JPH0119399B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides a group of novel saponins, novel saponins isolated from Jiaogulan, and methods for their production. “Gynostemma pentaphyllum Makino” in this invention is a plant of the Cucurbitaceae family, and its scientific name is Gynostemma pentaphyllum Makino.
It also includes plants that are botanically related to this. Related plants include Gynostemma brumanicum king ex tyaklavatei (Gynostemma brumanicum
burmanicum King ex Chakravarty), Gynostemma laxum cogun (Gynostemma
laxum Cogn.), Gynostemma Integrifoliola
Cogn.), etc., but the total saponin containing a large amount of saponins according to the present invention can be efficiently obtained from Jiaogulan from Hyogo Prefecture. The inventors of the present invention previously isolated novel saponins from Jiaogulan, elucidated their structures, and obtained a group of novel compounds by hydrolyzing these saponins. As a result of further research, they succeeded in isolating a new group of saponins from Jiaogulan that are different from previous saponins. Thus, according to the invention, the following general formula (): [In the formula, R ¹ is β-glucopyranosyl group, β-glucopyranosyl (1→2)-β-glucopyranosyl group, or β-glucopyranosyl (1→2)-α
-Arabinopyranosyl group; R ² is a hydroxymethyl group or formyl group; R ³ is a hydroxyl group or a hydrogen atom; R ⁴ is a β-glucopyranosyloxy group, α-rhamnopyranosyl (1→6)-β- Glucopyranosyloxy group, β-xylopyranosyl (1→6)-β-glucopyranosyloxy group, methyl group or hydroxy group; R ⁵ is methyl group, hydroxy group, hydroxymethyl group or β-glucopyranosyl Oxymethyl group; However, when R ¹ is a β-glucopyranosyl group, R ² is a hydroxymethyl group, R ³ is a hydrogen atom, and R ¹ is a β-glucopyranosyl (1→2)-α-arabinopyranosyl group In this case, R ² is a formyl group and R ³ is a hydrogen atom. The compound represented by the general formula () is a novel saponin isolated from Jiaogulan, and the specific names are listed below. 19,21-dihydroxy-3β-0-β-glucopyranosyl-20ξ-0-β-glucopyranosyl-
Dammar-24-ene Hereinafter referred to as gypenoside; 19,20ξ,21-trihydroxy-3β-0-[β
-Glucopyranosyl(1→2)-β-glucopyranosyl]-dammar-24-ene Hereinafter referred to as gypenoside; 19,20ξ-dihydroxy-3β-0-β-glucopyranosyl-21-0-β-glucopyranosyl-dammar-24-ene Hereinafter referred to as gypenoside; 19-oxo-20ξ,21-dihydroxy-3β-0
-[β-glucopyranosyl (1→2)-β-glucopyranosyl]-dammar-24-ene Hereinafter referred to as gypenoside; 19-oxo-21-hydroxy-3β-0-[β
-Glucopyranosyl (1→2)-β-glucopyranosyl]-20ξ-0-[α-rhamnopyranosyl (1→6)-β-glucopyranosyl]-dammar-24-ene Hereinafter referred to as gypenoside; 19-oxo-21-hydroxy- 3β-0-[β
-Glucopyranosyl (1→2)-β-glucopyranosyl]-20ξ-0-[β-xylopyranosyl (1→6)-β-glucopyranosyl]-dammar-24-ene Hereinafter referred to as gypenoside; 19-oxo-3β-0- [β-glucopyranosyl (1→2)-α-arabinopyranosyl]-20S
-0- [α-rhamnopyranosyl (1→6)-β-
glucopyranosyl]-dammar-24-ene Hereinafter referred to as gypenoside; 19-oxo-3β-0-[β-glucopyranosyl (1→2)-α-arabinopyranosyl]-20S
-0-[β-xylopyranosyl (1→6)-β-
glucopyranosyl]-dammar-24-ene Hereinafter referred to as gypenoside; 12β,19,20S-trihydroxy-3β-0-
[β-glucopyranosyl(1→2)-β-glucopyranosyl]-dammar-24-ene Hereinafter referred to as gypenoside; 12β,19,20R-trihydroxy-3β-0-
[β-glucopyranosyl(1→2)-β-glucopyranosyl]-dammar-24-ene Hereinafter referred to as gypenoside; 19-oxo-12β,20R-dihydroxy-3β-
