Radix pseudostellariae extract with high content of radix pseudostellariae cyclic peptide B and preparation method and cosmetic application of radix pseudostellariae extract

Abstract

The invention discloses a radix pseudostellariae extract with high content of radix pseudostellariae cyclic peptide B and a preparation method and cosmetic application of the radix pseudostellariae extract, and belongs to the technical field of cosmetics. The preparation method comprises the following steps: (1) crushing radix pseudostellariae, adding alcohol and performing ultrasonic treatment to obtain feed liquid; (2) adding a compound enzyme into the feed liquid to carry out enzymolysis, enzyme deactivation, solid-liquid separation, concentration and impurity removal to obtain an enzymatic hydrolysate; and (3) eluting the enzymatic hydrolysate with macroporous resin to obtain an eluent, and drying to obtain the product, the compound enzyme is prepared from pectinase, papain, beta-glucosidase, cellulase and xylanase according to the mass ratio of (5 to 10) to (0.5 to 1) to (3 to 7) to (0.3 to 1). The radix pseudostellariae extract disclosed by the invention is prepared by adopting an enzymolysis process, more active ingredients can be reserved, the content of radix pseudostellariae cyclic peptide B in the obtained extract is high, and the radix pseudostellariae extract has relatively good anti-inflammatory, soothing, repairing and anti-aging effects.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics. Specifically, it relates to a Pseudostellariae Radix extract with a high content of pseudostellarin B, a preparation method thereof, and a cosmetic use thereof. Background Art

[0002] Pseudostellariae Radix is the dried tuberous root of Pseudostellaria heterophylla Pseudostellaria heterophylla (Miq.) Pax ex Pax Hoffm.; it is dug during the withering of the stems and leaves in summer, washed, the fibrous roots are removed, slightly scalded in boiling water and then dried in the sun or directly dried in the sun. Pseudostellariae Radix has traditional effects such as replenishing qi and strengthening the spleen, promoting the production of body fluid and moistening the lungs; it is mainly used to treat symptoms such as weakness of the spleen and body tiredness, weakness after illness, deficiency of qi and yin, spontaneous sweating and thirst, dry cough due to lung dryness, etc.

[0003] Modern research shows that Pseudostellariae Radix mainly contains chemical components such as cyclopeptides, saponins, saccharides, amino acids, phospholipids, volatile oils, fatty acids, oils and fats, sterols, and trace elements, and has effects such as myocardial protection, immune regulation, antioxidant, hypoglycemic, anti-stress, anti-fatigue, anti-tumor, and antitussive, and is widely used in the fields of health foods, pharmaceuticals, and daily chemical industries.

[0004] Currently, the extraction methods for Pseudostellariae Radix are mostly water extraction processes, resulting in a large loss of active ingredients, which to a certain extent affects the efficacy. CN109528803A discloses a preparation method for a Pseudostellariae Radix extract, including extracting Pseudostellariae Radix with water and then freeze-drying the obtained Pseudostellariae Radix extract. This invention finds that freeze-drying after water extraction can reduce the loss of active ingredients in the extract to a certain extent, but the content of its cyclopeptide active ingredients is relatively low, especially the content of pseudostellarin B is only 0.0002 - 0.0003%.

[0005] Xiong Hejian et al. (Study on the in vitro antioxidant activity of Pseudostellariae Radix extract [J]. Acta Scientiarum Naturalium Universitatis Nankaiensis, December 2009, 42(6), 37 - 41) disclose that the water extract and alcohol extract of Pseudostellariae Radix have obvious antioxidant activities, and the alcohol extract is significantly better than the water extract, but they do not disclose the influence of different extraction processes on the content of pseudostellarin B in Pseudostellariae Radix. CN107929127A discloses a preparation method for a Pseudostellariae Radix extract, including the following steps: 1) Preparation of water-soluble substances of Pseudostellariae Radix: Weigh and crush the dried Pseudostellariae Radix medicinal materials, add water and soak overnight, then reflux and extract, filter to obtain a filtrate; 2) Preparation of non-polar substances of Pseudostellariae Radix: Use a eutectic solvent betaine-glycerol mixed solution as an extraction solvent to perform ultrasonic-assisted extraction of non-polar substances from the residues after water extraction of Pseudostellariae Radix, filter to obtain filtrate B; 3) Compound the water-soluble substances and non-polar substances of Pseudostellariae Radix: Combine filtrate A and filtrate B, and concentrate under reduced pressure on a rotary evaporator to obtain the Pseudostellariae Radix extract. Although this invention has a good extraction effect on cyclopeptides, it does not disclose the influence on the content of pseudostellarin B in Pseudostellariae Radix. Summary of the invention

[0006] The purpose of the present invention is to provide a Pseudostellariae Radix Extract with a high content of Pseudostellariae Radix Cyclopeptide B, a preparation method thereof and cosmetic uses thereof. The Pseudostellariae Radix Extract is prepared by enzymatic hydrolysis and can retain more active ingredients. The Pseudostellariae Radix Extract has a high content of Pseudostellariae Radix Cyclopeptide B and has good anti-inflammatory, anti-aging, soothing and repairing effects, and can be used as a functional raw material in cosmetics.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a Pseudostellariae Radix extract, which is prepared by enzymolysis of Pseudostellariae Radix as a substrate by a complex enzyme.

[0008] In some embodiments, the complex enzyme is pectinase, papain, β-glucosidase, cellulase and xylanase; preferably, the mass ratio of pectinase, papain, β-glucosidase, cellulase and xylanase is 5-10:0.5-1:1:3-7:0.3-1; further preferably, it is 7-9:0.5-0.7:1:4-6:0.4-0.6; further preferably, it is 8-9:0.6-0.7:1:4-5:0.5-0.6; and most preferably, it is 8:0.6:1:5:0.5.

[0009] In a second aspect, the present invention provides a method for preparing the above-mentioned Radix Pseudostellariae Extract, comprising the following steps: (1) crushing Pseudostellaria heterophylla, adding alcohol for ultrasonic treatment, recovering ethanol, and obtaining a feed liquid; (2) adding a composite enzyme to the feed liquid for enzymolysis, inactivating the enzyme, separating the solid and the liquid, concentrating and removing impurities, and obtaining an enzymolysis solution; (3) The enzymatic hydrolyzate is eluted through a macroporous resin to obtain an eluate, and the eluate is dried.

