WO2016019684A1 - 连翘苷/连翘脂素组合物在制备缓解或/和治疗病毒性疾病的药物或保健品中的应用 - Google Patents

连翘苷/连翘脂素组合物在制备缓解或/和治疗病毒性疾病的药物或保健品中的应用 Download PDF

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Publication number
WO2016019684A1
WO2016019684A1 PCT/CN2014/094710 CN2014094710W WO2016019684A1 WO 2016019684 A1 WO2016019684 A1 WO 2016019684A1 CN 2014094710 W CN2014094710 W CN 2014094710W WO 2016019684 A1 WO2016019684 A1 WO 2016019684A1
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Prior art keywords
forsythin
composition
forsylin
forsythiaside
virus
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PCT/CN2014/094710
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English (en)
French (fr)
Inventor
富力
鲁明明
付强
王凯乾
惠敏
刘正贤
柳洋
王硕
曲洪颖
于晓静
王海玲
盖鑫
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Individual
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Individual
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Priority to ES14899145T priority Critical patent/ES2839090T3/es
Priority to AU2014402938A priority patent/AU2014402938B2/en
Priority to KR1020177006088A priority patent/KR101939726B1/ko
Priority to JP2017505134A priority patent/JP6343389B2/ja
Priority to RU2017107136A priority patent/RU2655616C1/ru
Priority to CA2956988A priority patent/CA2956988C/en
Application filed by Individual filed Critical Individual
Priority to US15/502,000 priority patent/US10286002B2/en
Priority to EP14899145.8A priority patent/EP3178482B1/en
Publication of WO2016019684A1 publication Critical patent/WO2016019684A1/zh
Anticipated expiration legal-status Critical
Priority to US16/281,826 priority patent/US10881679B2/en
Ceased legal-status Critical Current

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    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • 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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • 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
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/40—Cyclodextrins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2022—Organic macromolecular compounds
    • A61K9/205—Polysaccharides, e.g. alginate, gums; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5036—Polysaccharides, e.g. gums, alginate; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • 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
    • 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
    • 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
    • A61P31/20—Antivirals for DNA viruses
    • 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
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention is in the field of medicinal chemistry, and in particular, the present invention relates to forsythin/forsylin compositions or mixtures and their use in the preparation of a medicament or nutraceutical for the alleviation or/and treatment of viral diseases.
  • Influenza is an acute, viral respiratory infection that seriously harms human health. Since the 21st century, the prevalence of SARS, H5N1 virus and influenza A (H1N1) virus has caused great harm to human beings. There is currently no natural medicine in the world for effective treatment of viral influenza and pneumonia.
  • Forsythia is the dry fruit of Forsythia suspensa (Thunb.) Vahl in Oleaceae, mainly distributed in Henan, Shanxi, Shaanxi, Shandong and other places in China, in addition, Hubei, Hebei, Sichuan and Gansu are also distributed. Commonly used for the treatment of acute wind-heat cold, swollen sore, lymph node tuberculosis, urinary tract infections and other diseases [1].
  • the main components of Forsythia suspensa are phillyrin and forsythin.
  • the forsythiaside is also called forsythiaside ((+)-phillygeninin).
  • the structural formula of the two components is as follows.
  • the main component of Forsythia suspensa is forsythin, which has pharmacological effects such as antiviral, antibacterial, antioxidative and free radical scavenging.
  • Forsythia saponin also one of the main active ingredients of Forsythia, has anti-oxidation, lowering blood lipids, scavenging free radicals, inhibiting bacteria, anti-tumor, anti-inflammatory and so on.
  • the antiviral pharmacological effects of forsythia extract and forsythin single component of unidentified components and several components have been studied, but no combination of forsythiaside and forsythin has been found.
  • the anti-viral pharmacological effects of the single component of forsythiaside has been studied, but no combination of forsythiaside and forsythin has been found. The anti-viral pharmacological effects of the single component of forsythiaside.
  • the object of the present invention is to provide a forsythin/forsylin composition which is prepared for the relief or/and treatment of viral diseases in view of the technical deficiencies existing in the process of treating, preventing or alleviating viral diseases.
  • the forsythiaside/forsylin composition of the present invention has a high performance and efficacy in relieving or/and treating viral diseases, and provides a new drug for the development of a drug for relieving or/and treating viral diseases. New approaches, new applications in the treatment, conditioning and mitigation of viral diseases or health foods, offer new ideas.
  • an aspect of the present invention provides a use of a forsythin/forsylin composition for the preparation of a medicament or a health care product for alleviating or/and treating a viral disease.
  • the viral diseases are influenza virus, parainfluenza virus, Coxsackie virus CoxA16, respiratory syncytial virus RSV, herpes simplex virus HSV-I, herpes simplex virus HSV-II, herpes simplex virus alone Viral diseases caused by CVB3, adenovirus ADV or enterovirus EV71, especially viral influenza caused by influenza virus, parainfluenza virus, Coxsackie virus CoxA16, respiratory syncytial virus RSV, pneumonia respiratory infection.
  • forsythin/forsylin composition has a strong inhibitory effect on viral influenza and pneumonia. Its efficacy is significantly better than the effect of forsythin or forsythin alone.
  • the weight ratio of forsythin to forsythin in the forsythin/forsylin composition is from 80 to 98:2 to 20, preferably from 90 to 98:2 to 10, further preferably 98:2.
  • the forsythin/forsylin composition further comprises a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are generally approved by health care professionals for this purpose and as inactive ingredients of the agent.
  • a compilation of pharmaceutically acceptable carriers can be found in the Handbook of Pharmaceutical excipients, 2nd edition, edited by A. Wade and PJ Weller; published by the American Phar-maceutical Association, Washington and The Pharmaceutical Press, London, 1994) found in the reference books.
  • the carrier comprises an excipient such as starch, water or the like; a lubricant such as magnesium stearate or the like; a disintegrating agent such as microcrystalline cellulose; a filler such as lactose; and a binder, Such as pregelatinized starch, dextrin, etc.; sweeteners; antioxidants; preservatives, flavors, spices, etc.
  • the medicament is in the form of tablets, capsules, pills, powders, granules, syrups, solutions, emulsions, injections, sprays, aerosols, gels, creams, cataplasms, rubber patches It is in the form of a plaster or a plaster.
  • the ratio of the total weight of forsythin to forsythin in the forsythin/forsylin composition to the weight of the pharmaceutically acceptable carrier is from 1:1 to 1:100, preferably 1: 10 is further preferably 1:5, still more preferably 1:2, still more preferably 1:1.
  • the content of the forsythin/forsylin composition is ⁇ 80%, preferably ⁇ 85%, further preferably ⁇ 88%, still more preferably ⁇ 90%, still more preferably ⁇ 99% .
  • the weight ratio of the two components in the forsythin/forsylin composition is from 2 to 98:98 to 2; preferably from 80:20 or 20:80, further preferably from 90:6. Or 6:90, still more preferably 98:2 or 2:98.
  • the forsythiaside/forsylin composition is prepared by using forsythin and forsythin as a monomer or by a solvent heating extraction method; Or a combination of forsypol and forsythin with a cyclodextrin or a cyclodextrin derivative to form a forsythiaside-forsylin-cyclodextrin composition.
  • the forsythiaside-forsylin-cyclodextrin composition is a mixture of forsythiaside and forsythin mixed with ⁇ -, ⁇ - or ⁇ -cyclodextrin or a derivative thereof. , or a complex formed by the physical and chemical treatment of forsythiaside and forsythin with ⁇ -, ⁇ - or ⁇ -cyclodextrin or a derivative thereof.
  • ratio of the weight of forsythin and forsythin in the forsythiaside-forsylin-cyclodextrin composition to the weight of the cyclodextrin or cyclodextrin derivative is 1:1-50.
  • the cyclodextrin is ⁇ - or ⁇ -, ⁇ -cyclodextrin;
  • the cyclodextrin derivative is hydroxyethyl ⁇ -cyclodextrin, 2,6-dimethyl ⁇ -cyclodextrin , 2,3,6-trimethyl ⁇ -cyclodextrin, 2,6-diethyl ⁇ -cyclodextrin, 2,3,6-triethyl ⁇ -cyclodextrin, maltosyl ⁇ -cyclodextrin Fine or sulfobutyl ether ⁇ -cyclodextrin, p-TsCl substituted ⁇ -cyclodextrin, 6-substituted ⁇ -CD p-toluenesulfonate ( ⁇ -cyclodextrin) -6-OTs), 2-oxohydroxypropyl- ⁇ -cyclodextrin, 2-position monosubstituted p-toluenesul
  • the forsythiaside-forsylin extraction composition is prepared by the following method:
  • the solvent described in steps 1), 3), 4) is methanol, ethanol, acetone, aqueous methanol or aqueous ethanol.
  • the aqueous methanol has a mass percentage concentration of 70-95%; and the aqueous ethanol has a mass percent concentration of 70-95%.
  • the temperature in the step 2) is at room temperature, preferably 10-35 ° C, further preferably 20-25 ° C; the standing time is 1-48 h; the volume and the original extract after the concentration of the extract in the step 2) The volume ratio is 0.1-0.5:1.
  • the temperature of the recrystallization treatment in the step 4) is room temperature, preferably from 10 to 35 ° C, more preferably from 20 to 25 ° C.
  • Another aspect of the present invention provides a medicament or health care product for alleviating or/and treating a viral disease, comprising forsythin and forsythiaside.
  • the ratio by weight of forsythin to forsythiaside is from 80 to 98:2 to 20, preferably from 90 to 98:2 to 10, and further preferably from 98:2.
  • the medicament or nutraceutical comprises a forsythin/forsylin composition and a pharmaceutically acceptable carrier.
  • the ratio of the total weight of forsythin to forsythin in the forsythin/forsylin composition to the weight of the pharmaceutically acceptable carrier is from 1:1 to 1:100, preferably 1: 10 is further preferably 1:5, still more preferably 1:2, still more preferably 1:1.
  • the ratio of the weight of the forsythiaside/forsylin composition to the total weight of the drug or nutraceutical is from 0.01 to 10:100, preferably from 0.1 to 10:100, further preferably from 1 to 10. :100.
  • the medicament is in the form of tablets, capsules, pills, powders, granules, syrups, solutions, emulsions, injections, sprays, aerosols, gels, creams, cataplasms, rubber patches It is in the form of a plaster or a plaster.
  • the medicine or health care product further includes dandelion extract, Radix Isatidis extract, Honeysuckle extract, Zhimu extract, Scrophularia sinensis extract, Prunella vulgaris extract, Reed root extract, light bamboo leaf extract, and alfalfa extract.
  • dandelion extract Radix Isatidis extract
  • Honeysuckle extract Zhimu extract
  • Scrophularia sinensis extract Prunella vulgaris extract
  • Reed root extract light bamboo leaf extract
  • alfalfa extract alfalfa extract.
  • the medicament can be prepared into various dosage forms by methods well known in the art, such as tablets, capsules, pills, powders, granules, syrups, solutions, emulsions, injections, sprays, aerosols, gels. , cream, cataplasm, rubber plaster or plaster.
  • the invention also provides a method for treating viral influenza and pneumonia diseases, comprising administering to a subject a therapeutically effective amount of a composition of forsythiaside and forsythin, the therapeutically effective amount of which is 0.1 to 50 mg/kg.d, preferably 0.3 to 30 mg/kg.d, further preferably 0.5 to 10 mg/kg.d.
  • therapeutically effective amount is an amount of a drug that is required to produce an effective effect; “therapeutically effective amount” is The adjustments and changes are ultimately determined by the medical staff, and the factors to be considered include the route of administration and the nature of the formulation, the recipient's weight, age, and the like, as well as the nature and severity of the condition being treated.
  • the present invention has the following distinct advantages:
  • the preparation method of the forsythin/forsylin composition of the invention is simple and suitable for industrial production, and the composition has obvious anti-influenza, pneumonia and other viruses, and the antiviral effect is significantly better than using forsythin alone.
  • the efficacy of forsythiaside is also significantly better than the current clinical antiviral drug, oseltamivir phosphate (Duffy); in addition, the forsythin/Forsylin composition has been found to be resistant to other viruses.
