Preparation method and application of nitraria tangutorum bobr. fermentation liquid with whitening and firming efficacy

Abstract

The application discloses a preparation method and application of a nitraria tangutorum bobr. fermentation liquor with whitening and firming effects, and the preparation method comprises the following steps: (1) crushing nitraria tangutorum bobr. fruits, adding water to obtain a fermentation substrate A, inoculating schizophyllum commune in the fermentation substrate A to perform preliminary fermentation, and obtaining nitraria tangutorum bobr. fruit fermentation liquor; (2) adding nitraria tangutorum bobr. leaves to the nitraria tangutorum bobr. fruit fermentation liquor to obtain a fermentation substrate B, and performing secondary fermentation in the fermentation substrate B by inoculating the schizophyllum commune again to obtain the nitraria tangutorum bobr. fermentation liquor. The fruits and leaves of nitraria tangutorum bobr. are subjected to stage fermentation, active substances in the nitraria tangutorum bobr. fruits are released in the first fermentation process, the nitraria tangutorum bobr. leaves are added to the nitraria tangutorum bobr. fruit fermentation liquor in the second fermentation process, active substances in the nitraria tangutorum bobr. leaves are further extracted, and decomposition of the active substances in the nitraria tangutorum bobr. leaves is avoided. The nitraria tangutorum bobr. fermentation liquor is subjected to multiple fermentation, release of the active substances in the nitraria tangutorum bobr. fermentation liquor is improved, ideal whitening, firming and other effects are achieved, and the nitraria tangutorum bobr. fermentation liquor has a good application prospect.

Description

Technical Field

[0001] This application belongs to the field of cosmetic preparation technology, and in particular relates to a method for preparing and applying a whitening and firming fermented liquid of Acanthopanax senticosus. Background Technology

[0002] In recent years, with the rapid development of my country's economy and the continuous improvement of economic level, people's living standards have been greatly improved, and the pace of life has also accelerated significantly. The fast-paced modern life and high-intensity work force young people to bear enormous work pressure, often staying up late, eating irregularly, and not getting enough rest. As a result, they are very prone to dry skin, dull complexion, and other problems. Therefore, unhealthy lifestyles and eating habits, as well as mental and work stress, have a great impact on the skin.

[0003] White thorn is mostly distributed in arid regions. Its fruit has high nutritional and medicinal value, possessing effects such as strengthening the stomach and spleen, nourishing yin and moistening the lungs, and promoting blood circulation and digestion. In folk medicine, it is mainly used to treat spleen and stomach weakness, indigestion, and neurasthenia. Modern scientific research has proven that white thorn contains abundant anthocyanins, polyphenols, flavonoids, and other components, which have excellent skin-care effects. However, current research focuses on extracting the active ingredients from white thorn using organic solvents. These organic solvents can easily damage the active ingredients during use, leading to reduced efficacy. Furthermore, organic solvents are prone to residue during use, causing side effects and skin irritation.

[0004] Therefore, there is an urgent need for an extraction method that can effectively utilize the active ingredients contained in white thorn and be applied to cosmetics, with excellent efficacy and no chemical residue. Summary of the Invention

[0005] The technical problem to be solved by this application is to overcome the shortcomings of existing technologies, which focus on extracting the effective components contained in white thorn using organic solvents, lacking effective utilization methods, and causing the product to be irritating due to the organic solvent extraction process. This application provides a method for preparing and applying a white thorn fermentation liquid with whitening and firming effects.

[0006] This application adopts the following technical solution to solve the above-mentioned technical problems:

[0007] This application provides a method for preparing a whitening and firming fermented liquid from *Acanthopanax senticosus*, which includes the following steps:

[0008] (1) After crushing the white thorn fruit, add water to obtain fermentation substrate A. Inoculate the fermentation substrate A with Schizophyllum commune for preliminary fermentation to obtain white thorn fruit fermentation liquid.

[0009] (2) White thorn leaves are added to the fermentation broth of white thorn fruit to obtain fermentation substrate B. The fermentation substrate B is then inoculated with the Schizophyllum commune for secondary fermentation to obtain the final product.

[0010] In some embodiments, in step (1), the mass ratio of the white thorn fruit to the water is 0.5 to 1:100, preferably 1:100.

[0011] In some embodiments, in step (1), the preparation method of the fermentation substrate A includes: mixing the white thorn fruit powder with the water evenly.

[0012] In some embodiments, step (1) further includes sterilization of the fermentation substrate A before use.

[0013] The sterilization method for the fermentation substrate A is generally high-temperature sterilization.

[0014] When the fermentation substrate is sterilized using the high-temperature sterilization method, the sterilization temperature can be a temperature commonly used in this type of operation in the art, preferably 110°C to 125°C, more preferably 110°C to 121°C, for example 121°C.

[0015] When the fermentation substrate is sterilized using the high-temperature sterilization method, the sterilization time can be the conventional time for this type of operation in the art, preferably 20-45 min, more preferably 25-40 min, for example 30 min.

[0016] In some embodiments, in step (1), the Schizophyllum commune includes specimens deposited at the China General Microbiological Culture Collection Center (CGMCC), with a deposit date of March 9, 2023, accession number CGMCC No. 40388, classified as Schizophyllum commune, and the depository is located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0017] In some embodiments, in step (1), the Schizophyllum commune is added in the form of the Schizophyllum commune liquid.

[0018] The preparation method of the *Schizophyllum commune* suspension includes: inoculating *Schizophyllum commune* into PDA liquid culture medium, then culturing it at 28°C and 180 rpm for 5 days, then spreading it onto PDA solid plates, and culturing it in a 28°C constant temperature incubator for 7 days, then picking a single colony and culturing it in 300 ml of PDA liquid culture medium at 28°C and 180 rpm for 7 days, which is then used as the *Schizophyllum commune* suspension for subsequent use.

