A composite plant extract and its preparation method and application

Abstract

This invention belongs to the technical field of composite plant extracts and discloses a composite plant extract, its preparation method, and application. The composite plant extract comprises wheat germ peptide and swetmarin, wherein the mass ratio of wheat germ peptide to swetmarin is 1:(0.8-1.2). The types and quantities of amino acids in the wheat germ peptide satisfy the following requirements: ① Glu+Lys+Pro+Val+His+Gly>32% and ② Gly+Ala+Val+Ile+Pro+Thr+Lys+His>62%. The composite plant extract provided by the present invention features high stability of swetmarin as an active ingredient. To repair and soothe problematic skin, wheat germ peptide and swetmarin can both increase the expression of targets in different pathways and synergistically promote target expression within the same pathway. Furthermore, wheat germ peptide and swetmarin can promote the cross-expression of targets within these pathways, synergistically enhancing the soothing and repairing effects on the skin in multiple dimensions.

Description

Technical Field

[0001] The present invention relates to the technical field of composite plant extracts, and more particularly 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-prone skin, and those with sensitive skin, the market has launched products with soothing and repairing effects. However, the soothing and repairing products currently on the market have the following main problems: (1) The stability of the active ingredients of the products is poor. The active ingredient of commonly used soothing products is mainly scutellaria baicalensis. Scutellaria baicalensis is mainly found in plants of the Gentianaceae family (such as Gentiana scabra, Swertia japonica, and Gentiana macrophylla). 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 an iridoid lactone glycoside, which is inherently unstable and easily degraded by ring opening. (2) The product's repair and soothing effect on damaged skin is relatively simple. It usually only focuses on the inhibitory effect on anti-inflammatory and anti-allergic targets, and does not consider 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 present invention provides a composite plant extract, wherein the active ingredient 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 the use of the composite plant extract in repairing and soothing daily chemical products.

[0007] To solve the above technical problems, the present invention provides a composite plant extract, comprising wheat germ peptide and swedemarin, wherein the mass ratio of wheat germ peptide to swedemarin 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: ① Glu+Lys+Pro+Val+His+Gly>32%; ② Gly+Ala+Val+Ile+Pro+Thr+Lys+His>62%.

[0008] The composite plant extract provided by the present invention has the following beneficial effects:

[0009] 1. Swertiamarin is highly stable

[0010] The composite plant extract provided by the present invention contains wheat germ peptides with specific ratios and amino acid profiles. When Glu+Lys+Pro+Val+His+Gly is greater than 32%, these amino acids form covalent bonds with metal ions through their amino and carboxyl groups, chelating the metal ions in swetiamarin. When Gly+Ala+Val+Ile+Pro+Thr+Lys+His is greater than 62%, the isoelectric points of these amino acids are all between pH 5.5 and 6.5, stabilizing the pH of swetiamarin. This effectively inhibits the degradation of swetiamarin and improves its stability. Experiments have shown that after standing at 48°C for one month, the color of the swetiamarin contained in the composite plant extract does not change, the degradation rate is less than 5%, and the pH drop of swetiamarin does not exceed 0.5, demonstrating excellent stability.

[0011] 2. The composite plant extract of the present invention repairs and soothes the skin through the following multiple pathways

[0012] (1) Antioxidant stress pathway

[0013] In the composite plant extract provided by this invention, wheat germ peptide can increase the levels of Nrf2 and Keap1 in the body, improving the body's ability to resist oxidative stress, while swertiamarin can synergistically enhance the increase in Nrf2 and Keap1 levels in the body, inhibiting antioxidant stress in the skin. Experiments have shown that the composite plant extract provided by this invention can increase Nrf2 and Keap1 expression by up to 130% and 175%, respectively.

[0014] (2) Neural hyperreflexia pathway

[0015] In the composite plant extract provided by the present invention, wheat germ peptide and swertiamarin synergistically increase microRNA-145 expression and inhibit PAK1 expression, thereby suppressing skin hyperresponsiveness. Experiments have shown that the composite plant extract provided by the present invention can increase microRNA-145 expression by up to 85% and inhibit PAK1 expression by up to 33%.

[0016] (3) Anti-inflammatory factor pathway

[0017] At inflammatory targets (such as TNFα and IL-1β), the swertiamarin in the composite plant extract provided by the present invention significantly inhibits the expression of inflammatory mediators TNFα and IL-1β. Wheat germ peptide stabilizes the presence of swertiamarin and provides a synergistic strengthening effect. The combination of the two has a significant anti-inflammatory effect on the skin. Experiments have shown that the composite plant extract provided by the present invention can inhibit the expression of inflammatory factors TNFα and IL-1β by up to 27% and 19%, respectively.

[0018] (4) Antioxidant pathway

[0019] Experiments have shown that both wheat germ peptides and swertiamarin in the composite plant extract provided by the present invention can significantly promote the expression of GCLC and scavenge excess free radicals. Furthermore, the experiments have shown that wheat germ peptides and swertiamarin have a synergistic effect on promoting GCLC expression, exerting excellent antioxidant effects on the skin. Experiments have also shown that the composite plant extract provided by the present invention can increase GCLC levels by up to 646%.

[0020] (5) Aquaporin AQP3 pathway

[0021] 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, improving skin dehydration, dryness, and itching, thereby moisturizing and soothing the skin. Experiments have shown that the composite plant extract provided by the present invention can increase AQP3 expression by up to 75%.

[0022] In addition, wheat germ peptide can increase the content of Nrf2 and keap1 in the body in the (1) anti-oxidative stress pathway mentioned above, and the increase in Nrf2 content 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), phosphoamidoadenine 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).

[0023] 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%.

[0024] 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.

[0025] The method for preparing wheat germ peptide of the present invention comprises the following steps:

[0026] S1. The wheat germ granule slurry was enzymatically hydrolyzed by adding moderate temperature amylase, and solid M1 was obtained by centrifugation;

[0027] 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.

[0028] Preferably, the enzymatic hydrolysis conditions in S1 are: adjusting the pH of the wheat germ granule slurry to 5-7, the enzymatic hydrolysis temperature to 50-60°C,

[0029] The amount of medium-temperature amylase added is 0.3%~0.5% of the mass of wheat germ particles.

[0030] Preferably, the S2 enzymatic hydrolysis conditions are: adjusting the solid M1 solution pH to 6-7, the enzymatic hydrolysis temperature to 50-60°C, the papaya egg

[0031] The amount of white enzyme added is 0.1%~0.2% of the mass of solid M1, and the amount of bromelain added is 0.05%~0.1% of the mass of solid M1.

