CN101385740A - Application of polysaccharide sulfate as preparation of anti-influenza virus medicine - Google Patents

Abstract

The invention belongs to the field of synthetic pharmacy, and relates to the application of polysaccharide sulfate as preparation of anti-influenza virus medicine. The invention discloses laver polysaccharide sulfate with a molecular weight range of 1,000 to 100,000 and a monosaccharide sulfate radical substitution degree of 0.5-3.0. As an application for preparing anti-influenza virus drugs, the monosaccharide sulfate with an average molecular weight range of 1,000-100,000 is prepared. Practical pharmaceutical formulations of laver polysaccharide sulfates with a degree of root substitution of 0.5-3.0. It is determined that these polysaccharide sulfates have synergistic anti-type A and B influenza viruses alone or in combination with existing anti-influenza drugs such as amantadine and ribavirin. the use of.

Description

Application of polysaccharide sulfate as preparation of anti-influenza virus medicine

technical field

The invention belongs to the field of synthetic pharmacy, and relates to the application of polysaccharide sulfate as preparation of anti-influenza virus drugs, in particular to marine red algal polysaccharide sulfate and α-D-(1→6)-dextran sulfate as preparation of anti-influenza virus drug application.

technical background

Influenza is an acute viral respiratory infectious disease caused by influenza virus that seriously endangers human health, and is known as one of the viral infectious diseases with the highest fatality rate. There have been several large-scale epidemics of influenza in history, which brought serious disasters to mankind. Although a variety of therapeutic drugs have been developed, the harm of influenza and the losses it causes cannot be ignored. influenza. my country is a place where influenza frequently occurs, and there are local outbreaks basically every year. For this reason, my country's Ministry of Health has listed influenza as one of the diseases that will be monitored during the Tenth Five-Year Plan period.

Influenza virus is a particle surrounded by two surface glycoproteins-hemagglutinin and neuraminidase, which belongs to the family of orthomyxoviruses. Antigen variation is its most significant biological feature. According to the influenza virus nucleoprotein (NP) and matrix protein (M) Antigen type differences can be divided into three types: A (A), B (B), and C (C). The type A influenza virus is divided into several subtypes (H _x Ny) according to the antigenic differences of hemagglutinin (HA) and neuraminidase (Neuraminidase, NA). H ₁ N ₁ and H ₃ N ₂ subtypes and type B influenza viruses are the main pathogens causing human influenza outbreaks and epidemics in the past decade.

Influenza vaccination is currently the only way to prevent influenza. Common influenza vaccines include inactivated vaccines, live attenuated vaccines, recombinant vaccines (or vector vaccines), subunit vaccines, synthetic oligopeptide vaccines and nucleic acid vaccines. Influenza vaccines currently in clinical use are inactivated vaccines that have lost their infectivity. Due to the variability of influenza virus antigens and the lack of reliable methods to effectively predict the periodicity of influenza occurrence and epidemics, the measures of influenza vaccination are still limited.

Drug research for the treatment of influenza has made great progress. At present, the anti-influenza virus drugs that have been applied and are being studied include amantadine drugs, neuraminidase inhibitors, and influenza virus receptor blockers. Amantadine and rimantadine are commonly used clinical treatment drugs, which are only effective against type A virus, but not effective against type B influenza virus, and are prone to drug resistance and neurotoxicity. In 1999, Zanamivir (GG167 or Relenza) and Oseltamivir (GS4104 or Tamiflu), which were approved by the US FDA for clinical use, are neuraminidase (NA) inhibitors, mainly used for early treatment, and are effective against influenza A and B. However, these two drugs are less effective against influenza with comorbidities, so they have not been widely used. In view of the harmfulness of influenza virus, research on drugs for the prevention and treatment of influenza virus has received special attention.

Polysaccharide sulfate refers to polysaccharides with sulfate groups on sugar hydroxyl groups, also known as sulfated polysaccharides or sulfated polysaccharides. In recent years, the antiviral effect of polysaccharides has attracted great attention in the medical field, especially the strong antiviral activity of polysaccharide sulfates, showing broad medicinal prospects.

According to relevant literature reports, the main mechanism of polysaccharides with strong anionic groups to resist enveloped viruses is to inhibit the adsorption and invasion of enveloped viruses to host cells, that is, polysaccharide sulfates bind to enveloped outer membrane proteins by electrostatic interaction, Occupies the site where the enveloped virus binds to the recipient cell, making the enveloped virus unable to infect the cell. Virus adsorption to the plasma membrane of cells is a receptor recognition process related to electrostatic interaction, which requires molecular affinity between the cell surface and the virus [Fenner, 1974]. Polysaccharide sulfate can combine with the cationic region of gp 120 of HIV-1 coat glycoprotein, occupy the receptor site between enveloped virus and host cell, thereby preventing the combination of virus and host cell [Zhou Liang 1997]. Studies of the inhibitory mode of action have shown that the adsorption of viruses to host cells is blocked by sulfated polyanions [Liebhaber 1963, Schulze 1964, Takemoto 1964, Baba 1988, Neyts 1992, Mitsuya 1988, Schols 1990]. Inhibition can be blocked or reversed in the presence of polycations such as DEAE-dextran and protamine. Polysaccharide sulfate also inhibits a certain step of virus entry into cells or replication after entry into cells [Zhou Liang 1997]. Influenza viruses are enveloped viruses like HIV.

