JP2005132810A - Physiologically active substance from nameko and/or maitake mushrooms and producing method and agency - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide EEM-P, a physiologically active substance from Nameko (Pholiota nameko) and/or Maitake (Grifola frondosa) mushroom, a method for producing the same and a medicinal agents containing the same. <P>SOLUTION: Nameko and/or Maitake mushrooms are extraction-treated with hot water or a lower alcohol and the extract is treated with molecular sieve to remove the low molecular fraction and the high molecular fraction and to collect the EEM-P substance. In this invention, the EEM-P, a production method, agents therefrom and physical and chemical properties of EEM-P are disclosed. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

Detailed Description of the Invention

Industrial application fields

  The present invention relates to a substance having a physiological activity such as anticancer activity, antiallergic activity, immunostimulatory activity, antioxidant activity, etc., obtained from an edible candy of sea cucumber and / or maitake, a method for producing the same, and the physiologically active substance. It relates to the preparations contained.

  The present inventors have been studying in detail the physiological activity of mosses for many years. As a result, anti-cancer action, immunostimulatory action, antioxidant action, blood pressure, blood glucose lowering action, etc. It is designed to show physiological activity. However, depending on the type of cocoon, although it is not active or very weak, if it works well, the effect is over-advertised and released.

The present inventors have studied the glucan which is a polysaccharide of sputum, knowing that the anticancer action of sputum is due to the contained polysaccharide, especially from the reliable research results based on a huge amount of research data. However, the pure simple polysaccharide, glucan, was found to be active by injection but not to be effective orally.
Administration by injection can only be performed in hospitals, and since oral administration is less painful for those receiving the administration, there is a strong need to provide an active ingredient for orally effective sputum.

Problems to be solved by the invention

  An object of the present invention is to provide a method for selecting moss containing a high concentration of substances having physiological activities such as anticancer action and immunostimulatory action by oral administration, and efficiently collecting those physiologically active substances. The present invention provides a preparation in which the obtained active substances are suitable for use.

Means for solving the problem

  As a result of various studies to solve the above-mentioned problems, the present inventors have found that fruit bodies and / or mycelia of maitake, which are used for food, are orally active active substances. And the extract of the moss with a hydrophilic solvent such as water or a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof contains an orally active active substance, These extracts were further processed by the molecular sieve method, and it was found that those having a certain molecular weight range excluding the low molecular part and the high molecular part showed excellent physiological activity, and the present invention was completed.

  That is, the present invention is to extract nameko and / or maitake mushrooms with water, a hydrophilic solvent or a mixed solvent thereof, and then treat these extracts by a molecular sieve method to obtain a molecular weight of 6,000 to 100, A bioactive substance EEM-P obtained by collecting 000 parts is provided.

Implementation symmetry

  In addition, the bioactive substance EEM-P of the present invention is a pulverized fruit body and / or mycelium of moss used for edible use such as nameko and / or maitake (hereinafter referred to as “茸”). Is extracted with water and / or a hydrophilic solvent. As the hydrophilic solvent used for this extraction, a lower alcohol having 1 to 4 carbon atoms is preferably used.

  Extraction of the fraction containing the physiologically active substance EEM-P from the cocoon, for example, by adding an appropriate amount of water or a hydrophilic solvent of alcohols to the cocoon as a raw material, for example, about 80 to 98 ° C., preferably water In this case, the extraction is performed at 90 to 98 ° C. under extraction under reflux for about 1 to 4 hours, preferably 2 to 4 hours. In the extraction, about 3 to 10 times by weight of solvent is used with respect to the weight of the soot.

  The extract obtained as described above can collect the extract as a solid by separating solid impurities by filtration or the like, if necessary, and then distilling off the solvent under reduced pressure.

  The solid obtained in this way is active by oral administration and can be used as it is. However, in order to increase the activity, the extract is subjected to molecular sieve treatment to remove unnecessary parts, and again under reduced pressure. A portion having a molecular weight of 6,000 to 100,000 obtained by concentrating to 1 is used as the bioactive substance EEM-P.

  Examples of molecular sieves used for the above purpose include molecular sieve membranes (flat membranes), hollow filtration membranes, dialysis membranes, molecular sieve chromatography, etc. The methods using these can be applied as appropriate.

  Hereinafter, taking a case where module type hollow filtration is used as an example, a fractionation acquisition method of the physiologically active substance EEM-P will be described as follows. That is, the water and / or hydrophilic solvent extract obtained by extraction as described above (a fraction containing the bioactive substance EEM-P) is first subjected to a low molecular fraction module such as AIP-3013, A low molecular weight material (a material having a molecular weight of 6,000 or less) is removed by treatment with AIP-2013 or AIP-1010 (manufactured by Asahi Kasei Kogyo Co., Ltd.). For example, by treating with AIP-3013, AIP-2013, or AIP-1010 (manufactured by Asahi Kasei) to remove the high molecular substance (substance having a molecular weight of 100,000 or more), the external liquid is again used with the low molecular fraction module. Concentrate. By doing so, a physiologically active substance EEM-P having a molecular weight of 6,000 to 100,000 can be obtained.

