WO2004099428A1

WO2004099428A1 - Extraction method of beta glucan from phellinus linteus through cell destruction and enzyme fermentation method

Info

Publication number: WO2004099428A1
Application number: PCT/KR2004/001055
Authority: WO
Inventors: Kwan-Ho Kim; Il-Geun Roh
Original assignee: Individual
Current assignee: Individual
Priority date: 2003-05-07
Filing date: 2004-05-06
Publication date: 2004-11-18
Anticipated expiration: 2005-11-07
Also published as: WO2004099427A1; KR20040096000A; AU2003256111A1

Abstract

Disclosed herein is a method for extracting and isolating crude β-glucan as a medically effective ingredient from Phellinus linteus in high yield. Specifically, the method increases the extraction rate of crude β-glucan from Phellinus linteus through degradation of the fruiting bodies of Phellinus linteus by sonication, destruction of cell walls using hot-water under high pressure, and enzyme fermentation.

Description

EXTRACTION METHOD OF BETA GLUCAN FROM PHELLINUS LINTEUS THROUGH CELL DESTRUCTION AND ENZYME FERMENTATION METHOD

Technical Field

The present invention relates to a method for efficiently extracting and isolating β-glucan from Phellinus linteus in high yield.

Background Art

Phellinus species are very rare perennial mushrooms in nature which grow on mulberry and broadleaved trees in the high reaches, and several tens of species have been identified to date. Representative Phellinus spp. are Phellinus linteus and Phellinus baumi which mainly grow on the trunk of mulberry and broadleaved trees. According to a recent report, Phellinus linteus which has been allowed only as a medical raw material can be used as a raw material for foods (see, an article from the Korean daily economic newspaper dated February 7, 2003). Like common mushrooms, Phellinus linteus contains a large quantity of nutrients beneficial to humans, such as potassium, calcium, magnesium, vitamin B2, B3 and C, fibroid materials, amino acids and the like, and in particular, β- glucan, which is a polysaccharide substance. β-glucan is a medicinal and therapeutically active ingredient. The fact that β-glucan has immunopotentiating effects and anticancer effects is found in many related literatures and Korean traditional herb medical books, including Dongeuibogam, Sinnongbonchogyeong and Hyangyakjipsungbang. Recent studies have revealed that Phellinus linteus has inhibitory effects against viruses, bacteria, fungi and parasites, inhibitory effects against tumors (cancers) at an inhibition level of 96.7%, and antioxidant effects against oxygen radicals and free radicals through immunopotentiating mechanisms. β-glucan contained in Phellinus linteus is present in cell walls in a protein-bound state, and is surrounded by a large amount of water-insoluble fibroid materials. Phellinus linteus includes fruiting bodies composed of a number of hard and thick cell layers. Korean Patent Laid-open No. 2003-30636 (published on April 18, 2003) discloses a method for producing a protein-polysaccharide by adding an organic substance and an organic nutrient to the fruiting bodies of a mushroom to obtain a fermentation mixture, inoculating the mixture with the mycelia of the ^■ basidiomycetes or yeast, followed by fermentation, hot-water extraction, dialysis, centrifugation and lyophilization.

According to this method, the texture of the fruiting bodies is softened by fermentation so that the extraction of the protein-polysaccharide is facilitated and thus the protein-polysaccharide is produced in high yield. However, since other fermentation metabolites as well as ingredients inherent to the mushroom are generated, it is difficult to collect functional ingredients of the mushroom in the natural state. Although the hard fruiting bodies are finely pulverized, they are too large in size to be used as substrates of an enzyme generated during fermentation by the inoculated mycelia of the basidiomycetes or yeast. In addition, since the fruiting bodies are highly insoluble, it is almost impossible to collect β-glucan present in the cell walls on a large scale. Furthermore, the fruiting bodies contain impurities other than β-glucan.

Korean Patent No. 308392 (published on August 28, 2001) discloses a method for producing an extract of natural Phellinus linteus by mixing pulverized - Phellinus linteus with a solvent, and boiling the mixture in a heating bath under reduced pressure. At this time, the mixture is subjected to super high frequency sonication for 24 hours in order to increase the extraction efficiency.

This method requires a long time for extraction, and uses an organic solvent such as 1,3-butylene chloride, propyleneglycol or isopropanol as an extracting solvent. Accordingly, the method is improper to separate highly polar β-glucan present in the cell walls on a large scale and to extract inherent ingredients of Phellinus linteus.