[0019] In some embodiments, in step (1), based on the water in the fermentation substrate A, the viable count of the *Schizophyllum commune* inoculated per unit mass of the water is 1 to 3 × 10⁻⁶.7 CFU / mL, preferably 3 × 10⁻⁶ 7 CFU / mL.

[0020] In some embodiments, in step (1), the preliminary fermentation is aerobic fermentation, and the conditions and methods of aerobic fermentation are conventional in the art, generally carried out in a constant temperature shaking chamber.

[0021] When the aerobic fermentation is carried out using the constant temperature shaking chamber, the rotation speed of the constant temperature shaking chamber is 150-200 rpm, preferably 180 rpm.

[0022] In some embodiments, in step (1), the temperature of the initial fermentation is 25–32°C, preferably 28°C.

[0023] In some embodiments, the initial fermentation time in step (1) is 2 to 4 days, preferably 3 days.

[0024] In some embodiments, step (1) further includes centrifugation to remove mycelium after initial fermentation.

[0025] The centrifugation speed can be a speed conventional for this type of operation in the art, preferably 3000-7000 rpm, more preferably 3500-6000 rpm, for example 5000 rpm.

[0026] The centrifugation time can be the conventional time for this type of operation in the art, preferably 20 to 45 minutes, more preferably 25 to 40 minutes, for example 30 minutes.

[0027] In some embodiments, in step (2), the white thorn leaf is white thorn leaf powder.

[0028] In some embodiments, in step (2), the mass ratio of the fermentation liquid of white thorn leaves to white thorn fruit is 0.5 to 1:100, preferably 0.5:100.

[0029] In some embodiments, in step (2), the preparation method of the fermentation substrate B includes: mixing the white thorn fruit powder with the white thorn fruit fermentation liquid evenly.

[0030] In some embodiments, step (2) further includes sterilization of the fermentation substrate B before use.

[0031] The sterilization method for the fermentation substrate A is generally high-temperature sterilization.

[0032] When the high-temperature sterilization method is used for sterilization, the sterilization temperature can be a temperature conventional for this type of operation in the art, preferably 100℃ to 110℃, and more preferably 110℃.

[0033] When the high-temperature sterilization method is used for sterilization, the sterilization time can be the conventional time for this type of operation in the art, preferably 20 to 45 minutes, more preferably 25 to 40 minutes, for example 30 minutes.

[0034] In some embodiments, in step (2), the secondary fermentation is aerobic fermentation. The conditions and methods of aerobic fermentation are conventional in the art, and aerobic fermentation is generally carried out in a constant temperature shaking chamber.

[0035] When the aerobic fermentation is carried out using the constant temperature shaking chamber, the rotation speed of the constant temperature shaking chamber is 150-200 rpm, preferably 180 rpm.

[0036] In some embodiments, in step (2), the temperature of the secondary fermentation is 25-32°C, preferably 28°C.

[0037] In some embodiments, the secondary fermentation time in step (2) is 2 to 4 days, preferably 3 days.

[0038] In some embodiments, in step (2), based on the water in the fermentation substrate B, the viable count of the *Schizophyllum commune* inoculated per unit mass of the water can be 0.5–1.5 × 10⁻⁶. 7 CFU / mL, preferably 1.5 × 10⁻⁶. 7 CFU / mL.

[0039] In some embodiments, step (2) further includes at least one of centrifugation, filtration to remove supernatant, and sterilization after the secondary fermentation.

[0040] The centrifugation speed can be a speed conventional for this type of operation in the art, preferably 3000-7000 rpm, more preferably 3500-6000 rpm, for example 5000 rpm.

[0041] The centrifugation time can be the conventional time for this type of operation in the art, preferably 20 to 45 minutes, more preferably 25 to 40 minutes, for example 30 minutes.

[0042] The filter membrane used for filtration has a thickness of 10–15 μm, preferably 15 μm.

[0043] The sterilization method mentioned above is generally high-temperature sterilization.

[0044] When the high-temperature sterilization method is used for sterilization, the sterilization temperature can be the temperature conventional for this type of operation in the art, preferably 100℃~110℃, and more preferably 110℃.

[0045] When the high-temperature sterilization method is used for sterilization, the sterilization time can be the conventional time for this type of operation in the art, preferably 20 to 45 minutes, more preferably 25 to 40 minutes, for example 30 minutes.

[0046] This application also provides a whitening and firming fermented liquid of Acanthopanax senticosus, which is prepared by the method described above for preparing a whitening and firming fermented liquid of Acanthopanax senticosus.

[0047] This application also provides the use of the whitening and firming fermented liquid of Acanthopanax senticosus, as described above, as a product, as an additive, or as a base in the preparation of topical skin agents.

[0048] In some embodiments, the whitening and firming fermented liquid of Acanthopanax senticosus is used as at least one of the following active ingredients in the topical skin agent: antioxidant active ingredient, anti-glycation active ingredient, anti-aging active ingredient, and whitening active ingredient.

[0049] The antioxidant component is an antioxidant component that has the ability to scavenge free radicals (DPPH).

[0050] The anti-glycation active ingredient is an anti-glycation active ingredient that inhibits the content of AGEs.

[0051] The anti-aging active ingredient is an anti-aging active ingredient that enhances SOD activity.

[0052] The whitening active ingredient is a whitening active ingredient that inhibits tyrosinase activity.

[0053] This application also provides a topical skin agent comprising the whitening and firming thorn ferment liquid as described above.

