Preparation method of pleione bulbocodioides extract and product and application of pleione bulbocodioides extract

Abstract

The invention relates to a pleione bulbocodioides extract preparation method, a pleione bulbocodioides extract product and application, the preparation method comprises the following steps: crushing pleione bulbocodioides, mixing the crushed pleione bulbocodioides with water, and carrying out ultrahigh pressure treatment to obtain a first mixture; carrying out subcritical extraction on the first mixture, and filtering to obtain an extracting solution; filtering the extracting solution through a nanofiltration membrane to obtain pleione bulbocodioides filtrate; and mixing the pleione bulbocodioides filtrate with a decolorizing agent, filtering, and drying the filtrate to obtain the pleione bulbocodioides decolorizing agent. The method disclosed by the invention is high in extraction efficiency, green and environment-friendly, the obtained extract is rich in active ingredients, can effectively activate the expression of silk polyprotein and paphiopetin, promote the repair of keratinocytes and inhibit sebaceous gland cells from secreting grease, and can maximize the barrier repair and oil control effects of the extract under a specific extraction process.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and relates to a preparation method of a Herba Allii subtilis extract, and its products and applications, and specifically to a preparation method of a Herba Allii subtilis extract with skin barrier repair and oil control effects, and its products and applications. Background Art

[0002] Pleione bulbocodioides (Franch.) Rolfe is a plant of the genus Pleione in the Orchidaceae family. Its pseudobulb is mainly used as medicine. It has the effects of clearing heat and detoxifying, resolving phlegm and dispersing nodules, and has high ornamental value. It mainly contains various ingredients such as phenanthrenes, bibenzyls, anthraquinones, flavonoids, polysaccharides, etc. According to patent literature reports, Pleione bulbocodioides has good anti-inflammatory, anti-tumor, neuroprotective, antihypertensive, anti-gout and antioxidant effects.

[0003] As the body's largest organ, the skin is the first line of defense against external damage. The skin barrier is composed of lipids and natural moisturizing factors between stratum corneum cells, as well as microorganisms attached to the skin surface, the sebum membrane, various chemicals, and the skin's neuroimmune system. Filaggrin (FLG) is an important component of the stratum corneum. It is synthesized by keratinocytes and is gradually degraded by enzymes during the migration of keratinocytes. It is degraded into small molecules required by the stratum corneum, such as natural moisturizing factors, which can maintain a certain level of moisture in the skin. Loricrin (LOR) is a positively charged, highly water-insoluble alkaline protein. It is expressed in the late stage of cell differentiation and begins to appear in the epidermal granular layer. It is a high-sulfur component in the transparent keratin granules. In the stratum corneum, loricrin is cross-linked to the keratin envelope by transglutaminase, becoming an important component of the keratin envelope and playing an important role in the barrier function of the epidermis. The skin's oil secretion is mainly controlled by sebaceous gland cells and is affected by many factors such as hormone levels, genetics, environment and lifestyle habits. Appropriate oil can protect the skin barrier, but excessive or insufficient secretion can cause skin problems.

[0004] Patent CN117849235 A discloses a method for extracting the Herba Lycoris Radiatae, in which the Herba Lycoris Radiatae is extracted using 60% ethanol, and the extract is extracted sequentially using petroleum ether, dichloromethane, ethyl acetate, and n-butanol. The obtained n-butanol extract is subjected to polyamide column chromatography to enrich the succinic acid dibenzyl ester glycoside compounds. However, the use of organic reagents in this process can easily cause residues of the organic reagents in the extract, increase the allergenic risk of the extract, and also easily cause environmental pollution.

[0005] Therefore, there is an urgent need to propose a safe and environmentally friendly preparation method for the barrier repair and oil control activity of the Herba Lycoris radiata extract, so as to expand the application of the Herba Lycoris radiata extract in barrier repair and oil control products. Summary of the Invention

[0006] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a method for preparing a Herba Lycoris Radiata extract, its products and applications, and specifically to provide a method for preparing a Herba Lycoris Radiata extract having skin barrier repair and oil control effects, its products and applications.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for preparing a Herba Lycoris Radiatae extract, the method comprising:

[0009] (1) crushing the orchid and mixing it with water, and performing ultrahigh pressure treatment to obtain a first mixture;

[0010] (2) extracting the first mixture using subcritical water, and filtering to obtain an extract;

[0011] (3) filtering the extract through a nanofiltration membrane to obtain a filtrate of the orchid;

[0012] (4) Mix the filtrate of the Herba Lycoris Radiatae with a decolorizing agent, filter the filtrate, and dry the filtrate to obtain the product.

