Extraction method of staphisagria cyclic peptide, product and application thereof

Abstract

The present application relates to the cosmetic technical field, specifically relates to a kind of stellaria media cyclic peptide extraction method and its product and application.Extraction method at least includes the following steps: stellaria media powder is extracted by eutectic solvent, enzymolysis, separation and purification, concentration, nanobubble freeze-drying, obtain the stellaria media cyclic peptide.The present application adopts betaine, malic acid and deionized water of specific molar ratio to construct ternary eutectic solvent (DES) system, match microwave pulse directional extraction, enzymolysis, ceramic membrane crossflow microfiltration separation and purification and nanobubble freeze-drying The joint action of together realizes stellaria media cyclic peptide high yield, high activity retention rate and form controllable, adapts the application requirement of different fields such as cosmetics, medicine.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, specifically to a method for extracting Wangbuliuxing cyclic peptides, its products, and their applications. Background Technology

[0002] Plant cyclic peptides are a class of cyclic compounds formed by the cyclization of peptide bonds or disulfide bonds. They have significant improvements over linear peptides in terms of stability, recognition of specific protein factors, transdermal absorption, and bioactivity, which makes them have broad application potential in the cosmetics industry.

[0003] Currently, the common methods for extracting plant cyclic peptides are ethanol / methanol reflux extraction or preparative liquid chromatography (HPLC) separation and purification. For example, Chinese invention patent application CN118373871A discloses a method for extracting and identifying plant cyclic peptides from *Hedyotis diffusa*. Fresh *Hedyotis diffusa* is chopped and soaked overnight in a 50% acetonitrile aqueous solution containing 1% formic acid. Subsequently, the crude extract is fractionated using a solid-phase extraction column with different ratios of acetonitrile / aqueous solution: 20%, 30%, 50%, and 80% acetonitrile aqueous solution containing 1% trifluoroacetic acid. The fractions are then injected into a semi-preparative reversed-phase high-performance liquid chromatograph for further separation. However, higher concentrations are usually required to provide a significant soft-focus effect, resulting in a thick formulation that may affect normal skin respiration and metabolic function, and is also expensive.

[0004] The literature "Advances in the Chemical and Bioactivity Research of Plant Peptides" reports that plant cyclic peptides are mostly extracted using weakly polar and moderately polar solvents, such as chloroform, methanol, ethyl acetate, and benzene. Commonly used methods for separating cyclic peptides include SDS-polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, gel filtration chromatography, ion exchange chromatography, and reversed-phase liquid chromatography.

[0005] However, the current technology has the following problems: First, the extraction solvent is highly toxic: the residue of the ethanol / methanol system affects the safety of the product and has poor selectivity for cyclic peptides, requiring multiple extractions; Second, there is serious coexistence of impurities: polysaccharides, colloids and cyclic peptides are cosoluble, which leads to complicated and costly subsequent purification steps; Third, the process is not consistent: many steps rely on manual intervention, resulting in poor batch stability. Summary of the Invention

[0006] To address the problems in the prior art, the first aspect of the present invention provides a method for extracting Vaccaria segetalis cyclic peptides, comprising at least the following steps: extracting Vaccaria segetalis powder with a co-crystallization solvent, enzymatically hydrolyzing, separating and purifying, concentrating, and freeze-drying with nanobubbles to obtain the Vaccaria segetalis cyclic peptides.

[0007] In one embodiment, the extraction method includes the following steps: S1. After mixing Wangbuliuxing powder with a eutectic solvent, microwave extraction was performed to obtain the extract. S2. Mix the extract with the compound enzyme and perform enzymatic hydrolysis for 25-35 minutes to obtain the enzymatic hydrolysate; S3. The enzymatic hydrolysate is separated by cross-flow microfiltration using a ceramic membrane, and then concentrated under vacuum to obtain a concentrated solution. S4. Mix the concentrated liquid with nanobubbles and freeze-dry to obtain the Wangbuliuxing cyclic peptide.

[0008] In this invention, *Vaccaria segetalis* is a plant belonging to the Caryophyllaceae family, specifically *Vaccaria sinica*. Vaccaria segetalis (Neck.) Garcke The dried, mature seeds.

