Pharmaceutical composition and application thereof

Abstract

The invention discloses a pharmaceutical composition and application thereof. The pharmaceutical composition contains the following components: homoplantaginin and amikacin which are stored independently or in a mixed manner of more than two of the homoplantaginin and the amikacin. According to the composition disclosed by the invention, homoplantaginin and amikacin are combined for use, and the homoplantaginin and amikacin play a synergistic effect, so that open wounds infected by methicillin-resistant staphylococcus (MRSA) can be effectively treated, and the effects of resisting inflammation, resisting oxidation and promoting healing are achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and in particular to a pharmaceutical composition and its applications. Background Art

[0002] The skin can protect the body from damage and microbial invasion and maintain body fluids, electrolytes and nutrients. Once the skin is severely damaged, it will seriously affect people's life and health. The treatment of open wounds is very important from both medical and aesthetic perspectives, mainly including four stages: hemostasis, inflammation, proliferation and remodeling. After an open wound occurs, bacterial infection is almost inevitable. Bacterial infection will directly initiate and maintain an inflammatory cascade reaction, leading to abnormal prolongation of inflammation, and inducing a hypoxic environment and continuous production of reactive oxygen species (ROS). The occurrence of the above abnormal reactions will lead to pathological scar hyperplasia. Therefore, it is necessary to combine antibacterial, antioxidant and anti-inflammatory effects to promote scar healing and avoid pathological scar hyperplasia.

[0003] In the existing treatment strategies for open wounds, the existing problems mainly include: ① slow wound healing; ② local bacterial infection and overactivation of inflammation; ③ abuse of antibiotics, etc. Among them, the persistent presence of the wound surface will increase the risk of bacterial infection, while local inflammation activation and excessive secretion will lead to non-healing of the wound. In addition, how to select effective antibacterial drugs while avoiding bacterial resistance caused by antibiotic abuse is also a major problem in the treatment of open wounds.

[0004] Therefore, it is necessary to develop a pharmaceutical composition with better effects for treating open wounds. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, in the first aspect of the present invention, a pharmaceutical composition is proposed. The pharmaceutical composition has the characteristics of antibacterial, anti-inflammatory, antioxidant and promoting healing, and can be used for the treatment of open wounds.

[0006] In the second aspect of the present invention, an application of the pharmaceutical composition is also provided.

[0007] According to a pharmaceutical composition provided by an embodiment of the first aspect of the present invention, the pharmaceutical composition contains the following components stored separately or in combination of two or more: homoplantaginin (CAS No. 17680-84-1) and amikacin (CAS No.: 37517-28-5).

[0008] The pharmaceutical composition according to the embodiment of the present invention has at least the following beneficial effects:

[0009] The composition of the present invention uses homoplantaginin and amikacin in combination, and the two play a synergistic effect, which can effectively treat open wounds infected with methicillin-resistant Staphylococcus aureus (MRSA), achieving the effects of anti-inflammatory, antioxidant and promoting wound healing.

[0010] According to a preferred embodiment of the present invention, the mass ratio of homoplantaginin to amikacin is 1:(0.1 - 10).

[0011] According to a preferred embodiment of the present invention, the mass ratio of homoplantaginin to amikacin is 1:(1 - 5).

[0012] According to a preferred embodiment of the present invention, the pharmaceutical composition further comprises a solvent.

[0013] According to a preferred embodiment of the present invention, the amount of the solvent is controlled such that the concentration of homoplantaginin is 0.5 - 4 μg / mL. For example, it includes 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL or a sub-range composed of any two of these values.

[0014] According to a preferred embodiment of the present invention, the amount of the solvent is controlled such that the concentration of amikacin is 0.5 - 5 μg / mL. For example, it includes 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL or a sub-range composed of any two of these values.

[0015] According to a preferred embodiment of the present invention, the solvent is selected from at least one of water, DMSO, and physiological saline.

[0016] According to a drug preparation provided in the second aspect of the present invention, it comprises the drug composition described in the first aspect of the present invention; and pharmaceutically acceptable excipients.

[0017] According to a preferred embodiment of the present invention, the dosage form of the drug preparation includes a solid dosage form, a semi-solid dosage form or a solution dosage form.

