A composition prepared based on plant cell culture and its application in anti-photoaging, whitening, firming and anti-wrinkle cosmetics

Abstract

The present invention relates to the technical field of cosmetics, and in particular to a composition prepared based on plant cell culture and its application in anti-photoaging, whitening, firming and anti-wrinkle cosmetics. The present invention uses raw materials (Schisandra chinensis, Borage, Polygonum cuspidatum, Bellis Perennis, Cryptomeria japonica, Kava pepper and Agave) with anti-photoaging, whitening, firming and anti-wrinkle effects, optimizes the dosage ratio of the raw materials, and uses plant cell culture technology to perform callus culture on the plant raw materials to obtain cell culture, and then uses Candida utilis CICC 31170 and Lactobacillus helveticus CICC 22818 for joint fermentation, and further adds ethanol to adjust the concentration to extract active ingredients, and then ultrafiltration is performed through an ultrafiltration membrane with a molecular weight cutoff of less than 3000Da, and finally after post-treatment, a composition is obtained. The composition prepared by the present invention has low irritation, excellent skin whitening effect, can give skin elasticity, and also has anti-wrinkle and anti-photoaging effects.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and in particular to a composition prepared based on plant cell culture and application thereof in anti-photoaging, whitening, firming and anti-wrinkle cosmetics. Background Art

[0002] With the continuous advancement of technology, the cosmetics industry is gradually developing in a safer, more efficient, green and environmentally friendly direction. As an important part of modern biotechnology, plant cell culture technology has shown great potential and application prospects in the screening, production and evaluation of cosmetic raw materials.

[0003] Plant cell culture technology is to place plant tissues (such as leaves, stems, roots, etc.) or cells in a culture medium containing appropriate nutrients under sterile conditions to induce their proliferation and differentiation, thereby obtaining a large number of cells or tissues with specific functions. These cells or tissues are rich in a variety of active ingredients, such as polyphenols, flavonoids, carotenoids, plant hormones, etc., and have a wide range of application value in cosmetics.

[0004] Although plant cell culture technology can be used to obtain cells or tissues containing a variety of active ingredients, applying this technology to the cosmetics field and obtaining cosmetics with excellent efficacy is challenging and is also a technical problem that needs to be solved at present. Summary of the invention

[0005] The present invention aims at the above-mentioned technical problems and proposes a technical solution of the present invention. The present invention relates to the field of cosmetic technology, and specifically to a composition prepared based on plant cell culture and its application in anti-photoaging, whitening, firming and anti-wrinkle cosmetics.

[0006] The invention adopts schisandra chinensis, borage, knotweed, daisy, cryptomeria fortunei, kava pepper and agave as raw materials to prepare the composition.

[0007] Among them, Schisandra chinensis is a plant with multiple pharmacological effects. Its extract can significantly inhibit the growth of Propionibacterium acnes and inhibit UVB-induced photoaging of fibroblasts.

[0008] The flavonoids contained in Borage have a certain central nervous system inhibitory effect, thus playing a sedative role. At the same time, its extract has a soothing effect on the skin. Using cosmetics containing Borage ingredients can promote skin cell renewal, enhance skin barrier function, reduce the damage of irritants to the skin, and achieve a soothing effect. The active ingredients in Borage extract can inhibit the production of inflammatory mediators, reduce inflammatory reactions, and thus play an anti-inflammatory role.

[0009] Polygonum cuspidatum contains active ingredients such as rhubarb and resveratrol. The extract of Polygonum cuspidatum can inhibit the activity of melanin. The extract can also promote the activity of macrophages, reduce melanin on the skin, and play a whitening role.

[0010] Daisy is a natural beauty product that contains a large amount of essential oils and active ingredients such as chrysanthemum pigments. It has the effect of removing melanin and softening epidermal cells, making the skin fair and attractive. Daisy extract can effectively inhibit the production of melanin in the skin and is suitable for dull skin with melanin deposition. In addition, daisy is also effective for skin problems such as sensitivity, erythema, dryness, eczema, peeling, and scars.

[0011] Extracts from Cryptomeria fortunei are raw materials for cosmetics, and are mainly used as skin conditioners. Extracts from Cryptomeria fortunei can reduce skin redness and irritation, while moisturizing the skin and increasing its elasticity. For dry skin, using extracts containing Cryptomeria fortunei can provide a more refreshing skin feel, lasting moisturizing, and multiple delays of skin oxidation, improves skin's self-repair ability, and comprehensively repairs the skin's metabolic cycle damaged by blue light, allowing the skin to enhance its resistance.

[0012] Kava extract has multiple effects in cosmetics, including improving skin hydration, reducing redness, erythema, and reducing skin stinging and itching. It is mainly used as a skin conditioner, with soothing, anti-inflammatory, antibacterial, anti-allergic and keratin softening effects, and is suitable for all types of skin, especially sensitive and allergic skin. Kava extract blocks the transmission of information by neurons by inhibiting the release of inflammatory mediators, thereby inhibiting the transmission of sensitivity and achieving a soothing effect. In addition, it can also reduce inflammatory reactions caused by contact with chemical compounds and ultraviolet rays. It contains a variety of kava lactones, which can control TNF-α to a certain extent, while inhibiting the activity of PEG2, effectively playing an anti-redness, analgesic, antipruritic and soothing role.

[0013] Agave is rich in polysaccharides, which can provide sufficient moisture and nutrients to help keep the skin hydrated and avoid dryness. Agave contains a large amount of vitamin E and collagen, which can moisturize the skin, promote the production and repair of skin cells, and make the skin more elastic and shiny. Agave is rich in natural antioxidants such as polyphenols and carotenoids, which can reduce free radical damage, prevent skin aging, and protect the skin from ultraviolet damage. The flavonoids in agave have anti-inflammatory and anti-inflammatory effects, which can help relieve skin allergies, eczema, acne and other problems. The fruit acid contained in agave can dissolve the aging keratin on the surface of the skin, help clean the pores, promote skin metabolism, and make the skin more tender and smooth.

