CN102389426A - Application of isoquinoline type alkaloids and derivatives thereof to preparation of medicine for inhibiting HPV (Human Papilloma Virus) infection - Google Patents
Application of isoquinoline type alkaloids and derivatives thereof to preparation of medicine for inhibiting HPV (Human Papilloma Virus) infection Download PDFInfo
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- CN102389426A CN102389426A CN2011102180791A CN201110218079A CN102389426A CN 102389426 A CN102389426 A CN 102389426A CN 2011102180791 A CN2011102180791 A CN 2011102180791A CN 201110218079 A CN201110218079 A CN 201110218079A CN 102389426 A CN102389426 A CN 102389426A
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Abstract
The invention relates to an application of isoquinoline type alkaloids and derivatives thereof to preparation of a medicine for inhibiting HPV (Human Papilloma Virus) infection, belonging to the field of natural medicine extract preparation and anti-virus and anti-tumor officinal application. The invention provides an application of corydaline, isocorydine, isoboldine, N-methyl hernovine, dicranostigma leptopodum or pharmaceutically acceptably salts or esters to the preparation of the medicine for treating the HPV infection.
Description
Technical field
The present invention relates to a kind of isoquinoline alkaloid and derivant thereof, and uses thereof, natural drug extract and pharmaceutical applications technical field belonged to.
Background technology
Cervical cancer is that the whole world is only second to the second largest malignant tumor that breast carcinoma causes women's death, and its characteristics are that grade malignancy is high, cure rate variance.It is the main high risk factor that causes the cervical cancer morbidity that human papilloma shape virus (HPV) infects, and there is the hypotype chimeric protein of two kinds of codings in it, and promptly E6 and E7 can promote that cell proliferation influences the host, regulate and control the variation of various GAP-associated protein GAPs.The target gene of HPV E6/E7 protein regulation is a NF-κ B molecule, and itself and NF-κ B transduction path is closely related, and these transcription factor can start replys GAP-associated protein GAP with inherent immunity and change apoptosis and cell proliferation process.And existing research shows that the morbidity of cervical cancer possibly be because human papilloma shape viral infection causes the unbalance of body antioncogene and proto-oncogene, thereby causes the oncogene high expressed and cause a disease.
Summary of the invention
The technical problem that the present invention will solve has provided a kind of as the isoquinoline alkaloid and the derivant thereof that suppress the HPV viral infection.
In order to solve the problems of the technologies described above, the invention provides following technical scheme:
Formula I or II chemical compound the application in the preparation of treatment HPV virus infective medicament of receptible salt or ester-formin;
Ⅰ
R
1Be H ,-OH ,-OCH
3,-O-C
1-C
8Alkyl ,-NH
2,-NO
2,-COOH ,-COOCH
3,-COOCH
2CH
3,-COO-C
1-C
8Alkyl ,-COO-fluoroalkyl, C
1-C
8Alkyl, C
3-C
6Cycloalkyl, C
1-C
8Carboxylic acid, C
1-C
8Carboxylate, 1-3 independent free halogen/HSCH
2-, CH
3SCH
2CH
2-;
R
2Be H ,-OH ,-OCH
3,-O-C
1-C
8Alkyl ,-NH
2,-NO
2,-COOH ,-COOCH
3,-COOCH
2CH
3,-COO-C
1-C
8Alkyl ,-COO-fluoroalkyl, C
1-C
8Alkyl, C
3-C
6Cycloalkyl, C
1-C
8Carboxylic acid, C
1-C
8Carboxylate, 1-3 independent free halogen/HSCH
2-, CH
3SCH
2CH
2-;
R
3Be H ,-OH ,-OCH
3,-O-C
1-C
8Alkyl ,-NH
2,-NO
2,-COOH ,-COOCH
3,-COOCH
2CH
3,-COO-C
1-C
8Alkyl ,-COO-fluoroalkyl, C
1-C
8Alkyl, C
3-C
6Cycloalkyl, C
1-C
8Carboxylic acid, C
1-C
8Carboxylate, 1-3 independent free halogen/HSCH
2-, CH
3SCH
2CH
2-;
R
4Be H ,-OH ,-OCH
3,-O-C
1-C
8Alkyl ,-NH
2,-NO
2,-COOH ,-COOCH
3,-COOCH
2CH
3,-COO-C
1-C
8Alkyl ,-COO-fluoroalkyl, C
1-C
8Alkyl, C
3-C
6Cycloalkyl, C
1-C
8Carboxylic acid, C
1-C
8Carboxylate, 1-3 independent free halogen/HSCH
2-, CH
3SCH
2CH
2-;
Ⅱ
R
1Be H ,-OH ,-OCH
3,-O-C
1-C
8Alkyl ,-NH
2,-NO
2,-COOH ,-COOCH
3,-COOCH
2CH
3,-COO-C
1-C
8Alkyl ,-COO-fluoroalkyl, C
1-C
8Alkyl, C
3-C
6Cycloalkyl, C
1-C
8Carboxylic acid, C
1-C
8Carboxylate, 1-3 independent free halogen/HSCH
2-, CH
3SCH
2CH
2-;
R
2Be H ,-OH ,-OCH
3,-O-C
1-C
8Alkyl ,-NH
