CN104823974A - Applications of caffeic acid alkyl ester - Google Patents
Applications of caffeic acid alkyl ester Download PDFInfo
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- CN104823974A CN104823974A CN201510173243.XA CN201510173243A CN104823974A CN 104823974 A CN104823974 A CN 104823974A CN 201510173243 A CN201510173243 A CN 201510173243A CN 104823974 A CN104823974 A CN 104823974A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to applications of caffeic acid alkyl ester, wherein the caffeic acid alkyl ester structure is represented by a formula (I), and R is H or C1-C10 alkyl. The present invention provides the applications of the caffeic acid alkyl ester on three gram-positive bacteria, two gram-negative bacteria, and one fungus, wherein the one or a plurality of materials selected from the caffeic acid and the caffeic acid alkyl ester (the alkyl carbon atom number is 1-10) are adopted to inhibit pathogenic bacteria, the effective concentrations for inhibiting the pathogenic bacteria are different, and the low concentration can effectively inhibit the pathogenic bacteria.
Description
Technical field
The invention belongs to microorganism prevention and control field, the caffeic acid Arrcostab (alkyl carbon atoms number is 1 ~ 10) particularly related to is preparing the application in broad-spectrum antibacterial agent.
Background technology
The pathogen such as bacterium, fungi microorganism often causes human body and animals and plants body tissue generation pathology, and the plant disease brought and food-borne pathogens and putrefying bacteria are global problems, and serious threat people's is healthy, and brings huge economic loss.
Plant disease: mulberry pseudomonas syringae (Pseudomonas syringae pv.mori), belonging to Gram-negative bacteria, is mulberry tree topmost bacterial disease mulberry epidemic disease, very big on the impact of output, all can cause the extensive underproduction of mulberry leaf every year; Phellinus (Phellinus igniarius), belongs to Basidiomycetes Aphyllophorales On Polyporaceae, and main parasitic, on the trunk of the trees such as mulberry, willow, birch, poplar, wood or stub, brings significant damage to each trees.
Food-borne pathogens: Escherichia coli (Escherichia coli, E.coli) Gram-negative brevibacterium, if when invading some positions of human body, can cause infection, as peritonitis, cholecystitis, cystitis and diarrhoea etc.; Staphylococcus aureus (Staphylococcusaureus) is a kind of Gram-positive spherical bacterial, it is modal pathogen in mankind's suppurative infection, causing many severe infections, is the common pathogenic bacteria causing food poisoning, is a kind of bacterium being unfavorable for human body.The infection caused by staphylococcus aureus accounts for second, is only second to Escherichia coli.
Putrefying bacteria: bacillus subtilis (Bacillus subtilis), gram-positive bacteria, is extensively distributed in the organic matter of soil and corruption, easily breeds in withered grass leaching juice; Tetrads (Micrococcus tetragenus), for gram positive coccus, extensively be present on the skin of humans and animals, people occasionally can be made to cause a disease, also be distributed widely on soil, water, plant and food, be important food spoilage bacterium, the corruption of the food such as meat, fish, aquatic product, bean product can be caused.
At present, main chemical bactericide and the preservative of adopting suppresses pathogen, although chemical bactericide and preservative energy effectively preventing pathogenic microorganism, but bring a lot of negative effect, such as environmental pollution, harm people's is healthy, aim being unfavorable for harmonious society etc.
Along with the raising day by day of people's life, people have recognized the impact that microorganism produces human body gradually, and start the various method of conscious employing and resist harmful microorganism, and searching has broad-spectrum antiseptic, using dosage is little, antibacterial effect is desirable antibacterial agent.Therefore, research and development can substitute novel, safety broad-spectrum bacteriostatic agent that traditional chemical bactericide and preservative reduced or remitted and controlled plant disease and food-borne pathogens and putrefying bacteria is of crucial importance and significant.
Synthetical prevention (IPM) is the ecological method that a kind of agricultural disease controls, and its main concept is that we make great efforts prevention, observation, just intervene with avirulence medicament if desired.Nearest Party and government are promoting " public plant protection " and " green prevention and control " theory energetically.Green prevention and control requires the behavior asset pricing arranging disease, regularly supervises, takes measures, solve disease problem, in Chemical control methods, advocates the material using organic (deriving from plant) material or natural origin.
Green prevention and control is the extension of IPM, and except control method, other everyway is very similar.The Disease management material that although comprehensive control of disease manages and green plant disease management risk of selection is minimum, green plant disease management requires higher on Disease management material, requires the material using organic material (plant) or natural origin.By applying the green prevention and control technology such as ecology regulation and control, biological control, physical control, Scientific Usage of Drugs; to reach protection bio-diversity; reduce the object that disease breaks out probability, it is also promote standardized production simultaneously, promotes the inevitable requirement of agricultural product quality and safety level.Reduce Pesticide use risk, the effective way of preserving the ecological environment.