[0009] In one embodiment, the eutectic solvent includes at least one of betaine, malic acid, and deionized water.

[0010] In a preferred embodiment, the eutectic solvent comprises betaine, malic acid, and deionized water, wherein the molar ratio of betaine, malic acid, and deionized water is 1:(1.8~2.2):(0.9~1.1). Examples include 1:1.8:0.9, 1:2:1, and 1:2.2:1.1.

[0011] In one embodiment, the preparation method of the eutectic solvent includes: mixing betaine, malic acid and deionized water, stirring at 55~65℃ for 20~30 min until a homogeneous transparent liquid is obtained, and measuring the conductivity at 25℃ to be 1.5~2.5 mS / cm to obtain the eutectic solvent.

[0012] In one embodiment, the mass ratio of the *Vaccaria segetalis* powder to the eutectic solvent is 1:(5-8). Examples include 1:5, 1:6, 1:7, and 1:8.

[0013] In one embodiment, the power density of the microwave extraction is 2~5W / mL, and the switching time ratio of the microwave pulse mode is (8~12)s:(3~6)s.

[0014] In one embodiment, the microwave extraction temperature is 55~65℃ and the time is 15~20 min. Preferably, the microwave extraction temperature is 60℃ and the time is 15 min.

[0015] The applicant constructed a ternary eutectic solvent (DES) system using a specific molar ratio of betaine, malic acid, and deionized water, and employed microwave pulsed direct extraction of Vaccaria segetalis cyclic peptides. In this system, on the one hand, betaine (hydrogen bond acceptor) and malic acid (hydrogen bond donor) form a stable eutectic structure through intermolecular hydrogen bonds. The addition of water precisely adjusts the system's conductivity to 1.5–2.5 mS / cm, ensuring a high degree of molecular polarity matching with the cyclic peptides and achieving selective dissolution. On the other hand, this DES system exhibits extremely low solubility for impurities with significant polarity differences, such as polysaccharides and colloids, reducing impurity co-dissolution at the source and resolving the interference problem caused by the indiscriminate dissolution of impurities in traditional ethanol / methanol systems. Simultaneously, pulsed microwave heating avoids localized overheating that could damage the cyclic peptide structure, while high-frequency vibration enhances the contact between the DES and the raw material, accelerating the release of cyclic peptides from plant cells and shortening the extraction time.

[0016] In one embodiment, the complex enzyme comprises cellulase and pectinase, wherein the mass ratio of cellulase to pectinase is (2.5~3.5):1. Examples include 2.5:1, 3:1, and 3.5:1.

[0017] In one embodiment, the amount of the compound enzyme added is 0.5-1% of the volume of the extract. Examples include 0.5%, 0.6%, 0.8%, and 1%.

[0018] In one embodiment, the pH value in step S2 is 4.5-5.0, and the enzymatic hydrolysis temperature is 35-45°C. Examples include 35°C, 40°C, and 45°C.

[0019] In one embodiment, the parameters for the ceramic membrane cross-flow microfiltration separation are: transmembrane pressure 0.18~0.22MPa, membrane surface flow rate corresponding to ceramic membrane rotation speed 550~650rpm, operating temperature 35~40℃, and feed solution pH value 5.0~5.5.

[0020] In one embodiment, the ceramic membrane has a pore size of 50-60 nm. Preferably, the ceramic membrane has a pore size of 50 nm.

[0021] Through extensive creative work, the applicant has unexpectedly discovered that, in the Wangbuliuxing-DES extraction system, after the extract is hydrolyzed by a compound enzyme, using a ceramic membrane with a specific pore size and matching specific process parameter windows—transmembrane pressure 0.18~0.22MPa, rotation speed 550~650rpm—can stably achieve high permeability (>96%) and efficient separation of cyclic peptides without relying on complex online monitoring and intelligent control systems, greatly simplifying equipment complexity and reducing costs. The compound enzyme directionally decomposes cellulose and pectin in plant cell walls, breaking the encapsulation structure of cyclic peptides and increasing their release rate; simultaneously, it degrades some soluble polysaccharides, reducing the viscosity of the extract and lessening the burden on subsequent membrane separation. The ceramic membrane with the specific pore size can precisely retain undigested solid impurities and large-molecule polysaccharides, allowing cyclic peptides to permeate; the optimized transmembrane pressure and membrane rotation speed create a stable cross-flow effect, avoiding membrane fouling and ensuring a cyclic peptide permeability >96%, improving the stability and continuity of the process and solving the problems of complex and costly traditional column chromatography purification steps.

