[1, 1apos; application of-biphenyl]-2, 4-diol in medicine for inhibiting melanoma cell proliferation

Abstract

The invention relates to the field of biological medicines, in particular to application of [1, 1 '-biphenyl]-2, 4-diol in drugs for inhibiting melanoma cell proliferation. The invention proposes that the [1, 1 '-biphenyl]-2, 4-diol can inhibit the proliferation of melanoma cells for the first time, and the product is low in price, has a very good inhibition effect on the proliferation of the melanoma cells, has a very good transdermal absorption effect, can deeply inhibit the melanoma cells, and can inhibit the proliferation of the melanoma cells within the concentration range of inhibiting the melanoma cells. No irritation or injury is caused to the skin. The invention provides a theoretical basis for research and development of drugs for treating melanoma cell proliferation.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of [1,1′-biphenyl]-2,4-diol in drugs for inhibiting melanoma cell proliferation. Background Art

[0002] Melanoma is a malignant tumor formed when melanocytes turn into melanoma cells. It often occurs in the skin and can also be seen in mucous membranes and internal organs. Melanoma is different from benign moles. The surrounding area of ​​melanoma is generally irregular and fan-shaped, with different pigmentation. The incidence and mortality of melanoma have gradually increased in recent decades. At present, the cause of melanoma is not yet fully understood. It is generally believed to be related to factors such as race, genetics, and long-term ultraviolet radiation. Initial melanoma cells only exist in the epidermis or mucosal epithelial layer, which is called malignant melanoma in situ. Melanoma cells break through the basal layer of the epidermis or the mucosal epithelial layer, enter the dermis, and even metastasize to more distant parts, such as lymph nodes, lungs, brain, etc., which is called invasive malignant melanoma. Currently, the main methods for treating melanoma are surgery, chemotherapy, and drug therapy. For example, BRAF inhibitors can bind to BRAF protein and block its abnormal activation, thereby inhibiting the growth and spread of melanoma cells. Many existing drugs that inhibit melanoma are relatively expensive. Finding relatively low-priced drugs that have higher and better effects in inhibiting melanoma is of great significance for the treatment of melanoma. Summary of the invention

[0003] The purpose of the present invention is to provide the use of [1,1'-biphenyl]-2,4-diol in drugs for inhibiting melanoma cell proliferation. [1,1'-biphenyl]-2,4-diol has a low price for inhibiting melanoma and has a higher and better inhibitory effect.

[0004] In order to achieve the above-mentioned invention object, the present invention adopts the following technical solutions:

[0005] The present invention provides an application of [1,1′-biphenyl]-2,4-diol in preparing a drug for inhibiting melanoma cell proliferation.

[0006] In the above technical solution, further, the melanoma cells are B16F10 cells.

[0007] In the above technical solution, further, the effective concentration of the [1,1′-biphenyl]-2,4-diol is >0.5 mM.

[0008] In the above technical solution, further, the effective concentration of the [1,1′-biphenyl]-2,4-diol is ≥1 mM.

[0009] In the above technical solution, further, the effective concentration of the [1,1′-biphenyl]-2,4-diol is ≥1 mM and ≤4 mM.

[0010] In the above technical solution, further, the drug uses [1,1′-biphenyl]-2,4-diol as an active ingredient.

[0011] In the above technical solution, further, the medicine is an external preparation.

[0012] Compared with the prior art, the present invention has the following beneficial effects: the present invention proposes for the first time that [1,1′-biphenyl]-2,4-diol can inhibit the proliferation of melanoma cells. [1,1′-biphenyl]-2,4-diol is inexpensive, has a good inhibitory effect on the proliferation of melanoma cells, and has a good transdermal absorption effect, can deeply inhibit melanoma cells, and has no irritation or damage to the skin within the concentration range of inhibiting melanoma cells. The present invention provides a theoretical basis for the research and development of drugs for treating melanoma cell proliferation. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The bar graph shows the effect of different concentration gradients of [1,1′-biphenyl]-2,4-diol on the proliferation of B16F10 cells;

