Application of isorhamnetin in treatment of influenza

A technology of isorhamnetin and aspect, applied in the application field of isorhamnetin in the treatment of influenza, can solve problems such as unreported, and achieve the effect of improving inflammation and deterioration

Pending Publication Date: 2022-03-11

THE FIRST AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV (GUANGZHOU RESPIRATORY CENT)

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AI-Extracted Technical Summary

Problems solved by technology

[0003] Isorhamnetin is a monomeric component of flavonoids. It is known that it has anti-inflammatory effects in various inflammatory diseases such as osteoarthritis, periodontitis and inflammatory bowel disease. However, thi...

Abstract

The invention relates to the technical field of medicines, in particular to application of isorhamnetin in the aspect of treating influenza. The invention provides a new application of isorhamnetin, namely the isorhamnetin is used for treating influenza, and the isorhamnetin can inhibit the replication of various subtype influenza viruses, has a remarkable curative effect on influenza, and can be applied to the treatment and prevention of influenza; the invention further provides another new application of the isorhamnetin, namely, the isorhamnetin is used for improving the inflammatory reaction caused by the influenza virus, and the isorhamnetin can inhibit abnormal expression of cell inflammatory factors mediated by the influenza virus and can be used for treating the inflammatory reaction caused by the influenza. The invention also provides another new application of the isorhamnetin, namely the isorhamnetin is used for treating influenza virus mediated inflammation amplified by the I-type interferon, so that the problem of inflammation deterioration caused by the I-type interferon is solved.

Application Domain

Organic active ingredientsAntivirals +1

Technology Topic

Influenza (flu)Molecular biology +6

Image

Examples

Experimental program(2)

Example Embodiment

[0032] Example 1 Isorhmnetin inhibition of influenza virus replication

[0033] 1. Experimental Materials

[0034] Cell: dog kidney cells (Madin Darby Canine Kidney, MDCK) cells

[0035] Virus: Influenza A virus strain PR8 (A / PR / 8/34, H1N1)

[0036] Drug and Reagent: fetal bovine serum (GIBCO, USA); MEM medium (GIBCO, USA); PBS (GIBCO, USA); of TPCK trypsin (Sigma, USA); the MTT kit (Sigma, USA); DMSO (U.S. Sigma); positive control oseltamivir, commercially available from pharmaceutical companies Bi; isorhamnetin.

[0037] 2. Drug Toxicity Test (MIT method)

[0038] MDCK cells taking the logarithmic growth phase were seeded into 96-well plates, each well of about 2.5 × 10 4 A, 24h until the cell monolayers grown, the medium was discarded, adding different dilutions of drug 100μL / hole, and normal cells blank control wells were added 100μL / hole MEM, at 37 [deg.] C and 5% CO 2 After culture was continued from 36 to 48 hours, each well was added a solution of MTT (5mg / mL) 20μL, at 37 ℃ with 5% CO 2 Incubator incubation was continued for 4 hours. Aspirate culture supernatant was added to each well 100μL dimethylsulphoxide (DMSO) low shaken for 10 minutes, the crystals were fully thawed. Select 490nm wavelength, absorbance of each well was measured by enzyme-linked immunoassay monitor. Inhibitory ratio was calculated, and the calculated 50% cytotoxic concentration of the drug half toxic concentration (the TC50) by Reed-Muench method.

[0039] The above-described inhibitory rate = [(mean OD value of control group - mean OD value of blank group) - (mean OD value of treatment group - mean OD value of blank group)] / (mean OD value of control group - mean OD value of blank group) × 100 %.

[0040] 3. Isorhmnetin of the H1N1 influenza virus CPE has a protective effect

[0041] Are eight experimental groups, divided into A, B, C, D, E, F, G, H group; wherein, A is the MDCK cells of normal control group, B is a group HINI MDCK cells + virus, C is 20μg / ml iso rhamnetin + MDCK cells group, D is a virus HINI + 20μg / ml + MDCK cells isorhamnetin group, E is HINI virus + 10μg / ml + MDCK cells isorhamnetin group, F is HINI virus + 5μg / ml + MDCK cells isorhamnetin group, G is HINI virus + 2.5μg / ml + MDCK cells isorhamnetin group, H is HINI virus + 1.25μg / ml + MDCK cells isorhamnetin group. After 72h, it was observed under microscope.

[0042] 4. Isorhmnetin inhibit influenza virus CPE experiments

[0043] MDCK cells (2.5 × 10 per well taken in exponential growth phase 4 ) Were seeded in 96 well plates, wait until the monolayer growth medium was aspirated, by Influenza virus A / PR / 8/34 (H1N1) (100TCID 50 ) Cellular effects 2 hours, containing 0.2% TPCK different dilutions of isorhamnetin, while setting the control group (group of normal cells, simplex virus group, normal cells + Isorhamnetin group), placed in 34 ℃, 5% CO 2 Incubated for 48 hours at ambient, the degree of cytopathic effect observed.