[0032] The method for preparing swertiamarin of the present invention comprises the following steps:

[0033] S1. The raw material containing swertiamarin was crushed and then subjected to ultrasonic reflux extraction and concentration to obtain an intermediate extract N1;

[0034] S2. The flocculant is added to the intermediate extract N1 for flocculation, followed by refrigeration, centrifugation, and concentration to obtain the intermediate extract N2;

[0035] S3 activated carbon was added to the intermediate extract N2 for decolorization, filtered and concentrated to obtain an intermediate extract N3;

[0036] S4. The intermediate extract N3 was sequentially adsorbed and eluted by a macroporous resin to obtain an intermediate extract N4;

[0037] S5. adding a molecularly imprinted polymer to the intermediate extract N4, performing molecularly imprinted polymer adsorption separation, and obtaining swertiamarin after elution and concentration.

[0038] 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.

[0039] 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 molecularly imprinted polymer is 1:(2-3).

[0040] The vacuum degree of the reduced pressure concentration in the present invention is ≥0.08.

[0041] The present invention also provides a method for preparing a composite plant extract, comprising the following steps:

[0042] Wheat germ peptide and swedemarin are added to butanediol in proportion, 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 swedemarin is prepared according to any of the above-mentioned swedemarin preparation methods.

[0043] The present invention also provides the use of the composite plant extract in preparing repairing or soothing daily chemical products.

[0044] Preferably, the soothing daily chemical product is selected from one of toner, cream, lotion, essence, facial mask, shampoo and shower gel. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is the calibration test spectrum of amino acid standards.

[0046] Figure 2 This is the amino acid test spectrum of the wheat germ peptide obtained in Example 1.

[0047] Figure 3 This is the amino acid test spectrum of the wheat germ peptide obtained in Example 2.

[0048] Figure 4 This is the amino acid test spectrum of the wheat germ peptide obtained in Example 3.

[0049] Figure 5 This is the amino acid test spectrum of the wheat germ peptide obtained in Comparative Example 1.

[0050] Figure 6 This is the amino acid test spectrum of the wheat germ peptide obtained in Comparative Example 2.

[0051] Figure 7 This is the amino acid test spectrum of the wheat germ peptide obtained in Comparative Example 3.

[0052] Figure 8 This is the amino acid test spectrum of the wheat germ peptide obtained in Comparative Example 4.

[0053] Figure 9 This is a WB image of skin tissue in PAK1 mice, a DNCB-induced dermatitis model.

[0054] Figure 10 This is a WB image of skin tissue of UVB light damage model Nrf2, Keap1, and GCLC mice. DETAILED DESCRIPTION

[0055] The present invention is further described in detail below in conjunction with specific embodiments.

[0056] 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.

[0057] The wheat germ peptides in all the examples and comparative examples were prepared at a concentration of 4000 ppm.

[0058] The flocculant composition in all the examples and comparative examples was 1% chitosan in 1% citric acid solution.

[0059] The specific preparation method of the molecularly imprinted materials described in all the examples and comparative examples comprises the following steps:

[0060] 1.1 Preparation of the dispersed phase: 4 mmol of acrylamide and 20 mmol of N,N'-methylenebisacrylamide were dissolved in 20 mL of water, and 1 mmol of the template molecule 2-(hydroxymethyl)-6-((4-ethoxynaphthalen-1-yl)oxy)-tetrahydro-2H-pyran-3,4,5-triol was added and uniformly dispersed. The mixture was magnetically stirred at room temperature for 0.5 h to form a prepolymer to obtain solution A. The structural formula of the 2-(hydroxymethyl)-6-((4-ethoxynaphthalen-1-yl)oxy)-tetrahydro-2H-pyran-3,4,5-triol is:

[0061]

[0062] 1.2 Preparation of the continuous phase: Dissolve 24 g of L4 polyoxyethylene lauryl ether (BRIJ 30) in 300 mL of n-hexane under a nitrogen atmosphere to form a continuous phase, which is solution B.

[0063] 1.3 Preparation of inverse dispersion system: Under magnetic stirring, slowly transfer solution A into solution B and stir at room temperature under nitrogen atmosphere for 60 min to obtain a white emulsion;

[0064] 1.4 Polymerization: 0.5 mL of 10% (w / v) potassium persulfate was added to the white emulsion to initiate the polymerization reaction. The reaction was continued at 50°C and 300 r / min for 2 h to obtain a polymer microsphere mixture.

[0065] 1.5 Post-treatment: Add ethanol to the polymer microsphere mixture to aggregate and precipitate the polymer. Filter the resulting polymer. Transfer the resulting polymer to a 240-mesh sieve, wash with water to remove fine powder, and dry. Place the dried polymer in a Soxhlet extractor and extract the template molecules with ethanol. Vacuum dry the polymer at 50-55°C to a constant weight. After drying, separate the imprinted material using sieves of varying mesh sizes to obtain molecularly imprinted polymers of varying sizes.

[0066] Wherein, the template molecule: 2-(hydroxymethyl)-6-((4-ethoxynaphthalen-1-yl)oxy)-tetrahydro-2H-pyran-3,4,5-triol, has the structural formula:

[0067]

[0068] The preparation method is specific and comprises the following steps:

[0069] (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 evaporation to obtain acetyl-protected glucose with a yield of 95%.

[0070] (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 (Product No.: Titan Technology 01375737) dropwise to the mixed solution at 0 °C. Continue stirring 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 it 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 over anhydrous sodium sulfate, and concentrate them under reduced pressure. Purify them by column chromatography to obtain the acetyl-protected template molecule with a yield of 88%.

[0071] (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% (mass fraction) 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%.

[0072] 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.

[0073] For details on the equipment and method for extracting swedemarin from medicinal materials in the preparation method of swedemarin described in all embodiments and comparative examples, please refer to the embodiment section of the Chinese patent application "Ultrasonic Extraction Equipment and Ultrasonic Extraction Method of Medicinal Materials" with application number: 202410591953.3.

[0074] In the wheat germ peptide preparation method described in S24 in all the embodiments and comparative examples, the total peptide testing method is the lowry method.

[0075] Example 1

[0076] A composite plant extract comprises wheat germ peptide and swetmarin, wherein the mass ratio of wheat germ peptide to swetmarin is 1:1.

[0077] Wherein, the preparation method of wheat germ peptide comprises the following steps:

[0078] S1. First, wheat germ granules were soaked in 10 times their mass of pure water at 200 rpm for 2 hours to obtain a wheat germ granule slurry. Then, the pH of the wheat germ granule slurry was adjusted to 6.5, the temperature was heated to 55°C, and 0.4% of the mass of the wheat germ granules was added with a mesophilic amylase, and enzymatic hydrolysis was carried out for 2 hours. Finally, the slurry was centrifuged at 7000 rpm, filtered, and the oil and water layers were discarded to obtain solid M1.

