Flavone selenium complexes and uses thereof

Abstract

This invention belongs to the field of pharmaceutical technology and discloses flavonoid selenium complexes and their applications. This study prepares flavonoid selenium complexes by reacting flavonoids with selenium dioxide, synthesizes a series of flavonoid selenium complexes, improves the antibacterial and anti-inflammatory effects of flavonoids, reduces the toxicity of flavonoids and selenium, and screens out flavonoid selenium complexes that can significantly improve the antibacterial activity of flavonoids. This provides a scientific basis for the structural modification of flavonoids and provides basic data and new ideas for the development and application of natural molecular antibacterial agents.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically, to flavonoid selenium complexes and their applications. Background Technology

[0002] Flavonoids are a class of secondary metabolites widely found in plants, possessing various biological activities such as antibacterial, anti-inflammatory, and antitumor activity. However, their high cytotoxicity, limited antibacterial activity, and poor absorption restrict their widespread clinical application.

[0003] The combination of flavonoids and metal ions helps reduce the cytotoxicity of flavonoids and enhance their biological activity. Dihydromyricetin and Fe have been reported to work together. 2+ Ca 2+ Zn 2+ Cu 2+ Ni 2+ Mn 2+ Compared to dihydromyricetin, the above complexes, containing metal ions, showed varying degrees of improvement in antioxidant capacity and free radical scavenging ability; luteolin, sulfuric acid, and metal salts (Zn) were also utilized. 2+ Cu 2+ Mn 2+ Co 2+ Four luteolin sulfate metal complexes were synthesized, among which luteolin and Cu were synthesized. 2+ The inhibitory effect of the complex on Staphylococcus aureus and Escherichia coli was significantly enhanced compared to that of luteolin. Existing studies show that the bioactivity of different types of flavonoids combined with different metal ions varies significantly. Even the same flavonoid compound exhibits different bioactivity when combined with different metal ions. Therefore, it is necessary to explore the bioactivity of new flavonoid compounds combined with metal ions to lay the foundation for the development of new compounds. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the above-mentioned problems existing in the prior art, and firstly, to provide the application of flavonoid selenium complex.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] The application of flavonoid selenium complexes in the preparation of formulations that reduce the in vitro cytotoxicity of flavonoids, wherein the flavonoid selenium complexes are prepared by complexation reaction of flavonoids and selenium dioxide; wherein the flavonoids are selected from rutin, baicalin, quercetin, 7,8-dihydroxyflavone, baicalin, myricetin, and dihydromyricetin; and wherein the cells are RAW267.4 and Caco-2.

[0007] Preferably, for RAW267.4 cells, the complexes obtained by complexing baicalein, quercetin, 7,8-dihydroxyflavone, baicalin, guarbanone, and dihydromyricetin with selenium dioxide, namely baicalein-selenium, quercetin-selenium, 7,8-dihydroxyflavone, baicalin-selenium, guarbanone-selenium, and dihydromyricetin-selenium, are less toxic to cells than baicalein, quercetin, 7,8-dihydroxyflavone, baicalin, guarbanone, and dihydromyricetin themselves.

[0008] Preferably, for Caco-2 cells, the complexes 7,8-dihydroxyflavone-selenium, jujube-selenium, and dihydromyricetin-selenium, obtained by complexing 7,8-dihydroxyflavone, jujube-selenium, and dihydromyricetin with selenium dioxide, respectively, are less toxic to cells than 7,8-dihydroxyflavone, jujube-selenium, and dihydromyricetin themselves.

[0009] This invention also provides the application of flavonoid selenium complex in the preparation of colistin synergistic antibacterial synergists, wherein the flavonoid selenium complex is prepared by complexation reaction of flavonoids and selenium dioxide; wherein the flavonoids are selected from rutin, baicalin, myricetin, and myricetin; and wherein the flavonoid selenium complex synergistically resists Salmonella typhimurium and Escherichia coli.

