Coleus forskohlii callus cell freeze-dried powder as well as preparation method and application thereof

Abstract

The invention discloses coleus forskohlii callus cell freeze-dried powder as well as a preparation method and application thereof, and relates to the technical field of cosmetics. The preparation method comprises the following steps: selecting petals of coleus forskohlii as explants, putting the explants into an induction culture medium, carrying out induction dedifferentiation to obtain calluses, obtaining coleus forskohlii callus cells by utilizing an in-vitro culture technology of infinite proliferation of dedifferentiated cells, and finally carrying out freeze-drying treatment on the coleus forskohlii callus cells to obtain the coleus forskohlii callus cell freeze-dried powder. According to the coleus forskohlii callus cell freeze-dried powder prepared by the invention, the clearance rate of DPPH free radicals can reach 33.31%, the inhibition rate of elastase can reach 47.11%, and the coleus forskohlii callus cell freeze-dried powder can obviously improve the skin compactness, the elasticity value and the skin roughness. Therefore, the coleus forskohlii callus cell freeze-dried powder prepared by the invention has a good anti-aging effect.

Description

Technical Field

[0001] The invention relates to the technical field of cosmetics, and in particular to a Coleus forskohlii callus cell freeze-dried powder, a preparation method and an application thereof. Background Art

[0002] Coleus barbatus (Andrews) Benth., also known as Coleus forskohlii, is a plant of the genus Coleus in the family Lamiaceae. Coleus forskohlii contains active ingredients such as terpenes, flavonoids, sterols and forskolin, which make it have important application value and development potential in the fields of medicine and cosmetics. Specifically, forskolin, as the key active ingredient in Coleus forskohlii, can activate adenylate cyclase and increase the level of the second messenger cyclic adenosine monophosphate (cAMP) in cells, thereby achieving the effect of strengthening the heart, lowering blood pressure and improving lung function; terpenoid compounds have antioxidant properties and can play an anti-thrombotic role. Flavonoids, sterols and terpenoid compounds also have antioxidant properties, and can work synergistically with each other to improve the skin's antioxidant defense system.

[0003] Skin aging is a complex physiological process that involves changes in mechanisms at multiple levels. For example, in terms of exogenous factors, factors such as ultraviolet radiation, environmental pollution, and stress can cause the skin to produce a large number of free radicals. These free radicals are highly active and can attack various biomacromolecules in skin cells, resulting in decreased skin elasticity and firmness. In terms of endogenous factors, as we age, cells enter a state of aging, accompanied by changes in hormone levels and metabolic disorders, which can increase the activity of elastase in the body. Excessive elastase can over-decompose elastic fibers, causing the skin to lose elasticity and firmness.

[0004] In the prior art, organic solvent extraction, column chromatography purification or microwave-assisted extraction are often used to extract the effective substances in the whole plant or root of Coleus forskohlii, and then the obtained Coleus forskohlii extract is used in the field of cosmetics. For example, CN111840201A discloses an anti-aging repair compound preparation containing stem cell factor and its preparation method, wherein the anti-aging repair compound preparation includes Coleus forskohlii root extract, and the forskolin unique to the root of Coleus forskohlii can activate the intracellular cyclic adenosine monophosphate level, thereby playing an anti-aging effect on the skin. CN118490592A discloses a sweet-scented osmanthus extract and its preparation method for anti-aging beauty and light wrinkle repair, wherein the sweet-scented osmanthus extract includes Coleus forskohlii root extract, and the Coleus forskohlii root extract is used in combination with other extracts to improve the anti-aging effect. Since skin aging involves a variety of complex mechanisms, and a single Coleus forskohlii extract is difficult to deal with in all directions, it is often necessary to be used in combination with other components to ensure the anti-aging effect. In addition, the active ingredients in the Coleus forskohlii extract are susceptible to external factors. For example, light, temperature changes and extended storage time may cause some of its active ingredients to decompose or transform, thereby reducing the activity of the extract.

