CN108888685A - Purposes and its action method of the position Lemna paucicostata TH-w to staphylococcus aureus - Google Patents
Purposes and its action method of the position Lemna paucicostata TH-w to staphylococcus aureus Download PDFInfo
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- CN108888685A CN108888685A CN201810837338.0A CN201810837338A CN108888685A CN 108888685 A CN108888685 A CN 108888685A CN 201810837338 A CN201810837338 A CN 201810837338A CN 108888685 A CN108888685 A CN 108888685A
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- staphylococcus aureus
- lemna paucicostata
- lemna
- paucicostata
- methicillin
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- 241000191967 Staphylococcus aureus Species 0.000 title claims abstract description 81
- 244000242291 Lemna paucicostata Species 0.000 title claims abstract description 76
- 238000000034 method Methods 0.000 title claims abstract description 32
- 230000009471 action Effects 0.000 title claims abstract description 16
- 238000010790 dilution Methods 0.000 claims abstract description 25
- 239000012895 dilution Substances 0.000 claims abstract description 25
- 238000003556 assay Methods 0.000 claims abstract description 6
- 238000003235 crystal violet staining Methods 0.000 claims abstract description 6
- 241000894006 Bacteria Species 0.000 claims description 34
- 235000013372 meat Nutrition 0.000 claims description 24
- 235000014347 soups Nutrition 0.000 claims description 24
- RJQXTJLFIWVMTO-TYNCELHUSA-N Methicillin Chemical compound COC1=CC=CC(OC)=C1C(=O)N[C@@H]1C(=O)N2[C@@H](C(O)=O)C(C)(C)S[C@@H]21 RJQXTJLFIWVMTO-TYNCELHUSA-N 0.000 claims description 20
- 229960003085 meticillin Drugs 0.000 claims description 20
- 239000000284 extract Substances 0.000 claims description 11
- 241000219095 Vitis Species 0.000 claims description 8
- 235000009754 Vitis X bourquina Nutrition 0.000 claims description 8
- 235000012333 Vitis X labruscana Nutrition 0.000 claims description 8
- 235000014787 Vitis vinifera Nutrition 0.000 claims description 8
- 230000001629 suppression Effects 0.000 claims description 8
- FFRBMBIXVSCUFS-UHFFFAOYSA-N 2,4-dinitro-1-naphthol Chemical compound C1=CC=C2C(O)=C([N+]([O-])=O)C=C([N+]([O-])=O)C2=C1 FFRBMBIXVSCUFS-UHFFFAOYSA-N 0.000 claims description 6
- 241001478240 Coccus Species 0.000 claims description 6
- 241000191940 Staphylococcus Species 0.000 claims description 5
- 230000015572 biosynthetic process Effects 0.000 claims description 5
- 230000012010 growth Effects 0.000 claims description 5
- 230000005764 inhibitory process Effects 0.000 claims description 5
- 230000008033 biological extinction Effects 0.000 claims description 2
- 230000001580 bacterial effect Effects 0.000 abstract description 18
- 239000001963 growth medium Substances 0.000 abstract description 8
- 239000000243 solution Substances 0.000 description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 17
- 230000032770 biofilm formation Effects 0.000 description 15
- 241000196324 Embryophyta Species 0.000 description 14
- 239000003814 drug Substances 0.000 description 14
- 206010041925 Staphylococcal infections Diseases 0.000 description 13
- 229940079593 drug Drugs 0.000 description 13
- 208000015688 methicillin-resistant staphylococcus aureus infectious disease Diseases 0.000 description 13
- 238000010586 diagram Methods 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 12
- 235000019441 ethanol Nutrition 0.000 description 9
- 230000002401 inhibitory effect Effects 0.000 description 9
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- 238000002835 absorbance Methods 0.000 description 6
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- 238000001514 detection method Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
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- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
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- 229910002027 silica gel Inorganic materials 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000000844 anti-bacterial effect Effects 0.000 description 3
- 229940088710 antibiotic agent Drugs 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- 238000002425 crystallisation Methods 0.000 description 3
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- 230000000694 effects Effects 0.000 description 3
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 3
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 3
- 239000010931 gold Substances 0.000 description 3
- 229910052737 gold Inorganic materials 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
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- 239000011347 resin Substances 0.000 description 3
- 229920005989 resin Polymers 0.000 description 3
- 208000035143 Bacterial infection Diseases 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- 239000003463 adsorbent Substances 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 208000022362 bacterial infectious disease Diseases 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 230000017531 blood circulation Effects 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
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- 238000012549 training Methods 0.000 description 2
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 206010010904 Convulsion Diseases 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 208000004232 Enteritis Diseases 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 206010062717 Increased upper airway secretion Diseases 0.000 description 1
