Application of licorice extract in oral whitening agent

Abstract

The invention discloses a preparation method of a licorice extract and application of the licorice extract in an oral whitening agent, and belongs to the technical field of foods and medicines. According to the method, the corresponding extract is prepared through extraction, refining and inclusion processes of liquorice, the core component glabridin can directly inhibit the activity of tyrosinase, release of proinflammatory factors is reduced, and the extract has the triple effects of whitening, resisting inflammation and resisting oxidation. Through a water-soluble dietary fiber and metal ion inclusion technology, the licorice extract and the resistant dextrin are compatible according to a specific ratio to prepare the oral whitening agent, and the oral whitening agent can effectively protect glabridin from being degraded in stomach and intestinal environments, realizes targeted release of intestinal tracts, and improves the stability and bioavailability of active ingredients. The process is simple and convenient, large-scale production is easy, and the product is safe and effective and has a good commercial prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of food and medicine, and particularly relates to licorice extract in the preparation of oral whitening agents and its application. BACKGROUND

[0002] As a traditional medicinal and edible material, licorice root is rich in glycyrrhizin, glabridin, licorice flavones and other active ingredients, and has unique development value. In China, there are three varieties of edible licorice: Ural licorice, swollen fruit licorice and light fruit licorice. Glabridin only exists in light fruit licorice and is also a recognized core whitening active ingredient. It can not only effectively inhibit tyrosinase activity and block melanin production at the source, but also has multiple effects such as free radical scavenging and anti-inflammatory. It has high safety and no obvious toxic and side effects. Compared with common ingredients such as glutathione, vitamin C and nicotinamide in the oral whitening market, it effectively avoids the defects of poor tolerance, single efficacy and questionable safety of long-term use, and is more suitable as an oral whitening raw material. However, the content of glabridin in natural plants is relatively low, and the conventional extraction process has problems such as low dissolution rate of active ingredients, poor stability and low bioavailability after oral administration, which limits its development and application in oral whitening products.

[0003] In the prior art, CN113616548A uses 2-sulfobutyl-β-cyclodextrin to complex glabridin, which improves the permeability of glabridin in the mucus layer and epithelial cells of the small intestine, but does not significantly improve the bioavailability in oral dosage forms. The prior art CN115040505A complexes glabridin with 2-sulfobutyl-β-cyclodextrin, and then forms a complex with chitosan through electrostatic interaction to enhance the mucosal adhesion, thereby improving the oral bioavailability to some extent, but there is still room for improvement. Therefore, it is of great industrial significance to develop a green preparation method based on licorice that can efficiently enrich and stabilize active ingredients such as glabridin and improve its bioavailability. SUMMARY

[0004] Although the prior art attempts to improve the oral absorption of glabridin through cyclodextrin complexation and mucosal adhesion strategies, the improvement in bioavailability is still very limited. Furthermore, although there are studies on the oral absorption of glabridin in the prior art, they mainly focus on pharmacokinetics and systemic exposure, and do not systematically evaluate its application from the perspective of oral whitening efficacy. The efficacy research on glabridin whitening mainly focuses on external preparations. The present application uses licorice extract in oral whitening agents. The present application prepares licorice extract oral whitening agents by combining licorice root extract with water-soluble dietary fiber and metal salt complexation technology. The oral whitening agent not only effectively inhibits tyrosinase, but also has anti-inflammatory effects, achieving a safe and efficient oral whitening effect.

[0005] The application discloses application of licorice extract in oral whitening agents, and specifically relates to the following: The preparation method of the oral whitening agent is as follows: licorice extract and resistant dextrin are mixed at a ratio of 1:15-25, and then stirred at 200-500 r / min for 10-30 min to obtain the oral whitening agent.

[0006] The resistant dextrin can improve the dispersibility and stability of the extract, and can enhance the absorption of the active ingredients in the intestinal tract.

[0007] The licorice extract: the licorice root is used as a source of core whitening active ingredients, and the components such as glabridin contained in the licorice root can directly and efficiently inhibit the activity of tyrosinase, and can block the generation of melanin from the source.

[0008] The preparation method of the licorice extract comprises the following steps: licorice root, coarse crushing, extraction, filtration, refining, concentration, inclusion, drying, fine crushing, mixing, screening and iron removal, gold exploration and packaging.

[0009] The coarse crushing treatment of the dried licorice root is as follows: 200-500 g of dried licorice root coarse powder is crushed at 1000-3000 r / min and sieved through a 30-40 mesh sieve; 3-5 times the amount of water is added, and the pH is adjusted to 5.0 by using a citric acid aqueous solution; 1-5 g of pectinase and 0.5-3 g of cellulase are added, and enzymolysis is carried out at 40-50 DEG C for 60-120 min; then the temperature is increased to 80-90 DEG C and kept for 10-15 min; after cooling to 25-30 DEG C, 0.1-1.0 g of lactobacillus plantarum is inoculated, and fermentation is carried out at 25-35 DEG C for 36-60 h; and then drying is carried out at 40-60 DEG C, and the powder is crushed at 2000-5000 r / min and sieved.

[0010] In the reflux extraction, the reflux temperature is set to 70-85 DEG C, each extraction is carried out for 1-3 h, and the extraction is repeated for 2-3 times.

[0011] The refining treatment is as follows: centrifugation is carried out at 3000-5000 r / min for 5-10 min, the refined licorice liquid is obtained by using an ultrafiltration membrane with a molecular weight cut-off of 5-10 kDa and a nanofiltration membrane system with a molecular weight cut-off of 200-500 Da.

[0012] In the concentration treatment, the refined licorice liquid is concentrated at 50-60 DEG C and under an absolute pressure of 0.02 to 0.08 MPa for 6-12 h to obtain the licorice concentrate.

