CN117379371A - Mugwort leaf volatile oil microemulsion gel and preparation method and application thereof - Google Patents

Mugwort leaf volatile oil microemulsion gel and preparation method and application thereof Download PDF

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CN117379371A
CN117379371A CN202310469922.6A CN202310469922A CN117379371A CN 117379371 A CN117379371 A CN 117379371A CN 202310469922 A CN202310469922 A CN 202310469922A CN 117379371 A CN117379371 A CN 117379371A
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volatile oil
mugwort leaf
leaf volatile
oil microemulsion
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武香香
朱鑫
江梦园
苗明三
曾华辉
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Henan University of Traditional Chinese Medicine HUTCM
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Henan University of Traditional Chinese Medicine HUTCM
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Abstract

The invention provides a mugwort leaf volatile oil microemulsion gel and a preparation method and application thereof, belonging to the technical field of medicine preparation. Comprising the following steps: the mugwort leaf volatile oil, a surfactant, medium chain triglyceride and water. The invention successfully induces a model of atopic dermatitis, and the percutaneous administration of the mugwort leaf volatile oil and the volatile oil microemulsion gel reveals that the mugwort leaf volatile oil microemulsion gel can effectively treat the atopic dermatitis, has better treatment effect than the mugwort leaf volatile oil and has no toxic or side effect in percutaneous administration through observing the weight, the back skin damage condition, the scratching frequency, the ear swelling degree, the organ coefficient, the immunological serum index, the back skin damage mRNA expression and the biochemical index of a mouse.

Description

Mugwort leaf volatile oil microemulsion gel and preparation method and application thereof
Technical Field
The invention relates to the technical field of medicine preparation, in particular to a mugwort leaf volatile oil microemulsion gel, and a preparation method and application thereof.
Background
The mugwort leaf is a dry leaf of Artemisia argyi Levl. Etvant of Compositae, and is one of the traditional Chinese medicines in China. The Chinese medicinal composition is often used in daily life, such as 'food moxa cake, drink moxa wine, wash Ai Zao'. Mugwort leaf is used as a medicine and first appears in Ming Yi Bie Lu of Liang Daitao Hongjing, and has the effects of being pungent and bitter in nature, warm, having little toxicity, entering spleen, liver and kidney meridians, warming meridians to stop bleeding, dispelling cold to stop pain, relieving cough and asthma, killing parasites and relieving itching. Ancient books "book of drawing and menstruation Ben Cao" describes mugwort leaves, and "… … decoction for external washing can treat damp sore, scabies, dispel dampness and relieve itching". The open filings of Ben Cao gang mu (compendium of materia Medica) recorded: moxa leaves are used as medicines, and moxa leaves can be used for moxibustion for all diseases. The "Yao Xian" in the cloud "goes into three yin to dispel cold and regulate blood, stop pain and regulate menstruation. Traditional medicine considers that the mugwort leaf can treat diseases such as deficiency-cold bleeding, lower abdomen crymodynia, infertility due to cold womb, cold stagnation channels and collaterals and the like, and can treat skin itch by external application. It is indicated for internal channels of Huang Di, typhoid treatises and jin Kui Yao LQ.
Mugwort leaf contains various bioactive components such as volatile oil, steroid terpenes, organic acid, flavonoid, polysaccharide, etc. The mugwort leaf volatile oil (Artemisia argyiessentialoils, AAEO) is a main effective component group of mugwort leaf, has the effects of resisting inflammation, relieving itching, resisting oxidation, inhibiting bacteria, enhancing organism immunity and the like, and has important development and utilization values. In recent years, more than 200 kinds of chemical components including monoterpenes and derivatives thereof, sesquiterpenes and derivatives thereof, and small amounts of aldehyde, ketone and phenolic compounds are detected by using a gas chromatography-mass spectrometry (GC-MS) technology. The monoterpene compounds are main components of the volatile oil of the mugwort leaf. Wherein the 3 compounds of eucalyptol, camphor and borneol are effective components of the mugwort leaf volatile oil which has anti-inflammatory effect.
Atopic Dermatitis (AD) is a chronic, recurrent, itchy and inflammatory skin disease. 80% of the patient families have a history of atopy, asthma, allergic rhinitis, etc. Worldwide, the incidence of AD has been on the rise and severely affects the quality of life of the patient, and creates a serious socioeconomic burden. The disease course of AD is chronic and continuous, the morbidity is higher, and no specific medicine is available, so that the disease course of AD becomes a clinical research hot spot, and the long-term management and relapse reduction are the key of AD treatment.
The exact pathogenesis of atopic dermatitis is currently unknown. It is believed that the action of allergens and microorganisms (mainly staphylococcus aureus and malassezia) on genetically susceptible individuals causes skin inflammation and immune dysfunction, leading to eczematoid skin and itching. Excessive scratching, improper washing, etc. in the development of the disease can in turn induce or aggravate skin inflammation. The main treatment for AD is topical administration of glucocorticoids or calcineurin inhibitors, but in some particularly severe cases immunosuppressants and uv radiation therapy are used. It is prone to telangiectasia, skin atrophy or complications such as pigmentation, secondary skin infection after long-term use, and has a high risk of recurrence after cessation of treatment. With the development of modern medical research, the treatment modes of atopic dermatitis are more and more, the curative effect is better and better, but the disease is still easy to recur, and all treatment modes have known toxic and side effects, particularly considering the influence of the physical and psychological development of teenagers and children, which are severely limited. It is critical to find effective and safe treatments to improve the clinical symptoms and reduce relapse in AD patients.
Disclosure of Invention
In order to solve the problems, the invention provides the mugwort leaf volatile oil microemulsion gel, and the preparation method and application thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides mugwort leaf volatile oil microemulsion, which comprises the following components in parts by weight:
1.5 to 1.7 parts of mugwort leaf volatile oil, 1.15 to 1.25 parts of surfactant, 0.15 to 0.25 part of medium chain triglyceride and 46.8 to 47.2 parts of water.
Preferably, the composition comprises the following components in parts by weight:
1.7 parts of mugwort leaf volatile oil, 1.15 parts of surfactant, 0.15 part of medium chain triglyceride and 47 parts of water.
Preferably, the surfactant comprises tween-80.
The invention also provides a preparation method of the mugwort leaf volatile oil, which comprises the following steps:
mixing the mugwort leaf volatile oil, the surfactant and the medium chain triglyceride to obtain a mixture;
mixing the obtained mixture with water, and emulsifying to obtain folium Artemisiae Argyi volatile oil microemulsion;
the temperature of the emulsification is 28-32 ℃.
Preferably, the rotational speed of the emulsification is 450-550 rpm.
The invention also provides the mugwort leaf volatile oil microemulsion gel, which comprises mugwort leaf volatile oil microemulsion and carbomer 940 solution; the mass ratio of the mugwort leaf volatile oil microemulsion to the carbomer 940 solution is 4:1;
The mass percentage of carbomer 940 in the carbomer 940 solution is 2%.
The invention also provides a preparation method of the mugwort leaf volatile oil microemulsion gel, which is characterized in that the mugwort leaf volatile oil microemulsion and carbomer 940 solution are mixed to obtain the mugwort leaf volatile oil microemulsion gel.
The invention also provides the mugwort leaf volatile oil microemulsion or the application of the mugwort leaf volatile oil microemulsion gel in preparing the medicine for treating atopic dermatitis.
The invention also provides the mugwort leaf volatile oil microemulsion or the application of the mugwort leaf volatile oil microemulsion gel in preparing medicaments for treating skin lesions.
The invention also provides the mugwort leaf volatile oil microemulsion or the application of the mugwort leaf volatile oil microemulsion gel in preparing medicines for treating ear swelling.
The beneficial effects of the invention are as follows:
the invention successfully induces a model of atopic dermatitis, and the percutaneous administration of the mugwort leaf volatile oil and the volatile oil microemulsion gel reveals that the mugwort leaf volatile oil microemulsion gel can effectively treat the atopic dermatitis, has better treatment effect than the mugwort leaf volatile oil and has no toxic or side effect in percutaneous administration through observing the weight, the back skin damage condition, the scratching frequency, the ear swelling degree, the organ coefficient, the immunological serum index, the back skin damage mRNA expression and the biochemical index of a mouse.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 shows the appearance of a mugwort leaf volatile oil microemulsion and gel;
in fig. 2, a is the appearance B of the microemulsion before centrifugation, and B is the appearance of the microemulsion after centrifugation;
FIG. 3 is a graph showing the diffusion of Sudan red and methylene blue in a microemulsion;
in fig. 4, the particle size distribution of the a mugwort leaf volatile oil microemulsion, the long-term stability of the B mugwort leaf volatile oil microemulsion,
zeta potential of AEEO-ME within 30 days of three batches C;
fig. 5 is a chromatogram, wherein a blank, B control, C mugwort leaf volatile oil test, 1: eucalyptol 2: camphor;
FIG. 6 is a graph showing eucalyptol-peak area curves for different concentrations;
FIG. 7 is a graph of camphor-peak area curves for different concentrations;
FIG. 8 shows skin conditions of mice at various time points;
FIG. 9 shows the cumulative penetration of eucalyptol and camphor in a mugwort leaf volatile oil microemulsion gel;
FIG. 10 is a schematic flow diagram of the construction of a BALB/c mouse AD model and drug intervention;
FIG. 11 is the effect of AAEO-MG on body weight of AD model mice;
FIG. 12 is a graph showing the effect of AAEO-MG on skin lesions in AD model mice;
FIG. 13 is a graph showing the effect of AAEO-MG on skin lesions in AD model mice;
FIG. 14 is a graph showing the effect of AAEO-MG on the number of scratching in AD model mice;
FIG. 15 is a graph showing the effect of AAEO-MG on ear swelling in AD model mice;
FIG. 16 is the effect of AAEO-MG on the immunological serum of AD model mice;
FIG. 17 is a graph showing the effect of AAEO-MG on the expression of back skin lesion factor mRNA in AD mice;
FIG. 18 shows the effect of AAEO-MG on biochemical markers in AD mice;
FIG. 19 is the effect of AAEO-MG on the skin pathology of AD mice;
FIG. 20 is the effect of AAEO-MG on the cardiac pathology in AD mice;
FIG. 21 is the effect of AAEO-MG on the spleen pathology of AD mice;
FIG. 22 is the effect of AAEO-MG on liver pathology in AD mice;
FIG. 23 shows the effect of AAEO-MG on renal pathology in AD mice.
