CN113499383B - Application of traditional Chinese medicine composition and/or pharmaceutical preparation containing traditional Chinese medicine composition in preparation of anti-inflammatory drugs or immunoregulation drugs - Google Patents
Application of traditional Chinese medicine composition and/or pharmaceutical preparation containing traditional Chinese medicine composition in preparation of anti-inflammatory drugs or immunoregulation drugs Download PDFInfo
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Abstract
The invention relates to the field of traditional Chinese medicines, and particularly provides an application of a traditional Chinese medicine composition and/or a pharmaceutical preparation containing the traditional Chinese medicine composition in preparing an inflammation medicine or an immunoregulation medicine, wherein the traditional Chinese medicine composition is obtained by repeatedly optimizing the composition and the content of a compound traditional Chinese medicine through clinical research and basic research, and ephedra, gypsum and radix bupleuri are used as monarch drugs in the formula; scutellaria baicalensis, sweet wormwood and rhizoma atractylodis are taken as ministerial drugs; herba Verbenae and herba Agastaches as adjuvant drugs; licorice root, radix Glycyrrhizae is used as a guiding drug for the recipe. The medicines have the effects of clearing away heat and toxic materials, ventilating the lung and relieving asthma, and play a positive role in regulating the immunity of an organism, particularly regulating abnormal quantity of immune cells, restoring the normal quantity of the immune cells, regulating unbalance of proinflammatory factors and inflammation-inhibiting factors, restoring the balance state of the proinflammatory factors and the inflammation-inhibiting factors, and preventing and/or treating diseases caused by abnormal immune function and abnormal immune function.
Description
Technical Field
The invention relates to the field of traditional Chinese medicines, and in particular relates to an application of a traditional Chinese medicine composition and/or a pharmaceutical preparation containing the traditional Chinese medicine composition in preparation of anti-inflammatory drugs or immunoregulation drugs.
Background
The immune system of the body plays an important role in eliminating cancerous tissues and pathogenic bacteria invasion, and cellular immunity is an important component of the immune system of the body. In cellular immunity, lymphoid T cells and lymphoid B cells are centrally located, and lymphoid T cells are largely classified into CD8 according to surface markers+Of cytotoxic T cells, CD4+Of helper T cells, CD4+CD25+Regulatory T cells of (3), and the like. The lymphocyte B cell is mainly divided into CD45RB cell, B1 cell, B2 cell and the like according to the surface marker, and various cytokines expressed by lymphocyte T cell and lymphocyte B cell include IL-IR, IL-2R, IL-4, IL-6, IL-10 and the like. Normally, the immune response of the body is tightly controlled to moderate the scale of the immune response, and T cells and B cells maintain the immune responsePlays an important role in the steady state.
Infectious diseases are common diseases and frequently encountered diseases in clinic, mainly comprise diseases such as parasitic infection, viral infection, mycoplasma infection, chlamydia infection, bacterial infection and the like, seriously threaten human health, and bring huge economic loss to the livestock industry all over the world. After the organism is infected with microbes such as bacteria and viruses, serious proinflammatory reaction and uncontrolled anti-inflammatory reaction often occur, so that cell factors are redundant, and a 'cell factor storm' is further caused. This excessive innate immune response often leads to severe tissue damage, induces sepsis, etc., and even death. In the case of influenza, the immune system of the body senses viral RNA and initiates a rapid signaling cascade, which leads the infected epithelial cells and immune cells of the body to produce about 15 cytokines to help protect against the virus. Interleukin-6 (IL-6) activates immune cells (T cells and macrophages) and eliminates cells infected with the virus, which is the major factor of this cytokine, and type I and type III Interferons (IFNs) influence viral RNA synthesis. This first wave cytokine, called the "primary storm," plays a key role in reducing viral replication in the initial stages, but also imbeds a vodka for subsequent overactivation of the immune system. T cells, when activated and differentiated, secrete a second wave of cytokines, of which interferon-gamma (IFN-gamma) is the dominant factor. This secondary storm is often fatal, when cytokine levels are exceeded for viral clearance, resulting in excessive inflammatory cell accumulation. If a large number of inflammatory cells accumulate in the alveoli, the oxygenation function is reduced, and respiratory failure is caused, which is also the reason for sudden exacerbation in part of influenza cases.
At present, the astaxanthin has an immunoregulation effect, and the expression of interleukin 6(IL-6) and ll-1B mRNAp65 induced by LPS is remarkably reduced by inhibiting nuclear transport of NFKB. Therefore, astaxanthin can be used for preventing or treating inflammation. The prior art also reports that lutein inhibits excessive Th1 cell-mediated immune responses and stimulates Th2 cell-mediated immune responses in patients during persistent infection and/or inflammation in patients. Th1 cell-mediated excessive immune responses are caused by autoimmune diseases as well as chronic viral and intracellular bacterial infections, such as psoriasis vulgaris, Multiple Sclerosis (MS), rheumatoid arthritis, Crohn's disease, insulin dependent diabetes mellitus, Tuberculosis (TB), acute graft versus virus-host disease (transplant rejection) and HIV viral infections. However, no relevant drugs are currently on the market.
In the past thousands of years, traditional Chinese medicine plays an important role in treating diseases and helps people overcome several times of epidemic diseases, so that the advantages of traditional Chinese medicine are fully exerted, and the development of effective anti-inflammatory or immunoregulatory traditional Chinese medicine becomes the next urgent subject to be solved.
Disclosure of Invention
Therefore, the invention aims to provide the application of the traditional Chinese medicine composition and/or the pharmaceutical preparation containing the traditional Chinese medicine composition in preparing anti-inflammatory drugs or immunoregulation drugs; the traditional Chinese medicine composition comprises, by weight, 7-12 parts of ephedra, 28-33 parts of gypsum, 8-13 parts of radix bupleuri, 13-18 parts of scutellaria baicalensis, 13-18 parts of sweet wormwood, 8-13 parts of rhizoma atractylodis, 28-33 parts of verbena, 13-18 parts of agastache rugosus and 8-13 parts of liquorice.
