CN111281959A - Volatile oil composition and preparation method of nano preparation thereof - Google Patents

Volatile oil composition and preparation method of nano preparation thereof Download PDF

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Publication number
CN111281959A
CN111281959A CN202010177526.2A CN202010177526A CN111281959A CN 111281959 A CN111281959 A CN 111281959A CN 202010177526 A CN202010177526 A CN 202010177526A CN 111281959 A CN111281959 A CN 111281959A
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Prior art keywords
volatile oil
oil
composition
polysaccharide
volatile
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王志杰
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Benxi Bereson Nano Biomedical Co Ltd
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Benxi Bereson Nano Biomedical Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/25—Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
    • A61K36/258—Panax (ginseng)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/08—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
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    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
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    • A—HUMAN NECESSITIES
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    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
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    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
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    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
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    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
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    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
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    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
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    • A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
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Abstract

The invention relates to a composition and a preparation method of a nanometer preparation thereof, in particular to a volatile oil composition, a composition added with polysaccharide and a preparation method of a micelle nanometer preparation thereof. The polysaccharide also has effect in enhancing immunoregulation. The preparation of the invention has no side effect in clinical tests, and is an antiviral compound preparation which is safe, efficient and suitable for multiple application scenes and administration modes.

Description

Volatile oil composition and preparation method of nano preparation thereof
Technical Field
The invention relates to a preparation method of an antibacterial and antiviral volatile oil composition and a nano preparation thereof, in particular to a preparation method of a micelle nano preparation.
Technical Field
Research shows that many volatile oils, including natural plant volatile oils, have certain killing or inhibiting effects on microorganisms and viruses. However, volatile oil components are volatile, the content of effective components can be obviously reduced along with the prolonging of the standing time, and toxic and side effects can be generated or increased due to improper compatibility of different volatile oils. Meanwhile, the volatile oil is insoluble in water, so that the application scenes of related preparations are limited due to the limitation of the product form of the volatile oil, for example, 2018093020180930 discloses an anti-haze spray with a bacteriostatic function, which comprises the following components in percentage by mass: 0.5-5% of chitosan quaternary ammonium salt, 0.5-5% of sodium hyaluronate, 3-5% of glycerol, 10-75% of ethanol and 20-80% of pure water, as well as CN106176960A, the invention discloses a natural antibacterial spray, which is prepared by heating and mixing chitosan oligosaccharide, berberine, laurocapram, lithospermum extract, tea tree essential oil, absolute ethyl alcohol and distilled water, wherein the dosage of each component is as follows by weight percent: 0.2-0.5% of chitosan oligosaccharide, 0.1-0.5% of berberine, 0.2-0.5% of laurocapram, 5-10% of lithospermum extract, 0.1-0.8% of tea tree essential oil, 40-60% of absolute ethyl alcohol and the balance of distilled water, and the technical scheme is not suitable for application in vivo or in mucosa areas, has large irritation due to the adoption of an ethanol organic solvent, does not have good physicochemical properties specific to an aqueous preparation except for possible side effects caused by the ethanol when being applied to the surface of an object, and cannot form a space distribution effect with good aerosol characteristics due to large-particle-size components of the volatile oil when being sprayed in a certain space. In addition, in the process of extracting polysaccharide, the current methods mainly comprise: traditional hot water extraction, ultrasonic extraction, microwave extraction, enzyme-assisted extraction, acid-base extraction, etc. The polysaccharide is extracted by a hot water extraction method, although the process is simple, the extraction temperature is higher, the extraction time is longer, the extraction rate is lower, water-soluble impurities are more, and the separation and purification are difficult; the polysaccharide is extracted by an ultrasonic extraction method, so that active substances are easily damaged, the extraction rate is low, and the method is not suitable for large-scale extraction; equipment required for extracting the ginseng polysaccharide by adopting a microwave extraction method is expensive, the microwave energy consumption is high, and the risk of potential radiation leakage harming human health is caused; the enzyme-assisted extraction method is adopted to carry out extraction conditions on the ginseng polysaccharide, the biological activity of the polysaccharide can be maintained to a certain extent, but a buffering agent is required to be added into an aqueous solvent to adjust the pH value so as to ensure the activity of a complex enzyme, the fluctuation of the activity of the enzyme along with the change of the pH value is large, the extraction rate is not stable enough, and the change of the pH value in the aqueous solvent can also cause the hydrolysis loss of the polysaccharide; the principle of extracting ginseng polysaccharide by using an acid-base method is that the cellular components of ginseng are damaged by acid and base, the bonding bond between sugar and protein is broken, and thus the extraction rate is improved, but the breakage of the glycosidic bond in the polysaccharide is promoted, and a container is corroded.