[0018] According to a preferred embodiment of the present invention, the liquid dosage form includes true solution types, colloid types, microparticle dosage forms, and suspension dosage forms.

[0019] According to a preferred embodiment of the present invention, the semi-solid dosage form includes ointment.

[0020] The third aspect of the present invention provides an application of the drug composition described in the first aspect of the present invention; or the drug preparation described in the second aspect of the present invention in the preparation of antibacterial drugs.

[0021] According to a preferred embodiment of the present invention, the bacterium includes methicillin-resistant Staphylococcus.

[0022] The fourth aspect of the present invention provides the use of the pharmaceutical composition according to the first aspect of the present invention; or the pharmaceutical preparation according to the second aspect of the present invention in the preparation of a medicament for treating and / or preventing open wounds.

[0023] According to a preferred embodiment of the present invention, the open wound is an open wound infected with methicillin-resistant Staphylococcus.

[0024] Definitions and general terms

[0025] "Pharmaceutically acceptable excipients" in the present invention include any solvent, solid excipient, diluent, binder, disintegrant, or other liquid excipient, dispersant, flavoring agent or suspending agent, surfactant, isotonic agent, thickening agent, emulsifying agent, preservative, solid binder, glidant or lubricant, etc., suitable for a particular target dosage form. As described in the following literature: In Remington: The Science and Practice of Pharmacy, 21st edition, 2005, ed. D. B. Troy, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988 - 1999, Marcel Dekker, New York. The combined content of the literature here shows that different excipients can be applied to the formulation of pharmaceutically acceptable compositions and their well-known preparation methods. Except for the range in which any conventional excipient is incompatible with the compounds of the present invention, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their use is also within the scope contemplated by the present invention.

[0026] Substances that can be used as pharmaceutically acceptable excipients include, but are not limited to, ion exchangers; aluminum; aluminum stearate; lecithin; serum proteins such as human serum albumin; buffering substances such as phosphates; glycine; sorbic acid; potassium sorbate; partial glyceride mixtures of saturated vegetable fatty acids; water; salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts; colloidal silicon; magnesium trisilicate; polyvinylpyrrolidone; polyacrylates; waxes; polyethylene-polypropylene-block polymers; lanolin; sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; gum powder; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; diol compounds such as propylene glycol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic salts; Ringer's solution; ethanol; phosphate buffer solution; and other non-toxic and suitable lubricants such as sodium lauryl sulfate and magnesium stearate; coloring agents; release agents; coating materials; sweeteners; flavoring agents; fragrances; preservatives and antioxidants.

[0027] Other features and advantages of the present invention will be set forth in the following description, and in part will be obvious from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the following description of embodiments in conjunction with the accompanying drawings, wherein:

[0029] Figure 1 is the in vitro biosafety experiment diagram of homoplantaginin at different concentrations;

[0030] Figure 2 is the in vivo biosafety experiment diagram of homoplantaginin at different concentrations;

[0031] Figure 3 is the microscopic diagram of the histological effects of homoplantaginin on the lungs, liver, spleen, kidneys and heart;

[0032] Figure 4 is the strong antioxidant experiment diagram of homoplantaginin;

[0033] Figure 5 is the anti-inflammatory effect experiment diagram of homoplantaginin;

[0034] Figure 6 is the wound healing promotion experiment diagram of homoplantaginin;

[0035] Figure 7It is an experimental diagram showing the synergistic antibacterial effect of homoplantaginin and amikacin;

[0036] Figure 8 It is an experimental diagram showing the synergistic clearance rate of biofilm by homoplantaginin and amikacin;

[0037] Figure 9 It is an experimental diagram showing the wound surface of mice effectively treated with homoplantaginin combined with amikacin for MRSA infection. Detailed implementation mode

[0038] The following are specific embodiments of the present invention, and the technical solutions of the present invention are further described in combination with the embodiments, but the present invention is not limited to these embodiments.

[0039] The reagents, methods and equipment used in the present invention, unless otherwise specified, are all conventional reagents, methods and equipment in the technical field.

[0040] The raw materials used in the embodiments of the present invention are as follows:

[0041] Antibiotics: Antibiotic powders are purchased from Shanghai Yuanye Bio-Technology Co., Ltd., dimethyl sulfoxide (DMSO) is purchased from Tianjin Pinxinnuo Bio-Technology Co., Ltd., and MH medium dry powder is purchased from Guangdong Huankai Microbial Sci & Tech Co., Ltd. The antibiotic powder is dissolved in DMSO to make a stock solution, stored at -20 °C, and diluted to the required concentration with MH broth when used.

