Aesculin-manganese complex as well as preparation method and application thereof

Abstract

The invention discloses an aesculetin-manganese complex as well as a preparation method and application thereof, and relates to the technical field of medicines. The structure of the aesculetin-manganese complex is as follows: Mn < 2 + > and O in aesculetin are coordinated to form a Mn-O bond, Mn is + 2 valence, and a Mn-O4 coordination form exists; the preparation method of the aesculetin-manganese complex comprises the following steps: mixing aesculetin with MnCl2, adding water, heating to react for 1-3 hours, and drying under reduced pressure to obtain a reaction product, namely the aesculetin-manganese complex. The invention also provides an application of the aesculetin-manganese complex in preparation of an antibacterial product. The aesculetin-manganese complex has the beneficial effects that the antibacterial activity of the aesculetin-manganese complex is superior to that of aesculetin, and the aesculetin-manganese complex is not easy to induce bacteria to generate drug resistance.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to a esculetin-manganese complex and a preparation method and application thereof. Background Art

[0002] Over the past few decades, the overuse of antibiotics has led to the emergence of highly resistant and contagious superbugs. These superbugs include methicillin-resistant Staphylococcus aureus (MRSA), drug-resistant tuberculosis, and drug-resistant Streptococcus pneumoniae. These bacteria have developed resistance to most of the antibiotics currently used in clinical practice, posing a major threat to human health. In addition to the irrational use of antibiotics, the serious lag in the development of new antibacterial drugs is also an important reason for the growing problem of bacterial resistance. At present, the research and development of antibacterial drugs is mainly based on the structural modification of various types of antibiotics on the market. In the past 20 years, the research and development of new antibacterial drugs with new chemical structures and mechanisms of action has almost stagnated. It can be seen that it is urgent to develop antibacterial drugs with novel structures, good antibacterial activity, and not easy to induce bacterial resistance.

[0003] Metal complexes have a completely different mechanism of action from traditional antibiotics in terms of antibacterial effects, mainly including: ① When a ligand with antibacterial activity combines with an essential metal ion to form a complex, the complex can enter the interior of the bacteria in a manner similar to a "Trojan horse" through natural channels related to the absorption, transport and storage of essential metal elements. Once these complexes enter the bacteria, they can inhibit or destroy the necessary life processes inside the bacteria, thereby killing the bacteria; ② Complexes composed of non-essential metal elements can compete with bacteria to absorb and utilize essential metal elements, thereby inhibiting the normal growth and division of bacteria to a certain extent. If this competition lasts long enough, the essential metal elements in the bacteria will be exhausted, causing the death of the bacteria; ③ After the metal ions and ligands are chelated, the delocalization of π electrons in the entire structure can be enhanced, thereby enhancing the binding ability of the metal complex to the cell membrane and making it easier for the metal complex to penetrate into the cell. At the same time, metal complexes can easily bind to electron-rich biomolecules, inactivate bacteria by interfering with enzyme activity, thereby improving antibacterial activity. Therefore, it is very necessary to develop a metal complex with antibacterial effects. Summary of the invention

[0004] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art and to provide a esculetin-manganese complex and a preparation method and application thereof.

[0005] The technical solution of the present invention is as follows: The first aspect of the present invention provides a esculetin-manganese complex having a structure of Mn 2+It coordinates with the O atom in esculetin to form a Mn-O bond, in which Mn is +2 and there is a Mn-O 4 Coordination form.

[0006] Optionally, it has at least one of the following structures: , , .

[0007] Optionally, the raw materials include aesculetin and Mn 2+ .

[0008] Optionally, the esculetin and Mn 2+ The feeding molar ratio is 1:6~8:1.

[0009] Optionally, the esculetin and Mn 2+ The feeding molar ratio is 2~8:1.

[0010] Optionally, the esculetin and Mn 2+ The feed molar ratio is 2:1.

[0011] The second aspect of the present invention provides a method for preparing the above-mentioned aesculetin-manganese complex, comprising the following steps: Aesculetin and MnCl 2 The mixture was mixed, water was added, the mixture was heated to react for 1 to 3 hours, and the mixture was dried under reduced pressure to obtain a reaction product, a esculetin-manganese complex.

[0012] Optionally, the heating temperature is 95-105 °C.

[0013] Optionally, the heating temperature is 95-100 °C.

[0014] The third aspect of the present invention provides a use of the above-mentioned aesculetin-manganese complex in the preparation of antibacterial products.

[0015] The present invention has at least one of the following beneficial effects: The fraxinotin-manganese complex was synthesized by hydrothermal method, and then its structure was characterized and its antibacterial activity was studied. Experiments have shown that the antibacterial activity of the fraxinotin-manganese complex against a variety of bacteria is significantly higher than that of fraxinotin, among which the antibacterial activity against Staphylococcus aureus is increased by 4 times. The antibacterial mechanism of the fraxinotin-manganese complex against Staphylococcus aureus may include inhibiting bacterial biofilm formation, inhibiting protein synthesis, reducing energy production, and inhibiting cell wall synthesis. More importantly, after 20 generations of continuous culture of Staphylococcus aureus with drug-containing medium, the minimum inhibitory concentration of the fraxinotin-manganese complex did not change, which suggests that the complex is not easy to induce bacterial resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In all the following figures, ESM represents aesculetin-manganese complex, and ES represents aesculetin.

