CN116687916A - A synergistic antibacterial and antifungal pharmaceutical composition containing natural products osthole and berberine - Google Patents

Abstract

The invention discloses a synergistic antibacterial and fungal pharmaceutical composition containing natural products osthole and berberine. The invention proves the effectiveness of the antibacterial activity of the osthole synergistically synergistic berberine through the minimum inhibitory concentration test of the checkerboard method, the antifungal activity experiment and the bacterial growth curve in vitro. The invention provides a new application of combined use of osthole and berberine in anti-bacteria and fungi. The composition belongs to a traditional Chinese medicine compound preparation and provides a new treatment strategy for clinical treatment of bacterial and fungal infectious diseases.

Description

A synergistic antibacterial and fungal agent containing the natural products osthole and berberine pharmaceutical composition

technical field

The invention belongs to the field of medicine, in particular to a natural pharmaceutical composition containing berberine and osthole, the pharmaceutical composition has obvious synergistic antibacterial and antifungal effects, and the composition is used in the preparation of antibacterial and fungal infection drugs in the application.

Background technique

Bacterial resistance has become a global public health problem. Especially in recent years, the drug resistance rate of some antimicrobial drugs has continued to rise, which has gradually become a new global public crisis. The development of new antibacterial drugs has become one of the important ways to curb bacterial drug resistance in clinical practice. In addition, clinical infectious diseases are often complex infections, that is, bacterial infections are often accompanied by fungal infections, and current antibiotic therapy can only target a single bacterial infection or a single fungal infection, and cannot treat both bacteria and fungi at the same time. The fungus exerts a therapeutic effect.

There are many kinds of active ingredients in plants and they come from a wide range of sources. According to their different active ingredients, they can be divided into alkaloids, organic acids, volatile oils, flavonoids, polyphenols, polysaccharides, saponins, tannins, etc. These substances may have antibacterial, bactericidal and antioxidant properties. In addition, traditional Chinese medicine often has multi-target characteristics and often has certain biological activities against different pathogenic bacteria. The discovery of antibacterial active substances from plants and the development of antibacterial drugs have become the current research hotspots in this field.

Berberine, also known as berberine, was originally an alkaloid extracted from the traditional Chinese medicine Chuan Shao, which can be extracted or artificially synthesized from a variety of herbs. Medical functions, including anti-inflammation, anti-oxidation, anti-microbial, immune regulation, blood sugar control, cardiovascular protection and other functions, are often used clinically to treat diarrhea, diabetes and cardiovascular diseases, and berberine has therapeutic effects on infectious diarrhea Good effect, the main form of clinical medication is berberine hydrochloride or berberine sulfate. At the same time, some in vitro studies have found that berberine also has certain antibacterial activity, among which the antibacterial activity against Gram-negative bacteria is weak, and the minimum inhibitory concentration (MIC) is in the range of 512-1024 μg/mL. Bacteria have strong antibacterial activity, and the minimum inhibitory concentration (MIC) is usually between 128-512μg/mL (Xia Shuai, Ma Liyan, Xie Miaorong. Study on antibacterial activity of berberine hydrochloride against Staphylococcus aureus in vitro[J]. Clinical and Journal of Experimental Medicine, 2022,21(07):673-678); in addition, berberine also has a certain therapeutic effect on fungal infections, as found in research, berberine has certain activity on Candida albicans, and the MIC is generally Between 64-128μg/mL (Yong Jiangyan, Wang Hai, Huang Xiaoxue et al. Effects 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), the MIC range of the activity against Cryptococcus neoformans is between 8-168μg/mL (Xu Jialong, Song Haolei, Chen Xiaoqin, etc. Berberine anti-Cryptococcus neoformans activity and mechanism of action[J] . Acta Microbiology, 2023, 63(04):1541-1550.).

