JPH0572921B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a lipopolysaccharide and a method for producing the same. More specifically, antitumor activity, immunomodulatory activity,
The present invention relates to a lipopolysaccharide having physiological activities such as cell activation activity and infection prevention activity, and a method for efficiently producing lipopolysaccharide from actinomycetes and/or related bacteria. [Prior art] Mycobacterium bovis BCG cells, anaerobic Propionibacterium cells, etc. have been used as immune enhancers and are known to have antitumor activity, but on the other hand, they have strong side effects. It is also clear that this phenomenon occurs, which poses an obstacle to clinical application. Therefore, various attempts have been made to extract active ingredients from these effective bacterial cells or related bacterial cells and to remove side-effect components. In particular, research on the bacterial body components and their activities of bacteria such as Mycobacterium, Propionibacterium, and Nocardia has been actively conducted, and various physiologically active components have been isolated and purified, and the components have been modified. Attempts have also been made to increase the activity.
Namely, cell wall skeleton components (JP-A No. 54-28813, etc.), muramyl dipeptide (JP-A-52-156812, etc.), modified products of muramyl dipeptide (JP-A-56-1989, etc.)
No. 49396, etc.), adjuvant active substances (Special Publication No. 1983-
409, etc.), hot water extract (Special Publication No. 48-43842, etc.),
Tumor immunotherapeutics containing lipopolysaccharide as an active ingredient (Japanese Patent Laid-Open No. 56-8320, etc.) have already been disclosed, and recently lipopolysaccharide and its production method (Japanese Patent Laid-Open No. 59-161320)
No.) was disclosed. [Problems to be solved by the invention] However, all of these lipopolysaccharides are not necessarily sufficient in terms of activity and side effects, and their production methods do not have very high yields, so further improvements are needed. It was wanted. [Means for solving the problems] The present inventors have discovered that the human tuberculosis bacterium Mycobacterium
Regarding bacterial bodies such as tuberculosis, Mycobacterium bovis, and the non-pathogenic anaerobic bacterium Propionibacterium acnes,
No side effects, antitumor activity, immunomodulatory activity,
Research has continued in order to obtain components that exhibit efficacy such as cell rejuvenation activity in higher yields and purify them into a form with higher specific activity. As a result, they discovered that the target product could be obtained in good yield by using a nonionic or ionic surfactant and further adding physical crushing to these bacterial cells, and crude lipopolysaccharide The present invention was completed based on the discovery that the specific activity could be increased several times by fractionating the protein using a combination of organic solvents and then removing protein using an immobilized protease. That is, the present invention provides a polysaccharide with a composition of 1 D-arabinose and 1 to 3 D-mannose.
A lipopolysaccharide consisting of 95 (W/W)% and 5 to 20 (W/W)% fatty acids, and bacteria obtained by culturing Mycobacterium and Propionibacterium genus bacteria are crushed. Alternatively, a lipopolysaccharide characterized by being extracted with a surfactant solution without crushing, purified by a combination of enzymatic digestion with amylase and amyloglycosidase, ultrafiltration to obtain a fraction with a molecular weight of 100,000 or more, and solvent fractionation. This relates to the method of manufacturing the body. The method for producing lipopolysaccharide of the present invention will be explained. Bacteria obtained by culturing aerobically or anaerobically in an appropriate medium are collected by sterilization or centrifugation and sterilized by an appropriate method, or directly sterilized and killed in the culture medium by an appropriate method. Get a body.
After thoroughly washing the bacterial cells, freeze-thaw, sonicate,
Alternatively, it is crushed by physical means such as a French press. The crushing may be carried out in an aqueous suspension or in a suspended state in an aqueous solution of a surfactant. If the crushed bacterial cells are in an aqueous suspension, add a surfactant, or if they are in the presence of a surfactant, stir at room temperature, remove bacterial debris by centrifugation, and prepare the extract. obtain. If the dead bacteria are not crushed, the dead bacteria are stirred in an aqueous surfactant solution and filtered or centrifuged to obtain an extract. A suitable organic solvent is added to the thus obtained extract to precipitate the crude polysaccharide. The obtained crude polysaccharide is emulsified in an appropriate buffered salt solution and treated with amylase and amyloglycosidase. After the enzymes are precipitated and removed by heat treatment, the crude polysaccharide expressed as a polymer is purified by ultrafiltration or molecular sieve column. The polysaccharide fraction is separated and freeze-dried. The obtained crude lipopolysaccharide is fractionated using a suitable combination of organic solvents such as pyridine-methanol, chloroform-methanol, etc. to remove mixed phospholipids and the like. If proteins are present, the purified lipopolysaccharide is obtained by dissolving in an appropriate salt solution, treating with an immobilized protease such as actinase E, and freeze-drying. The bacteria used may be of the genus Mycobacterium or Propionibacterium, and the bacteria used may have been degreased in advance with a combination of an appropriate organic solvent, or It may be treated with a protein-digesting enzyme. The lipopolysaccharide of the present invention has a white to slightly off-white powder appearance and is slightly cloudy and soluble in distilled water or physiological saline, but is insoluble in organic solvents. The composition of the substance of the present invention is a polysaccharide whose main constituent sugars are D-arabinose and D-mannose, to which _C14 to _C19 fatty acids are ester-bonded, and the polysaccharide is 80 to 95 (W/W). % and fatty acids 5-20 (W/W)%. In addition, this substance is thought to form a hydrophobic micelle structure in an aqueous solvent, and in various molecular weight measurement methods,
It behaves very much like a polymer. The lipopolysaccharide of the present invention can also be extracted with an alkaline aqueous solution or an alkaline buffer, and can be obtained by similar purification operations. However, with strong alkali or weak alkali heating, it is saponified and the molecular weight is about
12,000 arabinomannan, a mannan with a molecular weight of about 7,000, and alkali salts of fatty acids. [Effect] Next, regarding the physiological activity of the lipopolysaccharide obtained by the above procedure, the test results of antitumor activity, cell rejuvenation activity, polyclonal B cell activation ability, and phagocytic cell activation ability were examined. explain in detail. As test substances, lipopolysaccharides prepared in Examples 1, 2, and 3 described later were used, and as a control, a corresponding amount of physiological saline was used as appropriate. First, the test results of antitumor activity using tumor growth suppressive effect and tumor regression effect as indicators will be explained. Methylcholanthrene-induced syngeneic tumor MC-1 was inoculated subcutaneously into the upper abdomen of 8-week-old female C57BL/6 mice at 1×10 ⁵ mice per group, and 10 mice were implanted in each group.
On day 1, a sample dissolved in 0.2 ml of physiological saline is administered intraperitoneally, and on the 24th day after transplantation, the animals are sacrificed, the tumor is removed, and its weight is measured. The T/C value was calculated using the following formula to determine the tumor growth inhibitory effect. The control group received 0.2 ml of physiological saline on the same schedule. T/C (%)=average tumor weight of administration group/average tumor weight of control group×100 The results are shown in Table 1.

