation method and concentrate to a content of 1.7% of scutellaria glycoside to obtain a scutellaria glycoside mother liquor; wherein the mass ratio of the intermediate extract N4 converted into the mass of the input medicinal materials and the molecular imprinting polymer is 1:3.5.

[0059] The method for preparing the composite plant extract comprises the following steps: compounding wheat germ peptide mother solution, swertiamarin mother solution, 60% butanediol and pure water in proportion, sterilizing at 93° C. for 30 minutes, cooling to 40° C., and filtering through a 0.22 μm filter membrane to obtain the wheat germ composite.

[0060] Comparative Example 2 This comparative example is the second comparative example of the present invention, and is different from Example 1 in that: A composite plant extract comprises wheat germ peptide and swertiamarin, wherein the mass ratio of wheat germ peptide to swertiamarin is 1:0.7.

[0061] Wherein, the preparation method of wheat germ peptide comprises the following steps: S1. First, the wheat germ particles were soaked in 10 times the mass of pure water for 2 hours at 200 rpm to obtain wheat germ particle slurry; then, the pH of the wheat germ particle slurry was adjusted to 4.5, the temperature was heated to 45°C, and 0.25% of the mass of the wheat germ particles of medium-temperature amylase was added, and enzymolysis was performed for 2 hours; finally, centrifuged at 9000 rpm, filtered, and the oil layer and the water layer were discarded to obtain solid M1; S2. Add papain and bromelain to the solid M1 solution for enzymatic hydrolysis, centrifugation and ultrafiltration in sequence to obtain wheat germ peptide, which specifically comprises the following steps: S21. Add 20 times the mass of pure water to solid M1 and stir to dissolve; S22. The solid M1 solution was adjusted to pH 7.5, heated to 45 ° C, 0.075% of the mass of solid M1 papain and 0.15% of the mass of solid M1 bromelain were added, enzymatic hydrolysis for 2h, centrifuged at 9000rpm, filtered to obtain the supernatant M2; S23. The supernatant M2 is ultrafiltered using a 2000 Dalton ultrafiltration membrane at a pressure of 7 bar. When the retentate remains below 30%, 30% pure water is added and ultrafiltration is continued until the mass of the permeate is the same as that of M2, and the permeate M3 is taken; S24. The permeate M3 was nanofiltered using a 150 Dalton nanofiltration membrane, and the retentate was taken to a total peptide content of 1.8% to obtain a wheat germ peptide mother solution; Wherein, the preparation method of swertiamarin comprises the following steps: S1. The Swertia japonica was crushed and then subjected to reduced pressure ultrasonic reflux extraction for 2 h, with 60% ethanol as the extraction solvent, until the mass of the concentrate was 160% of the crude drug amount, to obtain the intermediate extract N1; S2. First, add 4.5% of the mass of the intermediate extract N1 as a flocculant, stir for 20 minutes, and refrigerate at 0-8℃ for 12 hours; secondly, Centrifuge at 8500rpm to obtain a clear liquid, and filter to obtain a supernatant; finally, concentrate the obtained supernatant under reduced pressure, and extract until the mass of the concentrated liquid is 265% of the crude drug amount to obtain an intermediate extract N2; the reduced pressure concentration process is: vacuum degree 0.075, temperature 60°C; S3. Add 1.2% of the intermediate extract N2 by mass and 150 mesh coconut shell activated carbon, stir evenly, and filter through a 0.45 μm membrane until clear. The filtrate was concentrated by membrane until the mass of the concentrate was 450% of the crude drug amount, and the intermediate extract N3 was obtained; wherein the membrane cut-off molecular weight was 150 Daltons and the pressure was 16 bar; S4. The intermediate extract N3 was added to the D101 macroporous resin column, and firstly subjected to dynamic adsorption at a flow rate of 1BV / h, then washed with 2BV of pure water at a flow rate of 1BV / h, and finally eluted with 3BV of 10% ethanol at a flow rate of 1BV / h to obtain the intermediate extract N4; wherein, the intermediate extract N3 was converted into a ratio of the input medicinal material mass to the macroporous resin dry weight of 1:8; S5. Add molecular imprinting polymer to the intermediate extract N4; first, wash the adsorbed molecular imprinting polymer with pure water twice the mass of the molecular imprinting polymer; then, elute with 95% ethanol 3.5 times the mass of the molecular imprinting polymer; finally, perform nanofiltration according to step S24 in the wheat germ peptide preparation method and concentrate to a content of 1.9% of scutellaria glycoside to obtain a scutellaria glycoside mother liquor; wherein the mass ratio of the intermediate extract N4 converted into the mass of the input medicinal materials to the molecular imprinting polymer is 1:5.

