CN107468682B - Application of mangiferin in preparation of antiviral drugs - Google Patents
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- YWQSXCGKJDUYTL-UHFFFAOYSA-N Mangiferin Natural products CC(CCC=C(C)C)C1CC(C)C2C3CCC4C(C)(C)CCCC45CC35CCC12C YWQSXCGKJDUYTL-UHFFFAOYSA-N 0.000 title claims abstract description 60
- 229940043357 mangiferin Drugs 0.000 title claims abstract description 60
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
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Abstract
本发明提供了芒果苷在制备抗病毒药物中的应用，包括芒果苷在制备治疗或预防鲤春病毒血症病毒病药物中的应用，包括芒果苷在制备治疗或预防斑点叉尾鮰病毒病药物中的应用。通过以一定浓度的芒果苷预处理FHM细胞或CCO细胞，再以鲤春病毒血症病毒或斑点叉尾鮰病毒感染，洗去游离的病毒，继续加入含有对应浓度芒果苷,24h后检测病毒的复制水平。结果显示，细胞经芒果苷处理后，这两种病毒的复制水平极显著降低，说明芒果苷具有抑制鲤春病毒血症病毒和斑点叉尾鮰病毒等水生病毒的作用。细胞毒性实验显示所使用浓度的芒果苷对FHM和CCO细胞均没有毒副作用，可以作为鲤春病毒血症病毒病和斑点叉尾鮰病毒病的治疗剂和预防剂。The present invention provides the application of mangiferin in the preparation of antiviral medicines, including the application of mangiferin in the preparation of medicines for treating or preventing carp viremia virus disease, including the application of mangiferin in the preparation of medicines for treating or preventing channel catfish virus disease. application. FHM cells or CCO cells were pretreated with a certain concentration of mangiferin, and then infected with carp spring viremia virus or channel catfish virus, the free virus was washed away, and the corresponding concentration of mangiferin was added, and the replication level of the virus was detected after 24 hours. . The results showed that after the cells were treated with mangiferin, the replication levels of these two viruses were significantly reduced, indicating that mangiferin has the effect of inhibiting the carp spring viremia virus and channel catfish virus and other aquatic viruses. Cytotoxicity experiments showed that mangiferin at the concentration used had no toxic and side effects on FHM and CCO cells, and could be used as a therapeutic and preventive agent for carp spring viremia virus disease and channel catfish virus disease.
Description
The application aims at the application date of 2015-7-24, and the application number of: 201510444307.5, title of the invention: application of mangiferin in preparing antiviral drugs is divided into cases.
Technical Field
The invention relates to application of mangiferin, in particular to application of mangiferin in preparing a medicine for resisting spring viremia of carp virus or channel catfish virus.
Background
Spring Viremia of Carp (SVCV) belongs to Rhabdoviridae (Rhabdoviridae) and vesicular stomatitis Virus (Vesiculovirus), and the Spring viremia of Carp caused by the Virus belongs to acute and hemorrhagic diseases with serious infectivity, which are listed as diseases which need to be declared by the International animal diseases Office (OIE), and are also listed as animal epidemic diseases in the famous records of first and second infectious diseases and parasitic diseases of the domestic animal of the people's republic of China (2008).
Ictalurus punctatus virus (CCV) belongs to Herpesvirales (Herpesvirales), Herpesviridae (Allophesviridae), and Ictalurus (Ictalurivims), and is a double-stranded DNA virus with an envelope. The channel catfish virus disease caused by the channel catfish virus disease can cause mass death of fish fries and fish species in farms, is listed as a disease which needs to be declared by the international animal and epidemic departments (OIE), and is also listed as a second animal epidemic disease in the first and second infectious diseases and parasitic disease records of the domestic animals of the people's republic of China (2008).
Diseases caused by the viruses are extremely harmful to the aquaculture industry in China, no effective medicine or treatment method exists temporarily, and once the diseases are outbreak, the diseases cause great loss to the aquaculture industry. The search for the medicine for resisting the spring viremia of carp virus and the channel catfish virus is particularly important and urgent.
Mangiferin (mangiferin), also known as mangiferin, chinonin, is a polyphenol compound with molecular formula C19H18O11Relative molecular weight 422. Mangiferin is mainly separated from leaves, fruits and barks of Mangifera indica and almond trees of Anacardiaceae, Gentiana manshurica and swertia mussotii of Gentianaceae, pyrrosia lingua of Hylocereus plant, and has been separated from vegetables in recent years. With the continuous and deep research on mangiferin, various pharmacological activities of relieving cough and asthma, eliminating phlegm, resisting inflammation and pain, protecting liver and benefiting gallbladder, resisting lipid peroxidation, resisting cancer, resisting diabetes, resisting bacteria and the like are continuously developed. However, no report of resisting aquatic animal viruses such as spring viremia of carp virus and channel catfish virus is found, so that the inventor researches whether mangiferin has the effect of resisting the spring viremia of carp virus and channel catfish virus, finds that the mangiferin can obviously inhibit the replication of the two viruses, is safe and nontoxic to cells under effective concentration, and has great significance in developing anti-aquatic virus drugs.
