WO2006026905A1 - A use of phyto-flavonoid compounds for preparing medicament against avian influenzal virus - Google Patents

A use of phyto-flavonoid compounds for preparing medicament against avian influenzal virus Download PDF

Info

Publication number
WO2006026905A1
WO2006026905A1 PCT/CN2005/001049 CN2005001049W WO2006026905A1 WO 2006026905 A1 WO2006026905 A1 WO 2006026905A1 CN 2005001049 W CN2005001049 W CN 2005001049W WO 2006026905 A1 WO2006026905 A1 WO 2006026905A1
Authority
WO
WIPO (PCT)
Prior art keywords
virus
group
influenzal
drug
avian
Prior art date
Application number
PCT/CN2005/001049
Other languages
French (fr)
Chinese (zh)
Inventor
Ren Huang
Original Assignee
Guangdong Laboratory Animals Monitoring Institute
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 Guangdong Laboratory Animals Monitoring Institute filed Critical Guangdong Laboratory Animals Monitoring Institute
Publication of WO2006026905A1 publication Critical patent/WO2006026905A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses

Definitions

  • This invention relates to the use of compounds, in particular to the use of plant flavonoids against avian influenza viruses. Background technique
  • Avian influenza is a serious infectious disease caused by influenza A viruses.
  • the infected animals are geese, chickens, ducks, pigeons and some wild birds.
  • Epidemiological studies have shown that the avian influenza virus is highly pathogenic and has a strong ability to spread. It not only causes huge losses to the poultry industry, but also has channels to spread the virus from chicken to humans.
  • the avian influenza virus belongs to the genus Influenza virus of the Orthomyxoviridae family and has a single negative strand RA.
  • the avian influenza virions are generally spherical and have two different shapes of protrusions on the surface: hemagglutinin (HA) and neuraminidase (NA) ).
  • HA and NA are the major glycoproteins on the surface of the virus. They have the specificity and variability of species (subtypes) and play an important role in the process of viral infection.
  • HA is the main antigenic component that determines the pathogenicity of a virus. It can induce a neutralizing antibody with a protective effect on the infected host, while the NA-induced corresponding antibody has no virus neutralization, but can reduce the virus proliferation and change the course of the disease.
  • 16 kinds of HA and 10 kinds of NA have been known, and different forms of random combinations may occur between different HAs and NAs, thereby constituting many different subtypes. It has been reported that at least 80' influenza virus subtypes have been found, most of which are non-pathogenic or low pathogenic, and the highly pathogenic subtypes are mainly strains containing H 5 and H 7 .
  • AI has always been regarded as the largest gene pool of human influenza, a new source of genetic mutations in human influenza, but in the natural state, the link between AI and human influenza needs to be breast-fed through intermediate hosts (such as pigs, horses, dolphins, etc.). Animals) to achieve.
  • intermediate hosts such as pigs, horses, dolphins, etc.
  • Animals to achieve.
  • H5N1 in Hong Kong, China, and H9N2 AIV in mainland China and Hong Kong in 1999 broke through the interspecies disorder for the first time. It even caused human death. It not only broke the natural conditions, but only H1, H2, H3 subtype influenza viruses can infect people. And the regularity of other mammals, and the addition of new strain subtypes (H5 and H9) to the human influenza epidemic, which in turn gives AIV a new public health significance.
  • neuraminidase inhibitors such as: Zanamivir and Oseltamivir; ion channel blockers such as Amantadine and rimantadine, which are considered to be The drug of choice for the treatment and prevention of influenza virus; and nucleoside drugs: ribavirin, ribavirin, also known as ribavirin.
  • Zanamivir is based on the complex structure of influenza virus NA and sialic acid. It is designed by computer molecular simulation. The thiol group in the structure and the amino acid of the active part of influenza virus NA pass hydrogen bond, electrostatic force and van der Waals force. Tightly bound to the enzyme, the intensity and selectivity are high; zanamivir also has a certain degree of binding to the influenza B virus.
  • the structure is as shown in formula (1).
  • Osmivir is an ethyl ester-type prodrug of GS4071, and its lipophilic 3-pentyl side chain has a strong affinity with the hydrophobic pocket of the active site of the influenza virus NA, blocking the influenza virus NA against virus-infected cells.
  • the cleavage of the surface sialic acid residues inhibits the release of viral particles from infected cells and is therefore a highly selective influenza virus NA inhibitor.
  • the structure is as shown in equation (11).
  • Amantadine hydrochloride English name: Amantadine Hydrochloride Tablets, its chemical name is: tricyclic [3, 3, 1, 13, 7] ⁇ -1-amine hydrochloride, its structural formula is (111).
  • Ribavirin (ribavirin), English name: Ribavirin Injection, the main chemical name: 1-b-D-ribofuranosyl-1,2,4,-triazole-3-carboxamide.
  • Molecular formula C8H12 ⁇ 405, molecular weight: 244.21.
  • the structural formula is as shown in formula (IV).
  • Neuraminidase inhibitors can effectively inhibit influenza A and B viruses, which are more expensive and restrict their use; ion channel blockers also have influenza A virus. Good inhibition, but there are neurotoxicity, long-term drug susceptibility to drug resistance, flu-like pandemic, and ineffectiveness of influenza B virus; ribavirin (ribavirin) caused by RNA and DNA viruses The infections are all effective, but such compounds have a certain teratogenic effect, which limits their clinical application. It is particularly urgent and important to find anti-avian flu drugs that are safe, effective, and have a wide range of sources.
  • Flavonoids have been reported to have antioxidant, antithrombotic, anticancer, anti-inflammatory, vascular vaso-regulating, anti-depressant, anti-anxiety, anti-cerebral ischemia, liver protection, analgesia, protection against peptic ulcer and anti-heart rhythm Abnormalities and anti-myocardial ischemia and other effects, but the application of plant flavonoids in the prevention and treatment of avian influenza virus has not been reported.
  • Flavonoids are a large class of low molecular natural plant constituents. They refer to a series of compounds in which two benzene rings are linked by a central three carbon chain. They all have the basic structure of C6-C3-C6, mainly including flavones.
  • the main structure is as shown in formula (V>) and its isoflavone class; xanthone (flavanone main structure such as formula (VI)) and its isomer isoflavone (isoflavanone); chalcone (chalcone main structure such as formula (VII)); mulberry (Xanthone main structure such as formula (VIII)); flavonol (flavonol main structure such as formula (IX)) and its isomer isoflavoneol (isoflavonol) Wait.
  • the chemical name of the plant flavonoid compound referred to in the present invention is 5,7-dihydroxyflavone, and the molecular structure is as shown in the formula (X), wherein R1 is H or OH; R2 is OH or 3,4,5-trihydroxyl
  • the structure of galloyl, 3,4,5-trihydroxybenzoyl is of formula (XI). It can be extracted from traditional Chinese medicine Rosaceae plants, for example, Rosa bracteata Wendl. contains plant flavonoids. Shuo Wei, also known as rose, monkey persimmon, etc., is an evergreen shrub, 2-5 meters high, widely distributed in Central China and South China.
  • the inventors of the present invention have shown that plant flavonoids can directly kill (injure) avian influenza virus, can be used for treating and preventing infection of avian influenza virus, and can be widely used in preparing drugs and additives for avian influenza virus. .
  • the plant flavonoid compound is used as an active ingredient
  • one or more pharmaceutically acceptable carriers may be added as needed.
  • the carrier includes conventional diluents, excipients, fillers, binders, wetting agents, disintegrating agents, absorption enhancers, surfactants, adsorption carriers, lubricants and the like in the pharmaceutical field.
  • the medicament of the present invention can be prepared into a plurality of pharmaceutical forms such as tablets, capsules, powders, granules, troches, suppositories, or oral liquids or sterile parenteral suspension liquid preparations by oral, injection or mucosal administration.
  • pharmaceutical forms such as tablets, capsules, powders, granules, troches, suppositories, or oral liquids or sterile parenteral suspension liquid preparations by oral, injection or mucosal administration.
  • injection forms such as large or small volume injections, freeze-dried powder needles or sterile powder dispensing agents.
  • the above various dosage forms of the drug can be prepared according to a conventional method in the field of pharmacy.
  • the anti-avian influenza virus drug or additive containing the plant flavonoid compound as an active ingredient is used in an amount of 20-250 mg/kg body weight, and the preferred dose is 60-180 mg/kg body weight ⁇ day.
  • plant flavonoids As a natural botanical component, plant flavonoids have obvious inhibitory effects on avian influenza virus, and are prepared into drugs or additives. They have the advantages of low toxicity, strong action, safety and effectiveness, and open up new ways for the prevention and control of avian influenza virus. It has important social value, economic value and broad application prospects.
  • Figure 1 is a comparison of the different compositional viability of experimental mice.
  • Figure 2 is a graph showing the relationship between virus proliferation and time in mouse lung.
  • Figure 3 is a graph showing changes in serum antibody of mice over time.
  • Example 1 Anti-H5N1 avian influenza virus effect of plant flavonoids in MDCK cell model
  • the present invention utilizes MDCK cells for the pharmacodynamic test of anti-H5N1 subtype avian influenza virus in vitro.
  • MDCK cells purchased from the China Center for Type Culture Collection of Wuhan University.
  • H5N1 avian influenza virus supplied by South China Agricultural University and identified by Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences. The strain number is: A/goose/Guangdong NH/2003(H5) (Source of isolation method: Chinese disease Prevention and Control Center, Avian Influenza Laboratory Test Method. Characterization of an Avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness. Kanta Subbarao, Alexander Klimov, et al. Science (1998), 279:393 -396).
  • the virus stock was diluted 100-fold, and the virus was inoculated into the 10-day-old chicken embryo allantoic cavity in a clean bench, 0.2 ml per embryo, sealed with paraffin, and incubated in a 37 ° C incubator.
  • the growth of chicken embryos was observed at 12h, and the embryos were turned twice a day.
  • the dead chicken embryos were discarded within 24 hours.
  • the chicken embryos that died after 24 hours were placed in a 4°C refrigerator. After 48 hours, all the chicken embryos were placed at 4°.
  • Plant flavonoids Provided by the Institute of Marine Medicine, School of Life Sciences, Sun Yat-sen University. 200 mg of the plant flavonoid compound was weighed and dissolved in 50 ml of the cell maintenance solution. After it was sufficiently dissolved, it was sterilized by filtration through a 0.22 ⁇ M filter, sterilized for 96 h (-), and dispensed at 4 ° C for storage.
  • the drug (phytoflavonoids and ribavirin) was serially diluted to 4 g/ml, ie 4 mg/ml, 2 mg/ml, 1 mg/ml, 50 ( ⁇ g/ml, 25 ( ⁇ g/ml, 1254 g/). 11 concentrations of ml, 62.5 g/ml, 31.25 g/ml, 16 g/ml, 8 g/ml, 4 g/ml. Add cell suspension 200 ⁇ 1 of cultured cells (concentration IX 105 cells/ml) to 96 wells. The microplate was cultured at 37 °C with 5 % C02.
  • the culture solution was aspirated, and the wells were washed 3 times with sterile PBS solution, and the above-mentioned different concentrations of the drug solution were added, 200 ⁇ l per well, each concentration.
  • Eight replicate wells were cultured in a 37 ° C 5% CO 2 incubator and the results were observed dynamically.
  • the cytopathic effect (CPE) produced by the drug was recorded, and the half poisoning concentration of the drug was calculated when CPE was no longer developed (about 48 hours).
  • TD50 and maximum non-toxic concentration (TD0)
  • TD0 is the starting concentration of the drug in the formal experiment.
  • the highly pathogenic H5N1 avian influenza virus had a TCID50 of 104.29/0.2 ml for MDCK cells, and 100 TCID50 for antiviral experiments, which was used to dilute the virus stock solution 195 times for cell experiments; the maximum of flavonoids and ribavirin
  • the non-toxic concentrations (TD0) were 125 g/ml, 10 ( ⁇ g/ml, and the half-toxic concentrations (TD50) were 500 g/ml and 1976 g/ml, respectively.
  • the coagulation test measures the titer of the virus to determine the degree of virus proliferation.
  • the results of the experiment shown in Table 1 indicate that the plant flavonoid compound has a direct killing effect on the avian influenza virus in MDCK cells, and its EC50 is 3.40 ⁇ 0.39 ⁇ ⁇ / ⁇ 1, the value is 147.
  • the blood coagulation assay showed that 2 g/ml was able to effectively inhibit the proliferation of the virus compared with the virus control group (P ⁇ 0.05). With the increase of the concentration, the inhibitory effect was more obvious, and it was completely complete at 16 g/ml. Inhibits the occurrence of cytopathic effects.
  • the cells in the group grew normally, and the results were determined when the CPE of the virus control group reached (+ + +). The experiment was repeated three times. At the end of each experiment, the cell plates were repeatedly frozen and thawed three times to lyse the cells, and the virus was released. The cell lysates of each concentration group were frozen in EF at -20 °C for the hemagglutination test, and the titer of the cells was measured to determine the degree of virus proliferation. The results are shown in Table 2.
  • MDCK cells were plated in 96-well plates at 200 ⁇ l per well (lX105/ml). When the cells are covered with a single layer, the growth solution is added. According to Table 3, different concentrations of plant flavonoids are prepared by using the cell maintenance solution. Each concentration is 8 replicate wells, 200 ⁇ l per well, at 37 ° C 5% CO 2 incubator After 2 hours of action, remove the drug-containing maintenance solution, wash it three times with sterile PBS to remove the free drug, add 200 TCID50 virus 200 ⁇ 1, and put it into the 37 ° C 5% CO 2 incubator for 2 hours, then remove the virus solution, using sterile PBS.
  • the maximum non-toxic concentration of plant flavonoids on MDCK cells is ⁇ ⁇ g/ml, and it shows good control effect on H5N1 subtype avian influenza virus within a certain concentration range below this concentration, and is not toxic to cells.
  • Example 2 Inhibition of H5N1 avian influenza virus by a plant flavonoid in a chicken embryo model.
  • Avian influenza virus can quickly cause chicken embryo death after inoculation of chicken embryos, and 10 day old chicken embryos are used as experimental materials.
  • the above plant flavonoid compound solution was used as a stock solution, and serially diluted with sterile physiological saline, and a total of six concentration gradients of 20 mg/ml, 10 mg/ml, 5 mg/ml, 2.5 mg/ml, 1.25 mg/ M, 0.625 mg / ml, inoculated in the 10-day-old chicken embryo allantoic cavity, 0.2 ml per embryo, 5 chicken embryos per dilution, 96 h at 37 ° C, continuous observation of chicken embryo growth and development, The maximum concentration of chicken embryos for 96 h was used as the maximum non-toxic dose of the drug. At the same time, the toxic effect of the positive drug ribavirin on chicken embryos was obtained. The ribavirin was diluted with a total of six concentrations of 100 mg/ml as the stock solution, and the same plant flavonoids were inoculated and observed. Normal saline control was set during the experiment.
  • TD0 non-toxic concentration
  • the virus stock solution was diluted by 10 times, and the virus was inoculated into the 10-day-old chicken embryo allantoic cavity in a clean bench, 0.2 ml per embryo, sealed with paraffin, and incubated in a 37 ° C incubator.
  • the growth of chicken embryos was observed at 12h, and the embryos were turned twice a day.
  • the dead chicken embryos were discarded within 24 hours.
  • the chicken embryos that died after 24 hours were placed in a 4°C refrigerator. After 48 hours, all the chicken embryos were placed at 4°.
  • the virus solution of 100EID50 was mixed with the different concentration of the drug solution according to Table 4, and after being treated at 37 °C for 2 hours, it was inoculated into the chicken embryo allantoic cavity, 0.2 ml per embryo, 5 chicken embryos per dilution, 37° C was incubated for 48 hours (24h death was non-specific death), and was placed in a refrigerator at 4 °C overnight.
  • the chicken embryo allantoic fluid was harvested the next day for hemagglutination test. The results are shown in Table 4. In the above experiments, the virus control and the normal control (salt saline) were set at the same time, and the positive drug ribavirin control was simultaneously performed.
  • the concentration of plant flavonoids was 10 mg/ml, 5 mg/ml, 2.5 mg/ml, 1.25 mg/ml, and 0.625 mg/ml, which could effectively kill the virus and protect the chicken embryo from Attack by virus (P ⁇ 0.01 compared with virus control group); concentration of 100mg/ml, 50mg/ml ribavirin solution can effectively kill avian influenza virus (compared with virus control group hemagglutinin titer, the former P ⁇ 0.01, the latter P ⁇ 0.05), and the effect of killing avian influenza virus at each concentration below 25mg/ml was not significant (P>0.05).
  • each embryo was inoculated with 0.1 ml of 100 EID50 avian influenza virus (5 chicken embryos per group), 37 °C for 2 h, and then 0.1 ml of each dilution of the drug was inoculated into the chicken embryo allantoic cavity, and incubated at 37 ° C for 48 h.
  • the refrigerator was overnight at 4 °C, and each chicken embryo allantoic fluid was harvested the next day for hemagglutination test.
  • Table 4 In the above experiments, both the virus control and the normal control (saline) were set, and the positive drug ribavirin control was performed simultaneously.
  • each chicken dilution was 5 chicken embryos, 37 °C 2h, then inoculated with 100EID50 avian influenza virus O.lml, incubated at 37 °C for 48h, into 4 °C The refrigerator was overnight, and the chicken embryo allantoic fluid was harvested the next day for hemagglutination test.
  • Table 4 In the above experiments, both the virus control and the normal control (saline) were set at the same time, and the positive drug ribavirin control was simultaneously performed.
  • the maximum non-toxic concentration of plant flavonoids on chicken embryos is 10 g/ml, and it has significant direct effects, therapeutic effects and certain preventive effects on H5N1 subtype avian influenza virus within a certain concentration range below this concentration. , non-toxic to chicken embryos.
  • Example 3 Test of flavonoids against mouse strain of H5N1 subtype avian influenza Experimental animals: 320 SPF ICR mice, weighing 20 ⁇ 2g, male and female; provided by Shanghai Slack Laboratory Animals Co., Ltd. (qualified Certificate No.: 0002710, License No.: SCXK (Shanghai) 2003— 003). Twenty mice were randomly selected before the experiment to confirm the infection of the avian influenza virus in the mice by hemagglutination test and hemagglutination inhibition test, and other respiratory infections were excluded by the test.
  • the animal experiment was carried out in the third-level biosafety laboratory of the Laboratory Animal Monitoring Institute of Guangdong province, which met the three-level biosafety laboratory standard. All items are autoclaved before entering the laboratory Or ultraviolet radiation or immersion disinfection, thoroughly pervaporate with potassium permanganate-formaldehyde solution before the experimental space experiment.
  • mice were randomly divided into 8 groups for observation of mouse survival rate, serum antibody, organ index and pathological changes, body weight and body temperature, respectively, high dose group of plant flavonoids (group I, 54 mg/ml).
  • Plant flavonoids in the middle dose group group II, 18mg/ml
  • plant flavonoids low dose group ⁇ group, 6mg/ml
  • very low dose group group IV, 2mg/ml
  • positive drug ribavirin control Group V group, 6 mg/ml
  • amantadine hydrochloride control group VI group, 2.5 mg/ml
  • virus control group VII group
  • normal control group VIII group
  • Another 120 animals were randomly divided into 8 groups.
  • the lungs and brain were used to isolate the virus, and the degree of proliferation of the virus in the lung and brain was observed.
  • the high dose group of plant flavonoids (group I, 54 mg/ml) and medium dose were respectively.
  • the amantadine hydrochloride control group (VI group, 2.5 mg/ml) ; the virus control group (VD group) and the normal control group (Shan group), 15 rats in each group.
  • Male and female are divided into words.
  • the animals were anesthetized with 0.1 ml/20 g of ketamine (20 mg/ml), and then the avian influenza virus allantoic fluid was instilled from the nasal cavity (the blood coagulation titer was 1: 256). Amount of virus-free chicken embryo allantoic fluid.
  • the animals in the I-IV group were intragastrically infused with high-dose (54mg/ml), medium-dose (18mg/ml), low-dose (6mg/ml), very low-dose (2mg/ml) flavonoids.
  • mice The clinical symptoms (including behavior, hair luster, respiration, appetite, etc.) and survival of each group of mice were observed every day.
  • the animals were found to die or die, and the animals were immediately sacrificed by eyeball removal.
  • the lungs, heart, liver, spleen and kidney were taken.
  • the tissue was accurately weighed (accurate to 0.1 g) and the liver, kidney, and lungs were fixed in 10% neutral buffered formalin.
  • mice in each experimental group used to isolate the virus were sacrificed 3 mice in each group on the 0th, 7th day of the experiment, and the lungs were aseptically taken.
  • the brain was placed in an EP tube. After homogenization, 0.5 ml of sterile physiological saline was added to each tube, and 0.2 ml of each was taken to inoculate 10 day old chicken embryos. After 48 hours of culture, the allantoic fluid was taken for virus hemagglutination test.
  • mice used to observe the survival rate, serum antibody, organ index and pathological changes, body weight, and body temperature of the mice were at 0, 3, 6, 9, 12,
  • the surviving mice were weighed and measured for body temperature for 15 days, and the changes in body weight and body temperature at each time point were calculated.
  • each time each method was collected by the tail-breaking method.
  • Four mice in the experimental group were treated with tail vein blood, and serum was separated for antibody analysis to understand the dynamic changes of antibody levels in mice and the effect of drugs on mouse antibody levels. Serum antibodies were tested using the hemagglutination inhibition test.
  • all rats were sacrificed. After liver, kidney, spleen, lung and heart were accurately weighed, liver, kidney and lung were fixed in 10% neutral buffered formalin.
  • mice 1 Comparison of mortality in mice 1 The survival of experimental mice is shown in Table 5. The results showed that the concentration of flavonoids in plants had significant inhibitory effects on avian influenza virus (P ⁇ 0.05).
  • the avian flu virus is widely distributed in many poultry and wild birds worldwide, as well as pigs, horses, and a variety of mammals such as whales, ferrets, and dolphins. Variability of its virulence The range is very large, depending on the virus type and subtype, animal species, age, concurrent infection, surrounding environment and host immune status.
  • the disease caused by avian influenza virus infection may be inconspicuous or mild transient syndrome, or it may be a disease with 100% morbidity and/or mortality. Therefore, for highly pathogenic avian influenza viruses, the ability to protect infected animals or humans from death is an indicator of drug effectiveness, which is also the purpose of research and development of drugs.
  • Figure I is a comparison of the different compositions of experimental mice.
  • the results show that compared with the virus control group, the high, middle and low concentration groups of plant flavonoids and the amantadine hydrochloride control group can significantly improve infected mice. Survival rate; the ribavirin control group, the very low dose group of plant flavonoids, although it can also improve the survival rate of mice to some extent, but there is no significant difference compared with the virus control group.
  • the high, medium and low dose groups of plant flavonoids have the same anti-avian influenza effect as amantadine and are superior to ribavirin.
  • Table 6 is a statistical table showing the survival rate, mortality, etc. of different groups of experimental mice, indicating that plant flavonoids can effectively protect mice from death caused by avian influenza infection within a certain dose range. VIII were high dose group, medium dose group, low dose group, very low dose group, ribavirin group, diamond sputum group, virus control group and normal control group.
  • mice The changes in virus proliferation in the lungs of mice as shown in Table 7 and Figure II are shown.
  • the 54 mg/ml group of the flavonoids, the 18 mg/ml group, the 6 mg/ml group and the amantadine group significantly inhibited the proliferation of the virus; on the seventh day, each drug group was It can significantly inhibit the proliferation of the virus; no virus was detected in each drug group except the low titer virus was detected in the brain control group.
  • Serum antibody levels in mice are shown in Table 8 and Figure III. Compared with the virus control group, the plant flavonoids 54 mg/ml group, the 18 mg/ml group and the amantadine group promoted the secretion of mouse antibodies, and the other drug groups had a slight effect on the secretion of mouse antibodies.
  • each drug group can effectively reduce the lung index of mice, and has little effect on other organ indexes; each drug group can effectively reduce the pathological changes of liver, kidney and lung.
  • the drug groups alleviated the weight loss of the mice three days before the experiment. After three days, each drug group could effectively promote the weight gain of the mice.
  • plant flavonoids can significantly improve the survival rate of model mice in a dose-dependent manner; it can effectively inhibit the proliferation of virus in the lungs of model mice, inhibit the invasion of the virus into the brain; can effectively activate the body fluids of the body
  • the immune system enables high-level titer expression of serum antibodies in the body; it can effectively promote the repair of lung, liver, kidney and other tissue and organ diseases caused by avian influenza virus infection; it can significantly inhibit the weight loss of model mice and promote weight Pick-up; can accelerate the normalization of the body temperature of the model mice and shorten the condition of the animal to some extent.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Virology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Pulmonology (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Epidemiology (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