[0062] The method for preparing the composite plant extract comprises the following steps: mixing wheat germ peptide mother solution, swertiamarin mother solution, 40% butanediol and pure water in proportion, sterilizing at 93°C for 30 minutes, cooling to less than 45°C, and filtering through a 0.22 μm filter membrane to obtain the wheat germ composite.

[0063] Comparative Example 3 This comparative example is the third comparative example of the present invention. The difference from Example 1 is that in the method for preparing wheat germ peptide, S22. the pH of the solid M1 solution is adjusted to 5.7, the heating temperature is 65°C, 0.5% of the mass of the solid M1 ficin is added, the solution is enzymolyzed for 2h, centrifuged at 7000rpm, filtered, and the supernatant M2 is obtained; S24. The permeate M3 is nanofiltered using a 150 Dalton nanofiltration membrane, and the retentate is taken. When the retentate remains below 30%, 30% pure water is added to the retentate mixture until the total peptide content of the retentate mixture reaches 1.9%, thereby obtaining the wheat germ peptide mother liquor.

[0064] Comparative Example 4 This comparative example is the fourth comparative example of the present invention. The difference from Example 1 is that the raw material for preparing wheat germ peptide is replaced with wheat grains. In S1, the wheat grain particles are crushed into 10-20 mesh particles, and 10 times the mass of pure water is added to soak for 2 hours, stirred at 200 rpm, the pH is adjusted to 5.0, the temperature is heated to 60°C, 0.3% of the mass of wheat grains is added to enzymolyze for 2 hours, centrifuged at 7000 rpm, filtered, and the oil layer and the water layer are discarded to obtain solid M1; S24. The permeate M3 is nanofiltered using a 150 Dalton nanofiltration membrane, and the retentate is taken. When the retentate remains below 30% by nanofiltration, 30% pure water is added to the retentate mixture until the total peptide content of the retentate mixture reaches 2.7%, thereby obtaining the wheat germ peptide mother liquor.

[0065] Comparative Example 5 This comparative example is the fifth comparative example of the present invention. Different from Example 1, in the method for preparing wheat germ peptide, the medium-temperature amylase enzymatic hydrolysis in step S1 is not performed; S24. The permeate M3 is nanofiltered using a 150 Dalton nanofiltration membrane, and the retentate is taken. When the retentate remains below 30%, 30% pure water is added to the retentate mixture until the total peptide content of the retentate mixture reaches 3.2%, thereby obtaining the wheat germ peptide mother liquor.

[0066] Comparative Example 6 This comparative example is the sixth embodiment of the present invention. The difference from the first embodiment is that in the method for preparing wheat germ peptide, papain is not added in step S22. S24. The permeate M3 is nanofiltered using a 150 Dalton nanofiltration membrane, and the retentate is taken. When the retentate remains below 30%, 30% pure water is added to the retentate mixture until the total peptide content of the retentate mixture reaches 2.4%, thereby obtaining the wheat germ peptide mother liquor.

[0067] Comparative Example 7 This comparative example is the seventh embodiment of the present invention. The difference from the first embodiment is that in the method for preparing wheat germ peptide, bromelain is not added in step S22. S24. The permeate M3 is nanofiltered using a 150 Dalton nanofiltration membrane, and the retentate is taken. When the retentate remains below 30% by nanofiltration, 30% pure water is added to the retentate mixture until the total peptide content of the retentate mixture reaches 2.5%, thereby obtaining the wheat germ peptide mother liquor.

[0068] Comparative Example 8 This comparative example is the eighth embodiment of the present invention. The difference from the first embodiment is that in the method for preparing wheat germ peptide, no protease is added in step S22. S24. The permeate M3 is nanofiltered using a 150 Dalton nanofiltration membrane, and the retentate is taken. When the retentate remains below 30%, 30% pure water is added to continue nanofiltration. The nanofiltration operation is repeated until the total peptide content of the retentate mixed liquid reaches 2.0%, thereby obtaining the wheat germ peptide mother liquor.

[0069] Comparative Example 9 This comparative example is the ninth embodiment of the present invention. The difference from the first embodiment is that the conventional alcohol extraction process is used to prepare swertiamarin, ultrasonic reflux extraction is not used, activated carbon adsorption is not added, and molecular imprinting polymer is not added. The specific preparation process includes the following steps: S1. After the herb of Swertia serrata was crushed, it was subjected to vacuum ultrasonic reflux extraction for 1 h, and 45% ethanol by mass was used as the extraction solvent to obtain the intermediate extract N1; S2. First, add 2% flocculant by weight of the intermediate extract N1, stir for 20 minutes, and refrigerate at 0-8°C for 18 hours; second, centrifuge at 8000rpm to obtain a clarified liquid, and filter to obtain the supernatant; finally, concentrate the obtained supernatant under reduced pressure until the mass of the concentrated liquid is 300% of the crude drug amount, to obtain the intermediate extract N2; the reduced pressure concentration process is: vacuum degree 0.08, temperature 75°C; S3. The intermediate extract N2 is added to the D101 macroporous resin column, and the intermediate extract N2 is converted into a ratio of the input medicinal material mass to the macroporous resin dry weight of 1:6, and dynamic adsorption is performed at a flow rate of 1BV / h, first washed with 2BV of pure water at a flow rate of 1BV / h, and then eluted with 3BV of 10% ethanol by volume at a flow rate of 1BV / h. The eluate is concentrated to a content of 2.3% of scutellaria ...

