Preparation method of red clover extract with low toxicity and high activity

Abstract

The invention belongs to the technical field of plant extracts, and particularly discloses a preparation method of a low-toxicity and high-activity red clover extract, which comprises the following steps: crushing dried whole red clover into raw material powder; adding an ethanol aqueous solution into the raw material powder, and concentrating to obtain a crude extract; adding a compound enzyme preparation into the crude extract, and carrying out enzymolysis; carrying out enzyme deactivation on the enzymatic hydrolysate, and inoculating a composite probiotic liquid; meanwhile, plant-derived exosomes are added, and fermentation is performed; and after fermentation is finished, adding a protein solution, adsorbing through macroporous resin, and concentrating and drying to obtain the red clover extract. According to the preparation method of the low-toxicity and high-activity red clover extract, efficient bioconversion from isoflavone glycoside to aglycone is realized through a synergistic process of alcohol extraction, probiotic fermentation, protein combination, enzymolysis and plant exosome loading, macromolecular impurities are removed, toxicity is reduced, and the yield of the red clover extract is increased. The plant exosome is used for improving the stability and skin permeability of active ingredients, and finally the functional raw material suitable for high-end skin care products is obtained.

Description

Technical Field

[0001] This invention relates to the field of plant extract technology, and in particular to a method for preparing a low-toxicity, high-activity red clover extract. Background Technology

[0002] Red clover is rich in isoflavones and polyphenols, possessing excellent antioxidant, anti-inflammatory, and skin microecological regulating effects. However, current extraction techniques mostly employ single solvent reflux or simple fermentation, which have the following technical drawbacks: 1. Insufficient release of bound state: Some isoflavones in red clover are tightly bound to cell wall polysaccharides and proteins, making them difficult to release by conventional alcohol extraction, resulting in low extraction rate.

[0003] 2. Insufficient activity conversion: Most natural isoflavones are glycosides, which have low skin absorption rates; and the residual protein impurities in crude extracts can easily cause skin allergies and have high toxicity.

[0004] 3. Delivery efficiency bottleneck: Active ingredients are easily degraded by skin metabolic enzymes when added directly, making it difficult to achieve an effective concentration.

[0005] Plant exosomes are nanoscale lipid bilayer-encapsulated structures secreted by plant cells, rich in plant-specific active ingredients, and possess advantages such as wide availability, simple preparation, low immunogenicity, and efficient transdermal delivery. Therefore, there is an urgent need for an integrated preparation process that combines plant exosomes to achieve efficient release of bound components, conversion of active structures, and low-irritation delivery. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing a low-toxicity and high-activity red clover extract. Through a synergistic process of "alcohol extraction-probiotic fermentation-protein binding-enzymatic hydrolysis-plant exosome loading", the method achieves efficient bioconversion of isoflavone glycosides to aglycones, removes macromolecular impurities, reduces toxicity, and utilizes plant exosomes to improve the stability and skin permeability of active ingredients, ultimately obtaining a functional raw material suitable for high-end skin care products.

[0007] To achieve the above objectives, the present invention provides a method for preparing a low-toxicity, high-activity red clover extract, comprising the following steps: Step 1, Pretreatment: Crush the dried whole red clover into 40-60 mesh, wash with deionized water, and dry at low temperature to obtain raw material powder; Step 2, multi-stage alcohol extraction: Add an aqueous ethanol solution to the raw material powder, reflux and extract twice at 50-60℃, each time for 1.5-2.5 hours, combine the filtrates, and concentrate to obtain crude extract; Step 3, compound enzymatic hydrolysis: Add compound enzyme preparation to crude extract, and the enzymatic hydrolysis time is 3-4 hours; Step 4, Ultrasonic-assisted fermentation: After inactivating the enzymes in the hydrolysate, inoculate it with compound probiotic liquid; at the same time, add plant-derived exosomes and place it in an ultrasonic field for ultrasonic fermentation; Step 5, purification and refining: After fermentation, add protein solution and stir at 40-50℃ for 1-2 hours. Then, adsorb through macroporous resin. First, wash with deionized water to remove impurities, proteins and sugars, then wash with 70% ethanol to remove the target component. After concentration and drying, red clover extract is obtained.