  • Efficacy can significantly inhibit Coxsackie virus CoxA16 and respiratory syncytial virus RSV, herpes simplex virus HSV-I and HSV-II and CVB3, adenovirus ADV, enterovirus EV71, can be used to treat the flu caused by the above virus Pneumonia, herpes zoster, myocarditis, hand, foot and mouth disease, upper respiratory tract infection, bronchiolitis, rash, meningitis and the like, so the present invention can be prepared for relieving or/and treating viral influenza and pneumonia, herpes zoster, High-efficiency natural medicines or health foods for myocarditis, hand, foot and mouth disease, upper respiratory tract infection, bronchiolitis, rash, meningitis and other diseases have opened up a new field for the application of forsythia medicinal materials.
  • the forsythin/forsylin composition of the present invention has anti-influenza and pneumonia virus effects, and its curative effect is significantly better than that of forsythin and forsythiaside alone, forsythin and forsythiaside Combined use in a specific ratio, with synergistic effect;
  • the forsythin/forsylin composition of the present invention inhibits Coxsackie virus CoxA16 and respiratory syncytial virus RSV, herpes simplex virus HSV-I and HSV-II and CVB3, adenovirus ADV, intestinal tract
  • the role of various viruses such as the virus EV71, forsythiaside/forsylin composition for the treatment of the above-mentioned virus caused by influenza and pneumonia, herpes zoster, myocarditis, hand, foot and mouth disease, upper respiratory tract infection, bronchiolitis, rash, meningitis And other diseases;
  • the forsythin/forsylin composition of the present invention has the effect of significantly inhibiting inflammation caused by the virus and enhancing the immunity of the patient; the pharmacological action of the forsythin/forsylin composition inhibiting viral influenza and pneumonia Strong, effective in relieving, conditioning and treating viral influenza and pneumonia, with quick effect, small side effects, good safety, long-term use, and good medicinal prospects;
  • the raw materials of the invention have rich sources, low cost, safe clinical use, simple preparation process, can be made into various dosage forms, and have small dosage and convenient use, so it is easy to promote;
  • the forsythin/forsylin composition of the present invention can be prepared by quantitatively preparing a monomer component or by extracting a forsythin/forsylin composition or the above forsythiaside/linkage.
  • the psyllid composition is combined with a derivative of ⁇ - or ⁇ -, ⁇ -cyclodextrin or cyclodextrin to form a composition, and the forsythin/forsylin composition and other activities can also be used.
  • the forsythin monomer is derived from Dalian Fusheng Natural
  • the product is produced by the Pharmaceutical Development Co., Ltd., and is determined by the high-performance liquid chromatography two-detector ultraviolet detector and the evaporative light scattering detector area normalization method. The purity is 99.5%, and the forsythiaside reference substance is determined by the Chinese pharmaceutical biological product content.
  • the composition was prepared in the following manner according to the weight ratio described in Table 2: (1) directly added to the cyclodextrin solution, or (2) Add directly to the cyclodextrin solution and stir well for 1-24h, (3) directly into the cyclodextrin solution and heat for 10-120 minutes, (4) directly added to the cyclodextrin solution and sonicated for 10-120 minutes (5) directly grinding with the cyclodextrin powder for 10-120 minutes, (6) uniformly mixing the forsythin/forsylin composition with the cyclodextrin powder, and sieving; (7) directly adding the cyclodextrin In the refined derivative solution, or (8) directly added to the cyclodextrin derivative solution and stirred well for 1-24h, (9) directly added to the cyclodextrin derivative solution and heated for 10-120 minutes, (10) Directly added to the cyclodextrin derivative solution and sonicated
  • excipients in Examples 5-24 are exemplified by the selection of ⁇ -cyclodextrin, and other cyclodextrin and cyclodextrin derivatives are suitable for use in the present invention, for example, 1) ⁇ -hydroxypropyl cyclodextrin, 2 ) hydroxyethyl ⁇ -cyclodextrin, 3) 2,6-dimethyl ⁇ -cyclodextrin, 4) 2,3,6-trimethyl ⁇ -cyclodextrin, 5) 2,6-diethyl --cyclodextrin, 6) 2,3,6-triethyl ⁇ -cyclodextrin, 7) maltosyl ⁇ -cyclodextrin, 8) sulfobutyl ether ⁇ -cyclodextrin, 9) p-methylbenzene Sulfonated chlorine (p-TsCl) substituted ⁇ -cyclodextrin, 10) 6-substituted ⁇ -CD
  • the forsythin/forsylin composition prepared in Example 1 was uniformly mixed with starch, and then granulated and talcum powder was added. After mixing with magnesium stearate, it was pressed into 10,000 tablets.
  • the granules of the forsythin/forsylin composition were prepared according to the following ratio:
  • the forsythin/forsylin composition prepared in Example 1 was uniformly mixed with the microcrystalline cellulose, and then granulated into bags to prepare 10,000 bags.
  • Capsules for preparation of forsythin/forsylin composition according to the following ratio:
  • the forsythin/forsylin composition prepared in Example 1 was uniformly mixed with starch and then filled into capsules to prepare 10,000 tablets.
  • the forsythin/forsylin composition was uniformly mixed with starch according to the weight ratio of Table 3, and then filled into capsules, each of which was made into 10,000 capsules.
  • the starting materials in the table can be replaced with the compositions of forsythiaside and forsythin prepared in Examples 5-29.
  • the forsythin/forsylin composition was uniformly mixed with the microcrystalline cellulose in the weight ratio of Table 4, and then granulated and bagged to prepare 10,000 bags.
  • the starting materials in the table can be replaced with the compositions of forsythiaside and forsythin prepared in Examples 5-29.
  • the forsythin/forsylin composition is uniformly mixed with the above-mentioned extract powder, and then uniformly mixed with starch to form granules, and talc powder and magnesium stearate are added and uniformly mixed, and then pressed into 10,000 tablets.
  • the forsythin/forsylin composition in the present embodiment can be replaced with the combination of forsythiaside and forsythin prepared in Examples 5-29.
  • the granules of the forsythin/forsylin composition were prepared according to the following ratio:
  • the forsythin/forsylin composition is uniformly mixed with the above-mentioned extract powder, and then uniformly mixed with the microcrystalline cellulose to form a granule bag, and 10,000 bags are prepared.
  • the forsythin/forsylin composition in the present embodiment can be replaced with the combination of forsythiaside and forsythin prepared in Examples 5-29.
  • Capsules for preparation of forsythin/forsylin composition according to the following ratio:
  • the forsythin/forsylin composition is uniformly mixed with the above-mentioned extract powder, and then uniformly mixed with the starch, and then filled into capsules to prepare 10,000 tablets.
  • the forsythin/forsylin composition in the present embodiment can be replaced with the combination of forsythiaside and forsythin prepared in Examples 5-29.
  • the forsythin/forsylin composition is uniformly mixed with the above-mentioned extract powder, and then uniformly mixed with the starch to form granules, and the talc powder and magnesium stearate are uniformly mixed and pressed to prepare 10000 tablets.
  • the forsythin/forsylin composition in the present embodiment can be replaced with the combination of forsythiaside and forsythin prepared in Examples 5-29.
  • the granules of the forsythin/forsylin composition were prepared according to the following ratio:
  • the forsythin/forsylin composition is uniformly mixed with the above-mentioned extract powder, and then uniformly mixed with the microcrystalline cellulose to form a granule bag, and 10,000 bags are prepared.
  • the forsythin/forsylin composition in the present embodiment can be replaced with the combination of forsythiaside and forsythin prepared in Examples 5-29.
  • Capsules for preparation of forsythin/forsylin composition according to the following ratio:
  • the forsythin/forsylin composition is uniformly mixed with the above-mentioned extract powder, and then uniformly mixed with the starch, and then filled into capsules to prepare 10,000 tablets.
  • the forsythin/forsylin composition in the present embodiment can be replaced with the combination of forsythiaside and forsythin prepared in Examples 5-29.
  • Forsythin / Forsythia Leptin Composition Forsythin / Forsythia Leptin Composition 1: White powder, produced by Dalian Fusheng Natural Medicine Development Co., Ltd., with forsythiaside and forsythiaside The ratio was determined by using 99.5% forsythin and 99.1% forsythin as a reference substance.
  • the content of the two monomer components in the forsythin/forsylin composition was 98%, of which forsythin/
  • the weight ratio of forsythin to forsythin in forsythiaside composition 1 is 98:2; the weight ratio of forsythin to forsythiaside in forsythin/forsylin composition 2 is 80:20 .
  • the above drugs are all dissolved in purified water, filtered, sterilized and dispensed, and stored at 4 ° C for the test.
  • Vero African green monkey kidney cell
  • Virus strain 1 influenza virus strain, parainfluenza virus strain, respiratory syncytial virus (RSV) strain: purchased from the Institute of Virology, Chinese Academy of Preventive Medicine; 2 Coxsackie virus B 3 (CVB 3 ) strain: derived from The United States, preserved for the teaching and research section; 3 Coxsackie virus A16 (CoxA16) strain, enterovirus EV71 strain: gifted by the National Sendai National Hospital, preserved in this teaching and research section; 4 adenovirus (AdV) from Bethune Medical University, a hospital pediatrics Laboratory; 5 herpes simplex virus type I (HSV-1) was purchased from the Ministry of Health Pharmaceutical and Biological Products.
  • RSV respiratory syncytial virus
  • Biosafety cabinet BHC-1300IIA/B3, AIRTECH; CO 2 incubator: MCO-18AIC, SANYO; inverted microscope: CKX41, OLYMPUS; electronic analytical balance: AR1140/C, DHAUS; medium: DMEM, HyClone; fetal bovine serum :HyClone; Trypsin: Gibco; MTT: Sigma; DMSO: Tianjin Beilian Fine Chemicals Development Co., Ltd.
  • Vero cells were subcultured for 1-2 days to make them into slices. The boundary was clear. When the stereoscopic effect and the refractive power were strong, trypsin digestion was performed. After the cell surface appeared a pinpoint-like pore, the digestive juice was aspirated, and several milliliters of the culture solution was blown away. The cells were counted and diluted with a culture solution (DMEM containing 10% fetal bovine serum) to about 5 ⁇ 10 7 /L, and then seeded in a 96-well culture plate until the cells were grown into a single layer.
  • DMEM containing 10% fetal bovine serum
  • Cytotoxicity test The drug was diluted at the concentrations shown in Table 1-1 for cytotoxicity assays.
  • viruses were diluted 10-fold to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 different dilutions and sequentially inoculated into a single layer of Vero cell 96-well culture plate. Above, 100 ⁇ L per well, 6 wells per dilution, and a normal cell control group. Incubate at 37 ° C, 5% CO 2 for 2 h, discard the virus solution, then add 100 ⁇ L of cell maintenance solution per well, and incubate at 37 ° C, 5% CO 2 .
  • the cytopathic effect was observed under the microscope on the third day, and the results were recorded on the 7th to 8th day, so that the highest dilution of 50% cell hole positive lesions was used as the end point, and the virus titer was calculated by the karber method.
  • the OD value was measured at 492 nm using the MTT colorimetric method to calculate the drug antiviral efficiency (ER%).
  • ANOVA was used to compare the significant differences in the antiviral efficacy of each drug in the SPSS 18.0 statistical software.
  • ER% (mean OD value of drug treatment group - average OD value of virus control group) / (average OD value of cell control group - average OD value of virus control group) ⁇ 100%
  • TC 0 The maximum non-toxic concentration (TC 0 ), half-toxicity concentration (TC 50 ) and drug anti-virus experimental concentration of each drug against Vero cells are shown in Table 1-2.
  • Table 1-3 show that the forsythin/forsylin composition has significant inhibitory effects on eight viruses (P ⁇ 0.01 or P ⁇ 0.001), for influenza virus, parainfluenza virus, and herpes simplex virus type I ( The effective rate of HSV-I), enterovirus EV71 and adenovirus (ADV) was 100%, and the curative effect was significantly better than forsythin and forsythiaside, indicating forsythin/forsylin composition. Synergistic effect.
