CN106309424B - The application of flavone compound - Google Patents
The application of flavone compound Download PDFInfo
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- CN106309424B CN106309424B CN201610695843.7A CN201610695843A CN106309424B CN 106309424 B CN106309424 B CN 106309424B CN 201610695843 A CN201610695843 A CN 201610695843A CN 106309424 B CN106309424 B CN 106309424B
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- 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
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Abstract
The present invention relates to pharmaceutical technology fields, the in particular to application of flavone compound.The present invention provides application of the flavone compound (I) in the inhibitor of preparation enterovirus, being experimentally confirmed the cytopathy that flavone compound (I) induces enterovirus has inhibiting effect, can improve the survival rate of virus infected cell；The duplication that can inhibit virus in vivo simultaneously, improves the survival rate of virus-infected animal, extends the life span of infection animal, alleviates the illness that virus infection causes, to have the function for the treatment of hand-foot-and-mouth disease and application value.
Description
Technical field
The present invention relates to pharmaceutical technology fields, the in particular to application of flavone compound.
Background technique
Hand-foot-and-mouth disease is children's infectious disease caused by virus, is the legal Class C infectious disease in China.Disease main infection 0-6
The infant in year, most commonly seen with 2-3 years old childhood infection, hand-foot-and-mouth disease early stage goes out in infant limb end and oral cavity etc.
Cause bleb or ulcer, infant recovery from illness mostly in 1-2 weeks.However, a small number of case disease progressions are rapid, it can be on a 1-5 days left sides of morbidity
It is right meningitis, aseptic isolator, brainstem encephalitis, encephalomyelitis, pulmonary edema and dyshaemia etc., the only a few case state of an illness occur
It is critical, it lethal can die, case can there are sequelae for survival.
According to WHO Report, about more than 20 kinds of the virus of hand-foot-and-mouth disease symptom can be caused, wherein with intestines
Road 71 types of virus (enterovirus 71, EV71) and coxsackievirus A16 (coxsachievirus A16, CoxA16)
Most commonly seen, the case load as caused by both virus infections accounts for about total 80% or more case load.Other can cause brothers mouthful
The virus of disease specifically includes that Coxsackie virus A group 4,5,7,9 and 10 types (CoxA4, CoxA5, CoxA7, CoxA9 and CoxA10),
The type of coxsackie virus group B 2 and 5 (CoxB2 and CoxB5) and echovirus (ECHO) etc..
Currently, still without the drug for being directly targeted virus, facing although being directed to the vaccine granted listing of EV71 virus
Bed mainly takes the strategy of symptomatic treatment；And broad-spectrum antiviral medicament Ribavirin has teratogenesis and inhibition when being applied to infant
The risk of growth and development.Therefore, compound provided by the invention can make up the deficiencies in the prior art in this regard.
Model is the carrier of compound activity and the mechanism of action, and hand-foot-and-mouth disease poison, which infects, can induce apoptosis of many kinds, packet
Include the pernicious embryo's rhabdomyoma cell (RD) of people, Neuroepithelioma Cells (SK-N-MC), human neuroblastoma cells (SK-N-
SH) spongioblast oncocyte (SF268), African green monkey kidney cell (Vero), human microvascular endothelial cell (mvec) and HeLa cell
Deng being usually used in the disease-resistant of cellular level since the sensibility of RD cell and Vero cell to virus is preferable in actual research
Poison research.In terms of animal model, the mouse of 1-7 age in days is commonly used for animal model to viral sensitivity.3- after mouse inoculation virus
Occur within 5 days refusing the symptoms such as cream and weight loss, the manifest symptoms such as stiff property paralysis occur in 6-8 days four limbs, according to disease after 9-13 days
Feelings self-healing or death.
Currently, common anti-hand-foot-and-mouth-disease drug has Ribavirin, acyclovir, Valaciclovir etc., but these medicine pairs
It acts on stronger.
In recent years, the research of Chinese medicine anti-hand-foot-and-mouth-disease gradually causes the concern of people, and due to many traditional Chinese medicinal components categories
In naturally extracting drug, have many advantages, such as Small side effects.
Flavanonol (Kanzonol) and neoflavane ketone (Shinflavanone) are the flavones extracted from Chinese medicinal plant
Constituents, pharmacological research show that the pure and mild neoflavane ketone of flavanone has that heart tonifying, diuresis, decompression, antibacterial, to enhance human body immune
Etc. a variety of effects.
The report that the pure and mild neoflavane ketone of flavanone is applied in preparation treatment hand-foot-and-mouth disease drug is had no at present.
Summary of the invention
In view of this, the present invention provides a kind of application of flavone compound.The purpose of the present invention is to provide flavonoids
Application of the pure and mild neoflavane ketone of compound (I), especially flavanone in the inhibitor of preparation enterovirus, present invention research
The cytopathy for showing that flavone compound (I) induces enterovirus has inhibiting effect, can improve depositing for virus infected cell
Motility rate, antiviral activity are obvious.In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides application of the flavone compound (I) in the inhibitor of preparation enterovirus；
Wherein, R=OH or H.
Work as R=OH, which is flavanonol；When R is H, which is new
Flavanones.
In some specific embodiments of the invention, the enterovirus is Coxsackie virus.
