CN104814973B - Application in the drug of diseases associated with inflammation of the Cappariloside A in preparation for treating influenza and influenza virus mediation - Google Patents

Abstract

The present invention provides Cappariloside A and is preparing the application in the drug for treating influenza or the diseases associated with inflammation mediated by influenza virus, provides a kind of new way for clinical treatment influenza virus property disease.The Cappariloside A has the function of that the progeny virus duplication for inhibiting influenza virus and the inflammatory factor for inhibiting influenza virus to mediate generate.The present invention also provides the drugs for treating influenza or the inflammation mediated by influenza virus, its active constituent of the drug includes previously described Cappariloside A.Cappariloside A has the characteristics that safe and effective, toxic side effect is small in terms for the treatment of influenza virus property disease.

Description

Cappariloside A is mediated in preparation for treating influenza and influenza virus Application in the drug of diseases associated with inflammation

Technical field

The invention belongs to pharmaceutical technology fields, and it is susceptible in preparation anti-current that the invention further relates to Cappariloside A Application in the drug of cytotoxic drug and the diseases associated with inflammation in preparation for treating influenza virus mediation.

Background technique

After influenza infection human body, other than viral caused damage itself, virus can also cause being immunized for host Reaction, discharges a large amount of inflammatory cell and inflammatory factor, causes inflammation storm, the damage of host is further aggravated.It grinds Study carefully and show: after influenza infection people, especially people infected with bird flu virus, inflammatory factor is horizontal significantly raised in blood.It is scorching The adjusting imbalance of inflammation factor is related to disease severity caused by influenza virus and Clinical Outcome.Also some researches show that inflammation because Son adjusts imbalance and has aggravated the pathogenic of H5N6, H7N9 et al. infection avian influenza virus, leads to acute respiratory distress syndrome (ARDS).Therefore now for disease treatment caused by influenza infection, in addition to antiviral other than itself, the inflammatory immune of host Adjusting also receives attention.The drug for being clinically used for the inflammatory reaction for inhibiting influenza virus to mediate at present is glucocorticoid, but It is the big using side effect of hormone, clinical use has dispute, and hormone can only adjust host itself, to virus itself without controlling Treatment effect.Many Chinese medicines have all been proved to have immunoregulation effect, and Radix Isatidis is one such Chinese medicine, have had research to demonstrate,prove The immune function of the adjustable host of effective component in bright Radix Isatidis, while the effective component of Radix Isatidis is also proved to press down The duplication of influenza virus processed.Therefore, influenza virus can be inhibited and inhibit influenza sick by separating to prepare and filter out from Radix Isatidis The effective component for the inflammatory reaction that poison mediates, finds its unique Pharmacodynamical mechanism, and obtain independent intellectual property right, for excavating ancestral State's medical treasure-house, the exploitation dynamics for increasing Chinese medicine are of great significance.

The structural formula of obtained Cappariloside A is separated and purified at present from northern Radix Isatidis are as follows:

At present about Cappariloside A (cappariloside A) Chemical Decomposition and Structural Identification report (such as Phytochemistry, 1999,50:1205-1208), but about Cappariloside A in resisiting influenza virus and inhibition The aborning application for the inflammatory factor that influenza virus mediates has not been reported.

Summary of the invention

The present invention provides the new opplication of Cappariloside A, i.e. Cappariloside A is in preparation for treating influenza Or it by the application in the drug of the diseases associated with inflammation of influenza virus mediation, is provided for clinical treatment influenza virus property disease a kind of new Approach.

Further, the Cappariloside A has the function of the progeny virus duplication for inhibiting influenza virus, specifically Such as inhibit the duplication of human influenza virus H1N1 (PR8 type strain) infection human airway epithelial cells (16HBE) progeny virus afterwards；Also have There is the inflammatory factor for inhibiting influenza virus to mediate to generate, it is specific as inhibited human influenza virus H1N1 (PR8 type strain) sense Contaminate mouse macrophage (RAW264.7) afterwards inflammatory factor albumen generate.The present invention also further provides for Cappariloside Application of the A in the drug that preparation has above-mentioned effect.Inflammatory factor includes but is not limited to IP-10, MIG, CCL-5, CCL-5/ RANTES。

The influenza virus includes but is not limited to first, influenza B virus and avian influenza virus；First, the influenza B disease Poison and avian influenza virus include but is not limited to human influenza virus H1N1 hypotype, H3N2 hypotype and INF Type B, further include bird flu Viral H7N3, H9N2 hypotype, human influenza virus's H1N1 hypotype includes new H1N1virus.

Further, the Cappariloside A can be from extracted form natural plant or chemically synthesized, the natural plant Blue, dimension medicine caper of for example northern plate of object etc..

Further, the drug can be made into any dosage form pharmaceutically allowed, and dosage form is, for example, tablet, capsule etc..

The present invention also provides a kind of for treating the drug of influenza or the inflammation mediated by influenza virus, its work of the drug Property ingredient includes previously described Cappariloside A.

The present invention provides a kind of drug safe and effective, toxic side effect is small for clinical treatment influenza virus property disease.