0-[β-glucopyranosyl(1→2)-β-glucopyranosyl]-dammar-24-ene Hereinafter referred to as Gypenoside L; 19,20R-dihydroxy-3β-0-[β-glucopyranosyl(1→2)-β- Glucopyranosyl]-dammar-24-ene Hereinafter referred to as gypenoside LI. These are extracted and separated into each component, for example, by the following method. First of all, Jiaogulan is extracted with water or a water-containing lower alcohol. Examples of water-containing lower alcohols include water-containing methanol and water-containing ethanol with a content of about 50 v/v% or less. This extraction is preferably carried out at or under heating. Incidentally, the raw material Jiaogulan may be finely chopped or defatted by a conventional method prior to extraction. When a hydrous lower alcohol is used as the extraction solvent, it is preferable to concentrate the extract to remove the alcohol content, then add an appropriate amount of water and subject it to the next treatment with a nonionic adsorption resin. The nonionic adsorption resin is preferably a highly porous one made of styrene-divinylbenzene copolymer, specifically Amberlite XAD.
-2 (manufactured by Rohm and Haas, USA), etc. are used. This treatment is conveniently carried out by passing the extract obtained above through a column packed with adsorption resin. This operation causes saponin to be adsorbed onto the resin. Next, the saponin adsorbed on the resin is eluted with a lower alcohol. As the lower alcohol used as the elution solvent, methanol (98% or more), ethanol (95% or more), etc. are preferable. Note that, prior to elution, it is preferable to wash the column with water or a water-containing lower alcohol of about 20 v/v%. The lower alcohol eluate obtained above is then treated with alumina. This treatment can also be easily performed using a column filled with alumina. Through this treatment, saponin is adsorbed onto alumina. Note that the lower alcohol eluate may be appropriately concentrated in advance prior to this treatment with alumina. The saponin adsorbed on the alumina is then eluted with a lower alcohol or a water-containing lower alcohol, preferably about 50% v/v of a water-containing lower alcohol. By concentrating this eluate, a mixture of gypenosides (total saponins) is obtained. The total saponin obtained as described above contains novel saponins that have not been previously known, and each of these components is separated and purified, for example, by the following method. That is, total saponin is dissolved in water, this aqueous solution is treated with a styrene-based adsorption resin, such as Servachrom XAD-2 (manufactured by Serva), and the adsorbed substance is eluted with methanol. The eluate is concentrated and then treated with a silica gel column. The saponin adsorbed on the silica gel is then eluted with chloroform/lower alcohol/water, preferably in the lower layer of chloroform/methanol/water (65:35:10), and the eluate is analyzed using thin layer chromatography (TLC) as an indicator. Fractionate into fractions 1-6. Fractions 1 to 6 are each treated with a silica gel column, and then the saponin adsorbed on the silica gel is treated with chloroform, lower alcohol, ethyl acetate, and water, preferably chloroform, methanol, and water.
Fractionate and elute with the lower layer of ethyl acetate/water (2:2:4:1). Concentrate each fraction eluate obtained above and further
When the above-mentioned silica gel column chromatography or high-performance liquid chromatography is performed using the TLC results as an index to separate and purify each of these gypenosides, the gypenosides previously discovered by the inventors of the present invention can be obtained. It will be done. That is, gypenoside is obtained from fraction 1, gypenoside is obtained from fraction 2, and gypenoside is obtained from fraction 3.