[0022] In one embodiment, the vacuum concentration conditions are: temperature 50~70℃, vacuum degree -0.07~-0.09MPa, and concentration to 1 / 10 of the original volume.

[0023] In one embodiment, the nanobubbles have a particle size of 50-150 nm and the injection volume is 5-15% of the concentrated liquid volume.

[0024] In one embodiment, the nanobubbles are prepared by pressurizing nitrogen to 0.8 MPa using a Venturi tube nanogenerator and then passing it through a ceramic filter with a pore size of 50-60 nm to obtain nanobubbles with a particle size ≤150 nm.

[0025] In one embodiment, step S4 includes: mixing the concentrate with nanobubbles, rapidly freezing at -40°C, and then vacuum freeze-drying until the moisture content of the material is ≤3%, to obtain the Wangbuliuxing cyclic peptide.

[0026] In a preferred embodiment, the parameters for the vacuum freeze-drying are: cold trap temperature -80℃, vacuum degree ≤10Pa, and heating rate during the sublimation drying stage 0.5℃ / min.

[0027] This invention employs nanobubble-assisted freeze-drying technology, injecting nitrogen nanobubbles with a particle size of 50-150 nm into the concentrate at 5-15% of the concentrate volume. During freeze-drying, the nanobubbles form micropores, creating a porous network structure in the freeze-dried powder. This significantly increases the specific surface area, solving the problem of slow reconstitution caused by large ice crystals in traditional freeze-dried products, reducing the reconstitution time from 180 s to 40 s. Furthermore, the nanobubble interface lowers the activation energy for ice crystal growth, reducing the ice crystal size from 50-100 μm in traditional processes to 5-10 μm. This reduces the damage to the cyclic peptide spatial structure caused by ice crystals, achieving a cyclic peptide activity retention rate of ≥98%. By adjusting the particle size and injection amount of the nanobubbles, the porosity and bulk density of the freeze-dried powder can be precisely controlled, adapting to the application needs of different fields such as cosmetics and pharmaceuticals.

[0028] The second aspect of this invention provides a method for extracting Wangbuliuxing cyclic peptides to obtain the product.

[0029] A third aspect of the present invention provides an application of the product in the preparation of cosmetics.

[0030] Beneficial effects 1. This invention uses a specific molar ratio of betaine, malic acid and deionized water to construct a ternary eutectic solvent (DES) system, which is then used in conjunction with microwave pulse directional extraction of Vaccaria segetalis cyclic peptides to avoid interference from impurities such as polysaccharides and improve the yield of plant cyclic peptides.

[0031] 2. The extract of this invention is hydrolyzed by a compound enzyme and then processed using a ceramic membrane with a specific pore size and specific process parameters. This allows for stable high permeability (>96%) and efficient separation of cyclic peptides without relying on complex online monitoring and intelligent control systems, greatly simplifying equipment complexity and reducing costs.

[0032] 3. The nanobubble-assisted freeze-drying technology of this invention introduces nitrogen nanobubbles to regulate the freeze-dried microstructure, reducing the damage of ice crystals to the spatial structure of cyclic peptides and achieving a cyclic peptide activity retention rate of ≥98%. By adjusting the particle size and injection amount of nanobubbles, the porosity and bulk density of the freeze-dried powder can be precisely controlled, adapting to the application needs of different fields such as cosmetics and pharmaceuticals.

