CN118236450A - Compound plant essential oil for inhibiting pathogenic fungi and preparation method thereof - Google Patents
Compound plant essential oil for inhibiting pathogenic fungi and preparation method thereof Download PDFInfo
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- CN118236450A CN118236450A CN202410387303.7A CN202410387303A CN118236450A CN 118236450 A CN118236450 A CN 118236450A CN 202410387303 A CN202410387303 A CN 202410387303A CN 118236450 A CN118236450 A CN 118236450A
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Abstract
The invention relates to a compound plant essential oil for inhibiting pathogenic fungi and a preparation method thereof, wherein the compound plant essential oil is matched and used according to a certain proportion, plays a role in drug effect jointly and continuously, can play a role in killing fungi by destroying cell membrane and cell wall structures or inhibiting respiration and energy metabolism and destroying organelles, and achieves the expected treatment or health care effect. The invention has the highest inhibition rate on the microsporum canis and the microsporum gypsilosis in the microsporum.
Description
Technical Field
The invention relates to the technical field of plant essential oil, in particular to compound plant essential oil for inhibiting pathogenic fungi and a preparation method thereof.
Background
Currently, treatments for dermatomycosis caused by fungi such as microsporum canis include topical and oral antifungal agents, such as itraconazole, ketoconazole, terbinafine, fluconazole, and the like. Itraconazole is an oral medicine for pets, but is easy to cause jaundice, anorexia and the like; ketoconazole can cause vomiting in dogs and cats, cause skin irritation, and can also cause skin atrophy, telangiectasia, pigmentation and secondary infection after long-term use; terbinafine is used as a short-acting drug, is generally well tolerated, but has obvious side effects, including adverse gastrointestinal reactions and cat face itching, and is forbidden for pets in lactation; fluconazole, when used as an antifungal agent, causes vomiting, diarrhea, liver and kidney dysfunction, rash, erythema and skeletal muscle problems such as joint swelling, muscle pain and weakness when used for a long period or at high doses. Several studies have shown that the acquired drug resistance caused by repeated use of these drugs gradually decreases the susceptibility of fungi such as microsporum canis to the drugs, thereby prolonging the course of the disease or exacerbating the disease.
The plant essential oil is a secondary metabolite of plants, and has antifungal, antibacterial, and antiviral effects. Research shows that the plant essential oil single and compound essential oils have stronger antifungal effect. However, the plant essential oil has strong volatility and short duration of efficacy, and the expected control effect is not achieved.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide the compound plant essential oil for inhibiting the pathogenic fungi and the preparation method thereof, wherein the compound plant essential oil is used for effectively inhibiting the pathogenic fungi, has a wide action target point based on the compound plant essential oil, effectively damages the cell wall of the fungi, enables exogenous active ingredients to permeate into the cells of the fungi, and damages organelles to play a role in killing the fungi.
The invention is realized by the following technical scheme: in one aspect, a compound plant essential oil for inhibiting pathogenic fungi is provided, the compound plant essential oil comprises cinnamon essential oil and citronella essential oil, and the cinnamon essential oil is as follows: the mass ratio of the citronella essential oil is 1:1-4, preferably cinnamon essential oil: the mass ratio of the citronella essential oil is 1:2.
According to the technical scheme, the cinnamon essential oil and the citronella essential oil can effectively inhibit the growth of fungi when being mixed at the concentration of 1:2 by taking the partial bacteriostasis concentration index (FICI) as a judgment standard, and the added effect is shown, so that the bacteriostasis activity of the compound essential oil is enhanced.
The cinnamon essential oil has special fragrance and sweet taste, is widely applied to various personal cleaning and nursing products, has the main chemical components of cinnamaldehyde, cinnamyl acetate, salicylaldehyde and the like, has the effects of dispelling wind and strengthening spleen, and has the effects of resisting fungi, relieving fever and pain, relieving itching and the like; the essential oil of citronella contains citronellol, citral, eugenol, zingiberene, etc., and has effects of relieving rigidity of muscles and activating collaterals, sterilizing, diminishing inflammation, relieving vomit, relieving itching, etc. The reasonable collocation of the two essential oils has extremely strong antibacterial and anti-inflammatory effects and itching relieving effects, and can obviously improve skin diseases caused by microsporidia canis and the like.
The compound plant essential oil for inhibiting the pathogenic fungi comprises tea tree essential oil and cinnamon essential oil: citronella essential oil: the mass ratio of the tea tree essential oil is 1 (2-4), namely (2-4); preferably, cinnamon essential oil: citronella essential oil: the mass ratio of the tea tree essential oil is 1:2:2.
According to the technical scheme, the essential oil mixed by cinnamon, citronella and tea tree essential oil can effectively inhibit the growth of fungi under the condition of minimum dosage.
The cinnamon essential oil has special fragrance and sweet taste, is widely applied to various personal cleaning and nursing products, has the main chemical components of cinnamaldehyde, cinnamyl acetate, salicylaldehyde and the like, has the effects of dispelling wind and strengthening spleen, and has the effects of resisting fungi, relieving fever and pain, relieving itching and the like; the essential oil of citronella contains citronellol, citral, eugenol, zingiberene, etc., and has effects of relieving rigidity of muscles and activating collaterals, sterilizing, diminishing inflammation, relieving vomit, relieving itching, etc. The reasonable collocation of the two essential oils has extremely strong antibacterial and anti-inflammatory effects and itching relieving effects, and can obviously improve skin diseases caused by microsporidian canis.
