WO2013023339A1

WO2013023339A1 - Secologanolic acid, pharmaceutical composition comprising same and use thereof

Info

Publication number: WO2013023339A1
Application number: PCT/CN2011/078333
Authority: WO
Inventors: 石建功; 张铁军; 孟红; 韩丰年; 马旭伟; 李竹兰
Original assignee: 甘肃普源医药科技有限公司
Priority date: 2011-08-12
Filing date: 2011-08-12
Publication date: 2013-02-21
Also published as: CN103889431A; CN103889431B

Abstract

A secologanolic acid used as a preventive agent and/or a therapeutic agent for diseases caused by bacteria. The secologanolic acid can resist various multiple antibiotic resistant bacteria including multidrug-resistant grampositive bacteria and gramnegative bacteria; and can be used in the preparation of drugs for the prevention and/or treatment of diseases caused by bacteria, especially multiple antibiotic resistant bacteria. Pharmaceutical compositions comprising secologanolic acid are included. A method for the prevention and/or treatment of diseases caused by bacteria, comprising the administration of a therapeutically effective amount of secologanolic acid and/or a pharmaceutical composition thereof to patients in need thereof.

Description

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. Intraperitoneal injection of 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. In addition, it is believed that 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.

Studies on the chemical constituents of honeysuckle showed that the honeysuckle contained organic acids, triterpenoid saponins, flavonoids and their glycosides, iridoid glycosides, volatile oils and the like. Although various components have been isolated and identified from honeysuckle, it is currently considered that 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. Although 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, the invention patent entitled "Honeysuckle Extract, Preparation Method and Application" 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. However, the invention has no clear research contents and results on the antibacterial action of the prepared honeysuckle extract. Summary of the invention

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.

It is still another object of the present invention to provide a method of preventing and/or treating a disease caused by bacteria. The above technical solution is achieved by the following:

In one aspect, the present invention provides an open sapogenin which is used as a prophylactic and/or therapeutic agent for a disease caused by bacteria.

Wherein the open-linked saponin has the structural formula (I):

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. According to a specific embodiment of the present invention, the saponin is prepared from the traditional Chinese medicine honeysuckle or the plant of the same origin, Lonicera japonica or the same genus. Specifically, the open-linked saponin of the present invention is prepared by a method comprising the following steps:

(1) pulverizing plant honeysuckle or its plant of origin Lonicera or other plants of the same genus, and then extracting with water and/or an aqueous solution of _Cr C ₆ alkanol having a content of not more than 95% by volume to obtain an extract;

(2) The 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;

(3) separating or purifying the precipitate or the solution concentrate obtained by the step (2) by chromatography, and collecting an eluate containing an iridoid compound, wherein the chromatographic method is selected from a macroporous adsorption resin column. One or more of chromatography, normal phase silica gel chromatography, and reverse phase silica gel chromatography;

Preferably, the preparation method of the open-linked saponin comprises the following steps:

(1) pulverizing the honeysuckle medicinal material and extracting it with 50% (vol/vol) aqueous ethanol solution to obtain an extract;

(2) 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;

(3) Dissolving and filtering the flow extract obtained in the step (2), and filtering the filtrate through a styrene macroporous adsorption resin color column, and then eluting with water, 20% (vol/vol) aqueous ethanol solution, and recovering 20%. (volume/volume) ethanol in the eluent of the aqueous ethanol solution;

More preferably, the preparation method further comprises purifying the eluate containing the iridoid compound obtained in the step (3) by gel chromatography;

Most preferably, 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, imipenem, minocycline, meropenem, penicillin, One or more of oxacillin, erythromycin, vancomycin, rifampicin and clindamycin. Preferably, 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.

Alternatively, 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.

In addition, 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.

Wherein, 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.

Alternatively, 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.

In another aspect, the present invention provides a pharmaceutical composition comprising the above-described kaimarinic acid; preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or a form Agent.

In still another aspect, 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.

Wherein, the bacteria are antibiotic resistant bacteria, preferably multiple antibiotic resistant bacteria.

Preferably, 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.

Alternatively, 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.

In addition, 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.

Preferably, 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.

