CN106822092B - A kind of pharmaceutical composition inhibiting infected by Aeromonas hydrophila - Google Patents
A kind of pharmaceutical composition inhibiting infected by Aeromonas hydrophila Download PDFInfo
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- CN106822092B CN106822092B CN201710095017.3A CN201710095017A CN106822092B CN 106822092 B CN106822092 B CN 106822092B CN 201710095017 A CN201710095017 A CN 201710095017A CN 106822092 B CN106822092 B CN 106822092B
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- honokiol
- aeromonas hydrophila
- dihydromyricetin
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- pharmaceutical composition
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- 241000607528 Aeromonas hydrophila Species 0.000 title claims abstract description 46
- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 19
- 239000008194 pharmaceutical composition Substances 0.000 title claims abstract description 19
- KJXSIXMJHKAJOD-LSDHHAIUSA-N (+)-dihydromyricetin Chemical compound C1([C@@H]2[C@H](C(C3=C(O)C=C(O)C=C3O2)=O)O)=CC(O)=C(O)C(O)=C1 KJXSIXMJHKAJOD-LSDHHAIUSA-N 0.000 claims abstract description 80
- VVOAZFWZEDHOOU-UHFFFAOYSA-N honokiol Natural products OC1=CC=C(CC=C)C=C1C1=CC(CC=C)=CC=C1O VVOAZFWZEDHOOU-UHFFFAOYSA-N 0.000 claims abstract description 54
- BYTORXDZJWWIKR-UHFFFAOYSA-N Hinokiol Natural products CC(C)c1cc2CCC3C(C)(CO)C(O)CCC3(C)c2cc1O BYTORXDZJWWIKR-UHFFFAOYSA-N 0.000 claims abstract description 52
- FVYXIJYOAGAUQK-UHFFFAOYSA-N honokiol Chemical compound C1=C(CC=C)C(O)=CC=C1C1=CC(CC=C)=CC=C1O FVYXIJYOAGAUQK-UHFFFAOYSA-N 0.000 claims abstract description 52
- KQILIWXGGKGKNX-UHFFFAOYSA-N dihydromyricetin Natural products OC1C(=C(Oc2cc(O)cc(O)c12)c3cc(O)c(O)c(O)c3)O KQILIWXGGKGKNX-UHFFFAOYSA-N 0.000 claims abstract description 40
- 239000003814 drug Substances 0.000 claims abstract description 36
- 229940079593 drug Drugs 0.000 claims abstract description 26
- 244000144972 livestock Species 0.000 claims abstract description 6
- 239000000203 mixture Substances 0.000 claims description 9
- IKMDFBPHZNJCSN-UHFFFAOYSA-N Myricetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC(O)=C(O)C(O)=C1 IKMDFBPHZNJCSN-UHFFFAOYSA-N 0.000 claims description 7
- 229940116852 myricetin Drugs 0.000 claims description 7
- PCOBUQBNVYZTBU-UHFFFAOYSA-N myricetin Natural products OC1=C(O)C(O)=CC(C=2OC3=CC(O)=C(O)C(O)=C3C(=O)C=2)=C1 PCOBUQBNVYZTBU-UHFFFAOYSA-N 0.000 claims description 7
- 235000007743 myricetin Nutrition 0.000 claims description 7
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 4
- 239000001257 hydrogen Substances 0.000 claims description 4
- 229910052739 hydrogen Inorganic materials 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 2
- 230000002265 prevention Effects 0.000 claims description 2
- 241000894006 Bacteria Species 0.000 abstract description 20
- 230000000694 effects Effects 0.000 abstract description 15
- 238000012360 testing method Methods 0.000 abstract description 11
- 230000002949 hemolytic effect Effects 0.000 abstract description 7
- 208000015181 infectious disease Diseases 0.000 abstract description 6
- 230000001717 pathogenic effect Effects 0.000 abstract description 6
- 230000001225 therapeutic effect Effects 0.000 abstract description 6
- 230000004927 fusion Effects 0.000 abstract description 5
- 206010059866 Drug resistance Diseases 0.000 abstract description 4
- 238000010171 animal model Methods 0.000 abstract description 4
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- 231100000331 toxic Toxicity 0.000 abstract description 4
- 230000002588 toxic effect Effects 0.000 abstract description 4
- 230000000845 anti-microbial effect Effects 0.000 abstract description 3
- 230000003385 bacteriostatic effect Effects 0.000 abstract description 2