[0013] The 5-15 g water-soluble dietary fiber is stirred in 800-1000 mL water at 40-50 DEG C and 200-500 r / min for 30-60 min; then the obtained licorice concentrate is added and centrifuged at 10000-20000 r / min to form a fog, which is added into a coagulation bath of 800-1000 mL metal salt to form an inclusion particle, which is collected by a screen and washed with water.

[0014] The water-soluble dietary fiber is any one of low-methoxyl pectin, gellan gum, and sodium alginate.

[0015] The metal salt is any one of calcium chloride and zinc sulfate.

[0016] In the drying process, the formed inclusion particle is subjected to spray drying treatment, and the inlet air temperature is set to 160-190 DEG C and the outlet air temperature is set to 75-90 DEG C.

[0017] In the iron-removing process by sieving, the particle is first sieved through an 80-100 mesh screen and simultaneously subjected to magnetic separation, and the magnetic induction intensity is set to be greater than or equal to 8000 GS. In the gold detection process, the sensitivity is set to be greater than or equal to 0.5 mm for iron and greater than or equal to 1.0 mm for non-iron.

[0018] Advantages of the present application: 1. The main raw material of the present application, i.e., the glycyrrhiza root, is rich in components such as glabridin, which can directly and efficiently inhibit the activity of tyrosinase. The glycyrrhiza root subjected to fermentation treatment can convert precursor components such as glycyrrhizin into active aglycone such as glycyrrhizin under the action of lactobacillus plantarum, which not only further enhances the tyrosinase inhibition capacity, but also synergistically plays a role of scavenging free radicals and inhibiting the release of pro-inflammatory factors, while reducing the inflammation-mediated excessive proliferation of melanocytes and pigmentation, thereby realizing the multi-target synergistic whitening effect.

[0019] 2. The present application utilizes water-soluble dietary fiber and metal ions to construct an intestinal targeting inclusion system, which remains stable in the stomach acid environment and the upper intestinal digestive environment, effectively blocks the degradation of core active components such as glabridin by digestive enzymes such as pepsin and trypsin, avoids the premature loss of active components in the upper digestive tract, realizes the targeted delivery of the active components to the absorption site in the intestine, and improves the oral bioavailability of the active components.

[0020] 3. The present application mixes licorice extract and resistant dextrin at a specific ratio, and the prepared oral whitening agent not only ensures the stability of the active components during gastrointestinal transport, but also greatly reduces the degradation and loss of the active components in the upper digestive tract. All components of the whitening agent are recognized as safe food raw materials, and have both whitening effect and safety in application. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Flow chart for preparing licorice root extract. DETAILED DESCRIPTION

[0022] The parameters of some substances in the examples and their sources are as follows: The variety of licorice root in the examples is the root of Glycyrrhiza glabra, and the place of origin is Azerbaijan.

[0023] Pectinase: CAS No. 9032-75-1; activity ≥5000 U / g; from Solabio; item No. P8130.

[0024] Cellulase: CAS No. 9012-54-8; activity ≥20000 U / g; from Solabio; item No. C8140.

[0025] Citric acid: CAS No. 77-92-9; food grade.

[0026] Preparation of citric acid aqueous solution: take 10 g of citric acid, dissolve with water and make up to 100 mL; when used, the pH value of the enzyme hydrolysis system is adjusted by adding citric acid aqueous solution dropwise.

[0027] Lactobacillus plantarum: strain No. ATCC 14917; commercially available; after conventional activation and expansion culture, the number of viable bacteria ≥1×10 10 CFU / g is put into use.

[0028] Gellan gum: CAS No. 71010-52-1; source: CP Kelco; item No. KELCOGEL CG-LA.

[0029] Low methoxyl pectin: CAS No. 9000-69-5; methoxyl content ≤30%, source: Sigma-Aldrich; item No. P9135.

[0030] Sodium alginate, CAS No. 9005-38-3; from Nanjing Songuan.

[0031] Ultrafiltration membrane: MWCO is 5-10 kDa; source: Jiangsu Jiugu.

[0032] Nanofiltration membrane: MWCO is 200-500 Da; model: NF270-400 / 34i; source: DuPont Water Solutions.

[0033] Three-dimensional mixer: model: SYH type; suitable for particle size difference ≤200 μm; source: Nanjing Hongchuang.

[0034] Example 1 A preparation method of licorice extract, specifically as follows: S1, coarse crushing Take 400 g of dried licorice root and crush it at 1000 r / min, then pass it through a 30-mesh sieve; then add 1200 g of water and adjust the pH to 5.0 with a citric acid solution; then add 3 g of pectinase and 2 g of cellulase, and enzymatically hydrolyze at 50°C for 90 min; then raise the temperature to 85°C and maintain for 15 min; then cool to 30°C, inoculate with 0.9 g of Lactobacillus plantarum, and ferment at 30°C for 48 h; then dry at 50°C; then crush at 3000 r / min and pass through a 40-mesh sieve to obtain a fermented licorice root coarse powder; S2, extraction Take 300 g of the fermented licorice root coarse powder and add 8 times the amount of 70 vol% ethanol water solution, heat to 80°C, and perform reflux extraction, collect the extract after 2 h; repeat the extraction 2 more times, combine the 3 licorice extracts, and reserve for use; S3, filtration Filter the licorice extract reserved for use through a 400-mesh sieve to obtain a licorice crude filtrate; S4, refining Centrifuge the licorice crude filtrate obtained at 3000 r / min for 10 min, pass it through a 10 kDa ultrafiltration membrane and a 300 Da nanofiltration membrane system in sequence, and collect the retentate to obtain a licorice refined liquid; S5, concentration Concentrate the licorice refined liquid at 60°C and 0.08 MPa absolute pressure for 12 h to obtain a licorice concentrate; S6, drying Then spray dry the licorice concentrate obtained, set the inlet air temperature to 180°C, the outlet air temperature to 85°C, and the atomizer speed to 25000 r / min to obtain a licorice extract dry powder; S7, fine crushing Take the licorice extract dry powder obtained and crush it at 3500 r / min for 5 min, then pass it through a 100-mesh sieve to obtain a fine powder; S8, mixing Put the licorice fine powder obtained into a three-dimensional mixer and mix at 15 r / min for 30 min to obtain a mixed powder; S9, sieving and iron removal Pass the licorice mixed powder through an 80-mesh sieve and simultaneously remove iron using a pipeline magnetic separator, set the magnetic induction intensity to ≥8000 GS; S10, metal detection and packaging Detect the licorice mixed powder after iron removal using a metal detector, set the sensitivity to iron ≥0.5 mm and non-iron ≥1.0 mm, and after passing the detection, seal and package in a polyethylene plastic bag, 50 g per bag, and store in the dark.

