Application of methyl coptisine in preparation of acne treatment medicine

Abstract

The invention discloses application of methyl coptisine in preparation of a propionibacterium acnes inhibitor and a medicine for treating acnes. By comparing the in-vitro inhibition effects of multiple common alkaloids of coptis chinensis on propionibacterium acnes, it is found that the bacteriostatic activity of methyl coptisine is higher than that of most coptis chinensis alkaloids including coptisine, berberine and palmatine, the minimum inhibitory concentration (MIC) of methyl coptisine is 43.75 g / ml, and the minimum bactericidal concentration (MBC) of methyl coptisine is 87.5 g / ml. Therefore, methyl coptisine is expected to be used for in-vitro inhibition of propionibacterium acnes and is expected to play a certain role in clinical treatment of acnes.

Description

Technical Field

[0001] The invention belongs to the pharmaceutical field and relates to a new pharmaceutical application of methyl coptisine, in particular to an application in preparing an in vitro inhibitor of Propionibacterium acnes and a drug for treating acne. Background Art

[0002] Acne is an inflammatory disease of the sebaceous glands and a common skin disease that can induce inflammation on the surface of the skin on the face, neck, chest and back. Acne is a chronic inflammatory skin disease with a complex pathogenesis, high incidence and easy recurrence. The lesions are often accompanied by papules, pustules, nodules, cysts and other types of rashes. Pustules and cysts can cause secondary pigmentation and even permanent scars, which seriously affect the physical and mental health of patients. Studies have found that Propionibacterium acnes is the main pathogenic bacteria of acne, followed by Staphylococcus, which induces local inflammatory reactions. Therefore, treatment can start from both antibacterial and anti-inflammatory aspects.

[0003] As a traditional Chinese herbal medicine, Coptis chinensis has been used to treat inflammation for a long time. A variety of alkaloids have been isolated from Coptis chinensis, including berberine, epiberberine, jatrorrhizine, coptis pine, methoxyberberine, methyl coptis pine, palmatine, berberythrine, chelerythrine, and nitrine. Studies have found that many components of Coptis chinensis have biological activities, including antioxidant, anti-inflammatory, antibacterial and other functions, and different Coptis chinensis alkaloids have different biological activities.

[0004] It has been confirmed that berberine has a broad-spectrum antibacterial effect, and has inhibitory effects on a variety of Gram-positive and Gram-negative bacteria and certain skin fungi. Yang Yong et al. (2007) compared the antibacterial effects of four berberine alkaloids and found that berberine, jatrorrhizine, coptisine and palmatine had the same antibacterial spectrum but had significant differences in antibacterial activity, among which berberine had the greatest antibacterial activity, and believed that the substitution groups on the A and D rings had an increasing or decreasing effect on the antibacterial activity of the original berberine compounds.

[0005] Propionibacterium acnes is a species of the genus Propionibacterium, a Gram-positive bacillus, and the main pathogen causing acne. It is named after the production of propionic acid by fermenting glucose. CN 110882246 A discloses the extraction method and application of coptis chinensis alkaloids with different biological activities, and obtains berberine, coptisine, magnoline, and palmatine with high purity. It is believed that palmatine can treat acne more effectively than berberine and coptisine.

[0006] Methyl coptisine is one of the main components of Coptis chinensis and belongs to the isoquinoline alkaloids. Its activity and function are still unclear. The applicant's previous research found that methyl coptisine can inhibit the activity of JNK2 kinase and thus be used for the treatment of psoriasis. In the present invention, we found that methyl coptisine can effectively inhibit Propionibacterium acnes and treat acne.

[0007]

[0008] Methylcopper alkaloids Summary of the invention The purpose of the present invention is to provide a new use of methyl coptisine in inhibiting Propionibacterium acnes and treating acne.

