JP2631686B2 - Novel steroid saponins, their production methods and novel uses of these derivatives - Google Patents

Description

Description: BACKGROUND OF THE INVENTION Prior Art The present invention relates to steroid saponins and a process for their preparation and novel uses of spirostanol derivatives derived from this steroid saponin.

Prior art Garlic is a perennial plant cultivated in China, Korea, Japan and other countries, and is generally known as tonic and tonic. It is used as

On the other hand, elephant garlic (Allium ampeloprasum L.) cultivated in the United States is also used, but it is also called giant garlic because of its large bulb compared to normal garlic, and it also has a weak garlic smell Therefore, it is also called odorless garlic.

By the way, it has been more than ten years since Saponin has made progress in research because of its difficulty in purification and isolation and its structural complexity.

The general characteristics of saponin are represented by its foaming properties in aqueous solution and its erythrocyte destruction (hemolysis), fish toxicity, and its ability to form complexes with cholesterol. Effects such as ulcers, promotion of lipid metabolism or antitumor have been found.

In addition, the drug absorption promoting action (JP-A-57-145816 and 58-145816)
(See Japanese Patent Application Laid-Open No. 57400/1987)
No. 488) has been reported.

By the way, in the study of garlic and its related compound elephant garlic, many reports have been recognized mainly on sulfur-containing compounds, but no report has been made on the steroid saponins obtained this time and related fields. .

SUMMARY OF THE INVENTION The present invention relates to a steroid saponin obtained from a plant of the genus Allium, a method for producing the same, and a novel use of a spirostanol derivative derived from the steroid saponin.

That is, the steroid saponin according to the present invention is represented by the following formula (1).

In the formula, R ₁ represents a hydrogen atom or a hydroxyl group, and R ₂ represents -β
-Galactose ⁴ -β-glucose, -β-galactose ⁴ -β-glucose ³ -β-glucose or -β-
Shows galactose ⁴ -β-glucose ( ³ -β-glucose) ² -β-glucose.

(1) The method for producing a steroid saponin represented by the above formula (1) is characterized in that the steroid saponin is obtained from a plant of the genus Allium.

The antifungal agent of the present invention contains a spirostanol derivative obtained by treating a steroid saponin represented by the above formula (1) with an acid or an enzyme, as an active ingredient.

Effects The steroid saponin in the present invention is a novel compound, and a new physiological activity can be expected in addition to the above-mentioned general saponin drug efficacy.

The method for producing these steroid saponins is described in Alli
By using a plant of the um genus as a raw material, industrial production is possible and it is extremely useful because it is considered to be highly safe.

Spirostanol derivatives obtained by treating these steroid saponins with acids or enzymes can be said to be useful for treating various infectious diseases because of their antifungal activity.

DETAILED DESCRIPTION OF THE INVENTION Steroidal saponin The steroidal saponin in the present invention is defined by the above formula (1)
And a salt may be formed with any compound as necessary. Specific examples of these compounds together with their physicochemical properties are as follows.

