CN112569221B - Use of Chibasu A in the preparation of medicines or skin care products for preventing and treating skin photoaging - Google Patents

Abstract

The invention discloses the use of chibain A in the preparation of medicines or skin care products for preventing and treating skin photoaging. Experimental results show that chibasuin A can improve the proliferation activity of HaCaT cells damaged by UVB; reduce the ROS content in HaCaT cells damaged by UVB , improve the antioxidant activity of UVB damaged HaCaT cells; reduce the apoptosis of UVB damaged HaCaT cells; and can significantly reduce the symptoms of skin photoaging. The experimental results show that the extract containing chilesin A can improve the proliferation activity of HaCaT cells damaged by UVB.

Description

Use of Chibasu A in the preparation of medicines or skin care products for preventing and treating skin photoaging

technical field

The invention relates to the use of chibasu A in the preparation of medicines or skin care products for preventing and treating skin photoaging.

Background technique

Aging is a process in which the body gradually loses function and goes to death. As the largest organ of the body, the skin is the defense barrier between the body and the external environment, and it can reflect aging more intuitively and obviously than other organs. Skin aging is usually divided into endogenous and exogenous aging, and exogenous aging is mainly caused by environmental factors. Ultraviolet (UV) radiation is the main environmental factor that causes skin aging. Long-term exposure of the skin to UV radiation will accelerate aging. Skin photoaging (also known as photoaging), severe cases even induce cancer.

Skin photoaging is a complex biochemical process, and its pathogenesis is related to mechanisms such as oxidative stress, inflammatory response, and high expression of matrix metalloproteinases. Oxidative stress is the initiating factor causing photoaging. A large number of reactive oxygen species (ROS), including superoxide anion, hydrogen peroxide, singlet oxygen, and hydroxyl radicals, are generated during photoaging. There is a relatively complete antioxidant system in the body, and the production and removal of ROS are in a balanced state. Excessive ROS leads to imbalance of redox system, which causes oxidative damage to intracellular lipids, proteins and nucleic acids, and finally triggers cell apoptosis and tissue and organ damage. Reducing the accumulation of ROS and maintaining the stability of the antioxidant internal environment is an important part of protecting the skin from UV damage.

Chibain A (also known as Melaleucain A, orogenin A) belongs to flavonoids, the full name is 5,7-dihydroxy-6-methoxy-2-phenyl-4H-1-benzofuran -4-ketone, mainly derived from the traditional Chinese medicine Eucommia. It has a wide range of pharmacological activities, such as anti-tumor, anti-inflammation, protection of blood vessels, protection of nerve cells, improvement of memory impairment, and anti-virus effects. So far, there has been no report on the use of chilesin A for the preparation of medicines or skin care products for preventing and treating skin photoaging.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art, and to provide the use of chilesin A in the preparation of medicines or skin care products for preventing and treating skin photoaging.

The second object of the present invention is to provide the use of the extract containing chilesin A in the preparation of medicines or skin care products for preventing and treating skin photoaging.

Technical scheme of the present invention is summarized as follows:

Use of Chibasu A in the preparation of medicines or skin care products for preventing and treating skin photoaging.

Use of the extract containing phyllosin A in the preparation of medicines or skin care products for preventing and treating skin photoaging.

The experimental results show that Chibalin A can improve the proliferation activity of HaCaT cells damaged by UVB; reduce the ROS content in HaCaT cells damaged by UVB, improve the antioxidant activity of HaCaT cells damaged by UVB; reduce the apoptosis of HaCaT cells damaged by UVB; and can Significantly reduces skin photoaging symptoms.

The experimental results show that the extract containing chilesin A can improve the proliferation activity of HaCaT cells damaged by UVB.

Description of drawings

Figure 1 is a graph showing the effect of radiation dose on the release of ROS in HaCaT cells, in which, A, blank control group B, group C with an irradiation dose of 30mJ/ ^cm2 , group D with an irradiation dose of 40mJ/ ^cm2 , group D with an irradiation dose of 50mJ/ ^cm2 E. In the irradiation dose of 60mJ/ ^cm2 group, the signal intensity of FL1 represents the content of ROS.