[0017] Figure 1 The antibacterial activity of ESM is shown in Figure 2. The final concentrations of ESM from top to bottom are 4.80, 2.40, 1.20 and 0.60 mmol / L (based on the molar mass of ES input during synthesis), the final concentrations of ES are 4.80, 2.40, 1.20 and 0.60 mmol / L, and the final concentrations of Mn 2+ The final concentration was 2.40 mmol / L.

[0018] Figure 2 The inhibitory effects of ESM synthesized at different molar ratios on Staphylococcus aureus, from top to bottom, the final concentrations of ESM are 4.80, 2.40, 1.20 and 0.60 mmol / L (based on the molar mass of ES input during synthesis) (When synthesizing ESM, ES and Mn 2+ The feeding molar ratio is 1:6~8:1).

[0019] Figure 3 This is the structure identification spectrum of ESM (A. UV-visible absorption spectra of ES and ESM in water; B. C 18 H 9 O 8 Theoretical isotope distribution of Mn (a) and measured isotope distribution (b); C. ES, ESM and MnCl 2 Thermogravimetric curves of ESM and MnO 2 EXAFS spectra of MnPc and Mn foil; E. K space fitting of ESM; F. R space fitting of ESM; G. Coordination bond of Mn in ESM) Figure 4 is the electron cloud density distribution of ES.

[0020] Figure 5 To quantify the calculated ESM (C 18 H 10 O 8 Mn), where white atoms are H atoms, gray atoms are C atoms, red atoms are O atoms, and purple atoms are Mn atoms.

[0021] Figure 6 The antibacterial mechanism of ESM against Staphylococcus aureus (A. The bactericidal kinetic curve of ESM against Staphylococcus aureus (MBC of ESM is 1.20 mmol / L); B. The inhibitory effect of ES and ESM on the biofilm formation of Staphylococcus aureus (Note: the concentration of ESM is 0.60 mmol / L, the concentration of ES is 0.60 mmol / L, and the concentration of Mn is 0.2+ The concentration was 2.40 mmol / L); C. The activity of AKP in the culture medium after the action of ES and ESM; D. The effect of ES and ESM on the lysis of rabbit red blood cells; E. The effect of ES and ESM on the lysis of rabbit red blood cells; F. The resistance of Staphylococcus aureus to ES, ESM and four antibiotics (the data differences were expressed by parameter test method)).

[0022] Figure 7 Transcriptomic analysis (A. Volcano plot of DEGs in ESM and ES groups; B. DEGs clustering heat map; C. GO enrichment analysis of DEGs; D. KEGG enrichment analysis of DEGs.) Figure 8 Proteomic analysis (A. Volcano plot of DEPs of ESM and ES; B. DEPs clustering heat map; C. GO enrichment analysis of DEPs; D. KEGG enrichment analysis of DEPs).

[0023] Fig. 9 Nine-quadrant plot for transcriptomic and proteomic association analysis of ES and ESM.

[0024] Fig.10 Diagram of the antibacterial mechanism of ESM. DETAILED DESCRIPTION

[0025] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0026] Example 1 1. This embodiment provides a method for preparing aesculetin-manganese complex (hereinafter referred to as "ESM") using aesculetin as a raw material, comprising the following steps: 0.060 mmol of esculetin (ES) (purchased from Chengdu Aifa Biotechnology Co., Ltd.) was weighed and mixed with 0.0075, 0.010, 0.015, 0.030, 0.060, 0.120, 0.240, and 0.480 mmol of MnCl, respectively. 2 The mixture was placed in a round-bottom flask, 10.0 mL of distilled water was added, the mixture was reacted at 98 °C for 2 h, and then dried under reduced pressure to obtain the reaction product ESM.

[0027] Second, this example also studied the antibacterial activity of ESM ESM was dissolved by ultrasonication in 2.50 mL of pure water containing 20% ​​DMSO, and 0.80 mL of the solution was diluted twice in sequence. 2Treat in the same way.

[0028] The method for determining the minimum bactericidal concentration (MBC) of a drug is as follows: The experimental bacteria were inoculated on solid culture medium and cultured at 37 °C for 24 h. A single colony was picked and placed in TBS liquid culture medium. The culture was shaken until the logarithmic growth phase and diluted to 105 CFU / mL with fresh TBS liquid culture medium.