Compound _{1 (R 1 = H, R} 2 = -Gal 4 -Glc (3 -Glc) 2 -Gl
c) Appearance: white powder Elementary _{analysis: C 57 H 96 O 30 .5H} 2 O _{^{polarimetry: ▲ [α] 27 D ▼}} -36.5 ° (C = 0.7, pyridine) ¹³ C NMR (pyridine -d _5): later tables 1 and 2 refer to ¹ H NMR (pyridine -d _5): anomeric proton 5.58 (1H, d, J = 7.0Hz), 5.31 (1H, d, J = 7.6Hz) 5.16 (1H, d, J = 7.6 Hz) 4.95 (1H, d, J = 7.3Hz) 4.82 (1H, d, J = 7.9Hz) compound _{2 (R 1 = OH, R} 2 = Gal 4 -Glc) appearance: white powder Elementary analysis: C ₄₅ H ₇₆ O ₂₁ .3H ₂ O _{^{polarimetry: ▲ [α] 21 D ▼}} -40.7 ° (C = 1.03, pyridine) ¹³ C NMR (pyridine -d _5): the following tables 1 and 2 refer to ¹ H NMR (pyridine -d _5): anomeric proton 5.27 (1H, d, J = 7.7Hz), 4.99 (1H, d, J = 7.7Hz) 4.82 (1H, d, J = 7.7Hz) compound _{3 (R 1 = OH, R} 2 = -Gal ⁴ -Glc ³ -Glc) appearance: white powder Elementary _{analysis: C 51 H 86 O 26 .2} 1 / 2H 2 O _{^{polarimetry: ▲ [α] 21 D ▼}} -25.1 ° (C = 1.05, pyridine) ¹³ C NMR (pyridine-d ₅ ): see Tables 1 and 2 below Irradiation ¹ H NMR (pyridine -d _5): anomeric proton 5.26 (1H, d, J = 8.1Hz), 5.23 (1H, d, J = 7.3Hz) 4.98 (1H, d, J = 7.3Hz), 4.81 (1H, d, J = 7.7 Hz) Note that Gal indicates β-galactose and Glc indicates β-glucose.

Allium genus plant The Allium genus plant referred to in the present invention is a lily family (Liliacea
e), a plant belonging to the genus Allium, specifically, Aryum.
Sativam. Linne (Allium sativum L.) and allium ampeloprasum L. (commonly called elephant garlic).

The part that can be used as a material for obtaining the target substance is particularly a bulb part (containing a split bulbous bulb formed inside), which can be frozen or extracted as it is. Can be offered.

Production method The production method of the present invention is characterized in that the production method is obtained from a plant belonging to the genus Allium. That is, it can be obtained by immersing in water or a water-containing or non-water-containing organic solvent that is miscible with water and performing extraction.

(A) Extraction Extraction can be performed by any method commonly used in the field of crude drugs. At this time, the organic solvent to be used as the extracting agent is a lower alcohol (having 1 to 1 carbon atoms).
Trihydric alcohol is common), water or water such as acetone or acetonitrile, and particularly preferable is a monohydric alcohol having 1 to 3 carbon atoms.

Extraction can be performed under warm or normal temperature,
It is preferable to perform immersion at 50 to 80 ° C. for about 2 to 4 hours. Also,
Needless to say, in order to increase the extraction efficiency, the target plant is preferably crushed.

(B) Fractionation After the extraction solvent was distilled off, the extract was subjected to column chromatography using a polystyrene-divinylbenzene copolymer resin, and then mixed with water and then water-containing or non-water-containing. Elute sequentially with possible organic solvents and collect fractions eluting with water-containing or non-water-soluble organic solvents that are miscible with water.

Here, the organic solvent includes, as described above, a water-miscible solvent such as a lower alcohol (a monohydric alcohol having 1 to 3 carbon atoms is common), acetone or acetonitrile, and particularly preferable is a carbon alcohol. 1 of Equations 1-3
It is a valence 4 alcohol.

(C) Purification by normal-phase and reverse-phase column chromatography After evaporating the solvent of the above fractions, the target substance, steroid saponin, is purified by normal-phase column chromatography and then by reverse-phase column chromatography. Obtainable.

Normal phase column chromatography here refers to column chromatography using a highly polar resin such as silicic acid or silica gel as a solid support, and the mobile phase is usually a non-phased material such as chloroform, dichloromethane, hexane or ethyl acetate. A solution having a smaller polarity than a solid phase carrier such as a mixture of a polar solvent and a water-containing or non-water-containing lower alcohol can be used. As a specific example of the above method, purification can be performed using silica gel chromatography [developing solvent: chloroform-methanol-water (6: 4: 1)] as described in Examples below.

