JP2002293803A - Antivirus agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel antivirus agent containing an active ingredient prepared from naturally occuring polysaccharides by the chemical modification. SOLUTION: The agent has a slufated fructan obtained by sulfating a fructan produced from a marine microorganism and its physiologically acceptable salts as the active ingredient.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a sulfated fructan having antiviral activity and an antiviral agent containing the same as an active ingredient.

[0002]

2. Description of the Related Art It has been reported that sulfated polysaccharides such as dextran sulfate and natural sulfate polysaccharide have an antiviral effect [H. Nakashima et al .: Antimicrobial Ag
ents and Chemotherapy, 31, 1524-1528 (1987) and Baba
(M. Baba) et al .: Antimicrobial Agents and Chemotherapy,
32, 1742-1745 (1988)]. However, to date, the activity of the above compounds when administered in vivo is extremely unstable, has not been put to practical use as an antiviral agent, and has been found to be effective for various sulfated polysaccharides having different structures and their chemical derivatives. Further search for viral activity is expected.

[0003] The present invention relates to a novel sulfated polysaccharide which expresses antiviral activity by chemically modifying a fractan produced by a marine microorganism to form a sulfated derivative, and an antiviral agent containing the substance as an active ingredient. Things.

The fructan used as a raw material of the present invention is a substance produced by a microorganism belonging to marine Pseudomonas [M. Matsuda: Academic report of Faculty of Agriculture, Kagawa University, 52, 57-61 (2000)] reference].

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel antiviral agent containing, as an active ingredient, a sulfate obtained by chemically modifying a polysaccharide.

[0005] The fructan is fructose beta (2
-6) It is characterized by having a linear Levan type structure consisting of bonds.

[0006]

Means for Solving the Problems In order to achieve the above object, the present inventors have sulfated polysaccharides produced by marine microorganisms, studied their antiviral activity, and conducted intensive studies. (Pseudomonas) The inventors have found that sulfated fructan produced by the genus (Pseudomonas) has antiviral activity, and have completed the present invention. Accordingly, the present invention relates to an antiviral agent containing as an active ingredient a sulphoxide of fructan produced by marine Pseudomonas sp. And a physiologically acceptable salt thereof.

[0007] The molecular weight of the fructan used in the present invention,
The types, constituent ratios, binding modes, and the like of the constituent sugars can be specified by various types of chromatography, methylation analysis, nuclear magnetic resonance analysis, and the like. Specifically, the following specific methods are exemplified.

Measurement of molecular weight For example, Asahi Pack GF
It is measured by size exclusion chromatography using an A-7M column (7.6 × 500 mm, Asahi Kasei). That is, a polysaccharide peak eluted at a flow rate of 0.4 mL / min is detected with a differential refractometer (RI) using 0.1 M sodium chloride as a mobile phase, and the molecular weight is calculated based on pullulan of various molecular weight sizes.

Constituent sugars and their constituent ratios The above polysaccharides are treated at 100 ° C, 12 ° C with 0.05M trifluoroacetic acid (TFA).
Wako-pack WBT-
It is measured using a 130E column (7.8 × 300 mm, Wako Pure Chemical Industries, Ltd.). That is, using water as a mobile phase, a sugar peak eluted at a flow rate of 0.5 mL / min is detected with a differential refractometer (RI). In addition, gas chromatography analysis is performed as an alditol acetate derivative.

Nuclear Magnetic Resonance Analysis The nuclear magnetic resonance analysis is carried out by dissolving a sample in D2O and performing a 13C-NMR spectrum at 100 MHz and 30 ° C. using a JEOL Alpha 400 spectrometer. For details of other analytical methods, see the literature [Mazda (M. Ma
tsuda) et al .: Journal of the Fisheries Society of Japan, 58, 1735-1741 (1992)]
May be performed by the method described in (1).

