CN109463402A - A kind of preparation method and application of cinnamomum camphora essential oil quorum-quenching agent - Google Patents
A kind of preparation method and application of cinnamomum camphora essential oil quorum-quenching agent Download PDFInfo
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- CN109463402A CN109463402A CN201811448758.6A CN201811448758A CN109463402A CN 109463402 A CN109463402 A CN 109463402A CN 201811448758 A CN201811448758 A CN 201811448758A CN 109463402 A CN109463402 A CN 109463402A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
- A01N65/08—Magnoliopsida [dicotyledons]
- A01N65/24—Lauraceae [Laurel family], e.g. laurel, avocado, sassafras, cinnamon or camphor
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Abstract
The present invention provides a kind of preparation method and application of the quorum-quenching agent of plants essential oil, the method includes the preparation of preparation and the quorum-quenching agent of essential oil.The present invention obtains cinnamomum camphora essential oil from camphor tree leaves, it is confirmed for the first time through testing, essential oil prepared by the present invention has the activity for inhibiting wild chromabacterium biolaceum intervention school-based, causes chromabacterium biolaceum not pathogenic, and essential oil will not cause chromabacterium biolaceum to generate survival pressure, and then generate drug resistance；Essential oil prepared by the present invention can inhibit the activity of Escherichia coli, chromabacterium biolaceum, staphylococcus aureus and pseudomonas aeruginosa, be a kind of novel antibacterial substance based on quorum-quenching effect.The present invention has many advantages, such as that raw material is easy to get, novel fine bacteria quorum sensing inhibitor preparation method is simple and reliable, bacteriostatic agent does not generate drug resistance and nonhazardous effect and feature to bacterium.
Description
Technical field
The invention belongs to biological pesticide technical fields, and in particular to a kind of system of cinnamomum camphora essential oil quorum-quenching agent
Preparation Method and application.
Background technique
In recent years, antibiotic and antibacterials are widely used during preventing and treating bacterial disease.Traditional antibacterials
By interfering the biochemical metabolism process of pathogen, its structure and function is influenced, has the function that inhibition and kills bacterium.Bacterium exists
It makes a variation rapidly under huge survival pressure, drug-resistant bacteria or superbacteria is caused to quickly increase, to the prevention and treatment of bacterial disease
Many difficulties are brought, therefore the new and effective antibacterial agent for developing low drug resistance is the direction of researchers' effort always.Bacterial community
Induction (Quorum sensing, QS) is a kind of regulatory mechanism of bacterial community communication, refers to that bacterium generates and secretes signal specific
Molecule, and changed by the concentration of signaling molecule to perceive the quantity of bacterium in ambient enviroment and change, when signaling molecule reaches one
When determining concentration threshold, starts or close the expression of related gene to adapt to the variation of environment, it is a large amount of individual in spy that QS assigns bacterium
The function of portion gene is co-expressed under fixed condition, to be in relatively in nature struggle for existence or with host's Interaction
Perch, to guarantee the existence and procreation of population.Such as the release of virulence factor, the shape of biomembrane in bacterium vital movement
The regulation of quorum sensing is all be unable to do without at, fluorescence generation, the formation of spore, the formation of antibiotic, DNA transcription etc..Study table
Bright, the quorum sensing inhibitor (Quorum sensing inhibitors, QSI) of plant source is in the feelings for not inhibiting bacterial growth
It is able to suppress the QS system of bacterium under condition, reduces the pathogenic of pathogen, can be played again with Induction of bacterial drug resistance is not easy
The advantages of preventing and treating bacteriosis.Therefore, the research and development that the plant source biocontrol agent of target is sensed as with bacterial community have become heat
Point.
Cinnamomum camphora (Cinnamomum camphora), it is Lauraceae Cinnamomum aiphyllium, is the master of the green broad-leaf forest of Subtropic of China
Want one of tree species, and the fragrant tree species of common greening.Root, skin, leaf and the fruit of camphor tree can be used as medicinal, and root, stem, leaf
Contain essential oil with fruit.To it, research finds that cinnamomum camphora essential oil is capable of the activity of strong inhibition chromabacterium biolaceum QS system in detail.Mesh
It is preceding there is not yet the compound have inhibit the active report of chromabacterium biolaceum QS, the antibacterial activity of camphor tree leaves essential oil whether and cinnamomum camphora
The quorum sensing inhibitory activity of blade essential oil is related still unclear.