[0010] In some embodiments, the amount of alcohol added in step (1) is 8-10 times the mass of Pseudostellaria heterophylla, preferably 9 times; the temperature of the alcohol is 40°C-50°C; preferably 45°C-50°C; the alcohol is an ethanol aqueous solution with a volume fraction of 30%-70%; preferably a volume fraction of 45%-70%.

[0011] In some embodiments, the ultrasound in step (1) is: ultrasound at 300W-500W for 12min-25min; preferably ultrasound at 400W for 20min.

[0012] In some embodiments, the complex enzyme in step (2) is pectinase, papain, β-glucosidase, cellulase and xylanase; preferably, the mass ratio of pectinase, papain, β-glucosidase, cellulase and xylanase is 5-10:0.5-1:1:3-7:0.3-1; more preferably 7-9:0.5-0.7:1:4-6:0.4-0.6; more preferably 8-9:0.6-0.7:1:4-5:0.5-0.6; most preferably 8:0.6:1:5:0.5.

[0013] In some embodiments, the amount of the complex enzyme added in step (2) is 0.1%-0.3% by weight of the alcohol; preferably 0.2%.

[0014] In some embodiments, the enzymatic hydrolysis conditions in step (2) are: enzymatic hydrolysis at 40°C-50°C for 2h-4h; preferably enzymatic hydrolysis at 45°C±2°C for 3h.

[0015] In some embodiments, the solid-liquid separation in step (2) is aimed at achieving separation of the solid phase and the liquid phase. Therefore, conventional technical means such as centrifugation, filtration or filter pressing can be used.

[0016] In some embodiments, the concentration and impurity removal in step (2) is to concentrate to a relative density of 1.10-1.12 at 60°C, refrigerate at -2°C to 4°C for 18h-30h, and filter to obtain an enzymatic hydrolyzate.

[0017] In some embodiments, the macroporous resin in step (3) is of model AB-8, D101 or HPD100, preferably AB-8.

[0018] In some embodiments, the elution in step (3) is as follows: the sample volume is 1BV-5BV, the sample is loaded at a flow rate of 0.5BV / h-4BV / h, and eluted with 2BV-8BV, 50%-80% ethanol at a flow rate of 1BV / h-5BV / h. Preferably, the sample volume is 2.5BV, the sample is loaded at a flow rate of 1BV / h, and eluted with 3BV, 70% ethanol at a flow rate of 2BV / h.

[0019] In some embodiments, the drying temperature in step (3) does not exceed 50°C, and is preferably freeze-drying, reduced pressure drying or spray drying.

[0020] In some embodiments, the method for preparing the Pseudostellaria heterophylla extract comprises the following steps: (1) Grind Pseudostellariae Radix, add 8-10 times the amount of alcohol (30%-70% ethanol aqueous solution by volume), and ultrasonicate at 300W-500W for 12min-25min to obtain a liquid; (2) Add a complex enzyme to the feed liquid, enzymatically hydrolyze at 40°C - 50°C for 2 h - 4 h, inactivate the enzyme after the reaction ends, separate the solid and liquid, concentrate the liquid phase to a relative density of 1.10 - 1.12 at 60°C, refrigerate at -2°C to 4°C for 18 h - 30 h, filter to remove impurities, and obtain the enzymolysis liquid. The addition amount of the complex enzyme is 0.1% - 0.3% of the weight of the alcohol; the complex enzyme is pectinase, papain, β - glucosidase, cellulase, and xylanase with a mass ratio of 5 - 10:0.5 - 1:1:3 - 7:0.3 - 1. (3) The enzymolysis liquid is eluted by macroporous resin. The volume of the sample loading liquid is 1 BV - 5 BV, the sample is loaded at a flow rate of 0.5 BV / h - 4 BV / h, and eluted with 2 BV - 8 BV of 50% - 80% ethanol at a flow rate of 1 BV / h - 5 BV / h to obtain the eluate, and the eluate is dried to obtain the product.

[0021] Thirdly, the present invention provides the use of the complex enzyme combination in increasing the content of heterophyllin B in the heterophylly falsestarwort root extract.

[0022] In some embodiments, the complex enzyme is pectinase, papain, β - glucosidase, cellulase, and xylanase; preferably, the mass ratio of pectinase, papain, β - glucosidase, cellulase, and xylanase is 5 - 10:0.5 - 1:1:3 - 7:0.3 - 1; more preferably 7 - 9:0.5 - 0.7:1:4 - 6:0.4 - 0.6; even more preferably 8 - 9:0.6 - 0.7:1:4 - 5:0.5 - 0.6; most preferably 8:0.6:1:5:0.5.

[0023] Fourthly, the present invention provides the use of the above - mentioned heterophylly falsestarwort root extract in the preparation of cosmetics.

[0024] The heterophylly falsestarwort root extract of the present invention has a high content of heterophyllin B and can be used as a functional raw material in cosmetics. For example, the efficacy of the functional raw material is at least one of anti - inflammation, soothing, repair, and anti - aging.

[0025] Fifthly, the present invention also provides a cosmetic comprising the above - mentioned heterophylly falsestarwort root extract.

[0026] In some embodiments, the cosmetic is in the form of emulsion, paste, water, powder, oil, or block.

[0027] In addition to the above-mentioned efficacy raw materials (Radix Pseudostellariae extract), the optional components of the cosmetics described in the present invention may also contain one or more optional components known or otherwise effective for use in personal care products, provided that these optional components are compatible with the physical and chemical properties of the basic components described herein, or do not unduly affect the stability, aesthetics or performance of the product. Non-limiting examples of such optional components are described in the "Technical Specifications for Cosmetics Safety, 2015 Edition", which are hereby incorporated by reference in their entirety.

[0028] The beneficial effects of the present invention are as follows: (1) In the present invention, pectinase, papain, β-glucosidase, cellulase and xylanase are used in combination to enzymatically hydrolyze the aqueous extract of Radix Pseudostellariae, which can retain more active ingredients. Compared with the prior art, the content of pseudostellarin B in the Radix Pseudostellariae extract prepared by the present invention is higher, reaching 0.13%-0.33%.

[0029] (2) Experiments have proved that the Radix Pseudostellariae extract prepared by the present invention has good safety and has good anti-inflammatory, soothing, repairing and anti-aging effects.

