JP5111732B2 - Inflammatory disease prevention and improvement agent - Google Patents

Description

  The present invention relates to an inflammatory disease prevention / amelioration agent, and more particularly to an inflammatory disease prevention / amelioration agent using a functional food.

When some external / internal stimulus is applied to a living body, an inflammatory reaction occurs as a biological defense reaction. This inflammatory response develops various inflammatory diseases through a complex process. For example, it has been reported that arthritis is caused by excessive activation of phospholipase A ₂ related to metabolism of arachidonic acid, platelet aggregation, generation of active oxygen and radicals, and the like.
An autoimmune disease is a phenomenon in which an immune system, which is a biological defense mechanism, attacks self-cells due to immune abnormalities and develops an inflammatory disease.

To date, non-steroidal anti-inflammatory analgesics have long been used as pharmacological treatments for inflammatory diseases, but they have side effects such as gastric mucosal damage and are insufficient in their effectiveness, and their roles are being reduced. is there. On the other hand, although steroids and immunosuppressive agents have certain effects, they are difficult to use for a long time because of strong side effects. For this reason, there is a growing interest in functional foods that are less likely to cause side effects.
For example, an inflammatory disease ameliorating agent (patent document 1) using a mixed culture of lactic acid bacteria and Saccharomyces cerevisiae, and an inflammatory disease preventing and treating agent using kale (patent document 2) have been developed.
JP 2004-91433 A JP 2004-43381A

However, in the above technique, the preventive or therapeutic effect for inflammatory diseases is not sufficient, and a better functional food has been demanded.
The present invention has been made in view of the above-mentioned problems of the prior art, and its purpose is derived from anti-inflammatory action and immunoregulatory action, and has excellent suppression and treatment effects on inflammatory diseases and produces side effects. The object is to provide an agent for preventing and improving inflammatory diseases, which has a low possibility.

In view of the above circumstances, as a result of intensive studies by the present inventors, it has been found that Pleurotus nebrodensis and / or an extract thereof are excellent in suppressing and treating inflammatory diseases, and the present invention has been completed. It was.
That is, the inflammatory disease preventive / ameliorating agent of the present invention is characterized by containing Pleurotus nebrodensis and / or an extract thereof as an active ingredient.
In the inflammatory disease preventive / ameliorating agent, Pleurotus nebrodensis is preferably a Pleurotus nebrodensis strain having an accession number of FERM P-19370.
In particular, water or methanol extract of Pleurotus nebrodensis is preferably used as an active ingredient.

  By using Pleurotus nebrodensis and / or its extract, the inflammatory disease prevention / amelioration agent of the present invention has no side effects and has an excellent inflammatory disease prevention and treatment effect.

Hereinafter, embodiments of the present invention will be described in detail.
The inflammatory disease prevention / amelioration agent of the present invention comprises Pleurotus nebrodensis and / or an extract thereof as an active ingredient.
In the present invention, Pleurotus nebrodensis which is particularly preferably used is a Pleurotus nebrodensis strain having a deposit number of FERM P-19370, which was dated May 22, 2003 and is one of the applicants of this patent. Deposited by the National Institute of Advanced Industrial Science and Technology.
The scientific properties are as follows.
1. Scientific properties ... Fungus characteristics: Forms white colonies under nutrient medium containing carbon and nitrogen sources. In addition, a clamp connection (a faint connection) is observed under an optical microscope.

2. Taxonomic position Basidiomycetes 3. Culture conditions <medium name>
SMYA medium (S: saccharose, M: malt extract, Y: yeast extract, A: agar)
S, A → commercial product M, Y → Difco (Difco)
<Composition of medium>
Per 1000 ml of medium
1% sucrose 10g
1% malt extract 10g
0.4% yeast extract 4g
2% agar 20g

<PH of medium>
5.0 to 7.0 (optimum pH 5.5)
<Bacteria sterilization conditions>
121 ° C 20 minutes <medium temperature>
28 ° C
<Culture period>
10 days <Oxygen demand>
Aerobic

4). Storage conditions Can be stored by freezing method.
<Freezing conditions>
-80 ° C
<Protective agent>
10-20% glycerin aqueous solution (optimum 20%)
<Restoration rate after freezing>
100% in 1 year, 99% in 3 years

5. Conditions for survival test <Restoration of microorganisms>
40 ° C
<Inoculation, culture, confirmation method>
According to the same conditions as the culture conditions.

