Extract of milk thistle and methods of making and using the same

Abstract

The application provides a milk vetch root extract and a preparation method and application thereof, and relates to the technical field of cosmetics. The preparation method of the milk vetch root extract provided by the application is mainly a method for preparing the milk vetch root extract through the enzymolysis of beta-glucosidase and bacillus subtilis fibrinolysin and the fermentation of lactobacillus sakei and yeast. The prepared milk vetch root extract can significantly improve the water content of the cuticle layer of the skin, reduce the red area of the skin, improve the cell anti-photoaging and antioxidant activities, and can be used in the preparation of cosmetics for skin redness removal, skin moisturizing, anti-photoaging, antioxidant and the like; and can also be used in wine brewing to improve the health care efficacy of the wine.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, and in particular to an extract of *Smilax glabra*, its preparation method, and its application. Background Technology

[0002] Millettia speciosa Champ., also known as beautiful cliff bean vine, South China Sea vine, golden bell root, and mighty cow, is a plant belonging to the genus Millettia in the legume family. It is widely distributed in tropical or subtropical regions.

[0003] In terms of traditional efficacy, the Qing Dynasty's "Essentials of Herbal Properties" and "Lu Chuan Materia Medica" both record that Niu Dali has the effects of clearing heat and detoxifying, promoting blood circulation and removing blood stasis, nourishing the kidneys, strengthening muscles and bones, moistening the lungs and replenishing qi. It is often used to treat injuries from falls and blows, coughs due to lung deficiency, and lumbar muscle strain.

[0004] Modern pharmacological studies have further confirmed that the roots, stems, leaves, and flowers of *Smilax glabra* are rich in bioactive components, especially the roots. Its main active substances include polysaccharides, flavonoids, phenolic acids, sterols, and various trace elements (such as Ca, Fe, Zn, Mg, Se, and Mn), which possess a variety of health benefits, including enhancing immunity, moisturizing the lungs and relieving coughs, regulating cardiovascular function, promoting cholesterol metabolism, anti-fatigue, anti-oxidation, anti-inflammation, liver protection, and anti-tumor effects.

[0005] In terms of extraction processes, traditional organic solvent extraction, ultrasonic-assisted extraction, and microwave-assisted extraction are commonly used. However, these methods still have many shortcomings: ① Traditional organic solvent extraction methods (such as reflux extraction, hot extraction, and Soxhlet extraction) are widely used, but they suffer from problems such as being time-consuming and labor-intensive, consuming large amounts of solvent, having low extraction efficiency, and poor reproducibility. Furthermore, solvents such as methanol, ethyl acetate, and acetone are prone to residues and have a certain degree of toxicity, which is detrimental to environmental protection and safe production. ② Ultrasonic-assisted extraction has simple equipment, is easy to operate, and has a short extraction time, but it requires high-quality instruments and a suitable factory environment, and it generates significant noise pollution. ③ Microwave-assisted extraction has advantages such as being time-saving, having high selectivity, requiring less solvent, and being suitable for industrial scale-up, but high-temperature conditions may destroy thermally unstable components and affect the bioactivity of the extract.

[0006] In terms of applied research, current research on the application and efficacy of Niu Dali is mostly concentrated in the fields of medicine and health products, while its application in cosmetics, topical preparations and brewing industry is still relatively weak. There is a lack of systematic formula development, process optimization and efficacy verification research, which limits the development and industrialization of its high value-added products.

[0007] In summary, *Smilax glabra* possesses significant medicinal and edible value, is rich in active ingredients, and offers diverse health benefits. However, its extraction process still faces bottlenecks in efficiency and safety, its application areas are relatively narrow, and further research into its efficacy mechanisms is needed.

[0008] In view of this, the present invention is hereby proposed. Summary of the Invention

[0009] The primary objective of this invention is to provide a method for preparing *Smilax glabra* extract to solve the aforementioned technical problems.

[0010] The second objective of this invention is to provide an extract of *Smilax glabra*.