[0009] To achieve the above purpose, the applicant first compared the inhibitory effects of various common alkaloids of Coptis chinensis on Propionibacterium acnes in vitro. The results showed that the antibacterial activity of methyl coptis alkaloids was higher than that of most Coptis chinensis alkaloids, including coptis alkaloids, berberine and bamipine, with a minimum inhibitory concentration (MIC) of 43.75µg / ml and a minimum bactericidal concentration (MBC) of 87.5µg / ml. Therefore, methyl coptis alkaloids are expected to be used for in vitro inhibition of Propionibacterium acnes. At the same time, given that Propionibacterium acnes is the main pathogen that causes acne, methyl coptis alkaloids are also expected to play a certain role in the clinical treatment of acne.

[0010] Furthermore, an acne animal model was established by intradermal injection of Propionibacterium acnes suspension in mice, and the effects of methyl coptisine on the clinical and pathological manifestations of the model animals were investigated. The results showed that methyl coptisine can effectively inhibit acne symptoms and pathological changes induced by Propionibacterium acnes, significantly reduce the size of acne and skin lesions, reduce the occurrence of rash and inflammatory cell infiltration, and has a good therapeutic effect on acne. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 : The inhibition zone diagram of four kinds of coptis alkaloids against Propionibacterium acnes. In the figure, 1 is methyl coptis alkaloid; 2 is coptis alkaloid; 3 is berberine; 4 is palmatine; 5 is negative control.

[0012] Figure 2 : The MIC and MBC of four coptis alkaloids against Propionibacterium acnes were investigated by micro-two-fold dilution method and colony counting method.

[0013] Figure 3 : Appearance of acne sites in each group of mice after modeling.

[0014] Figure 4 :Statistical curve of acne diameter in each group of mice. *P<0.05; ***P<0.005.

[0015] Figure 5:Statistical curve of acne height in each group of mice. *P<0.05; **P<0.01.

[0016] Figure 6 :Statistical curve of skin lesion scores of mice in each group. The severity of acne was evaluated according to Pillsbury's grading method, and the severity scores were as follows: 0: asymptomatic; 1: comedones; 2: papules; 3: inflammatory papules; 4: pustules; 5: nodules, cysts, scars; ****P<0.001.

[0017] Figure 7 : HE staining of acne skin on the back of mice in each group.

[0018] Figure 8 : The thickness of epidermis and dermis of acne in mice in each group. ***P<0.005; ****P<0.001. DETAILED DESCRIPTION

[0019] The present invention is described in detail below by specific examples. It should be noted that the specific examples are only used to explain the present invention and are not intended to limit the present invention. In addition, the various experimental operations involved in the embodiments are routine techniques in the art, and those of ordinary skill in the art can refer to various commonly used reference books, scientific and technological literature or relevant specifications, manuals, etc. before the application date of the present invention for implementation.

[0020] Example 1 Evaluation of the efficacy of methylcoptisine in inhibiting Propionibacterium acnes in vitro (1) Determination of the inhibition zone of four coptis alkaloids against Propionibacterium acnes Take out the Propionibacterium acnes (ATCC 6919) strain from the -80℃ refrigerator, thaw naturally, and evenly apply 100ul on the reinforced Clostridium solid culture medium, place it in a 37℃ constant temperature incubator, put it in an anaerobic bag and invert it for 48h. Take an appropriate amount of bacteria and suspend it in sterile PBS to make a bacterial solution, and adjust the concentration of the bacterial solution to 1.0×10 8 CFU / ml. Use a pipette to draw 100ul of bacterial solution onto the fortified Clostridium solid culture medium, pour in an appropriate amount of glass beads for coating the plate, cover the plate and shake the culture plate to ensure that the bacterial solution evenly covers the surface of the solid culture medium and then pour out the glass beads. After burning the tweezers, use the tweezers to pick up the drug sensitivity test paper and stick it on the plate and mark it. One piece is added with 20ul methyl coptisine (1.4mg / ml), one piece is added with 20ul coptisine (1.4mg / ml), one piece is added with 20ul berberine (1.4mg / ml), one piece is added with 20ul bamipine (1.4mg / ml), and one piece is added with 20ul sterile PBS as the negative control group. Each group of experiments was repeated 3 times. The plate was placed in an anaerobic zone in a 37℃ constant temperature incubator and incubated for 48h. Photos were taken and the size of the inhibition zone was recorded. Each group of experiments was repeated 3 times. The inhibitory effects of four coptis alkaloids on Propionibacterium acnes Figure 1 The diameters of the inhibition zones are shown in Table 1.

