A synergistic antibacterial and antifungal pharmaceutical composition containing natural products osthol and berberine

Abstract

This invention discloses a pharmaceutical composition containing the natural products osthol and berberine, exhibiting synergistic antibacterial and antifungal effects. The effectiveness of osthol in synergistically enhancing the antibacterial activity of berberine was demonstrated through checkerboard minimum inhibitory concentration (MIC) tests, antifungal activity experiments, and in vitro bacterial growth curves. This invention provides a novel application of the combined use of osthol and berberine in antibacterial and antifungal treatments. This composition belongs to the category of traditional Chinese medicine compound preparations, offering a new treatment strategy for the clinical treatment of bacterial and fungal infectious diseases.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to a natural pharmaceutical composition containing berberine and osthol, the pharmaceutical composition having significant synergistic antibacterial and antifungal effects, and the application of the composition in the preparation of drugs for antibacterial and antifungal infections. Background Technology

[0002] Antimicrobial resistance has become a global public health issue. Especially in recent years, the resistance rates to some antimicrobial drugs have been rising continuously, gradually becoming a new global public health crisis. Developing novel antimicrobial drugs has become one of the important ways to curb bacterial resistance in clinical practice. Furthermore, infectious diseases in clinical practice are often complex infections, meaning that bacterial infections are often accompanied by fungal infections. Current antibiotic therapies often only target a single bacterial infection or a single fungal infection, failing to simultaneously treat both bacteria and fungi.

[0003] Plants contain a wide variety of active ingredients from diverse sources. Based on their effective components, they can be classified into alkaloids, organic acids, volatile oils, flavonoids, polyphenols, polysaccharides, saponins, tannins, etc. These substances may have antibacterial, bactericidal, and antioxidant effects. In addition, traditional Chinese medicine often has multi-target characteristics and often exhibits certain biological activities against different pathogens. Discovering antibacterial active substances from plants and developing antibacterial drugs has become a current research hotspot in this field.

[0004] Berberine, also known as berberine alkaloid, was originally extracted from the traditional Chinese medicine Ligusticum chuanxiong. Currently, it can be extracted from various herbal plants or synthesized artificially. Its chemical structure is tetramethylpyrazine. It has been proven to have a variety of biological functions, including anti-inflammatory, antioxidant, antimicrobial, immunomodulatory, blood sugar control, and cardiovascular protection. Clinically, it is often used to treat diarrhea, diabetes, and cardiovascular diseases. Berberine has a good effect on the treatment of infectious diarrhea. Clinically, it is mainly used in the form of berberine hydrochloride or berberine sulfate. Meanwhile, some in vitro studies have found that berberine also has certain antibacterial activity, with weaker antibacterial activity against Gram-negative bacteria (MIC) ranging from 512 to 1024 μg / mL, and stronger antibacterial activity against Gram-positive bacteria (MIC usually between 128 and 512 μg / mL) (Xia Shuai, Ma Liyan, Xie Miaorong. Study on in vitro antibacterial activity of berberine hydrochloride against Staphylococcus aureus [J]. Journal of Clinical and Experimental Medicine, 2022, 21(07):673-678); In addition, berberine also has certain therapeutic effects on fungal infections, such as Studies have found that berberine has certain activity against Candida albicans, with a MIC generally between 64-128 μg / mL (Yong Jiangyan, Wang Hai, Huang Xiaoxue, et al. Effect of berberine hydrochloride combined with fluconazole on calcium homeostasis of drug-resistant Candida albicans [J]. Chinese Journal of Pathogenic Biology, 2020, 15(08):903-909), and the MIC range of activity against Cryptococcus neoformans is between 8-168 μg / mL (Xu Jialong, Song Haolei, Chen Xiaoqin, et al. Activity and mechanism of action of berberine against Cryptococcus neoformans [J]. Acta Microbiologica Sinica, 2023, 63(04):1541-1550.).

