JP2000229861A - Immunopotentiator, its production and usage - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an immunopotentiator capable of inducing the production of interleukin 12 and contributing to an indirect antitumor activity such as the strengthening of natural immunity and cell-mediated immunity by compounding a double strand RNA of an edible mushroom as an active ingredient. SOLUTION: This immunopotentiator contains a double strand RNA of an edible mushroom as an active ingredient. Further, to obtain the double stranded RNA, it is preferably isolated from an edible mushroom; for example, the RNA is contained mainly in a 80%-alcohol precipitated fraction obtained after an equal volume of ethyl alcohol is added to a hot water extract of the mushroom while stirring, and polysaccharides precipitated at 50%-alcohol are separated. Specifically, the extraction is carried out by adding a 1%-sodium dodecyl sulfate in an amount of 20 times and a tris-HCl buffer solution containing 100 mmol of sodium chloride to lyophilized powder of a mushroom such as Lentinus edodes. From the extract, a fraction precipitated by a 50%-concentration ethanol is removed by centrifugal separation, then a fraction insoluble in an 80%-concentration ethanol is collected, proteins are removed from the fraction, impurities are decomposed and removed, and subsequently the objective RNA is isolated and purified by chromatography or the like.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunostimulating agent by inducing the production of interleukin 12 using double-stranded RNA (double-stranded ribonucleic acid) contained in cultured mycelium and fruiting bodies of edible mushrooms and its production. And methods of using the immunostimulant.

[0002]

BACKGROUND ART Hot water extracts of various mushrooms are known to have an antitumor effect, and a large number of health foods and beverages using them as raw materials are commercially available. The active ingredient is considered to be mainly polysaccharide β-glucan. Among them, purified lentin derived from shiitake (trade name of Ajinomoto Co., Ltd.), crestin derived from Kawaratake (trade name of Kureha Chemical Industry Co., Ltd.), Suehirotake Derived schizophyllan (trade name of Taiwan Sugar Co., Ltd.) is used as a drug, and all have a host-mediated antitumor effect by enhancing or restoring T cells, which are a kind of lymphoid cells.

On the other hand, double-stranded RNA, which is a nucleic acid, is detected in various fungi such as mushrooms, and it has been reported that they induce interferon. Interferon is the oldest known cytokine and has been shown to exhibit a variety of biological activities such as an antitumor effect and an immune response regulating effect in addition to an antiviral effect.

The double-stranded RNA is a relatively stable molecule which is unlikely to be decomposed by heat or an enzyme, unlike single-stranded RNA such as ribosomal RNA which is present in a large amount in a cell or DNA derived from nucleus or cytoplasm. is there. Therefore, the hot water extract of mushrooms has pharmacological action of double-stranded RNA in addition to β-glucan.

[0005]

However, these polysaccharides and nucleic acids are known to induce the production of some cytokines such as interleukins and interferons, but their immunostimulatory effects are extremely weak. It is a disadvantage.

[0006] The inventors of the present invention have conducted intensive studies to solve the above-mentioned problems, and as a result, have studied an improved method for extracting mushrooms and a fractionation method. Interleukin 1 characterized by the activation of natural killer cells and the activation of helper T cells that can assist the T cells required for antibody production
2, which is a double-stranded RN contained in the mushroom
The present inventors have found that the immunostimulant is A, and have arrived at the immunostimulant of the present invention, its production method and its use.

[0007]

According to the present invention, the immunostimulant of the present invention is a double-stranded RNA contained in edible mushrooms.
As an active ingredient.

The method for producing an immunostimulant according to claim 2 of the present invention is characterized in that double-stranded RNA contained in edible mushrooms is isolated from the edible mushrooms.

Further, the method of using the immunostimulant according to claim 3 of the present invention relates to the method of using double-stranded RNA contained in edible mushrooms.
As an active ingredient, and induces the production of interleukin 12 when used.