Therefore, the preparation formula which completely maintains the killing effect of the volatile oil, has good physical and chemical properties in various application scenes in vivo and in vitro and can further increase the virus adsorption performance, and the preparation process which can realize low-cost mass production have good practical value. To date, no low-cost product with outstanding efficacy and safe and universal preparation performance exists in the prior art.
Disclosure of Invention
Researches prove that a plurality of volatile oils, in particular natural plant volatile oils such as ginseng oil, dandelion oil, honeysuckle oil, clove oil, peppermint oil, valerian oil, zedoary oil, patchouli oil, argyi leaf oil and the like have good antiviral and anti-inflammatory effects. Clinical and pharmacological researches show that the polysaccharide has the effects of improving immunity, resisting aging, resisting oxidation, diminishing inflammation, resisting tumors, reducing blood sugar, reducing blood pressure and the like, and also has adhesion and bacteriostasis effects. Aiming at the defects in the prior art, the invention provides a volatile oil composition and a preparation method of a nano preparation thereof, wherein the volatile oil of the composition exists in a stable 50-500 nano micelle form, and further can form a 100-250nm micelle form, the volatile oil has the characteristic of being close to an aqueous mixed solution, the particle size of most volatile oil molecular groups in the micelle preparation can be stably maintained in a preset range at normal temperature, and the volatility of the volatile oil molecular groups can be delayed to play a slow release role after the micelle preparation is further coated with polysaccharide. The volatile oil contained in the volatile oil composition can effectively inhibit bacteria and kill viruses, and the polysaccharide component has the functions of adhering and trapping viruses. Experiments show that after polysaccharide molecules are further combined based on the volatile oil molecular groups, the adhesion can be increased, the virus adhesion effect is achieved, particularly, the positively charged polysaccharide modification also has the effect of adsorbing bacteria or viruses, and related nano preparations can be used for trapping and killing viruses without risks in a body fluid environment. When atomized and sprayed in natural environment, the aerosol can be dispersed in the air for a long time, or dust particles are adhered and fall down, so that the quantity of toxic particles in the air is reduced, and the effect on the surface of an attached object can be achieved for a long time. The fermentation method for extracting the polysaccharide used in the preparation process of the nano preparation utilizes the reducing sugar in the leaching liquor as a carbon source, the protein and the free ammonia as nitrogen sources, and nutrient substances such as inorganic salt, vitamins and the like to grow in the growth process of yeast, and the polysaccharide can not be utilized by the yeast, so that the impurity removal and purification effects on the polysaccharide can be obtained, and the prepared polysaccharide has higher activity and better adhesion effect on viruses.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a composition comprises a volatile oil component and a stabilizer, wherein the volatile oil component contained in the composition is one or a mixture of a plurality of volatile oils, the proportion of the total α -unsaturated aldehyde contained in the volatile oil component to the total mass of the volatile oil component is not higher than 3.25 thousandths, the mass proportion of the volatile oil component in the composition is between 0.5 and 12 percent, the stabilizer contained in the composition is a surface active substance and comprises but not limited to polysorbate, sorbitan monolaurate, ethylene oxide addition compound, lauryl alcohol polyoxyethylene ether, coco acid diethanolamide, oleic acid monoglyceride, phospholipid, polyoxyethylene lanolin and the like, the mass proportion of the stabilizer in the composition is between 0.25 and 28 percent, and the interval range of the ratio of the stabilizer to the content of the volatile oil component in the composition within the mass proportion range is that the stabilizer is 0.5 to 8 parts and the volatile oil component is 1 part.