[0033] 4. The extraction method of Vaccaria segetalis cyclic peptide provided by this invention replaces the traditional ethanol / methanol system, develops a green, non-toxic, and biocompatible solvent, and ensures that the extract can be directly used in sensitive fields such as medicine and cosmetics. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention. Experimental methods not specifying specific conditions in the embodiments were performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0035] Example 1 The first aspect of this example provides a method for extracting cyclic peptides from Vaccaria segetalis, including the following steps: S1. Mix Wangbuliuxing powder and eutectic solvent at a mass ratio of 1:6 and place them in a microwave reactor for microwave extraction to obtain the extract. S2. After cooling the extract to 40°C, mix it with the compound enzyme and perform enzymatic hydrolysis for 30 minutes to obtain the enzymatic hydrolysate. S3. The enzymatic hydrolysate is separated by cross-flow microfiltration using a ceramic membrane, and then concentrated under vacuum to obtain a concentrated solution. S4. Nitrogen gas is pressurized to 0.8 MPa using a Venturi tube nanogenerator and then passed through a ceramic filter with a pore size of 50 nm to obtain nanobubbles with a particle size of 50 nm. The concentrated liquid is mixed with the nanobubbles, and then rapidly frozen at -40°C and vacuum freeze-dried until the moisture content of the material is ≤3% to obtain the Wangbuliuxing cyclic peptide.

[0036] The Wangbuliuxing powder was obtained from Ximei (Shanghai) Chemical Technology Co., Ltd.

[0037] The eutectic solvent is betaine, malic acid and deionized water, and the molar ratio of betaine, malic acid and deionized water is 1:1.8:0.9.

[0038] The preparation method of the eutectic solvent includes: mixing betaine, malic acid and deionized water, stirring at 60°C for 30 min until a homogeneous transparent liquid is obtained, and measuring the conductivity at 25°C to be 1.8 mS / cm to obtain the eutectic solvent.

[0039] The parameters for microwave extraction are: power density 5W / mL, microwave pulse mode switching time ratio 10s:5s, temperature 60℃, and time 15min.

[0040] The composite enzyme is cellulase and pectinase, and the mass ratio of cellulase to pectinase is 2.5:1.

[0041] The amount of the compound enzyme added is 0.8% of the volume of the extract.

[0042] The pH value in step S2 is 4.7, and the enzymatic hydrolysis temperature is 40℃.

[0043] The parameters for the ceramic membrane cross-flow microfiltration separation are: transmembrane pressure 0.18 MPa, membrane surface flow rate corresponding to ceramic membrane rotation speed 550 rpm, operating temperature 35℃, and feed solution pH 5.0.

[0044] The vacuum concentration conditions are: temperature 50℃, vacuum degree -0.07MPa, and concentration to 1 / 10 of the original volume.

[0045] The amount of nanobubbles injected is 5% of the volume of the concentrated liquid.

[0046] The parameters for the vacuum freeze-drying are: cold trap temperature -80℃, vacuum degree ≤10Pa, and heating rate during the sublimation drying stage 0.5℃ / min.

[0047] The second aspect of this example provides a method for extracting Wangbuliuxing cyclic peptides to obtain the product.

[0048] The third aspect of this example provides an application of the product in the preparation of cosmetics.

[0049] Example 2 The first aspect of this example provides a method for extracting cyclic peptides from Vaccaria segetalis, including the following steps: S1. Mix Wangbuliuxing powder and eutectic solvent at a mass ratio of 1:6 and place them in a microwave reactor for microwave extraction to obtain the extract. S2. After cooling the extract to 40°C, mix it with the compound enzyme and perform enzymatic hydrolysis for 30 minutes to obtain the enzymatic hydrolysate. S3. The enzymatic hydrolysate is separated by cross-flow microfiltration using a ceramic membrane, and then concentrated under vacuum to obtain a concentrated solution. S4. Nitrogen gas is pressurized to 0.8 MPa using a Venturi tube nanogenerator and then passed through a ceramic filter with a pore size of 50 nm to obtain nanobubbles with a particle size of 150 nm. The concentrated liquid is mixed with the nanobubbles, and then rapidly frozen at -40°C and vacuum freeze-dried until the moisture content of the material is ≤3% to obtain the Wangbuliuxing cyclic peptide.

[0050] The eutectic solvent is betaine, malic acid and deionized water, and the molar ratio of betaine, malic acid and deionized water is 1:2.2:1.1.