  • the forsythin/forsylin composition inhibits influenza, coxsackievirus A16 (CoxA16), respiratory syncytial virus (RSV), herpes simplex virus type I (HSV-I), adenovirus (ADV), intestine
  • the efficacy of EV71 and Coxsackie virus B 3 (CVB 3 ) was significantly better than that of the positive drug ribavirin (P ⁇ 0.01 or P ⁇ 0.001)
  • inhibition of influenza, Coxsackievirus A16 (CoxA16), respiratory syncytium Viral (RSV), herpes simplex virus type I (HSV-I), adenovirus (ADV), enterovirus EV71 and coxsackievirus B 3 (CVB 3 ) are significantly better than oseltamivir phosphate (P) ⁇ 0.05, or P ⁇ 0.01, P ⁇ 0.001).
  • the Kunming mice were provided by the Experimental Animal Center of the Bethune Medical Department of Jilin University, and the medical word No. 10-5219.
  • influenza virus and the parainfluenza virus were diluted 10 times to a virus solution having a concentration of 10 -1 , 10 -2 , 10 -3 , 10 -4 , and 10 -5 .
  • 120 Kunming mice 60 influenza virus and parainfluenza virus groups were randomly divided into 6 groups. The mice were lightly anesthetized with ether and infected with different dilutions of virus solution 0.03mL/only. At the same time, a blank control was set, and the physiological suspension was used instead of the viral suspension. Death and survival were observed and observed daily until 14 days after infection. The death within 24 hours of infection was non-specific death, not counted, and the Karber method calculated the LD50 of the virus solution.
  • mice were randomly divided into 27 groups, 10 in each group, for the determination of lung index and lung index inhibition rate of mice with influenza and parainfluenza virus infection by forsythin/forsylin composition. The test took 90 mice and repeated the test three times. Another 270 mice were randomly divided into 27 groups, 10 in each group, for the determination of hemagglutination titer of lung suspension virus by the combination of forsythiaside and forsythin; Only 3 trials were repeated.
  • mice Put a group of absorbent cotton in a beaker of 200 to 300 mL size, then pour the appropriate amount of ether (to make the cotton wool wet), pour the beaker containing the cotton wool, and place the mouse for anesthesia, see the mouse. Extremely excited, when apparently weak, the mice were placed supine, nasally infected with 15LD 50 influenza virus and parainfluenza virus 0.03ml/nostrils, and the normal control group was replaced with physiological saline instead of the virus suspension.
  • Forsythiaside/Forsythiamin composition 1 group, forsythin/Forsylin composition 2, forsythiaside group, forsythin group, ribavirin and oseltamivir phosphate control group The conventional intragastric administration was started on the day before the infection, and the forsythin/forsylin composition 1 and 2 were divided into high, medium and low dose groups, respectively, and the doses were 13.0, 6.5, 3.25 mg/kg, respectively.
  • the forsythiaside group was administered at a dose of 13 mg/kg, and the forsythiaside group was administered at a dose of 13 mg/kg.
  • Lung index (mouse lung weight / mouse body weight) ⁇ 100%
  • Pulmonary index inhibition rate (mean lung index in the infection model group - mean lung index in the experimental group) / mean lung index in the infection model group ⁇ 100%
  • the lungs of each group of mice on the 5th day after treatment were taken and homogenized by a homogenizer at a low temperature.
  • the physiological saline was diluted to 10% of the lung tissue suspension, and the supernatant was centrifuged and diluted to 0.2 ml/
  • the holes were dropped on the titration plate, 0.2 ml of 1% chicken red blood cell suspension was added to each well, and the mixture was allowed to stand at room temperature for 30 min, and the blood coagulation titer was observed.
  • the end point of red blood cell agglutination (++) is expressed as the titer of the suspension dilution factor.
  • the Kunming mice in the experimental group were infected with different concentrations of influenza virus and parainfluenza virus in 30 ⁇ L, and the mice in the third group (virus concentration 10 -1 group, 10 -2 group, 10 -3 group) before the third day of infection. There were different degrees of symptoms: shrub, shivering, and decreased diet. On the 5th day, the mice walked and waved; on the 6th day, the mice in the highest virus concentration group began to die, and the other groups continued on the 7th day after infection. Death has occurred. After the end of 14 days, the number of deaths of each group of mice was counted. The results are shown in Tables 1-4 and 1-5 below. Calculation of the LD 50 of influenza virus dilution was 10-2. 9, parainfluenza virus LD 50 of dilution of 10 -2. 5.
  • the Karber method calculates the LD 50 of the virus.
  • the LogLD 50 of the influenza virus is as follows:
  • the Karber method calculates the LD 50 of the virus.
  • the LogLD 50 of the parainfluenza virus is as follows:
  • the average lung index showed that compared with the infection model group, the normal control group, the forsythin group 13.0 mg/kg/d, the forsythiaside group 16.0 mg/kg/d, Three dose groups of forsythin/Forsylin composition 1 and 2 (3.25 mg/kg/d in the low dose group, 6.5 mg/kg/d in the middle dose group, 13.0 mg/kg/d in the high dose group), The lung index of the ribavirin group and the oseltamivir phosphate group was significantly lower (P ⁇ 0.05 or P ⁇ 0.01).
  • the forsythin/forsylin composition was in the range of 3.25-13.0 mg/kg/d.
  • the lung index was significantly decreased, and the inhibition rate of lung tissue lesion index was better than that of the forsythiaside group and forsythiaside group (P ⁇ 0.01 or P ⁇ 0.05). See Table 1-6, 1-7.
  • the blood group blood titer (InX) of the infected model group was 32.40 and 33.11, respectively, and the different concentrations of forsythiaside/forsylin composition 1 and 2 were treated for 5 days.
  • the hemagglutination titer of lung tissue virus decreased, compared with the infection model group, the difference was significant (P ⁇ 0.01), and their different dose groups had significantly lower blood coagulation titer than influenza virus.
  • the glycoside group and the forsythiaside group showed that the composition had synergistic effect, and the inhibition rate of virus reproduction was significantly higher than that of forsythiaside group and forsythiaside group (P). ⁇ 0.05-P ⁇ 0.001) Among them, the high, medium and low dose groups of forsythin/Forsythiaside compositions 1 and 2 significantly inhibited the hemagglutination titer of lung suspension in mice infected with influenza virus. Forsythiaside group and forsythiaside group (P ⁇ 0.01 ⁇ P ⁇ 0.001). The above test results are shown in Tables 1-8 and 1-9.
  • each test group virus control group * P ⁇ 0.05, ** P ⁇ 0.01; forsythin / forsythia visfatin composition Forsythin compared, # P ⁇ 0.05, ## P ⁇ 0.01, ### P ⁇ 0.001; Forsythin/Forsythiamin composition compared with forsythiaside, ⁇ P ⁇ 0.05, ⁇ P ⁇ 0.01, ⁇ P ⁇ 0.001.
  • Test Example 2 Antipyretic and anti-inflammatory tests of forsythin/and forsythiamin composition
  • Test animal Wistar rats weighing 120-250g, both male and female, certificate number: medical words 13-1225; Japanese big white rabbit, male, weighing 1.5 ⁇ 2.0kg. Certificate No.: No. 10-5115, which is supplied by Changchun High-tech Medical Animal Experimental Center, and animal feed is supplied by the Experimental Animal Department of Jilin University.
  • Forsythin / Forsythia Leptin Composition White powder, produced by Dalian Fusheng Natural Medicine Development Co., Ltd. It is made up of the combination of forsythin and forsythiaside. It is calibrated with 99.5% forsythin and 99.1% forsythin as control. There are two kinds of forsythin/forsylin composition. The monomer component content is 98%, wherein the forsythin/Forsylin composition 1 has a weight ratio of forsythin to forsythiaside of 98:2, and forsythin/forsylin composition 1 The weight ratio of telomere to forsythia is 80:20.
  • YLS-7A rat toe swelling measuring instrument Shandong Provincial Academy of Medical Sciences equipment station; 722 visible spectrophotometer: Shanghai Spectrometer Co., Ltd. production; portable digital thermometer: model WSC-411P, Shanghai Pudong No.
  • the amount of sweat secretion can be used to produce a purple reaction mechanism by using iodine and starch sweat, and the change of sweat secretion is observed.
  • the test took 500 Wistar rats, half male and half female, weighing 120-150 g. They were randomly divided into 50 groups according to body weight and sex, namely: control (0.5% carboxymethylcellulose) group, forsythin group, forsythiaside group, forsythin/forsylin composition group 1 and 2 Low, medium and high dose groups (2.5, 5, 10 mg/kg, respectively) and the positive drug pilocarpine (35 mg/kg) group, 10 in each group, 10 groups in each experiment, a total of 5 time periods (1, 5, 10, 15, 20min).
  • the rats were placed in a self-made rat fixed bag to expose the hind limbs.
  • the middle and high dose groups of forsythin/forsylin composition significantly promoted the secretion of sweat in the ankle and foot of rats at 10, 15 and 20 min after the application of B solution.
  • the effect (p ⁇ 0.05), the 2.5 mg/kg group of forsythin/forsylin composition was significantly promoted to the secretion of sweat in the ankle and foot of rats after 15 and 20 min after application of B solution (p ⁇ 0.05).
  • Their sweating effect is comparable to that of the positive drug pilocarpine, and it has the characteristics of slowly promoting the secretion of sweat in the foot and ankle of rats.
  • the high-dose group of forsythin/Forsylin composition was significantly better than the forsythiaside and forsythiaside after 10, 15 and 20 minutes after the application of B solution (p ⁇ 0.05).
  • the dose of the forsythin/Forsylin composition in the dose group was significantly better than that of forsythin and forsythiaside after 10 and 15 minutes after the application of B solution (p ⁇ 0.05).
  • the low-dose group of forsythin/Forsylin composition was significantly better than the forsythiaside and forsythiaside after 15 minutes after the application of B solution (p ⁇ 0.05).
  • the test took 300 Wistar rats, half male and half female, weighing 120-160 g. According to body weight and sex, they were randomly divided into 30 groups, namely: control group (0.5% carboxymethyl cellulose), forsythiaside, forsythin, forsythin/forsylin composition 1 and 2 were respectively divided into low, Medium and high (2.5, 5, 10 mg/kg) dose group and positive drug pilocarpine (35 mg/kg) group, 10 in each group, 3 times in each group. Except for the subcutaneous injection of pilocarpine solution, the other groups were administered by intragastric administration.
  • the control group was given 0.5% carboxymethylcellulose for 1 h
  • the positive drug group was given pilocarpine for 30 min
  • the forsythin, forsythiaside, forsythin/forsyric acid composition were administered 1 h after the administration.
  • the incidence of vacuoles was mainly observed, and statistically processed by X 2 test to compare the differences between the groups. The above test was repeated 3 times.
  • Percentage of vacuoles number of vacuolar sweat glands / number of observed sweat glands ⁇ 100%
  • mice Male Wistar rats weighing 180-200 g. Before the test, the normal anus temperature was measured twice with the WSC-411P portable digital thermometer (each time interval), and the average of the two measurements was taken as the normal body temperature of the rats. Then, 300 rats with body temperature of 36.5-38 °C were randomly divided into 30 groups according to body weight: model group (0.5% carboxymethylcellulose), forsythin/forsylin composition 1 and 2, respectively. For low, medium and high (2.5, 5, 10 mg/kg) dose group, forsythiaside group (13 mg/kg), forsythiaside group (13 mg/kg) and positive drug paracetamol (100 mg/kg) group, Group 10, each group was repeated 3 times.
  • Each group of rats was subcutaneously injected with 10% fresh beer yeast suspension 10 ml/kg. After the suspension of 10% fresh brewer's yeast for 6.0 h, the combination of forsythiaside and forsythin and paracetamol were intragastrically administered, and the model group was given an equal volume of 0.5% carboxymethylcellulose. The rectal temperature was measured at 1, 2, 3, and 4 h after administration, respectively. The changes of body temperature were observed, and the inter-group t-test was performed by the percentage of antipyretic, and the differences between the groups were compared. The above test was repeated a total of 3 times.
  • the blank control group was injected with normal saline 1ml/kg from the ear vein; the model control group and each drug group were injected with typhoid and paratyphoid vaccine 0.8ml/kg.
  • the rabbit's body temperature rises above 1 °C (about 1 ⁇ 1.5h, this experiment is limited to 1h)
  • the blank control group and the model group are intragastrically administered with 0.5% carboxymethylcellulose 1ml/kg.
  • the anus temperature was measured at 30, 60, 90, 120, 180 and 240 min after the drug, and the changes in body temperature were observed.
