JPH0236235B2 - TATONSGG1NOSEIZOHOHO - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a method for producing polysaccharide NSG-1. More specifically, the present invention relates to a method for producing polysaccharide NSG-1 having antitumor activity. In the present invention, polysaccharide NSG-1 is biosynthesized directly from carbohydrates by incubation, so it is possible to produce a large amount of extremely pure anti-tumor active polysaccharide NSG-1, thereby contributing to the medical world. There is something big here. (Prior technology) Mushroom fungi belonging to the Sclerotiniaceae family have a skeleton of D-glucopyranosyl residues with β-(1→3) bonds in the sclerotia formed on the roots and stems of plants of the Brassicaceae family. It is known that polysaccharides with one D-glucopyranosyl residue bonded to C-6 for every three residues are included (Kitahara et al. Gifu University Faculty of Agriculture Research Report 8100 (1957),
9139 (1958), Journal of the Japanese Society of Agricultural Chemistry 35468 (1961), and UENO etc. Sclerotinia libertiana (Sclerotinia libertiana). ) Hyphae (Agric.)
Boil.Chem., 44(2), 353-359, 1980) is sucrose, NaNO ₃ , MgSO ₄ .7H ₂ O, KH ₂ PO ₄ ,
It is obtained by the methanol precipitation method from the viscous culture solution obtained after shaking culture at 24-25°C for several days in a medium containing yeast extract. The above polysaccharide is usually called scleroglucan (on the chemistry of carbohydrates, P95, published by Tokyo Kagaku Doujin 1986, 7, 1).
It has antitumor activity (Pharmacia Review)
No.6P121 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 polysaccharides, the inventors discovered that Sclerotinia sclerothiolium, an institutionally preserved strain belonging to the family Sclerotiniaceae, Cultured mycelia of Sclerotinia solerotiorum) IFO9395 were added to a carbohydrate solution lacking nitrogen sources, nutrients necessary for the cultivation of normal microorganisms such as phosphorus, potassium, and magnesium, and whose pH was in the acidic range. When exposed to water, a large amount of polysaccharide NSG appears in the solution.
Knowing that 1 is produced, almost pure polysaccharide NSG can be produced by simply adding alcohol to the solution after removing the hyphae.
The antitumor activity was confirmed in animal tests, and the structure of polysaccharide NSG-1 was confirmed by physicochemical tests.
(1 → 3) Bound D-glucopyranosyl residue 3
Its structure is different from that of the polysaccharide, which has a branched structure of one D-glucopyranosyl residue and one D-glucopyranosyl residue for each
→3) 1 D-glucopyranosyl residue 1 at C-6 for every 2 D-glucopyranosyl residues bonded
It was confirmed that the polysaccharide has a structure with 5 branches. Bacterial strains belonging to the Sclerotiniaceae family used to produce the polysaccharide NSG-1 are classified as Ascomycetes, Ascomycetes, and Ascomycetes, according to the "Illustrated Encyclopedia of Primary Colors of Japanese Fungi" co-authored by Mutsuya Imazeki and Jiro Hongo (published by Yokusha in 1978). Takeme (Helotiales),
It is classified into the family Sclerotiniaceae and has the characteristics described in the same encyclopedia.It is isolated from nature and has retained its characteristics through purification and subculturing, or preserved strains in various preservation institutions, etc. By the method of the invention, polysaccharide NSG-1
It is a strain that has the ability to produce. As described above, this invention produces polysaccharide NSG-1 by enzymatically acting on carbohydrates such as glucose with the hyphae of an NSG-1-producing Sclerotinia strain belonging to the Sclerotiniaceae family. However, it has not been known to date that polysaccharides can be produced by such a method using bacterial strains belonging to the Sclerotiniaceae family. Now, as a representative example of a strain belonging to the Sclerotiniaceae family that can be used in this invention, a conserved strain of the genus Sclerotinia that has successfully produced the polysaccharide NSG-1 is Sclerotinia Sclerotiorum. ) Polysaccharide by IFO9395
Let me explain about NSG-1. Agar medium section (5 x 5 x 2 mm) containing mycelium from a preserved slope of Sclerotinia sclerotiorum IFO9395
For example, add 2 pieces of yeast extract 0.3%, Beptone 1
%, inoculated into 100 ml of medium containing 2% glucose.
Culture with shaking at 28°C for 4 days, and centrifuge the culture to separate mycelia. After thoroughly washing the mycelium to remove medium components, centrifuge it again, transfer the entire volume to a flask, and add water to make a total volume of 400 ml.
Separately prepare 40 ml of a homogeneous suspension. 60 ml of a carbohydrate solution containing 5 g of glucose, 0.5 g of citric acid, and whose pH has been adjusted to a test value of 2.5 to 6.0 (by NaOH).
When the solution is poured into 8 flasks and kept at 28°C for 4 days with shaking or stirring, polysaccharides are produced in the solution corresponding to each adjusted pH as shown in Table 1. That is, after holding at 28°C for 4 days, the contents were centrifuged to separate the mycelium and the separated liquid,
Adding 50 ml of ethanol to the separated solution will produce a precipitate. This precipitate is washed with ethanol and then vacuum dried to obtain a white flocculent material. As will be described later, the white flocculent substance obtained above is a polysaccharide NSG-1 (hereinafter often abbreviated as polysaccharide), which is β-glucan whose constituent sugar is glucose only, and which has antitumor activity.

