JPS6048995A - Novel saponin and its preparation - Google Patents

Abstract

NEW MATERIAL:The 21-beta-hydroxyhederagenin-3-0-( beta-D-glucopyranoside )-28-0-beta- D-glucopyranoside of formula (R<1> is hydroxymethyl, methyl or formyl; R<2> is beta-D- Gluropyranoside group of H; R<3> is H or hydroxy; R<4> is H or hydroxy), etc. USE:An antitussive, diuretic, skin moisturizer, and remedy for scald. PREPARATION:The ground part of underground part of a sponge gourd (Luffa cylindrica Roem) is extracted with water, lower alcohol or hydrated lower alcohol, and concentrated. The concentrate is suspended in water, made to contact with a porous crosslinked polystyrene resin-based absorbent having macro-network structure, and eluted with a lower alcohol or an aqueous solution of a lower alcohol. The eluted solution is concentrated, dissolved in a lower alcohol, and poured into a fat-soluble organic solvent, and the produced precipitate is dried under reduced pressure.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel saponins contained in loofah and a method for producing the same.

The "loofah" in this invention is a plant belonging to the Cucurbitaceae family, scientific name Rufa cylindrica loem (1-uf
fa cylindrica Roen+) and plants botanically related thereto.

Recently, there has been a report that oleanolic acid, palmitic acid, oleanolic acid-3-glucoside, and oleanolic acid-3-glucosyl-28-diglucoside were extracted from loofah confectionery.
je or Maier fa Medlca (1
'JankingPeop, R, China)
Chuno Tsao Yao; 1980゜11 (
2), 5564 (ch)).

The inventor of this invention conducted research on the ingredients contained in loofah! i fruit, nine new saponins [1-ucynocide Δ, B, C, D, , E
.

This invention was achieved by extracting ginsenosides Re and RO+ of known Korean ginseng saponins (referred to as F, G, H, and I).

The novel saponins of this invention are represented by 4-general formula (I): where R1 is a hydroxymethyl group, R2 is a β-D-glucopyranoside group, R3 is a hydroxy group, and R4 is a hydrogen atom, and the specific name is 21 -β-hydroxyhederagenin-3-0-(
β-D-glucopyranoside) -28-0-β-glucopyranoside, (Lucinoside A (LA+)): R1 is a hydroxymethyl group, R2 is a β-D-glucopyranoside group, R3 is a hydrogen atom and R4 is a hydroxy group,
The specific name is Alshnol'M-3-0-(β-D-glucopyranoside)-28-0-β-D glucopyranoside,
(Rusinoside B (LB2)); R1 is a methyl group, R2 is a β-D-glucopyranoside group, R3 is a hydroxy group, and R4 is a hydrogen atom, and the specific name is macaeriic acid-3-0-(
β-D-glucopyranoside) -28-0-β-D-glucopyranoside, [lucinoside C(LB+)): R1 is formyl group, R2 is β-D-glucopyranoside group, R3
is a hydrogen atom and R4 is a hydroxy group, and the specific name is 2-α
-Hydroxygypsogenin-3-〇-(β-D-glucopyranoside)-28-Q-β-ri-glucobyranoside)
-28-Q-β-D-1 Lucobyranoside, (Lucinoside D(1-D+)): R1 is a hydroxymethyl group, R2 is a β-D-glucopyranoside group, R3 and R4 are hydrogen atoms, and the specific name is hederagenin. -3-0-(β-D-glucopyranoside)-28-0-β-D-glu]pyranoside,
[Rusinoside E (1-02)): R1 is a formyl group, R2 is a β-D-glucopyranoside group, R3 and R4 are hydrogen atoms, and the specific name is gipsogenin-3-0-(β-D-
[glu]pyranoside)-28-0-β-D-glucopyranoside [lucinoside F (I-D2)): R1 is a methyl group, R2 is a β-D-glucopyranoside group,
R3 is a hydrogen atom and R4 is a hydroxy group, and the specific name is Sumatsuji l'1lt-3-0-(β-D-glucopyranoside)-28-Q-β-D-glucopyranoside, [Rusinoside G (LD4)): R1 is a methyl group, R2 is a β-D-glucopyranoside group,
and R3 and R4 are hydrogen atoms, and the specific name is oleanolic acid-3-0-(β-D-glucopyranoside)-28-Q-
β-D-glucopyranoside [Lucinoside H (LDa)]
: and R1 is a hydroxymethyl group, R2 and R3 are hydrogen atoms, and R4 is a hydroxy group, and the specific name is alshnolic acid-3
-0-(β-D-glucopyranoside), (Rusinoside I
(LE+)).

These new saponins as well as the known ginsenoside Re
and Rσ1 can be obtained by the following method.

