Preparation method and application of safflower extract containing flavonoids

Abstract

This application relates to the field of microbial transformation, and more specifically, to a method for preparing a safflower extract containing flavonoids and its application. Specifically, this application employs Escherichia coli and Yarrowia lipolytica to co-ferment safflower to prepare a safflower extract rich in flavonoids such as hydroxysafflower yellow, kaempferol, and quercetin. This extract can be used in foods, health products, and cosmetics, exhibiting significant antioxidant, anti-inflammatory, whitening, and anti-aging activities. It has the potential to improve skin health and is expected to become a substitute for plant-derived retinol.

Description

Technical Field

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

[0002] Phytorenin alcohols encompass a variety of ingredients, including Bidens pilosa extract, bakuchiol, milk thistle, Vigna aconitifolia seed extract, and kangaroo paw extract. While these can all be called phytorenin alcohols, they do not inherently contain vitamin A. Their key benefit lies in their ability to regulate the expression of collagen I and IV, elastin, fibroblast growth factor (FGF-β), tissue growth factor (TGF-β), matrix metalloproteinases (MMPs), and epidermal growth factor (EGFR). Currently, phytorenin alcohols include Bidens pilosa, milk thistle, and bakuchiol. Silymarin is one of the main components of milk thistle. Safflower contains chemically similar components to silymarin, including flavonoids, alkaloids, polysaccharides, lignans, polyacetylenes, spermidine, and sterols. Flavonoids and flavonoid glycosides are the primary active ingredients in safflower. Therefore, the flavonoids in safflower extract may be a promising candidate for phytorenin replacement.

[0003] Currently, methods for extracting flavonoids from safflower mainly include water extraction, microwave-assisted extraction, ultrasound-assisted water extraction, and cellulase extraction. However, these extraction methods are limited by environmental and seasonal variations (such as nutritional status and soil composition), making the preparation of safflower extracts complex and resulting in low flavonoid content. However, flavonoids in safflower extracts (such as hydroxysafflower yellow A) can reduce UV-induced MDA levels, effectively delaying photoaging. Flavonoids can also precisely regulate inflammation, rapidly alleviating seborrheic dermatitis. They modulate the skin microbiome, inhibiting Propionibacterium acnes and Staphylococcus aureus by over 90% without disrupting the skin's beneficial bacterial flora. They also inhibit melanin production through multiple targets and downregulate the expression of MITF and TRP-1 genes.

[0004] Therefore, there is an urgent need for a preparation method of safflower extract, which can increase the content of flavonoids in the safflower extract and make it more widely used in food, health products and cosmetics. Summary of the Invention

[0005] In order to solve or partially solve the problems existing in the related art, the present application provides a method for preparing safflower extract, which uses dual-bacteria cooperative fermentation to achieve a technological breakthrough, efficiently convert flavonoids, and improve flavonoid bioavailability. Through the synergistic action of the lipolytic enzyme of Yarrowia lipolytica and the β-glucosidase of Escherichia coli, the bound 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 in safflower (such as safflower glycoside), reduce the allergy rate, and significantly improve safety. Compared with the traditional ethanol extraction method, there is no organic solvent residue in the whole process, realizing green production.

[0006] On one hand, the present application provides a method for preparing a safflower extract containing flavonoid components, the specific steps comprising:

[0007] (1) Inoculate Escherichia coli and Yarrowia lipolytica into slant culture medium for activation;

[0008] (2) inoculating the activated Escherichia coli and Yarrowia lipolytica into a seed culture medium to obtain an Escherichia coli seed solution and a Yarrowia lipolytica seed solution;

[0009] (3) Mix safflower powder and water evenly, add to the fermentation medium, and first add Yarrowia lipolytica seed liquid for fermentation;

[0010] (4) After the fermentation in (3) is completed, Escherichia coli seed solution is added for co-cultivation to obtain a fermentation liquid;

[0011] (5) The fermentation liquid obtained in (4) is centrifuged, and the supernatant is taken and purified, eluted, concentrated, and dried in sequence to obtain a safflower extract containing flavonoid components.

