Preparation method of eliminating bacterial drug resistance amino plant sterol glycoside and application thereof

Abstract

The application discloses a preparation method and application of an amino phytosterol glycoside capable of eliminating bacterial drug resistance, and belongs to the technical field of medicines. The preparation method mainly comprises the following steps: obtaining the amino phytosterol glycoside by catalyzing 11-21 polymerization degree amino polysaccharide and phytosterol through an enzyme method. The amino phytosterol glycoside can eliminate the barrier protection of a bacterial biofilm, and can hinder the colony propagation of bacteria, reduce the bacterial abundance and improve the antibiotic drug potency in combination with the elimination effect of phytosterol on bacterial quorum sensing.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to a preparation method of an amino phytosterol glycoside for eliminating bacterial drug resistance and application thereof. BACKGROUND

[0002] Bacterial drug resistance refers to the resistance of bacteria to antibiotics or other antibacterial drugs, thereby reducing the efficacy of traditional antibiotics, which seriously affects the treatment effect and public health. Although the growth and reproduction of highly drug-resistant bacteria can be inhibited by high-concentration antibiotics, in actual application, due to the limitation of the human "treatment window", the amount of antibiotics is limited, and the concentration is much lower than the minimum inhibitory concentration (MIC). The bacterial biofilm provides a suitable environment for bacteria and produces a strong barrier effect, which promotes bacteria to adapt to antibiotic pressure, can strengthen the communication among bacteria in the community, transfer drug resistance genes, and form drug resistance, which makes the bacteria in the biofilm more stubborn and difficult to eliminate.

[0003] In order to overcome this problem, researchers began to look for antibiotic synergists, which are usually natural products, and are used with antibiotics Although it can increase the efficacy of antibiotics or repair the sensitivity of drug-resistant strains to antibiotics, but the research on bacterial drug resistance is mainly focused on new anti-drug antibiotics, and currently there are few studies on antibiotic synergists, only β-lactamase inhibitors have achieved clinical application, which limits the application of antibiotics.

[0004] Phytosterols are a class of functional ingredients naturally occurring in plants, which have the effects of reducing cholesterol, inhibiting tumors, antioxidant, anti-inflammatory, antibacterial, etc., and are widely used in medicine, food, cosmetics and personal care, agriculture and industry, etc. Many fields, such as a phytosterol-β-D-glucoside enzyme catalytic preparation method disclosed in Chinese patent CN201210474088.1, mainly reacts phytosterol with glucose under the action of free β-glucosidase to generate phytosterol glycoside, and water-soluble phytosterol-β-D-glucoside is obtained by column chromatography separation and purification. The technical scheme has the advantages of simple process and low cost, but it is only suitable for food industry production, therefore, how to eliminate bacterial drug resistance is an urgent problem to be solved. SUMMARY

[0005] In view of the defects of the prior art, the present application designs a preparation method of amino phytosterol glycoside, which can eliminate the barrier protection of bacterial biofilm and thus eliminate the drug resistance of bacteria.

[0006] In order to achieve the above purpose, the technical problem of the present application is solved by adopting the following technical scheme:

[0007] In one aspect, the application provides a preparation method of an amino phytosterol glycoside, comprising the following steps:

[0008] S1: preparing an amino polysaccharide solution and performing separation and purification;

[0009] S2: placing phytosterols in a solvent and performing ultrasonic oscillation, then adding the purified amino polysaccharide solution and performing enzymatic catalysis under the action of sterol glycosyltransferase, recovering the solvent by condensation reflux after the reaction is completed, and then drying to obtain the amino phytosterol glycoside;

[0010] The preparation reaction formula of the amino phytosterol glycoside is as follows:

[0011] ;

[0012] In the formula, n=7-17.

[0013] Preferably, in step S1, the amino polysaccharide is chitosan with a degree of polymerization of 11-21.

[0014] Preferably, in step S2, the solvent is one or more of diethyl ether, benzene, chloroform, ethyl acetate, carbon disulfide or petroleum ether.

[0015] Preferably, the phytosterols have cyclopentane perhydrophenanthrene as the main skeleton structure and contain an alcohol group.

[0016] Preferably, the phytosterols include sitosterol, campesterol, stigmasterol and brassicasterol.

[0017] Preferably, the mass ratio of the phytosterols to the amino polysaccharide is 1:(0.2-2), and the amount of the sterol glycosyltransferase accounts for 4-6% of the total weight of the reaction system.

[0018] The phytosterols are derived from corn germ oil, sesame oil, rapeseed oil, soybean, pine and the like.

[0019] Preferably, in step S2, the condensation recovery temperature is 80-90℃ and the time is 1-5h.