[0014] The invention uses raw materials (Schisandra chinensis, Borage, Polygonum cuspidatum, Bellis Perennis, Cryptomeria japonica, Kava pepper and Agave) with anti-photoaging, whitening, skin tightening and anti-wrinkle effects, optimizes the dosage ratio of the raw materials, and uses plant cell culture technology to perform callus culture on the plant raw materials to obtain cell culture, then adopts Candida utilis CICC 31170 and Lactobacillus helveticus CICC 22818 for joint fermentation, further adds ethanol to adjust the concentration to extract active ingredients, and then ultrafilters through an ultrafiltration membrane with a molecular weight cutoff lower than 3000Da, and finally undergoes post-treatment to obtain a composition.

[0015] Specifically, the present invention provides a composition prepared based on plant cell culture, and the preparation method of the composition comprises the following steps:

[0016] Step S1: material preparation and pretreatment: weigh the raw materials according to mass: 1-3 parts of Schisandra chinensis, 0.5-2 parts of Borago officinalis, 0.8-2 parts of Polygonum cuspidatum, 0.5-2.5 parts of Bellis Perennis, 0.5-2.2 parts of Cryptomeria fortunei, 0.4-2 parts of Kava Piper, and 0.5-3 parts of Agave; wash, drain, shear and sterilize the raw materials respectively, and then mix them to obtain pre-treated materials;

[0017] Step S2: Obtaining callus tissue: adding the pre-treated material to the culture medium, culturing at 20-25° C. in the dark for 4-6 weeks to obtain callus tissue;

[0018] Step S3: Obtaining cell culture: Dispersing callus tissue in culture medium, culturing at 22-30° C. in the dark for 5-8 weeks to obtain cell culture;

[0019] Step S4: obtaining the culture: filtering the cell culture, taking out the solid matter, and obtaining the culture;

[0020] Step S5: Fermentation: sterilize the culture, add water and carbon source to obtain a mixture, then inoculate the fermentation bacteria, and ferment at a temperature of 30-38°C in the dark for 32-50 hours to obtain a fermentation product; wherein the concentration of the fermentation bacteria in the mixture is 2.5×10 8 -6.8×10 8 cfu / mL, the fermentation bacteria consisted of Candida utilis CICC 31170 and Lactobacillus helveticus CICC 22818 in a live cell count ratio of 1.2-2.5:1;

[0021] Step S6: Extraction: sterilize the fermented product, then add ethanol, adjust the ethanol concentration, heat and keep warm and stir for 5-10 hours to obtain a stirred product;

[0022] Step S7: post-treatment: coarsely filter the stirred product, ultrafilter the coarse filtrate to obtain an ultrafiltrate, concentrate, dry and sterilize the ultrafiltrate to obtain the composition prepared based on plant cell culture.

[0023] Furthermore, in step S1, the parts of Schisandra chinensis, Borage, Polygonum cuspidatum, Bellis Perennis, Cryptomeria fortunei, Kava Piper and Agave that can be selected as raw materials may be roots, stems, branches, leaves, flowers and the like.

[0024] Furthermore, in step S1, the cleaning is performed using water.

[0025] Furthermore, in step S1, ultraviolet sterilization is used for sterilization.

[0026] Furthermore, in step S1, the mass ratio of knotweed to bellis perennis is 1:0.8-1.5.

[0027] Furthermore, in step S2, the culture medium composition is: MS culture medium, 10-22 g / L glucose, 0.3-1.1 mg / L indolebutyric acid, 0.2-1.0 mg / L indoleacetic acid, 0.5-0.9 mg / L adenine and 1-2.5 mg / L 6-benzylaminopurine.

[0028] Furthermore, in step S2, fresh culture medium is replaced every week of culture to continue the culture.

[0029] Furthermore, in step S3, the composition of the culture medium is: MS culture medium, 18-28 g / L glucose and 0.8-1.5 mg / L adenine.

[0030] Furthermore, in step S3, the culture medium is replaced with fresh culture medium every week of culture and culture is continued.

[0031] Furthermore, in step S4, filtering is performed using a filter membrane with a pore size less than 150 μm.

[0032] Furthermore, in step S5, the carbon source is at least one of glucose, sucrose, fructose, white sugar and brown sugar, and the amount of the carbon source is 2-5% of the mass of the mixture.

[0033] Furthermore, in step S5, the mass ratio of the culture medium to water is 1:20-60.

[0034] Furthermore, in step S5, the concentration of the fermentation bacteria in the mixture is 3×10 8 -6.2×10 8 cfu / mL.

[0035] Furthermore, in step S5, the fermentation bacteria are composed of Candida utilis CICC 31170 and Lactobacillus helveticus CICC22818 in a live bacteria count ratio of 1.4-2:1.

[0036] Furthermore, in step S6, the mass concentration of ethanol is adjusted to 20-45%.

[0037] Furthermore, in step S6, the insulation temperature is 40-55°C.

[0038] Furthermore, in step S7, the coarse filtration is performed using a filter membrane with a pore size greater than 0.2 μm.

[0039] Furthermore, in step S7, ultrafiltration is performed using an ultrafiltration membrane with a molecular weight cutoff lower than 3000Da; for example, the molecular weight cutoff is 2500Da, 2000Da or 1800Da, etc.

[0040] Furthermore, in step S7, the concentration is reduced pressure concentration. The reduced pressure concentration temperature is 55-64° C. The reduced pressure concentration is performed to 10-20% of the mass of the ultrafiltrate.

[0041] Furthermore, in step S7, the drying is freeze-drying, the freeze-drying temperature can be -45 to -20°C, and the freeze-drying is performed until the water content is no more than 8.5%.

[0042] Furthermore, in step S7, the sterilization is ultraviolet sterilization.

[0043] On the other hand, the present invention also provides the use of the composition prepared based on plant cell culture in the preparation of cosmetics. The cosmetics are anti-photoaging, whitening, firming and anti-wrinkle cosmetics.

