Pomegranate flower skin care extract as well as preparation method and application thereof

Abstract

The invention relates to the technical field of skin care, and discloses a pomegranate flower skin care extract and a preparation method and application thereof.The preparation method comprises the steps that firstly, a pomegranate flower whitening, antibacterial and anti-aging extract is obtained through microwave extraction, ultrasonic extraction and ethanol extraction; and carrying out compound enzyme enzymolysis, organic phase extraction and column chromatography separation to obtain a fourth pomegranate flower extract with a moisturizing function and fifth and sixth pomegranate flower extracts with antioxidant functions. The extracts with different effects of whitening, resisting aging, resisting bacteria, resisting oxidation, moisturizing and the like are obtained through staged extraction, limited pomegranate flower resources are fully utilized, the pomegranate flower extracts can be fully applied to the field of wash supplies, and the application scene of the pomegranate flower extracts is widened; the invention provides a preparation method of the pomegranate flower functional extract.

Description

Technical Field

[0001] The present invention relates to the technical field of skin care, and particularly relates to a pomegranate flower skin care extract, a preparation method thereof, and an application thereof. Background Art

[0002] The statements in this section only provide background information related to the disclosure of the present application and may not constitute prior art.

[0003] Pomegranate flower is the flower of Punica granatum L. of the genus Punica (Punicaceae). Pomegranate flower tastes sour, astringent, and warm in nature, and has the effects of clearing heat and detoxifying, strengthening the stomach and moistening the lungs, astringing the intestine and stopping bleeding, sterilizing and relieving pain, etc. Modern pharmacological research has found that pomegranate flower contains various biological components such as polyphenols, flavonoids, and polysaccharides, and also plays an important role in anti-inflammatory, liver protection, treatment of diabetes, etc. At present, methods such as water extraction and alcohol extraction are mainly used to obtain the above-mentioned pomegranate flower active substances.

[0004] The existing extraction methods mostly adopt the method of extracting a single substance at one time. Although a high-concentration certain type of pomegranate flower active substance can be purified, the extraction rate of the remaining active substances in pomegranate flower is relatively low; the pomegranate flower residue after extraction is directly discarded, resulting in the waste of the active substances in the pomegranate flower residue, and the utilization rate of pomegranate flower is relatively low, there is a waste of resources.

[0005] The various biological components such as polyphenols, flavonoids, and polysaccharides in pomegranate flower can synergistically exert their effects themselves, but the component ratios of polyphenols, flavonoids, and polysaccharides obtained by different extraction methods are different. Therefore, the effects of pomegranate flower extracts obtained by different methods are different. Re-compounding the separately extracted substances according to a ratio has problems such as complex operation and single substance type. Therefore, how to simply prepare an efficient pomegranate flower extract and make full use of pomegranate flower resources is worthy of exploration. Summary of the Invention

[0006] The purpose of the present invention is to: aiming at the problems of complex preparation and resource waste of the current pomegranate flower extract, provide a pomegranate flower skin care extract, a preparation method thereof, and an application thereof. By extracting the effective components of pomegranate flower in stages, pomegranate flower extracts with different functions are obtained, which not only broadens the utilization field of pomegranate flower extracts, but also makes full use of pomegranate flower resources.

[0007] The technical solution of the present invention is as follows:

[0008] A preparation method of a pomegranate flower skin care extract, comprising the following steps:

[0009] Step (1): Fresh pomegranate flowers are freeze-dried with liquid nitrogen and quickly ground into powder. According to the solid-liquid ratio of 1:25 g / mL, 70 - 80% ethanol solution is added as the extraction agent. First, it is extracted with a microwave device with a microwave power of 300 - 500 W and a microwave time of 30 - 60 seconds. Then, it is further crushed and broken by an ultrasonic device with an ultrasonic power of 400 - 500 W, a temperature of 50 - 60 °C, and an extraction time of 45 - 60 min. The residue is re-extracted once according to the above method. The two extracts are combined, filtered by suction, and the ethanol solution is removed by rotary evaporation at 60 °C to obtain the pomegranate flower whitening, antibacterial, and anti-aging extract, namely the first pomegranate flower extract. The obtained pomegranate flower residue is used for the next extraction step.

