Mulberry root bark extract for whitening and soothing products and preparation method of mulberry root bark extract

Abstract

The invention relates to the technical field of cosmetics, and particularly provides a preparation method and application of a mulberry root bark extract. According to the method, mulberry root bark is taken as a raw material, after being crushed, the mulberry root bark is subjected to enzymolysis by adopting a composite reagent under a specific PH condition, so that efficient conversion from mulberroside A to oxyresveratrol is realized, and the conversion rate is not lower than 99%. The purity of oxyresveratrol in the obtained extract is not lower than 99%, and the content of mulberroside A is not higher than 1%. And further performing microencapsulation treatment on a compounded wall material to prepare the whitening and soothing composition with the embedding rate of more than or equal to 99% and the transdermal rate of more than or equal to 50% in 24 hours. The method is green in process, high in conversion rate and suitable for industrial production, and the problems that oxidized resveratrol is poor in solubility and low in transdermal rate are effectively solved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of natural product extraction and cosmetics, and specifically relates to a method for efficiently extracting high-purity oxymorcozy from mulberry root bark, a mulberry root bark extract prepared by the method, a whitening and soothing composition containing the extract, and a preparation method and application thereof. BACKGROUND

[0002] Mulberry is a traditional tree species in China and is widely planted in China. Mulberry root bark has been recorded in ancient pharmacopoeias and is used to treat various diseases. Studies have shown that mulberry root bark is rich in various secondary metabolites and has a wide range of biological activities. However, the current development and utilization of mulberry root bark is relatively low, and it is mostly treated as agricultural waste or fuel, resulting in serious waste of resources.

[0003] In recent years, stilbene compounds have attracted market attention, and mulberry root bark has become a research hotspot due to its rich content of such components. Among them, oxymorcozy is one of the main stilbene compounds in mulberry root bark, which has antioxidant, anti-inflammatory, antiviral, and cell protection activities; mulbarin A, as a glycosylated derivative thereof, is substituted with glucose at 3'-OH and 4'-OH positions, and also exhibits antioxidant, anti-inflammatory, antiviral, and tyrosinase inhibition activities and pharmacological functions. Currently, research on mulberry root bark is mostly focused on flavonoids, polysaccharides, alkaloids, and other components, and there is relatively less research on the extraction and purification of stilbene compounds, especially mulbarin A and oxymorcozy. Existing methods usually extract the two separately, and the process flow is complex, and the separation of oxymorcozy mostly uses silica gel column chromatography with a petroleum ether-ethyl acetate mixed system for elution. Such methods are not only cumbersome to operate, but also harmful to the environment and operators, and are also difficult to apply to industrial production. In addition, although the bioconversion method for preparing oxymorcozy has certain effects, it still has problems such as long cycle, many impurities, and difficult purification.

[0004] For example, CN103194493A discloses a method for preparing oxymorcozy by using mulberry branch powder as a substrate and Aspergillus niger fermentation, but the process takes a long time from strain activation to the end of fermentation, and the operation is complex, and impurities are easily introduced during the fermentation process, increasing the difficulty of subsequent purification. CN104803830A uses organic solvents such as ethanol and ethyl acetate for multiple extractions and extractions, and combines a decolorization process to prepare high-purity oxymorcozy, but this method uses a large amount of solvent, which is not friendly to the environment and personnel, and the process flow is also relatively complex. CN114836482A prepares oxymorcozy by enzymatic hydrolysis of mulbarin A using β-glucanase, and the conversion rate is better than that of cellulase and xylanase, but the conversion rate is less than 96%, which limits its practical application.

[0005] On the other hand, oxidized resveratrol has poor solubility and low transdermal penetration, limiting its bioavailability and application in cosmetics. Microencapsulation technology is an effective means to improve the stability, solubility, and bioavailability of active ingredients, but current research on the microencapsulation of oxidized resveratrol is still relatively limited. Existing related patents, such as CN200920317642.9, propose resveratrol microcapsules using β-cyclodextrin and gum arabic as wall materials; CN201110116845.3 reports a method for preparing sustained-release microspheres of stilbene compounds by encapsulating genipin-crosslinked chitosan, which improves the stability of the compounds and achieves sustained release, but the encapsulation rate is only about 30%; CN201110142129.2 prepares water-soluble stilbene prodrugs through chemical modification, which improves water solubility and stability, but the process is complex and difficult to operate. CN104689771A uses β-cyclodextrin, chitosan, gum arabic, and maltodextrin as wall materials and microencapsulation process to prepare an oxidized resveratrol microcapsule product with high encapsulation efficiency, high sample loading capacity, good water solubility, and good stability. However, it still has not effectively solved problems such as stability and skin permeability.

