Preparation method and application of safflower extract containing flavonoid components

Abstract

The invention relates to the field of microbial conversion, in particular to a preparation method and application of a safflower extract containing flavonoid components. Specifically, escherichia coli and yarrowia lipolytica are adopted to jointly ferment safflower, so that the safflower extract rich in flavonoid components such as hydroxysafflor yellow, kaempferol and quercetin is prepared. The extract can be applied to food, health care products and cosmetics, shows remarkable antioxidant, anti-inflammatory, whitening and anti-aging activities, has the potential of improving skin health, and is expected to become a substitute of plant-derived alcohol A.

Description

Technical Field

[0001] This application belongs to the field of microbial fermentation, and specifically relates to a preparation method and application of safflower extract containing flavonoid components. Background Art

[0002] Plant A alcohol encompasses various components, such as Bidens pilosa extract, bakuchiol, silybum marianum, Vigna unguiculata seeds extract, Anigozanthos extract, etc. These can all be referred to as plant A alcohol, but they do not contain vitamin A itself. The key lies in that plant A alcohol can regulate the expression of collagen I and IV, elastin, fibroblast growth factor (FGF-β), tissue growth factor (TGF-β), matrix metalloproteinase (MMP), and epidermal growth factor (EGFR), etc. Currently, plant A alcohol components include Bidens pilosa, silybum marianum, and bakuchiol, etc. Among them, silymarin is one of the main components of silybum marianum. There are components with a chemical structure similar to silymarin in safflower, including flavonoids, alkaloids, polysaccharides, lignans, polyynes, spermidine, sterols, etc. Among them, flavonoids and flavonoid glycosides are the main active components of safflower. Therefore, the flavonoid components in safflower extract may be developed into plant A alcohol components to replace A alcohol.

[0003] Currently, the methods for extracting flavonoid components from safflower mainly include water extraction method, microwave-assisted extraction method, ultrasonic-assisted water extraction method, and cellulase method extraction, etc. However, these extraction methods are restricted by environmental and seasonal differences (such as nutritional status and soil composition), making the process complex when preparing safflower extract, and the content of flavonoid components in the finally obtained extract is low. And flavonoid components in safflower extract (such as hydroxysafflor yellow A) can reduce the MDA level induced by ultraviolet rays and effectively delay photoaging. Flavonoid components can also precisely regulate inflammation and quickly relieve seborrheic dermatitis. Regulate the skin microecology, with an inhibition rate of >90% against Propionibacterium acnes and Staphylococcus aureus, and without destroying the beneficial bacteria in the skin itself. Inhibit melanin production through multiple targets and simultaneously down-regulate the expression of MITF and TRP-1 genes.

[0004] Therefore, there is an urgent need for a preparation method of safflower extract to make the content of flavonoid components in safflower extract higher, so that its application in food, health products, and cosmetics is more extensive. Summary of the Invention

[0005] To solve or partially solve the problems existing in the related technologies, the present application provides a method for preparing a safflower extract. This method achieves a technological breakthrough through the synergistic fermentation of two bacteria, efficiently converts flavonoids, and improves the bioavailability of flavonoids. Through the synergistic action of the lipase of Yarrowia lipolytica and the β-glucosidase of Escherichia coli, the conjugated flavonoid glycosides in safflower are converted into free aglycones, and the total flavonoid content is increased from 1.1% in the traditional process to 2.7%. Deep degradation of allergens: The specific oxidoreductase expressed by Escherichia coli can degrade the allergenic components (such as safflower quercetin glycoside) in safflower, reduce the allergy rate, and significantly improve safety. Compared with the traditional ethanol extraction method, there is no organic solvent residue throughout the process, achieving green production.