[0013] Ultrahigh pressure pretreatment of the herb, Bletilla striata, rapidly increases and maintains the pressure within a short period of time to allow the solution to reach equilibrium, followed by rapid pressure relief to allow the active ingredients to diffuse rapidly. This simple and efficient method promotes rapid dissolution of the active ingredients after cell wall destruction, improving the extraction efficiency of bletilla striata glycosides, 2-O-glucosyl bletilla striata glycosides, and yamosin III.

[0014] Subcritical water extraction is performed on the medicinal materials of Dictamnus edulis after ultrahigh pressure pretreatment. By controlling the extraction temperature and pressure, continuous extraction of water-soluble components to fat-soluble components is achieved. Compared with traditional methods, this method has short extraction time, high extraction efficiency, is green and environmentally friendly, and has no pollution, and can be used for industrial production.

[0015] During the purification process of the extract, organic membrane separation technology is used, and organic membranes with different pore sizes are used to remove highly polar polysaccharide components; the process uses a combination of activated carbon and zeolite molecular sieves for decolorization to remove low-polarity fat-soluble pigments, which can further increase the content of the main active ingredients, and decolorization can make the garlic orchid extract meet its usage scenarios in cosmetic formulas.

[0016] Preferably, the ultrahigh pressure treatment is performed at a pressure of 300-400 MPa, a temperature of 20-40° C., and a time of 10-15 min.

[0017] The pressure can be selected as 300MPa, 320MPa, 340MPa, 360MPa, 380MPa, 400MPa, etc. The temperature is 20℃, 22℃, 25℃, 28℃, 30℃, 32℃, 35℃, 38℃, 40℃, etc. The time can be selected as 10min, 10.5min, 11min, 11.5min, 12min, 12.5min, 13min, 13.5min, 14min, 14.5min, 15min, 15.5min, 16min, etc. Other specific point values ​​within the above numerical range can be selected, so they will not be repeated here.

[0018] Preferably, the ratio of the garlic orchid to water is 1g: (10-20) mL, and the specific point values ​​in (10-20) can be selected from 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be repeated here.

[0019] Preferably, the subcritical extraction is carried out at a pressure of 1-2 MPa, a temperature of 110-160° C., and a time of 20-40 min.

[0020] The pressure can be selected as 1MPa, 1.2MPa, 1.4MPa, 1.6MPa, 1.8MPa, 2MPa, etc. The temperature can be selected as 110℃, 115℃, 120℃, 125℃, 130℃, 135℃, 140℃, 145℃, 150℃, 155℃, 160℃, etc. The time can be selected as 20min, 22min, 24min, 26min, 28min, 30min, 32min, 34min, 36min, 38min, 40min, etc. Other specific point values ​​within the above numerical range can be selected, so they will not be listed here.

[0021] Preferably, the extraction in step (2) is performed 2-3 times.

[0022] Preferably, the pore size of the nanofiltration membrane is 1-10 kDa, such as 1 kDa, 2 kDa, 4 kDa, 6 kDa, 8 kDa, 10 kDa, etc. Other specific point values ​​within the above numerical range can be selected and will not be described here one by one.

[0023] Preferably, the decolorizing agent comprises activated carbon and zeolite molecular sieve in a mass ratio of (1-3):1. Specific values ​​in (1-3) may be selected from 1, 1.2, 1.5, 1.8, 2, 2.2, 2.5, 2.8, 3, etc. Other specific values ​​within the above numerical range may be selected and will not be detailed here.

[0024] Preferably, the pore size of the zeolite molecular sieve is For example

[0025] Preferably, the temperature for mixing with the decolorizing agent is 40-70° C., and the time is 30-50 min.

[0026] The temperature can be selected as 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, etc., and the time can be selected as 30min, 35min, 40min, 45min, 50min, etc. Other specific point values ​​within the above numerical range can be selected, so they will not be listed here.

[0027] Preferably, the mass ratio of the filtrate of the orchid to the decolorizer is 100: (0.1-1), and the specific point values ​​in (0.1-1) can be selected from 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be repeated here.

[0028] In a second aspect, the present invention provides a Cymbidium orchid extract prepared according to the preparation method of the Cymbidium orchid extract described in the first aspect.

[0029] In the Herba Lysimachiae extract prepared by the invention, the content of 2-O-glucosyl bletilla striata glycoside is 2.0%-2.5%, the content of bletilla striata glycoside is 6.0%-7.9%, and the content of yamidine III is 2.0%-3.0%.

[0030] In a third aspect, the present invention provides a use of the Herba Lycoris radiata extract according to the second aspect in preparing a product having skin barrier repair or oil control effects.

[0031] The preparation method of the present invention adopts the specific processes of ultra-high pressure treatment, subcritical treatment, membrane separation and decolorization treatment to prepare a specific Herba Lycoris radiata extract, which has both barrier repair and oil control effects.

