CN117137971A - Oviductus Ranae composition and Chinese medicinal composition for relieving cough and asthma, and preparation method and application thereof - Google Patents
Oviductus Ranae composition and Chinese medicinal composition for relieving cough and asthma, and preparation method and application thereof Download PDFInfo
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- CN117137971A CN117137971A CN202311036373.XA CN202311036373A CN117137971A CN 117137971 A CN117137971 A CN 117137971A CN 202311036373 A CN202311036373 A CN 202311036373A CN 117137971 A CN117137971 A CN 117137971A
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- oviductus ranae
- enzymolysis
- cough
- asthma
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- 235000019643 salty taste Nutrition 0.000 description 1
- 230000035807 sensation Effects 0.000 description 1
- 235000019615 sensations Nutrition 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
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- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 239000008347 soybean phospholipid Substances 0.000 description 1
- 150000003432 sterols Chemical class 0.000 description 1
- 235000003702 sterols Nutrition 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
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- NRWCNEBHECBWRJ-UHFFFAOYSA-M trimethyl(propyl)azanium;chloride Chemical compound [Cl-].CCC[N+](C)(C)C NRWCNEBHECBWRJ-UHFFFAOYSA-M 0.000 description 1
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention belongs to the field of traditional Chinese medicines, and particularly relates to a toad oil composition for relieving cough and asthma, a traditional Chinese medicine composition, and a preparation method and application thereof. The oviductus Ranae composition is prepared from raw materials of forest frog oil, chinese forest frog skin, loquat leaf and liquorice. The traditional Chinese medicine composition is prepared into drug-loaded liposome by wrapping the oviductus ranae composition with soybean lecithin, cholesterol and cationic materials. The drug-loaded liposome has the effects of moistening lung and relieving cough, can relieve dry cough and asthma, and particularly has obvious relieving effect on cough caused by consumptive disease; can improve cough, expectoration, etc. caused by chronic bronchitis. The liposome preparation prepared by the invention is administered by pulmonary inhalation administration, has quick response, makes up for the blank of the administration of the oviductus Ranae aerosol, is matched with an atomizer for use, is convenient and safe, has high bioavailability, improves the conditions that the oviductus Ranae is difficult to absorb and easy to grease in the use process, has no adverse reaction, and is suitable for a wide range of people.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a toad oil composition for relieving cough and asthma, a traditional Chinese medicine composition, and a preparation method and application thereof.
Background
Respiratory diseases are common and frequently-occurring diseases, and are mainly caused by the diseases of trachea, bronchus, lung and chest, and the patients with light diseases are affected by cough, chest pain and respiration, and the patients with serious diseases are caused by dyspnea, hypoxia and even respiratory failure. Patients with respiratory diseases proliferate with changes in air pollution, environment, and climate. Respiratory diseases have attracted considerable attention from the world. Respiratory diseases have become one of the risk factors threatening human health. According to world health report issued by the world health organization in 2018, mortality from respiratory diseases is third in the global non-infectious disease mortality rank, next to cardiovascular diseases and tumors. Respiratory diseases have seriously compromised human health and are public health problems that need to be solved by the sustainable development of human society.
Oviductus Ranae, also called oviductus Ranae, is a dried oviduct product of female Rana chensinensis, recorded in Chinese pharmacopoeia in 1985, is a rare Chinese medicinal material in China, and has main production areas of Heilongjiang, jilin and Liaoning. The Chinese medicine considers that the oviductus ranae has flat oil quality, sweet and salty taste, and has the effects of tonifying kidney, replenishing essence, nourishing yin and moistening lung. Since ancient times, it has been known as a tonic and precious product, and has been known as tonic and soft gold, and oviductus Ranae has extremely high nutritive and medicinal values. Modern researches have found that oviductus ranae has various chemical components including proteins, amino acids, fatty acids, phospholipids, sterols, nucleosides, vitamins, 1-methylhydantoin and mineral elements, and has pharmacological effects including relieving cough, eliminating phlegm, resisting inflammation, resisting oxidation, resisting fatigue, resisting aging, resisting depression, resisting tumor, resisting osteoporosis, protecting liver, reducing blood lipid, regulating immunity, etc.
The oviductus Ranae has good effect of relieving cough and asthma and excessive phlegm cough, and has good health promoting effect. However, because the components of the oviductus Ranae are special and are confusing in the market in preparation and application, the development of the pharmaceutical application of the oviductus Ranae is obviously behind other traditional Chinese medicines at present, so that the pharmaceutical value of the oviductus Ranae is covered, the effective utilization of the oviductus Ranae is greatly reduced, and most of the oviductus Ranae products in the market are made of more food and health-care food, the effective components of the oviductus Ranae are almost and directly released, the bioavailability of the oviductus Ranae is hindered, the resource waste of the oviductus Ranae is caused, and therefore, the deep development of the pharmaceutical value of the oviductus Ranae is necessary, and the added value of the oviductus Ranae products is improved.
Disclosure of Invention
In order to solve the problems, the invention provides a oviductus Ranae composition and a traditional Chinese medicine composition for relieving cough and asthma, and a preparation method and application thereof. The oviductus ranae composition and the traditional Chinese medicine composition provided by the invention have obvious relieving effect on cough and asthma and excessive cough and phlegm.
The invention provides a toad oil composition for relieving cough and asthma, which comprises the following raw materials in parts by weight: 1 to 3 parts of Chinese forest frog oil, 2 to 6 parts of Chinese forest frog skin, 1 to 3 parts of loquat leaf and 1 to 3 parts of liquorice.
The invention also provides a preparation method of the cough and asthma relieving oviductus Ranae composition, which comprises the following steps: soaking the Chinese forest frog oil in citric acid with pH of 4.5, homogenizing, mixing the homogenate obtained by homogenizing with distilled water, and decocting to obtain an enzymolysis substrate;
performing first enzymolysis on the enzymolysis substrate by using pepsin to obtain pepsin enzymolysis liquid;
performing second enzymolysis on the pepsin enzymolysis liquid by using trypsin, centrifuging the obtained trypsin enzymolysis liquid to obtain supernatant, and freeze-drying to obtain the snow clam oil freeze-dried powder;
extracting effective components in the Chinese forest frog skin, the loquat leaf and the liquorice by using a polar alcohol solution to obtain a polar alcohol solution extract;
mixing the freeze-dried forest frog oil powder with the polar alcohol solution extract to obtain the forest frog oil composition.
Preferably, the mass-volume ratio of the Chinese forest frog oil to the citric acid with the pH value of 4.5 is 1g: 80-120 mL; the soaking time is 36 hours;
the volume ratio of the homogenate to the distilled water is 1: (3-5).
