WO2012139521A1 - 一种防治甲型流感的中药组合物及其制备方法和用途 - Google Patents

一种防治甲型流感的中药组合物及其制备方法和用途 Download PDF

Info

Publication number
WO2012139521A1
WO2012139521A1 PCT/CN2012/073992 CN2012073992W WO2012139521A1 WO 2012139521 A1 WO2012139521 A1 WO 2012139521A1 CN 2012073992 W CN2012073992 W CN 2012073992W WO 2012139521 A1 WO2012139521 A1 WO 2012139521A1
Authority
WO
WIPO (PCT)
Prior art keywords
virus
traditional chinese
influenza
chinese medicine
chinese medicinal
Prior art date
Application number
PCT/CN2012/073992
Other languages
English (en)
French (fr)
Inventor
汪鲲
Original Assignee
北京天福莱生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 北京天福莱生物科技有限公司 filed Critical 北京天福莱生物科技有限公司
Publication of WO2012139521A1 publication Critical patent/WO2012139521A1/zh

Links

Classifications

    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/08Solutions
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/11Pteridophyta or Filicophyta (ferns)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/28Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/28Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • A61K36/288Taraxacum (dandelion)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/68Plantaginaceae (Plantain Family)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/73Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/78Saururaceae (Lizard's-tail family)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/14Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12Antivirals
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12Antivirals
    • A61P31/14Antivirals for RNA viruses
    • A61P31/16Antivirals for RNA viruses for influenza or rhinoviruses

