CN109316487B - Extraction method and application of thoroughfare bitter orange ingredient for treating respiratory inflammation - Google Patents
Extraction method and application of thoroughfare bitter orange ingredient for treating respiratory inflammation Download PDFInfo
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Abstract
The invention relates to an extraction method of a component for treating respiratory inflammation in thoroughfare bitter orange and application thereof, wherein the extraction method specifically comprises the following steps: (1) thoroughfare fructus aurantii pulverizing part; (2) soaking the powder in an ethanol solution; (3) performing microwave irradiation; (4) drying under negative pressure; (5) extracting and filtering; (6) purifying by macroporous resin; (7) drying under reduced pressure. The extract obtained by the method effectively reduces the contact time of the powder and an ethanol solution, reduces oxidation, and enriches the active ingredients naringin and neohesperidin for treating respiratory inflammation.
Description
Technical Field
The invention relates to the field of medicines, in particular to an extraction method of a component for treating respiratory inflammation in thoroughfare bitter orange and application thereof.
Background
The Changshan Huyou is mainly produced in Zhejiang Quzhou region, is a unique citrus variety in China, and is a hybrid variety of Citrus grandis and Citrus sinensis in Citrus of Rutaceae. In 2016 for 8 months, dry immature fruits of Changshan Hu shaddock are brought into 2015 edition of Zhejiang Chinese medicine processing specifications under the name of 'Ququ bitter orange', formally become a traditional Chinese medicine, the main components of the traditional Chinese medicine are volatile components and flavonoid compounds, the main effects are regulating qi, relieving epigastric distention, activating stagnancy, relieving cough, reducing phlegm, clearing heat, detoxifying, promoting gastrointestinal function, resisting oxidation, reducing blood fat, reducing blood pressure and the like, and a lot of researches are carried out on the application of the extract of the Ququ bitter orange in the prior art.
CN108403817A relates to the use of a thoroughfare type fructus aurantii, a thoroughfare type fructus aurantii extract and a pharmaceutical composition containing the thoroughfare type fructus aurantii or the thoroughfare type fructus aurantii extract in the preparation of a medicament for protecting islet cells, restoring islet cell function, promoting insulin secretion or increasing the number of islet cells, wherein the extract is an alcohol extract of thoroughfare type fructus aurantii or a thoroughfare type fructus aurantii water extract, the extraction method comprises the following steps: (1) crushing thoroughfare bitter orange, and performing reflux extraction on the crushed product by using an alcohol solvent to obtain an extracting solution; (2) carrying out solid-liquid separation on the extracting solution, and recovering an alcohol solvent to obtain a concentrated solution; (3) drying the concentrated solution to obtain the thoroughfare bitter orange extract.
CN107714805A application of thoroughfare bitter orange extract in preparing traditional Chinese medicine preparation or functional food for preventing and treating respiratory inflammation. The preparation method of the thoroughfare bitter orange extract comprises the following steps: 1) taking thoroughfare bitter orange crushed particles, adding deionized water for soaking, distilling a soak solution, and collecting fractions for later use; 2) heating and refluxing the residue twice, filtering the two times, combining the filtrates, and concentrating under reduced pressure to obtain concentrated solution; 3) the concentrated solution is combined with the distillate to obtain the thoroughfare bitter orange extract, the main components of which are alkaloids and flavonoids, and the extract obtained by the method has complex components and can not know which components are effective components for preventing and treating respiratory inflammation.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide the extraction method and the application of the ingredients for treating respiratory inflammation in thoroughfare bitter orange.