[0030] (3) The preparation process of the present invention is simple, the conditions are mild, and the cost is low, which is suitable for large-scale popularization and application. Description of the Drawings

[0031] Figure 1 It is a representative diagram of neutrophils in the tail of zebrafish embryos, and the dark dots are the stained neutrophils; Figure 2 It is a schematic diagram of the measurement area of skin barrier damage in zebrafish embryos, and the skin barrier damage is marked with green fluorescence; Figure 3 It is a diagram of the test results of the skin barrier permeability of zebrafish embryos. Detailed Embodiments

[0032] The following description of the embodiments is only used to help understand the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art in this technical field, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention. The following description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but can be applied to a wider range that conforms to the principles and novel features disclosed herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0033] The present invention does not limit the source of the raw materials used. Unless otherwise specified, the raw materials used in the present invention are all ordinary commercially available products in this technical field. The Pseudostellaria heterophylla used in the present invention is produced in Guizhou, preferably from Huangping and Shibing.

[0034] Unless otherwise specified, all operations herein are carried out in a room temperature (20°C - 25°C) environment, and the solvent used is water. The multiple amount mentioned in the present invention is relative to the weight of the raw material. For example, in Example 1, 9 times the amount of alcohol, and the specific amount of alcohol used is 9 times the mass of Pseudostellaria heterophylla.

[0035] Next, the embodiments and comparative examples of the present invention are shown to explain the Pseudostellaria heterophylla extract of the present invention in more detail. However, the present invention is not limited thereto. Unless otherwise specified, the percentages in the present invention are all mass percentages. The pectinase in the present invention, product number YB30778; papain, product number YB49326; β-glucosidase, product number YB124915; cellulase, product number YB37524; xylanase, product number YB125042, are all purchased from Hunan Yunbang Biotechnology Co., Ltd.

[0036] Example 1 Preparation method of Pseudostellaria heterophylla extract The steps are as follows: (1) The Pseudostellaria heterophylla is crushed and passed through a 60-mesh sieve, added with 9 times the amount of alcohol (60% ethanol aqueous solution by volume, temperature 45°C), ultrasonicated at 400W for 20 min, and the ethanol is recovered to obtain a feed liquid; (2) Add a composite enzyme to the feed liquid, enzymatically hydrolyze at 45°C for 3 h, after the reaction ends, keep warm at 121°C for 15 min to inactivate the enzyme, centrifuge at 1500 rpm for 10 min to obtain a centrifuged liquid, concentrate the centrifuged liquid to a relative density of 1.10 - 1.12 at 60°C, refrigerate at 0°C for 24 h, and filter through a 0.22-μm acetic acid membrane to obtain an enzymatically hydrolyzed liquid; The addition amount of the complex enzyme is 0.2% of the weight of the alcohol; the complex enzyme is pectinase, papain, β-glucosidase, cellulase and xylanase with a mass ratio of 8:0.6:1:5:0.5; (3)The enzymolysis solution is eluted by AB-8 macroporous resin. The volume of the sample loading solution is 2.5 BV, and the sample is loaded at a flow rate of 1 BV / h. It is eluted with 3 BV of 70% ethanol at a flow rate of 2 BV / h to obtain an eluate. The eluate is dried under reduced pressure (50 °C, 0.05 Mpa) to obtain the product.

[0037] Example 2 Preparation method of Pseudostellariae Radix extract The steps are as follows: (1)Pseudostellariae Radix is pulverized and sieved through a 60-mesh sieve, added with 8 times the amount of alcohol (30% ethanol aqueous solution by volume, temperature 40 °C), and ultrasonicated at 300 W for 25 min. The ethanol is recovered to obtain a stock solution; (2)Add a complex enzyme to the stock solution, enzymolyze at 40 °C for 4 h, keep warm at 121 °C for 15 min to inactivate the enzyme after the reaction ends, centrifuge at 1500 rpm for 10 min to obtain a centrifugate. The centrifugate is concentrated to a relative density of 1.10 - 1.12 at 60 °C, refrigerated at 0 °C for 24 h, and filtered through a 0.22-μm acetic acid membrane to obtain an enzymolysis solution; The addition amount of the complex enzyme is 0.1% of the weight of the alcohol; the complex enzyme is pectinase, papain, β-glucosidase, cellulase and xylanase with a mass ratio of 5:0.5:1:3:0.3; (3)The enzymolysis solution is eluted by AB-8 macroporous resin. The volume of the sample loading solution is 1 BV, and the sample is loaded at a flow rate of 0.5 BV / h. It is eluted with 2 BV of 50% ethanol at a flow rate of 1 BV / h to obtain an eluate. The eluate is dried under reduced pressure (50 °C, 0.05 Mpa) to obtain the product.

[0038] Example 3 Preparation method of Pseudostellariae Radix extract The steps are as follows: (1)Pseudostellariae Radix is pulverized and sieved through a 60-mesh sieve, added with 10 times the amount of alcohol (70% ethanol aqueous solution by volume, temperature 50 °C), and ultrasonicated at 500 W for 12 min. The ethanol is recovered to obtain a stock solution; (2)Add a complex enzyme to the stock solution, enzymolyze at 50 °C for 2 h, keep warm at 121 °C for 15 min to inactivate the enzyme after the reaction ends, centrifuge at 1500 rpm for 10 min to obtain a centrifugate. The centrifugate is concentrated to a relative density of 1.10 - 1.12 at 60 °C, refrigerated at 0 °C for 24 h, and filtered through a 0.22-μm acetic acid membrane to obtain an enzymolysis solution; The addition amount of the complex enzyme is 0.3% of the weight of the alcohol; the complex enzyme is pectinase, papain, β-glucosidase, cellulase and xylanase with a mass ratio of 10:1:1:7:1; (3) The enzymatic hydrolysate is eluted with AB-8 macroporous resin. The volume of the sample loading solution is 5 BV, and the sample is loaded at a flow rate of 4 BV / h. Then, it is eluted with 8 BV of 80% ethanol at a flow rate of 5 BV / h to obtain the eluate. The eluate is dried under reduced pressure (50 °C, 0.05 Mpa) to obtain the product.

[0039] Comparative Example 1 Preparation method of Pseudostellaria heterophylla extract The difference between this comparative example and Example 1 lies in the different mass ratios of pectinase, papain, β-glucosidase, cellulase and xylanase.

