CN113559038A

CN113559038A - Nano antibacterial compound and preparation method thereof

Info

Publication number: CN113559038A
Application number: CN202110710712.2A
Authority: CN
Inventors: 许澎; 刘彩云
Original assignee: Hunan Yada Fenghui New Material Co ltd
Current assignee: Hunan Yada Fenghui New Material Co ltd
Priority date: 2021-06-25
Filing date: 2021-06-25
Publication date: 2021-10-29
Anticipated expiration: 2041-06-25
Also published as: CN113559038B

Abstract

The invention relates to the field of antibacterial materials, in particular to a nano antibacterial compound and a preparation method thereof, wherein the nano antibacterial compound is a nano antibacterial composition coated by liposome; the liposome consists of levo-polygalactose, lecithin, cholesterol and polyethylene glycol; the nano antibacterial compound prepared by the invention has broad-spectrum antibacterial activity and instant antibacterial activity, has good antibacterial effect on escherichia coli, staphylococcus aureus, aspergillus niger, diphtheroids, acnes and the like, can be stored for a long time, can be widely applied to the fields of skin care and cleaning, and can be added with essence water for use in spraying, facial masks and mouth wash.

Description

Nano antibacterial compound and preparation method thereof

Technical Field

The invention relates to the field of antibacterial materials, and particularly relates to a nano antibacterial compound and a preparation method thereof.

Background

Chitosan is a product of natural polysaccharide chitin with partial acetyl removed, has multiple physiological functions of biodegradability, biocompatibility, bacteriostasis, cancer resistance, lipid reduction, immunity enhancement and the like, can permeate into skin follicle pores, inhibits and kills harmful microorganisms such as mould, bacteria and the like hidden in the follicle pores, thereby eliminating acne and dermatitis caused by microorganism invasion, and simultaneously eliminating melanin, color spots and the like caused by microorganism accumulation.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the defects or the improvement needs of the prior art, the invention provides a nano antibacterial compound and a preparation method thereof.

The technical scheme adopted by the invention is as follows:

a nanometer antibacterial compound is a liposome-coated nanometer antibacterial composition;

the liposome consists of levo-polygalactose, lecithin, cholesterol and polyethylene glycol;

the nano bacteriostatic composition consists of chitosan-sodium tripolyphosphate nanoparticles and a bacteriostatic extract.

Further, the preparation method of the chitosan-sodium tripolyphosphate nanoparticle comprises the following steps:

s1: dissolving chitosan with acetic acid solution to obtain solution A;

s2: dissolving sodium tripolyphosphate in water to obtain a solution B;

s3: and adding the solution B into the solution A at a stirring speed of 500-800r/min, continuously stirring for 10-30min, and spray-drying the suspension to obtain the chitosan-sodium tripolyphosphate nanoparticles.

Further, the mass concentration of the acetic acid solution is 1-3%, the mass concentration of the solution A is 0.01-0.1%, and the mass concentration of the solution B is 0.01-0.1%.

Further, the antibacterial extract is magnolia bark extract, echinacea purpurea extract, ginger extract, garlic extract, onion extract, grape seed extract, olive leaf extract, coptis chinensis extract, datura flower extract, dandelion extract, angelica dahurica extract, bighead atractylodes rhizome extract, phellodendron bark extract, licorice extract, asarum extract, scutellaria baicalensis extract, bletilla striata extract, fructus arctii extract, burdock fruit extract, red peony root extract, forsythia suspensa extract, ephedra extract, andrographis paniculata extract, purslane extract, perilla seed extract, moutan bark extract, rehmannia root extract, sophora flavescens extract, subprostrate sophora extract, leonurus extract, plantain seed extract, gentian extract, dyers woad leaf extract, pulsatilla chinensis extract, groundsel extract, mint extract, dendrobe extract, anemarrhena extract, licorice root extract, lily root extract, lily bulb extract, lily bulb extract, lily extract, etc, Any one or more of sweet wormwood herb extract and blackberry lily extract.

