JP3444624B2 - Highly branched β-glucan, its production method and use - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a highly branched β-glucan, a method for producing the same and an infectious disease preventive agent or antitumor agent. The infectious disease preventive agent or antitumor agent of the present invention is useful as a drug, a food additive, a feed additive, or the like.

[0002]

2. Description of the Related Art Conventionally, the genus Aureobasidium(Aureoba
sidium sp.) produces β-1, 3-1, 6-D-glucan
Was known (Acta Chemica Scandinavia  1
7, 1351-1356 (1963), Agric. Biol. Chem.47(6), 11
67-1172 (1983)). These glucans have a phosphate group, phosphorus
Includes a goric acid group or sulfonic acid group to enhance activity
In order to remove these functional groups
There was a problem. On the other hand, gluca with multiple branches
Is also known (Chem.Pharm.Bull.,40, 2215 (199
2)), the main chain glucose without branching has many bonds of glucose alone.
There are a number of them, and this glucan is an antitumor
It had no activity.

[0003]

DISCLOSURE OF THE INVENTION The present inventors have paid attention to such a high molecular polysaccharide produced by the genus Aureobasidium and have tried to obtain a novel β-glucan having high physiological activity. As a result of repeated studies, the Aureobasidium pullulans IFO 4466 strain produced a novel glucan having a highly branched β-glucoside bond, and this glucan was orally administered as a polysaccharide consisting of glucose that does not bind to a phosphate group. The present invention has been completed by finding that they exhibit high antitumor activity and immunostimulatory activity.

Therefore, an object of the present invention is to provide a glucan having a novel highly branched β-glucoside bond. Moreover, the subject of this invention is Aureobasidium.
It is an object of the present invention to provide a novel method for producing β-glucan having a high degree of branching using the pullulans IFO4466 strain. A further object of the present invention is to provide an antitumor agent and an infectious disease preventive agent containing such a novel highly branched β-glucan as an active ingredient.

[0005]

[Means for Solving the Problems] That is, the present inventors have paid attention to the polysaccharides produced by the genus Aureobasidium, as described above, and used various microorganisms belonging to the genus Aureobasidium. After repeated studies on the production of Aureobasidium pullulans IFO 4
466 strain has a high yield and high physiological activity in a liquid medium containing xylose and vitamin C as essential components,
It was found to produce β-glucan with a highly branched β-glucoside bond.

The present invention will be described more specifically. For the genus Aureobasidium, refer to Riki Morinaga "Lecture / Classification of Fungi /
Identification '' (J. Antibact . Antifung . Agents . 18 (6) 295
-297 (1990), there are 14 varieties and 1 varieties, most of which are Aureobasidium pullulans.
pullulans) . There are two variants of Aureobasidium pullulans. These morphological features are that colonies are often covered on the smooth surface with a mass of viscous conidia and sparsely present aerial hyphae. Colors of colonies vary from light brown to yellow, pink or black. Hyphae are clear, often brown and thick-walled. Conidia-forming cells are transparent and branch on hyphae to form at the tip or in the middle. Conidia are produced densely on the conidia-forming cells by the budding method synchronously. The color is transparent and has a smooth wall, and it is a single cell with various shapes and sizes. Of these Aureobasidium pullulans, those isolated from nature, purified, subcultured, and retaining their traits, or strains deposited at depositary institutions,
Any strain can be used as long as it can produce the highly branched β-glucan of the present invention. However, it is preferable to use Aureobasidium pullulans IFO 4466, which has been deposited at the Fermentation Research Institute, in terms of the yield of the highly branched β-glucan and the ease of isolation.