In order to solve the problems, the technical scheme adopted by the invention is as follows:
a method for extracting a component for treating respiratory inflammation from thoroughfare bitter orange comprises the following steps:
(1) treating thoroughfare bitter orange: slicing thoroughfare bitter orange, adding the thoroughfare bitter orange into a pulverizer for pulverizing, adding ethyl acetate, stirring, filtering, performing freeze drying at the temperature of minus 5 to minus 15 ℃, and performing ultrasonic wall breaking treatment at the temperature of 40-60 ℃;
(2) powder infiltration: spraying 30-70% of ethanol into the powder obtained in the step (1), and fully soaking;
(3) microwave irradiation: performing microwave irradiation on the infiltrate obtained in the step (2);
(4) drying under negative pressure: drying the infiltrated substance subjected to microwave treatment in the step (3) under negative pressure to obtain dry powder;
(5) extracting and filtering: conveying the dried powder obtained in the step (4) by using inert gas, spraying 8-10 times of 30-70% ethanol liquid in opposite directions at high pressure, mixing, stirring for 20-40min, and filtering to obtain an extracting solution;
(6) and (3) purification: and (4) carrying out chromatography separation on the extracting solution obtained in the step (5) by using macroporous resin, and sequentially carrying out the following elution: eluting with distilled water, then eluting with 20% ethanol, finally eluting with 60-80% ethanol, and collecting 60-80% ethanol eluate;
(7) and (3) drying under reduced pressure: and (4) concentrating the eluent obtained in the step (6) under reduced pressure, and drying to obtain the extract.
As a further improvement of the invention, the addition amount of the ethyl acetate in the step (1) is 4-8 times of the weight of the thoroughfare bitter orange.
As a further improvement of the method, in the step (1), ultrasonic wall breaking is carried out, the frequency of ultrasonic waves is 30-50 kH, the pressure of inert gas is 150-200 MPa, the gas flow rate is 500-700 m/s, the feeding speed is 4-6 g/s, and the feeding pressure is 0.4-0.6 MPa.
As a further improvement of the invention, the microwave irradiation parameters of the step (1) are as follows: the power is 500-.
As a further improvement of the invention, the inert gas delivery pressure in the step (5) is 101KPa,
as a further improvement of the invention, the macroporous resin in the step (6) is D101 or AB-8 macroporous resin.
As a further improvement of the invention, in the step (6), the ethanol is eluted by distilled water, then the ethanol with the concentration of 20 percent is eluted, finally the ethanol with the concentration of 75 percent is eluted, and the ethanol eluent with the concentration of 75 percent is collected.
The preferable scheme of the invention is as follows:
the method specifically comprises the following steps:
(1) treating thoroughfare bitter orange: slicing thoroughfare bitter orange, adding the thoroughfare bitter orange into a pulverizer for pulverizing, adding 6 times of ethyl acetate, stirring and filtering at the temperature of minus 10 ℃, freeze-drying filter residue, and then carrying out ultrasonic wall breaking treatment at the temperature of 50 ℃, wherein the ultrasonic frequency is 40kH, the inert gas pressure is 170MPa, the gas flow rate is 600m/s, the feeding speed is 5g/s, and the feeding pressure is 0.5 MPa.
(2) Powder infiltration: spraying 70% ethanol to the powder obtained in the step (1) to fully soak the powder;
(3) microwave irradiation: and (3) performing microwave irradiation on the infiltrate obtained in the step (2), wherein the microwave power is 600w, the temperature is 60 ℃, and the time is 1.5 min.
(4) Drying under negative pressure: drying the infiltrated substance subjected to microwave treatment in the step (3) under negative pressure to obtain dry powder;
(5) extracting and filtering: conveying the dried powder obtained in the step (4) by using inert gas, wherein the conveying pressure of the inert gas is 101KPa, spraying the powder, carrying out high-pressure opposite spraying by using 70% ethanol liquid in an amount which is 9 times that of the powder, fully colliding and matching the powder and the ethanol, adjusting the spraying speed of the powder and the ethanol to ensure that the powder and the ethanol are matched and the powder and the ethanol are synchronously sprayed, mixing, stirring for 30min, and filtering to obtain an extracting solution;
(6) and (3) purification: carrying out chromatographic separation on the extracting solution obtained in the step (5) by using D101 macroporous resin, and sequentially carrying out the following elution: eluting with distilled water, eluting with 20% ethanol, eluting with 75% ethanol, and collecting 75% ethanol eluate.
(7) And (3) drying under reduced pressure: and (4) concentrating the eluent obtained in the step (6) under reduced pressure, and drying to obtain the extract.