Figure 2 is a graph showing the effect of chibasu A on the release of ROS in HaCaT cells damaged by UVB, in which, A, blank control group B, model group C, ^10-1 μM chibasu A group D, 1 μM chibasu A group E, 10 μM Chibalin A group, the signal intensity of FL1 represents the content of ROS.

Figure 3 is the graph of the effect of Chibasin A on the apoptosis of HaCaT cells damaged by UVB, in which, A, blank control group B, model group C, ^10-1 μM Chibasin A group D, 1 μM Chibasin A group E, In the 10 μM chilesin A group, the signal intensity of FL2 represents the amount of premature apoptotic cells, and the signal intensity of FL3 represents the amount of necrotic cells.

Fig. 4 is the HE staining picture of mouse skin, wherein, A is a blank control group, and B is a model group.

detailed description

Chibasu A, purchased from Nanjing Puyi Biotechnology Co., Ltd., content ≥ 98%.

HaCaT cells were purchased from Peking Union Medical College Cell Bank.

MEM medium, penicillin-streptomycin, 0.25% trypsin+0.02% EDTA, and PBS phosphate buffer were all purchased from Gibco, USA.

Fetal bovine serum FBS was purchased from Israel Biolnd Company.

Petri dish (60*15mm), purchased from Germany effendorf company.

Cell culture plates were purchased from Costar, USA.

CCK-8 kit was purchased from Dojindo Lab, Japan.

The complete culture solution is obtained by mixing MEM medium with 10% fetal bovine serum FBS and 1% penicillin-streptomycin.

ROS probe DCFH-DA (D6883) and DMSO were purchased from Sigma, USA.

SOD assay kit (WST-1 method), CAT assay kit (visible light method), GSH-PX assay kit (colorimetric method), MDA assay kit (TBA method), were purchased from Nanjing Jiancheng Bioengineering Institute .

PE-coupled Annexin V Apoptosis Detection Kit I (559763) was purchased from BD Company, USA.

Embodiment 1. The preparation of the extract containing chilesin A

Take 3.15 kg of dry Eucommia bark, use 27 L of ethanol aqueous solution with a volume concentration of 95% as the extraction solvent, heat and reflux for extraction twice, each time for 2.5 hours, combine the extracts and concentrate to half of the extraction solvent to obtain a concentrated solution 1; Add 6L of ethanol aqueous solution with a volume concentration of 60% to the dregs as an extraction solvent, heat and reflux for extraction for 2.5 hours, and concentrate the extract to half of the extraction solvent to obtain concentrated solution 2; after combining concentrated solution 1 and concentrated solution 2, evaporate the ethanol under reduced pressure Until the concentrated liquid has no ethanol smell, freeze-dry to obtain a solid extract. Take 200 g of the solid extract and add distilled water to obtain an aqueous extract solution with a mass concentration of 30%, which is then extracted with water-saturated petroleum ether and water-saturated chloroform to obtain 15.3 g of petroleum ether extract and 38.4 g of chloroform extract. Take 35g of chloroform extract and dissolve it in 80ml of chloroform, mix it with 75g of 100-200 mesh silica gel evenly, and let it dry naturally. Another 200g of 100-200 mesh silica gel was wet-packed into the column, and petroleum ether-acetone=9:1-7:3 gradient elution. Receive one fraction per 50 mL for a total of 105 fractions, labeled 1 to 105. The first gradient: petroleum ether-acetone=9:1, each 50mL is a fraction, a total of 20 fractions are connected, and 1L of solvent is used; the second gradient: petroleum ether-acetone=8:2, a total of 49 fractions are connected , using 2.45L of solvent; the third gradient: petroleum ether-acetone=7:3, a total of 36 fractions were connected, using 1.8L of solvent. After recovering the solvent, according to the different solubility of each fraction, the samples were dissolved with solvents such as chloroform and methanol respectively, and placed for further separation and purification. After being eluted by column chromatography, according to TLC thin-layer chromatographic analysis, a fraction with a large amount and relatively concentrated concentration was selected for processing. Separate and purify fractions with the same or similar components.