[0054] In some embodiments, the topical skin agent may further include active ingredients commonly used in the art, generally including at least one of moisturizing active ingredients, whitening active ingredients, anti-inflammatory active ingredients, anti-allergic active ingredients, and antioxidant active ingredients.

[0055] In some embodiments, the topical skin agent may be, in accordance with the conventions of the art, including but not limited to, face masks, serums, or toners.

[0056] In some embodiments, the whitening and firming fermented liquid of Acanthopanax senticosus accounts for 5% to 99% of the mass percentage of the topical skin agent, preferably 60% to 99%.

[0057] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of this application.

[0058] All reagents and raw materials used in this application are commercially available.

[0059] The positive and progressive effects of this application are as follows: This application uses a stepwise fermentation method to ferment the fruit and leaves of *Nitraria tangutorum* in multiple stages. During the first fermentation, the active substances in the fruit are effectively released. In the second fermentation, *Nitraria tangutorum* leaves are added to the fruit fermentation broth to further extract the active substances from the leaves, while avoiding the decomposition of these active substances. This application utilizes multiple fermentations to further enhance the release of active substances in the *Nitraria tangutorum* fermentation broth, achieving ideal anti-aging, antioxidant, whitening, and firming effects. Furthermore, the fermentation conditions are mild, showing promising application prospects. Simultaneously, the prepared product can be used directly in cosmetics or added to basic cosmetic formulations, exhibiting good antioxidant, anti-aging, and whitening effects. Attached Figure Description

[0060] This application can be better understood by referring to the description given below in conjunction with the accompanying drawings. These drawings, together with the detailed description below, are included in and form part of this specification, and are used to further illustrate preferred embodiments of the application and explain its principles and advantages.

[0061] in:

[0062] Figure 1 This is a diagram illustrating the DPPH free radical scavenging effect in the examples;

[0063] Figure 2 This is a diagram showing the effect of AGEs inhibition in the examples;

[0064] Figure 3 This is a diagram illustrating the effect of in vitro elastase inhibition in the examples;

[0065] Figure 4 This is a graph showing the relative expression levels of ELN mRNA in the examples;

[0066] Figure 5 This is a graph showing the relative expression levels of NRF2 mRNA in the examples.

[0067] Figure 6 This is a graph showing the SOD (superoxide dismutase) activity assay in the examples;

[0068] Figure 7 This is a graph showing the effect of tyrosinase activity inhibition in the examples. Detailed Implementation

[0069] The present application is further illustrated below by way of embodiments, but these embodiments are not intended to limit the scope of the present application. Experimental methods not specifically described in the following embodiments are performed according to conventional methods and conditions, or as selected in accordance with the product instructions.

[0070] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0071] The raw materials, white thorn fruit and white thorn leaves, used in the following examples are from Minqin County, Gansu Province.

[0072] Example 1

[0073] (1) After crushing the fruit of *Nitraria tangutorum*, 3g was weighed and added to 300g of water. The mixture was then sterilized at 121℃ for 30min and cooled to 25℃ to obtain fermentation substrate A. Subsequently, 3g of *Schizophyllum commune* seed culture was inoculated, wherein the viable count of the inoculated *Schizophyllum commune* was 3 × 10⁻⁶. 7 The concentration of CFU / mL was then increased, and aerobic fermentation was carried out at 28°C and 180 rpm for 3 days. The mycelium was then removed by centrifugation to obtain the fermentation broth of *Hippophae rhamnoides* fruit.

[0074] (2) 1.5g of white thorn leaf powder was added to 300g of white thorn fruit fermentation broth, then sterilized at 110℃ for 30min, and cooled to 25℃ to obtain fermentation substrate B. Then, 1.5g of *Schizophyllum commune* seed culture was inoculated and subjected to secondary aerobic fermentation at 28℃ and 180rpm for 2 days. The viable count of *Schizophyllum commune* inoculated was 1.5 × 10⁻⁶. 7 The concentration of CFU / mL was determined by centrifugation to remove mycelium after fermentation, followed by fine filtration through a 15μm filter membrane. The supernatant was then collected and sterilized at 110℃ for 30 minutes to obtain the final product.

[0075] Example 2

[0076] Compared with Example 1, the only difference is the initial fermentation time; the aerobic fermentation time is 2 days. All other conditions are the same as in Example 1, and the specific operation is as follows:

[0077] (1) After crushing the fruit of *Nitraria tangutorum*, 3g was weighed and added to 300g of water. The mixture was then sterilized at 121℃ for 30min and cooled to 25℃ to obtain fermentation substrate A. Subsequently, 3g of *Schizophyllum commune* seed culture was inoculated, wherein the viable count of the inoculated *Schizophyllum commune* was 3 × 10⁻⁶. 7 The concentration of CFU / mL was then increased, and aerobic fermentation was carried out at 28°C and 180 rpm for 2 days. The mycelium was then removed by centrifugation to obtain the fermentation broth of *Hippophae rhamnoides* fruit.

[0078] (2) 1.5g of white thorn leaf powder was added to 300g of white thorn fruit fermentation broth, then sterilized at 110℃ for 30min, and cooled to 25℃ to obtain fermentation substrate B. Then, 1.5g of *Schizophyllum commune* seed culture was inoculated and subjected to secondary aerobic fermentation at 28℃ and 180rpm for 2 days. The viable count of the inoculated *Schizophyllum commune* was 1.5 × 10⁻⁶. 7 The concentration was CFU / mL. After fermentation, the mycelium was removed by centrifugation, followed by fine filtration through a 15μm filter membrane. The supernatant was collected and finally sterilized at 110℃ for 30 minutes to obtain the final product.

