CN117180273A - Berberine synergistic SU3327 antibacterial activity and application thereof in preparation of anti-infective drugs - Google Patents

Abstract

The invention provides a novel application of berberine combined with an N-terminal kinase inhibitor SU3327 of C-JUN in preparing antifungal and bacterial infection medicines, and an application of a composition of SU3327 and berberine in preparing medicines with enhanced antifungal and bacterial infection efficacy. The invention not only proves the antibacterial activity of the berberine synergistic SU3327 through a chessboard minimum antibacterial concentration test and an in-vitro bacterial growth curve, but also proves the effectiveness of the combined use of the berberine and the SU3327 on the in-vivo treatment of the poultry candida albicans infection through clinical tests. The invention provides a new application of berberine synergistic SU3327 in treating antifungal and bacterial infection.

Description

Berberine synergistic SU3327 antibacterial activity and application thereof in preparation of anti-infective drugs

Technical Field

The invention relates to the field of medicines, in particular to the field of antibiosis, and particularly relates to an antibacterial activity of berberine synergistic SU3327 and application of berberine combined SU3327 in preparation of antibacterial medicines.

Background

Candida albicans (Candida albicans), an important conditionally pathogenic fungus, is usually present in the upper digestive tract of pigeons, is usually small in quantity in normal organisms and does not cause diseases, but is converted into pathogenic bacteria when the immune function or general defenses of the organisms are reduced or the normal flora structure is disordered, and causes harm to pigeons, especially the probability of young pigeons infection is particularly high. The disease is commonly called thrush, also known as mycotic stomatitis, candida stomatitis, sour crop disease and candidiasis, and is characterized in that a yellow-white cheese-like pseudofilm is generated on the mucosa of the esophagus and the crop of a diseased pigeon, and erosion and ulcer are visible after the pseudofilm is peeled off. When young pigeons are infected with Candida albicans, there are usually no obvious symptoms before the nest, obvious symptoms after the nest, obvious dry and hard, mental depression, loose feathers, unwilling to walk, reduced appetite and even abolished crop, poor growth, emaciation, and susceptibility to trichomonas disease, bacterial infection, as reported in the literature, candida albicans tasted secondary or co-infected with Staphylococcus aureus (Van Dyck, viela F, mathellie-Guinlet M, demulser L, hauben E, jabra-Rizk MA, vande Velde G, dufr E ne YF, krom BP, van Dijck P.adhesive of Staphylococcus aureus to Candida albicans During Co-Infection Promotes Bacterial Dissemination Through the Host Immune Response. Front Cell Informa 2, 10:624839), and eventually only as a residual pigeon. At present, in veterinary clinics, no effective therapeutic drug is directed against candida albicans infection or complex infection caused by candida albicans.

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.).

SU3327 (also known as halicin, hailixin, CAS No. 40045-50-9) is an N-terminal kinase inhibitor of C-JUN, the structure of which is shown in FIG. 1.SU3327 is a potent, selective and substrate-competitive JNK inhibitor with an IC50 of 0.7 μm. SU3327 also inhibits protein interactions between JNK and JIP at an IC50 value of 239 nM. SU3327 has low activity against p38α and Akt kinases. Previous studies have found SU3327 to be a potential antidiabetic agent (De SK, stebbins JL, chen LH, riel-Mehan M, machleidt T, dahl R, yuan H, emdadi A, barile E, chen V, murphy R, pellecchia M.design, synchesis, and structure-activity relationship of substrate competitive, selective, and in vivo active triazole and thiadiazole inhibitors of the c-Jun N-terminal kinase J Med chem.2009Apr 9;52 (7): 1943-52.). Another document has reported that SU3327 exhibits certain antibacterial activity against clinically common pathogenic bacteria such as enterococcus, pseudomonas aeruginosa, escherichia coli, staphylococcus aureus, etc. (Stokes JM, yang K, swanson K, jin W, cubillos-Ruiz A, donghia NM, macNair CR, french S, carfrae LA, bloom-Ackermann Z, tran VM, chiappino-Pepe A, badran AH, andrews IW, chord EJ, church GM, brown ED, jaakkola TS, barzila R, collins JJ.A Deep Learning Approach to Antibiotic discover.cell.202Apr 16;181 (2): 475-483.