[0044] Beneficial effects of the present invention:

[0045] The present invention uses raw materials (Schisandra chinensis, Borage, Polygonum cuspidatum, Bellis Perennis, Cryptomeria japonica, Kava pepper and Agave) with anti-photoaging, whitening, skin tightening and anti-wrinkle effects, optimizes the dosage ratio of the raw materials, and uses plant cell culture technology to perform callus culture on the plant raw materials to obtain cell culture, and then uses Candida utilis CICC 31170 and Lactobacillus helveticus CICC 22818 for joint fermentation, and further adds ethanol to adjust the concentration to extract active ingredients, and then ultrafiltration is performed through an ultrafiltration membrane with a molecular weight cutoff of less than 3000Da, and finally after post-treatment, a composition is obtained. The composition prepared by the present invention has low irritation, excellent skin whitening effect, can give skin elasticity, and also has anti-wrinkle and anti-photoaging effects.

[0046] The composition prepared by the invention has low irritation and is harmless to the skin.

[0047] The composition of the present invention is diluted to 5wt% and used for 1 month to increase the skin brightness value (L * ) is 3.957, and using it for 2 months can increase the skin brightness value (L * ) is 5.435; the composition of the present invention has a good skin whitening and lightening effect.

[0048] The present invention uses a fermentation method to prepare the composition, which can significantly improve the skin whitening effect of the composition. The present invention uses Candida utilis CICC 31170 and Lactobacillus helveticus CICC 22818 to ferment together to improve the whitening effect of the composition, which is better than the effect of using only one of them. The combination of the two for fermentation promotes the increase of the types and concentrations of active ingredients in the composition, thereby further improving the whitening effect of the composition.

[0049] The composition of the present invention uses knotweed and daisy to produce a compatibility relationship in increasing the whitening effect, so that the composition has a good whitening effect, especially when the mass ratio of knotweed to daisy is in the range of 1:0.8-1.5, the whitening effect of the obtained composition is better.

[0050] The present invention adopts plant cell culture technology to culture plant raw materials, adds ethanol for extraction, and uses ultrafiltration technology with a molecular weight cutoff lower than 3000Da, all of which can enhance the whitening and skin tightening effects of the composition.

[0051] The composition of the present invention has a good effect of increasing skin elasticity and imparting skin firming effect.

[0052] The present invention uses a fermentation method to prepare the composition, which can significantly improve the skin tightening effect of the composition. The present invention uses Candida utilis CICC 31170 and Lactobacillus helveticus CICC 22818 for co-fermentation, so that the skin tightening effect of the composition is better.

[0053] The present invention adopts Cryptomeria fortunei and Agave as raw materials to prepare the composition, so that the composition can have a good skin tightening effect.

[0054] The process of the invention is green, environmentally friendly and simple, and the prepared composition has excellent effects and good application prospects. DETAILED DESCRIPTION

[0055] The present invention will be described below in conjunction with specific embodiments, and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments are used to illustrate the present invention, rather than to limit the present invention.

[0056] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0057] 1. Raw material information

[0058] The raw materials used in the present invention were obtained by purchase, and the specific information is as follows:

[0059] Candida utilis CICC 31170: strain number 31170, purchased from China Food and Fermentation Industries Research Institute Co., Ltd.

[0060] Lactobacillus helveticus CICC 22818: The strain number is 22818, which was purchased from China Food Fermentation Industry Research Institute Co., Ltd.

[0061] MS medium: BM060-MS liquid medium (without agar), brand: Isejiu Bio.

[0062] 2. Preparation of the composition

[0063] Composition A: The preparation steps are as follows:

[0064] Step S1: material preparation and pretreatment: weigh the raw materials by mass: 2.2 parts of Schisandra chinensis, 1.5 parts of Borage, 1.5 parts of Polygonum cuspidatum, 1.8 parts of Bellis perennis, 1.8 parts of Cryptomeria japonica, 1.2 parts of Kava pepper and 1.4 parts of Agave, with the mass ratio of Polygonum cuspidatum to Bellis perennis being 1:1.2; wash the raw materials with water, drain, cut into pieces of about 0.8 cm × 0.8 cm × 0.8 cm, sterilize with ultraviolet light, mix, and obtain pre-treated materials. Among them, Schisandra chinensis, Polygonum cuspidatum, Cryptomeria japonica and Agave are all made of roots, and Borage, Bellis perennis and Kava pepper are all made of stems.

[0065] Step S2: Obtaining callus tissue: adding the pre-treated material to the culture medium, culturing at 22° C. in the dark for 5 weeks to obtain callus tissue; replacing the culture medium with fresh one week of culturing and continuing to culture under the same conditions; wherein the culture medium composition of this step is: MS culture medium, 20 g / L glucose, 0.5 mg / L indolebutyric acid, 0.8 mg / L indoleacetic acid, 0.75 mg / L adenine and 1.55 mg / L 6-benzylaminopurine.

[0066] Step S3: Obtaining cell culture: Disperse callus tissue in culture medium, culture at 28° C. in the dark for 7 weeks to obtain cell culture; replace with fresh culture medium every week and continue to culture under the same conditions; wherein the composition of the culture medium in this step is: MS medium, 25 g / L glucose and 1.35 mg / L adenine.

[0067] Step S4: Obtaining the culture: Filter the cell culture using a filter membrane with a pore size of 80 μm to remove the solid matter to obtain the culture.

[0068] Step S5: Fermentation: sterilize the culture with ultraviolet light, add water and carbon source glucose to obtain a mixture, then inoculate fermentation bacteria, ferment at a temperature of 37° C. in the dark for 42 hours to obtain a fermentation product.

