JPS6338325B2 - - Google Patents

Description

[Detailed description of the invention]

This invention relates to novel polysaccharides, particularly polysaccharides named Ganoderan A and B which can be isolated from Ganoderma lucidum, methods of isolation and uses thereof. Ganoderma, especially Polyporaceae
Ganoderma lucidum Karst.
It is widely known that it is used as an effective drug for chronic bronchitis, coronary insufficiency, hyperlipidemia, hypertension, neurasthenia, hepatitis, etc. However, nothing is known about the active ingredients of this reishi mushroom. The inventors of this invention obtained the knowledge that the water-soluble fraction contains a component that has a hypoglycemic effect,
Furthermore, as a result of separating and purifying this, two types of polysaccharides named Ganoderan A and B were obtained. As the Ganoderans-containing Reishi mushroom of the present invention, the most preferred is Ganoderma lucidum. Other related plants, Ganoderma neoyaponicum, Ganoderma
neo-japonicum Imazeki), Ganoderma tsugae, maril, Ganoderma
Ganoderma sessile (Tsugae Murrill), Ganoderma sessile
Murrill), Ganoderma
Kurteisi, Berg. Ganoderma curtissi
Berk.). The ganoderans of the present invention are obtained by extracting, separating, and purifying the fresh or dried material from the above-mentioned Ganoderma mushrooms or related plants, or by culturing the mycelia of the above-mentioned Moscanthus mushrooms or related plants, and then extracting the same. It can be obtained by separation and purification. Specifically, the ganoderanes of this invention can be isolated by the following method. First, the raw material Ganoderma lucidum is defatted without being defatted or using a conventional fat-soluble organic solvent, 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 concentrations ranging from about 1% to about 30% are used depending on the type of raw material. It will be done. Further, this extraction is promoted by heating, and the raw material is preferably pulverized. Next, the above extract is directly subjected to dialysis using a cellulose dialysis membrane or subjected to ultrafiltration treatment, or the extract is once concentrated by a method such as solvent distillation, or ethanol, methanol, etc. A precipitate of a substance having a hypoglycemic effect is prepared by adding a solvent, and a solution obtained by dissolving this concentrate or precipitate in water or the aqueous organic solvent is subjected to the dialysis or ultrafiltration. Then, the ganoderan mixture is obtained. The ganoderan mixture obtained as described above was subjected to the following fractionation treatment to obtain ganoderan A and B.
can be isolated into each simple substance. First, the above-mentioned ganoderane mixture is treated using the difference in electrical properties of each ganoderane to separate each ganoderane. For example, anion exchange resin
It is treated with DEAE Sephadex and DEAE Toyopearl and fractionated into a fraction containing Ganoderan A and a fraction containing Ganoderan B.
The two fractions thus obtained are subjected to the following molecular weight fractionation method. This fractionation method includes a gel filtration method using Sepharose, Sephadex, Sephacryl, agarose beads, etc., and a molecular weight cutoff of 14.
One example is a method using an ultrafiltration membrane of ~100,000.
The two fractions thus obtained earlier are processed to separate each ganoderane. Thus, this invention extracts the fresh or dried material of Cinnamon mushroom or related plants belonging to the family Arunocycolaceae without defatting or defatting it with water or an aqueous organic solvent at room temperature or by heating, and extracting the obtained extract as it is. Or, once the concentrate is made or a precipitate of the substance having hypoglycemic effect is made, this concentrate or a solution of the precipitate dissolved in water or an aqueous organic solvent is subjected to dialysis or ultrafiltration. Obtaining a ganoderan mixture containing ganoderan A and B, and further subjecting this ganoderan mixture to a fractionation treatment,
