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

A synergistic antibacterial and antifungal pharmaceutical composition containing natural products osthole and berberine Download PDF

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CN116687916A
CN116687916A CN202310683644.4A CN202310683644A CN116687916A CN 116687916 A CN116687916 A CN 116687916A CN 202310683644 A CN202310683644 A CN 202310683644A CN 116687916 A CN116687916 A CN 116687916A
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berberine
osthole
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staphylococcus aureus
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代重山
沈建忠
沈长奇
汤树生
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China Agricultural University
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    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
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    • A61P31/10—Antimycotics
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Abstract

The application discloses a synergistic antibacterial and antifungal pharmaceutical composition containing natural products osthole and berberine. The application proves the effectiveness of the antibacterial activity of the osthole synergistic berberine through a chessboard minimum antibacterial concentration test, an antifungal activity test and an in vitro bacterial growth curve. The application provides a new application of osthole and berberine in combination use in resisting bacteria and fungi, the composition belongs to a Chinese herbal compound preparation, and provides a new treatment strategy for clinically treating bacterial and fungal infectious diseases.

Description

A synergistic antibacterial and antifungal pharmaceutical composition containing natural products osthole and berberine
Technical Field
The application belongs to the field of medicines, and in particular relates to a natural pharmaceutical composition containing berberine and osthole, which has obvious synergistic antibacterial and antifungal effects and application of the composition in preparing antibacterial and antifungal medicines.
Background
Bacterial resistance has become a global public health problem. In particular, in recent years, the drug resistance of some antibacterial drugs is continuously increasing, and the drug resistance is becoming a new global public crisis. The development of novel antibacterial agents has become one of the important ways of clinically suppressing bacterial resistance. In addition, clinically infectious diseases are often complex infections, i.e., bacterial infections are often accompanied by fungal infections, whereas current antibiotic therapies are often only directed against a single bacterial infection or a single fungal infection, and cannot exert therapeutic effects on both bacteria and fungi at the same time.
The active ingredients in plants are various in variety and wide in source, and can be divided into alkaloid, organic acid, volatile oil, flavone, polyphenol, polysaccharide, saponin, tannin and the like according to the different active ingredients, wherein the substances possibly have antibacterial, sterilizing and antioxidant effects, in addition, traditional Chinese medicines often have multi-target characteristics and have certain biological activity on different pathogenic bacteria, and the research and development of antibacterial active substances from plants and further antibacterial medicines are becoming hot points of current research in the field.
Berberine, also called berberine, is an alkaloid extracted from the Chinese medicinal herb, rhizoma ligustici wallichii, can be extracted from various herbaceous plants or artificially synthesized at present, has a chemical structure of tetramethylpyrazine, has proved to have various biological functions including anti-inflammatory, antioxidant, antimicrobial, immune regulation and control, blood sugar control, cardiovascular protection and other functions, is often used for treating diarrhea, diabetes mellitus, cardiovascular diseases and other diseases clinically, has better effect on treating infectious diarrhea, and is mainly used for treating berberine hydrochloride or berberine sulfate clinically. Meanwhile, some in vitro researches show that berberine also has certain antibacterial activity, wherein the antibacterial activity on gram-negative bacteria is weaker, the Minimum Inhibitory Concentration (MIC) is in the range of 512-1024 mug/mL, the antibacterial activity on gram-positive bacteria is stronger, and the Minimum Inhibitory Concentration (MIC) is usually 128-512 mug/mL (Xia Shuai, ma Liyan, xie Miaorong. The research on the in vitro antibacterial activity of berberine hydrochloride on staphylococcus aureus [ J ]. J. Journal of clinical and experimental medicine, 2022,21 (07): 673-678); in addition, berberine has a certain therapeutic effect on fungal infection, and if researches show that berberine has a certain activity on candida albicans, the MIC is generally between 64-128 mug/mL (Yong Jiang Yan, wang Hai, huangxue, etc.. The combination of berberine hydrochloride and fluconazole has an effect on the calcium homeostasis of drug-resistant candida albicans [ J ]. J. China etiology journal, 2020,15 (08): 903-909), and the MIC for resisting cryptococcus neoformans is in the range of 8-168 mug/mL (Xu Jialong, song Haolei, chen Xiaoqin, etc.. Berberine has activity and action mechanism against cryptococcus neoformans [ J ]. Microbiology, 2023,63 (04): 1541-1550.).