Detailed Description
The invention provides mugwort leaf volatile oil microemulsion, which comprises the following components in parts by weight: 1.5 to 1.7 parts of mugwort leaf volatile oil, 1.15 to 1.25 parts of surfactant, 0.15 to 0.25 part of medium chain triglyceride and 46.8 to 47.2 parts of water.
The artemisia argyi volatile oil microemulsion provided by the invention comprises 1.5-1.7 parts by weight of artemisia argyi volatile oil, specifically 1.5 parts by weight, 1.6 parts by weight and 1.7 parts by weight. In the invention, the mugwort leaf volatile oil is an active substance, and can effectively treat atopic dermatitis. The source of the mugwort leaf volatile oil is not particularly limited, and the mugwort leaf volatile oil is prepared by adopting a commercial product or adopting a conventional preparation method, and in the specific embodiment of the invention, the mugwort leaf volatile oil is preferably purchased from Anhuin and biotechnology limited company, and the product number is 20210821.
The artemisia argyi volatile oil microemulsion provided by the invention comprises 1.15-1.25 parts by weight of surfactant, and specifically 1.15 parts by weight, 1.2 parts by weight and 1.25 parts by weight. In the present invention, the surfactant preferably includes tween-80.
The artemisia argyi volatile oil microemulsion provided by the invention comprises 0.15-0.25 part by weight of medium chain triglyceride, specifically 0.15 part by weight, 0.2 part by weight and 0.25 part by weight. In the invention, the medium chain triglyceride has the function of reducing the Ohnsonian ripening phenomenon and helping the formation of essential oil nano-particles; and the nanoemulsion can obtain a smaller particle size and better stability. In the present invention, therefore, the cosurfactant is preferably a medium chain triglyceride.
The artemisia argyi volatile oil microemulsion provided by the invention comprises 46.8-47.2 parts by weight of water, and specifically 47.2 parts by weight, 47.0 parts by weight and 46.8 parts by weight. In the present invention, the pure water is an aqueous phase.
The invention also provides a preparation method of the mugwort leaf volatile oil, which comprises the following steps:
mixing the mugwort leaf volatile oil, the surfactant and the medium chain triglyceride to obtain a mixture;
mixing the obtained mixture with water, and emulsifying to obtain folium Artemisiae Argyi volatile oil microemulsion;
The temperature of the emulsification is 28-32 ℃.
In the invention, the mugwort leaf volatile oil, the surfactant and the medium chain triglyceride are preferably uniformly mixed at the water bath temperature of 30 ℃. In the present invention, the temperature of the emulsification is 28 to 32 ℃, preferably 30 ℃. In the present invention, the rotational speed of the emulsification is preferably 450 to 550rpm, more preferably 500rpm. The invention preferably adds the mixture slowly into water for mixing and emulsifying.
The invention also provides the mugwort leaf volatile oil microemulsion gel, which comprises mugwort leaf volatile oil microemulsion and carbomer 940 solution; the mass ratio of the mugwort leaf volatile oil microemulsion to the carbomer 940 solution is 4:1; the mass percentage of carbomer 940 in the carbomer 940 solution is 2%. The carbomer 940 solution is preferably prepared by dissolving carbomer 940 in glycerol. The pH value of the mugwort leaf volatile oil microemulsion gel is preferably adjusted by using a triethanolamine solution with the volume percentage of 20%, and the pH value of the mugwort leaf volatile oil microemulsion gel is 5.56+/-0.12.
The invention also provides a preparation method of the mugwort leaf volatile oil microemulsion gel, which is characterized in that the mugwort leaf volatile oil microemulsion is mixed with carbomer 940 solution to obtain mugwort leaf volatile oil microemulsion gel.
The invention also provides the mugwort leaf volatile oil microemulsion or the application of the mugwort leaf volatile oil microemulsion gel in preparing the medicine for treating atopic dermatitis.
The invention also provides the mugwort leaf volatile oil microemulsion or the application of the mugwort leaf volatile oil microemulsion gel in preparing medicaments for treating skin lesions.
The invention also provides the mugwort leaf volatile oil microemulsion or the application of the mugwort leaf volatile oil microemulsion gel in preparing the medicine for treating ear swelling
The present invention will be described in detail with reference to examples for further illustration of the invention, but they should not be construed as limiting the scope of the invention.
Example 1
1 laboratory apparatus and materials
1.1 Experimental reagent
TABLE 1 Experimental reagents
Reagent name Manufacturer' s Goods number
Mugwort leaf volatile oil Anhui Beijing and Biotech Co.Ltd 20210821
Tween-80 TIANJIN KERMEL CHEMICAL REAGENT Co.,Ltd. T818929
Triglycerides (Triglycerides) TIANJIN FUYU FINE CHEMICAL Co.,Ltd. 56-81-5
Carbomer-940 BEIJING SOLARBIO TECHNOLOGY Co.,Ltd. C7650
Anhydrous methanol TIANJIN FUYU FINE CHEMICAL Co.,Ltd. 65-56-1
Eucalyptus extract reference substance (purity not less than 99%) Shanghai microphone Lin Biotechnology Co.Ltd C10778675
Camphor reference substance(purity is not less than 98%) SHANGHAI YUANYE BIOTECHNOLOGY Co.,Ltd. W08J7F8778
Triethanolamine salt Alatine L1912012
1.2 laboratory apparatus
Table 2 laboratory apparatus
Instrument name Manufacturer' s
Precision electronic scale Metrele-Toli instruments Shanghai Co., ltd
Intelligent temperature-control magnetic stirrer GONGYI CITY YUHUA INSTRUMENT Co.,Ltd.
Ultrasonic cleaner Zhengzhou primordial qi instrument
Particle size and potential analyzer Bruce Hai Wen
Low-temperature high-speed centrifugal machine BeckmanCoulter Co., germany
Absolute ethyl alcohol Sinopharm Group Chemical Reagent Co., Ltd.
Water bath kettle SHANGHAI YIHENG INSTR Co.,Ltd.
Gas chromatograph Agilent SpA of America
PH tester China Shanghai Instrument electric science instruments Co Ltd
Ultra-filtration tube Yi Kang Kemao
Purified water system Sigma-Aldrich Co
2 Experimental methods
2.1 preparation of the Artemisia argyi volatile oil microemulsion gel
2.1.1 prescription of the Artemisia argyi volatile oil microemulsion
An oil phase: tween-80 (Tween-80) 2.3%, medium Chain Triglyceride (MCT) 0.3%, folium Artemisiae Argyi volatile oil 3.4%;
aqueous phase: 94% of pure water (primary water);
2.1.2 preparation of the Artemisia argyi volatile oil microemulsion
The method of adding oil phase into water phase is adopted to prepare the mugwort leaf volatile oil microemulsion, and four factors including mugwort leaf volatile oil, MCT, tween-80 and emulsification temperature with different dosages are taken as single factors to prepare the mugwort leaf volatile oil microemulsion orthogonal experiment (Table 3). Nine test samples of different batches were prepared, designated as AY1, AY2, AY3, AY4, AY5, AY6, AY7, AY8, AY9, respectively, and conditions for preparing the microemulsion were screened using particle size and potential as evaluation indexes (table 4).
Weighing a certain amount of oil phase and water phase, firstly fully and uniformly mixing all the oil phase at the water bath of 30 ℃, putting a conical flask (built-in magnetic stirrer) filled with the water phase on a balanced constant-temperature magnetic stirrer (28 ℃,500 rpm) for balancing for 10 minutes, then slowly dripping the oil phase into the water phase, stirring for 30 minutes, and stopping to obtain the artemisia argyi volatile oil microemulsion.
TABLE 3 orthogonal test factors and level tables
TABLE 4 orthogonal test design table
2.1.3 preparation of the Artemisia argyi volatile oil microemulsion gel
The selection of the matrix type is particularly important for gel preparation, and according to the reference, carbomer-940 is the first choice of the gel matrix, a certain amount of carbomer-940 is weighed, and a certain amount of glycerol is added to prepare the carbomer 940 with the mass percent of 2%, so that the blank gel matrix is prepared. And adding the blank gel matrix with the mass percentage of 20% into the prepared mugwort oil microemulsion (namely, the mass ratio of the blank gel matrix to the mugwort oil microemulsion is 1:4), uniformly stirring, and regulating the pH (5.56+/-0.12) by using 20% of triethanolamine to obtain the mugwort leaf volatile oil microemulsion gel.