In a further aspect, 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.

Preferably, 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.

Alternatively, 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.

Alternatively, 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. After 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.

In addition, it was found that the extract of Lonicera japonicus containing higher content of Kailuan saponin showed outstanding efficacy in in vitro antibacterial and in vivo treatment of pneumonia, and the content was significantly positively correlated with the effect of extract, further indicating that Kailianmu The effect of guanylate. DRAWINGS Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings:

BRIEF DESCRIPTION OF THE DRAWINGS 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 present invention is further described in detail with reference to the preferred embodiments thereof.

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:

The 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).

In the examples, 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:

(1) Instrumentation Agilent 1100 liquid chromatograph, including quaternary pump, autosampler, DAD detector and Chemstation color program workstation;

( 2 ) Chromatographic conditions and system suitability test: The column is Prevail C ₁₈ 5μ(250ηιηιΧ4.6ηιηι) [Alltech, USA]; acetonitrile-1% aqueous glacial acetic acid (13: 87) is mobile phase, gradient elution; flow rate 0.9 ml/min, stop time 40 minutes, equilibration time 10 minutes, detection wavelength 242 nm. The number of theoretical plates should be no less than 1000 according to the JYH peak.

(3) 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.

Table 1: Background material for experimental strains of Klebsiella

Notes for Tables 1 and 2:

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.

Table 3 Pseudomonas aeruginosa background information

Remarks: R: resistance; S: sensitive; I: mediation.

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.

Table 4 Staphylococcus aureus background information

Notes to Tables 4 and 5:

R: resistance; S: sensitive; I: mediation.

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.

The 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.

Equipment used in the examples:

Constant temperature incubator: Shanghai Yuejin Medical Devices No. 1 Factory.

Purification Workbench: Jinan Longhong Company's products.

Spectrophotometer: produced by East China Electronic Tube Factory.

Microplate reader: Product of the Swiss company Leibo.

722 spectrophotometer: Shanghai Precision Scientific Instrument Co., Ltd. products.

-20 °C refrigerator: China Qingdao Debei company products.

-80 °C refrigerator: US FOMAS products. Centrifuge: Heraeus Benchtop Thermostat Centrifuge: Heraeus products.

Further, the laboratories conducting experiments in the examples were BSL-2 Laboratories and ABSL-2 Laboratories. Example 1 Preparation of Honeysuckle Extract

Take the 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).

Using Kailuan saponin as a reference, the HPLC extract was used to determine 52% of the extract of the honeysuckle extract N4 (see Figure 1 for chromatogram).

The 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. Honeysuckle extracts N3, N2 and Nl. Using open-labeled saponin as a reference, the HPLC method was used to determine the content of the extract of N. sinensis in N3, which is 79.3% (see Figure 2). 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

In this example, the 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. In vitro inhibition of Staphylococcus aureus and Staphylococcus epidermidis and Bacillus subtilis.

1. Preparation of activated strains and bacterial liquids

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.

2. Antibacterial test

The 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, ΙΟΟμΙ 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 ΙμΙ, 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

At the same time, set the bacterial liquid control, that is, the bacterial liquid plus the equal volume culture solution;

After incubating at 37 °C for 20 h, 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).

Specific distance = (above 50% inhibition rate -50%) / (higher than 50% inhibition rate - lower than 50% inhibition rate) Formula 1

Bacterial inhibition rate = (inhibition test OD value - drug control OD value) / (bacterial solution control OD value - blank control OD value) ... Formula 2

The results are shown in Tables 6 and 7.

In vitro antibacterial (gram-positive bacteria) test results

Nl l . lmg/ml 1.7mg/ml 1.4mg/ml

N2 2.6mg/ml 9.6mg/ml 1.9mg/ml

N3 5.2mg/ml 5.4mg/ml 4.9mg/ml

N4 25mg/ml 24mg/m 9.8mg/ml

It can be seen from the above results that 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. Among them, 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.

And from the above results, with the increase of the content of the saplings contained in the extract of honeysuckle, the antibacterial effect is more obvious, and the antibacterial effect of the pure sapogenin is the best. The content is obviously positively correlated with the effect of the extract, which further illustrates the antibacterial effect of the open sapogenin.