[0079] S2. Papain and bromelain were added to the solid M1 solution for enzymatic hydrolysis, followed by centrifugation and membrane separation to obtain wheat germ peptide, which specifically comprises the following steps:

[0080] S21. Add 20 times the mass of pure water to solid M1 and stir to dissolve;

[0081] S22. The solid M1 solution was adjusted to pH 6.5, heated to 55 ° C, 0.15% by mass of papain and 0.075% by mass of solid M1 were added to the solid M1, bromelain, enzymatic hydrolysis for 2h, centrifuged at 7000rpm, and filtered to obtain the supernatant M2;

[0082] S23. Ultrafiltration of supernatant M2 using a 2000 Dalton ultrafiltration membrane at a pressure of 5 bar. When the retentate remains below 30%, add 30% pure water and continue ultrafiltration until the permeate mass reaches the same mass as M2. The permeate M3 is then collected.

[0083] 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% pure water was added to the retentate mixture until the total peptide content reached 2.1%, thereby obtaining a wheat germ peptide mother liquor.

[0084] Wherein, the preparation method of swertiamarin comprises the following steps:

[0085] S1. The Swertia japonica herb was crushed and then subjected to reduced pressure ultrasonic reflux extraction for 1.2 h using 50% ethanol as the extraction solvent until the concentrate was 125% of the crude drug weight to obtain intermediate extract N1;

[0086] S2. First, add 3% flocculant by weight of the intermediate extract N1, stir for 20 minutes, and refrigerate at 0~8℃ for 24 hours; second, 7000rpm

[0087] Centrifuge to obtain a clear liquid, filter and take the supernatant; finally, concentrate the obtained supernatant under reduced pressure, extract until the concentrate mass is 225% of the crude drug amount, and obtain the intermediate extract N2; the reduced pressure concentration process is: vacuum degree 0.09, temperature 70 ° C;

[0088] S3. Add 0.75% of the intermediate extract N2 by mass of 250-mesh coconut shell activated carbon and stir at room temperature for 1h. Filter through a 0.45μm membrane until clarified. The filtrate is concentrated with a membrane to a concentrate mass of 350% of the crude drug amount to obtain an intermediate extract N3; wherein the membrane molecular weight cutoff is 150 Daltons and the pressure is 12 bar;

[0089] S4. Intermediate extract N3 was added to a D101 macroporous resin column and subjected to dynamic adsorption at a flow rate of 1 BV / h. The column was then washed with 2 BV of pure water at a flow rate of 1 BV / h, and finally eluted with 3 BV of 10% ethanol at a flow rate of 1 BV / h to obtain intermediate extract N4. The ratio of intermediate extract N3 to the dry weight of the macroporous resin was 1:6.

[0090] S5. Adding a molecularly imprinted polymer to the intermediate extract N4; first, washing the adsorbed molecularly imprinted polymer with pure water twice the mass of the molecularly imprinted polymer; then, eluting with 95% ethanol 4.5 times the mass of the molecularly imprinted polymer; finally, nanofiltration was performed using step S24 in the wheat germ peptide preparation method, and the content of swedemarin was concentrated to 2.1% to obtain a swedemarin mother liquor; the mass ratio of the intermediate extract N4 converted into the mass of the input medicinal material to the molecularly imprinted polymer was 1:2.5; the swedemarin content test was carried out according to the determination method of swedemarin in the 2020 edition of the Chinese Pharmacopoeia P140;

[0091] The method for preparing the composite plant extract comprises the following steps: compounding wheat germ peptide mother liquor, swedemarin mother liquor, 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.

[0092] The calculation method of the crude drug amount is: crude drug amount = the mass of the raw material input in the process ÷ the mass of the material obtained in this step × 100%.

[0093] Example 2

[0094] This embodiment is the second embodiment of the present invention, which differs from the first embodiment in that:

[0095] A composite plant extract comprises wheat germ peptide and swetmarin, wherein the mass ratio of wheat germ peptide to swetmarin is 1:1.2.

[0096] Wherein, the preparation method of wheat germ peptide comprises the following steps:

[0097] S1. First, wheat germ granules were soaked in 10 times their mass of pure water at 200 rpm for 2 hours to obtain a wheat germ granule slurry. Then, the wheat germ granule slurry was adjusted to pH 7, heated to 50°C, and enzymatically hydrolyzed for 2 hours by adding 0.5% of the mass of the wheat germ granules of a mesophilic amylase. Finally, the slurry was centrifuged at 8000 rpm, filtered, and the oil and water layers were discarded to obtain solid M1.

[0098] S2. Papain and bromelain are added to the solid M1 solution for enzymatic hydrolysis, followed by centrifugation and ultrafiltration to obtain wheat germ peptide, which specifically comprises the following steps:

[0099] S21. Add 20 times the mass of pure water to solid M1 and stir to dissolve;

[0100] S22. The solid M1 solution was adjusted to pH 7, heated to 60 ℃, and 0.2% by mass of solid M1 papain and 0.05% by mass of solid M1 bromelain were added, enzymatic hydrolysis for 2h, centrifuged at 8000rpm, and filtered to obtain the supernatant M2;

[0101] S23. Ultrafiltration of supernatant M2 using a 2000 Dalton ultrafiltration membrane at 6 bar. When the retentate remains below 30%, add 30% pure water and continue ultrafiltration until the permeate has the same mass as M2. The permeate M3 is collected.

[0102] 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% pure water was added to the retentate mixture to a total peptide content of 3.7% to obtain a wheat germ peptide mother solution.

[0103] Wherein, the preparation method of swertiamarin comprises the following steps:

[0104] S1. The gentiana root was crushed and then extracted under reduced pressure and ultrasonic reflux for 1.5 h using 55% ethanol as the extraction solvent until the concentrate was 150% of the crude drug to obtain the intermediate extract N1;

[0105] S2. First, add 4% flocculant of the intermediate extract N1 by weight, stir for 20 minutes, and refrigerate at 0~8℃ for 13 hours; second, 8000rpm

[0106] Centrifuge to obtain a clear liquid, filter and take the supernatant; finally, concentrate the obtained supernatant under reduced pressure, extract until the concentrate mass 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;

[0107] S3. Add 1% of 200-mesh coconut shell activated carbon to the intermediate extract N2 and stir evenly. Filter through a 0.45 μm membrane until clear.

[0108] The filtrate was concentrated by membrane extraction until the mass of the concentrate was 400% of the crude drug amount, to obtain the intermediate extract N3; wherein, the membrane molecular weight cut-off was 150 Daltons and the pressure was 15 bar;

[0109] S4. Intermediate extract N3 was added to an AB-8 macroporous resin column and first subjected to dynamic adsorption at a flow rate of 1 BV / h. The column was then washed with 2 BV of pure water at a flow rate of 1 BV / h. Finally, the column was eluted with 3 BV of 10% ethanol at a flow rate of 1 BV / h to obtain intermediate extract N4. The ratio of intermediate extract N3 (input medicinal material weight to macroporous resin dry weight) was 1:7.

[0110] 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 using step S24 in the wheat germ peptide preparation method and concentrate to a scutellaria glycoside content of 3.4% 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.

[0111] The method for preparing the composite plant extract comprises the following steps: mixing wheat germ peptide mother liquor, swedemarin mother liquor, 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.

