CN100435805C - Use of polysaccharose sulfate for preparing medicine against grippal virus - Google Patents
Use of polysaccharose sulfate for preparing medicine against grippal virus Download PDFInfo
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Abstract
The present invention discloses an application of a polysaccharide sulfate used as anti-influenza virus medicament, belongs to the field of synthetic pharmacology, and relates to an application of the polysaccharose sulfate used for preparing an anti-influenza virus medicament, particularly to an application of a ocean red algae polysaccharide sulfate and an alpha-D-(1 to 6)-dextran sulfate used for preparing the anti-influenza virus medicament. The present invention discloses the application of the polysaccharide sulfate in the anti-influenza virus medicament, and prepares the practical dosage forms of the alpha-D-(1 to 6)-dextran sulfate (the range of average molecular weight is from 1000 to 100000, and the substitution value of monosaccharide sulfate radical is from 0.2 to 3.0), the ocean red algae polysaccharide sulfate, comprising carrageenin polysaccharide sulfate (the range of the average molecular weight is from 1000 to 100000, and the substitution value of the monosaccharide sulfate radical is from 1.0 to 3.0), and porphyra polysaccharide sulfate (the range of the average molecular weight is from 1000 to 100000, and the substitution value of the monosaccharide sulfate radical is from 0.5 to 3.0). Thus, the purpose of the polysaccharide sulfates is determined to corporately resist A type and B type influenza viruses after the polysaccharide sulfates singly form the compounds or are combined with the existing anti-influenza medicaments comprising amantadine, ribavirin and the like to form compounds.
Description
Technical field
The invention belongs to the synthetic pharmacology field, relate to the application of polysaccharide sulfate, relate in particular to of the application of marine red alga polysaccharide sulfate and α-D-(1 → 6)-dextran sulfate as the preparation anti-influenza virus medicament as the preparation anti-influenza virus medicament.
Technical background
Influenza is a kind of acute viral respiratory infectious disease of the serious harm human health that causes of influenza virus, is called as one of the highest viral infectious of fatality rate.Influenza once had popularly on a large scale several times in history, had brought very grave disaster to the mankind.Although existing multiple medicine comes out, the hazardness of influenza and the loss that causes thereof still can not be ignored.Influenza.China is the multiple ground of influenza, and local eruption and prevalence is all arranged basically every year.For this reason, China's Ministry of Public Health has been classified influenza as one of disease of Tenth Five-Year Plan Period key monitoring.
Influenza virus is the microgranule that is surrounded by two kinds of surface glycoprotein-hemagglutinins and neuraminidase, belong to influenza virus family, antigenic variation is its most significant biological property, can be divided into first (A), second (B), third (C), three types according to influenza virus nucleoprotein (NP) and stromatin (M) antigenic type difference.Wherein A type influenza virus press hemagglutinin (Hemagglutinin, HA) and neuraminidase (Neuraminidase, antigenic specificity NA) are divided into some hypotype (H
xN
y).H
1N
1And H
3N
2Hypotype and Type B influenza virus are to cause that people's parainfluenza broke out and popular main pathogen in nearly ten years.
Influenza vaccinations are unique measures of present flu-prevention.Common influenza vaccines have inactivated vaccine, attenuated live vaccine, recombiant vaccine (or claiming carrier bacterin), subunit vaccine, synthetic oligopeptide vaccine and nucleic acid vaccine.The influenza vaccines of present clinical use are to lose infective inactivated vaccine.Because influenza antigen easily makes a variation, and influenza is taken place effectively to predict with popular periodicity the measure of therefore inoculating influenza vaccines still has limitation owing to still lack reliable method at present.
The drug research of treatment influenza has been obtained bigger progress.At present, the anti-influenza virus medicament of having used and having studied has diamantane (obsolete) amine medicine, neuraminidase inhibitor, influenza virus receptor blocking agent etc.Amantadine and rimantadine are clinical treatment medicines commonly used, and be only to A type virus effectively, invalid to the Type B influenza virus, and easily produce drug resistance and have neurotoxicity.Be used for clinical Zanamivir (GG167 or Relenza) by drugs approved by FDA in 1999 and Oseltamivir (GS4104 or Tamiflu) is neuraminidase (NA) mortifier, be mainly used in the early treatment, all effective to first, influenza B.Yet these two kinds of medicines are relatively poor to the influenza curative effect with complication, so these two kinds of medicines extensively are not applied as yet.In view of the hazardness of influenza virus, be subjected to special attention at the research of medicine of prevention and treatment influenza virus.
Polysaccharide sulfate is meant the polysaccharide that has sulfate group on the sugared hydroxyl, is also referred to as sulfated polysaccharide or controlling sulfate polyose.In recent years, the antivirus action of polysaccharide has caused the great attention of the world of medicine, and especially the strong antiviral activity of polysaccharide sulfate has shown wide prospect in medicine.
According to relevant bibliographical information, dominant mechanism with anti-tunicle virus of polysaccharose substance (Envelopedvirus) of reinforcing YIN-essence ionic group is to suppress absorption and the intrusion of tunicle virus to host cell, be that polysaccharide sulfate leans on electrostatic interaction to combine with the tunicle outer membrane protein, occupied tunicle virus and the bonded position of recipient cell, thereby made the tunicle virus can not infection cell.It is a kind of receptor identifying relevant with electrostatic interaction that virus is adsorbed onto the cellular plasm film, needs the molecule affinity [Fenner, 1974] between cell surface and virus.Polysaccharide sulfate can combine with the cation zone of the gp120 of HIV (human immunodeficiency virus) (Human Immunodeficiency Virus) HIV-1 shell glycoprotein, occupies tunicle virus and host cell receptor position, thereby stops combine [all beautiful 1997] of virus and host cell.To studies show that of inhibitory action pattern, virus to the absorption of host cell by the blocking-up of sulphuric acid polyanion [Liebhaber1963, Schulze 1964, Takemoto 1964, Baba 1988, Neyts 1992, Mitsuya 1988, Schols1990].When having polycation such as DEAE-glucosan and protamine, inhibitory action can be prevented from or reverse.The a certain step that polysaccharide sulfate also suppresses to duplicate after virus enters cell or enters cell [week beautiful 1997].Influenza virus and HIV etc. belong to tunicle virus.