When an extract of Pleurotus nebrodensis is used, the extraction method is not particularly limited, but a suitable method is described in detail below.
In the present invention, the fruiting body of Pleurotus nebrodensis is used. The fruiting body may be either raw or dried, but the dried fruiting body is desirable from the viewpoints of handleability, storage stability and extraction efficiency.
Prior to obtaining the extract, it is preferable to destroy the tissue of Pleurotus nebrodensis. This makes it possible to extract efficiently. Pleurotus nebrodensis tissues can be destroyed by grinding with various grinding mixers such as bead mills, Waring blenders, homogenizers, impact crushing with explosives, freezing, physical treatment such as ultrasonic treatment, sodium hydroxide, etc. There are chemical treatments such as alkaline treatment with aqueous solutions such as, treatment with enzymes having cell wall decomposing action such as cellulase and pectinase, and osmotic pressure treatment, and these can be carried out alone or in appropriate combination. Of these tissue destruction treatments, the pulverization treatment is particularly preferable.

In order to obtain the Pleurotus nebrodensis extract of the present invention, an extraction solvent such as water or a lower alcohol may be added to Pleurotus nebrodensis whose tissue has been destroyed as described above, and the mixture may be appropriately stirred and extracted. Here, water or methanol is particularly suitable as the extraction solvent. Moreover, although extraction operation may be performed under a normal pressure and normal temperature, it can also extract under 1-5 atmospheres, 60-150 degreeC pressurization, and a heating.
The recovery rate by hot water extraction is about 60% in terms of solid content with respect to the dry powder.
The extract obtained in this manner can be subjected to treatments such as concentration under reduced pressure, freeze drying, spray drying and the like to remove the extraction solvent and obtain a Pleurotus nebrodensis extract.

  The amount of intake when used as an inflammatory disease prevention / amelioration agent varies depending on symptoms and is not particularly limited, but it is sufficient if ingesting 1 to 30 g, particularly 10 to 20 g, in terms of Pleurotus nebrodensis dry powder per day for an adult Can be expected.

The inflammatory disease preventive / ameliorating agent of the present invention is usually administered as an internal medicine.
In the case of internal use, powders, tablets, capsules, granules, teas, suspending agents, flow extracts, liquids, syrups and the like can be prepared by conventional methods. In the formulation, usual formulation carriers such as excipients, binders, disintegrants, lubricants, coloring agents, flavoring agents, and flavoring agents may be used as necessary. it can. Moreover, a coating can also be given with a suitable coating agent etc. as needed.
For skin diseases, external application to the skin is also considered effective.

Inflammatory diseases in the present invention, e.g., systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, ulcerative colitis, primary biliary cirrhosis, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, weight disorders myasthenia, sympathetic ophthalmia, glomerulonephritis, other autoimmune diseases Hashimoto's disease such as, atopic dermatitis, contact dermatitis, skin myositis, rhinitis, hay fever, conjunctival inflammation, and the like.
The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto. Unless otherwise specified, the blending amount is expressed in mass% with respect to the system in which the component is blended.