[0008] Preferably, in step 2, a 60%-75% ethanol aqueous solution is added at a material-to-liquid ratio of 1:8-1:12 (g / mL).

[0009] Preferably, in step 3, the compound enzyme preparation includes cellulase and pectinase in a mass ratio of 2:1, and the amount added is 1.5%-2.5% of the mass of the raw material powder.

[0010] Preferably, in step 3, after adding the compound enzyme preparation, the pH is adjusted to 4.5-5.5, and the enzymatic hydrolysis temperature is controlled at 45-55℃.

[0011] Preferably, in step 4, the compound probiotics include plant lactic acid bacteria and brewer's yeast, with an inoculation amount of 5%-8% v / v and a volume ratio of 1:2.

[0012] Preferably, in step 4, the plant-derived exosomes include ginger exosomes, dandelion exosomes, and tremella exosomes, with an addition amount of 0.5%-1% w / v.

[0013] Preferably, in step 4, the ultrasonic power is 200-300W, the frequency is 20kHz, the fermentation temperature is 30-35℃, and the fermentation time is 48-72 hours.

[0014] Preferably, in step 4, the protein is hydrolyzed wheat protein or silk fibroin.

[0015] Preferably, in step 4, the method for preparing plant-derived exosomes is as follows: select fresh plant bodies, purify them by homogenization, differential centrifugation, and sucrose density gradient centrifugation to obtain exosome vesicles.

[0016] Preferably, the differential centrifugation employs a three-stage centrifugation strategy: 3000g, 4℃, 30 minutes to remove large cell debris; 10000g, 4℃, 30 minutes to remove large organelles; and 100000g, 4℃, 90 minutes to collect the exosome precipitate. The sucrose density gradient from bottom to top is 50%, 40%, 30%, 20%, and 10% sucrose solution. Centrifugation is performed at 100000g, 4℃ for 18 hours, and the exosome solution is collected. After dilution with phosphate buffer and ultrafiltration, high-purity exosomes are obtained, which are then lyophilized for later use. The lyophilization protectant is a compound formulation of 5% trehalose and 1mM ascorbic acid to ensure the activity and stability of the exosomes.

[0017] The advantages and beneficial effects of the above-mentioned method for preparing a low-toxicity, high-activity red clover extract are as follows: 1. This invention releases bound components by enzymatic hydrolysis of cell walls, transforms active structures through fermentation, and achieves efficient delivery and toxicity reduction of active ingredients through plant exosomes. At the same time, it enhances the application effect of extracts in skin care products by leveraging the efficacy of plant exosomes themselves.

[0018] 2. This invention utilizes a dual biotransformation process of probiotic fermentation and enzymatic hydrolysis, achieving an isoflavone glycoside conversion rate of over 90%, significantly increasing the content of highly active aglycones. The protein binding step effectively masks the irritant properties of isoflavones, and the fermentation process degrades some potentially sensitizing macromolecules, resulting in a significant improvement in the product's mildness.

[0019] The technical solution of the present invention will be further described in detail below through embodiments. Detailed Implementation

[0020] The technical solution of the present invention will be further described below through embodiments.

[0021] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0022] The following examples are not intended to limit the invention, but are only for illustration. Unless otherwise specified, the experimental methods used in the following examples are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0023] A method for preparing a low-toxicity, high-activity red clover extract includes the following steps: Step 1, Pretreatment: Crush the dried whole red clover into 40-60 mesh, wash with deionized water, and dry at low temperature to obtain raw material powder.

[0024] Step 2, multi-stage alcohol extraction: Add 60%-75% ethanol aqueous solution to the raw material powder at a material-to-liquid ratio of 1:8-1:12 (g / mL), reflux and extract twice at 50-60℃, each time for 1.5-2.5 hours, combine the filtrates, and concentrate to obtain crude extract.