Glucosan has α-D-(1 → 4), β-D-(1 → 4), α-D-(1 → 3), β-D-(1 → 3), α-D-multiple glycosidic bond connection types such as (1 → 6).Various dextran sulfates make it have different functions owing to the difference at molecular weight, molecular side chain, glycosidic bond type, thioester bond type, monosaccharide sulfuric ester number aspects such as (substitution values).As can be used as anticoagulant (US2715091, molecular weight are that 22000-200000, substitution value are up to 3.0); Anti-rotavirus (WO96/30027); AIDS virus resisting (US5861383 and EP0406512, molecular weight is 500000, is generally 75000; The EP0293826 molecular weight is 5000,8000,90000,200000; CN1206717A, β-D-(1 → 4)-dextran sulfate); Anti-herpes simplex virus (WO94/15624, molecular weight are 50000-500000, and glycosidic bond is α-D-(1 → 4) or β-D-(1 → 4)); Antitumor (CN1283632A, α-D-(1 → 3)-dextran sulfate) etc.The anti-influenza virus activity of α-D-(1 → 6)-glucosan (Dextran, dextran) sulfuric ester and other dextran sulfates does not appear in the newspapers.
The ear Eucheuma muricatum (Gmel.) Web.Van Bos. (Eucheume cottoni) that dashes forward is an important class red algae, and its main component is carrageenan (Carrageenan has the poly-D-galactose-3 of part of sulfuric acid ester group, 6-inner ether-galactose sulfuric ester).People such as Gerber in 1958 propose Sargassum first and contain the natural anti-virus active substance.Can protect fertilized ovum to avoid infecting of mumps virus and A type influenza virus from the natural polysaccharide that GelidiumCartilagenium extracts without chemical modification.The end of the eighties, scientist discovered that several polyanionic materials (natural or semisynthetic red seaweed polysaccharide sulfuric ester) are effective inhibitor that HIV (human immunodeficiency virus) (Human Immunodeficiency Virus) (HIV) is duplicated, caused the interest of people, from multiple red algae, also found herpes simplex virus HSV1 and the inhibited polysaccharide of HSV2 to the research of polysaccharide sulfate antivirus action.To finding that the Schizymenia extract has the antiretroviral activity, and water extract and fraction thereof are further discovered in 39 kinds of anti-murine leukemia virus screenings of California red algae, carrageenan is the antiviral activity composition really.In addition, mention the effect (WO94/15624) of carrageenan or its sulfuric ester anti-herpes simplex virus, the effect (WO88/06396 of anti-HIV in some patents, EPA293826), anti-HIV and other DNA and RNA viruses (GB2262531), anti-rotavirus (WO96/30027) etc., and with (EP0497341A2, molecular weight is greater than 1,000 ten thousand) such as the collaborative anti-herpes simplex virus of fibroblast growth factor, cytomegalovirus, influenza virus, respiratory syncytial virus, Sen Liji forest virus, HIV (human immunodeficiency virus) (Human Immunodeficiency Virus) and Moloney sarcoma viruses.The prominent anti-Type B influenza virus of Eucheuma muricatum (Gmel.) Web.Van Bos. polysaccharide sulfate of natural ear is not appeared in the newspapers; Prominent Eucheuma muricatum (Gmel.) Web.Van Bos. polysaccharide sulfate anti-A type of the ear that obtains specified molecular weight, specific monosaccharide sulfate radical substitution value through chemical process and Type B influenza virus are not appeared in the newspapers yet.
Porphyra haitanensis (Porphyra haitanensis) is to originate in the southern a kind of important economic red algae of China.Porphyra Polysaccharide has tangible anticoagulation, blood fat reducing, antithrombotic, cardiotonic, also have and suppress tumor, blood sugar lowering, antiinflammatory and prevent and treat effect such as ulcer, it is reported that Porphyra Polysaccharide also has the function of obvious enhancing cellular immunization and humoral immunization, but the research that relevant Porphyra Polysaccharide sulfuric ester is used for resisiting influenza virus does not appear in the newspapers.
In document of having reported and patent, about natural marine red seaweed polysaccharide (comprising carrageenan and Porphyra haitanensis polysaccharide) through chemical modification and handle that the mean molecule quantity make is 1000 to 100000, monosaccharide sulfate radical substitution value is 1.0 to 3.0 red seaweed polysaccharide sulfuric ester (Carrageenan polysaccharide sulfuric ester, Porphyra Polysaccharide sulfuric ester) and α-D-(1 → 6)-dextran sulfate to the independent inhibition or the coordinate repression of influenza A type and Type B virus, be not reported.
Summary of the invention
The objective of the invention is to the application of open polysaccharide sulfate in resisiting influenza virus, and (average molecular weight range is 1000-100000 to prepare α-D-(1 → 6)-dextran sulfate, monosaccharide sulfate radical substitution value 0.2-3.0) and marine red alga polysaccharide sulfate such as Carrageenan polysaccharide sulfuric ester (average molecular weight range is 1000-100000, monosaccharide sulfate radical substitution value 1.0-3.0) and the Porphyra Polysaccharide sulfuric ester (average molecular weight range is 1000-100000, monosaccharide sulfate radical substitution value 0.5-3.0) is practical pharmaceutical dosage form, determined that these polysaccharide sulfates have the purposes of anti-A type and Type B influenza virus alone or synergistically.
To achieve these goals, the technical solution used in the present invention is as follows:
(1) take by weighing the prominent Eucheuma muricatum (Gmel.) Web.Van Bos. of a certain amount of ear, pulverize the back and carry 3h in 95 ℃ of water, crude extract is collected supernatant after centrifugal, add an amount of KCl, the centrifugalize precipitate.Precipitate carries out post precipitation once more with ethanol after with water dissolution and filters, and filtrate is worn into fine powder in 50 ℃ of vacuum dryings, the carrageenan powder.