[0011] A third objective of this invention is to provide applications of the above-mentioned *Smilax glabra* extract.

[0012] To achieve the above objectives, the following technical solution is adopted: In a first aspect, the present invention provides a method for preparing an extract of *Smilax glabra*, comprising the following steps: a. Mix the powder of *Achyranthes bidentata* and β-glucosidase in water to obtain a mixed solution, and then obtain the first enzymatic hydrolysate after enzymatic hydrolysis. b. Add Bacillus subtilis plasmin to the first enzymatic hydrolysate, and obtain a second enzymatic hydrolysate after enzymatic hydrolysis. Then filter the second enzymatic hydrolysate, collect the filtrate and sterilize it. c. Mix the filtrate obtained in step b with the culture medium, ferment it with Lactobacillus sacchariformis, and obtain the first fermentation broth after sterilization and filtration. Then add culture medium to inoculate yeast for fermentation, and obtain the second fermentation broth after sterilization and filtration. d. After concentrating the second fermentation broth, crude extract of *Achyranthes bidentata* is obtained; e. After ethanol precipitation of the crude extract of *Smilax glabra*, collect the precipitate to obtain *Smilax glabra* extract.

[0013] As a further technical solution, the mass ratio of *Smilax glabra* powder to water in the mixed solution is 1:(5-15). The mass ratio of the β-glucosidase to the powder of *Smilax glabra* is 1:(20-40).

[0014] As a further technical solution, in step a, the pH of the enzymatic hydrolysis reaction is 4.5-5.5, the temperature is 40-55℃, and the time is 30-50 min.

[0015] As a further technical solution, the weight ratio of Bacillus subtilis fibrinolytic enzyme to Datura stramonium powder is 1:(10-30).

[0016] As a further technical solution, in step b, the pH of the enzymatic hydrolysis reaction is 5.0-7.0, the temperature is 40-55℃, and the time is 30-50 min.

[0017] As a further technical solution, the yeast is at least one of Candida bronchiol, Candida bumblebee, Pichia pastoris, or Saccharomyces cerevisiae.

[0018] As a further technical solution, the yeast is Candida Brønsted.

[0019] As a further technical solution, the concentration is to concentrate the second fermentation liquid to 1 / 10-1 / 2 of its original volume; As a further technical solution, the alcohol precipitation is carried out using 60wt%-80wt% ethanol.

[0020] Secondly, the present invention provides an extract of *Smilax glabra*, which is prepared by the above-described preparation method.

[0021] Thirdly, the present invention provides an application of *Smilax glabra* extract in any one of the following I-VI: I. Preparation of products for reducing skin redness; II. Prepare skin moisturizing products; III. Develop products to combat photoaging of the skin; IV. Preparation of antioxidant products; V. Preparation of cosmetics; VI. Brewing.

[0022] Compared with the prior art, the present invention has the following beneficial effects: The method for preparing *Achyranthes bidentata* extract provided by this invention mainly involves enzymatic hydrolysis by β-glucosidase and *Bacillus subtilis* plasminogen lysate, followed by fermentation with *Lactobacillus thuringiensis* and yeast in sake brewing. The prepared *Achyranthes bidentata* extract can significantly increase the moisture content of the stratum corneum, reduce the area of ​​skin redness, and enhance the anti-photoaging and antioxidant activity of cells. It can be used in the preparation of cosmetics for skin redness reduction, moisturizing, anti-photoaging, and antioxidant effects; it can also be used in brewing to enhance the health benefits of the wine. Detailed Implementation

