WO2013023339A1 - Acide sécologanolique, composition pharmaceutique à base de ce composé et utilisation - Google Patents

Acide sécologanolique, composition pharmaceutique à base de ce composé et utilisation Download PDF

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WO2013023339A1
WO2013023339A1 PCT/CN2011/078333 CN2011078333W WO2013023339A1 WO 2013023339 A1 WO2013023339 A1 WO 2013023339A1 CN 2011078333 W CN2011078333 W CN 2011078333W WO 2013023339 A1 WO2013023339 A1 WO 2013023339A1
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antibiotic
bacteria
bacterium
resistant
caused
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PCT/CN2011/078333
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English (en)
Chinese (zh)
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石建功
张铁军
孟红
韩丰年
马旭伟
李竹兰
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甘肃普源医药科技有限公司
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Priority to CN201180072835.2A priority Critical patent/CN103889431B/zh
Priority to PCT/CN2011/078333 priority patent/WO2013023339A1/fr
Publication of WO2013023339A1 publication Critical patent/WO2013023339A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Kailuanic acid pharmaceutical composition containing the same, and use thereof
  • the invention belongs to the field of pharmaceutical technology. Specifically, the present invention relates to the use of a plant extract obtained by extracting and purifying a traditional Chinese medicine honeysuckle or a plant of the same origin, Lonicera japonica or the same genus, for the preparation of an antibacterial agent. Background technique
  • “Honeysuckle” comes from the “Compendium of Materia Medica”, which is the collective name for Chinese herbal medicines and plants.
  • Plant honeysuckle also known as honeysuckle, is a perennial, semi-evergreen entwined woody vine of the family Lonicerae. Because the honeysuckle flower is initially white, and then turned yellow, it is named honeysuckle.
  • the medicinal material honeysuckle is the honeysuckle of the honeysuckle family and the dried flower buds or the first flowers of the same genus.
  • Honeysuckle has been praised as a good medicine for clearing away heat and detoxification since ancient times. It is one of the common antipyretic drugs commonly used in traditional Chinese medicine. It has been used in clinical practice for thousands of years and is deeply loved by doctors and patients. It is sweet and cold, fragrant, sweet and cold, without hurting the stomach, and it is fragrant and fragrant. Honeysuckle can not only dispel the wind and heat, but also clear the blood poison, used in a variety of heat and sexual diseases, such as body heat, rash, hair spots, heat sores, sore throat and so on, all have significant effects. In modern studies, the mechanism of heat-clearing and detoxifying honeysuckle was discussed. For example, honeysuckle has obvious antipyretic and anti-inflammatory effects.
  • honeysuckle extract 0.25g / kg can inhibit rat carrageenan foot swelling; injection of 30-40g / kg can reduce the degree of egg whiteness; 8g / kg, 2 times / day, also has obvious anti-exudation and anti-proliferation effects on rat croton oil granules.
  • honeysuckle has an important regulatory effect on the body's immune system. Honeysuckle decoction promotes the phagocytic function of leukocytes; intraperitoneal injection of honeysuckle injection also significantly promotes the phagocytic function of inflammatory cells.
  • honeysuckle contained organic acids, triterpenoid saponins, flavonoids and their glycosides, iridoid glycosides, volatile oils and the like.
  • the main active ingredient in honeysuckle is a chlorogenic acid compound; in addition, there is a volatile component linalool. Therefore, the quality of honeysuckle is usually evaluated by the content of chlorogenic acid.
  • the extraction and refining process of honeysuckle is mostly based on chlorogenic acid compounds, and a small part is based on volatile components.
  • chlorogenic acid has antibacterial and broad-spectrum antibacterial and antiviral effects, it is not the only antipyretic, bacteriostatic and antiviral component of honeysuckle, and chlorogenic acid can be inactivated by protein in vivo, its antibacterial and antibiotic resistance. The virus is not very effective. Moreover, the chlorogenic acid compound has Sensitization has also been recognized by more than a few researchers. Therefore, the active ingredients and mechanism of action of antipyretic, analgesic, anti-inflammatory, antibacterial and antiviral of honeysuckle are not fully understood.
  • Patent No. ZL200610083556.7 discloses a plant prepared from Lonicera japonica Thunb. or its plant origin Lonicera japonica or other plants of the same genus A method of extracting, and a honeysuckle extract prepared by the method and a pharmaceutical composition containing the extract.
