CN110004095B - Preparation method and application of bacillus pumilus antibacterial active substance - Google Patents
Preparation method and application of bacillus pumilus antibacterial active substance Download PDFInfo
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- CN110004095B CN110004095B CN201910373888.6A CN201910373888A CN110004095B CN 110004095 B CN110004095 B CN 110004095B CN 201910373888 A CN201910373888 A CN 201910373888A CN 110004095 B CN110004095 B CN 110004095B
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- ethyl acetate
- bacillus pumilus
- compound
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- petroleum ether
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Abstract
The invention relates to a preparation method and application of a bacillus pumilus antibacterial active substance. A strain of Bacillus Pumilus (BP) is preserved in the general microbiological center of China Committee for culture Collection of microorganisms in 3 months and 21 days in 2019, and the address is as follows: the collection number of the microorganism is CGMCC NO.17424 from Xilu No.1 of Beijing, Chaoyang, North Cheng, China academy of sciences; the invention also relates to an antibacterial active substance prepared by using the strain and related application. The invention extracts three fatty alcohol compounds with antibacterial activity from Bacillus pumilus (Bacillus pumilus) for the first time, but the three antibacterial active substances are not found in the existing Bacillus pumilus.
Description
Technical Field
The invention relates to a preparation method and application of a bacillus pumilus antibacterial active substance, belonging to the technical field of microbial technology and application.
Background
At present, the use of a large amount of antibiotics in livestock and poultry breeding causes drug resistance and veterinary drug residues, the quality of livestock products is seriously influenced, and soil and water pollution is caused by excretion of residues. Destroying the ecological environment and directly harming the human health. The environmental pollution causes deterioration of human living environment, and the food safety problem is increasingly highlighted.
The antibacterial active substance is a research hotspot at home and abroad in recent years, and can be widely applied to the aspects of biological feed, biocontrol, medicine, natural food preservative, cosmetics and the like. The bacillus can generate various antibacterial substances, has a unique antibacterial mechanism, has an inhibiting effect on gram negative bacteria, gram positive bacteria, mould and saccharomycetes, has the characteristics of broad-spectrum antibacterial, high efficiency, safety, difficulty in generating drug resistance, easiness in degradation and the like, and since Johnson and other reports that the bacillus subtilis can generate the antibacterial substances, scholars at home and abroad can separate various antibacterial substances from different bacilli in sequence, wherein the antibacterial substances comprise polypeptides, lipopeptides, antibacterial proteins and the like.
The bacillus pumilus is one of bacillus and widely exists in nature, and vegetative cells observed under a microscope are rod-shaped, gram-reaction-positive and capable of moving. The colony morphology is divided into two types, namely, a translucent type and an opaque type. The bacillus pumilus can secrete cellulose, lipase, xylanase, pectin lyase and the like with strong activity, and is favorable for degrading macromolecular substances. Can also produce antagonistic substances such as antibiotics, antibacterial proteins and the like, has wide antibacterial range and has inhibiting effect on various pathogenic bacteria.
At present, related technologies related to the production of Bacillus pumilus products exist in China, for example, Chinese patent document CN103563991A (application No. 201210271396.4) discloses a formulation of water dispersible granules of a rice blast biocontrol bacterium Bacillus pumilus bactericide and a preparation method thereof. The solid water dispersible granule contains bacillus brevis TW strain raw powder (2 x 10)12cfu/g) and auxiliary agents, wherein the auxiliary agents comprise carriers, dispersing agents, wetting agents, disintegrating agents, light protective agents and binding agents; the processing method comprises mixing the effective components and auxiliary probes in a certain proportion, and performing jet milling, granulation and drying to obtain the product.
