Use of a plant extract composition for the preparation of a preparation for promoting the expression of skin antibacterial peptides and cosmetics

Abstract

The application relates to an application of a plant extraction composition in preparing a preparation for promoting expression of skin antibacterial peptides and cosmetics. The plant extraction composition comprises: a Coptis chinensis and Magnolia officinalis extraction: extracted from a mixture of Coptis chinensis and Magnolia officinalis as an extraction raw material; and a Saussurea costus extraction: extracted from Saussurea costus as an extraction raw material.

Description

Technical Field

[0001] This application relates to the field of cosmetic technology, specifically to the application of a plant extract composition in the preparation of formulations that promote the expression of antimicrobial peptides in the skin and to cosmetics. Background Technology

[0002] Antimicrobial peptides are a class of peptides with diverse activities produced by the body's biological defense system. They can resist the invasion of foreign pathogens and are an important component of the body's own immunity. Initially, it was only discovered that these active polypeptides could exert a broad inhibitory effect on bacteria. Later, it was found that some antimicrobial peptides could also exhibit killing effects against fungi, protozoa, tumor cells, and viruses. Antimicrobial peptides have a broad antibacterial spectrum and multiple targets, making it difficult for microorganisms to develop resistance to them. Therefore, antimicrobial peptides hold promise as alternatives to traditional antibiotics. In addition to resisting foreign pathogens, the immunomodulatory function of antimicrobial peptides is receiving increasing attention. Antimicrobial peptides have small molecular weights and weak immunogenicity, making them important mediators of innate immunity. Antimicrobial peptides can inhibit pro-inflammatory cytokines, prevent endotoxemia caused by bacterial products, and inhibit the activity of certain enzymes that inhibit wound healing, thus preventing wound deterioration.

[0003] Defensins are among the most studied antimicrobial peptides, broadly classified into α-defensins and β-defensins based on their structural characteristics. β-defensins are polypeptides that can be expressed in mucosal epithelial cells such as skin, lungs, organs, kidneys, and reproductive organs. To date, six human β-defensins have been isolated and identified: human β-defensin-1 (hBD-1), human β-defensin-2 (hBD-2), human β-defensin-3 (hBD-3), human β-defensin-4 (hBD-4), human β-defensin-5 (hBD-5), and human β-defensin-6 (hBD-6).

[0004] Furthermore, LL-37 antimicrobial peptide possesses an α-helical structure and exhibits broad-spectrum antimicrobial activity and inflammatory regulatory functions in vivo. Previous studies have shown that when the skin experiences physiological damage or infection, it secretes defensins and LL-37 as antimicrobial peptides to induce antimicrobial activity and a systemic immune response, particularly inducing the differentiation and proliferation of epidermal glial cells, thereby promoting wound repair.

[0005] At low concentrations, hBD-2 can attract monocytes, macrophages, and neutrophils to migrate to the mucous membranes and skin invading by pathogenic microorganisms via the chemokine receptor CCR2, enhancing local defense function. hBD-3 plays an important role in maintaining skin barrier homeostasis. hBD-3 can enhance skin barrier function by directly increasing the expression of Claudin in tight junction proteins or enhancing the expression of atypical Rac1-activated protein kinase C, glycogen synthase kinase-3, and phosphatidylinositol 3-kinase, which are involved in the regulation of skin barrier function. β-defensins have significant antimicrobial effects and can effectively resist the invasion of bacteria, fungi, and viruses, serving as the first natural line of defense for humans. hBD-3 alleviates IL-4 and IL-13-mediated tight junction (TJ) barrier damage through keratinocyte autophagy activation (involving aryl hydrocarbon receptor (AhR) signaling). LL-37 is a regulator of keratinocyte apoptosis and can regulate cytokines and chemokines to enhance its antifungal effects. LL-37 can act as both an inflammatory inducer and an anti-inflammatory mediator, exerting various immunomodulatory effects. LL-37 can promote the production of the anti-inflammatory factor ILA-IRA and inhibit IL-32-induced inflammatory responses by inducing MKP-1 phosphorylation. Exogenous LL-37, without direct bactericidal activity, can induce a pro-inflammatory response, prompting neutrophils to clear *Pseudomonas aeruginosa* from the lungs. LL-37 exhibits chemotactic activity against various immune cells: it can chemotactically attract neutrophils, eosinophils, monocytes, and T cells. LL-37 can induce macrophage differentiation into M1-type cells with pro-inflammatory characteristics; after in vitro induction with LL-37, dendritic cells show significant upregulation of phagocytic capacity, promoting Th1 responses and releasing related inflammatory factors.

[0006] References:

[0007] (1) Zhou Hai, Wei Li, Ma Ping. Research progress on human β-defensin-2 [J]. Chongqing Medical Journal, 2014, 43(11):1386-1387.

[0008] (2) Zhao Xinfei, Tian Yan, He Congfen, et al. Research progress on endogenous antimicrobial peptides in the skin, human β-defensin and LL-37 [J]. Journal of Clinical Dermatology, 2023, 52(03):190-192. DOI:10.16761 / j.cnki.1000-4963.2023.03.020.

[0009] (3) Zhao Yadong, Qu Zhiguo, Li Lizhong. Research progress on human β-defensin [J]. Inner Mongolia Journal of Traditional Chinese Medicine, 2012, 31(05):101-102. DOI:10.16040 / j.cnki.cn15-1101.2012.05.087.

[0010] (4)Peng G, Tsukamoto S, Ikutama R, Nguyen HLT, Umehara Y, Trujillo-PaezJV, Yue H, Takahashi M, Ogawa T, Kishi R, Tominaga M, Takamori K, Kitaura J, Kageyama S, Komatsu M, Okumura K, Ogawa H, Ikeda S, Niyonsaba F.Humanβ-defensin-3attenuatesatopic dermatitis-like inflammation through autophagy activation and the arylhydrocarbon receptor signaling pathway.J Clin Invest.2022Sep 1;132(17):e156501.doi:10.1172 / JCI156501.PMID:35834333;PMCID:PMC9435650. Summary of the Invention

[0011] To address the problems existing in the prior art, this application provides an application of a plant extract composition in the preparation of an agent that promotes the expression of antimicrobial peptides in the skin, and a cosmetic product.

[0012] In a first aspect, embodiments of this application provide the use of a plant extract composition in the preparation of an agent that promotes the expression of antimicrobial peptides in the skin, the plant extract composition comprising:

[0013] Coptis chinensis and Magnolia officinalis extract: obtained by extraction from a mixture of Coptis chinensis and Magnolia officinalis;

[0014] Costus root extract: obtained by extracting Costus root from Costus root.

[0015] In some embodiments, the promotion of skin antimicrobial peptide expression is: promoting the expression of hBD-2, hBD-3 and LL-37 genes at the mRNA level.

[0016] In some embodiments, the mass ratio of the extraction raw materials Coptis chinensis, Magnolia officinalis and Aucklandia lappa is 1:(0.8-1.2):(0.8-1.2).

[0017] In some embodiments, the mass ratio of the extraction raw materials Coptis chinensis, Magnolia officinalis and Aucklandia lappa is 1:1:1.

[0018] In some embodiments, the plant extract composition is prepared by the following steps:

[0019] Coptis chinensis and Magnolia officinalis were mixed and subjected to subcritical extraction to obtain the first extract;

[0020] The first extract was cooled and crystallized, then dried to obtain the first extract product;

[0021] A mixture of costus root and organic alkali was extracted to obtain a second extract.

[0022] The second extract was adsorbed onto a resin and then desorbed with a polyol aqueous solution to obtain the desorbed solution.