[0005] Osthol, molecular formula: C 15 H 16 O3, also known as methoxyparsin or parsleyol methyl ether, is an important member of the total coumarins of Cnidium monnieri. Studies have found that osthol has strong anti-inflammatory and antioxidant activities. In vitro antibacterial activity studies of osthol have found that it has weak antibacterial activity, with a MIC greater than 1024 μg / mL against Escherichia coli and a MIC of 512 μg / mL against Staphylococcus aureus (Wu Xuejiao, Yan Boyu, Zhou Xurong, et al. Synthesis and antibacterial activity of osthol ether derivatives [J]. Chemical Research and Application, 2022, 34(07):1532-1537). Studies have found that osthol can inhibit pruritus induced by histamine and histamine receptor H1 agonist HTMT and histamine H4 receptor agonist VUF430, but has no significant inhibitory effect on pruritus induced by high doses of chloroquine. Its mechanism of action is related to the inhibition of TRPV1 channel (Yang Niu Niu. Study on TRPV1 participation in pruritus formation and the antipruritic mechanism of osthol [D]. Nanjing University of Chinese Medicine, Doctoral Dissertation, 2017). Summary of the Invention

[0006] One object of the present invention is to provide the use of osthol in the preparation of berberine antibacterial synergists.

[0007] The application is as follows: the use of osthol in the preparation of products that enhance the antibacterial and antifungal activities of berberine.

[0008] Another object of the present invention is to provide a natural pharmaceutical composition containing berberine and osthol.

[0009] In the natural pharmaceutical composition containing berberine and osthol provided by the present invention, the mass ratio of berberine to osthol is 0.9:(0.25-16), which is equivalent to a mass ratio of berberine hydrochloride to osthol of 1:(0.25-16).

[0010] Furthermore, the berberine is berberine or a pharmaceutically acceptable salt thereof, specifically berberine hydrochloride.

[0011] Furthermore, the active ingredients in the composition may be only osthol and berberine; or may further include other active ingredients.

[0012] Another object of the present invention is to provide the use of the above-described natural pharmaceutical composition containing berberine and osthol in the preparation of antibacterial and antifungal drugs.

[0013] Furthermore, the drug is an antibacterial and antifungal drug.

[0014] Furthermore, the fungus includes Candida albicans.

[0015] Furthermore, the bacteria include Gram-positive bacteria.

[0016] Furthermore, the Gram-positive bacteria include Staphylococcus aureus, Staphylococcus pseudointermediate, and Clostridium perfringens.

[0017] Further, the bacteria are Staphylococcus aureus; preferably, the Staphylococcus aureus includes methicillin-resistant Staphylococcus aureus.

[0018] The present invention also provides an antibacterial composition.

[0019] The antibacterial composition provided by the present invention comprises osthol and berberine as its active ingredients.

[0020] Furthermore, the mass ratio of berberine to osthol is 0.9:(0.25-16), which is equivalent to a mass ratio of berberine hydrochloride to osthol of 1:(0.25-16).

[0021] The active ingredients of the antibacterial composition may be only osthol and berberine; or may further include other active ingredients, which can be determined by those skilled in the art based on the antibacterial effect.

[0022] The above-mentioned antibacterial composition can kill and / or inhibit bacteria and fungi;

[0023] Furthermore, the fungus includes Candida albicans.

[0024] Furthermore, the bacteria include Gram-positive bacteria.

[0025] Furthermore, the Gram-positive bacteria include Staphylococcus aureus, Staphylococcus pseudointermediate, and Clostridium perfringens.

[0026] Further, the bacteria are Staphylococcus aureus; preferably, the Staphylococcus aureus includes methicillin-resistant Staphylococcus aureus.

[0027] The dosage form of the antibacterial composition is one of the following: tablets, ointments, creams, capsules, sustained-release tablets, controlled-release tablets, oral liquids, syrups, injections, pellets, or lyophilized powder for injection.

[0028] Furthermore, antibacterial products containing the above-mentioned antibacterial composition are also within the scope of protection of this invention.

[0029] The dosage form of the above-mentioned antibacterial products may be any one of the following: cream, ointment, tablet, suspension, capsule, sustained-release tablet, controlled-release tablet, oral liquid, syrup, injection dosage form, drop pill, or lyophilized powder injection dosage form.

