Application of myricetin in inhibiting microsporidia and preparing drugs for preventing or treating microsporidia

Abstract

The present invention discloses an application of myricetin for inhibiting microsporidia and preparing drugs related to the prevention or treatment of microsporidia. The present invention proposes for the first time that myricetin is used to inhibit microsporidia. Myricetin can efficiently bind to Orac1 protein, and inhibit the replication of the pathogen of silkworm microsporidia by blocking the function of Orac1 protein, thereby achieving the purpose of preventing or treating silkworm microsporidia. When myricetin is used in cells, the final concentration of 0.1 μM or more can inhibit silkworm microsporidia; when used to prepare drugs for the prevention or treatment of microsporidia, the concentration of 200 ppm or more can significantly reduce the incidence of silkworm microsporidia.

Description

Technical Field

[0001] The present invention relates to the fields of molecular biology and pathology, and in particular to the application of myricetin in the preparation of drugs related to the prevention or treatment of microparticle disease. Background Art

[0002] Pebrine disease is a devastating infectious disease caused by oral infection of silkworms by Nosema bombycis. Due to its long incubation period, especially the fact that it can be transmitted through embryos, it poses a huge threat to the production of silkworm seeds, the foundation of the sericulture industry. Once silkworm seed production companies sell poisonous silkworm seeds that exceed the standard to silkworm farmers for feeding, it is very easy to cause an outbreak of pebrine disease. Due to the strong prevention or treatment resistance of Nosema bombycis spores to the environment, it is easy to cause long-term pollution to the entire silkworm breeding area, causing economic losses to silkworm farmers. At present, the direct economic losses caused by pebrine disease in my country each year amount to more than 100 million yuan, and nearly half of the silkworm seed production expenditure of silkworm seed farms is used for the prevention and control of pebrine disease. At present, Nosema bombycis is the only statutory quarantine object for silkworm seed production in the world.

[0003] At present, the drugs used for the prevention and treatment of silkworm pebbles disease include the broad-spectrum insecticide chondroitin (active ingredient: albendazole) and the fungicide carbendazim (active ingredient: benzimidazole). However, both drugs are broad-spectrum drugs and have their own shortcomings. For example, the effective concentration of chondroitin for the prevention and treatment of silkworm pebbles disease must be above 1000ppm, and the drug must be used multiple times, which has a high economic cost. Carbendazim, which is used to prevent or treat silkworm pebbles disease for β-tubulin of silkworm microsporidia, was discovered more than 40 years ago. Its therapeutic effect is affected by factors such as pathogen infection coefficient, infection time, and medication time. It is currently banned in Europe and the United States because of its carcinogenicity. At the same time, in the process of testing drugs for silkworm pebbles disease, drugs that are effective on other species cannot be applied to silkworms. For example, Aspergillus fumigatus, which has a good effect on honey bee pebbles disease, has no effect on silkworm pebbles disease. Therefore, it is necessary to apply new strategies to discover new drugs for the prevention or treatment of silkworm pebbles disease. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide an application of myricetin in inhibiting microsporidia; another purpose of the present invention is to provide an application of myricetin in preparing a drug for preventing or treating microsporidia.

[0005] Technical solution: The application of the myrica chinensis glycosides described in the present invention in inhibiting intracellular microsporidia.

[0006] The structure of the myricetin is shown in (I), and the molecular formula is C 34 H 44 O 19, molecular weight: 756.71.

[0007]

[0008] According to the functional study of Orac1, a core gene of Bombyx mori Nosema, the present application discovered several Chinese herbal medicine monomers that have an inhibitory effect on the proliferation of Bombyx mori Nosema in the Chinese herbal medicine monomer database through protein structure modeling and virtual screening technology. The implementation confirmed that Myrica chinensis glycosides can specifically bind to Orac1, a core protein of Bombyx mori Nosema in silkworm cells, and inhibit the replication of Bombyx mori Nosema, the pathogen of Bombyx mori nematicide, by blocking the function of Orac1 protein, thereby achieving the purpose of preventing or treating Bombyx mori nematicide.