[0023] The desorption solution is mixed with the first extract to obtain the plant extract composition.

[0024] In some embodiments, the subcritical extraction of the mixture of Coptis chinensis and Magnolia officinalis to obtain the first extract includes:

[0025] Coptis chinensis and Magnolia officinalis were mixed and pulverized, then placed in a subcritical extraction tank. Deionized water was added for high-temperature and high-pressure extraction, and the mixture was filtered to obtain the first extract.

[0026] In some embodiments, the high-temperature and high-pressure extraction is performed at an extraction temperature of 105°C to 120°C and an extraction pressure of 1.3 to 1.5 standard atmospheres.

[0027] In some embodiments, the extraction of the second extract by mixing costus root and organic alkali includes:

[0028] The costus root powder was crushed, and then extracted with an organic alkali and deionized water at low temperature. After extraction, the mixture was filtered to obtain a second extract.

[0029] In some embodiments, the extraction temperature for the low-temperature extraction is 40°C to 55°C.

[0030] In some embodiments, the organic base includes one or more of betaine, L-carnitine, and stachydrine.

[0031] In some embodiments, the mass ratio of the costus root to the organic base is 1:(0.08-0.12).

[0032] In some embodiments, the resin used for resin adsorption includes one or both of polyamide resin and serpentine resin.

[0033] In some embodiments, the polyol includes one or more of glycerol, 1,3-propanediol, 1,3-butanediol, 1,2-propanediol, dipropylene glycol, and methylpropanediol.

[0034] In some embodiments, the mass concentration of the polyol aqueous solution is 55% to 65%.

[0035] In some embodiments, the mass ratio of the costus root to the polyol aqueous solution is 1:(11.76-16.66).

[0036] Secondly, embodiments of this application provide an formulation for promoting the expression of antimicrobial peptides in the skin, comprising a plant extract composition, wherein the plant extract composition comprises:

[0037] Coptis chinensis and Magnolia officinalis extract: obtained by extraction from a mixture of Coptis chinensis and Magnolia officinalis;

[0038] Costus root extract: obtained by extracting Costus root from Costus root.

[0039] in,

[0040] The method of administration for the formulation that promotes the expression of antimicrobial peptides in the skin is in vitro and / or in vivo.

[0041] Thirdly, embodiments of this application provide a cosmetic product, including an agent that promotes the expression of antimicrobial peptides in the skin according to embodiments of the second aspect of this application. Attached Figure Description

[0042] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments of this application will be briefly introduced below. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0043] Figure 1 The results of the HaCaT cytotoxicity experiment are shown in the embodiments of this application.

[0044] Figure 2-1 These are the experimental results of promoting hBD-2 expression at the mRNA level in the embodiments of this application.

[0045] Figure 2-2 These are the experimental results of promoting hBD-3 expression at the mRNA level in the embodiments of this application.

[0046] Figure 3 The results of the antimicrobial peptide LL-37 expression experiment at the mRNA level in the embodiments of this application are shown.

[0047] Figure 4 The results of the clinical acne periacne redness reduction test in the embodiments of this application are shown.

[0048] Figure 5 This is the result of a clinical acne volume change test in the embodiments of this application.

[0049] Figure 6 This is the result of a clinical skin oil content change test in the embodiments of this application.

[0050] Figure 7 The results of the clinical skin percutaneous water loss test in the embodiments of this application are shown.

[0051] Figure 8 This is a VISA image captured during clinical acne treatment in this application embodiment (the darker the color, the higher the relative height of the acne). Detailed Implementation

[0052] The features and exemplary embodiments of various aspects of this application will now be described in detail. Numerous specific details are set forth in the following detailed description to provide a comprehensive understanding of this application. However, it will be apparent to those skilled in the art that this application can be implemented without requiring some of these specific details. The following description of embodiments is merely intended to provide a better understanding of this application by illustrating examples. In the following description, at least some well-known structures and techniques are not shown to avoid unnecessarily obscuring the application; and, for clarity, the dimensions of some structures may be exaggerated. Furthermore, the features, structures, or characteristics described below can be combined in any suitable manner in one or more embodiments.

[0053] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. In the description of this application, it should be noted that, unless otherwise stated, "a plurality of" means two or more. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising..." does not exclude the presence of additional identical elements in the process, method, article, or apparatus that includes said element.

[0054] An embodiment of the first aspect of this application provides the use of a plant extract composition in the preparation of an agent that promotes the expression of antimicrobial peptides in the skin, wherein the plant extract composition comprises:

[0055] Coptis chinensis and Magnolia officinalis extract: obtained by extraction from a mixture of Coptis chinensis and Magnolia officinalis;

[0056] Costus root extract: obtained by extracting Costus root from Costus root.

[0057] In some embodiments, promoting the expression of skin antimicrobial peptides means promoting the expression of hBD-2, hBD-3, and LL-37 genes at the mRNA level.

[0058] The inventors discovered that a plant extract composition derived from Coptis chinensis, Magnolia officinalis, and Aucklandia lappa can effectively promote the expression of antimicrobial peptides in the skin, particularly human β-defensin-2 (hBD-2), human β-defensin-3 (hBD-3), and LL-37 antimicrobial peptides. Furthermore, the plant extract composition provided in this application is prepared and formulated using traditional Chinese medicine components, resulting in low irritation and fewer side effects. It can reduce the incidence of infections and complications caused by long-term hormone use, while traditional Chinese medicine has a good regulatory effect on the body's immune function and can repair the skin barrier.

[0059] In some embodiments, the mass ratio of the extraction raw materials Coptis chinensis, Magnolia officinalis and Aucklandia lappa is 1:(0.8-1.2):(0.8-1.2).

[0060] In some embodiments, the mass ratio of the raw materials Coptis chinensis, Magnolia officinalis and Aucklandia lappa is 1:1:1.

[0061] By limiting the proportions of each extractant within the above range, the resulting extract can have better compatibility, further enhancing the efficacy of the plant extract composition and enabling it to better promote the expression of antimicrobial peptides in the skin.

[0062] In some implementations, the plant extract composition is prepared by the following steps:

[0063] S100, take Coptis chinensis and Magnolia officinalis and perform subcritical extraction to obtain the first extract;

[0064] S200, the first extract is cooled and crystallized, then dried to obtain the first extract product;

[0065] S300, take Sichuan costus root and organic alkali and extract them to obtain the second extract;

[0066] S400, the second extract is adsorbed by resin and then desorbed by a polyol aqueous solution to obtain an eluent;

[0067] S500, the desorption solution is mixed with the first extract to obtain the plant extract composition.

[0068] In some embodiments, the mass ratio of Coptis chinensis, Magnolia officinalis and Aucklandia lappa is 1:(0.8-1.2):(0.8-1.2); in some embodiments, the mass ratio of the extraction raw materials Coptis chinensis, Magnolia officinalis and Aucklandia lappa is 1:1:1.

[0069] By limiting the mass ratio of Coptis chinensis, Magnolia officinalis, and Aucklandia lappa to the above range, the combined extraction of Coptis chinensis and Magnolia officinalis yields better results, which can further improve the purity and extraction rate of the extract. Furthermore, when the components are combined according to the above ratio, they can have a better synergistic effect, which is beneficial to improving the promotion of antimicrobial peptide expression in the skin.

[0070] In some embodiments, step S100 includes:

[0071] Coptis chinensis and Magnolia officinalis were mixed and pulverized, then put into a subcritical extraction tank, deionized water was added for high-temperature and high-pressure extraction, and the first extract was obtained by filtration.