[0029] Take a sterile 24-well plate, add 0.40 mL of the test solution and 1.60 mL of the bacterial solution to each well, and culture at 37 °C for 18 h. Then take 50.0 μL of the bacterial solution from the well plate, dilute it 1000 times with sterile PBS buffer according to the turbidity of the bacterial solution, and then take 50.0 μL to spread on the solid culture medium. After continuing to culture at 37 °C for 18 h, take it out, take pictures and record, and the lowest concentration without colony growth is the minimum bactericidal concentration (MBC). Each group of experiments is repeated 3 times.

[0030] The experimental results are as follows: The inhibitory effect of ESM on Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa is shown in Figure 1 .from Figure 1 It can be seen that the maximum concentration of Mn 2+ There was no obvious antibacterial activity against the three bacteria. Compared with ES, the antibacterial activity of ESM against the three bacteria was significantly enhanced. The antibacterial activity of ESM against Staphylococcus aureus was 4 times that of ES, and the antibacterial activity against Escherichia coli and Pseudomonas aeruginosa was 2 times that of ES.

[0031] Different ratios of ES and Mn 2+ The inhibitory effect of the complex formed on Staphylococcus aureus is as follows Figure 2 ES itself has a MBC of 4.80 mmol / L and has a certain antibacterial activity. 2+ After the reaction, the MBC was between 2.40 and 1.20 mmol / L, and the antibacterial activity was significantly enhanced. This phenomenon shows that Mn 2+ The introduction of Mn 2+ The interaction between ES and Mn changes its chemical properties, thereby enhancing its inhibitory effect on Staphylococcus aureus. 2+ When the reaction ratio was 2:1, the antibacterial activity of the reaction product was the highest. The reason may be that ES and Mn 2+ The complex structure formed with a ratio of 2:1 is the most stable and has the best antibacterial activity. 2+ At lower concentrations, less ESM is formed; 2+ When the concentration is too high, ES and Mn 2+It will coordinate in other forms and cannot form this stable structure, resulting in a decrease in antibacterial activity.

[0032] 3. This example also characterizes the structure of the prepared ESM, and the characterization method is as follows: ES and MnCl 2 The reaction was carried out in the above method with a molar mass ratio of 2:1. 0.1 mL of the reaction solution was transferred and mixed with 0.9 mL of pure water. 1.0 mL was taken and the absorption spectrum of 190 nm to 500 nm was scanned by UV-visible spectrophotometer. The UV-visible absorption spectrum of ES was scanned in the same way. (Results are shown in Figure 3 A) Take another 3.0 mL of the diluted reaction solution, centrifuge at 5000 r / min for 5 min, and detect the supernatant using UPLC-Q-TOF MS. Use infusion injection, ESI negative ion mode detection, mass detection range 100-1500, drying gas flow rate 800L / h, desolvation gas temperature 500 ℃. (Results see Figure 3 B) After the remaining reaction solution was dried under reduced pressure, the thermogravimetric curves of ES and ESM were measured using a thermogravimetric analyzer (TGA). The TGA analysis conditions were: nitrogen protection, a heating rate of 10 °C / min, and a measurement temperature range of 30-800 °C. The results are as follows: Figure 3 C. At the same time, the X-ray absorption fine structure (XAFS) spectrum was collected using the BL14W1 beamline of the Shanghai Synchrotron Radiation Source, as shown in Figure 3 As shown in D~G.

[0033] Gaussian 9.0 was used for quantitative calculations to infer the most likely structure of ESM. The B3LYP theoretical functional was used, the SMD pseudopotential was used for the central metal atom, and the def2-SVP basis set level was used for the surrounding atoms to optimize the geometric structure of ESM and analyze the vibration frequency. The energy minimum point structure without imaginary frequency was obtained, and the structure was displayed by Gauss-View6.0.

[0034] The experimental results are as follows: (1) UV-visible absorption spectrum of ESM ES and Mn 2+ The UV-visible absorption spectra of the supernatant after the reaction in hot water in different solvents are shown in Figure 3 A. From Figure 3 As can be seen from A, ES has two main absorption peaks at 205 nm and 345 nm, which are the absorption peaks of lactone group and 2-hydroxycinnamoyl group respectively. The UV-visible absorption spectra of ES and ESM in water basically overlap, and the peak shapes of the two are the same, but the absorbance of ESM is lower than that of ES at 203 nm and 344 nm.

[0035] (2) TOF MS detection results of ESM ES and Mn 2+ The supernatant after the reaction in hot water was detected by TOF MS, and the results are shown in Table 1. As can be seen from Table 1, in the ESI negative ion mode, incompletely reacted ES ( m / z =177.0188) and m / z The compound with a molecular weight of 407.9678 was compared with the isotope distribution ( Figure 3 B), it is speculated that this compound is ES and Mn 2+ The [MH] of the complex formed with a 2:1 (molar mass ratio) - Quasi-molecular ions.