The main components of the tea tree essential oil are terpinen-4-ol and 1,8 eucalyptol, have antibacterial, antiviral and antioxidant effects, have remarkable effects of treating inflammation, diarrhea and traumatic infection, and are the natural antibacterial agent with the strongest activity.
The compound plant essential oil for inhibiting pathogenic fungi is based on the compound plant essential oil for inhibiting pathogenic fungi, and further comprises peppermint essential oil and cinnamon essential oil: citronella essential oil: tea tree essential oil: the mass ratio of the peppermint essential oil is 1 (2-4) to 2 (2-4); preferably, cinnamon essential oil: citronella essential oil: tea tree essential oil: the mass ratio of the mint essential oil is 1:4:2:2.
The cinnamon essential oil has special fragrance and sweet taste, is widely applied to various personal cleaning and nursing products, has the main chemical components of cinnamaldehyde, cinnamyl acetate, salicylaldehyde and the like, has the effects of dispelling wind and strengthening spleen, and has the effects of resisting fungi, relieving fever and pain, relieving itching and the like; the essential oil of citronella contains citronellol, citral, eugenol, zingiberene, etc., and has effects of relieving rigidity of muscles and activating collaterals, sterilizing, diminishing inflammation, relieving vomit, relieving itching, etc. The reasonable collocation of the two essential oils has extremely strong antibacterial and anti-inflammatory effects and itching relieving effects, and can obviously improve skin diseases caused by microsporidian canis.
The main components of the tea tree essential oil are terpinen-4-ol and 1,8 eucalyptol, have antibacterial, antiviral and antioxidant effects, have remarkable effects of treating inflammation, diarrhea and traumatic infection, and are the natural antibacterial agent with the strongest activity.
The mint essential oil has cool smell, can be used as essential oil for both medicine and food, can be used as dishes and flavoring spice, has the effects of resisting bacteria, resisting oxidation, promoting wound healing, promoting hair growth and the like, and can be used for jointly inhibiting the growth of fungi such as microsporidian canis and the like, promoting the regeneration and reconstruction of blood vessels and the angiogenesis of hair follicles in cooperation with the above 3 plant essential oils, and rapidly repairing skin infection and hair loss caused by tinea.
Further comprises a slow release agent which is a pyrrolidone compound.
According to the technical scheme, polyvinylpyrrolidone is used as a carrier of the plant essential oil, the viscosity of the solution is properly increased, the solution is crosslinked with the effective components of the plant essential oil to form a net, and a film is formed after the solvent volatilizes, so that the purpose of delaying the release of the effective components of the plant essential oil is achieved.
Also provided is a method for preparing the compound plant essential oil for inhibiting pathogenic fungi, which comprises the following steps:
Step S1, weighing compound plant essential oil according to the prescription amount;
S2, dissolving the raw materials obtained in the step S1 in a solvent, adding a slow release agent, and uniformly stirring;
step S3: and (3) adding distilled water into the mixture obtained in the step (S2), and uniformly stirring to obtain the water-soluble polymer.
By adopting the technical scheme, the preparation method can effectively dissolve the plant essential oil, has large drug-loading capacity and strong adhesiveness of the matrix, is suitable for the multi-component and large-dose medication characteristics of the traditional Chinese medicine extract, and avoids the problems of quick volatilization of the plant essential oil and the like.
Further, in step S1, the compound plant essential oil includes cinnamon essential oil: citronella essential oil: tea tree essential oil: the mass ratio of the peppermint essential oil to the mint essential oil is 1 (2-4) to 2 (2-4).
Through the technical scheme, different components of the compound plant essential oil play roles in inhibiting fungal growth, and have the positive effects of resisting inflammation, promoting wound healing and the like.
Further, in step S2, the solvent is any one of tristyrylphenol polyoxyethylene ether, dimethyl sulfoxide, and propylene glycol.
By the technical scheme, the biocompatibility and stability of the plant essential oil can be improved, and the irritation to the skin, mucous membrane, focus and other parts is avoided.
Further, in the step S2, the slow release agent is polyvinylpyrrolidone, and the addition amount of the polyvinylpyrrolidone accounts for 3% -12% of the total amount of the plant essential oil solution.
According to the technical scheme, polyvinylpyrrolidone is selected as a carrier for the hydrophobic plant essential oil, so that the adhesive property and the moisture retention property of the hydrophobic plant essential oil to the skin can be improved, the hydrophobic plant essential oil has the advantages of comfort in use, small skin irritation and function of controlling the release of active ingredients.
Further, in step S3, the formulation of the obtained compound plant essential oil is selected from any one of spray, emulsion and paste.
According to the technical scheme, the spray is obtained according to the operation of the step S3, so that the medicine can directly reach the action part, and the spray is convenient to use, accurate in administration dosage, uniform in distribution and quick in effect; or adding dodecylphenol polyoxyethylene ether (OP-10) or tween 80 into the mixture obtained in the step S1, adding purified water, and stirring to obtain emulsion with high dispersion degree of liquid drops, rapid drug effect, absorption promotion, improved permeability to skin and mucous membrane, high bioavailability, and small irritation; or adding polyethylene glycol, lanolin or vaseline into the mixture obtained in the step S1, adding purified water or liquid paraffin, stirring to obtain paste, and accurately administering to target parts such as skin to exert drug effect and reduce injury of the drug to viscera.