[Table] From Table 1, when the initial PH is set to 2.5 to 6.0, polysaccharide production is observed in all cases, although the amount produced is small, and when the initial PH is set to 3.0 to 4.5, the amount produced is significant. It is recognized that a range of 3.4 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. acid and
Since mycelium is added to a carbohydrate solution containing NaOH to produce polysaccharides from glucose,
The production mechanism is clearly different from exopolysaccharide production by culture, and it can be recognized that the mycelium acts enzymatically on glucose used as a carbohydrate. As described above, in this invention, mycelium obtained by culturing bacteria belonging to the genus Sclerotinia is made to act enzymatically on carbohydrates, and various sugars are used as the carbohydrates in addition to the above-mentioned glucose. For example, monosaccharides or trisaccharides such as arabinose, xylose, fructose, mannose, galactose, maltose, sucrose, melibiose, lactose, and raffinose, various oligosaccharides such as fructooligo or galactooligo, starch, dextrin, and gum arabic. and their hydrolysates, other polyhydric alcohols such as glycerin and mannitol, and depending on the use, sweet potato or sugar beet molasses containing sucrose, glucose, fructose, raffinose, etc. However, it is preferable to use glucose, fructose, sucrose, etc. The above-mentioned carbohydrates may be used alone or in combination of two or more. To adjust the initial pH of a carbohydrate solution, citric acid,
Organic acids with strong buffering properties such as tartaric acid and lactic acid are used, but mineral acids such as hydrochloric acid and sulfuric acid as well as buffer solutions can also be used. Furthermore, as shown in Table 1, since the pH decreases over time, the pH may be adjusted with the above-mentioned acids midway through the process to maintain the initial pH. Contact between sugar and mycelium by shaking or stirring
It is carried out for 2 to 8 days in the range of 20 to 30℃, but 28±1℃, 3
±1 day 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 then thawed each time it is used. Furthermore, the 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 carbohydrate production will decrease slightly with repeated use, at least the same mycelium can be used for three times.
It can be used for multiple polysaccharide production. Next, in Table 1, the polysaccharide obtained at initial pH 3.5
The results of analysis of the composition and sugar structure of NSG-1 are as follows. (1) Composition analysis, Total sugar Total nitrogen Ash 95% or more Below detection limit Below detection limit However, total sugar content is determined by the phenol sulfuric acid method and is expressed as glucose. (2) As a result of reducing the hydrolyzate of the constituent sugar, trifluoroacetic acid (CF ₃ COOH) with sodium borohydride (NaBH ₄ ), and analyzing the alditol acetate derivative by gas chromatography, only glucose was clearly identified. , and other sugars such as fucose, xylose, mannose, and galactose are not detected. (3) Binding mode of constituent sugars The results of methylation analysis using the Hakomori method are 2, 3,
4,6-tetramethyl-OD-glycitol:2,4,6-trimethyl-OD-glycitol:2,4-dimethyl-OD-glycitol ratio of 1.0:0.9 to 1.1:0.9 to 1.1 It is known that this glucan has a branch of one 1→6-bonded D-glucopyranosyl residue for every two 1→3-bonded 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. 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, with a wave number of 880cm.
Since absorption (P) characteristic of β-glycosidic bond orientation is observed at ^-1 , it is recognized that it is a β-glycosidic bond structure. (6) ¹³ C-NMR spectrum The results of dissolving in deuterated dimethyl sulfoxide (DMSC-d ₆ ) and measuring with a JEOL-FX200 spectrometer at 60°C are shown in Figure 2.
C included in β (1 → 6) bond in the δ value range of 68 ppm
- Signal S ₁ including carbon assignment of 6 and δ value
C-3 included in β (1 → 3) bond in the 86ppm range
Signal S ₂ attributed to carbon and δ value 103ppm
The signal S ₃ assigned to the C-1 carbon of the β-bond in the region is characteristically recognized, and furthermore, the signal S ₂ is
Since it shows two peaks, it is known that the β-glucan structure has two β(1→3) bonds and one β(1→6) bond. From the above study results, this polysaccharide has β-1,3-linked D-glucopyranosyl residues as its main chain and this D