[0035] Example 2 A method for preparing a licorice extract, specifically as follows: S1, coarse crushing Take 400 g of dried licorice root and crush it at 1000 r / min through a 30-mesh sieve; then add 1200 g of water and adjust the pH to 5.0 with a citric acid solution; add 3 g of pectinase and 2 g of cellulase, and enzymatically hydrolyze at 50°C for 90 min; then raise the temperature to 85°C and maintain for 15 min; after cooling to 30°C, inoculate with 0.9 g of Lactobacillus plantarum and ferment at 30°C for 48 h; then dry at 50°C; then crush at 3000 r / min through a 40-mesh sieve to obtain a coarse powder of fermented licorice root; S2, Extraction Take 300 g of the coarse powder of fermented licorice root and add 8 times the amount of 70 vol% ethanol water solution; heat to 80°C and perform reflux extraction; collect the extract after 2 h; repeat the extraction 2 more times, and combine the 3 licorice extracts for use; S3, Filtration Filter the licorice extract through a 400-mesh sieve to obtain a crude licorice filtrate; S4, Refinement Centrifuge the crude licorice filtrate at 3000 r / min for 10 min, and then pass it through a 10 kDa ultrafiltration membrane and a 300 Da nanofiltration membrane system in sequence; collect the retentate to obtain a refined licorice extract; S5, Concentration Concentrate the refined licorice extract at 60°C and 0.08 MPa absolute pressure for 12 h to obtain a licorice concentrate; S6, Inclusion Take 15 g of sodium alginate in 1000 mL of water, and stir at 40°C and 500 r / min for 60 min; then add the licorice concentrate, and stir at 40°C and 300 r / min for 30 min; centrifuge at 15000 r / min to atomize the mixture, and spray it into a coagulation bath containing 1 L of a 0.01 wt% calcium chloride and 0.004 wt% zinc sulfate complex aqueous solution, and stir at 50 r / min for 40 min; collect the licorice extract inclusion particles using an 80-mesh sieve, and rinse them with water 3 times; S7, Drying Then disperse the licorice extract inclusion particles in 200 mL of water, and homogenize at 8000 r / min for 5 min; then perform spray drying, with an inlet temperature of 180°C, an outlet temperature of 85°C, and an atomizer speed of 25000 r / min; obtain a licorice extract dry powder; S8, Fine Crushing Take the licorice extract dry powder and crush it at 3500 r / min for 5 min through a 100-mesh sieve to obtain a fine powder; S9, Mixing Place the licorice fine powder into a three-dimensional mixer, and mix at 15 r / min for 30 min to obtain a mixed powder; S10, Sieving and Iron Removal The licorice powder mixture is passed through an 80-mesh sieve and simultaneously passes through a pipeline magnetic separator to remove iron, with a magnetic induction intensity of ≥8000GS. S11, Jintan and Packaging The iron-free licorice powder was tested with a metal detector with a sensitivity of ≥0.5mm for iron and ≥1.0mm for non-iron. After passing the test, it was sealed in polyethylene plastic bags, 50g per bag, and stored away from light.

[0036] Example 3 A method for preparing licorice extract is as follows: S1, coarse grinding Take 400g of dried licorice root and pulverize it at 1000r / min, then pass it through a 30-mesh sieve; add 1200g of water and adjust the pH to 5.0 with citric acid aqueous solution; add 3g of pectinase and 2g of cellulase, and enzymatically hydrolyze at 50℃ for 90min; then raise the temperature to 85℃ and hold for 15min; after cooling to 30℃, inoculate with 0.9g of Lactobacillus plantarum and ferment at 30℃ for 48h; then dry at 50℃; then pulverize it at 3000r / min and pass it through a 40-mesh sieve to obtain fermented licorice root coarse powder; S2, Extraction Take 300g of fermented licorice root powder and add 8 times the amount of 70vol% ethanol aqueous solution. Heat to 80℃ and reflux for extraction. Collect the extract after 2 hours. Repeat the extraction twice more and combine the licorice extracts from the three extractions for later use. S3, Filtering The prepared licorice extract was filtered through a 400-mesh filtration to obtain coarse licorice filtrate; S4, Refined The obtained licorice coarse filtrate was centrifuged at 3000 r / min for 10 min, and then passed through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa and a nanofiltration membrane with a molecular weight cutoff of 300 Da in sequence. The filtrate was collected to obtain licorice refined liquid. S5, Concentrated The licorice extract was concentrated at 60℃ and 0.08MPa absolute pressure for 12 hours to obtain licorice concentrate. S6, Enclosure Take 15g of gellan gum and place it in 1000mL of water. Stir at 40℃ and 500r / min for 60min. Then add the obtained licorice concentrate and stir at 40℃ and 300r / min for 30min. Centrifuge and atomize the mixture at 15000r / min, and spray it into a coagulation bath containing 1L of a composite aqueous solution of 0.01wt% calcium chloride and 0.004wt% zinc sulfate. Stir at 50r / min for 40min. Collect the licorice extract inclusion particles through an 80-mesh sieve and rinse with water three times. S7, Drying The obtained licorice extract inclusion particles were then dispersed in 200 mL of water and homogenized at 8000 r / min for 5 min; then spray dried with an inlet air temperature of 180℃, an outlet air temperature of 85℃, and an atomizer speed of 25000 r / min to obtain licorice extract powder. S8, Fine grinding The obtained licorice extract powder was pulverized at 3500 r / min for 5 min and then passed through a 100-mesh sieve to obtain fine powder. S9, Mixed The obtained licorice powder was placed in a three-dimensional mixer and mixed at 15 r / min for 30 min to obtain a mixed powder; S10, sieving and iron removal The licorice powder mixture is passed through an 80-mesh sieve and simultaneously passes through a pipeline magnetic separator to remove iron, with a magnetic induction intensity of ≥8000GS. S11, Jintan and Packaging The iron-free licorice powder was tested with a metal detector with a sensitivity of ≥0.5mm for iron and ≥1.0mm for non-iron. After passing the test, it was sealed in polyethylene plastic bags, 50g per bag, and stored away from light.