(1) Preparation method of sample (Pleurotus nebrodensis extract) (Fig. 1)
I. Hot water extract To 5 g of dried fruit body of Pleurotus nebrodensis with the accession number FERM P-19370, add 100 mL of water, extract at 85 ° C. for 2 hours, separate into an extract and a residue by filtration. Was lyophilized.
II. Ethanol Extract To 5 g of Pleurotus nebrodensis fruit body dry powder of accession number FERM P-19370, extraction was performed by adding 100 mL of ethanol, and the extract was separated into an extract and a residue by filtration, and the extract was freeze-dried.
III. Methanol extract
In the same manner as II, an extract was obtained using methanol instead of ethanol.
IV. Ethanol soluble content of hot water extract
In the same manner as I, a hot water extract was obtained, extraction was performed by adding 5 times the amount of ethanol, the extract was separated into an extract and a residue by filtration, and the extract was freeze-dried.
V. Ethanol insoluble in hot water extract
In the same manner as in I, a hot water extract was obtained, extraction was performed by adding 5 volumes of ethanol, the filtrate was separated into an extract and a residue, and the residue was lyophilized.

(2) Platelet aggregation inhibition test It has been clarified that arachidonic acid and its metabolites are involved in the development of various inflammatory skin diseases. Therefore, each of the above extracts was examined for PAF (platelet activating factor) and sodium arachidonic acid-induced platelet aggregation inhibitory effect.

(Test method)
Human blood was centrifuged at 1100 rpm for 20 minutes at room temperature to collect platelet-rich plasma (PRP), and further centrifuged at 3000 rpm for 5 minutes to collect platelet poor plasma (PPP). After pre-warming 223 μL of PRP at 37 ° C., 2 μL each of a 2% DMSO solution of the above extract as a test sample or ion-exchanged water as a control was added, and the mixture was further incubated at 37 ° C. for 3 minutes. 25 μL of an aqueous PAF solution (500 nM) was added.
The induced aggregation was measured using an aggregometer (manufactured by MCM Hematracer MCM Medical Co., Ltd.), and the maximum aggregation rate of the test sample (maximum value obtained from the aggregation curve of the test sample with a PPP value of 100) Was compared with the control maximum aggregation rate to evaluate the inhibitory effect on PAF-induced platelet aggregation.
In addition, the inhibitory effect on platelet aggregation for arachidonic acid was similarly evaluated by using a sodium arachidonic acid aqueous solution (500 μM) instead of the above PAF aqueous solution (FIG. 2).

The right side of FIG. 3 shows the inhibitory effect on platelet aggregation by PAF induction and the left side by arachidonic acid induction. All the extracts were confirmed to have a platelet aggregation inhibitory effect, but in particular, the hot water extract (Group A), the methanol extract (Group C), and the ethanol soluble part of the hot water extract (Group D) ) Was found to have an excellent inhibitory effect on platelet aggregation.
From the above, it is considered that platelet aggregation can be suppressed by ingesting Pleurotus nebrodensis and / or an extract thereof, and the effect of improving / preventing inflammatory diseases can be exhibited. For skin diseases, external application to the skin is also considered effective.

(3) Chemokine gene expression suppression test It has been clarified that chemokines such as interleukin-8 (IL-8) are strongly involved in the development of various inflammatory skin diseases. Therefore, suppression of chemokine expression is considered to be effective in improving and preventing these inflammatory disease symptoms.
Then, the chemokine gene expression inhibitory effect was investigated about each said extract.
(Test method)
Human dermal fibroblasts were cultured in a DCM medium (Dulbecco's modified Eagle medium) containing 10% fetal calf serum in a 6 cm diameter culture dish until they became confluent, and were used as experiments as test media.
To the test medium, each of the above extracts as a test sample or hydrocortisone () as a positive control was added. The final concentrations of the extract and hydrocortisone were 0.01% (dry mass) and 10 ⁻⁷ M, respectively.

Furthermore, tumor necrosis factor TNF-α (1 ng / ml), which is known to promote chemokine gene expression, was added and cultured at 37 ° C. for 6 hours.
Next, after isolating RNA from cells and synthesizing cDNA according to a conventional method, the expression level of IL-8 gene is measured by quantitative PCR (TaqMan PCR), and nothing is added to the test medium. The ratio with the expression level was calculated. The GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) gene was used as an internal standard gene, and the obtained data was corrected (FIG. 4).
Human skin fibroblasts are commercially available, for example, from Kurashiki Boseki Co., Ltd. Human fibroblasts may be cultured according to a conventional method for culturing animal cells, but a DMEM medium containing 10% fetal bovine serum is particularly preferable.