[0014] Figure 2 Line graph showing the effect of different concentration gradients of [1,1′-biphenyl]-2,4-diol on the proliferation of B16F10 cells;

[0015] Figure 3 The absorbance of different concentration gradients of [1,1′-biphenyl]-2,4-diol;

[0016] Figure 4 The maximum absorption peak wavelength of [1,1′-biphenyl]-2,4-diol at different concentration gradients;

[0017] Figure 5 Cumulative permeability of [1,1′-biphenyl]-2,4-diol over time T n ;

[0018] Figure 6 The picture is taken 14 days after applying 4mM [1,1′-biphenyl]-2,4-diol on the rabbit skin surface. DETAILED DESCRIPTION

[0019] The present invention is further described below in conjunction with specific embodiments, but the present invention is not limited in any way.

[0020] B16F10 cell culture, MTT detection of the effect of [1,1′-biphenyl]-2,4-diol on B16F10 cell proliferation; transdermal absorption test to detect the transdermal absorption rate of [1,1′-biphenyl]-2,4-diol; according to the "Safety Technical Specifications for Cosmetics" 2015 edition, test whether [1,1′-biphenyl]-2,4-diol is irritating to the skin.

[0021] Example 1 Effect of [1,1′-biphenyl]-2,4-diol on B16F10 cell proliferation

[0022] The structural formula of [1,1′-biphenyl]-2,4-diol is:

[0023]

[0024] Purchased from (Shanghai Yuanye Biotechnology Co., Ltd.).

[0025] method:

[0026] (i) Collect B16F10 cells in logarithmic phase, adjust the concentration of cell suspension to about 5000 cells per 100 μL, add 100 μL of cell suspension to each well of a 96-well plate; culture in a 37°C, 5% carbon dioxide incubator; after the cells adhere to the wall and cover about 80% of the bottom of the well, discard the supernatant.

[0027] (ii) Add 160 μL of culture medium (89% DMEM, 10% premium fetal bovine serum, 1% double-streptomycin), add 40 μL of different concentrations of [1,1′-biphenyl]-2,4-diol to the experimental group, and add them to a 96-well plate. The concentration gradient of [1,1′-biphenyl]-2,4-diol is 1 mM, 0.5 mM, 0.25 mM, 0.125 mM, 0.0625 mM, 0.03125 mM, and 0.015625 mM. Add 40 μL of 1× PBS to the control group. Set up 3 replicate wells for the experimental group and the control group respectively. Incubate at 5% carbon dioxide at 37°C for 48 hours and discard the supernatant.

[0028] (III) Add 10 μL MTT (5 mg / ml, i.e. 0.5% MTT), 90 μL DMEM medium, 5% carbon dioxide, 37°C, incubate for 4 hours, and discard the supernatant.

[0029] (iv) Add 100 μL of dimethyl sulfoxide, shake for 10 minutes, and measure the absorbance at 490 nm.

[0030] (V) Graphpad Prism was used to perform significant difference analysis.

[0031] Results: MTT assay was used to detect the effect of [1,1′-biphenyl]-2,4-diol at different concentration gradients of 1mM, 0.5mM, 0.25mM, 0.125mM, 0.0625mM, 0.03125mM, 0.015625mM, and 0mM on the proliferation of B16F10 cells. The absorbance at 0.5mM was 1.718, slightly lower than the absorbance of 1.818 at 0mM. The absorbance at 1mM was 0.524. The P value of adding 1mM [1,1′-biphenyl]-2,4-diol was 0.0003 compared with 0mM, indicating that there was a significant difference between the two. The inhibition rate at 1mM was about 71.1%. Starting from 0.5mM, the higher the concentration of [1,1′-biphenyl]-2,4-diol, the more obvious the effect on the proliferation of B16F10 cells. The P values ​​of the other groups of different concentration gradients of [1,1′-biphenyl]-2,4-diol were all greater than 0.99 compared with 0 mM, indicating that there was no significant difference in the effect of 0 to 0.5 mM [1,1′-biphenyl]-2,4-diol on the proliferation of B16F10 cells, and it can be considered that it has no effect on the proliferation of B16F10 cells.

