Malus micromalus flower extract as well as preparation method and application thereof

Abstract

The invention discloses a malus micromalus flower extract as well as a preparation method and application thereof, and the malus micromalus flower extract at least comprises the following components in percentage by mass: 1.5-8.5% of afzelin. The malus micromalus flower extract disclosed by the invention can be applied to preparation of cosmetics with tightening, soothing and repairing functions as an active ingredient.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, in particular to a malus fragrans flower extract, a preparation method and an application thereof. Background Art

[0002] As the body's barrier structure, the skin's core functions include preventing water loss, shielding against external stimuli, and maintaining microecological balance and immune response. Damage to this barrier can lead to collagen loss, resulting in decreased dermal support, sagging skin, and increased fine lines. Furthermore, imperfections in the barrier can lead to a series of reactions, including neurosensitivity and immune imbalance.

[0003] Skin sagging is closely related to the degradation of intracellular collagen and elastin. Collagen and elastin work together to maintain skin's firmness and elasticity: collagen provides the skin's support framework, while elastin imparts its elasticity and flexibility. These two proteins intertwine to form a complex network that enables the skin to maintain its firmness and elasticity. Excessive accumulation of ROS stimulates the expression of matrix metalloproteinases (MMPs), which degrade collagen and elastin, leading to a decrease in their synthesis rate and accelerated breakdown. This MMPs alters their conformation, impacting the skin's mechanical properties and causing skin sagging and increased wrinkling.

[0004] FLG (filagrin) and LOR (loricrin) are important indicators for evaluating the barrier repair efficacy of cosmetics. FLG is a key protein in the stratum corneum and a crucial molecule that connects keratin fibers in the human skin's stratum corneum. FLG monomers facilitate the regular aggregation of keratin fibers, forming a solid physical barrier in the outermost layer of the epidermis, preventing water loss and the invasion of external allergens. It can also be broken down into natural moisturizing factor (NMF), which maintains skin moisture and an acidic environment, reducing transepidermal water loss (TEWL) and antigen invasion. LOR, a major protein in the epidermal granular layer, is closely associated with stratum corneum formation and keratinocyte differentiation, helping to enhance the structural integrity of the stratum corneum.

[0005] Skin irritation often accompanies sensitive skin. The development of sensitive skin is a complex process involving the skin barrier, neurovascular system, and immune-inflammatory processes. Among these, the skin's immune-inflammatory response is a key factor in causing skin irritation (such as redness, swelling, stinging, sensitivity, and redness).

[0006] Therefore, maintaining the integrity of the barrier structure and function is the key to directly affecting the skin's firmness, soothing ability and self-repair efficiency.

[0007] Begonia Malus spp. ) is Rosaceae ( Rosaceae ) Apple ( MalusA small deciduous tree that blooms in March and April, it is cold- and drought-tolerant, making it suitable for cultivation in northern my country. Polyphenols and flavonoids are important secondary metabolites that produce orange petals in higher plants. Variations in these components during flowering result in different flower colors and fading patterns in different crabapple species. Crabapple flowers within the same genus contain flavonoids, polyphenols (such as chlorogenic acid and catechins), polysaccharides, volatile phenols, aldehydes, and terpenes (such as butylated hydroxytoluene, nonanal, and D-limonene).

[0008] Currently, the relevant technologies for crabapple extracts are concentrated on extraction from crabapple leaves and crabapple fruits, and there is no special extraction from crabapple flowers.

[0009] For example, CN104224969B discloses a weeping crabapple leaf extract, an extraction method and uses, which extracts components from the weeping crabapple leaves.

[0010] Moreover, many existing crabapple extract-related technologies are targeted at different crabapple varieties. At present, there is no extract-related technology specifically for the Chinese crabapple variety, especially the Chinese crabapple flower, and there is no relevant technology for the application of Chinese crabapple flower extract in the cosmetics field. Summary of the Invention

[0011] The object of the present invention is to provide a Malus micromalus This invention provides a specialized extraction process for Malus chinensis flowers, which can be applied in the cosmetics industry and offers advantages such as firming, soothing, and repairing effects.

[0012] The technical solution of the present invention: The crabapple flower extract contains at least 1.5%-8.5% of afudamicin by mass.

[0013] The above-mentioned Malus chinensis flower extract contains, by mass percentage, at least 0.5%-3% of chlorogenic acid, 1.0%-2.0% of rutin, 30.0%-79.0% of phloridzin, 0.1%-0.8% of astragaloside and 1.5%-8.5% of afudamicin.

[0014] The preparation method of the above-mentioned Malus chinensis flower extract comprises the following steps: A. Pretreatment: Place the flowers of Malus chinensis in an oven and dry them until they are constant in weight to obtain dried flowers, which are then crushed to obtain dried flower powder; B. Preparation of extract: Dried flower powder is extracted by reflux extraction to obtain a crude extract, using ethanol as the extraction reagent at a solid-liquid ratio of 1:10-30 (w / v); the crude extract is filtered and centrifuged to obtain an extract; C. Preparation of concentrated solution: vacuum concentrate the extract, add pure water to make up to volume, and centrifuge, collecting the supernatant after centrifugation to obtain the concentrated solution; D. Preparation of enriched solution: The concentrated solution is adsorbed on the resin according to the flow rate. After the adsorption is completed, the resin column bed is cleaned by passing deionized water through the column in a forward direction, and the product is desorbed by passing ethanol through the column in a forward direction to obtain the enriched solution; E. Concentration and drying into powder: the enriched liquid is vacuum concentrated and then spray-dried to obtain the Malus chinensis flower extract powder.