[0036] Table 1 Esculetin (ES) and Mn 2+ ESI-TOF MS detection results of the reaction solution (ESI negative ion mode) (3) Thermogravimetric analysis results of ESM At 20 mL / min of flowing N 2 The thermogravimetric curves of ES and ESM at 30-800 °C were measured under the protection of Figure 3 C). ES has two weight loss steps, the weight loss rate is 2.8% in the range of 100~150℃, and the weight loss rate is 67% in the range of 210~330℃. During the entire heating process of ES, the total weight loss rate is 83.19% and the residual mass is 16.81%. Figure 3 As shown in Figure C, ESM has three weight loss steps. The weight loss rate in the range of 50~230℃ is 6.79%, which is speculated to be the loss of two crystal waters (theoretical weight loss rate is 7.20%); the weight loss rate in the range of 210~330℃ is 27.3%, and the weight loss rate in the range of 360~700℃ is 24.8%. With the increase of temperature, the complex undergoes continuous degradation, the ligand gradually decomposes, the framework begins to collapse, and the final product is an oxide containing chlorine and manganese.

[0037] (4) X-ray absorption fine structure of ESM The K-edge X-ray absorption near-edge structure (XANES) spectrum of Mn is widely used to derive the coordination environment information of Mn in metal complexes with complex structures. 2 , MnPc, and ESM normalized Mn K-edge XANES spectra. X-ray absorption near-edge spectra ( Figure 3D) The ESM edge spectrum shows a pre-edge transition at 6542 eV, which is the same as that of MnPc (6542 eV) and far away from the Mn foil and MnO 2 This indicates that the Mn in the ESM is in the +2 oxidation state. EXAFS analysis at the Mn-K edge is used to study the coordination number of the Mn atoms and the bond distances between the Mn-O atoms. The EXAFS data are fitted to the K, R space to determine the local atomic structure and the relative bond lengths with respect to the absorbing atoms ( Figure 3 E, F, G). Structural parameters such as Mn–O bond distance (R), coordination number (CN), Debye–Waller factor (σ 2 ) and R factor, as shown in Table 2. By fitting the EXAFS spectrum of ESM, the amplitude reduction factor (S 0 2 ) was determined to be 0.96. The average coordination number of Mn-O in ESM is 4.1 ± 0.30, and the average bond length of Mn-O is 2.19 ± 0.02. In summary, Mn and O in ESM coordinate to form a Mn-O bond, Mn is + 2, and there is a Mn-O 4 Coordination form.

[0038] Table 2 - EXAFS fitting parameters of ESM (S 0 2 =0.96) (5) Quantitative calculation of ESM Since the exact structure of ESM cannot be directly detected by instruments, we use quantitative calculations to determine the possible structure of ESM. First, the geometric structure of ES is optimized and its electron cloud density is analyzed. The results show that ( Figure 4 ), the electronegativity is mainly concentrated on the phenolic hydroxyl groups at positions 6 and 7, and it is speculated that Mn in ESM 2+ It may be coordinated with the O atoms on the 6- and 7-hydroxyl groups of ES.

[0039] Based on the results of liquid chromatography, thermogravimetry and synchrotron radiation, the possible results of ESM were quantitatively calculated. When optimizing the structure, the def2-SVP basis set was used to apply all atoms except the central metal atom - the manganese atom, while the manganese atom used the SMD pseudopotential basis set. When calculating the energy, the solvation effect was considered, and the temperature and time design parameters of the reaction were referred to to calculate the optimal structure of ESM in the 2:1 case. As shown in Table 3 and Figure 5 As shown, Mn 2+ The structures of the two compounds are most stable when they are bonded to the 6-hydroxyl O atom in ES (compound A). The bonding energies of the two compounds are higher when they are bonded to the 6- and 7-hydroxyl O atoms (compound B), or to the 7-hydroxyl O atom (compound C), and the bond lengths are quite different. Therefore, it is speculated that C18 H 10 O 8 The optimal structure of Mn is Mn 2+ Bonded to the O atom in the 6-hydroxyl group of ES and two water molecules respectively ( Figure 5 ).

[0040] Table 3 Quantitative calculation results of ESM at different bonding sites Example 2 This example provides a study on the antibacterial mechanism of the ESM prepared in Example 1 against Staphylococcus aureus, and the specific method is as follows: 1. Study on the antibacterial activity of ESM: (1) Preparation of test solution According to the method of Example 1, ES: Mn 2+ =2:1 to synthesize ESM, dissolve it in 2.50 mL of distilled water containing 20% ​​DMSO, and use it as the mother solution for later use. When using, dilute it with purified water to the required concentration.

[0041] (2) Bactericidal kinetic curve of ESM The test solution was added to the bacterial suspension (10 5 CFU / mL), the final concentration of ESM was 1 / 2 ×, 1 ×, 2 ×, 4 × MBC, incubated at 37 °C, and the bacterial solution was removed at 0, 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, and 18 h, diluted with sterile PBS buffer, spread, and cultured at 37 °C for 18 h, and the number of colonies was recorded. Staphylococcus aureus treated with 20% DMSO aqueous solution was used as the control group.