- 238000002474 experimental method Methods 0.000 abstract description 2
- 238000000338 in vitro Methods 0.000 abstract description 2
- 239000000825 pharmaceutical preparation Substances 0.000 abstract description 2
- 238000010200 validation analysis Methods 0.000 abstract 1
- 230000001580 bacterial effect Effects 0.000 description 14
- 239000000243 solution Substances 0.000 description 9
- 241000252498 Ictalurus punctatus Species 0.000 description 7
- 238000005259 measurement Methods 0.000 description 6
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 5
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 5
- 239000000890 drug combination Substances 0.000 description 5
- 239000007789 gas Substances 0.000 description 5
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 4
- 244000052616 bacterial pathogen Species 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 230000002195 synergetic effect Effects 0.000 description 4
- 241000251468 Actinopterygii Species 0.000 description 3
- 230000000844 anti-bacterial effect Effects 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- OTLLEIBWKHEHGU-UHFFFAOYSA-N 2-[5-[[5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy]-3,4-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-3,5-dihydroxy-4-phosphonooxyhexanedioic acid Chemical compound C1=NC=2C(N)=NC=NC=2N1C(C(C1O)O)OC1COC1C(CO)OC(OC(C(O)C(OP(O)(O)=O)C(O)C(O)=O)C(O)=O)C(O)C1O OTLLEIBWKHEHGU-UHFFFAOYSA-N 0.000 description 2
- 101000991061 Homo sapiens MHC class I polypeptide-related sequence B Proteins 0.000 description 2
- 102100030300 MHC class I polypeptide-related sequence B Human genes 0.000 description 2
- 240000004580 Magnolia hypoleuca Species 0.000 description 2
- 230000008485 antagonism Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- LLEJIEBFSOEYIV-UHFFFAOYSA-N chelerythrine Chemical compound C1=C2OCOC2=CC2=CC=C3C4=CC=C(OC)C(OC)=C4C=[N+](C)C3=C21 LLEJIEBFSOEYIV-UHFFFAOYSA-N 0.000 description 2
- 229940000425 combination drug Drugs 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000002095 exotoxin Substances 0.000 description 2
- 231100000776 exotoxin Toxicity 0.000 description 2
- 238000003304 gavage Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 239000010445 mica Substances 0.000 description 2
- 229910052618 mica group Inorganic materials 0.000 description 2
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- 239000000304 virulence factor Substances 0.000 description 2
- 230000007923 virulence factor Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- SODWJACROGQSMM-UHFFFAOYSA-N 5,6,7,8-tetrahydronaphthalen-1-amine Chemical compound C1CCCC2=C1C=CC=C2N SODWJACROGQSMM-UHFFFAOYSA-N 0.000 description 1
- 208000031295 Animal disease Diseases 0.000 description 1
- 241000222122 Candida albicans Species 0.000 description 1
- RATMHCJTVBHJSU-UHFFFAOYSA-N Dihydrochelerythrine Natural products C1=C2OCOC2=CC2=C(N(C)C(O)C=3C4=CC=C(C=3OC)OC)C4=CC=C21 RATMHCJTVBHJSU-UHFFFAOYSA-N 0.000 description 1
- 206010021703 Indifference Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000219000 Populus Species 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 201000005010 Streptococcus pneumonia Diseases 0.000 description 1
- 241000193998 Streptococcus pneumoniae Species 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- GAMYVSCDDLXAQW-AOIWZFSPSA-N Thermopsosid Natural products O(C)c1c(O)ccc(C=2Oc3c(c(O)cc(O[C@H]4[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O4)c3)C(=O)C=2)c1 GAMYVSCDDLXAQW-AOIWZFSPSA-N 0.000 description 1
- 235000009392 Vitis Nutrition 0.000 description 1
- 241000219095 Vitis Species 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
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- 238000009395 breeding Methods 0.000 description 1
