JPS6230759B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is based on the formula Hopan-22-ol and the formula The present invention relates to a method for producing hopen-b represented by:

Hopan-22-ol and hopen-b obtained according to the present invention exhibit antiviral activity against viral infections in warm-blooded animals, and are therefore expected to be useful drugs.

Conventionally, hopan-22-ol and hopen-b are known to exist in plants such as odontocetes, bryophytes, and lichens, but in all of these cases the content is low, and these plants are It is difficult to obtain in large quantities and is not suitable for production on an industrial scale.

In addition, it has recently become clear that they exist in the metabolic products of microorganisms such as Methylococcus cabusulatus, Bacillus acidocaldarius, Acetobacter xylinum, and Acetobacter lancens, but the amount produced is small and is not suitable for industrial use. It is far from being produced on a large scale.

As a result of intensive research into the fermentative production of the above substances by microorganisms, the present inventors found that bacteria belonging to the genus Pseudomonas contain hopane-22-
It was found that large amounts of ol and hopene-b were accumulated.

According to the present invention, hopan-22-ol and hopen-b producing bacteria belonging to the genus Pseudomonas are cultured in a medium to produce hopan-22-ol and hopen-b.
Hopane produces and accumulates these substances, and collects these substances from the bacterial cells individually or as a mixture.
A method for producing 22-ol and hopene-b is provided.

To date, there is no knowledge that would allow us to predict that strains belonging to the genus Pseudomonas would produce hopane and hopene-based substances in metabolic products in proportions that would enable production on an industrial scale.

The hopan-22-ol and hopen-b producing bacteria belonging to the genus Pseudomonas suitable for use in the method of the present invention include Pseudomonas AM1.
(NCIB9133), Pseudomonas M27 [Biochem.J.
92 , 609 (1964)], Pseudomonas N842 Microtechnical Research Institute No.
3154 (Japanese Unexamined Patent Publication No. 52-47990), Protaminobacter Ruber [Journal of the Japanese Society of Agricultural Sciences, Volume 52, Page 477 (1978)]
Examples include: All of these strains themselves are known and available.

The culture medium in the present invention is not particularly limited; for example, as a carbon source, hydrous carbon such as glucose, organic acids such as citric acid, methanol, alcohols such as glycerin, etc. can be used, and as a nitrogen source, ammonium sulfate, urea, ammonium nitrate, Ammonium phosphate, ammonium chloride, ammonia gas, etc., inorganic substances such as phosphates, magnesium salts, calcium salts, iron salts, manganese salts, etc., and other trace metal salts as necessary, and growth-promoting substances such as amino acids, vitamins, and soybeans. Protein hydrolysates, yeast extracts, peptones, casamino acids, etc. can be used.

For culture, 20 to 40℃ at pH 5 to 8.
Culture with aerobic shaking or stirring for about 10 days.
The target substance can be collected by, for example, separating and collecting bacterial cells from a culture solution using a conventional method such as centrifugation or filtration, and then extracting the obtained bacterial cells with an organic solvent. In this case, the microbial cells can be either live microbial cells, dried microbial cells, a hydrolyzed product of microbial cells, or a microbial cell-treated product such as a product treated with ultrasonication. The extraction method is to add a mixture of methanol, caustic soda, and pyrogallol to the solution containing bacterial cells, and then to
A method of heating under reflux for ~3 hours, or methanol,
20°C to 80°C with organic solvents such as ethanol and acetone
Any method such as extraction can be adopted. The resulting extract or extract concentrate is extracted with a solvent such as hexane, the solvent layer is washed with water, dehydrated and concentrated, and this is added to a column such as silica gel and eluted with benzene or a hexane/ether mixture. Hopan-22-ol and hopen-b elute. After further purifying this fraction by thin layer chromatography, column chromatography, etc. as necessary,
When dissolved in a solvent such as ethyl acetate or hexane, and the resulting solution is left to cool, white crude crystals are obtained. Further repeating recrystallization yields the target substance as pure crystals. According to the present invention, hopan-22-ol and hopene-b can be supplied at low cost and in large quantities.