[0009] Furthermore, the microsporidia are Nosema bombycis or variant strains thereof.

[0010] Furthermore, the concentration of the myricetin used in cells is above 0.1 μM.

[0011] The invention discloses an application of the myrica chinensis glycosides in the preparation of a medicine for preventing or treating microparticle disease.

[0012] Furthermore, the microbacterial disease is Bombyx mori microbacterial disease.

[0013] Furthermore, the silkworm pebrine disease is caused by Nosema bombycis or its variant strains.

[0014] Furthermore, it is characterized in that the concentration of myricetin in the drug for preventing or treating microparticle disease is above 200 ppm.

[0015] Furthermore, the medicine also includes other pharmaceutically acceptable excipients.

[0016] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention proposes for the first time to use myricetin for inhibiting microsporidia. Myricetin can efficiently bind to Orac1 protein and inhibit the replication of the pathogen of silkworm microsporidia-M. bombyx mori by blocking the function of Orac1 protein, thereby achieving the purpose of preventing or treating silkworm microsporidia. When myricetin is used in cells, the final concentration of 0.1 μM or more can inhibit microsporidia in silkworms; when used to prepare drugs for preventing or treating microsporidia, the concentration of 200 ppm or more can significantly reduce the incidence of silkworm microsporidia. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a graph showing the genome copy number of Nosema bombycis in silkworm cells after treating silkworm cells with different concentrations of myricetin. DETAILED DESCRIPTION

[0018] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0019] Example 1 Inhibition experiment of different concentrations of myricetin on the growth of Nosema bombyx mori in cells

[0020] 1. Cell inoculation and preparation of storage solution of myricetin

[0021] (1) The silkworm ovary cell line BmN (stored in the Silkworm Physiology and Pathology Laboratory) was continuously cultured in TC100 insect cell culture medium containing 10% FBS (fetal bovine serum) at 27°C;

[0022] (2) Preparation of storage solution of myrica chinensis glycoside

[0023] Prepare a 10 mM stock solution of myricetin in DMSO;

[0024] (3) In vitro germination and infection of Nosema bombycis

[0025] Resuspend the spores of Nosema bombycis (stored in the Silkworm Physiology and Pathology Laboratory) with 1 ml of 0.1 mol / L KOH solution and incubate at 30°C for 30 min. Then, mix thoroughly with 12 ml of BmN cell suspension. After standing for 5 min, inject an equal amount into a 12-well cell culture plate and stand at 27°C for 1 h to allow it to fully adhere to the wall. Then, remove the culture medium, re-add fresh culture medium and seal and culture at 27°C.

[0026] 2. When the final concentration of myricetin was 0.1, 0.5 and 1 μM, the inhibition experiment on Bombyx mori Nosema was carried out in cells.

[0027] After in vitro germination and infection of Nosema bombycis, the culture medium was removed and fresh culture medium containing final concentrations of 0 (control), 0.1, 0.5 and 1 μM myricetin (prepared using the stock solution of myricetin in step (2)) was added. After culturing at 27°C for 72 hours, the cells were collected and total DNA was extracted using a DNA / RNA extraction kit (Tiangen Biochemical Technology Co., Ltd., DP422). The genome copy number of Nosema bombycis in the cells was detected using quantitative PCR. The results are shown in Table 1. Figure 1 The results show that myricetin can significantly inhibit the replication of Nosema bombyx mori when the concentration is above 0.1 μM.

[0028] Example 2 Experiment on the effect of different concentrations of myricetin on the incidence of microparticle disease

[0029] Silkworm varieties tested: Qiufeng × Baiyu (preserved by the Institute of Sericulture, Chinese Academy of Agricultural Sciences);

[0030] Age: Silkworms start at 4th instar;

[0031] Zone setting: Different concentrations of each treatment were repeated in 3 zones, with 50 silkworms in each zone.

[0032] Preparation of myrica sarmentosin solution: Use DMSO to prepare myrica sarmentosin solutions with final concentrations of 0, 10, 100, 200 and 1000 ppm.