[0015] In the aforementioned method for preparing the Malus chinensis flower extract, the specific contents of preparing the extract in step B are as follows: The extraction reagent is 10%-80% ethanol, the extraction temperature is 85°C, the number of extractions is 1-2, and each extraction time is 2-3 hours; after the crude extract is filtered, it is separated in a centrifuge for 20 minutes at a centrifugal force of 10,000 × g, and the precipitate is discarded to obtain the extract.

[0016] In the aforementioned method for preparing the Malus chinensis flower extract, the preparation of the concentrate described in step C is specifically as follows: The extract was vacuum concentrated to 4-10 times the weight of the dried flowers (v / w), and purified water was added to make the volume 8-15 times the weight of the dried flowers (v / w) to obtain a fixed volume crude solution; the fixed volume crude solution was centrifuged for 20 minutes at a centrifugal force of 10,000 × g and a temperature of 25°C; after centrifugation, the supernatant was collected to obtain a concentrated solution.

[0017] In the aforementioned method for preparing the Malus chinensis flower extract, the preparation of the enrichment solution described in step D is specifically as follows: The concentrated liquid is subjected to resin adsorption at a flow rate of 1-4 BV / h. The content of the target compound in the outlet liquid is detected every 1 BV to determine the adsorption state of the resin. The resins used include but are not limited to models including D101, LX-T83, HPD-100, and AB-8. After the resin adsorption is completed, the resin column bed is cleaned with deionized water in the forward direction. The cleaning flow rate is controlled at 1-6 BV / h and the cleaning flow rate is 4-12 BV to remove the residual liquid and some water-soluble impurities in the column bed. After the resin is washed with water, 2-10 BV of 30%-70% ethanol is used to pass through the column in the forward direction to desorb the product to obtain the enriched liquid. The desorption flow rate is controlled at 1-3 BV / h.

[0018] In the aforementioned method for preparing the Malus chinensis flower extract, the concentration and drying into powder described in step E is specifically as follows: The enriched solution was taken and concentrated under reduced pressure and vacuum at 50° C. to 1-4 times the weight of the dried flower (v / w); after concentration, the solution was spray-dried to obtain the Malus xifuensis flower extract powder.

[0019] The aforementioned Malus chinensis flower extract can be used as an active ingredient in the preparation of cosmetics with skin firming properties.

[0020] The aforementioned Malus chinensis flower extract can be used as an active ingredient in the preparation of cosmetics with skin-soothing properties.

[0021] The aforementioned Malus chinensis flower extract can be used as an active ingredient in the preparation of cosmetics for repairing skin.

[0022] In the aforementioned application of the Malus multiflorus flower extract, the mass fraction of the Malus multiflorus flower extract added to the cosmetic product is 0.01%-1.00%.

[0023] Compared with the prior art, the present application focuses on the flowers of Malus fragrans, wherein the flowers are dried in an oven, extracted with ethanol, concentrated in a vacuum, and fixed to volume with pure water. The flowers are then enriched by resin column chromatography, and finally concentrated in a vacuum and spray-dried to obtain a Malus fragrans flower extract powder. The extraction process of this application avoids the use of high-risk organic solvents such as n-hexane and ethyl acetate, making the entire process safer in the cosmetics industry; At the same time, the extraction process of the present application has been improved and optimized compared with the existing crabapple extraction process, thereby reducing some steps, greatly reducing production costs, and being more conducive to product implementation; Moreover, the Xifu Begonia flower extract prepared in the present application contains a variety of natural ingredients including dapoxetine, and the extract has great application value in the firming, soothing and repairing aspects of cosmetics; Therefore, the present invention provides a special extraction process for the crabapple flower, which has the advantages of being applicable to the cosmetics industry and having firming, soothing and repairing effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is a flow chart of the extraction method of the present invention; Figure 2 This is a liquid chromatogram of the afudamidin component of the standard product in the embodiment of the present invention; Figure 3 This is a liquid chromatogram of the afudamidin component of Example 1 in the present invention; Figure 4 This is a picture of the mean fluorescence intensity of elastin in Experiment 3 of the Examples of the present invention. DETAILED DESCRIPTION

[0025] The present invention will be further described below with reference to the accompanying drawings and examples, but they are not intended to limit the present invention.

