Preparation method of magnolia flower fermentation liquor and application of fermentation liquor in cosmetics

Abstract

The present invention discloses a magnolia flower fermentation broth, which is characterized in that the magnolia flower fermentation broth is obtained by fermenting the magnolia flower with a plant lactobacillus (Lactiplantarum), and the magnolia flower fermentation broth is obtained by fermenting the magnolia flower with the plant lactobacillus (Lactiplantarum). The fermentation method is simple and rapid, and the obtained magnolia flower fermentation liquor has skin care effects of whitening skin, relieving allergy, resisting aging and the like.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to a method for preparing magnolia flower fermentation liquid and application of the fermentation liquid in cosmetics. Background Art

[0002] Magnolia flower, also known as Wangchun flower, Yingchun flower, and Mubi flower, is the general name for the flower buds of Magnoliaceae plants. Magnolia flower varieties are complex, mainly Wangchun magnolia, Yulan magnolia and Wudang magnolia. Their flower buds can be used as medicine and are called "magnolia". Magnolia flower has a long history of use and is a traditional Chinese medicinal material in my country. It is generally picked before the flower buds open in early spring and can be used as medicine after drying. Studies have shown that magnolia flowers contain active ingredients such as volatile oils, lignans, and flavonoids, and exhibit good anti-inflammatory, anti-allergic, antibacterial, and antiviral pharmacological activities. Therefore, magnolia flowers are also often used clinically to treat diseases such as cold headaches, nasal congestion, sinusitis, and runny nose. However, traditional methods of using magnolia flowers, such as decoction and soaking, have problems such as low extraction rate of active ingredients and unstable efficacy, which limits its wider application.

[0003] In recent years, fermentation technology has attracted more and more attention as a green and efficient biotransformation method. Compared with traditional extraction methods, fermentation can not only improve the bioavailability of active ingredients, but also produce new active substances, reduce toxic side effects, and comply with the concept of sustainable development, thus having broad application prospects. However, there are few reports on the use of fermentation technology to carry out corresponding biotransformation of magnolia flowers and develop their innovative applications in the field of cosmetics and skin care products. Summary of the invention

[0004] Based on this, the present invention provides a magnolia flower fermentation liquid, which is obtained by fermenting the magnolia flower with Lactobacillus plantarum.

[0005] According to another aspect of the present invention, there is provided a method for preparing the above fermentation broth, the method comprising the following steps:

[0006] (1) Weighing an appropriate amount of magnolia flowers, crushing, and sieving to obtain magnolia flower powder;

[0007] (2) adding a culture medium into a container, adding the magnolia flower powder and seed liquid, and fermenting to obtain a crude fermentation liquid; and

[0008] (3) inactivating and purifying the crude fermentation broth to obtain the magnolia flower fermentation broth.

[0009] Furthermore, the sieve is a 10-mesh sieve.

[0010] Furthermore, the mass ratio of the magnolia flower powder to the culture medium is 0.005-0.1, such as 0.01-0.08, such as 0.03-0.08.

[0011] Furthermore, the volume ratio of the seed solution to the culture medium is 0.03 to 0.2, such as 0.04 to 0.15, such as about 0.06 to 0.1.

[0012] Furthermore, the culture medium is a liquid culture medium.

[0013] Furthermore, the container is a fermentation tank.

[0014] Further, the fermentation temperature is 20-45°C, such as 20-40°C, such as 25-40°C.

[0015] Furthermore, the fermentation pH is 2-7, such as 3-7, such as 4-7.

[0016] Furthermore, the fermentation rotation speed is 10 to 1000 r / min, such as 50 to 800 r / min, such as 100 to 600 r / min.

[0017] Furthermore, the fermentation time is 12 to 72 hours, such as 18 to 48 hours.

[0018] Furthermore, the inactivation temperature is 115-121°C.

[0019] Furthermore, the inactivation time is 10 to 30 minutes, for example, about 20 minutes.

[0020] Further, the purification includes filtering and sterilizing the crude fermentation broth.

[0021] Furthermore, the seed solution is obtained by culturing the Lactobacillus plantarum in a seed culture medium.

[0022] Furthermore, the Lactobacillus plantarum is activated Lactobacillus plantarum.

[0023] Furthermore, the seed culture medium is a liquid seed culture medium.

[0024] Furthermore, the Lactobacillus plantarum is isolated from fermentation slurry.

[0025] Furthermore, the plant lactobacillus (Lactiplantibacillus plantarum) is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, and its deposit number is CGMCC No. 28500. According to another aspect of the present invention, there is provided a use of the above fermentation broth in preparing cosmetics having whitening, soothing, anti-inflammatory and / or anti-aging effects.

[0026] According to another aspect of the present invention, there is provided a use of a composition comprising the above fermentation broth in preparing cosmetics having whitening, soothing, anti-inflammatory and / or anti-aging effects.

[0027] Furthermore, the whitening is to inhibit cellular melanin synthesis.

[0028] Furthermore, the anti-aging effect is achieved through one or more of the following ways: protecting type I collagen, inhibiting the production of MMP-1 and inhibiting the production of MMP-9.

[0029] Furthermore, the soothing and anti-inflammatory effects are achieved in one or more of the following ways: inhibiting the production of TNF-α and inhibiting the degranulation of mast cells.

[0030] Furthermore, the composition further comprises one or more auxiliary materials.

[0031] Furthermore, the auxiliary material is selected from one or more of the following: thickener, surfactant, co-surfactant, skin conditioner, preservative, flavor, lubricant, disintegrant, wetting agent, binder and filler.

[0032] Furthermore, the composition further comprises one or more whitening substances.

[0033] Beneficial effects of the present invention:

[0034] The invention utilizes Lactiplantibacillus plantarum separated from fermented slurry to ferment magnolia flowers. The method is simple and rapid. The obtained magnolia flower fermented liquid has skin care effects on skin whitening, soothing and anti-aging, and is suitable for popularization and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without exceeding the scope of protection required by the present invention.