The results are shown in FIG. Although all the extracts were confirmed to have an inhibitory effect on chemokine gene expression, in particular, the hot water extract (Group A), the methanol extract (Group C), and the ethanol soluble part of the hot water extract ( An excellent inhibitory action was observed in group D).
Hydrocortisone, which is a positive target, had a strong inhibitory effect, but because it is a steroid hormone, side effects such as tissue damage and ulceration may be caused. On the other hand, the Pleurotus nebrodensis extract of the present invention has a suppressive effect that is not as good as that of hydrocortisone, but is sufficient and can be used for a long time since there is no worry about side effects.
From the above, it is considered that chemokine gene expression can be suppressed by ingesting Pleurotus nebrodensis and / or its extract, and the effect of improving / preventing inflammatory diseases can be exhibited without side effects.

(4) In vivo test MRL / Mp-lpr / lp mice (MRL mice) develop acute and chronic inflammation simultaneously, naturally producing glomerulonephritis, granulomatous vasculitis, polyarthritis, lymphoma, autoantibody production Develops. This onset is similar to human systemic lupus erythematosus and rheumatoid arthritis, and is widely used as a model animal useful for analyzing the pathophysiology of inflammatory diseases and developing medicinal effects.
Therefore, using this MRL mouse and a normal ICR mouse as a comparison, the anti-inflammatory effect and the immunomodulating effect were tested on extracts I, III, and IV that were particularly effective among the above-mentioned extracts.

(1) Preliminary breeding of test mice (4-7 weeks old)
Four-week-old male MRL mice and male ICR mice purchased from Nippon Charles River Co., Ltd. were kept at room temperature 22 ± 1 ° C. and humidity 60 ± 10% with white fluorescent light for 12 hours a day. Light control was carried out (from 7 to 19 o'clock light period), and feed and tap water were freely ingested and pre-bred for 3 weeks.
After the preliminary breeding, the weight of each individual was measured to make 10 animals per group, MRL mice were classified into groups A to D so that the average values of body weights were almost equal, and ICR mice were set to group E.

2. Sample administration method (7-19 weeks old)
In the following manner, the extract was administered to each group of mice and further reared for 12 weeks to 19 weeks of age. The dose was adjusted so that an adult with a body weight of 60 kg corresponds to inoculation of 20 g per day in terms of fruiting body. All extracts were administered orally and were carried out daily from 10 am. After administration, sterilized water was freely ingested.
Solid feed (MF: manufactured by Oriental Yeast Co., Ltd.) and tap water were freely ingested in each group. The environment of the breeding room was a temperature of 20 ± 2 ° C., a humidity of 60 ± 10%, and white fluorescent lighting for 12 hours a day (7-19 o'clock light period).

(MRL mouse)
Group A: I. Hot water extract administration B group: III. Methanol extract administration C group: IV. Ethanol soluble fraction administration of hot water extract D group: No administration (ICR mice)
Group E: No administration

3. Blood test All mice were fasted the day before the last day of the experiment, and the next day, deep anesthesia (Nembutal, 45 mg / kg, ip) was taken, and blood was collected from the left ventricle as much as possible with a 20 G blood collection needle.
Using the collected blood, biochemical tests were performed on the following items. The obtained results were analyzed by Wilcoxon U-test for comparison between groups, and judged to have a significant difference with a risk rate within 5% (p <0.05). All values were expressed as average values and standard error values.

<About anti-inflammatory effect>
Lactate dehydrogenase (LDH) is a deviating enzyme that is widely distributed in the living body such as heart muscle, skeletal muscle, liver, kidney, erythrocyte, etc., and is released into the blood by increasing the permeability of the cell membrane of the tissue or cell destruction. The degree of inflammation can be determined from the increase in activity value.
Test method: LDH produces lactic acid using pyruvate as a substrate, and at the same time, reduced nicotinamide adenosine dinucleotide (NADH), which is a coenzyme, is changed to an oxidized form. Since NADH has an absorption maximum at 340 nm, the rate of decrease in absorbance was measured to calculate the activity value of LDH.

