JP5756158B2 - MIF secretion inhibitor - Google Patents

Description

  The present invention relates to a macrophage migration inhibitory factor (MIF) secretion inhibitor and an inflammatory disease ameliorating agent.

Macrophage migration inhibitory factor (hereinafter referred to as MIF) is one of the mediators of inflammation and immunity, and is a humoral factor that controls macrophage migration, collects macrophages at the site of inflammation, and enhances phagocytic ability. MIF is rheumatoid arthritis (Non-patent document 1: Expert Opin Ther Targets, 7, 2, 153-164, 2003), nephritis (Non-patent document 2: Expert Opin Ther Targets, 7, 2, 153-164, 2003), Atopic dermatitis (Non-patent document 3: Biochem Biophys Res Commun, 240, 11, 173-8, 1997), psoriasis (Non-patent document 4: BJ Dermatol, 141, 1061-66, 1999), ulcerative colitis, Sepsis, contact dermatitis (Non-patent document 5: Eur J Immunol, 33, 1478-87, 2003), inflammation caused by ultraviolet rays (Non-patent document 6: J Invest Dermatol, 112, 2, 210-15, 1999), delayed Acute / chronic inflammatory diseases (J End, 179, 15-23, 2003) such as sexual allergy (Non-patent Document 7: Pro Natl Acad Sci USA, 93, 7849-54, 1996), arthritis, graft rejection, tumor It is involved in the pathological processes of inflammatory diseases such as growth, angiogenesis, anemia, encephalomyelitis and the like, and it is expected to reduce these inflammatory diseases by inhibiting MIF.
Various active inhibitors of MIF have been developed (Patent Document 1: JP-T-2003-513065, Patent Document 2: JP-T-2005-500266, Patent Document 3: JP-T-2006-517777), MIF secretion inhibitors are not known.
The present inventor has focused on MIF secretion suppression and has continued research and development on MIF secretion inhibitors. However, the present inventor has previously proposed a technique that uses Himejoon or its extract as a MIF secretion inhibitor. Proposed in Japanese Patent Application No. 2008-022588.
Chinese maple is known as a folk medicine and has been used to treat coronary atherosclerosis and cerebrovascular diseases. Moreover, the antioxidant, anti-inflammatory, and fatty acid synthase inhibitory activity are known (nonpatent literature 8: J Chromato A, 1070, 211-214, 2006). However, the inhibitory effect of MIF secretion by Chinese maple is not known. Among them, it is not known at all that the leaves of Chinese maple maple have an excellent MIF secretion inhibitory effect.

Japanese translation of PCT publication No. 2003-513065 Japanese translation of PCT publication No. 2005-500026 JP-T-2006-517997 Expert Opin Ther Targets, 7, 2, 153-164,2003 Expert Opin Ther Targets, 7, 2, 153-164,2003 Biochem Biophys res Commun, 240, 11,173-8, 1997 B J Dermatol, 141, 1061-66, 1999 Eur J Immunol, 33, 1478-87, 2003 J Invest Dermatol, 112, 2, 210-15, 1999 Pro Natl Acad Sci USA, 93, 7849-54, 1996 J Chromato A, 1070, 211-214, 2006

  An object of the present invention is to provide a MIF secretion inhibitor.

The present inventors have found that a leaf extract of Acer truncatum (scientific name: Acer truncatum) has an excellent inhibitory effect on the secretion of macrophage migration inhibitory factor, thereby completing the present invention.
The main configuration of the present invention is as follows.
1. An inhibitor of macrophage migration inhibitory factor secretion, containing a leaf extract of Acer truncatum (scientific name: Acer truncatum).
2.1. A skin external preparation comprising the described macrophage migration inhibitory factor secretion inhibitor.
3.1. An ameliorating agent for inflammatory diseases comprising the described macrophage migration inhibitory factor secretion inhibitor.