[0006] On the one hand, the present application provides a method for preparing a safflower extract containing flavonoid components, and the specific steps include: (1) Inoculate Escherichia coli and Yarrowia lipolytica on a slant medium for activation; (2) Inoculate the activated Escherichia coli and Yarrowia lipolytica strains into a seed medium to obtain an Escherichia coli seed liquid and a Yarrowia lipolytica seed liquid; (3) Mix safflower powder evenly with water, add it to a fermentation medium, and first add the Yarrowia lipolytica seed liquid for fermentation; (4) After the fermentation in (3) ends, add the Escherichia coli seed liquid for co-cultivation to obtain a fermentation broth; (5) Centrifuge the fermentation broth obtained in (4), take the supernatant, and successively carry out purification, elution, concentration, and drying to obtain a safflower extract containing flavonoid components.

[0007] Further, the formula of the slant medium in (1) is: glucose 10 - 20 g / L, peptone 15 - 45 g / L, agar 15 - 20 g / L, yeast extract 5 - 10 g / L.

[0008] Further, the formula of the seed medium in (2) is: glucose / sucrose 5 - 30 g / L, peptone 10 - 20 g / L, manganese sulfate 0.1 - 10 g / L, ammonium sulfate 5 - 12 g / L, magnesium sulfate 1 - 10 g / L.

[0009] Further, the safflower powder in (3) is obtained by pulverizing safflower three times, passing through a 50-mesh sieve, and then carrying out vacuum drying.

[0010] Further, the formula of the fermentation medium in (3) is: glucose / xylose 120 - 300 g / L, peptone 15 - 45 g / L, ammonium sulfate 2 - 12 g / L, magnesium sulfate 1 - 6 g / L, manganese sulfate 1 - 6 g / L, rapeseed oil 20 - 110 g / L; The fermentation conditions are as follows: temperature 28 - 30°C, pH value 5.0 - 6.5, dissolved oxygen concentration 20% - 30%, rotation speed 200 - 800 rpm, and time 48 - 72 hours.

[0011] Furthermore, the co - culture conditions in (4) are: temperature 30°C, pH value 5.0 - 6.5, dissolved oxygen concentration 20% - 60%, rotation speed 200 - 800 rpm, and time 24 h.

[0012] Furthermore, the concentration method in (5) is vacuum concentration; the temperature of the vacuum is 60°C.

[0013] This application also provides the use of a safflower extract containing flavonoid components prepared according to the above - mentioned preparation method in foods, health products, and cosmetics.

[0014] Beneficial effects 1. The preparation method and application of a safflower extract provided by this application, by establishing a co - culture system of Escherichia coli and Yarrowia lipolytica, the two microorganisms interact with each other, and through the complementarity of metabolic pathways, synergistically promote the extraction and transformation of active ingredients in safflower. This method not only increases the yield of flavonoid compounds in the safflower extract but also enhances its biological activity.

[0015] 2. This application also provides the use of a safflower extract rich in flavonoid components such as hydroxysafflor yellow A and kaempferol in skin care products. This safflower extract can be used to prepare cosmetics with antioxidant, whitening, firming, and anti - inflammatory effects, or as a substitute for plant - derived retinol.

[0016] It should be understood that the above general description and the following detailed description are only exemplary and explanatory, and cannot limit this application. Brief description of the drawings

[0017] By describing the exemplary embodiments of this application in more detail in conjunction with the drawings, the above - mentioned and other objects, features, and advantages of this application will become more obvious. Among them, in the exemplary embodiments of this application, the same reference numerals generally represent the same components.

[0018] Figure 1 shows the melanin situation in the head of zebrafish; Figure 2 shows the effect of the safflower extract on melanin density; compared with the normal group, *** P <0.001; Figure 3 shows the comparison of the content of type I collagen in the test samples; compared with the blank group, *** P <0.001, ** P <0.01, * P< 0.05; ns indicates no significant difference.

[0019] Figure 4 It is the scavenging effect of safflower extract on DPPH.

[0020] Figure 5 It is the comparison of the content of IL-1β secreted by LPS-induced RAW264.7 cells by safflower extract; Compared with the blank group, ### P <0.001; compared with the model group, *** P <0.001.