The preparation method of the nano antibacterial compound comprises the following steps:

s1: preparing antibacterial extract nanoemulsion;

s2: dissolving levo-polygalactose, lecithin, cholesterol and polyethylene glycol in an organic solvent to form primary emulsion;

s3: adding cymene into PBS phosphate buffer solution as water phase;

s4: injecting antibacterial extract nanoemulsion into water phase, rapidly stirring, injecting colostrum, distilling under reduced pressure to remove organic solvent, adding chitosan-sodium tripolyphosphate nanoparticles, performing ultrasonic treatment for 30-60min, and standing at room temperature for 10-20 h.

Further, the preparation method of the antibacterial extract nanoemulsion in S1 comprises the following steps:

mixing tween 80 and glycerol, heating to 35-40 deg.C, stirring to clarify, adding isopropyl myristate, stirring for 10-30min, adding water, stirring for 10-30min, adding the antibacterial extract, and stirring to clarify.

Further, the mass ratio of tween 80, glycerol, isopropyl myristate and water is 4-6: 2-3: 1: 20-30.

Further, the organic solvent is any one of petroleum ether, dichloromethane, ethyl acetate and toluene.

Further, the mass ratio of the levorotatory polygalactose to the lecithin to the cholesterol to the polyethylene glycol is 1-2: 1-2: 1-2: 1-2.

Further, the mass ratio of the cymene to the PBS phosphate buffer solution is 1: 200-250.

The invention has the beneficial effects that:

the invention provides a nano bacteriostatic compound, which is a liposome-coated nano bacteriostatic composition, wherein liposomes are micro-vesicular bodies formed by encapsulating medicaments in lipid bilayer, the liposomes can be divided into small single-chamber liposomes, large single-chamber liposomes and multi-layer liposomes according to forms, lipid membranes mainly comprise phospholipid and cholesterol, the liposomes have the advantages of targeting and lymph directionality, corrosion inhibition, medicament toxicity reduction, medicament stability improvement and the like, the liposome serving as a carrier of medicaments or functional substances has huge application prospects in the fields of medicaments, foods and cosmetics, but the stability of the liposomes is the largest reason for limiting liposome industrialization, and comprises agglomeration caused by uneven particle size distribution in the preparation process, medicament leakage caused by instability of the lipid membranes, poor compounding capability with other surfactants and the like, and levo-polygalactose is prepared by the steps of, The liposome which can stably coat the nano antibacterial composition is compounded by lecithin, cholesterol and polyethylene glycol and has the excellent effects of good stability, simple preparation and low cost, the nano antibacterial composition can stably exist in the liposome, is not easy to agglomerate and precipitate and the like, chitosan is not easy to dissolve in water, is easy to form paste-like agglomerates after being heated, and can generate irritation by using an organic solvent, so the chitosan is prepared into chitosan-sodium tripolyphosphate nanoparticles, the chitosan-sodium tripolyphosphate nanoparticles and the antibacterial extract are coated by the liposome, the appearance of the chitosan in an aqueous solution is improved, the sterilization and bacteriostasis are more facilitated, the antibacterial extract is prepared by adding the antibacterial extract into a system in a nano-emulsion form, a homogeneous dispersion system of the antibacterial extract is facilitated to be formed, the antibacterial extract can still be stably distributed even after the organic solvent is removed by reduced pressure distillation, and the instability and the particles of the system caused by aggregation or gravity agglomeration can be well weakened, tests prove that the nano antibacterial compound prepared by the invention has broad-spectrum antibacterial activity and instant antibacterial activity, has good antibacterial effect on escherichia coli, staphylococcus aureus, aspergillus niger, diphtheroids, acnes and the like, can be stored for a long time, can be widely applied to the fields of skin care and cleaning, and can be added into essence water to achieve good using effect in spraying, facial masks and mouth wash.

Drawings

Fig. 1 is a photograph of an instant bacteriostatic performance test.

FIG. 2 is a schematic structural diagram of a nano bacteriostatic complex;

fig. 3 shows the appearances of the bacteriostatic nanocompositions prepared in example 1 and comparative example 3 after being stored at room temperature for 30 days, which indicates that the bacteriostatic nanocompositions prepared in the present invention have higher storage stability.