[0026] Example 1. Preparation method of Malus chinensis flower extract, such as Figure 1As shown, the steps are: A. Take 1000 g of Malus chinensis flowers and place them in an oven to a constant weight. Use a high-speed grinder to grind them and pass them through a 50-mesh sieve to obtain a uniform dry flower powder. B. Add the dried flower powder to 2 L of 10% ethanol solution and perform reflux extraction at 85°C for 3 h to obtain a crude extract. After filtering the crude extract, centrifuge it at 10,000 × g for 20 min to separate the precipitate. After separation, collect the supernatant to obtain the Malus xifuensis flower extract. C. First, concentrate the extract of Malus chinensis flowers in vacuum to 1.2 L, then add purified water to make up to 1.6 L; centrifuge at 10,000 × g for 20 min at room temperature, collect the supernatant to obtain the concentrate, and use it for subsequent enrichment with macroporous adsorption resin; D. The concentrate was adsorbed on AB-8 resin at a flow rate of 1 BV / h. After completion, the resin column bed was cleaned with deionized water in a forward direction at a flow rate of 5 BV / h. The water-soluble impurities were removed by washing for 10 BV. The product was then desorbed with 2 BV of 70% ethanol to obtain an enriched solution at a flow rate of 3 BV / h. E. The enriched solution was concentrated to about 200 mL under reduced pressure and vacuum at 50°C; and then spray-dried to obtain the Malus xifuensis flower extract powder.

[0027] The crabapple flower extract prepared by the preparation method contains 0.5%-3% of chlorogenic acid, 1.0%-2.0% of rutin, 30.0%-79.0% of phlorizin, 0.1%-0.8% of astragaloside and 1.5%-8.5% of afudamicin.

[0028] The malus xifu flower extract has the effect of tightening the skin when used in cosmetics.

[0029] The Chinese crabapple flower extract can be used in cosmetics to soothe the skin.

[0030] The Chinese crabapple flower extract can be used in cosmetics to repair the skin.

[0031] The mass fraction of the malus xifu flower extract added to the cosmetic product is 0.02%.

[0032] Example 2. The preparation method of the Malus chinensis flower extract is similar to that of Example 1, and the steps are as follows: A. Take 2500 g of crabapple flowers and place them in an oven to a constant weight. Then grind and sieve to obtain a uniform dry flower powder. B. Add the dried flower powder to 15 L of 60% ethanol solution at a solid-liquid ratio of 1:30 (w / v), perform reflux extraction, and perform extraction at 85°C for 3 hours to obtain a crude extract. Filter the crude extract, centrifuge, and separate the precipitate. After separation, collect the supernatant to obtain the Malus xifuensis flower extract. C. First, vacuum concentrate the Malus chinensis flower extract to 3500 mL, then add purified water to make the volume 10 times the weight of the dried flower (v / w). Centrifuge at 10,000 × g for 20 min at room temperature, and collect the supernatant to obtain the concentrate. D. The concentrate was adsorbed on LX-T83 resin at a flow rate of 3 BV / h. After completion, the resin column bed was cleaned with deionized water in the forward direction at a flow rate of 1 BV / h. The water-soluble impurities were removed by washing for 12 BV. The product was then desorbed with 5 BV of 40% ethanol to obtain an enriched solution at a flow rate of 2 BV / h. E. The enriched solution was concentrated to about 2000 mL under reduced pressure and vacuum at 50°C; and then spray-dried to obtain the Malus chinensis flower extract powder.

[0033] The crabapple flower extract prepared by the preparation method contains 0.5%-3% of chlorogenic acid, 1.0%-2.0% of rutin, 30.0%-79.0% of phlorizin, 0.1%-0.8% of astragaloside and 1.5%-8.5% of afudamicin.

[0034] The malus xifu flower extract has the effect of tightening the skin when used in cosmetics.

[0035] The Chinese crabapple flower extract can be used in cosmetics to soothe the skin.

[0036] The Chinese crabapple flower extract can be used in cosmetics to repair the skin.

[0037] The mass fraction of the malus xifu flower extract added to the cosmetic product is 0.02%.

[0038] Example 3. The preparation method of the Malus chinensis flower extract is similar to that of Example 1, and the steps are as follows: A. Take 4200 g of Malus chinensis flowers and place them in an oven to a constant weight. Grind them to obtain a uniform dry flower powder. B. Add the dried flower powder to 14 L of 80% ethanol solution at a material-liquid ratio of 1:20 (w / v), perform reflux extraction, and perform extraction at 85°C for 2 h to obtain crude extract A. Separate and collect the residue, then add it back to 7 L of 75% ethanol solution for a second extraction for 2 h to obtain crude extract B. Combine crude extracts A and B, filter, and centrifuge to separate the sediment. After separation, collect the supernatant to obtain the Malus chinensis flower extract. C. First, concentrate the extract of Malus chinensis flowers under vacuum to 7 L, then add purified water to make the volume 12 times the weight of the dried flowers (v / w); centrifuge at room temperature, and collect the supernatant to obtain the concentrate; D. The concentrate was adsorbed on LX-T83 resin at a flow rate of 2 BV / h. After completion, the resin column bed was cleaned with deionized water in a forward direction at a flow rate of 4 BV / h. 8 BV of water was used to remove water-soluble impurities. The product was then desorbed with 8 BV of 50% ethanol to obtain an enriched solution at a desorption flow rate of 2 BV / h. E. The enriched solution was concentrated to about 1400 mL under reduced pressure and vacuum at 50°C; and then spray-dried to obtain the Malus xifuensis flower extract powder.

[0039] The crabapple flower extract prepared by the preparation method contains 0.5%-3% of chlorogenic acid, 1.0%-2.0% of rutin, 30.0%-79.0% of phlorizin, 0.1%-0.8% of astragaloside and 1.5%-8.5% of afudamicin.

[0040] The malus xifu flower extract has the effect of tightening the skin when used in cosmetics.