In some specific embodiments of the invention, the Coxsackie virus is coxsackie virus A 16-type.
In other specific embodiments of the invention, the enterovirus is enterovirus EV 71 type.
In some specific embodiments of the invention, the active dose of the flavone compound (I) is 50~
200mg/kg/d。
The present invention also provides flavone compounds (I) in the disease of preparation prevention and/or treatment enterovirus infection
Application in drug；
Wherein, R=OH or H.
In some specific embodiments of the invention, the disease of the enterovirus infection is hand-foot-and-mouth disease.In this hair
In bright, the enterovirus is Coxsackie virus.Preferably, the Coxsackie virus is coxsackie virus A 16-type.The enteron aisle
Virus can also be enterovirus EV 71 type.
In some specific embodiments of the invention, the drug includes flavone compound (I) and can pharmaceutically connect
The auxiliary material received.
In some specific embodiments of the invention, the dosage form of the drug is that any one is clinically-acceptable oral
Form of administration, injecting medicine-feeding form or topical administration formulations.
In some specific embodiments of the invention, in the oral administered dosage form, the quality of the acetyl astragalin
Score is 17.5~88%.
In some specific embodiments of the invention, the oral or injection measurement of the drug is 0.5~2.5mg flavones
Class compound (I)/kg body weight/d.
In some specific embodiments of the invention, the drug is tablet, capsule, granule, pill, injection
Agent, soft extract, suspending agent, dispersing agent, syrup, suppository, gelling agent, aerosol, patch.
The present invention provides the application of flavone compound (I) in the inhibitor of preparation enterovirus, and the present invention tests knot
Fruit shows that flavone compound (I) has inhibition to the cytopathy of enterovirus EV 71 type, coxsackie virus A 16-type induction
Effect, can improve the survival rate of virus infected cell；It can inhibit the duplication of virus in vivo simultaneously, improve virus-infected animal
Survival rate, extend the life span of infection animal, alleviate the illness that virus infection causes, to have treatment hand-foot-and-mouth disease
Effect；And can the proliferation and diffusion of pre- anti-virus in vivo, protect normal tissue from damage, play prevention viral infection disease
Pathogenetic effect.
The beneficial effects of the present invention are:
1, the pure and mild neoflavane ketone of flavanone is a kind of natural small molecule compounds, and flavanonol is to enterovirus
EV71, coxsackie virus A 16, EC50Respectively 140.87 μm of ol/L, 148.04 μm of ol/L, SI are respectively 4.54,4.32；It is new yellow
Alkanone is to enterovirus EV 71, coxsackie virus A 16, EC50Respectively 35.89 μm of ol/L, 3.69 μm of ol/L, SI are respectively
37.69、3.51。
2, flavone compound (I) can inhibit to dose-dependant coxsackie virus A 16, enterovirus EV 71 virus sense
Dye is in dose-dependence.
3, alleviate symptom caused by virus, mitigate death caused by virus infection；Extend virus infected mice life span,
Improve mouse survival rate.
Specific embodiment
The invention discloses a kind of application of flavone compound, those skilled in the art can use for reference present disclosure, fit
When improvement realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications are for a person skilled in the art
It is it will be apparent that they are considered as being included in the present invention.Method of the invention and application passed through preferred embodiment into
Gone description, related personnel obviously can not depart from the content of present invention, in spirit and scope to method described herein and application
It is modified or appropriate changes and combinations, carrys out implementation and application the technology of the present invention.
The present invention provides application of the flavone compound (I) in the inhibitor of preparation enterovirus；
Wherein, R=OH or H.
In the present invention, the flavone compound (I) has structure shown in formula I, belongs to flavonoid glycoside, pharmacological research table
It is bright, the pure and mild neoflavane ketone of flavanone have heart tonifying, diuresis, decompression, it is antibacterial, a variety of effects such as human body is immune can be enhanced.
The flavone compound (I) is preferably that flavone compound (I) monomer and/or flavone compound (I) extract
Object, in flavone compound (I) extract, the content of monomer flavone compound (I) is preferably 90% or more, the present invention
It can conventionally extract and obtain from Chinese medicine or the prepared slices of Chinese crude drugs containing flavone compound (I), such as Radix Glycyrrhizae；It can also
Buy the commercial goods of flavone compound (I).Specifically, in an embodiment of the present invention, Chinese drug biology system can be used
Flavone compound (I) provided by product examine is fixed.
In the present invention, the enterovirus is Coxsackie virus.Preferably, the Coxsackie virus is Coxsackie virus
A16 type.
In other embodiments of the invention, the enterovirus is enterovirus EV 71 type.
The present invention also provides flavone compounds (I) in the disease of preparation prevention and/or treatment enterovirus infection
Application in drug.
The present invention provides application of the flavone compound (I) in preparation treatment hand-foot-and-mouth disease drug.Wherein, described
The virus for causing hand-foot-and-mouth disease includes enterovirus.Preferably, the virus for causing hand-foot-and-mouth disease includes Coxsackie virus
A16 type and/or enterovirus EV 71 type.
In the present invention, the anti-hand-foot-and-mouth-disease drug includes flavone compound (I) and pharmaceutically acceptable carrier.