[0010] This invention, through MIC and combined drug sensitivity experiments, revealed that rutin-selenium, baicalin-selenium, myricetin-selenium, and colistin significantly enhance the synergistic antibacterial activity, with rutin-selenium exhibiting the strongest synergistic antibacterial activity with colistin. Rutin-selenium may exert its antibacterial effect by reducing intracellular ATP and increasing bacterial inner membrane permeability.

[0011] Preferably, when the flavonoid compound is rutin, rutin and selenium dioxide are complexed to prepare rutin-selenium, and the concentration range of rutin-selenium synergistic with colistin for antibacterial effect is 2 μg / mL to 16 μg / mL.

[0012] This invention also provides the application of flavonoid selenium complex in the preparation of formulations that inhibit inflammatory factors, wherein the flavonoid selenium complex is prepared by a complexation reaction of flavonoids and selenium dioxide; wherein the flavonoids are selected from rutin, baicalin, and baicalein; and wherein the inflammatory factors are selected from NO, TNF-α, IL-1β, and IL-6.

[0013] This invention, through an in vitro LPS-induced RAW267.4 inflammation model experiment, found that Rut-Se can significantly inhibit the expression levels of inflammatory mediators IL-β and NO, and enhance the anti-inflammatory activity of Rut.

[0014] Preferably, when the flavonoid compound is rutin, rutin is complexed with selenium dioxide to prepare rutin-selenium, which inhibits the expression of NO and IL-1β; when the flavonoid compound is baicalin, baicalin is complexed with selenium dioxide to prepare baicalin-selenium, which inhibits the expression of NO and IL-1β; when the flavonoid compound is baicalein, baicalein is complexed with selenium dioxide to prepare baicalein-selenium, which inhibits the expression of NO and IL-6; when the flavonoid compound is myricetin, myricetin is complexed with selenium dioxide to prepare myricetin-selenium, which inhibits the expression of NO; ​​when the flavonoid compound is dihydromyricetin, dihydromyricetin is complexed with selenium dioxide to prepare dihydromyricetin-selenium, which inhibits the expression of NO.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] This invention provides the application of flavonoid selenium complexes. This study prepares flavonoid selenium complexes by reacting flavonoids with selenium dioxide, synthesizes a series of flavonoid selenium complexes, improves the antibacterial and anti-inflammatory effects of flavonoids, reduces the toxicity of flavonoids and selenium, and screens out flavonoid selenium complexes that can significantly improve the antibacterial activity of flavonoids. This provides a scientific basis for the structural modification of flavonoids and provides basic data and new ideas for the development and application of natural molecular antibacterial agents. Attached Figure Description

[0017] Figure 1 The structural formula for the reaction of rutin and selenium dioxide;

[0018] Figure 2 The rutin-selenium structure is characterized in Figure a (1H NMR spectrum), Figure b (Fourier transform infrared spectrum), and Figures c and d (X-ray photoelectron spectra).

[0019] Figure 3 The structural formula for the reaction of quercetin with selenium dioxide;

[0020] Figure 4 The structural formula for the reaction of baicalin with selenium dioxide;

[0021] Figure 5 The structural formula for the reaction of baicalin with selenium dioxide;

[0022] Figure 6 The structural formula for the reaction of myricetin with selenium dioxide;

[0023] Figure 7 The structural formula for the reaction of dihydromyricetin with selenium dioxide;

[0024] Figure 8 The structural formula for the reaction of 7,8-dihydroxyflavone with selenium dioxide;

[0025] Figure 9 The structural formula for the reaction of isoliquiritigenin with selenium dioxide;

[0026] Figure 10 The structural formula for the reaction of apigenin with selenium dioxide;

[0027] Figure 11 The structural formula for the reaction of kaempferol and selenium dioxide;

[0028] Figure 12 The cytotoxic effects of flavonoid selenium complex on RAW264.7 cells;