(2) take by weighing a certain amount of dry Thallus Porphyrae, soak and spend the night, it is ℃ freezing to smash postposition-20 to pieces, takes out the back microwave thawing, so repeat 3 times after, extract 30min in 60 ℃ of stirred in water bath, centrifugal, supernatant is through concentrating under reduced pressure, dry crude polysaccharides.Crude polysaccharides removes albumen through 732 cation exchange resin processes, obtains refining Porphyra Polysaccharide.
(3) (molecular weight is generally greater than 500,000 to take by weighing (1) prepared carrageenan or commercially available carrageenan, monosaccharide sulfate radical substitution value is 0.3-0.8) or (2) prepared Porphyra Polysaccharide, be made into the solution of 2-15%, sulphuric acid (or hydrochloric acid) concentration of 0.005%～5%w scope, 40～120 ℃ times effects 10～180 minutes.Reaction neutralizes with NaOH solution after finishing immediately, holds back filtration with ultrafilter membrane then, the Carrageenan polysaccharide or the Porphyra Polysaccharide of preparation specified molecular weight.
(4) (3) are resulting than the red seaweed polysaccharide of small-molecular weight or commercially available glucosan (Dextran, α-D-(1 → 6)-glucosan) handles through conventional pyridine-chlorosulfonic acid method, obtain the polysaccharide sulfate of specific monosaccharide sulfate radical substitution value, after adopting the ultrafiltration means to remove small-molecule substance, analyse precipitation with 2 times of amount dehydrated alcohol alcohol, precipitate is measured alcohol chromatographies with 2 times once more behind dissolved in distilled water, the gained precipitate obtains required polysaccharide sulfate through 60 ℃ of vacuum dryings, the impurity content of this polysaccharide sulfate powder is lower than 2%, and major impurity is NaCl and Na
2SO
4
(5) the polysaccharide sulfate powder is dressed up capsule to be prepared into peroral dosage form after drying in removing wet environment.Also the polysaccharide sulfate powder can be dissolved in normal saline according to desired concn, after 0.22 μ m membrane filtration degerming, promptly be prepared into pharmaceutical dosage form isosmoticity, that can be used for nasal mucosa (lung mucosa) absorption.
(6) adopt Embryo Gallus domesticus model and mouse experiment model, the polysaccharide sulfate sample prepared to (5) carries out the test of independent resisiting influenza virus, and after forming compound recipe with existing Tamiflu such as amantadine, virazole, rimantadine, Zanamivir (GG167 or Relenza) and Oseltamivir (GS4104 or Tamiflu), work in coordination with the test of resisiting influenza virus.Test shows, prepared polysaccharide sulfate has tangible anti-A type and Type B influenza virus activity, and with existing medicines such as amantadine, virazole by 1: 0.01-1: 100 content proportionings have synergism after forming compound recipe, can further strengthen the resisiting influenza virus effect, enumerate polysaccharide sulfate and amantadine, virazole form compound recipe by 5: 2 content proportioning resisiting influenza virus experiment effect among the embodiment.
Description of drawings
Fig. 1 is poly-D-galactose-3 for the structural formula that extracts the carrageenan that obtains from the prominent Eucheuma muricatum (Gmel.) Web.Van Bos. of ear involved in the present invention, and 6-inner ether-D-galactose has the part of sulfuric acid group.
Fig. 2 is the molecular structural formula of α-D-(1 → 6)-dextran sulfate involved in the present invention, and wherein substituent R can be H or SO
3H.When substituent R is α-D-(1 → 6)-glucosan during all for H.
Fig. 3 is the infrared spectrogram of α-D-(1 → 6)-glucosan.
Fig. 4 is the infrared spectrogram of α-D-(1 → 6)-dextran sulfate, wherein 1244cm
-1About be absorbed as S=O stretching vibration, 800cm
-1About be absorbed as the stretching vibration of C-O-S, illustrate that glucosan is by Sulfation.
The specific embodiment
The present invention is elaborated with reference to the following examples, but is not limited to this.
Embodiment 1: the preparation of the prominent Eucheuma muricatum (Gmel.) Web.Van Bos. polysaccharide (carrageenan) of ear
Take by weighing the prominent Eucheuma muricatum (Gmel.) Web.Van Bos. (available from Xiamen Aquatic product institute) of a certain amount of exsiccant ear, shred cleanly, add the hot water of 50 times of weight, 95 ℃ of agitating heating 3 hours.With centrifugal 10 minutes of liquid of extracting polysaccharide 5000rpm, collect supernatant.Add KCl, making final concentration is 5%, stirs evenly, and standing over night under 500rpm centrifugal 10 minutes, is separated insoluble matter.With the insoluble matter dissolved in distilled water, the ratio with 3: 1 (v/v) adds ethanol again, staticly settles 60 minutes, and 60 ℃ of vacuum dryings are worn into fine powder, must make with extra care the carrageenan powder.Prepared carrageenan molecule amount is higher than 500,000, and every monosaccharide sulfate radical substitution value is 0.45.
Embodiment 2: the preparation of Porphyra Polysaccharide
Take by weighing a certain amount of exsiccant porphyra haitanensis (place of production is the Pingtan, Fujian), shred cleanly, the distilled water immersion that adds 50 times spends the night, adopt bruisher that Thallus Porphyrae is smashed to pieces, put-20 ℃ freezing, take out the back microwave thawing, after so repeating 3 times, extract 30min, the centrifugal 20min of 3000rpm in 60 ℃ of stirred in water bath, supernatant is put in addition, the Thallus Porphyrae slag repeats to extract 2 times after adding distilled water, merges 3 times supernatant, 50 ℃ of concentrating under reduced pressure, lyophilizing gets crude polysaccharides.
The Thallus Porphyrae crude polysaccharides is made into 2% solution, adds sulphuric acid and regulate pH to 2.0.Polysaccharide solution regenerated 732 cation exchange resin columns of flowing through can effectively be removed protein.Collected post liquid, be concentrated into behind the 3%w/v with ethanol sedimentation polysaccharide.Precipitate obtains the Porphyra Polysaccharide powder through vacuum drying after the pulverizing.
