Huangqi callus extract and preparation method thereof, and cosmetic

Abstract

The application relates to the field of cosmetics, and relates to a radix astragali callus extract, a preparation method thereof and a cosmetic. The method for preparing the radix astragali callus extract comprises the following steps: culturing to obtain radix astragali callus under long-day conditions; and extracting the radix astragali callus by using propylene glycol as a solvent. The radix astragali callus is obtained by inoculation in an induction medium, the induction medium comprises 6-benzylaminopurine, naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid, the concentration of 6-benzylaminopurine is 0.5-2.0 mg / L based on the total volume of the induction medium, the concentration of naphthaleneacetic acid is 0.5-2.0 mg / L, and the concentration of 2,4-dichlorophenoxyacetic acid is 0.1-1.0 mg / L. The technical scheme has the advantages that the content and quality of active ingredients are significantly improved. The obtained extract has strong antioxidant and whitening activities, no other organic solvent residues, clear color, and has a good prospect as a cosmetic raw material.

Description

Technical Field

[0001] This application relates to the field of cosmetics, specifically to an extract of Astragalus membranaceus callus tissue, its preparation method, and cosmetics. Background Technology

[0002] Astragalus, a traditional Chinese medicine, contains saponins, flavonoids, and polysaccharides, whose active ingredients have been proven to possess various biological activities such as antioxidant, whitening, and immunomodulatory effects, showing broad application prospects in the pharmaceutical and cosmetic fields. Currently, the extraction of active ingredients from astragalus mainly relies on the roots of its wild or cultivated plants. However, this method is limited by problems such as long growth cycle, limited resources, and the content of active ingredients being greatly affected by environmental factors, making it difficult to meet the needs of standardized and large-scale production. Summary of the Invention

[0003] The purpose of this application is to provide an antioxidant component in Astragalus callus, its extraction method, and a cosmetic.

[0004] In a first aspect, this application provides a method for preparing Astragalus callus extract, comprising: Astragalus callus tissue was obtained by culturing under long-day conditions; the Astragalus callus tissue was extracted using propylene glycol as a solvent. The Astragalus callus tissue was obtained by inoculation into an induction medium containing 6-benzylaminopurine, naphthaleneacetic acid, and 2,4-dichlorophenoxyacetic acid. Based on the total volume of the induction medium, the concentration of 6-benzylaminopurine was 0.5-2.0 mg / L; the concentration of naphthaleneacetic acid was 0.5-2.0 mg / L; and the concentration of 2,4-dichlorophenoxyacetic acid was 0.1-1.0 mg / L.

[0005] The above-mentioned technical solution, through long-day cultivation, induction of Astragalus membranaceus callus tissue using an induction medium with a specific hormone ratio, and the first-ever use of propylene glycol as a solvent for extraction, demonstrates the synergistic effect of these technical features, significantly improving the content and quality of active ingredients. The specific long-day conditions combined with the optimized hormone ratio (synergistic effect of 6-benzylaminopurine, naphthaleneacetic acid, and 2,4-dichlorophenoxyacetic acid) effectively induce vigorous growth, loose structure, and high degree of dedifferentiation in Astragalus membranaceus callus tissue, resulting not only in a high callus yield but also in a significantly higher accumulation of antioxidant components such as total saponins compared to conventional dark culture methods. Furthermore, the extraction process of this technical solution is safe, efficient, and suitable for direct application in cosmetics. The extraction solvent used is propylene glycol, a commonly used moisturizer in cosmetics, avoiding the residue problems of traditional organic solvents (such as ethanol) and ensuring the safety of the extract. The extract obtained by this method is clear in color and can be used directly as a cosmetic raw material without complex solvent removal steps, simplifying subsequent processing procedures. Furthermore, the above-mentioned technical solution can obtain a highly active final product. The Astragalus callus extract prepared by this method has both excellent antioxidant activity (such as DPPH free radical scavenging rate) and whitening activity (such as tyrosinase inhibition rate). Its comprehensive biological activity is superior to the products prepared by traditional water extraction or alcohol extraction methods, providing an ideal choice for the development of highly efficient natural cosmetic raw materials.

[0006] In other embodiments of this application, culturing Astragalus callus tissue under long-day conditions includes: Astragalus callus was cultured under the following conditions: temperature 24℃~26℃, relative humidity 50%~70%, light intensity 1500 lux~2500 lux, and light duration 15h / d~20h / d.