Osthole, molecular formula: c (C) 15 H 16 O 3 Alias name: parslemol or Parslemol methyl ether is one of the important members of the total coumarin of fructus Cnidii. The research shows that osthole has strong anti-inflammatory and antioxidant activities. In vitro antibacterial activity research of osthole shows that osthole has weaker antibacterial activity, the MIC for Escherichia coli is more than 1024 mug/mL, the MIC for Staphylococcus aureus is 512 mug/mL (Wu Xuejiao, yan Ba, zhou Xurong, etc.. The osthole ether derivative is synthesized and has antibacterial activity [ J)]Chemical research and application 2022,34 (07): 1532-1537). Studies have found that osthole is capable of inhibiting itching induced by histamine and histamine receptor H1 agonist HTMT, histamine H4 receptor agonist VUF430, but has no significant inhibitory effect on high doses of chloroquine-induced itching, its mechanism of action is associated with inhibition of TRPV1 channels (Yang Niuniu. TRPV1 is involved in the formation of itching and in the study of the osthole antipruritic mechanism [ D)]University of south Beijing Chinese medicine, doctor's paper, 2017).
Disclosure of Invention
The application aims to provide an application of osthole in preparing berberine antibacterial synergists.
The application is as follows: application of osthole in preparing product for enhancing antibacterial activity and antifungal activity of berberine is provided.
It is another object of the present application to provide a natural pharmaceutical composition containing berberine and osthole.
In the natural pharmaceutical composition containing berberine and osthole, the mass ratio of the berberine to the osthole is 0.9 (0.25-16), namely the mass ratio of the berberine hydrochloride to the osthole is 1: (0.25-16).
Further, the berberine is berberine or pharmaceutically acceptable salt thereof, and specifically can be berberine hydrochloride.
Further, in the composition, the active ingredients thereof may be only osthole and berberine; other active ingredients may also be further included.
It is another object of the present application to provide the use of the above natural pharmaceutical composition containing berberine and osthole for preparing antibacterial and antifungal drugs.
Further, the medicine is a medicine for resisting bacterial infectious diseases and fungal infectious diseases.
Further, the fungus includes candida albicans.
Further, the bacteria include gram positive bacteria.
Further, the gram positive bacteria comprise staphylococcus aureus, pseudo-intermediate staphylococcus aureus, and clostridium perfringens.
Further, the bacterium is staphylococcus aureus; preferably, the staphylococcus aureus comprises methicillin-resistant staphylococcus aureus.
The application also provides an antibacterial composition.
The active ingredients of the antibacterial composition provided by the application comprise osthole and berberine.
Further, the mass ratio of the berberine to the osthole is 0.9 (0.25-16), namely the mass ratio of the berberine hydrochloride to the osthole is 1: (0.25-16).
The active ingredients of the antibacterial composition may be osthole and berberine only; other active ingredients may be further included as well, which may be determined by one skilled in the art based on the antimicrobial effect.
The antibacterial composition can kill and/or inhibit bacteria and fungi;
further, the fungus includes candida albicans.
Further, the bacteria include gram positive bacteria.
Further, the gram positive bacteria comprise staphylococcus aureus, pseudo-intermediate staphylococcus aureus, and clostridium perfringens.
Further, the bacterium is 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 injection.
In addition, antibacterial products containing the antibacterial composition also belong to the protection scope of the application.