Caffeic acid, has another name called Caffeic acid, and be a kind of common phenolic acid, caffeic acid is distributed widely in natural plants, belongs to the green prevention and control using and derive from plant or be directed to natural organic material.Caffeic acid Arrcostab refers to the large ester type compound containing caffeic acid basic structural unit, has biologically active more widely, as anticancer, and anti-inflammatory and antiviral etc., and environmentally safe, but report is had no in biological prevention and control.Caffeic acid and caffeic acid Arrcostab (alkyl carbon atoms number is 1 ~ 10) first Application are in the multiple pathogen of suppression, comprise fungi, gram-positive bacteria and Gram-negative bacteria, specifically comprise 5 kinds of bacteriums: mulberry pseudomonas syringae, Escherichia coli, bacillus subtilis, staphylococcus aureus and tetrads; 1 kind of fungi: Phellinus.
Summary of the invention
Technical problem: the present invention is directed to the problems referred to above, the invention provides a kind of caffeic acid Arrcostab in antibacterial application, caffeic acid Arrcostab (alkyl carbon atoms number is 1 ~ 10) is utilized to suppress the half lethal concentration of pathogen, this method effectively can suppress pathogen at low concentration caffeic acid Arrcostab, is a kind of effective ways preventing and treating cause of disease disease.
Technical scheme: caffeic acid Arrcostab is preparing the application in broad-spectrum antibacterial agent, described caffeic acid Arrcostab structure is such as formula shown in (I):
Wherein, R is H or C1 ~ C10 alkyl.
Described caffeic acid Arrcostab structure is preferably such as formula shown in (II):
Described is 0.001g/L-18g/L to suppression pathogen effective formula (I) compound concentration.
Embody rule method is: formula (I) compound is dissolved in cosolvent, and cosolvent comprises organic solvent and surfactant, and described organic solvent is acetone or dimethyl sulfoxide (DMSO), and adding concentration is 5% ~ 50% (v/v); Described surfactant is triton x-100 or tween, and adding concentration is 5% ~ 50% (v/v).
The active ingredient of described broad-spectrum antiseptic agent medicine is at least one in the caffeic acid Arrcostab of formula (I) structure.
Described suppression object comprises Gram-negative bacteria, gram-positive bacteria and fungi.
Described suppression object comprises mulberry pseudomonas syringae, Escherichia coli, bacillus subtilis, staphylococcus aureus, tetrads and Phellinus.
The fungistatic effect of caffeic acid Arrcostab measures, and comprises following methods:
(1) dull and stereotyped face-off method: adopt the different caffeic acid Arrcostab of agar punch method Preliminary detection to the inhibitory action of fungal pathogens;
Wherein, blank is the Phellinus diameter only adding culture medium flat plate; the negative blank of d is the Phellinus diameter that medium adds the flat board of bacterium liquid and variable concentrations cosolvent, and d caffeic acid Arrcostab is the Phellinus diameter that medium adds the flat board of bacterium liquid and variable concentrations caffeic acid Arrcostab.
(2) spectrophotometric method: adopt microplate reader to measure the bacterium liquid absorbance adding different caffeic acid Arrcostab, the different caffeic acid Arrcostab of Accurate Determining is to the inhibiting rate of pathogen and 503nhibiting concentration.
Wherein, blank is the absorbance of the test tube only adding medium, the negative blank of OD is the absorbance that medium adds the test tube of bacterium liquid and variable concentrations cosolvent, and OD caffeic acid Arrcostab is the absorbance that medium adds the test tube of bacterium liquid and variable concentrations caffeic acid Arrcostab.
Beneficial effect: utilize the Probit process of SPSS software to carry out probit analysis, calculates caffeic acid Arrcostab to the 503nhibiting concentration of pathogen, determines the size to pathogen inhibit activities.The invention provides the 503nhibiting concentration utilizing caffeic acid Arrcostab (alkyl carbon atoms number is 1 ~ 10) to suppress pathogen, this method display, can suppress pathogen effectively at low concentration caffeic acid Arrcostab.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read narrating content of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Embodiment 1
The present embodiment adopts the caffeic acid Arrcostab of spectrophotometry least concentration scope (to comprise methyl caffeate, NSC 619661, caffeic acid propyl ester, caffeic acid isopropyl ester, n-butyl caffeate, caffeic acid pentyl ester, caffeic acid isopentyl ester, the own ester of caffeic acid) inhibitory action to mulberry pseudomonas syringae, concrete grammar is as follows:
Mulberry pseudomonas syringae shaken cultivation under LB liquid nutrient medium 150r/min, 30 DEG C of conditions is spent the night, and obtains bacteria suspension.
Caffeic acid Arrcostab is dissolved in cosolvent, is mixed with the solution of 60g/L, carry out filtration sterilization process with the miillpore filter of 0.22 μm.
Preparation LB liquid nutrient medium, eachly in vitro puts 2mL liquid nutrient medium, caffeic acid Arrcostab is carried out filtration sterilization process with the miillpore filter of 0.22 μm, arranges the caffeic acid Arrcostab of variable concentrations.