[0042] Homoplantaginin: Commercial homoplantaginin reagent (Chengdu Phytos Standardization & Purification Biotechnology Co., Ltd., China) is purchased, dissolved in DMSO to prepare a 10 mg / mL stock solution, aliquoted and stored frozen at -20 °C. After thawing, it is diluted with physiological saline for use.

[0043] Biological safety verification:

[0044] The homoplantaginin stock solution is configured into working concentrations of 32, 64, 128, 256 and 512 μg / mL, and added to the healthy rabbit red blood cell suspension to test hemolysis, and added to Hela cells (human cervical cancer cell line) and HSF cells (human foreskin fibroblasts) to test cytotoxicity. The results are as Figure 1 shown, where Figure (1A) is the hemolysis experiment; Figure (1B) is the cytotoxicity experiment; Figure (1C) is the microscopic observation of the effect of homoplantaginin on cell morphology; at concentrations of 256 μg / mL and below, homoplantaginin did not significantly induce hemolysis or cell death (P > 0.05).

[0045] Six-week-old, 18-20 g specific pathogen-free male ICR mice were selected and divided into 4 groups of 5 mice each. The control group was injected with 200 μL of normal saline via the tail vein once a day for three consecutive days. The low-, medium-, and high-dose experimental groups were injected with 200 μL of 1 mg / mL, 3 mg / mL, and 6 mg / mL homoplantaginin saline solution via the tail vein, respectively, to achieve concentrations of 10 mg / kg, 30 mg / kg, and 60 mg / kg in the mice once a day for three consecutive days. The survival rate and body weight changes of the mice were recorded within 7 days. Heart, liver, spleen, lung, and kidney specimens were fixed, and whole blood was collected for routine blood, liver function, kidney function, and heart function analysis. All animal experiments were approved by the Ethics Committee of Central South University (No. CSU-2022-0433). The results are as Figure 2 shown, where Figure 2 (A) Survival curve; Figure 2 (B) Body weight change; Figure 2 (C) Proportion of peripheral blood cells; Figure 2 (D) Peripheral blood liver and kidney function and myocardial enzyme indexes; Figure 3 are the histological effects of homoplantaginin on the lungs, liver, spleen, kidneys, and heart observed under the microscope. Under the administration conditions of homoplantaginin at the test concentration, the survival time and body weight of the mice did not change significantly (P > 0.05), and their blood cell proportions, liver and kidney functions, and myocardial enzyme indexes were also not affected (P > 0.05). The immunohistochemical results showed that the structures of organs such as the lungs, liver, spleen, kidneys, and heart did not change significantly.

[0046] Both the above in vitro and in vivo experiments showed that homoplantaginin has good biosafety.

[0047] Furthermore, the antioxidant, anti-inflammatory, and wound-healing effects of homoplantaginin were verified.

[0048] Determination of free radical scavenging ability (ABTS method): The ABTS free radical scavenging ability kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd. Bacteria were collected into a centrifuge tube, and the supernatant was discarded after centrifugation. 5 million bacteria were taken and homogenized with 1 mL of 80% methanol extraction solution, and then transferred to a 2 mL centrifuge tube. Ultrasonic extraction was carried out at 60 °C and 200 - 300 W for 30 min, centrifuged at 12,000 rpm for 10 min, and the supernatant was retained. The ABTS working solution was added and incubated at room temperature for 2 - 6 min, and the absorbance was measured at a wavelength of 734 nm.

[0049] Determination of total antioxidant capacity (FRAP method): The total antioxidant capacity detection kit (FRAP method) was purchased from Shanghai Beyotime Biotechnology Co., Ltd. Bacterial samples were collected, added with pre-cooled PBS solution and sonicated, centrifuged at 12,000 g for 5 min at 4 °C, the supernatant was taken and added with FRAP working solution, and the absorbance was measured at a wavelength of 593 nm after incubation at 37 °C for 3 - 5 min.