Definitions

  • the present invention relates to a traditional Chinese medicine composition for preventing and treating influenza A and a preparation method thereof, and to the use of the composition for preventing influenza A.
  • influenza viruses There are three types of influenza viruses: type A (type A). Influenza virus infects mammals and birds; type B (type B) influenza virus only infects humans, and disease is usually milder than type A virus; type C (type C) flu The virus only infects humans and does not cause serious illness.
  • type A type A
  • influenza virus infects mammals and birds
  • type B type B
  • influenza virus only infects humans, and disease is usually milder than type A virus
  • type C type C flu The virus only infects humans and does not cause serious illness.
  • type A has the strongest attack power because the type A virus is densely covered with two kinds of weapons, and they can proliferate rapidly after the virus infects the organism.
  • This is hemagglutinin (H) and neuraminidase ( N) Two proteins. Among them, H has 15 subtypes and N has 9 subtypes. The combination of the two is different, and the toxicity and spread speed of the virus are also different. In theory, there are 135 types of type A viruses.
  • a wide variety of type A viruses can be transmitted from wild animals to livestock and poultry, and spread widely in chickens, ducks, and pigs. If the virus carried by these livestock and poultry is genetically altered, it is obtained in the population. The ability to transmit can cause great harm to human beings.
  • influenza viruses found in humans are only H 1 , H 2 , H 3 . Therefore, humans have been targeting these three viruses for prevention.
  • the H 5 type of avian influenza virus which has been popular among chickens in winter and spring, is a new face in type A. In 1979, humans infected with H 5 N 1 avian influenza virus died in Hong Kong, Vietnam and Thailand. Case. In addition, people have also discovered H 7 and H 9 viruses. Influenza A virus, including hundreds of different subtypes of influenza virus. The different naming of these subtypes comes from the difference between H and N.
  • H and N are two major types of proteins on the surface of nail-type influenza viruses.
  • H is Hemagglutinin, which acts like a key to help the virus open the host cell;
  • N is Neuraminidase, which destroys the receptors of cells and allows the virus to spread freely within the host.
  • influenza A virus can be arranged by 15 H-types and 9 N-types, such as H1N1 and H5N1. Even the same type A influenza virus may have a large difference in the spreadability and mortality of the virus due to changes in the gene sequence.
  • the H1N1 virus caused the 1918 Spanish flu. After 1918, H1N1 evolved separately in the crowd and the herd, which can be called human H1N1 and pig H1N1, respectively.
  • the new influenza A (H1N1) virus is most closely related to the pig H1N1 virus.
  • the H1N1 virus belongs to the Orthomyxoviridae family, Influenza virus A, and its genetic material is RNA.
  • Typical virus particles are spherical and have a diameter of 80 ⁇ ! ⁇ 120 nm, with a capsule.
  • There are many radial glycoproteins on the capsule namely hemagglutinin HA, neuraminidase NA and M2 protein.
  • the virion is a nucleocapsid with a helical symmetry and a diameter of 10 nm.
  • the H1N1 virus is a single-stranded negative-strand RNA virus, and the genome is about 13.6 kb. It consists of 8 separate segments of varying sizes.
  • influenza virus serotypes can be formed between different subtypes, the serotypes that cause human infection with swine influenza virus are mainly H1N1, H1N2 and 2.
  • Influenza A H1N1 is an influenza A virus carrying a H1N1 subtype swine influenza virus strain containing ribonucleic acid gene fragments of three influenza viruses, avian influenza, swine influenza and human influenza. It also has Asian swine flu and African pigs. Influenza virus characteristics. In March 2009, people in Japan and the United States were infected with influenza A (H1N1) virus. The clinical early symptoms after infection were similar to those of influenza. They had fever, cough, fatigue, loss of appetite, etc., and symptoms such as diarrhea and vomiting may occur. A small number of cases are severely ill and progress rapidly. Viral pneumonia can occur, combined with respiratory failure and multiple organ dysfunction. In severe cases, death can occur.
  • Avian influenza is a bird (poultry and wildfowl) infection caused by the Avian influenza virus.
  • Avian influenza virus infection can manifest as mild respiratory symptoms, gastrointestinal symptoms, and low mortality; or more severe systemic, hemorrhagic, septic symptoms, and higher mortality. This difference in symptoms is mainly determined by the toxicity of avian flu.
  • avian influenza According to the pathogenicity of avian influenza, avian influenza can be divided into highly pathogenic avian influenza, low pathogenic avian influenza and non-pathogenic avian influenza. Recently, avian influenza caused by H5N1 serotype at home and abroad is called high pathogenic avian influenza, and the morbidity and mortality are high and the damage is huge.
  • Avian influenza virus belongs to the genus of influenza A virus of the Orthomyxoviridae family.
  • the virions are 80 to 120 nm in diameter and are spherical, polymorphic or filamentous.
  • the nucleocapsid of the virus is helically symmetric.
  • the genome of the virus consists of 8 negative-stranded single-stranded RNAs.
  • the virus has a capsule with a layer of 12 ⁇ 14 nm.
  • the surface of the two different shapes is hemagglutinin (HA, rod-like, composed of a trimer of hemagglutinin molecules, agglutinating to red blood cells).
  • the virus is capable of agglutinating red blood cells in a variety of mammals and birds.
  • the virus can grow in chicken embryos and reach higher titers with lysed hemagglutinin.
  • the virus can also grow on chicken embryo fibroblasts (CEF). Most strains form plaques in CEF culture.
  • the growth of the virus can be determined by a blood cell adsorption assay.
  • H2 N 2 epidemic of avian influenza virus in Mexico in 1592 started to be less pathogenic and later pathogenic. Gradually enhanced, it was not until 1959 that the similarities and differences between H1N1 flu, avian flu and common flu were controlled. Showing symptoms. Often within 7 days. Death of influenza A (H1N1) can kill, but death rate of bird flu death
  • the S ⁇ - - 5 rate is less than 1%. Lower. The rate is over 60%.
  • Susceptible A H1N1 flu death 1 Older people, suffering from the liver, the majority of patients in the infected population have been aged! Among the chronic diseases such as kidneys and heart, the proportion of children most likely to be susceptible to the four types of people aged 13 to 45 years old is among the young adults. 1 dyeing, and frequent contact with the flow is higher, the condition is more
  • Tamiflu and Leganqing According to the recommendations of the Ministry of Health and WHO, the preferred Western medicine for anti-A flow is Tamiflu and Leganqing.
  • the manufacturer of Tamiflu is Swiss Roche; the manufacturer of Leganqing is British GlaxoSmithKline.
  • influenza V virus mutations occur in some countries, and individual patients develop resistance to Tamiflu. Duffy has clinical side effects on adolescents.
  • An object of the present invention is to provide a traditional Chinese medicine composition for preventing and treating influenza A, which is prepared from the following raw materials of traditional Chinese medicine by weight ratio:
  • the composition is prepared from the following weight ratios of traditional Chinese medicine raw materials:
  • the above-mentioned raw materials are subjected to conventional water or alcohol extraction, and the obtained herbal extracts are concentrated and dried, and have special effects on influenza A, by means of capsules, drinking water or spraying methods (the finer the droplets are as fine as possible) or by fumigation (air humidifier)
  • the drug enters the respiratory tract of a person or animal through the respiratory tract, and the doors and windows are closed for 30-60 minutes during spraying or fumigation.
  • Another object of the present invention is to provide a method for preparing the composition of the present invention, which comprises pulverizing the above ten traditional Chinese medicine raw materials into a coarse powder, adding the extraction tank, adding 6-8 times of purified water of the raw material, and opening the vacuum.
  • Auto-converter depressurize to a negative pressure of 0. 5-1MP, and heat to boiling, keep the micro-boiling state, double-effect concentrated reflux, dynamic extraction for 2-4 hours; collect the extract and concentrate under reduced pressure to a relative density of 1. 5-1. 6; Turn off the heater and gradually return to atmospheric pressure.
  • the spray drying is carried out at a temperature of 160 ° C for 10-20 minutes, the inlet air temperature is set to 150-200 ° C, the outlet air temperature is set to 50_100 ° C, the feed flow rate is 1. 2 L / min, to After all the spraying, continue to keep the air for 5 minutes, then open the hopper to discharge the dry powder.
  • compositions of the present invention can be prepared into various dosage forms according to conventional techniques in the art.
  • the dry powder is diluted with acetic acid and adjusted to a value of 4.0, potted, sterilized, packaged, or sprayed; or diluted 100 times with purified water, if necessary, can be added with flavoring and / or preservatives, and adjusted Total amount, potting, sterilization, packaging, that is, get oral liquid; or use No. 0 capsule filling, sterilization, packaging, that is, capsules.
  • the method of the present invention is only a preferred preparation process but is not limited thereto.