[0032] Example 2 Ultraviolet absorption detection of [1,1′-biphenyl]-2,4-diol

[0033] Method: The reference cell was placed in 1×PBS phosphate buffer and the baseline was scanned. After the baseline was stable, 7 concentration gradients of [1,1′-biphenyl]-2,4-diol solutions with 0.5mmol / L, 0.25mmol / L, 0.125mmol / L, 0.0625mmol / L, 0.03125mmol / L, 0.015625mmol / L and 0.0078125mmol / L, prepared with 1×PBS phosphate buffer as the solvent were detected.

[0034] Results: 1×PBS phosphate buffer was used as solvent to prepare 7 concentration gradients of [1,1′-biphenyl]-2,4-diol solutions for UV detection. 2 The maximum absorption wavelength is 0.9997, and the maximum absorption wavelength is 252nm. This step provides the ultraviolet detection wavelength for the next step of high performance liquid chromatography detection of [1,1′-biphenyl]-2,4-diol transdermal absorption and determination of the concentration of [1,1′-biphenyl]-2,4-diol in the receiving pool.

[0035] Example 3 Transdermal absorption

[0036] Methods: Franz diffusion cell was used as an in vitro transdermal absorption test model.

[0037] (1) Process the pigskin purchased from the market into slices less than 1 mm in thickness, soak them in physiological saline for 30 minutes, and remove the lipids in the pigskin;

[0038] (ii) Place the pigskin between the diffusion tank and the receiving tank and fix it with a clip. The effective diffusion area of ​​the pigskin is 0.94985 cm 2 . 15 mL of normal saline was added to the receiving pool, and 1 mL of [1,1′-biphenyl]-2,4-diol solution with 4 mmol / L solvent of normal saline was added to the diffusion pool of the experimental group. When the [1,1′-biphenyl]-2,4-diol in the diffusion pool and the receiving pool reached equilibrium, the concentration of [1,1′-biphenyl]-2,4-diol in the diffusion pool and the receiving pool was 0.25 mM. 1 mL of normal saline was added to the diffusion pool of the control group. The sampling port and the opening of the diffusion pool of the Franz diffusion pool were sealed with tin foil to prevent the solution from volatilizing. There were 3 groups in each of the experimental group and the control group;

[0039] (III) Place the Franz diffusion cell in a constant temperature magnetic heating stirrer at 37°C and stir at 400 r / min. Take samples at 0, 2, 4, 6, and 8 hours, with a sampling volume of 0.2 mL each time. After each sampling, add the corresponding volume of physiological saline;

[0040] (IV) The sample concentration in the receiving pool was detected by Agilent 1260Ⅱ (agilent 1260infinityⅡ), the chromatographic column was Poroshell 120EC-C18 (4.6×250mm, 4μm), the mobile phase was methanol (80%): water (20%), the flow rate was 1ml / min, the injection volume was 5μl, the column temperature was 25℃, and the detection wavelength was 252nm. The peak area of ​​0.25mM [1,1′-biphenyl]-2,4-diol was detected by high performance liquid chromatography. According to the peak area ratio being equal to the concentration ratio, the concentration of [1,1′-biphenyl]-2,4-diol in the receiving pool at 0 hours, 2 hours, 4 hours, 6 hours, and 8 hours was calculated, and the cumulative permeation amount Q was calculated. n (mg / cm 2 ) and cumulative transmittance T n (%);

[0041]

[0042] C n (mg / mL) is the sample concentration measured at the nth sampling point;

[0043] V n (mL) is the volume of the receiving solution at the nth sampling point (15mL);

[0044] C k (mg / mL) is the sample concentration measured at all sampling points before the nth sampling point;

[0045] V k(mL) is the sampling volume corresponding to all sampling points before the nth sampling point (0.2mL);

[0046] A(cm 2 ) is the effective diffusion area (0.94985cm 2 ).