[0070] Performance testing: Amino acid composition of wheat germ peptide: Hitachi's amino acid automatic analyzer (model: L8900) was used for detection. The detected components include Asp, Thr, Ser, Glu, Pro, Gly, Ala, Cys, Val, Met, Ile, Leu, Tyr, Phe, Lys, His, Arg. For details, see Figures 1 to 8 ,in Figure 1 It is the calibration test chart of amino acid standards. Figures 2 to 8 The amino acid test spectra of the wheat germ peptides obtained in Examples 1 to 3 and Comparative Examples 1 to 4 are shown respectively.

[0071] The specific detection method includes the following steps: take 2.0 mL of sample into a hydrolysis tube and add 2.0 mL of 6 mol / L hydrochloric acid. Continue to add 50 mg of phenol to the hydrolysis tube. Put it in a cryogen and cool it for 3-5 min, evacuate it, and fill it with nitrogen for three consecutive times. After sealing, hydrolyze it at 110 degrees for 22 hours, take it out and cool it to room temperature. Open the hydrolysis tube, accurately draw 1.0 mL of filtrate, transfer it to a test tube, blow it dry with a nitrogen blower at 60 degrees, dissolve the residue with 1.0 mL of water, and blow it dry again. Add 2.0 mL of pH sodium citrate buffer to the dried test tube to dissolve, shake and mix, draw the solution through a 0.22 um filter membrane, and transfer it to a 1.5 mL injection bottle. The sample injection volume is 10 μL, and the standard injection volume is 20 μL. Hydroxyproline and proline are detected at 440 nm, and other amino acids are detected at 570 nm.

[0072] The purity of wheat germ peptide is mainly calculated by the following formula: Purity = target component mass concentration ÷ dry matter mass concentration × 100%, The target ingredient is wheat germ peptide; the dry matter mass concentration is the mass of the test liquid dried to constant weight at 105°C ÷ the mass of the test liquid × 100%.

[0073] Table 1: Test results of purity and amino acid characteristics of wheat germ peptides obtained in various examples and comparative examples.

[0074] Table 1 Purity and amino acid characteristics of wheat germ peptides obtained in Examples and Comparative Examples

[0075] Note: “ / ” means not detected; X1=N1÷N0×100%, where N1=total concentration of Glu+Lys+Pro+Val+His+Gly, mg / mL; X2=N2÷N0×100%, where N2=total concentration of Gly+Ala+Val+Ile+Pro+Thr+Lys+His, mg / mL; N0=Asp+Thr+Ser+Glu+Pro+Gly+Ala+Cys+Val+Met+Ile+Leu+Tyr+Phe+Lys+His+Arg total concentration, mg / mL.

[0076] As can be seen from Table 1, compared with Comparative Examples 1 and 2, Example 1 shows that the process conditions have a relatively large influence on the purity of the wheat germ peptide mother liquor and the composition of the amino acids; compared with Comparative Example 4, Example 1 shows that the selection of wheat raw materials has a very large influence on the purity of the polypeptide mother liquor and the composition of the amino acids; compared with Comparative Examples 3 and Comparative Examples 5 to 8, Example 1 shows that the selection of enzyme preparations also has a very large influence on the purity of the polypeptide mother liquor and the composition of the amino acids.

[0077] Swertiamarin component detection: Swertiamarin content detection: refer to the determination method of swertiamarin in the 2020 edition of the Chinese Pharmacopoeia P140; Gentianapicrin content detection: refer to the determination method of gentiopicrin in the 2020 edition of the Chinese Pharmacopoeia P100; Total phenol test method: refer to GBT8313-2008 method 2; Swertiamarin purity is calculated by the following formula: Purity = target component mass concentration ÷ dry matter mass concentration × 100%, The target component is scutellaria baicalensis or gentiopicroside; the dry matter mass concentration is the mass of the test liquid dried to constant weight at 105°C ÷ the mass of the test liquid × 100%.

[0078] The test results of the swertiamarin components obtained in the examples and comparative examples are shown in Table 2.