[0036] Figure 1The figure is a schematic diagram of the results of the fermented liquor of Magnolia officinalis flower inhibiting the synthesis of melanin in A375 cells. BC: blank control (A375 cells), PC: positive control (0.5% w / w 377), compared with BC, * represents P < 0.05, ** represents P < 0.01.

[0037] Figure 2 The figure is a schematic diagram of the results of the magnolia flower fermentation liquid protecting type I collagen under UVA stimulation. BC: blank control (human skin fibroblasts), NC: negative control (9J / cm 2 UVA stimulation), PC: positive control (100 μg / mL VC + 7 μg / mL VE); compared with BC, # represents P < 0.05, ## represents P < 0.01; compared with NC, * represents P < 0.05, ** represents P < 0.01.

[0038] Figure 3 The result of inhibiting the synthesis of MMP-1 under UVA stimulation by the fermented liquor of Magnolia flower of the present invention is shown in the figure. BC: blank control (human skin fibroblasts), NC: negative control (UVA stimulation of 9J / cm2), PC: positive control (100μg / mL VC+7μg / mL VE); compared with BC, # represents P<0.05, ## represents P<0.01; compared with NC, * represents P<0.05, ** represents P<0.01.

[0039] Figure 4 The result of inhibiting the synthesis of MMP-9 under UVA stimulation by the fermented liquor of Magnolia flower of the present invention is shown in the figure. BC: blank control (human skin fibroblasts), NC: negative control (UVA stimulation of 9J / cm2), PC: positive control (100μg / mL VC+7μg / mL VE); compared with BC, # represents P<0.05, ## represents P<0.01; compared with NC, * represents P<0.05, ** represents P<0.01.

[0040] Figure 5 The figure is a schematic diagram of the results of the fermented liquid of Magnolia flower of the present invention inhibiting the production of TNF-α under LPS stimulation. BC: blank control (RAW264.7 cells), NC: negative control (0.00001% LPS), PC: positive control (0.01% dexamethasone); compared with BC, # represents P < 0.05, ## represents P < 0.01; compared with NC, * represents P < 0.05, ** represents P < 0.01.

[0041] Figure 6The result schematic diagram of the inhibition of mast cell degranulation by the fermented liquor of Magnolia officinalis flower under C48 / 80 stimulation of the present invention is shown in FIG. BC: blank control (mast cells), NC: negative control (100 μg / mL C48 / 80 stimulation), PC: positive control (1 mg / mL sodium cromoglycate); compared with BC, # represents P < 0.05, ## represents P < 0.01; compared with NC, * represents P < 0.05, ** represents P < 0.01. DETAILED DESCRIPTION

[0042] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0043] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those of ordinary skill in the field of the invention or the field in which the terms are used. Although any methods, conditions, substances or materials similar to or equivalent to those disclosed herein may be used in the practice of the present invention, preferred methods, conditions, substances or materials are described herein.

[0044] The present invention is intended to encompass all alternatives, variations and equivalents that may be included in the present invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0045] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0046] In the present invention, the term "comprising" is synonymous with "including". As used herein, the terms "comprising", "including", "having", "containing" or any other variation thereof are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus comprising the listed elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0047] As described in the background technology section, there is no related report in the prior art that the fermented magnolia flower liquid obtained by fermenting magnolia flower with Lactiplantibacillus plantarum has skin care effects such as whitening, soothing and anti-aging. In order to solve the above problems, the present invention provides a fermented magnolia flower liquid, which is obtained by fermenting magnolia flower with Lactiplantibacillus plantarum.

[0048] According to another aspect of the present invention, there is provided a method for preparing the above fermentation broth, the method comprising the following steps:

[0049] (1) Weigh an appropriate amount of magnolia flowers, crush them, and sieve them to obtain magnolia flower powder;

[0050] (2) adding a culture medium into a container, adding the magnolia flower powder and seed liquid, and fermenting to obtain a crude fermentation liquid; and

[0051] (3) inactivating and purifying the crude fermentation broth to obtain the magnolia flower fermentation broth.

[0052] In a preferred embodiment, the sieve is a 10 mesh sieve.

[0053] In the present invention, when a ratio, temperature, pH, rotation speed, time, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper preferred values ​​and lower preferred values, it should be understood that all ranges formed by any pairing of any range upper limit or preferred value and any range lower limit or preferred value are specifically disclosed, regardless of whether the range is disclosed separately. For example, when the range "0.01-0.08" is disclosed, the described range should be interpreted as including the range "0.01-0.08", "0.01-0.06", "0.01-0.04", "0.01-0.02", "0.02-0.08", "0.02-0.06", "0.02-0.04", "0.04-0.08", "0.04-0.06", "0.06-0.08" and the like. When a numerical range is described herein, unless otherwise stated, the range is intended to include its end values ​​and all integers and fractions within the range.

[0054] The present invention utilizes the metabolic activity of microorganisms to convert natural ingredients into more stable and more active compounds. Compared with traditional extraction methods, the fermentation process can not only improve the purity and stability of active ingredients, but also reduce dependence on raw materials and reduce the impact on the environment. Therefore, it is of great significance in the production of cosmetics.

[0055] In a preferred embodiment, the mass ratio of the magnolia flower powder to the culture medium is 0.005-0.1, such as 0.01-0.08, such as 0.03-0.08.

[0056] In a preferred embodiment, the volume ratio of the seed solution to the culture medium is 0.03-0.2, such as 0.04-0.15, such as about 0.06-0.1.

[0057] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.06" includes ±5% of 0.06, or from 0.057 to 0.063.

[0058] In a preferred embodiment, the culture medium is a liquid culture medium.

[0059] In a preferred embodiment, the container is a fermenter.

[0060] In a preferred embodiment, the fermentation temperature is 20-45°C, such as 20-40°C, such as 25-40°C.