2.2 evaluation of the microemulsion gel of the volatile oil of mugwort leaf
And evaluating the appearance, pH, viscosity, centrifugal stability, particle size and potential of the prepared mugwort leaf volatile oil microemulsion and microemulsion gel.
2.2.1 appearance Properties, pH, viscosity, and centrifugal stability
Taking 3 batches of different moxa oil microemulsions and microemulsion gels, and observing whether the appearance and shape of the moxa oil microemulsions and microemulsion gels meet the requirements. 1g of each was diluted with 15mL of double distilled water and measured at 25℃by a pH meter. Measurement of viscosity of microemulsion gel using Fungiab rotational viscometer. Taking 1.5mL of gel of different batches respectively, and measuring the gel at 3000 r.min -1 And accelerating centrifugation for 10min, and observing whether layering occurs.
2.2.2 staining method
3 different batches of artemisia argyi volatile oil microemulsions are taken, the dyes methylene (hydrophilic) and sudan III (lipophilic) are respectively dripped into the microemulsions, the type of the microemulsions is judged according to the diffusion speed of red or blue in the microemulsions, and if the diffusion speed of the red sudan III is higher than that of the blue methylene blue, the microemulsions are W/O type microemulsions, otherwise the microemulsions are O/W type microemulsions.
2.2.3 particle size
3 different batches of mugwort leaf volatile oil microemulsions are taken, diluted by 4 times by pure water, and the particle size, PDI and potential of the mugwort leaf volatile oil microemulsions are measured by a particle size meter at 25 ℃.
2.3 investigation of the content and methodology of the index ingredients of the mugwort leaf volatile oil microemulsion gel
2.3.1 gas chromatography conditions
The gas chromatography conditions were as follows: HP-5 column (30 m. Times.0.25 mm. Times.0.10 μm); the carrier gas is helium (He), the carrier gas is in a constant flow mode, and the flow rate of the column is 2.0mL/min; the sample injection amount is 1.0 mu L; the mode of the sample inlet is a non-shunt mode; the temperature of the sample inlet was 240 ℃. The temperature rise conditions are shown in Table 5.
TABLE 5 gas phase warming procedure
Rate (. Degree. C/min) Value (. Degree. C.) Holding time (min) Run time (min)
0 0 45 0 0
1 10 200 0 15.5
2 6 230 3 23.5
2.3.2 preparation of sample solutions
(1) Preparation of control solution
Respectively precisely weighing eucalyptol and camphor reference substance about 23.0mg and 19.80mg to 15mL in a centrifuge tube, dissolving with 10mL of n-hexane, swirling for 3min, mixing, diluting for 10 times, mixing, and filtering with 0.22 μm filter membrane to obtain reference substance mother liquor with concentration of 0.230mg/mL and 0.198 mg/mL.
(2) Preparation of test solution
Precisely weighing 2.008g of mugwort leaf volatile oil microemulsion gel, adding methanol to 10mL of the gel to fix the volume, uniformly mixing by vortex, carrying out ultrasonic treatment for 10min, centrifuging to obtain supernatant, extracting a proper volume by a syringe, and filtering with a 0.22 mu m filter membrane to obtain a sample solution.
(3) Preparation of negative test sample solution
Weighing the blank gel matrix into the same amount as the mugwort leaf volatile oil microemulsion gel, precisely adding methanol to 10mL to constant volume, performing vortex mixing uniformly, performing ultrasonic treatment for 10min, centrifuging to obtain supernatant, sucking a proper amount by a syringe, and filtering with a 0.22 mu m filter membrane to obtain a negative sample solution.
2.3.3 methodology investigation
(1) Investigation of the Linear relationship
Control solutions containing eucalyptol 115.00, 46.00, 23.00, 11.50, 4.60, 2.30, 1.15 μg/ml, camphor 198.00, 99.00, 39.60, 19.80, 9.90, 3.96, 1.98, 0.99 μg/ml were prepared according to the method under "2.3.2 (1)", and were sampled under the chromatographic conditions of "2.3.1" in this example, and peak areas of the two controls were determined. The abscissa (X) is the concentration of eucalyptol and camphor control, and the ordinate (Y) is the area of the peak obtained, and a linear regression curve is drawn.
(2) Sample content determination
A sample solution was prepared according to the method described in "2.3.2" of this example, and the eucalyptol and camphor contents in the samples were calculated based on the peak area and linear relationship.
(3) Encapsulation efficiency and drug loading rate
3 batches of micro-emulsion are prepared, 2mL of micro-emulsion is precisely measured, the micro-emulsion is placed in an inner tube of a 3000Da ultrafiltration tube, centrifugation is carried out at 4 ℃ for 30min at 3000r/min, a sample solution is prepared according to the method under the chapter ' 2.3.2 ', the peak areas of eucalyptol and camphor are recorded and the mass concentration is determined according to the standard curve substitution in the chapter ' 2.3.3 ' (2) ', the encapsulation efficiency is calculated, the formula is encapsulation efficiency = encapsulation dosage/total dosage x 100%, the drug loading is calculated again, and the formula is drug loading = encapsulation dosage/(lipid mass + composite emulsifier mass + encapsulation dosage) x 100%.
2.4 percutaneous experiments and researches on the microemulsion gel of the volatile oil of mugwort leaf
2.4.1 skin irritation test
8 normal female BALB/C mice (purchased from Fukan Biotechnology Co., ltd., beijing) were dehaired, and were discarded if skin damage occurred during dehairing. Skin irritation experiments were started 24h after dehairing. The gel was applied as an equal volume of the mugwort leaf volatile oil microemulsion, and evaluated according to table 6, and the presence or absence of skin irritation (erythema edema) after administration was observed at 2, 12, 24, 48 hours.
TABLE 6 skin irritation intensity grading Table
Score value Status of Evaluation
0～＜0.5 Free of erythema edema No irritation
0.5～＜2.0 Mild erythema and edema Light irritation
2.0～＜6.0 Obvious erythema edema Mid-irritation
＞6.0 Severe erythema edema Strong irritation
2.4.2 percutaneous permeation experiments
(1) Preparation of test sample
Prepared as in this example "2.1.2,2.1.3".
(2) Preparation of ex vivo skin
After the female BALB/C mice were sacrificed by cervical dislocation, the dorsal mouse hairs were removed, the dorsal skin was cut to a proper size, the adipose tissues and connective tissues of the dorsal skin of the mice were rubbed off with cotton balls, and washed clean with physiological saline. Finally, the aluminum foil is wrapped and then placed in a refrigerator at the temperature of minus 20 ℃ for dimensional preservation, and the aluminum foil is used within one week.
(3) Percutaneous permeability experiments
The preserved isolated skin is taken out of the refrigerator, naturally thawed, washed with normal saline, cut into proper size, and laid flat and fixed on the interface of Franz diffusion cell (dermis facing the receiving cell). Injecting 20% ethanol physiological saline (ultrasonic treatment) into the receiving tank, maintaining the temperature of the prepared receiving tank at (37+ -0.2) deg.C, and measuring 300 r.min -1 Is pre-equilibrated, taking care to allow the bubbles in the receiving liquid to escape as soon as possible. After 30min of pre-equilibration, the uniform artemisia argyi volatile oil microemulsion gel was equally applied to the skin supply area and the experiment was performed 3 times. 1mL of each sample was sampled at 2h, 4h, 6h, 8h, 12h, 24h, 36h, 48h, and after each sampling, an equal volume of a blank receiving solution containing 20% ethanol physiological saline was added to each sample at the same temperature, and bubbles in the receiving tank were removed. The received solution was extracted with 2ml of n-hexane, an appropriate volume of the extracted received solution was sucked by a syringe, and the solution was filtered through a membrane (0.22 μm) to obtain a sample of the received solution in each period, and the sample was analyzed under the chromatographic conditions under item "2.3.1" of this example to obtain the cumulative permeation amounts of eucalyptol and camphor.
3 results of experiments
3.1 preparation of the volatile oil microemulsion of mugwort leaf
The results in Table 7 are calculated from Table 4 as follows, K1 represents the sum of the amounts of volatile oils of A and of A when 1 (Table 4) is added, i.e., 1.5g (Table 3), and K1 is the average value, and so on. By combining the table 3 and the table 4, the A3B1C1D1 is the optimal process formula, namely, 1.7g of mugwort leaf volatile oil, 1.15g of surfactant (Tween-80), 0.15g of medium chain triglyceride MCT, the emulsification temperature is 28 ℃, and 47g of water.
TABLE 7 results of orthogonal test table
3.2 appearance Property, pH, viscosity, centrifuge stability
And (3) measuring appearance characteristics, pH value, viscosity and centrifugal stability of the sample prepared again according to the optimal process formula screened by the orthogonal test.
3.2.1 appearance Properties
The appearance of the microemulsion a in fig. 1 is a milky liquid, and the microemulsion diluted 4 times also has the tyndall phenomenon as shown in fig. 1B. The microemulsion gel was a white viscous semisolid having a uniform morphology, was not greasy, and had good abduction, as shown in fig. 1 (C-D).
3.2.2pH, viscosity, centrifuge stability
The pH values of the mugwort leaf volatile oil microemulsion and the microemulsion gel are respectively 5.43, 5.61 and 5.65, which are obtained by measurement by a pH meter, and meet the requirement of the topical skin external preparation on the pH value (the pH value of the skin is 4-7).