  • the t-test between groups was performed by the percentage of antipyretic, and the differences between the groups were compared.
  • Rabbits were injected with typhoid and paratyphoid vaccine for 1 hour, and the body temperature increased to about 1 °C, indicating that typhoid fever and paratyphoid vaccine can prepare rabbit fever model.
  • the body temperature of the model group continued to increase during the observation period of 300 min (p ⁇ 0.05-p ⁇ 0.001).
  • the high, medium and low doses of the forsythin/forsylin composition were compared.
  • the group had 30-240min, 60-240min, 90-240min after the drug had obvious antipyretic effect on fever caused by typhoid fever and paratyphoid vaccine (p ⁇ 0.05 ⁇ p ⁇ 0.001), and their curative effect was also better than that.
  • the forsythiaside group and the forsythiaside group (p ⁇ 0.01) showed that the forsythin/forsylin composition had obvious synergistic effect.
  • Table 2-8 The above test results are shown in Table 2-8.
  • Seventy male Wistar rats weighing 120-150 g were randomly divided into 7 groups according to body weight, namely: blank control (0.5% sodium carboxymethylcellulose) group, forsythin/forsylin composition 1
  • blank control (0.5% sodium carboxymethylcellulose
  • forsythin/forsylin composition 1 For the low, medium and high (2.5, 5 and 10 mg/kg) dose group, forsythiaside group, forsythiaside group and positive drug aspirin (200 mg/kg) group, 10 rats in each group.
  • Each group of the experiment was administered by sublingual injection.
  • the normal volume of the right hind paw of each group was determined by capillary amplification measurement before the experiment. In order to avoid errors, the measurement position should be fixed and operated by one person before and after administration.
  • the average of the two measurements was taken as the normal volume of the right hind paw of the rat before administration.
  • 0.1 ml of 1% carrageenan was injected subcutaneously into the right hind limb of the rat to cause inflammation.
  • the right hind paw volume was measured at 15, 30, 60, 120, 180, 240, 300 and 360 min after inflammation.
  • the difference between the groups was compared by the percentage difference (swelling rate) of the ankle volume before and after inflammation in rats, and the differences between the groups were compared.
  • the high dose group of forsythin/forsylin composition (10 mg/kg) was administered in the middle dose group (5 mg of forsythin/forsylin composition) within 15 min to 360 min after administration. /kg), low-dose group (2.5mg/kg) had significant inhibition on carrageenan-induced paw swelling in rats from 30min to 360min after administration (p ⁇ 0.05 or p ⁇ 0.01).
  • the curative effect was significantly better than that of forsythin 10 mg/kg group and forsythiaside 10 mg/kg group (p ⁇ 0.05 or p ⁇ 0.01), and the effects of each dose group of the above composition at 60 min and 240 min after administration were all effective.

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Abstract

一种连翘苷与连翘脂素的组合物在制备缓解或/和治疗病毒性疾病药物或保健品中的应用。

Description

连翘苷/连翘脂素组合物在制备缓解或/和治疗病毒性疾病的药物或保健品中的应用 技术领域
本发明属于药物化学领域，具体而言，本发明涉及连翘苷/连翘脂素组合物或混合物及其在制备用于缓解或/和治疗病毒性疾病的药物或保健品中的应用。
背景技术
流行性感冒是严重危害人类健康的急性、病毒性呼吸道传染病。二十一世纪以来，SARS、H5N1病毒和甲型H1N1流感病毒的流行，给人类带来极大的危害。目前世界上尚无有效治疗病毒性流感及肺炎的天然药物。
连翘为木犀科(Oleaceae)连翘属(Forsythia)植物连翘(Forsythia suspensa(Thunb.)Vahl)的干燥果实，主要分布于我国河南、山西、陕西、山东等地，此外，湖北、河北、四川、甘肃亦有分布。常用于治疗急性风热感冒、痈肿疮毒、淋巴结结核、尿路感染等症[1]。连翘的主要成分为连翘苷(phillyrin)和连翘苷元，连翘苷元又称连翘脂素((+)-phillygeninin)，两种成分的结构式如下。
Figure PCTCN2014094710-appb-000001
连翘主要成分为连翘苷，具有抗病毒、抗菌、抗氧化、清除自由基等药理作用。连翘脂素，亦为连翘的主要活性成分之一，具有抗氧化、降低血脂、清除自由基、抑菌、抗肿瘤、抗炎等作用。已发表文献中，主要对未明确成分和已明确几种成分的连翘提取物、连翘苷单一成分抗病毒药理作用进行了研究，而未见有人对连翘脂素和连翘苷的组合物、连翘脂素单一成分进行抗病毒药理作用研究。
发明内容
本发明的目的是针对现有在治疗、预防或缓解病毒性疾病的过程中存在的技术缺陷，提供一种连翘苷/连翘脂素组合物在制备用于缓解或/和治疗病毒性疾病的药物或保健品中的应用。本发明的连翘脂素/连翘苷组合物缓解或/和治疗病毒性疾病的性能和功效强，为研制用于缓解或/和治疗病毒性疾病的新药提供了 新的途径，即在治疗、调理和缓解病毒性疾病的药物或保健食品中的新应用提供了新的思路。
为实现上述目的，本发明一方面提供一种连翘苷/连翘脂素组合物在制备缓解或/和治疗病毒性疾病的药物或保健品中的应用。
其中，所述病毒性疾病为流感病毒、副流感病毒、柯萨奇病毒CoxA16、呼吸道合胞病毒RSV、单纯带状疱疹病毒HSV-I、单纯带状疱疹病毒HSV-II、单纯带状疱疹病毒CVB3、腺病毒ADV或肠道病毒EV71所致病毒性疾病，尤其是由流感病毒、副流感病毒、柯萨奇病毒CoxA16、呼吸道合胞病毒RSV所致的病毒性感冒、肺炎呼吸道传染病。
在筛选具有缓解或/和治疗病毒性流感及肺炎呼吸道传染病作用的天然活性成分的过程中，发明人发现连翘苷/连翘脂素组合物具有强烈的抑制病毒性流感及肺炎的作用，其疗效显著优于连翘苷或连翘脂素单独使用的效果。
其中，所述连翘苷/连翘脂素组合物中连翘苷与连翘脂素的重量份配比为80-98：2-20，优选为90-98：2-10，进一步优选为98：2。
特别是，所述连翘苷/连翘脂素组合物还包括药学上可接受的载体。
药学上可接受的载体通常被保健专家认可用于这一目的且作为药剂的非活性成分。有关药学上可接受的载体的汇编可以在《药物赋形剂手册》(Handbook of Pharmaceutical excipients，第2版，由A.Wade和P.J.Weller编辑；American Phar-maceutical Association出版，Washington and The Pharmaceutical Press,London,1994)等工具书中找到。
尤其是，所述的载体包括赋形剂，如淀粉、水等；润滑剂，如硬脂酸镁等；崩解剂，如微晶纤维素等；填充剂，如乳糖等；粘结剂，如预胶化淀粉、糊精等；甜味剂；抗氧化剂；防腐剂、矫味剂、香料等。
特别是，所述药物以片剂、胶囊剂、丸剂、散剂、颗粒剂、糖浆剂、溶液剂、乳剂、注射剂、喷雾剂、气雾剂、凝胶剂、霜剂、巴布剂、橡胶贴膏剂或贴膏剂形式存在。
其中，所述连翘苷/连翘脂素组合物中连翘苷与连翘脂素的总重量与药学上可接受的载体的重量之比为1:1～1:100，优选为1:10，进一步优选为1:5，更进一步优选为1:2，再更进一步优选为1:1。
特别是，所述的连翘苷/连翘脂素组合物含量≥80％，优选为≥85％，进一步优选为≥88％，更进一步优选为≥90％，再更进一步优选为≥99％。
特别是，所述的连翘苷/连翘脂素组合物中两种成分的重量比为：2～98：98～2；优选为80:20或20:80，进一步优选为90:,6或6:90，更进一步优选为98:2或2:98。
其中，所述连翘苷/连翘脂素组合物以连翘苷和连翘脂素以单体组成、或采用溶剂加热提取方法制备而成的连翘脂素-连翘苷提取组合物，或连翘脂素和连翘苷与环糊精或环糊精的衍生物组合而成连翘脂素-连翘苷-环糊精组合物。