Disclosure of Invention

In order to solve the problem of multiple drug-resistant gram-positive bacteria drug resistance and lack of effective drugs, the invention provides a natural product combination with obvious synergistic antibacterial effect;

the first aspect of the invention discloses a composition for resisting bacterial and fungal infection, and particularly adopts SU3327 in combination with berberine, which not only simply add functions, but also achieve synergistic antibacterial effect.

The minimum inhibitory concentration test and animal test of the chessboard method prove that the combination of SU3327 and berberine has obvious synergistic antibacterial and antifungal activity effects, and the bacteria in the test comprise staphylococcus aureus and methicillin-resistant staphylococcus aureus; the fungi in the test included candida albicans.

In a second aspect, the invention discloses a novel use of the above composition for the preparation of a medicament having antibacterial and antifungal efficacy.

Further, the above-mentioned drugs may be drugs against bacterial infectious diseases or drugs against fungal infectious diseases.

Further, the berberine is berberine or pharmaceutically acceptable salt thereof, and specifically can be berberine hydrochloride or berberine sulfate.

Further, the fungus is candida albicans.

Further, the bacteria are gram positive bacteria.

Further, the gram positive bacteria comprise staphylococcus aureus.

Further, the bacterium is staphylococcus aureus; preferably, the staphylococcus aureus comprises methicillin-resistant staphylococcus aureus.

Further, the fungus is candida albicans.

The medicine for resisting bacterial infection diseases or fungal infection diseases provided by the invention comprises SU3327 and berberine.

Further, the mass ratio of SU3327 to berberine is (1.25-10) (1.8-14.4).

The active ingredients in the medicine can be SU3327 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 above medicine can kill and/or inhibit bacteria and fungi;

the dosage form of the medicine 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, other antimicrobial products containing the above antimicrobial compositions are also within the scope of the present invention.

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.

The invention opens up a new application for the combined medication of SU3327 and berberine, namely the combination 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.

Drawings

FIG. 1 shows the results of a combined antimicrobial chessboard method of Berberine (Berberine) combined with SU3327 on methicillin-resistant Staphylococcus aureus.

FIG. 2 provides the synergistic antimicrobial chessboard results of Berberine (Berberine) in combination with SU3327 on Candida albicans ATCC 12301.

FIG. 3 is a representative view of a clinical spontaneous infection with Candida albicans infection cut-through.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention 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 invention 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.

The berberine used in the following experiments is berberine hydrochloride, namely berberine hydrochloride with CAS number 633-65-8, and the molecular formula is: C20H18ClNO4, molecular weight 372.82, purchased from Ab Ding Shiji company, purity not less than 98%. SU3327 was purchased from MCE reagent company, USA, purity not less than 99%. A certain amount of berberine hydrochloride is weighed to be prepared into an aqueous solution with the concentration of 128mg/mL of mother solution. SU3327 was formulated as a mother liquor with a mother liquor concentration of 40mg/mL using DMSO. All mother liquor was prepared and stored in a-20 degree refrigerator.

Pathogenic bacteria used in the following experiments:

methicillin-resistant staphylococcus aureus ATCC 33591 (MRSA 33591) is provided by the national veterinary drug safety evaluation center (beijing).

Candida albicans standard finger control bacteria ATCC12301 was purchased from Shanghai complex biotechnology company.

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 combination of SU3327 and berberine

Combination Index (FICI) determination of SU3327 and berberine hydrochloride combination against pathogenic bacteria: the combined antimicrobial effect of the combined application of SU3327 and berberine hydrochloride on staphylococcus aureus ATCC29213, methicillin-resistant staphylococcus aureus ATCC 33591 (MRSA 33591) was determined using a checkerboard method.