Fig. 4 is a graph of particle size analysis of the bacteriostatic nanocomplex prepared in example 1, wherein the bacteriostatic nanocomplex has a uniform size and is distributed between 50 nm and 200 nm.

Fig. 5 is an electron microscope analysis image of the bacteriostatic nanocomplex prepared in example 1, and it is shown that the bacteriostatic nanocomplex has a uniform size and is distributed between 50 nm and 200 nm.

Detailed Description

The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Example 1:

mixing 4g of tween 80 and 2g of glycerol, heating to 35 ℃, stirring until the mixture is clear, adding 1g of isopropyl myristate, continuously stirring for 20min, adding 20g of water, stirring for 20min, adding 0.5g of magnolia officinalis extract, stirring until the mixture is clear, obtaining magnolia officinalis extract nanoemulsion, dissolving 0.75g of chitosan in 1L of acetic acid solution with the mass concentration of 1% to obtain solution A, dissolving 0.75g of sodium tripolyphosphate in 1L of water to obtain solution B, adding the solution B into the solution A at the stirring speed of 800r/min, continuously stirring for 20min, spray-drying the suspension to obtain the chitosan-sodium tripolyphosphate nanoparticles, dissolving 1g of levo-polygalactose, 1g of lecithin, 1g of cholesterol and 1g of polyethylene glycol in 500mL of dichloromethane to form colostrum, adding 2g of cymene into 450mLPBS phosphate buffer solution, as the aqueous phase; injecting cortex Magnolia officinalis extract nanoemulsion into water phase, rapidly stirring, injecting colostrum, distilling under reduced pressure to remove organic solvent, adding chitosan-sodium tripolyphosphate nanoparticles, ultrasonic treating for 50min, and standing at room temperature for 15 hr.

Example 2:

mixing 4g of tween 80 and 2g of glycerol, heating to 40 ℃, stirring until the mixture is clear, adding 1g of isopropyl myristate, continuously stirring for 30min, adding 20g of water, further stirring for 30min, adding 0.5g of magnolia officinalis extract, stirring until the mixture is clear, obtaining magnolia officinalis extract nanoemulsion, dissolving 0.75g of chitosan in 1L of 2% acetic acid solution by mass concentration to obtain solution A, dissolving 0.75g of sodium tripolyphosphate in 1L of water to obtain solution B, adding the solution B into the solution A at the stirring speed of 600r/min, continuously stirring for 30min, spray-drying the suspension to obtain the chitosan-sodium tripolyphosphate nanoparticles, dissolving 1g of levo-polygalactose, 1g of lecithin, 1g of cholesterol and 1g of polyethylene glycol in 500mL of dichloromethane to form colostrum, adding 2g of cymene into 450mLPBS phosphate buffer solution, as the aqueous phase; injecting cortex Magnolia officinalis extract nanoemulsion into water phase, rapidly stirring, injecting primary emulsion, distilling under reduced pressure to remove organic solvent, adding chitosan-sodium tripolyphosphate nanoparticles, ultrasonic treating for 30min, and standing at room temperature for 20 hr.

Example 3:

mixing 4g of tween 80 and 2g of glycerol, heating to 40 ℃, stirring until the mixture is clear, adding 1g of isopropyl myristate, continuously stirring for 25min, adding 20g of water, stirring for 25min, adding 0.5g of magnolia officinalis extract, stirring until the mixture is clear, obtaining magnolia officinalis extract nanoemulsion, dissolving 0.75g of chitosan in 1L of acetic acid solution with the mass concentration of 1.5% to obtain solution A, dissolving 0.75g of sodium tripolyphosphate in 1L of water to obtain solution B, adding the solution B into the solution A at the stirring speed of 800r/min, continuously stirring for 25min, spray-drying the suspension to obtain the chitosan-sodium tripolyphosphate nanoparticles, dissolving 1g of levorotatory polygalactose, 1g of lecithin, 1g of cholesterol and 1g of polyethylene glycol in 500mL of dichloromethane to form colostrum, adding 2g of cymene into 450mL of phosphate buffer solution, as the aqueous phase; injecting cortex Magnolia officinalis extract nanoemulsion into water phase, rapidly stirring, injecting primary emulsion, distilling under reduced pressure to remove organic solvent, adding chitosan-sodium tripolyphosphate nanoparticles, ultrasonic processing for 40min, and standing at room temperature for 18 hr.

