CN108379278A - Application of the trifloroside in preparing anti HIV-1 virus infection medicine - Google Patents
Application of the trifloroside in preparing anti HIV-1 virus infection medicine Download PDFInfo
- Publication number
- CN108379278A CN108379278A CN201810431029.3A CN201810431029A CN108379278A CN 108379278 A CN108379278 A CN 108379278A CN 201810431029 A CN201810431029 A CN 201810431029A CN 108379278 A CN108379278 A CN 108379278A
- Authority
- CN
- China
- Prior art keywords
- trifloroside
- hiv
- cell
- virus
- added
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses application of the trifloroside in preparing anti HIV-1 virus infection medicine.Inventor, which studies, to be found, trifloroside has 1 activity of AntiHIV1 RT activity of wide spectrum, and has lower cytotoxicity, and possible mechanism of action, which is it, can specifically target the region of the N-terminal repetitive sequence of gp41, to inhibit the entrance of HIV, it is expected to be developed into the small molecule entry inhibitors of excellent AntiHIV1 RT activity.
Description
Technical field
The present invention relates to field of medicaments, and in particular to application of the trifloroside in preparing anti HIV-1 virus infection medicine.
Background technology
It is comprehensive that AIDS (Acquired immunodeficiency syndrome, AIDS) is also known as acquired immunodeficiency
Simulator sickness is serious by one kind caused by human immunodeficiency virus (Human Immunodeficiency Virus, HIV) infection
Single causal disease.Cut-off 2016, there are about 36,700,000 people's infected by HIV, 1,000,000 people die of AIDS and its correlation in the whole world
Property disease.AIDS includes mainly following three kinds of routes of transmission：Blood born spreads through sex intercourse and vertical transmission.Wherein, the same sex
And spreading through sex intercourse between the opposite sex has become aids transmission approach the most main, ratio is up to 80%, in some countries of Africa
More up to 90%.Currently, AIDS has become the first big communicable disease of the mankind, health and the society of the mankind seriously threaten
Meeting is stabilized.
Since HIV enters the first step that target cell is HIV infection host, it is to prevent and control effectively to prevent HIV from entering
Treat the key of AIDS.HIV envelope proteins are synthesized on the rough surfaced endoplasmic reticulum (RER) of target cell by amyloid protein precursor molecule, then
Become tripolymer by glycosylation in endoplasmic, is transported on golgiosome later, HIV envelope proteins are by the albumen of HIV
Enzyme hydrolysis is at two subunits of gp41 and gp120.Under the action of big vesica secreted by golgiosome, ripe gp120/
Gp41 tripolymers are transported to target cell membrane, participate in the assembling of the virion of a new round.The number of inhibition of HIV particle can be 6
~10h is doubled once, and the content of the free virus highest in the blood of human body can reach 108-9A/mL.Since gp41 is tool
There is the distinctive envelope protein of conserved sequence virus, therefore it has more advantage as drug target.
1987, neat Fu Duoding (AZT) was applied to AIDS patient as first anti-HIV-1 medicine.Currently,
The inverase of FDA approvals includes mainly following several major class：1) hiv reverse transcriptase inhibitor, including ucleosides and non-nucleoside
Reverse transcriptase inhibitor；2) hiv protease inhibitor；3) integrase inhibitor；4) HIV entrance/fusion inhibitor[10].It is clinical
On mostly use highly active antiretroviral therapy (Highly activate anti-retroviral therapy, HAART) or
Joint antiretroviral therapy (Combination antiretroviral therapy, cART), i.e., " cocktail therapy "
Treat AIDS.HAART therapies, which usually select 2~4 kinds of reverse transcriptase inhibitor and 2~4 kinds of protease inhibitors to combine, to be made
With, it is therefore an objective to the inverase synergistic corrosion virus for selecting different role mechanism avoids virus from generating quickly anti-medicine to single medicine
Property and influence therapeutic effect, to effectively inhibit HIV in the duplication of host, and repair the human immunity work(that has been destroyed of part
Can, greatly reduce the morbidity and mortality of virus.Therefore, HAART therapies are successfully lethal from one kind by AIDS
Property metabasis is the important breakthrough in AIDS preventing and controlling history at chronic controllability disease.But HAART therapies also have certain
Defect, such as：Expensive, toxic side effect is big, virus variation or the shortcomings that drug resistance, causes a large amount of AIDS patients cannot be after
It is continuous to use.Therefore it needs that new medicine and new strategy is clinically needed to prevent AIDS.HIV entry inhibitors due to
Antiviral activity can be played in virus replication early stage, and still effective to the drug resistant Strain of other inverases, therefore
Its development attracts attention.