[0005] Therefore, based on Coleus forskohlii, a lyophilized powder of Coleus forskohlii callus with higher biological activity is developed to ensure that it can effectively inhibit the activity of elastase and scavenge DPPH free radicals, thereby ensuring that even if a single component is used, a good anti-aging effect can still be achieved. Summary of the invention

[0006] In view of the above-mentioned prior art, the purpose of the present invention is to provide a lyophilized powder of Coleus forskohlii callus cells and a preparation method and application thereof. The present invention selects the petals of Coleus forskohlii as explants, places them in an induction medium and induces dedifferentiation to obtain callus tissue, and then utilizes the in vitro culture technology of unlimited proliferation of dedifferentiated cells to obtain Coleus forskohlii callus cells, and finally lyophilizes them to obtain lyophilized powder of Coleus forskohlii callus cells. The scavenging rate of the lyophilized powder of Coleus forskohlii callus cells prepared by the present invention can reach 33.31% for DPPH free radicals, and the inhibition rate of elastase can reach 47.11%, which can significantly improve skin firmness, elasticity value and smoothness. It can be seen that the lyophilized powder of Coleus forskohlii callus cells prepared by the present invention has good anti-aging effect.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention provides a method for preparing a lyophilized powder of Coleus forskohlii callus cells, comprising the following steps:

[0009] (1) pre-treating the petals of Coleus forskohlii and placing them in an induction medium for culturing to obtain Coleus forskohlii callus;

[0010] (2) placing the Coleus forskohlii callus in a proliferation medium for proliferation culture, filtering, and collecting the solid to obtain the Coleus forskohlii callus cells;

[0011] (3) The Coleus forskohlii callus cells are freeze-dried to obtain the Coleus forskohlii callus cell freeze-dried powder.

[0012] Preferably, in step (1), the specific operation of pre-treating the petals of Coleus forskohlii is:

[0013] Soak the washed petals of Coleus forskohlii in alcohol, and then rinse with water; then, place the rinsed petals of Coleus forskohlii in a calcium hypochlorite solution for disinfection, and then rinse and dry.

[0014] Further preferably, the mass fraction of the alcohol is 75%, and the immersion time is 25-35s; the mass fraction of calcium hypochlorite is 5%, and the disinfection treatment time is 3-5min.

[0015] Preferably, in step (1), the induction medium is composed of: MS medium, 1.0 mg / L NAA, 2.0 mg / L LBA, 30000 mg / L sucrose and 7000 mg / L agar.

[0016] Preferably, in step (1), during the culture process, the culture temperature is 23-27° C., the culture time is 18-22 days, and the culture is cyclically cultured under conditions of 16 h light and 7 h dark.

[0017] Preferably, in step (2), the proliferation medium is composed of: MS medium, 0.2 mg / L NAA, 1.0 mg / L LBA and 5000 mg / L AgNO3.

[0018] Preferably, in step (2), during the proliferation culture, the temperature is 23-27°C, the rotation speed is 110-130 r / min, the light treatment is 15-17 h per day, and subculture is performed once every 20 days.

[0019] Preferably, in step (2), during the filtration process, a ceramic membrane with a pore size of 400 nm is used for filtration.

[0020] Preferably, in step (3), the specific process of freeze-drying is:

[0021] The Coleus forskohlii callus cells are firstly subjected to a pre-freezing treatment, and then subjected to a first sublimation, a second sublimation, a third sublimation and a fourth sublimation in sequence to obtain the Coleus forskohlii callus cell freeze-dried powder.

[0022] Further preferably, during the pre-freezing treatment, the temperature is -40 to -50°C and the treatment time is 3-5 min; during the first sublimation process, the temperature is 0°C, the vacuum degree is 10-20 Pa, and the treatment time is 15-25 min; during the second sublimation process, the temperature is 3-7°C, the vacuum degree is 10-20 Pa, and the treatment time is 3-5 min; during the third sublimation process, the temperature is 10-20°C, the vacuum degree is 8-12 Pa, and the treatment time is 2 min.

[0023] The second aspect of the present invention provides a lyophilized powder of Coleus forskohlii callus cells prepared by the above preparation method.

[0024] Preferably, in the Coleus forskohlii callus cell freeze-dried powder, the content of Coleus forskohlii callus cells is 1.15-1.25 billion / g, and the total flavonoids content is 30-35%.

[0025] The third aspect of the present invention provides the use of the above-mentioned Coleus forskohlii callus cell freeze-dried powder in the preparation of anti-aging products.

[0026] Beneficial effects of the present invention:

[0027] The invention selects petals of Coleus forskohlii as explants, places the petals in an induction culture medium to induce dedifferentiation, obtains callus tissue, then uses an in vitro culture technology of unlimited proliferation of dedifferentiated cells to obtain Coleus forskohlii callus cells, and finally freeze-dries the petals to obtain Coleus forskohlii callus cell freeze-dried powder.