[0072] Coptis chinensis contains a relatively high amount of alkaloids such as berberine, coptisine, methylcoptisine, and palmatine, while Magnolia officinalis contains a relatively high amount of the active ingredient magnolol. When Coptis chinensis and Magnolia officinalis are pulverized and mixed for extraction, the alkaloids in Coptis chinensis can act as ion donors, and the magnolol in Magnolia officinalis can act as ion acceptors. The two can co-crystallize to form supramolecular compound crystals, thereby improving the extraction efficiency and purity of Coptis chinensis and Magnolia officinalis, resulting in a high-purity crystalline extract. After filtering and drying the obtained crystals, the first extract product is obtained.

[0073] In some embodiments, the extraction temperature of high-temperature and high-pressure extraction can be 105℃ to 120℃, and the extraction pressure can be 1.3 to 1.5 standard atmospheres. In this application, the standard atmosphere is 101.325 kPa.

[0074] Limiting the extraction temperature and pressure of Coptis chinensis and Magnolia officinalis to the above range can improve extraction efficiency and effectiveness, and increase the concentration of active ingredients in the extract.

[0075] In some embodiments, step S300 includes: taking Sichuan costus root powder, adding organic alkali and deionized water for low-temperature extraction, and filtering after extraction to obtain a second extract.

[0076] Costus root contains a significant amount of flavonoid active ingredients. When Costus root is pulverized and mixed with an organic base for extraction, the flavonoids in the Costus root act as ion acceptors, while the organic base acts as ion donors. The two can co-crystallize to form supramolecular compounds, thereby extracting the active ingredients from the Costus root and improving the extraction efficiency and product purity. During subsequent resin adsorption, since the adsorption resin does not adsorb organic bases, the organic bases in the second extract can be separated from the Costus root extract.

[0077] In some embodiments, the extraction temperature for low-temperature extraction can be between 40°C and 55°C. Within this temperature range, the purity and extraction efficiency of the extracted product can be improved.

[0078] In some embodiments, the organic base includes one or more combinations of betaine, L-carnitine, and stachydrine.

[0079] In some embodiments, the mass ratio of costus root to organic alkali can be 1:(0.08-0.12).

[0080] The aforementioned organic bases can form supramolecular compound crystals with the flavonoid active ingredients in Aucklandia lappa, which can further improve the extraction efficiency and the purity of the extracted product.

[0081] The second extract can be purified by resin adsorption to remove impurities such as organic bases. The resin used for adsorption can be one or more of polyamide resin and serpentine resin. These adsorption resins can effectively adsorb flavonoid active components in the second extract, while adsorbing little or no organic bases. Therefore, they can be used for the adsorption and separation of active components and organic bases in the second extract.

[0082] The active ingredients adsorbed by the adsorption resin are eluted with polyols. The polyols may include one or more of glycerol, 1,3-propanediol, 1,3-butanediol, 1,2-propanediol, dipropylene glycol, and methylpropanediol.

[0083] Polyols can act as solvents in plant extract compositions, enabling uniform dispersion of the components and improving the contact of the extract components on the skin surface.

[0084] In some embodiments, the mass concentration of the polyol aqueous solution can be 55% to 65%.

[0085] An embodiment of the second aspect of this application provides an formulation for promoting the expression of antimicrobial peptides in the skin, comprising a plant extract composition, the plant extract composition comprising:

[0086] Coptis and Magnolia officinalis extract: obtained by extraction from a mixture of Coptis chinensis and Magnolia officinalis;

[0087] Costus root extract: obtained by extracting Costus root from Costus root.

[0088] in,

[0089] The formulations that promote the expression of antimicrobial peptides in the skin are administered in vitro and / or in vivo.

[0090] An embodiment of the third aspect of this application provides a cosmetic product, including the formulation of the second aspect of this application that promotes the expression of antimicrobial peptides in the skin.

[0091] Example

[0092] The following specific embodiments illustrate the present invention. It should be noted that the embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0093] Example 1

[0094] A plant extract composition is prepared by the following method:

[0095] S100, take 7.5 kg of Coptis chinensis and 7.5 kg of Magnolia officinalis, mix and crush them, pass them through a 60-mesh sieve and put them into a subcritical extraction tank, add 100 kg of deionized water, extract at 125℃ and 1.5 times the standard atmospheric pressure for 2 hours, cool the extract to below 95℃, filter it with a 0.45 μm nylon membrane, let the filtrate crystallize for 24 hours, and collect the crystals by vacuum filtration;

[0096] S200: Take 7.5 kg of Sichuan costus root powder, pass it through a 60-mesh sieve, add 100 kg of deionized water and 0.75 kg of L-carnitine, extract at 50℃ for 2 h, filter the extract, collect the filtrate, add polyamide resin for static adsorption, filter with a 500-mesh filter bag to collect the resin, elute the resin with 100 kg of 60% 1,3-propanediol reflux solution for 1 h at 55℃, filter the eluent with a 500-mesh filter bag, and collect the filtrate.

[0097] S300: Add the crystals collected in step S100 to the filtrate collected in step S200, stir and dissolve at 60°C for 1 hour to form a transparent solution, filter with a 0.45μm nylon membrane to obtain the plant extract composition.

[0098] Example 2

[0099] A plant extract composition is prepared by the following method:

[0100] S100, take 6 kg of Coptis chinensis and 6 kg of Magnolia officinalis, mix and crush them, pass them through a 60-mesh sieve and put them into a subcritical extraction tank, add 100 kg of deionized water, extract at 125℃ and 1.5 times the standard atmospheric pressure for 2 hours, cool the extract to below 95℃, filter it with a 0.45 μm nylon membrane, let the filtrate crystallize for 24 hours, and collect the crystals by suction filtration.

[0101] S200, take 6kg of Sichuan costus root powder, pass through a 60-mesh sieve, add 100kg of deionized water and 0.65kg of L-carnitine, extract at 50℃ for 2h, filter the extract, collect the filtrate, add polyamide resin for static adsorption, filter with a 500-mesh filter bag to collect the resin, elute the resin with 100kg of 60% 1,3-propanediol aqueous solution for 1h at an elution temperature of 55℃, filter the eluent with a 500-mesh filter bag, and collect the filtrate;

[0102] S300: Add the crystals collected in step S100 to the filtrate collected in step S200, stir and dissolve at 60°C for 1 hour to form a transparent solution, filter with a 0.45μm nylon membrane to obtain the plant extract composition.

[0103] Example 3

[0104] A plant extract composition is prepared by the following method:

[0105] S100, take 8.5 kg of Coptis chinensis and 8.5 kg of Magnolia officinalis, mix and crush them, pass them through a 60-mesh sieve and put them into a subcritical extraction tank, add 100 kg of deionized water, extract at 125℃ and 1.5 times the standard atmospheric pressure for 2 hours, cool the extract to below 95℃, filter it with a 0.45 μm nylon membrane, let the filtrate crystallize for 24 hours, and collect the crystals by vacuum filtration;

[0106] S200: Take 8.5 kg of Sichuan costus root powder, pass it through a 60-mesh sieve, add 100 kg of deionized water and 0.85 kg of L-carnitine, extract at 50℃ for 2 h, filter the extract, collect the filtrate, add polyamide resin for static adsorption, filter with a 500-mesh filter bag to collect the resin, elute the resin with 100 kg of 60% 1,3-butanediol aqueous solution for 1 h at 55℃, filter the eluent with a 500-mesh filter bag, and collect the filtrate.

[0107] S300: Add the crystals collected in step S100 to the filtrate collected in step S200, stir and dissolve at 60°C for 1 hour to form a transparent solution, filter with a 0.45μm nylon membrane to obtain the plant extract composition.