[0069] The mass ratio of culture medium to water was 1:30, the carbon source accounted for 4.27% of the mass of the mixture, and the concentration of fermentation bacteria in the mixture was 4.50×10 8 cfu / mL, the fermentation bacteria consisted of Candida utilis CICC 31170 and Lactobacillus helveticus CICC22818 in a live cell count ratio of 2:1;

[0070] Step S6: Extraction: The fermented product was sterilized by ultraviolet light, and then ethanol was added to adjust the ethanol concentration to 32.5 wt %, and heated to 45° C. and stirred at 70 rpm for 6.8 hours to obtain a stirred product.

[0071] Step S7: post-treatment: the stirred product is coarsely filtered using a filter membrane with a pore size of 2 μm, the coarse filtrate is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 1800 Da to obtain an ultrafiltrate, the ultrafiltrate is concentrated under reduced pressure at 58° C. to 15.7% of the mass of the ultrafiltrate, freeze-dried at -30° C. to a water content of 6.12%, and sterilized with ultraviolet light to obtain the composition A.

[0072] Composition B: The preparation steps are as follows:

[0073] Step S1: material preparation and pretreatment: weigh the raw materials by mass: 3 parts of Schisandra chinensis, 2 parts of Borage, 1.8 parts of Polygonum cuspidatum, 1.5 parts of Bellis perennis, 1.0 parts of Cryptomeria japonica, 1.1 parts of Kava pepper and 2.2 parts of Agave tequila, with the mass ratio of Polygonum cuspidatum to Bellis perennis being 1:0.83; wash the raw materials with water, drain, cut into pieces of about 0.8 cm × 0.8 cm × 0.8 cm, sterilize with ultraviolet light, mix, and obtain pre-treated materials. Among them, Schisandra chinensis, Polygonum cuspidatum, Cryptomeria japonica and Agave are all made of roots, and Borage, Bellis perennis and Kava pepper are all made of stems.

[0074] Step S2: Obtaining callus tissue: adding the pre-treated material to the culture medium, culturing at 23° C. in the dark for 5 weeks to obtain callus tissue; replacing the culture medium with fresh one week of culturing and continuing to culture under the same conditions; wherein the culture medium composition of this step is: MS culture medium, 20 g / L glucose, 0.45 mg / L indolebutyric acid, 0.82 mg / L indoleacetic acid, 0.77 mg / L adenine and 1.6 mg / L 6-benzylaminopurine.

[0075] Step S3: Obtaining cell culture: Disperse callus tissue in culture medium, culture at 27° C. in the dark for 7 weeks to obtain cell culture; replace with fresh culture medium every week and continue to culture under the same conditions; wherein the composition of the culture medium in this step is: MS medium, 26.2 g / L glucose and 1.38 mg / L adenine.

[0076] Step S4: Obtaining the culture: Filter the cell culture using a filter membrane with a pore size of 80 μm to remove the solid matter to obtain the culture.

[0077] Step S5: Fermentation: sterilize the culture with ultraviolet light, add water and carbon source glucose to obtain a mixture, then inoculate fermentation bacteria, ferment at a temperature of 35° C. in the dark for 46 hours to obtain a fermentation product.

[0078] The mass ratio of culture medium to water was 1:32, the carbon source accounted for 4.35% of the mass of the mixture, and the concentration of fermentation bacteria in the mixture was 4.82×10 8 cfu / mL, the fermentation bacteria consisted of Candida utilis CICC 31170 and Lactobacillus helveticus CICC22818 at a live cell count ratio of 1.2:1;

[0079] Step S6: Extraction: The fermented product was sterilized by ultraviolet light, and then ethanol was added to adjust the ethanol concentration to 25 wt %, and heated to 48° C. and stirred at 70 rpm for 7.2 hours to obtain a stirred product.

[0080] Step S7: post-treatment: the stirred product is coarsely filtered using a filter membrane with a pore size of 2 μm, the coarse filtrate is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 1500 Da to obtain an ultrafiltrate, the ultrafiltrate is concentrated under reduced pressure at 58° C. to 16.8% of the mass of the ultrafiltrate, freeze-dried at -30° C. to a water content of 6.03%, and sterilized with ultraviolet light to obtain the composition B.

[0081] Composition C: Compared with composition A, the preparation of this composition does not use a fermentation process; specifically:

[0082] Step S5: fermentation: sterilize the culture with ultraviolet light, add water and carbon source glucose to obtain a mixture, and place it in the dark at a temperature of 37° C. for 42 hours to obtain material 1;

[0083] The mass ratio of culture medium to water was 1:30, and the carbon source accounted for 4.27% of the mass of the mixture;

[0084] Step S6: Extraction: sterilize material 1 with ultraviolet light, then add ethanol to adjust the ethanol concentration to 32.5wt%, heat and keep at 45°C and stir at 70rpm for 6.8 hours to obtain a stirred product;

[0085] The other preparation steps are the same as those of composition A.

[0086] Composition D: Compared with composition A, the preparation of this composition is only fermented with Candida utilis CICC 31170; specifically:

[0087] Step S5: fermentation: sterilize the culture with ultraviolet light, add water and carbon source glucose to obtain a mixture, then inoculate fermentation bacteria, ferment at a temperature of 37° C. in the dark for 42 hours to obtain a fermentation product;

[0088] The mass ratio of culture medium to water was 1:30, the carbon source accounted for 4.27% of the mass of the mixture, and the concentration of fermentation bacteria in the mixture was 4.50×10 8 cfu / mL, the fermentation bacteria was Candida utilis CICC 31170;

[0089] The other preparation steps are the same as those of composition A.

[0090] Composition E: Compared with composition A, the preparation of this composition is only fermented with Lactobacillus helveticus CICC 22818; specifically:

[0091] Step S5: Fermentation: sterilize the culture with ultraviolet light, add water and carbon source glucose to obtain a mixture, then inoculate fermentation bacteria, ferment at a temperature of 37° C. in the dark for 42 hours to obtain a fermentation product.

[0092] The mass ratio of culture medium to water was 1:30, the carbon source accounted for 4.27% of the mass of the mixture, and the concentration of fermentation bacteria in the mixture was 4.50×10 8 cfu / mL, the fermentation bacteria was Lactobacillus helveticus CICC 22818;

[0093] The other preparation steps are the same as those of composition A.

