CN113398189B - Application of traditional Chinese medicine composition in preparation of anti-influenza virus medicine - Google Patents
Application of traditional Chinese medicine composition in preparation of anti-influenza virus medicine Download PDFInfo
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- A61K36/18—Magnoliophyta (angiosperms)
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Abstract
The invention relates to the field of traditional Chinese medicines, and particularly provides application of a traditional Chinese medicine composition in preparation of a medicine for resisting influenza viruses. In addition, the four medicines of blackberry lily, subprostrate sophora, catechu and calyx seu fructus physalis are used as adjuvant medicines. Finally, liquorice is used as a guiding drug. In the recipe, atractylodes rhizome, rhizoma Atractylodis, herba Ephedrae, herba Agastaches, etc. have the effects of resolving dampness. The blackberrylily rhizome, the subprostrate sophora, the catechu and the calyx seu fructus physalis are used for clearing heat and relieving sore throat, the liquorice is used for harmonizing the effects of eliminating dampness and strengthening spleen and clearing heat and removing toxicity, and the active treatment effect on influenza virus infection is achieved.
Description
Technical Field
The invention relates to the field of traditional Chinese medicines, and in particular relates to application of a traditional Chinese medicine composition in preparation of a medicine for resisting influenza viruses.
Background
Influenza viruses, referred to as influenza viruses, are classified into types a, b and c, and influenza virus type d has also been found in recent years. The influenza virus can cause infection and morbidity of various animals such as human, pig, bird and the like, and is a pathogen of diseases such as human influenza, pig influenza, bird influenza and the like. Typical clinical symptoms of these diseases are fever, general pain, weakness, and respiratory symptoms such as sneezing, rhinorrhea, sore throat, and shortness of breath. Human influenza is mainly caused by influenza a and influenza b, both of which are infectious. Among them, influenza a H1N1 is a highly contagious acute respiratory disease in pigs, has a high incidence rate and a rapid transmission rate, and can be transmitted by direct or indirect contact. Herd epidemic may occur throughout the year, and interpersonal spread may also occur in some cases.
At present, common medicaments for clinically treating influenza virus are mainly neuraminidase inhibitors such as oseltamivir, zanamivir and the like, and the medicaments are favored by people due to good antiviral effect, but adverse reactions such as nausea, vomiting, bronchitis, insomnia, dizziness and the like can occur after the medicaments are taken. The traditional Chinese medicine has the characteristics of small side effect and overall treatment, so that a new traditional Chinese medicine with good treatment effect and small toxic and side effect still needs to be developed.
Disclosure of Invention
Therefore, the invention aims to provide the application of the traditional Chinese medicine composition in preparing the medicine for preventing and/or treating diseases caused by influenza viruses or influenza virus infection.
The traditional Chinese medicine composition comprises, by weight, 13-18 parts of rhizoma atractylodis, 8-13 parts of ephedra, 13-18 parts of agastache rugosus, 4-9 parts of subprostrate sophora, 8-13 parts of catechu, 13-18 parts of blackberry lily, 8-13 parts of calyx seu fructus physalis and 8-13 parts of liquorice.
Further, the traditional Chinese medicine composition comprises, by weight, 15 parts of rhizoma atractylodis, 10 parts of ephedra, 15 parts of agastache rugosus, 6 parts of subprostrate sophora, 10 parts of catechu, 15 parts of blackberry lily, 10 parts of calyx seu fructus physalis and 10 parts of liquorice; or 13 parts of rhizoma atractylodis, 8 parts of ephedra, 18 parts of agastache rugosus, 4 parts of subprostrate sophora, 8 parts of catechu, 18 parts of blackberry lily, 8 parts of calyx seu fructus physalis and 13 parts of liquorice; or,
18 parts of rhizoma atractylodis, 13 parts of ephedra, 13 parts of agastache rugosus, 9 parts of subprostrate sophora, 13 parts of catechu, 13 parts of blackberry lily, 13 parts of calyx seu fructus physalis and 8 parts of liquorice.
The rhizoma Atractylodis is selected from processed rhizoma Atractylodis, such as but not limited to bran-parched rhizoma Atractylodis and parched rhizoma Atractylodis, and more preferably bran-parched rhizoma Atractylodis. The herba Ephedrae can be, but is not limited to, raw herba Ephedrae. In the invention, the wrinkled giant hyssop is cablin potchouli herb, and the wrinkled giant hyssop is also called wrinkled giant hyssop. The Glycyrrhrizae radix can be conventional Glycyrrhrizae radix or Glycyrrhrizae radix product, such as radix Glycyrrhizae Preparata, Glycyrrhrizae radix decoction pieces, etc., preferably Glycyrrhrizae radix.
The preparation method of the traditional Chinese medicine composition comprises the steps of weighing rhizoma atractylodis, ephedra, agastache rugosus, blackberry lily, subprostrate sophora, catechu, calyx seu fructus physalis and liquorice according to the selected weight parts, mixing and extracting according to a conventional extraction method or respectively extracting according to a conventional extraction method and mixing.
Further, the conventional extraction method comprises one or more of decoction extraction, immersion extraction, percolation extraction, reflux extraction, ultrasonic extraction and steam distillation extraction.
In certain preferred embodiments, the extraction solvent is selected from water or an alcohol solution having a volume percent of 5-98%; the alcohol solution is an aqueous solution containing alcohol, and the alcohol can be ethanol or methanol.
In a particular embodiment, the number of extractions is at least 1, preferably 1-5, more preferably 1-3.
In a particular embodiment, the extraction time is at least 10min, preferably 20-240min, more preferably 30-120 min.