In March, 2003, U.S. FDA " fast channel " approval have listed first HIV entrance/fusion inhibitor enfuirtide
(Enfuvirtide, T-20), to contain 36 derived from the ends gp41C- d repetitive sequences (C-heptad repeat, CHR)
The polypeptide drug of amino acid can be combined the formation for inhibiting six helical structures of HIV with the N- terminal repeats of HIV.T-
20 can treat the AIDS patient that tolerance is generated to reverse transcriptase inhibitor and protease inhibitors well.But T-20 faces
Bed application also has a series of defects, including：Oral bioavilability extremely low (can only be administered by injection system), half-life period
Short, production cost high (90mg, twice daily), drug tolerance, dosage is big (each 100mg, 2 times/day), molecular weight it is big (>
4000Da), easily by the proteasome degradation of human endogenous's property, synthesis step is complicated and somewhat expensive etc..Currently, the peace that exploitation is novel
Entirely, effective and inexpensive HIV entrance/fusion inhibitor has become the hot spot of anti-AIDS drug treatment.
Trifloroside (Trilobatin) is derived from a kind of glycosylated dihydrochalcone in the leaf of Pasania cuspidata Sweet tea
Flavonoid micromolecule compound.It has been reported that and shows that trifloroside has anti-diabetic, anti-oxidant and anti-inflammatory bioactivity.Do not have
Research shows that trifloroside has AntiHIV1 RT activity infection activity.
Invention content
The purpose of the present invention is to provide application of the trifloroside in preparing anti HIV-1 virus infection medicine.
The technical solution used in the present invention is：
The application of trifloroside and its pharmaceutically acceptable derivates in preparing anti HIV-1 virus infection medicine.
As being further improved for above application, trifloroside pharmaceutically acceptable derivates are selected from its pharmaceutical salts, medicinal
Ester, medicinal ether.
As being further improved for above application, the pharmaceutically acceptable pharmaceutical salts of trifloroside are selected from potassium, sodium, calcium, zinc salt.
As being further improved for above application, the pharmaceutically acceptable medicinal ester of trifloroside is the medicinal ester of C1~C6.
As being further improved for above application, the pharmaceutically acceptable medicinal ether of trifloroside is the medicinal ether of C1~C6.
The beneficial effects of the invention are as follows：
Inventor the study found that trifloroside have wide spectrum Anti-HIV-1 Active, and have lower cytotoxicity, may
Mechanism of action to be it can specifically target the region of the N-terminal repetitive sequence of gp41, to inhibit the entrance of HIV.It is expected to out
Hair becomes excellent AntiHIV1 RT activity small molecule entry inhibitors.
Inventor has found micromolecular compound trifloroside (Trilobatin) for the first time by research, has preferable AntiHIV1 RT activity
Viral infectivity effectively can inhibit inhibition of HIV to enter target cell, to inhibit the infection of inhibition of HIV in early stage.
Inventor utilizes body outer clone viral infectivity drug screening cell model, experimental result to show that trifloroside can
(HIV-1 is infected to significantly inhibit tri- kinds of clonal virus strains of HIVSF162, HIV-1NL4-3And HIV-181A and NL4-3), and with dense
Spend dependence, IC50In low micromolar level.Time-of-addition experiments confirm trifloroside by acting on cell entry target
The preliminary stage blocking virus direct infection target cell of cell, effect maximum in the early stage inhibiting rate of cell entry cell
It is best.In order to further confirm that trifloroside is the early stage for acting on poisoning intrusion target cell, we have detected trifloroside pair
Functional pseudovirus HIV-1JR-FL(R5), HIV-1HXB2(X4) and the inhibiting effect of VSV-G.Trifloroside can dose-dependently press down
Two kinds of HIV-1 cape horn fever strains are made, action target spot may be the coating of HIV-1.But trifloroside is to VSV-G pseudovirus without apparent suppression
It makes and uses, show that trifloroside is specific effect in HIV-1 envelope proteins.Inventor passes through a kind of non-infectious experiment side
The inhibiting effect that method is used to detect trifloroside to the HIV-1 membrane glycoproteins Env Cell-Cell Fusions induced, as a result, it has been found that three leaves
Glycosides can inhibit the film fusion that HIV envelope glycoproteins Env is mediated.Pass through sandwich ELISA, natural N-PAGE gel and circular dichroism spectra etc.