[0032] Preferably, the products include cosmetics and toiletries.

[0033] In a fourth aspect, the present invention provides a use of the Herba Lycoris radiata extract according to the second aspect in the preparation of an FLG gene activator or a LOR gene activator.

[0034] According to the research results of the present invention, the extract of the Orchidaceae prepared by the present invention can activate the expression of FLG and LOR genes at the cellular level (in vitro level) and inhibit the secretion of oil in sebaceous gland cells. It is used to explore the expression process of FLG and LOR genes and the secretion of oil in sebaceous gland cells, that is, it is an application in the preparation of FLG and LOR gene activators or sebaceous gland cell oil secretion inhibitors.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The present invention provides a Herba Allii odoratis extract with enhanced barrier repair and oil-control efficacy. Specifically, the Herba Allii odoratis extract can effectively activate the expression of filaggrin and loricrin in keratinocytes, which is used to repair the skin barrier and promote scratch repair in keratinocytes. It can also effectively inhibit the secretion of sebaceous gland cells stimulated by 5α-dihydrotestosterone (DHT), showing significant oil-control efficacy. The extract obtained using a specific process has enhanced barrier repair and oil-control efficacy. This process uses only water as the extraction solvent throughout the entire process, making it safer and more environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the liquid chromatogram of 2-O-glucosyl bletilla striata glycoside standard.

[0038] Figure 2 This is the liquid chromatogram of Bletilla striata glycoside standard.

[0039] Figure 3 This is the liquid chromatogram of yamosin III standard.

[0040] Figure 4 This is a liquid chromatogram of the Herba Lycoris Radiatae extract described in Example 1.

[0041] Figure 5 This is the result of the scratch repair experiment of the blank group at 0h and 48h.

[0042] Figure 6 This is the result of the scratch repair experiment of the positive control group at 0h and 48h.

[0043] Figure 7 This is a graph showing the scratch repair test results at 0h and 48h in Example 1.

[0044] Figure 8 This is a graph showing the scratch repair test results at 0h and 48h in Example 2.

[0045] Figure 9 Graph showing the scratch repair test results at 0h and 48h in Example 3.

[0046] Figure 10 This is a graph showing the scratch repair test results at 0h and 48h in Example 4.

[0047] Figure 11 This is a graph showing the scratch repair test results at 0h and 48h in Example 5.

[0048] Figure 12 Graph showing the scratch repair test results at 0 h and 48 h in Example 6.

[0049] Figure 13This is a graph showing the scratch repair test results at 0h and 48h in Example 7.

[0050] Figure 14 This is a graph showing the scratch repair test results at 0h and 48h in Example 8.

[0051] Figure 15 Graph showing the scratch repair test results at 0 h and 48 h in Example 9.

[0052] Figure 16 Graph showing the scratch repair test results at 0 h and 48 h in Example 10.

[0053] Figure 17 This is a graph showing the scratch repair test results at 0h and 48h in Example 11.

[0054] Figure 18 This is a graph showing the scratch repair test results at 0h and 48h in Example 12.

[0055] Figure 19 This is a graph showing the scratch repair test results at 0h and 48h in comparative example 1.

[0056] Figure 20 This is a graph showing the scratch repair test results at 0h and 48h in group 2 of comparative example.

[0057] Figure 21 This is a graph showing the results of the scratch repair experiment at 0h and 48h in group 3 of comparative examples.

[0058] Figure 22 This is a graph showing the scratch repair test results of group 4 of the comparative example at 0h and 48h.

[0059] Figure 23 This is a graph showing the results of the scratch repair experiment at 0h and 48h in group 5 of the comparative example.

[0060] Figure 24 This is a graph showing the scratch repair test results for group 6 of comparative examples at 0h and 48h.

[0061] Figure 25 This is a graph showing the scratch repair test results for group 7 of comparative example 0h and 48h. DETAILED DESCRIPTION

[0062] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0063] The sources of the functional ingredients contained in the products involved in the following examples and comparative examples are as follows (only the functional ingredients are reflected, and the necessary auxiliary ingredients contained in other commercially available raw materials are not repeated):

[0064] The medicinal materials of the Herba Lycopodii involved in the following examples were purchased from Zhaotong Yunsheng Agricultural Science and Technology Development Co., Ltd.

