A chemical composition containing cinnamic acid, berberine or berberine hydrochloride and its application

Abstract

The application discloses a chemical composition containing cinnamic acid, berberine or berberine hydrochloride, and relates to the technical field of medicines.The chemical composition of the application is composed of a first component and a second component, wherein the first component is cinnamic acid, a traditional Chinese medicine extract containing cinnamic acid, berberine, berberine hydrochloride or a traditional Chinese medicine extract containing berberine and berberine hydrochloride, the second component is berberine, berberine hydrochloride, a traditional Chinese medicine extract containing berberine and berberine hydrochloride, zinc pyrithione or ketoconazole, and the first component and the second component are different in composition.The composition of the application has a synergistic effect of resisting Malassezia fungi, can be used for treating skin diseases such as dandruff, seborrheic dermatitis, pityriasis versicolor and Malassezia folliculitis caused by Malassezia fungal infection, can be used in external-use medicines for driving dandruff or treating seborrheic dermatitis, pityriasis versicolor and Malassezia folliculitis, and can produce a more durable and effective antibacterial effect.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a chemical composition containing cinnamic acid, berberine or berberine hydrochloride and its application. Background Technology

[0002] Malassezia fungi ( Malassezia Malassezia spp. is a lipophilic fungus and one of the common flora on the normal skin surface. However, under certain conditions, such as changes in the individual's immune system, disruption of the skin barrier function, or environmental factors, Malassezia fungi may overgrow and cause skin problems such as dandruff, seborrheic dermatitis, tinea versicolor, and Malassezia folliculitis. The most commonly used antifungal agents in clinical treatment and daily chemical products are ketoconazole and zinc pyrithione. Recent studies have shown that the emergence of drug-resistant Malassezia strains from humans and animals may lead to ineffectiveness of imidazole drugs, including ketoconazole (CAS No.: 65277-42-1) (Leong C, Kit JCW, Lee SM, et al. Azoleresistance mechanisms in pathogenic...). M . furfur [J]. Antimicrob Agents Chemother, 2021, 65(5):e01975-20). Due to its carcinogenic, teratogenic, and mutagenic toxicity, and its harmful effects on freshwater and marine animals, zinc pyrithione (CAS No.: 13463-41-7) has been banned from use in cosmetics by the European Union since March 2022. The emergence of imidazole-resistant strains and the trend of banning zinc pyrithione due to environmental concerns have increased the necessity of finding natural alternatives or partial alternatives to antifungal chemical drugs. Current research focuses on the inhibitory effect of plant essential oils on Malassezia (Jain S, Arora P, Nainwal LM. Essential oils as potential source of anti-dandruff agents: a review[J]. Comb Chem High Throughput Screen, 2022, 25(9):1411-1426). However, plant essential oils have the following undeniable drawbacks: they can irritate the skin, are volatile and affect efficacy, and some have unpleasant odors, all of which affect the practical application of plant essential oils in dandruff removal products.

[0003] Cinnamic acid (CAS No.: 140-10-3), also known as β-phenylacrylic acid or 3-phenyl-2-acrylic acid, is an organic compound. Its chemical formula is C9H8O2. This compound is usually isolated from cinnamon bark, cassia twigs, or benzoin, but can also be synthesized artificially. In the pharmaceutical industry, cinnamic acid is used to synthesize important drugs for treating coronary heart disease, such as lactic acid and nifedipine, as well as chlorophenylbutyric acid and cinnamylpiperazine. Furthermore, cinnamic acid has a significant inhibitory effect on the proliferation of lung adenocarcinoma cells and can be used as a component of anticancer drugs. It can also be used as a local anesthetic, bactericide, and hemostatic agent. In the food industry, cinnamic acid is mainly used as a flavoring agent, food additive, and preservative. It can be used as a flavoring agent to add aroma to food and beverages and extend the shelf life of food. In addition, cinnamic acid has been found to inhibit the formation of melanin tyrosinase and has a certain absorption effect on ultraviolet light, therefore it is added to cosmetics such as sunscreens. Literature reports that cinnamic acid has antifungal activity against Malassezia fungi (Baroni A, De Rosa R, De Rosa A, et al. New strategies in dandruff treatment: growth control of Malassezia ovalis[J].Dermatology. 2000, 201(4):332-336).