Glucan has α-D-(1→4), β-D-(1→4), α-D-(1→3), β-D-(1→3), α-D-(1 →6) and other types of glycosidic linkages. Various dextran sulfates have different functions due to differences in molecular weight, molecular side chains, glycosidic bond types, sulfuric acid bond types, and the number of monosaccharide sulfate esters (degree of substitution). For example, it can be used as an anticoagulant (US2715091, the molecular weight is 22000-200000, and the highest degree of substitution is 3.0); anti-rotavirus (WO96/30027); anti-AIDS virus (US5861383 and EP0406512, the molecular weight is 500000, generally 75000; EP0293826 Molecular weight is 5000, 8000, 90000, 200000; CN1206717A, β-D-(1→4)-glucan sulfate); anti-herpes simplex virus (WO94/15624, molecular weight is 50000-500000, glycosidic bond is α-D -(1→4) or β-D-(1→4)); anti-tumor (CN1283632A, α-D-(1→3)-glucan sulfate), etc. The anti-influenza virus activity of α-D-(1→6)-dextran (Dextran, dextran) sulfate and other dextran sulfates has not been reported.

Eucheume cottoni is an important class of red algae, the main component of which is carrageenan (poly D-galactose-3,6-intether-galactose sulfate with some sulfate groups) . In 1958, Gerber et al first proposed that seaweed contains natural antiviral active substances. Natural, chemically unmodified polysaccharides extracted from Gelidium Cartilagenium protect fertile eggs from mumps and influenza A viruses. In the late 1980s, scientists found that several polyanionic substances (natural or semi-synthetic red algae polysaccharide sulfate) were effective inhibitors of HIV replication, which aroused people's interest in the study of the antiviral effect of polysaccharide sulfate , Polysaccharides with inhibitory effects on herpes simplex viruses HSV1 and HSV2 were also found from a variety of red algae. In the screening of 39 kinds of California red algae against murine leukemia virus, it was found that the extract of Schizymenia had antiretroviral activity. Further research on the water extract and its fractions found that carrageenan was indeed an antiviral active ingredient. In addition, some patents mention the effect of carrageenan or its sulfate against herpes simplex virus (WO94/15624), anti-HIV (WO88/06396, EPA293826), anti-HIV and other DNA and RNA viruses (GB2262531), anti- Rotavirus (WO96/30027), etc., and cooperate with fiber growth factor to resist herpes simplex virus, cytomegalovirus, influenza virus, respiratory syncytial virus, forestry forest virus, HIV and Moloney sarcoma virus, etc. (EP0497341A2, molecular weight greater than 10 million). There is no report on the anti-type B influenza virus of natural Eucheuma auris polysaccharide sulfate; the anti-type A and B influenza virus of Eucheuma auris polysaccharide sulfate obtained by chemical processing with specific molecular weight and specific monosaccharide sulfate radical substitution degree is also available. None reported.

Porphyra haitanensis is an economically important red algae in southern my country. Porphyra polysaccharides have obvious anticoagulant, blood lipid-lowering, antithrombotic, and cardiotonic effects, and also have the effects of inhibiting tumors, lowering blood sugar, anti-inflammation, and preventing ulcers. It is reported that laver polysaccharides can also significantly enhance cellular immunity and humoral immunity. , but there is no report on the use of laver polysaccharide sulfate for anti-influenza virus.

In the reported literature and patents, the natural marine red algae polysaccharides (including carrageenan and Porphyra polysaccharides) are chemically modified and processed to produce polysaccharides with an average molecular weight of 1,000 to 100,000 and a monosaccharide sulfate radical substitution degree of 1.0 to 3.0. The individual or synergistic inhibitory effects of red algal polysaccharide sulfate (carrageenan polysaccharide sulfate, laver polysaccharide sulfate) and α-D-(1→6)-glucan sulfate on influenza A and B viruses, no see report.

Contents of the invention

The purpose of the present invention is to disclose the application of polysaccharide sulfate in anti-influenza virus, and prepare α-D-(1→6)-dextran sulfate (average molecular weight range is 1000-100000, monosaccharide sulfate radical substitution degree 0.2 —3.0) and marine red algae polysaccharide sulfate such as carrageenan polysaccharide sulfate (average molecular weight range is 1000-100000, monosaccharide sulfate radical substitution degree 1.0-3.0) and laver polysaccharide sulfate ester (average molecular weight range is 1000-100000, single The sugar sulfate radical substitution degree is 0.5-3.0) is a practical pharmaceutical dosage form, and it is determined that these polysaccharide sulfate esters have the use of single or synergistic anti-type A and type B influenza viruses.

In order to achieve the above object, the technical scheme adopted by the present invention is as follows:

(1) Weigh a certain amount of Eucheuma auris, crush it, and extract it in water at 95°C for 3 hours. The crude extract is centrifuged and the supernatant is collected, and an appropriate amount of KCl is added, and the precipitate is separated by centrifugation. The precipitate was dissolved in water, precipitated again with ethanol, and then filtered. The filtrate was vacuum-dried at 50° C. and ground into a fine powder to obtain carrageenan powder.

(2) Weigh a certain amount of dried seaweed, soak it in water overnight, mash it and freeze it at -20°C, take it out and microwave it to thaw, repeat this three times, stir and extract in a water bath at 60°C for 30 minutes, centrifuge, and reduce the supernatant Concentrate under pressure and dry to obtain crude polysaccharide. The crude polysaccharide was deproteinized by 732 cation exchange resin to obtain refined laver polysaccharide.

