CN104906117B - Application of the 1,6- diphosphofructoses in the drug of prevention infection of coated virus and virus inactivator is prepared - Google Patents
Application of the 1,6- diphosphofructoses in the drug of prevention infection of coated virus and virus inactivator is prepared Download PDFInfo
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Abstract
The invention discloses a kind of application of 1,6 diphosphofructose and its salt formed in the drug of prevention mankind's infection of coated virus and virus inactivator is prepared.We have rated the viricidal activity of 1,6 fructose diphosphate sodiums viruses a variety of to the mankind by detecting levels of replication of a variety of common virus of the mankind of 1,6 fructose diphosphate sodiums pretreatment on cell.1,6 fructose diphosphate sodium of the results show has dose-dependent viricidal activity to enveloped virus.Experiment confirms that the effect of killing the virus of 1,6 fructose diphosphate sodiums is temperature-independent.1,6 diphosphofructose is glycolysis mesostate, while plays adjustment effect to metabolism again.The compound can inactivate a variety of enveloped viruses, have exploitation into the drug of prevention mankind's infection of coated virus and the bright prospects of virus inactivator.
Description
Technical field
The invention belongs to pharmaceutical technology fields, are more particularly to a kind of fructose 1,6-diphosphate and its salt formed is being made
Application in the drug of standby prevention mankind's infection of coated virus and external virus inactivator.
Background technology
There is one layer of cyst membrane for deriving from host cell outside the nucleocapsid of enveloped virus.Cyst membrane is wrapped around outside stromatin
One layer of phospholipid bilayer tunic, this tunic derives from the cell membrane of host, also inlays the coating egg of virus-specific thereon
In vain.The dengue virus of flaviviridae（DENV）And Japanese B encephalitis virus（JEV）, Rhabdoviridae vesicular stomatitis virus
（VSV）And the Respiratory Syncytial Virus(RSV) of Paramyxoviridae（RSV）It is tunicary virus, is all that the common threat mankind are good for
The important pathogen body of health.
Dengue virus is widely current in global tropical and semi-tropical more than 60 a countries and regions, per year over 100000000 people by
Infection, more than 2,500,000,000 people are on the hazard.Dengue fever（dengue fever）Be dengue fever virus cause, her mosquito-borne one kind
Acute infectious disease.Clinical symptoms is hurried for onset, high fever, whole-body muscle, marrow and arthralgia, extremely tired, and part, which is suffered from, to be had
Fash, hemorrhagic tendency and enlargement of lymph nodes.Japanese B encephalitis（Encephalitis）It is the acute human as caused by Japanese B encephalitis virus
Poultry suffers from infectious disease altogether, clinically with the characteristics of the symptom and signs such as high fever, the disturbance of consciousness, twitch, respiratory failure and meningeal irritation sign,
Case fatality rate is about 25%.Dengue fever and encephalitis are propagated by mosquito medium, are popular in entire Asia, mainly in the east Asia torrid zone, Asia
The torrid zone and temperate countries.Encephalitis takes place mostly in summer and autumn, and children are susceptible to suffer from, and incidence accounts for more than 80%, is mostly less than 10 years old youngster
It is virgin.For japanese encephalitis virus, though having vaccine at present, there is no special antiviral therapies.Japanese encephalitis virus infects still
It is so a big threat of human health.The spread speed of influenza virus is fast, infectiousness is strong and can cause part in the short time
The outburst even whole world is very popular, and always is the emphasis of global concern so far from discovery.20th century, IAV cause big stream three times altogether
Row.The big influenza of Spain of wherein 1918-1920 outbursts influences maximum, causes global 50,000,000 people dead.1997, height caused a disease
Property bird flu appear in Hong Kong because the infection that outburst is dead and the mankind are dispersed in that poultry is destructive, while with very high
The death rate makes the mankind be absorbed in great fear.Although this two classes pathogen is in the infection sources, route of transmission and pathogenesis etc.
Aspect is entirely different, but human health is all made to be on the hazard and brings economic loss.
At present, clinically mainly carried out for disease caused by these enveloped viruses using interferon and Ribavirin disease-resistant
Poison treatment.However, drug is there are certain side effect, and relevant disease cannot be cured completely.In addition, these drugs is long-term
Many medicament-resistant mutation strains are generated using virus is caused.Therefore, it is badly in need of the exploitation effective drug of new type of safe to prevent or inactivate
Virus infection.