2,-NO
2,-COOH ,-COOCH
3,-COOCH
2CH
3,-COO-C
1-C
8Alkyl ,-COO-fluoroalkyl, C
1-C
8Alkyl, C
3-C
6Cycloalkyl, C
1-C
8Carboxylic acid, C
1-C
8Carboxylate, 1-3 independent free halogen/HSCH
2-, CH
3SCH
2CH
2-;
R
3Be H ,-OH ,-OCH
3,-O-C
1-C
8Alkyl ,-NH
2,-NO
2,-COOH ,-COOCH
3,-COOCH
2CH
3,-COO-C
1-C
8Alkyl ,-COO-fluoroalkyl, C
1-C
8Alkyl, C
3-C
6Cycloalkyl, C
1-C
8Carboxylic acid, C
1-C
8Carboxylate, 1-3 independent free halogen/HSCH
2-, CH
3SCH
2CH
2-;
R
4Be H ,-OH ,-OCH
3,-O-C
1-C
8Alkyl ,-NH
2,-NO
2,-COOH ,-COOCH
3,-COOCH
2CH
3,-COO-C
1-C
8Alkyl ,-COO-fluoroalkyl, C
1-C
8Alkyl, C
3-C
6Cycloalkyl, C
1-C
8Carboxylic acid, C
1-C
8Carboxylate, 1-3 independent free halogen/HSCH
2-, CH
3SCH
2CH
2-.
Preferably, corydaline, isocorydine, isoboldine, N-methyl hernovine, tinea alba premium alkali or the acceptable salt of their medicine or the ester-formin application in prevention or the preparation of treatment HPV virus infective medicament.
A kind of pharmaceutical composition, said composition comprise each the acceptable salt of chemical compound or ester-formin and the pharmaceutically-acceptable excipients or the carrier of claim 1-2 of drug effective dose.
The preferred salt form of chemical compound comprises those and medicine known in the art acceptable inorganic and the formed salt of organic acid among this paper.The salt form that uses mineral acid to make comprises treatment upward acceptable halate and sulfate, for example hydrochlorate, maleate, hydrobromate, sulfate, carbonate or bicarbonate.These salt forms can use the alkali compounds and the methods known in the art preparation of formula I or II.
The ester-formin of The compounds of this invention comprises straight chained alkyl ester or branched alkyl ester that contains 3 or 6 carbon atoms or the benzyl ester with 1-6 carbon atom, comprises methyl, ethyl, propyl group, butyl, 2-methyl-propyl and 1,1-dimethyl ethyl ester.The input alkyl that this paper uses comprises the carbochain of straight chain and side chain.Preferably, C1-C3 perfluoroalkyl substituent group is-CF3;-O-C1-C3 perfluoroalkyl substituent group is-OCF3 with-S-C1-C3 perfluoroalkyl substituent group is-SCF3.
Chemical compound of the present invention is the HPV viral inhibitors, therefore is used for treating, suppress, prevent or prevent those to relate to the process of the pathology of HPV viral infection, cervical cancer incidence and development at mammal, preferred human body.Therefore, The compounds of this invention is used to treat or prophylactic treatment HPV infects various skins and reproductive tract pathological changes or relevant therewith precancerous lesions of uterine cervix or the cervix uteri malignant change that causes.
Chemical compound of the present invention also is used to prevent and treat the pathological change that other cause because of HPV virus.
Different because of mechanism of action, The compounds of this invention can be united use with other antiviral drugs and ancillary drug thereof.
The compounds of this invention can the enhancing body autoimmune function.The compounds of this invention can be used for the treatment of human body immunity improving power aspect.
Treating, suppress, prevent or prevent the method for the listed every kind of disease of different animals of this paper is a part of the present invention.Every kind of method comprises The compounds of this invention or the acceptable salt of its medicine or the ester-formin and the various pharmaceutical carrier excipient forms of the mammal dose therapeutically effective that needs treatment.
The present invention also comprises compound structure formula I related among the present invention, the drug regimen that II is relevant.These combinations contain The compounds of this invention or the acceptable salt of its medicine or the ester-formin of dose therapeutically effective separately, perhaps with one or more pharmaceutical carriers or excipient mixed form mutually.The medicine of chemical compound or treatment effective dose are meant that the amount of said chemical compound can fully suppress to need hyperfibrinolysis in the mammal of treatment among this paper; Thereby to said symptom improvement is provided to a great extent, and then prevents, suppress or limit the pathologic basis of said disease or symptom.