(3) Weigh the carrageenan prepared by (1) or commercially available carrageenan (the molecular weight is generally greater than 500,000, the monosaccharide sulfate radical substitution degree is 0.3-0.8) or the laver polysaccharide prepared by (2), and make 2- The 15% solution acts on sulfuric acid (or hydrochloric acid) concentration in the range of 0.005% to 5%w at 40 to 120°C for 10 to 180 minutes. After the reaction is finished, neutralize with NaOH solution immediately, and then filter with an ultrafiltration membrane to prepare carrageenan polysaccharide or laver polysaccharide with specific molecular weight.

(4) Treat the red seaweed polysaccharide with smaller molecular weight obtained in (3) or commercially available dextran (Dextran, α-D-(1→6)-dextran) by conventional pyridine-chlorosulfonic acid method , to obtain polysaccharide sulfate with a specific degree of substitution of monosaccharide sulfate radicals. After removing small molecular substances by ultrafiltration, ethanol precipitation with 2 times the amount of absolute ethanol, the precipitate was dissolved in distilled water and then ethanol precipitated with 2 times the amount of ethanol. The obtained precipitate is vacuum-dried at 60° C. to obtain the desired polysaccharide sulfate. The impurity content of the polysaccharide sulfate powder is less than 2%, and the main impurities are NaCl and Na ₂ SO ₄ .

(5) The polysaccharide sulfate powder is dried and filled into capsules in a dehumidified environment to prepare an oral dosage form. The polysaccharide sulfate powder can also be dissolved in physiological saline according to the required concentration, and after being sterilized by filtration through a 0.22 μm membrane, it can be prepared into a pharmaceutical dosage form with isotonic pressure and can be used for nasal mucosa (pulmonary mucosa) absorption.

(6) adopt chicken embryo model and mouse experimental model, carry out the test of anti-influenza virus independently to the polysaccharide sulfate sample prepared in (5), and with amantadine, ribavirin, rimantadine, Zanamivir (GG167 or Relenza) and Oseltamivir (GS4104 or Tamiflu) and other existing anti-influenza drugs are compounded, and the synergistic anti-influenza virus test is carried out. Tests have shown that the prepared polysaccharide sulfate has obvious anti-type A and B influenza virus activity, and has synergistic effect after forming a compound with amantadine, ribavirin and other existing drugs at a content ratio of 1:0.01-1:100. The anti-influenza virus effect can be further enhanced. The anti-influenza virus experimental effect of polysaccharide sulfate, amantadine and ribavirin in a content ratio of 5:2 is listed in the embodiment.

Description of drawings

Fig. 1 is the structural formula of the carrageenan extracted from Eucheuma auricularia involved in the present invention, which is poly D-galactose-3,6-endether-D-galactose and has some sulfuric acid groups.

Fig. 2 is the molecular structure formula of α-D-(1→6)-glucan sulfate involved in the present invention, wherein the substituent R can be H or SO ₃ H. When the substituents R are all H, it is α-D-(1→6)-glucan.

Figure 3 is the infrared spectrum of α-D-(1→6)-glucan.

Figure 4 is the infrared spectrum of α-D-(1→6)-dextran sulfate, in which the absorption at about 1244cm ^-1 is S=O??? stretching vibration, and the absorption at about 800cm ^-1 is C—O?? ?—S stretching vibration, indicating that dextran has been sulfated.

Detailed ways

The present invention is illustrated in detail with reference to the following examples, but is not limited thereto.

Embodiment 1: the preparation of Eucheuma polysaccharide (carrageenan)

Weigh a certain amount of dry Eucheuma auris (purchased from Xiamen Fisheries Research Institute), cut into pieces and wash, add 50 times the weight of hot water, stir and heat at 95°C for 3 hours. The crude polysaccharide extract was centrifuged at 5000 rpm for 10 minutes, and the supernatant was collected. Add KCl to make the final concentration 5%, stir evenly, let stand overnight, and centrifuge at 5000 rpm for 10 minutes to separate insoluble matter. Dissolve the insoluble matter with distilled water, then add ethanol at a ratio of 3:1 (v/v), let it settle for 60 minutes, dry it under vacuum at 60°C, and grind it into a fine powder to obtain refined carrageenan powder. The molecular weight of the prepared carrageenan is higher than 500,000, and the substitution degree of sulfate group per monosaccharide is 0.45.

Embodiment 2: the preparation of laver polysaccharide

Weigh a certain amount of dry laver (produced in Pingtan, Fujian), cut it into pieces and wash it, add 50 times of distilled water to soak overnight, mash the laver with a masher, freeze it at -20°C, take it out and thaw it in microwave. After repeating 3 times, stir and extract in a 60°C water bath for 30 minutes, centrifuge at 3000rpm for 20 minutes, put the supernatant separately, add distilled water to the laver residue and repeat the extraction 2 times, combine the 3 supernatants, concentrate under reduced pressure at 50°C, and freeze-dry. get crude polysaccharides.

The laver crude polysaccharide was formulated into a 2% solution, and sulfuric acid was added to adjust the pH to 2.0. The polysaccharide solution flows through the regenerated 732 cation exchange resin column, which can effectively remove protein. The column solution was collected, concentrated to 3% w/v, and then the polysaccharide was precipitated with ethanol. The precipitate is vacuum-dried and pulverized to obtain laver polysaccharide powder.