特别是，所述连翘脂素-连翘苷-环糊精组合物选择连翘脂素和连翘苷与α-、β-或γ-环糊精或其衍生物相混合而成的混合物，或连翘脂素和连翘苷与α-、β-或γ-环糊精或其衍生物经物理、化学方法处理形成的复合物。
其中，所述连翘脂素-连翘苷-环糊精组合物中连翘苷和连翘脂素重量与环糊精或环糊精的衍生物的重量之比为1：1-50。
特别是，所述环糊精为α-或β-、γ-环糊精；所述环糊精衍生物为羟乙基β-环糊精、2,6-二甲基β-环糊精、2,3,6-三甲基β-环糊精、2,6-二乙基β-环糊精、2,3,6-三乙基β-环糊精、麦芽糖基β-环糊精或磺丁醚β-环糊精、对甲基苯磺酰氯(p-TsCl)取代的β-环糊精、6-位取代的β-CD对甲基苯磺酸酯(β-环糊精-6-OTs)、2-氧羟丙基-β-环糊精、2-位单取代的对甲基苯磺酸酯(β-环糊精-2-OTs)、β-环糊精对甲基苯磺酸酯(Tosyl-β-CD)、β-环糊精的星形大分子PCL-(Tos)7-β-CD。
其中，所述连翘脂素-连翘苷提取组合物按照如下方法提取制备而成：
1)采用溶剂对连翘叶或连翘果实进行加热回流提取2～3次，每次2～4小时；
2)提取液浓缩后静置，析出沉淀，获得连翘脂素和连翘苷混合物粗品；
3)将连翘脂素和连翘苷混合物粗品采用溶剂溶解后，静置，析出结晶，获得连翘脂素和连翘苷混合物；
4)用溶剂对连翘脂素和连翘苷混合物进行重结晶处理，得到连翘脂素-翘苷提取物。
特别是，步骤1)、3)、4)中所述溶剂为甲醇、乙醇、丙酮、含水甲醇或含水乙醇。
尤其是，所述含水甲醇的质量百分比浓度为70-95％；所述含水乙醇的质量百分比浓度为70-95％。
其中，步骤2)中静置的温度为室温，优选为10-35℃，进一步优选为20-25℃；静置时间为1-48h；步骤2)中提取液浓缩后的体积与原提取液的体积之比为0.1-0.5：1。
特别是，步骤4)中所述重结晶处理的温度为室温，优选为10-35℃，进一步优选为20-25℃。
本发明另一方面提供一种缓解或/和治疗病毒性疾病的药物或保健品，含有连翘苷和连翘脂素。
其中，所述连翘苷与连翘脂素的重量份配比为80-98：2-20，优选为90-98：2-10，进一步优选为98：2。
特别是，所述药物或保健品由连翘苷/连翘脂素组合物和药学上可接受的载体组成。
其中，所述连翘苷/连翘脂素组合物中连翘苷与连翘脂素的总重量与药学上可接受的载体的重量之比为1:1～1:100，优选为1:10，进一步优选为1:5，更进一步优选为1:2，再更进一步优选为1:1。
特别是，所述连翘脂素/连翘苷组合物的重量与所述药物或保健品的总重量之比为0.01～10：100，优选为0.1～10：100，进一步优选为1～10：100。
特别是，所述药物以片剂、胶囊剂、丸剂、散剂、颗粒剂、糖浆剂、溶液剂、乳剂、注射剂、喷雾剂、气雾剂、凝胶剂、霜剂、巴布剂、橡胶贴膏剂或贴膏剂形式存在。
特别是，所述药物或保健品中还包括蒲公英提取物、板蓝根提取物、金银花提取物、知母提取物、玄参提取物、夏枯草提取物、芦根提取物、淡竹叶提取物、栀子提取物、川贝提取物、苦丁茶提取物、鱼腥草提取物中的一种或多种。
所述药物可以采用本领域公知的方法制成各种剂型，如片剂、胶囊剂、丸剂、散剂、颗粒剂、糖浆剂、溶液剂、乳剂、注射剂、喷雾剂、气雾剂、凝胶剂、霜剂、巴布剂、橡胶贴膏剂或贴膏剂等。
本发明还提供了一种治疗病毒性流感及肺炎疾病的方法，包括向受试者给予治疗有效量的连翘脂素和连翘苷的组合物的药物组合物，其治疗有效量为0.1～50mg/kg.d，优选为0.3～30mg/kg.d，进一步优选为0.5～10mg/kg.d。
除非另外说明，本文所用的术语“治疗有效量”为需要产生有效作用的药物的用量；“治疗有效量”是可 以调整和变化的，最终由医务人员确定，其所考虑的因素包括给药途径和制剂的性质、接受者的体重、年龄等一般情况以及所治疗疾病的性质和严重程度。
与现有技术相比，本发明具有如下的明显优点：
本发明连翘苷/连翘脂素组合物的制备方法简便、适于工业化生产，组合物具有明显的抗流感、肺炎等多种病毒的疗效，且抗病毒效果显著优于单独使用连翘苷、连翘脂素的疗效，也明显优于目前临床最新抗病毒药——磷酸奥司他韦(达菲)；此外，还发掘了连翘苷/连翘脂素组合物具有抗其他病毒的疗效，能显著抑制柯萨奇病毒CoxA16及呼吸道合胞病毒RSV、单纯带状疱疹病毒HSV-I和HSV-II及CVB3、腺病毒ADV、肠道病毒EV71，可用于治疗上述病毒所致流感及肺炎、带状疱疹、心肌炎、手足口病、上呼吸道感染、毛细支气管炎、皮疹、脑膜炎等疾病，故本发明可制备成用于缓解或/和治疗病毒性流感及肺炎、带状疱疹、心肌炎、手足口病、上呼吸道感染、毛细支气管炎、皮疹、脑膜炎等疾病的高效天然药物或保健食品，为连翘药材的应用开拓了一个新的领域。
1、本发明的连翘苷/连翘脂素组合物具有抗流感及肺炎病毒的疗效，其疗效显著优于连翘苷、连翘脂素单独用药的疗效，连翘苷与连翘脂素按照特定比例组合使用，具有协同增效的作用；
2、本发明的连翘苷/连翘脂素组合物具有抑制柯萨奇病毒CoxA16及呼吸道合胞病毒RSV、单纯带状疱疹病毒HSV-I和HSV-II及CVB3、腺病毒ADV、肠道病毒EV71等多种病毒的作用，连翘苷/连翘脂素组合物治疗上述病毒所致流感及肺炎、带状疱疹、心肌炎、手足口病、上呼吸道感染、毛细支气管炎、皮疹、脑膜炎等疾病；
3、本发明的连翘苷/连翘脂素组合物具有显著抑制病毒所致炎症及增强患者的免疫力的功效；连翘苷/连翘脂素组合物抑制病毒性流感及肺炎的药理作用强，用于缓解、调理和治疗病毒性流感及肺炎的功效显著，见效快、毒副作用小、安全性好，能够长期服用，具有良好的药用前景；
4、本发明的产品原料来源丰富、价廉、临床使用安全，制备工艺简单，可制成各种剂型，且服量小，使用方便，因此易于推广；
5、本发明的连翘苷/连翘脂素组合物既可采用单体成分定量配制而成，也可通过提取制备成连翘苷/连翘脂素组合物，或上述连翘苷/连翘脂素组合物与一种α-或β-、γ-环糊精或环糊精的衍生物组合而成配方成组合物，还可采用连翘苷/连翘脂素组合物与其他活性成分(例如与蒲公英提取物、板蓝根提取物、金银花提取物、知母提取物、玄参提取物、夏枯草提取物、芦根提取物、淡竹叶提取物、栀子提取物、川贝提取物、苦丁茶提取物、鱼腥草提取物中的一种或多种)共同组方，制备治疗病毒性流感、肺炎及其他病毒性疾病的复方药物。
具体实施方式
下面通过具体实施方式来进一步描述本发明所述配方的有益效果。这些实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的配方思路、用途范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
实施例1-4 连翘苷/连翘脂素组合物的制备
分别取连翘苷和连翘脂素的两种单体成分粉末，两者可以按表1的重量比混合制备成连翘苷/连翘脂素组合物，连翘苷单体由大连富生天然药物开发有限公司生产，经高效液相色谱两种检测器紫外检测器和蒸发光散射检测器面积归一化法测定，其纯度为99.5％，并用中国药品生物制品含量测定用连翘苷对照品标定和确认其含量为99.5％；连翘脂素由大连富生天然药物开发有限公司生产，经高效液相色谱两种检测器紫外检测器和蒸发光散射检测器面积归一化法测定，其纯度为99.1％。
表1 实施例1-4的连翘苷/连翘脂素组合物的原料配比表
Figure PCTCN2014094710-appb-000002
实施例5-24 连翘苷/连翘脂素组合物的制备
取实施例1-4制备的连翘苷/连翘脂素组合物，以表2所述重量比、按以下方法制备组合物：(1)直接加入到环糊精溶液中，或(2)直接加入到环糊精溶液中并充分搅拌1-24h、(3)直接加入到环糊精溶液中并加热10-120分钟、(4)直接加入到环糊精溶液中并超声10-120分钟、(5)直接与环糊精粉末研磨10-120分钟、(6)使连翘苷/连翘脂素组合物与所述环糊精粉末混合均匀、过筛；(7)直接加入环糊精衍生物溶液中，或(8)直接加入到环糊精衍生物溶液中并充分搅拌1-24h、(9)直接加入到环糊精衍生物溶液中并加热10-120分钟、(10)直接加入到环糊精衍生物溶液中并超声10-120分钟、(11)直接与环糊精衍生物粉末研磨10-120分钟、(12)与环糊精衍生物粉末混合均匀、过筛。
表2  实施例5-24的原料配比和制备方法
Figure PCTCN2014094710-appb-000003
实施例5-24中的辅料以选用β-环糊精为例进行举例说明，其他环糊精、环糊精衍生物均适用于本发明，例如1)β-羟丙基环糊精、2)羟乙基β-环糊、3)2,6-二甲基β-环糊精、4)2,3,6-三甲基β-环糊精、5)2,6-二乙基β-环糊精、6)2,3,6-三乙基β-环糊精、7)麦芽糖基β-环糊精、8)磺丁醚β-环糊精、9)对甲基苯磺氯(p-TsCl)取代的β-环糊精、10)6-位取代的β-CD对甲基苯磺酸酯(β-环糊精-6-OTs)、11)2-氧羟丙基-β-环糊精、12)2-位单取代的对甲基苯磺酸酯(β-环糊精-2-OTs)、13)β-环糊精对甲基苯磺酸酯(Tosyl-β-CD)j，以及15)β-环糊精的星形大分子PCL-(Tos)7-β-CD。
实施例25 连翘苷和连翘脂素组合物的制备
干燥的连翘叶1kg，加入95％(m/m)乙醇10kg，加热回流提取2次，每次2小时，过滤，滤液减压浓缩至原体积的1/2，于25℃放置1h，析出沉淀；再用甲醇溶解，进行重结晶，析出沉淀；重复上述方法用甲醇重结晶，得连翘苷/连翘脂素组合物无定形粉末，采用HPLC测定连翘苷、连翘脂素的含量，分别为98％、2％。
实施例26 连翘苷和连翘脂素组合物的制备
干燥的连翘果实1kg，加入甲醇10kg，加热回流提取3次，每次4小时，过滤，滤液减压浓缩至原体积的1/10，于20℃放置48h，析出沉淀；再用乙醇溶解，进行重结晶，析出沉淀；重复上述方法用乙醇重结晶，得连翘苷/连翘脂素组合物无定形粉末，其所含连翘苷、连翘脂素的含量分别为95％、4％。
实施例27 连翘苷和连翘脂素组合物的制备
干燥的连翘叶1kg，加入70％(m/m)甲醇10kg，加热回流提取3次，每次3小时，过滤，滤液减压浓缩至原体积的1/3，于室温放置2h，析出沉淀；再用90％甲醇溶解之，进行重结晶，析出沉淀；重复上述方法用甲醇重结晶，得连翘苷/连翘脂素组合物无定形粉末，其所含连翘苷、连翘脂素的含量分别为88％、2％。
实施例28 连翘苷和连翘脂素组合物的制备
干燥的连翘果实1kg，加入无水乙醇10kg，加热回流提取2次，每次4小时，过滤，滤液减压浓缩至原体积的1/4，于室温放置24h，析出沉淀；再用丙酮溶解之，进行重结晶，析出沉淀；重复上述方法用丙酮重结晶，得连翘苷/连翘脂素组合物无定形粉末，其所含连翘苷、连翘脂素的含量分别为90％、6％。
实施例29 连翘苷和连翘脂素组合物的制备
干燥的连翘叶子1kg，加入丙酮10kg，加热回流提取3次，每次3小时，过滤，滤液减压浓缩至原体积的1/5，于室温放置10h，析出沉淀；再用70％乙醇溶解之，进行重结晶，析出沉淀；重复上述方法用70％乙醇重结晶，得连翘苷/连翘脂素组合物无定形粉末，其所含连翘苷、连翘脂素的含量分别为80％、5％。
实施例30 连翘苷和连翘脂素组合物片剂制备
按照如下配比制备连翘苷/连翘脂素组合物的片剂：
Figure PCTCN2014094710-appb-000004
按上述配比，取实施例1中制备的连翘苷/连翘脂素组合物与淀粉混合均匀后，制成颗粒，加入滑石粉 和硬脂酸镁混合均匀后压制成10000片。
实施例31 连翘苷/连翘脂素组合物颗粒剂制备
按照如下配比制备连翘苷/连翘脂素组合物的颗粒剂：
连翘苷/连翘脂素组合物(两者重量比为98:2)    100g
微晶纤维素                            10000g
