JPS60184025A - Polysaccharide, separation and use thereof - Google Patents

Abstract

NEW MATERIAL:The polysaccharide, ganoderane A [molecular weight, 9,300; specific rotation, [alpha]D=+58.8 deg. (C=0.19, water); infrared absorption spectrum (KBr), numax=3270 and 1013 (cm<-1>); <1>H nuclear magnetic resonance spectrum delta=1.12 (medium-broad), 2.07 (medium-broad), 4.86 (medium-borad); elemental analysis, C 41.79%, H 6.07%, N 0.00%. electrophoresis with glass fiber filter paper, 15.4cm (glucose 11.8cm); elution time of DEAE Toyopearl chromatography, 9.3hr] and ganoderane B. USE:Hypoglycemic agent. PREPARATION:Raw or dried REISHI (fruit body of Fomes japonicus, a bracket fungus of the genus Fomes, or its relating vegetables) is optionally defatted, and dialyzed to obtain a ganoderane mixture. It is administered as a hypoglycemic agent at a dose of 10-300mg daily in 2-3 divided doses.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel polysaccharides, particularly polysaccharides named Ganoderan A and B, which can be isolated from Reishi mushrooms, methods of isolation and uses thereof.

Ganoderma, especially Polyporaceae
e) belong to Ganoderma lucidum, Karst, Ganoderma lucid.
It is widely known that um Karst-) is used as an effective drug for chronic bronchitis, coronary insufficiency, hyperlipidemia, hypertension, neurasthenia, hepatitis, and the like. However, nothing is known about the active ingredients of this reishi mushroom.

The inventors of this invention discovered that the water-soluble fraction contained a component with a hypoglycemic effect, and as a result of separating and purifying this, two types of polysaccharides named Ganoderan A and B were obtained. Ta.

As the Ganoderans-containing Reishi mushroom of the present invention, the most preferred is Ganoderma lucidum. Other related plants, Ganoderma neoyabbonicum,
Imazeki-Ganodermaneo-japonic
um Imazeki), Ganoderma t.
Ganoderma cephacryl + Ganoderma 5e
ssile Murrill), Ganoderma curteisi, Perc Ganoder
Ma eurtissi Berk, ), etc.

The Ganoderans of this invention are obtained by extracting, separating and purifying the fresh or dried material of the above-mentioned near-green plants, or by culturing the hyphae of the above-mentioned stoneworks or related plants. It can be obtained by extraction, separation, and purification. Specifically, the ganoderanes of this invention can be isolated by the following method.

First, the raw material Reishi mushroom is defatted using a conventional fat-soluble organic solvent without being defatted, and then extracted with water or an aqueous organic solvent. Although water is sufficient for extraction, an aqueous organic solvent may be used to prevent spoilage of the extract and to promote extraction. Alternatively, both may be extracted.

As the organic solvent for the aqueous organic solvent, lower alcohols such as methanol and ethanol, and water-soluble organic solvents such as acetone are used, and the concentration ranges from about 1% to about 30% depending on the type of raw material. In addition, this extraction is promoted by heating, and the raw material is preferably pulverized.Next, the above-mentioned extract is directly subjected to dialysis using a cellulose dialysis membrane, or subjected to ultrafiltration treatment, or extracted. The liquid is first concentrated by a method such as solvent distillation, or a solvent such as ethanol or methanol is added to create a precipitate of a substance that has a hypoglycemic effect, and this concentrated liquid or precipitate is poured into water. Alternatively, the ganoderan mixture is obtained by subjecting the solution dissolved in the aqueous organic solvent to the dialysis or ultrafiltration.0 The ganoderan mixture obtained as described above is subjected to the following fractionation treatment. Ganoderan A and B'' can be isolated as single units.