[0029] Figure 13 The cytotoxic effect of flavonoid selenium complex on Caco-2 cells;

[0030] Figure 14 Analysis of the antibacterial activity of flavonoids and their selenium complexes combined with colistin against Salmonella;

[0031] Figure 15 Analysis of the time-killing curve of rutin-selenium synergistic colistin against Salmonella;

[0032] Figure 16 Analysis of the time-killing curve of baicalin-selenium synergistic colistin against Salmonella;

[0033] Figure 17 Analysis of the time-killing curve of myricetin-selenium synergistic colistin against Salmonella;

[0034] Figure 18 The inhibitory effect of flavonoids and flavonoid selenium complexes on NO in RAW264.7;

[0035] Figure 19 The anti-inflammatory effects of rutin and rutin-selenium on RAW264.7;

[0036] Figure 20 The anti-inflammatory effects of baicalin and baicalin-selenium on RAW264.7;

[0037] Figure 21 The anti-inflammatory effects of baicalin and baicalin-selenium on RAW264.7;

[0038] Figure 22 The anti-inflammatory effects of myricetin and myricetin-selenium on RAW264.7;

[0039] Figure 23 The anti-inflammatory effects of dihydromyricetin and dihydromyricetin-selenium on RAW264.7 were studied. Detailed Implementation

[0040] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0041] Example 1: Preparation and structural confirmation of flavonoid selenium complex

[0042] Based on the structure-activity relationship of flavonoids and the analysis of selenium complexation sites, 10 flavonoid selenium complexes were synthesized by complexing different flavonoids with 5-hydroxy and 4-carbonyl structures in ring A with selenium dioxide.

[0043] Taking rutin as an example, the preparation method of rutin-selenium complex is as follows: Accurately weigh 2.5 mmol of rutin (Rut) and place it in a double-necked round-bottom flask. Dissolve it in 25 mL of ethanol. After dissolution, adjust the pH of the solution to 2-3 with concentrated hydrochloric acid. Then weigh 1.25 mmol of selenium dioxide (SeO2) and dissolve it in 10 mL of ethanol. After ultrasonic dissolution, transfer it to a constant pressure dropping funnel and slowly add it dropwise to the double-necked round-bottom flask. Stir the reaction at 60℃ under nitrogen purging for 10 h. Monitor the reaction using a thin-layer chromatography plate to confirm whether the product has formed. Afterward, centrifuge the solution at 5000 rpm, discard the supernatant, wash the precipitate with ethanol, repeat 3-4 times, and then vacuum dry at room temperature and 0.8 MPa for 12 h to obtain the solid product rutin-selenium (Rut-Se, as shown in the image). Figure 1 The structure of Rut-Se was determined by characterization using proton nuclear magnetic resonance spectroscopy, Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy (e.g., Figure 2 ).

[0044] The synthesis methods for quercetin (Que), baicalin (Bal), baicalein (Bai), myricetin (Myr), dihydromyricetin (Dhy), 7,8-dihydroxyflavone (7,8-DHF), isoliquiritigenin (Iso), chrysin (Chy), and kaempferol (Kae) are similar to those of Rut. The compounds Que-Se, Bal-Se, Bai-Se, Myr-Se, Dhy-Se, 7,8-DHF-Se, Iso-Se, Chy-Se, and Kae-Se were isolated and purified. Their reaction structures are shown below. Figures 3 to 11 .

[0045] Example 2 Cell Viability Experiment

[0046] RAW264.7 and Caco-2 cells were revived using Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% streptomycin and penicillin antibiotics. After removing the cell cryopreservation tubes from liquid nitrogen, they were rapidly thawed in a 37°C water bath, and 2-3 mL of culture medium was added. The mixture was gently pipetted and mixed in a clean bench before being transferred to 5 mL sterile centrifuge tubes. The tubes were centrifuged at 1100 rpm for 5 min at room temperature, the supernatant was discarded, and 5 mL of DMEM containing 10% FBS and 1% penicillin antibiotics was added. The mixture was then pipetted and mixed before being transferred to cell culture flasks and cultured at 37°C and 5% CO2 for 24 h.

