CN104814947B - Application of the diterpene-kind compound in preventing and treating hepatitis virus medicament is prepared - Google Patents
Application of the diterpene-kind compound in preventing and treating hepatitis virus medicament is prepared Download PDFInfo
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- CN104814947B CN104814947B CN201510225636.0A CN201510225636A CN104814947B CN 104814947 B CN104814947 B CN 104814947B CN 201510225636 A CN201510225636 A CN 201510225636A CN 104814947 B CN104814947 B CN 104814947B
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- glaucocalyxin
- epoxides
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Abstract
The invention provides the application of diterpene-kind compound glaucocalyxin A, Minheryins I, the element of caudal lobe penta and glaucocalyxin A epoxides in preventing and treating hepatitis C virus cytotoxic drug is prepared and its application in the food and health products for preparing liver protection.The present invention is glaucocalyxin A, Minheryins I, the element of caudal lobe penta and glaucocalyxin A epoxides provide the purposes for preparing anti-hepatitis c virus (HCV) medicine, result of the test is found, glaucocalyxin A, Minheryins I, the element of caudal lobe penta and glaucocalyxin A epoxides have the anti-HCV activity of highly significant, available for the medicine, health products and food for preparing anti-hepatitis C virus.The present invention provides new source to find anti hepatitis C virus drug.
Description
Technical field
The present invention relates to pharmaceutical technology field, specifically, be diterpene-kind compound glaucocalyxin A, Minheryins I,
The application of the element of caudal lobe penta and glaucocalyxin A epoxides in preventing and treating hepatitis virus medicament is prepared.
Background technology
Glaucocalyxin A (Glaucocalyxin A, GLA) is planted from Labiatae (Labiatae) Rabdosia (Rabdosia)
A kind of diterpene-kind compound with the kaurene skeleton of 15- oxygen -16 isolated in thing rabdosia japonica, molecular formula is
C20H28O4, its structure is as shown in Equation 1.Research has shown that：Glaucocalyxin A have antitumor, antibacterial, inducing cell apoptosis, it is anti-oxidant,
The multiple biological activities such as DNA damage protection and CDCC.Minheryins I are to be extracted from plant rabdosia japonica
Obtained diterpene-kind compound, (scholar Yao etc., the anti-complement activity composition Study of rabdosia japonica is Chinese as shown in Equation 2 for its structure
J Chinese, 2013,28 (4):199-201).The element of caudal lobe penta is that obtained Diterpenes chemical combination is extracted from plant rabdosiaexcisa
Thing, its structure is as shown in Equation 3.Glaucocalyxin A epoxide structure formula is as shown in Equation 4, using glaucocalyxin A as raw material, through hydrogen peroxide
Or m-chloro-benzoic acid peroxide epoxidation is made.
Chinese patent literature CN201110041403.7 discloses application of the glaucocalyxin A in anticoagulation medicine is prepared, hair
Existing glaucocalyxin A can significantly extend mouse capillary clotting time and tail point bleeding time, and when significantly extending mouse fibrin ferment
Between, prothrombin time, activated partial thromboplastin time and blood plasma cover time.Chinese patent literature
CN201310535621.5 discloses application of the glaucocalyxin A derivative in treatment leukemia medicament is prepared.In May, 2006 Ji
Woods University Ph.D. Dissertation,《Rabdosiaexcisa Diterpenoids from bulbus and Anticancer Activities》To rabdosiaexcisa chemical composition and
Active anticancer is studied, and 16 dammara ene-type diterpene-kind compounds are divided into obtain from rabdosiaexcisa aerial part, wherein
The molecular formula of compound 5 is C26H42O9, by transplanted tumor model in Mice Body, to the antitumor of rabdosiaexcisa leaf total diterpene
Effect has carried out experimental study, as a result shows rabdosiaexcisa leaf total diterpene 100,200mg/kg to rat liver cancer H22, Lewis lungs
Cancer and melanoma B16With stronger growth inhibition effect.On the anti-HCV (hepatitis C virus) function of above-mentioned 4 kinds of compounds,
It yet there are no report.