Further, the traditional Chinese medicine composition comprises the following raw material medicines in parts by weight: 9 parts of ephedra, 30 parts of gypsum, 10 parts of radix bupleuri, 15 parts of scutellaria baicalensis, 15 parts of sweet wormwood, 10 parts of rhizoma atractylodis, 30 parts of verbena, 15 parts of agastache rugosus and 10 parts of liquorice;
or 7 parts of ephedra, 33 parts of gypsum, 8 parts of radix bupleuri, 13 parts of radix scutellariae, 13 parts of sweet wormwood, 13 parts of rhizoma atractylodis, 33 parts of verbena, 13 parts of agastache rugosus and 8 parts of liquorice;
or 12 parts of ephedra, 28 parts of gypsum, 13 parts of radix bupleuri, 18 parts of scutellaria baicalensis, 18 parts of sweet wormwood, 8 parts of rhizoma atractylodis, 28 parts of verbena, 15 parts of agastache rugosus and 13 parts of liquorice.
In certain preferred embodiments, the rhizoma Atractylodis is selected from processed rhizoma Atractylodis, such as but not limited to processed rhizoma Atractylodis, bran-parched rhizoma Atractylodis, charred 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.
In some preferred embodiments, the preparation method of the Chinese medicinal composition comprises weighing ephedra, gypsum, radix bupleuri, scutellaria baicalensis, artemisia apiacea, rhizoma atractylodis, verbena, agastache rugosus and liquorice according to selected parts by weight, mixing, extracting according to a conventional extraction method or extracting respectively 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 pharmaceutical preparation comprises the traditional Chinese medicine composition for clearing heat and removing toxicity or the traditional Chinese medicine composition for clearing heat and removing toxicity prepared by the preparation method, and optionally one or more pharmaceutically acceptable carriers.
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 preparation method of the pharmaceutical preparation comprises the following steps:
steam distillation step: weighing sweet wormwood, rhizoma atractylodis and agastache rugosus according to the selected weight parts, mixing, carrying out steam distillation extraction, and respectively collecting volatile oil and water extract;
water decoction: weighing ephedra, gypsum, radix bupleuri, scutellaria baicalensis, verbena 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 granules 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 mixing with the volatile oil to obtain medicinal preparation.
The mass ratio of the medicinal preparation to the adopted Chinese medicinal composition is 25-35: 126, 171, preferably 30: 144.
in certain preferred embodiments, the essential oils are mixed in a spray-on manner during the step of preparing the granules.
In certain preferred embodiments, the step of drying further comprises a step of pulverizing the granules.
In some preferred embodiments, the step of preparing the granules further comprises a drying step after the step of preparing the semi-finished pharmaceutical preparation.
In certain preferred embodiments, in the water decoction step, 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 sweet wormwood, 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, gypsum, radix bupleuri, scutellaria baicalensis, verbena and liquorice according to the selected weight parts, adding water, 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 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, drying, pulverizing, optionally adding adjuvant, making into semi-finished product, spraying the volatile oil, and mixing.
In a more preferred embodiment, the method for preparing the pharmaceutical formulation comprises the steps of:
weighing sweet wormwood, rhizoma atractylodis and agastache rugosus according to the selected weight parts, mixing, adding 5-10 times of water for steam distillation extraction for 0.5-2 hours, and respectively collecting volatile oil and water extract; weighing ephedra, gypsum, radix bupleuri, scutellaria baicalensis, verbena and liquorice according to the selected weight parts, adding water for decocting for 1-3 times, adding 5-10 times of water for each time, decocting for 0.5-2 hours for each time, combining water decoction liquids, 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 intermediate may be a granule, powder or tablet.
Further, the immunomodulation refers to the treatment of immune dysfunction.
In a preferred embodiment, the immune dysfunction is caused by a parasitic infection, a viral infection, a mycoplasma infection, a chlamydia infection, a bacterial infection, an autoimmune disease, or the like.
Preferably, the immune dysfunction is cytokine release syndrome, lymphohistiocytosis with hemophagic cells or cytokine storm syndrome.
In a preferred embodiment, the virus is at least one selected from the group consisting of influenza virus, SARS virus, MERS virus, 229E virus, NL63 virus, OC43 virus, HKU1 virus, and COVID-19 virus. Further, the influenza virus is an influenza a virus.
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. As a preferred embodiment, the immune cell is selected from CD4+T cell, CD8+One or more of T cells and CD45R B lymphocytes. As a preferred embodiment, the proinflammatory factor is selected from one or more of TNF-alpha, IL-1 beta, IL-6, IL-12p70, IFN-gamma, MCP-1. In a preferred embodiment, the anti-inflammatory agent is selected from one or more of IL-4 and IL-10.
By immunomodulation is meant the prevention and/or treatment of cytokine-related diseases or disorders. The diseases or conditions related to cytokines are respiratory tract inflammation, acute lung injury, acute respiratory distress syndrome, cytokine release syndrome and the like. Further, these diseases or conditions are caused by viral infections.
Specifically, the respiratory inflammation is selected from pharyngitis, laryngitis, tracheitis, bronchitis and/or pneumonia.
In addition, the traditional Chinese medicine composition or the medicine prepared from the pharmaceutical preparation can inhibit the expression of at least one of inflammatory factors TNF-alpha, IL-6, IL-1 beta, IL-12p70, IFN-gamma and MCP-1, and/or reduce the number of inflammatory infiltration cells.
The medicament prepared from the traditional Chinese medicine composition and/or the medicinal preparation can also inhibit the reduction of survival rate, survival time and/or weight reduction caused by influenza virus infection.