Each test tube is inoculated, and each bacterial strain is often planted caffeic acid Arrcostab and done 3 repetitions respectively.
The complete medium of inoculation is placed in shaken cultivation 24h under 30 DEG C of conditions.
Bacteria suspension in test tube is injected 96 orifice plates, application of sample 100 μ L, adopt microplate reader reading under 600nm.
Result shows, and the inhibitory action least concentration scope of caffeic acid Arrcostab to mulberry pseudomonas syringae is 0.009g/L ~ 0.04g/L, and inhibiting rate is 26.315% ~ 35.641%.
Embodiment 2
The present embodiment adopts the caffeic acid Arrcostab of spectrophotometry maximum concentration scope (to comprise methyl caffeate, NSC 619661, caffeic acid propyl ester, caffeic acid isopropyl ester, n-butyl caffeate, caffeic acid pentyl ester, caffeic acid isopentyl ester, the own ester of caffeic acid) inhibitory action to mulberry pseudomonas syringae, concrete grammar is as embodiment 1:
Mulberry pseudomonas syringae shaken cultivation under LB liquid nutrient medium 150r/min, 30 DEG C of conditions is spent the night, and obtains bacteria suspension.
Caffeic acid Arrcostab is dissolved in cosolvent, is mixed with the solution of 60g/L, carry out filtration sterilization process with the miillpore filter of 0.22 μm.
Preparation LB liquid nutrient medium, eachly in vitro puts 2mL liquid nutrient medium, caffeic acid Arrcostab is carried out filtration sterilization process with the miillpore filter of 0.22 μm, arranges the caffeic acid Arrcostab of variable concentrations.
Each test tube is inoculated, and each bacterial strain is often planted caffeic acid Arrcostab and done 3 repetitions respectively.
The complete medium of inoculation is placed in shaken cultivation 24h under 30 DEG C of conditions.
Bacteria suspension in test tube is injected 96 orifice plates, application of sample 100 μ L, adopt microplate reader reading under 600nm.
Result shows, and caffeic acid Arrcostab is to the inhibitory action maximum concentration scope 5.9g/L ~ 9.33g/L of mulberry pseudomonas syringae, and inhibiting rate is 51.682% ~ 69.364%.
Embodiment 3
The present embodiment adopts the caffeic acid Arrcostab of spectrophotometry optimal concentration scope (to comprise methyl caffeate, NSC 619661, caffeic acid propyl ester, caffeic acid isopropyl ester, n-butyl caffeate, caffeic acid pentyl ester, caffeic acid isopentyl ester, the own ester of caffeic acid) inhibitory action to mulberry pseudomonas syringae, calculate caffeic acid Arrcostab to the 503nhibiting concentration of mulberry pseudomonas syringae, concrete grammar is as embodiment 1:
Mulberry pseudomonas syringae shaken cultivation under LB liquid nutrient medium 150r/min, 30 DEG C of conditions is spent the night, and obtains bacteria suspension.
Caffeic acid Arrcostab is dissolved in cosolvent, is mixed with the solution of 60g/L, carry out filtration sterilization process with the miillpore filter of 0.22 μm.
Preparation LB liquid nutrient medium, eachly in vitro puts 2mL liquid nutrient medium, caffeic acid Arrcostab is carried out filtration sterilization process with the miillpore filter of 0.22 μm, arranges the caffeic acid Arrcostab of variable concentrations.
Each test tube is inoculated, and each bacterial strain is often planted caffeic acid Arrcostab and done 3 repetitions respectively.
The complete medium of inoculation is placed in shaken cultivation 24h under 30 DEG C of conditions.
Bacteria suspension in test tube is injected 96 orifice plates, application of sample 100 μ L, adopt microplate reader reading under 600nm.
Result shows, and caffeic acid Arrcostab is to the inhibitory action optimal concentration scope 0.9g/L ~ 3.8g/L of mulberry pseudomonas syringae, and inhibiting rate is 72.619% ~ 87.633%, and 503nhibiting concentration scope is 0.008g/L ~ 7.865g/L.
Embodiment 4
The present embodiment adopts the caffeic acid Arrcostab of spectrophotometry least concentration scope (to comprise methyl caffeate, NSC 619661, caffeic acid propyl ester, caffeic acid isopropyl ester, n-butyl caffeate, caffeic acid pentyl ester, caffeic acid isopentyl ester, the own ester of caffeic acid) to colibacillary inhibitory action, concrete grammar is as follows:
Escherichia coli shaken cultivation under LB liquid nutrient medium 150r/min, 30 DEG C of conditions is spent the night, and obtains bacteria suspension.
Caffeic acid Arrcostab is dissolved in cosolvent, is mixed with the solution of 60g/L, carry out filtration sterilization process with the miillpore filter of 0.22 μm.
Preparation LB liquid nutrient medium, eachly in vitro puts 2mL liquid nutrient medium, caffeic acid Arrcostab is carried out filtration sterilization process with the miillpore filter of 0.22 μm, arranges the caffeic acid Arrcostab of variable concentrations.
