CN106344575B - Application of the alkaloid compound in the drug of preparation treatment viral disease - Google Patents
Application of the alkaloid compound in the drug of preparation treatment viral disease Download PDFInfo
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/439—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom the ring forming part of a bridged ring system, e.g. quinuclidine
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Abstract
The invention belongs to pharmaceutical technology fields, provide application of the alkaloid compound in the drug of preparation treatment viral disease.The alkaloid compound is specially pyridine ketone in pyridine and Usu in Usu, can prepare or isolate and purify from natural products by chemical synthesis and obtains.Being experimentally confirmed alkaloid compound has inhibiting effect to the cytopathy of enterovirus, herpes simplex virus induction, the survival rate of virus infected cell can be improved, inhibit the duplication of virus, improve the survival rate of virus-infected animal, extend the life span of infection animal, alleviates the illness that virus infection causes.Therefore, it can be applied to the drug of preparation treatment viral disease.The treatment viral disease includes the alkaloid compound, and pharmaceutically acceptable carrier；The clinically-acceptable oral administered dosage form of the treatment viral disease, injecting medicine-feeding form or topical application.
Description
Technical field
The invention belongs to pharmaceutical technology fields more particularly to alkaloid compound in the medicine for preparing treatment viral disease
Application in object.
Background technique
Hand-foot-and-mouth disease (HFMD) is the disease of the common transmittable as caused by a variety of enteroviruses, mainly passes through alimentary canal, respiratory tract
Propagated with the approach such as close contact, fallen ill with infant based on.Hand-foot-and-mouth disease is mainly by RNA virus section enterovirus genus Coxsack
Virus A 16-type (CoxA 16) and enterovirns type 71 (EV 71) infection cause, the most serious with 71 type viruses.The main table of HFMD
It is now fever with myoclonia, stomatocace bleb and limb end blister sample fash, can seriously causes encephalitis, brain stem brain
A variety of diseases relevant to nervous system such as inflammation, polio sample paralysis, acute flaccid paralysis, harm are serious.Currently, to the greatest extent
Pipe is directed to the vaccine granted listing of EV71 virus, but still without the drug for being directly targeted virus, clinic is mainly taken to the ill
The strategy for the treatment of；And broad-spectrum antiviral medicament Ribavirin has teratogenesis when being applied to infant and inhibits the wind of growth and development
Danger.
Herpes simplex virus (HSV) belongs to Herpesvirus Alphaherpesvirinae, is a kind of tunicary linear double-strand
DNA virus enters in vivo through respiratory tract, oral cavity, eye, genital mucosa and damaged skin, sense big with host's variation range
The features such as dye position is extensive, destructible infection cell and latent infection.HSV mainly includes 1 type of herpesviral (HSV-1) and bleb
Viral 2 types (HSV-2) amphitypy, HSV-1 main infection mouth, eye, the skin of lip and mucous membrane and central nervous system, can cause skin
Skin herpe simplex, herpes labialis, serious corneal infection (keratitis) and inflammation of the central nervous system (encephalitis), caused by HSV-1
It may threat to life if encephalitis is not treated in time.HSV-2 is generally related with external genital organs infection and infection of newborn, can make
At genital herpes, infection of pregnant women HSV-2 easily miscarries or causes fetus congenital malformation and feeblemindedness, and newborn is passing through birth canal
It is even dead to occur high fever, expiratory dyspnea and central nervous system pathological change after being infected by HSV-2.Medical field research in recent years shows
HSV-1 and HSV-2 may be related with lip cancer and uterine neck carcinogenesis respectively, and the infection of HSV-2 external genital organs has become and is only second to
The sexually transmitted disease of AIDS viral infection, therefore HSV causes the new attention of medical field again.
The choice drug of clinical anti-HSV is acyclovir now, is known as in anti-HSV drug development history with its high-efficiency low-toxicity
Milestone, but its antiviral spectrum is narrow, is also easy to produce drug resistance and expensive, its clinical application is made to be very limited.In addition,
When the drug therapies such as idoxuridine, arabinosy ladenosine HSV infects, often there is drug resistance phenomenon, be difficult to clean off virus lays dormant.There are also some
Chemical classes drug is mostly applied because having stronger cytotoxicity or adverse reaction less.Since the gene of HSV is double-stranded DNA,
Integration easily occurs with host DNA, or forms latence, limits the effect of viral vaccine.
Therefore, find it is a kind of can effectively antagonism coxsackie virus A 16-type, enterovirus EV 71 type and/or herpesviral
Drug becomes those skilled in the art's urgent problem to be solved.
Summary of the invention
In view of this, the purpose of the present invention is to provide alkaloid compounds in the drug for preparing treatment viral disease
In application.