[0041] The Chinese crabapple flower extract can be used in cosmetics to soothe the skin.

[0042] The Chinese crabapple flower extract can be used in cosmetics to repair the skin.

[0043] The mass fraction of the malus xifu flower extract added to the cosmetic product is 0.02%.

[0044] Example 4. The preparation method of the Malus chinensis flower extract is similar to that of Example 1, and the steps are as follows: A. Take 6000 g of Malus chinensis flowers and place them in an oven to a constant weight, then grind them to obtain a uniform dry flower powder; B. Add the dried flower powder to 15 L of 45% ethanol solution at a material-liquid ratio of 1:15 (w / v), perform reflux extraction, and perform extraction at 85°C for 2 hours to obtain crude extract A. Separate and collect the residue, then add it back to 15 L of 35% ethanol solution for a second extraction, and perform extraction for 3 hours to obtain crude extract B. Crude extracts A and B are combined and filtered, and then centrifuged to separate the sediment. After separation, the supernatant is collected to obtain the extract of the Malus chinensis flower. C. First, concentrate the extract of Malus chinensis flowers under vacuum to 4000 mL, then add purified water to make the volume 15 times the weight of the dried flowers (v / w); centrifuge at room temperature, and collect the supernatant to obtain the concentrate; D. The concentrate was adsorbed on D101 resin at a flow rate of 4 BV / h. After completion, the resin column bed was cleaned with deionized water in a forward direction at a flow rate of 6 BV / h. Water-soluble impurities were removed by washing for 4 BV. The product was then desorbed with 10 BV of 30% ethanol to obtain an enriched solution at a desorption flow rate of 1 BV / h. E. The enriched solution was concentrated to about 1000 mL under reduced pressure and vacuum at 50°C; and then spray-dried to obtain the Malus chinensis flower extract powder.

[0045] The crabapple flower extract prepared by the preparation method contains 0.5%-3% of chlorogenic acid, 1.0%-2.0% of rutin, 30.0%-79.0% of phlorizin, 0.1%-0.8% of astragaloside and 1.5%-8.5% of afudamicin.

[0046] The malus xifu flower extract has the effect of tightening the skin when used in cosmetics.

[0047] The Chinese crabapple flower extract can be used in cosmetics to soothe the skin.

[0048] The Chinese crabapple flower extract can be used in cosmetics to repair the skin.

[0049] The mass fraction of the malus xifu flower extract added to the cosmetic product is 0.02%.

[0050] Verification of the composition and efficacy of the Xifu Begonia flower extract Experiment 1 Determination of total flavonoids and total polyphenols 1. Experimental methods: 1.1 Determination of total flavonoids content: Weigh 14.5 mg of rutin standard, dissolve it in 20 mL of dilute ethanol, and then dilute to 50 mL to prepare rutin working solution. Then, prepare 5% sodium nitrite solution, 10% aluminum nitrate solution, and sodium hydroxide (4.3 g / 100 mL) for the detection of total flavonoids in the Malus xifuensis flower extract.

[0051] The sample was prepared into a sample solution of about 10 mg / ml.

[0052] According to the flavonoid content determination operation table below, the corresponding solutions were added sequentially into a 25 mL volumetric flask. After all the systems were added, water was added to the scale, and the solution was shaken and allowed to stand for 15 minutes. 200 μL was taken into a 96-well plate and the absorbance was measured at a wavelength of 500 nm using a UV-visible spectrophotometer.

[0053] Table 1 Flavonoid content determination operation table 1.2 Determination of total polyphenol content: The experimental method for determining the total polyphenol content refers to the operating method of the "Spectrophotometric Method for Determination of Total Polyphenols in the 2022 Edition of the National Health Commission's Bulletin" and makes certain modifications.

[0054] Weigh 20 mg of gallic acid standard, add 10 mL of methanol, and dilute to 100 mL with pure water. Shake well, and store in the dark. This is referred to as the gallic acid standard stock solution (200 mg / L). Use pure water to prepare a series of standard working solutions of 0 mg / L, 40 mg / L, 80 mg / L, 120 mg / L, 160 mg / L, and 200 mg / L from the gallic acid standard stock solution.

[0055] Weigh 7 g of anhydrous sodium carbonate and dissolve it in pure water to make up to 100 ml to prepare a 7% sodium carbonate solution.

[0056] Samples were prepared and measured according to Examples 1-4.

[0057] The total polyphenol content of the sample was determined according to the total polyphenol content determination operation table in the table below. After all the systems were added, they were shaken evenly. After standing at room temperature for 90 min, 200 μL was taken into a 96-well plate and the absorbance was measured at a wavelength of 750 nm using a UV-visible spectrophotometer.

[0058] Table 2 Operation table for determination of total polyphenol content According to the experimental method for determining the content of total flavonoids, the standard curve of rutin is y = 0.0073x-0.0178, R 2 =0.9988; According to the experimental method for determining the total polyphenol content, the standard curve of gallic acid is y = 0.0023x-0.0021, R 2 =0.9999.

[0059] Experimental results Table 3 Total flavonoids and total polyphenols contents of samples from Examples 1, 2, 3, and 4 3. Experimental Conclusion Experimental tests have shown that the samples prepared by the preparation method described in the present invention have high contents of flavonoids and polyphenols, accounting for approximately 42.3%-45.6% and 41.5%-42.1% respectively, indicating that the preparation method can effectively enrich biologically active ingredients.