Specifically, methicillin-resistant staphylococcus aureus ATCC 33591 (MRSA 33591) strain was normally cultured using MHB broth medium. The chessboard method specifically comprises the following steps: SU3327 (as drug A) and berberine (as drug B) are diluted by MHB broth culture medium with the maximum concentration of 80 μg/mL and 128 μg/mL respectively, 50 μl of MHB broth culture medium containing two drugs with different concentrations is added along the transverse axis and the longitudinal axis of 96 microwell culture plates respectively, and then 50 μl of various pathogenic bacteria bacterial solutions are added respectively, so that the final bacterial count per well is 2×10 ⁵ CFU, incubation at 37℃for 24h, and observations were made. RecordingThe Minimum Inhibitory Concentrations (MIC) of the two drugs, when used alone and in combination, respectively, were calculated as FICI values (partial inhibitory concentration index) according to the following formula.

The results were as follows:

as shown in FIG. 1, the MICs of SU3327 and berberine hydrochloride used alone were > 20 μg/mL and > 128 μg/mL, respectively, for methicillin-resistant Staphylococcus aureus ATCC 33591 (MRSA 33591), the MICs of SU3327 and berberine hydrochloride were reduced to 5 μg/mL and 64 μg/mL, respectively, calculated according to the optimum formulation of the chessboard method to obtain the synergistic effect (i.e., the optimum formulation of SU3327 and berberine was 1:11.5), the synergy index FICI was 0.5, and it was determined that the synergistic effect was obtained.

Example 2 synergistic inhibition of Candida albicans by the combination of SU3327 and berberine

Combination Index (FICI) determination of SU3327 and berberine hydrochloride combination against pathogenic bacteria: the FICI value of the standard control strain ATCC12301 strain for Candida albicans was determined using the checkerboard method for the combined application of SU3327 and berberine hydrochloride. The specific operation is as follows: FICI 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.

Fici=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 (SU 3327 combination)/MIC (SU 3327 alone) of SU3327 and berberine.

The results are shown below:

as shown in FIG. 2, the MIC of each of SU3327 and berberine hydrochloride is 40 mug/mL and 128 mug/mL respectively, calculated according to the optimal proportion of the synergistic effect obtained by the chessboard method, the MIC of each of SU3327 and berberine is reduced to 2.5 mug/mL and 16 mug/mL (namely SU3327 and), and the synergistic index FICI is 0.1875, wherein the mass ratio of SU3327 to berberine hydrochloride is (1.25-10) and the mass ratio of SU3327 to berberine hydrochloride is (2-16) respectively, namely, the mass ratio of SU3327 to berberine hydrochloride is (1.25-10) and the mass ratio of SU3327 to berberine hydrochloride is (1.8-14.4), which indicates that the combined use of SU3327 and berberine has obvious synergistic antibacterial activity on candida albicans.

EXAMPLE 3 evaluation of the efficacy of SU3327 and berberine alone and in combination in the treatment of clinical poultry Candida albicans infection

(1) Grouping and handling of animals

The young pigeons for the experiment are provided by a young pigeon farm in Jiangmen city of Guangdong province. The experiment mainly uses clinic disease young pigeons with ages of 25 days to 28 days, and the main clinical symptoms are that the young pigeons have ulcers and yellow cheeses in oral cavities, and white disease ulcer-like lesions with different degrees of crop, lung and other organs are detected by cutting (see figure 3, representative cutting diagram of the diseased pigeons).

The weight of 450 g reaches the standard of marketing, and the young pigeons with the weight of less than 300 g generally have other diseases, so that the body organ is exhausted and can not be used as an experimental body, therefore, the ill pigeons are subjected to certain screening according to the experimental requirement, and finally 64 young pigeons with the weight of 300 g-400 g are selected, and the experiment is divided into 4 groups of 16 young pigeons.