Preferably, before the first enzymolysis with pepsin, the method further comprises: regulating the pH value of the enzymolysis substrate to 2.8-3.2; the feed liquid ratio of the pepsin to the enzymolysis substrate is 1g: 1500-2500 mL; the temperature of the first enzymolysis is 37 ℃ and the time is 2 hours;
the enzyme activity of the pepsin is more than or equal to 250U/mg.
Preferably, before the second enzymolysis with trypsin, the method further comprises: regulating the pH value of the pepsin enzymolysis liquid to 9.8-10.2; the feed liquid ratio of the trypsin to the pepsin enzymatic hydrolysate is 1g: 80-120 mL; the temperature of the second enzymolysis is 55 ℃ and the time is 6 hours;
the enzyme activity of the trypsin is 250.N.F.U/mg.
The invention also provides a traditional Chinese medicine composition for relieving cough and asthma, which comprises the following raw materials in parts by weight: 1 part of the oviductus ranae composition, 3.2 to 6.4 parts of soybean lecithin, 0.4 to 0.8 part of cholesterol and 0.04 to 0.08 part of cationic material.
The invention also provides a preparation method of the traditional Chinese medicine composition for relieving cough and asthma, which comprises the following steps:
dissolving soybean lecithin, cholesterol and a cationic material in a first organic solvent, and recovering the first organic solvent to obtain a first mixture;
mixing the second organic solvent with the oviductus Ranae composition, mixing the obtained second mixture with the first mixture, evaporating under reduced pressure, and performing ultrasound to obtain the Chinese medicinal composition for relieving cough and asthma;
the first organic solvent and the second organic solvent are one or more of dichloromethane, chloroform, methanol, diethyl ether, ethanol and acetone respectively.
The invention also provides application of the toad oil composition or the traditional Chinese medicine composition in preparing medicines for treating respiratory diseases.
Preferably, the respiratory disease includes at least one of cough, expectoration, pharyngitis, bronchial asthma and emphysema.
Preferably, the dosage form of the medicament comprises an aerosol or an inhaled water mist.
The beneficial effects are that: the oviductus Ranae composition for relieving cough and asthma is prepared from oviductus Ranae, chinese forest frog skin, folium eriobotryae and liquorice as raw materials, and the oviductus Ranae composition is wrapped by using liposome, so that the solubility of the medicine is improved, the stability is good, the problems that the existing oviductus Ranae is difficult to dissolve and the preparation is difficult are solved, and the blank of the administration of the oviductus Ranae mist is filled. Meanwhile, aerosol administration is used for treating respiratory diseases, so that the first pass elimination effect can be avoided, the effect is quick, the targeting is strong, and the stability and bioavailability of the oviductus ranae extract can be obviously improved. In addition, the adopted preparation method is the existing common method, and is convenient for industrial production. According to the embodiment, the traditional Chinese medicine composition provided by the invention can effectively prolong the residence time of the medicine in the lung, increase the medicine availability, and has better cough relieving and phlegm eliminating effects than other administration modes.
Drawings
FIG. 1 is a schematic diagram showing the effect of the ratio of components in cationic liposome on encapsulation efficiency and entrapment efficiency;
FIG. 2 is a graph showing the drug distribution in rats over time;
FIG. 3 is a schematic representation of total white blood cells following administration to each group of rats;
FIG. 4 is a graph showing eosinophil count following administration to each group of rats;
FIG. 5 is a graph showing lymphocyte counts after administration to each group of rats;
FIG. 6 is a graph showing neutrophil count following administration to each group of rats;
FIG. 7 is a graphical representation of IL-4 content in tissue homogenates following administration to rats in each group;
FIG. 8 is a graphical representation of IL-5 content in tissue homogenates following administration to rats in each group;
FIG. 9 is a graph showing IgE content in tissue homogenates after administration to rats in each group;
FIG. 10 is a schematic of the asthma-relieving latency after administration to each group of rats;
FIG. 11 is a graphical representation of asthma behavioural scores after each group of rats had been dosed;
FIG. 12 is a graphical representation of HE staining of lung tissue status after administration to each group of rats;
figure 13 is a graph showing the changes in cough latency, cough count and sputum excretion capacity following dosing in each group of mice.
Detailed Description
The invention provides a toad oil composition for relieving cough and asthma, which comprises the following raw materials in parts by weight: 1 to 3 parts of Chinese forest frog oil, 2 to 6 parts of Chinese forest frog skin, 1 to 3 parts of loquat leaf and 1 to 3 parts of liquorice.
The invention has no special requirements on the sources of the raw materials of the traditional Chinese medicine composition, and adopts commercial products well known to those skilled in the art.
The oviductus Ranae composition provided by the invention comprises 1-3 parts by mass of oviductus Ranae, preferably 1.2-2.8 parts by mass, more preferably 1.5-2.5 parts by mass, and most preferably 1.8-2.2 parts by mass. The Chinese forest frog is sweet and salty in flavor, and enters the lung meridian and kidney meridian. Is mainly used for tonifying kidney, replenishing vital essence, moistening lung and nourishing yin. For post-illness and postpartum weakness, cough and hematemesis due to tuberculosis, night sweat, listlessness, palpitation and insomnia.
The oviductus ranae composition provided by the invention comprises 2-6 parts of oviductus ranae, preferably 2.5-5.5 parts of oviductus ranae, more preferably 3-5 parts of oviductus ranae, and most preferably 3-4.5 parts of oviductus ranae. The mass part of the snow clam skin is preferably 2 times of the mass part of the snow clam oil. The skin has effects of nourishing yin and enhancing immunity
The oviductus ranae composition provided by the invention comprises 1-3 parts of loquat leaves, preferably 1.2-2.8 parts, more preferably 1.5-2.5 parts and most preferably 1.8-2.2 parts by mass of oviductus ranae. The mass part of the loquat leaf is preferably 0.5 times of the mass part of the Chinese forest frog oil. The loquat leaf is bitter in taste and slightly cold. Enter lung and stomach meridians. Clear lung and harmonize stomach, reduce qi and resolve phlegm. Is mainly used for treating lung heat phlegm cough, hemoptysis, epistaxis, stomach heat vomiting, dysphoria with smothery sensation and thirst.