[0006] Therefore, there is an urgent need to develop a mulberry root bark extract that has high extraction and conversion efficiency, is environmentally friendly, suitable for large-scale production, and can effectively improve the solubility, stability and transdermal properties of oxidized resveratrol. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing mulberry root bark extract that has a high conversion rate, is environmentally friendly, and is suitable for industrial production.

[0008] Another object of the present invention is to provide a high-purity oxidized resveratrol extract prepared by the above method that is almost free of morin A.

[0009] Another object of the present invention is to provide a whitening and soothing composition comprising the above-mentioned extract, which has the characteristics of high encapsulation rate, high transdermal rate, and high stability.

[0010] The present invention also aims to provide a method for preparing the above-mentioned whitening and soothing composition and its application in the preparation of cosmetics.

[0011] To achieve the above objectives, the present invention adopts the following technical solution:

[0012] In a first aspect, the present invention provides the application of mulberry root bark extract in the preparation of cosmetics for whitening and soothing, wherein the mulberry root bark extract contains oxidized resveratrol and contains almost no morin A.

[0013] Furthermore, the mass percentage of oxidized resveratrol in the mulberry root bark extract is not less than 99%.

[0014] Furthermore, the mass percentage of morin A in the mulberry root bark extract is no higher than 1%.

[0015] This invention provides a method for preparing mulberry root bark extract, comprising the following steps:

[0016] (a) Crush the mulberry root bark raw material, extract it, adjust the pH value of the extract, and concentrate it to obtain a concentrated solution;

[0017] (b) The concentrate is separated by a macroporous resin;

[0018] (c) Enzymatic hydrolysis reaction is carried out using a specific enzyme preparation under suitable temperature and pH conditions;

[0019] (d) The enzymatically hydrolyzed product was dissolved, decolorized, and allowed to stand to obtain an extract rich in oxidized resveratrol.

[0020] The enzyme preparation is a compound reagent containing β-glucosidase, rhamnosidase, and α-glucosidase.

[0021] In some embodiments, the composite reagent of the present invention further comprises diatomaceous earth.

[0022] In some embodiments, the mass percentages of the composite reagent β-glucosidase, rhamnosidase, α-glucosidase, and diatomaceous earth are 2:2:2:10.

[0023] In some embodiments, the mass percentages of the composite reagent β-glucosidase, rhamnosidase, α-glucosidase, and diatomaceous earth are 3:2:1:10.

[0024] On the other hand, the present invention provides the application of the above-mentioned mulberry root bark extract in the preparation of cosmetics for whitening and soothing. The present invention provides a whitening and soothing composition with oxidized resveratrol as the main active ingredient, almost free of morin A, and also containing α-cyclodextrin, carboxymethyl cellulose, hydroxypropyl-β-cyclodextrin, chitosan, and vitamin E.

[0025] In some embodiments, the whitening and soothing composition of the present invention uses oxidized resveratrol as the main active ingredient, contains almost no morin A, and also contains α-cyclodextrin, carboxymethyl cellulose, hydroxypropyl-β-cyclodextrin, chitosan, and epigallocatechin gallate. Preferably, the mass percentage of epigallocatechin gallate in the composition is 0.5%-1%.

[0026] In some embodiments, the whitening and soothing composition of the present invention is configured in the form of microcapsules, preferably, the encapsulation rate of oxidized resveratrol is not less than 99%, and its 24-hour transdermal rate is not less than 50%.

[0027] The beneficial effects of this invention are:

[0028] High conversion efficiency and selectivity: By specifically selecting and scientifically formulating compound enzymes and using readily available diatomaceous earth as a fixative for the enzyme reaction, the enzymatic hydrolysis process parameters were optimized, achieving a high-efficiency and targeted conversion of morin A to oxidized resveratrol with a conversion rate of over 99%, thus solving the problem of low raw material utilization.

[0029] Green and efficient: The method of this invention avoids the use of large amounts of harmful organic solvents and complex chromatographic separation columns. The process is simple, the conditions are mild, and the environment is friendly, making it more suitable for large-scale industrial production.

[0030] High encapsulation rate and high transdermal penetration rate: This invention is the first to combine high-purity oxidized resveratrol extract with a specific microencapsulation wall material system (a compound of α-cyclodextrin, carboxymethyl cellulose, hydroxypropyl-β-cyclodextrin, chitosan and epigallocatechin gallate) and process (the stability of the inclusion complex is improved through homogenization, freeze-drying and other technologies). This not only achieves a high encapsulation rate of nearly 100%, but also unexpectedly makes the 24-hour transdermal penetration rate of oxidized resveratrol reach more than 50%, which greatly improves its bioavailability and the whitening and soothing effects of the final product. This is something that has not been achieved by existing technologies.