Each test tube is inoculated, and each bacterial strain is often planted caffeic acid Arrcostab and done 3 repetitions respectively.
The complete medium of inoculation is placed in shaken cultivation 24h under 30 DEG C of conditions.
Bacteria suspension in test tube is injected 96 orifice plates, application of sample 100 μ L, adopt microplate reader reading under 600nm.
Result shows, and caffeic acid Arrcostab is 0.01g/L ~ 0.11g/L to colibacillary inhibitory action least concentration scope, and inhibiting rate is 18.207% ~ 35.860%.
Embodiment 5
The present embodiment adopts the caffeic acid Arrcostab of spectrophotometry maximum concentration scope (to comprise methyl caffeate, NSC 619661, caffeic acid propyl ester, caffeic acid isopropyl ester, n-butyl caffeate, caffeic acid pentyl ester, caffeic acid isopentyl ester, the own ester of caffeic acid) to colibacillary inhibitory action, concrete grammar is as embodiment 4:
Escherichia coli shaken cultivation under LB liquid nutrient medium 150r/min, 30 DEG C of conditions is spent the night, and obtains bacteria suspension.
Caffeic acid Arrcostab is dissolved in cosolvent, is mixed with the solution of 60g/L, carry out filtration sterilization process with the miillpore filter of 0.22 μm.
Preparation LB liquid nutrient medium, eachly in vitro puts 2mL liquid nutrient medium, caffeic acid Arrcostab is carried out filtration sterilization process with the miillpore filter of 0.22 μm, arranges the caffeic acid Arrcostab of variable concentrations.
Each test tube is inoculated, and each bacterial strain is often planted caffeic acid Arrcostab and done 3 repetitions respectively.
The complete medium of inoculation is placed in shaken cultivation 24h under 30 DEG C of conditions.
Bacteria suspension in test tube is injected 96 orifice plates, application of sample 100 μ L, adopt microplate reader reading under 600nm.
Result shows, and caffeic acid Arrcostab is 5.2g/L ~ 8.5g/L to colibacillary inhibitory action maximum concentration scope, and inhibiting rate is 25.643% ~ 48.689%.
Embodiment 6
The present embodiment adopts the caffeic acid Arrcostab of spectrophotometry optimal concentration scope (to comprise methyl caffeate, NSC 619661, caffeic acid propyl ester, caffeic acid isopropyl ester, n-butyl caffeate, caffeic acid pentyl ester, caffeic acid isopentyl ester, the own ester of caffeic acid) to colibacillary inhibitory action, concrete grammar is as follows:
Escherichia coli shaken cultivation under LB liquid nutrient medium 150r/min, 30 DEG C of conditions is spent the night, and obtains bacteria suspension.
Caffeic acid Arrcostab is dissolved in cosolvent, is mixed with the solution of 60g/L, carry out filtration sterilization process with the miillpore filter of 0.22 μm.
Preparation LB liquid nutrient medium, eachly in vitro puts 2mL liquid nutrient medium, caffeic acid Arrcostab is carried out filtration sterilization process with the miillpore filter of 0.22 μm, arranges the caffeic acid Arrcostab of variable concentrations.
Each test tube is inoculated, and each bacterial strain is often planted caffeic acid Arrcostab and done 3 repetitions respectively.
The complete medium of inoculation is placed in shaken cultivation 24h under 30 DEG C of conditions.
Bacteria suspension in test tube is injected 96 orifice plates, application of sample 100 μ L, adopt microplate reader reading under 600nm.
Result shows, and caffeic acid Arrcostab is 1.97g/L ~ 4.69g/L to colibacillary inhibitory action optimal concentration scope, and inhibiting rate is 68.245% ~ 86.312%, and 503nhibiting concentration scope is 0.001g/L ~ 7.382g/L.
Embodiment 7
The present embodiment adopts the caffeic acid Arrcostab of spectrophotometry least concentration scope (to comprise methyl caffeate, NSC 619661, caffeic acid propyl ester, caffeic acid isopropyl ester, n-butyl caffeate, caffeic acid pentyl ester, caffeic acid isopentyl ester, the own ester of caffeic acid) inhibitory action to bacillus subtilis, concrete grammar is as follows:
Bacillus subtilis shaken cultivation under LB liquid nutrient medium 150r/min, 30 DEG C of conditions is spent the night, and obtains bacteria suspension.
Caffeic acid Arrcostab is dissolved in cosolvent, is mixed with the solution of 60g/L, carry out filtration sterilization process with the miillpore filter of 0.22 μm.
Preparation LB liquid nutrient medium, eachly in vitro puts 2mL liquid nutrient medium, caffeic acid Arrcostab is carried out filtration sterilization process with the miillpore filter of 0.22 μm, arranges the caffeic acid Arrcostab of variable concentrations.