[0012] Furthermore, the formula of the slant culture 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.

[0013] Furthermore, the formula of the seed culture 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, and magnesium sulfate 1-10 g / L.

[0014] Furthermore, the safflower powder in (3) is obtained by grinding safflower three times, passing it through a 50-mesh sieve, and then vacuum drying it.

[0015] Furthermore, 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;

[0016] The fermentation conditions are: temperature 28-30° C., pH 5.0-6.5, dissolved oxygen concentration 20%-30%, rotation speed 200-800 rpm, and time 48-72 hours.

[0017] Furthermore, the co-cultivation conditions in (4) are: temperature 30°C, pH 5.0-6.5, dissolved oxygen concentration 20%-60%, rotation speed 200-800 rpm, and time 24 h.

[0018] Furthermore, the concentration method in (5) is reduced pressure concentration; the reduced pressure temperature is 60°C.

[0019] The present application also provides an application of the safflower extract containing flavonoid components prepared according to the above preparation method in food, health products and cosmetics.

[0020] Beneficial effects

[0021] This application provides a method for preparing a safflower extract and its application. By establishing a co-culture system of Escherichia coli and Yarrowia lipolytica, the two microorganisms interact and, through complementary metabolic pathways, synergistically promote the extraction and conversion of active ingredients from safflower. This method not only increases the yield of flavonoids in the safflower extract but also enhances its biological activity.

[0022] 2. The present application also provides an application of a safflower extract rich in flavonoid components such as hydroxysafflower yellow and kaempferol in skin care products. The safflower extract can be used to prepare cosmetics with antioxidant, whitening, firming, and anti-inflammatory effects, or as a substitute for plant-derived retinol.

[0023] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The above and other objects, features and advantages of the present application will become more apparent by describing in more detail the exemplary embodiments of the present application in conjunction with the accompanying drawings, wherein the same reference numerals generally represent the same components in the exemplary embodiments of the present application.

[0025] Figure 1 It is the melanin situation of zebrafish head;

[0026] Figure 2is the effect of safflower extract on melanin density; compared with the normal group,*** P <0.001;

[0027] Figure 3 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.

[0028] Figure 4 It is the scavenging effect of safflower extract on DPPH.

[0029] Figure 5 Comparison of safflower extract on IL-1β secretion in LPS-induced RAW264.7 cells;

[0030] Compared with the blank group, ### P <0.001; compared with the model group,*** P <0.001.

[0031] Figure 6 The effect of safflower extract on the secretion of IL-6 in RAW264.7 cells induced by LPS was compared with the blank group. ### P <0.001; compared with the model group,*** P <0.001;** P <0.01; ns indicates no significant difference. DETAILED DESCRIPTION

[0032] The following describes embodiments of the present application in more detail with reference to the accompanying drawings. Although the accompanying drawings illustrate embodiments of the present application, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments described herein. Rather, 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.

[0033] To overcome the limitations of traditional extraction methods, microbial fermentation conversion technology can be used. Yarrowia lipolytica is a non-traditional yeast that has the ability to produce large amounts of cytoplasmic acetyl-CoA and malonyl-CoA. These two compounds can serve as precursors for the synthesis of high-value natural products such as terpenes and flavonoids. Secondly, Yarrowia lipolytica has a strong ability to decompose hydrophobic compounds and can show high efficiency in the synthesis of flavonoids. It has a wide range of substrate utilization, and can utilize carbon sources such as glucose, glycerol, ethanol, and acetic acid, as well as organic acids such as citric acid and isocitric acid, and especially oils and alkanes. It also has strong stress resistance and can tolerate high salt, low temperature, and excessively high acid and alkaline environments. With the continuous application and development of synthetic biology and gene editing technologies in Yarrowia lipolytica, it can also be edited and modified. As a model organism, Escherichia coli has the characteristics of rapid growth and strong reproductive ability, and can produce large quantities of flavonoids in a short period of time. Secondly, compared with traditional extraction methods, it has lower costs. In the process of producing flavonoids, it does not produce large amounts of waste and pollutants and has little impact on the environment. Escherichia coli has a clear genetic background and is easy to genetically engineer, which can optimize the metabolic pathway of Escherichia coli in producing flavonoids.

