CN106924402B - Chinese medicinal compound preparation for preventing and treating influenza and viral pneumonia - Google Patents
Chinese medicinal compound preparation for preventing and treating influenza and viral pneumonia Download PDFInfo
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Abstract
The invention belongs to the field of traditional Chinese medicines, and particularly relates to a traditional Chinese medicine compound preparation for preventing and treating influenza and viral pneumonia, which is prepared from eight traditional Chinese medicinal materials including notopterygium root, rhizoma atractylodis, dandelion, scutellaria baicalensis, raw astragalus mongholicus, divaricate saposhnikovia root, houttuynia cordata and trichosanthes kirilowii. The compound preparation can be further used for preparing medicaments for preventing viral pneumonia induced by other viruses.
Description
Technical Field
The invention belongs to the field of traditional Chinese medicines, relates to a traditional Chinese medicine compound, in particular to a traditional Chinese medicine compound preparation for preventing and treating influenza and viral pneumonia, and particularly relates to a compound medicine for treating virus-induced acute lung injury.
Background
Viral pneumonia (viral pneumoconia) is a lung inflammation caused by a variety of viral infections, usually by the downward spread of upper respiratory tract viral infections, reported by studies. Research shows that viruses causing pneumonia are common to influenza viruses, and also comprise parainfluenza viruses, and cytomegalovirus, adenovirus, rhinovirus, coronavirus, certain enterovirus and the like can also cause viral pneumonia. Influenza a viruses are important pathogens causing respiratory diseases in humans and birds. The invasion of influenza virus into the bronchiole epithelium causes bronchiolitis, and the infection reaches the pulmonary interstitium and alveoli to cause pneumonia; on one hand, the influenza viruses are replicated in respiratory tract epithelial cells and vascular endothelial cells to damage lung tissues and destroy gas exchange barriers; on the other hand, a host immune mechanism is over-activated and out of control by releasing a large amount of cytokines through macrophages, segmented neutrophils, lymphocytes, endothelial cells and the like, resulting in acute lung injury (grand, wuqi, shore peak. international journal of respiration 2011, 14: 1094-. Research reports that in the process of body anti-virus infection immunity, nonspecific immunity mainly comprising macrophages, interferon and NK cells is acted in the early stage, and specific cellular immunity and humoral immunity are acted in the later stage; during influenza infection, monocytes/macrophages are activated to produce multiple cytokines that play important roles in the regulation of the immune and inflammatory responses of the body in the form of a network (mareli, labyrinthine, plum ghost, guan.
Influenza a H1N1 is an acute respiratory infectious disease caused by Influenza A Virus (IAV), which is a common pathogenic microorganism of viral pneumonia, and investigations have shown that people are generally susceptible to Influenza a H1N1 virus, usually manifested as flu-like symptoms, and few cases have rapidly progressed, and respiratory failure occurs, and serious patients can cause death. In severe clinical influenza, the facial Qian scales of traditional Chinese medicine think that the pathogenic factors of pestilence invade the human body from the mouth and nose, the disease is strong and violent, and the characteristics of rapid transmission and transformation are particularly emphasized that attacking the pathogenic factors does not hurt the body; when the cold-warm herbs are used together in the clinical syndrome, the body resistance is strengthened to achieve the pathogenic factor, the capability of the body to resist the pathogenic factor outside the defense system is improved, and then the spleen and stomach qi of the human body is protected; the application of the method is repeatedly practiced in the long-term clinical process, and the Qiang-Ying prescription is a traditional Chinese medicine compound used for preparing cold-warm medicines when special effects are obtained.
The main pathological change of influenza virus-induced viral pneumonia is gas exchange disorder at the alveolar level, and Acute Lung Injury (ALI) caused by virus infection can cause respiratory distress and severe hypoxemia of patients. Research shows that monocyte infiltration and pulmonary alveolar edema are the pathological bases for forming pulmonary capillary permeability increase and pulmonary edema, and viral infection induced lung injury is clinically manifested as dyspnea, which can seriously cause acute respiratory distress syndrome and even death. The invention proves that the Qiang-English decoction has a protective effect on mice with acute lung injury induced by influenza A virus.
The study at home and abroad is comprehensively carried out, and the drug effect of the Qiang Ying decoction on treating virus-induced acute lung injury and the drug research thereof are not reported. The inventor intends to provide a new medicinal application of the traditional Chinese medicine Qiang Ying decoction in preparing medicines for preventing and treating influenza A and viral pneumonia.
Disclosure of Invention
The invention aims to provide a traditional Chinese medicine compound preparation for preventing and treating influenza and viral pneumonia, in particular to a traditional Chinese medicine compound preparation for preventing and treating influenza A and viral pneumonia, and especially relates to a compound medicine for treating virus-induced acute lung injury.