[0079] Table 2 Swertiamarin components obtained in various examples and comparative examples

[0080] It can be seen from Table 2 that, compared with Comparative Examples 1 and 2, Example 1 shows that the process conditions have a relatively large influence on the purity of the mother liquor of deer sprouts, the purity of gentiopicroside, and the content of total phenols; compared with Comparative Example 9, Example 1 shows that the choice of process scheme has a very large influence on the purity of the mother liquor of deer sprouts, the purity of gentiopicroside, and the content of total phenols.

[0081] Stability test of swertiamarin in the composite plant extract: The composite plant extracts obtained in the embodiments and comparative examples were left to stand at 48° C. for one month to test the stability of swertiamarin. The test results are shown in Table 3.

[0082] Table 3 Stability test of swertiamarin in the composite plant extracts obtained in various examples and comparative examples

[0083] As can be seen from Table 3, the process conditions, process scheme, selection of enzyme preparations, and selection of wheat raw materials all have a great influence on the stability of swertiamarin, which is specifically reflected in the degree of degradation of the main component swertiamarin, the severity of discoloration of the solution, and the degree of pH decrease of the solution. The one with the greatest impact is Comparative Example 8, i.e., the scheme of not using protease to hydrolyze wheat germ, followed by Comparative Example 4, i.e., the scheme of using wheat grains as the source of polypeptide enzymatic hydrolysis. For polypeptides prepared from wheat grains, see Table 1, X1 accounts for more than 32%, but X2 accounts for far less than 62%, and the actual effect is worse than that of Comparative Examples 1 to 3, indicating that X1 and X2 must meet the conditions at the same time to achieve good technical effects.

[0084] The following single-factor experimental verification is carried out. The specific methods and results are shown in Table 4.

[0085] Table 4 Single factor experiment on stability test of swertiamarin

[0086] Remarks: 1. The scutellaria serrata and gentiopicroside used in the above table are both standard products purchased from Chengdu Mansite Biotechnology Co., Ltd.; the preparation process of the "scutellaria serrata extract" used in group (3) is the same as that in Example 1, step S4, the intermediate extract N3 is added to the permeate of the D101 macroporous resin column, concentrated to 300% of the crude drug amount, added with 95% ethanol, stirred and allowed to stand for 2 h, filtered through a 0.45 μm filter membrane to obtain a solid, washed twice with 95% ethanol (10 times the weight of the dry matter), and dried at 105°C to constant weight. After testing, the gentiopicroside content is less than 10 ppm, the scutellaria serrata content is less than 10 ppm, and the total phenol content is 207.4 ppm.

[0087] The degradation rate of scutellaria baicalensis = (C0-C1)÷C0×100%; C0 is the concentration of scutellaria baicalensis in the sample at the beginning of the stability test, and C1 is the concentration of scutellaria baicalensis in the sample at the end of the stability test.

[0088] From the comparison of groups (1) to (3), it can be seen that increasing the purity of scutellaria glycoside in the solution and controlling the relative content of gentiopicroside and the total phenol content can effectively reduce the degradation of scutellaria glycoside and alleviate discoloration. The reason is that the reducing ability of some total phenolic components is greater than that of scutellaria glycoside and is more easily oxidized, and gentiopicroside is also more susceptible to oxidation reactions because it has one more double bond than scutellaria glycoside. Finally, from the appearance, the solution is positively correlated with oxidation and discoloration. From the content detection, the content of scutellaria glycoside decreases more. The reason is that some aldehyde components produced by polyphenols or gentiopicroside, which are more easily oxidized, catalyze the oxidative degradation of scutellaria glycoside when they are oxidized faster than scutellaria glycoside.

[0089] From the comparison between groups (1) and (8), it can be seen that the addition of copper ions can greatly increase the degradation of scutellaria glycoside, which is related to the catalytic oxidation mechanism of copper ions such as the dissociation of dissolved oxygen and the promotion of diffusion. From the comparison between groups (6) and (7) and groups (1) and (2), it can be seen that the addition of chelating agents can reduce the degradation rate of scutellaria glycoside, proving that some metal ions in the extract promote the oxidative degradation of scutellaria glycoside.

[0090] From the comparison between groups (6) and (7) and groups (4) and (5), it can be seen that the inhibitory effect of adding wheat germ peptide in Example 1 on the degradation of scutellaria baicalensis is better than that of adding EDTANa2. In addition to the strong chelating effect of wheat germ peptide, there are two other reasons: first, wheat germ peptide in Example 1 has the effect of stabilizing pH; second, from the perspective of molecular structure, scutellaria baicalensis is an cyclopentane lactone glycoside. In addition to the unsaturated bond that will cause oxidative degradation, it will also undergo lactone ring-opening degradation. The stabilizing effect of wheat germ peptide in Example 1 on pH can slow down the rate of ring-opening degradation, thereby reducing the degradation rate.