  • the products prepared by the process of the invention have very good stability.
  • the traditional Chinese medicine composition of the present invention has a good curative effect on avian influenza (H5N1) and swine influenza (H1N1) which are commensurate with humans and animals, and has no side effects.
  • the traditional Chinese medicine of the invention has obvious inhibitory effects on Staphylococcus aureus, catarrhalis, influenza bacillus and pneumococci; inhibits various microorganisms, especially yeasts and molds; diuretic, analgesic, hemostasis, inhibits serous secretion , cough, promote tissue regeneration and other effects. It has a good effect on chronic bronchitis and has antitussive effect; it has a relieve effect on bronchospasm caused by histamine.
  • the traditional Chinese medicine of the invention has good curative effect on avian influenza (H5N1) and influenza A (H1N1) which are common to humans and animals, and relieves symptoms after 24-48 hours, and can be used as an air disinfectant.
  • Another object of the present invention is to provide a use of the composition of the present invention for the prevention and treatment of influenza A.
  • the influenza A is avian influenza (H5N1) and influenza A (H1N1).
  • Fig. 1 is a graph showing the inhibitory effect of the solvents of Q7 and Q7 of the present invention on influenza virus PR8 (H1N1);
  • Fig. 2 is an indirect immunofluorescence assay of the present invention for detecting the inhibitory effect of Q7 on PR8 virus (24 h after infection).
  • a Q7 10mg/ml;
  • b Q7 5mg/ml;
  • c Q7 2.5mg/ml;
  • d l.25mg/ml; e:0.625mg/ml.
  • f virus control 24 hours after virus infection, use small
  • the serum of the mouse infected with PR8 virus was used as a primary antibody, and the anti-murine FITC-labeled secondary antibody was used for indirect immunofluorescence detection. Poison.
  • Figure 3 Indirect immunofluorescence assay for the inhibitory effect of Q7 on PR8 virus (36 h after infection).
  • a Q7 10mg/ml ;
  • b Q7 5mg/ml ;
  • c Q7 2. 5mg/ ml ;
  • d 1. 25mg/ ml;
  • e 0. 625 mg/ml.
  • f virus control 24 hours after the virus infection, the serum of the PR8 virus was infected with the mouse as a primary antibody, and the virus was detected by indirect immunofluorescence using an anti-murine FITC-labeled secondary antibody.
  • Raw materials and dosage of traditional Chinese medicine 5% of bitter herbs, 10% of geranium, 5% of dandelion, 30% of houttuynia, 10% of plantain, 5% of rat grass, 10% of crane grass, 5% of eucalyptus, Eclipta 10 , phoenix grass 10%.
  • the above traditional Chinese medicine composition is weighed 100 kg, cleaned by 2-3 times of stirring, added to the extraction tank, added with purified water of 8 times the quality of the raw medicinal material, and the vacuum auto-converter is turned on, and the pressure is reduced to a negative pressure of 0. 67MP, and heated to boiling, kept in a slightly boiling state, double-effect concentrated reflux, dynamic extraction for 3 hours.
  • the collected extract was concentrated under reduced pressure to a relative density of 1.5. Turn off the heater and gradually return to atmospheric pressure.
  • the drying tower is preheated at 160 ° C for 15 minutes, the inlet air temperature is set to 170 ° C, the outlet air temperature is set to 80 ° C, and the feed flow rate is 1. 2 L / min. Continue to keep the air for 5 minutes and then open the hopper to discharge the dry powder.
  • Raw materials and dosage of traditional Chinese medicine 5% of bitter herbs, 20% of geranium, 5% of dandelion, 20% of houttuynia, 5% of plantain, 5% of rat grass, 10% of Agrimony, 15% of alfalfa, Eclipta prostrata 10%, 5% of phoenix.
  • Raw materials and dosage of traditional Chinese medicine 5% of bitter herbs, 20% of geranium, 5% of dandelion, 20% of houttuynia, 5% of plantain, 5% of rat grass, 10% of Agrimony, 15% of alfalfa, Eclipta prostrata 10%, 5% of phoenix.
  • Example 4 the drug of the present invention (hereinafter referred to as "Q7", which can be obtained from Example 2) is tested for inhibition of avian influenza virus (H5NK H9N2).
  • Q7 liquid medicine (drug of the present invention) is carried out on chicken embryos of 10 days old. Inhibition test for avian influenza virus (AIV).
  • the main components of the "Q7" liquid are plant polysaccharide, sunflower acetaldehyde, luteolin-glucoside, flavonoid glycosides, plant alcohol, lauric aldehyde, sunflower aldehyde, sunflower acid, etc., according to the embodiment 1 or 2 Got it.
  • the main ingredients are schizonepeta, peppermint, windproof, Bupleurum, perilla leaf, puerarin, platycodon, bitter almond, white peony, reed root and so on. Produced by Beijing Tongrentang Technology Development Co., Ltd. Pharmaceutical Factory, batch number 2110527.
  • HA price 512, toxic price 108.1EID50/0.1ml
  • AIV H5 subtype BP02 strain (chicken source), HA price 1: 256, toxic price 108.3ELD50/0. lml
  • DX strain (duck source), HA is 1: 512, the price of poison is 108.1ELD50/0. lml;
  • AIV H9 subtype and H5 subtype AIV was provided by Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences. NDVF48 strain and IBVM41 strain were purchased from China Veterinary Drug Surveillance Institute.
  • the virus is diluted with sterilized physiological saline for 10x, and the virus solution of 10-4, 10-5, 10-6, 10-7 is taken from 10-1 ⁇ 10-7, respectively.
  • the H5 subtype BP02 strain virus was diluted to 10-5 with physiological saline, and the virus content was
  • 3ELD50 I 0. lml will dilute the virus solution with 20x, 40x, 80x, 160x, 320x, respectively
  • the 640x diluted "Q7" was applied at room temperature.
  • the final concentrations of "Q7" were 40x, 80x, 160x, 320x, 640x, 1280x, respectively.
  • H5 subtype AIV strains were used in this test, and a commercially available Chinese medicine cold and heat-clearing granule was selected as a drug control.
  • the specific test method is to dilute the H5 subtype AIV BP/02 strain and the DX strain virus to 10-5, respectively, and the toxic amount is 0.13 and 103. 1ELD50, the diluted virus solution.
  • *Molecule is the number of diseased chicken embryos, and the denominator is the number of infected chicken embryos.
  • the number of eggs is the number of infected chicken embryos, and the denominator is the number of chicken embryos inoculated.
  • the laboratory-preserved PR8 (H1N1) influenza virus was diluted in a 10-fold gradient and infected into a single-layer MDCK cell ( 5 ⁇ 10 4 /well) in a 96-well plate, and the Q7 liquid was used in Opt i-MEM (containing lug /ml of TPCK-Trpsin) was used as a maintenance solution after virus infection after 100-fold dilution. After 48 hours, the proliferation of influenza virus in the maintenance solution was detected by hemagglutination. The results of the study showed that the maintenance solution containing Q7 significantly inhibited the proliferation of the virus on MDCK. The results are shown in Fig. 1. Also, take 100TCID 5 .
  • the concentration of Q7 was not significantly toxic to MDCK cells (about 12.5 mg/ml) as the starting concentration, and 2-fold gradient dilution was performed with Opti-MEM (TPCK-Trpsin containing lug/ml), and then different concentrations of Q7 pairs were determined. 100TCID 5 .
  • the inhibitory efficiency of the flu virus was determined by Q7 on 100 TCID 5 .
  • the EC50 value of the influenza virus, the value of EC50 was determined after 48 hours, and the blood coagulation price of the influenza virus in the maintenance solution containing different concentrations of Q7 was determined.
  • the EC50 value of Q7 to PR8 virus was calculated by SPSS software. 1. 8mg/ml.
  • the maintenance solution contained different concentrations of Q7, and 24 hours after the virus infection (Fig. 2) and 36 hours (Fig. 3), the serum of the PR8 virus was infected with the mouse as a primary antibody. Indirect immunofluorescence was used to detect virus by anti-mouse FITC-labeled secondary antibody, and Q7 inhibited PR8 virus.
  • Q7 was diluted 10 times and 5 times with PBS (pH 7.2), and then two groups of mice, 12 in each group. Each mouse was given 20 ul of diluted drug at intervals of 12 hours before infection (Q7 drug 5-fold dilution group), and each group was dissected 3 before the PR8 virus infection, and then the nasal lavage fluid was taken ( The nasal cavity of each mouse was washed with 1 ml of the culture medium for determination of whether or not there was an antiviral component. The experimental results showed that the nasal lavage fluid of the two groups of mice was 100 TCID 5 on MDCK cells. The PR8 virus has no inhibitory effect.
  • mice After 3 days of administration, 5LD 5 was used .
  • the mice were infected with the infected dose and the changes in body weight of the mice within 14 days after infection were recorded as shown.
  • the PR8 virus was diluted to 20 ul containing 5LD 5 .
  • the titer of the virus was then mixed with an equal volume of Q7 drug 5-fold dilution, and the mice were infected and the body weight of the mice was recorded within 14 days after infection.
  • three mice were dissected, and the lungs of each mouse were washed with 1 ml of antibiotic-containing PBS, and the lung virus titer of the mice was measured.
  • the experimental results are shown in Table 1.
  • mice are administered 3 days before infection, which can achieve a better preventive effect.
  • the mice lose weight after viral infection. Light.
  • the virus was pre-infected with the 5-fold dilution of Q7 and then infected.
  • the mice lost only a slight loss of body weight, and the body weight quickly began to recover, indicating that Q7 can inhibit the invasion of PR8 (H1N1) virus at a relatively high concentration. .
  • mice of different modes of administration were at 5LD 5 .