  • the invention discloses that the active ingredient in the prepared honeysuckle extract is Kailuan sylvestic acid and a derivative thereof, and the honeysuckle extract is found to have antipyretic, analgesic, anti-inflammatory, antibacterial and anti-viral properties. active.
  • the invention has no clear research contents and results on the antibacterial action of the prepared honeysuckle extract. Summary of the invention
  • An object of the present invention is to provide an open-linked sassafrasic acid which is used as a prophylactic and/or therapeutic agent for diseases caused by bacteria.
  • the saplings are prepared from honeysuckle or its plant-derived plant Lonicera japonica or other plants of the same species.
  • Another object of the present invention is to provide a pharmaceutical composition comprising the open-linked saponin. Still another object of the present invention is to provide use of the open-linked saponin or pharmaceutical composition for the preparation of a medicament for preventing and/or treating a disease caused by a bacterium.
  • the above technical solution is achieved by the following:
  • the present invention provides an open sapogenin which is used as a prophylactic and/or therapeutic agent for a disease caused by bacteria.
  • the honeysuckle extract described herein can be prepared according to the method disclosed in the patent ZL200610083556.7 The disclosure of this patent is hereby incorporated by reference in its entirety.
  • the saponin is prepared from the traditional Chinese medicine honeysuckle or the plant of the same origin, Lonicera japonica or the same genus.
  • the open-linked saponin of the present invention is prepared by a method comprising the following steps:
  • step (1) to give the extract concentrated under normal pressure or under reduced pressure and dried to give extract, or spray dried to obtain a powder, after redissolution with water, containing not more than 95% by volume aqueous C r C 6 alkanol Performing precipitation or sedimentation to obtain a precipitate or a solution concentrate;
  • the preparation method of the open-linked saponin comprises the following steps:
  • the extract obtained in the step (1) is concentrated under reduced pressure to obtain an extract, dissolved in water, filtered, and concentrated to dryness, dissolved in 95% (v/v) aqueous solution of ethanol, and distilled water is added to make the solution contain 75% ethanol. (volume/volume), filtered after standing, and the filtrate is recovered from the flow extract of ethanol;
  • the preparation method further comprises purifying the eluate containing the iridoid compound obtained in the step (3) by gel chromatography;
  • the preparation method further comprises purifying the eluate containing the iridoid compound obtained in the step (3) through a Sephadex LH-20 gel column, and collecting the water eluate.
  • the open-linked saponin of the present invention can be used for preparing a medicament for preventing and/or treating diseases caused by bacteria, the bacteria being antibiotic-resistant bacteria, preferably multi-antibiotic resistant bacteria; specifically, the antibiotic selection From ampicillin, penicillin, piperacillin, tazobactam, amoxicillin, clavulanic acid, cefazolin, cefuroxime, ceftriaxone, cefuroxime sodium, sulpiride, levofloxacin , cefotaxime, ceftazidime, imipenem, cefepime, cefoxitin, gentamicin, amikacin, ciprofloxacin, chloramphenicol, compound sulfamethoxazole, tetracycline, nitrofurantoin, ammonia South, ciprofloxacin, norfloxacin, amika, sulbactam, ticarcillin, clavulanic acid, tobramycin, star, imipe
  • the bacterium may be a multi-antibiotic-resistant Gram-negative bacterium; further preferably, the multiplex antibiotic-resistant Gram-negative bacterium is selected from the group consisting of Escherichia coli, Pseudomonas aeruginosa, Krebs One or more of Bacillus, Acinetobacter baumannii, Proteus, Enterobacter, Haemophilus influenzae, Klebsiella, and Kaposi; more preferably, the multiple antibiotic resistant Gram The negative bacteria are Escherichia coli, Pseudomonas aeruginosa and/or Klebsiella.
  • the bacterium is a Gram-positive bacterium resistant to multiple antibiotics; further preferably, the multiplex-antibiotic-resistant Gram-positive bacterium is selected from the group consisting of Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and suppuration One or more of Streptococcus group A, Streptococcus pneumoniae, Bacillus subtilis, and Staphylococcus epidermidis; more preferably, the multi-antibiotic resistant Gram-positive bacteria are methicillin-resistant Staphylococcus aureus , Staphylococcus aureus, Bacillus subtilis and/or Staphylococcus epidermidis.