[0037] Example 4 A method for preparing licorice extract is as follows: S1, coarse grinding Take 400g of dried licorice root and pulverize it at 1000r / min, then pass it through a 30-mesh sieve; add 1200g of water and adjust the pH to 5.0 with citric acid aqueous solution; add 3g of pectinase and 2g of cellulase, and enzymatically hydrolyze at 50℃ for 90min; then raise the temperature to 85℃ and hold for 15min; after cooling to 30℃, inoculate with 0.9g of Lactobacillus plantarum and ferment at 30℃ for 48h; then dry at 50℃; then pulverize it at 3000r / min and pass it through a 40-mesh sieve to obtain fermented licorice root coarse powder; S2, Extraction Take 300g of fermented licorice root powder and add 8 times the amount of 70vol% ethanol aqueous solution. Heat to 80℃ and reflux for extraction. Collect the extract after 2 hours. Repeat the extraction twice more and combine the licorice extracts from the three extractions for later use. S3, Filtering The prepared licorice extract was filtered through a 400-mesh filtration to obtain coarse licorice filtrate; S4, Refined The obtained licorice coarse filtrate was centrifuged at 3000 r / min for 10 min, and then passed through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa and a nanofiltration membrane with a molecular weight cutoff of 300 Da in sequence. The filtrate was collected to obtain licorice refined liquid. S5, Concentrated The licorice extract was concentrated at 60℃ and 0.08MPa absolute pressure for 12 hours to obtain licorice concentrate. S6, Enclosure Take 15g of low-methoxyl pectin and add it to 1000mL of water. Stir at 40℃ and 500r / min for 60min. Then add the obtained licorice concentrate and stir at 40℃ and 300r / min for 30min. Centrifuge and atomize the mixture at 15000r / min and spray it into a coagulation bath containing 1L of 0.01wt% calcium chloride aqueous solution. Stir at 50r / min for 40min. Collect the licorice extract inclusion particles through an 80-mesh sieve and rinse with water three times. S7, Drying The obtained licorice extract inclusion particles were then dispersed in 200 mL of water and homogenized at 8000 r / min for 5 min; then spray dried with an inlet air temperature of 180℃, an outlet air temperature of 85℃, and an atomizer speed of 25000 r / min to obtain licorice extract powder. S8, Fine grinding The obtained licorice extract powder was pulverized at 3500 r / min for 5 min and then passed through a 100-mesh sieve to obtain fine powder. S9, Mixed The obtained licorice powder was placed in a three-dimensional mixer and mixed at 15 r / min for 30 min to obtain a mixed powder; S10, sieving and iron removal The licorice powder mixture is passed through an 80-mesh sieve and simultaneously passes through a pipeline magnetic separator to remove iron, with a magnetic induction intensity of ≥8000GS. S11, Jintan and Packaging The iron-free licorice powder was tested with a metal detector with a sensitivity of ≥0.5mm for iron and ≥1.0mm for non-iron. After passing the test, it was sealed in polyethylene plastic bags, 50g per bag, and stored away from light.

[0038] Example 5 A method for preparing licorice extract is as follows: S1, coarse grinding Take 400g of dried licorice root and pulverize it at 1000r / min, then pass it through a 30-mesh sieve; add 1200g of water and adjust the pH to 5.0 with citric acid aqueous solution; add 3g of pectinase and 2g of cellulase, and enzymatically hydrolyze at 50℃ for 90min; then raise the temperature to 85℃ and hold for 15min; after cooling to 30℃, inoculate with 0.9g of Lactobacillus plantarum and ferment at 30℃ for 48h; then dry at 50℃; then pulverize it at 3000r / min and pass it through a 40-mesh sieve to obtain fermented licorice root coarse powder; S2, Extraction Take 300g of fermented licorice root powder and add 8 times the amount of 70vol% ethanol aqueous solution. Heat to 80℃ and reflux for extraction. Collect the extract after 2 hours. Repeat the extraction twice more and combine the licorice extracts from the three extractions for later use. S3, Filtering The prepared licorice extract was filtered through a 400-mesh filtration to obtain coarse licorice filtrate; S4, Refined The obtained licorice coarse filtrate was centrifuged at 3000 r / min for 10 min, and then passed through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa and a nanofiltration membrane with a molecular weight cutoff of 300 Da in sequence. The filtrate was collected to obtain licorice refined liquid. S5, Concentrated The licorice extract was concentrated at 60℃ and 0.08MPa absolute pressure for 12 hours to obtain licorice concentrate. S6, Enclosure Take 15g of low-methoxyl pectin and add it to 1000mL of water. Stir at 40℃ and 500r / min for 60min. Then add the obtained licorice concentrate and stir at 40℃ and 300r / min for 30min. Centrifuge and atomize at 15000r / min, and spray it into a coagulation bath containing 1L of 0.01wt% calcium chloride and 0.004wt% zinc sulfate composite aqueous solution. Stir at 50r / min for 40min. Collect the licorice extract inclusion particles through an 80-mesh sieve and rinse with water 3 times. S7, Drying The obtained licorice extract inclusion particles were then dispersed in 200 mL of water and homogenized at 8000 r / min for 5 min; then spray dried with an inlet air temperature of 180℃, an outlet air temperature of 85℃, and an atomizer speed of 25000 r / min to obtain licorice extract powder. S8, Fine grinding The obtained licorice extract powder was pulverized at 3500 r / min for 5 min and then passed through a 100-mesh sieve to obtain fine powder. S9, Mixed The obtained licorice powder was placed in a three-dimensional mixer and mixed at 15 r / min for 30 min to obtain a mixed powder; S10, sieving and iron removal The licorice powder mixture is passed through an 80-mesh sieve and simultaneously passes through a pipeline magnetic separator to remove iron, with a magnetic induction intensity of ≥8000GS. S11, Jintan and Packaging The iron-free licorice powder was tested with a metal detector with a sensitivity of ≥0.5mm for iron and ≥1.0mm for non-iron. After passing the test, it was sealed in polyethylene plastic bags, 50g per bag, and stored away from light.