Method, has detected the inhibiting effect that trifloroside is formed by simulation N peptides and C peptides Six helix bundle, and experimental result confirms three leaves
Glycosides can effectively inhibit viral gp41NHR and CHR to form six helical structures, but the gp41 film fusion nucleus hearty cords to having been formed
Structure does not act.
By surface plasma resonance (SPR) the experiment proves that trifloroside can be combined directly with polypeptide N36, pass through inhibition
The formation of six spirals of gp41 is to inhibit HIV-1 to enter target cell.Meanwhile playing the concentration model of antivirus action in trifloroside
Enclose interior to the basic no cytotoxicity of human normal cell, to sum up, trifloroside has in terms of pre- preventing HIV virus infection and high makes
With value and safety.Trifloroside is very likely developed into a kind of novel anti-HIV-1 small molecule entry inhibitors.Trifloroside
It derives from a wealth of sources, originating species Pasania cuspidata has very long applicating history in China as edible and medicinal plant, relatively
It is cheap, securely and reliably.
Description of the drawings
Fig. 1：Body outer clone virus infection model detects inhibiting effect of the trifloroside to HIV clonal virus；
Fig. 2：Time-of-addition experiment detection triflorosides act on the early stage of virus infected cell；
Fig. 3：Trifloroside is to HIV-1JR-FL, HIV-1HXB2With the detection trifloroside of VSV-G functionality pseudovirus infection abilities
Action target spot；
Fig. 4：Inhibiting effect of the trifloroside to the HIV-1 membrane glycoproteins Env Cell-Cell Fusions induced；
Fig. 5：The methods of sandwich ELISA, natural N-PAGE gel and circular dichroism spectra have detected trifloroside to simulation N peptides and C
Peptide is formed by the inhibiting effect of Six helix bundle；
Fig. 6：Surface plasma resonance experiment detection trifloroside is combined with polypeptide N36.
Specific implementation mode
Below in conjunction with experiment, the present invention is further described, but the scope of the present invention is not limited thereto.
Experiment one：Body outer clone virus infection model detects inhibiting effect of the trifloroside to HIV clonal virus
Experimental method：
1) gradient dilution trifloroside, with HIV-1SF162, HIV-1NL4-3And HIV-181A and NL4-3The isometric mixing of virus liquid
It is added in the TZM-b1 tissue culture plates being inoculated in advance after incubating 30min altogether, virus control wells and cell blank control wells is set；
2) TZM-b1 cell fluorescence element expression of enzymes situations are detected using Luciferase Assay Reagent box after virus infection 48h.
Experimental result as shown in Figure 1, trifloroside can dose-dependently inhibit three plants of HIV infection clone activity, it is right
HIV-1SF162, HIV-1NL4-3And HIV-181A and NL4-3The IC of virus50Value be respectively 11.83 ± 2.25,10.56 ± 2.02 and
10.69±3.76μg/mL.Wherein IC50For half inhibiting rate, indicate dense when trifloroside inhibits half inhibition of HIV infection cell
Degree.Illustrate that trifloroside has efficient and wide spectrum Anti-HIV-1 Active, is expected to develop into an ideal anti-HIV-1 medicines.