[0044] Recording severity cells following 6 criteria: "-" normal cell growth, no lesion occurs; "±" is less than 10% of the cytopathic cell monolayers; "+" represents about a cell monolayer lesions 25% of the cells; "++" for the entire cell disease accounts for about 50% of the monolayer; "+++" for the disease accounts for about 75% of the entire cell monolayer; "++++" is about cell disease more than 75% of the cell monolayers.

[0045] Calculated using Reed-Muench Method half maximal inhibitory concentration (IC 50 ), To select the selectivity index (SI) indicates the value, the SI is defined as TC50 / IC50, to indicate that the safety of the treatment. (SI <1 represents invalid: SI: 1 ~ 2 represents a highly toxic inefficient; SI> 2 represents a low toxicity and efficient).

[0046] 5. Experimental results

[0047] Cytotoxicity isorhamnetin: see figure 1 Half toxic concentration (the TC50) as measured by MTT test drug was 19.13μg / ml.

[0048] Isorhamnetin on H1N1 influenza virus CPE has a protective effect: A group of cells microscope sharp edges, clear boundary, are well adherent growth; Group B round cell swelling, accumulation, broken into pieces and other CPE off phenomena; compared to group B, C, and E ~ H group showed significantly high cell viability, with an increase in the concentration of isorhamnetin, cell fragmentation, loss and other CPE phenomenon gradually weakened, showing a dose-dependent manner. Visible when a dose of 1.25μg / ml and 2.5μg / ml CPE phenomenon significantly reduced. Isorhamnetin calculated IC50 of 2.65μg / ml, SI of 7.2.

[0049] Isorhamnetin inhibit influenza virus Pharmacodynamic: The results are shown Isorhmnetin inhibit the influenza virus A / PR / 8/34 (H1N1), IC50 of 2.65μg / ml, SI of 7.2.

Example Embodiment

[0050] Example 2 Inflammatory response caused by deflated viruses

[0051] Experimental material

[0052] Drug: 异李素.

[0053] Cell, virus: human lung adenocarcinoma 549 cell line (A549), H1N1 influenza virus (A / PR / 8/34, H1N1)

[0054] Reagents: DMEM medium; PBS, fetal bovine serum is purchased from GIBCO; TPCK is purchased from Guangzhou Jiejis; REAT-TIME PCR reagent purchases from Takarra, Cat # RR390A; Ripa lysl solution is purchased from US Sigma.

[0055] instrument:

[0056] Heracell 150i Constant temperature CO2 incubator, US THERMO;

[0057] BHC-1300IIA / B3 Secondary Bios Safety Cabinet, Suzhou Purification Equipment Co., Ltd .;

[0058] Leica DM3000 inverted microscope, Leica;

[0059] TGL-16M high-speed desktop frozen centrifuge, Germany Eppendorf;

[0060] Avanti J-26 high-speed frozen centrifuge, US Beckman Coulter;

[0061] Sigmaj-26 frozen centrifuge, Germany Sigma;

[0062] BS115-ESHK01 horizontal shaker, Taicang Science and Education Equipment Factory;

[0063] Al201 Electronic Balance, METTLER TOLEDO EQ (Shanghai) Co., Ltd.

[0064] Du800 UV and visible light shifting brightness meter, USBECKMAN;

[0065] JY-1000M electric heating constant temperature drum drying box, Wujiang Zhisheng oven equipment factory;

[0066] MultiScan MK3 type enzyme label, US THERMO;

[0067] Real-Time PCR, ABI7500;

[0068] Fluorescence microscope.

[0069] 2. Real-time fluorescent quantitative PCR experiment method

[0070] 3 × 10 per hole 5 / ml density A549 cell concentration inoculated into a 96-well cell plate, 2 ml of cell suspension per well was placed in an incubator, and cultured 24 hours to remove cell episodes after the cellular wall, add PBS to wash twice, add an epitudic flu Virus H1N1 (A / PR / 8/34 (H1N1) was incubated at 37 ° C for 2 hours. Add in TC 50 Different concentrations (10 μg / ml, 5 μg / ml) were continued to cultivate 24h, then discarded the cell culture supernatant in 6-well plates, washed twice with cold PBS, add Trizol reagent to extract RNA, followed by reverse The transcription kit reverses 500 ng of RNA into cDNA, using quantitative PCR to analyze the analysis of inflammatory factors, and determine the mRNA content as internal paramers, ultimately adopt 2 -ΔΔCt Formula Analysis of the expression of each sample of each sample. QPCR reaction conditions: denaturation 95 ° C, 30s; annealing 95 ° C, 5S (40 cycles); extension 60 ° C, 40S.

[0071] The primer sequence and probe sequences of the inflammatory factors used in real-time quantification of PCR reactions are as follows: primer name primer sequence (5 '- 3').

[0072] 3. Experimental results

[0073] See the results image 3 As shown, after the murray (10,5 μg / ml) intervention significantly inhibited the gene transcription level of the H1N1 influenza virus-induced inflammatory factor (TNF-α, IP-6, IP-10, IFN-α), which was present. Relational relationship; suggesting that 异鼠素干干流应应应应应应应应应应应应应应应

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