按上述配比，取实施例1中制备的连翘苷/连翘脂素组合物与微晶纤维素混合均匀后，制成颗粒装袋，制成10000袋。
实施例32 连翘苷/连翘脂素组合物胶囊剂制备
按照如下配比制备连翘苷/连翘脂素组合物的胶囊剂：
连翘苷/连翘脂素组合物(两者重量比为98:2)    250g
淀粉                                       2500g
按上述配比，取实施例1中制备的连翘苷/连翘脂素组合物与淀粉混合均匀后装胶囊，制成10000粒。
实施例33-36 连翘苷/连翘脂素组合物胶囊剂制备
实施例33-36中，连翘苷/连翘脂素组合物分别按表3的重量比与淀粉混合均匀后装胶囊，各制成10000粒胶囊。
表3
Figure PCTCN2014094710-appb-000005
表中原料，可替换为实施例5-29制备的连翘脂素和连翘苷的组合物。
实施例:37-40 连翘苷/连翘脂素组合物颗粒剂制备
实施例37-30中，连翘苷/连翘脂素组合物分别按表4的重量比与微晶纤维素混合均匀后，制成颗粒装袋，制成10000袋。
表4
Figure PCTCN2014094710-appb-000006
表中原料，可替换为实施例5-29制备的连翘脂素和连翘苷的组合物。
实施例41 连翘苷/连翘脂素组合物片剂制备
按照如下配比制备连翘苷/连翘脂素组合物的片剂：
Figure PCTCN2014094710-appb-000007
Figure PCTCN2014094710-appb-000008
按上述配比，连翘苷/连翘脂素组合物与上述提取物粉末混合均匀，再与淀粉混合均匀后制成颗粒，加入滑石粉和硬脂酸镁混合均匀后压制成10000片。其中，本实施例中的连翘苷/连翘脂素组合物可替换为实施例5-29制备的连翘脂素和连翘苷的组合物。
实施例42 连翘苷/连翘脂素组合物颗粒剂制备
按照如下配比制备连翘苷/连翘脂素组合物的颗粒剂：
Figure PCTCN2014094710-appb-000009
按上述配比，连翘苷/连翘脂素组合物与上述提取物粉末混合均匀，再与微晶纤维素混合均匀后制成颗粒装袋，制成10000袋。其中，本实施例中的连翘苷/连翘脂素组合物可替换为实施例5-29制备的连翘脂素和连翘苷的组合物。
实施例43 连翘苷/连翘脂素组合物胶囊剂制备
按照如下配比制备连翘苷/连翘脂素组合物的胶囊剂：
Figure PCTCN2014094710-appb-000010
按上述配比，连翘苷/连翘脂素组合物与上述提取物粉末混合均匀，再与淀粉混合均匀后装胶囊，制成10000粒。其中，本实施例中的连翘苷/连翘脂素组合物可替换为实施例5-29制备的连翘脂素和连翘苷的组合物。
实施例52 连翘苷/连翘脂素组合物片剂制备
按照如下配比制备连翘苷/连翘脂素组合物的片剂：
Figure PCTCN2014094710-appb-000011
按上述配比，连翘苷/连翘脂素组合物与上述提取物粉末混合均匀，再与淀粉混合均匀后制成颗粒，加入滑石粉和硬脂酸镁混合均匀后压，制成10000片。其中，本实施例中的连翘苷/连翘脂素组合物可替换为实施例5-29制备的连翘脂素和连翘苷的组合物。
实施例53 连翘苷/连翘脂素组合物颗粒剂制备
按照如下配比制备连翘苷/连翘脂素组合物的颗粒剂：
Figure PCTCN2014094710-appb-000012
按上述配比，连翘苷/连翘脂素组合物与上述提取物粉末混合均匀，再与微晶纤维素混合均匀后制成颗粒装袋，制成10000袋。其中，本实施例中的连翘苷/连翘脂素组合物可替换为实施例5-29制备的连翘脂素和连翘苷的组合物。
实施例54 连翘苷/连翘脂素组合物胶囊剂制备
按照如下配比制备连翘苷/连翘脂素组合物的胶囊剂：
Figure PCTCN2014094710-appb-000013
按上述配比，连翘苷/连翘脂素组合物与上述提取物粉末混合均匀，再与淀粉混合均匀后装胶囊，制成10000粒。其中，本实施例中的连翘苷/连翘脂素组合物可替换为实施例5-29制备的连翘脂素和连翘苷的组合物。
试验例1 连翘苷/连翘脂素组合物抗病毒试验
1体外抗病毒试验
1.1试验材料
(1)药物
①连翘苷，白色粉末，大连富生天然药物开发有限公司生产，经高效液相色谱两种检测器紫外检测器和蒸发光散射检测器面积归一化法测定，其纯度为99.5％，并用中国药品生物制品含量测定用连翘苷对照品标定和确认其含量为99.5％。
②连翘脂素，白色粉末，大连富生天然药物开发有限公司生产，经高效液相色谱两种检测器紫外检测器和蒸发光散射检测器面积归一化法测定，其纯度为99.1％；
③连翘苷/连翘脂素组合物：连翘苷/连翘脂素组合物1：白色粉末，大连富生天然药物开发有限公司生产，由连翘苷和连翘脂素两种单体配比而成，用99.5％连翘苷和99.1％连翘脂素作为对照品进行标定，连翘苷/连翘脂素组合物中两种单体成分含量均为98％，其中连翘苷/连翘脂素组合物1中连翘苷与连翘脂素的重量比98：2；连翘苷/连翘脂素组合物2中连翘苷与连翘脂素的重量比为80:20。
④利巴韦林注射液，无色透明液体，由河南润弘股份有限公司生产，产品批号：1206261，国药准字：H19993553，100mg/ml，作为本次试验阳性对照药物。
⑤磷酸奥司他韦，中国药品生物制品检定所。产品批号：101096-200901，100mg/支作为本次试验阳 性对照药物。
上述药品均用纯净水溶解，滤过，除菌分装，4℃备用，为本次试验待测药物。
(2)细胞株：Vero(非洲绿猴肾细胞细胞)由吉林大学基础医学院保存细胞株。
(3)病毒株：①流感病毒株、副流感病毒株、呼吸道合胞病毒(RSV)株：购于中国预防医学科学院病毒研究所；②柯萨奇病毒B3(CVB3)株：来源于美国,为本教研室保存；③柯萨奇病毒A16(CoxA16)株、肠道病毒EV71株：由日本仙台国立医院馈赠，于本教研室保存；④腺病毒(AdV)来源于白求恩医科大学一院儿科研究室；⑤单纯疱疹病毒I型(HSV-1)购于卫生部药品生物制品检定所。
(4)主要设备与试剂：
生物安全柜：BHC-1300ⅡA/B3，AIRTECH；CO2培养箱：MCO-18AIC，SANYO；倒置显微镜：CKX41，OLYMPUS；电子分析天平：AR1140/C，DHAUS；培养基：DMEM，HyClone；胎牛血清：HyClone；胰蛋白酶：Gibco；MTT：Sigma；DMSO：天津市北联精细化学品开发有限公司。
1.2试验方法
(1)细胞准备
Vero细胞传代培养1-2d，使之成片，界线清晰，立体感及折光度强时，用胰酶消化，待细胞面出现针尖样小孔，吸尽消化液，取数毫升培养液吹散细胞，计数，用培养液(含10％胎牛血清的DMEM)稀释至约5×107个/L后，接种于96孔培养板内，待细胞长成单层。
(2)药物毒性测定
细胞毒性试验：将药物按表1-1所示浓度进行稀释，用于细胞毒性测定。
表1-1  药物稀释参照表(单位：g/L)
Figure PCTCN2014094710-appb-000014
将上述用维持液(含2％胎牛血清的DMEM)稀释好的不同浓度的药品滴加于Vero单层细胞上，每孔0.2ml，每个浓度6个复孔，另设6孔正常对照(不加药物的正常对照组)和6孔空白对照(培养液)，置37℃，5％CO2培养箱中培养，每日置倒置显微镜观察CPE并记录。72h后，每孔加入MTT溶液20μL(5mg·mL－1)，继续孵育4h，吸弃各孔培养液，每孔加入100μL DMSO，振荡5min，492nm测定OD值，计算细胞存活率。在SPSS 18.0统计软件中，将细胞存活率进行Probit回归分析，计算药物对Vero细胞的最大无毒浓度(TC0)和半数毒性浓度(TC50)。
(3)各种病毒TCID50的测定
将各种病毒进行10倍递减稀释为10-1,10-2,10-3，10-4，10-5，10-6不同稀释度，按序接种于单层的Vero 细胞96孔培养板上，每孔100μL，每个稀释度6孔，同时设正常细胞对照组。置37℃，5％CO2中孵育2h，弃病毒液，随即每孔加细胞维持液100μL，置37℃，5％CO2中培养。第3天开始在显微镜下观察细胞病变结果，第7-8天判定结果并做好记录，以能使50％细胞孔发生阳性病变的最高稀释度作为终点，用karber法计算病毒滴度。
公式
Figure PCTCN2014094710-appb-000015
50％组织细胞感染量；XM：病毒最高浓度稀释度的对数；d：稀释度系数(倍数)的对数；Σpi：每个稀释度病变百分数的总和。)
(4)药物对病毒致细胞病变作用的影响
取已长满单层细胞的培养板，吸弃培养液，以100TCID50对应的病毒攻击量接种细胞，37℃，5％CO2培养箱吸附2h，加入特定浓度(最大无毒浓度左右)的各药液，每浓度6个复孔培养，200μL/孔。设利巴韦林注射液和磷酸奥司他韦为阳性药物对照组，同时设正常对照组(不加病毒不加药)和病毒对照组(加病毒但不加药物的对照组)，观察药物对病毒致CPE的影响。72h后，用MTT比色法，在492nm波长下测定OD值，计算药物抗病毒有效率(ER％)。在SPSS 18.0统计软件中用ANOVA法比较各药物抗病毒有效率之间的显著性差异。
ER％＝(药物处理组平均OD值－病毒对照组平均OD值)/(细胞对照组平均OD值－病毒对照组平均OD值)×100％
1.3试验结果
(1)各种病毒的TCID50
副流感病毒：
Figure PCTCN2014094710-appb-000016
流感病毒：
Figure PCTCN2014094710-appb-000017
CVB3：
Figure PCTCN2014094710-appb-000018
HSV-1：
Figure PCTCN2014094710-appb-000019
AdV：
Figure PCTCN2014094710-appb-000020
RSV：
Figure PCTCN2014094710-appb-000021
CoxA16：
Figure PCTCN2014094710-appb-000022
EV71：
Figure PCTCN2014094710-appb-000023
(2)药物毒性测定
1)药物对细胞毒性的测定
各药物对Vero细胞的最大无毒浓度(TC0)、半数毒性浓度(TC50)及药物抗病毒实验用浓度见表1-2。
表1-2  药物细胞毒性实验结果(单位：g/L)
Figure PCTCN2014094710-appb-000024
2)药物对病毒致细胞病变保护作用结果
药物抗各种病毒的有效率及ANOVA法单因素方差分析结果，详见表1-3。
表1-3  药物抗病毒有效率(ER％)统计表
Figure PCTCN2014094710-appb-000025
注：与病毒对照组相比，*P<0.05，**P<0.01；连翘苷/连翘脂素组合物与连翘苷相比，#P<0.05，##P<0.01；连翘苷/连翘脂素组合物与连翘脂素相比，▲P<0.05，▲▲P<0.01；连翘苷/连翘脂素组合物与利巴韦林相比相比，△P<0.05，△△P<0.01，△△△P<0.001；连翘苷/连翘脂素组合物与磷酸奥司他韦相比，●P<0.05，●●P<0.01，●●●P<0.001。
表1-3结果显示，连翘苷/连翘脂素组合物对8种病毒均有明显抑制作用(P<0.01或P<0.001)，对流感病毒、副流感病毒、单纯疱疹病毒Ⅰ型(HSV-I)、肠道病毒EV71及腺病毒(ADV)抗病毒的有效率均达到100％，且疗效明显优于连翘苷、连翘脂素，表明连翘苷/连翘脂素组合物具有协同增效作用。此外，连翘苷/连翘脂素组合物抑制流感、柯萨奇病毒A16(CoxA16)、呼吸道合胞病毒(RSV)、单纯疱疹病毒Ⅰ型(HSV-I)、腺病毒(ADV)、肠道病毒EV71及柯萨奇病毒B3(CVB3)的疗效明显优于阳性药利巴韦林(P<0.01或P<0.001)，抑制流感、柯萨奇病毒A16(CoxA16)、呼吸道合胞病毒(RSV)、单纯疱疹病毒I型(HSV-I)、腺病毒(ADV)、肠道病毒EV71及柯萨奇病毒B3(CVB3)的疗效上明显优于磷酸奥司他韦(P<0.05，或P<0.01、P<0.001)。