As the medium used in the present invention, a liquid medium containing carbon sources, nitrogen sources, phosphorus, potassium, magnesium and other nutrients necessary for culturing ordinary microorganisms is used. As the carbon source, at least xylose and vitamin C are used as essential components. Other than this, for example, glucose or sucrose can be used as the carbon source. The use ratio of carbon source is xylose 5-150 g / L, preferably 10-100 g / L, most preferably 20-60 g / L, vitamin C
0.01 to 100 g / L, preferably 0.1 to 60 g / L, most preferably 0.5 to 20 g / L are used. The proportion of components other than the carbon source used is NaNO ₃ 0.5 g / L to 20 g / L, preferably 1 to 10 g / L
, K ₂ HPO ₄ 0.05-10 g / L, preferably 0.1-5 g / L, KH ₂
PO ₄ 0~20g / L, preferably 0.5~5g / L, KCl 0.1~10g
/ L, 0.2~5g / L, MgSO 4 · 7H 2 O 0.05~5.0g / L, preferably _{0.1~2.0g / L, FeSO 4 · 7H} 2 O 0~5g / L, preferably 0.005～2.0G / L is used. Vitamin B ₁ can also be added to the liquid medium of the present invention. The culture is usually carried out at a temperature of 5 to 40 ° C for 1 to 10 days. It is preferably carried out under aeration. Thus, the high degree of branching β of the present invention
-Glucan is produced.

After completion of the culture, the culture is subjected to a means such as centrifugation to remove the cells from the culture, and the highly branched β-glucan of the present invention is collected from the culture supernatant. As a collection method, a method of adding an organic solvent to the culture supernatant to precipitate the highly branched β-glucan of the present invention can be preferably used. The organic solvent is not particularly limited, but for example, alcohol, ketone, nitrile, etc. are used. Specific examples thereof include ethanol, isopropyl alcohol, acetone and acetonitrile, with ethanol being particularly preferable. In addition to the highly branched β-glucan of the present invention, the obtained product usually contains impurities such as low molecular weight compounds, proteins, and water-insoluble glucan. In the present invention, when the highly branched β-glucan of the present invention is used as a food additive or a feed additive, the above product can be used as it is or after dried.

However, when it is used as an active ingredient of a drug or the like, it is dialyzed using a cellulose tube or the like to remove low molecular weight compounds, and an acidic substance such as trichloroacetic acid or picric acid, or n-butanol, n. -Precipitate and remove the protein using an organic solvent such as butanol in chloroform as a deproteinizing agent. Furthermore, to the precipitate of highly branched β-glucan, an alkaline aqueous solution of about 0.5N is added to dissolve the precipitate, insoluble glucan is removed by precipitation, and only water-soluble glucan is removed by acetic acid, citric acid, hydrochloric acid, sulfuric acid, etc. A highly branched glucan having a high degree of branching is obtained by neutralizing with the acid of The chemical used in the impurity removal operation can be removed by dialysis, gel permeation, ultrafiltration, etc. to obtain the highly branched β-glucan of the present invention with high purity.

The physicochemical properties of the highly branched β-glucan thus obtained are as follows.

(1) Constituent Monosaccharide To 50 mg of the above highly branched β-glucan, 2 ml of 1N sulfuric acid was added and heated for 8 hours for hydrolysis, followed by reduction with sodium borohydride according to a conventional method, and then pyridine and anhydrous. Acetylated with acetic acid and gas chromatography (column: 3 wt% ECNSS-M / chromosolve W temperature: 190
℃ Carrier gas: Nitrogen gas, Carrier gas flow rate: 3ml
/ Min), the specific rotation [α] _D ²⁵ was +
50 °, D-glucose [reference value [α] _D ²⁵ +52.8
(Hirokawa Shoten, “Organic Qualitative Analysis”, p. 276)], it was confirmed that 99% or more of glucose was glucose. Furthermore, the highly branched β-glucan of the present invention was enzymatically decomposed by exo β-glucanase obtained by purifying commercially available chelatase, and the decomposed sugar was examined by thin layer chromatography (TLC). This purified enzyme is allowed to act on laminarin consisting only of β-1,3 bond of glucose, and only glucose is detected when the decomposed sugar is examined by TLC. However, the decomposed sugar obtained from the highly branched β-glucan of the present invention Had the same Rf values as glucose and gentiobiose. Moreover, gentiobiose was 2 or more with respect to 1 glucose. In addition, the rate of degradation by the enzyme was 1/20 of the rate of degradation of laminarin.
It was revealed that the cleavage of -1,3 bond was sterically hindered by the branched chain. The thus obtained enzymatic decomposition product and the acid decomposition product are analyzed by HPLC (column: μBondaSphere-NH
₂ 5 [mu] 100 Å, solvent: 80% CH ₃ CN, flow rate: 0.8 ml / ml,
When the amount of decomposing sugar was quantified by (detection: by a differential refractometer), the decomposition rate by the enzyme was 1% or less of the decomposition rate by the acid, and it was shown that the decomposition was very difficult by the enzyme.