[0031] Good product stability: The microencapsulation form effectively protects oxidized resveratrol from the effects of light, heat and other factors, improving the product's stability in cosmetic systems.

[0032] Good market application prospects: The extract of this invention can be widely used in various forms of cosmetics such as solids, liquids, creams, and ointments. Attached Figure Description

[0033] Figure 1 The image shows the HPLC chromatogram of the products after the enzymatic hydrolysis reaction. The chromatographic peaks with retention times of 16.072 min and 25.006 min represent morin A and oxidized resveratrol, respectively.

[0034] Figure 2 The effect of each test substance on TNF-α expression (n=3).

[0035] Figure 3 The effect of each test substance on IL-1α expression (n=3).

[0036] Figure 4 The effect of each test substance on IL-1β expression (n=3).

[0037] Figure 5 The effect of each group of test substances on melanin production (n=3). Detailed Implementation

[0038] The present invention will be further described in conjunction with specific embodiments, but this does not limit the scope of the invention.

[0039] Unless otherwise specified, the methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents required in the following examples are commercially available.

[0040] Example 1: Preparation of Mulberry Root Bark Extract 1

[0041] (a) The dried mulberry roots were crushed to 10-20 mesh and extracted twice with pure water at 80℃ (material-liquid ratio 1:10, 1h each time). The pH of the extract was adjusted to 5.0 (citric acid adjustment) to prevent oxidation of the components. The filtrates were combined and concentrated to 1 / 5 volume. The content of morin A was found to be about 20% (on a dry basis) by HPLC.

[0042] (b) Using the concentrate from the previous step, load AB-8 macroporous resin at a rate of 6 BV (1 BV = 1 column volume). First wash with 3 BV of pure water, then elute with 20% ethanol for 2 BV, and finally elute with 60% ethanol for 3 BV. Collect the eluent and concentrate it under vacuum at 60°C to 1 / 4 volume. HPLC analysis showed that the content of morin A was approximately 60% (on a dry basis).

[0043] (c) Using the concentrate from the previous step, load NM400 polymer resin at a volume of 3 BV (1 BV = 1 column volume). First wash 3 BV with 40% ethanol, then elute 3 BV with 60% ethanol. Collect the eluent, concentrate and dry it under vacuum at 60°C, and the content of morin A was found to be about 95% (on a dry basis) by HPLC.

[0044] (d) Place 10g of the raw material from the previous step (containing approximately 95% morin A) into a reaction vessel, and add 1000ml of pure water.

[0045] Adjust the pH to 5.0 using 0.1M acetate-sodium acetate buffer. Add 0.6g of the complex enzyme (containing 0.3g β-glucosidase, 0.2g rhamnosidase, and 0.1g α-glucosidase), along with 1g of diatomaceous earth as a surface fixative to enhance enzyme stability and substrate contact rate. Maintain the temperature at 45℃±1℃, shake at 150rpm, and allow the reaction time to 4-6 hours. After the reaction, raise the temperature to 80℃ and inactivate the enzyme for 30 minutes. Cool to room temperature, let stand for 24 hours, filter, collect the unfiltered material (filter cake), and dry to obtain crude resveratrol oxide; (e) dissolve the crude resveratrol oxide obtained in the previous step in 4 times the volume of 70% ethanol, heat to 60°C, stir until completely dissolved, then add 10% solid content activated carbon, stir to decolorize for 1 hour, filter, cool the filtrate to 4°C, let stand for 24 hours to crystallize, filter and dry to obtain high-purity resveratrol oxide product, which, according to HPLC analysis, contains approximately 99.6% resveratrol oxide and approximately 0.4% morin A. Figure 1 As shown, the peak with a retention time of 16.072 min represents morin A, and the peak with a retention time of 25.006 min represents oxidized resveratrol. The enzymatic hydrolysis reaction can convert morin A to oxidized resveratrol with extremely high efficiency, converting almost all morin A in the material before the reaction into B, achieving a conversion rate of 99.58%. [Conversion rate = (1 - amount of morin A in the final product after enzymatic hydrolysis / amount of morin A before enzymatic hydrolysis) × 100%]

[0046] Example 2: Preparation of Mulberry Root Bark Extract 2

[0047] (a) Crush dried mulberry roots into 10-20 mesh and extract twice with pure water at 80℃ (material-to-liquid ratio 1:10, 1 hour each time). Adjust the pH of the extract to 5.0 (adjusted with citric acid) to prevent oxidation of components. Combine the filtrates and concentrate to 1 / 5 volume.

[0048] HPLC analysis revealed that the content of morin A was approximately 20% (on a dry basis).

[0049] (b) Using the concentrate from the previous step, load AB-8 macroporous resin at a rate of 6 BV (1 BV = 1 column volume). First use pure...