[0030] To address the problem of drug resistance in multidrug-resistant Gram-positive bacteria and the lack of effective drugs, this invention provides a natural product composition with significant synergistic antibacterial effects, specifically using osthol in combination with berberine. The two are not simply additive in function, but achieve synergistic antibacterial effects.

[0031] This invention demonstrates, through checkerboard minimum inhibitory concentration (MIC) tests, in vitro bacterial growth curves, and animal experiments, that the combination of osthol and berberine exhibits significant synergistic antibacterial and antifungal activity. The bacteria tested included Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and pseudostaphylococcus intermedia from canine and feline sources; the fungi tested included Candida albicans.

[0032] This invention opens up a new application for the combined use of osthol and berberine, namely, the combination has significant synergistic antibacterial and antifungal activity, and has important clinical application value in the treatment of multidrug-resistant Gram-positive bacterial infections and fungal infections.

[0033] This invention demonstrates that berberine combined with osthol exhibits significant synergistic antifungal and antibacterial effects, and that the combined use of the two drugs results in a marked synergistic effect. Furthermore, this combination is a pure traditional Chinese medicine formula, possessing high safety and a low likelihood of inducing drug resistance. It simultaneously combines antibacterial and antifungal activities, demonstrating significant advantages over currently available antibacterial or antifungal drugs, thus possessing important application value. Attached Figure Description

[0034] Figure 1 This is the result of the checkerboard method for the synergistic antibacterial effect of berberine combined with osthole against multiple strains of Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and pseudo-intermediate Staphylococcus in Example 1 of the present invention.

[0035] Figure 2 This is the checkerboard method result of the synergistic antibacterial effect of berberine combined with osthole against Candida albicans ATCC12301 in Example 2 of the present invention.

[0036] Figure 3 This is the synergistic bactericidal effect of berberine combined with osthole on MRSA33591 strain in Example 3 of the present invention.

[0037] Figure 4 This is the synergistic bactericidal result of berberine combined with osthole against clinically proven Staphylococcus pseudointermediate strain 31 in Example 4 of the present invention. Detailed Implementation

[0038] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0039] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0040] The osthol used in the following experiments was purchased from Chengdu Ruifensi Company, with a purity ≥99%. The berberine used in the following experiments was berberine hydrochloride, CAS number: 633-65-8, molecular formula: C20H18ClNO4, molecular weight: 372.82, purchased from Aladdin Reagent Company, with a purity ≥98%.

[0041] The pathogens used in the following experiments:

[0042] Staphylococcus aureus ATCC29213 was purchased from the China Institute of Veterinary Drug Control's Culture Collection Center.

[0043] Methicillin-resistant Staphylococcus aureus ATCC33591 (MRSA33591) was provided by the National Center for Veterinary Drug Safety Evaluation (Beijing).

[0044] The strains of *Staphylococcus intermedia* 31A, *Staphylococcus intermedia* 22-2, *Staphylococcus intermedia* I1847, *Staphylococcus intermedia* U5149, and *Staphylococcus intermedia* K3247-1 were preserved by the National Veterinary Drug Safety Evaluation Center of China Agricultural University.

[0045] The standard cytotoxic strain of Candida albicans, ATCC12301, was purchased from Shanghai Fuxiang Biotechnology Co., Ltd.

[0046] Staphylococcus aureus 02016 was preserved from the National Veterinary Drug Safety Evaluation Center of China Agricultural University.

[0047] Staphylococcus aureus 65577 was preserved from the National Veterinary Drug Safety Evaluation Center of China Agricultural University.

[0048] Staphylococcus aureus USA300 was provided by Professor Jianfeng Wang's research group at Jilin University (Liu Y, Shi D, Guo Y, Li M, Zha Y, Wang Q, Wang J. Drocorhodin Perochlorate Attenuates Staphylococcus aureus USA300 virulence by decreasing α-toxin expression. World Journal of Microbiol and Biotechnol).

[0049] 2017;33(1):17).

[0050] The Clostridium perfringens standard strain ATCC13124 was purchased from the American Culture Collection.

