CN117695331A

CN117695331A - Magnolia flower volatile oil, extraction method, quality detection method and Magnolia flower volatile oil microemulsion

Info

Publication number: CN117695331A
Application number: CN202311788800.XA
Authority: CN
Inventors: 果秋婷; 张小飞
Original assignee: Xianyang Vocational Technical College
Current assignee: Xianyang Vocational Technical College
Priority date: 2023-12-25
Filing date: 2023-12-25
Publication date: 2024-03-15

Abstract

The invention provides an extraction method of magnolia flower volatile oil. The invention also provides the application of the volatile oil prepared by the method in preparing medicines for nasal administration for treating allergic rhinitis. The invention also provides a method for detecting the volatile oil. The invention also provides the magnolia flower volatile oil microemulsion for treating allergic rhinitis. The magnolia flower volatile oil plays a role in treating allergic rhinitis by regulating related proteins on a PPAR-gamma/NF-kappa B signal pathway; the preparation process of the magnolia flower volatile oil microemulsion is stable, simple and controllable, has strong anti-inflammatory and antiallergic effects, and has definite curative effect on allergic rhinitis rat models, and good stability and safety.

Description

Magnolia flower volatile oil, extraction method, quality detection method and Magnolia flower volatile oil microemulsion

Technical Field

The invention relates to magnolia flower volatile oil, an extraction method, a quality detection method and magnolia flower volatile oil microemulsion thereof.

Background

Allergic Rhinitis (AR) is a worldwide health problem with an average global incidence of 10% to 25% and is considered to be "epidemic of the 21 st century". The prevalence of Chinese allergic rhinitis has also increased in the last decade. The prevalence of AR has a significant impact on a variety of factors including genetic factors, personal diet and living environment, and neuropsychiatric factors. Although the disease is far from endangering life health, the related uncomfortable symptoms caused by the disease, such as intermittent nasal obstruction of nose, allergic conjunctivitis of eyes and other organ symptoms, such as asthma, sore throat and other allergic phenomena, can cause a series of problems of sleep quality reduction, inappetence, emotional disorder and the like of patients, and seriously influence physical and mental health, work and study life conditions of people. In summary, the prevention and treatment of allergic rhinitis has become a public health problem commonly faced worldwide.

Flos Magnoliae has a variety of pharmacological effects, which have been demonstrated in experimental studies, but clinical drugs are focused on the treatment of nasal disease studies, with less attention paid to others. Recent studies of magnolia flower are currently mainly focused on volatile oils, in particular on their chemical composition and pharmacology. Volatile oil is a main active ingredient of magnolia flower with therapeutic value, has rich chemical components and wide pharmacological actions, is mainly researched and developed by anti-inflammatory and antiallergic actions, and is mainly used for treating respiratory diseases such as acute and chronic rhinitis, allergic rhinitis, nasosinusitis, asthma and the like clinically. However, the flos Magnoliae volatile oil has few practical applications in clinic, and the research on the action mechanism is not completely clear. The magnolia flower has more varieties and complex sources, the components and the content of the magnolia flower have larger differences due to the harvesting planting places, varieties and the like, and meanwhile, the study on the chemical characteristics of the magnolia flower is less, so that the identification and quality control of the magnolia flower or related products are difficult, and the utilization of medicinal material resources is influenced.

The chemical components of magnolia flower are mainly divided into two major categories, namely fat-soluble components and water-soluble components. Including volatile oils, lignans, alkaloids, etc. The flos Magnoliae volatile oil is used as main medicinal component of flos Magnoliae medicinal material, has abundant chemical components and wide medicinal effects, and mainly focuses on the research on anti-inflammatory and antiallergic effects. Is generally combined with cocklebur fruit, peppermint, asarum herb and other medicinal materials, is mainly used for treating rhinitis, nasosinusitis, allergic rhinitis and related diseases such as asthma and the like, has good treatment effect, and can obviously improve clinical uncomfortable symptoms.

Literature reports and a large number of experiments show that the magnolia flower volatile oil has obvious anti-inflammatory effect. Xiong Tianqin et al are based on the guinea pig allergic rhinitis model, and the study shows that: the flos Magnoliae volatile oil can relieve symptoms of allergic rhinitis of guinea pig such as nasal itching and sneeze, and relieve congestion and swelling of topical mucosa in nasal cavity. Wu Min the volatile oil of flos Magnoliae is prepared into nano liposome nose drops for clinical study of treating infantile allergic rhinitis, and observation and detection of curative effect. The results show that the total curative effect of the treatment group on the main symptoms such as sneeze, nasal obstruction and the like during the treatment period is better than that of the control group, and the nasal mucosa edema, pallor and turbinate swelling degree of the children patients are reduced to different degrees after the administration treatment. The researches of administration and the like find that the regulation and control disorder of Th1 and Th2 cells has a certain relation with the occurrence of allergic rhinitis, and the research results show that the magnolia flower volatile oil can reduce the release of histamine and maintain the dynamic balance state of Th1/Th2 by improving the level of interleukin-12 (IL-12) and interferon-gamma (IFN-gamma), thereby achieving the aim of treating the allergic rhinitis.

The traditional method for extracting volatile oil is a steam distillation method. The method uses water as solvent, can avoid the influence of certain organic solvents on volatile oil components or content in the extraction process, and has the characteristics of simple extraction equipment, simple and safe operation, low cost, no pollution to the environment and the like. However, steam distillation is only used for extracting volatile substances which can be distilled by steam without being destroyed, insoluble or poorly soluble in water, and the boiling point of these components is generally above 100 ℃.

Disclosure of Invention

The technical scheme of the invention provides magnolia flower volatile oil, an extraction method, a quality detection method and magnolia flower volatile oil microemulsion thereof.

The invention provides a method for extracting magnolia flower volatile oil, which is characterized by comprising the following steps of: the method takes magnolia flower as a raw material, adopts a cellulase enzymolysis auxiliary steam distillation method for extraction, and comprises the following steps:

a. weighing magnolia flower medicinal materials, crushing into coarse powder, adding water, adjusting the amount of cellulase to be 0.125-2.000%, adjusting the pH to be within the range of 5.0-5.5, and carrying out enzymolysis for 30-110 min at the temperature of 35-75 ℃;

b. adding the enzymolysis liquid obtained in the step a into a volatile oil extractor, and extracting volatile oil by adopting a steam distillation method.

Wherein the enzyme addition amount in the step a is 0.125-0.250%, the enzymolysis time is 45-65 min, and the enzymolysis temperature is 30-50 ℃.

Wherein the enzyme addition amount in the step a is 0.125%, the enzymolysis time is 65min, and the enzymolysis temperature is 50 ℃.

The volatile oil contains farnesol and eucalyptol, and the percentage content of the volatile oil is as follows:

farnesol is not less than 12.0%, and eucalyptol is not less than 8.0%.

The invention provides application of the volatile oil in preparing medicines for nasal administration for treating allergic rhinitis.

The invention provides a method for detecting volatile oil, which adopts GC-MS analysis and detection and comprises the following specific steps:

a. preparation of sample solution: accurately sucking 100 μl of volatile oil respectively, adding anhydrous diethyl ether to a 10mL brown volumetric flask, adding anhydrous sodium sulfate to remove water, sucking sample solution with 1mL syringe, filtering with 0.22 μm filter membrane, and placing filtrate in sample injection bottle;

b. GC-MS detection, chromatographic conditions are:

gas chromatography conditions: agilent HP-5ms (30 m×250 μm×0.25 μm) capillary column, high purity He, 3.0 μl sample injection amount, and flow rate of 1mL·min ^-1 The split ratio is 40:1, the temperature is programmed to be 55 ℃, the temperature is 8 ℃ and min-1 is programmed to be 176 ℃, and the holding time is as follows: 3min,20 ℃ min-1 to 250 ℃ for holding time: 20min;

mass spectrometry conditions: an EI ion source; electron energy 70ev; the ion source temperature is 230 ℃; MS quadrupole temperature 150 ℃; delaying the solvent for 3min; mass spectrum scanning mode full scanning, scanning range is 30-400 amu.