- 102000006833 Multifunctional Enzymes Human genes 0.000 description 1
- 108010047290 Multifunctional Enzymes Proteins 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 206010034133 Pathogen resistance Diseases 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 208000037581 Persistent Infection Diseases 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 241001355168 Tetrastigma Species 0.000 description 1
- 241001557415 Tetrastigma hemsleyanum Species 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
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- 229910052801 chlorine Inorganic materials 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 239000006781 columbia blood agar Substances 0.000 description 1
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- 208000001848 dysentery Diseases 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 125000001033 ether group Chemical group 0.000 description 1
- 239000002024 ethyl acetate extract Substances 0.000 description 1
- 229930003935 flavonoid Natural products 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 230000001408 fungistatic effect Effects 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
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- 210000000867 larynx Anatomy 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
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- 235000002639 sodium chloride Nutrition 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/87—Vitaceae or Ampelidaceae (Vine or Grape family), e.g. wine grapes, muscadine or peppervine
-
- 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
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- Health & Medical Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical & Material Sciences (AREA)
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- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
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Abstract
The invention discloses a kind of positions Lemna paucicostata TH-w to the purposes and its action method of staphylococcus aureus, by respectively to the position TH-t, the position TH-p, the position TH-a, the position TH-b, the position TH-w, the position TH-w1, the position TH-w2, the position TH-w3, TH-w4 is tested at position, and is compared, and having found optimal position is the position TH-w；The present invention cultivates staphylococcus aureus the position Lemna paucicostata TH-w with different culture medium doubling dilution concentration, then detects comparison bacterial biof iotalm Forming ability by crystal violet staining assay, finds out optimal concentration.
Description
Technical field
The present invention relates to be related to antibacterial field, especially use of the position Lemna paucicostata TH-w to staphylococcus aureus
Way and its action method.
Background technique
Bacterial biof iotalm (biofilm), which refers to, has been attached to life or lifeless object surface by bacterium extracellular macromolecule packet
It wraps up in, organized bacterial community, is the common existential mode of another kind for being different from free bacteria.Staphylococcus aureus
(Staphylococcus aureus, S.aureus), which is often secreted with exocellular polysaccharide, DNA and protein itself, forms " mushroom sample "
Membrane polymer biomembrane；Staphylococcus aureus often makes patient that Chronic persistent infection, drug resistance occur after forming biomembrane
It is 10~100 times stronger than planktonic bacteria, very big difficulty is brought to clinical anti-infective therapy.Bacterial biof iotalm formation the main reason for be
Abuse of antibiotics, extended residual, constantly by the stimulation of subinhibitory concentration antibiotic, make thin in the intracorporal staphylococcus aureus of people
Bacterium gene mutation causes drug resistance, and antibacterials traditional at present are difficult to effectively treat the sense that Staphylococcus Aureus Biofilm induces
Dye, and the exploitation of antibiotics is difficult to keep up with bacterial resistance paces.Therefore, the drug that microbial film is resisted in research and development has become recently
Microbiological art hot topic.
Radix tetrastigme is Vitaceae Tetrastigma, is plant tetratigma hemsleyanum (Tetrastigma hemsleyanum Diels
Et Gilg) dried root also known as hemsley rockvine root etc., be the endemic plant in China, be distributed mainly on the Southeast China people
Between common drug.The effective component of radix tetrastigme predominantly contains flavonoids chemical analysis, and cool in nature, acrid flavour, hardship are nontoxic, have
Clearing heat and detoxicating, promoting blood circulation and stopping pain, dispelling pathogenic wind and eliminating phlegm, promoting blood circulation and stopping pain and other effects, are usually used in hyperpyretic convulsion, and cold in children fever, larynx are itched swollen
Bitterly, the diseases such as cough, pneumonia, hepatitis, enteritis, dysentery.Modern pharmacological studies have shown that Lemna paucicostata combines antibiotic treatment
Bacterial infection disease curative effect is obvious.Therefore, the research and development for resisting the micro- filming of bacterium for effective component in radix tetrastigme have important
Clinical meaning.