[0050] In vitro, it was demonstrated by the ABTS experiment, and the results were as Figure 4 shown in A. When the concentrations of homoplantaginin were 10, 100, and 1000 μg / mL, its free radical scavenging abilities reached 2.98%, 29.53%, and 93.30% respectively, and its antioxidant ability exceeded that of the strong antioxidant Trolox (a vitamin E analogue) at the same concentration of 100 μg / mL, which was also confirmed in the FRAP experiment (P < 0.05, Figure 4 B). In addition, the intracellular reactive oxygen species concentration in Hela cells treated with homoplantaginin decreased significantly (P < 0.0001), and the degree of decrease was proportional to the concentration of homoplantaginin ( Figure 4 C, Figure 4 D). After the intervention of homoplantaginin, the detection result of intracellular MDA (lipid oxidation) also decreased significantly (P < 0.0001, Figure 4 E).

[0051] Mouse skin wound modeling and sample collection: SPF-grade 7-week-old (28±2.0 g) male ICR mice were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. and raised in the Animal Department of Xiangya School of Medicine, Central South University. All experimental operations were approved by the Experimental Animal Welfare and Ethics Committee of Central South University, approval number: CSU-2022-0433. After one week of adaptive cultivation, 30 experimental mice were randomly divided into 4 groups. Among them, 9 mice in each of the three groups were used for skin wound modeling, and the remaining 3 mice were used as the blank control group without skin resection treatment. The experimental mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution at a dose of 40 mg / kg. After anesthesia, the hair on the back of the experimental mice was removed with a hair clipper, and a small amount of depilatory cream was applied to remove the remaining short hair until the skin was completely exposed. After thoroughly cleaning the exposed skin with iodophor, a circular full-thickness skin wound with a diameter of about 8 mm was cut on the back of the mice with ophthalmic scissors. The wound and the surrounding skin were disinfected with iodophor. The day of modeling was recorded as day 1. The oil-in-water type medical cream matrix was purchased from Zhuyan Tree Raw Material Wholesale (Taobao) and used to prepare 0.25% and 0.5% w / w homoplantaginin creams, with the blank cream as the control. From day 1, 50 μL of the cream was evenly applied to the wound surface once a day. On day 3 and day 7, after anesthesia, the wound skin tissue and the surrounding 1-2 mm full-thickness skin were taken with sterile ophthalmic scissors. Part of it was fixed in 4% paraformaldehyde solution for pathological analysis, and the other part was quickly frozen in liquid nitrogen. Further, blood was collected by enucleating the eyeballs. After the blood samples were left standing at room temperature for 4 h, the serum was separated by centrifugation at 4°C and 4000 rpm for 10 min and stored at -80°C for the determination of serum oxidative damage indicators.

[0052] Assessment of oxidative stress status: The malondialdehyde (MDA) kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd., and the superoxide dismutase (SDO) kit and the micro reduced glutathione (GSH) kit were purchased from Nanjing Jiancheng Bioengineering Institute. Mouse serum was taken, and the contents of MDA, GSH, and SOD were measured according to the methods described in the kit instructions.

[0053] In vivo, oxidative stress status was evaluated by detecting oxidative indicators such as MDA, SOD, and GSH in the mouse model. The results are as Figure 4 shown in Figure F. After injection with different concentrations and different days of homoplantaginin, the in vivo oxidative stress level was significantly improved. The above results all indicate that homoplantaginin has strong antioxidant ability.

[0054] The anti-inflammatory effect of homoplantaginin has also been confirmed in a mouse model. An 8-mm-diameter full-thickness circular wound was made on the back of the mice with ophthalmic scissors, and a self-made homoplantaginin ointment was applied at a dose of 50 μL per day. On the 3rd and 7th days, the skin tissues of the wound surface and the surrounding 1-2 mm full-thickness skin tissues of the mice were collected by aseptic ophthalmic method, and RNA was extracted to detect the expression of various inflammatory factors. On the 3rd day after surgery, compared with the blank matrix group (M, without homoplantaginin), the expression levels of IL-6 in the wound skin of the low-concentration group (L, homoplantaginin concentration of 0.25%) and the high-concentration group (H, homoplantaginin concentration of 0.5%) decreased by 82.7% and 93.7% respectively, and the expression levels of IL-8 decreased by 90.5% and 94.0% respectively, as Figure 5 A, and the decrease was more obvious on the 7th day, as Figure 5 C, indicating that the inflammatory environment was improved. At the same time, after treatment with homoplantaginin in groups L and H, the expressions of cytokines VEGF, PDGF and TGF-β1 also increased significantly to varying degrees ( Figure 5 B, Figure 5 D), suggesting enhanced repair functions such as angiogenesis and wound healing.