Osthole, molecular formula: C ₁₅ H ₁₆ O ₃ , alias: methoxyparsrol or parsvelol, is one of the important members of the total coumarins of Cnidium mongolica. Studies have found that osthole has strong anti-inflammatory and antioxidant activities. The in vitro antibacterial activity study of osthole found that it has weak antibacterial activity, the MIC against Escherichia coli is greater than 1024 μg/mL, and the MIC against Staphylococcus aureus is 512 μg/mL (Wu Xuejiao, Yan Boyu, Zhou Xurong, etc. Osthole ethers Synthesis and Antibacterial Activity of Derivatives[J]. Chemical Research and Application, 2022,34(07):1532-1537). Studies have found that osthole can inhibit the itching induced by histamine and histamine receptor H1 agonist HTMT, and histamine H4 receptor agonist VUF430, but it has no obvious inhibitory effect on high-dose chloroquine-induced itching. It is related to the inhibition of TRPV1 channel (Yang Niuniu. TRPV1 participates in the formation of pruritus and the mechanism of osthole antipruritic [D]. Nanjing University of Traditional Chinese Medicine, doctoral dissertation, 2017).

Contents of the invention

An object of the present invention is to provide the application of osthole in the preparation of berberine antibacterial synergist.

The application is: the application of osthole in the preparation of products that enhance the antibacterial activity and antifungal activity of berberine.

Another object of the present invention is to provide a natural medicinal composition containing berberine and osthole.

In the natural medicinal composition containing berberine and osthole provided by the present invention, the mass ratio of said berberine and osthole is 0.9:(0.25～16), which is equivalent to berberine hydrochloride and osthole. The mass ratio of beddinglin is 1: (0.25-16).

Further, the berberine is berberine or a pharmaceutically acceptable salt thereof, specifically berberine hydrochloride.

Further, in the composition, the active ingredients may only be osthole and berberine; other active ingredients may also be further included.

Another object of the present invention is to provide the application of the above-mentioned natural medicine composition containing berberine and osthole in the preparation of antibacterial and antifungal medicines.

Further, the drug is a drug for anti-bacterial infectious diseases and anti-fungal infectious diseases.

Further, the fungi include Candida albicans.

Further, the bacteria include Gram-positive bacteria.

Further, the Gram-positive bacteria include Staphylococcus aureus, Staphylococcus aureus pseudointermedius and Clostridium perfringens.

Further, the bacteria are Staphylococcus aureus; preferably, the Staphylococcus aureus comprises methicillin-resistant Staphylococcus aureus.

The invention also provides an antibacterial composition.

In the antibacterial composition provided by the invention, its active ingredients include osthole and berberine.

Further, the mass ratio of berberine and osthole is 0.9:(0.25-16), which is equivalent to the mass ratio of berberine hydrochloride and osthole being 1:(0.25-16).

The active ingredients of the antibacterial composition may only be osthole and berberine; it may further include other active ingredients, which can be determined by those skilled in the art according to the antibacterial effect.

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

Further, the fungi include Candida albicans.

Further, the bacteria include Gram-positive bacteria.

Further, the Gram-positive bacteria include Staphylococcus aureus, Staphylococcus aureus pseudointermedius and Clostridium perfringens.

Further, the bacteria are Staphylococcus aureus; preferably, the Staphylococcus aureus comprises methicillin-resistant Staphylococcus aureus.

The dosage form of the antibacterial composition is one of tablet, ointment, cream, capsule, sustained-release tablet, controlled-release tablet, oral liquid, syrup, injection, dripping pill, and freeze-dried powder for injection.

In addition, antibacterial products containing the above antibacterial composition also belong to the protection scope of the present invention.

The dosage form of the above-mentioned antibacterial products can be any of the following: cream, ointment, tablet, suspension, capsule, sustained-release tablet, controlled-release tablet, oral liquid, syrup, dosage form of injection, dripping pill, freeze-dried powder One of the injection forms.