[0051] Clostridium perfringens strains 16, 12, 40, 26, and 38 were all preserved by the National Veterinary Drug Safety Evaluation Center (Beijing) of China Agricultural University.

[0052] Bacterial culture medium was used in the following experiments:

[0053] YPD medium: Weigh 20g glucose, 10g yeast extract, and 20g trypsin using an electronic balance, then add 800mL deionized water. Dissolve the solids completely on a magnetic stirrer, then bring the volume to 1L with deionized water and dispense into Erlenmeyer flasks. If preparing solid medium, add 2% agar as needed, and autoclave at 120℃ for 30 minutes. Then, store at room temperature for later use.

[0054] MHB broth culture medium was purchased from Beijing Luqiao Technology Co., Ltd., and the preparation method is as follows: weigh 25.0g into 1L of distilled water, heat to boiling until completely dissolved, autoclave at 121℃ for 15min, and set aside.

[0055] Liquid thioglycolate (FTG) composition (g / L): tryptone 15.0 g / L, yeast extract 5.0 g / L, sodium thioglycolate 0.5 g / L, glucose 5.0 g / L, sodium chloride 2.5 g / L, L-cysteine ​​0.5 g / L, resazurin 0.001 g / L, agar 0.75 g / L.

[0056] MHA culture medium was purchased from Beijing Luqiao Technology Co., Ltd., and the preparation method is as follows: weigh 38.0g into 1L of distilled water, heat to boiling until completely dissolved, autoclave at 121℃ for 15min, cool to 55℃ and pour into plates for later use.

[0057] Brain-Heart Infusion Culture Medium (BHI) was purchased from Beijing Luqiao Technology Co., Ltd., and the preparation method is as follows: Weigh 38.5g of this product, heat and stir to dissolve in 1000mL of distilled water, adjust the pH to 7.3, autoclave at 121℃ for 15 minutes, and set aside.

[0058] BHI solid culture medium was purchased from Beijing Luqiao Technology Co., Ltd., and the preparation method is as follows: Weigh 50.0g of this product into 1000mL of distilled water, heat to boiling until completely dissolved, autoclave at 121℃ for 20min, cool to 55℃ and pour into plates for later use.

[0059] Example 1: Evaluation of the antibacterial effect of combined use of osthol and berberine

[0060] Determination of the combined drug index (FICI) of osthol and berberine against pathogens: The checkerboard method was used to determine the combined antibacterial effects of osthol and berberine against Staphylococcus aureus ATCC29213, methicillin-resistant Staphylococcus aureus ATCC33591 (MRSA33591), Staphylococcus pseudointermediate 31A, Staphylococcus pseudointermediate 22-2, Staphylococcus pseudointermediate I1847, Staphylococcus pseudointermediate U5149, Staphylococcus pseudointermediate K3247-1, Staphylococcus aureus O2016, Staphylococcus aureus 65577, Staphylococcus aureus USA300, Clostridium perfringens 16, Clostridium perfringens 12, Clostridium perfringens 40, Clostridium perfringens 26, and Clostridium perfringens 38.

[0061] Specifically, strains such as Staphylococcus aureus ATCC29213, methicillin-resistant Staphylococcus aureus ATCC33591 (MRSA33591), Staphylococcus pseudointermediate 31A, Staphylococcus pseudointermediate 22-2, Staphylococcus pseudointermediate I1847, Staphylococcus pseudointermediate U5149, Staphylococcus pseudointermediate K3247-1, Staphylococcus aureus 02016, Staphylococcus aureus 65577, and Staphylococcus aureus USA300 were cultured normally using MHB broth medium. The checkerboard method is operated as follows: Osthol (as drug A) and berberine (as drug B) are serially diluted with MHB broth medium at maximum concentrations of 160 μg / mL and 256 μg / mL, respectively. 50 μL of MHB broth medium containing different concentrations of the two drugs is added along the horizontal and vertical axes of a 96-well microplate. Then, 50 μL of various pathogenic bacterial suspensions are added to each well, resulting in a final bacterial count of 2 × 10⁶ bacteria per well. 5 CFU was incubated at 37℃ for 18–24 h, and the results were observed. The minimum inhibitory concentrations (MICs) of the two drugs, used alone and in combination, were recorded, and the FICI values ​​(partial inhibitory concentration index) were calculated using the following formula.