[0010] Step (2): A complex enzyme containing cellulase and papain (the mass ratio of cellulase to papain is 1:1) with a mass fraction of 1.0% (mass of the complex enzyme × 100% / mass of the pomegranate flower residue) is dissolved in a potassium hydrogen phosphate - citric acid buffer solution with a pH of 6 - 7. The complex enzyme is pre-activated in a 50 °C water bath for 10 min before participating in enzymatic hydrolysis. According to the liquid-solid ratio of (15 - 20) mL:1 g, the pomegranate flower residue is added to the buffer solution mixed with the complex enzyme, and enzymatic hydrolysis is carried out in a 50 °C water bath for 100 - 120 min. After enzymatic hydrolysis, it is concentrated by suction filtration and rotary evaporation to 1 / 4 of the total volume, and then 4 times the volume of anhydrous ethanol is added, and it is left to stand at 4 °C for 24 h. The precipitate is collected to obtain the second pomegranate flower extract.

[0011] Step (3): An appropriate amount of pure water is added to the second pomegranate flower extract to dissolve it fully. Subsequently, a mixture of chloroform and n-butanol with a volume ratio of 3:1 equal to the volume of the aqueous phase is added, and after thorough mixing, the upper aqueous phase is collected. Then, petroleum ether equal to the volume of the obtained liquid is added, and after thorough mixing, the lower aqueous phase is collected to obtain the third pomegranate flower extract.

[0012] Step (4): The above-mentioned third pomegranate flower extract is separated by column chromatography on macroporous resin and eluted successively with three gradients of water, 30% ethanol, and 50% ethanol. After vacuum freeze-drying, the fourth pomegranate flower extract, the fifth pomegranate flower extract, and the sixth pomegranate flower extract are obtained respectively.

[0013] The fourth pomegranate flower extract is the pomegranate flower moisturizing extract; the fifth pomegranate flower extract is the pomegranate flower antioxidant extract; the sixth pomegranate flower extract is the pomegranate flower polyphenol extract.

[0014] A pomegranate flower skin care extract, comprising the following raw materials in parts by weight:

[0015] 2 - 4 parts of the first pomegranate flower extract, 3 - 8 parts of the fourth pomegranate flower extract, 1 - 2 parts of the fifth pomegranate flower extract, and 1 - 2 parts of the sixth pomegranate flower extract.

[0016] After extraction, the parts by weight of the pomegranate flower extracts at each stage are in the above ratios. After compounding the pomegranate flower extracts at each stage, the above-mentioned pomegranate flower skin care extract can be obtained, which exerts the overall effects of antioxidant, whitening, anti-aging and moisturizing.

[0017] The first pomegranate flower extract comprises components at the following concentrations: total phenolic compounds in pomegranate flower 0.48 - 0.51 mg GAE / gDW, total flavonoids in pomegranate flower 0.02 - 0.03 mg QE / g DW, anthocyanins in pomegranate flower 0.10 - 0.12 mg C3GE / g DW, and pomegranate flower polysaccharide 0.31 - 0.33 mg / mL.

[0018] The fourth pomegranate flower extract comprises components at the following concentrations: total phenolic compounds in pomegranate flower 0.022 - 0.025 mg GAE / gDW, total flavonoids in pomegranate flower 0.009 - 0.012 mg QE / g DW, anthocyanins in pomegranate flower 0.039 - 0.045 mg C3GE / g DW, and pomegranate flower polysaccharide 0.8 - 0.9 mg / mL.

[0019] The fifth pomegranate flower extract comprises components at the following concentrations: total phenolic compounds in pomegranate flower 0.39 - 0.41 mg GAE / gDW, total flavonoids in pomegranate flower 0.21 - 0.23 mg QE / g DW, anthocyanins in pomegranate flower 0.15 - 0.18 mg C3GE / g DW, and pomegranate flower polysaccharide 0.26 - 0.3 mg / mL.

[0020] The sixth pomegranate flower extract comprises components at the following concentrations: total phenolic compounds in pomegranate flower 0.62 - 0.69 mg GAE / gDW, total flavonoids in pomegranate flower 0.35 - 0.37 mg QE / g DW, anthocyanins in pomegranate flower 0.31 - 0.35 mg C3GE / g DW, and pomegranate flower polysaccharide 0.012 - 0.04 mg / mL.

[0021] On the other hand, the present invention provides an application of the first pomegranate flower extract in the preparation of washing and care products or health products with antioxidant, antibacterial, whitening or anti-aging effects.

[0022] Preferably, the antioxidant is to enhance the activities of catalase and superoxide dismutase. The antibacterial is to inhibit the growth of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. The whitening is to inhibit the activity of tyrosinase in vitro, reduce the content of skin melanin and increase skin brightness. The anti-aging is to enhance autophagy so as to enhance the anti-aging function.

[0023] On the other hand, the present invention provides an application of the fourth pomegranate flower extract in the preparation of moisturizing washing and care products.

[0024] Preferably, the washing and care products are, for example, skin care products, cosmetics, body wash, etc.