On the other hand, only 5~12% of ingredients of Phellinus linteus are extracted by hot-water extraction, and the remainder are not used. Considering that Phellinus linteus is an expensive oriental herb costing as much as hundreds or thousands of U.S. dollars per kg, the hot- water extraction must be reconsidered because it is uneconomical. The medicinal β-glucan is present in hard cell layers and is a highly water-insoluble macromolecule having a molecular weight on the order of 1,000,000 daltons. Since the quantification of the macromolecular β- ^• glucan has not been sufficiently investigated, the simple hot- water extraction must be reconsidered under this situation.

A number of studies on maximizing the extraction efficiency of medicinal ingredients, particularly β-glucan, from expensive Phellinus linteus are being actively undertaken. These studies seem to contribute to decreased economic burden to patients as a medicinal and edible material, and induction of scientific clinical results.

Brief Description the Drawings

The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

Figs. 1 to 5 are optical microscope images (1250x) showing changes in the morphology of the fruiting bodies of Phellinus linteus powders and residues obtained after extraction and centrifugation of the Phellinus linteus powders, which are obtained in Example 1 and Comparative Examples 1 and 2 of the present invention.

Specifically, Fig. 1 is an optical microscope image of Phelinus Linteus; Fig. 2 is an optical microscope image of pulverized Phellinus linteus; Fig. 3 is an optical microscope image of ultrasonically pulverized Phellinus linteus; Fig. 4 is an optical microscope image of hot-water treated Phellinus linteus under high pressure; and Fig. 5 is an optical microscope image of enzyme-treated Phellinus linteus.

Disclosure of the Invention

Technical Solution

The present invention has been made in view of the above problems, and it is an object of the present invention to provide a method for efficiently extracting and isolating β-glucan and other medically effective ingredients from Phellinus linteus. In the method of the present invention, the Phellinus linteus is subjected to sonication and the hard cell layers are degraded under high pressure hot-water extraction conditions, thereby forming a condition under which protease and cellulase can function, hi addition, since the content of soluble ingredients is determined by a dry process, the total extraction efficiency can be measured. Furthermore, the extraction efficiency of β-glucan can be quantitatively determined by an ultrafiltration method using the molecular size of β-glucan.

In order to accomplish the above object of the present invention, there is provided a method for extracting medically effective ingredients from Phellinus linteus, comprising the steps of: adding a pulverized Phellinus linteus powder into a distilled water of an amount ranging from 20 to 50 times based on the weight of the powder to make a mixture, and ultrasonicating the mixture; extracting the mixture with hot- water under a pressure of 2 to 3 atm, and filtering the extract through a membrane filter to obtain a filtrate; and enzyme-reacting the filtrate with cellulase and alkalase and subjecting to ultrafiltration on a membrane filter.

Advantageous effects

As apparent from the above description, the method for extracting

Phellinus linteus according to the present invention is very economical and quantitative. According to the method of the present invention, medically effective ingredients of expensive Phellinus linteus can be utilized in the various industrial fields such as pharmaceuticals, cosmetics and foods in an economic and efficient manner.

Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Best Mode for Carrying Out the Invention

The present invention will now be described in more detail with reference to the following examples. However, these examples are given for the purpose of illustration and are not to be construed as limiting the scope of the invention.

Comparative Example 1: Hot- water extraction of samples and ultrafiltration lOg of each of Phellinus linteus powder was extracted with about 300ml of distilled water at 100°C for 1, 2, 3 and 4 hours, followed by centrifugation. The resulting supernatant was concentrated with heating to a volume of 15ml. 5ml of the concentrate was separately dried to obtain a dry extract. The dry extract was weighed, and the result was multiplied by three. The obtained value was used as an extraction rate of the total dry extract. The results are summarized in Table 1 below. The remaining 10ml of the concentrate was subjected to ultrafiltration on a membrane filter having a pore size of 7,000 MWCO (Molecular Weight Cut Off). After 10ml of a phosphate buffer (pH 5.8) was added to the filtrate and dissolved, 1.2mg of cellulase (Fluka 22180 0.3 U/mg) was added thereto. The mixture was cultured at 60°C for one day. After the mixture was adjusted to pH 9.0 by addition of a potassium phosphate solution, it was subjected to an enzyme reaction with cellulose and 1ml of alkalase was added thereto. The resulting mixture was cultured at 60°C for one day. The obtained culture was subjected to ultrafiltration on a membrane filter having a pore size of 7,000 MWCO (Molecular Weight Cut Off). The obtained filtrate was concentrated. About 1ml of the resulting supernatant was again subjected to ultrafiltration using 20ml of distilled water. The obtained filtrate was concentrated. 1ml of the resulting supernatant was dried under reduced pressure to afford crude β-glucan as a dark brown solid. The crude β-glucan was weighed and the result was multiplied by 1.5. The obtained value was used as an extraction rate of the crude β-glucan. The results are summarized in Table 1 below.