[0062] Clostridium perfringens ATCC13124, Clostridium perfringens 16, Clostridium perfringens 12, Clostridium perfringens 40, Clostridium perfringens 26, and Clostridium perfringens 38 were streaked onto HA plates containing 5% defibrinated sheep blood and cultured in liquid thioglycolate (FTG) medium (pH = 7.1 ± 0.2) under a balanced gas environment containing 10% hydrogen, 10% carbon dioxide, and nitrogen. The checkerboard method was performed as follows: Osthol (drug A) and berberine (drug B) were serially diluted with MHB broth at maximum concentrations of 128 μg / mL and 128 μg / mL, respectively. 50 μL of MHB broth containing different concentrations of the two drugs was added along the horizontal and vertical axes of a 96-well microplate, respectively. Then, 50 μL of each pathogenic bacterial suspension was added to each well, resulting in a final bacterial count of 2 × 10⁶ bacteria per well.5 CFU was incubated at 37℃ for 24 hours, and the results were observed. The minimum inhibitory concentrations (MICs) of the two drugs, used alone and in combination, were recorded, and the FICI values ​​(partial inhibitory concentration index) were calculated according to the following formula.

[0063] FICI = MIC (Mutual Intake of Drug A in combination) / MIC (Mutual Intake of Drug A alone) + MIC (Mutual Intake of Drug B in combination) / MIC (Mutual Intake of Drug B alone). Judgment criteria: FICI ≤ 0.5, synergistic effect; 0.5 < FICI ≤ 1, additive effect; 1 < FICI ≤ 2, no effect; FICI > 2, antagonistic effect. That is: FICI of osthol and berberine = MIC (berberine in combination) / MIC (berberine alone) + MIC (osthol in combination) / MIC (osthol alone). The combined antibacterial results are as follows... Figure 1 As shown:

[0064] like Figure 1 As shown in Figure A, for Clostridium perfringens strain ATCC13124, the MICs of osthol and berberine used alone were >128 μg / mL and 64 μg / mL, respectively. After combined use, according to the optimal ratio for synergistic effect calculated by the checkerboard method, the MICs of osthol and berberine decreased to 16 μg / mL and 8 μg / mL, respectively (the optimal ratio of osthol and berberine (referring to berberine hydrochloride) is 2:1), and the synergistic index FICI was 0.1875.

[0065] like Figure 1 As shown in Figure B, for Clostridium perfringens 16, the MICs of osthol and berberine used alone were >128 μg / mL and 32 μg / mL, respectively. After combined use, according to the optimal ratio for synergistic effect calculated by the checkerboard method, the MICs of osthol and berberine decreased to 32 μg / mL and 4 μg / mL, respectively (the optimal ratio of osthol and berberine (referring to berberine hydrochloride) is 8:1), and the synergistic index FICI was 0.25.

[0066] like Figure 1 As shown in Figure C, for Clostridium perfringens 12, the MICs of osthol and berberine used alone were >128 μg / mL and 32 μg / mL, respectively. After combined use, according to the optimal ratio for synergistic effect calculated by the checkerboard method, the MICs of osthol and berberine decreased to 16 μg / mL and 4 μg / mL, respectively (the optimal ratio of osthol and berberine (referring to berberine hydrochloride) is 4:1), and the synergistic index FICI was 0.187.

[0067] like Figure 1As shown in Figure D, for Clostridium perfringens 40, the MICs of osthol and berberine used alone were >128 μg / mL and 16 μg / mL, respectively. After combined use, according to the optimal ratio for synergistic effect calculated by the checkerboard method, the MICs of osthol and berberine decreased to 32 μg / mL and 2 μg / mL, respectively (the optimal ratio of osthol and berberine (referring to berberine hydrochloride) is 16:1), and the synergistic index FICI was 0.25.