[0020] Preferably, in step S2, the enzymatic catalysis temperature is 30-50℃ and the time is 3-8h.

[0021] Preferably, the ultrasonic oscillation power is 2kW and the time is 1-3h.

[0022] In another aspect, the application provides the use of the amino phytosterol glycoside prepared by the preparation method of the amino phytosterol glycoside in preparing a product for eliminating bacterial drug resistance.

[0023] Compared with the prior art, the application has the following beneficial effects:

[0024] The present application designs a method for preparing amino phytosterol glycoside, which is obtained by catalyzing 11-21 degree of polymerization of amino polysaccharide and phytosterol by enzyme method, the amino polysaccharide as a glycoside bond donor can improve the water solubility of phytosterol, and as the only natural cationic polysaccharide, it can improve the van der Waals force of intermolecular binding.

[0025] The amino phytosterol glycoside can eliminate the barrier protection of bacterial biofilm due to the hydrophilic group and the solid sterol group and the carried cation, so as to eliminate the drug resistance of bacteria, and at the same time, the elimination effect of phytosterol itself on bacterial quorum sensing (homoserine lactone) is combined, which can hinder the bacterial colony reproduction of bacteria, reduce the bacterial abundance, and improve the antibiotic drug potency. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the present application, the drawings needed in the embodiments will be briefly introduced below, and obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained according to these drawings without creative labor for those skilled in the art.

[0027] Figure 1 It is the preparation flow chart of the amino phytosterol glycoside of the present application.

[0028] Figure 2 It is the synthesis route chart of the amino phytosterol glycoside of the present application.

[0029] Figure 3 It is the actual photo of MIC change of colistin sulfate combined with amino phytosterol glycoside to inhibit gram-negative bacteria.

[0030] Figure 4 It is the actual photo of MIC change of colistin sulfate, spectinomycin combined with amino phytosterol glycoside to inhibit gram-positive bacteria.

[0031] Figure 5 It is the actual photo of MIC change of spectinomycin combined with amino phytosterol glycoside to inhibit gram-negative bacteria.

[0032] Figure 6 It is the actual photo of MIC change of penicillin combined with amino phytosterol glycoside to inhibit gram-negative bacteria. DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0034] In the present application, if no special description is given, the numerical range is regarded as continuous and includes the minimum value and the maximum value of the range and each value between the minimum value and the maximum value. Further, when the range refers to an integer, each integer between the minimum value and the maximum value of the range is included. In addition, when multiple ranges are provided to describe a feature or a characteristic, the ranges can be combined. In other words, unless otherwise specified, all the ranges disclosed herein should be understood as including any and all sub-ranges falling within the range.

[0035] The test methods used in the following examples are conventional methods unless otherwise specified; the sterol glycosyltransferase (GT1) used is a product of UGT74AN2 gene (GenBank: MF942417.1) expressed in a competent cell (E. coli engineering bacteria) and directed evolution of the latter protein. The remaining materials, reagents, etc. are commercially available reagents and materials unless otherwise specified.

[0036] Example 1

[0037] A method for preparing an amino phytosterol glycoside, as shown in the formula, the method for preparing the chitosan amino phytosterol glycoside specifically includes the following steps: Figure 1

[0038] 1. Chitosan is prepared into a 5% chitosan solution with 1% acetic acid solution, and the pH is adjusted to 5.4 with sodium bicarbonate solution, and then chitosan is separated and purified by a weak acid cation resin chromatography column: a chromatography column with a length of 15 cm is used to separate chitosan, the loading speed is 4 mL / min, after loading, 1 mol / L ammonia water is used for elution, the elution flow rate is 2 mL / min, and the recovered product is chitosan with an n value (total monosaccharide number of sugar chain) of 9-19.

[0039] ​2, Put 5 g of phytosterol (campesterol) and 60 mL of ethyl acetate into a constant temperature ultrasonic reactor and ultrasonically shake for 2 h (2 kW), then filter and separate, and then put into a constant temperature stirring reactor, add 3.5 g of chitosan of step 1, 35 mL of ethyl acetate, and stir and mix, then add 5% of sterol glycosyltransferase (GT1) to the reaction system, and stir at constant temperature (stir at 40°C for 6 h), then filter and separate, and then put the residue into a reflux condensation reactor, evaporate and recover the solvent (85°C for 3 h), and then put the residue into a spray dryer, and add 10% of carrier white carbon black (silicon dioxide), to obtain amino phytosterol glycoside.

[0040] Example 2

[0041] Example 2 is different from example 1 in that step 2 is different, and the others are the same.