The employed exact dose of The compounds of this invention depends on symptom and the factors such as the order of severity, medication and employed excipient that comprise institute's administration object, the disease of treating.Can give said chemical compound, its salt or ester-formin and contain the pharmaceutical carrier and the excipient forms of said chemical compound, salt or ester-formin through any conventional route.Form of medication comprises oral administration, intravenously administrable, percutaneous drug delivery, through intestinal canal administration, topical administration.Institute's administered compound can be free or with the form of suitable pharmaceutical salt or ester.The compounds of this invention can work with any mode with the fibrinolysin contact when treatment hyperfibrinolysis and hemorrhage.They can be used with any usual manner of administration that is applicable to, perhaps as independent therapeutic dosage forms, or with the treatment preparation combining form use.They can be used separately, but preferably use with suitable pharmaceutical carrier or excipient.Pharmaceutical carrier is selected according to selected route of administration, and can make is solid, liquid or solid and mixtures of liquids.
Solid comprises powder, tablet, capsule and nanometer formulation.Solid carrier can be one or more materials that can be used as flavoring agent, lubricant, solubilizing agent, suspending agent, binding agent or tablet disintegrant.In powder, said carrier is pulverizing solid, it and active component powder mixes.In tablet, active component with have essential adhesion characteristic carrier with suitable mixed and be pressed into required shape and size.Gelatine capsule contains active component and dust carrier, and example is known lactose, starch, cellulose derivative, magnesium stearate and stearic acid etc.Similarly, can use the diluent tabletting.Tablet and capsule can be made sustained release preparation, discharge medicine continuously to continue a few hours.Tabletting can be with sweet tablet or film coating, makes and make label and air to broadcast to leave, perhaps carry out enteric coating with selectivity disintegrate in gastrointestinal tract to cover undesirable flavor.The liquid oral dosage form can contain coloring agent and correctives to increase patient's compliance.Suitable solid carrier is magnesium carbonate, magnesium stearate, Pulvis Talci, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, hydroxy methocel, sodium carboxymethyl cellulose, low melt wax, cocoa butter etc.Film-coat, become capsule material, nano material also can use with The compounds of this invention and salt thereof or ester-formin, term " compositions " is meant and comprises active component and become capsule material as prescription, is with or without other carriers.
The sterile liquid compositions comprises solution, suspension, emulsion, slurry agent, tincture and spirit.Chemical compound of the present invention dissolves in or is suspended in the medicine acceptable carrier, for example sterilized water for injection.When the chemical compound sufficiently soluble, they can directly be dissolved in the normal saline of use or inapplicable appropriate organic solvent such as Polyethylene Glycol.If desired, chemical compound can be dispersed in starch solution, sodium carboxymethyl cellulose solution or the suitable cyclodextrin aqueous solution.Be the composition of liquid medicine of sterile solution or suspension, can pass through muscle, subcutaneous, Intradermal, abdominal cavity, intravenous injection and use.In many cases, can use forms of liquid compositions to replace the Peroral solid dosage form administering mode.
The unit dosage forms of the said chemical compound of preferred preparation carries out the standard administration.Can UD be mixed with packing powder, phial or ampoule, and be preferably capsule or tablet form.The daily dose of reactive compound can be according to the frequency of the other drug of the character of the pharmacodynamic properties of concrete medicine, route of administration, patient's body weight, age and sex, health status, morbid state and the order of severity, treatment simultaneously, treatment and through making corresponding adjustment to blood substance concentration analysis and patient's recovery situation and to the reaction of treatment.The daily dose of active component is 0.1 to 100 mg/kg, and preferred daily dose is about 1.0 to about 10 mg/kg.The combination dosage form that is suitable for administration comprises about l mg to about 10 mg active component/units.In these pharmaceutical compositions, the common content of active component accounts for the 0.5-95 % of composition total weight.Active component can be with solid dosage forms for example capsule, tablet and form of powder, can also be with the sterile liquid formulations administration.
Chemical compound of the present invention can pass through following experimental technique, confirms that The compounds of this invention suppresses the ability of HPV virus multiplication:
1. extract and separate
Get dry Herba Dicranostigmatis Leptopodi (5.0 kg) and pulverize, with 95% ethanol extraction 3 times (each 7 days), decompression and solvent recovery gets extractum 200.0 g.Extractum is suspended in 2000 ml, 2% H
2SO
4In the aqueous solution, chloroform extraction part FA (12 g) successively, aqueous ph value is with ammonia modulation 9, chloroform extraction, extract FB (25 g); FB mixes appearance with silica gel 25 g, 500 g silica gel upper props, chloroform/methanol/triethylamine (10:1:0.1 is to 2:1:0.1) gradient elution, flow velocity, 10 ml/min; Thin layer chromatography detects, and merges same composition, gets 4 components, FB1 (5000 ml, 4.0 g; 100:1), and FB2 (4000 ml, 4.0 g, 50:1), FB3 (5000 ml; 2.0 g, 25:1) and FB4 (3000 ml, 3.5 g, 5:1); FB1 is with 40g silica gel, and chloroform/methanol/triethylamine 20:1:0.1,10:1:0.1,8:1:0.1,6:1:0.1 are mobile phase, and the upper prop eluting obtains different isoquinoline alkaloid chemical compounds respectively.
