CN102875551A - Chloro alkaloid compound and preparation and application thereof - Google Patents

Abstract

The invention relates to the fields of algicides, insecticides and bacteriostats, in particular to a chlorinated alkaloid compound derived from seaweed endophytic fungi and its preparation and application. The specific structural formula is shown in (I), and its preparation method is to inoculate Aspergillus versicolor (Aspergillus versicolor) DL-29 in a fungal liquid medium for fermentation and culture. After the fermentation product is purified, it is the chlorinated compound shown in formula (I). Alkaloid compounds. The chlorinated alkaloid compound obtained by the present invention has a half inhibitory concentration of 6.3 micrograms/milliliter obtained through an algae-inhibiting activity test, and the compound also has insecticidal and bacteriostatic activities.

Description

A kind of chlorinated alkaloid compound and its preparation and application

technical field

The invention relates to the fields of algicides, insecticides and bacteriostats, in particular to a chlorinated alkaloid compound derived from seaweed endophytic fungi and its preparation and application. the

Background technique

With the rapid development of the national economy, the problem of marine pollution has become increasingly serious, especially the increase in the degree of eutrophication, resulting in frequent occurrence of harmful red tides, which have brought serious harm to the marine environment, marine life and coastal aquaculture. Therefore, the development of harmful Red tide microalgae inhibitors have important application value. the

Since the 20th century, the extensive use of chemically synthesized pesticides has brought serious pollution to water bodies and soils. More than 2/3 of the pesticides have directly penetrated into the environment, and the residues are very serious, causing direct and potential harm to organisms and human health. . Compared with chemically synthesized pesticides, biological pesticides have the advantages of good environmental compatibility and less resistance. Their development and application are of great significance to human health, environmental protection and sustainable development of agriculture. the

Bacterial diseases remain an important type of disease in humans and other organisms. Due to the rapid mutation rate of pathogenic bacteria and the continuous emergence of drug resistance, it is still very urgent to develop new bacterial inhibitors. the

Contents of the invention

The object of the present invention is to provide a chlorinated alkaloid compound and its preparation and application. the

To achieve the above object, the technical solution adopted in the present invention is:

A chlorinated alkaloid compound, the chlorinated alkaloid compound is shown in formula (I)

The preparation method of the chlorinated alkaloid compound is to inoculate Aspergillus versicolor DL-29 in the fungal liquid medium for fermentation and culture, after the fermentation product is purified, it is the chlorinated alkaloid represented by the formula (I) Compound, said Aspergillus versicolor (Aspergillus versicolor) DL-29, which was preserved in China Center for Type Culture Collection CCTCC on November 25, 2011, and the preservation number is CCTCC M 2011421

Specific preparation steps:

1) Inoculate Aspergillus versicolor (Aspergillus versicolor) DL-29 in the fungal liquid medium and ferment for 10-60 days, filter, extract and concentrate the fermentation broth with ethyl acetate, extract the collected mycelium with an organic solvent, and then After extraction and concentration with ethyl acetate, the concentrate obtained from the fermentation broth and the concentrate obtained from the mycelia are combined to form the crude extract;

The Aspergillus versicolor (Aspergillus versicolor) DL-29 was preserved in the China Center for Type Culture Collection CCTCC on November 25, 2011, the preservation number is CCTCC M 2011421, and the address of the depository unit is: Wuhan University;

2) The crude extract in step 1) is subjected to silica gel column chromatography, gradient elution is performed with an organic solvent, the eluate is collected, and the eluate is detected by thin layer chromatography;

3) Collect the eluted components in step 2) with a gradient of eluent volume ratio of 3-0:1, and perform separation and purification of the collected eluted components by silica gel column chromatography, gel column chromatography and thin layer chromatography , purify and collect components with an Rf value of 0.6-0.7 to obtain a chlorinated alkaloid compound as shown in formula (I). the

The mycelium in step 1) is extracted with an organic solvent, and the organic solvent is one of heptane, hexane, pentane, chloroform, dichloromethane, ethyl acetate, acetone, isopropanol, propanol, ethanol or methanol Several kinds, when the several kinds of substances are combined, they are mixed in any proportion. the