Experiment two：Time-of-addition experiment detection triflorosides act on the early stage of virus infected cell
Experimental method：
Trifloroside inhibition HIV-1 enters the research (Time-of-addition) of target cell
1) by HIV-1SF162(CCR5 preferendums), HIV-1NL4-3The addition of (CXCR4 preferendums) type cape horn fever venom has been inoculated in advance
It in cell plates, is placed in incubator and cultivates 2h, suck supernatant, the fresh DMEM cultures containing 10%FBS are added in PBS board-washings 2 times
Base；
2) 50 μ L virus liquids are added per hole, with 1 × 10537 DEG C of/mL TZM-bl cells are incubated 0,0.5,1,2,4,6,8,
10,12, for 24 hours, at corresponding time point, per hole, (final concentration trifloroside is 50 μ g/mL, positive drug Maraviroc to 50 μ L drugs of addition
It is 15ng/mL for 6ng/mL, positive drug AZT)；
3) expression that Luciferase Assay Reagent box examining report gene luciferase is utilized after 48h calculates drug suppression
Rate processed.
As shown in Fig. 2, trifloroside is added before viral target cell infection, antiviral activity is kept experimental result.But
Trifloroside is added in different time points after viral target cell infection, and with gradually increasing for infection time, inhibiting rate is also gradual
Decline.When infection occurs 6 or 8 hours, effective inhibiting rate of trifloroside reduces by 50% or more.Similarly, HIV-1 enters suppression
Preparation positive control drug CCR5 accessory receptors inhibitor Maraviroc and CXCR4 accessory receptor inhibitor AMD3100 feels in virus
Contaminate target cell 1~6 hour is added, and antiviral activity is also remarkably decreased.It is above-mentioned statistics indicate that, trifloroside may be a kind of
HIV-1 entry inhibitors may act on the early stage of HIV-1 life cycles.
Experiment three：The experiment of the anti-functional pseudovirus infection activity of trifloroside shows that the action target spot of trifloroside is HIV-1
Coating
The detection of the anti-functional pseudovirus infection activity of trifloroside
1) phloretin is configured to the mother liquor of 200mg/mL with dimethyl sulfoxide (DMSO), put be formed on 4 DEG C it is spare；
2) when testing, the trifloroside and 50 μ L HIV-1 of 50 μ L concentration gradients are takenHXB2U87.CD4.CXCR4 is added in pseudovirus
Cell；Take the trifloroside and 50 μ L HIV-1 of 50 μ L concentration gradientsJR-FLU87.CD4.CCR5 cells are added in pseudovirus；Take 50 μ L dense
The trifloroside and 50 μ L VSV-G pseudovirus for spending gradient are added in U87.CD4.CXCR4 cells or U87.CD4.CCR5 cells；
3) in 37 DEG C, 5%CO2Under the conditions of cultivate 2 days, according to luciferase reporter gene kit specification method detect
The data of detection are analyzed in the expression of luciferase reporter gene luciferase.Positive drug is respectively
Maraviroc, AMD3100, T-20.
Experimental result is as shown in figure 3, HIV-1 pseudovirus can only carry out infecting for " single-wheel cell cycle ".In order to further
Confirm that trifloroside is the early stage for acting on poisoning intrusion target cell, trifloroside can dose-dependently inhibit two kinds of HIV-1
Cape horn fever strain, HIV-1JR-FLAnd HIV-1HXB2IC50Respectively 7.54 ± 1.72 μ g/mL, 1.99 ± 0.29 μ g/mL.Due to
Trifloroside is capable of the infection of the inhibition HIV-1 pseudovirus of specificity, and action target spot may be the coating of HIV-1.VSV-G cape horn fevers
Poison is built-up by the core plasmid of HIV-1 and the envelope plasmid of VSV-G viruses.If trifloroside can also inhibit VSV-
The activity of G pseudovirus, illustrate trifloroside be possible nonspecific action in HIV envelope proteins.As a result the three of 100 μ g/mL are shown
Leaf glycosides shows that trifloroside is specific effect in HIV-1 envelope proteins still without the apparent effect for inhibiting VSV-G pseudovirus.
Experiment four：Inhibiting effect of the trifloroside to the HIV-1 membrane glycoproteins Env Cell-Cell Fusions induced
The ratio that trifloroside inhibits Syncytium formation is detected, that is, inhibits CHO-WT cells (to transfect HIV-1 envelope glycoproteins
Gp160 subunits) film stablizes and the activity of the fusion of MT-2 target cells (Membrane surface expression CD4 receptors and CXCR4 accessory receptors).