(2) Glucose binding mode and degree of branching The highly branched β-glucan of the present invention was dissolved in dimethyl sulfoxide (DMSO) -d ₆ at 100 ° C, and the ¹³ C NMR spectrum was measured while the temperature was kept at 100 ° C. An example of the above is shown in FIG. As shown in FIG. 1, (i) δ value is in the 68 ppm range, peak S ₁ attributed to carbon of C-6 in glucose residue A shown in Chemical formula 3 is shown, and (ii) δ value is in the 86 ppm range. The peak S ₂ attributed to the carbon of C-3 in the glucose residue A and the glucose residue C, and (iii) the C in the glucose residues A, B and C shown in Chemical formula 3 in the δ value 103 ppm region -1 three signal peaks S ₃ attributable to carbon is observed. From this, the highly branched β-glucan of the present invention is β1 →
It is judged that the main chain consisting of A or C bound via 3 bonds is branched to the B bound via β1 → 6 bonds (Carbohydrate Polymers 2 , 135-144).
(1982)).

[0013]

[Chemical 2]

Further, when FIG. 1 is enlarged, as shown in FIG. 2, the C in the glucose residue C is in the δ value range of 60.5 to 60.8 ppm.
The intensity of the signal S _a attributable to the carbon of -6 to 1,
Since the intensity of the signal S _b attributed to the carbon C-6 of the glucose residue B in the δ value 61.0 ppm region is about 2, the high-branched β-glucan of the present invention has three main chain glucose residues. There are 2 branched glucose residues.

Further, FIG. 3 shows an enlarged spectrum of a region in which a signal attributed to C-3 carbon of β (1 → 3) bond is detected. In Fig. 3, the signal S with a δ value of 85.7 ppm
_c is HSQC-TOCSY [Ernst Two-dimensional NMR, published by Yoshioka Shobo, page 589 (1991) and Maruzen, edited by The Chemical Society of Japan, Vol. 5, pp. 133-137 (1991).
Year)], a correlation with the H-6 hydrogen signal of the glucose residue A (δ values 3.58 and 4.08 ppm) was observed, so it was assigned to the C-3 carbon of the glucose residue A (Car.
See bohydroate Polymers 2, 135-144 (1982)). The signal S _{d at the} remaining δ value of 86.2 ppm is C of glucose residue C.
-3 carbon. Further, the highly branched β-glucan of the present invention has a δ value of 85.9 ppm at the C-3 carbon which should be observed in a glucan such as scleroglucan or laminarin having a unit of a glucose residue C as a partial structure. Since the corresponding signal is not detected, the glucose residue C in the highly branched glucan of the present invention is C-3.
The glucose residue A is bound to the carbon side.
Since the signal intensity ratio of S _c and S _d is 2: 1,
The highly branched β-glucan of the present invention according to this example is composed of units GI and GII having the structure shown in Chemical formula 4, and GI and GI
It can be seen that the abundance ratio of I is 1: 1. For example DMS
High-branching degree of the present invention β-
Glucan includes a component soluble in DMSO and a component insoluble in DMSO. From the area intensity ratio of signals S _a and S _{b in} the spectrum shown in FIG. 4, the component soluble in DMSO has 9 glucose residues in the main chain. Is a β-glucan composed of 5 branched glucose residues, and the DMSO-insoluble component is a β-glucan composed of 3 branched glucose residues for 4 main-chain glucose residues. . That is, the highly branched β-glucan of the present invention has a GII unit content of 20 to 70%.
contains.

[0016]

[Chemical 3]

(3) Infrared absorption spectrum (KBr method) As shown in FIG. 5, there is an absorption (P) characteristic of β-glucoside bond orientation at a wavelength of 880 cm ^-1 .