Specific technical solution is as follows:
Application of the alkaloid compound shown in formula (I) in the drug of preparation treatment viral disease,
Wherein, R is-OH or=O.
When R is-OH, the alkaloid compound (I) is pyridine in Usu at this time；When R is=O, the at this time alkaloids
Closing object (I) is pyridine ketone in Usu.
Preferably, the virus is enterovirus and/or herpes simplex virus.
Preferably, the enterovirus is enterovirus EV 71 type.
Preferably, the enterovirus is Coxsackie virus.Further, the Coxsackie virus is Coxsackie virus
A16 type.
Preferably, the herpes simplex virus is herpes simplex virus type 1 and/or herpes simplex virus type 2.
In some embodiments, the virus is enterovirus, the activity of alkaloid compound shown in the formula (I)
Dosage is 50-200mg/kg/d.
Further, in some preferred embodiments, the virus is coxsackie virus A 16-type and/or enterovirus
EV71 type, the active dose of alkaloid compound shown in the formula (I) are 50-200mg/kg/d.
In some embodiments, the virus is herpes simplex virus, alkaloid compound shown in the formula (I)
Active dose is 30-200mg/kg/d.
Further, in some preferred embodiments, the virus is herpes simplex virus type 1 and/or herpe simplex
Viral 2 types, the active dose of alkaloid compound shown in the formula (I) are 30-200mg/kg/d.
Preferably, the drug includes alkaloid compound and pharmaceutically acceptable carrier shown in the formula (I).
Preferably, the drug is any one clinically-acceptable oral administered dosage form, injecting medicine-feeding form or external application
Form of administration.
Preferably, the drug is tablet, capsule, granule, pill, liquid preparation, soft extract, suspending agent, dispersion
Agent, syrup, suppository, gelling agent, aerosol, patch
The present invention provides the answering in the drug of preparation treatment viral disease of alkaloid compound shown in formula (I)
With, be experimentally confirmed alkaloid compound shown in formula (I) to enterovirus, herpes simplex virus induction cytopathy
Change has inhibiting effect, can improve the survival rate of virus infected cell, inhibits the duplication of virus, improves the life of virus-infected animal
Rate is deposited, the life span of infection animal is extended, alleviates the illness that virus infection causes.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1 is alkaloid compound (I) chemical structural formula.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical scheme in the embodiment of the invention is clearly and completely described,
Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based in the present invention
Embodiment, every other embodiment obtained by those of ordinary skill in the art without making creative efforts, all
Belong to the scope of protection of the invention.
The present invention provides the answering in the drug of preparation treatment viral disease of alkaloid compound shown in formula (I)
With alkaloid compound shown in the formula (I) is pyridine ketone in pyridine (Ussuriedine) in Usu and Usu
(Ussuriedinone)。
In an embodiment of the present invention, using pyridine in pyridine in the Usu of Nat'l Pharmaceutical & Biological Products Control Institute's offer and Usu
Ketone.
The present invention provides the answering in the drug of preparation treatment viral disease of alkaloid compound shown in formula (I)
With.Wherein, the virus is enterovirus and/or herpes simplex virus.Preferably, the enterovirus is enterovirus EV 71
Type.
Preferably, the enterovirus is Coxsackie virus.Further, the Coxsackie virus is Coxsackie virus
A16 type.
Preferably, the herpes simplex virus is herpes simplex virus type 1 and/or herpes simplex virus type 2.
In the present invention, the drug includes alkaloid compound and pharmaceutically acceptable carrier shown in formula (I).?
In the present invention, the pharmaceutically acceptable carrier preferably includes starch, low-substituted hydroxypropyl cellulose, superfine silica gel powder, tristearin
Sour magnesium, starch slurry, sucrose, dextrin, sodium carboxymethyl starch, talcum powder, polysorbate, polyethylene glycol, injection soybean lecithin and
One or more of glycerol for injection
In the present invention, the drug be any one clinically-acceptable oral administered dosage form, injecting medicine-feeding form or
Topical administration formulations.
Preferably, the drug is tablet, capsule, granule, pill, liquid preparation, soft extract, suspending agent, dispersion
Agent, syrup, suppository, gelling agent, aerosol, patch.
Preparation method of the present invention to the treatment viral disease drug comprising alkaloid compound shown in formula (I)
There is no special limitation, according to the common pharmaceutical methods of those skilled in the art.
The present invention uses protection of the pyridine to EV71 and CoxA16 virus infection Vero cell in external colorimetric method for determining Usu
Effect, the results showed that, pyridine has obvious inhibiting effect to EV71 and CoxA16 virus institute's cytopathogenic effect in vitro in Usu,
EC50 is respectively 245.23 μm of ol/L, 217.22 μm of ol/L, and SI is respectively 3.40,3.83.