[0021] Figure 6 It is the comparison of the content of IL-6 secreted by LPS-induced RAW264.7 cells by safflower extract; compared with the blank group, ### P <0.001; compared with the model group, *** P <0.001; ** P <0.01; ns indicates no significant difference. Specific implementation manners

[0022] The embodiments of the present application will be described in more detail with reference to the accompanying drawings. Although the embodiments of the present application are shown in the drawings, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to make the present application more thorough and complete, and to fully convey the scope of the present application to those skilled in the art.

[0023] To overcome the limitations of traditional extraction methods, microbial fermentation transformation technology can be utilized. As a non-conventional yeast, Yarrowia lipolytica has the ability to produce large amounts of cytoplasmic acetyl-CoA and malonyl-CoA, which can serve as synthetic precursors for high-value natural products such as terpenoids and flavonoids. Secondly, Yarrowia lipolytica has a strong ability to decompose hydrophobic compounds and can exhibit high efficiency in the synthesis of flavonoids; it has a wide range of substrate utilization, being able to utilize carbon sources such as glucose, glycerol, ethanol, acetic acid, etc., as well as organic acids such as citric acid and isocitric acid, and especially can utilize oils, alkanes, etc.; it also has strong stress resistance and can tolerate high salt, low temperature, and overly acidic or alkaline environments; with the continuous application and development of synthetic biology and gene editing technology in Yarrowia lipolytica, it can also be edited and modified. Escherichia coli, as a model organism, is characterized by rapid growth and strong reproductive ability, and can produce large amounts of flavonoids in a short time; secondly, compared with traditional extraction methods, it has lower costs; during the production of flavonoids, it does not produce a large amount of waste and pollutants and has little impact on the environment; Escherichia coli has a clear genetic background and is easy to genetically engineer, and the metabolic pathway for producing flavonoids in Escherichia coli can be optimized.

[0024] Due to its easy genetic manipulation and rapid growth, Escherichia coli is widely used in the production of flavonoids. Yarrowia lipolytica shows the potential to produce flavonoids due to its metabolism and high availability of key precursors. By co-culturing Escherichia coli and Yarrowia lipolytica, different metabolic pathways and enzyme systems can be utilized, and the combination of the two can complement the metabolic pathways and further optimize the interaction between microorganisms, enabling the efficient production of flavonoids.

[0025] Carthamus L. Carthamus L. plants belong to the Compositae family Asteraceae ), most of which are annuals, and fewer are biennials or perennials. There are about 18 - 20 species in this genus, mainly distributed in Central Asia, Southwest Asia, and the Mediterranean region. There are two species of this genus in China, namely Carthamus tinctorius L. Carthamus tinctorius L. and Carthamus lanatus Thunb. Carthamus Lanatus L. ), and these two species of Carthamus are used in this application.

[0026] Unless otherwise specified, the following experimental materials are all purchased from the market.

[0027] Example 1 Preparation of Safflower Extract Containing Flavonoid Components 1. A method for preparing safflower extract rich in flavonoid components, which uses Escherichia coli and Yarrowia lipolytica to co-ferment safflower, and the specific steps include: (1) Inoculate cryopreserved Escherichia coli and Yarrowia lipolytica on a slant medium for activation; The formula for the slant medium is: glucose 20 g / L, peptone 45 g / L, agar 18 g / L, yeast extract 7 g / L.

[0028] (2) Inoculate the activated Escherichia coli and Yarrowia lipolytica strains into the seed medium to obtain a mature seed solution. The formula for the seed medium is: glucose 30 g / L, peptone 20 g / L, manganese sulfate 10 g / L, ammonium sulfate 7 g / L, magnesium sulfate 10 g / L.

[0029] (3) Take an appropriate amount of safflower ( Carthamus tinctorius L. ) and crush it three times using a crusher, then perform sieving treatment (50 mesh), and subsequently perform vacuum drying to obtain safflower powder.

[0030] (4) Mix an appropriate amount of safflower powder evenly with water, add it to the fermentation medium, and first add the Yarrowia lipolytica seed culture solution. After stirring evenly, carry out fermentation under the conditions of a temperature of 28°C - 30°C, a pH value of 5.0 - 6.5, and a dissolved oxygen concentration of 20% - 30%. The rotation speed is between 200 - 800 rpm, and the fermentation time is 48 - 72 hours. The formula for the fermentation medium is: glucose / xylose 120 - 300 g / L, peptone 15 - 45 g / L, ammonium sulfate 2 - 12 g / L, magnesium sulfate 1 - 6 g / L, manganese sulfate 1 - 6 g / L, rapeseed oil 20 - 110 g / L.

