JPS58109423A - Anti-infective agent - Google Patents

Abstract

PURPOSE:To provide a low-toxic anti-infective agent, by using mannan obtained from the yeast belonging to Saccharomyces genus. CONSTITUTION:The objective agent contains mannan obtained from the yeast belonging to Saccharomyces genus (e.g. Saccharomyces cerevisiae), as an active component. Although the mannan obtained from wild yeast has branched structure, the mannan obtained from the mutant strain, Saccharomyces cerevisiae X-2180-1A-5, is a straight-chain molecule. The mannan can be extracted from the yeast cell by (1) culturing the yeast aerobically in a conventional liquid medium, (2) filtering the cultured product, (3) extracting the obtained cells with hot water, (4) removing proteins from the extract, (5) treating with a copper complex solution to obtain a coppermannan complex. (6) neutralizing the resultant aqueous solution of crude mannan with a base, and (7) dialyzing the product to obtain crude mannan. The crude mannan is subjected to column chromatography and eluted with water to obtain neutral mannan, which is then fractionated with saline water to obtain acidic mannan.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the use of mannan obtained from yeast belonging to the genus Satucharomyces, and more particularly to an anti-infective substance comprising the mannan.

Although infectious diseases have significantly decreased due to advances in chemotherapy, especially antibiotics, the number of bacteria that are resistant to chemotherapeutic agents has increased rapidly in recent years, necessitating the development of new antibiotics. There is. The present inventors took these current circumstances into consideration.

As a result of repeated research into therapeutic agents for infectious diseases from a different perspective, the present invention was completed by discovering that mannan, a yeast of the genus Satucharomyces, has anti-infective oil properties.

It was discovered by the inventors of the ζβ family that an antitumor effect has been observed in the yeast mannan of the genus Satucharomyces, and a patent has already been applied for, but it is not known that these mannans have anti-infective properties. It is completely new and based on knowledge.

Yeasts of the genus Satucharomyces used in the present invention include the following. That is, S accbaromyces Cer-vi
siae), Satucharomyces bairii (Sa-
ccharomyces bailii), Saccharomyces bayanus (Saccharomyces b)
ayanus), Saccharomyces bisporus (Sac
charomyces bisporus).

Saccharomyces sevarieri
ceschevalieri), Sa-ccharomyces rouxii
, Saccaromyces ubaru A
myces uvarum), Saccharomyces exigus
), Saccharomyces italicus (Saccha
romyces1talicus), Saccharomyces diastaticus (Saccharomyces diastaticus)
diastaticus), etc.

Among the yeasts mentioned above, the mannan obtained from the wild strain yeast has a branched structure, but the mannan obtained from the mutant strain Satucharomyces cerevisiae X-2180-IA-5 is linear. ,3
(See page 63, 1980).

Next, a method for extracting and purifying mannan from yeast cells of the genus Satucharomyces will be described.

Liquid culture is convenient for culturing yeast of the genus Satucharomyces in large quantities, and various medium sources used for the cultivation of ordinary microorganisms are used as the medium source. That is, carbon sources include glucose, galactose, sucrose, lactose, and maltose.

starch, starch hydrolyzate, dextrin, sugars such as molasses, organic acids such as citric acid, sodium citric acid, tartaric acid, sodium tartrate, acetic acid, fatty acids or their salts, glycerin, sorbitol, myo-inositol, ethanol, etc. Alcohol can be used. As a nitrogen source, any available nitrogen compound may be used.2For example, casamino acids, casein, casein hydrolyzate, peptone, meat extract, yeast extract, cornstarch liquor,
Soybean meal, fish meal, cottonseed meal and ammonium salts such as ammonium sulfate and ammonium chloride are used. Other nitrates such as sodium nitrate, potassium nitrate, and ammonium nitrate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, potassium chloride, calcium chloride, and ferric chloride.

Salts such as magnesium sulfate, sodium sulfate, potassium sulfate, and manganese sulfate are used as necessary.

Cultivation is usually carried out aerobically, and shaking or aeration is usually preferred. The culture temperature can be changed as appropriate within the range in which the bacteria grow, but it is 25 to 45°C, preferably 28 to 30°C.
It is. The culture time varies depending on various conditions, but is usually about 24 to 200 hours, and the culture is terminated at an appropriate time, taking into consideration the time when the number of bacterial cells reaches its maximum value.

To collect bacterial cells from the culture after completion of cultivation.

This is centrifuged to remove the lower bacterial cell portion, or the culture is filtered to remove the bacterial cells. Fresh and moist bacteria can be obtained by washing the separated bacterial cells with distilled water or physiological saline.

Next, a method for extracting and purifying mannan from yeast cells obtained by culturing will be described.

