CN115919892A - Preparation method for obtaining two pathogenic bacteria bacteriostatic complexes, two pathogenic bacteria bacteriostatic complexes and application thereof - Google Patents
Preparation method for obtaining two pathogenic bacteria bacteriostatic complexes, two pathogenic bacteria bacteriostatic complexes and application thereof Download PDFInfo
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- CN115919892A CN115919892A CN202211450018.2A CN202211450018A CN115919892A CN 115919892 A CN115919892 A CN 115919892A CN 202211450018 A CN202211450018 A CN 202211450018A CN 115919892 A CN115919892 A CN 115919892A
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Abstract
The invention discloses a preparation method for obtaining two pathogenic bacteria bacteriostatic complexes, the two pathogenic bacteria bacteriostatic complexes and application thereof, wherein the preparation method comprises the following steps: taking 1.0-1.5g of ferric sulfate or sodium sulfide, 3.0-3.6g of sodium acetate and 0.5-0.7g of organic sulfur compound, mixing, placing in 10-30ml of water, shaking uniformly, then placing in a high-temperature reaction kettle at 80-250 ℃ for reaction for 2.5-24h, centrifuging after the reaction is finished to obtain a supernatant and a precipitate, wherein the supernatant is a polysulfane compound, the precipitate is a polyferric sulfide compound, and the polysulfane compound and the polyferric sulfide compound have an antibacterial effect, can be divided into application in preparing medicaments for inhibiting candida albicans, staphylococcus aureus, trichophyton rubrum or escherichia coli, has a simple synthetic method and short chemical reaction time, provides support for development of novel antifungal agents and prevention and treatment of invasive fungi, and has good reference significance for preventing and treating common clinical drug-resistant bacteria.
Description
Technical Field
The invention relates to the field of pathogenic bacteria medicines, and particularly relates to a preparation method for obtaining two pathogenic bacteria bacteriostatic compounds, the two pathogenic bacteria bacteriostatic compounds and application thereof.
Background
After some pathogenic bacteria repeatedly contact some chemotherapeutic medicines, the reactivity of some pathogenic bacteria is continuously weakened, so that the pathogenic bacteria can resist the medicines without killing or inhibiting, and the resistance of the pathogenic bacteria to the medicines is called drug resistance or drug resistance. The main reason for generating drug resistance is that the drug dosage is insufficient or a certain drug is used for a long time, pathogenic bacteria generate drug resistance, the curative effect is reduced or completely lost, the clinical existing antibacterial drug varieties are limited, and the strategy for coping with the drug resistance of the pathogenic bacteria can be summarized into two types, one is to avoid the generation of the drug resistance of the pathogenic bacteria by reasonably using the existing antibacterial drug, the other is to enhance the research and development of a new antibacterial/bacteriostatic agent, and the research and development of the new bacteriostatic agent is long in research and development period and high in cost, but the method is regarded as a more effective coping method for a long time.
Disclosure of Invention
In view of this, the invention provides a preparation method for obtaining two pathogenic bacteria bacteriostatic complexes, the two pathogenic bacteria bacteriostatic complexes and application thereof, and provides a choice for developing novel bacteriostatic agents.
The technical scheme is as follows:
one of the objects of the invention is achieved by:
the preparation method for obtaining the bacteriostatic compound of two pathogenic bacteria is characterized by comprising the following steps: taking 1.0-1.5g of ferric sulfate or sodium sulfide, 3.0-3.6g of sodium acetate and 0.5-0.7g of organic sulfur compound, mixing, placing in 10-30ml of water, shaking uniformly, then placing in a high-temperature reaction kettle at 80-250 ℃ for reaction for 2.5-24h, and centrifuging after the reaction is finished to obtain supernatant and precipitate, wherein the supernatant is a polysulfane compound, and the precipitate is a polyferric sulfide compound, and both have an antibacterial effect.
By adopting the technical scheme, the obtained polysulfane compound can contain polysulfane with different chain lengths, and is not a single substance, the iron polysulfide compound contains iron sulfide with different chain lengths, and is also not a single substance, and the iron sulfide compound are both novel compounds, so that the iron sulfide compound and the iron sulfide compound are both called as 'compounds', the synthetic method is simple, the chemical reaction time is short, and the two obtained products have an antibacterial effect.