The present invention observes Lemna paucicostata to Staphylococcus Aureus Biofilm formation and point peptizaiton, discovery three
Leaf blueness extract can effectively inhibit and disperse the generation of Staphylococcus Aureus Biofilm, it was found that Lemna paucicostata is being combined
There is new medical usage in bacterial-infection resisting, can be researched and developed for some infection drug-fast bacteria patient's therapeutic agents and laboratory reference is provided.Again
To the position TH-t, the position TH-p, the position TH-a, the position TH-b, the position TH-w, the position TH-w1, the position TH-w2, the position TH-w3,
TH-w4 is tested at position respectively, it was found that the best position of fungistatic effect.
Summary of the invention
To solve the deficiencies in the prior art, the purpose of the present invention is to provide a kind of positions Lemna paucicostata TH-w to gold
The purposes and its action method of staphylococcus aureus, respectively to the position TH-t, the position TH-p, the position TH-a, the position TH-b, TH-w
Position, the position TH-w1, the position TH-w2, the position TH-w3, TH-w4 is tested at position, and is compared, and has found optimal portion
Position is the position TH-w and optimal action method.
In order to achieve the above objectives, the present invention adopts the following technical scheme that：
To the purposes of staphylococcus aureus, the position Lemna paucicostata TH-w inhibits gold at the position Lemna paucicostata TH-w
The growth of staphylococcus aureus.
The position Lemna paucicostata TH-w above-mentioned is to the purposes of staphylococcus aureus, to the portion Lemna paucicostata TH-w
Position susceptibility be：Methicillin Sensitive Staphylococcus aureus is greater than staphylococcus aureus type strain and is greater than methicillin-resistant
Staphylococcus aureus.
To the purposes of staphylococcus aureus, the position Lemna paucicostata TH-w inhibits gold at the position Lemna paucicostata TH-w
The formation of the biomembrane of staphylococcus aureus.
The position Lemna paucicostata TH-w is to the action method of staphylococcus aureus, by the position Lemna paucicostata TH-w
Staphylococcus aureus is cultivated with LB meat soup doubling dilution to 32.0mg/mL.
Action method of the position Lemna paucicostata TH-w above-mentioned to staphylococcus aureus, Lemna paucicostata TH-w
Position carries out 35 DEG C of culture 16- to Methicillin Sensitive Staphylococcus aureus with M-H meat soup doubling dilution to 32.0mg/mL
After 20h, Bacteria suppression rate is 0.88.
Action method of the position Lemna paucicostata TH-w above-mentioned to staphylococcus aureus, Lemna paucicostata TH-w
After position carries out 35 DEG C of culture 16-20h to methicillin-resistant staphylococcus aureus with M-H meat soup doubling dilution 32.0mg/mL,
Bacteria suppression rate is 0.34.
Action method of the position Lemna paucicostata TH-w above-mentioned to staphylococcus aureus, Lemna paucicostata TH-w
After position carries out 35 DEG C of culture 16-20h to staphylococcus aureus type strain with M-H meat soup doubling dilution 32.0mg/mL, bacterium
Inhibiting rate is 0.7.
Action method of the position Lemna paucicostata TH-w above-mentioned to staphylococcus aureus, Lemna paucicostata TH-w
Position is with LB meat soup doubling dilution 32.0mg/mL to Methicillin Sensitive Staphylococcus aureus, methicillin-resistant staphylococcus Portugal
After grape coccus carries out 37 DEG C of cultures for 24 hours, use crystal violet staining assay detection biomembrane absorbance A 570mm value for 3.1.