2.体内抗病毒试验
2.1实验材料
(1)实验动物
昆明种小鼠由吉林大学白求恩医学部实验动物中心提供，医动字第10-5219号。
(2)检测仪器、试剂
Figure PCTCN2014094710-appb-000026
2.2实验方法
(1)流感病毒和副流感病毒对小鼠半数致死量的测定
将流感病毒和副流感病毒(细胞裂解液)10倍递比稀释为10-1、10-2、10-3、10-4、10-5浓度的病毒液。取昆明种小鼠120只，流感病毒和副流感病毒组各60只，分别随机分成6组，乙醚轻度麻醉小鼠，滴鼻感染不同稀释度病毒液0.03mL/只。同时设空白对照，用生理盐水代替病毒悬液。以死亡和生存为观察指标，每天观察，直至感染后的14天。感染24h内死亡的为非特异死亡，不予统计，Karber法计算病毒液LD50。计算公式：
Figure PCTCN2014094710-appb-000027
[其中：LD50：半数致死量；XM：病毒最高浓度稀释度的对数；d：稀释度系数(倍数)的对数；Σpi：每个稀释度病变百分数的总和]。
(2)连翘苷/连翘脂素组合物抗流感病毒和副流感病毒感染所致肺炎的研究
1)试验动物及分组
取四周龄的昆明小鼠540只，进行2项试验。
先取小鼠270只，随机分成27组，每组10只，用于连翘苷/连翘脂素组合物对流感和副流感病毒感染小鼠肺指数和肺指数抑制率的测定试验；每次试验取小鼠90只，3次重复试验。另取小鼠270只，随机分成27组，每组10只，用于连翘脂素和连翘苷的组合物对肺悬液病毒血凝滴度的测定试验；每次试验取小鼠90只，3次重复试验。
2)感染方法
在200～300mL大小的烧杯内放入一团脱脂棉，然后倒入适量的乙醚(使脱脂棉变湿即可)，把装有脱脂棉的烧杯倒扣过来，把小鼠放入进行麻醉，见小鼠极度兴奋，明显呈无力样时，将小鼠仰卧，滴鼻感染15LD50流感病毒和副流感病毒0.03ml/鼻孔，正常对照组用生理盐水代替病毒悬液。
3)给药方法及给药剂量
连翘苷/连翘脂素组合物1组、连翘苷/连翘脂素组合物2组、连翘脂素组、连翘苷组、利巴韦林和磷酸奥司他韦对照组，分别于感染前一天开始常规灌胃给药，连翘苷/连翘脂素组合物1、2分别分为高、中、低剂量组，给药剂量分别为13.0、6.5、3.25mg/kg，连翘苷组给药剂量为13mg/kg、连翘脂素组给药剂量 为13mg/kg、利巴韦林阳性药给药剂量为58.5mg/kg、磷酸奥司他韦给药剂量为19.5mg/kg，每天一次，连续给药5d，正常对照组、病毒对照组灌服相同体积的生理盐水。
4)观察指标
①肺指数测定
小鼠用药后第5天，先禁食水8小时，称体重后摘眼球放血处死动物，打开胸腔摘出全肺，以生理盐水洗涤两次，用滤纸吸干表面水份，电子天平称肺重，按下列公式计算计算肺指数和肺指数抑制率：
肺指数＝(小鼠肺重/小鼠体重)×100％
肺指数抑制率＝(感染模型组平均肺指数-实验组平均肺指数)/感染模型组平均肺指数×100％
②肺悬液病毒血凝滴度测定
分别取治疗后第5天的各组小鼠肺，低温下置匀浆器研磨成匀浆，生理盐水稀释为10％的肺组织悬液，离心取上清，倍比稀释，按0.2ml/孔滴于滴定板上,每孔加入0.2ml 1％鸡红细胞悬液,混匀,置室温30min,观察记录血凝滴度。以红细胞凝集(++)时为終点，以悬液稀释倍数表示其滴度。
2.3试验结果及分析
(1)流感病毒和副流感病毒对小鼠半数致死量的测定结果
试验组昆明种小鼠分别被滴鼻感染不同浓度流感病毒、副流感病毒液30μL，感染第3天前3组(病毒浓度为10-1组、10-2组、10-3组)小鼠均出现不同程度的发病症状：耸毛、发抖、饮食减少等；第5天小鼠出现走路摇摆不定；第6天最高病毒浓度组小鼠开始出现死亡，其余各组于感染后第7天陆续出现死亡现象。观察14天结束后，统计各组小鼠死亡数目，结果见下表1-4、1-5。计算该流感病毒的LD50为稀释度10-2 . 9，副流感病毒的LD50为稀释度10-2 . 5。
表1-4  流感病毒半数致死量试验结果统计
Figure PCTCN2014094710-appb-000028
Karber法计算病毒的LD50。流感病毒的LogLD50如下：
Figure PCTCN2014094710-appb-000029
表1-5  副流感病毒半数致死量试验结果统计
Figure PCTCN2014094710-appb-000030
Karber法计算病毒的LD50。副流感病毒的LogLD50如下：
Figure PCTCN2014094710-appb-000031
(2)连翘苷/连翘脂素组合物抗流感病毒和副流感病毒感染所致肺炎的作用结果
①肺指数测定
流感病毒和副流感病毒感染小鼠后，平均肺指数结果显示：与感染模型组比较，正常对照组、连翘苷组13.0mg/kg/d、连翘脂素组16.0mg/kg/d、连翘苷/连翘脂素组合物1和2的3个剂量组(低剂量组3.25mg/kg/d、中剂量组6.5mg/kg/d、高剂量组13.0mg/kg/d)、利巴韦林组及磷酸奥司他韦组的肺指数明显降低(P<0.05或P<0.01)；其中，连翘苷/连翘脂素组合物在3.25～13.0mg/kg/d浓度范围内有明显保护作用，肺指数均明显降低、对肺组织病变指数的抑制率，其疗效明显优于优于与连翘苷组、连翘脂素组(P<0.01或P<0.05)，结果见表1-6、1-7。
表1-6  连翘苷/连翘脂素组合物对流感病毒感染小鼠肺指数的抑制率(n＝3)
Figure PCTCN2014094710-appb-000032
各试验组病毒对照组比较，*P<0.05，**P<0.01；连翘苷/连翘脂素组合物与连翘苷比较，#P<0.05,##P<0.01；连翘苷/连翘脂素组合物与连翘脂素比较，▲P<0.05，▲▲P<0.01。
表1-7  连翘苷/连翘脂素组合物对副流感病毒感染小鼠肺指数的抑制率(n＝3)
Figure PCTCN2014094710-appb-000033
Figure PCTCN2014094710-appb-000034
各试验组病毒对照组比较，*P<0.05，**P<0.01；连翘苷/连翘脂素组合物与连翘苷比较，#P<0.05,##P<0.01；连翘苷/连翘脂素组合物与连翘脂素比较，▲P<0.05。
②肺悬液病毒血凝滴度测定
流感病毒和副流感病毒感染小鼠后，感染模型组肺组织病毒血凝滴度(InX)分别为32.40和33.11，不同浓度连翘脂素/连翘苷组合物1和2用药治疗5天后，肺组织病毒血凝滴度均有所下降，与感染模型组比较，差异有显著性(P<0.01)，且它们的不同剂量组对流感、副流感病毒血凝滴度均明显低于连翘苷组、连翘脂素组(P<0.05～P<0.001)，表明组合物具有协同增效作用，其对病毒繁殖的抑制率均明显高于连翘苷组、连翘脂素组(P<0.05～P<0.001)其中，连翘苷/连翘脂素组合物1和2的高、中、低剂量组对流感病毒感染小鼠肺悬液血凝滴度的抑制率均显著高于连翘苷组、连翘脂素组(P<0.01～P<0.001)。上述试验结果详见表1-8、1-9。
表1-8  连翘苷/连翘脂素组合物对流感病毒感染小鼠肺悬液血凝滴度的影响(n＝3)
Figure PCTCN2014094710-appb-000035
表1-9  连翘苷/连翘脂素组合物对副流感病毒感染小鼠肺悬液血凝滴度的影响(n＝3)
Figure PCTCN2014094710-appb-000036
表1-8和1-9中，各试验组病毒对照组比较，*P<0.05，**P<0.01；连翘苷/连翘脂素组合物与连翘苷比 较，#P<0.05,##P<0.01，###P<0.001；连翘苷/连翘脂素组合物与连翘脂素比较，▲P<0.05,▲▲P<0.01，▲▲▲P<0.001。
试验例2 连翘苷/和连翘脂素组合物解热、抗炎试验
1.1试验材料
(1)试验动物Wistar大鼠，体重120～250g，雌雄兼用，合格证号：医动字第13-1225；日本大耳白兔，雄性，体重1.5～2.0kg。合格证号：医动字第10-5115，均由长春高新医学动物实验中心供给，动物饲料由吉林大学实验动物部供给。
(2)试验药品
①连翘苷，白色粉末，大连富生天然药物开发有限公司生产，经高效液相色谱两种检测器紫外检测器和蒸发光散射检测器面积归一化法测定，其纯度为99.5％，并用中国药品生物制品含量测定用连翘苷对照品标定和确认其含量为99.5％；
②连翘脂素，白色粉末，大连富生天然药物开发有限公司生产，经高效液相色谱两种检测器紫外检测器和蒸发光散射检测器面积归一化法测定，其纯度为99.1％；
③连翘苷/连翘脂素组合物：白色粉末，大连富生天然药物开发有限公司生产。由连翘苷与连翘脂素两种单体配比而成，用99.5％连翘苷和99.1％连翘脂素作为对照品进行标定，连翘苷/连翘脂素组合物中两种单体成分含量均为98％，其中连翘苷/连翘脂素组合物1中连翘苷与连翘脂素的重量比98：2，连翘苷/连翘脂素组合物1中连翘苷与连翘脂素的重量比80：20。
1.2主要仪器与试剂
YLS-7A大鼠足趾肿胀测量仪：山东省医学科学院设备站；722可见分光光度计：上海光谱仪器有限公司生产；便携式数字测温仪：型号WSC-411P，上海浦东三厂；毛果芸香碱：天津市人民制药厂，批号:20130112；组胺：上海生化所，批号：20130115；5-羟色胺：上海生化所，批号:20130623；伊文思蓝：上海化学试剂采购供应站，批号：20130217；扑尔敏片：长春经济开发区药业有限公司，批号：20130801；角叉菜胶：吉林省药物研究所，批号：20130502；扑热息痛片：辽源市百康药业有限责任公司，批号：20130512；阿司匹林片：白城万达药业有限公司，批号：20130305；啤酒酵母：北京奥博星生物技术责任有限公司，批号：2013020；伤寒、副伤寒疫苗：长春生物制品研究所，批号：20130216。
1.3统计处理
统计分析采用两样本比较的秩和检验、X2检验及t检验。
2.1连翘苷/连翘脂素组合物对大鼠足跖部汗液分泌影响胡试验(着色法)
(1)材料与方法
本试验根据大鼠足跖部肉垫上有汗腺分布，其汗液分泌的多少可利用碘与淀粉遇汗液即可产生紫色反应的机理，观测汗液分泌的变化。
试验取Wistar大鼠500只，雌雄各半，体重120～150g。按体重、性别随机分为50组，即：对照(0.5％羧甲基纤维素)组，连翘苷组、连翘脂素组、连翘苷/连翘脂素组合物组1和2的低、中、高剂量组(分别为2.5、5、10mg/kg)及阳性药毛果芸香碱(35mg/kg)组，每组10只，每次试验10组，共5个时间段的试验 (1、5、10、15、20min)。将大鼠置入自制的大鼠固定袋内，暴露双后肢。用棉签蘸取无水乙醇将其右足跖部污物轻轻擦洗干净。除毛果芸香碱溶液皮下注射外，其余各组均采用灌胃方式给药。给药后1h(毛果芸香碱组于给药后30min)，先将各组大鼠右足跖原有的和由于挣扎时所致的汗液用干棉签轻轻拭干，涂上和田-高垣氏试剂A液(取碘2g溶于100ml无水乙醇)，待充分干燥后，再薄薄涂上和田-高垣氏试剂B液(取可溶性淀粉50g、蓖麻油100ml均匀混合)。分别于涂B液后1、5、10、15及20min用放大镜仔细观察深紫色着色点(即汗点)出现的颜色和数量。试验结束后，按两样本比较的秩和检验进行统计学处理，比较各组间的差异。
(2)结果
与对照组比较，连翘苷/连翘脂素组合物的中、高剂量组(5、10mg/kg)于涂B液后10、15及20min对大鼠足跖部汗液分泌均有明显促进作用(p<0.05),连翘苷/连翘脂素组合物的2.5mg/kg组于涂B液后15及20min对大鼠足跖部汗液分泌均有明显促进作用(p<0.05),它们的发汗作用与阳性药毛果芸香碱相当，且具有缓缓促进大鼠足跖部汗液分泌的作用特点。连翘苷/连翘脂素组合物高剂量组于涂B液后10、15、20min后对大鼠足跖部汗液分泌的促进作用明显优于连翘苷和连翘脂素(p<0.05)，连翘苷/连翘脂素组合物中剂量组于涂B液后10、15min后对大鼠足跖部汗液分泌的促进作用明显优于连翘苷和连翘脂素(p<0.05)，连翘苷/连翘脂素组合物低剂量组于涂B液后15min后对大鼠足跖部汗液分泌的促进作用明显优于连翘苷和连翘脂素(p<0.05)。上述试验结果，表明连翘苷/连翘脂素组合物促进大鼠足跖部汗液分泌的作用明显优于连翘苷和连翘脂素，详见2-1、2-2、2-3、2-4、2-5。