1) CHO-WT cells are cultivated：
400 μM of glutamine synthetase inhibitor (MSX, L- amino is added inside A.GMEM-S culture mediums (10%FBS)
Sulfoxide methionine)；
The passage of B.CHO-WT cells is digested using the solution (PBS preparations) of 0.5mM EGTA and 0.5mM EDTA.
A.100mLGMEM-S the preparation of culture medium：
10mL 10×MEM
70.4mL pure water
7.5% sodium bicarbonates of 3.6mL
2mL 50 × nucleosides solution
10mL FBS
1mL 100 × (glutamic acid+asparagine solution)
The non-limiting amino acid of 1mL
1mL 100mM Sodium Pyruvates
1mL 5000U/mL mycillins
B.10mL the preparation of 50 × nucleosides solution：3.5mg adenosines, 3.5mg guanosines, 3.5mg cytidines, 3.5mg uridines,
1.2mg thymidines are dissolved in 10mL water；
C.10mL the preparation of 100 × glutamic acid+asparagine solution：60mg Pidolidones, 60mg altheines,
It is dissolved in 10mL water.
2) cell fusion that HIV-1 is mediated：
A. the 50 μ LCHO-WT cells of paving per hole, 4 × 105/ mL, with the trifloroside of the concentration gradient of 50 μ L of every hole 37 DEG C,
5%CO2Cultivate 30min；
B. the 100 μ LMT-2 cells of paving per hole, 2 × 105/ mL is added in mixture above in 37 DEG C, 5%CO2Culture 2 days；
C. 4 visual field observation CHO-WT cells and the plastidogenetic conjunction born of the same parents of MT-2 are picked out at random with inverted light microscope
Body calculates plasomidum number N, and calculating trifloroside with computer CalcuSyn softwares inhibits plasomidum IC50, inhibit the shape of plasomidum
At %=(1-N samples/N is positive) × 100.
Experimental result is as shown in figure 4, trifloroside can effectively inhibit the fusion of MT-2 and CHO-WT, IC50For 12.89 ±
1.03 μ g/mL, and it is in notable positive correlation to the inhibiting effect and dosage of cell fusion.HIV-1 entry inhibitors ADS-J1 makees
For positive drug (targeting envelope glycoprotein gp41), effective half inhibiting rate IC50For 1.78 ± 0.08 μ g/mL.Result above
Illustrate, trifloroside can inhibit the film fusion that HIV envelope glycoproteins Env is mediated.
Experiment five：The methods of sandwich ELISA, natural N-PAGE gel and circular dichroism spectra have detected trifloroside to simulating N peptides
The inhibiting effect of Six helix bundle is formed by with C peptides
Experimental method：
1) sandwich ELISA：
A) it is coated with：The p24 monoclonal antibodies (183-12H-5C) that 5 μ g/mL are added per hole in 96 half-pore plates (use PH=
9.6 phosphate buffers dissolve), it is placed in 4 DEG C of refrigerator overnights；Secondary daily PBS-T buffer solutions (containing 0.05%Tween-20) board-washing 3
It is secondary；
B) it closes：2% skim milk (PH=7.2, PBS dissolve) of 150 μ L is added per hole, 37 DEG C are incubated 1h, and PBS-T is washed
Plate 3 times；
C) it is loaded：P24 and 100 mixed liquors (1 of 5%Trition-X is added:1), 50 holes μ L/, 37 DEG C are incubated 1h, PBS-T
Board-washing 3 times；
D) add primary antibody：HIV-IgG (PBS, 1 μ g/mL), 50 holes μ L/, 37 DEG C of incubation 1h, PBS-T board-washings 3 times is added；
E) add secondary antibody：The goat anti-human igg that biotin labeling is added (is added 2% skimmed milk power and presses 1:10000 prepare), 50 μ
The holes L/, 37 DEG C of incubation 1h, PBS-T board-washings 3 times；
F) streptomysin marker：SA-HRP is added (10% sheep blood serum to be added and presses 1:10000 prepare), 50 holes μ L/, 37 DEG C incubate
Educate 1h, PBS-T board-washings 6 times；
G) it develops the color：3,3',5,5'-tetramethylbenzidine (TMB) solution of 50 μ L is added per hole, reacts 3~10min；
H) it terminates：1M H are added2SO4Terminate reaction, 25 holes μ L/ (when observing that blank control wells will develop the color).In enzyme
Mark instrument is measured with 450nm and is measured under wavelength (570nm reference wavelengths).
