CN112587515A

CN112587515A - Application of licorice flavonoids compounds in preparation of medicines for preventing and/or treating scars

Info

Publication number: CN112587515A
Application number: CN202011506527.3A
Authority: CN
Inventors: 张青; 彭伟; 汤丹丹; 吴纯洁; 刘佳
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-12-18
Filing date: 2020-12-18
Publication date: 2021-04-02

Abstract

The invention provides a new application of licorice flavonoids compounds in preparing a medicament for preventing and/or treating scars. Cell experimental research shows that glabridin in licorice flavonoids compounds can obviously inhibit proliferation and apoptosis of HFK cells of human scar fibroblasts, and can inhibit migration of the HFK cells; animal experiments also show that the glabridin can obviously inhibit scar indexes of the rabbit ear hypertrophic scar models. The glabridin has the potential of being developed into a medicinal preparation for treating scars, which has good curative effect and high safety, and has comprehensive development and application prospects.

Description

Application of licorice flavonoids compounds in preparation of medicines for preventing and/or treating scars

Technical Field

The invention relates to application of licorice flavonoids compounds in preparing a medicament for preventing and/or treating scars.

Background

Scar (scar) is a general term for the morphological and histopathological changes of the appearance of normal skin tissue caused by various traumas, and is an inevitable product in the process of repairing human traumas. When the growth of the scar exceeds a certain limit, various complications can occur, such as disfigurement, dysfunction of functional activities and the like, which bring great physical and mental pains to patients, in particular to the scar left after burns, scalds and serious trauma.

Glabridin, a flavonoid substance extracted from licorice, is known as "whitening gold" because of its strong whitening effect and is a whitening and anti-aging cherry for skin. Modern pharmacological research also finds that glabridin not only has the effects of resisting oxidation, resisting inflammation, reducing blood fat and protecting nerves, but also can show better anti-tumor effect in osteosarcoma, breast cancer, melanoma and the like by inhibiting tumor cell proliferation, transferring, inducing apoptosis and the like. Because of the obvious functions of eliminating free radicals and resisting oxidation, the glabridin can eliminate free radicals and muscle bottom melanin, and is also used as a fast, efficient and safe skin whitening and freckle removing cosmetic additive at present. No research report about the treatment research that glabridin can be independently applied to scars exists.

Disclosure of Invention

In order to solve the problems, the invention provides application of a licorice flavonoid compound in preparing a medicament for preventing and/or treating scars.

Further, the licorice flavonoids is glabridin.

Further, the medicine is a medicine for inhibiting the proliferation of human scar fibroblast HFK cells.

Further, the medicine is a medicine for inducing apoptosis of HFK (human scar fibroblast) cells.

Furthermore, the medicine is a medicine with the function of reducing the scar index of the wound surface.

Still further, the medicament is a medicament for the prevention and/or treatment of superficial scars.

Still further, the medicament is a medicament for preventing and/or treating hypertrophic scars.

Still further, the medicament is a medicament for the prevention and/or treatment of atrophic scars.

Further, the drug is a drug for preventing and/or treating keloid.

The invention relates to a new application of licorice flavonoids compounds in preparing medicaments for preventing and/or treating scars, which discovers that glabridin in the licorice flavonoids compounds can obviously inhibit the proliferation and induce the apoptosis of human scar fibroblast HFK cells and can inhibit the migration and invasion of the HFK cells through cell experimental research; animal experiments also show that the glabridin can obviously inhibit scar indexes of the rabbit ear hypertrophic scar models. The glabridin has the potential of being developed into a medicinal preparation for treating scars, which has good curative effect and high safety, and has comprehensive development and application prospects.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 inhibition of HFK scar fibroblast by glabridin

FIG. 2 apoptosis-inducing effect of glabridin on HFK scar fibroblasts

FIG. 3 inhibition of HFK scar fibroblast scratch healing by glabridin

FIG. 4 inhibition of HFK scar fibroblast migration by glabridin

Detailed Description

Example 1 cell assay

1 materials of the experiment

1.1 medicaments

The medicine of the invention is: glabridin (GD).