The invention also provides application of the traditional Chinese medicine composition and/or the medicinal preparation, wherein the traditional Chinese medicine composition and/or the medicinal preparation are used for preparing a medicament for regulating the intestinal flora; preferably, said modulating the intestinal flora comprises restoring the diversity of the intestinal flora and/or increasing the number of probiotics. Specifically, the intestinal flora adjustment comprises the adjustment of the intestinal flora structure of the influenza pneumonia mice, so that the level of the differential flora is adjusted back, and the number of probiotics in the intestinal tracts of the mice can be increased. For example, the abundance of the probiotic bacteria including the Acermanium incarnatum and the nodular bacteria is increased, so that the total ratio of the abundance of the probiotic bacteria including the Acermanium incarnatum and the lactobacillus is obviously improved. Wherein the traditional Chinese medicine composition is any one of the traditional Chinese medicine compositions or the traditional Chinese medicine composition prepared by any one of the preparation methods; the pharmaceutical preparation is any one of the pharmaceutical preparations described in the invention, or the pharmaceutical preparation prepared by any one of the preparation methods described in the invention.
The technical scheme of the invention has the following advantages:
1. the application of the traditional Chinese medicine composition provided by the invention is obtained by repeatedly optimizing the composition and the content of the compound traditional Chinese medicine through clinical research and basic research, wherein the traditional Chinese medicine composition is prepared by combining ephedra, gypsum and radix bupleuri as monarch drugs; scutellaria baicalensis, sweet wormwood and rhizoma atractylodis are taken as ministerial drugs; herba Verbenae and herba Agastaches as adjuvant drugs; licorice root, radix Glycyrrhizae is used as a guiding drug for the recipe. The medicines have the effects of clearing heat and removing toxicity, freeing lung and relieving asthma, can regulate body immunity, particularly regulate abnormal quantity of immune cells, restore the normal quantity of the immune cells, regulate imbalance of proinflammatory factors and inflammation-inhibiting factors, help the proinflammatory factors and the inflammation-inhibiting factors to restore the balanced state, and prevent and/or treat diseases caused by abnormal immune function and abnormal immune function.
Wherein, the ephedra, the gypsum and the bupleurum are combined to be monarch drug. Ma Huang is pungent, slightly bitter and warm in nature. It enters lung meridian and bladder meridian. Has the effects of inducing sweat, relieving exterior syndrome, dispersing lung qi, relieving asthma, inducing diuresis and relieving swelling. It is combined with Sheng Gao to clear lung heat and relieve dyspnea. Gypsum, Gypsum Fibrosum, is sweet and pungent in nature and cold in nature. It enters lung and stomach meridians. Has the effects of clearing heat, purging fire, relieving restlessness and quenching thirst. Bupleurum root, radix bupleuri is pungent and bitter in flavor, and slightly cold in nature. It enters liver, gallbladder and lung meridians. Has the efficacy of harmonizing exterior and interior.
Baikal skullcap root, sweet wormwood herb and rhizoma atractylodis are used as ministerial drugs. Baical skullcap root, radix Scutellariae is bitter in taste and cold in nature. It enters lung, gallbladder, spleen, large intestine and small intestine meridians. Has the effects of clearing heat, eliminating dampness, purging fire and removing toxicity. Sweet wormwood herb is bitter and pungent in flavor and cold in nature. It enters liver and gallbladder meridians. Has the efficacy of clearing deficiency heat. Can be used for warming pathogenic factors and damaging yin. Cang Zhu is pungent and bitter in flavor and warm in nature. It enters spleen, stomach and liver meridians. Has the effects of eliminating dampness, strengthening spleen, dispelling wind and dispelling cold. The scutellaria baicalensis and the rhizoma atractylodis can clear excess heat, the sweet wormwood can clear deficiency heat, and the three medicines can play a comprehensive heat clearing effect.
Verbena and ageratum are used as adjuvant drugs. Verbena is bitter in taste and cool. Enter liver and spleen meridians. Has the effects of clearing away heat and toxic materials, promoting blood circulation, removing blood stasis, inducing diuresis, and relieving edema. Huoxiang is pungent in flavor and warm in nature. It enters spleen, stomach and lung meridians. Has the effects of resolving dampness, activating spleen, removing dirt, regulating the middle warmer and relieving exterior syndrome.
The liquorice can not only clear away heat and toxic material, activate muscles with pungent and cool natured drugs, but also regulate drug properties, protect stomach and calm middle energizer.
The medicines have the effects of clearing heat and removing toxicity, ventilating and smoothing lung and relieving asthma and have good immunoregulation effect.
2. The nine traditional Chinese medicines in the formula of the traditional Chinese medicine composition provided by the invention supplement each other, but are not sufficient, if other traditional Chinese medicines with the effects of clearing heat and drying dampness such as coptis chinensis are adopted to replace the scutellaria baicalensis provided by the invention, or other traditional Chinese medicines with the effects of clearing deficiency heat such as phellodendron or bupleurum are adopted to replace the artemisia apiacea provided by the invention, the treatment effect on the coronavirus infection can be greatly influenced.
3. The traditional Chinese medicine composition can inhibit the increase of inflammatory factors TNF-alpha IL-6, IL-1 beta, IL-12p70, IFN-gamma and MCP-1 and can also inhibit CD4+Increased cell number of T cells and CD4+The reduction of the number of T cells and CD45R B lymphocytes prolongs the life days of virus infected patients or animals, has the effects of protecting the bodies infected by influenza virus, reducing the death rate, inhibiting the increase of lung index and reducing the infiltration of inflammatory cells, and shows that the traditional Chinese medicine composition has the function of immunoregulation, and can be used for preventing and/or treating the infection of the influenza virus and the diseases such as respiratory inflammation, acute lung injury, acute respiratory distress syndrome and the like caused by the infection of the influenza virus.
In addition, the traditional Chinese medicine composition can also regulate the intestinal flora structure of mice infected by influenza viruses.
4. 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.