The invention also provides application of the extract obtained by the method in preparing a medicament for treating respiratory inflammation.
Further, the respiratory inflammation is respiratory inflammation.
Adopt the produced beneficial effect of above-mentioned technical scheme to lie in:
the extraction method provided by the invention comprises the steps of infiltrating powder with ethanol, performing microwave radiation, performing reduced pressure drying, transferring effective components among the powder, spraying ethanol solution at high pressure, enabling the ethanol and the powder to impact at high speed in an inert atmosphere, promoting the dissolution of the effective components, and finally stirring for 20-40min to complete the whole extraction process, thereby effectively reducing the contact time of the powder and the ethanol solution, reducing oxidation, and enriching the effective components naringin and neohesperidin for treating respiratory inflammation.
The extract obtained by the extraction method provided by the invention can effectively treat respiratory inflammation, and is safe and reliable.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in detail and fully with reference to the following embodiments.
Example 1
A method for extracting a component for treating respiratory inflammation from thoroughfare bitter orange comprises the following steps:
(1) treating thoroughfare bitter orange: the preparation method comprises the steps of slicing 500g of thoroughfare bitter orange, adding the slices into a pulverizer to be pulverized, sieving the slices with a 60-mesh sieve, adding 6 times of ethyl acetate, stirring and filtering the slices at-10 ℃, freeze-drying filter residues, and then carrying out ultrasonic wall breaking treatment at 50 ℃, wherein the ultrasonic frequency is 40kH, the inert gas pressure is 170MPa, the gas flow rate is 600m/s, the feeding speed is 5g/s, and the feeding pressure is 0.5MPa, and all the obtained powder passes through the 600-mesh sieve.
(2) Powder infiltration: spraying 70% ethanol to the powder obtained in the step (1) to fully soak the powder;
(3) microwave irradiation: and (3) performing microwave irradiation on the infiltrate obtained in the step (2), wherein the microwave power is 600w, the temperature is 60 ℃, and the time is 1.5 min.
(4) Drying under negative pressure: drying the infiltrated substance subjected to microwave treatment in the step (3) under negative pressure to obtain dry powder;
(5) extracting and filtering: conveying the dried powder obtained in the step (4) by using inert gas, wherein the conveying pressure of the inert gas is 101KPa, spraying the powder, carrying out high-pressure opposite spraying by using liquid of 70% ethanol 9 times the amount of the powder, fully colliding and matching the powder at high pressure, mixing, stirring for 30min, and filtering to obtain an extracting solution;
(6) and (3) purification: carrying out chromatographic separation on the extracting solution obtained in the step (5) by using D101 macroporous resin, and sequentially carrying out the following elution: eluting with distilled water, eluting with 20% ethanol, eluting with 75% ethanol, and collecting 75% ethanol eluate.
(7) And (3) drying under reduced pressure: and (4) concentrating the eluent obtained in the step (6) under reduced pressure, and drying to obtain the extract.
Example 2
A method for extracting a component for treating respiratory inflammation from thoroughfare bitter orange comprises the following steps:
(1) treating thoroughfare bitter orange: the preparation method comprises the steps of slicing 500g of thoroughfare bitter orange, adding the slices into a pulverizer to be pulverized, sieving the slices with a 60-mesh sieve, adding 4 times of ethyl acetate, stirring and filtering the slices at-5 ℃, freeze-drying filter residues, and then carrying out ultrasonic wall breaking treatment at 50 ℃, wherein the ultrasonic frequency is 40kH, the inert gas pressure is 170MPa, the gas flow rate is 600m/s, the feeding speed is 5g/s, and the feeding pressure is 0.5MPa, and all the obtained powder passes through the 600-mesh sieve.
(2) Powder infiltration: spraying 60% ethanol to the powder obtained in the step (1) to fully soak the powder;
(3) microwave irradiation: and (3) performing microwave irradiation on the infiltrate obtained in the step (2), wherein the microwave power is 800w, the temperature is 60 ℃, and the time is 1 min.
