A Portulaca oleracea extract solution, its preparation method and application

Abstract

The present invention belongs to the technical field of extraction of bioactive components, and particularly relates to a method for preparing a purslane extract solution. Aiming at the problems that the pretreatment, extraction and decolorization processes of the existing purslane extract solution are cumbersome, resulting in low content of active ingredients and inability to carry out large-scale production, etc., the present invention adopts four steps of purslane biological fermentation, ceramic membrane sterilization and protein removal, ultrasonic-assisted non-ethanol extraction, and combined hollow cellulose membrane decolorization to prepare the purslane extract solution. This extraction method is simple and easy to operate, convenient for industrial production. The prepared purslane extract solution has stable properties, high contents of active ingredients such as flavonoids and alkaloids, has good anti-allergic effects and good safety, and can be widely applied to skin care and washing and care cosmetics.

Description

Technical Field

[0001] The invention belongs to the technical field of bioactive component extraction, and particularly relates to a purslane extract solution and a preparation method and application thereof. Background Art

[0002] Portulaca oleracea contains a variety of active ingredients, including flavonoids, alkaloids, and amino acids. It has the ability to clear heat and detoxify, cool blood, and stop bleeding. It is commonly used to treat skin conditions such as carbuncles, sores, and eczema. Modern pharmacological research has shown that Portulaca oleracea extract has anti-allergic, anti-inflammatory, and antibacterial properties, effectively alleviating skin problems such as sensitivity and itching. Consequently, it is widely used in skincare and cosmetics.

[0003] The existing purslane extract solution preparation technology generally uses enzymatic hydrolysis to pretreat purslane, such as CN102389385A and CN106890202A, which use cellulase to pretreat purslane. However, purslane contains rich metal ions including Ca 2+ Mg 2+ et al. (Chang Shanshan, Analysis of the main nutritional components of halophytic purslane and research on the antioxidant activity and antibacterial effect of its extracts [D]. Nanjing Normal University, 2013), while high concentrations of Ca 2+ Mg 2+ It has a great influence on the activity of cellulase endo-enzyme (Zhang Hongxin et al., Effect of metal ions on the activity of cellulase endo-enzyme and exo-enzyme [J]. Cellulose Science and Technology, 2011(4):6-13), which makes the existing enzymatic pretreatment method of purslane unable to achieve satisfactory results.

[0004] Existing purslane extract solutions generally use a certain ratio of ethanol / water as the extraction solvent during the preparation process (such as CN105640842A, CN108785341A, CN102389385A, etc.). However, ethanol increases the irritation of the extract to the skin. Even if ethanol is replaced with ethylene glycol / propylene glycol / butylene glycol solvents for re-dissolution during recovery, the problem of ethanol residue in the extract cannot be solved, and the process is complicated.

[0005] The existing preparation technologies for active ingredients of purslane generally use activated carbon for decolorization (for example, CN105640842A uses a compound of activated carbon and activated clay for decolorization, and CN106890202A uses activated carbon and diatomite for decolorization) or macroporous adsorption resin for decolorization and enrichment of active ingredients (for example, CN109419827A and CN10683359A use macroporous resin for decolorization and enrichment of flavonoid active ingredients). However, activated carbon will adsorb a large amount of components such as flavonoids and alkaloids during the decolorization process (Han Yongping, et al., Study on the adsorption properties of spherical activated carbon for several effective components of traditional Chinese medicine [J]. Chinese Traditional and Herbal Drugs, 2004, 35(05):510-513), resulting in the loss of active ingredients in the purslane extract solution; the adsorption and desorption processes of macroporous adsorption resin are cumbersome, and a large amount of acids and bases are used in the regeneration process, causing environmental pollution and being not conducive to large-scale production. Summary of the Invention

[0006] In view of the deficiencies in the prior art, the present invention provides a method for preparing a purslane extract solution. The prepared purslane extract solution has stable properties, high contents of active ingredients flavonoids and alkaloids, good anti-allergic efficacy and good safety. The method is simple and easy to implement, and is convenient for industrial production.

[0007] One of the purposes of the present invention is to provide a method for preparing a purslane extract solution, which includes the following steps: Ferment purslane by biological fermentation to obtain a fermentation broth. After the obtained fermentation broth is subjected to sterilization and protein removal treatment, solvent extraction and decolorization treatment are carried out to obtain a purslane extract solution, wherein the purslane fermentation uses fungal fermentation. Specifically, it includes the following steps:

[0008] (1) Uniformly mix purslane and water as a fermentation substrate, add fungi to the fermentation substrate for fermentation to obtain a fermentation broth;

[0009] (2) Perform a separation operation of sterilization and protein removal on the fermentation broth obtained in step (1) through a ceramic separation membrane to obtain a sterilized and protein-removed fermentation broth;

[0010] (3) Add a solvent to the sterilized and protein-removed fermentation broth obtained in step (2) for extraction;

[0011] (4) Perform decolorization treatment on the fermentation broth after extraction in step (3) using a hollow cellulose membrane to obtain a purslane extract solution.

[0012] Among them, in step (1), the mass ratio of purslane to water is 1:3 to 1:50, preferably 1:5 to 1:25; the fungus used is selected from the genus Fusarium, preferably at least one of Fusarium solani, Fusarium oxysporum, and Fusarium moniliforme, more preferably Fusarium solani; by mass percentage, the mass of the fungus is 0.1 to 20% of the total mass of purslane and water, preferably 1 to 10%; the fermentation substrate in step (1) is subjected to high-pressure sterilization before fermentation. Preferably, the high-pressure sterilization conditions are a pressure of 103.4 kPa, a temperature of 121.3 °C, and a time of 30 min; in step (1), the fermentation temperature during fermentation treatment is 25 to 35 °C, and the fermentation time is 12 to 36 h;