Further, the pathogenic fungi are one or more of trichophyton, epidermophyton and microsporophyte.
Through the technical scheme, the compound plant essential oil is found to be capable of effectively inhibiting the microsporidian canis and the microsporidian gypsum sample and the trichophyton mentagrophytes in the trichophyton, and the mechanism is that the plant essential oil active ingredient plays a role in bacteriostasis by destroying the cell membrane and the cell wall structure or inhibiting respiration and energy metabolism.
The invention has the beneficial effects that:
(1) The compound plant essential oil can jointly and continuously exert the drug effect under the condition that each component is less in quantity, thereby inhibiting the growth of fungi.
(2) The compound plant essential oil can play a bacteriostatic role by destroying the cell membrane and the structure of the cell wall or inhibiting respiration and energy metabolism. It has the highest inhibition rate on microsporum canis and microsporum gypsilosis in genus microsporum.
(3) The invention is most suitable for spray, and is convenient to use, accurate in administration dosage, small in administration dosage, uniform in distribution and quick in effect compared with emulsion and paste.
(4) Compared with chemical synthetic bactericides, the compound plant essential oil has the advantages of wide action target point, difficult generation of drug resistance, no residue and the like, has fragrant and pleasant smell, effectively avoids animal stress reaction caused by pungent smell, expands the application range of the plant essential oil, has obvious effect of inhibiting pathogenic fungi, and has wide application prospect in practical application.
Detailed Description
The invention takes the creation and application (231111111600) of a special efficient tie-resistant plant extract product developed in the key point of the Henan province of 2023 as support, and aims to select plant essential oil without toxic and side effects and drug resistance.
The present invention will be described in further detail with reference to specific embodiments in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions or under conditions recommended by the manufacturer. All percentages, ratios, proportions or parts are by weight unless otherwise indicated. The reagents and starting materials used in the examples and comparative examples of the present invention were commercially available unless otherwise specified.
Example 1
A preparation method of compound plant essential oil for inhibiting pathogenic fungi comprises the following specific preparation steps of:
step S1, at room temperature, according to cinnamon essential oil: the mass ratio of the citronella essential oil is 1:1-4, and the cinnamon essential oil and the citronella essential oil are accurately weighed;
Step S2, dissolving the cinnamon essential oil and the citronella essential oil obtained in the step S1 in a solvent, adding polyvinylpyrrolidone (PVP-K30), and uniformly stirring to obtain a mixture, wherein the solvent is preferably tristyrylphenol polyoxyethylene ether, dimethyl sulfoxide or propylene glycol;
step S3: and (3) adding distilled water into the mixture obtained in the step (S2), and uniformly stirring to obtain the compound plant essential oil.
Test example 1
(1) Antibacterial activity of single plant essential oil on fungi
Fungus is inoculated in a liquid culture medium (SDB) to prepare a fungus suspension, and the concentration of the fungus suspension is adjusted to be 10 5～106 CFU/mL. Diluting the essential oil solution by a double dilution method, quantitatively adding the diluted essential oil solution into a glucose agar culture medium (SDA) with the temperature of 45-55 ℃, cooling and solidifying the culture medium, uniformly coating the bacterial liquid on the culture medium, and placing the culture medium in a culture box with the temperature of 28 ℃ for 7d of incubation. Minimum Inhibitory Concentrations (MIC) of plant essential oils against microsporum canis and the like were observed and recorded.
TABLE 1 bacteriostatic Activity of Single plant essential oils against fungi
As can be seen from Table 1, both cinnamon essential oil and citronella essential oil are effective in inhibiting the growth of fungi. Wherein, the effect of the low-concentration cinnamon essential oil is more obvious.
(2) Cinnamon and citronella essential oil combined in vitro bacteriostasis test
The cinnamon essential oil and the citronella essential oil are respectively diluted to be 1/2MIC and 1/4MIC in a double way, and the solution is added into the ELISA plate by utilizing an orthogonal test design, and finally each hole contains 100 mu L of mixed essential oil and 100 mu L of 3 multiplied by 10 5 CFU/mL of bacterial suspension. The negative control was 100. Mu.L of the essential oil dilution and 100. Mu.LSDB, and the positive control was 200. Mu.L of the bacterial suspension. Culturing in a constant temperature incubator at 28deg.C for 7d. Each group was repeated 3 times. The results were recorded as MIC values for the formulated essential oils in terms of sterile growth.
The interaction of different compounds can produce 4 effects: synergistic, additive, independent, antagonistic. The experiment uses a part of bacteriostasis concentration index (FICI) as a judging basis of the effect of the essential oil combined antibacterial experiment. If FICI is less than or equal to 0.5, the compounded essential oil shows a synergistic effect; FICI is more than 0.5 and less than or equal to 1 and is added; FICI is more than 1 and less than or equal to 2 and has no relation effect; FICI > 2 is antagonism. The calculation formula is as follows:
MIC of MIC/a drug alone when combined with mic+b drug alone when combined with MIC/B drug alone when combined with fici=a
Table 2 antibacterial effect of compound plant essential oil on fungi
Note that: "+" indicates that the medium has colonies formed, and "-" indicates that the medium has no colonies formed.
