JPS60100594A - 3alpha,15beta-dihydroxy-5beta-cholanic acid and its production by microbial conversion process - Google Patents

Abstract

NEW MATERIAL:The compound of formula I (R is H or alkali metal) or its salt. USE:An agent for dissolving cholesterol gall stone. PREPARATION:Cunninghamella echinulate ST-22 strain (FERM-P No.7328) which is a mold belonging to Cunninghamella genus and capable of converting the 15beta- site of lithocholic acid (salt) to hydroxyl, is cultured, and the mold is made to contact with the lithocholic acid (salt) of formula II. The objective compound of formula I can be produced by recovering the 3alpha, 15beta-dihydroxy-5beta-cholanic acid (salt) converted from the lithocholic acid (salt).

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a 3α compound represented by the structural formula (1) shown in FIG.
, 15β-dihydride I'lxy5β-cholanic acid or a salt thereof, and the second invention related to this invention is the conversion of 3α,15β-dihydride from lithocholic acid or its salt by a method of microbial conversion. The present invention relates to a method for producing xy-5β-cholanic acid or a salt thereof.

3α,15β-dihydro+-nitocy 5β-cholanic acid as a new substance according to this invention and the second invention related thereto (hereinafter referred to as the present invention) is represented by the structural formula (1) shown in FIG. It is one of the dihydroxy bile acids consisting of colanic acid, which has carbo, 1-syl groups in the side chain and hydroxyl groups at the 3α and 15β positions.

This substance is ursodeoxycholic acid (3α,7β-dihydroxy-5
Like β-cholanic acid, it belongs to dihydroxy bile acids, and the hydroxyl groups in its molecular structure are oriented such that 3α hydroxyl groups are oriented on the lower side of the ring surface, and 15β hydroxyl groups are oriented on the upper side of the ring surface. This is similar to the orientation of the hydroxyl groups in ursodeoxycholic acid, and it is thought to have the same effect in terms of micelle-forming ability with lecithin and cholesterol, making it a new substance that can be used as a cholesterol gallstone dissolving agent. .

Regarding the production method of ursodeoxycholic acid, which is used as a gallstone dissolving agent, by the method of microbial conversion, ursodeoxycholic acid as a starting material is used as a gallstone dissolving agent.
A method for producing ursodeoxycholic acid as a target substance in a single step by contacting molds that succumb to .
No. 098.

Since such a microbial conversion method requires only one reaction step, it has the advantage that the reaction time is short and there are very few undesired side reactions, compared to conventional chemical synthesis methods. However, the disadvantage is that the types of dihydroxy bile acids that can be produced by this microbial conversion method are extremely limited, and to date, ursodeoxycol produced using the above-mentioned fungi belonging to the genus Fusarium has been used. Nothing other than acid is known.

Therefore, in order to increase the types of dihydroxy bile acids, which are gallstone dissolving agents, that can be produced by a microbial conversion method, the inventors of the present application have developed a dihydroxy bile acid that can be produced from monocholic acid or its salt by a microbial conversion method. In a search for a substance that has a gallstone-dissolving effect7), one of the filamentous fungi that succumbs to the genus Canninghamera (Harisushi Shizangetsuguha) has been found to be a new substance, 3α, 15β-dihydroxy, derived from lithocholic acid or its salts. The present inventors have discovered that it has the ability to convert into -5β-furanic acid or a salt thereof, and have completed the present invention.

That is, the object of the present invention is, in view of the problem of limitations in the types of gallstone dissolving agents that can be produced by conventional microbial conversion methods, to produce gallstone dissolving agents from tocholic acid or its salts as a starting material using microbial conversion methods. By making it possible to produce a new target substance, 3α,15β-dihydroxy-5β-cholanic acid or its salt, which is a bile acid, the above-mentioned drawbacks can be eliminated, and gallstone dissolution can be achieved by a method of microbial conversion. The aim is to increase the variety of drugs available, thereby increasing the degree of freedom in drug prescription.

The structure of the present invention in accordance with the above object is a novel substance specified by the structural formula shown in FIG. As shown in FIG. 2, the composition of the invention is as follows: one of the specific filamentous fungi belonging to the genus Cunninghamella is used as a starting material tocholic acid or a salt thereof (■).
642, 3α, 15β-dihydroxy-5 as a novel target substance was introduced by specifically introducing a hydroxyl group into the β-type at the carbon atom at the 15th position of the steroid ring.
The gist of the present invention is to produce β-cholanic acid or its compound ((I)).

Next, the configuration of the second invention will be detailed as follows.

