CN111602662B - Preparation method and application of tricobactin - Google Patents
Preparation method and application of tricobactin Download PDFInfo
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- CN111602662B CN111602662B CN202010575440.5A CN202010575440A CN111602662B CN 111602662 B CN111602662 B CN 111602662B CN 202010575440 A CN202010575440 A CN 202010575440A CN 111602662 B CN111602662 B CN 111602662B
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- PKVRCIRHQMSYJX-AIFWHQITSA-N trabectedin Chemical compound C([C@@]1(C(OC2)=O)NCCC3=C1C=C(C(=C3)O)OC)S[C@@H]1C3=C(OC(C)=O)C(C)=C4OCOC4=C3[C@H]2N2[C@@H](O)[C@H](CC=3C4=C(O)C(OC)=C(C)C=3)N(C)[C@H]4[C@@H]21 PKVRCIRHQMSYJX-AIFWHQITSA-N 0.000 description 1
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- YKUJZZHGTWVWHA-UHFFFAOYSA-N triptolide Natural products COC12CC3OC3(C(C)C)C(O)C14OC4CC5C6=C(CCC25C)C(=O)OC6 YKUJZZHGTWVWHA-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- 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
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
- A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
- A01N43/06—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom five-membered rings
- A01N43/12—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom five-membered rings condensed with a carbocyclic ring
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
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- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Environmental Sciences (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Pest Control & Pesticides (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Dentistry (AREA)
- Agronomy & Crop Science (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides a preparation method and application of a tricot-killing bacterium. The invention discovers for the first time that the trypsinicin (trypacin) has the activity of dissolving the algae cells, can be applied to dissolving the algae cells, particularly shows remarkable dissolving effect on the algae cells of the marine dunaliella (Chattonella marina), and can completely dissolve the algae cells within 15min when 100 mu g/mL of the trypsinicin is used according to the volume of the algae liquid containing the algae cells. Based on the activity of the trypanosicin for dissolving algae cells, the method can be used for red tide treatment, and further solves the problem that the ecological environment is damaged by mass propagation of algae.
Description
Technical Field
The invention relates to the field of compound preparation, and particularly relates to a preparation method and application of tricoxacin.
Background
Red tide (Red tide) refers to the harmful phenomenon of seawater discoloration caused by the excessive proliferation of some algae, protozoa or bacteria in the ocean under certain climatic conditions. At present, most of red tides are caused by algae, and due to the difference of algae species, seawater in an outbreak period can be in yellow, brown or green color or unchanged color besides red, so the red tides and fresh water blooms are also called Harmful Algal blooms (Harmful algae Bloom, HAB). The harm of red tide to marine life and human can be divided into three aspects, firstly, the red tide algae can consume oxygen in seawater, or the red tide algae cell directly blocks the respiratory organ of marine life to cause oxygen deficiency of marine fish and the like, so that a great amount of fish and shellfish die; secondly, some red tide algae can produce toxins, which can cause death of marine organisms, and some toxins are transferred upwards through a food chain and can even cause food poisoning and even death of human beings; finally, the mass propagation of red tide algae breaks the balance of the marine ecosystem, reducing the biodiversity in the sea.
The prevention and control of ocean red tides is a current worldwide problem. In recent years, as the outbreak frequency of red tides is accelerated, a great deal of research on the prevention and treatment measures of red tide disasters has been conducted by experts and scholars in many countries and regions. The biological control technology realizes algae control mainly through nutrition competition relationship among organisms, has the advantage of no pollution to the environment, and becomes the red tide control method with the greatest development prospect.