[0079] Example 3

[0080] Compared with Example 1, the only difference is the duration of the secondary fermentation; the aerobic fermentation time is 1 day, while other conditions are the same as in Example 1. The specific operation is as follows:

[0081] (1) After crushing the fruit of *Nitraria tangutorum*, 3g was weighed and added to 300g of water. The mixture was then sterilized at 121℃ for 30min and cooled to 25℃ to obtain fermentation substrate A. Subsequently, 3g of *Schizophyllum commune* seed culture was inoculated, wherein the viable count of the inoculated *Schizophyllum commune* was 3 × 10⁻⁶. 7 The concentration of CFU / mL was then increased, and aerobic fermentation was carried out at 28°C and 180 rpm for 2 days. The mycelium was then removed by centrifugation to obtain the fermentation broth of *Hippophae rhamnoides* fruit.

[0082] (2) 1.5g of white thorn leaf powder was added to 300g of white thorn fruit fermentation broth, then sterilized at 110℃ for 30min, and cooled to 25℃ to obtain fermentation substrate B. Then, 1.5g of *Schizophyllum commune* seed culture was inoculated and subjected to secondary aerobic fermentation at 28℃ and 180rpm for 1 day. The viable count of the inoculated *Schizophyllum commune* was 1.5 × 10⁻⁶. 7 The concentration was CFU / mL. After fermentation, the mycelium was removed by centrifugation, followed by fine filtration through a 15μm filter membrane. The supernatant was collected and finally sterilized at 110℃ for 30 minutes to obtain the final product.

[0083] Example 4

[0084] Compared with Example 1, the only difference is that the amount of white thorn leaf powder added to the secondary fermentation substrate is 0.6g, and the other conditions are the same as in Example 1. The specific operation is as follows:

[0085] (1) After crushing the fruit of *Nitraria tangutorum*, 3g was weighed and added to 300g of water. The mixture was then sterilized at 121℃ for 30min and cooled to 25℃ to obtain fermentation substrate A. Subsequently, 3g of *Schizophyllum commune* seed culture was inoculated, wherein the viable count of the inoculated *Schizophyllum commune* was 3 × 10⁻⁶. 7 The concentration of CFU / mL was then increased, and aerobic fermentation was carried out at 28°C and 180 rpm for 2 days. The mycelium was then removed by centrifugation to obtain the fermentation broth of *Hippophae rhamnoides* fruit.

[0086] (2) 0.6g of white thorn leaf powder was added to 300g of white thorn fruit fermentation broth, then sterilized at 110℃ for 30min, and cooled to 25℃ to obtain fermentation substrate B. Then, 1.5g of *Schizophyllum commune* seed culture was inoculated and subjected to secondary aerobic fermentation at 28℃ and 180rpm for 2 days. The viable count of the inoculated *Schizophyllum commune* was 1.5 × 10⁻⁶. 7 The concentration was CFU / mL. After fermentation, the mycelium was removed by centrifugation, followed by fine filtration through a 15μm filter membrane. The supernatant was collected and finally sterilized at 110℃ for 30 minutes to obtain the final product.

[0087] Example 1: DPPH free radical scavenging rate

[0088] DPPH is a stable nitrogen-centered free radical that appears purple in organic solvents and exhibits maximum absorption at 517 nm. In DPPH experiments, when antioxidants are added to a DPPH solution, they react with the DPPH free radical, capturing its unpaired electrons and causing it to lose its free radical properties, changing from purple to colorless. By comparing the changes in absorbance before and after the reaction, the antioxidant capacity of a sample can be quantitatively assessed. This method is simple, rapid, and widely used in the food, pharmaceutical, and cosmetic industries to evaluate the antioxidant performance of products. Therefore, the scavenging effect of a sample on DPPH free radicals can be evaluated by measuring changes in absorbance.

[0089] The specific experimental steps for the DPPH free radical scavenging experiment are as follows (the test solution is pre-diluted with water to a volume concentration of 25%):

[0090] (1) Take an equal volume (1 mL) of the test solution and 2 × 10 -4 Mix the mol / L DPPH solution thoroughly (tube A1);

[0091] (2) Take an equal volume (1 mL) of anhydrous ethanol (the solvent for the analyte) and 2 × 10⁻⁶ mol / L. -4 Mix the mol / L DPPH solution thoroughly (tube A2);

[0092] (3) Take an equal volume (1 mL) of anhydrous ethanol and mix it with the test solution (A3 tube);

[0093] (4) After reacting in the dark for 30 minutes, measure the absorbance values ​​of tubes A1, A2 and A3 at 517 nm; the clearance rate is calculated as follows: clearance rate = [(A2+A3)-A1] / A2×100%.

[0094] This experiment tested the DPPH free radical scavenging assay on the examples and comparative examples. The results are shown in Table 1 and... Figure 1 .

[0095] Table 1

[0096] Example 1 89.03 Example 2 88.16 Example 3 85.99 Example 4 86.17

[0097] The results showed that the DPPH free radical scavenging rate in Examples 1 to 4 was all above 85%, demonstrating a relatively ideal antioxidant effect.

[0098] Example 2: Anti-glycation

[0099] The main effects of advanced glycation end products (AGEs) on the skin are skin aging and dullness. AGEs are the end products of non-enzymatic glycation reactions (Maillard reactions), referring to stable covalent adducts formed spontaneously by macromolecules such as proteins, lipids, or nucleic acids with glucose or other reducing monosaccharides in the absence of enzymes. This test evaluates the ability of the test sample to clear advanced glycation end products by detecting the AGEs clearance rate through an AGEs clearance assay.

[0100] Simulated glycation inhibition system

[0101] A mixed solution containing 80 mg / mL bovine serum albumin and 240 mg / mL glucose was prepared using PBS and filtered through a 0.22 μm filter membrane as the 2x glycation reaction solution. The reaction systems for each group were prepared according to Table 3. The final concentration of bovine serum albumin in each reaction system was 40 mg / mL, and the final concentration of glucose was 120 mg / mL. The final concentrations of the samples or positive controls are shown in Table 2.