Each test tube is inoculated, and each bacterial strain is often planted caffeic acid Arrcostab and done 3 repetitions respectively.
The complete medium of inoculation is placed in shaken cultivation 24h under 30 DEG C of conditions.
Bacteria suspension in test tube is injected 96 orifice plates, application of sample 100 μ L, adopt microplate reader reading under 600nm.
Result shows, and the inhibitory action least concentration scope of caffeic acid Arrcostab to bacillus subtilis is 0.01g/L ~ 0.36g/L, and inhibiting rate is 15.374% ~ 33.264%.
Embodiment 8
The present embodiment adopts the caffeic acid Arrcostab of spectrophotometry maximum concentration scope (to comprise methyl caffeate, NSC 619661, caffeic acid propyl ester, caffeic acid isopropyl ester, n-butyl caffeate, caffeic acid pentyl ester, caffeic acid isopentyl ester, the own ester of caffeic acid) inhibitory action to bacillus subtilis, concrete grammar is as follows:
Bacillus subtilis shaken cultivation under LB liquid nutrient medium 150r/min, 30 DEG C of conditions is spent the night, and obtains bacteria suspension.
Caffeic acid Arrcostab is dissolved in cosolvent, is mixed with the solution of 60g/L, carry out filtration sterilization process with the miillpore filter of 0.22 μm.
Preparation LB liquid nutrient medium, eachly in vitro puts 2mL liquid nutrient medium, caffeic acid Arrcostab is carried out filtration sterilization process with the miillpore filter of 0.22 μm, arranges the caffeic acid Arrcostab of variable concentrations.
Each test tube is inoculated, and each bacterial strain is often planted caffeic acid Arrcostab and done 3 repetitions respectively.
The complete medium of inoculation is placed in shaken cultivation 24h under 30 DEG C of conditions.
Bacteria suspension in test tube is injected 96 orifice plates, application of sample 100 μ L, adopt microplate reader reading under 600nm.
Result shows, and the inhibitory action maximum concentration scope of caffeic acid Arrcostab to Bacillus subtillis is 4.681g/L ~ 9.872g/L, and inhibiting rate is 41.695% ~ 58.621%.
Embodiment 9
The present embodiment adopts the caffeic acid Arrcostab of spectrophotometry optimal concentration scope (to comprise methyl caffeate, NSC 619661, caffeic acid propyl ester, caffeic acid isopropyl ester, n-butyl caffeate, caffeic acid pentyl ester, caffeic acid isopentyl ester, the own ester of caffeic acid) inhibitory action to bacillus subtilis, concrete grammar is as follows:
Bacillus subtilis shaken cultivation under LB liquid nutrient medium 150r/min, 30 DEG C of conditions is spent the night, and obtains bacteria suspension.
Caffeic acid Arrcostab is dissolved in cosolvent, is mixed with the solution of 60g/L, carry out filtration sterilization process with the miillpore filter of 0.22 μm.
Preparation LB liquid nutrient medium, eachly in vitro puts 2mL liquid nutrient medium, caffeic acid Arrcostab is carried out filtration sterilization process with the miillpore filter of 0.22 μm, arranges the caffeic acid Arrcostab of variable concentrations.
Each test tube is inoculated, and each bacterial strain is often planted caffeic acid Arrcostab and done 3 repetitions respectively.
The complete medium of inoculation is placed in shaken cultivation 24h under 30 DEG C of conditions.
Bacteria suspension in test tube is injected 96 orifice plates, application of sample 100 μ L, adopt microplate reader reading under 600nm.
Result shows, and the inhibitory action optimal concentration scope of caffeic acid Arrcostab to bacillus subtilis is 2.54g/L ~ 4.86g/L, and inhibiting rate is 58.182% ~ 67.352%, and 503nhibiting concentration scope is 0.021g/L ~ 5.641g/L.
Embodiment 10
The present embodiment adopts the caffeic acid Arrcostab of spectrophotometry least concentration scope (to comprise methyl caffeate, NSC 619661, caffeic acid propyl ester, caffeic acid isopropyl ester, n-butyl caffeate, caffeic acid pentyl ester, caffeic acid isopentyl ester, the own ester of caffeic acid) inhibitory action to staphylococcus aureus, concrete grammar is as follows:
Staphylococcus aureus shaken cultivation under LB liquid nutrient medium 150r/min, 30 DEG C of conditions is spent the night, and obtains bacteria suspension.
Caffeic acid Arrcostab is dissolved in cosolvent, is mixed with the solution of 60g/L, carry out filtration sterilization process with the miillpore filter of 0.22 μm.
Preparation LB liquid nutrient medium, eachly in vitro puts 2mL liquid nutrient medium, caffeic acid Arrcostab is carried out filtration sterilization process with the miillpore filter of 0.22 μm, arranges the caffeic acid Arrcostab of variable concentrations.
Each test tube is inoculated, and each bacterial strain is often planted caffeic acid Arrcostab and done 3 repetitions respectively.