Chinese patent document CN105494441A (application No. 200710021509.1) discloses a wettable powder of Bacillus pumilus containing a spore germination agent and a preparation method thereof, wherein the wettable powder comprises the following raw materials in parts by weight: 40-70 parts of a bacillus pumilus fermentation liquid; filling: 20-30 parts of bentonite; wetting agent: 1-4 parts of polyethylene glycol; dispersing agent: 0.2-1 part of sodium dodecyl sulfate; spore germination agent: 0.1-0.5 part of L-alanine and 0.1-0.5 part of calcium 2, 6-pyridinedicarboxylate; ultraviolet protective agent: 0.5-2 parts of carboxymethyl cellulose and 0.2-1 part of beta-dextrin; drying the protective agent: 1-4 parts of maltose and 0.2-1 part of sodium glutamate.
Chinese patent document CN103205383A (application No. 201310128271.0) discloses a Bacillus pumilus (Bacillus pumilus) E14 CGMCC No. 6682. The invention also discloses a culture method of the bacillus pumilus E14, which comprises the following steps: inoculating Bacillus pumilus E14 strain into 2216E liquid culture medium, and culturing at 28 deg.C and 150rpm for 20 hr to obtain bacterial liquid. The bacillus pumilus E14 or the bacterial liquid obtained by the culture of the method has the bacteriostatic application of inhibiting Vibrio harveyi (Vibrio harveyi), and can be used for preparing shrimp and crab culture medicines or be added into shrimp and crab culture feeds as bacteriostatic medicines.
Chinese patent document CN102965299A (application No. 201210290374.2) discloses a fermentation process of Bacillus pumilus LD-b1 and application thereof in preventing and treating cucumber brown spot, cucumber gray mold, cucumber sclerotinia rot, apple rot, eggplant wilt, wheat scab, cabbage black spot and tomato gray mold. The influence of the bacillus pumilus fermentation liquor on tomato seed germination and seedling growth is studied, and the results show that the bacillus pumilus fermentation liquor has obvious promotion effects on tomato seed germination, root bud growth, plant height and plant weight of seedlings, wherein the effect of 50-time diluent is most obvious, and the effect of foliage spraying on a seedling application mode is obviously superior to root irrigation and seed soaking.
Chinese patent document CN105494441A (application No. 201510951106.4) discloses a wettable powder of Bacillus pumilus containing a spore germination agent and a preparation method thereof, wherein the wettable powder comprises the following raw materials in parts by weight: 40-70 parts of a bacillus pumilus fermentation liquid; filling: 20-30 parts of bentonite; wetting agent: 1-4 parts of polyethylene glycol; dispersing agent: 0.2-1 part of sodium dodecyl sulfate; spore germination agent: 0.1-0.5 part of L-alanine and 0.1-0.5 part of calcium 2, 6-pyridinedicarboxylate; ultraviolet protective agent: 0.5-2 parts of carboxymethyl cellulose and 0.2-1 part of beta-dextrin; drying the protective agent: 1-4 parts of maltose and 0.2-1 part of sodium glutamate.
The above techniques are focused on controlling plant diseases and promoting plant growth, and no reports are made on further studies on active substances of Bacillus pumilus.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a preparation method and application of a bacillus pumilus antibacterial active substance.
The technical scheme of the invention is as follows:
a strain of Bacillus Pumilus (BP) is preserved in the general microbiological center of China Committee for culture Collection of microorganisms in 3 months and 21 days in 2019, and the address is as follows: the collection number of the microorganism is CGMCC NO.17424, No. 3 of Xilu No.1 of Beijing, Chaoyang, Beijing, China academy of sciences.
The method for culturing the bacillus pumilus comprises the following steps:
(1) inoculating the bacillus pumilus into a seed culture medium, and culturing for 14-16 hours at 35-38 ℃ and 160-200 rpm to prepare a seed solution;
(2) inoculating the seed solution prepared in the step (1) into a fermentation medium according to the volume percentage of 3-5%, and culturing at the temperature of 28-32 ℃ and at the speed of 160-200 r/min for 45-50 h to prepare a fermentation liquid;
the fermentation medium comprises the following components per liter:
glucose 30.0g, K2HPO4×3H2O 7.0g，KH2PO4 3.0g，(NH4)2SO41.5g, NaCitrato 3H2O 0.5g，MgSO4·7H20.1g of O, 7.0-7.2 of pHs, and adding water to a constant volume of 1L.