The organic solvent in step 2) is petroleum ether-ethyl acetate, chloroform-ethyl acetate, petroleum ether-acetone, chloroform-acetone or chloroform-methanol. the

Step 3) The silica gel column chromatography eluent is petroleum ether-ethyl acetate with a volume ratio of 3-1:1; the gel column chromatography eluent is chloroform-methanol or chloroform with a volume ratio of 0-2:1 -Ethanol; The thin-layer chromatography developer is chloroform-ethyl acetate or petroleum ether-ethyl acetate with a volume ratio of 2-1:1. the

Step 3) The components with light yellow spots were collected during the silica gel column and gel column chromatography. the

The application of the chlorinated alkaloid compound, the chlorinated alkaloid compound is used for preparing an algastatic agent, an insecticide or a bacteriostatic agent. The chlorinated alkaloid compound is used for preparing a preparation for inhibiting the algae of Akashiwa. The chlorinated alkaloid compound is used for preparing an insecticidal preparation for Artemia. The chlorinated alkaloid compound is used for preparing the bactericidal preparation of Proteus mirabilis, Enterobacter cloacae or Bacillus cereus. the

The present invention has the following advantages: the present invention extracts and separates the chlorinated alkaloid compounds obtained through the fermentation of Aspergillus versicolor (Aspergillus versicolor) DL-29, which is isolated from the marine green algae Aspergillus versicolor (Aspergillus versicolor) DL-29. The half-inhibitory concentration is 6.3 μg/ml, and the compound also has insecticidal and bacteriostatic activities. the

Detailed ways

The present invention will be further elaborated below in conjunction with implementation examples. the

Example 1

The chlorinated alkaloid compound derived from endophytic fungi of seaweed is represented by formula (I). the

Example 2

The preparation method of the alkaloid compound shown in formula (I):

Take the fungus Aspergillus versicolor (Aspergillus versicolor) DL-29 strain that grew well on the plate, cut it into small pieces and inoculate it in PDB liquid medium, put 300mL medium in each 1L triangular flask, 50 bottles in total, and ferment statically at room temperature for 30 One-half volume of ethyl acetate was added to the fermented liquid to kill fungi, filtered, and the mycelia and fermented liquid were collected respectively. The PDB liquid medium consists of 200 milliliters of potato juice per liter, 20 grams of glucose, 5 grams of peptone, 3 grams of yeast extract, 500 milliliters of aged sea water, and 300 milliliters of distilled water. The fungus Aspergillus versicolor (Aspergillus versicolor) DL-29 strain was preserved in China Center for Type Culture Collection CCTCC on November 25, 2011, the preservation number is CCTCC M 2011421, the classification name is Aspergillus versicolor, and the strain number is DL-29;

Collect about 15 L of fermentation broth, extract three times with ethyl acetate, and concentrate under reduced pressure; after crushing the mycelia, extract three times with chloroform-methanol with a volume ratio of 1:1, then extract with ethyl acetate, and concentrate under reduced pressure; The results of layer chromatography detection (petroleum ether-ethyl acetate 20-0:1, sulfuric acid-anisaldehyde color development) were similar, and the concentrate of the two parts of the combined fermentation broth and mycelium was 29.4g of crude extract. the

The crude extract was subjected to 100-200 mesh silica gel column chromatography, and petroleum ether-ethyl acetate was used at a volume ratio of 100:0, 50:1, 20:1, 10:1, 5:1, 2:1 to 0: The gradient of 100 was used for elution, and the eluents were collected separately, and then detected by thin layer chromatography (TLC) (petroleum ether-ethyl acetate 20-0:1, anisaldehyde-sulfuric acid as the color reagent), according to the Rf value Judging and merging the same or similar parts to obtain 14 components (1-14). the