[0013] In step (2), the ceramic separation membrane is selected from inorganic ceramic membranes, preferably at least one of alumina, zirconia, titanium oxide, and silicon oxide; the pore size of the ceramic separation membrane is 0.01 to 8.0 microns, preferably 0.1 to 5.0 microns; in step (2), the ceramic separation membrane is used to separate and remove fermented microorganisms and miscellaneous proteins. The separation temperature of the ceramic separation membrane is 30 to 45 °C, and the separation pressure is 0.02 to 0.1 MPa;

[0014] In step (3), by mass percentage, the amount of the solvent added in step (3) is 5 to 10 times the total weight of the fermented liquid after removing bacteria and proteins obtained in step (2); the above solvent is a mixed solvent of alcohol and water, wherein the alcohol is selected from polyhydric alcohols, preferably at least one of ethylene glycol, propylene glycol, butylene glycol, glycerol, and polyethylene glycol. By mass percentage, the amount of alcohol in the solvent is 20 to 80% of the total weight of the mixed solvent; the extraction in step (3) is carried out under ultrasonic conditions, the extraction temperature is 30 to 50 °C, and the extraction time is 5 to 10 h;

[0015] In step (4), the decolorization treatment uses a combined hollow fiber membrane module. Among them, in the combined hollow fiber membrane module, the pore size of the filtration membrane is 0.05 to 0.1 micron, the filtration temperature is 30 to 40 °C, and the filtration pressure is 0.01 to 0.1 MPa; the pore size of the retention membrane is 0.001 to 0.02 micron, the retention temperature is 40 to 50 °C, and the retention pressure is 0.1 to 0.2 MPa.

[0016] The second object of the present invention is to provide a purslane extract solution obtained by the above preparation method. The flavonoid content in the above purslane extract solution is 80 to 95 mg / L, and the alkaloid content is 20 to 30 mg / L.

[0017] A third object of the present invention is to provide an application of the above-mentioned purslane extract solution in skin care cosmetics or washing and care cosmetics. Calculated by mass percentage, the content of the purslane extract solution in skin care cosmetics is 0.5-10%; the content of the purslane extract solution in washing and care cosmetics is 0.1-5.0%. In addition to the purslane extract solution, other commonly used components in cosmetics can be added to the above-mentioned skin care cosmetics and washing and care cosmetics. For example, some conventional oil phases, water phases and other additives can be included, such as emulsifiers, surfactants, antioxidants, moisturizers, fragrances, skin conditioners, etc. Some functional components can also be added. The application and dosage of these specific components can be selected according to the specific type of the cosmetics and can be added in a conventional amount.

[0018] The object of the present invention is achieved in the following manner:

[0019] (1) In order to improve the pretreatment effect of purslane, the present invention uses Fusarium solani (Mart.) isolated from Pinellia ternata roots (preservation number CGMCC No. 9580). The cellulase produced by this strain, especially the activity of cellobiase, is relatively strong, and the enzyme activity is less affected by metal ions. Pretreating purslane with Fusarium solani can effectively destroy the cell wall structure of purslane, making intracellular active ingredients more and more easily dissolved, and significantly improving the extraction efficiency.

[0020] (2) In order to improve the permeability of the solvent and the extraction rate of active ingredients, the present invention directly uses ethylene glycol / propylene glycol / butylene glycol / polyethylene glycol and water as extraction solvents. Although their permeability is not as good as that of ethanol and water, ultrasonic oscillation can greatly improve the permeability of the solvent, resulting in a significant increase in the extraction rate of active ingredients. Compared with the prior art using ethanol and water for extraction, the skin irritation caused by the inability to completely remove ethanol is avoided, and the extraction process is simplified.

[0021] (3) In order to improve the decolorization effect and overcome the loss of active ingredients in the purslane extract solution caused by the existing activated carbon decolorization technology, as well as the cumbersome steps of the macroporous adsorption resin decolorization technology, the large amount of acid and alkali used in the adsorption and desorption process and the resulting environmental pollution, the present invention combines the application of a hollow cellulose membrane to decolorize the purslane extract solution. The decolorization effect is good, the decolorization efficiency is high, and there is no loss of active substances, which is convenient for large-scale production.

[0022] Compared with the existing technology for preparing purslane extract solution, the present invention has the following beneficial effects:

[0023] (1) Using Fusarium solani (Mart.), preservation number CGMCC No. 9580, to pretreat Portulaca oleracea L. before fermentation can effectively destroy the cell wall structure of Portulaca oleracea L., overcoming the defect that the activity of cellulase is greatly affected by Ca 2+ and Mg 2+ in traditional cellulase pretreatment of Portulaca oleracea L.;

[0024] (2) Using inorganic ceramic membranes to remove bacteria and proteins can achieve bacteria and protein removal at a lower temperature, overcoming the damage of traditional high-temperature sterilization to the thermosensitive active components of Portulaca oleracea L., and effectively removing a part of miscellaneous proteins at the same time;

[0025] (3) Using ultrasonic-assisted extraction with ethylene glycol / propylene glycol / butylene glycol / polyethylene glycol and water can improve the permeability of the solvent, overcome the skin irritation caused by the incomplete removal of ethanol in traditional extraction with ethanol and water, and simplify the extraction process at the same time;

[0026] (4) Using a combined hollow fiber membrane for decolorization can not only overcome the loss of active substances such as flavonoids and alkaloids caused by decolorization with activated carbon in traditional processes, but also overcome the deficiencies of the traditional macroporous adsorption resin decolorization process, which is cumbersome, polluting and not conducive to large-scale production. Description of the Drawings

[0027] Figure 1 Determination of the flavonoid content of the Portulaca oleracea L. extract solution obtained in Examples 1-5 and Comparative Examples 1-3;

[0028] Figure 2 Determination of the alkaloid content of the Portulaca oleracea L. extract solution obtained in Examples 1-5 and Comparative Examples 1-3;

[0029] Figure 3 Inhibitory rate of the Portulaca oleracea L. extract solution obtained in Examples 1-5 and Comparative Examples 1-3 against hyaluronidase;

[0030] Figure 4 Hemolysis rate of the Portulaca oleracea L. extract solution obtained in Examples 1-5 and Comparative Examples 1-3 at different addition concentrations. Detailed Embodiments

[0031] The present invention will be specifically described below in conjunction with specific embodiments. It is necessary to point out here that the following embodiments are only for further illustration of the present invention and cannot be construed as limiting the protection scope of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the content of the present invention still fall within the protection scope of the present invention.

