CN105030675A - High-stability citionella oil nano lipidosome antibacterial agent and preparation method thereof - Google Patents
High-stability citionella oil nano lipidosome antibacterial agent and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the field of an antibacterial agent and a medicinal preparation or cosmetics, and particularly relates to a high-stability citionella oil nano lipidosome antibacterial agent and a preparation method thereof. The citionella oil nano lipidosome is prepared by citionella oil, soybean lecithin, cholesterol, surfactant, chitosan and gelatin. The preparation method comprises the steps: mixing the citionella oil, soybean lecithin and cholesterol with organic solvent to obtain a mixture I, decompressing and steam drying the mixture I to form a smooth film, dissolving the film product by virtue of a water-phase medium and surfactant, forming an emulsion in an ultrasonic manner, uniformly stirring and homogenizing the emulsion with chitosan and gelatin solution, and centrifuging and filtering by virtue of a microporous filter membrane, to obtain lipidosome with a nano-scale particle size. The preparation process is good in reproducibility, the encapsulation efficiency of the citionella oil multilayer nano lipidosome can reach up to 81.2 percent; moreover, the product is complete in shape, uniform in particle size and good in stability and antibacterial property.
Description
Technical field
The invention belongs to antibacterial and pharmaceutical preparation or cosmetic field, citronella oil nanometer liposome antibacterial being specifically related to a kind of high stability and preparation method thereof.
Background technology
Herba Cymbopogonis Citrari, also known as lemon grass (Cymbopogon citratus), is perennial herb, grass family cogon, warming up property of output quintessence oil; Main component is citral, orange blossom alkene, Folium Pruni alkene and cattle glycol; Herba Cymbopogonis Citrari nature and flavor are pungent, warm, have effect that dispelling wind dredging collateral, stomach function regulating are ventilated, refreshment is promoted the sexual maturity; Citronella oil, has certain anticorrosion, antibacterial, antioxidative ability, in addition, also there is the effects such as Tumor suppression, pain relieving, diuresis, allelopathic, have good antibacterial effect to various foodborne pathogens, and safely, have no side effect, be widely used in edible oil and fat processing, can Oxidation of Fat and Oils be prevented.
Domestic have some about the patent application of citronella oil in medical application: CN1970054A disclose a kind of from citronella oil, extract anticancer extract method and composition and purposes, the extracting method of a CN103361179A lemon grass (Cymbopogon citratus) quintessence oil, CN103651634A discloses citronella oil mosquito-driving liquid, mosquito expelling summer sleeping mat and preparation method thereof, and CN101229989 discloses a kind of method preparing high-purity beta-elemene from citronella oil by-product.
Although citronella oil is in medical science, foods and cosmetics industry is widely used, and has the features such as good sterilization, flavouring, but citronella oil is volatile, expose unstable in atmosphere, so method effectively of will finding reduces citronella oil volatilization degree in use, extend storage life.
In citronella oil is wrapped in by nano-lipid physical ability, and nanometer liposome can not damage the main active of citronella oil, nanometer liposome makes that the active component in citronella oil and external environment are isolated to come, and can reduce citronella oil volatility, extend storage life.But due to the slow releasing function of liposome, the liposome shelf-life of parcel quintessence oil is limited, so, chitosan and gelatin is selected to make the much higher layer liposome of stability, in addition, nanometer liposome, due to their subcellular fraction size, can strengthen the Passive intake mechanism of liposome, reduce matter transportation resistance, thus strengthen the effect such as antibacterial of quintessence oil.
Summary of the invention
Citronella oil nanometer liposome that the object of the invention is openly a kind of high stability and preparation method thereof, by citronella oil being wrapped in multi-layer nano liposome, to realize reducing citronella oil volatilization in use, thus reduce the waste of citronella oil, reach the object of long acting antibiotic and efficiency utilization.
A citronella oil nanometer liposome for high stability, citronella oil is wrapped in phospholipid bilayer, it is characterized in that: phospholipid bilayer is ground floor nanometer liposome, and be also provided with second layer nanometer liposome, the second layer is made up of chitosan.