[0034] Escherichia coli is widely used in flavonoid production due to its ease of genetic manipulation and rapid growth, while Yarrowia lipolytica has shown potential for flavonoid production due to its metabolic properties and high availability of key prerequisites. By co-culturing Escherichia coli and Yarrowia lipolytica, different metabolic pathways and enzyme systems can be utilized. The combination of the two can complement each other's metabolic pathways, further optimize the interaction between microorganisms, and achieve efficient production of flavonoids.

[0035] Carthamus tinctorius ( CarthamusL. ) Plants belong to the Asteraceae family ( Asteraceae ), most of them are annual, and fewer are biennial or perennial herbs. There are about 18 to 20 species in this genus, mainly distributed in Central Asia, Southwest Asia and the Mediterranean region. There are two species of this genus in my country, namely safflower ( Carthamus tinctorius L. ) and safflower ( Carthamus Lanatus L. ), used in this application are exactly these two safflowers.

[0036] Unless otherwise specified, the following experimental materials were purchased from the market.

[0037] Example 1 Preparation of safflower extract containing flavonoids

[0038] 1. A method for preparing a safflower extract rich in flavonoids, comprising fermenting safflower with Escherichia coli and Yarrowia lipolytica, the specific steps comprising:

[0039] (1) Inoculate frozen Escherichia coli and Yarrowia lipolytica into slant culture medium for activation;

[0040] The formula of the slant culture medium is: glucose 20 g / L, peptone 45 g / L, agar 18 g / L, and yeast extract 7 g / L.

[0041] (2) inoculating the activated Escherichia coli and Yarrowia lipolytica into the seed culture medium to obtain mature seed liquid;

[0042] The formula of the seed culture medium is: glucose 30g / L, peptone 20g / L, manganese sulfate 10g / L, ammonium sulfate 7g / L, and magnesium sulfate 10g / L.

[0043] (3) Take appropriate amount of safflower ( Carthamus tinctorius L. ), pulverized three times using a grinder, sieved (50 mesh), and then vacuum-dried to obtain safflower powder.

[0044] (4) Mix an appropriate amount of safflower powder and water, add it to the fermentation medium, and first add the Yarrowia lipolytica seed culture solution, stir evenly, and place it in the fermentation medium at a temperature of 28°C-30°C, a pH value of 5.0-6.5, and a dissolved oxygen concentration of 20%-30%. The fermentation is carried out at a speed between 200-800 rpm and a fermentation time of 48-72 hours.

[0045] 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, and rapeseed oil 20-110 g / L.

[0046] (5) After the fermentation in step (4) is completed, the E. coli seed solution is added and the culture is continued for 24 hours. During the co-culture, the fermentation is carried out at a temperature of 30°C, a pH value of 5.0-6.5, a dissolved oxygen concentration of 20%-60%, and a rotation speed of 200-800 rpm.

[0047] (6) The fermentation broth was collected and centrifuged, and the supernatant was taken and purified using macroporous resin AB-8, eluted with 95% ethanol, and the eluate was collected and concentrated at a concentration temperature of 60°C; finally, vacuum drying was performed to obtain safflower extract, which is rich in flavonoid components such as hydroxysafflower yellow and kaempferol.

[0048] Example 2 Preparation of safflower extract containing flavonoids

[0049] Different from Example 1, the culture method is adjusted as follows: Yarrowia lipolytica is first cultured at 28°C for 72 hours, and the supernatant is added to Escherichia coli for culture, and the culture temperature can be raised to 30-37°C.

[0050] Example 3 Preparation of safflower extract containing flavonoids

[0051] Different from Example 1, the culture method was adjusted so that Yarrowia lipolytica and Escherichia coli were cultured together at 30°C.