In the present invention, the pharmaceutically acceptable carrier preferably includes starch, low-substituted hydroxypropyl cellulose, superfine silica gel powder, hard
Fatty acid magnesium, starch slurry, sucrose, dextrin, sodium carboxymethyl starch, talcum powder, polysorbate, polyethylene glycol, injection soybean lecithin
One or more of with glycerol for injection；The anti-hand-foot-and-mouth-disease drug is any one clinically-acceptable oral administration agent
Type, injecting medicine-feeding form or topical administration formulations.Preferably, the anti-hand-foot-and-mouth-disease drug be tablet, capsule, granule,
Pill, injection, soft extract, suspending agent, dispersing agent, syrup, suppository, gelling agent, aerosol, patch.The oral administration
In the anti-hand-foot-and-mouth-disease drug of dosage form, the mass fraction of flavone compound (I) is 17.5~88%, more preferably 20~
80%, most preferably 25~75%.
In the present invention, the active dose of the flavone compound (I) is preferably 50-200mg/kg/d；More preferably
100-200mg/kg/d。
In the present invention, the daily oral dose of the anti-hand-foot-and-mouth-disease drug is 0.5~2.5mg flavone compound
(I)/kg body weight, more preferably 0.625~2.0mg flavone compound (I)/kg weight；The anti-hand-foot-and-mouth-disease drug
Daily injection dosage is 0.5~2.5mg flavone compound (I)/kg body weight, more preferably 0.625~2.0mg flavones
Class compound (I)/kg weight.
The present invention does not have special limit to the preparation method of the anti-hand-foot-and-mouth-disease drug comprising flavone compound (I)
System, according to the common pharmaceutical methods of those skilled in the art.
The present invention provides application of the flavone compound (I) in preparation treatment hand-foot-and-mouth disease drug, the present invention is used
Mtt assay determines flavone compound (I) to the inhibitory activity of coxsackie virus A 16-type and enterovirus EV 71 type, as a result
Show flavanonol to enterovirus EV 71, coxsackie virus A 16, EC50Respectively 140.87 μm of ol/L, 148.04 μm of ol/
L, SI are respectively 4.54,4.32；Neoflavane ketone is to enterovirus EV 71, coxsackie virus A 16, EC50Respectively 35.89 μm of ol/
L, 3.69 μm of ol/L, SI are respectively 37.69,3.51.
The present invention has been carried out flavanonol in a manner of stomach-filling (oral) and infected enterovirus EV 71 type induced mice
Curative effect test, the results showed that, flavanonol in dosage 50/100/200mg/kg/day gastric infusion 5 days, deposit by mouse
Motility rate is respectively 30%, 50% and 70%, and Death prevention rate is respectively 12.5%, 37.5% and 62.5%, positive control drug benefit
The mouse survival rate of Ba Weilin is 70%, Death prevention rate 62.5%；The present invention has carried out dihydro in a manner of stomach-filling (oral)
Flavonols tests the curative effect that coxsackie virus A 16-type induced mice infects, the results showed that, flavanonol is in dosage 50/
(oral) administration of stomach-filling 5 days when 100/200mg/kg/day, Death prevention rate is respectively 14.29%, 42.86% and 71.43%,
The mouse survival rate of positive control drug Ribavirin is 80%, Death prevention rate 71.43%,
The present invention has carried out the curative effect that flavanonol infects enterovirus EV 71 type induced mice in a manner of injecting
Test, the results showed that, flavanonol is in dosage 50/100/200mg/kg/day drug administration by injection 5 days, Death prevention rate difference
It is 33.33%, 55.56%, 77.78%；The present invention has carried out flavanonol to coxsackie virus A 16-type in a manner of injecting
The curative effect test of induced mice infection, the results showed that, flavanonol drug administration by injection in dosage 50/100/200mg/kg/day
5 days, Death prevention rate was respectively 37.50%, 50.00%, 87.50%.
As it can be seen that the pure and mild neoflavane ketone of flavanone and positive control drug Ribavirin are to coxsackie virus A 16-type and enteron aisle
The inhibitory effect of viral EV71 type is similar, and flavone compound (I) can inhibit the duplication of virus, improves virus-infected animal
Survival rate extends the life span of infection animal, alleviates the illness that virus infection causes, to have the work for the treatment of hand-foot-and-mouth disease
With.
In order to further illustrate the present invention, flavone compound provided by the invention (I) is being made with reference to embodiments
Application in standby treatment hand-foot-and-mouth disease drug is described in detail, but cannot be understood as the limit to the scope of the present invention
It is fixed.
Raw materials used and reagent is available on the market in the application of flavone compound provided by the invention.
Below with reference to embodiment, the present invention is further explained:
Protective effect of 1 flavanonol of embodiment to EV71 and CoxA16 virus infected cell
1, experimental material:
Coxsackie virus A 16-type (CoxA16) and enterovirns type 71 (EV71), by Chinese People's Liberation Army's military medicine
Academy of sciences microorganism epidemic research immunological investigation room provides, and is saved by Jiangsu Kang Yuan R&D of modern TCM institute, thin in Vero
Passage in born of the same parents' culture, -80 DEG C of preservations.
African green monkey kidney cell (Vero cell), is purchased from U.S.'s ATCC cell bank, by Jiangsu Kang Yuan R&D of modern TCM institute
It saves.