Fractions 43-56 were separated and purified, and the compound was identified as containing phyllomin A by using standard control and ¹ H NMR, with a mass content of 25%, concentrated, and freeze-dried to obtain an extract containing phyllocin A.

Example 2

Preparation of medicine (or skin care product) for preventing and treating skin photoaging with low dosage

Take by weight ratio:

Chibasu A 0.01%, Glyceryl Behenate 0.3%, Caprylic Triglyceride 0.15%, Soy Lecithin 0.3%, Polyoxyethylene (40) Stearate (Maize 52) 1.82%, Carbomer 9400.3 %, glycerin 4.6%, hydroxypropyl methylcellulose 0.3%, ethylparaben 0.3%, and the balance is deionized water.

Preparation:

Glyceryl behenate and triglyceride caprylate were dissolved in chloroform in a water bath at 75°C to obtain solution A;

Dissolve the formulated amount of soybean lecithin and chibalin A in absolute ethanol in a water bath at 75°C to obtain solution B;

Mix the two solutions of A and B to obtain solution C;

Dissolve the formulated amount of Meize 52 in deionized water in a water bath at 75°C to obtain solution D;

Slowly inject the solution C into the solution D under magnetic stirring (500r/min), and continue to stir until the organic solvent is removed to obtain the nanostructured lipid carrier of chibain A.

Carbomer 940, glycerin, hydroxypropyl methylcellulose, ethylparaben of formula quantity are dissolved in deionized water (the deionized water of formula quantity minus the deionized water used when preparing solution D) again, Swell at 4°C for 12 hours to obtain a blank gel.

Then the blank gel was stirred and mixed with the nanostructured lipid carrier of Chibasu A at a ratio of 1:1, and the pH was adjusted to 6.5 to obtain a low-dose drug (or skin care product) for preventing and treating skin photoaging.

How to use: Take an appropriate amount and apply it on the skin directly, once a day without washing.

Example 3

Preparation of medicine (or skin care product) for preventing and treating skin photoaging with low dosage

Use the extract containing phylloxene A to replace phylloxene A, and the others are the same as in Example 2 to obtain a low-dose medicine (or skin care product) for preventing and treating skin photoaging.

Example 4

Preparation of high-dose medicine (or skin care product) for preventing and treating skin photoaging

Take by weight ratio:

The Chibasu A in Example 2 is adjusted from 0.01% to 0.02%, and the rest are all the same as in Example 2 to prepare a high-dose medicine (or skin care product) for preventing and treating skin photoaging.

Example 5

Preparation of high-dose medicine (or skin care product) for preventing and treating skin photoaging

Use the extract containing phylloxene A to replace phylloxene A, and the rest are the same as in Example 4 to prepare a high-dose medicine (or skin care product) for preventing and treating skin photoaging.

Test example 1, the cell experiment of Chibasu A preventing and treating photoaging

(1) Establish a cell photodamage model

The UVB radiation skin damage model was simulated by using the SH2B ultraviolet phototherapy instrument of Sigma Company of the United States, with a spectrum of 280-320nm and an intensity of 12.8mW/cm ² . HaCaT cells in the logarithmic growth phase were taken, and the cell density was adjusted to 1×10 ⁵ cells/mL with complete culture medium, seeded in 96-well cell culture plates, and cultured in a 37°C, 5% CO ₂ cell incubator for 24 hours. Discard the culture medium in the 96-well plate, wash it twice with PBS, add 100 μL PBS to cover each well, and irradiate HaCaT cells with a UVB phototherapy device. The irradiation doses are 30, 40, 50, and 60 mJ/cm ² . PBS was added to the complete culture solution, and incubated in an incubator at 37° C. and 5% _CO for 24 hours. Cell viability was detected by CCK8 (see Table 1), and ROS release was measured by flow cytometry (see Table 2, FIG. 1 ).

Where As represents the absorbance of the experimental wells (representing the cells, CCK-8 solution and the wells irradiated by different doses of UVB), Ab represents the absorbance of the blank wells (represents the CCK-8 solution, no cells and UVB irradiated wells), and Ac represents the control Absorbance of wells (representing wells with cells and CCK-8 solution but no UVB irradiation).