(4) Drying under negative pressure: drying the infiltrated substance subjected to microwave treatment in the step (3) under negative pressure to obtain dry powder;
(5) extracting and filtering: conveying the dried powder obtained in the step (4) by using inert gas, wherein the conveying pressure of the inert gas is 101KPa, spraying the powder, carrying out high-pressure opposite spraying by using 8 times of 60% ethanol liquid, fully colliding and matching under high pressure, mixing, stirring for 40min, and filtering to obtain an extracting solution;
(6) and (3) purification: carrying out chromatographic separation on the extracting solution obtained in the step (5) by using D101 macroporous resin, and sequentially carrying out the following elution: eluting with distilled water, eluting with 20% ethanol, eluting with 80% ethanol, and collecting 80% ethanol eluate.
(7) And (3) drying under reduced pressure: and (4) concentrating the eluent obtained in the step (6) under reduced pressure, and drying to obtain the extract.
Example 3
A method for extracting a component for treating respiratory inflammation from thoroughfare bitter orange comprises the following steps:
(1) treating thoroughfare bitter orange: the preparation method comprises the steps of slicing 500g of thoroughfare bitter orange, adding the slices into a pulverizer to be pulverized, sieving the slices with a 60-mesh sieve, adding 4 times of ethyl acetate, stirring and filtering the slices at-10 ℃, freeze-drying filter residues, and then carrying out ultrasonic wall breaking treatment at 50 ℃, wherein the ultrasonic frequency is 40kH, the inert gas pressure is 170MPa, the gas flow rate is 600m/s, the feeding speed is 5g/s, and the feeding pressure is 0.5MPa, and all the obtained powder passes through the 600-mesh sieve.
(2) Powder infiltration: spraying 80% ethanol to the powder obtained in the step (1) to fully soak the powder;
(3) microwave irradiation: and (3) performing microwave irradiation on the infiltrate obtained in the step (2), wherein the microwave power is 500w, the temperature is 70 ℃, and the time is 1.5 min.
(4) Drying under negative pressure: drying the infiltrated substance subjected to microwave treatment in the step (3) under negative pressure to obtain dry powder;
(5) extracting and filtering: conveying the dried powder obtained in the step (4) by using inert gas, wherein the conveying pressure of the inert gas is 101KPa, spraying the powder, carrying out high-pressure opposite spraying by using 8 times of 80% ethanol liquid, fully colliding and matching under high pressure, mixing, stirring for 20min, and filtering to obtain an extracting solution;
(6) and (3) purification: carrying out chromatographic separation on the extracting solution obtained in the step (5) by using D101 macroporous resin, and sequentially carrying out the following elution: eluting with distilled water, eluting with 20% ethanol, eluting with 70% ethanol, and collecting 70% ethanol eluate.
(7) And (3) drying under reduced pressure: and (4) concentrating the eluent obtained in the step (6) under reduced pressure, and drying to obtain the extract.
Comparative example 1
(1) Treating the fructus aurantii: the preparation method comprises the steps of slicing 500g of thoroughfare bitter orange, adding the slices into a pulverizer to be pulverized, sieving the slices with a 60-mesh sieve, adding 6 times of ethyl acetate, stirring and filtering the slices at-10 ℃, freeze-drying filter residues, and then carrying out ultrasonic wall breaking treatment at 50 ℃, wherein the ultrasonic frequency is 40kH, the inert gas pressure is 170MPa, the gas flow rate is 600m/s, the feeding speed is 5g/s, and the feeding pressure is 0.5MPa, and all the obtained powder passes through the 600-mesh sieve.
(2) Extracting and filtering: adding 70% ethanol in an amount which is 9 times that of the powder obtained in the step (1) into the powder, and performing reflux extraction for 3 hours to obtain an extracting solution;
(3) and (3) purification: carrying out chromatographic separation on the extracting solution obtained in the step (5) by using D101 macroporous resin, and sequentially carrying out the following elution: eluting with distilled water, eluting with 20% ethanol, eluting with 75% ethanol, and collecting 75% ethanol eluate.