Embodiment 3: the preparation of specific molecular weight carrageenan polysaccharide

The carrageenan (or commercial carrageenan) prepared in Example 1 was formulated into a 5% glue solution, placed in a constant temperature water bath, and fully mixed. Add sulfuric acid to adjust the pH to 2.5, and react at a constant temperature of 90°C for 120 minutes. After the reaction, neutralize with 1N NaOH solution immediately, and then filter with ultrafiltration membranes with different molecular weight cut-offs to prepare carrageenan polysaccharides with different molecular weights. The prepared carrageenan polysaccharide with specific molecular weight is spray-dried and used for the preparation of carrageenan polysaccharide sulfate.

Embodiment 4: Preparation of α-D-(1→6)-dextran sulfate

Put a three-necked flask equipped with a condenser tube and a stirring device in a brine-ice bath, add 48ml of pyridine, stir to make it fully cooled, and add 13ml of chlorosulfonic acid with a dropping funnel. Quickly move the three-necked flask into a 100°C water bath, add 3.0 g of α-D-(1→6)-dextran with an average molecular weight of 20,000, and stir at 100°C for 1 hour. The reaction solution cooled to room temperature was poured into 200 ml of ice water, neutralized to pH 10.5 with 40% NaOH, allowed to stand and separated into layers, and the lower aqueous phase was separated with a separatory funnel. 4 times the volume of 95% ethanol was added to the water phase, and the dextran sulfate was precipitated, and the precipitate was collected by centrifugation at 3000 rpm for 10 minutes. The precipitate was dissolved in deionized water and dialyzed in deionized water for 48h. The dialyzed material was concentrated to 5% w/v in a vacuum rotary evaporator, 100 ml of absolute ethanol was added, allowed to stand for precipitation, and the precipitate was separated by centrifugation. Dissolve the precipitate with deionized water, then add absolute ethanol and centrifuge to separate the precipitate. Repeat the ethanol separation process three times. The final precipitate was vacuum-dried to obtain α-D-(1→6)-dextran sulfate. After determination, the average molecular weight is 45,000, and the average monosaccharide sulfate radical substitution degree is 2.13.

Embodiment 5: Preparation of small molecular weight laver polysaccharide sulfate

According to the method described in Example 3, the laver polysaccharide obtained in Example 2 was treated to obtain a low-molecular-weight laver polysaccharide ZSO with an average molecular weight of about 60,000 and a monosaccharide sulfate radical substitution degree of 0.50. Slowly add 25ml of chlorosulfonic acid into 120ml of anhydrous pyridine at a temperature of 10°C. Raise the reaction temperature rapidly, and add 15 grams of small molecular weight laver polysaccharide ZSO samples dispersed with pyridine under sufficient stirring conditions. React at 90°C for 4h. The temperature of the reaction solution was lowered to 10°C, and a saturated NaOH cold solution was added to adjust the pH to 10.5. After standing for 10 minutes to separate the layers, the upper organic phase and the lower aqueous phase were separated. The lower aqueous phase was concentrated in vacuo to 5% w/v, and isopropanol was added to make the final concentration of isopropanol 60% w/v. The precipitate was separated by centrifugation and washed three times with isopropanol. The precipitate washed with isopropanol was redissolved in deionized water and filtered with an ultrafiltration membrane with a molecular weight cut off of 5000 to remove small molecular weight impurities. When the solid content in the material concentrated by the ultrafiltration membrane reaches about 5% w/v, add 2 times the volume of isopropanol. The filtered precipitate was dried under vacuum (90° C., 0.005 MPa). The prepared sample ZSS has an average molecular weight of about 90,000 and an average monosaccharide sulfate radical substitution degree of 1.57.

Embodiment 6: Preparation of small molecular weight carrageenan polysaccharide sulfate

Degrade the large molecular weight carrageenan according to the method of Example 3, and prepare the smaller molecular weight carrageenan polysaccharide with an average molecular weight of 35000 by using the ultrafiltration membrane cut-off method.

In a 500ml three-necked flask, add 200ml of pyridine, and slowly add 20ml of chlorosulfonic acid at 10°C with sufficient stirring. After the addition of chlorosulfonic acid, the reaction solution was heated to 60° C., and 15 grams of carrageenan polysaccharide was added. Then the temperature was raised to 80° C. and stirred at constant temperature for 2 h. After the reaction was completed, the reaction liquid was cooled to 10° C., and neutralized to pH 9.0 with 40% NaOH cold solution. The reaction solution was allowed to stand to separate into layers, and the lower aqueous phase was separated with a separatory funnel. The aqueous phase feed solution was concentrated in vacuo to 5% w/v, and absolute ethanol was added to make the final concentration of ethanol 75%. After centrifugation, the precipitate was dissolved in deionized water, loaded into a dialysis membrane, and dialyzed in deionized water for 48 hours. The dialyzed material was then placed in a vacuum rotary drying device for concentration to a concentration of 5% w/v, and then absolute ethanol was added, and the final concentration of ethanol was 75%. The precipitate is dried and ground to obtain carrageenan polysaccharide sulfate. The average degree of substitution of monosaccharide sulfate radicals was determined to be 1.91.