[0060] Quantitative analysis of active ingredients in samples of Experiment 2 Liquid phase analysis was performed on the samples prepared in Examples 1, 2, 3, and 4 to analyze the bioactive components contained in the extracts.

[0061] Experimental methods Methanol and water (1% formic acid) were used as the mobile phase, and an Agilent Poroshell 120 SB-Aq (4.6*150 mm, 5 μm) column was used. A DAD detector was used for detection. The detection wavelengths were set to 257 nm, 260 nm, 266 nm, 285 nm, and 327 nm. Gradient elution was adopted. The elution program is shown in the following table: elution program for liquid chromatography detection of Malus chinensis flower extract.

[0062] Table 4 Elution procedure for liquid chromatography detection of Malus chinensis flower extract 2. Experimental Results Table 5 Liquid chromatography test results of Malus chinensis flower extract 3. Experimental Conclusion like Figure 2 、 3 As shown in Table 5, liquid phase analysis revealed that the Malus chinensis flower extract prepared in the embodiment of the present invention contained ingredients such as afudamicin, chlorogenic acid, phloridzin, rutin, quercetin, and astragalin. The chlorogenic acid content of the crabapple flower extract obtained by the method is between 0.5% and 3%, the rutin content is between 1.0% and 2.0%, the phloridzin content is between 30.0% and 79.0%, the astragalin content is between 0.1% and 0.8%, and the afudamicin content is between 1.5% and 8.5%. Among them, afudamicin was discovered for the first time in the flowers of Malus xifu, with a content of about 5.6%. So far, there have been no other reports on the detection and characterization of this component in Malus xifu.

[0063] Experiments 3, 4, and 5 intend to evaluate whether the extract samples have a firming effect by detecting the elastin transcription level, protein expression level, ROS clearance rate, and SOD enzyme activity in human dermal fibroblasts.

[0064] Experiment 3: Evaluation of the efficacy of improving elastin expression 1. Experimental Methods (1) MTT assay to detect the toxicity of samples to fibroblasts 1) Cell seeding: Cells were seeded into 96-well plates (5 × 10 3 / well), and cultured at 37°C, 5% CO2 for 24 h; 2) Administration: The sample group was treated with culture medium containing the sample at different concentrations (0.039%, 0.078%, 0.156%), while the normal control group and blank group were replaced with fresh culture medium and incubated at 37°C, 5% CO2 for 24 h. 3) After incubation, add MTT solution to each well and continue incubation for 4 h; 4) Remove the culture medium, add DMSO solution, shake to mix, and measure the absorbance at 490 nm.

[0065] (2) Determination of relative expression of elastin gene 1) Cell seeding: Fibroblasts were plated at a density of 5 × 10 3 Each well was inoculated into a 6-well plate and cultured in an incubator (37°C, 5% CO2) for 24 h; 2) Modeling: After the incubation, the culture medium was removed and washed 1-2 times with D-Hanks. Then, the sample group, negative control group and positive control group were irradiated with UVA (9 J / cm 2 ), the blank control group was not irradiated; 3) Administration: After modeling, the blank control group and the negative control group were replaced with fresh culture medium, and the positive group and the sample group were replaced with fresh culture medium containing the positive drug (TGF-β 100 ng / mL) and the test sample (Example 3 or 4), respectively. The cells were incubated at 37°C and 5% CO2 for 24 h. 4) Sample collection: After the culture is completed, the cells are sampled, the total RNA of each experimental group is extracted, cDNA is synthesized, and q-PCR is used for detection β-actin and gene expression of target genes; 5) Use β-actin As an internal reference for gene expression, the relative RNA expression of the target gene was calculated using the following formula.

[0066] , , .

[0067] (3) Determination of elastin expression 1) Cell seeding: Fibroblasts were seeded in 12-well plates (5 × 10 4 / well), and cultured in an incubator (37°C, 5% CO2) for 24 h; 2) Modeling and drug administration were the same as in (2) Determination of relative elastin gene expression; 3) After incubation, discard the solution and proceed with washing, fixation, permeabilization, and antibody incubation. Take photos under a fluorescence microscope and use ImageJ software to quantify the immunofluorescence results. S represents the mean fluorescence intensity. 2. Experimental Results (1) Effect of Begonia flower extract on fibroblast toxicity Table 6 Toxicity of the samples in the examples to fibroblasts According to the experimental results, the sample showed no cytotoxicity when the concentration was below 0.078%.

[0068] Combined with the results of the toxicity experiment in Experiment 8, the addition amount of 0.02% by mass was selected as the addition concentration of the example sample in the subsequent efficacy experiment. (2) Elastin gene expression test results Table 7 Detection results of elastin gene expression (3) Detection results of elastin expression Table 8 Detection results of mean fluorescence intensity of elastin 3. Experimental Conclusion According to the test results, the relative expression level and mean fluorescence intensity of the elastin gene in the negative control group were significantly downregulated compared with those in the blank control group (P<0.05), indicating that the stimulation conditions of this test were effective.