The invention provides a magnolia flower volatile oil microemulsion for treating allergic rhinitis, which is prepared from volatile oil, an emulsifying agent, an oil phase and an auxiliary emulsifying agent, wherein the emulsifying agent is one or more than two of Tween-80, tween-20, EL-40 and RH-40; the auxiliary emulsifier is one or more than two of polyethylene glycol 400, glycerol, absolute ethyl alcohol and 1, 2-propylene glycol; the oil phase is as follows: oleic acid, IPP, IPM, or a mixture of two or more thereof; wherein the volume ratio of the emulsifier to the mixed oil phase is as follows: 7-9:1-3; the volume ratio of the volatile oil to the oil phase is as follows: 1-3:1-3; the mass ratio of the emulsifier to the auxiliary emulsifier is as follows: 1-4:1-2.

Wherein the emulsifier is EL-40, the auxiliary emulsifier is polyethylene glycol 400, and the oil phase is IPM, wherein the mass ratio of EL-40 to polyethylene glycol 400 is 2:1.

Wherein the granularity of the flos magnoliae volatile oil microemulsion is 14.27+/-0.03 nm (n=3), and the average value of PDI is 0.0941 +/-0.31 (n=3); the Zeta potential of the essential oil microemulsion was measured to be-0.3585 + -0.12 mV (n=3).

The invention provides a method for preparing the microemulsion, which comprises the following steps:

a. preparing a mixed oil phase: mixing flos Magnoliae volatile oil and IPM at 30deg.C to obtain oil phase with Km value=2:1;

b. uniformly mixing EL-40 and PEG-400 according to the ratio of 2:1, and taking the mixture as a mixed emulsifier;

c. mixing the mixed emulsifier and the mixed oil phase in the mass ratio of 9:1 uniformly;

d. dripping the water phase into the mixed oil phase, adding the dripping edge, and stirring to prepare O/W type microemulsion;

wherein the mixed oil phase accounts for 2.63 percent, the water phase accounts for 73.69 percent of the total amount of the microemulsion, and the mixed emulsifier accounts for 23.68 percent.

The magnolia flower volatile oil plays a role in treating allergic rhinitis by regulating related proteins on a PPAR-gamma/NF-kappa B signal pathway; the prepared magnolia flower volatile oil microemulsion has stable, simple and controllable preparation process, strong anti-inflammatory and antiallergic effects, definite curative effect on allergic rhinitis rat models, and better stability and safety.

Drawings

The extraction of magnolia flower volatile oil by different methods shown in figure 1 shows that the yield of the magnolia flower volatile oil is @ oiln＝3)；

FIG. 2 is a graph of the response of the extraction process of Magnolia liliflora volatile oil to influencing factors;

FIG. 3 pseudo ternary area diagram formed by emulsifiern＝3)；

FIG. 4 pseudo ternary area diagram formed by the co-emulsifiern＝3)；

FIG. 5 pseudo ternary area diagram of the auxiliary oil phasen＝3)；

FIG. 6 pseudo-ternary area diagrams of different Km values [ ]n＝3)；

FIG. 7 pseudo ternary area diagram at different preparation temperaturesn＝3)；

FIG. 8 is a transmission electron microscope image;

FIG. 9 is a microemulsion type identification chart;

fig. 10 microemulsion particle size and Zeta potential profile (n=3);

FIG. 11 expression levels of IL-1. Beta., IL-6 and TNF-alpha. In rat serumn=6) (note: # #. Compared with the blank group, there was a significant difference (p < 0.01); * : significant differences (p < 0.05) compared to model group;

FIG. 12 expression level of protein related to nasal mucosa tissue of ratn=3); (note #: there was a significant difference (p < 0.05) compared to the blank group; there was a significant difference (p < 0.01) compared to the model group; there was a significant difference (p < 0.05) compared to the microemulsion formulation group;

FIG. 13 expression levels of IL-1. Beta., IL-6 and TNF-alpha. In the nasal wash of ratsn＝6)；

FIG. 14 shows the expression levels of IL-1. Beta., IL-6 and TNF-alpha. In rat serumn=6) (note: #: compared with the blank group, there was a significant difference (p < 0.05); * : significant differences (p < 0.05) compared to model group;

FIG. 15 shows the expression level and the band pattern of the protein related to the nasal mucosa tissue of ratsn=3) (note: #: compared with the blank group, there was a significant difference (p < 0.05); * : there was a significant difference (p < 0.05)) from the model group.

Detailed Description

Example 1 extraction Process of Magnolia volatile oil of the present invention

And selecting indexes such as farnesol, eucalyptol and oil extraction rate for comprehensive scoring.

1. Extracting flos Magnoliae volatile oil by different methods

Extracting flos Magnoliae volatile oil by 4 methods. The magnolia flower volatile oil is extracted through different extraction processes, and the extraction processes are optimized, so that the content of medicinal components and the extraction rate of volatile oil are improved, more time-saving, energy-saving, efficient and simple process parameters are obtained, and a basis is provided for determining the volatile oil extraction process parameters and controlling related quality standards.

1 Experimental method

1. Steam distillation method for extracting flos Magnoliae volatile oil

Weighing 50g flos Magnoliae (dried bud of Magnolia officinalis Magnolia denudate Desr. Of Magnoliaceae), pulverizing into coarse powder, placing into 2000mL round bottom flask, adding 10 times of water, and pulverizing ceramic chip (to prevent bumping); the round bottom flask is respectively connected with the volatile oil extractor and the condenser, and then is placed in an electric heating sleeve for slow heating, and the micro-boiling state is kept all the time in the extraction process; when the first drop of liquid is dropped out, recording oil quantities (5, 10, 20, 40, 60, 90, 120, 180, 240 and 300 minutes) obtained in different time periods until the volume of the volatile oil is not changed, stopping heating, standing and cooling, and collecting the volatile oil to obtain a sample X1.

2. Ultrasonic assisted steam distillation method for extracting flos Magnoliae volatile oil

Weighing 50g of flos Magnoliae (dried bud of Magnolia officinalis Magnolia denudate Desr. Of Magnoliaceae), pulverizing into coarse powder, placing in a 2000mL round bottom flask, adding 10 times of water, ultrasonic power 80%, and ultrasonic treating for 20min, and performing operation according to the method of step 1) from "round bottom flask is connected with volatile oil extractor and condenser tube respectively" to obtain sample X2.

3. Microwave extraction of flos Magnoliae volatile oil

Weighing 50g of flos Magnoliae (dried bud of Magnolia officinalis Magnolia denudate Desr. Of Magnoliaceae), pulverizing into coarse powder, placing in 2000mL round bottom flask, adding 10 times of water, setting microwave power 300w (optimized process result of earlier stage research), placing into broken ceramic chip (for preventing bumping), placing into ultrasonic microwave extractor, introducing into condenser tube, adding appropriate amount of water into thermal insulation jacket of volatile oil extractor, extracting until volatile oil volume is unchanged, stopping heating, standing, cooling, and collecting volatile oil to obtain sample X3.

4. Extraction of magnolia flower volatile oil by cellulase enzymolysis assisted steam distillation method

Weighing 50g of flos Magnoliae (dried bud of Magnolia officinalis Magnolia denudate Desr. Of Magnoliaceae), pulverizing into coarse powder, placing into a 2000mL round bottom flask, adding 10 times of water, cellulose 0.25%, adjusting pH to 5.0-5.5, performing enzymolysis for 30min at 35 deg.C (early stage research optimization process result), placing into broken ceramic chip (to prevent bumping), and operating under method of section "2.1" of the chapter from "round bottom flask is connected with volatile oil extractor and condenser tube" respectively, to obtain sample X4.

2. GC-MS analysis of volatile oils

2.1 preparation of samples

Precisely sucking 100 mu L of each of the volatile oil samples X1, X2, X3 and X4, fixing the volume of anhydrous diethyl ether to a 10mL brown volumetric flask, adding a proper amount of anhydrous sodium sulfate to remove water, sucking a sample solution by a 1mL syringe, passing through a filter membrane of 0.22 mu m, and placing the filtrate in a sample injection bottle for later use.