DMEM culture medium is purchased from Nanjing KaiJi Biology Science Development Co., Ltd, lot number: 20141024, cell growth medium
In contain 10% fetal calf serum, 1 × 105U/L penicillin, 100mg/L streptomysin.In cell maintenance medium in addition to containing 2% fetal calf serum,
Other same cell growth mediums.Ribavirin refines medicine purchased from medicine three is breathed out, and national drug standard H19993937, specification: 1ml:0.1g is criticized
Number: 140312.
M2e type microplate reader, Molecular Devices；Pipettor, eppendorf company；Biohazard Safety Equipment is purchased from
Heal Force company, model: HFsafe-1200；Carbon dioxide incubator is purchased from Thermo Scientific company, type
Number: FormaSteri-Cycle 371.
2, experimental method:
Vero cell (African green monkey kidney cell) presses 5 × 10 with the DMEM culture medium containing 2% fetal calf serum4A/ml concentration
96 well culture plates are inoculated with, every 100 μ l of hole sets 37 DEG C of 5%CO2Overnight incubation forms cell monolayer in incubator.After abandoning supernatant,
Cell is washed 1 time with PBS, to wash away remaining fetal calf serum, is added and is diluted to the DMEM culture medium without fetal calf serum
100TCID50Each strain virus (EV71 and CoxA16) after every hole 100 μ l, 35 DEG C of incubation 2h, discards virus liquid.With 2% tire ox blood
Flavanonol is configured to various concentration by clear DMEM culture medium, is separately added into Vero cell monolayer, if 3 multiple holes,
Vero cell is in 37 DEG C, 5%CO248h is cultivated in incubator, observes cytopathic phenomena daily.After 48h, after abandoning supernatant, it is added
100 μ l, 10% formaldehyde fixes 1h, and 0.1% (w/v) violet staining 15min, microplate reader 570nm measure absorbance.Experiment is heavy altogether
It is 3 times multiple.Viral suppression is calculated, and calculates flavanonol to two kinds of viral EC50(selection refers to by (medium effective concentration), SI
Number).
1 flavanonol In vitro antibacterial test (μM) of table
Table 1 is as the result is shown: flavanonol has obvious suppression to EV71 and CoxA16 virus institute's cytopathogenic effect in vitro
Production is used, EC50Respectively 140.87,148.04 μM, SI is respectively 4.54,4.32；Have and is applied to the sense of anti-hand-foot-and-mouth-disease poison
Contaminate the Pharmacodynamics in vitro basis for the treatment of.
The therapeutic effect infected EV71 is administered orally in 2 flavanonol of embodiment
1, experimental material:
Enterovirns type 71 (EV71) is immunized by Academy of Military Medicine, PLA's microorganism epidemic research
It learns research department to provide, is saved by Jiangsu Kang Yuan R&D of modern TCM institute, passed in Vero cell culture, -80 DEG C of preservations.
The 15-16 days pregnant mouse of BALB/c of SPF grades of pregnancies are purchased from Jiangning county's Qinglongshan experimental animal breeding field, raising
In IVC system (18~28 DEG C of temperature, 40~70% relative humidity, mechanical supply and exhaust；The light and shade period: 12h/12h；150~
300Lux illumination), take 3 age in days Neonatal Mouses to carry out experiment.
Ribavirin is purchased from and breathes out the purification medicine of medicine three, national drug standard H19993937, specification: 1ml:0.1g, lot number:
140312。
2, experimental method:
3 age in days BALB/c suckling mouses are randomly divided into normal group, model group, ribavirin granule group 100mg/kg/d, two by nest
Hydrogen flavonols low dose group 50mg/kg/d, middle dose group 100mg/kg/d, high dose group 200mg/kg/d, are removed just by every group 10
Outside normal control group, EV71 viral suspension (10 is injected intraperitoneally in remaining each group7TCID50) infected, 0.1mL/ only, each administration group with
Gastric infusion, each 0.1ml continuous 5 days, are put with after 75% ethyl alcohol sprinkling suckling mouse package padding after being administered or infect every time
It is raised jointly in the withdrawal of currency from circulation with female rat, Normal group and virus control group give distilled water.The life of suckling mouse is observed after infection daily
State is deposited, observes 14d altogether, and according to following scale, the severity 0 for calculating infection is divided: health；1 point: the back of a bow, perpendicular hair
(being observed after growing hair), thin, activity reduction etc.；2 points: hind leg diminished strength；3 points: unilateral hindlimb paralysis or paralysis；4 points: double
Side hindlimb paralysis or paralysis；5 points: dying or dead.It calculates each group death rate and increase in life span and is united with SPSS software
Meter analysis.
Influence (n=10) of the 2 flavanonol gastric infusion of table to EV71 infection BALB/c suckling mouse gradient of infection
P is compared with model group
Dead protective effect of the 3 flavanonol gastric infusion of table to EV71 infection Babl/c suckling mouse
P < 0.05 * P < 0.01, * is compared with model group
Table 2 can significantly slow EV71 and CoxA16 virus the results show that after each dosage group gastric infusion of flavanonol
Syntexis caused by suckling mouse is infected, hind leg diminished strength, the symptoms such as unilateral hindlimb paralysis, gradient of infection integral is compared with model group
All have significant difference；Table 3 the results show that the Death prevention rate of the high, medium and low dosage of flavanonol be respectively 62.50%,
37.50%, 12.5%, increase in life span is respectively 78.45%, 52.36%, 32.16%, shows each dosage group of flavanonol
Gradient of infection significantly reduces the EV71 virus infection suckling mouse death rate and gradient of infection, extends its life span, with model group ratio
Relatively there is significant difference, prompt flavanonol that there is therapeutic effect to the infection of brothers' Aphthovirus.