[0050] Wash with water for 3 BV, then elute with 20% ethanol for 2 BV, and finally elute with 60% ethanol for 3 BV. Collect the eluent and sterilize under vacuum at 60°C.

[0051] Concentrated to 1 / 4 volume, HPLC analysis revealed that the morin A content was approximately 60% (on a dry basis).

[0052] (c) Using the concentrate from the previous step, load NM400 polymer resin at a rate of 3 BV (1 BV = 1 column volume). First, wash the 3 BV with 40% ethanol, then elute the 3 BV with 60% ethanol. Collect the eluent, concentrate and dry it under vacuum at 60°C, and determine the HPLC value.

[0053] The content of morin A is approximately 95% (on a dry basis);

[0054] (d) Place 10g of the raw material from the previous step (containing approximately 95% morin A) into a reaction vessel, and add 1000ml of pure water.

[0055] Adjust the pH to 5.0 using 0.1M acetate-sodium acetate buffer. Add 0.6g of the complex enzyme (containing 0.2g β-glucosidase, 0.2g rhamnosidase, and 0.2g α-glucosidase), along with 1g of diatomaceous earth as a surface fixative to enhance enzyme stability and substrate contact rate. Maintain the temperature at 45℃±1℃, shake at 150rpm, and react for 4-6 hours. After the reaction, raise the temperature to 80°C and inactivate the enzyme for 30 minutes. Cool.

[0056] After cooling to room temperature and standing for 24 hours, filter, collect the unfiltered material (filter cake), and dry to obtain crude resveratrol oxide; (e) dissolve the crude resveratrol oxide obtained in the previous step in 4 times its volume of 70% ethanol, heat to 60°C, and stir until...

[0057] Completely dissolve the precipitate, then add activated carbon with a solid content of 10%, stir and decolorize for 1 hour, and filter. Cool the filtrate to 4°C, let it stand for 24 hours to crystallize, filter and dry to obtain a high-purity oxidized resveratrol product. HPLC analysis showed that the oxidized resveratrol content was approximately 98%, and the morin A content was approximately 2%.

[0058] The enzymatic hydrolysis reaction can convert morin A into oxidized resveratrol with a conversion rate of approximately 97.89%.

[0059] Example 3: Preparation of mulberry root bark extract 3;

[0060] (a) Crush dried mulberry roots into 10-20 mesh and extract twice with pure water at 80℃ (material-to-liquid ratio 1:10, 1 hour each time). Adjust the pH of the extract to 5.0 (adjusted with citric acid) to prevent oxidation of components. Combine the filtrates and concentrate to 1 / 5 volume.

[0061] HPLC analysis revealed that the content of morin A was approximately 20% (on a dry basis).

[0062] (b) Using the concentrate from the previous step, load AB-8 macroporous resin at a rate of 6 BV (1 BV = 1 column volume). First use pure...

[0063] Wash with water for 3 BV, then elute with 20% ethanol for 2 BV, and finally elute with 60% ethanol for 3 BV. Collect the eluent and sterilize under vacuum at 60°C.

[0064] Concentrated to 1 / 4 volume, HPLC analysis revealed that the morin A content was approximately 60% (on a dry basis).

[0065] (c) Using the concentrate from the previous step, load NM400 polymer resin at a rate of 3 BV (1 BV = 1 column volume). Wash the 3 BV with 40% ethanol, then elute the remaining 3 BV with 60% ethanol. Collect the eluent, concentrate and dry it under vacuum at 60°C, and determine the concentration by HPLC.

[0066] The content of morin A is approximately 95% (on a dry basis);

[0067] (d) Place 10g of the raw material from the previous step (containing approximately 95% morin A) into a reaction vessel, and add 1000ml of pure water.

[0068] The pH was adjusted to 5.0 using 0.1M acetate-sodium acetate buffer. 0.6g of β-glucosidase was added, along with 1g of diatomaceous earth as a surface fixative to enhance enzyme stability and substrate contact. The temperature was controlled at 45℃±1℃, with a shaking frequency of 150rpm, and the reaction time was 4-6 hours. After the reaction, the temperature was raised to 80℃ for 30 minutes to inactivate the enzyme. The mixture was cooled to room temperature, allowed to stand for 24 hours, filtered, and the unfiltered material (filter cake) was collected and dried to obtain crude oxidized resveratrol.

[0069] (e) Dissolve the crude resveratrol oxide obtained in the previous step in 4 times its volume of 70% ethanol, heat to 60°C, stir until completely dissolved, then add activated carbon with a solid content of 10%, stir and decolorize for 1 hour, and filter. Cool the filtrate to 4°C, let it stand for 24 hours to crystallize, filter and dry to obtain a high-purity resveratrol oxide product. HPLC analysis shows that the resveratrol oxide content is approximately 97%, and the morin A content is approximately 3%.