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- 229940095731 candida albicans Drugs 0.000 description 1
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- 150000001875 compounds Chemical class 0.000 description 1
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- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 229960003645 econazole nitrate Drugs 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 229930003944 flavone Natural products 0.000 description 1
- -1 flavone compound Chemical class 0.000 description 1
- 235000011949 flavones Nutrition 0.000 description 1
- 229940124307 fluoroquinolone Drugs 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 241000411851 herbal medicine Species 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 239000003120 macrolide antibiotic agent Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 229930182783 neolignan Natural products 0.000 description 1
- 230000001151 other effect Effects 0.000 description 1
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- 239000002504 physiological saline solution Substances 0.000 description 1
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- 238000010561 standard procedure Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 208000006379 syphilis Diseases 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
-
- 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
- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
- A61K31/05—Phenols
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Fodder In General (AREA)
Abstract
The present invention provides a kind of pharmaceutical compositions for inhibiting infected by Aeromonas hydrophila, are made of dihydromyricetin, honokiol, and the mass concentration ratio of the two is 1:2 to 1:16.Pass through bacteriostatic test, the test of checkerboard type Microdilution and time fusion experiment confirm that the pharmaceutical composition has collaboration bacteriostasis in vitro, it can inhibit Aeromonas hydrophila hemolytic activity in conjunction with hemolytic test discovery honokiol, it is pathogenic to reduce its, therapeutic effect can be played to aquatic livestock infected by Aeromonas hydrophila by combining dihydromyricetin by experimental animal infection model validation honokiol, the drug reduces the pathogenic of bacterium by honokiol while improving antimicrobial effect of dihydromyricetin, it is low with dosage, toxic side effect is small, residual quantity is low, the advantages that being not likely to produce drug resistance, it can be used as pharmaceutical preparation and feed addictive.
Description
Technical field
The invention belongs to technical field of aquaculture, and in particular to a kind of pharmaceutical composition for inhibiting infected by Aeromonas hydrophila
Object, for preventing and treating aquatic animal disease caused by infected by Aeromonas hydrophila.
Background technique
Cortex Magnoliae Officinalis also known as purple Piao, are a kind of common Chinese herbal medicines, are distributed mainly on the ground such as China Sichuan, Hubei.Cortex Magnoliae Officinalis
With promoting the circulation of qi dampness elimination, warm in pain and other effects, there is stronger bacteriostasis to common pathogenic bacteria.Neolignans are
The main chemical compositions of Cortex Magnoliae Officinalis, wherein the compounds such as magnolol, honokiol are the main component for generating pharmacological activity.Dihydro
Myricetin is the extract for extracting from vitis spp vine tea, is a kind of flavone compound, anti-oxidant, antitumor and anti-inflammatory
Etc. have preferable effect.But it has not yet to see related with dihydromyricetin, honokiol treatment Aeromonas hydrophila sense
Contaminate the report of drug.