[0040] The specific steps are as follows: (1) The Pseudostellaria heterophylla is crushed and sieved through a 60-mesh sieve, added with 9 times the amount of alcohol (60% ethanol aqueous solution by volume, temperature 45 °C), ultrasonicated at 400 W for 20 min, and solid-liquid separated to obtain the material liquid; (2) Add a compound enzyme to the material liquid, enzymatically hydrolyze at 45 °C for 3 h, keep the temperature at 121 °C for 15 min to inactivate the enzyme after the reaction ends, centrifuge at 1500 rpm for 10 min to obtain the centrifugate, concentrate the centrifugate to a relative density of 1.10 - 1.12 at 60 °C, refrigerate at 0 °C for 24 h, and filter through a 0.22-μm acetic acid membrane to obtain the enzymatic hydrolysate; The addition amount of the compound enzyme is 0.2% of the weight of the alcohol; the compound enzyme is pectinase, papain, β-glucosidase, cellulase and xylanase with a mass ratio of 1:0.2:2:10:2; (3) The enzymatic hydrolysate is eluted with AB-8 macroporous resin. The volume of the sample loading solution is 2.5 BV, and the sample is loaded at a flow rate of 1 BV / h. Then, it is eluted with 3 BV of 70% ethanol at a flow rate of 2 BV / h to obtain the eluate. The eluate is dried under reduced pressure (50 °C, 0.05 Mpa) to obtain the product.

[0041] Comparative Example 2 Preparation method of Pseudostellaria heterophylla extract The difference between this comparative example and Example 1 lies in the different mass ratios of pectinase, papain, β-glucosidase, cellulase and xylanase.

[0042] The specific steps are as follows: (1) The Pseudostellaria heterophylla is crushed and sieved through a 60-mesh sieve, added with 9 times the amount of alcohol (60% ethanol aqueous solution by volume, temperature 45 °C), ultrasonicated at 400 W for 20 min, and the ethanol is recovered to obtain the material liquid; (2) Add a compound enzyme to the material liquid, enzymatically hydrolyze at 45 °C for 3 h, keep the temperature at 121 °C for 15 min to inactivate the enzyme after the reaction ends, centrifuge at 1500 rpm for 10 min to obtain the centrifugate, concentrate the centrifugate to a relative density of 1.10 - 1.12 at 60 °C, refrigerate at 0 °C for 24 h, and filter through a 0.22-μm acetic acid membrane to obtain the enzymatic hydrolysate; The addition amount of the composite enzyme is 0.2% of the weight of the alcohol; the composite enzyme is pectinase, papain, β-glucosidase, cellulase and xylanase with a mass ratio of 15:3:0.8:0.5:0.2; (3) The enzymatic hydrolysate is eluted with AB-8 macroporous resin. The volume of the sample loading solution is 2.5 BV, and the sample is loaded at a flow rate of 1 BV / h. Elution is carried out with 3 BV of 70% ethanol at a flow rate of 2 BV / h to obtain an eluate. The eluate is dried under reduced pressure (50 °C, 0.05 Mpa) to obtain the product.

[0043] Comparative Example 3 Preparation method of Pseudostellariae Radix extract The difference between this comparative example and Example 1 is that: the enzymatic hydrolysis step is not included; The specific steps are as follows: (1) Pseudostellariae Radix is pulverized and sieved through a 60-mesh sieve, added with 9 times the amount of alcohol (60% ethanol aqueous solution by volume, temperature 45 °C), and ultrasonicated at 400 W for 20 min. The ethanol is recovered to obtain a feed liquid; (2) The feed liquid is centrifuged at 1500 rpm for 10 min to obtain a centrifugate. The centrifugate is concentrated to a relative density of 1.10 - 1.12 at 60 °C, refrigerated at 0 °C for 24 h, and filtered through a 0.22 μm acetic acid membrane to obtain a clear liquid; (3) The clear liquid is eluted with AB-8 macroporous resin. The volume of the sample loading solution is 2.5 BV, and the sample is loaded at a flow rate of 1 BV / h. Elution is carried out with 3 BV of 70% ethanol at a flow rate of 2 BV / h to obtain an eluate. The eluate is dried under reduced pressure (50 °C, 0.05 Mpa) to obtain the product.

[0044] I. Content of Pseudostellarin B in Pseudostellariae Radix extract For the Pseudostellariae Radix extracts provided in the examples and comparative examples, the content determination was carried out with reference to the 2010 edition of the Chinese Pharmacopoeia, and the content of Pseudostellarin B in the Pseudostellariae Radix extracts was detected by high performance liquid chromatography (HPLC). The results are shown in Table 1.

[0045] Table 1

[0046] The results show that: the content of Pseudostellarin B in the Pseudostellariae Radix extracts prepared in Examples 1 - 3 of the present invention reaches 0.13% - 0.33%.

[0047] Meanwhile, by comparing the single-factor experiments of Comparative Example 1, Comparative Example 2 and Example 1, it can be seen that when the mass ratios of pectinase, papain, β-glucosidase, cellulase and xylanase are changed outside the scope claimed in the present invention, the content of heterophyllin B in the prepared Pseudostellariae Radix extract is only 0.0018% - 0.0020%, indicating that the compounding of pectinase, papain, β-glucosidase, cellulase and xylanase with specific mass ratios in the present invention can significantly increase the content of heterophyllin B in the Pseudostellariae Radix extract.

[0048] II. L929 cell viability test In this experiment, a blank group (DMEM medium), a cell control group (DMEM medium + cells) and a sample group (DMEM medium + cells + Pseudostellariae Radix extracts prepared in Examples 1 - 3 and Comparative Examples 1 - 3) were set up for cell viability test, and each group had 3 parallel wells.

[0049] Mouse fibroblast L929 cells in the logarithmic growth phase were inoculated into 96-well plates at a density of 1×10 5 cells / mL. Incubate at 37 °C for 24 hours. Remove the culture medium, and add 100 μL of sample solutions with different concentrations to each well respectively, and continue to incubate at 37 °C for 12 hours. Add 20 μL of 5 mg / ml MTT solution to each well, mix well, and continue to culture at 37 °C for 2 hours. Discard the supernatant, and add 100 μL of isopropanol to each well. Under dark conditions at room temperature, oscillate at a rate of 100 r / min for 30 minutes. Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance of each well at a wavelength of 570 nm.

[0050] Calculate the cell viability according to the following formula, ; In the formula, OD (sample group) represents the absorbance of the sample group, OD (blank group) represents the absorbance of the blank group, and OD (cell control group) represents the absorbance of the cell control group.

[0051] The results are shown in Table 2 and Table 3.