[0039] Comparative Example 1 A method for preparing licorice extract is as follows: S1, coarse grinding Take 400g of dried licorice root and pulverize it at 1000r / min, then pass it through a 30-mesh sieve; add 1200g of water and adjust the pH to 5.0 with citric acid aqueous solution; add 3g of pectinase and 2g of cellulase, and enzymatically hydrolyze at 50℃ for 90min; then raise the temperature to 85℃ and hold for 15min; cool to 30℃, inoculate with 0.9g of Lactobacillus plantarum, and ferment at 30℃ for 48h; then dry at 50℃; then pulverize it at 3000r / min and pass it through a 40-mesh sieve to obtain fermented licorice root coarse powder; S2, Extraction Take 300g of the above-mentioned fermented licorice root powder and add 8 times the amount of 70 vol% ethanol aqueous solution. Heat to 80℃ and reflux for extraction. Collect the extract after 2 hours. Repeat the extraction twice more and combine the licorice extracts from the three extractions for later use. S3, Filtering The prepared licorice extract was filtered through a 400-mesh filtration to obtain coarse licorice filtrate; S4, Refined The obtained licorice coarse filtrate was centrifuged at 3000 r / min for 10 min, and then passed through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa and a nanofiltration membrane with a molecular weight cutoff of 300 Da in sequence. The filtrate was collected to obtain licorice refined liquid. S5, Concentrated The licorice extract was concentrated at 60℃ and 0.08MPa absolute pressure for 12 hours to obtain licorice concentrate. S6, Enclosure Take 15g of low-methoxyl pectin and add it to 1000mL of water. Stir at 40℃ and 500r / min for 60min. Then add the obtained licorice concentrate and stir at 40℃ and 300r / min for 30min. Centrifuge and atomize the mixture at 15000r / min and spray it into a coagulation bath containing 1L of 0.004wt% zinc sulfate composite aqueous solution. Stir at 50r / min for 40min. Collect the licorice extract inclusion particles through an 80-mesh sieve and rinse with water three times. S7, Drying The obtained licorice extract inclusion particles were then dispersed in 200 mL of water and homogenized at 8000 r / min for 5 min; then spray dried with an inlet air temperature of 180℃, an outlet air temperature of 85℃, and an atomizer speed of 25000 r / min to obtain licorice extract powder. S8, Fine grinding The obtained licorice extract powder was pulverized at 3500 r / min for 5 min and then passed through a 100-mesh sieve to obtain fine powder. S9, Mixed The obtained licorice powder was placed in a three-dimensional mixer and mixed at 15 r / min for 30 min to obtain a mixed powder; S10, sieving and iron removal The licorice powder mixture is passed through an 80-mesh sieve and simultaneously passes through a pipeline magnetic separator to remove iron, with a magnetic induction intensity of ≥8000GS. S11, Jintan and Packaging The iron-free licorice powder was tested with a metal detector with a sensitivity of ≥0.5mm for iron and ≥1.0mm for non-iron. After passing the test, it was sealed in polyethylene plastic bags, 50g per bag, and stored away from light.

[0040] Application Example 7-12 Take 100 mg of each of the products obtained in Examples 1-5 and Comparative Example 1, and mix them with 1.9 g of resistant dextrin. Stir at 300 r / min for 20 min at room temperature to obtain an oral whitening agent.

[0041] Test Example 1 Tyrosinase inhibitory activity The determination was carried out in accordance with the group standard "T / GDCA 006-2021 Test method for inhibition of tyrosinase activity by cosmetic raw materials (in vitro method)".

[0042] Solution preparation: Accurately weigh the licorice extract, glycyrrhizin, and positive controls ergothioneine and glutathione prepared in Examples 1-5 and Comparative Example 1, dissolve and dilute them in 0.1 mol / L pH 6.8 phosphate buffer to prepare five gradient concentrations of 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, and 160 μg / mL.

[0043] Reaction system: In a 96-well plate, add the following sequentially: 70 μL pH 7.4 phosphate buffer, 20 μL of sample solutions of different concentrations, and 40 μL 100 U / mL tyrosinase solution; mix well, incubate at 37 °C for 10 min, and then add 40 μL 2.5 mmol / L L-DOPA solution to start the reaction; L-DOPA serves as the substrate for the enzymatic reaction. Blank control group: 130 μL of pH 7.4 phosphate buffer and 40 μL of 2.5 mmol / L L-DOPA solution were mixed, without sample and tyrosinase; Sample background control group: 110 μL pH 7.4 phosphate buffer, 20 μL sample solution, 40 μL 2.5 mmol / L L-DOPA solution, without tyrosinase; Negative control group: 90 μL pH 7.4 phosphate buffer, 40 μL 100 U / mL tyrosinase solution, and 40 μL 2.5 mmol / L L-DOPA solution to initiate the reaction; Measurement: After continuing the reaction at 37℃ for 20 min, the absorbance (OD) value of each well was immediately measured using an ELISA reader at a wavelength of 475 nm. Calculation: Tyrosinase inhibition rate (%) = [1 - (OD)]样品 -OD 样品本底 ) / (OD 阴性对照 -OD 空白对照 ]×100%; IC 50 IC50 value calculation: Plot the logarithm of the sample concentration on the x-axis and the inhibition rate on the y-axis, perform linear regression, and calculate the sample concentration required to achieve an inhibition rate of 50%, i.e., IC50. 50 Values. The results are shown in Table 1.