A strain of a filamentous fungus belonging to the genus Cunninghamella that has the ability to 15β-hydroxylate lithocholic acid or its JLI, typically L.
A method for producing 3α, 15β-dihydroxy-5β-cholanic acid or its compound 1 using a microbial conversion method by contacting part or all of lithocholic acid or a salt thereof (referred to as strain 22) with lithocholic acid or a salt thereof. Yes, the salts refer to alkali metal salts such as sodium and potassium.
Specifically, the following methods are included.

+11 By culturing the above-mentioned specific filamentous fungus in a culture medium containing lithocholic acid or its salt, 3α,15β-dihydroxy-5β-cholanic acid or its salt is accumulated in the medium and recovered. how to.

(2) By bringing the fungal cells obtained by culturing the above-mentioned specific filamentous fungi in a culture medium in which they grow well into contact with lithocholic acid or its salt in a reaction solution, 3α, 15β
- A method for producing and recovering dihydroxy-5β-cholanic acid or a salt thereof.

Furthermore, in the method (1) above, lithocholic acid or its salt can be added at an appropriate stage of the culture process.

Next, in the method (2) above, all or part of the bacterial cells (spores) once separated from the culture medium may be introduced into a solution or suspension of lithocholic acid or its salt.

Alternatively, all or part of the isolated bacterial cells may be immobilized using polyacrylamide, calcium alginate, etc., and then exposed to lithocholic acid or a salt thereof for 1 hour.

Furthermore, in the method (2) above, it is desirable to add an organic substance as an energy source, typically glucose, galactose and other carbohydrates, casein hydrolyzate, yeast extract, etc., to the reaction solution at a low concentration.

Next, the growth form of the 5T-22 strain, which is an example of the microorganism used in the method according to the second invention related to the present invention, in a culture medium will be explained as follows.

The strain was cultured on potato dext 1:I-su agar (P'D A
>When inoculated into a medium and cultured at 27°C, the color of the colony changes from white to gray, and the hyphae grow to a height of 1.5 cm. Conidiophores are branched into two or more branches, and no whorls are observed. . The tip of the branched conidiophore is a small club-shaped vesicle with an average diameter of 30 μm and a medium width.
At its tip, there is a spherical conidium with a diameter of 9 to 15 μm.
nidia) has grown on it. Thorns are densely distributed on the surface of conidia, and their length is 2.5 μm. This ST-22 strain can grow at 40°C.

A microscopic photograph (magnification: 180
0x) is shown in Fig. 3.

Regarding the ST-22 strain having the above-mentioned growth form, - C Zycha et al.'s classification (Zycha+ll□Siepmanr+, R
,+Cunn1n hamella Matrucho
t 1903. In Mucorales (Zych
a+I1. +and Siepmann, R, +ed.
) + LOW 3301LEIIRE VERLAG V
ON J, CRAMUR 1969,), I tried to identify it and found that the growth form was Cunnin hamellaechinulata (Cunnin hamellaechinulata).
This strain was completely identical to that of Cunnin ha etinulata (Cunnin ha etinulata).
mella echinulata) and Cunnin hamella echinulata.
a) Deposited as strain 5T-22 at the Institute of Microbial Technology, Agency of Industrial Science and Technology (Feikoken Bacterium Deposit No. 7628).

The physiological properties of this ST-22 strain are as follows.

+l) Optimal growth conditions (pH 1 temperature) The optimum growth pi is 5.0 and the optimum growth temperature is 33°C.

(2) Growth range (pH, temperature) The pn range is 3 to 8.5, and the temperature range is 20 to 40°C.

Next, the collection route of the ST-22 strain will be explained as follows.

100ml of filamentous fungi isolated from the soil of Sambul in Pangkok region, Thailand was placed in a 500ml container containing medium for FI+1.
Each strain was inoculated into a 1-volume Sakaro flask and cultured with shaking at 27°C for 7 days. After the completion of Section 5, 20 ml of the culture solution was adjusted to pH 113 with 5N hydrochloric acid and extracted with 50 ml of ethyl acetate. After adding Glauber's salt to this extract and dehydrating it, it was concentrated under 4-vacuum using a rotary evaporator, and the concentrated liquid was qualitatively analyzed by chromatography, and a main spot was detected in the fraction corresponding to the Rf value of dihydroxy bile acid. We screened the strains that could be used.

As a result, 3α, 15β from lithocholic acid or its salt
Strain ST-22 was obtained as a strain having good conversion ability to -dihydroxy-5β-cholanic acid or its salt.