The invention has the beneficial effects that：
The present invention is respectively to the position TH-t, the position TH-p, the position TH-a, the position TH-b, the position TH-w, the position TH-w1,
The position TH-w2, the position TH-w3, TH-w4 is tested at position respectively, and is compared, and having found optimal position is the portion TH-w
Position；
The present invention is by the position Lemna paucicostata TH-w with different culture medium doubling dilution concentration to Staphylococcus aureus
Bacterium is cultivated, then detects comparison bacterial biof iotalm Forming ability by crystal violet staining assay, and finding out optimal concentration is 32mg/
mL。
Detailed description of the invention
Fig. 1 is that Lemna paucicostata of the invention inhibits MSSA-1 biofilm formation ability comparison diagram；
Fig. 2 is that Lemna paucicostata of the invention inhibits MSSA-2 biofilm formation ability comparison diagram；
Fig. 3 is that Lemna paucicostata of the invention inhibits MSSA-3 biofilm formation ability comparison diagram；
Fig. 4 is that Lemna paucicostata of the invention inhibits MSSA-4 biofilm formation ability comparison diagram；
Fig. 5 is that Lemna paucicostata of the invention inhibits MSSA-5 biofilm formation ability comparison diagram；
Fig. 6 is that Lemna paucicostata of the invention inhibits MRSA-1 biofilm formation ability comparison diagram；
Fig. 7 is that Lemna paucicostata of the invention inhibits MRSA-2 biofilm formation ability comparison diagram；
Fig. 8 is that Lemna paucicostata of the invention inhibits MRSA-3 biofilm formation ability comparison diagram；
Fig. 9 is that Lemna paucicostata of the invention inhibits MRSA-4 biofilm formation ability comparison diagram；
Figure 10 is that Lemna paucicostata of the invention inhibits MRSA-5 biofilm formation ability comparison diagram；
Figure 11 is after each position of Lemna paucicostata of the invention is cultivated in the culture solution of various concentration, with crystallization
Absorbance (A570mm) comparison diagram that purple decoration method detects；
Figure 12 is the extracting method schematic diagram of Lemna paucicostata of the invention.
Specific embodiment
Specific introduce is made to the present invention below in conjunction with the drawings and specific embodiments.
The separation of radix tetrastigme medicinal material extract
Extracting solution is divided by Lemna paucicostata according to preparation selected when extracting：The position TH-t, the position TH-p, TH-a
Position, the position TH-b, the position TH-w, the position TH-w1, the position TH-w2, the position TH-w3, the position TH-w4.
Material includes：Radix tetrastigme dried root is purchased from the Ningbo City state Yin pharmaceutical material and drug corporation；80% macroporous absorbent resin tree
Chromatograph；Yanaco micro melting point apparatus (thermometer does not correct)；Varian MAT-212 type mass spectrograph；Bruker-
Speckospin AC-600P type Nuclear Magnetic Resonance.Thin-layer chromatography silica gel plate and column chromatography silica gel (100~200 mesh, 200~300
Mesh) it is the production of Shandong Yantai Jiang You silica gel development company；(20~80 μm) of Sephadex LH-20 raw for Pharmacia company
It produces；ODS C18 reverse phase silica gel is the production of Merck company；Extraction ethyl alcohol；Petroleum ether, ethyl acetate, n-butanol are pharmaceutical grade,
Water is distilled water, remaining reagent is that analysis is pure.
As shown in figure 12, extracting method includes the following steps：
Lemna paucicostata extracts reference literature [such as Zhang Yujiong clustering and Principal Component Analysis research radix tetrastigme chlorine
Imitative position HPLC finger-print [J] Chinese patent drug, 2016,38 (3):607-612] it isolates and purifies to obtain, through high performance liquid chromatography
(HPLC) it detects.
The position TH-t, the position TH-p, the position TH-a, the position TH-b, the position TH-w extracting method be：Take radix tetrastigme dry
Medicinal material 500g, after adding 5 times of 80% ethanol waters of volume to impregnate for 24 hours, heating and refluxing extraction, totally 3 times, each 40min, merging is mentioned
Liquid is taken, is filtered, is recovered under reduced pressure and is evaporated, obtain the position total extract TH-t, yield 11.3%；Total extraction is taken, water is added to be suspended, point
Not Cai Yong 10 times of volumes petroleum ether (water saturation) carry out fractional extraction, respectively obtain petroleum ether part TH-p, yield 0.08%；
Ethyl acetate extract TH-a, yield 0.31%；Butanol position TH-b, yield 1.1%；And water position TH-w, yield 8.76%.