The bacterial cells obtained as described above are extracted with hot water. Examples of the extraction solvent include water or a dilute aqueous alkaline solution; however, in this case, if extraction is performed with a dilute alkaline solution, more glue/glue will be extracted than mannan, so water is preferred. The hot water extraction is preferably carried out under pressure (preferably 3 atmospheres) and at a temperature of 100 to 150°C. Extraction time is 1-5 hours. This extract is then dialyzed and concentrated, and after a protein removal operation is added, a water-soluble organic solvent is added to obtain the precipitated phase polysaccharide. Specific examples of water-soluble organic solvents used include ethanol, methanol, impropanol, and acetone. Next, dissolve this crude polysaccharide in a small amount of water to make a copper complex solution.

For example, a copper-mannan complex is obtained by treatment with Fehling's reagent, which is then treated with a strong acid type ion exchange resin to remove copper. Specifically, the strong acid type ion exchange resins used here include Amberly) IR120, Amberly) IRllg, and Amberlyte IR12.
1st prize is mentioned. The crude mannan aqueous solution obtained by decopper removal treatment is neutralized with a base, concentrated, and then subjected to running water dialysis.

The bases used here include 2, for example, soda carbonate,
Potassium carbonate, soda hydroxide, potassium hydroxide.

These include hydrogen carbonate and potassium hydrogen carbonate. The aqueous solution obtained by dialysis is concentrated and then subjected to protein removal treatment,
Then, it is poured into a water-soluble organic solvent and the precipitated powdery substance is collected. Neutral mannan can be obtained by eluting this crude mannan with water using column chromatography. Specifically, diethylaminoethyl Sephadex (DEAE) is used as a separation phase for column chromatography.
-sephadex), carboxymethylcello
- Suse77dex (CM-5ephadex),
Diethyl 7 Mi/x Chilcell a-ce (DEAE-ce
lulose) and carboxymethylcellulose (
CM-ce-11ulose) and the like.

Acidic mannan (acidic because it contains phosphorus) is obtained by separating neutral mannan and fractionating it with brine.

Various mannans are indicated by the following abbreviations.

Wild yeast neutral mannan: WNM Acidic mannan: WAN Mutant strain Linear neutral mannan: MNM Acidic mannan: MAM The chemical analysis results of these mannans are shown in Table 1.

The anti-infective agent containing mannan as an active ingredient in the present invention may be in the form of various preparations.

Specifically, it is suitable for use as an injection. Injectables are water-soluble, water-insoluble, and suspendable.

It is best used as emulsifiable and powder injections.

Water-soluble injections are made by dissolving mannan in an aqueous solvent.

It can be obtained by adding tonicity agents, stabilizers, preservatives, soothing agents, solubilizing agents, etc. as necessary, filling ampoules or bottles using a filling machine, sealing them, and sterilizing them.

Other injections can also be produced by conventional methods.

Next is the anti-infective effect of mannan obtained in this invention.

The effective amount, administration method, acute toxicity, etc. will be explained below.

(1) Method of anti-infective action Mannan was administered intraperitoneally to male ddY mice at a dose of 150 kg, 7 kg, 5 times starting 5 days before bacterial inoculation.

The presence or absence of survival was observed for 30 mouths. The bacteria used for infection was Staphylococcus aureus βH248 strain, and 8 x 10' cells were intravenously inoculated into mice.

Results Survival rate was 0% in control group (death on gold side in 4 days), WA
80 chi in M administration group, MNM, 70 chi in MAN group, WNM
The group is 60 inches long.

Mannan was found to have anti-infective effects, and acidic mannans tended to have stronger effects.

(2) Serum lysozyme increasing effect mannan 150 da/kl? Once a day for 5 o's male dd
After intraperitoneal administration to Y mice, serum lysozyme levels were measured. The serum lysozyme value in the control group was 74 μg/-
On the other hand, the mannan-administered group had WAM, WNM, MAM, and M
Almost the same as the NM group, 11.0-11.3μ9/rt
Increased tl and serum lysozyme were observed. From this, it seems that the anti-infective effect of mannan is due to the anti-bacterial effect of L-lysozyme.

(3) Acute toxicity MNM obtained in Example 1 was administered to ddY male mice (body weight 20±
2 g) administered subcutaneously, intraperitoneally, intravenously and orally;
Acute toxicity was observed after 24 hours. As a result, the acute toxicity value - (LD5. value) shown in 9th □ was obtained. (Table 3) Table 3 Acute toxicity value (LD5I) value Similar results were obtained for MAM, WNM, and WAM.

As shown above, the mannans of the present invention have extremely low toxicity and have been found to be very useful as anti-infective agents. The dosage of these mannans is 3o;j per day.
~300 da is appropriate (adult).

Next, reference examples and examples of the present invention will be shown.

The present invention is not limited to these in any way.