The content of the invention
The purpose of the present invention is that there is provided diterpene-kind compound, caudal lobe in rabdosia japonica for deficiency of the prior art
The application of diterpene-kind compound and glaucocalyxin A epoxides in preventing and treating hepatitis virus medicament is prepared in Rabdosia amethystoides.
Another purpose of the present invention is to provide described noval chemical compound glaucocalyxin A epoxides and its synthetic method.
To achieve the above object, the present invention is adopted the technical scheme that：
Diterpene-kind compound and glaucocalyxin A epoxides exist in diterpene-kind compound, rabdosiaexcisa in rabdosia japonica
Prepare the application in preventing and treating hepatitis virus medicament, in the rabdosia japonica diterpene-kind compound be glaucocalyxin A and
Minheryins I, its chemical constitution is respectively as shown in formula 1 and formula 2；Diterpene-kind compound is caudal lobe in the rabdosiaexcisa
Penta element, its chemical constitution is as shown in Equation 3；The chemical constitution of the glaucocalyxin A epoxides is as shown in Equation 4.
Described glaucocalyxin A, Minheryins I, the element of caudal lobe penta and glaucocalyxin A epoxides is preparing anti-third type liver
Application in scorching virus drugs.
Described glaucocalyxin A, Minheryins I, the element of caudal lobe penta and glaucocalyxin A epoxides suppresses HCV in preparation and entered
The application invaded in the medicine of target cell.
To realize above-mentioned second purpose, the present invention is adopted the technical scheme that：
The synthetic method of described glaucocalyxin A epoxides, using glaucocalyxin A as raw material, through hydrogen peroxide or m-chloro peroxide
Change benzoic acid epoxy chemical conversion glaucocalyxin A epoxides.
The invention has the advantages that：
The present invention is diterpene-kind compound glaucocalyxin A, Minheryins I, the element of caudal lobe penta and glaucocalyxin A epoxides
The purposes that one kind prepares anti-hepatitis c virus (HCV) medicine is provided, the third type liver originated by the cell culture set up in vitro
Scorching virus (HCVcc), using HCV easy infection cell human liver cancer Huh7 cell lines, to the HCV-Ab IgG of above-mentioned four kinds of diterpene-kind compounds
Activity has carried out experiment in vitro.As a result find, these four diterpene-kind compounds are respectively provided with significant anti-HCV activity, can especially press down
HCV processed invades target cell, available for the medicine for preparing anti-third type virus.The present invention be only diterpene-kind compound glaucocalyxin A,
Minheryins I, the element of caudal lobe penta and glaucocalyxin A epoxides provide new application, are also searching anti-hepatitis c virus medicine
Thing provides new source.
Embodiment
The embodiment provided with reference to embodiment the present invention elaborates.
The preparation (one) of the glaucocalyxin A epoxides of embodiment 1
Glaucocalyxin A 80mg, is added in 8ml ethanol, and 20%NaOH 1ml, H are added at room temperature2O21ml, stirring reaction
After 48h, TLC, raw material point disappears, and reaction is complete.Reaction solution is poured into 20ml water, CH is then used2Cl2Extraction, each 5ml,
Extraction 3 times.Merge CH2Cl2Extract, Na2SO43h is dried, Na is then filtered to remove2SO4, depressurize and boil off solvent.Residue silicon
Plastic column chromatography (petroleum ether：Ethyl acetate=3:2).Obtain target compound glaucocalyxin A epoxides.ESI-MS[M+H]+=
349.3；1H-NMR：3.16 (1H, d, J=6.0, H-17)；2.92 (1H, d, J=6.0, H-17)；4.30 (1H, dd, J=
11.4,3.6Hz,H-7)；4.98(1H,s,H-14)；2.29(1H,s,H-13).
The preparation (two) of the glaucocalyxin A epoxides of embodiment 2
Glaucocalyxin A 80mg, is added to 8ml CH2Cl2In, m-chloro-benzoic acid peroxide 532mg room temperatures are added at room temperature to be stirred
Mix after reaction 24h, TLC, raw material point disappears substantially, reaction is substantially completely.1 time is washed with protection solution of sodium bisulfite 5ml, so
Washed 3 times with water 5ml afterwards.CH2Cl2Liquid Na2SO43h is dried, Na is then filtered to remove2SO4, depressurize and boil off solvent.Residue silicon
Plastic column chromatography (petroleum ether：Ethyl acetate=3:2).Obtain target compound calyx A prime epoxides.