Embodiment 3: the preparation of specified molecular weight Carrageenan polysaccharide
The carrageenan (or commodity carrageenan) of embodiment 1 preparation is mixed with 5% glue, puts in the thermostat water bath, fully mixing.Add sulphuric acid and regulate pH to 2.5,90 ℃ of isothermal reactions 120 minutes.After reaction finished, the NaOH solution with 1N neutralized immediately, adopts the ultrafiltration membrance filter of PSPP then, the Carrageenan polysaccharide of preparation different molecular weight.The spray-dried processing of the Carrageenan polysaccharide of prepared specified molecular weight is used for the preparation of Carrageenan polysaccharide sulfuric ester.
Embodiment 4: the preparation of α-D-(1 → 6)-dextran sulfate
The three-neck flask of configuration condensing tube and agitating device is put in saline-ice bath, added pyridine 48ml, stir, make it abundant cooling, add chlorosulfonic acid 13ml with Dropping funnel.Rapidly three-neck flask is moved in 100 ℃ of water-baths, the adding mean molecule quantity is α-D-(1 → 6)-glucosan 3.0g of 20000, and 100 ℃ of constant temperature stir 1h.With being chilled in the reactant liquor impouring 200ml frozen water of room temperature, be neutralized to pH10.5 with 40%NaOH, standing demix is isolated lower floor's water with separatory funnel.Aqueous portion adds 95% ethanol of 4 times of volumes, and dextran sulfate is separated out precipitation, centrifugal 10 minutes of 3000rpm, collecting precipitation thing.Precipitate is dissolved in deionized water, and 48h dialyses in deionized water.Material after dialysis is concentrated into 5%w/v in vacuum rotary evaporator, add dehydrated alcohol 100ml, staticly settles the centrifugalize precipitate.With deionized water dissolution precipitation thing, add dehydrated alcohol centrifugalize precipitate again.Repeat alcohol and analyse separation process three times.With final primary sedimentation thing vacuum drying, make α-D-(1 → 6)-dextran sulfate.After measured, mean molecule quantity is 45000, and average monosaccharide sulfate radical substitution value is 2.13.
Embodiment 5: the preparation of small-molecular weight Porphyra Polysaccharide sulfuric ester
With after prepared Porphyra Polysaccharide is handled among the embodiment 2, obtain that mean molecule quantity is about 60000, monosaccharide sulfate radical substitution value is 0.50 small-molecular weight Porphyra Polysaccharide ZSO according to embodiment 3 described methods.Under 10 ℃ of temperature conditions, the 25ml chlorosulfonic acid is slowly added in the 120ml anhydrous pyridine.The rising reaction temperature is fully adding with the dispersive small-molecular weight Porphyra Polysaccharide of pyridine ZSO sample 15 grams under the stirring condition rapidly.Under 90 ℃ of conditions, react 4h.Reacting liquid temperature is reduced to 10 ℃, add saturated NaOH cold soln, pH is transferred to 10.5.Behind the standing demix 10min, separate upper organic phase and lower floor's water.Lower floor's water to 5%w/v, adds isopropyl alcohol through vacuum concentration, and the isopropyl alcohol final concentration is 60%w/v.Precipitate is used washed with isopropyl alcohol three times then through centrifugalize.Precipitate through washed with isopropyl alcohol is dissolved in the deionized water again, is 5000 ultrafiltration membrance filter with molecular cut off, removes small-molecular weight impurity.When solid content reaches about 5%w/v in the spissated material of ultrafilter membrane, add the isopropyl alcohol of 2 times of volumes.The precipitate that obtains after filtration dry under vacuum condition (90 ℃, 0.005MPa).The mean molecule quantity of prepared sample ZSS is about 90000, and average monosaccharide sulfate radical substitution value is 1.57.
Embodiment 6: the preparation of small-molecular weight Carrageenan polysaccharide sulfuric ester
Press the method degraded macromolecule carrageenan of embodiment 3, the method that adopts ultrafilter membrane to hold back prepare mean molecule quantity be 35000 than the small-molecular weight Carrageenan polysaccharide.
In the 500ml three-neck flask, add pyridine 200ml, at 10 ℃ with fully under the stirring condition, slowly add chlorosulfonic acid 20ml.After adding chlorosulfonic acid reactant liquor is heated to 60 ℃, adds Carrageenan polysaccharide 15 grams.Be warming up to 80 ℃ of constant temperature then and stir 2h.After reaction finishes reactant liquor is cooled to 10 ℃, is neutralized to pH9.0 with the 40%NaOH cold soln.The reactant liquor standing demix is isolated lower floor's water with separatory funnel.Aqueous phase liquid to 5%w/v, adds dehydrated alcohol through vacuum concentration, and the ethanol final concentration is 75%.Precipitate is dissolved in deionized water after centrifugalize, behind the dialyzer of packing into, 48h dialyses in deionized water.After the material of dialysis treatment places vacuum Rotary drying device to concentrate again, is concentrated into 5%w/v concentration, add dehydrated alcohol, the ethanol final concentration is 75%.The precipitate drying grinds, and makes the Carrageenan polysaccharide sulfuric ester.After measured, average monosaccharide sulfate radical substitution value is 1.91.
Embodiment 7 Carrageenan polysaccharide sulfuric ester resisiting influenza virus H
1N
1Drug efficacy study
A. press the method for embodiment 1, from the prominent Eucheuma muricatum (Gmel.) Web.Van Bos. of ear, extract carrageenan.Press the method for embodiment 3, the preparation mean molecule quantity is 35000 Carrageenan polysaccharide, and average monosaccharide sulfate radical substitution value is 0.45.
B. the Carrageenan polysaccharide with step a preparation carries out Sulfation.Make Carrageenan polysaccharide sulfuric ester K
1(mean molecule quantity 60000, average monosaccharide sulfate radical substitution value is 1.91).Prepare the K of 5000 μ g/ml
1Pharmaceutical preparation, cofabrication 2000 μ g/ml amantadine pharmaceutical preparatioies.By the dilution of 5 multiple proportions example, filtration sterilization prepares a series of variable concentrations samples.