[0023] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0024] In a first aspect, the present invention provides a method for preparing an extract of *Smilax glabra*, comprising the following steps: a. Mix the powder of *Achyranthes bidentata* and β-glucosidase in water to obtain a mixed solution, and then obtain the first enzymatic hydrolysate after enzymatic hydrolysis. b. Add Bacillus subtilis plasmin to the first enzymatic hydrolysate, and obtain a second enzymatic hydrolysate after enzymatic hydrolysis. Then filter the second enzymatic hydrolysate, collect the filtrate and sterilize it. c. Mix the filtrate obtained in step b with the culture medium, ferment it with Lactobacillus sacchariformis, and obtain the first fermentation broth after sterilization and filtration. Then add culture medium to inoculate yeast for fermentation, and obtain the second fermentation broth after sterilization and filtration. d. After concentrating the second fermentation broth, crude extract of *Achyranthes bidentata* is obtained; e. After ethanol precipitation of the crude extract of *Smilax glabra*, collect the precipitate to obtain *Smilax glabra* extract.

[0025] The method for preparing *Smilax glabra* extract provided by this invention is simple and convenient. The prepared *Smilax glabra* extract can significantly increase the moisture content of the stratum corneum of the skin, reduce the area of ​​skin redness, and improve the anti-photoaging and antioxidant activity of cells. It can be used in the preparation of cosmetics for skin redness reduction, moisturizing, anti-photoaging, and antioxidant effects; it can also be used in brewing to enhance the health benefits of the wine.

[0026] In some optional embodiments, the mass ratio of *Smilax glabra* powder to water in the mixed solution is 1:(5-15), for example, but not limited to 1:5, 1:10 or 1:15; The mass ratio of the β-glucosidase to the *Achyranthes bidentata* powder is 1:(20-40), for example, but not limited to 1:20, 1:30 or 1:40.

[0027] In some alternative embodiments, in step a, the pH of the enzymatic hydrolysis reaction is 4.5-5.5, for example, but not limited to 4.5, 5.0 or 5.5; The temperature for the enzymatic hydrolysis reaction is 40-55℃, for example, but not limited to 40℃, 50℃ or 55℃; The enzymatic hydrolysis reaction time is 30-50 minutes, for example, but not limited to 30 minutes, 40 minutes or 50 minutes.

[0028] In some alternative embodiments, the weight ratio of the Bacillus subtilis fibrinolytic enzyme to the powder of *Smilax glabra* is 1:(10-30), for example, but not limited to 1:10, 1:20 or 1:30.

[0029] In some alternative implementations, in step b, the pH of the enzymatic hydrolysis reaction is 5.0-7.0, for example, but not limited to 5.0, 6.0 or 7.0; The temperature for the enzymatic hydrolysis reaction is 40-55℃, for example, but not limited to 40℃, 50℃ or 55℃; The enzymatic hydrolysis reaction time is 30-50 minutes, for example, but not limited to 30 minutes, 40 minutes or 50 minutes.

[0030] In some alternative embodiments, the yeast is at least one of Candida Brønsted, Candida bumblebee, Pichia pastoris, or Saccharomyces cerevisiae, with Candida Brønsted preferred for fermentation.

[0031] In some alternative embodiments, the concentration is to concentrate the second fermentation broth to 1 / 10 to 1 / 2 of its original volume, for example, but not limited to 1 / 10, 1 / 5 or 1 / 2.

[0032] In some optional embodiments, the alcohol precipitation is performed using 60wt%-80wt% ethanol, preferably 70% ethanol.

[0033] Secondly, the present invention provides an extract of *Smilax glabra*, which is prepared by the above-described preparation method.

[0034] Thirdly, the present invention provides an application of *Smilax glabra* extract in any one of the following I-VI: I. Preparation of products for reducing skin redness; II. Prepare skin moisturizing products; III. Develop products to combat photoaging of the skin; IV. Preparation of antioxidant products; V. Preparation of cosmetics; VI. Brewing.

[0035] The extract of *Smilax glabra* provided by this invention can significantly increase the moisture content of the stratum corneum of the skin, reduce the area of ​​skin redness, and improve the anti-photoaging and antioxidant activity of cells. It can be used in the preparation of cosmetics for skin redness reduction, moisturizing, anti-photoaging, and anti-oxidation; it can also be used in winemaking to enhance the health benefits of wine.