Reference Example 1 Distilled water 1e was added to a 21-size Sakarokorpen, and 3g of casamino acid and 50.0g of glucose were added to it. S/, (-st extract 5L
Citric acid IL 5 g of citric acid, 1) Potassium dihydrogen citrate 0.5.5.9. Potassium chloride 0.425.9. Magnesium sulfate heptahydrate o, +25.9° Manganese sulfate 2.5
1+119. Calcium chloride dihydrate 0.125. ! 9.
Ferric chloride hexahydrate 2.5 ++ta, ammonium sulfate 3.7
4M and 10μ of inositol were added and dissolved. This was plugged with cotton and sterilized in an autoclave at 120°C for 15 minutes. Next, Satsukaromyces-Sel Hishi 7-engine X-21
80-IA-5 (Sa-ccharomyces c
erevisiae A culture containing the same bacterial cells was obtained. Centrifuge this at 3000r
Centrifuge for 0 minutes to obtain bacterial cells that separate into the lower layer.
This was thoroughly washed with 150 ml of physiological saline 1, centrifuged and lukewarm bacterial cells 2
Example 1 Satucharomyces cerevisiae X-21 obtained in Reference Example 1
80-IA-5 (8accX-2180-IA-5(8
accaro X-2180-IA-5) bacterial body 5k
Add 101 g of distilled water to 2 g at 135°C under pressure (3 atm).
Extraction is performed for a period of time, and after cooling, centrifugation is performed to separate the supernatant. On the other hand, 101 liters of distilled water is further added to the precipitate and the same extraction operation is carried out, and the resulting 1F clear liquid is combined with the first obtained supernatant liquid and concentrated until the total amount of L j becomes 41. Add this concentrate to 960 ml of chloroform-butanol (5:1).
Rinse 15 times in a trench where no precipitate will come out. Next, this concentrated solution is concentrated to 0.51, 21 parts of ethanol is added, and the precipitated powder is collected in a furnace. Dissolve this powder in water 11! 7. A slight excess of Fehling's test solution is added to this to remove the precipitated copper-mannan complex. This copper-mannan complex was washed with two aqueous solutions of dipotassium hydroxide (7), then suspended in distilled water and washed with Amberlite I
R120H"500 V was added, stirred for 45 minutes, filtered, and the filtrate was neutralized with soda carbonate and concentrated.

Then, after performing running water dialysis, Kinryu reached 80 +1 m.
This aqueous solution was poured into ethanol, the precipitated powder was collected in an oven, and then dissolved in water to make a 5% aqueous solution.
x length 100 crr*) and elute with water. After concentrating the eluate, dialysis is performed. Column chromatography, concentration, and dialysis were repeated twice, and the resulting dialyzed aqueous solution was concentrated and then poured into ethanol.
Obtain NM9.9. The characteristics of the city are as follows. Molecular weight: approximately 24,000. Optical rotation: [α] is also 0 = +80
．． 5° (C = 1.0. in H2O), constituents: sugar 99,
0 Ti; Nitrogen 0.1 Ti; Phosphorus 0.0 Ti.

3 Example 2 After eluting MNM, 0.05M and 0.1
It was removed by elution with M saline and 0.25M saline. The eluate was treated in the same manner as in Example 1 to obtain 15 g of MAM.

Example 3 Saccaromyces cerevisiae wild strain (Sacc-ha
romyces cerevisiae) cells were treated as in Examples 1 and 2 to obtain 9 g of WNM and 5 g of VANl.
I got g.

Example 4 MNM59 obtained in Example 1 was dissolved in sterile physiological saline to make a total volume of IO ml. This solution was aseptically dispensed into ampoules of 1 d each, and after thawing and closing, the mixture was placed in an autoclave at 121°C.
Heat sterilization was performed for 20 minutes to obtain a water-soluble injection.

4 Example 5 M A M obtained in Example 2 was added to a sterile 5% glucose solution for injection.
Dissolve 5.9 into 100ml, ? Knee (D
The solution was aseptically dispensed in 1 me portions into vials and dried under vacuum. In this way, a powder injection containing 50 μm of MAM in one vial was prepared.

Before use, dissolve in distilled water for injection.

Example 6 WNM5.9 obtained in Example 3 was ground aseptically.

This was aseptically suspended in soybean oil to make a total volume of 100 ml, and aseptically dispensed into ampoules in 1 ml portions, which were melted and sealed to obtain a non-aqueous suspension injection. Example 7 Obtained in Example 3. Dissolve WAM5p in 10ml of distilled water for injection, heat it in an autoclave at 121°G for 1Jn for 20 minutes, add 0.5p of polysorbate and soybean oil to make a total volume of 100mJ, and stir well to make an emulsion. This was aseptically dispensed into 1 ml ampoules and melted and sealed to obtain a water-in-oil emulsifiable injection.

patent applicant Grelan Pharmaceutical Co., Ltd.

Claims (1)

[Claims] Anti-infective substance consisting of mannan obtained from yeast belonging to the genus Satucharomyces