2. the RT-PCR method is observed the variation of gene expression:
Principle: can be used for gene expression dose in the detection of drugs patients before and after intervention cell, the content of RNA viruses and the directly clone and the cDNA sequence of specific gene are judged the inhibitory action that isocorydine is viral to HPV with this in the cell.
Step: with 5000/hole the cell kind is advanced in 96 orifice plates, cultivate 24 h after, discard old culture fluid, add isocorydine (750 mg/L contain different pharmaceutical concentration; 1500 mg/L, 3000 mg/L) intervene, cultivate 24h respectively, 48h; 72h, the RNA extraction test kit extracted total RNA with Takara at first, exhausts culture medium with the rifle head; The Buffer that in each hole, adds 125 μ l * 2, reuse rifle head exhaust Cell AmpTM Washing Buffer, and the back adds the Processing solution of 50 μ l in each hole; After blowing and beating Processing liquid repeatedly, move in the microcentrifugal tube 75 ℃ of water-bath 5min; The cell pyrolysis liquid that packing obtains is so that follow-up reverse transcription experiment is all set multiple hole for every group simultaneously.
RNA (5 μ g) is inverted the cDNA 10 μ l systems of recording into; Its reverse transcription system comprises: 5 * PrimeScript TM Buffer, 2 μ l; PrimeScript TM RT EnzymeMix I 0.5 μ l; 0ligo dT Primer (50 μ M) 0.5 μ l, Random 6 mers 0.5 μ l, RNase Free dH
2O 4.5 μ l, 37 ℃ of 15min of cDNA 2 μ l. reverse transcription conditions, 85 ℃ 5 seconds.Be that template is carried out specific amplification again with cDNA, its amplified reaction 20 μ l systems are by Tap enzyme 10 μ l, Forward primer0.4 μ l, Reverse primer 0.4 μ l, Rox 0.4 μ l, ddH
2O 6.8 μ l, and 2 μ l cDNA templates are formed.Need the primer sequence and the annealing temperature of amplifying target genes to provide by Table 1.β-actin is increased as contrast, after the fluorescent real time PCR reaction finishes, confirms threshold value by systematic analysis software, and then confirms respectively to react the CT value of sample to adopt 2 of relative quantification
-△ △ CTMethod is analyzed PCR result, confirms expression of gene difference between each sample.
Formula: △ CT=(C
TTarget-C
TActin)
△△CT=(C
TTarget-C
TActin)Time
x-(C
TTarget-C
TActin)Time
0
2
-△ △ CTWhat represent is the variation multiple of the expression of experimental group genes of interest with respect to matched group.
The size of table 1 primer and amplified production
3. Western Blot method is observed the HPV E6 of viral oncogene, the variation of E7 protein expression:
Principle: can observe the variation of protein expression through Western Blot method.
Step: total protein of cell extracts with RIPA and extracts, protein quantification according to Bradfords method with BSA as standard control.Survey its absorbance, the protein content in the calculation sample in 570 nm wavelength.The total protein sample of 50 g is placed in 100 ℃ of Buddhist decocting in water 10 min and carries out degeneration.Protein electrophoresis runs condensing glue with 80 V, and 100 V run separation gel, changes film and adopts 200 mA constant current ice bath 1-2 h; Perhaps 40 V voltages, 4 ℃ of 3h are specifically according to the size decision of molecular weight of albumen.After transfer film after immersion of the membrane with TBS once, into 5?% Skim milk blocking solution at 4 ℃ shaker closed 2h,? PVDF film is a good one with a diluted anti-p53,? HPV16/18? E6,? E7 phosphorylated and non-phosphorylated IκB from the U.S. Cell? Signalling? Techonology Company purchased two antibody incubation also used the CST.Protein band carries out video picture with the ECL luminescent solution, uses the X-ray magazine exposure image at last, and protein band is judged provides reference by the Marker that dyes molecular weight in advance.The result of Western Blotting analyzes the OD value of each band with Image-pro plus 6.0 softwares.
Description of drawings
Accompanying drawing is used to provide further understanding of the present invention, and constitutes the part of description, is used to explain the present invention with embodiments of the invention, is not construed as limiting the invention.In the accompanying drawings:
Fig. 1 Hela, the OD value figure after Siha, the Western Blotting result of C33A analyze with Image-pro plus 6.0.
The specific embodiment
Below the preferred embodiments of the present invention are described, should be appreciated that preferred embodiment described herein only is used for explanation and explains the present invention, and be not used in qualification the present invention.
Instance 1
Isocorydine suppresses the effect of HPV virus
Medicine name: isocorydine
Chemical name:
1,2,10-trimethoxy-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinolin-11-ol
Medicines structure:
Inquire into whether the HPV relevant mRNA of virus and protein expression exist inhibitory action in human cervical carcinoma Hela cell that isocorydine infects HPV and the Siha cell, thereby the evaluation isocorydine is in the using value of treating aspect the HPV infection.
