CN116421609A - Application of myricetin in inhibiting microsporidian and preparing medicine for preventing or treating corpuscle disease - Google Patents
Application of myricetin in inhibiting microsporidian and preparing medicine for preventing or treating corpuscle disease Download PDFInfo
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- 229940116852 myricetin Drugs 0.000 title claims abstract description 31
- PCOBUQBNVYZTBU-UHFFFAOYSA-N myricetin Natural products OC1=C(O)C(O)=CC(C=2OC3=CC(O)=C(O)C(O)=C3C(=O)C=2)=C1 PCOBUQBNVYZTBU-UHFFFAOYSA-N 0.000 title claims abstract description 31
- 235000007743 myricetin Nutrition 0.000 title claims abstract description 31
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- 235000007270 Gaultheria hispida Nutrition 0.000 description 2
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- 239000006013 carbendazim Substances 0.000 description 2
- JNPZQRQPIHJYNM-UHFFFAOYSA-N carbendazim Chemical compound C1=C[CH]C2=NC(NC(=O)OC)=NC2=C1 JNPZQRQPIHJYNM-UHFFFAOYSA-N 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
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- DCYOADKBABEMIQ-OWMUPTOHSA-N myricitrin Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1OC1=C(C=2C=C(O)C(O)=C(O)C=2)OC2=CC(O)=CC(O)=C2C1=O DCYOADKBABEMIQ-OWMUPTOHSA-N 0.000 description 2
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- HYZJCKYKOHLVJF-UHFFFAOYSA-N 1H-benzimidazole Chemical compound C1=CC=C2NC=NC2=C1 HYZJCKYKOHLVJF-UHFFFAOYSA-N 0.000 description 1
- 241001225321 Aspergillus fumigatus Species 0.000 description 1
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- 244000132436 Myrica rubra Species 0.000 description 1
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Abstract
The invention discloses an application of myricetin in inhibiting microsporidian and preparing a medicine for preventing or treating corpuscle diseases. The invention provides the application of the myricetin for inhibiting microsporidia for the first time, the myricetin can be combined with the Orac1 protein with high efficiency, and the replication of silkworm ichthyosis pathogen-silkworm microsporidia is inhibited by blocking the function of the Orac1 protein, so that the aim of preventing or treating silkworm ichthyosis is achieved. When the myricetin is used in cells, the final concentration reaches more than 0.1 mu M, so that the microsporidian of the silkworm can be inhibited; when the composition is used for preparing medicines for preventing or treating the corpuscle diseases, the incidence rate of the corpuscle diseases of the silkworm can be obviously reduced when the concentration reaches 200ppm or more.
Description
Technical Field
The invention relates to the fields of molecular biology and pathology, in particular to application of myricetin in preparing medicines for preventing or treating related to corpuscle diseases.
Background
Silkworm corpuscle disease is commonly a destructive infectious disease caused by the infection of silkworm microsporidian (Nosema bombycis) by the body of silkworms through the mouth feel. Because the incubation period is long, the method can be spread through embryo species, the production threat to basic silkworm species in the silkworm industry is huge, once silkworm species manufacturers sell out the silkworm species with toxin to silkworm farmers for feeding, the outbreak of silkworm microparticle diseases is easy to occur, and the spore of the silkworm microsporidian easily causes long-time pollution to the whole silkworm raising area due to strong prevention or treatment reversibility of the environment, so that the economic loss is brought to the silkworm farmers. At present, the direct economic loss caused by the corpuscle disease in China reaches hundreds of millions of yuan each year, nearly half of the production expenditure of silkworm seeds in a silkworm breeding field is used for preventing and controlling the corpuscle disease, and the microsporidian of the silkworm is the only legal quarantine object for the world silkworm seed production at present.
The existing medicines for preventing and treating silkworm corpuscle diseases comprise broad-spectrum insecticide enteroworm-cleaning (effective component: albendazole) and bactericide carbendazim (effective component: benzimidazole). However, the two medicines belong to broad-spectrum medicines, and have respective defects, for example, the effective concentration of the dosage of the enteromorpha for preventing and treating the silkworm ichthyosis is more than 1000ppm, and the medicines are needed to be used for multiple times, so that the economic cost is higher; the carbendazim of the silkworm microsporidian beta-tubulin is a medicament for preventing or treating silkworm corpuscle disease, which is discovered more than 40 years ago, the treatment effect is influenced by factors such as pathogen infection coefficient, infection time, administration time and the like, and the medicament is forbidden in European and American countries at present due to cancerogenicity. Meanwhile, in the process of drug examination aiming at the silkworm corpuscle disease, drugs with effects on other species cannot be applied to the silkworm, for example, aspergillus fumigatus with better curative effect on the bee corpuscle disease has no effect on the silkworm corpuscle disease, so that a new strategy is necessary to discover new drugs for preventing or treating the silkworm corpuscle disease.
Disclosure of Invention
The invention aims to: the invention aims to provide an application of myricetin in inhibiting microsporidian; the invention also aims to provide an application of the myricetin in preparing medicines for preventing or treating the microparticle diseases.
