JP3128147B2 - Immune system active substance and its production method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunologically active substance and a method for producing the same. More specifically, the present invention relates to a novel immune system active substance comprising a reishi extract-treated substance having excellent characteristics such as macrophage phagocytic activity and NK cell activity, and a method for producing the same.

[0002]

2. Description of the Related Art The living body has an immune system as a self-defense mechanism against foreign substances (non-self), and this immune system, along with the nervous system, is a skillful network that has great flexibility against external stimuli. By maintaining the homeostasis of the living body. If this homeostasis breaks down for some reason, one will fall into various diseases.

[0003] When a drug acts on a certain kind of disease, it can be considered that it directly affects the cause of the disease or indirectly by enhancing the self-defense function of the living body. Many drugs produced by chemical synthesis that have been developed and used so far have mostly aimed at the direct action of the former, while crude drugs in line with the traditions of Oriental medicine have largely been based on the latter indirect method. The purpose of the mechanism of action is to place an objective action, that is, activation and activation of the immune system and the like.

[0004] However, the mechanism of action of this crude drug has long been unknown. For example, Lingzhi is a fungus belonging to the genus Amanita mushroom, which has been used as a natural herbal medicine for the treatment of chronic bronchitis, coronary atherosclerotic heart disease, dyspnea, etc. Like other natural products and the like, which have antitumor activity as water-soluble polysaccharides, the mechanism of their action has been completely unclear even if causality is found as a result.

[0005] For this reason, even in the case of Reishi, whose characteristic effects have been recognized empirically, it is further utilized as an advanced technology in relation to the mechanism of action of the living body, and its application is developed. It was difficult to plan. The present invention
It was made in view of such circumstances, and was made to develop the physiological activity of Reishi, which is conventionally known, as a more advanced technology. Based on the technical knowledge of genetic engineering, cell engineering, and molecular biology that have been achieved, the provision of the reishi ingredient as a crude drug found as an immune system active substance,
The purpose is to provide a manufacturing method therefor.

[0006]

Means for Solving the Problems According to the present invention, there is provided, as an object of the present invention, a method comprising extracting an extraction residue obtained by extracting Ganoderma lucidum with a non-polar solvent with hot water or an aqueous alcohol solution, Further, the present invention provides an immunologically active substance, which is obtained by fractionating and purifying the substance as required. The present invention also provides a method for producing an immune system active substance according to the above means.

[0007] More specifically, the immune system active substance of the present invention is obtained by first removing soil and sand adhering to the reishi by washing with water or the like and drying it at a temperature of about 60 ° C.
Then, diethyl ether, dipropyl ether, n-
It is immersed in a non-polar solvent such as hexane or n-heptane to extract the non-polar solvent soluble matter. This extraction is equivalent to the previously proposed operation for removing bitter components of Ganoderma lucidum, and has a meaning as a process for removing non-polar solvent-soluble components including triterpenes. . This extraction is preferably performed at about the reflux temperature of the solvent, and is usually performed for about 2 to 6 hours.

Next, in the present invention, the extraction residue is subjected to hot water at 90 to 100 ° C.
Extraction is performed with an aqueous alcohol solution having a concentration of about 0 to 60%. As the alcohol aqueous solution, it is preferable to use an aqueous solution of ethanol, propanol, isopropanol, butanol, glycol, or the like, and particularly to an etal aqueous solution, and it is preferable to perform extraction at a temperature of about 40 to 70 ° C.

[0009] The extraction time is generally about several hours to 20 hours, and in the case of hot water extraction, it can be shorter. Of course, the extraction with the non-polar solvent is also
In the extraction of the extraction residue with hot water or an aqueous alcohol solution, the extraction time is appropriately selected depending on the amount of the extraction medium used.

[0010] By extracting the hot water and the aqueous alcohol solution, a Ganoderma lucidum extract can be obtained. The Ganoderma lucidum extract itself has an immune system activating action, increases macrophage proliferation, macrophage phagocytic ability, and NK. It shows the ability to activate cells. And in the present invention,
The extract can be fractionated and purified. For this fractionation purification, an appropriate means such as a well-known gel filtration method is employed.

The fraction of the fractionated and purified active substance shows a more selective and large immune system activity. Hereinafter, Examples will be described, and the immune system active substance of the present invention and a method for producing the same will be described in more detail.

[0012]

【Example】

Example 1 <a> Production of Extract Extract Reishi was washed with water to remove earth and sand, and was shredded and dried at a temperature of about 60 ° C. 500 g of this reishi was extracted with diethyl ether. After removing the ether-soluble component, 5 l of water was added to the extraction residue, and the mixture was extracted at a temperature of 90 to 100 ° C for 5 hours. After filtering the extract, 5 l of water was added to the residue, and the mixture was extracted twice in the same manner.