In order to solve the problem of drug resistance of multidrug-resistant Gram-positive bacteria and the lack of effective drugs, the present invention provides a natural product composition with obvious synergistic antibacterial and synergistic effects, specifically using osthole in combination with berberine, both of which not only It is a simple addition of functions, but a synergistic antibacterial effect.

The present invention proves that the combination of osthole and berberine has obvious synergistic antibacterial and antifungal activity through the minimum inhibitory concentration test of the checkerboard method, in vitro bacterial growth curves and animal experiments. The bacteria in the test include Staphylococcus aureus, resistant Methicillin Staphylococcus aureus, Staphylococcus pseudointermediates of canine and feline origin; fungi in the test included Candida albicans.

The present invention opens up a new application for the combination of osthole and berberine, that is, the composition has obvious synergistic antibacterial and antifungal activities, and can be used in the treatment of multidrug-resistant Gram-positive bacterial infections and fungal infections It has important clinical application value.

The invention proves that the combination of berberine and osthole has obvious synergistic antifungal and antibacterial effects, and the combined use of the two has obvious synergistic effect. In addition, this combination is a pure traditional Chinese medicine combination, which has high safety and is not easy to produce drug resistance. At the same time, it has the characteristics of antibacterial and antifungal activities. Has obvious advantages, therefore, has important application value.

Description of drawings

Fig. 1 is the result of the synergistic antibacterial checkerboard method of multiple bacterial strains such as berberine (Berberine) combined with osthole (Osthole) to Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Staphylococcus pseudointermedius in Example 1 of the present invention .

Fig. 2 is the result of the synergistic antibacterial checkerboard method of berberine (Berberine) combined with osthole (Osthole) against Candida albicans ATCC12301 in Example 2 of the present invention.

Fig. 3 is the result of synergistic sterilization of MRSA33591 strain by berberine (Berberine) combined with osthole (Osthole) in Example 3 of the present invention.

Fig. 4 is the synergistic sterilization result of berberine (Berberine) combined with osthole (Osthole) on clinical Staphylococcus pseudointermediate 31 strain in Example 4 of the present invention.

Detailed ways

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention. The examples provided below can be used as a guideline for those skilled in the art to make further improvements, and are not intended to limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

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

Pathogens used in the following experiments:

Staphylococcus aureus ATCC29213 was purchased from the Culture Collection Center of China Veterinary Drug Control Institute.

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

Staphylococcus pseudointermediates 31A, Staphylococcus pseudointermediates 22-2, Staphylococcus pseudointermediates I1847, Staphylococcus pseudointermediates U5149, Staphylococcus pseudointermediates K3247-1 and other strains were preserved from the National Veterinary Drug Safety Evaluation Center of China Agricultural University.

Candida albicans standard accuser ATCC12301 was purchased from Shanghai Fuxiang Biotechnology Company.

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

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

Staphylococcus aureus USA300 was provided by Wang Jianfeng's research group of Jilin University (LiuY, ShiD, GuoY, Li M, ZhaY, WangQ, WangJ.DracorhodinPerochlorate attenuates StaphylococcusaureusUSA300virulencebydecreasingα-toxinexpression.

2017;33(1):17).

The standard strain of Clostridium perfringens ATCC13124 was purchased from the American Type Culture Collection.

Clostridium perfringens 16, Clostridium perfringens 12, Clostridium perfringens 40, Clostridium perfringens 26, Clostridium perfringens 38 and other strains were approved by China Agricultural University National Veterinary Drug Safety Evaluation Center (Beijing) preservation.

Bacterial media were used in the following experiments:

YPD medium: First weigh 20g of glucose, 10g of yeast extract, and 20g of tryptone with an electronic balance, then add 800mL of deionized water, dissolve the solids completely on a magnetic stirrer, and then dilute to 1L with deionized water , packed in Erlenmeyer flasks, if preparing a solid medium, add 2% agar as needed, and sterilize with an autoclave at 120°C for 30 minutes, then place it at room temperature for later use.