2.2 Establishment of GC-MS conditions

The study adopts a gas chromatography-mass spectrometry (GC-MS) method to measure the chemical components of the magnolia flower volatile oil, and Data Analysis is carried out by applying Data Analysis software.

Gas chromatography conditions: agilent HP-5ms (30 m×250 μm×0.25 μm) capillary column, high purity He, 3.0 μl sample injection amount, and flow rate of 1mL·min ^-1 The split ratio is 40:1, the temperature is programmed to be 55 ℃, and the temperature is 8 ℃ and min ^-1 Heating to 176 deg.C (holding time: 3 min), 20 deg.C/min ^-1 Heating to 250deg.C (retention time: 20 min).

Mass spectrometry conditions: an EI ion source; electron energy 70ev; the ion source temperature is 230 ℃; MS quadrupole temperature; delaying the solvent for 3min; mass spectrum scanning mode full scanning, scanning range is 30-400 amu.

2.3 identification of Magnolia volatile oil Compounds

The resolved mass spectra were retrieved in standard NIST14 library and Database standard spectrum library and checked for matching, retention index and literature reported substance. Determination of Retention index n-alkane (C) was determined using the same gas chromatography conditions ₈ ～C ₄₀ ) According to the retention time of normal alkane, the retention index of each volatile compound in the magnolia flower volatile oil is calculated, and the formula is as follows:

wherein: t is t _R (x)、t _R (n)、t _R (n+1) represents the retention time, the carbon number n and the carbon number n+1 of the normal alkane to be measured and t, respectively _R (n)<t _R (x)<t _R (n+1)。

3. Results and analysis

3.1 extraction yield from different Processes

The extraction process is used for calculating the extraction rate after extracting the volatile oil, and the formula is as follows:

wherein the extraction rate by a steam distillation method is 3.00%; the extraction rate of the ultrasonic assisted steam distillation method is 3.40 percent; the extraction rate by the microwave method is 1.20%; the extraction rate of the cellulase enzymolysis auxiliary steam distillation method is 3.60 percent, and is shown in figure 1.

3.2 analysis of volatile oil composition of Magnolia flower

The composition of the four different extraction methods was identified using Database and NIST14 standard databases, respectively, and the relative amounts of the index components farnesol and eucalyptol were high, respectively, and were found to be steam distillation (11.108%, 9.349%), ultrasonic-assisted steam distillation (12.536%, 10.548%), microwave (6.647%, 16.884%), and cellulase-assisted steam distillation (12.298%, 8.803%), respectively, as shown in tables 1 to 4.

TABLE 1 analysis of volatile oil fractions of flos Magnoliae by steam distillation

TABLE 2 analysis of volatile oil fractions from flos Magnoliae by ultrasound assisted steam distillation

TABLE 3 analysis of volatile oil components of flos Magnoliae by microwave extraction

TABLE 4 analysis of volatile oil components from flos Magnoliae by cellulase enzymolysis assisted steam distillation

TABLE 5 analysis of common Components of Magnolia volatile oil extracted by different extraction methods

4 summary and discussion

The flos Magnoliae volatile oil has complex components, and has different components and contents by different extraction processes. According to the experimental results, the method shows that: the oil yield of the four different extraction methods is sequentially that the cellulase enzymolysis auxiliary steam distillation method (T4) > the ultrasonic auxiliary steam distillation method (T2) > the steam distillation method (T1) > the microwave method (T3). The four methods can improve the oil yield of the magnolia flower volatile oil, wherein the cellulase enzymolysis auxiliary steam distillation method is more remarkable, and compared with the steam distillation method of the traditional extraction method, the method is 1.2 times of that of the traditional extraction method. The total components of flos Magnoliae volatile oil prepared by the 4 methods are 16, which respectively account for 60.725% (steam distillation method), 66.344% (ultrasonic assisted steam distillation method), 72.890% (microwave method) and 64.870% (cellulase enzymolysis assisted steam distillation method) of the identified total components. The chemical components and relative contents of the volatile oil of magnolia flower can be found to have certain differences according to the extraction method, and the main differences are represented by the content differences of index components including farnesol and eucalyptol, namely, a steam distillation method, an ultrasonic-assisted steam distillation method and a cellulase enzymolysis-assisted steam distillation method, wherein the relative contents of farnesol are the first, the second is eucalyptol, and the first is eucalyptol in the microwave method, and the second is farnesol. In the extraction process, the magnolia flower volatile oil extracted by the other 3 methods except the microwave method can emit strong fragrance, the color is light yellow, the taste is not very strong in the extraction process by the microwave method, and the color of volatile oil liquid is between milky white and yellowish. In summary, the extraction process of the cellulase enzymolysis auxiliary steam distillation method is found to be optimal by comprehensively considering the content change of farnesol and eucalyptol and the oil yield as investigation indexes.

2. Optimization of magnolia flower volatile oil extraction process

And (3) performing single factor investigation on the influence factors such as enzyme adding amount, enzymolysis time, enzymolysis temperature and the like of the magnolia flower volatile oil by adopting a cellulase enzymolysis auxiliary steam distillation method, screening and verifying the technological parameters with great influence on the oil yield by combining with a Box-Behnken response surface, and optimizing the optimal extraction process.

1 Experimental method

1.1 Single factor investigation of Magnolia volatile oil extraction

Weighing 50g of flos Magnoliae crude powder, placing in 2000mL round bottom flask, adding 10 times of water and broken ceramic pieces (preventing bumping), respectively connecting volatile oil extractor and condenser tube, placing in electrothermal sleeve, and slowly heating to maintain micro-boiling state during extraction; recording oil obtaining amounts (5, 10, 20, 40, 60, 90, 120, 180, 240, 300 min) in different time periods until the first drop of liquid is dropped out, stopping heating when the volume of volatile oil in the extractor is not changed, standing and cooling, and collecting volatile oil.

1.1.1 Effect of enzyme addition amount

Firstly, the enzymolysis time is fixed for 30min, the enzymolysis temperature is 35 ℃, the enzyme addition amounts are respectively 0.125%, 0.250%, 0.500%, 1.000% and 2.000%, and the volatile oil is extracted according to the method under the section '2.1'.

1.1.2 Effect of enzymatic hydrolysis time

Firstly, fixing the enzyme adding amount of 0.250%, and extracting volatile oil according to the method of section 2.1, wherein the enzymolysis temperature is 35 ℃ and the enzymolysis time is 30min, 50min, 70min, 90min and 110min respectively.

1.1.3 Effect of enzymatic hydrolysis temperature

Firstly, fixing the enzyme adding amount to 0.250% and the enzymolysis time to 30min, wherein the enzymolysis temperature is 35 ℃, 45 ℃, 55 ℃, 65 ℃ and 75 ℃, and extracting volatile oil according to the method under the section '2.1'.

1.2 establishment of total composite score F

According to the earlier study, farnesol and eucalyptol are finally determined as anti-inflammatory index components, 10 minutes are used as total scores, the content of the index components accounts for 60 percent (30 percent of each of the two index components), the extraction rate accounts for 40 percent and are used as investigation indexes for weighted calculation, and the scores of the farnesol, the eucalyptol and the index components are overlapped to be used as total comprehensive scores F, so that the optimal extraction process is optimized. The calculation formula of the total composite score F is as follows:

F＝30％×X _{farnesol component content} +30％×X _{EucalyptolContent of ingredients} +40％×X _{Oil yield}

1.3Box-Behnken response surface method optimized optimal extraction process

On the basis of the Design of a Box-Behnken test, a 3-factor 3-level optimization test is designed by Design-expert.v-11 software by combining the result of a single-factor test, and the addition amount (A), the enzymolysis time (B) and the enzymolysis temperature (C) of enzyme are taken as independent variables, and the oil yield is taken as a response value. The factor level design is shown in table 6 below.