[0033] Control group: Nosema bombyx mori was diluted to 10 6 The suspension of spores / mL was used to soak mulberry leaves for 10 seconds, and then fed to the test silkworms for 12 hours after drying (infection). Then, normal mulberry leaves were fed to the silkworms until they matured, went to cocoons, and formed cocoons.

[0034] Experimental group: The purified Nosema bombyx mori was diluted to 10 6 The suspension of spores / mL was soaked in mulberry leaves, and the test silkworms were fed with the suspension for 12 hours after drying (infection). Then the poisoned silkworms were fed with medicated mulberry leaves (soaked in 0, 10, 100, 200, 1000 ppm myrica chinensis solution for 10 seconds) for 6 hours, and then fed with normal mulberry leaves. After that, the silkworms were fed with medicated mulberry leaves once a day, and fed with normal mulberry leaves at other times, and were raised until they matured, went to cocoons, and formed cocoons.

[0035] Six days after the moths emerged from the pupa, they were placed in a 60℃ oven and dried for 6 hours. Each moth was ground and examined under a microscope. During the experiment, dead silkworms and pupae were also examined under a microscope to see if they died from microsporidia infection. The number of silkworms that died due to other factors was deducted from the total number in each area and was not counted. Finally, the microsporidia infection rate in each area was investigated, and the treatment rate was calculated separately. The results are shown in Table 1. When the concentration of myricetin in the fed mulberry leaves was 0, the infection rate of silkworms was 100%; when the concentration of myricetin in the fed mulberry leaves was 100 ppm, the infection rate of microsporidia was 34.3% and the drug treatment rate was 65.7%, which proved that myricetin had a certain therapeutic effect; when the concentration of myricetin in the fed mulberry leaves was between 200 and 1000 ppm, the incidence of microsporidia was 0% and the drug treatment rate was 100%; however, when the concentration of myricetin in the fed mulberry leaves reached 1000 ppm, more than half of the silkworms died before cocooning, and no microsporidia were detected, indicating that this concentration of myricetin was toxic to silkworms.

[0036] Table 1 The therapeutic rate of different concentrations of myricetin against nematode disease in silkworms

[0037]

[0038] Example 3 Toxicity test of different concentrations of myricetin to silkworms

[0039] Silkworm varieties tested: Qiufeng × Baiyu (preserved by the Institute of Sericulture, Chinese Academy of Agricultural Sciences);

[0040] Age: Silkworms start at 4th instar;

[0041] Take 1000 silkworms, feed them with medicated mulberry leaves (soaked in 0, 100, 200, 1000ppm myrica chinensis solution) once a day, and feed them with normal mulberry leaves at other times, and raise them until they are mature, on cocoons, and cocoon. During the feeding period, record the growth of silkworms every day, record the number of dead or diseased silkworms, the number of cocoons, the number of moths, the number of dead cages and other life indicators, and record the total cocoon volume, cocoon layer volume, cocoon layer rate and other economic indicators. After the silkworm pupae emerge as moths, make the male and female moths mate and lay eggs, and investigate the egg volume and silkworm egg hatching rate. The results are shown in Table 2.

[0042] Table 2 Investigation of the toxic effects of myricetin on silkworms

[0043]

[0044]

[0045] It can be seen from the test data in Table 2 that the use of the myrica chinensis glycosides of the present invention to soak mulberry leaves as food for silkworms has no effect on the normal growth of silkworms compared with the control group (0ppm) within the effective use concentration (200-1000ppm), and there are no significant differences in the data of the pupae life rate (number of cocoons and number of moths), cocoon quality (total cocoon amount and cocoon layer rate) and egg quality (egg amount and hatching rate) of the silkworms.

Claims

1. A use of myricetin in the preparation of a drug for preventing or treating microsporidia caused by microsporidia infection, wherein the concentration of myricetin is 200-1000 ppm when the drug is used to prevent or treat microsporidia, and the microsporidia is Bombyx mori microsporidia, which is caused by Bombyx mori microsporidia. Nosema bombycis or its variant strains.

2. The use of myricetin according to claim 1 in the preparation of a drug for preventing or treating microparticle disease, characterized in that: The medicine also includes other pharmaceutically acceptable excipients.

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