[0007] In other embodiments of this application, the concentration of 6-benzylaminopurine is 1.0-2.0 mg / L, the concentration of naphthaleneacetic acid is 1.0-2.0 mg / L, and the concentration of 2,4-dichlorophenoxyacetic acid is 0.5-1.0 mg / L, based on the total volume of the induction medium.

[0008] In other embodiments of this application, the induction culture medium includes: MS powder, sucrose, ascorbic acid, polyvinylpyrrolidone, and agar; In other embodiments of this application, based on the total volume of the induction medium, the concentration of MS powder is 4 g / L to 5 g / L; the concentration of sucrose is 25 g / L to 35 g / L; the concentration of ascorbic acid is 0.05 g / L to 0.15 g / L; the concentration of polyvinylpyrrolidone is 3.0 g / L to 6.0 g / L; and the concentration of agar is 7.0 to 8.0 g / L.

[0009] In other embodiments of this application, the pH of the induction medium is 5.8-5.9.

[0010] In other embodiments of this application, propylene glycol is used as a solvent to extract Astragalus callus tissue, including: Propylene glycol with a volume fraction of 20%-80% was used to extract Astragalus callus tissue.

[0011] In other embodiments of this application, propylene glycol is used as a solvent to extract Astragalus callus tissue, including: The Astragalus callus tissue was extracted under the action of ultrasound, with an ultrasound power of 700W~750W.

[0012] In other embodiments of this application, propylene glycol is used as a solvent to extract Astragalus callus tissue, including: Astragalus callus tissue was extracted at a temperature of 50℃-70℃.

[0013] In other embodiments of this application, the extraction time of Astragalus callus is 20 min to 50 min.

[0014] Secondly, this application provides an extract of Astragalus callus tissue, which is prepared by any of the methods described in the first aspect above.

[0015] Thirdly, this application provides a cosmetic product comprising the Astragalus callus extract provided in the second aspect above.

[0016] In other embodiments of this application, the amount of Astragalus callus extract added to the cosmetic is 1.25% to 5.0%. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are some embodiments of this application, but not all embodiments.

[0018] Therefore, the following detailed description of the embodiments of this application is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments of the application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0019] Research has shown that using Astragalus callus tissue to produce active ingredients is considered a promising alternative. However, existing callus culture methods mostly employ dark culture or conventional hormone combinations, which generally suffer from slow callus growth, dense texture, uneven dedifferentiation, and low accumulation of target active substances (such as total saponins). Further research has revealed that in the extraction stage, current technologies primarily use water, ethanol, or other organic solvents to extract plant materials. These solvents have significant drawbacks when used as cosmetic raw materials: water extraction efficiency is low, while organic solvents such as ethanol may cause skin irritation and remain in the final product, affecting its safety and application range.

[0020] For cosmetic applications, there is a need to develop a new method for the efficient production of highly active Astragalus extract. This method should be able to stably obtain callus tissue with vigorous growth and high content of active ingredients, and use safe and efficient extraction solvents so that the product can be directly and safely used in cosmetics.

[0021] Based on the above research, this application provides a method for preparing Astragalus callus extract, comprising: Astragalus callus tissue was obtained by culturing under long-day conditions; the Astragalus callus tissue was extracted using propylene glycol as a solvent. The Astragalus callus tissue was obtained by inoculation into an induction medium containing 6-benzylaminopurine, naphthaleneacetic acid, and 2,4-dichlorophenoxyacetic acid. Based on the total volume of the induction medium, the concentration of 6-benzylaminopurine was 0.5-2.0 mg / L; the concentration of naphthaleneacetic acid was 0.5-2.0 mg / L; and the concentration of 2,4-dichlorophenoxyacetic acid was 0.1-1.0 mg / L.

[0022] The above-mentioned technical solution, through long-day cultivation, induction of Astragalus membranaceus callus tissue using an induction medium with a specific hormone ratio, and the first-ever use of propylene glycol as a solvent for extraction, demonstrates the synergistic effect of these technical features, significantly improving the content and quality of active ingredients. The specific long-day conditions combined with the optimized hormone ratio (synergistic effect of 6-benzylaminopurine, naphthaleneacetic acid, and 2,4-dichlorophenoxyacetic acid) effectively induce vigorous growth, loose structure, and high degree of dedifferentiation in Astragalus membranaceus callus tissue, resulting not only in a high callus yield but also in a significantly higher accumulation of antioxidant components such as total saponins compared to conventional dark culture methods. Furthermore, the extraction process of this technical solution is safe, efficient, and suitable for direct application in cosmetics. The extraction solvent used is propylene glycol, a commonly used moisturizer in cosmetics, avoiding the residue problems of traditional organic solvents (such as ethanol) and ensuring the safety of the extract. The extract obtained by this method is clear in color and can be used directly as a cosmetic raw material without complex solvent removal steps, simplifying subsequent processing procedures. Furthermore, the above-mentioned technical solution can obtain a highly active final product. The Astragalus callus extract prepared by this method has both excellent antioxidant activity (such as DPPH free radical scavenging rate) and whitening activity (such as tyrosinase inhibition rate). Its comprehensive biological activity is superior to the products prepared by traditional water extraction or alcohol extraction methods, providing an ideal choice for the development of highly efficient natural cosmetic raw materials.