[0047] RAW264.7 cells were passaged every 24 hours, while Caco-2 cells were passaged every 48 hours. Caco-2 cells required trypsin-assisted passage. Once cells reached 3-4 passages and were stable, they were inoculated at a rate of 1×10⁻⁶ cells / year. 5 -1×10 6 Cells were seeded at a concentration of [number] cells / mL in 24-well cell culture plates, with 500 μL of cell suspension added to each well. Cells were cultured at 37°C and 5% CO2 for 12 h. When the cell density reached approximately 60%, the supernatant was discarded, and 500 μL of DMEM medium (containing 10, 20, 40, 80, and 160 μM of flavonoids, selenium dioxide, flavonoid selenium, and a 1% DMSO blank control group) was added, with three replicates per group. After incubation at 37°C and 5% CO2 for 24 h, 50 μL of CCK-8 reagent was added to each well, and incubation continued for 1-2 h. An equal volume of the supernatant was then transferred to a 96-well plate. 450 The cell viability was calculated under the following conditions:

[0048] Cell viability (%) = (OD) 450 (Sample)-OD 450 (Blank)) / (OD 450 (Control)-OD 450 (Blank)×100%

[0049] Sample: Absorbance values ​​at OD450 of cell samples containing different concentrations of Rut, Bai, Bal, Myr, Dhy, 7,8-DHF, Iso, Kae, Que, Chy, Rut-Se, Bai-Se, Bal-Se, Myr-Se, Dhy-Se, 7,8-DHF-Se, Iso-Se, Kae-Se, Que-Se, Chy-Se and SeO2.

[0050] Control: Absorbance value at OD450 for cell samples containing 1% DMSO.

[0051] Blank: Absorbance value at OD450 of DMEM medium with CCK-8 reagent added.

[0052] from Figure 12 It can be seen that with the increase of flavonoid and flavonoid selenium complex concentration, the toxicity to RAW264.7 increases and the cell survival rate decreases. Between 10-40 μM, the cell survival rate of all 10 flavonoids and flavonoid selenium complexes is above 80%, showing little cell damage and high survival rate. In contrast, selenium dioxide exhibits significant cytotoxicity; at a concentration of 160 μM, the cell survival rate is only about 10%. Furthermore, the toxicity of flavonoid selenium complexes to RAW264.7 does not decrease significantly after flavonoid compounds are complexed with selenium. At a flavonoid concentration of 160 μM, the cell survival rates of Bai-Se, Que-Se, 7,8-DHF-Se, Bal-Se, and Dhy-Se are greater than those of the ligand flavonoids. This means that for RAW264.7, Bai-Se, Que-Se, 7,8-DHF-Se, Bal-Se, and Dhy-Se can reduce cytotoxicity compared to ligand flavonoids.

[0053] Cytotoxic effects on Caco-2: such as Figure 13 As shown, flavonoids and their corresponding flavonoid selenium complexes maintained cell viability above 80% at concentrations between 10 and 40 μM, indicating low toxicity. Across five concentration gradients, the cell viability of the flavonoid selenium complexes was consistently significantly higher than that of selenium dioxide, suggesting that the flavonoid selenium complexes can reduce the toxicity of selenium. Furthermore, Rut, Dhy, Bai, Iso, 7,8-DHF, Chy, and their corresponding flavonoid selenium complexes all maintained cell viability above 80% at 160 μM. 7,8-DHF-Se, Chy-Se, and Dhy-Se showed higher viability than their corresponding flavonoids, while Rut-Se, Bai-Se, and Iso-Se showed no significant difference in cell viability compared to their corresponding flavonoids. The above results indicate that at 160 μM, 7,8-DHF-Se, Chy-Se, and Dhy-Se reduced the cytotoxicity of the ligand flavonoids, while Rut-Se, Bai-Se, and Iso-Se showed no significant difference in cytotoxicity compared to the ligand flavonoids. Que-Se, Myr-Se, Bal-Se, and Kae-Se increased the toxicity of the ligand flavonoids.