The oviductus ranae composition provided by the invention comprises 1-3 parts of liquorice, preferably 1.2-2.8 parts of liquorice, more preferably 1.5-2.5 parts of liquorice and most preferably 1.8-2.2 parts of liquorice. The mass portion of the liquorice is preferably 0.5 times of the mass portion of the Chinese forest frog oil. The licorice is sweet in taste and flat in flavor. Enters spleen meridian, stomach meridian and lung meridian. Mainly treats the symptoms of harmonizing middle energizer and relieving urgency, moistening lung, detoxifying and harmonizing various medicines.
In the invention, the mass ratio of the forest frog oil to the loquat leaf to the liquorice in the forest frog oil composition is preferably 1:2:0.5:0.5.
the Chinese forest frog oil is a monarch drug, moistens lung to arrest cough and tonify consumptive disease; loquat leaves are ministerial drugs, clear lung and harmonize stomach, reduce qi and phlegm, and are compatible with oviductus Ranae to enhance the efficacy of moistening lung and relieving cough; glycyrrhrizae radix is adjuvant drug with effects of moistening lung, regulating middle warmer and relieving urgency to assist lung; the Chinese forest frog skin is used as a guiding drug to regulate the functions of the drugs and guiding the drugs into the channels.
The invention also provides a preparation method of the oviductus ranae composition for relieving cough and asthma, which comprises the following steps: soaking the Chinese forest frog oil in citric acid with pH of 4.5, homogenizing, mixing the homogenate obtained by homogenizing with distilled water, and decocting to obtain an enzymolysis substrate;
performing first enzymolysis on the enzymolysis substrate by using pepsin to obtain pepsin enzymolysis liquid;
performing second enzymolysis on the pepsin enzymolysis liquid by using trypsin, centrifuging the obtained trypsin enzymolysis liquid to obtain supernatant, and freeze-drying to obtain the snow clam oil freeze-dried powder;
extracting effective components in the Chinese forest frog skin, the loquat leaf and the liquorice by using a polar alcohol solution to obtain a polar alcohol solution extract;
mixing the freeze-dried forest frog oil powder with the polar alcohol solution extract to obtain the forest frog oil composition.
In the invention, the forest frog oil is soaked in citric acid with pH of 4.5 and homogenized to obtain homogenate.
In the invention, the ratio of the feed liquid of the Chinese forest frog oil to the citric acid with the pH of 4.5 is preferably 1g:80 to 120mL, more preferably 1g: 90-110 mL, most preferably 1g:100mL. The soaking time is preferably 36 hours; the homogenate is preferably obtained by co-homogenating the snow clam oil and the citric acid mixture. According to the invention, the snow clam oil is soaked in the citric acid with the pH of 4.5 and then homogenized, so that the snow clam oil can be fully foamed, and meanwhile, the damage of active ingredients in the snow clam oil is avoided.
After the homogenate is obtained, the homogenate is mixed with distilled water and decocted to obtain an enzymolysis substrate.
In the present invention, the volume ratio of the homogenate to distilled water is preferably 1: (3 to 5), more preferably 1: (3.5 to 4.5), most preferably 1:4, a step of; the decoction is preferably to boiling; the time of the decoction is preferably 1 to 3 hours, more preferably 1.5 to 2.5 hours, and most preferably 2 hours.
After an enzymolysis substrate is obtained, pepsin is utilized to carry out first enzymolysis on the enzymolysis substrate, so that pepsin enzymolysis liquid is obtained.
In the invention, the feed liquid ratio of the pepsin to the enzymolysis substrate is preferably 1g:1500 to 2500mL, more preferably 1g:1800 to 2200mL, most preferably 1g:2000mL; the temperature of the first enzymolysis is preferably 37 ℃; the time of the first enzymolysis is preferably 2 hours. After the first enzymolysis, the method preferably further comprises the step of inactivating enzyme; the enzyme deactivation is preferably performed by adopting high temperature enzyme deactivation, such as boiling a sample for 15min after enzymolysis to inactivate pepsin, and cooling to room temperature to obtain the pepsin enzymolysis liquid. According to the invention, the pepsin is utilized for the first enzymolysis, so that most of proteins in the oviductus ranae can be hydrolyzed into polypeptides, and the polypeptides are easier to absorb and prepare medicines.
The invention preferably further comprises the following steps before the first enzymolysis by pepsin: the pH of the substrate for enzymolysis is adjusted to 2.8 to 3.2, more preferably 2.9 to 3.1, and most preferably 3.0. The pH is preferably adjusted using 10mmol/L HCl solution.
After pepsin enzymatic hydrolysate is obtained, trypsin is utilized to carry out second enzymolysis on the pepsin enzymatic hydrolysate, so that trypsin enzymatic hydrolysate is obtained.
In the invention, the feed liquid ratio of the trypsin to the pepsin enzymolysis liquid is preferably 1g:80 to 120mL, more preferably 1g: 90-110 mL, most preferably 1g:100mL; the temperature of the second enzymolysis is preferably 55 ℃; the time of the second enzymolysis is preferably 6 hours. After the second enzymolysis, the method preferably further comprises the step of inactivating enzyme; the enzyme deactivation is preferably performed by adopting high temperature enzyme deactivation, such as the deactivation of trypsin by boiling a sample for 15min after enzymolysis, and then cooling to room temperature to obtain the trypsin enzymatic hydrolysate. The invention uses trypsin to carry out the second enzymolysis, which can hydrolyze denatured protein, fibrin, mucin and the like into polypeptide or amino acid, further hydrolyze protein components in the oviductus Ranae and improve the utilization rate of the oviductus Ranae.
The invention preferably further comprises, before the second enzymolysis by trypsin: the pH of the pepsin enzymatic hydrolysate is adjusted to 9.8-10.2, more preferably to 9.9-10.1, most preferably to 10.0. The pH is preferably adjusted using a 0.1mol/L NaOH solution.
After trypsin enzymatic hydrolysate is obtained, the trypsin enzymatic hydrolysate is centrifuged to obtain supernatant.
In the present invention, the centrifugation conditions are preferably 10000rpm for centrifugation for 15min. The pH of the trypsin enzymatic hydrolysate is preferably adjusted to 6.9-7.1, more preferably 7.0, before centrifugation. The pH adjustment is preferably carried out using 0.1mol/L NaOH.
After the supernatant is obtained, the invention carries out freeze drying on the supernatant to obtain the snow clam oil freeze-dried powder. The freeze-drying method is not particularly limited, and the freeze-drying method commonly used in the art can be adopted.
The invention utilizes polar alcohol solution to extract effective components in Chinese forest frog skin, loquat leaf and liquorice to obtain polar alcohol solution extract.