  • the diseases caused by the bacteria are infection-related diseases caused by bacteria, particularly infectious diseases caused by antibiotic-resistant bacteria, such as digestive system infections, blood system infections, respiratory infections, urinary tract infections, central nervous system infections, One or more of a bone joint infection, an ear, a mastoid and/or sinus infection, and a skin soft tissue infection; preferably, the disease caused by the bacteria is a respiratory infection caused by an antibiotic resistant bacteria.
  • the disease caused by the bacteria may be a respiratory infection caused by a multi-antibiotic-resistant Gram-negative bacterium; preferably, the disease caused by the bacterium is infectious by a multi-antibiotic-resistant Gram-negative bacterium Pneumonia; further preferably, the disease caused by the bacterium is a nosocomial pneumonia caused by a multi-antibiotic-resistant Gram-negative bacterium; more preferably, the disease caused by the bacterium is a multi-antibiotic-resistant Klebs Nosocomial infectious pneumonia caused by bacilli.
  • the disease caused by the bacterium is a respiratory infection caused by a multi-antibiotic-resistant Gram-positive bacterium; preferably, the disease caused by the bacterium is a pneumonia caused by a multi-antibiotic-resistant Gram-positive bacterium; further Preferably, the disease caused by the bacteria is pneumonia caused by methicillin-resistant Staphylococcus aureus.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the above-described kaimarinic acid; preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or a form Agent.
  • the present invention provides the use of the above-described kaimarinic acid or the above pharmaceutical composition for the preparation of a medicament for preventing and/or treating a disease caused by bacteria.
  • the bacteria are antibiotic resistant bacteria, preferably multiple antibiotic resistant bacteria.
  • the bacterium is a multi-antibiotic-resistant Gram-negative bacterium; further preferably, the multiplex antibiotic-resistant Gram-negative bacterium is selected from the group consisting of Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae , Acinetobacter baumannii, Proteus, Enterobacter, Haemophilus influenzae, Klebsiella pneumoniae And one or more of the genus of the genus; more preferably, the gram-negative bacteria resistant to the multiple antibiotics are Escherichia coli, Pseudomonas aeruginosa and/or Klebsiella.
  • the bacterium is a Gram-positive bacterium resistant to multiple antibiotics; further preferably, the multiplex-antibiotic-resistant Gram-positive bacterium is selected from the group consisting of Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and suppuration One or more of Streptococcus group A, Streptococcus pneumoniae, Bacillus subtilis, and Staphylococcus epidermidis; more preferably, the multi-antibiotic resistant Gram-positive bacteria are methicillin-resistant Staphylococcus aureus And / or Staphylococcus aureus.
  • the diseases caused by the bacteria are infection-related diseases caused by bacteria, particularly infectious diseases caused by antibiotic-resistant bacteria, such as digestive system infections, blood system infections, respiratory infections, urinary tract infections, central nervous system infections, One or more of a bone joint infection, an ear, a mastoid and/or sinus infection, and a skin soft tissue infection; preferably, the disease caused by the bacteria is a respiratory infection caused by the antibiotic resistant bacteria.
  • the disease caused by the bacteria is a respiratory infection caused by a multi-antibiotic-resistant Gram-negative bacteria; further preferably, the disease caused by the bacteria is an infection caused by a multi-antibiotic-resistant Gram-negative bacteria More preferably, the disease caused by the bacterium is a nosocomial pneumonia caused by a multi-antibiotic-resistant Gram-negative bacterium; and more preferably, the disease caused by the bacterium is a multi-antibiotic resistant Kreb Nosocomial pneumonia caused by Bacillus.
  • the disease caused by the bacterium is a respiratory infection caused by a multi-antibiotic-resistant Gram-positive bacterium; preferably, the disease caused by the bacterium is a pneumonia caused by a multi-antibiotic-resistant Gram-positive bacterium; further Preferably, the disease caused by the bacteria is pneumonia caused by methicillin-resistant Staphylococcus aureus.
  • the present invention provides a method of preventing and/or treating a disease caused by a bacterium, the method comprising administering to a patient in need thereof a therapeutically effective amount of the above-described kaimarisin and/or the combination of the above Things.
  • the bacteria are antibiotic resistant bacteria, preferably multiple antibiotic resistant bacteria.
  • the bacterium is a multi-antibiotic-resistant Gram-negative bacterium; further preferably, the multiplex antibiotic-resistant Gram-negative bacterium is selected from the group consisting of Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae , one or more of Acinetobacter baumannii, Proteus, Enterobacter, Haemophilus influenzae, Klebsiella pneumoniae and catarrhalis; more preferably, the multi-antibiotic resistant Gram negative The bacteria are Escherichia coli, Pseudomonas aeruginosa and/or Klebsiella.