The viscosity of the mugwort leaf volatile oil microemulsion gel is 658000, 606000 and 716000 mPa.s measured by a Fungiab rotary viscometer. Microemulsion of volatile oil of folium Artemisiae Argyi at 3000 r.min -1 After 10min, the appearance is still clear and transparent, and layering is avoided (as shown in figure 2), which shows that the prepared artemisia argyi volatile oil microemulsion has good centrifugal stability.
3.2.3 staining method
According to the principle of similar compatibility, the diffusion speed of methylene blue in the experiment is obviously higher than that of Sudan III, so that the artemisia argyi volatile oil microemulsion is in an oil-in-water O/W type. The results are shown in FIG. 3.
3.2.4 particle size
Preparing the moxa oil microemulsion and microemulsion gel, and measuring the particle size by using a Zeta potential and particle size analyzer. The average value of the particle sizes of three batches of micro-emulsion within 30 days is 67.21 +/-1.82 nm, PDI is 0.160+/-0.011 and electric potential is-15.10+/-2.22 mV, and the result is shown in figure 4.
3.3 content and methodological investigation
3.3.1 linear relationship investigation
The relevant chromatogram is shown in FIG. 5. The linear regression equations for eucalyptol and camphor are shown in Table 8, which illustrates that eucalyptol and camphor exhibit good linear relationships with peak area Y in the concentration ranges of 1.15-115 mug/mL and 0.99-198 mug/mL, respectively, and the standard curves are shown in FIGS. 6 and 7.
TABLE 8 Linear regression equation for eucalyptol and Camphora
Composition of the components Regression equation R 2 Linear range/(μg/ml)
Eucalyptus extract y＝16.177x+20.776 0.9999 1.15～115.00
Camphor y＝14.439x+18.116 0.9998 0.99～198.00
3.3.2 encapsulation and drug loading
The encapsulation efficiency and the drug loading rate of the eucalyptol and the camphor, which are measured according to the method of experiment 2.3.3 (3), are shown in table 9, and meet the requirements of Chinese pharmacopoeia.
TABLE 9 encapsulation efficiency and drug loading rate of blumea volatile oil microemulsion
3.4 percutaneous laboratory study
3.4.1 skin irritation test results
The skin irritation intensity scores of 8 mice are 0, and the skin erythema edema phenomenon does not occur. Skin conditions of one mouse were selected for different periods of time after application of the mugwort leaf volatile oil microemulsion gel, as shown in fig. 8.
3.4.2 percutaneous permeability test results
The cumulative transdermal amount Qn of the mugwort leaf volatile oil microemulsion gel at each time point is calculated by the following formula, wherein Qn is the cumulative permeation amount per unit area at the nth time point, A is the effective permeation area, cn is the drug mass concentration measured at the nth time point, V is the volume of the receiving tank, and Vi is the volume of each sampling.
Q n ＝(VC n +∑C i V i )/A
The cumulative transdermal amount per unit area of the mugwort leaf volatile oil microemulsion gel in each time period was analyzed and treated, and the results are shown in Table 10. The accumulated drug permeation quantity Qn (mug cm) -2 ) Plotted against time h, a graph of time versus cumulative permeation quantity Qn was obtained, and the results are shown in fig. 9. Wherein Cineole is eucalyptol and Camphor is Camphor.
TABLE 10 cumulative penetration of mugwort leaf volatile oil microemulsion gel eucalyptol and camphor
The study of example 1 shows that the instability and volatility of the volatile oil can be obviously improved after the volatile oil of the mugwort leaf is prepared into a micro-emulsion gel dosage form. Experimental results show that the prepared mugwort leaf volatile oil microemulsion gel has stable physicochemical properties and relatively stable component content. Therefore, the artemisia argyi volatile oil compound pain relieving micro-emulsion gel has good development potential as a novel transdermal external preparation.
Example 2
Pharmacodynamics research of mugwort leaf volatile oil microemulsion gel for treating atopic dermatitis
Atopic dermatitis (Atopic dermatitis, AD) is mainly manifested by dry skin itching and eczematoid dermatitis, and has high recurrence rate. The pathogenesis of atopic dermatitis is considered to be related to viscera dysfunction by ancient doctors, and the factors such as hereditary and immunological dysfunction are considered to aggravate the atopic dermatitis by modern medicine, but the pathogenesis of the atopic dermatitis involves complex immune response, so the specific mechanism has not been elucidated.
In order to deeply study the pathogenesis of atopic dermatitis, it is necessary to build an animal model conforming to the disease characteristics thereof. Construction of the mouse AD model by 2, 4-Dinitrochlorobenzene (DNCB) induction is the most common model method, and female BALB/C mice are often selected. In the present example, the therapeutic effect of the mugwort leaf volatile oil microemulsion gel on atopic dermatitis mice was investigated under the condition of changing the dosage form of mugwort leaf volatile oil, and the relevant action mechanism of mugwort leaf volatile oil for treating AD was analyzed.
1 Experimental materials and instruments
1.1 laboratory animals
56 female BALB/C mice, 20+ -2 g in weight, purchased from Beijing Fukang Biotechnology Co., ltd., license number: SCXK (jing) 2019-0008, experimental animal ethical examination number: DWLL202108004. Unified standardized feeding is performed at Henan traditional Chinese medicine university experimental animal experiment center, license number: SYXK (relaxation) 2021-0015. In the constant temperature (25+/-1 ℃) environment, the seeds are fed in separate cages and are eaten and drunk freely, and the seeds are illuminated for 12 hours.
1.2 Experimental reagents
Table 11 Experimental reagent
1.3 Experimental apparatus
Table 12 experimental equipment
2 Experimental methods
2.1 grouping of mice
The mice were weighed, ordered by body weight size from 1 to 56, and a random number table was generated by invoking a random number generator of the SPSS, randomly dividing 56 female BALB/c mice into 7 groups, i.e., normal group, model group, mugwort leaf volatile oil group (0.5 ml/kg), mugwort leaf volatile oil microemulsion group (i.e., optimal) (0.5 ml/kg), mugwort leaf volatile oil microemulsion gel (i.e., optimal) low dose group (0.25 ml/kg), mugwort leaf volatile oil microemulsion gel medium dose group (0.5 ml/kg), mugwort leaf volatile oil microemulsion gel high dose (1 ml/kg), 8 each. Are denoted as Control, model, AAEO, AAEO-ME, AAEO-MG (low), AAEO-MG (middle), and AAEO-MG (high), respectively.
2.2 construction of atopic dermatitis model
AD models were made for each of the remaining groups except for the normal control group. Modeling is carried out by adopting an induction method of repeated stimulation of 2, 4-Dinitrochlorobenzene (DNCB), and the construction of the BALB/c mouse AD model refers to an internationally recognized method. The skin of the back of the mouse is removed by depilatory cream before molding, and the area is about (2X 2 cm) 2 ). The matrix solution is prepared from acetone and olive oil in a volume ratio of 4:1, and DNCB solution with a mass ratio concentration of 1% and 0.1% is prepared by the matrix solution. Primary sensitization was performed on day 1 of molding by applying 1% DNCB solution to the dorsal skin area (150. Mu.l) and the dorsal skin (10. Mu.l) of the right ear of each group of mice except the normal group with a pipette for 2 consecutive days at 4-day intervals. Subsequently, 0.1% DNCB solution was applied to the dorsal skin preparation area (100 μl), the dorsal skin of the right ear (10 μl) of the mice on days 7, 9, 11, and 13 of the experiment, respectively, to re-stimulate the sensitized skin (secondary sensitization) and maintain AD-like inflammatory response while the same amount of 1:4 acetone and olive base solution was applied to the dorsal surface of the left ear of the mice as a control. The molding and drug administration flow chart is shown in fig. 10. Normal mice were coated with an equal volume of matrix solution at the same site at the same time point.
2.3 administration of drugs
After successful modeling on experiment day 13, the drug was administered from day 14 to day 21, once daily for 7 days. The normal group and the model group were smeared with sterile water at 0.5ml/d. Mice were not killed during the experiment, and were sacrificed 24h (day 22) after the last administration and the materials were available for use.
2.4 collection and processing of serum and tissue samples
Before taking materials, all mice are fasted and not forbidden for 12 hours, and after the body mass of the mice is weighed, eyeballs are picked up for blood taking. The blood sample was allowed to stand at room temperature for 1h, then centrifuged at 3000r/min for 15min at 4℃and the supernatant serum was aspirated with a pipette. Immediately after blood is taken from eyeballs, the mice are killed by a cervical dislocation method, the abdominal cavity is opened, the skin of the back of the mice, heart, liver, kidney, spleen and thymus tissues of the mice are rapidly taken out on an ice table, blood stains on the tissues are washed out by precooled normal saline, redundant water is sucked by filter paper, and then the quality of each viscera is weighed. Cutting part of liver, kidney and skin tissue, placing in a freezing tube, storing in a refrigerator at-80deg.C, and fixing the rest viscera with 4% paraformaldehyde.
2.5 Experimental index detection
2.5.1 mouse body weight
The weight of the mice was weighed and recorded every two days at the same time point during the experiment, and a weight curve of the mice was drawn.
2.5.2 evaluation of local skin lesions in mice
On days 0, 1, 8, 15 and 22 of the experiment, skin lesions were scored according to table 13, with four grades including inflammatory observations of erythema (bleeding), erythema (pimple), epidermolysis (scratch), scaling (dryness) and the like, and recorded by photographs. Each item is 3 minutes at most, the four indexes are added together, and the skin loss is not more than 12 minutes at most.