[0042] (3) Effects of ESM on bacterial cell wall Take logarithmic phase Staphylococcus aureus, centrifuge at 5000 r / min for 5 min, collect the cells, wash 3 times with PBS, and resuspend the cells with PBS (OD 600 nm =0.3), ESM, ES, and 20% DMSO aqueous solution were added to the bacterial suspension, incubated at 37 °C for 2 h, centrifuged at 5000 r / min for 10 min, and the supernatant was taken. According to the instructions of the AKP kit, the activity of bacterial AKP in the supernatant was determined. The blank control group was Staphylococcus aureus without sample treatment. The final concentrations of ESM were 1 / 2 ×, 1 ×, 2 ×, 4 ×MBC, and 8 ×MBC, and the final concentrations of ES were 9.60, 4.80, 2.40, 1.20, and 0.60 mmol / L, respectively. Each group of experiments was repeated 3 times.

[0043] (4) Inhibitory effect of ESM on bacterial biofilm Take 1.60 mL of logarithmic phase Staphylococcus aureus suspension (10 5 CFU / mL) into a sterile 24-well plate, and then 400 μL of ESM, ES and Mn were added respectively. 2+ 400 μL of saline solution containing 20% ​​DMSO was added to the control group, and the final concentration of ESM was 1 / 2× MBC, ES and Mn 2+ The final concentrations of 0.60 mmol / L and 0.30 mmol / L were respectively, and the cells were cultured at 37 °C for 48 h. The supernatant was discarded and the cells were washed twice with PBS to remove planktonic bacteria. The biofilm was fixed with methanol for 15 min, air-dried for 24 h, and stained with 0.1% crystal violet aqueous solution for 15 min. The excess staining solution was washed with deionized water, and then the crystal violet was redissolved in 33% (W / V) acetic acid aqueous solution. The OD value was measured using an enzyme reader (OD 595 nm ) was used to determine the absorbance and calculate the inhibition rate of biofilm.

[0044] Biofilm inhibition rate = (A 空白对照组 - A 实验组 ) / A 空白对照组 (5) Biosafety evaluation of ESM Red blood cells were extracted from fresh sterile rabbit blood and washed three times with sterile PBS (2000 r / min, 2 min). 50 μL of rabbit blood cells and 150 μL of the test solution were added to 1 mL of PBS, incubated at 37 °C for 30 min, centrifuged at 2000 r / min for 2 min), and the supernatant was taken and the absorbance was measured at 543 nm. 0.1% TriX-100 was used as a positive control, and the PBS group was used as a negative control to calculate the hemolysis rate.

[0045] Hemolysis rate (%) = (A 实验组 - A 阴性对照组 ) / ( A 阳性对照组 - A 阴性对照组 ) × 100%, HC 50 The values ​​were calculated using SPSS.

[0046] Normal human liver cells L-O2 in the logarithmic growth phase were inoculated into sterile 96-well culture plates, 200 μL per well, and cultured at 37 °C overnight, then the culture medium was discarded. 200 μL of culture medium containing ESM was added to each well (the final concentration of ESM was 100.00, 33.33, 11.11, 3.70, 1.85 μg / L, and the final concentration of DMSO was ≤ 1%), and 1% DMSO was used as a negative control. After 24 h of culture at 37 °C, the culture medium was discarded, and 20 μL of PBS solution containing MTT (the concentration of MTT was 5 mg / mL) was added to each well, and culture was continued for 4 h. The culture medium was discarded, 150 μL of DMSO was added to each well, and the absorbance value (A) of each well was measured at 490 nm by a microplate reader to calculate the inhibition rate of ESM on cells.

[0047] Cell inhibition rate (%) = (A 空白对照组 −A 实验组 ) / A 空白对照组 × 100%, IC 50 The values ​​were calculated using SPSS.

[0048] 2. ESM drug resistance test Take a sterile 96-well plate, add 40 μL of the test solution and 160 μL of the bacterial solution to each well, and culture at 37 °C for 18 h. The lowest concentration with flocculent turbidity visible to the naked eye in the well is the minimum inhibitory concentration (MIC). Record the MIC value and record it as the first generation experiment. Take the bacterial solution in the 1 / 2 × MIC well of the previous generation experiment and culture it to the mid-logarithmic phase, and then add the bacterial suspension (10 5 CFU / mL), add the test solution, incubate at 37 °C for 18 h, record the MIC value, and record it as the second generation experiment. Repeat the above experimental steps until the 20th generation. The final concentration of ESM (based on ES molar mass) in the first generation experiment was 4.80 mmol / L. The maximum final concentration of the positive control drugs (ciprofloxacin, gentamicin, ampicillin, and clindamycin) was 40 μg / mL.