Further, the citronella oil nanometer liposome of described a kind of high stability, is characterized in that: be also provided with third layer nanometer liposome, third layer is made up of gelatin.
Further, in second layer nanometer liposome, the concentration of chitosan is 0.2mg/mL.
Further, in third layer nanometer liposome, the concentration of gelatin is 0.4mg/mL.
The present invention is by citronella oil, and soybean lecithin, cholesterol, surfactant, citronella oil nanometer liposome made by chitosan and gelatin.
Soybean lecithin, cholesterol are that the group forming liposome wants composition, and cholesterol all has the effect regulating membrane fluidity, so investigate the proportioning change both it, on parameters such as the liposomal dispersion formed with or without impact; Surfactant increases liposome stability; Citronella oil concentration is determined according to envelop rate; The pH of Acetate Solution is in order to optimal dissolution chitosan; Chitosan and gelatin can increase stability.
Preparation method of the present invention is by citronella oil, soybean lecithin, cholesterol is mixed in organic solvent, evaporated under reduced pressure forms smooth thin film, add mixed solution dissolving films that aqueous media and surfactant form and ultrasonic become breast, centrifugal after get supernatant liquid and filter and obtain monolayer citronella oil nanometer liposome, it is characterized in that: monolayer citronella oil nanometer liposome and chitosan solution are stirred, by centrifugal and filtering with microporous membrane, obtaining particle diameter is nano level double-deck citronella oil nanometer liposome.
Further, double-deck citronella oil nanometer liposome and gelatin solution are stirred, by centrifugal and filtering with microporous membrane, obtaining particle diameter is nano level multilamellar citronella oil nanometer liposome.
Further, the mass ratio of soybean lecithin of the present invention and cholesterol is 5:1; The mass ratio of surfactant, citronella oil and cholesterol is: 1:3:4, can obtain the highest envelop rate under this condition.
Further, surfactant is PVP, and in mixed solution, the concentration of PVP is 1.0mg/mL.
The volume ratio of monolayer citronella oil nanometer liposome and chitosan solution is 1:10; The volume ratio of double-deck citronella oil nanometer liposome and gelatin solution is 1:10.
Organic solvent described in the present invention is chloroform.
Aqueous media used in the present invention is the acetate buffer solution according to Chinese Pharmacopoeia 2000 editions standard preparation, pH value 3.5 ~ 4.0.
Described chitosan solution is the Acetate Solution of chitosan, and concentration is 0.2mg/mL, and Acetate Solution is the acetate buffer solution according to Chinese Pharmacopoeia 2000 editions standard preparation, pH value 3.5 ~ 4.0, preferably 3.6, and can optimal dissolution chitosan.
Institute's gelatine solution is the Acetate Solution of gelatin, and concentration is 0.4mg/mL, and Acetate Solution is the acetate buffer solution according to Chinese Pharmacopoeia 2000 editions standard preparation, pH value 3.5 ~ 4.0.
In the present invention, the ground floor of liposome is phospholipid bilayer, i.e. artificial cell rete.
In the present invention, the second layer of liposome is made up of chitosan, and concentration is 0.2mg/mL.
In the present invention, the third layer of liposome is made up of gelatin, and concentration is 0.4mg/mL.
Accompanying drawing explanation
Fig. 1 is the envelop rate of citronella oil nanometer liposome.
Fig. 2 is particle diameter and the polydispersity coefficient PDI of citronella oil nanometer liposome.
Fig. 3 is multilamellar citronella oil nanometer liposome fluorescence microscopy figure.
Fig. 4 is multilamellar citronella oil nanometer liposome atomic force microscopy figure.
Fig. 5 multilamellar citronella oil nanometer liposome is to colibacillary anti-microbial property.
Fig. 6 multilamellar citronella oil nanometer liposome is to the anti-microbial property of staphylococcus aureus.
Table 1 is the Zeta potential of citronella oil nanometer liposome.
Detailed description of the invention
By example below, the specific embodiment of the present invention is described, but protection content of the present invention, be not only confined to this.
the envelop rate of embodiment 1 multilamellar citronella oil nanometer liposome
1 experiment material
Soybean lecithin; BR; Chemical Reagent Co., Ltd., Sinopharm Group.