Summary of the invention
The present invention is intended to provide a kind of novel, environmentally friendly inhibitor with inhibition bacterium QS system from plant,
Essential oil compounds obtained can be used in researching and developing novel antibacterial lead drug to prepare antibacterial agent, prevention and treatment bacterial disease.This
Invention extracts and prepares quorum-quenching agent from cinnamomum camphora plant using quorum-quenching activity as tracking means
And antibacterial agent.
To achieve the above object, the present invention adopts the following technical scheme:
A kind of preparation method of cinnamomum camphora essential oil quorum-quenching agent, specifically includes the following steps:
(1) camphor tree leaves pre-process: drying, be placed in spare in -20 DEG C of refrigerators after the camphor tree leaves of picking are cleaned；
(2) camphor tree leaves that step (1) is handled well are set in a round bottom flask, distilled water are added according to feed liquid mass ratio 1:10,
2h is extracted in distillation in extraction of essential oil device, collects essential oil with centrifuge tube after the completion of extraction, 1g anhydrous sodium sulfate is added in centrifuge tube
To remove extra moisture, obtaining has the active cinnamomum camphora essential oil of quorum-quenching.
(3) the cinnamomum camphora essential oil being prepared is diluted under sublethal concentration with methanol-water, i.e. acquisition cinnamomum camphora essential oil bacterium
Quorum sensing inhibitor.
Step (3) methanol-water is that first alcohol and water is uniformly mixed according to volume ratio 1:200.
Step (3) sublethal concentration is 2.5mg/mL.
Further, the above-mentioned cinnamomum camphora essential oil quorum-quenching agent being prepared is applied to preparation antibacterial guide
In drug.
The beneficial effects of the present invention are:
(1) the present invention is based on the bacterium QS inhibitor of cinnamomum camphora essential oil preparation is reduced significantly by the chromabacterium biolaceum of QS System-mediated
Element synthesis inhibits formation and the crowd hazards of the biomembrane by QS system regulation.
(2) the present invention is based on the bacterial inhibitors of cinnamomum camphora essential oil preparation to inhibit wild chromabacterium biolaceum, golden yellow significantly
The growth of staphylococcus, Escherichia coli and pseudomonas aeruginosa.
(3) present invention have prepare raw material easily obtain, the quorum-quenching that preparation method is simple and reliable, prepared
Agent does not generate the advantages that drug resistance and nonhazardous effect to bacterium.
Detailed description of the invention
Fig. 1 is that the quorum sensing of essential oil is extracted from cinnamomum camphora plant using quorum-quenching activity as tracking means
Active ingredients result figure.
Fig. 2 is influence of the various concentration cinnamomum camphora essential oil to chromabacterium biolaceum thalli growth.
Fig. 3 is influence of the various concentration cinnamomum camphora essential oil to violacein yield.
Fig. 4 is influence of the various concentration cinnamomum camphora essential oil to chromabacterium biolaceum biological membrane biological amount and its formation.
Fig. 5 is influence of the various concentration cinnamomum camphora essential oil to chromabacterium biolaceum crowd hazards.
Specific embodiment
Embodiment one: the screening of quorum-quenching agent
The substances such as polyphenol, polysaccharide, essential oil, alkaloid are extracted respectively from the blade, root system, branch of cinnamomum camphora plant, with bacterial flora
It is tracking means that body-sensing, which answers inhibitory activity, and screening has the active active component of quorum-quenching and effective component, from
It is separated in camphor tree leaves with the active cinnamomum camphora essential oil of obvious quorum-quenching.
Embodiment two: prepared by the extraction with the active cinnamomum camphora essential oil of quorum-quenching
The processing method of camphor tree leaves: it dries, is placed in spare in -20 DEG C of refrigerators after the camphor tree leaves of picking are cleaned.
It weighs the camphor tree leaves that 50g is handled well to set in a round bottom flask, distilled water is added according to feed liquid mass ratio 1:10, in
2h is extracted in distillation in extraction of essential oil device, collects essential oil with 2mL centrifuge tube after the completion of extracting, 1g anhydrous sodium sulfate is added in centrifuge tube
To remove extra moisture, obtaining has the active cinnamomum camphora essential oil of quorum-quenching.
Embodiment three: the quorum sensing inhibitory activity test of cinnamomum camphora essential oil
1. laboratory sample and experimental method
Select previously prepared wild type chromabacterium biolaceumChromobacterium violaceum ATCC31532,30 °C,
12 h are cultivated under the conditions of 150 rpm in LB liquid medium, obtain bacteria suspension (OD 600 ,0.9) stand-by.Heat solid LB culture
Base (contains 2% agar), is cooled to 50 °C or so by 1%(V/V) bacteria suspension is added, culture dish, 20mL/ ware are poured into after shaking up rapidly.