Control drugs: physiological saline.

1.2 cells

HFK human scar fibroblast

1.3 Primary reagents

Annexin V-FITC/PI apoptosis detection kit (Wuhan BOSTER bioengineering, Inc.); DMEM high-glucose medium (Gibco organism, usa); CCK8 kit (wuhan BOSTER organism); transwell cell (corning, usa); matrigel (BD company, usa).

1.4 Main Instrument

CO₂Thermostated cell culture chambers (Thermo Fisher, usa); enzyme-linked immunosorbent assay (BIO-RAD, USA); flow cytometry (BD corporation, usa).

2 method of experiment

2.1 cytotoxic Studies

The effect of glabridin on the survival rate of HFK cells was determined by the CCK8 method. HFK cells were digested in the logarithmic growth phase, resuspended in complete medium, and cultured in 96-well plates, to which 100. mu.l (about 2X 10) of cell suspension was added per well³One cell/well), 6 multiple wells are set, and the culture chamber is placed for 12 hours. Then, different concentrations of glabridin solution (final concentrations in the wells 2.5, 5, 10, 20, 40, 60, 80 and 100 μ g/ml) were used for incubation for 12h, 24h and 48 h. CCK8 (10. mu.l/well) was added in a dark room environment and incubation was continued for 1-2 hours. The absorbance values in the wells were determined using a fully automated microplate detector (detection wavelength 450nm) and the inhibition of cell proliferation/%:

2.2 flow cytometry apoptosis study

(1) HFK cells were seeded in 6-well plates (1X 10)⁵Perwell), incubated at 37 ℃ for 12 hours；

(2) After the cells adhere to the wall, adding glabridin (with final concentration of 10 mug/ml, 20 mug/ml and 40 mug/ml) into the holes for intervention treatment for 24 hours;

(3) after the treatment, the cells were digested with pancreatin without EDTA and collected;

(4) washing cells with PBS for 3 times, centrifuging, adding 500 μ l of binding buffer solution into cell sediment for resuspension, adding 5 μ l of annexin V-FITC, and continuously adding 5 μ l of PI for uniformly mixing;

(5) and (4) incubating the cells in a dark room for 10min at room temperature, detecting the apoptosis condition of the HFK cells on a flow cytometer, and recording the apoptosis rate/%.

2.3 scratch test

(1) HFK cells were seeded in 6-well plates (1X 10)⁵/well), incubated at 37 ℃ for 12 hours;

(2) after the cells are fully paved on the whole bottom of the hole, replacing a serum-free culture medium for hungry for 12h, performing cross scratch treatment on the bottom of the hole plate by using a 200-mu-l gun head, and adding glabridin (the final concentration in the hole is 5 mu g/ml, 10 mu g/ml and 20 mu g/ml) for intervention treatment for 24 h;

(3) after the treatment was completed, the cells were stained with crystal violet dye and photographed to record pictures.

2.4 migration and invasion experiments

The upper Transwell chamber was pretreated with/without Matrigel before the start of the experiment. And HFK cells were seeded into the upper chamber of the Transwell plate using serum-free medium and the lower chamber was supplemented with serum-containing medium. And simultaneously, randomly grouping the cells into a normal group and a glabridin dose group, adding glabridin (5 mu g/ml, 10 mu g/ml and 20 mu g/ml) with different concentrations to the rest of the cells except the normal group, continuously culturing for 24h, taking out the cells from the upper chamber, removing the cells on the inner side of the membrane at the bottom of the upper chamber by using a cotton swab, determining the cells on the outer side of the membrane as migrated (without using Matrigel)/invaded (using Matrigel), and carrying out crystal violet staining.

3. Results

The results of the inhibition rate of the glabridin with different concentrations on human scar fibroblast HFK cells are shown in figure 1, the induced apoptosis rate is shown in figure 2, the inhibition of the scar healing is shown in figure 3, and the inhibition of the migration is shown in figure 4.