The therapeutic effect infected CoxA16 is administered orally in 3 flavanonol of embodiment
1, experimental material:
Coxsackie virus A 16-type (Cox A16), is ground by Academy of Military Medicine, PLA's microorganism epidemic disease
The offer of immunological investigation room is provided, is saved by Jiangsu Kang Yuan R&D of modern TCM institute, is passed in Vero cell culture, -80 DEG C of guarantors
It deposits.
The 15-16 days pregnant mouse of BALB/c of SPF grades of pregnancies are purchased from Jiangning county's Qinglongshan experimental animal breeding field, raising
In IVC system (18~28 DEG C of temperature, 40~70% relative humidity, mechanical supply and exhaust；The light and shade period: 12h/12h；150~
300Lux illumination), take 3 age in days Neonatal Mouses to carry out experiment.
Flavanonol is made by oneself by Kangyuan Pharmaceutical Co., Ltd., Jiangsu Prov, purity 97.64%.Ribavirin, purchased from Kazakhstan
Medicine three refines medicine, national drug standard H19993937, specification: 1ml:0.1g, lot number: 140312.
2, experimental method:
3 age in days BALB/c suckling mouses are randomly divided into normal group, model group, ribavirin granule group 200mg/kg/d, two by nest
Hydrogen flavonols low dose group 50mg/kg/d, middle dose group 100mg/kg/d, high dose group 200mg/kg/d, are removed just by every group 10
Outside normal control group, CoxA16 viral suspension (10 is injected intraperitoneally in remaining each group7TCID50) infected, 0.1mL/, each administration group
With gastric infusion, each 0.1ml, continuous 5 days, after wrapping up padding with 75% ethyl alcohol sprinkling suckling mouse after being administered or infect every time,
It puts back in cage and is raised jointly with female rat, Normal group and virus control group give distilled water.Suckling mouse is observed after infection daily
Survival condition observes 14d altogether, and according to following scale, the severity 0 for calculating infection is divided: health；1 point: hunchbacked, perpendicular
Hair (being observed after growing hair), thin, activity reduction etc.；2 points: hind leg diminished strength；3 points: unilateral hindlimb paralysis or paralysis；4 points:
Bilateral hindlimb paralysis or paralysis；5 points: dying or dead.It calculates each group death rate and increase in life span and is carried out with SPSS software
Statistical analysis.
Influence (n=10) of the 4 flavanonol gastric infusion of table to CoxA16 infection BALB/c suckling mouse gradient of infection
P is compared with model group
Dead protective effect of the 5 flavanonol gastric infusion of table to CoxA16 infection Babl/c suckling mouse
P < 0.05 * P < 0.01, * is compared with model group
Table 4 can significantly slow CoxA16 virus infection cream the results show that after each dosage group gastric infusion of flavanonol
Syntexis caused by mouse, hind leg diminished strength, the symptoms such as unilateral hindlimb paralysis, gradient of infection integral all have compared with model group
Significant difference；Table 5 the results show that the Death prevention rate of the high, medium and low dosage of flavanonol be respectively 71.43%,
42.86%, 14.29%, increase in life span is respectively 80.45%, 55.37%, 35.18%, shows each dosage of flavanonol
Group gradient of infection significantly reduces the CoxA16 virus infection suckling mouse death rate and gradient of infection, extends its life span, with model
Group compares with significant difference, and flavanonol is prompted to have therapeutic effect to the infection of brothers' Aphthovirus.
Protective effect of the 4 neoflavane ketone of embodiment to EV71 and CoxA16 virus infected cell
1, experimental material:
Coxsackie virus A 16-type (Cox A16) and enterovirns type 71 (EV71), by Chinese People's Liberation Army's military medicine
Academy of sciences microorganism epidemic research immunological investigation room provides, and is saved by Jiangsu Kang Yuan R&D of modern TCM institute, thin in Vero
Passage in born of the same parents' culture, -80 DEG C of preservations.
African green monkey kidney cell (Vero cell), is purchased from U.S.'s ATCC cell bank, by Jiangsu Kang Yuan R&D of modern TCM institute
It saves.
DMEM culture medium is purchased from Nanjing KaiJi Biology Science Development Co., Ltd, lot number: 20141024, cell growth medium
In contain 10% fetal calf serum, 1 × 105U/L penicillin, 100mg/L streptomysin.In cell maintenance medium in addition to containing 2% fetal calf serum,
Other same cell growth mediums.Neoflavane ketone, is made by oneself by Kangyuan Pharmaceutical Co., Ltd., Jiangsu Prov, purity 98.97%.Li Bawei
Woods refines medicine, national drug standard H19993937, specification: 1ml:0.1g, lot number: 140312 purchased from medicine three is breathed out.