The present invention verifies the Cappariloside A for extracting from northern plate indigo plant using cytopathic-effect inhibition assay in the therapeutic mode And the inhibiting effect of chemically synthesized Cappariloside A infected by influenza duplication, it is positive right with Oseltamivir phosphate According to medicine.The inhibiting effect for observing toxicity and infected by influenza duplication of the Cappariloside A to mdck cell, utilizes MTT Method measures the toxic concentration (TC of half for extracting from the Cappariloside A of northern Radix Isatidis₅₀) it is 11.05mg/mL, chemical synthesis Cappariloside A the toxic concentration (TC of half₅₀) it is 2.5mg/ml.Cappariloside A extract is in prevention mould There are inhibiting effect, medium effective concentration (IC to Flu-A H1N1 (PR8) under formula and treatment mode₅₀) it is respectively 1.04mg/ ML and 5.2mg/mL, in the direct, mode in vain.And synthetics are only in the therapeutic mode to Flu-A H1N1 (PR8) There are inhibiting effect, medium effective concentration (IC₅₀) it is 0.743mg/mL, and without effect under avoidance mode.

The present invention acts on what the inflammatory factor that host cell inhibits influenza virus to mediate generated to Cappariloside A Effect is further explored, and stimulates people using the technique study Drug inhibition influenza virus of Real-time PCR and ELISA The expression of human airway epithelial cells (16HBE) inflammatory factor afterwards.Cappariloside A can reduce influenza virus as the result is shown The mRNA level in-site of chemotactic factor (CF) (IP-10, MIG, CCL-5 etc.) after H1N1 (PR8) is stimulated 16HBE cell 6 hours, and can reduce Influenza virus H1N1 (PR8) stimulate 16HBE cell 24 hours after chemotactic factor (CF) (IP-10, MIG, CCL-5 etc.) mRNA level in-site and Protein level.As a result it is also shown in the progeny virus that can have both inhibited influenza virus when drug concentration is 2mg/mL and 1mg/mL Duplication, the expression for the inflammatory factor that influenza virus can also be inhibited to mediate；The convection current when concentration is 0.5mg/ml and 0.25mg/mL The no inhibiting effect of the duplication of Influenza Virus, but can effectively inhibit the expression of inflammatory factor.For macrophage (RAW264.7), the inflammatory factor that drug can effectively inhibit influenza virus to mediate when concentration is 1mg/mL and 0.5mg/mL The expression of the protein level of (IP-10 and CCL-5/RANTES).

Detailed description of the invention

Fig. 1 is embodiment 2Cappariloside A chemical synthesis procedure chart；

Fig. 2A-Fig. 2 G is that drug presses down human airway epithelial cells (16HBE) drug toxicity and drug when acting on 24 hours The duplication of influenza virus progeny virus processed and the expression of the inflammatory factor mRNA for inhibiting influenza virus to mediate and albumen；

Fig. 3 A- Fig. 3 C is the expression for the inflammatory factor mRNA that drug inhibits influenza virus to mediate when acting on 6 hours.

Fig. 4 A- Fig. 4 F is that drug to macrophage (RAW264.7) drug toxicity and after drug effect 24 hours inhibits influenza The protein expression of virus-mediated inflammatory factor.

Specific embodiment

To be best understood from the present invention, the content of present invention is described further combined with specific embodiments below, but the present invention Protection content be not limited to following instance.

1 Cappariloside A monomer component of embodiment isolates and purifies, Structural Identification

Cappariloside A is isolated and purified using the existing isolation and purification method of the art, below to its point It is described below for reference from purification process: Radix Isatidis methanol extract is dissolved in water, use ethyl acetate, extracting n-butyl alcohol respectively, just Butanol fraction collects ethanol elution part through Sephadex LH-20 pillar layer separation through the further desugar of macroreticular resin D101, Fr.1~Fr.6 is obtained with methanol-water (0:100-100:0, volume ratio) gradient elution.Fr.3 is further pure through preparative HPLC Change obtains the crystallization of white powder sample.

The crystallization is as a result as follows through NMR, ESI-MS wave spectrum analysis:

ESIMS m/z:357.1058[M+Na]⁺(calcd for C₁₆H₁₈N₂O₆:334.1058).1H-NMR(DMSO- D6,400MHz) δ: 11.07 (1H, br, H-1), 7.19 (1H, s, H-2), 6.68 (1H, d, J=6.0Hz, H-5), 7.02 (1H, Br, H-6), 6.99 (1H, d, J=6.4Hz, H-7), 4.16 (2H, d, J=18.8Hz, H-8), 4.88 (1H, d, J=7.6Hz, β-glu-1 '), 3.36~3.80 (5H, m, β-glu-2 '~β-glu-6 ')；13C-NMR(DMSO-d6,100MHz)δ:123.3 (C-2), 104.2 (C-3), 117.1 (C-3a), 152.3 (C-4), 103.8 (C-5), 123.0 (C-6), 106.5 (C-7), 138.4(C-7a),15.4(C-8),120.7(C-9),101.8(β-glu-1’),74.0(β-glu-2’),77.2(β-glu- 3'),70.3(β-glu-4'),77.5(β-glu-5'),61.2(β-glu-6').Structural Identification are as follows: Cappariloside A, The structure referred in its structure and background technique above is consistent.