(4) Molecular weight (gel filtration method) Number average 10,000 to 5 million, preferably 500 to 5 million

(5) Color reaction: Maurish reaction, anthuron sulfate reaction, phenol sulfate reaction; positive ninhydrin reaction, burette reaction; negative.

From the above physicochemical properties, the chemical structure of the highly branched β-glucan of the present invention was determined as follows.

[0021]

[Chemical 4] (M is 80 to 30%, n is 20 to 70%)

The highly branched β-glucan of the present invention is β-1,3
With a glucose residue of the bond as the main chain, this glucose residue has a large number of β-1,6-bonded glucose residues branched, and there is virtually no unbranched glucose bond in the main chain. There are features.

The antitumor activity of the hyper-branched β-glucan of the present invention was tested by dissolving this glucan in physiological saline and orally or parenterally administering it to mice bearing cancer. It was possible to suppress the progression of the tumor and improve the survival rate. It also has immunostimulatory activity.

Therefore, the highly branched β-glucan of the present invention exhibits antitumor activity or immunostimulatory activity by oral or parenteral administration, and can be used as a medicine, a food additive or a feed additive. When used as a medicine, as an antitumor agent or an immunostimulatory agent, symptoms,
100-0.1 mg / day for adults, depending on age and sex,
It is preferable to administer 30 to 60 mg in several divided doses a day. This high-branched β-glucan can be used as it is as a drug, but is formulated as a mixture with a pharmaceutically acceptable diluent and / or another substance having other pharmacological action according to pharmaceutical practice. Can also be used in. The administration can be performed by oral administration, intravenous administration, intraperitoneal administration, enteral administration and the like. Therefore, it can be used as an appropriate agent suitable for such administration, for example, as a powder, granules, tablets, dragees, capsules, pills, suppositories, suspensions, solutions, emulsions, injections, aerosols and the like. it can. However, oral administration is particularly effective as a tumor metastasis-preventing agent.

When it is used as a food additive or a feed additive, the above-mentioned precipitated or highly branched β-glucan of the present invention is used as it is, or is mixed with a carrier, a bulking agent or the like which is commonly used for these additives. Then, it can be added to food or feed to impart antitumor activity to the food or feed or impart immunostimulatory activity to prevent or treat infectious diseases. For example, it is suitable as a feed additive for cattle, pigs, chickens, fish, birds, dogs, cats and the like.

Next, the present invention will be specifically described with reference to examples.

Example 1 Production of highly branched β-glucan 1. Cell culture Ou Leo Basi di Umm pullulan (Aureobasidium pull
ulans) Fermentation Research Institute Deposit No.IFO 4466 strain cultured in potato dextrose agar slant medium, the preserved strain, a liquid medium having the following composition (pH 5.0-6.0, preferably pH 5.5) 300 ml in a Sakaguchi flask was inoculated and aerated with stirring at a temperature of 20 to 30 ° C. for 2 to 3 days. Examples of compositions of the media used in the present invention xylose 30g vitamin _{C 6.0g NaNO 3 2.5g K 2 HPO} 4 0.4g KH 2 PO 4 2.0g KCl 0.5g MgSO 4 · 7H 2 O 0.5g FeSO 4 · 7H 2 O 0.01 g Vitamin B _{1 1} mg Distilled water 1 L Aureobasidium pullulan IFO 4466 strain uses pullulan (α-1,4-1,6-) when glucose or sucrose is used as sugar.
Glucan) is also produced, but when xylose is used as sugar, almost no pullulan is produced, and the highly branched β-glycan of the present invention is produced (see Table 1).

2. Production of highly branched β-glucan of the present invention The cells of 300 ml of the above culture solution were removed using a centrifuge, the same amount of ethanol was added to the obtained culture supernatant, and the mixture was stirred at room temperature for several hours. Next, centrifugation was performed, 300 ml of 0.5N NaOH was added to the obtained precipitate, and the mixture was stirred at room temperature to precipitate water-insoluble glucan, and this precipitate was removed by centrifugation. The liquid from which this insoluble matter was removed was dialyzed with a cellulose tube or the like. If it is necessary to remove a trace amount of protein, trichloroacetic acid is added to the crude precipitate aqueous solution before dialysis to precipitate and remove the protein.
Table 1 shows the amount of highly branched β-glucan produced.