[0004] Berberine (CAS No.: 2086-83-1), also known as berberine, has the chemical formula C. 20 H 18 NO4. Berberine hydrochloride (CAS No.: 633-65-8) is the hydrochloride salt of berberine, also known as berberine hydrochloride or berberine hydrochloride, with the chemical formula C. 20 H 18 ClNO4. Berberine and berberine hydrochloride are currently commonly used as antibacterial drugs, mainly for the treatment of gastroenteritis, bacterial dysentery and other intestinal infections caused by susceptible pathogens. Studies have reported that berberine and berberine hydrochloride have certain inhibitory activity against Malassezia (Zhang Zhihong, Sun Bo, Jin Yipeng, et al. In vitro drug sensitivity test of 17 active ingredients of traditional Chinese medicine against Malassezia canis [J]. Chinese Journal of Veterinary Medicine, 2010, 46(3):73-74.).

[0005] Although existing technologies have disclosed that cinnamic acid, berberine, and berberine hydrochloride have certain inhibitory activity against Malassezia fungi, none of them involve research on their combination, so their therapeutic value cannot be determined. Summary of the Invention

[0006] To address the above shortcomings, this invention provides a chemical composition containing cinnamic acid, berberine, or berberine hydrochloride, and its application. This composition can inhibit Malassezia, not only reducing the amount of antibacterial agent required but also producing an effective and long-lasting antibacterial effect. The specific technical solution is as follows: A chemical composition containing cinnamic acid, berberine, or berberine hydrochloride, comprising a first component and a second component, wherein the first component is cinnamic acid, a traditional Chinese medicine extract containing cinnamic acid, berberine, berberine hydrochloride, or a traditional Chinese medicine extract containing berberine and berberine hydrochloride, and the second component is berberine, berberine hydrochloride, a traditional Chinese medicine extract containing berberine and berberine hydrochloride, zinc pyrithione, or ketoconazole, wherein the first component and the second component have different compositions.

[0007] Preferably, when the first component is cinnamic acid and the second component is berberine, berberine hydrochloride, or a traditional Chinese medicine extract containing berberine and berberine hydrochloride, the weight ratio of the first component to the second component is 1:(16-32).

[0008] Preferably, when the first component is a traditional Chinese medicine extract containing cinnamic acid and the second component is berberine, berberine hydrochloride, or a traditional Chinese medicine extract containing berberine and berberine hydrochloride, the weight ratio of the first component to the second component is 1:(2-4).

[0009] Preferably, when the first component is cinnamic acid, a traditional Chinese medicine extract containing cinnamic acid, berberine, berberine hydrochloride, or a traditional Chinese medicine extract containing berberine and berberine hydrochloride, and the second component is zinc pyrithione, the weight ratio of the first component to the second component is (2.5-80):1.

[0010] Preferably, when the first component is cinnamic acid, a traditional Chinese medicine extract containing cinnamic acid, berberine, berberine hydrochloride, or a traditional Chinese medicine extract containing berberine and berberine hydrochloride, and the second component is ketoconazole, the weight ratio of the first component to the second component is (20-641):1.

[0011] Preferably, the traditional Chinese medicine containing cinnamic acid is cinnamon, cinnamon twig, cinnamon leaf, clove, or benzoin.

[0012] Preferably, the traditional Chinese medicine containing berberine and berberine hydrochloride is Coptis chinensis, Phellodendron chinense, three needles, or Mahonia fortunei.

[0013] Preferably, the herbal extract is an ethanol extract with an ethanol concentration of 1%-100%.

[0014] The present invention also provides the use of the above-described chemical composition in products for the prevention or treatment of skin diseases caused by Malassezia fungal infections.

[0015] Preferably, the skin diseases include dandruff, seborrheic dermatitis, tinea versicolor, and Malassezia folliculitis caused by Malassezia fungal infection; the product is a topical medicine.

[0016] The composition of the present invention exhibits good inhibitory effects on Malassezia fungi when its mass percentage in the drug ranges from 0.001% to 99%.

[0017] Compared with the prior art, the beneficial effects of the present invention are: The composition of this invention is a non-volatile chemical composition with no unpleasant odor, good safety for the skin, and synergistic antifungal activity against Malassezia fungi. It can be used to treat skin diseases such as dandruff, seborrheic dermatitis, tinea versicolor, and Malassezia folliculitis caused by Malassezia fungal infection. It can be used in topical medicines for treating dandruff, seborrheic dermatitis, tinea versicolor, and Malassezia folliculitis. Detailed Implementation

[0018] The specific embodiments of the present invention will be described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.

