Pharmaceutical combination of punicalagin

Abstract

The present invention relates to the use of punicalagin, such as anti-inflammatory and / or anti-Staphylococcus aureus, in particular anti-MRSA infection, including local and systemic infections, such as septicopyemia. Preferably, the medicine is combined with beta-lactam for use. The applicant discovers that PA can resist inflammation, has a unique staphylococcus aureus resisting mechanism, and is not easy to cause evolution and drug resistance of the staphylococcus aureus.

Description

BACKGROUND

[0001] Staphylococcus aureus (S. aureus) is a kind of bacteria that can cause a variety of infectious diseases in humans. S. aureus is the primary cause of bacterial infection-related deaths in about 135 countries and is the most common bacterial pathogen causing death in people over 15 years of age worldwide. In particular, methicillin-resistant S. aureus (MRSA) has a wide drug resistance spectrum and a high drug resistance rate. As the main pathogenic bacteria causing community-acquired and hospital-associated bacteremia, the mortality rate is as high as 15%-60%, making the treatment of MRSA a very difficult problem in clinical practice. With the further deterioration of antibiotic resistance worldwide, S. aureus infection has become a global public health threat. Related research reports on vancomycin-resistant MRSA strains suggest that controlling and blocking MRSA infection is an imminent global public health event. It is urgent to reverse the trend of antibiotic resistance, otherwise we will face a situation where there is no medicine to treat. In the post-antibiotic era, antibiotic replacement therapy is on the rise, and developing new antibiotic substitutes or implementing drug combination is one of the more promising implementation plans.

[0002] β-lactam antibiotics are commonly used but easily resistant antibiotics, such as cefoperazone (Cef). One solution to resistance is to combine it with a β-lactamase inhibitor, such as sulbactam (Sul).

[0003] Punicalagin (PA) is a new type of antibacterial compound. After extensive search of patents and other literature, there is little research on its mechanism of resistance to S. aureus. There are also few reports on the combination of PA and other antibiotics for the treatment of MRSA, and there is no report on the treatment of systemic infection of S. aureus. SUMMARY

[0004] The present applicant has found that PA has a unique mechanism of action against S. aureus (especially MRSA) and is not prone to cause the bacteria to evolve or become resistant. Through experiments, it has been found that PA can inhibit the proliferation of MRSA in vitro, slow down the generation and progression of resistance, and to some extent reduce the damage to cells and organisms after bacterial infection. In particular, the combination of PA and β-lactam antibiotics, especially Cef, has a significant synergistic inhibitory effect on the proliferation of MRSA. When using the same dose of Cef to inhibit bacterial growth and exert cell protection, PA can reduce the antibiotic usage by about 50%. In in vivo animal experiments, the combination of PA and Cef can significantly improve the pathological manifestations and inflammatory responses caused by resistant bacteria infection in vivo, alleviate the morphological damage and functional disorders of multiple organs caused by bacterial infection, and the overall therapeutic effect is similar to that of Cef + Sul standard treatment at the same dose, and has stronger activity in improving inflammatory response, inhibiting the generation and progression of bacterial resistance, especially in the aspect of antibiotic resistance gene transmission. PA helps to inhibit the transmission of related β-lactam antibiotic resistance genes through the intestinal-feces-environment pathway. The present application proves the inhibitory effect of PA and Cef on the proliferation of multi-drug resistant S. aureus through bacterial experiments, in vitro cell experiments and in vivo animal experiments, and because PA has high antibacterial activity and is not prone to induce resistance, it can be used as an ideal candidate drug or therapeutic adjuvant for future drug development.

[0005] Therefore, the present application discloses PA for preventing and / or treating S. aureus (especially MRSA) infection, including local and systemic infection, such as sepsis.

[0006] Specifically, the present application provides a combination of PA and β-lactam antibiotics for antibacterial activity, wherein the β-lactam antibiotics include penicillin, cephalosporin and atypical β-lactam antibiotics, the penicillin can be selected from penicillin G, amoxicillin and piperacillin, the cephalosporin can be selected from ceftriaxone, cefaclor, ceftizoxime, cefodizime, cefatroxel, cefazolin, cefdinir, cefixime, cefradine and cefoperazone, and the atypical β-lactam antibiotic can be cefoxitin or aztreonam.

[0007] Further, the present application provides a combination of PA and β-lactam antibiotics, which comprises PA and β-lactam antibiotics in a mixed or physically isolated form.

