CN117338795B - Honokiol sophorolipid complex and preparation method and application thereof - Google Patents
Honokiol sophorolipid complex and preparation method and application thereof Download PDFInfo
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- VVOAZFWZEDHOOU-UHFFFAOYSA-N honokiol Natural products OC1=CC=C(CC=C)C=C1C1=CC(CC=C)=CC=C1O VVOAZFWZEDHOOU-UHFFFAOYSA-N 0.000 title claims abstract description 61
- ZTOKUMPYMPKCFX-CZNUEWPDSA-N (E)-17-[(2R,3R,4S,5S,6R)-6-(acetyloxymethyl)-3-[(2S,3R,4S,5S,6R)-6-(acetyloxymethyl)-3,4,5-trihydroxyoxan-2-yl]oxy-4,5-dihydroxyoxan-2-yl]oxyoctadec-9-enoic acid Chemical compound OC(=O)CCCCCCC/C=C/CCCCCCC(C)O[C@@H]1O[C@H](COC(C)=O)[C@@H](O)[C@H](O)[C@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](COC(C)=O)O1 ZTOKUMPYMPKCFX-CZNUEWPDSA-N 0.000 title claims abstract description 59
- BYTORXDZJWWIKR-UHFFFAOYSA-N Hinokiol Natural products CC(C)c1cc2CCC3C(C)(CO)C(O)CCC3(C)c2cc1O BYTORXDZJWWIKR-UHFFFAOYSA-N 0.000 title claims abstract description 57
- FVYXIJYOAGAUQK-UHFFFAOYSA-N honokiol Chemical compound C1=C(CC=C)C(O)=CC=C1C1=CC(CC=C)=CC=C1O FVYXIJYOAGAUQK-UHFFFAOYSA-N 0.000 title claims abstract description 54
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- 238000010668 complexation reaction Methods 0.000 title description 2
- 239000000693 micelle Substances 0.000 claims abstract description 59
- 239000003960 organic solvent Substances 0.000 claims abstract description 25
- 239000012535 impurity Substances 0.000 claims abstract description 18
- 230000000844 anti-bacterial effect Effects 0.000 claims abstract description 16
- 230000036571 hydration Effects 0.000 claims abstract description 16
- 238000006703 hydration reaction Methods 0.000 claims abstract description 16
- 239000007864 aqueous solution Substances 0.000 claims abstract description 8
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 8
- 150000001875 compounds Chemical class 0.000 claims abstract description 7
- 230000000887 hydrating effect Effects 0.000 claims abstract description 6
- 239000012736 aqueous medium Substances 0.000 claims abstract description 4
- 238000004945 emulsification Methods 0.000 claims abstract description 3
- 239000011259 mixed solution Substances 0.000 claims abstract description 3
- 238000000034 method Methods 0.000 claims description 24
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 23
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 15
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical group ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 230000003385 bacteriostatic effect Effects 0.000 claims 1
- 238000013329 compounding Methods 0.000 claims 1
- 239000012982 microporous membrane Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 8
- 238000002156 mixing Methods 0.000 abstract description 6
- 238000000502 dialysis Methods 0.000 abstract description 3
- -1 honokiol sophorolipid compound Chemical class 0.000 abstract description 3
- 238000002474 experimental method Methods 0.000 abstract description 2
- 230000001580 bacterial effect Effects 0.000 description 23
- 238000005538 encapsulation Methods 0.000 description 11
- 239000003814 drug Substances 0.000 description 9
- 239000012528 membrane Substances 0.000 description 9
- 229940079593 drug Drugs 0.000 description 8
- 239000010408 film Substances 0.000 description 8
- 230000005764 inhibitory process Effects 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 239000000243 solution Substances 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 235000019441 ethanol Nutrition 0.000 description 6
- 230000032770 biofilm formation Effects 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- 239000011248 coating agent Substances 0.000 description 4
- 238000000576 coating method Methods 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 229920001992 poloxamer 407 Polymers 0.000 description 4
- 229940044476 poloxamer 407 Drugs 0.000 description 4
- 229920000053 polysorbate 80 Polymers 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 239000000872 buffer Substances 0.000 description 3
- 238000003235 crystal violet staining Methods 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 229920001993 poloxamer 188 Polymers 0.000 description 3
- 229940044519 poloxamer 188 Drugs 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000002390 rotary evaporation Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- CTKXFMQHOOWWEB-UHFFFAOYSA-N Ethylene oxide/propylene oxide copolymer Chemical compound CCCOC(C)COCCO CTKXFMQHOOWWEB-UHFFFAOYSA-N 0.000 description 2
- RVGRUAULSDPKGF-UHFFFAOYSA-N Poloxamer Chemical compound C1CO1.CC1CO1 RVGRUAULSDPKGF-UHFFFAOYSA-N 0.000 description 2
- 241000191967 Staphylococcus aureus Species 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 230000000845 anti-microbial effect Effects 0.000 description 2