5. According to the preparation method of the medicinal preparation provided by the invention, preferably, the three traditional Chinese medicines of sweet wormwood, rhizoma atractylodis and wrinkled gianthyssop are subjected to steam distillation extraction, the other raw materials are subjected to water decoction extraction, the water extract obtained by distillation and the water decoction are combined and concentrated, drying is carried out, the semi-finished product of the medicinal preparation is prepared, and then the volatile oil is sprayed in to obtain the finished product of the medicinal 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 the contiolled antipyretic mice 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 HE staining pattern of mice in large dose group of particles for clearing lung heat and removing toxicity in Experimental example 2 of the present invention;
FIG. 7 is a graph showing HE staining of mice in the dose group in the lung-heat-clearing and detoxifying granule in Experimental example 2 of the present invention;
FIG. 8 is a graph showing HE staining of mice in the small dose group of particles for clearing lung heat and removing toxicity in Experimental example 2 of the present invention.
Detailed Description
The following examples are provided to better understand the present invention, not to limit the best mode, and not to limit the content and protection scope of the present invention, and any product that is the same or similar to the present invention and is obtained by combining the present invention with other features of the prior art and the present invention 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 9g of raw ephedra herb, 30g of raw gypsum, 10g of radix bupleuri, 15g of radix scutellariae, 15g of sweet wormwood herb, 10g of rhizoma atractylodis stir-fried with bran, 30g of verbena, 15g of pogostemon cablin and 10g of raw liquorice.
The embodiment also provides a pharmaceutical preparation comprising the traditional Chinese medicine composition, and the preparation method comprises the following steps: weighing herba Artemisiae Annuae, rhizoma Atractylodis parched with bran and herba Agastaches according to the weight of the prescription of the Chinese medicinal composition, adding 10 times of water, extracting volatile oil for 2 hr by steam distillation, and collecting volatile oil for use; collecting the distilled water liquid in another container for later use; according to the prescription, raw ephedra herb, gypsum, radix bupleuri, radix scutellariae, verbena and raw liquorice are taken and decocted for three times by adding water, 10 times of water is added for the first time, the decoction is carried out for 1 hour, 8 times of water is added for the second time and the third time, the decoction is carried out for 0.5 hour respectively, the decoction liquids are combined and filtered, the filtrate is combined with the distilled water liquid, the concentrated solution is decompressed and concentrated to thick paste with the relative density of 1.20 to 1.25(50 ℃), the thick paste is dried and crushed, dextrin is added, a semi-finished product of particles is prepared, the semi-finished product is dried, the volatile oil collected by distillation is sprayed, and the mixture is mixed uniformly, so that 30g of the lung-clearing and detoxifying particles is obtained.
Example 2
This example provides a Chinese medicinal composition, which comprises Ephedra herb 7g, Gypsum Fibrosum 33g, Bupleurum root 8g, Scutellaria baicalensis 13g, Artemisia apiacea 13g, Atractylodes lancea 13g, Verbena officinalis 33g, Agastache rugosus 13g, and Glycyrrhiza uralensis 8 g.
The embodiment also provides a pharmaceutical preparation comprising 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 5 times of water, extracting volatile oil by steam distillation for 1h, and collecting volatile oil for use; collecting the distilled water solution in another container for later use; according to the prescription, weighing raw ephedra, gypsum, radix bupleuri, radix scutellariae, verbena and liquorice, adding water, decocting for three times, adding 20 times of water for the first time, decocting for 5 hours, adding 3 times of water for the second and third times, decocting for 0.5 hour respectively, combining decoction liquids, filtering, combining filtrate with the distilled water liquid, 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 granules for clearing away the lung-heat and toxic materials, tabletting by a dry method, and preparing into tablets.
Example 3
The embodiment provides a traditional Chinese medicine composition, which comprises 12g of raw ephedra herb, 28g of raw gypsum, 13g of radix bupleuri, 18g of radix scutellariae, 18g of sweet wormwood herb, 8g of rhizoma atractylodis, 28g of verbena, 15g of wrinkled gianthyssop herb and 13g of liquorice.
The embodiment also provides a pharmaceutical preparation comprising 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 prescription, raw ephedra, gypsum, radix bupleuri, radix scutellariae, verbena and liquorice are taken and decocted with water 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, the decoction liquids are combined and filtered, the filtrate is combined with the distilled water liquid, the concentrated solution is decompressed and concentrated into thick paste with the relative density of 1.20-1.25(50 ℃), the thick paste is dried and crushed, the thick paste is prepared into semi-finished products, the semi-finished products are dried, the volatile oil collected by distillation is sprayed, the mixture is mixed evenly, the granules for clearing away the lung-heat and toxic materials are obtained, and the granules for clearing away the lung-heat are filled into capsules to prepare capsules.
Experimental example 1 protective action of Lung-clearing and detoxifying granules 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): production, split charging enterprises: 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.
Granule for clearing lung and removing toxicity: prepared according to the method of example 1 of the invention.
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 particle group, a golden flower infection clearing particle group, a lung-heat clearing and detoxifying particle large dose group, a medium dose group and a small dose group according to sex and weight, 6 normal control groups and 20 rest groups.
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. Respectively pulverizing the dariffa, herba Lobeliae chinensis pestilence-clearing granules, flos Lonicerae heat-clearing granules, and lung-clearing and detoxicating granules, and dissolving in distilled water to obtain the final product with mass concentrations of 1.375mg/ml, 165mg/ml, and 137.5mg/ml, 550mg/ml, 275mg/ml, 137.5mg/ml of the test drug solution. After infection, the Daphne group, the Lianhua scourge-clearing granule group, the Jinhuaqinggan granule group, the lung-clearing and detoxifying granule large dose group, the medium dose group and the small dose group are respectively prepared into the dosage forms of 0.0275, 3.3, 2.75, 11, 5.5 and 2.75 g/kg-1·d-1The dosage of the composition is sequentially gavage to be administered to the test medicine solution, the gavage is performed once a day for 4 consecutive days according to the weight of 0.2mL/10 g/time, the survival condition of the mouse is observed every day from the 1 st day after infection, and the survival rate, the average survival number of days, the life prolonging number of days and the life prolonging rate of the mouse in 2 weeks are counted.