The compound preparation is prepared from eight Chinese medicinal materials of notopterygium root, rhizoma atractylodis, dandelion, scutellaria baicalensis, raw astragalus mongholicus, divaricate saposhnikovia root, houttuynia cordata and trichosanthes root; wherein, the notopterygium root is monarch, and can sweat to eliminate pathogenic factors, and dispel wind and dry dampness; dandelion, scutellaria baicalensis, houttuynia cordata and trichosanthes root are used as ministers to clear away heat, toxic materials and dampness; astragalus root, ledebouriella root and atractylodes rhizome are used as adjuvant and guiding drugs to replenish qi and strengthen superficies. The whole formula can sweat to reach pathogenic factors, clear away heat and toxic materials, tonify spleen excess and defense, sweat without violent fierce, reach pathogenic factors without damaging body resistance, clear away heat and toxic materials but protect spleen and stomach, and takes the guidance of 'deficiency of spleen and stomach and exhaustion of lung qi' pointed out in Lidongyuan 'differentiation theory of internal and external injury', the astragalus and the rhizoma atractylodis are used to express pathogenic factors, and then 'land without being attacked' is arranged firstly. The compound preparation centrally embodies the treatment ideas of taking internal and external factors into consideration, treating both principal and secondary aspects, tonifying in the middle of dispersing and nourishing the interior and dispersing the interior.
In the embodiment of the invention, the Chinese medicinal materials of the compound preparation are compatible according to the following weight proportion: 5g of notopterygium root; 10g of rhizoma atractylodis; 20g of dandelion; 10g of scutellaria baicalensis; 20g of astragalus; 5g of divaricate saposhnikovia root; 10g of houttuynia cordata; 15g of trichosanthes root, and further preparing the compound preparation freeze-dried powder.
Based on the fact that the severe influenza H1N1 belongs to the syndrome of exuberant pathogenic factors and healthy qi deficiency and deficiency-excess mixed in clinical influenza, and damp-heat toxin is the pathological factor, the traditional Chinese medicine is used for treating the severe influenza, and the traditional Chinese medicine is used for self-reinforcing qi, consolidating exterior, clearing heat, drying dampness and detoxifying.
The compound preparation is a traditional Chinese medicine aqueous extract, and an integral animal model test proves that the compound preparation has the drug effects of remarkably increasing the survival rate of virus-infected mice, improving the weight, reducing the inflammatory injury of the lung, reducing the virus titer and inhibiting the expression of inflammatory cytokines, chemokines and NF kappa B inflammatory transcription factors aiming at a mouse lung acute injury model induced by an H1N1 influenza A virus strain, and can remarkably treat the influenza virus-induced acute lung injury.
Experiments prove that the compound preparation can inhibit the replication of influenza virus in mouse lung and relieve inflammatory immune injury caused by virus infection; the action mechanism of the compound for protecting the virus-induced lung injury is related to antivirus and anti-inflammation, and the lung inflammation is inhibited by reducing the permeability of lung capillary vessels, reducing lymphocyte infiltration and recruiting inflammatory proteins.
The compound preparation can be used for treating influenza A and viral pneumonia induced by the influenza A.
In the present invention, the influenza a includes, but is not limited to, strains of H1N1, H3N2, and the like.
Furthermore, the compound preparation can be used for preparing medicines for preventing influenza and viral pneumonia.
In the invention, the viral pneumonia, including but not limited to pneumonia induced by influenza A virus, can also be used for viral pneumonia induced by other viruses.
Drawings
FIG. 1 shows the effect of the compound preparation of the present invention on the survival protection and body weight of H1N1 virus infection.
FIG. 2 shows the effect of the compound preparation of the present invention on the lung pathological morphology of H1N1 virus-infected mice;
wherein, A: a normal group; b: ribavirin group; c: the compound preparation group; d: disassembling the square group; e: and (4) model groups.
FIG. 3 shows the effect of the compound preparation soup of the present invention on the index of H1N1 virus infected mice.
FIG. 4 shows the effect of the compound preparation of the present invention on the pulmonary hemagglutination titer of H1N1 virus-infected mice.
FIG. 5 shows the effect of the compound preparation of the present invention on H1N1 virus infected mice homogenate TNF-alpha.
FIG. 6 shows the effect of the compound preparation of the present invention on H1N1 virus infected mice homogenate IL-6.
FIG. 7 shows the effect of the compound preparation of the invention on H1N1 virus infected mouse lung homogenate CCL 5.
FIG. 8 shows that the compound preparation soup of the present invention has the inhibition effect on the NF kappa B expression of the lung of the mouse infected by the H1N1 virus,
wherein, A: a normal group; b: ribavirin group; c: the compound preparation group; d: disassembling the square group; e: and (4) model groups.
