WO2024060536A1 - 一种中药提取物、药物及其检测方法 - Google Patents

一种中药提取物、药物及其检测方法 Download PDF

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WO2024060536A1
WO2024060536A1 PCT/CN2023/081598 CN2023081598W WO2024060536A1 WO 2024060536 A1 WO2024060536 A1 WO 2024060536A1 CN 2023081598 W CN2023081598 W CN 2023081598W WO 2024060536 A1 WO2024060536 A1 WO 2024060536A1
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parts
chinese medicine
peak
extract
atractylodes
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PCT/CN2023/081598
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English (en)
French (fr)
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肖伟
周恩丽
于桂芳
胡晗绯
王团结
章晨峰
曹亮
王振中
刘莉娜
李良
闫明
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江苏康缘药业股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/06—Aluminium, calcium or magnesium; Compounds thereof, e.g. clay
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/906—Zingiberaceae (Ginger family)
    • A61K36/9068—Zingiber, e.g. garden ginger
    • 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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the technical field of traditional Chinese medicine, and in particular to a traditional Chinese medicine extract, a medicine and a detection method thereof.
  • Viral pneumonia is a disease caused by upper respiratory tract viral infection and downward spread, causing lung inflammation and leading to pulmonary ventilation dysfunction.
  • the disease can occur throughout the year, but is mostly seen in winter and spring. It can occur in outbreaks or sporadically.
  • the main clinical manifestations are fever, headache, body aches, dry cough and pulmonary infiltration.
  • Pneumonia caused by influenza virus is the most common, and other pathogens including respiratory syncytial virus, adenovirus, and parainfluenza virus have also attracted widespread attention.
  • Viral infection mainly manifests as interstitial lung disease. It initially involves ciliated columnar epithelial cells and then invades other respiratory cells, including alveolar cells, mucous gland cells, and macrophages. Viruses replicate within cells and then release infectious virus to infect neighboring cells. Infected ciliated cells may undergo degenerative changes including granular degeneration, vacuolation, cell swelling and nuclear pyknosis, followed by necrosis and disintegration. Cellular debris collects in the airways and blocks the small airways, and swelling of the airways occurs. There was an obvious inflammatory reaction in the alveolar septa, with infiltration of lymphocytes, macrophages, and occasionally plasma cells and neutrophils and edema.
  • Necrotic and hemorrhagic fibrin thrombi may appear in the alveolar capillaries, and eosinophilic hyaline membranes may be seen in the alveoli. Severely infected patients may develop pulmonary edema, consolidation, hemorrhage, lung parenchymal necrosis, and atelectasis.
  • Influenza viruses and respiratory syncytial viruses are the most common viruses that cause upper respiratory tract infections.
  • Influenza virus belongs to the Orthomyxoviridae family and is an RNA virus. According to the antigenicity of the viral nucleoprotein and matrix protein, it is divided into three types: A, B, and C. Among them, influenza A virus mutates quickly and is highly virulent.
  • H1N1/FM1 belongs to the subtype A mouse lung-adapted strain of influenza virus and simulates influenza A virus to prepare lung infection damage.
  • Influenza A virus initiates infection by binding hemagglutinin to the cell surface of respiratory epithelial cells containing sialic acid receptors. After entering the cells, the viral genome begins to be transcribed and replicated.
  • Respiratory syncytial virus belongs to the Paramyxoviridae family and is an RNA virus. Pathogenesis involves interactions between the virus and affected host cell damage, inflammation, humoral and local immune responses, and hyperresponsiveness.
  • traditional Chinese medicine has significant antiviral efficacy, broad antiviral spectrum, and few toxic and side effects.
  • drugs have antipyretic and anti-inflammatory effects, and have multiple effects on infections caused by viruses, such as shortening the duration of fever, controlling the spread of inflammation, promoting the absorption of inflammation, etc., that is, multiple pathways, It works in many directions; it is precisely because of the diversification of the active ingredients of traditional Chinese medicine in this treatment process that it is difficult for viruses to develop resistance to it, which makes traditional Chinese medicine have obvious advantages in treating viral infectious diseases and has broad prospects for clinical use.
  • the object of the present invention is to make up for the shortcomings of the prior art and provide a traditional Chinese medicine extract with antiviral effect, which is characterized in that, in terms of weight ratio, the traditional Chinese medicine extract is made from traditional Chinese medicines including the following raw materials: 1 to 100 parts of Magnolia officinalis, 1 to 100 parts of betel nut, 1 to 100 parts of simmered grass fruit, 1 to 100 parts of ephedra, 1 to 100 parts of bitter almond, 1 to 100 parts of Qiang Huo, 1 to 100 parts of ginger, and patchouli.
  • the traditional Chinese medicine extract is made from traditional Chinese medicine including the following raw materials: 1 to 80 parts of magnolia bark, 1 to 80 parts of betel nut, 1 to 80 parts of simmered grass fruit, 1 to 60 parts of ephedra, and 1 to 60 parts of bitter almond. 1 to 80 parts of Qianhuo, 1 to 60 parts of ginger, 1 to 80 parts of patchouli, 1 to 60 parts of Perilla, 1 to 80 parts of Atractylodes, 1 to 160 parts of Poria, 1 to 120 parts of Atractylodes, and 1 to 80 parts of Gypsum.
  • the traditional Chinese medicine extract is made from traditional Chinese medicine including the following raw materials: 30 to 50 parts of magnolia bark, 20 to 30 parts of betel nut, 30 to 50 parts of simmered grass fruit, 20 to 30 parts of ephedra, and 20 to 30 parts of bitter almond.
  • 30 to 50 parts of Qiang Huo 30 to 50 parts of ginger
  • 30 to 50 parts of patchouli 20 to 30 parts of Perilla
  • 30 to 50 parts of Atractylodes 120 to 150 parts of Poria, 80 to 100 parts of Atractylodes, and 30 to 50 parts of Gypsum.
  • the traditional Chinese medicine extract is made from traditional Chinese medicine including the following raw materials: 50 parts of magnolia bark, 30 parts of betel nut, 50 parts of simmered grass fruit, 30 parts of ephedra, 30 parts of bitter almond, 50 parts of Qianghuo, 50 parts of ginger, 50 parts of patchouli, 30 parts of Perrin, 50 parts of Atractylodes, 150 parts of Poria, 100 parts of Atractylodes, 50 parts of gypsum, 30 parts of Jiao hawthorn, 50 parts of Jiao Liu Shenqu, 30 parts of Jiao malt, 50 parts of Dilong, 50 parts of Xu Changqing , 30 portions of Mianma Guanzhong, and 50 portions of Tinglizi.
  • the ephedrine content of the aforementioned extract is 0.5-1.5 mg/g, and the solid content transfer rate is 18-25%.
  • the procurin content of the aforementioned extract is 2-8 mg/g.
  • the preparation of the traditional Chinese medicine extract includes:
  • the extracts were combined, filtered, and the filtrate was concentrated to The relative density is 1.10-1.15, centrifugal filtration, and the filtrate is dried; wherein, the ephedrine content of the extract is 1.20-1.50 mg/g, the solid content transfer rate is 19-22%, and the purpurin content is 6- 8mg/g.
  • the preparation of the traditional Chinese medicine extract includes: weighing 15g of Magnolia officinalis, 9g of Betel nut, 9g of Szechuan grass fruit, 6g of ephedra, 9g of bitter almond, 15g of Astragalus root, 15g of ginger, 15g of patchouli, 9g of Peilan, and 15g of Atractylodes.
  • the similarity shall not be less than 0.90.
  • the control fingerprint contains the common peaks honokiol and magnolol.
  • the reference fingerprint pattern may be as shown in Figure 1, for example.
  • the similarity shall not be less than 0.90.
  • the control fingerprint contains the common peaks arecoline and arecoline.
  • the comparison fingerprint pattern may be as shown in Figure 2, for example.
  • the similarity of the fingerprint of the stewed grass fruit shall not be less than 0.90.
  • the control fingerprint contains the common peak protocatechuic acid.
  • the reference fingerprint pattern may be as shown in Figure 3, for example.
  • a method for preparing traditional Chinese medicine extracts which is characterized by: weighing Magnolia officinalis, Jiaobetel nut, Szechuancaoguo, Ephedra, Bitter almond, Qianhuo, ginger, patchouli, Peilan, Atractylodes, Poria, Atractylodes, Gypsum, and Jiaohaw.
  • Jiao Liushenqu, Jiao Malt, Dilong, Xu Changqing, Mianma Guanzhong, and Tinglizi were extracted twice by adding water. For the first time, add 6 times the amount of water and extract for 1.5 hours. For the second time, add 4 times the amount of water.
  • Extract for 1.0 h combine the extracts, filter, concentrate the filtrate to a relative density of 1.10-1.15, centrifuge, and dry the filtrate; wherein, the ephedrine content of the extract is 1.20-1.50 mg/g, and the solid content transfer rate is 19-22%, and the content of proanthoside is 6-8mg/g.
  • the fingerprint of Magnolia officinalis, Jiaobetel nut or Sichuan grass fruit is as defined before.
  • the present invention also proposes the application of the traditional Chinese medicine extract as described in the previous one in the preparation of drugs for treating influenza virus H1N1 or FM1, or respiratory syncytial virus.
  • the aforementioned traditional Chinese medicine extract is made from traditional Chinese medicine including the following raw materials: 50 parts of Magnolia officinalis, 30 parts of Betel nut, 30 parts of Sichuan grass fruit, 20 parts of ephedra, 30 parts of bitter almond, 50 parts of Qianghuo, 50 parts of ginger, Guangxi 50 parts of Patchouli, 30 parts of Peilan, 50 parts of Atractylodes, 150 parts of Poria, 100 parts of Atractylodes, 50 parts of gypsum, 30 parts of Jiao hawthorn, 30 parts of Jiao Liu Shenqu, 30 parts of Jiao malt, 50 parts of Dilong, 50 parts of Xu Changqing, 30 portions of Mianma Guanzhong and 50 portions of Tinglizi.
  • the present invention also proposes a medicine, which is prepared from the traditional Chinese medicine extract as described in the previous one and pharmaceutically acceptable auxiliary materials or additives.
  • the drug is selected from decoctions, granules, capsules, tablets, oral liquids, pills, soft capsules, dropping pills, tinctures, syrups, suppositories, gels, sprays, and injections.
  • the drug is in the form of granules, and the excipients or additives are preferably dextrin and sucralose.
  • the drugs may also include antiviral drugs for upper respiratory tract infections, such as ribavirin and/or oseltamivir phosphate.
  • the present invention also proposes a method for detecting the fingerprint of a traditional Chinese medicine extract or drug as described in the previous one, which is characterized in that the test solution is taken for HPLC detection.
  • the chromatographic conditions for HPLC detection include: using a C18 chromatographic column, using methanol is mobile phase A, and the phosphoric acid aqueous solution containing 0.1% concentration is mobile phase B.
  • the elution in the chromatographic conditions detected by HPLC is gradient elution.