As can be seen from table 2, cinnamon essential oil compared to the control group: when the quality ratio of the citronella essential oil is 1:1-1:4, no colony growth is seen on the culture medium. Cinnamon essential oil according to FICI decision criteria: the citronella essential oil has the additive effect when the mass ratio is 1:1 and 1:2, wherein the bacteriostatic effect is better when the mass ratio of the citronella essential oil and the citronella essential oil is 1:2.
Test example 2
(1) Screening of long-acting compound plant essential oil slow-release substances
Under the condition of room temperature, 1% -20% of PVP-K30, ethyl Cellulose (EC) and dextrin are respectively added into the compound plant essential oil obtained in the test example 1, uniformly stirred, uniformly mixed, and transparency, whether precipitation and layering phenomena exist or not are observed.
Table 3 observation of long-acting compound plant essential oil sustained-release substance properties
| Sustained release component | Transparency of the film | Precipitation | Layering |
| PVP-K30 | Clarifying | Without any means for | Without any means for |
| EC | Clarifying | Has the following components | Has the following components |
| Dextrin | Clarifying | Has the following components | Without any means for |
As shown in Table 3, the compound essential oil added with PVP-K30 has no precipitation or layering phenomenon and the solution is transparent, and the next test can be performed.
(2) Test for inhibiting fungi by long-acting compound plant essential oil
Quantitatively adding the long-acting compound plant essential oil in the step (1) into SDA at 45-50 ℃, coating fungus suspension in the step (1) of inoculation test example after the culture medium is cooled and solidified, setting a blank control group and an essential oil group without adding slow release substances, placing the blank control group and the essential oil group into an incubator for incubation for 15d, observing fungus growth conditions, and determining the concentration range of the slow release substances.
Table 4 antibacterial activity of long-acting compound plant essential oil
Note that: "+" indicates that the medium has colonies, "++" indicates that there are more colonies in the medium, "++" indicates that the medium is almost full of colonies, and "-" indicates that no colonies are present in the medium.
According to the measurement, compared with the compound plant essential oil without the slow-release substance, the compound plant essential oil with 1% -15% of PVP-K30 can effectively inhibit the growth of fungi, and the time for inhibiting the growth of fungi is prolonged by delaying the release of active ingredients of the essential oil.
(3) Compound plant essential oil stability test
According to cinnamon essential oil: the mass ratio of the citronella essential oil is 1:2, the cinnamon essential oil and the citronella essential oil are accurately weighed, uniformly mixed, added with a solvent and a slow release agent to prepare the essential oil spray, placed for 30d at 25 ℃ and 40 ℃ and minus 20 ℃, and whether the essential oil solution is layered is observed.
The results show that the prepared compound plant essential oil has no layering phenomenon.
Test example 3
(1) Compound plant essential oil skin sensitivity test
The skin sensibility test is carried out by selecting rats with the weight of 80-100 g, the compound plant essential oil determined in the test example 2 (3) is filled into a spray bottle and is respectively sprayed on the face, the ears, the limbs, the trunk and other parts of the rats, and the optimal concentration range of the compound essential oil is screened by combining with the clinical existing products.
Through observation, no abnormal symptoms are found at the spraying position of the compound plant essential oil on the rats.
(2) Determination of clinical effects in laboratory animals
And selecting adult rabbits with weight of 2-3 kg to carry out fungus skin infection test, carrying out drug treatment by using the compound plant essential oil, and judging the effect of the compound plant essential oil according to animal clinical manifestations.
Through observation, the compound plant essential oil has the effects of relieving and alleviating skin redness and swelling and inflammation caused by fungal infection, effectively prevents and treats fungal dermatophytosis, and is harmless to animals.
Therefore, the compound plant essential oil composed of cinnamon essential oil and citronella essential oil in a mass ratio of 1:2 has good inhibition effect on fungi of animals and in vitro.
Example 2
The compound plant essential oil for inhibiting pathogenic fungi comprises cinnamon essential oil, citronella essential oil and tea tree essential oil, and the preparation method comprises the following specific steps:
step S1, at room temperature, according to cinnamon essential oil: citronella essential oil: the mass ratio of the tea tree essential oil is 1 (2-4), namely (2-4), the cinnamon essential oil, the citronella essential oil and the tea tree essential oil are accurately weighed;
step S2, dissolving the cinnamon essential oil, the citronella essential oil and the tea tree essential oil obtained in the step S1 in a solvent, adding polyvinylpyrrolidone, and uniformly stirring to obtain a mixture, wherein the solvent is preferably tristyrylphenol polyoxyethylene ether, dimethyl sulfoxide or propylene glycol;
step S3: and (3) adding distilled water into the mixture obtained in the step (S2), and uniformly stirring to obtain the compound plant essential oil.
Test example 1
(1) Antibacterial activity of single plant essential oil on fungi
MIC values of tea essential oil for fungi were determined as in example 1, test example 1 (1).
The MIC values of the tea tree essential oil for microsporum canis, microsporum gypseum and trichophyton mentagrophytes were determined to be 25 mug/mL.