The present invention relates to a use of Phyto-flavonoid compounds for preparing medicaments against Avian influenzal virus. The Phyto-flavonoid compounds can directly kill Avian influenzal virus, treat and prevent infections caused by Avian influenzal virus, and can be used to prepare medicaments and additives against Avian influenzal virus. The effective dosages of the said medicaments and additives are 20-250mg/kg weight/day. Phyto-flavonoid compounds are natural components can be obtained from plants and they have obvious inhibitory effect on Avian influenzal virus. The medicaments and additives prepared by the Phyto-flavanoid compoundsof present invention have advantages such as safety and validity because of their lower toxicity and stronger activity. Thus, Phyto-flavonoid compounds of present invention offer a new use for treating and preventing Avian-influenzal virus.

Description

植物黄酮类化合物抗禽流感病毒的用途  Use of plant flavonoids against avian influenza virus
技术领域 Technical field
本发明涉及化合物的用途， 具体是涉及植物黄酮类化合物抗禽流 感病毒的用途。 背景技术  This invention relates to the use of compounds, in particular to the use of plant flavonoids against avian influenza viruses. Background technique
禽流感（Avian influenza, AI)是由 A型流感病毒引起的禽鸟类的 严重传染性疾病， 感染的动物是鹅、 鸡、 鸭、 鸽子以及一些野生鸟类 等。 流行病学研究显示， 禽流感病毒具有高致病性和很强的传播能力， 它不但会给养禽业带来巨大的损失， 而且已经有渠道使病毒从鸡传播 到人。 禽流感病毒属于正粘病毒科流感病毒属， 具有单股负链 R A， 禽流感病毒粒子一般为球形， 其表面有两种不同形状的突起： 血凝素 (HA)和神经氨酸酶 (NA)。 HA和 NA是病毒表面的主要糖蛋白，具有种 (亚型)的特异性和多变性， 在病毒感染过程中起着重要作用。 HA是决 定病毒致病性的主要抗原成分， 能诱发感染宿主产生具有保护作用的 中和抗体， 而 NA诱发的对应抗体无病毒中和作用， 但可减少病毒增 殖和改变病程。 迄今已知有 16种 HA和 10种 NA， 不同的 HA和 NA 之间可能发生不同形式的随机组合， 从而构成许许多多不同亚型。 据 报道现已发现的流感病毒亚型至少有 80'多种， 其 绝大多数属非致病 性或低致病性， 高致病性亚型主要是含 H5和 H7的毒株。 Avian influenza (AI) is a serious infectious disease caused by influenza A viruses. The infected animals are geese, chickens, ducks, pigeons and some wild birds. Epidemiological studies have shown that the avian influenza virus is highly pathogenic and has a strong ability to spread. It not only causes huge losses to the poultry industry, but also has channels to spread the virus from chicken to humans. The avian influenza virus belongs to the genus Influenza virus of the Orthomyxoviridae family and has a single negative strand RA. The avian influenza virions are generally spherical and have two different shapes of protrusions on the surface: hemagglutinin (HA) and neuraminidase (NA) ). HA and NA are the major glycoproteins on the surface of the virus. They have the specificity and variability of species (subtypes) and play an important role in the process of viral infection. HA is the main antigenic component that determines the pathogenicity of a virus. It can induce a neutralizing antibody with a protective effect on the infected host, while the NA-induced corresponding antibody has no virus neutralization, but can reduce the virus proliferation and change the course of the disease. To date, 16 kinds of HA and 10 kinds of NA have been known, and different forms of random combinations may occur between different HAs and NAs, thereby constituting many different subtypes. It has been reported that at least 80' influenza virus subtypes have been found, most of which are non-pathogenic or low pathogenic, and the highly pathogenic subtypes are mainly strains containing H 5 and H 7 .
AI历来被认为是人类流感的最大基因库， 是人流感病毒发生变异 的新基因来源， 不过在自然状态下， AI与人流感之间的联系需要通过 中间宿主 （如猪、 马、 海豚等哺乳动物） 来实现。 但 1997年中国香港 H5N1以及 1999年中国大陆、 香港 H9N2 AIV首次突破种间障碍直接 感染人甚至引起了人的死亡， 不但打破了自然条件下仅有 Hl、 H2、 H3亚型流感病毒可以感染人和其它哺乳动物的常规， 而且为人流感的 流行增添了新的毒株亚型（H5和 H9), 由此又赋予了 AIV全新的公共 卫生意义。  AI has always been regarded as the largest gene pool of human influenza, a new source of genetic mutations in human influenza, but in the natural state, the link between AI and human influenza needs to be breast-fed through intermediate hosts (such as pigs, horses, dolphins, etc.). Animals) to achieve. However, in 1997, H5N1 in Hong Kong, China, and H9N2 AIV in mainland China and Hong Kong in 1999 broke through the interspecies disorder for the first time. It even caused human death. It not only broke the natural conditions, but only H1, H2, H3 subtype influenza viruses can infect people. And the regularity of other mammals, and the addition of new strain subtypes (H5 and H9) to the human influenza epidemic, which in turn gives AIV a new public health significance.
目前， 对付禽流感尤其是高致病性禽流感病毒的感染， 除了用疫 苗进行紧急预防外， 还没有特效的防治药物。 用于抗流感的药物主要 有神经氨酸酶抑制剂如： 扎那米韦 （Zanamivir ) 及奥司米韦 (Oseltamivir)等； 离子通道阻滞剂如金刚烷胺 （Amantadine)和金刚 乙胺 （rimantadine), 它们被认为是治疗和预防流感病毒的首选药物； 以及核苷类药物： 三氮唑核苷即利巴韦林 (Ribavirin)亦称病毒唑。 At present, in response to the infection of avian influenza, especially the highly pathogenic avian influenza virus, there is no specific preventive drug other than emergency prevention with a vaccine. Mainly used for anti-influenza drugs There are neuraminidase inhibitors such as: Zanamivir and Oseltamivir; ion channel blockers such as Amantadine and rimantadine, which are considered to be The drug of choice for the treatment and prevention of influenza virus; and nucleoside drugs: ribavirin, ribavirin, also known as ribavirin.
扎那米韦是根据流感病毒 NA与唾液酸的复合物结构， 通过计算 机分子模拟设计而成， 结构中的胍基与流感病毒 NA活性部位的氨基 酸通过氢键、 静电力及范德华力的作用， 与酶紧密结合， 作用强度及 选择性均较高； 扎那米韦对 B型流感病毒也有一定程度的结合。 结构 式如式 （1)。  Zanamivir is based on the complex structure of influenza virus NA and sialic acid. It is designed by computer molecular simulation. The thiol group in the structure and the amino acid of the active part of influenza virus NA pass hydrogen bond, electrostatic force and van der Waals force. Tightly bound to the enzyme, the intensity and selectivity are high; zanamivir also has a certain degree of binding to the influenza B virus. The structure is as shown in formula (1).
奥司米韦是 GS4071的乙酯型前药，其亲脂性的 3-戊氧基侧链与流 感病毒 NA活性部位的疏水性口袋有较强的亲和力， 阻断了流感病毒 NA对病毒感染细胞表面的唾液酸残基的裂解，从而抑制了病毒颗粒从 感染细胞的释放， 因而是一种选择性高的流感病毒 NA抑制剂。 结构 式如式 （11)。  Osmivir is an ethyl ester-type prodrug of GS4071, and its lipophilic 3-pentyl side chain has a strong affinity with the hydrophobic pocket of the active site of the influenza virus NA, blocking the influenza virus NA against virus-infected cells. The cleavage of the surface sialic acid residues inhibits the release of viral particles from infected cells and is therefore a highly selective influenza virus NA inhibitor. The structure is as shown in equation (11).
Figure imgf000004_0001
Figure imgf000004_0001
盐酸金刚烷胺， 英文名： Amantadine Hydrochloride Tablets, 其化 学名称为： 三环 [3， 3, 1， 13,7]癸垸 -1-胺盐酸盐， 其结构式如式（111)。 Amantadine hydrochloride, English name: Amantadine Hydrochloride Tablets, its chemical name is: tricyclic [3, 3, 1, 13, 7] 癸垸 -1-amine hydrochloride, its structural formula is (111).
利巴韦林 (病毒唑)， 英文名： Ribavirin Injection, 主要成份化学名 称为： 1-b-D-呋喃核糖基 -1Η1,2,4，-三氮唑 -3-羧酰胺。 分子式： C8H12 Ν405, 分子量： 244.21。 结构式如式 （IV)。  Ribavirin (ribavirin), English name: Ribavirin Injection, the main chemical name: 1-b-D-ribofuranosyl-1,2,4,-triazole-3-carboxamide. Molecular formula: C8H12 Ν405, molecular weight: 244.21. The structural formula is as shown in formula (IV).
Figure imgf000004_0002
Figure imgf000004_0002
2 IV  2 IV
替换页（细则第 26条） 但这些药物都存在一定的局限性： 神经氨酸酶抑制剂类能有效地 抑制 A、 B 型流感病毒， 此类药物较为昂贵限制其推广使用； 离子通 道阻滞剂对 A型流感病毒也具有良好的抑制作用， 但存在神经毒性、 长期用药易产生耐药性， 易引起流感的大流行， 并对 B型流感病毒无 效等缺陷； 三氮唑核苷（病毒唑）对 RNA、 DNA病毒引起的感染均有 效， 但此类化合物具有一定的致畸作用， 限制了其在临床的应用。 寻 找安全、 有效、 来源广泛， 价廉低毒的抗禽流感药物也就显得尤为迫 切和重要。 Replacement page (Article 26) However, these drugs have certain limitations: Neuraminidase inhibitors can effectively inhibit influenza A and B viruses, which are more expensive and restrict their use; ion channel blockers also have influenza A virus. Good inhibition, but there are neurotoxicity, long-term drug susceptibility to drug resistance, flu-like pandemic, and ineffectiveness of influenza B virus; ribavirin (ribavirin) caused by RNA and DNA viruses The infections are all effective, but such compounds have a certain teratogenic effect, which limits their clinical application. It is particularly urgent and important to find anti-avian flu drugs that are safe, effective, and have a wide range of sources.
已报道黄酮类化合物具有抗氧化、 抗血栓、 抗癌、 抗炎、 调节血 管渗透性、 抗抑郁、 抗焦虑、 抗脑缺血、 护肝、 镇痛、 对消化性溃疡 的保护作用以及抗心律失常和抗心肌缺血等多种作用， 但植物黄酮类 化合物应用于禽流感病毒防治尚无报道。  Flavonoids have been reported to have antioxidant, antithrombotic, anticancer, anti-inflammatory, vascular vaso-regulating, anti-depressant, anti-anxiety, anti-cerebral ischemia, liver protection, analgesia, protection against peptic ulcer and anti-heart rhythm Abnormalities and anti-myocardial ischemia and other effects, but the application of plant flavonoids in the prevention and treatment of avian influenza virus has not been reported.
黄酮类化合物是一大类低分子天然植物成分， 通指两个苯环通过 中央三碳链相互联结而成的一系列化合物类，均具有 C6-C3-C6的基本 结构， 主要包括黄酮 （flavone主要结构如式 （V〉）及其异构体异黄酮 (isoflavone)类； 黄垸酮（flavanone主要结构如式 （VI) )及其异构体异 黄垸酮 (isoflavanone)类； 査尔酮 (chalcone主要结构如式（VII) )类； 桑 酮 （Xanthone主要结构如式 （VIII)); 黄酮醇 (flavonol主要结构如式 (IX) )类及其异构体异黄酮醇 (isoflavonol)类等。  Flavonoids are a large class of low molecular natural plant constituents. They refer to a series of compounds in which two benzene rings are linked by a central three carbon chain. They all have the basic structure of C6-C3-C6, mainly including flavones. The main structure is as shown in formula (V>) and its isoflavone class; xanthone (flavanone main structure such as formula (VI)) and its isomer isoflavone (isoflavanone); chalcone (chalcone main structure such as formula (VII)); mulberry (Xanthone main structure such as formula (VIII)); flavonol (flavonol main structure such as formula (IX)) and its isomer isoflavoneol (isoflavonol) Wait.
Figure imgf000005_0001
Figure imgf000005_0002
本发明中所称的植物黄酮类化合物的化学名称为 5，7-二羟基类黄 酮， 分子结构如式（X)， 其中 R1为 H或 OH; R2为 OH或 3，4,5-三羟 基苯甲酰 （galloyl), 3,4,5-三羟基苯甲酰的结构如式 （XI)。 可从传统 中药蔷薇科植物中提取得到， 例如硕苞蔷薇 （Rosa bracteata Wendl. ) 即含有植物黄酮类化合物。 硕苞蔷薇， 又名苞蔷薇、 猴柿等， 为常绿 灌木， 高 2-5米， 广泛分布于华中和华南地区。
Figure imgf000005_0001
Figure imgf000005_0002