In a specific embodiment, the ratio of the volume of the extraction solvent to the weight of the bulk drug is more than or equal to 2L/kg; preferably 3 to 20L/kg, more preferably 5 to 10L/kg.
In certain preferred embodiments, the purification treatment may be performed by conventional purification methods, such as, but not limited to, column chromatography, membrane filtration, and the like.
The traditional Chinese medicine composition is added or not added with a pharmaceutically acceptable carrier, and is prepared into a pharmaceutical preparation according to a conventional preparation method.
In certain preferred embodiments, the pharmaceutical formulation is selected from one of a liquid formulation, a solid formulation, or a semi-solid formulation.
In a specific embodiment, the pharmaceutical formulation is a gel, cream, tablet, capsule, powder, mixture, pill, granule, oral liquid, syrup, decoction, suppository, aerosol, patch, ointment, injection, spray, liniment, tincture, wet application, paste or lotion.
In a specific embodiment, the pharmaceutically acceptable carrier is selected from at least one of pharmaceutically acceptable solvents, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, tonicity adjusting agents, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adherents, integration agents, penetration enhancers, pH adjusting agents, buffers, plasticizers, surfactants, thickeners, encapsulation agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, polymeric matrix materials, and film-forming materials.
The mass ratio of the finished product of the pharmaceutical preparation to the adopted raw material medicines is 30-40: 75-115, preferably 36: 91. wherein the mass of the raw material medicines is the sum of the masses of all the raw material medicines.
The invention also provides a preparation method of the pharmaceutical preparation, which comprises the following steps:
steam distillation step: weighing rhizoma atractylodis and agastache rugosus according to the selected weight parts, mixing, performing steam distillation extraction, and respectively collecting volatile oil and water extract;
water decoction: weighing ephedra, blackberry lily, subprostrate sophora, catechu, calyx seu fructus physalis and liquorice according to the selected weight parts, adding water for decoction, carrying out solid-liquid separation, and collecting liquid;
the preparation steps of the pharmaceutical preparation are as follows: mixing the water extractive solution obtained by steam distillation and the liquid obtained by water decoction, concentrating, drying, optionally adding adjuvants, making into semi-finished product, and spraying the volatile oil to obtain medicinal preparation.
In certain preferred embodiments, drying is followed by a pulverization step.
In certain preferred embodiments, a drying step is included after the intermediate pharmaceutical preparation is formed.
In certain preferred embodiments, the solid-liquid separation is selected from filtration or centrifugation.
In certain preferred embodiments, the method of preparing the pharmaceutical formulation comprises the steps of:
weighing rhizoma atractylodis and agastache rugosus according to the selected weight parts, mixing, adding at least 2 times of water for steam distillation extraction for at least 10min, and respectively collecting volatile oil and water extract; weighing ephedra, blackberry lily, subprostrate sophora, catechu, calyx seu fructus physalis and liquorice according to the selected weight parts, adding water for decocting for at least 1 time, adding at least 2 times of water for each time, decocting for at least 10min for each time, combining water decoctions, carrying out solid-liquid separation, and collecting liquid; mixing the water extractive solution obtained by steam distillation and the liquid obtained by water decoction, concentrating, drying, pulverizing, optionally adding adjuvant, making into semi-finished product, spraying the volatile oil, and mixing. The quantity is the number of milliliters of water added into each gram of raw material medicine.
In a more preferred embodiment, the method for preparing the pharmaceutical formulation comprises the steps of:
weighing rhizoma atractylodis and agastache rugosus according to the selected weight parts, mixing, adding 5-15 times of water for steam distillation extraction for 0.5-5 hours, and respectively collecting volatile oil and water extract; weighing ephedra, blackberry lily, subprostrate sophora, catechu, calyx seu fructus physalis and liquorice according to the selected weight parts, adding water for decocting for 1-3 times, adding 5-15 times of water for each time, decocting for 0.5-5 hours for each time, combining water decoction, carrying out solid-liquid separation, and collecting liquid; mixing the water extractive solution obtained by steam distillation and the liquid obtained by water decoction, concentrating to relative density of 1.20-1.25(50 deg.C), drying, pulverizing, optionally adding adjuvant, making into semi-finished product, spraying the volatile oil, and mixing.
In certain preferred embodiments, the pharmaceutical preparation intermediate may be a granule intermediate, a powder intermediate, or a tablet intermediate.
Further, the influenza virus is an influenza a virus.
The diseases caused by the virus comprise respiratory tract inflammation, acute lung injury and acute respiratory distress syndrome.
The respiratory inflammation is selected from pharyngitis, laryngitis, tracheitis, bronchitis and/or pneumonia.
The medicine prepared by the traditional Chinese medicine composition can inhibit the expression of at least one of inflammatory factors TNF-alpha, IL-6 and IL-1 beta, and/or inhibit the reduction of IL-4 serving as the inflammatory factor, and/or reduce the number of inflammatory infiltration cells.
In addition, the medicine prepared from the traditional Chinese medicine composition and/or the medicinal preparation also has the effects of regulating immunity and blood coagulation function. The immune regulation refers to at least one of regulation of abnormal number of immune cells, regulation of imbalance of proinflammatory factors and inflammation suppressing factors.
In addition, the medicine prepared from the traditional Chinese medicine composition and/or the medicinal preparation can also inhibit the increase of pulmonary index caused by influenza virus infection, reduce pulmonary edema or pulmonary fluid accumulation and inhibit inflammatory reaction.
Wherein, the rhizoma atractylodis is used as a monarch drug in the prescription, has pungent, bitter and warm nature and taste, and enters spleen, stomach and liver channels, and is mainly used for treating abdominal distension, diarrhea, edema, beriberi atrophy 36484m, rheumatalgia and wind-cold type common cold. In the formula, rhizoma atractylodis is taken as a monarch drug to eliminate dampness and strengthen spleen, and expel wind and clear away cold.