According to the present invention, it is possible to provide a new therapeutic agent or preventive agent for atopic dermatitis in which abnormal secretion of MIF is observed.
In addition to atopic dermatitis, abnormal secretion and involvement of MIF are suggested, skin inflammation caused by psoriasis and ultraviolet rays, hemangioma, urticaria, sepsis, respiratory inflammation, ulcerative colitis, rheumatoid arthritis, nephritis, contact It is also expected to be effective in treating and preventing acute and chronic inflammatory diseases such as dermatitis and delayed allergy, arthritis, graft rejection, tumor growth, angiogenesis, anemia, encephalomyelitis, and other inflammation caused by bacterial infection. By locally prescribing the MIF secretion inhibitor as an external preparation for skin, the function of macrophage migration inhibitory factor can be suppressed, and the function of macrophages in the inflammatory site of skin can be suppressed.

FIG. 2 is a graph showing Table 1 of UVB-induced MIF secretion inhibition test results of Sinaia maple hot water extract using keratinocytes. Table 2 is a graph of the results of LPS-induced MIF secretion suppression test results of the hot water extract of Chinese maple maple using THP-1 cells. FIG. 3 is a graph showing the results of LPS-induced MIF secretion inhibition test results of Sinaia maple deethanol extract using THP-1 cells.

  The Chinese maple maple (scientific name: Acer truncatum) used in the present invention is a plant belonging to the genus Maple. In the present invention, leaves of Chinese corn maple are used, but dead leaves are particularly preferable. Dead leaves are those that fall naturally in the fall of deciduous trees, but leaves that have not fallen and the color of the leaves remaining on the branches may be yellow, red, or brown, which are not green. That is, any leaf that is naturally ripened may be used.

As an extract of the leaves of Chinese corn maple, the leaves or dead leaves of Chinese corn maple are crushed as they are, or dried and then crushed, water, alcohol such as ethanol, ether, acetone, 1,3-butylene glycol, 1 , 2-Pentanediol, Propylene glycol, Ethyl acetate and other crude extracts, and extracts obtained by stepwise purification of the crude extract with various types of chromatography such as partition extraction and column chromatography Includes fractions. These may be used alone or in combination of two or more. Although the leaves or dead leaves of Sinaia maple may be subjected to the extraction operation as they are, it is more efficient to perform the extraction after processing such as chopping, drying, and pulverization. Extraction can be performed by immersing in an extraction solvent. In order to increase the extraction efficiency, the extraction solvent can be stirred, crushed and homogenized in the extraction solvent, or pressure can be applied in the extraction solvent. The extraction temperature is suitably about 5 to 100 ° C., and the extraction time is about 5 minutes to 1 month. These conditions can be set as appropriate.
The Sinaia maple extract can be used as it is or in a state of being dissolved in an organic solvent such as water or ethanol, as a MIF secretion inhibitor, an inflammatory disease ameliorating agent, particularly a skin inflammatory disease ameliorating agent. Further, if necessary, the extraction solvent may be distilled off and the dried product may be used.

Sinaia maple leaf or dead leaf extract can be applied as a dry product in the range of 0.0001 to 1000 mg / day, and the amount is not limited to this range, and the amount is appropriately set according to the target, application form, and symptoms can do.
The blending amount of the Sinaia maple leaf or dead leaf extract of the present invention is preferably about 0.0001 to 10% by weight, but in a wide range up to 100% by weight depending on various conditions such as the dosage form to be used and the intended use. A compounding quantity can be set suitably.

The preparation of the present invention can be administered orally or parenterally.
The preparation of the present invention can be applied in the form of, for example, a solution such as an aqueous solution, an oil, an emulsion, and a liquid, a semi-solid such as a gel and a cream, and a solid such as a powder, granule, capsule, microcapsule, and solid. . It is prepared in these forms by conventionally known methods, and lotions, emulsions, gels, creams, ointments, plasters, haps, aerosols, suppositories, injections, powders, granules, tablets, pills , Various dosage forms such as syrups and lozenges. These can be applied to the body by applying, sticking, spraying, drinking and the like. Among these dosage forms, lotions, emulsions, creams, ointments, plasters, haps, aerosols and the like, which are external preparations for skin, are suitable.
Usually, these dosage forms and compositions can be produced according to the formulation method used in medicine.
Hereinafter, the present invention will be described in detail.