(n＝6)Table 1 Effect of different doses of UVB irradiation on cell viability

(n=6)

^*** P<0.001vs blank control group

(n＝3)Table 2 Effect of different doses of UVB irradiation on the release of cellular ROS

(n=3)

It can be seen from Tables 1 and 2 that when the radiation intensity is 60mJ/cm ² , the cell viability drops to 65.68%, and the ROS release reaches 215.61, indicating that the modeling is successful.

(2) Experimental drug: Oroxylin A

(3) Increase the proliferative activity of HaCaT cells damaged by UVB

experimental method

Take the HaCaT cells in the logarithmic growth phase, adjust the cell density to ¹ ×105 cells/mL with complete culture medium, inoculate them in a 96-well cell culture plate, and culture them in a 5% _CO2 incubator at 37°C until 96 The cells in the orifice plate grew to about 70%, and were randomly divided into normal group (blank control group), UVB irradiation group (model group), 10 ^-1 ~ 10μM Chibasin A solution group, 10 ^-1 -1μg/mL containing The extract solution group of Chibasu A was pre-administered for 24 hours. Both the blank control group and the UVB irradiation group were given complete culture solution during pre-administration. Aspirate and discard the culture medium in the 96-well plate, wash with PBS twice, add 100 μL PBS to cover each well, and irradiate the model group, 10 ^-1 ~ 10 μM Chibasin A solution group and 10 ^{-1 -1} μg/mL containing For the solution group of the extract of Chibalin A, the irradiation dose is 60mJ/cm ² . After the irradiation is completed, the PBS is discarded, and the complete culture solution is added. After incubation for 24 hours in an incubator at 37°C and 5% CO ₂ , the CCK8 method The cell proliferation activity was detected, and the results are shown in Table 3.

The molecular weight of Chibasu A is M=284.26g/mol, and the configuration of Chibasu A solution group of 10 ^-1 ~ 10μM is as follows:

Accurately weighed 1.42mg Chibaline A and dissolved in 500 μL DMSO solution to obtain 1×10 ⁴ μM Chibaline A stock solution

10 μM (2.8426 μg/mL) Chibasin A solution: take 1×10 ⁴ μM Chibasin A stock solution and dilute 1000 times with complete culture medium.

1 μM (2.8426×10 ^-1 μg/mL) chilesin A solution: take 1×10 ⁴ μM chilesin A stock solution and dilute it 10000 times with complete culture medium.

10 ^-1 μM (2.8426×10 ^-2 μg/mL) chilesin A solution: take 1×10 ⁴ μM chilesin A stock solution and dilute 100,000 times with complete culture medium.

The extract containing phyllotin A is a mixture, and the 10 ^-1 ~ 1 μg/mL extract solution containing phyllin A is configured as follows:

Accurately weighed 1.01mg of the extract containing fennelin A and dissolved it in 1mL of DMSO solution to obtain a stock solution of 1× ¹⁰³ μg/mL of the extract containing fennelin A

1 μg/mL extract solution containing phyllin A: take 1×10 ³ μg/mL stock solution of phyllin A-containing extract and dilute it 1000 times with complete culture solution.

10 ^-1 μg/mL extract solution containing chilesin A: take 1×10 ⁴ μM chiselin A stock solution and dilute it 10000 times with complete culture medium.

(n＝18)Table 3 Effect of phyllin A/extract containing phyllin A on cell viability after UVB irradiation

(n=18)

^### P<0.001vs blank control ^*** P<0.001vs model ^** P<0.01vs model It can be seen from Table 3 that Chibalin A or the extract containing Chibasu A acts on HaCaT cells damaged by UVB , can significantly increase its proliferative activity.