The prescription uses ephedra and agastache as ministerial drugs, wherein the ephedra is pungent, slightly bitter and warm, enters lung and bladder channels, and has the functions of sweating, dispelling cold, ventilating lung, relieving asthma, inducing diuresis and relieving swelling. It is used to treat common cold due to wind-cold, chest distress, cough, asthma, and edema. The ephedra herb is selected from the prescription for diffusing lung qi and relieving cough and asthma. Huo Xiang is pungent in flavor and slightly warm in nature, entering lung, spleen and stomach meridians. In the formula, agastache rugosus is taken as a ministerial drug with the purposes of dispersing the exterior and warming the middle-jiao to eliminate dampness.
In addition, the blackberry lily, the subprostrate sophora, the catechu and the calyx seu fructus physalis are used as adjuvant drugs, wherein the blackberry lily is bitter and cold and enters lung channels, and the blackberry lily is used as adjuvant drug to clear away heat and toxic material, eliminate phlegm and relieve sore throat. The subprostrate sophora is bitter and cold, enters lung and stomach meridians, and is selected to clear heat and relieve sore throat. Catechu, bitter and astringent, slightly cold, enters lung meridian, and uses Catechu to clear lung heat, resolve phlegm, nourish yin and promote fluid production. The calyx seu fructus physalis is bitter and cold, enters lung meridian, and has sore throat relieving and phlegm eliminating effects.
Finally, the liquorice is used as a guiding drug, and has sweet and mild properties and taste. It is a guiding drug for heart, lung, spleen and stomach meridians, and is prepared from Licorice root, radix Glycyrrhizae for harmonizing the actions of all the drugs, balancing cold and heat, tonifying middle-jiao energy, clearing lung-heat and relieving cough.
The technical scheme of the invention has the following advantages:
1. the traditional Chinese medicine composition provided by the invention is obtained by repeatedly optimizing the composition and content of the compound traditional Chinese medicine through clinical research and basic research, wherein the rhizoma atractylodis is used as a monarch drug, and the ephedra and the agastache are used as ministerial drugs in the formula. In addition, the four medicines of blackberry lily, subprostrate sophora, catechu and calyx seu fructus physalis are used as adjuvant medicines. Finally, liquorice is used as a guiding drug. In the recipe, atractylodes rhizome, rhizoma Atractylodis, herba Ephedrae, herba Agastaches, etc. have the effects of resolving dampness. The blackberrylily rhizome, the subprostrate sophora, the catechu and the calyx seu fructus physalis are used for clearing heat and relieving sore throat, the liquorice is used for harmonizing the effects of eliminating dampness and strengthening spleen and clearing heat and removing toxicity, and the active treatment effect on influenza virus infection is achieved.
The traditional Chinese medicine composition can also be used for treating the syndrome of pathogenic dampness stagnating in the lung, and the symptoms are as follows: fever, weakness, heaviness of the chest, muscular soreness, sore throat, chest oppression, gastric fullness, nausea, vomiting, poor appetite, loose stool or sticky stool. The tongue is pale red, the coating is white, thick, greasy or thin and yellow, and the pulse is slippery, rapid or soft, thus playing a positive role in clinically treating the influenza virus infection diseases.
2. The traditional Chinese medicine composition disclosed by the invention not only inhibits the increase of lung index and reduces inflammatory cell infiltration, but also inhibits the increase of inflammatory factors TNF-alpha, IL-6 and IL-1 beta, inhibits the reduction of IL-4 of the inflammatory factors, and also inhibits CD4+Reduction in cell number of T cells and CD8+The reduction of the number of T cells and the regulation of the immune function and the blood coagulation function of mice infected by the influenza virus indicate that the traditional Chinese medicine composition can be used for preventing and/or treating the influenza virus infection and diseases such as respiratory inflammation, acute lung injury and the like caused by the influenza virus infection.
3. The pharmaceutical preparation provided by the invention can be prepared into various dosage forms according to practical application, and can be externally used, injected or internally used, such as but not limited to various dosage forms of gels, creams, tablets, capsules, powder, mixtures, pills, granules, solutions, syrups, soft extracts, suppositories, aerosols, emplastrums, ointments, injections and the like. The granule is more convenient to use, has higher stability, is suitable for industrial production, and ensures the treatment effect and safety of the medicine.
4. The preparation method of the pharmaceutical preparation provided by the invention preferably carries out steam distillation extraction on the two traditional Chinese medicines of the rhizoma atractylodis and the agastache rugosus, the other raw materials are decocted with water for extraction, the water extract obtained by distillation and the water decoction are combined for concentration and drying to prepare a semi-finished pharmaceutical preparation, and then the volatile oil is sprayed in to obtain the finished pharmaceutical preparation.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a graph showing HE staining of a normal control group mouse in Experimental example 2 of the present invention;
FIG. 2 is a graph showing HE staining of mice in a model control group in Experimental example 2 of the present invention;
FIG. 3 is a HE staining pattern of Tamiflu group mice in experimental example 2 of the present invention;
FIG. 4 is a HE staining pattern of a Coptis japonica plague-clearing group mouse in Experimental example 2 of the present invention;
FIG. 5 is a HE staining pattern of a golden-mount group mouse in Experimental example 2 of the present invention;
FIG. 6 is a graph showing HE staining of mice in the large dose group of Cannabis virus particles in Experimental example 2 of the present invention;
FIG. 7 is a graph showing HE staining of mice in a dose group in Cannabis virus particles in Experimental example 2 of the present invention;
FIG. 8 is a graph showing HE staining of mice in a small dose group of Cannabis virus particles in Experimental example 2 of the present invention.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
All the medicinal materials are qualified by quality inspection and meet the standards of pharmacopoeia of the people's republic of China. In the extraction process, the amount of the extract is the milliliter of water added into each gram of the traditional Chinese medicinal materials.