Preferably, in step (1), the seed culture medium is LB broth, and the composition per liter is as follows:
10 g of peptone, 10 g of sodium chloride, 5g of yeast extract and water to reach the constant volume of 1000 ml.
The antibacterial active substance derived from the bacillus pumilus has the structural formula shown as follows:
compound I, name: 6,7- (. alpha. -hydroxy) diethyldodecane, english name: 6,7- (α -hydroxy) -diethyl-dodecane:
compound II, name: 4,5-dibutyl-1,2- (α -methyl) -cyclohexanedimethanol, english name: 4,5-dibutyl-1,2- (. alpha. -methyl) -cyclohexenedimethanol:
compound III, name: 4,5-dibutyl-1,2- (α -methyl) -cyclohexanedimethanone, english name: 4,5-dibutyl-1,2- (. alpha. -methyl) -cyclohexenedimethanol:
the preparation method of the antibacterial active substance from the bacillus pumilus comprises the following steps:
(i) carrying out solid-liquid separation on the fermentation liquor, taking supernatant, and concentrating to obtain concentrated solution;
(ii) (iii) extracting the concentrated solution prepared in the step (ii) with ethyl acetate, taking an extract phase, concentrating, separating by silica gel column chromatography, eluting with different petroleum ether, ethyl acetate and methanol solutions with different gradients, collecting the eluent with antibacterial activity of petroleum ether/ethyl acetate (50: 50), concentrating, and drying to obtain a concentrated dry product;
(iii) (III) subjecting the concentrated dried product obtained in step (II) to high performance liquid chromatography to obtain antibacterial active substances respectively as compound I, compound II and compound III.
Preferably, in the step (i), the solid-liquid separation is performed by centrifuging at 3500-4500 rpm for 25-35 minutes.
According to the invention, in the step (i), the concentration is preferably 8-12 times of the concentration of the original solution by rotary evaporation.
Preferably, in step (ii), the volume ratio of the concentrated solution to the ethyl acetate is 1: 1; preferably, the extraction times are 2-4.
According to the invention, in the step (ii), the silica gel column is 200-300 mesh silica gel column.
Preferably, in step (ii), the different gradients of the different solutions of petroleum ether, ethyl acetate and methanol are as follows, and are volume ratios:
petroleum ether, petroleum ether/ethyl acetate 75:25, petroleum ether/ethyl acetate 50: 50; petroleum ether/ethyl acetate 25: 75; petroleum ether/ethyl acetate 0: 100; ethyl acetate/methanol 75: 25; ethyl acetate/methanol 50: 50; ethyl acetate/methanol 25:75, ethyl acetate/methanol 0: 100.
According to the present invention, in the step (ii), the drying is performed by a nitrogen blow drying method.
Preferably, in step (iii), the separation conditions of high performance liquid chromatography are as follows:
the column is prepared by adopting YMC-Pack SIL column, 250mm multiplied by 10 mm; the mobile phase is 60% ethyl acetate and 40% n-butanol, the wavelength is 260nm, the sample amount is 5 μ L, the flow rate is 2.0mL/min, the column temperature is 25 deg.C, and the pressure is 40 bar.
The application of the compound I, the compound II and the compound III with antibacterial activity in preparing medicines for inhibiting pathogenic bacteria.
The compound I, the compound II and the compound III with antibacterial activity are applied to preparing antibacterial cosmetics.
The compound I, the compound II and the compound III with antibacterial activity are applied to the preparation of pesticides.
