CN112569221A - Application of phyllanthin A in 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 preparing medicines or skin care products for preventing and treating skin photoaging. The experimental results show that chibain A can increase the proliferation activity of UVB-damaged HaCaT cells; reduce the ROS content in UVB-damaged HaCaT cells , improve the antioxidant activity of UVB-damaged HaCaT cells; reduce the apoptosis of UVB-damaged HaCaT cells; and can significantly reduce skin photoaging symptoms. The experimental results show that the extract containing chibain A can increase the proliferation activity of UVB-damaged HaCaT cells.

Description

Application of phyllanthin A in preparation of medicines or skin care products for preventing and treating skin photoaging

Technical Field

The invention relates to application of a phyllanthin A in preparation of a medicine or a skin care product for preventing and treating skin photoaging.

Background

Aging is a process in which the body gradually loses function and dies. The skin is the biggest organ of the body, is the defense barrier of the body and the external environment, and can reflect aging more intuitively and obviously than other organs. Skin aging is usually divided into intrinsic and extrinsic aging, which is mainly caused by environmental factors. Ultraviolet (UV) radiation is the main environmental factor responsible for skin ageing, and prolonged exposure of the skin to UV radiation accelerates ageing, mainly manifested by surface roughness, a reduction in water content and elasticity, a reduction in collagen, and the formation of wrinkles, so-called photoaging (also called photoaging) of the skin, which, in the severe cases, even induces cancer.

Skin photoaging is a complex biochemical process, and its pathogenesis is related to oxidative stress, inflammatory reaction and high expression of matrix metalloproteinases. Oxidative stress is the initiating factor in causing photoaging. During photoaging, a large amount of Reactive Oxygen Species (ROS) including superoxide anion, hydrogen peroxide, singlet oxygen, hydroxyl radical, and the like are generated. Relatively well-established antioxidant systems exist in the body, and the production and elimination of ROS are in a balanced state. Excess ROS cause an imbalance in the redox system, thereby causing oxidative damage to intracellular lipids, proteins, and nucleic acids, ultimately triggering apoptosis and tissue organ damage. Reducing the accumulation of ROS and maintaining the stability of the antioxidant internal environment are important rings for protecting the skin from UV damage.

The oroxylin A (also called oroxylin A, oroxylin A) belongs to flavonoid compounds, is totally called 5, 7-dihydroxy-6-methoxy-2-phenyl-4H-1-benzofuran-4-ketone, and is mainly derived from traditional Chinese medicine eucommia. Has wide pharmacological activity, and has antitumor, antiinflammatory, blood vessel protecting, nerve cell protecting, memory disorder improving, and antiviral effects. So far, the reports of the application of the phyllanthin A in preparing medicaments or skin care products for preventing and treating skin photoaging are not seen.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides the application of the phyllanthin A in preparing the medicines or skin care products for preventing and treating the skin photoaging.

The second purpose of the invention is to provide the application of the extractive containing the phyllanthin A in preparing the medicines or skin care products for preventing and treating the skin photoaging.

The technical scheme of the invention is summarized as follows:

application of the phyllanthin A in preparing medicines or skin care products for preventing and treating skin photoaging is provided.

Application of an extract containing the phyllanthin A in preparing a medicament or a skin care product for preventing and treating skin photoaging.

The experimental result shows that the spilanthol A can improve the proliferative activity of UVB-damaged HaCaT cells; the ROS content in the UVB damaged HaCaT cells is reduced, and the antioxidant activity of the UVB damaged HaCaT cells is improved; reducing the apoptosis of HaCaT cells damaged by UVB; and can significantly alleviate the symptoms of skin photoaging.

The experimental results show that the extract containing the ever-increasing activity of the UVB-damaged HaCaT cells can be improved.

