WO2016169491A1 - Use of forsythin, derivatives thereof and composition of forsythin/forsythiaside in the preparation of drug for treating bacterial infections - Google Patents

Use of forsythin, derivatives thereof and composition of forsythin/forsythiaside in the preparation of drug for treating bacterial infections Download PDF

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Publication number
WO2016169491A1
WO2016169491A1 PCT/CN2016/079843 CN2016079843W WO2016169491A1 WO 2016169491 A1 WO2016169491 A1 WO 2016169491A1 CN 2016079843 W CN2016079843 W CN 2016079843W WO 2016169491 A1 WO2016169491 A1 WO 2016169491A1
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forsythin
forsythiaside
extract
drug
composition
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PCT/CN2016/079843
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French (fr)
Chinese (zh)
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富力
王凯乾
惠敏
鲁明明
刘正贤
王硕
付强
刘国友
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富力
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    • 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)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/63—Oleaceae (Olive family), e.g. jasmine, lilac or ash tree
    • A61K36/634—Forsythia

Definitions

  • the invention relates to the field of medicine, relates to a medicine or health food for treating bacterial infection, in particular to a medicine Chinese medicine forsythia and forsythia leaf extract component for relieving and/or treating bacterial infection medicine or health food.
  • Bacterial resistance to indoleamine antibiotics is the most common and important type of resistance to antibiotic resistance. At present, the most striking problem in Gram-negative bacilli is that bacteria are resistant to the third-generation cephalosporin and the new broad-spectrum-indolamine antibiotics. The main mechanism is the production of extended-spectrum-lactamase by bacteria.
  • ESBLs Extended spectrum-laetamases, ESBLs cause bacteria to be resistant to third-generation monolactam antibiotics and aztreonam.
  • ESBLs are a group of enzymes that are mediated by plasmids that hydrolyze most of the cephalosporin and monoamine antibiotics. Most EsBLs are inhibited by enzyme inhibitors such as tazobactam and clavulanic acid.
  • ESBLs are mainly divided into SHV, TEM, CTX-M, OXA and other types. In different countries, regions and hospitals, the genotypes of ESBLs are different due to different use of antibiotics. In China, CTX-M is the main type. Therefore, what kind of diseases are used for the bacterial infection of CTX-M ESBLs? The treatment of drugs has become a difficult point and key point in the treatment of ESBLS-producing bacteria in China.
  • Forsythia is one of the traditional traditional medicines used in traditional Chinese medicine. It is widely used and has a large dosage. It has the function of clearing away heat and detoxifying and dissipating free radicals. It is often formulated with other Chinese herbal medicines for various bacteria. Infection treatment. However, the specific active ingredients are still unclear. The inventors have arduously explored and prepared the active monomer compound forsythin, and chemically synthesized to prepare corresponding derivatives, and carried out a series of pharmacological activities. Studies have found that forsythin, forsythin derivatives, forsythin and forsythiaside compositions have the effect of inhibiting bacterial infections and enhancing the immune function of humans, opening up new therapeutic pathways for patients with bacterial infections.
  • the inventors have extracted the forsythin which is an effective component for treating bacterial infection from forsythia and forsythia leaves through a large number of modern scientific researches, and advanced synthesis and purification techniques, and further studied and synthesized forsythin derivatives. It can provide a highly effective and low-toxic drug for patients with bacterial infection.
  • the primary object of the present invention is to provide forsythiaside, forsythin derivatives, forsythin and forsythiaside compositions (ie, even for the technical problems existing in the application of drugs or health care products for treating bacterial infections.
  • the properties and efficacy of a disease caused by a bacterial infection by preventing, alleviating or/and treating a disease caused by a bacterial infection, and providing a new combination of forsythin, forsythiaside derivative, forsythin and forsythiaside Medicinal use, ie new applications in the treatment, conditioning, prevention and alleviation of bacterial infections or health foods.
  • an aspect of the present invention provides a forsythiaside, forsythin derivative, forsythin and forsythiaside composition for preparing a medicament or a health care product for preventing or/or treating a disease caused by a bacterial infection.
  • the medicament or health care product consists of forsythin, forsythin derivative or forsythin and forsythiaside composition and a pharmaceutically acceptable carrier.
  • the forsythiaside derivative is selected from the forsythiaside glucuronic acid derivative.
  • the forsythin derivative comprises 33-hydroxy-philoxyphilin-8-O- ⁇ -D-glucuronide, 9-hydroxy-Forsylin glucagon Acid glucoside (9-Hydroxy phillygenin-8-O- ⁇ -D-glucuronide), 33,34-methylenedioxy-Forsythine glucuronide (33,34-Methylenedioxy phillygenin-8-O- ⁇ -D-glucuronide), forsythiaside methyl glucuronide ((2R,3R,4R,5S)-methyl6-(5-((1R,4S)-4-(3,4-dimethoxyphenyl)hexahydrofuro[3 , 4-c]furan-1-yl)-2-methoxyphenoxy)-3,4,5-trihydroxytetra hydro-2H-pyran-2-carboxylate), forsythiaside sodium glucuronate (sodium(2R,3R, 4R
  • the weight ratio of forsythin to forsythiaside in the forsythiaside and forsythiaside composition is from 2 to 98:2 to 98; preferably from 2 to 10:90 to 98; further preferably It is 80:20 or 20:80, further preferably 90:10 or 10:90, still more preferably 98:2 or 2:98; still more preferably 98:2.
  • the content of the forsythin, forsythiaside derivative is ⁇ 1%, preferably ⁇ 30%, further preferably ⁇ 60%, still more preferably ⁇ 80%, still more preferably ⁇ 98%;
  • the content of forsythin and forsythiaside composition is ⁇ 1%, preferably ⁇ 30%, further preferably ⁇ 60%, still more preferably ⁇ 80%, still more preferably ⁇ 98%.
  • the content of forsythin and forsythin derivatives is from 1% to 98%; preferably from 30 to 80%; and the content of forsythin and forsythiaside compositions is from 1% to 98%; preferably 30 to 80%.
  • 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 Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994) found in the reference book.
  • 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, flavoring agents, spices, etc.;
  • the medicament is in the form of a tablet, a capsule, a pill, a powder, a granule, or a syrup.
  • the content of forsythin and forsythin derivatives is ⁇ 1%, preferably ⁇ 30%, further preferably ⁇ 60%, still more preferably ⁇ 80%, still more preferably ⁇ 98%;
  • the content of forsythin and forsythiaside composition is ⁇ 1%, preferably ⁇ 30%, further preferably ⁇ 60%, still more preferably ⁇ 80%, still more preferably ⁇ 98%.
  • Another aspect of the present invention provides a medicament or health care product comprising forsythiaside, forsythiaside derivative or forsythin and forsythiaside composition for preventing, alleviating and/or treating a disease caused by a bacterial infection.
  • the forsythiaside derivative is selected from the forsythiaglycine glucuronic acid derivative.
  • the forsythin derivative comprises 33-hydroxy-Forsylin glucuronide, 9-hydroxy-Forsylin glucuronide, 33,34-methylenedioxy-linked sulfonate Glucuronide, forsythiaside methyl glucuronate, forsythiaside sodium glucuronate, forsythias glucuronide, forsythiaglycine glucuronic acid; preferably 33-hydroxy-forsythia Lipid glucuronide, 9-hydroxy-Forsyric acid glucuronide, 33,34-methylenedioxy-Forsythine glucuronide, forsythias glucuronide methyl ester, even Sodium lipoate or sodium forsylate. Glucuronide.
  • the weight ratio of forsythin to forsythin in the forsythiaside and forsythiaside composition is from 2 to 98:2 to 98; preferably from 2 to 10:90 to 98; further preferably 80:20 or 20:80, further preferably 90:10 or 10:90, still more preferably 98:2 or 2:98; still more preferably 98:2.
  • the content of the forsythin or forsythin derivative is ⁇ 1%, preferably ⁇ 30%, further preferably ⁇ 60%, still more preferably ⁇ 80%, still more preferably ⁇ 98%
  • the content of forsythin and forsythiaside composition is ⁇ 1%, preferably ⁇ 30%, further preferably ⁇ 60%, still more preferably ⁇ 80%, still more preferably ⁇ 98%.
  • the content of the forsythin or forsythin derivative is ⁇ 1%, preferably from 1% to 98%; preferably from 30% to 80%, still more preferably 60%; forsythin and forsythiaside
  • the composition of the composition is ⁇ 1%, preferably 1% to 98%; preferably 30 to 80%, and still more preferably 60%.
  • the ratio of the weight of the forsythin, the forsythin derivative or the forsythin to the forsythiaside 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 1 to 10:100.
  • 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 selects forsythin and forsythin with alpha-, beta- or gamma-cyclodextrin a mixture of or a mixture thereof, or a complex formed by the treatment of forsythiaside and forsythin with ⁇ -, ⁇ - or ⁇ -cyclodextrin or a derivative thereof by physical or chemical treatment.
  • 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 or sulfobutylether ⁇ - cyclodextrin, p-TsCl substituted ⁇ -cyclodextrin, 6-substituted ⁇ -CD p-toluenesulfonate ( ⁇ -cyclodextrin-6-OTs) , 2-oxohydroxypropyl- ⁇ -cyclodextrin, 2-position monosubstituted p-toluenesulfonate ( ⁇ -cyclodextr
  • the medicine or health care product further comprises Andrographis paniculata extract, Silybum extract, Honeysuckle extract, Radix Isatidis extract, dandelion extract, Phellodendron extract, Rhizoma Coptidis extract, Astragalus membranaceus extract, Portulaca oleracea extract , Pulsatilla extract, garlic extract, Houttuynia cordata extract, wild chrysanthemum extract, Sophora flavescens extract, vitamin C and its derivatives or vitamin E and its derivatives.
  • the present invention has the following distinct advantages:
  • the present invention excavates new medicinal value for known compounds of forsythin, forsythin derivatives, forsythin/forsylin composition, and is used for preventing, alleviating and treating bacterial infections, and A drug or health food prepared to prevent, alleviate and/or treat bacterial infections, thereby opening up a new field for the application of Forsythia and Forsythia suspensa.
  • the known compounds of the present invention forsythin, forsythin derivatives, forsythin/forsylin composition have strong pharmacological action, and have remarkable effects for relieving, preventing and treating bacterial infections, and have quick effects and side effects. Small, safe, long-term, with 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 invention can be used for preparing a medicament for preventing, alleviating and treating diseases caused by bacterial infection by using a single component of forsythin, forsythin derivative or forsythin/forsylin composition active ingredient, and Osmotic glycosides, forsythin derivatives or forsythin/forsylin compositions and other active ingredients (eg with andrographis extract, cyclaea extract, honeysuckle extract, Radix isatidis extract, dandelion extract, cork extract) , Rhizoma Coptidis extract, Astragalus membranaceus extract, Portulaca oleracea extract, Pulsatilla extract, Garlic extract, Houttuynia cordata extract, Wild chrysanthemum extract, Sophora flavescens extract, Vitamin C and its derivatives
  • the compound or vitamin E and its derivatives are combined to prepare a compound medicine for preventing and treating bacterial infection.
  • Glucuronide is the same as the forsythiaside derivative described in the patent application (Application No.: 201510319303.4, Priority No.: 201510164294.6); forsythiaside methyl glucuronide, forsythiaside sodium glucuronate, forsythia
  • the fatty acid potassium gluconate, forsythiaside glucuronic acid is the same as in the patent application (Application No.: 201510320579.4, priority number: 201410825547.5).
  • the forsythin and the starch are uniformly mixed to form granules, and the talc powder and magnesium stearate are added and uniformly mixed, and then pressed into 10,000 pieces.
  • the forsythin and the starch are uniformly mixed and then filled into capsules to make 10,000 tablets.
  • the forsythin and the starch are uniformly mixed and then filled into capsules to make 10,000 tablets.
  • the 33-hydroxy-Forsythine glucuronide, the Pulsatilla extract, the vitamin C and the sucrose powder are uniformly mixed into a granule and then bagged to make 10,000 bags.
  • the 9-hydroxy-Forsysin glucuronide, the Pulsatilla extract, the vitamin C and the sucrose powder are uniformly mixed into granules and then bagged to make 10,000 bags.
  • the forsythin/forsylin composition was uniformly mixed with starch in the weight ratio of the following table, and then filled into capsules, each of which was made into 10,000 capsules.
  • the forsythin/forsylin composition was uniformly mixed with the microcrystalline cellulose in the weight ratio of the following table, and then granulated and bagged to prepare 10,000 bags.
  • Forsythiaside 245g, forsythiaside 5g and Sophora flavescens extract, Sijiqing extract, Andrographis paniculata extract powder are evenly mixed, and then mixed with starch and then filled into capsules to make 10,000 capsules.
  • Forsythiaside 1880g, forsythiaside 120g and the above extracts (Houttuynia, Soybean Meal, Sijiqing) powder are evenly mixed, and then mixed with starch and then filled with capsules to make 10,000 tablets.
  • Forsythin >98%), white powder, produced by Dalian Fusheng Natural Medicine Development Co., Ltd., determined by high-performance liquid chromatography two detector UV detector and evaporative light scattering detector area normalization method, the purity is 99.5%, and the content of Chinese forensic bio-products was determined and confirmed to be 99.5% with forsythia reference substance. Batch number: 20130303.
  • Micro-multiple inoculation instrument produced by Kunlun Drug Development Institute of Beijing Medical University.
  • Quantitative antibacterial experiments of forsythin were carried out using agar dilution method using M-H agar.
  • 900 mL of the solubilized sterile MH agar was dispensed into 10 flasks, 90 mL of molten sterile MH agar per flask, and 10 mL of the first tube was contained.
  • the 0.3% forsythin sterile distilled aqueous solution was placed in a first triangular flask containing 90 mL of medium, and after mixing, it was poured onto 7 plates (ie, plates), and each plate contained about 14 mL of drug (ie, even The agarose agar, which was prepared after coagulation, was the first drug-containing agar plate group, wherein: the first drug-containing agar plate group actually contained forsythin 3.0 mg/ml in each plate.
  • each plate in the 9th drug-containing agar plate group actually contained forsythin 0.0117 mg/ml; in the 10th drug-containing agar plate group, each plate actually contained forsythin as 0.0 mg/ml.
  • Table 1 The results of Table 1 indicate that forsythiaside has significant antibacterial effects against Staphylococcus aureus, yeast, Penicillium citrinum, and Aspergillus niger, and it is against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, yeast, and orange.
  • the MICs of mold, Aspergillus niger, and Aspergillus flavus were: 0.0469 mg/ml, 1.5 mg/ml, 1.5 mg/ml, 0.1875 mg/ml, 0.0469 mg/ml, 0.0469 mg/ml, and 0.1875 mg/ml.
  • the strains were the same as in Test Example 1, and were provided by the Microbiology Laboratory of Dalian Medical University.
  • Quantitative antibacterial experiments of forsythin were carried out using agar dilution method using M-H agar.
  • the sterile test tubes 30 were divided into 3 groups, 10 groups of each group were arranged in a test tube rack, and 10 mL of sterile distilled water was added to the 2 to 10 test tubes in the 10 test tubes of the first group; 10 mL of the derivative A solution was added to the first tube; 10 mL of the forsythin derivative A solution was added to the second tube, and after mixing, 10 mL of the mixture was taken from the second tube and added to the third tube. In a test tube, mix well and then add 10 mL of the mixture to the 4th test tube.
  • sterile workstation On a sterile workstation, aseptically, 900 mL of solubilized sterile MH agar was dispensed into 10 flasks, 90 mL of molten sterile MH agar per flask, and the first of the first set of tubes. 10 mL of forsythin derivative A solution contained in only one tube was placed in a first flask containing 90 mL of medium, and mixed and poured onto 7 plates (ie, plates), each plate containing about 14 mL.
  • the drug-containing (that is, forsythin derivative A) agar was prepared after solidification and was used as the first drug-containing agar plate group, wherein: the first drug-containing agar plate group actually contained forsythin derivative A 3.0 mg/in each plate. Ml.
