JPS63157994A - Production of polysaccharide nsg-1 - Google Patents

Abstract

PURPOSE:To obtain the titled compound useful as antitumor agent, etc., in high purity, by culturing a microbial strain capable of producing polysaccharide NSG-1, incubating the proliferated cell in a liquid containing a carbohydrate to produce polysaccharide NSG-1 and separating the product from the reaction liquid. CONSTITUTION:A microbial strain capable of producing polysaccharide NSG-1 [e.g. Sclerotinia sclerotiorum (IFO 9353), etc.] is cultured in a medium and the obtained primary seed strain is inoculated and cultured in a medium to obtain secondary seed strain. The strain is added to a jar fermenter, cultured in a mass and filtered to obtain cultured cells. The cell is filled together with a liquid containing a carbohydrate such as glucose, etc., into a jar fermenter and incubated. The objective polysaccharide NSG-1 produced in the reaction liquid is separated therefrom.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a method for producing polysaccharide NSG-1.

More specifically, the present invention provides polysaccharide NSG with antitumor activity.
The present invention relates to a method for manufacturing -1.

In the present invention, the polysaccharide NSG-1 is directly biosynthesized from carbohydrates by incubation, so it is possible to produce a large amount of extremely pure antitumor active polysaccharide N'5G-1, which will greatly contribute to the medical world. There is something.

(Prior art) Sclerotiniaceae
) The sclerotia formed on the roots and stems of plants in the Brassicaceae family by mushrooms belonging to the genus A. It is known that polysaccharides containing one D-glucopyranosyl residue are included (Kitahara Toki 1 University Faculty of Agriculture Research Report 8,100 (1957), 9,139 (1)
958), Journal of the Japanese Society of Agricultural Chemistry 35, 468 (1961
)) Furthermore, Sclerotinia libertiana (Sc
mycelia of lerotinia lj, bertiana) (Agric, Boil.

Chem, 44(2), 353-359.198
0) is sucrose, NaNO3, MgSO4・7H2
0, KH2PO4, 24 in medium containing yeast extract.
It is obtained by the methanol precipitation method from the viscous culture solution after shaking culture at ~25°C for several days.

The above polysaccharide is usually called scleroglucan (Carbohydrate Chemistry P95 published by Tokyo Kagaku Dojin on July 1, 1986) and has antitumor activity (Pharmacia Review Nα6 P12
1 Pharmaceutical Society of Japan).

(Problems to be Solved by the Invention) In the conventional method of separating and collecting polysaccharides from the sclerotia and the culture solution, many complicated purification methods are required to remove various components transferred from the sclerotia themselves and the culture solution. However, many problems remain to be solved in practical terms.

(Means for Solving the Problems) As a result of intensive research on how to easily obtain the above-mentioned useful polysaccharide, the inventors discovered that
Sclerothiolirum (Sclerotinia) is an institutionally preserved strain belonging to the genus Sclerotiniaceae.
tinia sclerotiorum) IFO93
95 cultured mycelia were added to a carbohydrate solution lacking nitrogen sources, phosphorus, potassium, magnesium, and other nutrients necessary for the cultivation of normal microorganisms, and whose pH was in the acidic range, and the mycelia were added to a carbohydrate solution that enzymatically acted on the carbohydrates. They found that a large amount of polysaccharide NSG-1 was produced in the solution when the hyphae were removed, and they were able to obtain almost pure polysaccharide NSG-1 by simply adding alcohol to the solution from which the hyphae had been removed. The antitumor activity can be confirmed by
Physicochemical tests revealed that the structure of polysaccharide NSG-1 was determined from the β-(1→3)-linked D-glucopyranosyl residue 3 obtained from the culture solution of Sufflerotinia libertiana.
Its structure is different from the branched polysaccharide, which has one D-glucopyranosyl residue for every two D-glucopyranosyl residues, and one D-glucopyranosyl residue for every two β-(l→3)-bonded D-glucopyranosyl residues. It was confirmed that the polysaccharide had a branched structure with one D-glucopyranosyl residue.

Acanthaceae (S) used for production of polysaccharide NSG-1
clerotiniaceae) were classified as Ascomycetes by Mutsuya Imazeki and Tsugube Hongo, "Illustrated Encyclopedia of Primary Colors of Japanese Fungi" (published by Yokusha in 1978).
es), the Helotiales order, and the Sclerotiniaceae family, and have the characteristics described in the encyclopedia, and those that have been isolated from nature, purified, and maintained through subculture, or These are strains that have the ability to produce polysaccharide NSG-1 by the method of the present invention, such as strains stored in various preservation institutions.