  • the gradient elution program is: 0 to 5 min, 0% A; 5 to 25min, 0% ⁇ 15%A; 25 ⁇ 60min, 15% ⁇ 55%A; 60 ⁇ 75min, 55% ⁇ 100%A; 75 ⁇ 80min, 100%A.
  • the test solution is a solution prepared from traditional Chinese medicine extracts or drugs for testing.
  • the preparation method can be, for example, taking 1 g of granules, placing it in a stoppered conical flask, adding 25 ml of water, and ultrasonic treatment for 30 minutes. Right now have to.
  • the chromatographic conditions include: the flow rate is 1.0 mL/min, the column temperature is 30°C, and the detection wavelength is 250 nm.
  • the relative retention time of each characteristic peak and the S peak should be calculated and should be within the prescribed value. Within ⁇ 10%, preferably within 5%, the relative retention time specified values are: 0.11 (peak 1), 0.27 (peak 2), 0.36 (peak 3), 0.38 (peak 4), 0.41 (peak 5), 0.84 (peak 6) ), 0.92 (Peak 7), 1.00 [Peak 8 (S)], 1.19 (Peak 9), 1.29 (Peak 10).
  • the present invention prepares a new Chinese medicine composition, improves the quality standard of the composition, and evaluates the effectiveness of the composition particles in treating viral upper respiratory tract infections by adopting an influenza virus H1N1/FM1 strain infection model and a respiratory syncytial virus infection model, indicating that the composition can reduce the viral load and inflammatory cytokines in lung tissue, indicating that it has an enhanced therapeutic effect on this type of common specific viral infection, providing a basis for further clinical research.
  • Figure 1 is the comparative fingerprint of Magnolia officinalis medicinal materials; Peak 6: honokiol Peak 7 (S): magnolol;
  • Figure 2 is the comparative fingerprint of areca nut medicinal materials; Peak 1: arecoline Peak 3 (S): arecoline;
  • Figure 3 is a comparative fingerprint of stewed tsaoko fruit medicinal material; Peak 1: protocatechuic acid;
  • Figure 4 is a full wavelength scan 3D image of the test solution
  • Figure 5 is the wavelength investigation chromatogram of the test solution
  • Figure 6 shows the chromatogram of mobile phase ratio investigation
  • Figure 7 is a chromatogram for investigating the preparation method of the test product
  • Figure 8 shows the chromatogram of durability inspection
  • Figure 9 is a chromatogram of precision investigation
  • Figure 14 compares the characteristic spectra and identification results of each characteristic peak; among them, 1-adenine; 2-uridine; 3-guanosine; 4-inosine; 5-5-hydroxymethylfurfural; 6-honokiside A; 7-bergamotol glucoside; 8-purpuroside; 9-6'-O-(trans-feruloyl)-purpuroside; 10-p-hydroxyphenylethyl anisate.
  • Oseltamivir is an inhibitor of the neuraminidase of the influenza virus and is mainly used clinically to treat influenza A and B.
  • Ribavirin is a broad-spectrum antiviral drug that can be used clinically to treat respiratory syncytial virus, influenza A and B, but it is not recommended by the FDA.
  • the present invention aims to provide a traditional Chinese medicine composition and its preparation method and application. The following will take commonly used influenza virus or respiratory syncytial virus drugs as examples and give a detailed description based on the experimental content.
  • the process is carried out under conventional conditions or conditions recommended by the manufacturer.
  • the raw materials or excipients used, as well as the reagents or instruments used, for which the manufacturer is not specified, are all conventional products that can be obtained commercially.
  • the total transfer rate refers to the ratio of the total amount of ephedrine hydrochloride and pseudoephedrine hydrochloride in the extract to the ephedrine hydrochloride and pseudoephedrine hydrochloride in the medicinal materials.
  • the experimental results show that the transfer rate of ephedrine hydrochloride and pseudoephedrine hydrochloride and the solid content transfer rate show an increasing trend as the amount of water added increases.
  • the amount of solvent increases from 4, 3 times to 6, 4 times, the transfer rate of each indicator increases more obviously.
  • the amount of water added continues to increase, the growth trend of each indicator is relatively gentle, but when the solvent is too high, it decreases.
  • the amount of water added is determined to be 6 times the amount for the first time and 4 times the amount for the second time.
  • the first time is to add 6 times the amount of water and extract for 1.5 hours.
  • the second time is to add 4 times the amount of water and extract for 1.0 hours.
  • the soaking times for the 3 portions are 0 and 1.0 hours, respectively. 30 and 60min.
  • the first extraction time is 60min, 75min, 90min, 105min, 120min.
  • the second extraction time is 30min, 45min, 60min, 75min, 90min respectively.
  • the experimental results show that the transfer rate of ephedrine hydrochloride and pseudoephedrine hydrochloride and the solid content transfer rate show an increasing trend with the increase of extraction time.
  • the extraction time increases from 60, 30 min to 90, 60 min, the growth trend of each indicator is more obvious, and the growth trend of each indicator gradually becomes gentle as the extraction time continues to increase. Therefore, the extraction time was selected to be 90 min for the first extraction and 60 min for the second extraction.
  • the optimal extraction process is determined to add water for two extractions.
  • the first time add 6 times the amount of water, and extract for 1.5 hours; the second time, add 4 times the amount of water. , extraction 1.0h.
  • Chromatographic conditions and system suitability test Use octadecylsilane bonded silica gel as the filler (Waters CORTECTS C 18 chromatographic column, 150mm ⁇ 4.6mm, 2.7 ⁇ m); use acetonitrile as the mobile phase A, and use 0.1% acetic acid solution as the mobile phase Phase B, perform gradient elution as specified in the table; column temperature is 35°C; flow rate is 0.8ml/min; detection wavelength is 230nm. The number of theoretical plates should not be less than 4000 based on the magnolol peak.
  • Preparation of reference solution Take appropriate amounts of magnolol and honokiol reference substances, weigh them accurately, add methanol to prepare a mixed solution containing 200 ⁇ g each per 1 ml, and you are ready.
  • test solution Take 1g of this product powder (passed through No. 3 sieve), weigh it accurately, place it in a stoppered Erlenmeyer flask, add 25ml of 50% methanol solution accurately, shake well, stopper tightly, soak for 24 hours, and filter. , take the continued filtrate and get it.
  • Determination method Accurately pipette 10 ⁇ l of reference solution and test solution respectively, inject into liquid chromatograph and measure.
  • the fingerprint of the test product should present chromatographic peaks with the same retention time as those of the reference material chromatographic peaks in Figure 1. According to the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, the similarity between the fingerprint of the test product and the control fingerprint should be calculated, and the similarity should not be less than 0.90.
  • Chromatographic conditions and system suitability test Use strong cation exchange bonded silica gel as filler (Thermo BioBasic SCX chromatographic column, column length is 25cm, inner diameter is 4.6mm, particle size is 5 ⁇ m); use acetonitrile-0.2% phosphoric acid solution (with ammonia water Adjust pH to 3.8) (65:35) as the mobile phase; column temperature is 35°C; flow rate is 1.2ml/min; detection wavelength is 210nm. The number of theoretical plates should not be less than 10,000 based on arecoline calculations.
  • Preparation of reference solution Take an appropriate amount of arecoline hydrobromide reference substance, weigh it accurately, add methanol to make a solution containing 50 ⁇ g per 1 ml, and you have it.
  • test solution Take 0.5g of this product powder (passed through No. 2 sieve), weigh it accurately, place it in a stoppered Erlenmeyer flask, accurately add 50 ml of 50% methanol solution, ultrasonicate for 30 minutes, let cool, and shake well. Filter and take the filtrate to obtain.
  • Determination method Accurately pipette 10 ⁇ l of reference solution and test solution respectively, inject into liquid chromatograph, measure, and record the chromatogram.
  • the fingerprint of the test product should show chromatographic peaks with the same retention time as those of the reference substance in Figure 2. According to the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, the similarity between the fingerprint of the test product and the control fingerprint should be calculated, and the similarity should not be less than 0.90.
  • Octadecylsilane bonded silica gel is used as the filler (Waters CORTECTS T3 chromatographic column, column length is 15cm, inner diameter is 4.6mm, particle size is 2.7 ⁇ m); acetonitrile is used as mobile phase A, with 0.1% formic acid solution is mobile phase B, and gradient elution is performed as specified in the table below; the column temperature is 30°C; the flow rate is 0.8ml/min; the detection wavelength is 254nm.
  • the number of theoretical plates should not be less than 10,000 based on the protocatechuic acid peak.
  • Preparation of reference solution Take an appropriate amount of protocatechuic acid reference substance, weigh it accurately, and add methanol to make a solution containing 200 ⁇ g per 1ml.
  • test solution Take 1g of the powder of this product (passed through No. 2 sieve), weigh it accurately, add 25ml of 50% methanol solution accurately, ultrasonicate for 30 minutes, let cool, shake well, filter, and take the filtrate to obtain .
  • Determination method Precisely draw 5 to 10 ⁇ l of the reference solution and 10 ⁇ l of the test solution, inject them into the liquid chromatograph, measure, and record the chromatogram.
  • the fingerprint of the test sample should show a chromatographic peak with the same retention time as the reference chromatographic peak in Figure 3. According to the similarity evaluation system of Chinese medicine chromatographic fingerprints, the similarity between the fingerprint of the test sample and the reference fingerprint should not be less than 0.90.
  • Recipe 50 parts of magnolia bark, 30 parts of betel nut, 30 parts of stewed grass fruit, 20 parts of ephedra, 30 parts of bitter almond, 50 parts of Qiang Huo, 50 parts of ginger, 50 parts of patchouli, 30 parts of Perrin, 50 parts of Atractylodes, 150 parts of Poria, 100 parts of Atractylodes, 50 parts of Gypsum, 30 parts of Jiao hawthorn, 30 parts of Jiao Liushenqu, 30 parts of Jiao malt, 50 parts of Dilong, 50 parts of Xu Changqing, 30 parts of Mianma Guanzhong, and 50 parts of Tinglizi.
  • Recipe 50 parts of magnolia bark, 30 parts of betel nut, 30 parts of stewed grass fruit, 20 parts of ephedra, 30 parts of bitter almond, 50 parts of Qiang Huo, 50 parts of ginger, 50 parts of patchouli, 30 parts of Perrin, 50 parts of Atractylodes, 150 parts of Poria, 100 parts of Atractylodes, 50 parts of Gypsum, 30 parts of Jiao hawthorn, 30 parts of Jiao Liushenqu, 30 parts of Jiao malt, 50 parts of Dilong, 50 parts of Xu Changqing, 30 parts of Mianma Guanzhong, and 50 parts of Tinglizi.