Cholesterol; AR; Chemical Reagent Co., Ltd., Sinopharm Group.
Chloroform; AR; Chemical Reagent Co., Ltd., Sinopharm Group.
Citronella oil; AR; France florihana quintessence oil.
PVP; GR; Chemical Reagent Co., Ltd., Sinopharm Group.
Chitosan; BR; Chemical Reagent Co., Ltd., Sinopharm Group.
Gelatin; BR; Chemical Reagent Co., Ltd., Sinopharm Group.
2 experimental techniques
1) preparation of monolayer citronella oil nanometer liposome
1. take 1g soybean lecithin, the citronella oil of 0.2g cholesterol and 150mg, add 50mL chloroform and make it dissolve.
2. in Rotary Evaporators, be evaporated to solvent evaporate to dryness, evaporating temperature is 10 ~ 30 DEG C, and round-bottomed flask inwall can form smooth thin film; Then products obtained therefrom is put into vacuum drying oven, 30 DEG C, drying 24 hours under vacuum state.
3. the PVP taking 0.05g, in 50mL acetate buffer, spreads under Ultrasonic Conditions, is then added in round-bottomed flask by the acetate buffer of PVP and carries out aquation under Ultrasonic Conditions.
4. by the mixed liquor after aquation in cell micronizing instrument with the 10s that works, gap 5s pulverize frequency 30min.
5. products obtained therefrom is carried out centrifugal, 4000rpm, 15min, get supernatant liquid.
6. being filtered by gained liquid 0.22 μm of filter membrane, obtain filtrate, is monolayer citronella oil nanometer liposome.
2) preparation of double-deck citronella oil nanometer liposome
1. according to the preparation method of above-mentioned monolayer citronella oil nanometer liposome, preparation is containing the monolayer nanometer liposome of 150mg citronella oil.
2. monolayer citronella oil nanometer liposome is dispersed in mix homogeneously in the Acetate Solution containing 0.2mg/mL chitosan; The volume ratio 1:10 of the Acetate Solution of monolayer citronella oil nanometer liposome and chitosan.
3. by gained mixed liquor in cell micronizing instrument with the 10s that works, the frequency of gap 5s pulverizes 30min.
4. products obtained therefrom is carried out centrifugal, 4000rpm, 15min, get supernatant liquid.
5. being filtered by gained liquid 0.22 μm of filter membrane, obtain filtrate, is double-deck citronella oil nanometer liposome.
3) preparation of multilamellar citronella oil nanometer liposome
1. according to the preparation method of above-mentioned double-deck citronella oil nanometer liposome, preparation is containing the double-layer nanometer liposome of 150mg citronella oil.
2. double-deck citronella oil nanometer liposome is dispersed in the Acetate Solution containing 0.4mg/mL gelatin and makes its mix homogeneously; The volume ratio of the Acetate Solution of double-deck citronella oil nanometer liposome and gelatin is 1:10.
3. by gained mixed liquor in cell micronizing instrument with the 10s that works, the frequency of gap 5s pulverizes 30min.
4. products obtained therefrom is carried out centrifugal, 4000rpm, 15min, get supernatant liquid.
5. being filtered by gained liquid 0.22 μm of filter membrane, obtain filtrate, is double-deck citronella oil nanometer liposome.
4) mensuration of envelop rate
Citronella oil is diluted with dehydrated alcohol, stepwise dilution becomes concentration to be respectively 0.1,0.2,0.4,0.6, the standard solution of 0.8mg/mL, then, draw 1 μ L standard solution respectively and carry out GC-MS analysis, automatic integration is carried out to the spectrum peak area of its main component citral, draws citral peak area-citronella oil essential oil concentration standard curve; First get 1mL citronella oil nanometer liposome sample, 13500rpm, centrifugal 3h, outwells supernatant, then adds 1mL ethanol demulsifier, ultrasonic 3h, finally with the centrifugal 15min of the rotating speed of 10000rpm, gets supernatant, analyzes for GC-MS.The liposomal samples prepared, carries out automatic integration to the face, peak of quintessence oil main constituent, then according to the standard curve drawn in step 1, calculates the content of plants essential oil in liposome.
