CN112870162A - Preparation method of peppermint oil nano emulsion - Google Patents
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Abstract
The invention discloses a preparation method of peppermint oil nano-emulsion, and relates to the technical field of preparation of bacteriostatic agent nano-emulsion. The peppermint oil nano emulsion prepared by the method is in a stable O/W type, and the bactericidal rate of the nano emulsion on gram-negative bacteria (escherichia coli) reaches 99%.
Description
Technical Field
The invention relates to the technical field of preparation of bacteriostatic agent nano emulsion, in particular to a production technology of peppermint oil nano emulsion.
Background
With the continuous change of human living environment, especially the outbreak of COVID-19, people pay more attention to the infection control of microorganisms, especially bacteria and viruses. Staphylococcus aureus and Escherichia coli are important pathogenic bacteria in daily life. At present, for the prevention and treatment of the bacteria, people generally use antibiotics such as beta-lactam (such as penicillin), cephalosporin or carbapenems, aminoglycosides, sulfonamides and the like to inhibit the bacteria; micro-or nano-materials containing inorganic materials such as zinc (Zn), silver (Ag), copper (Cu) and gold (Au) have also been developed for antibacterial use. With the wide use of the products, besides common side effects of human bodies, the problems of increasingly serious bacterial drug resistance and the like also exist. New antibacterial products have therefore been sought to replace the above mentioned products.
In recent years, plant essential oils of natural origin have attracted much attention as antibacterial substances, and peppermint oil is one of the most popular essential oils. The peppermint oil is one of the effective components of peppermint which is a herbaceous plant of the family Labiatae, has good treatment effect on migraine, dizziness and insomnia, and also has good antibacterial effect. But the mint essential oil has the defects of poor water solubility, high volatility and the like, so that the antibacterial effect of the mint essential oil is greatly limited. Therefore, developing a proper peppermint oil dosage form and fully playing the antibacterial action of the peppermint oil becomes one of the ways for improving the antibacterial effect of the peppermint oil.
The nano emulsion is an isotropic and thermodynamically stable colloidal dispersion, has the characteristics of small size, low viscosity, high transparency and the like, and is widely applied to the fields of food industry and medicine. The nano emulsion has more advantages in drug absorption than common emulsion, and not only can improve the bioavailability, but also can improve the solubility of water-insoluble drugs. In the preparation process of the nano emulsion, one phase can be dispersed on the other phase by adding a proper emulsifier, so that the unstable phenomenon of flocculation phase separation caused by the unstable mechanism of the nano emulsion is avoided.
Disclosure of Invention
The invention aims to disclose a preparation method of peppermint oil nano-emulsion with nano-scale particle size, good antibacterial property, high stability and low volatility.
Mixing and stirring peppermint oil and an emulsifier, adding deionized water, and treating a mixed system by a high-energy emulsification method to obtain peppermint oil nano emulsion; the peppermint oil, the emulsifier and the deionized water respectively account for 5-10%, 4-8% and 82-91% of the total feeding mass.
The concentration and the property of the emulsifier play an important role in the characteristics of the nano emulsion, and based on the spontaneous combination of the surfactant around essential oil molecules and insolubility of water, the invention can better exert the emulsification effect. The preparation method has the outstanding advantages that the emulsification is more complete, the prepared nano emulsion is uniformly dispersed in liquid drops, the system is stable, and the preparation method has the characteristics of high storage stability, low emulsifier dosage, simple processing and the like.
Coli is extremely sensitive to peppermint oil nanoemulsion. Since the peppermint oil nanoemulsion acts on bacterial cells to interrupt the balance between the production and consumption of active oxygen therein, oxidative stress occurs, followed by cell damage, resulting in lipid peroxidation, cell membrane rupture and leakage of a large amount of cell contents, resulting in bacterial cell death. Therefore, the antibacterial mechanism of the peppermint oil nanoemulsion may be due to the fact that lipophilic components of peppermint oil easily pass through cell membranes, so that the cell membranes are ruptured and bacteria die.