Fraction 14, which was eluted with petroleum ether-ethyl acetate 0:100 gradient, was separated by silica gel column, gel column and thin-layer chromatography. The eluent of silica gel column chromatography is petroleum ether-ethyl acetate at a volume ratio of 2:1, detected by TLC (chloroform-ethyl acetate 1:1, sulfuric acid-anisaldehyde for color development), and the components of light yellow spots are collected; During gel column chromatography, use chloroform-methanol with a volume ratio of 1:1 as the eluent, and detect with TLC (chloroform-ethyl acetate 1:1, sulfuric acid-anisaldehyde for color development), and collect the components with light yellow spots; thin layer Chloroform-ethyl acetate with a volume ratio of 1:1 was used as a developing solvent for analysis, and components with an Rf value of 0.6-0.7 were collected to obtain the compound (8.6 mg) shown in formula (I), which was detected by TLC (chloroform-ethyl acetate 1:1, sulfuric acid-anisaldehyde color development), a single, uniform light yellow spot, identified as a pure compound. After spectral analysis, its structure was identified as a new alkaloid, and its structural formula is shown in (I). the

The compound has the following physicochemical and spectroscopic properties:

Colorless gel, specific rotation [α] ²³ _D +17.8°(c0.18, MeOH); H NMR spectrum ( ¹ H-NMR, CDCl ₃ , 500MHz) δ _H 7.45,d(7.4),7.29, dd(7.2,7.4),7.44,dd(7.2,8.0),7.58,d(8.0),1.99,dd(13.6,1.6),2.48,d(13.6),6.29,s,3.93,s,2.54,ddd (8.8,8.8,8.8),3.00ddd(8.8,8.8,2.8),1.88,m,1.93,m,1.54,m,2.07,m,2.48,m,1.50,m,2.00,m,1.82,dd( 12.5,3.4), 1.26, s, 1.49, s; C NMR spectrum ( ¹³ C-NMR, CDCl ₃ , 125MHz) δ _C 184.4, qC, 86.2, qC, 135.2, qC, 123.0, CH, 126.9, CH, 131.1, CH, 121.3, CH, 152.7, qC, 38.0, _CH2 , 54.6, qC, 151.6, qC, 113.0 _, qC, 61.4, CH, 50.6, CH2, 21.4, _CH2 , 30.1, _CH2 , 58.4, CH, 27.4, _CH2 , 49.0, CH, 39.3, qC, 22.0, _CH3 , 27.2, _CH3 ; High resolution mass spectrum (HREIMS) [M] ⁺ m/z 427.1660, calculated 427.1663.

Example 3

The difference from Example 2 is that

Take the fungus Aspergillus versicolor (Aspergillus versicolor) DL-29 strain that grew well on the plate and inoculate it in JY liquid medium, put 300mL medium in each 1L Erlenmeyer flask, 50 bottles in total, ferment statically at room temperature for 20 days, add fermentation broth One-half volume of ethyl acetate kills fungi, filters, and collects mycelium and fermentation broth respectively. The JY liquid medium consists of 500 milliliters of Jerusalem artichoke tuber juice per liter, 10 grams of glucose, 2.0 grams of sodium nitrate, and 500 milliliters of aged seawater. the

Collect about 15L of fermentation broth, extract three times with ethyl acetate, and concentrate under reduced pressure; crush the mycelium, extract with ethanol three times, then extract with ethyl acetate, and concentrate under reduced pressure; the concentrate is detected by thin layer chromatography (petroleum ether- Ethyl acetate 20-0:1, sulfuric acid-anisaldehyde color development) the results are similar, the concentrate of the two parts of the combined fermentation broth and mycelia is 28.0g of crude extract. the

The crude extract was subjected to 100-200 mesh silica gel column chromatography with petroleum ether-acetone at a gradient of 100:0, 50:1, 20:1, 10:1, 5:1, 1:1 to 0:100 Elution, collect the eluents separately, and then detect with thin layer chromatography (TLC) (petroleum ether-ethyl acetate 20-0:1, sulfuric acid-anisaldehyde for color development), judge according to the Rf value, merge the same or similar section, to obtain 14 components (1-14). the