[0065] Example 1

[0066] This embodiment provides a method for preparing a Herba Lycoris radiata extract, the preparation method comprising:

[0067] (1) The medicinal material of the Herba Lycoris radiata was crushed and mixed with water, and then placed in an ultrahigh pressure device for ultrahigh pressure pretreatment. The treatment pressure was 350 MPa, the pressure holding time was 12 min, the temperature was 25 ° C, and the material-liquid ratio of the Herba Lycoris radiata to water was 1 g: 15 mL to obtain a Herba Lycoris radiata mixed solution;

[0068] (2) The mixed solution of the orchid was transferred to a subcritical extraction kettle, the extraction temperature was 140 ° C, the extraction pressure was 1.5 MPa, the extraction time was 30 min, the extraction times were 3 times, and the extracts obtained after filtration were combined;

[0069] (3) treating the extract obtained in step (2) with a 5 kDa nanofiltration membrane to obtain a membrane filtrate;

[0070] (4) 0.5 wt% of decolorant is added to the membrane filtrate, wherein the decolorant is activated carbon and zeolite molecular sieve (zeolite molecular sieve effective pore size is 2:1) in a mass ratio. ), stirred at 50°C for 40 min, filtered, concentrated and then dried to obtain the product.

[0071] Example 2

[0072] This embodiment provides a method for preparing a Herba Lycoris radiata extract, the preparation method comprising:

[0073] (1) The medicinal material of the Herba Lycoris radiata was crushed and mixed with water, and then placed in an ultrahigh pressure device for ultrahigh pressure pretreatment at a pressure of 300 MPa, a holding time of 15 min, a temperature of 40° C., and a material-liquid ratio of the Herba Lycoris radiata to water of 1 g:20 mL to obtain a Herba Lycoris radiata mixed solution;

[0074] (2) The mixed solution of the Orchidaceae was transferred to a subcritical extraction kettle, the extraction temperature was 120 ° C, the extraction pressure was 1 MPa, the extraction time was 40 min, the number of extractions was 3, and the extracts obtained after filtration were combined.

[0075] (3) treating the extract obtained in step (2) with a 1 kDa nanofiltration membrane to obtain a membrane filtrate;

[0076] (4) 0.9 wt% of decolorizing agent is added to the membrane filtrate, wherein the decolorizing agent is activated carbon and zeolite molecular sieve (zeolite molecular sieve effective pore size is 1:1) in a mass ratio. ), stirred at 40°C for 50 min, filtered, concentrated and then dried to obtain the product.

[0077] Example 3

[0078] This embodiment provides a method for preparing a Herba Lycoris radiata extract, the preparation method comprising:

[0079] (1) The medicinal material of the Herba Lycoris radiata was crushed and mixed with water, and then placed in an ultrahigh pressure device for ultrahigh pressure pretreatment, with a treatment pressure of 400 MPa, a holding time of 10 min, a temperature of 30° C., and a material-liquid ratio of the Herba Lycoris radiata to water of 1 g:10 mL to obtain a Herba Lycoris radiata mixed solution;

[0080] (2) The mixed solution of the orchid was transferred to a subcritical extraction kettle, the extraction temperature was 160 ° C, the extraction pressure was 2 MPa, the extraction time was 20 min, the extraction number was 3 times, and the extracts obtained after filtration were combined;

[0081] (3) treating the extract obtained in step (2) with a 10 kDa nanofiltration membrane to obtain a membrane filtrate;

[0082] (4) 0.2 wt% of decolorant is added to the membrane filtrate, wherein the decolorant is activated carbon and zeolite molecular sieve (zeolite molecular sieve effective pore size is 3:1) in a mass ratio. ), stirred at 70°C for 30 min, filtered, concentrated and then dried to obtain the product.

[0083] Example 4

[0084] This embodiment provides a method for preparing a Herba Lycoris radiata extract, which differs from Example 1 only in that step (1) is "crushing the Herba Lycoris radiata medicinal materials and mixing them with water, placing them in an ultrahigh pressure equipment for ultrahigh pressure pretreatment, with a treatment pressure of 200 MPa, a holding time of 12 min, a temperature of 25°C, and a material-liquid ratio of the Herba Lycoris radiata medicinal materials to water of 1 g:15 mL to obtain a Herba Lycoris radiata mixed solution", and other operations remain unchanged.

[0085] Example 5

[0086] This embodiment provides a method for preparing a Herba Lycoris radiata extract, which differs from embodiment 1 only in that step (2) is "transferring the Herba Lycoris radiata mixed solution to a subcritical extraction kettle, with an extraction temperature of 100°C, an extraction pressure of 0.8 MPa, an extraction time of 30 min, and three extractions, and combining and filtering the obtained extracts", and other operations remain unchanged.

[0087] Example 6

[0088] This embodiment provides a method for preparing a Herba Lycoris radiata extract, which differs from embodiment 1 only in that step (2) is "transferring the Herba Lycoris radiata mixed solution to a subcritical extraction kettle, with an extraction temperature of 180° C., an extraction pressure of 2.5 MPa, an extraction time of 30 min, and three extractions, and combining and filtering the extracts obtained", and other operations remain unchanged.