[0055] Cell migration rate determination: The effect of homoplantaginin on cell migration ability was preliminarily judged by a scratch assay. HeLa cells were seeded in 6-well plates (50×10 4 cells / well), gently shaken and cultured in the medium for 24 h. When the cell density reached 80%, a scratch was made with a sterile 100-μL pipette tip to simulate a physical wound surface. After gently rinsing with PBS, the serum-free medium containing homoplantaginin at final concentrations of 1, 50 and 100 μg / mL was replaced. Photos were taken under a fluorescence microscope, and the photo points were recorded and set as 0 h. The cells were continuously cultured for 24 h and 48 h, and fixed-point photos were taken respectively. Image J (Ver.1.38) was used to measure the pixels of the scratched area to quantitatively compare the cell migration speed and migration rate.

[0056] The migration rate of cells in in vitro culture is often used to evaluate the wound-healing potential of drugs. As Figure 6 shown in A, after treatment with 10, 100 and 1000 μg / mL homoplantaginin for 24 h, the cell migration rates increased by 3.74%, 9.40% (P < 0.05) and 13.5% (P < 0.01) respectively, and further increased after 48 h, reaching 16.15%, 24.31% (P < 0.05) and 32.22% (P < 0.01) respectively.

[0057] In the in vivo experiment of mice, as Figure 6As shown in Figure B, on the 5th day of treatment, the wounds of the mice in the L group and the H group became dry, and the edges of the wounds began to contract. In contrast, the M group had repeated inflammation, and the wounds were still moist on the 7th day. The wound healing rates of the L group and the H group reached 50% on the 5th day and the 6th day respectively, while the M group reached 50% only on the 9th day. On the 14th day after modeling, the wound healing rates of the M group, the L group and the H group were 77.12%, 93.49% (P < 0.0001) and 97.38% (P < 0.0001) ( Figure 6 C).

[0058] Histochemical staining: Take the full-thickness skin tissue of 1-2 mm around the wound skin tissue in the aforementioned mouse model, take the skin tissue fixed with 4% paraformaldehyde, perform ethanol dehydration, paraffin embedding, sectioning, baking, dewaxing and hydration treatments, and then perform HE and Masson staining to scan the pathological changes of the wound scabs and surrounding skin at different stages.

[0059] In addition, the immunohistochemical results showed ( Figure 6 D) that granulation tissue grew in the L group and the H group treated with homoplantaginin to fill the wound in the first three days. At this time, the skin tissue structure of the wound in the M group was broken, and a large number of inflammatory cells exuded. On the 7th day, it was observed that the wounds in the L group and the H group were further repaired, and the epidermal fibrous tissue began to mature and soften, while the wound in the M group was still severely damaged. The results of Masson trichrome staining were as Figure 6 shown in Figure E. After 14 days of intervention, the wounds in the L group and the H group were basically repaired, and new sebaceous glands and other accessory organs began to appear. The hair follicles in the dermis of the skin grew well, and the density of the newly formed hair follicles in the H group was the largest. The above results indicate that homoplantaginin has good ability to promote wound healing.

[0060] Example 1

[0061] This example provides a pharmaceutical composition, which includes homoplantaginin (Hom) and amikacin (AMK). The mass ratio of homoplantaginin to amikacin is 1:1.25; the concentration of homoplantaginin is prepared to be 4 μg / mL with normal saline, and the concentration of amikacin is prepared to be 5 μg / mL.