[0036] The present invention will be further illustrated below with specific embodiments and comparative examples. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0037] In the following examples, *Lactobacillus sacchariflorus* (product number: HZB690203; Wuhan Gray Algae Biotechnology Co., Ltd.); *Candida blakeana* (product number: HZB228837; Wuhan Gray Algae Biotechnology Co., Ltd.); and *Candida blakeana* (product number: HZB126908; Wuhan Gray Algae Biotechnology Co., Ltd.) were used.

[0038] Example 1 A preparation method of Millettia speciosa Champ extract, comprising the following steps: S1. Take Millettia speciosa Champ, crush it and pass through a 60-mesh sieve to obtain Millettia speciosa Champ powder.

[0039] S2. Add 10 times the amount of deionized water to the Millettia speciosa Champ powder, adjust the pH to 5.0 with citric acid-disodium hydrogen phosphate buffer solution, add β-glucosidase (the weight ratio of β-glucosidase to Millettia speciosa Champ powder is 1:30), and conduct enzymolysis at 50°C for 40 min to obtain enzymatic hydrolysate I; S3. Adjust the pH of the enzymatic hydrolysate I to 6.0 with citric acid-disodium hydrogen phosphate buffer solution, add subtilisin fibrinolytic enzyme (the weight ratio of subtilisin fibrinolytic enzyme to Millettia speciosa Champ powder is 1:20), and conduct enzymolysis at 50°C for 40 min to obtain enzymatic hydrolysate II; S4. Filter the enzymatic hydrolysate II, and inactivate the enzyme at 90°C for 10 min to obtain an enzymatic clear solution; S5. Sterilize the enzymatic clear solution, add a culture medium and Lactobacillus sakei for fermentation, and after fermentation is completed, sterilize and filter again to obtain a first fermented clear solution; S6. Add a culture medium and Candida blankii to the first fermented enzymatic clear solution for fermentation, and after fermentation is completed, sterilize and filter to obtain a second fermented clear solution; S7. Concentrate the second fermented enzymatic clear solution to 1 / 5 of the original volume, add ethanol to make the final ethanol concentration in the solution reach 70%, perform overnight alcohol precipitation, discard the supernatant, collect the precipitate and dry it to constant weight, to obtain the Millettia speciosa Champ extract of Example 1.

[0040] Example 2 A preparation method of Millettia speciosa Champ extract, comprising the following steps: S1. Take Millettia speciosa Champ, crush it and pass through a 60-mesh sieve to obtain Millettia speciosa Champ powder.

[0041] S2. Add 5 times the amount of deionized water to the Millettia speciosa Champ powder, adjust the pH to 4.5 with citric acid-disodium hydrogen phosphate buffer solution, add β-glucosidase (the weight ratio of β-glucosidase to Millettia speciosa Champ powder is 1:20), and conduct enzymolysis at 40°C for 50 min to obtain enzymatic hydrolysate I; S3. Adjust the pH of the enzymatic hydrolysate I to 5.0 with citric acid-disodium hydrogen phosphate buffer solution, add subtilisin fibrinolytic enzyme (the weight ratio of subtilisin fibrinolytic enzyme to Millettia speciosa Champ powder is 1:10), and conduct enzymolysis at 40°C for 50 min to obtain enzymatic hydrolysate II; S4. Filter the enzymatic hydrolysate II, and inactivate the enzyme at 90°C for 10 min to obtain an enzymatic clear solution; S5. Sterilize the enzymatic hydrolysate clear solution, add the culture medium and Lactobacillus sakei for fermentation, and after fermentation, perform sterilization and filtration again to obtain the first fermented enzymatic hydrolysate clear solution; S6. Add the culture medium and Candida blankii to the first fermented enzymatic hydrolysate clear solution for fermentation, and after fermentation, perform sterilization and filtration to obtain the second fermented enzymatic hydrolysate clear solution; S7. Concentrate the second fermented enzymatic hydrolysate clear solution to 1 / 2 of its original volume, add ethanol to make the final concentration of ethanol in the solution reach 80%, conduct overnight alcohol precipitation, discard the supernatant, collect the precipitate and dry it to constant weight to obtain the Millettia speciosa Champ extract of Example 2.