Fructose 1,6-diphosphate is the important intermediate product of glycolysis metabolism approach, while to the metabolism of carbohydrate, lipid again
Adjustment effect can be played, is considered as a kind of metabolic modifiers and energy-rich phosphate substrate.In clinicing aspect, fructose 1,6-diphosphate
It is widely used, the auxiliary available for myocarditis, angina pectoris, myocardial infarction, shock, the environment of ischemic hypoxia and hepatitis is controlled
It treats.But fructose 1,6-diphosphate is still blank out in the research of field of virology.Fructose 1,6-diphosphate structure is as follows：
The content of the invention
It is an object of the invention to make up the deficiencies in the prior art, be the provision of a kind of fructose 1,6-diphosphate and its
Application of the salt formed in the drug of prevention mankind's infection of coated virus and external virus inactivator is prepared, so as to be clinic
The prevention of relevant disease caused by upper mankind's infection of coated virus provides safely and effectively measure.Fructose 1,6-diphosphate and its institute's shape
Into salt the infection of enveloped virus can be effectively inactivated in non-toxic scope, prevention enveloped virus can be further developed as and drawn
The drug of the relevant disease risen and external virus inactivator, are with a wide range of applications.
In order to realize above-mentioned purpose, the technical solution adopted by the present invention is：
A kind of 1,6- diphosphofructoses and its salt formed are preparing the drug and disease of prevention mankind's infection of coated virus
Application in malicious inactivator.
Preferably, the enveloped virus is Japanese B encephalitis virus（JEV）, dengue virus（DENV）, respiratory tract close
Cellular virus（RSV）And influenza A virus（IAV）Each hypotype.
Comprise the following steps：
1. fructose 1,6-diphosphate sodium salt kills enveloped virus Activity determination, step is：
A. fructose 1,6-diphosphate sodium salt is real to mdck cell, VERO cells, BHhK-21 cells and Hep-2 cytotoxicities
It tests：
Mdck cell, VERO cells, BHhK-21 cells and Hep-2 cells press 8 × 103It is thin that a cells/well is inoculated in 96 holes
It is spare after cell attachment in born of the same parents' culture plate.With culture medium by fructose 1,6-diphosphate sodium salt since 20mg/mL, 2 times of gradients according to
It is secondary to be diluted to 9 concentration, the culture medium of drug containing is directly appended in adherent various cells, three holes of every group of repetition are put
In 37 DEG C, 5% CO2It is cultivated in incubator.37 DEG C, 5% CO will be placed in added with the cell of drug2When culture 48 ~ 96 is small in incubator
Afterwards, using the survival rate of Alamarblue activity detection kits detection cell.
The results show fructose 1,6-diphosphate sodium salt is very low to the toxicity of these cells, half toxic concentration（CC50）
More than 2000 μ g/mL, security is fine.
Fructose 1,6-diphosphate sodium salt detects the viricidal activity of enveloped virus：
(1) influenza virus liquid (6.5 × 105 TCID50/ mL) it is (whole with the fructose 1,6-diphosphate sodium salt of 3 times of gradient dilutions
Concentration is respectively 1000 μ g/mL, 333 μ g/mL, 111 μ g/mL, 37 μ g/mL, 12.3 μ g/mL, 4.1 μ g/mL, 1.37 μ g/mL)
When 37 DEG C of preincubates 4 are small, mixture is then diluted into 100 times of postoperative infection mdck cells；Control group be then viral stock with it is each
Drug is respectively when 37 DEG C of preincubates 4 are small, and mixing infects mdck cell after each 50 times of dilution.Virus infected cell 1 is dropped back when small
Fall supernatant, washed twice with PBS, remove unadsorbed viral and remaining drug.Then fresh culture is changed in 37 DEG C of continuation
Cultivate 24 it is small when after, collect supernatant measure neuraminidase (NA) activity, calculate inhibiting rate.
（2）Japanese B encephalitis virus JEV-Luc liquid（8×106pfu/mL）With dengue virus DENV-Luc liquid（4×107
pfu/mL）Respectively with the fructose 1,6-diphosphate sodium salt of 3 times of gradient dilutions（Final concentration be respectively 1000 μ g/mL, 333 μ g/mL,
111μg/mL、37μg/mL、12.3μg/mL、4.1μg/mL、1.37μg/mL）When 37 DEG C of preincubates 4 are small, then by virus and medicine
Object biased sample dilutes 100 times of postoperative infection BHK-21 cells, when 37 DEG C of cultures 1 are small.Equally, cell is washed twice with PBS, is removed not
After the viral and remaining drug of absorption, be changed to fresh culture continue culture 48 it is small when, detect cell fluorescence element enzymatic activity, meter
Calculate inhibiting rate.
（3）Bubble stomatitis virus VSV-GFP virus liquids（4×107pfu/mL）With 1,6-, bis- phosphorus of 3 times of gradient dilutions
Tart fruit sugar sodium salt（Final concentration is respectively 1000 μ g/mL, 333 μ g/mL, 111 μ g/mL, 37 μ g/mL, 12.3 μ g/mL, 4.1 μ g/
mL、1.37μg/mL）When 37 DEG C of preincubates 4 are small.Virus and drug mixture are diluted into 100 times of postoperative infection BhK-21 cells,
Supernatant is removed after when 37 DEG C of cultures 1 are small.Cell is washed twice with PBS, is removed unadsorbed viral and remaining drug, is then changed to
Fresh culture.Continue culture 12 it is small when after, it is strong by the fluorescence of high intension image checking and quantitative analysis green fluorescent protein
Degree calculates inhibiting rate.