[0031] (5) After the fermentation in step (4) is completed, add the Escherichia coli seed solution and continue to culture for 24 h. During the co - culture, carry out fermentation under the conditions of a temperature of 30°C, a pH value of 5.0 - 6.5, and a dissolved oxygen concentration of 20% - 60%. The rotation speed is between 200 - 800 rpm.

[0032] (6) Collect the fermentation broth and perform centrifugation. Take the supernatant and purify it using macroporous resin AB - 8, elute it with 95% ethanol, collect the eluate and concentrate it. The concentration temperature is 60°C; finally, perform vacuum drying to obtain the safflower extract, which is rich in flavonoid components such as hydroxysafflor yellow A and kaempferol.

[0033] Example 2 Preparation of Safflower Extract Containing Flavonoid Components Different from Example 1, adjust the culture method so that Yarrowia lipolytica is first cultured at 28°C for 72 hours, take its supernatant and add Escherichia coli for culture, and the culture temperature can be raised to 30 - 37°C.

[0034] Example 3 Preparation of Safflower Extract Containing Flavonoid Components Different from Example 1, adjust the culture method so that both Yarrowia lipolytica and Escherichia coli are cultured together at 30°C.

[0035] Example 4 Preparation of Safflower Extract Containing Flavonoid Components Differing from Example 1, the safflower variety was adjusted to Carthamus tinctorius L. var. acanthophyllus C. Winkl. Carthamus Lanatus L. )

[0036] For Comparative Example 1, all other steps were the same as those in Example 1 except for the following experimental steps.

[0037] 1) Adjust the formula of the slant medium in step (1) of Example 1 to: glucose 15 g / L, peptone 25 g / L, agar 20 g / L, yeast extract 10 g / L; 2) Adjust the formula of the slant medium in step (1) of Example 1 to: glucose 10 g / L, peptone 15 g / L, agar 15 g / L, yeast extract 5 g / L.

[0038] Comparative Example 2 All other steps were the same as those in Example 1 except for the following experimental steps.

[0039] 1) Adjust the formula of the seed medium in step (2) of Example to: glucose 5 g / L, peptone 18 g / L, manganese sulfate 6 g / L, ammonium sulfate 12 g / L, magnesium sulfate 3 g / L; 2) Adjust the formula of the seed medium in step (2) of Example to: sucrose 25 g / L, peptone 10 g / L, manganese sulfate 0.1 g / L, ammonium sulfate 5 g / L, magnesium sulfate 1 g / L.

[0040] Comparative Example 3 All other steps were the same as those in Example 1 except for the following experimental steps.

[0041] Adjust the types of macroporous resins in step (6) of Example 1 to D101, HPD-600, HPD-100, AB-8.

[0042] Comparative Example 4 A method for preparing safflower extract rich in flavonoid components, using Escherichia coli and Yarrowia lipolytica to co-ferment safflower, and the specific steps include: (1) Inoculate cryopreserved Yarrowia lipolytica into a slant medium for activation; the formula of the slant medium is: glucose 20 g / L, peptone 45 g / L, agar 18 g / L, yeast extract 7 g / L.

[0043] (2) Inoculate the activated Yarrowia lipolytica strain into a seed medium to obtain a mature seed solution; the formula of the seed medium is: glucose 30 g / L, peptone 20 g / L, manganese sulfate 10 g / L, ammonium sulfate 7 g / L, magnesium sulfate 10 g / L.

[0044] (3) Take an appropriate amount of safflower, pulverize it three times using a pulverizer, and perform sieving treatment (50 mesh), then carry out vacuum drying to obtain safflower powder.