[0013] The extract was concentrated and freeze-dried to obtain an extract of Lingzhi hot water. <B> as evaluation 8-week-old male mice of macrophage over di proliferative capacity (Maitoji E down activity), ddY, C3H / He, DB
A / 2, C57BL / 6, B10, B10A, DBA /
1J, BALB / c strain mice were purchased from Sankyo Lab Service, bred in a constant temperature and humidity breeding room with free access to commercially available solid samples and tap water, and used.

Lymphocytes were collected from the spleens of C3H / He and DBA / 2 mice using the Ficoll-coray method, and Lingzhi extract was added to 1,2,5,10,25,50,10.
The suspension was suspended in RPM11640 containing 10% FCS added to a concentration of 0 μg / ml, and adjusted to 1 × 10 ⁶ cells / ml. This was cultured in a 5% CO ₂ incubator for 24 hours, and 0.5 μCi of ³ H-TTP was added thereto, followed by further culturing for 3 hours. After completion of the culture, use a glass filter (GF) using an immunodot blotter (Attou).
-C: Whatman) cells are sucked fixed on, physiological saline
1 ml of brine , 500 μl of 5% TCA, 70% ethanol,
The filter was washed with 500 μl, and the amount of tritium incorporated into the cells was measured using a liquid scintillation counter (Aloka) to examine cell proliferation.

In this way, C3H / He, DBA /
After spleen cells of both mouse strains were cultured for 24 hours in a culture solution containing Reishi extract, the uptake of ³ H-TTP was observed. As shown in FIG. 1, the mitogenic activity was observed at a concentration of 1 μg / ml. And peaked at 10-15 μg / ml. The mitogenic activity gradually decreased at more than 20 μg / ml, and markedly decreased at more than 50 μg / ml. It should be noted that no significant difference was found in the mitogen activity of the Ganoderma lucidum extract in both C3H / He and DBA / 2 lines.

As is apparent from FIG. 1, it is found that the Ganoderma lucidum hot water extract has mitogenic activity on lymphocytes in vitro. This suggests that lymphocytes are one of the points of action of Ganoderma lucidum extract on the immune system. <C> Evaluation of macrophage phagocytic ability 2 ml of sterilized liquid paraffin was administered intraperitoneally to mice.
Three days later, cells exuded into the peritoneal cavity were collected. This is PRM
After three washes with I1640, the resulting cells were washed with 10% F
Suspend to PRMI1640 with CS and 10cm
The cells were cultured in a plastic dish (1058 Petri dish: Alcon) for 1 hour. The culture dish was washed three times with PRMI1640 to remove floating cells and was used as a macrophage. Reishi extract concentration 1,2,5,10,25,50,10
0 Pg / ml of dissolved RPMI1640 containing 10% FCS to the added to a concentration, the concentration of the recovered macrophages was adjusted to ^{1 × 10 6 ce ll s /} ml.
This was incubated for 24 hours at 5% C _2, 37 ℃, suspended La Te'<br/> Kusubizu the (dimater 1μm) to a final concentration of 0.01%, and incubated for another 15 minutes. Next, the dishes were thoroughly washed with RPMI1640, and the cells were fixed with 2% formalin solution, stained with ethidium bromide, and the ratio of macrophages phagocytic to latex beads (Sekisui) was measured by a fluorescence microscope.

As shown in Table 1 below, Reishi extract increased macrophage phagocytosis.

[0018]

[Table 1]

Example 2 <a> Fractionation and Purification of Extract Extract 500 g of Reishi was extracted with ether to remove ether-soluble components, and 5 l of water was added thereto.
Extracted at 5 ° C. for 5 hours. After filtering the extract, 5 l
And the extraction was repeated twice more. Next, the filtrate was collected, concentrated under reduced pressure, and concentrated to about 1.5 l.
And 500 ml of ethyl acetate, and the filtrate was washed, and then washed with n-butanol. 200 ml of the aqueous layer thus obtained was applied to Sephadex G-75 (65 × 50
0 mm) and eluted with 2% acetic acid. The eluate was collected in 16 ml portions and freeze-dried. Each fraction name and 1
The yield per 6 ml is as shown in Table 2.

[0020]

[Table 2]

[0021] <b> Maitoji E down activity rating 8 Shureiyu was collected only lymphocytes using Ficoll-Conray method from spleens of mice of both strains of C3H / the He or DBA / 2. Next, RPM11640 containing 10% FCS was applied to each fraction of Reishi extract to a concentration of 10 μg / ml.
Lysed and recovered lymphocytes are 1 × 10 ⁶ cells / ml
It was adjusted to become. This was cultured in a 5% CO ₂ incubator for 24 hours, and then 0.5 μCi of ³ H-TT
P was added, and the cells were further cultured for 3 hours. After completion of the culture, the cells were suction-fixed on a glass filter using an immunodot plotter, and 1 ml of physiological saline , 500 μl of 5% TCA,
The filter was washed with 500 μl of 70% ethanol, and the amount of tritium incorporated into the cells was measured using a liquid scintillation counter to evaluate cell proliferation.