Example 1
The embodiment provides a traditional Chinese medicine composition which comprises 15g of bran-fried rhizoma atractylodis, 10g of raw ephedra herb, 15g of pogostemon cablin, 6g of subprostrate sophora, 10g of catechu, 15g of blackberry lily, 10g of calyx seu fructus physalis and 10g of raw liquorice.
The embodiment provides a pharmaceutical preparation containing the traditional Chinese medicine composition, and the preparation method comprises the following steps: weighing bran-fried rhizoma Atractylodis and herba Agastaches according to the weight of the Chinese medicinal composition, adding 10 times of water, extracting volatile oil by steam distillation for 2 hr, and collecting volatile oil; collecting the distilled water solution in another container for later use; according to the formula, raw ephedra, blackberry lily, subprostrate sophora, catechu, calyx seu fructus physalis and raw liquorice are taken and decocted with water for three times, 10 times of water is added for the first time and decocted for 1h, 8 times of water is added for the second time and the third time respectively and decocted for 0.5h respectively, the decoction liquids are combined and filtered, the filtrate is combined with the distilled water liquid, the mixture is decompressed and concentrated into thick paste with the relative density of 1.20-1.25(50 ℃), the thick paste is dried, crushed and added with dextrin to prepare a semi-finished product of particles, the semi-finished product is dried, the volatile oil collected by distillation is sprayed, and the mixture is mixed evenly, so that 36g of xanthium toxin-removing particles is obtained.
Example 2
The embodiment provides a traditional Chinese medicine composition which comprises 13g of bran-fried rhizoma atractylodis, 8g of raw ephedra herb, 18g of wrinkled gianthyssop herb, 4g of subprostrate sophora, 8g of catechu, 18g of blackberry lily, 8g of calyx seu fructus physalis and 13g of liquorice.
The embodiment provides a pharmaceutical preparation containing the traditional Chinese medicine composition, and the preparation method comprises the following steps: weighing bran-fried rhizoma atractylodis, agastache rugosus, raw ephedra herb, blackberry lily, subprostrate sophora, catechu, calyx seu fructus physalis and liquorice according to the weight of the prescription of the traditional Chinese medicine composition, adding water for decocting for 2 times, adding 10 times of water for the first time, decocting for 6 hours, adding 5 times of water for the second time, decocting for 0.5 hour, combining the decoction, filtering, combining the filtrate with the distilled water, concentrating under reduced pressure to obtain thick paste with the relative density of 1.20-1.25(50 ℃), drying, crushing, preparing into semi-finished granules, drying, spraying the volatile oil collected by distillation, mixing uniformly to obtain the xanthium and ephedra toxin-resolving granules, tabletting by a dry method, and preparing into tablets.
Example 3
The embodiment provides a traditional Chinese medicine composition which comprises 13g of bran-fried rhizoma atractylodis, 8g of raw ephedra herb, 18g of wrinkled gianthyssop herb, 4g of subprostrate sophora, 8g of catechu, 18g of blackberry lily, 8g of calyx seu fructus physalis and 13g of liquorice.
The embodiment provides a pharmaceutical preparation containing the traditional Chinese medicine composition, and the preparation method comprises the following steps: weighing herba Artemisiae Annuae, rhizoma Atractylodis and herba Agastaches according to the weight of the prescription of the Chinese medicinal composition, adding 20 times of water, extracting volatile oil by steam distillation for 3 hr, and collecting volatile oil; collecting the distilled water solution in another container for later use; according to the formula, raw ephedra, gypsum, radix bupleuri, astragalus mongholicus, verbena and liquorice are added with water and decocted twice, 3 times of water is added for the first time and decocted for 0.5 hour, 15 times of water is added for the second time and decocted for 3 hours, decoction liquids are combined and filtered, filtrate is combined with the distilled water liquid, the filtrate is concentrated under reduced pressure to thick paste with the relative density of 1.20-1.25(50 ℃), dried and crushed to prepare a semi-finished product, the semi-finished product is dried, the volatile oil collected by distillation is sprayed, the mixture is mixed uniformly to obtain xanthium and ephedra toxin removing granules, and the xanthium and ephedra toxin removing granules are filled into capsules to prepare capsules.
Experimental example 1 protective action of Cannabis virus particles on influenza virus infected mice
1. Experimental Material
Lian Hua Qing Wen granules: beijing, Kyoto pharmaceutical Co. The national standard of medicine Z20100040. Specification: each bag is 6 g. Production batch number: 2001053.
golden flower refreshing granules: jue Chang (Beijing) pharmaceutical Co. The national drug standard Z20160001. Specification: 5g (equivalent to 17.3g of decoction pieces) per bag, production lot number: 20200101.
oseltamivir phosphate capsules (tamiflu): roche Registration ltd. production, split charging enterprise: shanghai Roche pharmaceuticals, Inc. Registration certificate number of imported drugs: h20140344; subpackage approval document No.: the national medicine standard J20140121. Product batch number: and (M1050). Subpackage batch number: SH 0077. Specification: 75 mg/pellet (calculated as oseltamivir). times.10 pellets.
Cang ma toxin-resolving granule: prepared according to the method of the invention example 1.