  • the bacterium is a Gram-positive bacterium resistant to multiple antibiotics; further preferably, the multiplex-antibiotic-resistant Gram-positive bacterium is selected from the group consisting of Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and suppuration Streptococcus group A, Streptococcus pneumoniae, Bacillus subtilis and Staphylococcus epidermidis More preferably, the multi-antibiotic resistant Gram-positive bacteria are methicillin-resistant Staphylococcus aureus and/or Staphylococcus aureus.
  • the diseases caused by the bacteria are infection-related diseases caused by bacteria, particularly infectious diseases caused by antibiotic-resistant bacteria, such as digestive system infections, blood system infections, respiratory infections, urinary tract infections, central nervous system infections, One or more of a bone joint infection, an ear, a mastoid and/or sinus infection, and a skin soft tissue infection; preferably, the disease caused by the bacteria is a respiratory infection caused by the antibiotic resistant bacteria.
  • the disease caused by the bacteria is a respiratory infection caused by a multi-antibiotic-resistant Gram-negative bacteria; further preferably, the disease caused by the bacteria is an infection caused by a multi-antibiotic-resistant Gram-negative bacteria More preferably, the disease caused by the bacterium is a nosocomial pneumonia caused by a multi-antibiotic-resistant Gram-negative bacterium; and more preferably, the disease caused by the bacterium is a multi-antibiotic resistant Kreb Nosocomial pneumonia caused by Bacillus.
  • the disease caused by the bacterium is a respiratory infection caused by a multi-antibiotic-resistant Gram-positive bacterium; preferably, the disease caused by the bacterium is a pneumonia caused by a multi-antibiotic-resistant Gram-positive bacterium; further Preferably, the disease caused by the bacteria is pneumonia caused by methicillin-resistant Staphylococcus aureus.
  • chlorogenic acid and its derivatives are considered to have antibacterial and antiviral activities, the study of the substance of the heat-clearing and detoxifying substance of honeysuckle has ignited a climax. Many research institutions or laboratories have devoted themselves to the separation and extraction of the active constituents of honeysuckle.
  • the inventors have found through extensive experiments that the saponin has the function of inhibiting and/or killing bacteria, especially antibiotic-resistant bacteria, in vitro and in vivo, including significant neutralizing bacterial endotoxin and Antipyretic effect.
  • the bacteria include Gram-negative bacteria and Gram-positive bacteria, and the effect on Gram-negative bacteria is more prominent than that of Gram-positive bacteria.
  • the half-inhibitory concentration of Kailuoside from some Gram-negative bacteria, such as Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa is about 1 mg/ml, against Gram
  • the half-bacterial concentration of positive bacteria, such as Bacillus also reached 0.92 mg/ml. All of these indicate that the saponin has a strong antibacterial effect on multi-drug resistant bacteria.
  • Fig. 1 is a HPLC chromatogram of the honeysuckle extract N4 prepared in Example 1 of the present invention, which was determined to contain 52.6% by weight of open-linked saponin.
  • Fig. 2 is a HPLC chromatogram of the honeysuckle extract N3 prepared in Example 1 of the present invention, which was determined to contain 79.3% by weight of open-linked saponin.
  • Fig. 3 is a HPLC chromatogram of the honeysuckle extract N2 prepared in Example 1 of the present invention, which was determined to contain 84.7% by weight of open-linked sassafras.
  • Fig. 4 is a HPLC chromatogram of the honeysuckle extract N1 prepared in Example 1 of the present invention, which was determined to contain 90.67% by weight of open-linked saponin.
  • Figure 5 is a HPLC chromatogram of the open-linked saponin standard used in the examples of the present invention, and the purity was 98.03% (by weight). The best way to implement the invention
  • the experimental methods in the following examples are conventional methods unless otherwise specified.
  • the raw materials, reagent materials, etc. used in the following examples can be purchased from conventional biochemical reagent stores or pharmaceutical companies without special instructions. among them:
  • honeysuckle medicinal materials used in the examples were purchased from Beijing Tongrentang Chain Pharmacy, and the place of origin was Henan. It was processed by Peizhou Jingyi Traditional Chinese Medicine Pieces Factory, batch number 200502014, and was identified as the Lonicera japonica Thunb. Dry flower buds.