(4) And (3) drying under reduced pressure: and (4) concentrating the eluent obtained in the step (6) under reduced pressure, and drying to obtain the extract.
Comparative example 2
A method for extracting a component for treating respiratory inflammation from thoroughfare bitter orange comprises the following steps:
(1) treating thoroughfare bitter orange: 500g of thoroughfare bitter orange slices are added into a pulverizer to be pulverized, 6 times of ethyl acetate is added, the filter residue is subjected to freeze drying under the condition of stirring and filtering at the temperature of minus 10 ℃, then ultrasonic wall breaking treatment is carried out at the temperature of 50 ℃, the ultrasonic frequency is 40kH, the inert gas pressure is 170MPa, the gas flow rate is 600m/s, the feeding speed is 5g/s, the feeding pressure is 0.5MPa, and the obtained powder passes through a 600-mesh sieve.
(2) Extracting and filtering: conveying the powder obtained in the step (1) by using inert gas, wherein the conveying pressure of the inert gas is 101KPa, spraying the powder, carrying out high-pressure opposite spraying by using 70% ethanol liquid in an amount which is 9 times that of the powder, fully colliding and matching the powder at high pressure, mixing, stirring for 30min, and filtering to obtain an extracting solution;
(6) and (3) purification: carrying out chromatographic separation on the extracting solution obtained in the step (5) by using D101 macroporous resin, and sequentially carrying out the following elution: eluting with distilled water, eluting with 20% ethanol, eluting with 75% ethanol, and collecting 75% ethanol eluate.
(7) And (3) drying under reduced pressure: and (4) concentrating the eluent obtained in the step (6) under reduced pressure, and drying to obtain the extract.
Comparative example 3
A method for extracting a component for treating respiratory inflammation from thoroughfare bitter orange comprises the following steps:
(1) treating thoroughfare bitter orange: 500g of thoroughfare bitter orange slices are added into a pulverizer to be pulverized, 6 times of ethyl acetate is added, the filter residue is subjected to freeze drying under the condition of stirring and filtering at the temperature of minus 10 ℃, then ultrasonic wall breaking treatment is carried out at the temperature of 50 ℃, the ultrasonic frequency is 40kH, the inert gas pressure is 170MPa, the gas flow rate is 600m/s, the feeding speed is 5g/s, the feeding pressure is 0.5MPa, and the obtained powder passes through a 600-mesh sieve.
(2) Powder infiltration: spraying 70% ethanol to the powder obtained in the step (1) to fully soak the powder;
(3) microwave irradiation: and (3) performing microwave irradiation on the infiltrate obtained in the step (2), wherein the microwave power is 600w, the temperature is 60 ℃, and the time is 1.5 min.
(4) Drying under negative pressure: drying the infiltrated substance subjected to microwave treatment in the step (3) under negative pressure to obtain dry powder;
(5) extracting and filtering: soaking the dried powder obtained in the step (4) in 70% ethanol 9 times the weight of the dried powder, stirring for 30min, and filtering to obtain an extracting solution;
(6) and (3) purification: carrying out chromatographic separation on the extracting solution obtained in the step (5) by using D101 macroporous resin, and sequentially carrying out the following elution: eluting with distilled water, eluting with 20% ethanol, eluting with 75% ethanol, and collecting 75% ethanol eluate.
(7) And (3) drying under reduced pressure: and (4) concentrating the eluent obtained in the step (6) under reduced pressure, and drying to obtain the extract.
Comparative example 4
A method for extracting a component for treating respiratory inflammation from thoroughfare bitter orange comprises the following steps:
(1) treating thoroughfare bitter orange: adding 500g of thoroughfare bitter orange slices into a pulverizer to be pulverized, sieving the slices by a 60-mesh sieve, and then carrying out ultrasonic wall breaking treatment at the temperature of 50 ℃, wherein the ultrasonic frequency is 40kH, the inert gas pressure is 170MPa, the gas flow rate is 600m/s, the feeding speed is 5g/s, and the feeding pressure is 0.5MPa, and all the obtained powder passes through the 600-mesh sieve.