C. get the H that China national influenza center provides
1N
1The influenza virus strain infects chick embryo allantoic cavity, and 37 ℃ of constant temperature are hatched.After 3 days, get chick embryo allantoic cavity liquid, carry out virus concentration and measure.The preparation virus concentration is the H of 100TCID
1N
1The influenza virus sample.
D. get 9 age in days chicken embryo, medicine sample and the viral sample with above-mentioned steps a, b preparation successively injects the embryo egg, every embryo egg 0.2ml rapidly.5 embryo eggs are used in every kind of drug level test.Set virus-positive matched group and normal saline negative control group simultaneously.37 ℃ of constant temperature are hatched, and observe the Embryo Gallus domesticus survival condition for three days on end.
E. after the embryo egg of d processing is hatched 3 days set by step, place 4 ℃ of refrigerator overnight.Collect embryo egg allantoic cavity liquid next day, carry out the blood clotting experiment, measure H
1N
1Virus titer.
Experiment shows (see Table 1, table 2), K
1Sample has tangible anti-H
1N
1The effect of influenza virus.When laboratory sample concentration reached 4.26 μ g/ embryo eggs, viral suppression ratio can reach more than 50%.K
1Anti-H
1N
1The drug effect of influenza virus is better than amantadine, and toxicity is extremely low, its therapeutic index TI
50Apparently higher than amantadine.
Table 1 Carrageenan polysaccharide sulfuric ester K
1Resisiting influenza virus H
1N
1Experimental result
Table 2 Carrageenan polysaccharide sulfuric ester K
1With amantadine antiviral H
1N
1Drug effect relatively
F. Carrageenan polysaccharide sulfuric ester K
1Anti-H with amantadine
1N
1The cooperative effect of influenza virus
The formulation content proportioning is 5: 2 Carrageenan polysaccharide sulfuric ester K
1With amantadine mixed solution, wherein K
1Content is 5000 μ g/ml, and amantadine content is 2000 μ g/ml.D implements set by step, measures the anti-H of hybrid medicine
1N
1The cooperative effect of influenza virus, experimental result is shown in table 2.Table 2 shows that the mixing of two kinds of medicines is used, and helps to improve anti-H
1N
1The effect of virus, therapeutic index increases; Toxicity during two kinds of medicament mixed and amantadine are suitable when using separately, do not observe two kinds of synergetic phenomenons of drug toxicity.
Embodiment 8 Carrageenan polysaccharide sulfuric ester K
2Anti-Type B influenza virus drug efficacy study
A. prepare Carrageenan polysaccharide sulfuric ester K by embodiment 7 methods
2(mean molecule quantity about 40000, per unit monosaccharide sulfate radical substitution value 1.75).Prepare 5000 μ g/mlK
2Pharmaceutical preparation by the dilution of 2 multiple proportions example, is made a series of medicine samples.
B. prepare B influenza virus (being provided by national influenza center) sample by embodiment 7 methods, virus concentration is 100TCID.
C. pressing embodiment 7 steps d implements.
D. the embryo degree of c processing is hatched set by step, places 4 ℃ of refrigerator overnight.Collect embryo egg allantoic cavity liquid next day, carry out the blood clotting experiment, measure the Type B virus titer.Table 3 shows, K
2The effect of independent anti-Type B influenza virus is comparatively obvious.When drug dose was 100 μ g/ embryo eggs, viral suppression ratio reached more than 90%; During drug dose 12.5 μ g/ embryo eggs, viral suppression ratio reaches 75%.
Table 3K
2Anti-Type B hirst's hemagglutination experimental result
Embodiment 9 Carrageenan polysaccharide sulfuric ester K
2Anti-H
3N
2The influenza virus pharmacodynamic experiment
A. press the prepared Carrageenan polysaccharide sulfuric ester K of embodiment 8
2Sample.Prepare the sample of 5000 μ g/ml, prepare 2000 μ g/ml amantadine samples.Dilute one by one by 2 multiple proportions examples.Amantadine is a control drug.
B. prepare H by embodiment 7 methods
3N
2Influenza virus sample, virus concentration are 100TCID.
C, d all implement by embodiment 8.Experimental data is shown in table 4.
Table 4 shows, Carrageenan polysaccharide sulfuric ester sample K
2Have and suppress H more significantly
3N
2The effect of influenza viruse attack host cell.
Table 4 Carrageenan polysaccharide sulfuric ester K
2Antiviral H
3N
2Experimental result
Embodiment 10 Carrageenan polysaccharide sulfuric ester K
3Resisiting influenza virus H
1N
1The drug efficacy study of Mus lung adapted strain
A. adopt commercially available carrageenan powder, the method for pressing embodiment 3, the preparation mean molecule quantity is 4000 Carrageenan polysaccharide, average monosaccharide sulfate radical substitution value is 0.45.
B. press the method for embodiment 6, the Carrageenan polysaccharide that step a is prepared carries out Sulfation, makes Carrageenan polysaccharide sulfuric ester K
3(mean molecule quantity is 7000, and average monosaccharide sulfate radical substitution value is 2.14).
C. prepare the K of 5000 μ g/ml
3The pharmaceutical preparation sample.Prepare 2000 μ g/ml virazole pharmaceutical preparation samples.Dilute one by one by 4 multiple proportions examples, make a series of samples.Virazole is a control drug.
D. influenza virus H
1N
1Mus lung adapted strain (being provided by national influenza center) is after the embryo egg infects enrichment, and making virus concentration is that 100TCID virus is for sample.
E. get 18-20 gram mice, first infective virus adopts the administration of collunarium mode behind the 2h.Be administered twice every day, successive administration 7 days.Put to death mice after 7 days, claim its body weight, dissect then, get its lung, claim lung heavy.Other establishes blank negative control group, virus-positive matched group and virazole matched group.
