CN114304193A - Application of flammulina velutipes mushroom dreg extract in preparation of herbicide - Google Patents
Application of flammulina velutipes mushroom dreg extract in preparation of herbicide Download PDFInfo
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- CN114304193A CN114304193A CN202210056088.3A CN202210056088A CN114304193A CN 114304193 A CN114304193 A CN 114304193A CN 202210056088 A CN202210056088 A CN 202210056088A CN 114304193 A CN114304193 A CN 114304193A
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Abstract
The invention belongs to the technical field of pesticide herbicides, and discloses an application of a flammulina velutipes mushroom dreg extract in preparation of herbicides, wherein the extraction method of the flammulina velutipes mushroom dreg extract comprises the following steps: s1, weighing dried needle mushroom dregs, adding ethyl acetate to extract to obtain paste, and mixing with silica gel to obtain a mixture; s2, placing the mixture obtained in the step S1 in a mixed solvent, and carrying out chromatographic separation through a filtration separation column to obtain an extract; the flammulina velutipes mushroom dreg extract achieves the purpose of weeding by inhibiting seed germination and root growth. The invention discovers the herbicidal activity effect of the flammulina velutipes mushroom dreg extract for the first time, has obvious effect of inhibiting the growth of plants, is used for developing herbicide pesticide products, greatly reduces the research and development cost of new pesticides, fills the blank of related fields of new component pesticides, greatly reduces the possibility of teratogenesis and carcinogenesis of organisms compared with artificially and purely synthesized new compounds, has no pollution, is environment-friendly and can greatly improve the product quality.
Description
Technical Field
The invention belongs to the technical field of pesticide herbicides, and particularly relates to an application of a needle mushroom fungus dreg extract in preparation of a herbicide.
Background
With the increasing environmental awareness of people, society puts forward higher and higher requirements on pesticide products. The difficulty of developing new products suitable for marketing is continuously increased, high development cost hinders many manufacturers, but huge profits brought by development and application of new pesticide products attract attention of many institutes and manufacturers all the time. The active discovery and search of compounds with biological activity and novel structures, and the development of new species with independent intellectual property rights are the key points for creating new pesticides and are the interest and research hotspots of all the current pesticide companies and research institutions.
In the aspect of practicability, the market of pesticides has continuously increased in 2005-2019, and the market share of herbicides and insecticides is also continuously increased. Relatively speaking, herbicides increase steadily, insecticides increase more slowly, and are surpassed by fungicides. Despite the impact of the development of transgenic crops on herbicide products, herbicides are still needed and are the most important means of crop protection, and the market share of the development of herbicides has been on the rise throughout the year.
The technical defects of the current domestic herbicide research and development are as follows: not only the new products are few, the new pesticides with independent intellectual property rights are less, and a great deal of manpower and financial resources are invested in the development work of the new pesticides aiming at the dilemma of the domestic pesticide market and the disputes of domestic scientific research institutes and manufacturers, but the effect is very little. The main reason for this is that it is difficult to screen for suitable active substances. Currently, active substance screening works mainly depend on sea selection, one type is to perform activity tests one by one after researching and creating new compounds, the other type is to perform biological activity tests after separating and purifying from various natural products, then select substances with higher activity as backup materials to perform research and development works, and often, a proper material is difficult to obtain from tens of thousands of substances, so that high development cost hinders many manufacturers.
Disclosure of Invention
The invention aims to find that the flammulina velutipes mushroom dreg extract can inhibit the growth activity of plants, so as to provide a new choice in the aspect of biological herbicide pesticides and lay a solid foundation for the development of new varieties of pesticides.
The first purpose of the invention is to provide the application of the flammulina velutipes mushroom dreg extract in preparing herbicides, and the extraction method of the flammulina velutipes mushroom dreg extract comprises the following steps:
s1, weighing dried needle mushroom dregs, and adding ethyl acetate to extract to obtain paste; removing acetic acid by using sodium carbonate, and mixing with 80-100 meshes of silica gel to obtain a mixture;
s2, placing the mixture obtained in the step S1 in a mixed solution of petroleum ether and ethyl acetate, and separating by a filtration separation column to obtain an extract of primary separation.