In the present invention, the polar alcohol solution preferably includes an ethanol solution or a methanol solution, more preferably 75% ethanol. The invention can utilize polar alcohol solution to extract the Chinese forest frog skin, the loquat leaf and the liquorice respectively, or can extract the Chinese forest frog skin, the loquat leaf and the liquorice after mixing; the extraction conditions of the respective extraction or the extraction after mixing are the same, and the extraction after mixing is described as an example. The feed liquid ratio of the mixture of the Chinese forest frog skin, the loquat leaf and the liquorice to the polar alcohol solution is preferably 1g:3 to 5mL, more preferably 1g:3.5 to 4.5mL, most preferably 1g:4mL; the extraction is preferably reflux extraction for 1-3 times; the extraction time is preferably 1 to 3 hours each time, more preferably 1.5 to 2.5 hours each time, and most preferably 1.5 to 2 hours each time. The extraction can fully extract the effective components of the medicinal materials, and improves the treatment effect of the medicine.
The preparation of the Chinese forest frog oil freeze-dried powder and the polar alcohol solution extract is not time sequential. The invention extracts the oviductus Ranae and the traditional Chinese medicine composition compatible with the oviductus Ranae respectively, and ensures the dissolution rate of the effective parts of the medicine.
After the Chinese forest frog oil freeze-dried powder and the polar alcohol solution extract are obtained, the Chinese forest frog oil freeze-dried powder and the polar alcohol solution extract are mixed to obtain the Chinese forest frog oil composition. The mixing of the invention preferably mixes the obtained snow clam oil freeze-dried powder and the polar alcohol solution extract.
The invention also provides a traditional Chinese medicine composition for relieving cough and asthma, which comprises the following raw materials in parts by weight: 1 part of the oviductus ranae composition, 3.2 to 6.4 parts of soybean lecithin, 0.4 to 0.8 part of cholesterol and 0.04 to 0.08 part of cationic material. The mass part ratio of the soybean lecithin, the cholesterol and the cationic material is preferably 80:10:1.
the invention also provides a preparation method of the traditional Chinese medicine composition for relieving cough and asthma, which comprises the following steps: dissolving soybean lecithin, cholesterol and a cationic material in a first organic solvent, and recovering the first organic solvent to obtain a first mixture; mixing the second organic solvent with oviductus Ranae composition, mixing the second mixture with the first mixture, evaporating under reduced pressure, and ultrasound to obtain the Chinese medicinal composition for relieving cough and asthma. The invention preferably comprises soybean lecithin, cholesterol, cationic material and oviductus Ranae composition according to 80:10:1: 20.
The invention dissolves soybean lecithin, cholesterol and cationic material in a first organic solvent, and recovers the first organic solvent to obtain a first mixture. In the present invention, the first organic solvent is preferably one or more of dichloromethane, chloroform, methanol, diethyl ether, ethanol and acetone, and more preferably dichloromethane. According to the invention, the soybean lecithin, the cholesterol and the cationic material are dissolved in the first organic solvent, so that the soybean lecithin, the cholesterol and the cationic material are conveniently and fully contacted and fused, and the successful preparation of the drug-loaded liposome nano-film is facilitated. The mixing conditions are not particularly limited, and the mixing conditions are usually used in the art. The amount of the first organic solvent is not critical, and the addition is preferably stopped after the soybean lecithin, cholesterol and cationic material are completely dissolved.
The method for recovering the first organic solvent is preferably reduced pressure evaporation, and the step of reduced pressure evaporation is not critical, and the method is usually used in the field. The invention removes the first organic solvent to a residual volume of 1mL of solution.
After the first mixture is obtained, the second organic solvent and the oviductus Ranae composition are mixed, the obtained second mixture and the first mixture are mixed, evaporated under reduced pressure and subjected to ultrasound, and the traditional Chinese medicine composition for relieving cough and asthma is obtained. In the present invention, the second organic solvent is preferably one or more of dichloromethane, chloroform, methanol, diethyl ether, ethanol and acetone, and more preferably dichloromethane. The second organic solvent of the present invention is preferably identical to the first organic solvent.
The invention firstly dissolves the soybean lecithin, cholesterol and cationic materials in the first organic solvent, removes the first organic solvent and mixes the first organic solvent with the second mixture, thereby promoting the full dissolution of the oviductus Ranae composition and the first mixture.
The conditions of reduced pressure evaporation and ultrasound are not particularly limited, and the method is carried out by adopting a treatment method commonly used in the field.
The cationic material of the present invention is preferably trimethyl-2, 3-dioleyloxypropyl ammonium chloride (DOTMA), trimethyl-2, 3-dioleyloxypropyl ammonium bromide (DOTAP), dimethyl-2, 3-dioleyloxypropyl-2- (2-spermamoylamino) ethylammonium bromide (DOSPA), trimethyldodecylammonium bromide (DTAB), trimethyltetradecylammonium bromide (TTAB), trimethylhexadecylammonium bromide (CTAB), dimethyldioctadecyl ammonium bromide (DDAB), dimethyl-2-hydroxyethyl-2, 3-dioleyloxypropyl ammonium bromide (DORI), dimethyl-2-hydroxyethyl-2, 3-dioleyloxypropyl ammonium bromide (DORIE), dimethyl-3-hydroxypropyl-2, 3-dioleyloxypropyl ammonium bromide (DOHP), dimethyl-4-hydroxybutyl-2, 3-dioleyloxypropyl ammonium bromide (DOAB), dimethyl-5-hydroxypentyl-2, 3-dioleyloxypropyl ammonium bromide (DOHPE), dimethyl-2-hydroxyethyl-2, 3-dioleyloxypropyl ammonium bromide (DORIE), dimethyl-2-dioleyloxypropyl-2, 3-dioleyloxypropyl ammonium bromide (DORIE), 3-Ditetraalkoxypropylammonium (DMRIE), N- (2-arginyl formyl) -N ', N' -dioctadecyl glycinamide, 1, 2-dioleoyl-3-succinyl-sn-glycerolcholine ester (DOGS), 3β - [ N- (N ', N' -dimethylaminoethyl) carbamoyl ] cholesterol (DC-Chol), lipopoly-L-lysine (LPLL), and Stearylamine (SA). The invention uses the positive liposome prepared by the positive material as a medicine carrier, so that the solubility and stability of the oviductus ranae can be greatly improved. Meanwhile, due to the fat-soluble effect of the cationic liposome, the encapsulation efficiency of the oviductus ranae is increased, and the medicine amount of the oviductus ranae extract reaching the lung surface is improved. Compared with the oral administration, the liposome administration of the medicine can increase the distribution and residence time of the medicine in lung tissues, improve the medicine concentration, reduce the administration dosage and further enhance the treatment effect of the oviductus ranae extract.