[0042] Put 3 g of phytosterol (stigmasterol) and 60 mL of ethyl acetate into a constant temperature ultrasonic reactor and ultrasonically shake for 2 h (2 kW), then filter and separate, and then put into a constant temperature stirring reactor, add 5 g of chitosan of step 1, 35 mL of ethyl acetate, and stir and mix, then add 6% of sterol glycosyltransferase (GT1) to the reaction system, and stir at constant temperature (stir at 30°C for 3 h), then filter and separate, and then put the residue into a reflux condensation reactor, evaporate and recover the solvent (80°C for 1 h), and then put the residue into a spray dryer, and add 10% of carrier white carbon black (silicon dioxide), to obtain amino phytosterol glycoside.

[0043] Example 3

[0044] Example 3 is different from example 1 in that step 2 is different, and the others are the same.

[0045] Put 8 g of phytosterol (campesterol) and 60 mL of ethyl acetate into a constant temperature ultrasonic reactor and ultrasonically shake for 2 h (2 kW), then filter and separate, and then put into a constant temperature stirring reactor, add 2 g of chitosan of step 1, 35 mL of ethyl acetate, and stir and mix, then add 4% of sterol glycosyltransferase (GT1) to the reaction system, and stir at constant temperature (stir at 50°C for 6 h), then filter and separate, and then put the residue into a reflux condensation reactor, evaporate and recover the solvent (90°C for 5 h), and then put the residue into a spray dryer, and add 10% of carrier white carbon black (silicon dioxide), to obtain amino phytosterol glycoside.

[0046] Test Example 1

[0047] Analysis of the effect of amino phytosterol glycoside on drug potency (TTC method)

[0048] 1. Sterilization:

[0049] Put the MH broth medium, PBS buffer, sterile water, matching gun head, centrifugal tube into the high pressure sterilization pot at 121℃ for 25min, 96 well plate in the ultraclean table ultraviolet sterilization for 30min.

[0050] 2. Preparation of antibiotic solution:

[0051] First, dissolve the antibiotic (drug) in sterile phosphate buffer to prepare a solution with a concentration of 51.2mg / mL, vortex to mix and filter. The filtrate is physically sterilized through a filter membrane with a diameter of 0.22μm, and is divided into sterile centrifuge tubes and stored in a -20℃ freezer, protected from light.

[0052] 3. Preparation of amino phytosterol glycoside solution:

[0053] Take 0.5g of amino phytosterol glycoside prepared in Example 1 in a centrifuge tube, add 15mL of pure water to dissolve, the concentration of the amino phytosterol glycoside solution is 80mM, shake to mix and filter. The filtrate is physically sterilized through a filter membrane with a diameter of 0.22μm, and is divided into sterile centrifuge tubes and stored in a -20℃ freezer, protected from light.

[0054] 4. Put the bacteria (gram-negative bacteria or gram-positive bacteria) into a centrifuge tube containing 4mL of MH broth, and place it in a 37℃ shaking bed at 180rpm for 4h. Then take out the centrifuge tube and dilute the incubated bacteria 100 times with MH broth, about 10 6 CFU / mL, ready for use.

[0055] 5. Add 180μL of MH broth medium to A1-H1 wells of the sterile 96 well plate, and add 100μL of MH broth medium to the remaining wells. Then add 10μL of antibiotic to A1-H1 wells, mix evenly by blowing, and then transfer 100μL from A1-H1 to A2-H2, mix evenly, and then transfer 100μL from A2-H2 to A3-H3, and so on, until A12-H12.

[0056] Add 10μL of amino phytosterol glycoside solution to columns 1-3 and 7-9 of the sterile 96 well plate. Add 100μL of the diluted bacteria solution from step 4 to A1-H12 wells of the sterile 96 well plate. Compare the same strain to determine the change in minimum inhibitory concentration (MIC) with or without the addition of amino phytosterol glycoside.

[0057] 6. Place the inoculated 96 well plate in a 35℃ ordinary air incubator for 20h to determine the results.

[0058] The red color metabolic product in the hole with bacterial activity, the change of red color in the hole indicates the change of bacterial activity in the hole. The results are shown in Table 1 below, for more intuitive to see the MIC change, with sulfobacillus acid, spectinomycin, penicillin as an example, 96-well plate is taken, as shown in Figures 3-6

[0059] Table 1 MIC change of different antibacterial drugs combined with amino phytosterol glycoside

[0060]

[0061] Figures 3-6 K88 is K88 toxin producing E. coli, gold is Staphylococcus aureus, and enteritis is Salmonella enteritidis. Figure 3 、 4 Sulfur is sulfobacillus acid, and sulfur + shell is sulfobacillus acid combined with phytosterol chitosan glycoside. Figure 5 Big view is spectinomycin, and big view + shell is spectinomycin combined with phytosterol chitosan glycoside. Figure 6 Blue is penicillin, and blue + shell is penicillin combined with phytosterol chitosan glycoside.