[0068] like Figure 1 As shown in Figure E, for Clostridium perfringens 26, the MICs of osthol and berberine used alone were >128 μg / mL and 32 μg / mL, respectively. After combined use, according to the optimal ratio for synergistic effect calculated by the checkerboard method, the MICs of osthol and berberine decreased to 32 μg / mL and 4 μg / mL, respectively (the optimal ratio of osthol and berberine (referring to berberine hydrochloride) is 8:1), and the synergistic index FICI was 0.25.

[0069] like Figure 1 As shown in Figure F, for Clostridium perfringens 38, the MICs of osthol and berberine used alone were >128 μg / mL and 32 μg / mL, respectively. After combined use, according to the optimal ratio for synergistic effect calculated by the checkerboard method, the MICs of osthol and berberine decreased to 32 μg / mL and 2 μg / mL, respectively (the optimal ratio of osthol and berberine (referring to berberine hydrochloride) is 16:1), and the synergistic index FICI was 0.187.

[0070] For Clostridium perfringens, when the two are used in combination, the optimal ratio of osthol to berberine (referring to berberine hydrochloride) is 2-16:1;

[0071] like Figure 1 As shown in Figure G, for Staphylococcus aureus ATCC29213, the MICs of osthol and berberine used alone were >160 μg / mL and 64 μg / mL, respectively. After combined use, according to the optimal ratio for synergistic effect calculated by the checkerboard method, the MICs of osthol and berberine decreased to 10 μg / mL and 8 μg / mL, respectively (the optimal ratio of osthol and berberine (referring to berberine hydrochloride) is 1.25:1), and the synergistic index FICI was 0.156.

[0072] like Figure 1As shown in Figure H, against methicillin-resistant Staphylococcus aureus ATCC33591 (MRSA33591), the MICs of osthol and berberine used alone were >160 μg / mL and >256 μg / mL, respectively. Calculated using the checkerboard method to obtain the optimal ratio for synergistic effect, the MICs of osthol and berberine decreased to 20 μg / mL and 8 μg / mL, respectively (the optimal ratio of osthol to berberine (referring to berberine hydrochloride) is 2.5:1), with a synergistic index (FICI) of 0.078.

[0073] like Figure 1 As shown in Figure I, the MICs of osthol and berberine used alone against *Staphylococcus intermedia* 31A were >160 μg / mL and 128 μg / mL, respectively. Calculated using the checkerboard method to obtain the optimal ratio for synergistic effect, the MICs of osthol and berberine decreased to 10 μg / mL and 8 μg / mL, respectively (the optimal ratio of osthol to berberine (referring to berberine hydrochloride) is 1.25:1), with a synergistic index (FICI) of 0.093.

[0074] like Figure 1 As shown in Figure J, for *Staphylococcus intermedia* 22-2, the MICs of osthol and berberine used alone were >160 μg / mL and 64 μg / mL, respectively. Calculated according to the optimal ratio for synergistic effect using the checkerboard method, the MICs of osthol and berberine decreased to 20 μg / mL and 8 μg / mL, respectively (the optimal ratio of osthol to berberine (referring to berberine hydrochloride) is 2.5:1), and the synergistic index FICI was 0.1875.

[0075] like Figure 1 As shown in Figure K, for *Staphylococcus intermedia* I1847, the MICs of osthol and berberine used alone were >160 μg / mL and 64 μg / mL, respectively. Calculated according to the optimal ratio for synergistic effect using the checkerboard method, the MICs of osthol and berberine decreased to 10 μg / mL and 8 μg / mL, respectively (the optimal ratio of osthol to berberine (referring to berberine hydrochloride) is 1.25:1), with a synergistic index (FICI) of 0.156.

[0076] like Figure 1 As shown in Figure L, for *Staphylococcus intermedia* U5149, the MICs of osthol and berberine used alone were >160 μg / mL and 128 μg / mL, respectively. Calculated according to the optimal ratio for synergistic effect using the checkerboard method, the MICs of osthol and berberine were reduced to 10 μg / mL and 8 μg / mL, respectively (the optimal ratio of osthol to berberine (referring to berberine hydrochloride) is 1.25:1), with a synergistic index (FICI) of 0.093.