【table】

[Table] As a result of this test, it was found that the lipopolysaccharides of the present invention all have extremely excellent growth-inhibiting and regression effects on mouse syngeneic tumors. Next, the test results for bacterial larvaization activity will be explained. Normal splenocytes from 8-week-old C57BL/6 female mice were taken out, and each lipopolysaccharide obtained in each example was
The cell activation effect of the lipopolysaccharide was determined by culturing with 10 μg of the lipopolysaccharide and measuring the amount of ³ H-thymidine taken up by the cells. As a control, cells cultured with a corresponding amount of physiological saline were used. Each test group was tested using cells from the same individual animal,
The same test was repeated for 5 individual animals. The results of the test are shown in Table 2. As a result of this test, it was found that all lipopolysaccharides of the present invention significantly enhance thymidine uptake into mouse splenocytes and have excellent cell activation and cell rejuvenation effects.

[Table] Next, the results of the polyclonal B cell activation test will be explained. Normal splenocytes from 10-week-old CDF ₁ female mice were taken out, and 10 μg of each lipopolysaccharide obtained in each example was collected.
The activity was evaluated by counting anti-trinitrophenylated horse red blood cell plaque-forming cells.
As a control, cells cultured with a corresponding amount of physiological saline were used. The test was carried out using cells from the same individual animal in each test group, and the test was repeated on five individual animals. The test results are shown in Table 3.

[Table] As a result of this test, it was found that all the lipopolysaccharides of the present invention have polyclonal B cell activation ability. Next, the test results of phagocyte activation effect will be explained. 10 μg of each lipopolysaccharide obtained in each example (dissolved in 0.2 ml of physiological saline) was administered intraperitoneally to 8-week-old BALB/c male mice, and after 2 days, the ascites was removed.
1 x 10 ^{3 syngeneic tumor (Meth) A cells were added to 2 x 10 4 cells} of the peritoneal cavity that adhered to the plastic shear, and the cells were incubated in a medium supplemented with fetal bovine serum.
After 24 hours of incubation, 3H-thymidine was added and further incubated for 16 hours.
After culturing for a period of time, the effect of activated macrophages on the growth of tumor cells was determined based on the amount of 3H uptake, and based on this, the ability of the sample to activate macrophages was determined. Each group consisted of 10 animals, and the control group was administered with a corresponding amount of physiological saline. The activation rate was calculated using the following formula. Activation rate (%)=average uptake of control group−average uptake of administration group/average uptake of control group×100 The results of the test are shown in Table 4.