[0049] 3. Multi-omics analysis of ESM antibacterial mechanism Take the logarithmic phase Staphylococcus aureus and dilute the bacterial solution to 10 with fresh TBS liquid culture medium. 5 CFU / mL, set aside. ESM (final concentration of 0.90 mmol / L), ES (final concentration of 0.90 mmol / L), and blank control group (DMSO final concentration of 0.75%) were added to the diluted bacterial solution, three samples were paralleled in each group, incubated at 37 °C for 5 h, centrifuged at 4 °C 5000 r / min for 10 min, the bacteria were collected and quickly frozen in liquid nitrogen, and transferred to -80 °C for storage. The samples were tested by Beijing Novogene Technology Co., Ltd. for transcriptomics and proteomics.

[0050] The above experimental results are as follows: 1. Antimicrobial activity of ESM (1) Bactericidal kinetic curve of ESM The bactericidal kinetic curves of ESM at different concentrations against Staphylococcus aureus after 0, 1, 2, 3, 4, 5, and 6 h of action are shown in the figure. Figure 6 As shown in A. As can be seen from the figure, the bactericidal rate of ESM is dependent on concentration and time. 1 / 2 × MBC ESM has no inhibitory effect on Staphylococcus aureus. When MBC ESM acts for 0-7 hours, the number of bacteria in the culture medium is not significantly different from that of the control group. After the 7th hour, the number of colonies begins to decrease rapidly. At the 14th hour, all Staphylococcus aureus are killed. For 2 × MBC and 4 × MBC ESM, the number of bacteria in the culture medium reaches the maximum at the 4th hour and 2nd hour, respectively, and all bacteria are killed at the 8th hour and 5th hour.

[0051] (3) Effects of ESM on cell wall Alkaline phosphatase (AKP) exists between the bacterial cell wall and cell membrane. It is not secreted into the bacterial culture medium under normal circumstances. Only when the bacterial cell wall is damaged will AKP leak into the bacterial culture medium. Therefore, the integrity of the bacterial cell wall can be observed by detecting the activity of AKP in the bacterial culture medium. Figure 6 B It can be seen that Mn 2+ It does not increase the activity of AKP in the culture medium, which indicates that Mn 2+ The cell wall will not be damaged. The AKP activity in the culture medium treated with ES and ESM increased with the increase of concentration, and at the same concentration, the AKP activity of the ESM group was significantly stronger than that of the ES group.

[0052] (4) Inhibitory effect of ESM on biofilm Previous experiments have shown that 1 / 2 × MBC ESM has no inhibitory effect on Staphylococcus aureus, so this concentration was selected to investigate its inhibitory effect on the biofilm formation of Staphylococcus aureus. The results are as follows: Figure 6 C. Compared with the control group, ES and Mn 2+ It has a certain inhibitory effect on the formation of biofilm, and ESM has a more significant inhibitory effect on the formation of biofilm.

[0053] (5) Biosafety of ESM In order to understand the toxicity of ESM, we used sterile rabbit blood as the test object and calculated the HC of ESM on rabbit red blood cells. 50 The biosafety of ESM can be evaluated by using the value of Figure 6As shown in D, the hemolytic effects of ES and ESM were significantly weaker than the positive control Trix-100, and at the same concentration, the hemolytic effect of ESM was generally lower than that of ES. The hemolytic rate of 2 × MBC ESM was 10.62%, and the hemolytic rate of ES was 16.97% at the same concentration. The HC 50 The value was greater than 200 μg / mL. ESM has good biocompatibility and has no obvious toxic effect on red blood cells. To explore the cytotoxicity of the compounds, the MTT method was used to test the effects of the compounds on the proliferation of normal liver cells L-O2. The inhibition of ES and ESM on L-O2 was shown in Figure 2. Figure 6 As shown in E, the results show that the IC of ESM for L-O2 50 When the value is greater than 100 μM, ESM has less inhibitory effect on normal hepatocyte proliferation and lower toxicity.

[0054] 2. ESM resistance Four antibiotics used clinically to treat Staphylococcus aureus infections, gentamicin (aminoglycoside), ampicillin (penicillin), clindamycin (lincomycin) and ciprofloxacin (quinolone), were used as positive controls to study whether long-term exposure of ESM to Staphylococcus aureus would induce drug resistance. Figure 6 F It can be seen that Staphylococcus aureus developed resistance to gentamicin, ciprofloxacin, ampicillin and clindamycin in the 2nd, 3rd, 5th and 12th generations, respectively. After 20 generations of culture, the MICs of the four antibiotics increased to 16 times (ciprofloxacin), 8 times (ampicillin and gentamicin), 4 times (clindamycin) and 2 times (ES) of the initial generation, respectively, but the MIC value of ESM did not change, which indicates that Staphylococcus aureus is not easy to develop resistance to ESM.