The present invention provides a method for isolating polysaccharides, which is characterized by separating the individual components of Ganoderan A and B having the properties described below. 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 in the case of oral administration for adults, it is 10 to 300 mg, preferably 30 to 100 mg, of ganoderans per day.
The effect can be exerted by administering the drug in 2 to 3 divided 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 method and dosage form are usually oral forms such as powders, tablets including sublingual tablets, emulsions, capsules, medicines, granules, and liquids (including liquid extracts, syrups, etc.). There is. It may also be in the form of an injection. 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. To give some specific examples, excipients in powders and other powders for internal use include lactose, starch,
Examples include dextrin, calcium phosphate, calcium carbonate, synthetic and natural aluminum silicates, magnesium oxide, dried aluminum hydroxide, magnesium stearate, sodium bicarbonate, dried yeast, and the like. In addition, transdermal absorption agents prepared by adding ordinary excipients are also included in the present invention. Although the water extract of Reishi 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 present invention will be explained by examples and animal experiments. Example: Thoroughly dry the fruiting body of a mature stone mushroom, wash approximately 10 kg of it with cold water, thoroughly drain the water, dry it, and then apply it to a crusher. Add and extract for about 3 hours under boiling. Repeat this extraction three times.
The obtained extracts were combined and evaporated to dryness under reduced pressure at a temperature of 50 to 60°C to obtain 0.6 kg of extract (A) as a brown powder. 100 g of this extract (A) was dissolved in 400 ml of water, and the solution was filtered by suction to remove insoluble matter. This liquid is used as the internal liquid and the water from step 2 is used as the external liquid.
Dialysis was performed using Tube 36/32 (cellulose dialysis tube manufactured by Visking, which passes particles with a size of 24 angstroms or less) for 10 days, replacing the external water once a day. The obtained dialyzed fluid was subjected to chromatography using a DEAE Toyopearl column (2.2 cmφ x 45 cm), first eluted with water 2, then 1M sodium chloride aqueous solution 2, and water eluted fraction G-1. and NaCl aqueous solution elution fraction G-2
I got it. Fraction G-1 was evaporated to dryness under reduced pressure at 50-60°C to obtain 5 g of powder. Dissolve 2 g of this powder in 40 ml of water and add it to a Sepharose 6B column (4.0 cmφ×
95 cm) and eluted with 0.1 M NaCl solution (flow rate 2 ml/mm) to obtain multiple fractions of 20 ml each, among which fractions containing ganoderane were selected as follows and combined under reduced pressure. 50
Concentrate to 100ml at ~60°C. That is, 0.8 ml of water is added to 0.2 ml of each fraction, 1 ml of a 2% phenol-containing aqueous solution is added, and 5 ml of sulfuric acid is added, and after standing for 30 minutes, λmax:
The absorbance at 490 nm was measured, and the fraction with the highest absorbance was selected. The above concentration (100 ml) was put into a Visking tube 3.6/32 as the inner solution, and dialysis was performed using 2 water as the outer solution, replacing the outer solution once a day for 3 days (3 days dialysis). The obtained internal solution was concentrated at 50 to 60°C under reduced pressure to 40 ml. The above concentrate (40 ml) was subjected to chromatography on a DEAE Toyopearl column (2.2 cmφ 2ml/min)
Gradient elution was performed over 20 hours. Fraction of eluate obtained in gradient elution (20
ml fraction), select a ganoderan-containing fraction in the same manner as above, combine the fractions, and boil under reduced pressure for 50~
It was concentrated to 100 ml at 60°C. 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 ml at 50 to 60° C. under reduced pressure. This concentrated solution was applied to a Sephacryl S-200 column (4.0
cmφ×95cm) column chromatography,
A solution of 30.3 g of trishydroxymethylaminomethane in saline (73.05 g of NaCl in 2.5 g of water) was eluted with 0.1 M Tris-HCl buffer whose pH was adjusted to 7.0 with 15-20% hydrochloric acid (flow rate 2 ml/min). death,