Preferably, S2 further includes: performing thin-layer chromatography and reduced pressure concentration on the extract to obtain substances with different polarities of W1, W2, W3 and W4, mixing W4 with 200-300 meshes of silica gel, performing gradient washing on the mixed liquid of petroleum ether and ethyl acetate, and performing chromatographic separation on the washed mixed liquid through a filtration separation column to obtain a secondary separated extract W4-1.
Preferably, the extract or the extract W4-1 achieves the purpose of weeding by inhibiting seed germination and root growth.
Preferably, the extract or W4-1 is diluted with water and used, or the extract is added to prepare a herbicide preparation.
Preferably, the dilution is carried out by 0 to 2000 times with water.
Preferably, in S1, the mass-to-volume ratio of the needle mushroom dregs to the ethyl acetate is 1g:10 mL.
Preferably, in S1, the mass ratio of the paste to the silica gel is 1: 1-1.5.
Preferably, in S2, the volume ratio of the petroleum ether to the ethyl acetate is 5: 1.
Preferably, the mass ratio of W4 to silica gel is 1: 1-1.5.
Preferably, when the extract W4-1 is prepared, the volume ratio of the petroleum ether to the ethyl acetate is 2: 1.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention discovers the herbicidal activity effect of the flammulina velutipes mushroom residue extract for the first time, has obvious effect of inhibiting plant growth, has the potential of being developed into commodities when being used for developing herbicide pesticide products, provides a new choice for developing domestic new pesticide products, greatly reduces the research and development cost of new pesticides, has high economic benefit, makes up the blank of related fields of new pesticides and lays a foundation for the research and development of products with independent property rights in China.
(2) The flammulina velutipes mushroom residue extract adopted by the invention is a natural product, is a biological active substance compared with a new artificially purely synthesized compound, has the advantages of greatly reduced possibility of teratogenesis, carcinogenesis and mutagenesis of organisms, no pollution, environmental friendliness, capability of greatly improving the product quality and increasing the additional value of agricultural products, good social and ecological benefits and ideal biopesticide products.
Drawings
FIG. 1 shows crude extracts of the invention in mobile phase petroleum ether: a thin layer chromatography silica gel plate diagram at an ethyl acetate ratio of 2: 1;
FIG. 2 is a diagram of thin layer chromatography silica gel plates at low and high concentrations of a crude extract of the present invention;
FIG. 3 shows the germination and growth conditions of the crude extract of the present invention on the 5 th day of the Chinese cabbage seed;
FIG. 4 shows the root growth of the crude extract of the present invention on the 5 th day of the Chinese cabbage seed;
FIG. 5 shows the germination and growth of various crude extracts of the present invention on day 5 of Brassica campestris seeds;
FIG. 6 shows the germination and growth of the extract of the present invention on day 5 of the pakchoi seeds.
Detailed Description
In order to make the technical solutions of the present invention better understood and implemented by those skilled in the art, the present invention is further described below with reference to the following specific embodiments and the accompanying drawings, but the embodiments are not meant to limit the present invention. According to the invention, a cabbage experiment represents a herbicide screening experiment, and the cabbage cruciferae plants are easy to obtain seeds, stable in germination rate and fast in growth, so that the growth inhibition effect of the flammulina velutipes mushroom residue extract as a herbicidal active substance on the cruciferae plants is researched by taking the cabbages as a material.
Example 1
The application of the flammulina velutipes mushroom dreg extract in preparing the herbicide is that the extract is separated and purified from the flammulina velutipes mushroom dreg, and the separation and purification comprises the following steps:
s1, weighing 1.5Kg of dry mushroom dregs (the water content of the fresh mushroom dregs is 55 percent), and extracting by 15L of ethyl acetate to obtain 114.13g of paste; taking 42.21g of paste, and removing acetic acid by using sodium carbonate to obtain 32g of paste; mixing 32g of paste with 32g (80-100 meshes) of silica gel;
s2, passing the mixture obtained in S1 through a mixer made of petroleum ether: washing the mixed solution with the volume ratio of ethyl acetate of 5:1 to obtain mixed liquid, weighing 600g (200 meshes) of silica gel, filling the silica gel into a filtration and separation column with the diameter of 8cm, and carrying out chromatographic separation on the mixed liquid through the filtration and separation column to obtain a primary separated crude extract.