[0025] On the other hand, the present invention provides the application of the fifth pomegranate flower extract in the preparation of antioxidant skin care products or health care products.

[0026] On the other hand, the present invention provides the application of the sixth pomegranate flower extract in the preparation of antioxidant skin care products or health care products.

[0027] Compared with the existing technology, the beneficial effects of the present invention are as follows:

[0028] 1. A pomegranate flower skin care extract and its preparation method adopt a staged extraction method to separately extract various effective components in pomegranate flowers in batches, obtaining a variety of extracts with different active effects; not only reducing the waste of pomegranate flower resources, fully extracting the effective active components in pomegranate flowers, but also providing a preparation idea for developing pomegranate flower extract products with more effects and broadening the application prospects of pomegranate flowers;

[0029] 2. A pomegranate flower skin care extract, its preparation method and application. In this application, not only the first pomegranate flower extract with whitening, antibacterial and anti-aging effects is extracted from pomegranate flowers, but also the moisturizing extract of pomegranate flowers (the fourth pomegranate flower extract) is obtained. After use, it can significantly improve the water content of the skin; the fifth pomegranate flower extract and the sixth pomegranate flower extract can exert strong antioxidant properties, and each extract can be separately applied in products with various effects; or integrally applied to overall improve the content of pomegranate flower active ingredients and the utilization rate of pomegranate flowers;

[0030] 3. A pomegranate flower skin care extract, its preparation method and application. By precisely separating and extracting the pomegranate flower active substance group, it provides an innovative raw material solution for the development of cosmetics and functional health care products with targeted effects. Description of the Drawings

[0031] Figure 1 The inhibition rate of tyrosinase by each pomegranate flower extract at different concentrations;

[0032] Figure 2 The cytotoxicity results of the first pomegranate flower extract;

[0033] Figure 3 The antioxidant effect of the first pomegranate flower extract on keratinocytes; Note: A is the CAT activity of the first pomegranate flower extract at different concentrations on HaCaT cells; B is the SOD activity of the first pomegranate flower extract at different concentrations on HaCaT cells; compared with the blank group and the control group (galactose), ****P < 0.0001, ***P < 0.001, **P < 0.01.

[0034] Figure 4Autophagy effect of the first pomegranate flower extract on keratinocytes; Note: A represents the autophagy ability of cells in the blank group; B represents the autophagy ability of cells in the control group (galactose group); C represents the autophagy ability of cells in the first pomegranate flower extract (62.5 μg / mL) group; D represents the autophagy ability of cells in the first pomegranate flower extract (125 μg / mL) group; E represents the statistical chart of fluorescence area. Compared with the blank group and the control group (galactose group), and the first pomegranate flower extract at different concentrations, ****P < 0.0001; The scale bar in the figure is 100 μm;

[0035] Figure 5 To verify the moisturizing effect of the fourth pomegranate flower extract and the whitening effect of the first pomegranate flower extract through human experiments; Note: A represents the change in skin moisture content after applying the fourth pomegranate flower extract; B represents the change in skin melanin content after applying the first pomegranate flower extract; C represents the change in skin heme content after applying the first pomegranate flower extract; D represents the change in skin glossiness after applying the first pomegranate flower extract. Specific embodiments

[0036] The specific embodiments listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions made to the embodiments described below are also within the scope of the present invention. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention. Conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. All reagents or instruments not indicating the manufacturer can be obtained as conventional products through commercial purchase. To better illustrate the present invention, numerous specific details are given in the following specific embodiments. Those skilled in the art should understand that the present invention can be implemented without some specific details. In other embodiments, methods, means, equipment, and steps well-known to those skilled in the art are not described in detail to highlight the gist of the present invention.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. Without special instructions, the units used in this specification are all international standard units, and the numerical values and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0038] The characteristics and performance of the present invention will be further described in detail below in combination with the embodiments.

[0039] Example 1

[0040] Fresh pomegranate flowers were freeze-dried with liquid nitrogen and quickly ground and pulverized. A 70% ethanol solution was added as an extractant at a solid-liquid ratio of 1:25 g / mL. The flowers were first extracted with a microwave device at a microwave power of 400 W and a microwave time of 60 seconds. The flowers were then further pulverized and broken with an ultrasonic device at a power of 500 W, a temperature of 60°C, and an extraction time of 45 minutes. The residue was extracted once again according to the above method, and the two extracts were combined, filtered, and the ethanol solution was removed by rotary evaporation at 60°C to obtain a pomegranate flower whitening, antibacterial and anti-aging extract, i.e., the first pomegranate flower extract. The obtained pomegranate flower residue was used for the next step of extraction.