Example 2 animal testing

1. Medicine

1.1 preparation of Glabridin solid Dispersion cream

1) Taking the mass ratio of 1: dissolving glabridin and PVP (polyvinyl pyrrolidone) of 4 in appropriate amount of ethanol, mixing, performing rotary evaporation in water bath at 60 deg.C to remove solvent, drying the viscous substance in a constant temperature drying oven at 60 deg.C for 24 hr, pulverizing, and sieving with 80 mesh sieve to obtain glabridin solid dispersion;

2) taking stearic acid, glyceryl monostearate and liquid paraffin, placing in an evaporation dish, heating in a water bath to 70-80 ℃ to melt into an oil phase, and keeping the temperature at 75 ℃; heating purified water, glycerol and triethanolamine in water bath to the same temperature to obtain water phase, adding the water phase into the oil phase under stirring, and cooling under stirring to obtain blank matrix containing stearic acid: glyceryl monostearate: liquid paraffin: glycerol: triethanolamine: 3:7:8:5: 1;

3) adding glabridin solid dispersoid with different weight into blank matrix to obtain glabridin solid dispersible cream with content of 0.5%, 1% and 2% for test.

1.2 control drugs: blank matrix

2. Rabbit ear hyperplastic scar model

2.1 preparation of Rabbit ear wound

After the pentobarbital sodium is adopted to anaesthetize the rabbits, blood vessels are avoided at the inner sides of the rabbit ears, a hole puncher is adopted to manufacture a circular wound surface with the diameter of about 0.7cm, the whole layer of skin is completely cut off, the perichondrium is thoroughly scraped, the cartilage is reserved, the wound surface is not treated, and the wound surface is naturally healed. 6 circular wounds were prepared per rabbit ear, with 1.0cm spacing between wounds. The 12 wounds per rabbit were randomly divided into a drug administration group (0.5%, 1%, 2%) and a control group, and the drug administration was started 7 days after the operation, once a day, and the scar index evaluation was performed on the 14 th, 21 th, and 28 th days after the drug administration.

2.2 scar index detection

The skin thickness at the scar and adjacent normal skin thickness were measured with a vernier caliper to a precision of 0.01mm at 14, 21 and 28 days post-surgery.

3 results and analysis

The scar index results of the rabbit ear hypertrophic scar model are shown in table 1, and the glabridin can obviously reduce the scar index of the rabbit ear wound surface from the table 1.

TABLE 1 Effect of glabridin solid Dispersion cream on Rabbit ear hypertrophic scar index

In conclusion, experiments prove that the glabridin in the licorice flavonoids compounds can obviously inhibit the proliferation and induce the apoptosis of HFK cells of human scar fibroblasts, can inhibit the migration and invasion of the HFK cells, and has obvious effect on reducing scar indexes of wounds.

Claims

1. Application of licorice flavonoids compounds in preparing medicaments for preventing and/or treating scars is provided.

2. Use according to claim 1, characterized in that said licorice flavonoid is glabridin.

3. Use according to claim 1 or 2, wherein the medicament is a medicament for inhibiting proliferation of human scar fibroblast HFK cells.

4. Use according to claim 1 or 2, wherein the medicament is a medicament for inducing apoptosis of human scar fibroblast HFK cells.

5. Use according to claim 1 or 2, wherein the medicament is a medicament for reducing the scar index of a wound.

6. Use according to claim 1 or 2, characterized in that the medicament is a medicament for the prevention and/or treatment of keloids.

7. Use according to claim 1 or 2, characterized in that the medicament is a medicament for the prevention and/or treatment of atrophic scars.

8. Use according to claim 1 or 2, characterized in that the medicament is a medicament for the prevention and/or treatment of hypertrophic scars.

9. Use according to claim 1 or 2, characterized in that the medicament is a medicament for the prevention and/or treatment of superficial scars.