Therefore, the peppermint oil nano emulsion prepared by the method is in a stable O/W type, and the bactericidal rate of the nano emulsion on gram-negative bacteria (escherichia coli) reaches 99%.
Further, the high-energy emulsification method provided by the invention is used for shearing for 3-7 min at a rotating speed of 8000-12000 r/min. Practice has shown that the shearing time is too short and incomplete shearing may occur during the preparation. When the shearing time is too long, the probability of liquid drop collision is increased, and the fusion of the liquid drops is promoted. The selection time is 3-7 min determined by repeated tests, and the nano-emulsion with small particle size can be obtained within the shearing time range. And the rotating speed is preferably 8000-12000 r/min, so that proper emulsification energy can be provided, and better emulsification effect is ensured.
More preferably, the high energy emulsification method is shearing at a rotational speed of 10000 r/min for 5 min; the peppermint oil, the emulsifier and the deionized water respectively account for 10%, 8% and 82% of the total feeding mass; and the emulsifier consists of Tween-60 and EL-20, wherein the Tween-60 accounts for 6% of the total feeding mass, and the EL-20 accounts for 2% of the total feeding mass. Can prepare the peppermint oil nanoemulsion with high stability and small particle size reaching the nano level.
The purity of the charged peppermint oil was 98 wt%. Researches show that the antibacterial efficacy of the prepared peppermint oil nanoemulsion is directly related to the purity of peppermint essential oil. The main component of the mint essential oil is menthol, and the mint essential oil with low purity contains high content of fusel, so that the nano-emulsion has poor bacteriostatic effect. The invention can only reach 99% of bacteriostasis rate by using the high-purity mint essential oil to prepare the nano emulsion.
The emulsifier mainly comprises polyoxyethylene sorbitan monooleate and castor oil polyoxyethylene ether, wherein the polyoxyethylene sorbitan monooleate accounts for 3-6% of the total mass of the emulsifier, and the castor oil polyoxyethylene ether accounts for 1-2% of the total mass of the emulsifier.
The invention has the beneficial effects that the invention starts from the characteristics of peppermint oil, adopts a high-energy emulsification technology, uses peppermint essential oil as an oil phase, selects polyoxyethylene sorbitan monooleate (Tween-60) and castor oil polyoxyethylene ether (EL-20) as an emulsifier, and prepares the bacteriostatic nano-emulsion by a high-energy emulsification method. The system changes oil solubility of oleum Menthae Dementholatum, increases its solubility, and is beneficial for inhibiting bacteria; meanwhile, the strong volatility and instability of the traditional mint essence are avoided through high-energy emulsification; the O/W type peppermint oil nano-emulsion is used for replacing the traditional peppermint oil, so that the using amount of the peppermint oil is reduced, and the defects of easy volatilization, poor water solubility and the like of the peppermint oil are overcome. Compared with the traditional emulsion, the nano emulsion has smaller particle size, is uniformly dispersed, greatly improves the stability of the emulsion and is easy to improve the storage stability; the invention prepares the peppermint oil into the sprayable peppermint oil nano-emulsion with strong bacteriostatic property for the first time.
Drawings
FIG. 1 is a graph of the morphological analysis of E.coli without treatment with the peppermint oil nanoemulsion.
FIG. 2 is a diagram of the morphological analysis of E.coli treated with the peppermint oil nanoemulsion.
Figure 3 is a back-scattered light scanning spectrum of the peppermint oil nanoemulsion.
Detailed Description
Firstly, a preparation process:
example 1:
preparing an emulsifier: 1.2 g of Tween-60 and 0.4 g of EL-20 were mixed to obtain 1.6 g of an emulsifier.