As mentioned above, this invention
The method is characterized in that poly@NSG-1 is produced by enzymatically acting on carbohydrates such as glucose by the hyphae of a strain that produces the polysaccharide NSG-1 belonging to the group Rotiniaceae), and the product is separated and collected. However, it has not been previously known that polysaccharides can be produced by such a method using a strain belonging to the Sclerotiniaceae family.

Now, as a representative example of a strain belonging to the family Sclerotiniaeeae that can be used in this invention, the preserved strain Sclerotinia sfulerothiorirum (Sclerotiniae), which successfully produced the polysaccharide NSG-1, is used.
rotinia Sclerotiorum) IFO9
Polysaccharide NSG-1 produced by No. 395 will be explained.

Sclerotinia sclerotiorum
) Two agar medium sections (5 x 5 x 2 mm) containing mycelium from the preservation slope of IFO9395 were added with yeast extract 0, for example.
．． Medium 10 containing 3%, peptone 1%, glucose 2%
The mycelia were inoculated into 0 and cultured with shaking at 28° C. for 4 days, and the culture was centrifuged to separate 4 mycelia. After thoroughly washing the mycelium to remove medium components, it is centrifuged again and the entire volume is transferred to a flask and water is added to make a total volume of 400 J to form a uniform suspension. Separately prepare 40 mQ of this suspension. Glucose 5
g, containing 0.5 g of citric acid and adjusting the pH to 2.5 to 6.0 in advance.
Carbohydrate solution adjusted (by NaOH) to the test value of 60+n
Dispense the amount addressed to Q into 8 flasks and incubate at 28℃ for 4 hours.
When kept under shaking or stirring for days.

Polysaccharides are produced in solution corresponding to each preparation P)l as shown in Table 1.

That is, after holding at 28° C. for 4 days, the contents are centrifuged to separate the mycelium and a separated liquid, and when ethanol 50+++Q is added to the separated liquid, a precipitate is produced. This precipitate is washed with ethanol and then vacuum dried to obtain a white flocculent material.

As described below, the white flocculent substance obtained above is a β-glucan whose only constituent sugar is glucose, and is a polysaccharide NSG-1 (hereinafter often abbreviated as polysaccharide) that has antitumor activity.
It is.

Table 1 Initial pH Final pH Polysaccharide (mg/100mQ separated liquid) 2.
5 2.6 25.93.0
2.9 109.73.5 2.
7 213.34.0 3.0
168.14.5 3.3
159.35.0 3.5
59.85.5 4.0 12
．． 16.0 4.7 32.2 From Table 1, when the initial pH is set to 2.5 to 6.0, there is a reserve amount of production, but polysaccharide production is observed in all cases, and the initial pH is set to 3. It is recognized that when the value is set to .0 to 4.5, the amount produced is significant, and a range of 3.5 to 4.5 is particularly preferable.

As mentioned above, this invention does not contain nitrogen sources or inorganic components such as phosphorus, potassium, and magnesium, which are normally essential for culturing microorganisms, and contains only glucose as a carbohydrate, and a small amount of citric acid for pH adjustment. Since polysaccharide is produced from glucose by adding mycelium to a carbohydrate solution containing acid and NaOH, the production mechanism is clearly different from exopolysaccharide production by culture, and the mycelium is This can be recognized as an enzymatic action on the glucose used as a carbohydrate.

Thus, in this invention, soufflerotinia (Scl
Mycelia obtained by culturing bacteria belonging to the genus Erotinia are enzymatically applied to carbohydrates, and in addition to the above-mentioned glucose, various sugars can be used, such as arabinose, xylose, fructose, Monosaccharides or trisaccharides such as mannose, galactose, maltose, sucrose, melibiose, lactose, raffinose, various oligosaccharides represented by fructooligo or galactooligo, polymeric substances such as starch, dextrin, gum arabic, etc., and their hydration Decomposition products, other polyhydric alcohols such as glycerin and mannitol, or depending on the use, sucrose, glucose, fructose,
Examples include sweet potato or sugar beet molasses containing raffinose etc., but preferably glucose, fructose,
Sucrose and the like are preferably used, and the carbohydrates listed above may be used alone or in combination of two or more.

To adjust the initial pH of a carbohydrate solution, organic acids with strong buffering properties such as citric acid, tartaric acid, and lactic acid are usually used, but hydrochloric acid,
In addition to mineral acids such as sulfuric acid, buffers can also be used. Furthermore, as shown in Table 1, a decrease in pH is observed over time, so in order to maintain the initial pH, the pH may be adjusted with the above-mentioned acids midway through the process.

Contact between sugar and mycelium by shaking or stirring is 20 to 3
It is carried out at 0°C for 2 to 8 days, but 28±1°C for 3±1 days is more preferable.