The extracting method at the position TH-w1 is：It takes the water position TH-w to carry out macroporous adsorbent resin chromatography, first uses pure water
It is eluted, removes sugar, reuse 10%, 30%, 60%, 95% ethanol water and successively elute respectively；Wherein, 10% second
Alcohol aqueous strip solution is recovered under reduced pressure be evaporated after obtain water position desugar flow point TH-w1, yield 0.1%；
The extracting method at the position TH-w2 is：It takes the water position TH-w to carry out macroporous adsorbent resin chromatography, first uses pure water
It is eluted, removes sugar, reuse 10%, 30%, 60%, 95% ethanol water and successively elute respectively；Wherein, 30% second
Alcohol aqueous strip solution obtains water position desugar flow point TH-w 1g, yield 0.17% after being evaporated；
The extracting method at the position TH-w3 is：It will subtract after radix tetrastigme medicinal material boiling after 80% ethyl alcohol extracts (TH-t)
It pushes back receipts to be evaporated, obtains 5.4 grams of dehydrate；
The extracting method at the position TH-w4 is：It will subtract after radix tetrastigme medicinal material boiling after 80% ethyl alcohol extracts (TH-t)
It pushes back receipts to be evaporated, reuses the successively desugar respectively of 10%, 30%, 60%, 95% ethanol water, wherein obtain 10% second
Alcohol water position desugar flow point.
As a result as shown in the table：
The result of 1 Different Extraction Method of table extraction radix tetrastigme
Test 1：Verifying each position of Lemna paucicostata is to the most strong position of Staphylococcus Aureus Biofilm rejection ability
TH-w soldier takes section；
Materials and methods
Bacterial strain：By the 5 plants of Methicillin Sensitive Staphylococcus aureus left and taken in this microbial room conventional detection
(methicillin-sensitive Staphylococcus aureus, MSSA), 5 plants of methicillin-resistant staphylococcus grape balls
Bacterium (methicillin-re.sistant Staphylococcus aureus, MRSA).
Instrument includes with reagent：VITEK MS full automatic microorganism Mass Spectrometer Method system (France bioMerieux)；
96 porocyte culture plates (GIBCO company of the U.S.)；Multi-functional microwell plate detects enzyme-linked instrument (BioTek company of the U.S.)；M-H meat soup
With LB meat soup (Hangzhou shore and company)；
Experimental method includes：
Staphylococcus Aureus Biofilm model foundation；Picking staphylococcus aureus single bacterium colony is in 4mL LB culture medium
In, 35 DEG C of incubators are transferred to, for 24 hours in the culture of 120r/min state, take out adjustment bacterial concentration to 0.5 Maxwell turbidity unit.
With LB culture solution 1:After the 100 above-mentioned bacteria suspensions of dilution, 100 μ l is taken to be added in 96 orifice plates, then plus 100 μ l LB culture solutions, to be not added
200 μ l LB culture solutions of bacterium solution make blank control, and 35 DEG C of stationary cultures detect biomembrane for 24 hours, after 48h and 72h respectively.It is all
Experiment is repeated 3 times, and sets 3 multiple holes every time.
Staphylococcus Aureus Biofilm film forming ability detection process is：Using crystal violet staining assay detection bacterium biomembrane
Forming ability, fresh overnight culture single bacterium colony is in 4ml LB culture medium on picking Columbia Blood Agar plate, and 35 DEG C
120r/min cultivates 18h.It draws 10 μ l bacterium solutions and carries out secondary culture into fresh 4mL LB culture medium.It is repeated 3 times.It will passage
The bacterial concentration obtained after culture is adjusted to 0.5 Maxwell turbidity unit, and 10 μ l bacterium solutions is taken to be added to containing 190 μ l LB culture solutions
In 96 porocyte culture plates, every plant of strain does 3 multiple holes, makees blank control so that 200 μ l LB culture solutions of bacterium solution are not added.35℃
Stationary culture 6h.After biofilm formation, upper layer flcating germ is sucked, adds 200 μ l crystal violet solutions to dye 15min, discards dye
Color liquid is simultaneously rinsed with PBS and washes away planktonic bacteria 3 times, and after dry plus 200 μ l, 95% ethyl alcohol is to dissolve the crystallization in conjunction with bacterium
Purple surveys absorbance (A570nm) value at 570nm with enzyme-linked instrument.With+3 times of blank control wells of blank control wells mean absorbance values
Standard deviation is as cut off value (Ac).If A570nm≤Ac of experimental group, film forming ability is negative；If A, A570nm>Ac, then
Film forming ability is positive.