[0091] From the comparison of groups (9) to (16), it can be seen that different process conditions, schemes, enzyme preparation types, and raw material sources of wheat germ peptides will affect the stabilizing effect of wheat germ peptides on scutellaria baicalensis. The purity, molecular weight, and amino acid composition of wheat germ peptides are important influencing factors. Among them, the amino acid composition contained in X1 is related to chelation. These amino acids may form covalent bonds with metal ions through their amino and carboxyl groups to achieve chelation effects, and the oxygen atoms of the carboxyl or carbonyl groups in these amino acid compositions may participate in coordination and enhance chelation. The amino acid composition contained in X2 is related to pH stabilization, and its characteristic is that the isoelectric points of these amino acids are all pH = 5.5~6.5.

[0092] Application Example 1: Effects of compound plant extracts on inhibiting high-grade neuralgia and anti-inflammatory factors Test of the soothing effect of compound plant extracts on DNCB-induced dermatitis model 1. Experimental Materials Positive control drug: hydrocortisone butyrate (Uzoler), Tianjin Jinyao Pharmaceutical Co., Ltd., batch number: 23051017. 18-20g BALB / C mice, purchased from Shanghai Jiesijie Experimental Animal Co., Ltd. 1-Chloro-2,4-dinitrotoluene (DNCB), Tokyo Chemical Industry Co., Ltd., batch number: WT6CA-VO.

[0093] 2. Experimental Methods Mice were randomly divided into groups, with 8 mice in each group. Model preparation: After 1 week of adaptive feeding, all mice were depilated using an electric pusher and depilatory cream, with the depilatory area of ​​the abdomen being 1*2 cm2 and the depilatory area of ​​the back being 2*3 cm2. On the first day of modeling, 7% DNCB acetone solution was applied to the depilated area of ​​the abdomen for sensitization, and the second sensitization was performed at the same location 3 days later; on the eighth day of modeling, 0.5% DNCB acetone solution was applied to the back for stimulation, and the stimulation was performed every 5 days for the first 15 days, and every other day after 15 days, for 16 consecutive stimulations. The drug administration of each group is shown in Table 5.

[0094] Table 5: Drug administration and dosage of each group

[0095] Starting from the first day of model preparation, the blank group and the model group did not receive any intervention. The positive control group applied Uzhuoer to the back lesions once a day; the sample group applied the composite plant extract obtained in the embodiment or the comparative example to the back lesions once a day. For 45 consecutive days. If the hair of the mice grows during the medication period, a depilatory cream is applied externally to supplement the hair removal. Samples were collected on the 45th day, and the skin lesion scores and appearance photos of each group were recorded. The back skin of each group of mice was taken to determine the levels of TNF-α and IL-1β in the tissues, qPCR was used to detect micro RNA-145, and WB was used to detect the PAK1 protein level. The WB image of the skin tissue of PAK1 mice in the DNCB-induced dermatitis model is shown in Fig. 9 The test concentration of DSM1 and DSM2 was 4%, and the test concentration of the implementation and comparative examples was 2%.

[0096] 3. Data Processing and Analysis GraphPad prism8 software was used for analysis, and the data of each group were expressed as mean ± standard deviation, and the inter-group comparison was performed using student t-test. Compared with the model group, P < 0.05 or 0.01 was considered statistically significant. The results of the test on the inhibitory effect of the composite plant extracts obtained in the embodiments and comparative examples on TNF-α and IL-1β factors and the expression levels of micro RNA-145 and PAK1 are shown in Table 7, and the significant comparison between the groups is shown in Table 9.

[0097] Application Example 2: Antioxidant Effects of Compound Plant Extracts Testing of composite plant extracts on UVB-induced skin photoaging model 1. Experimental Materials Kangfuxin solution (positive control) (Sichuan Good Doctor Panxi Pharmaceutical Co., Ltd., batch number: 231104); KM mice 18-20 g, purchased from Shanghai Jiesijie Experimental Animal Co., Ltd.; UV light therapy device: model: SS01B, manufacturer: Sigma.

[0098] 2. Experimental Methods Mice were randomly divided into groups (blank, model, positive control, and test groups), with 12 mice in each group. The back of the mice was depilated, with an area of ​​4 cm × 2 cm. Gauze was spread flat on the exposed skin of the mice 30 minutes before irradiation with the ultraviolet light therapy device. The drugs were administered at 9 o'clock every morning. No drugs were applied externally in the blank group, 1 mL of normal saline was evenly applied externally in the model group, and 1 mL of drugs were evenly applied externally in the positive control and sample groups for 10 minutes. The drug administration of each group is shown in Table 6.