- 230000004888 barrier function Effects 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 230000001804 emulsifying effect Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 239000010409 thin film Substances 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 208000037581 Persistent Infection Diseases 0.000 description 1
- 208000035415 Reinfection Diseases 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 238000009635 antibiotic susceptibility testing Methods 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- 230000003305 autocrine Effects 0.000 description 1
- 238000002815 broth microdilution Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 206010014665 endocarditis Diseases 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000007764 o/w emulsion Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 210000001215 vagina Anatomy 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7024—Esters of saccharides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
- A61K31/05—Phenols
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Animal Behavior & Ethology (AREA)
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- Molecular Biology (AREA)
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- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
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Abstract
The invention provides a honokiol sophorolipid compound and a preparation method and application thereof, wherein the compound comprises honokiol and sophorolipid according to the mass ratio of 1: 6-8. The complex is obtained by one of the following preparation modes: in the mode 1, honokiol and sophorolipid are dissolved in an organic solvent I, then an organic mixed solution is added into an aqueous medium for mixed emulsification, the organic solvent is removed for hydration after micelle formation, and finally impurities are removed; mode 2, dissolving honokiol and sophorolipid in an aqueous solution containing an organic solvent II, removing the organic solvent II after micelle formation, hydrating, and finally removing impurities; mode 3, mixing honokiol, sophorolipid and water, and finally removing impurities; in the mode 4, honokiol and sophorolipid are dissolved in an aqueous solution containing an organic solvent III, water is adopted for dialysis, and finally impurities are removed from the dialysate. Experiments show that the compound has good biological film removing effect and antibacterial effect.
Description
Technical Field
The invention relates to the technical field of antibacterial products, in particular to a honokiol sophorolipid compound and a preparation method and application thereof.
Background
Bacterial biofilms are 3D structures formed by the encapsulation of bacterial communities by an autocrine extracellular polymeric matrix (EPS), also known as "microbial cities. The physical barrier of EPS prevents penetration of the antimicrobial agent and protects the internal bacteria from external conditions. Among them, biofilm-producing staphylococcus aureus (s.aureus) is the most common conditional pathogen, and can cause refractory recurrent infections, such as persistent infections of wound surfaces, endocarditis, and further serious infections caused by colonization of implant material surfaces (e.g., catheters, joint prostheses). At present, due to implementation of forbidden measures, the acceleration of searching for antibiotic substitutes is an urgent need.
Disclosure of Invention
The invention aims to provide a honokiol sophorolipid complex, a preparation method and application thereof, so as to explore the potential of the complex in bacteriostasis and antibiosis.
In order to achieve the technical purpose, the technical scheme of the invention is as follows:
In a first aspect, the present invention provides a honokiol sophorolipid complex, comprising honokiol and sophorolipid in a mass ratio of 1: 6-8.
In a second aspect, the invention provides a method for preparing the honokiol sophorolipid complex, which adopts one of the following preparation modes:
in the mode 1, honokiol and sophorolipid are dissolved in an organic solvent I, then an organic mixed solution is added into an aqueous medium for mixed emulsification, the organic solvent is removed for hydration after micelle formation, and finally impurities are removed;
Mode 2, dissolving honokiol and sophorolipid in an aqueous solution containing an organic solvent II, removing the organic solvent II after micelle formation, hydrating, and finally removing impurities;
mode 3, mixing honokiol, sophorolipid and water, and finally removing impurities;
in the mode 4, honokiol and sophorolipid are dissolved in an aqueous solution containing an organic solvent III, water is adopted for dialysis, and finally impurities are removed from the dialysate.