[0047]

[0048] M (mg) is the mass of the added sample.

[0049] Results: The [1,1′-biphenyl]-2,4-diol in the diffusion cell and the receiving cell reached equilibrium at 0.25 mM. The peak area of ​​0.25 mM [1,1′-biphenyl]-2,4-diol detected by HPLC was 179.723 mAu·S, and 0.25 mM was 0.0465 mg / mL. Based on the peak area ratio being equal to the concentration ratio, the concentration of [1,1′-biphenyl]-2,4-diol in the sample solution at 0, 2, 4, 6, and 8 hours was calculated. The cumulative permeation amount Q was calculated according to the formula: n (mg / cm 2 ) and cumulative transmittance T n (%). The cumulative transmittance is linearly correlated with time. After 8 hours, the cumulative transmittance of [1,1′-biphenyl]-2,4-diol T n It is 36.1%.

[0050] Table 1 Transdermal absorption of [1,1′-biphenyl]-2,4-diol at different times

[0051]

[0052] It can be seen from the results in Table 1 that the concentration of [1,1′-biphenyl]-2,4-diol penetrating the skin is increasing from 2 hours to 8 hours. The transdermal absorption of [1,1′-biphenyl]-2,4-diol is not high. Melanoma is located in the epidermis of the skin. A higher concentration of [1,1′-biphenyl]-2,4-diol can stay in the epidermis of the skin for a longer time and be slowly absorbed into the human body. The concentration of the drug entering the body is low and stable, which can better inhibit the proliferation of melanoma cells.

[0053] Example 4 Skin irritation detection

[0054] Methods: Multiple skin irritation tests were conducted according to the procedures of skin irritation / corrosion test in "Technical Specification for Safety of Cosmetics" (2015 edition).

[0055] (I) Before the experiment, the hair on one side of the spine of the test animals was trimmed, the hair removal area was 3cm×3cm, and the application area was 2.5cm×2.5cm; rabbits No. 1 and No. 2 were the control group, and rabbits No. 3 and No. 4 were the test group.

[0056] (ii) Take 0.5 mL of 4 mM [1,1′-biphenyl]-2,4-diol and apply it to one side of the skin, apply a patch, and apply it once a day for 14 days;

[0057] (III) Starting from the second day, the hair should be cut before each application, and the residual test substance should be washed with water. The results should be observed after one hour and scored according to Table 2. The test group is treated the same as the control group except for the application of the drug. Result evaluation: Calculate the average score of each animal per day according to the following formula, and determine the skin irritation intensity according to Table 2.

[0058]

[0059] Table 2 Skin irritation reaction scores

[0060]

[0061] Table 3 Skin irritation intensity classification

[0062]

[0063]

[0064] Results: Four rabbits were given the drug for 14 consecutive days. There was no erythema or edema at the drug administration site. 4 mM [1,1′-biphenyl]-2,4-diol had no irritating effect on the skin.

[0065] For any technician familiar with the art, without departing from the scope of the technical solution of the present invention, the technical content disclosed above can be used to make many possible changes and modifications to the technical solution of the present invention, or modified into equivalent embodiments with equivalent changes. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention should still fall within the scope of protection of the technical solution of the present invention.

Claims

1. Application of [1,1'-biphenyl]-2,4-diol in the preparation of drugs for inhibiting melanoma cell proliferation.

2. The use according to claim 1, characterized in that: The melanoma cells are B16F10 cells.

3. The use according to claim 1, characterized in that: The effective concentration of the [1,1'-biphenyl]-2,4-diol is >0.5 mM.

4. The use according to claim 3, characterized in that: The effective concentration of the [1,1'-biphenyl]-2,4-diol is ≥1 mM.

5. The use according to claim 4, characterized in that: The effective concentration of the [1,1'-biphenyl]-2,4-diol is ≥1 mM and ≤4 mM.

6. The use according to claim 1, characterized in that: The drug uses [1,1'-biphenyl]-2,4-diol as an active ingredient.

7. The use according to claim 1, characterized in that: The medicine is an external preparation.

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