In the method of this invention, the above-ground or underground parts of the raw material loofah are either not degreased or extracted after degreasing using a common fat-soluble organic solvent such as benzene or n-hexane. .

Next, extraction is performed in the following manner to obtain crude hechima sabonin.

(a) The raw material is heated under reflux and filtered with water or a lower alcohol such as methanol or ethanol or a water-containing lower alcohol in an amount of about 2 to 5 times its weight to obtain an extract. This extraction operation may be repeated as necessary. The extracts are combined and concentrated and the concentrate is suspended in water. The resulting suspension was treated with a porous cross-linked polystyrene resin adsorbent with a large network structure (e.g. Amberlite XAD-1,
Amberlite These adsorbents are used in an amount of 20 to 300 times the content of saponin m, preferably 40
~150 (8ffi) is used.Then, the resin is thoroughly washed with water, and then eluted and concentrated with a lower alcohol such as ethanol or methanol, or water containing about 30% or more lower alcohol, and the concentrate is converted into lower alcohol 8-alcohol. Crude loofah saponin is obtained by dissolving and injecting into a fat-soluble organic S* such as ether or n-hexane, and drying the resulting precipitate under reduced pressure.

(b) The raw material is heated with about 2 to 5 times its weight of water or a lower alcohol such as methanol, or a water-containing lower alcohol such as water-containing methanol or water-containing ethanol, and filtered to obtain an extract. This extraction operation may be repeated as necessary. The extracts are combined and concentrated, and the concentrate is either directly dissolved in n-butanol, added with water and shaken, and distributed, or mixed with water and a fat-soluble organic solvent (ether, n-hexane, etc.). Dissolve the concentrate in n-butanol, add water, shake and distribute, concentrate the obtained n-butanol, and dissolve the concentrate in lower alcohol. The crude loofah sabonin is obtained by dissolving it in water, injecting it into a fat-soluble organic solvent such as ether, and drying the resulting precipitate under reduced pressure.

The crude loofah sabonin obtained as described above contains lucinosides A, B, and C of the above-mentioned novel saponin substances.

It contains D, E, F, G, I-1 and (2) as well as the known substances ginsenoside Re and Rol, and each of these components is separated by the following method.

That is, - the above-mentioned crude loofah sabonin is dissolved in a lower alcohol such as methanol or ethanol in an amount of 3 to 7 times its weight, and the solution is subjected to reverse phase silica gel column chromatography. For example, C19 packing material for separation (35-105μ
Using a column packed with 100% Δ-Tars (manufactured by Japan), the elution solvent was a solvent pre-solvent whose concentration was varied from 40% lower alcohol to 70% lower alcohol from the lower alcohol-water mixture described above. Chromatography with gradient elution. The obtained eluate was subjected to thin-layer chromatography [e.g., chloroform/methanol/water (i5: 35:
10 Developed in the lower layer of the mixture, sprayed with 30% sulfuric acid, and heated to 105°C.
The mixture was heated for 5 minutes to develop color) and divided into the following seven fractions, and these fractions were evaporated to dryness.

Fraction number Saponin contained 1 Lucinoside A, Ginzenoside Re and RCl+ 2 Lucinoside B and 0 3 Lucinoside D 5 Lucinoside F and 0 6 Lucinoside I 7 Lucinoside I For the M1 fraction, first chloroform: methanol: water (20: 4: 0.1 Column chromatography on silica gel eluting with > yields ginsenoside Ra1 and a mixture of ginsenoside Re and lucinoside A. The latter mixture is then further diluted with chloroform:methanol:ethyl acetate:water (2:2:4:
Ginsenoside Re is separated into lucinoside by column chromatography on silica gel eluting with 1).

For the Ntl, 2 fraction, the 1.
1 - A mixed solution of dinoside B and lucinoside C was added to 151, and this was further mixed with chloroform:methanol:ethyl acetate:water (2:
Lucinoside B and C are separated by column chromatography on silica gel, eluting with 2:/l:1).

Fractions 3.4.6 and 7 were subjected to column chromatography on silica gel eluting with chloroborum:methanol:water (20:4:0.1) to extract lucinoside D1 and lucinoside E from each fraction.
, lucinoside F1, lucinoside ■, and lucinoside 1'' are separated.

The fV&11.5 fraction is first chromatographed on silica gel eluting with chloroform:methanol:water (20:4:0.1). The obtained eluate was applied to, for example, a Nucleosil 3O-C10 (30±4μ, manufactured by M Nagl, West Germany) column (7 mm x 61c+).
Lucinoside F and Lucinoside G are separated by high performance liquid chromatography using a 30% acetonyl acetate solution as a mobile phase.