Advantageous effects
1. The invention discovers a Bacillus Pumilus (BP) strain for the first time, wherein the strain can generate three fatty alcohol compounds with antibacterial activity, and the three antibacterial active substances are not found in the existing Bacillus pumilus;
2. the invention discloses three compounds with antibacterial activity of enol for the first time, the compounds have strong effect of inhibiting pathogenic bacteria, and the preparation method adopting microbial fermentation has the advantages of short production period, stable product and high content, and the compounds can be widely applied to the production of pesticides, medicines and cosmetics.
Drawings
FIG. 1 is a graph showing the results of mass spectrometric detection of Compound I;
FIG. 2 is a graph showing the results of hydrogen spectrum detection of Compound I;
FIG. 3 is a graph showing the results of carbon spectrum detection of Compound I;
FIG. 4 is a graph showing the results of mass spectrometric detection of Compound II;
FIG. 5 is a graph showing the results of hydrogen spectrum detection of Compound II;
FIG. 6 is a graph showing the results of carbon spectrum detection of Compound II;
FIG. 7 is a graph showing the results of mass spectrometric detection of Compound III;
FIG. 8 is a graph showing the results of hydrogen spectrum detection of Compound III;
FIG. 9 is a graph showing the results of carbon spectrum detection of Compound III;
FIG. 10 is a photograph showing the results of the experiment of the bacteriostatic activity of the concentrated dried substance obtained in the step (ii) of example 3;
FIG. 11 is a photograph showing the results of the bacteriostatic activity test of the three compounds prepared in example 3;
wherein: 1. zone of inhibition of compound I; 2. the zone of inhibition of compound II; 3. the zone of inhibition of compound III;
FIG. 12 is a graph of the bacteriostatic results of compound I against different strains;
FIG. 13 is a graph of the bacteriostatic results of compound II against different strains;
FIG. 14 is a graph of the bacteriostatic results of compound III against different strains;
Detailed Description
To further illustrate the present invention, the technical solutions of the present invention will be described in more detail with reference to the following specific embodiments, but the examples are only for illustration and not intended to limit the present invention, and any alternatives or modifications based on the teachings of the present invention are within the protection scope of the present invention.
Biological material
Bacillus Pumilus (BP) is preserved in China general microbiological culture Collection center (CGMCC for short, the address: No. 3 of Xilu No.1 of Beijing university of Chaoyang, China institute of microbiology) at 21.3.2019, and the preservation number is CGMCC NO. 17424.
Bacillus pumilus BP-5 purchased from China culture Collection of microorganisms with the following numbering: CGMCC 1.8167.
Example 1
A Bacillus Pumilus (BP) strain is preserved in China general microbiological culture Collection center (CGMCC for short, the address: No. 3 of Xilu No.1 of Beijing Kogyo Xinyang district, China academy of sciences microbiological research institute) in 3 months and 21 days in 2019, and the preservation number is CGMCC NO. 17424.
The biological characteristics of the strain are as follows: the bacterium belongs to the genus Bacillus, and the bacterium is rod-shaped and gram-positive. The growth on LB inclined plane is transparent, and can produce antibacterial active ingredient.
Example 2
A method for culturing Bacillus pumilus short (Bacillus pumilus) BP comprises the following steps:
(1) inoculating the Bacillus pumilus into a seed culture medium, and culturing at 37 deg.C and 180 rpm for 16 hr to obtain a seed solution;
the seed culture medium is LB broth culture medium, and each liter of the seed culture medium comprises the following components:
10 g of peptone, 10 g of sodium chloride, 5g of yeast extract and water to a constant volume of 1000 ml;
(2) inoculating the seed solution prepared in the step (1) into a fermentation culture medium according to the volume percentage of 3%, and culturing at 30 ℃ and 180 r/min for 48h to prepare a fermentation liquid;
the fermentation medium comprises the following components per liter:
glucose 30.0g, K2HPO4×3H2O 7.0g，KH2PO4 3.0g，(NH4)2SO41.5g, NaCitrato 3H2O 0.5g，MgSO4·7H20.1g of O, 7.0-7.2 of pHs, and adding water to a constant volume of 1L.