After sample loading detection by a thin layer chromatography silica gel plate, combining the tubes according to different polarities of substances, and concentrating under reduced pressure to obtain 4 parts of isolate samples, which are specifically shown in the following table 1:
TABLE 1 samples of isolates after concentration under reduced pressure
Among the four isolate samples shown in table 1, isolates W2, W3 and W4 were dissolved with ethyl acetate and tested by thin layer chromatography on silica gel plate; the results are shown in FIGS. 1 and 2, respectively.
FIG. 1 shows the mushroom residue isolates W2, W3 and W4 in the mobile phase of petroleum ether: the results of thin layer chromatography silica gel plate with ethyl acetate ratio of 2:1 are shown in FIG. 1A, which also shows that the needle mushroom residue isolate W4 was separated in mobile phase petroleum ether: thin layer chromatography silica gel plate results for relatively high concentrations (loading) at a 2:1 ethyl acetate ratio as shown in the left band of FIG. 1B and relatively low concentrations (loading) as shown in the right band of FIG. 1B. FIG. 2 shows the mushroom residue isolate in mobile phase petroleum ether: thin layer chromatography silica gel plate result when ethyl acetate ratio is 5: 1; wherein, FIG. 2a shows the results of thin layer chromatography silica gel plate with low concentration of Flammulina velutipes (Fr.) Sing residues W2, W3 and W4, and FIG. 2b shows the results of thin layer chromatography silica gel plate with high concentration of Flammulina velutipes (Fr.) Sing residues isolate W2, W3 and W4.
As can be seen from the results of the thin layer chromatography silica gel plate shown in FIGS. 1 and 2, the mixture extracted from the mushroom dregs of Flammulina velutipes is well separated according to the polarity difference;
s3, dissolving the 2.22gW4 sample obtained in the S2 by using 25mL of ethyl acetate solution, and then carrying out reduced pressure concentration at the temperature of 30-40 ℃ and under the pressure of less than 0.09MPa to obtain 1.4g of sample; then 1.4g of the sample was mixed with 1.4g of silica gel (200 mesh);
s4, subjecting the mixture obtained in S3 to gradient washing by using petroleum ether and ethyl acetate according to the ratio of 2:1, 1:1 and 1:2, finally washing the rest part by using ethyl acetate, weighing 30g (200 meshes) of silica gel, filling the silica gel into a filtration separation column with the diameter of 3cm, subjecting the mixed liquid to chromatographic separation by using the filtration separation column to obtain an extract W4-1 for secondary separation, and performing chromatographic separation; collecting the mixture in a tube of 20mL, and specifically displaying the following table 2;
TABLE 2 active sample from the second separation
| Corresponding separation tube | Eluent |
| 1 to 26 tubes | Petroleum ether: ethyl acetate (2:1) |
| 27-45 pipe | Petroleum ether: ethyl acetate (1:1) |
| 46-56 pipe | Petroleum ether: ethyl acetate (1:2) |
| 57 tube | Rinsing with ethyl acetate |
| 58 tube | Methanol rinse |
According to the loading on the silica gel plate, the 21-36 tubes were combined to obtain a relatively pure fraction W4-1, yielding 0.55 g of material. And performing structural identification as a sample. The results are shown in FIG. 2c for thin layer chromatography silica gel plates. In fig. 2c, the mobile phase is petroleum ether: the ethyl acetate ratio was 2:1 (a little acetic acid was added), the left band was W4, the right band was 21-36 tubes, and the concentrated material W4-1 was combined.
Example 2
And (3) directly extracting needle mushroom fungus dregs to obtain a needle mushroom fungus dreg crude extract sample, performing an activity experiment test, and observing the conditions of inhibiting seed germination and growth.