[0019] Example 1: Determination of the minimum bactericidal concentration (MBC) of each component in the composition. (1) Prepare 1.50×10⁻⁶ saline solution. 8 CFU / mL of Malassezia furfur ( Malassezia furfur (MF) suspension, accurately pipette an appropriate amount of the suspension, and dilute it 1500 times with liquid culture medium to obtain a concentration of 0.10 × 10⁻⁶. 6 A fungal suspension containing CFU / mL.

[0020] (2) The stock solutions of cinnamon ethanol extract (70% ethanol extract of the remaining medicinal material residue after volatile oil extraction of cinnamon), Coptis chinensis ethanol extract (70% ethanol extract of Coptis chinensis), cinnamic acid, berberine, berberine hydrochloride, ketoconazole and zinc pyrithione (prepared by dissolving in 50% DMSO, concentration of 50.00 mg / mL) were diluted with liquid culture medium to prepare sample solutions with concentrations of 10.00, 10.00, 10.00, 10.00, 10.00, 0.25 and 0.25 mg / mL, respectively. In addition, a 10.00% DMSO solution was prepared with liquid culture medium as a blank control solution for later use.

[0021] (3) The checkerboard microdilution method was used to determine MBC. 100 L of sample solution and fungal suspension of different concentrations were added to the drug treatment group, and 100 L of blank control solution and fungal suspension were added to the fungal control group. The final concentration of MF in each well was 0.05 × 10⁻⁶. 6 The final concentration of DMSO was 5.00% (CFU / mL). Three replicates were set up for each concentration of each sample, and the samples were incubated at 30°C for 24 h after addition. One culture dish was used for each concentration, and the solid culture medium in the dish was divided into four equal sectors and numbered: sectors 1-3 were for parallel drug administration, and sector 4 was for fungal control. The culture medium in the 96-well plate was thoroughly mixed, and 10 μL was precisely pipetted into each well and placed into the numbered solid culture medium. The mixture was spread evenly with an inoculation loop and incubated at 30°C for 24 h. Fungal growth was observed. If fungal control sector showed MF growth, while no MF growth was observed in any of the three sectors of the drug administration group, that concentration was considered the MBC of the sample.

[0022] (4) Results of MBC measurement: MF colonies were present on the control fan of each culture dish, indicating that the inoculation concentration of MF was appropriate. The order of MBC size for each sample was: Coptis chinensis ethanol extract (5000.00 μg / mL) > berberine = berberine hydrochloride (2500.00 μg / mL) > cinnamon ethanol extract (1250.00 μg / mL) > cinnamic acid (156.25 μg / mL) > zinc pyrithione (62.50 μg / mL) > ketoconazole (7.81 μg / mL). Therefore, the order of inhibitory activity against MF was: ketoconazole > zinc pyrithione > cinnamic acid > cinnamon ethanol extract > berberine = berberine hydrochloride > Coptis chinensis ethanol extract. The MBC detection results of the samples are shown in Table 1.

[0023] Table 1. MBC test results for each sample ( n = 3) Note: - indicates no MF growth, + indicates MF growth.

[0024] Example 2: Determination of the combined bactericidal concentration index (FBCI) (1) Based on the MBC results, the combined minimum bactericidal concentration (CMBC) was determined using a 96-well plate culture combined with solid medium inoculation method. Each sample solution was mixed in pairs and added to a 96-well plate. The sample addition and subsequent processing methods were the same as those in section "1". Finally, the combined bactericidal effect was evaluated using FBCI, calculated as follows: FBCI = MBC A联用 / MBC A单用 + MBC B联用 / MBC B单用 In the above formula, A and B are two different fungicides respectively, MBC A单用 and MBC B单用 are the MBC values of A and B when used alone, respectively. MBC A联用 is the CMBC value of A when A and B are used in combination, and MBC B联用 is the CMBC value of B when A and B are used in combination. Antagonism occurs when FBCI > 2; unrelated effect when 1 < FBCI ≤ 2; additive effect when 0.5 < FBCI ≤ 1; synergistic effect when FBCI ≤ 0.5.