Example 3
A preparation method of an antibacterial active substance derived from Bacillus pumilus BP comprises the following steps:
(i) centrifuging the fermentation liquor prepared in the example 2 for 35 minutes under the condition of 3500 rpm, taking supernatant, concentrating the supernatant to 8 times of the concentration of the original solution by adopting rotary evaporation to prepare concentrated solution;
(ii) extracting the concentrated solution prepared in the step (ii) by ethyl acetate, wherein the volume ratio of the concentrated solution to the ethyl acetate is 1:1, extracting an extraction phase for 4 times, combining the extraction phases, concentrating, separating by 200-mesh silica gel column chromatography, eluting by different gradient with different solutions of petroleum ether, ethyl acetate and methanol respectively, collecting the eluent with antibacterial activity of petroleum ether/ethyl acetate (50: 50), concentrating, and drying by nitrogen blowing to prepare a concentrated dry product;
different gradients of different solutions of the petroleum ether, the ethyl acetate and the methanol are as follows, and the gradients are volume ratios: petroleum ether, petroleum ether/ethyl acetate 75:25, petroleum ether/ethyl acetate 50: 50; petroleum ether/ethyl acetate 25: 75; petroleum ether/ethyl acetate 0: 100; ethyl acetate/methanol 75: 25; ethyl acetate/methanol 50: 50; ethyl acetate/methanol 25:75, ethyl acetate/methanol 0: 100;
detecting the antibacterial activity of Escherichia coli by agar diffusion method, as shown in FIG. 10;
(iii) (III) subjecting the concentrated dried product obtained in step (II) to high performance liquid chromatography to obtain antibacterial active substances respectively as compound I, compound II and compound III;
the high performance liquid chromatography separation conditions are as follows:
the column was prepared using YMC-Pack SIL column, 250mm × 10mm I.D, S-5 μm,12nm, SL 12S-2510WT, Ser. No. 114EA80134; the mobile phase was 60% ethyl acetate (Eta-00060458HPLC) and 40% n-butanol (a-He-00010273HPLC), wavelength 260nm, sample size 5. mu.L, flow rate 2.0mL/min, column temperature 25 ℃ and pressure 40 bar.
Respectively detecting the mass spectrum, the hydrogen spectrum and the carbon spectrum of the compound I, the compound II and the compound III, wherein the detection results are shown in figures 1-9; from the above results, compound I, named: 6,7- (. alpha. -hydroxy) diethyldodecane, english name: 6,7- (alpha-hydroxy) -diethyl-dodecane, the structural formula of which is as follows:
compound II, name: 4,5-dibutyl-1,2- (α -methyl) -cyclohexanedimethanol, english name: 4,5-dibutyl-1,2- (a-methyl) -cyclohexenedimethanol, the structural formula is as follows:
compound III, name: 4,5-dibutyl-1,2- (α -methyl) -cyclohexanedimethanone, english name: 4,5-dibutyl-1,2- (a-methyl) -cyclohexenedimethanol, the structural formula is as follows:
escherichia coli inhibition experiments were performed on the three compounds, respectively, and the concentrations of the experimental compounds were 2.6mg/mL, 3mg/mL, and 3.2mg/mL, respectively, and the results were shown in FIG. 11 after dilution according to the experiments.