TABLE 6 response surface design factor level Table

2. Results and analysis

2.1 Single factor investigation results

2.1.1 Effect of enzyme addition on volatile oil extraction Rate

As shown in Table 7, the content percentage of the index components farnesol and eucalyptol and the oil yield were used as evaluation indexes, and the optimal enzyme addition amount was in the range of 0.125% -0.250%.

TABLE 7 extraction of Magnolia volatile oil-index component content and oil yield with different enzyme addition amounts

2.1.2 influence of enzymolysis temperature on volatile oil extraction rate

As shown in Table 8, the content percentage of the index components including farnesol and eucalyptol and the oil yield are used as evaluation indexes, and the optimal enzymolysis temperature range is 45-55 ℃.

TABLE 8 extraction of Magnolia volatile oil-index component content and oil yield at different enzymolysis temperatures

2.1.3 Effect of enzymolysis time on volatile oil extraction Rate

As shown in Table 9, the content percentage of the index components including farnesol and eucalyptol and the oil yield are used as evaluation indexes, and the optimal enzymolysis time range is 30-50 min.

TABLE 9 extraction of Magnolia volatile oil-index component content and oil yield at different enzymolysis times

2.2 Box-Behnken response surface analysis

2.2.1 response surface design results

Each factor and level were input to Design-Expert v-11 software, 17 sets of data were fitted for experiments, and three significant factors of enzyme addition (a), enzyme hydrolysis time (B), and enzyme hydrolysis temperature (C) were subjected to response surface test designs, and the results are shown in table 10.

Performing analysis of variance on experimental data by adopting Design-Expert v-11 software, and fitting the experimental data to obtain a multiple quadratic regression model equation:

Y＝3.01+1.7816A+0.0659B+0.0985C-0.0101AB-0.0112AC-0.0003BC-0.4030A ² -0.0003B ² -0.0007C ²

the results are shown in table 11, model p=0.0002<0.001, the chosen quadratic regression equation model is very significant and can be used to predict the outcome of the experiment. The mismatch term showed no significant effect, indicating that the error had no significant effect on the experiment. Determining the coefficient R ² = 0.9687, adjustment coefficient R ² adj= 0.8285 means that the 82.85% change in response value comes from the variable.

TABLE 10 response surface method test design and results

TABLE 11 Box-Behnken response surface test analysis of variance

Note that: * Representing significant (P < 0.05), representing extremely significant (P < 0.001)

2.2.2 influence factor analysis of response surface

The interactions between the factors can be illustrated by the three-dimensional plot of the response surface and the corresponding contour plot, as shown in FIG. 2, the contour is almost elliptical, indicating that the enzyme addition, the enzyme hydrolysis time, and the enzyme hydrolysis temperature interact with each other to varying degrees. The optimal test conditions obtained according to the analysis result of the software are as follows: the enzyme addition amount is 0.1250%, the enzymolysis time is 65.1252min, the enzymolysis temperature is 53.7056 ℃, and the comprehensive score prediction result of the volatile oil is 7.94769. Considering the actual situation, the optimal test conditions are adjusted as follows: the enzyme addition amount is 0.125%, the enzymolysis time is 65min, and the enzymolysis temperature is 50 ℃.

2.2.3 response surface test verification

Three parallel experiments were performed with the optimal experimental conditions adjusted and the volume of 3 volatile oils extracted was measured. The comprehensive scores (n=3) are 7.9663, 8.0031 and 8.1769 respectively, the average value is 8.0487 +/-0.1125, the test result is close to the predicted value 7.9476, the preferable technological parameters in the test are stable and reliable, and a reference can be provided for predicting the extraction process of the magnolia flower volatile oil.

3 knots

Taking the relative content of the oil yield and the index components as a common evaluation index, and examining single factors. The enzyme addition amount is between 0.125% and 0.250%, the relative component content of farnesol and eucalyptol is higher, the oil yield is also relatively higher, and when the enzyme addition amount is 0.125%, the relative component content of farnesol reaches 19.527%. The comprehensive score is higher when the enzymolysis time is 30-50 min, wherein the difference of farnesol and the oil yield is not obvious, and the obvious difference is represented by that the content of relative components of eucalyptol is higher than that of the eucalyptol when the enzymolysis is carried out for 50min, the comprehensive score is higher than that of other enzymolysis temperatures when the enzymolysis temperature is 55 ℃.

The section extracts magnolia flower volatile oil by a cellulase enzymolysis method assisted steam distillation method, examines the single factor influence of the volatile oil, designs a Box-Behnken response surface, analyzes the interaction among the enzyme addition amount, the enzymolysis time and the enzymolysis temperature, and optimizes the optimal test condition: the enzyme addition amount is 0.125%, the enzymolysis time is 65min, and the enzymolysis temperature is 50 ℃. The results of three parallel extraction show that the experiment has better repeatability, can improve the oil yield, has accurate and reliable process parameters, and provides guidance significance for large-scale extraction of the magnolia flower volatile oil.

In summary, the extraction process of the magnolia flower volatile oil comprises the following steps: weighing 50g of flos Magnoliae (dried bud of Magnolia officinalis Magnolia denudate Desr. Of Magnoliaceae), pulverizing into coarse powder, placing in 2000mL round bottom flask, adding 10 times of water, cellulose 0.125%, adjusting pH to 5.0-5.5, performing enzymolysis for 65min at 50deg.C, placing into broken ceramic chip (for preventing bumping), connecting volatile oil extractor and condenser tube to the round bottom flask, and slowly heating in electric heating jacket to keep micro-boiling state. Under the extraction process, the total comprehensive score of the magnolia flower volatile oil is highest and is 8.0487 +/-0.1125, so that the extraction process is the optimal extraction process.

Test example 2 preparation of the Magnolia volatile oil microemulsion preparation of the present invention

The microemulsion is a transparent or semitransparent solution system with lower viscosity and thermodynamic stability, which is formed by water phase, oil phase, surfactant and cosurfactant according to a certain proportion, can be divided into water-in-oil (W/O), oil-in-water (O/W) and biphasic continuous microemulsion at present, and has great development potential as a novel drug carrier. Because the magnolia flower volatile oil is considered to be fat-soluble and is insoluble in the aqueous traditional Chinese medicine volatile oil, the magnolia flower volatile oil is combined with a microemulsion drug delivery system in the chapter, and the influence of each factor on the microemulsion is inspected on the premise of reducing the emulsifier as much as possible, so that the small amount of the emulsifier is screened, and meanwhile, the prescription composition and the proportion are stable.

1. Study on microemulsion preparation process

The formation of the microemulsion is influenced by various factors, and in the research of this section, the factors such as an emulsifier, an oil phase, an auxiliary emulsifier, a Km value, a temperature and the like are mainly examined, and the optimal technological composition of the magnolia volatile oil microemulsion preparation is determined by adding water for titration by using a pseudo ternary phase diagram classical method and by changing the size of the area for forming the microemulsion and related parameters.

1 Experimental method

1.1 preparation of Magnolia volatile oil microemulsion

The preparation process was studied in a preliminary experiment to preliminarily fix the prescription quality at 5.0g. Experiments are carried out on the volatile oil and the mixed emulsifier according to a certain proportion (a series of gradient change ratios of 9:1 to 1:9), ultrapure water is added dropwise while stirring each time, and a mixed system shows a series of state changes along with continuous addition of an aqueous phase; and determining the critical point of the microemulsion by measuring the conductivity change, and drawing a pseudo ternary phase diagram according to the percentage of each component of the critical point when the peak point of the conductivity starts to show a descending trend.

1.2 screening of Magnolia volatile oil microemulsion solution System

1.2.1 investigation of emulsifiers

Emulsifiers are a class of surfactants that have both hydrophilic and lipophilic properties. It is generally considered that the lower the "hydrophilic-lipophilic balance (HLB value)" is, the more lipophilic it is; conversely, the higher the HLB value, the more hydrophilic. Different emulsifying agents have different HLB values, and proper emulsifying agents are needed to obtain better emulsifying effect. Therefore, tween-80, tween-20, EL-40 and RH-40 are selected as emulsifying agents to examine the influence of the emulsifying agents on the formation of the magnolia flower volatile oil microemulsion.