[0052] Example 4 Preparation of safflower extract containing flavonoids

[0053] The difference from Example 1 is that the species of safflower is adjusted to be safflower ( Carthamus Lanatus L. ).

[0054] Comparative Example 1 Except for the following experimental steps, the remaining steps are the same as those in Example 1.

[0055] 1) The formula of the slant culture medium in step (1) of Example 1 was adjusted to: glucose 15 g / L, peptone 25 g / L, agar 20 g / L, and yeast extract 10 g / L;

[0056] 2) The formula of the slant culture medium in step (1) of Example 1 was adjusted to: glucose 10 g / L, peptone 15 g / L, agar 15 g / L, and yeast extract 5 g / L.

[0057] Comparative Example 2

[0058] Except for the following experimental steps, the remaining steps are the same as those in Example 1.

[0059] 1) The formula of the seed culture medium in step (2) of the embodiment was adjusted to: glucose 5 g / L, peptone 18 g / L, manganese sulfate 6 g / L, ammonium sulfate 12 g / L, magnesium sulfate 3 g / L;

[0060] 2) The formula of the seed culture medium in step (2) of the embodiment was adjusted to: sucrose 25 g / L, peptone 10 g / L, manganese sulfate 0.1 g / L, ammonium sulfate 5 g / L, and magnesium sulfate 1 g / L.

[0061] Comparative Example 3

[0062] Except for the following experimental steps, the remaining steps are the same as those in Example 1.

[0063] The models of the macroporous resins in step (6) of Example 1 were adjusted to D101, HPD-600, HPD-100, and AB-8.

[0064] Comparative Example 4

[0065] A method for preparing a safflower extract rich in flavonoids comprises co-fermenting safflower with Escherichia coli and Yarrowia lipolytica, and specifically comprising the following steps:

[0066] (1) The frozen Yarrowia lipolytica was inoculated into a slant culture medium for activation; the formula of the slant culture medium was as follows: glucose 20 g / L, peptone 45 g / L, agar 18 g / L, and yeast extract 7 g / L.

[0067] (2) The activated Yarrowia lipolytica strain was inoculated into a seed culture medium to obtain a mature seed solution; the formula of the seed culture medium was as follows: 30 g / L glucose, 20 g / L peptone, 10 g / L manganese sulfate, 7 g / L ammonium sulfate, and 10 g / L magnesium sulfate.

[0068] (3) Take an appropriate amount of safflower, grind it three times using a grinder, sieve it (50 mesh), and then vacuum dry it to obtain safflower powder.

[0069] (4) Mix an appropriate amount of safflower powder and water evenly, add it to the fermentation medium, and first add the culture medium of Yarrowia lipolytica seed bacteria. After stirring evenly, place it under the conditions of temperature 28℃-30℃, pH value 5.0-6.5, dissolved oxygen concentration of 20%-30% for fermentation, rotation speed between 200-800rpm, and fermentation time for 48-72 hours; the formula of the fermentation medium is: glucose / xylose 120-300g / L, peptone 15-45g / L, ammonium sulfate 2-12g / L, magnesium sulfate 1-6g / L, manganese sulfate 1-6g / L, rapeseed oil 20-110g / L.

[0070] (5) The fermentation broth was collected and centrifuged, and the supernatant was taken and purified using macroporous resin AB-8, eluted with 95% ethanol, and the eluate was collected and concentrated at a concentration temperature of 60°C; finally, vacuum drying was performed to obtain safflower extract.

[0071] Example 5: Determination and identification of the safflower extract obtained in Example 1

[0072] 1. Determination of total flavonoids content in sample solution

[0073] The sodium nitrite-aluminum nitrate-sodium hydroxide colorimetric method was used for determination, with rutin as the reference substance, and the absorbance was measured at 510 nm. A 0.2 mg / mL rutin reference solution was prepared by dissolving the rutin reference substance in 70% ethanol. 1 mL of the safflower extract obtained in Example 1 was weighed into a 10 mL volumetric flask, and 0.5 mL of a 5% sodium nitrite solution was added. The mixture was shaken and allowed to stand for 6 min. 0.5 mL of a 10% aluminum nitrate solution was then added. The mixture was shaken and allowed to stand for 6 min. 4 mL of a 4% sodium hydroxide solution was then added. The mixture was made up to volume with 70% ethanol, shaken, allowed to stand for 15 min, and the absorbance was measured at 510 nm.