The complete medium of inoculation is placed in shaken cultivation 24h under 30 DEG C of conditions.
Bacteria suspension in test tube is injected 96 orifice plates, application of sample 100 μ L, adopt microplate reader reading under 600nm.
Result shows, and the inhibitory action least concentration scope of caffeic acid Arrcostab to staphylococcus aureus is 0.01g/L ~ 0.52g/L, and inhibiting rate is 21.204% ~ 37.346%.
Embodiment 11
The present embodiment adopts the caffeic acid Arrcostab of spectrophotometry maximum concentration scope (to comprise methyl caffeate, NSC 619661, caffeic acid propyl ester, caffeic acid isopropyl ester, n-butyl caffeate, caffeic acid pentyl ester, caffeic acid isopentyl ester, the own ester of caffeic acid) inhibitory action to staphylococcus aureus, concrete grammar is as follows:
Staphylococcus aureus shaken cultivation under LB liquid nutrient medium 150r/min, 30 DEG C of conditions is spent the night, and obtains bacteria suspension.
Caffeic acid Arrcostab is dissolved in cosolvent, is mixed with the solution of 60g/L, carry out filtration sterilization process with the miillpore filter of 0.22 μm.
Preparation LB liquid nutrient medium, eachly in vitro puts 2mL liquid nutrient medium, caffeic acid Arrcostab is carried out filtration sterilization process with the miillpore filter of 0.22 μm, arranges the caffeic acid Arrcostab of variable concentrations.
Each test tube is inoculated, and each bacterial strain is often planted caffeic acid Arrcostab and done 3 repetitions respectively.
The complete medium of inoculation is placed in shaken cultivation 24h under 30 DEG C of conditions.
Bacteria suspension in test tube is injected 96 orifice plates, application of sample 100 μ L, adopt microplate reader reading under 600nm.
Result shows, and the inhibitory action maximum concentration scope of caffeic acid Arrcostab to staphylococcus aureus is 7.63g/L ~ 10.47g/L, and inhibiting rate is 28.245% ~ 36.312%.
Embodiment 12
The present embodiment adopts the caffeic acid Arrcostab of spectrophotometry optimal concentration scope (to comprise methyl caffeate, NSC 619661, caffeic acid propyl ester, caffeic acid isopropyl ester, n-butyl caffeate, caffeic acid pentyl ester, caffeic acid isopentyl ester, the own ester of caffeic acid) inhibitory action to staphylococcus aureus, concrete grammar is as follows:
Staphylococcus aureus shaken cultivation under LB liquid nutrient medium 150r/min, 30 DEG C of conditions is spent the night, and obtains bacteria suspension.
Caffeic acid Arrcostab is dissolved in cosolvent, is mixed with the solution of 60g/L, carry out filtration sterilization process with the miillpore filter of 0.22 μm.
Preparation LB liquid nutrient medium, eachly in vitro puts 2mL liquid nutrient medium, caffeic acid Arrcostab is carried out filtration sterilization process with the miillpore filter of 0.22 μm, arranges the caffeic acid Arrcostab of variable concentrations.
Each test tube is inoculated, and each bacterial strain is often planted caffeic acid Arrcostab and done 3 repetitions respectively.
The complete medium of inoculation is placed in shaken cultivation 24h under 30 DEG C of conditions.
Bacteria suspension in test tube is injected 96 orifice plates, application of sample 100 μ L, adopt microplate reader reading under 600nm.
Result shows, and the inhibitory action optimal concentration scope of caffeic acid Arrcostab to staphylococcus aureus is 3.55g/L ~ 6.58g/L, and inhibiting rate is 60.257% ~ 75.369%, and 503nhibiting concentration scope is 0.011g/L ~ 9.842g/L.
Embodiment 13
The present embodiment adopts the caffeic acid Arrcostab of spectrophotometry least concentration scope (to comprise methyl caffeate, NSC 619661, caffeic acid propyl ester, caffeic acid isopropyl ester, n-butyl caffeate, caffeic acid pentyl ester, caffeic acid isopentyl ester, the own ester of caffeic acid) inhibitory action to tetrads, concrete grammar is as follows:
Tetrads shaken cultivation under LB liquid nutrient medium 150r/min, 30 DEG C of conditions is spent the night, and obtains bacteria suspension.
Caffeic acid Arrcostab is dissolved in cosolvent, is mixed with the solution of 60g/L, carry out filtration sterilization process with the miillpore filter of 0.22 μm.
Preparation LB liquid nutrient medium, eachly in vitro puts 2mL liquid nutrient medium, caffeic acid Arrcostab is carried out filtration sterilization process with the miillpore filter of 0.22 μm, arranges the caffeic acid Arrcostab of variable concentrations.
Each test tube is inoculated, and each bacterial strain is often planted caffeic acid Arrcostab and done 3 repetitions respectively.
The complete medium of inoculation is placed in shaken cultivation 24h under 30 DEG C of conditions.