The mycelium of a strain belonging to the genus Sclerotinia may be used immediately after being isolated after culturing, or it may be stored frozen and thawed each time it is used. Furthermore, used mycelium that has been used for polysaccharide production can be added to a newly prepared carbohydrate solution and used for polysaccharide production, and although the amount of polysaccharide produced will decrease slightly with repeated use, the same mycelium can be used at least three times. can be used for polysaccharide production.

Next, in Table 1, polysaccharide NS obtained at initial pH 3.5
The results of analyzing the composition and sugar structure of G-1 are as follows.

(1) Composition analysis, total sugar content, total nitrogen, ash content of 95% or more, below detection limit, below detection limit, however, total sugar content was determined by the phenol-sulfuric acid method and is expressed as glucose.

(2) As a result of reducing the hydrolyzate of constituent sugar, trifluoroacetic acid (CF3COOH) with sodium borohydride (NaBH), and analyzing its alditol acetate derivative by gas chromatography, only glucose was clearly detected. However, other sugars such as fucose, xylose, mannose, and galactose are not detected.

(3) Binding mode of constituent sugars The result of methylation analysis using the Hakomori method is 2,3,4.6-titramethyl-〇-D-glycidol: 2,4.6-
Trimethyl-〇-D-glycidol: 2.4-dimethyl-〇-D-glycitol is 1.0: 0.9-1.1
: Since it is obtained with a ratio of 0.9 to 11, it is known that the glucan has a branch of one 1→6 bond D-glucopyranosyl residue for every two 1→3 bond D-glucopyranosyl residues.

(4) Smith decomposition products Glucose and glycerin were detected as products by complete Smith decomposition, and glycerin was detected from the external dialysis fluid of the relaxed Smith decomposition product, and only glucose was detected from the hydrolyzed internal fluid.1 It is known that this glucan has a structure of →3 bonds and a branch at C-6.

(5) Infrared absorption spectrum The results measured by the KBr method using a Hitachi 215 model infrared spectrophotometer are shown in Figure 1.
Since absorption (P) characteristic of glucoside bond orientation is observed, it is recognized that it is a β-glucoside bond structure.

(6) 13C-NMR spectrum dissolved in heavy dimedimethyl sulfoxide MSO-d, ) and measured by JEOL-FX200 spectrometer.
The results measured at 0°C are shown in Figure 2, with a δ value of 6 gpp.
The signal S□ includes the assignment of C-6 carbon included in the β (1 → 6) bond in the m region, and the β (1 → 3
) Signal S2 attributed to C-3 carbon included in the bond
Since the signal S3 attributed to the C-1 carbon of the β-bond with a δ value of 1103 pp is characteristically recognized, and furthermore, the signal S2 shows two peaks, indicating that there are two β (1 → 3) bonds. A β-glucan structure with one β(1→6) is known.

From the results of the above two studies, this polysaccharide has β-1,3-linked D
It is recognized that it is β-glucan, which has a main chain of -glucopyranosyl residues and one β-1,6-linked glucopyranosyl residue as a branched structure for every two D-glucopyranosyl residues, and has the following properties. show.

(a) Elemental analysis values, etc. C: 42.2-44.0%, H: 5.9-6.2%
, N: Below the quantification limit, halogen and sulfur are not quantified.

(b) Molecular weight 0.2 mol NaOH/8 mol urea equilibrium Sepharose CL
The molecular weight distribution range was 10G by gel filtration chromatography using a -4B (Pharmacia Japan) column.
~io'.

(c) Melting point It undergoes blackening thermal decomposition at about 230°C.

(d) Specific rotation [α
]D indicates 10±56.

(e) Solubility: Easily soluble in water, alkali, and dimethyl sulfoxide (DMSO), but insoluble in organic solvents such as ethyl alcohol, methyl alcohol, ether, and acetone.

(f) The color reaction Molisch reaction, Anthrone sulfuric acid reaction, and phenol sulfuric acid reaction were all positive, and the ninhydrin reaction and Biuret reaction were both negative, indicating the presence of proteins and peptides).

(g) Basic, acidic, and neutral aqueous solutions A 1% aqueous solution exhibits a neutral range (pH 6 to 6.5).

The polysaccharide obtained by the method of the present invention is a β-glucan having the above-mentioned physical and chemical properties and exhibits antitumor activity as described below, so it is useful for medicinal purposes, and the manufacturing method itself is extremely simple. According to this invention, a polysaccharide with antitumor activity is provided at a low cost, and this polysaccharide can be used for intraperitoneal administration, intravenous administration, intratumoral administration, and oral administration, and can be expected to be used in various ways. It is.

The production method of the present invention and the pharmacological action of the polysaccharide obtained thereby will be specifically explained below.