(2) Determination of in-vitro antibacterial activity of cinnamon, citronella and tea tree compound essential oil
The inhibition effect of the compound essential oil on fungi was measured by the method of example 1, test example 1 (2).
Table 5 antibacterial effect of Compound plant essential oil on fungi
| Treatment group | Cinnamon essential oil | Citronella essential oil | Tea tree essential oil | Colony growth |
| Bacterial suspension | 0 | 0 | 0 | + |
| 1 | 3.12 | 6.25 | 6.25 | – |
| 2 | 3.12 | 6.25 | 12.5 | – |
| 3 | 3.12 | 12.5 | 12.5 | – |
Note that: "+" indicates that the medium has colonies formed, and "-" indicates that the medium has no colonies formed.
From Table 5, it is clear that the cinnamon essential oil, citronella essential oil and tea tree essential oil are mixed in a mass ratio of 1:2:2, 1:2:4 and 1:4:4, which can effectively inhibit the growth of fungi.
Test example 2
(1) Test for inhibiting fungi by long-acting compound plant essential oil
Quantitatively adding 1% -15% PVP-K30-added compound plant essential oil into SDA at 45-50 ℃, coating fungus suspension in inoculation test example 1 (1) after the culture medium is cooled and solidified, setting a blank control group and an essential oil group without adding slow release substances, placing the blank control group and the essential oil group into an incubator for incubation for 15d, observing fungus growth conditions, and determining the concentration range of the slow release substances.
Table 6 antibacterial activity of long-acting compound plant essential oil
Note that: "+" indicates that the medium has colonies, "++" indicates that there are more colonies in the medium, "++" indicates that the medium is almost full of colonies, and "-" indicates that no colonies are present in the medium.
According to the measurement, compared with the compound plant essential oil without adding the slow-release substance, the compound plant essential oil with 3-15% of PVP-K30 can effectively prolong the growth time of fungi inhibition.
(2) Compound plant essential oil stability test
Accurately weighing the essential oils according to the mass ratio of the cinnamon essential oil to the citronella essential oil to the tea tree essential oil of 1:2:2, 1:2:4 and 1:4:4, uniformly mixing, adding a solvent and a slow release agent to prepare an essential oil spray, standing for 30d at 25 ℃, 40 ℃ and-20 ℃, and observing whether the essential oil solution is layered.
The results show that the prepared compound plant essential oil has no layering phenomenon.
Test example 3 safety and skin sensitivity test of Compound plant essential oil
The compound plant essential oil determined in test example 1 (3) is prepared into solutions with different concentrations by selecting rats with weight of 80-100 g, and is filled into a spray bottle to be sprayed on the face, ears, limbs, trunk and other parts of the rats respectively, and the safety of the compound essential oil is verified by combining with the clinical existing products.
Through observation, no abnormal symptoms are found at the spraying position of the compound plant essential oil on the rats.
Test example 4 determination of clinical effects in laboratory animals
Adult dogs are selected for fungus skin infection, safety and fungus inhibition effect tests, compound plant essential oil with different concentrations is used for drug treatment, and the effectiveness of the compound plant essential oil is judged according to animal clinical manifestations and recovery conditions of infected parts.
According to the measurement, the essential oil spray prepared by mixing cinnamon, citronella and tea tree essential oil in a mass ratio of 1:2:2 has no stimulation to experimental animals, and the animals have no phenomenon of evasion, so that the compound plant essential oil can effectively prevent and treat skin diseases caused by fungal infection.
And as a result of the comprehensive test, the mass ratio of the three components is 1:2:2, the dosage of the essential oil is minimum, and the control effect on the fungal growth and the fungal infection of animals can be exerted.
Example 3
The compound plant essential oil for inhibiting pathogenic fungi also comprises cinnamon essential oil, citronella essential oil, tea tree essential oil and peppermint essential oil, and the preparation method comprises the following steps:
step S1, at room temperature, according to cinnamon essential oil: citronella essential oil: tea tree essential oil: the mass ratio of the mint essential oil is 1 (2-4), 2-4, 1-4, and the cinnamon essential oil, the citronella essential oil, the tea tree essential oil and the mint essential oil are accurately weighed;
Step S2, dissolving the cinnamon essential oil, the citronella essential oil, the tea tree essential oil and the peppermint essential oil obtained in the step S1 in a solvent, adding polyvinylpyrrolidone, and uniformly stirring to obtain a mixture, wherein the solvent is preferably tristyrylphenol polyoxyethylene ether or dimethyl sulfoxide;
step S3: and (3) adding distilled water into the mixture obtained in the step (S2), and uniformly stirring to obtain the compound plant essential oil.
Test example 1
(1) Determination of fungus growth inhibition effect of single plant essential oil
Fungus is selected and inoculated on SDB to prepare fungus suspension, and the concentration of the fungus suspension is adjusted to be 10 5～106 CFU/mL. Diluting the essential oil solution by a double dilution method, quantitatively adding the diluted essential oil solution into SDA at 50 ℃, after the culture medium is cooled and solidified, absorbing the bacterial liquid, uniformly coating the bacterial liquid on the culture medium, and placing the culture medium in a constant temperature incubator at 28 ℃ for incubation for 7d. MIC of plant essential oil to fungi such as microsporidian canis was observed and recorded.