  • each plate set contained 7 plates.
  • Each plate contains approximately 14 mL of the drug-containing (ie, forsythin derivative A) agar, which is set aside after solidification.
  • the forsythiaside derivative B-containing agar plate group was prepared according to the preparation method of forsythiaside derivative A drug-containing agar plate group, except that the forsythiaside derivative B drug in the second group of test tubes was added to the second group of triangular flasks. Except for the liquid, the rest is the same as step 3-1).
  • the forsythiaside derivative C-containing agar plate group was prepared according to the preparation method of forsythiaside derivative A drug-containing agar plate group, except that the forsythiaside derivative C drug in the third group of test tubes was added to the third group of triangular flasks. Except for the liquid, the rest is the same as step 3-1).
  • Forsythin derivative A, forsythin derivative B and forsythin derivative C against Staphylococcus aureus, Escherichia coli The results of the bacteriostatic effects of Bacillus licheniformis, yeast, Penicillium citrinum, Aspergillus niger, and Aspergillus flavus are shown in Table 2-4.
  • the results of Table 2-4 show that the forsythin derivatives A, B, and C have significant antibacterial effects against Staphylococcus aureus, yeast, Penicillium citrinum, and Aspergillus niger.
  • Forsythiaside derivative A is golden yellow.
  • the MICs of Staphylococcus, Escherichia coli, Bacillus subtilis, yeast, Penicillium citrinum, Aspergillus niger, and Aspergillus flavus are: 0.0234 mg/ml, 0.75 mg/ml, 0.75 mg/ml, 0.0938 mg/ml, 0.0469 mg/ml, 0.0234 mg/ml, 0.0938 mg/ml.
  • the MICs of forsythin derivative B against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, yeast, Penicillium citrinum, Aspergillus niger, and Aspergillus flavus are: 0.0938 mg/ml, 0.75 mg/ml, 0.75 mg/ml, 0.0938 Mg/ml, 0.0938 mg/ml, 0.0938 mg/ml, 0.0469 mg/ml.
  • the MIC of forsythin derivative C against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, yeast, Penicillium citrinum, Aspergillus niger, and Aspergillus flavus is: 0.0469 mg/ml, 1.5 mg/ml, 1.5 mg/ml, 0.1875 Mg/ml, 0.0469 mg/ml, 0.0469 mg/ml, 0.1875 mg/ml.
  • Forsythin/Forsythiamin composition A forsythin (content > 98%), white powder, produced by Dalian Fusheng Natural Medicine Development Co., Ltd., batch number: 20130303; forsythiaside (content > 98%), white Powder, produced by Dalian Fusheng Natural Medicine Development Co., Ltd., batch number: 20130301; the ratio of forsythin to forsythin is 98:2.
  • Forsythin/Forsythiamin B forsythin (>98%), white powder, Dalian Fusheng Natural Medicine Development Co., Ltd., batch number: 20130303; forsythias (content > 98%), white Powder, Dalian Fusheng Natural Medicine Development Co., Ltd. production, batch number: 20130301; the ratio of forsythin to forsythiaside is 89:11.
  • the strains were the same as in Test Example 1, and were provided by the Microbiology Laboratory of Dalian Medical University.
  • Quantitative antibacterial experiments of forsythin were carried out using agar dilution method using M-H agar.
  • sterile tubes were divided into 2 groups, 10 groups in each group were distributed in the tube rack, and 10 mL of sterile distilled water was added to the 2 to 10 tubes in the first group of 10 tubes; Add 10 mL of forsythiaside composition A solution to the first tube; then add 10 mL of forsythin/Forsylin composition A solution to the second tube, mix and mix from the second tube. Add 10 mL of the mixture in the test tube to the third tube, mix well, then add 10 mL of the mixture to the fourth tube. After mixing, take 10 ml of the mixture from the fourth tube and count to the fifth. In the test tube, mix well, and then serially dilute to the 9th tube and mix, remove 10 mL and discard. In the 10th test tube, only sterile distilled water was used, and sterile distilled water in the 10th test tube was used as a control. Thus, 1 to 9 tubes contain 10 mL each of different concentrations of the drug solution.
  • sterile workstation On a sterile workstation, aseptically, 900 mL of solubilized sterile MH agar was dispensed into 10 flasks, 90 mL of molten sterile MH agar per flask, and the first of the first set of tubes. 10 mL of forsythin/forsylin composition A solution contained in a test tube was placed in a first flask containing 90 mL of medium, and mixed and poured onto 7 plates (i.e., plates).
  • Each plate contains approximately 14 mL of drug-containing (ie, forsythin/Forsylin composition A)
  • the lipid which was prepared after coagulation, was the first drug-containing agar plate group, wherein: the first drug-containing agar plate group actually contained forsythin/forsylin composition A 3.0 mg/ml per plate.
  • each plate set contained 7 plates.
  • Each plate contained approximately 14 mL of a drug-containing (ie, forsythin/Forsylin composition A) agar, which was solidified and used.
  • each plate in the second drug-containing agar plate group actually contained forsythin/forsylin composition A 1.5 mg/ml; each plate in the third drug-containing agar plate group actually contained forsythin/forsylin Composition A was 0.75 mg/ml; each plate in the fourth drug-containing agar plate group actually contained forsythin/Forsylin composition A was 0.375 mg/ml; each plate in the fifth drug-containing agar plate group Actually containing forsythin/Forsylin composition A is 0.1875 mg/ml; in the sixth drug-containing agar plate group, each plate actually contains forsythin/Forsylin composition A is 0.0938 mg/ml; Each plate in the drug-containing agar plate group actually contained forsythin/forsylin composition A was 0.0469 mg/ml; in the eighth drug-containing agar plate group, each plate actually contained forsythin/forsylin Composition A was 0.0234 mg
  • the forsythiaside/forsylin composition B-containing agar plate group was prepared according to the preparation method of forsythiaside/forsylin composition A drug-containing agar plate group, except that the second group was added to the second group of triangular flasks. The procedure was the same as in step 3-1) except for the forsythiaside/forsylin composition B in the test tube.
  • the MIC of forsythin/forsylin composition A against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, yeast, Penicillium citrinum, Aspergillus niger, and Aspergillus flavus is: 0.0234 mg/ml, 0.375 mg /ml, 0.375 mg/ml, 0.0469 mg/ml, 0.0234 mg/ml, 0.0234 mg/ml, 0.0469 mg/ml.
  • the MIC of forsythin/forsylin composition B against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, yeast, Penicillium citrinum, Aspergillus niger, and Aspergillus flavus was: 0.0938 mg/ml, 0.75 mg/ml, 0.75 Mg/ml, 0.0938 mg/ml, 0.0938 mg/ml, 0.0938 mg/ml, 0.0469 mg/ml.
  • forsythiaside, forsythin derivatives, forsythin and forsythiaside compositions have the effect of inhibiting bacteria.
  • This test result has important guiding significance for guiding clinical prevention of common pathogenic bacteria infection. .
  • Forsythia saponin (content >98%), white powder, produced by Dalian Fusheng Natural Medicine Development Co., Ltd., batch number: 20130301.
  • Quantitative antibacterial experiments of forsythin were performed using agar dilution method using M-H agar.
  • 900 mL of the solubilized sterile MH agar was dispensed into 10 flasks, 90 mL of molten sterile MH agar per flask, and 10 mL of the first tube was contained.
  • the 0.3% forsypolin sterile distilled aqueous solution was placed in a first flask containing 90 mL of medium, and after mixing, it was poured onto 7 plates (ie, plates), and each plate contained about 14 mL of drug (ie, Forsythiaside) agar, after solidification, was used as the first drug-containing agar plate group, wherein: the first drug-containing agar plate group actually contained forsythin 3.0 mg/ml in each plate.

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Abstract

Provided is a use of forsythin, derivatives thereof and a composition of forsythin/forsythiaside in the preparation of a drug for treating bacterial infections. Experiments have proven that the efficacy of forsythin, forsythin derivatives, the composition of forsythin and forsythiaside for treating bacterial infections is significant, quick, has few toxic side effects, and is a safe, efficient, stable drug with a simple preparation process for treating bacterial infections, is suitable for industrial production, easily popularized, and provides a new drug source for preventing, relieving and treating bacterial infections and the complications thereof.

Description

连翘苷、其衍生物、连翘苷/连翘脂素组合物在制备治疗细菌感染药物中的应用Application of forsythin, its derivative, forsythin/forsylin composition in the preparation of medicine for treating bacterial infection 技术领域Technical field
本发明涉及医药领域，涉及一种治疗细菌感染的药物或保健食品，特别涉及一种中药连翘及连翘叶提取成分在缓解和/或治疗细菌感染药物或保健食品中的应用。The invention relates to the field of medicine, relates to a medicine or health food for treating bacterial infection, in particular to a medicine Chinese medicine forsythia and forsythia leaf extract component for relieving and/or treating bacterial infection medicine or health food.
背景技术Background technique
在全球范围内,抗生素耐药性己成为一个令人担忧的普遍性问题,导致与感染性疾病相关的发病率和死亡率及其治疗费用的不断上升,并且,细菌对新出现的抗菌药物产生耐药性的周期越来越短,已经成为困扰临床医生的一大难题,如何解决抗生素耐药性问题是广大科研工作者和临床医生不可回避的重要课题。细菌对-内酞胺类抗生素耐药是抗生素耐药中最为普遍和重要的耐药类型。目前革兰阴性杆菌中最引人注目的问题是细菌对第三代头抱菌素和新的广谱-内酞胺类抗生素耐药,其主要机制是细菌产超广谱-内酰胺酶(extended spectrum-laetamases,ESBLs)导致细菌对第三代一内酰胺类抗生素和氨曲南耐药。ESBLs是一组由质粒介导,能水解绝大多数头饱菌素、单酞胺类抗生素的酶,大多数EsBLs能被酶抑制剂如他唑巴坦和克拉维酸抑制。ESBLs主要分为SHV、TEM、CTX－M、OXA和其它型。在不同的国家、地区和医院,由于抗生素的使用不同,ESBLs的基因型各不相同,在我国主要是以CTX－M型为主.因而对产CTX－M型ESBLs细菌感染的疾病采用何种药物进行治疗就成了我国目前治疗产ESBLS细菌的难点和重点"Globally, antibiotic resistance has become a worrying universal problem, leading to an increase in morbidity and mortality associated with infectious diseases and the cost of treatment, and the emergence of new antibacterial agents by bacteria. The cycle of drug resistance is getting shorter and shorter, which has become a major problem that plagues clinicians. How to solve the problem of antibiotic resistance is an important issue that most researchers and clinicians cannot avoid. Bacterial resistance to indoleamine antibiotics is the most common and important type of resistance to antibiotic resistance. At present, the most striking problem in Gram-negative bacilli is that bacteria are resistant to the third-generation cephalosporin and the new broad-spectrum-indolamine antibiotics. The main mechanism is the production of extended-spectrum-lactamase by bacteria. Extended spectrum-laetamases, ESBLs) cause bacteria to be resistant to third-generation monolactam antibiotics and aztreonam. ESBLs are a group of enzymes that are mediated by plasmids that hydrolyze most of the cephalosporin and monoamine antibiotics. Most EsBLs are inhibited by enzyme inhibitors such as tazobactam and clavulanic acid. ESBLs are mainly divided into SHV, TEM, CTX-M, OXA and other types. In different countries, regions and hospitals, the genotypes of ESBLs are different due to different use of antibiotics. In China, CTX-M is the main type. Therefore, what kind of diseases are used for the bacterial infection of CTX-M ESBLs? The treatment of drugs has become a difficult point and key point in the treatment of ESBLS-producing bacteria in China.
连翘是中药临床常用传统药物之一，应用广泛，用量巨大，它具有清热"解毒"消肿散结"清除自由基的功能，临床上常与其他的中草药制成配方，用于各类细菌感染治疗。但是，其中具体活性成分尚不清楚，发明者经过艰苦探索，制备提取了其中活性单体化合物连翘苷，并经化学合成，制备了相应的衍生物，进行了一系列的药理活性研究，发现连翘苷、连翘苷衍生物、连翘苷与连翘脂素组合物具有抑制细菌感染，增强人体免疫功能活性，为细菌感染患者开辟了新的治疗途经。 Forsythia is one of the traditional traditional medicines used in traditional Chinese medicine. It is widely used and has a large dosage. It has the function of clearing away heat and detoxifying and dissipating free radicals. It is often formulated with other Chinese herbal medicines for various bacteria. Infection treatment. However, the specific active ingredients are still unclear. The inventors have arduously explored and prepared the active monomer compound forsythin, and chemically synthesized to prepare corresponding derivatives, and carried out a series of pharmacological activities. Studies have found that forsythin, forsythin derivatives, forsythin and forsythiaside compositions have the effect of inhibiting bacterial infections and enhancing the immune function of humans, opening up new therapeutic pathways for patients with bacterial infections.
Figure PCTCN2016079843-appb-000001
Figure PCTCN2016079843-appb-000001
本发明人通过大量的现代科学研究，采用先进的分离纯化技术从连翘及连翘叶中提取其治疗细菌感染的有效成分连翘苷，并经进一步研究合成制备了连翘苷衍生物。可以为细菌感染患者提供一种高效低毒的药物。The inventors have extracted the forsythin which is an effective component for treating bacterial infection from forsythia and forsythia leaves through a large number of modern scientific researches, and advanced synthesis and purification techniques, and further studied and synthesized forsythin derivatives. It can provide a highly effective and low-toxic drug for patients with bacterial infection.
发明内容Summary of the invention
本发明的首要目的是针对现有治疗细菌感染的药物或保健品在应用过程中存在的技术问题，提供连翘苷、连翘苷衍生物、连翘苷与连翘脂素组合物(即连翘苷/连翘脂素)预防、缓解或/和治疗细菌感染所导致的疾病的性能和功效，并提供连翘苷、连翘苷衍生物、连翘苷与连翘脂素组合物新的药用用途，即在治疗、调理、预防和缓解细菌感染的药物或保健食品中的新应用。The primary object of the present invention is to provide forsythiaside, forsythin derivatives, forsythin and forsythiaside compositions (ie, even for the technical problems existing in the application of drugs or health care products for treating bacterial infections. The properties and efficacy of a disease caused by a bacterial infection by preventing, alleviating or/and treating a disease caused by a bacterial infection, and providing a new combination of forsythin, forsythiaside derivative, forsythin and forsythiaside Medicinal use, ie new applications in the treatment, conditioning, prevention and alleviation of bacterial infections or health foods.
为实现上述目的，本发明一方面提供一种连翘苷、连翘苷衍生物、连翘苷与连翘脂素组合物在制备预防或/和治疗细菌感染所致疾病的药物或保健品中的应用。In order to achieve the above object, an aspect of the present invention provides a forsythiaside, forsythin derivative, forsythin and forsythiaside composition for preparing a medicament or a health care product for preventing or/or treating a disease caused by a bacterial infection. Applications.
在筛选具有预防、缓解、治疗细菌感染作用的天然活性成分的过程中，发明人发现连翘的化学成分中连翘苷、连翘苷衍生物、连翘脂素具有强烈的抑制细菌感染的作用。 In the process of screening natural active ingredients for preventing, alleviating and treating bacterial infections, the inventors found that forsythiaside, forsythin derivatives and forsythin have strong inhibitory effects on bacterial infections in the chemical constituents of Forsythia suspensa. .
其中，所述药物或保健品由连翘苷、连翘苷衍生物或连翘苷与连翘脂素组合物和药学上可接受的载体组成。Wherein the medicament or health care product consists of forsythin, forsythin derivative or forsythin and forsythiaside composition and a pharmaceutically acceptable carrier.
特别是，所述连翘苷衍生物选择连翘脂素葡萄糖醛酸衍生物。In particular, the forsythiaside derivative is selected from the forsythiaside glucuronic acid derivative.