  • Recipe 50 parts of magnolia bark, 30 parts of betel nut, 30 parts of stewed grass fruit, 20 parts of ephedra, 30 parts of bitter almond, 50 parts of Qiang Huo, 50 parts of ginger, 50 parts of patchouli, 30 parts of Perrin, 50 parts of Atractylodes, 150 parts of Poria, 100 parts of Atractylodes, 50 parts of Gypsum, 30 parts of Jiao hawthorn, 30 parts of Jiao Liushenqu, 30 parts of Jiao malt, 50 parts of Dilong, 50 parts of Xu Changqing, 30 parts of Mianma Guanzhong, and 50 parts of Tinglizi.
  • Test drug Granules of Preparation Example 1 were provided by Jiangsu Kangyuan Pharmaceutical Co., Ltd.
  • Oseltamivir phosphate granules (Kewei): produced by Yichang Dongguang Yangtze Pharmaceutical Co., Ltd., ingredients: Each bag of granules contains 15 mg of oseltamivir phosphate.
  • Ribavirin granules produced by Sichuan Baili Pharmaceutical Co., Ltd., ingredients: the main ingredient of this product is ribavirin. Specifications: 50mg.
  • Influenza A (H1N1) virus FM1 strain was purchased from the American Type Biological Collection (ATCC). It was routinely passaged in the ABSL-2 laboratory of our institute and stored at -80°C for later use.
  • ATCC American Type Biological Collection
  • Preparation of medicinal solution Before the test, take the granules and add distilled water to a constant volume. The dosage is 26.4g crude drug/kg/d, and administered by gavage at a rate of 0.2mL/10g body weight/time, once/day for 4 consecutive days.
  • mice during the test was 27.5mg/kg/d, which is equivalent to the same clinical dose for humans. During administration, it was administered by gavage at 0.2mL/10g body weight/time, once/day, continuously. 4 days.
  • Ribavirin The dosage of ribavirin in mice during the test was 82.5 mg/kg/d, and the dosage was 0.2 mL/10 g body weight/time by gavage, once/day for 4 consecutive days.
  • mice weighing 14 ⁇ 1g, half male and half female, were randomly divided into 7 groups according to weight class, namely normal control group, model control group, oseltamivir phosphate group, ribavirin, and composition group. , oseltamivir phosphate group + composition group, ribavirin + composition group.
  • the mice were lightly anesthetized with isoflurane and infected intranasally with 15 LD 50 influenza virus liquid (H1N1/FM1 strain), 35 ⁇ L each.
  • Administration was started on the day of infection, with 0.2 mL/10 g administered once a day for 4 consecutive days.
  • the normal control group and the model control group were administered with distilled water under the same conditions.
  • the mice in each group were weighed; the lungs were weighed by autopsy, and lung tissue was taken for HE pathological examination to calculate the lung index and lung index inhibition rate.
  • Lung index (%) lung wet weight (g)/body weight (g) ⁇ 100
  • “+++” There is a large area of inflammation and mucus exudation in the alveolar interstitium of the mouse lung tissue.
  • the exudative inflammation is mainly lymphocytes, with a small number of lobulated nuclei and eosinophils. The cells are uneven in size and clustered. There is a large amount of pink mucus.
  • the perivascular inflammation was obvious, the inflammation around the bronchioles in the lungs was localized, the intraluminal membrane cells proliferated, and the perivascular inflammation was severe.
  • composition particles of the present invention on lung index and lung index inhibition rate
  • the research results show that after the mice were infected with the FM1 strain of influenza A H1N1 virus, the lung index of the mice in the model control group increased significantly, which was significantly different from the normal control group (P ⁇ 0.01); administration was started on the day of infection After 4 days of treatment with the composition particles, the lung index of the oseltamivir phosphate group, the composition group, the oseltamivir phosphate group+composition group, and the ribavirin+composition group was significantly reduced, which was significantly higher than that of the model control group. The difference indicates that the composition has a significant synergistic effect on antiviral drugs used for upper respiratory tract infections in anti-influenza virus.
  • Oseltamivir phosphate granules (Kewei): produced by Yichang Dongguang Yangtze Pharmaceutical Co., Ltd., ingredients: Each bag of granules contains 15 mg of oseltamivir phosphate.
  • Ribavirin Granules Produced by Sichuan Baili Pharmaceutical Co., Ltd. Ingredients: The main ingredient of this product is ribavirin. Specification: 50 mg.
  • Respiratory syncytial virus (RSV) strains were purchased from the American Type Biological Collection (ATCC). It was routinely passaged in the ABSL-2 laboratory of our institute and stored at -80°C for later use.
  • ATCC American Type Biological Collection
  • Preparation of liquid medicine Take the granules, add distilled water, and adjust the volume to 40 mL.
  • the dosage is 26.4 g of crude drug/kg/d.
  • the dosage is 0.2 mL/10 g of body weight/time, once/day for 4 consecutive days.
  • mice during the test was 27.5mg/kg/d, which is equivalent to the same clinical dose for humans. During administration, it was administered by gavage at 0.2mL/10g body weight/time, once/day. 4 days in a row.
  • mice during the test was 82.5 mg/kg/d.
  • the dosage was 0.2 mL/10 g body weight/time by gavage, once/day for 4 consecutive days.
  • Composition group 1 Preparation example 1 granules 13.2g crude drug/kg/d + oseltamivir phosphate 13.7mg/kg/d, the administration method is the same as above.
  • Composition group 2 Preparation example 1 granules 13.2g crude drug/kg/d + ribavirin 41.2mg/kg/d, the administration method is the same as above.
  • mice weighing 14 ⁇ 1g, half male and half male, were randomly divided into 7 groups according to weight class, namely normal control group, model control group, oseltamivir phosphate group, ribavirin, and combination Composition group, oseltamivir phosphate group + composition group, ribavirin + composition group, 10 animals in each group. Except for the normal control group, the mice were lightly anesthetized with isoflurane and intranasally infected with 100 TCID 50 RSV virus liquid, 45 ⁇ L each. Administration was started on the day of infection, with 0.2 mL/10 g administered once a day for 4 consecutive days.
  • mice in each group were weighed; their lungs were weighed by autopsy, and lung tissue was taken to detect inflammatory factors, and the lung index and lung index inhibition rate were calculated.
  • Lung index (%) lung wet weight (g)/body weight (g) ⁇ 100
  • Table 2 show that after intranasal infection of mice with respiratory syncytial virus (RSV), the lung index of the mice in the model group increased significantly, which was significantly different from the normal control group (P ⁇ 0.01); administration was started on the day of infection After 4 days of treatment with the composition particles, the lung index of the oseltamivir phosphate group, the composition group, the oseltamivir phosphate group+composition group, and the ribavirin+composition group was significantly reduced, which was significantly higher than that of the model control group. The difference indicates that the composition has a certain synergistic effect on antiviral drugs used for upper respiratory tract infections in anti-respiratory syncytial virus.
  • RSV respiratory syncytial virus
  • Mettler Toledo XP6 electronic analytical balance Mettler Corporation
  • Mettler Toledo AL204 electronic analyzer Analytical balance Mettler
  • KQ500DB CNC ultrasonic cleaner Kunshan Ultrasonic Instrument Co., Ltd.
  • HH digital constant temperature water bath Changzhou Guoyu Instrument Manufacturing Co., Ltd.
  • Milli-Q ultrapure water meter Millipore Corporation of the United States;
  • Reagents methanol (Merck Co., Ltd., Merida Co., Ltd., USA, chromatographically pure); acetic acid (L07203503, Merck Fisher Scientific Co., Ltd.), other reagents are of analytical grade.
  • Sample Particles prepared by the method of Preparation Example 1, the batch numbers are: 200201, 200202, 200203, 200204, 200205, 200206, 200207, 200208, 200209, 200210, 200211, 200212, 200213, 200214, 200601, 2006 02, 200603, of which 200602 batches These are methodological research batches, all provided by the Traditional Chinese Medicine Research and Development Department of Jiangsu Kangyuan Pharmaceutical Co., Ltd.
  • the proposed chromatographic conditions are: using octadecylsilane bonded silica gel as the filler (Waters Atlantis T3, column length: 25cm, inner diameter: 4.6mm, particle size: 5 ⁇ m); methanol as the mobile phase A, and 0.1% phosphoric acid is mobile phase B, and gradient elution is performed as specified in the table below; the flow rate is 1.0ml per minute; the column temperature is 30°C; the detection wavelength is 250mn.
  • the number of theoretical plates should not be less than 10,000 based on the peaks of purpurin.
  • test solution Take an appropriate amount of this product, grind it finely, take about 1g, make 4 parts in total, weigh accurately, place in a stoppered conical flask, add water and 25ml of 50% methanol accurately, and ultrasonicate for 30 minutes respectively. Reflux for 30 minutes, shake well, centrifuge, and take the supernatant to obtain 4 portions of test solution.
  • the proposed treatment method for the test sample is: take an appropriate amount of this product, grind it finely, take about 1g, weigh it accurately, place it in a stoppered conical flask, add 25ml of water accurately, and ultrasonic treatment (power 500W, frequency 40kHz) 30 minutes, shake well, centrifuge and take the supernatant.
  • Stability test Take the same test solution and inject samples at 0h, 3h, 6h, 9h, 12h, 18h, 24h, 30h, and 36h. Each injection is 10 ⁇ l and measured. ) is the reference peak, and the relative retention times of the calibrated 10 characteristic peaks are calculated. The results showed that the RSD values of the relative retention times of the 10 characteristic peaks were all less than 0.5%, indicating that the test solution had good stability within 36 hours. See Table 6 and Figure 11.
  • the chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as the filler (Waters Atlantis T3, column length 25cm, inner diameter 4.6mm, particle size 5 ⁇ m); methanol as mobile phase A, 0.1% Phosphoric acid is mobile phase B, and gradient elution is performed as specified in the table; the flow rate is 1.0ml per minute; the column temperature is 30°C; the detection wavelength is 250mn.
  • the number of theoretical plates should not be less than 10,000 based on the peaks of purpurin.
  • Preparation of reference solution Take an appropriate amount of procurin reference substance, weigh it accurately, add 50% methanol to make a solution containing 0.15mg per 1ml, and you have it.
  • test solution Take an appropriate amount of this product, grind it finely, take about 1g, weigh it accurately, place it in a stoppered Erlenmeyer flask, add 25ml of water accurately, ultrasonicate (power 500W, frequency 40kHz) for 30 minutes, shake well. Centrifuge and take the supernatant.
  • Determination method Precisely draw 10 ⁇ l each of the reference solution and the test solution, inject them into the liquid chromatograph, measure, and record the chromatogram, and you have it.
  • the relative retention time value is : 0.11 (Peak 1), 0.27 (Peak 2), 0.36 (Peak 3), 0.38 (Peak 4), 0.41 (Peak 5), 0.84 (Peak 6), 0.92 (Peak 7), 1.00 [Peak 8 (S) ], 1.19 (peak 9), 1.29 (peak 10).
  • the comparison characteristic map is shown in Figure 14.