[0099] The blank group was not given UVB intervention. The other groups were given medication once a day before irradiation for 7 consecutive days. The irradiation distance was determined to be 20 cm from the mouse back, and the UVB irradiation intensity was 13 mW / cm2 for one week. The irradiation time on the first and last day was 10 min, and the remaining 5 days were 15 min, with a total irradiation dose of 74.1 J / cm2.

[0100] A large amount of pigmentation, scaling, erythema, and wrinkles appeared in the irradiated area on the back of the mice, and even ulceration, scabs, and leather-like touch appeared locally, indicating that the model was successful. Photos were taken to record the changes in the skin appearance of each group in the experiment. After the end of the experiment, that is, on the 8th day, the mice were killed, and the expression levels of NRF2, KEAP1, and GCLC proteins were detected by Wb. The WB images of the skin tissues of the UVB light-damaged model Nrf2, Keap1, and GCLC mice are shown in the figure. Fig.10 The test concentration of DSM1 and DSM2 was 4%, and the test concentration of the implementation and comparative examples was 2%.

[0101] Table 6 Modeling and drug administration of light-damaged mice

[0102] 3. Data Processing and Analysis GraphPad prism8 software was used for analysis, and the data of each group were expressed as mean ± standard deviation, and the inter-group comparison was performed using student t-test distribution. Compared with the model group, P < 0.05 was considered statistically significant. The test results of the expression levels of NRF2, KEAP1, and GCLC proteins obtained by the composite plant extracts obtained in the embodiments and comparative examples are shown in Table 8, and the significance comparison between the groups is shown in Table 9.

[0103] Application Example 3: Promotion of the expression of water channel protein AQP3 by compound plant extracts Detection of single-cell fluorescence intensity of AQP3 by compound plant extracts 1. Experimental Materials The cells used in the experiment were keratinocytes HaCaT; the main reagents were: trypsin (Sigma 20201031-0404, USA), PBS (Boster 700857135, Wuhan), double antibody (Sigma 20200804-0341, USA), fetal bovine serum (Sijiqing 18100501, Zhejiang), 4% paraformaldehyde, Tween 20, TritonX-100, BSA, AQP3 primary antibody (rabbit anti-), fluorescently labeled secondary antibody (rabbit anti-), DAPI.

[0104] 2. Experimental Methods Sample grouping: HaCaT cells treated with plant extracts, of which DSM1 and DSM2 were tested at a concentration of 1%, and the concentration of the implementation and comparative examples was 0.5%. Control information: ① Blank control group: HaCaT cells cultured normally without treatment. ② Model control group: HaCaT cells treated with LPS.

[0105] S1. Resuscitate the cryopreserved HaCaT cells and inoculate them into cell culture flasks at an appropriate density. Add DMEM medium (10% FBS) and culture them in a cell culture incubator at 37°C and 5% CO2.

[0106] S2. Wash HaCaT cells in the logarithmic growth phase twice with PBS, add trypsin to digest, add DMEM medium (10% FBS) to terminate digestion, centrifuge, discard the supernatant, add DMEM medium (10% FBS) to prepare a cell suspension. Add 2 mL of cell suspension to each well of a 6-well plate with a coverslip and culture in a cell culture incubator.

[0107] S3. When the confluence of HaCaT cells reached about 40%, the model control group was treated with LPS. Under the same conditions, the sample group was treated with the test concentration for 24 hours.

[0108] S4. Each well was fixed with paraformaldehyde at room temperature, 0.5% TritonX-100 was prepared, and the membrane was permeabilized at room temperature. After washing with PBS, the membrane was blocked at room temperature and the blocking solution was discarded. The primary antibody was incubated overnight, and the fluorescent secondary antibody was incubated in the dark. After washing with PBS, DAPI was incubated at room temperature in the dark. The slides were removed, blocked and fixed with the sealing solution overnight, and the images were taken under a fluorescence microscope for data analysis.

[0109] 3. Data processing GraphPad Prism 8.0 software was used for statistical processing of data. All experimental data were expressed as mean ± SEM, and statistical methods such as t-test or one-way analysis of variance were used to analyze the data.

[0110] Judgment criteria: Test the relative expression rate of aquaporin AQP3 (%). Compared with the model control group, the relative expression rate of aquaporin AQP3 in keratinocytes in the sample group increased, and the result *p<0.05, the difference (significant), indicating that the sample has the effect of promoting the production of aquaporin AQP3 at this test concentration. The test results of the expression level of aquaporin AQP3 obtained by the composite plant extracts of the examples and comparative examples are shown in Table 8, and the significance comparison between the groups is shown in Table 9.