Preferably:
the organic sulfur content in the organic sulfur compound is more than or equal to 10 percent.
The organic sulfur compound is cystine, cysteine or methionine.
1.21g of ferric sulfate, 3.6g of sodium acetate and 0.625g of organic sulfur compound are mixed and put into 20ml of water to be shaken up, then put into a high-temperature reaction kettle at 200 ℃ to react for 24 hours, and after the reaction is finished, the polysulfane compound and the polyferric sulfide compound are obtained by centrifugation.
The organic sulfur compound is derived from animal blood.
The second purpose of the invention is realized by the following steps:
the key point of the pathogenic bacteria inhibiting compound is that the compound is prepared according to the preparation method, so that the polysulfane compound is obtained.
The key point of the pathogenic bacteria inhibiting compound is that the compound is prepared according to the preparation method, so as to obtain the polyferric sulfide compound.
By adopting the technical scheme, the obtained polysulfane compound and iron polysulfide compound can be independently used for preparing medicines for inhibiting pathogenic bacteria, and a new choice is provided for developing novel bacteriostatic agents.
The third purpose of the invention is realized by the following steps:
the application of a pathogenic bacteria inhibiting compound is characterized in that: the polysulfane compound or the polyferric sulfide compound prepared by the preparation method or the pathogenic bacteria inhibiting compound is applied to preparation of medicines for inhibiting pathogenic bacteria.
Preferably:
the pathogenic bacteria are Candida albicans, staphylococcus aureus, trichophyton rubrum or Escherichia coli.
Compared with the prior art, the invention has the beneficial effects that: the compound is a polysulfane compound or a polyferric sulfide compound, both of which have obvious bacteriostatic action and provide a choice for developing novel bacteriostatic agents.
Drawings
FIG. 1 is a particle size test chart and an appearance chart of the prepared polysulfane compound.
Detailed Description
The present invention will be further described with reference to the following examples and the accompanying drawings.
1. Preparation of bacteriostatic compound for pathogenic bacteria
Example 1, a bacteriostatic complex of pathogenic bacteria was prepared as follows:
taking 1.0 g of ferric sulfate, 3.5g of sodium acetate and 0.6g of cystine, wherein the proportion of organic sulfur in the cystine is 25.8%, mixing, uniformly shaking in 10ml of water, then putting into a high-temperature reaction kettle at 250 ℃ for reaction for 2.5h, centrifuging at 4000 rpm after the reaction is finished for 7 minutes to obtain supernatant and precipitate, wherein the supernatant is a polysulfane compound, and washing the precipitate with water and ethanol to obtain the polyferric sulfide compound.
Example 2, a pathogenic bacteria inhibiting composition was prepared as follows:
taking 1.5g of ferric sulfate, 3.0g of sodium acetate and 0.5g of cysteine, wherein the percentage of organic sulfur in the cysteine is 26.4%, mixing, uniformly shaking in 20ml of water, then putting into a high-temperature reaction kettle at 80 ℃ for reaction for 13h, centrifuging for 7 min at 4000 rpm after the reaction is finished to obtain supernatant and precipitate, wherein the supernatant is a polysulfane compound, and washing the precipitate with water and ethanol to obtain the polyferric sulfide compound.
Example 3, a bacteriostatic pathogenic bacterial complex was prepared as follows:
taking 1.21g of ferric sulfate, 3.6g of sodium acetate and 0.625g of methionine, wherein the proportion of organic sulfur in the methionine is 21.5%, mixing, placing in 20ml of water, shaking up, then placing in a high-temperature reaction kettle at 200 ℃ for reaction for 24h, centrifuging for 7 min at 4000 rpm after the reaction is finished to obtain supernatant and precipitate, wherein the supernatant is a polysulfane compound, and washing the precipitate with water and ethanol to obtain the polyferric sulfide compound.