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Abstract

本发明公开了一种中药提取物，所述中药提取物由包括以下原料的中药制成：厚朴1～100份、焦槟榔1～100份、煨草果1～100份、麻黄1～100份、苦杏仁1～100份、羌活1～100份、生姜1～100份、广藿香1～100份、佩兰1～100份、苍术1～100份、茯苓1～100份、白术1～100份、石膏1～100份、焦山楂1～100份、焦六神曲1～100份、焦麦芽1～100份、地龙1～100份、徐长卿1～100份、绵马贯众1～100份、葶苈子1～100份，其中，所述提取物的麻黄碱含量为0.5-1.5mg/g，固含物转移率为18-25％。

Description

一种中药提取物、药物及其检测方法 技术领域
本发明涉及中药技术领域，特别涉及一种中药提取物、药物及其检测方法。
背景技术
病毒性肺炎是由上呼吸道病毒感染、向下蔓延引发肺部炎症，导致肺换气功能障碍的疾病。该病一年四季均可发生，但大多见于冬、春季节，可暴发流行、亦可散在发生。临床主要表现为发热、头痛、全身酸痛、干咳及肺部浸润等。以流感病毒引发的肺炎最常见，其他病原体包括呼吸道合胞病毒、腺病毒、副流感病毒也引起了广泛关注。
病毒感染主要表现为肺间质病变。最初累及纤毛柱状上皮细胞，然后侵及其他呼吸道细胞，包括肺泡细胞、黏液腺细胞及巨噬细胞。病毒在细胞内复制，然后释放出感染性病毒感染相邻细胞。被感染的纤毛细胞可出现退行性变包括颗粒变性，空泡形成，细胞肿胀和核固缩，继而坏死和崩解。细胞碎片聚集在气道内和阻塞小气道，并出现呼吸道肿胀。肺泡间隔有明显的炎症反应，伴淋巴细胞、巨噬细胞浸润，偶有浆细胞和中性粒细胞浸润和水肿。肺泡毛细血管内可出现坏死和出血的纤维蛋白血栓，肺泡可见嗜酸性透明膜。重症感染者可出现肺水肿、实变、出血，肺实质坏死，肺不张。
流感病毒和呼吸道合胞病毒是引起上呼吸道感染最常见的病毒。流感病毒属于正黏病毒科，是RNA病毒，按照病毒核蛋白和基质蛋白的抗原性不同，分为甲、乙、丙3型。其中甲型流感病毒变异快、毒力强。H1N1/FM1属于流感病毒亚甲型鼠肺适应株，模拟甲型流感病毒制备肺感染损伤。甲型流感病毒通过血凝素结合呼吸道上皮细胞含有唾液酸受体的细胞表面启动感染，进入细胞后，病毒基因组开始转录和复制。复制出的大量子代病毒颗粒通过呼吸道黏膜扩散开始感染其他细胞，从而诱发细胞因子风暴，导致全身炎症反应，进而出现急性呼吸窘迫综合征、休克及多脏器功能衰竭。呼吸道合胞病毒属副粘病毒科，是RNA病毒。发病机制包括病毒与宿主受累细胞损伤、炎症、体液和局部免疫反应及高反应性之间的相互作用。
目前据各种资料及临床使用证实，中药抗病毒疗效显著，抗病毒谱广,毒副作用小的特点。而且，其在抗病毒同时，许多药物兼有解热、抗炎等作用，对病毒引起的感染具有多重作用，如缩短发热的时间、控制炎症的扩散、促进炎症的吸收等，即多途径、多方位起作用；正是由于这种治疗过程中中药有效成分的多元化，病毒难以对其产生抗药性，使得中药在治疗病毒感染性疾病方面具有明显的优势，临床使用前景广阔。
发明内容
本发明的目的在于弥补现有技术的不足，提供一种具有抗病毒作用的中药提取物，其特征在于，以重量比计，所述中药提取物由包括以下原料的中药制成： 厚朴1～100份、焦槟榔1～100份、煨草果1～100份、麻黄1～100份、苦杏仁1～100份、羌活1～100份、生姜1～100份、广藿香1～100份、佩兰1～100份、苍术1～100份、茯苓1～100份、白术1～100份、石膏1～100份、焦山楂1～100份、焦六神曲1～100份、焦麦芽1～100份、地龙1～100份、徐长卿1～100份、绵马贯众1～100份、葶苈子1～100份。
进一步地，所述中药提取物由包括以下原料的中药制成：厚朴1～80份、焦槟榔1～80份、煨草果1～80份、麻黄1～60份、苦杏仁1～60份、羌活1～80份、生姜1～60份、广藿香1～80份、佩兰1～60份、苍术1～80份、茯苓1～160份、白术1～120份、石膏1～80份、焦山楂1～50份、焦六神曲1～80份、焦麦芽1～60份、地龙1～80份、徐长卿1～80份、绵马贯众1～60份、葶苈子1～80份。
进一步地，所述中药提取物由包括以下原料的中药制成：厚朴30～50份、焦槟榔20～30份、煨草果30～50份、麻黄20～30份、苦杏仁20～30份、羌活30～50份、生姜30～50份、广藿香30～50份、佩兰20～30份、苍术30～50份、茯苓120～150份、白术80～100份、石膏30～50份、焦山楂20～30份、焦六神曲30～50份、焦麦芽20～30份、地龙30～50份、徐长卿30～50份、绵马贯众20～30份、葶苈子30～50份。
进一步地，所述中药提取物由包括以下原料的中药制成：厚朴50份、焦槟榔30份、煨草果50份、麻黄30份、苦杏仁30份、羌活50份、生姜50份、广藿香50份、佩兰30份、苍术50份、茯苓150份、白术100份、石膏50份、焦山楂30份、焦六神曲50份、焦麦芽30份、地龙50份、徐长卿50份、绵马贯众30份、葶苈子50份。
进一步地，前述提取物的麻黄碱含量为0.5-1.5mg/g，固含物转移率为18-25％。
进一步地，前述提取物的紫花前胡苷含量为2-8mg/g。
进一步地，所述中药提取物的制备包括：
称取厚朴、焦槟榔、煨草果、麻黄、苦杏仁、羌活、生姜、广藿香、佩兰、苍术、茯苓、白术、石膏、焦山楂、焦六神曲、焦麦芽、地龙、徐长卿、绵马贯众、葶苈子，分别加水提取两次，第一次加6倍量水，提取1.5h，第二次加4倍量水，提取1.0h，合并提取液，过滤，滤液浓缩至相对密度1.10～1.15，离心滤过，滤液干燥；其中，所述提取物的麻黄碱含量为1.20-1.50mg/g，固含物转移率为19-22％，紫花前胡苷含量为6-8mg/g。
进一步地，所述中药提取物的制备包括：称取厚朴15g、焦槟榔9g、煨草果9g、麻黄6g、苦杏仁9g、羌活15g、生姜15g、广藿香15g、佩兰9g、苍术15g、茯苓45g、白术30g、石膏15g、焦山楂9g、焦六神曲9g、焦麦芽9g、地龙15g、徐长卿15g、绵马贯众9g、葶苈子15g；分别加水提取两次，第一次加6倍量水，提取1.5h，第二次加4倍量水，提取1.0h，合并提取液，过滤，滤液浓缩至相对密度1.10～1.15，离心 滤过，滤液干燥。
进一步地，所述厚朴的指纹图谱和对照指纹图谱相比，相似度不得低于0.90。其中，对照指纹图谱包含共有峰和厚朴酚、厚朴酚。所述对照指纹图谱例如可以是图1。
进一步地，所述焦槟榔的指纹图谱和对照指纹图谱相比，相似度不得低于0.90。其中，对照指纹图谱包含共有峰槟榔次碱、槟榔碱。所述对照指纹图谱例如可以是图2。
进一步地，所述煨草果的指纹图谱和对照指纹图谱相比，相似度不得低于0.90。其中，对照指纹图谱包含共有峰原儿茶酸。所述对照指纹图谱例如可以是图3。