Experimental technique:
1. the RT-PCR method is observed the variation of gene expression
With 5000/hole the cell kind is advanced in 96 orifice plates, cultivate 24 h after, discard old culture fluid, add isocorydine (750 mg/L contain different pharmaceutical concentration; 1500 mg/L, 3000 mg/L) intervene, cultivate 24 h respectively, 48 h; 72 h, the RNA extraction test kit extracted total RNA with Takara at first, exhausts culture medium with the rifle head; The Buffer that in each hole, adds 125 μ l * 2, reuse rifle head exhaust Cell AmpTM Washing Buffer, and the back adds the Processing solution of 50 μ l in each hole; After blowing and beating Processing liquid repeatedly, move in the microcentrifugal tube 75 ℃ of water-bath 5 min; The cell pyrolysis liquid that packing obtains is so that follow-up reverse transcription experiment is all set multiple hole for every group simultaneously.
RNA (5 μ g) is inverted the cDNA 10 μ l systems of recording into; Its reverse transcription system comprises: 5 * PrimeScript TM Buffer, 2 μ l; PrimeScript TM RT EnzymeMix I 0.5 μ l; 0ligo dT Primer (50uM) 0.5 μ l, Random 6 mers 0.5 μ l, RNase Free dH
2O 4.5 μ l, 37 ℃ of 15min of cDNA 2 μ l. reverse transcription conditions, 85 ℃ 5 seconds.Be that template is carried out specific amplification again with cDNA, its amplified reaction 20 μ l systems are by Tap enzyme 10 μ l, Forward primer0.4 μ l, Reverse primer 0.4 μ l, Rox 0.4 μ l, ddH
2O 6.8 μ l, and 2 μ l cDNA templates are formed.Need the primer sequence and the annealing temperature of amplifying target genes to provide by Table 1.B-actin is increased as contrast, after the fluorescent real time PCR reaction finishes, confirms threshold value by systematic analysis software, and then confirms respectively to react the CT value of sample to adopt 2 of relative quantification
-△ △ CTMethod is analyzed PCR result, confirms expression of gene difference between each sample.
Table 2 1500 mg/L isocorydine alkali treatments are to the expression relative value (2 of HeLa cell HPV18 E6/E7 mRNA
-△ △ CT)
Table 3 1500 mg/L isocorydine alkali treatments are to SiHa cell HPV16E6/E7, the expression relative value (2 of mRNA
-△ △ CT)
The result is shown in table 2, table 3, and it shows, when the isocorydine administration concentration is 1000 mg/L; 2000 mg/L; 24 h after administration respectively during 3000 mg/L extract total RNA from three kinds of cells on the 48 h time points, with specific primer to host gene HPV-16 E6 and E7; HPV-18 E6 and E7; P53; β-actin carries out the RT-PCR specificity extension self-increasing reaction.From interpretation of result, the expression of dosing intervention group Siha cell HPV-16 E6 and E7 more intervention group is expressed not lowly, and E6 be described, E7 isocorydine treatment Siha group with do not treat Siha and organize that to compare expression lower.HPV-18 E6 and E7 compares with non-intervention group at dosing intervention group Hela cell and decreases, and explains that isocorydine makes E6 in the Hela cell, and the positive degree of E7 gene expression reduces.HPV-16 and HPV-18 E6, the E7 expression of gene is detected for HPV virus depression effect through isocorydine.
2. Western Blot method is observed the isocorydine inhibition HPV E6 of viral oncogene, the effect of E7 protein expression:
Principle: can observe the variation of protein expression through Western Blot method.
Step: total protein of cell extracts with RIPA and extracts, protein quantification according to Bradfords method with BSA as standard control.Survey its absorbance, the protein content in the calculation sample in 570 nm wavelength.The total protein sample of 50 g is placed in 100 ℃ of Buddhist decocting in water 10 min and carries out degeneration.Protein electrophoresis runs condensing glue with 80 V, and 100 V run separation gel, changes film and adopts 200 mA constant current ice bath 1-2 h; Perhaps 40 V voltages, 4 ℃ of 3 h is specifically according to the size decision of molecular weight of albumen.After transfer film after immersion of the membrane with TBS once, into 5?% Skim milk blocking solution at 4 ℃ shaker closed 2? H,? PVDF film is a good one with a diluted anti-p53,? HPV16/18? E6, ? E7 phosphorylated and non-phosphorylated IκB from the U.S. Cell? Signalling? Techonology Company purchased two antibody incubation also used the CST.Protein band carries out video picture with the ECL luminescent solution, uses the X-ray magazine exposure image at last, and protein band is judged provides reference by the Marker that dyes molecular weight in advance.The result of Western Blotting analyzes the OD value of each band with Image-pro plus 6.0 softwares, and is as shown in Figure 1.
The result shows, three-type-person's cervical cancer cell is respectively with 1000 mg/L, 2000 mg/L, and 3000 mg/L administration concentration are intervened 24 h; During 48 h, extract protein sample, detect Hela, Siha with Western blotting; Target protein E6 in the C33A cell, E7, p53 and I κ B-a; Phospho-I κ B-a, NF-κ B p65, Phospho-NF-κ B p65 level changes.The power of gray scale appears in destination protein, and protein content what are used for reflecting.