Preferably, in the above embodiment 1, the first organic solvent is chloroform.
Preferably, in the mode 2, the second organic solvent is at least one selected from methanol, ethanol and acetonitrile.
Preferably, in the mode 4, the organic solvent three is at least one selected from methanol, ethanol and acetonitrile.
Preferably, in the modes 1 to 4, the impurity removal is performed by using a microporous filter membrane of 0.22 μm.
Preferably, in the mode 1 or the mode 2, the hydration process includes: and adding normal saline or PBS buffer solution with pH of 7.4 into the compound from which the first or second organic solvent is removed, heating to 40-70 ℃, and hydrating for 5-1 h.
Preferably, the hydration temperature is raised to 50-60 ℃ and the hydration time is 5 min-15 min.
In a third aspect, the invention provides application of the honokiol sophorolipid complex or the honokiol sophorolipid complex obtained by the preparation method in preparation of antibacterial products.
The invention provides a honokiol sophorolipid complex and a preparation method and application thereof. Experiments show that the compound has good bacterial biofilm removal effect and antibacterial effect.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and do not constitute a limitation on the invention. In the drawings:
FIG. 1 shows the surface tension curves of aqueous solutions of sophorolipids at various concentrations.
FIG. 2 is a graph showing the effect of sophorolipid loading on the encapsulation efficiency of honokiol-sophorolipid micelles.
FIG. 3 shows the inhibition of S.aureus by treatment groups of different concentrations.
Fig. 4 shows the antimicrobial effect of different treatment groups.
FIG. 5 shows the inhibition effect of different treatment groups on S.aureus biofilm formation.
FIG. 6 shows the effect of different treatment groups on S.aureus biofilm clearance.
FIG. 7 shows the antibacterial effect of different treatment groups on the biofilm.
Detailed Description
In the description of the present invention, it is to be noted that the specific conditions are not specified in the examples, and the description is performed under the conventional conditions or the conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The invention provides a honokiol sophorolipid compound, which is prepared by mixing honokiol (purchased from Shanxi Jinkangtai biological film technology Co., ltd.) and sophorolipid (purchased from ancient new technology Co., ltd.) according to a mass ratio of 1: 6-1:8. The preparation method of the compound adopts one of the following steps:
In the mode 1, honokiol and sophorolipid are dissolved in an organic solvent containing chloroform, then the organic solvent is added into an aqueous medium for mixing and emulsifying to form an oil-in-water (O/W) emulsion, the sophorolipid rearranges to form micelles in the emulsifying process, then the organic solvent is removed and hydrated, and finally impurities are removed through a microporous filter membrane with the size of 0.22 mu m.
In the mode 2, honokiol and sophorolipid are dissolved in an aqueous solution containing at least one of methanol, ethanol and acetonitrile, the sophorolipid automatically forms micelles in the stirring process, finally, an organic reagent is removed through rotary evaporation, and impurities are removed through hydration and a 0.22 mu m microporous filter membrane.
Mode 3, evenly mixing honokiol and sophorolipid in water, stirring by 12 h, and finally removing impurities through a 0.22 mu m microporous filter membrane.
And 4, dissolving honokiol and sophorolipid in at least one aqueous solution containing methanol, ethanol and acetonitrile, filling into a dialysis bag, dialyzing with water, and finally removing impurities through a microporous filter membrane with the size of 0.22 mu m.
In the above manner, the hydration process includes: and adding normal saline or PBS buffer solution with pH of 7.4 into the compound from which the first or second organic solvent is removed, heating to 40-70 ℃, and hydrating for 5-1 h. Preferably, the hydration temperature is raised to 50-60 ℃ and the hydration time is 5 min-15 min.
The following will describe embodiments of the present invention in detail by referring to examples, so that the implementation process of how to apply the technical means to solve the technical problems and achieve the technical effects of the present invention can be fully understood and implemented.