[0112] Example 3

[0113] This embodiment is the third embodiment of the present invention, and is different from the first embodiment in that:

[0114] A composite plant extract comprises wheat germ peptide and swetmarin, wherein the mass ratio of wheat germ peptide to swetmarin is 1:0.8.

[0115] Wherein, the preparation method of wheat germ peptide comprises the following steps:

[0116] S1. First, wheat germ granules were soaked in 10 times their mass of pure water at 200 rpm for 2 hours to obtain a wheat germ granule slurry. Then, the pH of the wheat germ granule slurry was adjusted to 5, the temperature was heated to 60°C, and 0.3% of the mass of the wheat germ granules was added with a mesophilic amylase, and enzymatic hydrolysis was carried out for 2 hours. Finally, the slurry was centrifuged at 6000 rpm, filtered, and the oil and water layers were discarded to obtain solid M1.

[0117] S2. Papain and bromelain are added to the solid M1 solution for enzymatic hydrolysis, followed by centrifugation and ultrafiltration to obtain wheat germ peptide, which specifically comprises the following steps:

[0118] S21. Add 20 times the mass of pure water to solid M1 and stir to dissolve;

[0119] S22. The solid M1 solution was adjusted to pH 6, heated to 50 ℃, and 0.1% by mass of solid M1 papain and 0.1% by mass of solid M1 bromelain were added, enzymatic hydrolysis for 2h, centrifuged at 6000rpm, and filtered to obtain the supernatant M2;

[0120] S23. Ultrafiltration of supernatant M2 using a 2000 Dalton ultrafiltration membrane at 4 bar. When the retentate remains below 30%, add 30% pure water and continue ultrafiltration until the permeate has the same mass as M2. The permeate is then collected as M3.

[0121] 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 to a total peptide content of 3.2% to obtain a wheat germ peptide mother liquor;

[0122] Wherein, the preparation method of swertiamarin comprises the following steps:

[0123] S1. The herbal material Gentiana macrophylla was crushed and then subjected to reduced pressure ultrasonic reflux extraction for 1 h using 45% ethanol as the extraction solvent until the concentrate was 100% of the crude drug amount to obtain the intermediate extract N1;

[0124] 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, at 6000 rpm

[0125] Centrifuge to obtain a clear liquid, filter and take the supernatant; finally, concentrate the obtained supernatant under reduced pressure, extract until the concentrate mass 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;

[0126] S3. Add 0.5% of the intermediate extract N2 by mass to 300 mesh coconut shell activated carbon and stir evenly. Filter through a 0.45 μm membrane until clear.

[0127] The filtrate was concentrated by membrane extraction until the mass of the concentrate was 300% of the crude drug amount, to obtain the intermediate extract N3; wherein, the membrane molecular weight cut-off was 150 Daltons and the pressure was 10 bar;

[0128] S4. Intermediate extract N3 was added to a D101 macroporous resin column and first subjected to dynamic adsorption at a flow rate of 1 BV / h. The column was then washed with 2 BV of pure water at a flow rate of 1 BV / h. Finally, the column was eluted with 3 BV of 10% ethanol at a flow rate of 1 BV / h to obtain intermediate extract N4. The ratio of intermediate extract N3 to the dry weight of the macroporous resin was 1:5.

[0129] 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 four times the mass of the molecular imprinting polymer; finally, perform nanofiltration using step S24 in the wheat germ peptide preparation method and concentrate to a scutellaria glycoside content of 2.8% 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 1:3.

[0130] The method for preparing the composite plant extract comprises the following steps: mixing wheat germ peptide mother liquor, swedemarin mother liquor, 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.

[0131] Comparative Example 1

[0132] This comparative example is the first comparative example of the present invention, and is different from Example 1 in that:

[0133] A composite plant extract comprises wheat germ peptide and swetmarin, wherein the mass ratio of wheat germ peptide to swetmarin is 1:1.3.

[0134] Wherein, the preparation method of wheat germ peptide comprises the following steps:

[0135] S1. First, wheat germ granules were soaked in 10 times their mass of pure water at 200 rpm for 2 hours to obtain a wheat germ granule slurry. Then, the pH of the wheat germ granule slurry was adjusted to 7.5, the temperature was heated to 65°C, and 0.6% of the mass of the wheat germ granules was added with a mesophilic amylase, and enzymatic hydrolysis was carried out for 2 hours. Finally, the slurry was centrifuged at 5000 rpm, filtered, and the oil and water layers were discarded to obtain solid M1.

[0136] S2. Papain and bromelain are added to the solid M1 solution for enzymatic hydrolysis, followed by centrifugation and ultrafiltration to obtain wheat germ peptide, which specifically comprises the following steps:

[0137] S21. Add 20 times the mass of pure water to solid M1 and stir to dissolve;

[0138] S22. The solid M1 solution was adjusted to pH 5.5, heated to 65 ° C, 0.25% by mass of papain and 0.04% by mass of bromelain were added to the solid M1, enzymatic hydrolysis for 2h, centrifuged at 5000rpm, and filtered to obtain the supernatant M2;

[0139] S23. Ultrafilter the supernatant M2 using a 2000 Dalton ultrafiltration membrane at a pressure of 3.5 bar. When the retentate remains below 30%, add 30% pure water and continue ultrafiltration until the permeate has the same mass as M2. Collect the permeate M3.

[0140] 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 liquor;

[0141] Wherein, the preparation method of swertiamarin comprises the following steps:

[0142] S1. The Swertia japonica herb was crushed and then subjected to reduced pressure ultrasonic reflux extraction for 45 min using 40% ethanol as the extraction solvent until the concentrate was 90% of the crude drug, obtaining intermediate extract N1;

[0143] S2. First, add 1.5% of the mass of the intermediate extract N1 to the flocculant, stir for 20 minutes, and refrigerate at 0-8℃ for 12 hours; secondly,

[0144] 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 extracted until the mass of the concentrated liquid was 180% of the crude drug amount, thereby obtaining the intermediate extract N2; the reduced pressure concentration process was as follows: vacuum degree 0.07, temperature 80°C;

[0145] S3. Add 0.4% of the intermediate extract N2 by mass to 350 mesh coconut shell activated carbon and stir evenly. Filter through a 0.45 μm membrane until clear.

[0146] The filtrate was concentrated by membrane extraction until the mass of the concentrate was 250% of the crude drug amount, to obtain the intermediate extract N3; wherein, the membrane molecular weight cut-off was 150 Daltons and the pressure was 8 bar;

[0147] S4. Intermediate extract N3 was added to a D101 macroporous resin column and first subjected to dynamic adsorption at a flow rate of 1 BV / h. The column was then washed with 2 BV of pure water at a flow rate of 1 BV / h. Finally, the column was eluted with 3 BV of 10% ethanol at a flow rate of 1 BV / h to obtain intermediate extract N4. The ratio of intermediate extract N3 to the dry weight of the macroporous resin was 1:4.