特别是，所述连翘苷衍生物包括33-羟基-连翘脂素葡萄糖醛酸苷(33-Hydroxy phillygenin-8-O-β-D-glucuronide)、9-羟基-连翘脂素葡萄糖醛酸苷(9-Hydroxy phillygenin-8-O-β-D-glucuronide)、33，34-亚甲基二氧-连翘脂素葡萄糖醛酸苷(33,34-Methylenedioxy phillygenin-8-O-β-D-glucuronide)、连翘脂素葡萄糖醛酸甲酯((2R,3R,4R,5S)-methyl6-(5-((1R,4S)-4-(3,4-dimethoxyphenyl)hexahydrofuro[3,4-c]furan-1-yl)-2-methoxyphenoxy)-3,4,5-trihydroxytetra hydro-2H-pyran-2-carboxylate)、连翘脂素葡萄糖醛酸钠(sodium(2R,3R,4R,5S)-6-(5-((1R,4S)-4-(3,4-dimethoxyphenyl)hexahydrofuro[3,4-c]furan-1-yl)-2-methoxyphenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylate)、连翘脂素葡萄糖醛酸钾(potassium(2R,3R,4R,5S)-6-(5-((1R,4S)-4-(3,4-dimethoxyphenyl)hexahydrofuro[3,4-c]furan-1-yl)-2-methoxyphenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylate)、连翘脂素葡萄糖醛酸((2R,3R,4R,5S)-6-(5-((1R,4S)-4-(3,4-dimethoxyphenyl)hexahydrofuro[3,4-c]furan-1-yl)-2-methoxyphenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid)；优选为33-羟基-连翘脂素葡萄糖醛酸苷、9-羟基-连翘脂素葡萄糖醛酸苷、33，34-亚甲基二氧-连翘脂素葡萄糖醛酸苷、连翘脂素葡萄糖醛酸甲酯、连翘脂素葡萄糖醛酸钠或连翘脂素葡萄糖醛酸钾。In particular, the forsythin derivative comprises 33-hydroxy-philoxyphilin-8-O-β-D-glucuronide, 9-hydroxy-Forsylin glucagon Acid glucoside (9-Hydroxy phillygenin-8-O-β-D-glucuronide), 33,34-methylenedioxy-Forsythine glucuronide (33,34-Methylenedioxy phillygenin-8-O-β -D-glucuronide), forsythiaside methyl glucuronide ((2R,3R,4R,5S)-methyl6-(5-((1R,4S)-4-(3,4-dimethoxyphenyl)hexahydrofuro[3 , 4-c]furan-1-yl)-2-methoxyphenoxy)-3,4,5-trihydroxytetra hydro-2H-pyran-2-carboxylate), forsythiaside sodium glucuronate (sodium(2R,3R, 4R,5S)-6-(5-((1R,4S)-4-(3,4-dimethoxyphenyl)hexahydrofuro[3,4-c]furan-1-yl)-2-methoxyphenoxy)-3,4, 5-trihydroxytetrahydro-2H-pyran-2-carboxylate), forsythiaside potassium gluconate (potassium(2R,3R,4R,5S)-6-(5-((1R,4S)-4-(3, 4-dimethoxyphenyl)hexahydrofuro[3,4-c]furan-1-yl)-2-methoxyphenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylate), forsythiaside glucuronic acid (( 2R,3R,4R,5S)-6-(5-((1R,4S)-4-(3,4-dimethoxyphenyl)hexahydro Furo[3,4-c]furan-1-yl)-2-methoxyphenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid); preferably 33-hydroxy-Forsythine Acid glycoside, 9-hydroxy-Forsyric acid glucuronide, 33,34-methylenedioxy-forsylin glucuronide, forsythiaside methyl glucuronide, forsythiaside glucose Sodium aldate or forsythiaside potassium glucuronide.
特别是，所述连翘苷与连翘脂素组合物中连翘苷与连翘脂素的重量份配比为2～98：2～98；优选为2-10:90-98；进一步优选为80:20或20:80，进一步优选为90:10或10:90，更进一步优选为98:2或2:98；再进一步优选为98:2。In particular, the weight ratio of forsythin to forsythiaside in the forsythiaside and forsythiaside composition is from 2 to 98:2 to 98; preferably from 2 to 10:90 to 98; further preferably It is 80:20 or 20:80, further preferably 90:10 or 10:90, still more preferably 98:2 or 2:98; still more preferably 98:2.
其中，所述的连翘苷、连翘苷衍生物的含量≥1％，优选为≥30％，进一步优选为≥60％，更进一步优选为≥80％，再更进一步优选为≥98％；连翘苷与连翘脂素组合物含量≥1％，优选为≥30％，进一步优选为≥60％，更进一步优选为≥80％，再更进一步优选为≥98％。Wherein, the content of the forsythin, forsythiaside derivative is ≥1%, preferably ≥30%, further preferably ≥60%, still more preferably ≥80%, still more preferably ≥98%; The content of forsythin and forsythiaside composition is ≥1%, preferably ≥30%, further preferably ≥60%, still more preferably ≥80%, still more preferably ≥98%.
特别是，所述的连翘苷、连翘苷衍生物含量为1％～98％；优选为30～80％；连翘苷与连翘脂素组合物含量为1％～98％；优选为30～80％。In particular, the content of forsythin and forsythin derivatives is from 1% to 98%; preferably from 30 to 80%; and the content of forsythin and forsythiaside compositions is from 1% to 98%; preferably 30 to 80%.
特别是，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 Pharmaceutical Association出版，Washington and The Pharmaceutical Press,London,1994)等工具书中找到。 In particular, 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 Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994) found in the reference book.
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；In particular, 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, flavoring agents, spices, etc.;
其中，所述药物以片剂、胶囊剂、丸剂、散剂、颗粒剂、糖浆剂形式存在。Wherein, the medicament is in the form of a tablet, a capsule, a pill, a powder, a granule, or a syrup.
特别是，所述的连翘苷、连翘苷衍生物含量≥1％，优选为≥30％，进一步优选为≥60％，更进一步优选为≥80％，再更进一步优选为≥98％；连翘苷与连翘脂素组合物含量≥1％，优选为≥30％，进一步优选为≥60％，更进一步优选为≥80％，再更进一步优选为≥98％。In particular, the content of forsythin and forsythin derivatives is ≥1%, preferably ≥30%, further preferably ≥60%, still more preferably ≥80%, still more preferably ≥98%; The content of forsythin and forsythiaside composition is ≥1%, preferably ≥30%, further preferably ≥60%, still more preferably ≥80%, still more preferably ≥98%.
本发明另一方面提供一种含有连翘苷、连翘苷衍生物或连翘苷与连翘脂素组合物的预防、缓解和/或治疗细菌感染导致的疾病的药物或保健品。Another aspect of the present invention provides a medicament or health care product comprising forsythiaside, forsythiaside derivative or forsythin and forsythiaside composition for preventing, alleviating and/or treating a disease caused by a bacterial infection.
其中，所述连翘苷衍生物选择连翘脂素葡萄糖醛酸衍生物。Wherein the forsythiaside derivative is selected from the forsythiaglycine glucuronic acid derivative.
特别是，所述连翘苷衍生物包括33-羟基-连翘脂素葡萄糖醛酸苷、9-羟基-连翘脂素葡萄糖醛酸苷、33，34-亚甲基二氧-连翘脂素葡萄糖醛酸苷、连翘脂素葡萄糖醛酸甲酯、连翘脂素葡萄糖醛酸钠、连翘脂素葡萄糖醛酸钾、连翘脂素葡萄糖醛酸；优选为33-羟基-连翘脂素葡萄糖醛酸苷、9-羟基-连翘脂素葡萄糖醛酸苷、33，34-亚甲基二氧-连翘脂素葡萄糖醛酸苷、连翘脂素葡萄糖醛酸甲酯、连翘脂素葡萄糖醛酸钠或连翘脂素葡萄糖醛酸钾。In particular, the forsythin derivative comprises 33-hydroxy-Forsylin glucuronide, 9-hydroxy-Forsylin glucuronide, 33,34-methylenedioxy-linked sulfonate Glucuronide, forsythiaside methyl glucuronate, forsythiaside sodium glucuronate, forsythias glucuronide, forsythiaglycine glucuronic acid; preferably 33-hydroxy-forsythia Lipid glucuronide, 9-hydroxy-Forsyric acid glucuronide, 33,34-methylenedioxy-Forsythine glucuronide, forsythias glucuronide methyl ester, even Sodium lipoate or sodium forsylate. Glucuronide.
其中，所述连翘苷与连翘脂素组合物中连翘苷与连翘脂素的重量份配比为2～98：2～98；优选为2-10:90-98；进一步优选为80:20或20:80，进一步优选为90:10或10:90，更进一步优选为98:2或2:98；再进一步优选为98:2。Wherein the weight ratio of forsythin to forsythin in the forsythiaside and forsythiaside composition is from 2 to 98:2 to 98; preferably from 2 to 10:90 to 98; further preferably 80:20 or 20:80, further preferably 90:10 or 10:90, still more preferably 98:2 or 2:98; still more preferably 98:2.
特别是，所述的连翘苷、连翘苷衍生物的含量≥1％，优选为≥30％，进一步优选为≥60％，更进一步优选为≥80％，再更进一步优选为≥98％；连翘苷与连翘脂素组合物含量≥1％，优选为≥30％，进一步优选为≥60％，更进一步优选为≥80％，再更进一步优选为≥98％。In particular, the content of the forsythin or forsythin derivative is ≥1%, preferably ≥30%, further preferably ≥60%, still more preferably ≥80%, still more preferably ≥98% The content of forsythin and forsythiaside composition is ≥1%, preferably ≥30%, further preferably ≥60%, still more preferably ≥80%, still more preferably ≥98%.
尤其是，所述的连翘苷、连翘苷衍生物的含量≥1％，优选为1％～98％；优选为30～80％，更进一步优选为60％；连翘苷与连翘脂素组合物含量≥1％，优选为1％～98％；优选为30～80％，更进一步优选为60％。In particular, the content of the forsythin or forsythin derivative is ≥1%, preferably from 1% to 98%; preferably from 30% to 80%, still more preferably 60%; forsythin and forsythiaside The composition of the composition is ≥ 1%, preferably 1% to 98%; preferably 30 to 80%, and still more preferably 60%.
特别是，所述连翘苷、连翘苷衍生物或连翘苷与连翘脂素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。In particular, the ratio of the weight of the forsythin, the forsythin derivative or the forsythin to the forsythiaside 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 1 to 10:100.
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。Wherein 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.
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。In particular, the forsythiaside-forsylin-cyclodextrin composition selects forsythin and forsythin with alpha-, beta- or gamma-cyclodextrin a mixture of or a mixture thereof, or a complex formed by the treatment of forsythiaside and forsythin with α-, β- or γ-cyclodextrin or a derivative thereof by physical or chemical treatment.
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。Wherein the 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.
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-环糊精、2,6-二甲基-环糊精、2,3,6-三甲基-环糊精、2,6-二乙基-环糊精、2,3,6-三乙基-环糊精、麦芽糖基-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。In particular, the cyclodextrin is α- or β-, γ-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 or sulfobutylether β - cyclodextrin, p-TsCl substituted β-cyclodextrin, 6-substituted β-CD p-toluenesulfonate (β-cyclodextrin-6-OTs) , 2-oxohydroxypropyl-β-cyclodextrin, 2-position monosubstituted p-toluenesulfonate (β-cyclodextrin-2-OTs), β-cyclodextrin p-toluenesulfonic acid Starch macromolecule PCL-(Tos)7-β-CD of ester (Tosyl-β-CD), β-cyclodextrin.
特别是，所述药物或保健品中还包括穿心莲提取物，四季青提取物，金银花提取物，板蓝根提取物，蒲公英提取物，黄柏提取物，黄连提取物，黄芩提取物，马齿苋提取物，白头翁提取物，大蒜提取物，鱼腥草提取物，野菊花提取物，苦参提取物、维生素C及其衍生物或维生素E及其衍生物。In particular, the medicine or health care product further comprises Andrographis paniculata extract, Silybum extract, Honeysuckle extract, Radix Isatidis extract, dandelion extract, Phellodendron extract, Rhizoma Coptidis extract, Astragalus membranaceus extract, Portulaca oleracea extract , Pulsatilla extract, garlic extract, Houttuynia cordata extract, wild chrysanthemum extract, Sophora flavescens extract, vitamin C and its derivatives or vitamin E and its derivatives.
与现有技术相比，本发明具有如下的明显优点：Compared with the prior art, the present invention has the following distinct advantages:
1、本发明对已知化合物连翘苷、连翘苷衍生物、连翘苷/连翘脂素组合物发掘了新的药用价值，将其用于预防、缓解、治疗细菌感染，并可制备成预防、缓解和/或治疗细菌感染的药物或保健食品，从而为连翘及连翘叶药材的应用开拓了一个新的领域。1. The present invention excavates new medicinal value for known compounds of forsythin, forsythin derivatives, forsythin/forsylin composition, and is used for preventing, alleviating and treating bacterial infections, and A drug or health food prepared to prevent, alleviate and/or treat bacterial infections, thereby opening up a new field for the application of Forsythia and Forsythia suspensa.
2、本发明的系列试验研究证明已知化合物连翘苷、连翘苷衍生物、连翘苷/连翘脂素组合物具有显著的缓解、治疗细菌感染的功效。2. A series of experimental studies of the present invention demonstrate that the known compounds fortuitin, forsythiaside derivatives, and forsythin/forsylin compositions have significant efficacy in relieving and treating bacterial infections.
3、本发明的已知化合物连翘苷、连翘苷衍生物、连翘苷/连翘脂素组合物药理作用强，用于缓解、预防、治疗细菌感染的功效显著，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。3. The known compounds of the present invention, forsythin, forsythin derivatives, forsythin/forsylin composition have strong pharmacological action, and have remarkable effects for relieving, preventing and treating bacterial infections, and have quick effects and side effects. Small, safe, long-term, with good medicinal prospects.
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。4. 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.
5、本发明既可采用单一成分的连翘苷、连翘苷衍生物或连翘苷/连翘脂素组合物活性成分制备预防、缓解、治疗细菌感染所致疾病的药物，又可采用连翘苷、连翘苷衍生物或连翘苷/连翘脂素组合物与其它活性成分(例如与穿心莲提取物，四季青提取物，金银花提取物，板蓝根提取物，蒲公英提取物，黄柏提取物，黄连提取物，黄芩提取物，马齿苋提取物，白头翁提取物，大蒜提取物，鱼腥草提取物，野菊花提取物，苦参提取物、维生素C及其衍生 物或维生素E及其衍生物)共同组方，制备预防、治疗细菌感染的复方药物。5. The invention can be used for preparing a medicament for preventing, alleviating and treating diseases caused by bacterial infection by using a single component of forsythin, forsythin derivative or forsythin/forsylin composition active ingredient, and Osmotic glycosides, forsythin derivatives or forsythin/forsylin compositions and other active ingredients (eg with andrographis extract, cyclaea extract, honeysuckle extract, Radix isatidis extract, dandelion extract, cork extract) , Rhizoma Coptidis extract, Astragalus membranaceus extract, Portulaca oleracea extract, Pulsatilla extract, Garlic extract, Houttuynia cordata extract, Wild chrysanthemum extract, Sophora flavescens extract, Vitamin C and its derivatives The compound or vitamin E and its derivatives are combined to prepare a compound medicine for preventing and treating bacterial infection.
具体实施方式detailed description
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，这些实施例包括了本发明的连翘苷、连翘苷衍生物的胶囊、片剂、软胶囊剂的药效学试验。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。The beneficial effects of the formulations of the present invention are further described below by way of specific embodiments which include pharmacodynamic tests of forsythin, forsythiaside derivative capsules, tablets, and soft capsules of the present invention. These examples are merely illustrative and are not intended to limit the scope of the invention in any way. It should be understood that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the invention, and such modifications and substitutions fall within the scope of the present invention.