Example 1
Preparation of Supported magnolol-sophorolipid micelle
1.1 Determination of Critical Micelle Concentration (CMC) of sophorolipids
The method comprises the following steps: the sophorolipids were distributed with distilled water at a concentration of 1. Mu.g/ml, 2. Mu.g/ml, 3. Mu.g/ml, 5. Mu.g/ml, 8. Mu.g/ml, 10. Mu.g/ml, 15. Mu.g/ml, 20. Mu.g/ml, 30. Mu.g/ml, respectively. Surface tension at different concentrations is detected by a pull-tab method, and the surface tension is plotted on the ordinate and the concentration on the abscissa. The CMC is calculated by fitting the data points to find the inflection point. The pull-tab method is a conventional method for testing the surface tension of micelles in the art and is not described in detail herein.
Results: as shown in FIG. 1, the surface tension rapidly decreases with the increase of the concentration, the surface tension starts to slowly decrease until the surface tension is stable with the increase of the concentration, and the sophorolipid is found to have an inflection point at 3.586 mug/ml through the fitting result, and the surface tension value is almost unchanged. Therefore, the CMC value of the sophorolipids is 3.586. Mu.g/ml. The sophorolipid CMC value is lower and the ability to form micelles is easier.
1.2 Preparation method of honokiol-sophorolipid micelle
The method comprises the following steps: the honokiol-sophorolipid micelle is prepared by a film hydration method. Precisely weighing 30.0 mg and magnolol, respectively dissolving together with sophorolipids of 90 mg, 120 mg, 150mg, 180 mg, 210 mg and 240 mg in absolute ethanol, stirring for 10min to make them fully dissolved. The ethanol was then removed by rotary evaporation at 55℃and finally a thin film was formed on the bottom forming wall of the round bottom flask. 20ml normal saline or ph=7.4 PBS buffer was added and hydrated at 55 ℃ for 10 min. After hydration is completed, removing impurities through a microporous filter membrane with the size of 0.22 mu m after the temperature of the powder drops to room temperature, and determining a final prescription by taking the medicine encapsulation efficiency as an investigation index.
Encapsulation efficiency (%) =amount of drug in micelle/amount of drug administered x 100%,
Drug loading (%) =drug amount in micelle/drug loaded micelle total amount x 100%,
Results: as shown in FIG. 2, as the amount of sophorolipid dosed increases, so does the encapsulation efficiency of honokiol. When 180 mg sophorolipids were used, the encapsulation efficiency was 98.55%. The addition amount of sophorolipid is continuously increased, and the encapsulation efficiency is not obviously increased.
1.3 Comparison of the Effect of different vectors
The method comprises the following steps: respectively preparing honokiol-sophorolipid micelle, honokiol-tween 80 micelle, honokiol-poloxamer 188 micelle and honokiol-poloxamer 407 micelle by the film hydration method in 1.2, precisely weighing 30.0 mg parts and 4 parts of magnolol, respectively dissolving together with 180 mg sophorolipid, tween 80, poloxamer 188 and poloxamer 407 in absolute ethyl alcohol, and stirring for 10 min to enable the honokiol-sophorolipid micelle, the honokiol-tween 80 micelle and the honokiol-poloxamer 407 micelle to be fully dissolved. The ethanol was then removed by rotary evaporation at 55℃and finally a thin film was formed on the bottom forming wall of the round bottom flask. 20 ml normal saline or ph=7.4 PBS buffer was added and hydrated at 55 ℃ for 10 min. After hydration is completed, impurities are removed through a microporous filter membrane of 0.22 mu m after the temperature of the powder is reduced to room temperature, and then the powder is packaged and placed at rest under a low-temperature condition.
Results: the prepared honokiol-sophorolipid micelle has good stability, does not precipitate at the low temperature of 4 ℃, but has poor stability, namely honokiol-tween 80 micelle, honokiol poloxamer 188 micelle and honokiol poloxamer 407 micelle, and can precipitate.
1.4 Prescription verification
The method comprises the following steps: 3 parts of honokiol-sophorolipid micelles were repeatedly prepared according to the method 1.2 described above and their encapsulation efficiency was measured, wherein honokiol was 30.0 mg and sophorolipid was 180mg.
Results: the encapsulation efficiency and drug loading rate of micelles were measured under the same conditions (table 1), and as a result, the average encapsulation efficiency SD (%) was 97.41 ±0.70%. The result shows that the honokiol-sophorolipid micelle is prepared according to the preparation method, the encapsulation rate is high, and the preparation method is stable and reliable.