Example 4:

a preparation method of a nano antibacterial compound bacterium comprises the following steps:

mixing 4g of tween 80 and 2g of glycerol, heating to 37 ℃, stirring until the mixture is clear, adding 1g of isopropyl myristate, continuously stirring for 30min, adding 20g of water, further stirring for 30min, adding 0.5g of magnolia officinalis extract, stirring until the mixture is clear, obtaining magnolia officinalis extract nanoemulsion, dissolving 0.75g of chitosan by using 1L of 2% acetic acid solution by mass concentration to obtain solution A, dissolving 0.75g of sodium tripolyphosphate by using 1L of water to obtain solution B, adding the solution B into the solution A at the stirring speed of 800r/min, continuously stirring for 20min, spray-drying the suspension to obtain the chitosan-sodium tripolyphosphate nanoparticles, dissolving 1g of levo-polygalactose, 1g of lecithin, 1g of cholesterol and 1g of polyethylene glycol in 500mL of dichloromethane to form colostrum, adding 2g of cymene into 450mLPBS phosphate buffer solution, as the aqueous phase; injecting cortex Magnolia officinalis extract nanoemulsion into water phase, rapidly stirring, injecting primary emulsion, distilling under reduced pressure to remove organic solvent, adding chitosan-sodium tripolyphosphate nanoparticles, ultrasonic treating for 50min, and standing at room temperature for 10 hr.

Example 5:

mixing 4g of tween 80 and 2g of glycerol, heating to 40 ℃, stirring until the mixture is clear, adding 1g of isopropyl myristate, continuously stirring for 30min, adding 20g of water, further stirring for 30min, adding 0.5g of magnolia officinalis extract, stirring until the mixture is clear, obtaining magnolia officinalis extract nanoemulsion, dissolving 0.75g of chitosan by using 1L of acetic acid solution with the mass concentration of 3% to obtain solution A, dissolving 0.75g of sodium tripolyphosphate by using 1L of water to obtain solution B, adding the solution B into the solution A at the stirring speed of 800r/min, continuously stirring for 30min, spray-drying the suspension to obtain the chitosan-sodium tripolyphosphate nanoparticles, dissolving 1g of levo-polygalactose, 1g of lecithin, 1g of cholesterol and 1g of polyethylene glycol in 500mL of dichloromethane to form colostrum, adding 2g of cymene into 450mLPBS phosphate buffer solution, as the aqueous phase; injecting cortex Magnolia officinalis extract nanoemulsion into water phase, rapidly stirring, injecting primary emulsion, distilling under reduced pressure to remove organic solvent, adding chitosan-sodium tripolyphosphate nanoparticles, ultrasonic treating for 60min, and standing at room temperature for 20 hr.

Example 6:

mixing 4g of tween 80 and 2g of glycerol, heating to 35 ℃, stirring until the mixture is clear, adding 1g of isopropyl myristate, continuously stirring for 15min, adding 20g of water, further stirring for 15min, adding 0.5g of magnolia officinalis extract, stirring until the mixture is clear, obtaining magnolia officinalis extract nanoemulsion, dissolving 0.75g of chitosan by using 1L of acetic acid solution with the mass concentration of 1% to obtain solution A, dissolving 0.75g of sodium tripolyphosphate by using 1L of water to obtain solution B, adding the solution B into the solution A at the stirring speed of 600r/min, continuously stirring for 15min, spray-drying the suspension to obtain the chitosan-sodium tripolyphosphate nanoparticles, dissolving 1g of levo-polygalactose, 1g of lecithin, 1g of cholesterol and 1g of polyethylene glycol in 500mL of dichloromethane to form colostrum, adding 2g of cymene into 450mLPBS phosphate buffer solution, as the aqueous phase; injecting cortex Magnolia officinalis extract nanoemulsion into water phase, rapidly stirring, injecting primary emulsion, distilling under reduced pressure to remove organic solvent, adding chitosan-sodium tripolyphosphate nanoparticles, ultrasonic treating for 40min, and standing at room temperature for 20 hr.