[0102] Table 2. AGEs Removal Test Reaction System

[0103]

[0104] After thorough mixing, the mixture was incubated at 55°C for 4 days. PBS was used as a negative control instead of the sample, 10% aminoguanidine hydrochloride solution as a positive control, and PBS was used instead of the glycosylation reaction solution as a control system. After the reaction, the incubated solution was cooled to room temperature, centrifuged at 2000 rpm for 5 minutes, and the supernatant was filtered through a 0.22 μm filter. 200 μL of the reaction solution was then added to each well of a 96-well plate, and the AGEs inhibition rate was calculated using a fluorescence microplate reader at an excitation wavelength of 320 nm and an emission wavelength of 460 nm, according to the following formula.

[0105]

[0106] In the formula:

[0107] A - Fluorescence intensity of the saccharification system with added test substance;

[0108] B - Fluorescence intensity of the PBS solution containing the test substance;

[0109] The results are shown in Table 3 and Figure 2 .

[0110] Table 3

[0111] Example 1 95.38 Example 2 94.37 Example 3 92.34 Example 4 93.72

[0112] The results showed that the AGEs inhibition rate in Examples 1 to 4 was all above 90%, demonstrating a relatively ideal anti-glycation effect.

[0113] Example 3: In vitro elastase inhibition

[0114] Elastin plays a crucial role in maintaining youthful skin, and its loss is a major cause of skin aging. Inhibiting elastase activity can effectively slow down elastin breakdown and maintain skin elasticity. Elastase catalyzes the hydrolysis of hydroxyl groups in the polypeptide bonds of various amino acids. Therefore, when it reacts with the substrate N-succinyl-alanine-alanine-alanine-p-nitroaniline, a colored substance is produced, the absorbance of which can be measured at 406 nm using a spectrophotometer. Based on the change in absorbance, the inhibitory effect of the test substance on elastase activity can be calculated, and the anti-wrinkle and firming effects of the test substance can be evaluated.

[0115] The reaction was performed in test tubes. 50 μL of 1 μmol / L elastase solution was added to each well, along with 400 μL of sample. The reaction was incubated in a constant temperature water bath at 25°C for 15 min. After the reaction was complete, if precipitation occurred, centrifugation was performed. The absorbance was measured at 406 nm in a 96-well plate (see Table 4).

[0116] Table 4

[0117]

[0118] Elastase activity inhibition rate:

[0119]

[0120] In the formula:

[0121] T - Absorbance of the sample group, i.e., the absorbance of the solution after the sample affects the reaction between elastase and substrate; T0 - Absorbance of the sample background group;

[0122] C-Enzyme reaction group absorbance, i.e., the absorbance of elastase and substrate reaction without sample; C0-Solvent background group absorbance.

[0123] The results are shown in Table 5 and Figure 3 .

[0124] Table 5

[0125]

[0126]

[0127] The results showed that the in vitro elastase inhibition rate in Examples 1-4 was all above 90%.

[0128] Example 4: Relative expression level of ELN mRNA

[0129] Cells were treated with a 2% sample concentration, followed by RNA extraction from HaCaT cells using the TriQuick total RNA extraction reagent according to the manufacturer's instructions. First-strand cDNA synthesis was performed using the FastQuant cDNA kit. qPCR Master Mix and qRT-PCR were used to detect the cDNA obtained from further reverse transcription. Specific primer sequences are shown in Table 6 below, and test results are shown in Tables 7 and 8. Figure 4 .

[0130] Table 6. Primer Sequence List for qRT-PCR

[0131]

[0132] Table 7

[0133]

[0134] The results showed that, compared with the model group, the relative expression level of the ELN gene in Examples 1-4 was significantly increased, indicating that the examples could significantly enhance the anti-aging effect. Figure 4 **p < 0.01 indicates a statistically significant difference compared to the model group, representing a significant decrease; ### p < 0.001 indicates a highly statistically significant difference compared to the control group.

[0135] Example 5: Relative expression level of NRF2 mRNA

[0136] Cells were treated with a 2% sample concentration, followed by RNA extraction from HaCaT cells using the TriQuick total RNA extraction reagent according to the manufacturer's instructions. First-strand cDNA synthesis was performed using the FastQuant cDNA kit. qPCR Master Mix and qRT-PCR were used to detect the cDNA obtained from further reverse transcription. Specific primer sequences are shown in Table 8 below, and test results are shown in Tables 9 and 9. Figure 5 .

[0137] Table 8. Primer Sequence List for qRT-PCR

[0138]

[0139] Table 9

[0140]

[0141] The results showed that, compared with the model group, the relative expression level of the NRF-2 gene in Examples 1-4 was significantly increased, indicating that the examples could enhance the anti-aging effect. Figure 5 **p < 0.01 indicates a statistically significant difference compared to the model group, representing a significant decrease; ### p < 0.001 indicates a highly statistically significant difference compared to the control group.

[0142] Example 6: SOD (superoxide dismutase) activity assay

[0143] This experiment used Hacat cells from the Chinese Scientific Cell Bank to verify the cellular SOD enzyme activity of the products prepared in the above examples and comparative examples.

[0144] 1. The HaCaT cell density is 500,000 cells / well (6-well plate). When the cell density reaches 80%, the culture medium is aspirated.

[0145] 2. Clean twice with PBS, then add 1mL of PBS, cover the blank hole with aluminum foil, and place it in a UVB lamp box for 30s.

[0146] 3. After irradiation, remove PBS, then wash once with PBS, add 2 mL of 2% sample and serum-free solution, and the positive control is 50 μg / mL VC.