(2) Powder infiltration: spraying 70% ethanol to the powder obtained in the step (1) to fully soak the powder;
(3) microwave irradiation: and (3) performing microwave irradiation on the infiltrate obtained in the step (2), wherein the microwave power is 600w, the temperature is 60 ℃, and the time is 1.5 min.
(4) Drying under negative pressure: drying the infiltrated substance subjected to microwave treatment in the step (3) under negative pressure to obtain dry powder;
(5) extracting and filtering: conveying the dried powder obtained in the step (4) by using inert gas, wherein the conveying pressure of the inert gas is 101KPa, spraying the powder, carrying out high-pressure opposite spraying by using liquid of 70% ethanol 9 times the amount of the powder, fully colliding and matching the powder at high pressure, mixing, stirring for 30min, and filtering to obtain an extracting solution;
(6) and (3) purification: carrying out chromatographic separation on the extracting solution obtained in the step (5) by using D101 macroporous resin, and sequentially carrying out the following elution: eluting with distilled water, eluting with 20% ethanol, eluting with 75% ethanol, and collecting 75% ethanol eluate.
(7) And (3) drying under reduced pressure: and (4) concentrating the eluent obtained in the step (6) under reduced pressure, and drying to obtain the extract.
Examples of effects
After drying the extracts obtained in examples 1 to 3 and comparative examples 1 to 4 to a constant weight, the extracts were weighed and the mass of the extract was recorded. According to chromatographic conditions: agilent Zorbax SB C18 column (250 mm. times.4.6 mm, 5 μm); the mobile phase is acetonitrile-0.02% phosphoric acid water solution (20: 80), the flow rate is 1.0 m L min-1, the sample injection volume is 25 μ L, the detection wavelength is 280 nm, the column temperature is 35 ℃, and the content of hesperidin and neohesperidin is measured by an external standard method, which is shown in Table 1.
Table 1 component detection and quality determination of extracts
Application example the present invention is effective in inhibiting bronchitis
Experimental animals: the method comprises the following steps of randomly dividing 60 healthy Kunming mice with the mass of 18-22 g into three groups of 20 mice, wherein the three groups of mice are respectively a negative control group, a test group and a positive control group.
And (3) interference scheme:
negative control group: the physiological saline was administered twice daily in the morning and afternoon, respectively.
Test groups: 1g/kg of the extract obtained in example 1 was administered twice a day in the morning and afternoon, respectively.
Positive control group: the rolipram is 10mg/kg twice a day in the morning and in the afternoon respectively.
The test method comprises the following steps: intraperitoneal injection of female mice 10 μ g ovalbumin with aluminum hydroxide 1: 1 mixture 0.2ml was sensitized, and the administration time was 0, 7 and 14 days. After 8 days and 10 days of sensitization, 1.0% egg protein is made into aerosol, and the aerosol is sprayed for 50min to induce airway inflammation. After the first sensitization, interference was given orally until 23 days after the first sensitization, and 24 hours after the last inflammation induction, bronchoalveolar cells were washed with 1.5ml of phosphate buffer (ph7.2), and then a bronchoalveolar washing solution was collected, and the number of leukocytes in the washing solution was measured with a hematology analyzer. The bronchitis inhibition (%) was calculated based on the number of leukocytes in the negative control group (physiological saline). The results obtained using rolipram as a control are shown in Table 2
TABLE 2 Effect on the inhibitory Rate of bronchitis: (
n＝20)
It can be seen that the extracts provided herein have significant airway inflammation inhibitory activity.
Clinical examples
1 general data:
64 outpatient respiratory inflammation patients of a traditional Chinese medicine hospital in Henan province from 1 month to 10 months in 2018 are selected and randomly divided into 32 patients of a treatment group and a control group. Treatment groups: 14 men and 18 women with a mean disease course of 24.6 + -5.7 h and a mean age of 32.04 + -6.48 years. Control group: 16 men and 16 women with a mean disease course of 25.9 + -3.9 h and a mean age of 33.51 + -5.65 years. The difference between the two groups of cases in disease course, age, respiration, pulse, body temperature, height, etc. has no statistical significance (p is more than 0.05).