Preparation of Sinaia maple maple ethanol extract 100 g of dried Syringa maple leaf was soaked in 6 L of 99.5% ethanol for 1 week, and the solvent of the obtained ethanol extract was distilled off to obtain 2 g of Saga maple maple extract. Got.

Preparation of Sinaia maple maple hot water extract 100 g of dried Sinaia maple leaf was placed in 6 L of distilled water and boiled at 100 ° C. for 10 minutes. The obtained hot water extract was freeze-dried to obtain 9 g of Chinese corn maple extract.

Preparation of Sinaia maple maple leaf ethanol extract 100 g of a dry maple leaf maple leaf soaked in 6 L of 99.5% ethanol for 1 week, and the solvent of the obtained ethanol extract was distilled off to give 1 g of cinnamon maple maple leaf. An extract was obtained.

Preparation of Sinaia maple maple leaf hot water extract 100 g of dried Sinaia maple maple leaf extract was placed in 6 L of distilled water and boiled at 100 ° C. for 10 minutes. The obtained hot water extract was freeze-dried to obtain 4 g of Chinese corn maple extract.

1. MIF secretion inhibition test of Chinese corn maple extract UVB-induced MIF secretion inhibition test using keratinocytes It is known that MIF is strongly expressed in keratinocytes, one of the constituent cells of the skin. It is known that MIF secretion is promoted by irradiating UVB to keratinocytes (J Invest Dermatol, 112, 2, 210-15, 1999). Therefore, it was examined whether or not the Sinaia maple extract extracts MIF secretion when keratinocytes are irradiated with UVB.
Human keratinocytes were seeded in a φ35 mm dish at 7.0 × 104 cells / cm 2 and cultured in KBM / KGM medium (manufactured by LONZA) for 5 days. Change to KBM / KGM medium (BPE (-)) containing each sample, and after overnight culture, in the presence of HANKS (-), UVB was irradiated at 1.0 W / m2, 20 mJ / m2, The KBM / KGM medium (BPE (-)) was replaced and cultured for 6 hours. The culture supernatant was collected, and the amount of MIF in the supernatant was measured using the hMIF ELISA kit according to a conventional method, and the amount of MIF secretion was calculated. Furthermore, the cells were lysed with Cell Lysis Buffer, the protein in the lysate was measured with a protein assay kit, and the amount of protein per dish was calculated. The sample was not added and UVB (-) treatment was taken as 100%, and the MIF secretion rate per unit protein at the time of sample addition was compared. The results are shown in Table 1. The results of Table 1 are graphed and shown in FIG. In addition, as a result of comparing the protein amount of the control and the sample-added cells in this test, there was no difference in the protein amount between the two, and no cytotoxicity of each sample was observed in this test system.

枯れていないシナヤグルマカエデの熱水抽出物（実施例２）を添加したdishでも、コントロール（ＭＩＦ分泌率860％）と比較してＭＩＦ分泌率が492％と低くなり、ＭＩＦ分泌を抑制する効果が認められたが、枯れたシナヤグルマカエデの熱水抽出物（実施例４）を添加したdishのＭＩＦ分泌率は365％とより低い値であり、実施例２の３／４に抑制されている。
このことから、シナヤグルマカエデにおいては、特に枯葉の抽出物の方がＭＩＦ分泌を抑制する効果に優れることが分かった。

Even with dish to which a hot water extract of unripe Chinese maple maple (Example 2) was added, the MIF secretion rate was lowered to 492% compared to the control (MIF secretion rate 860%), and the effect of suppressing MIF secretion However, the MIF secretion rate of dish to which a hot water extract of withered Chinese maple map (Example 4) was added was a lower value of 365%, which was suppressed to 3/4 of Example 2. Yes.
From this, it was found that the extract of dead leaves is particularly effective in suppressing MIF secretion in Chinese maple maple.

  MIF secretion was remarkably suppressed by adding cinnamon maple maple leaf extract to keratinocytes. Sinaia maple dead leaves against skin inflammatory diseases such as skin damage / inflammation caused by UV rays, atopic dermatitis, psoriasis, contact dermatitis, etc. The extract can be expected to have a better improvement effect.