The dosage form of the antibacterial product can be any one of the following: one of cream, ointment, tablet, suspension, capsule, sustained release tablet, controlled release tablet, oral liquid, syrup, injection, dripping pill and lyophilized powder for injection.
In order to solve the problems of multiple drug-resistant gram-positive bacteria drug resistance and lack of effective drugs, the application provides a natural product composition with obvious synergistic antibacterial effect, and particularly adopts osthole combined with berberine, which not only simply adds functions, but also achieves the synergistic antibacterial effect.
According to the application, the combination of osthole and berberine has obvious synergistic antibacterial and antifungal activity through a chessboard minimum inhibitory concentration test, an in-vitro bacterial growth curve and an animal test, and bacteria in the test comprise staphylococcus aureus, methicillin-resistant staphylococcus aureus and canine/feline pseudo-intermediate staphylococcus; the fungi in the test included candida albicans.
The application opens up a new application for the combined medication of osthole and berberine, namely the composition has obvious synergic antibacterial and antifungal activity, and has important clinical application value in the treatment of multi-drug resistant gram positive bacterial infection and fungal infection.
The application proves that the berberine and the osthole have obvious synergistic antifungal and antibacterial effects, and have obvious synergistic effects after being used together. In addition, the combination is a pure traditional Chinese medicine combination, has the characteristics of higher safety, difficult generation of drug resistance and the like, and simultaneously has the characteristics of antibacterial activity, antifungal activity and the like, and has obvious advantages compared with antibacterial drugs or antifungal drugs used in the current market, thus having important application value.
Drawings
FIG. 1 shows the results of a synergistic antimicrobial chessboard method of Berberine (Berberine) in combination with Osthole (Osthole) against a plurality of strains of Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, staphylococcus pseudointermedia, etc., in example 1 of the present application.
FIG. 2 shows the results of a synergistic antimicrobial chessboard method of Berberine (Berberine) in combination with Osthole (Osthole) to Candida albicans ATCC12301 in example 2 of the present application.
FIG. 3 shows the synergistic sterilization results of Berberine (Berberine) in combination with Osthole (Osthole) on MRSA33591 strain in example 3 of the present application.
FIG. 4 shows the synergistic bactericidal effect of Berberine (Berberine) in combination with Osthole (Osthole) on clinical pseudo-middle staphylococcus 31 strain in example 4 of the present application.
Detailed Description
The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Osthole used in the following experiments was purchased from Chengduremix, with a purity of 99% or more. The berberine used in the following experiments is berberine hydrochloride with CAS number 633-65-8 and molecular formula: C20H18ClNO4, molecular weight 372.82, purchased from Ab Ding Shiji company, purity not less than 98%.
Pathogenic bacteria used in the following experiments:
staphylococcus aureus ATCC29213 was purchased from the chinese veterinary drug administration collection.
Methicillin-resistant staphylococcus aureus ATCC33591 (MRSA 33591) is provided by the national veterinary drug safety evaluation center (beijing).
Strains such as the staphylococcus pseudointermedia 31A, the staphylococcus pseudointermedia 22-2, the staphylococcus pseudointermedia I1847, the staphylococcus pseudointermedia U5149, the staphylococcus pseudointermedia K3247-1 and the like are stored from national veterinary drug safety evaluation center of China agricultural university.
Candida albicans standard finger control bacteria ATCC12301 was purchased from Shanghai complex biotechnology company.
Staphylococcus aureus 02016 was saved from national veterinary drug safety evaluation center of the university of agriculture in china.
Staphylococcus aureus 65577 is preserved from national veterinary drug safety evaluation center of China agricultural university.
Staphylococcus aureus USA300 is supplied by the university of gillin group Wang Jianfeng topic (LiuY, shiD, guoY, li M, zhaY, wangQ, wangJ.DracorhodinPerochlorateattenuatesStaphylococcusaureus USA300 virus core by de-treatment alpha-toxin x pressure.