[0042] Example 3 A preparation method of Millettia speciosa Champ extract, comprising the following steps: S1. Take Millettia speciosa Champ, crush it and pass through a 60-mesh sieve to obtain Millettia speciosa Champ powder.

[0043] S2. Add 15 times the amount of deionized water to the Millettia speciosa Champ powder, adjust the pH to 5.5 with citric acid-disodium hydrogen phosphate buffer solution, add β-glucosidase (the weight ratio of β-glucosidase to Millettia speciosa Champ powder is 1:40) and conduct enzymatic hydrolysis at 55°C for 30 min to obtain enzymatic hydrolysate I; S3. Adjust the pH of the enzymatic hydrolysate I to 7.0 with citric acid-disodium hydrogen phosphate buffer solution, add Bacillus subtilis fibrinolysin (the weight ratio of Bacillus subtilis fibrinolysin to Millettia speciosa Champ powder is 1:30) and conduct enzymatic hydrolysis at 55°C for 30 min to obtain enzymatic hydrolysate II; S4. Filter the enzymatic hydrolysate II, and inactivate the enzyme at 90°C for 10 min to obtain an enzymatic hydrolysate clear solution; S5. Sterilize the enzymatic hydrolysate clear solution, add the culture medium and Lactobacillus sakei for fermentation, and after fermentation, perform sterilization and filtration again to obtain the first fermented enzymatic hydrolysate clear solution; S6. Add the culture medium and Candida blankii to the first fermented enzymatic hydrolysate clear solution for fermentation, and after fermentation, perform sterilization and filtration to obtain the second fermented enzymatic hydrolysate clear solution; S7. Concentrate the second fermented enzymatic hydrolysate clear solution to 1 / 10 of its original volume, add ethanol to make the final concentration of ethanol in the solution reach 60%, conduct overnight alcohol precipitation, discard the supernatant, collect the precipitate and dry it to constant weight to obtain the Millettia speciosa Champ extract of Example 3.

[0044] Example 4 The difference from Example 1 is that Candida blankii is replaced with Candida bombicola.

[0045] Comparative Example 1 The difference from Example 1 is that Comparative Example 1 only adopts ordinary water extraction and alcohol precipitation, without the enzymatic hydrolysis and fermentation steps, and the specific steps are as follows: S1. Take Radix Millettiae Speciosae, crush it and pass through a 60-mesh sieve to obtain Radix Millettiae Speciosae powder; S2. Add 10 times the amount of deionized water to the Radix Millettiae Speciosae powder, heat and reflux for extraction for 80 min, filter and sterilize to obtain a crude extract; S3. Concentrate the crude extract to 1 / 5 of its original volume, add ethanol to make the final concentration of ethanol in the solution reach 70%, perform overnight alcohol precipitation, discard the supernatant, collect the precipitate and dry it to constant weight to obtain Comparative Example 1; Comparative Example 2 The difference from Example 1 is that Comparative Example 2 only undergoes enzymatic hydrolysis with a complex enzyme (β-glucosidase + Bacillus subtilis fibrinolysin) without fermentation.

[0046] Comparative Example 3 The difference from the Example is that Comparative Example 3 only undergoes fermentation with complex bacteria (Lacticaseibacillus sakei + Candida blankii) without enzymatic hydrolysis with a complex enzyme.

[0047] Comparative Example 4 The difference from Example 1 is that Comparative Example 4 only undergoes enzymatic hydrolysis with a single enzyme (β-glucosidase) and fermentation with complex bacteria (Lacticaseibacillus sakei + Candida blankii).

[0048] Comparative Example 5 The difference from Example 1 is that Comparative Example 5 only undergoes enzymatic hydrolysis with a single enzyme (Bacillus subtilis fibrinolysin) and fermentation with complex bacteria (Lacticaseibacillus sakei + Candida blankii).