The chemical synthesis of 2 Cappariloside A of embodiment

A kind of method that synthesis Cappariloside A is provided below for reference, is prepared in accordance with the following steps Cappariloside A (being mass percentage if the percentage composition occurred in the embodiment is not illustrated):

1) N is prepared, N- dimethyl (4- benzyloxy -1H- indol-3-yl) methylamine (corresponds to compound 2 in Fig. 1)

In 1000mL round-bottomed flask be added 1,4- dioxane (100mL), glacial acetic acid (100mL) and 37% hydration first 40% dimethylamine agueous solution and Isosorbide-5-Nitrae-dioxy six of 4- benzyloxy indole is successively added dropwise at 0 DEG C in aldehyde (3.83mL, 50.5mmol) Ring solution (11.16g is dissolved in 100mL Isosorbide-5-Nitrae-dioxane) after being added dropwise, is warmed to room temperature stirring 36 hours.To reaction solution Middle addition 500mL water adjusts pH value between 8-9 with 10% sodium hydrate aqueous solution, the white precipitate of precipitation is filtered, filter Cake is successively washed with water and ethyl acetate, obtains white solid 13.2g, yield 94%.

It is as a result as follows through detecting:¹H NMR(400MHz,DMSO)δ10.91(s,1H),10.91(s,1H),7.56(d,J =7.1Hz, 2H), 7.41 (t, J=7.2Hz, 2H), 7.34 (d, J=7.0Hz, 1H), 7.04 (s, 1H), 6.95 (s, 2H), 6.55(s,1H),5.15(s,2H),3.63(s,2H),2.07(s,6H).

¹³C NMR(126MHz,DMSO)δ153.68,138.40,138.13,128.75,128.04,127.97,123.80, 122.05,177.83,112.14,105.47,100.69,69.53,55.30,45.10.

MS m/z(ESI)289.1[M+Na]⁺

2) 2- (4- benzyloxy -1H- indol-3-yl) acetonitrile (corresponding to compound 4 in Fig. 1) is prepared

Compound 2 (6.61g, 28.25mmol) is dissolved in the in the mixed solvent (v/v=8/15) of methylene chloride and toluene 230mL is added dropwise iodomethane (7.02mL, 56.49mmol), is stirred at room temperature 12 hours.It is evaporated reaction dissolvent, is dissolved the residue in Trimethylsilyl cyanide (4.91mL, 42.37mmol) and tetrabutyl ammonium fluoride solution 85mL (1N is successively added dropwise in 130mL tetrahydrofuran In THF, 85mmol), it is stirred at room temperature 12 hours.It is evaporated reaction dissolvent, the dissolution of 200mL methylene chloride is added, is washed with water (100mL), anhydrous sodium sulfate is dry, is spin-dried for solvent, and silica gel column chromatography purifies (petroleum ether: ethyl acetate=2:1), obtains product 2.9g, yield 47.4%.

It is as a result as follows through detecting:¹H NMR (400MHz, DMSO) δ 11.11 (s, 1H), 7.57 (d, J=7.5Hz, 2H), 7.42-7.31(m,3H),7.23(s,1H),7.05–6.93(m,2H),6.65–6.52(m,1H),5.21(s,2H),4.05(s, 2H).

¹³C NMR(126MHz,DMSO)δ153.10,138.47,128.86,128.11,127.84,123.35,123.07, 120.33,116.62,105.77,104.08,101.11,69.67,15.67.

MS m/z(ESI)271.0[M+Na]⁺

3) 2- (4- hydroxyl -1H- indoles l-3- yl) acetonitrile (corresponding to compound 5 in Fig. 1) is prepared

Compound 4 (2.62g, 10mmol) is dissolved in 50mL methanol, 10% palladium carbon (0.26g) is added, it is big with one The hydrogen reducing of air pressure 10 hours.Palladium carbon is filtered out, reaction dissolvent is evaporated, obtains product 1.36g, yield 80%.

It is as a result as follows through detecting:¹H NMR(400MHz,DMSO)δ10.91(s,1H),9.52(s,1H),7.13(s, 1H), 6.92-6.76 (m, 2H), 6.35 (d, J=7.4Hz, 1H), 4.04 (s, 2H)

¹³C NMR(126MHz,DMSO)δ151.95,138.90,123.16,122.60,120.47,116.24,104.11, 103.69,103.65,15.47.

MS m/z(ESI)195.0[M+Na]⁺

4) it prepares 4- (2 ', 3 ', 4 ', 6 ' -- four acetoxyl group-α-D- glucopyranoses) indole -3-acetonitrile and (corresponds to Fig. 1 Middle compound 6)

By compound 5 (1.72g, 10mol), tetra- acetoxyl group-alpha-D- glucopyranose bromide of 2,3,4,6- (8.2g, 20mmol), benzyl tributyl ammonium chloride (0.6g, 2mmol) and potassium carbonate (6.9g, 50mmol) are dissolved in water and chloroform is mixed It closes in solution (v/v=1/10,44mL), is stirred at room temperature.TLC is monitored after raw material fully reacting (about 24 hours), is added dropwise 10% Salt acid for adjusting pH value separates organic layer to 7, is successively washed with saturated sodium bicarbonate (30mL) and saturated salt solution (30mL) organic Layer, is evaporated reaction dissolvent, and column chromatography purifies (petroleum ether: ethyl acetate=1:1), obtains product 4.3g, yield 83%.