The chemical name of the plant flavonoid compound referred to in the present invention is 5,7-dihydroxyflavone, and the molecular structure is as shown in the formula (X), wherein R1 is H or OH; R2 is OH or 3,4,5-trihydroxyl The structure of galloyl, 3,4,5-trihydroxybenzoyl is of formula (XI). It can be extracted from traditional Chinese medicine Rosaceae plants, for example, Rosa bracteata Wendl. contains plant flavonoids. Shuo Wei, also known as rose, monkey persimmon, etc., is an evergreen shrub, 2-5 meters high, widely distributed in Central China and South China.
Figure imgf000006_0001
Figure imgf000006_0001
发明内容 Summary of the invention
本发明的目的是提供植物黄酮类化合物抗禽流感病毒的用途。 本发明发明人的研究表明， 植物黄酮类化合物能直接杀灭 （伤） 禽流感病毒， 可以用于治疗和预防禽流感病毒的感染， 能在制备抗禽 流感病毒的药物和添加剂中得到广泛应用。  It is an object of the present invention to provide the use of plant flavonoids against avian influenza viruses. The inventors of the present invention have shown that plant flavonoids can directly kill (injure) avian influenza virus, can be used for treating and preventing infection of avian influenza virus, and can be widely used in preparing drugs and additives for avian influenza virus. .
在以植物黄酮类化合物为活性成分的药物中， 需要的时候还可以 加入一种或多种药学上可接受的载体。 所述载体包括药学领域常规的 稀释剂、 赋形剂、 填充剂、 粘合剂、 湿润剂、 崩解剂、 吸收促进剂、 表面活性剂、 吸附载体、 润滑剂等。  In the case where the plant flavonoid compound is used as an active ingredient, one or more pharmaceutically acceptable carriers may be added as needed. The carrier includes conventional diluents, excipients, fillers, binders, wetting agents, disintegrating agents, absorption enhancers, surfactants, adsorption carriers, lubricants and the like in the pharmaceutical field.
本发明的药物可通过口服、 注射或粘膜给药， 可以制成片剂、 胶 囊、 粉剂、 颗粒、 锭剂、 栓剂， 或口服液或无菌胃肠外悬液液体制剂 等多种药物形式， 还有大或小容量注射剂、 冻干粉针或无菌粉分装剂 等针剂形式。 上述各种剂型的药物均可以按照药学领域的常规方法制 备。  The medicament of the present invention can be prepared into a plurality of pharmaceutical forms such as tablets, capsules, powders, granules, troches, suppositories, or oral liquids or sterile parenteral suspension liquid preparations by oral, injection or mucosal administration. There are also injection forms such as large or small volume injections, freeze-dried powder needles or sterile powder dispensing agents. The above various dosage forms of the drug can be prepared according to a conventional method in the field of pharmacy.
4  4
替换页（细则第 26条） 将植物黄酮类化合物以词料添加剂的形式加入到禽类饲料中， 可 以提高禽类预防禽流感病毒感染的能力。 Replacement page (Article 26) The addition of plant flavonoids to poultry feed in the form of a word additive enhances the ability of birds to prevent avian influenza virus infection.
以植物黄酮类化合物为主要成分的抗禽流感病毒的药物或添加 剂， 其有效用量为 20— 250mg/kg体重，天； 优选剂量为 60— 180mg/kg 体重 ·天。  The anti-avian influenza virus drug or additive containing the plant flavonoid compound as an active ingredient is used in an amount of 20-250 mg/kg body weight, and the preferred dose is 60-180 mg/kg body weight·day.
植物黄酮类化合物作为一种天然植物成分， 对禽流感病毒具有明 显的抑制作用， 制备成药物或添加剂， 具有毒性小、 作用强、 安全有 效等优点， 为禽流感病毒的防治开辟新的途径， 具有重要的社会价值、 经济价值和广阔的应用前景。 附图说明  As a natural botanical component, plant flavonoids have obvious inhibitory effects on avian influenza virus, and are prepared into drugs or additives. They have the advantages of low toxicity, strong action, safety and effectiveness, and open up new ways for the prevention and control of avian influenza virus. It has important social value, economic value and broad application prospects. DRAWINGS
图 1为实验小鼠不同组成活率比较图  Figure 1 is a comparison of the different compositional viability of experimental mice.
图 2为小鼠肺内病毒增殖随时间变化曲线  Figure 2 is a graph showing the relationship between virus proliferation and time in mouse lung.
图 3为小鼠血清抗体随时间变化曲线 具体实施方式  Figure 3 is a graph showing changes in serum antibody of mice over time.
实施例一、 植物黄酮类化合物在 MDCK细胞模型中的抗 H5N1禽 流感病毒作用  Example 1. Anti-H5N1 avian influenza virus effect of plant flavonoids in MDCK cell model
大量研究表明，禽流感病毒能在 MDCK细胞中产生较明显的病变， 本发明利用 MDCK细胞进行体外抗 H5N1亚型禽流感病毒的药效学试 验。  Numerous studies have shown that avian influenza virus can produce more obvious lesions in MDCK cells. The present invention utilizes MDCK cells for the pharmacodynamic test of anti-H5N1 subtype avian influenza virus in vitro.
MDCK细胞： 购于武汉大学中国典型培养物保藏中心。  MDCK cells: purchased from the China Center for Type Culture Collection of Wuhan University.
高致病性 H5N1 禽流感病毒： 由华南农业大学提供， 并经中国农 科 院 哈 尔 滨 兽 医 研 究 所 鉴 定 ， 毒 株 编 号 为 ： A/goose/Guangdong NH/2003(H5) (分离方法来源： 中国疾病预防控制 中心， 《禽流感实验室检测方法》。 Characterization of an Avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness. Kanta Subbarao, Alexander Klimov, et al. Science (1998), 279:393 -396)。  Highly pathogenic H5N1 avian influenza virus: supplied by South China Agricultural University and identified by Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences. The strain number is: A/goose/Guangdong NH/2003(H5) (Source of isolation method: Chinese disease Prevention and Control Center, Avian Influenza Laboratory Test Method. Characterization of an Avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness. Kanta Subbarao, Alexander Klimov, et al. Science (1998), 279:393 -396).
将病毒原液作 100倍稀释， 并在超净工作台中将病毒接种于 10日 龄的鸡胚尿囊腔中， 每胚 0.2ml， 石蜡封口， 放于 37°C温箱中孵育， 每 12h观察鸡胚生长情况， 并每天翻胚 2次， 将 24h内死亡的鸡胚弃去， 24h后死去的鸡胚放入 4°C冰箱， 48小时后将所有的鸡胚均放入 4°C冰 箱过夜， 次日无菌收集尿囊液， 3000rpm离心 15min， 取上清进行血凝 试验， 以血凝滴度大于 1 : 256的病毒液作实验材料， 分装 -20 °C贮存备 用。 The virus stock was diluted 100-fold, and the virus was inoculated into the 10-day-old chicken embryo allantoic cavity in a clean bench, 0.2 ml per embryo, sealed with paraffin, and incubated in a 37 ° C incubator. The growth of chicken embryos was observed at 12h, and the embryos were turned twice a day. The dead chicken embryos were discarded within 24 hours. The chicken embryos that died after 24 hours were placed in a 4°C refrigerator. After 48 hours, all the chicken embryos were placed at 4°. C refrigerator overnight, the next day aseptic collection of allantoic fluid, centrifuged at 3000rpm for 15min, the supernatant was taken for hemagglutination test, the virus liquid with a blood coagulation titer greater than 1: 256 was used as experimental material, and stored at -20 °C for storage.
植物黄酮类化合物： 由中山大学生命科学院海洋药物研究所提供。 称取 200mg植物黄酮类化合物溶于 50ml细胞维持液中，待其充分 溶解后， 用 0.22μΜ滤膜过滤除菌，无菌试验 96h(-)， 分装， 4°C贮存备 用。  Plant flavonoids: Provided by the Institute of Marine Medicine, School of Life Sciences, Sun Yat-sen University. 200 mg of the plant flavonoid compound was weighed and dissolved in 50 ml of the cell maintenance solution. After it was sufficiently dissolved, it was sterilized by filtration through a 0.22 μM filter, sterilized for 96 h (-), and dispensed at 4 ° C for storage.
1、 植物黄酮类化合物和病毒唑对 MDCK细胞的最大无毒浓度 将浓度为 1 X 105个 /ml的 MDCK细胞加入 96孔板， 每孔 200μ ， 细胞铺满单层后，用维持液稀释病毒，按 10倍递减，从病毒原液至 10-9， 每个浓度 8个复孔， 正常细胞对照加维持液， 放入 37°C5 %C02细胞 培养箱中孵育， 观察细胞病变效应 （cytopathic effect ， CPE) , 釆用 Reed-Muench法计算 TCID50。 进行植物黄酮类化合物抗 H5N1亚型禽 流感病毒细胞实验时以 100TCID50为病毒接种量。  1. Maximum non-toxic concentration of plant flavonoids and ribavirin on MDCK cells MDCK cells at a concentration of 1×105 cells/ml were added to a 96-well plate at 200 μ per well. After the cells were covered with a monolayer, the virus was diluted with a maintenance solution. Decrease by 10 times, from virus stock to 10-9, 8 replicate wells per concentration, normal cell control plus maintenance solution, and incubate in a 37 °C 5% C02 cell incubator to observe the cytopathic effect. CPE) , TC Calculate TCID50 using the Reed-Muench method. When the plant flavonoid compound was tested against the H5N1 subtype avian influenza virus cell test, 100 TCID50 was used as the virus inoculum.
依次将药物 （植物黄酮类化合物和病毒唑） 对倍稀释至 4 g/ml， 即 4mg/ml、 2mg/ml、lmg/ml、50(^g/ml、25(^g/ml、1254g/ml、62.5 g/ml、 31.25 g/ml、 16 g/ml、 8 g/ml、 4 g/ml共 11个浓度。 将培养细胞 （浓 度 I X 105个 /ml) 的细胞悬液 200μ1加入 96孔微量培养板， 37°C 5 % C02培养， 待细胞铺满单层后， 吸出培养液， 用无菌 PBS液洗板孔 3 次， 加入上述不同浓度的药液， 每孔 200μ1， 每个浓度 8个复孔， 然后 在 37°C 5 %C02培养箱中培养并动态观察结果， 记录由药物产生的细 胞病变效应 （CPE) , 计算 CPE不再进展时 （约 48小时） 药物的半数 中毒浓度 （TD50) 和最大无毒浓度 （TD0)。 并以 TD0 为正式实验时 的药物起始浓度。  The drug (phytoflavonoids and ribavirin) was serially diluted to 4 g/ml, ie 4 mg/ml, 2 mg/ml, 1 mg/ml, 50 (^g/ml, 25 (^g/ml, 1254 g/). 11 concentrations of ml, 62.5 g/ml, 31.25 g/ml, 16 g/ml, 8 g/ml, 4 g/ml. Add cell suspension 200μ1 of cultured cells (concentration IX 105 cells/ml) to 96 wells. The microplate was cultured at 37 °C with 5 % C02. After the cells were covered with a single layer, the culture solution was aspirated, and the wells were washed 3 times with sterile PBS solution, and the above-mentioned different concentrations of the drug solution were added, 200 μl per well, each concentration. Eight replicate wells were cultured in a 37 ° C 5% CO 2 incubator and the results were observed dynamically. The cytopathic effect (CPE) produced by the drug was recorded, and the half poisoning concentration of the drug was calculated when CPE was no longer developed (about 48 hours). (TD50) and maximum non-toxic concentration (TD0), and TD0 is the starting concentration of the drug in the formal experiment.