2017；33(1):17)。
Clostridium perfringens standard strain ATCC13124 was purchased from the american type culture collection.
Strains of clostridium perfringens 16, clostridium perfringens 12, clostridium perfringens 40, clostridium perfringens 26, clostridium perfringens 38, etc. were all maintained by the national veterinary drug safety evaluation center of the university of china (beijing).
Bacterial culture media was used in the following experiments:
YPD medium: firstly, weighing 20g of glucose, 10g of yeast extract and 20g of tryptone by using an electronic balance, then adding 800mL of deionized water, completely dissolving the solid on a magnetic stirrer, then fixing the volume to 1L by using the deionized water, subpackaging in a conical flask, adding 2% of agar according to the requirement if preparing a solid culture medium, sterilizing at 120 ℃ for 30min by using an autoclave, and standing at normal temperature for later use.
MHB broth medium was purchased from beijing land bridge technologies, inc, and was formulated as follows: weighing 25.0g in 1L distilled water, heating and boiling to dissolve completely, and sterilizing at 121deg.C for 15 min.
Liquid thioglycolate medium (FTG) composition (g/L): 15.0g/L of tryptone, 5.0g/L of yeast powder, 0.5g/L of sodium thioglycolate, 5.0g/L of glucose, 2.5g/L of sodium chloride, 0.5g/L of L-cystine, 0.001g/L of resazurin and 0.75g/L of agar.
MHA medium was purchased from beijing land bridge technology, inc, and the preparation method was as follows: 38.0g of the mixture is weighed in 1L of distilled water, heated and boiled until the mixture is completely dissolved, autoclaved for 15min at 121 ℃, cooled to 55 ℃ and poured into a flat plate for standby.
Brain heart infusion medium (BHI) was purchased from beijing land bridge technologies, inc, and the preparation method was as follows: 38.5g of the product is weighed, heated, stirred and dissolved in 1000mL of distilled water, the pH is regulated to 7.3, and the mixture is autoclaved at 121 ℃ for 15 minutes for standby.
BHI solid medium was purchased from Beijing Luqiao technology Co., ltd, and the preparation method was as follows: weighing 50.0g of the product in 1000mL of distilled water, heating and boiling until the product is completely dissolved, sterilizing at 121 ℃ for 20min under high pressure, cooling to 55 ℃ and pouring the product into a flat plate for later use.
EXAMPLE 1 evaluation of antibacterial Effect of osthole and berberine in combination
Combination Index (FICI) of osthole and berberine for pathogenic bacteria: the combined antimicrobial effect of osthole and berberine combination on strains of staphylococcus aureus ATCC29213, methicillin-resistant staphylococcus aureus ATCC33591 (MRSA 33591), pseudo-intermediate staphylococcus 31A, pseudo-intermediate staphylococcus 22-2, pseudo-intermediate staphylococcus I1847, pseudo-intermediate staphylococcus U5149, pseudo-intermediate staphylococcus K3247-1, staphylococcus aureus O2016, staphylococcus aureus 65577, staphylococcus aureus USA300, clostridium perfringens 16, clostridium perfringens 12, clostridium perfringens 40, clostridium perfringens 26, clostridium perfringens 38, and the like was measured using the checkerboard method.
Specifically, strains such as staphylococcus aureus ATCC29213, methicillin-resistant staphylococcus aureus ATCC33591 (MRSA 33591), pseudo-intermediate staphylococcus 31A, pseudo-intermediate staphylococcus 22-2, pseudo-intermediate staphylococcus I1847, pseudo-intermediate staphylococcus U5149, pseudo-intermediate staphylococcus K3247-1, staphylococcus aureus 02016, staphylococcus aureus 65577, staphylococcus aureus USA300, and the like were normally cultured using MHB broth culture medium. The chessboard method specifically comprises the following steps: the osthole (serving as medicine A) and berberine (serving as medicine B) are respectively diluted by using MHB broth culture medium in multiple ratio with the highest concentration of 160 mug/mL and 256 mug/mL, 50 mug of MHB broth culture medium containing two medicines with different concentrations is respectively added along the transverse axis and the longitudinal axis of a 96-micropore culture plate, then 50 mug of various pathogenic bacteria bacterial liquids are respectively added, and the final bacterial count of each hole is 2 multiplied by 10 5 CFU, culturing at 37 ℃ for 18-24 h, and observing the result. The Minimum Inhibitory Concentrations (MIC) of each of the two drugs were recorded for single and combined use, and FICI values (partial inhibitory concentration index) were calculated according to the following formula.