Example 4, a bacteriostatic complex of pathogenic bacteria was prepared as follows:
taking 1.3 g of sodium sulfide, 3.3g of sodium acetate and 0.7g of dried pig blood (the organic sulfur content in fresh pig blood is 1.6-320 mu g/mL, the organic sulfur content after drying is generally more than 10%, and the organic sulfur content in the dried pig blood used in the embodiment is 12%), mixing, placing in 30mL of water, shaking uniformly, then placing in a high-temperature reaction kettle at 150 ℃ for reaction for 10h, centrifuging for 7 minutes at 4000 rpm after the reaction is finished to obtain a supernatant and a precipitate, wherein the supernatant is a polysulfane compound, and washing the precipitate by using water and ethanol to obtain the polyferric sulfide compound.
Taking the polysulfane compound prepared in example 3 as an example, the particle size of the prepared polysulfane compound is measured by placing the compound on a malvern laser particle sizer, the average particle size of the prepared polysulfane compound is about 183nm (figure 1A), the solution is clear, transparent and brown and orange (figure 1B), and the solution is stored in a refrigerator at 4 ℃ for later use.
2. Experiment for inhibiting bacteria
Taking the polysulfane compound prepared in example 3 as an example, the compound is respectively tested for drug sensitivity experiment, minimum inhibitory concentration, minimum bactericidal concentration and treatment effect on pathogenic bacteria; the iron polysulfide compound prepared in example 3 was also used as an example to test its drug sensitivity test and therapeutic effect on pathogenic bacteria. The experimental results of the polysulfane compound and the polyferric sulfide compound prepared in other examples are not much different from those of example 3, and are not repeated herein.
2.1 drug sensitivity test (paper filter diffusion method)
A diffusion test was carried out using a polysulfane complex or a polyferric sulfide complex, and the YPD solid medium was uniformly spread to a concentration of 10 5 CFU/mL Candida albicans/Staphylococcus aureus/Trichophyton rubrumPlacing bacteria/Escherichia coli, filter paper sheet into plate, placing the plate upside down in 30 deg.C incubator, culturing for 24 hr, observing polythiol complex to corresponding pathogenic bacteria (concentration is 10) 5 CFU/mL), with sterile physiological saline as a blank control, the experiment was repeated three times.
2.2 determination of the Minimum Inhibitory Concentration (MIC)
YPD liquid medium diluted the bacterial liquid to turbidity of 3.5X 10 5 CFU/mL was used for the determination of MIC values of the polysulfane complex for the isolated strains by broth dilution with minimal amounts of water, and the experiments were repeated three times.
2.3 determination of Minimum Bactericidal Concentration (MBC)
On the basis of MIC, the culture solution without bacteria growth in the wells is further inoculated to a fresh YPD culture medium, if bacteria grow again, the concentration only has bacteriostatic action, if bacteria grow aseptically, the concentration is considered to have bactericidal action, and is recorded as the lowest bactericidal concentration (MBC).
2.4 therapeutic Effect of Polysulfane Complex or iron polysulfide Complex on skin diseases of mice infected with Candida albicans, staphylococcus aureus and Trichophyton rubrum
For each pathogenic bacterium, 18 Kunming mice, 22-28g, were selected, purchased from the animal center of the southwest university of medical science, and were raised and tested in the laboratories of the animal medical colleges of the southwest university. The experimental mouse is fed every day at regular time, the animal room is cleaned every day to keep sanitary, and good feeding environment and conditions are provided.
The experimental mice are continuously inoculated with 3 days of the immunosuppressant dexamethasone to reduce the body immunity of the mice. A10X 10mm wound was made by lacerating the dermis of the mouse and a 6.8X 10mm pipette was used to aspirate 8 And (3) 500 mu L of CFU/mL pathogenic bacteria liquid is applied to the wound and evenly coated. Randomly grouping successfully established pathogenic bacteria infected mice (n = 6), and respectively obtaining a disease model group, a polysulfane compound or polyferric sulfide compound treatment group and a sterile normal saline group with the use concentration of 6.24 g.L -1 The polysulfane compound or iron polysulfide compound was applied in 500 μ L to create a treatment group, while the sterile saline group was applied in 500 μ L only at the wound.
3. Results of the experiment
3.1 drug susceptibility test
The results are shown in Table 1.
The content of the medicament used on the filter paper sheet in the medicament sensitivity test is calculated by the mass of the organic sulfur compound in the reaction raw material. For the present experiment, the drug was prepared from example 3, and the polysulfane complex and iron polysulfide complex were calculated on the mass of methionine.