The mouse lung varying index is the heavy ratio with body weight of mouse lung, and expression mice pneumonopathy behind influenza virus infection becomes the weightening finish index.Negative control group is 0.686, and positive controls is 0.850.Carrageenan sulfated ester K3 can obtain obvious antiviral effect, its lung varying index close with negative control group (seeing Table 5) by the administration of collunarium mode.Experiment shows that Nasal Mucosa Absorption is a kind of good administering mode.
The carrageenan sulfated ester K of table 5
3The drug effect of resisiting influenza virus Mus lung adapted strain relatively
Embodiment 11 Carrageenan polysaccharide sulfuric ester K
1The Embryo Gallus domesticus toxicity test
A. prepare the Carrageenan polysaccharide sulfuric ester K of 30000 μ g/ml
1Pharmaceutical preparation solution.Cofabrication 5000 μ g/ml amantadine pharmaceutical preparation solution.Dilute filtration sterilization one by one by 5 multiple proportions examples.Every embryo egg allantoic cavity injects the 0.5ml sample.5 embryo eggs of per sample (p.s.) experiment.
B. above-mentioned Embryo Gallus domesticus is hatched in 37 ℃ of constant temperature.Observe chicken embryo death or developmental state continuously.Inoculating Embryo Gallus domesticus amount dead in back 24 hours is non-specific death, is not included in the statistical data.
Laboratory observation is to Carrageenan polysaccharide sulfuric ester K
1Drug dose when reaching 3000 μ g/ eggs, lethality appears in Embryo Gallus domesticus.K
1Half lethal dose LD
50Be 9706 μ g/ embryo eggs.And the half lethal dose LD of control drug amantadine
50Be 1866 μ g/ embryo eggs.During less than 2000 μ g/ embryo eggs, do not observe K in dosage
1Chick embryo development is had harmful effect, and after experiment embryo egg developed into chicken, also not observing the chicken growth had abnormal phenomena.Experiment shows, K
1Extremely low to chick embryo development toxicity.
C. prepare 10000 μ g/ml K
1With 5000 μ g/ml amantadine composite samples, by 5 multiple proportions example stepwise dilution, filtration sterilization.Carry out the collaborative toxicity test of 9 age in days embryo egg medicines.Find that the lethal lowest concentration of drug of Embryo Gallus domesticus is: K
12000 μ g/ embryo eggs and amantadine 1000 μ g/ embryo eggs; The half lethal concentration of hybrid medicine is LD
50For: K
17386.86 μ g/ embryo egg and amantadine 1866 μ g/ embryo eggs.The toxicity of hybrid medicine is not observed two kinds of drug toxicity superposition phenomenon mainly from amantadine.
Embodiment 12 Carrageenan polysaccharide sulfuric ester K
3Mouse toxicity experiment
Prepare the K of 60000 μ g/ml with normal saline
3Solution, filtration sterilization.
Get 18-20 gram mice and adopt the administration of filling stomach mode, investigate K
3Toxicity to mice.Divide every day and irritated stomach, each perfusion 0.5ml at interval in 8 hours in the morning, afternoon.Continuous medicine-filling was not observed mice in 7 days and is caused death, and did not observe mice internal organs abnormal phenomena and other abnormal phenomenas yet.
Get 18-20 gram mice and adopt the administration of collunarium mode.Dosage 120 μ g/dkg divide the morning, administration in 8 hours at interval in afternoon.Successive administration 7 days is not observed mice and is caused death, and does not observe other abnormal phenomenas of mice yet.
The drug efficacy study of embodiment 13 α-D-(1 → 6)-dextran sulfate resisiting influenza virus
A. the method by embodiment 4 prepares α-D-(1 → 6)-dextran sulfate sample L
1Sample L
1Mean molecule quantity about 45000, monosaccharide sulfate radical substitution value 2.13.Prepare 5000 μ g/ml L
1Pharmaceutical preparation.The amantadine of cofabrication 2000 μ g/ml (or virazole) pharmaceutical preparation is control drug.Dilute one by one by 5 multiple proportions examples, filtration sterilization, make a series of variable concentrations for test agent.
B. get the A type influenza virus (H that China national influenza center provides
1N
1And H
3N
2) and Type B influenza virus strain, infecting chick embryo allantoic cavity respectively, 37 ℃ of constant temperature are hatched.After 3 days, get chick embryo allantoic cavity liquid, carry out virus concentration and measure.The preparation virus concentration is A type and the Type B influenza virus sample that is 100TCID.
C. get 9 age in days chicken embryo, medicine sample and the viral sample with above-mentioned steps a, b preparation successively injects the embryo egg, every embryo egg 0.2ml rapidly.5 embryo eggs are used in every kind of drug level test.Set virus-positive matched group and normal saline negative control group simultaneously.37 ℃ of constant temperature are hatched, and observe the Embryo Gallus domesticus survival condition for three days on end.
D. after the embryo egg that step c handles is hatched 3 days, place 4 ℃ of refrigerator overnight.Collect embryo egg allantoic cavity liquid next day, carry out the blood clotting experiment, measure virus concentration.Experimental result (table 6 and table 7) shows α-D-(1 → 6)-dextran sulfate L
1Has tangible anti-influenza virus activity, to H
1N
1The concentration that the half of virus suppresses virus is 15.61 μ g/ embryo eggs, to H
3N
2The concentration that the half of virus suppresses virus is 12.60 μ g/ embryo eggs, and the concentration that the half of Type B influenza virus is suppressed virus is 8.75 μ g/ embryo eggs.
Table 6 α-D-(1 → 6)-dextran sulfate L
1With amantadine resisiting influenza virus H
1N
1The blood clotting experimental result
E. α-D-(1 → 6)-dextran sulfate L
1Drug efficacy study with the collaborative resisiting influenza virus of amantadine (or virazole)
The preparation proportioning is α-D-(1 → 6)-dextran sulfate L of 5: 2
1With the mixed solution of amantadine (or virazole), wherein L
1Content is 5000 μ g/ml, and amantadine (or virazole) content is 2000 μ g/ml.Dilute one by one by 2 multiple proportions examples, make α-D-(1 → the 6)-dextran sulfate L1 of a series of variable concentrations and the hybrid medicine sample of amantadine (or virazole).