Clostridium perfringens ATCC13124, clostridium perfringens 16, clostridium perfringens 12, clostridium perfringens 40, clostridium perfringens 26, clostridium perfringens 38 were streaked on 5% defibrinated sheep blood HA plates and cultured with liquid thioglycolate (FTG) medium (ph=7.1±0.2) under mixed gas conditions containing 10% hydrogen, 10% carbon dioxide, and nitrogen balance. The chessboard method specifically comprises the following steps: the osthole (serving as medicine A) and berberine (serving as medicine B) are respectively diluted by MHB broth culture medium in multiple ratio with 128 mug/mL and 128 mug/mL as the highest concentration, 50 mug of MHB broth culture medium containing two medicines with different concentrations is respectively added along the transverse axis and the longitudinal axis of a 96-micropore culture plate, then 50 mug of various pathogenic bacteria bacterial liquids are respectively added, and the final bacterial count of each hole is 2 multiplied by 10 5 CFU, incubation at 37℃for 24h, and observations were made. The Minimum Inhibitory Concentrations (MIC) of each of the two drugs were recorded for single and combined use, and FICI values (partial inhibitory concentration index) were calculated according to the following formula.
FICI = MIC-combination/MIC-combination alone + MIC-combination/MIC-combination alone, criteria: FICI is less than or equal to 0.5, and the synergism is achieved; FICI is more than 0.5 and less than or equal to 1, and the addition effect is achieved; the FICI is less than 1 and less than or equal to 2, and the effects are irrelevant; FICI > 2, antagonism. Namely: fici=mic (berberine combination)/MIC (berberine alone) +mic (osthole combination)/MIC (osthole alone) of osthole and berberine. The combined antibacterial results are shown in fig. 1:
as shown in FIG. 1A, for Clostridium perfringens ATCC13124 strain, the MICs of osthole and berberine used alone were > 128 μg/mL and 64 μg/mL, respectively, and after the combination, the MICs of osthole and berberine were reduced to 16 μg/mL and 8 μg/mL, respectively, calculated as the optimum ratio for achieving the synergistic effect according to the checkerboard method (the optimum ratio of osthole and berberine referred to as berberine hydrochloride was 2:1), and the synergy index FICI was 0.1875.
As shown in FIG. 1B, for clostridium perfringens 16, the MIC of osthole and berberine used alone was > 128 μg/mL and 32 μg/mL, respectively, and after combined use, the MIC of osthole and berberine was reduced to 32 μg/mL and 4 μg/mL, respectively, calculated as the optimum ratio for achieving synergistic effect according to the chessboard method (optimum ratio of osthole and berberine referred to as berberine hydrochloride was 8:1), and the synergy index FICI was 0.25.
As shown in FIG. 1C, for clostridium perfringens 12, the MIC of osthole and berberine used alone was > 128 μg/mL and 32 μg/mL, respectively, and after combined use, the MIC of osthole and berberine was reduced to 16 μg/mL and 4 μg/mL, respectively, calculated as the optimum ratio for achieving synergistic effect according to the chessboard method (optimum ratio of osthole and berberine referred to as berberine hydrochloride was 4:1), and the synergy index FICI was 0.187.