[0070] The enzymatic hydrolysis reaction can convert morin A into oxidized resveratrol with a conversion rate of approximately 96.84%.

[0071] Surprisingly, the composite reagent of β-glucosidase, rhamnosidase, α-glucosidase and diatomaceous earth in a mass percentage ratio of 3:2:1:10 exhibits an extremely excellent ability to convert morin A, with an enzymatic hydrolysis rate of over 99%.

[0072] Example 4: Preparation of Whitening and Soothing Microcapsules 1

[0073] 1. Preparation of encapsulation material solution: 20g α-cyclodextrin, 20g carboxymethyl cellulose, 30g hydroxypropyl-β-cyclodextrin, and 20g chitosan were added to 1000mL of deionized water and stirred in a 50℃ water bath until completely dissolved;

[0074] 2. Preparation of inclusion complex suspension: Dissolve 9.5g of high-purity oxidized resveratrol (mulberry root bark extract 1 prepared in Example 1) and 0.5g of epigallocatechin gallate (EGCG) in 50mL of 95% ethanol, stir until completely dissolved, and slowly drop the solution into the inclusion material solution while maintaining a constant temperature of 50℃ and stirring for 3 hours (300r / min) until the inclusion complex suspension is formed;

[0075] 3. The inclusion complex suspension was transferred to a high-pressure homogenizer and homogenized three times at a pressure of 1100 BAR to further improve the stability of the inclusion complex.

[0076] 4. The inclusion complex suspension, which has been homogenized under high pressure, is concentrated under vacuum (0.1 MPa) at 60°C until the solid content is ≥20%.

[0077] 5. Transfer the concentrated solution to a freeze dryer and freeze-dry at -50℃ and a vacuum of 10Pa to obtain oxidized resveratrol microcapsules. The product is a white powder, odorless, easily soluble in water, with an encapsulation rate of 99.2%. The encapsulation rate is calculated as follows: The oxidized resveratrol content ratio in the microcapsules is determined using HPLC. The microcapsules are dissolved in pure water at room temperature with a mass concentration of 5%. The initial total oxidized resveratrol amount M is calculated by multiplying the total added mass by the content. After complete dissolution, the solution is centrifuged at 10,000 rpm for 30 minutes. The total mass of the centrifuged liquid is weighed, and the oxidized resveratrol content in the liquid is determined using HPLC to calculate the total encapsulated oxidized resveratrol amount A. The encapsulation rate of the oxidized resveratrol microcapsules is then calculated using the following formula:

[0078] Inclusion rate (%) = [Total amount of oxidized resveratrol included A / Initial total amount of oxidized resveratrol M] × 100%

[0079] Example 5: Preparation of Whitening and Soothing Microcapsules 2

[0080] 1. Preparation of encapsulation material solution: 20g α-cyclodextrin, 20g carboxymethyl cellulose, 30g hydroxypropyl-β-cyclodextrin, and 20g chitosan were added to 1000mL of deionized water and stirred in a 50℃ water bath until completely dissolved;

[0081] 2. Preparation of inclusion complex suspension: Dissolve 9.5g of high-purity oxidized resveratrol (mulberry root bark extract 1 prepared in Example 1) and 0.5g of vitamin E in 50mL of 95% ethanol, stir until completely dissolved, and slowly drop the solution into the inclusion material solution while maintaining a constant temperature of 50℃ and stirring for 3 hours (300r / min) until an inclusion complex suspension is formed;

[0082] 3. The inclusion complex suspension was transferred to a high-pressure homogenizer and homogenized three times at a pressure of 1100 BAR to further improve the stability of the inclusion complex.

[0083] 4. The inclusion complex suspension, which has been homogenized under high pressure, is concentrated under vacuum (0.1 MPa) at 60°C until the solid content is ≥20%.

[0084] 5. The concentrated solution was transferred to a freeze dryer and freeze-dried at -50°C and a vacuum of 10 Pa to obtain oxidized resveratrol microcapsules. The microcapsules were a white, odorless powder, readily soluble in water, with an encapsulation rate of 92.7%.

[0085] Example 6: Preparation of Whitening and Soothing Microcapsules 3

[0086] 1. Preparation of encapsulation material solution: 10g α-cyclodextrin, 10g carboxymethyl cellulose, 40g hydroxypropyl-β-cyclodextrin, and 30g chitosan were added to 1000mL of deionized water and stirred in a 50℃ water bath until completely dissolved;

[0087] 2. Preparation of inclusion complex suspension: 9.5g of high-purity oxidized resveratrol (mulberry root bark extract 1 prepared in Example 1) and 0.5g of EGCG were dissolved in 50mL of 95% ethanol and stirred until completely dissolved. The solution was then slowly added dropwise to the inclusion material solution while maintaining a constant temperature of 50°C and stirring for 3 hours (300r / min) until an inclusion complex suspension was formed.

[0088] 3. The inclusion complex suspension was transferred to a high-pressure homogenizer and homogenized three times at a pressure of 1100 BAR to further improve the stability of the inclusion complex.

[0089] 4. The inclusion complex suspension, which has been homogenized under high pressure, is concentrated under vacuum (0.1 MPa) at 60°C until the solid content is ≥20%.

[0090] 5. The concentrated solution was transferred to a freeze dryer and freeze-dried at -50°C and a vacuum of 10 Pa to obtain oxidized resveratrol microcapsules. The microcapsules were a white, odorless powder, readily soluble in water, with an encapsulation efficiency of 83.6%.

[0091] Example of effectiveness: Microcapsule performance testing

[0092] Stability tests, transdermal absorption tests, anti-inflammatory and soothing efficacy tests, and whitening efficacy tests were conducted on whitening and soothing microcapsules 1, 2, and 3.

[0093] Experiment 1: Stability Test

[0094] 1. Experimental Objective and Principle

[0095] By simulating the extreme condition of long-term heating, the changes in the mass characteristics of whitening and soothing microcapsules 1, 2, and 3 (5% mass concentration) aqueous solutions over time were tested to explore the thermal stability of each group of test substances under heating conditions.

[0096] 2. Experimental Procedure

[0097] Take 90g of each of the following whitening and soothing microcapsules (1, 2, and 3) at a concentration of 5% (w / v): dissolve them separately in approximately 1800ml of water. Add 1% preservative (0.8% hexanediol + 1% caprylyl glycol) and shake well. Dispense the prepared 5% aqueous solution of the test substance into 30ml plastic bottles for each of the following testing factors: room temperature and 50℃ heating. Label each bottle accordingly and record the changes over 30 days.

[0098] 2.4 Indicator Determination

[0099] The determination of product content changes is shown in Table 1.

[0100] Table 1. Judgment of Content Changes

[0101]

[0102] 2.5 Results Analysis

[0103] As shown in Table 2, after heating at 50℃ for 30 days, the content of whitening and soothing microcapsules 1 decreased by approximately 2% compared to the unheated sample solution, while the solution remained clear and showed no significant change in appearance or color. The content of whitening and soothing microcapsules 2 decreased to some extent, by approximately 8%, and the solution became slightly turbid, with the color tending to darken, ending in a deep yellow. Compared to the unheated sample solution, the content of whitening and soothing microcapsules 3 decreased significantly, the solution became turbid with precipitation, and the color darkened significantly, ending in brown. In conclusion, by comparing the solution states of the three experimental groups after 30 days of heating, the solution of whitening and soothing microcapsules 1 is more stable and more applicable.

[0104] Table 2. Statistical table of changes in each experimental group over 30 days.

[0105]

[0106] Experiment 2: Transdermal Transfusion Rate Test

[0107] 1. Experimental Objective

[0108] The transdermal absorption efficiency of whitening and soothing microcapsule 1, whitening and soothing microcapsule 2, and whitening and soothing microcapsule 3 solutions was evaluated through in vitro transdermal absorption experiments, and the feasibility of their transdermal absorption was studied and compared.

[0109] 2. Experimental Procedure

[0110] Skin was harvested from the back of a healthy rat, and subcutaneous adipose tissue was removed. The skin was washed with physiological saline to ensure a clean surface. The skin was then fixed between the donor and recipient chambers of the Franz diffusion cell, ensuring a tight skin fit.

[0111] Phosphate-buffered saline (PBS, pH 7.4) and ethanol were mixed at a ratio of 7:3 (v / v) to prepare the receptor phase. The receptor phase was then added to the receptor chamber of the diffusion cell, ensuring complete skin immersion.

[0112] To ensure the experiment proceeded smoothly and to maintain consistent concentrations of active ingredients across all experimental groups, the final concentrations of Whitening and Soothing Microcapsules 1, 2, and 3 were all set at 500 μg / ml.

[0113] The diffusion cell was placed in a 37°C constant-temperature water bath to maintain a constant temperature for the acceptor phase. A certain amount of acceptor phase solution was taken from the acceptor chamber at time points of 0, 2h, 4h, 8h, 12h, 16h, 20h, and 24h. The absorbance was measured at 326nm using a UV-Vis spectrophotometer, and the concentration of the test substance was calculated. After each sampling, an equal amount of fresh acceptor phase was added to maintain a constant volume of the acceptor phase.