Then:
.
The envelop rate of 3 citronella oil nanometer liposomes
Envelop rate is the most important index evaluating Liposomal formulation quality, is also the key that can liposome play the features such as, low toxicity efficient compared with ordinary preparation; As seen from Figure 1, the envelop rate of monolayer citronella oil nanometer liposome is 31.6%, the envelop rate of double-deck citronella oil nanometer liposome is 49.1%, the envelop rate of multilamellar citronella oil nanometer liposome is maximum, be 81.2%, therefore prepare the envelop rate that multilamellar citronella oil nanometer liposome can significantly improve liposome.
the particle diameter of embodiment 2 multilamellar citronella oil nanometer liposome and polydispersity coefficient PDI
1 experiment material
1. monolayer citronella oil nanometer liposome.
2. double-deck citronella oil nanometer liposome.
3. multilamellar citronella oil nanometer liposome.
2 experimental techniques
Produce with Brooker Hai Wen instrument company of the U.S., model is particle diameter and the polydispersity coefficient PDI value that the high concentration laser particle analyzer of BI-9000 measures citronella oil nanometer liposome, and institute's test sample product are put into sample cell and directly measured.
The particle diameter of 3 citronella oil nanometer liposomes and polydispersity coefficient PDI
Polydispersity coefficient PDI directly reflects the stability of citronella oil nanometer liposome, is therefore Primary Reference index, and the PDI of liposome belongs to best in 0 ~ 0.3 scope, poor in 0.3 ~ 0.7 scope, but can accept, as PDI>0.8, not consider; As shown in Figure 2, the particle diameter of monolayer citronella oil nanometer liposome is 146.1nm, PDI is 0.368, and the particle diameter of double-deck citronella oil nanometer liposome is 198.3nm, PDI is 0.299, and the particle diameter of multilamellar citronella oil nanometer liposome is 229.7nm, PDI is 0.211; The polydispersity coefficient PDI of multilamellar citronella oil nanometer liposome is minimum, therefore prepares the stability that multilamellar citronella oil nanometer liposome can significantly improve liposome.
the Zeta potential of embodiment 3 citronella oil nanometer liposome
1 experiment material
1. monolayer citronella oil nanometer liposome.
2. double-deck citronella oil nanometer liposome.
3. multilamellar citronella oil nanometer liposome.
2 experimental techniques
Measure with the potentiometer that the model that Malvern Instr Ltd. of Britain produces is ZetasirernanoZSZeta, directly liposomal samples to be measured is put into potentiometer and measure.
The Zeta potential of 3 citronella oil nanometer liposomes
The Zeta potential of table 1 citronella oil nanometer liposome
| Citronella oil nanometer liposome | Zeta potential |
| Monolayer | -22.9mV |
| Double-deck | -30.6mV |
| Multilamellar | -41.2mV |
Zeta potential also directly can reflect the stability of citronella oil nanometer liposome, therefore be also Primary Reference index, the larger explanation liposome of the absolute value more stability of the Zeta potential of liposome, the absolute value of Zeta potential belongs to unstable, the liposome comparatively stability when being greater than 30 in 0 ~ 30 scope; As shown in table 1, three kinds of citronella oil nanometer liposomes are all electronegative, the Zeta potential of monolayer citronella oil nanometer liposome is-22.9mV, it is unstable that its absolute value is less than 30 liposomees, the Zeta potential of double-deck citronella oil nanometer liposome is-30.6mV, and it is more stable that its absolute value is greater than 30 liposomees, and the Zeta potential of multilamellar citronella oil nanometer liposome is-41.2mV, its maximum absolute value, liposome is the most stable.
the fluorescence microscope of embodiment 4 multilamellar citronella oil nanometer liposome
1 experiment material
Multilamellar citronella oil nanometer liposome.
2 experimental techniques
With Leca instrument company produce model be the fluorescence microscope of TCS-SP5, directly liposomal samples to be measured is put into fluorescence microscope and observes.
Fluorescence microscope sample-pretreating method:
(1) preparation (A liquid) of multilamellar citronella oil nanometer liposome sample: get 1mL citronella oil liposome, 0.5mL methanol and the mixing of 0.5mL chloroform.