B, c, d carry out the Embryo Gallus domesticus experiment set by step.Experimental result shows that α-D-(1 → 6)-dextran sulfate and amantadine (or virazole) have collaborative resisiting influenza virus effect.By synergism, reduce the EC of α-D-(1 → 6)-dextran sulfate and amantadine
50Value directly improves the medicine antiviral effect, and experimental result sees Table 7a, 7b, 7c.α-D-(1 → 6)-dextran sulfate L
1When using separately to the toxicity of Embryo Gallus domesticus less than amantadine, and L
1Behind amantadine composition compound recipe, do not observe two drug toxicity superposition phenomenon yet.
Table 7a α-D-(1 → 6)-dextran sulfate L
1With amantadine resisiting influenza virus H
1N
1Drug effect relatively
Table 7b α-D-(1 → 6)-dextran sulfate L
1Resisiting influenza virus H
3N
2Drug effect
Table 7c α-D-(1 → 6)-dextran sulfate L
1The drug effect of anti-Type B influenza virus
A. prepare α-D-(1 → 6)-dextran sulfate L of 5000 μ g/ml
1The preparation sample.Prepare 2000 μ g/ml virazole pharmaceutical preparation samples.Dilute one by one by 4 multiple proportions examples, make a series of samples.Virazole is a control drug.
B. pressing embodiment 10 steps d implements.
C. get the 18-20g mice, first infective virus, behind the 2h respectively by oral or collunarium mode administration.Be administered twice every day, and dosage is 120 μ g/dkg, successive administration 7 days.Virazole is a control drug.Put to death mice after 7 days, measure the mouse lung varying index.
α-D-(1 → 6)-dextran sulfate L
1By the administration of collunarium mode, its lung varying index is close with negative control group, illustrates that antiviral effect is obvious, and oral way administering effect not too significantly (seeing Table 8).
Table 8 α-D-(1 → 6)-dextran sulfate L
1The drug effect of resisiting influenza virus Mus lung adapted strain relatively
Press embodiment 12 described methods, investigate α-D-(1 → 6)-dextran sulfate L
1Toxicity to mice.Adopt and irritate the administration of stomach mode and adopt the administration of collunarium mode, successive administration was not all observed mice in 7 days and is caused death, and did not observe mice internal organs abnormal phenomena and other abnormal phenomenas yet, and α-D-(1 → 6)-dextran sulfate L is described
1Toxicity to mice is minimum.
The drug efficacy study of the resisiting influenza virus of embodiment 15 Porphyra Polysaccharide sulfuric esters
A. (mean molecule quantity is 60000 to the Porphyra Polysaccharide sulfuric ester ZSO that will be obtained by embodiment 5, monosaccharide sulfate radical substitution value is 0.50) and Porphyra Polysaccharide sulfuric ester ZSS (mean molecule quantity is 90000, and monosaccharide sulfate radical substitution value is 1.57) be mixed with 5000 μ g/ml pharmaceutical preparation samples.Preparation amantadine control drug preparation sample, concentration is 2000 μ g/ml.Said sample is all diluted one by one by 2 multiple proportions examples, and a series of variable concentrations samples are made in filtration sterilization.
B. press embodiment 13 step b, preparation A type influenza virus (H
1N
1And H
3N
2) and Type B influenza virus sample.Carry out virus concentration and measure, the preparation virus concentration is the viral sample of 100TCID.
C. pressing embodiment 13 step c implements.
D. after the embryo egg that step c handles is hatched 3 days, place 4 ℃ of refrigerator overnight.Collect embryo egg allantoic cavity liquid next day, carry out the blood clotting experiment, measure virus concentration.
Porphyra Polysaccharide sulfuric ester ZSO has certain anti-H
1N
1Virus activity, EC
50About 28.9 μ g/ embryo eggs.Porphyra Polysaccharide sulfuric ester ZSS has stronger anti-H
1N
1Virus activity.Behind Sulfation, the EC of ZSS sample
50Be 13.1 μ g/ embryo eggs, show that improving monosaccharide sulfate radical substitution value behind the Sulfation helps to improve anti-H
1N
1The activity of influenza virus.
Porphyra Polysaccharide sulfuric ester ZSS has stronger anti-influenza type A virus H
3N
2Drug effect with the Type B influenza virus.When every embryo egg dosage is 62.5 μ g/ embryo eggs, to H
3N
2Reach 51.40% and 68.75% (seeing Table 9) respectively with the suppression ratio of Type B influenza virus.
Table 9a Porphyra Polysaccharide sulfuric ester ZSS resisiting influenza virus H
1N
1The blood clotting experimental result
Table 9b Porphyra Polysaccharide sulfuric ester ZSS resisiting influenza virus H
3N
2The blood clotting experimental result
The blood clotting experimental result of the anti-Type B influenza virus of table 9c Porphyra Polysaccharide sulfuric ester ZSS
E. carry out the Embryo Gallus domesticus toxicity test of ZSS according to the step of embodiment 11.ZSS is 2122.49 μ g/ embryo eggs to the median lethal dose(LD 50) LD50 of Embryo Gallus domesticus, shows that ZSS is minimum to chick embryo development toxicity.
Claims (10)
1. average molecular weight range is 1000 to 100000, and monosaccharide sulfate radical substitution value is the application of the Carrageenan polysaccharide sulfuric ester of 1.0-3.0 as the preparation anti-influenza virus medicament.
2. according to the application of the described Carrageenan polysaccharide sulfuric ester of claim 1 as the preparation anti-influenza virus medicament, it is characterized in that: described Carrageenan polysaccharide sulfuric ester mean molecule quantity is 60000, and monosaccharide sulfate radical substitution value is 1.91.
3. according to the application of the described Carrageenan polysaccharide sulfuric ester of claim 1 as the preparation anti-influenza virus medicament, it is characterized in that: described Carrageenan polysaccharide sulfuric ester mean molecule quantity is 40000, and monosaccharide sulfate radical substitution value is 1.75.