[0008] It should be noted that the active molecules mentioned in the present application can be provided in a pharmaceutically acceptable alternative form, such as a pharmaceutically acceptable salt thereof, an analogue thereof, a metabolite thereof. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1 Intervention of PA, Cef and Sul on bacterial growth

[0010] Figure 2 , after successive induction of passage by different concentrations of PA, the inhibitory effect of PA on the growth of each generation of MRSA 600 value, the Punicalagin 256 μg / mL curve in the figure partially coincides with the Control curve.

[0011] Figure 3 , after successive induction of passage by different concentrations of PA, the inhibitory rate of PA on each generation of MRSA at different time points

[0012] Figure 4 , the inhibitory effect of different drugs on each generation of MRSA after successive induction

[0013] Figure 5 , heat map of relative expression of cell inflammatory factors and macrophage classification biomarkers (using the non-intervention group as control and GAPDH as internal reference gene)

[0014] Figure 6 , body weight changes of each group in the abdominal infection experiment

[0015] Figure 7 , mouse liver HE staining chart

[0016] Figure 8 , mouse colon HE staining chart (cross section)

[0017] Significance analysis involved in each chart:

[0018] ns: no significant difference, *: P≤0.05, **: P≤0.01, ***: P≤0.001. DETAILED DESCRIPTION

[0019] The embodiments of the present application will be described in detail below in conjunction with experimental examples, but the following experimental examples are only used to illustrate the present application, and should not be considered as limiting the scope of the present application. The MRSA bacteria used in the experiment are preserved in the laboratory of the present team. Unless otherwise specified, the reagents, biological materials, culture media and solutions used below are commonly used in the art, publicly available or commercially available.

[0020] Experiment 1: Drug synergy test

[0021] Experimental method: microdilution method was used to detect the minimum inhibitory concentration (MIC) of the laboratory MRSA strain to different antibiotics; chessboard method was used to detect the fractional inhibitory concentration index (FICI) of PA and different antibiotics to MRSA.

[0022] I. Determination of MIC

[0023] The test antibiotic or compound PA was diluted twofold in a 96-well plate to a final concentration of 512 μg / mL–0.125 μg / mL, with 100 μL of solution per well. 100 μL of MHB (Muller-Hinton broth) medium was added to each well in the control group, and 100 μL of a 2×10⁻⁶ concentration was added to each well in the experimental group. 6 MRSA bacterial suspensions at CFU / mL were incubated at 35℃ for 24 hours. The control group was compared with the control group, and the minimum drug concentration (MIC) was defined as the concentration at which no statistically significant difference in OD value was observed. The results are shown in Table 1 below.

[0024] MIC (μg / mL) of a compound or antibiotic

[0025]

[0026] II. Determination of FICI

[0027] (1) Adjust the bacterial concentration to 2×10 6 CFU / mL.

[0028] (2) Label the 96-well plate as rows A-G and columns 1-9. Add 200 μL of MHB medium to each well in column 1 as a blank control. Add 50 μL of 4×MIC antibiotic solution to each well in columns 2. Serially dilute the solution 2-fold in each well from columns 3 to 8, so that the antibiotic concentrations in columns 2 to 8 are 4×MIC, 2×MIC, and 1×MIC, respectively. and Add 50 μL of 4×MIC, 2×MIC, and 1×MIC solutions sequentially to each well in rows A through G. MIC and Add 100 μL of PA solution with a concentration of 2 × 10⁻⁶ to each of the above wells. 6 With a bacterial culture of CFU / mL, rows A to G and columns 2 to 8 each constitute a 1×MIC. The concentration gradient was set, and 100 μL of bacterial solution and 100 μL of MHB medium were added to each well in column 9 as a control group.

[0029] (3) Construct 96-well plates for the combined experiments of PA and various antibiotics according to the above steps, and measure OD. 600 The value is calculated according to the FICI formula:

[0030]

[0031] In the formula The ratio of the MIC value obtained when antibiotics are used in combination with PA to the MIC value obtained when antibiotics are used alone. The ratio of the MIC value of PA combined with antibiotics to the MIC value of PA alone. According to the standard of FICI < 0.5 for synergistic effect; 0.5 < FICI < 1 for additive effect; 1 < FICI < 2 for irrelevant effect; and FICI > 2 for antagonistic effect, the subsequent drugs are identified and screened.

[0032] The experimental results are shown in Table 2.

[0033] Table 2 FICI determination results of each antibiotic combined with PA

[0034]

[0035]

[0036] Experiment 2: In vitro bacteriostatic effect determination of PA combination

[0037] Experimental method:

[0038] (1) Prepare MRSA bacterial solution with a concentration of 2 x 10 6 CFU / mL.