[0010]

DETAILED DESCRIPTION OF THE INVENTION Interleukin 12 (hereinafter IL)
-12) is mainly produced from macrophages, is known as an activator of natural killer cells that nonspecifically binds to and destroys cancer cells, and is a helper T cell (Th0) that assists cell-mediated immunity. It is known to be involved in the differentiation into Th1 type and control of its balance (Th1 / Th2), and indirect antitumor activities such as enhancement of innate immunity and cell-mediated immunity have attracted attention.

[0011] Regarding the acquisition of such IL-12, the inventors of the present invention have added to a hot water extract of mushrooms an ethyl alcohol having an equal solubility while stirring to remove 80% of polysaccharide precipitated at 50%. It was found that the precipitated fraction had significant IL-12 inducing activity.

The 80% precipitated fraction is dissolved in Tris-HCl buffer and added to anion exchange chromatography. The active fraction eluted with 0.5 M NaCl has an absorption maximum at 260 nm. RNase was matched with the spectrum of nucleic acid and positive for orcinol reaction.
(Ribonuclease) resistance and a change in the spectrum in the UV region in a formaldehyde solution revealed that the RNA was double-stranded RNA.

That is, 20 times the volume of 1% sodium dodecyl sulfate, 100
Tris-HCl buffer containing mM sodium chloride (pH
7.0), and the mixture was extracted. The fraction precipitated with 50% ethanol was removed by centrifugation, and the 80% ethanol-insoluble fraction was collected.
protein is removed by the Sevag method using inase K), and then nucleic acid DNA and single-stranded RNA as impurities are removed.
To DNase I (deoxyribonuclease I), R
After decomposing and removing with Nase A (ribonuclease A), DEAE (ethylenediaminetetraacetic acid) -cel
ulose anion exchange chromatography or methylated albumin column chromatography.
NA (ribonucleic acid) is isolated and purified.

The RNA isolated as described above is positive for the orcinol reaction, and it is clear from the resistance to RNase and the change in the spectrum in the UV region in the formaldehyde solution that the RNA is double-stranded RNA. An immunostimulant containing the single-stranded RNA as an active ingredient can be obtained.

In this immunostimulant, the double-stranded RNA, which is an active ingredient, is involved in an immune response to a living body and induces the production of IL-12, which is a kind of cytokine. It contributes to indirect antitumor activity such as enhancement against cellular immunity.

Preferred mushrooms to be targeted by the immunostimulant of the present invention include, in addition to the shiitake mushrooms described above, edible mushrooms such as Kawaratake, Suehirotake, Maitake, Matsuouji, Enokitake, and Pleurotus, and the double-stranded RN.
A can be obtained from cultured mycelia and fruiting bodies of these mushrooms.

FIG. 1 shows the elution pattern of shiitake mushroom double-stranded RNA isolated by the above-mentioned methylated albumin column chromatography.

[FIG. 2] shows the results of DEAE-cellulos.
The elution pattern of double-stranded RNA when the shiitake mushroom double-stranded RNA was isolated by e-column chromatography is shown in FIG.
260 indicates a nucleic acid, and A620 indicates a polysaccharide obtained by an anthrone reaction.

FIG. 3 is an ultraviolet spectrum of shiitake mushroom double-stranded RNA.

[Fig. 4] shows the RN of shiitake mushroom double-stranded RNA.
The black circles indicate resistance to case A.
RNase A was allowed to act in the presence of 3 moles of NaCl, and the open circles represent RNase A in the presence of 0.015 moles of NaCl.
The degree of decomposition was determined from the increase in absorbance (A260) caused by the action of se A.

FIG. 5 shows the reaction between double-stranded RNA and formalin, and the ultraviolet absorption curve was examined over time at a temperature of 37 ° C., showing a known double-stranded RNA (derived from rice dwarf virus). And single-stranded RNA (tobacco mosaic virus).

In this figure, since the double-stranded RNA in column A does not react with formalin, the RNA in other columns B and C
UV absorption is less changed than in the case of

[0023]

It is already known that virus particles and their double-stranded RNA induce interferon. However, direct administration of cytokines such as interferon is not practical because of multiple physiological activities and strong side effects of cytokines. Has also been difficult. On the other hand, mushrooms contain a cryptoptic virus (unidentified) that has no pathogenicity,
In Shiitake mushroom, it is detected in all varieties. However, it is important that the components of edible mushrooms that are taken daily have an inducing effect, and IL-12 is not found in existing pharmaceuticals such as lentinan derived from shiitake mushroom.
The activity by inducing the production is expected to be fully useful as a novel immunostimulant.