[0028] The lyophilized powder of Coleus forskohlii callus cells prepared by the present invention can reach a scavenging rate of 33.31% for DPPH free radicals, and can reach a 47.11% inhibition rate for elastase, and can significantly improve skin firmness, elasticity value and smoothness. It can be seen that the lyophilized powder of Coleus forskohlii callus cells prepared by the present invention has good anti-aging effect. Therefore, it can be used as a raw material of cosmetics with anti-aging effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : The callus cell diagram of Coleus forskohlii obtained in Example 1;

[0030] Figure 2 : Physical picture of the freeze-dried powder of Coleus forskohlii callus cells prepared in Example 1;

[0031] Figure 3 : The corresponding wrinkle scores before and after using the cell sample / ordinary sample in Test Example 4;

[0032] Figure 4: The corresponding skin firmness F4 values ​​before trial and after using the cell sample / ordinary sample in Test Example 4;

[0033] Figure 5 : The corresponding skin total elasticity R2 value before trial and after using the cell sample / ordinary sample in Test Example 4;

[0034] Figure 6 : The corresponding skin smoothness values ​​before trial and after using the cell sample / ordinary sample in Test Example 4;

[0035] Figure 7 : Comparison of the improvement of facial wrinkles on human body before and after 28 days of using freeze-dried powder of Coleus forskohlii callus cells in Experimental Example 5;

[0036] Figure 8 : Comparison of facial smoothness improvement before and after using the Coleus forskohlii extract for 28 days in Test Example 5. DETAILED DESCRIPTION

[0037] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0038] In the prior art, Coleus forskohlii extract is often used as a raw material for preparing products with anti-aging effects, but skin aging is caused by the combined effects of endogenous and exogenous factors, involving multi-level mechanism changes. However, a single Coleus forskohlii extract is difficult to deal with all aspects, and it is often necessary to combine it with other components to ensure the anti-aging effect. In addition, the active ingredients in the Coleus forskohlii extract are easily affected by external factors.

[0039] Based on this, the present invention provides a Coleus forskohlii callus cell freeze-dried powder. The preparation method thereof is: selecting the petals of Coleus forskohlii as explants, pre-treating the petals and placing them in an induction medium to culture callus tissue, then placing the callus tissue in a proliferation medium for proliferation culture to obtain callus tissue cells, and finally freeze-drying the petals to obtain the Coleus forskohlii callus cell freeze-dried powder.

[0040] Specifically, petals are selected as explants and placed in an induction medium to induce callus tissue. The induction medium is composed of MS medium, NAA, LBA, sucrose and agar. NAA (naphthaleneacetic acid) is used to induce cell elongation and division, promote dedifferentiation of explants, restore the ability of differentiated cells to divide, and form callus tissue; LBA (indolebutyric acid) is an endogenous auxin that can promote cell division and growth, induce the formation of root primordium, regulate hormone balance, and improve the quality and vitality of callus tissue. Sucrose is used as a carbon source to provide energy for callus tissue to meet the energy requirements of cell growth, division and various physiological metabolic activities. Subsequently, the callus tissue is placed in a proliferation medium for proliferation culture to obtain callus tissue cells, wherein the proliferation medium is composed of MS medium, 0.2 mg / L NAA, 1.0 mg / L LBA and 0.5% AgNO3. As plant growth regulators, NAA and LBA can induce and promote cell division to form callus tissue. AgNO3 can inhibit the biosynthesis and physiological effects of ethylene, indirectly regulate the balance of plant hormones, promote cell division and growth, and improve the callus induction rate and quality. At the same time, it can also promote organogenesis and prevent browning. Finally, the callus cells are freeze-dried to obtain the final product.

[0041] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0042] The experimental materials used in the embodiments of the present invention are all conventional experimental materials in the art and can be purchased through commercial channels.

[0043] Example 1: Preparation of lyophilized powder of Coleus forskohlii callus cells

[0044] (1) Take 50 petals of Coleus forskohlii, wash them with running water, soak them in 75% alcohol by mass for 30 seconds, and then rinse them with water three times. Then, place the rinsed petals in 5% calcium hypochlorite for disinfection for 4 minutes, rinse them with water three times, and absorb the moisture on the surface of the petals with filter paper;

[0045] The pretreated petals of Coleus forskohlii are placed in an induction medium, and cultured at 25° C. for 20 days. During the culture period, the culture is cycled under 16 hours of light and 7 hours of darkness to obtain Coleus forskohlii callus, wherein the induction medium is composed of: MS medium, 1.0 mg / L NAA, 2.0 mg / L LBA, 30000 mg / L sucrose and 7000 mg / L agar;