（4）Adenovirus hominis（ADV）, enterovirns type 71（EV71-GFP）Respectively with the 1,6- diphosphonic acid of 3 times of gradient dilutions
Fructose sodium salt（Final concentration be respectively 1000 μ g/mL, 333 μ g/mL, 111 μ g/mL, 37 μ g/mL, 12.3 μ g/mL, 4.1 μ g/mL,
1.37μg/mL）After when 37 DEG C of preincubates 4 are small, VERO cells are infected with MOI=1 and MOI=0.2 after 100 times of dilution.37 DEG C of trainings
Support 1 it is small when after remove supernatant, washed twice with PBS, remove unadsorbed viral and remaining drug.Then it is changed to fresh culture
Continue culture 48 it is small when after, pass through high intension examine imaging system survey and quantitative analysis cell lesion（cytopathic effect,
CPE）Or the fluorescence intensity of green fluorescent protein, calculate inhibiting rate.
（5）Respiratory Syncytial Virus(RSV) RSV and the 1,6- fructose diphosphate sodiums diluted（Final concentration is respectively 1000 μ g/
mL、333μg/mL、111μg/mL、37μg/mL、12.3μg/mL、4.1μg/mL、1.37μg/mL）When 37 DEG C of preincubates 3 are small
Afterwards, with 100TCID after diluting 100 times50Infect Hep-2 cells.Supernatant is removed after when 33 DEG C of cultures 1 are small, is washed twice, gone with PBS
Fall unadsorbed viral and remaining drug.Then be changed to fresh culture 33 DEG C continue culture 96 it is small when after, pass through
CellTiter-Glo kit detection cells activity calculates the inhibiting rate of cytoprotection rate and drug to virus.
The results show fructose 1,6-diphosphate sodium salt has dosage to enveloped viruses such as JEV, DENV, RSV and influenza viruses
The viricidal activity of dependence.However, fructose 1,6-diphosphate sodium salt does not show but to kill to nonenveloped virus ADV and EV71
Virus activity.Illustrate that fructose 1,6-diphosphate sodium salt has enveloped virus the effect of killing the virus of wide spectrum.
2. fructose 1,6-diphosphate sodium salt temperature-independent kills influenza activity detection, step is：
Influenza A virus [A/PuertoRico/8/34 (H1N1)] liquid（6.5×105 TCID50/mL）With 3 times of gradients
Diluted 1,6- fructose diphosphate sodiums（Final concentration is respectively 1000 μ g/mL, 333 μ g/mL, 111 μ g/mL, 37 μ g/mL, 12.3
μg/mL）Respectively when 4 DEG C, 25 DEG C, 37 DEG C and 42 DEG C incubations 4 are small, virus and the mixture of drug are then diluted 100 times
Postoperative infection mdck cell.Supernatant is removed after when virus infected cell 1 is small, is washed twice with PBS, removes unadsorbed virus and residual
Drug.Then be changed to fresh culture 37 DEG C continue culture 24 it is small when after, collect supernatant measure NA activity, calculate inhibit
Rate.
The result shows that the effect of killing the virus of fructose 1,6-diphosphate sodium salt infected by influenza is temperature-independent.1,6- bis- phosphorus
Tart fruit sugar sodium salt is better than the inactivating efficacy of 4 DEG C and 25 DEG C in 37 DEG C and 42 DEG C of inactivating influenza virus effects.
Compared with prior art, the present invention haing the following advantages and effect：
1. clinically, it is scarce to be widely used in myocarditis, angina pectoris, myocardial infarction, part for fructose 1,6-diphosphate sodium salt
The auxiliary treatment of anoxemia and hepatitis, and be the intermediate product of human body glycometabolism approach, it is small to the toxic side effect of human body.
2. fructose 1,6-diphosphate sodium salt under the conditions of avirulent, can effectively inactivate JEV, DENV, RSV and influenza
The infection of each hypotype of virus, can be made into the antiviral drugs of wide spectrum.
3. using modern common drug preparation means, piece can be made using fructose 1,6-diphosphate sodium salt as active ingredient
Any one such as agent, capsule, granule, oral liquid, sustained release preparation, controlled release preparation, nanometer formulation or injection can pharmaceutically connect
The dosage form received.
4. fructose 1,6-diphosphate sodium salt has the function of inactivation of viruses, external virus inactivator can be prepared into.