From fraction 4, gypenoside is obtained, from fraction 5, gypenoside is obtained, and from fraction 6, gypenoside is obtained. Next, each residual liquid of the above fractions 2, 3, 4, and 6 was concentrated, and each of these was
Treated with 30C18 (nucleocil30C ₁₈ , 30±4μ, manufactured by M. Nagel, West Germany) column 7 mm x 61 cm x 2,
The saponin adsorbed on the column was then eluted with 35-45% ethanol to obtain the gypenosides of the present invention. That is, gypenoside, L and LI from fraction 2,
Gypenoside from fraction 3,
, and the fraction 4
From fraction 6, gypenoside and gypenoside were obtained. It has also been found that the gypenoside of the present invention can be obtained by subjecting a known gypenoside to an enzymatic hydrolysis reaction. That is, by hydrolyzing gypenoside and gypenoside with a cellulase enzyme, the gypenoside and gypenoside of the present invention were obtained, respectively. The thus obtained compound of general formula () is hydrolyzed by contacting with an enzyme.
At least one sugar molecule can be eliminated from the substitution of sugars at positions 20, 20, and 21. This is 3
To explain the substituent at the position as an example, a glucopyranosyl-glucopyranosyl group or a glucopyranosyl-arabinopyranosyl group is a glucopyranosyl group,
It can be hydrolyzed to an arabinopyranosyl group or a hydrogen atom, respectively. The same applies to 20th and 21st place. The enzyme used is selected from hydrolases that have the ability to cleave O-glucoside bonds. Preferred examples include hesperidinase, maltase, cellulase, takadiastase, and the like. Furthermore, naringinase,
Examples include pectinase, amylase, emulsin, and the like. These enzymes do not need to be purified products and may be crude. The amount of enzyme used varies depending on the type of enzyme, but for example, when using cellulase, about 600 to 1000 units may be used per 1 g of the compound of formula (). It is preferable to carry out the reaction under acidic conditions, and it is effective to carry out the reaction in a buffer solution having a pH of about 4, for example. It may be preferable to vary the acidic conditions somewhat depending on the type of enzyme. The reaction temperature is preferably room temperature or slightly elevated temperature. Generally carried out at 30-40°C.
However, there is no particular limitation. The reaction time varies depending on the type of enzyme, degree of purification, reaction temperature, desired degree of hydrolysis, etc. Generally, a reaction time of about 2 to 3 hours to about 1 week is used. According to this invention, one sugar molecule can be selectively removed by appropriately selecting conditions. Of course, it is preferable to stop the hydrolysis so that one or more sugar molecules remain. In addition, if multiple hydrolysis products are obtained,
Each product can be separated by employing the same technique as used for purifying and separating the extract of Jiaogulan vulgaris mentioned above. Furthermore, in this invention, the compounds of general formula () may be used as a mixture depending on the case. The compound of general formula () in this invention has an anti-ulcer effect, particularly an effect on gastrointestinal ulcers,
It is a new substance with potential as a medicine. Next, the present invention will be explained with reference to Examples. Example 1 Production of total saponin fraction 1.2 tons of Gypsophila (aerial part) collected in Mihara-gun, Hyogo Prefecture were extracted twice with 4 times the amount of water under hot conditions. The re-extracts were combined (approximately 9.6 tons), concentrated to an appropriate amount, and then spray-dried to obtain approximately 25 kg of pale yellow-brown powder extract. 200 g of this was dissolved in 2 parts of water, and the solution was passed through a column filled with 500 ml of styrene adsorption resin Serverchrome XAD-2 (100 to 200 μm, manufactured by Server Co., Ltd.). After washing the adsorption area with four parts of water and then four parts of 20% methanol, elution was carried out with two parts of methanol, and the methanol eluate was concentrated to dryness under reduced pressure to obtain 13.5 g of a yellowish brown powder. Yellowish brown powder obtained by repeating this method
Dissolve 30g in methanol 1 and add alumina [70~
290 mesh, neutral, manufactured by Woelm]
It was passed through a column packed with 500 g. 50 suction parts
Eluted at approximately 20% methanol. The eluate was concentrated to dryness under reduced pressure, and the total saponin fraction was obtained as a pale yellow powder.