[0046] (2) placing the callus of Coleus forskohlii in a proliferation culture medium, and performing proliferation culture at 25° C. and 120 r / min, during the proliferation culture period, ensuring that the light treatment is 16 hours per day, and performing subculture every 20 days. After the culture is completed, filtering and collecting the solid, which is the callus cell of Coleus forskohlii; wherein the proliferation culture medium is composed of: MS medium, 0.2 mg / L NAA, 1.0 mg / L LBA and 5000 mg / L AgNO3;

[0047] (3) freeze-drying the Coleus forskohlii callus cells to obtain freeze-dried powder of Coleus forskohlii callus cells; wherein the specific operation of the freeze-drying treatment is: pre-freezing the Coleus forskohlii callus cells at -45°C for 4 minutes, treating them at 0°C and 15Pa for 20 minutes for the first sublimation, treating them at 5°C and 15Pa for 4 minutes for the second sublimation, and treating them at 15°C and 10Pa for 2 minutes for the third sublimation.

[0048] The Coleus forskohlii callus cells prepared in this example are as follows: Figure 1 As shown, the lyophilized powder of Coleus forskohlii callus cells is as follows Figure 2 As shown,

[0049] Example 2: Preparation of lyophilized powder of Coleus forskohlii callus cells

[0050] (1) Take 50 petals of Coleus forskohlii, wash them with running water, soak them in 75% alcohol by mass for 25 seconds, and then rinse them with water three times. Then, place the rinsed petals in 5% calcium hypochlorite for disinfection for 3 minutes, rinse them with water three times, and absorb the moisture on the surface of the petals with filter paper;

[0051] The pretreated petals of Coleus forskohlii are placed in an induction medium, and cultured at 23° C. for 22 days. During the culture period, the culture is cycled under conditions of 16 hours of light and 7 hours of darkness to obtain Coleus forskohlii callus, wherein the induction medium is composed of: MS medium, 1.0 mg / L NAA, 2.0 mg / L LBA, 30000 mg / L sucrose and 7000 mg / L agar;

[0052] (2) placing the callus of Coleus forskohlii in a proliferation culture medium, and performing proliferation culture at 23° C. and 110 r / min, during the proliferation culture period, ensuring that the light treatment is 15 hours per day, and performing subculture every 20 days. After the culture is completed, filtering and collecting the solid, which is the callus cell of Coleus forskohlii; wherein the proliferation culture medium is composed of: MS medium, 0.2 mg / L NAA, 1.0 mg / L LBA and 5000 mg / L AgNO3;

[0053] (3) freeze-drying the Coleus forskohlii callus cells to obtain freeze-dried powder of Coleus forskohlii callus cells; wherein the specific operation of the freeze-drying treatment is: pre-freezing the Coleus forskohlii callus cells at -40°C for 5 minutes, treating them at 0°C and 10Pa for 25 minutes for the first sublimation, treating them at 3°C ​​and 20Pa for 5 minutes for the second sublimation, and treating them at 20°C and 8Pa for 2 minutes for the third sublimation.

[0054] Example 3: Preparation of lyophilized powder of Coleus forskohlii callus cells

[0055] (1) Take 50 petals of Coleus forskohlii, wash them with running water, soak them in 75% alcohol by mass for 35 seconds, and then rinse them with water three times. Then, place the rinsed petals in 5% calcium hypochlorite for disinfection for 5 minutes, rinse them with water three times, and use filter paper to absorb the moisture on the surface of the petals;

[0056] The pretreated petals of Coleus forskohlii are placed in an induction medium, and cultured at 25° C. for 20 days. During the culture period, the culture is cycled under 16 hours of light and 7 hours of darkness to obtain Coleus forskohlii callus, wherein the induction medium is composed of: MS medium, 1.0 mg / L NAA, 2.0 mg / L LBA, 30000 mg / L sucrose and 7000 mg / L agar;

[0057] (2) placing the callus of Coleus forskohlii in a proliferation culture medium, and performing proliferation culture at 27° C. and 130 r / min, during the proliferation culture period, ensuring that the light treatment is 17 hours per day, and performing subculture every 20 days. After the culture is completed, filtering and collecting the solid, which is the callus cell of Coleus forskohlii; wherein the proliferation culture medium is composed of: MS medium, 0.2 mg / L NAA, 1.0 mg / L LBA and 5000 mg / L AgNO3;

[0058] (3) The Coleus forskohlii callus cells are freeze-dried to obtain freeze-dried Coleus forskohlii callus cell powder.