[0077] like Figure 1As shown in Figure M, for *Staphylococcus intermedia* K3247-1, the MICs of osthol and berberine used alone were >160 μg / mL and 128 μg / mL, respectively. Calculated according to the optimal ratio for synergistic effect using the checkerboard method, the MICs of osthol and berberine were reduced to 20 μg / mL and 8 μg / mL, respectively (the optimal ratio of osthol to berberine (referring to berberine hydrochloride) is 2.5:1), and the synergistic index FICI was 0.125.

[0078] like Figure 1 As shown in Figure N, for Staphylococcus aureus 02016, the MICs of osthol and berberine used alone were >160 μg / mL and >128 μg / mL, respectively. Calculated according to the optimal ratio for synergistic effect using the checkerboard method, the MICs of osthol and berberine were reduced to 20 μg / mL and 32 μg / mL, respectively (the ratio of osthol to berberine (referring to berberine hydrochloride) was 0.625:1), with a synergistic index FICI of 0.187.

[0079] like Figure 1 As shown in Figure O, for Staphylococcus aureus 65577, the MICs of osthol and berberine used alone were >160 μg / mL and 64 μg / mL, respectively. According to the optimal ratio for synergistic effect calculated by the checkerboard method, the MICs of osthol and berberine were reduced to 20 μg / mL and 8 μg / mL, respectively (the ratio of osthol to berberine (referring to berberine hydrochloride) was 2.5:1), and the synergistic index FICI was 0.187.

[0080] like Figure 1 As shown in Figure P, for Staphylococcus aureus USA300, the MICs of osthol and berberine used alone were >160 μg / mL and 64 μg / mL, respectively. According to the optimal ratio for synergistic effect calculated by the checkerboard method, the MICs of osthol and berberine were reduced to 20 μg / mL and 8 μg / mL, respectively. The ratio of osthol to berberine (referring to berberine hydrochloride) was 2.5:1, and the synergistic index FICI was 0.187.

[0081] For Staphylococcus aureus, when used in combination, the optimal ratio of osthol to berberine (referring to berberine hydrochloride) is 0.625-2.5:1.

[0082] For Staphylococcus pseudointermediate, when used in combination, the optimal ratio of osthol to berberine (referring to berberine hydrochloride) is 1.25-2.5:1.

[0083] The above experimental results show that the combined use of osthol and berberine has a significant synergistic antibacterial effect against bacteria including Staphylococcus aureus, Staphylococcus pseudointermediate, and Clostridium perfringens, with FICI values ​​ranging from 0.078 to 0.25.

[0084] Example 2: Synergistic inhibitory effect of combined use of osthol and berberine on Candida albicans

[0085] Determination of the combination drug index (FICI) of osthol and berberine against pathogens: The FICI value of the combined application of osthol and berberine against the standard accusation strain ATCC12301 of Candida albicans was determined using the checkerboard method. The specific procedure was as follows: The FIC between different drugs was determined using the checkerboard broth dilution method. Single fungal colonies were picked and cultured in Sabouraud dextrose broth at 27°C for 48 hours. The fungal turbidity was adjusted to 0.5 using a McFarland turbidimeter, and then diluted to 1.0 × 10³ CFUs / mL with Sabouraud dextrose medium. 100 μL of Sabouraud dextrose medium was added to a 96-well U-shaped plate. The corresponding concentration of the drug was added to the first 10 wells of the eighth row, and serially diluted upwards to the second row. Other test drugs were added to the wells of the first column, and serially diluted from left to right to the ninth column. 100 μL of the diluted test bacterial solution was added to each of columns 1-10. Columns 11 and 12 show the negative and positive controls, respectively, containing only Sabouraud dextrose medium and the test bacterial culture. After incubating the 96-well plate at 27°C for 24 hours, the results were read. The lowest drug concentration that inhibits fungal growth and is visible to the naked eye was taken as the MIC value of the drug.