MHB broth medium was purchased from Beijing Land Bridge Technology Co., Ltd., and the preparation method was as follows: Weigh 25.0 g into 1 L of distilled water, heat and boil until completely dissolved, and autoclave at 121 °C for 15 min, and set aside.

Liquid thioglycolate medium (FTG) composition (g/L): tryptone 15.0g/L, yeast powder 5.0g/L, sodium thioglycolate 0.5g/L, glucose 5.0g/L, sodium chloride 2.5 g/L, L-cystine 0.5g/L, resazurin 0.001g/L, agar 0.75g/L.

The MHA medium was purchased from Beijing Land Bridge Technology Co., Ltd., and the preparation method was as follows: Weigh 38.0 g into 1 L of distilled water, heat and boil until completely dissolved, autoclave at 121 °C for 15 min, cool to 55 °C and pour onto a plate for later use.

Brain Heart Infusion Medium (BHI) was purchased from Beijing Land Bridge Technology Co., Ltd., and the preparation method was as follows: Weigh 38.5 g of this product, dissolve it in 1000 mL of distilled water with heating and stirring, adjust the pH to 7.3, and autoclave at 121 °C for 15 minutes. spare.

BHI solid medium was purchased from Beijing Luqiao Technology Co., Ltd. The preparation method is as follows: Weigh 50.0 g of this product in 1000 mL of distilled water, heat and boil until completely dissolved, autoclave at 121 °C for 20 min, cool to 55 °C and pour on a plate for later use.

Embodiment 1, osthole and berberine combined use antibacterial effect evaluation

Determination of combined drug index (FICI) of osthole and berberine on pathogenic bacteria: using checkerboard method to determine the effect of combined application of osthole and berberine on Staphylococcus aureus ATCC29213, methicillin-resistant Staphylococcus aureus ATCC33591 ( MRSA33591), Staphylococcus pseudointermediates 31A, Staphylococcus pseudointermediates 22-2, Staphylococcus pseudointermediates I1847, Staphylococcus pseudointermediates U5149, Staphylococcus pseudointermediates K3247-1, Staphylococcus aureus O2016, Staphylococcus aureus 65577, gold Combined antibacterial effect of Staphylococcus aureus USA300, Clostridium perfringens 16, Clostridium perfringens 12, Clostridium perfringens 40, Clostridium perfringens 26, Clostridium perfringens 38 and other strains .

Specifically, Staphylococcus aureus ATCC29213, Methicillin-resistant Staphylococcus aureus ATCC33591 (MRSA33591), Staphylococcus pseudointermediate 31A, Staphylococcus pseudointermediate 22-2, Staphylococcus pseudointermediate I1847, Staphylococcus pseudointermediate U5149, pseudointermediate Staphylococcus Staphylococcus K3247-1, Staphylococcus aureus 02016, Staphylococcus aureus 65577, Staphylococcus aureus USA300 and other strains were normally cultured in MHB broth medium. The specific operation of the checkerboard method is as follows: osthole (as drug A) and berberine (as drug B) were respectively at the highest concentration of 160 μg/mL and 256 μg/mL, diluted in multiples with MHB broth medium, respectively along 96 μg/mL. Add 50 μL of MHB broth medium containing different concentrations of the two drugs to the horizontal axis and vertical axis of the well culture plate, and then add 50 μL of various pathogenic bacteria solutions respectively, so that the final number of bacteria in each well is 2×10 ⁵ CFU, and culture at 37°C for 18 ~24h, observe the results. Record the minimum inhibitory concentration (MIC) of the two drugs when they are used alone or in combination, and calculate the FICI value (partial inhibitory concentration index) according to the following formula.