[0054] Example 3: Antibacterial experiment of flavonoid selenium complex and its combination with colistin

[0055] I. Determination of the minimum inhibitory concentration of flavonoid selenium complexes

[0056] Streak *Salmonella typhimurium*, *Escherichia coli*, *Staphylococcus aureus*, *Klebsiella pneumoniae*, and *Enterococcus faecalis* onto LB agar plates and incubate overnight at 37°C. Single colonies are picked and transferred to MH broth, then incubated for 4 hours at 180 rpm and 37°C in a shaker. 100 μL of LPM broth (simulating the macrophage intracellular environment) or MH broth is added to columns 2-11 of a 96-well plate. 180 μL of broth is added to the first column, followed by 20 μL of flavonoids and flavonoid selenium complex. Each treatment group is repeated in triplicate. 100 μL of each drug is serially diluted from column 1 to column 12 using a 300 μL multichannel pipette. The cultured bacterial culture is then diluted to 1×10⁻⁶. 6 Add 100 μL of diluted bacterial solution to each well at approximately CFU / mL. The positive control group is given only bacterial solution without any drug, while the blank control group is given only LPM or MH broth. After incubation at 37°C for 16 hours, the results can be observed.

[0057] II. Antibacterial effect of flavonoid selenium complex combined with colistin against Salmonella.

[0058] 1. Checkerboard method for flavonoid selenium complexes

[0059] Dilute the original bacterial culture to 1×10 using LPM broth. 7 CFU / mL, add 50 μL LPM broth to 8 rows and 8 columns of a 96-well plate. Add 50 μL of a prepared flavonoid and flavonoid selenium solution to the first row, serially dilute to the seventh row, and then add 50 μL of a prepared CS solution serially diluted to 7 columns, from low to high concentration, to columns 1-7 of the 96-well plate. Add 100 μL of bacterial culture to the last 8 rows and 8 columns, incubate at 37℃ for 16 h, and measure OD using a microplate reader. 600 Observe the experimental results and use the fractional inhibitory concentration index (FICI) as the basis for judging the combined drug susceptibility test. The FICI calculation method is as follows:

[0060]

[0061] FICI ≤ 0.5 indicates a synergistic effect; 0.5 < FICI ≤ 1 indicates an additive effect; 1 < FICI ≤ 2 indicates an unrelated effect; and FICI > 2 indicates an antagonistic effect.

[0062] 2. Sterilization curve determination

[0063] Salmonella was revived onto LB agar plates and cultured overnight before being inoculated into MH broth and cultured for 4 hours at 180 rpm and 37°C in a shaker for subsequent experiments. 4 mL of LPM broth was added to 15 mL sterile EP tubes, including single-drug groups (flavonoids, flavonoid selenium, selenium dioxide, and different concentrations of CS); combination groups (flavonoids combined with CS, flavonoid selenium combined with CS); a positive control group (no drugs added, only bacterial culture); and a blank control group (broth added only to avoid broth contamination affecting experimental results). Each tube contained the same concentration of prepared drug and an equal volume of 1×10⁻⁶ final concentration. 6 Salmonella culture at CFU / mL was prepared, with a final volume of 4 mL per tube. The tubes were placed in a shaker at 180 rpm and 37°C. At 0, 3, 9, and 24 h, 100 μL of each tube was taken and diluted with phosphate-buffered saline (PBS) solution. Gradient drop plate counting was performed, and the change in bacterial count over time was recorded.