[0039] (2) Prepare Sul, PA, and Cef solutions with a concentration of 20 μg / mL; prepare Cef solution with a concentration of 40 μg / mL; prepare Cef+PA mixed solutions with concentrations of (5+20), (10+20), (15+20), and (20+20) μg / mL, respectively; and prepare Cef+Sul mixed solution with a concentration of (20+20) μg / mL.

[0040] (3) Take centrifuge tubes, set up 10 groups as shown in Table 3, and the final concentration of the bacterial solution in each group is 1 x 10 6 CFU / mL. Incubate in a constant temperature shaker, take samples at 2 h, 4 h, 6 h, 8 h, and 12 h, respectively, dilute with PBS, and plate, and perform colony counting the next day. The experimental results are shown in Table 3. Figure 1

[0041] Table 3 In vitro bacteriostatic experiment grouping and composition

[0042]

[0043] Note: * The concentration of the bacterial solution is 2 x 10 6 CFU / mL.

[0044] Experiment 3: Drug resistance induction experiment

[0045] Experimental method:

[0046] ① Prepare MRSA bacterial solution with a concentration of 2 x 10 6 CFU / mL.​

[0047] ② Prepare PA solutions with concentrations of 512 (2×MIC), 256 (1×MIC), 128, 64, 32, 16, and 8 μg / mL;

[0048] ③ Take centrifuge tubes and add 3 mL of PA solution of different concentrations, then add an equal amount of 2 × 10⁻⁶. 6 The bacterial suspension was mixed with CFU / mL and incubated on a constant temperature shaker for 12 hours before sampling. After bacterial subculturing, the above steps were repeated to obtain MRSA bacteria that multiplied under different concentrations of PA pressure for nine consecutive generations.

[0049] ④ Based on the microdilution method for determining MIC, a blank control group, an MRSA group, and intervention groups with different concentrations of PA were set up in a 96-well plate. After incubation at a constant temperature for 24 hours, OD was measured. 600 Record the absorbance value.

[0050] ⑤ Following the method in Experiment 2, the plate coating method was used to take samples at 2h, 4h, 6h, 8h, and 12h to determine the inhibition rate:

[0051]

[0052] In the formula, ρ0 is the initial concentration of MRSA, ρ 实验组 and ρ 对照组 These represent the colony counts of the experimental group and the control group at a specific time point.

[0053] ⑥ Prepare separate solutions of 40 μg / mL Cef, (20+20) μg / mL Cef+PA, and (20+20) μg / mL Cef+Sul. Following the method described in step ③ of this experiment, subculture the bacteria under each drug intervention to obtain nine consecutive generations of MRSA strains grown under different drug interventions. MIC and OD values ​​were then determined using the microdilution method. 600 value.

[0054] Experimental results are as follows Figures 2-4 As shown, the experimental results indicate that MRSA exhibits partial adaptation to continuous antibiotic intervention, while it can effectively prevent the development of resistance to the natural active small molecule compound PA under continuous action.

[0055] Experiment 4: PA Intervention Cell Experiment

[0056] Experimental methods:

[0057] ① Cell culture: Human monocytic leukemia cells (THP-1 cell line) were selected for cell passage.

[0058] ② Induction of differentiation: THP-1 suspension cells were induced with phorbol ester (PMA), and the morphology and adhesion of THP-1 macrophages were observed.

[0059] ③ Cell intervention: Prepare THP-1 macrophages and MRSA bacterial culture, and set up 8 experimental groups as shown in Table 4.

[0060] ④ Cell viability calculation: After setting the intervention, the cells were incubated, and the approximate cell viability was calculated according to the standard formula. The results are shown in Table 5.

[0061] ⑤ Perform cell intervention experiments as per experimental steps ② and ③. After 6 hours, extract total RNA from cells and use a qPCR kit to quantitatively detect inflammatory factors and typical biomarkers for macrophage typing (IL-1β, IL-6, IL-10, TNF-α, CD80, CD86, CD163, and CD206) as well as the internal reference gene GAPDH. The anti-inflammatory effect is as follows: Figure 5 As shown.

[0062] Table 4 Grouping of Cell Intervention Experiments

[0063]

[0064]

[0065] Note: * Cell density is 2×10 5 cells / mL # The bacterial concentration is 2×10 6 CFU / mL.