  • the open-linked saponin used in the examples which is used as a standard for the determination of extract content and as a drug for pharmacological experiments (this tube is called NO), and is a natural medicinal chemistry laboratory of the Institute of Materia Medica, Chinese Academy of Medical Sciences. It was prepared according to the method disclosed in the patent ZL200610083556.7, and the determined content was 98.03% (the chromatogram is shown in Fig. 5).
  • the HPLC method was used for the determination of the content of the open-linked saponin in the honeysuckle extract, and the following instruments and conditions were specifically used:
  • Reagents Chromatography with acetonitrile; purified water; analytically pure acetic acid.
  • the strains used in the examples were Escherichia coli, Pseudomona aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, and Staphylococcus epidermidis.
  • the clinical isolate which is a multi-drug resistant strain, is provided by the Clinical Laboratory of the Fourth Hospital of Jinan City, Shandong province. The drug resistance is shown in Tables 1, 2, 3, 4, and 5 below.
  • Bacillus subtilis is provided by the Institute of Microbiology, Chinese Academy of Sciences.
  • R resistance
  • S sensitive
  • I mediation (moderate resistance).
  • AMP ampicillin
  • PIP piperacillin
  • TZP piperacillin/tazobactam
  • AMC amoxicillin/clavulanic acid
  • CZO cefazolin
  • CXM cefuroxime
  • CTX cefotaxime
  • CAZ ceftazidime
  • CRO ceftriaxone
  • IPM imipenem
  • FEP cefepime
  • FOX cefoxitin
  • GEN gentamicin
  • AMK amikacin
  • CIP ciprofloxacin
  • CHL chloramphenicol
  • SXT compound sulfamethoxazole
  • TCY tetracycline
  • NIT nitrofurantoin.
  • AK amikacin
  • ATM aztreonam
  • AMP ampicillin
  • CRO ceftriaxone
  • CLS Shupushen
  • CXM cefuroxime sodium
  • CAZ ceftazidime
  • CTX cefotaxime
  • CFP Cefoperazone
  • CIP ciprofloxacin
  • CN Qingda
  • FEP cefepime
  • LEV levooxyfluoride
  • NOR norfloxacin
  • PIP piperacillin
  • SAM amika/sulbactam
  • TIM ticarcillin / clavulanic acid
  • TOB refractory
  • TZP special star
  • IPM imipenem
  • MH minocycline
  • MEM meropenem.
  • PEN penicillin
  • OXA oxacillin
  • SAM ampicillin/penicillin
  • FOX cefoxitin
  • GEN gentamicin
  • AMK amikacin
  • CIP ciprofloxacin
  • VAN vancomycin
  • RIF rifampicin
  • SXT compound sulfamethoxazole
  • TCY tetracycline
  • CLI clindamycin
  • NIT nitrofurantoin.
  • tissue sectioning operation used in the examples is based on "Pathology Technology", People's Medical Publishing House, 1st edition, 39-41; Hematoxylin-Eosin staining operation according to "Pathology Technology", People's Medical Publishing House, No. 1 edition, 41-43 pages.
  • Constant temperature incubator Shanghai Yuejin Medical Devices No. 1 Factory.
  • Spectrophotometer produced by East China Electronic Tube Factory.
  • Microplate reader Product of the Swiss company Leibo.
  • honeysuckle medicinal material 500g
  • coarsely pulverize it and extract it twice with 50% (vol/vol) ethanol aqueous solution with a weight of 13 times dry weight of the honeysuckle medicinal material, and extract for 1 hour each time.
  • the extracts were combined, concentrated to a thick extract under reduced pressure, dissolved in 450 ml of distilled water, cooled to room temperature, allowed to stand for 24 hours, and filtered to give a clear solution.
  • the clear solution was concentrated to dryness under reduced pressure, and 1600 ml of a 95% (v/v) aqueous solution of ethanol was added thereto, and the mixture was stirred well, and distilled water was slowly added thereto to make the solution contain 75% (v/v) of ethanol, allowed to stand for 24 hours, and filtered. The filtrate was collected, and the ethanol was recovered under reduced pressure to a flow extract.
  • the flow extract was added with water (500 ml), dissolved and filtered, and the filtrate was passed through a pretreated styrene SP-825 macroporous adsorption resin color column, followed by 5 times the amount of resin column volume of water, and then 6 20% (vol/vol) of the volume of the resin column was eluted with an aqueous ethanol solution, and the ethanol was recovered under reduced pressure to give an alcohol-free taste, and lyophilized to obtain 4.31 g of honeysuckle extract (Hollywood extract N4).