Description of the drawings
Fig. 1 is fructose 1,6-diphosphate chemical structural formula.
Fig. 2 kills influenza activity detects schematic diagram for fructose 1,6-diphosphate sodium salt temperature-independent.
Specific embodiment
In order to better understand the content of the present invention, present invention is made furtherly with reference to specific implementation method
It is bright, but the protection content of the present invention is not limited to following embodiment.
Embodiment 1:Fructose 1,6-diphosphate sodium salt detects the viricidal activity of each subtype influenza virus.
1. experiment material
1.1 cells, virus
Dog kidney cells MDCK（ATCC CCL-34）It is preserved by this laboratory.Influenza A H1N1 hypotypes（A/
PuertoRico/8/1934 and A/human/Hubei/1/2009）, H3N2 hypotypes（A/ Human /Hubei/3/2005）、
H6N6 hypotypes（A/Duck/Hubei/5/2010）, H7N8 hypotypes（A/Duck/Hubei/ 216/ 1983）With H1N1 hypotype Buddha's warrior attendants
Alkanamine persister（A/Human/WSN/33(S31N)）It is that this laboratory preserves, expands to obtain by chicken embryo.Instar chicken embryo on the 10th
Purchased from Beijing Cimmeria Wei Tong experimental animals Technology Co., Ltd..
1.2 reagent
DMEM culture mediums are purchased from GIBCO companies；Alamarblue activity detection kits are purchased from Promega companies；Nerve
Propylhomoserin zymolyte MUNANA is purchased from Sigma-Aldrich.Fructose 1,6-diphosphate sodium salt is purchased from Aladdin.
1.3 laboratory apparatus
Multiple labeling microtiter plate reader（PerkinElmer）For Bole's Products；Cell incubator is Thermo companies
Product.
2. experimental method and result
2.1 fructose 1,6-diphosphate sodium salts detect the cytotoxicity of mdck cell.
（1）By mdck cell according to 8 × 103A cells/well is inoculated in 96 porocyte culture plates, using containing 10% tire
Cow's serum（FBS）, the penicillin of 100U/mL and the DMEM culture mediums of streptomysin.Cell is in 37 DEG C, 5% CO2 humidified incubators
Culture.
（2）With culture medium by fructose 1,6-diphosphate sodium salt since 20mg/ml, 2 times of gradients be diluted to successively 9 it is dense
Degree, the culture medium of drug containing is directly appended in adherent mdck cell, three holes of every group of repetition are placed in 37 DEG C, 5% CO2
It is cultivated in incubator.
（3）48 it is small when after, utilize Alamarblue activity detection kits detection drug cytotoxicity.With
PerkinElmer multiple labeling plate readers（Envison 2102 Multilabel Reader）Read fluorescence signal.
（4）Each group of data is calculated using being free of drug control group as standard group, calculation formula=medicine group/non-dosing
Object * 100.As a result average value and standard deviation are calculated by 5 softwares of GraphPad Prism.
Utilize step（4）In result of calculation, by software statistics, statistical result is shown in Table 1.The results show 1,6- diphosphonic acid
Fructose sodium salt is to the CC of the cytotoxicity of mdck cell50For 2800 μ g/mL.
Detection of the 2.2 fructose 1,6-diphosphate sodium salts to each subtype influenza virus inactivating efficacy.
（1）Influenza A H1N1 hypotypes（A/PuertoRico/8/1934 and A/Human/Hubei/1/2009）、
H3N2 hypotypes（A/ Human /Hubei/3/2005）, H6N6 hypotypes（A/Duck/Hubei/5/2010）, H7N8 hypotypes（A/
Duck/Hubei/ 216/ 1983）With H1N1 hypotype amantadine persisters（A/Human/WSN/33(S31N)）Virus storage
The liquid fructose 1,6-diphosphate sodium salt with 3 times of gradient dilutions respectively（Final concentration is respectively 1000 μ g/mL, 333 μ g/mL, 111 μ g/
mL、37μg/mL、12.3μg/mL、4.1μg/mL、1.37μg/mL）When 37 DEG C of preincubates 4 are small.Then by virus and drug
Mixture dilutes 100 times of postoperative infection mdck cells；Control group is then that viral stock is incubated in advance at 37 DEG C respectively with each acute drug
Educate 4 it is small when, mixing infects mdck cell immediately after each 50 times of dilution.
（2）Supernatant is removed after when virus infected cell 1 is small, twice is washed with PBS and removes unadsorbed viral and remaining medicine
Then object is changed to fresh culture and continues to cultivate.
（3）Cultivate 24 it is small when after, collect supernatant measure NA（Neuraminidase）Activity calculates inhibiting rate.