As shown in FIG. 1D, for clostridium perfringens 40, the MIC of osthole and berberine used alone was > 128 μg/mL and 16 μg/mL, respectively, and after combined use, the MIC of osthole and berberine was reduced to 32 μg/mL and 2 μg/mL, respectively, calculated as the optimum ratio for achieving synergistic effect according to the chessboard method (16:1 for osthole and berberine, respectively), and the synergy index FICI was 0.25.
As shown in FIG. 1E, for clostridium perfringens 26, the MIC of osthole and berberine used alone was > 128 μg/mL and 32 μg/mL, respectively, and after combined use, the MIC of osthole and berberine was reduced to 32 μg/mL and 4 μg/mL, respectively, calculated as the optimum ratio for achieving synergistic effect according to the chessboard method (optimum ratio of osthole and berberine referred to as berberine hydrochloride was 8:1), and the synergy index FICI was 0.25.
As shown in F in FIG. 1, for clostridium perfringens 38, the MIC of osthole and berberine used alone was > 128 μg/mL and 32 μg/mL, respectively, and after combined use, the MIC of osthole and berberine was reduced to 32 μg/mL and 2 μg/mL, respectively, calculated as the optimum ratio for achieving synergistic effect according to the chessboard method (16:1 for osthole and berberine, indicated as berberine hydrochloride), and the synergy index FICI was 0.187.
Aiming at clostridium perfringens, when the two are combined, the optimal ratio of osthole to berberine (berberine hydrochloride) is 2-16:1;
as shown in FIG. 1G, the MIC of osthole and berberine used alone was > 160 μg/mL and 64 μg/mL, respectively, for Staphylococcus aureus ATCC29213, and after the combined use, the MIC of osthole and berberine was reduced to 10 μg/mL and 8 μg/mL, respectively, according to the optimum ratio of the synergistic effect obtained by the chessboard method (the optimum ratio of osthole and berberine (referred to as berberine hydrochloride) was 1.25:1), and the synergy index FICI was 0.156.
As shown in FIG. 1H, the MIC of osthole and berberine used alone was > 160 μg/mL and > 256 μg/mL for methicillin-resistant Staphylococcus aureus ATCC33591 (MRSA 33591), respectively, and was reduced to 20 μg/mL and 8 μg/mL (osthole and berberine (berberine hydrochloride) respectively, calculated as the optimum ratio for achieving synergistic effect according to the chessboard method), and the synergy index FICI was 0.078.
As shown in FIG. 1I, for pseudo-intermediate staphylococci 31A, the MICs of osthole and berberine used alone were > 160 μg/mL and 128 μg/mL, respectively, calculated as the optimum ratio for achieving synergistic effect according to the chessboard 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 (referred to as berberine hydrochloride) was 1.25:1), and the synergy index FICI was 0.093.
As shown in J in FIG. 1, for pseudo-intermediate staphylococci 22-2, the MICs of osthole and berberine used alone were > 160 μg/mL and 64 μg/mL, respectively, calculated according to the optimum formulation for achieving synergistic effect by chessboard method, the MICs of osthole and berberine were reduced to 20 μg/mL and 8 μg/mL, respectively (the optimum formulation of osthole and berberine (referred to as berberine hydrochloride) was 2.5:1), and the synergy index FICI was 0.1875.
As shown in K in FIG. 1, for pseudo-intermediate staphylococci I1847, the MICs of osthole and berberine used alone were > 160 μg/mL and 64 μg/mL, respectively, calculated according to the optimum formulation for achieving synergistic effect by chessboard method, the MICs of osthole and berberine were reduced to 10 μg/mL and 8 μg/mL, respectively (the optimum formulation of osthole and berberine (referred to as berberine hydrochloride) was 1.25:1), and the synergy index FICI was 0.156.