It is worth noting that this study specifically used multi-drug resistant bacteria, suggesting that the strong antibacterial effect of open-chain saponin on multi-drug resistant bacteria has important clinical significance. 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)

Experimental animals: Kunming breeder, male, 16~18g, purchased from the Animal Center of Shandong University.

1, animal grouping

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.

2, model preparation

Under the anesthesia of ether, the inoculum was slowly dropped into the nasal cavity of the animal, and the body was erected to make the inoculation. The fluid enters the tracheobronchial. The normal control group was inoculated with bacterial culture medium 50 μΙ, 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).

3, intervention treatment

The drug-resistant control group (gentamicin group) was intramuscularly injected with gentamicin daily at 400 units/unit (200μ) 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 (200μ∑) daily, once a day, the dosage is shown in Table 8. ;

Normal control group and model control group were injected with normal saline 200μΙ^ once a day for 2 days; continuous intervention for 6 days.

Table 8 Honeysuckle II Extract o Take N1 dose

o

O

1

Each experimental group (except the anti-drug control group) was administered with tail vein II pulse o on the first 1-4 days, 5-6 曰 o

It is administered by intraperitoneal injection.

4, animal observation results

T τ

Compared with the normal control group, all animals had a reduced diet and a messy fur, and the model group, the test drug 'J, the dose group, and the drug-resistant control group were particularly significant. II II

O O

On the fifth day, there were 2 deaths in the model group, 2 deaths in the drug-resistant control group, and 1 death in the low-dose group of the test drug. The dead animals were immediately weighed and dissected, and the lung tissues were fixed with formalin and prepared for illness. Scientific examination.

5, weight detection and necropsy

Two hours after the last dose on day 6, the animals were sacrificed by ethereal anesthesia under ether anesthesia, weighed and dissected.

1) Body weight analysis

The results are shown in Table 9 below.

Table 9 Results of weight analysis

Normal group and each group model group and each group drug group and each group group mean ( X ) standard deviation ( S )

Compare Compare Normal group 21.5439 0.79959

Model group 18.5274 2.21512 High dose 20.0214 1.81394

Medium dose 20.3523 0.89170 2→4: p=0.032 3→4: ρ=0.614 Low dose 20.2090 1.77033 3→5 : ρ=0.827 Drug resistance group 20.2331 1.34141 1→6: p=0.020 2→6: ρ=0.090 3→ 6: ρ=0.790 Sensitive group 20.0686 0.88956 2→10: ρ=0.064 3→10: ρ=0.943

2) necropsy

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

1

Mission); O

Honeysuckle Extract N1 Efficacy Model Group II II

In the II dose II o o group, 2 animals had red lung tissue, or o o O O

See the purple-red spots, the other 8 animals lung tissue meat o o o observation similar to the normal control animals, occasionally the surface is distributed with red spots;

(

In the N1 middle dose group, 4 animals showed T τ red n i color and purple red spots. The lung tissues of the other 6 animals were similar to the high dose group.

II II

In the dose group, the lung tissue of the animal was red, and it was purple-red.

N1 small O spots, similar to model group animals;

The lung tissue of the drug-resistant control group (gentamicin) was similar to that of the model group;

In the sensitive drug control group (ceftazidime), the lung tissue of the animal was white with occasional red spots.

6, animal lung pathology examination

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 ):

A. Thin branch, peri-infiltration (percentage of parts):

0 = none; 1 = a little ( <25 % ); 2 = many (25 % ~ 75 %); 3 = all ( >75 %).

B. Qualitative of bronchioles and bronchial infiltration:

0 = no: occasionally mild infiltration, or lymphoid cell mass around the bronchi is found in normal animals; 1 = light: abnormal, often accompanied by intermittent rings; 2 = moderate: complete ring or crescent ring , with a thickness of <5 cells; 3 = severe: a complete ring with >5 to 10 cell thicknesses. C. Bronchiole/bronchial lumen exudation:

0 = no; 1 = mild: ≤ 25% closed in the moon; 2 = severe: ≥ 25% closed in the moon.