Component 14, that is, the component eluted with petroleum ether-acetone 0:100 gradient, was separated by silica gel column, gel column and thin layer chromatography. The eluent of silica gel column chromatography is petroleum ether-ethyl acetate at a volume ratio of 2:1, detected by TLC (chloroform-ethyl acetate 1:1, sulfuric acid-anisaldehyde for color development), and the components of light yellow spots are collected; During gel column chromatography, use chloroform-methanol with a volume ratio of 2:1 as the eluent, and detect with TLC (chloroform-ethyl acetate 1:1, sulfuric acid-anisaldehyde for color development), and collect the components with light yellow spots; thin layer Chloroform-ethyl acetate with a volume ratio of 1:1 is used as a developing solvent for analysis, and components with an Rf value of 0.6-0.7 are collected to obtain a chlorinated alkaloid compound represented by formula (I). the

Example 4

Red tide microalgae inhibition experiment:

Take I. akashiwo algae in the exponential growth phase, dilute it to a certain initial algae cell density (5×10 ⁴ cells/mL) with sterilized f/2 medium, and set aside for later use. The compound to be tested was dissolved in dimethyl sulfoxide (DMSO) to prepare a mother solution for later use. The final test volume of each well of the Costar96-well plate is 200 microliters, which is composed of 199 microliters of algae liquid and 1 microliter of the sample solution to be tested. The final concentration of DMSO in the experimental algae liquid is 0.5%, and three parallel samples are set for each concentration. Shake 3 times a day during the culture period, observe the algal cell count under the microscope every 24 hours with a hemocytometer (or plankton counting box), measure the concentration of algal cells (unit/mL), and calculate the EC ₅₀ value for 24 hours.

Experimental results: the half-inhibitory concentration of the alkaloid compounds obtained in the above-mentioned examples on I. akashiwo algae was 6.3 micrograms per milliliter. the

Example 5

Insecticidal activity test:

Hatching of Artemia eggs: Take 100 mg of Artemia eggs and put them in a 500ml beaker, add 400ml of artificial seawater, inflate slowly with a small air pump, and incubate at room temperature for 24 hours, remove egg shells and unhatched eggs, and Artemia The larvae were continued to be cultivated for 24 hours and set aside. the

Artemia biological lethal method: according to the improved method of Solis, take a 96-well cell culture plate, add 190 microliters of artificial seawater containing about 10 Artemia larvae to each hole, and make a test culture plate. Set three parallel wells for the blank control group and the sample group of each concentration, add 10 microliters of solvent dimethyl sulfoxide (DMSO) to the blank control group, and add 10 microliters of the two-and-a-half times shown in formula (I) to the sample group Solution of terpenoids (DMSO as solvent). After incubation at room temperature for 24 hours, the number of dead individuals of Artemia was detected and counted under a binocular dissecting microscope. the

The biological lethal activity of Artemia is expressed in terms of corrected mortality, and the calculation formula is as follows:

Adjusted mortality rate = (survival rate of control group - survival rate of treatment group) / survival rate of control group × 100%

Experimental results: the lethality of the alkaloid compound obtained in the above example to Artemia was 27.5% at 100 μg/ml. the

Example 6

Antibacterial activity test:

Specifically: the bacterial culture medium used in the experiment is LB culture medium, soak the bacterial suspension of Proteus mirabilis, Enterobacter cloacae and Bacillus cereus with sterile cotton swabs respectively, evenly spread on the culture medium, and the test sample is dissolved in DMSO In the medium, the concentration is 6.0 mg/ml, take 5 microliters of sample and add it to a sterile filter paper piece with a diameter of 5 mm (30 micrograms per piece); and use the filter paper piece with the same volume of DMSO as a negative control, and use chloramphenicol as a negative control. Antibacterial positive control, each in three parallels. Place the plate culture medium with the sample at 28°C for 24 hours, observe the experimental results, and measure the diameter of the inhibition zone if there is an inhibition zone. the