1.2.2 investigation of co-emulsifiers

The co-emulsifier can adjust the HLB value of the emulsifier to provide greater stability in fine emulsion polymerization. Therefore, polyethylene glycol 400, glycerol, absolute ethyl alcohol and 1, 2-propylene glycol are selected as auxiliary emulsifying agents, the influence of the auxiliary emulsifying agents on the formation of the magnolia flower volatile oil microemulsion is examined, and the optimal auxiliary emulsifying agents are screened out.

1.2.3 investigation of oil phase

In the early pre-experiments, it was found that if only the magnolia flower volatile oil is used as the oil phase, the microemulsion cannot form a uniform transparent state, so that the addition of the auxiliary oil phase is considered, oleic acid, IPP and IPM are respectively used as mixed oil phases with the magnolia flower volatile oil according to the proportion of 1:1, and the influence of the oleic acid, IPP and IPM on the formation of the magnolia flower volatile oil microemulsion is examined.

1.3 screening of preparation conditions

1.3.1 Effect of Km value

The Km value is the mass ratio of emulsifier to co-emulsifier, and the ability to form a microemulsion will vary with Km value. Thus, the effect on microemulsion formation was examined herein for Km values of 4:1, 3:1, 2:1, 1:1, respectively, and the best Km values were selected.

1.3.2 influence of temperature

For the traditional Chinese medicine volatile oil, the preparation temperature has influence on the traditional Chinese medicine volatile oil. Therefore, based on the preliminary basic prescription establishment, the micro-emulsion state at 25 ℃, 30 ℃, 40 ℃ and 50 ℃ is inspected and the optimal preparation temperature is selected.

2 results and analysis

2.1 screening of optimal emulsifiers

The preparation temperature is preliminarily set at 25 ℃, IPM is set in an auxiliary oil phase, and the auxiliary oil phase is mixed with magnolia volatile oil in proportion, and the emulsifying capacity of four different emulsifying agents of Tween-80, tween-20, EL-40 and RH-40 is examined according to the method of section 2.1. The recorded data is imported into Origin 8.0 drawing software to draw a pseudo ternary phase diagram, the investigation result is shown in the following figure 3, and the areas forming the micro-emulsion areas are respectively S _EL-40 ＝0.00351＞S _Tween-80 ＝0.00204＞S _RH-40 ＝0.00196＞S _Tween-20 =0.00009. The result shows that when EL-40 is used as the emulsifier, the area of the formed microemulsion is maximum, the emulsifying effect is optimal, and meanwhile, the microemulsion has clear, transparent and uniform appearance, and the measured particle size meets the requirement.

TABLE 12 influence of emulsifiers on the preparation of microemulsionsn＝3)

2.2 screening of optimal Co-emulsifiers

The influence of polyethylene glycol 400, glycerol, absolute ethanol and 1, 2-propylene glycol as auxiliary emulsifiers on emulsion synthesis was studied on the basis of the determination that EL-40 is used as an emulsifier, the 1:1 ratio of IPM to magnolia volatile oil is used as a mixed oil phase, the initial provisional setting of Km is 2:1, the preparation temperature is 25 ℃, and the result is shown in the following figure 4.

The areas for forming the micro-emulsion areas are respectively S _PEG-400 ＝0.00044＞S _{1, 2-propanediol} ＝0.00034＞S _Glycerol ＝0.00027＞S _{Absolute ethyl alcohol} The results of the process are shown by the formula of (i) 0.00013, the formed microemulsion has the largest area and strong emulsification assisting capability when the polyethylene glycol 400 is used as the emulsification assisting agent, and meanwhile, the microemulsion has clear, transparent and uniform appearance, and the measured particle size meets the requirements.

TABLE 13 influence of co-emulsifier on the preparation of microemulsionsn＝3)

2.3 screening of optimal auxiliary oil phases

According to the screening result, the emulsifier is EL-40, the co-emulsifier is polyethylene glycol 400, the Km value is 2:1, and the mixture is prepared at 25 ℃, oleic acid, IPP and IPM are respectively mixed with flos Magnoliae volatile oil at a ratio of 1:1 to serve as mixed oil phases, the influence on the preparation of the microemulsion is observed, the auxiliary oil phase with the smallest influence on the flos Magnoliae volatile oil is selected, as shown in figure 5, the areas for forming the microemulsion areas are respectively S _IPM ＝0.00394＞S _IPP ＝0.00278＞S _{Oleic acid} =0.00233. The result shows that the IPM is used as an auxiliary oil phase, the area of the formed microemulsion is maximum, and meanwhile, the microemulsion has clear, transparent and uniform appearance, and the measured particle size meets the requirement.

TABLE 14 microemulsion preparation with auxiliary oil phaseInfluence ofn＝3)

2.4 screening of optimal Km values

According to the above screening results, the emulsifier, co-emulsifier and co-oil phase were respectively designated as EL-40, polyethylene glycol 400 and IPM, and when they were disposed at 25 ℃, the effect on the preparation of the microemulsion was examined when Km was 4:1, 3:1, 2:1, 1:1, respectively, and the optimum Km values were screened as shown in fig. 6.

The areas for forming the micro-emulsion areas are respectively S _2:1 ＝0.00232＞S _1:1 ＝0.00135＞S _4:1 ＝0.00028＞S _3:1 The results showed that the Km value was 2:1, the area of the formed microemulsion was maximum, and the microemulsion appearance was clear, transparent and uniform, and the measured particle size was satisfactory.

TABLE 15 influence of different Km values on the preparation of microemulsions [ ]n＝3)

2.5 screening of optimal preparation temperature

According to the screening result of the earlier influencing factors, the optimal prescription proportion is determined, the influence of the microemulsion preparation at 25 ℃, 30 ℃, 40 ℃ and 50 ℃ is examined, and the optimal preparation temperature is screened. The areas for forming the micro-emulsion areas are respectively S _30℃ ＝0.00097＞S _25℃ ＝0.00070＞S _40℃ ＝0.00044＞S _50℃ As a result of=0.00006, see fig. 7, the area of the formed microemulsion was maximum when the microemulsion preparation was prepared at 30 ℃, and the microemulsion appearance was clear, transparent and uniform, and the measured particle size was satisfactory.

TABLE 16 microemulsion at different preparation temperaturesInfluence ofn＝3)

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4 summary and discussion

The flos Magnoliae volatile oil belongs to fat-soluble liquid, is not easily dissolved in water and has poor bioavailability. The volatile oil is considered to be prepared into the microemulsion, so that the problems of water insolubility, uniform quality of liquid preparation and the like can be solved; the microemulsion is used as a carrier to prepare other dosage forms, thereby playing the roles of improving bioavailability and increasing stability.

Therefore, the preparation method adopts a water-drop titration method to prepare the magnolia flower volatile oil microemulsion preparation, draws a pseudo ternary phase diagram, compares the area and the size of the formed microemulsion, observes factors such as the appearance, the particle size and the like of the microemulsion, and screens out the optimal components for preparing the magnolia flower volatile oil microemulsion preparation. The conductivity value is measured, the water adding amount is accurately controlled, the change trend of conductivity in the preparation process of the microemulsion is discussed, the optimal formula is optimized, and the optimal preparation conditions are obtained: the emulsifier is EL-40, the auxiliary emulsifier is polyethylene glycol 400, the flos Magnoliae volatile oil and IPM are mixed to form an oil phase, the Km value=2:1, the preparation temperature is 30 ℃, the mass ratio of the total emulsifier to the mixed oil phase is 9:1, wherein the mixed oil phase accounts for 2.61%, the water phase accounts for 71.22% of the total amount of the microemulsion, and the mixed emulsifier accounts for 26.17%. The prepared microemulsion is O/W type, and the appearance of the microemulsion is clear, transparent and uniform, and meets the quality requirement of the microemulsion.