Plain and glaucocalyxin A epoxides the anti-third type hepatovirus of the glaucocalyxin A of embodiment 3, Minheryins I, caudal lobe penta
Experiment
First, Experimental agents, reagent and material
1. compound：Glaucocalyxin A, Minheryins I, the element of caudal lobe penta and glaucocalyxin A epoxides
2. cell line Huh7, human hepatoma cell strain (is referred to：Yimin Tong,Yongzhe Zhu,Xueshan Xia,
Yuan Liu,et al.Tupaia CD81,SR-BI,Claudin-1,and Occludin Support Hepatitis C
Virus Infection,JOURNAL OF VIROLOGY,2011；85(6):2793–2802；Jin Zhong,Pablo
Gastaminza, Guofeng Cheng, et al.Robust hepatitis C virus infection in vitro,
PNAS,2005；102(26):9294-9299).
3. cell line 293T, human embryonic kidney cell line (is referred to：Yimin Tong,Yongzhe Zhu,Xueshan Xia,
Yuan Liu,et al.Tupaia CD81,SR-BI,Claudin-1,and Occludin Support Hepatitis C
Virus Infection,JOURNAL OF VIROLOGY.2011；85(6):2793–2802；Graham,F.L.,Smiley,
J.,Russell,W.C.&Nairn,R.J.Gen.Virol 1977；36:59–74).
4. cell culture fluid, is prepared, it is blue or green containing 10% hyclone, 0.03% glutamine, nonessential amino acid, ammonia benzyl
Mycin and streptomysin 100U/ml, adjust pH to 7.4.
5. cell dissociation buffer, is prepared, it contains 0.25% trypsase, is prepared with phosphate buffer.
6.HCVcc：The infectious HCV of cell culture (is referred to：Yimin Tong,Yongzhe Zhu,
Xueshan Xia,Yuan Liu,et al.Tupaia CD81,SR-BI,Claudin-1,and Occludin Support
Hepatitis C Virus Infection,JOURNAL OF VIROLOGY,Mar.2011；85(6):2793–2802；Jin
Zhong,Pablo Gastaminza,Guofeng Cheng,et al.Robust hepatitis C virus infection
In vitro, PNAS, 2005；102(26):9294-9299).
2nd, experimental method：
(1) HCVcc preparation
1. virus amplification
J6, JFH-1 are fitted together to HCVcc (105Ffu/ml), 50 μ l infection is taken to be inoculated in the cells of Huh 7.5 of 24 orifice plates, next day
Liquid is changed, then according to cell density Secondary Culture, cell growth state is observed, treats cytopathy caused by viral fast breeding
After change effect (CPE) occurs, the culture supernatant of the 7th~20 day is collected, take is used for virus titer measure in right amount, and remaining 8,000rpm
Centrifugation 5min abandon after cell fragment packing be stored in -70 DEG C it is standby.
2. titration of virus
With blank Huh 7.5 (1 × 104Cells/ holes), after 12h, culture supernatant is abandoned, 100 μ l are added per hole through 10 times of ladders
The HCVcc supernatants of dilution are spent, 5h is incubated altogether, renews fresh DMEM full nutrient solutions and continues to cultivate 72h, row immuno-fluorescence assay
HCV positive cells, primary antibody uses 1:The HCV antibody positive patients serums of 100 dilutions, secondary antibody is 1:The FITC mark sheep of 100 dilutions
Anti-human igg.In fluorescence microscopy Microscopic observation luminescent cell, and record last hole that gfp positive cell can be observed
Interior gfp positive cell number and corresponding dilution gradient, calculate focus forming unit/ml (ffu/ml) numerical value,
HCVcc titres are represented with this.
(2) the infective detections of HCVcc
The Huh7 cells in exponential phase are taken, adjustment cell concentration is 1 × 105Individual/ml, takes the orifice plate of 100 μ l kinds 96；
Be separately added into four kinds of compounds and HCVcc after culture 24h, compound is by 0.2,1,5,20ug/ml concentration dilutions, 37 degree are cultivated
Compound is removed after 4 hours and HCVcc mixes liquid, culture medium is changed and continues to cultivate；Immunofluorescence test is carried out after 48h, it is aobvious in fluorescence
Each hole HCVcc positive colony numbers are read under micro mirror.