  In general, the molecular sieve method differs depending on the shape of the molecule and may differ from the molecular weight obtained by other methods. Accordingly, the molecular weight in the present invention means the molecular sieve method, and more precisely, the low molecular weight molecular weight was measured using AIP-3013 or a low molecular fraction module equivalent thereto. Value. Similarly, the molecular weight on the high molecular weight side is a value measured using AIP-3013 or a low molecular weight fraction module equivalent thereto.

  Although the bioactive substance EEM-P of moss obtained in this manner was described, the yield of the bioactive substance from cocoons varies depending on the culture conditions when the koji mycelium is used as a raw material, It is necessary to select the optimal culture temperature. In addition, physicochemical properties may be slightly different depending on the type of cocoon.

  For example, a bioactive substance EEM-P (hereinafter referred to as EEM-PN) produced from nameko as a raw material exhibits the physicochemical properties shown in Example 1 below. Moreover, the bioactive substance EEM-P (hereinafter referred to as EEM-PM) produced using maitake as a raw material exhibits the physicochemical properties shown in Example 2 below.

  Thus, even if there is a slight difference in physicochemical properties, as shown in Example 3 below, each has a higher physiological activity than the simple hot water extract of each cocoon. Therefore, it can be said that they possess common properties.

  Although the biologically active substance EEM-P described above can exert physiological activity by oral administration, the oral intake for exerting the desired effect varies depending on the age, weight, purpose, etc. of the person, In particular, EEM-P per day for an adult is in the range of 200 to 5000 mg, preferably 1000 to 3000 mg, and it is appropriate to take this amount divided into several times a day.

  The physiologically active substance EEM-P of the present invention can be used as tablets, granules, capsules, and liquids by a conventional method. However, if it is left in the air as it is, it may absorb moisture and change color or change its quality. It is preferable to use as tablets with The film coating material used for this purpose is preferably soy peptide, shell resin material, etc., and other film coating materials may not be able to sufficiently prevent moisture absorption and may cause discoloration and alteration. obtain.

  The present invention will be described more specifically with reference to the following examples. In the examples, the anticancer effect was shown as the physiological activity. However, it goes without saying that the substance of the present application is not limited to the anticancer property and has the aforementioned physiological activity.

Example 1
After pulverizing 1000 grams of nameko finely, hot water extraction is performed, and a precipitate is removed from the obtained extract with a PS-88 membrane (manufactured by Otsuka Business Co., Ltd.). Using this hot water extract, a fraction containing a high molecular weight substance (a substance having a molecular weight of 100,000 or more) was concentrated using a low molecular fraction module-type hollow filtration membrane, AIP-3013 (manufactured by Asahi Kasei Corporation). . The concentrated solution was removed from the polymer material (molecular weight of 100,000 or more) using a module-type hollow filtration membrane for polymer fractionation, AIP-3013 (manufactured by Asahi Kasei Kogyo Co., Ltd.), and the external solution was again removed from the polymer solution with a low molecular fraction. Concentrated using module AIP-3013. In this way, a bioactive substance EEM-PN having a molecular weight of 4,000 to 100,000 was obtained.
[Physicochemical properties of EEM-PN]
(1) UV absorption (measurement of UV absorption at 370 to 190 nm in an aqueous solution):
Maximum value: 258.0 nm
(2) Infrared absorption (measured by ATR method):
Maximum value (cm ⁻¹ ): 3235, 1556, 1396, 1033
(The absorption curve is shown as FIG. 1)
(3) ¹ H-NMR (measured in D ₂ O):
The chart is shown in Fig. 3.
(4) Measured in ¹³ C-NMRD ₂ O):
Figure 5 shows the chart.
(5) Protein content 42.9%
(6) Sugar content 19.1%
(7) Composition ratio of main sugars Glucose: galactose: mannose = 3: 1: 1
(8) Amino acid composition Aspartic acid: 12.2%, Glutamic acid: 13.9%, Glycine: 10.9%
, Alanine: 11.3%, valine: 6.8%, serine: 6.5%, leucine: 6.
9%.
The thus obtained EEM-PN could be made into a solid powder by lyophilization, and further tableted to form an orally administrable tablet.

Example 2
Extract 1,000 grams of maitake with refluxing with hot water to obtain 107 grams of hot water extract. This was dissolved in water, then put into a dialysis tube and dialyzed to obtain a fraction containing a polymer substance (molecular weight of 60,000 or more) in the dialysis inner membrane. The fraction containing the polymer substance is treated with a molecular sieve membrane PM-10 to remove the polymer substance (molecular weight of 50,000 or more), freeze-dried, and a physiologically active substance having a molecular weight of 6,000 to 50,000. A material with a high EEM-PM content was obtained. This was used as a powder.
[Physicochemical properties of EEM-PM]
(1) UV absorption (measurement of UV absorption at 370 to 190 nm in an aqueous solution):
Maximum value: 258.4 nm
(2) Infrared absorption (measured by ATR method):
Maximum value (cm ⁻¹ ): 3220, 1572, 1395, 987
(The absorption curve is shown in FIG. 2)
(3) ¹ H-NMR (measured in D ₂ O):
Figure 4 shows the chart.
(4) Protein content 64.8%
(5) Sugar content 15.7%
(6) Composition ratio of main sugars Glucose: galactose: mannose = 1: 2: 1
(7) Amino acid composition Aspartic acid: 12.6%, glutamic acid: 13.2%, glycine 11.2%, alanine: 10.1%, valine: 6.7%, serine: 6.6%, leucine: 6 .7%.