[0147] 4. Place in a 37℃ incubator and incubate for 24 hours;

[0148] 5. Discard the supernatant;

[0149] 6. Wash twice with pre-cooled PBS, add 100 μL of SOD sample preparation solution, pipette to fully lyse the cells, centrifuge at 12,000 g for 5 minutes at 4°C, and take the supernatant as the sample to be tested.

[0150] 7. Reagent preparation

[0151] 1) Preparation of WST-8 / enzyme working solution: Mix 60-40 μl of SOD detection buffer, 320 μl of WST-8 and 40 μl of enzyme solution evenly to prepare WST-8 / enzyme working solution.

[0152] 2) Preparation of reaction start-up working solution: Dilute 1 μl of reaction start-up solution (40X) with 39 μl of SOD detection buffer solution and mix well to obtain the reaction start-up working solution.

[0153] 8. Sample determination

[0154] Set up the sample wells and various blank control wells using a 96-well plate according to the table below. Add the test samples and other solutions in the order listed in the table. After adding the reaction start-up working solution, mix thoroughly. Incubate at 37°C for 30 minutes. Measure the absorbance at 450 nm.

[0155] Calculation formula: Inhibition percentage = (A blank control 1 - A sample) / (A blank control 1 - A blank control 2) × 100%

[0156] SOD enzyme activity units in the test sample = inhibition percentage / (1 - inhibition percentage) units

[0157] The calculation results are shown in Table 10 and Figure 6 .

[0158] Table 10

[0159] control group 1.19 Model group 0.81 Vitamin C (positive control) 1.15 Example 1 1.12 Example 2 0.98 Example 3 1.18 Example 4 0.94

[0160] The results showed that the SOD activity in Examples 1-4 was significantly increased compared with the model group, indicating that the examples could increase the content of anti-aging active ingredients. Figure 6 In this context, **p < 0.01 indicates a statistically significant difference compared to the model group, meaning a significant decrease; ***p < 0.001 indicates an extremely statistically significant difference compared to the model group, meaning an extremely significant decrease. ### p < 0.001 indicates a highly statistically significant difference compared to the control group.

[0161] Example 7: In vitro whitening test

[0162] Tyrosinase is a key enzyme in the biosynthesis of melanin in the skin. It acts on dopa to form dopaquinone, which then spontaneously undergoes a series of reactions to finally form melanin. Tyrosinase can catalyze the conversion of dopa to dopaquinone in a phosphoric acid solution at pH 6.8, and the absorbance can be measured at 475 nm using a spectrophotometer.

[0163] Raw materials that inhibit tyrosinase activity can reduce the conversion of dopa to dopaquinone, thereby lowering the absorbance. The inhibitory effect of the raw materials on tyrosinase activity is evaluated based on changes in absorbance.

[0164] Cultured cells

[0165] After B16 cells in the logarithmic growth phase were passaged and adhered, they were cultured directly in DMEM medium containing α-MSH (final concentration 0.5 μM); when the cells reached 80-90% confluence (culture dish), 8 ml of PBS was added to wash the cells, and this process was repeated once.

[0166] Collect cells

[0167] (Collect one cell pellet per large dish) Add 2 mL of PBS, scrape the cells off using a cell scraper, and collect them into a 15 mL centrifuge tube. Simultaneously, rinse the culture dish with PBS and transfer the liquid to the 15 mL centrifuge tube. Centrifuge at 1000 rpm for 5-10 min, then carefully discard the supernatant (retain 1 mL). Gently resuspend the cells and transfer them to a 1 mL Eppendorf tube. Centrifuge at 1000 rpm for 5-10 min, discard the supernatant, and retain the cell pellet for later use. Note: Avoid leaving any liquid in the Eppendorf tube. If not used for an extended period, freeze at -80°C.

[0168] Lysed cells

[0169] Add 500 μL of cell lysis buffer to each tube of cells, vortex to mix, and lyse at -20°C for 30 min.

[0170] 4. Collect cell lysate

[0171] After lysis, centrifuge at 10000g for 30 min at 4°C, and transfer the supernatant to a 1.5ml Eppendorf tube for further processing.

[0172] 5. Protein content determination

[0173] Take 10 μL of cell lysate and dilute it 10-fold with PBS to a volume of 100 μL for protein content determination (final incubation at 37°C with cap for 30 min). Prepare standard solutions according to the manufacturer's instructions, construct a curve, and calculate the protein content in the cell lysate. Finally, dilute the protein content of the cell lysate with PBS (pH 6.8) to the required concentration of 3.5 mg / mL.

[0174] The tyrosinase activity inhibition experiment was performed as shown in the table below, with the contents added sequentially to 1.5 ml Eppendorf tubes.

[0175] PBS phosphate buffer (pH 6.8).

[0176]

[0177] Calculate the tyrosinase inhibition rate:

[0178]

[0179] (1) In the formula:

[0180] T—Absorbance of the sample tube, i.e., the absorbance of the solution after the sample reacts with tyrosinase;

[0181] T0—Sample background absorbance;

[0182] C—The average absorbance of the enzyme reaction tube, i.e. the absorbance of the tyrosinase and dopa reaction without the addition of sample;

[0183] C0 — Solvent background absorbance.

[0184] The results are shown in Table 11 and Figure 7 .

[0185] Table 11

[0186] Example 1 74.49 Example 2 67.08 Example 3 71.60 Example 4 63.37

[0187] The results showed that the tyrosinase activity inhibition rate in Examples 1-4 remained stable at over 60%, exhibiting a relatively ideal whitening effect.