Detailed Description
Example 1 preparation of a compound formulation lyophilized powder of the present invention:
taking 5g of traditional Chinese medicine notopterygium root; 10g of rhizoma atractylodis; 20g of dandelion; 10g of scutellaria baicalensis; 20g of astragalus; 5g of divaricate saposhnikovia root; 10g of houttuynia cordata; 15g of trichosanthes root, which is prepared into traditional Chinese medicine decoction pieces according to a conventional method;
placing the Chinese medicinal decoction pieces in an electric decocting pot, adding mineral water 5 times the amount of the medicinal materials, soaking for 1 hr, boiling with strong fire for 30min, and filtering with gauze. Decocting the residue with 6 times of mineral water, boiling for 20min, and filtering; mixing the two filtrates, vacuum concentrating by rotary evaporator (0.095MPa, 50 deg.C), concentrating, and evaporating to obtain extract; and putting the extract into a freeze dryer (133Pa and 25 ℃), preparing freeze-dried powder with the extraction rate of 24 percent, subpackaging, and freezing and storing at the temperature of minus 80 ℃.
Example 2 animal experiments
BALB/C mice 60 (16-18g) were randomized into 5 groups by body weight (A, B, C, D, E): group A is a normal control group, group B is an H1N1 virus model group, group C is a compound preparation group (40mg/kg), group D is a prescription-splitting control group (mg/kg), group E is 100mg/kg of ribavirin, and 12 groups are selected; all animals except group A were anesthetized with ether and infected by nasal drip with 10LD50H1N1 virus solution 30 μ L, group A nose drop infection 1640 culture solution 30 μ L as control; C. d, E group was administered 2 hours after H1N1 by gavage with doses of Qiangying Fang and ribavirin of 100 mg/kg; and simultaneously, the group A and the group B are administered with 0.5 percent CMC by intragastric administration as normal and virus control, the administration is carried out once a day for seven days continuously, the administration is stopped for 14 days, the weight, the survival number and the death number of animals are recorded every day, and the life protection rate and the weight change of the drug to the severe influenza infected mice are calculated.
Example 3 animal experiments
BA40 LB/C mice (16-18g), experimental groups, infection dose and dosing schedule as in example 2; all animals except group A were anesthetized with ether and infected by nasal drip with 10LD5030 mu L of H1N1 virus solution, wherein the doses of Qiangying prescription, Chuanfang prescription and ribavirin are 100mg/kg respectively after C, D, E groups of patients are infected with H1N1 and then are administered by intragastric administration 2 hours; simultaneously, the group A and the group B are administrated by 0.5% CMC by intragastric administration once a day for four days continuously, after the H1N1 virus attacks for 96 hours, the weight is weighed, eyeballs are picked for blood sampling, and a whole lung specimen is weighed and recorded after being treated conventionally; wherein the upper right lung lobe is placed in 10% formalin; rinsing the middle and lower lobes of the right lung and the lower lobes of the right lung with normal saline, and storing at-80 deg.C; and (3) after freezing the preserved lung tissues, using precooled PBS, homogenizing in an ice bath by using a homogenizer, harvesting homogenate, centrifuging, collecting supernatant, subpackaging, and storing at-80 ℃ for detecting indexes such as inflammatory cytokines and chemotactic factors.
Example 4 survival protection test of severe influenza virus H1N 1A infected mice with this compound preparation:
a BALB/C mouse is selected, an influenza virus H1N 1A is used for nasal drip infection, an influenza virus induced severe lung injury model is established, administration is carried out for 7 days, medicine stopping and observation are carried out for 7 days, and the life protection effect of the compound preparation on a severe influenza virus infected mouse in a 14-day observation period is observed.
Example 5 treatment of influenza A H1N1 to induce acute Lung injury
A BALB/C mouse is selected, influenza A virus H1N1 is dripped into the nose for infection, an acute lung injury model induced by the influenza virus is established, the compound preparation is administrated by gastric gavage, the lung inflammatory injury degree of animals, and indexes such as inflammatory cytokine, chemotactic factor, NF kappa B protein expression and the like are observed after 96 hours, and the result proves that the compound preparation has obvious protective effect on the acute lung injury of the mouse induced by the influenza A virus H1N1 after the compound preparation is orally taken.