[0108] Example 4

[0109] A plant extract composition is prepared by the following method:

[0110] S100: Take 7.5 kg of Coptis chinensis and 7.5 kg of Magnolia officinalis, mix and crush them, pass them through a 60-mesh sieve and put them into a subcritical extraction tank. Add 100 kg of deionized water and extract at 125℃ and 1.5 times the standard atmospheric pressure for 2 hours. Cool the extract to below 95℃, filter it with a 0.45 μm nylon membrane, let it crystallize for 24 hours, and collect the crystals by suction filtration.

[0111] S200: Take 7.5 kg of Sichuan costus root powder, pass it through a 60-mesh sieve, add 100 kg of deionized water and 0.75 kg of betaine, extract at 50℃ for 2 h, filter the extract, collect the filtrate, add polyamide resin for static adsorption, filter with a 500-mesh filter bag to collect the resin, elute the resin with 100 kg of 60% 1,3-propanediol aqueous solution for 1 h at 55℃, filter the eluent with a 500-mesh filter bag, and collect the filtrate.

[0112] S300: Add the crystals collected in step S100 to the filtrate collected in step S200, stir and dissolve at 60°C for 1 hour to form a transparent solution, filter with a 0.45μm nylon membrane to obtain the plant extract composition.

[0113] Example 5

[0114] A plant extract composition is prepared by the following method:

[0115] S100, take 7.5 kg of Coptis chinensis and 7.5 kg of Magnolia officinalis, mix and crush them, pass them through a 60-mesh sieve and put them into a subcritical extraction tank, add 100 kg of deionized water, extract at 125℃ and 1.5 times the standard atmospheric pressure for 2 hours, cool the extract to below 95℃, filter it with a 0.45 μm nylon membrane, let it crystallize for 24 hours, and collect the crystals by suction filtration.

[0116] S200: Take 7.5 kg of Sichuan costus root powder, pass it through a 60-mesh sieve, add 100 kg of deionized water and 0.75 kg of L-carnitine, extract at 50℃ for 2 h, filter the extract, collect the filtrate, add polyamide resin for static adsorption, filter with a 500-mesh filter bag to collect the resin, elute the resin with 120 kg of 65% 1,3-propanediol aqueous solution for 1 h at 55℃, filter the eluent with a 500-mesh filter bag, and collect the filtrate.

[0117] S300: Add the crystals collected in step S100 to the filtrate collected in step S200, stir and dissolve at 60°C for 1 hour to form a transparent solution, filter with a 0.45μm nylon membrane to obtain the plant extract composition.

[0118] Example 6

[0119] A plant extract composition is prepared by the following method:

[0120] S100: Take 7.5 kg of Coptis chinensis and 9.0 kg of Magnolia officinalis, mix and crush them, pass them through a 60-mesh sieve and put them into a subcritical extraction tank. Add 100 kg of deionized water and extract at 125℃ and 1.5 times the standard atmospheric pressure for 2 hours. Cool the extract to below 95℃, filter it with a 0.45 μm nylon membrane, let it crystallize for 24 hours, and collect the crystals by suction filtration.

[0121] S200: Take 9.0 kg of Sichuan costus root powder, pass it through a 60-mesh sieve, add 100 kg of deionized water and 0.75 kg of L-carnitine, extract at 50℃ for 2 h, filter the extract, collect the filtrate, add polyamide resin for static adsorption, filter with a 500-mesh filter bag to collect the resin, elute the resin with 90 kg of 55% 1,3-propanediol aqueous solution for 1 h at 55℃, filter the eluent with a 500-mesh filter bag, and collect the filtrate.

[0122] S300: Add the crystals collected in step S100 to the filtrate collected in step S200, stir and dissolve at 60°C for 1 hour to form a transparent solution, filter with a 0.45μm nylon membrane to obtain the plant extract composition.

[0123] Comparative Example 1

[0124] S100, take 7.5 kg each of Coptis chinensis and Magnolia officinalis, crush them, pass them through a 60-mesh sieve and put them into a subcritical extraction tank, add 100 kg of deionized water, extract at 125℃ and 1.5 times the standard atmospheric pressure for 2 hours, cool the extract to below 95℃, filter it with a 0.45 μm nylon membrane, let it crystallize for 24 hours, and collect the crystals by suction filtration.

[0125] S200: The crystals collected in step S100 are added to 100 kg of 60% 1,3-propanediol aqueous solution and stirred at 60°C for 1 h to form a transparent solution. The solution is then filtered through a 0.45 μm nylon membrane to obtain comparative composition 1.

[0126] Comparative Example 2

[0127] S100, take 7.5 kg of Coptis chinensis, crush it, pass it through a 60-mesh sieve and put it into a subcritical extraction tank, add 1700 kg of deionized water, extract at 125℃ and 1.5 times the standard atmospheric pressure for 2 hours, cool the extract to below 95℃, filter it with a 0.45 μm nylon membrane, let it crystallize for 24 hours, and collect the crystals by suction filtration.

[0128] S200: Take 7.5 kg of Sichuan costus root powder, pass it through a 60-mesh sieve, add 100 kg of deionized water and 0.75 kg of L-carnitine, extract at 90℃ for 2 h, filter the extract, collect the filtrate, add polyamide resin for static adsorption, filter with a 500-mesh filter bag to collect the resin, elute the resin with 100 kg of 760% 1,3-butanediol for 1 h at an elution temperature of 55℃, filter the eluent with a 500-mesh filter bag, and collect the filtrate.

[0129] S300, the crystals collected in step S100 are added to the filtrate collected in step S200, and stirred at 60°C for 1 hour to form a transparent solution. The solution is then filtered through a 0.45 μm nylon membrane to obtain comparative composition 2.

[0130] Comparative Example 3

[0131] S100, take 7.5 kg of Magnolia officinalis, crush it, pass it through a 60-mesh sieve, add 100 kg of deionized water and extract it at high temperature for 2 h, filter it through a 0.45 μm nylon membrane, let the filtrate crystallize for 24 h, and collect the crystals by vacuum filtration.

[0132] S200: Take 7.5 kg of Sichuan costus root powder, pass it through a 60-mesh sieve, add 100 kg of deionized water and 0.75 kg of L-carnitine, extract at 90℃ for 2 h, filter the extract, collect the filtrate, add polyamide resin for static adsorption, filter with a 500-mesh filter bag to collect the resin, elute the resin with 100 kg of 60% 1,3-propanediol aqueous solution for 1 h at 55℃, filter the eluent with a 500-mesh filter bag, and collect the filtrate.

[0133] S300: Add the crystals collected in step S100 to the filtrate of S200, stir and dissolve at 60°C for 1 hour to form a transparent solution, filter with a 0.45μm nylon membrane to obtain comparative composition 3.

[0134] Comparative Example 4

[0135] S100: Take 7.5 kg of Coptis chinensis and 7.5 kg of Magnolia officinalis, crush them, pass them through a 60-mesh sieve, add 100 kg of deionized water and extract at 100℃ for 2 h, then filter using a 5 μm polypropylene membrane; let the filtrate crystallize for 24 h, and collect the crystals by vacuum filtration.

[0136] S200: The crystals collected in step S100 are added to 100 kg of 60% 1,3-propanediol aqueous solution and stirred at 60°C for 1 h to form a transparent solution. The solution is then filtered through a 0.45 μm nylon membrane to obtain comparative composition 4.

[0137] Comparative Example 5

[0138] S100: Take 7.5 kg of Coptis chinensis and 7.5 kg of Magnolia officinalis, crush them, pass them through a 60-mesh sieve, add 100 kg of deionized water and extract at 100℃ for 2 h, then filter using a 5 μm polypropylene membrane; let the filtrate crystallize for 24 h, and collect the crystals by vacuum filtration.