The invention also provides the application of the toad oil composition or the traditional Chinese medicine composition in preparing medicines for treating respiratory diseases. The respiratory diseases comprise at least one of cough, expectoration, pharyngitis, bronchial asthma and emphysema. The dosage forms of the medicament of the invention preferably comprise aerosols or inhaled mists. The invention is administered by aerosol or inhalation water mist, the particle size is 2-5 μm, and the invention has good stability, solves the technical problems of difficult dissolution and preparation of the traditional oviductus Ranae, and fills the blank of the administration of the oviductus Ranae mist. Meanwhile, aerosol administration is used for treating respiratory diseases, so that the first pass elimination effect can be avoided, the effect is quick, the targeting is strong, and the stability and bioavailability of the oviductus ranae extract can be obviously improved. The adopted preparation method is the existing common method, and is convenient for industrial production.
The invention firstly extracts the oviductus Ranae and the traditional Chinese medicine composition compatible with the oviductus Ranae respectively, ensures the dissolution rate of the effective parts of the medicine, constructs cationic liposome, adds the oviductus Ranae extract and ethanol extracts of the oviductus Ranae, loquat leaf and liquorice into a liposome membrane to prepare the composition liposome containing the oviductus Ranae extract, and treats respiratory diseases by a lung administration mode. The oviductus Ranae composition prepared by the invention can be effectively used for pulmonary administration treatment, improves the stability and bioavailability of the oviductus Ranae, has convenient application, quick response and no toxic or side effect, and improves the compliance of patients.
For further explanation of the present invention, the following description will be given in detail of the cough and asthma relieving oviductus Ranae composition, the traditional Chinese medicine composition, and the preparation method and application thereof, with reference to the accompanying drawings and examples, but they are not to be construed as limiting the scope of the present invention.
Example 1
5g of Chinese forest frog oil, 10g of Chinese forest frog skin, 2.5g of loquat leaf and 2.5g of liquorice are taken, 5g of Chinese forest frog oil is soaked in 500mL of citric acid solution with pH of 4.5 for 36 hours, then homogenized, 2000mL of distilled water is added after homogenization, the mixture is decocted for 2 hours, cooled to room temperature, the pH is adjusted to 3.0 by using 10mmol/L of HCl solution, and pepsin is adopted as follows: the volume of the material is 1g:2000mL of pepsin (the enzyme activity is more than or equal to 250U/mg) is added for enzymolysis for 2 hours at 37 ℃, the sample is boiled for 15 minutes for enzyme inactivation after enzymolysis, and after cooling to room temperature, the pH is adjusted to 10.0 by using 0.1mol/L NaOH solution, and according to trypsin: the volume of the material is 1g: adding trypsin (enzyme activity is greater than or equal to 250. N.F.U/mg) into 100mL of the solution according to the mass-volume ratio, carrying out enzymolysis for 6 hours at 55 ℃, boiling a sample for 1min after enzymolysis, inactivating the enzyme, cooling to room temperature, regulating pH to 7.0 by using 0.1mol/L NaOH, centrifuging for 15min at 10000rpm, collecting supernatant, and freeze-drying to obtain 1.63g of snow clam oil freeze-dried powder.
10g of Chinese forest frog skin, 2.5g of loquat leaf and 2.5g of liquorice are put into a round bottom flask, 60ml of 75% ethanol solution is added, after soaking for 1 hour, reflux extraction is carried out for 2 times, the first time is 2 hours, the second time is 1.5 hours, and ethanol is recovered, thus obtaining 4.82g of ethanol extract.
The oviductus ranae composition is prepared by fully mixing 1.63g of the Chinese forest frog oil freeze-dried powder and 4.82g of ethanol extract.
Respectively weighing 80mg of soybean lecithin, 10mg of cholesterol and 1mg of cationic material (dioleoyl propyl trimethylammonium chloride and 3 beta- [ N- (N ', N' -dimethylaminoethyl) carbamoyl ], placing in a eggplant-shaped bottle, adding dichloromethane vortex to completely dissolve the materials, rotationally decompressing and evaporating, recovering an organic solvent until the concentration of the organic solvent is about 1mL, taking out, adding 0.5mL of dichloromethane and 20mg of the toad oil composition, vortex uniformly mixing, placing on a rotary evaporator, continuously decompressing and evaporating until a dry uniform lipid film is formed on the bottle wall, taking out the eggplant-shaped bottle, adding 3mL of PBS solution with concentration of 0.01mmol/L, washing the film, and hydrating the film at 25 ℃ for 1h to form a liposome primary emulsion, carrying out water bath ultrasonic treatment on the liposome primary emulsion for 2min to obtain a Chinese medicinal composition (namely the toad oil composition liposome) for relieving cough and asthma.
Finished product properties: the product is in the form of milky white, fine and uniform liquid.
Particle size measurement: the liposome particle size is detected by a laser particle size analyzer, the average particle size is 21.52 mu m, and the polydispersity is 0.46.
Example 2-1
Preparation optimization of cationic liposome:
the weight ratio of soybean lecithin and cholesterol, soybean lecithin and the oviductus Ranae composition prepared in example 1 was optimized according to the encapsulation efficiency and entrapment efficiency of the oviductus Ranae composition. The influence of the ratio (1:1, 2:1, 4:1, 8:1, 16:1) of soybean lecithin and cholesterol on the encapsulation efficiency and encapsulation efficiency of the oviductus Ranae composition is examined, and the encapsulation efficiency and encapsulation efficiency of the oviductus Ranae composition in the obtained traditional Chinese medicine composition are measured according to the total content of amino acids.
(encapsulation efficiency% = (drug content in liposome/drug dose) x 100%.
Entrapment efficiency% = (drug content in liposomes/total drug loaded liposomes) ×100%)
The experimental results are shown in table 1 and the first row bar chart of fig. 1. The specific preparation conditions of the different schemes are as follows:
(1) Soybean lecithin: cholesterol (1:1)
The preparation method was the same as in example 1 except that the soybean lecithin was added in an amount of 10 mg.
(2) Soybean lecithin: cholesterol (2:1)
The preparation method was the same as in example 1 except that the soybean lecithin was added in an amount of 20 mg.
(3) Soybean lecithin: cholesterol (4:1)
The preparation method was the same as in example 1 except that the soybean lecithin was added in an amount of 40 mg.
(4) Soybean lecithin: cholesterol (8:1)
The preparation method is the same as in example 1.