一种中药提取物的制备方法，其特征在于：称取厚朴、焦槟榔、煨草果、麻黄、苦杏仁、羌活、生姜、广藿香、佩兰、苍术、茯苓、白术、石膏、焦山楂、焦六神曲、焦麦芽、地龙、徐长卿、绵马贯众、葶苈子，分别加水提取两次，第一次加6倍量水，提取1.5h，第二次加4倍量水，提取1.0h，合并提取液，过滤，滤液浓缩至相对密度1.10～1.15，离心滤过，滤液干燥；其中，所述提取物的麻黄碱含量为1.20-1.50mg/g，固含物转移率为19-22％，紫花前胡苷含量为6-8mg/g。其中，所述厚朴、焦槟榔或煨草果的指纹图谱如前所定义。本发明还提出了如前任一所述的中药提取物在制备治疗抗流感病毒H1N1或FM1，或呼吸道合胞病毒药物中的应用。
进一步地，前述中药提取物由包括以下原料的中药制成：厚朴50份、焦槟榔30份、煨草果30份、麻黄20份、苦杏仁30份、羌活50份、生姜50份、广藿香50份、佩兰30份、苍术50份、茯苓150份、白术100份、石膏50份、焦山楂30份、焦六神曲30份、焦麦芽30份、地龙50份、徐长卿50份、绵马贯众30份、葶苈子50份。
本发明还提出了一种药物，该药物由如前任一所述的中药提取物与药学上可接受的辅料或添加剂制得。
具体地，该药物选自汤剂、颗粒剂、胶囊剂、片剂、口服液、丸剂、软胶囊、滴丸、酊剂、糖浆剂、栓剂、凝胶剂、喷雾剂、注射剂。
进一步地，所述药物为颗粒剂，所述辅料或添加剂优选为糊精和三氯蔗糖。
进一步地，所述药物还可以包括用于上呼吸道感染的抗病毒药物，例如可以是利巴韦林和/或磷酸奥司他韦。
本发明还提出了检测如前任一所述的中药提取物或药物的指纹图谱的方法，其特征在于，取供试品溶液进行HPLC检测，HPLC检测的色谱条件包括：采用C18色谱柱，以甲醇为流动相A，含有0.1％浓度的磷酸水溶液为流动相B，所述HPLC检测的色谱条件中的洗脱为梯度洗脱，该梯度洗脱程序为：0～5min，0％A；5～25min，0％～15％A；25～60min，15％～55％A；60～75min，55％～100％A；75～80min，100％A。
所述供试品溶液为由中药提取物或药物所制得的用于检测的溶液，制备方法例如可以是，取颗粒剂1g，置具塞锥形瓶中，加水25ml，超声处理30分钟，即 得。
进一步地，所述色谱条件包括：所述流速为1.0mL/min，所述柱温为30℃，检测波长250nm。
进一步地，所述指纹图谱如图14所示。
具体地，所述指纹图谱中，供试品特征图谱中应呈现10个特征峰，与参照物峰相应的峰为S峰，计算各特征峰与S峰的相对保留时间，应在规定值的±10％优选5％之内，相对保留时间规定值为：0.11(峰1)、0.27(峰2)、0.36(峰3)、0.38(峰4)、0.41(峰5)、0.84(峰6)、0.92(峰7)、1.00[峰8(S)]、1.19(峰9)、1.29(峰10)。
本发明制备了新的中药组合物，提升了组合物的质量标准，而且通过采用流感病毒H1N1/FM1株感染模型、呼吸道合胞病毒感染模型，评价组合物颗粒治疗病毒性上呼吸道感染的有效性，表明组合物可以降低肺组织中病毒载量及炎性细胞因子，表明对该类常见的特定病毒感染有增强治疗的作用，为进一步临床研究提供依据。
附图说明
图1为厚朴药材对照指纹图谱；峰6：和厚朴酚峰7(S)：厚朴酚；
图2为槟榔药材对照指纹图谱；峰1：槟榔次碱峰3(S):槟榔碱；
图3为煨草果药材对照指纹图谱；峰1:原儿茶酸；
图4为供试品溶液全波长扫描3D图；
图5为供试品溶液波长考察色谱图；
图6为流动相比例考察色谱图；
图7为供试品制备方法考察色谱图；
图8为耐用性考察色谱图；
图9为精密度考察色谱图；
图10重复性考察色谱图；
图11稳定性考察色谱图；
图12 20批制剂特征图谱叠加图；
图13对照特征图谱；
图14对照特征图谱及各特征峰指认结果；其中，1-腺嘌呤；2-尿苷；3-鸟苷；4-肌苷；5-5-羟甲基糠醛；6-厚朴苷A；7-佛手酚葡萄糖苷；8-紫花前胡苷；9-6'-O-(反式阿魏酰基)-紫花前胡苷；10-茴香酸对羟基苯乙酯。
具体实施方式
奥司他韦是流感病毒的神经氨酸酶的抑制药，临床上主要用来治疗甲型、乙型流感。利巴韦林为广谱抗病毒药，临床上可以用来治疗呼吸道合胞病毒、甲型和乙型流感，但FDA不太推荐。本发明则旨在提供一种中药组合物及其制备方法和应用。以下将以常用的流感病毒或呼吸道合胞病毒药物为例，结合实验内容进行具体描述。
特别需要指出的是，针对本发明所做出的类似的替换和改动对本领域技术人 员来说是显而易见的，它们都被视为包括在本发明。相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。
本发明如未注明具体条件者，均按照常规条件或制造商建议的条件进行，所用原料药或辅料，以及所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实验例1制备工艺考查
1.提取溶剂考察
称取厚朴15g、焦槟榔9g、煨草果9g、麻黄6g、苦杏仁9g、羌活15g、生姜15g、广藿香15g、佩兰9g、苍术15g、茯苓45g、白术30g、石膏15g、焦山楂9g、焦六神曲9g、焦麦芽9g、地龙15g、徐长卿15g、绵马贯众9g、葶苈子15g为1份，加水煎煮两次，第一次提取1.5h，第二次提取1.0h。合并提取液，过滤，滤液浓缩至相对密度1.10～1.15，离心滤过，滤液真空干燥，喷雾干燥粉碎，得中间体组合物。不同提取溶剂体积的麻黄碱转移率和固含物转移率检测结果如下：
计算公式：

其中，总转移率是指提取物中盐酸麻黄碱和盐酸伪麻黄碱的总量与药材中盐酸麻黄碱和盐酸伪麻黄碱的比值。
实验结果表明，盐酸麻黄碱和盐酸伪麻黄碱转移率及固含物转移率随着加水量的增加，呈递增趋势。当溶媒倍量由4，3倍增加到6，4倍时，各指标转移率增加较为明显，当加水量继续增加，各指标增长趋势较为平缓，但溶剂过高时反而有所下降。综合考虑能效及指标成分转移率，确定加水量为第一次加6倍量，第二次加4倍量。
2.浸泡时间考察
另称取厚朴15g、焦槟榔9g、煨草果9g、麻黄6g、苦杏仁9g、羌活15g、生姜15g、广藿香15g、佩兰9g、苍术15g、茯苓45g、白术30g、石膏15g、焦山楂9g、焦六神曲9g、焦麦芽9g、地龙15g、徐长卿15g、绵马贯众9g、葶苈 子15g为1份，共3份，加水煎煮两次，第一次加6倍量水、提取1.5h，第二次加4倍量水、提取1.0h，3份浸泡时间分别为0、30和60min。合并提取液，过滤，滤液浓缩至相对密度1.10～1.15，离心滤过，滤液真空干燥，喷雾干燥粉碎，得中间体组合物。
考察浸泡时间对盐酸麻黄碱和盐酸伪麻黄碱转移率及固含物转移率的影响。实验结果如下：
计算公式：

实验结果表明，浸泡时间对盐酸麻黄碱和盐酸伪麻黄碱总转移率及固含物转移率影响较小。因此，确定提取工艺选择不浸泡，直接进行提取。
3.提取时间考察
另称取厚朴15g、焦槟榔9g、煨草果9g、麻黄6g、苦杏仁9g、羌活15g、生姜15g、广藿香15g、佩兰9g、苍术15g、茯苓45g、白术30g、石膏15g、焦山楂9g、焦六神曲9g、焦麦芽9g、地龙15g、徐长卿15g、绵马贯众9g、葶苈子15g为一份，共5份，分别加水提取2次，第一次加6倍量水，第二次加4倍量水，第一次提取时间分别为60min、75min、90min、105min、120min，第二次提取时间分别为30min、45min、60min、75min、90min，考察提取时间对盐酸麻黄碱和盐酸伪麻黄碱转移率及固含物转移率的影响，实验结果如下：
计算公式：