[0052] Table 2

[0053] The results show that compared with the blank group, at concentrations of 2.50 mg / mL, 5.00 mg / mL and 100 mg / mL, the cell viability increased significantly, (P value < 0.001 or < 0.0001). It indicates that the Pseudostellariae Radix extract prepared in Example 1 has a cell proliferation effect at the above concentrations.

[0054] Table 3

[0055] The results showed that compared with the blank group, the cell viability in the groups of Examples 1 - 3 was significantly increased (P value < 0.001); while there was no significant change in the cell viability in the groups of Comparative Examples 1 - 3 (P value > 0.05), indicating that at a concentration of 5.00 mg / mL, the extracts of Pseudostellaria heterophylla prepared in Examples 1 - 3 all had cell proliferation effects, while the extracts of Pseudostellaria heterophylla prepared in Comparative Examples 1 - 3 had no cell proliferation effects.

[0056] III. Expression of SIRT1 longevity protein in L929 cells In this experiment, a blank group (DMEM medium), a model group (DMEM medium + cells + H2O2), and a sample group (DMEM medium + cells + H2O2 + extracts of Pseudostellaria heterophylla prepared in Examples 1 - 3 and Comparative Examples 1 - 3) were set up, with 3 parallel wells in each group.

[0057] Mouse fibroblast L929 cells in the logarithmic growth phase were inoculated into 96 - well plates at a density of 1×10 5 cells / mL. They were incubated at 37°C for 24 hours. The culture medium was removed, and 100 μL of sample solutions with different concentrations were added to each well, and then incubated at 37°C for another 12 hours. The expression of SIRT1 protein was tested using an ELISA kit for SIRT1.

[0058] The results are shown in Table 4.

[0059] Table 4

[0060] The results showed that compared with the model group, the expression levels of SIRT1 protein in the cells of the groups of Examples 1 - 3 were significantly increased (P value < 0.0001 or 0.01); while there was no significant change in the expression levels of SIRT1 protein in the cells of the groups of Comparative Examples 1 - 3 (P value > 0.05), indicating that at a concentration of 5.00 mg / mL, the extracts of Pseudostellaria heterophylla prepared in Examples 1 - 3 had the effect of enhancing the expression of SIRT1 protein in cells.

[0061] IV. Expression of phosphorylated STAT3D protein in L929 cells In this experiment, a blank group (DMEM medium), a model group (DMEM medium + cells + H2O2), and a sample group (DMEM medium + cells + H2O2 + extracts of Pseudostellaria heterophylla prepared in Examples 1 - 3 and Comparative Examples 1 - 3) were set up, with 3 parallel wells in each group.

[0062] Mouse fibroblast L929 cells in the logarithmic growth phase were inoculated at a density of 1×10 5Inoculate at a density of cells / mL into a 96-well plate. Incubate at 37 °C for 24 hours. Remove the culture medium, add 100 μL of sample solutions with different concentrations to each well respectively, and continue to incubate at 37 °C for 12 hours. Use the P-STAT3 Elisa kit to test the expression of P-STAT3 protein.

[0063] The results are shown in Table 5.

[0064] Table 5

[0065] The results showed that: compared with the model group, the expression levels of P-STAT3 protein in the cells of Examples 1-3 groups were significantly decreased (P value < 0.0001 or 0.05); while there were no significant changes in the expression levels of P-STAT3 protein in the cells of Comparative Examples 1-3 groups (P value > 0.05), indicating that at a concentration of 5.00 mg / mL, the Pseudostellaria heterophylla extracts prepared in Examples 1-3 had an inhibitory effect on the expression of P-STAT3 protein in cells.

[0066] V. Anti-lipid aging and anti-oxidative stress of nematodes Cell test model: Caenorhabditis elegans.

[0067] Test method for anti-lipid aging of nematodes: After synchronization of nematodes, L4-stage nematodes were continuously intervened with the sample at 20 mg / mL for 10 d, 20 mg / mL of vitamin C was used as a positive control, each group was parallel three times, and after anesthesia with sodium azide, they were transferred to a 2% (mass fraction) agarose gel pad, and fluorescence images were obtained under a fluorescence microscope and the fluorescence intensity was analyzed.

[0068] The results are shown in Table 6.

[0069] Table 6

[0070] The results showed that: compared with the blank group, the relative fluorescence intensity of lipofuscin in the nematodes of Examples 1-3 groups was significantly decreased (P value < 0.0001 or 0.01); while there were no significant changes in the relative fluorescence intensity of lipofuscin in the nematodes of Comparative Examples 1-3 groups (P value > 0.05), indicating that at a concentration of 20 mg / mL, the Pseudostellaria heterophylla extracts prepared in Examples 1-3 had an inhibitory effect on the expression of lipofuscin in nematodes.

[0071] Test method for anti-oxidative stress of nematodes: After synchronization of nematodes, L4-stage nematodes were transferred to the above blank group, positive control group or sample group for 5 d respectively, then transferred to a new NGM plate, and the nematodes were exposed to a plate containing hydrogen peroxide. Calculate the survival rate under stress.

[0072] The results are shown in Table 7.

[0073] Table 7

[0074] The results showed that: compared with the blank group, the average lifespan of nematodes in the groups of Examples 1 - 3 increased significantly (P value < 0.05); while there was no significant change in the average lifespan of nematodes in the positive control group and the groups of Comparative Examples 1 - 3 (P value > 0.05), indicating that at a concentration of 20 mg / mL, the extracts of Pseudostellaria heterophylla prepared in Examples 1 - 3 had the effect of prolonging the lifespan of nematodes under oxidative stress conditions.

[0075] VI. Anti - aging test on zebrafish 6.1 Promotion test of zebrafish Sirt1 gene expression Sirt1 is a NAD⁺ (nicotinamide adenine dinucleotide) - dependent deacetylase, which is involved in a variety of cell biological functions mainly through the deacetylation of a variety of non - histone and histone proteins, and is a recognized longevity gene. Studies have found that when the Sirt1 gene in mammals and zebrafish is knocked out or defective, there will be an increase in reactive oxygen species (ROS), chronic inflammation, and a shorter lifespan; while promoting the expression of the Sirt1 gene can improve metabolism and the overall health level of organisms. Therefore, the anti - aging efficacy of a sample can be evaluated by testing whether it can promote the expression of the Sirt1 gene.