[0061] In a preferred embodiment, the pH of the fermentation is 2-7, such as 3-7, such as 4-7.

[0062] In a preferred embodiment, the fermentation rotation speed is 10-1000 r / min, such as 50-800 r / min, such as 100-600 r / min.

[0063] In a preferred embodiment, the fermentation time is 12 to 72 hours, such as 18 to 48 hours.

[0064] In a preferred embodiment, the inactivation temperature is 115-121°C.

[0065] In a preferred embodiment, the inactivation time is 10 to 30 minutes, such as about 20 minutes.

[0066] In a preferred embodiment, the purification comprises filtering and sterilizing the crude fermentation broth.

[0067] In a preferred embodiment, the seed liquid is obtained by culturing the Lactobacillus plantarum in a seed culture medium.

[0068] In a preferred embodiment, the Lactobacillus plantarum is activated Lactobacillus plantarum.

[0069] In a preferred embodiment, the seed culture medium is a liquid seed culture medium.

[0070] In a preferred embodiment, the Lactiplantibacillus plantarum is isolated from fermentation slurry.

[0071] In a preferred embodiment, the Lactobacillus plantarum is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration, and its deposit number is CGMCC No.28500.

[0072] For other information about the above-mentioned strains used in the present invention, please refer to the information disclosed in application number 202410502170.3 and publication number CN118165889A.

[0073] According to another aspect of the present invention, there is provided a use of the fermentation broth in preparing cosmetics having whitening, soothing, anti-inflammatory and / or anti-aging effects.

[0074] According to another aspect of the present invention, there is provided a use of a composition comprising the above fermentation broth in preparing cosmetics having whitening, soothing, anti-inflammatory and / or anti-aging effects.

[0075] In a preferred embodiment, the whitening is to inhibit cellular melanin synthesis.

[0076] In a preferred embodiment, the anti-aging effect is achieved through one or more of the following ways: protecting type I collagen, inhibiting the production of MMP-1, and inhibiting the production of MMP-9.

[0077] In a preferred embodiment, the soothing and anti-inflammatory effect is achieved through one or more of the following means: inhibiting the production of TNF-α and inhibiting the production of mast cell degranulation.

[0078] In a preferred embodiment, the composition further comprises one or more auxiliary materials.

[0079] In a preferred embodiment, the auxiliary material is selected from one or more of the following: thickener, surfactant, co-surfactant, skin conditioner, preservative, flavor, lubricant, disintegrant, wetting agent, binder and filler.

[0080] In a preferred embodiment, the composition further comprises one or more whitening substances.

[0081] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions or conditions recommended by the manufacturer.

[0082] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0083] The above features mentioned in the present invention or the features mentioned in the embodiments can be combined in any way. All the features disclosed in this patent specification can be used in combination with any combination form, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0084] Example

[0085] Preparation Example

[0086] Biochemical incubator (LRH-150, Shanghai Yiheng), autoclave (SYQ-DSX-280B, Shanghai Shen'an), clean bench (SW-CJ-2F, Suzhou Purification), centrifuge (Hermle, Z216M), fully automatic double water purifier (Shanghai Jinke Industry, SZ-93), anaerobic box (ANAEROPACK, Mitsubishi Gas), rotary evaporator (R-206, Shanghai Shensheng), circulating water vacuum pump (SHZ-D, Shanghai Yuhua), MRS broth culture medium (Beijing Luqiao), MRS agar culture medium (Oxoid).

[0087] The magnolia flowers used in this example were purchased from Shanghai Jiangqiao Wholesale Market, and are a dried sample of magnolia flowers produced in Anhui. They were identified as the flower buds of the plant magnolia Yulania denudata (Desr.) DLFu of the genus Magnolia of the Magnoliaceae family. They were placed in a ventilated and dry place for storage. The white plum blossoms used in this example were purchased from Shanghai Jiangqiao Wholesale Market, and are a dried sample of white plum blossoms produced in Anhui. They were identified as the flower buds of the plant green calyx plum Prunus mumef.viridicalyx (Makino) TYChen of the Rosaceae family. They were placed in a ventilated and dry place for storage.

[0088] Example 1

[0089] The magnolia flowers were crushed by a grinder and passed through a 10-mesh screen. The crushed magnolia flowers were then added to the fermentation medium at a mass fraction of 3%, and the activated lactobacillus seed solution was added at an inoculum amount of 6% of the volume percentage of the fermentation medium. The fermentation temperature was 25°C, the pH was 4.0, the speed was 100r / min, and the fermentation time was 18h. After the fermentation was completed, the prepared fermentation liquid was inactivated and purified multiple times to obtain magnolia flower fermentation liquid A.

[0090] Example 2

[0091] The magnolia flower is crushed by a grinder and passed through a 10-mesh screen, and then the crushed magnolia flower is added according to 8% of the mass fraction of the fermentation medium, and then the activated lactobacillus seed liquid is added according to the inoculation amount of 10% of the volume percentage of the fermentation medium, the fermentation temperature is 40°C, the pH is 7.0, the speed is 600r / min, and the fermentation time is 48h. After the fermentation is completed, the prepared fermentation liquid is inactivated and purified multiple times to obtain magnolia flower fermentation liquid B.

[0092] Comparative Example 1

[0093] The magnolia flowers were crushed by a grinder and passed through a 10-mesh screen. The crushed magnolia flowers were then added according to 15% of the mass fraction of the fermentation medium, and the activated lactobacillus seed liquid was added according to an inoculum amount of 6% of the volume percentage of the fermentation medium. The fermentation temperature was 25°C, the pH was 4.0, the speed was 100r / min, and the fermentation time was 18h. After the fermentation was completed, the prepared fermentation liquid was inactivated and purified multiple times to obtain magnolia flower fermentation liquid C.