[0148] S5. adding a molecularly imprinted polymer to the intermediate extract N4; first, washing the adsorbed molecularly imprinted polymer with pure water twice the mass of the molecularly imprinted polymer; then, eluting with 95% ethanol 5.5 times the mass of the molecularly imprinted polymer; finally, performing nanofiltration using step S24 in the wheat germ peptide preparation method and concentrating to a scutellaria glycoside content of 1.7% 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 molecularly imprinted polymer is 1:3.5.

[0149] The method for preparing the composite plant extract comprises the following steps: compounding wheat germ peptide mother liquor, swertiamarin mother liquor, 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 complex.

[0150] Comparative Example 2

[0151] This comparative example is the second comparative example of the present invention, and is different from Example 1 in that:

[0152] A composite plant extract comprises wheat germ peptide and swetmarin, wherein the mass ratio of wheat germ peptide to swetmarin is 1:0.7.

[0153] Wherein, the preparation method of wheat germ peptide comprises the following steps:

[0154] S1. First, wheat germ granules were soaked in 10 times their mass of pure water at 200 rpm for 2 hours to obtain a wheat germ granule slurry. Then, the pH of the wheat germ granule slurry was adjusted to 4.5, the temperature was heated to 45°C, and 0.25% of the mass of the wheat germ granules was added with a mesophilic amylase, and enzymatic hydrolysis was carried out for 2 hours. Finally, the slurry was centrifuged at 9000 rpm, filtered, and the oil and water layers were discarded to obtain solid M1.

[0155] S2. Papain and bromelain are added to the solid M1 solution for enzymatic hydrolysis, followed by centrifugation and ultrafiltration to obtain wheat germ peptide, which specifically comprises the following steps:

[0156] S21. Add 20 times the mass of pure water to solid M1 and stir to dissolve;

[0157] S22. The solid M1 solution was adjusted to pH 7.5, heated to 45 ° C, 0.075% of papain and 0.15% of bromelain by mass of solid M1 were added, enzymatic hydrolysis for 2h, centrifuged at 9000rpm, and filtered to obtain the supernatant M2;

[0158] S23. Ultrafilter the supernatant M2 using a 2000 Dalton ultrafiltration membrane at 7 bar. When the retentate remains below 30%, add 30% pure water and continue ultrafiltration until the permeate has the same mass as M2. Collect the permeate M3.

[0159] 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 liquor;

[0160] Wherein, the preparation method of swertiamarin comprises the following steps:

[0161] S1. The Swertia japonica herb was crushed and then subjected to reduced pressure ultrasonic reflux extraction for 2 h using 60% ethanol as the extraction solvent until the concentrate was 160% of the crude drug weight to obtain intermediate extract N1;

[0162] S2. First, add 4.5% of the mass of the intermediate extract N1 to the flocculant, stir for 20 minutes, and refrigerate at 0-8℃ for 12 hours; secondly,

[0163] The mixture was centrifuged at 8500 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 extracted until the mass of the concentrated liquid was 265% of the crude drug amount, thereby obtaining the intermediate extract N2; the reduced pressure concentration process was as follows: vacuum degree 0.075, temperature 60°C;

[0164] S3. Add 1.2% of the intermediate extract N2 by mass to 150 mesh coconut shell activated carbon and stir evenly. Filter through a 0.45 μm membrane until clear.

[0165] The filtrate was concentrated by membrane extraction until the mass of the concentrate was 450% of the crude drug amount, to obtain the intermediate extract N3; wherein, the membrane molecular weight cut-off was 150 Daltons and the pressure was 16 bar;

[0166] S4. Intermediate extract N3 was added to a D101 macroporous resin column and first subjected to dynamic adsorption at a flow rate of 1 BV / h. The column was then washed with 2 BV of pure water at a flow rate of 1 BV / h. Finally, the column was eluted with 3 BV of 10% ethanol at a flow rate of 1 BV / h to obtain intermediate extract N4. The ratio of intermediate extract N3 to the dry weight of the macroporous resin was 1:8.

[0167] S5. adding a molecularly imprinted polymer to the intermediate extract N4; first, washing the adsorbed molecularly imprinted polymer with pure water twice the mass of the molecularly imprinted polymer; then, eluting with 95% ethanol 3.5 times the mass of the molecularly imprinted polymer; finally, performing nanofiltration using step S24 in the wheat germ peptide preparation method and concentrating to a scutellaria glycoside content of 1.9% 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 molecularly imprinted polymer is 1:5.

[0168] The method for preparing the composite plant extract comprises the following steps: compounding wheat germ peptide mother liquor, swedemarin mother liquor, 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 complex.

[0169] Comparative Example 3

[0170] 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 by ficin is added, the solution is enzymatically hydrolyzed for 2 h, centrifuged at 7000 rpm, and filtered to obtain the supernatant M2;

[0171] 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 1.9%, thereby obtaining the wheat germ peptide mother liquor.

[0172] Comparative Example 4

[0173] 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, adjusted to pH = 5.0, heated to 60°C, and 0.3% of the mass of wheat grain warm amylase is added for enzymatic hydrolysis for 2 hours. The mixture is centrifuged at 7000 rpm, filtered, and the oil layer and the water layer are discarded to obtain solid M1.

[0174] 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 2.7%, thereby obtaining the wheat germ peptide mother liquor.

[0175] Comparative Example 5

[0176] This comparative example is the fifth comparative example of the present invention. The difference from Example 1 is that in the method for preparing wheat germ peptide, the medium-temperature amylase enzymatic hydrolysis in step S1 is not performed;

[0177] 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.

[0178] Comparative Example 6

[0179] This comparative example is the sixth embodiment of the present invention. Unlike Example 1, in the method for preparing wheat germ peptide, papain is not added in step S22.

[0180] 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.

[0181] Comparative Example 7

[0182] This comparative example is the seventh embodiment of the present invention. Unlike Example 1, in the method for preparing wheat germ peptide, bromelain is not added in step S22.

[0183] 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.

[0184] Comparative Example 8

[0185] This comparative example is the eighth embodiment of the present invention. The difference from Example 1 is that in the method for preparing wheat germ peptide, no protease is added in step S22.

[0186] S24. Nanofiltration is performed on the permeate M3 using a 150 Dalton nanofiltration membrane. 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 mixture reaches 2.0%, thereby obtaining the wheat germ peptide mother liquor.

[0187] Comparative Example 9

[0188] This comparative example is the ninth embodiment of the present invention. Unlike Example 1, the conventional alcohol extraction process is used to prepare swertiamarin, without ultrasonic reflux extraction, activated carbon adsorption, or molecularly imprinted polymer. The specific preparation process includes the following steps:

[0189] S1. After crushing the herb, the herb was extracted under reduced pressure and ultrasonic reflux for 1 hour using 45% ethanol as the extraction solvent to obtain intermediate extract N1.