TABLE 7 antibacterial Activity of unilateral plant essential oils
(2) Determination of antibacterial activity of compound plant essential oil on fungi
The single plant essential oils in Table 7 were respectively twice diluted to 1/2MIC, 1/4MIC, 1/8MIC, mixed by orthogonal design method, and mixed with bacterial suspension at a volume ratio of 1:1. And determining the optimal concentration range of the compound essential oil according to the growth condition of fungi. The formula and antibacterial activity of the compound plant essential oil are shown in table 8.
Table 8 Compound plant essential oil composition and antibacterial activity
Note that: "+" indicates that the medium has colonies formed, and "-" indicates that the medium has no colonies formed.
The results show that the compositions 1,2 and 4 can effectively inhibit the growth of fungi such as microsporidia canium.
(3) Animal skin sensitivity test
And (3) selecting rats with 6-8 weeks of age for skin sensitivity test, applying the compound plant essential oil determined in the step (2) to the rats, and screening the optimal concentration range of the compound essential oil according to the clinical existing products.
In conclusion, the compound plant essential oil is prepared from cinnamon essential oil, citronella essential oil, tea tree essential oil and peppermint essential oil according to the mass ratio of 1 (2-4) to 2 (2-4) through experiments (1), (2) and (3), can inhibit the growth of fungi, and is harmless to animals.
Test example 2
(1) Test for inhibiting fungi by long-acting compound plant essential oil
At room temperature, quantitatively adding 3% -15% PVP-K30-added compound plant essential oil into SDA at 45-50 ℃, coating fungus suspension in inoculation test example 1 (1) after a culture medium is cooled and solidified, setting a blank control group and an essential oil group without adding slow release substances, placing the blank control group and the essential oil group into an incubator for incubation for 15d, observing fungus growth conditions, and determining the concentration range of the slow release substances.
Table 9 Long-acting compound plant essential oil antibacterial activity
Note that: "+" indicates that the medium has colonies, "++" indicates that there are more colonies in the medium, "++" indicates that the medium is almost full of colonies, and "-" indicates that no colonies are present in the medium.
According to the measurement, compared with the compound plant essential oil without the slow-release substance, the compound plant essential oil with 3-12% PVP-K30 can effectively inhibit the growth of fungi, and the time for inhibiting the growth of fungi is prolonged by delaying the release of active ingredients of the essential oil for about 3-5 days.
(3) Compound plant essential oil stability test
According to the mass ratio of cinnamon essential oil, citronella essential oil and tea tree essential oil of 1 (2-4) to 2 (2-4), accurately weighing the essential oils, uniformly mixing, adding a solvent and a sustained release substance to prepare an essential oil spray, standing for 30d at 25 ℃, 40 ℃ and-20 ℃, and observing whether the essential oil solution is layered.
The results show that the prepared compound plant essential oil has no layering phenomenon.
(4) Long-acting compound plant essential oil animal skin sensitivity test
Selecting rats with weight of 80-100 g for skin sensitivity test, preparing the compound plant essential oil determined in the step (1) into spray respectively spraying on the face, ears, limbs, trunk and other parts of the rats, and screening the optimal concentration range of the compound essential oil by combining with the clinical existing products.
Through observation, rats added with PVP-K30 compound plant essential oil have no abnormal symptoms.
Experimental example 3 determination of clinical effects of experimental animals
The method is characterized in that adult rabbits with the weight of 2.5-3 kg are selected for fungus skin infection test, compound plant essential oils with different concentrations are used for drug treatment, and the safety of the compound plant essential oils is judged according to animal clinical manifestations and the like.
The results show that the compound plant essential oil with the composition of the cinnamon essential oil, the citronella essential oil and the tea tree essential oil and the mint essential oil with the mass ratio of 1 (2-4) to 2-4) can effectively prevent and treat fungal dermatophytosis and is harmless to animals.
Test example 4: antifungal clinical application of compound plant essential oil
According to the mass ratio of cinnamon essential oil, citronella essential oil and tea tree essential oil to mint essential oil of 1 (2-4) to 2 (2-4), accurately weighing each essential oil, dissolving in a solvent to prepare an essential oil spray, and filling into a 100-500 mL spray bottle. The dogs and cats with different degrees of dermatophytosis (manifested by symptoms of dehairing, dander, red swelling, pimple, etc.) were treated. Spraying the medicine to the focus part for 2-3 times daily for 7-10 days.
The compound plant essential oil spray has good treatment effect on animals suffering from dermatophytosis. The plant essential oil spray is used for 3-7 days, so that the scratching phenomenon of animals is reduced, and the itching symptom is improved; skin redness and swelling, depilation and desquamation are reduced or eliminated; the hair removal site grows fine hair gradually as the treatment progresses. The medicine is taken for 10 days, 40 animals are completely cured, and the cure rate is 80%; 10 symptoms are reduced or obviously improved, and the effective protection rate is 100%.
Thus, it is evident from the comparative analysis of test example 1-test example 4 that the essential oil spray prepared by dissolving cinnamon essential oil, citronella essential oil, tea tree essential oil and peppermint essential oil in a solvent in a mass ratio of 1 (2-4): 2 in this example has a good antifungal effect.