[0076] In this experiment, a blank control group (fish embryo culture medium), a positive control group (glutathione), and a test substance group (the extract of Pseudostellaria heterophylla provided in Example 1) were set up. Specifically: Blank control group: Randomly select 36 zebrafish and evenly distribute them into 24 - well plates, with 12 zebrafish and 2.5 mL of fish embryo culture medium in each well; Positive control group: Randomly select 36 zebrafish and evenly distribute them into 24 - well plates, with 3 wells, 12 zebrafish and 2.5 mL of glutathione working solution (prepare a 0.08 mg / mL glutathione solution with fish embryo culture medium) in each well.

[0077] Test substance group: Randomly select 36 zebrafish and evenly distribute them into 24 - well plates, with 3 wells, 12 zebrafish and 2.5 mL of test substance solution (prepare a 0.2% extract solution of Pseudostellaria heterophylla in Example 1 with fish embryo culture medium) in each well.

[0078] Place them in an incubator at 28℃ ± 1℃ and culture until 24 h ± 1 h after fertilization. Collect 12 zebrafish from each well into a 1.5 mL tube, remove the solution, add 0.5 mL of RNAlater solution ((Invitrogen; AM7020)), and store frozen.

[0079] RNA was extracted from zebrafish, and RNA was synthesized into cDNA using the PrimeScript RT reagent Kit with gDNA Eraser (Takara; Catno. RR047A). Real-time PCR amplification was performed using the SYBR Premix Ex Taq Kit (Takara; Cat no. RR420A). β-actin was used as the housekeeping gene, and Ct was used as the amplification result. The relative expression level of Sirt1 was calculated and statistically analyzed. Using 2 -△△Ct , the gene expression promotion rate of Sirt1 was calculated according to the following formula.

[0080] ; In the formula, 2 -△△Ct 受试物 : relative expression level of the target gene in the test substance group; 2 -△△Ct 空白对照组 : relative expression level of the target gene in the blank control group.

[0081] The results are shown in Table 8.

[0082] Table 8

[0083] The results showed that the extract of Pseudostellaria heterophylla prepared in Example 1 had a 75% promotion rate on the expression of the Sirt1 gene in zebrafish at an added concentration of 0.2%, indicating that the extract of Pseudostellaria heterophylla prepared by the present invention can promote the expression of the Sirt1 gene in zebrafish and has an anti-aging effect.

[0084] 6.2. Promotion test of zebrafish elastin gene (Elna) expression Elastin in human skin is a protein that enables many tissues in the body to maintain their original shape after stretching and contraction and enables the skin to return to its original shape after being squeezed. As people age, the elastin fibers in the skin gradually break and are lost, causing the skin to wrinkle and lose its elasticity. Compared with human skin, zebrafish skin is highly similar to human skin in other structures except that the epidermis is composed of differentiated epidermal cells and mucus layer instead of the stratum corneum. The elastin gene is highly conserved. Elastin in the human body is synthesized by the Eln gene, and the corresponding gene in zebrafish is Elna. By testing whether the sample can promote the expression of the Elna gene in zebrafish, it can be judged whether the sample has the effect of promoting elastin regeneration, helping the skin to resist wrinkles and achieving skin firming. Many domestic and foreign cosmetics achieve the effects of skin anti-wrinkle and firming by promoting elastin regeneration.

[0085] In this experiment, a blank control group (fish embryo culture medium), a positive control group (acetyl hexapeptide-8), and a test substance group (the extract of Pseudostellaria heterophylla provided in Example 1) were set up. Specifically: Blank control group: Randomly select 36 zebrafish and evenly distribute them into 24-well plates, with 12 zebrafish and 2.5 mL of fish embryo culture medium in each well; Positive control group: Randomly select 36 zebrafish and evenly distribute them into 24-well plates. Set up 3 wells, with 12 zebrafish and 2.5 mL of acetyl hexapeptide-8 working solution (prepare a 0.8 g / L acetyl hexapeptide-8 solution with fish embryo culture medium) in each well.

[0086] Test substance group: Randomly select 36 zebrafish and evenly distribute them into 24-well plates. Set up 3 wells, with 12 zebrafish and 2.5 mL of test substance solution (prepare a 0.2% extract solution of Pseudostellaria heterophylla with fish embryo culture medium) in each well.

[0087] Place them in an incubator at 28°C ± 1°C and culture until 24 h ± 1 h after fertilization. Collect 12 zebrafish in each well into 1.5 mL tubes, remove the solution, add 0.5 mL of RNAlater solution ((Invitrogen; AM7020)), and store frozen.

[0088] Extract RNA from zebrafish, synthesize cDNA from RNA using a PrimeScript RT kit containing gDNA Eraser (Takara; Catno. RR047A), perform real-time PCR amplification using a SYBR Premix Ex Taq kit (Takara; Cat no. RR420A), use β-actin as the housekeeping gene, Ct as the amplification result, calculate the relative expression level of the elastin gene (Elna) and conduct statistical analysis, using 2 -△△Ct , calculate the gene expression promotion rate of Elna according to the following formula.

[0089] ; In the formula, 2 -△△Ct 受试物 : Relative expression level of the target gene in the test substance group; 2 -△△Ct 空白对照组 : Relative expression level of the target gene in the blank control group.

[0090] The results are shown in Table 9.

[0091] Table 9

[0092] The results show that: The promotion rate of the extract of Pseudostellaria heterophylla prepared in Example 1 on the expression of the Elna gene in zebrafish is 20% at an added concentration of 0.2%, indicating that the extract of Pseudostellaria heterophylla prepared in the present invention can promote the expression of the Elna gene in zebrafish, has the effect of promoting elastin regeneration, and can achieve the effects of anti-wrinkle and firming.

[0093] 6.3 Promotion Test for the Expression of Zebrafish Type I Collagen Gene Collagen is the most abundant extracellular matrix protein in the human body, and type I collagen is the most abundant protein in the skin. The distribution of type I collagen in zebrafish is the same as that in humans and shows a high degree of conservation with humans. Their innate expression significantly decreases at 6 days after fertilization and becomes very low between 10 and 12 days after fertilization. By testing the expression of zebrafish type I collagen genes (col1a1a, col1a1b, and col1a2), comparing the changes in the relative expression levels of zebrafish type I collagen genes between the treatment group and the blank control group, and calculating the promotion rate of type I collagen gene expression, the anti-wrinkle and firming effects of raw materials or products can be evaluated.