Table 1

Comparative Example 2: Hot- water extraction of samples under high pressure and ultrafiltration

About 300ml of distilled water was added to lOg of each of Phellinus linteus powder, and each mixture was extracted under 2 atm and at 120°C for 1, 2, 3 and 4 hours. The following procedure was carried out in the same manner as in Comparative Example 1. The results are summarized in Table 2 below.

Table 2

10. Example 1: Ultrasonic hot-water extraction of samples under high pressure and ultrafiltration

About 300ml of distilled water was added to lOg of each of Phellinus linteus powder, and each mixture was subjected to ultrasonic irradiation using a Power 6, Sonifier Cell Disrupter 350 manufactured by Branson Sonic Power Co.

15 for 10, 20, 30 and 60 minutes. Thereafter, the extraction was performed under 2 atm at 120°C for 3 hours. The following procedure was carried out in the same manner as in Comparative Example 1. The results are summarized in Table 3 below.

20 Table 3

As can be seen from the extraction conditions and extraction rates of crude β-glucan in Phellinus linteus shown in Tables 1 to 3, the maximum extraction rate (34.6%) of the total dry extract and the maximum extraction rate (5.2%o) of crude β-glucan in Example 1 (a combination of ultrasonic extraction for degradation of fruiting bodies and hot-water extraction under high pressure) were higher than those in Comparative Example 1 (hot-water extraction) and Comparative Example 2 (hot-water extraction under high pressure). From these results, the extraction rate of the total dry extract and the extraction rate of crude β-glucan having anticancer activities in Example 1 are 5 and 12 times as high as those in the hot-water extraction, respectively.

Determination of content of crude β-glucan in dry extract prepared in Example 1 The content of crude β-glucan in the dry extracts prepared in Example 1 was determined in accordance with methods of Mannors, D. J. (1973, The structure of a β-(l,6)-D-glucan, from yeast cell walls. Biochem. J. 135, 19-30) and Catley, B. J. (1988. Isolation and analysis of cell walls Yeast (A practical approach). IRL press, pp. 163-183). In the present invention, the content of crude β-glucan in the dry extracts was determined by measuring the glucose content in glucan fractions obtained through chemical treatment using a NaOH solution.

Referring to Figs. 1 to 5, changes in the morphology of Phellinus linteus according to the respective steps are shown. That is, Figs. 1 to 5 are optical microscope images (1250x) showing changes in the morphology of the fruiting bodies of the Phellinus linteus powders and the residues obtained after extraction and centrifugation of the Phellinus linteus powders, which were obtained in Example 1 and Comparative Examples 1 and 2. Specifically, Fig. 1 is an optical microscope image of Phelinus Linteus; Fig. 2 is an optical microscope image of the pulverized Phellinus linteus; Fig. 3 is an optical microscope image of the ultrasonically pulverized Phellinus linteus; Fig. 4 is an optical microscope image of the hot-water treated Phellinus linteus under high pressure; and Fig. 5 is an optical microscope image of the enzyme-treated Phellinus linteus.

From Figs. 1 to 5, it was confirmed that as the steps were proceeded, the fruiting bodies of Phellinus linteus were sufficiently loosened so as to be easy to extract.

Claims

Claims:

1. A method of extracting medically effective ingredients from Phellinus linteus, comprising the steps of: adding a pulverized Phellinus linteus powder into a distilled water of an amount ranging from 20 to 50 times based on the weight of the powder to make a mixture, and ultrasonicating the mixture; extracting the mixture with hot-water under a pressure of 2 to 3 atm, and filtering the extract through a membrane filter to obtain a filtrate; and enzyme-reacting the filtrate with cellulase and alkalase and subjecting to ultrafiltration on a membrane filter.

2. The method according to claim 1, wherein the medically effective ingredient is crude β-glucan.

3. A composition comprising medically effective ingredients exfracted by the method according to claim 1.