Table 1 validates the test results
Example 2
Study on antibacterial effect of honokiol-sophorolipid micelle
2.1 Bacterial culture
Individual colonies of staphylococcus aureus were picked on TSA dishes and placed in 6mL TSB broth tubes, which were then shaken in a 100rpm water bath shaker at 37 ℃ for 10 h to stationary phase, and the inoculum was diluted to 1.5 x 10 7 CFU/mL with TSB broth for use.
2.2MIC values
The method comprises the following steps: MIC values were determined with reference to broth dilution in the antimicrobial susceptibility test performance standard (CLSL). 100 μl of TSB medium was added to each well of a 96-well plate; then 100. Mu.L of 128. Mu.g/mL honokiol was added to the first well of each row, mixed well thoroughly, 100. Mu.L was aspirated from the first well to the second well, 100. Mu.L was discarded to the last 1 well, and finally 100. Mu.L of bacterial liquid was added to each well. The final concentrations were set at 32. Mu.g/mL, 16. Mu.g/mL, 8. Mu.g/mL, 4. Mu.g/mL, 2. Mu.g/mL, and positive and negative control groups with only bacterial solution and no bacterial solution were set, and 3 replicates were performed for each treatment. After 10h of stationary culture at 37 ℃, the culture broth was observed visually, and the lowest concentration well for sterile growth was the MIC of the drug. The antibacterial test of sophorolipid and honokiol-sophorolipid micelle was repeated in the same manner, and MIC values of sophorolipid and honokiol-sophorolipid micelle were obtained.
Results: results show that MIC values of honokiol, sophorolipid and honokiol-sophorolipid micelle are respectively 16 [ mu ] g/ml, >32 [ mu ] g/ml and 8 [ mu ] g/ml. Therefore, the antibacterial effect of honokiol-sophorolipid micelle is superior to that of honokiol and sophorolipid.
2.3 Antibacterial Rate
The method comprises the following steps: and (3) processing by a method of 2.2, finally measuring an OD value at 600 nm by using an enzyme-labeled instrument, and calculating the bacteriostasis rate according to the following formula. Antibacterial ratio (%) =od Yang (Yang) −ODSample /OD Yang (Yang) −OD Yin type vagina ×100%
Results: as shown in FIG. 3, the antibacterial rates of honokiol-sophorolipid composite micelle, honokiol and sophorolipid are respectively 99.50+/-0.09%, 61.04+/-7.60% and 30.01+/-4.23% at the concentration of 8 mug/mL. From this, it can be seen that the antibacterial effect of honokiol-sophorolipid micelles is better than that of free honokiol and sophorolipid.
2.4 Bacterial plate coating
The method comprises the following steps: after treatment by method 2.2. Then, the bacterial solution in each well was diluted by the same factor, 50. Mu.l of the diluted bacterial solution was applied to a plate with 6-8 coating strains, and then observed.
Results: as shown in FIG. 4, by comparing with the positive comparison, the colony count was significantly reduced visually with the increase in concentration of honokiol-sophorolipid micelles, while the colony count was also reduced visually with the increase in concentration of honokiol and sophorolipid, but no honokiol-sophorolipid micelles were significantly reduced visually. The result shows that the honokiol-sophorolipid micelle has better antibacterial effect.
Example 3
Honokiol-sophorolipid micelle antibacterial biological membrane capability
3.1 Inhibition of bacterial biofilm formation
The method comprises the following steps: micelle inhibition of s.aureus biofilm formation was studied using crystal violet staining. Firstly, uniformly mixing bacterial liquid with honokiol, sophorolipid and honokiol-sophorolipid micelle, so that the final concentrations of honokiol, sophorolipid and honokiol-sophorolipid micelle are respectively 1 mug/ml, 2 mug/ml and 4 mug/ml. Then, each group was incubated with 2 ml mixed liquids at 37℃in a 12-well plate for 12 h, and a positive control group with only bacterial liquid and a negative control group without bacterial liquid were set, and each treatment was repeated for 3 times. Finally, the S.aureus biofilm surface plankton was rinsed with PBS buffer (pH=7.4), naturally dried, stained with 1% crystal violet, and decolorized with 33% acetic acid. The absorbance values were then measured using a microplate reader at 570 nm to evaluate the inhibition of s.aureus biofilm formation by micelles.