[0094] In this experiment, a blank control group (fish embryo culture medium), a positive control group (acetyl hexapeptide-8), and a test substance group (the extract of Pseudostellaria heterophylla provided in Example 1) were set up. Specifically: Blank control group: Randomly select 36 zebrafish and evenly distribute them into a 24-well plate, with 12 zebrafish and 2.5 mL of fish embryo culture medium in each well; Positive control group: Randomly select 36 zebrafish and evenly distribute them into a 24-well plate, with 3 wells, 12 zebrafish and 2.5 mL of acetyl hexapeptide-8 working solution (prepare a 0.8 g / L acetyl hexapeptide-8 solution with fish embryo culture medium) in each well.

[0095] Test substance group: Randomly select 36 zebrafish and evenly distribute them into a 24-well plate, with 3 wells, 12 zebrafish and 2.5 mL of test substance solution (prepare a 0.2% extract solution of Pseudostellaria heterophylla with fish embryo culture medium) in each well.

[0096] Place them in an incubator at 28°C ± 1°C and culture until 24 h ± 1 h after fertilization. Collect 12 zebrafish in each well into a 1.5 mL tube, remove the solution, add 0.5 mL of RNAlater solution ((Invitrogen; AM7020)), and store frozen.

[0097] Extract RNA from zebrafish, synthesize cDNA from RNA using a PrimeScript RT kit with gDNA Eraser (Takara; Catno. RR047A), perform real-time PCR amplification using a SYBR Premix Ex Taq kit (Takara; Cat no. RR420A), use β-actin as the housekeeping gene, and Ct as the amplification result, calculate the relative expression level of type I collagen gene and conduct statistical analysis. Using 2 -△△Ct , calculate the promotion rate of type I collagen gene expression according to the following formula.

[0098] ; wherein, 2 -△△Ct 受试物 : the relative expression level of the target gene in the test substance group; 2 -△△Ct 空白对照组 : the relative expression level of the target gene in the blank control group.

[0099] The results are shown in Table 10.

[0100] Table 10

[0101] Note: Compared with the blank control group, **P < 0.01.

[0102] The results show that the promoting rates of the Pseudostellaria heterophylla extract prepared in Example 1 on the gene expressions of zebrafish col1a1a, col1a1b, and col1a2 are 62%, 56%, and 56% respectively at the addition concentration of 0.2%, indicating that the Pseudostellaria heterophylla extract prepared by the present invention can promote the expression of zebrafish type I collagen gene, has the effect of promoting the regeneration of type I protein, and can achieve the effects of anti-wrinkle and firming.

[0103] VII. Neutrophil Test on Zebrafish Embryos Soothing means helping to improve the state of skin irritation, etc. When the skin is stimulated by physical, chemical, or pathogen invasion, an immune response will occur, with discomfort such as redness and swelling, showing signs of sensitive skin. At this time, immune white blood cells (including neutrophils and macrophages) in the skin will aggregate to phagocytize and remove harmful substances such as infections, foreign bodies, or damaged cells in the skin. Inhibiting the aggregation of white blood cells at the stimulated site can soothe the skin irritation state. The neutrophils of zebrafish embryos (the main immune white blood cells) are highly similar to human neutrophils in morphology, biochemistry, and physiological functions. Therefore, the test results obtained from establishing a stimulated zebrafish model have good derivability for human skin efficacy. The neutrophils of zebrafish embryos can be observed by in vivo visualization using a transgenic line with fluorescently labeled neutrophils or Sudan black staining. In this experiment, a model in which copper sulfate is used to induce the stimulation of neuromast cells in the lateral line region of zebrafish embryos to cause neutrophil aggregation is used for testing. The changes in the number of neutrophils in the lateral line region of fish embryos in the test substance group and the model control group are compared, and the neutrophil aggregation inhibition rate is calculated to evaluate whether the raw material, formula, or product has the effect of soothing irritation to achieve a soothing effect.

[0104] In this experiment, a blank control group, a model control group, a positive control group, and a test substance group (the Pseudostellaria heterophylla extract provided in Example 1) are set up. Specifically: Blank control group: Randomly select 24 zebrafish embryos into a 3-cm culture dish, and add 5 mL of fish embryo culture medium; Model control group: Randomly select 24 zebrafish embryos into a 3-cm culture dish, and add 5 mL of fish embryo culture medium containing 10 μM anhydrous copper sulfate; Positive control group: Randomly select 24 zebrafish embryos into a 3-cm culture dish, and add 5 mL of fish embryo culture medium containing 10 μM anhydrous copper sulfate and 10 μM indomethacin; Test substance group: Randomly select 24 zebrafish embryos into a 3-cm culture dish, and add 5 mL of fish embryo culture medium containing 10 μM anhydrous copper sulfate and 0.2% test substance).

[0105] Place them in an incubator at 28°C ± 1°C for 40 - 45 min. After fixing the fish embryos in each test group in paraformaldehyde for at least 1 h, treat the fish embryos with PBST 3 times, 5 min each time, and then treat them with 50% ethanol for 3 min. After staining the fish embryos with Sudan black staining solution at room temperature for 1 h, wash them with 70% ethanol 4 times, 5 min each time, and then treat them with PBST 2 times, 5 min each time. Treat the fish embryos with bleaching solution for 10 min. If it is treated in a test tube, keep the lid open. Then treat them with 70% ethanol solution for 5 min, PBST for 1 min, Clearance Solution 1 for 15 min, Clearance Solution 2 for 10 min, and then treat them with PBST for 3 min. Place the fish embryos on their sides. Then place them under a stereomicroscope to take pictures of the tails of the fish embryos, and calculate the number of neutrophils in the three-quarter tail lateral line area starting from the anus of each fish embryo (see Figure 1 ) Calculate the neutrophil aggregation inhibition rate: ; In the formula, S is the average value of the number of neutrophils in the fish embryos of the test substance group, with the unit of grains per fish (grains / fish); M is the average value of the number of neutrophils in the fish embryos of the model control group, with the unit of grains per fish (grains / fish); B is the average value of the number of neutrophils in the fish embryos of the blank control group, with the unit of grains per fish (grains / fish).

[0106] The results are shown in Table 11.

[0107] Table 11

[0108] Note: Compared with the model control group, ***P < 0.01.

[0109] The results showed that the inhibition rate of neutrophil aggregation in the zebrafish embryos of the model control group was 0, indicating no soothing effect. The inhibition rate of indomethacin at 10 μM on neutrophil aggregation in zebrafish embryos was 39%, and the inhibition rate of the Radix Pseudostellariae extract prepared in Example 1 on neutrophil aggregation in zebrafish embryos at a concentration of 0.2% was 92%. This indicated that the Radix Pseudostellariae extract prepared in Example 1 of the present invention could significantly inhibit the aggregation of neutrophils in zebrafish embryos and had a soothing effect.