The present invention has carried out what enterovirus EV 71 type induced mice was infected in pyridine in Usu in a manner of stomach-filling (oral)
Curative effect test, the results showed that, pyridine stomach-filling in dosage 50mg/kg/day, 100mg/kg/day, 200mg/kg/day is given in Usu
Medicine 5 days, the survival rate of mouse was respectively 50%, 60% and 90%, and Death prevention rate is respectively 44.44%, 55.56% and
77.78%, the mouse survival rate of positive control drug Ribavirin is 70%, Death prevention rate 66.67%.The present invention is with stomach-filling
The mode of (oral) has carried out pyridine in Usu and has tested the curative effect that coxsackie virus A 16-type induced mice infects, the results showed that, crow
Pyridine is in dosage 50mg/kg/day, 100mg/kg/day, 200mg/kg/day gastric infusion 5 days in Soviet Union, the survival rate point of mouse
Not Wei 40%, 60% and 70%, Death prevention rate is respectively 33.33%, 55.56% and 66.67%, positive control drug Li Bawei
The mouse survival rate of woods is 60%, Death prevention rate 55.56%；The inhibitory effect of pyridine and positive control drug Ribavirin in Usu
It is similar, dead mouse caused by virus can be alleviated to some extent, extend the mouse survival time, and can be inhibited caused by virus
Inflammatory factor release, alleviates the symptoms such as inflammation.
Meanwhile the present invention infects carefully herpes virus hominis HSV-1 and HSV-2 using pyridine ketone in external colorimetric method for determining Usu
The protective effect of born of the same parents.The result shows that pyridine ketone is in vitro to I type, II type herpes virus hominis's institute's cytopathogenic effect with bright in Usu
Aobvious inhibiting effect, EC50 is respectively 15.74 μm of ol/L, 17.58 μm of ol/L, and SI is respectively 9.8,8.8.
The present invention determines in Usu pyridine ketone to I type people's blister in such a way that stomach-filling (oral) and external application (skin) are administered respectively
The therapeutic effect of the infection of guinea pig skin caused by exanthema virus, the results showed that, pyridine ketone can obviously slow down guinea pig skin infection in Usu
Symptom shortens cure time.The present invention also determines in Usu in such a way that stomach-filling (oral) and mucous membrane (vagina) are administered respectively
Therapeutic effect of the pyridine ketone to mouse vagina inflammation caused by II type herpes virus hominis, the results showed that, pyridine ketone is to mouse vagina in Usu
Inflammation has apparent therapeutic effect, and can significantly improve survival rate and the Average Survival day of mouse, extending life rate.
In order to further illustrate the present invention, with reference to embodiments to pyridine in pyridine in Usu provided by the invention and shellfish Usu
Application of the ketone in the drug of preparation treatment viral disease is described in detail, but cannot be understood as protecting the present invention
The restriction of range.Unless otherwise specified, reagent used in following embodiment is commercially available.
The external anti-hand-foot-and-mouth-disease cytotoxic activity experiment of pyridine in 1 Usu of embodiment
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.Pyridine in Usu, is made by oneself by Kangyuan Pharmaceutical Co., Ltd., Jiangsu Prov, purity 98.04%.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
Vero cell uses the DMEM culture medium containing 2% fetal calf serum to press 5 × 104A/ml concentration is inoculated with 96 well culture plates, often
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, it is added and is diluted to 100TCID with the DMEM culture medium without fetal calf serum50Each strain virus
(EV71 and CoxA16) after every hole 100 μ l, 35 DEG C of incubation 2h, discards virus liquid.It will with the DMEM culture medium of 2% fetal calf serum
Pyridine is configured to various concentration in Usu, is separately added into Vero cell monolayer, if 3 multiple holes, Vero cell is in 37 DEG C, 5%CO2
48h is cultivated in incubator, observes cytopathic phenomena daily.After 48h, after abandoning supernatant, the fixed 1h of 100 μ l, 10% formaldehyde is added,
0.1% (w/v) violet staining 15min, microplate reader 570nm measure absorbance.Experiment is repeated 3 times altogether.Calculate pyridine pair in Usu
Two kinds of viral CC50(half toxic concentration), EC50(medium effective concentration), SI (selection index).
Pyridine anti-EV71 and CoxA16 virus activity test result in vitro in 1 Usu of table
Table 1 the results show that in Usu pyridine it is inhibited to EV71 and CoxA16 virus institute cytopathogenic effect in vitro,
Its EC50Respectively 245.23 μM, 217.22 μM, SI are respectively 3.40,3.83.Show that pyridine has applied to anti-brothers mouthful in Usu
The Pharmacodynamics in vitro basis of viral (EV71 and CoxA16) treatment of viral infections.