[Table] The results of this test confirmed that the lipopolysaccharide of the present invention is a substance that promotes the activation of macrophages in mice. [Examples] The present invention will be explained in more detail with reference to Examples, but the present invention is not limited in any way by these Examples. Example 1 Mycobacterium tuberculosis Aoyama B
（Mycobacterium tuberculosis strain Aoyama
B) was cultured in Sorton's medium at 37°C for 5 weeks, heat sterilized at 100°C for 20 minutes, filtered and washed with water.
-100 aqueous solution (approximately 5 times the wet bacterial weight),
After dispersing by stirring and ultrasonication, the bacterial cells are further crushed using a French press (1500 Kg/cm ² ). After centrifugation at 40,000 x g for 30 minutes, 9 times the amount of ethyl alcohol was added to the supernatant obtained, stirred, and the precipitated fraction was collected by centrifugation at 5,000 x g for 15 minutes, followed by ethyl alcohol. Wash with ethyl ether and dry. The resulting precipitate is treated with amylase and amyloglycosidase in an acetate buffer, heated and then centrifuged to remove the denatured enzyme. Fractions with a molecular weight of 100,000 or more are concentrated by ultrafiltration and freeze-dried to obtain crude lipopolysaccharide. The procedure of dissolving this substance in pyridine and adding methyl alcohol to precipitate it is repeated three times to remove the solvent-soluble fraction, and then the procedure of washing with chloroform-methyl alcohol is repeated to remove the solvent-soluble fraction. do. Further, the precipitate was dissolved in a phosphate buffer and shaken with actinase E immobilized on Sepharose 4B in the phosphate buffer, the immobilized enzyme was removed, the solution was desalted by dialysis, and lyophilized. Obtain lipopolysaccharide. The yield was 0.3% of the wet bacterial weight. As a result of the analysis, the composition of this substance was 91% (w/w) of a polysaccharide whose constituent sugars were D-arabinose:D-mannose (1:2.5), mainly palmitic acid,
It was a lipopolysaccharide consisting of tuberculosis stearic acid and 9 (w/w)% saturated fatty acids of stearic acid. Example 2 Mycobacterium bovis BCG
(Mycobacterium bovis BCG) was cultured in Sauton medium at 37℃ for 5 weeks, and the resulting bacterial cells were washed with water, washed with acetone several times, degreased, dried, and suspended in water. crush using a French press (1500Kg/cm ² ). Final concentration 1.5 (W/
W) Add Triton X-100 so that it becomes %,
Continue stirring for 24 hours at room temperature. Centrifugation (40000×
g, 30 minutes) is treated in the same manner as in Example 1 to obtain lipopolysaccharide. The yield was about 1% of the dry bacterial cells. As a result of analysis, a polysaccharide whose main constituent sugar is D-arabinose:D-mannose (1:3)88
(W/W)% and 12% (W/W) of saturated fatty acids mainly consisting of palmitic acid and tuberculostearic acid. Example 3 Propionibacterium acnes C-7
(Propionibacterium acnes C-7) was statically cultured under anaerobic conditions for 4 days in a modified thioglycolic acid liquid medium supplemented with meat extract, and the bacterial cells obtained by centrifugation were washed with water. % sodium lauryl sulfate solution, treated with a 20 KHz ultrasonic cell disrupter for 5 minutes x 6 times, and then centrifuged (40000 x g, 30 minutes) to obtain a supernatant. Perform the same procedure to obtain lipopolysaccharide. The yield was 0.8% of the wet bacterial weight. As a result of analysis, polysaccharide 89 (W/
W)%, mainly C ₁₅ to C ₁₈ fatty acids 11 (W/W)
% composition. [Effect of the invention] The lipopolysaccharide according to the present invention is obtained from the order Actinomycetes and related bacteria, and contains D-arabinose and D-
It is a lipopolysaccharide in which fatty acids, mainly palmitic acid and tuberculostearic acid, are ester-bonded to a polysaccharide whose main constituent sugar is mannose, and it has antitumor effects, cell rejuvenation ability, It is an excellent substance with activities such as immunoenhancing effects, and is expected to be put to practical use as a tumor immunity enhancer or immunomodulator. Furthermore, the production method of the present invention allows lipopolysaccharide to be obtained from bacterial cells in a high yield through relatively simple operations, and is therefore advantageous for industrial production.

Claims (1)

[Scope of Claims] 1 80-95% (W/W) of a polysaccharide having a composition of 1 D-arabinose and 1-3 D-mannose;
A lipopolysaccharide consisting of 5-20 (W/W)% fatty acids. 2. Bacteria obtained by culturing Mycobacterium and Propionibacterium bacteria are extracted using a surfactant solution, enzymatic digestion with amylase and amyloglucosidase, and ultrafiltration to obtain a fraction with a molecular weight of 100,000 or more. A polysaccharide with a composition of 1 D-arabinose and 1 to 3 D-mannose, purified by a combination of method and solvent fractionation, 80-95% (W/W)
A method for producing a lipopolysaccharide, the method comprising obtaining a lipopolysaccharide comprising 5% to 20% (W/W) of fatty acids. 3 After crushing the bacterial cells obtained by culturing Mycobacterium and Propionibacterium bacteria, they were extracted with a surfactant solution, enzymatically digested with amylase and amyloglycosidase, and the molecular weight
Purified by a combination of ultrafiltration and solvent fractionation to obtain more than 100,000 fractions, a polysaccharide with a composition of 1 D-arabinose and 1 to 3 D-mannose 80 to 95
(W/W)% and fatty acid 5-20 (W/W)%.