2 method of treatment
Treatment groups: the extract provided in example 1 of the present invention was administered twice a day, 1g each time.
Control group: cefixime, administered as described.
3 Observation indicators and methods
3.1 evaluation criteria for clinical efficacy of disease
The effect is shown: within 5 days of treatment, the clinical manifestations of cough, fever, pharyngalgia and the like disappear;
the method has the following advantages: after 5 days of treatment, the cough, the pharyngalgia and the nasal discharge are all improved, and the fever is improved;
and (4) invalidation: after 5 days of treatment, the cough, the pharyngalgia and the nasal discharge are not improved, and the fever phenomenon exists.
3.2 inflammatory factor detection
The patient takes 5mL of peripheral blood before and after treatment with an empty stomach, separates serum, and measures the content of IL-2 (interleukin-2), IL-6 (interleukin-6), IL-8 (interleukin-8) and TNF-alpha (tumor necrosis factor-alpha) by adopting a double antibody sandwich ABC-ELISA method. Anti-human IL-2, IL-6, IL-8 and TNF-alpha monoclonal antibodies are coated on an enzyme label plate, IL-2, IL-6, IL-8 and TNF-alpha in a standard product and a sample are combined with the monoclonal antibodies, biotin-labeled anti-human IL-2, IL-6, IL-8 and TNF-alpha antibodies are respectively added to form an immune complex which is connected on the plate, peroxidase-labeled avidin is combined with biotin, an enzyme substrate TMB is added to generate color, and TMB stop solution is added to change the color. The OD values were measured at different wavelengths, and the concentration of the substance in the specimen was determined by plotting a standard curve.
4 statistical analysis
SPSS 18.0 statistical software was used for analysis of results and for data measurement "
"means, t-test; the technical data are tested by adopting X2.
5 analysis of results
5.1 clinical efficacy comparison of two groups of patients
After 5 days of treatment, the total effective rate of the patients in the treatment group is 96.9 percent, which is obviously higher than 84.4 percent of that in the control group, and the treatment group has statistical significance, which is shown in table 3.
TABLE 3 comparison of the efficacy of the two groups of patients
5.2 comparison of inflammatory response indexes before and after two groups of treatment
The statistics of inflammatory response indexes before and after treatment are shown in table 4.
TABLE 4 comparison of inflammatory response indexes before and after treatment of two groups: (
，pg/mL）
As can be seen from the results, the treatment groups had differences in the IL-2, IL-6, IL-8 and TNF- α indexes before and after treatment, and the control group and the treatment groups had differences in the IL-2, IL-6, IL-8 and TNF- α indexes after treatment. The extract obtained in the example 1 of the application can obviously reduce the levels of serum inflammatory factors IL-2, IL-6, IL-8 and TNF-alpha of a patient.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. A method for extracting a component for treating respiratory inflammation in thoroughfare bitter orange is characterized by comprising the following steps:
(1) treating thoroughfare bitter orange: slicing thoroughfare bitter orange, adding the thoroughfare bitter orange into a pulverizer for pulverizing, adding ethyl acetate, stirring, filtering, performing freeze drying at the temperature of minus 5 to minus 15 ℃, and performing ultrasonic wall breaking treatment at the temperature of 40-60 ℃;
(2) powder infiltration: spraying 30-70% of ethanol into the powder obtained in the step (1), and fully soaking;
(3) microwave irradiation: performing microwave irradiation on the infiltrate obtained in the step (2);
(4) drying under negative pressure: drying the infiltrated substance subjected to microwave treatment in the step (3) under negative pressure to obtain dry powder;
(5) extracting and filtering: conveying the dried powder obtained in the step (4) by using inert gas, spraying 8-10 times of 30-70% ethanol in opposite directions at high pressure, mixing, stirring for 20-40min, and filtering to obtain an extracting solution;
(6) and (3) purification: and (4) carrying out chromatography separation on the extracting solution obtained in the step (5) by using macroporous resin, and sequentially carrying out the following elution: eluting with distilled water, then eluting with 20% ethanol, finally eluting with 60-80% ethanol, and collecting 60-80% ethanol eluate;
(7) and (3) drying under reduced pressure: and (4) concentrating the eluent obtained in the step (6) under reduced pressure, and drying to obtain the extract.