[0041] Disodium hydrogen phosphate-citric acid buffer with pH 6.8 was used as the extraction solvent, and a composite enzyme with a mass fraction of 1% (the mass ratio of cellulase to papain was 1:1) was added at a liquid-to-solid ratio of 20:1 (mL / g). After pre-activation in a 50°C water bath for 10 minutes, 40g of pomegranate flower residue was added, and enzymolysis was performed in a 50°C water bath for 120 minutes. After enzymolysis, the mixture was concentrated by suction filtration and rotary evaporation to 1 / 4 of the total volume, and then 4 times the volume of anhydrous ethanol was added, and the mixture was placed at 4°C for 24 hours, and the precipitate was collected to obtain a second pomegranate flower extract. An appropriate amount of pure water was added to the second pomegranate flower extract to fully dissolve it. Subsequently, 1 / 4 volume of chloroform and n-butanol were added to the aqueous phase, and the upper aqueous phase was collected after sufficient mixing. Petroleum ether was then added to the resulting liquid, and the mixture was fully mixed, and the lower aqueous phase was collected to obtain a third pomegranate flower extract.

[0042] The third pomegranate flower extract is separated by column chromatography using a macroporous resin, and eluted using three gradients of water, 30% ethanol and 50% ethanol in sequence. After vacuum freeze-drying, a fourth pomegranate flower extract, a fifth pomegranate flower extract and a sixth pomegranate flower extract are obtained respectively.

[0043] The fourth pomegranate flower extract is a pomegranate flower moisturizing extract; the fifth pomegranate flower extract is a pomegranate flower antioxidant extract; and the sixth pomegranate flower extract is a pomegranate flower polyphenol extract.

[0044] Detection of common components (total flavonoids, total phenols, anthocyanins and polysaccharide content) in the first pomegranate flower extract, the fourth pomegranate flower extract, the fifth pomegranate flower extract and the sixth pomegranate flower extract:

[0045] 1. Determination of total flavonoids content

[0046] After mixing 80 μL of distilled water with 6 μL of 5% (mass fraction) NaNO2 and 6 μL of 10% (mass fraction) AlCl3, 20 μL of the extract (1 mg / mL, freeze-dried powder dissolved in distilled water) was added. After incubation at 25 °C for 6 min, 80 μL of 4% (mass fraction) NaOH was added, and incubation was continued at 25 °C for 30 min under light-proof conditions. Subsequently, the absorbance was measured at 405 nm using a microplate reader. The total flavonoid content was expressed as quercetin equivalents per gram of dry weight (mg QE / g DW).

[0047] 2. Determination of total phenolic content

[0048] 16 μL of the extract (1 mg / mL, freeze-dried powder dissolved in distilled water) was mixed with 60 μL of Folin-Ciocalteu reagent at 25 °C for 5 min. Subsequently, 60 μL of 60 g / L Na2CO3 was added, and incubation was carried out at 25 °C for 90 min. The absorbance was measured at 725 nm using a microplate reader. The total phenolic content was expressed as gallic acid equivalents per gram of dry weight (mg GAE / g DW).

[0049] 3. Determination of anthocyanin content

[0050] The sample extract (1 mg / mL, freeze-dried powder dissolved in distilled water) was diluted 10-fold with buffer solutions of pH 1.0 and pH 4.5 respectively and placed in different containers for equilibration. After a 15-minute equilibration period, the absorbance of each solution was measured at wavelengths of 520 nm and 700 nm respectively, with distilled water as the blank control. The differential absorbance of the diluted sample was calculated by the following formula:

[0051] A = (A 520 - A 700 ) pH1.0 - (A 520 - A 700 ) pH4.5

[0052] On this basis, the calculation of anthocyanin content was based on the molar extinction coefficient (ε) and molecular weight (MW) of cyanidin-3-O-glucoside. The specific parameters were: ε = 2.69×10 4 L mol -1 cm -1 , MW = 449.2 g mol -1 . The anthocyanin content was expressed as cyanidin-3-O-glucoside equivalents per gram of dry weight (mg C3GE / g DW).

[0053] 4. Determination of polysaccharide content

[0054] Take a clean test tube with a stopper, accurately transfer 0.2 mL of sample extract, and add 0.3 mL of 5% phenol solution in an ice water bath. Do not touch the wall of the test tube. After shaking well, quickly add 1.0 mL of concentrated sulfuric acid. Do not touch the wall of the test tube. After adding, quickly rotate the test tube on ice to accelerate heat release. It is best to release no heat or a small amount of heat. After shaking well and adding the stopper, place it in a boiling water bath for 20 minutes. After taking it out, cool it with cold water for 10 minutes. Take 200 μL from each tube and add it to a 96-well plate. Make three replicates for each tube of sample, measure the absorbance at the maximum absorption wavelength of 490 nm, and calculate the total sugar concentration in the sample according to the standard curve. The regression equation is Y=6.8544X+0.2704, R 2 =0.9907.