[0094] Comparative Example 2

[0095] The magnolia flowers were crushed by a grinder and passed through a 10-mesh screen. The crushed magnolia flowers were then added to the fermentation medium at a mass fraction of 3%, and the activated lactobacillus seed solution was added at an inoculum amount of 2% of the fermentation medium volume percentage. The fermentation temperature was 25°C, the pH was 4.0, the speed was 100r / min, and the fermentation time was 18h. After the fermentation was completed, the prepared fermentation liquid was inactivated and purified multiple times to obtain magnolia flower fermentation liquid D.

[0096] Comparative Example 3

[0097] The magnolia flowers were crushed by a grinder and passed through a 10-mesh screen. The crushed magnolia flowers were then added to the fermentation medium at a mass fraction of 3%, and the activated lactobacillus seed solution was added at an inoculum amount of 6% of the volume percentage of the fermentation medium. The fermentation temperature was 65°C, the pH was 4.0, the speed was 100r / min, and the fermentation time was 18h. After the fermentation was completed, the prepared fermentation liquid was inactivated and purified multiple times to obtain magnolia flower fermentation liquid E.

[0098] Comparative Example 4

[0099] The magnolia flowers were crushed by a grinder and passed through a 10-mesh screen. The crushed magnolia flowers were then added to the fermentation medium at a mass fraction of 3%, and the activated lactobacillus seed solution was added at an inoculum amount of 6% of the volume percentage of the fermentation medium. The fermentation temperature was 25°C, the pH was 9.0, the speed was 100 r / min, and the fermentation time was 18 hours. After the fermentation was completed, the prepared fermentation liquid was inactivated and purified multiple times to obtain magnolia flower fermentation liquid F.

[0100] Comparative Example 5

[0101] The magnolia flowers are crushed by a grinder and passed through a 10-mesh screen. The crushed magnolia flowers are then added according to 3% of the mass fraction of the fermentation medium, and the activated lactobacillus seed liquid is added according to an inoculum amount of 6% of the volume percentage of the fermentation medium. The fermentation temperature is 25°C, the pH is 4.0, the speed is 1200r / min, and the fermentation time is 18h. After the fermentation is completed, the prepared fermentation liquid is inactivated and purified multiple times to obtain the magnolia flower fermentation liquid G.

[0102] Comparative Example 6

[0103] The magnolia flowers were crushed by a grinder and passed through a 10-mesh screen. The crushed magnolia flowers were then added to the fermentation medium at a mass fraction of 3%, and the activated lactobacillus seed solution was added at an inoculum amount of 6% of the fermentation medium volume percentage. The fermentation temperature was 25°C, the pH was 4.0, the speed was 100r / min, and the fermentation time was 96h. After the fermentation was completed, the prepared fermentation liquid was inactivated and purified multiple times to obtain the magnolia flower fermentation liquid H.

[0104] Comparative Example 7

[0105] The magnolia flowers were crushed by a grinder and passed through a 10-mesh screen. The crushed magnolia flowers were then added at 3% of the mass fraction of the fermentation medium, and the sterile seed liquid culture medium system was added at an inoculation amount of 6% of the volume percentage of the fermentation medium. The fermentation temperature was 25°C, the pH was 4.0, the speed was 100r / min, and the fermentation time was 18h. After the fermentation was completed, the prepared fermentation liquid was inactivated and purified multiple times to obtain magnolia flower fermentation liquid I.

[0106] Comparative Example 8

[0107] The white plum blossoms were crushed by a grinder and passed through a 10-mesh screen. The crushed white plum blossoms were then added to the fermentation medium at a mass fraction of 3%, and the activated lactobacillus seed solution was added at an inoculum amount of 6% of the volume percentage of the fermentation medium. The fermentation temperature was 25°C, the pH was 4.0, the speed was 100r / min, and the fermentation time was 18h. After the fermentation was completed, the prepared fermentation liquid was inactivated and purified multiple times to obtain the white plum blossom fermentation liquid J.

[0108] Efficacy Example

[0109] Efficacy Example 1: Determination of the inhibition rate of melanin synthesis in A375 cells (whitening)

[0110] 1.1 Experimental instruments and reagents

[0111] Biological safety cabinet (Sujing Antai, BSC-1004IIA2), inverted fluorescence microscope (Leica, DMi8), vortex oscillator (Shanghai Hanno, XH-D), CO2 incubator (Thermo Scientific), microplate reader (Thermo Scientific), 24-well plate (Coming Incorporated), 96-well plate (Coming Incorporated), 6 cm culture dish (Coming Incorporated), DMSO (Sigma), NaOH (Sinopharm), cell lysis buffer (Gibco), DMEM medium (Gibco), double antibody Pen Strep (Gibco), fetal bovine serum (Gibco), 0.05% Trypsin-EDTA (Gibco), PBS (Vivacell), MTT (Sigma).

[0112] 1.2 Experimental Methods

[0113] Cell seeding: 1×10 5 A375 cells (Stem Cell Bank, Chinese Academy of Sciences) were seeded into 24-well plates at a seeding density of 10 cells / well and incubated overnight in an incubator (37° C., 5% CO 2 ).

[0114] Liquid preparation: Prepare the working solution of the test substance according to the experimental design (Table 1). Take 50 mg of 377, nonapeptide-1, and glabridin powder, respectively, and add 10 mL of DMEM medium (containing 10% fetal bovine serum) to obtain a cell culture solution with a concentration of 0.5% (w / w) of 377, nonapeptide-1, and glabridin; take 50 μL of magnolia flower fermentation liquid AI and white plum flower fermentation liquid J, respectively, and add 10 mL of DMEM medium (containing 10% fetal bovine serum) to obtain a cell culture solution with a concentration of 0.5% (w / w) of magnolia flower fermentation liquid AI and white plum flower fermentation liquid J; take 100 μL of magnolia flower fermentation liquid AI and white plum flower fermentation liquid J, respectively, and add 10 mL of DMEM medium (containing 10% fetal bovine serum) to obtain a cell culture solution with a concentration of 1% (w / w) of magnolia flower fermentation liquid AI and white plum flower fermentation liquid J;

[0115] Table 1 Pharmacodynamics Example 1 Experimental Design

[0116]

[0117] Administration: After 24 hours of culture, according to the experimental design in Table 1, the groups were administered. 1 mL of cell culture medium was added to each well of the blank control group; 1 mL of culture medium containing 0.5% (w / w) 377 was added to each well of the positive control group; 1 mL of culture medium containing the test substance at the corresponding concentration was added to each well of the sample group; after the administration, the 24-well plate was placed in an incubator (37°C, 5% CO2) and cultured for 24 hours.