[0011]

Embodiments of the present invention will be described below. The sulfated fructan of the present invention can be obtained by sulfated fructan produced by a marine microorganism using an ordinary method [for example, Takano (R.Ta
kano) et al .: Journal of Carbohydrate Chemistry, 15, 44
9-457 (1996)].

The fructan used as a raw material for the present invention is, for example, Pseudomonas SP IW-1 (Pseud
omonas sp. IW-1) (M. Matsuda: (2000)).

As a medium for culturing a microorganism for producing the raw material fructan used in the present invention, it is preferable to use a medium having good growth of the microorganism and good production of polysaccharides. Sexual Pseudomonas (Pseudom
Onas) is not particularly limited as long as it can grow a microorganism belonging to the present invention and contains an appropriate amount of a carbon source, a nitrogen source, an inorganic salt, and a trace nutrient that produce the polysaccharide.

[0014] Glucose, fructose, sucrose and the like are used as the carbon source. As the nitrogen source, synthetic compounds such as nitrates and ammonium salts, and natural organic substances such as polypeptone, yeast extract, meat extract, and amino acids are used. Phosphates, potassium salts, sodium salts and the like are used as the inorganic salts. If necessary, iron salts, calcium salts, manganese salts and the like can be added to the medium. In addition, yeast extract, various vitamins, and the like are used as trace nutrient sources.

The state of the medium may be either solid or liquid. In the case of liquid culture, high yields of fructan can be obtained by aeration or shaking culture.

The pH at the time of culturing is not particularly limited as long as the microorganism can grow and can produce the polysaccharide, but a pH of 6.5 to 7.5 is usually appropriate. Even at the culture temperature,
Although not particularly limited, usually 25 to 35 ° C is appropriate. As the culturing time, a period in which the production of the polysaccharide used in the present invention reaches the maximum is selected, and usually 3 to 4 days is appropriate.

As a method for collecting the fructan used in the present invention from the culture obtained by the above-mentioned culture method, a conventionally known method can be employed. For example, first,
After removing cells from the culture by centrifugation or filtration, an organic solvent such as methanol, ethanol, isopropanol, or acetone is added to the obtained culture solution to cause precipitation. Next, after dissolving the precipitate in water, dialysis is performed on the water, and the inner dialysis solution is dried by a method such as ventilation drying, hot air drying, spray drying, drum drying, reduced pressure drying, freeze drying, etc. Collect sugars.

In addition to the above-mentioned collecting method, a method may be adopted in which components other than polysaccharides are removed from the culture solution by ultrafiltration, and the obtained concentrated solution is subjected to the above-mentioned drying step.
Further, if necessary, a highly purified product can be obtained by purifying the polysaccharide according to a general purification method. As the purification method, various column chromatography such as ion exchange and gel filtration, precipitation and salting out with a quaternary ammonium salt, precipitation with an organic solvent, and the like are employed.

Further, the fructan used as a raw material of the present invention, which is collected from the culture solution by the above-described microbial culture, is characterized in that it has a levan-type structure consisting of beta (2-6) linkage of D-fructose. .

The number of repetitions of the D-fructose unit of the present fructan is not particularly limited, but is usually about 3,000 to 4,000.

[0021] The novel sulfated fructan according to claim 1 can be obtained by sulfating the above-mentioned fructan by a conventional method.

The number of repeating D-fructose units of the present sulfated fructan is not particularly limited, but is usually about 300 to 400.

The sulfated fructan of the present invention may be in the form of a pharmacologically acceptable salt. Such salts include alkali metals such as sodium and potassium salts,
Examples thereof include calcium salts and aluminum salts, but are not limited thereto.

The sulphoxides thus obtained exhibit excellent antiviral activity and, in addition to agents for preventing and treating infections in the fields of medicines and cosmetics, also in the field of agriculture since the raw materials are natural polysaccharides. The use as is possible. As described below, the antiviral activity of this substance is determined by, for example, allowing the sulfated fructan to act on RPMI8226 cultured cells infected with Herpes simplex virus and measuring the cell number after 3 to 4 days of culture. Antiviral activity can be measured.