[0094] Composition F: Compared with composition A, this composition does not use knotweed, but the amount of daisy is changed to 3.3 parts; specifically:

[0095] Step S1: material preparation and pretreatment: weigh the raw materials by mass: 2.2 parts of Schisandra chinensis, 1.5 parts of Borage, 3.3 parts of Bellis Perennis, 1.8 parts of Cryptomeria japonica, 1.2 parts of Kava and 1.4 parts of Agave; wash the raw materials with water, drain, cut into pieces of about 0.8cm×0.8cm×0.8cm, sterilize with ultraviolet light, mix, and obtain pre-treated materials. Among them, Schisandra chinensis, Polygonum cuspidatum, Cryptomeria japonica and Agave are all made of roots, and Borage, Bellis Perennis, and Kava are all made of stems;

[0096] The other preparation steps are the same as those of composition A.

[0097] Composition G: Compared with composition A, this composition does not use daisy, but the amount of knotweed is changed to 3.3 parts; specifically:

[0098] Step S1: material preparation and pretreatment: weigh the raw materials by mass: 2.2 parts of Schisandra chinensis, 1.5 parts of Borage, 3.3 parts of Polygonum cuspidatum, 1.8 parts of Cryptomeria fortunei, 1.2 parts of Kava and 1.4 parts of Agave; wash the raw materials with water, drain, cut into pieces of about 0.8cm×0.8cm×0.8cm, sterilize with ultraviolet light, mix, and obtain pre-treated materials. Among them, Schisandra chinensis, Polygonum cuspidatum, Cryptomeria fortunei and Agave are all made of roots, and Borage, Perennial daisy and Kava are all made of stems;

[0099] The other preparation steps are the same as those of composition A.

[0100] Composition H: Compared with composition A, the amount ratio of knotweed and bellis perennis in the preparation of this composition is changed; specifically:

[0101] Step S1: material preparation and pretreatment: weigh the raw materials by mass: 2.2 parts of Schisandra chinensis, 1.5 parts of Borage, 0.7 parts of Polygonum cuspidatum, 2.6 parts of Bellis perennis, 1.8 parts of Cryptomeria japonica, 1.2 parts of Kava pepper and 1.4 parts of Agave, with the mass ratio of Polygonum cuspidatum to Bellis perennis being 1:3.7; wash the raw materials with water, drain, cut into pieces of about 0.8cm×0.8cm×0.8cm in size, sterilize with ultraviolet light, mix, and obtain pre-treated materials. Among them, Schisandra chinensis, Polygonum cuspidatum, Cryptomeria japonica and Agave are all made from roots, and Borage, Bellis perennis and Kava pepper are all made from stems;

[0102] The other preparation steps are the same as those of composition A.

[0103] Composition I: Compared with composition A, the amount ratio of knotweed and bellis perennis in the preparation of this composition is changed; specifically:

[0104] Step S1: material preparation and pretreatment: weigh the raw materials by mass: 2.2 parts of Schisandra chinensis, 1.5 parts of Borage, 2.2 parts of Polygonum cuspidatum, 1.1 parts of Bellis perennis, 1.8 parts of Cryptomeria japonica, 1.2 parts of Kava pepper and 1.4 parts of Agave, with the mass ratio of Polygonum cuspidatum to Bellis perennis being 1:0.5; wash the raw materials with water, drain, cut into pieces of about 0.8cm×0.8cm×0.8cm in size, sterilize with ultraviolet light, mix, and obtain pre-treated materials. Among them, Schisandra chinensis, Polygonum cuspidatum, Cryptomeria japonica and Agave are all made from roots, and Borage, Bellis perennis and Kava pepper are all made from stems;

[0105] The other preparation steps are the same as those of composition A.

[0106] Composition J: Compared with composition A, the composition does not contain Cryptomeria fortunei; specifically:

[0107] Step S1: material preparation and pretreatment: weigh the raw materials by mass: 2.2 parts of Schisandra chinensis, 1.5 parts of Borago officinalis, 1.5 parts of Polygonum cuspidatum, 1.8 parts of Bellis perennis, 1.2 parts of Kava pepper, and 3.2 parts of Agave, with the mass ratio of Polygonum cuspidatum to Bellis perennis being 1:1.2; wash the raw materials with water, drain, cut into pieces of about 0.8 cm × 0.8 cm × 0.8 cm in size, sterilize with ultraviolet light, mix, and obtain pre-treated materials. Among them, Schisandra chinensis, Polygonum cuspidatum, Cryptomeria fortunei, and Agave are all made from roots, and Borago officinalis, Bellis perennis, and Kava pepper are all made from stems;

[0108] The other preparation steps are the same as those of composition A.

[0109] Composition K: Compared with composition A, the preparation of this composition does not contain agave; specifically:

[0110] Step S1: material preparation and pretreatment: weigh the raw materials by mass: 2.2 parts of Schisandra chinensis, 1.5 parts of Borage, 1.5 parts of Polygonum cuspidatum, 1.8 parts of Bellis perennis, 3.2 parts of Cryptomeria fortunei and 1.2 parts of Kava Piper, with the mass ratio of Polygonum cuspidatum to Bellis perennis being 1:1.2; wash the raw materials with water, drain, cut into pieces of about 0.8cm×0.8cm×0.8cm in size, sterilize with ultraviolet light, mix, and obtain pre-treated materials. Among them, Schisandra chinensis, Polygonum cuspidatum, Cryptomeria fortunei and Agave are all made from roots, and Borage, Bellis perennis and Kava Piper are all made from stems;

[0111] The other preparation steps are the same as those of composition A.