[0044] Table 1 Results of inhibitory activity test

[0045] IC 50 The smaller the value, the stronger the inhibitory effect of the prepared licorice extract on tyrosinase.

[0046] Compared with Comparative Example 1, Examples 1-5 all exhibited varying degrees of tyrosinase inhibitory activity. Among them, the licorice extract of Example 1 may have promoted the dissolution of flavonoid active ingredients such as glycyrrhizin by disrupting the cell wall structure of licorice root through the synergistic enzymatic hydrolysis of pectinase and cellulase. The subsequent fermentation with Lactobacillus plantarum may have produced glycosidases that hydrolyzed some saponins such as glycyrrhizic acid into their aglycone glycyrrhetinic acid, and may have enhanced the dual tyrosinase inhibitory and anti-inflammatory activities of the extract by transforming other glycosides or metabolites.

[0047] Furthermore, the inhibitory activity of Examples 2-5 is superior to that of the unencapsulated Example 1. This may be because the inclusion system can form a stable gel network or complex around hydrophobic and unstable active molecules such as glycyrrhizin, effectively reducing their aggregation, precipitation, or degradation. This allows them to contact and act on tyrosinase in a higher concentration and more stable form within the reaction system. Examples 3-5 are all polysaccharide-metal ion gel systems, with Example 5 incorporating low-methoxyl pectin-Zn. 2+ / Ca 2+ The system performed best, possibly because calcium ions can bind to the carboxyl groups on the pectin molecular chain, optimizing the three-dimensional network structure of pectin and improving the encapsulation rate and controlled release performance of glycyrrhizin. In addition, the additional zinc ions can act as a competitive inhibitor of copper ions at the active site of tyrosinase, directly interfering with the enzyme's catalytic function by occupying the enzyme's active site, thus achieving a dual synergy of physical inclusion and chemical enzyme activity inhibition.

[0048] In comparison, Example 5 showed better inhibitory activity than the positive control ergothioneine, while the positive control glutathione also showed strong inhibitory activity in this system. However, the inclusion system of Example 5 integrates multiple mechanisms such as targeted delivery of active ingredients, improved physical stability, and zinc ion-assisted inhibition, thus demonstrating a more efficient and stable potential whitening effect in blocking melanin production, improving skin tone, and preventing pigmentation.

[0049] Test Example 2 Anti-inflammatory activity evaluation Preliminary preparation: RAW264.7 mouse macrophage cell line was used and cultured in DMEM complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. The cells were obtained from the American Type Culture Collection cell bank and were model RAW264.7 (TIB-71). TM ); 1% penicillin-streptomycin solution was sourced from Sigma-Aldrich, catalog number: P4333; DMEM medium was sourced from Corning, catalog number: 10-013-CV; When the cells reach 80% confluence, use 1×10 5 Cells / well were seeded into 96-well plates and cultured for 24 hours; After synchronization, the cells were replaced with 100 μL / well serum-free DMEM medium and incubated for 2 h to synchronize them. Pretreatment: Take an appropriate amount of the oral whitening agent prepared in Examples 7-12, dissolve and dilute it in serum-free DMEM medium to three concentrations of 1, 10, and 50 μg / mL; discard the synchronization medium and perform the following group intervention: The serum-free DMEM medium is from Corning, catalog number: 17-205-CV, low sugar type, without L-glutamine and sodium pyruvate; Test group: Application example 7-12: Add 100 μL of sample solution and 100 μL of 1 μg / mL lipopolysaccharide; Positive control group: Dexamethasone, prepared with the same culture medium and the same gradient concentrations; 100 μL of positive control solution and 100 μL of 1 μg / mL lipopolysaccharide were added. Negative control group: only 200 μL of the same serum-free DMEM medium used in the sample treatment was added; Model control group: Add 100 μL of 1 μg / mL lipopolysaccharide and 100 μL of the same serum-free DMEM medium used in the sample treatment; Culture and collection: After culturing at 37℃ and 5% CO2 for 24 h, the cell supernatant was collected and stored at -80℃ for later use; Cytokine detection: The concentrations of TNF-α and IL-6 in the supernatant were determined using an ELISA kit from R&D Systems, batch number 202501. The procedure was performed according to the kit instructions. The TNF-α detection kit was catalog number DY410, and the IL-6 detection kit was catalog number DY406. The OD value was measured at 450 nm using a Thermo Scientific Multiskan FC microplate reader, and the cytokine concentration was calculated using a standard curve.

[0050] Calculation: Inhibition rate (%) = [1 - (sample cytokine concentration / model control group cytokine concentration)] × 100%.

[0051] IC50 calculation: Plot the logarithm of sample concentration on the x-axis and the inhibition rate on the y-axis, perform linear regression, and calculate the sample concentration required to achieve a 50% inhibition rate, i.e., IC50. 50 Values. The results are shown in Table 2.

[0052] Table 2 Results of anti-inflammatory activity test

[0053] Ultraviolet radiation can activate the skin's immune system, releasing inflammatory factors TNF-α and IL-6. These cytokines can directly stimulate melanocyte proliferation. Among them, TNF-α can rapidly initiate an inflammatory cascade and stimulate keratinocytes to release melanocyte-promoting substances such as stem cell factors, prolonging the stimulation of melanocytes and making pigmentation spots more difficult to fade. IL-6, as a key pro-inflammatory factor, can upregulate the expression of tyrosinase in melanocytes and is an important reason why ultraviolet radiation causes skin inflammation and pigmentation.