2. Experimental methods
(1) Animal grouping and administration
146 ICR mice are randomly divided into a normal control group, a model control group, a Duffy control group, a Lianhua scourge-clearing granule group, a golden flower infection-clearing granule group, a cang pockmark chemical poison granule (CMHD) high dose group, a medium dose group and a small dose group according to sex and weight, 6 normal control groups are used, and 20 other groups are used.
Mice in each group were lightly anesthetized with ether and nasally infected 1 time, 35 μ L/mouse/time with 15 drops of LD50H1N1/FM1 virus. Grinding the Daphne, Lianhua scourge-clearing granules and the golden flower Qinggan granules respectively, and dissolving the ground Daphne, golden flower Qinggan granules and the golden flower Qinggan granules in distilled water to prepare tested drug solutions with the mass concentrations of 1.375mg/ml, 165mg/ml and 137.5mg/ml respectively, grinding the xanthate poisoning granules in the embodiment 1 of the invention, and dissolving the grinded xanthate poisoning granules in the distilled water to prepare tested drug solutions with the mass concentrations of 550mg/ml, 275mg/ml and 137.5mg/ml respectively. The infected duffy group, the copperleaf antipyretic granule group, the golden flower antipyretic granule group, the cannabelism particle large dose group, the cannabelism particle medium dose group and the cannabelism particle small dose group are respectively 0.0275, 3.3, 2.75, 11, 5.5 and 2.75 g/kg-1·d-1Sequentially gavage the corresponding test drug solution, gavage the stomach once a day for 4 consecutive days according to 0.2mL/10g body weight/time, observing the survival condition of the mouse every day from the 1 st day after infection, and counting the survival rate, the average survival number of days, the number of life prolonging days and the life prolonging rate of the mouse for 2 weeks.
(2) Analysis of Experimental data
Calculating the survival rate, average survival days and life prolonging rate of the mouse according to the following formula;
survival rate is the total number of the animals surviving in each group/the total number of the animals in each group multiplied by 100 percent;
the average survival days are the sum of actual survival days of each group/20;
the life extension rate is (average survival days of the administration group-average survival days of the model control group)/average survival days of the model control group × 100%.
3. Results of the experiment
TABLE 1 protective Effect of Cannabis virus particles on influenza A virus strain FM1 infection-induced mouse death model
Note: compared with the model control group,**P<0.01，*P<0.05. the dosage g/kg/d refers to the amount of drug per kg of mice per day administered by gavage based on the mass of the drug formulation.
The results showed that the mortality of the model control group mice was 95% when normal mice were infected with influenza A virus strain FM 1. On the day of infection, dosing was started for 4 consecutive days in each dosing group. The cang ma hua du granules are large, and the mortality of mice in a medium-dose group is reduced; the average survival days are prolonged compared with the model control group, and have no significant difference compared with the model control group (P > 0.05). The death rate of the mice in the Tamiflu group is 80%, the average survival days is prolonged by more than 2 days compared with that of a model control group, and the significant difference is shown compared with that of the model control group (P < 0.01). The death rate of mice in the lianhua antipyretic granule group and the Jinhua antipyretic granule group is reduced, the average survival days are prolonged compared with the model control group, and the mice have no significant difference (P is more than 0.05) compared with the model control group. The death protection effect of the large and medium dose group of cang ma hua toxin-removing granules on influenza mice is similar to that of the lian hua scourge-clearing granules and the golden flower cold-clearing granules.
Experimental example 2 drug efficacy test of Cannabis virus particles on influenza virus infected mice
1. Experimental Material
The same as in experimental example 1.
2. Experimental methods
(1) Animal grouping and administration
80 ICR mice are randomly divided into a normal control group, a model control group, a Duffy control group, a Lianhua scourge particle group, a golden flower infection particle group, a cang pockmarked drug particle large dose group, a medium dose group and a small dose group according to sex and weight, and each group comprises 10 mice.
After completion of the grouping, each group of mice was lightly anesthetized with ether and nasally infected with 15 drops of LD50H1N1/FM1 virus 1 times, 35. mu.L/mouse/time. Taking daphne and copperleaf antipyretic granules,The golden flower influenza-clearing particles are respectively crushed and then dissolved in distilled water to prepare tested medicine solutions with the mass concentrations of 1.375mg/ml, 165mg/ml and 137.5mg/ml, the xanthate poisoning particles in the embodiment 1 of the invention are crushed and then dissolved in distilled water to prepare tested medicine solutions with the mass concentrations of 550mg/ml, 275mg/ml and 137.5mg/ml, and the infected duffy group, the honeysuckle influenza-clearing particle group, the xanthate poisoning particle large dose group, the xanthate poisoning particle medium dose group and the xanthate poisoning particle small dose group are respectively 0.0275, 3.3, 2.75, 11, 5.5 and 2.75 g.kg-1·d-1Sequentially gavage the corresponding test drug solutions, and gavage the drugs once a day for 4 days according to the weight of 0.2mL/10 g/time.
(2) Weighing and calculating the pulmonary index and pulmonary index inhibition
After weighing on day 5, mice were bled from the orbital venous plexus (3 drops collected in 10mL of 1 XPBS, the remaining blood was stored at-80 ℃ after serum collection). Dissecting mouse, collecting lung, weighing, fixing left lung lobe in 4% paraformaldehyde solution, and storing the rest lung at-80 deg.C. Dead mice prior to the end of the experiment were not included in the statistics. The pulmonary index and the pulmonary index inhibition rate were calculated according to the following formulas. And intestinal feces of each group of mice were aseptically collected and stored in a refrigerator at-80 ℃.
Lung index ═ lung wet weight (g)/bulk mass (g) × 100%;
the lung index inhibition rate is (model control lung index-administration lung index)/(model control lung index-normal control lung index) × 100%.