[0190] S2. First, add a flocculant at 2% by weight of the intermediate extract N1, stir for 20 minutes, and refrigerate at 0-8°C for 18 hours. Next, centrifuge at 8000 rpm to obtain a clarified liquid, and filter the supernatant. Finally, concentrate the resulting supernatant under reduced pressure until the concentrate contains 300% of the crude drug, obtaining the intermediate extract N2. The reduced pressure concentration process is as follows: vacuum degree 0.08, temperature 75°C.

[0191] S3. Add the intermediate extract N2 to a D101 macroporous resin column. The intermediate extract N2 is converted to a 1:6 ratio of the input medicinal material mass to the macroporous resin dry weight. Dynamic adsorption is performed at a flow rate of 1 BV / h. The column is first washed with 2 BV of pure water at a flow rate of 1 BV / h, followed by elution with 3 BV of 10% ethanol at a flow rate of 1 BV / h. The eluate is concentrated according to the method of step S24 of the wheat germ peptide preparation method to a swertiamarin content of 2.3%, thereby obtaining a swertiamarin mother liquor.

[0192] Performance testing:

[0193] Wheat germ peptide amino acid composition: Hitachi automatic amino acid analyzer (model: L8900) was used for detection. The detection 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 patterns of the wheat germ peptides obtained in Examples 1 to 3 and Comparative Examples 1 to 4 are shown respectively.

[0194] The specific detection method includes the following steps: Place 2.0 mL of sample in a hydrolysis tube and add 2.0 mL of 6 mol / L hydrochloric acid. Add 50 mg of phenol to the hydrolysis tube. Place in a freezer and cool for 3-5 minutes. Then evacuate and refill with nitrogen three times. Seal the tube and hydrolyze at 110°C for 22 hours. Remove the tube and cool to room temperature. Open the hydrolysis tube and accurately pipette 1.0 mL of the filtrate into a test tube. Blow dry with a nitrogen blower at 60°C. Dissolve the residue in 1.0 mL of water and blow dry again. Add 2.0 mL of pH sodium citrate buffer to the dried test tube and dissolve it. After vortexing, mix thoroughly. Pipette the solution through a 0.22 μm filter and transfer to a 1.5 mL injection vial. The sample injection volume is 10 μL, and the standard injection volume is 20 μL. Hydroxyproline and proline are detected at 440 nm, while other amino acids are detected at 570 nm.

[0195] The purity of wheat germ peptide is mainly calculated by the following formula:

[0196] Purity = target component mass concentration ÷ dry matter mass concentration × 100%,

[0197] The target ingredient is wheat germ peptide; the dry matter concentration is the mass of the test liquid dried to constant weight at 105°C divided by the mass of the test liquid × 100%.

[0198] Table 1: Test results of the purity and amino acid characteristics of the wheat germ peptides obtained in various examples and comparative examples.

[0199] Table 1 Purity and amino acid characteristics of wheat germ peptides obtained in Examples and Comparative Examples

[0200]

[0201] Note: “ / ” means not tested;

[0202] X1=N1÷N0×100%, where N1=total concentration of Glu+Lys+Pro+Val+His+Gly, mg / mL;

[0203] X2=N2÷N0×100%, where N2=total concentration of Gly+Ala+Val+Ile+Pro+Thr+Lys+His, mg / mL;

[0204] N0=Asp+Thr+Ser+Glu+Pro+Gly+Ala+Cys+Val+Met+Ile+Leu+Tyr+Phe+Lys+His+Arg total concentration, mg / mL.

[0205] 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 impact on the purity and amino acid composition of the wheat germ peptide mother liquor; compared with Comparative Example 4, Example 1 shows that the selection of wheat raw materials has a very large impact on the purity and amino acid composition of the polypeptide mother liquor; compared with Comparative Example 3 and Comparative Examples 5 to 8, Example 1 shows that the selection of enzyme preparation also has a very large impact on the purity and amino acid composition of the polypeptide mother liquor.

[0206] Swertiamarin component detection: Swertiamarin content detection: refer to the determination method of swertiamarin in the 2020 edition of the Chinese Pharmacopoeia P140; Gentiopicroside content detection: refer to the determination method of gentiopicroside 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:

[0207] Purity = target component mass concentration ÷ dry matter mass concentration × 100%,

[0208] The target component is scutellariamarin or gentiopicroside; the dry matter concentration is the mass of the test liquid dried to constant weight at 105°C divided by the mass of the test liquid × 100%.

[0209] The test results of the swertiamarin components obtained in each example and comparative example are shown in Table 2.

[0210] Table 2 Swertiamarin components obtained in various examples and comparative examples

[0211]

[0212] 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 swede 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 swede sprouts, the purity of gentiopicroside, and the content of total phenols.

[0213] Stability test of swedemarin in the composite plant extract: The composite plant extracts obtained in each example and comparative example were allowed to stand at 48° C. for one month to test the stability of swedemarin. The test results are shown in Table 3.

[0214] Table 3 Stability test of swertiamarin in the composite plant extracts obtained in various examples and comparative examples

[0215]

[0216] As can be seen from Table 3, the process conditions, process scheme, selection of enzyme preparation 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. Among them, 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. The polypeptides prepared from wheat grains, as shown in Table 1, account for X1>32%, but the proportion of X2 is 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.

[0217] The following single-factor experimental verification is carried out. The specific methods and results are shown in Table 4.

[0218] Table 4 Single factor experiment on the stability test of swertiamarin

[0219]

[0220] Notes: 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 hours, filtered with 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 <10 ppm, the scutellaria serrata content is <10 ppm, and the total phenol content is 207.4 ppm.

[0221] The degradation rate of scutamarin = (C0-C1)÷C0×100%; C0 is the concentration of scutamarin in the sample at the beginning of the stability test, and C1 is the concentration of scutamarin in the sample at the end of the stability test.

[0222] 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 power 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 surface, it can be seen that 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 oxidize faster than scutellaria glycoside.

[0223] 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 glycosides, 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 glycosides, proving that some metal ions in the extract promote the oxidative degradation of scutellaria glycosides.

[0224] From the comparison between groups (6), (7) and groups (4), (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: one is that wheat germ peptide in Example 1 has the effect of stabilizing pH, and the other is that from the perspective of molecular structure, scutellaria baicalensis is a cyclic ether terpene 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 ring-opening degradation rate, thereby reducing the degradation rate.

[0225] 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 stabilization 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, and the oxygen atoms of the carboxyl or carbonyl groups in these amino acid compositions may participate in the coordination effect and enhance the chelation effect. The amino acid composition contained in X2 is related to pH stabilization. Its characteristic is that the isoelectric points of these amino acids are all pH = 5.5~6.5.