14.2g was obtained. The general properties of the total saponin obtained were as follows. (i) A yellowish-white to brown odorless powder with a slightly bitter taste, easily soluble in methanol and dilute methanol, soluble in water and ethanol, insoluble in benzene, chloroform, ether, hexane and petroleum ether, (ii) 1% The aqueous solution is neutral, (iii) Infrared absorption spectrum [IR (KBr) νmax]
(iv) 4.0 (broad), 1.6 (broad), 1.2 (broad) and 0.9 (broad) ppm for nuclear magnetic ^resonance spectra (PMR, C ₅ D ₅ N); (v) generates persistent small bubbles when added to water and shaken; (vi) Lieberman reaction and Zarkowski reaction are positive; and (vii) first signal when subjected to thin layer chromatography under the following conditions It develops reddish-purple spots as shown in the figure. Plate: Silica gel 60F ₂₅₄ (thickness 0.25 mm, manufactured by Merck) Developing solvent: Chloroform/methanol/water (volume ratio 65:35:10) Lower layer Developing distance: 10 cm Detection: 1% cerium sulfate/10% sulfuric acid mixed solution After spraying, heat at 105℃ for 5 minutes. Dissolve 30 g of the total saponin fraction in the minimum amount of methanol, mix well with 60 g of silica gel, and evaporate the solvent to dryness under reduced pressure. 500g of the product was layered on a dry-packed column, and developed and eluted using a lower layer of chloroform/methanol/water (65:35:10). TLC the eluate at appropriate times [Adsorbent: silica gel, solvent system: chloroform/methanol/water (65:35:
100) Lower layer, detection: 30% sulfuric acid], fractionated into fractions 1 to 6, and concentrated to dryness under reduced pressure. The yield was 8.6 g for fraction 1, 5.6 g for fraction 2, and 5.6 g for fraction 3. 5.1 g, fraction 4 was 5.4 g, fraction 5 was 2.8 g, and fraction 6 was 2.2 g. Each fraction was dissolved in the minimum amount of methanol again according to the silica gel column chromatography described above, mixed well with twice the amount of silica gel, the solvent was evaporated to dryness under reduced pressure, and then 50 times the amount of silica gel was evaporated to dryness. It was layered in a packed column. Develop and elute in the lower layer of chloroform, methanol, water (70:30:10), perform the same operation on the eluate, and develop in the lower layer of chloroform, methanol, ethyl acetate, water (2:2:4:1). It eluted. This purification operation by silica gel column chromatography was repeated again for each fraction, 120 mg of gypenoside and 1.5 g of crude gypenoside were obtained from fraction 1, and gypenoside was obtained from fraction 2.
1.2g, gypenoside from fraction 3
80mg and gypenoside 350mg, gypenoside 850mg from fraction 4, gypenoside 250mg from fraction 5, fraction 6
3000 mg of crude gypenoside was obtained from each. Next, each solution of the above fractions 2, 3, 4, and 6 was evaporated to dryness, and each of the obtained solids was 0.5 to
Dissolve 1.0g in 30% ethanol and apply this solution to Nucleosil 30C ₁₈ (30±4μ) column 7mm x 61cm x 2.
It was injected into a book and developed and eluted with 35-45% ethanol.
As a result, from the residual liquid of fraction 2, 40% each of gypenoside, L, and LI were found.
mg, 40mg, 45mg, 30mg were obtained, and gypenoside was obtained from the residual liquid of fraction 3.
and are 45mg, 300mg, 25mg, respectively.
30 mg of gypenoside and 35 mg of gypenoside were obtained from the residual liquid of fraction 4.
40 mg and 35 mg of gypenoside were obtained from the residual liquid of fraction 6.
Obtained. The physical properties of the obtained gypenosides are shown in Table 1, Table 2a,
Shown in Table 2b.

[Table] * Using pyridine

【table】

[Table] Example 2 Enzymatic hydrolysis of gypenoside 2000 mg of gypenoside was dissolved in 0.005 M-NaH ₂ PO ₄
Dissolve cellulase (practical
Add 200mg of grade type I (manufactured by Sigma) to 37~
The mixture was stirred at 38°C for 2 hours. Next, the reaction solution was passed through a Serverchrome XAD-2 (30 ml) column, and 500 ml of water was added.