(3) HE staining method for detecting pathological changes of lung tissues of mice
Dehydrating the fixed mouse lung by using 75 percent ethanol water solution by volume percentage, then using dimethylbenzene for transparence, and then embedding the lung by using paraffin; cutting the embedded wax block into slices of 4-6 microns by using a slicing machine, flattening in hot water, sticking the slices to a glass slide, and then drying in a constant temperature oven at 45 ℃; the sections were stained with hematoxylin-eosin (HE); the HE-stained sections were placed under an optical microscope to observe pathological changes of lung tissues and photographed.
3. Results of the experiment
(1) Pulmonary index and pulmonary index inhibition rate
TABLE 2 Effect of Cannabis virus particles on pulmonary index of influenza Virus infected mice
Note: comparison with model control group**P<0.01，***P<0.001。
The test results show that when the H1N1/FM1 virus strain is used to infect the mice, the lung index of the mice is increased, and the mice have significant difference compared with a normal control group (P < 0.001). After the corresponding drugs of each administration group are respectively administered, the lung index values of the mice show different change trends. The lung index of 3 dose groups of cannabelism particles is reduced, and the large dose group has significant difference (P <0.01) compared with a model control group. The lung index of the Taffy group is most reduced, and has significant difference compared with a model control group (P < 0.001). The lung indexes of the lianhua antipyretic granule group and the Jinhua antipyretic granule group are both reduced, and the lung indexes are remarkably different from those of a model control group (P <0.05 and P < 0.01).
(2) HE staining results
As shown in fig. 1-8, extensive lung injury, including tissue necrosis and extensive inflammatory cell infiltration, was observed in mice of the model control group. Compared with the lung of the mouse of the model control group, the lung inflammatory infiltration of the mouse treated by the cang ma hua du granules is less, and the improvement degree is equivalent to that of the lian hua qing wen granules group and the jinhua qing gan granules group. The lung tissue structure of the Taffy mice is relatively complete and clear. The lung tissue structure conditions of the mice in the Lianhua scourge-clearing group and the Jinhua Qinggan granule group are improved, but are different from those in the Taffy group.
(3) Small knot
The cang ma hua du particles can obviously inhibit the increase of lung index and relieve the lung inflammation caused by influenza virus, and have obvious treatment effect. The efficacy of the cang ma hua du granules is better than the efficacy of the medium and small doses in the large dose. The drug effect of the large-dose group is equivalent to that of the lianhua antipyretic granule group and the golden flower refreshing granule group.
Experimental example 3 Effect of Cannabis virus particles on inflammatory response in influenza Virus-infected mice
1. Experimental methods
Detecting the expression level of the inflammatory factors of the lung tissues of the mice by a liquid phase protein chip method.
Investigating the influence of the xanthate poisoning particles on the expression of the inflammatory factor in the serum of the mouse, detecting the expression level of the inflammatory factor in the serum of the mouse by adopting an ELISA method, and collecting blood from each group of animals in the experimental example 1 through the orbital venous plexus in the step (2). Respectively adopting Mouse TNF-alpha ELISAKIT, Mouse IL-1 beta ELISA KIT, Mouse IL-6ELISA KIT and Mouse IL-4ELISA KIT KITs (provided by Shanghai enzyme-linked immunosorbent assay technology Co., Ltd.), and carrying out the detection process strictly according to the KIT operation instructions. The kit was allowed to equilibrate at room temperature for 60 minutes. The enzyme labeling coating plate is provided with a standard sample hole, a sample hole and a blank hole. The standard wells were each filled with 50. mu.L of standard at different concentrations. 40 mu L of the sample diluent is added into the sample hole, and then 10 mu L of the sample to be detected is added respectively (the final dilution of the sample is 5 times). The blank control hole is not added with the sample and the enzyme labeling reagent, and the operation of the other steps is the same. Adding sample to the bottom of the plate hole of the enzyme label, keeping the sample from touching the hole wall as much as possible, and gently shaking and mixing the sample and the hole wall. Add enzyme labeling reagent 100. mu.L to each well except for blank wells. Sealing the plate with a sealing plate film, and then incubating for 1h at 37 ℃; washing the plate, discarding the liquid, spin-drying, filling washing liquid into each hole, standing for 30S, discarding, repeating for 5 times, and patting to dry; adding 50 μ L of color-developing agent A into each well, adding 50 μ L of color-developing agent B, shaking gently, mixing, and developing at 37 deg.C in dark for 15 min. Stop solution (50. mu.L/well) was added to stop the reaction (blue color turned yellow immediately). The microplate reader detects the absorbance (OD value) of each well at 450 nm. And finally calculating the concentration value of each sample by fitting a curve.