The specific gravity value parameter of the volatile oil adopted by the volatile oil component or the fully mixed volatile oil mixture at the natural environment of 20 ℃ is generally selected to be within the range of 0.82-1.096, so that the standard reaching rate of the product during preparation of the nano micelle preparation is guaranteed, and further, the parameter index of the specific gravity value range of 0.89-0.99 at the natural environment of 20 ℃ is preferably selected. Experiments show that when the mass ratio of the volatile oil components is between 2% and 8%, a better adjustment space can be obtained among killing effect, cost and process stability, and a mathematical model for simulating mass production shows that the mass ratio of 5.5% to 6% is a preferred proportioning interval of compatibility schemes of several common volatile oils. The volatile oil component compatibility scheme which is supported by data of professional virus detection institutions and aims at stabilizing the SI value of coronavirus to more than 10 comprises a volatile oil component series scheme which is a combination of two or more of ginseng oil, zedoary turmeric oil, argy wormwood leaf oil, patchouli oil and peppermint oil, and a volatile oil component series scheme which is a combination of two or more of atractylodes rhizome oil, peppermint oil, clove oil, dandelion oil and honeysuckle oil. Although the method for proportioning the volatile oil by using the surfactant exists in the prior art, only the research on the nano micelle preparation with the particle size of 175nm as the median is concentrated, and the technical scheme of the invention is a technical scheme which is found out through a large number of experiments and in a plurality of physical and chemical parameters which influence each other, has obvious beneficial effects, can realize mass production and is suitable for commercial use.
Further, the composition also comprises polysaccharide, the mass ratio of the polysaccharide is in a range of 0.2-7.5%, preferably in a range of 0.5-5%, and the polysaccharide is preferably positively charged polysaccharide, such as a ginseng polysaccharide or American ginseng polysaccharide or chitosan. In the preparation process flow set by the invention, the molecular groups of the polysaccharide are wrapped or attached to the periphery of the volatile oil molecular groups in a large proportion, and the positively charged polysaccharide molecules also have the function of adsorbing pathogens while adhering peripheral particles, so as to assist discrete volatile oil molecules to take effect on the pathogens, wherein the outer layer of the partially and completely wrapped polysaccharide is gradually damaged and is also one of slow-release and long-acting microscopic mechanisms.
The technical scheme of the invention also comprises a process method for preparing the nano micelle preparation of the related composition, which comprises the following steps: step one, preparing one volatile oil or a mixture of a plurality of volatile oils according to the mass ratio of 2-8% to obtain a volatile oil base solution; step two, taking a stabilizer with a proportion of 0.5-8: 1 relative to the total amount of the volatile oil within a range that the total amount is not more than 28% by mass, mixing the stabilizer with the volatile oil base solution, heating to 75-90 ℃, and fully stirring; step three, adding polysaccharide according to the mass ratio of 0.5-5%, adding hot water at 75 ℃ according to the mass ratio of 15%, fully stirring and standing for 15-20 minutes; step four, adding a proper amount of phosphate buffer solution with pH8.5; step five, hydrating at 75 ℃ for 0.2-0.75h, thus obtaining the micelle preparation of the composition in a scheme with a certain component proportion, wherein the particle size of the molecular group of more than 80 percent of the active ingredients in the preparation is in the range of 50-500 nm. According to the preparation method, the process conditions of individual links can be adjusted according to the actual compatibility of the components, for example, the step two is changed into the step of mixing the normal-temperature stabilizer and the essential oil base solution in the range that the total amount is not more than 28% by mass and the proportion of the normal-temperature stabilizer is 0.5-8: 1 of the total amount of the essential oil, fully stirring and standing the mixture, and meanwhile, the step four is changed into the step of adding a proper amount of water or phosphate buffer solution with the pH value of 7.4 and the like. On the basis of the various preparation methods, the third step can be changed into a step of adding polysaccharide according to the mass ratio of 0.5-5%, fully stirring and standing, and the fifth step is changed into a step of hydrating at 60 ℃ for 0.25-1h and the like. The technical scheme of the invention can accurately control the molecular group particle size of the active ingredients of the micelle preparation of the composition in a certain component proportion scheme, and experiments prove that the standard that the proportion of the active ingredients of the micelle preparation in the range of 100nm-250nm is not less than 75 percent can be reached. The technical solution of the present invention is further illustrated by the following examples.