[0023] The above technical solution provides an optimized whole-chain approach from callus culture to extraction and preparation, ultimately achieving the comprehensive benefits of efficiently preparing highly active, safe, and directly applicable Astragalus callus antioxidant extract.

[0024] Further optionally, and exemplaryly, in some embodiments of this application, based on the total volume of the induction medium, the concentration of 6-benzylaminopurine is 0.5 mg / L, 0.6 mg / L, 0.8 mg / L, 1.0 mg / L, 1.2 mg / L, 1.5 mg / L, 1.8 mg / L, 1.9 mg / L, 2.0 mg / L, or a range between any two of the aforementioned values; the concentration of naphthaleneacetic acid is 0.5 mg / L, 0.6 mg / L, 0.8 mg / L, 1.0 mg / L, 1.2 mg / L, 1.5 mg / L, 1.8 mg / L, 1.9 mg / L, 2.0 mg / L, or a range between any two of the aforementioned values; and the concentration of 2,4-dichlorophenoxyacetic acid is 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, or a range between any two of the aforementioned values. mg / L, 1.0 mg / L, or any range between the two aforementioned values.

[0025] Further optionally, in some embodiments of this application, the concentration of 6-benzylaminopurine is 0.8 mg / L - 2.0 mg / L; the concentration of naphthaleneacetic acid is 0.8 mg / L - 2.0 mg / L; and the concentration of 2,4-dichlorophenoxyacetic acid is 0.2 mg / L - 1.0 mg / L, based on the total volume of the induction medium.

[0026] Further optionally, in some embodiments of this application, the concentration of 6-benzylaminopurine is 1.0 mg / L - 2.0 mg / L, the concentration of naphthaleneacetic acid is 1.0 mg / L - 2.0 mg / L, and the concentration of 2,4-dichlorophenoxyacetic acid is 0.5 mg / L - 1.0 mg / L, based on the total volume of the induction medium.

[0027] Furthermore, in some embodiments of this application, culturing Astragalus callus tissue under long-day conditions includes: Astragalus callus was cultured under the following conditions: temperature 24℃~26℃, relative humidity 50%~70%, light intensity 1500 lux~2500 lux, and light duration 15h / d~20h / d.

[0028] Exemplarily, in some embodiments of this application, culturing Astragalus membranaceus callus under long-day conditions includes: Astragalus callus was cultured under the following conditions: temperature 24℃, 24.5℃, 25℃, 25.5℃, 26℃; relative humidity 50%, 55%, 60%, 65%, 70% or any two of the aforementioned values; light intensity 1500 lux, 1600 lux, 1800 lux, 2000 lux, 2200 lux, 2300 lux, 2400 lux, 2500 lux or any two of the aforementioned values; and light duration 15 h / d, 15.5 h / d, 16 h / d, 16.5 h / d, 17 h / d, 17.5 h / d, 18 h / d, 18.5 h / d, 19 h / d, 19.5 h / d, 20 h / d or any two of the aforementioned values.

[0029] Furthermore, in some embodiments of this application, the induction culture medium includes: MS powder, sucrose, ascorbic acid, polyvinylpyrrolidone, and agar.

[0030] Further optionally, based on the total volume of the induction medium, the concentration of MS powder is 4 g / L to 5 g / L; the concentration of sucrose is 25 g / L to 35 g / L; the concentration of ascorbic acid is 0.05 g / L to 0.15 g / L; the concentration of polyvinylpyrrolidone is 3.0 g / L to 6.0 g / L; and the concentration of agar is 7.0 to 8.0 g / L.