[0112] Composition L: The preparation of this composition does not contain steps S2 and S3 compared to composition A; specifically:

[0113] Step S1: material preparation and pretreatment: weigh the raw materials by mass: 2.2 parts of Schisandra chinensis, 1.5 parts of Borage, 1.5 parts of Polygonum cuspidatum, 1.8 parts of Bellis perennis, 1.8 parts of Cryptomeria japonica, 1.2 parts of Kava pepper and 1.4 parts of Agave, with the mass ratio of Polygonum cuspidatum to Bellis perennis being 1:1.2; wash the raw materials with water, drain, crush to a particle size of less than 2 mm, sterilize with ultraviolet light, mix, and obtain pre-treated materials. Among them, Schisandra chinensis, Polygonum cuspidatum, Cryptomeria japonica and Agave are all made of roots, and Borage, Bellis perennis and Kava pepper are all made of stems.

[0114] Step S2: Fermentation: water and carbon source glucose are added to the pre-treated material to obtain a mixture, and then fermentation bacteria are inoculated and fermented at a temperature of 37° C. in the dark for 42 hours to obtain a fermentation product.

[0115] The mass ratio of pre-treated materials to water was 1:30, the carbon source was 4.27% of the mass of the mixture, and the concentration of fermentation bacteria in the mixture was 4.50×10 8cfu / mL, the fermentation bacteria consisted of Candida utilis CICC 31170 and Lactobacillus helveticus CICC22818 in a live cell count ratio of 2:1;

[0116] Step S3: Extraction: The fermented product was sterilized by ultraviolet light, and then ethanol was added to adjust the ethanol concentration to 32.5 wt %, and heated to 45° C. and stirred at 70 rpm for 6.8 hours to obtain a stirred product.

[0117] Step S4: post-treatment: the stirred product is coarsely filtered using a filter membrane with a pore size of 2 μm, the coarse filtrate is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 1800 Da to obtain an ultrafiltrate, the ultrafiltrate is concentrated under reduced pressure at 58° C. to 15.7% of the mass of the ultrafiltrate, freeze-dried at -30° C. to a water content of 6.12%, and sterilized with ultraviolet light to obtain the composition.

[0118] Composition M: The preparation of this composition does not contain step S6 compared to composition A; specifically:

[0119] Step S1: material preparation and pretreatment: weigh the raw materials by mass: 2.2 parts of Schisandra chinensis, 1.5 parts of Borage, 1.5 parts of Polygonum cuspidatum, 1.8 parts of Bellis perennis, 1.8 parts of Cryptomeria japonica, 1.2 parts of Kava pepper and 1.4 parts of Agave, with the mass ratio of Polygonum cuspidatum to Bellis perennis being 1:1.2; wash the raw materials with water, drain, cut into pieces of about 0.8 cm × 0.8 cm × 0.8 cm, sterilize with ultraviolet light, mix, and obtain pre-treated materials. Among them, Schisandra chinensis, Polygonum cuspidatum, Cryptomeria japonica and Agave are all made of roots, and Borage, Bellis perennis and Kava pepper are all made of stems.

[0120] Step S2: Obtaining callus tissue: adding the pre-treated material to the culture medium, culturing at 22° C. in the dark for 5 weeks to obtain callus tissue; replacing the culture medium with fresh one week of culturing and continuing to culture under the same conditions; wherein the culture medium composition of this step is: MS culture medium, 20 g / L glucose, 0.5 mg / L indolebutyric acid, 0.8 mg / L indoleacetic acid, 0.75 mg / L adenine and 1.55 mg / L 6-benzylaminopurine.

[0121] Step S3: Obtaining cell culture: Disperse callus tissue in culture medium, culture at 28° C. in the dark for 7 weeks to obtain cell culture; replace with fresh culture medium every week and continue to culture under the same conditions; wherein the composition of the culture medium in this step is: MS medium, 25 g / L glucose and 1.35 mg / L adenine.

[0122] Step S4: Obtaining the culture: Filter the cell culture using a filter membrane with a pore size of 80 μm to remove the solid matter to obtain the culture.

[0123] Step S5: Fermentation: sterilize the culture with ultraviolet light, add water and carbon source glucose to obtain a mixture, then inoculate fermentation bacteria, ferment at a temperature of 37° C. in the dark for 42 hours to obtain a fermentation product.

[0124] The mass ratio of culture medium to water was 1:30, the carbon source accounted for 4.27% of the mass of the mixture, and the concentration of fermentation bacteria in the mixture was 4.50×10 8 cfu / mL, the fermentation bacteria consisted of Candida utilis CICC 31170 and Lactobacillus helveticus CICC22818 in a live cell count ratio of 2:1;

[0125] Step S6: Post-treatment: sterilize the fermented product with ultraviolet light, and coarsely filter the stirred product using a filter membrane with a pore size of 2 μm. Ultrafilter the coarse filtrate through an ultrafiltration membrane with a molecular weight cutoff of 1800 Da to obtain an ultrafiltrate. Concentrate the ultrafiltrate under reduced pressure at 58° C. to 15.7% of the mass of the ultrafiltrate, freeze-dry at -30° C. to a water content of 6.12%, and sterilize with ultraviolet light to obtain the composition.

[0126] Composition N: Compared with composition A, the preparation of this composition does not include ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 1800Da; specifically:

[0127] Step S7: post-treatment: filtering the stirred product using a filter membrane with a pore size of 0.5 μm, concentrating the filtrate under reduced pressure at 58° C. to 15.7% of the mass of the filtrate, freeze-drying at -30° C. to a water content of 6.12%, and sterilizing with ultraviolet light to obtain the composition;

[0128] The other preparation steps are the same as those of composition A.

[0129] Composition O: This composition was prepared by ultrafiltration using an ultrafiltration membrane with a different molecular weight cutoff compared to composition A; specifically:

[0130] Step S7: post-treatment: the stirred product is coarsely filtered using a filter membrane with a pore size of 2 μm, the coarse filtrate is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 4500 Da to obtain an ultrafiltrate, the ultrafiltrate is concentrated under reduced pressure at 58° C. to 15.7% of the mass of the ultrafiltrate, freeze-dried at -30° C. to a water content of 6.12%, and sterilized with ultraviolet light to obtain the composition;

[0131] The other preparation steps are the same as those of composition A.