Ganoderan-containing fractions were selected from the 20 ml fractions obtained and combined in the same manner as described above, and concentrated to 100 ml under reduced pressure at 50-60°C. The resulting concentrated solution was dialyzed 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 A (90 mg) as a white powder. Ganoderan A had the properties described below and was confirmed to be a single substance, and Ganoderan B, which will be described below, was similarly confirmed. The above fraction G-2 was heated at 50 to 60°C under reduced pressure.
Concentrate to 100 ml and subject this concentrated solution to dialysis for 3 days in the same manner as above, and the resulting internal solution was heated at 50 to 60°C under reduced pressure.
It was concentrated to 40ml. The obtained concentrate (40 ml) was subjected to chromatography using a Sepharose 6B column → dialysis → chromatography using a DEAE Toyopearl column → dialysis in the same manner as for Fraction G-1. 100ml of the obtained internal solution was heated to 40ml under reduced pressure at 50-60℃.
Concentrate to
The mixture was subjected to chromatography using 0.05 M phosphate buffer (PH7.0) (flow rate 0.5 ml/min) to obtain fractions of 10 ml each. These fractions were examined in the same manner as above, and fractions containing ganoderane were selected and combined, and this was heated under reduced pressure for 50 to 50 minutes.
It was concentrated to 100ml at 60°C. This concentrated solution was subjected to dialysis for 3 days in the same manner as above, and the resulting internal solution was
Ganoderan B is evaporated to dryness at ~60°C as a white powder.
(180mg) was obtained. Each of the ganoderans obtained as described above has the following properties (residues other than C, H, and N in the elemental analysis below are 0). (a) Ganoderan A Molecular weight (gel permeation method): 9300 Specific optical rotation: [α] _D +58.8° (c0.19, water) Infrared absorption spectrum (KBr) νmax (cm ^-1 ):
3270, 1013 ¹ H 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 paper electrophoretic mobility: 15.4 cm (glucose 11.8 cm) DEAE Toyopearl chromatography elution time : 9.3 hours (b) Ganoderan B Molecular weight (gel filtration method): 3600 Specific optical rotation: [α] _D −33.3° (c0.20, water) Infrared absorption spectrum (KBr) νmax (cm ^-1 ):
3210, 1028 ¹ H nuclear magnetic resonance spectrum δ: 4.40 (Nakahiro) Elemental analysis value: C, 42.94; H, 5.82; N, 1.26% Glass fiber paper electrophoretic mobility: 16.1 cm (glucose 11.8 cm) DEAE Toyo Pearl Chromatography Elution time by graphi: 10.3 hours Among the above properties, the molecular weight, glass fiber paper electrophoretic mobility, and elution time by DEAE Toyopearl chromatography were measured as follows. () Molecular weight Each ganoderan was subjected to gel filtration using Cephalyl S-200 to determine its retention capacity, and the molecular weight was calculated from a standard curve prepared using Dextran T series. Molecular weight Cephacrylganoderane A 9300 Cephacryl S-200 〃 B 3600 〃 -200 () Glass fiber paper electrophoretic mobility Glass fiber paper (Watmann GF/C,
15 x 40cm) indicates the following moving distance (cm). (Conditions: alkaline borate buffer PH9.3,
450V, 2 hours) Travel distance Ganoderan A 15.4 〃 B 16.1 Glucose 11.8 () Elution time of DEAE Toyopearl chromatography DEAE Toyopearl column (2.2cmφ x 45cm)
using 0.05M Tris-HCl buffer (PH
8.0) for the first 5 hours, and then added sodium to the same buffer (0-7M,
20 hours) and perform elution (1 ml/min),
Give the following elution times (hours). Elution time Ganoderan A 9.3 B 10.3 Next, the pharmacological effects of the ganoderans of this invention will be shown. Pharmacological effect test Blood sugar level 140-170mg/dl weight, normal 25-30g
The ganoderans of the present invention were intravenously administered to each group of five DD mice at various doses, and the relative blood glucose level (% of control) was measured after a certain period of time. Shown in the table. As a result, it is clear that the relative blood sugar level in the ganoderan group was lower than that in the control group.