结果表明， 高致病性 H5N1禽流感病毒对 MDCK细胞的 TCID50 为 104.29/0.2ml， 进行抗病毒实验时用 100TCID50 即将病毒原液稀释 195 倍后用于细胞实验； 植物黄酮类化合物及病毒唑的最大无毒浓度 (TD0) 分别为 125 g/ml， 10(^g/ml， 半数中毒浓度 （TD50) 分别为 500 g/ml、 1976 g/ml。 2、 植物黄酮类化合物对 MDCK细胞内病毒的直接杀伤作用 先将表 1中不同浓度的植物黄酮类化合物溶液和 100TCID50病毒 液等体积混合， 作用 2h后接种于长满单层细胞的 96 孔板中， 每孔 200μ1, 每个浓度 8个复孔， 放入 37°C5 %C02培养箱中培养。 每 12h 观察细胞生长情况， 设病毒对照组及正常细胞对照组， 待细胞对照生 长正常， 病毒对照 CPE达 （ + + + ) 以上时判定结果， 拍照。 实验重 复三次， 每次实验结束时将细胞板反复冻融三次， 使细胞裂解， 病毒 释放， 分别收集每次实验各浓度组细胞裂解液于 EP管中， -20°C中冻 存， 进行血凝试验， 测其效价以判定病毒的增殖程度， 结果如表 1 所 实验结果表明， 植物黄酮类化合物对 MDCK细胞内禽流感病毒具 有直接杀伤作用， 其作用的 EC50为 3.40±0.39μβ/πι1， Ή值为 147。血 凝测定显示： 与病毒对照组相比， 2 g/ml即能有效抑制病毒的增殖（P <0.05)， 随着浓度的增加， 其抑制效果愈加明显， 至 16 g/ml时已能 完全抑制细胞病变的发生。 The results showed that the highly pathogenic H5N1 avian influenza virus had a TCID50 of 104.29/0.2 ml for MDCK cells, and 100 TCID50 for antiviral experiments, which was used to dilute the virus stock solution 195 times for cell experiments; the maximum of flavonoids and ribavirin The non-toxic concentrations (TD0) were 125 g/ml, 10 (^g/ml, and the half-toxic concentrations (TD50) were 500 g/ml and 1976 g/ml, respectively. 2. Direct killing effect of plant flavonoids on MDCK intracellular virus First, the different concentrations of plant flavonoid solution in Table 1 and 100TCID50 virus solution were mixed in equal volume. After 2 hours, they were inoculated into a 96-well plate filled with monolayer cells. Medium, 200 μl per well, 8 replicate wells per concentration, placed in a 37 ° C 5% CO 2 incubator. The growth of the cells was observed every 12 hours. The virus control group and the normal cell control group were set up until the cell control grew normally. When the virus control CPE reached (+ + +), the results were determined and photographed. The experiment was repeated three times. At the end of each experiment, the cell plates were repeatedly frozen and thawed three times to lyse the cells, and the virus was released. The cell lysates of each concentration group were collected in EP tubes, frozen at -20 ° C, and blood was taken. The coagulation test measures the titer of the virus to determine the degree of virus proliferation. The results of the experiment shown in Table 1 indicate that the plant flavonoid compound has a direct killing effect on the avian influenza virus in MDCK cells, and its EC50 is 3.40±0.39μ β / Πι1, the value is 147. The blood coagulation assay showed that 2 g/ml was able to effectively inhibit the proliferation of the virus compared with the virus control group (P < 0.05). With the increase of the concentration, the inhibitory effect was more obvious, and it was completely complete at 16 g/ml. Inhibits the occurrence of cytopathic effects.
表 1. 植物黄酮类化合物 I对 MDCK细胞内病毒的直接杀伤作用 Table 1. Direct killing effect of plant flavonoids I on MDCK intracellular virus
药物浓度 Log 培养 病变孔数 （病变百分率） 血凝效价  Drug concentration Log culture Number of lesions (percentage of lesions) Blood coagulation titer
( g/ml) 孔数 I II III log(x +s)  ( g/ml) Number of holes I II III log(x +s)
125 2.097 8 0 (0) 0 (0) 0 (0) ―  125 2.097 8 0 (0) 0 (0) 0 (0) ―
62.5 1.796 8 0 (0) 0 (0) 0 (0) ―  62.5 1.796 8 0 (0) 0 (0) 0 (0) ―
31.25 1.495 8 0 (0) 0 (0) 0 (0) ―  31.25 1.495 8 0 (0) 0 (0) 0 (0) ―
16 1.204 8 0 (0) 0 (0) 0 (0) ―  16 1.204 8 0 (0) 0 (0) 0 (0) ―
8 0.903 8 1(12.5) 1(12.5) 1(12.5) 0.50±0.17 * * 8 0.903 8 1(12.5) 1(12.5) 1(12.5) 0.50±0.17 * *
4 0.602 8 3(37.5) 2(25) 3(37.5) 1.11 ±0.15 * *4 0.602 8 3(37.5) 2(25) 3(37.5) 1.11 ±0.15 * *
2 0.301 8 5(62.5) 4(50) 5(62.5) 1.41 ±0.16 *2 0.301 8 5(62.5) 4(50) 5(62.5) 1.41 ±0.16 *
1 0 8 8(100) 7(87.5) 8(100) 2.21 ±0.171 0 8 8(100) 7(87.5) 8(100) 2.21 ±0.17
0.5 -0.301 8 8(100) 8(100) 8(100) 2.51 ±0·140.5 -0.301 8 8(100) 8(100) 8(100) 2.51 ±0·14
0.25 -0.602 8 8(100) 8(100) 8(100) 2.41 ±0.30 阳性对照 8 8(100) 8(100) 8(100) 2.61 ±0.15 阴性对照 8 0(0) 0(0) 0(0) ― 0.25 -0.602 8 8(100) 8(100) 8(100) 2.41 ±0.30 Positive control 8 8(100) 8(100) 8(100) 2.61 ±0.15 Negative control 8 0(0) 0(0) 0( 0) ―
**Ρ<0.01 ; **Ρ<0.05。  **Ρ<0.01; **Ρ<0.05.
3、 植物黄酮类化合物对 MDCK细胞病毒感染的治疗作用 将 MDCK细胞在 96孔板长成单层后， 接种 20(VL100TCID50的 禽流感病毒于各孔中，放入 37°C5%C02培养箱中感染 2h后去病毒液， 用无菌 PBS洗三次以去除游离的残余病毒， 再按表 2加不同浓度的植 物黄酮类化合物，每孔 20(^L，每个浓度设 8个复孔，放入 37°C5%C02 培养箱继续培养。 每 12h观察细胞生长情况， 设正常细胞对照组和病 毒对照组。 待正常细胞对照组细胞生长正常， 病毒对照组细胞 CPE达 ( + + + ) 以上时判定结果， 拍照。 实验重复三次， 每次实验结束时 将细胞板反复冻融三次， 使细胞裂解， 病毒释放， 分别收集每次实验 各浓度组细胞裂解液于 EP管中， -20°C中冻存， 进行血凝试验， 测其 效价以判定病毒的增殖程度， 结果如表 2所示。 3, the therapeutic effect of plant flavonoids on MDCK cell virus infection MDCK cells in a 96-well plate into a single layer, inoculated 20 (VL100TCID50 The avian influenza virus was placed in each well and placed in a 37 ° C 5% CO 2 incubator for 2 h to remove the virus solution. Wash with sterile PBS three times to remove the free residual virus, and then add different concentrations of plant flavonoids according to Table 2. 20 (^L per well, 8 replicate wells per concentration, and continue to culture in a 5% CO2 incubator at 37 ° C. The cell growth was observed every 12 h, and the normal cell control group and the virus control group were set. The cells in the group grew normally, and the results were determined when the CPE of the virus control group reached (+ + +). The experiment was repeated three times. At the end of each experiment, the cell plates were repeatedly frozen and thawed three times to lyse the cells, and the virus was released. The cell lysates of each concentration group were frozen in EF at -20 °C for the hemagglutination test, and the titer of the cells was measured to determine the degree of virus proliferation. The results are shown in Table 2.
结果表明， 当植物黄酮类化合物浓度为 0.5 g/ml时， MDCK细胞 的 CPE达 （++++)， 可见细胞完全脱落并聚集； 浓度为 16 g/ml时， CPE为 （++); 浓度为 31.25 g/ml时， 细胞形态与正常对照细胞无异。 经计算， 植物黄酮类化合物对病毒感染细胞治疗作用的 EC50为 4.01 ±0.58 g/ml， TI值为 124.69。血凝测定显示：与病毒对照组相比， 2 g/ml 即能有效抑制病毒的增殖（P<0.05)， 随着浓度的增加， 其抑制效果愈 加明显， 至 31.25 g/ml时已能完全抑制细胞病变的发生。  The results showed that when the concentration of plant flavonoids was 0.5 g/ml, the CPE of MDCK cells reached (++++), and the cells were completely detached and aggregated; when the concentration was 16 g/ml, CPE was (++); At a concentration of 31.25 g/ml, the morphology of the cells was identical to that of normal control cells. The EC50 of the therapeutic effect of plant flavonoids on virus-infected cells was calculated to be 4.01 ± 0.58 g/ml and the TI value was 124.69. Hemagglutination assay showed that compared with the virus control group, 2 g/ml could effectively inhibit the proliferation of the virus (P<0.05). With the increase of concentration, the inhibitory effect was more obvious, and it was completely complete at 31.25 g/ml. Inhibits the occurrence of cytopathic effects.
表 2. 植物黄酮类化合物对 MDCK细胞病毒感染的治疗作用结果 药物浓度 Log 培养 病变孔数 （病变百分率） 血凝效价 g/ml 孔数 I II III log(x ±s) Table 2. Results of therapeutic effects of plant flavonoids on MDCK cell virus infection Drug concentration Log culture Number of lesions (% of lesions) Blood coagulation titer g/ml Number of wells I II III log(x ± s)
125 2.097 8 0 (0) 0(0) 0(0) ― 125 2.097 8 0 (0) 0(0) 0(0) ―
62.5 1.796 8 0 (0) 0(0) 0(0) ―  62.5 1.796 8 0 (0) 0(0) 0(0) ―
31.25 1.495 8 0 (0) 0(0) 0(0) ―  31.25 1.495 8 0 (0) 0(0) 0(0) ―
16 1.204 8 1(12.5) 1(12.5) 0(0) 0.30±0.13 * * 16 1.204 8 1(12.5) 1(12.5) 0(0) 0.30±0.13 * *
8 0.903 8 2(25) 3(37.5) 2(25) 1.01±0.17 * *8 0.903 8 2(25) 3(37.5) 2(25) 1.01±0.17 * *
4 0.602 8 3(37.5) 4(50) 3(37.5) 1.21±0.15 *4 0.602 8 3(37.5) 4(50) 3(37.5) 1.21±0.15 *
2 0.301 8 5(62.5) 5(62.5) 5(62.5) 1.61±0·16 *2 0.301 8 5(62.5) 5(62.5) 5(62.5) 1.61±0·16 *
1 0 8 7(87.5) 7(87.5) 6(75) 1.91±0·171 0 8 7(87.5) 7(87.5) 6(75) 1.91±0·17
0.5 -0.301 8 8(100) 8(100) 8(100) 2.51±0.190.5 -0.301 8 8(100) 8(100) 8(100) 2.51±0.19
0.25 -0.602 8 8(100) 8(100) 8(100) 2.51±0.17 病毒对照 ― 8 8(100) 8(100) 8(100) 2.61±0.18 正常对照 ― 8 0(0) 0(0) 0(0) ― 0.25 -0.602 8 8(100) 8(100) 8(100) 2.51±0.17 Virus Control - 8 8(100) 8(100) 8(100) 2.61±0.18 Normal Control - 8 0(0) 0(0) 0(0) ―
4、 植物黄酮类化合物对 MDCK细胞病毒感染的预防作用 接 MDCK细胞于 96孔板中， 每孔 200μ1 (lX105/ml) 待各孔细 胞铺满单层时， 去生长液， 按表 3加用细胞维持液配制的不同浓度的 植物黄酮类化合物， 每个浓度 8个复孔， 每孔 200μ1， 在 37°C 5%C02 培养箱中作用 2h后， 去含药维持液， 用灭菌 PBS 洗涤三次， 以去除 游离药物，加入 100TCID50的病毒 200μ1，放入 37°C5%C02培养箱中 感染 2h后去病毒液， 用无菌 PBS洗涤三次， 除去残余游离病毒， 各孔 补充 20(^L维持液放入 37°C5%C02培养箱中继续培养， 每 12h观察 细胞生长情况， 同时设病毒对照和正常细胞对照。 待细胞对照生长正 常， 病毒对照 CPE达 （ + + + ) 以上时判定结果， 拍照。 实验重复三 次， 每次实验结束时将细胞板反复冻融三次， 使细胞裂解， 病毒释放， 分别收集每次实验各药物浓度细胞裂解液于 EP管中， -20°C中冻存， 进行血凝试验， 测其效价以判定病毒的增殖程度， 结果如表 3所示。 4. Prevention of MDCK cell virus infection by plant flavonoids. MDCK cells were plated in 96-well plates at 200 μl per well (lX105/ml). When the cells are covered with a single layer, the growth solution is added. According to Table 3, different concentrations of plant flavonoids are prepared by using the cell maintenance solution. Each concentration is 8 replicate wells, 200 μl per well, at 37 ° C 5% CO 2 incubator After 2 hours of action, remove the drug-containing maintenance solution, wash it three times with sterile PBS to remove the free drug, add 200 TCID50 virus 200μ1, and put it into the 37 ° C 5% CO 2 incubator for 2 hours, then remove the virus solution, using sterile PBS. Wash three times, remove residual free virus, add 20 ml of each well to 37 ° C 5% CO 2 incubator to continue the culture, observe the cell growth every 12h, and set the virus control and normal cell control. Normally, the virus was compared with the CPE (+ + +) to determine the result, and the photograph was taken. The experiment was repeated three times, and the cell plate was repeatedly frozen and thawed three times at the end of each experiment to lyse the cells, release the virus, and collect the concentration of each drug in each experiment. The cell lysate was frozen in an EP tube at -20 ° C, subjected to a hemagglutination test, and the titer thereof was measured to determine the degree of virus proliferation. The results are shown in Table 3.
结果表明， 当植物黄酮类化合物浓度为 l g/ml 时， MDCK细胞 CPE为 （++++); 浓度为 8 g/ml时， MDCK细胞 CPE为 （++); 浓度 为 31.25 g/ml时， 细胞形态与正常对照细胞无异， 表明植物黄酮类化 合物对禽流感病毒具有明显的预防作用。 经计算， 其作用的 EC50 为 5。54±0/74 g/ml， TI值为 90.25。 血凝试验结果表明： 与病毒对照组相 比， 4 g/ml 即能有效抑制病毒的增殖 （P<0.05)， 随着浓度的增加， 其抑制效果愈加明显，至 31.25 g/ml时已能完全抑制细胞病变的发生。  The results showed that when the concentration of plant flavonoids was lg/ml, the CPE of MDCK cells was (++++); when the concentration was 8 g/ml, the CPE of MDCK cells was (++); when the concentration was 31.25 g/ml The cell morphology is no different from that of normal control cells, indicating that plant flavonoids have obvious preventive effects against avian influenza virus. It has been calculated to have an EC50 of 5.54 ± 0/74 g/ml and a TI value of 90.25. The results of hemagglutination test showed that 4 g/ml could effectively inhibit the proliferation of virus compared with the virus control group (P<0.05). With the increase of concentration, the inhibitory effect was more obvious, and it was able to achieve the effect at 31.25 g/ml. Completely inhibit the occurrence of cytopathic effects.
表 3. 植物黄酮类化合物对 MDCK细胞病毒感染的预防作用结果 药物浓度 Log 培养 病变孔数 （病变百分率） 血凝效价  Table 3. Results of prophylactic effect of plant flavonoids on MDCK cell virus infection Drug concentration Log culture Number of lesions (% of lesions) Blood coagulation titer
孔数 I II III log(x ±s) Number of holes I II III log(x ±s)
125 2.097 8 0 (0) 0 (0) 0 (0) ― 125 2.097 8 0 (0) 0 (0) 0 (0) ―