[0188] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof in this application are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0189] Although this application has been disclosed above through the description of specific embodiments, it should be understood that those skilled in the art can devise various modifications, improvements, or equivalents to this application within the spirit and scope of the appended solutions. Such modifications, improvements, or equivalents should also be considered to be included within the scope of protection claimed in this application.

Claims

1. A method for preparing a whitening and firming fermented liquid from *Acanthopanax senticosus*, characterized in that, The steps include the following: (1) After crushing the fruit of *Nitraria tangutorum*, water was added to obtain fermentation substrate A. *Schizophyllum commune* was inoculated into fermentation substrate A for preliminary fermentation to obtain *Nitraria tangutorum* fermentation broth. The *Schizophyllum commune* includes strains preserved at the China General Microbiological Culture Collection Center (CGMCC), with a preservation date of March 9, 2023, preservation number CGMCC No. 40388, and classified as *Schizophyllum commune* (…). Schizophyllum commune The depository is China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing, 100101, China. The mass ratio of the white thorn fruit to the water is 0.5~1:

100. The initial fermentation temperature is 25~32℃, and the initial fermentation time is 2~3 days. (2) White thorn leaves are added to the fermentation liquid of white thorn fruit to obtain fermentation substrate B. The fermentation substrate B is then inoculated with the Schizophyllum commune for secondary fermentation. The mass ratio of white thorn leaves to white thorn fruit fermentation liquid is 0.5~1:100, the temperature of the secondary fermentation is 25~32℃, and the time of the secondary fermentation is 2~4 days.

2. The method for preparing the whitening and firming fermented liquid of *Acanthopanax senticosus* as described in claim 1, characterized in that, The preparation method of the whitening and firming fermented liquid of Acanthopanax senticosus meets at least one of the following conditions: In step (1), the preparation method of the fermentation substrate A includes: mixing white thorn fruit powder with water evenly; In step (1), the fermentation substrate A is further subjected to sterilization before use; When the fermentation substrate A is sterilized using a high-temperature sterilization method, the sterilization temperature is 110℃~125℃; the sterilization time is 20~45min.

3. The method for preparing the whitening and firming fermented liquid of *Acanthopanax senticosus* as described in claim 2, characterized in that, The preparation method of the whitening and firming fermented liquid of Acanthopanax senticosus meets at least one of the following conditions: In step (1), the mass ratio of the white thorn fruit to the water is 1:100; When the fermentation substrate A is sterilized using the high-temperature sterilization method, the sterilization temperature is 110℃~121℃ and the sterilization time is 25~40min.

4. The method for preparing the whitening and firming fermented liquid of Acanthopanax senticosus as described in claim 3, characterized in that, The preparation method of the whitening and firming fermented liquid of Acanthopanax senticosus meets at least one of the following conditions: When the fermentation substrate A is sterilized using the high-temperature sterilization method, the sterilization temperature is 121°C and the sterilization time is 30 minutes.

5. The method for preparing the whitening and firming fermented liquid of *Acanthopanax senticosus* as described in claim 1, characterized in that, The preparation method of the whitening and firming fermented liquid of Acanthopanax senticosus meets at least one of the following conditions: In step (1), the *Schizophyllum commune* is added in the form of *Schizophyllum commune* bacterial solution; In step (1), based on the water in the fermentation substrate A, the viable count of the *Schizophyllum commune* inoculated in the water is 1~3×10⁻⁶. 7 CFU / mL.

6. The method for preparing the whitening and firming fermented liquid of *Acanthopanax senticosus* as described in claim 5, characterized in that, In step (1), based on the water in the fermentation substrate A, the viable count of the *Schizophyllum commune* inoculated in the water is 3 × 10⁻⁶. 7 CFU / mL.

7. The method for preparing the whitening and firming fermented liquid of *Acanthopanax senticosus* as described in claim 1, characterized in that, The preparation method of the whitening and firming fermented liquid of Acanthopanax senticosus meets at least one of the following conditions: In step (1), the preliminary fermentation is aerobic fermentation, which is carried out in a constant temperature shaking box. In step (1), after the initial fermentation, the operation of centrifuging to remove the residue and mycelium is also included; the centrifugation speed is 3000~7000 rpm and the centrifugation time is 20~45 min.

8. The method for preparing the whitening and firming fermented liquid of Acanthopanax senticosus as described in claim 7, characterized in that, The preparation method of the whitening and firming fermented liquid of Acanthopanax senticosus meets at least one of the following conditions: In step (1), when the aerobic fermentation is carried out using a constant temperature shaking box, the rotation speed of the constant temperature shaking box is 150~200 rpm; In step (1), the initial fermentation temperature is 28°C; In step (1), the initial fermentation time is 3 days; In step (1), the centrifugation speed is 3500~6000 rpm and the centrifugation time is 25~40 min.

9. The method for preparing the whitening and firming fermented liquid of *Acanthopanax senticosus* as described in claim 8, characterized in that, The preparation method of the whitening and firming fermented liquid of Acanthopanax senticosus meets at least one of the following conditions: In step (1), the rotation speed of the constant temperature oscillation chamber is 180 rpm; In step (1), the centrifugation speed is 5000 rpm and the centrifugation time is 30 min.

10. The method for preparing the whitening and firming fermented liquid of *Acanthopanax senticosus* as described in claim 1, characterized in that, The preparation method of the whitening and firming fermented liquid of Acanthopanax senticosus meets at least one of the following conditions: In step (2), the white thorn leaves are white thorn leaf powder; In step (2), the preparation method of the fermentation substrate B includes: mixing the white thorn leaf powder with the white thorn fruit fermentation liquid evenly; In step (2), before the fermentation substrate B is used, a sterilization operation is also included. The sterilization method for the fermentation substrate B is high-temperature sterilization. When the high-temperature sterilization method is used for sterilization, the sterilization temperature is 100℃~110℃ and the sterilization time is 20~45min. In step (2), the secondary fermentation is aerobic fermentation, which is carried out in a constant temperature shaking box. In step (2), the water in the fermentation substrate B is used as a base, and the viable count of the *Schizophyllum commune* inoculated in the water is 0.5~1.5×10⁻⁶. 7 CFU / mL.