Disclosure of Invention
The invention aims to provide application of mangiferin in preparing medicaments for treating or preventing spring viraemia of carp, and comprises application of mangiferin in inhibiting spring viraemia of carp in vitro.
The invention also aims to provide application of mangiferin in preparing a medicament for treating or preventing channel catfish virus diseases, and the application of mangiferin in inhibiting channel catfish virus in vitro.
The last purpose of the invention is to provide the application of mangiferin in preparing medicines for simultaneously treating or preventing the spring viremia of carp virus disease and the channel catfish virus disease.
In order to achieve the purpose, the invention adopts the following technical measures:
the mangiferin is used for carrying out cell level antiviral experiments, FHM cells and CCO cells are respectively pretreated by using the mangiferin with a certain concentration, the carp spring viremia virus with 0.1MOI and channel catfish virus are infected, free viruses are washed away, the mangiferin with the corresponding concentration is continuously added, and the replication level of the viruses is detected after 24 hours. The results show that after the cells are treated by mangiferin, the replication levels of the two viruses are remarkably reduced, and the mangiferin has the effect of inhibiting aquatic viruses such as spring viremia of carp virus and channel catfish virus. Cytotoxicity experiments show that mangiferin with the used concentration has no toxic or side effect on FHM and CCO cells, and can be used as a therapeutic agent and a prophylactic agent for spring viremia of carp virus diseases and channel catfish virus diseases. Compared with the prior art, the invention has the following advantages:
1. the mangiferin can be used for preventing and treating viruses in the field of aquatic products for the first time, and a new treatment way and means are provided for treating spring viremia of carp virus diseases and channel catfish virus diseases.
2. Mangiferin can inhibit replication of SVCV and CCV, and is nontoxic to cells, and is a low-toxicity and high-efficiency antiviral drug.
3. By utilizing the modern common pharmaceutical preparation means, mangiferin can be used as an active ingredient to prepare any pharmaceutically acceptable dosage form such as tablets, capsules, granules, oral liquid, sustained release preparations, controlled release preparations, nano preparations, injections and the like.
Drawings
FIG. 1 shows the toxicity test results of different concentrations of mangiferin on FHM cells in example 1 of the present invention.
FIG. 2 shows the results of SVCV inhibition experiments using different concentrations of mangiferin in example 2 of the present invention.
FIG. 3 shows the results of toxicity test of different concentrations of mangiferin on CCO cells in example 1 of the present invention.
FIG. 4 shows the results of CCV inhibition experiments with different concentrations of mangiferin used in example 3 of this invention.
Detailed Description
The application of mangiferin of the present invention is specifically illustrated by the following examples. These examples are merely illustrative of the invention and are not intended to be limiting. The technical scheme of the invention is the conventional technology in the field if not particularly stated.
Example 1:
experiment of cytotoxicity of mangiferin
The MTT method is adopted to detect the toxicity of the medicine, and the specific method comprises the following steps: collecting Fat head carp cell (FHM) cells or CCO cells (channel catfish ovarian cells) in logarithmic growth phase, adjusting the concentration of cell suspension, adding the cell suspension into a 96-well plate, adding 200 mu L of culture solution into each well, wherein the cell density is 1000-; 5% CO2Incubating at 28 ℃ until cells adhere to the wall (about 6-8h), adding mangiferin with concentration gradient, setting 6 multiple holes for each gradient, incubating for 48h, and observing the cell state under an inverted microscope; adding 20 μ L MTT solution (5mg/ml, i.e. 0.5% MTT) into each well, continuing to culture for 4h, terminating the culture, and carefully sucking up the culture solution in each well; adding 150 μ L dimethyl sulfoxide into each well, placing on decolorizing shaker, oscillating at low speed for 10min to dissolve the crystal completely, measuring absorbance of each well at OD490nm of ELISA detector, and calculating cell survival rate.
The experiment was also set with zero wells (medium, MTT, DMSO) and control wells (cells, solvent of corresponding concentration, medium, MTT, DMSO).
The results are shown in fig. 1, the cell survival rate of FHM cells treated by mangiferin with various concentrations for 24h in the experiment has no significant change compared with the control group, which indicates that mangiferin with the used concentration has no toxic effect on FHM cells.
The results are shown in fig. 3, the cell survival rate of CCO cells treated by mangiferin concentrations for 24h in the experiment has no significant change compared with the control group, which indicates that mangiferin concentrations used have no toxic effect on CCO cells.