[0059] The specific operation of freeze-drying treatment is as follows: the callus cells of Coleus forskohlii are placed at -50°C for pre-freezing treatment for 3 minutes, treated at 0°C and 20Pa for 15 minutes for the first sublimation, treated at 7°C and 10Pa for 3 minutes for the second sublimation, and treated at 10°C and 12Pa for 2 minutes for the third sublimation.

[0060] Comparative Example 1: Coleus forskohlii extract

[0061] Propylene glycol is heated to 85° C., mixed with petals of Coleus forskohlii and immersed for 24 hours, wherein the solid-liquid ratio of propylene glycol to petals of Coleus forskohlii is 1 g:100 mL. After the immersion is completed, the filtered liquid is separated and purified to obtain the Coleus forskohlii extract.

[0062] Test Example 1: DPPH free radical scavenging test

[0063] The lyophilized powder of Coleus forskohlii callus cells prepared in Example 1 was mixed with sterile water to prepare a lyophilized solution of Coleus forskohlii callus cells with a mass fraction of 1%. At the same time, the Coleus forskohlii extract prepared in Comparative Example 1 was mixed with sterile water to prepare a 1% Coleus forskohlii extract. The lyophilized solution of Coleus forskohlii callus cells and the Coleus forskohlii extract were used as sample solutions to perform a DPPH free radical scavenging test. The specific steps are as follows:

[0064] (1) According to the addition amount of each reagent in Table 1, a DPPH free radical scavenging test reaction system was constructed;

[0065] Table 1 Addition amount of each group of reagents in the DPPH free radical scavenging test reaction system

[0066]

[0067] (2) The reaction system was placed at room temperature for 20 min. After the reaction was completed, 200 μL of each reaction solution was added to a 96-well plate, the absorbance value (OD value) was read at 517 nm, and the DPPH free radical scavenging rate was calculated. The results are shown in Table 2, where the DPPH free radical scavenging rate calculation formula is as follows:

[0068] DPPH free radical scavenging rate (%) = [1-(OD T1 -OD T2 ) / (OD C1 -OD C2 )]×100%;

[0069] In the formula, OD T1 is the absorbance value of T1 group at 517nm, OD T2 is the absorbance value of T2 group at 517nm, OD C1 is the absorbance value of group C1 at 517 nm, OD C2 is the absorbance value of group C1 at 517 nm.

[0070] Table 2 DPPH free radical scavenging rate

[0071] Group sample DPPH free radical scavenging rate (%) Example 1 Coleus forskohlii callus cell freeze-dried solution 33.31 Comparative Example 1 Coleus forskohlii petal extract 25.99

[0072] One of the important causes of aging is the damage to cells by free radicals. Specifically, free radicals are constantly produced in the human body under normal physiological processes and external stimuli. Free radicals have extremely strong oxidative ability and will attack biological macromolecules in cells, leading to cell function decline and apoptosis, and then inducing aging of the body. If the excessive free radicals in the body can be removed, the oxidative damage to cells by free radicals can be reduced, and the normal structure and function of cells can be protected, the aging process of the body can be delayed. It can be seen that the DPPH free radical scavenging rate can reflect the anti-aging effect, and the higher the DPPH free radical scavenging rate, the better the anti-aging effect.

[0073] It can be seen from Table 2 that the DPPH radical scavenging rate of the corresponding solution of the lyophilized powder of the petals of Coleus forskohlii prepared by the present invention can reach 33.31%, while the DPPH radical scavenging rate of the corresponding solution of the Coleus forskohlii petals extract prepared in Comparative Example 1 is only 25.99%. It can be seen that the DPPH radical scavenging rate of the lyophilized powder of the petals of Coleus forskohlii prepared by the present invention is much higher than that of the Coleus forskohlii petals extract prepared in Comparative Example 1, and therefore, the lyophilized powder of the petals of Coleus forskohlii prepared by the present invention has excellent anti-aging effect.