Aeromonas hydrophila is a kind of a kind of conditionity pathogenic bacteria distributed widely in nature, is a variety of aquatic animals
Primary pathogenic bacteria, be that a kind of typical people-beast-fish suffers from pathogenic bacteria altogether.Recently as people to the day of Aquatic products consumption
Benefit increases, and the event of infected by Aeromonas hydrophila people happens occasionally, and produces serious threat to human health.On the other hand,
Since the development of culture fishery in recent years causes antibiotics largely to use in the breeding process, lead to bacterial drug resistance
It aggravates, therefore the treatment means based on antibiotic face great limitation.Therefore new antibacterial mechanisms are found for aquatic products
It is very urgent to cultivate medicament research and development.Recent study finds that the exotoxin of Aeromonas hydrophila secretion is that it leads to various diseases
Main cause, wherein gas lysin is one of the most important virulence factor of Aeromonas hydrophila secretion, is risen in its pathogenic course
Key effect, the strain pathogenic strength for not expressing the Aeromonas hydrophila of gas lysin, which is remarkably decreased, even to disappear.Therefore, with gas lysin
Have great importance for drone design drug to the treatment of infected by Aeromonas hydrophila.By the study find that, dihydromyricetin
Element joint honokiol is improved bacteriostatic activity, is had in conjunction with honokiol using the Mlc that can reduce dihydromyricetin
The effect for inhibiting the expression of gas lysin reduces causing a disease for bacterium by honokiol while improving antimicrobial effect of dihydromyricetin
Property, improve the survival rate of infection fish model.
Summary of the invention
The purpose of the invention is to provide a kind of pharmaceutical composition for inhibiting infected by Aeromonas hydrophila, the drugs
Composition is made of dihydromyricetin, honokiol, is dropped while improving antimicrobial effect of dihydromyricetin by honokiol
Low bacterium it is pathogenic.
Another object of the present invention is to be that the pharmaceutical composition for providing a kind of inhibition infected by Aeromonas hydrophila is being made
The influence of application in the standby drug for preventing or treating aquatic livestock Aeromonas hydrophila, experimental animal model survival rate confirms two
Hydrogen myricetin joint honokiol can play therapeutic effect to aquatic livestock infected by Aeromonas hydrophila, which has dosage
It is low, toxic side effect is small, residual quantity is low, is not likely to produce the advantages that drug resistance, can be used as pharmaceutical preparation and feed addictive.
In order to achieve the above purpose, the invention adopts the following technical scheme:
A kind of pharmaceutical composition inhibiting infected by Aeromonas hydrophila, it is characterised in that: the pharmaceutical composition is by two
The mass concentration ratio of hydrogen myricetin, honokiol composition, dihydromyricetin and honokiol is 1:2 to 1:16.
Preferably, inhibiting the quality of dihydromyricetin and honokiol in the pharmaceutical composition of infected by Aeromonas hydrophila
Concentration ratio is 1:2 to 1:8.
Preferably, inhibiting the quality of dihydromyricetin and honokiol in the pharmaceutical composition of infected by Aeromonas hydrophila
Concentration ratio is 1:2.
It is a kind of inhibit infected by Aeromonas hydrophila pharmaceutical composition preparation prevention or treatment the thermophilic aqueous vapor list of aquatic livestock
Application in the drug of born of the same parents bacterium, for infect Aeromonas hydrophila aquatic livestock can every 8h primary, dihydromyricetin is administered orally
The dosage of element is 6.25-50mg/kg, and the dosage of honokiol is 100mg/kg, can reach best cure rate.
Compared with prior art, the present invention has the following advantages and beneficial effects: the composition dosage is low, treatment effect
Fruit is good, is not likely to produce drug resistance, and two kinds of ingredients are Chinese medical extract, do not have toxic side effect to fish body, not medicament residue
Risk.
Detailed description of the invention
Fig. 1 is honokiol and dihydromyricetin various combination mode (alone or in combination) to Aeromonas hydrophila bacterial strain
The time fusion of MS201509.
Fig. 2 is effect of the honokiol to different Aeromonas hydrophila bacterial strain gas lysin hemolytic activities.