[0024]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in more detail with reference to embodiments. <Example 1> Frozen shiitake mushroom fruit body was 1% of equal volume
SDS and 0.2% 2-mercaptoetha
STE buffer (100 mM NaC
l, milled with 50 mmol Tris.HCl (pH 7.5) and 1 mmol EDTA.

[0025] This milled product (10,000 xg) was centrifuged for 10 minutes, an equal volume of water-saturated phenol / isoamyl alcohol (24: 1) was added to the obtained supernatant, and the mixture was inverted and mixed. And transfer it to a new container,
The protein removal with water-saturated phenol / isoamyl alcohol was repeated.

The polysaccharide mixed in the aqueous layer was composed of an equal volume of 2.5 mol K ₂ HPO ₄ and 0.05 volume of 33.3% H ₃ P.
This was carried out by adding O ₄ and an equal volume of 2-methoxyethanol and removing the lower layer separated into two layers by centrifugation.

The upper layer thus obtained has twice the amount of ethanol
To precipitate all nucleic acids, add a small amount of TE buffer
(10 mol Tris · HCl pH 7.5, 1 mmol
 EDTA) and 100 μg / ml DNase
 I and 10 mmol MgCl _TwoCompletely degrades DNA
Followed by RNa in the presence of 300 mmol NaCl.
RNA is degraded by se A (100 μg / ml)
After decomposing protein with Proteinase K
And removed by water-saturated phenol / isoamyl alcohol treatment
Protein.

After the protein removal, the nucleic acid is recovered by precipitation with ethanol, and the contaminating nucleic acid, protein and polysaccharide are removed by methylated albumin-celite column chromatography to isolate and purify double-stranded RNA. did. As a result, about 50 μg of double-stranded RNA was recovered from 1 kg of raw shiitake mushroom.

Example 2 A 20-fold amount of an extraction buffer (0.7 M NaCl, 1% CTAB (Cetyltrimet)) was added to a powder of lyophilized shiitake mushroom or cultured mycelium.
ylammonium bromide), 50 mmol Tris · HCl pH 7.5, 1 mmol ED
TA) was added and the mixture was kept at 60 ° C. for 1 hour, and stirred several times during that time.

An equal volume of chloroform / isoamyl alcohol (24: 1) was added thereto, and the mixture was further stirred for 30 minutes, centrifuged at 10,000 × g for 10 minutes, and the supernatant was recovered. Collected.

In the same manner as in Example 1, DNA and RNA were decomposed and removed with DNase I and RNase A, respectively. The double-stranded RNA was isolated and purified by diethylaminoethylcellulose (DEAE-cellulose) column chromatography.

That is, the nucleic acid was converted to 50 mM Tris
・ DEAE- equilibrated by dissolving in HCl pH 7.5
After adding to the cellulose column chromatography and washing off the non-adsorbed fraction, the NaCl concentration was 0 to 0.
Elution was carried out stepwise at concentrations ranging from 1.0 mol (0 mol without the use of NaCl), and fractions eluting with 0.5 mol NaCl were collected and collected by ethanol precipitation. Thereby, about 25 μg of double-stranded RNA from 50 g of dried shiitake mushroom
Could be recovered.

Example 3 A double-stranded RNA isolated and purified from a fruit body of Lentinula edode was used in physiological saline (0.85% NaC).
In l), the mixture was dissolved to a predetermined concentration and sterilized by filtration.

[0034] Macrophages derived from mice were transformed with RP
It was prepared to be 1 × 10 ⁵ cells / ml in MI1640 medium, and 900 μl thereof was dispensed into a 24-well microplate.

[0035] 100 µl of this sample was aseptically added to the cells and cultured in a 5% CO ₂ culture vessel at 37 ° C for 48 hours. The culture supernatant was collected by centrifugation, and the cytokines IL-1β, T
NF-α and IL-12 were quantified. The cytokines IL-1β, TNF-α and IL were similarly prepared using saline, laminarin and lentinan as controls.
-12 was quantified. The results are shown in [Table 1].