[0025] Step 3, compound enzymatic hydrolysis: Add compound enzyme preparation to crude extract, adjust pH to 4.5-5.5, control enzymatic hydrolysis temperature at 45-55℃, and enzymatic hydrolysis time at 3-4 hours; compound enzyme preparation includes cellulase and pectinase in a mass ratio of 2:1, and the amount added is 1.5%-2.5% of the raw material powder mass.

[0026] Step 4, Ultrasonic-Assisted Fermentation: After inactivating the enzymes in the hydrolysate, inoculate with a compound probiotic solution. The compound probiotics include plant-based lactic acid bacteria and brewer's yeast, with an inoculation amount of 5%-8% v / v and a volume ratio of 1:2. Simultaneously, add plant-derived exosomes, including ginger exosomes, dandelion exosomes, and tremella exosomes, at an addition amount of 0.5%-1% w / v. Place in an ultrasonic field with an ultrasonic power of 200-300W, a frequency of 20kHz, a fermentation temperature of 30-35℃, and a fermentation time of 48-72 hours.

[0027] The preparation method of plant-derived exosomes is as follows: Fresh plant bodies are selected and purified by homogenization, differential centrifugation, and sucrose density gradient centrifugation to obtain exosome vesicles. Differential centrifugation employs a three-stage centrifugation strategy: 3000g, 4℃, 30 minutes to remove large cell debris; 10000g, 4℃, 30 minutes to remove large organelles; and 100000g, 4℃, 90 minutes to collect the exosome precipitate. The sucrose density gradient from bottom to top consists of 50%, 40%, 30%, 20%, and 10% sucrose solutions. Centrifugation is performed at 100000g, 4℃ for 18 hours. The exosome solution is collected, diluted with phosphate buffer, and ultrafiltered to obtain high-purity exosomes. These are then lyophilized for later use. The lyophilization protectant is a compound formulation of 5% trehalose and 1mM ascorbic acid to ensure the activity and stability of the exosomes.

[0028] Step 5, purification and refining: After fermentation, add protein solution, which is hydrolyzed wheat protein or silk fibroin. Stir and react at 40-50℃ for 1-2 hours, then adsorb through macroporous resin. First, wash with deionized water to remove impurities, proteins and sugars, then wash with 70% ethanol to remove the target component. After concentration and drying, red clover extract is obtained.

[0029] Example 1 A method for preparing a low-toxicity, high-activity red clover extract includes the following steps: (1) Pretreatment: Take 10 kg of dried whole red clover, crush it to 50 mesh, rinse it three times with deionized water, and dry it at 60℃ until the moisture content is ≤8% to obtain raw material powder.

[0030] (2) Multi-stage alcohol extraction: Add 70% ethanol aqueous solution to the raw material powder at a material-to-liquid ratio of 1:10 (g / mL), and reflux extract twice at 55℃ for 2 hours each time. Combine the two extraction filtrates and concentrate under reduced pressure at 60℃ and a vacuum of 0.08MPa until there is no ethanol odor to obtain crude extract.

[0031] (3) Compound enzymatic hydrolysis: Add cellulase and pectinase (mass ratio 2:1) to the crude extract, the amount of which is 2% of the mass of the raw material powder. Adjust the pH of the crude extract to 5.0 with citrate-sodium citrate buffer, place it in a constant temperature shaker at 50℃, rotate at 180 r / min, and enzymatically hydrolyze for 3.5 hours.

[0032] (4) Ultrasonic-assisted fermentation: The enzyme hydrolysate was placed in a 95℃ water bath for 15 minutes to inactivate the enzymes. After cooling to 32℃, a mixed bacterial culture with an inoculum of 6% (v / v) (Lactobacillus plantarum LP6:Saccharomyces cerevisiae Y-1=1:2) was inoculated. At the same time, 0.8% (w / v) of Tremella fuciformis exosomes were added. The mixture was placed in an ultrasonic reactor with an ultrasonic power of 250W and a frequency of 20kHz, and intermittent ultrasonication (3s ultrasonication, 2s pause). Fermentation was carried out at 32℃ for 60 hours, with stirring every 12 hours to ensure that the exosomes and fermentation broth were fully mixed.