[0069] Compared with the negative control group, the relative expression level and mean fluorescence intensity of elastin gene in the positive control group were significantly increased (P<0.05), indicating that this experimental system is stable and reliable. The relative expression level and mean fluorescence intensity of the elastin gene in fibroblasts after treatment in the sample groups of Examples 3 and 4 were significantly upregulated compared with those in the negative control group (P<0.05); This shows that the sample prepared according to the present invention has a firming effect.

[0070] Experiment 4 ROS clearance rate detection 1. Experimental Methods (1) MTT assay for toxicity of samples to keratinocytes (HaCaT) was performed according to the method in Experiment 3. (2) ROS clearance rate determination 1) Cell seeding: Keratinocytes (HaCaT) were plated at a density of 1×10 5 pc / well were seeded in 12-well plates and cultured at 37°C, 5% CO2 for 24 h; 2) Administration: After the incubation period, the culture medium was removed and the cells were rinsed 1-2 times with D-Hanks. Fresh culture medium was added to the blank control group and the negative control group, and fresh culture medium containing N-acetyl-L-cysteine ​​and the sample of Example 3 or 4 was added to the positive control group and the sample group, respectively. The cells were incubated at 37°C, 5% CO2 for 24 h. 3) Modeling: After incubation, the culture medium was removed and washed 1-2 times with D-Hanks, and then the modeling was performed. The sample group, negative control group, and positive control group were exposed to 80 mJ / cm 2 UVB ultraviolet light irradiation, while the blank control group was placed in the dark without radiation treatment; 4) Detection: After staining the cells using a reactive oxygen species detection kit, photograph them under a fluorescence microscope. Analyze the fluorescence intensity (S) of each cell group using ImageJ, and calculate the ROS clearance rate according to the formula.

[0071] 2. Experimental Results (1) Effect of Begonia flower extract on keratinocyte toxicity Table 9 Toxicity of Example Samples to Human Immortalized Keratinocytes (HaCaT) According to the experimental results, the sample showed no cytotoxicity when the concentration was below 0.039%.

[0072] Combined with the results of the toxicity experiment in Experiment 8, the addition amount of 0.02% by mass was selected as the addition concentration of the example sample in the subsequent efficacy experiment. (2) ROS scavenging activity test results Table 10 ROS scavenging activity test results 3. Experimental Conclusion According to the test results, the average fluorescence intensity of ROS in the negative control group was significantly increased compared with the blank control group (P<0.05), indicating that the stimulation conditions of this test were effective.

[0073] The mean fluorescence intensity of ROS in the positive control group was significantly weakened compared with that in the negative control group (P<0.05), indicating that this experimental system is stable and reliable.

[0074] The mean fluorescence intensity of ROS in keratinocytes after treatment in the sample groups of Examples 3 and 4 was significantly reduced compared with that in the negative control group (P<0.05), indicating that the samples had an antioxidant effect.

[0075] Experiment 5: SOD activity detection 1. Experimental Methods (1) The MTT assay was used to detect the toxicity of the samples to keratinocytes (HaCaT) with reference to the results in Experiment 4.

[0076] (2) SOD activity determination 1) Cell seeding: Keratinocytes (HaCaT) were plated at 6×10 5 Each well was inoculated into a 6-well plate and cultured in an incubator (37°C, 5% CO2) for 24 h; 2) Modeling: After incubation, the culture medium was removed and washed three times with D-Hanks. 1 ml of D-Hanks was added to each culture dish to submerge the cells. Each group was exposed to 80 mJ / cm 2 UVB modeling; the blank control group was placed in a dark place without radiation treatment; 3) Administration: After modeling, the cells were gently rinsed once or twice with D-Hanks; the blank control group and the negative control group were replaced with fresh culture medium, the positive control group was replaced with fresh culture medium containing N-acetyl-L-cysteine, and the sample group was replaced with fresh culture medium of the sample (Example 3 or 4), and incubated at 37°C, 5% CO2 for 24 h. 4) Cell collection: After the culture is completed, collect cell samples for subsequent SOD activity determination (refer to the SOD enzyme activity kit instructions for specific procedures).

[0077] Experimental results Table 11 SOD activity test results 3. Experimental Conclusion According to the test results, the SOD activity of the negative control group was significantly reduced compared with the blank control group (P<0.05), indicating that the stimulation conditions were effective. Compared with the negative control group, the SOD activity of the positive control group increased significantly (P<0.05), indicating that this experimental system is stable and reliable; Compared with the negative control group, the SOD activity in the keratinocytes treated with the sample groups of Examples 3 and 4 was significantly increased (P<0.05), indicating that the samples had an antioxidant effect.

[0078] Experiment 6: Evaluation of the efficacy of improving the expression of filaggrin and loricrin 1. Experimental Methods (1) The toxicity test of samples on human immortalized keratinocytes (HaCaT) was performed by MTT assay, referring to Experiment 4.

[0079] (2) Determination of relative expression levels of filaggrin and loricrin genes 1) Cell seeding: Keratinocytes were plated at 3×10 5 Each well was inoculated into a 6-well plate and cultured in an incubator (37°C, 5% CO2) for 24 h; 2) Administration: After the incubation period, the culture medium was removed and the cells were rinsed 1-2 times with D-Hanks. Fresh culture medium was added to the blank control group, fresh culture medium containing the sample of Example 3 or 4 was added to the sample group, and fresh culture medium containing WY-14643 (100 μM) was added to the positive control group. The cells were incubated at 37°C, 5% CO2 for 24 h. 3) After the culture is complete, cells are collected and processed and gene expression is calculated using the same procedures as in Experiment 3.