The therapeutic effect that enterovirus EV 71 is infected in pyridine in 2 Usu 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 SPF grades of pregnancies pregnant mouse of BALB/c on the 15th~16 is 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 200mg/kg/d, crow by nest
Pyridine low dose group 50mg/kg/d in Soviet Union, pyridine middle dose group 100mg/kg/d in Usu, pyridine high dose group 200mg/kg/ in Usu
D, every group 10, in addition to Normal group, EV71 viral suspension (10 is injected intraperitoneally in remaining each group7TCID50) infected,
0.1mL/ only, continuous 5 days, sprayed with 75% ethyl alcohol after administration or infection every time with gastric infusion, each 0.1ml by each administration group
After spilling suckling mouse package padding, puts back in cage and raised jointly with female rat, Normal group and virus control group give distilled water.Infection
The survival condition for observing suckling mouse daily afterwards, 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 (being observed after growing hair), thin, activity reduction etc.；2 points: hind leg diminished strength；3 points: after unilateral
Acroparalysis or paralysis；4 points: bilateral hindlimb paralysis or paralysis；5 points: dying or dead.It calculates each group death rate and life extends
Rate, and it is for statistical analysis with SPSS software.Calculation formula is as follows:
Influence (n=10) of the pyridine to EV71 infection suckling mouse gradient of infection in 2 Usu of table
| Group | Dosage (mg/kg/d) | 0 point | 1 point | 2 points | 3 points | 4 points | 5 points | P |
| Normal group | - | 10 | 0 | 0 | 0 | 0 | 0 | - |
| Model group | - | 0 | 0 | 0 | 0 | 1 | 9 | - |
| Ribavirin group | 100 | 0 | 3 | 2 | 1 | 1 | 3 | < 0.01 |
| High dose group | 200 | 0 | 3 | 3 | 1 | 1 | 2 | < 0.01 |
| Middle dose group | 100 | 0 | 2 | 1 | 1 | 1 | 5 | < 0.01 |
| Low dose group | 50 | 0 | 1 | 1 | 1 | 2 | 5 | < 0.05 |
Note: P be and model group ratio
Dead protective effect (n=10) of the pyridine to EV71 infection suckling mouse in 3 Usu of table
Note: * * indicates P < 0.01 compared with model group
Table 2 can significantly slow EV71 virus infection suckling mouse and draw the results show that in Usu after each dosage group gastric infusion of pyridine
The syntexis risen, hind leg diminished strength, the symptoms such as unilateral hindlimb paralysis, gradient of infection integral all has significant compared with model group
Sex differernce.Table 3 the results show that in Usu the Death prevention rate of the high, medium and low dosage of pyridine be respectively 77.78%, 55.56%,
44.44%, increase in life span is respectively 79.45%, 64.28%, 50.32%, shows that each dosage group of pyridine obviously subtracts in Usu
Few EV71 virus infection suckling mouse death rate and gradient of infection, extend its life span, with significant difference compared with model group,
Prompt pyridine in Usu that there is therapeutic effect to the infection of brothers' Aphthovirus.
The therapeutic effect that coxsackie virus A 16-type is infected in pyridine in 3 Usu 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 SPF grades of pregnancies pregnant mouse of BALB/c on the 15th~16 is 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 200mg/kg/d, crow by nest
Pyridine low dose group 50mg/kg/d in Soviet Union, pyridine middle dose group 100mg/kg/d in Usu, pyridine high dose group 200mg/kg/ in Usu
D, every group 10, in addition to Normal group, CoxA16 viral suspension (10 is injected intraperitoneally in remaining each group7TCID50) infected,
0.1mL/ only, continuous 5 days, sprayed with 75% ethyl alcohol after administration or infection every time with gastric infusion, each 0.1ml by each administration group
After spilling suckling mouse package padding, puts back in cage and raised jointly with female rat, Normal group and virus control group give distilled water.Infection
The survival condition for observing suckling mouse daily afterwards, 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 (being observed after growing hair), thin, activity reduction etc.；2 points: hind leg diminished strength；3 points: after unilateral
Acroparalysis or paralysis；4 points: bilateral hindlimb paralysis or paralysis；5 points: dying or dead.Calculate each group death rate and increase in life span
And it is for statistical analysis with SPSS software.