Isocorydine has suppressed the E6 of HPV viral oncogene, E7 (HPV-16 and HPV-18) expression, thus influenced proteic expression with the closely-related p53 of its malignancy of tumor degree; Because isocorydine is to HPV-16/HPV-18 E6; The difference of two kinds of different subtype proteins of E7 self influence, and p53 changes of expression level cause the activation of NF-κ B signal path jointly; After NF-κ B path activates, the phosphorylation site of activation NF-κ B and then.
Isocorydine has suppressed the E6 of HPV viral oncogene on mRNA and protein level, E7 (HPV-16 and HPV-18) expresses, and influence and the proteic expression of the closely-related p53 of its malignancy of tumor degree.Because isocorydine is to HPV16/18 E6; Two kinds of different subtype proteins of E7 self influence is different, and the p53 changes of expression level, starts NF-κ B signal path jointly and activates; Finally cause apoptosis of tumor cells, whole process life period-dose dependent.Visible dna fragmentation fracture in the apoptosis generating process lander ladder band occurs, and heterochromatin increases in the nucleus, reunites, and apoptotic body etc. appears in fragmentation, all can be respectively by DNA Lander and Electronic Speculum is observed arrives.Cell proliferation, immunne response, angiogenesis etc. all receive the regulation and control of nuclear factor NF-κ B, have great importance for apoptosis.This research shows that isocorydine can suppress the E6 of HPV viral oncogene, the activation of the NF-κ B signal path that E7 starts, and prove at gene transcription level and protein level.Our result demonstrates isocorydine, and viral inhibiting performance is because block N F-κ B phosphorylation site is realized with the effect of having reduced I κ Ba to HPV.
Isocorydine can suppress the breeding of HPV virus, and it is that isocorydine has suppressed the E6 of HPV viral oncogene as mechanism, E7 (HPV-16 and HPV-18) horizontal expression; Thereby influenced proteic expression with the closely-related p53 of its malignancy of tumor degree; Because isocorydine is to HPV16/18 E6, the difference of two kinds of different subtype proteins of E7 self influence, and p53 changes of expression level; Cause the activation of NF-κ B signal path jointly; After the activation of NF-κ B path, follow the phosphorylation site of activation NF-κ B, thereby suppress tumor cell proliferation.
Instance 2
Capaurine alkali suppresses the effect of HPV virus
Medicine name: capaurine alkali
Chemical name:
(6aS)-2,10,11-Trimethoxy-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinolin-1-ol
Medicines structure:
Inquire into whether the HPV relevant mRNA of virus and protein expression exist inhibitory action in human cervical carcinoma Hela cell that capaurine alkali infects HPV and the Siha cell, thereby evaluation capaurine alkali is in the using value of treating aspect the HPV infection.
Experimental technique:
1. the RT-PCR method is observed the variation of gene expression
With 5000/hole the cell kind is advanced in 96 orifice plates, cultivate 24 h after, discard old culture fluid, add capaurine alkali (750 mg/L contain different pharmaceutical concentration; 1500 mg/L, 3000 mg/L) intervene, cultivate 24 h respectively, 48 h; 72 h, the RNA extraction test kit extracted total RNA with Takara at first, exhausts culture medium with the rifle head; The Buffer that in each hole, adds 125 μ l * 2, reuse rifle head exhaust Cell AmpTM Washing Buffer, and the back adds the Processing solution of 50 μ l in each hole; After blowing and beating Processing liquid repeatedly, move in the microcentrifugal tube 75 ℃ of water-bath 5min; The cell pyrolysis liquid that packing obtains is so that follow-up reverse transcription experiment is all set multiple hole for every group simultaneously.
RNA (5 μ g) is inverted the cDNA 10 μ l systems of recording into; Its reverse transcription system comprises: 5 * PrimeScript TM Buffer, 2 μ l; PrimeScript TM RT EnzymeMix I 0.5 μ l; Oligo dT Primer (50uM) 0.5 μ l, Random 6 mers 0.5 μ l, RNase Free dH
2O 4.5 μ l, 37 ℃ of 15min of cDNA 2 μ l. reverse transcription conditions, 85 ℃ 5 seconds.Be that template is carried out specific amplification again with cDNA, its amplified reaction 20 μ l systems are by Tap enzyme 10 μ l, Forward primer0.4 μ l, Reverse primer 0.4 μ l, Rox 0.4 μ l, ddH
2O 6.8 μ l, and 2 μ l cDNA templates are formed.Need the primer sequence and the annealing temperature of amplifying target genes to provide by Table 1.B-actin is increased as contrast, after the fluorescent real time PCR reaction finishes, confirms threshold value by systematic analysis software, and then confirms respectively to react the CT value of sample to adopt 2 of relative quantification
-△ △ CTMethod is analyzed PCR result, confirms expression of gene difference between each sample.