(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 action of Lung-clearing and detoxifying particles on mouse death model caused by influenza A virus FM1 strain infection
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 result shows that the mortality rate of the model control group mouse is 95 percent when the influenza A virus strain FM1 is used to infect the normal mouse. On the day of infection, dosing was started for 4 consecutive days in each dosing group. The mortality rate of mice in three dose groups of the lung clearing and detoxifying granules is reduced; the life prolonging rate is 9.32-14.91%; compared with a model control group, the average survival days of the large-dose group and the medium-dose group are prolonged by more than 1 day, and have significant difference (P is less than 0.05) compared with the model control group, which shows that the lung-clearing and detoxifying particles can treat or prevent diseases caused by influenza virus infection, and lays a foundation for clinical application.
Experimental example 2 cellular immune effect test of Lung-clearing and detoxifying particles on influenza virus-infected mice
1. Experimental Material
The same as in experimental example 1.
2. Experimental methods
(1) Grouping and administration of animals
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 clearing particle group, a lung-heat clearing and detoxifying particle large dose group, a middle 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. Respectively crushing the duffy, the honeysuckle antipyretic particles, the golden flower antipyretic particles and the lung-heat clearing and detoxifying particles, and dissolving the crushed particles in distilled water to prepare tested medicine solutions with the mass concentrations of 1.375mg/ml, 165mg/ml, 137.5mg/ml, 550mg/ml, 275mg/ml and 137.5 mg/ml. After infection, the Daphne group, the Lianhua scourge-clearing granule group, the Jinhuaqinggan granule group, the lung-clearing and detoxifying granule large dose group, the medium dose group and the small dose group are respectively prepared into the dosage forms of 0.0275, 3.3, 2.75, 11, 5.5 and 2.75 g/kg-1·d-1The dosage of (A) is sequentially administered by intragastric administration, and the administration is performed 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 of blood were collected in 10mL of 1 XPBS, and the remaining blood was collected as serum and stored at-80 ℃). 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 micrometers by using a slicing machine, flattening in hot water, attaching to a glass slide, and then placing in a 45 ℃ thermostat for drying; 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.
(4) Liquid phase protein chip method for detecting mouse lung tissue inflammatory factor expression level
Weighing 50mg of lung tissues (lungs preserved at-80 ℃ after each group of animals are treated in the step (2), randomly selecting 6 lung tissues for detection in each group), and adding 250 mu L of cell lysate; homogenizing with a tissue homogenizer, centrifuging at 12000r/min at 4 deg.C for 10min, and collecting supernatant. The BCA Protein quantification Kit (Pierce BCA Protein Assay Kit supplied by Thermo, lot number VG298889) measures the total Protein concentration of each sample. Samples were diluted with PBS (1X) to a final concentration of 10mg total protein/mL and stored at-80 ℃.
The detection reagent was diluted for use according to the protocol of a multifactorial detection kit (supplied by Thermo, cat # PPX-12). To each well of a 96-well plate, 50 μ L of the pre-mixed microspheres were added. The 96-well plate was placed in a magnetic separator plate to ensure that the plate was firmly clamped. And standing the plate for 2min to allow the microspheres to settle. The magnetic plate was then quickly inverted and the liquid in the well plate was poured out. Adding 150 μ L of washing buffer (WashBuffer) in the kit of 1X to each well, standing for 30s, then inverting the magnetic plate, and pouring out the liquid in the well plate; in the inverted state, the residual liquid on the surface of the orifice plate was adsorbed with a paper towel. Add 25. mu.L of universal assay buffer (Universal assay buffer) from the kit to each well; adding 25 mu L of standard substance or sample into the designated hole; add 25. mu.L of Universal assay buffer (Universal assay buffer) to the blank; the plate was sealed, incubated with shaking at 500rpm for 30min at room temperature, and allowed to stand overnight at 4 ℃. The next day, the cells were removed and incubated at 500rpm with shaking at room temperature for 30 min. Washing the plate, repeating for 4 times, and sucking out residual liquid; adding 100 mu L of antibody into each hole, and incubating for 2h at room temperature; washing the plate, repeating for 3 times, and sucking out residual liquid; to each well was added 25. mu.L of the detection antibody mixture in the 1X kit, the well plate was sealed with a new sealing film, the 96 well plate was removed from the magnetic separation plate, and it was shaken at 500rpm in a well plate shaker at room temperature for 30 min. Washing the plate, repeating for 3 times, and sucking out residual liquid; adding 50. mu.L phycoerythrin-labeled streptavidin (SA-PE) to each well, and sealing the well plate with a new sealing film; the 96-well plate was removed from the magnetic separation plate and placed in a well plate shaker at 500rpm for 30min at room temperature. Washing the plate, repeating for 3 times, and sucking out residual liquid; add 120. mu.L of the reagent solution (reading buffer) in the kit to each well; sealing the orifice plate with a new sealing membrane; taking the 96-well plate out of the magnetic separation plate, and placing the 96-well plate in a well plate oscillator to oscillate at the room temperature of 500rpm for 5 min; the sealing membrane was gently removed and placed in a Luminex200 instrument for reading.
And fitting the standard curve by adopting a five-parameter nonlinear regression mode to calculate a concentration value.
The detection indexes of the kit comprise TNF-alpha, IL-1 beta, IL-6, IFN-gamma, IL-12p70 and MCP-1.
(5) Flow cytometry detection of mouse peripheral blood lymphocytes
Dripping 3 drops of blood into 10ml PBS (each group of animals is subjected to orbital venous plexus blood collection in the step (2), randomly selecting 8 blood samples in each group for detection), and mixing uniformly; 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.