[0055] The contents of common components (total flavonoids, total phenols, anthocyanins and polysaccharides) in the first pomegranate flower extract, the fourth pomegranate flower extract, the fifth pomegranate flower extract and the sixth pomegranate flower extract were measured as follows:

[0056] The first pomegranate flower extract comprises the following components in concentrations: 0.48-0.51 mg GAE / gDW of pomegranate flower total phenols, 0.02-0.03 mg QE / g DW of pomegranate flower total flavonoids, 0.10-0.12 mg C3GE / g DW of pomegranate flower anthocyanidins and 0.31-0.33 mg / mL of pomegranate flower polysaccharides.

[0057] The fourth pomegranate flower extract includes components with the following concentrations: pomegranate flower total phenol 0.022-0.025 mg GAE / gDW, pomegranate flower total flavonoids 0.009-0.012 mg QE / g DW, pomegranate flower anthocyanidins 0.039-0.045 mg C3GE / g DW and pomegranate flower polysaccharides 0.8-0.9 mg / mL.

[0058] The fifth pomegranate flower extract includes components with the following concentrations: pomegranate flower total phenol 0.39-0.41 mg GAE / gDW, pomegranate flower total flavonoids 0.21-0.23 mg QE / g DW, pomegranate flower anthocyanidins 0.15-0.18 mg C3GE / g DW and pomegranate flower polysaccharides 0.26-0.3 mg / mL.

[0059] The sixth pomegranate flower extract includes components in the following concentrations: pomegranate flower total phenol 0.62-0.69 mg GAE / gDW, pomegranate flower total flavonoids 0.35-0.37 mg QE / g DW, pomegranate flower anthocyanidins 0.31-0.35 mg C3GE / g DW and pomegranate flower polysaccharides 0.012-0.04 mg / mL.

[0060] Example 2 Determination of antioxidant activity of pomegranate flower extract

[0061] Using DPPH and ABTS + The antioxidant capacity of purified extracts of different components of fresh pomegranate flowers was evaluated by the free radical scavenging rate parameters.

[0062] 1. Determination experiment of DPPH free radical scavenging ability

[0063] Using DPPH solution (0.15 mmol / L) as the substrate, it was divided into three groups: blank group A (160 μL of methanol and 40 μL of DPPH solution), sample group B (160 μL of sample solution and 40 μL of DPPH solution), and sample control group C (160 μL of sample solution and 40 μL of methanol). After preparing the solutions, they were added to the 96-well plate in sequence. The sample concentrations were set at 500, 250, 125, 62.5, 31.25, 15.625 μg / mL. After reacting for 30 min in the dark to allow sufficient reaction, the absorbance value was measured at 517 nm. The calculation formula for the DPPH free radical scavenging rate is:

[0064] DPPH scavenging rate (%) = [A - (B - C)] / A × 100%

[0065] Note: A is the absorbance of the blank control group; B is the absorbance of the sample to be tested; C is the absorbance of the sample control group.

[0066] 2. ABTS + Determination experiment of free radical scavenging ability

[0067] Mix 7 mM ABTS solution and 2.45 mM potassium persulfate solution (1:1), and react at 4 °C in the dark for 10 - 12 hours to form ABTS + stock solution, and then dilute the stock solution with absolute ethanol to make the absorbance measured at 734 nm reach (0.7 ± 0.05) as the substrate. It was divided into three groups: blank group A (160 μL of absolute ethanol and 40 μL of ABTS + solution), sample group B (160 μL of sample solution and 40 μL of ABTS + solution), sample control group C (160 μL of sample solution and 40 μL of absolute ethanol), and they were added to the 96-well plate in sequence. After reacting for 30 min in the dark to allow sufficient reaction, the absorbance value was measured at 734 nm. The calculation formula for the ABTS + free radical scavenging rate is:

[0068] ABTS + scavenging rate (%) = [A - (B - C)] / A × 100%

[0069] Note: A is the absorbance of the blank control group; B is the absorbance of the sample to be tested; C is the absorbance of the sample control group. The results are expressed as IC 50 values, as shown in Table 1.