D. Perivascular infiltration, (percentage of parts):

0 = none; 1 = less (<10%); 2 = many (10% ~ 50%); 3 = most (>50%)

E. Substantial pneumonia:

0 = no; 1 = mild: plaque-like substantial infiltration; 2 = severe: substantial infiltration of plaques and fusion. Calculation of the score = A + 3 (B + C) + D + E = 0-26 points.

The pathological test results of each experimental group are shown in Table 10. Table 10 Animal pathology test score results

( ±s, n=10)

It can be seen from the above results that the 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.

Claims

1. Open-linked saponin for use as a prophylactic and/or therapeutic agent for diseases caused by bacteria.

The open-linked sassafrasic acid according to claim 1, wherein the kaichasin is extracted from honeysuckle or its plant of origin, Lonicera japonica or other plants.

The open-linked saponin according to claim 1, wherein the kainic acid is prepared by a method comprising the following steps:

(2) in 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 of C _r C ₆ alkanol The aqueous solution is precipitated or sedimented to obtain a precipitate or a solution concentrate;

(3) separating or purifying the precipitate or the solution concentrate obtained by the step (2) by chromatography, and collecting an eluate containing an iridoid compound, wherein the chromatographic method is selected from a macroporous adsorption resin column. One or more of chromatography, normal phase silica gel chromatography, and reverse phase silica gel chromatography.

4. The open-linked sapogenin according to claim 3, wherein the method comprises:

The open-linked sapogenin according to claim 1, wherein the bacterium is an antibiotic-resistant bacterium, preferably a multi-drug resistant bacteria;

Wherein the antibiotic is selected from the group consisting of ampicillin, penicillin, piperacillin, tazobactam, amoxicillin, clavulanic acid, cefazolin, cefuroxime, ceftriaxone, cefuroxime sodium, sulphate Deep, Levooxyfluoride, cefotaxime, ceftazidime, imipenem, cefepime, cefoxitin, gentamicin, amikacin, ciprofloxacin, chloramphenicol, co-trimoxazole, tetracycline, nitrofurantoin , aztreonam, ciprofloxacin, norfloxacin, amika, sulbactam, ticarcillin, clavulanic acid, tobramycin, star, imipenem, minocycline, meropenem , one or more of penicillin, oxacillin, erythromycin, vancomycin, rifampicin and clindamycin.

The open-linked sapogenin according to claim 5, wherein the bacterium is a multi-antibiotic-resistant Gram-negative bacterium;

Preferably, the multi-antibiotic-resistant Gram-negative bacteria are selected from the group consisting of Escherichia coli, Pseudomonas aeruginosa, Klebsiella, Acinetobacter baumannii, Proteus, Enterobacter, Haemophilus influenzae One or more of the genus Klebsiella and the genus Kapta;

Further preferably, the multi-antibiotic resistant Gram-negative bacteria are Escherichia coli, Pseudomonas aeruginosa and/or Klebsiella.

The open-linked sapogenin according to claim 5, wherein the bacterium is a Gram-positive bacterium resistant to multiple antibiotics;

Preferably, the multi-antibiotic-resistant Gram-positive bacteria are selected from the group consisting of Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Streptococcus pyogenes A group, Streptococcus pneumoniae, Bacillus subtilis and Staphylococcus epidermidis One or more

Further preferably, the multi-antibiotic resistant Gram-positive bacteria are methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, Bacillus subtilis and/or Staphylococcus epidermidis.

The open-linked sapogenin according to claim 1, wherein the disease caused by the bacterium is an infection-causing disease caused by bacteria, particularly an infectious disease caused by an antibiotic-resistant bacterium, such as a digestive system. One or more of infection, blood system infection, respiratory infection, urinary tract infection, central nervous system infection, bone and joint infection, ear, mastoid and/or sinus infection, and skin and soft tissue infection;

Preferably, the disease caused by the bacteria is a respiratory infection caused by an antibiotic resistant bacteria.

