JPS6358839B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a new method for producing Echinoside A, which is useful as an antifungal agent. Echinoside A is a patent application filed in 1982.
As described in No. 160181, it is an oligoglycoside with antifungal activity isolated from Actinopyga echinites JAEGER, which belongs to the genus Actinopyga of the sea cucumber (Holothuroidea), and has the following structure. It is expressed by the formula. The properties of Echinocide A are as follows. (1) Molecular formula (molecular weight) C ₅₄ H ₈₇ O ₂₆ SNa (1206) (2) Elemental analysis C ₅₄ H ₈₇ O ₂₆ SNa・2H ₂ O Calculated value C52.16; H7.38; S2.58 Experimental value C52 .41; H7.37; S2.42 (3) Melting point 228-230°C (80% EtOH) (4) [α] ¹² _D −6.0° (pyridine, C=1.36) (5) For wavelengths longer than 210 nm Does not exhibit ultraviolet absorption. (6) IR (KBr, cm ^-1 ) 3380 (vs ^* ), 2940 (s),
2850 (sh), 1760 (sh), 1745 (s), 1635 (s),
1460(s), 1385(s), 1260(s), 1070(s),
945 (w), 895 (w), 820 (w), 780 (w) *vs Very strong s Strong m Moderate w Weak (7) CHCl ₃ , not soluble in acetone. Poorly soluble in methanol, ethanol, and water. Soluble in aqueous methanol and aqueous ethanol. Soluble in DMSO and pyridine. (8) Color reaction (TLC) Color reagent Ehrlich...negative BCG...negative Anthrone...blue 2,4-DNP...orange 1%Ce (SO ₄ ) ₂・10%H ₂ SO ₄ ...reddish-purple potassium rhodizonate test...Positive (orange) (9) It has no odor and is a neutral substance. (10) Colorless needle crystals. (11) CD (MeOH, c=0.196), [θ] ₂₄₈ 0, [θ] ₂₂₃
-4000 (neg, max), [θ] ₂₁₆ 0, [θ] ₂₁₂ +4500
(end absorption) (12) Rf 0.08 TLC Kieselgel 60F ₂₅₄ developing solvent CHCl ₃ −MeOH−H ₂ O=65:
35:10 (lower layer) As a result of further research, the present inventors found that Bohadschia, which is different from the genus Actinopiga,
Genus Bohadschia graeffei SEM
We succeeded in extracting echinoside A at an extremely high yield using PER), and based on this, we completed the present invention. That is, the present invention is a method for producing echinocide A, which is characterized by extracting the body wall of the sea cucumber with a solvent. Solvent extraction is performed using water and/or a hydrophilic organic solvent that can dissolve Echinoside A, such as lower alcohols (eg, methanol, ethanol, propanol), acetone, and the like. The amount of solvent used is 1 part of the body wall of the sea cucumber.
For example, in the case of methanol, it is usually about 1 to 5 kg. It is preferable to perform extraction with an organic solvent in a water-containing state, but the water normally contained in the sea cucumber body wall can fulfill this role. Extraction is generally carried out efficiently by thermal extraction, usually at a temperature at or below the boiling point of the solvent, particularly conveniently carried out under reflux. The extract is concentrated under reduced pressure and the residue is partitioned with a mixture of water and an organic solvent (eg, butanol) that is immiscible with water and capable of dissolving Echinoside A. The ratio of water and water-insoluble organic solvent in the mixed solution is usually about 1:1, but can be adjusted as necessary. Echinoside A can be obtained in high yield by concentrating the organic solvent layer obtained by the above distribution under reduced pressure and then purifying the residue. Examples of purification methods include recrystallization and column chromatography. For purification by recrystallization, it is preferable to first remove the soluble portion by washing the concentrated residue with a suitable solvent or a mixture thereof in which Echinoside A is difficult to dissolve, and then perform a normal recrystallization operation. As the cleaning liquid, for example, a mixture of chloroform-methanol-water is conveniently used. The mixed solution is usually prepared in a volume ratio of 1 part water to 3 to 5 parts chloroform and 10 to 20 parts methanol. The washing is preferably carried out at room temperature or under cooling with stirring. After washing, the insoluble part (echinoside A) is collected by ordinary filtration and subjected to recrystallization. Examples of the recrystallization solvent include a mixture of chloroform-methanol-water, aqueous methanol, aqueous ethanol, and aqueous acetonitrile. When using the above-mentioned liquid mixture, the mixing ratio (volume ratio) is usually about 1 to 3 chloroform and 4 to 8 methanol to 1 part water. The recrystallization operation can be carried out according to a conventional method. According to the method of the present invention, echinoside A can be produced in crystalline form in a high yield simply by the above-mentioned solvent manipulation, and is extremely advantageous from an industrial standpoint. In a preferred embodiment of this method, for example, the body wall of a sea cucumber is thermally extracted with methanol, the concentrated residue of the extract is distributed between water and butanol, the butanol layer is concentrated, and the residue is mixed with chloroform, methanol, and water. There is a method for producing echinoside A in which echinoside A is crystallized by purification using a mixed solution. Echinoside A produced by this method has an excellent antifungal effect and has a broad antibacterial spectrum, so it can be used as an antifungal agent, such as an antifungal agent, an antitinea fungus agent, and an anticandida agent. It is useful for treating and preventing conditions such as athlete's foot (for example, athlete's foot) and candidiasis. Since Echinoside A has low toxicity, it can be used orally for the above purposes, but it is usually preferable to use it parenterally as an external preparation. When used as such an antifungal agent for external use, Echinoside A may be applied directly in the form of a fine powder or in the form of an extract containing Echinoside A, but it is usually applied in the form of a pharmaceutical composition with an appropriate carrier. It is preferable to use it in Pharmaceutical compositions as antifungal agents can be prepared, for example, by dissolving or dispersing Echinoside A in a suitable liquid carrier (e.g. a solvent) or by mixing it with a suitable solid carrier (e.g. diluent, filler). , or adsorbed thereto, and if necessary, further admixed with suitable additives such as emulsifiers, dispersants, suspending agents, spreading agents, penetrating agents, wetting agents, etc.
By adding mucilage agents, stabilizers, etc., the formulation can be made into solutions, granules, emulsions, suspensions, ointments, powders, sprays, pastes, poultices, etc. Specific examples of liquid carriers used include water, alcohols (eg, methyl alcohol, ethyl alcohol, glycol, glycerin, etc.), glycol ethers (eg, ethylene glycol monomethyl ether, diethylene glycol monomethyl ether, etc.), and the like. Specific examples of solid carriers include waxes (eg, polyethylene glycol 4000, spermaceti, wax, etc.). The amount of active ingredient in the antifungal agent is not limited, but when used for the purpose of treating athlete's foot, it is usually about 0.01 to 70% by weight of Echinoside A, more preferably about 0.1 to 70% by weight, based on the entire preparation.
It is 5% by weight. In addition, the antifungal agent in the present invention is similar to other known antifungal agents, such as keratolytic agents such as salicylic acid, other antibacterial agents such as undecylenic acid, griseofulvin, fuezathion, etc.
Other antibiotics such as penicillins, cephalosporins, streptomycin, fradiomycin, and tetracyclines may also be included. The above-mentioned antifungal agents are applied to the affected area once to several times a day in accordance with conventional methods, such as by coating or spraying. The present invention will be explained in more detail below using test examples and examples, but the scope of the present invention is not limited thereto. Test Example Antibacterial activity test of Echinocide A The minimum growth-inhibiting concentration of Echinocide A against the following test bacteria was determined using the multiple dilution method using an agar medium. The assay medium was a 1% glucose bouillon agar medium (PH6.0), and the test compound was dissolved in methanol and then diluted with sterilized water. The concentration in the medium is 1/
I adjusted it to be 10. Each test bacterium was cultured at 28°C for 3 days using the bacterial liquid smear inoculation method. The test results are shown in Table 1.