Influence (n=10) of the pyridine to CoxA16 infection suckling mouse gradient of infection in 4 Usu of table
Note: P be and model group ratio
Influence (n=10) of the pyridine to CoxA16 infection suckling mouse death protective effect in 5 Usu of table
Note: * indicates that P < 0.05, * * indicate P < 0.01 compared with model group
Table 4 can significantly slow CoxA16 virus infection suckling mouse the results show that in Usu after each dosage group gastric infusion of pyridine
Caused syntexis, hind leg diminished strength, the symptoms such as unilateral hindlimb paralysis, gradient of infection integral all has aobvious compared with model group
Write sex differernce.Table 5 the results show that in Usu the Death prevention rate of the high, medium and low dosage of pyridine be respectively 55.56%, 66.67%,
33.33%, increase in life span is respectively 74.21%, 65.28%, 30.11%, shows each dosage group gradient of infection of pyridine in Usu
The CoxA16 virus infection suckling mouse death rate and gradient of infection are significantly reduced, extends its life span, has compared with model group
Significant difference prompts pyridine in Usu to have therapeutic effect to the infection of brothers' Aphthovirus.
Protective effect of the pyridine ketone to herpes virus hominis's HSV-1 and HSV-2 infection cell in 4 Usu of embodiment
1, experimental material
Cell and virus: African green monkey kidney cell (Vero cell) is purchased from U.S.'s ATCC cell bank, by the Jiangsu Kang Yuan modern times
Traditional Chinese medicine research institute saves.SM44 plants of chimpanzee agent (HSV-1), Sav plants of herpesvirusⅡtype (HSV-2), by the Chinese Academy of Sciences
Wuhan institute of viruses provides, and 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.
Reagent: acyclovir is purchased from Hubei KeYi Pharmacentic Co., Ltd..DMEM culture medium is purchased from Nanjing Kai Jisheng
Object development in science and technology Co., Ltd, lot number: 20141024,10% fetal calf serum, 1 × 10 are contained in cell growth medium5U/L penicillin,
100mg/L streptomysin.In cell maintenance medium in addition to containing 2% fetal calf serum, other same cell growth mediums.
2, experimental method
(1) toxicity test of the pyridine ketone to Vero cell in Usu
Vero cell uses the DMEM culture medium containing 2% fetal calf serum to press 5 × 104A/ml concentration is inoculated with 96 well culture plates, often
100 μ l of hole sets overnight incubation in 37 DEG C of 5%CO2 incubators and forms cell monolayer.After abandon growth-promoting media, 100 μ l series are added in every hole
Pyridine ketone (25 μM, 50 μM, 100 μM, 200 μM, 400 μM) and positive control medicine acyclovir in the Usu of ratio of two term diluted concentration
The maintaining liquid (the DMEM culture solution containing 2% calf serum) of (25 μM, 50 μM, 100 μM, 200 μM, 400 μM), each dilution is set
4 multiple holes, while setting up normal cell controls.37 DEG C of 5%CO2 are set, observe cytopathy daily, 20 μ l are added in every hole after 3d
MTS, cultivates 2h in 37 DEG C of incubators, and microplate reader surveys the OD value at 490nm.Various concentration drug is calculated as follows to cell
Survival rate determines half toxic concentration CC50。
Cell survival rate (%)=(experimental port OD value/control wells OD value) × 100%
(2) inhibiting effect of the pyridine ketone to herpes virus hominis HSV-1 and HSV-2 in Usu
Vero cell uses the DMEM culture medium containing 2% fetal calf serum to press 5 × 104A/ml concentration is inoculated with 96 well culture plates, often
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, the DMEM culture medium without fetal calf serum is added and is diluted to 100TCID50HSV-1/HSV-2 disease
Poison after every hole 100 μ l, 35 DEG C of incubation 2h, discards virus liquid.With the DMEM culture medium of 2% fetal calf serum by pyridine ketone in Usu and
Positive control medicine acyclovir is configured to various concentration, is separately added into Vero cell monolayer, if 3 multiple holes, Vero cell
In 37 DEG C, 5%CO248h is cultivated in incubator, observes cytopathic phenomena daily.After 48h, after abandoning supernatant, 100 μ l are added
10% formaldehyde fixes 1h, and 0.1% (w/v) violet staining 15min, microplate reader 570nm measure absorbance.Experiment is repeated 3 times altogether.
Pyridine ketone is to two kinds of viral EC in calculating Usu50(medium effective concentration), SI (selection index).