[0066] Table 5. Cell survival rate (%) of each intervention group after 6 hours )

[0067]

[0068] Experiment 5: In vivo antibacterial activity of PA

[0069] Laboratory animals: 4-week-old male ICR rats, SPF grade.

[0070] In vivo MRSA infection model of systemic sepsis: Mice injected intraperitoneally with MRSA bacterial solution exhibited pathological conditions such as conjunctival congestion, erect hair, curled-up body, reduced activity, loose stools, and significant weight loss, which can be considered as successful model establishment.

[0071] Experimental groups: As shown in the table below, each group consisted of 10 mice.

[0072] Table 6 Grouping of in vivo animal experiments

[0073]

[0074] Experimental method:

[0075] ① According to the experimental grouping: A group of each mouse intraperitoneal injection of 500 μL sterile saline, B ~ H group of mice in each group of intraperitoneal injection of bacteria 500 μL, 1 h after A group and B group of mice injected with 500 μL sterile saline, C ~ H group of mice according to the experimental grouping injection of equal amounts of drugs, the 25th and the 49th again to implement intervention. Injection of bacteria every 12 h observation of physiological activity of mice, during the control of the amount of feed (, record weight and physiological activity changes, collection of feces, observation of feed and water consumption.

[0076] ③ ② from the start of the experiment 72 h, all mice were anesthetized and cervical dislocation method to kill mice, dissection of mice, blood, intestine, liver, spleen, kidney, lung and fresh feces, etc. to observe and record the morphological changes of organs (such as abnormal size, abscess formation, weight changes, etc.), weighing to calculate the organ coefficient:

[0077]

[0078] ④ Bacterial load determination: 0.1 g of organs / 100 μL of blood, grinding into homogenate. After 10-fold dilution of homogenate, take 100 μL evenly coated on Baird-Parker (gold staphylococcus differential medium) plate, after culture colony counting, the number of colonies within 30 to 300 is considered to be valid count, calculate the bacterial load in organs (CFU / g), the formula is:

[0079]

[0080] Experimental results (1) basic physiological changes of experimental animals:

[0081] Weight changes such as Figure 6As shown, PA treatment group can effectively reduce the weight drop caused by MRSA infection within 24h, and the combined drug treatment group (G and H group) has a significantly higher effect on weight loss than single drug treatment group. From the physiological point of view, the control group of mice has normal activity, good food, water and other physiological activities, and no obvious abnormalities, while the MRSA group of mice has typical conjunctival congestion and occlusion, rough hair, body curl, significantly decreased activity, and generally appears to have a rectal adhesive pad of loose stool and visible blood stool phenomenon, some mice have convulsions, convulsions and other nervous system dysfunction, and the average daily food intake of mice is much lower than 5g, suggesting a more serious multi-organ bacterial infection feature. PA and Cef drug single treatment group has limited improvement on the above conditions, while the drug combination group, especially the G group and the H group, has a larger range of pathological changes, and no obvious conjunctival congestion, loose stool and decreased activity, suggesting effective treatment. The DAI score of each group of mice was scored, and the scoring standard is shown in Table 7, and the results are consistent with the physiological activity changes of the observed mice. The drug combination group has obvious advantages in reducing the DAI score.

[0082] Table 7 DAI score standard

[0083]

[0084]

[0085] Experimental results (2) organ coefficient as shown in Table 8:

[0086] Table 8 organ coefficient of each group (%)

[0087]

[0088] Experimental results (3) organ bacterial load:

[0089] Table 9 organ bacterial load of each group of animals log 10 CFU / g)

[0090]

[0091]

[0092] Experimental results (4) organ pathological changes:

[0093] The drug alone treatment group can make the colon shortening part caused by MRSA infection to be extended, and the drug combination group can make the colon to restore the length. The difference in colon length between group G and group E has statistical significance, and the colon length of group G is longer than that of group E (P<0.001). The kidney morphology is observed. The MRSA infection group has obvious abscess formation, and part of the ulcer and blood exudation appear. The kidney of the PA group has local small abscess and slight blood exudation. The difference in kidney morphology between the other groups is small. The liver morphology is observed. The liver of the control group is brown, the edge is smooth, and under the microscope, it has luster, no blood exudation and tissue structure damage. The liver of the MRSA infection group has a large amount of blood exudation, the color is deepened, and small abscess formation appears. The liver of the PA group is darker than that of the control group, and there is local small tissue damage and slight blood exudation. The difference in morphology between the other groups is small, and the color is close to normal.