[0074] Example 2: Elastase inhibition assay

[0075] The lyophilized powder of Coleus forskohlii callus cells prepared in Example 1 was mixed with sterile water to prepare a lyophilized solution of Coleus forskohlii callus cells with a mass fraction of 1%. At the same time, the Coleus forskohlii extract prepared in Comparative Example 1 was mixed with sterile water to prepare a 1% Coleus forskohlii extract. The lyophilized solution of Coleus forskohlii callus cells and the Coleus forskohlii extract were used as sample solutions to perform an elastase inhibition test. The specific steps are as follows:

[0076] (1) Solution configuration:

[0077] Dilute 1 mol / L Tris-HCl solution by 20 times with water to obtain 50 mmol / L Tris-HCl solution;

[0078] Weigh 0.001 g of elastase and add it to 10 mL of 50 mmol / L–HCl solution. After it is completely dissolved, add 10 mL of water to obtain a 200 mU / mL elastase solution.

[0079] To 0.002 g of AAPAN was added 10 mL of 50 mmol / L Tris-HCl solution, and after complete dissolution, 20 mL of water was added to obtain a 4.43 mmol / L AAPANN solution, where AAPANN is succinyl-alanine-alanine-alanine-p-nitroaniline, purchased from MacLean;

[0080] (2) Construction of elastase inhibition test reaction system

[0081] Referring to the addition amount of each reagent in Table 3, an elastase inhibition test reaction system was constructed. The specific steps were as follows: after mixing the Tris-HCl solution and the elastase solution, the mixture was treated at 25°C for 15 minutes, and then the AAPAN solution was added. After mixing evenly, the mixture was color developed at room temperature for 10 minutes, and then the absorbance was measured at a wavelength of 590 nm to calculate the elastase inhibition rate. The results are shown in Table 4.

[0082] Table 3 Addition amount of each reagent in the elastase inhibition test reaction system

[0083] A B C D Tris-HCl solution (mL) 2 3 1 2 Sample solution (mL) 0 0 1 1 Elastase solution (mL) 1 0 1 0 AAPAN solution (mL) 1 1 1 1 Parallel times 3 3 3 3

[0084] The calculation formula of elastase inhibition rate (%) is as follows:

[0085]

[0086] In the formula, A is the absorbance of the control group; B is the absorbance of the blank group; C is the absorbance of the experimental group; and D is the absorbance of the sample blank group.

[0087] Table 4 Elastase inhibition rate

[0088]

[0089]

[0090] Elastase is an enzyme that can specifically degrade elastin. As a vital protein in human skin and other tissues, it gives skin and other tissues elasticity and retraction ability. During the aging process, the activity of elastase increases, thereby accelerating the decomposition of elastin, which in turn damages skin tissue and function and promotes the appearance of aging symptoms. The elastase inhibition rate can intuitively reflect the strength of its protective effect on elastin, and is then used to evaluate the anti-aging effect of the product. Specifically, the higher the elastase inhibition rate, the lower the degradation rate of elastin, which allows elastin to remain in the skin tissue for a longer time, thereby reflecting a better anti-aging effect.

[0091] As can be seen from Table 4, the elastase inhibition rate of the corresponding solution of the lyophilized powder of the petals of Coleus forskohlii prepared by the present invention can reach 47.11%, while the elastase inhibition rate of the corresponding solution of the Coleus forskohlii extract prepared in Comparative Example 1 is only 33.63%. It can be seen that the elastase inhibition rate of the lyophilized powder of the petals of Coleus forskohlii prepared by the present invention is much higher than that of the Coleus forskohlii extract prepared in Comparative Example 1, and therefore, the lyophilized powder of the petals of Coleus forskohlii prepared by the present invention has excellent anti-aging effect.

[0092] Test Example 3:

[0093] The lyophilized powder of Coleus forskohlii callus cells prepared in Example 1 was mixed with sterile water to prepare a lyophilized solution of Coleus forskohlii callus cells with a mass fraction of 1% as a cell sample. At the same time, the Coleus forskohlii extract prepared in Comparative Example 1 was mixed with sterile water to prepare a 1% Coleus forskohlii petal extract as a common sample. The cell samples and the common samples were respectively taken to test the anti-aging effect on human face.

[0094] In this test case, 60 subjects aged 21-45 years were selected and randomly divided into 2 groups, of which:

[0095] Treatment group 1: using normal samples;

[0096] Treatment group 2: using cell samples;

[0097] Test instruments: German CK Cutometer dual MPA580, VISIA7.

[0098] Test environment requirements: Temperature: 21.0℃±1.0℃; Humidity: 50%±10%.

[0099] The validity period of the test sample is 28 days.

[0100] Observation and test part: face.

[0101] The subjects in each treatment group used the common sample / cell sample three times a day, with a dosage of 1.5 mL each time, for 28 consecutive days.