Example 7 Study on the Drug Efficacy of Carrageenan Polysaccharide Sulfate Against Influenza Virus H ₁ N ₁

a. according to the method of embodiment 1, extract carrageenan from Eucheuma auricularis. Carrageenan polysaccharides with an average molecular weight of 35,000 were prepared according to the method of Example 3, and the average degree of substitution of monosaccharide sulfate groups was 0.45.

b. Carrageenan polysaccharide prepared in step a is carried out sulfation. Carrageenan polysaccharide sulfate K ₁ (average molecular weight 60,000, average monosaccharide sulfate group substitution degree 1.91) was obtained. A _K1 pharmaceutical preparation of 5000 μg/ml was prepared, and a 2000 μg/ml amantadine pharmaceutical preparation was prepared at the same time. Dilute by 5 times, filter and sterilize, and prepare a series of samples with different concentrations.

c. Take the H ₁ N ₁ influenza virus strain provided by the China National Influenza Center, infect the allantoic cavity of chicken embryos, and incubate at a constant temperature of 37°C. Three days later, the allantoic cavity fluid of chicken embryos was taken to measure the virus concentration. Prepare H ₁ N ₁ influenza virus samples with a virus concentration of 100 TCID.

d. Get 9-day-old chicken embryo eggs, and quickly inject the drug sample and virus sample prepared in the above steps a and b into the embryo eggs successively, 0.2ml per embryo egg. Five embryonated eggs were used for each drug concentration test. At the same time, a virus positive control group and a normal saline negative control group were set. Incubate at a constant temperature of 37°C, and observe the survival of chicken embryos for 3 consecutive days.

e. After incubating the embryo eggs treated according to step d for 3 days, place them in a refrigerator at 4°C overnight. The next day, the allantoic cavity fluid of embryonated eggs was collected, and the hemagglutination test was carried out to determine the H ₁ N ₁ virus titer.

Experiments show (see Table 1 and Table 2) that the _K1 sample has obvious anti _{- H1N1} influenza virus effect. When the experimental sample concentration reaches 4.26 μg/embryonic egg, the virus inhibition rate can reach more than 50%. The efficacy of K ₁ against H ₁ N ₁ influenza virus is superior to that of amantadine, and its toxicity is extremely low, and its therapeutic index TI ₅₀ is significantly higher than that of amantadine.

Table 1 Experimental results of carrageenan polysaccharide sulfate K ₁ against influenza virus H ₁ N ₁

K ₁ dosage (μg/embryonic egg) Virus inhibition rate % 1 1000 98.2 2 200 87.5 3 40 85.6 4 8 75.0 5 1.6 42.8

Table 2 Comparison of efficacy of carrageenan polysaccharide sulfate K ₁ and amantadine against virus H ₁ N ₁

K ₁ amantadine K ₁ + amantadine LD ₅₀ (μg/embryonic egg) 7386.7 1866.07 7386.87+1866.7 EC ₅₀ (μg/embryonic egg) 4.26 5.68 3.38+1.35 TI ₅₀ 1734.0 328.53 2185.46+1382.7

f. Synergistic effect of carrageenan polysaccharide sulfate K ₁ and amantadine against H ₁ N ₁ influenza virus

Prepare a mixed solution of carrageenan polysaccharide sulfate K ₁ and amantadine with a content ratio of 5:2, wherein the content of K ₁ is 5000 μg/ml, and the content of amantadine is 2000 μg/ml. According to step d, the synergistic effect of the mixed drugs against _H1N1 influenza virus was determined, _and the experimental results are shown in Table 2. Table 2 shows that the mixed use of the two drugs helps to improve the effect of anti- _H1N1 virus, and the therapeutic index increases; the toxicity of _the two drugs mixed is comparable to that of amantadine when used alone, and the two drugs have not been observed The phenomenon of toxic superposition.

Example 8 Carrageenan Polysaccharide Sulfate K ₂ Anti-Influenza B Virus Drug Efficacy Study

a. Carrageenan polysaccharide sulfate K ₂ was prepared according to the method in Example 7 (the average molecular weight is about 40,000, and the degree of substitution of sulfate per unit of monosaccharide is 1.75). A 5000 μg/ml _K2 pharmaceutical preparation was prepared and diluted by 2 times to prepare a series of pharmaceutical samples.

b. Prepare a sample of influenza B virus (provided by the National Influenza Center) according to the method in Example 7, and the virus concentration is 100 TCID.

c. Implement according to step d of Example 7.

d. Incubate the embryos treated according to step c, and place them in a 4°C refrigerator overnight. The next day, the allantoic cavity fluid of embryonated eggs was collected, and the hemagglutination test was carried out to determine the titer of type B virus. Table 3 shows that the effect of K ₂ alone against influenza B virus is more obvious. When the drug dosage is 100 μg/embryo egg, the virus inhibition rate reaches over 90%; when the drug dosage is 12.5 μg/embryo egg, the virus inhibition rate reaches 75%.

Table 3 K ₂ anti-influenza virus hemagglutination test results

Dosage of drug K ₂ (μg/embryonic egg) Virus suppression rate (%) 1 200 95.53 2 100 90.54 3 50 82.14 4 25 78.57 5 12.5 75.0

Example 9 Pharmacodynamic experiment of carrageenan polysaccharide sulfate K ₂ against H ₃ N ₂ influenza virus

a. Carrageenan polysaccharide sulfate K ₂ sample prepared by Example 8. A 5000 μg/ml sample and a 2000 μg/ml amantadine sample were prepared. Serially diluted by 2 times. Amantadine was used as the control drug.

b. Prepare the H ₃ N ₂ influenza virus sample according to the method in Example 7, and the virus concentration is 100 TCID.

c, d are all implemented according to embodiment 8. The experimental data are shown in Table 4.