[0045] (4) Mix an appropriate amount of safflower powder evenly with water, add it to the fermentation medium, and first add the culture solution of Yarrowia lipolytica seed bacteria. After stirring evenly, place it under the conditions of a temperature of 28°C - 30°C, a pH value of 5.0 - 6.5, and a dissolved oxygen concentration of 20% - 30% for fermentation, with the rotation speed between 200 - 800 rpm and the fermentation time of 48 - 72 hours; the formula of the fermentation medium is: glucose / xylose 120 - 300 g / L, peptone 15 - 45 g / L, ammonium sulfate 2 - 12 g / L, magnesium sulfate 1 - 6 g / L, manganese sulfate 1 - 6 g / L, rapeseed oil 20 - 110 g / L.

[0046] (5) Collect the fermentation broth for centrifugation, take the supernatant, purify it using macroporous resin AB - 8, elute it with 95% ethanol, collect the eluate for concentration, and the concentration temperature is 60°C; finally, carry out vacuum drying to obtain the safflower extract.

[0047] Example 5 measures and identifies the safflower extract obtained in Example 1 above 1. Determination of the total flavonoid content in the sample solution The determination is carried out by the sodium nitrite - aluminum nitrate - sodium hydroxide colorimetric method. Using rutin as the reference substance, the absorbance value is measured at 510 nm. After dissolving the rutin reference substance with 70% ethanol, a 0.2 mg / mL rutin reference substance solution is prepared. Weigh 1 mL of the safflower extract obtained in Example 1 into a 10 mL volumetric flask, add 0.5 mL of 5% sodium nitrite solution respectively, shake well, stand for 6 min, then add 0.5 mL of 10% aluminum nitrate solution, shake well, stand for 6 min, then add 4 mL of 4% sodium hydroxide solution, make up the volume with 70% ethanol, shake well, stand for 15 min, and then measure the absorbance at 510 nm.

[0048] 2. Determination of the content of hydroxysafflor yellow A (HSYA) (1) Use octadecylsilane - bonded silica gel as the filler; use methanol - acetonitrile - 0.7% phosphoric acid solution (26:2:72) as the mobile phase; the detection wavelength is 403 nm. The number of theoretical plates calculated based on the hydroxysafflor yellow A peak should not be less than 3000.

[0049] (2) Preparation of the reference substance solution Weigh accurately 1.32 mg of hydroxysafflor yellow A reference substance, place it in a 10 mL volumetric flask, make up the volume with 25% methanol, shake well, and that's it.

[0050] (3) Accurately pipette 10 μl each of the reference solution and the test solution, inject them into the liquid chromatograph, and measure to obtain the results.

[0051] 3. Determination of kaempferol and quercetin contents (1) Use octadecylsilane chemically bonded silica gel as the filler; use methanol - 0.7% phosphoric acid solution (45:55) as the mobile phase; detection wavelength: 370 nm (kaempferol), 360 nm (quercetin); under these chromatographic conditions, quercetin and kaempferol are well separated and can achieve baseline separation from other components. The number of theoretical plates based on quercetin is 10400, and the resolution is greater than 1.2.

[0052] (2) Preparation of the reference solution Weigh accurately 18.30 mg of quercetin reference substance, place it in a 50 mL volumetric flask, add methanol to dissolve and dilute to the mark, shake well, and obtain the quercetin reference solution; weigh accurately 17.20 mg of kaempferol reference substance, place it in a 50 mL volumetric flask, add methanol to dissolve and dilute to the mark, shake well, and obtain the kaempferol reference solution.

[0053] (3) Accurately pipette 10 μl each of the reference solution and the test solution, inject them into the liquid chromatograph, and measure to obtain the results.

[0054] Table 1 Sample data 3. Evaluate the whitening effect of safflower extract Experimental principle: The most important action pathway for the whitening effect of skin care products is to inhibit the activity of tyrosinase, thereby reducing the production of melanin. The melanin regulation mechanism in zebrafish skin is highly conserved with that of humans, and the production rate of melanin is fast and can be clearly observed by the naked eye under a microscope. Through image processing technology, the degree of melanin reduction by skin care products can be quantified, thereby realizing the evaluation of the whitening effect.