(2) preparation (B liquid) of fluorescent dye DIL: the DIL of 0.1mL is dissolved in the dichloromethane of 0.1mL.
(3) get A liquid 0.5mL and B liquid 50 μ L and put into the mixing of little centrifuge tube, concussion evenly.
(4) above-mentioned mixing material is put into vacuum drying oven, a dry night.
(5) get dry centrifuge tube, add 0.5mL ultra-pure water, shake 30min on the oscillator.
(6) room temperature places 3h.
(7) drop on microscope slide and observe.
The fluorescence microscope of 3 multilamellar citronella oil nanometer liposomes
The microphotograph photographed as can be seen from above fluorescence microscope, after liposome dyeing, presents circle, disperses more even.
the atomic force microscope observation of embodiment 5 multilamellar citronella oil nanometer liposome
1 experiment material
Multilamellar citronella oil nanometer liposome.
2 experimental techniques
With Agilent Technologies of the U.S. produce model be the atomic force microscope of Agilent5500, directly liposomal samples to be measured is put into atomic force microscope to observe, atomic force pre-treating method gets plants essential oil liposomal samples 10 μ L to drop in 10min on mica sheet, then the liquid on surface is absorbed with liquid-transfering gun, drip 10 μ L ultra-pure water 30s again, repeated washing 3 times, ventilation leaves standstill 3h, observes under being positioned over atomic force microscope.
The atomic force microscope observation of 3 multilamellar citronella oil nanometer liposomes
The microphotograph photographed as can be seen from above atomic force microscope, liposome presents circle, disperses more even.
the anti-microbial property of embodiment 6 multilamellar citronella oil nanometer liposome
1 experiment material
1. monolayer citronella oil nanometer liposome (preserving 7 days, 30 days, 60 days, 90 days).
2. double-deck citronella oil nanometer liposome (preserving 7 days, 30 days, 60 days, 90 days).
3. multilamellar citronella oil nanometer liposome (preserving 7 days, 30 days, 60 days, 90 days).
2 experimental techniques
Adopt the method for plate culture count, with escherichia coli (
escherichiacoli) and staphylococcus aureus (
staphylococcusaureus) measure the remaining bacterium number of citronella oil nanometer liposome for pattern bacterium, by escherichia coli and S. aureus Inoculate in fluid medium, be placed in respectively gas bath shaking table 37 DEG C, shake cultivation 24 ~ 48h under 150rpm condition, obtain the antibacterial of exponential phase, get in the test tube that the escherichia coli that are in logarithmic (log) phase in right amount and staphylococcus aureus add containing a certain amount of sterile phosphate buffer respectively that (bacteria concentration is about 10
5~ 10
6cfu/mL), and then in test tube, add the various citronella oil nanometer liposomes that concentration is 10%, separately get two test tubes respectively containing above two kinds of bacterium simultaneously and also add equivalent sterilized water (not adding citronella oil nanometer liposome) wherein in contrast, each test tube is all placed in gas bath shaking table at 37 DEG C, concussion reaction 24h under 150rpm condition, appropriate culture fluid of getting respectively at different time points carries out ten times of gradient dilutions to suitable concentration, then pipetting 100 μ L diluents drips on sterile solid plating medium, coating evenly, put into 37 DEG C of constant temperature and humidity incubators afterwards and be inverted cultivation, plate count is carried out after 24 ~ 48h, thus to evaluating the antibacterial activity of each citronella oil nanometer liposome, do three repetitions, results averaged.
The anti-microbial property of 3 multilamellar citronella oil nanometer liposomes
The change of the antibacterial activity of the citronella oil nanometer liposome of different storage life also can reflect the stability of liposome indirectly, therefore carried out anti-microbial property evaluation to preservation 7 days, the various citronella oil nanometer liposomes of 30 days, 60 days, 90 days, result as shown in Figure 5, Figure 6; Holding time, when being 7 days, the antibacterial activity of monolayer citronella oil nanometer liposome, double-deck citronella oil nanometer liposome, multilamellar citronella oil nanometer liposome was all identical, all shows good antibacterial effect to escherichia coli and staphylococcus aureus; Holding time, when being 30 days, the antibacterial effect of monolayer citronella oil nanometer liposome obviously reduced and double-deck citronella oil nanometer liposome and multilamellar citronella oil nanometer liposome all show good antibacterial effect; Holding time be 60 days and 90 days time, monolayer citronella oil nanometer liposome does not show antibacterial effect, and the antibacterial effect of double-deck citronella oil nanometer liposome obviously reduces, and multilamellar citronella oil nanometer liposome keeps good antibacterial effect always.