Drawings

FIG. 1 is a graph showing the effect of radiation dose on the amount of ROS released in HaCaT cells, wherein A, blank control group B, and radiation dose are 30mJ/cm²Group C, irradiation dose 40mJ/cm²Group D, irradiation dose 50mJ/cm²Group E, irradiation dose 60mJ/cm²Group, FL1 Signal StrengthRepresents the ROS content.

FIG. 2 is a graph of the effect of spilanthol A on the amount of ROS released in UVB-damaged HaCaT cells, wherein A, blank control B, model groups C, 10^-1μ M, 1 μ M, 10 μ M, FL1 signal intensity represents ROS content.

FIG. 3 is a graph of the effect of trilobatin A on UVB-damaged HaCaT apoptosis, wherein A, blank control B, model C, 10^-1mu.M of the trilobetin group A D, 1. mu.M of the trilobetin group A E, 10. mu.M of the trilobetin group A, the FL2 signal intensity represents the amount of the withered cells, and the FL3 signal intensity represents the amount of the necrotic cells.

FIG. 4 is a graph of HE staining of mouse skin, wherein A is blank control group and B is model group.

Detailed Description

The content of the thousand-leaf element A is more than or equal to 98 percent, and the thousand-leaf element A is purchased from Nanjing Puyi biotechnology limited.

HaCaT cells, purchased from beijing synergetics cell bank.

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

Fetal bovine serum FBS, available from Biolnd, israel.

Petri dishes (60 x 15mm), from effendorf, Germany.

Cell culture plates, purchased from Costar, usa.

CCK-8 kit, available from Dojindo Lab, Japan.

And (3) adding 10% fetal bovine serum FBS and 1% penicillin-streptomycin into MEM culture medium, and mixing to obtain the complete culture solution.

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

SOD determination kit (WST-1 method), CAT determination kit (visible light method), GSH-PX determination kit (colorimetric method), and MDA determination kit (TBA method) were purchased from Nanjing institute of bioengineering.

PE-coupled Annexin V apoptosis detection kit I (559763) was purchased from BD corporation, USA.

Example 1 preparation of an extract containing a thousand leaf elements A

Taking 3.15kg of dried eucommia bark, using 27L of 95% ethanol water solution in volume concentration as an extraction solvent, heating and refluxing for extraction for 2 times, each time for 2.5h, combining the extracting solutions, and concentrating to half of the extraction solvent to obtain a first concentrated solution; adding 6L of 60% ethanol water solution as extraction solvent into the residue, heating and reflux-extracting for 2.5 hr, and concentrating the extractive solution to half of the extraction solvent to obtain second concentrated solution; mixing the first concentrated solution and the second concentrated solution, evaporating ethanol under reduced pressure until the concentrated solution has no ethanol smell, and freeze drying to obtain solid extract. Adding distilled water into 200g of the solid extract to obtain an extract water solution with the mass concentration of 30%, and sequentially extracting with water-saturated petroleum ether and water-saturated chloroform to obtain 15.3g of petroleum ether extract and 38.4g of chloroform extract. 35g of chloroform extract is taken and dissolved by 80ml of chloroform, and is evenly mixed with 75g of 100-mesh 200-mesh silica gel and is naturally dried. And loading 200g of 100-200 mesh silica gel into a column by a wet method, and performing gradient elution on petroleum ether-acetone (9: 1-7: 3). One stream is received every 50mL for a total of 105 streams, indicated 1 to 105 respectively. A first gradient: petroleum ether-acetone (9: 1) in 50mL portions, and 20 portions are added in total, and the solvent is used for 1L; a second gradient: petroleum ether-acetone (8: 2) was added to 49 fractions in total, and 2.45L of solvent was added; a third gradient: petroleum ether-acetone ═ 7:3, a total of 36 fractions were collected, and 1.8L of solvent was added. After the solvent is recovered, the sample is dissolved in solvents such as chloroform and methanol according to the solubility of each fraction, and then left for further separation and purification. After elution by column chromatography, fractions of large and relatively concentrated fractions were selected for processing according to TLC thin layer chromatography. Separating and purifying fractions with the same or similar components.