[0032] The sources of the raw materials used in the examples are as follows:

[0033] Portulaca oleracea L., Beijing Tongrentang Group Co., Ltd.

[0034] Aluminum oxide ceramic separation membrane, Shanghai Kaixin Separation Technology Co., Ltd.

[0035] Hollow fiber membrane, Tianjin WoChi Technology Co., Ltd.

[0036] Alcohol solvents (ethanol, ethylene glycol, propylene glycol, butylene glycol, polyethylene glycol), Shandong Yousuo Chemical Technology Co., Ltd.

[0037] Fusarium solani (Depositary Institution: China General Microbiological Culture Collection Center, Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, Date of Deposit: September 1, 2014, Deposit Number: CGMCC No. 9580, Taxonomic Nomenclature: Fusarium solani)

[0038] Comparative Example 1

[0039] Fermentation with yellow rice wine yeast solution (Xu Danni et al., Research on the content of active ingredients and cosmetic efficacy in the fermentation broth of Portulaca oleracea L. [J]. Hubei Agricultural Sciences, 2017, 56(05): 924-926)

[0040] The specific preparation process is as follows: Weigh 0.5 g of powdered Portulaca oleracea L. into a 100 mL conical flask, add deionized water (the ratio of material to liquid is 1:20), sterilize at 121 °C under high pressure for 25 min, cool, then inoculate 5% of yellow rice wine yeast solution, culture in an air shaker at 28 °C and 180 r / min for 48 h, and then centrifuge at 4800 r / min for 20 min. The supernatant is the fermentation broth of Portulaca oleracea L.

[0041] Comparative Example 2

[0042] (1) Biological fermentation of Portulaca oleracea L.: Crush the above-ground part of Portulaca oleracea L. through a 40-mesh sieve, uniformly mix the dry powder of Portulaca oleracea L. and water according to the mass ratio of 5:100 as the fermentation substrate, sterilize at a pressure of 103.4 kPa and a temperature of 121 °C for 30 min, cool, and then inoculate Fusarium solani into the mixed solution at an inoculation amount of 3% of the mass of the fermentation substrate, and ferment at a fermentation temperature of 25 °C for 12 h;

[0043] (2) Sterilization and protein removal by inorganic ceramic membrane: Remove the fermented microorganisms and miscellaneous proteins from the fermentation broth in step (1) under the conditions that the pore size of the ceramic separation membrane is 0.2 μm, the separation temperature is 30 °C, and the pressure is 0.02 MPa.

[0044] (3) Ultrasonic-assisted extraction: Add a mixed solvent of ethanol and water to the sterilized and protein-removed fermentation broth obtained in (2). The volume ratio of ethanol to water in the mixed solvent is 7:3, and the amount of the mixed solvent used is 5 times the weight of the sterilized and protein-removed fermentation broth. Perform ultrasonic extraction for 5 hours under the conditions of an extraction temperature of 30 °C and an ultrasonic frequency of 50 kHz;

[0045] (4) Combined hollow fiber membrane decolorization: Under the conditions of controlling the pore size of the filtration membrane to be 0.05 μm, the filtration temperature to be 30 °C, the filtration pressure to be 0.01 MPa, the pore size of the retention membrane to be 0.001 μm, the retention temperature to be 40 °C, and the retention pressure to be 0.1 MPa, use the combined hollow fiber membrane to decolorize the fermentation broth in (3) to prepare the purslane extract solution of the present invention.

[0046] Comparative Example 3

[0047] (1) Purslane biological fermentation: Crush the above-ground part of purslane and pass it through a 40-mesh sieve. Uniformly mix purslane dry powder and water at a mass ratio of 5:100 as the fermentation substrate. Sterilize at a pressure of 103.4 kPa and a temperature of 121 °C for 30 min. After cooling, inoculate Fusarium solani on the mixed solution at an inoculation amount of 3% of the mass of the fermentation substrate. Ferment at a fermentation temperature of 25 °C for 12 hours;

[0048] (2) Inorganic ceramic membrane sterilization and protein removal: Remove the fermentation microorganisms and miscellaneous proteins from the fermentation broth in step (1) under the conditions of a pore size of the ceramic separation membrane of 0.2 μm, a separation temperature of 30 °C, and a pressure of 0.02 MPa;

[0049] (3) Ultrasonic-assisted extraction: Add a mixed solvent of ethylene glycol, propylene glycol, ethanol and water to the sterilized and protein-removed fermentation broth obtained in (2). The sum of the mass percentages of ethanol and water in the mixed solvent is 30%, the volume ratio of ethanol to water is 7:3, and the volume ratio of ethylene glycol to propylene glycol is 3:7. The amount of the mixed solvent used is 5 times the weight of the sterilized and protein-removed fermentation broth. Perform ultrasonic extraction for 5 hours under the conditions of an extraction temperature of 30 °C and an ultrasonic frequency of 50 kHz;

[0050] (4) Combined hollow fiber membrane decolorization: Under the conditions of controlling the pore size of the filtration membrane to be 0.05 μm, the filtration temperature to be 30 °C, the filtration pressure to be 0.01 MPa, the pore size of the retention membrane to be 0.001 μm, the retention temperature to be 40 °C, and the retention pressure to be 0.1 MPa, use the combined hollow fiber membrane to decolorize the fermentation broth in (3) to prepare the purslane extract solution of the present invention.