M2e type microplate reader, Molecular Devices；Pipettor, eppendorf company；Biohazard Safety Equipment is purchased from
Heal Force company, model: HFsafe-1200；Carbon dioxide incubator is purchased from Thermo Scientific company, type
Number: FormaSteri-Cycle 371.
2, experimental method:
After neoflavane ketone toxicity test Vero cell is digested with pancreatin, adjustment cell density to 1 × 105A/ml, every hole
100 μ l are inoculated with 96 well culture plates, set 37 DEG C of 5%CO2Supernatant is abandoned after cultivating 2h in incubator, 200 μ l ketone containing neoflavane are added
(DMSO is molten) culture medium (drug 4 times of dilutions, 7 gradients, 2.5%FBS from 400 μM), if control group (containing 1/1000DMSO),
Every group of 3 multiple holes, MTS reagent box measures cell viability after cultivating 72h, and 490nm detects absorbance, calculates each dosage neoflavane ketone
To cytotoxicity.In GraphPad Prism5 statistical software, each drug dose and poisonous effect are constructed with nonlinear regression
Relation curve calculates neoflavane ketone to the maximal non-toxic concentration (TC of Vero cell0) and half toxic concentration (TC50)。
Influence of the neoflavane ketone to EV71 and CoxA16 cytopathic effect takes the culture plate for having covered with cell monolayer, inhales
Culture solution is abandoned, with 100TCID50Cell is added in corresponding virus attack amount, and every 100 μ l, Vero cell of hole, which is used, contains 2% fetal calf serum
DMEM culture medium press 5 × 104A/ml concentration is inoculated with 96 well culture plates, every hole 100 μ l, 37 DEG C of 5%CO2Incubator adsorbs 2h
Afterwards, virus liquid is discarded.Neoflavane ketone is configured to maximal non-toxic concentration with the DMEM culture medium of 2% fetal calf serum, single layer is added
In Vero cell, if 6 multiple holes.Ribavirin is positive control medicine, at the same set cell controls group (be not added viral not dosing),
Virus control group (add virus but drug is not added) and blank control group (cell-free, viral not dosing is not added).37 DEG C, 5%CO2Training
It supports MTS reagent box after cultivating 72h in case and measures cell viability, 490nm detects absorbance A.Neoflavane ketone is calculated according to the following formula
To the inhibiting rate of virus.
Viral suppression=(A490 drug-A490 virus)/(normal-A490 virus of A490) × 100%
EC50The DMEM culture medium containing 2% fetal calf serum is used to press 5 × 10 with SI measurement Vero cell4A/ml concentration inoculation 96
Well culture plate, every 100 μ l of hole, sets 37 DEG C of 5%CO2Overnight incubation forms cell monolayer in incubator.It, will with PBS after abandoning supernatant
Cell washs 1 time, to wash away remaining fetal calf serum, is added and is diluted to 100TCID with the DMEM culture medium without fetal calf serum50
Virus after every hole 100 μ l, 35 DEG C of incubation 2h, discards virus liquid.Neoflavane ketone is matched with the DMEM culture medium of 2% fetal calf serum
Various concentration is made, is separately added into Vero cell monolayer, if 3 multiple holes, Vero cell is in 37 DEG C, 5%CO2It is trained in incubator
48h is supported, observes cytopathic phenomena daily.After 48h, after abandoning supernatant, 100 μ l, 10% formaldehyde fixed 1h, 0.1% (w/v) is added
Violet staining 15min, microplate reader 570nm measure absorbance.Experiment is repeated 3 times altogether.Viral suppression is calculated, and is calculated new yellow
EC of the alkanone to two kinds of influenza virus cells50(medium effective concentration) and SI (selection index).
6 neoflavane ketone In vitro antibacterial test (μM) of table
Inhibiting rate (%) of the 7 neoflavane ketone of table to EV71 and Cox A16 cytopathogenic effect
*P < 0.05,**P < 0.01, compared with virus control；△P < 0.05,△△P < 0.01, compared with Ribavirin
Table 6 is as the result is shown: neoflavane ketone has certain inhibiting effect, EC to enterovirus type EV71, CoxA16 virus50
Respectively 35.89,37.69 μM, SI is respectively 3.69,3.51；Table 7 is as the result is shown: neoflavane ketone is right under maximal non-toxic concentration
EV71 and Cox A16 has significant statistical difference by apparent inhibiting effect compared with virus control group.Experimental result table
Bright neoflavane ketone is inhibited to the above strain replication in vitro, improves the survival rate of virus infected cell.
The therapeutic effect that 5 neoflavane ketone of embodiment infects enterovirus EV 71
1, experimental material:
Enterovirns type 71 (EV71) is immunized by Academy of Military Medicine, PLA's microorganism epidemic research
It learns research department to provide, is saved by Jiangsu Kang Yuan R&D of modern TCM institute, passed in Vero cell culture, -80 DEG C of preservations.
The 15-16 days pregnant mouse of BALB/c of SPF grades of pregnancies are purchased from Jiangning county's Qinglongshan experimental animal breeding field, raising
In IVC system (18~28 DEG C of temperature, 40~70% relative humidity, mechanical supply and exhaust；The light and shade period: 12h/12h；150~
300Lux illumination), take 3 age in days Neonatal Mouses to carry out experiment.