Separating and purifying the fraction 43-56, and performing mass transfer with standard substance,¹H NMR proves that the compound contains the phyllanthin A, the mass content of the phyllanthin A is 25%, and the compound is concentrated, frozen and dried to obtain the extract containing the phyllanthin A.

Example 2

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

Taking the following components in percentage by weight:

0.01% of phyllanthin A, 0.3% of glyceryl behenate, 0.15% of caprylic triglyceride, 0.3% of soybean lecithin, 1.82% of polyoxyethylene (40) stearate (maize 52), 9400.3% of carbomer, 4.6% of glycerol, 0.3% of hydroxypropyl methyl cellulose, 0.3% of ethylparaben and the balance of deionized water.

The preparation method comprises the following steps:

dissolving the behenic glyceride and the caprylic triglyceride in the formula according to the formula ratio in chloroform at the temperature of 75 ℃ in a water bath to obtain a solution A;

dissolving the soybean lecithin and the spilanthol A in the formula ratio in absolute ethyl alcohol at the temperature of 75 ℃ in a water bath to obtain a solution B;

A. mixing the two solutions B to obtain a solution C;

dissolving the formula amount of maize 52 in deionized water at the temperature of 75 ℃ in a water bath to obtain solution D;

slowly injecting the solution C into the solution D under magnetic stirring (500r/min), and continuously stirring until the organic solvent is removed to obtain the nano-structure lipid carrier of the phyllanthin A.

And dissolving the formula amount of carbomer 940, glycerol, hydroxypropyl methylcellulose and ethylparaben in deionized water (the deionized water used for preparing the solution D is subtracted from the formula amount of deionized water), and swelling at 4 ℃ for 12h to obtain the blank gel.

And then respectively stirring and uniformly mixing the blank gel and the nano-structure lipid carrier of the spilanthol A according to the ratio of 1:1, and adjusting the pH value to 6.5 to obtain the low-dose medicament (or skin care product) for preventing and treating the skin photoaging.

The using method comprises the following steps: the appropriate amount of the external preparation is directly applied to the skin once a day without cleaning.

Example 3

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

The extract containing the phyllanthin A is used for replacing the phyllanthin A, and the medicine (or the skin care product) for preventing and treating the skin photoaging with low dosage is obtained in the same way as the example 2.

Example 4

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

Taking the following components in percentage by weight:

the spilanthol A in example 2 is adjusted from 0.01% to 0.02%, and the rest is the same as that in example 2 to prepare the high-dose medicament (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

The same procedures as in example 4 were repeated except that the extract containing the phyllanthin A was used in place of the phyllanthin A to prepare a high-dose pharmaceutical (or skin care) for preventing and treating skin photoaging.

Test example 1 cell experiment of preventing and treating photoaging by Qiafolin A

(1) Establishing cell photodamage model

An SH2B type ultraviolet phototherapy instrument of Sigma company in the United states is adopted to simulate a UVB radiation skin damage model, the spectrum is 280-320 nm, and the intensity is 12.8mW/cm². Taking HaCaT cells in logarithmic growth phase, adjusting cell density to 1 × 10 with complete culture medium⁵one/mL, seeded in 96-well cell culture plates at 37 ℃ in 5% CO₂Culturing in a cell culture box for 24 h. Removing culture medium from 96-well plate, washing with PBS for 2 times, covering each well with 100 μ L PBS, and irradiating HaCaT cells with UVB phototherapy at doses of 30, 40, 50, and 60mJ/cm²After irradiation, PBS was discarded, and complete medium was added at 37 ℃ with 5% CO₂After incubation in the incubator for 24h, cell viability was measured by the CCK8 method (see Table 1), and ROS release amount was measured by flow-assay (see Table 2, FIG. 1).