[0094] The results of liver HE staining are shown in Figure 7 The results of colon HE staining are shown in Figure 8

[0095] Experimental results (5) determination of liver inflammatory factors:

[0096] Table 10 average expression of mouse liver genes relative to control group (GAPDH as internal reference gene)

[0097]

[0098] Experimental results (7) survival analysis:

[0099] Experimental method:

[0100] ① 4-week-old ICR male mice, SPF level.

[0101] ② Set the same experimental groups as the above infection model, and increase the bacterial liquid concentration to 2x10 9 CFU / mL, and inject 500 μL of bacterial liquid into the abdominal cavity of the mice except the control group. After 1 h of bacterial infection, the mice are treated with drugs according to the experimental grouping, and the drug administration is performed once every 24 h. The survival and death of the mice are recorded for 144 h.

[0102] Experimental results: the 7-day survival rate of the MRSA infection group is only 12.5%, and the death phenomenon is concentrated within 72 h after the experiment starts. The 7-day survival rate of the PA treatment group is 25%, and the 7-day survival rate of the Cef (20 mg / kg) treatment group is increased to 62.5%. The 7-day survival rate of the combined treatment F group is 75%, and the death phenomenon is concentrated within 36 h. The 36 h protection rate is good, and the 7-day survival rate of the combined treatment G and H groups is 100%.

[0103] Experiment 6: PA anti-MRSA mechanism research

[0104] ​6.1 Srap L-lectin module binding

[0105] Serine-rich platelet adhesion factor (SraP) is a kind of cell wall anchored protein, and the L-Lectin module in the ligand binding region of SraP protein can specifically bind to the sialylated receptor (Neu5Ac) on the cell membrane surface.

[0106] Experimental method:

[0107] (1) SraP L-Lectin conservative analysis

[0108] (2) CADD technology targets SraP L-Lectin module: molecular docking is performed, the position of Neu5Ac binding to L-lectin is referred to, and the binding effect of PA and L-lectin is evaluated.

[0109] (3) The binding of SraP L-Lectin and PA is detected by surface plasmon resonance (SPR)

[0110] Experimental results:

[0111] (1) We randomly selected 48 strains of MRSA preserved in the laboratory, including 24 clinical isolates, 23 food isolates and 1 community-associated strain (MW2), and detected the conservation of the L-Lectin module carried by them. The results showed that the L-Lectin module was highly conserved among S. aureus strains; the key amino acid sites interacting with Neu5Ac were completely conserved in different strains.

[0112] (2) Molecular docking found that PA was more likely to bind to L-ectin module than Neu5Ac. At the same time, PA can occupy the spatial position of L-Lectin binding to Neu5Ac, thereby blocking the binding of L-Lectin and Neu5Ac.

[0113] (3) SPR measured the KD of PA and L-Lectin module as 5.656 x 10 -7 M, which is much smaller than that of Neu5Ac and L-Lectin.

[0114] 6.2 RNA-seq sequencing result mining and verification

[0115] Experimental method:

[0116] PA antibiotic was used to induce MW2 bacteria according to the conventional method, a control group was set, and RNA was extracted for transcriptomic sequencing and analysis.

[0117] Experimental results:

[0118] (1) After PA treatment, the expression levels of MW2 genes changed significantly, with 286 genes significantly down-regulated and 264 genes significantly up-regulated.

[0119] (2) KEGG annotation was performed on the genes in this transcriptome, and 100 KEGG pathways were obtained, among which the pyrimidine metabolism pathway and the Caulobacter cell cycle pathway were significantly enriched (P < 0.05), and the enrichment of other pathways was not significant (P > 0.05).

[0120] (3) After PA intervention, the expression levels of many virulence genes of MW2, including surface proteins and exogenous toxins, changed significantly. For example, the expression levels of cell wall anchor proteins related to adhesion such as spa, clfB, eap, sraP, fnbB, etc. were significantly down-regulated, and the expression levels of exogenous toxin genes such as alpha toxin gene hly and leukocidin-related gene lukS-PV were also significantly down-regulated.

[0121] (4) According to the transcriptomic results, it was found that after PA treatment, the expression levels of multiple genes in the pyrimidine metabolism pathway of MW2 were down-regulated, especially in the de novo synthesis pathway of pyrimidine nucleotides. qRT-PCR was used to verify these differentially expressed genes, and the results showed that after PA treatment, pyrB, pyrC, pyrD, pyrE, pyrF, carA, etc. involved in the de novo pathway of pyrimidine nucleotides were down-regulated, and the transcriptomic and qRT-PCR detection results were highly consistent.