[0051] The preparation method of the eutectic solvent includes: mixing betaine, malic acid and deionized water, stirring at 60°C for 30 min until a homogeneous transparent liquid is obtained, and measuring the conductivity at 25°C to be 2.5 mS / cm to obtain the eutectic solvent.

[0052] The parameters for microwave extraction are: power density 5W / mL, microwave pulse mode switching time ratio 10s:5s, temperature 60℃, and time 15min.

[0053] The composite enzyme is cellulase and pectinase, and the mass ratio of cellulase to pectinase is 3.5:1.

[0054] The amount of the compound enzyme added is 0.8% of the volume of the extract.

[0055] The pH value in step S2 is 4.7, and the enzymatic hydrolysis temperature is 40℃.

[0056] The parameters for the ceramic membrane cross-flow microfiltration separation are: transmembrane pressure 0.22 MPa, membrane surface flow rate corresponding to ceramic membrane rotation speed 650 rpm, operating temperature 40℃, and feed solution pH 5.5.

[0057] The vacuum concentration conditions are: temperature 70℃, vacuum degree -0.09MPa, and concentration to 1 / 10 of the original volume.

[0058] The amount of nanobubbles injected is 15% of the volume of the concentrated liquid.

[0059] The parameters for the vacuum freeze-drying are: cold trap temperature -80℃, vacuum degree ≤10Pa, and heating rate during the sublimation drying stage 0.5℃ / min.

[0060] The second aspect of this example provides a method for extracting Wangbuliuxing cyclic peptides to obtain the product.

[0061] The third aspect of this example provides an application of the product in the preparation of cosmetics.

[0062] Example 3 The first aspect of this example provides a method for extracting cyclic peptides from Vaccaria segetalis, including the following steps: S1. Mix Wangbuliuxing powder and eutectic solvent at a mass ratio of 1:6 and place them in a microwave reactor for microwave extraction to obtain the extract. S2. After cooling the extract to 40°C, mix it with the compound enzyme and perform enzymatic hydrolysis for 30 minutes to obtain the enzymatic hydrolysate. S3. The enzymatic hydrolysate is separated by cross-flow microfiltration using a ceramic membrane, and then concentrated under vacuum to obtain a concentrated solution. S4. Nitrogen gas is pressurized to 0.8 MPa using a Venturi tube nanogenerator and then passed through a ceramic filter with a pore size of 50 nm to obtain nanobubbles with a particle size of 80 nm. The concentrated liquid is mixed with the nanobubbles, and then rapidly frozen at -40°C and vacuum freeze-dried until the moisture content of the material is ≤3% to obtain the Wangbuliuxing cyclic peptide.

[0063] The eutectic solvent is betaine, malic acid, and deionized water, and the molar ratio of betaine, malic acid, and deionized water is 1:2:1.

[0064] The preparation method of the eutectic solvent includes: mixing betaine, malic acid and deionized water, stirring at 60°C for 30 min until a homogeneous transparent liquid is obtained, and measuring the conductivity at 25°C to be 2 mS / cm to obtain the eutectic solvent.

[0065] The parameters for microwave extraction are: power density 5W / mL, microwave pulse mode switching time ratio 10s:5s, temperature 60℃, and time 15min.

[0066] The composite enzyme is cellulase and pectinase, and the mass ratio of cellulase to pectinase is 3:1.

[0067] The amount of the compound enzyme added is 0.8% of the volume of the extract.

[0068] The pH value in step S2 is 4.7, and the enzymatic hydrolysis temperature is 40℃.

[0069] The parameters for the ceramic membrane cross-flow microfiltration separation are: transmembrane pressure 0.2 MPa, membrane surface flow rate corresponding to ceramic membrane rotation speed 600 rpm, operating temperature 38℃, and feed solution pH 5.3.

[0070] The vacuum concentration conditions are: temperature 60℃, vacuum degree -0.08MPa, and concentration to 1 / 10 of the original volume.

[0071] The amount of nanobubbles injected is 12% of the volume of the concentrated liquid.

[0072] The parameters for the vacuum freeze-drying are: cold trap temperature -80℃, vacuum degree ≤10Pa, and heating rate during the sublimation drying stage 0.5℃ / min.