4. according to the application of the described Carrageenan polysaccharide sulfuric ester of claim 1 as the preparation anti-influenza virus medicament, it is characterized in that: described Carrageenan polysaccharide sulfuric ester mean molecule quantity is 7000, and monosaccharide sulfate radical substitution value is 2.14.
5. according to the application of the described Carrageenan polysaccharide sulfuric ester of claim 1 as the preparation anti-influenza virus medicament, it is characterized in that: described influenza virus is an A type influenza virus.
6. according to the application of the described Carrageenan polysaccharide sulfuric ester of claim 1 as the preparation anti-influenza virus medicament, it is characterized in that: described influenza virus is the Type B influenza virus.
7. according to the application of the described Carrageenan polysaccharide sulfuric ester of claim 1 as the preparation anti-influenza virus medicament, it is characterized in that: described Carrageenan polysaccharide sulfuric ester is made medicament separately.
8. according to the application of the described Carrageenan polysaccharide sulfuric ester of claim 1 as the preparation anti-influenza virus medicament, it is characterized in that: described Carrageenan polysaccharide sulfuric ester and Tamiflu compatibility are made medicament.
9. according to the application of the described Carrageenan polysaccharide sulfuric ester of claim 8 as the preparation anti-influenza virus medicament, it is characterized in that: described Carrageenan polysaccharide sulfuric ester and amantadine or virazole are by weight 1: 0.01-1: 100 form compound recipe.
10. according to the application of the described Carrageenan polysaccharide sulfuric ester of claim 9 as the preparation anti-influenza virus medicament, it is characterized in that: described Carrageenan polysaccharide sulfuric ester and amantadine or virazole formed compound recipe by weight 5: 2.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031384005A CN100435805C (en) | 2003-05-26 | 2003-05-26 | Use of polysaccharose sulfate for preparing medicine against grippal virus |
| CN2008101664201A CN101385740B (en) | 2003-05-26 | 2003-05-26 | Use of polysaccharide sulfate as anti influenza virus medicine |
| CN2008101664220A CN101385741B (en) | 2003-05-26 | 2003-05-26 | Use of polysaccharide sulfate as anti influenza virus medicine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031384005A CN100435805C (en) | 2003-05-26 | 2003-05-26 | Use of polysaccharose sulfate for preparing medicine against grippal virus |
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|---|---|---|---|
| CN2008101664220A Division CN101385741B (en) | 2003-05-26 | 2003-05-26 | Use of polysaccharide sulfate as anti influenza virus medicine |
| CN2008101664201A Division CN101385740B (en) | 2003-05-26 | 2003-05-26 | Use of polysaccharide sulfate as anti influenza virus medicine |
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| Publication Number | Publication Date |
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| CN100435805C true CN100435805C (en) | 2008-11-26 |
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| CN2008101664201A Expired - Fee Related CN101385740B (en) | 2003-05-26 | 2003-05-26 | Use of polysaccharide sulfate as anti influenza virus medicine |
| CN2008101664220A Expired - Fee Related CN101385741B (en) | 2003-05-26 | 2003-05-26 | Use of polysaccharide sulfate as anti influenza virus medicine |
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| CN1318451C (en) * | 2005-09-09 | 2007-05-30 | 北京工业大学 | Sulfated polysaccharides and its preparation method and uses |
| CN100494222C (en) * | 2006-04-30 | 2009-06-03 | 华南理工大学 | Preparation method of beta-1,4-glucan-6,2,3-sulfate |
| DE602007005697D1 (en) * | 2006-12-05 | 2010-05-12 | Marinomed Biotechnologie Gmbh | USE OF CARRAGEENAN FOR THE TREATMENT OF RHINOVIRUS INFECTIONS |
| CN102821770A (en) * | 2010-02-22 | 2012-12-12 | 玛丽诺瓦股份有限公司 | Anti-viral formulations |
| CN102516323B (en) * | 2011-10-25 | 2014-05-07 | 中国海洋大学 | Low molecular weight carrageenan oligosaccharide and preparation method and application thereof |
| CN104721221B (en) * | 2015-01-29 | 2017-08-04 | 广州暨南生物医药研究开发基地有限公司 | Eucheuma gelatinae polysaccharide is used to prepare the purposes in preventing respiratory viruses medicine |
| CN105535927A (en) * | 2016-01-29 | 2016-05-04 | 山东中海制药有限公司 | Medicine used for treating influenza, upper respiratory infection and viral pneumonia |
| CN107149609B (en) * | 2017-06-08 | 2019-11-08 | 重庆三峡医药高等专科学校 | Application of the laver amylose in the drug of preparation treatment hand-foot-and-mouth disease poison infection |
| CN108484792B (en) * | 2018-05-24 | 2020-09-25 | 山东谷雨春生物科技有限公司 | Dextran sulfate and method for preparing dextran sulfate |
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| JPS6153220A (en) * | 1984-08-22 | 1986-03-17 | Sapporo Breweries Ltd | Novel polysaccharide ron substance, its preparation and antitumor agent, immuno-regulating agent and preventive and remedy for infectious disease, containing said substance as active component |
| WO2000009075A2 (en) * | 1998-08-14 | 2000-02-24 | Galenica Pharmaceuticals, Inc. | Chemically modified saponins and the use thereof as adjuvants |
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| 多糖抗病毒作用研究进展III.卡拉胶及其抗病毒作用. 王长云.生物工程进展，第20卷第3期. 2000 |
| 多糖抗病毒作用研究进展III.卡拉胶及其抗病毒作用. 王长云.生物工程进展，第20卷第3期. 2000 * |
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| Publication number | Publication date |
|---|---|
| CN101385741A (en) | 2009-03-18 |
| CN1552346A (en) | 2004-12-08 |
| CN101385740B (en) | 2011-06-08 |
| CN101385741B (en) | 2012-05-16 |
| CN101385740A (en) | 2009-03-18 |
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