On the other hand, reverse-phase column chromatography is performed by chemically bonding a silane having a hydrocarbon residue having 1 to 18 carbon atoms such as dimethylsilane, octadecylsilane, and octylsilane to the above silica gel or the styrene-divinylbenzene system. This refers to column chromatography using a low-polarity resin such as a copolymer or a gel filtration agent as a solid support, and any solvent having a higher polarity than the above-mentioned solid support can be used as a mobile phase.

For example, a water-containing or non-water-containing lower alcohol or a mixed solution of water and an organic solvent miscible with water such as acetone or acetonitrile can be used, but the former water-containing or non-water-containing lower alcohol is preferably used. As used herein, the lower alcohol is generally a monohydric alcohol having 1 to 3 carbon atoms as described above.

Spirostanol derivative The spirostanol derivative according to the present invention is obtained by treating the steroid saponin represented by the above formula (1) with an acid or an enzyme. Specific examples of these compounds are spirostanol derivatives represented by the following formula.

In the formula, R ₁ represents a hydrogen atom or a hydroxyl group, and R ₂ represents -β
-Galactose ⁴ -β-glucose, -β-galactose ⁴ -β-glucose ³ -β-glucose or -β-
Shows galactose ⁴ -β-glucose ( ³ -β-glucose) ² -β-glucose.

Further, specific examples of the above series of compounds together with their scientific properties are as follows.

Compound _{4 (R 1 = H, R} 2 = -Gal 4 -Glc (3 -Glc) 2 -Gl
c) Appearance: White powder Light intensity: ▲ [α] ²⁷ _D ▼ -62.6 ゜ (C = 0.4, chloroform-methanol (10: 1)) ¹³ C NMR (pyridine-d ₅ ): See Tables 1 and 2 above ¹ H NMR (pyridine -d _5): anomeric proton 5.59 (1H, d, J = 7.3Hz), 5.31 (1H, d, J = 7.6Hz) 5.16 (1H, d, J = 7.9Hz) 4.95 (1H, d, J = 7.6 Hz) Compound 5 (R ₁ = OH, R ₂ = -Gal ⁴ -Glc) Appearance: colorless needle-like crystal Melting point: 249 to 251 ° C. Light intensity: ▲ [α] ¹⁷ _D ▼ -48.3 ゜ ( C = 1.10, pyridine) ¹³ C NMR (pyridine-d ₅ ): see Tables 1 and 2 above ¹ H NMR (pyridine-d ₅ ): anomeric proton 5.26 (1H, d, J = 7.7 Hz), 4.98 (1H) , d, J = 7.7 Hz) Compound 6 (R ₁ = OH, R ₂ = -Gal ⁴ -Glc ³ -Glc) Appearance: colorless needle crystal Melting point:> 300 ° C. Light intensity: ▲ [α] ¹⁷ _D ▼- 52.5 ° (C = 1.12, pyridine) ¹³ C NMR (pyridine -d _5): table 2 reference ¹ H NMR (pyridine -d _5): anomeric proton 5.26 1H, d, J = 7.7Hz, 5.23 (1H, d, J = 7.7Hz), 4.97 (1H, d, J = 7.7Hz) It is needless to say that may be formed.

The acid used here is hydrochloric acid, sulfuric acid, acetic acid, formic acid,
It is an arbitrary inorganic or organic acid such as phthalic acid, and β-glycosidase or β-glucosidase can be used as the enzyme.

Antifungal agent The antifungal agent according to the present invention comprises the above-mentioned series of spirostanol derivatives as active ingredients. This antifungal agent is particularly useful in mammals, including humans, for local fungal infections by species of the genus Candida, Trichophyton, Microsporum or Epidermophyton, or Candida. It can also be used to treat mucosal infections caused by Albicans. Also, for example, Candida. Albicans, cryptococcus. Neoformans, Aspergillus. Flavas, Aspergillus. It can be used to treat systemic fungal infections by fungi of the genus Fumigatus, Coccidioides, Paracoccidioides, Histoplasma or Blastomyces.