表2-1  连翘苷/连翘脂素组合物对正常大鼠足跖部汗液分泌的影响(着色法)
Figure PCTCN2014094710-appb-000037
表2-2  连翘苷/连翘脂素组合物对正常大鼠足跖部汗液分泌的影响(着色法)
Figure PCTCN2014094710-appb-000038
表2-3  连翘苷/连翘脂素组合物对正常大鼠足跖部汗液分泌的影响(着色法)
Figure PCTCN2014094710-appb-000039
表2-4  连翘苷/连翘脂素组合物对正常大鼠足跖部汗液分泌的影响(着色法)
Figure PCTCN2014094710-appb-000040
Figure PCTCN2014094710-appb-000041
表2-5  连翘苷/连翘脂素组合物对正常大鼠足跖部汗液分泌的影响(着色法)
Figure PCTCN2014094710-appb-000042
汗点等级评定标准：
“-”大鼠足跖肉垫表面无汗点；“+”大鼠足跖肉垫表面偶见汗点，汗点面积约占足跖表面的10％以下；“++”大鼠足跖肉垫表面散在分布汗点，汗点面积约占足跖表面的11-40％；“+++”大鼠足跖肉垫表面多处分布汗点，汗点面积约占足跖表面的41-70％；“++++”大鼠足跖肉垫表面均匀分布汗点，汗点面积约占足跖表面的71％以上。
各试验组病毒对照组比较，*P<0.05；连翘苷/连翘脂素组合物与连翘苷比较，#P<0.05；连翘苷/连翘脂素组合物与连翘脂素比较，▲P<0.05。
2.2连翘苷/连翘脂素组合物对大鼠足跖部汗液分泌的影响(组织形态观察法)
(1)材料与方法
本试验根据大鼠汗腺兴奋时，除汗液分泌增加外，汗腺上皮细胞内的形态也随之改变。光学显微镜下可见汗腺上皮细胞空胞数目增多、扩大。这种扩大的空泡在电子显微镜下则是汗腺上皮细胞内线粒体肿胀、破裂，融合和分泌小泡扩大，通过对大鼠足跖部汗腺上皮组织形态学观察，即可了解汗腺的分泌活动。
试验取Wistar大鼠300只，雌雄各半，体重120～160g。按体重、性别随机分为30组，即：对照组(0.5％羧甲基纤维素)连翘脂素、连翘苷、连翘苷/连翘脂素组合物1和2分别分为低、中、高(2.5、5、10mg/kg)剂量组及阳性药毛果芸香碱(35mg/kg)组，每组10只，每组试验3次。除毛果芸香碱溶液皮下注射给药外，其余各组均采用灌胃方式给药。对照组给予0.5％羧甲基纤维素1h后、阳性药组给予毛果芸香碱30min后，以及连翘苷、连翘脂素、连翘苷/连翘脂素组合物均给药后1h，于齐踝关节处瞬时截断右 后肢，随即取下右足跖部肉垫，置10％甲醛溶液中，按常规方法固定、脱水、包埋、切片、HE染色，光学显微镜下观察各组大鼠足跖部汗腺上皮细胞内变化，主要观察空泡发生率，并通过X2检验进行统计学处理，比较各组间的差异。上述试验重复3次。
空泡发生百分率＝空泡汗腺数/观察汗腺数×100％
(2)结果
与对照组比较，连翘苷/连翘脂素组合物1和2的2.5、5、10mg/kg组对大鼠足跖部汗液分泌均有极显著的促进作用(p<0.001)；其中，它们的低、中、高剂量组(2.5、5、10mg/kg)的疗效显著优于连翘苷及连翘脂素(p<0.001或p<0.01)，表明连翘苷/连翘脂素组合物具有协同增效作用。试验结果详见表2-6；
表2-6  连翘苷/连翘脂素组合物对大鼠足跖部汗液分泌的影响(组织形态观察法，n＝3)
Figure PCTCN2014094710-appb-000043
与对照组比较，**p<0.01，***p<0.001；连翘苷/连翘脂素组合物与连翘苷比较，##p<0.01，###p<0.001；连翘苷/连翘脂素组合物与连翘脂素比较，▲▲p<0.01，▲▲▲p<0.001；
2.3连翘苷/连翘脂素组合物对啤酒酵母致大鼠发热的影响
(1)材料与方法
雄性Wistar大鼠，体重180～200g。试验前分别用WSC-411P型便携式数字测温仪测量正常肛温2次(每次间隔一定时间)，取两次测量的平均值作为大鼠的正常体温。然后选体温在36.5～38℃的大鼠300只，按体重随机分为30组：模型组(0.5％羧甲基纤维素)，连翘苷/连翘脂素组合物1、2组分别分为低、中、高(2.5、5、10mg/kg)剂量组、连翘苷组(13mg/kg)、连翘脂素组(13mg/kg)及阳性药扑热息痛(100mg/kg)组，每组10只，每组重复试验3次。各组大鼠均背部皮下注射10％鲜啤酒酵母混悬液10ml/kg致热。给10％鲜啤酒酵母混悬液6.0h后，连翘脂素和连翘苷的组合物及扑热息痛均灌胃给药，模型组灌胃等体积0.5％羧甲基纤维素。分别于给药后1、2、3及4h测量肛温。观察体温变化情况，并通过解热百分率进行组间t检验处理，比较各组间的差异。上述试验共重复3次。
Figure PCTCN2014094710-appb-000044
(2)结果
各组大鼠皮下注射10％鲜啤酒酵母混悬液6h后，体温升高均在1.5℃左右，与致热前比较差异显著(p<0.001)，表明啤酒酵母致大鼠发热模型建立成功。与模型组比较，连翘苷/连翘脂素组合物1和2的中、高剂量组于药后1、2、3及4h，以及低剂量组于药后2、3及4h对啤酒酵母混悬液所致大鼠发热均有明显的降温作用(p<0.05～p<0.001)；同时，连翘苷/连翘脂素组合物1和2的不同剂量组的降温疗效极显著优于连翘苷组、连翘脂素组(p<0.001或p<0.01)，表明它们具有明显协同增效作用。上述试验结果见表2-7。
2.4连翘苷/连翘脂素组合物对伤寒、副伤寒疫苗致家兔发热的影响
(1)材料与方法
雄性日本大耳白兔，体重1.5～2.0kg。试验前分别用WSC-411P型便携式数字测温仪测量正常肛温2次，取均值作为正常体温。然后选体温在38～39.6℃的日本大耳白兔198只，按体重随机分为33组，即：空白对照(生理盐水)组，模型对照(0.5％羧甲基纤维素)组，连翘苷/连翘脂素组合物1、2组分为低、中、高(1.25、2.5、5mg/kg)剂量组、连翘苷组、连翘脂素组及阳性药扑热息痛(50mg/kg)组。每组6只大白兔，各试验均重复3次。将兔置固定器内固定。空白对照组由耳缘静脉注射生理盐水1ml/kg；模型对照组和各药物组耳缘静脉注射伤寒、副伤寒疫苗0.8ml/kg。待兔体温升高超过1℃时(约需1～1.5h，本实验限定在1h)，空白对照组和模型组灌胃0.5％羧甲基纤维素1ml/kg，给药组灌胃连翘脂素和连翘苷的组合物和扑热息痛。于药后30、60、90、120、180及240min测量肛温，观察体温变化情况，并通过解热百分率进行组间t检验处理，比较各组间的差异。
Figure PCTCN2014094710-appb-000045
(2)结果
家兔耳缘静脉注射伤寒、副伤寒疫苗1h后，体温升高在1℃左右，表明伤寒、副伤寒疫苗可制备家兔发热模型。与空白对照组比较，模型组的体温在300min观察期内持续升高(p<0.05～p<0.001)，与模型组比较，连翘苷/连翘脂素组合物的高、中、低剂量组于药后30～240min、60～240min、90～240min对伤寒、副伤寒疫苗所致家兔发热有明显的解热作用(p<0.05～p<0.001)，且它们的疗效也明显优于连翘苷组、连翘脂素组(p<0.01)，表明连翘苷/连翘脂素组合物具有明显的协同增效作用。上述试验结果见表2-8。
2-5连翘苷/连翘脂素组合物对角叉菜胶所致大鼠足跖肿胀影响
(1)材料与方法
取体重120～150g的雄性Wistar大鼠70只，按体重随机分为7组，即：空白对照(0.5％羧甲基纤维素钠)组，连翘苷/连翘脂素组合物1组分为低、中、高(2.5、5及10mg/kg)剂量组、连翘苷组、连翘脂素组及阳性药阿司匹林(200mg/kg)组，每组10只。实验各组均采用舌下静脉注射方式给药。实验前用毛细管放大测量法测定各组大鼠右后足的正常体积。为避免误差，应固定测量位置，给药前后均由1人操作。取两次测量的平均值作为给药前大鼠右后足的正常体积。给药后立即在大鼠右后肢足跖皮下注射1％角叉菜胶0.1ml致炎。 测定致炎后15、30、60、120、180、240、300及360min的右后足跖体积。并通过大鼠致炎前后足跖体积的差值百分率(肿胀率)进行组间t检验处理，比较各组间的差异。
Figure PCTCN2014094710-appb-000046
结果
与空白对照组比较，连翘苷/连翘脂素组合物的高剂量组(10mg/kg)给药后于15min至360min内、连翘苷/连翘脂素组合物的中剂量组(5mg/kg)、低剂量组(2.5mg/kg)均于给药后30min至360min内均对角叉菜胶所致大鼠足跖肿胀均有明显的抑制作用(p<0.05或p<0.01)，其疗效明显优于连翘苷10mg/kg组及连翘脂素10mg/kg组(p<0.05或p<0.01)，且上述组合物的各剂量组在给药后60min、240min的疗效皆极显著优于连翘脂素组(p<0.01)。以上试验结果表明，连翘苷/连翘脂素组合物中连翘苷与连翘脂素组合使用具有明显的协同增效作用，详见表2-9。
Figure PCTCN2014094710-appb-000047
Figure PCTCN2014094710-appb-000048
Figure PCTCN2014094710-appb-000049

Claims (10)

  1. 一种连翘苷/连翘脂素组合物在制备用于缓解或/和治疗病毒性疾病的药物或保健品中的应用。
  2. 如权利要求1所述的应用，其特征是所述病毒性疾病为流感病毒、副流感病毒、柯萨奇病毒CoxA16、呼吸道合胞病毒RSV、单纯带状疱疹病毒HSV-I、单纯带状疱疹病毒HSV-II、单纯带状疱疹病毒CVB3、腺病毒ADV或肠道病毒EV71所致病毒性疾病。
  3. 如权利要求1或2所述的应用，其特征是所述连翘苷/连翘脂素组合物中连翘苷与连翘脂素的重量份配比为80～98：2～20。
  4. 如权利要求1或2所述的应用，其特征是所述连翘苷/连翘脂素组合物还包括药学上可接受的载体。
  5. 如权利要求4所述的应用，其特征是所述连翘苷/连翘脂素组合物中连翘苷与连翘脂素的总重量与药学上可接受的载体的重量之比为1:1～1:100。
  6. 如权利要求1或2所述的应用，其特征是所述连翘苷/连翘脂素组合物以连翘苷和连翘脂素以单体组成、或采用溶剂加热提取方法制备而成的连翘脂素-连翘苷提取组合物，或连翘脂素和连翘苷与环糊精或环糊精的衍生物组合而成连翘脂素-连翘苷-环糊精组合物。
  7. 如权利要求4所述的的应用，其特征是所述连翘脂素-连翘苷-环糊精组合物选择连翘脂素和连翘苷与α-、β-或γ-环糊精或其衍生物相混合而成的混合物，或连翘脂素和连翘苷与α-、β-或γ-环糊精或其衍生物经物理、化学方法处理形成的复合物。
  8. 如权利要求1或2所述的应用，其特征是所述药物以片剂、胶囊剂、丸剂、散剂、颗粒剂、糖浆剂、溶液剂、乳剂、注射剂、喷雾剂、气雾剂、凝胶剂、霜剂、巴布剂、橡胶贴膏剂或贴膏剂形式存在。
  9. 一种缓解或/和治疗病毒性疾病的药物或保健品，其特征是含有连翘苷和连翘脂素。
  10. 如权利要求9所述的药物或保健品，其特征是所述连翘苷与连翘脂素的重量份配比为80～98：2～20。
PCT/CN2014/094710 2014-08-07 2014-12-23 连翘苷/连翘脂素组合物在制备缓解或/和治疗病毒性疾病的药物或保健品中的应用 Ceased WO2016019684A1 (zh)

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