Accurately weighing 0.5g of crude extract sample, dissolving in 15mL of ethyl acetate, respectively sucking 1.0 mL, 0.5 mL and 0.25mL of ethyl acetate solution, placing on a filter paper, placing the filter paper in a labeled glass culture dish after the ethyl acetate is completely volatilized, then injecting 20mL of water into each culture dish, taking about 30 Chinese cabbage seeds as a contrast with clear water, repeating for 3 times each treatment, placing in a constant-temperature incubator at 25 ℃ for culture, and observing the germination condition of the seeds.
TABLE 4 Germination and growth status of active samples on 5 th day of Brassica campestris seeds
As shown in the results of Table 4, the germination inhibition of the active sample on the Chinese cabbage seeds is remarkably shown in figure 3, the root growth is remarkably inhibited after the Chinese cabbage seeds germinate and sprout under the condition of 600-time concentration, the germination rate is 85 percent as shown in figure 4; under the condition of 1200 times concentration, the length of the root system is obviously shorter than that of the control group; compared with the control, the active substance has obvious influence on the growth of the root system under the concentration of 2400 times. The test result shows that the active sample contains active substances for inhibiting seed germination, and the inhibition effect is obvious.
Example 3
Performing activity tests on flammulina velutipes mushroom residue isolates W2, W3 and W4 obtained in the primary separation in example 1 respectively; the specific method comprises the following steps: dissolving about 0.1g of needle mushroom fungus dreg isolate with 10mL of ethyl acetate respectively, taking 1mL of the ethyl acetate solution, dripping the ethyl acetate solution on dry filter paper (weighed in advance), weighing again to determine the mass of substances on the filter paper after the ethyl acetate is volatilized, putting the filter paper into a culture dish, adding 20mL of purified water, putting 30 white rapeseed grains into the culture dish, culturing in a constant-temperature incubator at 27 ℃, observing and recording the germination condition after 5 days. The experiment was set up with filter paper plus purified water as a blank Control (CK) and repeated 3 times per treatment.
As shown in FIG. 5, it is clear that germination was observed in the culture dish when the blank reference CK and the needle mushroom residue isolates W2 and W3 were treated at 1000 times; in contrast, the seeds treated with the needle mushroom residue isolate W4, such as the blank reference CK, did not germinate; the results showed that the needle mushroom residue isolate W4 had activity of inhibiting seed germination.
Example 4
Concentration gradient activity test is carried out on W4-1 (needle mushroom fungus dreg extract) separated in the embodiment 1 of the invention, and the conditions of inhibiting seed germination and growth are observed.
Weighing 0.2g W4-1 substance, dissolving in 10mL ethyl acetate, respectively sucking 1.0, 0.5 and 0.25mL ethyl acetate solution, placing on a filter paper, placing the filter paper in a labeled glass culture dish after the ethyl acetate is completely volatilized, then injecting 20mL water and about 30 Chinese cabbage seeds into each culture dish, using clear water as a contrast, repeating for 3 times each treatment, placing in a constant temperature incubator at 25 ℃ for culture, and observing the germination condition of the seeds.
TABLE 5 germination and growth conditions of Flammulina velutipes (Fr.) Sing residue extract W4-1 on 5 th day of Chinese cabbage seed
| 1000 times of | 2000 times of | 4000 times of | CK | |
| Suppression of conditions | Inhibiting growth | Inhibiting growth | Is free of | Is free of |
| Germination rate | 26.7％ | 67.78％ | 100％ | 100％ |
As shown in the results of the table 5 and the figure 6, the flammulina velutipes mushroom dreg extract W4-1 has obvious inhibition on the germination of the seeds of the pakchoi, and the seeds of the pakchoi grow slowly after sprouting under the condition of 1000 times of concentration, and the germination rate is 26.7%; under the condition of 2000 times concentration, the germination rate of the Chinese cabbage seeds is 67.78%, and the root length is obviously shorter than that of a control group; compared with a control, the active substance basically has no influence on the germination of the pakchoi under the condition of 4000 times of concentration.