It is as a result as follows through detecting:¹H NMR (400MHz, DMSO) δ 11.20 (s, 1H), 7.26 (d, J=2.3Hz, 1H), 7.09-7.01 (m, 7.6Hz, 2H), 6.64 (d, J=6.9Hz, 1H), 5.76 (d, J=7.9Hz, 1H), 5.47 (t, J= 9.5Hz, 1H), 5.22 (dd, J=9.6,8.0Hz, 1H), 5.02 (t, J=9.7Hz, 1H), 4.38-4.25 (m, 1H), 4.21 (m,1H),4.06(m,1H),3.92(s,2H),2.01(m,12H).

¹³C NMR(126MHz,DMSO)δ170.36,170.07,169.94,169.81,150.35,138.58,124.15, 122.72,120.10,116.56,107.23,103.69,103.28,96.56,72.74,71.38,71.16,68.68, 62.14,20.97,20.89,20.87,20.76,14.98.

MS m/z(ESI)525.1[M+Na]⁺

5) 2- (4- ((2R, 3S, 4S, 5S, 6R) -3,4,5- trihydroxy -6- (methylol)-tetrahydro -2H- pyrans)-is prepared 1H- indol-3-yl) acetonitrile (corresponding to compound 7 in Fig. 1)

Compound 6 (1g, 2mmol) is dissolved in the 12mL mixed solvent of tetrahydrofuran, first alcohol and water (v/v/v=2/3/1) In, 0.5g lithium hydroxide is added, reacts at room temperature 12 hours.After TLC monitors raw material fully reacting, it is evaporated reaction dissolvent, silicagel column Column chromatography purifies (petroleum ether: ethyl acetate=1:10), obtains product 0.32g, yield 50%.

Through detecting, product is Cappariloside A, and testing result is as follows:¹H NMR(400MHz,DMSO)δ11.08 (s, 1H), 7.20 (s, 1H), 7.09-6.90 (m, 2H), 6.69 (d, J=6.8Hz, 1H), 5.31 (d, J=5.5Hz, 1H), 5.11 (d, J=4.6Hz, 1H), 5.03 (d, J=5.1Hz, 1H), 4.90 (d, J=7.7Hz, 1H), 4.59 (t, J=5.6Hz, 1H),4.29–3.83(m,2H),3.75-3.71(m,1H),3.53-3.47(m,1H),3.41–3.35(m,1H),3.31–3.17 (m,1H).

¹³C NMR(126MHz,DMSO)δ152.34,138.45,123.28,123.01,120.63,117.16,106.53, 104.26,103.86,101.84,77.56,77.24,74.04,70.32,61.28,15.39.

MS m/z(ESI)357.1[M+Na]⁺

Test example, which is provided below, confirms new effect possessed by Cappariloside A, for furtheing elucidate The present invention, and be not meant to limit the scope of the invention.

In-vitro Inhibitory Effect of the 1 Cappariloside A of test example to different subtype influenza virus

1.1. material

1.1.1 U.S. GIBCO company, article No. Y0001337 Oseltamivir phosphate: drug-positive comparison medicine: are purchased from；Experiment Drug -- Cappariloside A: Radix Isatidis raw medicinal herbs is provided by Guangzhou Baiyunshan Heji Huangpu Chinese Medicine Co., Ltd., is picked up from black Longjiang grand celebration and Fuyang isatis root GAP base are accredited as woaded blue by Chinese Academy of Sciences south China plant Yuan Yehua state researcher The root of Isatis infigotica Fort., i.e. Radix Isatidis.It is extracted according to embodiment 1, purifies, identify, is obtained Cappariloside A extract.Cappariloside A chemical synthetic drug is made according to embodiment 2.

1.1.2 cell dog kidney cells (MDCK) is quoted from the American Type Culture Collection committee, Chinese Academy of Sciences cell bank

1.1.3 strains of influenza viruses H1N1virus PR8 plants (A/PR/8/34, H1N1), A type H3N2 influenza virus Aichi plants (A/Aichi/2/68, H3N2) are purchased from American classic culture collecting center (ATCC)；New Influenza A H1N1 disease Strain (A/Guangzhou/GIRD07/09, H1N1, Genebank No.HM014332.1), influenza B virus

It (B/Guangzhou/GIRD08/09) is this room clinical separation strain, influenza A virus H7N3 (A/Duck/ Guangdong/1994, H7N3), H9N2 (A/Chicken/Guangdong/1996, H9N2) is by Agricultural University Of South China's veterinary science Institute professor Chen Jianxin give.Above-mentioned strain influenza virus expands in the chick embryo allantoic cavity by being inoculated in 9 ages in days, 35 DEG C of incubations 2d harvests allantoic fluid.Virus is titrated with mdck cell, the half infection of these strains is determined with cytopathic-effect inhibition assay (CPE) Measure (TCID₅₀/ 100 μ L) as viral original titer.