The results show fructose 1,6-diphosphate sodium salt energy dose-dependant ground each hypotype of inactivating influenza virus, is united by software
Meter, the IC of each hypotype of fructose 1,6-diphosphate infected by influenza50For 45 ~ 130 μ g/mL, 1 the results are shown in Table.
Embodiment 2：Fructose 1,6-diphosphate sodium salt is to the viricidal activity of dengue virus and Japanese B encephalitis virus
Detection.
1. experiment material
1.1 cells and virus
Hamster kidney cell（BHK-21）It is preserved for this laboratory.
Japanese B encephalitis virus (JEV-Luc) and dengue virus (DENV-Luc)：To carry Rellina
The Japanese B encephalitis virus and dengue virus of luciferase reporter genes, by Wuhan institute of viruses of the Chinese Academy of Sciences ripple research
Member's present.
1.2 reagent
DMEM culture mediums and FBS are purchased from GIBCO companies；Alamarblue activity detection kits are purchased from Promega companies；RenillaLuciferase Assay Reagent box is purchased from Promega companies.
1.3 laboratory apparatus
Multiple labeling microtiter plate reader（PerkinElmer）；High intension cytoanalyze（PerkinElmer）；
Megafuge 1.0R types refrigerated centrifuges and cell incubator are Thermo Products.
2. experimental method and result
2.1 1,6- fructose diphosphate sodiums detect the cytotoxicity of BHK-21 cells.
（1）By BHK-21 cells according to 8 × 103A cells/well is inoculated in 96 porocyte culture plates, using containing 10%
The penicillin of FBS, 100U/mL and the DMEM culture mediums of streptomysin are cultivated in 37 DEG C, the humidified incubator of 5% CO2.
（2）With culture medium by fructose 1,6-diphosphate sodium salt since 20mg/ml, 2 times of gradients be diluted to successively 9 it is dense
Degree, the culture medium of drug containing is directly appended in adherent BHK-21 cells, three holes of every group of repetition, be placed in 37 DEG C, 5%
CO2It is cultivated in incubator.
（3）48 it is small when after, utilize Alamarblue activity detection kits detection drug cytotoxicity.With
PerkinElmer multiple labeling plate readers（Envison 2102 Multilabel Reader）Read fluorescence signal.
（4）Each group of data is calculated using being free of drug control group as standard group, calculation formula=medicine group/non-dosing
Object * 100.As a result average value and standard deviation are calculated by 5 softwares of GraphPad Prism.
Step（4）In result of calculation, by software counting statistics, the results are shown in Table 1.The results show fructose 1,6-diphosphate
Sodium salt is to the CC of the cytotoxicity of BHK-21 cells50For 7090 μ g/mL.
2.2 fructose 1,6-diphosphate sodium salts detect the viricidal activity of dengue virus and Japanese B encephalitis virus
（1）JEV-Luc（8×106pfu/ml）With DENV-Luc（4×106pfu/ml）Respectively with 3 times of gradient dilutions
1,6- fructose diphosphate sodiums（Final concentration be respectively 1000 μ g/mL, 333 μ g/mL, 111 μ g/mL, 37 μ g/mL, 12.3 μ g/mL,
4.1μg/mL、1.37μg/mL）100 times are diluted when 37 DEG C of preincubates 4 are small, then by virus and drug biased sample, postoperative infection
BHK-21 cells；Control group is then viral stock with each acute drug respectively when 37 DEG C of preincubates 4 are small, each 50 times of dilution
Mixing infects BHK-21 cells immediately afterwards.
（2）Supernatant is removed after when virus infected cell 1 is small, is washed twice with PBS, removes unadsorbed viral and remaining medicine
Then object is changed to fresh culture and continues to cultivate.
（3）After when 37 DEG C of cultures 48 are small, intracellular luciferase activity is measured, calculates inhibiting rate.Cell PBS to be measured
It washes 2 times, then adding in lysate with the method for Renilla Luciferase Assay Reagent box specifications makes cell fully crack, so
In the substrate that lysate sample addition has been diluted afterwards, it is detected on luciferase detector.Calculation formula is：Inhibiting rate
（%）= 100 -（Sample well-blank control）/（Enzyme activity control-blank control）* 100%.
Experimental result inactivates Japanese B encephalitis virus and Dengue with showing fructose 1,6-diphosphate sodium salt energy dose-dependant
Virus.Fructose 1,6-diphosphate sodium salt is to the IC of Japanese B encephalitis virus and dengue virus50Respectively 112 and 6.8 μ g/mL,
It the results are shown in Table 1.
Embodiment 3：Fructose 1,6-diphosphate sodium salt is to vesicular stomatitis virus（VSV）Viricidal activity detection.
1. experiment material
1.1 cells, virus
Hamster kidney cell（BHK-21）It is preserved for this laboratory.
VSV-GFP：To carry the vesicular stomatitis virus of GFP reporter genes, presented by Wuhan University professor Chen Mingzhou.