3. Results of the experiment
(1) Pulmonary index and pulmonary index inhibition rate
TABLE 2 Effect of Lung-clearing and detoxifying particles on pulmonary index of influenza Virus-infected mice
Note: comparison with model group**P<0.01，***P<0.001。
The test results show that the lung index of the model mice infected by the H1N1/FM1 virus strain is obviously increased and has a 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 the lung-clearing and detoxifying particles is reduced, and the lung index of the 3 dose groups of the lung-clearing and detoxifying particles is significantly different from that of a model control group (P <0.001 and P < 0.01). 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 influenza-clearing granule group are both reduced, and have significant difference (P <0.01) compared with a model control group. The lung index inhibition rate of the large-dose group of lung clearing and detoxifying particles is close to that of the Tamiflu group, and the lung index inhibition rate of the medium-dose and small-dose groups of lung clearing and detoxifying particles is equivalent to that of the honeysuckle antipyretic particle group and the golden flower antipyretic particle group.
(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 of the mouse treated by the lung-heat clearing and detoxifying particles has less inflammatory infiltration, and the improvement degree is equivalent to that of the lianhua antipyretic particles group and the golden flower antipyretic particles 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. The lung-clearing and detoxifying particles are used for treating pneumonia lesions caused by influenza virus infection.
(3) Inflammatory factor expression levels
TABLE 3 Effect of Lung-clearing and detoxifying particles on the expression level of lung cytokine in mice infected with influenza A virus strain FM 1: (
n＝6)
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:
1) the expression level of TNF-alpha in the lung tissue homogenate of the mouse of the model control group is obviously increased, and the TNF-alpha expression level has significant difference (P <0.01) 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 shows a reduced trend. TNF-alpha expression of 3 dose groups of the lung-heat clearing and detoxifying particles is in negative correlation with dose size, and has significant difference compared with a model control group (P <0.001, P <0.01, P < 0.05). The TNF-alpha expression of the Tamiflu group and the golden flower Qinggan granule group is reduced, and the significant difference (P <0.05) is compared with that of a model control group. The TNF-alpha expression level of the desmoplastic granule group is not significantly different from that of the model control group (P > 0.05).
2) The IL-1 beta expression level in the lung tissue of the mouse of the model control group is obviously increased, and the obvious difference is compared with that of the normal control group (P < 0.05). After the mice of each administration group are respectively administered with the corresponding drugs, the IL-1 beta expression level shows a decreasing trend. The large-dose and medium-dose groups of the lung-heat clearing and detoxifying granules have reduced IL-1 beta expression, and have significant difference compared with the model control group (P <0.05, P < 0.01). The expression level of IL-1 beta in the duffy group is reduced, and the significant difference is compared with that in a model control group (P < 0.05). The IL-1 beta expression levels of the Lianhua scourge-clearing granule group and the golden flower infection-clearing granule group have no significant difference (P is more than 0.05) compared with that of a model control group.
3) The IL-6 expression level in the lung tissue of the mouse of the model control group is obviously increased, and the expression level is obviously different from that of the normal control group (P <0.05, P < 0.01). After the mice of each administration group are respectively administered with the corresponding drugs, the expression level of IL-6 shows a trend of decreasing. The IL-6 expression of 3 dose groups of the lung-heat clearing and detoxifying granules is reduced, and the granules have significant difference compared with a model control group (P <0.05, P < 0.01). The IL-6 expression of the Tamiflu group is reduced, and the significant difference is compared with that of a model control group (P < 0.05). The IL-6 expression levels of the paniculate swallowwort 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 the model control group.
4) The expression level of IL-12P70 in the lung tissue of the mouse in the model control group is obviously increased, and has a significant difference compared with the normal control group (P < 0.01). After the mice in each administration group are respectively administered with the corresponding drugs, the expression level of IL-12p70 shows a trend of decreasing. The expression level of IL-12P70 in 3 dose groups of the lung-heat clearing and detoxifying granules is in negative correlation with the dose size, and the large and medium doses have significant difference compared with a model control group (P < 0.05). The expression level of IL-12P70 in the duffy group is reduced, and the significant difference is compared with that in the model control group (P < 0.05). The IL-12P70 expression levels of the Lianhua Qingwen granule group and the Jinhua Qinggan granule group are only reduced, and have no significant difference compared with the model control group (P is more than 0.05).
5) The IFN-gamma expression level in the lung tissue of the mouse in the model control group is obviously increased, and the significant difference is compared with that in the normal control group (P < 0.05). The expression level of IL-12p70 showed a tendency to decrease after mice of each administration group were administered with the corresponding drugs. The expression level of IL-12P70 in 3 dose groups of the lung-heat clearing and detoxifying granules is in negative correlation with the dose size, and the large dose group has significant difference compared with a model control group (P < 0.05). The expression levels of IL-12P70 in the duffy group, the copperleaf antipyretic granule group and the golden flower influenza-clearing granule group are all reduced, and the significant difference (P <0.05) is compared with that in the model control group.
6) The MCP-1 expression level in the lung tissue of the mouse in the model control group is obviously increased, and the MCP-1 expression level is obviously different from that of the lung tissue in the normal control group (P < 0.05). After the mice of each administration group are respectively administered with the corresponding drugs, the expression level of MCP-1 shows a decreasing trend. The lung-heat clearing and detoxifying particle large dose group has significant difference (P <0.05) compared with the model control group. The expression level of IL-12P70 in the Tamiflu group and the golden flower Qinggan granule group is significantly different from that in the model control group (P < 0.05). The group with the honeysuckle antipyretic granules only has a reduced trend and has no significant difference compared with the model control group (P > 0.05).
The granule has effects of inhibiting inflammatory factor expression and resisting influenza virus. In the tested indexes, the inhibition effect of a large-dose group is equivalent to that of tamiflu and is superior to that of honeysuckle plague clearing granules and golden flower cold clearing granules; the inhibiting effect of the medium-dose group is superior to that of the flower plague-clearing granules and the golden flower cold-clearing granules.