1.1.4 reagent D MSO (SIGMA company, the U.S.)；MEM (GIBCO company, the U.S.) lot number: 8114414；Fetal calf serum (GIBCO company, the U.S.) lot number: 10099；PBS (GIBCO company, the U.S.) lot number: 20150122；1mg/ml TPCK lot number: 20140220 purchased from this Biotechnology Co., Ltd of prompt times of Guangzhou.MTT (3- (4,5- dimethylthiazole -2) -2) is purchased from Genebase。

1.2. experimental method

1.2.1 drug dissolves Cappariloside A, (extract and chemical synthetic drug including extracting from northern Radix Isatidis Object) with DMSO dissolution be configured to working stocks (concentration 500mg/mL), set 4 DEG C of preservations；Positive drug Oseltamivir phosphate is used 4 DEG C of postposition preservations are filtered in the dissolution of DMEM culture solution.

1.2.2. Drug toxicity trails (mtt assay)

By every hole about 2.5 × 10⁴Mdck cell is inoculated into 96 orifice plates, and rear after cells grow up to the individual layer, discards culture for 24 hours Liquid, is added 100 hole μ L/ of drug of different dilutions, and blank control and normal cell controls hole are added 100 μ L/ hole MEM, and 37 DEG C, 5%CO₂Continue culture 36-48 hours, every hole adds 20 μ L of MTT solution (5mg/mL), sets 37 DEG C, 5%CO₂Continue incubation 4 in incubator Hour.It inhales and abandons culture supernatant, every hole adds 100 μ L dimethyl sulfoxides (DMSO), low-speed oscillation 10 minutes, melts crystal sufficiently Solution.490nm wavelength is selected, each hole absorbance value is measured on enzyme linked immunological monitor.Inhibiting rate is calculated according to the following formula, and Calculating 50% toxic concentration with Reed-Muench method is the toxic concentration (TC of drug half₅₀).Inhibiting rate=[(normal to organize average OD Value-blank group mean OD value)-(administration group mean OD value-blank group mean OD value)]/(normally organize mean OD value-blank Group mean OD value) × 100%.

1.2.3. vitro Drug anti-influenza virus activity is tested

Cappariloside is studied with cytopathic-effect inhibition assay (Cytopathic Effect Reduction Method) A (including extracting from northern Radix Isatidis and chemical synthetic drug) is to the inhibiting effect of different subtype influenza virus.Cover with the 96 of single layer Mdck cell viral adsorption in porocyte culture plates 2 hours, concurrently sets positive drug and cell controls.(1) treatment mode: Various concentration drug (in addition to virus and cell controls) is added in 37 DEG C of 5%CO in cell viral adsorption 2h₂It is cultivated under environment.(2) Protected mode: cell incubation drug 2h, be added viruses adsorption 2h after, change the serum free medium of the pancreatin containing TPCK, 37 DEG C, 5%CO₂It is cultivated under environment.(3) it Direct Model: is inoculated in cell, sets after virus and 37 DEG C of incubation 1h of drug of various concentration In 4 DEG C of incubation 1h, inhales and abandon supernatant, change 37 DEG C of serum free medium, 5%CO containing TPCK₂8h is cultivated under environment.

There is lesion degree and records by following 6 grade standard in cell :-normal for cell growth, no lesion occurs；± it is cell Lesion is less than the 10% of entire cell monolayer；+ it is that cytopathy accounts for about the 25% of entire cell monolayer；++ account for about for cytopathy The 50% of entire cell monolayer；+++ it be the 75%:++++ that cytopathy accounts for about entire cell monolayer is that cytopathy accounts for about entirely 75% or more of cell monolayer.With Reed-Muench method calculation of half inhibitory concentration (IC₅₀), and to select index SI to indicate (SI =TC₅₀/IC₅₀), SI > 2 indicates low toxicity efficiently；SI:1~2 indicates that high poison is inefficient；SI < 1 indicates invalid.

1.3. experimental result

1.3.1 cytotoxicity

Toxic concentration (the TC of half of test medicine (extracting from northern Radix Isatidis) is measured using mtt assay₅₀) it is 11.05mg/mL； Toxic concentration (the TC of the half of test medicine (chemical synthetic drug)₅₀) it is 2.5mg/mL.It is shown in Table 1.

1.3.2 vitro Drug inhibits influenza virus drug effect

Cappariloside A (extract) is under avoidance mode and treatment model to influenza A virus as the result is shown H1N1 (PR8) has inhibiting effect, and acts under avoidance mode most by force, ineffective under direct binding mode；And chemical synthesis Drug only has the drug effect for inhibiting influenza virus in the therapeutic mode, without effect under avoidance mode.Further experiment result table Bright Cappariloside A (less due to extracting object amount, so completing following experiment using synthetics) is in treatment model Under to a variety of subtype influenza virus, including A type, influenza B virus, influenza A virus includes human influenza virus and bird flu Virus has inhibiting effect.See Table 2 for details.

To influenza under the external different role mode of 1 Cappariloside A of table (including extract and chemical synthetic drug) The drug effect of virus

Note: a: protected mode；B: treatment mode；C: Direct Model .NT: expression does not detect.M5:Cappariloside A Abbreviation

2 Cappariloside A of table is in vitro to different subtype influenza virus drug action

Note: selection index (SI), SI > 2 indicate low toxicity efficiently；SI:1~2 indicates that high poison is inefficient；SI < 1 indicates invalid.M5: The abbreviation of Cappariloside A

The inflammatory reaction experiment that 2 Cappariloside A of test example inhibits influenza virus to mediate in human airway epithelial cells

2.1 experimental material

2.1.1 drug given the test agent: Cappariloside A, abbreviation M5 (for the chemical synthetic drug of embodiment 2).

2.1.2 cell human airway epithelial cells (16HBE) and dog renal epithelial cell (MDCK) are purchased from Chinese Academy of Sciences Shanghai Cell bank.

2.1.3 the common H1N1virus of Strain (A/PR/8/34, H1N1) is purchased from ATCC, with Reed- Muench method measures its infection multiplicity (M.O.I), using M.O.I=1 as virus titer infection cell.

2.1.4 reagent D MEM culture medium (lot number: 8114176)；PBS (lot number: 20150122), fetal calf serum (lot number: 10099) it is purchased from GIBCO company；TPCK (lot number: 20140220) purchased from this company of prompt times of Guangzhou.ELISA kit purchase is certainly RayBio company, Cat ELH-IP-10；ELH-CCL-5.Reat-time PCR reagent is bought from TaKarRa company, Cat^# RR390A.RIPA lysate is purchased from U.S. sigma company.NP albumen primary antibody is purchased from SANTA CRUZ BIOTECHNOLOGY company (sc-80481).GADPH primary antibody is purchased from Cell Signalling company (#2118).Goat anti-rabbit igg secondary antibody and goat resist small Mouse IgG secondary antibody is purchased from U.S. KPL company.

2.2 experimental method

2.2.1 16HBE cell culture

Normal human airway epithelial cells (16HBE) are inoculated in 100mm culture dish, with 10%DMEM high glucose medium, In 37 DEG C, 5%CO₂, cultivated in cell incubation case under the conditions of saturated humidity, grow into the logarithmic proliferation phase to cell When, collect cell.After being counted to the cell of collection, with every hole 2 × 10⁵The density of a/mL is inoculated in diameter 15.6mm's Microscopically observation cell is adherent in 24 orifice plate Tissue Culture Dish, after 24 hours, well-grown.Change 1%DMEM high glucose medium Virus in next step and medicine irritation experiment are carried out after cell starvation is cultivated 24 hours, and when adding stimulant, cell culture medium is changed At the culture medium for being free of fetal calf serum.

2.2.2 virus and medicine irritation

After adherent 24 hours of cell, PBS is rinsed 2 times, with the DMEM without serum that Influenza virus H1N1 (PR8) is dilute It releases to M.O.I=1,0.5mL virus liquid is added in every hole, and 37 DEG C 2 hours adherent.Still contain 0.4ug/mL with without fetal calf serum The DMED of TPCR enzyme dilutes drug, and every hole is added 1mL, collects cell supernatant and cell after effect 6 hours or 24 hours RNA。

2.2.3 detection drug is to 16HBE cell toxicity test (mtt assay)

16HBE cell is with every hole 2 × 10⁵The density of a/mL is inoculated in the 96 orifice plate Tissue Culture Dish of diameter 6.94mm, Microscopically observation cell is adherent after 24 hours, well-grown.The DMEM2 times of gradient dilution drug without serum, effect 48 are small Shi Hou, mtt assay (with above-mentioned) detect drug toxicity.

2.2.4 the measurement (cytopathic-effect inhibition assay: CPE method) of cell supernatant virus titer

Cell supernatant carries out 10 times of gradient dilutions with the MEM of the enzyme of TPCR containing 1.5ug/mL, is added in cell, and 48 hours The lesion of cell is observed afterwards.(method is same as above).

2.2.5 inflammatory factor protein ELISA method detects in cell culture medium

It is operated according to ELISA kit product description

1., reagent take out balance to room temperature, sample is added, is incubated at room temperature 2.5 hours or 4 DEG C overnight；

2., Washing buffer rinse 4 times；

3., be added dilute biotinylated antibdy100 μ L room temperature effect 1 hour；

4., Washing buffer rinse 5 times, the Streptavidin solution100 μ L that has diluted, room temperature is added Effect 45 minutes；

5., be added TMB developing solution, react at room temperature 30 minutes；The Stop Solution that 500 μ L are added terminates reaction, all-wave Long microplate reader reads (450nm), and the content of inflammatory factor albumen in sample is calculated according to standard curve.

2.2.616HBE the extraction of RNA in cell

1., in each hole be added 1mL Trizol lysate, then collect cell RNA.12000g, under conditions of 4 DEG C It is centrifuged 10min, takes supernatant in the centrifuge tube of 1.5ml；

2., 200uL chloroform is added in supernatant, use lower concussion on hand is allowed to mix well into emulsion form, be incubated at room temperature 3min.Sample 12000g is centrifuged 15min under conditions of 4 DEG C, then carefully takes out centrifuge tube and be placed on ice, avoid rocking, with suction Head slowly draws supernatant liquid (300uL), avoids being drawn onto middle white albumin layer, be placed in a new centrifuge tube；

3., into pipe be added 0.5mL isopropanol, mix, be incubated at room temperature 10min.It is placed in 12000g in centrifuge again, 4 DEG C Under conditions of be centrifuged 10min.；

4., abandon supernatant, 75% ethyl alcohol (being matched with the dehydrated alcohol of DEPC water and 100%) for preparing in advance is added, concussion is set 5min is centrifuged under conditions of 7500g in centrifuge, 4 DEG C.Upper layer ethyl alcohol is outwelled, and centrifuge tube is kept to be in inversion state, to Ethyl alcohol just volatilizees dry；

5., after ethyl alcohol is dry, suitable DEPC water is added (depending on the amount with the RNA extracted).Reverse transcription is provided It uses.2.2.7RT-PCR reaction

1., the instrument that is detected using RNA, the successively concentration of the RNA in the good sample of Detection and Extraction.