[0118] Detection: 72 hours after the third liquid change, measure the melanin content of the cells. Wash the cells twice with 1×PBS, remove the PBS, add 200μL 0.25% trypsin to each well to digest the cells, and digest them in a CO2 incubator for 5 minutes. Add 2mL PBS twice to blow the cells, collect the cells into a centrifuge tube, and centrifuge at 4000rpm for 5 minutes. Try to suck out the residual PBS in the centrifuge tube, add 200μL of melanin extract to each tube, shake evenly, and heat in an 80℃ water bath for 1h. After cooling, centrifuge the droplets on the tube wall moderately, blow evenly, aspirate 150μL of solution in each centrifuge tube, transfer to a 96-well plate, and use an enzyme reader to detect the absorbance (OD) of each well at 405nm.

[0119] The relative inhibition rate of melanin was calculated.

[0120] Melanin inhibition rate (%) = [1-(OD of drug well-OD of blank well) / (OD of cell control well-OD of blank well)] × 100

[0121] 1.3 Experimental Results

[0122] like Figure 1As shown, compared with the BC group, the 0.5% (w / w) concentration of 377 in the PC group significantly increased the melanin inhibition rate, indicating that the experimental system is effective. Compared with the BC group, the common whitening monomer raw materials nonapeptide-1 and glabridin in the test group significantly improved the melanin inhibition rate at a concentration of 0.5%; compared with the BC group, the magnolia flower fermented liquor A and B described in Examples 1 and 2 improved the melanin inhibition rate at a concentration of 0.5%, and significantly improved the melanin inhibition rate at a concentration of 1%, which was close to the inhibition rate of nonapeptide-1 and glabridin at a concentration of 0.5%, indicating that the magnolia flower fermented liquor A and magnolia flower fermented liquor B have the ability to inhibit melanocytes from synthesizing melanin and have whitening effect; while in Comparative Examples 1-8, the magnolia flower fermented liquor CI and white plum blossom fermented liquor J had no statistically significant differences between the groups at concentrations of 0.5% and 1% compared with the BC group (P>0.05), and therefore did not show the ability to inhibit melanocytes from synthesizing melanin; it proves that only within the scope of the preparation method described in the claims, the prepared magnolia flower fermented liquor has whitening effect.

[0123] Efficacy Example 2: Protection of Type I Collagen (UVA) (Anti-aging) --- Determination of Type I Collagen Content in Fibroblasts Stimulated by UVA

[0124] 2.1 Experimental instruments and reagents

[0125] Biological safety cabinet (Sujing Antai, BSC-1004IIA2), CO2 incubator (Thermo Scientific), micro-oscillator (Qilin Bell, TS-92), ultra-low temperature refrigerator (-80℃, Thermo), low-speed centrifuge (bioridge), precision balance (ME204 Mettler), inverted microscope (Leica, DMi8), cell counter (Thermo), microplate reader (ThermoScientific), adjustable pipette (eppendorf), PBS (Viva cell), trypsin (Gibco), newborn calf serum (Gibco), low-glucose DMEM medium (Gibco), DMSO (Sigma), Human Collagen I alpha 1 ELISA detection kit (Thermo), type I collagen primary antibody, secondary antibody (Thermo), vitamin C (Aladdin), vitamin E (Aladdin), etc.

[0126] 2.2 Experimental Methods

[0127] Cell inoculation: Human skin fibroblasts (Stem Cell Bank, Chinese Academy of Sciences) were inoculated into 24-well plates at a density of 45% to 60% confluence 24 hours after inoculation, and incubated overnight in an incubator (37° C., 5% CO 2 ).

[0128] Solution preparation: Referring to the solution preparation method of Example 1, prepare the working solution of the test substance according to the experimental design (Table 2).

[0129] Table 2 Pharmacodynamics Example 2 Experimental Design

[0130]

[0131] Drug administration: According to the experimental design in Table 2, when the cell plating rate in the 24-well plate reaches 40% to 60%, wash with PBS, and then culture and expose to 9J / cm 2 After exposure, the treated cells were washed with PBS and then divided into groups for drug administration. The sample group was added with culture medium containing the sample, and the positive control group was added with culture medium containing the positive control. They were exposed to different test substances for 24 hours. The dosage per well was 1.0 mL, and 3 replicates were set for each group. The blank control group BC group was added with cell culture medium and treated in the same way, but covered with aluminum foil to avoid ultraviolet exposure.

[0132] Detection: Cell supernatant collection: After the incubation, collect 1 mL of cell culture supernatant in a 1.5 mL sterile centrifuge tube and store it in a -80°C ultra-low temperature refrigerator. Determine the type I collagen content according to the operating instructions of the ELISA detection kit.

[0133] Result analysis: Calculation of CollagenⅠ content: After subtracting the OD450 value of the blank well from the OD450 value of the standard and sample, the average value of the three parallel wells was calculated. Using the professional curve making software Curve Expert, the concentration of the standard was used as the ordinate and the OD450 value was used as the abscissa to draw the standard curve, and the regression equation was obtained. The OD450 value measured by the sample group was substituted into the equation to calculate the CollagenⅠ protein content of each group.