First, the above-mentioned ganoderane mixture is treated using the difference in electrical properties of each ganoderane to separate each ganoderane. That is, it is treated with an anion exchange resin such as DEAE Sephadex, DEAE) and Ganoderan A.
and a fraction containing Ganoderan B. The two fractions thus obtained are then subjected to molecular weight fractionation. This fractionation method includes gel filtration using Sepharose, Sephadex, Sephacryl, agarose beads, etc.
A method using ~100,000 ultrafiltration membranes is used to treat and separate each ganoderan.

It will be done. Thus, the present invention is based on the two previously obtained Fragrances, which belong to the family Arunocysaceae. □The fresh or dried material of the Physcomitrella sp. Extraction is carried out by heating, and the resulting extract is made into a concentrate or a precipitate of a substance that has a hypoglycemic effect, and the concentrate or precipitate is mixed with water or an aqueous organic solvent. Ganoderan A
A polysaccharide isolation method characterized by obtaining a ganoderan mixture containing ganoderan A and B, and further subjecting this ganoderan mixture to a fractionation treatment to separate the individual components of ganoderan A and B having the characteristics described below. It provides:

It has been found that the polysaccharide obtained as described above has an excellent hypoglycemic effect and almost no side effects as described below, and is extremely useful as a hypoglycemic agent. Thus, the present invention provides a hypoglycemic agent containing as an active ingredient a polysaccharide consisting of one or a mixture of Ganoderan A and B having the properties described below.

The dosage of the hypoglycemic agent of this invention varies depending on the medical condition, but when administered orally to adults, the daily dose of ganoderan is 90%.
The efficacy can be exerted by administering 10 to 300 kg, preferably 30 to 100 kg, in 2 to 3 doses.

The hypoglycemic agent according to the present invention consists of a single ganoderane or a mixture thereof and a solid or liquid excipient. The administration methods and dosage forms are usually powders, tablets including sublingual tablets, emulsions, capsules, drugs, granules, and liquids (including liquid extracts, syrups, etc.).
There are forms of oral administration such as It may also be in the form of an injection. The solid or liquid excipients used here include:
Those known in the art are used. It may be desirable to formulate the compound to contain the required amount of the compound for a single dose, such as those described above.

Excipients in powders and other powders for internal use include lactose, starch, dextrin, calcium phosphate, calcium carbonate, synthetic and natural aluminum silicates, magnesium oxide, dry aluminum hydroxide, stearin, to name a few specific examples. Examples include magnesium acid, sodium bicarbonate, and dried yeast.

In addition, transdermal absorption agents prepared by adding ordinary excipients are also included in the present invention.

Although the aqueous extract of Rhein is used as a health food, Ganoderan A and B of the present invention can be used as a health food in an amount that does not exhibit a medicinal therapeutic effect and is sufficient to maintain health. Suitable dosage forms include liquids, capsules, soft capsules, granules, and tea preparations.

Next, the invention will be explained by examples and animal experiments.

Example: Thoroughly dry the mature fruiting bodies of Cinnamon mushrooms, wash approximately 9 out of 10 of them lightly with cold water, thoroughly drain the water, and dry.
Approximately 10
Add 0 t of deionized water and extract under boiling for about 3 hours.

Repeat this extraction three times.

The obtained extracts were combined and evaporated and dried under reduced pressure at a temperature of 50 to 60°C to obtain a brown powder extract powder of 0.6 powder.

100 f of this extract (A) was dissolved in 400 m of water, and the solution was aspirated and filtered to remove insoluble matter.The F solution was used as the internal liquid and 2 t of water was used as the external liquid. Dialysis was performed using Visking cellulose dialysis tubing (which passes through particles with a size of 24 angstroms or less) for 10 days, replacing the external water once a day.

The obtained dialyzed fluid was passed through a DEAE) Yopal column (2, 2
cmφ
Fraction G-2 eluted with aC1 aqueous solution was obtained.

Fraction G-1 was evaporated to dryness under reduced pressure at 50-60°C to obtain powder 52. This powder 2? was dissolved in 40-m water and subjected to chromatography on a Sepharose 6B column (4,0αφ×95σ) and eluted with 0.1M NaCl solution at a flow rate of 2'/= ) 201!1! A plurality of fractions were obtained, and the ganoderan-containing fractions were selected and combined as follows, and the mixture was heated at 50 to 60°C under reduced pressure.
It was concentrated to 0-.