Table 4 shows that carrageenan polysaccharide sulfate sample K ₂ has a more significant effect of inhibiting H ₃ N ₂ influenza virus from attacking host cells.

Table 4 Experimental results of carrageenan polysaccharide sulfate K ₂ against virus H ₃ N ₂

Dosage of drug K ₂ (μg/embryonic egg) Virus suppression rate (%) 1 200 85.24 2 100 80.15 3 50 69.42 4 25 64.29 5 12.5 60.15

Example 10 Study on the Drug Efficacy of Carrageenan Polysaccharide Sulfate K ₃ Against Influenza Virus H ₁ N ₁ Mouse Lung-adapted Strain

a. Using commercially available carrageenan powder, according to the method of Example 3, a carrageenan polysaccharide with an average molecular weight of 4000 was prepared, and the average degree of substitution of monosaccharide sulfate groups was 0.45.

b. According to the method of Example 6, the carrageenan polysaccharide prepared in step a was sulfated to obtain carrageenan polysaccharide sulfate K ₃ (the average molecular weight was 7000, and the average degree of substitution of monosaccharide sulfate groups was 2.14).

c. Prepare a 5000 μg/ml _K3 pharmaceutical preparation sample. A 2000 μg/ml ribavirin pharmaceutical preparation sample was prepared. Dilute successively by 4 times to make a series of samples. Ribavirin was used as the control drug.

d. Influenza virus H ₁ N ₁ murine lung-adapted strain (provided by the National Influenza Center) was infected and enriched by embryonated eggs, and the virus concentration of 100 TCID virus was prepared as a sample.

e. Take 18-20 grams of mice, firstly infect the virus, and administer it by nasal drops after 2 hours. Dosing twice a day for 7 consecutive days. After 7 days, the mice were sacrificed, their body weights were weighed, and then their lungs were dissected, and their lungs were weighed. A blank negative control group, a virus positive control group and a ribavirin control group were also set up.

The mouse lung change index is the ratio of the mouse lung weight to the body weight, and it represents the weight gain index of the lung lesions of the mice infected with the influenza virus. The negative control group was 0.686, and the positive control group was 0.850. Carrageenan sulfate K ₃ administered through nasal drops can achieve obvious antiviral effect, and its lung change index is similar to that of the negative control group (see Table 5). Experiments have shown that nasal mucosal absorption is a good way of administration.

Table 5 Comparison of efficacy of carrageenan sulfate K ₃ against murine lung-adapted strains of influenza virus

blank negative control virus positive control Ribavirin Carrageenan Polysaccharide Sulfate K ₃ Dosage μg/d·kg 120 120 Pulmonary Change Index % 0.686 0.850 0.822 0.680

Example 11 Chicken Embryo Toxicity Test of Carrageenan Polysaccharide Sulfate _K1

a. prepare the pharmaceutical preparation solution of the carrageenan polysaccharide sulfate K ₁ of 30000 μ g/ml. At the same time, a 5000 μg/ml amantadine drug preparation solution was prepared. Dilute successively according to 5 times ratio, filter and sterilize. Inject 0.5ml of sample into the allantoic cavity of each embryo egg. Five embryonated eggs were tested for each sample.

b. Incubate the above-mentioned chicken embryos at a constant temperature of 37°C. The death or development of chicken embryos was observed continuously. The number of chick embryos that died within 24 hours after inoculation was non-specific death and was not included in the statistical data.

It was observed that when the dosage of carrageenan polysaccharide sulfate K ₁ reached 3000 μg/embryonic egg, chicken embryos were lethal. The LD ₅₀ of K ₁ was 9706 μg/embryonic egg. The semi-lethal dose LD ₅₀ of the control drug amantadine was 1866 μg/embryonic egg. When the dose was less than 2000 μg/embryonic egg, no adverse effects of K ₁ on the development of chicken embryos were observed, and no abnormal growth of chicks was observed after the experimental embryonic eggs developed into chicks. Experiments show that K ₁ has very low toxicity to chicken embryo development.

c. Prepare a mixed sample of 10000 μg/ml K ₁ and 5000 μg/ml amantadine, dilute it step by step according to the ratio of 5 times, and filter to sterilize. The 9-day-old embryo-egg drug synergistic toxicity experiment was carried out. It was found that the lowest drug concentration lethal to chicken embryos was: K ₁ 2000μg/embryo egg and amantadine 1000μg/embryo egg; the semi-lethal concentration of mixed drugs was LD ₅₀ : K ₁ 7386.86μg/embryo egg and amantadine 1866μg/embryo Egg. The toxicity of the mixed drugs mainly came from amantadine, and no superimposed toxicity of the two drugs was observed.

Example 12 Mouse Toxicity Test of Carrageenan Polysaccharide Sulfate _K3

Prepare 60000 μg/ml _K3 solution with physiological saline, and filter to sterilize.

Take 18-20 grams of mice and administer them by gavage to investigate the toxicity of K ₃ to mice. Every day, the morning and afternoon are divided into 8 hours for intragastric administration, and each time 0.5ml of medicine is administered. No death of mice was observed after continuous drug administration for 7 days, and no abnormalities of internal organs and other abnormalities of mice were observed.

Take 18-20 grams of mice and administer them by nasal drops. The dosage is 120 μg/d·kg, and the administration is divided into morning and afternoon at an interval of 8 hours. After continuous administration for 7 days, no death of mice was observed, nor other abnormal phenomena of mice were observed.