(5) Soybean lecithin: cholesterol (16:1)
The preparation method was the same as in example 1 except that the soybean lecithin was added in an amount of 160 mg.
TABLE 1 Effect of Soybean lecithin and cholesterol addition ratio on encapsulation efficiency and encapsulation efficiency of oviductus Ranae composition
Soybean lecithin and cholesterol ratio | 1:1 | 2:1 | 4:1 | 8:1 | 16:1 |
Encapsulation efficiency (%) | 47.26 | 50.08 | 68.37 | 80.45 | 78.55 |
Efficiency of load (%) | 13.22 | 13.85 | 13.62 | 14.28 | 14.09 |
Example 2-2
The effect of the ratio of soy lecithin to oviductus Ranae composition (1:1, 2:1, 3:1, 4:1, 5:1) on the encapsulation efficiency and entrapment efficiency of oviductus Ranae compositions, the results are shown in Table 2 and the second bar chart of FIG. 1.
(1) Soybean lecithin: oviductus Ranae composition (1:1)
The preparation method was the same as in example 1 except that the soybean lecithin was added in an amount of 20 mg.
(2) Soybean lecithin: oviductus Ranae composition (2:1)
The preparation method was the same as in example 1 except that the soybean lecithin was added in an amount of 40 mg.
(3) Soybean lecithin: oviductus Ranae composition (3:1)
The preparation method is the same as in example 1.
(4) Soybean lecithin: oviductus Ranae composition (4:1)
The preparation method was the same as in example 1 except that the soybean lecithin was added in an amount of 160 mg.
(5) Soybean lecithin: oviductus Ranae composition (5:1)
The preparation method was the same as in example 1 except that the soybean lecithin was added in an amount of 320 mg.
TABLE 2 influence of the addition ratio of soybean lecithin and oviductus Ranae composition on encapsulation efficiency and encapsulation efficiency of oviductus Ranae composition
Soybean lecithin and oviductus Ranae composition ratio | 1:1 | 2:1 | 3:1 | 4:1 | 5:1 |
Encapsulation efficiency (%) | 25.96 | 52.43 | 75.34 | 76.29 | 76.11 |
Efficiency of load (%) | 12.18 | 13.24 | 14.89 | 15.23 | 15.18 |
Examples 2 to 3
The effect of the ratio of oviductus Ranae composition to cholesterol (1:0.5, 1:1, 1:2, 1:3, 1:4) on the encapsulation efficiency and entrapment efficiency of oviductus Ranae composition is shown in Table 3 and the third bar chart of FIG. 1.
(1) Oviductus Ranae composition: cholesterol (1:0.5)
The preparation method is the same as in example 1.
(2) Oviductus Ranae composition: cholesterol (1:1)
The preparation was carried out in the same manner as in example 1 except that the amount of cholesterol added was 20 mg.
(3) Oviductus Ranae composition: cholesterol (1:2)
The preparation was carried out in the same manner as in example 1 except that the amount of cholesterol added was 40 mg.
(4) Oviductus Ranae composition: cholesterol (1:3)
The preparation was carried out in the same manner as in example 1 except that the amount of cholesterol added was 60 mg.
(5) Oviductus Ranae composition: cholesterol (1:4)
The preparation was carried out in the same manner as in example 1 except that the amount of cholesterol added was 80 mg.
TABLE 3 influence of the ratio of oviductus Ranae composition to cholesterol on encapsulation efficiency and encapsulation efficiency of oviductus Ranae composition
As can be seen from fig. 1 and tables 1 to 3, the mass fraction of soybean lecithin can directly affect the encapsulation efficiency and entrapment effect of the oviductus ranae composition, and the mass ratio of soybean lecithin to cholesterol is 8: the encapsulation efficiency and the encapsulation effect are best in the step 1; the mass ratio of the soybean lecithin to the oviductus Ranae composition is 4: the encapsulation efficiency and the encapsulation effect are best in the 1 st time. The mass fraction of cholesterol has no obvious influence on the encapsulation efficiency and the encapsulation effect of the toad oil composition, and the encapsulation efficiency of the toad oil composition are respectively about (72+/-1) percent and (14+/-1) percent within the evaluation range of the invention, so that the cholesterol is used as an auxiliary matrix to help improve the encapsulation effect of the liposome. From experimental results, the mass ratio of the oviductus ranae composition to the cholesterol is 2: the effect is better when 1. With the improvement of the mass ratio of the soybean lecithin, the encapsulation efficiency and the encapsulation effect of the soybean lecithin are improved, and the improvement of the cholesterol ratio does not greatly improve the encapsulation efficiency and the encapsulation effect of the oviductus Ranae composition. Therefore, the mass ratio of the soybean lecithin, the cholesterol and the oviductus ranae composition is finally determined to be 4:0.5:1.
example 3
In vivo distribution experiment in rats
The nile red reagent was loaded into the liposome of the oviductus Ranae composition prepared in example 1 according to the procedure of the specification. 90 Wistar rats are divided into a nile red solution group, a nile red + oviductus Ranae liposome atomizing group and a nile red + oviductus Ranae liposome oral group, each group comprising 30 rats. Nile red solution group rats were given with Nile red solution, nile red + toad fat plastid atomizing group rats were given with Nile red + the traditional Chinese medicine composition solution prepared in example 1, and Nile red + toad fat plastid oral group rats were given with Nile red + the traditional Chinese medicine composition solution prepared in example 1 by intragastric administration.
And (3) putting the nile red solution group and the nile red and oviductus Ranae liposome atomization group rats into a quantitative atomization administration device, wherein the administration dosage is 0.5mg/kg, and the atomization administration is completed within 2 min. The oral administration dose of Nile red and oviductus Ranae liposome is 0.5mg/kg. After administration, 6 rats were harvested at 0.5h, 1h, 2h, 4h, and 8h, respectively, and after anesthesia with pentobarbital, blood was taken from the abdominal aorta, and serum was isolated. Taking brain, heart, liver, spleen, lung and kidney, flushing blood on the surface with physiological saline, sucking the blood with filter paper, and weighing 5g of each tissue respectively. Adding precooled PBS for homogenizing to prepare tissue homogenate. The serum and tissue homogenate obtained by separation are detected and analyzed by a fluorescence spectrophotometer.
The results are shown in FIG. 2 and Table 4.