TABLE 1
The result of drug sensitivity experiments shows that the polysulfane compound has inhibitory effect on candida albicans, staphylococcus aureus and trichophyton rubrum fungi and also has obvious bacteriostatic effect on escherichia coli, wherein the candida albicans, staphylococcus aureus and trichophyton rubrum fungi are moderately sensitive to 2mg/mL of the polysulfane compound (based on the weight of organic sulfur compounds), and the escherichia coli shows high sensitivity to the polysulfane compound; the poly-ferric sulfide compound has inhibiting effect on various pathogenic bacteria, and fungi such as candida albicans, staphylococcus aureus and trichophyton rubrum have surface sensitivity on 2mg/mL poly-ferric sulfide compound (by weight of organic sulfur compound), and escherichia coli has high sensitivity on 2mg/mL poly-ferric sulfide compound (by weight of organic sulfur compound); all pathogens appeared to be insensitive to sterile saline.
3.2MIC and MBC values
The MIC value of the polysulfane compound to Candida albicans is detected by adopting a 96-well plate for micro-dilution, and the MIC result shows that the MIC of the polysulfane compound to Candida albicans is 0.78mg/mL by taking sterile physiological saline as a control through three repeated experiments, a culture solution of non-growing bacteria in the MIC hole is inoculated into a fresh YPD liquid culture medium for incubation, the minimum concentration of aseptic growth is 3.12mg/mL, namely the Minimum Bactericidal Concentration (MBC) of the polysulfane compound to Candida albicans is 3.12mg/mL.
The MIC value of the polysulfane compound to staphylococcus aureus is 0.75mg/mL, and the MBC is 3.02mg/mL; the MIC for trichophyton rubrum is 0.80mg/mL, and the MBC is 3.33mg/mL; the MIC for E.coli was 0.68mg/mL and the MBC was 2.97mg/mL.
The MIC and MBC values in the experimental results are both based on the mass of the organic sulfur compound in the reaction raw materials, and the mass of methionine in the experiment.
3.3 treatment of skin diseases in mice infected with Candida albicans, staphylococcus aureus and Trichophyton rubrum by polysulfane complex or iron polysulfane complex
(1) Compared with the normal saline group, the wounds of the Candida albicans infected group are obviously enlarged (P < 0.001) on the 3 rd day, and a large amount of yellow-green fluorescence is found around the wounds under a Wood's lamp, which indicates that the Candida albicans is propagated in a large amount. After day 4, the wound was dilated and pain after palpation (mice appeared excited, manic). The wound size change is not obvious from 6 days to 9 days (P is more than 0.05), the wound gradually shrinks from 10 days to 13 days (P is less than 0.01), the wound is self-healed, the crust peels off, the wound is smooth, yellow-green fluorescence is not found under a wood lamp, and the back hair gradually grows out. On the 6 th day of treatment by the polysulfane compound, scabbing and falling off are caused, yellow-green fluorescence disappears at wounds and the periphery, yellow-green fluorescence is not found under a wood lamp, and the hairs grow out gradually; and after the polyferric sulfide compound is used for treating for 7 days, scabs are removed, yellow-green fluorescence disappears from the wound and the periphery, and yellow-green fluorescence is not found under a wood lamp, so that the hair grows gradually.
(2) Compared with the normal saline group, the wound of the staphylococcus aureus infected group is obviously enlarged at day 3 (P < 0.001), and a large amount of staphylococcus aureus proliferates around the wound under a microscope. After day 4, the wound was dilated and pain after palpation (mice appeared excited, manic). The wound size change is not obvious from 5 days to 8 days (P is more than 0.05), the wound gradually shrinks from 9 days to 12 days (P is less than 0.01), the wound is self-healed, the crust peels off, the wound is smooth, no staphylococcus aureus is found under a microscope, and the back hair gradually grows out. On the 6 th day of the treatment of the polysulfane compound, scabbing and falling off are caused, the staphylococcus aureus on the wound and the periphery disappears, the staphylococcus aureus is not found under a microscope, and the hair grows out gradually; and after the iron polysulfide compound is used for treating for 7 days, scabs fall off, staphylococcus aureus on wounds and the periphery disappears, and the hair grows out gradually.