[0132] 3. Effect Verification

[0133] 1. Skin patch test

[0134] The above composition was diluted with deionized water to a concentration of 5wt% as a sample, and then a skin patch test was performed. The specific experimental steps were: volunteers with sensitive skin aged between 25 and 45 were selected and randomly divided into 15 groups, with 6 people in each group; a 40mm 2 , a spot tester with a depth of about 1mm, put the test substance into the small chamber of the spot tester, and the amount is 0.02mL. The test substance is a cosmetic, and the spot tester with the test substance is applied to the back of the subject with a low-allergenic tape, and the palm is gently pressed to evenly apply it to the skin. After 24 hours, the test substance spot tester is removed. After the test substance spot tester is removed and the indentation disappears, observe the skin reaction according to the standards in Table 1 (skin reaction grading standards for skin occlusion patch test) and record the observation results. The specific results are shown in Table 2.

[0135] Table 1: Skin occlusion patch test skin reaction grading criteria

[0136]

[0137] Table 2: Skin patch test results

[0138] Test / dilute to 5wt% Level 0 people Level 1 Number of people Level 2 Number of People Level 3 Number of People Level 4 Number of People Composition A 8 0 0 0 0 Composition B 8 0 0 0 0 Composition C 8 0 0 0 0 Composition D 8 0 0 0 0 Composition E 8 0 0 0 0 Composition F 8 0 0 0 0 Composition G 8 0 0 0 0 Composition H 8 0 0 0 0 Composition I 8 0 0 0 0 Composition J 8 0 0 0 0 Composition K 8 0 0 0 0 Composition L 8 0 0 0 0 Composition M 8 0 0 0 0 Composition N 8 0 0 0 0 Composition O 8 0 0 0 0

[0139] It can be seen from the test results in Table 2 that the composition prepared by the present invention has low irritation and is harmless to the skin.

[0140] 2. Whitening test

[0141] The above composition was diluted to 5wt% with deionized water to obtain a test sample, which was then tested in a whitening experiment. The specific test method is: volunteers aged between 25 and 45 were selected and randomly divided into 15 groups, with 3 male and 3 female volunteers in each group; each group of volunteers used it once in the morning and once in the evening, with 8 drops of the test sample used each time, and the method of use was to apply it evenly on the face for 2 months. Before using the sample and after 1 month and 2 months of using the sample, 5 points were randomly selected on the cheeks of the volunteers to test the color brightness value L* using a test instrument (Finland Delfin Skin Color Meter, model: Finland Skin Color Catch), and the average value was taken to calculate the change value △L in the cheek skin color brightness * , which is used to characterize the skin whitening effect of the composition. * The calculation formula is the average L after using the sample * - Average L before using the sample * The test results are shown in Table 3.

[0142] Table 3: Whitening effect test results

[0143] Test / dilute to 5wt% △L* after 1 month of use △L* after 2 months of use Composition A 3.957 5.435 Composition B 3.928 5.398 Composition C 3.052 4.265 Composition D 3.764 5.186 Composition E 3.821 5.261 Composition F 3.793 5.224 Composition G 3.834 5.274 Composition H 3.865 5.316 Composition I 3.891 5.350 Composition J 3.935 5.407 Composition K 3.947 5.422 Composition L 3.376 4.686 Composition M 3.704 5.109 Composition N 3.628 5.013 Composition O 3.809 5.247

[0144] The test results in Table 3 show that the composition of the present invention can be diluted to 5wt% and used for one month to increase the skin brightness value (L * ) is 3.957, and using it for 2 months can increase the skin brightness value (L * ) is 5.435; the composition of the present invention has a good skin whitening and lightening effect.

[0145] The test results of compositions A and C show that the present invention uses fermentation to prepare the composition, which can significantly improve the skin whitening effect of the composition. The test results of compositions A, D, and E show that the present invention uses Candida utilis CICC31170 and Lactobacillus helveticus CICC 22818 for co-fermentation to enhance the whitening effect of the composition, which is better than the effect of using only one of them. The possible reason is that the combination of the two for fermentation mutually promotes the increase of the types and concentrations of active ingredients in the composition, so that the whitening effect of the composition is further enhanced.

[0146] The test results of compositions A, F, G, H, and I show that the use of knotweed and daisy in the composition of the present invention produces a compatible relationship in increasing the whitening effect, so that the composition has a good whitening effect, especially when the mass ratio of knotweed to daisy is in the range of 1:0.8-1.5, the whitening effect of the obtained composition is better.

[0147] The present invention adopts plant cell culture technology to culture plant raw materials, adds ethanol for extraction, and uses an ultrafiltration process with a molecular weight cutoff lower than 3000Da, all of which can enhance the whitening effect of the composition, and the above content can be obtained through the test results of compositions A, L, M, and N.

[0148] 3. Skin tightening effect test

[0149] In the whitening test, three points on the cheeks of volunteers were randomly selected before using the sample and after using the sample for 1 month and 2 months, and the skin elasticity R2 value was tested using a test instrument (Cutometer MPA 580) (the closer the R2 value is to 1, the better the skin elasticity); the R2 value is the ratio of the rebound amount of the skin without negative pressure to the maximum stretching amount under negative pressure; this parameter is used to characterize the skin tightening effect of the composition. The results are shown in Table 4.

[0150] Table 4:

[0151] Test / dilute to 5wt% R2 value before use Use 1 month R2 value Use 2-month R2 value Composition A 0.6935 0.7370 0.7503 Composition B 0.6954 0.7385 0.7517 Composition C 0.6916 0.7258 0.7343 Composition D 0.7001 0.7416 0.7539 Composition E 0.6925 0.7346 0.7472 Composition F 0.7013 0.7445 0.7576 Composition G 0.6892 0.7323 0.7453 Composition H 0.6927 0.7363 0.7494 Composition I 0.6885 0.7319 0.7449 Composition J 0.7007 0.7431 0.7558 Composition K 0.6948 0.7376 0.7505 Composition L 0.6871 0.7246 0.7349 Composition M 0.6903 0.7312 0.7432 Composition N 0.6984 0.7385 0.7501 Composition O 0.6962 0.7381 0.7507

[0152] The contents of Table 4 were converted to calculate the increase in skin elasticity value R2 after using the sample for 1 month and 2 months compared with before using the sample. The results are shown in Table 5.