As shown in L in FIG. 1, for pseudo-intermediate staphylococci U5149, the MICs of osthole and berberine used alone were > 160 μg/mL and 128 μg/mL, respectively, calculated according to the optimum formulation for obtaining synergistic effect by chessboard method, the MICs of osthole and berberine were reduced to 10 μg/mL and 8 μg/mL, respectively (the optimum formulation of osthole and berberine (referred to as berberine hydrochloride) was 1.25:1), and the synergy index FICI was 0.093.
As shown in M in FIG. 1, for pseudo-intermediate staphylococci K3247-1, the MICs of osthole and berberine used alone were > 160 μg/mL and 128 μg/mL, respectively, calculated as the optimum ratio for achieving synergistic effect according to the chessboard method, the MICs of osthole and berberine were reduced to 20 μg/mL and 8 μg/mL, respectively (the optimum ratio of osthole and berberine (referred to as berberine hydrochloride) was 2.5:1), and the synergy index FICI was 0.125.
As shown in N in FIG. 1, for Staphylococcus aureus 02016, the MIC of osthole and berberine used alone was > 160 μg/mL and > 128 μg/mL, respectively, calculated according to the optimum formulation for obtaining synergistic effect by chessboard method, the MIC of osthole and berberine was reduced to 20 μg/mL and 32 μg/mL, respectively (the formulation of osthole and berberine (referred to as berberine hydrochloride) was 0.625:1), and the synergy index FICI was 0.187.
As shown in FIG. 1O, the MIC of osthole and berberine used alone was > 160 μg/mL and 64 μg/mL, respectively, for Staphylococcus aureus 65577, calculated as the optimum ratio for achieving synergistic effect according to the chessboard method, the MIC of osthole and berberine was reduced to 20 μg/mL and 8 μg/mL, respectively (the ratio of osthole and berberine (referred to as berberine hydrochloride) was 2.5:1), and the synergy index FICI was 0.187.
As shown by P in FIG. 1, for Staphylococcus aureus USA300, the MICs of osthole and berberine used alone were > 160 μg/mL and 64 μg/mL, respectively, calculated according to the optimum ratio for achieving synergistic effect by chessboard method, the MICs of osthole and berberine were reduced to 20 μg/mL and 8 μg/mL, respectively, the ratio of osthole and berberine (referred to as berberine hydrochloride) was 2.5:1, and the synergy index FICI was 0.187.
Aiming at staphylococcus aureus, when the two are combined, the optimal ratio of osthole to berberine (berberine hydrochloride) is 0.625-2.5:1;
aiming at pseudo-intermediate staphylococcus aureus, when the two are combined, the optimal ratio of osthole to berberine (berberine hydrochloride) is 1.25-2.5:1.
The above test results show that the combined use of osthole and berberine has obvious synergistic antibacterial effect, and the bacteria specifically comprise staphylococcus aureus, pseudo-middle staphylococcus and clostridium perfringens, and the FICI is in the range of 0.078 to 0.25.
Example 2 synergistic inhibition of Candida albicans by the combination of osthole and berberine
Combination Index (FICI) of osthole and berberine for pathogenic bacteria: the FICI value of the standard control strain ATCC12301 strain against Candida albicans was determined using the checkerboard method in combination with osthole and berberine. The specific operation is as follows: FIC between different drugs was determined using a checkerboard broth dilution method. Single colonies of the fungi were picked in Sa glucose broth and grown at 27℃for 48h. The fungus turbidity is regulated to 0.5 by a Mitsubishi turbidimeter, and diluted to 1.0X103 CFUs/mL by a Satsubishi glucose culture medium for later use. 100 μl of glucose-over-the-air medium was added to the 96-well U-plate, and the corresponding concentrations of drug were added to the first 10 wells of the eighth row, followed by upward multiple dilution to the second row. Other drugs to be tested are added to each well of the first row and diluted from left to right in turn to the ninth row. And adding 100 mu L of diluted bacteria liquid to be tested into 1-10 columns. Columns 11 and 12 are negative and positive controls containing only the glucose medium and the bacterial liquid to be tested, respectively. After leaving the 96-well plate at 27℃for 24 hours for stationary culture, the results were read as MIC values for the drugs at the lowest drug concentration that inhibits fungal growth that was visually distinguishable.