As a culture medium for isolation of bacterial strains, 40g of glucose and 3g of 5KCI per 1°C of distilled water.

tall, pn42g, Mg5O+・71120
0.5g, CaC12(1,51;, Fe5Oc 7H
t0 10mg, Zn5O< 10mg5Mn504
H1■, CuS0.110l11. NaMo0.10
mg, 6'l mother kiss 1g.

and Nori Tokono as a conversion reaction substrate! ) A medium containing 1 g of acid was used.

Next, embodiments of the present invention will be described as follows.

<Example 1> Dextrin 4 (Ig, asparagine 12 g, KCI 3 g, K11.+10°2 to 1 e of distilled water)
g, yeast extract Ig, CaCl20.5g, Mg
5 (L・711.0 0.5g, Fe50.・7H,
O]Omg, ZnS0410mg, Mn5041On
+g,,CuS0.10mgSNaMoOt10mg
and 1 g of lithocholic acid 67! A liquid medium of 10 μm was placed in a 10 i capacity fermenter, and the ST-22 strain, which had been cultured with shaking in a Sakalo flask at 27°C for 48 hours using a medium with the same composition, was added to the liquid medium in the fermenter at a concentration of 5%. The culture was cultured aerobically for 4 days at a stirring speed of 300 rpm and an aeration rate of 1 vvm while adjusting the culture temperature to 33° C. and ρ118. After the culture was completed, the culture solution was adjusted to pH 3 with 5N hydrochloric acid, extracted with 12j2 ethyl acetate, and dehydrated by adding Glauber's salt to this extract. After leaving it for another night, filter the Glauber's salt and collect the filtrate: 1. Concentrate with a vaporator, 1060■
1v of oily substance. Add this to silica gel (Wako Gel C)
-200) - adsorbed on a column packed with 300g,
Ethyl acetate: 2.2.4-trimethylpentane: Vinegar F
Elute with a mixed solvent consisting of 'IQ (5:5:1),
Both eluted fractions containing the target substance were collected and concentrated using an evaporator.

Furthermore, it was adsorbed in a column packed with 75 g of silica gel,
Chloroform: Acetone: Acetic acid (250: so: 3
), and the eluted fractions containing the target substance were collected and concentrated to dryness using an evaporator. Further, the sample obtained from the above fraction was crystallized from a small amount of ethyl acetate-hexane solvent to obtain 660 ml of crude crystals. This was recrystallized in ethyl acetate to obtain white crystals No. 59.

The purification yield was 61.7%.

<Example 2> The concentration of tocholic acid in the medium of Example 1 was 0, Ig/j!
Strain ST-22 was cultured for 48 hours in the closed medium at pH 7, aeration rate of 1 vvm, and stirring speed of 300 rpm. The bacterial cells were collected with gauze, washed with pH 7 phosphate buffer, and then mixed with KzllPO+ Ig, glucose
10g, yeast extract 0.5g, and lithocholic acid 0.
2 g, distilled water 11 (pH 8,0),
Suspend to a bacterial cell concentration of 20 g/It, 27
The conversion reaction was carried out for 60 hours at °C under oxygen bubbling.

Thereafter, 0.12 g (yield: 60%) of 3α,15β-dihydroxy-5β-cholanic acid crystals were recovered per 1 i of the reaction solution by the same operation as in Example 1.

The crystals recovered in Examples 1 and 2 of the present invention were chemically identified using the Hh' method listed below.

txt Mass spectrometry molecular ion peak is observed at 392 (M), so the molecular weight is 392.

(2) Elemental analysis Elemental analysis values are C: 7:(,38%, H: 10.30
%, O: 16.32%, and the molecular weight is 392, so the 111 constant molecular formula of this substance is C,411,04.

(3) The results of measurement using the infrared absorption Svector Nujol method are shown in FIG. Stretching vibration of hydroxyl group at 3280 cm, 170
Absorption indicating carbonyl stretching vibration is observed at 0 cm'.

(4) Light intensity (α3.) = -4-3,4° (c:, = 1.0
, HtOll) (5) Melting point is 185.5-186.5°C.

(6) C-N M R Sample measurement in heavy methanol solution went.

178.0ppm (sl C00tl (24th place) 72
．． 4 (dl C11(011) (15th or 3rd place) 70.6 (dl CH(O)l) (3rd place or 15th place)
position) 62.4 (dl C1+ (14th position or r),
7th place) 58.0 (d) C11 (14th place 0r 17th place) 43, 6 1s) 35.9 fsl 43.6-33.0 (d) 4 pieces 42.9-21.8ftllo book 24.0 (q ) 18 , 9 (ql 15 , 2 (ql (71'H-NMR (Figure 5)) Sample measurements were performed in a heavy benzene: heavy methanol (10:1) solution.