Biofilm formation inhibits test to include the following steps：Picking single bacterium colony is placed in 37 DEG C of 200r/min trainings in LB meat soup
16h is supported, 3000g is centrifuged after 20min and adjusts bacteria suspension concentration to 1 Maxwell turbidity unit with physiological saline.By Lemna paucicostata
TH-w sections are used LB meat soup doubling dilution to various concentration (0.078~l0mg/mL), draw 198 μ l respectively and 96 orifice plates are added, with not
Add the LB meat soup of radix tetrastigme TH-w extract as negative control, then is separately added into the spare bacteria suspension of 2 μ l to every hole.37 DEG C of trainings
After supporting for 24 hours, 3 times are rinsed to remove flcating germ with physiological saline.(1) violet staining：It is blotted with micropipettor residual in micropore
Extraction raffinate body, each hole are added 200 μ l (5g/L) crystal violet solutions, 15min are shaken on oscillator, abandon dye liquor, and physiological saline is rinsed 3 times and gone
Except unbonded crystal violet solution, 200 μ l, 95% ethanol solution is added in each hole after air-drying, to elute the crystallization in conjunction with biomembrane
Purple stands the absorbance (A570mm) after 20min at multi-functional enzyme-linked instrument detection 570nm.(2) negative control hole and plus people not
The radix tetrastigme TH-w extract experimental port of same concentration (0.078~l0mg/mL) carries out flat-plate bacterial colony dilution and counts：It swims to removal
200 μ l sterile salines, sonic oscillation (frequency are respectively added in the micropore of bacterium：40kHz；Power：200W) 15min makes to be adhered to
Hole wall and the biofilm dispersion of bottom hole are at floating state, with taking 10 μ l to dilute bacteria suspension even spread after physiological saline doubling dilution
In sheep blood agar plate, the plate count colony counts with 5~20 bacterium colonies are chosen after incubator culture overnight.Experiment weight
It is 3 times multiple, 3 multiple holes are set every time.
Interpretation of result：
The analysis of staphylococcus aureus film forming ability：5 plants of staphylococcus aureus strains and 5 plants of reference cultures can be formed
Biomembrane.MSSA cultivates biomembrane absorbance A 570mm value, that is, reach to peak value when for 24 hours, is 1.63 ± 0.47；Distinguish when 48h and 72h
It is 1.18 ± 0.83,1.56 ± 0.44.MRSA film formation time is longer, for 24 hours when A570mm value be 0.38 ± 0.15；Extinction when 48h
Spending A570mm is 1.27 ± 0.36, reach to peak value；1.11 ± 0.76 are reduced to when 72h.
As the result of Fig. 1-10 can be learnt：Each Lemna paucicostata can inhibit the biomembrane of staphylococcus aureus
Formation, the inhibiting effect that TH-w sections of Lemna paucicostata forms Staphylococcus Aureus Biofilm is the most obvious.
Various concentration and the different Lemna paucicostatas for extracting section is added simultaneously during forming biomembrane；Through three leaves
MSSA group and MRSA group biofilm formation after after green extract TH-w sections of inductions 24 are significantly lower than control group, and TH-w is prompted to inhibit
Obviously, through there is statistical difference (P between comparative group<0.05)；Same concentrations and different strain act on A570 value and control group when for 24 hours
Compare the reduction of biofilm formation ability conspicuousness, more has statistical difference (P between each same concentrations group<0.05).Identical drug
The inhibiting effect difference that the different radix tetrastigme extraction section of concentration forms Staphylococcus Aureus Biofilm is obvious, there is system between group
Meter learns difference (P<0.05).The inhibition that identical drug concentration Lemna paucicostata forms different Staphylococcus Aureus Biofilms
Difference between the effects are obvious, more there is statistical difference (P between group<0.05).
Result as Figure 1-10 shows may indicate that：
The position Lemna paucicostata TH-t, the position TH-p, the position TH-a, the position TH-b, the position TH-w, the position TH-w1,
The position TH-w2, the position TH-w3, the position TH-w4 each concentration (0.078-32mg/mL) to each 5 plants of methicillin-sensitivity golden yellow
Staphylococcus and methicillin-resistant staphylococcus aureus biomembrane have inhibiting effect, to the inhibiting effect of all bacterial strains with
Drug concentration increase bacteriostasis enhancing.There is drug concentration dependence.