[0054] The anti-inflammatory effects of Application Examples 7-11 of this invention are all higher than those of Application Example 12. Application Example 12 may have suffered from poor stability / insertion protection due to the poor stability of the inclusion system used, resulting in insufficient stability and reduced bioavailability of some active ingredients in simulated digestion environments or cell experiments. Application Example 7, on the other hand, may have limited its overall anti-inflammatory efficacy due to its single component, failing to form a multi-target, multi-pathway anti-inflammatory component network. In contrast, Application Examples 8-11 achieved stable delivery and synergistic effects of active ingredients by combining fermentation-pretreated raw materials with specific inclusion technologies.

[0055] Further application of Example 11 showed better results, possibly due to the stable gel network formed by low-methoxyl pectin and metal ions, ensuring the protection and targeted delivery of core active ingredients such as glycyrrhizin in the gastrointestinal environment. Secondly, glycyrrhizin itself not only inhibits tyrosinase but also regulates inflammatory signaling pathways such as NF-κB, directly reducing the transcription and expression of pro-inflammatory factors. The introduced metal ions, acting as cross-linking agents in the inclusion network, can also serve as intracellular anti-inflammatory regulators, inhibiting the nuclear translocation of NF-κB and curbing the excessive production of pro-inflammatory factors TNF-α and IL-6 at the source of the signaling pathway, thereby blocking inflammation-mediated melanin synthesis. Applications of Examples 7-11 showed poorer inhibitory effects compared to the positive control dexamethasone, but with better safety. Dexamethasone is a synthetic glucocorticoid; long-term use may cause immunosuppression and metabolic side effects. Licorice extract, as a food-grade medicinal material, is suitable for long-term daily health maintenance and oral anti-inflammatory and skin-whitening solutions for the management of chronic low-grade inflammation.

[0056] Test Example 3 In vitro simulated digestion and intestinal targeted release performance evaluation Simulated gastric juice: Dissolve 1.0 g of 250 U / mg pepsin in 0.1 mol / L hydrochloric acid solution and adjust the pH to 2.0; Simulate the first stage of intestinal fluid: Dissolve 1.0g of 4×USP pancreatic enzyme in phosphate buffer and adjust the pH to 6.8; Simulated second-stage intestinal fluid: To simulate the distal intestinal environment, a phosphate buffer containing 0.5 w / v% bile salts and 1 w / v% lipase was prepared and the pH was adjusted to 7.4; the lipase was derived from Rhizopus oryzae and had an enzyme activity ≥10000 U / g. Test: Accurately weigh 100 mg of each sample from Application Examples 7-12 and place them in a dialysis bag with a molecular weight cutoff of 10 kDa; Gastric stage: Place the dialysis bag into 100 mL of simulated gastric fluid and incubate at 37 °C and 100 r / min for 2 h with shaking. Intestinal first stage: Remove the dialysis bag, gently rinse the surface with water, transfer it into 100mL of simulated intestinal fluid, and continue incubation for 3 hours under the same conditions; Second stage of intestinal digestion: Remove the dialysis bag, rinse it again, and finally transfer it into 100mL of simulated intestinal fluid. Incubate it under the same conditions for 5 hours to bring the total digestion time to 10 hours. Sampling and determination: At 2h, 5h, 7h and 10h, 1mL of sample was taken from the release medium and replenished with the corresponding fresh medium at the same temperature and volume. After the sample was filtered through a 0.45μm microporous membrane, the concentration of glycyrrhizin in the filtrate was determined by HPLC, and the cumulative release rate of glycyrrhizin at each time point was calculated. The results are shown in Table 3.

[0057] cumulative release rate of glycyrrhizin Q n (%) = (C) n ×V+∑ i=1 n-1 C i ×V i ) / M×100% Among them: Q n : The cumulative release rate at time point n; C n : The concentration of glycyrrhizin in the release medium (μg / mL) at the nth time point; V: Total volume of the release medium (mL); C i : The concentration of glycyrrhizin in the release medium (μg / mL) at the i-th time point; V i : The volume (mL) of the sample taken at the i-th time point; ∑ i=1 n-1 Ci×Vi: The sum of the total amount of glycyrrhizin extracted at all previous time points (μg); M: Total mass (μg) of glycyrrhizin in the sample contained in the dialysis bag.

[0058] Table 3. Results of cumulative release test of glycyrrhizin.

[0059] Application Example 7 may suffer from premature and rapid release of glycyrrhizin due to its free state and lack of effective inclusion protection, making it susceptible to degradation or protonation dissolution in the acidic environment of the stomach, potentially reducing bioavailability. In contrast, Application Examples 8-11 utilize a cross-linked gel network of water-soluble dietary fiber and metal ions, maintaining structural stability in the gastrointestinal environment and effectively blocking the erosion of core components such as glycyrrhizin by digestive enzymes and acidic environments, thus achieving protective sustained release.

[0060] Applications of the inclusion process, including Examples 8-11 and 12, show that glycyrrhizin is encapsulated within a gel network, maintaining a stable gel structure in the gastrointestinal environment. In a simulated distal intestinal environment, due to factors such as increased pH and the presence of bile salts, the gel network gradually relaxes or undergoes responsive degradation, thereby achieving continuous and controlled release of the active ingredient in the primary absorption area of ​​the intestine. In Example 11, fermented licorice root extract forms a robust gel with a pectin-metal ion network, providing a protective mechanism in the stomach and upper intestine. Pectin is sensitive to pH and ionic environments, minimizing ineffective degradation and waste in the upper digestive tract under changing intestinal conditions, ensuring the active ingredient reaches and acts on the intestinal absorption site and related immunomodulatory sites in a highly efficient manner. This inhibits inflammation-induced melanin production at its source, specifically by inhibiting TNF. α, IL The release of inflammatory factors such as type 6 reduces stimulation of melanocytes and avoids ineffective metabolism and waste in the upper digestive tract.