The current research shows that the algae-lysing bacteria exist in the ocean, and the algae-lysing bacteria and the red tide algae are in a symbiotic system, so that on one hand, the algae-lysing bacteria provide nutrition for the growth and the propagation of the algae; on the other hand, the growth of algae can be inhibited through direct or indirect action, and even the algae cells are cracked to show the algae killing effect. The restriction relationship between algicidal bacteria and red tide algae provides a new way for preventing and controlling red tide by microorganisms. Solutions of the present discoveryThe algae extracellular algae-lysing active substance includes pigments, proteins, amino acids, polypeptides, hydroxylamine, antibiotics, alkaloids[1]。
Aspergillus fumigatus (Aspergillus fumigatus) is a fungus belonging to Aspergillus of Tricholomataceae of Ascomycetales, has a wide source, and the current literature reports mainly focus on plant endophytic environments and marine environments, wherein the marine environments mainly comprise marine animals, marine sediment and marine plants. Shinggu Fumiquinazoles A-C isolated from the gastrointestinal tract of the marine rough Pseudobulbus Pseudosciaenae (Pseudolaris japonica) was moderately cytotoxic to cultured lymphoid leukemia cell P-388[2]. Most of 8 aspergillus fumigatus separated from plant tissues and soil samples of mangrove such as Linhaipeng show certain inhibitory activity to staphylococcus aureus and candida albicans, and a small number of strains show certain cytotoxicity[3]。
Trypacin (trypticin) was first isolated in 1963 from the fermentation of Aspergillus fumigatus and was defined as a natural product against Trypanosoma cruzi and Toxoplasma gondii[4]The chemical structure of which was determined in 1965 by Turner's spectroscopic data[5]And in 2005 some of the location assignments of carbon spectra were modified[6]. The related experiments show that the substance exists only in the spores of the mold[7]. The cone trexamectin has been shown to have a wide range of activity, but there is currently no report on the activity of cone trexamectin in lysing algal cells.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to overcome the problem of red tide caused by mass propagation of algae in the prior art, thereby providing the application of the trypanosicidin in dissolving algae cells and the preparation method of the trypanosicidin.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
in one aspect, the invention provides the use of a spinosyn for lysing algal cells.
Further, the algae cells are cells of marine dunaliella carlsbergii.
Further, the algae fine powder is prepared byThe volume of the algae liquid of the cells is calculated, the dosage of the tricot-killing bacterin is not less than 100 mu g/mL, and the concentration of the algae cells in the algae liquid is 1.0 multiplied by 104～2.0×104one/mL.
Further, the said aspergillin is isolated from a metabolite of Aspergillus sp.LW1 fungus with the deposit number: CCTCC No. M2019453.
In another aspect, the present invention provides a method for preparing a spinosyn, comprising:
fermenting Aspergillus sp.LW1 to obtain fermentation culture;
and (3) sequentially performing crude extraction, separation and purification on the fermentation culture to obtain the tricopeptide.
Further, the step of obtaining the fermentation culture comprises:
inoculating Aspergillus sp.LW1 into a seed culture medium, culturing at 20-30 ℃ for 30-60h at 250rpm of 100 and preferably at 28 ℃ for 48h at 180rpm to obtain a seed solution, wherein raw materials of the seed culture medium comprise potatoes, glucose, sea salt and deionized water, the potatoes are 200g, the glucose is 20g, the sea salt is 30g, and the deionized water is metered to 1L in the seed culture medium;
adding the seed solution into a fermentation culture medium, standing and fermenting for 10-35 days at 20-30 ℃, preferably for 30 days at 28 ℃ to obtain a fermentation culture, wherein the raw materials of the fermentation culture medium comprise rice, sea salt and deionized water, the rice is 80g and the sea salt is 3g based on 120mL of deionized water.
Further, the step of crude extraction comprises:
ultrasonically extracting the fermentation culture with ethanol, and filtering to obtain ethanol extract;
concentrating the ethanol extract to remove the solvent to obtain an ethanol concentrate;
dissolving the ethanol concentrate with deionized water, extracting with ethyl acetate, recovering ethyl acetate layer, and concentrating under reduced pressure to obtain crude extract.
Further, in the step of obtaining the ethanol extract, the extraction is repeated 2-5 times, preferably, 4 times;
in the step of obtaining an ethanol concentrate, the concentration is performed at 45 ℃;
in the step of obtaining the crude extract, the volume ratio of the deionized water to the ethyl acetate is 1:1, repeating the extraction 2-5 times, preferably 3 times.
Further, the separation and purification comprises:
sequentially carrying out forward silica gel column chromatographic separation and Sephadex LH-20 chromatographic separation on a crude extract obtained by crude extraction to obtain a chromatographic separation component;
and carrying out forward HPLC separation and purification on the chromatographic separation component.