实验结果表明，盐酸麻黄碱和盐酸伪麻黄碱转移率及固含物转移率随着提取时间的增加，呈递增趋势。当提取时间自60，30min增加到90，60min时，各指标增长趋势较为明显，提取时间继续增加各指标增长趋势逐渐平缓。因此，提取时间选择第一次提取90min，第二次提取60min。
4.提取工艺验证
为验证优选的提取工艺，按单因素实验优选结果进行放大验证试验。在前期实验中，提取物的几个主要成分波动较大，导致疗效受到了影响，因此通过加强从原料到制备工艺的控制，以期实现提取物的有效、可控。称取厚朴15g、焦槟榔9g、煨草果9g、麻黄6g、苦杏仁9g、羌活15g、生姜15g、广藿香15g、佩兰9g、苍术15g、茯苓45g、白术30g、石膏15g、焦山楂9g、焦六神曲9g、焦麦芽9g、地龙15g、徐长卿15g、绵马贯众9g、葶苈子15g为1份，共称取12份(一些重要的药物厚朴、槟榔和煨草果均建立了特有的药材及饮片指纹图谱控制标准，处方药材均符合最新药典标准的规定)，分别加水提取两次，第一次加6倍量水，提取1.5h，第二次加4倍量水，提取1.0h。每4份合并作为1组，共3组。测定盐酸麻黄碱和盐酸伪麻黄碱转移率、固含物转移率以及其它重要指标，考察工艺的稳定性和可行性，实验结果如下：
验证结果显示，优选的工艺稳定、可行，重现性良好，因此确定的最佳提取工艺为加水提取两次，第一次加6倍量水，提取1.5h；第二次加4倍量水，提取1.0h。
5、厚朴、槟榔和煨草果药材及饮片指纹图谱控制标准
5.1厚朴药材及饮片指纹图谱控制标准
色谱条件与系统适用性试验 以十八烷基硅烷键合硅胶为填充剂(Waters CORTECTS C18色谱柱，150mm×4.6mm，2.7μm)；以乙腈为流动相A，以0.1％乙酸溶液为流动相B，按下表中的规定进行梯度洗脱；柱温为35℃；流速为0.8ml/min；检测波长为230nm。理论板数按厚朴酚峰计算应不低于4000。

参照物溶液的制备 取厚朴酚、和厚朴酚对照品适量，精密称定，加甲醇制成每1ml各含200μg的混合溶液，即得。
供试品溶液的制备 取本品粉末(过三号筛)1g，精密称定，置具塞锥形瓶中，精密加入50％甲醇溶液25ml，摇匀，密塞，浸渍24小时，滤过，取续滤液，即得。
测定法 分别精密吸取参照物溶液和供试品溶液各10μl，注入液相色谱仪，测定。
供试品指纹图谱中应呈现与参照物色谱峰图1保留时间相同的色谱峰。按中药色谱指纹图谱相似度评价系统，供试品指纹图谱与对照指纹图谱经相似度计算，相似度不得低于0.90。
5.2槟榔药材及饮片指纹图谱控制标准
色谱条件与系统适用性试验 以强阳离子交换键合硅胶为填充剂(Thermo BioBasic SCX色谱柱，柱长为25cm，内径为4.6mm，粒径为5μm)；以乙腈-0.2％磷酸溶液(用氨水调节pH至3.8)(65:35)为流动相；柱温为35℃；流速为1.2ml/min；检测波长为210nm。理论板数按槟榔碱计算应不低于10000。
参照物溶液的制备 取氢溴酸槟榔碱对照品适量，精密称定，加甲醇制成每1ml含50μg的溶液，即得。
供试品溶液的制备 取本品粉末(过二号筛)0.5g，精密称定，置具塞锥形瓶中，精密加入50％甲醇溶液50ml，超声处理30分钟，放冷，摇匀，滤过，取续滤液，即得。
测定法 分别精密吸取参照物溶液和供试品溶液各10μl，注入液相色谱仪，测定，记录色谱图，即得。
供试品指纹图谱中应呈现与参照物色谱峰图2保留时间相同的色谱峰。按中药色谱指纹图谱相似度评价系统，供试品指纹图谱与对照指纹图谱经相似度计算，相似度不得低于0.90。
5.3煨草果药材及饮片指纹图谱控制标准
色谱条件与系统适用性 以十八烷基硅烷键合硅胶为填充剂(Waters CORTECTS T3色谱柱，柱长为15cm，内径为4.6mm，粒径为2.7μm)；以乙腈为流动相A，以0.1％甲酸溶液为流动相B，按下表的规定进行梯度洗脱；柱温为30℃；流速为0.8ml/min；检测波长为254nm。理论板数按原儿茶酸峰计算应不低于10000。

参照物溶液的制备 取原儿茶酸对照品适量，精密称定，加甲醇制成每1ml含200μg的溶液，即得。
供试品溶液的制备 取本品粉末(过二号筛)1g，精密称定，精密加入50％甲醇溶液25ml，超声处理30分钟，放冷，摇匀，滤过，取续滤液，即得。
测定法 分别精密吸取参照物溶液5～10μl和供试品溶液10μl，注入液相色谱仪，测定，记录色谱图，即得。
供试品指纹图谱中应呈现与参照物色谱峰图3保留时间相同的色谱峰。按中药色谱指纹图谱相似度评价系统，供试品指纹图谱与对照指纹图谱经相似度计算，相似度不得低于0.90。
实验例2制剂的制备
制备例1中药复方颗粒
组方：厚朴50份、焦槟榔30份、煨草果30份、麻黄20份、苦杏仁30份、羌活50份、生姜50份、广藿香50份、佩兰30份、苍术50份、茯苓150份、白术100份、石膏50份、焦山楂30份、焦六神曲30份、焦麦芽30份、地龙50份、徐长卿50份、绵马贯众30份、葶苈子50份。
该中药组合物颗粒剂的制备方法：
取上述药材，加入水回流提取2次，第一次加入6倍水，提取1.5h，第二次加入4倍水，提取1.0h，合并提取液，过滤，滤液浓缩至相对密度1.10～1.15，离心滤过，滤液真空干燥，喷雾干燥粉碎，得中间体组合物，加入三氯蔗糖和糊精，混匀，得颗粒剂。
制备例2中药复方胶囊
组方：厚朴50份、焦槟榔30份、煨草果30份、麻黄20份、苦杏仁30份、羌活50份、生姜50份、广藿香50份、佩兰30份、苍术50份、茯苓150份、白术100份、石膏50份、焦山楂30份、焦六神曲30份、焦麦芽30份、地龙50份、徐长卿50份、绵马贯众30份、葶苈子50份。
该中药组合物胶囊的制备方法：
取药材加入水回流提取2次，第一次加入6倍水，提取1.5h，第二次加入4倍水，提取1.0h，合并提取液，过滤，滤液浓缩至相对密度1.10～1.15，离心滤过，滤液真空干燥，喷雾干燥，制粒，制成胶囊。
制备例3中药复方片剂
组方：厚朴50份、焦槟榔30份、煨草果30份、麻黄20份、苦杏仁30份、羌活50份、生姜50份、广藿香50份、佩兰30份、苍术50份、茯苓150份、白术100份、石膏50份、焦山楂30份、焦六神曲30份、焦麦芽30份、地龙50份、徐长卿50份、绵马贯众30份、葶苈子50份。
该中药组合物片剂的制备方法：
取药材加入水回流提取2次，第一次加入6倍水，提取1.5h，第二次加入4倍水，提取1.0h，合并提取液，过滤，滤液浓缩至相对密度1.10～1.15，离心滤过，滤液真空干燥，喷雾干燥，得浸膏粉加入微晶纤维素等辅料，制成片剂。
实验例3本发明组合物颗粒对流感病毒H1N1/FM1株感染的治疗作用
1试验材料
1.1受试药物 制备例1的颗粒，江苏康缘药业股份有限公司提供。
1.2阳性对照药
1.2.1磷酸奥司他韦颗粒(可威)：宜昌东阳光长江药业股份有限公司生产，成分：每袋颗粒内含15毫克磷酸奥司他韦。
1.2.2利巴韦林颗粒：四川百利药业有限责任公司生产，成分：本品主要成分为利巴韦林。规格：50mg。
1.3试剂
1.4仪器