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

TABLE 1 Effect of different doses of UVB irradiation on cell viability

(n＝6)

^***P<0.001vs blank control group

TABLE 2 Effect of cellular ROS Release amount after different UVB doses

(n＝3)

As can be seen from tables 1 and 2, the radiation intensity was 60mJ/cm²When the cell activity is reduced to 65.68%, and the ROS release amount reaches 215.61, the molding is successful.

(2) Experimental drugs: qiafolinin A (Oroxylin A)

(3) Increasing proliferative activity of UVB-damaged HaCaT cells

Experimental methods

Taking HaCaT cells in logarithmic growth phase, adjusting the cell density to 1 × 10 with complete culture medium⁵one/mL of the cells were plated in 96-well cell culture plates at 37 ℃ in 5% CO₂Culturing in a cell culture box until about 70% of cells in a 96-well plate grow, and randomly dividing into a normal group (blank control group), a UVB irradiation group (model group) and 10^-110 μ M of the trilysin A solution group, 10^-11 μ g/mL of the group of the spilanthol A-containing extract solutions, pre-administered for 24 h. Complete culture was given to both the white control group and the UVB-irradiated group at the time of predose. Removing culture medium from 96-well plate, washing with PBS for 2 times, covering each well with 100 μ L PBS, and irradiating model group and model group with UVB phototherapy apparatus 10^-110 μ M of the thousand phyllin A solution group and 10^-11 μ g/mL of a solution containing a patchouli A extract at an irradiation dose of 60mJ/cm²After irradiation, PBS was discarded, and complete medium was added at 37 ℃ with 5% CO₂After incubation in the incubator for 24h, the cell proliferation activity was measured by the CCK8 method, and the results are shown in Table 3.

The molecular weight M of the trilobatin A is 284.26g/mol，10^-1The 10 μ M trilysin A solution group was configured as follows:

precisely weighing 1.42mg of the phyllanthin A, and dissolving in 500 mu of LDMSO solution to obtain 1 × 10⁴Mu M Qiaphyllin A stock solution

10 μ M (2.8426 μ g/mL) of a solution of trilobatin A: take 1X 10⁴Diluting the mu M of the thousand-leaf element A stock solution by 1000 times with complete culture solution to obtain the finished product.

1μM(2.8426×10^-1μ g/mL) of a solution of trilobatin a: take 1X 10⁴Diluting the mu M of the thousand-leaf element A stock solution by 10000 times with complete culture solution to obtain the finished product.

10^-1μM(2.8426×10^-2μ g/mL) of a solution of trilobatin a: take 1X 10⁴Diluting the mu M of the thousand leaf element A stock solution with 100000 times of the complete culture solution to obtain the finished product.

The extract containing spilanthol A is mixture, 10^-1About 1 μ g/mL of the spilanthol a-containing extract solution was prepared as follows:

precisely weighing 1.01mg of the extract containing the spilanthol A, and dissolving in 1ml of the MSO solution to obtain 1 × 10³Mu g/mL stock solution of spilanthol A-containing extract

1 μ g/mL of the spilanthol a-containing extract solution: take 1X 10³Diluting the stock solution of the extract containing the orophyllin A by 1000 times by using complete culture solution to obtain the orophyllin A-containing extract.

10^-1μ g/mL of the spilanthol a-containing extract solution: take 1X 10⁴Diluting the mu M of the thousand-leaf element A stock solution by 10000 times with complete culture solution to obtain the finished product.

TABLE 3 Effect of the Qiaphyllin A/extract containing Qiaphyllin A on the Activity of cells after UVB irradiation

(n＝18)

^###P<0.001vs blank control^***P<0.001vs model^**P<0.01vs model As can be seen from Table 3, the action of Qiafolin A or an extract containing Qiafolin A on UAfter the HaCaT cells with VB damage can obviously improve the proliferative activity of the HaCaT cells.