[0080] (3) Determination of filaggrin and loricrin expression 1) Cell seeding: Immortalized human keratinocytes (HaCaT) were seeded in 6-well plates (6 × 10 5 / well), and cultured in an incubator (37°C, 5% CO2) for 24 h; 2) Administration: The administration method is the same as in Experiment 4 (2) above; 3) After incubation, cells were collected and lysed. ELISA kit instructions were used for detection, and the upregulation rate was calculated using the following formula.

[0081] Experimental results (1) Detection results of relative expression levels of filaggrin and loricrin genes Table 12 Detection results of filaggrin gene expression Table 13 Detection results of loricrin gene expression (2) Detection results of filaggrin and loricrin expression Table 14 Detection results of filaggrin expression Table 15 Detection results of loricrin expression 3. Experimental Conclusion Compared with the blank control group, the relative gene expression levels and protein expression levels of filaggrin and loricrin (P<0.05) in the positive control group were significantly upregulated (P<0.05), indicating that this experimental system is stable and reliable.

[0082] Compared with the blank control group, the relative gene expression levels and protein expression levels of filaggrin and loricrin in the fibroblasts treated with the sample groups of Examples 3 and 4 were significantly upregulated, demonstrating that the samples have a repairing effect.

[0083] Experiment 7 TRPV1 Detection of relative gene expression 1. Experimental Methods (1) The toxicity test of samples on human immortalized keratinocytes (HaCaT) was performed by MTT assay, referring to Experiment 4.

[0084] (2) TRPV1 Determination of relative gene expression 1) Cell seeding: Keratinocytes (HaCaT) were plated at 5×10 3 Each well was inoculated into a 6-well plate and cultured at 37°C, 5% CO2 for 24 h; a blank control group (0 mM CAP + 0 mM sample), a negative control group (15 μmol / L CAP + 0 mM sample), a positive control group (15 μmol / L CAP + 15.6 μg / mL 4-tert-butylcyclohexanol), and a sample group (15 μmol / L CAP + sample from Example 3 or 4) were set up respectively; 2) Modeling and drug administration: After the incubation period, the cells were gently rinsed once or twice with D-Hanks. Fresh culture medium was added to the blank control group, fresh culture medium containing CAP (15 μmol / L) was added to the negative control group, fresh culture medium containing the corresponding concentration of sample (Example 3 or 4) and CAP (15 μmol / L) was added to the sample group, and fresh culture medium containing 4-tert-butylcyclohexanol (15.6 μg / mL) and CAP (15 μmol / L) was added to the positive control group. The cells were cultured at 37°C, 5% CO2 for 24 h. 3) Collect cells, process, and calculate gene expression using the same procedures as in Experiment 3.

[0085] Experimental results Table 16 TRPV1 Gene relative expression detection results 3. Experimental Conclusion According to the test results, compared with the blank control group, the negative control group TRPV1 The relative expression of genes was significantly upregulated (P<0.05), indicating that the stimulation conditions were effective; Compared with the negative control group, the positive control group TRPV1 The relative expression of genes was significantly downregulated (P<0.05), indicating that this experimental system is stable and reliable; Compared with the negative control group, the keratinocytes in the sample groups of Examples 3 and 4 were TRPV1 The relative expression levels of the genes were significantly downregulated (P<0.05), with downregulation rates of 26% and 32%, respectively, proving that the samples have soothing effects.

[0086] Effects of 8 samples in Experiment 8 on TNF-α, IL-1α and NO levels in monocytes and macrophages of mice after LPS modeling 1. Experimental Methods (1) MTT assay to detect the toxicity of samples to mouse mononuclear macrophages (Raw264.7) 1) Cell seeding: Cells were seeded into 96-well plates (5 × 10 3 / well), and cultured at 37°C, 5% CO2 for 24 h; 2) Administration: The sample group was added with culture medium containing the sample (Example 3 or 4) at the corresponding concentration, the normal control group was replaced with fresh culture medium, and the blank group was added with blank culture medium. The cells were incubated at 37°C, 5% CO2 for 24 h. 3) After incubation, add MTT solution to each well and continue incubation for 4 h; 4) Remove the culture medium, add DMSO solution, shake to mix, and measure the absorbance at 490 nm.

[0087] (2) Detection of TNF-α, IL-1α and NO content in macrophages after LPS modeling 1) Cell inoculation: Macrophages were seeded at a density of 3 × 10 5 Each well was inoculated into a 6-well plate and cultured at 37°C, 5% CO2 for 24 h; a blank control group, a negative control group (0.5 μg / mL LPS), a positive control group (0.5 μg / mL LPS + 0.01% dexamethasone), and a sample group (0.5 μg / mL LPS + Example 3 or Example 4 sample) were set up respectively; 2) LPS modeling and drug administration: According to the experimental groups, the modeling and drug administration groups were treated with a certain final concentration of LPS and drug (37°C, 5% CO2) and cultured for 24 h ± 2 h; 3) Cell supernatant collection: After incubation, collect the cell culture supernatant into a 1.5 mL sterile centrifuge tube and assay for TNF-α, IL-1α, and NO levels (perform the assay according to the kit instructions).