[0033] The preparation method of Tremella fuciformis exosomes is as follows: Fresh Tremella fuciformis fruiting bodies are selected, homogenized, purified by differential centrifugation, and sucrose density gradient centrifugation to obtain exosome vesicles. Differential centrifugation employs a three-stage centrifugation strategy: 3000g, 4℃, 30 minutes to remove large cell debris; 10000g, 4℃, 30 minutes to remove large organelles; and 100000g, 4℃, 90 minutes to collect the exosome precipitate. The sucrose density gradient from bottom to top consists of 50%, 40%, 30%, 20%, and 10% sucrose solutions. Centrifugation is performed at 100000g, 4℃ for 18 hours. The exosome solution is collected, diluted with phosphate buffer, and ultrafiltered to obtain high-purity exosomes. These are then lyophilized for later use. The lyophilization protectant is a compound formulation of 5% trehalose and 1mM ascorbic acid to ensure the activity and stability of the exosomes.

[0034] (5) Purification and refining: After fermentation, add hydrolyzed wheat protein solution, stir and react at 45°C for 1.5 hours, then adsorb through macroporous resin, first wash with deionized water to remove impurity proteins and sugars, then wash with 70% ethanol to remove the target component, concentrate and dry to obtain red clover extract.

[0035] Example 2 A method for preparing a low-toxicity, high-activity red clover extract includes the following steps: (1) Pretreatment: Take 10 kg of dried whole red clover, crush it to 60 mesh, rinse it three times with deionized water, and dry it at 60℃ until the moisture content is ≤8% to obtain raw material powder.

[0036] (2) Multi-stage alcohol extraction: Add 65% ethanol aqueous solution to the raw material powder at a material-to-liquid ratio of 1:8 (g / mL), and reflux extract twice at 60℃ for 2.5 hours each time. Combine the two extraction filtrates and concentrate under reduced pressure at 60℃ and a vacuum degree of 0.08MPa until there is no ethanol odor to obtain crude extract.

[0037] (3) Compound enzymatic hydrolysis: Add cellulase and pectinase (mass ratio 2:1) to the crude extract, the amount of which is 1.5% of the raw material powder mass. Adjust the pH of the crude extract to 4.5 with citrate-sodium citrate buffer, place it in a constant temperature shaker at 55℃, rotate at 180 r / min, and enzymatically hydrolyze for 4 hours.

[0038] (4) Ultrasonic-assisted fermentation: The enzyme hydrolysate was placed in a 95℃ water bath for 15 minutes to inactivate the enzymes. After cooling to 32℃, a mixed bacterial culture of 8% (v / v) (Lactobacillus plantarum LP6:Saccharomyces cerevisiae Y-1=1:2) was inoculated. Ginger exosomes were added at the same time at a rate of 0.5% (w / v). The mixture was placed in an ultrasonic reactor with an ultrasonic power of 300W and a frequency of 20kHz. Intermittent ultrasonication was performed (3 seconds of ultrasonication followed by 2 seconds of pause). Fermentation was carried out at 35℃ for 55 hours, with stirring every 12 hours to ensure that the exosomes and fermentation broth were fully mixed.

[0039] The preparation method of ginger exosomes is as follows: Fresh ginger rhizomes are selected, homogenized, purified by differential centrifugation and sucrose density gradient centrifugation to obtain exosome vesicles. Differential centrifugation adopts a three-stage centrifugation strategy: 3000g, 4℃, 30 minutes to remove large cell debris; 10000g, 4℃, 30 minutes to remove large organelles; and 100000g, 4℃, 90 minutes to collect the exosome precipitate. The sucrose density gradient from bottom to top is 50%, 40%, 30%, 20%, 10% sucrose solution, centrifuged at 100000g, 4℃ for 18 hours, and the exosome solution is collected. After dilution with phosphate buffer and ultrafiltration, high-purity exosomes are obtained, which are then lyophilized for later use. The lyophilization protectant is a compound formulation of 5% trehalose and 1mM ascorbic acid to ensure the activity and stability of the exosomes.