62.5 1.796 8 0 (0) 0 (0) 0 (0) ―  62.5 1.796 8 0 (0) 0 (0) 0 (0) ―
31.25 1.495 8 0 (0) 0 (0) 0 (0) ―  31.25 1.495 8 0 (0) 0 (0) 0 (0) ―
16 1.204 8 2 (25) 1(12.5) 2 (25) 0.80±0.14 * * 16 1.204 8 2 (25) 1(12.5) 2 (25) 0.80±0.14 * *
8 0.903 8 3(37.5) 3(37.5) 3(37.5) 1.10±0.17* *8 0.903 8 3(37.5) 3(37.5) 3(37.5) 1.10±0.17* *
4 0.602 8 5(62.5) 4(50) 5(62.5) 1.41±0.15 *4 0.602 8 5(62.5) 4(50) 5(62.5) 1.41±0.15 *
2 0.301 8 7(87.5) 6(75) 7(87.5) 2.01±0·192 0.301 8 7(87.5) 6(75) 7(87.5) 2.01±0·19
1 0 8 8(100) 7(87.5) 8(100) 2.61±0.181 0 8 8(100) 7(87.5) 8(100) 2.61±0.18
0.5 -0.301 8 8(100) 8(100) 8(100) 2.61±0.170.5 -0.301 8 8(100) 8(100) 8(100) 2.61±0.17
0.25 -0.602 8 8(100) 8(100) 8(100) 2,51±0.13 病毒对照 8 8(100) 8(100) 8(100) 2·61±0.18 正常对照 8 0(0) 0(0) 0(0) ― 5、 植物黄酮类化合物对 MDCK细胞的毒性 0.25 -0.602 8 8(100) 8(100) 8(100) 2,51±0.13 Virus control 8 8(100) 8(100) 8(100) 2·61±0.18 Normal control 8 0(0) 0( 0) 0(0) ― 5. Toxicity of plant flavonoids to MDCK cells
植物黄酮类化合物对 MDCK细胞的最大无毒浓度为 ΙΟΟ μ g/ml， 并在此浓度以下的一定浓度范围内对 H5N1 亚型禽流感病毒表现出良 好的防治作用， 对细胞无毒性。 实施例二、 植物黄酮类化合物在鸡胚模型中对 H5N1 禽流感病毒 的抑制作用。  The maximum non-toxic concentration of plant flavonoids on MDCK cells is ΙΟΟ μ g/ml, and it shows good control effect on H5N1 subtype avian influenza virus within a certain concentration range below this concentration, and is not toxic to cells. Example 2 Inhibition of H5N1 avian influenza virus by a plant flavonoid in a chicken embryo model.
禽流感病毒接种鸡胚后能很快引起鸡胚的死亡， 用 10日龄鸡胚作 为实验材料。  Avian influenza virus can quickly cause chicken embryo death after inoculation of chicken embryos, and 10 day old chicken embryos are used as experimental materials.
1、 植物黄酮类化合物和病毒唑对鸡胚的最大无毒浓度  1. The maximum non-toxic concentration of plant flavonoids and ribavirin on chicken embryos
准确称取 2g植物黄酮类化合物溶于 100ml生理盐水中， 待充分溶 解后， 用 0.22 μ Μ滤膜过滤除菌， 无菌试验 96h(-)后分装， 4Ό保存备 用。  Accurately weigh 2g of plant flavonoids dissolved in 100ml of normal saline, and after fully dissolving, filter and sterilize with 0.22 μ Μ filter membrane, sterilize test 96h (-), then pack and store for 4Ό.
以上述植物黄酮类化合物溶液作为原液， 用灭菌生理盐水作系列 倍比稀释， 共作六个浓度梯度即 20mg/ml、 10 mg/ml、 5 mg/ml、 2.5 mg/ml、 1.25 mg/ml、 0.625 mg/ml, 接种于 10日龄鸡胚尿囊腔中， 每胚 0.2ml, 每个稀释度接种 5个鸡胚， 37°C孵育 96 h， 连续观察鸡胚生长 发育情况， 以鸡胚存活 96h的最大浓度作为药物的最大无毒剂量。 同 时作阳性药物病毒唑对鸡胚的毒性作用，病毒唑以 100mg/ml为原液依 次倍比稀释共六个浓度， 接种和观察同植物黄酮类化合物。 实验时设 正常生理盐水对照。  The above plant flavonoid compound solution was used as a stock solution, and serially diluted with sterile physiological saline, and a total of six concentration gradients of 20 mg/ml, 10 mg/ml, 5 mg/ml, 2.5 mg/ml, 1.25 mg/ M, 0.625 mg / ml, inoculated in the 10-day-old chicken embryo allantoic cavity, 0.2 ml per embryo, 5 chicken embryos per dilution, 96 h at 37 ° C, continuous observation of chicken embryo growth and development, The maximum concentration of chicken embryos for 96 h was used as the maximum non-toxic dose of the drug. At the same time, the toxic effect of the positive drug ribavirin on chicken embryos was obtained. The ribavirin was diluted with a total of six concentrations of 100 mg/ml as the stock solution, and the same plant flavonoids were inoculated and observed. Normal saline control was set during the experiment.
结果表明， 植物黄酮类化合物和病毒唑对鸡胚的最大无毒浓度 (TD0) 分别为 10mg/ml、 lOOmg/mL  The results showed that the maximum non-toxic concentration (TD0) of plant flavonoids and ribavirin on chicken embryos was 10 mg/ml and 100 mg/mL, respectively.
将病毒原液按 10倍递减进行稀释， 并在超净工作台中将病毒接种 于 10日龄的鸡胚尿囊腔中， 每胚 0.2ml， 石蜡封口， 放于 37°C温箱中 孵育， 每 12h观察鸡胚生长情况， 并每天翻胚 2次， 将 24h内死亡的 鸡胚弃去， 24h后死去的鸡胚放入 4°C冰箱， 48小时后将所有的鸡胚均 放入 4°C冰箱过夜， 次日无菌收集尿囊液， 3000rpm离心 15min， 取上 清进行血凝试验， 计算病毒对鸡胚的 EID50。 进行植物黄酮类化合物 抗 H5N1亚型禽流感病毒鸡胚实验时以 100EID50为病毒接种量。  The virus stock solution was diluted by 10 times, and the virus was inoculated into the 10-day-old chicken embryo allantoic cavity in a clean bench, 0.2 ml per embryo, sealed with paraffin, and incubated in a 37 ° C incubator. The growth of chicken embryos was observed at 12h, and the embryos were turned twice a day. The dead chicken embryos were discarded within 24 hours. The chicken embryos that died after 24 hours were placed in a 4°C refrigerator. After 48 hours, all the chicken embryos were placed at 4°. C refrigerator overnight, the next day aseptic collection of allantoic fluid, centrifuged at 3000 rpm for 15 min, the supernatant was taken for hemagglutination test, and the EID50 of the virus on the chicken embryo was calculated. When the plant flavonoid compound was tested against the H5N1 subtype avian influenza virus chicken embryo, 100 EID50 was used as the virus inoculum.
结果表明， 病毒对鸡胚的 EID50为 EID50=105..67/0.2ml， 药物抗 病毒实验时用 100EID50即将病毒稀释 2344倍。。 The results showed that the EID50 of the virus on the chicken embryo was EID50=105..67/0.2ml, drug resistance In the virus experiment, the virus was diluted 2344 times with 100EID50. .
2、 直接杀伤作用组  2, direct killing effect group
用 100EID50的病毒液与按表 4的不同浓度药物溶液等体积混匀， 37°C作用 2h后， 接种于鸡胚尿囊腔中， 每胚 0.2ml， 每稀释度 5个鸡 胚， 37°C孵育 48h (24h死亡者为非特异性死亡）， 入 4°C冰箱过夜， 次 日收获各鸡胚尿囊液， 作血凝试验， 结果如表 4所示。 以上实验均同 时设病毒对照及正常对照（生理盐水），阳性药物病毒唑对照同时进行。  The virus solution of 100EID50 was mixed with the different concentration of the drug solution according to Table 4, and after being treated at 37 °C for 2 hours, it was inoculated into the chicken embryo allantoic cavity, 0.2 ml per embryo, 5 chicken embryos per dilution, 37° C was incubated for 48 hours (24h death was non-specific death), and was placed in a refrigerator at 4 °C overnight. The chicken embryo allantoic fluid was harvested the next day for hemagglutination test. The results are shown in Table 4. In the above experiments, the virus control and the normal control (salt saline) were set at the same time, and the positive drug ribavirin control was simultaneously performed.
结果表明， 在直接杀伤作用组中， 植物黄酮类化合物 浓度为 lOmg/mK 5mg/ml、 2.5mg/ml、 1.25mg/ml、 0.625mg/ml 时均能有效地 杀伤病毒， 使鸡胚免于受病毒的攻击 （与病毒对照组比较 P<0.01 ); 浓度为 100mg/ml、 50mg/ml病毒唑溶液均能有效地杀伤禽流感病毒（与 病毒对照组血凝素效价比较，前者 P<0.01、后者 P<0.05 )，而 25mg/ml 以下各浓度杀伤禽流感病毒效果不显著 （P〉0.05)。  The results showed that in the direct killing group, the concentration of plant flavonoids was 10 mg/ml, 5 mg/ml, 2.5 mg/ml, 1.25 mg/ml, and 0.625 mg/ml, which could effectively kill the virus and protect the chicken embryo from Attack by virus (P<0.01 compared with virus control group); concentration of 100mg/ml, 50mg/ml ribavirin solution can effectively kill avian influenza virus (compared with virus control group hemagglutinin titer, the former P< 0.01, the latter P <0.05), and the effect of killing avian influenza virus at each concentration below 25mg/ml was not significant (P>0.05).
3、 治疗作用组  3, treatment group
先每胚分别接种 100EID50禽流感病毒 0.1ml (每组 5个鸡胚）， 37 °C2h，再将不同稀释度的药物每胚 0.1ml接种于鸡胚尿囊腔中， 37°C孵 育 48h， 4°C冰箱过夜， 次日分别收获各鸡胚尿囊液， 作血凝试验， 结 果如表 4所示。 以上实验均同时设病毒对照及正常对照 （生理盐水）， 阳性药物病毒唑对照同时进行。  First, each embryo was inoculated with 0.1 ml of 100 EID50 avian influenza virus (5 chicken embryos per group), 37 °C for 2 h, and then 0.1 ml of each dilution of the drug was inoculated into the chicken embryo allantoic cavity, and incubated at 37 ° C for 48 h. The refrigerator was overnight at 4 °C, and each chicken embryo allantoic fluid was harvested the next day for hemagglutination test. The results are shown in Table 4. In the above experiments, both the virus control and the normal control (saline) were set, and the positive drug ribavirin control was performed simultaneously.
结果表明， 先感染病毒再用药， 植物黄酮类化合物各浓度均能有 效地抑制禽流感病毒在鸡胚内的增殖； 而 25mg/ml以下的病毒唑则对 抑制禽流感病毒效果不显著 （P>0.05)。  The results showed that the concentration of flavonoids in plants could effectively inhibit the proliferation of avian influenza virus in chicken embryos, while the concentration of flavonoids below 25mg/ml was not significant in inhibiting avian influenza virus (P> 0.05).
4、 预防作用组  4. Prevention role group
将不同稀释度的药物 0.1ml 接种鸡胚尿囊腔， 每个药物稀释度 5 个鸡胚， 37°C2h， 再分别接种 100EID50禽流感病毒 O.lml, 37°C孵育 48h， 入 4°C冰箱过夜， 次日收获鸡胚尿囊液， 作血凝试验， 结果如表 4所示。 以上实验均同时设病毒对照及正常对照 （生理盐水）， 阳性药 物病毒唑对照同时进行。  0.1ml of different dilutions of the drug were inoculated into the chicken embryo allantoic cavity, each chicken dilution was 5 chicken embryos, 37 °C 2h, then inoculated with 100EID50 avian influenza virus O.lml, incubated at 37 °C for 48h, into 4 °C The refrigerator was overnight, and the chicken embryo allantoic fluid was harvested the next day for hemagglutination test. The results are shown in Table 4. In the above experiments, both the virus control and the normal control (saline) were set at the same time, and the positive drug ribavirin control was simultaneously performed.
结果表明， 在先用药物处理鸡胚再接种病毒后， 可见植物黄酮类 化合物 10mg/ml、 5mg/ml、 2.5mg/ml组能有效预防禽流感病毒对鸡胚 的感染 （PO.01 ); 病毒唑组中， 除 100 mg/ml 组能有效预防禽流感病 毒对鸡胚的感染（PO.01)夕卜， 其余各剂量组均不能有效预防禽流感病 毒对鸡胚的感染 （P>0.05)。 The results showed that the plant flavonoids 10mg/ml, 5mg/ml and 2.5mg/ml could effectively prevent the infection of chicken embryos (PO.01) after the chicken embryos were treated with the drug. In the ribavirin group, the 100 mg/ml group can effectively prevent avian influenza Infection of chicken embryos (PO.01), the other dose groups were not effective in preventing infection of chicken embryos by avian influenza virus (P>0.05).
表 4. 实验组不同给药方式观察结果 Table 4. Observations of different modes of administration in the experimental group
Figure imgf000014_0001
Figure imgf000014_0001
(mg/ml) 直接作用 治疗作用 预防作用 植物黄酮 10 0.12±0.17* * 0.54±0.25 * * 0.36±0.25 * * 类化合物 5 0.18±0.17* * 0.66 ±0.25 * * 0.96 + 0.25 * *  (mg/ml) Direct action Therapeutic effect Preventive effect Phytoflavonoids 10 0.12±0.17* * 0.54±0.25 * * 0.36±0.25 * * Class of compounds 5 0.18±0.17* * 0.66 ±0.25 * * 0.96 + 0.25 * *
2.5 0.18±0.27 * * 0.66+0.25 * * 1.51±0.56* 2.5 0.18±0.27 * * 0.66+0.25 * * 1.51±0.56*
1.25 0·36±0.25 * * 1.14±0·57* * 1.75±0.75 1.25 0·36±0.25 * * 1.14±0·57* * 1.75±0.75
0.625 0.66±0.25* * 1.51±0.71 * 2.41 ±0.22 病毒唑 100 0.30±0.30 * * 0.78±0.17 * * 0.66±0.25 * *  0.625 0.66±0.25* * 1.51±0.71 * 2.41 ±0.22 ribavirin 100 0.30±0.30 * * 0.78±0.17 * * 0.66±0.25 * *
50 1.45 + 1.07 * 1.45±0.91 * 1.69 + 0.46  50 1.45 + 1.07 * 1.45±0.91 * 1.69 + 0.46