[0089] Example 7

[0090] This embodiment provides a method for preparing a Herba Lycopodii extract, which differs from Example 1 only in that step (3) is "treating the extract obtained in step (2) using a 12 kDa nanofiltration membrane to obtain a membrane filtrate", and other operations remain unchanged.

[0091] Example 8

[0092] This embodiment provides a method for preparing a Herba Lycopodii extract, which differs from Example 1 only in that step (3) is "treating the extract obtained in step (2) using a 0.5 kDa nanofiltration membrane to obtain a membrane filtrate", and other operations remain unchanged.

[0093] Example 9

[0094] This embodiment provides a method for preparing a Herba Lycopersicum var. uniflorum extract, which differs from Example 1 only in that step (4) is "adding 0.5 wt % of activated carbon to the membrane filtrate, stirring at 50° C. for 40 min, filtering, concentrating, and then drying to obtain the extract", and the other operations remain unchanged.

[0095] Example 10

[0096] This embodiment provides a method for preparing a Herba Lycoris radiata extract, which differs from embodiment 1 only in that step (4) is to add 0.5 wt% of zeolite molecular sieve (pore size 0.1 wt%) to the membrane filtrate. ), stirred at 50°C for 40 min, filtered, concentrated and then dried to obtain ". The rest of the operations remain unchanged.

[0097] Example 11

[0098] This embodiment provides a method for preparing a Herba Lycoris radiata extract, which differs from embodiment 1 only in that step (4) is "adding 1.2 wt% of a decolorizing agent to the membrane filtrate, wherein the decolorizing agent is a mixture of activated carbon and zeolite molecular sieve (the effective pore size of the zeolite molecular sieve is 2:1) in a mass ratio of 1. ), stirred at 50°C for 40 min, filtered, concentrated and then dried to obtain ". The rest of the operations remain unchanged.

[0099] Example 12

[0100] This embodiment provides a method for preparing a Herba Lycoris radiata extract, which differs from embodiment 1 only in that step (4) is "adding 0.05 wt% of a decolorizing agent to the membrane filtrate, wherein the decolorizing agent is a mixture of activated carbon and zeolite molecular sieve (the effective pore size of the zeolite molecular sieve is 2:1) in a mass ratio of 0.05 wt% ... ), stirred at 50°C for 40 min, filtered, concentrated and then dried to obtain ". The rest of the operations remain unchanged.

[0101] Comparative Example 1

[0102] This comparative example provides a preparation method of a Herba Lycoris radiata extract, which differs from Example 1 only in that step (1) is "crushing the Herba Lycoris radiata medicinal material and mixing it with water, with the material-liquid ratio of the Herba Lycoris radiata medicinal material to water being 1 g:15 mL to obtain a Herba Lycoris radiata mixed solution", and other operations remain unchanged.

[0103] Comparative Example 2

[0104] This comparative example provides a preparation method of a Herba Lycoris Radiata extract, which differs from Example 1 only in that step (1) is "crushing the Herba Lycoris Radiata medicinal materials and mixing them with water, with a solid-liquid ratio of Herba Lycoris Radiata medicinal materials to water being 1 g:15 mL, ultrasonicating three times for 1 h each time at an ultrasonic power of 600 W to obtain a Herba Lycoris Radiata mixed solution", and other operations remain unchanged.

[0105] Comparative Example 3

[0106] This comparative example provides a preparation method of a Herba Lycopodii extract, the preparation method comprising:

[0107] (1) The medicinal material of the Herba Lycoris radiata was crushed and mixed with water, and then placed in an ultrahigh pressure device for ultrahigh pressure pretreatment. The treatment pressure was 350 MPa, the pressure holding time was 12 min, the temperature was 25 ° C, and the material-liquid ratio of the Herba Lycoris radiata to water was 1 g: 15 mL to obtain a Herba Lycoris radiata mixed solution;

[0108] (2) treating the mixed solution of the orchid with a 5 kDa nanofiltration membrane to obtain a membrane filtrate;

[0109] (3) 0.5 wt% of decolorant is added to the membrane filtrate, wherein the decolorant is activated carbon and zeolite molecular sieve (zeolite molecular sieve effective pore size is 2:1) in a mass ratio. ), stirred at 50°C for 40 min, filtered, concentrated and then dried to obtain the product.

[0110] Comparative Example 4

[0111] This comparative example provides a method for preparing a Herba Lycoris radiata extract, which differs from Example 1 only in that step (2) is "extracting the Herba Lycoris radiata mixed solution by heating and refluxing for 30 minutes and three times, and combining and filtering the obtained extracts", and other operations remain unchanged.