The technical scheme is as follows: the application of the myricetin in inhibiting microsporidian in cells is provided.
The structure of the myricetin is shown as (I), and the molecular formula is C 34 H 44 O 19 Molecular weight: 756.71.
according to functional research of a silkworm microsporidian core gene Orac1, a plurality of Chinese herbal medicine monomers with inhibition effect on the proliferation of the silkworm microsporidian are found in a Chinese herbal medicine monomer database through protein structure modeling and virtual screening technology, and implementation proves that the myricetin can be specifically combined with the silkworm microsporidian core protein Orac1 in a silkworm cell, and the replication of silkworm microsporidian pathogen-silkworm microsporidian is inhibited through blocking the function of the Orac1 protein, so that the aim of preventing or treating the silkworm microsporidian is achieved.
Further, the microsporidian is Nosema bombycis or a variant strain thereof.
Further, the concentration of the myricetin used in the cells is more than 0.1 mu M.
The invention relates to application of myricetin in preparing medicines for preventing or treating corpuscle diseases.
Further, the ichthyosis is silkworm ichthyosis.
Further, the silkworm corpuscle disease is caused by Nosema bombycis or a variant strain thereof.
Further, the method is characterized in that the concentration of the myricetin in the medicine for preventing or treating the corpuscle disease is more than 200 ppm.
Further, the medicament also comprises other pharmaceutically acceptable auxiliary materials.
The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages: the invention provides the myricetin for inhibiting microsporidia for the first time, the myricetin can be combined with the Orac1 protein with high efficiency to inhibit the replication of silkworm ichthyosis pathogen-silkworm microsporidia by blocking the function of the Orac1 protein, thereby achieving the purpose of preventing or treating silkworm ichthyosis. When the myricetin is used in cells, the final concentration reaches more than 0.1 mu M, so that the microsporidian of the silkworm can be inhibited; when the composition is used for preparing medicines for preventing or treating the corpuscle diseases, the incidence rate of the corpuscle diseases of the silkworm can be obviously reduced when the concentration reaches 200ppm or more.
Drawings
FIG. 1 is a graph showing the results of genome copy numbers of microsporidian of silkworm in cells after treatment of silkworm cells with different concentrations of myricetin.
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings.
EXAMPLE 1 experiment of different concentrations of Changshan glycoside of Myrica rubra with respect to inhibition of microsporidian of Bombyx mori in cells
1. Cell inoculation and preparation of stock solution of myricetin
(1) Continuously culturing silkworm ovary cell line BmN (preserved in physiological and pathological laboratory) in TC100 insect cell culture medium containing 10% FBS (fetal bovine serum) at 27deg.C;
(2) Preparation of stock solution of myricetin
Preparing a storage solution of 10mM myricetin by using DMSO;
(3) In vitro germination and infection of silkworm microsporidian
The microsporidian spores of silkworm (stored in the physiological and pathological laboratory of silkworm) were resuspended in 1ml of 0.1mol/L KOH solution and incubated at 30℃for 30min before being thoroughly mixed with 12ml of BmN cell suspension; after standing for 5min, injecting the same amount into a 12-hole cell culture plate, standing at 27 ℃ for 1h, removing the culture medium after the cell culture plate is completely adhered, and sealing and culturing at 27 ℃ after adding fresh culture medium again.
2. Experiments on inhibition of microsporidian from silkworm in cells when final concentrations of myricetin are 0.1,0.5 and 1 mu M.
After in vitro germination and infection of the silkworm microsporidian, the culture medium is removed, fresh culture medium containing 0 (control), 0.1,0.5 and 1 mu M of myricitrin (prepared by using the stock solution of the myricitrin in the step (2)) is added again, cells are respectively collected after culturing for 72 hours at 27 ℃, total DNA is extracted by using a DNA/RNA extraction kit (Tiangen Biochemical Co., ltd., DP 422), and the genome copy number of the silkworm microsporidian in the cells is detected by using a quantitative PCR technology. As shown in FIG. 1, the concentration of the myricetin is more than 0.1 μm, and the replication of microsporidian of silkworm can be remarkably inhibited.
Example 2 experiment of the Effect of different Bayberry Dichroside concentrations on the incidence of Cryptosporidium
Test silkworm variety: autumn-abundant x white jade (saved by the national academy of agricultural science, silkworm industry research institute);
age: 4-year-old silkworm;
setting a region: the treatments were repeated for 3 zones at different concentrations, 50 silkworms per zone.
Preparing a waxberry antifebrile glycoside solution: final concentrations of 0, 10, 100, 200 and 1000ppm myricetin solutions were formulated with DMSO.
Control group: diluting microsporidian of Bombyx mori to 10 with sterile water 6 spore/mL suspension was used to soak mulberry leaves for 10 seconds, and after air-drying, the test silkworms were fed for 12 hours (infection). Then feeding normal mulberry leaves, and feeding the mulberry leaves until the mulberry leaves are cooked, cocooning and cocooning.