Table 4 1500 mg/L capaurine alkali treatments are to the expression relative value (2 of HeLa cell HPV18E6/E7 mRNA
-△ △ CT)
Table 5 1500 mg/L capaurine alkali treatments are to SiHa cell HPV16E6/E7, the expression relative value (2 of mRNA
-△ △ CT)
The result is shown in table 4, table 5, and it shows, when capaurine alkali administration concentration is 1000 mg/L; 2000 mg/L; 24 h after administration respectively during 3000 mg/L extract total RNA from three kinds of cells on the 48 h time points, with specific primer to host gene HPV-16 E6 and E7; HPV-18 E6 and E7; P53; β-actin carries out the RT-PCR specificity extension self-increasing reaction.From interpretation of result, the expression of dosing intervention group Siha cell HPV-16 E6 and E7 more intervention group is expressed not lowly, and E6 be described, E7 capaurine alkali treatment Siha group with do not treat Siha and organize that to compare expression lower.HPV-18 E6 and E7 compare with non-intervention group at dosing intervention group Hela cell and decrease, and explain that capaurine alkali makes E6 in the Hela cell, and the positive degree of E7 gene expression reduces.HPV-16 and HPV-18 E6, the E7 expression of gene is detected for HPV virus depression effect through capaurine alkali.
2. Western Blot method is observed the capaurine alkali inhibition HPV E6 of viral oncogene, the effect of E7 protein expression:
Principle: can observe the variation of protein expression through Western Blot method.
Step: total protein of cell extracts with RIPA and extracts, protein quantification according to Bradfords method with BSA as standard control.Survey its absorbance, the protein content in the calculation sample in 570 nm wavelength.The total protein sample of 50 g is placed in 100 ℃ of Buddhist decocting in water 10 min and carries out degeneration.Protein electrophoresis runs condensing glue with 80 V, and 100 V run separation gel, changes film and adopts 200 mA constant current ice bath 1-2 h; Perhaps 40 V voltages, 4 ℃ of 3h are specifically according to the size decision of molecular weight of albumen.After transfer film after immersion of the membrane with TBS once, into 5?% Skim milk blocking solution at 4 ℃ shaker closed 2? H,? PVDF film is a good one with a diluted anti-p53,? HPV16/18? E6, ? E7 phosphorylated and non-phosphorylated IκB from the U.S. Cell? Signalling? Techonology Company purchased two antibody incubation also used the CST.Protein band carries out video picture with the ECL luminescent solution, uses the X-ray magazine exposure image at last, and protein band is judged provides reference by the Marker that dyes molecular weight in advance.The result of Western Blotting analyzes the OD value of each band with Image-pro plus 6.0 softwares.
Three-type-person's cervical cancer cell is respectively with 1000 mg/L, 2000 mg/L, and 3000 mg/L administration concentration are intervened 24h, during 48h; Extract protein sample, detect Hela, Siha with Western blotting; Target protein E6 in the C33A cell, E7, P53 and I κ B-a; Phospho-I κ B-a, NF-κ B p65, Phospho-NF-κ B p65 level changes.The power of gray scale appears in destination protein, and protein content what are used for reflecting, the result shows similar like instance 1.
Capaurine alkali can suppress the breeding of HPV virus on mRNA and protein level, it has suppressed the E6 of HPV viral oncogene as mechanism for capaurine alkali, E7 (HPV-16 and HPV-18) horizontal expression.
What should explain at last is: the above is merely the preferred embodiments of the present invention; Be not limited to the present invention; Although the present invention has been carried out detailed explanation with reference to previous embodiment; For a person skilled in the art, it still can be made amendment to the technical scheme that aforementioned each embodiment put down in writing, and perhaps part technical characterictic wherein is equal to replacement.All within spirit of the present invention and principle, any modification of being done, be equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (9)