[0031] For example, in some embodiments of this application, based on the total volume of the induction medium, the concentration of MS powder is 4 g / L, 4.1 g / L, 4.2 g / L, 4.3 g / L, 4.4 g / L, 4.5 g / L, 4.6 g / L, 4.7 g / L, 4.8 g / L, 4.9 g / L, 5 g / L, or any two of the aforementioned values; the concentration of sucrose is 25 g / L, 26 g / L, 28 g / L, 30 g / L, 32 g / L, 33 g / L, 34 g / L, 35 g / L, or 35 g / L. g / L or any two of the aforementioned values; ascorbic acid concentrations of 0.05 g / L, 0.06 g / L, 0.07 g / L, 0.08 g / L, 0.09 g / L, 0.10 g / L, 0.12 g / L, 0.13 g / L, 0.14 g / L, 0.15 g / L or any two of the aforementioned values; polyvinylpyrrolidone concentrations of 3.0 g / L, 3.2 g / L. The concentrations are 3.5 g / L, 3.8 g / L, 4.0 g / L, 4.2 g / L, 4.5 g / L, 4.8 g / L, 5.0 g / L, 5.2 g / L, 5.5 g / L, 5.8 g / L, 6.0 g / L, or any two of the aforementioned values; the agar concentrations are 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0 g / L, or any two of the aforementioned values.

[0032] Furthermore, in some embodiments of this application, the pH of the induction culture medium is 5.8-5.9.

[0033] For example, in some embodiments of this application, the pH value of the induction culture medium is 5.8, 5.85, 5.9, or any two of the aforementioned values.

[0034] Furthermore, in some embodiments of this application, propylene glycol is used as a solvent to extract Astragalus callus tissue, including: Propylene glycol with a volume fraction of 20%-80% was used to extract Astragalus callus tissue.

[0035] For example, in some embodiments of this application, propylene glycol with a volume fraction of 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or any two of the aforementioned values ​​is used to extract Astragalus callus tissue.

[0036] Further, optionally, in some embodiments of this application, propylene glycol is used as a solvent to extract Astragalus callus tissue, including: Propylene glycol with a volume fraction of 30%-60% was used to extract Astragalus callus tissue.

[0037] Furthermore, in some embodiments of this application, propylene glycol is used as a solvent to extract Astragalus callus tissue, including: The Astragalus callus tissue was extracted under the action of ultrasound, with an ultrasound power of 700W~750W.

[0038] Exemplary, in some embodiments of this application, propylene glycol is used as a solvent to extract Astragalus callus tissue, including: Under the action of ultrasound, Astragalus callus tissue was extracted, with ultrasound power of 700W, 705W, 710W, 715W, 720W, 725W, 730W, 735W, 740W, 745W, 750W or any two of the aforementioned values.

[0039] Furthermore, in some embodiments of this application, propylene glycol is used as a solvent to extract Astragalus callus tissue, including: The Astragalus callus tissue was extracted under the action of ultrasound, with an ultrasound power of 710W~740W.

[0040] Furthermore, in some embodiments of this application, propylene glycol is used as a solvent to extract Astragalus callus tissue, including: Astragalus callus tissue was extracted at a temperature of 50℃-70℃.

[0041] Exemplary, in some embodiments of this application, propylene glycol is used as a solvent to extract Astragalus callus tissue, including: Astragalus callus was extracted at temperatures ranging from 50℃, 52℃, 53℃, 54℃, 55℃, 56℃, 58℃, 60℃, 62℃, 65℃, 68℃, 70℃, or any two of the aforementioned values.

[0042] Further, optionally, in some embodiments of this application, propylene glycol is used as a solvent to extract Astragalus callus tissue, including: Astragalus callus was extracted at a temperature of 55℃-60℃.

[0043] Exemplarily, further, in some embodiments of this application, a method for preparing Astragalus callus extract includes the following steps: Step S1: Obtaining Astragalus callus tissue.

[0044] Furthermore, in some embodiments of this application, the step of obtaining Astragalus callus tissue includes: (1) Seed pretreatment, disinfection and sowing: Select fresh, plump Astragalus seeds, soak them in sterile water overnight, then evenly place them on a damp gauze and incubate in the dark. After 2 days, discard any moldy seeds, collect the remaining seeds, and sterilize them.

[0045] In a clean bench, disinfect the surface with 75% ethanol for 30-60 seconds, rinse off the ethanol with sterile water, then disinfect with 5% sodium hypochlorite solution for 8-12 minutes, and rinse with sterile water 4-5 times.

[0046] Sterilized Astragalus seeds were sown on 1 / 2 MS solid medium without any growth regulators and cultured in the dark. After germination, they were transferred to long-day culture. Long-day culture conditions: temperature 25℃, relative humidity 60%, light intensity 2000 lux, and photoperiod 16 h / d.