[0102] 1. Wrinkle score

[0103] The wrinkle scores of the subjects were tested before trial and after 28 days of use. The average value was taken as the wrinkle score of each group, and the change rate was calculated to reflect the anti-aging effect of the sample. The results are as follows: Figure 3 shown.

[0104] The calculation formula of the change rate is:

[0105] Change rate = [(wrinkle score after trial - wrinkle score before trial) / wrinkle score before trial] × 100%;

[0106] The wrinkle score is a comprehensive indicator that reflects the size, area and density of wrinkles. The smaller the value, the less obvious the wrinkles. Figure 3 (a) It can be seen that before using the cell sample, the wrinkle score was 35.74; after using the cell sample for 28 days, the wrinkle score was 18.34, and the change rate was -48.68% compared with before the trial. Figure 3(b) It can be seen that before using the common sample, the wrinkle score was 35.45, and after using the common sample for 28 days, the wrinkle score was 19.01, and the change rate was -46.37% compared with before the trial. It can be seen that the freeze-dried powder of Coleus forskohlii callus cells prepared by the present invention can significantly improve skin wrinkles, and its effect of improving skin wrinkles is better than that of the Coleus forskohlii petal extract.

[0107] 2. Skin firmness (F4 value)

[0108] The skin firmness (F4 value) of the subjects before trial and after 28 days of use was tested, and the average value was taken as the skin firmness (F4 value) of each group, and the change rate was calculated to reflect the anti-aging effect of the sample. The results are as follows Figure 4 shown.

[0109] The calculation formula of the change rate is:

[0110] Change rate = [(skin firmness F4 value after trial - skin firmness F4 value before trial) / skin firmness F4 value before trial] * 100%.

[0111] Skin firmness (F4 value) is a comprehensive indicator of wrinkle size, area and density. The smaller the value, the less obvious the wrinkles. Figure 4 (a) It can be seen that before using the cell sample, the skin firmness F4 value was 9.34, and after using the cell sample for 28 days, the skin firmness F4 value was 5.03, with a change rate of -46.15%; Figure 4 (b) It can be seen that before using the common sample, the skin firmness F4 value was 8.46, and after using the common sample for 28 days, the skin firmness F4 value was 4.78, and the change rate was -43.50%. It can be seen that the freeze-dried powder of Coleus forskohlii callus cells prepared by the present invention can improve skin firmness.

[0112] 3. Total skin elasticity (R2 value)

[0113] The total skin elasticity (R2 value) of the subjects before trial and after 28 days of use was tested, and the average value was taken as the total skin elasticity (R2 value) of each group, and the change rate was calculated to reflect the anti-aging effect of the sample. The results are as follows Figure 5 As shown. The calculation formula of the change rate is:

[0114] Change rate = [(total skin elasticity R2 value after trial - total skin elasticity R2 value before trial) / total skin elasticity R2 value before trial] × 100%.

[0115] The skin elasticity value (R2 value) reflects the total elasticity of the skin, indicating the degree to which the skin can recover within a cycle after being deformed by negative pressure. The larger the value, the greater the elasticity. Figure 5(a) It can be seen that before using the cell sample, the R2 value of the total skin elasticity was 56.99. After using the cell sample for 28 days, the R2 value of the total skin elasticity was 79.78, and the change rate was 39.99%. Figure 5 (b) It can be seen that before using the common sample, the skin total elasticity R2 value was 71.83, and after using the common sample for 28 days, the skin total elasticity R2 value was 81.81, and the rate of change was 13.89%. This shows that the freeze-dried powder of Coleus forskohlii callus cells prepared by the present invention can significantly improve the total elasticity of the skin.

[0116] 4. Skin roughness (SEr)

[0117] The skin roughness (SEr) of the subjects was tested before trial and after 28 days of use. The average value was taken as the skin roughness (SEr) of each group, and the change rate was calculated to reflect the anti-aging effect of the sample. The results are as follows: Figure 6 shown.

[0118] The calculation formula of the change rate is:

[0119] Change rate = [(skin roughness after trial - skin roughness before trial) / skin roughness before trial] × 100%.

[0120] The smaller the value of skin roughness (SEr), the better the skin. Figure 6 (a) It can be seen that before using the cell sample, the skin roughness SEr value was 5.52. After using the cell sample for 28 days, the skin roughness SEr value was 2.74, and the change rate was -50.36%. Figure 6 (b) It can be seen that before using the common sample, the skin roughness SEr value was 6.13, and after using the common sample for 28 days, the skin roughness SEr value was 3.77, and the change rate was -38.50%. It can be seen that the freeze-dried powder of Coleus forskohlii callus cells prepared by the present invention can significantly reduce skin roughness, and the effect is better than that of the Coleus forskohlii extract prepared in Comparative Example 1.