Results: as shown in FIG. 5, honokiol, sophorolipid and honokiol-sophorolipid complex micelle have a dose-dependent increase in the effect of inhibiting the formation of S. The inhibition rate of honokiol and sophorolipid to the biological film reaches 57.16 +/-2.73% and 42.81 +/-15.21% respectively at 4 mug/ml. At the same concentration and with magnolol-sophorolipid micelle inhibition 91.10 + -0.63% S.aureus biofilm formation. Thus, the preparation method of micelle helps to significantly improve the inhibition effect on the formation of the S.
3.2 Ability to clear mature bacterial biofilm
The method comprises the following steps: the study of removal of s.aureus biofilm was performed by using crystal violet staining. The 2ml broth was incubated at 37℃in a 12-well plate for 24: 24h, and the medium was discarded. Adding 2ml g/ml of honokiol, 16 g/ml and 32 g/ml of honokiol, sophorolipid and honokiol-sophorolipid micelle, incubating at 37 ℃ for 12h, setting a positive control group with only bacterial liquid and a negative control group without bacterial liquid, and repeating each treatment for 3 times. Finally, flushing the plankton on the surface of the S.aureus biological film with PBS, naturally airing, dyeing with 1% crystal violet, and decoloring with 33% acetic acid. The absorbance values were then measured using a microplate reader at 570 nm to evaluate the clearance of the micelles to the s.aureus biofilm.
Results: as shown in FIG. 6, the effect of eliminating S.aureus biofilm by honokiol, sophorolipid and honokiol-sophorolipid micelle was seen to increase in a dose-dependent manner by the crystal violet staining results. The sophorolipid has a certain EPS clearance effect, and the clearance efficiency of the biological membrane is 46.08% +/-8.71% at the concentration of 32 mug/ml. The honokiol can clear the bacterial biofilm of 58.05 +/-5.53% at the same concentration. However, the clearance rate of the biomembrane of honokiol-sophorolipid micelle can reach 87.36% +/-1.39% at the concentration of 32 mu g/ml. This also demonstrates that the physical barrier structure of the biofilm prevents the antimicrobial from exerting therapeutic effects, and that the ability to clear bacterial biofilm is significantly increased when drug-loaded micelles are prepared.
3.3 Plate coating of bacteria within biological film
The method comprises the following steps: 2ml bacterial liquid is taken to incubate 24 h at 37 ℃ of a 12-well plate, and the culture medium is discarded. Adding honokiol, sophorolipid and honokiol-sophorolipid micelle with the concentration of 2ml being 8 mug/ml, 16 mug/ml and 32 mug/ml. Incubation was performed at 37℃for 12 h, and a positive control group with only bacterial solution and a negative control group without bacterial solution were set, each treatment being repeated 3 times. Finally, the planktonic bacteria were rinsed with PBS. The biofilm in each well was then equally diluted with PBS, 50 μl was taken from the diluted solution, coated on a plate with 6-8 coating strains, and then observed.
Results: as shown in FIG. 7, by comparing with the positive comparison, the number of colonies in the biofilm was significantly reduced visually with the increase in concentration of honokiol-sophorolipid micelles, and the number of colonies in the biofilm was also reduced correspondingly with the increase in concentration of honokiol and sophorolipid, but the reduction was not significantly seen with the naked eye. The honokiol-sophorolipid micelle is favorable for delivering the antibacterial agent, improves the antibacterial effect of the antibacterial agent on bacteria in the biological film, and realizes the capability of eradicating the bacterial biological film.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (6)
1. The honokiol sophorolipid complex is characterized in that honokiol and sophorolipid are mixed according to the mass ratio of 1:6-8, compounding and forming;
the preparation method of the honokiol sophorolipid complex adopts one of the following preparation modes:
in the mode 1, honokiol and sophorolipid are dissolved in an organic solvent I, then an organic mixed solution is added into an aqueous medium for mixed emulsification, the organic solvent is removed for hydration after micelle formation, and finally impurities are removed;
Mode 2, dissolving honokiol and sophorolipid in an aqueous solution containing an organic solvent II, removing the organic solvent II after micelle formation, hydrating, and finally removing impurities;
in the above-described mode 1 or the above-described mode 2, the hydration process includes: adding normal saline or PBS buffer solution with pH=7.4 into the compound with the first or second organic solvent removed, heating to 40-70 ℃, and hydrating for 5 min-1h;
In the mode 1 or the mode 2, MIC values of honokiol and sophorolipid micelles are 8 μg/ml.