2) natural N-PAGE gel：
With glue：
A. separation gel (18%) is prepared：The deionized water of 0.75mL, the 30% of 3mL acrylamide, 1.25mL
1.5MTris-HCl (pH=8.8), 10% ammonium persulfate solution of 25 μ L, 2.5 μ L TEMED (N, N, N ', N '-tetramethyls
Ethylenediamine)；
B. it is poured into plastic plate after mixing, then being slowly added to absolute ethyl alcohol on separation gel upper layer keeps glue surface smooth, prevents glue
Liquid oxidation by air.Room temperature about 30min is set, absolute ethyl alcohol above is discarded；
C. gluing (5%) is prepared：The deionized water of 1.15mL, 0.5M Tris-HCl (pH 6.8), the 0.35mL of 0.5mL
30% acrylamide, 10% ammonium persulfate solution of 15 μ L, 2.5 μ L TEMED；
D. it is poured into plastic plate after mixing, is stably inserted into the 10 hole sample combs of 0.1cm, sets room temperature about 20min.
Prepare sample：The drug of concentration gradient is incubated half an hour at 37 DEG C in N36, and the C34 being subsequently added into is total at 37 DEG C
With incubation half an hour.The final concentration of N36 and C34 is all 100 μM；
Loading：Take 15 μ L mixtures and 2 × Tris glycine buffers (by 1:1 ratio) be uniformly mixed, later 10 ×
The gel comb hole of 0.1cm is added the sample that 15 μ L are handled well per hole, voltage 120V electrophoresis one and a half hours under room temperature；
Dyeing and decoloration：It is then small with destainer decoloration about 1 with coomassie brilliant blue staining about 1 hour (depending on the circumstances)
When (depend on the circumstances), finally taken pictures with FluorChem8800 gel imagers.
3) influence that circular dichroism detector (CD) detection trifloroside forms gp41 Six helix bundles (alpha-helix) structure
With PBS dissolvings N36, C34, trifloroside (1mg/mL) and positive drug ADS-J1 (600 μ g/mL)；
N36's (final concentration of 10 μM) reacts half an hour with positive drug or trifloroside at 37 DEG C；
C34 (final concentration of 10 μM), 37 DEG C of common incubation half an hour are added；
It is equipped with：Sample cell：1mm, sample size：200 μ L, wave-length coverage：190nm~260nm, length scanning speed：50nm/
Min, wave are wide：5nm, slit：0.1nm, time constant：4s.
Experimental result as shown in figure 5, ELISA the result shows that compared with positive drug ADS-J1, trifloroside energy dose-dependant
It reduces gp41 6-HB structures to property to be formed, IC50It is 21.56 ± 8.73 μ g/mL.N-PAGE as a result, it has been found that, trifloroside energy dosage
Inhibit to dependence the formation of gp41 6-HB bands.With the increase of trifloroside concentration, the band brightness of 6-HB continuously decreases, should
As a result similar to positive control medicine ADS-J1.Circular dichroism spectra the results show that trifloroside (1mg/mL) can significantly inhibit N36 and
C34 forms α-helixstructure.But trifloroside cannot reverse the α-helixstructure formed.Positive drug ADS-J1 (600 μ
G/mL it) acts on similar with trifloroside.
Experiment six：Surface plasma resonance experiment (SPR) detection trifloroside is combined with polypeptide N36
1) point sample：The point sample under the conditions of humidity 37.2%, 25 DEG C of temperature, a concentration of 10mM triflorosides are fixed on 3D photo-crosslinkings
On chip；
2) detection polypeptide N36 interacts with trifloroside：Polypeptide N36 is as mobile phase, a concentration of 500 μM, 250 μM, and 125
μM reaction condition is as follows：25 DEG C of association reaction temperature, buffer solution are 1 × PBS, 2 μ L.s of mobile phase sample introduction speed-1, binding time
300s, Dissociation time 300s, liquid of living again are Glycine-HCl (PH=2.0), and live again 3 μ L.s of flow velocity-1, live again time 300s；
3) data analysis：PlexeraDE softwares analyze experimental data and the mapping of 5.0 softwares of GraphPad Prism.