Neoflavane ketone, is made by oneself by Kangyuan Pharmaceutical Co., Ltd., Jiangsu Prov, purity 98.97%.Ribavirin, purchased from Kazakhstan medicine
Three purification medicines, national drug standard H19993937, specification: 1ml:0.1g, lot number: 140312.2, experimental method:
3 age in days BALB/c suckling mouses are randomly divided into normal group, model group, Ribavirin group 100mg/kg/d, neoflavane by nest
Ketone low dose group 50mg/kg/d, middle dose group 100mg/kg/d, high dose group 200mg/kg/d, remove normal control by every group 10
Group is outer, and enterovirus EV 71 virus liquid (10 is injected intraperitoneally in remaining each group7TCID50) infected, 0.1mL/ only, each administration group with
Gastric infusion, each 0.1ml continuous 5 days, are put with after 75% ethyl alcohol sprinkling suckling mouse package padding after being administered or infect every time
It is raised jointly in the withdrawal of currency from circulation with female rat, Normal group and virus control group give distilled water.The life of suckling mouse is observed after infection daily
State is deposited, observes 14d altogether, and according to following scale, the severity 0 for calculating infection is divided: health；1 point: the back of a bow, perpendicular hair
(being observed after growing hair), thin, activity reduction etc.；2 points: hind leg diminished strength；3 points: unilateral hindlimb paralysis or paralysis；4 points: double
Side hindlimb paralysis or paralysis；5 points: dying or dead.It calculates each group death rate and increase in life span and is united with SPSS software
Meter analysis.
Influence (n=10) of the 8 neoflavane ketone therapeutic administratp of table to enterovirus EV 71 infection BALB/c suckling mouse gradient of infection
*P < 0.05,**P < 0.01, compared with model group
Table 8 can significantly slow enterovirus EV 71 the results show that after each dosage group progress therapeutic of neoflavane ketone
Syntexis caused by suckling mouse is infected, hind leg diminished strength, the symptoms such as unilateral hindlimb paralysis, gradient of infection integral is compared with model group
All have significant difference.
9 neoflavane ketone therapeutic administratp enterovirus EV 71 of table infects suckling mouse survival rate and life span (n=10)
*P < 0.05,**P < 0.01, compared with model group
Table 9 the results show that the Death prevention rate of the high, medium and low dosage of neoflavane ketone be respectively 77.78%, 55.56%,
33.33%, increase in life span is respectively 48.96%, 40.39%, 28.89%, shows each dosage group gradient of infection of neoflavane ketone
The enterovirus EV 71 infection suckling mouse death rate and gradient of infection are significantly reduced, its life span is extended, is had compared with model group
There is significant difference, prompts neoflavane ketone that there is therapeutic effect to the infection of enterovirus EV 71.
The therapeutic effect that 6 neoflavane ketone of embodiment infects coxsackie virus A 16-type
1, experimental material:
Coxsackie virus A 16-type (Cox A16), is ground by Academy of Military Medicine, PLA's microorganism epidemic disease
The offer of immunological investigation room is provided, is saved by Jiangsu Kang Yuan R&D of modern TCM institute, is passed in Vero cell culture, -80 DEG C of guarantors
It deposits.
The 15-16 days pregnant mouse of BALB/c of SPF grades of pregnancies are purchased from Jiangning county's Qinglongshan experimental animal breeding field, raising
In IVC system (18~28 DEG C of temperature, 40~70% relative humidity, mechanical supply and exhaust；The light and shade period: 12h/12h；150~
300Lux illumination), take 3 age in days Neonatal Mouses to carry out experiment.
Neoflavane ketone, is made by oneself by Kangyuan Pharmaceutical Co., Ltd., Jiangsu Prov, purity 98.97%.Ribavirin, purchased from Kazakhstan medicine
Three purification medicines, national drug standard H19993937, specification: 1ml:0.1g, lot number: 140312.2, experimental method:
3 age in days BALB/c suckling mouses are randomly divided into normal group, model group, Ribavirin group 100mg/kg/d, neoflavane by nest
Ketone low dose group 50mg/kg/d, middle dose group 100mg/kg/d, high dose group 200mg/kg/d, remove normal control by every group 10
Group is outer, and coxsackie virus A 16-type (Cox A16) virus liquid (10 is injected intraperitoneally in remaining each group7TCID50) infected, 0.1mL/
Only, each administration group continuous 5 days, sprays suckling mouse with 75% ethyl alcohol after administration or infection every time with gastric infusion, each 0.1ml
After wrapping up padding, puts back in cage and raised jointly with female rat, Normal group and virus control group give distilled water.It is daily after infection
The survival condition for observing suckling mouse, observes 14d, and according to following scale, the severity 0 for calculating infection is divided altogether: health；1
Point: the back of a bow, perpendicular hair (observing after growing hair), thin, activity are reduced etc.；2 points: hind leg diminished strength；3 points: unilateral hindlimb paralysis
Or paralysis；4 points: bilateral hindlimb paralysis or paralysis；5 points: dying or dead.It calculates each group death rate and increase in life span is used in combination
SPSS software is for statistical analysis.