[0055] 3. Possible antibacterial mechanisms of ESM (1) Transcriptomic analysis In order to gain a deeper understanding of why ESM has a stronger inhibitory effect on S. aureus than ES, we used transcriptomics technology to analyze and compare the differences in the effects of ES and ESM on S. aureus at the gene level. Figure 7A) It can be seen that compared with the ES treatment group, the ESM treatment group has 694 differentially expressed genes (DEGs) ( | log2(FoldChange) |>0&p<0.05), of which 341 DEGs were significantly upregulated and 353 DEGs were significantly downregulated. After comparative analysis, we found that there was consistency in gene expression between the Control group and the ESM group. Similarly, there was similarity in gene expression between the ESM group and the ES group, and between the Control group and the ES group. However, it is worth noting that the expression levels of the same gene in the three groups of ESM, ES, and Control are not the same, showing obvious differences ( Figure 7 B).

[0056] The GO database is an internationally standardized functional classification system consisting of three categories: biological process (BP), cellular component (CC), and molecular function (MF). The GO database was used to annotate and enrich the DEGs of the ESM and ES treatment groups ( Figure 7 C), it can be seen that in the biological process category, most DEGs are related to tRNA, metabolism and amino acids; in terms of cellular components, the most significantly enriched DEGs are related to cell membrane; in terms of molecular function, DEGs are mainly concentrated in aminoacyl-tRNA ligase activity, RNA and tRNA catalytic activity, adenylate and other nucleotide binding activities involved in RNA structure.

[0057] KEGG enrichment analysis was used to analyze and identify DEGs, and several pathways closely related to the antibacterial process of ESM were found ( Figure 7 D). After ESM treatment, the differentially expressed genes were mainly enriched in pathways involved in bacterial aminoacyl-tRNA biosynthesis, cationic antimicrobial peptide (CAMP) resistance, citric acid cycle (TCA cycle), biosynthesis of various other secondary metabolites, fatty acid biosynthesis, valine, leucine and isoleucine degradation, arginine biosynthesis, dicarboxylic acid metabolism, fatty acid metabolism, propionate metabolism, and phosphotransferase system pathways.

[0058] (2) Proteomic analysis We also used proteomics technology to analyze and compare the differences in the antibacterial mechanisms of ES and ESM at the protein level. A total of 1,784 proteins were identified in the samples, including 1,784 quantified proteins. Screening for significantly differentially expressed proteins (DEPs) was performed according to the criteria of expression fold change of more than 1.2 times (upregulation greater than 1.2 times or downregulation less than 0.83 times) and p<0.05. Compared with the ES treatment group, the expression levels of 158 proteins in the ESM treatment group changed significantly, of which 33 proteins were significantly upregulated and 125 proteins were significantly downregulated ( Figure 8A). After comparative analysis, we found that there was consistency in the expression of some proteins between the Control group and the ESM treatment group. Similarly, there was similarity in protein expression between the ESM group and the ES group, and between the Control group and the ES group. However, it is worth noting that the expression levels of the same gene in the three groups of ESM, ES, and Control are not the same, showing obvious differences ( Figure 8 B).

[0059] Compared with the ES treatment group, the ESM treatment group had 5 significantly enriched items in "biological process" (BP), namely: cellular amino acid metabolism process, siderophore biosynthesis process, carboxylic acid metabolism process, cellular amino acid biosynthesis process, and cofactor biosynthesis process; and had significant enrichment in "molecular function" (MF) for iron-sulfur cluster binding activity, siderophore transmembrane transporter activity, amidase activity, carboxyl or carbamoyl transferase activity, hydrolase activity, and single-stranded DNA binding activity ( Figure 8 C).

[0060] Compared with the ES treatment group, the up-regulated significantly differentially expressed proteins (DEPs) in the ESM treatment group were mainly concentrated in protein pathways related to Staphylococcus aureus infection, D-alanine metabolism, cationic antimicrobial peptide (CAMP) resistance, glycerol metabolism, quorum sensing, pyruvate metabolism, two-component system, ABC transport, etc.; the down-regulated DEPs were concentrated in the biosynthesis of secondary metabolites, biosynthesis of antibiotics, tyrosine metabolism, metabolic pathways, arginine biosynthesis, chloroalkane and chloroalkene degradation, naphthalene degradation, lysine degradation, arginine and proline metabolism, lysine biosynthesis pathway ( Figure 8 D).

[0061] (3) Transcriptomics and proteomics association analysis Due to a large number of transcriptional and translational regulatory systems, all living cells can rapidly adjust their transcriptomes and proteomes in response to changing environmental conditions. Therefore, a single omics analysis cannot fully reveal the relevant regulatory mechanisms. From a biological point of view, mRNA levels represent the intermediate state of gene expression and can represent the potential protein expression. The transcriptome can achieve higher throughput at lower consumption and can provide more detailed information to some extent; proteins are direct functional executors, and therefore, have irreplaceable advantages in measuring protein expression levels. The joint analysis of the two omics can not only provide insight into the laws and nature of life activities at different omics levels, but also reveal the mutual regulatory effects or associations between the two, which can better understand biological processes.