[0062] Example 2

[0063] The synergistic antibacterial effect of homoplantaginin and amikacin

[0064] First, the antibacterial ability of homoplantaginin was tested using the MRSA standard strain ATCC 43300 and 8 clinical isolates of Staphylococcus aureus (clinical strains), respectively. The Staphylococcus aureus ATCC 43300 was provided by the Department of Laboratory Medicine, the Second Xiangya Hospital of Central South University. The other clinically isolated Staphylococcus aureus strains were collected by the Department of Laboratory Medicine, the Second Xiangya Hospital of Central South University and identified by an automatic microbial identification / susceptibility analyzer. All strains were stored in a -80 °C refrigerator. The frozen strains were subcultured on Columbia blood agar plates three times in a row to obtain stable characteristics for subsequent experiments. The results are shown in Figure 7 A-7B and Table 1. Homoplantaginin showed antibacterial effects against both the MRSA standard strain and clinical strains. However, after 24 h of treatment, the bacterial concentration rebounded, indicating that homoplantaginin alone could not completely inhibit MRSA( Figure 7 C).

[0065] Table 1: Treatment of MRSA clinical strains with 0.5 μg / mL homoplantaginin

[0066]

[0067] Secondly, the checkerboard method was used to investigate the antibacterial effects of combinations of different types of antibiotics and homoplantaginin.

[0068] Experimental procedure of the checkerboard method: The checkerboard dilution method was used for the combined susceptibility test of homoplantaginin and commonly used clinical antibiotics. Each antibacterial drug was serially diluted with MH broth starting from 2-fold the MIC concentration, and homoplantaginin was serially diluted starting from 16 μg / mL. 50 μL of each was respectively arranged in the rows and columns of a 96-well plate, and then 100 μL of a bacterial suspension with a concentration of 1×10 6 CFU / mL was added, and the mixture was cultured overnight at 37 °C. The OD450 results were measured using a microplate reader. The fractional inhibitory concentration index (FICI) = MIC A (in combination) / MIC A (alone) + MIC B (in combination) / MIC B (alone). The judgment criteria are as follows: FICI ≤ 0.5 indicates synergy; 0.5 < FICI ≤ 1 indicates addition; 1 < FICI ≤ 4 indicates no interaction; 4 < FICI indicates antagonism. The results of the checkerboard method experiment are as follows:

[0069] Table 2: Combined antibacterial effects of homoplantaginin and commonly used clinical antibiotics

[0070]

[0071]

[0072] From Table 2 and Figure 7 the data of D, when amikacin and homoplantaginin are used in combination, they have a synergistic effect, and the concentrations of the two are 4 μg / mL and 5 μg / mL respectively to achieve the antibacterial effect. As Figure 7 shown in E, after the combined use of homoplantaginin and amikacin, the growth of bacteria can be effectively inhibited and will not rebound after 24 h. When other commonly used clinical antibiotics are used together with homoplantaginin, they all show additive or irrelevant effects.

[0073] An important reason for the delayed healing of open wounds infected with MRSA is that MRSA is likely to form a biofilm with strong resistance and difficult to remove on the wound surface. In this invention, vancomycin (VAN) is used as a control, and the drug composition (4 μg / mL homoplantaginin and 5 μg / mL amikacin) in Example 1 of this invention is used to evaluate the clearance efficiency of the biofilm. The results are as Figure 8 shown in A and Figure 8 B. The use of homoplantaginin alone can effectively inhibit biofilm formation, but its ability to disperse the already formed biofilm is limited. When combined with amikacin, it not only significantly inhibits biofilm formation (P < 0.05), but also the clearance rate of the already formed biofilm reaches 72.16% (P < 0.001). The PCR results show that the combined use of drugs can change the expression of genes related to biofilm formation ( Figure 8 C).

[0074] Example 3

[0075] This example provides a pharmaceutical preparation, which consists of a mixed ointment containing 0.4% homoplantaginin and 0.5% amikacin;

[0076] The combination of homoplantaginin and amikacin is used for the treatment of open wounds.

[0077] 0.4% homoplantaginin ointment: The oil-in-water type medical cream matrix is purchased from Zhuyan Tree Raw Material Wholesale (Taobao), and the homoplantaginin cream is prepared according to a volume ratio of 0.4%.

[0078] 0.5% amikacin ointment: The oil-in-water type medical cream matrix is purchased from Zhuyan Tree Raw Material Wholesale (Taobao), and the amikacin cream is prepared according to a volume ratio of 0.5%.

[0079] The mixed ointment containing 0.4% homoplantaginin and 0.5% amikacin: The oil-in-water type medical cream matrix is purchased from Zhuyan Tree Raw Material Wholesale (Taobao), and the mixed ointment is prepared, and the volume percentages of homoplantaginin and amikacin are 0.4% and 0.5% respectively (where the mass ratio of homoplantaginin and amikacin is 1:1.25).