[0226] Application Example 1: Effects of compound plant extracts on inhibiting high-risk neurological reactions and anti-inflammatory factors

[0227] Test of the soothing effect of compound plant extracts on DNCB-induced dermatitis model

[0228] 1. Experimental Materials

[0229] Positive control drug: hydrocortisone butyrate (Euzol), Tianjin Jinyao Pharmaceutical Co., Ltd., batch number: 23051017. 18-20 g BALB / C mice were purchased from Shanghai Jiesijie Laboratory Animal Co., Ltd. 1-Chloro-2,4-dinitrotoluene (DNCB), Tokyo Chemical Industry Co., Ltd., batch number: WT6CA-VO.

[0230] 2. Experimental Methods

[0231] Mice were randomly divided into groups of 8. Model preparation: After one week of adaptive feeding, all mice were depilated using an electric hair remover and depilatory cream. The abdominal area was depilated (1 x 2 cm²) and the back area (2 x 3 cm²). On the first day of modeling, mice were sensitized with 7% DNCB in acetone solution on the depilated area of ​​the abdomen. Three days later, the same area was sensitized again. On the eighth day of modeling, mice were challenged with 0.5% DNCB in acetone solution on the back. Challenges were repeated every five days for the first 15 days, and every other day thereafter, for a total of 16 challenges. The medication schedule for each group is shown in Table 5.

[0232] Table 5: Administration and dosage of each group

[0233]

[0234] 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 Uzol on the skin lesions on the back once a day; the sample group applied the composite plant extract obtained in the embodiment or the comparative example on the skin lesions on the back 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. Sampling was done 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 level of PAK1 protein. The WB image of the skin tissue of PAK1 mice in the DNCB-induced dermatitis model is shown in the figure. Figure 9 The test concentration of DSM1 and DSM2 was 4%, and the test concentration of the implementation and comparative examples was 2%.

[0235] 3. Data Processing and Analysis

[0236] GraphPad Prism 8 software was used for analysis. Data were expressed as mean ± standard deviation, and intergroup comparisons were performed using the Student t-test. Statistically significant differences were considered when compared with the model group, with a P value < 0.05 or 0.01. The results of the inhibitory effects of the composite plant extracts obtained in the Examples and Comparative Examples on TNF-α and IL-1β, and on the expression levels of microRNA-145 and PAK1, are shown in Table 7. The intergroup significance comparisons are shown in Table 9.

[0237] Application Example 2: Antioxidant Effects of Compound Plant Extracts

[0238] Testing of composite plant extracts on UVB-induced skin photoaging model

[0239] 1. Experimental Materials

[0240] Kangfuxin solution (positive control) (Sichuan Good Doctor Panxi Pharmaceutical Co., Ltd., batch number: 231104); KM mice (18-20 g) were purchased from Shanghai Jiesijie Experimental Animal Co., Ltd.; ultraviolet light therapy device: model: SS01B, manufacturer: Sigma.

[0241] 2. Experimental Methods

[0242] Mice were randomly divided into groups (blank, model, positive control, and test groups), with 12 mice per group. Hair was removed from the back of the mice, covering an area of ​​4 cm × 2 cm. Thirty minutes before UV light therapy, gauze was placed flat on the exposed skin of the mice. Dosing was performed at 9:00 AM each day. The blank group received no medication, the model group was evenly coated with 1 mL of saline, and the positive control and sample groups were evenly coated with 1 mL of medication for 10 minutes. The dosing schedule for each group is shown in Table 6.

[0243] The blank group received no UVB treatment. The remaining groups received medication once daily before irradiation for seven consecutive days. The irradiation distance was 20 cm from the back of the mice, and the UVB intensity was 13 mW / cm². This lasted for one week. The irradiation duration was 10 minutes on the first and last day, and 15 minutes on the remaining five days, for a total irradiation dose of 74.1 J / cm².

[0244] The irradiated areas on the back of the mice showed a large amount of pigmentation, scales, erythema, and wrinkles, and even ulceration, scabs, and a leathery feel in some areas, indicating that the model was successful. Photos were taken to record the changes in the skin appearance of each group during the experiment. After the end of the experiment, on the 8th day, the mice were sacrificed and the expression levels of NRF2, KEAP1, and GCLC proteins were detected by Western blotting. The WB images of the skin tissue of the UVB light-damaged model Nrf2, Keap1, and GCLC mice are shown in the figure. Figure 10 The test concentration of DSM1 and DSM2 was 4%, and the test concentration of the implementation and comparative examples was 2%.

[0245] Table 6 Light-damaged mouse modeling and drug administration regimen

[0246]

[0247] 3. Data Processing and Analysis

[0248] GraphPad Prism 8 software was used for analysis. Data are presented as mean ± standard deviation, and intergroup comparisons were performed using the Student t-test. Statistically significant differences were considered when compared with the model group, with a P < 0.05. The results of the composite plant extracts obtained in the Examples and Comparative Examples on the expression levels of NRF2, KEAP1, and GCLC proteins are shown in Table 8. The intergroup significance comparisons are shown in Table 9.

[0249] Application Example 3: Promotion of Aquaporin AQP3 Expression by Compound Plant Extracts

[0250] Detection of AQP3 single cell fluorescence intensity by compound plant extracts

[0251] 1. Experimental Materials

[0252] The cells used in the experiment were keratinocytes HaCaT; the main reagents included: 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, Triton X-100, BSA, AQP3 primary antibody (rabbit anti-), fluorescent-labeled secondary antibody (rabbit anti-), and DAPI.

[0253] 2. Experimental Methods

[0254] Sample groups: HaCaT cells treated with plant extracts, with DSM1 and DSM2 tested at 1% concentration, and the experimental and comparative samples tested at 0.5% concentration. Controls: ① Blank control group: HaCaT cells cultured normally without treatment. ② Model control group: HaCaT cells treated with LPS.

[0255] S1. Resuscitate cryopreserved HaCaT cells and seed them into cell culture flasks at an appropriate density. Add DMEM medium (10% FBS) and culture in a cell culture incubator at 37°C and 5% CO2.

[0256] S2. Wash HaCaT cells in the logarithmic growth phase twice with PBS, then trypsinize them. Add DMEM (10% FBS) to terminate digestion and centrifuge. Discard the supernatant and add DMEM (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.

[0257] 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 experimental concentration for 24 hours.

[0258] S4. Fix each well with paraformaldehyde at room temperature, prepare 0.5% Triton X-100, and permeabilize at room temperature. Wash with PBS, block at room temperature, and discard the blocking solution. Incubate with the primary antibody overnight and incubate with the fluorescent secondary antibody in the dark. Wash with PBS and incubate with DAPI at room temperature in the dark. Remove the slides, block with mounting solution, and fix overnight. Image and analyze the data under a fluorescence microscope.

[0259] 3. Data Processing

[0260] 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 for data analysis.