Specific embodiment
Embodiment 1: dihydromyricetin is used alone or in combination antibacterial dense to the minimum of Aeromonas hydrophila with honokiol
Degree measurement
The minimum suppression that micro-dilution method measurement dihydromyricetin, the honokiol recommended using CLSI are used alone or in combination
Bacteria concentration (MICs).The Aeromonas hydrophila bacterial strain of 7 kinds of separate sources involved in the present invention is saved by the Changjiang river aquatic products research institute,
Title is shown in Table 1.It the steps include:
(1) by configured drug, doubling dilution, dihydromyricetin concentration are respectively 128 μ in 96 porocyte culture plates
g/mL,64μg/mL,32μg/mL,16μg/mL,8μg/mL,4μg/mL,2μg/mL,1μg/mL,0.5μg/mL,0.25μg/mL；
Honokiol concentration is respectively 512 μ g/mL, 256 μ g/mL, 128 μ g/mL, 64 μ g/mL, 32 μ g/mL, 16 μ g/mL, 8 μ g/mL, 4
μ g/mL, 2 μ g/mL, 1 μ g/mL, every 100 μ L of hole injection volume；
(2) single bacterium of 7 bacterial strains of aseptic inoculation Aeromonas hydrophila drops down onto 30 DEG C of overnight incubations in MH culture medium respectively, from
It is 0.5 maxwell reduced turbidity that the heart, which takes thallus sterile saline adjustment concentration, is then diluted to 1 × 10 with culture medium6CFU/mL,
Bacterium solution is added in 96 porocyte culture plates, its ultimate density is made to reach 5 × 105It is right that the negative and positive is respectively set in CFU/mL
According to group (negative control group is not added drug and bacterium solution is not added, and positive controls only add bacterium solution that drug is not added), each drug in triplicate,
Observation is as a result, be determined as the medicine with the lowest concentration of drug of not bacterial growth after cultivating 18-24h in 30 DEG C of biochemical cultivation cases
The minimum inhibitory concentration of object.
The results are shown in Table 1, and dihydromyricetin, honokiol, which are used alone, distinguishes the MIC range of Aeromonas hydrophila
For 4-16 μ g/mL and 32-128 μ g/mL.
1 honokiol of table is with dihydromyricetin (alone or in combination) to the minimal inhibitory concentration of tested Aeromonas hydrophila
Embodiment 2: the function and effect that checkerboard type Microdilution test measurement dihydromyricetin and honokiol are used in combination
Combine honokiol to thermophilic aqueous vapor unit cell according to the CLSI standard method measurement various concentration dihydromyricetin announced
The inhibiting effect of bacterium Ah01.It the steps include:
(1) different pharmaceutical for being used to test by two kinds respectively combines the doubling dilution in sterile centrifugation tube, makes dihydromyricetin
Plain concentration is respectively 128 μ g/mL, 64 μ g/mL, 32 μ g/mL, 16 μ g/mL, 8 μ g/mL, 4 μ g/mL, 2 μ g/mL, 1 μ g/mL, 0.5 μ
g/mL,0.25μg/mL；Honokiol concentration be respectively 512 μ g/mL, 256 μ g/mL, 128 μ g/mL, 64 μ g/mL, 32 μ g/mL,
16 μ g/mL, 8 μ g/mL, 4 μ g/mL, are then respectively added dihydromyricetin, the magnolia obovata of 50 μ L into 96 porocyte culture plates respectively
Phenol reduces dihydromyricetin concentration from left to right (1-10) successively, and honokiol concentration successively reduces from top to bottom (A-H)；
(2) be added 100 μ L of Aeromonas hydrophila bacterium solution into 96 porocyte culture plates, make bacterium solution final concentration of 5 ×
105CFU/mL, feminine gender is respectively set, and (negative control group is not added drug and bacterium solution is not added, and positive controls only add with positive controls
Drug is not added in bacterium solution), each drug in triplicate, is observed after 18-24h is cultivated in 30 DEG C of biochemical cultivation cases as a result, record medicine
MIC value both after object use in conjunction；The later antibacterial activity of Drug combination passes through Mlc index FICI
(fractioanl inhibitory concerntration index) is evaluated: FICI=MICA medicine joint/MICA medicine
Individually+MICB medicine joint/MICB prescription is only.It is judged to acting synergistically (Synergism, SYN) when FICI≤0.5；0.5 < FICI
It is determined as unrelated effect (indifference, IND) when≤4；Be determined as when FICI > 4 for antagonism (antagonism,
ANT)。
As shown in table 1, dihydromyricetin MIC range is 1-16 μ g/mL, honokiol MIC after two kinds of Drug combinations
Range is 8-32 μ g/mL.In addition to IHB01 bacterial strain, honokiol joint dihydromyricetin has collaboration bacteriostasis in vitro, can
To reduce the usage amount of two kinds of drugs, toxic side effect and residual quantity are reduced.The drug combination MIC concentration data obtained according to table 1
It is found that the mass concentration ratio of dihydromyricetin and honokiol is 1:2,1:4,1:8,1 in combination after two kinds of Drug combinations:
16, four kinds of various combinations have collaboration bacteriostasis to 6 Aeromonas hydrophila bacterial strains in addition to IHB01.Press down with collaboration
The drug concentration combination of bacterium effect can have stronger inhibiting effect to Aeromonas hydrophila, advantageously reduce drug using dense
Degree improves function and effect, can be used as alternative drug concentration combination.Dihydro poplar in the composition is primarily determined according to this scheme
Syphilis and honokiol mass concentration ratio are 1:2 to 1:16.