TABLE 13 skin loss scoring criteria
2.5.3 measurement of the number of mouse scratches
The number of scratching per mouse was calculated according to the reference: when the mice leave the bottom of the cage with the hind paws, scratch the skin of the application sites such as ears, heads and the like until the hind paws land or lick the hind paws, the complete scratching action is regarded as 1 time. The scratching time is recorded as 2 scratching times in more than 3s, and the mice are manually interfered after 3s, so that the scratching activity is stopped.
2.5.4 ear swelling thickness and organ coefficients
After the mice were sacrificed, holes were punched in the corresponding parts of the right auricle skin lesions and the left auricle (using a mouse ear punch with a diameter of 0.6 cm) to obtain ear tissue specimens, which were individually labeled, and weighed on an analytical balance to calculate the ear swelling degree. Ear swelling degree = right ear mass (mg) -left ear mass (mg). Heart, liver, kidney, spleen and thymus were dissected and organ coefficients were measured. Organ coefficient=organ weight (g)/volume mass (g) ×100%.
2.5.5 immune molecule serological index
The levels of IGE and IL-1 beta in the serum of the mice are detected by an enzyme-labeled method according to the instruction of ELISA kit. Serum was taken under item "2.4" of this example, and the specific procedure was as follows:
(1) Preparation before detection
The kit was taken out of the refrigerator 20 minutes in advance and allowed to stand at room temperature (18 to 25 ℃). If the kit needs to be used for multiple times, only the enzyme-labeled strip and the reagent needed by the experiment are taken out, and the rest strip and the reagent are stored according to the specified conditions.
Washing liquid: taking out the concentrated washing liquid from the kit, if the concentrated washing liquid has crystallization phenomenon, opening a water bath kettle to be positioned at 40 ℃, heating the concentrated washing liquid to cause crystallization to be completely dissolved, diluting the concentrated washing liquid by double distilled water, wherein the dilution ratio is the concentrated washing liquid: double distilled water=20:1. The washing liquid is required to be prepared in situ.
Standard working solution: centrifuging the freeze-dried standard substance in a micro centrifuge for 1 minute, adding 1mL of standard substance and sample diluent into the freeze-dried standard substance, screwing a tube cover, standing for 1-2 minutes, vibrating uniformly by a low-speed vortex instrument, centrifuging in the low-speed centrifuge to remove bubbles generated in the vortex process, avoiding foaming, and preparing 2000pg/mL of standard substance working solution. The standard working fluid ratios of 2000pg/mL were diluted to 1000, 500, 250, 125, 62.5, 31.25,0pg/mL according to the reagent specifications.
Biotinylated antibody working solution: the amount of biotinylated antibody working fluid required (100. Mu.l per well) was calculated before the experiment was started, and more reagent was prepared. About 20 minutes before use, the concentrated biotinylated antibody is diluted with a biotinylated antibody diluent (to be mixed by vortex shaking centrifugation), and the dilution ratio is the concentrated biotinylated antibody: biotinylated antibody dilution=1:99.
Enzyme conjugate working solution: the amount of enzyme conjugate working solution required (100. Mu.l per well) was calculated before the start of the experiment, and more reagent was used. About 20 minutes before use, the concentrated HRP enzyme conjugate was diluted with enzyme conjugate diluent (mixed by vortex shaking centrifugation) in a ratio of concentrated HRP enzyme conjugate: enzyme conjugate dilution=1:99.
(2) The operation steps are as follows:
1) Standard holes, blank holes and sample holes are respectively set on the ELISA plates according to the specification of the kit. Standard holes are added with standard working solution diluted in advance in sample preparation, blank holes are added with standard and sample diluent reagents, sample holes are respectively added with samples to be tested according to groups, and each hole is added with 100 mu l of reagents. The plates were covered with a plate patch and incubated in an incubator at 37℃for one and a half hours. Prompting: the sample adding time should not be too long, and the gun head should not touch the bottom of the ELISA plate as much as possible during sample adding.
2) And taking out the ELISA plate from the constant temperature incubator at 37 ℃, removing the plate paste of the ELISA plate, throwing the liquid in the plate vertically downwards, and beating the liquid on the absorbent paper. 100. Mu.L of biotinylated antibody working solution was added to each well in sequence, and the ELISA plate was attached and incubated in a 37℃incubator for 1 hour.
3) And taking the ELISA plate out of the incubator, removing the plate paste, throwing the liquid in the plate vertically downwards, and beating the plate on the water absorbing paper. Each well was filled with 350. Mu.L of wash solution in the order of addition, soaked for about 1 minute, and the solution was thrown vertically downward from the plate and tapped clean on absorbent paper. This procedure was repeated for 3 total washes.
4) 100 mu L of enzyme conjugate working solution is added into each hole in sequence, an enzyme label plate is added with a plate paste, and the mixture is placed into a constant temperature incubator at 37 ℃ for incubation for 30 minutes.
5) And taking the ELISA plate out of the incubator, removing the plate paste, throwing the liquid in the plate vertically downwards, and beating the plate on the water absorbing paper. The plate was washed 3 times.
6) And adding 90 mu L of substrate solution to each hole according to the sample adding sequence of the substrate solution, adding a plate patch to an ELISA plate, and incubating for 15 minutes in a constant temperature incubator at 37 ℃ in a dark place. The microplate reader was turned on 15 minutes in advance for preheating.
7) The reaction was stopped by adding 50. Mu.L of stop solution to each well in the order of addition of the substrate solution.
8) The absorbance of each well was measured immediately with an enzyme-labeled instrument at a wavelength of 450 nm.
(3) And respectively calculating the average OD values of the blank hole, the standard substance and the sample compound hole, and subtracting the average OD value of the blank hole from the average OD value of the standard substance and the sample compound hole to obtain a correction value. And (3) fitting a standard curve by taking the standard substance concentration as an abscissa and the OD value as an ordinate, and calculating the concentration of each group of samples. 2.5.6 Back skin injury inflammatory factor mRNA expression
The frozen skin tissues of three mice under the randomly selected item "2.4" of each group were used for extracting total RNAs for real-time fluorescence quantitative PCR detection.
1) A proper amount of BALB/C mouse skin tissue in liquid nitrogen was placed in a homogenate tube. Trizol of lmL was added to each of the homogenization tubes and the mixture was thoroughly ground, and the ground mixture was allowed to stand at room temperature for 5 minutes. Transfer to an enzyme-free centrifuge tube, add 200. Mu.L chloroform solution, cover the sample tube tightly, shake vigorously for 15s, then leave it to stand at room temperature for 10-15min.
2) The three-layered cleavage mixture was visualized by centrifugation in a refrigerated centrifuge at 12000rpm for 10min at 4℃and RNA was a colorless clear solution in the upper layer, which was carefully aspirated into a clean enzyme-free centrifuge tube with an enzyme-free gun head (note that no intermediate white floc was aspirated).
3) Then 500. Mu.L of isopropanol was added thereto, and the mixture was stirred and mixed well, and allowed to stand at room temperature for 10 minutes. Centrifugation was carried out under the same conditions for 10min, at which time a white gelatinous pellet was attached to the bottom of the centrifugation tube, which was the extracted RNA.
4) The supernatant was poured off, 1mL of 75% ethanol (DEPC water and absolute ethanol) was added, and after mixing, the precipitate was washed and centrifuged at 5000rpm at 4℃for 5min. The second elution was performed under the same conditions as above (note that this time the EP tube was placed in the centrifuge in the opposite direction to the first one, so that a more complete elution was possible). The direction of the EP tube in the centrifuge was again reversed (no more ethanol was added), 7500g/min, centrifuged for 3min at 4℃and the residual ethanol was discarded with a 10. Mu.L white gun head (note RNA not sucked to the bottom of the tube), the lid was opened and the residual ethanol was evaporated at room temperature.
5) 20. Mu.L of DEPC water was added for solubilization, and then the concentration and purity of RNA were measured using a Nanodrop system.
6) Reverse transcription is performed to remove genomic DNA.
Gently mix (gently blow mix with a pipette or gently mix with a vortex mixer at minimum speed), then centrifuge pellet the liquid and react at 42 ℃ for 2min. Then 5x superscript rtmix (4 μl) was added directly, carefully mixed well, and the second step was performed: the reaction was carried out at 50℃for 15min and at 85℃for 5min.
7) After the completion of reverse transcription, 180uLDEPC water was added to dilute the mixture into 200uL to obtain a cDNA template. Searching corresponding gene source codes according to NCBI database, wherein the primer sequences are as follows:
TABLE 14 primer sequences
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Gene expression was detected in an ABI 7500fast instrument using a PowerUp TM SYBRTMGreenPCRMasterMix kit, the Q-PCR reaction system is shown in the following Table:
TABLE 15 reaction system
Reagent(s) Volume of
PowerUp TM SYBR TM GreenPCRMasterMix 5μL
Upstream and downstream primers 1μL
cDNA template 4μL
The amplification reaction conditions for the ABI 7500fast were: pre-denaturation procedure: cycling procedure from 50deg.C for 2min to 95deg.C for 2 min: 95 ℃ for 15s to 60 ℃ for 1min, 40 cycles total, annealing procedure: 95℃for 15s to 60℃for 1min. GAPDH is used as reference gene, 2 is used -ΔΔCt The relative quantification of each gene was analyzed by calculation.