【table】

【table】

Claims (1)

[Claims] 1. The following characteristics: (1) It is a polysaccharide, (2) It has a hypoglycemic effect, (3) Each substance has (a) Ganoderan A molecular weight (gel filtration method): 9300 ratio Optical rotation: [α] _D +58.8° (c0.19, water) Infrared absorption spectrum (KBr) νmax (cm
^-1 ): 3270, 1013 ¹ H 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 paper electrophoretic mobility: 15.4cm
(glucose 11.8 cm) DEAE Toyopearl chromatography elution time: 9.3 hours (b) Ganoderan B Molecular weight (gel filtration method): 3600 Specific rotation: [α] _D −33.3° (c0.20, water) Infrared absorption Spectrum (KBr) νmax (cm
^-1 ): 3210, 1028 ¹ H nuclear magnetic resonance spectrum δ: 4.40 (Nakahiro) Elemental analysis values: C, 42.94; H, 5.82; N, 1.26
% Glass fiber paper electrophoretic mobility: 16.1cm
(Glucose 11.8 cm) A polysaccharide consisting of Ganoderan A and/or Ganoderan B having an elution time of DEAE Toyopearl chromatography: 10.3 hours. 2. Extracting the fruiting body of Ganoderma or related plants belonging to the family Arunococcaceae, fresh or dried, with or without defatting treatment, with water or an aqueous organic solvent, or with both, The obtained extract is used as it is, or the concentrate is made into a precipitate of a substance that has a hypoglycemic effect, and the concentrate or precipitate is dissolved in water or an aqueous organic solvent. A ganoderan mixture containing ganoderan A and B having the following properties is obtained by external filtration, and this ganoderan mixture is further subjected to fractionation treatment to have the following properties: (1) It is a polysaccharide: (2) Hypoglycemic (3) Individual substances (a) Ganoderan A Molecular weight (gel filtration method): 9300 Specific rotation: [α] _D +58.8° (c0.19, water) Infrared absorption spectrum (KBr) νmax (cm
^-1 ): 3270, 1013 ¹ H 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 paper electrophoretic mobility: 15.4cm
(glucose 11.8 cm) DEAE Toyopearl chromatography elution time: 9.3 hours (b) Ganoderan B Molecular weight (gel filtration method): 3600 Specific rotation: [α] _D −33.3° (c0.20, water) Infrared absorption Spectrum (KBr) νmax (cm
^-1 ): 3210, 1028 ¹ H nuclear magnetic resonance spectrum δ: 4.40 (Nakahiro) Elemental analysis values: C, 42.94; H, 5.82; N, 1.26
% Glass fiber paper electrophoretic mobility: 16.1cm
(Glucose 11.8 cm) DEAE Toyopearl chromatography elution time: 10.3 hours A polysaccharide isolation method characterized by separating individual components of Ganoderan A or B. 3. Ganoderma lucidium (Karst.), Ganoderma neoyaponicum (Imazeki), Ganoderma tsugae (Maril), Ganoderma lucidum (Karst.), Ganoderma lucidum (Karst.), Ganoderma lucidium (Imazeki), Ganoderma lucidium (Imazeki), Ganoderma lucidum (Karst.), Ganoderma lucidium (Karst.), Ganoderma lucidium (Karst.)
2. The isolation method according to claim 2, wherein the isolation method is from Ganoderma curteisi, Berg. 4. The isolation method according to claim 2, wherein the reishi mushroom is Ganoderma lucidum, Karst. 5. 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. 6. The isolation method according to claim 2, wherein the fractionation treatment comprises a molecular weight fractionation treatment and an ion exchange resin treatment. 7 The following properties: (1) It is a polysaccharide, (2) It has a hypoglycemic effect, (3) Each substance has (a) Ganoderan A Molecular weight (gel filtration method): 9300 Specific optical rotation: [α] _D +58.8° (c0.19, water) Infrared absorption spectrum (KBr) νmax (cm
^-1 ): 3270, 1013 ¹ H 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 paper electrophoretic mobility: 15.4cm
(glucose 11.8 cm) DEAE Toyopearl chromatography elution time: 9.3 hours (b) Ganoderan B Molecular weight (gel filtration method): 3600 Specific rotation: [α] _D −33.3° (c0.20, water) Infrared absorption Spectrum (KBr) νmax (cm
^-1 ): 3210, 1028 ¹ H nuclear magnetic resonance spectrum δ: 4.40 (Nakahiro) Elemental analysis values: C, 42.94; H, 5.82; N, 1.26
% Glass fiber paper electrophoretic mobility: 16.1cm
(Glucose 11.8 cm) DEAE Toyopearl chromatography elution time: 10.3 hours A hypoglycemic agent containing a polysaccharide consisting of Ganoderan A and/or B as an active ingredient.