1.5试验动物
1.6病毒株
甲型H1N1流感病毒FM1毒株购自美国标准生物品收藏中心(ATCC)。由本所ABSL-2试验室常规传代，-80℃保存备用。
2剂量设计及药物配制
2.1制备例1：
药液配制：试验前取颗粒定容加入蒸馏水，定容，用量为26.4g生药/kg/d，按0.2mL/10g体重/次灌胃给药，1次/日，连续4天。
2.2磷酸奥司他韦：试验时小鼠用量为27.5mg/kg/d，相当于人临床等倍剂量，给药时按0.2mL/10g体重/次灌胃给药，1次/日，连续4天。
2.3利巴韦林：试验时小鼠用量为82.5mg/kg/d，给药时按0.2mL/10g体重/次灌胃给药，1次/日，连续4天。
2.4组合物组1：制备例1 13.2g生药/kg/d+磷酸奥司他韦13.7mg/kg/d，给药方式同上。
2.5组合物组2：即制备例1 13.2g生药/kg/d+利巴韦林41.2mg/kg/d，给药方式同上。
3试验方法
取ICR小鼠70只，体重14±1g，雌雄各半，按体重等级随机分为7组，分别为正常对照组、模型对照组、磷酸奥司他韦组、利巴韦林、组合物组、磷酸奥司他韦组+组合物组、利巴韦林+组合物组。除正常对照组外，将小鼠用异氟烷轻度麻醉，以15个LD50流感病毒液(H1N1/FM1株)滴鼻感染，每只35μL。感染当天开始给药，每次按0.2mL/10g灌胃，每天1次，连续4天，正常对照组和模型对照组在同等条件下以蒸馏水灌胃。第5天各组小鼠称体重；解剖称肺重，取肺组织进行HE病理检测，计算肺指数及肺指数抑制率。
肺指数(％)＝肺湿重(g)/体重(g)×100
镜下标准：
“－”：小鼠肺间质未见有渗出，未见有瘀血，组织间质细胞未见有增大，肺内细支气管周围未见有炎症，组织结构正常。
“+”：小鼠肺组织、肺泡间质未见有明显渗出性炎症，未见有大面积瘀血，肺内细支气管周围有少量局限性炎症，以淋巴为主。
“++”：小鼠肺组织肺泡间质未见有大面积炎性及粘液渗出，有局限性小片状粘液渗出，肺细支气管周围有局限性炎症，以淋巴为主，腔内膜细胞有局限性增多(节段性)。
“+++”：小鼠肺组织肺泡间质内有大面积炎性及粘液渗出，渗出炎症以淋巴细胞为主，有少量分叶核及嗜酸细胞，细胞大小不均匀，成团状聚集，并有多量粉色粘液。血管周围炎症明显，肺内细支气管周围炎症局限性，腔内膜细胞增生，血管周围炎症较重。
统计方法：结果采用组间比较t检验进行统计学处理。
4本发明组合物颗粒对肺指数及肺指数抑制率的影响
研究结果(表1)显示：采用甲型H1N1流感病毒FM1株病毒感染小鼠后，模型对照组小鼠肺指数明显增高，与正常对照组比较有显著差异(P<0.01)；感染当天开始给予组合物颗粒治疗4天，磷酸奥司他韦组、组合物组、磷酸奥司他韦组+组合物组、利巴韦林+组合物组的肺指数显著降低，与模型对照组比较有明显差异，表明组合物对用于上呼吸道感染的抗病毒药物在抗流感病毒上有明显的增效作用。
表1组合物颗粒对流感病毒H1N1/FM1株感染小鼠肺指数的影响
注：与正常对照组比较##P<0.01；与模型对照组比较，**P<0.01，*P<0.05。
实验例4本发明组合物颗粒对呼吸道合胞病毒感染的治疗作用
1试验材料
1.1受试药物 制备例1颗粒，江苏康缘药业股份有限公司提供。
1.2阳性对照药
1.2.1磷酸奥司他韦颗粒(可威)：宜昌东阳光长江药业股份有限公司生产，成分：每袋颗粒内含15毫克磷酸奥司他韦。
1.2.2利巴韦林颗粒：四川百利药业有限责任公司生产，成分：本品主要成分为利巴韦林。规格：50mg。
1.3试剂
1.4仪器
1.5试验动物
1.6病毒株
呼吸道合胞病毒(RSV)毒株购自美国标准生物品收藏中心(ATCC)。由本所ABSL-2试验室常规传代，-80℃保存备用。
2剂量设计及药物配制
2.1制备例1：
药液配制：取颗粒，加入蒸馏水，定容至40mL，用量为26.4g生药/kg/d，给药时按0.2mL/10g体重/次灌胃给药，1次/日，连续4天。
2.2磷酸奥司他韦颗粒：试验时小鼠用量为27.5mg/kg/d，相当于人临床等倍剂量，给药时按0.2mL/10g体重/次灌胃给药，1次/日，连续4天。
2.3利巴韦林颗粒：试验时小鼠用量为82.5mg/kg/d，给药时按0.2mL/10g体重/次灌胃给药，1次/日，连续4天。
2.4组合物组1：即制备例1颗粒13.2g生药/kg/d+磷酸奥司他韦13.7mg/kg/d，给药方式同上。
2.5组合物组2：即制备例1颗粒13.2g生药/kg/d+利巴韦林41.2mg/kg/d，给药方式同上。
3试验方法
取BALB/C小鼠70只，体重14±1g，雌雄各半，按体重等级随机分为7组，分别为正常对照组、模型对照组、磷酸奥司他韦组、利巴韦林、组合物组、磷酸奥司他韦组+组合物组、利巴韦林+组合物组，每组10只。除正常对照组外，将小鼠用异氟烷轻度麻醉，以100TCID50RSV病毒液滴鼻感染，每只45μL。感染当天开始给药，每次按0.2mL/10g灌胃，每天1次，连续4天，正常对照组和模型对照组在同等条件下以蒸馏水灌胃。第5天各组小鼠称体重；解剖称肺重，取肺组织进行炎性因子检测，计算肺指数及肺指数抑制率。
肺指数(％)＝肺湿重(g)/体重(g)×100
4实验结果
4.1组合物颗粒对肺指数及肺指数抑制率的影响
研究结果(表2)显示：采用呼吸道合胞病毒(RSV)滴鼻感染小鼠后，模型组小鼠肺指数明显增高，与正常对照组比较有显著差异(P<0.01)；感染当天开始给予组合物颗粒治疗4天，磷酸奥司他韦组、组合物组、磷酸奥司他韦组+组合物组、利巴韦林+组合物组的肺指数显著降低，与模型对照组比较有明显差异，表明组合物对用于上呼吸道感染的抗病毒药物在抗呼吸道合胞病毒上有一定的增效作用。
表2组合物颗粒对呼吸道合胞病毒感染小鼠肺指数的影响
注：与正常对照组比较##P<0.01；与模型对照组比较，*P<0.05。
4.2对肺组织中细胞因子IL-33的影响
将肺组织从-80℃低温冰箱中取出，用高效蛋白裂解液(已加入PMSF及蛋白酶抑制剂)处理肺组织后离心，取蛋白上清。将蛋白样品放置于冰盒中交予莱兹生物技术有 限公司进行高通量液相蛋白芯片检测小鼠肺组织中各个细胞因子情况，具体检测步骤详见附件。
采用呼吸道合胞病毒(RSV)滴鼻感染小鼠后，模型组小鼠肺组织细胞因子IL-33含量明显增高，与正常对照组比较有显著差异(P<0.01)；感染当天开始给予组合物颗粒治疗4天，磷酸奥司他韦组、组合物组、磷酸奥司他韦组+组合物组、利巴韦林+组合物组的IL-33含量明显降低，与模型组比较有显著差异(P<0.01，P<0.05)。见表3。
表3组合物颗粒对呼吸道合胞病毒感染小鼠肺组织细胞因子的影响
注：与正常对照组比较##P<0.01，#P<0.05；与寒湿疫毒肺炎模型组比较，**P<0.01。
本研究中，将组合物颗粒26.4g生药/kg/d(1/2倍)小鼠灌胃给药，1次/日，连续3～4天，采用流感病毒H1N1/FM1株感染模型、呼吸道合胞病毒感染模型，评价组合物颗粒治疗病毒性上呼吸道感染的有效性，结果显示：组合物联合抗病毒药物可以降低肺组织中病毒载量及炎性细胞因子，表明对该类常见的特定病毒感染有治疗作用，利用了中药本身的优势来进一步促进和巩固其它药物的抗病毒的效果，为进一步临床研究提供依据。
实验例5 本发明组合物指纹图谱研究
仪器与试药
仪器：Agilent 1260高效液相色谱仪，DAD紫外检测器；Agilent 1260高效液相色谱仪，VWD紫外检测器；Agilent 1290超高压液相色谱仪，DAD紫外检测器，Agilent 6538 Q-TOF-MS质谱检测器，电喷雾(ESI)离子源，美国安捷伦公司；Thermo Fisher Ultimate3000高效液相色谱仪；
Mettler Toledo XP6电子分析天平，梅特勒公司；Mettler Toledo AL204型电子分 析天平，梅特勒公司；KQ500DB数控超声波清洗仪，昆山超声仪器有限公司；HH数显恒温水浴锅，常州国宇仪器制造有限公司；Milli-Q超纯水仪，美国密理博公司；
对照品：腺嘌呤，99.4％，批号B0002908，北京曼哈格生物科技有限公司；尿苷，99.4％，批号B0008580，北京曼哈格生物科技有限公司；鸟苷，96.1％，批号B0006767，北京曼哈格生物科技有限公司；肌苷，99.2％，批号140669-202007，中国食品药品检定研究院；5-羟甲基糠醛，95.0％，批号8610，上海诗丹德标准技术有限公司；厚朴苷A，99.1％，批号7611，上海诗丹德标准技术有限公司；佛手酚葡萄糖苷，98％，批号CFS202002，武汉天植生物技术有限公司；紫花前胡苷，99.6％，批号111821-201604，中国食品药品检定研究院；6'-O-(反式阿魏酰基)-紫花前胡苷，98％，批号CFS202002，武汉天植生物技术有限公司；茴香酸对羟基苯乙酯，99.6％，批号A05GB156933，上海源叶生物科技有限公司；
试剂：甲醇(默克股份有限公司、美国迈瑞达公司，色谱纯)；乙酸(L07203503，默克飞世尔科技有限公司)，其他试剂均为分析纯。
样品：制备例1方法制备的颗粒，批号为：200201、200202、200203、200204、200205、200206、200207、200208、200209、200210、200211、200212、200213、200214、200601、200602、200603，其中200602批为方法学研究批次，均由江苏康缘药业股份有限公司中药研发部提供。
1色谱条件选择
取本品适量，研细，取约2g，精密称定，置具塞锥形瓶中，精密加入水50ml，超声处理30分钟，摇匀，离心，取上清液，即得供试品溶液。
1.1检测波长的选择
取供试品溶液，采用DAD进行全波长扫描，结果可见250nm条件下色谱峰信息较丰富，响应较均匀，基线较平稳，因此选取250nm作为特征图谱的检测波长。见附图4、图5。
1.2流动相的选择
基于本品水提工艺特点，采用对水溶性成分具有较好分离效果的Waters Atlantis T3(4.6×250mm，5μm)C18色谱柱，采用甲醇-0.1％磷酸作为流动相系统，250nm作为检测波长，考察不同流动相梯度的分离效果，结果梯度程序Ⅳ各峰分离度较好，保留时间合适，基线较为平稳，因此，优选梯度程序Ⅳ作为流动相洗脱程序。梯度洗脱程序如下，结果见附图6。