本发明中所述连翘苷衍生物33-羟基-连翘脂素葡萄糖醛酸苷、9-羟基-连翘脂素葡萄糖醛酸苷、33，34-亚甲基二氧-连翘脂素葡萄糖醛酸苷与专利申请(申请号：201510319303.4，优先权号：201510164294.6)中所述连翘苷衍生物相同；连翘脂素葡萄糖醛酸甲酯、连翘脂素葡萄糖醛酸钠、连翘脂素葡萄糖醛酸钾、连翘脂素葡萄糖醛酸与专利申请(申请号：201510320579.4，优先权号：201410825547.5)中的相同。The forsythin derivative 33-hydroxy-forsylin glucuronide, 9-hydroxy-forsylin glucuronide, 33,34-methylenedioxy-forsylin in the present invention Glucuronide is the same as the forsythiaside derivative described in the patent application (Application No.: 201510319303.4, Priority No.: 201510164294.6); forsythiaside methyl glucuronide, forsythiaside sodium glucuronate, forsythia The fatty acid potassium gluconate, forsythiaside glucuronic acid is the same as in the patent application (Application No.: 201510320579.4, priority number: 201410825547.5).
实施例1 连翘苷片剂Example 1 Forsythin tablets
1、按照如下配比准备原料1. Prepare raw materials according to the following ratio
Figure PCTCN2016079843-appb-000002
Figure PCTCN2016079843-appb-000002
2、将连翘苷和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。2. The forsythin and the starch are uniformly mixed to form granules, and the talc powder and magnesium stearate are added and uniformly mixed, and then pressed into 10,000 pieces.
实施例2 连翘苷胶囊剂Example 2 Forsythin capsules
1、按照如下配比准备原料1. Prepare raw materials according to the following ratio
连翘苷(纯度94％)             200gForsythin (purity 94%) 200g
淀粉                         1000gStarch 1000g
2、将连翘苷和淀粉混合均匀后装胶囊，制成10000粒。2. The forsythin and the starch are uniformly mixed and then filled into capsules to make 10,000 tablets.
实施例3 连翘苷胶囊剂Example 3 Forsythin capsules
1、按照如下配比准备原料1. Prepare raw materials according to the following ratio
连翘苷(纯度98％)             500gForsythin (purity 98%) 500g
淀粉                         1000gStarch 1000g
2、将连翘苷和淀粉混合均匀后装胶囊，制成10000粒。 2. The forsythin and the starch are uniformly mixed and then filled into capsules to make 10,000 tablets.
实施例4 连翘苷片剂Example 4 Forsythin tablets
1、按照如下配比准备原料1. Prepare raw materials according to the following ratio
Figure PCTCN2016079843-appb-000003
Figure PCTCN2016079843-appb-000003
2、将连翘苷、穿心莲提取物、维生素C和淀粉混合均匀后，制成颗粒，加入滑石粉和硬脂酸镁混合均匀后压制成10000片。2. After mixing forsythin, andrographolide extract, vitamin C and starch, it is made into granules, and talc powder and magnesium stearate are added and uniformly mixed, and then pressed into 10,000 tablets.
实施例5 连翘苷胶囊剂Example 5 Forsythin capsules
1、按照如下配比准备原料1. Prepare raw materials according to the following ratio
Figure PCTCN2016079843-appb-000004
Figure PCTCN2016079843-appb-000004
2、将连翘苷、金银花提取物、维生素C和淀粉混合均匀后装胶囊，制成10000粒。2. The forsythin, honeysuckle extract, vitamin C and starch are uniformly mixed and then filled into capsules to make 10,000 tablets.
实施例6 连翘苷颗粒剂Example 6 Forsythia granules
1、按照如下配比准备原料1. Prepare raw materials according to the following ratio
Figure PCTCN2016079843-appb-000005
Figure PCTCN2016079843-appb-000005
2、将连翘苷、黄芩提取物、维生素C和蔗糖粉混合均匀制成颗粒后装袋，制成10000袋。2. Mixing forsythin, astragalus extract, vitamin C and sucrose powder into granules and then bagging them into 10,000 bags.
实施例7 连翘苷口服液Example 7 Forsythin Oral Solution
1、按照如下配比准备原料1. Prepare raw materials according to the following ratio
Figure PCTCN2016079843-appb-000006
Figure PCTCN2016079843-appb-000006
Figure PCTCN2016079843-appb-000007
Figure PCTCN2016079843-appb-000007
2、取连翘苷、蒲公英提取物、马齿苋提取物，用乙醇溶解后加入葡萄糖糖浆，最终加去离子水至100ml，即得。2, taking forsythin, dandelion extract, purslane extract, dissolved in ethanol, add glucose syrup, and finally add deionized water to 100ml, that is.
实施例8 33-羟基-连翘脂素葡萄糖醛酸苷片剂Example 8 33-Hydroxy-Forsyricin Glucuronide Tablets
1、按照如下配比准备原料1. Prepare raw materials according to the following ratio
Figure PCTCN2016079843-appb-000008
Figure PCTCN2016079843-appb-000008
2、将33-羟基-连翘脂素葡萄糖醛酸苷和淀粉混合均匀后，制成颗粒，加入滑石粉和硬脂酸镁混合均匀后压制成10000片。2. The 33-hydroxy-Forsysin glucuronide and the starch were uniformly mixed, and then granulated. The talc powder and magnesium stearate were added and uniformly mixed, and then pressed into 10,000 tablets.
实施例9 33-羟基-连翘脂素葡萄糖醛酸苷胶囊剂Example 9 33-Hydroxy-Forsythine Glucuronide Capsules
1、按照如下配比准备原料1. Prepare raw materials according to the following ratio
33-羟基-连翘脂素葡萄糖醛酸苷(纯度60％)   200g33-hydroxy-Forsyricin Glucuronide (60% purity) 200g
淀粉                                     1000gStarch 1000g
2、将33-羟基-连翘脂素葡萄糖醛酸苷和淀粉混合均匀后装胶囊，制成10000粒。2. The 33-hydroxy-Forsythine glucuronide and starch were uniformly mixed and then filled into capsules to prepare 10,000 tablets.
实施例10 33-羟基-连翘脂素葡萄糖醛酸苷胶囊剂Example 10 33-Hydroxy-Forsythine Glucuronide Capsules
1、按照如下配比准备原料1. Prepare raw materials according to the following ratio
33-羟基-连翘脂素葡萄糖醛酸苷(纯度98％)   500g33-hydroxy-Forsyricin Glucuronide (purity 98%) 500g
淀粉                                     1000gStarch 1000g
2、将33-羟基-连翘脂素葡萄糖醛酸苷和淀粉混合均匀后装胶囊，制成10000粒。2. The 33-hydroxy-Forsythine glucuronide and starch were uniformly mixed and then filled into capsules to prepare 10,000 tablets.
实施例11 33-羟基-连翘脂素葡萄糖醛酸苷片剂Example 11 33-Hydroxy-Forsyricin Glucuronide Tablets
1、按照如下配比准备原料1. Prepare raw materials according to the following ratio
Figure PCTCN2016079843-appb-000009
Figure PCTCN2016079843-appb-000009
2、将33-羟基-连翘脂素葡萄糖醛酸苷、金银花提取物、维生素C和淀粉混合均匀后制粒， 加入滑石粉和硬脂酸镁混匀后压制成10000片。2. Mix 33-hydroxy-Forsythin glucuronide, honeysuckle extract, vitamin C and starch, and then granulate. The talc powder and magnesium stearate were added and mixed, and then pressed into 10,000 pieces.
实施例12 33-羟基-连翘脂素葡萄糖醛酸苷胶囊剂Example 12 33-Hydroxy-Forsythine Glucuronide Capsules
1、按照如下配比准备原料1. Prepare raw materials according to the following ratio
Figure PCTCN2016079843-appb-000010
Figure PCTCN2016079843-appb-000010
2、将33-羟基-连翘脂素葡萄糖醛酸苷、板蓝根提取物、维生素C和淀粉混合均匀后装胶囊，制成10000粒。2. The 33-hydroxy-Forsythine glucuronide, Radix Isatidis extract, vitamin C and starch were uniformly mixed and then filled into capsules to make 10,000 tablets.
实施例13 33-羟基-连翘脂素葡萄糖醛酸苷颗粒剂Example 13 33-Hydroxy-Forsythine Glucuronide Granules
1、按照如下配比准备原料1. Prepare raw materials according to the following ratio
Figure PCTCN2016079843-appb-000011
Figure PCTCN2016079843-appb-000011
2、将33-羟基-连翘脂素葡萄糖醛酸苷、白头翁提取物、维生素C和蔗糖粉混合均匀制成颗粒后装袋，制成10000袋。2. The 33-hydroxy-Forsythine glucuronide, the Pulsatilla extract, the vitamin C and the sucrose powder are uniformly mixed into a granule and then bagged to make 10,000 bags.
实施例14 33-羟基-连翘脂素葡萄糖醛酸苷口服液Example 14 33-Hydroxy-Forsythine Glucuronide Oral Solution
1、按照如下配比准备原料1. Prepare raw materials according to the following ratio
Figure PCTCN2016079843-appb-000012
Figure PCTCN2016079843-appb-000012
2、取33-羟基-连翘脂素葡萄糖醛酸苷、白头翁提取物、金银花提取物，用乙醇溶解后加入葡萄糖糖浆，最终加去离子水至100ml，即得。2, taking 33-hydroxy-Forsysin glucuronide, Pulsatilla extract, honeysuckle extract, dissolved in ethanol, add glucose syrup, and finally add deionized water to 100ml, that is.
实施例15 9-羟基-连翘脂素葡萄糖醛酸苷片剂Example 15 9-Hydroxy-Forsyricin Glucuronide Tablets
1、按照如下配比准备原料1. Prepare raw materials according to the following ratio
Figure PCTCN2016079843-appb-000013
Figure PCTCN2016079843-appb-000013
Figure PCTCN2016079843-appb-000014
Figure PCTCN2016079843-appb-000014
2、将9-羟基-连翘脂素葡萄糖醛酸苷和淀粉混合均匀后，制成颗粒，加入滑石粉和硬脂酸镁混合均匀后压制成10000片。2. The 9-hydroxy-Forsysin glucuronide and the starch were uniformly mixed, and then granulated. The talc powder and magnesium stearate were added and uniformly mixed, and then pressed into 10,000 tablets.
实施例16 9-羟基-连翘脂素葡萄糖醛酸苷胶囊剂Example 16 9-Hydroxy-Forsythin Glucuronide Capsules
1、按照如下配比准备原料1. Prepare raw materials according to the following ratio
9-羟基-连翘脂素葡萄糖醛酸苷(纯度97％)   200g9-hydroxy-Forsylin, glucuronide (purity 97%) 200g
淀粉                           1000gStarch 1000g
2、将9-羟基-连翘脂素葡萄糖醛酸苷和淀粉混合均匀后装胶囊，制成10000粒。2. The 9-hydroxy-Forsysin glucuronide and starch are uniformly mixed and then encapsulated to make 10,000 tablets.
实施例17 9-羟基-连翘脂素葡萄糖醛酸苷胶囊剂Example 17 9-Hydroxy-Forsythine Glucuronide Capsules
1、按照如下配比准备原料1. Prepare raw materials according to the following ratio
9-羟基-连翘脂素葡萄糖醛酸苷(纯度98％)   500g9-hydroxy-Forsythin Glucuronide (purity 98%) 500g
淀粉                                1000gStarch 1000g
2、将9-羟基-连翘脂素葡萄糖醛酸苷和淀粉混合均匀后装胶囊，制成10000粒。2. The 9-hydroxy-Forsysin glucuronide and starch are uniformly mixed and then encapsulated to make 10,000 tablets.
实施例18 9-羟基-连翘脂素葡萄糖醛酸苷片剂Example 18 9-Hydroxy-Forsythine Glucuronide Tablets
1、按照如下配比准备原料1. Prepare raw materials according to the following ratio
Figure PCTCN2016079843-appb-000015
Figure PCTCN2016079843-appb-000015
2、将9-羟基-连翘脂素葡萄糖醛酸苷、金银花提取物、维生素C和淀粉混合均匀后制粒，加入滑石粉和硬脂酸镁混匀后压制成10000片。2. The 9-hydroxy-Forsysin glucuronide, honeysuckle extract, vitamin C and starch were uniformly mixed and granulated, and talc powder and magnesium stearate were added and mixed, and then pressed into 10,000 tablets.
实施例19 9-羟基-连翘脂素葡萄糖醛酸苷胶囊剂Example 19 9-Hydroxy-Forsythine Glucuronide Capsules
1、按照如下配比准备原料1. Prepare raw materials according to the following ratio
Figure PCTCN2016079843-appb-000016
Figure PCTCN2016079843-appb-000016
2、将9-羟基-连翘脂素葡萄糖醛酸苷、板蓝根提取物、维生素C和淀粉混合均匀后装胶囊，制成10000粒。2. The 9-hydroxy-Forsythine glucuronide, Radix Isatidis extract, vitamin C and starch were uniformly mixed and then filled into capsules to make 10,000 tablets.
实施例20 9-羟基-连翘脂素葡萄糖醛酸苷颗粒剂Example 20 9-Hydroxy-Forsythine Glucuronide Granules
1、按照如下配比准备原料1. Prepare raw materials according to the following ratio
Figure PCTCN2016079843-appb-000017
Figure PCTCN2016079843-appb-000017
2、将9-羟基-连翘脂素葡萄糖醛酸苷、白头翁提取物、维生素C和蔗糖粉混合均匀制成颗粒后装袋，制成10000袋。2. The 9-hydroxy-Forsysin glucuronide, the Pulsatilla extract, the vitamin C and the sucrose powder are uniformly mixed into granules and then bagged to make 10,000 bags.
实施例21 9-羟基-连翘脂素葡萄糖醛酸苷口服液Example 21 9-Hydroxy-Forsythine Glucuronide Oral Solution
1、按照如下配比准备原料1. Prepare raw materials according to the following ratio
Figure PCTCN2016079843-appb-000018
Figure PCTCN2016079843-appb-000018
2、取9-羟基-连翘脂素葡萄糖醛酸苷、白头翁提取物、鱼腥草提取物，用乙醇溶解后加入葡萄糖糖浆，最终加去离子水至100ml，即得。2. Take 9-hydroxy-Forsysin glucuronide, Pulsatilla extract, Houttuynia cordata extract, dissolve it with ethanol, add glucose syrup, and finally add deionized water to 100ml.
实施例22 33，34-亚甲基二氧-连翘脂素葡萄糖醛酸苷片剂Example 22 33,34-Methylenedioxy-Forsythine Glucuronide Tablets
1、按照如下配比准备原料1. Prepare raw materials according to the following ratio
Figure PCTCN2016079843-appb-000019
Figure PCTCN2016079843-appb-000019
2、将33，34-亚甲基二氧-连翘脂素葡萄糖醛酸苷和淀粉混合均匀后，制成颗粒，加入滑石粉和硬脂酸镁混合均匀后压制成10000片。2. After mixing 33,34-methylenedioxy-docasanthin glucuronide and starch, the mixture was granulated, and talc powder and magnesium stearate were added to be uniformly mixed, and then pressed into 10,000 tablets.
实施例23 33，34-亚甲基二氧-连翘脂素葡萄糖醛酸苷胶囊剂Example 23 33,34-Methylenedioxy-Forsythine Glucuronide Capsules
1、按照如下配比准备原料1. Prepare raw materials according to the following ratio
33，34-亚甲基二氧-连翘脂素葡萄糖醛酸苷(纯度97％)   200g 33,34-Methylenedioxy-Forsythin Glucuronide (purity 97%) 200g
淀粉                          1000gStarch 1000g
2、将33,34-亚甲基二氧-连翘脂素葡萄糖醛酸苷和淀粉混合均匀后装胶囊，制成10000粒。2. The 33,34-methylenedioxy- forsyric acid glucuronide and the starch were uniformly mixed and then filled into capsules to prepare 10,000 tablets.