(4) Reduce the ROS content in HaCaT cells damaged by UVB

Experimental method: Take HaCaT cells in the logarithmic growth phase and adjust the density to 1×10 ⁵ cells/mL in complete culture medium, plant them in 60mm culture dishes, and culture them in a cell culture incubator at 37°C and 5% CO ₂ . After the cells in the culture dish grew to about 60%, they were randomly divided into a normal group (blank control group), a UVB irradiation group (model group), and a 10 ^-1 ~ 10 μM Chibasin A solution group, and pre-administered for 24 hours. Both the blank control group and the model group were given complete culture medium during pre-administration. Discard the culture medium in the culture dish, wash with PBS twice, add 100 μL of PBS to cover each well, irradiate the model group and the Chibasin A solution group with various concentrations with a UVB phototherapy instrument, and the irradiation dose is 60mJ/cm ² , after the irradiation is completed, discard Remove PBS, add complete culture solution, and incubate in an incubator at 37°C and 5% CO ₂ for 24 hours, and then detect the ROS level in HaCaT cells by flow cytometry. The results are shown in Table 4, Figure 2.

(n＝3)Table 4 Effects of chilesin A on ROS levels in HaCaT cells after UVB irradiation

(n=3)

^### P<0.001vs blank control ***P<0.001vs model It can be seen from Table 4 that Chibasin A can significantly reduce the ROS content of HaCaT cells damaged by UVB.

(5) Improve the antioxidant activity of UVB damaged HaCaT cells - superoxide dismutase

Experimental method: Take HaCaT cells in the logarithmic growth phase and adjust the density to 1×10 ⁵ cells/mL in complete culture medium, plant them in 60mm culture dishes, and culture them in a cell culture incubator at 37°C and 5% CO ₂ . After the cells in the culture dish grew to about 60%, they were randomly divided into a normal group (blank control group), a UVB irradiation group (model group), and a 10 ^-1 ~ 10 μM Chibasin A solution group, and pre-administered for 24 hours. Both the blank control group and the model group were given complete culture medium during pre-administration. Discard the culture medium in the culture dish, wash it twice with PBS, add 100 μL of PBS to cover each well, irradiate the model group and the Chibasin A solution group with various concentrations with a UVB phototherapy instrument, and the irradiation dose is 60mJ/cm ² . After the irradiation is completed, discard Remove PBS, add complete culture solution, incubate in an incubator at 37°C and 5% CO ₂ for 24 hours, then use a kit to detect the level of superoxide dismutase (SOD), the results are shown in Table 5.

Operate according to the instructions of the SOD determination kit (WST-1 method).

(n＝3)Table 5 Effects of chilesin A on SOD activity in HaCaT cells after UVB irradiation

(n=3)

^## P<0.01vs blank control *P<0.05vs model**P<0.01vs model

It can be seen from Table 5 that after Chibalin A acts on UVB-damaged HaCaT cells, it can significantly increase its SOD activity.

(6) Improve the antioxidant activity of UVB damaged HaCaT cells - catalase

Experimental method: Take HaCaT cells in the logarithmic growth phase and adjust the density to 1×10 ⁵ cells/mL in complete culture medium, plant them in 60mm culture dishes, and culture them in a cell culture incubator at 37°C and 5% CO ₂ . After the cells in the culture dish grew to about 60%, they were randomly divided into a normal group (blank control group), a UVB irradiation group (model group), and a 10 ^-1 ~ 10 μM Chibasin A solution group, and pre-administered for 24 hours. Both the blank control group and the model group were given complete culture medium during pre-administration. Discard the culture medium in the culture dish, wash with PBS twice, add 100 μL of PBS to cover each well, irradiate the model group and the Chibasin A solution group with various concentrations with a UVB phototherapy instrument, and the irradiation dose is 60mJ/cm ² , after the irradiation is completed, discard PBS was removed, complete culture solution was added, and after incubation in an incubator at 37°C and 5% CO ₂ for 24 hours, the catalase (CAT) level was detected using a kit, and the results are shown in Table 6.

Operate according to the instructions of the CAT assay kit (visible light method)

(n＝3)Table 6 Effects of chilesin A on CAT activity in HaCaT cells after UVB irradiation

(n=3)

^## P<0.01vs blank control *P<0.05vs model

It can be seen from Table 6 that after Chibalin A acts on HaCaT cells damaged by UVB, it can significantly increase its CAT activity.