The detailed groupings and dosing were as follows:

placebo control group: a corresponding solvent control, 1mL of sodium carboxymethyl cellulose also 0.5% was given.

Berberine hydrochloride in combination with SU3327 treatment group: oral administration, in accordance with SU3327 to berberine hydrochloride ratio 1: the ratio of 6.4 was followed, and finally, SU3327 was administered at a dose of 3.12mg/kg body weight and berberine hydrochloride was administered at a dose of 20mg/kg body weight (corresponding to 18mg/kg body weight).

Berberine alone group: the berberine hydrochloride solution is orally administered to each pigeon, and the dosage is 20mg/kg body weight, which is equivalent to 18mg/kg body weight of berberine.

SU3327 treatment group alone: each pigeon was dosed orally with SU3327 solvent at a dose of 3.12mg/kg body weight.

The treatment period is 10 days, 1 time every two days, and total 5 times.

(2) Therapeutic Effect evaluation criteria

(1) Sensory judgment

Observing the mental state, the color change of the false oral membrane, the crop plumpness and the body type emaciation degree of the pigeon;

and observing the fecal condition of pigeons.

Judging the recovery degree of pigeons:

complete recovery: normal feeding, full body shape recovery and reaching the market standard;

semi-rehabilitation: normal feeding, incomplete recovery of body shape, and failure of marketability;

marasmus disease pigeon: the crop has no feed, emaciation, and poor mental state.

For pigeons with complete recovery, half recovery and emaciation, one pigeon is randomly extracted from each group for dissection, and the oral cavity, the crop and each viscera are observed.

(3) Test results

The treatment effect is shown in table 1, the placebo control group shows no obvious improvement of pigeon symptoms, wherein 6 pigeons die and the recovery amount is 0 pigeons; SU3327 treatment group, 1 death, 7 recovery number, recovery rate 43.75%; the number of the berberine single treatment groups is 8, and the recovery rate is 50%; SU3327 combined with berberine treatment group, recovery number is 14, recovery rate is 87.5%. The results show that the SU3327 and berberine combined treatment has obvious advantages when being singly treated, and the mass ratio of the SU3327 to the berberine is 1:5.8.

TABLE 1 evaluation of the Effect of SU3327 in combination with berberine on treatment of Candida albicans infection in pigeons

The above examples of the present disclosure are merely examples for clearly illustrating the present disclosure and are not limiting of the embodiments of the present disclosure. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. Any modifications, equivalent substitutions, improvements, etc. that fall within the spirit and principles of the present disclosure are intended to be included within the scope of the claims of the present disclosure.

Claims (9)

1. An antibacterial and antifungal composition comprises berberine and N-terminal kinase inhibitor SU3327 of C-JUN.

2. Use of a composition according to claim 1 for the preparation of a medicament having antibacterial and antifungal efficacy.

3. The composition of claim 1 or the use of claim 2, wherein the bacteria are gram positive bacteria.

4. The composition of claim 1 or the use of claim 2, wherein the bacteria comprise staphylococcus aureus.

5. The composition of claim 1 or the use of claim 2, wherein the bacteria are one or more of staphylococci; preferably, one or more of the staphylococci is resistant to the presence of methicillin Lin Duochong.

6. The composition of claim 1 or the use of claim 2, wherein the fungus is one or more of candida; preferably, the candida is candida albicans.

7. The composition according to claim 1 or the use according to claim 2, wherein the mass ratio of SU3327 and berberine is (1.25-10): (1.8-14.4) or 1:11.5.

8. the composition of claim 1 or the use of claim 2, wherein the final animal therapeutic dose of SU3327 and berberine is 3.12mg/kg and 18mg/kg body weight, respectively, in a ratio of 1:5.8.

9. the use according to claim 2, wherein the pharmaceutical dosage form is one of a tablet, a cream, an ointment, a sustained release tablet, a controlled release tablet, an oral liquid, a syrup, an injection dosage form, a dripping pill, and a lyophilized powder injection dosage form.