[0055] Experimental protocol: Divide zebrafish into three groups, namely the normal control group, the positive group, and the test product group. The normal control group is not treated, the positive group is given 0.1% niacinamide, and the test product group is added with a certain concentration of safflower extract (prepared according to the preparation method in Example 1). After incubation for a period of time, take pictures of the heads of zebrafish, and use advanced image processing software to analyze the melanin signal intensity of the heads of zebrafish.

[0056] It can be seen from Figure 1 that compared with the normal group, in the experimental groups of safflower extract at different concentrations, the melanin in the heads of zebrafish was significantly reduced, revealing its whitening effect.

[0057] Perform statistical analysis on the melanin density of each group, as Figure 2As shown, compared with the normal group, safflower extracts at different concentrations were able to significantly reduce the melanin density ( P <0.001), indicating its whitening effect.

[0058] 4. Evaluate the up-regulation rate of type I collagen content by safflower extract Experimental principle: Type I collagen is one of the main components of the extracellular matrix of dermal cells. Type I procollagen is synthesized intracellularly by dermal fibroblasts and secreted extracellularly. After the terminal procollagen peptidase acts and the telopeptides are separated, they polymerize to form collagen fibers. Human dermal fibroblasts can be used as a cell model to study the increase of type I collagen content by cosmetics. By measuring the up-regulation rate of type I collagen content after administration of the test substance and in the blank control and after administration of the test substance, it is evaluated whether the test substance has an effect in promoting collagen synthesis.

[0059] Experimental protocol: The experiment was divided into three groups: normal control group, positive group, and test article group. The normal control group was not treated. The positive group was given TGF-β1 at 100 ng / mL. The test article group was added with safflower extracts at different concentrations (prepared by the method of Example 1). After incubation for a period of time, the type I collagen content was measured by enzyme-linked immunosorbent assay (ELISA).

[0060] The content and up-regulation rate of type I collagen are as Figure 3 shown: After intervention, the positive control group (TGF-β1 100 ng / mL) significantly increased the type I collagen content in human dermal fibroblasts HSF ( P <0.001); The test sample, safflower extract, increased the type I collagen content in human dermal fibroblasts HSF in the concentration range of 50 μg / mL to 250 μg / mL ( P <0.001). It shows that safflower extract has firming and anti-wrinkle effects in the concentration range of 50 μg / mL to 250 μg / mL.

[0061] 5. Determine the antioxidant activity of safflower extract by DPPH method Experimental principle: DPPH is a stable free radical in organic solvents. Its alcohol solution is purple and needs to be stored at low temperature in the dark. It has a single electron, so it can accept an electron or a hydrogen ion and has a maximum absorption at a wavelength of 517 nm. When there is a free radical scavenger, the single electron of DPPH is captured and its color becomes lighter, and the absorbance value at the maximum light absorption wavelength decreases, and the degree of decrease shows a linear relationship. The decrease in the absorbance level indicates an increase in antioxidant activity, so as to evaluate the antioxidant ability of the test sample. This antioxidant ability is expressed by the inhibition rate. The greater the inhibition rate, the stronger the antioxidant property.

[0062] Experimental protocol: Weigh a certain amount of DPPH powder and dissolve it in absolute ethanol. Take 1 mL of safflower extract test solutions with concentrations of 100, 200, 300, 400, and 500 μg / mL respectively into stoppered test tubes, add 5 mL of DPPH solution, shake well, and measure the absorbance at a wavelength of 517 nm after 30 minutes.

[0063] It can be seen from Figure 4 that as the concentration of the safflower extract increases, the DPPH radical scavenging rate also increases, indicating that there is a dose-dependent relationship between the DPPH radical scavenging activity of the safflower extract.

[0064] 6. Evaluate the anti-inflammatory activity of the safflower extract. The safflower extract can inhibit the production of inflammatory factors induced by lipopolysaccharide Experimental principle: LPS-induced RAW264.7 is a classic cell model for studying inflammatory factors. LPS binds to antigen recognition receptors on the surface of macrophages and can induce macrophages to secrete various inflammatory factors such as TNF-α, IL-1β, and IL-6.