Detailed Description
Example 1 preparation method of a volatile oil composition and a nano-micelle preparation thereof
A method for preparing a nano micelle preparation of a volatile oil composition comprises the following process steps:
the method comprises the following steps: method for extracting ginseng polysaccharide by yeast fermentation
Washing fresh ginseng with clear water, removing silt, drying, crushing a dried sample by a crusher, sieving by a 80-mesh sieve, mixing ginseng powder and distilled water in a ratio of 1: 20, uniformly stirring, placing in a constant-temperature water bath, extracting at 95 ℃ for 3h, centrifuging to obtain a supernatant, diluting the supernatant by 10 times for later use to obtain a ginseng fermentation stock solution, placing 100mL of the fermentation stock solution in a 250mL conical flask, sealing a cotton plug, sterilizing at 115 ℃ for 20min, inoculating and fermenting, wherein the yeast inoculation amount is 5%, the fermentation time is 48h, the fermentation rotation speed is 150r/min, the pH is 6, centrifuging after the fermentation is finished, and taking the supernatant to obtain ginseng polysaccharide for later use;
step two: mixing 1% of ginseng oil, 1% of zedoary turmeric oil, 1% of patchouli oil, 1% of blumea oil, 1% of honeysuckle flower oil, 0.5% of dandelion oil and 0.25% of peppermint oil, and then fully stirring to obtain a mixed volatile oil base solution (the specific gravity measured at 20 ℃ is 0.9430) for later use;
step three: mixing 16% polysorbate with the volatile oil base solution, heating to 75-90 deg.C, and stirring;
step four: adding 5% of prepared panaxan into the above mixed solution, adding 15% of 75 deg.C hot water, stirring, and standing for 15-20 min;
step five: adding a proper amount of phosphate buffer solution with pH8.5;
step six: hydrating at 75 deg.C for fifteen minutes.
And (3) detecting the nano particle size:
taking a proper amount of the nano micelle preparation of the volatile oil composition, and detecting the nano micelle preparation three times by using a laser instrument. The detection result of the content is shown in table-1, the mixed solution is micelle, the proportion of the molecular group with the particle size of 50nm to 500nm is 91.6%, the average particle size is 186.6 +/-2.0, the PDI is less than 0.3, and the particle size distribution of the nano micelle is uniform.
TABLE-1 particle size distribution Range of volatile oil nanocomposites
Figure BDA0002411305150000071
And (3) testing the bacteriostatic effect:
the agar dilution method is to add different dosages of the volatile oil micelles prepared in this example into melted and cooled quantitative MH agar to prepare plates containing different decreasing concentrations (6,12,24,48,96,192,384,768 μ g/mL) of antibacterial agent, and the inoculation concentrations are all 2 × 106The MBC of the medicine is determined by observing the growth condition of bacteria after incubation for 24 hours in/mL test bacteria (three strains of Klebsiella pneumoniae, staphylococcus aureus and escherichia coli). The highest dilution of the drug for 50% and 90% of the same bacterial strains without growth is MIC50And MIC90. The results are shown in Table-2, and the volatile oil micelles prepared in this example have a significant inhibitory effect on bacterial proliferation.
TABLE-2 MIC and MBC assay results for volatile oil nanocomposites
Figure BDA0002411305150000072
Effect test of coronavirus killing or proliferation inhibition:
taking Vero cells to grow into single layers in 96-hole cell culture plates respectively, removing supernatant culture solution, washing with serum-free culture medium solution for three timesAdding 10 μ L/well of 10TCID50(10-1) influenza virus solution, at 40 deg.C and 5% CO2: adsorbing in incubator for 2 hr, discarding virus solution, adding the volatile oil micelles with concentration of 1.5, 3,6,12,24, 48,96,192 μ g/mL, respectively, and culturing. 3 multiple wells are set, and virus control and normal cell control are set at the same time. Placing influenza virus, parainfluenza virus, and respiratory syncytial virus group into incubator, rotary culturing, observing pathological change effect (CPE) the next day, culturing for 7d, and observing CPE once every day. And (4) judging the end point by taking the lowest concentration of the medicine with the CPE completely inhibited (grade 0) as the antiviral effect, and judging the result. The results are shown in Table-3, and the volatile oil micelles prepared in this example have significant killing or proliferation inhibitory effects on viruses.
TABLE-3 in vitro anti-influenza virus, parainfluenza virus, respiratory syncytial virus test results of volatile oil nano-composition
Figure BDA0002411305150000081
Example 2 preparation method of volatile oil composition and nano micelle preparation thereof
A method for preparing a nano micelle preparation of a volatile oil composition comprises the following process steps:
the method comprises the following steps: preparing a proper amount of chitosan for later use;
step two: taking 3 g of honeysuckle oil (the specific gravity measured at 20 ℃ is 0.9113) as essential oil base liquid for later use;
step three: adding 24 g of sorbitan monolaurate into the volatile oil base solution, fully stirring and slowly heating to 75-90 ℃;
step four: adding 3 g of chitosan into the mixed solution, adding 15 g of hot water at 75 ℃ (± 5 ℃), fully stirring and standing for 15 minutes;
step five: adding about 55 g of phosphate buffer solution with the pH value of 8.5 into the mixed solution to obtain 100 g of mixed solution;
step six: hydrating the mixed solution at 75 deg.C (+ -5 deg.C) for 0.2h to obtain the final product.