[0049] Comparative Example 6 The difference from Example 1 is that Comparative Example 6 only undergoes enzymatic hydrolysis with a complex enzyme (β-glucosidase + Bacillus subtilis fibrinolysin) and fermentation with a single strain (Lacticaseibacillus sakei).

[0050] Comparative Example 7 The difference from Example 1 is that Comparative Example 7 only undergoes enzymatic hydrolysis with a complex enzyme (β-glucosidase + Bacillus subtilis fibrinolysin) and fermentation with a single strain (Candida blankii).

[0051] Test Example, Performance Test Test 1 Redness-reducing and moisturizing effects of Radix Millettiae Speciosae extract 1. The raw materials for preparing the test samples are shown in Table 1.

[0052] Table 1 Preparation of Radix Millettiae Speciosae essence water

[0053] The preparation method of the Radix Millettiae Speciosae essence water is as follows: S1: Mix and dissolve butanediol, p-hydroxyacetophenone and Carbomer 980, add half of the amount of hot water and then homogenize; after homogenization, the resulting phase is Phase A; S2: Dissolve the test sample in half of the remaining hot water, add it to phase A, stir and homogenize to obtain the finished product.

[0054] 2. Testing Methods Test area: face Frequency of use: Use daily; once in the morning and once in the evening. Directions for use: After cleansing, apply an appropriate amount of this product to the face and gently massage until absorbed.

[0055] Test procedure: as shown in Table 2.

[0056] Table 2 Test Procedure

[0057] (5) Evaluation methods As shown in Table 3.

[0058] Table 3 Evaluation Methods

[0059] (6) Analytical methods Rate of change (%) = (D) 28 -D0) / D0×100%.

[0060] Note: D0 represents the baseline skin parameters before using the product; D 28 This indicates the skin parameter values ​​after 28 days of product use.

[0061] 3. Test Results The test results are shown in Table 4.

[0062] Table 4 Test Results

[0063] As shown in Table 4: (1) The rate of change of moisture content in the stratum corneum of the skin is ranked as follows: ① The samples prepared by combining enzymatic hydrolysis with two-stage fermentation (Examples 1-3) showed the best results and were superior to other treatments.

[0064] ② Next were the samples that combined single-enzyme hydrolysis with two-stage fermentation (Comparative Example 4 and Comparative Example 5). Among them, the effect of Comparative Example 5 was better than that of Comparative Example 4, indicating that the hydrolysis effect of Bacillus subtilis plasminase was better than that of β-glucosidase.

[0065] ③ The samples were again subjected to enzymatic hydrolysis combined with single-strain fermentation (Comparative Example 6 and Comparative Example 7). The effect of Comparative Example 7 was better than that of Comparative Example 6, indicating that the fermentation effect of Candida Brønsted is better than that of Lactobacillus thuringiensis in sake.

[0066] ④The following samples were in the following order: samples with only two-stage fermentation (Comparative Example 3), samples with only complex enzyme hydrolysis (Comparative Example 2), and samples with simple extraction (Comparative Example 1).

[0067] The above results indicate that both enzymatic hydrolysis and fermentation can effectively increase the moisture content of the stratum corneum of the skin, and the combined effect of enzymatic hydrolysis and two-stage fermentation is the most significant.

[0068] (2) The ranking of the rate of change of the red area ratio is consistent with the ranking of the rate of change of the moisture content of the stratum corneum.

[0069] In summary, the combined use of compound enzyme hydrolysis and two-stage fermentation can increase skin hydration and reduce the area of ​​redness on the skin, making it suitable for use in moisturizing, redness-reducing, soothing, and repairing products.