[0111] Table 7 Test of the composite plant extracts obtained in the examples and comparative examples for inhibiting inflammation and high-grade nervous system reactions

[0112] Remarks: Example 1-DSM1: The wheat germ peptide mother solution obtained in step 6 of Example 1 was added with pure water to prepare a solution containing 4000 ppm of total peptide; Example 1-DSM2: The scutellaria glycoside mother solution obtained in step 16 of Example 1 was added with pure water to prepare a solution containing 4000 ppm of scutellaria glycoside; compared with the model group, * in the test group is P < 0.05, ** is P < 0.01; compared with the blank group, # is P < 0.05, ## is P < 0.01 Table 8 Tests on antioxidant and aquaporin-enhancing properties of the composite plant extracts obtained in the examples and comparative examples

[0113] Notes are the same as Table 7.

[0114] As can be seen from Tables 7 and 8, compared with the model group, the embodiment can significantly inhibit the expression of inflammatory mediators TNFα and IL-1β to achieve an anti-inflammatory effect; it can significantly increase the expression of NrF2 and Keap1, play an anti-oxidative stress role, and relieve the damage caused by excessive oxidative stress of the skin; it can significantly increase the expression of GCLC to achieve an antioxidant effect and reduce the damage of free radicals to the skin; it can significantly increase the expression of aquaporin AQP3, improve the moisturizing effect and repair effect of the skin, and promote faster recovery of problem skin; it can significantly increase the expression of miR-145 and reduce the expression of PAK1, thereby effectively improving the skin sensitivity, skin itching and other discomforts caused by excessive innervation of the epidermal nerves of the problem skin. In summary, the composition of the present invention has the effects of simultaneous anti-inflammatory, anti-oxidation, anti-oxidative stress, moisturizing, and inhibiting nerve hyperreaction, and has a more comprehensive skin soothing effect compared with the existing technology.

[0115] The difference between Comparative Examples 1 and 2 and Example 1 is that the process conditions are different. Compared with the model group, there is no significant difference in some pathways or targets between Comparative Examples 1 and 2, and the effect is not as good as Example 1, indicating that the difference in process conditions has a certain influence on the soothing and repairing effect. The difference between Comparative Example 3 and Example 1 is that the type of enzyme preparation for wheat germ enzymatic hydrolysis is different, and the result is that the effect is not significant in most pathways, and the effect is obviously not as good as Example 1; and the difference between Example 4 and Example 1 is that wheat germ is replaced with wheat grains, and the result is that the effect is not significant in most pathways, indicating that the source of raw materials also plays a decisive role.

[0116] Table 9 Significant comparison between groups

[0117] Note: Compared with the two groups, * means P < 0.05, ** means P < 0.01, × means P ≥ 0.05, / means no test was done DSM1 and DSM2 are single-component compositions of Example 1. The test concentrations of DSM1 and DSM2 during the test are double those of Example 1, so that the total active substance concentrations of the three groups during the test are consistent. From the significant comparisons of the 1st, 2nd, and 3rd rows of Table 9, it can be seen that in the expression pathway of inhibiting inflammatory mediators TNFα and IL-1β, there are significant differences between DSM1 vs. DSM2 and DSM1 vs. Example 1, while there is no significant difference between DSM2 vs. Example 1, indicating that DSM2, i.e., swertiamarin, plays a more important role in this pathway, and DSM1 and DSM2 have a synergistic effect. After compounding, the dosage is reduced to achieve the same effect as using swertiamarin alone.

[0118] In terms of the expression of NrF2 and Keap1, the pathway targets for inhibiting oxidative stress, there were significant differences between DSM1 vs DSM2 and DSM2 vs Implementation 1, but there was no significant difference between DSM1 vs Implementation 1, indicating that DSM1, i.e. wheat germ peptide, plays a more important role in this pathway, and that DSM1 and DSM2 have a synergistic effect. After compounding, reducing the dosage can achieve the same effect as using wheat germ peptide alone.

[0119] In terms of the effect on the antioxidant pathway GCLC, there is a significant difference between DSM1 and DSM2, indicating that wheat germ peptide plays a more important role, and Example 1 has significant differences on both DSM1 and DSM2, indicating that swertiamarin and wheat germ peptide also have significant synergistic effects.

[0120] In the expression of aquaporin AQP3, there are significant differences between DSM1 vs DSM2 and DSM2 vs Example 1, while there is no significant difference between DSM1 vs Example 1, indicating that DSM1, i.e., wheat germ peptide, plays a more important role in this pathway, and DSM1 and DSM2 have a synergistic effect. After compounding, the dosage is reduced to achieve the effect of no statistical difference with wheat germ peptide alone; in inhibiting the neural hyperreaction pathway, i.e., increasing the expression of miR-145 and reducing the expression of PAK1, there is no significant difference between DSM1 vs DSM2, while DSM1 and DSM2 are significantly different from Example 1, indicating that in this pathway, swertiamarin and wheat germ peptide have a significant synergistic effect. In summary, swertiamarin and wheat germ peptide have both synergistic and complementary effects and synergistic effects in terms of comprehensive soothing effects.