Example 13 Study on the efficacy of α-D-(1→6)-dextran sulfate against influenza virus

a. Prepare α-D-(1→6)-dextran sulfate sample L ₁ according to the method in Example 4. The average molecular weight of sample _L1 is about 45,000, and the degree of substitution of monosaccharide sulfate group is 2.13. Prepare 5000 μg/ml L ₁ drug formulation. At the same time, a 2000 μg/ml amantadine (or ribavirin) drug preparation was prepared as a control drug. Diluted successively by 5 times, and sterilized by filtration to make a series of test samples with different concentrations.

b. Infect the allantoic cavity of chicken embryos with influenza A virus (H ₁ N ₁ and H ₃ N ₂ ) and influenza B virus strains provided by the National Influenza Center of China, and incubate at a constant temperature of 37°C. Three days later, the allantoic cavity fluid of chicken embryos was taken to measure the virus concentration. The preparation virus concentration is A type and B type influenza virus sample that are all 100TCID.

c. Get 9-day-old chicken embryo eggs, and quickly inject the drug sample and virus sample prepared in the above steps a and b into the embryo eggs successively, 0.2ml per embryo egg. Five embryonated eggs were used for each drug concentration test. At the same time, a virus positive control group and a normal saline negative control group were set. Incubate at a constant temperature of 37°C, and observe the survival of chicken embryos for 3 consecutive days.

d. After incubating the embryonated eggs treated in step c for 3 days, place them in a refrigerator at 4° C. overnight. The next day, the allantoic cavity fluid of embryonated eggs was collected, and the hemagglutination test was carried out to determine the virus concentration. The experimental results (Table 6 and Table 7) show that α-D-(1→6)-dextran sulfate L ₁ has obvious anti-influenza virus activity, and the half-inhibitory virus concentration of H ₁ N ₁ virus is 15.61 μg per embryonated egg, the half-inhibitory virus concentration of H ₃ N ₂ virus was 12.60 μg/embryo egg, and the half-inhibitory virus concentration of B influenza virus was 8.75 μg/embryo egg.

Table 6 Hemagglutination test results of α-D-(1→6)-dextran sulfate L ₁ and amantadine against influenza virus H ₁ N ₁

e. Study on the synergistic anti-influenza virus efficacy of α-D-(1→6)-glucan sulfate L ₁ and amantadine (or ribavirin)

Prepare a mixed solution of α-D-(1→6)-dextran sulfate L ₁ and amantadine (or ribavirin) with a ratio of 5:2, wherein the content of L ₁ is 5000 μg/ml, amantadine (or ribavirin) content is 2000μg/ml. A series of mixed drug samples of α-D-(1→6)-dextran sulfate L ₁ and amantadine (or ribavirin) with different concentrations were prepared by serially diluting in a 2-fold ratio.

Carry out chicken embryo experiment according to steps b, c, d. The experimental results show that α-D-(1→6)-glucan sulfate and amantadine (or ribavirin) have a synergistic anti-influenza virus effect. Through the synergistic effect, the EC ₅₀ value of α-D-(1→6)-glucan sulfate and amantadine was reduced, and the antiviral effect of the drug was directly improved. The experimental results are shown in Tables 7a, 7b, and 7c. α-D-(1→6)-dextran sulfate L ₁ is less toxic to chicken embryos than amantadine when used alone, and when L ₁ is combined with amantadine, no superimposed toxicity of the two drugs is observed .

Table 7a Comparison of the efficacy of α-D-(1→6)-dextran sulfate L ₁ and amantadine against influenza virus H ₁ N ₁

L ₁ amantadine L ₁ + amantadine LD ₅₀ (μg/embryonic egg) 6427.2 1866.07 6427.2+1866.07 EC ₅₀ (μg/embryonic egg) 15.61 5.68 5.24+2.09 TI ₅₀ 411.7 328.5 1226.5+892.8

Table 7b The efficacy of α-D-(1→6)-dextran sulfate L ₁ against influenza virus H ₃ N ₂

L ₁ Ribavirin L ₁ + ribavirin EC ₅₀ (μg/embryonic egg) 12.60 17.07 9.78+14.88

Table 7c α-D-(1→6)-dextran sulfate L ₁ anti-influenza virus efficacy

L ₁ Ribavirin L ₁ + ribavirin EC ₅₀ (μg/embryonic egg) 8.75 12.68 5.71+9.69

Example 14 Study on the efficacy of α-D-(1→6)-dextran sulfate L ₁ against influenza virus H ₁ N ₁ murine lung-adapted strain

a. Prepare 5000 μg/ml α-D-(1→6)-glucan sulfate L ₁ preparation sample. A 2000 μg/ml ribavirin pharmaceutical preparation sample was prepared. Dilute successively by 4 times to make a series of samples. Ribavirin was used as the control drug.

b. Implement according to step d of Example 10.

c. Take 18-20g mice, firstly infect the virus, and then administer the virus orally or nasally after 2 hours. Administered twice a day, the dosage was 120 μg/d·kg, for 7 consecutive days. Ribavirin was used as the control drug. After 7 days, the mice were sacrificed, and the lung degeneration index of the mice was measured.

α-D-(1→6)-glucan sulfate L ₁ was administered through nasal drops, and its lung change index was similar to that of the negative control group, indicating that the antiviral effect was obvious, while the effect of oral administration was not significant ( See Table 8).