Claims (10)
1. the citronella oil nanometer liposome of a high stability, citronella oil is wrapped in phospholipid bilayer, it is characterized in that: phospholipid bilayer is ground floor nanometer liposome, is also provided with second layer nanometer liposome, the second layer is made up of chitosan, to improve envelop rate, stability and anti-microbial property.
2. the citronella oil nanometer liposome of a kind of high stability as claimed in claim 1, is characterized in that: be also provided with third layer nanometer liposome, third layer is made up of gelatin, improves envelop rate, stability and anti-microbial property further.
3. the citronella oil nanometer liposome of a kind of high stability as claimed in claim 1, is characterized in that: in second layer nanometer liposome, the concentration of chitosan is 0.2mg/mL.
4. the citronella oil nanometer liposome of a kind of high stability as claimed in claim 2, is characterized in that: in third layer nanometer liposome, the concentration of gelatin is 0.4mg/mL.
5. the preparation method of the citronella oil nanometer liposome of a kind of high stability as claimed in claim 1, by citronella oil, soybean lecithin, cholesterol is mixed in organic solvent, evaporated under reduced pressure forms smooth thin film, add mixed solution dissolving films that aqueous media and surfactant form and ultrasonic become breast, get supernatant liquid filtration after centrifugal and obtain monolayer citronella oil nanometer liposome, it is characterized in that: monolayer citronella oil nanometer liposome and chitosan solution are stirred, by centrifugal and filtering with microporous membrane, obtaining particle diameter is nano level double-deck citronella oil nanometer liposome.
6. the preparation method of the citronella oil nanometer liposome of a kind of high stability as claimed in claim 5, it is characterized in that: further, double-deck citronella oil nanometer liposome and gelatin solution are stirred, by centrifugal and filtering with microporous membrane, obtaining particle diameter is nano level multilamellar citronella oil nanometer liposome.
7. the preparation method of the citronella oil nanometer liposome of a kind of high stability as claimed in claim 5, is characterized in that: the mass ratio of soybean lecithin and cholesterol is 5:1; The mass ratio of surfactant, citronella oil and cholesterol is: 1:3:4, can obtain the highest envelop rate under this condition; Described organic solvent is chloroform.
8. the preparation method of the citronella oil nanometer liposome of a kind of high stability as claimed in claim 5, is characterized in that: surfactant is PVP, in mixed solution, the concentration of PVP is 1.0mg/mL; Aqueous media used is the acetate buffer solution according to Chinese Pharmacopoeia 2000 editions standard preparation, pH value 3.5 ~ 4.0.
9. the preparation method of the citronella oil nanometer liposome of a kind of high stability as claimed in claim 5, is characterized in that: the volume ratio of monolayer citronella oil nanometer liposome and chitosan solution is 1:10; Described chitosan solution is the Acetate Solution of chitosan, and concentration is 0.2mg/mL, and Acetate Solution is the acetate buffer solution according to Chinese Pharmacopoeia 2000 editions standard preparation, pH value 3.5 ~ 4.0, preferably 3.6, and can optimal dissolution chitosan.
10. the preparation method of the citronella oil nanometer liposome of a kind of high stability as claimed in claim 6, is characterized in that: the volume ratio of double-deck citronella oil nanometer liposome and gelatin solution is 1:10; Institute's gelatine solution is the Acetate Solution of gelatin, and concentration is 0.4mg/mL, and Acetate Solution is the acetate buffer solution according to Chinese Pharmacopoeia 2000 editions standard preparation, pH value 3.5 ~ 4.0.
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| Publication number | Priority date | Publication date | Assignee | Title |
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