FIC = MIC (combination a)/MIC (single a) +mic (combination B)/MIC (single B), criteria: FICI is less than or equal to 0.5, and the synergism is achieved; FICI is more than 0.5 and less than or equal to 1, and the addition effect is achieved; the FICI is less than 1 and less than or equal to 2, and the effects are irrelevant; FICI > 2, antagonism. Namely: fici=mic (berberine combination)/MIC (berberine alone) +mic (osthole combination)/MIC (osthole alone) of osthole and berberine.
As shown in FIG. 2, the MIC of the osthole and the berberine used alone to ATCC12301 is respectively more than 128 mug/mL and 128 mug/mL, calculated according to the optimal proportion of the synergistic effect obtained by the chessboard method, the MIC of the osthole and the berberine is respectively reduced to 8 mug/mL and 32 mug/mL, and the synergistic index FICI is 0.28, which indicates that the combined use of the osthole and the berberine has obvious synergistic antibacterial activity to candida albicans (aiming at candida albicans, and the optimal proportion of the osthole and the berberine (aiming at berberine hydrochloride) is 0.25:1 when the osthole and the berberine are combined).
Example 3 synergistic Sterilization curve of osthole in combination with berberine
3.1 test materials
Osthole and berberine are the same as in example 1.
3.2 in vitro Sterilization Curve test
MRSA33591 and pseudo-intermediate staphylococcal strain 31A were cultured in BHI broth for 6 hours, and then an equal volume of DMSO (final concentration of 0.5%), berberine, osthole and berberine mixed solution were added.
(1) For MRSA33591 strain, the groupings and dosages were as follows:
control group (control): 0.5% DMSO;
berberine group (Berberine): the final concentration of berberine is 16 mug/mL;
osthole group (Osthole): the final concentration of osthole is 40 mug/mL;
berberine and osthole combination group: the final concentration of berberine is 16 mug/mL, and the final concentration of osthole is 40 mug/mL.
Then, 100. Mu.L of the bacterial solutions were applied to 10cm diameter MHA agar plates at 1h, 3h, 6h, 12h and 24h, respectively, and colony counting was performed after overnight incubation.
(2) For the clinical pseudo-middle staphylococcus strain 31A, the groupings and dosages were as follows:
control group (control): 0.5% DMSO;
berberine group (Berberine): the final concentration of berberine is 16 mug/mL;
osthole group (Osthole): the final concentration of osthole is 20 mug/mL;
berberine and osthole combination group: the final concentration of berberine is 16 mug/mL, and the final concentration of osthole is 20 mug/mL.
Then, 100. Mu.L of the bacterial solutions were applied to 10cm diameter MHA agar plates at 1h, 3h, 6h, 12h and 24h, respectively, and colony counting was performed after overnight incubation.
3.3 test results
As shown in fig. 3, berberine and osthole alone had no significant effect on bacterial colony count at 24h compared to the control group for MRSA33591 strain. When berberine and osthole are combined for treatment, the colony number is 0CFU/mL at 1h, and 0Log at 24h 10 CFU/mL。
As shown in fig. 4, the berberine and osthole alone treatment had no significant effect on the bacterial colony count at 1h to 24h compared to the control group for the clinical pseudo-middle staphylococcus strain 31A strain. When berberine and osthole are treated in combination, the colony number is 1.91Log at 1h 10 CFU/mL, 3,6, 12 and 24h, colony numbers were 0Log 10 CFU/mL。
These data indicate that the combined treatment of berberine and osthole has obvious synergistic bactericidal function.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.

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.
CN202310683644.4A 2023-06-09 2023-06-09 A synergistic antibacterial and antifungal pharmaceutical composition containing natural products osthole and berberine Pending CN116687916A (en)

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