[0064] like Figures 14 to 17 As shown, by testing the minimum inhibitory concentration (MIC) of 10 flavonoid selenium complexes, flavonoid selenium complexes synergistically with colistin were screened. It was found that compared with the corresponding flavonoids, rutin-selenium, baicalin-selenium, and myricetin-selenium significantly reduced the MIC of Salmonella typhimurium and Escherichia coli, and synergistically with colistin significantly enhanced the anti-Salmonella activity of flavonoids. Among them, rutin-selenium and colistin (16 μg / mL) showed the best synergistic antibacterial effect, completely inhibiting Salmonella growth within 3 hours. Compared with the Salmonella group, the synergistic antibacterial effect of rutin-selenium and colistin (2 μg / mL) within 24 hours reduced the MIC by 3 log10 (CFU / mL), demonstrating a significant synergistic antibacterial effect. The combined use of rutin-selenium and colistin significantly enhanced the intracellular permeability of Salmonella cells and reduced intracellular ATP content, achieving antibacterial activity.

[0065] Table 1. Determination of minimum inhibitory concentration (MH broth)

[0066]

[0067]

[0068] Table 2. Determination of minimum inhibitory concentration (LPM broth)

[0069]

[0070] Example 4: Evaluation of the anti-inflammatory effect of flavonoid selenium complex

[0071] I. In vitro experiments

[0072] 1. Effects of flavonoid-selenium on NO, a pro-inflammatory mediator in RAW264.7

[0073] NO detection principle: NO produced in cells is oxidized to nitrite (NO3). 2- This reagent reacts with Griess' chromogenic reagent to form a red azo compound. The absorbance of the red azo compound at 540 nm is measured using a microplate reader and a spectrophotometer. Simultaneously, the absorbance of a sodium nitrate standard solution is used as a reference to calculate the nitrite content in the sample, thus indirectly obtaining the change in NO content. The specific operating steps are as follows:

[0074] (1) Drug incubation treatment: RAW264.7 cells in the logarithmic growth phase were passaged, and the cell concentration was diluted to 1×10⁻⁶. 5 -1×10 6 Cells were seeded at a concentration of 100 cells / mL in 24-well cell culture plates. 500 μL of cell suspension was added to each well. The plates were incubated at 37°C and 5% CO2 for 12 h. The cell concentration was observed under a microscope until it reached approximately 60%. The cell supernatant was then discarded. 500 μL of DMEM solution containing 10 μM and 20 μM flavonoids and corresponding 5 μM and 10 μM flavonoid selenium were added to the sample treatment groups, respectively. 500 μL of DMEM medium containing 100 μM nitric oxide synthase inhibitor (S-Methylisothiourea Sulfate, SMT) was added to the positive control group. 500 μL of DMEM medium was added to the blank control group. Each sample was divided into 3 replicate wells. After incubation at 37°C and 5% CO2 for 2 h, except for the blank control group, 10 μL of LPS at a final concentration of 1 μg / mL was added to each well of the remaining plates. The plates were incubated at 37°C and 5% CO2 for another 16 h.

[0075] (2) NO content determination: Take out the 24-well plate, shake it gently to mix, take 50 μL of supernatant from each well and add it to the 96-well plate. Then add 50 μL of solvent I from the NO detection kit, followed by 50 μL of solvent II. After standing at room temperature in the dark for 10 min, measure the absorbance at 540 nm using an ELISA reader.

[0076] (3) Determination of sodium nitrite (NaNO2) standard curve: Dilute the NaNO2 of the given concentration in the kit to a gradient sample concentration range, measure the OD value at 540 nm, plot the NaNO2 concentration on the x-axis and the OD value on the y-axis to obtain the standard curve between the two, find the first regression equation, and then calculate the NaNO2 content, thereby obtaining the NO content in the sample solution.