2. Quality evaluation of flos Magnoliae volatile oil microemulsion preparation

The microemulsion is used as nano-scale droplets, has the characteristics of transparency, stability, perfect absorption, targeted drug release and the like, is a novel ideal drug release carrier, has the effect of improving the curative effect of the drug, and has wide development prospect. Therefore, the quality system of the magnolia flower volatile oil microemulsion is primarily evaluated by researching the basic properties, the physicochemical parameters and the like of the microemulsion.

1 Experimental method

1.1 observing the appearance of the microemulsion

Taking 15 mu L of microemulsion sample, dripping the sample into a carbon-coated supporting film copper net, and sucking the residual microemulsion sample solution by using filter paper after the sample stays for about 8-12 min. After complete drying, dropping in TEM phosphotungstic acid solution, dyeing for 90s, sucking the residual dye liquor with filter paper, placing it on the filter paper for drying for 8h, and then observing the appearance by using a transmission electron microscope.

1.2 identification of microemulsion types

Judging the type of the microemulsion by observing the diffusion rates of the water-soluble dye and the oil-soluble dye in the microemulsion, wherein when the diffusion rate of the water-soluble dye is faster than that of the oil-soluble dye, the microemulsion is O/W type; otherwise, the flow is of a W/O type; the dyes used in this study were methylene blue (water-soluble) and sudan III (oil-soluble) dyes, respectively.

1.3 measurement of the physicochemical parameters of the microemulsion

1.3.1 measurement of the pH of the microemulsion

Taking 3 batches of microemulsions with different preparation time, sampling 1.0mL each, diluting with purified water, and measuring the pH value of the microemulsions at room temperature.

1.3.2 measurement of microemulsion particle size, zeta potential

Taking 3 batches of microemulsions with different preparation time, respectively diluting 1.0mL of each sample with 10 times of water, and measuring the granularity and the potential of the microemulsions by using a Markov particle sizer.

1.4 measurement of microemulsion stability

1.4.1 physical stability of the microemulsion

Taking 3 parts of equivalent microemulsion into a centrifuge tube, and placing the microemulsion into a rotating speed of 10000 r.min ^-1 Centrifuging for 15min in a high-speed centrifuge, and observing the transparency of the microemulsion and whether layering occurs, so as to judge the centrifugal stability of the microemulsion.

1.4.2 microemulsion accelerated stability test

And (3) placing 3 parts of the magnolia flower volatile oil microemulsion in an equal amount into a weighing bottle for sealing, and preserving for 3 months under the conditions that the temperature is 30+/-2 ℃ and the humidity is 65+/-5%, wherein at the end of each month, indexes such as appearance, conductivity, particle size and the like of the microemulsion are sampled and detected, so that the stability of the microemulsion is judged.

2 results and analysis

2.1 appearance Properties of the microemulsion

The magnolia flower volatile oil microemulsion is placed under a transmission electron microscope, see fig. 8, and the scanning result shows that: the flos Magnoliae volatile oil microemulsion is round, has clear outline, and has uniform particle diameter.

2.2 identification of microemulsion types

As can be intuitively observed from fig. 9, the diffusion rate of methylene blue (blue) is significantly faster than that of sudan III (red), and the prepared microemulsion is judged to be O/W type.

2.3 measurement of the physicochemical parameters of the microemulsion

2.3.1 pH of microemulsion

The final average value of the pH value of the flos magnoliae volatile oil microemulsion measured at room temperature is 6.33+/-0.23 (n=3), and the flos magnoliae volatile oil microemulsion is weak acid microemulsion.

2.3.2 microemulsion particle size and Zeta potential thereof

The particle size of the flos Magnoliae volatile oil microemulsion was 14.27+ -0.03 nm (n=3), the average value of PDI was 0.0941 + -0.31 (n=3), and the Zeta potential of the volatile oil microemulsion was-0.3585 + -0.12 mV (n=3) as shown in FIG. 10.

2.4 stability study of microemulsion

2.4.1 appearance Properties of the microemulsion

The flos Magnoliae volatile oil microemulsion preparation prepared according to the process of screening out the optimal prescription has uniform appearance, and is clear and transparent. 3 parts of flos Magnoliae volatile oil microemulsion with physical stability are placed in a high-speed centrifuge at a rotational speed of 10000 r.min < -1 >, and after centrifugation for 15min, the appearance is clear and transparent, and no layering phenomenon occurs, so that the flos Magnoliae volatile oil microemulsion has good centrifugal stability.

2.4.2 accelerated stability test

Placing the magnolia flower volatile oil microemulsion for 3 months in an environment with the temperature of 30+/-2 ℃ and the humidity of 60+/-5%, sampling and detecting at the end of each month, and indicating that the centrifugal stability of the microemulsion is stable, the appearance is clear and transparent, the microemulsion is uniform, no separation and layering phenomenon exists, and the conductivity, the pH value and the particle size have no obvious change along with the extension of the placing time, so that the microemulsion is relatively stable under the experimental condition.

TABLE 17 accelerated test results [ ]n＝3)

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3 summary and discussion

The section observes the shape of the microemulsion through a transmission electron microscope, and discovers that the surface of the microemulsion is round and uniform in particle size distribution. In determining the type of microemulsion, methylene blue was found to diffuse significantly faster than sudan III, and was therefore identified as an O/W type microemulsion. The result of measuring the physicochemical parameters of the microemulsion shows that the pH value of the microemulsion is stable, the particle size is consistent, and the requirements are met. In the stability test of the microemulsion, the microemulsion is found to be still in a clear and transparent state in appearance by high-speed centrifugation, and no delamination is found, and no unstable phenomena such as oil-water separation and the like are caused. In the accelerated stability test process, stability investigation for 3 months is carried out, and pH value, particle size and conductivity change are respectively inspected, so that the result shows that the microemulsion is clear and uniform liquid in the investigation period, and other physicochemical parameters have no obvious change.

The technology for preparing the magnolia flower volatile oil microemulsion is feasible, the method is accurate and repeatable, and a good foundation is laid for developing a novel magnolia flower volatile oil microemulsion preparation.

The following demonstrates the beneficial effects of the invention by pharmacodynamic tests.

Test example 1 treatment efficacy comparison of Magnolia volatile oil microemulsion preparation and oil solution group

The therapeutic effect of the microemulsion preparation and the volatile oil solution is examined by establishing a allergic rhinitis rat model, the secretion of IL-6, TNF-alpha and IL-1 beta in serum is detected, and the expression level of related proteins is detected by an immunohistochemical technology.

1 materials and instruments

1.1 animals

SPF-class male Wistar rats, 30 in total, weighing 110-120 g, provided by the Chengdu experimental animal center, animal license: SCXK 2020-030. Animals were kept in a traditional Chinese medicine pharmacology laboratory at Shaanxi university of traditional Chinese medicine, and experiments were carried out after 5 days of adaptive breeding in an environment with a temperature of 20+ -2 ℃ and a relative humidity of 65+ -2% and a good ventilation.

2 Experimental methods

2.1 establishing a model of allergic rhinitis in rats

And (3) carrying out a molding experiment by using the molding method under the second section '2.1' of the fifth chapter.

2.2 grouping and administration

After adaptive feeding for 5 days, rats are relatively uniform in quality, so that the rats are divided into 5 groups according to a random distribution principle, 6 groups of rats are respectively provided with a volatile oil solution group, a microemulsion preparation group, a model group, a blank group and an auxiliary Shu Liang (fluticasone propionate nasal spray) group, wherein the volatile oil solution is prepared into 0.25% of oil solution by olive oil for nasal administration. After one week of priming, the drug intervention was performed for 3 weeks on the second week, and the same concentration microemulsion preparation group and oil solution group were instilled into the nasal cavities on both sides of the test rats, 100 μl of each nostril, and the same positive control group was given the same volume of auxiliary Shu Liang (fluticasone propionate nasal spray) (specification: 120 spray), and the blank group and model group were instilled with nasal daily, an equal dose of physiological saline once a day.

2.3 observing the behavioural status of animals

The behavioral state of the rats was observed.