2.5.7 biochemical level detection
All mice were weighed 24 hours after the last dose, and the "2.4" frozen liver and kidney were removed for biochemical index determination. Biochemical indexes are detected by an AST kit (liver tissue), an ALT kit (liver tissue), a CRE kit (kidney tissue) and a BUN kit (kidney tissue) respectively.
Taking a proper amount of frozen liver tissue, weighing, adding pre-cooled physiological saline (tissue mass (g): physiological saline capacity (ml) =1:9) according to the liver tissue mass, grinding into 10% of liver tissue homogenate by a high-speed low-temperature tissue grinder, sucking a trace amount of 10% of liver tissue homogenate, and diluting into 1% of liver homogenate by physiological saline for later use. Protein content in liver tissue was calculated by BCA method. The specific detection method comprises the following steps:
(1) AST kit for detecting glutamic-oxaloacetic transaminase in liver tissue
1) Principle of the kit: glutamic-oxaloacetic transaminase converts alpha-ketoglutarate and aspartic acid to amino and keto groups, producing glutamic acid and oxaloacetic acid. During the reaction, oxaloacetic acid can spontaneously release from the carboxyl group to yield pyruvic acid. The pyruvic acid further reacts with 2, 4-dinitrophenylhydrazine to obtain 2, 4-dinitrophenylhydrazone, which can display reddish brown when meeting alkali. And (3) measuring the OD value of each hole at 510nm by using an enzyme-labeled instrument, and checking a standard curve to obtain the activity unit of the enzyme.
2) Assays were performed in 96-well plates and were grouped as assay wells, control wells. The measurement wells were filled with 20. Mu.L of 37℃preheated substrate solution and 5. Mu.L of the sample to be measured, and the control wells were filled with 20. Mu.L of 37℃preheated substrate solution. The solution in the 96-well plate is repeatedly blown and evenly mixed and then placed in a constant temperature incubator at 37 ℃ for 30min.
3) Adding 20 mu L of 2, 4-dinitrophenylhydrazine into the sample hole; to the control wells, 20. Mu.L of 2, 4-dinitrophenylhydrazine and 5. Mu.L of sample were added. After adding the solution, repeatedly blowing and beating uniformly, and placing in a constant temperature incubator at 37 ℃ for 20min.
4) 200 mu L of 0.4mol/L sodium hydroxide solution is added into each of the sample hole and the control hole, the mixture is placed on a horizontal rotator to shake for 10min, the mixture is placed at room temperature for 15min, the OD value of each hole (absolute OD value = sample hole OD value-control hole OD value) is measured at the wavelength of 510nm, and the standard curve calculation is substituted, so that the corresponding AST activity unit is obtained.
5) Preparation of a standard curve: 3 multiplex wells were made for each concentration, and 5. Mu.L of 0.1mol/L phosphate buffer, 20. Mu.L 2, 4-dinitrophenylhydrazine solution, and 0, 2,4, 6, 8. Mu.L of 2. Mu. Mol/L sodium pyruvate standard solution, 20, 18, 16, 14, 12. Mu.L matrix buffer were added to each well in a concentration gradient. After gentle stirring, incubation was carried out in a 37℃incubator for 20min, 200. Mu.L of 0.4mol/L sodium hydroxide solution was added to each well. After mixing by gentle shaking, the wells were left at room temperature for 15min and the OD of each well was measured at a wavelength of 510 nm. When a standard curve is made, the abscissa is the absolute OD value, and the ordinate is the Kanji unit.
(2) ALT kit for detecting glutamic pyruvic transaminase in serum of each group
1) The principle of the kit is that glutamic-pyruvic transaminase converts alanine and a-ketoglutarate into pyruvic acid and glutamic acid at 37 ℃ and pH of 7.4. After 30min of reaction, adding hydrochloric acid solution of 2, 4-dinitrophenylhydrazine, stopping the reaction, and simultaneously, enabling the 2, 4-dinitrophenylhydrazine to react with carbonyl in keto acid chemically to obtain pyruvic acid phenylhydrazone. Phenylhydrazone appeared reddish brown in the presence of alkaline solution, OD value was measured at 510nm with a microplate reader, and enzyme activity was calculated.
2) Assays were performed in 96-well plates, grouped as sample wells, control wells. To the assay wells, 20. Mu.L of a matrix solution preheated at 37℃and 5. Mu.L of a sample to be measured were added, and to the control wells, 20. Mu.L of a matrix solution preheated at 37℃were added. The solution was repeatedly blown and mixed uniformly, and then placed in a constant temperature incubator at 37℃for 30 minutes.
3) Adding 20 μl of 2, 4-dinitrophenylhydrazine to the sample well; control wells were added with 20. Mu.L of 2, 4-dinitrophenylhydrazine and 5. Mu.L of sample. After adding the solution, repeatedly blowing and beating uniformly, and placing in a constant temperature incubator at 37 ℃ for 20min.
4) To the sample wells and control wells, 200 μl of 0.4mol/L sodium hydroxide solution was added to each well, and the wells were placed on a horizontal rotator and shaken for 10min, and placed at room temperature for 15min, and the OD value of each well was measured at a wavelength of 510nm (absolute OD value=sample well OD value-control well OD value), and the standard curve was checked, and the corresponding AST activity units were calculated.
5) Preparation of a standard curve: the solution was divided into 6 concentration gradients, 3 wells were made for each concentration, and 5. Mu.L of 0.1mol/L phosphate buffer, 20. Mu.L 2, 4-dinitrophenylhydrazine solution, and 0, 2,4, 6, 8, 10. Mu.L of 2pmol/L sodium pyruvate standard solution, 20, 18, 16, 14, 12, 10. Mu.mol/L matrix buffer were added to each well. After gentle stirring, incubation was carried out in a 37℃incubator for 20min, followed by 200. Mu.L of 0.4mol/L sodium hydroxide solution per well. After shaking slightly and evenly, the wells were left at room temperature for 15min and the OD of each well was measured at a wavelength of 510nm using an ELISA reader. And the abscissa is the absolute OD value, the ordinate is the Kanji unit, and a standard curve is manufactured to obtain a cubic binary equation and an R value thereof.
(3) Cre kit for detecting creatinine content in serum of each group
1) Principle of kit measurement: creatinine hydrolyzed by creatinine amidohydrolase generates creatine, which is hydrolyzed by creatinine amidohydrolase to form sarcosine and urea, and the sarcosine is catalyzed by sarcosine oxidase to form glycine, formaldehyde and hydrogen peroxide. Hydrogen peroxide and 2,4- (6-triiodo-3-hydroxybenzoic acid) and 4-aminoantipyrine, under the catalysis of peroxidase, produced a reddish compound, and the OD was measured at 546 nm.
2) The detection operation is carried out in a 96-well plate and is divided into a sample well, a standard well and a blank well. 6 mu L of a sample to be detected is added into a sample hole, 6 mu L of a standard substance is added into a standard hole, 6 mu L of double distilled water is added into a blank hole, then 180 mu L of enzyme solution A is added into each hole, the mixture is incubated for 5min in a constant temperature incubator at 37 ℃, and the absorbance value of each hole is measured to be Al at 546 nm.
3) Then, 60. Mu.L of enzyme solution B was added to each well, and the mixture was placed in a constant temperature incubator at 37℃for 5 minutes, and the absorbance value of each well measured at 546nm in the microplate reader was designated as A2.
4) The result calculation formula: creatinine content (umol/L) = [ (sample A2-186/246×sample A1) - (blank A2-186/246×blank A1) ]/[ (standard A2-186/246×standard A1) - (blank A2-186/246×blank A1) ]×standard concentration
(4) BUN kit for detecting urea nitrogen content in serum
1) Principle of kit measurement: under the conditions of heat and strong acid, urea nitrogen can condense with diacetyl oxime to a red biazine compound, which has an OD value measured at 520 nm.
2) This operation was performed in 5mL EP tubes, which were divided into sample tubes, standard, blank tubes. 20 mu L of a sample is added into a sample tube, 20 mu L of urea nitrogen standard substance is added into a standard tube, 20 mu L of double distilled water is added into a blank tube, and then lmL of a reagent I and 1mL of a reagent II application liquid are added into each tube.
3) After mixing the tubes well, incubating in boiling water for 15min, taking out and immediately cooling in ice water. OD was measured at 520nm in 1cm optical path.
4) The result calculation formula: urea nitrogen content (mmol/L) = (sample OD value-blank OD value)/(standard OD-blank OD value) ×standard concentration×dilution
2.5.8 pathological histomorphology
After the mice were sacrificed, heart, spleen, liver, kidney, and skin tissues of the mice were carefully peeled off, and after fixation with 4% paraformaldehyde for 24 hours, HE staining experiments were performed. The pathological structure was observed under an optical microscope.
1) Embedding and slicing: taking out, placing in a full-automatic dehydrator, dehydrating, embedding paraffin, cutting into 4 μm slices, placing in a 43 deg.C water bath, spreading, attaching, draining, placing in a 65 deg.C sheet baking machine for 10-15min, and placing in a sheet rack for use.
2) Tissue dewaxing: placing in a 65 ℃ oven to melt wax for 30min, and immediately placing in xylene I for 5min, xylene II for 5min and toluene II for 10min.