[0134] 2.3 Experimental Results

[0135] like Figure 2As shown, compared with the BC group, the type I collagen concentration of the NC group was significantly reduced (P<0.01), and compared with the NC group, 100μg / mL VC+7μg / mL VE in the PC group significantly increased the type I collagen concentration (P<0.01), indicating that the experimental system is effective. Compared with the NC group, the magnolia flower fermented liquor A and B described in Examples 1 and 2 increased the type I collagen concentration at a concentration of 0.5%, and significantly increased the type I collagen concentration at a concentration of 1%, indicating that the magnolia flower fermented liquor A and B have the ability to protect the type I collagen of fibroblasts under UVA stimulation and have anti-aging effects; while in Comparative Examples 1-8, the magnolia flower fermented liquor CI and the white plum blossom fermented liquor J were compared with the NC group at concentrations of 0.5% and 1%, and the differences between the groups were not statistically significant (P>0.05), so they did not show the ability to protect type I collagen; it proves that only within the scope of the preparation method described in the claims, the magnolia flower fermented liquor prepared has anti-aging effects.

[0136] Efficacy Example 3: Inhibition of MMP-1 Synthesis (UVA) (Anti-aging) --- Determination of MMP-1 Content in Fibroblasts Stimulated by UVA

[0137] 3.1 Experimental instruments and reagents

[0138] Biological safety cabinet (Sujing Antai, BSC-1004IIA2), CO2 incubator (Thermo Scientific), micro-oscillator (Qilin Bell, TS-92), ultra-low temperature refrigerator (-80℃, Thermo), low-speed centrifuge (bioridge), precision balance (ME204 Mettler), inverted microscope (Leica, DMi8), cell counter (Thermo), microplate reader (ThermoScientific), adjustable pipette (eppendorf), PBS (Viva cell), trypsin (Gibco), newborn calf serum (Gibco), low-glucose DMEM medium (Gibco), DMSO (Sigma), human MMP-1 ELISA kit (Sigma), vitamin C (Aladdin), vitamin E (Aladdin), etc.

[0139] 3.2 Experimental Methods

[0140] Cell inoculation: Human skin fibroblasts (Stem Cell Bank, Chinese Academy of Sciences) were inoculated into 24-well plates at a density of 45% to 60% confluence 24 hours after inoculation, and incubated overnight in an incubator (37° C., 5% CO 2 ).

[0141] Solution preparation: Referring to the solution preparation method of Example 1, prepare the working solution of the test substance according to the experimental design (Table 3).

[0142] Table 3 Pharmacodynamics Example 3 Experimental Design

[0143]

[0144] Drug administration: According to the experimental design in Table 3, when the cell plating rate in the 24-well plate reaches 40% to 60%, wash with PBS, and then culture and expose to 9J / cm 2 After exposure, the treated cells were washed with PBS and then divided into groups for drug administration. The sample group was added with culture medium containing the sample, and the positive control group was added with culture medium containing the positive control. They were exposed to different test substances for 24 hours. The dosage per well was 1.0 mL, and 3 replicates were set for each group. The blank control group BC group was added with cell culture medium and treated in the same way, but covered with aluminum foil to avoid ultraviolet exposure.

[0145] Detection: Cell supernatant collection: After the incubation, collect 1 mL of cell culture supernatant in a 1.5 mL sterile centrifuge tube and store it in a -80°C ultra-low temperature refrigerator. Determine the MMP-1 protein content according to the operating instructions of the ELISA detection kit.

[0146] Result analysis: The MMP-1 protein content was calculated according to the operating instructions of the ELISA detection kit.

[0147] 3.3 Experimental Results

[0148] like Figure 4 As shown, compared with the BC group, the MMP-1 concentration of the NC group was significantly increased (P<0.01), and compared with the NC group, 100μg / mL VC+7μg / mL VE in the PC group significantly reduced the MMP-1 concentration (P<0.01), indicating that the experimental system is effective. Compared with the NC group, the magnolia flower fermented liquor A and B described in Example 1 and Example 2 reduced the MMP-1 concentration at a concentration of 0.5%, and significantly reduced the MMP-1 concentration at a concentration of 1%, indicating that the magnolia flower fermented liquor A and B have the ability to inhibit the synthesis of MMP-1 protein by fibroblasts under UVA stimulation, and have anti-aging effects; while in Comparative Examples 1-8, the magnolia flower fermented liquor CI and white plum blossom fermented liquor J at 0.5% and 1% concentrations compared with the NC group, the differences between the groups were not statistically significant (P>0.05), so they did not show the ability to inhibit the synthesis of MMP-1; it proves that only within the scope of the preparation method described in the claims, the magnolia flower fermented liquor prepared has anti-aging effects.

[0149] Efficacy Example 4: Inhibition of MMP-9 Synthesis (UVA) (Anti-aging) --- Determination of MMP-9 Content in Fibroblasts Stimulated by UVA

[0150] 4.1 Experimental instruments and reagents

[0151] Biological safety cabinet (Sujing Antai, BSC-1004IIA2), CO2 incubator (Thermo Scientific), micro-oscillator (Qilin Bell, TS-92), ultra-low temperature refrigerator (-80℃, Thermo), low-speed centrifuge (bioridge), precision balance (ME204 Mettler), inverted microscope (Leica, DMi8), cell counter (Thermo), microplate reader (ThermoScientific), adjustable pipette (eppendorf), PBS (Viva cell), trypsin (Gibco), newborn calf serum (Gibco), low-glucose DMEM medium (Gibco), DMSO (Sigma), human MMP9 ELISA kit (Sigma), vitamin C (Aladdin), vitamin E (Aladdin), etc.

[0152] 4.2 Experimental Methods

[0153] Cell inoculation: Human skin fibroblasts (Stem Cell Bank, Chinese Academy of Sciences) were inoculated into 24-well plates at a density of 45% to 60% confluence 24 hours after inoculation, and incubated overnight in an incubator (37° C., 5% CO 2 ).