[0070] Table 1 Radical Scavenging Capabilities of Different Purified Extracts from Fresh Pomegranate Flowers against DPPH and ABTS +

[0071]

[0072] Results: According to the data in Table 1, the smaller the IC 50 value, the stronger the radical scavenging capabilities of DPPH and ABTS + , that is, the higher the antioxidant activity. After column chromatography separation, the fifth pomegranate flower extract had the lowest IC 50 value, indicating that it had the strongest ability to scavenge DPPH and ABTS + radicals and had the best antioxidant activity. Followed by the sixth pomegranate flower extract, whose antioxidant ability was slightly inferior to that of the fifth pomegranate flower extract. Then came the first pomegranate flower extract, whose antioxidant ability was slightly inferior to that of the sixth pomegranate flower extract. Generally speaking, the above three series of extracts had strong antioxidant abilities. While the fourth pomegranate flower extract had the highest IC 50 value, indicating that it had the weakest ability to scavenge DPPH and ABTS + radicals and had a lower antioxidant activity.

[0073] Example 3 Tyrosinase Activity Inhibition Experiment of Pomegranate Flower Extracts

[0074] Mix 75 μL of the first pomegranate flower extract, the fourth pomegranate flower extract, the fifth pomegranate flower extract, and the sixth pomegranate flower extract with 25 μL of tyrosinase solution respectively, incubate at 37 °C for 10 min, and then add 100 μL of levodopa solution to initiate the reaction. Measure the absorbance at 475 nm, and the results are as follows Figure 1 shown.

[0075] Tyrosinase Inhibition Rate (%) = [(Ab - A) - (Cb - C)] / (Cb - C) × 100

[0076] where Ab is the absorbance of the tyrosinase solution towards the sample solution, A is the absorbance of the sodium phosphate buffer (pH 6.8) towards the sample solution; Cb is the absorbance of the sodium phosphate buffer (pH 6.8) towards the tyrosinase solution, and C is the absorbance of the sodium phosphate buffer (pH 6.8).

[0077] It can be seen from Figure 1 that the first extract exhibited the strongest tyrosinase inhibitory ability.

[0078] Example 4 Cytotoxicity Determination of the First Pomegranate Flower Extract

[0079] The inoculation density of keratinocytes (HaCaT) is 5×10 5 ​Cells / well were seeded in 96-well plates. After culturing for 24 hours, cells were treated with different concentrations of the first pomegranate flower extract for 24 h, and a blank control group was set up. The cells were treated with the CCK8 method for 1 h, and the absorbance was measured at 450 nm. Three parallels were made for each group. The results are as Figure 2 shown. Cell survival rate (%) = (absorbance of each group / absorbance of the blank group) × 100%.

[0080] As Figure 2 can be seen, the first pomegranate flower extract showed different degrees of cytotoxicity at different concentrations. When the concentration was 1000 μg / mL, the cytotoxicity reached 30%; when it was 500 μg / mL, it was 22%; when it was 250 μg / mL, it was 8%; when it was 125 μg / mL, it was 1%. At lower concentrations, the cytotoxicities of 62.5 μg / mL, 31.25 μg / mL, 15.625 μg / mL, and 7.8125 μg / mL were 2%, 7%, 3%, and 2% respectively. Generally speaking, the concentrations below 125 μg / mL all showed cytotoxicity below 10%. Therefore, 125 μg / mL and 62.5 μg / mL were selected for subsequent experiments.

[0081] Example 5 Cell experiment to detect the effect of the first pomegranate flower extract on the antioxidant capacity of living cells

[0082] Keratinocytes (HaCaT) were seeded in 6-well culture plates containing DMEM, 5×10 5 cells / well. After culturing for 24 hours, cells were treated with different concentrations of the first pomegranate flower extract for 24 h, and galactose was used as a control. Then the cells in each well were collected, and the activities of catalase (CAT) and superoxide dismutase (SOD) in the cell suspension were detected according to the kit instructions. The results are as follows Figure 3 shown.

[0083] Results: In HaCaT cells with the addition of different concentrations of the first pomegranate flower extract, the activities of SOD and CAT in HaCaT cells also increased continuously. In addition, as the concentration of the extract increased, SOD (superoxide dismutase) in HaCaT cells showed a significant dose-dependent enhancement trend. This indicates that the first pomegranate flower extract may enhance the antioxidant defense ability by scavenging reactive oxygen species (ROS) in cells, thereby improving the oxidative stress state of cells.

[0084] Example 6 Effect of the first pomegranate flower extract on autophagy of keratinocytes

[0085] An autophagy experiment was conducted on keratinocytes (HaCaT). The keratinocytes (HaCaT) were inoculated in a 6-well culture plate containing DMEM. After 24 hours of culture, keratinocytes were treated with different concentrations of the first pomegranate flower extract for 24 hours, and galactose was used as a control. Then, the cells in each well were collected, and the bright green dansylcadaverine (MDC)-labeled particles were detected according to the instructions of the autophagy staining detection kit. The results are as Figure 4 shown.