[0040] (5) Purification and refining: After fermentation, silk fibroin solution is added and stirred at 40°C for 2 hours. Then, it is adsorbed through macroporous resin. First, deionized water is used to wash away impurities, proteins and sugars, and then 70% ethanol is used to wash away the target components. After concentration and drying, red clover extract is obtained.

[0041] Example 3 A method for preparing a low-toxicity, high-activity red clover extract includes the following steps: (1) Pretreatment: Take 10 kg of dried whole red clover, crush it to 40 mesh, rinse it three times with deionized water, and dry it at 60℃ until the moisture content is ≤8% to obtain raw material powder.

[0042] (2) Multi-stage alcohol extraction: Add 75% ethanol aqueous solution to the raw material powder at a material-to-liquid ratio of 1:12 (g / mL), and reflux extract twice at 55℃ for 2 hours each time. Combine the two extraction filtrates and concentrate under reduced pressure at 60℃ and 0.08MPa until there is no ethanol odor to obtain crude extract.

[0043] (3) Compound enzymatic hydrolysis: Add cellulase and pectinase (mass ratio 2:1) to the crude extract, the amount of which is 2.5% of the raw material powder mass. Adjust the pH of the crude extract to 5.5 with citrate-sodium citrate buffer, place it in a constant temperature shaker at 50℃, rotate at 180 r / min, and enzymatically hydrolyze for 3.5 hours.

[0044] (4) Ultrasonic-assisted fermentation: The enzyme hydrolysate was placed in a 95℃ water bath for 15 minutes to inactivate the enzymes. After cooling to 32℃, a mixed bacterial culture of 5% (v / v) (Lactobacillus plantarum LP6:Saccharomyces cerevisiae Y-1=1:2) was inoculated. At the same time, 1% (w / v) of dandelion exosomes were added. The mixture was placed in an ultrasonic reactor with an ultrasonic power of 200W and a frequency of 20kHz. Intermittent ultrasonication was performed (3s of ultrasonication followed by 2s of pause). Fermentation was carried out at 30℃ for 60 hours, with stirring every 12 hours to ensure that the exosomes and fermentation broth were fully mixed.

[0045] The preparation method of dandelion exosomes is as follows: Fresh whole dandelion plants were selected, homogenized, purified by differential centrifugation, and sucrose density gradient centrifugation to obtain exosome vesicles. Differential centrifugation employed a three-stage centrifugation strategy: 3000g, 4℃, 30 minutes to remove large cell debris; 10000g, 4℃, 30 minutes to remove large organelles; and 100000g, 4℃, 90 minutes to collect the exosome precipitate. The sucrose density gradient consisted of 50%, 40%, 30%, 20%, and 10% sucrose solutions from bottom to top. Centrifugation was performed at 100000g, 4℃ for 18 hours, and the exosome solution was collected. After dilution with phosphate buffer and ultrafiltration, high-purity exosomes were obtained, lyophilized, and stored for later use. The lyophilization protectant was a compound formulation of 5% trehalose and 1mM ascorbic acid to ensure the activity and stability of the exosomes.

[0046] (5) Purification and refining: After fermentation, add hydrolyzed wheat protein solution, stir and react at 50°C for 1 hour, then adsorb through macroporous resin, first wash with deionized water to remove impurity proteins and sugars, then wash with 70% ethanol to remove the target component, concentrate and dry to obtain red clover extract.

[0047] Comparative Example 1 (Traditional alcohol extraction method) A method for preparing red clover extract includes the following steps: (1) Pretreatment: Take 10 kg of dried whole red clover, crush it to 50 mesh, rinse it three times with deionized water, and dry it at 60℃ until the moisture content is ≤8% to obtain raw material powder.

[0048] (2) Multi-stage alcohol extraction: 70% ethanol aqueous solution was added to the raw material powder at a material-to-liquid ratio of 1:10 (g / mL), and the mixture was refluxed twice at 55℃ for 2 hours each time. The filtrates from the two extractions were combined and concentrated under reduced pressure at 60℃ and a vacuum of 0.08 MPa until no ethanol odor remained, yielding a crude extract. This crude extract was then adsorbed through a macroporous resin, and impurities such as proteins and sugars were first removed by washing with deionized water. After concentration and drying, the red clover extract was obtained.

[0049] Comparative Example 2 (Simple Enzymatic Hydrolysis) A method for preparing red clover extract includes the following steps: (1) Pretreatment: Take 10 kg of dried whole red clover, crush it to 50 mesh, rinse it three times with deionized water, and dry it at 60℃ until the moisture content is ≤8% to obtain raw material powder.

[0050] (2) Multi-stage alcohol extraction: Add 70% ethanol aqueous solution to the raw material powder at a material-to-liquid ratio of 1:10 (g / mL), and reflux extract twice at 55℃ for 2 hours each time. Combine the two extraction filtrates and concentrate under reduced pressure at 60℃ and a vacuum of 0.08MPa until there is no ethanol odor to obtain crude extract.

[0051] (3) Compound enzymatic hydrolysis: Cellulase and pectinase (mass ratio 2:1) were added to the crude extract at 2% of the raw material powder mass. The pH of the crude extract was adjusted to 5.0 with citrate-sodium citrate buffer, and the extract was placed in a constant temperature shaker at 50℃ and a rotation speed of 180 r / min for 3.5 hours. The extract was then adsorbed through macroporous resin. First, deionized water was used to remove impurities, proteins, and sugars, and then 70% ethanol was used to elute the target components. After concentration and drying, the red clover extract was obtained.

[0052] Comparative Example 3 (Simple Fermentation Method) A method for preparing a low-toxicity, high-activity red clover extract includes the following steps: (1) Pretreatment: Take 10 kg of dried whole red clover, crush it to 50 mesh, rinse it three times with deionized water, and dry it at 60℃ until the moisture content is ≤8% to obtain raw material powder.

[0053] (2) Multi-stage alcohol extraction: Add 70% ethanol aqueous solution to the raw material powder at a material-to-liquid ratio of 1:10 (g / mL), and reflux extract twice at 55℃ for 2 hours each time. Combine the two extraction filtrates and concentrate under reduced pressure at 60℃ and a vacuum of 0.08MPa until there is no ethanol odor to obtain crude extract.

[0054] (3) Ultrasonic-assisted fermentation: The crude extract was inoculated with a mixed bacterial solution (Lactobacillus plantarum LP6: Saccharomyces cerevisiae Y-1=1:2) at an inoculation amount of 6% (v / v) and placed in an ultrasonic reactor. The ultrasonic power was 250W, the frequency was 20kHz, and the ultrasonic treatment was intermittent (3s ultrasonic treatment, 2s pause). Fermentation was carried out at 32℃ for 60 hours, and the mixture was stirred once every 12 hours.

[0055] (4) Purification and refining: After fermentation, the red clover extract is obtained by adsorption through macroporous resin, followed by washing with deionized water to remove impurities, proteins and sugars, and then concentration and drying.

[0056] The performance of the red clover extracts prepared by the methods of Example 1 and Comparative Examples 1-4 was verified, and the verification results are shown in Tables 1 and 2.

[0057] Table 1 Test Results

[0058] Table 2 Results of antioxidant properties and transdermal absorption

[0059] As shown in Tables 1 and 2, a lower IC50 value indicates stronger antioxidant capacity. The antioxidant activity of Example 1 is close to that of Vitamin C, and significantly superior to that of traditional extracts.