2. The extraction method of a component for treating respiratory inflammation in a thoroughfare bitter orange according to claim 1, wherein the amount of ethyl acetate added in step (1) is 4-8 times the weight of the thoroughfare bitter orange.
3. The extraction method of the component for treating the respiratory inflammation in the thoroughfare bitter orange according to claim 1, wherein in the step (1), the wall is broken by ultrasonic waves, the ultrasonic frequency is 30-50 kH, the pressure of inert gas is 150-200 MPa, the gas flow rate is 500-700 m/s, the feeding speed is 4-6 g/s, and the feeding pressure is 0.4-0.6 MPa.
4. The extraction method of a component for treating respiratory inflammation in thoroughfare fructus aurantii according to claim 1, wherein the microwave irradiation parameters of step (3) are as follows: the power is 500-.
5. The extraction method of a component for treating respiratory inflammation in thoroughfare fructus aurantii according to claim 1, wherein the inert gas delivery pressure in step (5) is 101KPa, and the spraying pressure of ethanol is 200-300 MPa.
6. The extraction method of a component for treating respiratory inflammation in thoroughfare fructus aurantii according to claim 1, wherein the macroporous resin in the step (6) is D101 or AB-8 macroporous resin.
7. The extraction method of a component for treating respiratory inflammation in thoroughfare fructus aurantii according to claim 1, wherein in the step (6), the component is eluted with distilled water, then eluted with 20% ethanol, and finally eluted with 75% ethanol, and then the 75% ethanol eluate is collected.
8. The extraction method of the component for treating respiratory inflammation in the thoroughfare bitter orange according to claim 1, which comprises the following steps:
(1) treating thoroughfare bitter orange: slicing thoroughfare bitter orange, adding the thoroughfare bitter orange into a pulverizer for pulverizing, adding 6 times of ethyl acetate, stirring and filtering at the temperature of minus 10 ℃, freeze-drying filter residue, and then carrying out ultrasonic wall breaking treatment at the temperature of 50 ℃, wherein the ultrasonic frequency is 40kH, the inert gas pressure is 170MPa, the gas flow rate is 600m/s, the feeding speed is 5g/s, and the feeding pressure is 0.5 MPa;
(2) powder infiltration: spraying 30-70% of ethanol into the powder obtained in the step (1), and fully soaking;
(3) microwave irradiation: performing microwave irradiation on the infiltrate obtained in the step (2), wherein the microwave power is 500-800w, the temperature is 50-70 ℃, and the time is 1-2 min;
(4) drying under negative pressure: drying the infiltrated substance subjected to microwave treatment in the step (3) under negative pressure to obtain dry powder;
(5) extracting and filtering: conveying the dried powder obtained in the step (4) by using inert gas, wherein the conveying pressure of the inert gas is 101KPa, spraying the powder, carrying out high-pressure opposite spraying by using liquid of 60% ethanol in an amount which is 9 times that of the powder, fully colliding and matching under high pressure, mixing, stirring for 30min, and filtering to obtain an extracting solution;
(6) and (3) purification: carrying out chromatographic separation on the extracting solution obtained in the step (5) by using D101 macroporous resin, and sequentially carrying out the following elution: eluting with distilled water, then eluting with 20% ethanol, then eluting with 75% ethanol, and collecting 75% ethanol eluate;
(7) and (3) drying under reduced pressure: and (4) concentrating the eluent obtained in the step (6) under reduced pressure, and drying to obtain the extract.
9. Use of an extract obtained by the extraction method according to any one of claims 1 to 8 in the preparation of a medicament for the treatment of respiratory inflammation.
10. The use of claim 9, wherein the respiratory inflammation is respiratory inflammation.
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