[0061] Test Example 4 Evaluation of skin whitening and anti-pigmentation effects on human skin Participants: This test was a single-arm, self-controlled before-and-after efficacy evaluation study. A total of 34 healthy Chinese women who met the inclusion criteria were recruited, and 30 of them completed all tests and were included in the data analysis; their ages ranged from 25 to 49 years, and their average skin type was Fitzpatrick III; all participants signed informed consent forms, and the trial protocol complied with ethical principles. Instructions for use: Subjects should take one sachet (2g) orally 30-60 minutes before lunch daily. For example, in cases 7-12, the whitening agent should be dissolved in 200-300mL of warm water and taken continuously for 28 days. Evaluation indicators: Measurements were taken before use and 28 days after use under conditions of 21±1℃ and 50±10%RH. Skin melanin and hemoglobin index: MI values ​​were measured on specific pigmented areas of the face using the Mexameter® MX18; Skin pigmentation analysis: Using the VISIA® CR facial image acquisition system and IPP software, the proportion of facial pigmentation area was analyzed. The lower the proportion, the more obvious the improvement in pigmentation. Skin color and luster: The L* value of the skin brightness on the back was measured using a Colorimeter® CL400 colorimeter; skin luster was analyzed using a VISIA® CR. UV-induced pigmentation model: An artificial UV irradiation model and a darkened area were established on the back of the subjects. The melanin MI value in this area was measured again 28 days after sample use to evaluate the product's protective effect against UV-induced pigmentation. The results are shown in Tables 4 and 5.

[0062] Subject self-assessment: After 28 days of use, subjects' subjective feelings about skin tone, pigmentation, radiance, and overall skin condition were assessed using a standardized questionnaire. The results are shown in Table 6.

[0063] Table 4 Evaluation Results of Skin Whitening Efficacy in Human Skin

[0064] Table 5. Evaluation results of anti-pigmentation efficacy of human skin.

[0065] Table 6. Subjects' self-assessment results

[0066] The lower the MI value, the less melanin content there is, and the more obvious the whitening effect.

[0067] The lower the percentage of facial pigmentation area, the more obvious the improvement in pigmentation.

[0068] The higher the brightness value of the skin on the back, the brighter and whiter the skin tone, and the better the brightening effect.

[0069] The MI value of the UV-induced darkening area on the back reflects the ability to protect against and repair UV-induced pigmentation.

[0070] The GU value of skin radiance reflects the improvement in skin condition.

[0071] Application examples 7-11 showed whitening effects, improved radiance, and reduced pigmentation caused by UV exposure after 28 days of use; and no adverse reactions occurred.

Claims

1. Application of licorice extract in oral whitening agents.

2. The application as described in claim 1, characterized in that: The preparation method of the licorice extract includes the following steps: licorice root → coarse crushing → extraction → filtration → refining → concentration → encapsulation → drying → fine crushing → mixing → sieving and iron removal → metal detection → packaging; The coarse grinding process involves coarsely grinding, fermenting, and drying the licorice root. The extraction process involves reflux extraction of crude licorice root powder with an ethanol aqueous solution, and combining the extracts to obtain a licorice extract. The refining process involves filtering the licorice extract and then performing centrifugation and membrane filtration to obtain a refined licorice extract. The inclusion process involves mixing concentrated licorice extract with a water-soluble dietary fiber solution, atomizing the mixture, and then introducing it into a metal salt coagulation bath to form inclusion particles. The drying process involves spray drying the resulting inclusion particles.

3. The application as described in claim 2, characterized in that: The coarse grinding process is as follows: take 200-500g of dried licorice root coarse powder and grind it at 1000-3000r / min, then pass it through a 30-40 mesh sieve; add 3-5 times the amount of water and adjust the pH to 5.0 with citric acid aqueous solution; add 1-5g of pectinase and 0.5-3g of cellulase, and enzymatically hydrolyze at 40-50℃ for 60-120min; then raise the temperature to 80-90℃ and maintain it for 10-15min; after cooling to 25-30℃, inoculate with 0.1-1.0g of Lactobacillus plantarum and ferment at 25-35℃ for 36-60h; then dry at 40-60℃, grind at 2000-5000r / min and sieve.

4. The application as described in claim 2, characterized in that: In the extraction process, the reflux extraction temperature is 70-85℃, each extraction lasts 1-3 hours, and the extraction is repeated 2-3 times; the concentration of the ethanol aqueous solution is 60-80 vol.

5. The application as described in claim 2, characterized in that: The purification process involves centrifuging at 3000-5000 r / min for 5-10 min, then filtering and collecting the retentate through an ultrafiltration membrane with a molecular weight cutoff of 5-10 kDa and a nanofiltration membrane with a molecular weight cutoff of 200-500 Da.

6. The application as described in claim 2, characterized in that: In the inclusion process, 5-15g of water-soluble dietary fiber is stirred in 800-1000mL of water at 40-50℃ and 200-500r / min for 30-60min; then the obtained licorice concentrate is added, and after centrifugation and atomization at 10000-20000r / min, it is added to a coagulation bath of 800-1000mL of metal salt to form licorice extract inclusion particles, which are collected by sieve and rinsed with water.

7. The application as described in claim 2 or 6, characterized in that: The water-soluble dietary fiber is any one of low-methoxyl pectin, gellan gum, and sodium alginate; the metal salt is any one of calcium chloride and zinc sulfate.

8. The application as described in claim 2, characterized in that: In the drying process, the inlet air temperature for spray drying is set to 160-190℃, and the outlet air temperature is set to 75-90℃.

9. The application as described in claim 2, characterized in that: In the iron removal process, the iron is first passed through an 80-100 mesh sieve and magnetic separation is performed simultaneously, with a magnetic induction intensity of ≥8000GS.

10. The application as described in claim 2, characterized in that: In the gold detection process, the sensitivity is set as follows: iron ≥ 0.5 mm, non-ferrous metals ≥ 1.0 mm.

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