[0060] The total isoflavone aglycone content of the extract in Example 1 was three times that of the traditional alcohol extraction (Comparative Example 1), and the chickpea sprout A content (35.2 mg / g) was 2.8 times that of Comparative Example 1; the total polyphenol content (58.9 mg / g) was significantly higher than all comparative examples. This demonstrates that the synergistic effect of "compound enzymatic hydrolysis + ultrasound-assisted fermentation + plant exosomes" can effectively disrupt the cell wall structure of red clover, releasing isoflavones and polyphenols bound to polysaccharides and proteins. This solves the technical pain point of insufficient release of bound components in traditional extraction. Moreover, the addition of plant exosomes did not affect the extraction of target components; on the contrary, the plant exosome structure protected the active ingredients and reduced degradation during the extraction process.

[0061] Antioxidant Activity: Example 1 showed the lowest DPPH scavenging IC50 value (18.6 μg / mL), significantly lower than Comparative Example 1 (45.2 μg / mL) and Comparative Example 4 (42.5 μg / mL), indicating that the extract prepared by the method of this invention has extremely strong antioxidant activity. The core reason is that the complex enzyme releases more highly active free isoflavones and polyphenols, and the fermentation process converts glycoside-type isoflavones into more easily absorbed aglycone forms. Furthermore, the polysaccharide components carried by the plant exosomes themselves form a synergistic antioxidant effect with isoflavones and polyphenols, further enhancing the activity. Plant exosomes are nanoscale lipid bilayer vesicle structures secreted by plant cells, carrying various bioactive substances such as miRNAs, plant active peptides, polysaccharides, lipids, and polyphenols. They possess both natural carrier characteristics and their own biological activity, enabling intercellular signal transduction and delivery of active substances. The lipid bilayer structure can completely encapsulate active substances, isolating them from external factors such as light, temperature, and oxidation, thus preventing the active ingredients from becoming inactive. At the same time, it slows down the release of ingredients, achieving long-lasting skincare and improving the stability and duration of action of the ingredients.

[0062] Transdermal delivery efficiency: The transdermal absorption rate of Example 1 (32.8%) is 4 times that of Comparative Example 1, highlighting the delivery advantages of plant exosomes. Plant exosomes, as nanoscale carriers, can penetrate the skin's stratum corneum barrier to efficiently deliver isoflavones and polyphenols to the epidermis and dermis, avoiding degradation of active ingredients by skin metabolic enzymes. This solves the technical bottleneck of low transdermal absorption and poor bioavailability of traditional extracts, which is one of the core differences between this invention and existing technologies.

[0063] Comparative Example 1 shows that ultrasound-assisted fermentation can promote the full combination of plant exosomes and active ingredients in the fermentation broth, further improving the stability and delivery efficiency of active ingredients. Comparative Example 1 and all comparative examples demonstrate that the series process of "multi-stage alcohol extraction-compound enzymatic hydrolysis-ultrasound-assisted fermentation-exosomes" is not a simple superposition of technologies, but rather forms a synergistic effect, solving multiple technical pain points of existing technologies such as low extraction rate, weak activity, high toxicity, and poor delivery efficiency.

[0064] Table 3 Skin irritation test (human patch test, n=30)

[0065] Table 3 shows that, after protein binding and exosome encapsulation, no skin irritation was observed in Example 1, even at high concentrations, demonstrating its low toxicity and mildness. Comparative Examples 1-3, without exosome and protein treatment, showed significantly greater irritation compared to Example 1.

[0066] Therefore, this invention employs the aforementioned method for preparing a low-toxicity, high-activity red clover extract, utilizing a five-in-one process of alcohol extraction, fermentation, protein binding, enzymatic hydrolysis, and exosome loading. This successfully overcomes the technical bottlenecks of low activity, high irritation, and difficulty in transdermal absorption of red clover extract. The resulting product possesses high aglycone content, low toxicity, high stability, and excellent transdermal absorption performance, showing broad application prospects in the high-end skincare product field.