Mixing 2.0 g of peppermint oil and 1.6 g of emulsifier to prepare a mixture of the emulsifier and the oil, and stirring for 12 hours; then 16.4g of deionized water is added into the mixed system, and the mixture is sheared for 7min at the rotating speed of 8000 r/min, thus obtaining the O/W type peppermint oil nano emulsion.
Example 2:
preparing an emulsifier: 1.2 g of Tween-60 and 0.4 g of EL-20 were mixed to obtain 1.6 g of an emulsifier.
Mixing 2.0 g of peppermint oil and 1.6 g of emulsifier to prepare a mixture of the emulsifier and the oil, and stirring for 12 hours; then 16.4g of deionized water is added into the mixed system, and then the mixture is sheared for 7min at the rotating speed of 10000 r/min by a high-energy emulsification method, thus obtaining the O/W type peppermint oil nano emulsion.
Example 3:
preparing an emulsifier: 1.2 g of Tween-60 and 0.4 g of EL-20 were mixed to obtain 1.6 g of an emulsifier.
Mixing 2.0 g of peppermint oil and 1.6 g of emulsifier to prepare a mixture of the emulsifier and the oil, and stirring for 12 hours; then 16.4g of deionized water is added into the mixed system, and then the mixture is sheared for 7min at the rotating speed of 12000 r/min, and the O/W type peppermint oil nano emulsion is obtained.
Example 4:
preparing an emulsifier: 0.9 g of Tween-60 and 0.3 g of EL-20 were mixed to obtain 1.2 g of an emulsifier.
Mixing 2.0 g of peppermint oil and 1.2 g of emulsifier to prepare a mixture of the emulsifier and the oil, and stirring for 12 hours; then 16.8g of deionized water is added into the mixed system, and the mixture is cut for 3 min by a high-energy emulsification method at the rotating speed of 8000 r/min, thus obtaining the O/W type peppermint oil nano emulsion.
Example 5:
preparing an emulsifier: 0.9 g of Tween-60 and 0.3 g of EL-20 were mixed to obtain 1.2 g of an emulsifier.
Mixing 2.0 g of peppermint oil and 1.2 g of emulsifier to prepare a mixture of the emulsifier and the oil, and stirring for 12 hours; then 16.8g of deionized water is added into the mixed system, and then the mixture is sheared for 3 min at the rotating speed of 10000 r/min by a high-energy emulsification method, thus obtaining the O/W type peppermint oil nano emulsion.
Example 6:
preparing an emulsifier: 0.9 g of Tween-60 and 0.3 g of EL-20 were mixed to obtain 1.2 g of an emulsifier.
Mixing 2.0 g of peppermint oil and 1.2 g of emulsifier to prepare a mixture of the emulsifier and the oil, and stirring for 12 hours; then 16.8g of deionized water is added into the mixed system, and then the mixture is sheared for 3 min at the rotating speed of 12000 r/min, and the O/W type peppermint oil nano emulsion is obtained.
Example 7:
preparing an emulsifier: 0.6g of Tween-60 and 0.2 g of EL-20 were mixed to obtain 0.8 g of an emulsifier.
Mixing 2.0 g of peppermint oil and 0.8 g of emulsifier to prepare a mixture of the emulsifier and the oil, and stirring for 12 hours; then adding 17.2g of deionized water into the mixed system, and then shearing for 5min at the rotating speed of 8000 r/min to obtain the O/W type peppermint oil nano emulsion.
Example 8:
preparing an emulsifier: 0.6g of Tween-60 and 0.2 g of EL-20 were mixed to obtain 0.8 g of an emulsifier.
Mixing 2.0 g of peppermint oil and 0.8 g of emulsifier to prepare a mixture of the emulsifier and the oil, and stirring for 12 hours; then adding 17.2g of deionized water into the mixed system, and then shearing for 5min at the rotating speed of 10000 r/min by a high-energy emulsification method to obtain the O/W type peppermint oil nano emulsion.