[0070] Test 2: Protective effect of *Smilax glabra* extract on photoaging of HaCaT cells 1. Testing Methods Hacat cells were loaded at 1.5 × 10⁻⁶. 5 Hacat cells were seeded at a density of 1 / ml in 96-well plates and incubated for 24 h using a standard incubator. After incubation, the old culture medium was aspirated, and the cells were washed twice with PBS. A small amount of PBS was then added (leaving only a thin layer of PBS in the culture plate to keep the cells moist), and Hacat cells were irradiated with an LED UV lamp. After irradiation, the PBS was aspirated, and each well was immediately filled with culture medium containing 0.1% of the sample (the extract of *Achyranthes bidentata* from the example or comparative example) and a negative control was set up by adding only complete culture medium. The cells were then incubated for another 24 h using a standard incubator. After incubation, the old culture medium was aspirated, and 100 μL of 10% CCK8 solution was added to each well of the 96-well plate. The cells were incubated for 30 min to 4 h using a standard incubator, and then the OD value of each well was measured at 450 nm using a microplate reader. Cell viability was calculated as (sample group OD - background group OD / model group OD - background group OD) × 100%.

[0071] 2. Test Results The test results are shown in Table 5.

[0072] Table 5. Protective effect of each sample against UVB-induced photoaging of HaCaT cells.

[0073] As shown in Table 5, in the UVB-induced photoaging cell model, only the samples of Examples 1-3, Comparative Examples 4 and 5 showed a cell survival rate higher than 80% after treatment, indicating that these three samples have good anti-photoaging effects.

[0074] Further analysis revealed that the cell viability of Comparative Examples 4 and 5 (both subjected to enzymatic hydrolysis and two-stage fermentation) was significantly higher than that of Comparative Example 2 (compound enzymatic hydrolysis only), Comparative Example 3 (two-stage fermentation only), Comparative Example 6, and Comparative Example 7 (compound enzymatic hydrolysis + single-strain fermentation), indicating that enzymatic hydrolysis + two-stage fermentation can significantly improve the photoaging resistance of the samples.

[0075] It is noteworthy that the cell viability of Examples 1-3 (prepared by combined enzymatic hydrolysis and two-stage fermentation) exceeded 90%, significantly higher than that of Comparative Examples 4 and 5. This indicates a synergistic effect between enzymatic hydrolysis and two-stage fermentation, which can further enhance the anti-photoaging effect of *Achyranthes bidentata* extract.

[0076] Test 3: Antioxidant effect of *Smilax glabra* extract 1. Testing Methods Weigh 7.884 mg of 1,1-diphenyl-2-trinitrophenylhydrazine powder and dilute to 100 mL with ethanol. Shake well to obtain a 0.2 mmol DPPH solution. Weigh different samples and dilute them to a 5% concentration for testing. Use a pipette to transfer 500 μL of each sample into a plastic centrifuge tube, add 500 μL of DPPH solution, shake well, and protect from light for 30 min. Use ethanol as a blank control. To avoid protein flocculation in the filtrate affecting the experimental results, centrifuge in the dark for the last 10 min of the reaction, and use the supernatant for analysis.

[0077] 200 μL of absorbance was taken from each well of a 96-well plate, and the absorbance was measured at 517 nm. The experiment was performed in triplicate. The specific calculation formula is as follows: DPPH removal rate = (1 - D1 / D2) × 100%; In the formula, D1 is the absorbance of the test sample or positive control; D2 is the absorbance of the blank control.

[0078] 2. Test Results The antioxidant results of each test sample are shown in Table 6.

[0079] Table 6. Antioxidant test results for each sample.

[0080] As shown in Table 6, the antioxidant trend is similar to that of Test 1 and Test 2: ① The sample prepared by combining enzymatic hydrolysis with two-stage fermentation (Example 1) showed the best results and was superior to other treatments.

[0081] ② Next were samples with single-enzyme hydrolysis combined with two-stage fermentation (Comparative Example 4, Comparative Example 5), samples with compound enzyme hydrolysis combined with single-strain fermentation (Comparative Example 6, Comparative Example 7), samples with only two-stage fermentation (Comparative Example 3), samples with only compound enzyme hydrolysis (Comparative Example 2), and samples with simple extraction (Comparative Example 1).