[0036]

[Table 1]

From the results shown in Table 1, production of TNF-α was observed in Lentinum of shiitake mushroom, which is a β-glucan, but no induction activity of IL-1β or IL-12 was observed.
It can be seen that laminarin has no activity to induce any cytokine. On the other hand, the double-stranded RNA isolated and purified from shiitake mushroom exhibited an activity of inducing IL-1β and TNF-α and inducing IL-12 production.

Example 4 The double-stranded RNA similarly isolated and purified from Shiitake mushroom and Maitake mushroom was tested for its ability to produce IL-12 in a mouse-derived macrophage cell line (TtT / M8
Table 2 shows the results of comparison using J774.1) and J774.1).

[0039]

[Table 2]

From Table 2, it can be seen that the production amount differs depending on the mushroom-derived macrophages, but that both the double-stranded RNAs of shiitake and maitake have high IL-12 production ability. In addition, it has been found that clinical improvement is expected with a very small amount of production of IL-12, and the effectiveness of the extract is clear.

[0041]

The immunostimulant of the present invention contains double-stranded RNA contained in edible mushrooms such as shiitake mushroom as an active ingredient, and its activity is stable even at a heat treatment at a temperature of 120 ° C., is relatively simple, and is inexpensive. In addition, its usefulness is remarkably high because it can be produced efficiently and efficiently, and remarkably induces the production of IL-12 which is a kind of cytokine.

On the other hand, IL-
Direct administration of 12 has been considered difficult to put into practical use due to blood inhibitors and antagonists, and unexpected side effects, but the inducer has sufficient practical expectations.

The endogenous factor that induces IL-12 is known as interferon γ (IFN-γ).
Such an IL-12-inducing substance is not known as a natural product, and the double-stranded RNA contained in mushrooms has no pathogenicity in the mushroom itself, and has been used for food for many years and has adverse effects such as toxicity. Nor. Therefore, it can be said that finding a substance having high activity in the edible mushrooms that are consumed daily is useful from the viewpoint of prevention of cancer and various infectious diseases.

[Brief description of the drawings]

FIG. 1 shows the elution pattern of the double-stranded RNA of the present invention isolated by methylated albumin column chromatography.

FIG. 2: Double-stranded RN of the present invention when double-stranded RNA is isolated by DEAE cellulose column chromatography
1 shows an elution pattern of A.

FIG. 3 is an ultraviolet spectrum of shiitake mushroom double-stranded RNA obtained by the present invention.

FIG. 4 shows the RNa of shiitake mushroom double-stranded RNA obtained by the present invention.
It is a graph which shows resistance to seA.

FIG. 5 shows the reaction between double-stranded RNA and formalin, and the ultraviolet absorption curve was examined over time at a temperature of 37 ° C.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:91) (72) Inventor Tatsunori Kuramo 3-39-15 Showa-cho, Maebashi-shi, Gunma Gunma University School of Medicine Health Within the department (72) Inventor Tadashi Nakamura 3-39-15 Showa-cho, Maebashi-shi, Gunma Prefecture Gunma University Faculty of Medicine Department of Health Sciences (72) Inventor Hideko Kazama 3-39-15 Showa-cho, Maebashi-shi, Gunma Prefecture Gunma University School of Medicine F term (reference) 4B064 AG02 CA10 CD16 CD22 DA01 DA13 4C057 AA05 BB02 DD01 MM02 4C086 AA01 AA02 EA17 EA18 MA01 MA04 NA14 ZB07

Claims (3)

[Claims] 1. An immunostimulator comprising a double-stranded RNA contained in edible mushrooms as an active ingredient. 2. The double-stranded RNA contained in edible mushrooms,
A method for producing an immunopotentiator, comprising isolating from the edible mushroom. 3. A method of using an immunostimulant, comprising using double-stranded RNA contained in edible mushrooms as an active ingredient, and inducing the production of interleukin 12 by using the double-stranded RNA.