[0028]

[Table 1]

The physicochemical properties of the thus-obtained highly branched β-glucan were examined, and it was in agreement with that of the previously described highly branched β-glucan.

[0030]

Example 2 The antitumor activity of the highly branched β-glucan obtained in Example 1 was measured. ICR mouse (female, about 30g)
Sarcoma 180 allograft was transplanted subcutaneously in the groin area at 5 × 10 ⁶ cells in 14 or 7 animals. Food and water were available ad libitum. Highly branched β-glucan of the present invention 7 days after transplantation 2.
Dissolve 5 mg in 1 ml of physiological saline and add 40 mg per kg of body weight.
Was intraperitoneally administered once a day. The tumor was excised 5 weeks after the transplantation, the weight was measured, and comparison was made with a control group to which only physiological saline was administered. The results are shown in Table 2. As shown in Table 2, high antitumor activity was observed by intraperitoneal administration of the highly branched β-glucan of the present invention.

[0031]

[Table 2]

[0032]

Example 3 The immunostimulatory activity of the highly branched β-glucan obtained in Example 1 was measured. ICR mouse (female, about 30g)
3 mg of highly branched β-glucan of the present invention was added to 1 ml of physiological saline.
It was dissolved in and was intraperitoneally administered so as to be 20 mg per kg body weight. Food and water were available ad libitum. Two days or three days after the start of administration, the spleen was extracted and its weight and cell number were measured. Further, the abdominal exudate cells and blood were taken out and the number of abdominal exudate cells and the number of cells in blood were measured. The phagocytic activity of the peritoneal exudate cells was also measured by measuring the uptake ability of the fluorescently labeled beads and the lysosomal acid phosphatase activity using the peritoneal exudate cells. Then, similarly to Example 2, the comparison with the control group was performed. The results are shown in Table 3 ~
6 shows. As shown in Tables 3 to 6, when the hyper-branched β-glucan of the present invention was intraperitoneally administered at 40 mg / kg of body weight, the weight of the spleen was doubled on the second day after the administration as compared with the case of no administration (control). However, the number of cells also increased 1.8 times. The numbers of peritoneal exudate cells and blood lymphocytes increased 3.4 times and 1.4 times on the third day after administration, respectively. It was found that the phagocytic activity was increased by the 1.2-fold increase in the macrophage bead uptake ability of the abdominal exudate cells and the 2.2-fold increase in the phosphatase activity. From these, it is judged that the immunostimulatory activity was remarkably enhanced.

[0033]

[Table 3]

[Table 4]

[Table 5]

[Table 6]

[0034]

[Example 4] 7-8 ICR mice (female, about 30 g) were allografted with the tumor Sarcoma 180 subcutaneously transplanted into the abdomen 5 × 10 ⁶ cells, and immediately after that, the highly branched β-glucan used in Example 2 was used. The above physiological saline was forcibly orally administered or intraperitoneally administered for breeding. The feed and water were freely ingested, the body weight was measured 5 weeks after the administration of glucan, the tumor was excised, and the weight was measured. And it compared with the control group like Example 2. The results are shown in Tables 7-9.

[0035]

[Table 7]

[Table 8]

[Table 9]

As can be seen from Tables 7 to 9, oral administration of the highly branched β-glucan of the present invention at 90 mg / kg body weight suppressed the growth of solid tumors to 61% as compared with no administration (control), Moreover, the life extension rate was increased to 71%, compared with 13% in the control. The tumorigenicity of the ascitic fluid was 88% in the control, while the tumorigenicity could be reduced to 29%, and the body weight could be increased by 1.6 times compared with the case of no administration. The increase in body weight is due to the increase in weight of the spleen responsible for immunization, and thus it is considered that the immunostimulatory activity is significantly enhanced.

[0037]

Example 5 (1) 5 mg of highly branched β-glucan obtained in Example 1 was dissolved in 10 ml of physiological saline to prepare an orally administrable antitumor agent or immunopotentiator. (2) 5 mg of highly branched β-glucan obtained in Example 1 was mixed with 50 mg of lactose, mannitol and glucose, and the mixture was tableted to give tablets. (3) Precipitation of the highly branched β-glucan obtained in Example 1 20
g to 1 kg of bovine mixed feed to enhance bovine immunity,
Prevented infectious diseases.