Further, the step of separating by forward silica gel column chromatography comprises: dissolving the crude extract with methanol, mixing, loading into a column, eluting with dichloromethane, concentrating the obtained eluent to remove the solvent, and obtaining dichloromethane eluate;
in the step of chromatographic separation of Sephadex LH-20, comprising: dissolving the dichloromethane eluate with methanol, loading, eluting with eluents with elution volumes of 210mL, 160mL and 120mL in sequence, collecting eluents corresponding to the elution volumes in sequence, and taking the eluents corresponding to the elution volumes of 120mL as chromatographic separation components, wherein the eluents adopt chloroform and methanol in a volume ratio of 1: 1;
in the step of forward HPLC separation and purification, the chromatographic column is: zorbax Rx-SIL normal phase chromatography column (250 mm. times.4.6 mm, 5 μm), mobile phase: ethanol and cyclohexane in a volume ratio of 15:85, and the elution mode is as follows: isocratic elution, and collecting the components with retention time of 8min to obtain the final product.
The technical scheme of the invention has the following advantages:
1. the invention discovers for the first time that the trypsinicin (trypacin) has the activity of dissolving the algae cells, can be applied to dissolving the algae cells, particularly shows remarkable dissolving effect on the algae cells of the marine dunaliella (Chattonella marina), and can completely dissolve the algae cells within 15min when 100 mu g/mL of the trypsinicin is used according to the volume of the algae liquid containing the algae cells. Based on the activity of the trypanosicin for dissolving algae cells, the method can be used for red tide treatment, and further solves the problem that the ecological environment is damaged by mass propagation of algae.
2. The invention provides a preparation method of a tricolor, which is used for separating and obtaining the tricolor in Aspergillus sp.LW1, the prior preparation method of the tricolor adopts a reverse phase system, uses methanol, water or acetonitrile and water as eluent, and causes the problems of low sample loading amount and difficult sample recovery, while the embodiment of the invention adopts a normal phase system, and carries out forward silica gel column chromatography separation, Sephadex LH-20 chromatography separation and forward HPLC separation and purification on a crude extract obtained by crude extraction in sequence, and has the advantages of larger sample loading amount and easier solvent recovery.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The raw materials or equipment used are all conventional products which can be obtained commercially, including but not limited to the raw materials or equipment used in the examples of the present application.
The invention provides an application of cone aspergillin in dissolving algae cells. Wherein the algae cell can be marine dunaliella shield cell. Based on the volume of the algae solution containing algae cells, the consumption of the echinococcin is not less than 100 mug/mL, and the concentration of the algae cells in the algae solution is 1.0 multiplied by 104～2.0×104one/mL.
The spinosad may be isolated from a metabolite of the Aspergillus sp.lw1 fungus. The fungus is preserved in China center for type culture Collection with the preservation number: CCTCC No. M2019453, the preservation date is: 6 and 13 months in 2019.
Specifically, the invention also provides a preparation method of the ecteinascidin, which comprises the following steps: fermenting Aspergillus sp.LW1 to obtain fermentation culture; and (4) sequentially performing crude extraction, separation and purification on the fermentation culture to obtain the tricopeptide.
As a preferred embodiment of the present invention, the step of obtaining a fermentation culture comprises: inoculating Aspergillus sp.LW1 into seed culture medium, culturing at 20-30 deg.C at 100-250rpm for 30-60 hr, preferably at 28 deg.C at 180rpm for 48 hr to obtain seed solution; adding the seed solution into fermentation culture medium, and standing and fermenting at 20-30 deg.C for 10-35 days, preferably at 28 deg.C for 30 days to obtain fermentation culture.
Alternatively, the raw materials of the seed culture medium comprise potato, glucose, sea salt and deionized water, and can be prepared according to the following method: slicing potato 200g, boiling for 30min, filtering to obtain juice, adding glucose 20g, sea salt 30g, deionized water to constant volume to 1L, mixing, and autoclaving at 121 deg.C for 30min with autoclave. The raw materials of the fermentation medium comprise rice, sea salt and deionized water, and can be prepared according to the following method: mixing rice 80g, sea salt 3g, and deionized water 120mL, and autoclaving at 121 deg.C for 20min with autoclave.