Clostridium perfringens ATCC13124, Clostridium perfringens 16, Clostridium perfringens 12, Clostridium perfringens 40, Clostridium perfringens 26, Clostridium perfringens 38 used in the Streak on 5% defibrinated sheep blood HA plate, adopt liquid thioglycolate (FTG) medium (pH=7.1±0.2), culture under mixed gas conditions containing 10% hydrogen, 10% carbon dioxide, and nitrogen balance. The specific operation of the checkerboard method is as follows: osthole (as drug A) and berberine (as drug B) were respectively at the highest concentration of 128 μg/mL and 128 μg/mL, diluted in multiples with MHB broth medium, respectively along 96 μg/mL. Add 50 μL of MHB broth medium containing different concentrations of the two drugs to the horizontal axis and vertical axis of the well culture plate, and then add 50 μL of various pathogenic bacteria solutions respectively, so that the final number of bacteria per well is 2×10 ⁵ CFU, and culture at 37°C for 24 hours ,Observation results. Record the minimum inhibitory concentration (MIC) of the two drugs when they are used alone or in combination, and calculate the FICI value (partial inhibitory concentration index) according to the following formula.

FICI=MIC A drug combined/MIC A drug alone + MIC B drug combined/MIC B drug alone, judging criteria: FICI≤0.5, synergistic effect; 0.5<FICI≤1, additive effect; 1﹤FICI≤2, Irrelevant effect; FICI>2, antagonistic effect. Namely: FICI of osthole and berberine=MIC (combination of berberine)/MIC (single use of berberine)+MIC (combination of osthole)/MIC (single use of osthole). The combined antibacterial results are shown in Figure 1:

As shown in Figure 1, A, for Clostridium perfringens ATCC13124 strain, the MICs of osthole and berberine used alone are >128 μg/mL and 64 μg/mL, respectively. After combined use, a synergistic effect is obtained according to the checkerboard method According to the optimal ratio calculation, the MICs of osthole and berberine are respectively reduced to 16 μg/mL and 8 μg/mL (the optimal ratio of osthole and berberine (referring to berberine hydrochloride) is 2:1 ), the synergy index FICI is 0.1875.

As shown in Figure 1 B, against Clostridium perfringens 16, the MICs of osthole and berberine used alone were >128 μg/mL and 32 μg/mL, respectively. After combined use, the synergistic effect was obtained according to the chessboard method According to the optimal ratio calculation, the MICs of osthole and berberine were reduced to 32 μg/mL and 4 μg/mL respectively (the optimal ratio of osthole and berberine (referring to berberine hydrochloride) is 8:1) , the synergy index FICI is 0.25.

As shown in C in Figure 1, against Clostridium perfringens 12, the MICs of osthole and berberine alone were >128 μg/mL and 32 μg/mL, respectively. According to the optimal ratio calculation, the MICs of osthole and berberine were reduced to 16 μg/mL and 4 μg/mL respectively (the optimal ratio of osthole and berberine (referring to berberine hydrochloride) is 4:1) , the synergy index FICI is 0.187.

As shown in D in Figure 1, against Clostridium perfringens 40, the MICs of osthole and berberine alone were >128 μg/mL and 16 μg/mL, respectively. According to the optimal ratio calculation, the MICs of osthole and berberine were reduced to 32 μg/mL and 2 μg/mL respectively (the optimal ratio of osthole and berberine (referring to berberine hydrochloride) is 16:1) , the synergy index FICI is 0.25.

As shown in E in Figure 1, against Clostridium perfringens 26, the MICs of osthole and berberine alone were >128 μg/mL and 32 μg/mL, respectively. According to the optimal ratio calculation, the MICs of osthole and berberine were reduced to 32 μg/mL and 4 μg/mL respectively (the optimal ratio of osthole and berberine (referring to berberine hydrochloride) is 8:1) , the synergy index FICI is 0.25.

As shown in F in Figure 1, against Clostridium perfringens 38, the MICs of osthole and berberine alone were >128 μg/mL and 32 μg/mL, respectively. According to the optimal ratio calculation, the MICs of osthole and berberine were reduced to 32 μg/mL and 2 μg/mL respectively (the optimal ratio of osthole and berberine (referring to berberine hydrochloride) is 16:1) , the synergy index FICI is 0.187.