[0139] S200: Take 7.5 kg of Sichuan costus root powder, pass it through a 60-mesh sieve, add 100 kg of deionized water and 0.75 kg of L-carnitine, extract at 90℃ for 2 h, filter the extract, collect the filtrate, add polyamide resin for static adsorption, filter with a 500-mesh filter bag to collect the resin, elute the resin with 100 kg of 60% 1,3-propanediol aqueous solution for 1 h at 55℃, filter the eluent with a 500-mesh filter bag, and collect the filtrate.

[0140] S300: Add the crystals collected in step S100 to the filtrate of S200, stir and dissolve at 60°C for 1 hour to form a transparent solution, filter with a 0.45μm nylon membrane to obtain comparative composition 5.

[0141] Comparative Example 6

[0142] S100, take 7.5 kg of Coptis chinensis and 7.5 kg of Magnolia officinalis, crush them, put them into a subcritical extraction tank through a 60-mesh sieve, add 100 kg of deionized water, extract at 125℃ and 1.5 times the standard atmospheric pressure for 2 hours, cool the extract to below 95℃, filter it with a 0.45 μm nylon membrane, let it crystallize for 24 hours, and collect the crystals by suction filtration.

[0143] S200: Take 7.5 kg of Sichuan costus root powder, pass it through a 60-mesh sieve, add 100 kg of deionized water, extract at 90℃ for 2 h, filter the extract, collect the filtrate, add polyamide resin for static adsorption, filter with a 500-mesh filter bag to collect the resin, elute the resin with 100 kg of 60% 1,3-propanediol aqueous solution for 1 h at an elution temperature of 55℃, filter the eluent with a 500-mesh filter bag, and collect the filtrate.

[0144] S300, the crystals collected in step S100 are added to the filtrate collected in step S200, and stirred at 60°C for 1 hour to form a transparent solution. The solution is then filtered through a 0.45 μm nylon membrane to obtain the comparative composition 6.

[0145] Comparative Example 7

[0146] S100, take 7.5 kg of Coptis chinensis and 7.5 kg of Magnolia officinalis, crush them, add 100 kg of deionized water and extract at 100℃ for 2 h, filter with 0.45 μm nylon membrane, let the filtrate crystallize for 24 h, and collect the crystals by vacuum filtration;

[0147] S200: Take 7.5 kg of Sichuan costus root powder, pass it through a 60-mesh sieve, add 100 kg of deionized water, extract at 90℃ for 2 h, filter the extract, collect the filtrate, add polyamide resin for static adsorption, filter with a 500-mesh filter bag to collect the resin, elute the resin with 100 kg of 60% 1,3-propanediol aqueous solution for 1 h at an elution temperature of 55℃, filter the eluent with a 500-mesh filter bag, and collect the filtrate.

[0148] S300, the crystals collected in step S100 are added to the filtrate collected in step S200, and stirred at 60°C for 1 hour to form a transparent solution. The solution is then filtered through a 0.45 μm nylon membrane to obtain comparative composition 7.

[0149] Comparative Example 8

[0150] A composition is prepared by the following method:

[0151] S100: Take 5.5 kg of Coptis chinensis and 5.5 kg of Magnolia officinalis, mix and crush them, pass them through a 60-mesh sieve and put them into a subcritical extraction tank. Add 100 kg of deionized water and extract at 125℃ and 1.5 times the standard atmospheric pressure for 2 hours. Cool the extract to below 95℃ and filter it with a 0.45 μm nylon membrane. Let the filtrate crystallize for 24 hours and collect the crystals by suction filtration.

[0152] S200: Take 5.5 kg of Sichuan costus root, crush it, pass it through a 60-mesh sieve, add 100 kg of deionized water and 0.55 kg of L-carnitine, extract at 50℃ for 2 h, filter the extract, collect the filtrate, add polyamide resin for static adsorption, filter with a 500-mesh filter bag to collect the resin, elute the resin with 100 kg of 60% 1,3-propanediol aqueous solution for 1 h at 55℃, filter the eluent with a 500-mesh filter bag, and collect the filtrate.

[0153] S300, the crystals collected in step S100 are added to the filtrate collected in step S200, and stirred at 60°C for 1 hour to form a transparent solution. The solution is then filtered through a 0.45 μm nylon membrane to obtain comparative composition 8.

[0154] Comparative Example 9

[0155] A composition is prepared by the following method:

[0156] S100: Take 9.0 kg of Coptis chinensis and 9.0 kg of Magnolia officinalis, mix and crush them, pass them through a 60-mesh sieve and put them into a subcritical extraction tank. Add 100 kg of deionized water and extract at 125℃ and 1.5 times the standard atmospheric pressure for 2 hours. Cool the extract to below 95℃ and filter it with a 0.45 μm nylon membrane. Let the filtrate crystallize for 24 hours and collect the crystals by suction filtration.

[0157] S200: Take 9.0 kg of Sichuan costus root powder, pass it through a 60-mesh sieve, add 100 kg of deionized water and 1.08 kg of L-carnitine, extract at 50℃ for 2 h, filter the extract, collect the filtrate, add polyamide resin for static adsorption, filter with a 500-mesh filter bag to collect the resin, elute the resin with 100 kg of 60% 1,3-propanediol aqueous solution for 1 h at 55℃, filter the eluent with a 500-mesh filter bag, and collect the filtrate;

[0158] S300, the crystals collected in step S100 are added to the filtrate collected in step S200, and stirred at 60°C for 1 hour to form a transparent solution. The solution is then filtered through a 0.45 μm nylon membrane to obtain the comparative composition 9.

[0159] Comparative Example 10

[0160] A composition is prepared by the following method:

[0161] S100: Take 7.5 kg of Coptis chinensis and 10 kg of Magnolia officinalis, mix and crush them, pass them through a 60-mesh sieve and put them into a subcritical extraction tank. Add 100 kg of deionized water and extract at 125℃ and 1.5 times the standard atmospheric pressure for 2 hours. Cool the extract to below 95℃ and filter it with a 0.45 μm nylon membrane. Let the filtrate crystallize for 24 hours and collect the crystals by suction filtration.

[0162] S200, take 5kg of Sichuan costus root powder, pass through a 60-mesh sieve, add 100kg of deionized water and 0.4kg of L-carnitine, extract at 50℃ for 2h, filter the extract, collect the filtrate, add polyamide resin for static adsorption, filter with a 500-mesh filter bag to collect the resin, elute the resin with 100kg of 60% 1,3-propanediol aqueous solution for 1h at 55℃, filter the eluent with a 500-mesh filter bag, and collect the filtrate;

[0163] S300, the crystals collected in step S100 are added to the filtrate collected in step S200, and stirred at 60°C for 1 hour to form a transparent solution. The solution is then filtered through a 0.45 μm nylon membrane to obtain comparative composition 10.

[0164] Comparative Example 11

[0165] A composition is prepared by the following method:

[0166] S100, take 7.5 kg of Coptis chinensis and 7.5 kg of Magnolia officinalis, mix and crush them, pass them through a 60-mesh sieve and put them into a subcritical extraction tank, add 100 kg of deionized water, extract at 125℃ and 1.5 times the standard atmospheric pressure for 2 hours, cool the extract to below 95℃, filter it with a 0.45 μm nylon membrane, let the filtrate crystallize for 24 hours, and collect the crystals by vacuum filtration;

[0167] S200: Take 7.5 kg of Sichuan costus root, crush it, pass it through a 60-mesh sieve, add 100 kg of deionized water and 0.5 kg of L-carnitine, extract at 60℃ for 2 h, filter the extract, collect the filtrate, add polyamide resin for static adsorption, filter with a 500-mesh filter bag to collect the resin, elute the resin with 130 kg of 70% 1,3-propanediol aqueous solution for 1 h at an elution temperature of 55℃, filter the eluent with a 500-mesh filter bag, and collect the filtrate;

[0168] S300, the crystals collected in step S100 are added to the filtrate collected in step S200, and stirred at 60°C for 1 hour to form a transparent solution. The solution is then filtered through a 0.45 μm nylon membrane to obtain comparative composition 11.