Landscapes

  • Health & Medical Sciences (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Botany (AREA)
  • Microbiology (AREA)
  • Medical Informatics (AREA)
  • Biotechnology (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Mycology (AREA)
  • Virology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Pulmonology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicinal Preparation (AREA)

Description

一种防治甲型流感的中药组合物及其制备方法和用途 技术领域
本发明涉及用于防治甲型流感的中药组合物及其制备方法, 也涉及该组 合物在防治甲型流感中的应用。
背景技术
流感病毒有三种类型: 甲型 (A型) 流感病毒感染哺乳动物以及鸟类; 乙型(B型)流感病毒只感染人类, 疾病的产生通常较甲型病毒温和; 丙型 ( C型) 流感病毒只感染人类, 并不会引起严重的疾病。
人感染流感病毒之后, 体内产生抗体, 抗体有阻碍病毒活动、 保护身体 不受侵害的作用。 经过发烧、 浑身酸痛、 出汗等过程, 大多数流感患者可以 自愈, 但有其他疾病的患者, 由于自身免疫力差, 病情会恶化。 每个流感流 行季节大约会有 1万人因流感死亡。
在 3种流感病毒中, A型攻击力最强, 原因是 A型病毒表面密布两种武 器, 在病毒感染生物后它们能够迅速增殖, 这就是血细胞凝集素(H )和神 经氨酸苷酶 (N ) 两种蛋白质。 其中, H有 1 5个亚型, N有 9个亚型。 二 者组合不同, 病毒的毒性和传播速度也不相同。 从理论上说, A型病毒可以 有 1 3 5种类型。
种类多样的 A型病毒可以从野生动物传给家畜家禽等, 在鸡、 鸭、 猪等 身上广泛传播。 这些家畜家禽携带的病毒如果发生基因变异, 获得在人群中 传染的能力, 会对人类造成巨大危害。
长时间以来, 在人身上发现的流感病毒只有 H 1、 H 2、 H 3型。 因此, 人类一直以这 3种病毒为目标进行预防。而去冬今春在鸡群中大流行的禽流 感病毒 H 5型在 A型中属于新面孔, 1 9 9 7年在香港、 越南和泰国都出现 了人类感染 H 5 N 1型禽流感病毒死亡的病例。 另外人们还发现了 H 7、 H 9型病毒。 甲型流感病毒, 包括上百种不同亚型的流感病毒。 对于这些亚型 的不同命名, 来自于 H和 N的不同。
所谓 H和 N, 是指甲型流感病毒表面的两大类蛋白质。 H是红细胞凝集 素 (Hemagglutinin) , 其作用像一把钥匙, 帮助病毒打开宿主细胞的大门; N 是神经氨酸苷酶 (Neuraminidase) , 能够破坏细胞的受体, 使病毒在宿主体 内自由传播。
根据 H和 N的形态, 甲型流感病毒可由 15种 H型和 9种 N型进行排列 组合, 比如 H1N1和 H5N1等。 即使是同一种甲型的流感病毒, 也可能因为基 因序列的变化, 在病毒的传播性、 致死率等方面出现很大差异。
造成 1918年西班牙大流感的就是一种 H1N1病毒。 1918年以后, H1N1 在人群与猪群中分别进化, 可分别称之为人 H1N1和猪 H1N1。 而此次新型的 甲型 H1N1流感病毒与猪 H1N1病毒的关系最为密切。
甲型 H1N1病毒属于正粘病毒科(Orthomyxoviridae), 甲型流感病毒属 ( Influenza virus A), 其遗传物质为 RNA。 典型病毒颗粒呈球状, 直径为 80 ηπ!〜 120 nm, 有囊膜。 囊膜上有许多放射状排列的突起糖蛋白, 分别是 血凝素 HA、 神经氨酸酶 NA和 M2蛋白。 病毒颗粒内为核衣壳, 呈螺旋状对 称, 直径为 10nm。甲型 H1N1病毒为单股负链 RNA病毒,基因组约为 13. 6 kb, 由大小不等的 8 个独立片段组成。尽管不同亚型之间可以组成很多种流感病 毒血清型, 但是可造成人感染猪流感病毒的血清型主要有 H1N1、 H1N2 和 謂 2。
甲型 H1N1流感病毒是 A型流感病毒,携带有 H1N1亚型猪流感病毒毒株, 包含有禽流感、 猪流感和人流感三种流感病毒的核糖核酸基因片段, 同时拥 有亚洲猪流感和非洲猪流感病毒特征。 2009年 3月日本和美国等先后发生人 感染甲型 H1N1流感病毒, 人感染后的临床早期症状与流感类似, 有发烧、 咳嗽、疲劳、食欲不振等, 还可以出现腹泻和呕吐等症状。少数病例病情重, 进展迅速, 可出现病毒性肺炎, 合并呼吸衰竭、 多脏器功能损伤, 严重者可 以死亡。
禽流感是由 A型禽流行性感冒病毒引起的一种禽类(家禽和野禽)传染 病。 禽流感病毒感染后可以表现为轻度的呼吸道症状、 消化道症状, 死亡率 较低; 或表现为较严重的全身性、 出血性、 败血性症状, 死亡率较高。 这种 症状上的不同, 主要是由禽流感的毒型决定的。
根据禽流感致病性的不同, 可以将禽流感分为高致病性禽流感、 低致病 性禽流感和无致病性禽流感。 最近国内外由 H5N1血清型引起的禽流感称高 致病性禽流感, 发病率和死亡率都很高, 危害巨大。
禽流感病毒(Avian influenza virus, AIV)属于正粘病毒科的 A型流 感病毒属的成员。 病毒粒子的直径为 80〜120纳米, 呈球形、 多形性或长丝 状。 病毒的核衣壳呈螺旋对称。 病毒的基因组由 8个负链的单股 RNA组成。 本病毒有囊膜, 上有一层 12〜14纳米的纤突, 两种不同形状的表面纤突是 血凝素 (HA, 棒状, 为血凝素分子的三聚体构成, 对红细胞有凝集性) 和神 经氨酸酶 (NA, 蘑菇状, 为神经氨酸酶分子的四聚体构成, 能将吸附在细胞 表面的病毒粒子解脱下来), 两者都是糖蛋白, 纤突的一端镶嵌在病毒的脂 质囊膜中, 囊膜下面有一层膜蛋白。 本病毒能凝集多种哺乳动物和禽类的红 细胞。
本病毒能在鸡胚中生长, 并达到较高滴度, 具有裂解的血凝素。 病毒还 能在鸡胚成纤维细胞 (CEF) 上生长。 大多数毒株在 CEF培养中形成空斑。 可以通过血细胞吸附试验测定病毒的生长。
在家禽中短期传播后, 有时致病性低的病毒可发生突变, 变成致病性高 的病毒。在 1 9 8 3— 1 9 8 4年美国禽流感病毒 H 5 N 2流行期间, 开始 时病死率不高, 但在 6个月内其致病性显著增强 病死率接近 9 0 %。 为控 制流行宰杀了超过 1 7 0 0万只家禽, 经济损失接近 6 5 0 0万美元。 1 9 9 9 - 2 0 0 1年意大利流行期间, 开始 H 7 N 1病毒的致病性也较低, 但 在 9个月内变成致病性非常强的病毒。 死亡和宰杀的家禽超过 1 3 0 0万 只。 控制流行的常规措施包括对感染养殖场进行检疫, 宰杀受感染或接触过 病禽的家禽; 目的是预防感染扩散至其他养殖场及防止禽流感病毒在该国家 禽中 '安家落户' 。 除了高度接触传染性外, 禽流感病毒很容易通过机械方 法(例如污染的设备、 车辆、 饲料、 笼子或衣服) 从一个养殖场传至另一个 养殖场。 排出体外后, 高度致病性病毒可在环境中存活很长时间, 尤其是环 境温度较低时。 严格的卫生措施可为养殖场提供一定程度的保护。 如不立即 采取措施控制流行, 并进行严密监视, 流行可以持续数年。 例如, 1 9 9 2 年发生在墨西哥的禽流感病毒 H 5 N 2流行, 开始致病性较低, 以后致病性 逐渐增强, 直至 1 9 9 5年才被控制 甲型 H1N1流感、 禽流感与普通流感的异同