3) Hydration, namely placing the glass slide in absolute ethyl alcohol I for 3min, absolute ethyl alcohol II for 3min, 95% ethyl alcohol I for 5min, 95% ethyl alcohol I for 3min and 90% ethyl alcohol for 3min, and washing the glass slide with flowing water to clean (the glass slide cannot be washed, and the glass slide is easy to damage).
4) Dyeing: placing in hematoxylin for 15min, repeating the washing step, differentiating with 1% hydrochloric acid alcohol for 2s, washing with flowing alkaline soapy water, staining with eosin for 8min, and repeating the washing step.
5) Dehydrating: placing in 95% ethanol for 6-10s, absolute ethanol I for 1min, absolute ethanol II for 1min, xylene I for 2min, and toluene I for 2min.
6) Sealing piece: the neutral resin is used for sealing the sheet, so that bubbles are avoided in the process, and the sheet is naturally dried. (the neutral resin is sticky and can be mixed with the xylene according to the proportion of 1:1, so that the effect is better).
7) The radiograph was observed under a microscope to observe the change in tissue morphology.
2.6 statistical analysis
Statistical analysis of the data was performed using the SPASS 21.0 software. The data are expressed as mean.+ -. Standard deviation (x.+ -. S), the comparison between groups is single factor analysis of variance (onegay-ANOVA), and the comparison between groups is LSD-t test. Check level α=0.05. The data graphs and statistics were processed using GraphPadPrism9 software.
3 results of experiments
3.1 influence of AAEO-MG on the weight of mice in the AD model
There was no significant difference in pre-dose body weight for each group of mice. After the end of the administration, the quality of mice in the model group, AAEO-ME group and AAEO-MG group was significantly reduced (P < 0.05) compared to the normal group, and the quality of mice in the AAEO group, AAEO-ME group and AAEO-MG group was significantly increased (P < 0.01) compared to the model group. Two-by-two comparisons were made between groups, where AAEO groups were statistically significant (P < 0.01) with AAEO-MG (middle) and AAEO (low) groups were statistically significant (P < 0.01).
TABLE 16 influence of AAEO-MG on body weight of AD model mice (x.+ -. S, n=8)
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Note that: in comparison with the normal group, * P＜0.05， *** p is less than 0.001; in comparison with the set of models, # P＜0.05， ## P＜0.01， ### P＜0.001。
3.2 Effect of AAEO-MG on skin lesions in mice of the AD model
The skin damage score was recorded every 2 days during the experiment according to the scoring criteria of table 15, and the mice skin damage score graph was plotted as shown in fig. 12. The mice in the other groups had increased skin loss scores (P < 0.05) compared to the normal group and significantly decreased AAEO, AAEO-ME and AAEO-MG dosing groups (P < 0.01) compared to the AD model group. The AAEO-MG (low) and the AAEO-MG (high) groups were statistically significant (P < 0.01), and the AAEO group and the AAEO-MG group (except the AAEO-MG (low)) were statistically significant (P < 0.001).
As shown in fig. 13, the skin tissue structure of the mice in the blank group was substantially normal. Skin lesions such as erythema, dryness, scaling, pimple, crusting and the like were seen in the skin of the mice of the model group. After the microemulsion and the gel are coated for 7d, the administration groups have different degrees of administration effects. The AAEO group mice had partial erythema and scaling on the backs. The AAEO-ME group showed significant erythema. The mice in each dose group of AAEO-MG had a small amount of scale on the backs and no significant erythema. Wherein AAEO-MG (high) group skin is smoother.
TABLE 17 influence of AAEO-MG on skin lesions in AD micen＝8)
Grouping Skin damage scoring
Normal group 0.00±0.00
Model group 11.71±0.31 ***
Mugwort leaf volatile oil group 6.31±1.06 ***###
Folium artemisiae argyi volatile oil microemulsion group 3.75±1.75 ***###
Folium artemisiae argyi volatile oil microemulsion gel low-dose group 4.88±1.36 ***###
Dosage group in mugwort leaf volatile oil microemulsion gel 3.63±1.60 ***###
High dose set of artemisia argyi volatile oil microemulsion gel 2.63±1.92 ***###
Note that: in comparison with the normal group, * P＜0.05， *** p is less than 0.001; in comparison with the set of models, # P＜0.05， ## P＜0.01， ### P＜0.001。
3.3 Effect of AAEO-MG on the symptoms of scratching in mice of the AD model
The number of mouse scratches was substantially identical to the skin lesions of the mice. The number of scratching times was somewhat reflective of the modeling status of AD-like mice, as shown in table 18. After the treatment by the medicine, the symptoms of the modeling areas of the mice in other groups are obviously improved except the normal group and the model group, the erythema area and punctate exudation are reduced, and the surface state of the skin is obviously improved. FIG. 14 shows that the number of scratches per group was significantly increased (P < 0.05) compared to the normal group, and significantly decreased (P < 0.001) compared to the model group.
TABLE 18 influence of AAEO-MG on the number of scratching in AD micen＝8)/>
Note that: in comparison with the normal group, * P＜0.05， ** P＜0.01， *** p is less than 0.001; in comparison with the set of models, # P＜0.05， ## P＜0.01， ### P＜0.001。
3.4 Effect of AAEO-MG on ear swelling degree in AD model mice
The right ears of different groups of mice are externally coated with DNCB solution with the concentration of 0.2%, and after the experiment is finished, the ears with the same area of the left auricle and the right auricle of the mice are respectively weighed and weighed. Ear swelling degree = right ear mass (mg) -left ear mass (mg). As shown in table 19, fig. 15, the auricle of the right ear of the model group mice was significantly thickened compared to the ear swelling degree of the normal group, and significant significance was achieved (P < 0.01). The AAEO (middle) and AAEO (high) groups showed reduced ear swelling compared to the model group, and the difference was statistically significant (P < 0.01).
TABLE 19 influence of AAEO-MG on ear swelling degree in AD micen＝8)
Grouping Number of scratching times
Normal group 0.65±0.14
Model group 12.80±1.17 ***
Mugwort leaf volatile oil group 10.70±1.12 **##
Folium artemisiae argyi volatile oil microemulsion group 10.75±0.69
Folium artemisiae argyi volatile oil microemulsion gel low-dose group 10.67±0.66
Dosage group in mugwort leaf volatile oil microemulsion gel 9.55±1.01 **##
High dose set of artemisia argyi volatile oil microemulsion gel 9.19±1.16 **##
Note that: in comparison with the normal group, * P＜0.05， ** p is less than 0.01; in comparison with the set of models, # P＜0.05， ## P＜0.01。
3.5 influence of AAEO-MG on organ coefficients of AD model mice
After the experiment is finished, mice are sacrificed to obtain the weights of all organs, and the organ coefficients are calculated. As shown in table 20, the organ coefficients of liver, kidney and thymus were not significantly significant compared with the normal group. There was an increasing trend but no statistical significance compared to the model group. The heart and spleen coefficients of AD model mice were elevated compared to the normal group (P < 0.01), demonstrating that the onset of atopic dermatitis is immune-related.
TABLE 20 influence of AAEO-MG on organ coefficients of AD micen＝8)/>
Note that: in comparison with the normal group, ** P＜0.01
3.6 Effect of AAEO-MG on the immunological serum of mice in the AD model
As shown in fig. 16, the level of IgE, IL-1 β was significantly elevated in the peripheral blood of mice in the model group (P < 0.001) compared to the normal group, indicating successful modeling of AD model mice. Compared with the model group, the AAEO-ME group and the AAEO-MG group can obviously reduce the peripheral blood IgE level (P < 0.05), which shows that the mugwort leaf volatile oil can reduce the inflammatory symptoms of mice and reduce the production and release of IgE and IL-1 beta.
TABLE 21 influence of AAEO-MG on the immunological serum of AD micen＝8)
Grouping IgE IL-1β
Normal group 16.69±3.89 28.10±3.20
Model group 46.64±5.00 *** 65.57±5.23 ***
Mugwort leaf volatile oil group 37.00±4.82 ***## 45.02±1.58 ***###
Folium artemisiae argyi volatile oil microemulsion group 34.87±7.32 ***### 42.88±3.87 ***###
Folium artemisiae argyi volatile oil microemulsion gel low-dose group 26.38±5.27 **### 41.28±3.09 ***###
Dosage group in mugwort leaf volatile oil microemulsion gel 25.10±4.17 *### 33.34±3.63 ###
High dose set of artemisia argyi volatile oil microemulsion gel 21.92±5.20 ### 34.34±3.16 ###
Note that: in comparison with the normal group, * P＜0.05， ** p is less than 0.01; in comparison with the set of models, # P＜0.05， ## P＜0.01， ### P＜0.001
3.7 Effect of AAEO-MG on expression of inflammatory factor on Back skin lesions in AD model mice
As shown in FIG. 17, the levels of TNF-. Alpha., IL-6, IL-4, IL-5, IL-13, IFN-. Gamma.were all significantly elevated (P < 0.05) in the back skin of the mice in the atopic dermatitis model group compared to the normal group. The differences were statistically significant in that TNF-. Alpha., IL-6, IL-4, IL-5, IL-13 levels were significantly reduced (P < 0.01) in the back skin of mice in the AAEO group, AAEO-ME group, AAEO-MG (low), AAEO-MG (middle) and AAEO-MG (high) groups, as compared to the model group. The comparison between groups showed that the levels of IL-5 and IL-13 were decreased (P < 0.05) in the dorsal skin of mice in the AAEO-MG (middle) group and AAEO-MG (high) group, as compared with those in the AAEO group. IL-6, IL-4 levels were reduced (P < 0.05) in the dorsal skin of mice in the AAEO-MG (high) group compared to the AAEO-ME group.