[0021] Table 1: Diameters of inhibition zones of four Rhizoma Coptidis alkaloids against Propionibacterium acnes (mm)

[0022] Note: The diameter of the inhibition zone is expressed as mean ± standard deviation, in millimeters. NA means no inhibition zone.

[0023] The inhibition zone test showed that the four alkaloids had great differences in their antibacterial abilities against Propionibacterium acnes. Overall, methyl coptisine had a stronger antibacterial effect, followed by coptisine and berberine.

[0024] (2) MIC and MBC determination of four coptis alkaloids against Propionibacterium acnes Take out the Propionibacterium acnes (ATCC 6919) strain from the -80℃ refrigerator, thaw naturally, and evenly apply 100ul on the reinforced Clostridium solid culture medium, place it in a 37℃ constant temperature incubator, put it in an anaerobic bag and invert it for 48h. Take an appropriate amount of bacteria and suspend it in the reinforced Clostridium liquid culture medium to make a bacterial solution. The MIC and MBC of methylcoptisine, coptisine, berberine and bamipine against Propionibacterium acnes were investigated by micro-two-fold dilution method and colony counting method.

[0025] First, add 100ul of culture medium to the 96-well plate, then add 100ul of the corresponding drug to the first well and pipette 100ul to the next well, dilute in turn, discard 100μL of the last well, then add 100μL of the adjusted bacterial solution and shake and mix. The final concentration of the bacterial solution is 1.4×10 6 CFU / ml, the final drug concentration of the first well added is 0.7mg / mL, set up a positive control well without drug addition and a negative control well without bacteria, and repeat 3 times per well. Place in a 37℃ constant temperature incubator, put in an anaerobic bag and invert for 48h. Starting from the initial drug concentration, select 7 drug dilution concentrations, draw 100ul liquid culture from the well and inoculate it on the corresponding agar culture plate, place in a 37℃ constant temperature incubator, put in an anaerobic bag and invert for 48~72h, and use the minimum drug concentration that inhibits 80% bacterial growth as the MIC to judge the MIC of the corresponding drug, and the test is measured in parallel 3 times. When the number of colonies is less than 5, the drug concentration is used as its MBC, and the test is measured in parallel 3 times.

[0026] The inhibitory effects of four coptis alkaloids on Propionibacterium acnes Figure 2 The MIC and MBC results are shown in Table 2.

[0027] Table 2: MIC and MBC (µg / ml) of four Rhizoma Coptidis alkaloids against Propionibacterium acnes

[0028] The micro-dilution method and colony counting method showed that the four coptis alkaloids had antibacterial effects on Propionibacterium acnes, but the antibacterial strengths were different. According to the analysis of plate coating counts of different drugs and different concentrations, methyl coptis alkaloids had a stronger antibacterial effect, followed by coptis alkaloids and berberine.

[0029] Example 2: Verification of the efficacy of methylcoptisine in treating acne (1) Establishing an acne animal model Adult Balb / c mice (7 weeks old) were shaved on their backs 1 day in advance and randomly divided into four groups, with 5 mice in each group: negative control group (CON), acne group (ACNE), methyl coptisine group (WOR), and erythromycin group (EM). The acne mouse model was established by intradermally injecting 100ul of Propionibacterium acnes suspension (1×10 8 CFU / ml), 20ul of artificial sebum was applied to the injection site immediately after injection. The second day after the mouse model was established, the application of the drug began. The dosage of methyl coptisine was 30 mg (0.07%) applied to the acne site on the back of each mouse, and the dosage of erythromycin was 30 mg (0.1%) applied to the back of each mouse. The negative treatment group and the acne group were treated with the same amount of Carbomer 940. The drug was applied continuously for 7 days. After the mice were killed, the acne skin tissue on the back of the mice was collected for subsequent experiments.