Among the saponins isolated in this way, 12-dinocyto A, B, C, D, E, F, G, H and ■ are all new substances, and have antitussive, diuretic, moisturizing skin, and recovery from burns. It is useful because of its effects. As for these saponins, individual saponins or a mixture thereof can be used as an active ingredient.

In addition, by using the method of the present invention, not only can useful new saponins as described above be obtained from inexpensive raw materials such as loofah, but also 20-8- Useful ginsenosides Re and R (I
This is an extremely useful invention.

Next, the invention will be explained by way of examples.

Example 1 (i) Extraction of crude loofah saponin from loofah. Crude loofah saponin was extracted by the following two methods.

(a) The dry matter of the entire above-ground part of the loofah plant is 5k (l is 1ω
Cut into large pieces, methanol 251! and heated under reflux for 1 hour. The above operation was performed twice more on the obtained residue, all extracts were combined and filtered, and methanol was distilled off under reduced pressure at a temperature not exceeding 60°C to obtain 75 g of methanol extract.
get. Add 300 xl of water to this dry extract to suspend it, inject this suspension into a cylindrical column filled with Amberlite XDA21Q using water, and then add 20xl of water.
The solution was allowed to flow down at a flow rate of 1 minute until the color of the effluent disappeared (3 g). Next, methanol (3Ω) was allowed to flow down at a flow rate of 10y1/min for elution. The eluates were combined and the methanol was distilled off under reduced pressure at a temperature not exceeding 60°C to obtain a greenish brown residue 150.

This residue was dissolved in 50 xl of methanol and 5 ml of ether.
00 ml, and the precipitate was filtered off. This operation 2
The whole precipitate was dried under reduced pressure at 40°C several times to obtain 10 g of light brown crude Hejima sabonin.

(b) Loofah ground” Some whole plant dried matter 7,3k (]
Cut into 1-mark size pieces, add methanol 40Q, and heat under reflux for 1 hour. Repeat the above operation two more times with the resulting residue.
All extracts were combined and filtered, and methanol was distilled off under reduced pressure at a temperature not exceeding 60°C. The resulting residue was mixed with 1 part of water.
The suspension was suspended in Ether 2Q, shaken and distributed, and the aqueous layer was taken and further shaken and distributed with Ether 1Q to remove chlorophyll by transfer to the ether layer. The resulting aqueous layer was taken and dried under reduced pressure without evaporation at a temperature not exceeding 60°C.
2 g of brown residue were obtained. Add this residue to 500 xi of water.
and shake-extracted twice with 1Q of n-butanol.

The n-butanol layer is separated and shaken with 200 g of water to transfer and separate contaminating sugars to the aqueous layer. The n-butanol layer is distilled under reduced pressure at a temperature not exceeding 60 DEG C., and the residue is dissolved in 50 ml of methanol, poured into 500 ml of ether, and the precipitate is filtered off. This precipitate was dried under reduced pressure at 40°C to obtain light brown crude Hejima saponin 12.5 (I).

7 g of crude loofah saponin was dissolved in 35% methanol 3001, and the solution was passed through a column packed with preparative C19 packing material (for reverse phase, 35-105μ, manufactured by Nippon Waters Co., Ltd.) 14011i. This column was subjected to gradient elution using 40% methanol → 70% methanol, and the eluate was subjected to thin layer chromatography (thin layer of silica gel G (15-Merck AG, West Germany), chloroborum/methanol/water, 6
Developed in the lower layer of 5:35:10 and sprayed with 30% sulfuric acid.
Heat for 5 minutes at 5°C to develop color] to form the following seven 7s.
The mixture was divided into fractions and each fraction was evaporated to dryness.

(Continued in the margin below) = 16- Fraction number Containing saponin 1 Lucinoside A, ginzenoside Re and Rot 2 Lucinoside B and 0 3 Lucinoside D 4 Lucinoside E and F 5 Lucinoside F and G 6 Lucinoside ■ 7 Lucinoside H Fraction 1 (0,35G) and fraction 2 (0
, 25 g) were placed in a silica gel column (60-230 mesh, manufactured by Merck & Co., Ltd., packed with 35 g and 25 (L), respectively), and the columns were filled with chloroform:
Fractional elution was performed with methanol:water (20:4:0.1). Thus from fraction 1 ginsenoside RO1
(95m Q > and lucinoside A and ginsenoside Re
A mixture (100 mg) of lucinoside B and lucinoside C (7) was obtained from fraction 2 (100 mΩ
) was obtained.

The mixture of lucinoside A and ginzenoside Re and the mixture of lucinoside B and C were each mixed with silica gel (10
g, 60-230 meshes, Merurisha! 11), and the columns were eluted with chloroborum:methanol:ethyl acetate:water (2:2+4:1), and lucinoside A (25ma) and ginsenoside Re (50ma) were added to the column.
a ) and lucinoside B (30+110) and lucinoside C (30+n O) were obtained, respectively.