2. LPS-induced MIF secretion inhibition test using THP-1 cells It is known that MIF secretion from macrophages is promoted by bacterial infection or stimulation of foreign substances. THP-1 cells, which are human monocytic leukemia cells, which are macrophage model cells, secrete MIF upon stimulation with LPS in a bacterial infection model (FEBS Let 551, 78-88, 2003). Therefore, it was examined whether or not the extract of Chinese maple maple suppresses MIF secretion of THP-1 cells stimulated with LPS.

THP-1 cells were cultured in 1% serum / RPMI medium for 3 hours, seeded at 1 × 10 6 cells / well (12 well plate) in the same medium, and each sample was added. 45 minutes after the addition of each sample, LPS was added at a final concentration of 10 μg / ml and cultured for 4 hours. After culture, the cells were centrifuged at 400 × g, the cell supernatant was collected, the amount of MIF in the supernatant was measured according to a conventional method using an hMIF ELISA kit, and the amount of MIF secretion was calculated. The sample was not added and LPS (-) treatment was taken as 100%, and the MIF secretion rate at the time of sample addition was compared.
Table 2 shows the results when the samples of Example 2 and Example 4 were added. The results of Table 2 are graphed and shown in FIG.

枯れていないシナヤグルマカエデ熱水抽出物（実施例２）を添加したwellでは、コントロールがＭＩＦ分泌率１５８％であるのに対し、ＭＩＦ分泌率が１１８％であった。
これに対し、シナヤグルマカエデ枯葉熱水抽出物（実施例４）を添加したwellでは、ＭＩＦ分泌率が６９％と抑えられ、実施例２と比べて著しいＭＩＦ分泌抑制効果が認められた。

In the wells to which the unburned Chinese corn maple hot water extract (Example 2) was added, the MIF secretion rate was 158% while the MIF secretion rate was 118%.
On the other hand, in the well to which Sinaia maple dead leaf hot water extract (Example 4) was added, the MIF secretion rate was suppressed to 69%, and a remarkable MIF secretion inhibitory effect was observed compared to Example 2.

Since the extracts of Example 1 and Example 3 were added after dissolved in DMSO (dimethyl sulfoxide), the same amount of DMSO was added, no sample was added, and LPS (−) treatment was set to 100%, and the sample was added at the time of sample addition. Comparison of MIF secretion rate was performed.
Table 3 shows the results when the samples of Example 1 and Example 3 were added. The results of Table 3 are graphed and shown in FIG.

枯れていないシナヤグルマカエデエタノール抽出物（実施例１）を添加したwellでは、ＤＭＳＯがＭＩＦ分泌率１４６％であるのに対し、ＭＩＦ分泌率が１５１％であり、ＭＩＦ分泌を抑制する効果は全く認められなかった。
これに対し、シナヤグルマカエデ枯葉エタノール抽出物（実施例３）を添加したwellでは、ＭＩＦ分泌率が４５％と抑えられ、実施例１と比べて著しいＭＩＦ分泌抑制効果が認められた。

In the well to which the cinnamon red maple ethanol extract (Example 1) was added, DMSO had a MIF secretion rate of 146%, whereas the MIF secretion rate was 151%, and the effect of suppressing MIF secretion was completely I was not able to admit.
On the other hand, in the wells to which Sinaia maple maple leaf ethanol extract (Example 3) was added, the MIF secretion rate was suppressed to 45%, and a remarkable MIF secretion inhibitory effect was observed compared to Example 1.

  MIF secretion was remarkably suppressed by adding Sinaia maple leaf extract to THP-1 cells. With respect to inflammation caused by bacterial infections such as acne and sepsis, and chronic inflammatory diseases such as invasive inflammation, allergies, and rheumatoid arthritis, Sinaia maple leaf extract can be expected to be better prevented or improved.

Claims (1)

A skin external preparation for inhibiting the secretion of macrophage migration inhibitory factor , including a macrophage migration inhibitory factor secretion inhibitor , comprising as an active ingredient water or ethanol extract of dead leaves of Acer truncatum (scientific name: Acer truncatum).

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