[0073] The second aspect of this example provides a method for extracting Wangbuliuxing cyclic peptides to obtain the product.

[0074] The third aspect of this example provides an application of the product in the preparation of cosmetics.

[0075] Comparative Example 1 This example provides a method for extracting cyclic peptides from Vaccaria segetalis, including the following steps: Step 1, Drying and Grinding: After washing the seeds of Vaccaria segetalis, dry them at a low temperature (≤60°C), then grind them into fine powder using a Chinese medicine pulverizer and pass them through a 50-mesh sieve; Step 2, Alcohol Extraction: High-concentration methanol (85%) was used as the solvent. Reflux extraction: The mixture was refluxed three times in a constant-temperature water bath, each time for 1.5 hours. Step 3, Preliminary purification of crude extract: The combined extracts are concentrated under reduced pressure at a low temperature of 40°C using a rotary evaporator, and the solvent is recovered to obtain a viscous paste or solid. Step 4, Deep Separation and Purification: Conventional Column Chromatography (CC): Stationary Phase: Silica Gel Column. Gradient elution was performed using a dichloromethane-methanol system based on the polarity of the cyclic peptide.

[0076] Procedure: Fractions containing cyclic peptides were collected by monitoring with TLC (thin-layer chromatography), and then combined and concentrated.

[0077] Comparative Example 2 This example provides a method for extracting cyclic peptides from Vaccaria segetalis, including the following steps: Step 1, Drying and Grinding: After washing the seeds of Vaccaria segetalis, dry them at a low temperature (≤60°C), then grind them into fine powder using a Chinese medicine pulverizer and pass them through a 50-mesh sieve; Step 2, Alcohol Extraction: High-concentration methanol (85%) was used as the solvent. Reflux extraction: The mixture was refluxed three times in a constant-temperature water bath, each time for 1.5 hours. Step 3, Preliminary purification of crude extract: The combined extracts were concentrated under reduced pressure at a low temperature of 40°C using a rotary evaporator, the solvent was recovered, and a viscous paste or solid was obtained. The paste was then suspended in water and extracted multiple times with ethyl acetate.

[0078] Step 4, Deep Separation and Purification: High-Performance Preparative Liquid Chromatography (MPLC / HPLC): A preparative liquid chromatography system equipped with a reversed-phase C18 preparative column was used. The mobile phase consisted of methanol-water gradient elution (with 0.1% formic acid added to improve peak shape).

[0079] Procedure: After dissolving the crude product from the previous step, the sample is injected and monitored by a UV detector. The target peak is automatically collected to obtain the cyclic peptide.

[0080] Performance testing The Wangbuliuxing cyclic peptides prepared in each example and comparative example were determined by high performance liquid chromatography (HPLC) under the following chromatographic conditions: Chromatographic column: C18 (ODS) column (4.6 mm × 250 mm, 5 μm), column temperature: 30 ℃, detection wavelength: 220 nm, flow rate: 1.0 mL / min; injection volume: 10 μL; mobile phase A: aqueous solution containing 0.1% trifluoroacetic acid (TFA); mobile phase B: acetonitrile (ACN) solution containing 0.1% trifluoroacetic acid (TFA); gradient elution conditions are shown in Table 1.

[0081] Table 1

[0082] Sample preparation: Accurately weigh approximately 100.0 mg of Vaccaria segetalis extract powder, place it in a 10 ml volumetric flask, add an appropriate amount of pure water to dissolve it, sonicate at 40℃ until the powder is completely dissolved, cool it, add pure water to make up to the mark, filter it through a 0.22 μm microporous membrane, and take the filtrate as the test solution.

[0083] Plotting the standard curve: Accurately weigh an appropriate amount of Wangbuliuxing cyclic peptide reference standard, dissolve it in 70% methanol and dilute to volume to prepare a reference standard stock solution with a concentration of 0.9 mg / mL. Inject 0.2 μl, 0.5 μl, 0.8 μl, 1 μl, 1.4 μl, 1.7 μl, and 2 μl of the reference standard solution sequentially under the above chromatographic conditions, and record the peak area of ​​Wangbuliuxing cyclic peptide. Plot the injection volume of the reference standard (X, μg) on ​​the x-axis and the peak area (Y) on the y-axis to perform linear regression and establish a standard curve: Y = 3313.5X + 202.94, R² 2 =0.9996.