The antifungal agent according to the present invention is useful not only for treating fungal infections in animals, but also for plant fungi caused by antifungals.

The antifungal agent can be used alone or in combination with liquid or solid pharmaceutical auxiliary ingredients such as excipients, binders,
And a diluent, and can be administered orally or parenterally in any form such as powder, granules, capsules, injections, ointments, aerosols, and liquids.

Further, other medicines may be prepared as needed. The dose may be adjusted according to the age, body weight and symptoms. However, orally, 10 mg to 10 g, preferably 50 mg per day per adult
About 5g.

Experimental Example Manufacturing Method Experimental Example 1 After 4 kg of garlic bulb was frozen and crushed in methanol 6, the extract was heated and extracted at a bath temperature of about 70 ° C. After filtration, the residue was further extracted by heating with methanol 2. After combining the extracts, methanol was distilled off, and water was added to adjust the total amount to about 3.

 Use this extract as MCI GEL  By CHP 20P (Mitsubishi Kasei)
Column chromatography, water, 20% methanol
Elute sequentially with methanol and methanol.
8.6 g of a crude saponin fraction was obtained from the obtained fraction. To this mixture
About silica gel chromatography [Developing solvent:
Roform-methanol-water (6: 4: 1)]
A crude fraction containing compound 1 was obtained (0.6 g). In addition, the book
Per fraction, MCI GEL  Reversed-phase column chromatography with CHP 20P
Perform the chromatography (developing solvent: 80% methanol),
The obtained fraction (0.49 g) was mixed with a solution of acetone: water (2: 5).
After heating at 95 ° C for 4 hours, the solvent was distilled off to obtain Compound 1.
(0.45 g).

Experimental Example 2 2 kg of garlic bulbs were crushed in water 3 and brought to room temperature.
And extracted overnight with MCI GEL as in Experimental Example 1.
The crude saponin fraction 5.
0 g was obtained. For this mixture, use silica gel chromatography.
Raffy [Developing solvent: chloroform-methanol-water
(7: 3: 0.5)] and MCI GEL  Reverse phase by CHP 20P
Chromatography (developing solvent: 90% methanol)
The compound 4 (26 mg) was obtained.

Experimental Example 3 5 kg of elephant garlic bulb was frozen, crushed in methanol 3, and then heated and extracted at a bath temperature of about 80 ° C.
After filtration, the residue was further extracted twice with methanol 3 while heating. After combining the extracts, methanol was distilled off.

Extract the extract with Amberlite XAD-2 (Organo)
And eluted sequentially with 30% methanol, methanol and chloroform, to obtain 12.9 g of a crude saponin fraction from the fraction eluted with methanol.
About this fraction, silica gel chromatography [Developing solvent: chloroform-methanol-water (7: 3: 0.5 → 6:
4: 1)] and fractionated into fractions Nos. 1-3. Among them, No. 3 was subjected to silica gel chromatography [developing solvent: chloroform-methanol-water (7: 3: 0.5)] to obtain two fractions.

 Furthermore, for each fraction, silica gel chromatography
Graphy and MCI GEL  Chromatography with CHP 20P
Repeat the feed to make a solution of acetone-water (1: 2)
After heating at 00 ° C., the solvent was distilled off, and Compound 2 (0.13 g) and
And compound 3 (0.53 g).

Experimental Example 4 Elephant garlic 2.9 kg in ethyl acetate 2.5
After crushing, the ethyl acetate layer was removed and the resulting residue was
Heat extraction was performed three times with methanol 3. Combine the extracts
After that, the solvent was distilled off under reduced pressure.
Light XAD-2 to give crude saponin fraction (8.08g)
Was. This fraction is subjected to silica gel chromatography [Development
Dissolution: chloroform-methanol-water (7: 3: 0.5 or
6: 4: 1)] and Lichroprep Rp−
8 (Merck) (Developing solvent: 8
0% methanol), compound 5 (1.28 g) and
Compound 6 (0.15 g) was obtained.