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

As shown in Figure 1 G, against Staphylococcus aureus ATCC29213, the MICs of osthole and berberine used alone are >160 μg/mL and 64 μg/mL, respectively. After combined use, the best synergistic effect can be obtained according to the checkerboard method According to the ratio calculation, the MICs of osthole and berberine were respectively reduced to 10 μg/mL and 8 μg/mL (the optimal ratio of osthole and berberine (referring to berberine hydrochloride) was 1.25:1), synergistic The index FICI is 0.156.

As shown in H in Figure 1, against methicillin-resistant Staphylococcus aureus ATCC33591 (MRSA33591), the MICs of osthole and berberine alone were >160 μg/mL and >256 μg/mL, respectively, and the synergy was obtained according to the checkerboard method. The optimal ratio calculation of effect, the MIC of osthole and berberine is reduced to 20 μ g/mL and 8 μ g/mL respectively (the optimal ratio of osthole and berberine (referring to berberine hydrochloride) is 2.5: 1), the synergy index FICI is 0.078.

As shown in I in Figure 1, for Staphylococcus pseudointermediate 31A, the MICs of osthole and berberine used alone are >160 μg/mL and 128 μg/mL respectively, calculated according to the optimal ratio of the synergistic effect obtained by the checkerboard method, The MICs of osthole and berberine were reduced to 10 μg/mL and 8 μg/mL respectively (the optimum ratio of osthole and berberine (referring to berberine hydrochloride) was 1.25:1), and the synergy index FICI was 0.093 .

As shown in J in Figure 1, against Staphylococcus pseudointermediate 22-2, the MICs of osthole and berberine used alone were >160 μg/mL and 64 μg/mL, respectively, and the optimal ratio of synergistic effect was obtained according to the checkerboard method Calculated, the MICs of osthole and berberine were reduced to 20 μg/mL and 8 μg/mL (the optimal ratio of osthole and berberine (referring to berberine hydrochloride) was 2.5:1), synergy index FICI is 0.1875.

As shown in Figure 1 K, against Staphylococcus pseudointermediate I1847, the MICs of osthole and berberine used alone are >160 μg/mL and 64 μg/mL, respectively, calculated according to the optimal ratio of the synergistic effect obtained by the checkerboard method, The MICs of osthole and berberine were reduced to 10 μg/mL and 8 μg/mL respectively (the optimal ratio of osthole and berberine (referring to berberine hydrochloride) was 1.25:1), and the synergy index FICI was 0.156 .

As shown in L in Figure 1, against Staphylococcus pseudointermediate U5149, the MICs of osthole and berberine alone were >160 μg/mL and 128 μg/mL, respectively, calculated according to the optimal ratio of the synergistic effect obtained by the checkerboard method, The MICs of osthole and berberine were reduced to 10 μg/mL and 8 μg/mL respectively (the optimum ratio of osthole and berberine (referring to berberine hydrochloride) was 1.25:1), and the synergy index FICI was 0.093 .

As shown in Figure 1 M, against Staphylococcus pseudointermediate K3247-1, the MICs of osthole and berberine used alone are >160 μg/mL and 128 μg/mL respectively, and the optimal ratio of synergistic effect is obtained according to the checkerboard method Calculated, the MICs of osthole and berberine were reduced to 20 μg/mL and 8 μg/mL (the optimal ratio of osthole and berberine (referring to berberine hydrochloride) was 2.5:1), synergy index FICI is 0.125.

As shown by N in Figure 1, against Staphylococcus aureus 02016, the MICs of osthole and berberine used alone are >160 μg/mL and >128 μg/mL, respectively, calculated according to the optimal ratio of the synergistic effect obtained by the checkerboard method , the MICs of osthole and berberine were reduced to 20 μg/mL and 32 μg/mL respectively (the ratio of osthole and berberine (referring to berberine hydrochloride) was 0.625:1), and the synergy index FICI was 0.187.