实施例24 33，34-亚甲基二氧-连翘脂素葡萄糖醛酸苷胶囊剂Example 24 33,34-Methylenedioxy-Forsythine Glucuronide Capsules
1、按照如下配比准备原料1. Prepare raw materials according to the following ratio
33，34-亚甲基二氧-连翘脂素葡萄糖醛酸苷(纯度98％)   500g33,34-Methylenedioxy-Forsythine Glucuronide (purity 98%) 500g
淀粉                           1000gStarch 1000g
2、将33，34-亚甲基二氧-连翘脂素葡萄糖醛酸苷和淀粉混合均匀后装胶囊，制成10000粒。2. The 33,34-methylenedioxy- forsyric acid glucuronide and the starch were uniformly mixed and then filled into capsules to prepare 10,000 tablets.
实施例25 33，34-亚甲基二氧-连翘脂素葡萄糖醛酸苷片剂Example 25 33,34-Methylenedioxy-Forsythine Glucuronide Tablets
1、按照如下配比准备原料1. Prepare raw materials according to the following ratio
Figure PCTCN2016079843-appb-000020
Figure PCTCN2016079843-appb-000020
2、将33，34-亚甲基二氧-连翘脂素葡萄糖醛酸苷、鱼腥草提取物、维生素C和淀粉混合均匀后制粒，加入滑石粉和硬脂酸镁混匀后压制成10000片。2. Mix 33,34-methylenedioxy-docasanthin glucuronide, Houttuynia cordata extract, vitamin C and starch into granules, add talc powder and magnesium stearate, mix and compress In 10,000 pieces.
实施例26 33，34-亚甲基二氧-连翘脂素葡萄糖醛酸苷胶囊剂Example 26 33,34-Methylenedioxy-Forsythin Glucuronide Capsules
1、按照如下配比准备原料1. Prepare raw materials according to the following ratio
Figure PCTCN2016079843-appb-000021
Figure PCTCN2016079843-appb-000021
2、将33，34-亚甲基二氧-连翘脂素葡萄糖醛酸苷、板蓝根提取物、维生素C和淀粉混合均匀后装胶囊，制成10000粒。2. The 33,34-methylenedioxy-Forsythine glucuronide, Radix Isatidis extract, vitamin C and starch were uniformly mixed and then filled into capsules to make 10,000 tablets.
实施例27 33，34-亚甲基二氧-连翘脂素葡萄糖醛酸苷颗粒剂Example 27 33,34-Methylenedioxy-Forsythine Glucuronide Granules
1、按照如下配比准备原料1. Prepare raw materials according to the following ratio
Figure PCTCN2016079843-appb-000022
Figure PCTCN2016079843-appb-000022
Figure PCTCN2016079843-appb-000023
Figure PCTCN2016079843-appb-000023
2、将33，34-亚甲基二氧-连翘脂素葡萄糖醛酸苷、白头翁提取物、维生素C和蔗糖粉混合均匀制成颗粒后装袋，制成10000袋。2. The 33,34-methylenedioxy- forsythin glucuronide, the pulsatilla extract, the vitamin C and the sucrose powder are uniformly mixed into a granule and then bagged to make 10,000 bags.
实施例28 33，34-亚甲基二氧-连翘脂素葡萄糖醛酸苷颗粒剂Example 28 33,34-Methylenedioxy-Forsythine Glucuronide Granules
1、按照如下配比准备原料1. Prepare raw materials according to the following ratio
Figure PCTCN2016079843-appb-000024
Figure PCTCN2016079843-appb-000024
2、将33，34-亚甲基二氧-连翘脂素葡萄糖醛酸苷、白头翁提取物、维生素C和蔗糖粉混合均匀制成颗粒后装袋，制成10000袋。2. The 33,34-methylenedioxy- forsythin glucuronide, the pulsatilla extract, the vitamin C and the sucrose powder are uniformly mixed into a granule and then bagged to make 10,000 bags.
实施例29 33，34-亚甲基二氧-连翘脂素葡萄糖醛酸苷口服液Example 29 33,34-Methylenedioxy-Forsythine Glucuronide Oral Solution
1、按照如下配比准备原料1. Prepare raw materials according to the following ratio
Figure PCTCN2016079843-appb-000025
Figure PCTCN2016079843-appb-000025
2、取33，34-亚甲基二氧-连翘脂素葡萄糖醛酸苷、白头翁提取物、鱼腥草提取物，用乙醇溶解后加入葡萄糖糖浆，最终加去离子水至100ml，即得。2, taking 33,34-methylenedioxy- forsyric acid glucuronide, pulsatilla extract, Houttuynia cordata extract, dissolved in ethanol, add glucose syrup, and finally add deionized water to 100ml, that is .
实施例30 连翘苷和连翘脂素组合物片剂制备Example 30 Tablet Preparation of Forsythin and Forsythia Compound Composition
1、按照如下配比制备连翘苷/连翘脂素组合物的片剂：1. Tablets for the preparation of forsythin/forsylin composition according to the following ratio:
Figure PCTCN2016079843-appb-000026
Figure PCTCN2016079843-appb-000026
2、取连翘苷490g、连翘脂素10g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。2. Take 490g of forsythin and 10g of forsythiaside and mix it evenly with starch, make granules, add talc powder and magnesium stearate and mix well, then press into 10000 pieces.
实施例31 连翘苷/连翘脂素组合物颗粒剂制备 Example 31 Preparation of Forsythin/Forsylin Composition Granules
1、按照如下配比制备连翘苷/连翘脂素组合物的颗粒剂：1. Preparation of granules of forsythin/forsylin composition according to the following ratio:
连翘苷/连翘脂素组合物(两者重量比为98:2)               100gForsythin/Forsythia Leptin Composition (both weight ratio 98:2) 100g
微晶纤维素                                            10000gMicrocrystalline cellulose 10000g
2、取连翘苷98g、连翘脂素2g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2. After taking 98 g of forsythin, 2 g of forsythiaside and microcrystalline cellulose, the mixture was granulated and bagged to make 10,000 bags. Example 32 Preparation of Forsythin/Forsylin Composition Capsules
1、按照如下配比制备连翘苷/连翘脂素组合物的胶囊剂：1. Capsules for preparing forsythin/forsylin composition according to the following ratio:
连翘苷/连翘脂素组合物(两者重量比为98:2)               250gForsythin/Forsythia Leptin Composition (both weight ratio 98:2) 250g
淀粉                                                  2500gStarch 2500g
2、取连翘苷245g、连翘脂素5g与淀粉混合均匀后装胶囊，制成10000粒。2. Take 245g of forsythin, 5g of forsythiaside and starch and mix it evenly, then pack it into 10,000 capsules.
实施例33-36 连翘苷/连翘脂素组合物胶囊剂制备Example 33-36 Preparation of forsythin/forsylin composition capsules
实施例33-36中，连翘苷/连翘脂素组合物分别按下表的重量比与淀粉混合均匀后装胶囊，各制成10000粒胶囊。In Examples 33-36, the forsythin/forsylin composition was uniformly mixed with starch in the weight ratio of the following table, and then filled into capsules, each of which was made into 10,000 capsules.
Figure PCTCN2016079843-appb-000027
Figure PCTCN2016079843-appb-000027
实施例:37-40 连翘苷/连翘脂素组合物颗粒剂制备Example: Preparation of 37-40 forsythin/forsylin composition granules
实施例37-30中，连翘苷/连翘脂素组合物分别按下表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。In Examples 37 to 30, the forsythin/forsylin composition was uniformly mixed with the microcrystalline cellulose in the weight ratio of the following table, and then granulated and bagged to prepare 10,000 bags.
Figure PCTCN2016079843-appb-000028
Figure PCTCN2016079843-appb-000028
实施例41 连翘苷/连翘脂素组合物片剂制备Example 41 Preparation of Forsythin/Forsylin Composition Tablets
1、按照如下配比制备连翘苷/连翘脂素组合物的片剂：1. Tablets for the preparation of forsythin/forsylin composition according to the following ratio:
Figure PCTCN2016079843-appb-000029
Figure PCTCN2016079843-appb-000029
Figure PCTCN2016079843-appb-000030
Figure PCTCN2016079843-appb-000030
2、称取连翘苷490g、连翘脂素10g与黄芩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。2. Weigh 490g of forsythin, 10g of forsythiaside and the extract of Astragalus membranaceus, and mix it evenly with the starch to make granules. Add talc powder and magnesium stearate and mix well to make 10000 tablets.
实施例42 连翘苷/连翘脂素组合物颗粒剂制备Example 42 Preparation of Forsythin/Forsylin Composition Granules
1、按照如下配比制备连翘苷/连翘脂素组合物的颗粒剂：1. Preparation of granules of forsythin/forsylin composition according to the following ratio:
Figure PCTCN2016079843-appb-000031
Figure PCTCN2016079843-appb-000031
2、取连翘苷245g、连翘脂素5与野菊花提取物、黄芩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。2. Take 245g forsythin and forsythiaside 5 and mix with wild chrysanthemum extract and Astragalus membranaceus extract powder, then mix with microcrystalline cellulose to make granules and make 10,000 bags.
实施例43 连翘苷/连翘脂素组合物胶囊剂制备Example 43 Preparation of Forsythin/Forsylin Composition Capsules
1、按照如下配比制备连翘苷/连翘脂素组合物的胶囊剂：1. Capsules for preparing forsythin/forsylin composition according to the following ratio:
Figure PCTCN2016079843-appb-000032
Figure PCTCN2016079843-appb-000032
2、连翘苷245g、连翘脂素5g与苦参提取物、四季青提取物、穿心莲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。2. Forsythiaside 245g, forsythiaside 5g and Sophora flavescens extract, Sijiqing extract, Andrographis paniculata extract powder are evenly mixed, and then mixed with starch and then filled into capsules to make 10,000 capsules.
实施例44 连翘苷/连翘脂素组合物片剂制备Example 44 Preparation of Forsythin/Forsylin Composition Tablets
1、按照如下配比制备连翘苷/连翘脂素组合物的片剂：1. Tablets for the preparation of forsythin/forsylin composition according to the following ratio:
Figure PCTCN2016079843-appb-000033
Figure PCTCN2016079843-appb-000033
2、取连翘苷490g、连翘脂素10g与四季青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。2. Take 490g of forsythin, 10g of forsythiaside and the powder of Sijiqing extract evenly, then mix it with starch and make it into granules. Add talc powder and magnesium stearate and mix it evenly to make 10000 pieces.
实施例45 连翘苷/连翘脂素组合物颗粒剂制备 Example 45 Preparation of Forsythin/Forsylin Composition Granules
1、按照如下配比制备连翘苷/连翘脂素组合物的颗粒剂：1. Preparation of granules of forsythin/forsylin composition according to the following ratio:
Figure PCTCN2016079843-appb-000034
Figure PCTCN2016079843-appb-000034
2、取连翘苷900g、连翘脂素100g与上述提取物(鱼腥草、板蓝根)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。2. Take 900 g of forsythin and 100 g of forsythiaside and mix with the above extracts (Houttuynia cordata, Radix Isatidis) powder, and mix them with microcrystalline cellulose to make granules and bag into 10,000 bags.
实施例46 连翘苷/连翘脂素组合物胶囊剂制备Example 46 Preparation of Forsythin/Forsylin Composition Capsules
1、按照如下配比制备连翘苷/连翘脂素组合物的胶囊剂：1. Capsules for preparing forsythin/forsylin composition according to the following ratio:
Figure PCTCN2016079843-appb-000035
Figure PCTCN2016079843-appb-000035
2、连翘苷1880g、连翘脂素120g与上述提取物(鱼腥草、草豆蔻、四季青)粉末混合均匀，再与淀粉混合均匀后装胶囊，制成10000粒。2. Forsythiaside 1880g, forsythiaside 120g and the above extracts (Houttuynia, Soybean Meal, Sijiqing) powder are evenly mixed, and then mixed with starch and then filled with capsules to make 10,000 tablets.
试验例1 连翘苷抗菌作用的实验研究Test Example 1 Experimental study on the antibacterial effect of forsythin
1 实验材料1 Experimental materials
1.1 药物与试剂1.1 Drugs and reagents
连翘苷(含量＞98％)，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％。批号：20130303。Forsythin (>98%), white powder, produced by Dalian Fusheng Natural Medicine Development Co., Ltd., determined by high-performance liquid chromatography two detector UV detector and evaporative light scattering detector area normalization method, the purity is 99.5%, and the content of Chinese forensic bio-products was determined and confirmed to be 99.5% with forsythia reference substance. Batch number: 20130303.
1.2 菌种1.2 strains
(1)细菌枯草芽孢杆菌(Bacillus subtilis)、金黄色葡萄球菌(Staphylococcus aureus)、大肠杆菌(Escherichia coli)，(1) Bacillus subtilis, Staphylococcus aureus, Escherichia coli,
(2)霉菌黑曲霉(Aspergillus niger)、黄曲霉(Aspergillus flavus)，桔青霉(Penicillium citrinum)，(2) Aspergillus niger, Aspergillus flavus, Penicillium citrinum,
(3)酵母(Saccharomyces cerevisiae)。(3) Yeast (Saccharomyces cerevisiae).
以上菌种均由大连医科大学微生物实验室提供。The above strains were all provided by the Microbiology Laboratory of Dalian Medical University.
1.3 实验仪器及培养基 1.3 Experimental instruments and media
微量多点接种仪：北京医科大学昆仑药物发展研究所生产。Micro-multiple inoculation instrument: produced by Kunlun Drug Development Institute of Beijing Medical University.
培养基:Mueller-Hinton琼脂(法国事物梅里埃公司,简称M-H琼脂)。Medium: Mueller-Hinton agar (French-Mertier, M-H agar).
1.4实验方法1.4 Experimental methods
采用琼脂稀释法，用M-H琼脂进行连翘苷的定量抗菌实验。Quantitative antibacterial experiments of forsythin were carried out using agar dilution method using M-H agar.
1)药液稀释：取无菌试管10只排列于试管架中，于第2～10只试管中分别加入无菌蒸馏水10mL；接着取0.3％连翘苷无菌0.5％CMC-Na溶液(称取连翘苷3g，用适量的0.5％CMC-Na溶液混悬后，加0.5％CMC-Na溶液至1000ml，充分混匀后使用。)10mL加入到第1只试管中；然后再取10mL的0.3％连翘苷无菌0.5％CMC-Na溶液加入到第2只试管中，混匀后从第2只试管中取10mL混合液加入到第3只试管内，再混合均匀后取10mL混合液加入到第4只试管中，混匀后再从第4只试管中取10ml混合液计入到第5只试管中，混匀，如此连续稀释至第9只试管混匀后，取出10mL弃掉。第10只试管中只有无菌蒸馏水，以第10只试管中的无菌蒸馏水作为对照。这样1～9管含有不同浓度药液各10mL。1) Dilution of the drug solution: 10 sterile test tubes are arranged in the test tube rack, and 10 mL of sterile distilled water is added to the 2nd to 10th test tubes respectively; then 0.3% forsythin sterile 0.5% CMC-Na solution is taken. Take 3 g of forsythin, suspend it with an appropriate amount of 0.5% CMC-Na solution, add 0.5% CMC-Na solution to 1000 ml, mix well and use.) 10 mL is added to the first tube; then 10 mL is taken. 0.3% forsythin sterile 0.5% CMC-Na solution was added to the second tube, and after mixing, 10 mL of the mixture was taken from the second tube and added to the third tube. After mixing, 10 mL of the mixture was taken. Add to the 4th test tube, mix well, then take 10ml of the mixture from the 4th test tube into the 5th test tube, mix well, and then serially dilute to the 9th test tube and mix, remove 10mL and discard it. . In the 10th test tube, only sterile distilled water was used, and sterile distilled water in the 10th test tube was used as a control. Thus, 1 to 9 tubes contain 10 mL each of different concentrations of the drug solution.