[0169] Test section

[0170] 1. Appearance and stability test

[0171] The appearance of the compositions prepared in each example and comparative example was observed, and their stability under different environments for 3 months was recorded, as detailed in Table 1.

[0172] Table 1

[0173]

[0174] According to the test results in Table 1, the compositions obtained using traditional water extraction methods or with excessive amounts of raw materials generally have a darker, deep yellow color. Furthermore, compositions with a high content of *Saussurea costus* extract have a strong, pungent odor. In contrast, the composition of this application, a blend of *Coptis chinensis* and *Magnolia officinalis* extracts with *Saussurea costus* extract, has no pungent odor. This is crucial for plant extract compositions used as cosmetic ingredients. Additionally, the addition of *Saussurea costus* extract improves the stability of the *Coptis chinensis* and *Magnolia officinalis* blend, reducing discoloration and precipitation under high temperature or light conditions. Moreover, the composition obtained using subcritical extraction technology has a significantly milder odor and better stability compared to compositions obtained using traditional water extraction techniques.

[0175] 2. Cell safety

[0176] Experimental Methods: HaCaT cells are a human immortalized epidermal cell line. The cytotoxicity of HaCaT cells can serve as a reference for skin safety. Normal cells have a high metabolic rate; succinate dehydrogenase in their mitochondria can reduce tetrazolium salts to colored crystalline substances, which deposit around the cells. This change can be measured by the OD (optical density) value using a microplate reader. By comparing the OD value with that of the blank control group, the relative growth of the cells can be determined (OD value is directly proportional to cell viability). After treating the samples with a 0.5% aqueous solution of the plant extract composition, the viability of HaCaT cells was determined using a CCK-8 assay. Cell viability above 75% was considered "non-toxic." The experimental results are as follows: Figure 1 .

[0177] Combination Figure 1 The experimental results show that the plant extract compositions in Examples 1 to 6 have no cytotoxicity to HaCaT cells, the compositions in Comparative Examples 5, 6, 8 to 11 have no cytotoxicity to HaCaT cells, the compositions in Comparative Examples 1 to 4 have cytotoxicity to HaCaT cells, and the composition in Comparative Example 7 has weak cytotoxicity to HaCaT cells. The above experimental results indicate that the combined extract of the three raw materials Coptis chinensis, Magnolia officinalis and Aucklandia lappa provided in this application can significantly reduce the cytotoxicity to HaCaT cells.

[0178] 3. Experiments on promoting the expression of defensins hBD-2 and hBD-3 at the mRNA level

[0179] 3.1 Extraction of total RNA from cells

[0180] (1) After discarding the culture medium, add Trizol total RNA extraction reagent at a ratio of 3 mL / 10 cm diameter culture dish to adherent cells with 80%-90% confluence and good growth status. Repeatedly pipet until the liquid is completely transparent and let stand at room temperature for 5 min.

[0181] (2) Add chloroform at a ratio of 200 μL chloroform / 1 ml Trizol, invert the EP tube for 50 seconds, and let it stand at room temperature for 3 minutes.

[0182] (3) Centrifuge at 4℃, 12000 rpm, for 15 min.

[0183] (4) Transfer the upper aqueous phase to another EP tube, add an equal volume of isopropanol and mix well, and let stand at room temperature for 10 min.

[0184] (5) Centrifuge at 4℃, 12000 rpm, for 10 min, carefully pour off the liquid, and let the RNA settle at the bottom of the tube.

[0185] (6) Wash the RNA precipitate with 1 ml of 75% ethanol (DEPC water, freshly prepared) and gently shake the centrifuge tube to suspend the precipitate.

[0186] (7) Centrifuge at 4℃, 8000 rpm, for 5 min. After discarding the ethanol, invert the EP tube onto absorbent paper and let the RNA precipitate dry at room temperature for about 10 min.

[0187] (8) RNA samples can be dissolved in 30 μL H2O (adjust as needed depending on the amount of RNA), TE buffer, or 0.5% SDS (sodium dodecyl sulfate) and stored at -20℃ (preferably -80℃). Note: H2O, TE, or 0.5% SDS must be treated with DEPC and autoclaved.

[0188] 3.2 RNA concentration determination and reverse transcription

[0189] (1) Instrument: Micro-ultraviolet spectrophotometer

[0190] (2) RNA concentration unit: ng / μL; after the assay, the RNA template was diluted to 500 ng / μL;

[0191] (3) The RT-PCR reverse transcription system is as follows (reagent company: TAKARA):

[0192] Element Volume (μL) 5x Prime Script Buffer(for Real Time) 4 Prime Script RT Enzyme Mix I 1 Random 6-mers 2 Total RNA (500 ng / uL) 2 <![CDATA[RNase Free dH2O]]> 11

[0193] The reverse transcription program for cDNA was: 37°C for 15 min; 85°C for 5 s; 4°C cycle; Total: approximately 15 min.

[0194] qRCR steps:

[0195] Centrifuge the primers at 12000 rpm for 5 min; dissolve them in water according to the recommended dosage and mix well to achieve a concentration of 100 μM.

[0196] Take a new EP tube, add 5 μL of forward primer and 5 μL of reverse primer, then add 40 μL of deionized water and mix well to obtain primers with a concentration of 10 μM.

[0197] The concentration of reverse transcribed cDNA was determined, and the cDNA was diluted with deionized water to 200 ng / μL.

[0198] The reverse transcription system is as follows:

[0199]

[0200]

[0201] The total volume for real-time PCR is 10 μL per tube.

[0202] Program: Pre-denaturation 95°C, 5 min, 1 cycle; Denaturation 95°C, 10 sec Annealing / Extension 60°C, 30 sec, 40 cycles; Melting curve stage, 1 cycle.

[0203]

[0204] For detailed experimental data, please refer to Figure 2-1 , Figure 2-2 .

[0205] Combination Figure 2-1 , 2-2The experimental results show that the plant extract compositions in Examples 1, 7, and 2 at appropriate concentrations can all promote the expression of hBD-2 and hBD-3 at the mRNA level. At the same concentration of 0.125%, Comparative Examples 7 and 2 showed no significant effect in promoting the expression of hBD-2 and hBD-3 at the mRNA level, while Example 1 showed a significant effect. At the same concentration of 0.25%, Comparative Examples 7, 2, and 1 all significantly promoted the expression of hBD-2 and hBD-3 at the mRNA level. The data indicate that the 0.125% and 0.25% concentrations of Example 1 significantly promoted the expression of hBD-2 and hBD-3 at the mRNA level compared to Comparative Examples 7 and 2 at the same concentrations. Furthermore, Comparative Example 7 showed a slightly better effect than Comparative Example 2, meaning that when the plant extract composition only contains Coptis chinensis and Aucklandia lappa, its effect in promoting the expression of hBD-2 and hBD-3 at the mRNA level is weaker than the composition containing Coptis chinensis, Aucklandia lappa, and Magnolia officinalis. The combination of Coptis chinensis, Magnolia officinalis, and Aucklandia lappa extracted using supercritical and supramolecular technologies showed significantly better results in promoting the expression of hBD-2 and hBD-3 at the mRNA level than the traditional water-extracted combination of Coptis chinensis, Magnolia officinalis, and Aucklandia lappa.