Viral suppression (%)=[test hole (drug+virus) be averaged A570 value-virus control wells be averaged A570 value]/
(cell control well be averaged A570 value-virus control wells be averaged A570 value) × 100%
SI=CC50/EC50
Antiviral activity of 6 drug of table to HSV-1/HSV-2
Table 6 is as the result is shown: pyridine ketone in vitro has herpes virus hominis HSV-1 and HSV-2 institute cytopathogenic effect in Usu
Obvious inhibiting effect, EC50Respectively 15.74 μM, 17.58 μM, SI are respectively 9.8,8.8.Show in Usu that pyridine ketone has to answer
Pharmacodynamics in vitro basis for the treatment of anti-human herpesvirus infection.
The therapeutic effect that pyridine ketone topical administration infects guinea pig skin I type herpes virus hominis (HSV-1) in 5 Usu of embodiment
1, experimental material
Reagent: Virless Cream is produced by Changzhou Yabang Pharmaceutical & Chemical Co., Ltd..Animal: Dunkin-Harthey
White hair cavy is purchased from Wuhan Biological Products Inst., Ministry of Public Health
Virus: SM44 plants of 1 type of herpesviral (HSV-1) is provided by Wuhan Virology Institute,Chinan academy of Sciences, by Jiang Sukang
Edge R&D of modern TCM institute saves, and passes in Vero cell culture, -80 DEG C of preservations.
2, experimental method
Dunkin-Harthey white hair cavy, weight 300-350g, male feed 3 green vegetables and feed daily.Experiment shares 12
Cavy, is divided into 4 groups by every group 3, and grouping is respectively acyclovir group, agent in pyridine ketone in pyridine ketone high dose group, Usu in Usu
Pyridine ketone low dose group in amount group, Usu, every according to circumstances selects 6 dermatotome modelings, administrations, and model group group is with the PBS of equivalent
Solution (containing 0.1%Tween-80) replaces, and after yellow Jackets anesthesia, guinea pig back is lost hair or feathers according to a conventional method, cleans, disappear
Guinea pig back is sloughed fur part with marker pen and is divided into 4 dermatotomes by poison.Center intracutaneous injection 10 in each dermatotome7PFU/
Ml HSV-1 strain virus, per injection 0.1ml.During anesthesia, animal feeding 2h in 30 DEG C of incubators.In acyclovir group
4 dermatotomes apply acyclovir, and in addition 2 dermatotomes apply PBS solution (model group).Pyridine ketone in pyridine ketone high dose group, Usu in Usu
Pyridine ketone in the Usu of every group of 4 dermatotomes painting various dose of pyridine ketone low dose group in middle dose group, Usu, in addition 2 dermatotomes apply PBS
Solution, smearing medical fluid every time is 30 μ l acyclovir, until waiting pharmaceutical dryings, smears medicine 2 times daily, successive administration 5d.Day by day
Observe cavy diseased region recovery from illness situation.For the effect of pharmaceutical, the score system (table established by Alenius and Oberg
2), after to cavy inoculation HSV-1 virus, the region of inoculation is pressed symptom score 1 time daily.Scoring with me when bleb occurs
Uncle's number display scores when bleb starts dry doubling incrustation and uses Roman number instead, all to carry out with scoring equal double blind, will infect
All Arab and Rome (III/II/I respectively represents 3/2/1 point) scoring number are added together the note accumulated in the process
Point.
The scoring system of 7 cavy HSV of table infection
Therapeutic effect of the pyridine ketone topical administration to human simplex virus cutaneous infection of guniea pigs in 8 Usu of table
Note: P < 0.05 * * * P < 0.001, * * P < 0.01, * is compared with model group
Table 8 compared with the dermatotome for smearing solvent, smears Usu the results show that after guinea pig skin inoculation HSV-1 virus
In pyridine ketone dermatotome HSV infection score significantly reduce, present dose-dependence, and have statistical difference, but smear
The cure time of pyridine ketone cavy does not change significantly in Usu.Show that pyridine ketone infects the HSV-1 of guinea pig skin in Usu
With therapeutic effect, infection symptoms can be slowed down, but not can be shortened cure time.
The therapeutic effect infected guinea pig skin I type herpes virus hominis (HSV-1) is administered orally in pyridine ketone in 6 Usu of embodiment
1, experimental material
Reagent: acyclovir is purchased from Hubei KeYi Pharmacentic Co., Ltd..Animal: Dunkin-Harthey white hair globefish
Mouse is purchased from Wuhan Biological Products Inst., Ministry of Public Health.
Virus: SM44 plants of 1 type of herpesviral (HSV-1) is provided by Wuhan Virology Institute,Chinan academy of Sciences, by Jiang Sukang
Edge R&D of modern TCM institute saves, and passes in Vero cell culture, -80 DEG C of preservations.