1.2 reagent
DMEM culture mediums and FBS are purchased from GIBCO companies；Alamarblue activity detection kits are purchased from Promega companies.
1.3 laboratory apparatus
High intension cytoanalyze（PerkinElmer）；The advanced inverted microscope Axio observer A1 of Zeiss
(Carl Zeiss )。
2. experimental method and result
2.1 1,6- fructose diphosphate sodiums detect the cytotoxicity of BHK-21 cells（See embodiment 2）.
2.2 fructose 1,6-diphosphate sodium salts kill vesicular stomatitis virus Activity determination.
（1）VSV-GFP viral stocks（4×107pfu/mL）With the 1,6- fructose diphosphate sodiums of 3 times of gradient dilutions
（Final concentration is respectively 1000 μ g/mL, 333 μ g/mL, 111 μ g/mL, 37 μ g/mL, 12.3 μ g/mL, 4.1 μ g/mL, 1.37 μ g/
mL）100 times of postoperative infection BhK-21 cells are diluted when 37 DEG C of preincubates 4 are small, then by virus and drug biased sample；Control group
It is then viral stock with each acute drug respectively when 37 DEG C of preincubates 4 are small, mixing infects BHK- immediately after each 50 times of dilution
21 cells.
（2）Supernatant is removed after when virus infected cell 1 is small, is washed twice with PBS, removes unadsorbed viral and remaining medicine
Then object is changed to fresh culture and continues to cultivate.
（3）Cultivate 12 it is small when after, pass through high intension detection and quantitative analysis green fluorescent protein fluorescence intensity, calculate suppression
Rate processed.Inhibiting rate（%）=（Viral hole fluorescence intensity-sample well fluorescence intensity）/ virus hole fluorescence intensity * 100%.
Experimental result inactivates vesicular stomatitis virus, 1,6- bis- phosphorus with showing fructose 1,6-diphosphate sodium salt energy dose-dependant
Tart fruit sugar sodium salt is to the IC of vesicular stomatitis virus50For 12 μ g/mL, 1 the results are shown in Table.
Embodiment 5：Fructose 1,6-diphosphate sodium salt detects the viricidal activity of adenovirus ADV and enterovirus EV 71.
1. experiment material
1.1 cells, virus
VERO cells preserve for this laboratory.
Adenovirus（Adenovirus）By Wuhan Virology Institute,Chinan academy of Sciences, microbial bacteria seed culture of viruses resource in application
The heart is presented.EV71-GFP viruses：With EV71-GFP plasmid as template, in-vitro transcription obtains full-length genome RNA after linearisation, then uses
Amplification obtains possessing infective virion after liposome transfection VERO cells.
1.2 reagent
DMEM culture mediums and FBS are purchased from GIBCO companies；Alamarblue activity detection kits are purchased from Promega companies.
1.3 laboratory apparatus
High intension cytoanalyze（PerkinElmer）；The advanced inverted microscope Axio observer A1 of Zeiss
(Carl Zeiss )。
2. experimental method and result
2.1 1,6- fructose diphosphate sodiums detect the cytotoxicity of VERO cells.
（1）By VERO cells according to 8 × 103A cells/well is inoculated in 96 porocyte culture plates, using containing 10%
The penicillin of FBS, 100U/mL and the DMEM culture mediums of streptomysin are at 37 DEG C, 5% CO2Humidified incubator in cultivate.
（2）With culture medium by fructose 1,6-diphosphate sodium salt since 20mg/ml, 2 times of gradients be diluted to successively 9 it is dense
Degree, the culture medium of drug containing is directly appended in adherent VERO cells, three holes of every group of repetition are placed in 37 DEG C, 5% CO2
It is cultivated in incubator.
（3）Cultivate 48 it is small when after, utilize Alamarblue activity detection kits detection drug cytotoxicity.With
PerkinElmer multiple labeling plate readers（Envison 2102 Multilabel Reader）Read fluorescence signal.
（4）Each group of data is calculated using being free of drug control group as standard group, calculation formula=medicine group/non-dosing
Object * 100.As a result average value and standard deviation are calculated by 5 softwares of GraphPad Prism.
Experimental result show fructose 1,6-diphosphate sodium salt 1000 μ g/mL to VERO cells be do not have it is virose, as a result
It is shown in Table 1.
2.2 fructose 1,6-diphosphate sodium salts kill the detection of ADV and EV71 virus activities.
（1）The 1,6- fructose diphosphate sodiums of ADV and EV71-GFP viral stocks and 3 times of gradient dilutions（Final concentration point
It Wei not 1000 μ g/mL, 333 μ g/mL, 111 μ g/mL, 37 μ g/mL, 12.3 μ g/mL, 4.1 μ g/mL, 1.37 μ g/mL）It is pre- at 37 DEG C
Be incubated 4 it is small when.Then virus and drug biased sample are diluted into 100 times of postoperative infection VERO cells；Control group is then viral storage
Liquid storage and each acute drug are respectively when 37 DEG C of preincubates 4 are small, and mixing infects VERO cells immediately after each 50 times of dilution.