Example 4
A preparation method of an antibacterial active substance derived from Bacillus pumilus comprises the following steps:
(i) centrifuging the fermentation liquor prepared in the embodiment 2 for 25 minutes under the condition of 4500 rpm, taking supernate, concentrating the supernate to 12 times of the concentration of the original solution by adopting rotary evaporation to prepare concentrated solution;
(ii) extracting the concentrated solution prepared in the step (ii) by ethyl acetate, wherein the volume ratio of the concentrated solution to the ethyl acetate is 1:1, extracting the extract phase for 2 times, combining the extract phases, concentrating, separating by 200-mesh silica gel column chromatography, eluting by different gradient with different solutions of petroleum ether, ethyl acetate and methanol respectively, collecting the eluent with antibacterial activity of petroleum ether/ethyl acetate (50: 50), concentrating, and drying by nitrogen blowing to prepare a concentrated dry product;
different gradients of different solutions of the petroleum ether, the ethyl acetate and the methanol are as follows, and the gradients are volume ratios: petroleum ether, petroleum ether/ethyl acetate 75:25, petroleum ether/ethyl acetate 50: 50; petroleum ether/ethyl acetate 25: 75; petroleum ether/ethyl acetate 0: 100; ethyl acetate/methanol 75: 25; ethyl acetate/methanol 50: 50; ethyl acetate/methanol 25:75, ethyl acetate/methanol 0: 100;
(iii) (III) subjecting the concentrated dried product obtained in step (II) to high performance liquid chromatography to obtain antibacterial active substances respectively as compound I, compound II and compound III;
the high performance liquid chromatography separation conditions are as follows:
the column is prepared by adopting YMC-Pack SIL column, 250mm multiplied by 10 mm; the mobile phase is 60% ethyl acetate and 40% n-butanol, the wavelength is 260nm, the sample amount is 5 μ L, the flow rate is 2.0mL/min, the column temperature is 25 deg.C, and the pressure is 40 bar.
The three compounds separated were tested as in example 3.
Comparative example 1
The existing bacillus pumilus (BP-5) is purchased from China culture collection of microorganisms, and the numbering is as follows: CGMCC 1.8167.
The preparation was carried out according to the method of example 2-3, and as a result, the relevant compounds I to III (fatty alcohol-based compounds) were not obtained.
Examples of the experiments
The antibacterial test method comprises the following steps: by detecting OD of pathogenic bacteria at different time intervals600To determine the growth of the pathogenic bacteriaAnd judging whether the antibacterial effect is achieved or not. The specific operation steps are as follows:
before the test, the pathogenic bacteria liquid is prepared into 105cfu/mL, on an ultra-clean workbench, adding 100 mu L of pathogenic bacteria liquid into each EP tube according to the test dosage, centrifuging at a high speed for 1min, removing the supernatant and retaining the bacteria, adding 900 mu L of fresh LB broth culture medium, marking and correspondingly adding 100 mu L of a sample to be tested, and uniformly mixing. OD was measured at 0 hour and 15 hours, respectively600And recording the data. Each set of experiments was repeated 3 times.
Through detection, the bacteriostatic results of the compound I aiming at different strains are shown in figure 12; the bacteriostatic results of the compound II against different strains are shown in FIG. 13; the bacteriostatic results of compound III against different strains are shown in fig. 14.
Claims (16)
1. A strain of Bacillus Pumilus (BP) is preserved in the general microbiological center of China Committee for culture Collection of microorganisms in 3 months and 21 days in 2019, and the address is as follows: the collection number of the microorganism is CGMCC NO.17424, No. 3 of Xilu No.1 of Beijing, Chaoyang, Beijing, China academy of sciences.
2. The method for culturing Bacillus pumilus of claim 1, comprising the steps of:
(1) inoculating the bacillus pumilus into a seed culture medium, and culturing for 14-16 hours at 35-38 ℃ and 160-200 rpm to prepare a seed solution;
(2) inoculating the seed solution prepared in the step (1) into a fermentation medium according to the volume percentage of 3-5%, and culturing at the temperature of 28-32 ℃ and at the speed of 160-200 r/min for 45-50 h to prepare a fermentation liquid;
the fermentation medium comprises the following components per liter:
glucose 30.0g, K2HPO4×3H2O 7.0g，KH2PO4 3.0g，(NH4)2SO41.5g, NaCitrato 3H2O 0.5g，MgSO4·7H20.1g of O, 7.0-7.2 of pHs, and adding water to a constant volume of 1L.