[0062] Test example 2

[0063] The amino phytosterol glycoside prepared in Example 1 is combined with amoxicillin, doxycycline, florfenicol, tylosin and other antibiotics, and the MIC change of the antibiotics alone and the antibiotics combined with amino phytosterol glycoside is detected by TTC method to analyze the influence of amino phytosterol glycoside on bacterial (E. coli, Clostridium perfringens) resistance. The strain types of E. coli and Clostridium perfringens involved in TTC detection and their specific sources and resistance phenotypes are shown in Table 2 as follows:

[0064] Table 2 Sources and resistance of E. coli and Clostridium perfringens

[0065]

[0066] Table 3 MIC of E. coli after combination of antibiotics and amino phytosterol glycoside

[0067]

[0068] The lower the MIC, the more effective the antibiotic can inhibit the growth and reproduction of bacteria, and the higher the MIC, the higher the resistance of the bacteria to the antibiotic, affecting the treatment effect. As can be seen from Table 3, the combination of antibiotics amoxicillin, doxycycline, doxycycline, tylosin and amino phytosterol glycoside can basically lower the MIC value of E. coli than the single use of antibiotics amoxicillin, doxycycline, doxycycline and tylosin.​

[0069] Table 4 MIC of Clostridium perfringens after combination of antibiotics and amino phytosterol glycoside

[0070]

[0071] As shown in Table 4, the MIC of E. coli inhibited by the combination of antibiotic amoxicillin and amino phytosterol glycoside is much lower than that of the antibiotic amoxicillin alone, which indicates that the combination of antibiotic and amino phytosterol glycoside can inhibit E. coli at a lower concentration, and thus the amino phytosterol glycoside of the present application can improve the potency of antibiotic drugs.

[0072] In summary, the amino phytosterol glycoside is prepared by catalysis of sterol glycosyltransferase (GT1) using amino polysaccharide and phytosterol, and it is found by TTC method that the amino phytosterol glycoside can eliminate the drug resistance of Gram-positive bacteria represented by Clostridium perfringens to penicillin represented by amoxicillin, and has obvious synergistic effect on the bacteriostatic performance of various antibiotics (enrofloxacin, colistin sulfate, amoxicillin, doxycycline, florfenicol, tylosin, etc.).

[0073] In addition, it should be understood that although the present specification is described in terms of embodiments, not every embodiment contains only one independent technical solution, and the specification is described in this way only for clarity, and those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that those skilled in the art can understand.

Claims

1. A process for the preparation of an amino phytosterol glycoside, characterized by, The preparation method comprises the following steps: S1: preparing an amino polysaccharide solution and performing separation and purification; S2: placing phytosterol in an ethyl acetate solvent and performing ultrasonic oscillation, then adding the purified amino polysaccharide solution, and performing enzymatic catalysis under the action of sterol glycosyltransferase, condensing and recovering the solvent after the reaction is completed, and then drying to obtain an amino phytosterol glycoside; the phytosterol is campesterol or stigmasterol; the sterol glycosyltransferase is GenBank: MF942417.1; The preparation reaction formula of the amino phytosterol glycoside is as follows: ； In the formula, R= n = 7-17.

2. The process for the preparation of an aminophytosterol glycoside according to claim 1, characterized in that, The mass ratio of the phytosterol to the amino polysaccharide is 1: (0.2-2); the amount of the sterol glycosyltransferase accounts for 4-6% of the total weight of the reaction system.

3. The process for the preparation of an aminophytosterol glycoside according to claim 1, characterized in that, In step S2, the condensation recovery temperature is 80-90 DEG C, and the time is 1-5 h.

4. The process for the preparation of an aminophytosterol glycoside according to claim 1, characterized in that, In step S2, the enzymatic catalysis reaction temperature is 30-50 DEG C, and the time is 3-8 h.

5. The process for the preparation of an aminophytosterol glycoside according to claim 1, characterized in that, The ultrasonic oscillation power is 2 kW, and the time is 1-3 h.

6. The amino phytosterol glycoside prepared by the preparation method of the amino phytosterol glycoside according to any one of claims 1-5 is used for preparing a drug for improving bacterial sensitivity; the bacteria are Escherichia coli, Clostridium perfringens, Staphylococcus aureus or Salmonella enteritidis.

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