It is active using filter paper enzyme research quorum-quenching, method particularly includes: the filter paper that diameter is 6mm is placed on above-mentioned
In the culture medium prepared, the cinnamomum camphora essential oil of 15 μ L is injected toward filter paper, culture dish is sealed with sealing film and is placed under the conditions of 30 DEG C
It is observed after cultivating 24 h.Inhibit whether bacterium in circle survives to verify quorum sensing, takes the bacterium in inhibiting to enclose solid respectively
It is verified after 24 h of scribing line culture on body LB culture medium, having bacteria living i.e. proves that the bacterium in circle is inhibited not to be killed.
2. experimental result
Attached drawing 1 is that cinnamomum camphora essential oil inhibits the active experimental result picture of chromobacterium violaceum quorum sensing.In the week of cinnamomum camphora essential oil smaple
Two circles different with transparency are enclosed, transparent inner ring color is inhibition zone, and it is that quorum sensing inhibits circle that outer ring is more muddy.It takes
After bacterium scribing line culture for 24 hours in muddiness circle, there is bacterium colony growth, illustrate that the bacterium in the circle is not killed.This result
Show that cinnamomum camphora essential oil has the effect of quorum sensing inhibition, while also having the function of antibacterial.
Example IV: cinnamomum camphora essential oil tests the minimal inhibitory concentration (MIC) and growth curve of chromabacterium biolaceum
1. laboratory sample and experimental method
Minimal inhibitory concentration uses double dilution method, slightly changes.The 1.5mL centrifuge tube after sterilizing is taken, is added in every pipe
150 μ L of LB liquid medium, and the cinnamomum camphora essential oil of addition 80 mg/mL of final concentration mixes dilution into the first hole, then hole-specifically
Twice of dilution, is made into the culture solution of a series of samples concentration (80-0.625mg/mL), is then added into every hole for trying wild purple
Color bacillus bacteria suspension (OD 600 ,0.9)5 µL；And cultivate centrifuge tube under the conditions of 30 °C, 150 rpm, culture is taken after 24 h
Culture solution afterwards places it in 96 orifice plates, measures absorbance at 600 nm using iMark microplate reader and determines bacterial activity, and sees
The minimum sample concentration examined in solution without obvious thalli growth is minimum inhibitory concentration.
Wild type chromabacterium biolaceum (Chromobacterium violaceumATCC31532) in advance in 30 °C, 150rpm
Under the conditions of in LB liquid medium activation culture 12h.Containing 1%(V/V) Liquid Culture of the 20mL of chromabacterium biolaceum bacteria suspension
In base, the various concentration after methanol-water dilutes is added, and (MIC, 1/2 MIC, 1/4 MIC, 1/8 MIC, wherein MIC is 2.5mg/
ML cinnamomum camphora essential oil 1.2mL) is activated in 30 °C, the shaking table of 150r/min, is measured 3h-48h'sOD 600 Light absorption value.
2. experimental result
It is 2.5mg/mL that cinnamomum camphora essential oil, which inhibits the MIC of chromabacterium biolaceum,.
Attached drawing 2 is the growth curve result figure that cinnamomum camphora essential oil handles lower chromabacterium biolaceum.Phase (0-3h) before treatment, it is each to handle
Group is in the growth retardation phase with chromabacterium biolaceum growth in control group；Purple when 3-6h, under each concentration camphor tree leaves essential oil processing
Color bacillus and control group (control) are all in growth index phase, and no significant difference；Phase (>=6h) after treatment, control group
(Control) and in essential oil processing group, the growing state of chromabacterium biolaceum is totally consistent, no significant difference.The result shows that in the examination
It tests under concentration, cinnamomum camphora essential oil acts on the growth unrestraint of chromabacterium biolaceum.
Embodiment five: the generation of cinnamomum camphora essential oil inhibition chromabacterium biolaceum pigment
1. laboratory sample and experimental method
Tested using double dilution method, by 240mL after methanol-water dilutes various concentration (MIC, 1/2MIC, 1/4MIC,
1/8MIC, wherein MIC be 2.5mg/mL) cinnamomum camphora essential oil, be placed in containing 1%(v/v) wild chromabacterium biolaceum bacterium solution and 4mL LB liquid
In the test tube of body culture medium, after test mixed liquor is cultivated for 24 hours under the conditions of 30 °C, violacein is measured.Measurement side
Method is as follows: drawing the above-mentioned cultured bacterium solution of 1 mL in 1.5 mL centrifuge tubes, 12000 g, 4 °C of 20 min of centrifugation make bacterium
Body and mycetin are precipitated to centrifugation bottom of the tube；Supernatant is removed, 1 mL dimethyl sulfoxide is added in centrifuge tube, the concussion that is vortexed makes
Mycetin is completely dissolved in dimethyl sulfoxide；12000 g room temperatures are centrifuged 3 min again, remove thallus and clast；It draws
200 μ L supernatants, measure in microplate readerOD 600 Absorbance value.Experiment is repeated 3 times.