Example 4
A compound plant essential oil for inhibiting pathogenic fungi comprises cortex Cinnamomi essential oil, citronella essential oil, tea tree essential oil, and herba Menthae essential oil; the preparation method comprises the following specific steps:
step S1, accurately weighing cinnamon essential oil, citronella essential oil, tea tree essential oil and peppermint essential oil according to table 10 at room temperature;
Step S2, according to the table 10, dissolving cinnamon essential oil, citronella essential oil, tea tree essential oil and peppermint essential oil obtained in the step S1 in a solvent, adding PVP-K30, and uniformly stirring to obtain a mixture;
step S3: and (3) adding distilled water into the mixture obtained in the step (S2), and uniformly stirring to obtain the compound plant essential oil.
Table 10 additive amount (mg) of each substance in the compound plant essential oil solution
Test example 1 determination of antibacterial Activity of Compound plant essential oil
Fungus colonies are picked and inoculated to SDB to prepare fungus suspension, and the concentration of the fungus suspension is adjusted to 10 5～106 CFU/mL. Dripping essential oil into SDA at 50deg.C, cooling and solidifying the culture medium, absorbing 100 μl of bacterial liquid, uniformly coating on the culture medium, and incubating in a constant temperature incubator at 28deg.C for 10d. Fungal growth was observed and recorded.
Table 11 inhibition effect of Compound plant essential oil on fungi
Note that: "+" indicates that the medium has colonies formed, and "-" indicates that the medium has no colonies formed.
From Table 11, it can be seen that the compound essential oil composed of cinnamon essential oil, citronella essential oil, tea tree essential oil and peppermint essential oil in a mass ratio of 1:4:2:2 can effectively inhibit the growth of fungi.
Test example 2: antifungal clinical application of compound plant essential oil
The plant essential oil solution prepared in test example 1 was filled into spray bottles. The liquid spray is used for spraying dogs and cats which are 15 cases and have been diagnosed with fungal dermatophytosis for 7-10 days in 3-10 months, and the improvement of the affected parts of animals is observed and recorded.
The results show that: 10 sick animals are all cured, and the cure rate is 100 percent. The compound plant essential oil has good effect on treating fungal skin diseases.
Therefore, the compound plant essential oil composed of cinnamon essential oil, citronella essential oil, tea tree essential oil and peppermint essential oil in a mass ratio of 1:4:2:2 can effectively inhibit the growth of fungi, and has good control effect on diseased animals.
Example 5
A compound plant essential oil for inhibiting pathogenic fungi comprises cortex Cinnamomi essential oil, citronella essential oil, tea tree essential oil, and herba Menthae essential oil; the preparation method comprises the following specific steps:
Step S1, accurately weighing cinnamon essential oil, citronella essential oil, tea tree essential oil and peppermint essential oil according to table 12 at room temperature;
Step S2, according to the table 12, dissolving cinnamon essential oil, citronella essential oil, tea tree essential oil and peppermint essential oil obtained in the step S1 in a solvent, adding PVP-K30, and uniformly stirring to obtain a mixture;
step S3: and (3) adding distilled water into the mixture obtained in the step (S2), and uniformly stirring to obtain the compound plant essential oil.
Table 12 additive amount (mg) of each substance in the compound plant essential oil solution
Test example 1 determination of antibacterial Activity of Compound plant essential oil
Fungus colonies are picked and inoculated to SDB, fungus suspension is prepared by shaking culture, and the concentration of the fungus suspension is adjusted to 10 5～106 CFU/mL. Dripping essential oil into SDA, mixing, absorbing 100 μl of bacterial liquid after the culture medium is solidified, spreading on the culture medium, and incubating in a constant temperature incubator at 28deg.C for 10d. Fungal growth was observed and recorded.
Table 13 inhibition of fungi by compound plant essential oils
Note that: "+" indicates that the medium has colonies formed, and "-" indicates that the medium has no colonies formed.
From Table 13, it is clear that the compound plant essential oil composed of cinnamon essential oil, citronella essential oil, tea tree essential oil and peppermint essential oil in a mass ratio of 1:2:2:2 can effectively inhibit the growth of Microsporum gypseum and Trichophyton mentagrophytes; the inhibition effect on microsporum canis is relatively weak.
Experimental example 2 determination of clinical effects of experimental animals
And selecting an adult rabbit with a weight of 2-3 kg to perform a fungus skin infection test, and when the skin of the adult rabbit is red and swollen, hair is lost or scratched, and the like, performing intervention by using the compound plant essential oil of the test example 1, and judging the effect of the compound plant essential oil according to the clinical manifestation of animals.
The compound plant essential oil is sprayed for 7-10 days, so that the skin redness and swelling of adult rabbits disappear, the itching symptom is fully improved, the hair falling part slowly grows out, desquamation is not seen, and other stress reactions are avoided.
Therefore, the compound plant essential oil composed of cinnamon essential oil, citronella essential oil, tea tree essential oil and peppermint essential oil in a mass ratio of 1:2:2:2 has good inhibition effect on fungi in vitro and animals.