2. Results of the experiment
TABLE 3 Cannabis toxin-resolving granule for type AInfluence of influenza virus FM1 strain on pulmonary cytokine expression level of mice infected with the virus
Note: compared with the model control group,*P<0.05，**P<0.01。
mice were infected with the H1N1/FM1 strain and the results showed that: the expression level of TNF-alpha in the serum of the mouse of the model control group is obviously increased, and the significant difference (P <0.001) is compared with that of the normal control group. After the mice of each administration group are respectively administered with the corresponding drugs, the expression level of TNF-alpha presents different change trends. The TNF-alpha expression levels of large, medium and small dose groups of xanthate and ephedra toxin granules are reduced, and have significant differences compared with a model control group (P is less than 0.001, P is less than 0.01, and P is less than 0.05). The expression level of TNF-alpha in the Murphy group, the Lian Hua Qingwen granule group and the Jinhua Qinggan granule group is reduced, and the significant difference is compared with that in the model control group (P <0.05)
② the expression quantity of IL-1 beta in the serum of the mouse of the model control group is obviously increased, and has significant difference (P <0.001) compared with the normal control group. After the mice of each administration group are respectively administered with the corresponding drugs, the expression level of the IL-1 beta shows different change trends. The IL-1 beta expression of the large and medium dose groups of the cannabis virus particles is reduced, and the large and medium dose groups of the cannabis virus particles have significant differences compared with a model control group (P <0.001 and P < 0.01). The IL-1 beta expression level of the xanthate and ephedra toxin granule small dose group is not reduced, and has no significant difference compared with a model control group (P > 0.05). The IL-1 beta expression of the Murphy group, the Lian Hua Qingwen granule group and the Jinhua Qinggan granule group is reduced, and has significant difference compared with the model control group (P <0.001, P <0.01)
③ the expression level of IL-6 in the serum of the mouse of the model control group is obviously increased, and has significant difference (P <0.001) compared with the normal control group. After the mice of each administration group are respectively administered with the corresponding drugs, the expression level of IL-6 shows different trend. The IL-6 expression of the large and medium dose groups of the cannabis virus particles is reduced, and the large and medium dose groups of the cannabis virus particles have significant differences compared with a model control group (P is less than 0.001 and P is less than 0.05). The IL-6 expression of the Tamiflu group is reduced, and the significant difference is compared with that of a model control group (P < 0.001). The IL-6 expression levels of the panicled flower antipyretic granule group and the golden flower antipyretic granule group are only reduced, and have no significant difference (P is more than 0.05) compared with that of a model control group.
And fourthly, the expression level of IL-4 in the serum of the mouse in the model control group is obviously reduced, and the obvious difference (P <0.001) is obtained compared with that in the normal control group. After the mice of each administration group are respectively administered with the corresponding drugs, the expression level of IL-4 shows a trend of increasing. The expression level of IL-4 in 3 dose groups of cannabelism particles is increased, and the 3 dose groups have significant difference compared with a model control group (P <0.001, P <0.01 and P < 0.05). The IL-4 expression levels of the Murphy group, the Lian Hua Qingwen granule group and the Jinhua Qinggan granule group are increased, and have significant differences compared with a model control group (P <0.001)
Experimental example 4 Effect of Cannabis virus particles on immune function of influenza virus-infected mice
1. Experimental methods
Flow cytometry detected peripheral blood lymphocytes from mice. 3 drops of blood (from each group of animals in experimental example 2, 6 blood samples were randomly selected from each group, and blood was collected from orbital venous plexus in step (2)) were dropped into 10ml of PBS and mixed; centrifuging at 2000r/min for 15min at 4 ℃; sucking out the supernatant by a pipette, adding 1mL of erythrocyte lysate into the red precipitate, blowing, stirring uniformly, and adding 10mL of PBS to stop the lysis after the liquid is red, transparent and clear; centrifuging at 2000r/min for 5min at 4 ℃; discarding the supernatant, sucking the red suspension, and resuspending the precipitate with 5ml PBS; centrifuging at 2000r/min for 5min at 4 ℃; discard the supernatant, add 500L of blocking solution (PBS containing 5% FBS), resuspend, and transfer to 1.5mLEP tube; centrifuging at 2000r/min for 5min at 4 ℃; removing supernatant, adding 50 μ L of antibody (PE-labeled anti-mouse CD45R antibody, PerCPCy5.5-labeled anti-mouse CD4 antibody, APC-labeled anti-mouse CD8a antibody 0.3 μ L each), and incubating at 4 deg.C in dark for 30 min; adding 1ml PBS for resuspension, and centrifuging at 4 ℃ at 2000r/min for 5 min; and (4) throwing off the supernatant, adding 2% paraformaldehyde fixing solution, resuspending the cells, and storing at 4 ℃ in a dark place. And (6) performing detection on the machine.
2. Results of the experiment
TABLE 4 Effect of Cannabis virus particles on the percentage of peripheral blood lymphocytes in mice infected with influenza Virus
Note: comparison with model control group**P<0.01，*P<0.05。
The test results show that the material has the characteristics of,
the mouse is infected with FM1 virus strain, and the peripheral blood CD4 of the model control group mouse+The proportion of T cells is increased, and the T cells have significant difference (P) compared with a normal control group<0.05). After the corresponding drugs were administered to each administration group, the peripheral blood CD4 was obtained+The T cell ratio shows a reduced trend. Cannabis toxin-removing granule 3 dose groups CD4+The T cell ratio has significant difference compared with the model control group (P)<0.05). The duffy group, the copperleaf antipyretic group and the golden flower influenza-clearing group have no significant difference (P) compared with the model control group>0.05)。
② model control group mouse peripheral blood CD8+The proportion of T cells is reduced, and the T cells have significant difference (P) compared with a normal control group<0.05). CD8 after the mice of each administration group were administered the corresponding drugs+The T cell ratios showed different trends. Large and medium dose group of cang-ma toxin-removing granules CD8+The T cell ratios all tend to be increased, and have significant difference (P) compared with the model control group<0.05). Golden flower Qinggan granule group CD8+The proportion of T cells is increased, and the T cells have significant difference (P) compared with the model control group<0.05). Daphne group and Lianhua scourge-clearing granule group CD8+The T cell ratio only has rising trend, and has no significant difference compared with a model control group (P)>0.05)。
③ the proportion of CD45R B lymphocytes in the peripheral blood of the mice of the model control group is reduced, and the difference is more significant compared with the normal control group (P < 0.01). After the mice of each administration group are respectively administered with the corresponding drugs, the CD45R cell ratio shows a rising trend. The proportion of CD45R cells in 3 dose groups of cannabelism particles is dose-dependent, and the large and medium doses have significant difference compared with a model control group (P <0.05 and P < 0.01). The ratio of CD45R cells in the duffy group and the copperleaf antipyretic granule group is increased, and the significant difference is compared with that in the model control group (P <0.01, P < 0.001). The CD45R cell ratio of the golden flower influenza virus particle group is only increased compared with that of a model control group, and no significant difference exists (P > 0.05).