[0067] 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 preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a low-toxicity, high-activity red clover extract, characterized in that: Includes the following steps: Step 1, Pretreatment: Crush the dried whole red clover into 40-60 mesh, wash with deionized water, and dry at low temperature to obtain raw material powder; Step 2, multi-stage alcohol extraction: Add an aqueous ethanol solution to the raw material powder, reflux and extract twice at 50-60℃, each time for 1.5-2.5 hours, combine the filtrates, and concentrate to obtain crude extract; Step 3, compound enzymatic hydrolysis: Add compound enzyme preparation to crude extract, and the enzymatic hydrolysis time is 3-4 hours; Step 4, Ultrasonic-assisted fermentation: After inactivating the enzymes in the hydrolysate, inoculate it with compound probiotic liquid; at the same time, add plant-derived exosomes and place it in an ultrasonic field for ultrasonic fermentation; Step 5, purification and refining: After fermentation, add protein solution and stir at 40-50℃ for 1-2 hours. Then, adsorb through macroporous resin. First, wash with deionized water to remove impurities, proteins and sugars, then wash with 70% ethanol to remove the target component. After concentration and drying, red clover extract is obtained.

2. The method for preparing a low-toxicity, high-activity red clover extract according to claim 1, characterized in that: In step 2, add 60%-75% ethanol aqueous solution at a material-to-liquid ratio of 1:8-1:12 (g / mL).

3. The method for preparing a low-toxicity, high-activity red clover extract according to claim 1, characterized in that: In step 3, the compound enzyme preparation includes cellulase and pectinase in a mass ratio of 2:1, and the amount added is 1.5%-2.5% of the mass of the raw material powder.

4. The method for preparing a low-toxicity, high-activity red clover extract according to claim 1, characterized in that: In step 3, after adding the compound enzyme preparation, adjust the pH to 4.5-5.5 and control the enzymatic hydrolysis temperature at 45-55℃.

5. The method for preparing a low-toxicity, high-activity red clover extract according to claim 1, characterized in that: In step 4, the compound probiotics include plant lactic acid bacteria and brewer's yeast, with an inoculation amount of 5%-8% v / v and a volume ratio of 1:

2.

6. The method for preparing a low-toxicity, high-activity red clover extract according to claim 1, characterized in that: In step 4, plant-derived exosomes include ginger exosomes, dandelion exosomes, and tremella exosomes, with an addition amount of 0.5%-1% w / v.

7. The method for preparing a low-toxicity, high-activity red clover extract according to claim 1, characterized in that: In step 4, the ultrasonic power is 200-300W, the frequency is 20kHz, the fermentation temperature is 30-35℃, and the fermentation time is 48-72 hours.

8. The method for preparing a low-toxicity, high-activity red clover extract according to claim 1, characterized in that: In step 4, the protein is hydrolyzed wheat protein or silk fibroin.

9. The method for preparing a low-toxicity, high-activity red clover extract according to claim 1, characterized in that: In step 4, the preparation method of plant-derived exosomes is as follows: select fresh plant bodies, purify them by homogenization, differential centrifugation, and sucrose density gradient centrifugation to obtain exosome vesicles.

10. The method for preparing a low-toxicity, high-activity red clover extract according to claim 9, characterized in that: The differential centrifugation employed a three-stage centrifugation strategy: 3000g, 4℃, 30 minutes to remove large cell debris; 10000g, 4℃, 30 minutes to remove large organelles; and 100000g, 4℃, 90 minutes to collect the exosome precipitate. A sucrose density gradient from bottom to top consisted of 50%, 40%, 30%, 20%, and 10% sucrose solutions. Centrifugation was performed at 100000g, 4℃ for 18 hours, and the exosome solution was collected. After dilution with phosphate buffer and ultrafiltration, high-purity exosomes were obtained, lyophilized, and stored for later use. The lyophilization protectant was a compound formulation of 5% trehalose and 1mM ascorbic acid to ensure the activity and stability of the exosomes.

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