Experimental group: diluting purified microsporidian of Bombyx mori with sterile water to 10 6 spore/mL suspension, soaked mulberry leaf, dried in the air and fed to test silkworms for 12 hours (infection). Then feeding the detoxified silkworm with medicated mulberry leaves (respectively using 0, 10, 100, 200, 1000ppm of bayberry dichroa glucoside solution for 10 seconds) for 6 hours, and reutilizingFeeding normal mulberry leaves, feeding the medicated mulberry leaves for 1 time every day, feeding the normal mulberry leaves at other times, and feeding the normal mulberry leaves until the silkworm is cooked, cocooning and cocooning.
And (5) after emerging moths are 6 days, placing the moths into a 60 ℃ oven for drying for 6 hours, and grinding and microscopic examination of the moths. In the experimental process, the dead silkworms and the dead pupae are also checked by a microscope to determine whether the silkworms and the dead pupae are dead from microspore infection, and the number of the silkworms dead due to other factors is subtracted from the total number of each region, so that statistics are not carried out. Finally, the infection rate of microsporidia in each region was investigated, and the treatment rate was calculated separately. The results are shown in Table 1. When the fed mulberry She Zhongyang Mei Changshan glycoside concentration is 0, the infection rate of the silkworms is 100%; when the fed mulberry She Zhongyang Mei Changshan glycoside concentration is 100ppm, the infection rate of the corpuscle disease is 34.3 percent, and the drug treatment rate is 65.7 percent, which proves that the bayberry dichroa glycosides have certain treatment effect; when the fed mulberry She Zhongyang Mei Changshan glycoside concentration is 200-1000ppm, the incidence rate of the microparticle disease is 0% and the drug treatment rate is 100%; however, when the concentration of the fed mulberry She Zhongyang Mei Changshan glycoside reached 1000ppm, more than half of silkworms died before cocooning, and microsporidian was not detected, indicating that the concentration of myricetin was toxic to silkworms.
TABLE 1 therapeutic Rate of different concentrations of Bayberry dichroa glycosides against silkworm sarcoidosis
Example 3 toxicity experiments on silkworms with different Bayberry Dichrooside concentrations
Test silkworm variety: autumn-abundant x white jade (saved by the national academy of agricultural science, silkworm industry research institute);
age: 4-year-old silkworm;
taking 1000 silkworms, feeding the silkworms 1 time a day with medicated mulberry leaves (respectively soaked in 0, 100, 200 and 1000ppm of waxberry antifebrile glycoside solution), feeding normal mulberry leaves at other times, and feeding the silkworms to mature silkworms, cocooning and cocooning. The growth condition of domestic silkworms is recorded every day in the raising period, and the life indexes such as the number of dead silkworms or sick silkworms, the number of cocoons, the number of chemical moths, the number of dead cages and the like are recorded, and the economic indexes such as the whole cocoon quantity, the cocoon layer rate and the like are recorded. After the silkworm pupas emerge out of the moth, mating male and female moths, spawning, and investigating the egg quantity and the silkworm egg hatching rate. The results are shown in Table 2.
TABLE 2 investigation of toxicity effects of Bayberry dichroa glycosides on silkworm
As can be seen from the test data in Table 2, the mulberry leaf soaked with the myricetin of the present invention is fed to silkworms, and the normal growth of silkworms is not affected compared with the control group (0 ppm) in the effective use concentration (200-1000 ppm), and the silkworm pupa vital rate (cocooning number and moth number), cocoon quality (whole cocoon quantity and cocoon layer rate) and egg quality (egg quantity and hatching rate) data of silkworms are not significantly different.
Claims (8)
1. An application of myricetin in inhibiting microsporidian is provided.
2. The use of bayberry dichroa febrifuga glycoside according to claim 1 for inhibiting microsporidian, wherein the microsporidian is Nosema bombycis of silkworm or a variant strain thereof.
3. The use of bayberry dichroa glycosides in inhibiting microsporidian according to claim 1, wherein the concentration of bayberry dichroa glycosides is above 0.1 μm.
4. Application of myricetin in preparing medicine for preventing or treating microparticle diseases caused by microsporidian infection is provided.
5. The use of bayberry dichroin according to claim 4 for the preparation of a medicament for the prevention or treatment of a disease of the corpuscle, wherein the disease of the corpuscle is a silkworm corpuscle.
6. The use of bayberry dichroa glycosides in the manufacture of a medicament for preventing or treating a disease of micro-particles, according to claim 5, wherein the disease of micro-particles of silkworm is caused by Nosema bombycis of silkworm or a variant strain thereof.
7. The use of myricetin in preparing a medicament for preventing or treating a microparticle disease according to claim 4, wherein the concentration of myricetin in the medicament for preventing or treating a microparticle disease is 200ppm or more.
8. The use of bayberry dichroa glycosides according to claim 4 in the manufacture of a medicament for the prevention or treatment of micro-particulate disease, said medicament further comprising other pharmaceutically acceptable excipients.
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