[0114] After the experiment, rat skin was collected. The skin was thoroughly washed with physiological saline to remove any residual drug. The skin was then minced, and the test substance was extracted from the skin using methanol. The concentration of the test substance in the skin was determined using a UV-Vis spectrophotometer. 3. Data Analysis

[0115] Calculate the cumulative permeation (Qn) and transdermal rate of the test substance.

[0116]

[0117] In the formula:

[0118] Cn—The concentration of the test substance in the acceptor phase at the nth sampling;

[0119] V—Acceptor phase volume;

[0120] Vi – Volume of each sample

[0121] Ci — the concentration of the test substance in the acceptor phase during each sampling.

[0122] Transdermal penetration rate (%) = [Cumulative penetration amount (Qn) ÷ Initial addition amount] × 100%

[0123] 5. Results Analysis

[0124] The data show that the transdermal permeability of each test group increased over time, with a faster rate of increase in the initial stage (0–16 hours) and a slower rate of increase in the later stage (16–24 hours). The transdermal permeability of whitening and soothing microcapsules 1, 2, and 3 were 52.1%, 43.8%, and 28.3%, respectively. Among them, whitening and soothing microcapsules 1 showed a significantly improved transdermal permeability, thus enhancing the bioavailability of topical skin administration.

[0125] Table 3 Comparison of transdermal permeability of test substances in each group (mean, n=3)

[0126]

[0127] Experiment 3: Determination of Anti-inflammatory and Soothing Efficacy

[0128] 1. Experimental Objective and Principle

[0129] Lipopolysaccharide (LPS) is a lipid and polysaccharide complex, mainly extracted from the cell walls of Gram-negative bacteria, and possesses strong immunogenicity. LPS can stimulate skin keratinocytes to secrete pro-inflammatory cytokines (TNF-α, IL-1β, IL-1α, etc.). This test uses the expression levels of TNF-α, IL-1α, and IL-1β as the evaluation standard to compare the anti-inflammatory and soothing abilities of whitening and soothing microcapsules 1, 2, and 3.

[0130] 2. ELIAS method for measuring inflammatory factors

[0131] HaCaT cells were used at a rate of 1×10 5 Cells were seeded at a density of [number] cells / mL in 6-well plates, with 1 mL of complete culture medium (DMEM high-glucose medium + 10% FBS + 1% penicillin antibiotics) added to each well. The plates were incubated overnight at 37°C with 5% CO2 to allow cell attachment. After cell attachment, different treatment groups of drugs were added according to the experimental design, and the plates were cultured for another 24 hours. After culture, the cell culture supernatant was collected, centrifuged at 2000×g for 10 minutes to remove cell debris, and the supernatant was aliquoted and stored at -20°C for later use.

[0132] Thaw the frozen supernatant at room temperature, mix thoroughly, and set aside. Determine the concentrations of TNF-α, IL-1α, and IL-1β in the supernatant according to the instructions of each ELIAS kit (TNF-α, IL-1α, IL-1β). Measure the absorbance at a wavelength of 450 nm.

[0133] 3. Repeatability

[0134] The absolute difference between at least three independent measurements obtained under repeatability conditions shall not exceed 20% of the arithmetic mean.

[0135] 4. Data Processing

[0136] All experimental data were analyzed using t-tests, using the mean and standard deviation of three measurements. If the p-value in the t-test was less than 0.05, statistical significance was considered. #, p < 0.05; ##, p < 0.01; ###, p < 0.001 (calculated compared to the control group). *, p < 0.05; **, p < 0.01 (calculated compared to the induced group).

[0137] 5. Results Analysis

[0138] like Figures 2-4 As shown, compared with the blank group, the expression of inflammatory factors TNF-α, IL-1α, and IL-1β was significantly increased in the LPS-induced group (P < 0.001); compared with the LPS-induced group, the expression of TNF-α, IL-1α, and IL-1β was significantly reduced in the whitening and soothing microcapsule 1, whitening and soothing microcapsule 2, and whitening and soothing microcapsule 3 experimental groups after treatment (P < 0.05). Considering the inhibitory effect of each test group on the expression level of inflammatory factors, whitening and soothing microcapsule 1 at the same test concentration was superior to other experimental groups, effectively reducing the expression of inflammatory factors and exhibiting better anti-inflammatory and soothing effects.

[0139] Experiment 4: Whitening Efficacy Test

[0140] 1. Experimental Objective and Principle

[0141] Tyrosinase catalyzes the conversion of tyrosine into dopa, which in turn forms melanin. This test will measure the melanin in cells to directly evaluate the whitening efficacy of Whitening and Soothing Microcapsules 1, 2, and 3.