The results of the nano particle size test are shown in table-4, and the mixed solution is in a micelle state, wherein the proportion of the molecular groups with the particle size of 50nm to 150nm is 83.4%. The average particle size is 102.5 +/-1.6, the PDI is less than 0.3, and the particle size distribution of the nano micelle is uniform.
TABLE-4 particle size distribution Range of the volatile oil nanocomposites
Figure BDA0002411305150000091
And (3) testing the bacteriostatic effect:
the agar dilution method is adopted, and different dosages of the volatile oil micelles prepared in the embodiment are added into melted and cooled quantitative MH agar to prepare plates containing different decreasing concentrations (3,6,12,24, 48,96,192,384,768 mu g/mL) of the antibacterial agent, and the inoculation concentrations are 2 multiplied by 106The MBC of the medicine is determined by observing the growth condition of bacteria after incubation for 24 hours in/mL test bacteria (three strains of Klebsiella pneumoniae, staphylococcus aureus and escherichia coli). The highest dilution of the drug for 50% and 90% of the same bacterial strains without growth is MIC50And MIC90. The results are shown in Table-2, and the volatile oil micelles prepared in this example have a significant inhibitory effect on bacterial proliferation.
The test result of the bacteriostatic effect is shown in table-5, and the volatile oil micelle prepared in the embodiment has a certain inhibitory effect on bacterial proliferation.
TABLE-5 MIC and MBC assay results for volatile oil nanocomposites
Figure BDA0002411305150000092
Figure BDA0002411305150000101
Effect test of coronavirus killing or proliferation inhibition:
respectively growing Vero cells into single layer in 96-well cell culture plate, discarding supernatant culture solution, washing with serum-free culture medium solution for three times, adding 10 μ L/well of 10TCID50(10-1) influenza virus solution, 40 deg.C, and 5% CO2: adsorbing in incubator for 2 hr, discarding virus solution, adding the volatile oil micelles with concentration of 1.5, 3,6,12,24, 48,96,192 μ g/mL, respectively, and culturing. 3 multiple wells are set, and virus control and normal cell control are set at the same time. Placing influenza virus, parainfluenza virus, and respiratory syncytial virus group into incubator, rotary culturing, observing pathological change effect (CPE) the next day, culturing for 7d, and observing CPE once every day. And (4) judging the end point by taking the lowest concentration of the medicine with the CPE completely inhibited (grade 0) as the antiviral effect, and judging the result. As shown in Table-6, the volatile oil micelles obtained in this example had significant effects on killing or inhibiting the proliferation of viruses.
TABLE-6 test results of in vitro anti-influenza virus, parainfluenza virus and respiratory syncytial virus of volatile oil nano composition
Figure BDA0002411305150000102
Example 3 preparation method of volatile oil composition and nano micelle preparation thereof
A method for preparing a nano micelle preparation of a volatile oil composition comprises the following process steps:
the method comprises the following steps: preparing proper amount of American ginseng polysaccharide for later use;
step two: mixing 4 g of clove oil, 2.5 g of honeysuckle oil, 1 g of dandelion oil, 1 g of peppermint oil and 0.5 g of atractylodes rhizome oil, and fully stirring to obtain 9 g of volatile oil base liquid (the specific gravity measured at the environment of 20 ℃ is 0.953) for later use;
step three: adding 28 g of coconut diethanolamide into the essential oil base liquid, fully stirring and standing;
step four: adding 2 g of American ginseng polysaccharide into the mixed solution, adding 15 g of hot water at 75 ℃ (± 5 ℃), fully stirring and standing for 15 minutes;
step five: adding about 46 g of phosphate buffer solution with the pH value of 7.4 into the mixed solution to obtain 100 g of mixed solution;
step six: hydrating the mixed solution at 75 deg.C (+ -5 deg.C) for 45 min.
The results of the nano-particle size test are shown in table-7, and the mixed solution is in a micelle state, wherein the proportion of the molecular groups with the particle size of 100nm to 250nm is 83.4%. The average particle size is 173.8 +/-1.9, the PDI is less than 0.3, and the particle size distribution of the nano micelle is uniform.