25 1.99 ±0.79 2.17 + 0.58 1.99 ±0.46 25 1.99 ±0.79 2.17 + 0.58 1.99 ±0.46
12.5 2.35 ±0.25 2.41 ±0.21 2.23±0.3512.5 2.35 ±0.25 2.41 ±0.21 2.23±0.35
6.25 2.41 ±0.21 2.35±0.25 2.23 ±0.17 病毒对照 2.53±0·17 2.53±0.17 2.41 ±0.21 正常对照 - - - 由此可见， 植物黄酮类化合物在三种作用方式中对禽流感病毒的 效果均优于阳性对照药病毒唑。 6.25 2.41 ±0.21 2.35±0.25 2.23 ±0.17 Virus control 2.53±0·17 2.53±0.17 2.41 ±0.21 Normal control - - - It can be seen that flavonoids are excellent in avian influenza virus in three modes of action. In the positive control drug ribavirin.
5、 植物黄酮类化合物对鸡胚的毒性  5. Toxicity of plant flavonoids to chicken embryos
植物黄酮类化合物对鸡胚的最大无毒浓度为 10 g/ml， 并在此浓 度以下的一定浓度范围内对 H5N1 亚型禽流感病毒均表现出显著的直 接作用、 治疗作用及一定的预防作用， 对鸡胚无毒性。 实施例三、 植物黄酮类化合物抗 H5N1亚型禽流感的小鼠试验 实验动物： SPF级 ICR小鼠 320只， 体重 20±2g ， 雌雄各半； 由 上海斯莱克实验动物有限责任公司提供 (合格证号: 0002710,许可证号： SCXK (沪） 2003— 003)。 实验前随机抽取 20只小鼠用血凝试验及血 凝抑制试验确认鼠群中无禽流感病毒的感染， 并经检验排除其它呼吸 道病源感染。  The maximum non-toxic concentration of plant flavonoids on chicken embryos is 10 g/ml, and it has significant direct effects, therapeutic effects and certain preventive effects on H5N1 subtype avian influenza virus within a certain concentration range below this concentration. , non-toxic to chicken embryos. Example 3: Test of flavonoids against mouse strain of H5N1 subtype avian influenza Experimental animals: 320 SPF ICR mice, weighing 20±2g, male and female; provided by Shanghai Slack Laboratory Animals Co., Ltd. (qualified Certificate No.: 0002710, License No.: SCXK (Shanghai) 2003— 003). Twenty mice were randomly selected before the experiment to confirm the infection of the avian influenza virus in the mice by hemagglutination test and hemagglutination inhibition test, and other respiratory infections were excluded by the test.
动物实验在广东省实验动物监测所三级生物安全实验室进行， 符 合三级生物安全实验室标准。 所有物品在进入实验室前均经高压消毒 或紫外线照射或浸泡消毒， 实验空间实验前用高锰酸钾-甲醛液彻底薰 菡。 The animal experiment was carried out in the third-level biosafety laboratory of the Laboratory Animal Monitoring Institute of Guangdong Province, which met the three-level biosafety laboratory standard. All items are autoclaved before entering the laboratory Or ultraviolet radiation or immersion disinfection, thoroughly pervaporate with potassium permanganate-formaldehyde solution before the experimental space experiment.
将 160只动物随机分为 8组， 用于观察小鼠存活率、 血清抗体、 脏器指数及病理变化、 体重、 体温， 分别为植物黄酮类化合物高剂量 组 （I组， 54mg/ml)， 植物黄酮类化合物中剂量组 （II组， 18mg/ml)， 植物黄酮类化合物低剂量组 （ΠΙ组， 6mg/ml)， 极低剂量组 （IV组， 2mg/ml), 阳性药物病毒唑对照组 （V组， 6mg/ml)、 盐酸金刚烷胺对 照组 （VI组， 2.5mg/ml)， 病毒对照组 （VII组） 和正常对照组 （VIII 组）， 每组 20只， 雌雄各半， 分笼饲养。  160 animals were randomly divided into 8 groups for observation of mouse survival rate, serum antibody, organ index and pathological changes, body weight and body temperature, respectively, high dose group of plant flavonoids (group I, 54 mg/ml). Plant flavonoids in the middle dose group (group II, 18mg/ml), plant flavonoids low dose group (ΠΙ group, 6mg/ml), very low dose group (group IV, 2mg/ml), positive drug ribavirin control Group (V group, 6 mg/ml), amantadine hydrochloride control group (VI group, 2.5 mg/ml), virus control group (VII group) and normal control group (VIII group), 20 rats in each group, male and female , caged to raise.
另 120只动物随机分为 8组， 取肺及脑用于分离病毒， 观察病毒 在肺、 脑内的增殖程度， 分别为植物黄酮类化合物高剂量组 （ I组， 54mg/ml)、 中剂量组 （II组， 18mg/ml)、 低剂量组 （III组， 6mg/ml)、 极低剂量组（IV组， 2mg/ml);阳性药物病毒唑对照组（ V组， 6mg/ml); 盐酸金刚烷胺对照组 （VI组， 2.5mg/ml); 病毒对照组 （VD组） 和正常 对照组 （珊组）， 每组 15只。 雌雄分笼词养。 Another 120 animals were randomly divided into 8 groups. The lungs and brain were used to isolate the virus, and the degree of proliferation of the virus in the lung and brain was observed. The high dose group of plant flavonoids (group I, 54 mg/ml) and medium dose were respectively. Group (group II, 18 mg/ml), low dose group (group III, 6 mg/ml), very low dose group (group IV, 2 mg/ml) ; positive drug ribavirin control group (group V, 6 mg/ml) ; The amantadine hydrochloride control group (VI group, 2.5 mg/ml) ; the virus control group (VD group) and the normal control group (Shan group), 15 rats in each group. Male and female are divided into words.
实验 I -VE组动物用 0.1ml/20g***（20mg/ml)肌肉注射麻醉后 从鼻腔滴入含禽流感病毒尿囊液 （血凝效价为 1 : 256) ΙΟΟμ^ 珊组 则滴入同样量的无病毒鸡胚尿囊液。 2h后， I一 IV组组动物分别经胃 灌入高剂量 (54mg/ml) 、 中剂量（18mg/ml)、 低剂量 (6mg/ml)、 极低剂 量（2mg/ml)浓度的植物黄酮类化合物 0.2ml; V组动物经胃灌入病毒 唑（6mg/ml) 0.2ml; VI组经胃灌入盐酸金刚烷胺（2.5mg/ml) 0.2ml; VII组、 環组动物经胃灌入 0.5%的羧甲基纤维素钠 0.2ml。 所有灌胃操 作均在清晨清醒状态下进行， 每天 1次， 连续灌胃 5天。 各组动物每 日饲以标准日粮， 自由饮水， 12h光照、 黑暗间隔。 每 2天更换垫料， 为避免交叉感染， 接病毒组和正常对照组相对隔离饲养。 实验连续观 察 15天。每天观察各组小鼠的临床症状（包括行为、毛发光泽、呼吸、 食欲等） 及生存情况， 发现动物濒死或死亡、 立即采用眼球摘除法处 死动物， 取肺、 心、 肝、 脾、 肾等组织， 准确称重 （精确到 0.1g)， 并 将肝、 肾、 肺固定于 10%中性缓冲***液。  In the experimental I-VE group, the animals were anesthetized with 0.1 ml/20 g of ketamine (20 mg/ml), and then the avian influenza virus allantoic fluid was instilled from the nasal cavity (the blood coagulation titer was 1: 256). Amount of virus-free chicken embryo allantoic fluid. After 2h, the animals in the I-IV group were intragastrically infused with high-dose (54mg/ml), medium-dose (18mg/ml), low-dose (6mg/ml), very low-dose (2mg/ml) flavonoids. Compound 0.2 ml; V group animals were intragastrically injected with ribavirin (6mg/ml) 0.2ml; Group VI was intragastrically injected with amantadine hydrochloride (2.5mg/ml) 0.2ml; Group VII, ring group animals by gastric irrigation 0.2 ml of sodium carboxymethylcellulose was added in an amount of 0.2 ml. All gavage operations were performed in the morning and awake, once a day, for 5 days. Animals in each group were fed a standard diet daily, free to drink water, 12 hours light, dark interval. The litter was changed every 2 days. In order to avoid cross infection, the virus group and the normal control group were kept in isolation. The experiment was observed continuously for 15 days. The clinical symptoms (including behavior, hair luster, respiration, appetite, etc.) and survival of each group of mice were observed every day. The animals were found to die or die, and the animals were immediately sacrificed by eyeball removal. The lungs, heart, liver, spleen and kidney were taken. The tissue was accurately weighed (accurate to 0.1 g) and the liver, kidney, and lungs were fixed in 10% neutral buffered formalin.
将感染病毒并幵始用药的当天作为 0天， 用于分离病毒的各实验 组小鼠在实验的第 0 3 4 7天各组分别处死 3只小鼠， 无菌取肺及 脑， 入 EP管中， 匀浆后每管加入无菌生理盐水 0.5ml， 分别各取 0.2ml 接种 10日龄鸡胚， 培养 48小时后取尿囊液作病毒血凝实验。 The day when the virus was infected and the drug was started for 0 days, the mice in each experimental group used to isolate the virus were sacrificed 3 mice in each group on the 0th, 7th day of the experiment, and the lungs were aseptically taken. The brain was placed in an EP tube. After homogenization, 0.5 ml of sterile physiological saline was added to each tube, and 0.2 ml of each was taken to inoculate 10 day old chicken embryos. After 48 hours of culture, the allantoic fluid was taken for virus hemagglutination test.
将感染病毒并开始用药的当天作为 0天， 用于观察小鼠存活率、 血清抗体、脏器指数及病理变化、体重、体温的各组小鼠在第 0、 3、 6、 9、 12、 15天对存活的各小鼠分别称重及测量体温， 计算各时间点的体 重、 体温变化情况， 并在第 1、 4、 7、 11 天及实验结束时， 通过断尾 法每次采集各实验组四只小鼠尾静脉血， 分离血清作抗体分析， 了解 小鼠体内抗体水平的动态变化及药物对小鼠抗体水平的影响。 血清抗 体的检测采用血凝抑制试验方法。 实验结束时将所有鼠均剖杀， 取肝、 肾、 脾、 肺、 心准确称重后，将肝、 肾、 肺固定于 10%中性缓冲福尔马 林液中。  The day when the virus was infected and started to be used as 0 days, the mice used to observe the survival rate, serum antibody, organ index and pathological changes, body weight, and body temperature of the mice were at 0, 3, 6, 9, 12, The surviving mice were weighed and measured for body temperature for 15 days, and the changes in body weight and body temperature at each time point were calculated. At the 1st, 4th, 7th, and 11th days and at the end of the experiment, each time each method was collected by the tail-breaking method. Four mice in the experimental group were treated with tail vein blood, and serum was separated for antibody analysis to understand the dynamic changes of antibody levels in mice and the effect of drugs on mouse antibody levels. Serum antibodies were tested using the hemagglutination inhibition test. At the end of the experiment, all rats were sacrificed. After liver, kidney, spleen, lung and heart were accurately weighed, liver, kidney and lung were fixed in 10% neutral buffered formalin.
计算每组小鼠死亡率、 药物保护率％: Calculate the mortality and drug protection rate of each group of mice :
药物保护率 ==病毒对照组小鼠死亡率一试验组小鼠死亡率 效果指数==病毒对照组死亡数 /药物组死亡数  Drug protection rate == virus control group mouse mortality - test group mice mortality effect index == virus control group deaths / drug group deaths
病理组织学观察- 小鼠肺、 肝、 肾等组织常规固定， 石蜡包埋， 切片 5-8μηι， HE染 色后镜检观察脏器病理变化。  Histopathological observation - The lung, liver, kidney and other tissues of the mice were routinely fixed, embedded in paraffin, sliced 5-8μηι, HE stained and examined for pathological changes of organs.
1、 小鼠死亡率比较 1 实验小鼠生存情况如表 5所示， 结果表明， 植物黄酮类化合物各 浓度组对禽流感病毒均有显著的抑制作用 （P<0.05 )。  1. Comparison of mortality in mice 1 The survival of experimental mice is shown in Table 5. The results showed that the concentration of flavonoids in plants had significant inhibitory effects on avian influenza virus (P<0.05).
表 5. 各组小鼠生存情况  Table 5. Survival of mice in each group
组别 0d Id 2d 3d 4d 5d 6d 7d 8d 9d  Group 0d Id 2d 3d 4d 5d 6d 7d 8d 9d
高剂量组 20 20 20 19 17 16 16 16 16 16 16 中剂量组 20 20 20 19 17 16 16 16 16 16 16 低剂量组 20 20 20 18 16 14 14 14 14 14 14 极低剂量组 20 20 20 16 12 10 10 10 10 10 10 病毒唑组 20 20 20 16 14 12 12 12 12 12 12 金刚垸胺组 20 20 20 18 16 15 15 15 15 15 15 病毒对照组 20 20 20 17 12 8 8 8 8 8 8 正常对照组 20 20 20 20 20 20 20 20 20 20 20  High dose group 20 20 20 19 17 16 16 16 16 16 16 Medium dose group 20 20 20 19 17 16 16 16 16 16 16 Low dose group 20 20 20 18 16 14 14 14 14 14 14 Very low dose group 20 20 20 16 12 10 10 10 10 10 10 ribavirin group 20 20 20 16 14 12 12 12 12 12 12 rimine group 20 20 20 18 16 15 15 15 15 15 15 virus control group 20 20 20 17 12 8 8 8 8 8 8 Normal control group 20 20 20 20 20 20 20 20 20 20 20