[0074] 2. Determination of hydroxysafflor yellow A (HSYA) content

[0075] (1) Octadecylsilane bonded silica gel was used as the filler; methanol-acetonitrile-0.7% phosphoric acid solution (26:2:72) was used as the mobile phase; the detection wavelength was 403 nm. The theoretical plate number calculated based on the hydroxysafflor yellow A peak should be no less than 3000.

[0076] (2) Preparation of reference solution: Accurately weigh 1.32 mg of hydroxysafflor yellow A reference solution and place it in a 10 mL volumetric flask. Add 25% methanol to the volume and shake well.

[0077] (3) Use precise pipette to pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and measure.

[0078] 3. Determination of Kaempferol and Quercetin Content

[0079] (1) Octadecylsilane bonded silica gel was used as the filler; methanol-0.7% phosphoric acid solution (45:55) was used as the mobile phase; the detection wavelengths were 370 nm (kaempferol) and 360 nm (quercetin). Under these chromatographic conditions, quercetin and kaempferol were well separated and baseline-separated from the other components. The theoretical plate number for quercetin was 10,400, and the resolution was greater than 1.2.

[0080] (2) Preparation of reference solution: Accurately weigh 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; accurately weigh 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.

[0081] (3) Use precise pipette to pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and measure.

[0082] Table 1 Sample data

[0083]

[0084] 3. Evaluate the whitening efficacy of safflower extract

[0085] Experimental principle: The main pathway of the whitening effect of skin care products is achieved by inhibiting the activity of tyrosinase, thereby reducing the production of melanin. The melanin regulation mechanism in zebrafish skin is highly conserved with that in humans, and the melanin is produced quickly, which can be clearly observed with the naked eye under a microscope. Image processing technology can be used to quantify the degree to which skin care products reduce melanin, thereby realizing the evaluation of whitening efficacy.

[0086] Experimental plan: Zebrafish were divided into three groups: a normal control group, a positive control group, and a test sample group. The normal control group received no treatment, the positive control group received 0.1% niacinamide, and the test sample group received a certain concentration of safflower extract (prepared according to the preparation method in Example 1). After incubation for a period of time, the zebrafish heads were photographed and the melanin signal intensity in the zebrafish heads was analyzed using advanced image processing software.

[0087] Depend on Figure 1 It can be seen that compared with the normal group, the melanin in the zebrafish head was significantly reduced in the experimental groups with different concentrations of safflower extract, revealing that it has a whitening effect.

[0088] The melanin density of each group was statistically analyzed. Figure 2 As shown, compared with the normal group, different concentrations of safflower extract can significantly reduce the melanin density ( P <0.001), indicating that it has whitening effect.

[0089] 4. Evaluation of the upregulation rate of type I collagen content by safflower extract

[0090] Principle: Type I collagen is a major component of the dermal extracellular matrix. Dermal fibroblasts synthesize type I procollagen intracellularly and secrete it extracellularly. Terminal procollagen peptidase dissociates the telopeptides and polymerizes to form collagen fibrils. Human dermal fibroblasts can be used as a cell model to study cosmetics that increase type I collagen content. By measuring the rate of increase in type I collagen content after administration of the test substance, compared to a blank control and the test substance, the efficacy of the test substance in promoting collagen synthesis is evaluated.

[0091] Experimental plan: The experiment was divided into three groups: a normal control group, a positive group, and a test sample group. The normal control group received no treatment, the positive group received 100 ng / mL of TGF-β1, and the test sample group received varying concentrations of safflower extract (prepared by the method in Example 1). After incubation for a period of time, the type I collagen content was measured using an enzyme-linked immunosorbent assay (ELISA).