[0030] (2) Effects of methylcoptisine treatment on acne induced by Propionibacterium acnes Figure 3 The following are the appearance pictures of the acne sites in each group of mice after modeling. In order to verify the therapeutic effect of methyl coptisine on acne, the thickness and diameter of the acne were also measured during the treatment period ( Figure 4-5 ), and the severity of acne was scored ( Figure 6 During the treatment, the skin of mice in the acne group (ACNE) was swollen, and the swelling gradually worsened, developing into pustular acne. In contrast, the swelling of mice in the methyl coptisine group (WOR) and erythromycin group (EM) was significantly reduced on the 6th and 7th days. From the results of acne skin lesion scores, compared with the acne group (ACNE), the methyl coptisine group (WOR) and erythromycin group (EM) were significantly reduced on the 6th and 7th days, with significant differences, indicating that methyl coptisine has a therapeutic effect on acne.

[0031] (3) Pathological analysis Skin removal and fixation: The mice were killed by cervical dislocation, and the acne skin on the back of the mice was removed with sterile dissecting tools. A part of the acne skin on the back of the mice was placed in a pre-prepared fixative (4% tissue cell fixative) and fixed for 24 hours for HE staining.

[0032] Dehydration and transparency: Place the pre-fixed tissue in an embedding box, rinse with running water (to remove the fixative in the tissue) for 30 minutes, and dehydrate it in alcohol of different concentrations. Generally, alcohol from low to high concentrations is used as a dehydrating agent to gradually remove the water in the tissue block. The tissue block is then placed in xylene, a transparent agent that is soluble in both alcohol and paraffin, for transparency. Xylene is used to replace the alcohol in the tissue block before it can be immersed in wax and embedded.

[0033] Wax immersion and embedding: Place the transparent tissue block in the melted paraffin and put it into a wax melting box to keep it warm. After the paraffin is completely immersed in the tissue block, embed it and cool and solidify it into a block.

[0034] Slice, spread and bake: Fix the embedded wax block on the slicer and slice it into thin slices, usually 5-8 microns thick. The slices are often wrinkled and need to be ironed flat in heated water, then attached to the slide and dried in a 45℃ constant temperature oven.

[0035] Staining: Use xylene to remove the paraffin in the slices, stain them in a hematoxylin aqueous solution for several minutes, separate them in acid water and ammonia water for several seconds each, rinse them in running water for 1 hour, then put them in distilled water for a while, dehydrate them in 70% and 90% alcohol for 10 minutes each, and stain them in alcohol eosin staining solution for 2-3 minutes. Dehydrate the stained slices with pure alcohol, then make them transparent with xylene, drip gum on the transparent slices, and cover them with coverslips for sealing.

[0036] The effect of methyl coptisine on the acne mouse model induced by Propionibacterium acnes was investigated by using acne skin pathological sections. Acne is characterized by a large number of inflammatory cell infiltrations and vascular dilation in the dermis and subcutaneous tissues, as well as obvious thickening of the epidermis and dermis, which is a typical acne skin phenotype. Methyl coptisine can significantly reduce the number of inflammatory cell infiltrations and reduce the thickness of the epidermis and dermis ( Figure 7-8 ). In conclusion, these results suggest that methylcoppertone is an effective treatment for acne.

[0037] Discussion: The current research on Coptis chinensis alkaloids mainly focuses on the substitution of the A and D rings. The present invention confirms that methyl coptis chinensis alkaloids have stronger inhibitory activity against Propionibacterium acnes than other alkaloids, and can thus be better used in the treatment of acne. This may be related to the electrophilic substitution on the isoquinoline ring, which leads to enhanced permeability to the bacterial cell membrane and destructiveness to the cell wall. The specific mechanism needs further exploration.

Claims

1. Use of methyl coptisine in the preparation of an in vitro inhibitor of Propionibacterium acnes.

2. Use of methyl coptisine in the preparation of medicines for treating acne.

3. The use according to claim 2, characterized in that: The medicine is for external use.

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