[0088] Q: TNF-α content, IL-1α content, NO content 2. Experimental Results (1) Effect of Begonia flower extract on macrophage cytotoxicity Table 17 Toxicity of samples to macrophages (Raw264.7) According to the experimental results, the sample showed no cytotoxicity when the concentration was below 0.0195%; Combined with the results of the toxicity experiment in Experiment 3, an addition concentration of 0.02% by mass was selected as the addition concentration for subsequent experiments.

[0089] (2) TNF-α, IL-1α, and NO content test results Table 18 TNF-α relative expression detection results Table 19 IL-1α relative expression detection results Table 20 Test results of NO generation inhibition 3. Experimental conclusions: Compared with the blank control group, the TNF-α, IL-1α and NO contents in the negative control group were significantly increased (P<0.05), indicating that the stimulation conditions were effective. Compared with the negative control group, the TNF-α, IL-1α and NO contents in the positive control group were significantly decreased (P<0.05), indicating that the experimental system was stable and reliable. Compared with the negative control group, the sample groups of Examples 3 and 4 significantly inhibited the production of TNF-α, IL-1α and NO in the treated macrophages (P<0.05), demonstrating that the samples have soothing effects.

Claims

1. Malus chinensis flower extract, characterized by: Calculated by mass percentage, it contains at least 1.5%-8.5% of afdapoxetine.

2. The Malus chinensis flower extract according to claim 1, wherein: Calculated by mass percentage, it contains at least 0.5%-3% of chlorogenic acid, 1.0%-2.0% of rutin, 30.0%-79.0% of phlorizin, 0.1%-0.8% of astragaloside and 1.5%-8.5% of afudamicin.

3. A method for preparing a Malus chinensis flower extract, characterized in that: The steps include: A. Pretreatment: Place the flowers of Malus chinensis in an oven and dry them until they are constant in weight to obtain dried flowers, which are then crushed to obtain dried flower powder; B. Preparation of extract: Dried flower powder is extracted by reflux extraction to obtain a crude extract, using ethanol as the extraction reagent at a solid-liquid ratio of 1:10-30 (w / v); the crude extract is filtered and centrifuged to obtain an extract; C. Preparation of concentrated solution: vacuum concentrate the extract, add pure water to make up to volume, and centrifuge, collecting the supernatant after centrifugation to obtain the concentrated solution; D. Preparation of enriched solution: The concentrated solution is adsorbed on the resin at a certain flow rate. After adsorption, the resin column bed is cleaned by passing deionized water through the column in a forward direction, and the product is desorbed by passing ethanol through the column in a forward direction to obtain the enriched solution; E. Concentration and drying into powder: the enriched liquid is vacuum concentrated and then spray-dried to obtain the Malus chinensis flower extract powder.

4. The method for preparing the Malus chinensis flower extract according to claim 3, wherein The preparation of the extract described in step B is specifically as follows: The extraction reagent is 10%-80% ethanol, the extraction temperature is 85°C, the number of extractions is 1-2, and the extraction time for each time is 2-3 hours; after the crude extract is filtered, it is separated by a centrifuge for 20 minutes at a centrifugal force of 10,000 × g, and the precipitate is discarded to obtain the extract.

5. The method for preparing the Malus chinensis flower extract according to claim 3, wherein The preparation of the concentrated solution described in step C is specifically as follows: The extract was vacuum concentrated to 4-10 times the weight of the dried flowers (v / w), and purified water was added to make the volume 8-15 times the weight of the dried flowers (v / w) to obtain a fixed volume crude solution; the fixed volume crude solution was centrifuged for 20 min at a centrifugal force of 10,000 × g and a temperature of 25°C; after centrifugation, the supernatant was collected to obtain a concentrated solution.

6. The method for preparing the Malus chinensis flower extract according to claim 3, wherein: The preparation of the enrichment solution described in step D is specifically as follows: The concentrate was adsorbed on resin at a flow rate of 1-4 BV / h, with a resin to dry flower mass ratio of 2:1 (v / m); After the resin adsorption is completed, the resin column bed is cleaned with deionized water in the forward direction. The cleaning flow rate is controlled at 1-6 BV / h and the cleaning time is 4-12 BV. After the resin is washed with water, 2-10 BV of 30%-70% ethanol is used to pass through the column in the forward direction to desorb the product to obtain the enriched liquid. The desorption flow rate is controlled at 1-3 BV / h.

7. Use of the Malus xifuii flower extract according to any one of claims 1 to 6 as an active ingredient in the preparation of cosmetics having skin-tightening effects.

8. Use of the Malus niger flower extract according to any one of claims 1 to 6 as an active ingredient in the preparation of skin-soothing cosmetics.

9. Use of the Malus niger flower extract according to any one of claims 1 to 6 as an active ingredient in the preparation of cosmetics for repairing skin.

10. Use of the Malus niger flower extract according to any one of claims 7 to 9 as an active ingredient in the preparation of cosmetics, characterized in that: The mass fraction of the malus xifu flower extract added to the cosmetic product is 0.01%-1.00%.

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