Violacein inhibiting rate=(blank control light absorption value-sample light absorption value)/blank control light absorption value × 100%
2. experimental result
Attached drawing 3 is the result figure that cinnamomum camphora essential oil inhibits chromabacterium biolaceum pigment to generate.In the bacterium solution handled through essential oil, violacein
Content be below control group (CK), and reduce (figure -3A) with the increase of concentration for the treatment of.Quantitative result (figure -3B) is aobvious
Show: the inhibiting rate of the lower violacein of various concentration essential oil (MIC, 1/2MIC, 1/4MIC, 1/8MIC) processing is respectively 63%,
56.23%,51.08%,38%.It can thus be concluded that going out: camphor tree leaves essential oil can obviously inhibit the conjunction of violacein in chromabacterium biolaceum
At, and with the increase of essential oil concentration, inhibiting effect is gradually increased.
Embodiment six: cinnamomum camphora essential oil inhibits chromabacterium biolaceum biofilm formation
1. laboratory sample and experimental method
Using 96 orifice plates after sterilizing, fluid nutrient medium 100mL, 1%(v/v are contained in each hole) chromabacterium biolaceum stoste and through first
The cinnamomum camphora essential oil of various concentration (MIC, 1/2 MIC, 1/4 MIC, 1/8 MIC, wherein MIC is 2.5mg/mL) after alcohol-water dilution
20 μ L, are cultivated for 24 hours under the conditions of 30 DEG C.Specifically: culture solution is poured out, is cleaned repeatedly with sterile PBS buffer, every hole is added
200 μ L methanol fix 15min, and 200 μ L concentration are added as the crystal violet solution of 1%(w/v) in every hole after drying, and room temperature dyes 5mim,
The violet staining liquid in culture hole is poured out, is rinsed well extra dyestuff with flowing water, after being completely dried, every hole is added
200 μ L, 33% glacial acetic acid acts on 30min, microplate reader measurement in 30 °C of insulating boxsOD 600 Light absorption value.
Microscopic biomembrane: the coverslip that specification is 1 × 1cm is placed in the 6 orifice plates bottom after sterilizing, Mei Gekong
In contain fluid nutrient medium 5mL, the cinnamomum camphora essential oil of 1% chromabacterium biolaceum stoste and various concentration (MIC and 1/4 MIC), in 30 °C
Under the conditions of cultivate for 24 hours.Coverslip is covered on clean coverslip after crystallized purple dyeing by processing method with embodiment five,
It is subsequently placed under optical microscopy and observes.
2. experimental result
Attached drawing 4 is the suppression result figure that cinnamomum camphora essential oil inhibits chromabacterium biolaceum biological membrane biological amount and its formation.Quantitative result is shown
The biofilm biomass of (figure -4A), chromabacterium biolaceum are reduced with the increase of camphor tree leaves essential oil concentration.Various concentration (MIC, 1/
2MIC, 1/4MIC, 1/8MIC) cinnamomum camphora essential oil processing under, the inhibiting rate of biomembrane is respectively as follows: 77.64%, 69.21%, 64.03% and
26.83%.As shown in figure -4B, cinnamomum camphora essential oil can also inhibit the formation of chromabacterium biolaceum biomembrane, raw in control group (Control)
The distribution of object film is more intensive and aggregation is blocking；The biomembrane distribution of chromabacterium biolaceum is loose in camphor tree leaves essential oil processing group and cannot
It is linked to be block.
Embodiment seven: the crowd hazards of cinnamomum camphora essential oil inhibition chromabacterium biolaceum
1. laboratory sample and experimental method
Swarming culture medium: include 1%(w/v) bacteriological peptone, 0.5%(w/v) sodium chloride, 0.5%(w/v) glucose and
0.5%(w/v) agar.Heating solid medium makes its thawing, and 1.2mL is added when culture medium temperature is decreased to about 50 DEG C through first
Concentration is the cinnamomum camphora essential oil smaple of MIC, 1/2 MIC, 1/4 MIC, 1/8 MIC after alcohol-water dilution, and pours into culture dish, wait train
It after supporting base cooling, takes above-mentioned 5 μ L point of bacteria suspension to culture medium central, then takes sealed membrane sealing culture dish and in 30 °C of conditions
Lower culture 18h, wherein MIC is 2.5mg/mL.