(6) Mouse peripheral blood lymphocytes
TABLE 4 Effect of Lung-clearing and detoxifying particles on the percentage of peripheral blood lymphocytes from mice infected with influenza Virus
Note: comparison with model group**P<0.01，*P<0.05。
The test results show that the material has the characteristics of,
1) infecting mice with FM1 virus strain, and controlling mice with peripheral blood CD4+The proportion of T cells is increased, and the T cells have significant difference (P) compared with a normal control group<0.001). After the corresponding drugs were administered to each administration group, the peripheral blood CD4 was obtained+The T cell proportion shows a reduced trend. 3 dose groups of lung-heat clearing and detoxifying granules CD4+The T cell ratio has significant difference compared with the model control group (P)<0.01). The Tamiflu group, the copperleaf antipyretic group and the golden flower antipyretic group have significant differences (P) compared with the model control group<0.001)。
2) CD8 in peripheral blood of model control group mouse+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. 3 dose groups of lung-heat clearing and detoxifying granules CD8+The T cell ratio has a rising trend, wherein the large dose group has a significant difference (P) compared with the model control group<0.05). JINHUAQINGGAN granule group CD8+The proportion of T cells is increased, and the T cells have significant difference (P) compared with the model control group<0.01). Lianhua scourge clearing granule group CD8+The proportion of T cells is increased, and no significant difference is generated compared with a model control group (P)>0.05). Tamiflu group CD8+T cell proportion, no significant difference compared with model control group (P)>0.05)。
3) The proportion of CD45R B lymphocytes in peripheral blood of mice in a model control group is reduced, and the mice have a significant difference compared with a normal control group (P < 0.05). 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 the lung-heat clearing and detoxifying particles is dose-dependent, and has significant difference compared with a model control group (P <0.05, 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 not significantly different from that of the model control group (P > 0.05).
The lung-heat clearing and detoxifying particles have the function of immunoregulation and have dose-effect relationship; the action degree of the large-dose group is superior to that of the lianhua antipyretic granules and the golden flower antipyretic granules. The effect degree of the medium-dose group is equivalent to that of the lianhua antipyretic granules and the golden flower antipyretic granules.
Experimental example 3 Effect of Lung-clearing and detoxifying particles on intestinal flora structure of influenza Virus-infected mice
The microbial diversity is based on an IlluminaHiSeq sequencing platform, and a small fragment library is constructed for sequencing by using a double-ended sequencing (Paired-End) method. Species composition of the sample can be revealed by performing splicing filtration on Reads, OTUs (operational taxonomicunits) clustering, and performing species annotation and abundance analysis; further alpha diversity analysis (AlphaDiversity), beta diversity analysis (BetaDiversity) and significant species diversity analysis were performed to mine the differences between samples.
1. Experimental methods
Fecal samples from each group of mice (feces from each group of animals in Experimental example 2 that were stored at-80 ℃ after being treated in Experimental procedure (2)) were collected and subjected to microbial diversity analysis by Beijing Baimaike Biotech Co.
In this project, the analysis of microbial diversity mainly comprises the following operations: 1) filtering and splicing double ends of an original sequencing sequence to obtain an optimized sequence (Tags); 2) clustering the optimized sequences, dividing the OTUs, and obtaining species classification according to the sequence composition of the OTUs; 3) based on the OTU analysis result, performing taxonomic analysis on the samples at each classification level to obtain community structure diagrams of the samples at phyla, class, order, family, genus and taxonomic level; 4) the species diversity in a single sample is researched through Alpha diversity analysis, Ace, Chao1, Shannon and Simpson indexes of each sample under the 97% similarity level are counted, and a sample dilution curve and a grade abundance curve are drawn; 5) the differences in species diversity (colony composition and structure) of different samples were compared by Beta diversity analysis. Obtaining sample NMDS analysis, sample PCA, PCoA images (with grouping information), box line images based on various distances and the like under corresponding distances according to the distance matrix; 6) the use of LEfSe software can search for a Biomarker 7 with statistical difference among different groups, and through 16S functional gene prediction analysis, the gene function prediction is carried out on the sample and the functional gene abundance is calculated.
2. Main test results
(1) Effect on the relative abundance of the intestinal flora
TABLE 5 relative abundance of fecal Enterobacteriaceae (M%) in each group of mice
Wherein, -represents relative abundance values arranged outside of the 1 st to 3 rd names.
At the phylum level, the abundance ratio of the firmicutes and bacteroidetes of the model control group was significantly reduced compared to the normal control group. The firmicutes and bacteroidetes are reported in the literature to be closely related to obesity. The intestinal firmicutes are more than bacteroidetes, which results in more effective absorption of heat from food and easy weight gain. The experimental result that the body weight of the model control group mice is obviously reduced after the model control group mice are infected with the virus supposes that the reduction of the abundance of the firmicutes bacteria can further cause the reduction of the energy absorbed by the influenza mice by food, and aggravates the severity of the influenza. The lung-clearing and detoxifying particle group is at phylum level, the abundance ratio of the firmicutes and the bacteroidetes is reduced to be close to 1, and the growth of the two dominant bacteria is still in a balanced state.
The first dominant bacterial strain of the granule group for clearing lung and detoxifying is verrucomicrobia. Subsequent experimental results show that the lung-clearing detoxifying particles are singly enriched in the genus Exmenon in the phylum Micromyces verrucosus. It is known that akkermansia is inversely related to many health problems. That is, the less Ackermann bacteria, the more easily inflammation and type 2 diabetes occur. Conversely, if the more icorman bacteria are in the gut, the less inflammation and type 2 diabetes the animal will experience. The more abundant people of Effman's fungus, also the better to the immunotherapy of cancer. Recent studies report that Ackermans bacteria can also promote the barrier function of the intestinal tract and relieve the symptoms of experimental colitis in mice. It can be seen that the bacterium should have a high potential as part of a probiotic.
TABLE 6 relative abundance of fecal Enterobacter (%)
Wherein, -represents relative abundance values arranged outside of the 1 st to 10 th names.
At the genus level, infection with influenza virus increases the abundance of bacteroides and ruminococcus in mice, and the segmented filamentous bacteria and lactobacillus in probiotics are reduced, causing serious dysbacteriosis. The use of the lung-clearing and detoxifying particles improves the disorder state caused by influenza. For influenza mice, the lung clearing and detoxifying particles can increase the number of probiotics including Acermann species bacterium and arthroepidium bacterium.