[0261] Judgment Criteria: The relative expression rate (%) of aquaporin AQP3 was tested. Compared with the model control group, the relative expression rate of aquaporin AQP3 in keratinocytes in the sample group was increased, and the result was significantly different (*p < 0.05), indicating that the sample, at this test concentration, has the effect of promoting the production of aquaporin AQP3. The results of the composite plant extracts obtained in the Examples and Comparative Examples on the expression level of aquaporin AQP3 are shown in Table 8, and the significance comparison between the groups is shown in Table 9.

[0262] Table 7 Test of the composite plant extracts obtained in the examples and comparative examples for inhibiting inflammation and high-pressure nervous system reactions

[0263]

[0264] Note: Example 1-DSM1: The wheat germ peptide mother liquor 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 swedemarin mother liquor obtained in step 16 of Example 1 was added with pure water to prepare a solution containing 4000 ppm of swedemarin; compared with the model group, * indicates P < 0.05, ** indicates P < 0.01; compared with the blank group, # indicates P < 0.05, ## indicates P < 0.01

[0265] Table 8 Antioxidant and Aquaporin Enhancement Tests of Composite Plant Extracts Obtained from Examples and Comparative Examples

[0266]

[0267] Notes are the same as Table 7.

[0268] 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, achieving an anti-inflammatory effect; can significantly increase the expression of NrF2 and Keap1, play an anti-oxidative stress role, and alleviate the damage caused by excessive oxidative stress of the skin; can significantly increase the expression of GCLC to achieve an antioxidant effect, reduce the damage of free radicals to the skin; can significantly increase the expression of the water channel protein AQP3, improve the moisturizing effect and repair effect of the skin, and promote faster recovery of problem skin; can significantly increase the expression of miR-145, 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 to the existing technology.

[0269] The difference between Comparative Examples 1 and 2 and Example 1 lies in the different process conditions. Compared with the model group, Comparative Examples 1 and 2 showed no significant differences in certain pathways or targets, and the effects were inferior to those of Example 1, indicating that differences in process conditions have a certain impact on the soothing and repairing efficacy. Comparative Example 3 differs from Example 1 in the type of enzyme preparation used for wheat germ enzymatic hydrolysis, resulting in no significant effect on most pathways, and the effect was significantly inferior to that of Example 1. Example 4 differs from Example 1 in that wheat germ was replaced with wheat grains, and the results were also insignificant in most pathways, indicating that the source of the raw materials also plays a decisive role.

[0270] Table 9 Significant comparison between groups

[0271]

[0272] Note: When comparing two groups, * means P < 0.05, ** means P < 0.01, × means P ≥ 0.05, / means no test was performed

[0273] DSM1 and DSM2 are the single-ingredient components of Example 1. During testing, the concentrations of DSM1 and DSM2 were double those of Example 1, resulting in consistent total active ingredient concentrations across the three groups. The significance comparisons in rows 1, 2, and 3 of Table 9 show that significant differences were observed between DSM1 and DSM2, and between DSM1 and Example 1, in inhibiting the expression of the inflammatory mediators TNFα and IL-1β, while no significant differences were observed between DSM2 and Example 1. This suggests that DSM2, or swertiamarin, plays a more important role in this pathway, and that DSM1 and DSM2 have a synergistic effect. After compounding, the dosage can be reduced to achieve the same effect as swertiamarin alone.

[0274] In terms of the expression of NrF2 and Keap1, the pathway targets for inhibiting oxidative stress, there were significant differences between DSM1 and DSM2, and between DSM2 and Example 1, while there was no significant difference between DSM1 and Example 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, the dosage can be reduced to achieve the same effect as using wheat germ peptide alone.

[0275] 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 a significant synergistic effect.

[0276] In terms of the expression of the water channel protein AQP3, there were significant differences between DSM1 and DSM2, and between DSM2 and Example 1, while there was no significant difference between DSM1 and Example 1, indicating that DSM1, or wheat germ peptide, plays a more important role in this pathway, and that DSM1 and DSM2 have a synergistic effect. After compounding, the dosage is reduced to achieve an effect that is statistically indifferent to the use of wheat germ peptide alone. In terms of inhibiting the neural hyperresponsiveness pathway, that is, increasing the expression of miR-145 and reducing the expression of PAK1, there was no significant difference between DSM1 and DSM2, while both DSM1 and DSM2 were significantly different from Example 1, indicating that swertiamarin and wheat germ peptide have a significant synergistic effect in this pathway. In summary, swertiamarin and wheat germ peptide have both synergistic and complementary effects and synergistic effects in terms of comprehensive soothing efficacy.

[0277] In summary, the composite plant extract provided by the present invention contains swertiamarin and wheat germ peptide, with swertiamarin being highly stable. For problematic skin, the composite plant extract can synergistically repair and soothe problematic skin by providing multi-pathway benefits: anti-oxidative stress, suppression of hypersensitivity reactions, inflammation, antioxidant activity, and increased aquaporin expression.

[0278] The above is only an embodiment 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 and still fall within the scope of patent protection.

Claims

1. A composite plant extract, characterized in that The invention comprises wheat germ peptide and swetmarin, wherein the mass ratio of the wheat germ peptide to the swetmarin 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%; The wheat germ peptide preparation method comprises the following steps: S1. The wheat germ granule slurry was enzymatically hydrolyzed by adding moderate temperature amylase, and solid M1 was obtained 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. 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 adding the medium-temperature amylase in an amount of 0.3%-0.5% of the mass of the wheat germ particles. The S2 enzymatic hydrolysis conditions are: adjusting the pH of the solid M1 solution to 6-7, the enzymatic hydrolysis temperature to 50-60° C., the added amount of papain to be 0.1%-0.2% of the mass of the solid M1, and the added amount of bromelain to be 0.05%-0.1% of the mass of the solid M1. The method for preparing swertiamarin comprises the following steps: S1. The raw material containing swertiamarin was crushed and then subjected to ultrasonic reflux extraction and concentration 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 activated carbon was added to the intermediate extract N2 for decolorization, filtered and concentrated to obtain an intermediate extract N3; S4. The intermediate extract N3 was sequentially adsorbed and eluted by a macroporous resin to obtain an intermediate extract N4; S5. adding a molecularly imprinted polymer to the intermediate extract N4, performing molecularly imprinted polymer adsorption separation, and obtaining swertiamarin after elution and concentration. 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. The ratio of the S4 intermediate extract N3 converted into the weight 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 weight of the input medicinal materials to the molecularly imprinted polymer is 1:(2-3).

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 greater than 90%. The gentiopicroside content in the swertiamarin is less than 1%, the total phenol content is less than 0.05%, and the purity is greater than 90%.

3. 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, sterilized, cooled and filtered in sequence to obtain a composite plant extract; the wheat germ peptide is prepared according to the wheat germ peptide preparation method of the composite plant extract according to claim 1; the swedemarin The compound plant extract is prepared according to the method for preparing the swertiamarin in claim 1.

4. Use of the composite plant extract according to claim 1 in the preparation of repairing and soothing daily chemical products.

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