(7) Improve the antioxidant activity of UVB damaged HaCaT cells - glutathione peroxidase

Experimental method: Take HaCaT cells in the logarithmic growth phase and adjust the density to 1×10 ⁵ cells/mL in complete culture medium, plant them in 60mm culture dishes, and culture them in a cell culture incubator at 37°C and 5% CO ₂ . After the cells in the culture dish grew to about 60%, they were randomly divided into a normal group (blank control group), a UVB irradiation group (model group), and a 10 ^-1 ~ 10 μM Chibasin A solution group, and pre-administered for 24 hours. Both the blank control group and the model group were given complete culture medium during pre-administration. Discard the culture medium in the culture dish, wash it twice with PBS, add 100 μL of PBS to cover each well, irradiate the model group and the Chibasin A solution group with various concentrations with a UVB phototherapy instrument, and the irradiation dose is 60mJ/cm ² . After the irradiation is completed, discard PBS was removed, complete culture solution was added, and after incubation in an incubator at 37°C and 5% _CO for 24 hours, the kit was used to detect the level of glutathione peroxidase (GSH-Px). The results are shown in Table 7.

Operate according to the instructions of the GSH-P _X assay kit (colorimetric method)

(n＝3)Table 7 Effects of chilesin A on GSH-Px activity in HaCaT cells after UVB irradiation

(n=3)

^## P<0.01vs blank control *P<0.05vs model**P<0.01vs model

It can be seen from Table 7 that after Chibalin A acts on HaCaT cells damaged by UVB, it can significantly increase the activity of GSH-P _X.

(8) Improve the antioxidant activity of HaCaT cells damaged by UVB-malondialdehyde

Experimental method: Take HaCaT cells in the logarithmic growth phase and adjust the density to 1×10 ⁵ cells/mL in complete culture medium, plant them in 60mm culture dishes, and culture them in a cell culture incubator at 37°C and 5% CO ₂ . After the cells in the culture dish grew to about 60%, they were randomly divided into a normal group (blank control group), a UVB irradiation group (model group), and a 10 ^-1 ~ 10 μM Chibasin A solution group, and pre-administered for 24 hours. Both the blank control group and the model group were given complete culture medium during pre-administration. Discard the culture medium in the culture dish, wash with PBS twice, add 100 μL of PBS to cover each well, irradiate the model group and the Chibasin A solution group with various concentrations with a UVB phototherapy instrument, and the irradiation dose is 60mJ/cm ² , after the irradiation is completed, discard PBS was removed, complete culture solution was added, and after incubation in an incubator at 37°C and 5% CO ₂ for 24 hours, a kit was used to detect the level of malondialdehyde (MDA). The results are shown in Table 8.

Operate according to the instructions of the MDA assay kit (TBA method)

(n＝3)Table 8 Effects of chilesin A on MDA content in HaCaT cells after UVB irradiation

(n=3)

^## P<0.01vs blank control *P<0.05vs model

It can be known from Table 8 that the MDA content of HaCaT cells damaged by UVB can be significantly reduced after Chibalin A acts on them.

(9) Reduce the apoptosis of HaCaT cells damaged by UVB

Experimental method: Take HaCaT cells in the logarithmic growth phase and adjust the density to 1×10 ⁵ cells/mL in complete culture medium, plant them in 60mm culture dishes, and culture them in a cell culture incubator at 37°C and 5% CO ₂ . After the cells in the culture dish grew to about 60%, they were randomly divided into a normal group (blank control group), a UVB irradiation group (model group), and a 10 ^-1 ~ 10 μM Chibasin A solution group, and pre-administered for 24 hours. Both the blank control group and the model group were given complete medium during pre-administration. Discard the culture medium in the culture dish, wash with PBS twice, add 100 μL of PBS to cover each well, irradiate the model group and the Chibasin A solution group with various concentrations with a UVB phototherapy instrument, and the irradiation dose is 60mJ/cm ² , after the irradiation is completed, discard Remove PBS, add complete culture medium, incubate in an incubator at 37°C and 5% CO ₂ for 24 hours, discard the cell culture medium, rinse twice with PBS, add 0.25% trypsin (containing 0.02% EDTA) to digest for 5 minutes , When the cell protrusions are observed under a microscope to become round, the digestion is terminated. The cells were collected by centrifugation and washed twice with PBS, and the cells were resuspended with 1×Binding Buffer (reagent in the kit), and the cell density was adjusted to 1×10 ⁶ cells/mL. Take an appropriate amount of cell suspension and add it to the kit PEAnexin V and 7-AAD, mix well, incubate at room temperature in the dark for 15 minutes, and use the Flow cytometer to complete the sample loading test within 1 hour. The results are shown in Table 9, Figure 3.