The open-linked sapogenin according to claim 8, wherein the disease caused by the bacterium is a respiratory infection caused by a multi-antibiotic-resistant Gram-negative bacterium;

Further preferably, the disease caused by the bacterium is an infectious pneumonia caused by a multi-antibiotic-resistant Gram-negative bacterium;

More preferably, the disease caused by the bacterium is a nosocomial pneumonia caused by a multi-antibiotic-resistant Gram-negative bacterium;

Still preferably, the disease caused by the bacteria is caused by multiple antibiotic-resistant Klebsiella In-hospital infectious pneumonia.

The open-linked sapogenin according to claim 8, wherein 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 bacteria is pneumonia caused by multiple antibiotic-resistant Gram-positive bacteria;

Further preferably, the disease caused by the bacteria is pneumonia caused by methicillin-resistant Staphylococcus aureus.

A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the kaimuthic acid according to any one of claims 1 to 10;

Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or excipient.

The open-broad saponin according to any one of claims 1 to 10 or the pharmaceutical composition according to claim 11 in the preparation of a medicament for preventing and/or treating a disease caused by bacteria use.

The use according to claim 12, characterized in that the bacterium is an antibiotic-resistant bacterium, preferably a multi-antibiotic-resistant bacterium.

14. The use according to claim 13, wherein the bacterium is a multi-antibiotic resistant Gram-negative bacterium;

The use according to claim 13, wherein the bacterium is a multi-antibiotic-resistant Gram-positive bacterium;

Further preferably, the multiplex-antibiotic-resistant Gram-positive bacterium is methicillin-resistant Staphylococcus aureus and/or Staphylococcus aureus.

The use according to claim 12, wherein the disease caused by the bacteria is an infection-causing disease caused by bacteria, particularly an infection-like disease caused by an antibiotic-resistant bacteria, such as a digestive system infection, a blood system infection, One or more of respiratory infections, urinary tract infections, central nervous system infections, bone and joint infections, ear, mastoid and/or sinus infections, and skin and soft tissue infections; Preferably, the disease caused by the bacteria is a respiratory infection caused by the antibiotic resistant bacteria.

The use according to any one of claims 16 to 16, wherein the disease caused by the bacterium is a respiratory infection caused by a multi-antibiotic-resistant Gram-negative bacterium;

Still preferably, the disease caused by the bacteria is nosocomial pneumonia caused by multiple antibiotic-resistant Klebsiella.

The use according to claim 16, wherein the disease caused by the bacterium is a respiratory infection caused by a multi-antibiotic-resistant Gram-positive bacterium;

A method for preventing and/or treating a disease caused by a bacterium, characterized in that the method comprises administering to a patient in need thereof a therapeutically effective amount of the opening according to any one of claims 1 to 10. A saponin and/or a pharmaceutical composition according to claim 11.

20. The method according to claim 19, wherein the bacteria are antibiotic resistant bacteria, preferably multiple antibiotic resistant bacteria.

21. The method according to claim 20, wherein the bacterium is a multi-antibiotic resistant Gram-negative bacterium;

22. The method according to claim 20, wherein the bacterium is a multi-antibiotic resistant Gram-positive bacterium;

Preferably, the multi-antibiotic-resistant Gram-positive bacteria are selected from the group consisting of Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Streptococcus pyogenes A group, Streptococcus pneumoniae, Bacillus subtilis and Staphylococcus epidermidis One or more Further preferably, the multi-antibiotic-resistant Gram-positive bacteria are methicillin-resistant Staphylococcus aureus and/or Staphylococcus aureus.

The method according to claim 19, wherein the disease caused by the bacteria is an infection-causing disease caused by bacteria, particularly an infectious disease caused by an antibiotic-resistant bacteria, such as a digestive system infection, a blood system infection, One or more of a respiratory infection, a urinary tract infection, a central nervous system infection, a bone and joint infection, an ear, a mastoid and/or sinus infection, and a skin soft tissue infection; preferably, the disease caused by the bacterium is caused by the disease Respiratory infections caused by antibiotic-resistant bacteria.

24. The method according to claim 23, wherein the disease caused by the bacterium is a respiratory infection caused by a multi-antibiotic-resistant Gram-negative bacterium;

The method according to claim 23, wherein the disease caused by the bacteria is a respiratory infection caused by a multi-antibiotic-resistant Gram-positive bacteria;