TABLE-7 particle size distribution Range of the volatile oil nanocomposites
Figure BDA0002411305150000111
And (3) testing the bacteriostatic effect:
the agar dilution method was used to add different doses of the volatile oil micelles prepared in this example to melted and cooled quantitive MH agar to prepare plates containing varying decreasing concentrations (9,18,36,72, 144,288,576, 1152. mu.g/mL) of the antimicrobial drug, all at 2X 106The MBC of the medicine is determined by observing the growth condition of bacteria after incubation for 24 hours in/mL test bacteria (three strains of Klebsiella pneumoniae, staphylococcus aureus and escherichia coli). The highest dilution of the drug for 50% and 90% of the same bacterial strains without growth is MIC50And MIC90. The results are shown in Table-2, and the volatile oil micelles prepared in this example have a significant inhibitory effect on bacterial proliferation.
The test result of the bacteriostatic effect is shown in table-8, and the volatile oil micelle prepared in the embodiment has an obvious inhibition effect on bacterial proliferation.
TABLE-8 MIC and MBC assay results for volatile oil nanocomposites
Figure BDA0002411305150000121
Effect test of coronavirus killing or proliferation inhibition:
respectively growing Vero cells into single layer in 96-well cell culture plate, discarding supernatant culture solution, washing with serum-free culture medium solution for three times, adding 10 μ L/well of 10TCID50(10-1) influenza virus solution, 40 deg.C, and 5% CO2: adsorbing in incubator for 2 hr, discarding virus solution, adding the volatile oil micelles with concentration of 1.5, 4.5, 9,18,36,72, 144,288. mu.g/mL of the culture medium. 3 multiple wells are set, and virus control and normal cell control are set at the same time. Placing influenza virus, parainfluenza virus, and respiratory syncytial virus group into incubator, rotary culturing, observing pathological change effect (CPE) the next day, culturing for 7d, and observing CPE once every day. And (4) judging the end point by taking the lowest concentration of the medicine with the CPE completely inhibited (grade 0) as the antiviral effect, and judging the result. As shown in Table-9, the volatile oil micelles obtained in this example had significant effects on killing or inhibiting the proliferation of viruses.
TABLE-9 results of in vitro anti-influenza virus, parainfluenza virus, respiratory syncytial virus test on volatile oil nano-composition
Figure BDA0002411305150000131
Example 4 preparation method of a volatile oil composition and a nano-micelle preparation thereof
A method for preparing a nano micelle preparation of a volatile oil composition comprises the following process steps:
the method comprises the following steps: preparing proper amount of ginseng polysaccharide for later use;
step two: mixing 4 g of honeysuckle oil, 3 g of zedoary turmeric oil, 2 g of patchouli oil, 2 g of argy wormwood leaf oil and 1 g of peppermint oil, fully stirring, and standing to obtain 12 g of volatile oil base solution (the specific gravity measured at 20 ℃ in an environment is 0.9636) for later use;
step three: adding 6 g of lauryl alcohol polyoxyethylene ether into the volatile oil base liquid, fully stirring and slowly heating to 75-90 ℃;
step four: adding 5 g of ginseng polysaccharide into the mixed solution, fully stirring and standing for 15 minutes;
step five: adding about 77 g of phosphate buffer solution with the pH value of 8.5 into the mixed solution to obtain 100 g of mixed solution;
step six: hydrating the mixture at 60 deg.C for 0.25-1 h.
The results of the nano-particle size test are shown in table-10, and the mixed solution is in a micelle state, wherein the proportion of the molecular groups with the particle size of 100nm to 250nm is 86.1%. The average particle size is 209.9 +/-2.8, the PDI is less than 0.3, and the nano micelle has uniform particle size distribution.
TABLE-10 particle size distribution Range of the volatile oil nanocomposites
Figure BDA0002411305150000141
And (3) testing the bacteriostatic effect:
the agar dilution method is adopted, and different dosages of the volatile oil micelles prepared in the embodiment are added into melted and cooled quantitative MH agar to prepare plates containing different decreasing concentrations (3,6,12,24, 48,96,192,384,768 mu g/mL) of the antibacterial agent, and the inoculation concentrations are 2 multiplied by 106The MBC of the medicine is determined by observing the growth condition of bacteria after incubation for 24 hours in/mL test bacteria (three strains of Klebsiella pneumoniae, staphylococcus aureus and escherichia coli). The highest dilution of the drug for 50% and 90% of the same bacterial strains without growth is MIC50And MIC90. As shown in Table-11, the volatile oil micelles obtained in this example had a significant inhibitory effect on bacterial proliferation.