[0065] Experimental protocol: Divide the experiment into three groups, namely the normal control group, the model group, and the test article group. The normal control group is not treated, the model group is given 1 μg / ml LPS, and the test article group is added with safflower extracts at different concentrations. After incubation for a period of time, the contents of inflammatory factors IL-6 and IL-1β are measured by enzyme-linked immunosorbent assay (ELISA).

[0066] It can be seen from Figure 5 and Figure 6 that the levels of IL-1β and IL-6 in the serum of the model group are significantly increased ( P <0.01). Safflower extracts (25, 50, 100, and 250 μg / mL) can significantly reduce the level of IL-1β ( P <0.01). Compared with the model group, safflower extracts (50, 100, and 250 μg / mL) can significantly reduce the level of IL-6 ( P <0.01). The results show that the safflower extract can inhibit the production of inflammatory cytokines induced by LPS, indicating that the safflower extract has anti-inflammatory effects.

[0067] Example 6 Application of safflower extract in skin care products.

[0068] Taking Example 1 as the preferred example, the safflower extract containing flavonoid components prepared in Example 1 is added to skin care products to prepare an essence with anti-aging efficacy. The formula list is shown in Table 2: Table 2 Anti-aging essence formula list Based on the anti-aging essence formula in Table 2, two test samples were prepared respectively: Sample 1: containing 1% safflower extract (original formula); Sample 2: replacing 1% safflower extract with 0.3% A-alcohol, and keeping the rest of the concentrations and ingredients unchanged.

[0069] Human efficacy test: Inclusion criteria: aged 30 - 60 years old; having fine lines or wrinkles at the corners of the eyes, meeting the Asian grading of 1 - 6 in "Skin Aging Atlas"; the subjects have not participated in other clinical studies in the past three months; Exclusion criteria: using antihistamines in the past week or immunosuppressants in the past month; using any anti-inflammatory drugs on the test site in the past two months; insulin-dependent diabetes patients; patients with respiratory diseases under treatment; lactating or pregnant women; people with allergic constitution, allergic dermatitis, etc., having a history of skin diseases or diseases; people under dermatological treatment, or subjects who have taken hydroxy acids, whitening agents, and anti-aging drugs within one month; having skin manifestations such as large birthmarks, scratches, leukoplakia, pigmented moles, keloid scars, etc. in the test area that affect the test; in addition, people judged by the test leader as not suitable as subjects for this test.

[0070] Instruments: Visia-CR type facial image analyzer (Canfield Company, USA); skin elasticity test probe Cutometer (MPA580, CK Company, Germany) Test method: An auto-control trial was adopted. After cleansing their faces in the morning and evening every day, the subjects applied the essence on their faces; Sample 1: prepared according to the formula in Table 2 (containing 1% safflower extract); Sample 2: on the basis of the formula in Table 2, replacing 1% safflower extract with 0.3% A-alcohol, gently massaging until fully absorbed, twice a day for 28 days. The human efficacy test was conducted on the subjects on the 0th (D0) and 28th days (D28) respectively.

[0071] Thirty qualified subjects were selected. Before the formal test, their faces were cleaned with warm water and dried with a lint-free paper towel. After sitting still for at least 30 minutes in an environment with a temperature of 20°C - 22°C and a relative humidity of 40% - 60%RH, the initial values were tested and recorded by professional personnel, and the same tests were conducted and recorded again 28 days after using the products.

[0072] Skin wrinkle and roughness test The Visia-CR type facial image analyzer was used to measure the average value of local wrinkles and the roughness value, and observe the changes in local wrinkles and roughness values before and after using the product. The smaller each measured value is, the more obvious the wrinkle improvement effect is.

[0073] Skin elasticity test At each visit time point, the R2 value of the test area was measured using a skin elasticity tester. The larger the R2 value, the better the skin elasticity.