, or 0.2 yd of each 7 lux! 0.8 to
- add water, add 2-thiphenol-containing aqueous solution 1-,
Furthermore, after adding 5 ml+ of sulfuric acid and leaving it for 30 minutes, λma
The absorbance at x:490 nm was measured, and the fraction with the highest absorbance was selected.

The concentrated solution (i o 0sd) was put into a Visking tube 3.6732 as the inner solution, and 2 tons of water was used as the outer solution. Dialysis was performed by exchanging the outer solution once a day for 3 days. Dialysis) 0 The obtained internal solution was dialysis under reduced pressure from 50 to
It was concentrated to 40 mg at 60°C.

The above concentrate (40ml) was subjected to chromatography on a DEAE Jobar column (2.2cmφ呵-
) gradient elution was performed over 20 hours. From the eluate fraction (20d fraction) obtained by gradient elution, cupran-containing fractions were selected and combined in the same manner as described above, and the mixture was heated to 100 °C at 50 to 60 °C under reduced pressure.
It was concentrated to -.

The obtained concentrated solution was subjected to dialysis for 3 days in the same manner as above, and the obtained internal solution was concentrated to 40 m at 50 to 60° C. under reduced pressure.

This concentrated solution was applied to a Sephacryl S-200 column (4,0c
mφX 95 cm ) column chromatography, and a solution of 30.32 trishydroxymethylaminomethane in brine (a solution of 23.05 f NaCl in 2.5 t of water) was adjusted to pH 7.0 with 15-20% hydrochloric acid. Elute with adjusted 0, 1 M) lj sous-acid buffer (
The flow rate was 2"/rig) L, and the ganoderan-containing fractions were selected from the obtained 20me fractions in the same manner as above, and the mixture was heated to 100% at 50 to 60°C under reduced pressure.
Concentrated to the point of

The obtained concentrate was dialyzed for 3 days in the same manner as above, and the obtained internal solution was evaporated to dryness under reduced pressure at 50 to 60°C to obtain Ganoderan A (90 my) as a white powder.

Ganoderan A was confirmed to have the properties described below and is a single substance, and Ganoderan B, which will be described below, was similarly confirmed.

The fraction G-2 was heated to 100° C. under reduced pressure at 50 to 60°C.
The concentrated solution was subjected to dialysis for 3 days in the same manner as described above, and the resulting internal solution was concentrated to 40.degree. C. under reduced pressure at 50 to 60.degree.

Regarding the obtained concentrate (40d), fraction G-
As in case 1, the mixture was subjected to chromatography using a Sepharose 6B column → dialysis → DEAE) chromatography using a Yopar column → dialysis. Obtained internal fluid 100
m was concentrated under reduced pressure at 50 to 60°C to 40°C, and applied to a Chromadog 2 filter on a Sephadex G50 column (4,0crnφX95crn) in 0.05M phosphate buffer pH 7.
At a flow rate of 0.5'/=), 10 fractions were obtained. The fractions were examined in the same manner as above, and the ganoderan-containing fractions were selected and combined, which was concentrated to 100°C under reduced pressure at 50-60°C.

This concentrated solution was subjected to dialysis for 3 days in the same manner as above, and the resulting internal solution was evaporated to dryness under reduced pressure at 50 to 60°C to obtain Ganoderan B (180 cm) as a white powder.

Each of the ganoderans obtained as described above has the following properties (C, H in the elemental analysis below).
, the remainders other than N are 0).

b) Ganoderan A Molecular weight (gel filtration method): 9300 Specific optical rotation: [α) p +58.80 (c O, 19, water) Infrared absorption spectrum (KBr) νn1aX (cr
n-1): 3270.1013 1H nuclear magnetic resonance spectrum δ: 1.12. (Nakahiro).