Table 9 Effects of chilesin A on apoptosis of HaCaT cells after UVB irradiation (x±SD) (n=3)

^### P<0.001vs blank control**P<0.01vs model*P<0.05vs model

It can be seen from Table 9 that after Chibalin A acts on HaCaT cells damaged by UVB, it can significantly reduce the level of apoptosis.

Test example 2, the animal experiment of Chibasu A preventing and treating photoaging

(1) Establish photoaging animal model

The hair on the back of the mouse was first removed with scissors and an electric shaver, and the back skin was fully exposed. After the mouse was fixed with a self-made mouse fixer, the mouse model was irradiated by the SS-03AB phototherapy apparatus. Irradiate 5 days a week, rest 2 days, and irradiate for 9 weeks to build a model. The total radiation dose of UVB: 9.45J/cm ² , and UVA: 94.5J/cm ² . After modeling, the back skin of the successfully modeled mice was obviously thickened, the skin surface was rough, the skin was dry and hard, and obvious wrinkles were visible. The results of HE staining are shown in Figure 4.

(2) Experimental grouping

Divide into 5 groups at random, are respectively: blank control group, 8, prepare skin, device is fixed, do not irradiate; Model group, 8, prepare skin, device is fixed, irradiate; The quality of Chibasu A in the medicine (or skin care product) of skin photoaging can be replaced with deionized water) group, 8, prepare skin, device is fixed, back skin is given blank preparation locally, irradiates; Low dose Chibasu A preparation group (prepared in Example 2), 8 rats, skin preparation, fixed device, local administration of low-dose Chibasu A preparation on the back skin, irradiation; high-dose Chibasu A preparation group (prepared in Example 4), 8 rats , skin preparation, device fixation, local administration of high-dose Chibasu A preparation on the back skin, and irradiation.

(3) Effect of Chibasu A preparation on skin moisture of photoaging mice induced by UV irradiation

The probe of the skin moisture content tester (Cornemeter CM 825, Courage and Khazaka, Germany) is based on the principle of capacitance for measurement. The dielectric constant of water is 81, and the dielectric constant of other substances is usually less than 7. Water is the substance with the highest dielectric constant on the skin. When the water content changes, the capacitance value of the skin also changes, so the water content of the skin surface can be analyzed by measuring the skin capacitance value. The measured value is a relative value. The unit is represented by C.U. (Corneometer Units).

In the test, after testing the use of Chibasu A preparation, the change of skin moisture content. Specifically: the blank control group was not irradiated, and the model group, the blank preparation group, the high-dose Chibasu A preparation group, and the low-dose Chibasu A preparation group were all irradiated with ultraviolet rays. See the establishment of photoaging animal models for the irradiation conditions. Irradiate 5 days a week, and measure the skin moisture content on the 6th day (ie 6d, 12d, 18d, 24d, 30d, 36d, 42d, 48d, 54d) every week after the 5th day of irradiation. The skin MMV values of the test areas were measured using a skin moisture content tester, and each test area was tested 3 times to obtain the average value. The change of MMV value of skin moisture content is reflected in the test period, and the moisture content of the experimental area changes with time. The larger the value, the greater the moisture content, and vice versa, the smaller the moisture content. The measurement results are shown in Table 10.

Table 10 Effect of Chibasin A Preparation on UV Radiation-Induced Photoaging Mouse Skin Moisture (x ± SD) (n=8)

^### P<0.001vs blank control group ^# P<0.05vs blank control group***P<0.001vs model group**P<0.01vs model group*P<0.05vs model group

It can be seen from Table 10 that during the test period, the skin moisture MMV values of the test area of the Chibasu A preparation group were higher than those of the model group and the blank preparation group. It shows that Chibasu A preparation can increase the water content of photoaging skin and improve dry symptoms of photoaging skin during the experimental period.