[0051] Example 1

[0052] (1) Biological fermentation of Portulaca oleracea: The above-ground part of Portulaca oleracea is crushed and sieved through a 40-mesh sieve. The dry powder of Portulaca oleracea and water are evenly mixed at a mass ratio of 5:100 as the fermentation substrate. Under a pressure of 103.4 kPa and a temperature of 121 °C, it is sterilized for 30 min. After cooling, Fusarium solani is inoculated into the mixed solution at an inoculation amount of 3% of the mass of the fermentation substrate, and fermented at a fermentation temperature of 25 °C for 12 hours;

[0053] (2) Removal of bacteria and proteins by inorganic ceramic membrane: The fermentation broth in step (1) is removed of fermentation microorganisms and miscellaneous proteins under the conditions that the pore size of the ceramic separation membrane is 0.2 μm, the separation temperature is 30 °C, and the pressure is 0.02 MPa;

[0054] (3) Ultrasonic-assisted extraction: A mixed solvent of ethylene glycol, propylene glycol and water is added to the fermentation broth after removal of bacteria and proteins obtained in (2). The sum of the mass percentages of ethylene glycol and propylene glycol in the mixed solvent is 30%, the volume ratio of ethylene glycol to propylene glycol is 3:7, and the amount of the mixed solvent used is 5 times the weight of the fermentation broth after removal of bacteria and proteins. Ultrasonic extraction is carried out at an extraction temperature of 30 °C and an ultrasonic frequency of 50 kHz for 5 hours;

[0055] (4) Decolorization by combined hollow fiber membrane: Under the conditions that the pore size of the filtration membrane is controlled at 0.05 μm, the filtration temperature is 30 °C, the filtration pressure is 0.01 MPa, the pore size of the retention membrane is 0.001 μm, the retention temperature is 40 °C, and the retention pressure is 0.1 MPa, the combined hollow fiber membrane is used to decolorize the fermentation broth in (3) to prepare the Portulaca oleracea extract solution of the present invention.

[0056] Example 2

[0057] (1) Biological fermentation of Portulaca oleracea: The above-ground part of Portulaca oleracea is crushed and sieved through a 40-mesh sieve. The dry powder of Portulaca oleracea and water are evenly mixed at a mass ratio of 10:100 as the fermentation substrate. Under a pressure of 103.4 kPa and a temperature of 121 °C, it is sterilized for 30 min. After cooling, Fusarium solani is inoculated into the mixed solution at an inoculation amount of 5% of the substrate mass, and fermented at a fermentation temperature of 30 °C for 24 hours;

[0058] (2) Removal of bacteria and proteins by ceramic membrane: The fermentation broth in step (1) is removed of fermentation microorganisms and miscellaneous proteins under the conditions that the pore size of the ceramic separation membrane is 1.0 μm, the separation temperature is 35 °C, and the pressure is 0.04 MPa;

[0059] (3) Ultrasonic-assisted extraction: Add a mixed solvent of ethylene glycol, butylene glycol, and water to the sterilized and protein-removed fermentation broth obtained in (2). The sum of the mass percentages of ethylene glycol and butylene glycol in the mixed solvent is 40%, the volume ratio of ethylene glycol to propylene glycol is 3:7, the amount of the mixed solvent used is 5 times the weight of the sterilized and protein-removed fermentation broth, and ultrasonic extraction is carried out for 6 hours under the conditions of an extraction temperature of 40 °C and an ultrasonic frequency of 100 kHz;

[0060] (4) Combined hollow fiber membrane decolorization: Under the conditions of controlling the pore size of the filtration membrane to be 0.06 μm, the filtration temperature to be 35 °C, the filtration pressure to be 0.06 MPa, the pore size of the retention membrane to be 0.005 μm, the retention temperature to be 45 °C, and the retention pressure to be 0.15 MPa, use the hollow fiber membrane in combination to decolorize the fermentation broth in (3) to prepare the purslane extract solution of the present invention.

[0061] Example 3

[0062] (1) Purslane biological fermentation: Crush the above-ground part of purslane and pass it through a 40-mesh sieve. Uniformly mix purslane dry powder and water as the fermentation substrate according to the mass ratio of purslane dry powder to water of 15:100. Sterilize at a pressure of 103.4 kPa and a temperature of 121 °C for 30 min. After cooling, inoculate Fusarium solani at an inoculation amount of 8% of the substrate mass into the mixed solution, and ferment at a fermentation temperature of 32 °C for 30 hours;

[0063] (2) Ceramic membrane sterilization and protein removal: Remove the fermentation microorganisms and miscellaneous proteins from the fermentation broth in step (1) under the conditions of a pore size of the ceramic separation membrane of 2.0 μm, a separation temperature of 42 °C, and a pressure of 0.08 MPa;

[0064] (3) Ultrasonic-assisted extraction: Add a mixed solvent of propylene glycol, butylene glycol, and water to the sterilized and protein-removed fermentation broth obtained in (2). The sum of the mass percentages of propylene glycol and butylene glycol in the mixed solvent is 60%, the volume ratio of ethylene glycol to propylene glycol is 3:7, the amount of the mixed solvent used is 5 times the weight of the sterilized and protein-removed fermentation broth, and ultrasonic extraction is carried out for 8 hours under the conditions of an extraction temperature of 42 °C and an ultrasonic frequency of 150 kHz;

[0065] (4) Combined hollow fiber membrane decolorization: Under the conditions of controlling the pore size of the filtration membrane to be 0.08 μm, the filtration temperature to be 38 °C, the filtration pressure to be 0.08 MPa, the pore size of the retention membrane to be ước 0.012 μm, the retention temperature to be 4óC, and the retention pressure to be 0.18 MPa, use the hollow fiber membrane in combination to decolorize the fermentation broth in (3) to prepare the purslane extract solution of the present invention.

[0066] Example 4

[0067] (1) Purslane biofermentation: The aerial part of purslane was crushed and passed through a 40-mesh sieve. Purslane dry powder and water were uniformly mixed at a mass ratio of 15:100 as a fermentation substrate. The mixture was sterilized at a pressure of 103.4 kPa and a temperature of 121°C for 30 min. After cooling, the mixture was inoculated with Fusarium solani at an inoculum amount of 10% of the substrate mass. The mixture was fermented at a fermentation temperature of 35°C for 36 hours.

[0068] (2) Ceramic membrane sterilization and protein removal: The fermentation broth in step (1) is subjected to the conditions of a ceramic separation membrane with a pore size of 5.0 μm, a separation temperature of 45° C., and a pressure of 0.1 MPa to remove fermentation microorganisms and foreign proteins;

[0069] (3) Ultrasonic-assisted extraction: a mixed solvent of ethylene glycol, propylene glycol, butylene glycol and water was added to the sterilized and deproteinized fermentation broth obtained in (2), wherein the sum of the mass percentages of ethylene glycol, propylene glycol and butylene glycol in the mixed solvent was 80%, the volume ratio of ethylene glycol, propylene glycol and butylene glycol was 2:3:5, and the amount of the mixed solvent was 10 times the weight of the sterilized and deproteinized fermentation broth. Ultrasonic extraction was performed at an extraction temperature of 50°C and an ultrasonic frequency of 200 kHz for 10 hours;

[0070] (4) Decolorization by hollow fiber membrane combination: The fermentation broth in (3) was decolorized by using a hollow fiber membrane in combination under the conditions of controlling the pore size of the filtration membrane to 0.1 μm, the filtration temperature to 40°C, the filtration pressure to 0.1 MPa, the pore size of the retention membrane to 0.02 μm, the retention temperature to 50°C, and the retention pressure to 0.2 MPa to prepare the purslane extract solution of the present invention.

[0071] Example 5

[0072] (1) Purslane biofermentation: The aerial part of purslane was crushed and passed through a 40-mesh sieve. Purslane dry powder and water were uniformly mixed at a mass ratio of 20:100 as a fermentation substrate. The mixture was sterilized at a pressure of 103.4 kPa and a temperature of 121°C for 30 min. After cooling, the mixture was inoculated with Fusarium solani at an inoculum amount of 10% of the substrate mass. The mixture was fermented at a fermentation temperature of 35°C for 36 hours.

[0073] (2) Ceramic membrane sterilization and protein removal: The fermentation broth in step (1) is subjected to the conditions of a ceramic separation membrane with a pore size of 5.0 μm, a separation temperature of 45° C., and a pressure of 0.1 MPa to remove fermentation microorganisms and foreign proteins;

[0074] (3) Ultrasonic-assisted extraction: Add a mixed solvent of ethylene glycol, propylene glycol, butylene glycol, polyethylene glycol and water to the sterilized and protein-removed fermentation broth obtained in (2). The sum of the mass percentages of ethylene glycol, propylene glycol, butylene glycol and polyethylene glycol in the mixed solvent is 80%. The volume ratio of ethylene glycol, propylene glycol, butylene glycol to polyethylene glycol is 2:2:3:3. The amount of the mixed solvent used is 10 times the weight of the sterilized and protein-removed fermentation broth. Perform ultrasonic extraction for 10 hours under the conditions of an extraction temperature of 50 °C and an ultrasonic frequency of 200 kHz;

[0075] (4) Combined hollow fiber membrane decolorization: Under the conditions of controlling the pore size of the filtration membrane to be 0.1 μm, the filtration temperature to be 40 °C, the filtration pressure to be 0.1 MPa, the pore size of the retention membrane to be 0.02 μm, the retention temperature to be 50 °C, and the retention pressure to be 0.2 MPa, use the hollow fiber membrane in combination to decolorize the fermentation broth in (3) to prepare the purslane extract solution of the present invention.

[0076] Example 6 Determination of total flavonoid and total alkaloid contents in the purslane extract solution and evaluation of anti-allergic and anti-irritant activities

[0077] Experimental materials: Purslane extract solutions prepared in Comparative Examples 1-3 and Examples 1-5

[0078] 1. Determination of total flavonoid content in the purslane extract solution

[0079] 1.1 Instruments and reagents

[0080] UV-265 type ultraviolet spectrophotometer (Shimadzu Corporation, Japan).

[0081] Rutin reference substance, National Institute for the Control of Pharmaceutical and Biological Products; ethanol, sodium nitrite, aluminum nitrate, and sodium hydroxide are all of analytical grade and commercially available; the experimental water is double-distilled water.

[0082] 1.2 Experimental method

[0083] (1) Preparation of the standard curve

[0084] Accurately weigh 100 mg of rutin reference substance, dissolve it with 70% ethanol, and make the volume up to 500 mL in a volumetric flask. Shake well. Accurately pipette 10 mL and place it in a 100 mL volumetric flask, and make the volume up with water to obtain a standard solution of 0.2 mg / mL. Accurately pipette 0.4 mL, 0.8 mL, 1.2 mL, 1.6 mL, 2.0 mL, and 2.4 mL of the standard solution respectively into 10 mL volumetric flasks, add water to 2.4 mL, add 0.4 mL of 5% sodium nitrite, shake well, let stand for 6 min, add 0.4 mL of 10% aluminum nitrate, shake well, let stand for 6 min, add 4.0 mL of 4.3% sodium hydroxide, then make the volume up to the mark with water, shake well, let stand for 15 min, using the reagent blank as the reference. Use a UV spectrophotometer to measure the absorbance at a wavelength of 500 nm (full wavelength scan, with the maximum absorption at 500 nm), and obtain the standard curve linear regression equation y = 76.941A - 0.478 for the concentration C (μg / mL) and the absorbance A, with the correlation coefficient R 2 = 0.9998.

[0085] (2) Determination of total flavonoid content

[0086] Accurately pipette 1.0 mL of the flavonoid extract into a 10 mL volumetric flask, add water to 2.4 mL, add 0.4 mL of 5% sodium nitrite, shake well, let stand for 6 min, add 0.4 mL of 10% aluminum nitrate, shake well, let stand for 6 min, add 4.0 mL of 4.3% sodium hydroxide, then make the volume up to the mark with water, shake well, let stand for 15 min, using the reagent blank as the reference. Use a UV spectrophotometer to measure the absorbance at a wavelength of 500 nm, substitute it into the regression equation, and calculate the total flavonoid content.