[0074] Statistical analysis The experimental results were compared through statistical test methods to determine whether there were statistical differences; the data were statistically analyzed using SPSS. If the data were normally distributed, the T-test method was used for statistical analysis; if the data were non-normally distributed, the rank sum test method was used for statistical analysis; all statistical methods used two-tailed tests, and the significance level α = 0.05.

[0075] Analysis test results of skin indicators: Table 3 Comparative analysis of anti-aging indicators of Sample 1 (containing safflower extract) at different times (mean ± standard deviation, n = 30) Table 4 Comparative analysis of anti-aging indicators of Sample 2 (containing A-alcohol) at different times (mean ± standard deviation, n = 30) Note: The significance annotation method "ns" indicates no statistical difference; * P > 0.05 indicates a significant difference; ** P < 0.01 indicates a significant difference.

[0076] According to the experimental results, it can be seen that Sample 1 and Sample 2 provided by the present invention have obvious effects of improving skin elasticity, and also have significant effects in reducing skin wrinkles and skin roughness, which can be beneficial for anti-aging and improving skin health conditions.

[0077] The embodiments of the present application have been described above. The above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations are obvious to those of ordinary skill in the art in the technical field without departing from the scope and spirit of the described embodiments. The selection of the terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to the technologies in the market, or to enable other ordinary technical personnel in the technical field to understand the embodiments disclosed herein.

Claims

1. A preparation method of safflower extract containing flavonoid components, characterized in that, The specific steps include: (1) Inoculate Escherichia coli and Yarrowia lipolytica on a slant medium for activation; (2) Inoculate the activated Escherichia coli and Yarrowia lipolytica strains into a seed medium to obtain an Escherichia coli seed solution and a Yarrowia lipolytica seed solution; (3) Mix safflower powder evenly with water, add it to a fermentation medium, and add the Yarrowia lipolytica seed solution for fermentation; (4) After the fermentation in (3) ends, add the Escherichia coli seed solution for co-cultivation to obtain a fermentation broth; (5) Centrifuge the fermentation broth obtained in (4), take the supernatant, and successively carry out purification, elution, concentration, and drying to obtain a safflower extract containing flavonoid components.

2. The preparation method according to claim 1, characterized in that, The formula of the slant medium in (1) is: glucose 10 - 20 g / L, peptone 15 - 45 g / L, agar 15 - 20 g / L, yeast extract 5 - 10 g / L.

3. The preparation method according to claim 1, characterized in that, The formula of the seed medium in (2) is: glucose or sucrose 5 - 30 g / L, peptone 10 - 20 g / L, manganese sulfate 0.1 - 10 g / L, ammonium sulfate 5 - 12 g / L, magnesium sulfate 1 - 10 g / L.

4. The preparation method according to claim 1, characterized in that, The safflower in (3) includes plants of the genus Carthamus in the family Asteraceae.

5. The preparation method according to claim 1, characterized in that, The safflower powder in (3) is obtained by pulverizing safflower three times, passing through a 50-mesh sieve, and then carrying out vacuum drying.

6. The preparation method according to claim 1, wherein The formula of the fermentation medium in (3) is: glucose or xylose 120 - 300 g / L, peptone 15 - 45 g / L, ammonium sulfate 2 - 12 g / L, magnesium sulfate 1 - 6 g / L, manganese sulfate 1 - 6 g / L, rapeseed oil 20 - 110 g / L; The conditions for the fermentation are: temperature 28 - 30 °C, pH value 5.0 - 6.5, dissolved oxygen concentration 20% - 30%, rotation speed 200 - 800 rpm, time 48 - 72 h.

7. The preparation method according to claim 1, characterized in that, The conditions for the co-cultivation in (4) are: temperature 30 °C, pH value 5.0 - 6.5, dissolved oxygen concentration 20% - 60%, rotation speed 200 - 800 rpm, time 24 h.

8. The preparation method according to claim 1, characterized in that, The concentration method in (5) is vacuum concentration; the temperature of the vacuum concentration is 60 °C.

9. Use of a safflower extract containing flavonoid components prepared by the preparation method according to claim 1 in food, health products, and cosmetics.

Images