TABLE-11 MIC and MBC assay results for volatile oil nanocomposites
Figure BDA0002411305150000142
Effect test of coronavirus killing or proliferation inhibition:
respectively growing Vero cells into single layer in 96-well cell culture plate, discarding supernatant culture solution, washing with serum-free culture medium solution for three times, adding 10 μ L/well of 10TCID50(10-1) influenza virus solution, 40 deg.C, and 5% CO2: adsorbing in incubator for 2 hr, discarding virus solution, adding the volatile oil micelles with concentration of 1.5, 3,6,12,24, 48,96,192 μ g/mL, respectively, and culturing. 3 multiple wells are set, and virus control and normal cell control are set at the same time. Placing influenza virus, parainfluenza virus, and respiratory syncytial virus group into incubator, rotary culturing, observing pathological change effect (CPE) the next day, culturing for 7d, and observing CPE once every day. The lowest concentration of the drug with CPE completely inhibited (0 grade) is taken as the antiviral effect judgment end point for judgmentAnd (6) determining the result. As shown in Table-12, the volatile oil micelles obtained in this example had significant effects on killing or inhibiting the proliferation of viruses.
TABLE-12 results of in vitro anti-influenza virus, parainfluenza virus, respiratory syncytial virus test on volatile oil nano-composition
Figure BDA0002411305150000151
Example 5 preparation method of a volatile oil composition and a nano-micelle preparation thereof
A method for preparing a nano micelle preparation of a volatile oil composition comprises the following process steps:
the method comprises the following steps: preparing proper amount of ginseng polysaccharide for later use;
step two: mixing 5 g of zedoary turmeric oil and 2 g of patchouli oil, fully stirring, standing, and obtaining 7 g of volatile oil base fluid (the specific gravity measured at the temperature of 20 ℃ is 0.971) for later use;
step three: adding 17 g of polyoxyethylene lanolin into the volatile oil base liquid, fully stirring and standing;
step four: adding 1 g of ginseng polysaccharide into the mixed solution, fully stirring and standing for 15 minutes;
step five: adding about 75 g of water to the mixed solution to obtain 100 g of mixed solution;
step six: hydrating the mixture at 60 deg.C for 0.25-1 h.
The results of the nano-particle size test are shown in table-13, and the mixed solution is in a micelle state, wherein the proportion of the molecular groups with the particle size of 100nm to 250nm is 84.9%. The average particle size is 195.6 +/-0.8, the PDI is less than 0.3, and the nano micelle has uniform particle size distribution.
TABLE-13 particle size distribution Range of the volatile oil nanocomposites
Figure BDA0002411305150000161
And (3) testing the bacteriostatic effect:
the agar dilution method is to mix different agentsAmounts of the volatile oil micelles prepared in this example were added to a melted and cooled aliquot of MH agar to form plates containing varying concentrations of the antibacterial agent (7,14,28,56, 70, 112,224,448, 896. mu.g/mL) at 2X 10 concentrations6The MBC of the medicine is determined by observing the growth condition of bacteria after incubation for 24 hours in/mL test bacteria (three strains of Klebsiella pneumoniae, staphylococcus aureus and escherichia coli). The highest dilution of the drug for 50% and 90% of the same bacterial strains without growth is MIC50And MIC90. As shown in Table-14, the volatile oil micelles obtained in this example had a significant inhibitory effect on bacterial proliferation.
TABLE-14 MIC and MBC assay results for volatile oil nanocomposites
Figure BDA0002411305150000162
Effect test of coronavirus killing or proliferation inhibition:
respectively growing Vero cells into single layer in 96-well cell culture plate, discarding supernatant culture solution, washing with serum-free culture medium solution for three times, adding 10 μ L/well of 10TCID50(10-1) influenza virus solution, 40 deg.C, and 5% CO2: adsorbing in incubator for 2 hr, discarding virus solution, adding the volatile oil micelles with concentration of 1.4, 3.5, 7,14,28,56, 70, and 112 μ g/mL respectively, and culturing. 3 multiple wells are set, and virus control and normal cell control are set at the same time. Placing influenza virus, parainfluenza virus, and respiratory syncytial virus group into incubator, rotary culturing, observing pathological change effect (CPE) the next day, culturing for 7d, and observing CPE once every day. And (4) judging the end point by taking the lowest concentration of the medicine with the CPE completely inhibited (grade 0) as the antiviral effect, and judging the result. As shown in Table-15, the volatile oil micelles obtained in this example had significant effects on killing or inhibiting the proliferation of viruses.