Optionally, as the Aspergillus sp.lw1 fungus is cryopreserved, prior to its inoculation into the seed medium, it further comprises: the cryopreserved aspergillus sp.lw1 was spread into a recovery medium for recovery and cultured at 28 ℃ for 3 days. The recovery culture medium comprises the following raw materials of potato, glucose, sea salt, agar and deionized water, wherein 200g of potato, 20g of glucose, 30g of sea salt and 15g of agar are counted by 1L of recovery culture medium, and deionized water is added to the recovery culture medium to fix the volume to 1L; the preparation method comprises the following steps: boiling potato 200g slice for 30min, filtering to obtain juice, adding glucose 20g, sea salt 30g, agar 15g, adding deionized water to constant volume to 1L, mixing, and autoclaving at 121 deg.C for 30min with autoclave.
As a preferred embodiment of the present invention, the step of crude extraction comprises: ultrasonically extracting the fermentation culture with ethanol, and filtering to remove residue to obtain ethanol extract; concentrating the ethanol extract to remove the solvent to obtain ethanol concentrate; dissolving the ethanol concentrate with deionized water, extracting with ethyl acetate, recovering ethyl acetate layer, and concentrating under reduced pressure to obtain crude extract.
Optionally, in the step of obtaining the ethanol extract, the extraction is repeated 2-5 times, preferably 4 times; in the step of obtaining an ethanol concentrate, the concentration is performed at 45 ℃; in the step of obtaining the crude extract, the volume ratio of the deionized water to the ethyl acetate is 1:1, repeating the extraction 2-5 times, preferably 3 times.
As a preferred embodiment of the present invention, the separation and purification comprises: sequentially carrying out forward silica gel column chromatographic separation and Sephadex LH-20 chromatographic separation on a crude extract obtained by crude extraction to obtain a chromatographic separation component; and carrying out forward HPLC separation and purification on the chromatographic separation component.
Optionally, the step of forward silica gel column chromatography separation comprises: dissolving the crude extract with methanol, loading, eluting with dichloromethane, concentrating the obtained eluate, and removing solvent to obtain dichloromethane eluate; in the step of Sephadex LH-20 chromatographic separation, comprising: dissolving a dichloromethane eluate with methanol, loading the dichloromethane eluate, sequentially eluting with eluents with elution volumes of 210mL, 160mL and 120mL, sequentially collecting eluents corresponding to the elution volumes, and taking the eluents corresponding to the elution volumes of 120mL as chromatographic separation components, wherein the eluent is chloroform and methanol in a volume ratio of 1: 1; in the step of forward HPLC separation and purification, the chromatographic column is: zorbax Rx-SIL normal phase chromatography column (250 mm. times.4.6 mm, 5 μm), mobile phase: ethanol and cyclohexane in a volume ratio of 15:85, and the elution mode is as follows: isocratic elution, flow rate: 1mL/min, and collecting the components with the retention time of 8min to obtain the triptolide.
The technical solution provided by the present invention will be described in detail with reference to specific examples.
Example 1
The preparation method of the trypanostatin comprises the following steps:
firstly, fermenting strains
1. Preparation of culture Medium
(1) Preparing recovery culture medium
Slicing potato 200g, boiling for 30min, filtering to obtain juice, mixing glucose (Shanghai raw product) 20g, sea salt (Guangdong salt industry group) 30g, agar (Shanghai raw product) 15g, adding deionized water to constant volume to 1L, and autoclaving at 121 deg.C for 30min with autoclave (GR85DP, Xiamen instruments Co., Ltd.).
(2) Preparing seed culture medium
Slicing potato 200g, boiling for 30min, filtering to obtain juice, adding glucose 20g, sea salt 30g, deionized water to constant volume to 1L, mixing, and autoclaving at 121 deg.C for 30min with autoclave.
(3) Preparing a fermentation medium
Adding rice 80g, sea salt 3g, and deionized water 120mL into 500mL conical flask, mixing well, and autoclaving at 121 deg.C for 20min with autoclave to obtain the final product.