[0112] Comparative Example 5

[0113] This comparative example provides a preparation method of a Herba Lycopersicum var. uniflorum extract, which differs from Example 1 only in that step (2) is "ultrasonic extraction of the Herba Lycopersicum var. uniflorum mixed solution is performed 3 times, each time for 30 min, with an ultrasonic power of 600 W, and the extracts obtained after filtration are combined", and other operations remain unchanged.

[0114] Comparative Example 6

[0115] This comparative example provides a preparation method of a Herba Lycopersicum var. uniflorum extract, which differs from Example 1 only in that step (2) is "extracting the Herba Lycopersicum var. uniflorum mixed solution by microwave, with a microwave power of 400 W, an extraction temperature of 70° C., three extractions, each for 30 min, and combining and filtering the obtained extracts", and other operations remain unchanged.

[0116] Comparative Example 7

[0117] This comparative example provides a method for preparing the extract of Orchidaceae, which differs from Example 1 only in that step (4) is "concentrating the membrane filtrate and then drying it to obtain the extract", and other operations remain unchanged.

[0118] Test Example 1

[0119] The following determinations of the contents of bletilla striata glycoside, 2-O-glucosyl bletilla striata glycoside and yamidine III were performed using a liquid phase method, as follows:

[0120] Standards of bletilla striata glycoside, 2-O-glucosyl bletilla striata glycoside and yaminol III were purchased from Wuhan Tianzhi Biotechnology Co., Ltd. The detection conditions were octadecyl bonded silica gel as the filler column, acetonitrile-0.1% phosphoric acid water (5:95) as the mobile phase, the flow rate was 0.6 mL / min, the detection wavelength was 203 nm, and the column temperature was 30°C. The liquid chromatograms of the standards of 2-O-glucosyl bletilla striata glycoside, bletilla striata glycoside and yaminol III are shown in Figure 2. Figure 1 、 Figure 2 and Figure 3 As shown, the liquid chromatogram of the Herba Lycoris radiata extract prepared in Example 1 is as shown in FIG. Figure 4 shown.

[0121] The contents of bletilla striata glycoside, 2-O-glucosyl bletilla striata glycoside and yamidine III were determined for the extracts of the Orchidaceae obtained in Examples 1-12 and Comparative Examples 1-7. The results are shown in Table 1:

[0122] Table 1

[0123]

[0124] As can be seen from the data in Table 1, in the extract of the Orchidaceae obtained under the preparation process of the present invention, the proportions of 2-O-glucosyl bletilla striata glycoside, bletilla striata glycoside and yamidine III are relatively high. It can be seen from Examples 1-3 and Examples 4-8 that by controlling the pressure of ultrahigh pressure pretreatment, the temperature and pressure of subcritical extraction and the pore size of the organic membrane, different enrichments of the three components can be achieved;

[0125] It can be seen from Example 1, Examples 9-12 and Comparative Example 7 that the type and amount of the decolorizing agent will affect the content of 2-O-glucosyl bletilla striata glycoside, bletilla striata glycoside and yamin III. Zeolite molecular sieve can achieve the effect of synergistic removal of small molecular pigments with activated carbon, further increasing the content of the components. Excessive addition of the decolorizing agent will cause excessive adsorption of small polar components and cause component loss. If the amount is too little, the decolorization is insufficient, reducing the decolorization effect.

[0126] It can be seen from Example 1 and Comparative Examples 1-6 that the extraction process of the combination of ultra-high pressure pre-treatment and subcritical extraction can dissolve and break the wall of the three components to the greatest extent, achieve the effect of enrichment, and further affect the barrier repair and oil control effects.

[0127] Test Example 2

[0128] Barrier repair efficacy test

[0129] Test method:

[0130] HaCat human keratinocytes were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences. Fluorescence quantitative PCR was used to determine the relative mRNA expression of FLG and LOR barrier-related genes. HaCaT cells in the logarithmic growth phase with good morphology were seeded in 24-well plates at a seeding density of 1 × 10 5 Cells / well were incubated in an incubator for 24 hours. A blank control group (untreated cells) and a sample group (the extract of Orchidaceae obtained in Examples 1-12 and Comparative Examples 1-7, with an extract concentration of 120 μg / mL) were set up, with three replicates for each concentration. The treated cells were then incubated in a 37°C, 5% CO2 incubator for another 24 hours.

[0131] After the culture, RNA was extracted and its content was determined. Reverse transcription was performed to obtain cDNA, and fluorescence quantitative PCR was performed. The experimental data were processed using GraphPad Prism10 software. The gene expression upregulation rate was calculated as follows:

[0132]

[0133] The results of up-regulation of FLG and LOR gene expression are shown in Table 2 .