(4) Reducing ROS content in UVB-damaged HaCaT cells

The experimental method comprises the following steps: taking HaCaT cells in logarithmic growth phase, adjusting the HaCaT cells to 1 x 10 in complete culture solution⁵seeds/mL were plated in 60mm dishes at 37 ℃ in 5% CO₂Culturing in a cell culture box. When the cells in the culture dish grow to about 60%, the cells are randomly divided into a normal group (blank control group), a UVB irradiation group (model group) and 10^-110 μ M of the group of the thousand phyllin A solution, pre-dosed for 24 h. The control group and the model group were given complete culture medium when pre-dosed. Removing culture solution from the culture dish, washing with PBS for 2 times, covering each well with 100 μ L PBS, irradiating model group and each concentration of the solution group of trilobatin A with UVB phototherapy at irradiation dose of 60mJ/cm²After irradiation, PBS was discarded, and complete medium was added at 37 ℃ with 5% CO₂After incubation in the incubator of (1) for 24h, the ROS levels in HaCaT cells were measured by flow cytometry, and the results are shown in table 4 and fig. 2.

TABLE 4 Effect of trilobatin A on ROS levels in HaCaT cells following UVB irradiation

(n＝3)

^###P<0.001vs blank control<0.001vs model as can be seen from Table 4, spilanthol A significantly reduced ROS levels after acting on UVB-damaged HaCaT cells.

(5) Antioxidant activity-superoxide dismutase for improving UVB damage HaCaT cells

The experimental method comprises the following steps: taking HaCaT cells in logarithmic growth phase, adjusting the HaCaT cells to 1 x 10 in complete culture solution⁵seeds/mL were plated in 60mm dishes at 37 ℃ in 5% CO₂Culturing in a cell culture box. When the cells in the culture dish grow to about 60%, the cells are randomly divided into a normal group (blank control group), a UVB irradiation group (model group) and 10^-110 μ M of the group of the thousand phyllin A solution, pre-dosed for 24 h. The control group and the model group were given complete culture medium when pre-dosed. Removing culture solution from the culture dish, washing with PBS for 2 times, covering each well with 100 μ L PBS, irradiating model group and each concentration of the solution group of trilobatin A with UVB phototherapy at irradiation dose of 60mJ/cm²After irradiation, PBS was discarded, and complete medium was added at 37 ℃ with 5% CO₂After incubation for 24h in the incubator, the superoxide dismutase (SOD) level was detected using the kit, and the results are shown in Table 5.

The procedures were carried out according to the instructions of the SOD assay kit (WST-1 method).

TABLE 5 Effect of trilobatin A on SOD activity in HaCaT cells after UVB irradiation

(n＝3)

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

As shown in table 5, when the spilanthol a acts on the HaCaT cells damaged by UVB, the SOD activity of the spilanthol a can be significantly improved.

(6) Catalase for improving antioxidant activity of HaCaT cells damaged by UVB

The experimental method comprises the following steps: taking HaCaT cells in logarithmic growth phase, adjusting the HaCaT cells to 1 x 10 in complete culture solution⁵seeds/mL were plated in 60mm dishes at 37 ℃ in 5% CO₂Culturing in a cell culture box. After the cells in the culture dish grow to about 60 percent, randomly dividing the cells into a normal group (blank control group) and a UV groupB irradiation group (model group), 10^-110 μ M of the group of the thousand phyllin A solution, pre-dosed for 24 h. The control group and the model group were given complete culture medium when pre-dosed. Removing culture solution from the culture dish, washing with PBS for 2 times, covering each well with 100 μ L PBS, irradiating model group and each concentration of the solution group of trilobatin A with UVB phototherapy at irradiation dose of 60mJ/cm²After irradiation, PBS was discarded, and complete medium was added at 37 ℃ with 5% CO₂After incubation for 24h, the Catalase (CAT) level was determined using the kit, and the results are shown in Table 6.

Operating according to CAT determination kit (visible light method) instructions

TABLE 6 Effect of trilobatin A on CAT Activity in HaCaT cells after UVB irradiation

(n＝3)

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

As shown in table 6, when the spilanthol a acts on the HaCaT cells damaged by UVB, the CAT activity of the spilanthol a can be significantly improved.