The cannabelism particles regulate the proportion of T lymphocytes, show a certain immunoregulation effect, have obvious effect in large and medium doses and are superior to a small dose group. The antipyretic granule containing flos Nelumbinis and flos Lonicerae has effects in increasing the ratio of T lymphocyte, and regulating immunity.
In conclusion, the traditional Chinese medicine composition or the cang ma hua du granules have obvious treatment effect on influenza virus pneumonia model mice, the treatment effect basically has dose-effect relationship, and the effect of the cang ma hua du granules on treating influenza in a large-dose group and the effect of the cang ma hua qingwen granules and the effect of the cang ma hua qing granules on treating influenza are superior to those of a medium-dose group and a small-dose group. The cannabelism particles can inhibit lung inflammation, improve the pathological state of the lung of an infected mouse, inhibit the expression level of the lung inflammatory cytokines of an influenza virus pneumonia mouse, improve the immune state of the influenza virus pneumonia mouse and inhibit the expression of key proteins of an inflammation pathway.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (10)
1. The application of a Chinese medicinal composition in preparing a medicament for preventing and/or treating diseases caused by influenza virus or influenza virus infection is characterized in that the Chinese medicinal composition comprises the following raw material medicaments of 13-18 parts of rhizoma atractylodis, 8-13 parts of ephedra, 13-18 parts of agastache rugosus, 4-9 parts of subprostrate sophora, 8-13 parts of catechu, 13-18 parts of blackberry lily, 8-13 parts of calyx seu fructus physalis and 8-13 parts of liquorice in parts by weight; the influenza virus is influenza A virus.
2. The application of claim 1, wherein the traditional Chinese medicine composition comprises, by weight, 15 parts of rhizoma atractylodis, 10 parts of ephedra, 15 parts of agastache rugosus, 6 parts of subprostrate sophora, 10 parts of catechu, 15 parts of blackberry lily, 10 parts of calyx seu fructus physalis and 10 parts of liquorice; or,
13 parts of rhizoma atractylodis, 8 parts of ephedra, 18 parts of agastache rugosus, 4 parts of subprostrate sophora, 8 parts of catechu, 18 parts of blackberry lily, 8 parts of calyx seu fructus physalis and 13 parts of liquorice; or,
18 parts of rhizoma atractylodis, 13 parts of ephedra, 13 parts of agastache rugosus, 9 parts of subprostrate sophora, 13 parts of catechu, 13 parts of blackberry lily, 13 parts of calyx seu fructus physalis and 8 parts of liquorice.
3. The use according to claim 1 or 2, wherein the preparation is prepared by weighing rhizoma atractylodis, ephedra, agastache rugosus, blackberry lily, subprostrate sophora, catechu, calyx seu fructus physalis and liquorice according to selected parts by weight, mixing and extracting according to a conventional extraction method or extracting respectively according to a conventional extraction method and mixing.
4. The use of claim 3, wherein the conventional extraction method comprises one or more of decoction extraction, maceration extraction, percolation extraction, reflux extraction, ultrasonic extraction and steam distillation extraction; the extraction solvent is selected from water or 5-98% alcohol solution; the extraction times are at least 1 time; the extraction time is at least 10 min; the ratio of the volume of the extraction solvent to the weight of the raw material medicine is more than or equal to 2L/kg.
5. The use of claim 1 or 2, wherein the pharmaceutical preparation is prepared from the Chinese medicinal composition according to a conventional preparation method with or without a pharmaceutically acceptable carrier.
6. The use according to claim 5, wherein the pharmaceutical formulation is a gel, cream, tablet, capsule, powder, mixture, pill, granule, oral liquid, syrup, decoction, suppository, aerosol, patch, ointment, injection, spray, liniment, tincture, cataplasm, paste or lotion.
7. The use according to claim 5, wherein the pharmaceutically acceptable carrier is selected from at least one of pharmaceutically acceptable solvents, solubilizers, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, tonicity adjusting agents, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adherents, integration agents, permeation enhancers, pH adjusting agents, buffers, plasticizers, surfactants, thickeners, encapsulation agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, polymeric matrix materials, and film-forming materials.
8. The use according to claim 5, wherein the process for the preparation of the pharmaceutical formulation comprises the steps of:
steam distillation step: weighing rhizoma atractylodis and agastache rugosus according to the selected weight parts, mixing, performing steam distillation extraction, and respectively collecting volatile oil and water extract;
water decoction: weighing ephedra, blackberry lily, subprostrate sophora, catechu, calyx seu fructus physalis and liquorice according to the selected weight parts, adding water for decoction, carrying out solid-liquid separation, and collecting liquid;
the preparation steps of the pharmaceutical preparation are as follows: mixing the water extractive solution obtained by steam distillation and the liquid obtained by water decoction, concentrating, drying, optionally adding adjuvants, making into semi-finished product, and spraying the above volatile oil to obtain medicinal preparation.
9. Use according to claim 1 or 2, wherein the disease caused by influenza virus is selected from respiratory tract inflammation, acute lung injury and/or acute respiratory distress syndrome.
10. Use according to claim 9, wherein the inflammation of the respiratory tract is selected from pharyngitis, laryngitis, tracheitis, bronchitis and/or pneumonia.
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