[0092] The results of type I collagen content and upregulation rate are as follows Figure 3As shown: After intervention, the positive control group (TGF-β1100ng / 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 within the concentration range of 50μg / mL~250μg / mL ( P <0.001). This indicates that safflower extract has firming and anti-wrinkle effects within the concentration range of 50μg / mL to 250μg / mL.

[0093] 5. Determination of the antioxidant activity of safflower extract by DPPH method

[0094] Principle: DPPH is a stable free radical in organic solvents. Its alcoholic solution is purple and must be stored at low temperatures and away from light. It has a single electron and can therefore accept an electron or hydrogen ion, with maximum absorption at a wavelength of 517 nm. In the presence of a free radical scavenger, DPPH's single electron is captured, causing its color to lighten. The absorbance at the wavelength of maximum absorption decreases linearly, and a decrease in absorbance indicates an increase in antioxidant activity, thereby evaluating the antioxidant capacity of the test sample. This antioxidant capacity is expressed as an inhibition rate; a higher inhibition rate indicates a stronger antioxidant capacity.

[0095] Experimental plan: Weigh a certain amount of DPPH powder and dissolve it in anhydrous ethanol. Take 1 mL of safflower extract test solution with a concentration of 100, 200, 300, 400, and 500 μg / mL in a stoppered test tube, add 5 mL of DPPH solution, shake well, and measure the absorbance at a wavelength of 517 nm after 30 minutes.

[0096] Depend on Figure 4 It can be seen that as the concentration of safflower extract increases, the DPPH free radical scavenging rate also increases, indicating that the DPPH free radical scavenging activity of safflower extract has a dose-dependent relationship.

[0097] 6. Evaluate the anti-inflammatory activity of safflower extract, which can inhibit the production of inflammatory factors induced by lipopolysaccharide

[0098] Experimental Principle: LPS-induced RAW264.7 cells are a classic cell model for studying inflammatory factors. LPS binds to antigen-recognition receptors on the surface of macrophages, inducing macrophages to secrete multiple inflammatory factors, including TNF-α, IL-1β, and IL-6.

[0099] Experimental plan: The experiment was divided into three groups: a normal control group, a model group, and a test sample group. The normal control group received no treatment, the model group received 1 μg / ml LPS, and the test sample group received varying concentrations of safflower extract. After incubation, the levels of inflammatory factors IL-6 and IL-1β were measured using enzyme-linked immunosorbent assay (ELISA).

[0100] Depend on Figure 5 、 Figure 6 It can be seen that the levels of IL-1β and IL-6 in the serum of the model group were significantly increased ( P <0.01). Safflower extract (25, 50, 100 and 250 μg / mL) can significantly reduce IL-1β levels ( P <0.01). Compared with the model group, safflower extract (50, 100 and 250 μg / mL) significantly reduced the level of IL-6 ( P <0.01). The results showed that safflower extract could inhibit the production of inflammatory cytokines induced by LPS, indicating that safflower extract has anti-inflammatory effects.

[0101] Example 6 Application of safflower extract in skin care products.

[0102] Taking Example 1 as a preferred embodiment, the safflower extract containing flavonoids prepared in Example 1 was added to a skin care product to prepare an essence with anti-aging effects. The formula is shown in Table 2:

[0103] Table 2 Anti-aging essence formula

[0104]

[0105] Based on the anti-aging essence formula in Table 2, two test samples were prepared: Sample 1: containing 1% safflower extract (original formula); Sample 2: 1% safflower extract was replaced with 0.3% retinol, and the other concentrations and ingredients remained unchanged.