2, experimental method
Dunkin-Harthey white hair cavy, weight 300-350g, male feed 3 green vegetables and feed daily.Experiment shares 12
Cavy, is divided into 4 groups, grouping is respectively acyclovir group, the high, medium and low dosage group of pyridine ketone in Usu, every basis by every group 3
Situation selects 6 dermatotome modelings, administrations.After yellow Jackets anesthesia, guinea pig back is lost hair or feathers according to a conventional method, is cleaned, disinfection,
Guinea pig back is sloughed fur part with marker pen and is divided into 4 dermatotomes.Center intracutaneous injection 10 in each dermatotome7PFU/ml
HSV-1HS-1 strain virus, per injection 0.1ml.During anesthesia, animal feeding 2h in 30 DEG C of incubators.After modeling
The high, medium and low dosage group of pyridine ketone carries out gastric infusion according to dosage described in table 4 in acyclovir group and Usu, and every every time
0.2ml, model group are replaced, successive administration 5d with the PBS solution (containing 0.1%Tween-80) of equivalent.Day by day cavy lesion is observed
Position recovery from illness situation.In order to which the effect of pharmaceutical is inoculated with by the score system that Alenius and Oberg is established to cavy
After HSV-1 virus, the region of inoculation is pressed symptom score 1 time daily.Scoring when bleb occurs is shown with Arabic numerals, works as blister
When rash starts dry doubling incrustation, scores and use Roman number instead, it is all to carry out with scoring equal double blind, by all I in course of infection
Uncle and Rome scoring number are added together scoring of being accumulated.
The therapeutic effect to human simplex virus cutaneous infection of guniea pigs is administered orally in pyridine ketone in 9 Usu of table
Note: P < 0.05 * * * P < 0.001, * * P < 0.01, * is compared with model group
Table 9 compared with the dermatotome of placebo, takes orally Ah former times the results show that after guinea pig skin inoculation HSV-1 virus
The score of the HSV infection of Luo Wei group and recovery from illness number of days significantly reduce；Similar, compared with the dermatotome of placebo, take orally crow
The score of the HSV infection of pyridine ketone group and recovery from illness number of days significantly reduce in Soviet Union, dose-dependence are presented, and have statistics poor
It is different, show that pyridine ketone has therapeutic effect to the HSV-1 infection of guinea pig skin in Usu, can slow down infection symptoms, shorten recovery from illness
Time.
Pyridine ketone mucosa delivery infects yin caused by II type herpes virus hominis (HSV-2) to mouse vagina in 7 Usu of embodiment
The therapeutic effect of road inflammation
1, experimental material
Reagent: Virless Cream is produced by Changzhou Yabang Pharmaceutical & Chemical Co., Ltd..
Animal: BALB/c mouse, female, Jiangning county Qinglongshan animal reproduction field.
Virus: Sav plants of herpes virus type 2 (HSV-2) is provided by Wuhan Virology Institute,Chinan academy of Sciences, by Jiang Sukang
Edge R&D of modern TCM institute saves, and passes in Vero cell culture, -80 DEG C of preservations.
2, experimental method
BALB/c mouse female 50, is randomly divided into 6 groups by weight average, Normal group, model group, acyclovir group
With the high, medium and low dosage group of pyridine ketone in Usu.Except Normal group, every group of mouse will be adsorbed with virus liquid with ophthalmology tweezers
The gelfoam fritter of 0.03ml (10LD50) is sent into mouse vagina, and Normal group uses sterile PBS solution, mouse infection 2h instead
Each administration group smears the high, medium and low dosage emulsifiable paste of pyridine ketone in Virless Cream or Usu in intravaginal and external genital respectively afterwards
Treatment, administration number of days are 10 days, and model group awards smearing emulsifiable paste matrix.Dead mouse number is observed, life number of days calculates mouse and deposits
Motility rate, average life day, extending life rate.
Therapeutic effect (n=10, x ± s) of the pyridine ketone mucosa delivery to Balb/c mouse HSV-2 vaginitis in 10 Usu of table
Note: P < 0.05 * * * P < 0.001, * is compared with model group
Table 10 compared with the control group, smears pyridine ketone in Usu the results show that after mouse vagina inoculation HSV-2 virus
There is certain therapeutic effect to vaginitis caused by mouse vagina infection HSV-2 virus, and is in dose-dependence, each dosage
Group can significantly improve survival rate and the Average Survival day of mouse, extending life rate.
The oral administration of pyridine ketone infects yin caused by II type herpes virus hominis (HSV-2) to mouse vagina in 8 Usu of embodiment
The therapeutic effect of road inflammation
1, experimental material:
Acyclovir is purchased from Hubei KeYi Pharmacentic Co., Ltd..
Animal: BALB/c mouse, female, Jiangning county Qinglongshan animal reproduction field.