2. Fermentation of bacterial species
Coating the Aspergillus sp.LW1 strain preserved at low temperature in a recovery culture medium for recovery, and culturing at 28 ℃ for 3 days; inoculating the recovered strain into seed culture medium with inoculating loop in super clean bench (SW-CJ-2FD, Sujing Antai), and culturing at 28 deg.C and 180rpm for 48 hr to obtain seed solution; inoculating the seed solution into a fermentation culture medium, wherein the inoculation amount is 5 mL/bottle, and standing and fermenting for 30 days at 28 ℃ to obtain a fermentation culture.
Separation of dicentrin
1. Crude extraction
Putting 6000g of the obtained fermentation culture into a conical flask, adding ethanol (analytically pure, manufactured by Xiong science corporation) to immerse the fermentation culture, cutting the fermentation culture into small blocks by using a shovel blade, adding ethanol to a constant volume of 400mL, performing ultrasonic treatment for 1h, filtering, repeatedly extracting for 4 times according to the above method, and combining the filtrates to obtain an ethanol extract; concentrating the ethanol extract at 45 deg.C with rotary evaporator to remove ethanol to obtain ethanol concentrate; dissolving the ethanol concentrate with deionized water, diluting to 3L, extracting with ethyl acetate (analytically pure, manufactured by Sjogren science corporation) for 3 times, recovering ethyl acetate layer, and concentrating under reduced pressure to obtain crude extract.
2. Separating and purifying
(1) Forward silica gel column chromatographic separation
Mixing samples: dissolving 60g of the crude extract with 60mL of methanol, adding the dissolved crude extract into 120g of dry silica gel after complete dissolution, uniformly stirring, and placing in a fume hood for volatilizing to obtain sample-mixing silica gel;
column assembling: weighing 300g of silica gel (200-300 meshes, Nintentangyou silica gel development Co., Ltd.), soaking and stirring uniformly with 1000mL of dichloromethane (analytically pure, Xilonga science Co., Ltd.), loading into a glass column (10cm × 100cm), and obtaining a silica gel column after the silica gel is settled;
loading: adding the sample-mixing silica gel into a filled silica gel column, and then adding 60g of protective silica gel for covering;
and (3) elution: the eluate was collected by eluting 5 column volumes (each column volume was 600mL) with dichloromethane, and the eluate was concentrated at 45 ℃ with a rotary evaporator (RV10, ai ka (IKA) instruments ltd) to remove the solvent to give a dichloromethane eluate.
(2) Sephadex LH-20 chromatographic separation
The methylene chloride eluate was completely dissolved in a small amount of methanol, and then loaded on a Sephadex LH-20 column (20 cm. times.200 cm, Pharmacia) and purified with chloroform: methanol 1: and 1(v/v) is used as an eluent, elution is sequentially carried out by using elution volumes of 210mL, 160mL, 120mL and 100mL, eluents A5-1, A5-2, A5-3 and A5-4 corresponding to the elution volumes are sequentially collected, wherein an obvious dark spot can be seen by ultraviolet light with the wavelength of 254nm through thin-layer chromatography (TLC) of the A5-3, the obvious dark spot is developed into magenta through concentrated sulfuric acid-vanillin, and the eluent A5-3 is taken as a chromatographic separation component.
(3) Forward HPLC separation and purification
A5-3 was purified by forward HPLC using a column (Instrument: Essentia LC-16P, Shimadzu) which was: zorbax Rx-SIL normal phase chromatography column (4.6 mm. times.250 mm, particle size 5 μm, Agilent technologies, Ltd.), isocratic elution, and collection of A5-3-1 fraction (29.3mg, retention time t)R＝8min)。
Structural identification of III, A5-3-1
And (3) carrying out structural analysis test on the collected A5-3-1 flow parts to obtain the following physicochemical property data:
yellowish powder (dichloromethane), dark spots under a 254nm ultraviolet lamp (ZF-20D, Shanghai jibifiai scientific analysis Instrument Co., Ltd.), and magenta color development of vanillin sulfate. ESI-MS (LC-MS 6120, Agilent technologies, Ltd.) gave the excimer ion peak M/z344.9[ M + H []+Determining the molecular mass to be 344;1H NMR(600MHz，CDCl3d6, AVANCE III 600MHZ, Bruker) and13C NMR(150MHz，CDCl3d6, AVANCE III 600MHZ, Bruker) data are given in Table 1.