(7) Glutathione peroxidase capable of improving antioxidant activity of HACaT cells damaged by UVB

The experimental method comprises the following steps: taking HaCaT cells in logarithmic growth phase, adjusting the HaCaT cells to 1 x 10 in complete culture solution⁵seeds/mL were plated in 60mm dishes at 37 ℃ in 5% CO₂Culturing in a cell culture box. When the cells in the culture dish grow to about 60%, the cells are randomly divided into a normal group (blank control group), a UVB irradiation group (model group) and 10^-110 μ M of the group of the thousand phyllin A solution, pre-dosed for 24 h. The control group and the model group were given complete culture medium when pre-dosed. Culture by aspirationPlating culture solution, washing with PBS for 2 times, covering each well with 100 μ L PBS, irradiating model group and each concentration of the solution group of the thousand leaf element A with UVB phototherapy at irradiation dose of 60mJ/cm²After irradiation, PBS was discarded, and complete medium was added at 37 ℃ with 5% CO₂After incubation for 24h in the incubator of (1), the kit was used to detect the level of glutathione peroxidase (GSH-Px), and the results are shown in Table 7.

By GSH-P_XInstruction operation of determination kit (colorimetric method)

TABLE 7 Effect of trilobatin A on GSH-Px viability in HaCaT cells after UVB irradiation

(n＝3)

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

As can be seen from Table 7, when orophyllin A acts on UVB-damaged HaCaT cells, GSH-P thereof can be significantly improved_XAnd (4) vitality.

(8) Malondialdehyde for improving UVB-damaged HaCaT cell antioxidant activity

The experimental method comprises the following steps: taking HaCaT cells in logarithmic growth phase, adjusting the HaCaT cells to 1 x 10 in complete culture solution⁵seeds/mL were plated in 60mm dishes at 37 ℃ in 5% CO₂Culturing in a cell culture box. When the cells in the culture dish grow to about 60%, the cells are randomly divided into a normal group (blank control group), a UVB irradiation group (model group) and 10^-110 μ M of the group of the thousand phyllin A solution, pre-dosed for 24 h. The control group and the model group were given complete culture medium when pre-dosed. Removing culture medium from the culture dish, washing with PBS for 2 times, adding 100 μ L PBS to each well, covering, irradiating model group and each concentration of a solution of trilobatin A with UVB phototherapy apparatusGroup, irradiation dose 60mJ/cm²After irradiation, PBS was discarded, and complete medium was added at 37 ℃ with 5% CO₂After incubation for 24h in the incubator of (1), Malondialdehyde (MDA) level was detected using the kit, and the results are shown in table 8.

According to the instructions of MDA determination kit (TBA method)

TABLE 8 Effect of trilobatin A on MDA content in HaCaT cells after UVB irradiation

(n＝3)

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

As shown in table 8, when the spilanthol a acts on the HaCaT cells damaged by UVB, its MDA content can be significantly reduced.

(9) Reducing apoptosis in UVB-damaged HaCaT cells

The experimental method comprises the following steps: taking HaCaT cells in logarithmic growth phase, adjusting the HaCaT cells to 1 x 10 in complete culture solution⁵seeds/mL were plated in 60mm dishes at 37 ℃ in 5% CO₂Culturing in a cell culture box. When the cells in the culture dish grow to about 60%, the cells are randomly divided into a normal group (blank control group), a UVB irradiation group (model group) and 10^-110 μ M of the group of the thousand phyllin A solution, pre-dosed for 24 h. Both the control group and the model group were given complete medium at the time of predose. Removing culture solution from the culture dish, washing with PBS for 2 times, covering each well with 100 μ L PBS, irradiating model group and each concentration of the solution group of trilobatin A with UVB phototherapy at irradiation dose of 60mJ/cm²After irradiation, PBS was discarded, and complete medium was added at 37 ℃ with 5% CO₂After incubation for 24h, the cell culture medium was aspirated, washed 2 times with PBS, digested for 5min with 0.25% pancreatin (containing 0.02% EDTA), and the digestion was stopped when the cell bulge became round under microscope. The cells were collected by centrifugation, washed 2 times with PBS, resuspended in 1 XBinding Buffer (reagent in kit), and conditionedDensity of 1X 10⁶one/mL. Adding a proper amount of cell suspension into the kit PEAnnexin V and 7-AAD, mixing uniformly, incubating for 15min at room temperature in a dark place, and completing the sample loading detection within 1h by using a Flow cytometer, wherein the result is shown in Table 9 and fig. 3.