综上考察，拟定色谱条件为：以十八烷基硅烷键合硅胶为填充剂(Waters Atlantis T3，柱长为25cm，内径为4.6mm，粒径为5μm)；以甲醇为流动相A，以0.1％磷酸为流动相B，按下表中的规定进行梯度洗脱；流速为每分钟1.0ml；柱温为30℃；检测波长为250mn。理论板数按紫花前胡苷峰计应不低于10000。
2供试品制备方法选择
为优选合理、简便的供试品溶液制备方法，对提取溶剂、提取方式考察。
供试品溶液制备：取本品适量，研细，取约1g，共4份，精密称定，置具塞锥形瓶中，分别精密加入水、50％甲醇25ml，分别超声处理30分钟、回流30分钟，摇匀，离心，取上清液，即得4份供试品溶液。
结果：取各供试品溶液，按上述色谱条件进行测定，结果显示采用水为提取溶剂所得的色谱图25min之前的极性较大的色谱峰响应较高，整体色谱峰较为丰富，响应较均匀，结合产品的水提工艺特点，优选水作为提取溶剂。回流与超声两种方式得到的色谱图色谱峰的丰富程度与响应无明显差异，考虑操作的方便性，优选超声提取方式。见附图7。
综上考察，拟定供试品处理方法为：取本品适量，研细，取约1g，精密称定，置具塞锥形瓶中，精密加入水25ml，超声处理(功率500W，频率40kHz)30分钟，摇匀，离心，取上清液，即得。
3耐用性考察
考察不同的波长(245nm、250nm、255nm)、流速(0.8ml/min、0.9ml/min、1.0ml/min、1.1ml/min、1.2ml/min)、柱温(25℃、28℃、30℃、32℃、35℃)、色谱柱不同批号{柱1：Waters Atlantis T3(4.6×250mm，5μm)，序列号01813017014031；柱2：Waters Atlantis T3(4.6×250mm，5μm)，序列号01813017014008}、仪器(仪器1：Agilent 1260、仪器2：Ultimate3000)对选定的10个特征峰分离效果的影响。
结果显示，在波长250±5nm、流速0.8ml/min～1.2ml/min、柱温25℃～35℃范围 内，以及使用不同批号的色谱柱、不同品牌的仪器，选定的特征峰均可得到较好的分离效果，说明方法耐用性良好。见附图8。
4方法学验证
精密度考察：按上述方法制备供试品溶液，精密吸取同一供试品溶液10μl，连续进样6次，测定，以紫花前胡苷(峰8)为参照峰，对标定的10个特征峰的相对保留时间进行计算。结果显示，10个特征峰的相对保留时间的RSD值均小于0.5％，表明仪器的精密度良好。见表4、附图9。
重复性考察：按上述方法平行制备6份供试品溶液，测定。以紫花前胡苷(峰8)为参照峰，对标定的10个特征峰的相对保留时间进行计算。结果显示，10个特征峰的相对保留时间的RSD值均小于0.5％，表明该方法重复性良好。见表5，附图10。
稳定性考察：取同一供试品溶液，分别于第0h、3h、6h、9h、12h、18h、24h、30h、36h进样，每次进样10μl，测定，以紫花前胡苷(峰8)为参照峰，对标定的10个特征峰的相对保留时间进行计算。结果显示，10个特征峰的相对保留时间的RSD值均小于0.5％，表明供试品溶液36小时内稳定性较好。见表6，附图11。
表4特征图谱精密度考察计算结果
表5重复性考察计算结果
表6稳定性考察计算结果
5多批次测定及对照特征图谱生成
依据上述方法进行14批临床实践用样品、3批中试样品及3批工艺验证样品的测定，选定10个峰为特征峰，以20批制剂生成对照指纹图谱，以紫花前胡苷参照物峰相应的峰为S峰，计算各特征峰的相对保留时间，各峰应在规定值的±10％以内，规定值为：0.11(峰1)、0.27(峰2)、0.36(峰3)、0.38(峰4)、0.41(峰5)、0.84(峰6)、0.92(峰7)、1.00(峰8)、1.19(峰9)、1.29(峰10)。多批次结果见附图12、表7，对照特征图谱见附图13。
表7 20批制剂相对保留时间结果

6特征图谱质量标准
色谱条件与系统适用性试验以十八烷基硅烷键合硅胶为填充剂(Waters Atlantis T3，柱长为25cm，内径为4.6mm，粒径为5μm)；以甲醇为流动相A，以0.1％磷酸为流动相B，按下表中的规定进行梯度洗脱；流速为每分钟1.0ml；柱温为30℃；检测波长为250mn。理论板数按紫花前胡苷峰计应不低于10000。
参照物溶液的制备 取紫花前胡苷对照品适量，精密称定，加50％甲醇制成每1ml含0.15mg的溶液，即得。
供试品溶液的制备 取本品适量，研细，取约1g，精密称定，置具塞锥形瓶中，精密加入水25ml，超声处理(功率500W，频率40kHz)30分钟，摇匀，离心，取上清液，即得。
测定法 分别精密吸取参照物溶液和供试品溶液各10μl，注人液相色谱仪，测定，记录色谱图，即得。
供试品特征图谱中应呈现10个特征峰，与参照物峰相应的峰为S峰，计算各特征峰的相对保留时间，应在规定值的±10％之内，相对保留时间规定值为：0.11(峰1)、0.27(峰2)、0.36(峰3)、0.38(峰4)、0.41(峰5)、0.84(峰6)、0.92(峰7)、1.00[峰8(S)]、1.19(峰9)、1.29(峰10)。对照特征图谱见附图14。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (12)

  1. 一种中药提取物，其特征在于，以重量比计，所述中药提取物由包括以下原料的中药制成：厚朴1～100份、焦槟榔1～100份、煨草果1～100份、麻黄1～100份、苦杏仁1～100份、羌活1～100份、生姜1～100份、广藿香1～100份、佩兰1～100份、苍术1～100份、茯苓1～100份、白术1～100份、石膏1～100份、焦山楂1～100份、焦六神曲1～100份、焦麦芽1～100份、地龙1～100份、徐长卿1～100份、绵马贯众1～100份、葶苈子1～100份；其中，所述提取物的麻黄碱含量为0.5-1.5mg/g，固含物转移率为18-25％。
  2. 根据权利要求1所述的中药提取物，其特征在于，所述中药提取物由包括以下原料的中药制成：厚朴1～80份、焦槟榔1～80份、煨草果1～80份、麻黄1～60份、苦杏仁1～60份、羌活1～80份、生姜1～60份、广藿香1～80份、佩兰1～60份、苍术1～80份、茯苓1～160份、白术1～120份、石膏1～80份、焦山楂1～50份、焦六神曲1～80份、焦麦芽1～60份、地龙1～80份、徐长卿1～80份、绵马贯众1～60份、葶苈子1～80份；其中，所述提取物的紫花前胡苷含量为2-8mg/g。
  3. 根据权利要求1或2所述的中药提取物，其特征在于，所述中药提取物由包括以下原料的中药制成：厚朴30～50份、焦槟榔20～30份、煨草果30～50份、麻黄20～30份、苦杏仁20～30份、羌活30～50份、生姜30～50份、广藿香30～50份、佩兰20～30份、苍术30～50份、茯苓120～150份、白术80～100份、石膏30～50份、焦山楂20～30份、焦六神曲30～50份、焦麦芽20～30份、地龙30～50份、徐长卿30～50份、绵马贯众20～30份、葶苈子30～50份。
  4. 根据权利要求3所述的中药提取物，其特征在于，所述中药提取物由包括以下原料的中药制成：厚朴50份、焦槟榔30份、煨草果30份、麻黄20份、苦杏仁30份、羌活50份、生姜50份、广藿香50份、佩兰30份、苍术50份、茯苓150份、白术100份、石膏50份、焦山楂30份、焦六神曲30份、焦麦芽30份、地龙50份、徐长卿50份、绵马贯众30份、葶苈子50份。
  5. 根据权利要求1-4任一所述的中药提取物，其特征在于，所述中药提取物的制备包括：
    称取厚朴、焦槟榔、煨草果、麻黄、苦杏仁、羌活、生姜、广藿香、佩兰、苍术、 茯苓、白术、石膏、焦山楂、焦六神曲、焦麦芽、地龙、徐长卿、绵马贯众、葶苈子，分别加水提取两次，第一次加6倍量水，提取1.5h，第二次加4倍量水，提取1.0h，合并提取液，过滤，滤液浓缩至相对密度1.10～1.15，离心滤过，滤液干燥；其中，所述提取物的麻黄碱含量为1.20-1.50mg/g，固含物转移率为19-22％，紫花前胡苷含量为6-8mg/g。
  6. 根据权利要求5所述的中药提取物，其特征在于，所述中药提取物的制备包括：
    称取厚朴15g、焦槟榔9g、煨草果9g、麻黄6g、苦杏仁9g、羌活15g、生姜15g、广藿香15g、佩兰9g、苍术15g、茯苓45g、白术30g、石膏15g、焦山楂9g、焦六神曲9g、焦麦芽9g、地龙15g、徐长卿15g、绵马贯众9g、葶苈子15g；分别加水提取两次，第一次加6倍量水，提取1.5h，第二次加4倍量水，提取1.0h，合并提取液，过滤，滤液浓缩至相对密度1.10～1.15，离心滤过，滤液干燥。
  7. 权利要求1-6任一项所述的中药提取物在制备治疗抗流感病毒H1N1或FM1，或呼吸道合胞病毒药物中的应用。
  8. 一种药物，其特征在于，该药物由1-6任一项所述的中药提取物与药学上可接受的辅料或添加剂制得。
  9. 根据权利要求6所述的药物，其特征在于，所述药物为颗粒剂，所述辅料或添加剂优选为糊精和三氯蔗糖。
  10. 检测权利要求1-6任一所述的中药提取物或权利要求6所述的药物的指纹图谱的方法，其特征在于，取供试品溶液进行HPLC检测，HPLC检测的色谱条件包括：采用C18色谱柱，以甲醇为流动相A，含有0.1％浓度的磷酸水溶液为流动相B，所述HPLC检测的色谱条件中的洗脱为梯度洗脱，该梯度洗脱程序为：0～5min，0％A；5～25min，0％～15％A；25～60min，15％～55％A；60～75min，55％～100％A；75～80min，100％A。
  11. 根据权利要求10所述的方法，其特征在于，所述色谱条件包括：所述流速为1.0mL/min，所述柱温为30℃，检测波长250nm。
  12. 根据权利要求10或11所述的方法，其特征在于，所述指纹图谱中，供试品特征图谱中应呈现10个特征峰，与参照物峰相应的峰为S峰，计算各特征峰与S峰的相对保留时间，应在规定值的±10％之内，相对保留时间规定值为：0.11(峰1)、0.27 (峰2)、0.36(峰3)、0.38(峰4)、0.41(峰5)、0.84(峰6)、0.92(峰7)、1.00[峰8(S)]、1.19(峰9)、1.29(峰10)。
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