- Recognized as a β-glucan with a branched structure of one β-1,6-linked glucopyranosyl residue for every two glucopyranosyl residues, and exhibits the following properties. (a) Elemental analysis values, etc. C: 42.2-44.0%, H: 5.9-6.2%, N: below the limit of quantification, halogen and sulfur are not quantified. (b) Molecular weight The molecular weight distribution range is 10 ⁶ to 10 ⁷ as determined by gel filtration chromatography using a 0.2 mol NaOH/8 mol urea equilibrium Sepharose CL-4B (Pharmacia Japan) column. (c) Melting point: Blackens and thermally decomposes at approximately 230°C. (d) Specific rotation [α] _D at a concentration of 0.1 g/100 ml in water at 20°C
shows 10±5°. (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) Color reaction Moritsch reaction, Anthrone sulfuric acid reaction, and phenol sulfuric acid reaction are all positive, and both the ninhydrin reaction and Buillet's reaction are negative, which does not indicate the presence of proteins or peptides. (g) Water-soluble basic, acidic, and neutral 1% aqueous solution exhibits a neutral range (PH6 to 6.5). The polysaccharide obtained by the method of this invention has the above-mentioned physical properties,
Since it is a β-glucan with various chemical properties and exhibits antitumor activity as described below, it is useful medicinally, and the manufacturing method itself is extremely simple. Therefore, according to the present invention, antitumor active polysaccharides can be produced at low cost. This polysaccharide can be used for intraperitoneal administration, intravenous administration, intratumoral administration, and oral administration, and can be expected to be used in various other ways. The production method of the present invention and the pharmacological action of the polysaccharide obtained thereby will be specifically explained below. Example 1 Two 5 x 5 x 2 mm sections from the subculture medium of Sclerotinia sclerotiorum IFO9395 were extracted with yeast extract.
25 inoculated into 50 ml of autoclaved medium containing 0.3% polypeptone, 1% polypeptone, and 2% glucose.
C. for 1 week and use this as a primary inoculum.
Then autoclaved medium with the same composition as above
Inoculate 5 ml of the primary inoculum into 100 ml, culture with shaking at 25°C for 4 days, and use this as the secondary inoculum.Next, take medium 6 with the same composition as above into a 10-volume jar fermenter, and inoculate it with the above secondary inoculum. Add two bottles (200 ml) of the inoculum and culture at 28°C for 3 days with an aeration rate of 3/min.
423.2 mg of culture was obtained. The entire content was suction filtered through filter paper (No. 2), thoroughly washed with water, water was removed, and the entire amount was mixed with 5% glucose, citric acid, and
Add to carbohydrate solution 6 containing 0.5% and adjusted to pH 3.5 with 5N-NaOH, aeration rate 3/min, stirring number
After processing for 40 hours at 250 rpm and a temperature of 28°C, the contents were suction filtered using filter paper (No. 2), washed with cold water, and ethanol 5.8 was added to the filtrate to form a precipitate. Dissolve the precipitate obtained by filtration with a net in 1 part of warm water at 80 to 90°C, and after cooling, add 1 part to ethanol. Add the resulting precipitate to 1 part of hot water at 80 to 90°C, and heat at 20KHz, 140W,
Dissolved under ultrasonication for 1 hour.
The entire amount of the separated liquid obtained by centrifugation at 3000 rpm for 5 minutes was freeze-dried to obtain 1.972 g of white cotton-like polysaccharide. This polysaccharide exhibited the physical and chemical properties described above. Example 2 In Example 1, the wet mycelium that had been filtered after the sugar treatment was added to a 10-capacity jar fermenter containing the sugar solution 6 shown in Example 1, and the mixture was stirred at an aeration rate of 3/min. After processing at several 250 rpm for 69 hours at a temperature of 28°C, the contents were suction filtered using filter paper (No. 2), washed with cold water, 5.9% of ethanol was added to 5.9% of the filtrate to form a precipitate, and filtered using a nylon net as above. The precipitate obtained was dissolved in 1 part of warm water at 80 to 90°C, and after cooling, 1 part of ethanol was added. It was 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 cells were treated in the same manner as in Example 2, 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 27-30g)
Allograft tumor sarcoma 180 in groups of 10 animals
5 x 10 ⁶ tumor cells were transplanted subcutaneously in the groin area, 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. A comparison was made with a control group to which only physiological saline was administered, and the results shown in Table 2 were obtained.