Figure imgf000007_0001
表现出病症。 常在 7天以内。 死 亡 甲型 H1N1 流感死亡 可以致死, 但死亡率 人患禽流感死亡
S ^- - 5 率不到 1%。 较低。 率超过 60%。 易 感 甲型 H1N1 流感致死 1 老年人, 患有肝脏、 在已发现的感染 人群 的患者年龄绝大多 ! 肾脏、 心脏等慢性病 病例中, 13 岁以 数在 20岁至 45岁之 1 的四类人群最易感 下的儿童所占比 间, 属于青壮年。 1 染, 以及经常接触流 例较高, 病情较
1 感人群的医护人员, 重,其属于易感人
1 儿童。 群。 防 治 中国已研制可预防 已研制出可预防流感 目前尚无适合人 疫苗 的疫苗, 已对易感人 i 的疫苗, 接种时间多 群大规模接种的 群开始接种. 为每年 lo-ii 月中 预防禽流感的疫 旬, 每年接种 1次。 苗,但各国正在积 极研制中。
抗甲流首选药物
根据国家卫生部和世卫组织的推荐, 抗甲流首选西药是达菲和乐感清, 达菲的生产厂家是瑞士罗氏; 乐感清的生产厂家是英国葛兰素史克。
有报道一些国家发生甲流病毒变异, 个别患者出现对达菲的耐药性, 达 菲在临床上对青少年会产生副作用。
发明内容 本发明的一个目的是提供一种防治甲型流感的中药组合物,该组合物由 下列重量比的中药原料制备而成:
苦菜 5-25%、黄蜀葵 10-30%、蒲公英 5-25%、鱼腥草 10_50%,车前草 5-35% 克, 鼠曲草 5-25%克, 仙鹤草 10-30%, 枇杷叶 3-30%, 旱莲草 10_40%, 凤尾 草 3-25%。
优选地, 该组合物由下列重量比的中药原料制备而成:
苦菜 5-10%,黄蜀葵 10-20%,蒲公英 5-10%,鱼腥草 20-30%,车前草 5_10% 克, 鼠曲草 5-10%克, 仙鹤草 10-20%, 枇杷叶 5-15%, 旱莲草 10_20%, 凤尾 草 5-15%。
将上述原料进行常规水或醇提取, 浓縮干燥得到的中草药提取物, 对甲 型流感有特效, 通过胶囊、 饮水或喷雾方法(雾滴愈细愈好)或以熏蒸方式 (空气加湿器)使药物通过呼吸道进入人或动物呼吸道, 喷雾或熏蒸时门窗 关闭 30-60分钟。
本发明另一个目的是提供一种本发明组合物的制备方法, 该方法包括将 以上十味中药原料粉碎成粗粉, 加入提取罐中, 加入生药材质量 6-8倍的纯 化水, 开启真空自动转换器, 减压至负压 0. 5-1MP, 并加热至沸, 保持微沸 状态, 双效浓縮回流, 动态提取 2-4小时; 收集提取液减压浓縮至相对密度 1. 5-1. 6;关闭加热器,逐渐恢复至大气压。进行喷雾干燥,先将干燥塔 160°C 预热 10-20分钟, 将进风温度设定为 150-200°C, 出风温度设定 50_100°C, 入料流量 1. 2L/分, 至全部喷完后, 继续保持进风 5分钟后打开出料斗出料 得到干燥粉剂。
随后,可以按照本领域的常规技术,将本发明的组合物制备成各种剂型。 例如干燥粉剂用醋酸稀释 loo倍并调节 ra值至 4.0, 灌封, 灭菌, 包装, 即 得喷雾剂; 或用纯净水稀释 100倍, 如果需要可加入调味剂和 /或防腐剂, 并调整总量, 灌封, 灭菌, 包装, 即得口服液; 或用 0号胶囊灌装, 灭菌, 包装, 即得胶囊剂。
需要说明的是,本发明所述的方法只是一种优选的制备工艺但并不限于 此。 由本发明方法制备的产品具有很好的稳定性。
多年来, 采用本发明的中药组合物, 对人畜共患的禽流感 (H5N1)、 猪 流感(H1N1)有很好的疗效, 未见任何毒副作用。 本发明的中药对金黄色葡 萄球菌、 卡他球菌、 流感杆菌、 肺炎球菌有明显抑制作用; 对多种微生物有 抑制作用, 特别对酵母菌和霉菌; 有利尿、 镇痛、 止血、 抑制浆液分泌、 止 咳、 促进组织再生等作用。 治疗慢性气管炎有良效、 有镇咳作用; 对组织胺 引起的支气管痉挛有缓解作用。 本发明的中药对人畜共患的禽流感(H5N1)、 甲流感 (H1N1) 有很好的疗效, 24-48小时解除症状, 可作为空气消毒剂使 用。
为此,本发明的另一目的是提供一种本发明组合物在防治甲型流感中的 应用。 所述甲型流感为禽流感 (H5N1)、 甲流感 (H1N1)。
附图说明
图 1是本发明的 Q7以及 Q7的溶剂对流感病毒 PR8 (H1N1)的抑制效果; 图 2是本发明的间接免疫荧光法检测 Q7对 PR8病毒的抑制效果 (感染 后 24h)。 a: Q7 10mg/ml;b: Q7 5mg/ml ; c : Q7 2.5mg/ml;d:l.25mg/ml; e:0.625mg/ml. f: virus control在病毒感染后 24小时, 用小鼠感染 PR8病 毒后的血清作为一抗, 用抗鼠的 FITC标记的二抗进行间接免疫荧光检测病 毒。
图 3. 间接免疫荧光法检测 Q7对 PR8病毒的抑制效果 (感染后 36h)。 a : Q7 10mg/ml ; b: Q7 5mg/ml ; c: Q7 2. 5mg/ ml ; d : 1. 25mg/ ml;
e : 0. 625mg/ml. f : virus control在病毒感染后 24小时, 用小鼠感染 PR8病 毒后的血清作为一抗, 用抗鼠的 FITC标记的二抗进行间接免疫荧光检测病 毒。 具体实施方案
实施例 h 喷雾剂
中药原料及用量: 苦菜 5%, 黄蜀葵 10%, 蒲公英 5%, 鱼腥草 30%, 车前 草 10%, 鼠曲草 5%, 仙鹤草 10%, 枇杷叶 5%, 旱莲草 10, 凤尾草 10%。
制法: 1. 以上中药组合物经称量 100公斤, 清洗, 加入提取罐中, 分别 进行常规水或醇提取, 浓縮干燥得到干燥粉剂;
或者 2.以上中药组合物经称量 100公斤,经 2-3次搅拌清洗干净,加入 提取罐中, 加入生药材质量 8倍的纯化水, 开启真空自动转换器, 减压至负 压 0. 67MP, 并加热至沸, 保持微沸状态, 双效浓縮回流, 动态提取 3小时。 收集提取液减压浓縮至相对密度 1. 5。 关闭加热器, 逐渐恢复至大气压。 进 行喷雾干燥, 先将干燥塔 160°C预热 15分钟, 将进风温度设定为 170°C, 出 风温度设定 80°C, 入料流量 1. 2L/分, 至全部喷完后, 继续保持进风 5分钟 后打开出料斗出料得到干燥粉剂即可。
取干燥粉剂, 加入 99公斤醋酸, 加纯净水调节 ra至 1¾3. 6即成本发明 喷雾剂。 通过空气加湿器喷雾 (雾滴愈细愈好) 或以熏蒸方式 (如电饭锅) 使药物通过呼吸道进入人或动物呼吸道,喷雾或熏蒸时门窗关闭 30-60分钟。 本发明喷雾剂禽流感、 甲流感有特效, 24-48小时解除症状。 实施例 2: 口服液
中药原料及用量: 苦菜 5%, 黄蜀葵 20%, 蒲公英 5%, 鱼腥草 20%, 车前 草 5%, 鼠曲草 5%克, 仙鹤草 10%, 枇杷叶 15%, 旱莲草 10%, 凤尾草 5%。
制法同实施例 1 ;
取干燥粉剂 200克, 加入 1800ml纯净水, 如果需要可加入调味剂和 /或 防腐剂, 即成本发明口服液。 防治甲流感, 每人每天饮用 200ml, 连用 3天。 实施例 3, 胶囊剂
中药原料及用量: 苦菜 5%, 黄蜀葵 20%, 蒲公英 5%, 鱼腥草 20%, 车前 草 5%, 鼠曲草 5%克, 仙鹤草 10%, 枇杷叶 15%, 旱莲草 10%, 凤尾草 5%。