2.4 animal sample sampling method

2.4.1 serum collection

After the last administration treatment, the patients were fasted for 24 hours (not to be water-inhibited) and were treated by intraperitoneal injection of pentobarbital sodium (30-60 mg. Kg ^-1 ) The rat was anesthetized, the abdomen was cut, blood was collected after peeling off the exposed abdominal aorta, and the blood sample was left at room temperature for 2 hours at 4000 r.min ^-1 Centrifuging at-80deg.C for 10min, collecting supernatant, packaging, and storing in frozen state at-80deg.C.

2.4.2 stripping nasal mucosal tissue

The nasal mucosa tissue material taking method.

2.5 ELISA (enzyme-Linked immuno sorbent assay) for detecting content expression of related inflammatory factors in rat serum and nasal rinse solution

Specific experimental procedures were performed according to the instructions of the kit.

2.6 immunohistochemical detection of related protein expression level in rat nasal mucosal tissue

And detecting the content of the related protein.

3 results and analysis

3.1 behavioral observations

Rats in the blank group were free to move and had good mental status, while rats in the other groups had different degrees of scratching the nasal face and sneezing, and the scores before and after modeling were shown in Table 18.

Table 18 evaluation score table of behavioural before and after modeling

Note that: ^### : compared with the blank group, there was a significant difference (p < 0.001); ^*** : compared with the model group, the model group has obvious difference (p < 0.001)

3.2ELISA measurement of the expression level of the related inflammatory factors in rat serum

As shown in FIG. 11, the levels of IL-1β, IL-6 and TNF- α in the serum were significantly elevated (p < 0.01) in the model group compared to the blank group. Serum IL-1 beta, IL-6 and TNF-alpha levels were significantly reduced (p < 0.05) in the adjuvant Shu Liang and dosed groups compared to the model group.

TABLE 19 secretion of related inflammatory factors in rat serum

Note that: ^## : compared with the blank group, there was a significant difference (p < 0.01); ^* : compared with the model group, has the advantages ofThe difference in the origins (p < 0.05)

3.3 immunohistochemical detection of the expression level of related proteins in nasal mucosal tissue of rat

As shown in FIG. 12, there was no statistical significance in the differences in NF- κB-p65, p-NF- κB-p65, and PPAR- γ protein expression in the adjuvant Shu Liang, oil solution, and microemulsion treatment groups compared to the blank groups. Compared with the model group, the auxiliary Shu Liang group, the oil solution group and the microemulsion treatment group respectively show significant decrease and significant increase trend (p < 0.05) on NF-kappa B-p65, p-NF-kappa B-p65 and PPAR-gamma proteins; compared with the volatile oil microemulsion preparation, the related proteins of the oil solution group have significant differences (p is less than 0.05), which suggests that the magnolia flower volatile oil microemulsion preparation can play a therapeutic role in PPAR-gamma/NF-kappa B signaling pathway by regulating NF-kappa B-p65, p-NF-kappa B-p65 and PPAR-gamma.

TABLE 20 expression level of protein related to nasal mucosa tissue of ratn＝3)/>

Note that: ^## : compared with the blank group, there was a significant difference (p < 0.01); ^* : compared with the model group, the model group has obvious difference (p < 0.05)

4. Summary and discussion

The behavioral results show that the positive medicine control group, the volatile oil solution group and the microemulsion preparation group can relieve uncomfortable symptoms such as sneeze, rhinocnesmus and the like of the allergic rhinitis rats to different degrees; the ELISA is used for detecting relevant inflammatory factors, and the results show that compared with a model group, the levels of IL-1 beta, IL-6 and TNF-alpha in serum are obviously reduced, and the results have obvious differences; the detection results of related proteins such as PPAR-gamma, NF-kappa B-p65 and p-NF-kappa B-p65 show that: compared with the model group, the protein expression levels of NF- κB-p65 and p-NF- κB-p65 in the microemulsion preparation group and the oil solution group are obviously reduced, and the PPAR-gamma protein expression level is obviously increased; the oil solution group related proteins all had significant differences compared to the microemulsion formulation group. The volatile oil solution group can exert therapeutic effects, but belongs to oily liquid, and administration is difficult and the dosage is difficult to control. In the actual operation process, the oil solution is directly dripped on the nose of the rat, so that the nasal cavity of the rat can be stimulated and blocked, and the nasal cavity is led to bleed; the volatile oil is easily dissolved in the microemulsion matrix, and can be prepared into microemulsion preparation, and the administration of the volatile oil can not irritate the nose of a rat to cause uncomfortable symptoms.

Test example 2 preliminary study of pharmacodynamics of Magnolia volatile oil microemulsion preparation

The section adopts a rat model of allergic rhinitis by taking magnolia volatile oil microemulsion as a tested medicament on the basis of combining the optimization of the extraction process of magnolia volatile oil, the screening of the preparation process of volatile oil microemulsion and the network pharmacology prediction of weight with an RNA-seq technology, and carries out pharmacodynamics evaluation on the magnolia volatile oil microemulsion according to indexes such as behaviours, serum, nasal lavage liquid, inflammatory factor measurement in nasal mucosa tissues and the like.

1. Animals

SPF-class male Wistar rats, 36 in total, weighing 110-120 g, provided by the Chengdu experimental animal center, animal license: SCXK 2020-030. Animals were kept in a traditional Chinese medicine pharmacology laboratory at Shaanxi university of traditional Chinese medicine, and experiments were carried out after 5 days of adaptive breeding in an environment with a temperature of 20+ -2 ℃ and a relative humidity of 65+ -2% and a good ventilation.

2 Experimental methods

2.1 establishing a model of allergic rhinitis in rats

2.2 grouping and administration

After the adaptive feeding for 5 days, the quality of the rats is uniform, so that the rats are divided into 6 groups according to a random distribution principle, and each group is divided into 6 groups, namely, a volatile oil microemulsion high dose group, a medium dose group, a low dose group, a model group, a blank group and a positive medicament group. After one week of priming, the drug intervention was performed for 3 weeks at the second week, the volatile oil microemulsion was dropped into the bilateral nasal cavities of the test rats, 100mL of each lateral nasal cavity, and the same volume of auxiliary Shu Liang (fluticasone propionate nasal spray) (specification: 120 spray) was administered to the same positive drug group, and the blank group and model group were dropped with equal doses of physiological saline once a day.

2.3 observing the behavioural status of animals

The behavioral state of the rats was observed as in the method under section "2.1.2" of chapter five.

2.4 animal sample sampling method

2.4.1 serum collection

Under the same section Zhang Di, "2.4.1".

2.4.2 collecting nasal rinse

Immediately after blood sampling, the rat throat is plugged by cotton wool soaked in liquid paraffin, so as to prevent flushing fluid from flowing out; cutting the skin of the neck of the rat, peeling off the air outlet pipe, cutting a V-shaped bevel, and inserting the rubber pipe into the bevel; injecting 1mL of flushing liquid, repeating for three times, collecting combined flushing liquid, and treating at 4000 r.min ^-1 Centrifuging at-80deg.C for 10min, collecting supernatant, packaging, and storing in frozen state.

2.4.3 nasal mucosal tissue removal

The method for obtaining nasal mucosa tissue under the item "2.1.3" in the second chapter of the fifth chapter.

2.4.4 ELISA (enzyme-Linked immuno sorbent assay) for detecting content expression of related inflammatory factors in rat serum and nasal rinse solution

2.4.5 Western Blot method for detecting related protein expression quantity in rat nasal mucosa tissue

Adding nasal mucosa tissue into lysate, homogenizing in homogenizer, repeatedly grinding on ice for several times to make tissue sufficiently ground, and splitting for 30min to obtain supernatant.

(1) Protein standard solutions of different concentrations were prepared according to the BSA kit protocol, and the actual concentration of the sample (. Mu.g. Mu.L-1) was determined and then frozen at-80 ℃.