  • the HPLC extract was used to determine 52% of the extract of the honeysuckle extract N4 (see Figure 1 for chromatogram).
  • honeysuckle extract N4 was further purified by Sephadex LH-20 gel column, including elution with water, and the sample liquid was collected, recovered under reduced pressure, and lyophilized, and the obtained product was purified by the gel column several times to obtain respectively.
  • N2 contains open hibiscus Glycoside 84.7% (see Figure 3 for chromatogram); N1 contains 90.67% of open sapogenin (see Figure 4 for chromatogram).
  • Example 2 Inhibition of clinically resistant strains by honeysuckle extract and Kailuan saponin
  • honeysuckle extracts Nl, N2, N3, N4 and Kailuan saponin (NO) prepared in Example 1 were tested for clinically resistant strains of Escherichia coli, Pseudomonas aeruginosa and Klebsiella.
  • NO Kailuan saponin
  • the above six strains stored in lyophilization were inoculated into a nutrient broth liquid medium. Take about 4ml of broth culture medium, add 1ml of liquid strain, incubate at 37 °C for 20 hours, observe Escherichia coli, Klebsiella, Staphylococcus aureus, Staphylococcus epidermidis; Bacillus subtilis, patina False order The bacterium grows on the surface to form a bacterial membrane. The inoculation culture was repeated to obtain an activated strain.
  • test sample honeysuckle extracts Nl, N2, N3, N4 and Kailuan saponin (NO) were diluted with PBS to a concentration of 40 mg/ml, and then serially diluted by 2 times (1:2, 1 : 4). , 1 : 8, 1 : 16, 1 : 32, 1 : 64, 1 : 128 ).
  • the activated bacterial suspension was diluted to a concentration equivalent to 0.5 McMurray standard, and after dilution with broth medium 1:10, ⁇ broth was added to each well.
  • the stock solution and the different concentrations of the antibacterial drug solution after dilution are separately added to the sterilized 96-well plate, and the first to eighth holes are added with the drug solution, and each well is ⁇ , at this time, the drug concentration of the first hole to the eighth hole 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625 mg/mL
  • the microplate reader was tested at 630 nm, and the inhibition rate of bacteria was calculated by Formula 2, and the obtained data was substituted into Formula 1 to calculate the specific distance, and the ratio was less than 50% of the disease rate virus dilution index.
  • Half of the bacteriostatic concentration was obtained (Reed-Muench method).
  • Bacterial inhibition rate (inhibition test OD value - drug control OD value) / (bacterial solution control OD value - blank control OD value) ...
  • the honeysuckle extract and the open-linked saponin-like extract having a certain content of the open-shelled saponin have certain inhibitory effects on the above-mentioned clinical resistant bacteria and spores, and The effect on Gram-negative bacteria is more pronounced than that of Gram-positive bacteria.
  • the half-inhibitory concentration of saponin from Klebsiella, Escherichia coli and Pseudomonas aeruginosa was about 1 mg/ml, and the half inhibitory concentration against Gram-positive Bacillus Also reached 0.92 mg / ml.
  • Example 3 Observation of the therapeutic effect of honeysuckle extract on a mouse-Klebsiella pneumonia model This example observes the in vivo effect of the honeysuckle extract N1 prepared in Example 1 on a mouse-Klebsiella pneumonia model.
  • the bacterial test strain used in this example is Klebsiella spp. in Table 1, and is specifically isolated from the sputum sample of clinical pneumonia patients of the Fourth People's Hospital of Jinan City. The drug resistance is shown in Table 1.
  • Test drug Honeysuckle extract N 1 , milky white crystalline powder.
  • Gentamicin Sulfate Injection (Specification: 2ml 80,000 units; purchased from Tianjin Pharmaceutical Co., Ltd., production batch number 09082732)
  • Ceftazidime for injection (Specification: lg; purchased from Shandong Weiqida Pharmaceutical Co., Ltd., production batch number 20081204)
  • Kunming mice were randomly divided into normal control group, model control group, honeysuckle extract N1 curative model group, large, medium and small dose groups, drug resistant control group (gentamicin) and sensitive drug control group (ceftazidime). There were 10 animals in each group and a total of 70 animals.