制法同实施例 1
取干燥粉剂用 0号胶囊灌装, 每颗胶囊 0. 5克, 灭菌, 包装, 即得。 防 治甲流感, 每人每天服用 3-5颗, 连用 3天。 实施例 4, 本发明药物(以下简称" Q7 ", 可由实施例 2制得)对禽流感病毒 (H5NK H9N2 ) 的抑制试验在 10日龄鸡胚上进行 "Q7 "药液(本发明药物) 对禽流感病毒(AIV)的抑制试验。采用 6个稀释度(1 : 40、 1: 80、 1: 160、 1: 320、 1: 640、 1: 1280)、 5个病毒株 (禽流感 H5亚型 BP02株及 DX株、 禽流感 H9亚型、 和不同时间(20〜50分钟) 的三因子交叉试验。 结果表明, 1: 40〜1: 640倍稀释的 " Q7"对禽流感 H5亚型病毒和 1: 40〜1: 320倍稀 释的 Q7对 AIVH9亚型病毒的抑制作用最好, 可达到 100%。
1 材料与方法
1.1 材料
1.1.1 试验用药
1.1.1.1 "Q7"药液 主要成分为植物多糖、 葵酰乙醛、 木犀草黄素一葡 萄糖甙、 黄酮甙、 植物 醇、 月桂醛、 葵醛、 葵酸等, 按照实施例 1或 2制 得。
1.1.1.2 感冒清热颗粒 主要成份为荆芥穗、 薄荷、 防风、 柴胡、 紫苏 叶、 葛根、 桔梗、 苦杏仁、 白芷、 芦根等。 由北京同仁堂科技发展股份有限 公司制药厂生产, 批号 2110527。
1.1.2 病毒 AIV H9 亚型毒株, 血凝价(HA 价)为 1: 512, 毒价 108.1EID50/0.1ml; AIV H5 亚型 BP02 株 (鸡源), HA 价 1: 256, 毒价 108.3ELD50/0. lml, DX株(鸭源), HA为 1: 512,毒价为 108.1ELD50/0. lml;
AIV H9亚型及 H5亚型 AIV由中国农科院哈尔滨兽医研究所提供, NDVF48株 及 IBVM41株均购自中国兽药监察所。
1.1.3 鸡胚 10日龄 SPF鸡胚, 由北京实验动物中心引入。
1.2 方法
1.2.1 "Q7"溶液的配制
将 "Q7"药液用灭菌生理盐水分别稀释 20 x、 40x、 80x、 160x、 320x、 640x 和 1280x, 室温放置备用。
1.2.2 "Q7"对鸡胚的毒性试验 将 20 x、 40x、 80x、 160x、 320x、 640x、 1280x稀释的 "Q7 "溶液分别接种 10日龄 SPF鸡胚尿囊腔内, 每个稀释度接种 5枚, 每胚 0. 2ml, 同时设不接 种的鸡胚作为空白对照。 将所有的 SPF鸡胚置 370C继续孵育 120小时, 每 隔 4小时照蛋一次。 24小时以内死亡的鸡胚弃去不计, 记录 24小时〜 120 小时鸡胚死亡情况, 120小时后所有鸡胚逐个进行剖捡,观察鸡胚发育情况。 1. 2. 3 "Q7 "对 AIVH9亚型 AIV的抑制试验
先将病毒用灭菌生理盐水作 10x递进稀释,从 10- 1〜10— 7,取 10— 4、 10-5, 10-6, 10-7四个稀释度的病毒液, 病毒含量分别为 104. 1、 103. 1、
102. 1、 101. 1EID50 I 0. lml,每个稀释度的病毒液分别与等量 20x、 40x、 80x、 160x、 320x、 640x稀释的 "Q7 "溶液 ("Q7 " 的实际最终浓度为 40x、 80x、 160x、 320x、 640x、 1280x)在室温下(200C左右)感作 30〜45分钟后, 接 种于 10日龄 SPF鸡胚尿囊腔, 每胚 0. 2ml , 每一样品接种 5枚鸡胚, 接种后 在 370C条件下继续孵育, 每隔 4小时照蛋一次。接种后 24小时内死亡的鸡 胚为非特异性死亡。 24〜120小时的鸡胚无论死活均逐个剖检观察病变并测 定其尿囊液的 HA价, 凡 HA^ l : 128的判为感染, 否则为不感染, 并根据感 染鸡胚与接种鸡胚的比值计算鸡胚的感染比例。 同时设 10— 4、 10-5, 10 一 6、 10— 7四个稀释度的病毒对照组, 每个稀释度接种 5枚 SPF鸡胚, 接种 后的观察及 HA价检测同上。
1. 2. 4 "Q7 "对 H5亚型 AIV的抑制试验
1. 2. 4. 1 "Q7 "对 H5亚型 AIV的抑制试验一
先将 H5 亚型 BP02 株病毒用生理盐水稀释成 10— 5, 病毒含量为
103. 3ELD50 I 0. lml将稀释的病毒液分别与 20x、 40x、 80x、 160x、 320x、 640x稀释的 "Q7 "在室温下作用, "Q7 " 终浓度分别为 40x 、 80x 、 160x 、 320x 、 640x、 1280x,分别作用 20分钟及 40分钟后接种于 10日龄 SPF鸡 胚尿囊腔, 每胚 0. 2ml, 每组样品接种 5枚鸡胚, 另外设病毒对照组, 接种 后的观察及 HA价检测同 1. 2. 3。
1. 2. 4. 2 "Q7 "对 H5亚型 AIV的抑制试验二
为保证试验的可靠性, 我们在试验一的基础之上, 进行了本试验。 本试 验使用了 2个 H5亚型 AIV毒株, 并且选用了市售中药感冒清热颗粒作为药 物对照。 具体试验方法是分别将 H5亚型 AIV BP/02株及 DX株病毒用生理盐 水分别稀释成 10-5 , 含毒量每 0. lml分别为 103. 3及 103. 1ELD50, 将稀释 的病毒液分别与 20 x、 40 x、 80 x、 160 x、 320 x稀释的 Q7及 20 x、 40 x、 80 x、 160 x、 320 x稀释的感冒清热颗粒, 在室温下作用, Q7及感冒清热 颗粒最终浓度分别为 40 x、 80 x、 160 x、 320 x、 640 x, 作用 50分钟后, 接种于 10日龄 SPF鸡胚尿囊腔, 每胚 0. 2ml, 每组样品接种 5枚鸡胚, 另 外设病毒对照组及不同稀释度感冒清热颗粒对照组,接种后的观察及判定同
1. 2. 3项。
2 结果
2. 1 "Q7 "对鸡胚的毒性试验
接种后 24小时内, 40x及 80x "Q7 "溶液接种组各有一枚鸡胚出现非特 异性死亡, 160x "Q7 "溶液接种组在接种后 62小时死亡一枚, 经剖检鸡胚 内有两个胎儿, 属于畸型, 亦为非特异性死亡。 其余鸡胚孵化至 120小时剖 捡, 鸡胚活力、 胎儿发育等均正常, 表明 "Q7 "对鸡胚无任何毒副作用。 试 验结果见表 1。 表 1 "Q7"对鸡胚的毒性试验
Figure imgf000016_0001
*分子是病变鸡胚数, 分母是接种鸡胚数
2.2不同浓度的 "Q7"对 AIV H9亚型的抑制作用
分别接种 04.1、 103.1、 102.1、 101.1EID50 I 0. lml H9亚型病毒液的 对照组鸡胚, 除 101.1EID50/0. lml组有两枚鸡胚未死亡外, 其余鸡胚均于 接种后 84小时内死亡, 对所有死亡鸡胚包括未死亡的这两枚鸡胚尿囊液进 行血凝试验, 血凝价均大于 1: 128 (未死亡的两枚鸡胚血凝价均为 1: 512), 且血凝可被 H9 亚型阳性血清抑制, 证明确系 AIV H9 亚型阳性。 除 101.1EID50/0. lml病毒液与终浓度为 1: 1280 的 "Q7"溶液作用试验组鸡 胚以及 102.1EID50/0. lml病毒液与终浓度为 1: 640的 "Q7"溶液试验组各 有一枚鸡胚感染外, 其余各试验组鸡胚均健活, 无血凝价, 说明 1: 40〜1: 320的 "Q7"在 30〜45分钟内对 AIVH9亚型的保护率可达到 100%, 1: 640 及 1: 1280时保护率为 80%。 结果见表 2。 表 2 不同浓度的 "Q7"对 AIV H9亚型的抑制作用