[0122] (5) After PA treatment, the expression levels of surface protein genes such as sraP, fnbB, sdrD, spa, clfB, etc. were significantly down-regulated, and the expression of the auxiliary regulatory factor agrB was significantly up-regulated, and the qRT-PCR verification results were consistent with the transcriptomic results.

[0123] (6) After PA treatment, the expression levels of penicillin-binding protein genes pbp2, mecA, fmt1, penicillin-binding protein-related factor reuC, and peptidoglycan biosynthesis-related genes murF, fmhC were significantly down-regulated.

[0124] 6.3: Spread of antibiotic resistance genes of intestinal flora

[0125] Experimental method:

[0126] After collecting the feces of mice in Experiment 5, DNA was extracted and qPCR technology was used to detect the expression amount of drug resistance genes.

[0127] Experimental results:

[0128] As shown in Table 11, compared with the MRSA infection group, after PA intervention, the structural gene mecA encoding penicillin-binding protein PBP2a, pbpA and pbp2 and related regulatory genes mecR1 and femA all decreased, and the expression amount of β-lactam antibiotic binding protein fmt1 also decreased, suggesting that PA intervention reduced the resistance of β-lactam antibiotics represented by methicillin, and also inhibited the development of potential drug resistance. The expression amount of the related drug resistance genes in the Cef intervention group was higher than that in the MRSA infection group and the PA intervention group, and thus was positively correlated with the development of drug resistance. Moreover, the expression amount of the drug resistance genes in the drug combination group with or without PA intervention was consistent with the above results. For example, after PA and Cef intervention, the expression amount of the drug resistance genes was lower than that in the drug combination group without PA intervention, suggesting that PA intervention inhibited the spread of antibiotic resistance genes.

[0129] Table 11 Expression amount of genes relative to the MRSA infection group (using 16S rRNA as the internal reference gene)

[0130]

Claims

1. punicalagins and β - the use of a lactam antibiotic for the preparation of a pharmaceutical composition for the treatment of inflammation and / or for the treatment of Staphylococcus aureus, β - the lactam antibiotic is selected from the group consisting of penicillins selected from the group consisting of penicillin G, amoxicillin, piperacillin and / or pharmaceutically acceptable salts thereof, cephalosporins selected from the group consisting of ceftriaxone, cefaclor, ceftazidime, cefodizime, cefditoren pivoxil, cefazolin, cefdinir, cefixime, ceftizoxime, cefoperazone and / or pharmaceutically acceptable salts thereof and non-typical beta-lactam antibiotics.

2. Use according to claim 1, wherein the compound is ###0001### or a pharmaceutically acceptable salt thereof. The atypical β - the lactam antibiotic is selected from the group consisting of cefoxitin or aztreonam and / or a pharmaceutically acceptable salt thereof.

3. Use according to claim 1 or 2, characterised in that The Staphylococcus aureus is MRSA.

4. Use according to claim 1, characterized in that, The pharmaceutical composition is used for treating sepsis.

5. The use according to claim 4, wherein the compound is ###0002### The sepsis is caused by MRSA.

6. Use according to any of the preceding claims, characterised in that The medicine is used for treating local or systemic infection.

7. The use according to claim 1, wherein the compound is ###0002### The medicine can reduce the expression of proinflammatory cytokines or / and M1 macrophage biomarkers, and / or, increase the expression of anti-inflammatory cytokines or / and M2 macrophage biomarkers, the proinflammatory cytokines can be selected from IL-1β, IL-6, TNF-β and TNF-α, the anti-inflammatory cytokine can be IL-10, the M1 macrophage biomarker can be selected from CD80 and CD86, and the M2 macrophage biomarker can be selected from CD163 and CD206.

8. Use according to any of the preceding claims, characterised in that In the pharmaceutical composition, the angoroside and β - mixed or physically isolated from the lactam antibiotic.

9. a pharmaceutical composition comprising maslinic acid and β - a pharmaceutical composition of a lactam antibiotic, β - the lactam antibiotic is selected from the group consisting of penicillins selected from the group consisting of amoxicillin, piperacillin and / or pharmaceutically acceptable salts thereof, cephalosporins selected from the group consisting of cefaclor, ceftizoxime, cefotetan, cefditoren pivoxil, cefazolin, cefdinir, cefixime, ceftizoxime, cefoperazone and / or pharmaceutically acceptable salts thereof and aztreonam.

10. A pharmaceutical composition according to the preceding claim, characterized in that its punicalagins and β - mixed or physically separated from lactam antibiotics.

Images