[0206] 4. Experiment on the expression of antimicrobial peptide LL-37 at the mRNA level

[0207] 4.1 Extraction of total RNA from cells

[0208] (1) After discarding the culture medium, add Trizol to the adherent cells with 80-90% confluence and good growth status in a 10cm diameter culture dish at a rate of 3mL / 10cm diameter culture cell. Repeatedly pipette until the liquid is completely transparent and let stand at room temperature for 5min.

[0209] (2) Add chloroform at a ratio of 200 μL chloroform / 1 ml Trizol, invert the EP tube for 50 seconds, and let it stand at room temperature for 3 minutes.

[0210] (3) Centrifuge at 4℃, 12000 rpm, for 15 min.

[0211] (4) Transfer the upper aqueous phase to another EP tube, add an equal volume of isopropanol and mix well (about 200 μL-300 μL), and let stand at room temperature for 10 min.

[0212] (5) Centrifuge at 4℃, 12000 rpm, for 10 min, carefully pour off the liquid, and let the RNA settle at the bottom of the tube.

[0213] (6) Wash the RNA precipitate with 1 ml of 75% ethanol (DEPC water, freshly prepared) and gently shake the centrifuge tube to suspend the precipitate.

[0214] (7) Centrifuge at 4℃, 8,000 rpm, for 5 min. After discarding the ethanol, invert the EP tube onto absorbent paper to dry the RNA precipitate at room temperature for about 10 min.

[0215] (8) RNA samples can be dissolved in 30 μL H2O (adjust as needed depending on the amount of RNA), TE buffer, or 0.5% SDS and stored at -20℃ (preferably -80℃). Note: H2O, TE, or 0.5% SDS must be treated with DEPC and autoclaved.

[0216] 4.2 RNA concentration determination and reverse transcription

[0217] (1) Instrument: Micro-ultraviolet spectrophotometer

[0218] (2) RNA concentration unit: ng / μL; after the assay, the RNA template was diluted to 500 ng / μL;

[0219] (3) The RT-PCR reverse transcription system is as follows (reagent company: TAKARA):

[0220] Element Volume (μL) 5x Prime Script Buffer(for Real Time) 4 Prime Script RT Enzyme Mix I 1 Random 6-mers 2 Total RNA (500 ng / uL) 2 RNase-Free dH2O 11

[0221] The reverse transcription program for cDNA was: 37°C for 15 min; 85°C for 5 s; 4°C cycle; Total: approximately 15 min.

[0222] qRCR steps:

[0223] Centrifuge the primers at 12000 rpm for 5 min; dissolve them in water according to the recommended dosage and mix well to achieve a concentration of 100 μM.

[0224] Take a new EP tube, add 5 μL of forward primer and 5 μL of reverse primer, then add 40 μL of deionized water and mix well to obtain primers with a concentration of 10 μM.

[0225] The concentration of reverse transcribed cDNA was determined, and the cDNA was diluted with deionized water to 200 ng / μL.

[0226] The reverse transcription system is as follows:

[0227] Element Volume (μL) HieffqPCR SYBR Green Master Mix(Low Rox PLus)(2X) 5 (F+R) primers 1 Template cDNA (200 ng / μL) 2 Sterile ultrapure water 2

[0228] The total volume for real-time PCR is 10 μL per tube.

[0229] Program: Pre-denaturation 95°C 5min, 1 cycle; Denaturation 95°C 10sec, Annealing / Extension 60°C 30sec, 40 cycles; Melting curve stage, 1 cycle.

[0230]

[0231] For detailed experimental data, please refer to Figure 3 .

[0232] Combination Figure 3 The experimental results show that Comparative Example 2 at concentrations of 0.125% and 0.25% did not promote the expression of LL-37 at the mRNA level, while Comparative Example 7 and Example 1 at the same concentrations promoted the expression of LL-37 at the mRNA level. Specifically, Comparative Example 7 at a concentration of 0.25% and Example 1 at concentrations of 0.125% and 0.25% showed significant promoting effects, and the promoting effect of Example 1 at a concentration of 0.125% was comparable to that of Comparative Example 7 at a concentration of 0.25%. In other words, the combination containing only Coptis chinensis and Aucklandia lappa had a weaker effect on promoting the expression of LL-37 at the mRNA level, while the combination containing Coptis chinensis, Aucklandia lappa, and Magnolia officinalis had a stronger effect. The combination of Coptis chinensis, Aucklandia lappa, and Magnolia officinalis obtained by traditional water extraction had a weaker effect on promoting the expression of LL-37 at the mRNA level than the combination of Coptis chinensis, Aucklandia lappa, and Magnolia officinalis obtained by subcritical and supramolecular extraction techniques at the same concentrations.

[0233] 5. Clinical efficacy in treating acne

[0234] 1) Subject selection

[0235] 1.1 The inclusion criteria for subjects are as follows:

[0236] Healthy men or women aged 18-40;

[0237] People with acne on their face;

[0238] The facial skin should be free of blemishes, moles, birthmarks, or other conditions that could affect the test.

[0239] The subjects did not have any serious chronic wasting diseases (such as asthma, diabetes, etc.);

[0240] Individuals with no allergic diseases and no history of allergies to cosmetics or other topical preparations (products);

[0241] Those who comply with the trial requirements, voluntarily participate in the trial, and sign a written informed consent form;

[0242] Those who have not participated in other clinical trials in the past month;

[0243] I have not used any acne products or received any acne treatment (such as medication, injections, laser treatments, or other cosmetic procedures) in the past month.

[0244] 1.2 The exclusion criteria for subjects are as follows:

[0245] Women who are planning to become pregnant, are pregnant, are breastfeeding, or are within six months postpartum;

[0246] Individuals with severe systemic diseases, immunodeficiency, or autoimmune diseases;

[0247] Those who have used antihistamines in the past week or immunosuppressants in the past month;

[0248] Those who have used any anti-inflammatory drugs on the test site within the past two months;

[0249] The test site has participated in other clinical trials within the past three months;

[0250] Those with pustules, nodules, or cystic acne on the face;

[0251] People who are currently receiving dermatological treatment;

[0252] Other clinical assessments deemed unsuitable for participation in the trial.

[0253] 1.3 Other precautions for subjects:

[0254] During the testing period, the test site should use the test product provided by the testing institution, and no other products with the same number of shifts or that may affect the test results should be used;

[0255] During the testing period, it is forbidden to ingest any preparations that may affect or potentially affect the test results (such as anti-inflammatory drugs, injectable drugs, etc.). If it is necessary to ingest them, the testing institution should be notified in advance.

[0256] During the testing period, participants should maintain a regular lifestyle and pay close attention to their diet.

[0257] During the testing period, cosmetic procedures or treatments that may affect the test results, such as laser spot removal or skin resurfacing, are prohibited.

[0258] 1.4 Subject withdrawal criteria:

[0259] During the testing process, if the test subject experiences an adverse reaction, is lost to follow-up without cause, or violates the testing protocol (such as using other methods to affect the test results), the test results may be affected.

[0260] In cases involving cosmetics or medications that may have an impact, or other special circumstances, and if a dermatologist determines that the subject is no longer suitable to continue the test, the subject will be asked to withdraw.

[0261] 2) Test site

[0262] Areas of the face with acne.