TABLE 4 in vivo distribution of experimental data in rats
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As shown in the results of fig. 2 and 4, the accumulation of nile red in each organ gradually decreased with time, and the content of nile red in the lung of the encapsulated liposome solution of the oviductus ranae composition was significantly increased as compared with the nile red solution, and significantly higher than that of the oral liposome of the oviductus ranae composition. The content of the nile red in other organs and tissues is equivalent, and the nile red content in the lung after 8 hours is more than 50%, so that the residence time of the medicine in the lung is obviously prolonged, and the treatment effect in the lung is improved.
Meanwhile, the in vivo tissue distribution research results show that the Chinese medicinal composition atomization group can obviously improve the stability of the oviductus Ranae in rats, prolong the residence time of the medicament in the lung and improve the bioavailability of the medicament.
Example 4
The Chinese medicinal composition prepared in example 1 was subjected to in vivo animal efficacy experimental study
Preparation of liposome solution not encapsulating the oviductus Ranae composition prepared in example 1: the liposome preparation method of example 1 was performed according to the same procedure as in example 1 except that the oviductus Ranae composition was not added, and only 80mg of soybean lecithin, 10mg of cholesterol and 1mg of cationic material were used, and the mixture was placed in a eggplant-shaped bottle, and methylene chloride was added and vortexed to completely dissolve the above; evaporating under reduced pressure, recovering organic solvent until a dried lipid film is formed on the bottle wall, taking out the eggplant-shaped bottle, adding 3mL of PBS solution with the concentration of 0.01mmol/L for washing the film, and hydrating for 1h at 25 ℃ to form liposome primary emulsion. The liposome primary emulsion was sonicated in a water bath for 2min to obtain liposomes not encapsulating the oviductus Ranae composition prepared in example 1.
Experimental method
SPF grade SD rats were randomly divided into 7 groups: a blank group (CO), a model group (MO), a positive drug group (DE), a liposome-atomized group of oviductus Ranae composition (AT-OR-LI), an oral liposome-atomized group of oviductus Ranae composition (AT-OR-LI), an atomized group of oviductus Ranae composition (AT-OR, i.e., oviductus Ranae composition not encapsulated with liposomes), and an oral group of oviductus Ranae composition (IG-OR, i.e., oviductus Ranae composition not encapsulated with liposomes). After one week of adaptive rearing of 8 animals per group, asthma models were prepared.
Except for the blank control group, all rats were prepared with ovalbumin in combination with aluminum hydroxide to form an asthma model. The rats of each group were sensitized by subcutaneously injecting 20 μg ovalbumin and 1mg aluminum hydroxide mixture at 0d, 7d and 14d of the experiment; the asthma was stimulated by inhalation of the 1% ovalbumin suspension at 21d, 23d, 25d, 27d for 15min. Meanwhile, the rats in the blank group were subcutaneously injected with an equivalent amount of physiological saline in experiments 0d, 7d, and 14d, and the 21d, 23d, 25d, and 27d were inhaled with an equivalent amount of atomized physiological saline.
After the model preparation is successful, the blank control group and the model group are atomized and given to liposome solution of the toad oil composition prepared in the non-entrapped example 1; AT-OR-LI group nebulization gives 1mg/kg of the solution of the Chinese medicinal composition prepared in example 1; the group IG-OR-LI was given by gavage 1mg/kg of the solution of the Chinese medicinal composition prepared in example 1, the group AT-OR was given by atomization 1mg/kg of the solution of the oviductus Ranae composition prepared in example 1, the group IG-OR was given by gavage 1mg/kg of the solution of the oviductus Ranae composition prepared in example 1, and the group DE was given by intraperitoneal injection 2mg/kg of dexamethasone. The administration treatment was started at 14d of a mixed solution of 20. Mu.g of ovalbumin and 1mg of aluminum hydroxide by subcutaneous injection, and the administration was continued for 14d.
After the last dose of each group of mice, the rat's asthma-inducing latency was recorded and asthmatic behavioural scoring was performed. After the end of the experiment, the animals were anesthetized with sodium pentobarbital (40 mg/kg), alveolar lavage fluid was collected, and the total number of White Blood Cells (WBC), eosinophils (Eos), lymphocytes (Lym) and neutrophils (Neu) in the alveolar lavage fluid were detected by a fully automatic blood cell analyzer. Picking right leaf tissue of the lung, fixing the right leaf tissue in 10% paraformaldehyde solution, and performing HE staining after fixing completely; the left lung leaf is homogenized, supernatant is collected, and the IL-4, IL-5 and IgE content in the homogenized tissue is measured by ELISA method.
The results are shown in FIGS. 3 to 12 and Table 5.
Asthma behavioural scoring criteria: no obvious shortness of breath, 0 minutes; the respiratory rate is accelerated, nasal wings are not found to fan, abdominal respiration is achieved, and the respiratory rate is 0.5 minute; dyspnea (nasal fan, abdominal breathing), 1.0 score; dyspnea with cough, 1.5 min; dyspnea with cough and wheeze (less than 3 times/min), 2.0 minutes; dyspnea, frequent cough (greater than 3 times/min), 2.5 minutes; opening mouth breath, or deep breath, apnea with cyanosis of eyes, ears and lips, 3.0 minutes; unstable standing for 3.5 minutes; shock, fall, sudden death of 4.0 minutes.
TABLE 5 animal in vivo efficacy experimental data (asthma)
As shown in fig. 3-12 and table 5, compared with the model group, the liposome atomization group of the oviductus ranae composition can obviously improve the asthma behavioural score of asthmatic rats, prolong the asthma inducing latency period, repair the inflammatory injury of the lung of asthmatic rats and reduce IL-4, IL-5 and IgE in the lung tissues; the number of WBC, eos, lym, neu cells in the alveolar lavage fluid is reduced, and the treatment effect of the toad oil composition liposome atomization group on various indexes of asthmatic rats is better than that of the toad oil composition liposome oral group, the toad oil composition atomization group and the toad oil composition oral group, so that the toad oil composition liposome has better treatment effect on asthmatic rats.
Example 5
In vivo animal efficacy experimental study of the Chinese medicinal composition prepared in example 1
Grouping: the oviductus Ranae composition liposome spray group (AT-OR-LI), oviductus Ranae composition liposome oral group (IG-OR-LI), oviductus Ranae composition spray group (AT-OR), oviductus Ranae composition oral group (IG-OR) and negative control group (RE).