2. The honokiol sophorolipid complex according to claim 1, wherein in the mode 1, the organic solvent one is chloroform.
3. The honokiol sophorolipid complex according to claim 1, wherein in the mode 2, the organic solvent II is at least one selected from methanol, ethanol and acetonitrile.
4. The honokiol sophorolipid complex according to claim 1, wherein in the modes 1-2, the impurity removal is performed by using a microporous membrane of 0.22 μm.
5. The honokiol sophorolipid complex according to claim 1, wherein the hydration is carried out at a temperature of 50-60 ℃ for 5-15 min.
6. Use of a honokiol sophorolipid complex according to any one of claims 1-5 in the preparation of a bacteriostatic and antibacterial product.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104163755A (en) * | 2014-08-07 | 2014-11-26 | 云南中医学院 | Method for preparing magnolol and honokiol from Mangnolia officinalis leaves |
| CN104173324A (en) * | 2014-07-22 | 2014-12-03 | 吉林大学 | Application of honokiol in inhibiting staphylococcus aureus biofilm |
| WO2019023323A1 (en) * | 2017-07-25 | 2019-01-31 | Dsm Ip Assets B.V. | Use of sophorolipids |
| WO2021068614A1 (en) * | 2019-10-06 | 2021-04-15 | 吉林大学 | Application of honokiol and magnolol in preparing mcr-1 enzyme inhibitor |
| KR20210068195A (en) * | 2019-11-29 | 2021-06-09 | 주식회사 마크로케어 | Purification method of lactonic sophorolipid |
| CN113632910A (en) * | 2021-08-06 | 2021-11-12 | 江苏省农业科学院 | Eugenol-sophorolipid nano emulsion and preparation method and application thereof |
| US11220699B1 (en) * | 2020-08-12 | 2022-01-11 | Advanced Biocatalytics Corporation | Compositions and methods for enhancing efficiencies of microbial-derived biosurfactants |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9585903B2 (en) * | 2013-09-30 | 2017-03-07 | Council Of Scientific & Industrial Research | Pharmaceutical composition comprising sophorolipid in combination with an antibiotic |
| WO2016013026A1 (en) * | 2014-07-23 | 2016-01-28 | Council Of Scientific & Industrial Research | Curcumin-sophorolipid complex |
-
2023
- 2023-10-30 CN CN202311415680.9A patent/CN117338795B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104173324A (en) * | 2014-07-22 | 2014-12-03 | 吉林大学 | Application of honokiol in inhibiting staphylococcus aureus biofilm |
| CN104163755A (en) * | 2014-08-07 | 2014-11-26 | 云南中医学院 | Method for preparing magnolol and honokiol from Mangnolia officinalis leaves |
| WO2019023323A1 (en) * | 2017-07-25 | 2019-01-31 | Dsm Ip Assets B.V. | Use of sophorolipids |
| WO2021068614A1 (en) * | 2019-10-06 | 2021-04-15 | 吉林大学 | Application of honokiol and magnolol in preparing mcr-1 enzyme inhibitor |
| KR20210068195A (en) * | 2019-11-29 | 2021-06-09 | 주식회사 마크로케어 | Purification method of lactonic sophorolipid |
| US11220699B1 (en) * | 2020-08-12 | 2022-01-11 | Advanced Biocatalytics Corporation | Compositions and methods for enhancing efficiencies of microbial-derived biosurfactants |
| CN113632910A (en) * | 2021-08-06 | 2021-11-12 | 江苏省农业科学院 | Eugenol-sophorolipid nano emulsion and preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 内酯型槐糖脂生物表面活性剂性能评价;潘洪哲;包木太;林军章;刘涛;宋永亭;李希明;;油气地质与采收率;20130925(05);全文 * |
| 槐糖脂生物表面活性剂的结构特征及理化性质初探;宋丹丹 等;《环境化学》;20111231;第8卷;摘要、2.3小节、图5、表3 * |
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