C-reactive protein (CRP) is a typical acute phase reaction protein, which is a plasma protein that increases when there is inflammation or tissue necrosis in the body. The degree of inflammation can be determined from the increase in activity value.
Test method: The CRP value was measured by an immunoturbidimetric method (Pure Auto SCRP ^™ : Daiichi Kagaku) using a spectrophotometer. Since the antigen-antibody complex obtained by adding a specific antibody against CRP and mouse serum produces turbidity, the intensity of the turbidity was measured as a change in absorbance.

Rheumatonoid factor (RF) is found in the blood of patients with rheumatoid polyarthritis and is a group of autoantibodies against immunoglobulins. In rheumatoid arthritis (RA), it is believed that B lymphocytes present in peripheral blood and affected areas produce RF. Therefore, the degree of inflammation can be known from the increase in RF value.
Test method: RF value measured RF reacting with heat-denatured γ-globulin by immunoturbidimetry (Nippon Pharmaceutical Co., Ltd.). Since the composite produced turbidity, the intensity of the turbidity was measured as a change in absorbance.

FIG. 6 shows the measurement results (anti-inflammatory effect) of LDH, CRP and RF.
In ICR mice, LDH values were low and CRP and RF values were zero.
In contrast, these values were higher in MRL mice. However, in MRL mice, these parameters were reduced in the extract administration group (Groups A to C) compared to the non-administration group (Group D).
Thus, the anti-inflammatory effect of Pleurotus nebrodensis was confirmed from the values of LDH, CRP and RF.

<About immunomodulating action>
CD4 and CD8 are one of the surface antigen molecules present on lymphocytes. CD4 forms a complex with a major histocompatibility class II (MHC-II) antigen and has a function of instructing antigen recognition. CD8 has a complex with a major histocompatibility class I (MHC-I) antigen. It has the function of forming and assisting antigen recognition. The balance (CD4 / CD8) decreases in an immunodeficient state, and increases in autoimmune diseases.
Test method: CD4 / CD8 was measured as follows.
T cells were isolated from the spleen tissue, lysed with Lysing Regent, and passed through a nylon mesh. Using OKT4 (CD4) and OKT8 (CD8), they were reacted with helper / inducer T cells and suppressor / cytotoxic T lymphocytes. The experiment was carried out by reacting with T cells with a combination of OKT4 (FITC label) and OKT8 (PE label) labeled monoclonal antibody, and analyzed with a flow cytometer (Profile; Coulter, Hialeah, FL, USA).

RF-IgG is a group of autoantibodies against IgG (immunoglobulin that is most frequently present in blood and is the center of antigen-antibody reaction). RF-IgG is often observed in RA with strong extra-articular symptoms, and is more related to vasculitis than arthritis, and from that value, immune abnormalities can be predicted.
Test method: RF-IgG value was measured by immunoturbidimetry (Nippon Pharmaceutical Co., Ltd.) for RF reacting with heat-denatured γ-globulin. Since the composite produced turbidity, the intensity of the turbidity was measured as a change in absorbance.

FIG. 7 shows the measurement results (immunomodulatory action) of CD4 / CD8 and RF-IgG.
As described above, CD4 / CD8 is a value at which an increase is observed in an autoimmune disease or the like. This value was higher in MRL mice compared to normal ICR mice. However, in the MRL mice, a decrease was observed in the extract administration group (Groups A to C) compared to the non-administration group (Group D).
Moreover, although RF-IgG is an autoantibody group, this value was 0 in ICR mice. In contrast, MRL mice had higher RF-IgG values. However, in the MRL mice, a decrease was observed in the extract administration group (Groups A to C) compared to the non-administration group (Group D).
Thus, the immunoregulatory effect of Pleurotus nebrodensis was confirmed from CD4 / CD8 and RF-IgG.