Table 9 influence of trilobatin a on HaCaT apoptosis after UVB irradiation (x ± SD) (n ═ 3)

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

As shown in table 9, when the spilanthol a acts on the HaCaT cells damaged by UVB, the level of apoptosis can be significantly reduced.

Experimental example 2 animal experiment for preventing and treating photoaging by using Qiafolin A

(1) Establishing photoaging animal model

The method comprises the steps of firstly removing the hair on the back of a mouse by using scissors and an electric shaver, fully exposing the skin on the back of the mouse, fixing the mouse by using a self-made mouse fixer, and irradiating the mouse by using an SS-03AB type phototherapy instrument to form a model. Irradiating for 5 days per week, resting for 2 days, and irradiating for molding for 9 weeks, wherein the total radiation dose is UVB total dose: 9.45J/cm²Total UVA dose: 94.5J/cm². After the modeling is finished, the skin on the back of the successfully modeled mouse is obviously thickened, the skin surface is rough, the skin is dry and hard, and obvious wrinkles can be seen, and the HE dyeing result is shown in figure 4.

(2) Experiment grouping

Randomly divided into 5 groups, respectively: blank control group, 8, skin preparation, device fixation, no irradiation; model group, 8, skin preparation, device fixation and irradiation; blank preparations (obtained by replacing the quality of the spilanthol A in the low-dose medicament (or skin care product) for preventing and treating skin photoaging prepared in example 2 with deionized water) 8 of the groups, preparing skin, fixing the device, locally administering the blank preparations to the skin on the back, and irradiating; low dose of the trilobatin a formulation group (prepared in example 2), 8, skin prepped, device fixed, low dose of the trilobatin a formulation given topically to the dorsal skin, irradiated; group of high dose of a preparation of a thousand leaf elements a (prepared in example 4), 8, prepared skin, fixed device, topical administration of high dose of a preparation of thousand leaf elements a to the skin of the back, irradiated.

(3) Effect of the Broccophyllin A preparation on UV irradiation-induced skin moisture in photoaged mice

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

In the experiment, the change in skin moisture content after the administration of the trilobatin A formulation was tested. Specifically, the blank control group is not irradiated, the model group, the blank preparation group, the high-dose patchouli A preparation group and the low-dose patchouli A preparation group are all irradiated by ultraviolet rays, and the irradiation condition is found in the establishment of the photoaging animal model. The skin hydration was measured 5 days a week, and every week after the end of 5 days of irradiation, i.e., day 6 (i.e., days 6d, 12d, 18d, 24d, 30d, 36d, 42d, 48d, 54 d). The MMV values of the skin were measured for the test areas using a skin moisture content tester, respectively, and the average was taken 3 times per test area. The change of the MMV value of the skin moisture content is reflected in the test period, and the moisture content of the experimental area changes regularly along with time. The larger the value, the larger the moisture content, and conversely, the smaller the moisture content. The measurement results are shown in table 10.

Table 10 effect of a trilobatin a formulation on UV irradiation induced skin moisture (x ± SD) (n ═ 8) in photoaged mice

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

As can be seen from table 10, the MMV values for skin moisture were higher in the test zones using the trilobatin a formulation group than in the model and blank formulation groups during the test period. The results show that the spilanthol A preparation can increase the water content of photoaging skin and improve the dry symptom of the photoaging skin in the experimental period.