[0025] (2) Determination results of FBCI: The FBCI detection results of the samples are shown in Table 2: Table 2 FBCI test results ( n = 3) It can be seen from the experimental results that cinnamic acid has synergistic antibacterial effect with Coptis chinensis ethanol extract, berberine and berberine hydrochloride, and all FBCI values are 0.5; Cinnamon ethanol extract has synergistic antibacterial effect with Coptis chinensis ethanol extract, berberine and berberine hydrochloride, and all FBCI values are 0.5; Coptis chinensis ethanol extract, cinnamon ethanol extract, cinnamic acid, berberine and berberine hydrochloride all have synergistic antibacterial effects with the positive drugs ketoconazole and zinc pyrithione, and all FBCI values are 0.5. In clinical practice, synergistic antibacterial effect is an important means to avoid the generation of drug-resistant bacteria. It can be seen that the combined use of the present invention can not only reduce the dosage of antibacterial agents, but also avoid the generation of drug-resistant bacteria, thereby producing a more durable and effective antibacterial effect, and the antibacterial effect is obviously better than that of using antibacterial agents alone. Compared with ketoconazole, the combination medication of the present invention can reduce the generation of drug resistance; compared with zinc pyrithione, the combination medication of the present invention can not only reduce the generation of drug resistance, but also reduce the dosage of zinc pyrithione, which can relatively reduce the possible adverse consequences.

[0026] Example 3 Cytotoxicity test on human immortalized keratinocytes (HaCaT) (1) Accurately pipette an appropriate amount of the stock solution of each sample and dilute it appropriately with liquid culture medium. The final concentrations of HaCaT administered to each sample were 10.00, 20.00, 40.00, 80.00, 160.00, and 200.00 μg / mL, respectively. The concentration of DMSO in the 200.00 µg / mL sample solution was 0.40%. HaCaT was cultured in DMEN medium containing 10% FBS and 1% penicillin-streptomycin at 37 ℃ in a 5% CO2 incubator. HaCaT samples in good logarithmic growth phase were then cultured at 2 × 10⁻⁶ cells / mL. 4 Cells were seeded per well in a 96-well plate and cultured for 12 h. 100 μL of each sample solution was precisely added. A control group (containing 0.40% DMSO) and a blank control group (containing no DMSO) were also set up, with the culture medium serving as the blank control. Each group was configured in triplicate. After 24 h of cell treatment, the OD value was measured at 450 nm using the CCK-8 assay to calculate the inhibition rate. The half-maximal inhibitory rate (IC50) was calculated using SPSS 23 statistical software. 50 The inhibition rate is calculated using the following formula: Inhibition rate (%) = [1 - (OD administration - OD blank control) / (OD cell control - OD blank control)] × 100% (2) Results of cytotoxicity assays against HaCaT The cell control group contained 0.40% DMSO, and its OD value was not significantly different from that of the blank cell group without DMSO. F = 5.73, P >0.05), indicating that 0.40% DMSO had no significant effect on the growth of HaCaT. Within the concentration range of 10.00~200.00 μg / mL, the ethanol extracts of cinnamon, ethanol extracts of coptis, and cinnamic acid showed such weak inhibitory activity that the IC50 could not be calculated. 50 Zinc pyrithione, however, has such strong inhibitory activity that its IC50 cannot be calculated. 50 The order of cytotoxicity of each sample to HaCaT cells is as follows: zinc pyrithione (IC50) 50 <10.00 μg / mL)> Berberine hydrochloride (IC50) 50 = 27.83 ± 0.66 μg / mL) > Ketoconazole (IC50) 50 = 28.22 ± 0.14 μg / mL) > Berberine (IC50) 50 =73.11 ± 1.61 μg / mL) > Coptis chinensis ethanol extract (IC50) 50 >200.00 μg / mL) >Cinnamon ethanol extract (IC50) 50 >200.00 μg / mL) >Cinnamic acid (IC50) 50>200.00 μg / mL).

[0027] The results showed that compared to existing drugs (zinc pyrithione and ketoconazole), berberine, ethanol extract of Coptis chinensis, ethanol extract of cinnamon, and cinnamic acid had lower toxicity and higher safety to human skin keratinocytes. Furthermore, compared to existing drugs, zinc pyrithione and ketoconazole, when combined with other components, could reduce their dosage and overall lower toxicity. Berberine hydrochloride had toxicity to human skin keratinocytes comparable to ketoconazole.

[0028] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A composition for preventing and treating infections caused by Malassezia fungi, characterized in that, The composition consists of cinnamic acid and berberine hydrochloride in a weight ratio of 1:

16.

2. The use of the composition of claim 1 in the preparation of a product for the prevention or treatment of skin diseases caused by Malassezia fungal infections.

3. The application according to claim 2, characterized in that, The skin diseases mentioned include dandruff, seborrheic dermatitis, tinea versicolor, and Malassezia folliculitis caused by Malassezia fungal infections; the product mentioned is a topical medication.

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