[0134] Table 2

[0135] Group FLG gene expression upregulation rate LOR gene expression upregulation rate Example 1 114.21％ 243.63％ Example 2 111.36％ 227.47％ Example 3 107.34％ 225.19％ Example 4 42.10％ 60.01％ Example 5 50.31％ 57.10％ Example 6 55.29％ 65.37％ Example 7 35.71％ 53.37％ Example 8 23.01％ 39.72％ Example 9 57.34％ 70.46％ Example 10 55.36％ 60.27％ Example 11 40.21％ 45.29％ Example 12 39.01％ 46.17％ Comparative Example 1 15.29％ 18.46％ Comparative Example 2 20.78％ 21.53％ Comparative Example 3 4.19％ 8.17％ Comparative Example 4 18.60％ 25.71％ Comparative Example 5 12.06％ 19.37％ Comparative Example 6 23.17％ 28.07％ Comparative Example 7 29.17％ 42.42％

[0136] It can be seen from the data in Table 2 that the extract of the orchid obtained by the specific process of the present invention has an excellent barrier repair effect. It can be seen from Examples 1-3 and Examples 4-6 that controlling the temperature and pressure of ultra-high pressure pretreatment and subcritical extraction can effectively control the content of 2-O-glucosyl bletilla striata glycoside, bletilla striata glycoside, and yam indole III, and at the same time maximize the barrier repair effect; it can be seen from Examples 1-3 and Examples 7-8 that by controlling the pore size of the organic membrane, the enrichment of small molecule components 2-O-glucosyl bletilla striata glycoside, bletilla striata glycoside, and yam indole III can be achieved. If the pore size of the organic membrane is too large, impurities such as polysaccharides will be introduced, and a certain barrier effect can be exerted, but the effect is not optimal; it can be seen from Examples 1-3, Examples 9-12 and Comparative Example 7 that the type and amount of activated carbon will affect the barrier effect. Only when the activated carbon and zeolite molecular sieve are combined and the addition amount is within a certain range can the barrier effect be maximized; at the same time, the combination of ultra-high pressure and subcritical extraction can maximize the barrier effect of the extract to the greatest extent.

[0137] Test Example 3

[0138] Scratch repair efficacy test

[0139] Test method: HaCat human keratinocytes are derived from the Shanghai Cell Bank of the Chinese Academy of Sciences. A cell scratch plug was placed vertically in the middle of each 12-well cell culture plate seeded with cells, 70 μL of cell suspension was added to the two chambers of each plug, 200 μL of cell suspension was used to moisten the area around the plug, and the cells were cultured at 5% CO2 and 37°C for the required time; the plug was gently removed with sterilized tweezers, the cells were gently rinsed one to two times with 1 mL of PBS solution and all PBS was aspirated; 1 mL of the test substance of appropriate concentration was accurately added, a blank control group (serum-free DMEM culture medium), a positive control group (epidermal growth factor EGF solution concentration of 10,000 IU / mL) and different groups of sample groups (concentration of 120 μg / mL) were set up, the cell growth status at the scratch site at 0 hours and 48 hours was observed under a microscope, and photographed and recorded respectively. The experimental results of each group are shown as follows: Figure 5-25 As shown, the healing area was calculated using ImageJ software.

[0140] Healing rate calculation formula:

[0141]

[0142] The cell repair efficacy of the extracts of the Herba Lycoris radiata obtained in Examples 1-12 and Comparative Examples 1-7 was evaluated, and the results are shown in Table 3:

[0143] Table 3

[0144]

[0145]

[0146] As can be seen from the data in Table 3, the extract of the Orchidaceae extract obtained by the specific process of the present invention has an excellent scratch repair effect. It can be seen from Examples 1-3 and Examples 4-6 that controlling the temperature and pressure of ultrahigh pressure pretreatment and subcritical extraction can fully dissolve 2-O-glucosyl bletilla striata glycoside, bletilla striata glycoside, and yamin III, thereby maximizing the scratch repair effect. It can be seen from Examples 1-3 and Examples 7-8 that by controlling the pore size of the organic membrane, the small molecule components 2-O-glucosyl bletilla striata glycoside, bletilla striata glycoside, and yamin III can be fully dissolved. The enrichment of organic membranes, the large pore size of which will lead to the introduction of impurities such as polysaccharides, can also play a certain scratch repair effect, but the effect is not optimal; it can be seen from Examples 1-3, Examples 9-12 and Comparative Example 7 that the type and amount of activated carbon will affect the repair effect. Only when the activated carbon and zeolite molecular sieve are combined and the addition amount is within a certain range can the scratch repair effect be maximized; at the same time, the use of different pretreatment and extraction methods will also affect the scratch repair effect; at the same time, the combination of ultrahigh pressure and subcritical extraction can maximize the repair effect of the extract.