Then wash with 20% methanol 1 and then methanol
It eluted at 200ml. The eluate was concentrated under reduced pressure to obtain about 160 mg of hydrolyzate. Dissolve this in the minimum amount of methanol, mix well with 300 mg of silica gel, evaporate the solvent to dryness under reduced pressure, layer it in a column dry-packed with 10 g of silica gel, and mix it with chloroform, methanol, ethyl acetate, water (2: 2:4:1) was developed and eluted in the lower layer. Examine the eluate by TLC at appropriate times,
The gypenoside fractions were separated and concentrated to dryness under reduced pressure to obtain 35 mg of gypenoside. Example 3 Enzymatic hydrolysis of gypenoside Gypenoside and 200 mg each were treated in the same manner as in Example 2 to obtain 25 mg of gypenoside from gypenoside and 45 mg of gypenoside from gypenoside.

Claims (1)

[Claims] 1 General formula () [In the formula, R ¹ is β-glucopyranosyl group, β-glucopyranosyl (1→2)-β-glucopyranosyl group, or β-glucopyranosyl (1→2)-α
-Arabinopyranosyl group; R ² is a hydroxymethyl group or formyl group; R ³ is a hydroxyl group or a hydrogen atom; R ⁴ is a β-glucopyranosyloxy group, α-rhamnopyranosyl (1→6)-β- Glucopyranosyloxy group, β-xylopyranosyl (1→6)-β-glucopyranosyloxy group, methyl group or hydroxy group; R ⁵ is methyl group, hydroxy group, hydroxymethyl group or β-glucopyranosyl Oxymethyl group; However, when R ¹ is a β-glucopyranosyl group, R ² is a hydroxymethyl group, R ³ is a hydrogen atom, and R ¹ is a β-glucopyranosyl (1→2)-α-arabinopyranosyl group In this case, R ² is a formyl group, R ³ is a hydrogen atom, and R ₁ is β
- Glucopyranosyl (1 → 2) - β-glucopyranosyl group, when R ₂ is a formyl group and R ₃ is a hydrogen atom, R ₄ is α-rhamnopyranosyl (1 → 6) - β
- glucopyrnonyloxy group, β-xylopyranosyl (1→6)-β-glucopyranosyloxy group, or methyl group]. 2 The compound represented by the general formula () is 19,21-dihydroxy-3β-0-β-glucopyranosyl-
20ξ-0-β-glucopyranosyl-dammar-24
-Ene;19,20ξ,21-trihydroxy-3β-0
-[β-glucopyranosyl (1→2)-β-glucopyranosyl]-dammar-24-ene; 19,20ξ
-dihydroxy-3β-0-β-glucopyranosyl-21-0-β-glucopyranosyl-dammar-
24-ene; 19-oxo-21-hydroxy-3β-
0-[β-glucopyranosyl (1 → 2)-β-glucopyranosyl]-20ξ-0-[α-rhamnopyranosyl (1 → 6)-β-gucopyranosyl]-dammar-24-ene; 19-oxo-21-hydroxy-
3β-0-[β-glucopyranosyl (1→2)-
β-glucopyranosyl]-20ξ-0-[β-xylopyranosyl (1→6)-β-glucopyranosyl]-dammar-24-ene; 19-oxo-3β-
0-[β-glucopyranosyl (1 → 2)-α-arabinopyranosyl]-20S-0-[α-rhamnopyranosyl (1 → 6)-β-glucopyranosyl]-
Danmar-24-ene;19-oxo-3β-0-
[β-glucopyranosyl (1→2)-α-arabinopyranosyl]-20S-0-[β-xylopyranosyl (1→6)-β-glucopyranosyl]-dammar-24-ene; 12β,19,20S- Trihydroxy-3β-0-[β-glucopyranosyl (1→2)
-β-glucopyranosyl]-dammar-24-ene; 12β,19,20R-trihydroxy-3β-0-
[β-glucopyranosyl (1→2)-β-glucopyranosyl]-dammar-24-ene; 19-oxo-12β,20R-dihydroxy-3β-0- [β-glucopyranosyl (1→2)-β-glucopyranosyl]- Dammar-24-ene 19,20R-dihydroxy-3β-0-[β-glucopyranosyl (1→2)
-β-glucopyranosyl]-dammar-24-ene.