8. One-dimensional spectrum ■ 3.98ppIIIH-C-011 (+n13.5
2ppm H-C-011++++l From J value 3rd place 1.00ppm H, Ct+1 19th place 0.92ppm
Hs C 忰) 18th place 0.88ppm H, C(I Li 21st place 0.65ppm
Lock HC (d, d) - ■ Since 'H at the 18th position shifts down the magnetic field due to the appropriate sound, it is the hydroxyl group 12β, 15β or 168th position.

8. Two-dimensional spectrum ■ 3.98 ppm 1H is coupled with 2.211.1.36 and 0.65 ppm 'I(.

■ 2.28ppm and 1.3(5pt+H) of steel are coupled to each other, and are more geminal than the other.

The presence of a hydroxyl group at the 12th position is ruled out since the 1H value of 3.98ppI11 is between f-lene and methylene. 4゜■ 1H at 0.65ppm is further coupled with 'H at 1.82ppm. This "I-1" becomes 'H' at the 8th position when the hydroxyl group is at the 15th position, and at the 20th position when the hydroxyl group is at the 16th position.

■ Methyl at position 21 (0.88ppm) is 1.55p
It is coupled with pm's '1-1 (20th place).

Therefore, from ■ and ■, it was found that '+1 of 1.82 ppm was at the 8th position, so the hydroxyl group was located at the 15th position.

From the above, the molecular formula is C24+14 from mass spectrometry and elemental analysis.
0 o4 and that the substrate is lithocholic acid, it is determined that hydroxylation has occurred, and further,
``From the C-NMR spectrum, it was confirmed that the hydroxide was dihydro IJ bile acid.
According to the H-NMR spectrum, this crystal has 3α, 15β-
It was identified as dihydroxy-5β-=1lanic acid.

[Brief explanation of the drawing]

Figure 1 shows 3α and 15β as new substances according to this invention.
FIG. 2 is an explanatory diagram showing the structural formula of -dihydroxy-5β-cholanic acid, and FIG. 2 is an explanatory diagram showing the structural formula of -dihydroxy-5β-cholanic acid, and FIG. 3α, 15β-
FIG. 2 is an explanatory diagram showing a reaction formula leading to dihydroxy-5β-cholanic acid or a salt thereof. FIG. 3 is a microscopic photograph (11 B (10 times)) showing the growth form of Cunninghamella etinulata 5T-22 strain, which is an example of the microorganism according to the second invention.
The figure shows the target substance 3α,15β-dihydroxy-5β
FIG. 5 is an infrared absorption spectrum of -colanic acid, and FIG. 5 is its °II-NMR spectrum. Patent applicant: Yakult Hon R1 Figure 1 (R is 7), #Hara+Mamu (J sword young full bend,) Figure 2 (R is Mizunana Kyoko or Alkashi Metal) (If) (1 ) Engraving of the drawings (no change in content) Figure 3 Procedural amendment (method) Kazuo Wakasugi, Commissioner of the Japan Patent Office 1. Indication of the case 1988 Patent Application No. 20T26B 2. Title of the invention 3α, 15β-dihydroxy- 5β-Colanic acid and its production method by microbial conversion 3 Relationship with the case of the person making the amendment Patent applicant 4, attorney 6 Number of inventions increased by amendment 0

Claims (1)

[Scope of Claims] 3α,15β-dihydroxy-5β-furanic acid or a salt thereof represented by the following formula: (wherein R is a hydrogen atom or an alkali metal.) (2) A step of cultivating a filamentous fungus belonging to the genus Canninghamella that has the ability to 15β-hydroxylate lithocholic acid or a salt thereof; 3α, 15β- by microbial conversion characterized by comprising a step of contacting the lithocholic acid or the salt thereof, and a step of recovering the 3α, 15β-dihydroxy-5β-cholanic acid or the salt thereof converted from the lithocholic acid or the salt thereof. A method for producing dihydroxy-5β-furanic acid or a salt thereof. (3) 15β for the above lithocholic acid or its salt
Claim 1, wherein the step of contacting a filamentous fungus belonging to the genus Cunninghamera having hydroxylation ability with lithocholic acid or a salt thereof is a step carried out in a culture medium for culturing the filamentous fungus. 3 by microbial transformation described in Section 2
A method for producing α,15β-dihydroxy-5β-cholanic acid or a salt thereof. (4) further comprising the steps of collecting a filamentous fungus belonging to the genus Canninghamella that has the ability to 15β-hydroxylate lithocholic acid or a salt thereof from the culture medium, and preparing a reaction solution in which the filamentous fungus is suspended. 3α,15β-dihydroxy-5β-furanic acid or How to make that salt.