Figure imgf000016_0002
101.1 30 0/5 0/4 0/5 0/4 0/5 1/4 5/5
Figure imgf000017_0001
2.3 "Q7"对 H5亚型 AIV的抑制试验。
2.3.1 "Q7"对 H5亚型 AIV的抑制试验一
表 3 不同浓度的 "Q7"对 H5亚型 AIV BP02株的抑制作用
Figure imgf000017_0002
*分子为感染鸡胚数, 分母为接种鸡胚数
由表 3可见, 不同浓度的 "Q7"与含毒量为 103.3ELD50/0. lml的稀释 的 AIV H5亚型毒株感作 20分钟, 1: 40〜1: 640的 "Q7"试验组均 100% 保护, 而 1: 1280的 " Q7"试验组的保护率仅为 20%; 而相同条件下病毒与 "Q7"药液感作 40分钟时, 除 1: 1280 "Q7" 感作组有两个鸡胚感染外, 其余各组均为 100%保护, 而病毒对照组鸡胚均于接种后 32小时死亡, 尿囊 液 HA价为 1: 256。 以上结果说明 1: 40〜1: 640的 " Q7"在 20〜40分钟内 能 100%的抑制 AIV H5病毒。
2.3.2 "Q7"对 H5亚型 AIV的重复抑制试验 表 4 "Q7"对 H5亚型 AIV的重复抑制试验结果
Figure imgf000017_0003
* 分子为感染鸡胚数, 分母为接种鸡胚数。
从表 4可见, 1 : 40- 1: 640稀释的 Q7与 H5亚型 AIV BP/02株及 DX 株作用 50分钟后, 均可 100%抑制这两种病毒在鸡胚内的复制, 保护率达 100% , 而相同稀释度的感冒清热颗粒与 BP/02株作用后接种鸡胚, 没有任 何作用。
3. 结论
3. 1 以不同浓度的 "Q7 "药液接种鸡胚, 观察其对鸡胚的毒性作用, 结果 证明 1 : 20〜1: 1280稀释的 "Q7"接种 0. 2ml对鸡胚无毒性作用, 接种后 的鸡胚均生长发育正常。
3. 2 "Q7"对 H9亚型 AIV抑制试验证明, 1 : 40〜1: 320稀释的 "Q7"药 液与含毒量为 101. 1〜104. 1 EID50/0. lml的 H9亚型 AIV在室温条件下作用 30〜45分钟, 其抑制作用达 100%, 而 1 : 640及 1 : 1280为 80〜100%。 尽管 10-4, 10— 5稀释的病毒液和不同浓度的 Q7感作组所使用的病毒含量 (含 毒量分别为 104. 1、 103. 1 EID50/0. lml )与 10— 6、 10— 7稀释的病毒液和 Q7感作组所使用的病毒含量(含毒量分别为 102. 1、 101. 1EID50/0. lml )高 10〜1000倍, 但前两个病毒含量试验组的鸡胚保护率达 100%, 而后两个试 验组未达到 100%。分析原因, 可能是药物作用时间所致, 前者的作用时间比 后者长约 10〜15分钟。看来, 本药物的作用时间对于能否有效抑制 H9亚型 AIV是十分重要的。
3. 3 "Q7"对 H5亚型 AIV有明显抑制作用, 1 : 40〜1: 640的 " Q7"在室 温条件下作用 20〜50分钟, 可完全抑制病毒在鸡胚内的增殖。 而相同稀释 度的市售中药一感冒清热颗粒与 H5亚型 AIV作用后接种鸡胚,无任何作用。 实例 5, Q7 (本发明药物) 对甲型流感病毒 PR8 (H1N1)的抑制效果
1. Q7在 MDCK细胞上对 PR8 (H1N1)病毒的抑制效果研究
将实验室保存的 PR8 (H1N1 ) 流感病毒以 10倍梯度稀释后, 感染 96孔 板中已长成单层的 MDCK细胞(5xl04个 /孔),将 Q7液体用 Opt i -MEM (含 lug/ml 的 TPCK-Trpsin)做 100倍稀释后作为病毒感染后的维持液, 48小时后, 用 血凝的方法检测维持液中流感病毒增殖情况。 研究结果表明, 含 Q7的维持 液, 可显著抑制病毒在 MDCK上的增殖, 结果参见附图 1所示。 另外,以 100TCID5。的 PR8病毒感染 96孔板 MDCK细胞后, Q7用 Opti-MEM (含 lug/ml的 TPCK-Trpsin) 100倍稀释作为病毒感染细胞的维持液, 感染 后每间隔 12h更换新鲜的含 TQ的培养液, 测定不同时间收集的培养液上清 中病毒效价, 结果如图 5所示。药物组, 24小时内在培养上清中未检测到病 毒, 而对照组在感染后的 12 小时, 培养液上清中的病毒含量即达到 102 5TCID5。。
2. Q7对 PR8 (H1N1)病毒的 EC50值的确定
以 Q7对 MDCK细胞无明显毒性的浓度 (约 12. 5mg/ml ) 作为起始浓度, 用 Opti-MEM (含 lug/ml的 TPCK-Trpsin)进行 2倍梯度稀释, 然后测定不同 浓度的 Q7对 100TCID5。的流感病毒的抑制效率, 确定 Q7对 100TCID5。的流感 病毒的 EC50值, EC50的值通过 48小时后测定含不同浓度的 Q7的维持液中流 感病毒的血凝价, 通过 SPSS软件计算得到 Q7对 PR8病毒的 EC50值约为 1. 8mg/ml。
3. 间接免疫荧光法检测不同浓度的 Q7对 PR8 (H1N1)病毒的抑制效果
以 100TCID5。的 PR8病毒感染 MDCK细胞后, 维持液中含不同浓度的 Q7, 分别在病毒感染的 24小时(附图 2)和 36小时(附图 3), 用小鼠感染 PR8病 毒后的血清作为一抗, 用抗鼠的 FITC标记的二抗进行间接免疫荧光检测病 毒, Q7对 PR8病毒的抑制效果。
4. Q7在小鼠体内抑制 PR8 (H1N1)病毒的效果研究
将 Q7用 PBS (pH7. 2 )分别做 10倍和 5倍稀释, 然后取两组小鼠, 每组 12只。 每只小鼠在感染前的 72小时每间隔 12小时滴鼻 20ul稀释好的药物 (Q7药物 5倍稀释组), 并在 PR8病毒感染前, 每组解剖 3只, 然后取其鼻 腔冲洗液 (每只小鼠的鼻腔用 1ml 的培养液冲洗), 用于测定其中是否有抗 病毒成分。实验结果表明,两组小鼠的鼻腔冲洗液在 MDCK细胞上对 100TCID5。 的 PR8病毒均无抑制效果。
给药 3天后, 以 5LD5。/只的感染剂量感染小鼠,记录感染后 14天内小鼠 的体重变化, 如图所示。 另外, 将 PR8病毒稀释到 20ul 含 5LD5。的病毒的滴 度, 然后与 Q7药物 5倍稀释液等体积混合后感染小鼠, 记录感染后 14天内 小鼠的体重变化。 另外在病毒感染后的第三天, 解剖 3只小鼠, 每只小鼠的 肺用 lml含抗生素的 PBS冲洗, 测定小鼠的肺部病毒效价, 实验结果见表 1 所示。从实验结果可以看出, Q7用 PBS进行 5倍稀释后, 在感染前 3天给小 鼠用药, 可以达到一个比较好的预防的效果, 小鼠在病毒感染后体重丢失较 轻。 将病毒预先与 Q7的 5倍稀释液混合后再感染小鼠, 小鼠体重仅发生轻 微丢失, 而且体重很快开始恢复, 表明 Q7 在比较高的浓度下可以抑制 PR8 (H1N1)病毒的侵入机体。
表 1 不同给药方式的小鼠在以 5LD5。PR8 (H1N1)感染小鼠后的第 3天肺部病毒
Q7液体 5倍稀释感染 Q7液体 5倍稀释与病 5LD5。病毒感染小鼠 给药方式
前 3天滴鼻 毒混合滴鼻 的对照 肺部病毒量
2. 7 1. 0 2. 5
( logTCIDso)