（2）Virus infected cell 37 DEG C culture 1 it is small when after remove supernatant, washed twice with PBS, remove unadsorbed virus
With remaining drug, then it is changed to fresh culture and continues to cultivate.
（3）After when 37 DEG C of cultures 48 are small, pass through the detection of high intension and quantitative analysis cell lesion（cytopathic
effect, CPE）Or the fluorescence intensity of green fluorescent protein (GFP), calculate inhibiting rate.Inhibiting rate（%）=（Viral hole fluorescence
Intensity-sample well fluorescence intensity）/ virus hole fluorescence intensity * 100%.
Experimental result shows that fructose 1,6-diphosphate sodium salt cannot inactivate ADV and EV71.
Embodiment 6：Fructose 1,6-diphosphate sodium salt detects the viricidal activity of Respiratory Syncytial Virus(RSV).
1. experiment material
1.1 cells, virus
Hep-2 cells preserve for this laboratory.
Respiratory Syncytial Virus(RSV)（RSV）It is presented by Wuhan University.
1.2 reagent
DMEM culture mediums and FBS are purchased from GIBCO companies；Alamarblue and CellTiter Glo cytoactive detections try
Agent box is purchased from Promega companies.
1.3 laboratory apparatus
Multiple labeling microtiter plate reader（PerkinElmer）；High intension cytoanalyze（PerkinElmer）；Cell is trained
It is Thermo Products to support case.
2. experimental method and result
2.1 1,6- fructose diphosphate sodiums detect the cytotoxicity of Hep-2 cells.
（1）By Hep-2 cells according to 8 × 103A cells/well is inoculated in 96 porocyte culture plates, using containing 10%
The penicillin of FBS, 100U/mL and the DMEM culture mediums of streptomysin are at 37 DEG C, 5% CO2Humidified incubator in cultivate.
（2）With culture medium by fructose 1,6-diphosphate sodium salt since 20mg/ml, 2 times of gradients be diluted to successively 9 it is dense
Degree, the culture medium of drug containing is directly appended in adherent Hep-2 cells, three holes of every group of repetition are placed in 37 DEG C, 5% CO2
It is cultivated in incubator.
（3）Cultivate 48 it is small when after, utilize Alamarblue activity detection kits detection drug cytotoxicity.With
PerkinElmer multiple labeling plate readers（Envison 2102 Multilabel Reader）Read fluorescence signal.
（4）Each group of data is calculated using being free of drug control group as standard group, calculation formula=medicine group/non-dosing
Object * 100.As a result average value and standard deviation are calculated by 5 softwares of GraphPad Prism.
The results show fructose 1,6-diphosphate 1000 μ g/mL to Hep-2 cells be do not have it is virose.
2.2 fructose 1,6-diphosphate sodium salts kill Respiratory Syncytial Virus(RSV)（RSV）Activity determination.
（1）The 1,6- fructose diphosphate sodiums of RSV viral stocks and 3 times of gradient dilutions（Final concentration is respectively 1000 μ g/
mL、333μg/mL、111μg/mL、37μg/mL、12.3μg/mL、4.1μg/mL、1.37μg/mL）When 37 DEG C of preincubates 3 are small,
Virus and drug biased sample are diluted into 100 times of postoperative infection Hep-2 cells again（Virus infection titer is 100TCID50）；Control group
It is then viral stock with each acute drug respectively when 37 DEG C of preincubates 3 are small, mixing infects Hep- immediately after each 50 times of dilution
2 cells.
（2）Virus infected cell 33 DEG C culture 1 it is small when after remove supernatant, cell is washed twice with PBS, is removed unadsorbed
Viral and remaining drug, be then changed to fresh culture and continue to cultivate.
（3）33 DEG C continue culture 96 it is small when after, by CellTiter-Glo detect cytoactive, calculate cytoprotection rate,
And then learn inhibiting rate of the fructose 1,6-diphosphate sodium salt to RSV viricidal activities.Inhibiting rate（%）=（Sample well-virus hole）/
（Cell hole-virus hole）* 100%.
Experimental result inactivates Respiratory Syncytial Virus(RSV), 1,6- bis- phosphorus with showing fructose 1,6-diphosphate sodium salt energy dose-dependant
Sour levulosazone inhales the IC of road syncytial virus50For 105 μ g/mL, 1 the results are shown in Table.