Example 9:
preparing an emulsifier: 0.6g of Tween-60 and 0.2 g of EL-20 were mixed to obtain 0.8 g of an emulsifier.
Mixing 2.0 g of peppermint oil and 0.8 g of emulsifier to prepare a mixture of the emulsifier and the oil, and stirring for 12 hours; and then adding 17.2g of deionized water into the mixed system, and then shearing for 5min at the rotating speed of 12000 r/min by a high-energy emulsification method to obtain the O/W type peppermint oil nano emulsion.
Secondly, the stability of the peppermint oil nano emulsion is investigated:
the method comprises the following steps: scanning once every 10 min within 12 h by using a Tubiscan multiple light scattering stabilizer, wherein the increment of back scattering light delta BS% <0.2%, and the test result shows that: each O/W type peppermint oil nanoemulsion was a stable nanoemulsion.
The second method comprises the following steps: the stability of the peppermint oil nanoemulsion was further examined by aging at low temperature (4 + -2 deg.C), high temperature (54 + -2 deg.C) and normal temperature (25 + -2 deg.C), respectively, and the results were: the O/W type peppermint oil nano-emulsion does not have phase separation phenomenon after one year.
Thirdly, the mint oil nanoemulsion has the effect of inhibiting gram-negative bacteria (escherichia coli):
coli single colonies were inoculated in 5ml LB liquid medium at 37 ℃ and 180rpm for 15 h. After 24 h, 50. mu.L of the broth was diluted with 4950. mu.L of fresh LB broth and incubated at 37 ℃ and 180rpm for 3 h to OD600Up to 0.7.
Mixing 100 μ L of inoculum with 900 μ L H2O mix served as control.
Mixing 100 μ L of the strain with the prepared oleum Menthae Dementholatum nanoemulsion, incubating at 37 deg.C for 24 h, and checking the result.
The indoor bacteriostasis experiment and agar dilution method are adopted to carry out bacteriostasis experiment on gram-negative bacteria (escherichia coli), and the bacteriostasis result is 99%. The method improves the defects of poor volatility and water solubility of oleum Menthae Dementholatum, and improves antibacterial rate.
The Control method comprises the following steps: bacteria (100. mu.L) were mixed with water (900. mu.L) for 30 min and 2.5% glutaraldehyde fixed at 4 ℃ overnight. Then washed 3 times with water and dehydrated with a gradient of ethanol (30%, 50%, 70%, 80%, 90%, 95% and 100%). Drying with supercritical fluid, spraying gold with gold spraying instrument, and analyzing the shape of bacteria with scanning electron microscope, as shown in FIG. 1.
A Treated method: bacteria (100 μ L) were mixed with peppermint oil nanoemulsion (100 μ L) in water (800 μ L) for 30 min, and 2.5% glutaraldehyde was fixed at 4 ℃ overnight. Then washed 3 times with water and dehydrated with a gradient of ethanol (30%, 50%, 70%, 80%, 90%, 95% and 100%). After drying with supercritical fluid, spraying gold with a gold spraying instrument, and analyzing the shape of the bacteria with a scanning electron microscope, as shown in fig. 2.
The Control group did not add the peppermint oil nanoemulsion and it can be seen from figure 1: observing the complete shape of the escherichia coli under a scanning electron microscope; the Treated group is Treated by adding the peppermint oil nanoemulsion, and the rupture of the cell membrane of the escherichia coli is serious and a large amount of contents flow out under the scanning electron microscope of figure 2, so that the escherichia coli is dead. The bactericidal effect of the peppermint oil nanoemulsion on escherichia coli is reflected more intuitively.
The fresh undiluted O/W type peppermint oil nanoemulsion prepared in each of the above examples was placed in a sample bottle (22 mm) respectively. The nanoemulsion was scanned every 10 minutes using Tubiscan and 12 h at 25 ℃. Changes in droplet diameter and phase separation in the nanoemulsion were monitored by measuring the increase in backscattering (Delta BS,%) over time at different sample heights and the scanning spectra of the peppermint oil nanoemulsion of FIG. 3 were obtained.