[0080] Re - establish a mouse wound infection model to evaluate the efficacy of homoplantaginin combined with amikacin. After cutting out a complete skin injury with a diameter of 5 mm, take 50 μL of MRSA standard strain solution (ATCC 43300, concentration 1.5×10 8 CFU / mL) and apply it to the surface of the wound, and repeat the application once the next day. From the 3rd day, apply 0.4% homoplantaginin ointment, 0.5% amikacin ointment, and a mixed ointment containing 0.4% homoplantaginin and 0.5% amikacin to the wound once a day, 50 μL each time. Mice with skin injuries but without applying bacterial solution are used as the baseline group, mice with skin injuries and infected with MRSA are applied with a matrix ointment without drugs as the negative control group, and mice with skin injuries and MRSA infections are applied with 0.9% vancomycin ointment as the positive control. After continuous drug administration for 3 days, excise the entire wound skin and 1 - 2 mm of normal skin tissue around the wound for immunofluorescence staining, calculate the bacterial load in the wound, and collect peripheral whole blood of the mice simultaneously.

[0081] The results are as Figure 9 shown in A. After 3 days of continuous treatment, the bacterial load in the combined - medication group decreased significantly (P < 0.01), indicating that homoplantaginin combined with amikacin also has good anti - MRSA effects in vivo.

[0082] Figure 9 B shows the peripheral blood test results of the infection models of mice in different groups. Among them, the white blood cell count, neutrophil count, and C - reactive protein concentration in the combined - medication group are all significantly lower than those in the blank control group, similar to the effect of vancomycin, indicating that the inflammatory response in the mice is effectively inhibited. And Figure 9 in C, different treatment groups all have a certain antioxidant capacity, but only the combined - medication group significantly increases the GSH concentration in the peripheral blood of mice (P < 0.01). As Figure 9 shown in D, after the combined treatment of homoplantaginin and amikacin, the expression levels of IL - 6 and TNF - α at the wound site both decrease significantly, and the effect is even better than that of vancomycin. As Figure 9 shown in E, on the 3rd day of treatment, the wound defect and bacterial infection in the negative control group with infection but without drug treatment are still very serious, and the arrangement of collagen fibers is disordered. While the combined medication can accelerate the healing of wounds infected with MRSA.

[0083] In summary, the combined use of homoplantaginin and amikacin in the present invention can effectively treat open wounds infected with MRSA, achieving the effects of anti - inflammation, anti - oxidation, and promoting wound healing.

[0084] The above has made a detailed description in combination with the embodiments of the present invention, but the present invention is not limited to the above - mentioned embodiments. Within the knowledge scope of those of ordinary skill in the art to which it pertains, various changes can also be made without departing from the purpose of the present invention.

Claims

1. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the following components stored independently or in combination of two or more: homoplantaginin and amikacin.

2. The pharmaceutical composition according to claim 1, wherein The mass ratio of homoplantaginin to amikacin is 1:(0.1 - 10).

3. The pharmaceutical composition according to claim 1 or 2, characterized in that, The mass ratio of homoplantaginin to amikacin is 1:(1 - 5).

4. The pharmaceutical composition according to claim 1, characterized in that, The pharmaceutical composition further includes a solvent.

5. The pharmaceutical composition according to claim 4, characterized in that, The amount of the solvent is controlled such that the concentration of homoplantaginin is 0.5 - 4 μg / mL.

6. The pharmaceutical composition according to claim 4, wherein The amount of the solvent is controlled such that the concentration of amikacin is 0.5 - 5 μg / mL.

7. A pharmaceutical preparation, characterized in that, It includes the pharmaceutical composition according to any one of claims 1 - 6; and a pharmaceutically acceptable excipient.

8. Use of the pharmaceutical composition according to any one of claims 1 - 6; or the pharmaceutical preparation according to claim 7 in the preparation of antibacterial drugs.

9. Use of the pharmaceutical composition according to any one of claims 1 - 6; or the pharmaceutical preparation according to claim 7 in the preparation of drugs for treating and / or preventing open wounds.

10. The application according to claim 9, characterized in that The open wound is a wound infected by methicillin-resistant Staphylococcus aureus.

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