2. experimental result
Attached drawing 5 is the result figure that cinnamomum camphora essential oil inhibits chromabacterium biolaceum crowd hazards.Chromabacterium biolaceum is in the form of flagellum to external migration.
In the experimental group that various concentration (MIC and 1/4MIC) cinnamomum camphora essential oil is added, the migration diameter of chromabacterium biolaceum is bright compared with the control
It is aobvious to become smaller, therefore crowd hazards weaken, MIC concentration becomes apparent from the depression effect of chromabacterium biolaceum crowd hazards.
Embodiment eight: the biocidal property test of cinnamomum camphora essential oil
1. laboratory sample and experimental method
Test strain be wild chromabacterium biolaceum (Chomobacterium violaceinATCC31532), staphylococcus aureus
(Staphylococcus aureus ATCC25933), Escherichia coli (Escherichia coli) and verdigris ATCC25922
Pseudomonas alba (Pseudomonas aeruginosaPAO1), with 150 rpm, 30 °C of condition trainings in LB liquid medium
12 h of supporting is spare, and cinnamomum camphora essential oil is successively diluted to 50%, 25% and 12.5% concentration with methanol.Test uses plate
Punch method: heating solid LB media (contains 2% agar), is cooled to 50 °C or so by 1%(v/v) above-mentioned bacteria suspension is added
(OD 600 ,0.9) culture dish is poured into after, shaking up rapidly.After culture medium is cooling, with the punch after sterilizing at culture medium edge
Locate every 120 ° of rotation to make a hole, be repeated 3 times.25 μ L various concentrations (100%, 50%, 25% and 12.5%) are successively added in hole
Cinnamomum camphora essential oil.Culture dish is respectively placed in after being trained in insulating box, measurement antibacterial circle diameter (including foramen primum diameter 6mm).With first
Alcoholic solution is control.
2. experimental result
The results are shown in Table 1 for biocidal property of the camphor tree leaves essential oil to bacterium, camphor tree leaves essential oil to chromabacterium biolaceum, Escherichia coli,
Staphylococcus aureus and pseudomonas aeruginosa have more apparent inhibiting effect, and inhibiting effect is with essential oil concentration
Increase and increases.Essential oil under same concentrations is significantly greater than Escherichia coli, purple bar to the inhibiting effect of pseudomonas aeruginosa
Bacterium and Staphylococcus aureus illustrate that three kinds of bacterial strains are different to the susceptibility of camphor tree leaves essential oil, and pseudomonas aeruginosa is to perfume (or spice)
The susceptibility of camphor tree leaf piece essential oil is higher.
The evaluation of 1 cinnamomum camphora essential oil biocidal property of table
Note: table indicates significant difference (P < 0.05) of the same sample under various concentration with the difference letter in a line.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
Claims (4)
1. a kind of preparation method of cinnamomum camphora essential oil quorum-quenching agent, which is characterized in that specifically includes the following steps:
(1) camphor tree leaves pre-process: drying, be placed in spare in -20 DEG C of refrigerators after the camphor tree leaves of picking are cleaned；
(2) camphor tree leaves that step (1) is handled well are set in a round bottom flask, distilled water are added according to feed liquid mass ratio 1:10,
2h is extracted in distillation in extraction of essential oil device, collects essential oil with centrifuge tube after the completion of extraction, 1g anhydrous sodium sulfate is added in centrifuge tube
To remove extra moisture, obtaining has the active cinnamomum camphora essential oil of quorum-quenching；
(3) the cinnamomum camphora essential oil being prepared is diluted under sublethal concentration with methanol-water, i.e. acquisition cinnamomum camphora essential oil bacterial community
Incude inhibitor.
2. a kind of preparation method of cinnamomum camphora essential oil quorum-quenching agent according to claim 1, which is characterized in that step
Suddenly (3) described methanol-water is that first alcohol and water is uniformly mixed according to volume ratio 1:200.
3. a kind of preparation method of cinnamomum camphora essential oil quorum-quenching agent according to claim 1, which is characterized in that step
Suddenly (3) described sublethal concentration is 2.5mg/mL.
4. a kind of preparation method of cinnamomum camphora essential oil quorum-quenching agent is in preparation antibacterial as described in claim 1-3 is any
Application in lead drug.
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