To the position TH-t, the position TH-p, the position TH-a, the position TH-b, the position TH-w, the position TH-w1, the position TH-w2,
The position TH-w3, the position TH-w4 it is most sensitive be Methicillin Sensitive Staphylococcus aureus, and to methicillin-resistant staphylococcus
Staphylococcus acts on the inhibition difference of weaker two pairs of staphylococcus aureuses up to 30% or more.
Test is learnt：The position Lemna paucicostata TH-t, the position TH-p, the position TH-a, the position TH-b, the position TH-w, TH-
The position w1, the position TH-w2, the position TH-w3, the position TH-w are with M-H meat soup doubling dilution to 32.0mg/mL to 5 plants of golden yellow Portugals
After grape coccus MSSA carries out 35 DEG C of culture 16-20h, it is most sensitive with the position TH-w that bacterial membrane generates inhibiting effect.
The position Lemna paucicostata TH-t, the position TH-p, the position TH-a, the position TH-b, the position TH-w, the position TH-w1,
The position TH-w2, the position TH-w3, the position TH-w are with M-H meat soup doubling dilution to 32.0mg/mL to 5 plants of staphylococcus aureuses
After MRSA carries out 35 DEG C of culture 16-20h, it is most sensitive with the position TH-w that bacterial membrane generates inhibiting effect.
It follows that the inhibition that different Lemna paucicostatas generate the biomembrane of staphylococcus aureus MSSA and MRSA
With with the position TH-w sensitivity.
Test 2：Lemna paucicostata inhibits bacteriagrowthtest, finds out the position Lemna paucicostata TH-w culture medium times
Than diluted optimal concentration；
Materials and methods
Bacterial strain includes：Methicillin Sensitive Staphylococcus aureus (the methicillin- that this micro- raw room saves
Sensitive Staphylococcus aureus, MSSA-29213) 1 plant, methicillin-resistant staphylococcus aureus
1 plant of (methicillin-re.sistant Staphylococcus aureus, MRSA-BAA1029) and purchase are simultaneously micro- by this
1 plant of the staphylococcus aureus type strain (ATCC 259231) that biotron saves.
Instrument includes with reagent：VITEK MS full automatic microorganism Mass Spectrometer Method system (France bioMerieux)；
96 porocyte culture plates (GIBCO company of the U.S.)；Multi-functional microwell plate detects enzyme-linked instrument (BioTek company of the U.S.)；M-H meat soup
With LB meat soup (Hangzhou shore and company)；
Experimental method includes：
Inhibit bacteriagrowthtest process be：Inhibit staphylococcus aureus growth and right carrying out Lemna paucicostata
It is found in bacterial biof iotalm inhibition test, each position of Lemna paucicostata is most strong to Staphylococcus Aureus Biofilm rejection ability
Position is that TH-w soldier takes section.Therefore, this research selects the position TH-w for the trial drug of this research.It will be with the radix tetrastigme got ready
The position extract TH-w is with M-H meat soup doubling dilution (32.0,8.0,2.0,0.5,0.125,0.03,0.0075mg/mL), with not
Add the M.H meat soup of any drug as blank control.Every hole is separately added into 100 μ l in 96 orifice plates, prepares Microdilution disk.It will
259231 strain suspensions of staphylococcus aureus MSSA-29213, MRSA-BAA1029 and ATCC are adjusted to 0.5 Maxwell turbidity list
Position makees 1:20 dilutions, make its bacterial content 5 × 106CFU/mL takes the 10 above-mentioned bacteria suspensions of μ l to be added to the Microdilution prepared
In disk.35 DEG C of culture 16-20h, the lowest concentration of drug that finding of naked eye can completely inhibit bacterial growth are judged as minimum antibacterial dense
Spend (MIC) value.Experiment is repeated 3 times, and sets 3 multiple holes every time.
Interpretation of result：
Result as shown in figure 11 may indicate that：
Each concentration (0.078-20mg/mL) at the position Lemna paucicostata TH-w is equal to three plants of staphylococcus aureus growths
There is inhibiting effect, to the inhibiting effect of all bacterial strains as the increase bacteriostasis of drug concentration enhances.Have drug concentration according to
Lai Xing.
Most sensitive to the position TH-w is Methicillin Sensitive Staphylococcus aureus, is secondly staphylococcus aureus mark
Quasi- strain, and methicillin-resistant staphylococcus aureus is acted on most weak.