[0038]

According to the present invention, a novel β-glucan having a high degree of branching can be mass-produced with a high yield in a simple culture. And since the obtained highly branched β-glucan has high antitumor activity and immunostimulatory activity by oral administration, it is used as a preventive or therapeutic drug for human cancer or livestock, pet animals,
It is useful as a preventive or therapeutic drug for infectious diseases of cultured fish.

[Brief description of drawings]

FIG. 1 shows a two-dimensional NMR spectrum of highly branched β-glucan.

FIG. 2 shows an expanded spectrum of the two-dimensional NMR of FIG.

FIG. 3 shows an expanded spectrum of the two-dimensional NMR of FIG.

FIG. 4 shows NMR spectra of DMSO-soluble and insoluble components of highly branched β-glucan.

FIG. 5: Infrared absorption spectrum of highly branched β-glucan
(KB _r method) is shown.

FIG. 6 shows the relationship between administration of the highly branched β-glucan and the survival rate in Example 4.

[Explanation of symbols]

○ Control ◇ Oral administration (90mg / kg) ◆ Oral administration (180mg / kg) □ Intraperitoneal administration (25mg / kg)

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI A61K 31/715 ADZ A61K 31/715 ADZ C12P 19/04 C12P 19/04 // (C12P 19/04 C12R 1: 645 C12R 1: 645) (72) Inventor Kazuhiro Yagishita 8 Chidori-cho, Naka-ku, Yokohama, Kanagawa Japan Central Research Laboratory (56) Reference JP-A-5-39303 (JP, A) JP-A-6-114255 ( JP, A) JP 62-201901 (JP, A) Agric. Biol. Chem. 1983, Vol. 47, No. 6, p. 1167-1172 Acta Chemica Scandinavica, 1963, Vol. 17, p. 1351-1356 Chem. Pharm. Bull. 1992, Vol. 40, No. 8, p. 2215-2218 Carbohydrate Research, 1976, Vol. 47, p. 99-104 Carbohydrate Research, 1981, Vol. 89, p. 121-135 Agric. Biol. Chem. 1968, Vol. 32, No. 10, p. 1261-1269 (58) Fields investigated (Int. Cl. ⁷ , DB name) C08B 37/00 C12P 19/04 CA (STN) JSTPlus (JOIS) REGISTRY (STN) WPI (DIALOG)

Claims (5)

(57) [Claims] 1. A β-glucan represented by the following structural formula, having a number average molecular weight of 10,000 to 5,000,000 (measured by a gel filtration method) and having a high degree of branching. [Chemical 1] (However, in the formula, m represents 80 to 30% and n represents 20 to 70%.) 2. Aureo
basidium pulluans ) A highly branched β-glucan having the following physicochemical properties, which can be obtained by adding an organic solvent to the culture supernatant of the IFO 4466 strain to cause precipitation. 1) Number average molecular weight 10,000 to 5,000,000 (measured by gel filtration method), 2) Infrared absorption spectrum (KBr method), β at wavelength 880 cm ^-1
-There is a characteristic absorption in the glucoside bond orientation. 3) It has a signal in the ¹³ C NMR spectrum at i) δ values around 68 ppm, 86 ppm and 103 ppm. ii) The intensity of the signal with a δ value of 61 ppm is 1.2 to 3.0 times that of 60.5 to 60.8 ppm. iii) The intensity of the signal with a δ value of 85.7 ppm is
It is 1.2 to 3.0 times. 3. A liquid medium containing xylose and vitamin C as essential components is inoculated with Aureobasidium pulluans IFO4466 strain and cultured, and the resulting culture supernatant is highly branched. 2. The highly branched β-glucan according to claim 1, wherein β-glucan is collected.
Glucan manufacturing method. 4. An infectious disease preventive agent comprising the highly branched β-glucan according to claim 1 or 2 as an active ingredient. 5. An antitumor agent comprising the highly branched β-glucan according to claim 1 or 2 as an active ingredient.