[0154] Solution preparation: Referring to the solution preparation method of Example 1, prepare the working solution of the test substance according to the experimental design (Table 4).

[0155] Table 4 Pharmacodynamics Example 4 Experimental Design

[0156]

[0157] Drug administration: According to the experimental design in Table 4, when the cell plating rate in the 24-well plate reaches 40% to 60%, wash with PBS, and then culture and expose to 9J / cm 2 After exposure, the treated cells were washed with PBS and then divided into groups for drug administration. The sample group was added with culture medium containing the sample, and the positive control group was added with culture medium containing the positive control. They were exposed to different test substances for 24 hours. The dosage per well was 1.0 mL, and 3 replicates were set for each group. The blank control group BC group was added with cell culture medium and treated in the same way, but covered with aluminum foil to avoid ultraviolet exposure.

[0158] Detection: Cell supernatant collection: After the incubation, collect 1 mL of cell culture supernatant in a 1.5 mL sterile centrifuge tube and store it in a -80°C ultra-low temperature refrigerator. Determine the MMP-9 protein content according to the operating instructions of the ELISA detection kit.

[0159] Result analysis: The MMP-9 protein content was calculated according to the operating instructions of the ELISA detection kit.

[0160] 4.3 Experimental Results

[0161] like Figure 4 As shown, compared with the BC group, the MMP-9 concentration of the NC group was significantly increased (P<0.01), and compared with the NC group, 100μg / mL VC+7μg / mL VE in the PC group significantly reduced the MMP-9 concentration (P<0.01), indicating that the experimental system is effective. Compared with the NC group, the magnolia flower fermented liquor A and B described in Example 1 and Example 2 reduced the MMP-9 concentration at a concentration of 0.5%, and significantly reduced the MMP-9 concentration at a concentration of 1%, indicating that the magnolia flower fermented liquor A and B have the ability to inhibit the synthesis of MMP-9 protein by fibroblasts under UVA stimulation, and have anti-aging effects; while in Comparative Examples 1-8, the magnolia flower fermented liquor CI and white plum blossom fermented liquor J were compared with the NC group at concentrations of 0.5% and 1%, and the differences between the groups were not statistically significant (P>0.05), so they did not show the ability to inhibit the synthesis of MMP-9; it proves that only within the scope of the preparation method described in the claims, the magnolia flower fermented liquor prepared has anti-aging effects.

[0162] Efficacy Example 5, TNF-α Content Determination (Soothing)

[0163] 5.1 Experimental instruments and reagents

[0164] Biosafety cabinet (Sujing Antai, BSC-1004IIA2), inverted fluorescence microscope (Leica, DMi8), micro-oscillator (Qilin Bell, TS-92). High-glucose DMEM culture medium (Gibco), fetal bovine serum (Gibco), PBS (Gibco), MTT (Sigma), trypsin (Gibco), bacterial lipopolysaccharide LPS (AbMole). Human TNF alpha ELISA kit (Thermo).

[0165] 5.2 Experimental Methods

[0166] Cell seeding: 1×10 5 RAW264.7 cells (Stem Cell Bank, Chinese Academy of Sciences) were seeded into 24-well plates at a seeding density of 10 cells / well and incubated overnight in an incubator (37° C., 5% CO 2 ).

[0167] Solution preparation: Referring to the solution preparation method of Example 1, prepare the working solution of the test substance according to the experimental design (Table 5).

[0168] Table 5 Pharmacodynamics Example 5 Experimental Design

[0169]

[0170]

[0171] Drug administration: According to the experimental design in Table 5, when the cell plating rate in the 24-well plate reaches 40% to 60%, culture medium containing samples is added in groups, with 3 replicate wells in each group. The 24-well plate is placed in an incubator (37°C, 5% CO2) and cultured for 24 hours.

[0172] Detection: After culturing for 24 hours, the cell supernatant was collected and the TNF-α content was determined according to the operating instructions of the ELISA detection kit.

[0173] Result analysis: TNF-α content was calculated according to the operating instructions of the ELISA detection kit.

[0174] 5.3 Experimental Results

[0175] like Figure 5 As shown, compared with the BC group, the TNF-α concentration of the NC group was significantly increased (P<0.01), and compared with the NC group, 0.01% dexamethasone in the PC group significantly reduced the TNF-α concentration (P<0.01), indicating that the experimental system is effective. Compared with the NC group, the magnolia flower fermented liquor A and B described in Example 1 and Example 2 significantly reduced the TNF-α concentration at 0.5% and 1%, indicating that the magnolia flower fermented liquor A and B have the ability to inhibit the production of TNF-α under LPS stimulation and have a soothing effect; while in Comparative Examples 1-8, the magnolia flower fermented liquor CI and white plum blossom fermented liquor J at 0.5% and 1% concentrations compared with the NC group, the differences between the groups were not statistically significant (P>0.05), so they did not show the ability to inhibit the production of TNF-α; it proves that only within the scope of the preparation method described in the claims, the magnolia flower fermented liquor prepared has a soothing effect.

[0176] Efficacy Example 6: Inhibition of Mast Cell Degranulation (C48 / 80) (Shumin) --- Determination of Mast Cell Degranulation Rate Stimulated by C48 / 80

[0177] 6.1 Experimental instruments and reagents

[0178] Biosafety cabinet (Sujing Antai, BSC-1004IIA2), inverted fluorescence microscope (Leica, DMi8), CO2 incubator (Thermo Scientific), vortex oscillator (Hanno, XH-D). High-glucose DMEM culture medium (Gibco), fetal bovine serum (Gibco), PBS (Gibco), trypsin (Gibco).

[0179] 6.2 Experimental Methods

[0180] Cell inoculation: Mast cells (Stem Cell Bank, Chinese Academy of Sciences) were inoculated into 24-well plates at a density that reached 45% to 60% confluence 24 hours after inoculation, and incubated overnight in an incubator (37° C., 5% CO 2 ).