Test is learnt：The position Lemna paucicostata TH-w is with M-H meat soup doubling dilution to 32.0mg/mL to golden yellow grape
After coccus MSSA-29213 carries out 35 DEG C of culture 16-20h, Bacteria suppression rate is 0.88.
The position Lemna paucicostata TH-w is with M-H meat soup doubling dilution 32.0mg/mL to staphylococcus aureus MRSA-
After BAA1029 carries out 35 DEG C of culture 16-20h, Bacteria suppression rate is 0.34.
The position Lemna paucicostata TH-w is with M-H meat soup doubling dilution 32.0mg/mL to staphylococcus aureus
After ATCC259231 carries out 35 DEG C of culture 16-20h, Bacteria suppression rate is 0.7.
It follows that the position Lemna paucicostata TH-w is 32.0mg/mL with the optimal concentration of culture medium doubling dilution.
The present invention provides a kind of Lemna paucicostata TH-w to the purposes and its action method of staphylococcus aureus, respectively
To the position TH-t, the position TH-p, the position TH-a, the position TH-b, the position TH-w, the position TH-w1, the position TH-w2, the position TH-w3,
TH-w4 is tested at position, and is compared, and having found optimal position is the position TH-w；The present invention is by Lemna paucicostata TH-
Staphylococcus aureus is cultivated with different culture medium doubling dilution concentration in the position w, then is examined by crystal violet staining assay
Comparison bacterial biof iotalm Forming ability is surveyed, optimal concentration is found out.
The basic principles, main features and advantages of the invention have been shown and described above.The technical staff of the industry should
Understand, the above embodiments do not limit the invention in any form, all obtained by the way of equivalent substitution or equivalent transformation
Technical solution is fallen within the scope of protection of the present invention.
Claims (8)
1. the position Lemna paucicostata TH-w is to the purposes of staphylococcus aureus, which is characterized in that Lemna paucicostata TH-w
The growth of position inhibition staphylococcus aureus.
2. the position Lemna paucicostata TH-w according to claim 1 exists to the purposes of staphylococcus aureus, feature
In the susceptibility to the position Lemna paucicostata TH-w is：Methicillin Sensitive Staphylococcus aureus is greater than golden yellow grape
Coccus type strain is greater than methicillin-resistant staphylococcus aureus.
3. the position Lemna paucicostata TH-w is to the purposes of staphylococcus aureus, which is characterized in that Lemna paucicostata TH-w
Position inhibits the formation of the biomembrane of staphylococcus aureus.
4. the position Lemna paucicostata TH-w is to the action method of staphylococcus aureus, which is characterized in that extract radix tetrastigme
Staphylococcus aureus is cultivated with LB meat soup doubling dilution to 32.0mg/mL at the position object TH-w.
5. the position Lemna paucicostata TH-w according to claim 4 is to the action method of staphylococcus aureus, special
Sign is that the position Lemna paucicostata TH-w is with M-H meat soup doubling dilution to 32.0mg/mL to methicillin-sensitivity golden yellow Portugal
After grape coccus carries out 35 DEG C of culture 16-20h, Bacteria suppression rate is 0.88.
6. the position Lemna paucicostata TH-w according to claim 4 is to the action method of staphylococcus aureus, special
Sign is that the position Lemna paucicostata TH-w is with M-H meat soup doubling dilution 32.0mg/mL to methicillin-resistant staphylococcus grape ball
After bacterium carries out 35 DEG C of culture 16-20h, Bacteria suppression rate is 0.34.
7. the position Lemna paucicostata TH-w according to claim 4 is to the action method of staphylococcus aureus, special
Sign is, the position Lemna paucicostata TH-w with M-H meat soup doubling dilution 32.0mg/mL to staphylococcus aureus type strain into
After 35 DEG C of culture 16-20h of row, Bacteria suppression rate is 0.7.
8. the position Lemna paucicostata TH-w according to claim 4 is to the action method of staphylococcus aureus, special
Sign is that the position Lemna paucicostata TH-w is with LB meat soup doubling dilution 32.0mg/mL to methicillin-sensitivity golden yellow grape
After coccus, methicillin-resistant staphylococcus aureus carry out 37 DEG C of cultures for 24 hours, biomembrane extinction is detected using crystal violet staining assay
Spending A570mm value is 3.1.
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