Bacteria suspension in test tube is injected 96 orifice plates, application of sample 100 μ L, adopt microplate reader reading under 600nm.
Result shows, and the inhibitory action least concentration scope of caffeic acid Arrcostab to tetrads is 0.01g/L ~ 0.92g/L, and inhibiting rate is 30.267% ~ 45.764%.
Embodiment 14
The present embodiment adopts the caffeic acid Arrcostab of spectrophotometry maximum concentration scope (to comprise methyl caffeate, NSC 619661, caffeic acid propyl ester, caffeic acid isopropyl ester, n-butyl caffeate, caffeic acid pentyl ester, caffeic acid isopentyl ester, the own ester of caffeic acid) inhibitory action to tetrads, concrete grammar is as follows:
Tetrads shaken cultivation under LB liquid nutrient medium 150r/min, 30 DEG C of conditions is spent the night, and obtains bacteria suspension.
Caffeic acid Arrcostab is dissolved in cosolvent, is mixed with the solution of 60g/L, carry out filtration sterilization process with the miillpore filter of 0.22 μm.
Preparation LB liquid nutrient medium, eachly in vitro puts 2mL liquid nutrient medium, caffeic acid Arrcostab is carried out filtration sterilization process with the miillpore filter of 0.22 μm, arranges the caffeic acid Arrcostab of variable concentrations.
Each test tube is inoculated, and each bacterial strain is often planted caffeic acid Arrcostab and done 3 repetitions respectively.
The complete medium of inoculation is placed in shaken cultivation 24h under 30 DEG C of conditions.
Bacteria suspension in test tube is injected 96 orifice plates, application of sample 100 μ L, adopt microplate reader reading under 600nm.
Result shows, and the inhibitory action maximum concentration scope of caffeic acid Arrcostab to tetrads is 5.92g/L ~ 9.76g/L, and inhibiting rate is 48.624% ~ 58.996%.
Embodiment 15
The present embodiment adopts the caffeic acid Arrcostab of spectrophotometry optimal concentration scope (to comprise methyl caffeate, NSC 619661, caffeic acid propyl ester, caffeic acid isopropyl ester, n-butyl caffeate, caffeic acid pentyl ester, caffeic acid isopentyl ester, the own ester of caffeic acid) inhibitory action to tetrads, concrete grammar is as follows:
Tetrads shaken cultivation under LB liquid nutrient medium 150r/min, 30 DEG C of conditions is spent the night, and obtains bacteria suspension.
Caffeic acid Arrcostab is dissolved in cosolvent, is mixed with the solution of 60g/L, carry out filtration sterilization process with the miillpore filter of 0.22 μm.
Preparation LB liquid nutrient medium, eachly in vitro puts 2mL liquid nutrient medium, caffeic acid Arrcostab is carried out filtration sterilization process with the miillpore filter of 0.22 μm, arranges the caffeic acid Arrcostab of variable concentrations.
Each test tube is inoculated, and each bacterial strain is often planted caffeic acid Arrcostab and done 3 repetitions respectively.
The complete medium of inoculation is placed in shaken cultivation 24h under 30 DEG C of conditions.
Bacteria suspension in test tube is injected 96 orifice plates, application of sample 100 μ L, adopt microplate reader reading under 600nm.
Result shows, and the inhibitory action optimal concentration scope of caffeic acid Arrcostab to tetrads is 2.15g/L ~ 6.23g/L, and inhibiting rate is 61.645% ~ 81.104%, and 503nhibiting concentration scope is 0.026g/L ~ 9.218g/L.
Embodiment 16
The caffeic acid Arrcostab that the present embodiment adopts dull and stereotyped face-off method to detect least concentration scope (comprises methyl caffeate; NSC 619661; caffeic acid propyl ester; caffeic acid isopropyl ester; n-butyl caffeate, caffeic acid pentyl ester, caffeic acid isopentyl ester; the own ester of caffeic acid) inhibitory action to Phellinus, concrete grammar is as follows:
Phellinus is seeded in PDA solid culture medium (potato 300g; Glucose 20g; Agar 17g; Distilled water 1000mL; PH nature) cultivate 10 days, until cover with flat board under 30 DEG C of conditions.
Dull and stereotyped face-off method: by caffeic acid, methyl caffeate, NSC 619661, caffeic acid propyl ester, caffeic acid isopropyl ester, n-butyl caffeate, caffeic acid pentyl ester, caffeic acid isopentyl ester, the own ester of caffeic acid, caffeic acid heptyl ester, caffeic acid monooctyl ester, caffeic acid ester in the ninth of the ten Heavenly Stems, caffeic acid ester in the last of the ten Heavenly stems and CAPE are dissolved in cosolvent, make the solution of variable concentrations, carry out filtration sterilization process with the miillpore filter of 0.22 μm.
The PDA solid culture medium of preparation variable concentrations caffeic acid Arrcostab, does blank with cosolvent, is inoculated in medium by the Phellinus of card punch formed objects.