Example 6
A compound plant essential oil for inhibiting pathogenic fungi comprises cortex Cinnamomi essential oil, citronella essential oil, tea tree essential oil, and herba Menthae essential oil; the preparation method comprises the following specific steps:
step S1, accurately weighing cinnamon essential oil, citronella essential oil, tea tree essential oil and peppermint essential oil according to table 14 at room temperature;
Step S2, according to the table 14, dissolving cinnamon essential oil, citronella essential oil, tea tree essential oil and peppermint essential oil obtained in the step S1 in a solvent, adding PVP-K30, and uniformly stirring to obtain a mixture;
step S3: and (3) adding distilled water into the mixture obtained in the step (S2), and uniformly stirring to obtain the compound plant essential oil.
Table 14 additive amount (mg) of each substance in the compound plant essential oil solution
Test example 1 determination of antibacterial Activity of Compound plant essential oil
Fungus colony is inoculated on SDB for enrichment culture, and the concentration of fungus liquid is adjusted to 10 5～106 CFU/mL. Mixing essential oil with SDA at proper temperature, absorbing 100 μl of bacterial liquid after the culture medium is solidified, uniformly coating on the culture medium, and incubating in a constant temperature incubator at 28deg.C for 10d. The effect of plant essential oils on fungal growth was observed and recorded.
Table 15 inhibition activity of compound plant essential oil against fungi
Note that: "+" indicates that the medium has colonies formed, and "-" indicates that the medium has no colonies formed.
From Table 15, it is known that the compound plant essential oil composed of cinnamon essential oil, citronella essential oil, tea tree essential oil and peppermint essential oil in a mass ratio of 1:4:4:2 can effectively inhibit the growth of microsporidian canis and microsporidian gypsum; has weak inhibition effect on trichophyton mentagrophytes.
Experimental example 2 determination of clinical effects of experimental animals
And (3) selecting an adult rabbit with a weight of 2-3 kg for a fungus skin infection test, spraying a lesion part when the adult rabbit skin is red and swollen, hair is lost, desquamation or scratching is performed, and judging the antibacterial effect of the compound plant essential oil according to the animal clinical manifestation.
Through observation, the compound plant essential oil is sprayed for 7-10 days, the animal has no scratch, the skin is red, swollen and disappeared, and the hair and the desquamation are obviously improved.
Therefore, the compound plant essential oil composed of cinnamon essential oil, citronella essential oil, tea tree essential oil and peppermint essential oil in a mass ratio of 1:4:4:2 has good inhibition effect on fungi in vitro and animals.
In conclusion, the cinnamon essential oil, the citronella essential oil, the tea tree essential oil and the mint essential oil are selected to be matched according to a certain proportion for use, and the slow-release substance is added, so that the cinnamon essential oil has a good antifungal effect.
Finally, it should be noted that: the foregoing description is only illustrative of the preferred embodiments of the present invention, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described, or equivalents may be substituted for elements thereof, and any modifications, equivalents, improvements or changes may be made without departing from the spirit and principles of the present invention.
Claims (9)
1. The compound plant essential oil for inhibiting pathogenic fungi is characterized by comprising cinnamon essential oil and citronella essential oil, wherein the cinnamon essential oil is as follows: the mass ratio of the citronella essential oil is 1:1-4.
2. A compound plant essential oil for inhibiting pathogenic fungi, which is characterized by comprising tea tree essential oil and cinnamon essential oil based on the compound plant essential oil for inhibiting pathogenic fungi according to claim 1: citronella essential oil: the mass ratio of the tea tree essential oil is 1 (2-4) to 2-4.
3. A compound plant essential oil for inhibiting pathogenic fungi, which is characterized by further comprising peppermint essential oil and cinnamon essential oil based on the compound plant essential oil for inhibiting pathogenic fungi according to claim 2: citronella essential oil: tea tree essential oil: the mass ratio of the peppermint essential oil is 1 (2-4) to 2.
4. The compound plant essential oil for inhibiting pathogenic fungi according to any of claims 1-3, further comprising a slow release agent, wherein the slow release agent is a pyrrolidone compound.
5. A method for preparing the compound plant essential oil for inhibiting pathogenic fungi according to claim 4, which is characterized by comprising the following steps:
Step S1, weighing compound plant essential oil according to the prescription amount;
S2, dissolving the raw materials obtained in the step S1 in a solvent, adding a slow release agent, and uniformly stirring;
step S3: and (3) adding distilled water into the mixture obtained in the step (S2), and uniformly stirring to obtain the water-soluble polymer.
6. The method for preparing a compound plant essential oil for inhibiting pathogenic fungi according to claim 5, wherein in the step S2, the solvent is any one of tristyrylphenol polyoxyethylene ether, dimethyl sulfoxide and propylene glycol.
7. The method for preparing compound plant essential oil for inhibiting pathogenic fungi according to claim 5, wherein in the step S2, the slow release agent is polyvinylpyrrolidone, and the addition amount of polyvinylpyrrolidone is 3% -12% of the total amount of the plant essential oil solution.
8. The method of preparing a compound plant essential oil for inhibiting a pathogenic fungus according to claim 5, wherein in step S3, the formulation of the obtained compound plant essential oil is selected from any one of a spray, an emulsion and a paste.
9. A compound plant essential oil for inhibiting pathogenic fungi according to any of claims 1-3, wherein the pathogenic fungi is one or more of trichophyton, epidermophyton and microsporophyte.
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| Publication Number | Publication Date |
|---|---|
| CN118236450A true CN118236450A (en) | 2024-06-25 |
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