[0153] Table 5: Increase in skin elasticity R2 at different times

[0154] Test / dilute to 5wt% Use 1 month R2 value to increase value Use the 2-month R2 value for increased value Composition A 0.0435 0.0568 Composition B 0.0431 0.0563 Composition C 0.0342 0.0427 Composition D 0.0415 0.0538 Composition E 0.0421 0.0547 Composition F 0.0432 0.0563 Composition G 0.0431 0.0561 Composition H 0.0436 0.0567 Composition I 0.0434 0.0564 Composition J 0.0424 0.0551 Composition K 0.0428 0.0557 Composition L 0.0375 0.0478 Composition M 0.0409 0.0529 Composition N 0.0401 0.0517 Composition O 0.0419 0.0545

[0155] It can be seen from the test results in Tables 4-5 that the composition of the present invention has a good effect of increasing skin elasticity and imparting skin firming effect.

[0156] The present invention uses a fermentation method to prepare the composition, which can significantly improve the skin tightening effect of the composition. The test results of compositions A, D, and E show that the present invention uses Candida utilis CICC 31170 and Lactobacillus helveticus CICC22818 for co-fermentation, so that the skin tightening effect of the composition is better.

[0157] The test results of compositions A, J and K show that the present invention uses Cryptomeria fortunei and Agave as raw materials to prepare the composition, which can make the composition have a better skin tightening effect.

[0158] The present invention adopts plant cell culture technology to culture plant raw materials, adds ethanol for extraction, and uses an ultrafiltration process with a molecular weight cutoff lower than 3000Da, all of which can enhance the whitening effect of the composition, and the above content can be obtained through the test results of compositions A, L, M, and N.

[0159] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A composition prepared based on plant cell culture, characterized in that: The composition preparation method comprises the following steps: Step S1: material preparation and pretreatment: weigh the raw materials according to mass: 1-3 parts of Schisandra chinensis, 0.5-2 parts of Borago officinalis, 0.8-2 parts of Polygonum cuspidatum, 0.5-2.5 parts of Bellis Perennis, 0.5-2.2 parts of Cryptomeria fortunei, 0.4-2 parts of Kava Piper, and 0.5-3 parts of Agave; wash, drain, shear and sterilize the raw materials respectively, and then mix them to obtain pre-treated materials; Step S2: Obtaining callus tissue: adding the pre-treated material to the culture medium, culturing at 20-25° C. in the dark for 4-6 weeks to obtain callus tissue; Step S3: Obtaining cell culture: Dispersing callus tissue in culture medium, culturing at 22-30° C. in the dark for 5-8 weeks to obtain cell culture; Step S4: obtaining the culture: filtering the cell culture, taking out the solid matter, and obtaining the culture; Step S5: Fermentation: sterilize the culture, add water and carbon source to obtain a mixture, then inoculate the fermentation bacteria, and ferment at a temperature of 30-38°C in the dark for 32-50 hours to obtain a fermentation product; wherein the concentration of the fermentation bacteria in the mixture is 2.5×10 8 -6.8×10 8 cfu / mL, the fermentation bacteria consisted of Candida utilis CICC 31170 and Lactobacillus helveticus CICC 22818 in a live cell count ratio of 1.2-2.5:1; Step S6: Extraction: sterilize the fermented product, then add ethanol, adjust the ethanol concentration, heat and keep warm and stir for 5-10 hours to obtain a stirred product; Step S7: post-treatment: coarsely filter the stirred product, ultrafilter the coarse filtrate to obtain an ultrafiltrate, concentrate, dry and sterilize the ultrafiltrate to obtain the composition prepared based on plant cell culture; In step S1, the mass ratio of knotweed to bellis is 1:0.8-1.5; In step S6, the mass concentration of ethanol is adjusted to 20-45%; In step S7, the coarse filtration is performed using a filter membrane with a pore size of 2 μm, and the ultrafiltration is performed using an ultrafiltration membrane with a molecular weight cutoff of 1800 Da.

2. The composition prepared based on plant cell culture according to claim 1, characterized in that: In step S5, the fermentation bacteria are composed of Candida utilis CICC 31170 and Lactobacillus helveticus CICC 22818 in a live bacteria ratio of 1.4-2:

1.

3. The composition prepared based on plant cell culture according to claim 1, characterized in that: In step S5, the concentration of the fermentation bacteria in the mixture is 3×10 8 -6.2×10 8 cfu / mL.

4. The composition prepared based on plant cell culture according to claim 1, characterized in that: In step S5, the carbon source is at least one of glucose, sucrose, fructose, white sugar and brown sugar, and the amount of the carbon source is 2-5% of the mass of the mixture.

5. The composition prepared based on plant cell culture according to claim 1, characterized in that: In step S5, the mass ratio of the culture medium to water is 1:20-60.

6. The composition prepared based on plant cell culture according to claim 1, characterized in that: The culture medium used in step S2 is composed of: MS culture medium, 10-22 g / L glucose, 0.3-1.1 mg / L indolebutyric acid, 0.2-1.0 mg / L indoleacetic acid, 0.5-0.9 mg / L adenine and 1-2.5 mg / L 6-benzylaminopurine; The composition of the culture medium used in step S3 is: MS culture medium, 18-28 g / L glucose and 0.8-1.5 mg / L adenine.

7. Use of the composition prepared based on plant cell culture according to any one of claims 1 to 6 in the preparation of anti-photoaging, whitening, firming and anti-wrinkle cosmetics.

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