[0047] (2) Callus induction: Aseptic seedlings of Astragalus membranaceus cultured for 14-28 days under long-day conditions were used as material. The hypocotyls were cut into 0.5-1.0 cm segments as explants and inoculated onto the induction medium. After 3 days of dark culture, the culture was transferred to long-day culture. The induction medium contained the following components: MS powder 4.74 g / L, sucrose 25-35 g / L, 6-benzylaminopurine (6-BA) 0.5-2.0 mg / L, naphthaleneacetic acid (NAA) 0.5-2.0 mg / L, 2,4-dichlorophenoxyacetic acid (2,4-D) 0.1-1.0 mg / L, ascorbic acid 0.1 g / L, polyvinylpyrrolidone 5.0 g / L, and agar 7.0-8.0 g / L; the pH of the medium was 5.8-5.9.

[0048] (3) Subculture and proliferation of callus: Callus formation begins approximately 14 days after explant inoculation. The well-grown Astragalus membranaceus callus obtained through induction is cut into small pieces and inoculated onto the above-mentioned culture medium for subculture and proliferation. The preferred inoculation density is 8-12 callus / 50 cm². 2 The subculture cycle is 10-20 days. The specific induction conditions of this invention can produce pale yellow-green, loosely structured, and vigorously growing Astragalus membranaceus callus tissue.

[0049] Step S2: Preparation of propylene glycol extract from Astragalus callus.

[0050] Furthermore, in some embodiments of this application, vigorously growing Astragalus callus tissue is taken and homogenized. Using propylene glycol of different volume fractions as solvent, the Astragalus callus tissue is extracted using ultrasound-assisted extraction technology. The ultrasound power is 720W, the extraction temperature is 60℃, and the extraction time is 30 min. After extraction, the tissue is centrifuged at 6000-12000 rpm for 5-15 min, and the supernatant is taken as the Astragalus callus tissue propylene glycol extract.

[0051] Some embodiments of this application provide an Astragalus callus extract, which is prepared using the method for preparing Astragalus callus extract provided in any of the foregoing embodiments.

[0052] Some embodiments of this application provide a cosmetic product comprising the Astragalus callus extract provided in any of the foregoing embodiments.

[0053] In other embodiments of this application, the amount of Astragalus callus extract added to the cosmetic is 1.25% to 5.0%.

[0054] For example, in the cosmetic, the amount of Astragalus callus extract added is 1.25%, 1.30%, 1.35%, 1.40%, 1.45%, 1.50%, 1.85%, 2.0%, 2.15%, 2.5%, 2.8%, 3.0%, 3.5%, 3.8%, 4.0%, 4.5%, 4.8%, 5.0%, or any two of the aforementioned values.

[0055] This application describes the induction of Astragalus membranaceus callus under long-day conditions. The explant material used is readily available, and the obtained Astragalus membranaceus callus is pale yellow-green, exhibits high dedifferentiation, good looseness, and rapid growth. This application uses propylene glycol, a moisturizing agent directly applicable to cosmetics, as a solvent to prepare the Astragalus membranaceus callus extract. This method is time-efficient, low-cost, and highly safe, leaving no other reagent residues. This application is the first to use propylene glycol as a solvent to extract active ingredients from Astragalus membranaceus callus, demonstrating strong antioxidant and whitening activities, and showing promising potential as a cosmetic raw material.

[0056] The features and performance of this application will be further described in detail below with reference to embodiments: Astragalus callus tissues numbered 1-9 were prepared. Detailed process parameters for the preparation of Astragalus callus tissues numbered 1-9 are shown in Table 1. The preparation steps are as follows: (1) Select fresh and plump Astragalus seeds, soak them in sterile water overnight, and then place them evenly on a damp gauze for dark incubation. After 2 days, discard the moldy seeds, collect the remaining seeds and disinfect them.

[0057] (2) Disinfect the surface with 75% ethanol for 50 seconds in a clean bench, rinse off the ethanol with sterile water, then disinfect with 5% sodium hypochlorite solution for 10 minutes, and rinse with sterile water 5 times.

[0058] (3) The sterilized Astragalus seeds were sown on 1 / 2 MS solid medium without any growth regulators and cultured in the dark. After germination, they were transferred to long-day culture for 20 days to obtain sterile Astragalus seedlings. Long-day culture conditions: temperature 25℃, relative humidity 60%, light intensity 2000 lux, and light duration 16 h / d.