TABLE 1A 5-3-1 NMR data
Data reported in the literature[6]The nuclear magnetic data of the compound is consistent with that of the compound of the treprostinil (tryptcidin), so that the A5-3-1 is determined to be the treprostinil (tryptcidin), and the structure of the compound is shown as follows:
example 2
The preparation of the tricot-icides is as in example 1, except that: in the step of obtaining the seed solution, culturing for 30h at 250rpm and 30 ℃; in the step of obtaining the fermentation culture, standing and fermenting at 20 ℃ for 35 days; in the step of obtaining the ethanol extract, the extraction is repeated for 2 times; in the step of obtaining a crude extract, the extraction was repeated 2 times. After identification, the method is adopted to obtain the trypanostatin (trypsinin).
Example 3
The preparation of the tricot-icides is as in example 1, except that: in the step of obtaining the seed solution, culturing at 100rpm and 20 ℃ for 60 h; in the step of obtaining the fermentation culture, the fermentation is performed for 10 days at 30 ℃; in the step of obtaining the ethanol extract, the extraction is repeated for 5 times; in the step of obtaining a crude extract, the extraction was repeated 5 times. After identification, the method is adopted to obtain the trypanostatin (trypsinin).
Experimental example identification of algal-lysing Activity of Fungicide Trimycin
Culturing marine Cardunaliella algae cells (provided by aquatic organism research institute of river-south university, Guangzhou) in light culture box (LRH-400, Thanghong medical equipment Co., Ltd., Shaoguan city) at 22 deg.C under 12 hr light, 12 hr dark, and light quantum flux density of 50 μmol/m2And s, shaking the algae every day when the illumination is carried out for 5-6 h until all the living algae are suspended, wherein the shaking is not too violent.
Counting the algae cells in the ocean Kadun algae liquid by using a plankton frame (DSJ-01 research grade, Xiamendengtong instruments and equipments Co., Ltd.), measuring the concentration of the algae cells, shaking uniformly, and subpackaging the algae liquid to a 24-hole plate with 1 mL/hole (adjusting the concentration of the algae cells in the algae liquid to 1.0-2.0 × 10)4one/mL); dissolving a sample to be detected with the monacolin into a sample solution of 100 mug/muL by using DMSO, adding 1 muL of the prepared sample solution into 1mL of the algae solution in an experimental group, so that the final concentration of the monacolin in the algae solution is 100 mug/mL, adding 1 muL of DMSO into 1mL of the algae solution in a control group, and setting the experimental group and the control group to be 3 parallels respectively; placing the 24-hole plate in a light incubator for culture; counting the number of algae cells by using a floating counting plate at 15min and 120min, wherein the final result of the experimental group and the control group is the average value of the number of the algae cells of 3 parallel groups, and calculating the algae dissolving rate: (control group alga concentration-experimental group alga concentration)/control group alga concentration, the result of alga lysing rate is shown in table 2.
TABLE 2 results of the Trypticidin algae-lysing Activity test (100. mu.g/mL)
As can be seen from Table 2, the concentration of algal cells in 1mL of algal solution (algal cell concentration of 1.0 to 2.0X 10)4One per mL) can dissolve all algae cells within 15min when 100 mu g of the trypanostatin is added, and the algae dissolving rate is up to 100%. The cone aspergillin is proved to have algae-lysing activity and can be used for lysing algae cells. Based on the activity of the trypanosicin for dissolving algae cells, the method can be used for red tide treatment, and further solves the problem that the ecological environment is damaged by mass propagation of algae.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Reference documents:
[1]An X,Zhang B,Zhang H,et al.Discovery of an algicidal compound from Brevibacterium sp.BS01 and its effect on a harmful algal bloom-causing species,Alexandrium tamarense[J].Frontiers in microbiology,2015,6:1235.
[2]Numata A,Takahashi C,Matsushita T,et al.Fumiquinazolines,novel metabolites of a fungus isolated from a saltfish[J].Tetrahedron letters,1992,33(12):1621-1624.
[3] mundau, Linhaipeng, Zhang Shihua, Manzhuang, Hongkui.8 isolation and identification of biologically active mangrove fungus Aspergillus fumigatus [ J ] tropical crops journal, 2010,31(09):1641-1646.