[0087] 1.3 Experimental results

[0088] As Figure 1 shown are the determination results of the flavonoid content of the purslane extract solutions prepared in Comparative Examples 1 - 3 and Examples 1 - 5, which are 78.43 mg / L, 79.00 mg / mL, 81.36 mg / mL, 89.49 mg / L, 91.20 mg / L, 89.78 mg / L, 91.8 mg / L, and 90.8 mg / L respectively. It can be seen that compared with the common extraction methods, the content of flavonoids in the purslane extract solution prepared by the present invention is higher.

[0089] 2. Determination of the total alkaloid content of the purslane extract solution

[0090] 2.1 Instruments and reagents

[0091] UV-265 type UV spectrophotometer; PHS-3A type pH meter; caffeine, Jiangsu Institute for Drug Control; bromocresol green, potassium hydrogen phthalate, Sinopharm Chemical Reagent Co., Ltd.

[0092] 2.2 Experimental methods

[0093] (1) Preparation of standard solution

[0094] Caffeine was prepared into an alcohol solution with a mass concentration of 1.0 mg / mL, and bromocresol green buffer solution: the concentration was 7.2*10 -4 mol / L, pH = 4.42. Accurately weigh 125 mg of bromocresol green and dissolve it in 25 mL of 0.1 mol / L NaOH solution. Add 2.55 g of potassium hydrogen phthalate and dissolve it in a small amount of distilled water. Transfer the solution to a 250 mL volumetric flask. Dilute to the mark with distilled water, shake well, and calibrate the pH using a pH meter to 4.42.

[0095] (2) Determination of total alkaloids

[0096] Use a graduated pipette to accurately transfer 0.08 mL of the refined alkaloid into a 10 mL colorimetric tube, add 1 mL of bromocresol green buffer solution and 2 mL of anhydrous ethanol solution respectively, dilute to the scale with distilled water, shake well, and use a 1 cm colorimetric cell with the reagent blank as a reference to measure its absorbance at a wavelength of 634 nm.

[0097] 2.3 Experimental Results

[0098] like Figure 2 The figures show the alkaloid content determination results of the Portulaca oleracea extract solutions prepared in Comparative Examples 1 to 3 and Examples 1 to 5, which are 19.67 mg / L, 20.06 mg / mL, 20.83 mg / mL, 24.86 mg / L, 25.10 mg / L, 24.93 mg / L, 25.08 mg / L, and 26.43 mg / L, respectively. It can be seen that compared with the commonly used extraction method, the Portulaca oleracea extract solution prepared by the present invention has a higher alkaloid content.

[0099] 3. Evaluation of the anti-allergic activity of Portulaca oleracea extract solution - Hyaluronidase inhibition test

[0100] 3.1 Instruments and reagents

[0101] UV-265 ultraviolet spectrophotometer; hyaluronidase, Sigma; sodium hyaluronate, Solarbio; CaCl2, acetylacetone, and Ellie's reagent, Sinopharm Chemical Reagent Co., Ltd.

[0102] 3.2 Experimental methods

[0103] The Elson-Morgan method was used for determination. 0.1 mL of 0.25 mmol / L CaCl2 solution and 0.5 mL of hyaluronidase solution were taken and incubated at 37 °C for 20 min; 0.5 mL of the sample solution was added and incubation was continued at 37 °C for 20 min; 0.5 mL of sodium hyaluronate solution was added and incubated at 37 °C for 30 min, then left at room temperature for 5 min; 0.1 mL of 0.4 mol / L NaOH solution and 0.5 mL of acetylacetone solution were added, heated in a boiling water bath for 15 min and immediately cooled with ice water for 5 min; 1.0 mL of Ehrlich reagent was added and diluted with 3.0 mL of absolute ethanol, left for 20 min for color development, and the absorbance value was measured with a spectrophotometer.

[0104]

[0105] Where: A is the absorbance (ABS value) of the control solution (using acetate buffer solution instead of the sample solution); B is the ABS value of the control blank solution (using acetate buffer solution instead of the sample solution and enzyme solution); C is the ABS value of the test sample solution; D is the ABS value of the test sample blank solution (using acetate buffer solution instead of the enzyme solution). During the experiment, the A group of test samples were first scanned at wavelengths in the range of 450 nm to 700 nm to determine the maximum absorption wavelength, and then deionized water was used as the reference, and the ABS values were measured at this maximum absorption wavelength respectively.

[0106] 3.3 Experimental results

[0107] Hyaluronic acid is a component in the tissue matrix that restricts the diffusion of water and other extracellular substances. After being hydrolyzed by hyaluronidase, the intercellular space becomes non-viscous, causing cell degranulation and the exudation of newly synthesized mediators, and exerting a biological effect leading to the occurrence of immediate hypersensitivity reactions. As Figure 3 shown, the inhibition rates of sodium hyaluronidase for Comparative Examples 1 - 3 and Examples 1 - 5 were 90.97%, 91.88%, 93.56%, 96.72%, 95.98%, 96.54%, 95.83% and 94.58% respectively. It can be seen that the purslane extract solution prepared by the present invention can significantly reduce type I skin hypersensitivity reactions and has good anti-skin sensitivity efficacy.

[0108] 4. Safety evaluation experiment of purslane extract solution - erythrocyte hemolysis experiment

[0109] 4.1 Instruments and reagents

[0110] UV-265 type ultraviolet spectrophotometer: ZHTY-50V shaker, Shanghai Zhichu Instrument Co., Ltd.; HH-2 digital display constant temperature water bath, Jintan Chenyang Electronic Instrument Factory; fresh rabbit blood, taken from the Experimental Animal Center of Jiangsu Provincial Academy of Chinese Medicine;

[0111] Citric acid, trisodium citrate, glucose, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, sodium dodecyl sulfate (SDS), etc. were all purchased from Sinopharm Chemical Reagent Co., Ltd.