[0263] 3) Testing instruments

[0264] Instrument type and model:

[0265] Tewameter TM300 Courage-Khazaka transdermal water loss test probe, Germany;

[0266] VISIA 7 Canfield skin analyzer, USA;

[0267] Antera 3D Miravex 3D Image Acquisition System, Ireland;

[0268] Sebumeter SM815 Courage-Khazaka, Germany; Skin oil testing probe.

[0269] 4) Testing the environment

[0270] Temperature 21℃±1℃;

[0271] Humidity 50% ± 10% RH;

[0272] 5) Inspection process

[0273] 5.1 Subject Screening

[0274] Recruit subjects as required and obtain informed consent; ask subjects relevant questions according to the inclusion and exclusion criteria, and record the results together with the test screening information.

[0275] 5.2 Preparations before testing

[0276] On the day of the visit, no products should be applied to the test area. Subjects should use a facial cleansing product provided by the laboratory that has no efficacy to cleanse their face and sit quietly in a constant temperature and humidity room for 30 minutes. During this time, they should not drink hot water or eat hot food, and the test area should not come into contact with water or be touched.

[0277] 5.3 Initial Value Test

[0278] All participants underwent a pre- and post-control design, sitting quietly in a temperature- and humidity-controlled room for 30 minutes before being tested using relevant instruments. A unique code was assigned and recorded, designated as the initial value "D". O ".

[0279] 5.4 Follow-up Test

[0280] All subjects were followed up on days 1, 3, and 7. On the days of the follow-up, no products were applied to the test area. Subjects used a uniform facial cleansing product provided by the laboratory that had no efficacy to cleanse their faces. After sitting quietly in a constant temperature and humidity room for 30 minutes, they were tested using a facial image acquisition device and a skin color tester. A unique code was assigned and recorded as the test value "D1", "D3", and "D7".

[0281] 5.5 Adverse Reaction Management

[0282] If a subject experiences an adverse skin reaction while using the test sample, use should be discontinued immediately and appropriate medical protective measures should be taken. The adverse reaction event should be recorded.

[0283] 5.6 Data Analysis

[0284] Statistical analysis was performed on the measured values ​​for each test area, including quantity, mean, and standard deviation. The differences between the baseline value DO and D1, D3, and D7 for each test area were calculated.

[0285] The sample applied is a gel, and its components are shown in Table 2 below:

[0286] Table 2

[0287]

[0288] The results of the redness reduction a value test were referenced. Figure 4 The results of the pimple volume experiment were referenced. Figure 5 Skin oil content results are referenced Figure 6 The results of transepidermal water loss of the skin were referenced. Figure 7 Acne treatment VISA chart reference Figure 8 .

[0289] from Figure 4 The results of the change rate of the a-value show that both the control and sample groups can reduce redness around acne, which is consistent with the skin's own repair and regulatory functions. From the change rate results, the redness reduction effect of Examples 1 to 6 is significantly better than that of the comparative and control groups. In Examples 1, the redness value around acne was reduced by more than 13% on day 3 and by more than 25% on day 7. In the comparative groups, the redness value around acne was reduced by 6%–12% on day 3 and by 11%–23% on day 7.

[0290] from Figure 5 The results of the acne volume change rate showed that both the control group and the sample group could reduce acne, which is consistent with the skin's own repair and regulatory functions. Looking at the change rate results, the acne-reducing effects of Examples 1 to 6 were significantly better than those of the comparative and control groups. In Examples 1, the acne volume decreased by 30%–45% on day 3 and by 45%–55% on day 7. In the comparative group, the acne volume decreased by 4%–25% on day 3 and by 8%–40% on day 7.

[0291] from Figure 6 Based on the results of the change rate of skin oil content, the acne-removing effects of Examples 1 to 6 were significantly better than those of the comparative and control groups. On the first day of Examples 1, the skin oil content was reduced by 16% to 19%; on the third day of Examples 3, the skin oil content was reduced by 25% to 30%; and on the seventh day, the skin oil content was reduced by 37% to 40%. In contrast, on the first day of the comparative example, the skin oil content was reduced by 4% to 14%; on the third day of Examples 3, the skin oil content was reduced by 14% to 23%; and on the seventh day, the skin oil content was reduced by 13% to 32%.

[0292] from Figure 7 The results of the change rate of transepidermal water loss showed that both the control and sample groups reduced transepidermal water loss over time, which is consistent with the skin's own repair and regulatory functions. From the change rate results, the acne-removing effects of Examples 1 to 6 were significantly better than those of the comparative and control groups. In Examples 1, transepidermal water loss around acne was reduced by 5%–8% on day 1; by more than 22%–23% on day 3; and by more than 29%–34% on day 7. In the comparative group, transepidermal water loss around acne was reduced by 1%–5% on day 1; by 9%–21% on day 3; and by 15%–28% on day 7.

[0293] from Figure 8 The results of acne changes captured by VISA on the face showed that the size of the acne gradually decreased over time, which is consistent with the skin's natural repair and regulatory functions. In Comparative Example 10, after gel application, a slight reduction in acne size and elevation (acne height) was observed on day 7, but the VISA image still showed a darker color in the center of the acne, indicating that it remained raised. In Sample Group (Example 1), after gel application, a significant reduction in acne size and elevation (acne height) was observed on day 3, and the VISA image on day 7 showed that the center of the acne had completely lost its dark color, meaning the elevation had disappeared. These VISA images indicate that Example 1 has a better acne-removing effect than Comparative Example 10.

[0294] Although this application has been described with reference to preferred embodiments, various modifications can be made thereto and components can be replaced with equivalents without departing from the scope of this application. In particular, the technical features mentioned in the various embodiments can be combined in any manner, provided there is no structural conflict. This application is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. Use of a plant extract composition in the preparation of an anti-acne preparation, characterized in that, The application is to promote the expression of skin antibacterial peptides. The plant extraction composition is prepared by the following steps: mixing and crushing Coptis chinensis and Magnolia officinalis, putting them into a subcritical extraction tank, adding deionized water to extract under high temperature and high pressure, filtering to obtain a first extraction liquid, cooling and crystallizing the first extraction liquid, and drying to obtain a first extraction product, the mass of Coptis chinensis and Magnolia officinalis is 7.5 kg, and the mass of deionized water is 100 kg. Cortex costus and organic alkali are mixed to extract at low temperature to obtain a second extraction liquid; the second extraction liquid is subjected to resin adsorption and then desorbed with a polyhydric alcohol aqueous solution to obtain a desorption liquid, the organic alkali includes betaine or L-carnitine, the mass of Cortex costus is 7.5 kg, the mass of deionized water is 100 kg, and the mass of the organic alkali is 0.75 kg. The desorption liquid is mixed with the first extraction product to obtain the plant extraction composition.

2. Use according to claim 1, characterized in that, The promotion of the expression of skin antibacterial peptides is to promote the expression of hBD-2, hBD-3 and LL-37 genes at the mRNA level.

3. Use according to claim 1, characterized in that, The extraction temperature of the high temperature and high pressure extraction is 105-120 DEG C, and the extraction pressure is 1.3-1.5 standard atmospheres.

4. Use according to claim 1, characterized in that, The extraction temperature of the low temperature extraction is 40-55 DEG C.

5. The use according to claim 1, characterized in that, The resin used for the resin adsorption includes one or both of polyamide resin and snake cage resin; and / or The polyhydric alcohol includes one or more of glycerol, 1,3-propanediol, 1,3-butanediol, 1,2-propanediol, dipropylene glycol and methyl propylene glycol; and / or The mass concentration of the polyhydric alcohol aqueous solution is 55%-65%; and / or The mass ratio of Cortex costus to the polyhydric alcohol aqueous solution is 1:(11.76-16.66).

Images