Virus: Sav plants of herpes virus type 2 (HSV-2) is provided by Wuhan Virology Institute,Chinan academy of Sciences, by Jiang Sukang
Edge R&D of modern TCM institute saves, and passes in Vero cell culture, -80 DEG C of preservations.
2, experimental method:
BALB/c mouse female 50, is randomly divided into 6 groups by weight average, Normal group, model group, acyclovir group
With the high, medium and low administration group of pyridine ketone in Usu.Except Normal group, every group of mouse will be adsorbed with virus liquid with ophthalmology tweezers
The gelfoam fritter of 0.03ml (10LD50) is sent into mouse vagina, and Normal group uses sterile PBS solution, mouse infection 2h instead
The high, medium and low dosage of pyridine ketone in acyclovir and Usu is given in stomach-filling to each administration group respectively afterwards, and administration number of days is 10 days, model group
Physiological saline is given in stomach-filling.Dead mouse number is observed, life number of days calculates mouse survival rate, average life day, extending life
Rate.
Therapeutic effect (n=10, x ± s) of the pyridine ketone oral administration to Balb/c mouse HSV-2 vaginitis in 11 Usu of table
Note: P < 0.05 * * * P < 0.001, * * P < 0.01, * is compared with model group
Table 11 compared with the control group, takes orally pyridine ketone in Usu the results show that after mouse vagina inoculation HSV-2 virus
There is therapeutic effect to vaginitis caused by mouse vagina infection HSV-2 virus, and is in dose-dependence, each dosage group
Significantly improve survival rate and the Average Survival day of mouse, extending life rate.
Pyridine ketone is preparing the application in capsule medicine in 9 Usu of embodiment
Pyridine ketone in the Usu 350g and 32g starch, 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 pyridine ketone in Usu.3 times a day, often
Secondary 1.
Pyridine is preparing the application in granules medicine in 10 Usu of embodiment
Pyridine in the Usu 350g and 1000g sucrose and 500g dextrin are mixed, are conventionally made in 1000 packet Usus
Pyridine granule.3 times a day, 1 tablet each time.
Pyridine ketone is preparing the application in tablet medicine in 11 Usu of embodiment
Pyridine ketone in the Usu 350g and 50g starch, 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, pyridine ketone tablet in Usu.3 times a day, often
Secondary 1.
Application of the pyridine in preparation pill medicine in 12 Usu of embodiment
Pyridine in the Usu 350g and 12g polyethylene glycol-6000,80.5g Tween-80, appropriate liquid paraffin are mixed, pressed
Pyridine pill 1000 in Usu are made in more solito.3 times a day, 1 tablet each time.
Pyridine ketone is preparing the application in injection medicine in 13 Usu of embodiment
By pyridine ketone in the Usu 200g and 15g injection soybean lecithin, 25g glycerol for injection, water for injection is settled to
Pyridine ketone injection 1000 in Usu is conventionally made in 1000mL.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 (10)
1. application of the alkaloid compound shown in formula (I) in the drug of preparation treatment viral disease,
Wherein, R is-OH or=O；The viral disease is hand-foot-and-mouth disease or herpe simplex disease.
2. application according to claim 1, which is characterized in that the virus is enterovirus and/or herpes simplex virus.
3. application according to claim 2, which is characterized in that the enterovirus is enterovirus EV 71 type.
4. application according to claim 2, which is characterized in that the enterovirus is Coxsackie virus.
5. application according to claim 4, which is characterized in that the Coxsackie virus is coxsackie virus A 16-type.
6. application according to claim 2, which is characterized in that the herpes simplex virus be herpes simplex virus type 1 or
Herpes simplex virus type 2.
7. application according to claim 1, which is characterized in that the virus is enterovirus, biology shown in the formula (I)
The active dose of alkaloid compound is 50-200mg/kg/d.
8. application according to claim 1, which is characterized in that the virus is herpes simplex virus, shown in the formula (I)
The active dose of alkaloid compound is 30-200mg/kg/d.
9. application according to claim 1, which is characterized in that the drug includes alkaloids shown in the formula (I)
Close object and pharmaceutically acceptable carrier；
The dosage form of the drug is any one clinically-acceptable oral administered dosage form, injecting medicine-feeding form or topical administration agent
Type.
10. application according to claim 9, which is characterized in that the drug be tablet, capsule, granule, pill,
Soft extract, suspending agent, syrup, suppository, gelling agent, aerosol, patch.
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| CN102327571A (en) * | 2011-09-30 | 2012-01-25 | 四川大学 | New cancer-resisting use of fritillaria cirrhosa total alkaloids and compounds contained in fritillaria cirrhosa total alkaloids |
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