[0059] (4) Using the aseptic seedlings of Astragalus membranaceus obtained in step (3) above as material, the hypocotyl was cut into 0.5 cm segments as explants and inoculated on various induction culture media. After dark culture for 3 days, it was transferred to long-day culture. The growth status of each callus tissue was recorded, as detailed in Table 1.

[0060] Each induction medium contained 4.74 g / L MS powder, 30 g / L sucrose, 0.1 g / L ascorbic acid, 5.0 g / L polyvinylpyrrolidone, and 7.0-8.0 g / L agar; the pH of the medium was 5.8-5.9. The differences between the various induction media are detailed in Table 1 below.

[0061] Table 1. Effects of different culture media on Astragalus membranaceus callus formation

[0062] As can be seen from the data in the table above: Based on the total volume of the induction medium, the concentration of 6-benzylaminopurine is 0.5-2.0 mg / L; the concentration of naphthaleneacetic acid is 0.5-2.0 mg / L; and the concentration of 2,4-dichlorophenoxyacetic acid is 0.1-1.0 mg / L. Within this range, and under long-day conditions, callus tissue can be effectively induced.

[0063] Furthermore, explants cultured in media 9, 6, and 3 showed superior performance, with callus emergence rates ranging from 66.29% to 76.84%. This indicates that, based on the total volume of the induction medium, the concentrations of 6-benzylaminopurine (0.5-2.0 mg / L), naphthaleneacetic acid (2 mg / L), and 2,4-dichlorophenoxyacetic acid (0.1-1.0 mg / L), within this range, and under long-day conditions, are more conducive to callus culture.

[0064] Furthermore, the callus emergence rates of explants in media 9 and 6 were superior, ranging from 75.56% to 76.84%. This indicates that, based on the total volume of the induction medium, the concentrations of 6-benzylaminopurine (1-2.0 mg / L), naphthaleneacetic acid (2 mg / L), and 2,4-dichlorophenoxyacetic acid (0.1-0.5 mg / L), under long-day conditions, are more conducive to callus culture, inducing dense, green callus, and promoting abundant proliferation.

[0065] Example 1 A method for preparing Astragalus callus extract is provided, comprising the following steps: The Astragalus callus tissue obtained above (serial number 3) was cut into small pieces and inoculated onto the culture medium corresponding to serial number 3 for subculture and proliferation. The culture conditions were maintained under long-day conditions, and the culture medium was replaced with fresh medium every 14 days for 28 days; vigorous Astragalus callus tissue was obtained.

[0066] Vigorously growing Astragalus callus tissue was collected and homogenized. Using propylene glycol as the extraction solvent, ultrasonic-assisted extraction was performed on the Astragalus callus tissue at a power of 720W, an extraction temperature of 60℃, and an extraction time of 30 min. After extraction, the tissue was centrifuged at 10000 rpm for 10 min, and the supernatant was collected as the propylene glycol extract of the Astragalus callus tissue.

[0067] Example 2 A method for preparing Astragalus callus extract is provided, which differs from Example 1 in that it uses Astragalus callus tissue No. 6 and its corresponding culture medium for subculture and proliferation.

[0068] Example 3 A method for preparing Astragalus callus extract is provided, which differs from Example 1 in that it uses Astragalus callus tissue (serial number 9) and its corresponding culture medium for subculture and proliferation.

[0069] Examples 4-9 A method for preparing Astragalus callus extract is provided, which differs from Example 2 in the concentration of the extractant, as detailed in Table 2.

[0070] Comparative Examples 1-3 A method for preparing Astragalus callus extract is provided, which differs from Examples 1-3 in the culture conditions, as detailed in Table 2.

[0071] Comparative Example 4-5 A method for preparing Astragalus callus extract is provided, which differs from Example 2 in the concentration of the extractant, as detailed in Table 2.

[0072] Table 2

[0073] The performance of the samples obtained from the above embodiments and comparative examples was tested: 1. Determination of total saponins in Astragalus callus tissue obtained from each example and comparative example.

[0074] The method for determining total saponins is as follows: Take 10 g of Astragalus callus tissue obtained from each example or comparative culture, add 75% ethanol solution at a material-to-liquid ratio of 1:30 (g / mL), and extract with ultrasonic assistance. The ultrasonic power is 720 W, the extraction temperature is 60℃, and the extraction time is 30 min. After extraction, take 1 mL of the supernatant of each sample into a test tube, add 1 mL of 8% vanillin anhydrous ethanol solution, mix thoroughly, place in an ice bath, slowly add 10 mL of 72% sulfuric acid solution, shake well, and incubate in a 62℃ water bath for 20 min. After removal, immediately cool to room temperature in an ice bath. Measure the absorbance value at 544 nm using an ELISA reader. Plot a standard curve regression equation with the concentration of astragaloside A standard solution as the x-axis and the absorbance value as the y-axis, and calculate the total saponin content in each sample based on this curve.

[0075] The test results are shown in Table 3.

[0076] Table 3

[0077] As can be seen from the results in Table 3 above: The Astragalus callus tissue prepared in the various embodiments of this application has a high total saponin content. The total saponin content is 1.88~1.97 mg / g.

[0078] Furthermore, comparing Examples 1-3 with Comparative Examples 1-3, it can be seen that when the culture conditions are long-day culture, the total saponin content is significantly increased compared with dark culture.

[0079] 2. The properties of the Astragalus callus extracts prepared in each embodiment and comparative example were tested.

[0080] (1) The test method for DPPH free radical scavenging rate is as follows: DPPH·, also known as 1,1-diphenyl-2-trinitrophenylhydrazine, is a very stable nitrogen-centered free radical. Its stability primarily stems from the steric hindrance of the three benzene rings through resonance stabilization, preventing the unpaired electrons on the central nitrogen atom from performing their proper electron pairing function. Its anhydrous ethanol solution is purple, exhibiting maximum absorption at 517 nm, with absorbance showing a linear relationship with concentration. Adding free radical scavengers can bind to or replace DPPH·, reducing the number of free radicals, decreasing absorbance, and lightening the solution color, thus allowing evaluation of its free radical scavenging ability.

[0081] The specific experimental steps are as follows: Each example or comparative sample was diluted 20 times with water to obtain the test sample; 2.0 mL of each test sample and 2.0 mL of DPPH solution were respectively pipetted into a stoppered test tube and mixed well; the reaction was carried out in the dark for 30 min, and the absorbance value at a wavelength of 517 nm was measured.

[0082]

[0083] In the formula: A0: No sample added, DPPH added; A1: Add sample, add DPPH; A2: Sample added, no DPPH added.

[0084] The test results of the DPPH free radical scavenging rate of the fermentation products of each embodiment and comparative example are shown in Table 5.

[0085] (2) The method for testing tyrosinase inhibition rate is as follows: Referring to the group standard T / SHRH 015-2018 "Cosmetics - Test Method for Tyrosinase Activity Inhibition", tyrosinase can catalyze the conversion of dopa to dopaquinone in a phosphate solution with pH=6.8, and the absorbance can be measured at 475 nm using a spectrophotometer. Prepare a phosphate buffer solution to dilute the test sample to a 5% concentration, and prepare a 1 mg / ml levodopa solution and a 100 KU tyrosinase solution. After adding reagents according to the composition shown in the table below and allowing the reaction to proceed fully, measure the absorbance at 475 nm.

[0086] Calculation formula based on tyrosinase inhibition rate: Tyrosinase inhibition rate = (1 - (B - B') / (A - A')) × 100%.

[0087] Table 4

[0088] The test results are shown in Table 5.

[0089] Table 5

[0090] As can be seen from the results in Table 5 above: Compared with Comparative Example 4 (Aqueous extract of Astragalus callus tissue), it can be seen that the DPPH free radical scavenging rate and tyrosinase inhibition rate of each embodiment of this application are significantly improved, which shows that the solution of the embodiment of this application can improve the antioxidant and whitening effects of Astragalus callus tissue extract.

[0091] Furthermore, comparing Examples 4-9, it can be seen that the DPPH free radical scavenging rate and tyrosinase inhibition rate of extracts with different propylene glycol volume fractions show a trend of first increasing and then decreasing with increasing propylene glycol concentration. Further, optionally, when the concentration of the propylene glycol solution is 50%~60%, the extracted Astragalus callus extract exhibits stronger antioxidant activity and tyrosinase inhibition rate.

[0092] 3. Investigate the effect of Astragalus callus extract obtained in Example 3 on melanin inhibition rate. Experimental methods: Cell experiments Specific testing steps for cell experiments: Logarithmic growth phase mouse melanoma cells (B16 cells) were seeded into 24-well plates at a density of 20,000 cells / well and incubated for 24 h. Subsequently, the test samples were diluted to the appropriate concentration with DMEM medium containing 2% fetal bovine serum (FBS) and added to the 24-well plates, and incubated for 24 h.