[4]Balan J,Ebringer L,Nemec P,et al.Antiprotozoal antibiotics.II.Isolation and cbaracterisation of trypacidin,a new antibiotic,active against Trypanosoma cruzi and Toxoplasma gondii.[J].The Journal of antibiotics,1963,16:157-160.
[5]Turner W B.1232.The production of trypacidin and monomethylsulochrin by Aspergillus fumigatus[J].Journal of the Chemical Society(Resumed),1965:6658-6659.
[6] Luchunshua, yellow flare, sinking hair, secondary metabolite of Aspergillus fumigatus var. fumigatus (English) [ J ]. Chinese Natural medicine, 2005(05): 269) 271.
[7]Parker G F,Jenner P C.Distribution of trypacidin in cultures of Aspergillus fumigatus[J].Applied microbiology,1968,16(8):1251.
Claims (5)
1. Application of the cone aspergillin in dissolving algae cells.
2. The use of claim 1, wherein the algal cells are cells of marine dunaliella.
3. The use according to claim 1 or 2, wherein the amount of the triconazole is not less than 100 μ g/mL based on the volume of the algal solution containing the algal cells, and the concentration of the algal cells in the algal solution is 1.0 x 104～2.0×104one/mL.
4. Use according to claim 1, wherein the conicidin is isolated from a metabolite of the Aspergillus sp.lw1 fungus with the deposit number: CCTCC No. M2019453.
5. A method for preparing a tricot-killing agent, which is characterized by comprising the following steps:
fermenting Aspergillus sp.LW1 to obtain fermentation culture;
sequentially performing crude extraction, separation and purification on the fermentation culture to obtain the tricolor;
the step of obtaining the fermentation culture comprises:
inoculating Aspergillus sp.LW1 into a seed culture medium, and culturing at 20-30 ℃ for 30-60h at 250rpm of 100-;
adding the seed solution into a fermentation culture medium, standing and fermenting for 10-35 days at 20-30 ℃ to obtain a fermentation culture, wherein the raw materials of the fermentation culture medium comprise rice, sea salt and deionized water, and the rice is 80g and the sea salt is 3g based on 120mL of deionized water;
the crude extraction step comprises:
ultrasonically extracting the fermentation culture with ethanol, and filtering to obtain ethanol extract;
concentrating the ethanol extract to remove the solvent to obtain an ethanol concentrate;
dissolving the ethanol concentrate with deionized water, extracting with ethyl acetate, recovering ethyl acetate layer, and concentrating under reduced pressure to obtain crude extract;
repeating the extracting step for 2-5 times to obtain ethanol extract;
in the step of obtaining an ethanol concentrate, the concentration is performed at 45 ℃;
in the step of obtaining the crude extract, the volume ratio of the deionized water to the ethyl acetate is 1:1, repeatedly extracting for 2-5 times;
the separation and purification comprises the following steps:
sequentially carrying out forward silica gel column chromatographic separation and Sephadex LH-20 chromatographic separation on a crude extract obtained by crude extraction to obtain a chromatographic separation component;
carrying out forward HPLC separation and purification on the chromatographic separation component;
in the step of separating by forward silica gel column chromatography, the method comprises the following steps: dissolving the crude extract with methanol, mixing, loading into a column, eluting with dichloromethane, concentrating the obtained eluent to remove the solvent, and obtaining dichloromethane eluate;
in the step of chromatographic separation of Sephadex LH-20, comprising: dissolving the dichloromethane eluate with methanol, loading, eluting with eluents with elution volumes of 210mL, 160mL and 120mL in sequence, collecting eluents corresponding to the elution volumes in sequence, and taking the eluents corresponding to the elution volumes of 120mL as chromatographic separation components, wherein the eluents adopt chloroform and methanol in a volume ratio of 1: 1;
in the step of forward HPLC separation and purification, the chromatographic column is: zorbax Rx-SIL normal phase chromatography column (250 mm. times.4.6 mm, 5 μm), mobile phase: ethanol and cyclohexane in a volume ratio of 15:85, and the elution mode is as follows: isocratic elution, and collecting the components with retention time of 8min to obtain the final product.
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