TABLE 22 influence of AAEO-MG on mRNA expression of Back skin lesion factor in AD micen＝3)
Grouping TNF-α IL-6 IL-4 IL-5 IL-13 IFN-γ
Normal group 0.97±0.22 1.00±0.03 0.93±0.08 1.10±0.12 0.70±0.16 1.67±0.07
Model group 1.62±0.20 ** 1.26±0.09 * 2.20±0.12 *** 2.23±0.19 *** 1.60±0.19 *** 2.43±0.20 *
Mugwort leaf volatile oil group 0.88±0.27 ## 0.86±0.05 ### 1.86±0.09 ***# 1.74±0.08 **# 0.89±0.11 ## 2.14±0.19
Folium artemisiae argyi volatile oil microemulsion group 0.86±0.14 ## 1.01±0.07 # 1.84±0.10 ***# 1.62±0.30 *## 0.86±0.09 ## 1.73±0.11 #
AAEO-MG low dose group 0.71±0.13 ### 0.90±0.17 ## 1.45±0.20 **### 1.52±0.10 ## 0.60±0.03 ### 1.95±0.40
Dosage group in AAEO-MG 0.57±0.13 #### 0.94±0.06 ## 1.16±0.07 ### 1.31±0.13 ### 0.66±0.11 ### 1.58±0.22 ##
AAEO-MG high dose group 0.57±0.14 #### 0.70±0.05 *#### 1.00±0.09 ### 1.15±0.05 ### 0.53±0.67 ### 1.82±0.24
Note that: in comparison with the normal group, * P＜0.05， ** p is less than 0.01; in comparison with the set of models, # P＜0.05， ## P＜0.01， ### P＜0.001
3.8 influence of AAEO-MG on the Biochemical index of mice in the AD model
Glutamic-pyruvic transaminase (AST), glutamic-pyruvic transaminase (ALT), urea nitrogen (BUN), creatinine (CRE) are indicators for judging liver and kidney functions, respectively. The four indexes are higher than the normal value, and the damage of liver and kidney functions is indicated. The results in Table 23 and FIG. 18 show that the BUN and CRE contents of the AD model group were significantly higher than those of the normal group (P < 0.05), and that the AST and ALT contents tended to rise. The AST and BUN contents of the AAEO-MG (low) and AAEO-MG (high) groups are significantly lower than those of the AD model group (P < 0.05). The CRE content of the AAEO-MG (high) group was significantly lower than that of the AD model group (P < 0.01). The AST, ALT, BUN, CRE content of the remaining groups was not significant to the normal and model groups.
TABLE 23 influence of AAEO-MG on Biochemical index of AD micen＝8)
Grouping AST(U/L) ALT(U/L) BUN(mmol·L -1 ) CRE(μmol·L -1 )
Normal group 14.67±1.53 27.78±4.02 3.88±0.54 14.43±2.40
Model group 17.05±2.06 30.45±4.58 5.61±0.75 ** 17.94±1.38 *
Mugwort leaf volatile oil group 14.83±1.67 27.95±2.05 4.94±0.78 15.58±0.88
Folium artemisiae argyi volatile oil microemulsion group 14.96±1.06 29.94±1.55 4.68±0.55 16.23±1.75
Folium artemisiae argyi volatile oil microemulsion gel low-dose group 13.45±1.45 ## 29.50±3.05 4.46±0.96 # 16.27±1.39
Dosage group in mugwort leaf volatile oil microemulsion gel 14.95±1.78 27.53±3.52 4.60±0.70 15.91±2.32
High dose set of artemisia argyi volatile oil microemulsion gel 13.01±1.02 ## 26.68±2.57 3.85±0.69 ## 15.06±2.13 ##
Note that: in comparison with the normal group, * P＜0.05， ** p is less than 0.01; in comparison with the set of models, # P＜0.05， ## P＜0.01。
3.9 influence of AAEO-MG on pathological histomorphology of mice in AD model
After the experiment is finished, the skin at the back of the mouse and organs such as heart, spleen, liver and kidney are taken, and HE pathological sections are prepared and observed under a lens, and the tissue morphology changes are shown in figures 19-23.
HE section of skin (x 200) shows: the back skin structure and the morphology of the normal mice are normal, the boundary between the epidermis layer and the dermis layer is obvious, and each layer of the epidermis has no inflammatory reaction and has no phenomena of vasodilation and inflammatory cell infiltration. The skin epidermis of the back of the mice in the model group is hyperkeratosis and hypokeratosis, the thickening of the thorn layer is obviously accompanied with spongiform edema, the blood vessel expansion and congestion phenomenon appears in the shallow dermis layer, and a large amount of inflammatory cells infiltrate, and obvious chronic additive change is realized. The administration groups have different degrees of skin injury reduction, wherein the reduction of the skin injury inflammation of the AAEO-MG low, medium and high dose groups is more obvious, the infiltration of inflammatory cells is reduced, and the spongiform edema is reduced. The AAEO and AAEO-ME groups had a relatively greater inflammatory cell infiltration than the AAEO-MG group, but were lighter than the model group.
HE section of heart (x 200) shows: the normal group, the model group, the AAEO-ME group, the AAEO-MG (low) group, the AAEO-MG (middle) group and the AAEO-MG (high) group have clear myocardial transverse lines, normal cell morphology and good myocardial fiber arrangement.
HE section of spleen (x 200) shows: the normal group spleen has clear red marrow and white marrow boundary, the lymphocyte arrangement is compact, the boundary of the model group is fuzzy, and the lymphocyte arrangement is dispersed. The spleen state and model group of AAEO group, AAEO-ME group, AAEO-MG (low) group, AAEO-MG (middle) group, and AAEO-MG (high) group are preferable.
HE section of liver (x 200) shows: normal group, model group, AAEO-ME group, AAEO-MG (low) group, AAEO-MG (middle) group, AAEO-MG (high) liver cell has no edema, liver cable is orderly arranged, and liver lobule structure is clear.
HE section of kidney (x 200) shows: the glomeruli of the normal group, model group, AAEO-ME group, AAEO-MG (low) group, AAEO-MG (middle) group, AAEO-MG (high) group were intact and normal in structure.
Example 2 successfully induced the model of atopic dermatitis, the percutaneous administration of the mugwort leaf volatile oil and the volatile oil microemulsion gel revealed that the mugwort leaf volatile oil microemulsion gel can effectively treat atopic dermatitis, has better treatment effect than mugwort leaf volatile oil and has no toxic or side effect in percutaneous administration through the observation of the weight, back skin damage condition, scratching times, ear swelling degree and organ coefficient, immunological serum index, back skin damage mRNA expression and biochemical index of mice.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims (10)

1. The artemisia argyi volatile oil microemulsion is characterized by comprising the following components in parts by weight:
1.5 to 1.7 parts of mugwort leaf volatile oil, 1.15 to 1.25 parts of surfactant, 0.15 to 0.25 part of medium chain triglyceride and 46.8 to 47.2 parts of water.
2. The mugwort leaf volatile oil microemulsion of claim 1, comprising the following components in parts by weight:
1.7 parts of mugwort leaf volatile oil, 1.15 parts of surfactant, 0.15 part of medium chain triglyceride and 47 parts of water.
3. The mugwort essential oil of claim 1 or 2, wherein the surfactant comprises tween-80.
4. A method for preparing the mugwort essential oil as claimed in any of claims 1 to 3, comprising the steps of:
mixing the mugwort leaf volatile oil, the surfactant and the medium chain triglyceride to obtain a mixture;
mixing the obtained mixture with water, and emulsifying to obtain folium Artemisiae Argyi volatile oil microemulsion;
The temperature of the emulsification is 28-32 ℃.
5. The method according to claim 4, wherein the rotational speed of the emulsification is 450 to 550rpm.
6. A mugwort leaf volatile oil microemulsion gel, which is characterized by comprising mugwort leaf volatile oil microemulsion according to any one of claims 1-3 and carbomer 940 solution; the mass ratio of the mugwort leaf volatile oil microemulsion to the carbomer 940 solution is 4:1;
the mass percentage of carbomer 940 in the carbomer 940 solution is 2%.
7. A method for preparing the mugwort leaf volatile oil microemulsion gel according to claim 6, which is characterized in that mugwort leaf volatile oil microemulsion and carbomer 940 solution are mixed to obtain mugwort leaf volatile oil microemulsion gel.
8. Use of a mugwort leaf volatile oil microemulsion according to any one of claims 1 to 3 or a mugwort leaf volatile oil microemulsion gel according to claim 6 for the manufacture of a medicament for the treatment of atopic dermatitis.
9. Use of a mugwort leaf volatile oil microemulsion according to any one of claims 1 to 3 or a mugwort leaf volatile oil microemulsion gel according to claim 6 for the manufacture of a medicament for the treatment of skin lesions.
10. Use of a mugwort leaf volatile oil microemulsion according to any one of claims 1 to 3 or a mugwort leaf volatile oil microemulsion gel according to claim 6 for the manufacture of a medicament for the treatment of ear swelling.
CN202310469922.6A 2023-04-27 2023-04-27 Mugwort leaf volatile oil microemulsion gel and preparation method and application thereof Pending CN117379371A (en)

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