Comparative example 1:

comparative example 1 is substantially the same as example 1 except that magnolia bark extract is added directly.

dissolving 0.75g of chitosan in 1L of acetic acid solution with the mass concentration of 1% to obtain solution A, dissolving 0.75g of sodium tripolyphosphate in 1L of water to obtain solution B, adding the solution B into the solution A at the stirring speed of 800r/min, continuously stirring for 20min, spray-drying the suspension to obtain the chitosan-sodium tripolyphosphate nanoparticles, dissolving 1g of levo-polygalactose, 1g of lecithin, 1g of cholesterol and 1g of polyethylene glycol in 500mL of dichloromethane to form colostrum, and adding 2g of cymene into 450mL of PBS phosphate buffer solution to serve as a water phase; adding cortex Magnolia officinalis extract into water phase, rapidly stirring, injecting primary emulsion, distilling under reduced pressure to remove organic solvent, adding chitosan-sodium tripolyphosphate nanoparticles, ultrasonic treating for 50min, and standing at room temperature for 15 hr.

Comparative example 2:

comparative example 2 is essentially the same as example 1 except that chitosan and sodium tripolyphosphate are added separately.

mixing 4g of tween 80 and 2g of glycerol, heating to 35 ℃, stirring until the mixture is clear, then adding 1g of isopropyl myristate, continuing to stir for 20min, then adding 20g of water, stirring for 20min, then adding 0.5g of magnolia officinalis extract, stirring until the mixture is clear to obtain magnolia officinalis extract nanoemulsion, dissolving 1g of levo-polygalactose, 1g of lecithin, 1g of cholesterol and 1g of polyethylene glycol in 500mL of dichloromethane to form colostrum, and adding 2g of cymene into 450mL of PBS phosphate buffer solution to serve as a water phase; injecting cortex Magnolia officinalis extract nanoemulsion into water phase, rapidly stirring, injecting primary emulsion, distilling under reduced pressure to remove organic solvent, adding chitosan and sodium tripolyphosphate, ultrasonic treating for 50min, and standing at room temperature for 15 hr.

Comparative example 3:

comparative example 3 is substantially the same as example 1 except that the magnolia bark extract is added directly and the chitosan and sodium tripolyphosphate are added separately.

dissolving 1g of levo-polygalactose, 1g of lecithin, 1g of cholesterol and 1g of polyethylene glycol in 500mL of dichloromethane to form colostrum, and adding 2g of cymene into 450mLPBS phosphate buffer solution to serve as a water phase; adding cortex Magnolia officinalis extract into water phase, rapidly stirring, injecting primary emulsion, distilling under reduced pressure to remove organic solvent, adding chitosan and sodium tripolyphosphate, ultrasonic treating for 50min, and standing at room temperature for 15 hr.

And (3) testing the instant antibacterial performance:

(1) preparing a flat plate from a freshly prepared non-resistant LB culture medium;

(2) inoculating E.coli into 10ml liquid culture medium in advance, and culturing for 8 hours at 200-300 rpm of a shaking table;

(3) experimental group 1: 1ml of the nano bacteriostatic complex prepared in example 1 + 10. mu.L of E.coli;

experimental group 2: 1ml of the nano bacteriostatic complex prepared in comparative example 1 +10 μ L of escherichia coli;

experimental group 3: 1ml of the nano bacteriostatic complex prepared in comparative example 2 +10 μ L of escherichia coli;

experimental group 4: 1ml of the nano bacteriostatic complex prepared in comparative example 3 +10 μ L of escherichia coli;

the nano antibacterial compound prepared in the embodiment 1 of the invention has an obvious antibacterial effect, the comparative example 1 is the second time, the comparative example 2 is the second time, and the rapid antibacterial effect of the comparative example 3 is the worst.