Example 1 NH ₄ H ₂ PO ₄ 0.4%, KH ₂ PO ₄ 0.2%, Na ₂ HPO ₄ .
_12H2O0.3 %, _MgSO4・_7H2O0.02 %, _CaCl2・
_2H2O0.001 %, _FeSO4・_7H2O0.0005 %,
_MnSO4・_nH2O0.0005 %, _CoSO4・_7H2O0.0001 %
Pseudomonas AM1 was added to 10 500ml triangular bottles containing 100ml each of medium (PH7.2) containing 1ml of methanol per 100ml of medium.
(NCIB9133), added 1 ml of methanol each on the 2nd and 3rd day, cultured with shaking at 30℃ for 4 days, and centrifuged to obtain 11 g of bacterial paste.
I got it. The obtained wet bacterial cells were mixed with 30 ml of water and methanol.
200ml, 4g pyrogallol and 30g caustic soda
The mixture was heated under reflux at 85°C for 1 hour. After cooling,
Extraction was carried out three times with 200 ml of hexane, and the hexane layer was taken, washed with water, dried over Glauber's salt, and concentrated to dryness. The residue is dissolved in hexane, and this solution is developed with an ether/hexane mixture using a silica gel column to collect fractions containing hopen-b and hopen-22-ol, respectively. Each fraction is further purified by thin layer chromatography. Each fraction is eluted from the thin layer with ether and dried. Repeated recrystallization three times with ethyl acetate yielded 3 mg of hopene-b crystals and 7.5 mg of hopane-22-ol crystals.
I got mg.

Example 2 Pseudomonas M27 was grown in the same medium as in Example 1.
[Biochem. J. 92 , 609 (1964)] was cultured and centrifuged to obtain 53 g of bacterial cell paste. 400 ml of acetone was added to the bacterial cell paste, and the mixture was heated at 55°C for 2 hours to perform extraction three times. The acetone extracts were combined, concentrated to dryness, dissolved in hexane, and subjected to silica gel column chromatography, thin layer chromatography, and crystallization in the same manner as in Example 1. hopane-b crystal 1 mg and hopane-22
-30 mg of all crystals were obtained.

Example 3 Pseudomonas N842 Microken No. 3154 (Japanese Unexamined Patent Publication No. 52-47990) was added to medium 15 consisting of 2% glucose, 1% heptone and 1% yeast extract (PH6.5).
was cultured for 48 hours in 300 ml of a medium with the same composition and then inoculated as a seed. 48 at 30°C with aeration of 15 air per minute using a 30 volume jar fermenter.
Wet bacterial cell paste is made by centrifugation after agitating culture for a period of time.
Obtained 300g. The obtained wet bacterial cells were mixed with 300 ml of water, 2 methanol, 40 g of pyrogallol and caustic soda.
300g and heated under reflux at 85°C for 1 hour. After cooling, the mixture was extracted three times with hexane (2), and the hexane layer was taken, washed with water, dried over Glauber's salt, and concentrated to dryness. The dry matter was dissolved in hexane, the solution was developed using a silica gel column with an ether/hexane mixture, and hopene-b and hopane-22-
Each fraction containing ol is collected. After each fraction was purified again by silica gel column chromatography, the solvent was distilled off and the dried product was recrystallized three times from ethyl acetate. Hopen-b
24 mg of crystals and 180 mg of hopan-22-ol crystals were obtained.

Example 4 Protaminobacter ruber [Journal of Japan Society of Agricultural Sciences, Vol. 52, p. 447 (1978)] was cultured in the same medium as in Example 1, and 34 g of bacterial cell paste was obtained by centrifugation. 400 ml of acetone was added to the bacterial cell paste, and the mixture was heated at 55°C for 2 hours to perform extraction three times. The acetone extracts were combined, concentrated to dryness, dissolved in hexane, and subjected to silica gel column chromatography, thin layer chromatography, and crystallization in the same manner as in Example 1. Hopen-b crystal 4.4mg
and 11.2 mg of hopan-22-ol crystals were obtained.

Claims (1)

[Claims] 1 Cultivate hopan-22-ol and hopen-b-producing bacteria belonging to the genus Pseudomonas in a medium to produce and accumulate hopan-22-ol and hopen-b, and extract these substances from the bacterial cells individually or as a mixture. A method for producing hopan-22-ol and hopen-b, the method comprising collecting hopan-22-ol and hopen-b.