<Influence on blood protein>
Total protein is a collective term for approximately 100 types of blood proteins, which are combined with carbohydrates and lipids to form complex proteins.
The A / G ratio is the ratio of albumin (Alb), which is a plasma protein, and total globulin (Glob), and its value depends on the relative variation of Alb and Glob.
The total protein value and A / G ratio are used as an index of protein metabolism in the body, and the general state of the whole body can be estimated.
Test method: These values were analyzed by an automatic chemical analyzer (Auto Lab, Radio lmmuno Assay method).

FIG. 8 shows the measurement results of total protein and A / G (for effects on blood protein).
Compared to normal ICR mice, MRL mice had higher total protein values and lower A / G values. However, in the MRL mice, these parameters improved in the extract administration group (Groups A to C) as compared with the non-administration group (Group D), which is almost the same as that of the normal rats (Group E). It became equal.
From this, it was confirmed that protein metabolism was improved in addition to the anti-inflammatory effect and the immune regulation effect.

<Influence on renal function>
Urea nitrogen is nitrogen in urea, which is the final metabolite of protein. Urea is a nitrogen component produced through the urea cycle in the liver, and is released into the blood and excreted from the kidney.
Creatinine is one of the non-protein nitrogen compounds in the blood and is excreted in the urine via the kidney like urea and uric acid.
Uric acid is the final metabolite of purines, which are nucleic acid constituents produced in the liver, muscle, and bone marrow. Uric acid is broken down in tissues, and a small amount is excreted in bile / intestine, and most (60-80%) is excreted in urine.
Measurements of urea nitrogen, creatinine, and uric acid are used as indicators of renal function.
Test method: These values were analyzed by an automatic chemical analyzer (Auto Lab, Radio lmmuno Assay method).

FIG. 9 shows the results of measurement of urea nitrogen, creatinine, and uric acid (for effects on renal function).
Compared with normal ICR mice, MRL mice had higher uric acid levels and lower urea nitrogen and creatinine levels. However, in the MRL mice, the extract administration group (Groups A to C) shows an improvement in these parameters compared to the non-administration group (Group D), approaching the values of normal rats (Group E). There was a trend.
From this, in addition to the anti-inflammatory effect and the immune regulation effect, it was confirmed that the renal function was also improved.

As described above, also in in vivo tests, it was confirmed that Pleurotus nebrodensis and / or its extract have a suppressive and therapeutic effect on the onset of inflammatory diseases derived from both anti-inflammatory and immunomodulatory effects. .
In addition, especially the extraction part with a high effect was a hot-water extract (and its ethanol soluble part), and a methanol extract. In this test, the active ingredient was not elucidated, but there was a clear difference in activity between the methanol extract and the ethanol extract, and in the organic solvent soluble part of the hot water extract. Since the activity was observed, it is considered effective for elucidating the active ingredient and the mechanism of action.

It is a figure which shows the preparation method of the extract used for this invention. It is a figure which shows the test method of the platelet aggregation inhibitory effect in this invention. It is a figure which shows the test result of the platelet aggregation inhibitory effect in this invention. It is a figure which shows the test method of the chemokine gene expression inhibitory effect in this invention. It is a figure which shows the test result of the chemokine gene expression inhibitory effect in this invention. It is a figure which shows the result (anti-inflammatory effect) of the blood test in this invention. It is a figure which shows the result (immunity adjustment effect | action) of the blood test in this invention. It is a figure which shows the result (influence on blood protein) of the blood test in this invention. It is a figure which shows the result (influence on a renal function) of the blood test in this invention.

Claims (2)

Inflammatory disease prevention improving agent characterized by comprising an accession number FERM P-19370 in Pureurotasu Neburodenshisu (Pleurotus nebrodensis) strains and / or its extract as an active ingredient. Prevention in improving agents, inflammatory disease prevention improving agent characterized by containing, as an active ingredient, said Pureurotasu Neburodenshisu (Pleurotus nebrodensis) water or methanol extract of strain of claim 1.

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