[0112] 4.1.1 Prepare PBS buffer (phosphate buffer) with a pH of 7.4 using the above reagents, and prepare citric acid buffer solution with a trisodium citrate concentration of 66 mmol / L and a citric acid concentration of 44 mmol / L; prepare SDS with a mass percentage concentration of 0.1% using PBS buffer; dilute the purslane extract solution 10 times with PBS to prepare the test sample.

[0113] 4.1.2 Preparation of red blood cell suspension (RBC)

[0114] Take fresh rabbit blood and place it in a polyethylene plastic container, add citric acid buffer solution according to the ratio of rabbit blood to citric acid buffer solution of 1:9 and mix well. Immediately keep the mixed blood sample in an incubator at a temperature of 21 - 22 °C for storage. Aliquot the collected blood sample into 10 mL sterile polyethylene centrifuge tubes, centrifuge at 3000 rpm for 15 min at room temperature, carefully aspirate and discard the supernatant, and then wash the RBC twice with 2.5 times PBS buffer (3000 rpm, 15 min).

[0115] 4.1.3 Analysis of RBC integrity

[0116] Prepare a 1.0 mg / mL SDS solution with PBS. Add 20 μL, 40 μL, 60 μL, 80 μL, and 100 μL of SDS solution to EP tubes in sequence, make up to 975 μL with PBS, and quickly add 25 μL of RBC suspension. Place each tube on a shaker, incubate at 180 rpm at room temperature for 10 min, and terminate the incubation. Centrifuge at 10000 rpm for 1 min, take 200 μL of the supernatant and measure the absorbance at a wavelength of 530 nm. The results show that the semi-hemolysis amount of the freshly prepared RBC suspension is about 90 μg / mL, indicating good RBC integrity.

[0117] 4.2 Experimental method

[0118] Prepare test solution samples, add the test samples to EP tubes according to the amount in 400 μL respectively, then make up to 975 μL with PBS, and quickly add 25 μL of RBC suspension. Place each tube on a shaker, incubate at 180 rpm at room temperature for 10 min, and terminate the incubation. Centrifuge at 10000 rpm for 1 min, take 200 μL of the supernatant and measure the absorbance value at a wavelength of 530 nm. Negative control: The hemolysis rate of 975 μL PBS + 25 μL RBC is 0%; positive control: The hemolysis rate of 975 μL water + 25 μL RBC is 100%; generally, when the hemolysis rate is higher than 20%, it indicates that the sample has strong irritation.

[0119] 4.3 Experimental Results

[0120] The erythrocyte hemolysis experiment is an alternative experiment to the in vivo eye irritation test. Based on measuring the amount of hemoglobin leaked from erythrocytes, the degree of cell membrane damage is evaluated. The correlation analysis between the two shows good consistency. At different added concentrations of the purslane extract solution, the hemolysis rate is less than 4%, as shown in Figure 4 , indicating that the purslane extract solution prepared by the present invention has low irritation and high safety, and can be safely added to cosmetics.

[0121] Example 7 A Soothing and Revitalizing Milk Containing Purslane Extract

[0122] This soothing and revitalizing milk is prepared from Phase A, Phase B, Phase C, Phase D, Phase E and Phase F. Phase A, Phase B, Phase C, Phase D, Phase E and Phase F include the following raw materials:

[0123] Phase A: A mixture of cetearyl alcohol and PEG-20 stearate, cetearyl alcohol, polysorbate-60, polydimethylsiloxane, mineral oil, cyclopentasiloxane, etc.;

[0124] Phase B: Xanthan gum, trehalose, carbomer, glycerin, propylene glycol, water, polyethylene glycol-160M, titanium dioxide, betaine, a mixture of glycerol polymethacrylate and PVM / MA copolymer;

[0125] Phase C: Water and triethanolamine;

[0126] Phase D: Pure lavender hydrosol; A mixed extract of the purslane extract, chamomile extract and centella asiatica extract obtained in Example 1; Ginseng root extract, angelica extract, lemon peel oil, mint essence and phenoxyethanol;

[0127] Phase E: Menthol and butanediol;

[0128] Phase F: CI 19140 and caramel color.

[0129] Table 1 Formula of the Soothing and Revitalizing Milk Containing Purslane Extract

[0130]

[0131]

[0132] Production process: Stir and heat Phase A to 90 °C and keep warm for 10 minutes. Stir and heat Phase C to 50 °C, and combine Phase A and Phase C. Stir and heat Phase B and Phase D to 50 °C, and emulsify Phase A, Phase B, Phase C and Phase D in an emulsifying device for 30 minutes. Cool to 60 °C, add the substances of Phase E and Phase F, stir evenly and cool to 40 °C for discharging. After passing the inspection and testing, it is packaged.

[0133] Example 8 Evaluation of the Whitening Effect of a Soothing and Energizing Milk Containing Purslane Extract

[0134] 1) Test materials

[0135] Tyrosinase (Aladdin Reagent Co., Ltd.), potassium dihydrogen phosphate, disodium hydrogen phosphate (Sinopharm Chemical Reagent Co., Ltd.), L-DOPA (Abcam, USA);

[0136] 2) Experimental instruments

[0137] 722E type ultraviolet-visible spectrophotometer (Shanghai Spectrum Instruments Co., Ltd.); HH-6 type digital display constant temperature water bath (Jintan Jerry Electric Co., Ltd.); BSA224S type electronic analytical balance (Beijing Chuqi Instrument Co., Ltd.).

[0138] 3) Experimental method

[0139] Take the soothing and energizing milk prepared in Example 7 and dissolve it in phosphate buffered saline PBS (pH = 7), and dilute it into soothing and energizing milk solutions with different mass concentrations of 1.25 mg·mL -1 、2.5 mg·mL -1 、5 mg·mL -1 、10 mg·mL -1 、20 mg·mL -1 of the soothing and energizing milk solution.