Table 8 Comparison of efficacy of α-D-(1→6)-dextran sulfate L ₁ against murine lung-adapted strains of influenza virus

According to the method described in Example 12, the toxicity of α-D-(1→6)-glucan sulfate L ₁ to mice was investigated. Administering by intragastric administration and nasal drop administration, no mouse death was observed in continuous administration for 7 days, and abnormal viscera and other abnormal phenomena of mice were not observed, indicating that α-D-(1→6 )-dextran sulfate L ₁ is minimally toxic to mice.

Example 15 Anti-influenza virus efficacy research of laver polysaccharide sulfate

a. the laver polysaccharide sulfate ZSO (average molecular weight is 60000, monosaccharide sulfate radical substitution degree is 0.50) and laver polysaccharide sulfate ester ZSS (average molecular weight is 90000, monosaccharide sulfate radical substitution degree is 1.57) preparation obtained by embodiment 5 into 5000μg/ml pharmaceutical preparation sample. A sample of the amantadine control drug preparation was prepared with a concentration of 2000 μg/ml. The above samples were diluted successively according to the ratio of 2 times, and sterilized by filtration to make a series of samples with different concentrations.

b. According to step b of Example 13, prepare samples of influenza A virus (H ₁ N ₁ and H ₃ N ₂ ) and influenza B virus. To measure the virus concentration, a virus sample with a virus concentration of 100 TCID was prepared.

c. Implement according to step c of Example 13.

d. After incubating the embryonated eggs treated in step c for 3 days, place them in a refrigerator at 4°C overnight. The next day, the allantoic cavity fluid of embryonated eggs was collected, and the hemagglutination test was carried out to determine the virus concentration.

Porphyra polysaccharide sulfate ZSO has certain anti-H ₁ N ₁ virus activity, EC ₅₀ is about 28.9 μg/embryonic egg. Porphyra polysaccharide sulfate ZSS has strong anti-H ₁ N ₁ virus activity. After sulfation, the EC ₅₀ of the ZSS sample was 13.1 μg/embryonic egg, indicating that increasing the degree of substitution of monosaccharide sulfate radicals after sulfation helps to improve the activity against H ₁ N ₁ influenza virus.

Laver polysaccharide sulfate ZSS has strong anti-influenza A virus H ₃ N ₂ and influenza B virus efficacy. When the dose per embryo egg was 62.5 μg/embryo egg, the inhibitory rates to H ₃ N ₂ and type B influenza virus reached 51.40% and 68.75% respectively (see Table 9).

Table 9a Hemagglutination test results of laver polysaccharide sulfate ZSS against influenza virus H ₁ N ₁

Table 9b Hemagglutination test results of laver polysaccharide sulfate ZSS against influenza virus H ₃ N ₂

Drug dosage (μg/embryo egg) Virus inhibition rate % 123 1000.0500.0250.0 97.2584.3881.25 4 125.0 56.25 5 62.5 51.40

Table 9c Hemagglutination test results of Laver polysaccharide sulfate ZSS against influenza B virus

Drug dosage (μg/embryo egg) Virus inhibition rate % 1 1000.0 97.85 2 500.0 96.29 3 250.0 85.94 4 125.0 73.80 5 62.5 68.75

e. Carry out the chicken embryo toxicity test of ZSS according to the steps of Example 11. The LD50 of ZSS to chicken embryos was 2122.49 μg/embryonic egg, indicating that ZSS had minimal toxicity to chicken embryo development.

Claims (9)

1. The molecular weight range is 1000 to 100000, and the laver polysaccharide sulfate ester of monosaccharide sulfate group substitution degree is 0.5-3.0, as the application of preparation anti-influenza virus medicine. 2. according to the application of Porphyra laver polysaccharide sulfate according to claim 1 as the preparation of anti-influenza virus medicine, it is characterized in that: the average molecular weight of said Porphyra laver polysaccharide sulfate ester is 60000, and the degree of substitution of monosaccharide sulfate is 0.50. 3. according to the application of the Porphyra laver polysaccharide sulfate according to claim 1 as the preparation of anti-influenza virus drugs, it is characterized in that: the average molecular weight of the Porphyra laver polysaccharide sulfate ester is 90000, and the degree of substitution of monosaccharide sulfate is 1.57. 4. according to claim 1, the laver polysaccharide sulfate is used as the preparation of anti-influenza virus medicine, characterized in that: the influenza virus is type A influenza virus. 5. According to the application of the Porphyra laver polysaccharide sulfate according to claim 1 as the preparation of anti-influenza virus medicine, it is characterized in that: the influenza virus is type B influenza virus. 6. According to the application of the Porphyra laver polysaccharide sulfate according to claim 1 as the preparation of anti-influenza virus medicine, it is characterized in that: the described polysaccharide sulfate is made into a medicament separately. 7. According to the application of Porphyra laver polysaccharide sulfate according to claim 1 as the preparation of anti-influenza virus drugs, it is characterized in that: said Porphyra laver polysaccharide sulfate ester is compatible with anti-influenza drugs to make a medicament. 8. According to the application of the laver polysaccharide sulfate ester according to claim 7 as preparing anti-influenza virus drugs, it is characterized in that: the laver polysaccharide sulfate ester and amantadine or ribavirin are in a weight ratio of 1:0.01-1 : 100 form a compound recipe. 9. According to the application of the laver polysaccharide sulfate according to claim 8 as the preparation of anti-influenza virus drugs, it is characterized in that: the laver polysaccharide sulfate and amantadine or ribavirin form a compound in a weight ratio of 5:2 .

Images