TABLE-15 in vitro anti-influenza virus, parainfluenza virus, respiratory syncytial virus test results of volatile oil nano composition
Figure BDA0002411305150000171
The above examples are only illustrative embodiments for illustrating the technical solutions of the present invention, but the embodiments of the present invention are not limited by the above examples, and any other changes, modifications, substitutions, combinations, simplifications, or alterations made without departing from the spirit and principle of the present invention should be regarded as equivalents or substitutions of the technical solutions of the present invention, and shall be included in the protection scope of the present invention.

Claims (11)

1. The composition comprises a volatile oil component and a stabilizer, and is characterized in that the volatile oil component is a volatile oil or a mixture of a plurality of volatile oils, the volatile oil component contains α -unsaturated aldehyde with the total content not higher than 3.25 per thousand of the mass of the volatile oil component, the composition contains the volatile oil component with the mass ratio ranging from 0.5% to 12%, the stabilizer is a surface active substance such as sorbitan monocinnamate, ethylene oxide addition product, polyoxyethylene lauryl ether, cocofatty acid diethanolamide, polysorbate, oleic acid monoglyceride, phospholipid, polyoxyethylene lanolin and the like, the composition contains the stabilizer with the mass ratio ranging from 0.25% to 28%, and the ratio of the mass ratio of the stabilizer to the volatile oil is 0.5-8: 1.
2. The composition of claim 1, wherein: the composition also comprises polysaccharide, and the mass ratio of the composition to the polysaccharide is between 0.2% and 7.5%.
3. The composition of claim 1, wherein: the specific gravity of the volatile oil component at 20 ℃ in natural environment ranges from 0.82 to 1.096.
4. The composition of claim 1, wherein: the mass ratio of the volatile oil component is in the range of 2-8%, and the preferable range is in the range of more than 5.5% and less than 6%.
5. The composition of claim 1, wherein: the specific gravity of the volatile oil component at 20 ℃ in natural environment ranges from 0.89 to 0.99.
6. The composition of claim 1, wherein: the volatile oil component comprises a mixture of two or more of ginseng oil, zedoary turmeric oil, blumea oil, patchouli oil and peppermint oil, or a mixture of two or more of atractylodes rhizome oil, peppermint oil, clove oil, dandelion oil and honeysuckle oil.
7. The composition of claim 2, wherein: the mass ratio of the polysaccharide ranges from 0.5% to 5%.
8. The composition of claim 2, wherein: the polysaccharide is a positively charged polysaccharide, such as ginseng polysaccharide or American ginseng polysaccharide or chitosan.
9. A method of preparing a nano-formulation of a composition according to claims 4-8, characterized in that: the method comprises the following process steps of preparing one volatile oil or a mixture of a plurality of volatile oils according to the mass ratio of 2-8% to obtain a volatile oil base solution, mixing a stabilizer which is 0.5-8: 1 of the total amount of the volatile oil with the volatile oil base solution in the range that the total amount is not more than 28% by mass, heating to 75-90 ℃, and fully stirring, adding polysaccharide according to the mass ratio of 0.5-5% in the step three, adding 75-DEG C hot water according to the mass ratio of 15%, fully stirring, standing for 15-20 minutes, adding a proper amount of phosphate buffer solution with the pH of 8.5 in the step four, and hydrating at 75 ℃ for 0.2-0.75h in the step five to obtain the volatile oil.
10. A method of preparing a nano-formulation of a composition according to claim 8, characterized in that: and step two, mixing a normal-temperature stabilizer and the essential oil base solution in a proportion of 0.5-8: 1 relative to the total amount of the essential oil within a range that the total amount is not more than 28% by mass, fully stirring, standing, and adding a proper amount of water or phosphate buffer solution with the pH value of 7.4.
11. A process for the preparation of a nano-formulation of a composition according to claim 8 or 9, characterized in that: step three, adding polysaccharide according to the mass ratio of 0.5-5%, fully stirring and standing, and step five, hydrating at 60 ℃ for 0.25-1 h.
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