Example 3
A powder was prepared using the bioactive substance EEM-PN or PM obtained in Example 1 or 2, and the following anticancer test was performed. The anticancer activity was determined by comparing the survival rate in the anticancer test with that of the control group. The results are shown in Table 1.
(Anti-cancer test)
Live cells of Lewis lung cancer were transplanted subcutaneously into BDF1 female mice, and from the following day, the physiologically active substance EEM-PM or PN obtained in each example was suspended once a day in 500 mg / kg in purified water and orally administered for 20 days. It was administered. For the control group administered with purified water only, the group treated with the hot water extract of sputum and the group administered with the physiologically active substance obtained in Example 1 or 2, the survival rate was calculated from the average survival days of the mice in each group. The results are shown in Table 1.

The invention's effect

    According to the present invention, a substance having high physiological activity can be easily obtained from edible sea cucumber and / or maitake, and a product useful as a medicine or health food can be obtained.

FIG. 1 is an infrared absorption curve of the bioactive substance EEM-PN obtained in Example 1 measured by the ATR method. FIG. 2 is an infrared absorption curve of the bioactive substance EEM-PM obtained in Example 2 measured by the ATR method. FIG. 3 is a ¹ H-NMR (D ₂ O) chart of the bioactive substance EEM-PN obtained in Example 1. FIG. 4 is a chart of ¹ H-NMR (D ₂ O) of the bioactive substance EEM-PM obtained in Example 2. FIG. 5 shows the bioactive substance EEM-PN obtained in Example 1.¹³C-NMR (D ₂O) chart.

Claims (11)

Nameko and / or maitake mushrooms are extracted with water, a hydrophilic solvent or a mixed solvent thereof, and then this extract is treated by a molecular sieve method to obtain a molecular weight of 6,000 to 100,000 in an aqueous solution. A bioactive substance EEM-P.P. Obtained by collecting a portion where the maximum value of ultraviolet absorption is 255 to 260 nm. The bioactive substance EEM-P. 2 according to claim 1, wherein the hydrophilic solvent is a lower alcohol having 1 to 4 carbon atoms. Nameko is extracted with water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, and then processed by a molecular fluid method, and has a molecular weight of 6,000 to 100,000, and absorbs ultraviolet rays in an aqueous solution. The maximum absorption wave number of infrared absorption is 3235, 1556, 1396, 1033 cm ⁻¹ (both include errors within ± 50 ⁻¹ ), and the protein content is 38% to 48%. The sugar content is 14% to 24%, and the amino acid composition (molar ratio) is aspartic acid: 12.2%, glutamic acid: 13.9%, glycine: 10.9%, alanine: 11.3. %, Valine: 6.8%, serine: 6.5%, leucine: 6.9% (all include errors within ± 3%), and physiological activity mainly consisting of glucose as a constituent sugar Quality EEM-PN. Extraction of maitake with water, lower alcohols having 1 to 4 carbon atoms or a mixed solvent thereof, followed by treatment by molecular sieve method, molecular weight of 6,000 to 100,000, absorption of ultraviolet rays in aqueous solution The maximum absorption wave number of infrared absorption is 3220, 1572, 1395, and 987 cm ⁻¹ (all including errors within ± 50 ⁻¹ ), and the protein content is 60% to 70%. The sugar content is 10% to 20%, and the amino acid composition (molar ratio) is aspartic acid: 12.6%, glutamic acid: 13.2%, glycine 11.2%, alanine: 10.1% , Valine: 6.7%, serine: 6.6%, leucine: 6.7% (both including errors within ± 3%), mainly composed of glucose and galactose Bioactive substance EEM-PM. Nameko and / or maitake mushrooms are extracted with water, a hydrophilic solvent or a mixed solvent thereof, and then this extract is treated by a molecular sieve method to fractionate a portion having a molecular weight of 6,000 to 100,000. A process for producing a physiologically active substance EEM-P. The method for producing a bioactive substance EEM-P according to claim 5, wherein the hydrophilic solvent is a lower alcohol having 1 to 4 carbon atoms. The method for producing a physiologically active substance EEM-P according to claim 5 or 6, wherein the molecular sieve method is a method using a molecular sieve membrane (flat membrane), a hollow filtration membrane, a dialysis membrane, or a molecular chromatography. An agent comprising the physiologically active substance according to any one of claims 1 to 4 as an active ingredient. The agent according to claim 8, which is an anticancer agent. The agent according to claim 8 or 9, which is a tablet, powder, granule, capsule or liquid. The agent according to claim 8, 9 or 10, which is a tablet coated with a film coating agent of soybean peptide and / or shell resin.

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