Influence (n=of the 10 neoflavane ketone therapeutic administratp of table to coxsackie virus A 16-type infection BALB/c suckling mouse gradient of infection
10)
*P < 0.05,**P < 0.01, compared with model group
Table 10 can significantly slow Coxsackie virus the results show that after each dosage group progress therapeutic of neoflavane ketone
A16 type infects syntexis caused by suckling mouse, hind leg diminished strength, the symptoms such as unilateral hindlimb paralysis, gradient of infection integral and model
Group, which compares, all has significant difference.
Coxsackie virus A 16-type infection suckling mouse survival rate and life span (n=10) after 11 neoflavane ketone therapeutic administratp of table
*P < 0.05,**P < 0.01, compared with model group
Table 11 the results show that the Death prevention rate of the high, medium and low dosage of neoflavane ketone be respectively 87.50%, 50.00%,
37.50%, increase in life span is respectively 41.43%, 28.04%, 22.12%, shows each dosage group gradient of infection of neoflavane ketone
Coxsackie virus A 16-type (Cox A16) infection suckling mouse death rate and gradient of infection are significantly reduced, its life span is extended, with
Model group compares with significant difference, and neoflavane ketone is prompted to there is treatment to make coxsackie virus A 16-type (Cox A16) infection
With.
7 flavanonol of embodiment is preparing the application in capsule medicine
The pure and mild 32g starch of 350g flavanone, 6g low-substituted hydroxypropyl cellulose, 4.5g superfine silica gel powder, 1.5g is stearic
Sour magnesium and the mixing of appropriate 10% starch slurry, are packed into capsule, obtain capsule preparations 1000 of flavanonol.3 times a day, often
Secondary 1.
8 neoflavane ketone of embodiment is preparing the application in granules medicine
350g neoflavane ketone and 1000g sucrose and 500g dextrin are mixed, 1000 packet neoflavanes are conventionally made
Ketone granule.3 times a day, 1 tablet each time.
9 flavanonol of embodiment is preparing the application in tablet medicine
The pure and mild 50g starch of 350g flavanone, 7.5g sodium carboxymethyl starch, 0.8g talcum powder, 50g dextrin, 0.8g is hard
Fatty acid magnesium and the suitable mixing of appropriate 10% starch slurry, are conventionally made 1000, flavanonol tablet.3 times a day, often
Secondary 1.
Application of the 10 neoflavane ketone of embodiment in preparation pill medicine
350g neoflavane ketone and 12g polyethylene glycol-6000,80.5g Tween-80, appropriate liquid paraffin are mixed, pressed
Neoflavane ketone pill 1000 is made in more solito.3 times a day, 1 tablet each time.
11 flavanonol of embodiment is preparing the application in injection medicine
By the pure and mild 15g injection soybean lecithin of 200g flavanone, 25g glycerol for injection, water for injection is settled to
1000mL is conventionally made flavanonol injection 1000.One time a day, every time 1,250mL is at least used
Intravenous drip after the dilution of 5% glucose injection.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (7)
1. application of the flavone compound (I) in the inhibitor of preparation enterovirus；
Formula (I)
Wherein, R=OH or H；
The enterovirus is coxsackie virus A 16-type or enterovirus EV 71 type.
2. application according to claim 1, which is characterized in that the active dose of the flavone compound (I) be 50 ~
200 mg/kg/d。
3. application of the flavone compound (I) in the drug of preparation prevention and/or the disease for the treatment of enterovirus infection；
Formula (I)
Wherein, R=OH or H；
The enterovirus is coxsackie virus A 16-type or enterovirus EV 71 type.
4. application according to claim 3, which is characterized in that the disease of the enterovirus infection is hand-foot-and-mouth disease.
5. application according to claim 4, which is characterized in that the drug includes flavone compound (I) and pharmaceutically
Acceptable auxiliary material；
The dosage form of the drug is any one clinically-acceptable oral administered dosage form, injecting medicine-feeding form or topical administration system
Agent.
6. application according to claim 5, which is characterized in that in the oral administered dosage form, the flavone compound
(I) mass fraction is 17.5 ~ 88%.
7. according to the described in any item applications of claim 3 to 6, which is characterized in that the oral or injection dosage of the drug is
0.5 ~ 2.5mg flavone compound (I)/kg body weight/d.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998009652A1 (en) * | 1996-09-06 | 1998-03-12 | Otsuka Pharmaceutical Co., Ltd. | COMPOSITION, ANTIMICROBIAL AGENT, INFECTION PREVENTIVE, AND FOOD AGAINST $i(HELICOBACTER PYLORI) |
| CN101711796A (en) * | 2008-10-06 | 2010-05-26 | 大百汇生物科技（深圳）有限公司 | Application of scullcapflavone in preparing medicament for treating enterovirus infection |
-
2016
- 2016-08-19 CN CN201610695843.7A patent/CN106309424B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998009652A1 (en) * | 1996-09-06 | 1998-03-12 | Otsuka Pharmaceutical Co., Ltd. | COMPOSITION, ANTIMICROBIAL AGENT, INFECTION PREVENTIVE, AND FOOD AGAINST $i(HELICOBACTER PYLORI) |
| CN101711796A (en) * | 2008-10-06 | 2010-05-26 | 大百汇生物科技（深圳）有限公司 | Application of scullcapflavone in preparing medicament for treating enterovirus infection |
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