2., according to testing result, sample is diluted to suitable concentration.

3., prepare the reaction system of RT-PCR, upper machine.According to designed program reaction before.After reaction, will CDNA is saved in -80 DEG C.

Note: the condition of RT-PCR reaction

37℃ 15min

85℃ 5s

4℃ forever

2.2.8 QPCR reacts

1., take out RT product, sonde method detection.

2., prepare reaction system, be added in reacting hole, sample be then added, mixed, upper machine reaction.

3., QPCR reaction condition

95℃ 30s,

95 DEG C of 5s (40 reaction cycles)

60℃ 40s.

4., primer sequence

IP-10 (people): L:5'-GAAATTATTCCTGCAAGCCAATTT-3'

R:5'-TCACCCTTCTTTTTCAT-TGTAGCA-3'

Probe primer: 5'-FAM-TCCACGTGTTGAGATCA-3'MGB

MIG (people): L:5'-TCTTGCTGGTTCTGATTGGAGTG-3'

R:5'-GATAGTCCCTTGGTTGGTGCTG-3'

Probe primer: 5'-FAM-CAGGAACAGCGACCCTTTCTCACTACTGG-3 ' BHQ-1

CCL5 (people): L:5'-CAGCAGTCGTCTTTGTCACC-3'

R:5'-GTTGATGTACTCCCGAACCC-3'

Probe primer: 5'-FAM-CGCCAAGTGTGTGCCAACCC-3 ' TAMRA

Internal reference (GAPDH): L:5'-GAAGGTGAAGGTCGGAGTC-3'

R:5'-GAAGATGGTGATGGGATTTC-3'

Probe primer: 5 '-FAM-CAAGCTTCCCGTTCTCAGCC-3 ' TAMRA

2.2.9 influenza NP protein immunoblot experiment

1., RIPA lysate extract cell protein；

2. electrophoresis: 100V 10min；150V 50min, or bromophenol blue is run to bottom；

3., transferring film: isolate separation gel, glue cut according to destination protein clip size, and shear pvdf membrane and filter paper (size For filter paper > pvdf membrane > glue), pvdf membrane is activated with methanol, the pvdf membrane after activation is put into togerther electricity with filter paper and is turned in liquid, It is placed according to the sequence of " sponge-filter paper-gel-PDF film-filter paper-sponge ", production " sandwich " folder (pays attention to every layer Between cannot have bubble while paying attention to electrode direction)." sandwich " made is folded up in electric turn trough.It is put in slot simultaneously Enter ice bag cooling, it is excessively high with heat production when anti-electric turn.Transferring film condition: 300mA, 80min；

4., closing: after electricity turns, take the film out, perform label and be placed in 5% milk confining liquid, general room temperature envelope Close 1 hour or 4 DEG C overnight；

5., primary antibody be incubated for: after closing, with 2.5% antibody diluent by destination protein primary antibody dilute (1:1000), Room temperature is incubated for 2h or 4 DEG C overnight；

6., secondary antibody is incubated for: after primary antibody is incubated for, wash film 3 times, each 10min with TBST washing lotion, it is dilute that secondary antibody be then added It releases liquid (1:3000), room temperature is incubated for 1h；TBST washing lotion washes film 3 times, each 10min；

7., exposure: by volume 1:1 prepare ECL luminescent solution A and B, exposed using instrument Tanon-5200.

2.3Experimental result

2.3.1 drug is to 16HBE cell drug toxicity

Mtt assay detects drug effect after 16HBE cell 48 hours, and 4mg/mL concentration versus cell toxicity is big, and 2mg/mL pairs Cell slightly has toxicity, but to 16HBE cytotoxic below 1mg/mL concentration.See Fig. 2A

2.3.2 the inhibition of the progeny virus duplication of infected by influenza and inhibition influenza virus are situated between after drug effect 24 hours The expression for the inflammatory factor led

Drug concentration, which acts on 16HBE cell for 2mg/mL and 1mg/mL, can inhibit the filial generation disease of influenza virus for 24 hours The duplication of poison, concentration are suppression of the 2mg/mL to the inhibiting rate of progeny virus close to 100%, when concentration is 1mg/mL to progeny virus Rate processed is close to 60%, but when concentration is 0.5mg/mL acts on (Fig. 2 B) to the basic unrestraint of progeny virus.And 2mg/mL~ 0.5mg/mL can obviously inhibit the generation of inflammatory factor (such as IP-10, MIG, CCL-5 etc.) mRNA and albumen, in drug concentration For the expression quantity that can also lower about 30% when 0.5mg/mL to IP-10 albumen, also have about 50% to the expression quantity of CCL-5 albumen Lower and (is compared with virus group)；And expression (Fig. 2 C- of inflammatory factor can be also reduced using drug (without virus stimulation is added) merely Fig. 2 F).It is the expression that can significantly inhibit the NP albumen of influenza virus that protein immunoblot result, which also shows drug in 1mg/mL, Drug concentration be 0.5mg/mL be the expression that can also inhibit the NP albumen of influenza virus, and drug concentration be 0.25mg/mL when press down The drug effect of influenza NP protein processed is unobvious (Fig. 2 G).The result shows that detection drug -- Cappariloside A can be effective The inflammatory reaction for inhibiting influenza virus and influenza virus to mediate.