[0082] Test 4: Application of *Smilax glabra* extract in the brewing industry (1) Preparation method of Niu Dali wine S1. Raw material processing: Prepare *Achyranthes bidentata* extract (powder or granules) according to the embodiments of the present invention for later use.

[0083] S2. Steaming and adding yeast: Put the extract of *Smilax glabra* (powder / granules) into a steaming device and steam it for 2.5-3.5 hours. After cooling, add yeast in a ratio of (80-120):1 and mix well to obtain the mixture.

[0084] S3. Fermentation: Put the above-mentioned mixture into a container and ferment for 70-280 hours at room temperature, away from light and in a well-ventilated place to obtain fermented material.

[0085] S4. Extraction and distillation: Mix the above fermented material with rice wine at a ratio of 1:1 and extract at room temperature for 10-15 days; obtain Niudali distilled spirits of different precision through distillation.

[0086] To obtain a better taste and health benefits from traditional Chinese medicine distilled spirits, other food and medicinal ingredients can be added during fermentation, such as sorghum, corn, wheat, rice bran, hawthorn, wolfberry, codonopsis, cistanche, and dendrobium officinale.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing an extract of *Smilax glabra*, characterized in that, Includes the following steps: a. Mix the powder of *Achyranthes bidentata* and β-glucosidase in water to obtain a mixed solution, and then obtain the first enzymatic hydrolysate after enzymatic hydrolysis. b. Add Bacillus subtilis plasmin to the first enzymatic hydrolysate, and obtain a second enzymatic hydrolysate after enzymatic hydrolysis. Then filter the second enzymatic hydrolysate, collect the filtrate and sterilize it. c. Mix the filtrate obtained in step b with the culture medium, ferment it with Lactobacillus sacchariformis, and obtain the first fermentation broth after sterilization and filtration. Then add culture medium to inoculate yeast for fermentation, and obtain the second fermentation broth after sterilization and filtration. d. After concentrating the second fermentation broth, crude extract of *Achyranthes bidentata* is obtained; e. After ethanol precipitation of the crude extract of *Smilax glabra*, collect the precipitate to obtain *Smilax glabra* extract.

2. The preparation method according to claim 1, characterized in that, In the mixed solution, the mass ratio of *Smilax glabra* powder to water is 1:(5-15). The mass ratio of the β-glucosidase to the powder of *Smilax glabra* is 1:(20-40).

3. The preparation method according to claim 1, characterized in that, In step a, the pH of the enzymatic hydrolysis reaction is 4.5-5.5, the temperature is 40-55℃, and the time is 30-50 min.

4. The preparation method according to claim 1, characterized in that, The weight ratio of Bacillus subtilis fibrinolytic enzyme to Datura stramonium powder is 1:(10-30).

5. The preparation method according to claim 1, characterized in that, In step b, the pH of the enzymatic hydrolysis reaction is 5.0-7.0, the temperature is 40-55℃, and the time is 30-50 min.

6. The preparation method according to claim 1, characterized in that, The yeast is at least one of Candida bronchiol, Candida bumblebee, Pichia pastoris, or Saccharomyces cerevisiae.

7. The preparation method according to claim 6, characterized in that, The yeast strain is Candida Brønkii.

8. The preparation method according to claim 1, characterized in that, The concentration is to concentrate the second fermentation broth to 1 / 10-1 / 2 of its original volume; The alcohol precipitation is performed using 60wt%-80wt% ethanol.

9. An extract of *Smilax glabra*, characterized in that, It is prepared by the preparation method according to any one of claims 1-8.

10. The use of the *Smilax glabra* extract according to claim 9 in any one of the following I-VI: I. Preparation of products for reducing skin redness; II. Prepare skin moisturizing products; III. Develop products to combat photoaging of the skin; IV. Preparation of antioxidant products; V. Preparation of cosmetics; VI. Brewing.

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