[0147] Test Example 4

[0148] Oil control efficacy test

[0149] Test method:

[0150] The SZ95 sebaceous gland cells used in this experiment were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences. 5α-dihydrotestosterone (DHT) was used as an induction condition for lipid droplets, and isotretinoin was used as a positive marker. The oil-control efficacy of the test samples was evaluated by examining their inhibitory effects on sebum secretion.

[0151] Test method: press 1×10 5 Cells were seeded into 24-well plates at a seeding density of 100 cells / well and incubated overnight in an incubator (37°C, 5% CO2). When the cell plating rate in the 24-well plate reached 40% to 60%, the cells were divided into groups for drug administration: a blank group (cultured with culture medium only, without any treatment), a negative control group (treated with 10% DMSO solution), a positive control group (treated with 3 μg / mL isotretinoin), and a sample group (treated with 120 μg / mL dissolved in 10% DMSO). All groups, except the blank group, were induced with DHT. Three replicates were set up for each group and the cells were cultured in an incubator (37°C, 5% CO2) for 24 hours. The culture medium was discarded and the cells were rinsed with PBS. After fixation, cells were stained with Nile red for 15 minutes, rinsed, and recorded using a fluorescence microscope. Fluorescence intensity was quantified using Image Pro Plus software.

[0152] Grease inhibition rate calculation formula:

[0153]

[0154] The cell oil control efficacy of the Herba Lycoris radiata extracts obtained in Examples 1-12 and Comparative Examples 1-7 was evaluated, and the results are shown in Table 4:

[0155] Table 4

[0156]

[0157]

[0158] It can be seen from the data in Table 4 that the Herba Lysimachiae extract obtained by the specific process of the present invention has an excellent oil-control effect. It can be seen from Examples 1-3 and Examples 4-6 that controlling the temperature and pressure of ultrahigh pressure pretreatment and subcritical extraction can fully dissolve 2-O-glucosyl bletilla striata glycoside, bletilla striata glycoside, and yamidine III, thereby maximizing the oil-control effect. It can be seen from Examples 1-3 and Examples 7-12 that different organic membrane pore sizes and the combination and addition amount of activated carbon will also affect the oil-control effect of the extract. At the same time, different ultrahigh pressure pretreatment and extraction methods will also affect the oil-control effect.

[0159] The applicant declares that the present invention illustrates the preparation method of a Herba Lycoris radiata extract, its product, and application through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials of the product of the present invention, addition of auxiliary ingredients, selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

[0160] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0161] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for preparing a Herba Lycoris Radiatae extract, characterized in that: The preparation method comprises: (1) crushing the orchid and mixing it with water, and performing ultrahigh pressure treatment to obtain a first mixture; (2) extracting the first mixture using subcritical water, and filtering to obtain an extract; (3) filtering the extract through a nanofiltration membrane to obtain a filtrate of the orchid; (4) Mix the filtrate of the Herba Lycoris Radiatae with a decolorizing agent, filter the filtrate, and dry the filtrate to obtain the product.

2. The method for preparing the Herba Lycoris Radiatae extract according to claim 1, wherein: The ultra-high pressure treatment is performed under a pressure of 300-400 MPa, a temperature of 20-40° C., and a time of 10-15 minutes.

3. The method for preparing the Herba Lycoris Radiatae extract according to claim 1, wherein: The ratio of the monocotyledon to water is 1 g: (10-20) mL.

4. The method for preparing the Herba Lycoris Radiatae extract according to claim 1, wherein: The subcritical extraction pressure is 1-2 MPa, the temperature is 110-160° C., and the time is 20-40 min.

5. The method for preparing the Herba Lycoris Radiatae extract according to claim 1, wherein: The pore size of the nanofiltration membrane is 1-10 kDa.

6. The method for preparing the Herba Lycoris radiata extract according to claim 1, wherein: The decolorizing agent comprises activated carbon and zeolite molecular sieve in a mass ratio of (1-3):

1.

7. The method for preparing the Herba Lycoris radiata extract according to claim 1, wherein: The mass ratio of the filtrate of the orchid to the decolorizing agent is 100:(0.1-1).

8. The Herba Lycoris radiata extract prepared by the method for preparing the Herba Lycoris radiata extract according to any one of claims 1 to 7.

9. Use of the Herba Lycoris Radiatae extract according to claim 8 in preparing products having skin barrier repairing or oil control effects.

10. Use of the Orchidaceae extract with skin barrier repairing or oil-controlling effects according to claim 9 in the preparation of an FLG gene activator or a LOR gene activator.

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