(4) Effect of Chibasu A preparation on skin sensitivity of photoaging mice induced by UV irradiation

Tissue Viability Imager (Tissue Viability Imager TiVi 700, WheelsBridge AB, Sweden) is based on the fact that the linearly polarized light emitted by the white light illuminator after passing through the polarizing filter reaches the skin surface to be analyzed, and most of the linearly polarized light is directly reflected by the skin surface After being blocked by the optical filter of the detector, only part of it irregularly passes through the lower layer of the skin, and the polarized light absorbed by the red blood cells passes through the optical filter and enters the detector. In the polarized light reaching the detector, the green light component is weakened due to the absorption of red blood cells in the blood vessel, and the red component is almost unchanged, while other tissues around the red blood cells absorb green light and red light basically the same. In this way, the state of blood vessel microcirculation in the deep layer of the skin can be obtained, and the red blood cell concentration value of each point in the image, that is, the TiVi value can be obtained. The TiVi 700 system uses the characteristics of red blood cells in blood vessels to absorb green light to obtain the distribution and aggregation images of red blood cells in skin tissue. The content of red blood cells represents skin sensitivity.

In the test, the change of skin sensitivity was tested after using Chibasu A preparation. Specifically: the blank control group was not irradiated, and the model group, the blank preparation group, the high-dose Chibasu A preparation group, and the low-dose Chibasu A preparation group were all irradiated with ultraviolet rays. See the establishment of photoaging animal models for the irradiation conditions. Irradiate 5 days a week, and measure skin sensitivity on the 6th day (ie 6d, 12d, 18d, 24d, 30d, 36d, 42d, 48d, 54d) every week after the 5th day of irradiation. The measurement results are shown in Table 11.

(n＝8)Table 11 Effect of Chibasin A preparation on skin sensitivity of photoaging mice induced by UV irradiation

(n=8)

^### P<0.001vs blank control group ***P<0.001vs model group**P<0.01vs model group*P<0.05vs model group

It can be seen from Table 11 that during the test period, the skin sensitivity values of the test areas of the Chibasu A preparation group were lower than those of the model group and the blank preparation group. It shows that the chibasu A preparation can reduce the sensitivity of photoaging skin and improve the hypersensitivity of photoaging skin in the experimental period.

(5) Effect of Chibatin A preparation on skin melanin content of photoaging mice

The image was taken by the tissue viability imager (Tissue Viability Imager TiVi 700, WheelsBridgeAB, Sweden) in (4), and the melanin content was analyzed by TiVi97 pigment analysis software.

In the test, the change of skin melanin content was tested after using Chibatin A preparation. Specifically: the blank control group was not irradiated, and the model group, the blank preparation group, the high-dose Chibasu A preparation group, and the low-dose Chibasu A preparation group were all irradiated with ultraviolet rays. See the establishment of photoaging animal models for the irradiation conditions. Irradiate 5 days a week, and measure skin melanin content on the 6th day (ie, 6d, 12d, 18d, 24d, 30d, 36d, 42d, 48d, 54d) every week after the 5th day of irradiation. The measurement results are shown in Table 12.

(n＝8)Table 12 Effect of Chibasin A preparation on skin melanin content of photoaging mice induced by UV irradiation

(n=8)

^### P<0.001vs blank control group ^## P<0.01vs blank control group***P<0.001vs model group**P<0.01vs model group*P<0.05vs model group

It can be seen from Table 12 that during the test period, the melanin content in the test area of the Chibasu A preparation group was lower than that of the model group and the blank preparation group. It shows that the chibasin A preparation can reduce the melanin content of photoaging skin and improve the pigmentation problem of photoaging skin during the experimental period.

Experiments have proved that using Chibasu A and common pharmaceutical excipients to make gels, creams, creams, emulsions or waters to make medicines or skin care products for preventing and curing skin photoaging can increase the water content of photoaging skin. Reduces its sensitivity and lightens hyperpigmentation.

The above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Anyone skilled in the art can easily think of changes or substitutions within the technical scope disclosed in the present invention. Should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be determined by the protection scope of the claims.

Claims (1)

1. The use of Chibasu A as the only active ingredient in the preparation of medicines or skin care products for preventing and treating skin photoaging.

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