[0121] In summary, the composite plant extract provided by the present invention contains swertiamarin and wheat germ peptide, wherein swertiamarin has high stability. For problematic skin, the composite plant extract can synergistically repair and soothe problematic skin through multiple pathways of anti-oxidative stress, inhibition of neural hyperreaction, inhibition of inflammation, anti-oxidation, and increase of aquaporin expression.

[0122] The above is only an implementation method of the invention, and does not limit the scope of patent protection. Those skilled in the art can make non-substantial changes or substitutions based on the invention, which still fall within the scope of patent protection.

Claims

1. A composite plant extract, characterized in that: It comprises wheat germ peptide and swertiamarin, wherein the mass ratio of wheat germ peptide to swertiamarin is 1:(0.8-1.2); the types and quantities of amino acids in the wheat germ peptide satisfy ①Glu+Lys+Pro+Val+His+Gly>32% and ②Gly+Ala+Val+Ile+Pro+Thr+Lys+His>62%.

2. The composite plant extract according to claim 1, characterized in that: The molecular weight of the wheat germ peptide is 150-2000 Daltons, and the purity is >90%; the gentiopicroside content in the swertiamarin is <1%, the total phenol content is <0.05%, and the purity is >90%.

3. The composite plant extract according to claim 1, characterized in that: The method for preparing wheat germ peptide comprises the following steps: S1. adding medium temperature amylase to the wheat germ granule slurry for enzymatic hydrolysis, and obtaining solid M1 by centrifugation; S2. Add papain and bromelain to the solid M1 solution for enzymatic hydrolysis, and then centrifuge and membrane treat in sequence to obtain wheat germ peptide.

4. The composite plant extract according to claim 3, characterized in that: The enzymatic hydrolysis conditions in S1 are: adjusting the pH of the wheat germ particle slurry to 5-7, the enzymatic hydrolysis temperature to 50-60° C., and the amount of medium-temperature amylase added to be 0.3%-0.5% of the mass of the wheat germ particles.

5. The composite plant extract according to claim 3, characterized in that: The S2 enzymolysis conditions are: adjusting the solid M1 solution pH to 6-7, the enzymolysis temperature to 50-60° C., the amount of papain added to be 0.1%-0.2% of the mass of the solid M1, and the amount of bromelain added to be 0.05%-0.1% of the mass of the solid M1.

6. The composite plant extract according to claim 1, characterized in that: The method for preparing swertiamarin comprises the following steps: S1. The raw material containing swertiamarin was crushed, extracted by ultrasonic reflux, and concentrated to obtain an intermediate extract N1; S2. The flocculant is added to the intermediate extract N1 for flocculation, followed by refrigeration, centrifugation, and concentration to obtain the intermediate extract N2; S3. The activated carbon is added to the intermediate extract N2 for decolorization, and the intermediate extract N3 is obtained by filtering and concentrating; S4. The intermediate extract N3 is sequentially adsorbed and eluted by a macroporous resin to obtain an intermediate extract N4; S5. adding molecular imprinting polymer to the intermediate extract N4, performing molecular imprinting polymer adsorption separation, and obtaining swertiamarin after elution and concentration.

7. The composite plant extract according to claim 6, characterized in that: The amount of flocculant added to the S2 is 2% to 4% of the mass of the intermediate extract N1; the concentration method is reduced pressure concentration, and the concentration temperature is 65 to 75° C.; the amount of activated carbon added to the S3 is 0.5% to 1% of the mass of the intermediate extract N2.

8. The composite plant extract according to claim 6, characterized in that: The S4 intermediate extract N3 is converted into the input medicinal material mass and the macroporous resin dry weight ratio of 1: (5-7); the S5 intermediate extract N4 is converted into the input medicinal material mass and the molecular imprinting polymer mass ratio of 1: (2-3).

9. A method for preparing a composite plant extract, characterized in that: The following steps are involved: Wheat germ peptide and swedemarin are added to butanediol in proportion for compounding, sterilized, cooled and filtered in sequence to obtain a composite plant extract; the wheat germ peptide is prepared according to the method for preparing wheat germ peptide in the composite plant extract according to any one of claims 3 to 5; the swedemarin is prepared according to the method for preparing swedemarin in the composite plant extract according to any one of claims 6 to 8.

10. Use of the composite plant extract according to any one of claims 1 to 9 in the preparation of repairing and soothing daily chemical products.

Images