As shown by O in Figure 1, against Staphylococcus aureus 65577, the MICs of osthole and berberine used alone are >160 μg/mL and 64 μg/mL, respectively, calculated according to the optimal ratio of the synergistic effect obtained by the checkerboard method, The MICs of osthole and berberine were reduced to 20 μg/mL and 8 μg/mL respectively (the ratio of osthole and berberine (referring to berberine hydrochloride) was 2.5:1), and the synergy index FICI was 0.187.

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

For Staphylococcus aureus, when the two are used in combination, the optimal ratio of osthole and berberine (referring to berberine hydrochloride) is 0.625-2.5:1;

For pseudointermediate Staphylococcus aureus, when the two are used in combination, the optimal ratio of osthole and berberine (referring to berberine hydrochloride) is 1.25-2.5:1.

The above test results show that the combined use of osthole and berberine has an obvious synergistic antibacterial effect on bacteria including Staphylococcus aureus, Staphylococcus pseudointermediate and Clostridium perfringens, and the FICI is in the range of 0.078 to 0.25.

Embodiment 2, the synergistic inhibitory effect of osthole and berberine combined use to Candida albicans

Determination of combined drug index (FICI) of osthole and berberine against pathogenic bacteria: The FICI value of the combined use of osthole and berberine against Candida albicans standard accuser strain ATCC12301 was determined by checkerboard method. The specific operation is as follows: the FIC between different drugs is determined using the checkerboard broth dilution method. Pick a single fungal colony in Sabouraud dextrose broth, and culture the fungus at 27°C for 48h. Adjust the fungal turbidity to McFarland turbidity 0.5 with a McFarland turbidimeter, and dilute it to 1.0×103 CFUs/mL with Sabouraud dextrose medium for later use. Add 100 μL of Sabouraud dextrose medium to a 96-well U-plate, add the corresponding concentration of drug to the first 10 wells in the eighth row, and sequentially dilute upward to the second row. Add other drugs to be tested in each well of the first column, and then serially dilute to the ninth column from left to right. Add 100 μL of the diluted bacterial solution to be tested to columns 1-10. Columns 11 and 12 are negative and positive controls containing only Sabouraud dextrose medium and the bacteria solution to be tested, respectively. Place the 96-well plate at 27°C for static culture for 24 hours, read the results, and take the lowest drug concentration that can be distinguished by the naked eye to inhibit fungal growth as the MIC value of the drug.

FIC＝MIC(combination of drug A)/MIC(single use of drug A)+MIC(combination of drug B)/MIC(single use of drug B), judging criteria: FICI≤0.5, synergistic effect; 0.5<FICI≤1, summation Effect; 1﹤FICI≤2, irrelevant effect; FICI>2, antagonistic effect. Namely: FICI of osthole and berberine=MIC (combination of berberine)/MIC (single use of berberine)+MIC (combination of osthole)/MIC (single use of osthole).

As shown in Figure 2, the MICs of osthole and berberine alone to ATCC12301 are >128 μg/mL and 128 μg/mL, respectively. According to the optimal ratio of synergistic effects obtained by the checkerboard method, the MICs of osthole and berberine The MICs were reduced to 8 μg/mL and 32 μg/mL, respectively, and the synergy index FICI was 0.28, indicating that the combined use of osthole and berberine has obvious synergistic antibacterial activity against Candida albicans (for Candida albicans, when the two are used in combination, The optimum ratio of osthole and berberine (referring to berberine hydrochloride) is: 0.25:1).

Embodiment 3, the synergistic bactericidal curve of osthole combined with berberine

3.1 Test material

Osthole and berberine are with embodiment 1.

3.2 In vitro bactericidal curve test

MRSA33591 and Staphylococcus pseudointermediate strain 31A were cultured in BHI broth for 6 hours, and then an equal volume of DMSO (0.5% final concentration), berberine, osthole, and a mixture of osthole and berberine were added.