[0086] FIC = MIC(Drug A combined) / MIC(Drug A alone) + MIC(Drug B combined) / MIC(Drug B alone). Judgment criteria: FICI ≤ 0.5, synergistic effect; 0.5 < FICI ≤ 1, additive effect; 1 < FICI ≤ 2, irrelevant effect; FICI > 2, antagonistic effect. That is: FICI of osthol and berberine = MIC(berberine combined) / MIC(berberine alone) + MIC(osthol combined) / MIC(osthol alone).

[0087] Figure 2 As shown, the MICs of osthol and berberine alone against ATCC12301 were >128 μg / mL and 128 μg / mL, respectively. Calculations based on the optimal ratio for synergistic effect using the checkerboard method reduced the MICs of osthol and berberine to 8 μg / mL and 32 μg / mL, respectively, with a synergistic index (FICI) of 0.28. This indicates that the combined use of osthol and berberine has significant synergistic antibacterial activity against Candida albicans (for Candida albicans, the optimal ratio of osthol to berberine (referring to berberine hydrochloride) when used in combination is 0.25:1).

[0088] Example 3: Synergistic bactericidal curve of osthol combined with berberine

[0089] 3.1 Test Materials

[0090] Osthol and berberine are the same as in Example 1.

[0091] 3.2 In vitro bactericidal curve test

[0092] After culturing MRSA33591 and pseudo-Staphylococcus intermediate strain 31A in BHI broth for 6 hours, equal volumes of DMSO (final concentration 0.5%), berberine, osthol, and a mixture of osthol and berberine were added.

[0093] (1) For MRSA33591 strain, the grouping and dosage are as follows:

[0094] Control group: 0.5% DMSO;

[0095] Berberine group: The final concentration of berberine was 16 μg / mL;

[0096] Osthole group: The final concentration of osthole was 40 μg / mL;

[0097] Berberine and osthol combined group: final concentration of berberine was 16 μg / mL, and final concentration of osthol was 40 μg / mL.

[0098] Then, 100 μL of bacterial culture was taken at 1 h, 3 h, 6 h, 12 h and 24 h and spread onto MHA agar plates with a diameter of 10 cm. After incubation overnight, colony counting was performed.

[0099] (2) For clinical pseudostaphylococcal intermediate strain 31A, the grouping and dosage are as follows:

[0100] Control group: 0.5% DMSO;

[0101] Berberine group: The final concentration of berberine was 16 μg / mL;

[0102] Osthole group: The final concentration of osthole was 20 μg / mL;

[0103] Berberine and osthol combined group: final concentration of berberine was 16 μg / mL, and final concentration of osthol was 20 μg / mL.

[0104] Then, 100 μL of bacterial culture was taken at 1 h, 3 h, 6 h, 12 h and 24 h and spread onto MHA agar plates with a diameter of 10 cm. After incubation overnight, colony counting was performed.

[0105] 3.3 Test Results

[0106] like Figure 3As shown, for strain MRSA33591, at 24 h, berberine and osthol treatment alone had no significant effect on bacterial colony count compared to the control group. When berberine and osthol were treated together, the colony count was 0 CFU / mL at 1 h and remained at 0 Log until 24 h. 10 CFU / mL.

[0107] like Figure 4 As shown, for the clinical *Staphylococcus pseudointermediate* strain 31A, compared with the control group, treatment with berberine and osthol alone had no significant effect on bacterial colony count from 1 h to 24 h. When berberine and osthol were treated together, the colony count at 1 h was 1.91 Log. 10 CFU / mL, and the colony count was 0 Log at 3, 6, 12 and 24 h. 10 CFU / mL.

[0108] These data indicate that the combined treatment with berberine and osthol has a significant synergistic bactericidal effect.

[0109] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Application of natural drug compositions containing berberine and osthol in the preparation of drugs against Clostridium perfringens; In the composition, the ratio of osthol to berberine hydrochloride is 2:

1.

2. Application of natural pharmaceutical compositions containing berberine and osthol in the preparation of anti-Staphylococcus aureus drugs; In the composition, the ratio of osthol to berberine hydrochloride is 1.25-2.5:

1.

3. Application of natural drug compositions containing berberine and osthol in the preparation of anti-Candida albicans drugs; The ratio of osthol to berberine hydrochloride is 0.25:1.

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