2.07 (Nakahiro), 4.86 (Nakahiro) Elemental analysis value: C, 41,79; H, 6,07: N, 0
．． 00% 0% glass fiber aerophoretic mobility: 15.4c
m (glucose 11.8 m) DEAE) Jobar chromatography elution time: 9.
3 hours) Ganoderan B Molecular weight (gel filtration method): 3600 Specific rotation: [αID 33.30 (c O, 20, water)
Infrared absorption spectrum (KBr) νmax (cy-
1) :3210.1028 1H nuclear magnetic resonance spectrum δ: 4.40 (Nakahiro) Elemental analysis values: C, 42,94:H, 5,82:N, 1.26
% gelatin birch paper electrophoretic mobility: 16.1 cm
(Glucose 11.8 crn) DEAE) Jobar chromatography elution time: 10
．． Among the above characteristics, the molecular weight and elution time of glass fino (-filter paper electrophoresis and DEAE) Jovar chromatography were measured as follows.

(2) Molecular weight Each ganoderan was subjected to gel filtration using Sephacryl S-200 to determine its retention capacity, and the molecular weight was calculated from a standard curve prepared using Dextman T series.

Molecular weight Sephacryl Ganodera yA 9300 Sephacryl S-200# used for measurement
B 3600 -200 ■) Glass fiber F paper electrophoretic mobility Glass fiber F paper (Whatman GF/C.

15 x 40 cm), the following moving distances (m) are shown. (Conditions: alkaline borate buffer pH 9,3°450
V, 2 hours) Distance traveled Ganoderan A I5.4 B 16. ．． 1 Glucose 11.8 1 [) DEAE) Jobar chromatography elution time DEAE) Yopal column (2.2 cmφX 45
cm) using 0.05 M Tris-HCl buffer (
Elution was carried out for the first 5 hours at pHs, o), and then sodium chloride was added to the same buffer (0-7 M, 20
When performing elution (1 mj/s-) with 1 mj/s-, the following elution time (hour) is given.

Elution time Ganoderan A9.3 B10.3 Next, the pharmacological effects of the ganoderans of this invention will be shown. Pharmacological effect test Five normal DD mice with a blood sugar level of 140 to 170 /dt↓ of 25 to 30f were grouped. C1 The ganoderanes of the present invention were intracavitally administered to each of these groups at various doses, and the relative blood sugar levels (ch relative to the control) after a certain period of time were measured and are shown in the table below. As a result, it is clear that the relative blood glucose level was lower in the ganoderan group than in the control group.

Claims (1)