[0025]

EXAMPLES Hereinafter, the present invention will be described in more detail by reference examples, examples, and test examples, but the present invention is not limited thereto. The raw material fructan used in the present invention is described in the above-mentioned Mazda [M. Matsuda: (2000)], but the production method will be described in more detail by reference examples.

Reference Example A medium prepared from seawater having a composition of peptone 0.5% and yeast extract 0.1% was sterilized in an autoclave at 121 ° C. for 20 minutes. Pseudomonas sp. (10 mL), and cultured with shaking at 28 ° C. for 72 hours. Then
The preculture was inoculated into the above sterile medium (200 mL) containing 3% sucrose in a 500 mL Erlenmeyer flask, and cultured at 28 ° C. for 72 hours with shaking. After the culture, the culture-finished solution was centrifuged to remove the cells, and twice the amount of ethanol was added to the supernatant to obtain a white precipitate. The precipitate was collected, dissolved in water (200 mL), and a 5% cetyltrimethylammonium bromide aqueous solution was gradually added until no new precipitate was formed. Was precipitated. After removing this complex by filtration, potassium thiocyanate was added to remove the remaining cetyltrimethylammonium bromide as a precipitate,
To this solution, twice the amount of ethanol was added to precipitate the polysaccharide. The obtained precipitate was dissolved in water, dialyzed, and freeze-dried to obtain a polysaccharide (about 2 g / L of medium).

The polysaccharide thus obtained was checked for homogeneity by using cellulose acetate membrane electrophoresis, and D-fructose was confirmed to be β-fructose by sugar composition analysis and nuclear magnetic resonance (NMR) analysis. (2-6) It was confirmed that the levan type was bound [Mazda (M.Ma
tsuda): (2000)]. FIG. 1 shows a 13C-NMR analysis diagram.

Example The fructan obtained in Reference Example was
After dissolving in 50 mL of N, N-dimethylformamide (DMF),
Dicyclohexylcarbodiimide (DCC) -DM under nitrogen stream
F solution (3 g / 40 mL), followed by sulfuric acid-DMF solution (0.2 mL / 5 mL)
Was added, and the mixture was stirred at 4 ° C. for 3 hours to perform a sulfation reaction. After cooling by adding ice, the reaction was stopped by neutralization with a 0.5 M sodium hydroxide solution, and dialysis was performed. After the precipitate generated during the dialysis was removed by filtration (Advantech No. 5B, Celite 503 filter aid), the filtrate was dialyzed and freeze-dried to obtain a sulfated fructan. The obtained sulfated fructan was confirmed to be the substance of the present invention by component analysis, nuclear magnetic resonance analysis and the like. FIG. 2 shows a nuclear magnetic resonance analysis diagram.

Properties of sulfated fructans Determination of molecular weight
The relative average molecular weight of the sulfated fructan was confirmed by comparing the elution position in GPC-HPLC with pullulan of various molecular weight sizes using an Asahi Pack GFA-7M column (Asahi Kasei). The eluate used was 0.1 M sodium chloride (flow rate: 0.4 mL / min), and the detection was performed with an RI detector. Figure of the elution pattern
See Figure 3.

The molecular weight was changed from 7.0 × 105 of the raw material to 5.6 × 104 of the product by the sulfation of the raw material fractan, indicating that the glycoside bond of the raw material fractan was unstable with respect to the present sulfation reaction. Was.

Analysis of Sulfate Group Content The sulfate group was quantified by separating sulfate ions released after acid hydrolysis with Shimpak I.
Using C-A1 column and CDD-6A electric conductivity detector (Shimadzu)
Measured by HPLC.

As a result, the number of sulfate groups per D-fructose unit was 0 for the raw material fractane and 3 for the sulfated fractane of the present invention. Sulfation was noted.

Infrared absorption spectrum (IR) The infrared absorption spectrum of the solid sample was measured by a potassium bromide tablet method using a JASCO A-302 type infrared spectrometer. The result is shown in FIG.