[0181] Solution preparation: Referring to the solution preparation method of Example 1, prepare the working solution of the test substance according to the experimental design (Table 6).

[0182] Table 6 Pharmacodynamics Example 6 Experimental Design

[0183]

[0184] Drug administration: According to the experimental design in Table 6, when the cell plating rate in the 24-well plate reaches 40% to 60%, group drug administration is carried out, the dosage per well is 1.0 mL, and each group has 3 replicate wells. The culture is continued in the incubator (37°C, 5% CO2) for 2 hours.

[0185] Detection: Collect samples, discard the supernatant, and then divide and administer the drugs. The blank control group BC is added with cell culture medium, the sample group is added with culture medium containing C48 / 80 and samples, the positive control group is added with culture medium containing C48 / 80 and positive control, and the negative control group is added with culture medium containing C48 / 80. The dosage per well is 1mL, and 3 replicates are set for each group. After adding the sample, C48 / 80 is stimulated for 45 minutes, and the reaction is terminated by ice bath. After the culture is completed, the degranulation of cells in each group is observed under an inverted microscope and photographed.

[0186] Result analysis: Image Pro Plus software was used to count and calculate the cell degranulation rate.

[0187] The mast cell degranulation rate was calculated.

[0188] Degranulation rate (%) = [total number of degranulated cells / (total number of degranulated cells + total number of non-degranulated cells)] × 100

[0189] 6.3 Experimental Results

[0190] like Figure 6As shown, compared with the BC group, the mast cell degranulation rate of the NC group was significantly increased (P<0.01), and compared with the NC group, 1 mg / mL of sodium cromoglycate in the PC group significantly reduced the mast cell degranulation rate (P<0.01), indicating that the experimental system is effective. Compared with the NC group, the magnolia flower fermented liquor A and B described in Example 1 and Example 2 significantly reduced the mast cell degranulation rate at a concentration of 0.5% and 1%, indicating that the magnolia flower fermented liquor A and B have the ability to inhibit the generation of mast cell degranulation under the stimulation of C48 / 80, and have a soothing effect; while in Comparative Examples 1-8, the magnolia flower fermented liquor CI and white plum blossom fermented liquor J at a concentration of 0.5% and 1% compared with the NC group, the differences between the groups were not statistically significant (P>0.05), so they did not show the ability to inhibit the generation of mast cell degranulation rate; it is proved that only within the scope of the preparation method described in the claims, the magnolia flower fermented liquor prepared has a soothing effect.

[0191] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, changes or deformations made by those skilled in the art based on the ideas of the present invention, the specific implementation methods and application scope of the present invention, all belong to the scope of protection of the present invention. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A fermented liquid of magnolia flower, characterized in that: The fermented liquid is obtained by fermenting magnolia flower with Lactobacillus plantarum.

2. A method for preparing the fermentation broth according to claim 1, characterized in that: The method comprises the following steps: (1) Weighing an appropriate amount of magnolia flowers, crushing, and sieving to obtain magnolia flower powder; (2) adding a culture medium into a container, adding the magnolia flower powder and seed liquid, and fermenting to obtain a crude fermentation liquid; and (3) inactivating and purifying the crude fermentation broth to obtain the magnolia flower fermentation broth.

3. The method according to claim 2, characterized in that The sieve is a 10-mesh sieve; Preferably, the mass ratio of the magnolia flower powder to the culture medium is 0.005 to 0.1, such as 0.01 to 0.08, such as 0.03 to 0.08; More preferably, the volume ratio of the seed solution to the culture medium is 0.03 to 0.2, such as 0.04 to 0.15, such as about 0.06 to 0.1; Also preferably, the culture medium is a liquid culture medium.

4. The method according to claim 2, characterized in that: The container is a fermentation tank; Preferably, the fermentation temperature is 20-45°C, such as 20-40°C, such as 25-40°C; Preferably, the pH of the fermentation is 2 to 7, such as 3 to 7, such as 4 to 7; More preferably, the fermentation speed is 10 to 1000 r / min, such as 50 to 800 r / min, such as 100 to 600 r / min; More preferably, the fermentation time is 12 to 72 hours, such as 18 to 48 hours.

5. The method according to claim 2, characterized in that: The inactivation temperature is 115-121°C; Preferably, the inactivation time is 10 to 30 minutes, for example, about 20 minutes; More preferably, the purification comprises filtering and sterilizing the crude fermentation broth.

6. The method according to claim 2, characterized in that The seed solution is obtained by culturing the Lactobacillus plantarum in a seed culture medium; Preferably, the Lactobacillus plantarum is an activated Lactobacillus plantarum; More preferably, the seed culture medium is a liquid seed culture medium; Preferably, the Lactobacillus plantarum is isolated from fermentation slurry; Particularly preferably, the Lactobacillus plantarum is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, and its deposit number is CGMCC No.28500.

7. Use of the fermentation broth according to claim 1 in preparing cosmetics having whitening, soothing, anti-inflammatory and / or anti-aging effects.

8. Use of a composition comprising the fermentation broth according to claim 1 in preparing cosmetics having whitening, soothing, anti-inflammatory and / or anti-aging effects.

9. The use according to claim 7 or 8, characterized in that The whitening is to inhibit the synthesis of cellular melanin; Preferably, the anti-aging is achieved by one or more of the following ways: protecting type I collagen, inhibiting the production of MMP-1, and inhibiting the production of MMP-9; Preferably, the soothing and anti-inflammatory effects are achieved by one or more of the following means: inhibiting the production of TNF-α and inhibiting mast cell degranulation.

10. The use according to claim 8, characterized in that The composition further comprises one or more excipients; Preferably, the excipient is selected from one or more of the following: thickener, surfactant, co-surfactant, skin conditioner, preservative, flavor, lubricant, disintegrant, wetting agent, binder and filler; Also preferably, the composition further comprises one or more whitening substances.

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