Broad spectrum bacteriostatic performance test

Respectively inoculating Escherichia coli, Staphylococcus aureus, Aspergillus niger, diphtheroids and acne bacillus into sterilized solid slant culture medium, culturing at 37 deg.C for 24 hr, dissolving the slant culture in sterile water, shaking, mixing, diluting by 10 times, and making into product with content of about 10⁵Taking the bacterial suspension per mL, uniformly coating the bacterial suspension on a sterilized nutrient agar plate by a coating method, dripping a drop of the nano antibacterial compound prepared in the examples 1-6 and the comparative examples 1-3 at the center of the nutrient agar plate, putting a culture dish into a constant temperature incubator at 37 ℃ for culturing for 24 hours, measuring the diameter D1(mm) of a bacteriostatic zone, taking another blank group of drops of physiological saline, carrying out the same treatment, measuring the diameter D2(mm) of the bacteriostatic zone, and calculating the bacteriostatic rate (%).

The bacteriostasis rate is [ (D2-D1)/D2 ]. times of 100%

The results are shown in table 1 below:

table 1:

as can be seen from table 1 above, the nano antibacterial compound prepared by the present invention has broad spectrum antibacterial activity, and has good antibacterial activity against escherichia coli, staphylococcus aureus, aspergillus niger, diphtheroids, acnes, etc.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Claims

1. A nano bacteriostatic composite is characterized in that the nano bacteriostatic composite is a nano bacteriostatic composition coated by liposome;

2. The nano bacteriostatic complex as claimed in claim 1, wherein the chitosan-sodium tripolyphosphate nanoparticle is prepared by the following method:

s1: dissolving chitosan with acetic acid solution to obtain solution A;

s2: dissolving sodium tripolyphosphate in water to obtain a solution B;

3. The nano bacteriostatic complex as claimed in claim 2, wherein the mass concentration of the acetic acid solution is 1-3%, the mass concentration of the solution A is 0.01-0.1%, and the mass concentration of the solution B is 0.01-0.1%.

4. The nano bacteriostatic complex of claim 1, wherein the bacteriostatic extract is magnolia bark extract, echinacea purpurea extract, ginger extract, garlic extract, onion extract, grape seed extract, olive leaf extract, coptis chinensis extract, datura flower extract, dandelion extract, angelica dahurica extract, atractylodes macrocephala koidz extract, phellodendron bark extract, licorice extract, asarum herb extract, scutellaria baicalensis extract, bletilla striata extract, fructus arctii extract, burdock fruit extract, red peony root extract, forsythia suspensa extract, ephedra extract, andrographis paniculata extract, purslane extract, perilla seed extract, moutan bark extract, rehmanniae radix extract, sophora flavescens extract, subprostrate sophora extract, leonurus extract, plantain seed extract, gentian extract, isatis leaf extract, pulsatilla chinensis extract, senecio scandens extract, morinda officinalis extract, and kaki extract, morinda officinalis extract, any one or more of mint extract, dendrobium extract, rhizoma anemarrhenae extract, sweet wormwood extract and blackberry lily extract.

5. The preparation method of the nano bacteriostatic complex as claimed in claims 1 to 4, which is characterized by comprising the following steps:

s1: preparing antibacterial extract nanoemulsion;

s3: adding cymene into PBS phosphate buffer solution as water phase;

6. The method for preparing a nano bacteriostatic complex as claimed in claim 5, wherein the method for preparing the bacteriostatic extract nanoemulsion of S1 comprises the following steps:

7. The preparation method of the nano bacteriostatic complex as claimed in claim 6, wherein the mass ratio of tween 80, glycerol, isopropyl myristate and water is 4-6: 2-3: 1: 20-30.

8. The method for preparing a nano bacteriostatic complex according to claim 5, wherein the organic solvent is any one of petroleum ether, dichloromethane, ethyl acetate and toluene.

9. The preparation method of the nano bacteriostatic compound according to claim 5, wherein the mass ratio of the levo-polygalactose, lecithin, cholesterol and polyethylene glycol is 1-2: 1-2: 1-2: 1-2.

10. The method for preparing a nano bacteriostatic complex as claimed in claim 5, wherein the mass ratio of the cymene to the PBS phosphate buffer solution is 1: 200-250.