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

Control group (control): 0.5% DMSO;

Berberine group (Berberine): the final concentration of berberine was 16 μg/mL;

Osthole group (Osthole): the final concentration of osthole is 40 μg/mL;

Berberine and osthole combined group: the final concentration of berberine was 16 μg/mL, and the final concentration of osthole was 40 μg/mL.

Then at 1h, 3h, 6h, 12h and 24h, 100 μL of bacterial liquid was spread on MHA agar plates with a diameter of 10 cm, and the colonies were counted after culturing overnight.

(2) For clinical pseudointermediate Staphylococcus strain 31A bacteria, the grouping and dosage are as follows:

Control group (control): 0.5% DMSO;

Berberine group (Berberine): the final concentration of berberine was 16 μg/mL;

Osthole group (Osthole): the final concentration of osthole is 20 μg/mL;

Berberine and osthole combined group: the final concentration of berberine was 16 μg/mL, and the final concentration of osthole was 20 μg/mL.

Then at 1h, 3h, 6h, 12h and 24h, 100 μL of bacterial liquid was spread on MHA agar plates with a diameter of 10 cm, and the colonies were counted after culturing overnight.

3.3 Test results

As shown in Figure 3, for the MRSA33591 strain, at 24h, compared with the control group, the individual treatment of berberine and osthole had no significant effect on the number of bacterial colonies. When berberine and osthole were jointly treated, the number of colonies was 0 CFU/mL at the first hour of treatment, and the number of colonies was 0 Log ₁₀ CFU/mL until 24 hours.

As shown in Figure 4, for the clinical pseudointermediate Staphylococcus strain 31A strain, compared with the control group, berberine and osthole alone had no significant effect on the number of bacterial colonies from 1 h to 24 h. When berberine and osthole were jointly treated, the number of colonies was 1.91 Log ₁₀ CFU/mL at the first hour of treatment, and the number of colonies was 0 Log ₁₀ CFU/mL at the 3rd, 6th, 12th and 24th hours.

These data indicated that the combined treatment of berberine and osthole had obvious synergistic bactericidal function.

The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experiments, the present invention can be practiced in a wider range under equivalent parameters, concentrations and conditions. While specific embodiments of the invention have been shown, it should be understood that the invention can be further modified. In a word, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by using conventional techniques known in the art and departing from the disclosed scope of this application.

Claims (10)

1. Application of osthole in preparing berberine antibacterial synergist is provided.

2. The use according to claim 1, characterized in that: the application is the application of osthole in preparing products for enhancing the antibacterial activity and antifungal activity of berberine.

3. A natural pharmaceutical composition containing berberine and osthole, wherein the mass ratio of berberine to osthole is 0.9 (0.25-16).

4. The pharmaceutical composition according to claim 2, wherein: the berberine is berberine or pharmaceutically acceptable salt thereof;

in the composition, the active ingredients are osthole and berberine only or further comprise other active ingredients.

5. Use of a natural pharmaceutical composition comprising berberine and osthole according to claim 3 or 4 for the preparation of antibacterial and antifungal agents.

6. The use according to claim 5, characterized in that: the medicine is a medicine for resisting bacterial infectious diseases and fungal infectious diseases;

the fungus comprises candida albicans;

the bacteria include gram positive bacteria.

7. The use according to claim 6, characterized in that: the gram positive bacteria comprise staphylococcus aureus, pseudo-intermediate staphylococcus aureus, and clostridium perfringens.

8. An antibacterial composition comprises osthole and berberine as active ingredients, wherein the mass ratio of the berberine to the osthole is 0.9 (0.25-16).

9. The antimicrobial composition of claim 8, wherein: the antibacterial composition has a killing and/or inhibiting effect on bacteria and fungi;

the fungus comprises candida albicans;

the bacteria include gram positive bacteria.

10. An antimicrobial composition according to claim 9, wherein: the gram positive bacteria comprise staphylococcus aureus, pseudo-intermediate staphylococcus aureus, and clostridium perfringens.