The experimental result also shows that the lung clearing and detoxifying particles reduce the abundance of the lactobacillus, probably because the drug is specifically enriched with the Acermann species, so that the Acermann species becomes the first dominant bacteria in the intestinal tract, and the growth of the lactobacillus is weakened to a certain extent. However, the use of the lung clearing and detoxifying particles leads to the total abundance ratio of the probiotics including the Acermann species and the lactobacillus to be obviously improved. The experimental results indicate that the lung-clearing and detoxifying particles can play a role in resisting influenza viruses by adjusting the level of intestinal differential flora and enriching the quantity of probiotics. This effect may be closely related to the directed enrichment of akkermansia.
(3) Significance analysis of differences between groups
The normal control group, the model control group and the lung-heat clearing and detoxifying granule group are compared with each other, and the normal control group is significantly enriched in Lactobacillus (Lactobacillus), and bacillus (Bacillus), and has significant difference (LDA >4) with other two groups. The model control group uncultured _ bacterium _ f _ mycobacteriaceae, prevotellaceae (prevotellaceae), Bacteroides (Alloprevotella), Bacteroides (Bacteroides), Ruminococcaceae (Ruminococcaceae) were significantly enriched and significantly different from the other two groups (LDA > 4). Particle group for clearing lung heat and removing toxicity wart microbials (Verrucomicrobia), Verrucomicrobiae (Verrucomicrobiae), verrucomicriales (Verrucomicrobiales), akkermanaceae (akkermansiae), Akkermansia (Akkermansia) were significantly enriched and significantly different compared to the other two groups (LDA > 4).
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 (14)
1. The application of a Chinese medicinal composition in preparing anti-inflammatory drugs or immunoregulation drugs is characterized in that the Chinese medicinal composition is composed of the following raw material medicaments of 7-12 parts of ephedra herb, 28-33 parts of gypsum, 8-13 parts of radix bupleuri, 13-18 parts of radix scutellariae, 13-18 parts of sweet wormwood herb, 8-13 parts of rhizoma atractylodis, 28-33 parts of verbena, 13-18 parts of wrinkled gianthyssop herb and 8-13 parts of liquorice; by immunomodulation is meant the prevention and/or treatment of immune dysfunction and inflammation caused by influenza a virus infection.
2. The application of claim 1, wherein the traditional Chinese medicine composition comprises, by weight, 9 parts of ephedra herb, 30 parts of gypsum, 10 parts of bupleurum, 15 parts of scutellaria baicalensis, 15 parts of artemisia apiacea, 10 parts of rhizoma atractylodis, 30 parts of verbena, 15 parts of agastache rugosus and 10 parts of liquorice;
or 7 parts of ephedra, 33 parts of gypsum, 8 parts of radix bupleuri, 13 parts of radix scutellariae, 13 parts of sweet wormwood, 13 parts of rhizoma atractylodis, 33 parts of verbena, 13 parts of agastache rugosus and 8 parts of liquorice;
or 12 parts of ephedra, 28 parts of gypsum, 13 parts of radix bupleuri, 18 parts of scutellaria baicalensis, 18 parts of sweet wormwood, 8 parts of rhizoma atractylodis, 28 parts of verbena, 15 parts of agastache rugosus and 13 parts of liquorice.
3. The use of claim 1 or 2, wherein the Chinese medicinal composition is prepared by weighing ephedra, gypsum, radix bupleuri, scutellaria baicalensis, artemisia apiacea, rhizoma atractylodis, verbena, agastache rugosus and liquorice according to selected parts by weight, mixing, 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 medicament is prepared from the Chinese medicinal composition with or without pharmaceutically acceptable adjuvants according to conventional preparation method.
6. The use of claim 5, wherein the medicament is a tablet, capsule, powder, pill, granule, oral liquid, syrup, or electuary.
7. Use according to claim 5, wherein the medicament is a cocktail.
8. The use according to claim 5, wherein the pharmaceutically acceptable excipients are selected from at least one of pharmaceutically acceptable solvents, solubilizers, emulsifiers, colorants, binders, disintegrants, fillers, wetting agents, tonicity adjusting agents, stabilizers, flavoring agents, preservatives, suspending agents, coating materials, anti-adherents, integration agents, penetration enhancers, pH adjusting agents, buffers, plasticizers, surfactants, thickeners, encapsulation agents, humectants, flocculants and deflocculants, filter aids, release retardants, and polymeric matrix materials.
9. The use according to claim 5, wherein the process for the preparation of the medicament comprises the steps of:
steam distillation step: weighing sweet wormwood, rhizoma atractylodis and agastache rugosus according to the selected weight parts, mixing, carrying out steam distillation extraction, and respectively collecting volatile oil and water extract;
water decoction: weighing ephedra, gypsum, radix bupleuri, scutellaria baicalensis, verbena 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 mixing with above volatile oil to obtain the final product.
10. Use according to claim 1 or 2, wherein the immune dysfunction is cytokine release syndrome, lymphohistiocytosis with haemophagocytic cells or cytokine storm syndrome.
11. The use of claim 1 or 2, wherein said immunomodulation is at least one of modulating an abnormal number of immune cells, modulating an imbalance of pro-inflammatory and anti-inflammatory factors.
12. The use according to claim 11, wherein said immune cells are selected from the group consisting of CD4+T cell, CD8+One or more of T cells and CD45R B lymphocytes;
the proinflammatory factors are selected from one or more of TNF-alpha, IL-1 beta, IL-6, IL-12p70, IFN-gamma and MCP-1;
the inflammation-inhibiting factor is selected from one or more of IL-4 and IL-10.
13. Use according to claim 1, wherein said inflammation is selected from respiratory tract inflammation, acute lung injury, acute respiratory distress syndrome or cytokine release syndrome.
14. Use according to claim 13, wherein the inflammation of the respiratory tract is selected from pharyngitis, laryngitis, tracheitis, bronchitis and/or pneumonia.
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