2)含药平板制备2) Preparation of drug-containing plates
在无菌操作台上按照无菌操作方法，将已经溶化的无菌M-H琼脂900mL,分装于10个三角瓶中,每瓶90mL熔融的无菌M-H琼脂,将第1只试管中含有的10mL的0.3％连翘苷无菌蒸馏水溶液放入到含90mL培养基的第1个三角瓶中，混合后平均倾倒至7个平板(即平皿)中，每个平板约含14mL含药(即连翘苷)琼脂，凝固后备用，为第1含药琼脂平板组，其中：第1含药琼脂平板组每个平板中实际含连翘苷3.0mg/ml。On a sterile workstation, according to the aseptic method, 900 mL of the solubilized sterile MH agar was dispensed into 10 flasks, 90 mL of molten sterile MH agar per flask, and 10 mL of the first tube was contained. The 0.3% forsythin sterile distilled aqueous solution was placed in a first triangular flask containing 90 mL of medium, and after mixing, it was poured onto 7 plates (ie, plates), and each plate contained about 14 mL of drug (ie, even The agarose agar, which was prepared after coagulation, was the first drug-containing agar plate group, wherein: the first drug-containing agar plate group actually contained forsythin 3.0 mg/ml in each plate.
第2-10只试管内的溶液按照与第1只试管内的溶液同样的方法倾倒平板，制成第2-10含药平板组，每个平板组含有7个平板，每个平板约含14mL含药(即连翘苷)琼脂，凝固后备用。第2含药琼脂平板组中每个平板实际含连翘苷1.5mg/ml；第3含药琼脂平板组中每个平板实际含连翘苷为0.75mg/ml；第4含药琼脂平板组中每个平板实际含连翘苷为0.375mg/ml；第5含药琼脂平板组中每个平板实际含连翘苷为0.1875mg/ml；第6含药琼脂平板组中每个平板实际含连翘苷为0.0938mg/ml；第7含药琼脂平板组中每个平板实际含连翘苷为0.0469mg/ml；第8含药琼脂平板组中每个平板实际含连翘苷为0.0234mg/ml；第9含药琼脂平板组中每个平板实际含连翘苷为0.0117mg/ml；第10含药琼脂平板组中每个平板实际含连翘苷为0.0mg/ml。The solutions in the 2-10th test tubes were poured into the same plate as the solution in the first test tube to prepare a 2-10 drug-containing plate set, each plate set containing 7 plates, each plate containing about 14 mL. Drug-containing (ie forsythin) agar, ready for use after solidification. In the second drug-containing agar plate group, each plate actually contained forsythin 1.5 mg/ml; in the third drug-containing agar plate group, each plate actually contained forsythin at 0.75 mg/ml; the fourth drug-containing agar plate group Each plate actually contained forsythin at 0.375 mg/ml; in the fifth drug-containing agar plate group, each plate actually contained forsythin at 0.1875 mg/ml; in the sixth drug-containing agar plate group, each plate actually contained Forsythin was 0.0938 mg/ml; in the 7th drug-containing agar plate group, each plate actually contained forsythin at 0.0469 mg/ml; in the 8th drug-containing agar plate group, each plate actually contained forsythin at 0.0234 mg. /ml; each plate in the 9th drug-containing agar plate group actually contained forsythin 0.0117 mg/ml; in the 10th drug-containing agar plate group, each plate actually contained forsythin as 0.0 mg/ml.
3)用多点接种仪分别吸取各种菌液并将菌液点种于含不同药物浓度的琼脂平板上。于35℃培养18～24h,观察菌体生长情况，生长菌体为阳性(+)，不生长菌体为阴性(一)，以不生长菌体的最低浓度为最低抑菌浓度(MIC)。 3) Using a multi-point inoculation instrument to separately absorb various bacterial liquids and plant the bacterial liquid on agar plates containing different drug concentrations. The cells were cultured at 35 ° C for 18-24 hours, and the growth of the cells was observed. The growth cells were positive (+), the non-growth cells were negative (1), and the lowest concentration of non-growth cells was the minimum inhibitory concentration (MIC).
2 实验结果2 Experimental results
金黄色葡萄球菌、大肠杆菌、枯草芽孢杆菌、酵母、桔青霉、黑曲霉、黄曲霉抑菌效果测定结果如表1所示。The results of the bacteriostatic effects of Staphylococcus aureus, Escherichia coli, Bacillus subtilis, yeast, Penicillium citrinum, Aspergillus niger, and Aspergillus flavus are shown in Table 1.
表1 连翘苷对7种细菌的MICTable 1 MIC of forsythin on seven bacteria
Figure PCTCN2016079843-appb-000036
Figure PCTCN2016079843-appb-000036
注：-表示无菌生长；+表示菌体生长少；++表示菌体生长较多；+++表示菌体生长多，下同Note: - indicates sterile growth; + indicates less growth of bacteria; ++ indicates more growth of bacteria; +++ indicates more growth of bacteria, the same below
由表1的研究结果表明,连翘苷对金黄色葡萄球菌、酵母菌、桔青霉菌、黑曲霉菌等抗菌作用显著，其对金黄色葡萄球菌、大肠杆菌、枯草芽孢杆菌、酵母、桔青霉、黑曲霉、黄曲霉的MIC为：0.0469mg/ml、1.5mg/ml、1.5mg/ml、0.1875mg/ml、0.0469mg/ml、0.0469mg/ml、0.1875mg/ml。The results of Table 1 indicate that forsythiaside has significant antibacterial effects against Staphylococcus aureus, yeast, Penicillium citrinum, and Aspergillus niger, and it is against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, yeast, and orange. The MICs of mold, Aspergillus niger, and Aspergillus flavus were: 0.0469 mg/ml, 1.5 mg/ml, 1.5 mg/ml, 0.1875 mg/ml, 0.0469 mg/ml, 0.0469 mg/ml, and 0.1875 mg/ml.
试验例2 连翘苷衍生物抗菌作用的实验研究Test Example 2 Experimental study on the antibacterial effect of forsythin derivatives
1 实验材料1 Experimental materials
1.1 药物与试剂1.1 Drugs and reagents
33-羟基-连翘脂素葡萄糖醛酸苷(连翘苷衍生物A)，(含量＞98％)，白色粉末，大连富生天然药物开发有限公司生产，批号：20130301。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98.5％。33-hydroxy-Forsyricin Glucuronide (Forsythine Derivative A), (>98%), white powder, produced by Dalian Fusheng Natural Medicine Development Co., Ltd., batch number: 20130301. The purity was determined by the high-performance liquid chromatography two detector UV detector and the evaporative light scattering detector area normalization method, and the purity was 98.5%.
9-羟基-连翘脂素葡萄糖醛酸苷(连翘苷衍生物B)，(含量＞98％)，白色粉末，大连富生天然药物开发有限公司生产，批号：20130302。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99.2％。 9-Hydroxy-Forsysin Glucuronide (Forsythine Derivative B), (>98%), white powder, produced by Dalian Fusheng Natural Medicine Development Co., Ltd., batch number: 20130302. It was determined by high-performance liquid chromatography two detector UV detector and evaporative light scattering detector area normalization method, and its purity was 99.2%.
33，34-亚甲基二氧-连翘脂素葡萄糖醛酸苷(连翘苷衍生物C)，(含量＞98％)，白色粉末，大连富生天然药物开发有限公司生产，批号：20130301。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98.7％。33,34-Methylenedioxy-Forsyricin Glucuronide (Forsythine Derivative C), (>98%), white powder, produced by Dalian Fusheng Natural Medicine Development Co., Ltd., batch number: 20130301. It was determined by high-performance liquid chromatography two detector UV detector and evaporative light scattering detector area normalization method, and its purity was 98.7%.
1.2 菌种1.2 strains
菌种与试验例1相同，均由大连医科大学微生物实验室提供。The strains were the same as in Test Example 1, and were provided by the Microbiology Laboratory of Dalian Medical University.
1.3 实验仪器及培养基1.3 Experimental instruments and media
与试验例1相同。The same as Test Example 1.
1.4 实验方法1.4 Experimental methods
采用琼脂稀释法，用M-H琼脂进行连翘苷的定量抗菌实验。Quantitative antibacterial experiments of forsythin were carried out using agar dilution method using M-H agar.
1)配制连翘苷衍生物药液1) Preparation of forsythin derivative liquid
称取33-羟基-连翘脂素葡萄糖醛酸苷3g，用适量的0.5％CMC-Na溶液混悬后，加0.5％CMC-Na溶液至1000ml，充分混匀，制得0.3％33-羟基-连翘脂素葡萄糖醛酸苷无菌0.5％CMC-Na溶液(简称连翘苷衍生物A药液)，备用；Weigh 33 g of 33-hydroxy-Forsylin glucuronide, suspend it with an appropriate amount of 0.5% CMC-Na solution, add 0.5% CMC-Na solution to 1000 ml, and mix well to obtain 0.3% 33-hydroxyl. - forsythiaside glucuronide sterile 0.5% CMC-Na solution (referred to as forsythin derivative A solution), spare;
称取9-羟基-连翘脂素葡萄糖醛酸苷3g，用适量的0.5％CMC-Na溶液混悬后，加0.5％CMC-Na溶液至1000ml，充分混匀，制得0.3％9-羟基-连翘脂素葡萄糖醛酸苷无菌0.5％CMC-Na溶液(简称连翘苷衍生物B药液)，备用；Weigh 9g of 9-hydroxy-Forsylin glucuronide, suspend it with an appropriate amount of 0.5% CMC-Na solution, add 0.5% CMC-Na solution to 1000ml, mix well, and obtain 0.3% 9-hydroxyl - Forsythiaside glucuronide sterile 0.5% CMC-Na solution (referred to as forsythin derivative B solution), spare;
称取33，34-亚甲基二氧-连翘脂素葡萄糖醛酸苷3g，用适量的0.5％CMC-Na溶液混悬后，加0.5％CMC-Na溶液至1000ml，充分混匀，制得0.3％33，34-亚甲基二氧-连翘脂素葡萄糖醛酸苷无菌0.5％CMC-Na溶液(简称连翘苷衍生物C药液)，备用。Weigh 33g of 33,34-methylenedioxy-forsylin glucuronide, suspend it with an appropriate amount of 0.5% CMC-Na solution, add 0.5% CMC-Na solution to 1000ml, mix well. 0.3% 33,34-methylenedioxy-forsylin glucuronide sterile 0.5% CMC-Na solution (referred to as forsythin derivative C solution), spare.
2)药液稀释：2) Dilution of liquid:
取无菌试管30只分成3组，每组10只分布排列于试管架中，在第一组的10只试管中于第2～10只试管中分别加入无菌蒸馏水10mL；接着取连翘苷衍生物A药液10mL加入到第1只试管中；再取10mL连翘苷衍生物A药液加入到第2只试管中，混匀后从第2只试管中取10mL混合液加入到第3只试管内，再混合均匀后取10mL混合液加入到第4只试管中，混匀后再从第4只试管中取10ml混合液计入到第5只试管中，混匀，如此连续稀释至第9只试管混匀后，取出10mL弃掉。第10只试管中只有无菌蒸馏水，以第10只试管中的无菌蒸馏水作为对照。这样1～9管含有不同浓度药液各10mL。The sterile test tubes 30 were divided into 3 groups, 10 groups of each group were arranged in a test tube rack, and 10 mL of sterile distilled water was added to the 2 to 10 test tubes in the 10 test tubes of the first group; 10 mL of the derivative A solution was added to the first tube; 10 mL of the forsythin derivative A solution was added to the second tube, and after mixing, 10 mL of the mixture was taken from the second tube and added to the third tube. In a test tube, mix well and then add 10 mL of the mixture to the 4th test tube. After mixing, take 10 ml of the mixture from the 4th tube and count it into the 5th tube, mix well, and serially dilute to After the 9th tube was mixed, 10 mL was taken out and discarded. In the 10th test tube, only sterile distilled water was used, and sterile distilled water in the 10th test tube was used as a control. Thus, 1 to 9 tubes contain 10 mL each of different concentrations of the drug solution.
第二组试管中除了加入的药液是连翘苷衍生物B药液之外，其它操作方法与第一组试管的药液进行稀释方法相同；第三组试管中除了加入的药液是连翘苷衍生物C药液之外，其它操作方法与第一组试管的药液进行稀释方法相同。 In addition to the forsythiaside derivative B solution, the other methods of operation are the same as those of the first group of test tubes; the third group of tubes except the added solution is In addition to the scutellarin derivative C solution, other methods of operation are the same as those for the first group of test tubes.
3)含药平板制备3) Preparation of drug-containing plates
3-1)连翘苷衍生物A含药琼脂平板组3-1) Forsythin derivative A drug-containing agar plate group
在无菌操作台上按照无菌操作方法，将已经溶化的无菌M-H琼脂900mL,分装于10个三角瓶中,每瓶90mL熔融的无菌M-H琼脂,将第一组试管中的第1只试管中含有的10mL的连翘苷衍生物A药液放入到含90mL培养基的第1个三角瓶中，混合后平均倾倒至7个平板(即平皿)中，每个平板约含14mL含药(即连翘苷衍生物A)琼脂，凝固后备用，为第1含药琼脂平板组，其中：第1含药琼脂平板组每个平板中实际含连翘苷衍生物A 3.0mg/ml。On a sterile workstation, aseptically, 900 mL of solubilized sterile MH agar was dispensed into 10 flasks, 90 mL of molten sterile MH agar per flask, and the first of the first set of tubes. 10 mL of forsythin derivative A solution contained in only one tube was placed in a first flask containing 90 mL of medium, and mixed and poured onto 7 plates (ie, plates), each plate containing about 14 mL. The drug-containing (that is, forsythin derivative A) agar was prepared after solidification and was used as the first drug-containing agar plate group, wherein: the first drug-containing agar plate group actually contained forsythin derivative A 3.0 mg/in each plate. Ml.
第一组试管中的第2-10只试管内的溶液按照与第1只试管内的溶液同样的方法倾倒平板，制成第2-10含药平板组，每个平板组含有7个平板，每个平板约含14mL含药(即连翘苷衍生物A)琼脂，凝固后备用。第2含药琼脂平板组中每个平板实际含连翘苷衍生物A1.5mg/ml；第3含药琼脂平板组中每个平板实际含连翘苷衍生物A为0.75mg/ml；第4含药琼脂平板组中每个平板实际含连翘苷衍生物A为0.375mg/ml；第5含药琼脂平板组中每个平板实际含连翘苷衍生物A为0.1875mg/ml；第6含药琼脂平板组中每个平板实际含连翘苷衍生物A为0.0938mg/ml；第7含药琼脂平板组中每个平板实际含连翘苷衍生物A为0.0469mg/ml；第8含药琼脂平板组中每个平板实际含连翘苷衍生物A为0.0234mg/ml；第9含药琼脂平板组中每个平板实际含连翘苷衍生物A为0.0117mg/ml；第10含药琼脂平板组中每个平板实际含连翘苷衍生物A为0.0mg/ml。The solutions in the 2-10th test tubes in the first set of tubes were poured into the same plate as in the first test tube to prepare a 2-10 drug-containing plate set, and each plate set contained 7 plates. Each plate contains approximately 14 mL of the drug-containing (ie, forsythin derivative A) agar, which is set aside after solidification. In the second drug-containing agar plate group, each plate actually contained forsythiaside derivative A 1.5 mg/ml; in the third drug-containing agar plate group, each plate actually contained forsythin derivative A was 0.75 mg/ml; Each of the plates in the drug-containing agar plate group actually contained forsythin derivative A was 0.375 mg/ml; in the fifth drug-containing agar plate group, each plate actually contained forsythin derivative A was 0.1875 mg/ml; Each tablet in the drug-containing agar plate group actually contained forsythiaside derivative A was 0.0938 mg/ml; in the seventh drug-containing agar plate group, each plate actually contained forsythin derivative A was 0.0469 mg/ml; In each of the 8 drug-containing agar plates, the actual contains forsythiaside derivative A was 0.0234 mg/ml; in the 9th drug-containing agar plate group, each plate actually contained forsythin derivative A was 0.0117 mg/ml; Each plate in the 10-medicated agar plate group actually contained forsythin derivative A at 0.0 mg/ml.