[0086] Results: In HaCaT cells treated with different concentrations of the first pomegranate flower extract, the autophagy ability of HaCaT cells also increased continuously. The first pomegranate flower extract can effectively enhance the autophagy of HaCaT cells. This research result provides an experimental basis for the application of pomegranate flower in the fields of skin health and anti-aging.

[0087] Example 7 Antibacterial effect of the first pomegranate flower extract

[0088] The filter paper method was used to screen for microbial activity, and the sizes of the inhibition zones of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa were observed. The results are shown in Table 2.

[0089] Table 2 Antibacterial effect of the first pomegranate flower extract

[0090]

[0091] Note: The judgment criteria for the inhibition zone experiment: When the diameter of the inhibition zone d > 15 mm, it is highly sensitive; when 15 mm > d > 10 mm, it is moderately sensitive; when 10 mm > d > 6 mm, it is lowly sensitive; when the diameter of the inhibition zone d < 6 mm, it indicates no antibacterial effect.

[0092] Results: The first pomegranate flower extract has a moderately sensitive antibacterial effect on Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa.

[0093] Example 8 Moisturizing effect of the fourth pomegranate flower extract and whitening effect of the first pomegranate flower extract

[0094] I. Moisturizing effect of the fourth pomegranate flower extract

[0095] Thirty subjects aged 20 - 55 were selected and applied with 125 μg / ml of the fourth pomegranate flower extract once in the morning and once in the evening. The observation period was 30 days. A multi-functional skin tester was used to measure the moisture content of the skin before and 30 days after application. The results are as Figure 5 shown.

[0096] From Figure 5A It can be seen that before using the pomegranate flower moisturizing extract, the moisture content of the skin was 35.42CU. After continuous application for 30 consecutive days, the moisture content increased significantly to 40.15CU, an increase of 4.73CU compared to before use. This result shows that the pomegranate flower moisturizing extract can effectively increase the moisture content of the skin and play a significant moisturizing role.

[0097] 2. The whitening effect of pomegranate flower extract

[0098] 30 subjects aged 20-55 were selected and 125ug / ml of the first pomegranate flower extract was applied once in the morning and evening for 30 days. The skin moisture was tested with a multifunctional skin tester before and after 30 days of use. The results are as follows Figure 5 As shown. Figure 5 B shows that before using the first pomegranate flower extract, the melanin content of the skin was 165.7AU, and after continuous application for 30 days, the melanin content dropped to 158.7AU, a decrease of 7.0AU. This result shows that the first pomegranate flower extract can effectively reduce melanin deposition and has a certain whitening effect. Figure 5 C shows that the skin hemoglobin content was 212.4AU before use, and after 30 days of use it dropped to 199.4AU, a decrease of 13.0AU. This change shows that the first pomegranate flower extract reduces the red blood phenomenon, makes the skin tone more even, and presents a healthier state. Figure 5 D shows that the skin glossiness was 5.7AU before use, and it increased to 6.4AU after using the first pomegranate flower extract for 30 days, an increase of 0.7AU. This shows that the first pomegranate flower extract can improve the skin glossiness, making the skin more translucent and shiny. Overall, the first pomegranate flower extract has a positive effect on whitening, improving skin tone uniformity and improving skin glossiness, showing its broad application prospects in the field of skin care.