(3) result (being shown in Table 1)
The inhibitory activity (%) of 1 four kinds of compound on intracellular culture hepatitis C virus (HCVcc) of table
| Compound | 20μg/ml | 5μg/ml | 1μg/ml |
| Glaucocalyxin A | (-) | (-) | 62.5 |
| Minheryins I | 51 | 36 | 26 |
| The element of caudal lobe penta | (-) | (-) | 74.5 |
| Glaucocalyxin A epoxides | 52 | 18.2 | 5.4 |
| Glaucocalyxin C | N | N | N |
| Glaucocalyxin E | N | N | N |
| Oridonin | N | N | N |
Note：(-) represents cell death under the concentration；N represents inactive.
All had from 1,4 compounds of table and significantly inhibit hepatitis C virus (HCVcc) effect, illustrate that it has anti-third type
Hepatitis viruse and hepatoprotective effect.It is above-mentioned test result indicates that, glaucocalyxin A (1), Minheryins I (2), caudal lobe penta plain (3) and
Glaucocalyxin A epoxides (4) has significant antihepatitis C virus activity, available for the medicine for preparing anti-hepatitis c virus
Thing and the medicine of liver protection.
HCV (HCVcc) model that the present invention is originated by the cell culture set up in vitro, it is susceptible using HCV
Cell human liver cancer Huh7 cell lines are contaminated, to diterpene-kind compound glaucocalyxin A, Minheryins I, the element of caudal lobe penta and glaucocalyxin A
The anti-HCV activity of epoxides has carried out experiment in vitro.As a result find, it has significant anti-HCV activity, can especially suppress
HCV invades target cell.Inventor is found surprisingly that glaucocalyxin A ring during being chemically modified to glaucocalyxin A simultaneously
Oxide equally has hepatitis C virus inhibitory action.Therefore these four diterpene-kind compounds can be used for preparing anti-hepatitis C virus HCV
Medicine.
Meanwhile, inventor from rabdosia japonica dry aerial parts be extracted compound Glaucocalyxin C, Glaucocalyxin E (referring to
Document：The 3rd three director's conference nd Annual Meeting special editions of Chinese Medicine Education Associations,《Rabdosia japonica chemical composition and anti-swollen
Knurl and anti-HBV effect research》, 2013；Document：Anti-infective pharmacy,《Rabdosia japonica chemical composition and bioactivity
Recent Research》, 2011) and compound Oridonin, and according to above-mentioned experimental method test these three compounds for
The inhibitory activity of hepatitis C virus, as a result shows that this three classes compound is acted on the equal unrestraint of hepatitis C virus.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, on the premise of the inventive method is not departed from, can also make some improvement and supplement, and these are improved and supplement also should be regarded as
Protection scope of the present invention.
Claims (4)
1. diterpene-kind compound and glaucocalyxin A epoxides are being made in diterpene-kind compound, rabdosiaexcisa in rabdosia japonica
Application in standby anti hepatitis C virus drug, it is characterised in that diterpene-kind compound is blue calyx first in the rabdosia japonica
Element and Minheryins I, its chemical constitution is respectively as shown in formula 1 and formula 2；Diterpene-kind compound is in the rabdosiaexcisa
The element of caudal lobe penta, its chemical constitution is as shown in Equation 3；The chemical constitution of the glaucocalyxin A epoxides is as shown in Equation 4：
2. application according to claim 1, it is characterised in that described glaucocalyxin A, Minheryins I, the element of caudal lobe penta
The application in preventing and treating hepatitis C medicine is being prepared with glaucocalyxin A epoxides.
3. a kind of glaucocalyxin A epoxides, it is characterised in that the glaucocalyxin A epoxide chemistry structure is as follows：
4. the synthetic method of glaucocalyxin A epoxides as claimed in claim 3, it is characterised in that using glaucocalyxin A as original
Material, glaucocalyxin A epoxides is melted into through hydrogen peroxide or m-chloro-benzoic acid peroxide epoxy.
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