Fraction 3 (0,35(+), 4 (la)
, 6 (0,501 J) and 7 (0,30 CI) were placed in columns filled with silica gel (60-230 mesh, manufactured by Merck & Co., Ltd.) at 35 G, 100 CI, 50 (+ and 30 (L), respectively, and chloroform. Fractional elution with methanol/water (20:4:0.1) yields lucinoside D (15+11(J)) from fraction 3 and lucinoside E (263+11(J) from fraction 4).
) and lucinoside F (415 ma), from fraction 6 lucinoside I (10111 g), and from fraction 7 lucinoside 1''(105111<1
) were obtained respectively.

Fraction 5 (0,250) was dissolved in silica gel (25 (
1) was placed in a column filled with 1), the column was eluted with the lower layer of chloroform:methanol:water (20:4:0.1), and the concentrate was subjected to high performance liquid chromatography [
Nippon Waters ALC/GPC type high performance liquid chromatograph two columns are Nucleosil 30・C,9 (30±
4μ, made by West German M. Nagel) filling (7mmφ x 61
); mobile phase was 30% acetonitrile) to obtain lucinoside F (55n+g) and lucinoside G (55 mg).

The physical properties of the saponins that are the subject of this invention are as follows:
, as shown in Tables 2 and 3.

(Margin below, continued on next page.) Tokukai Sho GO-48995 (7) (4) -〇Flash- qri () Evil-〇Koichi Ding 42 ηoo-Φ size Φ prisoner ■QN size■Q

Claims (1)

[Claims] 1.21-β-hydroxyhederagenin-3-〇-(β
-D-glucopyranoside) -28-Q-β-D-glucopyranoside: Arjunol ff-3-0-(β-D-
macaerin FIQ-3-0-(β-D-glucopyranoside)-28-Q-β-D-glucopyranoside; 2-α-
Hydroxygypsogenin-3-0-(β-D-glucopyranoside)-28-0-β-D-glucopyranoside; Hederagenin-3-0-(β-D-glucopyranoside)
-28-Q-β-D-glucopyranoside; Kypsogenin-3-0-(β-D-glucopyranoside)-28-Q-
〇-〇-glucopyranoside; maslinic acid-3-0-(β
-D-glucopyranoside)-28-0-β-D-glucopyranoside: Oleanolic acid-3-0-(β-D-glucopyranoside)-28-Q-β-D-glucopyranoside; and Alshnol M-3-〇- A novel saponin selected from the group consisting of β-D-glucopyranoside. 2. The above-ground or underground parts of loofah (Rufa cylindrica, loem) are degreased, but the following method: (a) Extract with water, lower alcohol, or water-containing lower alcohol, and add the concentrate to water. After suspending and contacting the suspension with a porous cross-linked polystyrene resin adsorbent with a large network structure, it is eluted with a lower alcohol or lower alcohol-containing water, and the eluate is concentrated to form a concentrate. Dissolve it in a lower alcohol and inject it into a fat-soluble organic solvent and dry the resulting precipitate under reduced pressure, or (b) extract it with water, a lower alcohol, or a water-containing lower alcohol, and add the concentrate directly to n-butanol and water. Alternatively, the concentrate obtained by partitioning into water or a fat-soluble organic solvent and concentrating the water portion is distributed between n-butanol and water, and the n-butanol portion is dried under reduced pressure to remove the remaining The product was dissolved in a lower alcohol and poured into a fat-soluble organic solvent, and the resulting precipitate was dried under reduced pressure to obtain crude Hejima sabonin.The crude Hejima sabonin was dissolved in a lower alcohol, and the solution was subjected to reverse-phase silica gel column chromatography. 21-β-hydroxyhederagenin-3-0-(β-D
-glucopyranoside)-28-Q-β-D-glucopyranoside; alshnol IQ-3-0-(β-D-glucopyranoside)-28-Q-β-D-glucopyranoside; macaerin l-3-0-(β- D-glucopyranoside)
-28-0-β-D-glucopyranoside; 2-α-hydroxypsogenin-3=O-(β-D-glucopyranoside) -28-0-β-D-glucopyranoside; hederagenin-3-0-(β- D-glucopyranoside)-28-
0-β-D-glucopyranoside; Gibsogenin-3-0-(β-D-glucopyranoside)
-28-0-β-D-glucopyranoside; maslinic acid-
3-0-(β-D-glucopyranoside)-28-Q-β
-D-glucopyranoside; oleanolic acid-3-0-(
β-D-glucopyranoside)-28-0-β-D-glucopyranoside; and Alshnol M-3-0-β-D-glucopyranoside; and ginsenoside Re and ginsenoside R (saponins characterized by obtaining 1+) Manufacturing method.