禽流感病毒广泛分布于世界范围内的诸多家禽和野禽中， 同时也 感染猪、 马以及鲸鱼、 雪貂,海豚等多种哺乳动物。 其致病力的变化范 围很大， 随病毒型及亚型、 动物种属、 龄期、 并发感染、 周围环境及 宿主免疫状态的不同而不同。 禽流感病毒感染引发的疾病可能是不明 显的或是温和的一过性的综合症，也可能是 100%发病率和 /或死亡率的 疾病。 因此对于高致病性禽流感病毒而言， 能够保护受感染动物或人 类免于死亡就成为药物有效性的一个评价指标， 这也是研究和开发药 物的目的。 The avian flu virus is widely distributed in many poultry and wild birds worldwide, as well as pigs, horses, and a variety of mammals such as whales, ferrets, and dolphins. Variability of its virulence The range is very large, depending on the virus type and subtype, animal species, age, concurrent infection, surrounding environment and host immune status. The disease caused by avian influenza virus infection may be inconspicuous or mild transient syndrome, or it may be a disease with 100% morbidity and/or mortality. Therefore, for highly pathogenic avian influenza viruses, the ability to protect infected animals or humans from death is an indicator of drug effectiveness, which is also the purpose of research and development of drugs.
图 I是实验小鼠不同组成活率比较图， 结果表明， 与病毒对照组 相比， 植物黄酮类化合物的高、 中、 低浓度组及盐酸金刚烷胺对照组 均能显著地提高感染小鼠的存活率； 病毒唑对照组、 植物黄酮类化合 物的极低剂量组尽管也能一定程度地提高小鼠的存活率， 但与病毒对 照组比较无明显差异。与传统的抗病毒药病毒唑及盐酸金刚烷胺相比， 植物黄酮类化合物的高、 中、 低剂量组抗禽流感病毒效果与金刚烷胺 相当而优于病毒唑。  Figure I is a comparison of the different compositions of experimental mice. The results show that compared with the virus control group, the high, middle and low concentration groups of plant flavonoids and the amantadine hydrochloride control group can significantly improve infected mice. Survival rate; the ribavirin control group, the very low dose group of plant flavonoids, although it can also improve the survival rate of mice to some extent, but there is no significant difference compared with the virus control group. Compared with the traditional antiviral drugs ribavirin and amantadine hydrochloride, the high, medium and low dose groups of plant flavonoids have the same anti-avian influenza effect as amantadine and are superior to ribavirin.
表 6是实验小鼠不同组的存活率、 死亡率等的数据统计表， 表明 在一定剂量范围内， 植物黄酮类化合物能有效保护小鼠免受禽流感感 染所引起的死亡，表中 I一 VIII分别为高剂量组、中剂量组、低剂量组、 极低剂量组、 病毒唑组、 金刚垸组、 病毒对照组和正常对照组。  Table 6 is a statistical table showing the survival rate, mortality, etc. of different groups of experimental mice, indicating that plant flavonoids can effectively protect mice from death caused by avian influenza infection within a certain dose range. VIII were high dose group, medium dose group, low dose group, very low dose group, ribavirin group, diamond sputum group, virus control group and normal control group.
表 6.植物黄酮类化合物对禽流感小鼠模型的保护作用  Table 6. Protective effects of plant flavonoids on avian influenza mouse model
I II III IV V VI ffl 存活率 80% * 80% * 70% * 50% 45% 70% * 40% 100% 死亡率 20% 20% 30% 50% 55% 30% 60% 0% 药物保护率 40% 40% 30% 10% 5% 30% - - 效果指数 3 3 2 1.2 1.1 2 - - 与病毒对照组相比 * P<0.05。  I II III IV V VI ffl Survival rate 80% * 80% * 70% * 50% 45% 70% * 40% 100% Mortality 20% 20% 30% 50% 55% 30% 60% 0% Drug protection rate 40% 40% 30% 10% 5% 30% - - Effect index 3 3 2 1.2 1.1 2 - - Compared with the virus control group * P < 0.05.
2、 小鼠脏器的病毒浓度比较  2. Comparison of virus concentrations in mouse organs
小鼠肺内病毒增殖随时间变化情况如表 7和图 II所示。 与病毒对 照组比较， 实验第四天， 植物黄酮类化合物的 54mg/ml 组、 18mg/ml 组、 6mg/ml组及金刚烷胺组能显著抑制病毒的增殖； 第七天， 各药物 组均能显著抑制病毒的增殖； 脑内除病毒对照组检出低滴度病毒外， 各药物组均未检出病毒。  The changes in virus proliferation in the lungs of mice as shown in Table 7 and Figure II are shown. Compared with the virus control group, on the fourth day of the experiment, the 54 mg/ml group of the flavonoids, the 18 mg/ml group, the 6 mg/ml group and the amantadine group significantly inhibited the proliferation of the virus; on the seventh day, each drug group was It can significantly inhibit the proliferation of the virus; no virus was detected in each drug group except the low titer virus was detected in the brain control group.
表 7. 各实验组肺内病毒增殖随时间动态变化 is) 时 间 （天） Table 7. Dynamic changes of virus proliferation in the lungs of each experimental group by is) Time (days)
组别 0 3 4 7  Group 0 3 4 7
高剂量组 - 1.054±0.125 0.955±0.213 * * 0.451±0.213 * 中剂量组 - 1.063±0.175 1.054±0.172 * 0.602±0.00 * 低剂量组 - 1.204±0.214 1.205±0.117 * 0.753±0.213 * 极低剂量组 - 1.076±0.187 1.654±0.213 1.806±0.00 * 病毒唑组 - 1.078±0.242 1.355±0.213 * 0.905±0.00 * 金刚烷胺组 - 1.055±0.157 1.204±0.152 * 0.451±0.213 * 病毒对照组 - 1.241±0.274 1.957±0.213 2.559±0.213 正常对照组 - - - - High dose group - 1.054 ± 0.125 0.955 ± 0.213 * * 0.451 ± 0.213 * Medium dose group - 1.063 ± 0.175 1.054 ± 0.172 * 0.602 ± 0.00 * Low dose group - 1.204 ± 0.214 1.205 ± 0.117 * 0.753 ± 0.213 * Very low dose group - 1.076 ± 0.187 1.654 ± 0.213 1.806 ± 0.00 * ribavirin group - 1.078 ± 0.242 1.355 ± 0.213 * 0.905 ± 0.00 * amantadine group - 1.055 ± 0.157 1.204 ± 0.152 * 0.451 ± 0.213 * virus control group - 1.241 ± 0.274 1.957 ±0.213 2.559±0.213 normal control group - - - -
-表示血凝试验阴性，与病毒对照组比较 * P<0.05 ， * * P<0.01 o 3、 小鼠血清抗体水平比较 - indicates a negative hemagglutination test, compared with the virus control group * P < 0.05, * * P < 0.01 o 3. Comparison of serum antibody levels in mice
小鼠体内血清抗体水平如表 8和图 III所示。 与病毒对照组比较， 植物黄酮类化合物 54 mg/ml组、 18mg/ml组及金刚烷胺组能促进小鼠 抗体的分泌， 其余用药组对小鼠抗体的分泌影响轻微。  Serum antibody levels in mice are shown in Table 8 and Figure III. Compared with the virus control group, the plant flavonoids 54 mg/ml group, the 18 mg/ml group and the amantadine group promoted the secretion of mouse antibodies, and the other drug groups had a slight effect on the secretion of mouse antibodies.
表 8. 各实验组小鼠血清抗体水平情况 ±s)  Table 8. Serum antibody levels in mice of each experimental group ± s)
时 间 （天）  Time (days)
组别 0 4 7 11 15 高剂量组 - 0.301±0.246 0.828±0.288 * 1.656±0.174 * 2.182±0.151 中剂量组 - 0.376±0.151 0.903±0.246 * 1.731±0.151 * 2.333±0.151 低剂量组 - - 0.376±0.151 1.279±0.288 2.032±0.288 极低剂量组 - - 0.376±0.151 1.129±0.288 1.731±0.288 病毒唑组 - - 0.452±0.174 1.129±0.151 1.957±0.174 金刚垸胺组 - 0.151±0.174 0.828±0.288 * 1.731±0.288 * 2.333±0.151 病毒对照组 - - 0.376±0.150 0.978±0.151 1.881±0.151 正常对照组 - - - - - 一表示血凝抑制试验阴性， 与病毒对照组比较 * P<0.05。  Group 0 4 7 11 15 High dose group - 0.301 ± 0.246 0.828 ± 0.288 * 1.656 ± 0.174 * 2.182 ± 0.151 medium dose group - 0.376 ± 0.151 0.903 ± 0.246 * 1.731 ± 0.151 * 2.333 ± 0.151 low dose group - - 0.376 ± 0.151 1.279±0.288 2.032±0.288 Very low dose group - - 0.376 ± 0.151 1.129 ± 0.288 1.731 ± 0.288 ribavirin group - - 0.452 ± 0.174 1.129 ± 0.151 1.957 ± 0.174 Adamantamine group - 0.151 ± 0.174 0.828 ± 0.288 * 1.731 ± 0.288 * 2.333 ± 0.151 virus control group - - 0.376 ± 0.150 0.978 ± 0.151 1.881 ± 0.151 Normal control group - - - - - One indicates a negative hemagglutination inhibition test, compared with the virus control group * P < 0.05.
4、 小鼠脏器指数比较  4, mouse organ index comparison
实验结束时不同小鼠平均脏器系数比较如表 9所示。 与病毒 组比较， 各药物组均能有效降低小鼠肺指数， 对其它脏器指数的影响 不大； 各药物组均能有效减轻肝、 肾、 肺等脏器的病变。  The comparison of the average organ coefficients of different mice at the end of the experiment is shown in Table 9. Compared with the virus group, each drug group can effectively reduce the lung index of mice, and has little effect on other organ indexes; each drug group can effectively reduce the pathological changes of liver, kidney and lung.
表 9.实验结束时不同组小鼠平均脏器系数比较（ ±s )  Table 9. Comparison of mean organ coefficients of different groups of mice at the end of the experiment (±s)
- - - - - 高剂量组 0.70±0.06 * * 5廉0.29 0.37±0.06 1.54±0.11 0.50±0.07 中剂量组 0.77±0.06 * * 5.89±0.35 0.45±0.06 1.58±0.12 0.46±0.05 低剂量组 0.81±0.04 * * 5.85±0.38 0.45±0.04 1.62±0.14 0.49±0.05 极低剂量组 0.92±0.08 * 6.31±0.33 0.4H0.08 1.84±0.11 0.51±0.07 病毒唑组 0.98±0.06 * 6.45±0.76 0.5±0.05 1.76±0.11 0.48±0.05 金刚垸胺组 0.83±0.06 * * 5.71±0.44 0.44±0.06 1.41±0.10 0.51±0.05 病毒对照组 1.45±0.10 6.20±0.42 0.48±0.07 1.46±0.19 0.50±0.08 正常对照组 0.69±0.04 5.80±0.20 0.39±0.04 1.39±0.06 0.50±0.06 与病毒对照组比较 * P<0.05， * * P<0.01 o - - - - - High dose group 0.70±0.06 * * 5 cheap 0.29 0.37±0.06 1.54±0.11 0.50±0.07 medium dose group 0.77±0.06 * * 5.89±0.35 0.45±0.06 1.58±0.12 0.46±0.05 low dose group 0.81±0.04 * * 5.85± 0.38 0.45±0.04 1.62±0.14 0.49±0.05 Very low dose group 0.92±0.08 * 6.31±0.33 0.4H0.08 1.84±0.11 0.51±0.07 ribavirin group 0.98±0.06 * 6.45±0.76 0.5±0.05 1.76±0.11 0.48±0.05 Amygdalin group 0.83±0.06 * * 5.71±0.44 0.44±0.06 1.41±0.10 0.51±0.05 Virus control group 1.45±0.10 6.20±0.42 0.48±0.07 1.46±0.19 0.50±0.08 Normal control group 0.69±0.04 5.80±0.20 0.39± 0.04 1.39±0.06 0.50±0.06 compared with the virus control group * P<0.05, * * P<0.01 o
5、 小鼠体重比较  5, mouse weight comparison
与病毒对照组比较， 实验前三天， 各药物组均能减缓小鼠体重的 减轻， 三天后各药物组均能有效促进小鼠体重的回升。  Compared with the virus control group, the drug groups alleviated the weight loss of the mice three days before the experiment. After three days, each drug group could effectively promote the weight gain of the mice.
6、 小鼠体温比较  6, mouse body temperature comparison
与病毒对照组比较， 实验前三天， 各用药组对小鼠体温的影响不 明显， 三天后各药物组均能有效促进小鼠体温的回升。  Compared with the virus control group, the effects of the drug groups on the body temperature of the mice were not obvious three days before the experiment. After three days, each drug group could effectively promote the recovery of the body temperature of the mice.
结果表明： 植物黄酮类化合物能显著提高模型小鼠的存活率且呈 剂量依赖关系； 能有效地抑制病毒在模型小鼠肺内的增殖， 抑制病毒 对脑的侵袭； 能有效地激活机体的体液免疫***， 使机体内血清抗体 呈高滴度表达； 能有效地促进禽流感病毒感染所致的肺， 肝、 肾等组 织器官病变的修复； 能显著抑制模型小鼠体重的下降及促进体重的回 升； 能在一定程度上加速模型小鼠体温恢复正常和缩短动物的病情。  The results showed that: plant flavonoids can significantly improve the survival rate of model mice in a dose-dependent manner; it can effectively inhibit the proliferation of virus in the lungs of model mice, inhibit the invasion of the virus into the brain; can effectively activate the body fluids of the body The immune system enables high-level titer expression of serum antibodies in the body; it can effectively promote the repair of lung, liver, kidney and other tissue and organ diseases caused by avian influenza virus infection; it can significantly inhibit the weight loss of model mice and promote weight Pick-up; can accelerate the normalization of the body temperature of the model mice and shorten the condition of the animal to some extent.
7、 植物黄酮类化合物对小鼠的毒性  7. Toxicity of plant flavonoids to mice
实验小鼠的急性毒性实验表明， 所使用的有效抗 H5N1 亚型禽流 感病毒浓度均对小鼠无毒。  Acute toxicity experiments in experimental mice showed that the effective anti-H5N1 subtype avian influenza virus used was not toxic to mice.