Experimental results: the alkaloid compounds obtained above have a bacteriostatic zone diameter of 7.0 mm, 6.5 mm and 6.0 mm respectively to Proteus mirabilis, Enterobacter cloacae and Bacillus cereus, and have the ability to inhibit Proteus mirabilis, Enterobacter cloacae or cereus activity of Bacillus spp. the

Claims (11)

1. chloro alkaloid compound, it is characterized in that: the chloro alkaloid compound as shown in the formula (I)

2. the preparation method of a chloro alkaloid compound claimed in claim 1, it is characterized in that: aspergillus versicolor (Aspergillus versicolor) DL-29 is inoculated in fermentation culture in the fungi liquid substratum, behind the tunning purifying, be the chloro alkaloid compound shown in the formula (I), described aspergillus versicolor (Aspergillus versicolor) DL-29, it is stored in Chinese Typical Representative culture collection center C CTCC on November 25th, 2011, and deposit number is CCTCC M 2011421

3. by the preparation method of chloro alkaloid compound claimed in claim 2, it is characterized in that concrete preparation process:

1) aspergillus versicolor (Aspergillus versicolor) DL-29 was inoculated in the fungi liquid substratum static fermentation 10-60 days, filter, fermented liquid is concentrated through ethyl acetate extraction, the mycelium organic solvent extraction of collecting, then concentrated through ethyl acetate extraction again, fermented liquid gained enriched material and mycelium gained enriched material merge, and are crude extract;

Described aspergillus versicolor (Aspergillus versicolor) DL-29, it is stored in Chinese Typical Representative culture collection center C CTCC on November 25th, 2011, and deposit number is CCTCC M 2011421;

2) crude extract of getting in the step 1) carries out silica gel column chromatography, carries out gradient elution with organic solvent, collects elutriant, and elutriant detects through thin-layer chromatography;

3) collect step 2) in the elution fraction of effluent volume than 3-0:1 gradient, the elution fraction of collecting is carried out silica gel column chromatography, gel filtration chromatography and thin-layer chromatography separation and purification, it is the 0.6-0.7 component that purifying is collected the Rf value, namely gets chloro alkaloid compound as shown in the formula (I).

4. press the preparation method of chloro alkaloid compound claimed in claim 3, it is characterized in that: mycelium organic solvent extraction in the step 1), organic solvent are in heptane, hexane, pentane, chloroform, methylene dichloride, ethyl acetate, acetone, Virahol, propyl alcohol, ethanol or the methyl alcohol one or more.

5. by the preparation method of chloro alkaloid compound claimed in claim 3, it is characterized in that: step 2) in organic solvent be petroleum ether-ethyl acetate, chloroform-ethyl acetate, sherwood oil-acetone, chloroform-acetone or chloroform-methanol.

6. by the preparation method of chloro alkaloid compound claimed in claim 3, it is characterized in that: the petroleum ether-ethyl acetate that the described silica gel column chromatography elutriant of step 3) is volume ratio 3-1:1; The gel filtration chromatography elutriant is chloroform-methanol or the chloroform-ethanol of volume ratio 0-2:1; The thin-layer chromatography developping agent is that volume ratio is chloroform-ethyl acetate or the petroleum ether-ethyl acetate of 2-1:1.

7. by the preparation method of chloro alkaloid compound claimed in claim 3, it is characterized in that: the component of collecting faint yellow spot when the described silicagel column of step 3) and gel filtration chromatography.

8. the application of a chloro alkaloid compound claimed in claim 1 is characterized in that: described chloro alkaloid compound is for the preparation of algae-inhibiting agent, sterilant or fungistat.

9. by the application of chloro alkaloid compound claimed in claim 8, it is characterized in that: described chloro alkaloid compound is for the preparation of the preparation that suppresses heterosigma akashiwo.

10. by the application of chloro alkaloid compound claimed in claim 8, it is characterized in that: described chloro alkaloid compound is for the preparation of the pesticide preparation of halogen worm.

11. by the application of chloro alkaloid compound claimed in claim 8, it is characterized in that: described chloro alkaloid compound is for the preparation of the biocide preparation of Proteus mirabilis, enterobacter cloacae or bacillus cereus.