【table】

[Table] As is clear from Table 2, the polysaccharide of this 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 this invention. . Test Example 2 Syngeneic transplantation of the polysaccharide obtained in Example 1
As a result of testing the activity against Meth A fibrosarcoma and breast cancer MM46, as shown in Table 3, antitumor activity was also observed against syngeneic tumors.

[Table] (Effects of the invention) When the method of this invention is used, it is possible to obtain a pure product very easily 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, and can be used in various dosage forms and is extremely useful as a drug.

[Brief explanation of drawings]

FIG. 1 shows the infrared absorption spectrum of the polysaccharide of the present invention, and FIG. 2 shows the ¹³ C-NMR spectrum of the same polysaccharide in dimethyl sulfoxide.

Claims (1)

[Scope of Claims] 1. Belongs to the genus Sclerotinia and has the following physicochemical properties, and has a β-1,3 bond D
- Cultivate a polysaccharide NSG-1 producing strain that has glucopyranosyl residues as a main chain and one β-1,6-linked glucopyranosyl residue as a branched structure for every two D-glucopyranosyl residues, and the resulting culture. Incubating the bacterial cells in a carbohydrate-containing solution,
Polysaccharide NSG-1 is produced, and polysaccharide is extracted from the reaction solution.
Polysaccharide NSG characterized by collecting NSG-1
-1 manufacturing method. (1) Infrared absorption spectrum (KBr method) There is an absorption (P) characteristic of β-glycosidic bond orientation at a wave number of 880 cm ^-1 . (2) ¹³ C-NMR spectrum (in DMSO-d ₆ ) Signal S ₁ including the assignment of C-6 carbon included in the β (1 → 6) bond in the δ value range of 68 ppm, δ value
C-3 included in β (1 → 3) bond in the 86ppm range
A signal showing two peaks attributed to the carbon of
There is a characteristic signal of S ₂ and a signal S ₃ attributed to the C-1 carbon of the β bond with a δ value in the 103 ppm range. (3) Elemental analysis values: C42.2-44.0%, H5.9-6.2%,
Below the N quantification limit. (4) Molecular weight (gel filtration method): 10 ⁶ to 10 ⁷ range. (5) Blackening thermal decomposition at melting point of approximately 230℃. (6) Specific rotation: [α] _D 10±5° (C=0.1, H ₂ O). (7) Solubility: Easily soluble in water, alkali, and dimethyl sulfoxide. Insoluble in organic solvents such as ethyl alcohol, methyl alcohol, ether, and acetone. (8) Color reaction: Moritsch reaction, Anthrone sulfuric acid reaction, and phenol sulfuric acid reaction are positive. Ninhydrin reaction and Biuretz reaction were negative. (9) Basic, acidic, and neutral aqueous solutions A 1% aqueous solution exhibits a neutral range (PH6 to 6.5). 2 The polysaccharide NSG-1 producing strain is Sclerotinia
Sclerotinia sclerotiorum
A method for producing polysaccharide NSG-1 according to claim 1, which is IFO 9395.