FIG. 4 is an IR spectrum of the complete O-sulfated fructan of the present invention prepared in the examples. The strong absorption of 1250 Kaiser is attributed to sulfate. For the assignment of IR spectra, reference was made to literature [Atsuya Harada et al .: Integrated Polysaccharide Science, Above, Kodansha, 297-414 (1973)].

Test Example Measurement of antiviral activity Human herpes simplex virus type 1 (Herpessimplex virus typ)
e 1, abbreviated as HSV-1), human parainfluenza virus type 2 (abbreviated as Parainfluenza virus type 2, PFluV-2), human respiratory virus type A (Respiratory s
yncytial virus type A, abbreviated as RSV-A), human acquired immunodeficiency virus type 1 (Human immunodeficiency v
The antiviral activity was tested for irus type 1, abbreviated as HIV-1) and human influenza virus type A, abbreviated as FluV-A. The host cells were used to grow RPMI8226 cells cultured in RPMI1640 medium containing 2% fetal calf serum (FCS) for HSV-1 and RPMI1640 containing 2% FCS.
HMV-II cells cultured in culture medium were grown for growth of PFluV-2, G-HeLa cells grown in G1-Dulbecco medium containing 2% FCS were grown for growth of RSV-A, and RPMI1640 medium containing 10% FCS. MDCK cells
For growth of FluV-A, cultured in RPMI1640 medium containing 10% FCS
MT-4 cells were used for HIV-1 propagation, respectively. Virus-infected cells (1 × 104 / well) were added to 96-well microtiter plates along with samples at various concentrations. Simultaneously, non-virus-infected cells were cultured with the sample in order to determine the cytotoxicity of the sample to host cells. 35 ℃ in 5% CO2 incubator, HSV-1
96 hours, PFluv-2 120 hours, RSV 168 hours, HIV-1 12
After culturing FluV-A for 120 hours and FluV-A for 120 hours, the number of viable cells was measured by the MTT method. [R. Pauwels et al .: Jour
nal of Virological Methods, 20, 309-321 (1988)]. The antiviral activity was expressed by a sample concentration (EC50) that protects against 50% of cytotoxicity due to virus infection, and the cytotoxicity was expressed by a 50% cytotoxicity concentration (CC50) of the sample. The results of the antiviral activity and cytotoxicity tests are shown in Table 1 below.

[0036]

[Table 1]

From these results, it can be seen that the compound according to the present invention has antiviral activity. Also 100 cytotoxic
The value was more than microgram / mL. As described above, the compound according to the present invention has a low cytotoxicity and a wide range of antiviral activity, and is therefore effective as a drug highly safe for living organisms.

[0038]

The substance according to the present invention is a novel substance, has an antiviral effect, is easy to manufacture, and can be supplied at low cost.

[Brief description of the drawings]

FIG. 1 is a 13 C-NMR spectrum of a fructan.

FIG. 2 is a 13C-NMR spectrum of the sulfated fructan of the present invention.

FIG. 3 is a GPC-HPLC elution diagram of a sulfated fructan of the present invention.

FIG. 4 is an infrared absorption spectrum diagram of the sulfated fructan of the present invention.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:38) F-term (Reference) 4B064 AF11 CA02 CE04 CE06 CE17 DA01 4C086 AA01 AA02 EA26 MA01 MA04 NA14 ZB33 4C090 AA04 AA05 AA09 BA42 BB40 BB52 BB63 BB65 BB95 BC20 CA42 DA23

Claims (3)

[Claims] 1. An antiviral agent comprising a sulfated fructan or a pharmaceutically acceptable salt thereof. 2. A method for producing a sulfated fructan, which comprises sulfated fructan to produce the sulfated polysaccharide according to claim 1, and collecting the sulfated polysaccharide. 3. The sulfated fructan according to claim 1, wherein the fructan is produced by Pseudomonas sp. IW-1.