[0099] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. A preparation method of a pomegranate flower skin care extract, characterized in that, It includes the following steps: Step (1): Freeze-dry fresh pomegranate flowers with liquid nitrogen and quickly grind them into powder. Add 70 - 80% ethanol solution as an extractant according to the solid-liquid ratio of 1:25 g / mL. First, extract with a microwave device with a microwave power of 300 - 500 W and a microwave time of 30 - 60 seconds. Then, further crush and break the cell walls with an ultrasonic device with an ultrasonic power of 400 - 500 W, a temperature of 50 - 60 °C, and an extraction time of 45 - 60 min. Re-extract the residue once according to the above method. Combine the two extracts, filter by suction, and rotary evaporate to remove the ethanol solution to obtain a pomegranate flower whitening, antibacterial, and anti-aging extract, that is, the first pomegranate flower extract; Step (2): A composite enzyme containing cellulase and papain with a mass fraction of 1.0% is dissolved in a potassium hydrogen phosphate-citric acid buffer solution with a pH of 6 - 7. After pre-activating in a 50 °C water bath for 10 min, add the remaining pomegranate flower residue from step (1) to the buffer solution mixed with the composite enzyme according to the liquid-solid ratio of (15 - 20) mL:1 g, and enzymatically hydrolyze in a 50 °C water bath for 100 - 120 min. After enzymatic hydrolysis, filter by suction and concentrate by rotary evaporation to 1 / 4 of the total volume. Then add 4 times the volume of anhydrous ethanol, place it at 4 °C and let it stand for 24 h, collect the precipitate to obtain the second pomegranate flower extract; Step (3): Add an appropriate amount of pure water to the second pomegranate flower extract to dissolve it fully; Subsequently, add an equal volume of a mixture of chloroform and n-butanol to the aqueous phase, mix well, and collect the upper aqueous phase; then add an equal volume of petroleum ether to the obtained liquid, mix well, and collect the lower aqueous phase to obtain the third pomegranate flower extract; Step (4): Subject the above third pomegranate flower extract to column chromatography separation on macroporous resin, and elute successively with three gradients of water, 30% ethanol, and 50% ethanol. After vacuum freeze-drying, obtain the fourth pomegranate flower extract, that is, the pomegranate flower moisturizing extract, the fifth pomegranate flower extract, that is, the pomegranate flower antioxidant extract, and the sixth pomegranate flower extract, that is, the pomegranate flower polyphenol extract respectively.

2. The preparation method of a pomegranate flower skin care extract according to claim 1, characterized in that, In step (2), the ratio of cellulase to papain is 1:1, and the mass fraction of the composite enzyme is (the mass of the composite enzyme × 100%) / the mass of the pomegranate flower residue.

3. The preparation method of a pomegranate flower skin care extract according to claim 2, characterized in that, In step (3), the volume ratio of the chloroform to the n-butanol is 3:

1.

4. A pomegranate flower skin care extract, characterized in that, The raw materials are composed of the following parts by weight: 2 - 4 parts of the first pomegranate flower extract, 3 - 8 parts of the fourth pomegranate flower extract, 1 - 2 parts of the fifth pomegranate flower extract, and 1 - 2 parts of the sixth pomegranate flower extract.

5. Use of the first pomegranate flower extract in the preparation of antioxidant, antibacterial, whitening or anti-aging washing and care products or health products, characterized in that, The first pomegranate flower extract includes components with the following concentrations: total phenolic content in pomegranate flower 0.48 - 0.51 mg GAE / g DW, total flavonoid content in pomegranate flower 0.02 - 0.03 mg QE / g DW, anthocyanin content in pomegranate flower 0.10 - 0.12 mg C3GE / g DW, and polysaccharide content in pomegranate flower 0.31 - 0.33 mg / mL.

6. The application according to claim 5, wherein The antioxidant activity is to enhance the activities of catalase and superoxide dismutase; the antibacterial activity is to inhibit the growth of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa; the whitening activity is to inhibit the activity of tyrosinase in vitro, reduce the content of skin melanin, and increase skin brightness; the anti-aging activity is to enhance autophagy and thus enhance the anti-aging function.

7. The application according to claim 5, wherein The washing and care products are cosmetics, skin care products, body wash, or facial cleanser.

8. Use of the sixth pomegranate flower extract in the preparation of antioxidant washing and care products or health care products, characterized in that, The sixth pomegranate flower extract comprises components with the following concentrations: total phenolic content in pomegranate flower 0.62 - 0.69 mg GAE / g DW, total flavonoid content in pomegranate flower 0.35 - 0.37 mg QE / g DW, anthocyanin content in pomegranate flower 0.31 - 0.35 mg C3GE / g DW, and pomegranate flower polysaccharide 0.012 - 0.04 mg / mL.

9. Use of the fifth pomegranate flower extract in the preparation of antioxidant washing and care products or health products, characterized in that, The fifth pomegranate flower extract comprises components with the following concentrations: total phenolic content in pomegranate flower 0.39 - 0.41 mg GAE / g DW, total flavonoid content in pomegranate flower 0.21 - 0.23 mg QE / g DW, anthocyanin content in pomegranate flower 0.15 - 0.18 mg C3GE / g DW, and pomegranate flower polysaccharide 0.26 - 0.3 mg / mL.

10. Use of the fourth pomegranate flower extract in the preparation of moisturizing washing and care products, characterized in that, The fourth pomegranate flower extract comprises components with the following concentrations: total phenolic content in pomegranate flower 0.022 - 0.025 mg GAE / g DW, total flavonoid content in pomegranate flower 0.009 - 0.012 mg QE / g DW, anthocyanin content in pomegranate flower 0.039 - 0.045 mg C3GE / g DW, and pomegranate flower polysaccharide 0.8 - 0.9 mg / mL.

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