For experimental result as shown in fig. 6, after polypeptide N36 sample introductions, there is response signal in trifloroside, and is rapidly achieved flat
Weighing apparatus, when buffer solution flows through chip surface instead of sample solution, the N36 being incorporated on chip is gradually dissociated, response signal by
Gradually return to baseline level.With the increase of N36 concentration, response signal is also gradually increasing.Using PlexeraDE softwares to data into
The affinity constant of row analysis fitting, trifloroside and N36 are KD=1.73 × 10-7M.Therefore, trifloroside and N36 have it is relatively strong
Affinity.
Experiment seven：Trifloroside is to HIV target cells and the basic no cytotoxicity of effector cell
1) TZM-bl, U87-CD4-CCR5, U87-CD4-CXCR4, MT-2 and CHO-WT cell are after pancreatin digests, 100 μ
The holes L/ 1 × 105/ mL cells are added in 96 porocyte culture plates, 37 DEG C, 5%CO2Overnight incubation；
2) next day, the trifloroside of concentration gradient was added in cell, another that 50 μ L of blank cultures are added per 50 μ L of hole, with
DMEM blank cultures are as blank control, 37 DEG C, 5%CO2Cultivate 48h.
3) supernatant is discarded, the MTT solution of 0.5mg/mL is added, and (mother liquor, which with PBS is dissolved into 5mg/mL and is placed in 4 DEG C of refrigerators, to be protected
Deposit), 100 holes μ L/, 37 DEG C, 5%CO2It is protected from light culture 4h.
4) supernatant is discarded, DMSO, 150 holes μ L/ are added；5~10min is vibrated to blue crystallization dissolving.
5) absorbance (measurement wavelength is 570nm) is read with microplate reader, the CC of trifloroside is calculated according to software50Value.
Experimental result is as shown in table 1.
The cytotoxicity (n=3, Mean ± SD) of 1 trifloroside of table
As shown in Table 1：Trifloroside is to HIV target cells and the basic no cytotoxicity of effector cell
Wherein, CC50：Concentration needed for cause half cytotoxicity, i.e. drug make concentration when half cell-lethal.Trifloroside is sent out
Wave the concentration i.e. IC of antivirus action50, the far smaller than concentration of trifloroside (even if cell-lethal) toxic to cell, i.e. CC50
>>IC50.I.e. drug play antivirus action when concentration versus cell be avirulent.
Claims (5)
1. the application of trifloroside and its pharmaceutically acceptable derivates in preparing anti HIV-1 virus infection medicine.
2. application according to claim 1, it is characterised in that：It is medicinal that trifloroside pharmaceutically acceptable derivates are selected from its
Salt, medicinal ester, medicinal ether.
3. application according to claim 2, it is characterised in that：The pharmaceutically acceptable pharmaceutical salts of trifloroside be selected from potassium, sodium,
Calcium, zinc salt.
4. application according to claim 2, it is characterised in that：The pharmaceutically acceptable medicinal ester of trifloroside is C1~C6's
Medicinal ester.