1 fructose 1,6-diphosphate sodium salt of table is to enveloped virus broad-spectrum killing virus Activity determination
IC50:Inhibiting rate is 50% drug concentration
CC50:Cytotoxicity is 50% drug concentration
7 fructose 1,6-diphosphate sodium salt temperature-independent of embodiment kills influenza activity detection.
1. experiment material
1.1 cells, virus
Dog kidney cells MDCK（ATCC CCL-34）It is preserved by this laboratory.Influenza A H1N1 hypotypes（A/
PuertoRico/8/1934）It is preserved for this laboratory, expands to obtain by chicken embryo.Instar chicken embryo on the 10th is tieed up purchased from Beijing Cimmeria
Logical experimental animal Technology Co., Ltd..
1.2 reagent
DMEM culture mediums are purchased from GIBCO companies；Neuraminidase substrate MUNANA is purchased from Sigma-Aldrich.1,6- bis-
Phosphofructose sodium salt is purchased from Aladdin.
1.3 laboratory apparatus
Multiple labeling microtiter plate reader（PerkinElmer）For Bole's Products；Cell incubator is Thermo companies
Product；Electric heating constant temperature sink is purchased from the permanent Science and Technology Ltd. in Shanghai one.
2. experimental method and result
（1）Influenza virus H1N1 hypotype（A/PuertoRico/8/1934）（6.5×105 TCID50/ml）With 3 times
The fructose 1,6-diphosphate sodium salt of gradient dilution（Final concentration be respectively 1000 μ g/mL, 333 μ g/mL, 111 μ g/mL, 37 μ g/mL,
12.3μg/mL）Respectively 4 DEG C, 25 DEG C, 37 DEG C, 42 DEG C be incubated 4 it is small when, then by virus and the mixture of drug dilution 100
Times postoperative infection mdck cell.
（2）Supernatant is removed after when virus infected cell 1 is small, twice is washed with PBS and removes unadsorbed viral and remaining medicine
Then object is changed to fresh culture and continues to cultivate at 37 DEG C.
（3）24 it is small when after, collect supernatant measure NA（Neuraminidase）Activity calculates inhibiting rate.
Experimental result is as shown in Fig. 2, the effect of killing the virus of fructose 1,6-diphosphate sodium salt infected by influenza is temperature-independent
's.In the case of 4 DEG C, fructose 1,6-diphosphate infected by influenza does not kill the virus effect.When temperature is 25 DEG C, 1,6-
Diphosphofructose shows the weaker effect of killing the virus.However, in the case of 37 DEG C and 42 DEG C, fructose 1,6-diphosphate kills
Virus function significantly improves.
As it will be easily appreciated by one skilled in the art that the foregoing is merely illustrative of the preferred embodiments of the present invention, not to
The limitation present invention, all any modification, equivalent and improvement made within the spirit and principles of the invention etc., should all include
Within protection scope of the present invention.
Claims (5)
1.1,6- diphosphofructoses and its salt formed are preparing prevention mankind's enveloped virus sense as antiviral activity ingredient
Application in the drug of dye.
2.1,6- diphosphofructoses and its salt formed are preparing prevention mankind's enveloped virus sense as antiviral activity ingredient
It is applied in the virus inactivator of dye.
3. application as described in claim 1, it is characterised in that：Mankind's enveloped virus is Japanese B encephalitis virus
(JEV), dengue virus (DENV), vesicular stomatitis virus (VSV), Respiratory Syncytial Virus(RSV) (RSV) or influenza virus.
4. application as claimed in claim 2, it is characterised in that：Mankind's enveloped virus is Japanese B encephalitis virus
(JEV), dengue virus (DENV), vesicular stomatitis virus (VSV), Respiratory Syncytial Virus(RSV) (RSV) or influenza virus.
5. application as described in claim 1, it is characterised in that：The drug be with 1,6- diphosphofructoses or its formed
Salt as active constituents of medicine, any pharmaceutically acceptable dosage form is made.
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| CN1265397A (en) * | 2000-01-17 | 2000-09-06 | 欧阳平凯 | Preparation of magnesium fructose-1,6-diphosphate |
| CN101904856A (en) * | 2010-08-06 | 2010-12-08 | 南京南农高科兽药研究所有限公司 | Application of 1,6-diphosphofructose and derivative thereof in preparing animal medical health care products |
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|---|---|---|---|---|
| CN1265397A (en) * | 2000-01-17 | 2000-09-06 | 欧阳平凯 | Preparation of magnesium fructose-1,6-diphosphate |
| CN101904856A (en) * | 2010-08-06 | 2010-12-08 | 南京南农高科兽药研究所有限公司 | Application of 1,6-diphosphofructose and derivative thereof in preparing animal medical health care products |
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| WO2020106853A1 (en) * | 2018-11-20 | 2020-05-28 | Exosome Diagnostics, Inc. | Compositions and methods for internal controls of microvesicle isolations |
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