Experimental Example 5 Compound 1 (0.25 g) was converted to β-glucosidase (0.25 g, a
0.1 M acetate buffer (p.L.Bio Chemical)
H4.3) After reacting for about 2 hours at 37 ° C in 50 ml, add 50 ml of water to the reaction solution.
Add MCI GEL  Fill the column with CHP 20P
And elute sequentially with water, 50% methanol and methanol
The fraction eluted with methanol was distilled off under reduced pressure to give Compound 4
Was obtained (0.17 g).

Confirmation of Structure Compound I is positive for Erich reaction and is presumed to be florstanol saponin consisting of 4 molecules of glucose and 1 molecule of galactose from acid hydrolysis and NMR. As already reported, as shown in Experimental Example 5, compound 4 obtained from compound 1 was converted to spirostanol saponin by β-glucosidase to give spirostanol saponin. From the results, it is considered that spirostanol saponin is composed of three molecules of glucose and one molecule of galactose.

As a partial hydrolyzate of compound 4 with 5% sulfuric acid, β
-Chlorogenin, β-chlorogenin 3-0-β-galactopyranoside and β-chlorogenin 3-0-glucopyranosyl (1 → 4) -β-galactopyranoside were identified, and alditol derived from compound 4 From the examination of the sugar binding mode of the acetate pair by GC-MS, Compound 4
At the 3-position of β-chlorogenin [β-Glc (1 →
2)]-[β-Glc (1 → 3)]-β-Glc (1 → 4)-
It was determined that the saponin bound to the sugar unit of β-Gal.

Therefore, Compound 1 was a proto form of Compound 4, and was determined to be florstanol saponin with β-glucopyranose bound at position 26 thereof.

Structural analysis was also performed on compounds 2, 3, 5, and 6 by the same method, and the structures were determined in the same manner.

Antibacterial Activity The steroid saponin derivatives 4 to 6 according to the present invention were examined for growth inhibitory activity against Candida albicans according to a conventional method (liquid dilution method). The results are as shown in Table 3 below.

 ──────────────────────────────────────────────────の Continued on front page (72) Inventor Norihiro Hayashi 1624 Shimodate, Koda-cho, Takada-gun, Hiroshima Prefecture

Claims (4)

(57) [Claims] 1. A steroid saponin represented by the following formula: In the formula, R ₁ represents a hydrogen atom or a hydroxyl group, and R ₂ represents -β-
Galactose ^4-- β-glucose, -β-galactose
⁴ -β-glucose ³ -β-glucose or -β-galactose ⁴ -β-glucose ( ³ -β-glucose)
¹ shows ² -β-glucose. 2. A method for producing a steroid saponin represented by the following formula, which is obtained from a plant of the genus Allium. In the formula, R ₁ represents a hydrogen atom or a hydroxyl group, and R ₂ represents -β-
Galactose ^4-- β-glucose, -β-galactose
⁴ -β-glucose ³ -β-glucose or -β-galactose ⁴ -β-glucose ( ³ -β-glucose)
¹ shows ² -β-glucose. 3. An antifungal agent comprising, as an active ingredient, a spirostanol derivative obtained by treating a steroid saponin represented by the following formula with an acid or an enzyme. In the formula, R ₁ represents a hydrogen atom or a hydroxyl group, and R ₂ represents -β-
Galactose ^4-- β-glucose, -β-galactose
⁴ -β-glucose ³ -β-glucose or -β-galactose ⁴ -β-glucose ( ³ -β-glucose)
¹ shows ² -β-glucose. 4. The antifungal agent according to claim 3, wherein the spirostal derivative is represented by the following formula: In the formula, R ₁ represents a hydrogen atom or a hydroxyl group, and R ₂ represents -β-
Galactose ^4-- β-glucose, -β-galactose
⁴ -β-glucose ³ -β-glucose or -β-galactose ⁴ -β-glucose ( ³ -β-glucose)
¹ shows ² -β-glucose.