[0077] 2. Effects of flavonoid-selenium on the expression levels of inflammatory factors in RAW264.7

[0078] (1) Drug incubation treatment: RAW264.7 in the logarithmic phase was selected and its concentration was diluted to 1×10. 5 -1×106 Cells were seeded at a density of 1 / mL into 12-well cell culture plates, with 1 mL of cell suspension added to each well. The plates were cultured in a cell culture incubator for 12 h, and the supernatant was discarded. For the sample treatment group, 500 μL of DMEM medium with a final concentration of 20 μM flavonoids, 10 μM flavonoid selenium, and 10 μM SeO2 was added to each well. 20 μM curcumin (CUR) was used as the positive control group, while the LPS control group and the blank control group were in DMEM medium with 1% DMSO added. Three replicate groups were set up. After incubation at 37°C with 5% CO2 for 3 h, the plates were removed. Except for the blank control group, 10 μL of LPS with a final concentration of 1 μg / mL was added to each well of the other plates, and the plates were incubated for another 6 h.

[0079] (2) Cell lysis: Discard the supernatant, add 500 μL of TRIzol Reagent to each well to lyse the cells, let stand for 5 min, observe that the cell lysate is jelly-like, then thoroughly pipette and transfer to 1.5 mL enzyme-free tube.

[0080] (3) Chloroform extraction: Add 200 μL of chloroform to each tube, shake vigorously at room temperature to mix into a light red emulsion, let stand at room temperature for 3 min, and then centrifuge at 12000 rpm, 4℃ for 15 min.

[0081] (4) Isopropanol extraction: After centrifugation, the solution is divided into three layers. Take 200 μL of the supernatant and place it in a new enzyme-free tube. Be careful not to take the middle layer. Then add an equal amount of 200 μL of isopropanol solvent to each tube, gently invert it 10 times, place it on ice and let it stand for 10 min, then centrifuge for 10 min at 12000 rpm and 4℃.

[0082] (5) Washing with 75% ethanol: Prepare a 75% ethanol solution with enzyme-free water, centrifuge and discard the supernatant. Add 700 μL of 75% ethanol solution to each tube, gently invert the tube 10 times, and a white precipitate will be seen suspended. Centrifuge at 12000 rpm, 4℃ and 15 min. Repeat the above steps to wash once more, and finally centrifuge empty once. Take the remaining 75% ethanol from the test tube, place it on ice, open the cap and let it stand to dry for 5 min. Then add 30-50 μL of enzyme-free water to dissolve, mix thoroughly by blowing and place on ice.

[0083] (6) RNA concentration determination: The single-stranded RNA mode of the concentration meter was used to measure and record the OD of 1 μL of sample RNA. 260 / OD 280 The value should be kept between 1.8 and 2.0; then the quality of the extracted RNA is determined based on the brightness and diffusion of the gel electrophoresis bands.

[0084] (7) DNA removal: After the system is prepared, mix it by pipetting, centrifuge at a short speed, and run the PCR program at 42°C for 2 min.

[0085] (8) Reverse transcription: After the system was prepared, it was thoroughly mixed by pipetting and centrifugation at a short speed. The PCR program was then set to 37℃ for 15 min and 85℃ for 5 s. The reverse transcription product was then stored at -20℃. Real-time quantitative PCR was then performed to determine the product.

[0086] The results showed that rutin-selenium, baicalin-selenium, baicalin-selenium, myricetin-selenium, and dihydromyricetin-selenium significantly reduced NO levels compared with their corresponding flavonoids. Among them, baicalin-selenium and rutin-selenium enhanced the inhibition of the expression level of the pro-inflammatory factor IL-1β, while baicalin-selenium enhanced the inhibition of the expression level of IL-6.

Claims

1. The application of flavonoid selenium complexes in the preparation of colistin synergistic antibacterial synergists, characterized in that, The flavonoid selenium complex is prepared by a complexation reaction of flavonoids and selenium dioxide; the flavonoids are selected from rutin, baicalin, and myricetin; the flavonoid selenium complex synergistically resists Salmonella typhimurium with colistin; the concentration range of colistin is 2 μg / mL to 16 μg / mL; The structural formula of the flavonoid selenium complex is as follows: ， or , or .

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