(3) Compared with the saline group, the wound of the trichophyton rubrum infected group is obviously enlarged on the 4 th day (P < 0.001), and a large amount of light blue fluorescence around the wound is found under a wood's lamp, so that the trichophyton rubrum is greatly propagated. After day 5, the wound dilated and pain after palpation (mice appeared excited, manic). The wound size change is not obvious from 7 days to 10 days (P is more than 0.05), the wound gradually shrinks from 11 days to 15 days (P is less than 0.01), the wound is self-healed, the crust peels off, the wound is smooth, light blue fluorescence is not found under a wood lamp, and the back hair gradually grows out. On the 7 th day of the treatment of the polysulfane compound, scabbing and falling off are caused, light blue fluorescence disappears at the wound and the periphery, the light blue fluorescence is not found under a wood lamp, and the hair grows out gradually; and after 8 days of treatment by adopting the polyferric sulfide compound, scabbing and falling off occur, light blue fluorescence disappears around the wound, and light blue fluorescence is not found under a wood lamp, so that the hair grows gradually.
The invention adopts a thermal reaction system of a high-temperature reaction kettle, has simpler synthesis method and shorter chemical reaction time, is suitable for large-scale production, synthesizes the novel polysulfane compound and the polyferric sulfide compound for resisting invasive fungi, provides support for the development of novel antifungal agents and the prevention and treatment of invasive fungi, and has good reference significance for preventing the prevention and treatment of clinically common drug-resistant bacteria.
Finally, it should be noted that the above-mentioned description is only a preferred embodiment of the present invention, and those skilled in the art can make various similar representations without departing from the spirit and scope of the present invention.
Claims (9)
1. A preparation method for obtaining two bacteriostatic complexes of pathogenic bacteria is characterized by comprising the following steps: taking 1.0-1.5g of ferric sulfate or sodium sulfide, 3.0-3.6g of sodium acetate and 0.5-0.7g of organic sulfur compound, mixing, placing in 10-30ml of water, shaking up, then placing in a high-temperature reaction kettle at 80-250 ℃ for reaction for 2.5-24h, and centrifuging after the reaction is finished to obtain supernatant and precipitate, wherein the supernatant is a polysulfane compound, and the precipitate is a polyferric sulfide compound.
2. The preparation method for obtaining bacteriostatic complexes of two pathogenic bacteria according to claim 1, characterized in that: the organic sulfur content in the organic sulfur compound is greater than or equal to 10%.
3. The preparation method for obtaining a bacteriostatic complex of two pathogenic bacteria according to claim 1 or 2, characterized in that: the organic sulfur compound is cystine, cysteine or methionine.
4. The preparation method for obtaining bacteriostatic complexes of two pathogenic bacteria according to claim 3, characterized in that: 1.21g of ferric sulfate, 3.6g of sodium acetate and 0.625g of organic sulfur compound are mixed and then placed into 20ml of water to be shaken up, then the mixture is placed into a high-temperature reaction kettle at 200 ℃ to react for 24 hours, and after the reaction is finished, the polysulfane compound and the polyferric sulfide compound are obtained by centrifugation.
5. The preparation method for obtaining bacteriostatic complexes of two pathogenic bacteria according to claim 2, characterized in that: the organosulfur compounds are derived from animal blood.
6. A pathogenic bacteriostatic complex, characterized by being prepared according to the preparation method of any one of claims 1 to 5, thereby obtaining a polysulfane complex.
7. A bacteriostatic pathogenic bacterium complex, characterized by being prepared in the manner as described in any one of claims 1 to 5, thereby obtaining a polyferric sulfide complex.
8. The application of a pathogenic bacteria-inhibiting compound is characterized in that: use of a polysulfane complex or polyferric sulfide complex prepared by a method according to any one of claims 1 to 5, or a pathogenic bacteriostatic complex according to claim 6, or a pathogenic bacteriostatic complex according to claim 7, for the preparation of a medicament for inhibiting a pathogen.
9. Use of a pathogenic bacteriostatic complex according to claim 8, characterized in that: the pathogenic bacteria are Candida albicans, staphylococcus aureus, trichophyton rubrum or Escherichia coli.
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