【table】

[Table] Example Production of Echinocide A Black sea cucumber Bohadschia graeffei (SEM
After cutting the body walls of 120 (14 kg) PER) into small pieces, they were heat-extracted (3 times) with methanol (20).
Residue obtained by concentrating methanol under reduced pressure (70g)
Partition water-butanol (ca. 10-ca. 10). The butanol layer was concentrated under reduced pressure, and chloroform (400 ml), methanol (1500 ml), and water (100 ml) were added to the resulting residue (230 g), and the mixture was heated at room temperature (15°C).
After stirring for 24 hours, filter the insoluble portion (130 g). 6 g of the insoluble portion was recrystallized from chloroform (80 ml) - methanol (240 ml) - water (40 ml) to obtain 5 g of echinoside A (45% from n-BuOH ext.). Echinocide A obtained from the above sea cucumber
is mixed with standard echinocide A obtained from Actinopyga echinites (Jaeger), [α] _D ,
¹³ Identified by CNMR.

Claims (1)

[Claims] 1. A method for producing echinocide A, which comprises extracting the body wall of a sea cucumber with a solvent. 2. In the method described in claim 1,
Extract the body wall of the sea cucumber with a hydrophilic organic solvent,
A method for producing echinoside A, which comprises distributing the extract between water and a water-insoluble organic solvent, and then crystallizing echinoside A from the organic solvent layer. 3. In the method described in claim 1,
The body wall of the sea cucumber is thermally extracted with methanol, the concentrated residue of the extract is distributed between water and butanol, the butanol layer is concentrated, and the residue is purified with a mixture of chloroform, methanol, and water to crystallize echinoside A. Method for producing echinocide A.