[0110] VIII. Test on the permeability of the skin barrier of zebrafish embryos Twenty-four 72-h zebrafish embryos were exposed to a solution of Radix Pseudostellariae extract at a formulated concentration of 0.2%. Meanwhile, a blank control group and a model control group were set up. After 1 h of exposure, the fish embryos were co-treated with sodium dodecyl sulfate solution (SDS) + sample solution for 40 min, and then stained with sodium fluorescein for 30 min. Fluorescence photography was used to measure the fluorescence intensity of the caudal fin and statistical analysis was performed.

[0111] Specifically, in this experiment, a blank control group, a model control group, a positive control group, and a test substance group (Radix Pseudostellariae extract provided in Example 1) were set up. Among them, Blank control group: Twenty-four zebrafish embryos were randomly selected and placed into a 6-well plate, and 6 mL of fish embryo culture medium was added; Model control group: Twenty-four zebrafish embryos were randomly selected and placed into a 6-well plate, and 6 mL of fish embryo culture medium was added; Positive control group: Twenty-four zebrafish embryos were randomly selected and placed into a 6-well plate, and 6 mL of ectoin working solution (150 mg / L, prepared with fish embryo culture medium) was added; Test substance group: Twenty-four zebrafish embryos were randomly selected and placed into a 6-well plate, and 6 mL of test substance solution (fish embryo culture medium containing 0.2% Radix Pseudostellariae extract provided in Example 1) was added.

[0112] After culturing at 28°C ± 1°C for 1 h ± 0.1 h, the blank control group was changed to 6 mL of new fish embryo culture medium, and the other groups were changed to 6 mL of fish embryo culture medium containing 0.05 g / L SDS and placed at 28°C ± 1°C for 40 min. The fish embryos were washed 3 times and then stained with 1 g / L sodium fluorescein solution (prepared with fish embryo culture medium) for 30 min.

[0113] The photos were opened with analysis software, and the caudal fin area of each fish embryo (see Figure 2 ) was marked, and the "average signal intensity" in the test results was selected as the fluorescence intensity.

[0114] Calculate the inhibition rate of skin barrier permeability: ; Wherein, S is the average fluorescence intensity of the caudal fins of the fish embryos in the test substance treatment group; M is the average fluorescence intensity of the caudal fins of the fish embryos in the model control group; B is the average fluorescence intensity of the caudal fins of the fish embryos in the blank control group.

[0115] The results are as Figure 3 shown in

[0116] Table 12

[0117] Note: Compared with the model control group, ***P < 0.01.

[0118] The results show that the inhibition rate of the skin barrier permeability of the zebrafish embryos in the model control group is 0, indicating no repair effect. The inhibition rate of the skin barrier permeability of the zebrafish embryos by ectoine at 150 mg / L is 41%. The inhibition rate of the skin barrier permeability of the zebrafish embryos by the Pseudostellaria heterophylla extract prepared in Example 1 at a concentration of 0.2% is 30%. This shows that the Pseudostellaria heterophylla extract prepared in Example 1 of the present invention can significantly reduce the skin permeability of the zebrafish embryos and has the effect of repairing the skin barrier, which can support the repair efficacy.

[0119] The above is a further description of the present invention in combination with specific embodiments, but these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

Claims

1. A method for preparing a Pseudostellariae Radix extract with a high content of pseudostellarin B, characterized in that, The steps include: (1) crushing Pseudostellaria heterophylla, adding alcohol for ultrasonic treatment, recovering ethanol, and obtaining a feed liquid; (2) adding a composite enzyme to the feed liquid for enzymolysis, inactivating the enzyme, separating the solid and the liquid, concentrating and removing impurities, and obtaining an enzymolysis solution; (3) The enzymatic hydrolyzate is eluted through a macroporous resin to obtain an eluate, which is then dried; The complex enzyme in step (2) is pectinase, papain, β-glucosidase, cellulase and xylanase in a mass ratio of 5-10:0.5-1:1:3-7:0.3-1; The model of the macroporous resin in step (3) is at least one of AB-8, D101 and HPD100.

2. The preparation method according to claim 1, wherein In step (1), the amount of alcohol added is 8-10 times the mass of Pseudostellaria heterophylla, the temperature of the alcohol is 40°C-50°C, and the alcohol is an ethanol aqueous solution with a volume fraction of 30%-70%; The ultrasound in step (1) is: ultrasound at 300W-500W for 12min-25min.

3. The preparation method according to claim 1, wherein The amount of the complex enzyme added in step (2) is 0.1%-0.3% by weight of the alcohol; The enzymatic hydrolysis conditions in step (2) are 40°C-50°C for 2h-4h; The concentration and impurity removal in step (2) is to concentrate to a relative density of 1.10-1.12 at 60°C, refrigerate at -2°C to 4°C for 18h-30h, filter and remove impurities to obtain an enzymatic hydrolyzate.

4. The preparation method according to claim 1, characterized in that, The mass ratio of the pectinase, papain, beta-glucosidase, cellulase and xylanase is 7-9:0.5-0.7:1:4-6:0.4-0.

6.

5. The preparation method according to claim 1, wherein, The elution in step (3) is as follows: the sample volume is 1BV-5BV, the sample is loaded at a flow rate of 0.5BV / h-4BV / h, and elution is performed with 2BV-8BV, 50%-80% ethanol at a flow rate of 1BV / h-5BV / h.

6. The Pseudostellariae Radix extract prepared by the preparation method according to any one of claims 1 to 5.

7. Use of a combined complex enzyme in increasing the content of pseudostellarin B in the extract of Pseudostellaria heterophylla, characterized in that, The complex enzyme is pectinase, papain, beta-glucosidase, cellulase and xylanase; the mass ratio of the pectinase, papain, beta-glucosidase, cellulase and xylanase is 5-10:0.5-1:1:3-7:0.3-1.

8. Use of the Pseudostellariae Radix extract prepared by the preparation method according to any one of claims 1 to 5 in the preparation of cosmetics.

9. The use according to claim 8, characterized in that, The cosmetic is in the form of milk, paste, water, powder, oil or block.

10. A cosmetic, characterized in that, The invention relates to a Pseudostellaria heterophylla extract prepared by the preparation method according to any one of claims 1 to 5.

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