[0121] Test Example 5: Human Experiment

[0122] The lyophilized powder of Coleus forskohlii callus cells prepared in Example 1 was mixed with sterile water to prepare a lyophilized solution of Coleus forskohlii callus cells with a mass fraction of 1%. Meanwhile, the Coleus forskohlii extract prepared in Comparative Example 1 was mixed with sterile water to prepare a 1% Coleus forskohlii extract. The lyophilized solution of Coleus forskohlii callus cells and the Coleus forskohlii extract were applied to human facial skin for 28 days, and the facial wrinkles before and after use were measured with a Visia skin detector. The results are as follows: Figure 7 and Figure 8 As shown. It is used three times a day at fixed times in the morning, noon and evening, with each dose of 1.5 mL.

[0123] Depend on Figure 7 and Figure 8 It can be seen that the anti-aging effect of using the freeze-dried solution of Coleus forskohlii callus cells is better than that of the Coleus forskohlii extract.

[0124] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing freeze-dried powder of Coleus forskohlii callus cells, characterized in that: The following steps are involved: (1) pre-treating the petals of Coleus forskohlii and placing them in an induction medium for culturing to obtain Coleus forskohlii callus; (2) placing the Coleus forskohlii callus in a proliferation medium for proliferation culture, filtering, and collecting the solid to obtain the Coleus forskohlii callus cells; (3) The Coleus forskohlii callus cells are freeze-dried to obtain the Coleus forskohlii callus cell freeze-dried powder.

2. The method for preparing the freeze-dried powder of Coleus forskohlii callus cells according to claim 1, characterized in that: In step (1), the specific operation of pre-treating the petals of Coleus forskohlii is: The washed petals of Coleus forskohlii are soaked in alcohol and then rinsed with water; the rinsed petals of Coleus forskohlii are then placed in a calcium hypochlorite solution for disinfection, and then rinsed and dried; wherein the soaking time is 25-35 seconds and the disinfection time is 3-5 minutes.

3. The method for preparing the freeze-dried powder of Coleus forskohlii callus cells according to claim 1, characterized in that: In step (1), the composition of the induction medium is: MS medium, 1.0 mg / L NAA, 2.0 mg / L LBA, 30000 mg / L sucrose and 7000 mg / L agar.

4. The method for preparing the freeze-dried powder of Coleus forskohlii callus cells according to claim 1, characterized in that: In step (1), during the culture process, the culture temperature is 23-27° C., the culture time is 18-22 h, and the culture is cyclically carried out under the conditions of 16 h light and 7 h dark.

5. The method for preparing the freeze-dried powder of Coleus forskohlii callus cells according to claim 1, characterized in that: In step (2), the proliferation medium is composed of: MS medium, 0.2 mg / L NAA, 1.0 mg / L LBA and 5000 mg / L AgNO3.

6. The method for preparing the freeze-dried powder of Coleus forskohlii callus cells according to claim 1, characterized in that: In step (2), during the proliferation culture, the temperature is 23-27°C, the rotation speed is 110-130 r / min, the light treatment is 15-17 hours per day, and subculture is performed once every 20 days.

7. The method for preparing the freeze-dried powder of Coleus forskohlii callus cells according to claim 1, characterized in that: In step (3), the specific process of freeze-drying is as follows: The Coleus forskohlii callus cells are firstly subjected to a pre-freezing treatment, and then subjected to a first sublimation, a second sublimation, a third sublimation and a fourth sublimation in sequence to obtain the Coleus forskohlii callus cell freeze-dried powder.

8. The method for preparing the freeze-dried powder of Coleus forskohlii callus cells according to claim 7, characterized in that: During the pre-freezing process, the temperature is -40 to -50°C and the processing time is 3 to 5 minutes; During the first sublimation process, the temperature is 0°C, the vacuum degree is 10-20Pa, and the processing time is 15-25min; During the second sublimation process, the temperature is 3-7°C, the vacuum degree is 10-20Pa, and the processing time is 3-5min; During the third sublimation process, the temperature was 10-20°C, the vacuum degree was 8-12 Pa, and the processing time was 2 min.

9. The lyophilized powder of Coleus forskohlii callus cells prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the freeze-dried powder of Coleus forskohlii callus cells according to claim 9 in the preparation of anti-aging products.

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