Flat board is cultivated 8 days as the incubator of 30 DEG C, measures Phellinus growth diameter, compare diameter with the flat board of blank.
Result shows, and the inhibitory action least concentration scope of caffeic acid Arrcostab to Phellinus is 0.01g/L ~ 0.09g/L, and inhibiting rate is 18.315% ~ 37.950%.
Embodiment 17
The caffeic acid Arrcostab that the present embodiment adopts dull and stereotyped face-off method to detect maximum concentration scope (comprises methyl caffeate; NSC 619661; caffeic acid propyl ester; caffeic acid isopropyl ester; n-butyl caffeate, caffeic acid pentyl ester, caffeic acid isopentyl ester; the own ester of caffeic acid) inhibitory action to Phellinus, concrete grammar is as follows:
Phellinus is cultivated 10 days, until cover with flat board under being seeded in PDA solid culture medium 30 DEG C of conditions.
Caffeic acid Arrcostab is dissolved in cosolvent, is mixed with the solution of 60g/L, carry out filtration sterilization process with the miillpore filter of 0.22 μm.
The PDA solid culture medium of preparation variable concentrations caffeic acid Arrcostab, does blank with cosolvent, is inoculated in medium by the Phellinus of card punch formed objects.
Flat board is cultivated 8 days as the incubator of 30 DEG C, measures Phellinus growth diameter, compare diameter with the flat board of blank.
Result shows, and the inhibitory action maximum concentration scope of caffeic acid Arrcostab to Phellinus is 8g/L ~ 18g/L, and inhibiting rate is 30.457% ~ 46.824%.
Embodiment 18
The caffeic acid Arrcostab that the present embodiment adopts dull and stereotyped face-off method to detect optimal concentration scope (comprises methyl caffeate; NSC 619661; caffeic acid propyl ester; caffeic acid isopropyl ester; n-butyl caffeate, caffeic acid pentyl ester, caffeic acid isopentyl ester; the own ester of caffeic acid) inhibitory action to Phellinus, concrete grammar is as follows:
Phellinus is cultivated 10 days, until cover with flat board under being seeded in PDA solid culture medium 30 DEG C of conditions.
Caffeic acid Arrcostab is dissolved in cosolvent, is mixed with the solution of 60g/L, carry out filtration sterilization process with the miillpore filter of 0.22 μm.
The PDA solid culture medium of preparation variable concentrations caffeic acid Arrcostab, does blank with cosolvent, is inoculated in medium by the Phellinus of card punch formed objects.
Flat board is cultivated 8 days as the incubator of 30 DEG C, measures Phellinus growth diameter, compare diameter with the flat board of blank.
Result shows, and the inhibitory action optimal concentration scope of caffeic acid Arrcostab to Phellinus is 5g/L ~ 7g/L, and inhibiting rate is 56.328% ~ 68.957%, and 503nhibiting concentration scope is 0.001g/L ~ 12.879g/L.
Embodiment 19
The present embodiment detects optimum caffeic acid Arrcostab and (comprises methyl caffeate, NSC 619661, caffeic acid propyl ester, caffeic acid isopropyl ester, n-butyl caffeate, caffeic acid pentyl ester, caffeic acid isopentyl ester, the own ester of caffeic acid) inhibitory action to various gram-positive bacteria, Gram-negative bacteria and fungi, method as embodiment 1, embodiment 16.
Result shows, and n-butyl caffeate is the strongest to the inhibitory action of each bacterial strain in all caffeic acid Arrcostabs, and inhibiting rate is 78.132% ~ 91.612%, and 503nhibiting concentration scope is 0.001g/L ~ 9.762g/L, and n-butyl caffeate structural formula is such as formula (II)
Claims (7)
1. caffeic acid Arrcostab is preparing the application in broad-spectrum antibacterial agent, and described caffeic acid Arrcostab structure is such as formula shown in (I):
Wherein, R is H or C1 ~ C10 alkyl.
2. application according to claim 1, is characterized in that described caffeic acid Arrcostab structure is such as formula shown in (II):
3. application according to claim 1, is characterized in that described is 0.001g/L-18g/L to suppression pathogen effective formula (I) compound concentration.
4. application according to claim 1, it is characterized in that formula (I) compound is dissolved in cosolvent, cosolvent comprises organic solvent and surfactant, and described organic solvent is acetone or dimethyl sulfoxide (DMSO), and adding concentration is 5% ~ 50% (v/v); Described surfactant is tritonx-100 or tween, and adding concentration is 5% ~ 50% (v/v).
5. application according to claim 1, is characterized in that the active ingredient of described broad-spectrum antiseptic agent medicine is at least one in the caffeic acid Arrcostab of formula (I) structure.
6. application according to claim 1, is characterized in that described suppression object comprises Gram-negative bacteria, gram-positive bacteria and fungi.
7. application according to claim 6, is characterized in that described suppression object comprises mulberry pseudomonas syringae, Escherichia coli, bacillus subtilis, staphylococcus aureus, tetrads and Phellinus.
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