(2) SDS-PAGE: respectively preparing upper layer glue and lower layer glue according to different proportions, respectively loading samples according to the sequence, adding Marker and nasal mucosa sample proteins, and regulating voltage and current for electrophoresis;

(3) Film transfer (ice bath cooling): the membranes were transferred to the transfer solution in the following order: sponge-2 pieces of filter paper-sponge-NC membrane-2 pieces of filter paper-sponge;

(4) Immunocompetence: transferring the membrane into a sealing liquid, and shaking and sealing on a room temperature shaking table for 70min;

(5) Adding an antibody: after dripping the primary antibody, incubating overnight at 4 ℃, washing three times with TBST on a decolorizing shaking table at room temperature for 5min each time the next day; adding a secondary antibody, incubating for 50min at room temperature, and washing with TBST on a decolorizing shaking table at room temperature for three times, each time for 5min;

(6) Finally, the film was placed in ECL reagent and luminescence was shown.

3 results and analysis

3.1 behavioral observations

The behavioral results are shown in Table 21, and the scores after modeling of the model group, the administration group and the auxiliary Shu Liangzu are not statistically different and are obviously higher than those of the blank group; the scores of the administration group and the auxiliary Shu Liang group are obviously reduced after treatment, and the obvious difference is provided, so that the magnolia flower volatile oil microemulsion and the auxiliary Shu Liang (fluticasone propionate nasal spray) can relieve the nasal inflammatory response of the rats with allergic rhinitis.

Table 21 score table for behavioral evaluation before and after modeling

3.2ELISA measurement of the expression level of the related inflammatory factors in the nasal rinse solution and serum of rats

As shown in FIGS. 13 to 14, the levels of IL-1β, IL-6 and TNF- α in nasal rinse, serum were all significantly elevated in the model group compared to the blank group. The levels of IL-1 beta, IL-6 and TNF-alpha in the nasal wash, serum were significantly reduced in the positive drug control and the dosed treatment compared to the model group.

TABLE 22 secretion of related inflammatory factors in nasal rinse solution of rats

TABLE 23 secretion of related inflammatory factors in rat serum

3.3 analysis of the expression level of the related proteins in nasal mucosal tissue of rat

As shown in FIG. 15, there was no statistical significance in the difference in NF- κB-p65, p-NF- κB-p65 and PPAR- γ protein expression in the positive drug control group, the oil solution group and the microemulsion treatment group, as compared with the blank group. Compared with the model group, the positive control drug group, the oil solution group and the microemulsion treatment group show significant decrease and significant increase trend (p < 0.05) of NF-kB-p 65, p-NF-kB-p 65 and PPAR-gamma protein respectively, which suggests that the magnolia flower volatile oil microemulsion preparation can play a treatment role in PPAR-gamma/NF-kB signal path by regulating NF-kB-p 65, p-NF-kB-p 65 and PPAR-gamma.

TABLE 24 expression level of inflammation-related protein in nasal mucosal tissue of rat [ (]n＝3)

Note that: ^# : compared with the blank group, there was a significant difference (p < 0.05); ^* : compared with the model group, the model group has significant difference (p < 0.05); ^& : compared with the positive control group, (p < 0.05)

4 summary and discussion

The expression conditions of PPAR-gamma/NF- κB signal pathway related proteins PPAR-gamma, NF- κB-p65 and p-NF- κB-p65 are studied by establishing a allergic rhinitis rat model, and how the pathway regulates and controls the related proteins to play a role in treating allergic rhinitis is analyzed. According to the change of the behavioral state of the rat, the change of the expression level of inflammatory factors in serum and nasal wash of the rat measured by ELISA, the condition of detecting the expression level of related proteins by Western Blot experiment and the like, the pharmacodynamics process of the magnolia flower volatile oil microemulsion for treating allergic rhinitis is primarily studied.

The combination of early differential gene sequencing results and "weight" network pharmacology prediction results, as well as the reports of related literature, suggest that the PPAR-gamma/NF- κB signaling pathway plays an important therapeutic regulatory mechanism in the treatment of allergic rhinitis. The behavioral results show that the microemulsion preparation group and the positive drug control group can relieve uncomfortable symptoms such as sneeze, rhinocnesmus and the like of the allergic rhinitis rats to different degrees; the ELISA is used for detecting relevant inflammatory factors, and the results show that compared with a model group, the levels of IL-1 beta and IL-6 TNF-alpha in nasal lavage and serum are obviously reduced, and the differences are obvious (p is less than 0.05); western Blot experiments respectively detect related proteins such as PPAR-gamma, NF-kappa B-p65, p-NF-kappa B-p65 and the like in terms of PPAR-gamma/NF-kappa B signal channels, and the results show that: compared with the model group, the protein expression levels of NF- κB-p65 and p-NF- κB-p65 are obviously reduced (p < 0.05), and the PPAR-gamma protein expression level is obviously increased (p < 0.05).

The invention discloses a magnolia flower volatile oil microemulsion preparation which can treat allergic rhinitis by regulating and controlling the expression quantity of related proteins such as IL-1 beta, IL-6, TNF-alpha, PPAR-gamma, NF-kappa B-p65, p-NF-kappa B-p65 and the like in a PPAR-gamma/NF-kappa B signal channel.

Claims

1. A method for extracting magnolia flower volatile oil is characterized by comprising the following steps of: the method takes magnolia flower as a raw material, adopts a cellulase enzymolysis auxiliary steam distillation method for extraction, and comprises the following steps:

2. The extraction method according to claim 1, characterized in that: the enzyme addition amount in the step a is 0.125-0.250%, the enzymolysis time is 45-65 min, and the enzymolysis temperature is 30-50 ℃.

3. The extraction method according to claim 2, characterized in that: the enzyme addition amount in the step a is 0.125%, the enzymolysis time is 65min, and the enzymolysis temperature is 50 ℃.

4. The volatile oil prepared by the extraction method of any one of claims 1 to 3, characterized in that: the volatile oil contains farnesol and eucalyptol, and the percentage content of the volatile oil is as follows:

Farnesol is not lower than 12.0% w/w and eucalyptol is not lower than 8.0% w/w.

5. Use of the volatile oil of claim 4 in the manufacture of a medicament for nasal administration for the treatment of allergic rhinitis.

6. A method of detecting the volatile oil of claim 4, wherein: the method adopts GC-MS analysis and detection, and comprises the following specific steps:

b. GC-MS detection, chromatographic conditions are:

gas chromatography conditions: agilent HP-5ms (30 m×250 μm×0.25 μm) capillary column, high purity He, 3.0 μl sample injection amount, and flow rate of 1mL·min ^-1 The split ratio is 40:1, the temperature is programmed to be 55 ℃, the temperature is 8 ℃ and min-1 is programmed to be 176 ℃, and the holding time is as follows: 3min,20 ℃ mAnd (3) raising the temperature of the in-1 to 250 ℃ for a holding time: 20min;

7. A flos magnoliae volatile oil microemulsion for treating allergic rhinitis is characterized in that: the oil phase is prepared from the volatile oil, an emulsifier, an oil phase and a co-emulsifier according to claim 4, wherein the emulsifier is one or more than two of tween-80, tween-20, EL-40 and RH-40; the auxiliary emulsifier is one or more than two of polyethylene glycol 400, glycerol, absolute ethyl alcohol and 1, 2-propylene glycol; the oil phase is as follows: oleic acid, IPP, IPM, or a mixture of two or more thereof; wherein the volume ratio of the emulsifier to the mixed oil phase is as follows: 7-9:1-3; the volume ratio of the volatile oil to the oil phase is as follows: 1-3:1-3; the mass ratio of the emulsifier to the auxiliary emulsifier is as follows: 1-4:1-2.

8. The microemulsion of claim 7 wherein: the emulsifier is EL-40, the auxiliary emulsifier is polyethylene glycol 400, and the oil phase is IPM, wherein the mass ratio of the EL-40 to the polyethylene glycol 400 is 2:1.

9. The microemulsion according to claim 7 or 8, wherein: the granularity of the flos magnoliae volatile oil microemulsion is 14.27+/-0.03 nm (n=3), and the average value of PDI is 0.0941 +/-0.31 (n=3); the Zeta potential of the essential oil microemulsion was measured to be-0.3585 + -0.12 mV (n=3).

10. A method of preparing the microemulsion of claim 8 or 9, characterized by: it comprises the following steps: