Extract of callus based on flower water culture medium, and preparation method and application thereof

Abstract

The present application relates to the skin care technology field, especially based on the flower water as the culture medium's callus extract and preparation method and application;Based on the flower water as the culture medium's callus extraction method, including using the high mountain fire Chuancao leaf blade to induce the primary callus;Through the subculture screening out the loose callus;The loose callus is dispersed and then is transferred into the liquid culture medium and is cultured;The preparation method of liquid culture medium: the deionized water in B5 culture medium formula is replaced with the same weight of peony flower water, and the basic culture medium is obtained, and then 2,4-dichlorophenoxyacetic acid, sucrose, L-glutamine and hydrolyzed milk protein are added to the basic culture medium;Using flower water as culture medium to cultivate high mountain fire Chuancao callus, promote high mountain fire Chuancao callus to produce active substances under the premise of not using silver nitrate and other harmful substances to human body, and then improve the technical effect of skin care products on skin cell repair.

Description

Technical Field

[0001] This invention relates to the field of skincare technology, and in particular to callus extracts based on floral water as a culture medium, their preparation methods, and applications. Background Technology

[0002] Edelweiss callus extract can be used for skin cell repair and hair cuticle repair. However, the repair effect of normally cultivated Edelweiss callus extract is not ideal. Existing technologies generally add stimulants or inducers such as silver nitrate and salicylic acid to the culture medium to promote the production of higher concentrations of active substances in the callus, in order to achieve better repair effects. For example, Chinese patent 201811628974.9 discloses a method for simultaneously increasing the total flavonoids and various organic acids in Edelweiss callus. It describes that by simultaneously adding two synergistically effective inducers, AgNO3 aqueous solution and salicylic acid (SA) 95% ethanol solution, the content of total flavonoids, chlorogenic acid, and leonuric acid in Edelweiss callus and suspension cells can be increased.

[0003] However, the Edelweiss callus extract obtained by this method inevitably contains silver ions. Long-term use of its products may lead to silver deposition, which in turn may cause harm such as skin irritation, flora imbalance, or even heavy metal poisoning. Summary of the Invention

[0004] To address the shortcomings of existing technologies, one objective of this invention is to provide a method for extracting callus tissue based on floral water as a culture medium. Another objective is to provide a callus tissue extract based on floral water as a culture medium. A third objective is to provide the application of the callus tissue extract based on floral water as a culture medium in skincare products.

[0005] One of the objectives of this invention is achieved through the following technical solution: A method for callus extraction based on flower water as a culture medium includes the following steps: Step 1: Induce primary callus tissue using Edelweiss leaves; Step 2: Select loose callus tissue through subculture. Step 3: After dispersing the loose callus tissue, transfer it to a liquid culture medium for further culture. Preparation method of liquid culture medium: Replace the deionized water in the B5 culture medium formula with an equal weight of peony flower water (flower water, also known as hydrolate, is the condensed aqueous solution produced during the distillation and extraction of essential oils from plants) to obtain the basal culture medium. Then, add 2,4-dichlorophenoxyacetic acid, sucrose, L-glutamine and hydrolyzed milk protein to the basal culture medium to obtain the final concentrations of 2,4-dichlorophenoxyacetic acid 2.0 mg / L, sucrose 30 g / L, L-glutamine 0.1 mg / L and hydrolyzed milk protein 0.2 mg / L, respectively. Step 4: After passing through a 40-mesh sieve, the callus filtrate is obtained. The callus filtrate is then subjected to ultrafiltration for decolorization, desalting, and impurity removal, and dried to obtain a callus extract based on flower water as the culture medium.

[0006] Specifically, in step one, the method for inducing primary callus is as follows: take leaves of Edelweiss, disinfect them, cut them into 0.5cm segments as explants, and then inoculate the explants onto the induction medium and culture them in the dark at 20-25℃ for 14-20 days to obtain primary callus.

[0007] Specifically, the induction medium is obtained by adding sucrose and agar to B5 medium, with a final concentration of 30 g / L sucrose and 8 g / L agar.

[0008] Specifically, in step two, the subculture method is as follows: select loose tissue blocks from the primary callus, transfer them to the subculture medium, subculture once every 10-14 days, and obtain loose callus after three consecutive subcultures.

[0009] Specifically, the method for preparing peony flower water is as follows: peony flowers are placed in a steam distillation kettle, steam is introduced into the lower air inlet of the steam distillation kettle, the steam is discharged from the upper air outlet of the steam distillation kettle and condensed by a condenser, and the distillate is peony flower water.

[0010] Specifically, the water vapor temperature at the air inlet is ≥105℃, the medium temperature in the condenser is ≤8℃, the distillation time is 2-2.5h, and the distillate production rate is 25kg / h±5%.

[0011] The second objective of this invention is achieved through the following technical solution: The callus extract obtained by the above preparation method is based on flower water as a culture medium.

[0012] The third objective of this invention is achieved through the following technical solution: Callus extracts based on floral water culture medium are used in skincare products.

[0013] Specifically, the skincare product comprises the following components in parts by weight: The contents of the callus extract (3 parts), glutathione (1 part), carnosine (0.5 parts), butylene glycol (5 parts), and compound preservative (0.5 parts) were based on flower water as the culture medium.

[0014] Specifically, the compound preservative is composed of equal weight mixtures of hexanediol, ethylhexylglycerin, and disodium EDTA.

[0015] The beneficial effects of this invention are as follows: using floral water as a culture medium to cultivate Edelweiss callus tissue, promoting the production of active substances in Edelweiss callus tissue without using harmful substances such as silver nitrate, thereby enhancing the technical effect of skin care products on skin cell repair; secondly, this invention also has the effect of anti-aging and anti-wrinkle by regulating the expression of ECM in skin cells; finally, this invention also has the effect of repairing hair cuticles. Attached Figure Description

[0016] Figure 1 Before and after photos of one of the volunteers in a hair cuticle repair experiment using skincare products.

[0017] Figure 2 Before and after photos of another volunteer using skincare products in an experiment to repair hair cuticles. Detailed Implementation

[0018] The following is a further explanation with reference to specific implementation methods: Example

[0019] Preparation of peony flower water: 20 kg of fresh peony flowers were kneaded for 15 minutes and then placed in a steam distillation kettle. Steam was introduced through the lower inlet of the kettle and exited through the upper outlet, where it was condensed in a condenser. The distillate was peony flower water. The steam temperature at the inlet was 105°C, the condenser medium temperature was 8°C, the distillation time was 2.5 hours, and the distillate yield was 25 kg / h ± 5%. The distillate weighed 61 kg and was cooled to room temperature to obtain peony flower water.

[0020] Preparation of induction medium: Sucrose and agar were added to B5 medium to obtain the final concentration of 30 g / L sucrose and 8 g / L agar.

[0021] Preparation of subculture medium: 2,4-Dichlorophenoxyacetic acid, 6-benzylaminopurine, sucrose, L-glutamine, and hydrolyzed milk protein were added to B5 medium to obtain the final concentrations of 2,4-dichlorophenoxyacetic acid 2.0 mg / L, 6-benzylaminopurine 0.2 mg / L, sucrose 30 g / L, L-glutamine 0.1 mg / L, and hydrolyzed milk protein 0.2 mg / L.

[0022] Preparation of liquid culture medium: The deionized water in the B5 medium formulation was replaced with an equal weight of peony flower water to obtain the basal medium. Then, 2,4-dichlorophenoxyacetic acid, sucrose, L-glutamine, and hydrolyzed milk protein were added to the basal medium to obtain the final concentrations: 2.0 mg / L for 2,4-dichlorophenoxyacetic acid, 30 g / L for sucrose, 0.1 mg / L for L-glutamine, and 0.2 mg / L for hydrolyzed milk protein. 6-Benzylaminopurine was not added to the liquid culture medium to prevent cell aggregation. No inducing agents such as silver nitrate were added.

[0023] Primary callus induction: Leaves of Edelweiss were cleaned and disinfected, and cut into 0.5cm segments to obtain explants; the explants were inoculated on induction medium; and cultured in the dark at 25℃ for 15 days. Pale yellow callus tissue formed at the cut of the explants, which is the primary callus tissue.

[0024] Subculture: Select loose callus blocks from the primary callus and transfer them to the subculture medium. Subculture once every 10 days for a total of 3 times to obtain loose callus.

[0025] Suspension culture: Two parts by weight of loose callus tissue were mixed with 10 parts by weight of liquid culture medium. The loose callus tissue was further dispersed into small particles by pipetting. The mixture was then cultured in the dark on a shaker at 25°C for 7 days. Afterward, the mixture was removed and allowed to settle. The supernatant was discarded, and the mixture was transferred to a bioreactor. 60 parts by weight of liquid culture medium were added, and the mixture was cultured for 12 days to obtain cell slurry. The aeration rate during culture was 0.3 m³ / h. 3 / h, stirring frequency is 5r / min.

[0026] Preparation of callus extract based on flower water as culture medium: The cell sap was passed through a 40-mesh sieve to obtain callus filtrate. The callus filtrate was then subjected to ultrafiltration for decolorization, desalting and impurity removal, and drying to obtain callus extract based on flower water as culture medium.

[0027] Preparation of skincare products: The skincare products comprise the following components in parts by weight: The culture medium consisted of 3 parts callus extract, 1 part glutathione, 0.5 parts carnosine, 5 parts butylene glycol, and 0.5 parts compound preservative. The compound preservative was composed of equal weight mixtures of hexanediol, ethylhexylglycerin, and disodium EDTA.

[0028] Comparative Example 1 The only difference from the examples is the composition of the liquid culture medium. In this comparative example, the liquid culture medium was prepared by adding 2,4-dichlorophenoxyacetic acid, sucrose, L-glutamine, and hydrolyzed milk protein to B5 medium, resulting in final concentrations of 2.0 mg / L 2,4-dichlorophenoxyacetic acid, 30 g / L sucrose, 0.1 mg / L L-glutamine, and 0.2 mg / L hydrolyzed milk protein.

[0029] Comparative Example 2 Compared to Comparative Example 1, the only difference was the addition of silver nitrate and salicylic acid to the liquid culture medium on day 7 of the "12-day culture after adding 60 parts by weight of liquid culture medium" method. The final concentrations were 10 μM silver nitrate and 20 μM salicylic acid. Silver nitrate is an inhibitor of ethylene activity, and salicylic acid is a signaling molecule. The synergistic effect of the two can significantly activate key enzymes in the flavonoid and phenolic acid synthesis pathways. Obviously, this method is a common preparation method in the existing technology.

[0030] Comparative Example 3 Preparation of skincare products: The skincare products comprise the following components in parts by weight: The mixture contained 3 parts of Edelweiss extract (purchased from Lanzhou Waterles Biotechnology Co., Ltd.), 1 part of glutathione, 0.5 parts of carnosine, 5 parts of butylene glycol, and 0.5 parts of a compound preservative. The compound preservative was composed of equal weight mixtures of hexanediol, ethylhexylglycerin, and disodium EDTA.

[0031] Cell proliferation experiment HFF-1 fibroblasts were used at a concentration of 1×10 4 Cells were seeded at a density of 100 cells / mL into culture flasks containing the skincare products of Examples 1-3 and Comparative Examples 1-3, and cultured at 37°C and 5% CO2 for 1 hour. Non-adherent cells were then aspirated and transferred to the same culture flasks containing the skincare products of Examples 1-3 and Comparative Examples 1-3, and cultured at 37°C and 5% CO2 for 18 hours. Once cell confluence reached 80%, the supernatant was discarded, and the resulting precipitate was washed twice with PBS. The precipitate was then seeded with the skincare products of Examples 1-3 at a rate of 1×10⁻⁶. 6 Cells were resuspended at a density of cells / mL and then transferred to an incubator for culture at 37°C and 5% CO2, designated as generation P1. When the cells were passaged to P8, the cell proliferation in each culture flask was detected using an MTT assay kit (catalog number 20311, purchased from Shanghai Shangbao Biotechnology Co., Ltd.). The results are shown in Table 1.

[0032] Table 1 Cell proliferation status OD value 0.801±0.31* 0.612±0.19** 0.790±0.28 0.546±0.34** Note: Compared with Comparative Example 2, *P<0.05, **P<0.01.

[0033] As shown in Table 1, the skincare product obtained by this invention, without the addition of silver nitrate or other inducing agents, achieves the technical effect of enhancing cell proliferation (its efficacy is comparable to that of existing technologies) by replacing the deionized water in the liquid culture medium with peony flower water, which is beneficial for skin cell repair. Furthermore, by comparing Comparative Examples 1-3, it can be found that Comparative Example 1, lacking peony flower water, showed poor results, while Comparative Example 3, using edelweiss extract instead of edelweiss callus extract, not only failed to improve the technical effect but also inhibited cell proliferation.

[0034] Cellular ECM Regulation Experiment 3T3 mouse fibroblasts were diluted with cell culture medium and cultured to a density of 50%. 200 μL of the diluted medium was seeded into 96-well plates and incubated in a CO2 incubator for 24 h. The culture medium in the 96-well plates was discarded, and drug administration was performed. Culture medium containing the skincare products from Examples 1-3 (at a concentration of 1.25 mg / mL) was added to the test wells, and normal cell culture medium was added to the blank / solvent control wells (200 μL per well). After drug administration, the 96-well plates were incubated in a CO2 incubator for 24 h. After incubation, 200 μL of cell culture supernatant was collected into a 1.5 mL sterile centrifuge tube and stored at -80°C. The expression of mouse type I collagen was detected according to the instructions of the mouse type I collagen protease-linked immunosorbent assay kit. The upregulation rate of type I collagen expression was calculated.

[0035] The results are shown in Table 2.

[0036] Table 2. Statistical table of upregulation rate of fibroblast expression. Increased expression rate of type I collagen (%) 10.92 6.85 10.17 5.67 Experiments show that the skincare product obtained by this invention has a better effect on regulating ECM expression in skin cells compared with existing technologies. In contrast, Comparative Example 1 showed a significant decrease in regulation results due to the lack of stimulation from inducers such as silver nitrate. Comparative Example 3, which used Edelweiss extract to replace Alpine Edelweiss callus extract, did not show good results, indicating that whole plant extract or aerial part extract of Edelweiss cannot replace callus extract.

[0037] Hair cuticle repair experiment The skincare product obtained in the examples was used as a hair conditioner by volunteers. Before and after shampooing with this skincare product, see [link to product description]. Figure 1 and Figure 2 .

[0038] The embodiments and descriptions above are merely illustrative of the principles and preferred embodiments of the present invention. Various changes and modifications may be made to the present invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed.

Claims

1. A method for extracting callus tissue based on flower water as a culture medium, comprising the following steps: Step 1: Induce primary callus tissue using Edelweiss leaves; Step 2: Select loose callus tissue through subculture. Step 3: After dispersing the loose callus tissue, transfer it to a liquid culture medium for further culture. Preparation method of liquid culture medium: Replace the deionized water in the B5 culture medium formula with an equal weight of peony flower water to obtain the basal culture medium. Then, add 2,4-dichlorophenoxyacetic acid, sucrose, L-glutamine and hydrolyzed milk protein to the basal culture medium to obtain the final concentrations of 2,4-dichlorophenoxyacetic acid 2.0 mg / L, sucrose 30 g / L, L-glutamine 0.1 mg / L and hydrolyzed milk protein 0.2 mg / L. Step 4: After passing through a 40-mesh sieve, the callus filtrate is obtained. The callus filtrate is then subjected to ultrafiltration, decolorization, desalting, impurity removal, and drying to obtain a callus extract based on flower water as the culture medium.

2. The method for callus extraction based on flower water as a culture medium according to claim 1, characterized in that: In step one, the method for inducing primary callus is as follows: take leaves of Edelweiss, disinfect them, cut them into 0.5cm segments as explants, and then inoculate the explants onto the induction medium and culture them in the dark at 20-25℃ for 14-20 days to obtain primary callus.

3. The method for callus extraction based on flower water as a culture medium according to claim 2, characterized in that: The induction medium was obtained by adding sucrose and agar to B5 medium, with a final concentration of 30 g / L sucrose and 8 g / L agar.

4. The method for callus extraction based on flower water as a culture medium according to claim 3, characterized in that: In step two, the subculture method is as follows: select loose tissue blocks from the primary callus, transfer them to the subculture medium, subculture once every 10-14 days, and obtain loose callus after three consecutive subcultures.

5. The method for callus extraction based on flower water as a culture medium according to claim 4, characterized in that: The method for preparing peony flower water is as follows: peony flowers are placed in a steam distillation kettle, steam is introduced into the lower air inlet of the steam distillation kettle, the steam is discharged from the upper air outlet of the steam distillation kettle and condensed by a condenser, and the distillate is peony flower water.

6. The method for callus extraction based on flower water as a culture medium according to claim 5, characterized in that: The water vapor temperature at the air inlet is ≥105℃, the medium temperature in the condenser is ≤8℃, the distillation time is 2-2.5h, and the distillate production rate is 25kg / h±5%.

7. A callus extract based on flower water as a culture medium obtained by the callus extraction method according to claim 6.

8. The use of the callus extract based on floral water as a culture medium according to claim 7 in skin care products.

9. The use according to claim 8, characterized in that: The skincare product comprises the following components in parts by weight: The contents of the callus extract (3 parts), glutathione (1 part), carnosine (0.5 parts), butylene glycol (5 parts), and compound preservative (0.5 parts) were based on flower water as the culture medium.

10. The use according to claim 9, characterized in that: The compound preservative is composed of equal weight mixtures of hexanediol, ethylhexylglycerin and disodium EDTA.

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