[0106] Human efficacy test:

[0107] Inclusion criteria: Age 30-60 years; fine lines or wrinkles around the eyes, meeting the Skin Aging Atlas Asian classification of 1-6; participants have not participated in other clinical studies in the past three months;

[0108] Exclusion criteria: Use of antihistamines in the past week or immunosuppressants in the past month; use of any anti-inflammatory drugs in the test area in the past two months; insulin-dependent diabetic patients; patients with respiratory diseases being treated; breastfeeding or pregnant women; people with allergies, allergic dermatitis, etc., with skin diseases or a history of diseases; people receiving dermatological treatment, or subjects who have taken hydroxy acids, whitening drugs, and anti-aging drugs within one month; large birthmarks, scratches, white spots, pigmented moles, keloids, and other skin manifestations that affect the test in the test area; in addition, people who are judged by the trial leader to be unsuitable as subjects for this trial.

[0109] Instruments: Visia-CR facial image analyzer (Canfield, USA); Cutometer (MPA580, CK, Germany)

[0110] Test method:

[0111] In a self-controlled, before-and-after study, participants applied the serum to their face every morning and evening after cleansing. Sample 1: prepared according to the formula in Table 2 (containing 1% safflower extract); Sample 2: based on the formula in Table 2, replacing the 1% safflower extract with 0.3% retinol. The serum was gently applied until fully absorbed, twice daily for 28 days. Efficacy testing was conducted on subjects on Day 0 (D0) and Day 28 (D28).

[0112] 30 qualified subjects were screened and cleaned their faces with warm water and dried with dandruff-free facial tissue before the formal test. After sitting quietly for at least 30 minutes in an environment with a temperature of 20℃~22℃ and a relative humidity of 40%~60%RH, professionals conducted an initial value test and recorded it. The same test was conducted and recorded again after 28 days of using the product.

[0113] Skin wrinkle and roughness test

[0114] The average value and roughness of local wrinkles were measured using a Visia-CR facial image analyzer, and the changes in local wrinkles and roughness before and after using the product were observed. The smaller the measured values, the more obvious the wrinkle improvement effect.

[0115] Skin elasticity test

[0116] At each visit 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.

[0117] Statistical analysis

[0118] The experimental results were compared by 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. The statistical methods all used two-tailed tests, and the test level a=0.05.

[0119] Skin index analysis test results:

[0120] Table 3 Comparative analysis of anti-aging indicators of sample 1 (containing safflower extract) at different times (mean ± standard deviation, n=30)

[0121]

[0122] Table 4 Comparative analysis of anti-aging indicators of sample 2 (containing A alcohol) at different times (mean ± standard deviation, n=30)

[0123]

[0124] Note: The significance marking method "ns" means no statistical difference; * P >0.05, indicating significant difference; ** P <0.01, indicating a significant difference.

[0125] According to the experimental results, Samples 1 and 2 provided by the present invention have the effect of significantly improving skin elasticity, and also have a significant effect in reducing skin wrinkles and skin roughness, which can be beneficial for resisting aging and improving skin health.

[0126] 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 will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is selected to best explain the principles of the embodiments, their practical applications, or improvements to the technology in the market, or to enable other persons skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for preparing a safflower extract containing flavonoids, characterized in that: The specific steps include: (1) Inoculate Escherichia coli and Yarrowia lipolytica into slant culture medium for activation; (2) inoculating the activated Escherichia coli and Yarrowia lipolytica into a seed culture medium to obtain an Escherichia coli seed solution and a Yarrowia lipolytica seed solution; (3) Mix safflower powder and water evenly, add to fermentation medium, and add Yarrowia lipolytica seed liquid for fermentation; (4) After the fermentation in (3) is completed, Escherichia coli seed solution is added for co-cultivation to obtain a fermentation liquid; (5) The fermentation liquid obtained in (4) is centrifuged, and the supernatant is collected and purified, eluted, concentrated, and dried in sequence to obtain a safflower extract containing flavonoid components.

2. The preparation method according to claim 1, characterized in that The formula of the slant culture 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 culture 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 tinctorius in the Asteraceae family.

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

6. The preparation method according to claim 1, characterized in that 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 fermentation conditions are: temperature 28-30° C., pH 5.0-6.5, dissolved oxygen concentration 20%-30%, rotation speed 200-800 rpm, and time 48-72 hours.

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

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

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

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