Embodiment 3: the dihydromyricetin that minute killing curve further verifies various combination is combined with honokiol
The synergistic effect used
In order to verify obtained synergistic effect in embodiment 2, the connection with synergistic effect is sought according to test method
Sharing dihydromyricetin and honokiol concentration ratio after medicine is 1:2, the pharmaceutical composition of 1:8,1:16, using MS201509 bacterial strain as
Representative strain carries out time fusion measurement.Bacteria Culture is packed as 4 bottles, every bottle of 10mL when to OD600 being about 0.3, respectively
The following drug combined: 1/2MIC honokiol, 1/2MIC dihydromyricetin, 1/2MIC honokiol+1/2MIC dihydro is added
Myricetin, negative control, see Table 2 for details.30 DEG C of shaken cultivations, the coated plate after 0,12,24,36 and 48h takes bacterium solution and dilutes,
Use tricks rolling counters forward.Criterion: under the logarithm of the stronger medicine group bacterial population of the logarithm specific activity of drug combination group bacterial population
Drop is judged to having collaboration bacteriostasis when being more than or equal to 2.As shown in Figure 1, the combination A (matter of dihydromyricetin and honokiol
Amount concentration ratio is 1:2) occur cooperateing with bacteriostasis after 24h to MS201509 bacterial strain, combine B (dihydromyricetin and magnolia obovata
The mass concentration ratio of phenol is 1:8) occur cooperateing with bacteriostasis, combination C (dihydromyricetin and and thickness after 48h to MS201509
The mass concentration ratio of plain phenol is 1:16) occur cooperateing with bacteriostasis after 24h to MS201509.Thus result can be seen that three
Kind different components all have collaboration bacteriostasis to MS201509 bacterial strain.The program is verified again when dihydromyricetin in composition
When the mass concentration ratio of element and honokiol is 1:2 to 1:16, both drugs have collaboration bacteriostasis.
2 time fusion various combination drug concentration table of table
Note: according to magazine rans such as J Antimicrob Chemother, two kinds of drugs add when time fusion is tested
The concentration for being added to each test group is the 1/2 of the MIC surveyed when being used alone, and the MIC after being not used in combination, therefore is made herein
Drug ratios and concentration are not correspond to the ratio combined in title.Document: Synergistic Activity of
Econazole-Nitrate and Chelerythrine against Clinical Isolates of Candida
albicans,Iranian Journal of Pharmaceutical Research(2014),13(2):567-573.；
Comparative evaluation of synergy of combinations ofβ-lactams with
fluoroquinolones or a macrolide in Streptococcus pneumonia,J Antimicrob
Chemother 2011；66:845–849.