As shown in FIG. 2, a high activity was observed in Fr. 1, which is a fraction having the largest molecular weight. In addition, Con A of FIG. 2 is 10 μg / ml of concanavalin A.
Is added as a positive control. Sephade × G-75 used for fractionation has a particle diameter of 4
0 to 120 μm and the fractionation area is molecular weight of 1,000 to 50,0
Given that the 00 or so, component showing a Maitoji E emission activity is that several tens of thousand to several hundreds of thousand molecular weight, is believed to have been eluted as Fr. 1 for that.

This Fr.1 enhanced the macrophage phagocytic ability. Liquid paraffin 2ml intraperitoneally injected to C3h / the He or DBA / 2 mice evaluation 8 Shureiyu of cytokine production by <c> macrophages were recovered intraperitoneal exudate macrophages after 3 days. Next, 100 ng of LPS and a reishi extract extracted with hot water were added to RPMI1640 containing 10% FCS at a concentration of 10 μg / ml, and the recovered macrophages were adjusted to 1 × 10 ⁶ cells / ml. This is placed in a 5% CO ₂ 37 ° C incubator for 2 hours.
After culturing for 4 hours, the suspended matter was removed using a 0.2 μm membrane filter, and this was used as an activity measurement sample. Next, thymus-derived lymphocytes were collected from ddy 5-week-old mice by the Ficoll-conray method. Next, the activity measurement sample was analyzed using RPMI1640 containing 10% FCS.
Serial dilution was performed to adjust the lymphocytes to 1 × 10 ⁶ cells / ml. This was cultured in a 5% CO ₂ 37 ° C. incubator for 24 hours, and 0.5 μCi of ³ H-TTP was added thereto, followed by further culturing for 3 hours. After the culture, the cells are suction-fixed on a glass filter using an immunodot blotter,
The filter was washed with 1 ml of physiological saline , 500 μl of 5% TCA, and 500 μl of 70% ethanol, and the amount of tritium incorporated into the cells was measured using a liquid scintillation counter to examine cell proliferation.

[0024] Evaluation of the cytokine production amount, as shown in FIG. 3, there is high activity in Fr. 1, also Fr.
4 also showed a high activity although there was a difference depending on the mouse strain. Macrophages activate T cells by antigen presentation, and themselves are important effector cells in innate immunity, and are currently recognized as antitumor active substances such as Bicibanil (OK-432) and Krestin (P-SK). Has also been shown to act on macrophages and exhibit antitumor activity. Activated macrophages are thought to release cytokines, eicosanoids, active oxygen and the like to regulate the immune system. Among these, IL-1 and TNF-α are major cytokines produced by macrophages. I
L-1 has various biological activities, acts on T cells and NK cells, and promotes IL-2 receptor expression. In addition, TNF-
α is a factor obtained from the serum of a mouse injected with BCG and then injected with endotoxin, which necroses certain tumors in vivo. In addition, TNF-α exhibits cytotoxicity against many cancer cells in vitro.

[0025] From this point of view, looking at the site <br/> mosquitoes in production ability of the street of the, Fr. For 1, as already seen, because they exhibit a high mitogenic activity, high in this impact activity It seems to indicate. On the other hand, Fr.
In the case of No. 4, the mitogen activity was low, and further examination was conducted. As a result, it was confirmed that Fr. 4 contributed to the activation of NK (Natural Killer) cells due to the high ability to produce cytokines . .

The molecular weight of about 10,000 to 20,000
Fr. 4 was also found to have an effect of suppressing leukopenia.

[0027]

[Effect of the Invention] As described by the present invention, more particularly, as ganoderma extract, macrophage proliferation, enhancement of macrophage phagocytosis, activation of NK cells by activation of cytokine-producing capacity, more leukopenia suppress such A novel substance having an immune system activity of the present invention is provided.

[Brief description of the drawings]

1 is a measurement diagram of Maitoji E down activity ganoderma extract of the present invention.

FIG. 2 is a diagram showing the measurement of mitogenic activity of fractionated and purified fractions of Ganoderma lucidum extract.

FIG. 3 is a measurement diagram of cytokine-producing capacity by macrophages.

──────────────────────────────────────────────────続 き Continued on the front page (58) Fields surveyed (Int. Cl. ⁷ , DB name) A61K 35/84 A61P 37/04 C07G 17/00 BIOSIS (DIALOG) CA (STN) MEDLINE (STN)

Claims (3)

(57) [Claims] 1. An immunologically active substance obtained by extracting an extraction residue after extracting Ganoderma lucidum with a non-polar solvent with hot water or an aqueous alcohol solution. 2. An immunologically active substance obtained by extracting an extraction residue after extracting Ganoderma lucidum with a non-polar solvent and extracting it with hot water or an aqueous alcohol solution, followed by fractionation and purification. 3. A method for extracting Ganoderma lucidum with a non-polar solvent,
A method for producing an immunologically active substance, comprising extracting the obtained extraction residue with hot water or an aqueous alcohol solution and, if necessary, purifying it by fractionation.