(4) Effect of the Broccophyllin A preparation on UV irradiation-induced skin sensitivity of photoaged mice

A Tissue Viability Imager (Tissue Viability Imager TiVi 700, wheellsbridge AB, sweden) is based on that linearly polarized light emitted by a white light illuminator passes through a polarizing filter and reaches the surface of the skin to be analyzed, most of the linearly polarized light is blocked by the filter of a detector after being directly reflected by the surface of the skin, only part of the linearly polarized light randomly passes through the underlying layer of the skin, and the polarized light absorbed by red blood cells passes through the filter and enters the detector. Of the polarized light reaching the detector, the green component is attenuated by the absorption of the red blood cells in the blood vessel, the red component is hardly altered, and the green and red light is absorbed by other tissue surrounding the red blood cells substantially the same. Thus, the blood vessel microcirculation state of the deep layer of the skin can be obtained, and the red blood cell concentration value, namely the TiVi value, of each point in the image is obtained. The TiVi 700 system utilizes the characteristic of red blood cells in blood vessels to absorb green light to obtain a distribution aggregation image of red blood cells in skin tissues, and the content of the red blood cells represents skin sensitivity.

In the experiment, the change in skin sensitivity after using the trilobatin a formulation was tested. Specifically, the blank control group is not irradiated, the model group, the blank preparation group, the high-dose patchouli A preparation group and the low-dose patchouli A preparation group are all irradiated by ultraviolet rays, and the irradiation condition is found in the establishment of the photoaging animal model. The skin sensitivity was measured 5 days a week, and every week after the end of 5 days of irradiation, i.e., day 6 (i.e., day 6d, 12d, 18d, 24d, 30d, 36d, 42d, 48d, 54 d). The measurement results are shown in Table 11.

TABLE 11 Effect of the trilysin A formulations on UV irradiation-induced skin sensitivity of photoaged mice

(n＝8)

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

As can be seen from table 11, the skin sensitivity values of the test zones using the trilobatin a formulation group were lower than those of the model and blank formulations during the test period. The results show that the spilanthol A preparation can reduce the sensitivity of the photoaged skin and improve the high sensitivity of the photoaged skin in the experimental period.

(5) Effect of Qiafolin A preparation on melanin content in skin of photoaged mice

Images were taken using the Tissue Viability Imager (Tissue Viability Imager TiVi 700, wheellsbridge AB, sweden) in (4) and the melanin content was analyzed using the TiVi97 pigment analysis software.

In the experiment, the change in skin melanin content after using the trilobatin a formulation was tested. Specifically, the blank control group is not irradiated, the model group, the blank preparation group, the high-dose patchouli A preparation group and the low-dose patchouli A preparation group are all irradiated by ultraviolet rays, and the irradiation condition is found in the establishment of the photoaging animal model. The skin melanin content was measured 5 days a week, and 6 days (i.e., 6d, 12d, 18d, 24d, 30d, 36d, 42d, 48d, 54d) after 5 days of irradiation. The measurement results are shown in table 12.

TABLE 12 Effect of the trilysin A preparation on the melanin content of the skin of UV irradiation-induced photoaged mice

(n＝8)

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

As can be seen from table 12, the melanin content in the test area using the trilobatin a formulation group was lower than that of the model and blank formulation groups during the test period. The results show that the spilanthol A preparation can reduce the melanin content of the photoaging skin and improve the pigmentation problem of the photoaging skin in the experimental period.

Experiments prove that the medicament or the skin care product for preventing and treating skin photoaging, which is prepared from the phyllanthin A and pharmaceutically common auxiliary materials, can improve the water content of photoaging skin, reduce the sensitivity of the photoaging skin and relieve pigmentation.

The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Claims (2)

1. Use of chibain A in the preparation of medicines or skin care products for preventing and treating skin photoaging. 2. The use of the extract containing chibain A in the preparation of medicines or skin care products for preventing and treating skin photoaging.

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