Embodiment 4: influence of the honokiol to Aeromonas hydrophila supernatant hemolytic activity
5 bottles are packed as when by the Bacteria Culture of separate sources to OD600 being about 0.3, being separately added into honokiol keeps its dense
Degree reaches 16 μ g/mL, 8 μ g/mL, 4 μ g/mL, 2 μ g/mL, 1 μ g/mL and 0 μ g/mL.Continue culture to OD600 be 2.0 when collection
Bacterium solution, 12000rpm centrifugation 1min take the hemolytic activity of supernatant measurement supernatant.As shown in Fig. 2, with honokiol concentration
The haemocylolysis for increasing supernatant gradually weakens, and is in significant dose dependent, and the drug has broad spectrum activity, to all tests
Bacterial strain is effective.It can be found that honokiol passes through the table of inhibition Aeromonas hydrophila haemolytic exotoxin by hemolytic experiment result
Its is pathogenic up to reducing, and honokiol concentration is higher, and it is stronger to the inhibiting effect of Aeromonas hydrophila virulence factor, therefore
The ratio that honokiol is improved in combination can play better therapeutic effect.
Embodiment 5: honokiol combines dihydromyricetin to the experimental therapy of channel catfish infected by Aeromonas hydrophila
Test carries out in 100cm × 50cm × 60cm aquarium, changes water daily and maintains water temperature at 25 DEG C or so.
The healthy channel catfish of 200g or so establishes artificial challenge's model by the way that Aeromonas hydrophila Ah01 is injected intraperitoneally after anaesthetizing.
2h starts to gavage following pharmaceutical composition (being shown in Table 3) to infection fish after artificial challenge, and every 8h is administered once.Positive controls gavage phase
The physiological saline of same volume is as control.Every group uses 20 tail of channel catfish, and start recording death condition, is as a result shown in after infection
Table 4.Should the result shows that, the channel catfish of artificial challenge Aeromonas hydrophila is different through dihydromyricetin, honokiol drug
The death rate caused by can be significantly reduced after the treatment of ratio composition of medicine because of bacterium infection, survival rate are higher than exclusive use and thickness
Plain phenol or dihydromyricetin.The mass concentration when dihydromyricetin and honokiol in composition is determined by animal model test
When than for 1:2 to 1:16, there is therapeutic effect to channel catfish infected by Aeromonas hydrophila.In order to improve therapeutic effect, dihydro
The mass concentration ratio of myricetin and honokiol preferred 1:2 to 1:8, to the thermophilic aqueous vapor unit cell of channel catfish when concentration ratio is 1:2
The therapeutic effect of bacterium infection is best.
Various combination drug concentration table in 3 animal model test of table
Note: in order to reduce test grouping, two kinds of drugs are all made of maximum concentration when being used alone.
4 different pharmaceutical of table combines the influence to the channel catfish infected by Aeromonas hydrophila death rate
Claims (4)
1. a kind of pharmaceutical composition for inhibiting infected by Aeromonas hydrophila, it is characterised in that: the pharmaceutical composition is by dihydro
The mass concentration ratio of myricetin, honokiol composition, dihydromyricetin and honokiol is 1:2 to 1:16.
2. the pharmaceutical composition according to claim 1 for inhibiting infected by Aeromonas hydrophila, it is characterised in that: described two
The mass concentration ratio of hydrogen myricetin and honokiol is 1:2 to 1:8.
3. the pharmaceutical composition according to claim 1 for inhibiting infected by Aeromonas hydrophila, it is characterised in that: described two
The mass concentration ratio of hydrogen myricetin and honokiol is 1:2.
4. the pharmaceutical composition described in claim 1 for inhibiting infected by Aeromonas hydrophila is in preparation prevention or treatment aquatic livestock
Application in the drug of infected by Aeromonas hydrophila.
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| CN105560217A (en) * | 2016-03-01 | 2016-05-11 | 周斌 | Application of honokiol in acute myelocytic leukemia |
| CN105726522A (en) * | 2016-01-28 | 2016-07-06 | 西北农林科技大学 | Application of magnolol in killing fish parasitic protozoa and preparation thereof |
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| CN105726522A (en) * | 2016-01-28 | 2016-07-06 | 西北农林科技大学 | Application of magnolol in killing fish parasitic protozoa and preparation thereof |
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