Claims

权 利 要 求 书
1、 一种防治甲型流感的中药组合物, 该组合物由下列重量百分比的中 药原料制备而成:
苦菜 5-25%,黄蜀葵 10-30%,蒲公英 5-25%,鱼腥草 10_50%,车前草 5-35%, 鼠曲草 5-25%,仙鹤草 10-30%,枇杷叶 3-30%,旱莲草 10_40%,凤尾草 3_25%, 所述中药原料的重量百分比之和为 100%。
2、 根据权利要求 1 的组合物, 该组合物是由下列重量比的中药原料制 备而成的:
苦菜 5-10%,黄蜀葵 10-20%,蒲公英 5-15%,鱼腥草 10_30%,车前草 5_10% 克, 鼠曲草 5-20%克, 仙鹤草 10-20%, 枇杷叶 3-15%, 旱莲草 1-10%, 凤尾 草 3-15%。
3、 根据权利要求 1或 2的任一组合物, 所述组合物为喷雾剂、 口服液 或胶囊。
4、 根据权利要求 1或 2的组合物的制备方法, 该方法包括将以上十味 中药原料粉碎成粗粉, 加入提取罐中, 加入生药材质量 6-8倍的纯化水, 开 启真空自动转换器, 减压至负压 0. 5-1MP, 并加热至沸, 保持微沸状态, 双 效浓縮回流,动态提取 2-4小时;收集提取液减压浓縮至相对密度 1. 5-1. 6; 关闭加热器,逐渐恢复至大气压。进行喷雾干燥,先将干燥塔 160°C预热 10-20 分钟, 将进风温度设定为 150-200°C, 出风温度设定 50-100°C, 入料流量 1. 2L/分, 至全部喷完后, 继续保持进风 5分钟后打开出料斗出料得到干燥 粉剂。
5、 根据权利要求 4的方法, 其特征在于进一歩将干燥粉剂用醋酸稀释 100倍并调节 pH值至 4. 0, 灌封, 灭菌, 包装, 即得喷雾剂。
6、 根据权利要求 4的方法, 其特征在于进一歩将干燥粉剂用纯净水稀 释 100倍, 如果需要可加入调味剂和 /或防腐剂, 并调整总量, 灌封, 灭菌, 包装, 即得口服液。
7、 根据权利要求 4的方法, 其特征在于进一歩将干燥粉剂用 0号胶囊 灌装, 灭菌, 包装, 即得胶囊剂。
PCT/CN2012/073992 2011-04-15 2012-04-13 一种防治甲型流感的中药组合物及其制备方法和用途 WO2012139521A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN2011100950956A CN102166276B (zh) 2011-04-15 2011-04-15 一种防治甲型流感的中药组合物及其制备方法和用途
CN201110095095.6 2011-04-15

Publications (1)

Publication Number Publication Date
WO2012139521A1 true WO2012139521A1 (zh) 2012-10-18

Family

ID=44487719

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2012/073992 WO2012139521A1 (zh) 2011-04-15 2012-04-13 一种防治甲型流感的中药组合物及其制备方法和用途

Country Status (2)

Country Link
CN (1) CN102166276B (zh)
WO (1) WO2012139521A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102166276B (zh) * 2011-04-15 2013-03-27 北京天福莱生物科技有限公司 一种防治甲型流感的中药组合物及其制备方法和用途
CN105125864A (zh) * 2015-08-21 2015-12-09 江志鑫 一种防治甲型h1n1流感的药物组合物及其制备工艺

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1302638A (zh) * 2000-01-01 2001-07-11 同济医科大学附属同济医院 一种清热解毒抗感染中药及其制备方法
CN102166276A (zh) * 2011-04-15 2011-08-31 北京天福莱生物科技有限公司 一种防治甲型流感的中药组合物及其制备方法和用途

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1302638A (zh) * 2000-01-01 2001-07-11 同济医科大学附属同济医院 一种清热解毒抗感染中药及其制备方法
CN102166276A (zh) * 2011-04-15 2011-08-31 北京天福莱生物科技有限公司 一种防治甲型流感的中药组合物及其制备方法和用途

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LI KAI ET AL.: "The Mechanism of Prevention and Cure Avian Influenza Virus with Chinese Herbal Medicine", JOURNAL OF ZHANGJIAKOU AGRICULTURAL COLLEGE, vol. 20, no. 2, June 2004 (2004-06-01), pages 23 - 26 *
LI YUE ET AL.: "Study on Traditional Chinese Medicine Treatment to Flu", JOURNAL OF LIAONING UNIVERSITY OF TCM, vol. 12, no. 5, May 2010 (2010-05-01), pages 125 - 128 *
WANG ZHENGLIN: "Discussion on Traditional Chinese Medicine Treatment to Influenza A", CJGMCM, vol. 25, no. 5, May 2010 (2010-05-01), pages 875 - 876 *

Also Published As

Publication number Publication date
CN102166276A (zh) 2011-08-31
CN102166276B (zh) 2013-03-27

Similar Documents

Publication Publication Date Title
Yasmin et al. Herbal extracts as antiviral agents
US8591962B2 (en) Composition for preventing and treating influenza-virus-induced diseases
US8795742B2 (en) Chinese traditional medicine composition for treatment of avian influenza, method for preparation and application thereof
EP2444098B1 (en) Traditional chinese medicine composition for treating h1n1 swine influenza, preparation method and use thereof
CN101111608A (zh) 降低疾病传染性的组合物和方法
CN103687599A (zh) 氯喹或氯丙嗪或其衍生物或它们的混合物在制备用于治疗和/或预防肺感染和损伤的药物中的用途
US20120164253A1 (en) Anti-influenza viral composition containing bark or stem extract of alnus japonica
WO2007046643A1 (en) Composition comprising an extract of pine needle for preventing and treating animal disease caused by viruses and the use thereof
KR20080084992A (ko) 엘더베리 추출물의 용도
KR20160115381A (ko) 유백피 추출물을 유효성분으로 함유하는 항바이러스용 조성물
WO2012139521A1 (zh) 一种防治甲型流感的中药组合物及其制备方法和用途
US20090061027A1 (en) Composition For The Prevention and Treatment Of Common Cold Diseases
CN113288967A (zh) 一种中药组合物在制备治疗或预防冠状病毒感染的药物中的应用
CN103239518B (zh) 治疗禽流感的中药组合物
KR101837445B1 (ko) 구맥 추출물을 유효성분으로 함유하는 선천면역 증진 및 항바이러스용 조성물
TWI763916B (zh) 藍綠藻萃取物及其製備方法與用途
CN106957826B (zh) 一种病毒灭活剂及其应用
RU2653388C1 (ru) Вакцинный штамм вируса гриппа А/17/НЬЮ ЙОРК/15/5364 (H1N1) pdm09 для производства живой гриппозной интраназальной вакцины для взрослых и для детей
JP5173813B2 (ja) インフルエンザの予防および処置のための薬剤
WO2007046642A1 (en) Composition comprising an extract of pine needle for preventing and treating human disease caused by viruses and the use thereof
WO2007066991A1 (en) Composition comprising an extract of celosia for preventing and treating human disease caused by viruses
Sainhi et al. A Review Article on Phytochemicals New Line of Treatment of Sars Covid-19
CN112294868B (zh) 用于病毒灭活与卫生消毒、激活机体免疫能力产生的药物
El-Hamid et al. In vitro antiviral activity of commercial products of herbal extracts against highly pathogenic avian influenza (H5N1) virus.
Amjad et al. In vivo concurrent efficacy of purified immunoglobulins and licorice (Glycyrrhiza glabra) root extracts against Newcastle disease virus.

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12770606

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12770606

Country of ref document: EP

Kind code of ref document: A1