[0062] The transcriptomic and proteomic analysis structures of the ES and ESM treatment groups were analyzed by correlation analysis. Although proteins are the products of gene expression, gene expression on proteins is a co-translational and post-mRNA translational effect of different environments, resulting in gene expression levels not always being completely correlated. Although there was no significant correlation between transcriptomic and proteomic expression at the overall level, a total of 37 DEPs (7 upregulated and 30 downregulated) were identified, and their inferred protein expression trends were the same as their corresponding DEGs. In addition, we performed a nine-quadrant analysis ( Fig. 9 ) to examine the relationship between proteins and RNAs detected under ESM treatment. In this study, genes were highly enriched in quadrant 5, followed by quadrants 6 and 7. The proteins and RNAs enriched in quadrants 6, 8, and 9 were related to RNAs with high abundance, which may be due to regulation or protein accumulation at the post-transcriptional or translational level. The enrichment of proteins and RNAs in quadrant 5 indicated that these proteins and RNAs were non-differentially co-expressed. In quadrants 1, 2, and 4, the abundance of a few proteins was lower than that of the related RNAs. In addition, relatively few DEGs and DEPs showed the same expression pattern in quadrants 3 and 7. The functional analysis of genes and proteins with similar expression trends and related functions is shown in Table 4.

[0063] Table 4 Significantly differentially expressed proteins and corresponding significantly differentially expressed transcripts between ESM and ES The results of transcriptomic and proteomic analyses showed that ESM can affect the life process of bacteria through various mechanisms, ultimately leading to bacterial death. These mechanisms mainly include: (1) ESM can activate the autolytic enzymes lytM and atl of Staphylococcus aureus, leading to the upregulation of gene and protein expression of these enzymes, thereby increasing the autolysis of bacterial cell walls, resulting in cell wall damage and leakage of bacterial contents, thus causing bacterial death; (2) ESM can also inhibit the gene expression of various amino acid ligases in the bacterial protein synthesis process, such as thrs, cysS, thrC, metE, dapD, etc. These enzymes are key components of bacterial protein synthesis. Their inhibition leads to the blockage of bacterial protein synthesis and a significant reduction in protein content. The clp protein forms the ClpP protease complex together with ATPases such as clpX and clpA. Through its protease activity, it can recognize and degrade proteins that cannot be correctly folded or have aged in bacterial cells. This protein degradation helps maintain the homeostasis of proteins inside bacteria and remove abnormal proteins, which is crucial for the survival and adaptation of bacterial cells to the environment. The downregulation of clpP expression leads to disordered intracellular protein metabolism. This further weakens the survival ability of the bacteria and eventually leads to their death; (3) ESM also inhibits a variety of enzymes involved in bacterial energy metabolism and biofilm formation. These enzymes are important links in bacterial energy synthesis and metabolism. Their inhibition leads to insufficient bacterial energy supply, further affecting their survival and reproduction. (4) manA contributes to the formation of Staphylococcus aureus biofilm. ESM can inhibit its mRNA and protein expression, thereby affecting the formation of Staphylococcus aureus biofilm; (5) ESM can inhibit the expression of the norB gene. The norB efflux pump is a member of the MFS family, which leads to increased resistance of Staphylococcus aureus to hydrophilic quinolones, tetracyclines, etc. ESM inhibits the expression of the norB gene, which may be an important reason why ESM is not easy to induce drug resistance in Staphylococcus aureus. The antibacterial mechanism of ESM is as follows: Fig.10 As shown, these mechanisms work synergistically, making ESM a new type of antibacterial drug with great research value and development potential.

[0064] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A esculetin-manganese complex, characterized in that: Its structure is Mn 2+ It coordinates with the O atom in quinoline to form a Mn-O bond, in which Mn is +2 and there is a Mn-O4 coordination form.

2. The aesculetin-manganese complex according to claim 1, characterized in that It has at least one of the following structures:

3. The aesculetin-manganese complex according to claim 1, characterized in that The raw materials for preparation include aesculetin and Mn 2+ .

4. The aesculetin-manganese complex according to claim 3, characterized in that The aesculetin and Mn 2+ The feeding molar ratio is 1:6 to 8:

1.

5. The aesculetin-manganese complex according to claim 3, characterized in that The aesculetin and Mn 2+ The feeding molar ratio is 2 to 8:

1.

6. The aesculetin-manganese complex according to claim 3, characterized in that The aesculetin and Mn 2+ The feed molar ratio is 2:

1.

7. A method for preparing the aesculetin-manganese complex according to any one of claims 1 to 6, characterized in that: The following steps are involved: The esculetin and MnCl2 are mixed, water is added, the mixture is heated to react for 1 to 3 hours, and dried under reduced pressure to obtain a reaction product, the esculetin-manganese complex.

8. The preparation method according to claim 7, characterized in that: The heating temperature is 95-105°C.

9. The preparation method according to claim 7, characterized in that: The heating temperature is 95-100°C.

10. Use of the aesculetin-manganese complex according to any one of claims 1 to 6 in the preparation of antibacterial products.

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