3-2)连翘苷衍生物B、C含药琼脂平板组3-2) Forsythin derivatives B, C drug-containing agar plate group
按照连翘苷衍生物A含药琼脂平板组的配制方法制备连翘苷衍生物B含药琼脂平板组，除了向第二组三角瓶中加入第二组试管中的连翘苷衍生物B药液之外，其余与步骤3-1)相同。The forsythiaside derivative B-containing agar plate group was prepared according to the preparation method of forsythiaside derivative A drug-containing agar plate group, except that the forsythiaside derivative B drug in the second group of test tubes was added to the second group of triangular flasks. Except for the liquid, the rest is the same as step 3-1).
按照连翘苷衍生物A含药琼脂平板组的配制方法制备连翘苷衍生物C含药琼脂平板组，除了向第三组三角瓶中加入第三组试管中的连翘苷衍生物C药液之外，其余与步骤3-1)相同。The forsythiaside derivative C-containing agar plate group was prepared according to the preparation method of forsythiaside derivative A drug-containing agar plate group, except that the forsythiaside derivative C drug in the third group of test tubes was added to the third group of triangular flasks. Except for the liquid, the rest is the same as step 3-1).
4)用多点接种仪分别吸取各种菌液并将菌液点种于含不同药物浓度的琼脂平板上。于35℃培养18～24h,观察菌体生长情况，生长菌体为阳性(+)，不生长菌体为阴性(一)，以不生长菌体的最低浓度为最低抑菌浓度(MIC)。4) Using a multi-point inoculation instrument to separately absorb various bacterial liquids and plant the bacterial liquid on agar plates containing different drug concentrations. The cells were cultured at 35 ° C for 18-24 hours, and the growth of the cells was observed. The growth cells were positive (+), the non-growth cells were negative (1), and the lowest concentration of non-growth cells was the minimum inhibitory concentration (MIC).
2 实验结果2 Experimental results
连翘苷衍生物A、连翘苷衍生物B和连翘苷衍生物C对金黄色葡萄球菌、大肠杆菌、枯 草芽孢杆菌、酵母、桔青霉、黑曲霉、黄曲霉抑菌效果测定结果如表2-4所示。Forsythin derivative A, forsythin derivative B and forsythin derivative C against Staphylococcus aureus, Escherichia coli The results of the bacteriostatic effects of Bacillus licheniformis, yeast, Penicillium citrinum, Aspergillus niger, and Aspergillus flavus are shown in Table 2-4.
表2 连翘苷衍生物A对7种细菌的MICTable 2 MIC of forsythin derivative A against seven bacteria
Figure PCTCN2016079843-appb-000037
Figure PCTCN2016079843-appb-000037
表3 连翘苷衍生物B对7种细菌的MICTable 3 MIC of forsythin derivative B against seven bacteria
Figure PCTCN2016079843-appb-000038
Figure PCTCN2016079843-appb-000038
表4 连翘苷衍生物C对7种细菌的MICTable 4 MIC of forsythin derivative C against 7 bacteria
Figure PCTCN2016079843-appb-000039
Figure PCTCN2016079843-appb-000039
由表2-4的研究结果表明,连翘苷衍生物A、B、C对金黄色葡萄球菌、酵母菌、桔青霉菌、黑曲霉菌等抗菌作用显著，连翘苷衍生物A对金黄色葡萄球菌、大肠杆菌、枯草芽孢杆菌、酵母、桔青霉、黑曲霉、黄曲霉的MIC为：0.0234mg/ml、0.75mg/ml、0.75mg/ml、0.0938mg/ml、0.0469mg/ml、0.0234mg/ml、0.0938mg/ml。连翘苷衍生物B对金黄色葡萄球菌、大肠杆菌、枯草芽孢杆菌、酵母、桔青霉、黑曲霉、黄曲霉的MIC为：0.0938mg/ml、0.75mg/ml、0.75mg/ml、0.0938mg/ml、0.0938mg/ml、0.0938mg/ml、0.0469mg/ml。The results of Table 2-4 show that the forsythin derivatives A, B, and C have significant antibacterial effects against Staphylococcus aureus, yeast, Penicillium citrinum, and Aspergillus niger. Forsythiaside derivative A is golden yellow. The MICs of Staphylococcus, Escherichia coli, Bacillus subtilis, yeast, Penicillium citrinum, Aspergillus niger, and Aspergillus flavus are: 0.0234 mg/ml, 0.75 mg/ml, 0.75 mg/ml, 0.0938 mg/ml, 0.0469 mg/ml, 0.0234 mg/ml, 0.0938 mg/ml. The MICs of forsythin derivative B against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, yeast, Penicillium citrinum, Aspergillus niger, and Aspergillus flavus are: 0.0938 mg/ml, 0.75 mg/ml, 0.75 mg/ml, 0.0938 Mg/ml, 0.0938 mg/ml, 0.0938 mg/ml, 0.0469 mg/ml.
连翘苷衍生物C对金黄色葡萄球菌、大肠杆菌、枯草芽孢杆菌、酵母、桔青霉、黑曲霉、黄曲霉的MIC为：0.0469mg/ml、1.5mg/ml、1.5mg/ml、0.1875mg/ml、0.0469mg/ml、0.0469mg/ml、0.1875mg/ml。The MIC of forsythin derivative C against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, yeast, Penicillium citrinum, Aspergillus niger, and Aspergillus flavus is: 0.0469 mg/ml, 1.5 mg/ml, 1.5 mg/ml, 0.1875 Mg/ml, 0.0469 mg/ml, 0.0469 mg/ml, 0.1875 mg/ml.
试验例3 连翘苷/连翘脂素组合物抗菌作用的实验研究Test Example 3 Experimental study on antibacterial effect of forsythin/forsylin composition
1 实验材料1 Experimental materials
1.1 药物与试剂1.1 Drugs and reagents
连翘苷/连翘脂素组合物A，连翘苷(含量＞98％)，白色粉末，大连富生天然药物开发有限公司生产，批号：20130303；连翘脂素(含量＞98％)，白色粉末，大连富生天然药物开发有限公司生产，批号：20130301；连翘苷与连翘脂素的重量之比为98：2。Forsythin/Forsythiamin composition A, forsythin (content > 98%), white powder, produced by Dalian Fusheng Natural Medicine Development Co., Ltd., batch number: 20130303; forsythiaside (content > 98%), white Powder, produced by Dalian Fusheng Natural Medicine Development Co., Ltd., batch number: 20130301; the ratio of forsythin to forsythin is 98:2.
连翘苷/连翘脂素组合物B，连翘苷(含量＞98％)，白色粉末，大连富生天然药物开发有限公司生产，批号：20130303；连翘脂素(含量＞98％)，白色粉末，大连富生天然药物 开发有限公司生产，批号：20130301；连翘苷与连翘脂素的重量之比为89：11。Forsythin/Forsythiamin B, forsythin (>98%), white powder, Dalian Fusheng Natural Medicine Development Co., Ltd., batch number: 20130303; forsythias (content > 98%), white Powder, Dalian Fusheng Natural Medicine Development Co., Ltd. production, batch number: 20130301; the ratio of forsythin to forsythiaside is 89:11.
1.2 菌种1.2 strains
菌种与试验例1相同，均由大连医科大学微生物实验室提供。The strains were the same as in Test Example 1, and were provided by the Microbiology Laboratory of Dalian Medical University.
1.3 实验仪器及培养基1.3 Experimental instruments and media
与试验例1相同。The same as Test Example 1.
1.4 实验方法1.4 Experimental methods
采用琼脂稀释法，用M-H琼脂进行连翘苷的定量抗菌实验。Quantitative antibacterial experiments of forsythin were carried out using agar dilution method using M-H agar.
1)配制连翘苷/连翘脂素组合物药液1) Preparation of forsythin/forsylin composition
称取连翘苷/连翘脂素组合物A 3g，用适量的0.5％CMC-Na溶液混悬后，加0.5％CMC-Na溶液至1000ml，充分混匀，制得0.3％连翘苷/连翘脂素组合物A无菌0.5％CMC-Na溶液(简称连翘苷/连翘脂素组合物A药液)，备用；Weigh the forsythin/forsylin composition A 3g, suspend it with an appropriate amount of 0.5% CMC-Na solution, add 0.5% CMC-Na solution to 1000ml, mix well, and obtain 0.3% forsythin / Forsythia saponin composition A sterile 0.5% CMC-Na solution (referred to as forsythin / forsythia sap composition A liquid), spare;
称取连翘苷/连翘脂素组合物B 3g，用适量的0.5％CMC-Na溶液混悬后，加0.5％CMC-Na溶液至1000ml，充分混匀，制得0.3％连翘苷/连翘脂素组合物B无菌0.5％CMC-Na溶液(简称连翘苷/连翘脂素组合物B药液)，备用；Weigh the forsythin/Forsythiamin composition B 3g, suspend it with an appropriate amount of 0.5% CMC-Na solution, add 0.5% CMC-Na solution to 1000ml, mix well to obtain 0.3% forsythin / Forsythia saponin composition B sterile 0.5% CMC-Na solution (referred to as forsythin / forsythia sap composition B liquid), spare;
2)药液稀释：2) Dilution of liquid:
取无菌试管20只分成2组，每组10只分布排列于试管架中，在第一组的10只试管中于第2～10只试管中分别加入无菌蒸馏水10mL；接着取连翘苷/连翘脂素组合物A药液10mL加入到第1只试管中；再取10mL连翘苷/连翘脂素组合物A药液加入到第2只试管中，混匀后从第2只试管中取10mL混合液加入到第3只试管内，再混合均匀后取10mL混合液加入到第4只试管中，混匀后再从第4只试管中取10ml混合液计入到第5只试管中，混匀，如此连续稀释至第9只试管混匀后，取出10mL弃掉。第10只试管中只有无菌蒸馏水，以第10只试管中的无菌蒸馏水作为对照。这样1～9管含有不同浓度药液各10mL。20 sterile tubes were divided into 2 groups, 10 groups in each group were distributed in the tube rack, and 10 mL of sterile distilled water was added to the 2 to 10 tubes in the first group of 10 tubes; Add 10 mL of forsythiaside composition A solution to the first tube; then add 10 mL of forsythin/Forsylin composition A solution to the second tube, mix and mix from the second tube. Add 10 mL of the mixture in the test tube to the third tube, mix well, then add 10 mL of the mixture to the fourth tube. After mixing, take 10 ml of the mixture from the fourth tube and count to the fifth. In the test tube, mix well, and then serially dilute to the 9th tube and mix, remove 10 mL and discard. In the 10th test tube, only sterile distilled water was used, and sterile distilled water in the 10th test tube was used as a control. Thus, 1 to 9 tubes contain 10 mL each of different concentrations of the drug solution.
第二组试管中除了加入的药液是连翘苷/连翘脂素组合物B药液之外，其它操作方法与第一组试管的药液进行稀释方法相同。In addition to the forsythiaside/Forsylin composition B solution, the other methods of operation were the same as those of the first group of test tubes.
3)含药平板制备3) Preparation of drug-containing plates
3-1)连翘苷/连翘脂素组合物A含药琼脂平板组3-1) Forsythin/Forsyxin composition A drug-containing agar plate set
在无菌操作台上按照无菌操作方法，将已经溶化的无菌M-H琼脂900mL,分装于10个三角瓶中,每瓶90mL熔融的无菌M-H琼脂,将第一组试管中的第1只试管中含有的10mL的连翘苷/连翘脂素组合物A药液放入到含90mL培养基的第1个三角瓶中，混合后平均倾倒至7个平板(即平皿)中，每个平板约含14mL含药(即连翘苷/连翘脂素组合物A)琼 脂，凝固后备用，为第1含药琼脂平板组，其中：第1含药琼脂平板组每个平板中实际含连翘苷/连翘脂素组合物A 3.0mg/ml。On a sterile workstation, aseptically, 900 mL of solubilized sterile MH agar was dispensed into 10 flasks, 90 mL of molten sterile MH agar per flask, and the first of the first set of tubes. 10 mL of forsythin/forsylin composition A solution contained in a test tube was placed in a first flask containing 90 mL of medium, and mixed and poured onto 7 plates (i.e., plates). Each plate contains approximately 14 mL of drug-containing (ie, forsythin/Forsylin composition A) The lipid, which was prepared after coagulation, was the first drug-containing agar plate group, wherein: the first drug-containing agar plate group actually contained forsythin/forsylin composition A 3.0 mg/ml per plate.
第一组试管中的第2-10只试管内的溶液按照与第1只试管内的溶液同样的方法倾倒平板，制成第2-10含药平板组，每个平板组含有7个平板，每个平板约含14mL含药(即连翘苷/连翘脂素组合物A)琼脂，凝固后备用。第2含药琼脂平板组中每个平板实际含连翘苷/连翘脂素组合物A1.5mg/ml；第3含药琼脂平板组中每个平板实际含连翘苷/连翘脂素组合物A为0.75mg/ml；第4含药琼脂平板组中每个平板实际含连翘苷/连翘脂素组合物A为0.375mg/ml；第5含药琼脂平板组中每个平板实际含连翘苷/连翘脂素组合物A为0.1875mg/ml；第6含药琼脂平板组中每个平板实际含连翘苷/连翘脂素组合物A为0.0938mg/ml；第7含药琼脂平板组中每个平板实际含连翘苷/连翘脂素组合物A为0.0469mg/ml；第8含药琼脂平板组中每个平板实际含连翘苷/连翘脂素组合物A为0.0234mg/ml；第9含药琼脂平板组中每个平板实际含连翘苷/连翘脂素组合物A为0.0117mg/ml；第10含药琼脂平板组中每个平板实际含连翘苷/连翘脂素组合物A为0.0mg/ml。The solutions in the 2-10th test tubes in the first set of tubes were poured into the same plate as in the first test tube to prepare a 2-10 drug-containing plate set, and each plate set contained 7 plates. Each plate contained approximately 14 mL of a drug-containing (ie, forsythin/Forsylin composition A) agar, which was solidified and used. Each plate in the second drug-containing agar plate group actually contained forsythin/forsylin composition A 1.5 mg/ml; each plate in the third drug-containing agar plate group actually contained forsythin/forsylin Composition A was 0.75 mg/ml; each plate in the fourth drug-containing agar plate group actually contained forsythin/Forsylin composition A was 0.375 mg/ml; each plate in the fifth drug-containing agar plate group Actually containing forsythin/Forsylin composition A is 0.1875 mg/ml; in the sixth drug-containing agar plate group, each plate actually contains forsythin/Forsylin composition A is 0.0938 mg/ml; Each plate in the drug-containing agar plate group actually contained forsythin/forsylin composition A was 0.0469 mg/ml; in the eighth drug-containing agar plate group, each plate actually contained forsythin/forsylin Composition A was 0.0234 mg/ml; each plate in the 9th drug-containing agar plate group actually contained forsythin/Forsylin composition A was 0.0117 mg/ml; each plate in the 10th drug-containing agar plate group The actual forsythin/Forsylin composition A was 0.0 mg/ml.
3-2)连翘苷/连翘脂素组合物B含药琼脂平板组3-2) Forsythin/Forsythiamin B composition A drug agar plate set
按照连翘苷/连翘脂素组合物A含药琼脂平板组的配制方法制备连翘苷/连翘脂素组合物B含药琼脂平板组，除了向第二组三角瓶中加入第二组试管中的连翘苷/连翘脂素组合物B药液之外，其余与步骤3-1)相同。The forsythiaside/forsylin composition B-containing agar plate group was prepared according to the preparation method of forsythiaside/forsylin composition A drug-containing agar plate group, except that the second group was added to the second group of triangular flasks. The procedure was the same as in step 3-1) except for the forsythiaside/forsylin composition B in the test tube.
4)用多点接种仪分别吸取各种菌液并将菌液点种于含不同药物浓度的琼脂平板上。于35℃培养18～24h,观察菌体生长情况，生长菌体为阳性(+)，不生长菌体为阴性(一)，以不生长菌体的最低浓度为最低抑菌浓度(MIC)。4) Using a multi-point inoculation instrument to separately absorb various bacterial liquids and plant the bacterial liquid on agar plates containing different drug concentrations. The cells were cultured at 35 ° C for 18-24 hours, and the growth of the cells was observed. The growth cells were positive (+), the non-growth cells were negative (1), and the lowest concentration of non-growth cells was the minimum inhibitory concentration (MIC).