Experimental method
The ammonia water cough-inducing method is used for detecting the cough times of mice: mice were taken in 50 groups of 10 randomly: the oviductus Ranae composition liposome spray group (AT-OR-LI), oviductus Ranae composition liposome oral group (IG-OR-LI), oviductus Ranae composition spray group (AT-OR), oviductus Ranae composition oral group (IG-OR), and negative control group (RE). Wherein 1mg/kg of the Chinese medicinal composition solution prepared in example 1 was given by AT-OR-LI group atomization, 1mg/kg of the Chinese medicinal composition solution prepared in example 1 was given by IG-OR-LI group lavage, 1mg/kg of the oviductus Ranae composition solution prepared in example 1 was given by AT-OR group atomization, 1mg/kg of the oviductus Ranae composition solution prepared in example 1 was given by IG-OR group lavage, and an equal volume of liposome solution without the oviductus Ranae composition entrapped was given by negative control group atomization, the administration was carried out 1 time per day for 7 days, and after the last administration of each group for 2 hours, mice were placed in a cough-inducing instrument, and cough of the mice was induced with 25% ammonia water. The incubation period of cough in mice was observed and recorded as the number of coughs in 3 min.
Phenol red method for detecting sputum excretion effect of mice: mice were taken in 50 groups of 10 randomly: the oviductus Ranae composition liposome spray group (AT-OR-LI), oviductus Ranae composition liposome oral group (IG-OR-LI), oviductus Ranae composition spray group (AT-OR), oviductus Ranae composition oral group (IG-OR), and negative control group (RE). Wherein 1mg/kg of the Chinese medicinal composition solution prepared in example 1 was given by AT-OR-LI group atomization, 1mg/kg of the Chinese medicinal composition solution prepared in example 1 was given by IG-OR-LI group lavage, 1mg/kg of the oviductus Ranae composition solution prepared in example 1 was given by AT-OR group atomization, 1mg/kg of the oviductus Ranae composition solution prepared in example 1 was given by IG-OR group lavage, and an equal volume of liposome solution without the oviductus Ranae composition entrapped was given by negative control group atomization, the administration was carried out 1 time per day for 7 days, and after each group was given for 2 hours last, 0.1ml/10g of phenol red with a volume fraction of 0.5% was injected intraperitoneally. Mice were sacrificed after 0.5h, the trachea was isolated, a section of trachea was removed at the same position, the trachea was flushed with 5% sodium bicarbonate solution for 5 consecutive times, the eluate was collected, absorbance was measured at 546nm wavelength, and phenol red concentration in the tracheal rinse was calculated.
The experimental results are shown in fig. 13 and table 6.
TABLE 6 animal in vivo efficacy experimental data (cough)
As shown in fig. 13 and table 6, the liposome spray group of the oviductus ranae composition can effectively prolong the incubation period of mice cough induced by ammonia water, reduce the times of the mice cough, and enhance the phlegm-expelling capacity of the mice. The effects are better than the oral group of the liposome of the oviductus Ranae composition, the atomized group of the oviductus Ranae composition and the oral group of the oviductus Ranae composition, and the oral group of the oviductus Ranae composition has better relieving effects on relieving cough and eliminating phlegm.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (10)
1. The oviductus ranae composition for relieving cough and asthma is characterized by comprising the following raw materials in parts by weight: 1 to 3 parts of Chinese forest frog oil, 2 to 6 parts of Chinese forest frog skin, 1 to 3 parts of loquat leaf and 1 to 3 parts of liquorice.
2. The method for preparing the cough and asthma relieving oviductus Ranae composition as claimed in claim 1, which is characterized by comprising the following steps: soaking the Chinese forest frog oil in citric acid with pH of 4.5, homogenizing, mixing the homogenate obtained by homogenizing with distilled water, and decocting to obtain an enzymolysis substrate;
performing first enzymolysis on the enzymolysis substrate by using pepsin to obtain pepsin enzymolysis liquid;
performing second enzymolysis on the pepsin enzymolysis liquid by using trypsin, centrifuging the obtained trypsin enzymolysis liquid to obtain supernatant, and freeze-drying to obtain the snow clam oil freeze-dried powder;
extracting effective components in the Chinese forest frog skin, the loquat leaf and the liquorice by using a polar alcohol solution to obtain a polar alcohol solution extract;
mixing the Chinese forest frog oil freeze-dried powder and the polar alcohol solution extract to obtain the Chinese forest frog oil composition.
3. The preparation method according to claim 2, wherein the mass-to-volume ratio of the Chinese forest frog oil to the citric acid with the pH of 4.5 is 1g: 80-120 mL; the soaking time is 36 hours;
the volume ratio of the homogenate to the distilled water is 1: (3-5).
4. The method according to claim 2, wherein the first enzymolysis with pepsin is preceded by: regulating the pH value of the enzymolysis substrate to 2.8-3.2; the feed liquid ratio of the pepsin to the enzymolysis substrate is 1g: 1500-2500 mL; the temperature of the first enzymolysis is 37 ℃ and the time is 2 hours;
the enzyme activity of the pepsin is more than or equal to 250U/mg.
5. The method according to claim 2, wherein the second enzymatic hydrolysis with trypsin is preceded by: regulating the pH value of the pepsin enzymolysis liquid to 9.8-10.2; the feed liquid ratio of the trypsin to the pepsin enzymatic hydrolysate is 1g: 80-120 mL; the temperature of the second solution is 55 ℃ and the time is 6h;
the enzyme activity of the trypsin is 250.N.F.U/mg.
6. The traditional Chinese medicine composition for relieving cough and asthma is characterized by comprising the following raw materials in parts by weight: the oviductus Ranae composition of claim 1, soybean lecithin 3.2-6.4, cholesterol 0.4-0.8 and cationic material 0.04-0.08.
7. The method for preparing the traditional Chinese medicine composition according to claim 6, which comprises the following steps: dissolving soybean lecithin, cholesterol and a cationic material in a first organic solvent, and recovering the first organic solvent to obtain a first mixture;
mixing the second organic solvent with the oviductus Ranae composition, mixing the obtained second mixture with the first mixture, evaporating under reduced pressure, and performing ultrasound to obtain the Chinese medicinal composition for relieving cough and asthma;
the first organic solvent and the second organic solvent are one or more of dichloromethane, chloroform, methanol, diethyl ether, ethanol and acetone respectively.
8. The use of a oviductus Ranae composition according to claim 1 or a oviductus Ranae composition obtained by a method according to any one of claims 1 to 5 or a Chinese medicinal composition according to claim 6 or a Chinese medicinal composition obtained by a method according to claim 7 in the preparation of a medicament for treating respiratory diseases.
9. The use according to claim 8, wherein the respiratory disease comprises at least one of cough, expectoration, pharyngitis, bronchial asthma and emphysema.
10. The use according to claim 8, wherein the pharmaceutical dosage form comprises an aerosol or an inhaled mist.
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