  • the normal control group was inoculated with bacterial culture medium 50 ⁇ , and the other groups of animals were inoculated with Klebsiella bacterial liquid 5 ( ⁇ L (freshly expanded bacterial solution diluted to 1.5 ⁇ 10 8 CFU/ml).
  • the drug-resistant control group (gentamicin group) was intramuscularly injected with gentamicin daily at 400 units/unit (200 ⁇ ) once a day;
  • the sensitive drug control group (ceftazidime) was injected daily with 30mg/200ml, once a day;
  • the honeysuckle extract N1 efficacy model group was injected with honeysuckle extract N1 (200 ⁇ ) daily, once a day, the dosage is shown in Table 8. ;
  • Normal control group and model control group were injected with normal saline 200 ⁇ once a day for 2 days; continuous intervention for 6 days.
  • the normal control group showed that the lung tissue was milky white and shiny
  • the lungs of the model control group showed blood red color, purple-red spots or plaques, and sometimes the whole lung lobe showed consolidation (the T T sinking in formalin) or the atelectasis of individual lung lobe (the lobe atrophied into small
  • the lung tissue of the animal was red, and it was purple-red.
  • the lung tissue of the drug-resistant control group was similar to that of the model group;
  • the lung tissue of the animal was white with occasional red spots.
  • the paraffin-embedded HE (hematoxylin-eosin) stained tissue sections were examined by microscopy. The scores were scored according to the following tissue case scoring system. The results are shown in Table 10.
  • Pathology scoring system (References: [l] Martin RJ, Chu HW, Honour JM, Airway inflammation and bronchial hypersponsiveness after mycoplasma pneumoniaw infection in a murine model J Am J Reapir Cell Mol Biol. 2001.24:577; [2] Xiaohong , Xin Deli, Hou Ancun et al. Establishment of a mouse model of Mycoplasma pneumoniae pneumonia and application of histopathological scoring method, Chongqing Medical 2004: vol 33(9): 1338-1340 ):
  • honeysuckle extract N1 has significant anti-Klebsiella pneumoniae-mouse pneumonia effect in vivo, and has a dose-effect relationship, further indicating that its active ingredient is open-linked saponin The antibacterial effect of acid.

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Abstract

La présente invention concerne l'acide sécologanolique utilisé en tant qu'agent préventif et/ou agent thérapeutique pour des maladies provoquées par des bactéries. L'acide sécologanolique peut résister à diverses bactéries résistantes à de multiples antibiotiques y compris des bactéries Gram positif et des bactéries Gram négatif résistantes à de multiples médicaments; et ledit acide sécologanolique peut être utilisé dans la préparation de médicaments destinés à la prévention et/ou au traitement de maladies provoquées par des bactéries, spécialement des bactéries résistantes à de multiples antibiotiques. L'invention concerne également des compositions pharmaceutiques renfermant de l'acide sécologanolique. L'invention a également pour objet une méthode de prévention et/ou de traitement de maladies provoquées par des bactéries, comprenant l'administration d'une quantité thérapeutiquement efficace d'acide sécologanolique et/ou d'une composition à base de ce composé à des patients en ayant besoin.
PCT/CN2011/078333 2011-08-12 2011-08-12 Acide sécologanolique, composition pharmaceutique à base de ce composé et utilisation WO2013023339A1 (fr)

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CN201180072835.2A CN103889431B (zh) 2011-08-12 2011-08-12 开联番木鳖苷酸、包含其的药物组合物及其用途
PCT/CN2011/078333 WO2013023339A1 (fr) 2011-08-12 2011-08-12 Acide sécologanolique, composition pharmaceutique à base de ce composé et utilisation

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CN106380493A (zh) * 2016-08-29 2017-02-08 中山大学 一种分离纯化开联番木鳖苷酸的方法

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CN101085795A (zh) * 2006-06-07 2007-12-12 石家庄汉康生化药品有限公司 金银花提取物,其制备方法和应用

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CN101085795A (zh) * 2006-06-07 2007-12-12 石家庄汉康生化药品有限公司 金银花提取物,其制备方法和应用

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106380493A (zh) * 2016-08-29 2017-02-08 中山大学 一种分离纯化开联番木鳖苷酸的方法
CN106380493B (zh) * 2016-08-29 2019-03-29 中山大学 一种分离纯化开联番木鳖苷酸的方法

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CN103889431B (zh) 2016-07-06

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