2 实验结果2 Experimental results
连翘苷/连翘脂素组合物A、连翘苷/连翘脂素组合物B对金黄色葡萄球菌、大肠杆菌、枯草芽孢杆菌、酵母、桔青霉、黑曲霉、黄曲霉抑菌效果测定结果如表5、6所示。Anti-bacterial effect of forsythin/forsylin composition A, forsythin/forsylin composition B on Staphylococcus aureus, Escherichia coli, Bacillus subtilis, yeast, Penicillium citrinum, Aspergillus niger, and Aspergillus flavus The measurement results are shown in Tables 5 and 6.
表5 连翘苷/连翘脂素组合物A对7种细菌的MICTable 5 MIC of forsythin/forsylin composition A against 7 bacteria
Figure PCTCN2016079843-appb-000040
Figure PCTCN2016079843-appb-000040
Figure PCTCN2016079843-appb-000041
Figure PCTCN2016079843-appb-000041
表6 连翘苷/连翘脂素组合物B对7种细菌的MICTable 6 MIC of forsythin/forsylin composition B against 7 bacteria
Figure PCTCN2016079843-appb-000042
Figure PCTCN2016079843-appb-000042
由表5、6的研究结果表明,连翘苷/连翘脂素组合物A、连翘苷/连翘脂素组合物B对金黄色葡萄球菌、酵母菌、桔青霉菌、黑曲霉菌等抗菌作用显著，连翘苷/连翘脂素组合物A对金黄色葡萄球菌、大肠杆菌、枯草芽孢杆菌、酵母、桔青霉、黑曲霉、黄曲霉的MIC为：0.0234mg/ml、0.375mg/ml、0.375mg/ml、0.0469mg/ml、0.0234mg/ml、0.0234mg/ml、0.0469mg/ml。The results of the studies in Tables 5 and 6 indicate that forsythin/forsylin composition A, forsythin/forsylin composition B against Staphylococcus aureus, yeast, Penicillium citrinum, Aspergillus niger, etc. The antibacterial effect is remarkable. The MIC of forsythin/forsylin composition A against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, yeast, Penicillium citrinum, Aspergillus niger, and Aspergillus flavus is: 0.0234 mg/ml, 0.375 mg /ml, 0.375 mg/ml, 0.0469 mg/ml, 0.0234 mg/ml, 0.0234 mg/ml, 0.0469 mg/ml.
连翘苷/连翘脂素组合物B对金黄色葡萄球菌、大肠杆菌、枯草芽孢杆菌、酵母、桔青霉、黑曲霉、黄曲霉的MIC为：0.0938mg/ml、0.75mg/ml、0.75mg/ml、0.0938mg/ml、0.0938mg/ml、0.0938mg/ml、0.0469mg/ml。The MIC of forsythin/forsylin composition B against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, yeast, Penicillium citrinum, Aspergillus niger, and Aspergillus flavus was: 0.0938 mg/ml, 0.75 mg/ml, 0.75 Mg/ml, 0.0938 mg/ml, 0.0938 mg/ml, 0.0938 mg/ml, 0.0469 mg/ml.
根据上述试验结果可知，连翘苷、连翘苷衍生物、连翘苷与连翘脂素组合物均具有抑制细菌的作用，这一试验结果对指导临床预防常见致病菌感染具有重要指导意义。According to the above test results, forsythiaside, forsythin derivatives, forsythin and forsythiaside compositions have the effect of inhibiting bacteria. This test result has important guiding significance for guiding clinical prevention of common pathogenic bacteria infection. .
试验例4 连翘脂素抗菌作用的实验研究 Test Example 4 Experimental study on the antibacterial effect of forsythin
1 实验材料1 Experimental materials
1.1 药物与试剂1.1 Drugs and reagents
连翘脂素(含量＞98％)，白色粉末，大连富生天然药物开发有限公司生产，批号：20130301。Forsythia saponin (content >98%), white powder, produced by Dalian Fusheng Natural Medicine Development Co., Ltd., batch number: 20130301.
1.2 菌种1.2 strains
与试验例1相同Same as test example 1
1.3 实验仪器及培养基1.3 Experimental instruments and media
与试验例1相同Same as test example 1
1.4 实验方法1.4 Experimental methods
采用琼脂稀释法，用M-H琼脂进行连翘脂素的定量抗菌实验。Quantitative antibacterial experiments of forsythin were performed using agar dilution method using M-H agar.
1)药液稀释：取无菌试管10只排列于试管架中，于第2～10只试管中分别加入无菌蒸馏水10mL；接着取0.3％连翘脂素无菌0.5％CMC-Na溶液(称取连翘脂素3g，用适量的0.5％CMC-Na溶液混悬后，加0.5％CMC-Na溶液至1000ml，充分混匀后使用。)10mL加入到第1只试管中；然后再取10mL的0.3％连翘脂素无菌0.5％CMC-Na溶液加入到第2只试管中，混匀后从第2只试管中取10mL混合液加入到第3只试管内，再混合均匀后取10mL混合液加入到第4只试管中，混匀后再从第4只试管中取10ml混合液计入到第5只试管中，混匀，如此连续稀释至第9只试管混匀后，取出10mL弃掉。第10只试管中只有无菌蒸馏水，以第10只试管中的无菌蒸馏水作为对照。这样1～9管含有不同浓度药液各10mL。1) Dilution of the drug solution: 10 sterile tubes are arranged in the test tube rack, and 10 mL of sterile distilled water is added to the 2nd to 10th tubes; then 0.3% forsythiaside sterile 0.5% CMC-Na solution is taken ( Weigh 3 g of forsythin, suspend it with an appropriate amount of 0.5% CMC-Na solution, add 0.5% CMC-Na solution to 1000 ml, mix well and use.) 10 mL is added to the first tube; then take 10 mL of 0.3% forsypolin sterile 0.5% CMC-Na solution was added to the second tube, and after mixing, 10 mL of the mixture was taken from the second tube and added to the third tube, and then mixed and taken. Add 10 mL of the mixture to the 4th test tube, mix well, then take 10 ml of the mixture from the 4th test tube into the 5th test tube, mix well, and then serially dilute to the 9th test tube, mix and remove Discard 10mL. In the 10th test tube, only sterile distilled water was used, and sterile distilled water in the 10th test tube was used as a control. Thus, 1 to 9 tubes contain 10 mL each of different concentrations of the drug solution.
2)含药平板制备2) Preparation of drug-containing plates
在无菌操作台上按照无菌操作方法，将已经溶化的无菌M-H琼脂900mL,分装于10个三角瓶中,每瓶90mL熔融的无菌M-H琼脂,将第1只试管中含有的10mL的0.3％连翘脂素无菌蒸馏水溶液放入到含90mL培养基的第1个三角瓶中，混合后平均倾倒至7个平板(即平皿)中，每个平板约含14mL含药(即连翘脂素)琼脂，凝固后备用，为第1含药琼脂平板组，其中：第1含药琼脂平板组每个平板中实际含连翘脂素3.0mg/ml。On a sterile workstation, according to the aseptic method, 900 mL of the solubilized sterile MH agar was dispensed into 10 flasks, 90 mL of molten sterile MH agar per flask, and 10 mL of the first tube was contained. The 0.3% forsypolin sterile distilled aqueous solution was placed in a first flask containing 90 mL of medium, and after mixing, it was poured onto 7 plates (ie, plates), and each plate contained about 14 mL of drug (ie, Forsythiaside) agar, after solidification, was used as the first drug-containing agar plate group, wherein: the first drug-containing agar plate group actually contained forsythin 3.0 mg/ml in each plate.
第2-10只试管内的溶液按照与第1只试管内的溶液同样的方法倾倒平板，制成第2-10含药平板组，每个平板组含有7个平板，每个平板约含14mL含药(即连翘脂素)琼脂，凝固后备用。第2含药琼脂平板组中每个平板实际含连翘脂素1.5mg/ml；第3含药琼脂平板组中每个平板实际含连翘脂素为0.75mg/ml；第4含药琼脂平板组中每个平板实际含连翘脂素为0.375mg/ml；第5含药琼脂平板组中每个平板实际含连翘脂素为0.1875mg/ml；第6含药琼脂平板组中每个平板实际含连翘脂素为0.0938mg/ml；第7含药琼脂平板组中每个平板实际含连翘脂素为0.0469mg/ml；第8含药琼脂平板组中每个平板实际含连翘脂素为0.0234mg/ml； 第9含药琼脂平板组中每个平板实际含连翘脂素为0.0117mg/ml；第10含药琼脂平板组中每个平板实际含连翘脂素为0.0mg/ml。The solutions in the 2-10th test tubes were poured into the same plate as the solution in the first test tube to prepare a 2-10 drug-containing plate set, each plate set containing 7 plates, each plate containing about 14 mL. Drug-containing (ie, forsythiaside) agar, ready for use after solidification. In the second drug-containing agar plate group, each plate actually contained forsythin 1.5 mg/ml; in the third drug-containing agar plate group, each plate actually contained forsythin was 0.75 mg/ml; the fourth drug-containing agar Each plate in the plate group actually contained forsypolin of 0.375 mg/ml; in the fifth drug-containing agar plate group, each plate actually contained forsythin 0.1875 mg/ml; in the sixth drug-containing agar plate group, The actual plate contains forsythiaside of 0.0938 mg/ml; in the seventh drug-containing agar plate group, each plate actually contains forsythiaside of 0.0469 mg/ml; in the eighth drug-containing agar plate group, each plate actually contains Forsythin is 0.0234mg/ml; In the 9th drug-containing agar plate group, each plate actually contained forsythin 0.0117 mg/ml; in the 10th drug-containing agar plate group, each plate actually contained forsythin 0.0 mg/ml.
3)用多点接种仪分别吸取各种菌液并将菌液点种于含不同药物浓度的琼脂平板上。于35℃培养18～24h,观察菌体生长情况，生长菌体为阳性(+)，不生长菌体为阴性(一)，以不生长菌体的最低浓度为最低抑菌浓度(MIC)。3) Using a multi-point inoculation instrument to separately absorb various bacterial liquids and plant the bacterial liquid on agar plates containing different drug concentrations. The cells were cultured at 35 ° C for 18-24 hours, and the growth of the cells was observed. The growth cells were positive (+), the non-growth cells were negative (1), and the lowest concentration of non-growth cells was the minimum inhibitory concentration (MIC).
2 实验结果2 Experimental results
金黄色葡萄球菌、大肠杆菌、枯草芽孢杆菌、酵母、桔青霉、黑曲霉、黄曲霉抑菌效果测定结果如表7所示。The results of the bacteriostatic effects of Staphylococcus aureus, Escherichia coli, Bacillus subtilis, yeast, Penicillium citrinum, Aspergillus niger, and Aspergillus flavus are shown in Table 7.
表7 连翘脂素对7种细菌的MICTable 7 MIC of forsythin for 7 bacteria
Figure PCTCN2016079843-appb-000043
Figure PCTCN2016079843-appb-000043
注：-表示无菌生长；+表示菌体生长少；++表示菌体生长较多；+++表示菌体生长多，下同Note: - indicates sterile growth; + indicates less growth of bacteria; ++ indicates more growth of bacteria; +++ indicates more growth of bacteria, the same below
由表7的研究结果表明,当连翘脂素达到3mg/ml时，其对金黄色葡萄球菌、酵母菌、桔青霉菌、黑曲霉菌等细菌的生长也没有抑制作用。因此可知连翘苷基本没有抗菌的作用。 The results of the study in Table 7 indicate that when forsythiaside reaches 3 mg/ml, it does not inhibit the growth of bacteria such as Staphylococcus aureus, yeast, Penicillium citrinum, and Aspergillus niger. Therefore, it can be seen that forsythin has substantially no antibacterial effect.

Claims (10)

  1. 连翘苷、连翘苷衍生物、连翘苷与连翘脂素组合物在制备用于预防和/或治疗细菌感染的药物或保健品中的应用。Use of forsythin, forsythin derivatives, forsythin and forsythiaside compositions for the preparation of a medicament or health supplement for the prevention and/or treatment of bacterial infections.
  2. 根据权利要求1所述的应用，其特征是所述药物由连翘苷、连翘苷衍生物或连翘苷与连翘脂素组合物和药学上可接受的载体组成。The use according to claim 1, wherein the drug consists of forsythin, forsythin derivative or forsythin and forsythiaside composition and a pharmaceutically acceptable carrier.
  3. 根据权利要求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、溶液剂形式存在。The use according to claim 1 or 2, characterized in that the medicament is in the form of tablets, capsules, pills, powders, granules, syrups, solutions.
  4. 根据权利要求1或2所述的应用，其特征是所述的连翘苷、连翘苷衍生物的纯度为≥1％；连翘苷与连翘脂素组合物的含量≥1％。The use according to claim 1 or 2, wherein the forsythin and forsythin derivatives have a purity of ≥ 1%; and the forsythin and forsythiaside compositions have a content of ≥ 1%.
  5. 根据权利要求4所述的应用，其特征是所述的连翘苷、连翘苷衍生物的纯度为1％～98％；连翘苷与连翘脂素组合物的含量为1％～98％。The use according to claim 4, wherein the forsythin and forsythin derivatives have a purity of from 1% to 98%; and the forsythin and forsythiaside compositions have a content of from 1% to 98%. %.
  6. 一种预防或/和治疗细菌感染的药物或保健品，其特征是含有连翘苷、连翘苷衍生物或连翘苷与连翘脂素组合物。A medicament or health care product for preventing or/and treating a bacterial infection, which comprises a forsythin, a forsythiaside derivative or a forsythiaside and a forsythiaside composition.
  7. 根据权利要求6所述的药物或保健品，其特征是所述的连翘苷、连翘苷衍生物的纯度为≥1％；连翘苷与连翘脂素组合物的含量≥1％。The pharmaceutical or nutraceutical according to claim 6, wherein said forsythin and forsythin derivatives have a purity of ≥ 1%; and the forsythin and forsythiaside compositions have a content of ≥ 1%.
  8. 根据权利要求6所述的药物或保健品，其特征是所述连翘苷、连翘苷衍生物或连翘苷与连翘脂素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。The pharmaceutical or nutraceutical according to claim 6, wherein the weight of the forsythin, forsythin derivative or forsythin and forsythiaside composition is the total weight of the drug or health care product. The ratio is 0.01-10:100.
  9. 根据权利要求6所述的药物或保健品，其特征是所述连翘苷与连翘脂素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。A pharmaceutical or nutraceutical according to claim 6 wherein said forsythin and forsythiaside compositions are selected from forsythiaside and forsythin and alpha-, beta- or gamma-cyclodextrin or A mixture of derivatives, or a complex formed by the treatment of forsythiaside and forsythin with α-, β- or γ-cyclodextrin or a derivative thereof by physical or chemical treatment.
  10. 根据权利要求6所述的药物或保健品，其特征是还包括穿心莲提取物，四季青提取物，金银花提取物，板蓝根提取物，蒲公英提取物，黄柏提取物，黄连提取物，黄芩提取物，马齿苋提取物，白头翁提取物，大蒜提取物，鱼腥草提取物，野菊花提取物，苦参提取物、维生素C及其衍生物或维生素E及其衍生物。 The medicine or health care product according to claim 6, further comprising an extract of Andrographis paniculata, Silybum extract, Honeysuckle extract, Radix Isatidis extract, dandelion extract, Cortex extract, Rhizoma Coptidis extract, Astragalus extract, Portulaca oleracea extract, Pulsatilla extract, garlic extract, Houttuynia cordata extract, wild chrysanthemum extract, Sophora flavescens extract, vitamin C and its derivatives or vitamin E and its derivatives.
PCT/CN2016/079843 2015-04-23 2016-04-21 Use of forsythin, derivatives thereof and composition of forsythin/forsythiaside in the preparation of drug for treating bacterial infections WO2016169491A1 (en)

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