[0142] 2. Experimental Procedure

[0143] (I) Cell Culture

[0144] 3.0×10 5B16 cells were seeded in 24-well plates and cultured in DMEM medium for 24 hours. Cells were pretreated with 50 nM α-melanocyte-stimulating hormone (α-MSH) for 24 hours. They were then treated with 2 μM hydrogen peroxide (H2O2) for 1 hour to induce oxidative stress, followed by incubation in fresh DMEM for 6 hours. The oxidative stress-induced B16 cells were then incubated with 10 μg / ml and 12.5 μg / ml of the test substance (dissolved in DMEM) for 48 hours, respectively.

[0145] (II) Measurement of melanin

[0146] To determine melanin content, B16 cells were first washed with phosphate-buffered saline (PBS) at pH 7.4, then lysed with 100 μl of lysis buffer containing 1N sodium hydroxide (NaOH), 1% Triton X-100, and 1 mM benzyl sulfonyl fluoride (PMSF), and incubated at 60°C for 1 hour. The absorbance of the lysis buffer was measured at 405 nm. The relative percentage of melanin production in each experimental group was calculated with the melanin production of untreated cells as 100%.

[0147] 5.4 Repeatability

[0148] The absolute difference between at least three independent measurements obtained under repeatability conditions shall not exceed 20% of the arithmetic mean.

[0149] 5.5 Data Processing

[0150] All experimental data were analyzed using Student's t-tests, using the mean and standard deviation of three measurements. A p-value less than 0.05 was considered statistically significant. Compared to the control group, # P < 0.05 ## P < 0.01, ### P < 0.001; compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001.

[0151] 5.6 Results Analysis

[0152] like Figure 5 As shown, compared with the blank group, the H2O2-induced group significantly enhanced melanin production (P < 0.001). Compared with the induction group, whitening and soothing microcapsules 1, 2, and 3 at doses of 10 and 12.5 μg / mL showed a dose-dependent effect on melanin production, with significant inhibitory effects (P < 0.001). At the same active ingredient concentration, whitening and soothing microcapsule 1 showed a more pronounced inhibitory effect on melanin production in cells compared with whitening and soothing microcapsules 2 and 3.

[0153] In summary, this invention successfully provides a green and efficient conversion pathway from mulberry bark waste to high-value cosmetic raw materials, and successfully solves the core bottleneck problem in the application of oxidized resveratrol, demonstrating enormous market application potential.

[0154] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. 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. Such 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. Non-essential improvements, adjustments or substitutions made by those skilled in the art based on the content of this specification are all within the scope of protection claimed by the present invention.

Claims

1. The application of mulberry root bark extract in the preparation of cosmetics for whitening and soothing, characterized in that, The mulberry root bark extract contains oxidized resveratrol and almost no morin A.

2. The application according to claim 1, characterized in that, The mass percentage of oxidized resveratrol in the mulberry root bark extract is not less than 99%.

3. The application according to claim 1 or 2, characterized in that, The mass percentage of morin A in the mulberry root bark extract is no higher than 1%.

4. A method for preparing mulberry root bark extract, characterized in that, The preparation method includes the following steps: (a) Crush the mulberry root bark raw material, extract it, adjust the pH value of the extract, and concentrate it to obtain a concentrated solution; (b) The concentrate is separated by a macroporous resin; (c) Enzymatic hydrolysis reaction is carried out using a specific enzyme preparation under suitable temperature and pH conditions; (d) The enzymatically hydrolyzed product was dissolved, decolorized, and allowed to stand to obtain an extract rich in oxidized resveratrol. The enzyme preparation is a compound reagent containing β-glucosidase, rhamnosidase, and α-glucosidase.

5. The preparation method according to claim 4, characterized in that, The composite reagent also contains diatomaceous earth.

6. The preparation method according to claim 5, characterized in that, The mass percentages of the composite reagent β-glucosidase, rhamnosidase, α-glucosidase and diatomaceous earth are 3:2:1:10 or 2:2:2:

10.

7. A whitening and soothing composition, characterized in that, The composition contains oxidized resveratrol, contains almost no morin A, and the remainder are cosmetically acceptable excipients.

8. The whitening and soothing composition according to claim 7, characterized in that, The composition has oxidized resveratrol as the main active ingredient, contains almost no morin A, and also contains α-cyclodextrin, carboxymethyl cellulose, hydroxypropyl-β-cyclodextrin, chitosan and epigallocatechin gallate.

9. The whitening and soothing composition according to claim 7, characterized in that, The composition has oxidized resveratrol as the main active ingredient, contains almost no morin A, and also contains α-cyclodextrin, carboxymethyl cellulose, hydroxypropyl-β-cyclodextrin, chitosan and vitamin E.

10. The whitening and soothing composition according to any one of claims 7 to 9, characterized in that, The composition is formulated as a microcapsule.

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