Example 1 Sclerotinia sclerotiorum
) 5 x 5 x 2 m from subculture medium of IFO9395
Two sections of M.m were inoculated into 50 mQ of an autoclaved medium containing 0.3% yeast extract, 1% polypeptone, and 2% glucose, and were left to stand at 25° C. for one week to serve as a primary inoculum. Next, 5tQ of primary inoculum was inoculated into 100mQ of autoclaved medium having the same composition as above, and 25
℃ for 4 days with shaking and use this as a secondary inoculum.Next, medium 6Q having the same composition as above was taken into a jar fermenter with a capacity of 10 fl, and 2 wood portions (20
0 mQ) and cultured at 28°C for 3 days with an aeration rate of 3Q/mjn, and the mycelium dry weight was 4 per 100 mf of culture solution.
23.2 mg of culture was obtained.

The entire content was suction filtered through a filter paper (Nn2), thoroughly washed with water, water was removed, and the entire content was mixed with 5% glucose and 0.5% citric acid, which was placed in a jar fermentor with a capacity of 10Q. % and adjusted to pH 3.5 with 5N-NaOH, and treated at aeration rate of 3Q/min, stirring number of 25 rpm, and temperature of 28°C for 40 hours, and then filtered the contents with filter paper (Nα2). After suction filtration and washing with cold water, add 5.8Q of ethanol to 5.8Q of filtrate to form a precipitate, filter through a 50-100 mesh nylon net, and dissolve the obtained precipitate in 1μ of warm water at 80-90℃. death,
After cooling, add ethanol IQ to reduce the resulting precipitate to 80~
In addition to hot water IQ at 90°C, it was dissolved under ultrasonication at 20KHz, 140υ, for 1 hour, and then heated at 3000r, p.
The entire amount of the separated liquid obtained by centrifugation at 5 minutes was freeze-dried to obtain white cotton-like polysaccharide 1.972E.

This polysaccharide exhibited the physical and chemical properties described above.

Example 2 In Example 1, the wet mycelium that had been filtered after the carbohydrate treatment was added to the ILQ capacity jar fermentor containing 6Q of the carbohydrate solution shown in Example 1, and the aeration rate was 3 Q/min.
After processing for 69 hours at a stirring number of 250 r, p, m and a temperature of 28°C, the contents were suction filtered using filter paper (Nα2), washed with cold water, and 5.9 fl of ethanol was added to the filtrate 5.9Q to form a precipitate. Similarly, the precipitate obtained by filtration with a nylon net was dissolved in 1 ρ of warm water at 80-90°C, and after cooling, ethanol IQ was added. The mixture was dissolved and treated in the same manner as in Example 1 to obtain 1.595 g of white flocculent polysaccharide. The polysaccharide obtained here also showed the same properties as in Example 1.

Example 3 The same procedure as in Example 2 was carried out, except that the mycelium was filtered after the carbohydrate treatment and then frozen and preserved, and the carbohydrate treatment time was changed to 120 hours. 0.830 g of white flocculent polysaccharide was obtained.

Test Example 1 The polysaccharide obtained in Example 1 was tested for its pharmacological action.

IRC-strain 6-week-old mouse (male, weight 27-30 g) 1
5 x 10 cells of allografted Sarcoma 180 tumor cells were subcutaneously transplanted into the groin area of 0 animals as one group, and from the next day, the above polysaccharide dissolved in physiological saline was administered by various administration methods and routes, and 5 weeks after transplantation. The tumor was removed and its weight was measured, and a comparison was made with a control group to which only physiological saline was administered, and the results shown in Table 2 were obtained.

As is clear from Table 2, the polysaccharide of the present invention has a high inhibitory effect by any route of administration, and in particular, the inhibition rate of 50% or more was observed by oral administration, which is a major feature of the polysaccharide of the present invention.

Test Example 2 Regarding the polysaccharide obtained in Example 1, syngeneic transplanted tumor Meth
As a result of testing the activity against A fibrosarcoma and breast cancer MM46, as shown in Table 3, antitumor activity was also observed against syngeneic tumors.

119 = (Effects of the invention) When the method of this invention is used, it can be obtained very easily as a pure product without using complicated purification means, resulting in mass production at low cost, and the obtained polysaccharide has antitumor effects against various tumors. It exhibits activity and can be administered orally in addition to injection, so it can be used in various dosage forms and is extremely useful as a drug.

[Brief explanation of the drawing]

FIG. 1 shows the infrared absorption spectrum of the polysaccharide of the present invention, and FIG. 2 shows the "C-NMR spectrum" of the same polysaccharide in dimethyl sulfoxide.

Claims (1)

[Claims] Polysaccharide NSG-1-producing bacteria were cultured, and the resulting cultured cells were incubated in a carbohydrate-containing solution to produce polysaccharide NSG-1.
1. A method for producing polysaccharide NSG-1, which comprises producing polysaccharide NSG-1 and collecting polysaccharide NSG-1 from the reaction solution.