3. The culture method according to claim 2, wherein in the step (1), the seed culture medium is LB broth, and the composition per liter is as follows:
10 g of peptone, 10 g of sodium chloride, 5g of yeast extract and water to reach the constant volume of 1000 ml.
4. An antibacterial active substance derived from the Bacillus pumilus (Bacillus pumilus) BP of claim 1, having the structural formula shown below:
compound II, name: 4,5-dibutyl-1,2- (α -methyl) -cyclohexanedimethanol:
compound III, name: 4,5-dibutyl-1,2- (α -methyl) -cyclohexanedimethanone:
5. a method for preparing an antibacterial active substance from Bacillus pumilus, which is characterized by comprising the following steps:
(i) carrying out solid-liquid separation on the fermentation liquor in the claim 2, taking supernatant, and concentrating to obtain concentrated solution;
(ii) (iii) extracting the concentrated solution prepared in the step (ii) with ethyl acetate, taking an extract phase, concentrating, separating by silica gel column chromatography, eluting with different petroleum ether, ethyl acetate and methanol solutions with different gradients, collecting the eluent with antibacterial activity of petroleum ether/ethyl acetate (50: 50), concentrating, and drying to obtain a concentrated dry product;
(iii) (III) subjecting the concentrated dry product obtained in step (II) to high performance liquid chromatography to obtain antibacterial active substances, namely compound I, compound II and compound III;
the compound I is named as: 6,7- (. alpha. -hydroxy) diethyldodecane:
compound II, name: 4,5-dibutyl-1,2- (α -methyl) -cyclohexanedimethanol:
compound III, name: 4,5-dibutyl-1,2- (α -methyl) -cyclohexanedimethanone:
6. the method according to claim 5, wherein in the step (i), the solid-liquid separation is performed by centrifugation at 3500 to 4500 rpm for 25 to 35 minutes.
7. The method according to claim 5, wherein in the step (i), the concentration is performed by rotary evaporation to 8 to 12 times of the concentration of the original solution.
8. The method of claim 5, wherein in step (ii), the volume ratio of the concentrate to the ethyl acetate is 1: 1.
9. The method according to claim 5, wherein the number of times of extraction in the step (ii) is 2 to 4.
10. The preparation method according to claim 5, wherein in the step (ii), the silica gel column is 200-300 mesh silica gel column.
11. The preparation method according to claim 5, wherein in the step (ii), different gradients of different solutions of petroleum ether, ethyl acetate and methanol are as follows, and are volume ratios:
petroleum ether, petroleum ether/ethyl acetate 75:25, petroleum ether/ethyl acetate 50: 50; petroleum ether/ethyl acetate 25: 75; petroleum ether/ethyl acetate 0: 100; ethyl acetate/methanol 75: 25; ethyl acetate/methanol 50: 50; ethyl acetate/methanol 25:75, ethyl acetate/methanol 0: 100.
12. The method according to claim 5, wherein in the step (ii), the drying is performed by nitrogen blow drying.
13. The method according to claim 5, wherein in step (iii), the separation conditions of high performance liquid chromatography are as follows:
the column is prepared by adopting YMC-Pack SIL column, 250mm multiplied by 10 mm; the mobile phase is 60% ethyl acetate and 40% n-butanol, the wavelength is 260nm, the sample amount is 5 μ L, the flow rate is 2.0mL/min, the column temperature is 25 deg.C, and the pressure is 40 bar.
14. The use of compounds II, III with antibacterial activity according to claim 4 for the preparation of a medicament for the inhibition of pathogenic bacteria.
15. Use of the compounds II, III having antibacterial activity according to claim 4 for the preparation of antibacterial cosmetics.
16. The use of compounds II, III with antibacterial activity according to claim 4 for the preparation of pesticides.
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| CN112708646A (en) * | 2020-12-28 | 2021-04-27 | 昆明理工大学 | Preparation method of Pyrophen compound |
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