- Formula I or II chemical compound the application in the preparation of treatment HPV virus infective medicament of receptible salt or ester-formin;ⅠR 1Be H ,-OH ,-OCH 3,-O-C 1-C 8Alkyl ,-NH 2,-NO 2,-COOH ,-COOCH 3,-COOCH 2CH 3,-COO-C 1-C 8Alkyl ,-COO-fluoroalkyl, C 1-C 8Alkyl, C 3-C 6Cycloalkyl, C 1-C 8Carboxylic acid, C 1-C 8Carboxylate, 1-3 independent free halogen/HSCH 2-, CH 3SCH 2CH 2-;R 2Be H ,-OH ,-OCH 3,-O-C 1-C 8Alkyl ,-NH 2,-NO 2,-COOH ,-COOCH 3,-COOCH 2CH 3,-COO-C 1-C 8Alkyl ,-COO-fluoroalkyl, C 1-C 8Alkyl, C 3-C 6Cycloalkyl, C 1-C 8Carboxylic acid, C 1-C 8Carboxylate, 1-3 independent free halogen/HSCH 2-, CH 3SCH 2CH 2-;R 3Be H ,-OH ,-OCH 3,-O-C 1-C 8Alkyl ,-NH 2,-NO 2,-COOH ,-COOCH 3,-COOCH 2CH 3,-COO-C 1-C 8Alkyl ,-COO-fluoroalkyl, C 1-C 8Alkyl, C 3-C 6Cycloalkyl, C 1-C 8Carboxylic acid, C 1-C 8Carboxylate, 1-3 independent free halogen/HSCH 2-, CH 3SCH 2CH 2-;R 4Be H ,-OH ,-OCH 3,-O-C 1-C 8Alkyl ,-NH 2,-NO 2,-COOH ,-COOCH 3,-COOCH 2CH 3,-COO-C 1-C 8Alkyl ,-COO-fluoroalkyl, C 1-C 8Alkyl, C 3-C 6Cycloalkyl, C 1-C 8Carboxylic acid, C 1-C 8Carboxylate, 1-3 independent free halogen/HSCH 2-, CH 3SCH 2CH 2-;ⅡR 1Be H ,-OH ,-OCH 3,-O-C 1-C 8Alkyl ,-NH 2,-NO 2,-COOH ,-COOCH 3,-COOCH 2CH 3,-COO-C 1-C 8Alkyl ,-COO-fluoroalkyl, C 1-C 8Alkyl, C 3-C 6Cycloalkyl, C 1-C 8Carboxylic acid, C 1-C 8Carboxylate, 1-3 independent free halogen/HSCH 2-, CH 3SCH 2CH 2-;R 2Be H ,-OH ,-OCH 3,-O-C 1-C 8Alkyl ,-NH 2,-NO 2,-COOH ,-COOCH 3,-COOCH 2CH 3,-COO-C 1-C 8Alkyl ,-COO-fluoroalkyl, C 1-C 8Alkyl, C 3-C 6Cycloalkyl, C 1-C 8Carboxylic acid, C 1-C 8Carboxylate, 1-3 independent free halogen/HSCH 2-, CH 3SCH 2CH 2-;R 3Be H ,-OH ,-OCH 3,-O-C 1-C 8Alkyl ,-NH 2,-NO 2,-COOH ,-COOCH 3,-COOCH 2CH 3,-COO-C 1-C 8Alkyl ,-COO-fluoroalkyl, C 1-C 8Alkyl, C 3-C 6Cycloalkyl, C 1-C 8Carboxylic acid, C 1-C 8Carboxylate, 1-3 independent free halogen/HSCH 2-, CH 3SCH 2CH 2-;R 4Be H ,-OH ,-OCH 3,-O-C 1-C 8Alkyl ,-NH 2,-NO 2,-COOH ,-COOCH 3,-COOCH 2CH 3,-COO-C 1-C 8Alkyl ,-COO-fluoroalkyl, C 1-C 8Alkyl, C 3-C 6Cycloalkyl, C 1-C 8Carboxylic acid, C 1-C 8Carboxylate, 1-3 independent free halogen/HSCH 2-, CH 3SCH 2CH 2-.
- 2. corydaline, isocorydine, isoboldine, N-methyl hernovine, tinea alba premium alkali or the acceptable salt of their medicine or the ester-formin application in prevention and the preparation of treatment HPV virus infective medicament.
- 3. application according to claim 1 and 2 is characterized in that: the acceptable salt of said medicine comprises the formed salt of the acceptable mineral acid of medicine.
- 4. application according to claim 3 is characterized in that: the formed salt of said mineral acid comprises hydrochlorate, hydrobromate, sulfate, carbonate, bicarbonate, maleate.
- 5. application according to claim 1 and 2 is characterized in that: the acceptable ester of said medicine comprises the straight chained alkyl ester with 1-6 carbon atom, or contains the branched alkyl ester of 3 or 6 carbon atoms, or benzyl ester.
- 6. application according to claim 5 is characterized in that: the acceptable ester of said medicine comprises methyl, ethyl, propyl group, butyl, 2-methyl-propyl and 1,1-dimethyl ethyl ester.
- 7. application according to claim 1 and 2 is characterized in that: said fluoroalkyl substituent group comprises the substituent C1-C3 perfluoroalkyl of perfluoroalkyl substituent group-CF3;-O-C1-C3 perfluoroalkyl substituent group-OCF3 and-S-C1-C3 perfluoroalkyl substituent group-SCF3.
- 8. pharmaceutical composition, said composition comprise each the acceptable salt of chemical compound or ester-formin and the pharmaceutically-acceptable excipients or the carrier of claim 1-2 of drug effective dose.
- 9. pharmaceutical composition according to claim 9 is characterized in that: said drug regimen is powder, tablet, capsule and nanometer formulation; Or solution, suspension liquor, emulsion agent, slurry agent, tincture and spirit; Or solid-liquid mix preparation.
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| CN114015802A (en) * | 2021-12-10 | 2022-02-08 | 海南师范大学 | SSR (simple sequence repeat) primer for endangered semi-mangrove plant mallotus japonicus and application of SSR primer |
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