[Claims] / The following characteristics: 1) It is a polysaccharide, 2) It has a hypoglycemic effect, 3) Each substance is a) Ganoderan A Molecular weight (gel filtration method): 9300 Specific optical rotation: [α) +58.8° (c O, 19, water) Infrared absorption spectrum # (KBr) νmax (crn-1
): 3270. 013 1H nuclear magnetic resonance spectrum δ: 1.12 (Nakahiro), 2
2.07 (Nakahiro), 4.86 (Nakahiro) Elemental analysis value: C, 41,79; H, 6,07; N,
0.00T Glass fiber Oki paper electrophoretic mobility: 15.4
crn (glucose 11.8 cm ) DEAE ) Jobar chromatography elution time = 9
．． 3 hours) Ganoderan B Molecular weight (gel permeation method): 3600 Specific rotation: [α) D-33,3° (co, 20. Water) Infrared absorption spectrum (KBr) νmax (σ-1): 3
210. 1028 1H nuclear magnetic resonance spectrum δ: 4.40 (Nakahiro) Elemental analysis value: C, 42,94:H, 5,82:N, 1.26
%Glass fiber P paper electrophoretic mobility: 16.1crn
(Glucose 11.86n) DEAE) Yopal chromatography elution time = 1
A polysaccharide consisting of Ganoderan A and/or Ganoderan B having 0.03 hours. Fresh or dried fruiting bodies of Ganoderma or related plants belonging to the family Arunocarinaceae, that is, Ganoderma lucidum, are extracted with water or an aqueous organic solvent, or both, with or without defatting treatment. The extracted solution as it is, or the concentrated solution or the precipitate of a substance that has a hypoglycemic effect is prepared and the concentrated solution or precipitate is dissolved in water or an aqueous organic solvent, and then dialysis or A ganoderan mixture containing ganoderan A and B having the following properties is obtained by ultrafiltration, and this ganoderan mixture is further subjected to fractionation treatment to obtain the following properties: 1) polysaccharide content, 2) hypoglycemia 3) Each substance has a) Ganoderan A Molecular weight (gel filtration method): 9300 Specific optical rotation: [α)n + 58.80 (c O, 19,
Water) Infrared absorption spectrum (KBr) vmax (m
-'): 3270.1013 1H nuclear magnetic resonance spectrum δ: 1.12 (Nakahiro). 2.07 (Nakahiro), 4.86 (Nakahiro) Elemental analysis value: C, 41,79; H, 6,07: N, 0
．． 00% fiberglass, bar oki paper Aerophoretic mobility: 15.4
crn (glucose 11.80) DEAE) Yopal chromatography elution time: 9
．． 3 hours) Ganoderan B Molecular weight (gel filtration method): 3600 Specific rotation: [α) p 33.30 (c O, 20, water)
Infrared absorption spectrum (KBr) vmax (crn)
. 3210.1028 1H nuclear magnetic resonance spectrum δ: 4.40 (Nakahiro) Elemental analysis values: C, 42,94:H, 5,82:N, 1.26
%Glass fiber filter paper electrophoretic mobility 1116.1 cIn
(Glucose 11.8 cm) DEAE) Yopal chromatography bathing time: 10
．． A method for isolating polysaccharides, characterized in that the individual components of Ganoderan A or B are separated for 3 hours. 3. Ganoderma lucidium (Karst), Ganoderma neoyabonicum (Imazeki), Ganoderma tsugae (Maril), Ganoderma secile (Maril), or Tsuyakamaki (Ganoderma secile, Marill), which belongs to the family Sarnococcinaceae. Isolation method according to claim 2, which is Ganoderma culteisi (Berg.) 4! , Reishi belongs to the family Salmonaceae (
Ganoderma luchidoum, Karst). j. The isolation method according to claim 2, wherein the aqueous organic solvent is an aqueous solution containing a lower aliphatic alcohol, acetone, or other water-soluble organic solvent. A. The isolation method according to claim 2, wherein the fractionation treatment includes a molecular weight fractionation treatment and an ion exchange resin treatment. Z The following properties: ■) It is a polysaccharide, 2) It has a hypoglycemic effect, 3) The individual substances are i) Ganoderan A Molecular weight (gel filtration method): 9300 Specific optical rotation: [α) p + 58. 80 (c O, 19,
Water) Infrared absorption spectrum (KBr) νmf1 x (C
1n-"): 3270.1013 1H nuclear magnetic resonance spectrum δ: 1.12 (Nakahiro). 2.07 (Nakahiro), 4.86 (Nakahiro) Elemental analysis value: C, 41, 79; H, 6,07:N,0
．． 00% glass fiber r paper aerophoretic mobility: 1s, 4c
+++ (glucose 11.8 cIn) DEAE) Yopal chromatography elution time: 9.
3 hours) Ganoderan B Molecular weight (gel filtration method): 3600 Specific rotation: [α) D-33,3° (co, 20. Water) Infrared absorption spectrum (Knr) vmax (6n-')
:3210.1028 1H nuclear magnetic resonance spectrum δ: 4.40 (Nakahiro) Elemental analysis value: C, 42,94: H, 5,82; N, 1.
26T glass fiber filter paper electrophoretic mobility: 16.1c
m (glucose 11.8 m) DEAE) Yopal chromatography elution time = 1
Hypoglycemic agent Q containing a polysaccharide consisting of Ganoderan A and/or B as an active ingredient