5. application according to claim 2, it is characterised in that：The pharmaceutically acceptable medicinal ether of trifloroside is C1~C6's
Medicinal ether.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810431029.3A CN108379278A (en) | 2018-05-08 | 2018-05-08 | Application of the trifloroside in preparing anti HIV-1 virus infection medicine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810431029.3A CN108379278A (en) | 2018-05-08 | 2018-05-08 | Application of the trifloroside in preparing anti HIV-1 virus infection medicine |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108379278A true CN108379278A (en) | 2018-08-10 |
Family
ID=63071293
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810431029.3A Pending CN108379278A (en) | 2018-05-08 | 2018-05-08 | Application of the trifloroside in preparing anti HIV-1 virus infection medicine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108379278A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101874824A (en) * | 2009-05-01 | 2010-11-03 | 常州高新技术产业开发区三维工业技术研究所有限公司 | Active extract containing trilobatin and application thereof |
| CN104711307A (en) * | 2015-04-09 | 2015-06-17 | 佛山市金骏康健康科技有限公司 | Higher value application method for shaddock agricultural waste resource |
| CN106822239A (en) * | 2016-12-17 | 2017-06-13 | 郑州郑先医药科技有限公司 | It is a kind of to treat Chinese and western medicinal composition of the infection of the upper respiratory tract and preparation method thereof |
-
2018
- 2018-05-08 CN CN201810431029.3A patent/CN108379278A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101874824A (en) * | 2009-05-01 | 2010-11-03 | 常州高新技术产业开发区三维工业技术研究所有限公司 | Active extract containing trilobatin and application thereof |
| CN104711307A (en) * | 2015-04-09 | 2015-06-17 | 佛山市金骏康健康科技有限公司 | Higher value application method for shaddock agricultural waste resource |
| CN106822239A (en) * | 2016-12-17 | 2017-06-13 | 郑州郑先医药科技有限公司 | It is a kind of to treat Chinese and western medicinal composition of the infection of the upper respiratory tract and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 曾宪彪等: "木姜叶柯总黄酮大鼠口服吸收研究", 《西北药学杂志》 * |
| 李胜华等: "多穗柯中黄酮类成分研究", 《中草药》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| De Clercq et al. | Potent and selective inhibition of human immunodeficiency virus (HIV)-1 and HIV-2 replication by a class of bicyclams interacting with a viral uncoating event. | |
| Liu et al. | Different from the HIV fusion inhibitor C34, the anti-HIV drug Fuzeon (T-20) inhibits HIV-1 entry by targeting multiple sites in gp41 and gp120 | |
| Lee-Huang et al. | Discovery of small-molecule HIV-1 fusion and integrase inhibitors oleuropein and hydroxytyrosol: Part I. Integrase inhibition | |
| Jiang et al. | Development of HIV entry inhibitors targeted to the coiled-coil regions of gp41 | |
| Pan et al. | HIV-1 gp41 fusion intermediate: a target for HIV therapeutics | |
| Ikuta et al. | Expression of human immunodeficiency virus type 1 (HIV-1) gag antigens on the surface of a cell line persistently infected with HIV-1 that highly expresses HIV-1 antigens | |
| KR100950520B1 (en) | Synthetic or natural peptides binding protein phosphatase 2a, identification method and uses | |
| An et al. | Amino acid derivatives of the (−) enantiomer of gossypol are effective fusion inhibitors of human immunodeficiency virus type 1 | |
| Lin et al. | Characterization of siamycin I, a human immunodeficiency virus fusion inhibitor | |
| AU2007314456A1 (en) | Proteins for use in diagnosing and treating infection and disease | |
| Li et al. | Maleic anhydride-modified chicken ovalbumin as an effective and inexpensive anti-HIV microbicide candidate for prevention of HIV sexual transmission | |
| US6268484B1 (en) | HIV-vaccines | |
| Montefiori et al. | Complement-mediated, infection-enhancing antibodies in plasma from vaccinated macaques before and after inoculation with live simian immunodeficiency virus | |
| JPH11139977A (en) | Nef action suppressant | |
| CN108379278A (en) | Application of the trifloroside in preparing anti HIV-1 virus infection medicine | |
| EP1481006A2 (en) | Universal antimicrobial treatment | |
| US10336817B2 (en) | Therapeutic composition of camel milk | |
| Fiorentini et al. | Synthetic peptide AT20 coupled to KLH elicits antibodies against a conserved conformational epitope from a major functional area of the HIV-1 matrix protein p17 | |
| CN102160870B (en) | Application of poly(4-styrenesulfonic acid-co-maleic acid) sodium and medicinal composition thereof | |
| KR101138930B1 (en) | Composition for preventing and treating ???? comprising ??? ?? protein | |
| Swanson | Molecular engineering of a banana lectin that inhibits hiv-1 replication | |
| US6924353B2 (en) | Inhibitors for RNA viruses | |
| Kim et al. | Enhancing HIV-1 neutralization by increasing the local concentration of MPER-directed bnAbs | |
| Arendrup et al. | HIV neutralizing antibodies: development and association with HIV related disease | |
| Pušnik | Helper T cell and B cell responses during the natural course of HIV infection |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180810 |
|
| RJ01 | Rejection of invention patent application after publication |