Therefore, the application discovers for the first time that the flammulina velutipes mushroom dregs and the flammulina velutipes mushroom dreg extract have obvious plant growth inhibition effect, and the flammulina velutipes mushroom dreg extract has the weeding activity effect, so that the flammulina velutipes mushroom dreg extract is used for developing herbicide pesticide products, thereby not only providing a new choice for developing domestic new pesticide products, but also laying a solid foundation for developing domestic independent intellectual property new varieties of pesticides. In addition, the flammulina velutipes mushroom residue extract is a natural product, compared with a new compound which is synthesized purely artificially, the possibility of teratogenesis, carcinogenesis and mutagenesis of organisms is greatly reduced, and the flammulina velutipes mushroom residue extract is an ideal biopesticide product.
While the present invention has been described with respect to preferred embodiments, additional variations and modifications will occur to those embodiments once the basic inventive concepts are known to those skilled in the art. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (10)
1. The application of the flammulina velutipes mushroom dreg extract in preparing the herbicide is characterized in that the extraction method of the flammulina velutipes mushroom dreg extract comprises the following steps:
s1, weighing dried needle mushroom dregs, and adding ethyl acetate to extract to obtain paste; removing acetic acid by using sodium carbonate, and mixing with 80-100 meshes of silica gel to obtain a mixture;
s2, placing the mixture obtained in the step S1 in a mixed solvent of petroleum ether and ethyl acetate, and carrying out chromatographic separation through a filtration separation column to obtain an extract of primary separation.
2. The use of needle mushroom pomace extract according to claim 1 in preparing herbicides, wherein S2 further comprises: carrying out thin-layer chromatography and reduced pressure concentration on the extract to sequentially obtain substances with different polarities of W1, W2, W3 and W4, mixing W4 with 200-300 meshes of silica gel, carrying out gradient washing on the mixed liquid of petroleum ether and ethyl acetate, and carrying out chromatographic separation on the washed mixed liquid through a filtration separation column to obtain a secondary separated extract W4-1.
3. The application of the flammulina velutipes mushroom dreg extract in preparing the herbicide as claimed in claim 2, wherein the extract or the extract W4-1 achieves the purpose of weeding by inhibiting seed germination and root growth.
4. The use of the flammulina velutipes mushroom residue extract as claimed in claim 2 in the preparation of herbicides, wherein the extract or the extract W4-1 is diluted with water for use, or the extract W4-1 is added for preparation of herbicide formulations.
5. The application of the flammulina velutipes dreg extract in preparing the herbicide according to claim 4 is characterized in that the flammulina velutipes dreg extract is diluted to 0-2000 times by water.
6. The application of the flammulina velutipes mushroom dreg extract in preparing the herbicide as claimed in claim 1, wherein in S1, the mass-to-volume ratio of the flammulina velutipes mushroom dreg to the ethyl acetate is 1g:10 mL.
7. The application of the flammulina velutipes mushroom dreg extract in preparing the herbicide as claimed in claim 1, wherein in S1, the mass ratio of the paste to the silica gel is 1: 1-1.5.
8. The application of the flammulina velutipes mushroom dreg extract in preparing the herbicide as claimed in claim 1, wherein in S2, the volume ratio of the petroleum ether to the ethyl acetate is 5: 1.
9. The application of the flammulina velutipes mushroom residue extract in preparing the herbicide as claimed in claim 2, wherein the mass ratio of W4 to silica gel is 1: 1-1.5.
10. The application of the flammulina velutipes mushroom dreg extract in preparing the herbicide as claimed in claim 2, wherein the volume ratio of the petroleum ether to the ethyl acetate is 2:1 when preparing the extract W4-1.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115299315A (en) * | 2022-08-10 | 2022-11-08 | 武汉市农业科学院 | Matrix for preventing and controlling farmland weeds and preventing and controlling method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115299315A (en) * | 2022-08-10 | 2022-11-08 | 武汉市农业科学院 | Matrix for preventing and controlling farmland weeds and preventing and controlling method thereof |
| CN115299315B (en) * | 2022-08-10 | 2024-02-13 | 武汉市农业科学院 | Matrix for farmland weed prevention and control and prevention and control method thereof |
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