Example 6 method for detecting anti-inflammatory related research indexes of virus-induced acute lung injury in mice
1) Index of lung
Weighing the animals on the fourth day of virus infection, weighing the lungs in the conventional treatment, and taking the lung weight as the lung wet weight; calculating the lung index, wherein the lung index is lung wet weight (mg)/body weight (g), and the experimental result shows that compared with a model group, the compound preparation can obviously inhibit the increase of the lung index of the mouse (P < 0.01);
2) determination of pulmonary homogenate hemagglutination potency
According to the principle that hemagglutinin HA on the influenza virus can perform agglutination reaction with chicken red blood cells, the strength of virus virulence is evaluated; taking lung homogenate, centrifuging at high speed to obtain supernatant, mixing the homogenate obtained by equal-time serial dilution with 1% of chicken erythrocyte in a U-shaped 96-well plate, standing and observing, and recording the dilution at which the chicken erythrocyte agglutinates, namely the hemagglutination titer of the lung tissue homogenate, wherein the experimental result shows that compared with a model group, 20mg/kg and 40mg/kg of the compound preparation can remarkably inhibit the titer rise of hemagglutinin in virus-infected mouse lungs (P <0.05, P <0.01), which indicates that the compound preparation can effectively inhibit the replication of virus in mouse lungs;
3) assay of TNF-alpha, IL-6, CCL5 in Lung homogenate
Homogenizing mouse lung, high speed centrifuging to obtain supernatant, packaging, and freezing. The ELISA method is applied, the homogenate supernatant is determined according to the specifications of TNF-alpha, IL-6 and CCL5 kit, and the result shows that compared with a model group, the compound preparation can obviously reduce the increase of the levels of proinflammatory factor/chemokine TNF-alpha, IL-6 and CCL5 in the lung of a mouse (P is less than 0.05);
4) detection of lung tissue NFkB protein expression
Placing the cut white lung tissue slices with the thickness of 4um on a slide frame, placing the slide frame on an oven, baking the cut white lung tissue slices for 30 minutes, dewaxing the cut white lung tissue slices, adding water, soaking the slide frame in 0.01M PBS for 5 minutes, then placing the slide frame in EDTA antigen repairing solution PH8.0 heated in water bath at the temperature of 97 ℃ for antigen repairing for 12 minutes, soaking the slide frame placed at room temperature in PBS for 5 minutes, then placing the slide frame in PBS for soaking for 5 minutes, and then placing the slide frame in 3% H2O2For 10 minutes in methanol solution; soaking the slide holder in PBS for 5 min, repeating for 3 times, wiping off excessive water on the slide with absorbent paper, adding 100ul serum, incubating at 37 deg.C for 30min, wiping off excessive serum, adding phosphorylated NFkB primary antibody, incubating at 4 deg.C overnight, rewarming at room temperature for 45 min, and soaking in PBSAfter soaking for 5 minutes and repeating for 3 times, HRP-labeled secondary antibody was added dropwise and incubated at 37 ℃ for 30 minutes. Washing with PBS for 5 min, repeating for 3 times, dropwise adding DAB for color development, re-staining with hematoxylin for 30-45 s, washing, differentiating with 0.5-1% hydrochloric acid alcohol for 1-2 s, rapidly washing in running water, and placing slide racks in 70% alcohol, 80% alcohol, 90% alcohol, and 100% alcohol respectively; xylene, xylene; the neutral gum sealing piece is used, and the observation result under the mirror shows that the influenza A virus infection can cause a large amount of p-NFkB expression, which indicates that the oral administration of the compound preparation can obviously inhibit the expression of inflammatory transcription factors.
The experimental result shows that the compound preparation has obvious treatment effect on acute lung injury induced by influenza virus H1N 1; can significantly improve the survival rate of mice infected by severe influenza virus, reduce the pulmonary index (the wet weight/body weight ratio of lung tissues) and the titer of hemagglutinin, inhibit the release of cytokines and chemokines (including TNF-alpha, IL-6 and CCL5) in the lung, and inhibit the expression of inflammatory nuclear transcription factor NF kappa B.
Claims (2)
1. The Chinese medicinal compound preparation for preventing and treating influenza and viral pneumonia is characterized by comprising the following Chinese medicinal materials in part by weight: 5g of notopterygium root; 10g of rhizoma atractylodis; 20g of dandelion; 10g of scutellaria baicalensis; 20g of astragalus; 5g of divaricate saposhnikovia root; 10g of houttuynia cordata; 15g of radix trichosanthis;
the influenza is influenza A caused by H1N1 virus, and the viral pneumonia is pneumonia induced by the influenza A virus H1N 1.
2. The compound Chinese medicinal preparation for preventing and treating influenza and viral pneumonia according to claim 1, wherein the compound preparation is prepared into lyophilized powder.
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| 芪众颗粒预防老年人上呼吸道感染的临床研究;苏中昊等;《广州中医药大学学报》;20100131;第27卷(第1期);第1-5、9页 * |
| 顾植山对当前甲型H1N1流感疫病防治的几点建议;顾植山;《浙江中医药大学学报》;20090531;第33卷(第3期);第297-299页，尤其是第298页3.1节 * |
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