11. The method for preparing the whitening and firming fermented liquid of *Acanthopanax senticosus* as described in claim 10, characterized in that, The preparation method of the whitening and firming fermented liquid of Acanthopanax senticosus meets at least one of the following conditions: In step (2), the mass ratio of the fermentation liquid of white thorn leaves to white thorn fruit is 0.5:100; In step (2), when the high-temperature sterilization method is used for sterilization, the sterilization temperature is 110°C and the sterilization time is 25~40 min; In step (2), when the aerobic fermentation is carried out using a constant temperature shaking box, the rotation speed of the constant temperature shaking box is 150~200 rpm; In step (2), the temperature of the secondary fermentation is 28°C; In step (2), the secondary fermentation time is 3 days; In step (2), based on the water in the fermentation substrate B, the viable count of the *Schizophyllum commune* inoculated in the water is 1.5 × 10⁻⁶. 7 CFU / mL.

12. The method for preparing the whitening and firming fermented liquid of *Acanthopanax senticosus* as described in claim 11, characterized in that, The preparation method of the whitening and firming fermented liquid of Acanthopanax senticosus meets at least one of the following conditions: In step (2), when the high-temperature sterilization method is used for sterilization, the sterilization time is 30 minutes. In step (2), when the aerobic fermentation is carried out using the constant temperature shaking box, the rotation speed of the constant temperature shaking box is 180 rpm.

13. The method for preparing the whitening and firming fermented liquid of *Acanthopanax senticosus* as described in any one of claims 10-12, characterized in that, The secondary fermentation process also includes at least one of the following operations: centrifugation, filtration to remove supernatant, and sterilization.

14. The method for preparing the whitening and firming fermented liquid of *Acanthopanax senticosus* as described in claim 13, characterized in that, The preparation method of the whitening and firming fermented liquid of Acanthopanax senticosus meets at least one of the following conditions: The centrifuge speed is 3000~7000 rpm; The centrifugation time is 20-45 minutes; The filter membrane used for filtration has a thickness of 10~15μm; The sterilization method after secondary fermentation is high-temperature sterilization. When the high-temperature sterilization method is used to sterilize the secondary fermentation, the sterilization temperature is 100℃~110℃ and the sterilization time is 20~45min.

15. The method for preparing the whitening and firming fermented liquid of *Acanthopanax senticosus* as described in claim 14, characterized in that, The preparation method of the whitening and firming fermented liquid of Acanthopanax senticosus meets at least one of the following conditions: The centrifuge speed is 3500~6000 rpm; The centrifugation time is 25-40 minutes; The filter membrane used for filtration is 15 μm. When the high-temperature sterilization method is used for sterilization after secondary fermentation, the sterilization temperature after secondary fermentation is 110°C, and the sterilization time after secondary fermentation is 25~40 minutes.

16. The method for preparing the whitening and firming fermented liquid of *Acanthopanax senticosus* as described in claim 15, characterized in that, The preparation method of the whitening and firming fermented liquid of Acanthopanax senticosus meets at least one of the following conditions: The centrifuge speed is 5000 rpm; The centrifugation time was 30 minutes. When the high-temperature sterilization method is used for sterilization after the secondary fermentation, the sterilization time after the secondary fermentation is 30 minutes.

17. A whitening and firming fermented liquid of Acanthopanax senticosus, which is prepared by the method of preparing whitening and firming fermented liquid of Acanthopanax senticosus as described in any one of claims 1-16.

18. The application of a whitening and firming fermented liquid of *Elaeagnus pungens* as a product, as an additive, or as a base in the preparation of topical skin agents.

19. The application of the whitening and firming fermented liquid of *Acanthopanax senticosus* as described in claim 18, used directly as a product, as an additive, or as a base in the preparation of topical skin agents, characterized in that... The whitening and firming fermented liquid of Acanthopanax senticosus is at least one of the antioxidant active ingredient, anti-glycation active ingredient, anti-aging active ingredient, and whitening active ingredient in the topical skin agent.

20. The application of the whitening and firming fermented liquid of *Acanthopanax senticosus* as described in claim 19, used directly as a product, as an additive, or as a base in the preparation of topical skin agents, characterized in that... The antioxidant component is an antioxidant component that can scavenge free radicals (DPPH). The anti-glycation active ingredient is an anti-glycation active ingredient that inhibits the content of AGEs; The anti-aging active ingredient is an anti-aging active ingredient that enhances SOD activity; The whitening active ingredient is a whitening active ingredient that inhibits tyrosinase activity.

21. A topical skin agent comprising the whitening and firming fermented liquid of Acanthopanax senticosus as described in claim 17.

22. The topical skin agent as described in claim 21, characterized in that, The topical skin agent meets at least one of the following conditions: The topical skin agent also includes at least one of the following active ingredients: moisturizing active ingredient, whitening active ingredient, anti-inflammatory active ingredient, anti-allergic active ingredient, and antioxidant active ingredient; The topical skin agents include face masks, serums, or toners; The whitening and firming fermented liquid of Acanthopanax senticosus accounts for 5% to 99% of the mass of the topical skin agent.

23. The topical skin agent as described in claim 22, characterized in that, The whitening and firming fermented liquid of Acanthopanax senticosus accounts for 60% to 99% of the mass percentage of the topical skin agent.

Images