As can be seen from fig. 3: the peppermint oil nanoemulsion was more stable under droplet growth or gravity separation conditions because the Delta BS (%) change in the sample bottle was very small, fluctuating above and below the baseline, with a backscattered light increase Δ BS% <0.2%, indicating that the peppermint oil nanoemulsion was stable.
Claims (7)
1. A preparation method of peppermint oil nano-emulsion is characterized by comprising the following steps: mixing and stirring the peppermint oil and the emulsifier, adding deionized water, and treating the mixed system by a high-energy emulsification method to obtain the peppermint oil nano emulsion; the peppermint oil, the emulsifier and the deionized water respectively account for 5-10%, 4-8% and 82-91% of the total feeding mass.
2. The method of preparing a peppermint oil nanoemulsion according to claim 1, wherein: the high-energy emulsification method is to shear at a rotating speed of 8000-12000 r/min for 3-7 min.
3. The method of preparing a peppermint oil nanoemulsion according to claim 2, wherein: the high-energy emulsification method is to shear for 5min at the rotating speed of 10000 r/min.
4. The method of preparing a peppermint oil nanoemulsion according to claim 3, wherein: the peppermint oil, the emulsifier and the deionized water respectively account for 10%, 8% and 82% of the total feeding mass.
5. The method of claim 4, wherein the peppermint oil nanoemulsion is prepared by: the emulsifier is composed of Tween-60 and EL-20, wherein the Tween-60 accounts for 6% of the total feeding mass, and the EL-20 accounts for 2% of the total feeding mass.
6. The method of preparing a peppermint oil nanoemulsion according to claim 1, wherein: the purity of the charged peppermint oil was 98 wt%.
7. The method of preparing a peppermint oil nanoemulsion according to claim 1, wherein: the emulsifier mainly comprises polyoxyethylene sorbitan monooleate and castor oil polyoxyethylene ether, wherein the polyoxyethylene sorbitan monooleate accounts for 3-6% of the total mass of the emulsifier, and the castor oil polyoxyethylene ether accounts for 1-2% of the total mass of the emulsifier.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113317431A (en) * | 2021-06-11 | 2021-08-31 | 扬州大学 | Oil-in-water carvacrol nanoemulsion and preparation method thereof |
| CN114041503A (en) * | 2021-11-03 | 2022-02-15 | 重庆第二师范学院 | Probiotics activity maintaining method and application of probiotics activity maintaining method in solid lipid food |
| CN114288244A (en) * | 2021-12-09 | 2022-04-08 | 扬州大学 | Zanthoxylum oil nano-emulsion and preparation method and application thereof |
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| CN102511511A (en) * | 2011-12-09 | 2012-06-27 | 江南大学 | Nano-antibacterial emulsion containing mint oil and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102511511A (en) * | 2011-12-09 | 2012-06-27 | 江南大学 | Nano-antibacterial emulsion containing mint oil and preparation method thereof |
Non-Patent Citations (1)
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| QI LIU等: "Preparation of peppermint oil nanoemulsions: Investigation of stability, antibacterial mechanism and apoptosis effects", 《COLLOIDS AND SURFACES B: BIOINTERFACES》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113317431A (en) * | 2021-06-11 | 2021-08-31 | 扬州大学 | Oil-in-water carvacrol nanoemulsion and preparation method thereof |
| CN114041503A (en) * | 2021-11-03 | 2022-02-15 | 重庆第二师范学院 | Probiotics activity maintaining method and application of probiotics activity maintaining method in solid lipid food |
| CN114288244A (en) * | 2021-12-09 | 2022-04-08 | 扬州大学 | Zanthoxylum oil nano-emulsion and preparation method and application thereof |
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Application publication date: 20210601 |