[0140] Table 2. Composition of the soothing and energizing milk sample solution

[0141]

[0142] Prepare soothing and energizing milk sample solutions according to the 4 different compositions in Table 2 for the above soothing and energizing milk solutions with different mass concentrations. Specific operation: Add PBS (pH = 7), the above soothing and energizing milk solutions with different mass concentrations, and tyrosinase solution (100 U / mL) to the test tubes in sequence, respectively prepare 4 groups of soothing and energizing milk sample solutions, mix evenly, place them in a 37°C water bath for constant temperature reaction for 10 min, quickly add 0.5 mL of L-DOPA solution (1.5 mmol / L) respectively, mix evenly, react for 15 min, measure the absorbance value (A) at 475 nm, and record the absorbance values of each reaction group (groups 1-4 in Table 2) as A1, A2, A3, and A4 respectively, and set arbutin as the control. Calculate the inhibition rate of the soothing and energizing milk solution with different concentrations on tyrosinase according to the following formula:

[0143]

[0144] 4) Experimental results

[0145] Table 3. Inhibitory effects of the soothing vitality milk sample solution and arbutin on tyrosinase

[0146]

[0147] As shown in Table 3, within the test concentration range, the inhibition rates of the soothing vitality milk sample solution and arbutin on tyrosinase are proportional to the concentration. When the concentration of the sample solution is 20 mg·mL -1 -1, the inhibition rate of the soothing vitality milk sample solution on tyrosinase is as high as 76.71%, and its inhibition rate is 1.25 times that of arbutin at the same concentration, indicating that the purslane extract obtained by the extraction method of the present invention can become an effective skin care product with whitening effect and no toxic and side effects on the human body.

Claims

1. A method for preparing a purslane extract solution, comprising the following steps: The purslane is subjected to biological fermentation to obtain a fermentation broth. After the obtained fermentation broth is subjected to sterilization and protein removal treatment, solvent extraction and decolorization treatment are carried out to obtain a purslane extract solution. Among them, the purslane fermentation adopts fungal fermentation, and the fungus is Fusarium solani.

2. The preparation method according to claim 1, wherein Specifically, it includes the following steps: (1) Uniformly mix purslane and water as a fermentation substrate, add a fungus to the fermentation substrate for fermentation to obtain a fermentation broth; (2) Carry out a separation operation of sterilization and protein removal on the fermentation broth obtained in step (1) through a ceramic separation membrane to obtain a sterilized and protein-removed fermentation broth; (3) Add a solvent to the sterilized and protein-removed fermentation broth obtained in step (2) for extraction; (4) Use a hollow cellulose membrane to carry out decolorization treatment on the fermentation broth after extraction in step (3), and the purslane extract solution is obtained.

3. The preparation method according to claim 2, wherein In step (1): The mass ratio of the purslane to water is 1:3 to 1:50; and / or, Calculated by mass percentage, the mass of the fungus is 0.1% to 20% of the total mass of the purslane and water; and / or, The fermentation substrate is subjected to high-pressure sterilization treatment before fermentation; and / or, The fermentation temperature is 25°C to 35°C; and / or, The fermentation time is 12h to 36h.

4. The preparation method according to claim 3, characterized in that, In step (1): The mass ratio of the purslane to water is 1:5 to 1:25; and / or, Calculated by mass percentage, the mass of the fungus is 1% to 10% of the total mass of the purslane and water.

5. The preparation method according to claim 2, wherein In step (2): The ceramic separation membrane is selected from inorganic ceramic membranes; and / or, The pore size of the ceramic separation membrane is 0.01 to 8.0 microns; and / or, The separation temperature is 30°C to 45°C; and / or, The separation pressure is 0.02 to 0.1 MPa.

6. The preparation method according to claim 5, characterized in that, In step (2): The ceramic separation membrane is selected from at least one of alumina, zirconia, titanium oxide, and silicon oxide; and / or, The pore size of the ceramic separation membrane is 0.1 to 5.0 microns.

7. The preparation method according to claim 2, characterized in that, In step (3): The solvent is a mixed solvent of alcohol and water; and / or, Calculated by mass percentage, the solvent dosage is 5 to 10 times the total weight of the sterilized and protein-removed fermentation broth obtained in step (2); and / or, The extraction temperature is 30°C to 50°C; and / or, The extraction time is 5 to 10h; and / or, The extraction is carried out under ultrasonic conditions.

8. According to the preparation method described in claim 7, it is characterized in that The alcohol is selected from polyhydric alcohols; and / or, Calculated by mass percentage, the alcohol dosage in the solvent is 20% to 80% of the total weight of the solvent.

9. According to the preparation method described in claim 8, it is characterized in that The alcohol is selected from at least one of ethylene glycol, propylene glycol, butylene glycol, glycerol, and polyethylene glycol.

10. According to the preparation method described in claim 2, it is characterized in that The decolorization treatment in step (4) adopts a combined hollow fiber membrane module.

11. According to the preparation method described in claim 10, it is characterized in that In the described combined hollow fiber membrane module, the pore diameter of the filtration membrane is 0.05 - 0.1 μm, the filtration temperature is 30 - 40 °C, and the filtration pressure is 0.01 - 0.1 MPa; the pore diameter of the retention membrane is 0.001 - 0.02 μm, the retention temperature is 40 - 50 °C, and the retention pressure is 0.1 - 0.2 MPa.

12. The purslane extract solution obtained by the preparation method according to any one of claims 1 to 11, characterized in that, In the described Portulaca oleracea extract solution, the flavonoid content is 80 - 95 mg / L, and the alkaloid content is 20 - 30 mg / L.

13. Use of the Portulaca oleracea extract solution according to claim 12 in the preparation of skin care cosmetics or washing and care cosmetics.

14. The use according to claim 13, wherein Calculated by mass percentage, the content of the Portulaca oleracea extract solution in the skin care cosmetics is 0.5 - 10%; and / or Calculated by mass percentage, the content of the Portulaca oleracea extract solution in the washing and care cosmetics is 0.1 - 5.0%.

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