[0093] B16 cells were washed twice with PBS after sample treatment. The PBS was removed, and 100 μL of 0.25% trypsin was added to digest the cells. The cells were resuspended in 0.5 mL of PBS and collected into centrifuge tubes. The cells were centrifuged at 4000 r / min for 5 min. The PBS residue in the centrifuge tubes was removed, and 200 μL of melanin extraction solution (1 mol / L NaOH solution containing 10% DMSO) was added to each tube. The mixture was stirred and heated in an 80°C water bath for 1 h. After cooling, the cells were centrifuged moderately, and 150 μL of the supernatant from each centrifuge tube was collected and the absorbance was measured at 405 nm.

[0094] Calculate the melanin inhibition rate using the following formula.

[0095] Melanin inhibition rate (%) = [1 [(Sample average absorbance / Blank average absorbance)] × 100 The experimental results are shown in Table 6.

[0096] Table 6

[0097] The experimental data in Table 6 above show that: Compared to the control group, the Astragalus callus extract provided in this application showed a significant inhibitory effect on melanin biosynthesis in B16 cells in a dose-dependent manner. When the concentration of the callus extract was 2.50-5%, the inhibition rate of melanin biosynthesis was significantly increased, reaching 23.94%-28.54%, which was significantly higher than that of the control group.

[0098] In summary, this application induced and proliferated Astragalus membranaceus callus to obtain Astragalus membranaceus callus tissue, and for the first time used propylene glycol as a solvent combined with ultrasound-assisted extraction technology to extract Astragalus membranaceus callus tissue into a propylene glycol extract. The obtained extract exhibits strong antioxidant and whitening activities, has no other organic solvent residues, and is clear in color, showing good potential as a cosmetic raw material.

[0099] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for preparing Astragalus callus extract, characterized in that, include: Culture of Astragalus callus under long-day conditions; The Astragalus callus was extracted using propylene glycol at a volume fraction of 20%-80%; The Astragalus callus tissue was obtained by inoculation into an induction medium, the composition of which was: 6-benzylaminopurine, naphthaleneacetic acid, 2,4-dichlorophenoxyacetic acid, MS powder, sucrose, ascorbic acid, polyvinylpyrrolidone, and agar. Based on the total volume of the induction medium, the concentrations of the following were: 6-benzylaminopurine: 0.5-2.0 mg / L; naphthaleneacetic acid: 0.5-2.0 mg / L; 2,4-dichlorophenoxyacetic acid: 0.1-1.0 mg / L; MS powder: 4 g / L-5 g / L; sucrose: 25 g / L-35 g / L; ascorbic acid: 0.05 g / L-0.15 g / L; polyvinylpyrrolidone: 3.0 g / L-6.0 g / L; agar: 7.0-8.0 g / L; and the pH of the induction medium was 5.8-5.

9. The cultivation of Astragalus callus tissue under long-day conditions includes: Astragalus callus was cultured under the following conditions: temperature 24℃~26℃, relative humidity 50%~70%, light intensity 1500 lux~2500 lux, and light duration 15h / d~20h / d.

2. The method for preparing Astragalus callus extract according to claim 1, characterized in that, Based on the total volume of the induction medium, the concentration of 6-benzylaminopurine is 1.0-2.0 mg / L; the concentration of naphthaleneacetic acid is 1.0-2.0 mg / L; and the concentration of 2,4-dichlorophenoxyacetic acid is 0.5-1.0 mg / L.

3. The method for preparing Astragalus callus extract according to any one of claims 1-2, characterized in that, Extraction of the Astragalus callus tissue using propylene glycol as a solvent includes: The Astragalus callus tissue was extracted under the action of ultrasound, with an ultrasound power of 700W~750W.

4. The method for preparing Astragalus callus extract according to claim 1, characterized in that, Extraction of the Astragalus callus tissue using propylene glycol as a solvent includes: The Astragalus callus tissue was extracted at a temperature of 50℃-70℃.

5. The method for preparing Astragalus callus extract according to claim 1, characterized in that, The extraction time for the Astragalus callus was 20 min to 50 min.

6. An extract of Astragalus membranaceus callus, characterized in that, It was prepared by the method for preparing Astragalus callus extract according to any one of claims 1 to 5.

7. A cosmetic product, characterized in that, Includes the Astragalus callus extract as described in claim 6.

8. The cosmetic product according to claim 7, characterized in that, In the cosmetic product, the amount of Astragalus callus extract added is 1.25% to 5.0%.

Images