[0084] Content calculation: Inject 10 μL of the test solution for analysis. Substitute the measured peak area of ​​the target cyclic peptide into the regression equation of the standard curve to calculate the content of Wangbuliuxing cyclic peptide in the test solution. The content of Wangbuliuxing cyclic peptide in the sample is calculated according to the following formula: Content of cyclic peptides in Vaccaria segetalis:

[0085] In the formula: Y: The chromatographic peak area of ​​the sample.

[0086] V: Sample injection volume (unit: μL).

[0087] T: Total concentration of the sample (unit: mg / mL) Note: The unit of T in the formula is mg / mL, which is consistent with the implicit cyclic peptide concentration unit μg / μL (i.e., mg / mL), so they can be directly divided without additional conversion.

[0088] The test results are shown in Table 2.

[0089] Table 2

[0090] As can be seen from the data in Table 2, compared with the comparative example which used traditional solvents and traditional extraction methods, the example achieved a high yield of Wangbuliuxing cyclic peptides through the combined effects of DES directional extraction, enzymatic hydrolysis, ceramic membrane cross-flow microfiltration separation and purification, and nanobubble freeze drying.

Claims

1. A method for extracting cyclic peptides from Vaccaria segetalis, characterized in that, At least the following steps are included: The *Vaccaria segetalis* powder was extracted with a co-crystallized solvent, enzymatically hydrolyzed, separated and purified, concentrated, and freeze-dried with nanobubbles to obtain the *Vaccaria segetalis* cyclic peptide.

2. The method for extracting Wangbuliuxing cyclic peptides according to claim 1, characterized in that, The extraction method includes the following steps: S1. After mixing Wangbuliuxing powder with a eutectic solvent, microwave extraction was performed to obtain the extract. S2. Mix the extract with the compound enzyme and perform enzymatic hydrolysis for 25-35 minutes to obtain the enzymatic hydrolysate; S3. The enzymatic hydrolysate is separated by cross-flow microfiltration using a ceramic membrane, and then concentrated under vacuum to obtain a concentrated solution. S4. Mix the concentrated liquid with nanobubbles and freeze-dry to obtain the Wangbuliuxing cyclic peptide.

3. The method for extracting Wangbuliuxing cyclic peptides according to claim 2, characterized in that, The eutectic solvent includes at least one of betaine, malic acid, and deionized water.

4. The method for extracting Wangbuliuxing cyclic peptides according to claim 3, characterized in that, The eutectic solvent includes betaine, malic acid and deionized water, and the molar ratio of betaine, malic acid and deionized water is 1:(1.8~2.2):(0.9~1.1).

5. The method for extracting Wangbuliuxing cyclic peptides according to claim 2, characterized in that, The power density of the microwave extraction is 2~5W / mL, and the switching time ratio of the microwave pulse mode is (8~12)s:(3~6)s.

6. The method for extracting Wangbuliuxing cyclic peptides according to claim 2, characterized in that, The complex enzyme includes cellulase and pectinase, and the mass ratio of cellulase to pectinase is (2.5~3.5):

1.

7. The method for extracting Wangbuliuxing cyclic peptides according to claim 2, characterized in that, The parameters for the ceramic membrane cross-flow microfiltration separation are: transmembrane pressure 0.18~0.22MPa, membrane surface flow rate corresponding to ceramic membrane rotation speed 550~650rpm, operating temperature 35~40℃, and feed solution pH 5.0~5.

5.

8. The method for extracting Wangbuliuxing cyclic peptides according to claim 2, characterized in that, The nanobubbles have a particle size of 50-150 nm and are injected in an amount of 5-15% of the volume of the concentrated liquid.

9. A product obtained by the extraction method of Vaccaria segetalis cyclic peptide according to any one of claims 1 to 8.

10. An application of the product according to claim 9, characterized in that, It is used in the preparation of cosmetics.

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