Example 2:
the application of mangiferin in preparing medicaments for treating or preventing spring viremia of carp virus comprises the following steps:
inoculating FHM to a 12-well plate, absorbing and removing the culture solution after a monolayer of 70-80% grows, adding complete culture solution containing 100uM, 50uM, 25uM, 12.5uM mangiferin and PBS for pretreatment for 6h, absorbing cells by using 100 mu L of SVCV of 0.1MOI, incubating for 1h at 28 ℃, washing free viruses, continuously adding complete culture medium containing mangiferin and PBS at corresponding concentrations, and continuously culturing for 24h in a 28 ℃ and 5% CO2 incubator (three independent repeat groups are arranged for each concentration treatment). And collecting cells after 24h to extract total RNA, and performing fluorescent quantitative detection on the SVCV-G gene expression level after reverse transcription. The primer sequence for quantitatively detecting the SVCV-G gene is TGCTGTGTTGCTTGCACTTATYT/TCAAACKAARGACCGCATTTCG. SVCV codifies 5 structural proteins, such as nucleoprotein (N), phosphoprotein (P), membrane protein (M), glycoprotein (G), and RNA-dependent RNA polymerase (L-protein), wherein the glycoprotein encoded by the G gene determines the host range of SVCV, is related to virus virulence, and is often used as a target for detecting viral load.
As shown in FIG. 2, compared with the control group, the expression of SVCV-G gene was significantly reduced (P < 0.05) at a mangiferin concentration of 25uM, which was 31.38% lower than the control group. When the mangiferin concentration is 50uM and 100uM, the gene expression level is greatly reduced (P is less than 0.01), and 36.57% and 67.42% are respectively reduced compared with the control group. Indicating that mangiferin can inhibit the replication of SVCV and has concentration dependence.
Example 3:
the application of mangiferin in preparing the medicine for treating or preventing channel catfish virus diseases comprises the following steps:
inoculating CCO cells into a 12-well plate, sucking and removing culture solution after a monolayer of 70-80% grows, adding complete culture solution containing 100uM, 50uM, 25uM, 12.5uM mangiferin and PBS for pretreatment for 6h, sucking and removing the culture solution, adsorbing the cells by using CCV 100 uL of 0.1MOI, incubating for 1h at 28 ℃, washing free viruses, continuously adding complete culture medium containing mangiferin and PBS at corresponding concentrations, and continuously culturing for 24h in an incubator of 28 ℃ and 5% CO2 (three independent repeat groups are arranged for each concentration treatment). And collecting cells after 24h to extract total RNA, and performing fluorescent quantitative detection on the CCV-ORF46 gene expression level after reverse transcription. The primer sequence for quantitatively detecting the CCV-ORF46 gene is GTCTATTTTGGAGGCTGC/ATTCGGGTTATCGGTGGC. CCV-ORF46 encodes the major structural protein of CCV, which is involved in viral entry and is often used as a target for viral load detection.
The results are shown in fig. 4, compared with the control group, the replication level of CCV is significantly reduced (P < 0.05) when the mangiferin concentration is 50uM, and is reduced by 41.43% compared with the control group, and the replication level of CCV is very significantly reduced (P < 0.01) when the mangiferin concentration is 100uM, and is reduced by 76.07% compared with the control group. The result shows that after the mangiferin is used for treating the CCO cells, the CCV (cell-mediated isothermal amplification) resistance effect of the cells can be obviously improved, and the concentration dependence is realized.
The experiments prove that mangiferin obviously reduces the replication level of spring viremia of carp virus and channel catfish virus, and is safe and nontoxic to FHM cells and CCO cells. The mangiferin can be used for preparing medicines for preventing and treating spring viremia of carp or/and channel catfish virus diseases.
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| CN111166737B (en) * | 2019-10-21 | 2022-04-01 | 江苏农牧科技职业学院 | Application of mangiferin |
| CN114533799B (en) * | 2022-03-17 | 2023-03-14 | 湖南师范大学 | Application of callicarpa nudiflora in antiviral aspect of freshwater fishes |
| CN116617238B (en) * | 2023-05-29 | 2024-08-09 | 中国科学院水生生物研究所 | Application of Chinese herbal medicine saffron glycoside I in fish antiviral |
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| CN101991026B (en) * | 2010-11-24 | 2012-02-29 | 广州海因特生物技术有限公司 | Complex immunity reinforcing agent for improving stress resistance of black carp and preparation method thereof |
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Non-Patent Citations (3)
| Title |
|---|
| Mangiferin activates Nrf2-antioxidant response element signaling without reducing the sensitivity to etoposide of human myeloid leukemia cells in vitro;Ben-ping Zhang等;《Acta Pharmacologica Sinica》;20131230;第35卷;257-266 * |
| Up-regulation of nuclear factor E2-related factor 2 upon SVCV infection;Yi Yang等;《Fish & Shellfish Immunology》;20140717;第40卷;245-252 * |
| 芒果苷药理研究进展;廖洪利 等;《天津药学》;20050430;第17卷(第2期);50-52 * |
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