2.3.3 the inhibition for the inflammatory factor expression that infected by influenza mediates after drug effect 6 hours

Be that 1mg/mL~0.25mg/mL can effectively inhibit IP-10 in concentration after drug effect 6 hours, MIG and The mRNA expression of CCL-5, even concentration also has about 30% inhibition to the mRNA level in-site of IP-10 when being 0.25mg/mL Effect also has about 40% inhibiting effect (comparing with virus group) to MIG mRNA level in-site, but when concentration is 0.125mg/mL Expression unrestraint effect to inflammatory factor, is shown in Fig. 3 A- Fig. 3 C.

The inflammatory reaction experiment that 3 Cappariloside A of test example inhibits influenza virus to mediate in macrophage

3.1 experimental material

3.1.1 drug is the same as test example 2

3.1.2 cell mouse macrophage (RAW264.7) is purchased from Shanghai Cell Bank of the Chinese Academy of Sciences.

3.1.3 the common H1N1virus of Strain (A/PR/8/34, H1N1) is purchased from ATCC, with Reed- Muench method measures its infection multiplicity (M.O.I), using M.O.I=0.1 as virus titer infection cell.

3.1.4 reagent E LISA kit is bought from RayBio company, Cat ELM-IP-10；ELM-CCL-5, remaining is same On.

3.2 experimental method

3.2.1RAW264.7 cell culture

Same 2.2.1

3.2.2 virus and medicine irritation

Ibid

3.2.3 inflammatory factor protein ELISA method detects in cell culture medium

Ibid

3.3Experimental result

3.3.1 drug is to 16HBE cell drug toxicity

Mtt assay detects drug effect after RAW264.7 cell 48 hours, and 2mg/mL concentration versus cell slightly has toxicity, 1mg/ ML or less is to cytotoxic.See Fig. 4 A.

3.3.2 the expression for the inflammatory factor albumen for inhibiting influenza virus to mediate after drug effect 24 hours

Drug concentration acts on RAW264.7 cell 24 hours for 2mg/mL and 1mg/mL can obviously inhibit influenza virus The duplication of progeny virus, inhibiting rate be greater than 80%, and drug concentration be 0.5mg/mL when infected by influenza progeny virus unrestraint It acts on (Fig. 4 B, Fig. 4 C).Meanwhile drug concentration is that 1mg/mL and 0.5mg/mL can significantly inhibit inflammatory factor (such as IP- 10, CCL-5 etc.) generation of albumen, to the albumen inhibiting rate of IP-10 and CCL-5 close to 1 times when drug concentration is 1mg/mL, medicine Object concentration also has apparent inhibiting effect to IP-10 and CCL-5 expressing quantity when being 0.5mg/mL, (and virus group phase Than)；And the expression (comparing with normal group) of inflammatory factor can be also reduced using drug (without virus stimulation is added) merely, while It can inhibit the expression of their mRNA.But the infected by influenza when drug concentration is 0.25mg/mL and 0.125mg/mL The inflammatory reaction unrestraint of mediation acts on (Fig. 4 D, Fig. 4 E, Fig. 4 F).The result shows that in macrophage, drug to be detected -- Cappariloside A, abbreviation M5 can inhibit influenza virus progeny virus in high concentration (2mg/mL~1mg/mL) simultaneously Duplication and the inflammatory reaction for inhibiting influenza virus to mediate；Effectively influenza virus can be inhibited to mediate at low concentration (0.5mg/mL) Inflammatory reaction.

The above described is only a preferred embodiment of the present invention, limitation in any form not is done to the present invention, therefore All contents without departing from technical solution of the present invention, it is made to the above embodiment according to the technical essence of the invention any simply to repair Change, equivalent variations and modification, all of which are still within the scope of the technical scheme of the invention.

Claims (5)

1.Cappariloside A is preparing the application in the drug for treating influenza, feature as sole active agent It is, the drug is the drug that the inflammatory factor for having the function of that influenza virus is inhibited to mediate generates, the inflammatory factor choosing From IP-10, MIG, CCL-5 or CCL-5/RANTES；The influenza virus be common H1N1virus A/PR/8/34, Ichi plants of A type H3N2 influenza virus A, new H1N1virus strain or influenza B virus.

2. application according to claim 1, which is characterized in that the Cappariloside A has in preparation inhibits stream The effect of the progeny virus duplication of Influenza Virus and/or the drug with the inflammatory factor generation for inhibiting influenza virus mediation Middle application.

3. application according to claim 1, which is characterized in that the Cappariloside A is from extracted form natural plant Or it is chemically synthesized.

4. application according to claim 3, which is characterized in that the natural plants are in Radix Isatidis, dimension medicine caper One or two.

5. application according to claim 1, which is characterized in that the drug is made the dosage form pharmaceutically allowed, and described dose Type is tablet or capsule.