Claims

权利要求书 Claim
、 植物黄酮类化合物在制备抗禽流感病毒药物中的应用。 The use of plant flavonoids in the preparation of anti-avian influenza virus drugs.
、 根据权利要求 1所述的应用， 其特征在于： 所述药物的有效剂0—250mg/kg体重 ·天。 The use according to Claim 1, characterized in that the effective agent of the drug is 0-250 mg/kg body weight·day.
、 根据权利要求 2所述的应用， 其特征在于： 所述药物的有效剂0— 180mg/kg体重 ·天。 The use according to claim 2, characterized in that the effective agent of the drug is 0-180 mg/kg body weight·day.
、 植物黄酮类化合物作为伺料添加剂的应用。 The use of plant flavonoids as a feed additive.
、 根据权利要求 4所述的应用， 其特征在于： 所述添加剂的有效 20— 250mg/kg体重 ·天。 The use according to claim 4, characterized in that: the additive is effective from 20 to 250 mg/kg body weight per day.
、 根据权利要求 5所述的应用， 其特征在于： 所述药物的有效剂0— 180mg/kg体重 ·天。 The use according to claim 5, characterized in that the effective agent of the drug is 0-180 mg/kg body weight·day.
PCT/CN2005/001049 2004-09-07 2005-07-15 A use of phyto-flavonoid compounds for preparing medicament against avian influenzal virus WO2006026905A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CNB2004100745928A CN1301711C (en) 2004-09-07 2004-09-07 Use of vegetable flavonoid
CN200410074592.8 2004-09-07

Publications (1)

Publication Number Publication Date
WO2006026905A1 true WO2006026905A1 (en) 2006-03-16

Family

ID=35930211

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2005/001049 WO2006026905A1 (en) 2004-09-07 2005-07-15 A use of phyto-flavonoid compounds for preparing medicament against avian influenzal virus

Country Status (2)

Country Link
CN (1) CN1301711C (en)
WO (1) WO2006026905A1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101703151B (en) * 2009-06-18 2013-03-20 北京华牧伟业科技有限公司 Application of coordination compound of flavone and trace elements in feedstuff industry

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1323354A2 (en) * 2001-12-28 2003-07-02 Nor-Feed A/S Natural, intestinally active feed additive

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1066185A (en) * 1991-04-30 1992-11-18 黎元鑫 Multi-function capsules for poultry influenza
CN1180781C (en) * 2002-07-11 2004-12-22 江苏正大天晴药业股份有限公司 Bupleurum flavone composition for resisiting influenza virus and its prepn.
CN1191262C (en) * 2003-02-12 2005-03-02 复旦大学 Cranesbill total flavonoid extract and use in preparation of anti-virus medicine

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1323354A2 (en) * 2001-12-28 2003-07-02 Nor-Feed A/S Natural, intestinally active feed additive

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DEQUAN Z ET AL: "SURVEY ON RESEARCH AND APPLICATION OF BIOFLAVONOIDS.", FOOD AND FERMENTATION INDUSTRIES., vol. 25, no. 6, 1999, pages 52 - 57 *
XIE B X ET AL: "EFFECT OF FLAVONOID ON LIPID METABOLISM AND PRODUCTION PERFORMANCE OF BROILERS.", ACTA ZOONUTRIMENTA SINICA., vol. 14, no. 4, October 2002 (2002-10-01), pages 49,53 - 58 *

Also Published As

Publication number Publication date
CN1301711C (en) 2007-02-28
CN1718182A (en) 2006-01-11

Similar Documents

Publication Publication Date Title
EP1954258B1 (en) Materials and methods for treating influenza viral infections with a cysteamine compound
Lee et al. Anti-influenza virus activity of green tea by-products in vitro and efficacy against influenza virus infection in chickens
Chen et al. Activity of andrographolide and its derivatives against influenza virus in vivo and in vitro
Reichling Antiviral and virucidal properties of essential oils and isolated compounds–A scientific approach
CN101111608A (en) Compositions and methods for reducing the transmissivity of illnesses
CN100444734C (en) A compound antivirus sprayer and preparation method thereof
CN112472698B (en) Application of bromophenol-pyrazoline compounds in treatment of porcine coronavirus diseases
KR20120118707A (en) A composition comprising the extract of galla rhois or the compounds isolated therefrom showing inhibiting activity of novel influenza, avian influenza, or sars syndrome
CN1548056A (en) Application of chitin, chitosan and their derivatives in preparing antiviral
Huang et al. Research on the indirect antiviral function of medicinal plant ingredient quercetin against grouper iridovirus infection
EP1976543A1 (en) Use of elderberry extract
TWI453026B (en) Use of anisomeles indica (l.) kuntze extract and purified products thereof against influenza virus
CA2465062C (en) Preventive and/or therapeutic agent for viral infection
Chakraborty et al. Fighting the flu: a brief review on anti-influenza agents
JP2023026528A (en) Cyanobacterial extract, method for preparing the same and uses thereof
WO2006026905A1 (en) A use of phyto-flavonoid compounds for preparing medicament against avian influenzal virus
WO2009089534A2 (en) Compositions and methods for the treatment of viral infections caused by influenza virus
JP2010528053A (en) Substances and methods for treating influenza infections
CN102166276B (en) Traditional Chinese medicine composition for preventing and treating influenza A and preparation method and application thereof
Goel et al. Ocimum sanctum: in vitro antiviral potential against animal viruses
JP5173813B2 (en) Drugs for the prevention and treatment of influenza
Conly et al. Ode to oseltamivir and amantadine?
El-Hamid et al. In vitro antiviral activity of commercial products of herbal extracts against highly pathogenic avian influenza (H5N1) virus.
KR102578233B1 (en) Composition for preventing or treating avian influenza virus infection comprising Angelica dahurica, Curcuma longa and Resina Pini extracts
CN100421673C (en) Pharmaceutical use of glycoconjugates CHB for preventing and treating avian flu

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU LV MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase