JP6144564B2 - Inflammation prevention agent - Google Patents
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- JP6144564B2 JP6144564B2 JP2013152357A JP2013152357A JP6144564B2 JP 6144564 B2 JP6144564 B2 JP 6144564B2 JP 2013152357 A JP2013152357 A JP 2013152357A JP 2013152357 A JP2013152357 A JP 2013152357A JP 6144564 B2 JP6144564 B2 JP 6144564B2
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Description
本発明は、炎症予防剤に関し、詳細には、血中遊離脂肪酸低下作用又は炎症性サイトカイン発現抑制作用による炎症予防作用が有効性を示す、炎症予防剤に関する。本発明が予防する「炎症」は、主として、脂肪で産生する炎症性サイトカイン及び遊離脂肪酸が健常時より有意に多い状態を意味する。この「炎症」により、炎症性サイトカインが全身に回り、他の臓器の炎症シグナルを活性化して動脈硬化やがんを誘導するリスクが高まるとされている。また、この「炎症」により、脂肪組織とマクロファージのパラクリン調節系が継続して発生し、脂肪組織を増悪する「悪循環」が形成されることから、この「炎症」は慢性的な炎症であると言える。 The present invention relates to an inflammation preventive agent, and in particular, relates to an inflammation preventive agent that exhibits an effective anti-inflammatory effect by reducing blood free fatty acids or suppressing inflammatory cytokine expression. “Inflammation” prevented by the present invention mainly means a state in which inflammatory cytokines and free fatty acids produced in fat are significantly higher than normal. This "inflammation" is said to increase the risk that inflammatory cytokines will spread throughout the body and activate inflammatory signals in other organs to induce arteriosclerosis and cancer. In addition, this “inflammation” causes the paracrine regulation system of adipose tissue and macrophages to continue to develop, forming a “vicious circle” that exacerbates the adipose tissue. Therefore, this “inflammation” is a chronic inflammation. I can say that.
炎症は、種々の外因性又は内因性の組織障害刺激に対する生体防御応答の１つである。近年話題のメタボリックシンドローム（内臓脂肪症候群）の主要な機序は、インスリン抵抗性、腹部肥満、炎症と考えられており、内臓脂肪が蓄積し、脂肪細胞が大きくなると、マクロファージ等の免疫細胞が集まって慢性的な炎症が起こり、この炎症が動脈硬化や糖尿病等につながるおそれがある。また、肥満により肥大した脂肪細胞からは遊離脂肪酸が分泌され、これがインスリン抵抗性を生じさせると考えられており、血液中の遊離脂肪酸量の増加は、炎症性疾患の発症・進展に深く関与している。 Inflammation is one of the biodefense responses to various exogenous or endogenous tissue damage stimuli. In recent years, the main mechanisms of metabolic syndrome (visceral fat syndrome) are considered to be insulin resistance, abdominal obesity, and inflammation. When visceral fat accumulates and fat cells grow, immune cells such as macrophages gather. Chronic inflammation may occur, and this inflammation may lead to arteriosclerosis, diabetes, and the like. Moreover, free fatty acids are secreted from fat cells enlarged due to obesity, which is thought to cause insulin resistance, and the increase in the amount of free fatty acids in the blood is deeply involved in the onset and progression of inflammatory diseases. ing.
炎症性疾患の多くは、生体内における炎症性サイトカインの制御異常が関連していると考えられている。炎症性サイトカインは、炎症を促進するサイトカインの総称で、IL-6、IL1-β等が炎症性サイトカインに分類される。炎症性サイトカインは、各種刺激によって血球から産生されるが、一方で、炎症を抑制する働きを有する抗炎症性サイトカイン（IL-10、IL-4等）も血球から産生され、健常時には、両サイトカインの産生量が適宜調整されているため、生体内における炎症反応は過剰になることなく正常範囲内に制御されている。 Many inflammatory diseases are considered to be associated with abnormal regulation of inflammatory cytokines in vivo. Inflammatory cytokine is a general term for cytokines that promote inflammation, and IL-6, IL1-β and the like are classified as inflammatory cytokines. Inflammatory cytokines are produced from blood cells by various stimuli. On the other hand, anti-inflammatory cytokines (IL-10, IL-4, etc.) that suppress inflammation are also produced from blood cells. Therefore, the inflammatory reaction in the living body is controlled within the normal range without becoming excessive.
また近年、脂肪細胞、特に内臓脂肪と動脈硬化性疾患との関連が明らかになってきている。脂肪細胞は重量で身体の１０％以上を占める巨大な組織であり、中性脂肪としてエネルギーを貯蔵するのみならず、アディポサイトカイン（脂肪組織由来生理活性物質）を分泌することにより、生体ホメオスタシスの維持に積極的に参与していると考えられている。特許文献１には、アディポサイトカインの一種であるアディポネクチン（Adpn）の分泌促進作用と脂肪前駆細胞の分化抑制作用及び脂肪細胞の脂肪蓄積抑制との関係、並びに、抗肥満関連遺伝子群（PPARγ、Adpn、Leptin、aP2）の遺伝子発現変動及び血中Adpnと中性脂肪との関係が示唆されており、アディポネクチンの分泌を誘導することが動脈硬化、肥満、糖尿病等の予防・治療に有効であること、及び、特定の植物から抽出された特定成分がAdpnの分泌を誘導することが開示されており、該特定成分として、レゾルシノール誘導体を含有するものが開示されている。
In recent years, the relationship between adipocytes, particularly visceral fat, and arteriosclerotic diseases has become clear. Adipose cells are a huge tissue that occupies more than 10% of the body by weight. In addition to storing energy as neutral fat, adipocytokine (adipose tissue-derived bioactive substance) is secreted to maintain biological homeostasis. It is believed that they are actively participating.
また特許文献２には、合成されたアルキルレゾルシノール及びその塩が活性酸素除去能を有することが開示されており、これを用いた皮膚外用剤も開示されている。尚、アルキルレゾルシノールに関して特許文献２に開示されているのは、抗酸化作用に関するものであり、炎症性サイトカインが関連する炎症に関しては記載されていない。 Patent Document 2 discloses that the synthesized alkylresorcinol and a salt thereof have an ability to remove active oxygen, and a skin external preparation using the same is also disclosed. In addition, what is indicated by patent document 2 regarding the alkyl resorcinol is related with an antioxidant effect | action, and is not described regarding the inflammation which an inflammatory cytokine relates.
本発明は、天然物由来で長期服用しても安心な炎症予防剤に関する。 The present invention relates to an inflammation preventive agent that is derived from natural products and can be used safely for a long time.
本発明は、イネ科植物種子のアルコール抽出物の分配クロマトグラフィーのピーク成分を有効成分として含有し、該ピーク成分に、下記一般式（Ｉ）で表されるアルキルレゾルシノールを複数種含む、特定アルキルレゾルシノール混合物が含有されている炎症予防剤である。 The present invention contains a peak component of partition chromatography of an alcohol extract of a grass seed as an active ingredient, and the peak component contains a plurality of alkylresorcinols represented by the following general formula (I). It is an inflammation preventive agent containing a resorcinol mixture.
本発明の炎症予防剤は、種々の炎症性疾患、特に、血中遊離脂肪酸量の増加又は炎症性サイトカイン発現上昇に起因する炎症性疾患の予防に有効である。特に本発明の炎症予防剤は、脂肪細胞の炎症性サイトカイン発現（産生）及び遊離脂肪酸産生量を低減させ、その結果、全身の臓器の炎症シグナル活性化を抑制し、動脈硬化やがんを誘導するリスクを低減させる。また、本発明の炎症予防剤の有効成分（アルキルレゾルシノール）は、植物由来のものであるため、安全性が高く、副作用のおそれが少ないので、本発明の炎症予防剤は、使用上の問題がなく有利である。 The inflammation preventive agent of the present invention is effective in preventing various inflammatory diseases, in particular, inflammatory diseases caused by an increase in the amount of free fatty acids in blood or an increase in expression of inflammatory cytokines. In particular, the inflammation preventive agent of the present invention reduces the expression (production) of inflammatory cytokines and production of free fatty acids in adipocytes, and as a result, suppresses inflammatory signal activation in organs throughout the body and induces arteriosclerosis and cancer. Reduce the risk of In addition, since the active ingredient (alkylresorcinol) of the inflammation preventive agent of the present invention is derived from a plant, it has high safety and is less likely to cause side effects. Therefore, the inflammation preventive agent of the present invention has a problem in use. Is advantageous.
本発明の炎症予防剤は、イネ科植物種子のアルコール抽出物の分配クロマトグラフィーのピーク成分を有効成分として含有する。このピーク成分（本発明の炎症予防剤の有効成分）には、上記一般式（Ｉ）で表されるアルキルレゾルシノールを複数種含む、特定アルキルレゾルシノール混合物が含有されている。この特定アルキルレゾルシノール混合物は、血中遊離脂肪酸量の低下作用及び／又は炎症性サイトカインの発現抑制作用を有する。 The inflammation preventive agent of the present invention contains, as an active ingredient, a peak component of partition chromatography of an alcoholic extract of a grass plant seed. This peak component (the active ingredient of the inflammation preventive agent of the present invention) contains a specific alkyl resorcinol mixture containing a plurality of alkyl resorcinols represented by the general formula (I). This specific alkyl resorcinol mixture has a blood free fatty acid content reducing action and / or an inflammatory cytokine expression suppressing action.
アルキルレゾルシノールは、天然の非イソテルぺノイド系フェノール性両親媒性化合物であるレゾルシノール脂質として、種々の植物に含まれていることが知られており、アルキルレゾルシノールの給源としては、イネ科植物以外にも、例えば、ウルシ科、イチョウ科、ヤマモガシ科、ヤブコウジ科、サクラソウ科、ニクズク科、アヤメ科、サトイモ科、キク科のヨモギ、マメ科等が知られている。これらの植物の中でも、イネ科植物は、可食性有効成分としてのアルキルレゾルシノールの研究が進んでいること等から、本発明においては、アルキルレゾルシノールの給源として、イネ科植物を採用している。 Alkylresorcinol is known to be contained in various plants as a resorcinol lipid, which is a natural non-isoterpenoid phenolic amphiphilic compound. In addition, for example, Urushiaceae, Ginkgoaceae, Porcupineaceae, Yabukodiidae, Primulaceae, Stigmaceae, Iridaceae, Araceae, Artemisia, Asteraceae, Legume, and the like are known. Among these plants, since gramineous plants have been studied for alkylresorcinol as an edible active ingredient, in the present invention, gramineous plants are employed as a source of alkylresorcinol.
アルキルレゾルシノールの給源として利用可能なイネ科植物としては、特に制限されないが、例えば、小麦、デュラム小麦、ライ麦、ライ小麦、大麦、オーツ麦、はと麦、トウモロコシ、イネ、ヒエ、アワ、キビ等の穀類が挙げられ、これら１種を単独で又は２種以上を組み合わせて用いることができる。これらの穀類の中でも、高い活性が得られる点から、特に小麦、デュラム小麦等のコムギ属の植物が好ましく、小麦が更に好ましい。イネ科植物種子としては、任意の形態のイネ科植物種子で良く、例えば、イネ科植物種子（好ましくは種子外皮；糟糠類）そのもの；当該イネ科植物種子を切断、粉砕若しくは粉末化したもの；当該イネ科植物種子を乾燥したもの；当該イネ科植物種子を乾燥後粉砕若しくは粉末化したもの等でも良い。イネ科植物種子外皮を含む好適な例としては、ふすま、末粉、籾殻、ぬか等が挙げられる他、外皮を伴った種子も挙げられる。 Examples of grasses that can be used as a source of alkylresorcinol are not particularly limited. For example, wheat, durum wheat, rye, rye wheat, barley, oats, potato, corn, rice, millet, millet, millet, etc. These cereals can be mentioned, and one of these can be used alone or in combination of two or more. Among these cereals, wheat plants such as wheat and durum wheat are particularly preferable, and wheat is more preferable because high activity is obtained. The gramineous plant seed may be any form of gramineous plant seed, for example, gramineous plant seed (preferably seed hull; moss) itself; a product obtained by cutting, crushing or pulverizing the gramineous plant seed; It may be a product obtained by drying the grass plant seed; a product obtained by drying or crushing or powdering the grass seed. Preferable examples including grass seed hulls include bran, powder, rice husk, bran and the like, and seeds with hulls.
イネ科植物種子のアルコールによる抽出方法は特に制限されないが、例えば、上記各種形態のイネ科植物種子をアルコール中に浸漬、攪拌又は還流する方法の他、超臨界流体抽出法等が挙げられる。前者の方法の場合、抽出温度は２〜１００℃が好ましく、抽出時間は３０分〜７２時間が好ましく、アルコール使用量は、イネ科植物種子１００質量部に対し５０〜２０００質量部が好ましい。 The method of extracting grass seeds with alcohol is not particularly limited, and examples thereof include a supercritical fluid extraction method and the like in addition to a method of immersing, stirring, or refluxing various kinds of grass seeds in the above-mentioned various forms. In the former method, the extraction temperature is preferably 2 to 100 ° C., the extraction time is preferably 30 minutes to 72 hours, and the amount of alcohol used is preferably 50 to 2000 parts by mass with respect to 100 parts by mass of the grass seed.
イネ科植物種子の抽出に用いられるアルコールとしては、例えば、メタノール、エタノール、ｎ−プロパノール、イソプロパノール、ｎ−ブタノール等の１価の低級アルコール（好ましくは炭素原子数１〜４のもの）、及び１，３−ブチレングリコール、プロピレングリコール、グリセリン等の多価アルコール等の室温（２５℃）で液体であるアルコールが挙げられる。これらのアルコールの中でも、操作性や環境性の点から、エタノールが好ましい。尚、イネ科植物種子の抽出に用いられるアルコールとしては、アルコール以外の水性成分（水、純水、蒸留水、水道水、酸性水、アルカリ水、中性水等）が含まれている含水エタノールを用いることもできる。含水アルコール中のアルコール含有量は、通常７０体積％以上、好ましくは８０体積％以上、より好ましくは９０体積％以上である。 Examples of the alcohol used for the extraction of grass seeds include monovalent lower alcohols (preferably those having 1 to 4 carbon atoms) such as methanol, ethanol, n-propanol, isopropanol, and n-butanol, and 1 , 3-butylene glycol, propylene glycol, polyhydric alcohols such as glycerin, and the like, which are liquids at room temperature (25 ° C.). Among these alcohols, ethanol is preferable from the viewpoint of operability and environmental properties. In addition, as alcohol used for the extraction of gramineous plant seeds, water-containing ethanol containing aqueous components (water, pure water, distilled water, tap water, acidic water, alkaline water, neutral water, etc.) other than alcohol Can also be used. The alcohol content in the hydrous alcohol is usually 70% by volume or more, preferably 80% by volume or more, more preferably 90% by volume or more.
本発明において、イネ科植物種子のアルコール抽出物の精製に用いられる分配クロマトグラフィーは、本発明の炎症予防剤の有効成分（特定アルキルレゾルシノール混合物）が得られる手法であればその種類は問わないが、移動相として非水系溶媒を用いる順相クロマトグラフィー法が好ましく、オープンカラム法、中圧カラム法、高速液体クロマトグラフィー等の公知の方法を適宜選択することができる。 In the present invention, the partition chromatography used for purification of the alcoholic extract of the grass seed is not limited as long as it is a technique that can obtain the active ingredient (specific alkyl resorcinol mixture) of the inflammation preventive agent of the present invention. A normal phase chromatography method using a non-aqueous solvent as a mobile phase is preferable, and a known method such as an open column method, a medium pressure column method, or a high performance liquid chromatography can be appropriately selected.
分配クロマトグラフィーにおける移動相としては、メタノール、エタノール、ｎ−プロパノール、イソプロパノール、ｎ−ブタノール等の１価の低級アルコール（好ましくは炭素原子数１〜４のもの）、及び１，３−ブチレングリコール、プロピレングリコール、グリセリン等の多価アルコール等の室温（２５℃）で液体であるアルコール；ジエチルエーテル、プロピルエーテル等のエーテル；酢酸ブチル、酢酸エチル等のエステル；アセトン、エチルメチルケトン等のケトン；ヘキサン；塩化メチレン；アセトニトリル；並びにクロロホルム等が挙げられ、これら溶媒の１種を単独で又は２種以上を組み合わせて用いることができる。複数の溶媒を組み合わせて移動相とする場合、分配クロマトグラフィーの実施中（イネ科植物種子のアルコール抽出物の精製中）において、複数の溶媒の混合比を一定にするイソクラクティックモードでも良く、あるいは該混合比を変化させるグラジエントモードでも良い。 As a mobile phase in partition chromatography, monovalent lower alcohols (preferably having 1 to 4 carbon atoms) such as methanol, ethanol, n-propanol, isopropanol, n-butanol, and 1,3-butylene glycol, Alcohols that are liquid at room temperature (25 ° C.) such as polyhydric alcohols such as propylene glycol and glycerine; ethers such as diethyl ether and propyl ether; esters such as butyl acetate and ethyl acetate; ketones such as acetone and ethyl methyl ketone; hexane Methylene chloride; acetonitrile; and chloroform and the like. One of these solvents can be used alone, or two or more can be used in combination. When a plurality of solvents are combined into a mobile phase, an isocratic mode in which the mixing ratio of the plurality of solvents is constant during partition chromatography (purification of the alcoholic extract of the grass seed) may be used. Alternatively, a gradient mode in which the mixing ratio is changed may be used.
分配クロマトグラフィーにおける担体としては、目的とする有効成分を担持−放出できる担体であればいずれも用いることができるが、一般的にはシリカゲル、ポリアクリルアミドゲル、デキストランゲル等を挙げることができる。 Any carrier can be used as the carrier in the partition chromatography as long as it can carry and release the target active ingredient, and generally includes silica gel, polyacrylamide gel, dextran gel and the like.
イネ科植物種子のアルコール抽出物の分配クロマトグラフィーにおける検出波長は、１７０〜３２０ｎｍであれば良く、好ましくは１９０〜２８０ｎｍである。 The detection wavelength in the partition chromatography of the alcoholic extract of the grass plant seed may be 170 to 320 nm, preferably 190 to 280 nm.
イネ科植物種子（好ましくはコムギ属の植物）のアルコール抽出物（好ましくはエタノール抽出物）の精製に好適な分配クロマトグラフィーの例として、下記分配クロマトグラフィーＡ及びＢが挙げられる。
・分配クロマトグラフィーＡ：担体としてシリカゲル及び移動相としてヘキサン−酢酸エチル混合溶媒を用いた中圧カラム法（中圧クロマトグラフィー）を用い、且つその分配クロマトグラフィーの実施中に、移動相を「ヘキサン−酢酸エチル混合溶媒においてヘキサンの含有割合が相対的に高いもの」から「ヘキサン−酢酸エチル混合溶媒においてヘキサンの含有割合が相対的に低いもの」へと変化させ（即ち、「ヘキサン大−少」へのグラジエントモードで用い）、且つ検出波長２５４ｎｍでのピーク成分を分取する。
・分配クロマトグラフィーＢ：担体としてシリカゲル及び移動相としてメタノールを用いた高速液体クロマトグラフィー（ＨＰＬＣ）を用い、且つ検出波長２１５ｎｍでのピーク成分を分取する。
Examples of partition chromatography suitable for purification of an alcohol extract (preferably an ethanol extract) of a grass seed (preferably a plant of the genus Wheat) include the following partition chromatography A and B.
Partition chromatography A: using a medium pressure column method (medium pressure chromatography) using silica gel as a carrier and a hexane-ethyl acetate mixed solvent as a mobile phase, and during the execution of the partition chromatography, -Changed from "relatively high hexane content in ethyl acetate mixed solvent" to "relatively low hexane content in hexane-ethyl acetate mixed solvent" And a peak component at a detection wavelength of 254 nm is fractionated.
Partition chromatography B: Using high performance liquid chromatography (HPLC) using silica gel as a carrier and methanol as a mobile phase, a peak component at a detection wavelength of 215 nm is fractionated.
本発明の炎症予防剤における有効成分の含有量、即ち、イネ科植物種子のアルコール抽出物の分配クロマトグラフィーのピーク成分（特定アルキルレゾルシノール混合物）の含有量は、血中遊離脂肪酸量の増加又は炎症性サイトカイン発現上昇に起因する炎症性疾患の予防効果をより確実に奏させるようにする観点から、本発明の炎症予防剤中、好ましくは７０質量％以上、更に好ましくは７５質量％以上である。上記有効成分（特定アルキルレゾルシノール混合物）の含有量は１００質量％、即ち、本発明の炎症予防剤は上記有効成分（特定アルキルレゾルシノール混合物）のみから構成されていても良い。 The content of the active ingredient in the inflammation preventive agent of the present invention, that is, the content of the peak component (particular alkylresorcinol mixture) in the partition chromatography of the alcoholic extract of the gramineous plant seeds increases the amount of free fatty acid in the blood or inflammation From the viewpoint of more surely exerting a preventive effect on inflammatory diseases caused by increased expression of sex cytokines, it is preferably 70% by mass or more, more preferably 75% by mass or more in the inflammation preventive agent of the present invention. Content of the said active ingredient (specific alkyl resorcinol mixture) is 100 mass%, ie, the inflammation preventive agent of this invention may be comprised only from the said active ingredient (specific alkyl resorcinol mixture).
本発明の炎症予防剤の有効成分であり、イネ科植物種子のアルコール抽出物の分配クロマトグラフィーのピーク成分に含有されている、特定アルキルレゾルシノール混合物について説明すると、特定アルキルレゾルシノール混合物は、上記一般式（Ｉ）で表されるアルキルレゾルシノールを複数種含む。 The specific alkyl resorcinol mixture, which is an active ingredient of the inflammation preventive agent of the present invention and is contained in the peak component of the partition chromatography of the alcoholic extract of the grass seed, will be described. A plurality of alkylresorcinols represented by (I) are included.
上記一般式（Ｉ）におけるＲ1に関し、炭素原子数１５〜２５の飽和アルキル基としては、代表例として、ｎ−ペンタデシル、ｎ−ヘプタデシル、ｎ−ノナデシル、ｎ−ヘンイコシル、ｎ−トリコシル、ｎ−ペンタコシル、ｎ−ヘプタコシル等の直鎖状のものが挙げられ、これらの他に、分岐状又は環状のものでも良い。これらの中でも、炭素原子数１５〜２３の飽和アルキル基が好ましい。 Regarding R 1 in the above general formula (I), typical examples of the saturated alkyl group having 15 to 25 carbon atoms include n-pentadecyl, n-heptadecyl, n-nonadecyl, n-henicosyl, n-tricosyl, n- Examples include linear ones such as pentacosyl and n-heptacosyl, and besides these, branched or cyclic ones may be used. Among these, a saturated alkyl group having 15 to 23 carbon atoms is preferable.
また、上記一般式（Ｉ）におけるＲ1に関し、炭素原子数１５〜２５の不飽和アルキル基としては、上記の炭素原子数１５〜２５の飽和アルキル基に対応するものが挙げられる。不飽和アルキル基に含まれる不飽和結合の数及び位置に特に制限はない。 Moreover, regarding R 1 in the general formula (I), examples of the unsaturated alkyl group having 15 to 25 carbon atoms include those corresponding to the saturated alkyl group having 15 to 25 carbon atoms. There is no restriction | limiting in particular in the number and position of the unsaturated bond contained in an unsaturated alkyl group.
また、上記一般式（Ｉ）におけるＲ2は水素原子であることが好ましく、また、Ｒ1はＲ2に対してパラ位に結合していることが好ましい。 In the general formula (I), R 2 is preferably a hydrogen atom, and R 1 is preferably bonded to R 2 at the para position.
上記特定アルキルレゾルシノール混合物に含まれ得るアルキルレゾルシノールの具体例としては、以下のものが挙げられる。
１，３−ジヒドロキシ−５−ｎ−ペンタデシルベンゼン（Ｃ１５：０）
１，３−ジヒドロキシ−５−ｎ−ヘプタデシルベンゼン（Ｃ１７：０）
１，３−ジヒドロキシ−５−ｎ−ノナデシルベンゼン（Ｃ１９：０）
１，３−ジヒドロキシ−５−ｎ−ヘンイコシルベンゼン（Ｃ２１：０）
１，３−ジヒドロキシ−５−ｎ−トリコシルベンゼン（Ｃ２３：０）
１，３−ジヒドロキシ−５−ｎ−ペンタコシルベンゼン（Ｃ２５：０）
Specific examples of the alkyl resorcinol that can be contained in the specific alkyl resorcinol mixture include the following.
1,3-dihydroxy-5-n-pentadecylbenzene (C15: 0)
1,3-dihydroxy-5-n-heptadecylbenzene (C17: 0)
1,3-dihydroxy-5-n-nonadecylbenzene (C19: 0)
1,3-dihydroxy-5-n-henicosylbenzene (C21: 0)
1,3-dihydroxy-5-n-tricosylbenzene (C23: 0)
1,3-dihydroxy-5-n-pentacosylbenzene (C25: 0)
上記特定アルキルレゾルシノール混合物の好ましい一例として、下記６種類のアルキルレゾルシノールを含有するものが挙げられる。本発明者らの知見によれば、下記６種類のアルキルレゾルシノールは、血中遊離脂肪酸低下作用及び炎症性サイトカイン発現抑制作用に特に優れ、血中遊離脂肪酸量の増加又は炎症性サイトカイン発現上昇に起因する炎症性疾患の予防に有効である。
１）上記一般式（Ｉ）におけるＲ1が炭素原子数１５の飽和又は不飽和のアルキル基であるアルキルレゾルシノール（以下、ＡＲ１５ともいう）。
２）上記一般式（Ｉ）におけるＲ1が炭素原子数１７の飽和又は不飽和のアルキル基であるアルキルレゾルシノール（以下、ＡＲ１７ともいう）。
３）上記一般式（Ｉ）におけるＲ1が炭素原子数１９の飽和又は不飽和のアルキル基であるアルキルレゾルシノール（以下、ＡＲ１９ともいう）。
４）上記一般式（Ｉ）におけるＲ1が炭素原子数２１の飽和又は不飽和のアルキル基であるアルキルレゾルシノール（以下、ＡＲ２１ともいう）。
５）上記一般式（Ｉ）におけるＲ1が炭素原子数２３の飽和又は不飽和のアルキル基であるアルキルレゾルシノール（以下、ＡＲ２３ともいう）。
６）上記一般式（Ｉ）におけるＲ1が炭素原子数２５の飽和又は不飽和のアルキル基であるアルキルレゾルシノール（以下、ＡＲ２５ともいう）。
A preferable example of the specific alkyl resorcinol mixture is one containing the following six types of alkyl resorcinol. According to the findings of the present inventors, the following 6 types of alkylresorcinol are particularly excellent in blood free fatty acid lowering action and inflammatory cytokine expression suppressing action, and are caused by an increase in blood free fatty acid content or an increase in inflammatory cytokine expression. It is effective in preventing inflammatory diseases.
1) Alkyl resorcinol (hereinafter, also referred to as AR15) in which R 1 in the general formula (I) is a saturated or unsaturated alkyl group having 15 carbon atoms.
2) Alkyl resorcinol (hereinafter, also referred to as AR17) in which R 1 in the above general formula (I) is a saturated or unsaturated alkyl group having 17 carbon atoms.
3) Alkyl resorcinol (hereinafter, also referred to as AR19) in which R 1 in the above general formula (I) is a saturated or unsaturated alkyl group having 19 carbon atoms.
4) Alkyl resorcinol (hereinafter, also referred to as AR21) in which R 1 in the general formula (I) is a saturated or unsaturated alkyl group having 21 carbon atoms.
5) Alkyl resorcinol (hereinafter, also referred to as AR23) in which R 1 in the general formula (I) is a saturated or unsaturated alkyl group having 23 carbon atoms.
6) Alkyl resorcinol (hereinafter, also referred to as AR25) in which R 1 in the general formula (I) is a saturated or unsaturated alkyl group having 25 carbon atoms.
ＡＲ１５として特に好ましいものは、Ｒ1が炭素原子数１５の飽和アルキル基、Ｒ2が水素原子であるものであり、具体的には、１，３−ジヒドロキシ−５−ｎ−ペンタデシルベンゼン（Ｃ１５：０）が挙げられる。
ＡＲ１７として特に好ましいものは、Ｒ1が炭素原子数１７の飽和アルキル基、Ｒ2が水素原子であるものであり、具体的には、１，３−ジヒドロキシ−５−ｎ−ヘプタデシルベンゼン（Ｃ１７：０）が挙げられる。
ＡＲ１９として特に好ましいものは、Ｒ1が炭素原子数１９の飽和アルキル基、Ｒ2が水素原子であるものであり、具体的には、１，３−ジヒドロキシ−５−ｎ−ノナデシルベンゼン（Ｃ１９：０）が挙げられる。
ＡＲ２１として特に好ましいものは、Ｒ1が炭素原子数２１の飽和アルキル基、Ｒ2が水素原子であるものであり、具体的には、１，３−ジヒドロキシ−５−ｎ−ヘンイコシルベンゼン（Ｃ２１：０）が挙げられる。
ＡＲ２３として特に好ましいものは、Ｒ1が炭素原子数２３の飽和アルキル基、Ｒ2が水素原子であるものであり、具体的には、１，３−ジヒドロキシ−５−ｎ−トリコシルベンゼン（Ｃ２３：０）が挙げられる。
ＡＲ２５として特に好ましいものは、Ｒ1が炭素原子数２５飽和アルキル基、Ｒ2が水素原子であるものであり、具体的には、１，３−ジヒドロキシ−５−ｎ−ペンタコシルベンゼン（Ｃ２５：０）が挙げられる。
Particularly preferred as AR15 is one in which R 1 is a saturated alkyl group having 15 carbon atoms and R 2 is a hydrogen atom. Specifically, 1,3-dihydroxy-5-n-pentadecylbenzene (C15 : 0).
Particularly preferred as AR17 is one in which R 1 is a saturated alkyl group having 17 carbon atoms and R 2 is a hydrogen atom. Specifically, 1,3-dihydroxy-5-n-heptadecylbenzene (C17 : 0).
Particularly preferred as AR19 is one in which R 1 is a saturated alkyl group having 19 carbon atoms and R 2 is a hydrogen atom. Specifically, 1,3-dihydroxy-5-n-nonadecylbenzene (C19 : 0).
Particularly preferred as AR21 is one in which R 1 is a saturated alkyl group having 21 carbon atoms and R 2 is a hydrogen atom. Specifically, 1,3-dihydroxy-5-n-henecosylbenzene ( C21: 0).
Particularly preferred as AR23 is one in which R 1 is a saturated alkyl group having 23 carbon atoms and R 2 is a hydrogen atom. Specifically, 1,3-dihydroxy-5-n-tricosylbenzene (C23 : 0).
Particularly preferred as AR25 is one in which R 1 is a saturated alkyl group having 25 carbon atoms and R 2 is a hydrogen atom. Specifically, 1,3-dihydroxy-5-n-pentacosylbenzene (C25 : 0).
ＡＲ１５、ＡＲ１７、ＡＲ１９、ＡＲ２１、ＡＲ２３及びＡＲ２５の含有量は、血中遊離脂肪酸量の増加又は炎症性サイトカイン発現上昇に起因する炎症性疾患を効果的に予防する観点から、それぞれ、下記範囲内にあることが好ましい。
ＡＲ１５の含有量は、上記特定アルキルレゾルシノール混合物中、好ましくは０．１〜１０．０質量％、更に好ましくは０．１〜５．０質量％、特に好ましくは０．５〜１．５質量％である。
ＡＲ１７の含有量は、上記特定アルキルレゾルシノール混合物中、好ましくは１．０〜２０．０質量％、更に好ましくは５．０〜１５．０質量％、特に好ましくは８．０〜１２．０質量％である。
ＡＲ１９の含有量は、上記特定アルキルレゾルシノール混合物中、好ましくは２５．０〜４０．０質量％、更に好ましくは２７．５〜３７．５質量％、特に好ましくは３０．０〜３５．０質量％である。
ＡＲ２１の含有量は、上記特定アルキルレゾルシノール混合物中、好ましくは４０．０〜５５．０質量％、更に好ましくは４２．５〜５２．５質量％、特に好ましくは４５．０〜５０．０質量％である。
ＡＲ２３の含有量は、上記特定アルキルレゾルシノール混合物中、好ましくは１．０〜１５．０質量％、更に好ましくは２．５〜１２．５質量％、特に好ましくは５．０〜１０．０質量％である。
ＡＲ２５の含有量は、上記特定アルキルレゾルシノール混合物中、好ましくは０〜５．０質量％、更に好ましくは０〜２．０質量％、特に好ましくは０〜１．５質量％である。
The content of AR15, AR17, AR19, AR21, AR23, and AR25 is within the following ranges from the viewpoint of effectively preventing inflammatory diseases caused by an increase in blood free fatty acid content or inflammatory cytokine expression. Preferably there is.
The content of AR15 is preferably 0.1 to 10.0% by mass, more preferably 0.1 to 5.0% by mass, and particularly preferably 0.5 to 1.5% by mass in the specific alkylresorcinol mixture. It is.
The content of AR17 is preferably 1.0 to 20.0% by mass, more preferably 5.0 to 15.0% by mass, and particularly preferably 8.0 to 12.0% by mass in the specific alkylresorcinol mixture. It is.
The content of AR19 is preferably 25.0 to 40.0% by mass, more preferably 27.5 to 37.5% by mass, and particularly preferably 30.0 to 35.0% by mass in the specific alkylresorcinol mixture. It is.
The content of AR21 is preferably 40.0 to 55.0% by mass, more preferably 42.5 to 52.5% by mass, and particularly preferably 45.0 to 50.0% by mass in the specific alkylresorcinol mixture. It is.
The content of AR23 is preferably 1.0 to 15.0% by mass, more preferably 2.5 to 12.5% by mass, and particularly preferably 5.0 to 10.0% by mass in the specific alkylresorcinol mixture. It is.
The content of AR25 is preferably 0 to 5.0% by mass, more preferably 0 to 2.0% by mass, and particularly preferably 0 to 1.5% by mass in the specific alkylresorcinol mixture.
上記特定アルキルレゾルシノール混合物は、ＡＲ１５、ＡＲ１７、ＡＲ１９、ＡＲ２１、ＡＲ２３及びＡＲ２５以外の他のアルキルレゾルシノールの１種以上を含有していても良い。この他のアルキルレゾルシノールとしては、例えば、上記一般式（Ｉ）におけるＲ1が炭素原子数２７の飽和又は不飽和のアルキル基であるアルキルレゾルシノール（以下、ＡＲ２７ともいう）が挙げられる。ＡＲ２７として特に好ましいものは、Ｒ1が炭素原子数２７の飽和アルキル基、Ｒ2が水素原子であるものであり、具体的には、１，３−ジヒドロキシ−５−ｎ−ヘプタコシルベンゼン（Ｃ２７：０）が挙げられる。 The specific alkyl resorcinol mixture may contain one or more alkyl resorcinols other than AR15, AR17, AR19, AR21, AR23 and AR25. Examples of the other alkylresorcinol include alkylresorcinol (hereinafter, also referred to as AR27) in which R 1 in the general formula (I) is a saturated or unsaturated alkyl group having 27 carbon atoms. Particularly preferred as AR27 is one in which R 1 is a saturated alkyl group having 27 carbon atoms and R 2 is a hydrogen atom. Specifically, 1,3-dihydroxy-5-n-heptacosylbenzene ( C27: 0).
また、上記特定アルキルレゾルシノール混合物は、アルキルレゾルシノール以外の他の成分を含んでいても良く、このアルキルレゾルシノール以外の他の成分の含有量は、上記特定アルキルレゾルシノール混合物中、好ましくは３０質量％以下である。 The specific alkyl resorcinol mixture may contain other components other than the alkyl resorcinol, and the content of other components other than the alkyl resorcinol is preferably 30% by mass or less in the specific alkyl resorcinol mixture. is there.
上記特定アルキルレゾルシノール混合物の好ましい一例として、次の組成を有するものが挙げられる。即ち、ＡＲ１５として１，３−ジヒドロキシ−５−ｎ−ペンタデシルベンゼン（Ｃ１５：０）を１．２質量％、ＡＲ１７として１，３−ジヒドロキシ−５−ｎ−ヘプタデシルベンゼン（Ｃ１７：０）を１０．９質量％、ＡＲ１９として１，３−ジヒドロキシ−５−ｎ−ノナデシルベンゼン（Ｃ１９：０）を３３．９質量％、ＡＲ２１として１，３−ジヒドロキシ−５−ｎ−ヘンイコシルベンゼン（Ｃ２１：０）を４６．４質量％、ＡＲ２３として１，３−ジヒドロキシ−５−ｎ−トリコシルベンゼン（Ｃ２３：０）を７．５質量％、及びＡＲ２５として１，３−ジヒドロキシ−５−ｎ−ペンタコシルベンゼン（Ｃ２５：０）を０．１質量％含有する特定アルキルレゾルシノール混合物である。 A preferable example of the specific alkyl resorcinol mixture is one having the following composition. That is, 1.2% by mass of 1,3-dihydroxy-5-n-pentadecylbenzene (C15: 0) as AR15 and 1,3-dihydroxy-5-n-heptadecylbenzene (C17: 0) as AR17 10.9% by mass, AR19 as 1,3-dihydroxy-5-n-nonadecylbenzene (C19: 0) 33.9% by mass, AR21 as 1,3-dihydroxy-5-n-heneicosylbenzene ( C21: 0) 46.4% by weight, AR23 as 1,3-dihydroxy-5-n-tricosylbenzene (C23: 0) as 7.5% by weight, and AR25 as 1,3-dihydroxy-5-n -A specific alkyl resorcinol mixture containing 0.1% by mass of pentacosylbenzene (C25: 0).
本発明の炎症予防剤は、イネ科植物種子のアルコール抽出物の分配クロマトグラフィーのピーク成分（上記特定アルキルレゾルシノール混合物）、並びに必要に応じて薬学的に許容される種々の担体、賦形剤、その他の添加剤、その他の成分を含有するものである。本発明の炎症予防剤は、常法により製剤化することができ、その場合、本発明の炎症予防剤の剤型は、錠剤、散剤、顆粒剤、カプセル剤等の経口剤である。また、本発明の炎症予防剤に含有可能な「その他の成分」としては、その他の薬効作用を有する成分、抗炎症薬、各種ビタミン類、生薬、ミネラル類等が挙げられる。 The inflammation preventive agent of the present invention comprises a peak component (particular mixture of the above specific alkylresorcinol) of an alcohol extract of a grass seed, and various pharmaceutically acceptable carriers and excipients as necessary. It contains other additives and other components. The inflammation preventive agent of the present invention can be formulated by a conventional method. In this case, the dosage form of the inflammation preventive agent of the present invention is an oral preparation such as a tablet, powder, granule or capsule. Examples of the “other components” that can be contained in the inflammation preventive agent of the present invention include other medicinal components, anti-inflammatory drugs, various vitamins, herbal medicines, minerals, and the like.
本発明の炎症予防剤中の有効成分（特定アルキルレゾルシノール混合物）の含有量は、炎症予防剤の剤型、投与又は摂取する者の症状や年齢性別等によって適宜変化させることができ、人を対象とする場合、通常、本発明の炎症予防剤の有効成分の投与量又は摂取量が１人（６０ｋｇ換算で）１日当たり０．５〜５０００ｍｇとなるように含有させることが好ましい。 The content of the active ingredient (specific alkyl resorcinol mixture) in the inflammation preventive agent of the present invention can be appropriately changed depending on the dosage form of the inflammation preventive agent, the symptom or age sex of the person to be administered or ingested, etc. In general, the dosage or intake of the active ingredient of the inflammation-preventing agent of the present invention is preferably contained so that it is 0.5 to 5000 mg per person (in terms of 60 kg) per day.
本発明の炎症予防剤は、人又は動物に直接投与若しくは摂取させても良いし、又は飲食品若しくは動物用飼料に添加する等して間接的に投与又は摂取させても良い。後者の場合、炎症予防剤の添加対象である飲食品としては、パン類、麺類、タブレット、キャンディー等の菓子類、清涼飲料、ジュース、栄養ドリンク等の飲料等が挙げられるが、これらに限定されるものではなく、また、炎症予防剤の飲食品又は動物用飼料への添加時期も、特に制限されるものではなく、飲食品又は動物用飼料の製造工程中に添加しても良く、製造された飲食品又は動物用飼料に添加しても良い。 The inflammation preventive agent of the present invention may be directly administered or ingested to a human or animal, or may be indirectly administered or ingested by adding it to food or drink or animal feed. In the latter case, foods and drinks to which the inflammation preventive agent is added include confectionery such as breads, noodles, tablets, and candy, beverages such as soft drinks, juices, and nutritional drinks, but are not limited thereto. In addition, the timing of addition of the inflammation preventive agent to the food or drink or animal feed is not particularly limited, and may be added during the production process of the food or drink or animal feed. It may be added to food and drink or animal feed.
本発明の炎症予防剤は、特に、高脂肪食に添加して人又は動物に摂取させるのが効果的である。高脂肪食としては、例えば、動物用飼料として販売されているＤ１２４９２（脂肪含量３０質量％、リサーチダイエット社製）が挙げられる。 The inflammation preventive agent of the present invention is particularly effective when it is added to a high fat diet and ingested by a human or animal. Examples of the high-fat food include D12492 (fat content 30% by mass, manufactured by Research Diet) sold as animal feed.
以下、実施例及び試験例を挙げて、本発明を更に詳細に説明するが、本発明はこれらの実施例及び試験例により制限されるものではない。 EXAMPLES Hereinafter, although an Example and a test example are given and this invention is demonstrated further in detail, this invention is not restrict | limited by these Examples and a test example.
〔実施例〕
下記＜抽出精製法＞により、小麦ふすま（イネ科植物種子）から特定アルキルレゾルシノール混合物を得、これを炎症予防剤とした。小麦ふすまから得られた特定アルキルレゾルシノール混合物（実施例の炎症予防剤）の組成は次の通り。
・１，３−ジヒドロキシ−５−ｎ−ペンタデシルベンゼン（Ｃ１５：０）１．２質量％。
・１，３−ジヒドロキシ−５−ｎ−ヘプタデシルベンゼン（Ｃ１７：０）１０．９質量％。
・１，３−ジヒドロキシ−５−ｎ−ノナデシルベンゼン（Ｃ１９：０）３３．９質量％。
・１，３−ジヒドロキシ−５−ｎ−ヘンイコシルベンゼン（Ｃ２１：０）４６．４質量％。
・１，３−ジヒドロキシ−５−ｎ−トリコシルベンゼン（Ｃ２３：０）７．５質量％。
・１，３−ジヒドロキシ−５−ｎ−ペンタコシルベンゼン（Ｃ２５：０）０．１質量％。
〔Example〕
A specific alkylresorcinol mixture was obtained from wheat bran (Poaceae seed) by the following <Extraction and purification method>, and this was used as an inflammation preventive agent. The composition of the specific alkyl resorcinol mixture obtained from wheat bran (inflammation preventive agent in Examples) is as follows.
-1,3-dihydroxy-5-n-pentadecylbenzene (C15: 0) 1.2 mass%.
-1,3-dihydroxy-5-n-heptadecylbenzene (C17: 0) 10.9 mass%.
-1,3-dihydroxy-5-n-nonadecylbenzene (C19: 0) 33.9 mass%.
-1,3-dihydroxy-5-n-henicosylbenzene (C21: 0) 46.4 mass%.
-7.5 mass% of 1, 3- dihydroxy-5-n-tricosyl benzene (C23: 0).
-1,3-dihydroxy-5-n-pentacosylbenzene (C25: 0) 0.1 mass%.
＜抽出精製法＞
小麦ふすまに質量で５倍量のエタノールを添加して、６００ｒｐｍ、室温の条件で、１６時間撹拌抽出した。抽出物を濾過して不要物を除きエタノール抽出液を回収した後、エタノールを留去し、小麦ふすま（イネ科植物種子）のエタノール抽出物を得た。次いで、このエタノール抽出物を中圧クロマトグラフィーによって精製した。中圧クロマトグラフィー条件は下記の通りである。溶出開始後３１〜３６分に出現するピーク成分を回収して、溶媒留去し、目的とする特定アルキルレゾルシノール混合物を得た。
（中圧クロマトグラフィーの条件）
・カラム：シリカゲル（インジェクトカラム３Ｌ、ハイフラッシュカラム５Ｌ、６０Å、４０μｍ、山善株式会社製）
・移動相：ヘキサン／酢酸エチル混合溶媒（体積比）＝９０／１０にて９分、８０／２０にて１５分、６０／４０にて１６分
・検出波長：２５４ｎｍ
<Extraction purification method>
Ethanol in an amount of 5 times by mass was added to wheat bran, and the mixture was extracted by stirring for 16 hours at 600 rpm and room temperature. The extract was filtered to remove unnecessary substances and an ethanol extract was collected, and then the ethanol was distilled off to obtain an ethanol extract of wheat bran (Poaceae seed). The ethanol extract was then purified by medium pressure chromatography. Medium pressure chromatography conditions are as follows. The peak component appearing 31 to 36 minutes after the start of elution was collected, and the solvent was distilled off to obtain the desired specific alkylresorcinol mixture.
(Medium pressure chromatography conditions)
Column: silica gel (injection column 3 L, high flash column 5 L, 60 mm, 40 μm, manufactured by Yamazen Corporation)
・ Mobile phase: Hexane / ethyl acetate mixed solvent (volume ratio) = 9/10 at 90/10, 15 minutes at 80/20, 16 minutes at 60/40 ・ Detection wavelength: 254 nm
尚、上記＜抽出精製法＞におけるエタノール抽出物の精製は、中圧クロマトグラフィーに代えて、ＨＰＬＣによって行うこともできる。その場合、エタノール抽出物にメタノールを添加して該エタノール抽出物の濃度が２００ｕｇ／ｍｌのメタノール添加液を調製し、該メタノール添加液を、孔径０．４５μｍのフィルターを通過させ、その通過分を、ＨＰＬＣの試料とする。ＨＰＬＣの条件は下記の通り。
（ＨＰＬＣの条件）
・カラム：シリカゲル（ＯＤＳ−８０Ａ、５μｍ、４．６×２５０ｍｍ、ジーエルサイエンス株式会社製）
・ガードカラム：ＯＤＳ−８０Ａ、５μｍ、４．６×５０ｍｍ、
・カラム温度：３０℃
・移動相：メタノール１００％
・検出波長：２１５ｎｍ
The purification of the ethanol extract in <Extraction and purification method> can also be performed by HPLC instead of medium pressure chromatography. In that case, methanol is added to the ethanol extract to prepare a methanol addition solution having a concentration of 200 ug / ml, and the methanol addition solution is passed through a filter having a pore size of 0.45 μm. , HPLC sample. HPLC conditions are as follows.
(HPLC conditions)
Column: silica gel (ODS-80A, 5 μm, 4.6 × 250 mm, manufactured by GL Sciences Inc.)
Guard column: ODS-80A, 5 μm, 4.6 × 50 mm,
-Column temperature: 30 ° C
-Mobile phase: 100% methanol
・ Detection wavelength: 215 nm
〔試験例〕
実施例の炎症予防剤について、下記試験により、高脂肪食負荷マウスの体内での炎症性疾患への影響を調べた。その結果を図１〜図５に示す。図１〜図５中、「コントロール」は、普通食を摂餌したマウス群、「高脂肪食」は、高脂肪食を摂餌したマウス群、「ＡＲｓ」は、実施例の炎症予防剤（特定アルキルレゾルシノール混合物）が添加された高脂肪食を摂餌したマウス群である。尚、図１〜図４において、＊＊（アスタリスクが２つ）は、コントロールと高脂肪食との間における有意差が、有意水準ｐ＜０．０１で認められたことを示し、＊（アスタリスクが１つ）は、該有意差が、有意水準ｐ＜０．０５で認められたことを示す。また、＋＋（プラスが２つ）は、ＡＲｓと高脂肪食との間における有意差が、有意水準ｐ＜０．０１で認められたことを示し、＋（プラスが１つ）は、該有意差が、有意水準ｐ＜０．０５で認められたことを示す。
[Test example]
About the inflammation preventive agent of an Example, the influence on the inflammatory disease in the body of a high fat diet load mouse | mouth was investigated by the following test. The results are shown in FIGS. In FIG. 1 to FIG. 5, “control” is a group of mice fed with a normal diet, “high fat diet” is a group of mice fed with a high fat diet, and “ARs” is an inflammation preventive agent of Examples ( It is a group of mice fed a high fat diet to which a specific alkyl resorcinol mixture) was added. 1 to 4, ** (two asterisks) indicates that a significant difference between the control and the high fat diet was recognized at a significance level p <0.01, and ** (asterisk Indicates that the significant difference was recognized at the significance level p <0.05. Further, ++ (two pluses) indicates that a significant difference between ARs and the high fat diet was recognized at a significance level p <0.01, and + (one plus) represents the significance. The difference is observed at the significance level p <0.05.
＜血中遊離脂肪酸低下作用及び炎症性サイトカイン発現抑制作用の確認試験＞
MCP-1は単球・リンパ球の遊走を引き起こすケモカインであり、脂肪組織へのマクロファージの浸潤を誘導している。脂肪組織へ浸潤したマクロファージがTNF-αを産生し、それが脂肪細胞に作用すると、遊離脂肪酸の過剰な放出が誘導される。誘導された遊離脂肪酸がマクロファージに浸潤すると、TNF-α産生を更に亢進する。このようにMCP-1とTNF-αと遊離脂肪酸とによって形成される、脂肪組織とマクロファージとの相互作用（パラクリン調節系）が悪循環を形成し、慢性炎症が発症する。その結果、IL-6やIL-1βといった炎症性サイトカインが脂肪組織から産生され、血液に入って全身に送られる。これらが動脈硬化やがんを誘導するリスクを増強させている。炎症予防剤の摂取が脂肪組織の慢性炎症に与える影響を確認するために、実験用マウスを複数匹用い、これらを普通食摂取群と、高脂肪食摂取群と、高脂肪＋炎症予防剤摂取群との３群に分け、各群の血中遊離脂肪酸量、及び炎症性サイトカインの遺伝子発現量（MCP-1量、IL-6量、IL-1β量及びTNF-α量）をそれぞれ測定した。確認試験のより具体的な手順は次の通り。
<Confirmation test of blood free fatty acid lowering action and inflammatory cytokine expression inhibitory action>
MCP-1 is a chemokine that causes monocyte / lymphocyte migration, and induces macrophage infiltration into adipose tissue. Macrophages that infiltrate adipose tissue produce TNF-α, which acts on adipocytes, leading to excessive release of free fatty acids. When induced free fatty acids infiltrate macrophages, TNF-α production is further enhanced. Thus, the interaction (paracrine regulatory system) between adipose tissue and macrophages formed by MCP-1, TNF-α and free fatty acids forms a vicious circle, and chronic inflammation develops. As a result, inflammatory cytokines such as IL-6 and IL-1β are produced from adipose tissue and enter the blood and sent to the whole body. These increase the risk of inducing arteriosclerosis and cancer. In order to confirm the effects of intake of inflammation preventive agents on chronic inflammation of adipose tissue, several experimental mice were used, and these were consumed with normal diet intake group, high fat diet intake group, and high fat + inflammation preventive agent intake Divided into 3 groups, each group measured the amount of free fatty acid in blood and the expression level of inflammatory cytokine genes (MCP-1, IL-6, IL-1β and TNF-α). . The specific procedure for the confirmation test is as follows.
固形飼料として、普通食としてＤ１２４５０Ｂ（リサーチダイエット社製）、高脂肪食としてＤ１２４９２（リサーチダイエット社製）を用意し、また別途、高脂肪食であるＤ１２４９２に実施例の炎症予防剤（特定アルキルレゾルシノール混合物）を０．４質量％添加したもの（ＡＲｓ添加高脂肪食）を用意し、これらを滅菌した。C57BL/6系統のマウス（１６週齢の雄性、日本エスエルシー株式会社）を明期１２時間、暗期１２時間の明暗サイクル下（0:00点灯、12:00消灯）で３週間馴化飼育した後、これらのマウスを普通食摂餌群（６ケージ）と、高脂肪食摂餌群（６ケージ）と、ＡＲｓ添加高脂肪食摂餌群（６ケージ）との３群に分け、３週間自由摂食させた。自由摂食期間終了後、マウスを解剖し、血液及び精巣周辺の脂肪組織をサンプリングし、血中遊離脂肪酸量並びにMCP-1量、IL-6量、IL-1β量及びTNF-α量をそれぞれ測定した。血中遊離脂肪酸量の測定は、Free Fatty Acid Quantification Kit（BioVision K612-100）を用い、プロトコール通りに実施した。MCP-1、IL-6、IL-1β及びTNF-α量の遺伝子発現解析は下記解析法により行った。 As a solid feed, D12450B (manufactured by Research Diet) is prepared as a normal food, and D12492 (manufactured by Research Diet) is prepared as a high-fat food. What added 0.4 mass% (mixture) (ARs addition high fat food) was prepared, and these were sterilized. C57BL / 6 strain mice (16-week-old male, Japan SLC Co., Ltd.) were bred for 3 weeks under a light / dark cycle of 12 hours light period and 12 hours dark period (lights on at 0:00, lights off at 12:00). Thereafter, these mice were divided into three groups, a normal diet group (6 cages), a high fat diet group (6 cages), and an ARs-added high fat diet group (6 cages) for 3 weeks. Ad libitum. At the end of the free feeding period, the mouse was dissected, the blood and the fat tissue around the testis were sampled, and the amount of free fatty acids in the blood and the amounts of MCP-1, IL-6, IL-1β and TNF-α were measured. It was measured. Measurement of the amount of free fatty acid in blood was carried out according to the protocol using the Free Fatty Acid Quantification Kit (BioVision K612-100). Gene expression analysis of MCP-1, IL-6, IL-1β and TNF-α levels was performed by the following analysis method.
＜MCP-1、IL-6、IL-1β及びTNF-αの遺伝子発現解析法＞
１．RNA抽出：精巣周辺脂肪組織を100mg測り、RNeasy Lipid Tissue MiniKit (Qiagen 74804)及びRNase-Free DNase set (Qiagen 79254) を用い、プロトコール通りにRNAを抽出した。Nano Drop ND-1000（Thermo Fisher Scientific）を用いて吸光度の測定を行い、260/280が2.0未満若しくは260/230が1.5未満のサンプルは、RNeasy MinElute Cleanup Kit (Qiagen 74204)を使用して精製を行った。
２．逆転写反応：こうして抽出したRNAを鋳型に、サーマルサイクラーを用いて、「３０℃１０分→４２℃４０分→９９℃５分」の条件で逆転写反応を行い、cDNA合成反応液を作成した。逆転写反応の反応条件を下記表１に示す。
３．ＰＣＲ反応：このcDNA合成反応液を用い、リアルタイムPCR機（7900HT：Applied Biosystems）を使用して、「５０℃、２分→９５℃、１０分→〔９５℃、１５秒→６０℃、１分〕×４５サイクル」の条件で遺伝子発現解析を行った（N=3）。ＰＣＲ反応の反応条件を下記表２に示す。プライマーは以下の配列のβ-Actinを用いた。そして、β-Actinと標的遺伝子のスレッシュホールドライン（0.256）でのCt値とを算出して次式に当てはめ、標的遺伝子（MCP-1、IL-6、IL-1β及びTNF-α）の発現量を算出した。
相対的遺伝子発現量＝2^（β-ActinのCt値−標的遺伝子のCt値）
<Method for analyzing gene expression of MCP-1, IL-6, IL-1β and TNF-α>
1. RNA extraction: 100 mg of testicular adipose tissue was measured, and RNA was extracted according to the protocol using RNeasy Lipid Tissue MiniKit (Qiagen 74804) and RNase-Free DNase set (Qiagen 79254). Absorbance is measured using Nano Drop ND-1000 (Thermo Fisher Scientific) and samples with 260/280 less than 2.0 or 260/230 less than 1.5 are purified using the RNeasy MinElute Cleanup Kit (Qiagen 74204). went.
2. Reverse transcription reaction: Using the extracted RNA as a template, a reverse cycle reaction was performed using a thermal cycler under the conditions of “30 ° C. 10 minutes → 42 ° C. 40 minutes → 99 ° C. 5 minutes” to prepare a cDNA synthesis reaction solution. . The reaction conditions for the reverse transcription reaction are shown in Table 1 below.
3. PCR reaction: Using this cDNA synthesis reaction solution, using a real-time PCR machine (7900HT: Applied Biosystems), “50 ° C., 2 minutes → 95 ° C., 10 minutes → [95 ° C., 15 seconds → 60 ° C., 1 minute] ] × 45 cycles ”was performed for gene expression analysis (N = 3). The reaction conditions for the PCR reaction are shown in Table 2 below. The primer used was β-Actin having the following sequence. Then, β-Actin and the Ct value at the target gene threshold line (0.256) are calculated and applied to the following formula to express the target genes (MCP-1, IL-6, IL-1β and TNF-α) The amount was calculated.
Relative gene expression = 2 ^ (β-Actin Ct value-Target gene Ct value)
・β-Actin：
Forward 5'-CGTCATTGCACGAAGACACAA-3'
Reverse 5'-CCTGGTCCACCATTTTAAGGC-3'
・MCP-1(monocytechemoattractant protein-1) ：
Forward 5'-CCACTCACCTGCTGCTACTCAT-3'
Reverse 5'-TGGTGATCCTCTTGTAGCTCTCC-3'
・IL-6(interleukin-6)：
Forward 5'-ACAACCACGGCCTTCCCTACTT-3'
Reverse 5'-CACGATTTCCCAGAGAACATGTG-3'
・IL-1β(interleukin-1β)：
Forward 5'-CACAGGAGCAACGACAAAATACCTGTG-3'
Reverse 5'-TCTTCTTTGGGTATTGCTTGG-3'
・TNF-α(tumor necreosis factor-α)：
Forward 5'-ACCCTCACACTCAGATCATCTTC-3'
Reverse 5'-TGGTGGTTTGCTACGACGT-3'
・ Β-Actin:
Forward 5'-CGTCATTGCACGAAGACACAA-3 '
Reverse 5'-CCTGGTCCACCATTTTAAGGC-3 '
・ MCP-1 (monocytechemoattractant protein-1):
Forward 5'-CCACTCACCTGCTGCTACTCAT-3 '
Reverse 5'-TGGTGATCCTCTTGTAGCTCTCC-3 '
・ IL-6 (interleukin-6):
Forward 5'-ACAACCACGGCCTTCCCTACTT-3 '
Reverse 5'-CACGATTTCCCAGAGAACATGTG-3 '
・ IL-1β (interleukin-1β):
Forward 5'-CACAGGAGCAACGACAAAATACCTGTG-3 '
Reverse 5'-TCTTCTTTGGGTATTGCTTGG-3 '
・ TNF-α (tumor necreosis factor-α):
Forward 5'-ACCCTCACACTCAGATCATCTTC-3 '
Reverse 5'-TGGTGGTTTGCTACGACGT-3 '
図１から明らかなように、高脂肪食摂餌群は、普通食摂餌群（コントロール）に比べて、血中遊離脂肪酸量が有意に増加しており、このことから、高脂肪食の摂取が、血中遊離脂肪酸量の増加を促進し、遊離脂肪酸が脂肪以外の組織でも炎症シグナルを活性化し、これに起因する炎症性疾患（例えば慢性の炎症性疾患である動脈硬化）を引き起こす原因となることが明らかである。しかし、このような炎症性疾患の原因となり得る高脂肪食に実施例の炎症予防剤を所定量添加した飼料を摂餌した群（ＡＲｓ）は、高脂肪食摂餌群に比べて血中遊離脂肪酸量が少なく、コントロールのそれと略同レベルであった。これにより、実施例の炎症予防剤による血中遊離脂肪酸低下作用が確認され、該炎症予防剤により、他の臓器での炎症シグナルの活性化が予防された。 As is clear from FIG. 1, the amount of free fatty acid in the blood was significantly increased in the high-fat diet group compared to the normal diet group (control). However, it promotes an increase in the amount of free fatty acids in the blood, and free fatty acids activate inflammatory signals in tissues other than fat and cause inflammatory diseases (for example, arteriosclerosis, which is a chronic inflammatory disease). It is clear that However, the group (ARs) that fed a high-fat diet that could cause such inflammatory diseases with a feed in which a predetermined amount of the inflammation preventive agent of the example was added was more free in the blood than the high-fat diet group. The amount of fatty acid was low, almost the same as that of the control. Thereby, the blood free fatty acid lowering action by the inflammation preventive agent of the Example was confirmed, and the inflammation preventive agent prevented activation of inflammatory signals in other organs.
また、図２及び図３から明らかなように、高脂肪食摂餌群は、普通食摂餌群（コントロール）に比べて、炎症性サイトカインであるMCP-1及びIL-6の発現量が有意に増加しており、このことから、高脂肪食の摂取が、炎症性サイトカイン発現を上昇させ、炎症性サイトカインが脂肪以外の組織でも炎症シグナルを活性化し、これに起因する炎症性疾患を引き起こす原因となることが明らかである。しかし、ＡＲｓは、高脂肪食摂餌群に比べて炎症性サイトカイン発現量が少なく、コントロールのそれと略同レベルであった。これにより、実施例の炎症予防剤による炎症性サイトカイン発現抑制作用が確認され、該炎症予防剤により、他の臓器での炎症シグナルの活性化が予防された。また、IL-1β及びTNF-αにおいては統計的な有意差は得られなかったものの、図４及び図５から明らかなように、同じく炎症性サイトカインであるMCP-1及びIL-6と同じ挙動を示した。 As is clear from FIG. 2 and FIG. 3, the high-fat diet-fed group has significant expression levels of inflammatory cytokines MCP-1 and IL-6 compared to the normal-fed diet group (control) As a result, ingestion of a high-fat diet increases inflammatory cytokine expression and causes inflammatory cytokines to activate inflammatory signals in tissues other than fat and cause inflammatory diseases resulting therefrom. It is clear that However, ARs had less inflammatory cytokine expression than the high-fat diet group, and were at the same level as that of the control. Thereby, the inflammatory cytokine expression inhibitory effect by the inflammation preventive agent of an Example was confirmed, and activation of the inflammatory signal in another organ was prevented by this inflammation preventive agent. In addition, although no statistically significant difference was obtained in IL-1β and TNF-α, the same behavior as MCP-1 and IL-6, which are also inflammatory cytokines, is evident from FIGS. 4 and 5. showed that.
尚、実施例の炎症予防剤について、下記解析法により、アディポネクチン分泌促進作用の有無を調べた。その結果を図６に示す。図６中の「コントロール」、「高脂肪食」及び「ＡＲｓ」はそれぞれ上記の通りである。図６から明らかなように、３つの群の間でアディポネクチン分泌量に有意な差は見られず、このことから、実施例の炎症予防剤はアディポネクチン分泌促進作用を有しておらず、特許文献１に記載の如きアディポネクチン分泌促進剤とは作用効果が異なるものであることが明らかである。 In addition, about the inflammation preventive agent of an Example, the presence or absence of the adiponectin secretion promoting effect was investigated by the following analysis method. The result is shown in FIG. “Control”, “high fat diet”, and “ARs” in FIG. 6 are as described above. As is clear from FIG. 6, there is no significant difference in the amount of adiponectin secreted among the three groups. From this, the inflammation preventive agents of the examples do not have an adiponectin secretion promoting action. It is clear that the effect is different from the adiponectin secretion promoter as described in 1.
＜アディポネクチンの遺伝子発現解析法＞
上記＜MCP-1、IL-6、IL-1β及びTNF-αの遺伝子発現解析法＞と同様に、RNA抽出、逆転写反応及びＰＣＲ反応を順次実施し、標的遺伝子（Adpn）の発現量を算出した。
・β-Actin：
Forward 5'-CGTCATTGCACGAAGACACAA-3'
Reverse 5'-CCTGGTCCACCATTTTAAGGC-3'
・Adpn（Adiponectin）：
Forward 5'-ATGGCAGAGATGGCACTCCT-3'
Reverse 5'-CCTTCAGCTCCTGTCATTCCA-3'
<Adiponectin gene expression analysis method>
In the same manner as in the above <Method for analyzing gene expression of MCP-1, IL-6, IL-1β and TNF-α>, RNA extraction, reverse transcription reaction and PCR reaction were sequentially performed, and the expression level of the target gene (Adpn) was determined. Calculated.
・ Β-Actin:
Forward 5'-CGTCATTGCACGAAGACACAA-3 '
Reverse 5'-CCTGGTCCACCATTTTAAGGC-3 '
・ Adpn (Adiponectin):
Forward 5'-ATGGCAGAGATGGCACTCCT-3 '
Reverse 5'-CCTTCAGCTCCTGTCATTCCA-3 '
Claims (3)
上記特定アルキルレゾルシノール混合物は、１）下記一般式（Ｉ）におけるＲ 1 が炭素原子数１５の飽和又は不飽和のアルキル基であるアルキルレゾルシノールを０．１〜１０．０質量％、２）下記一般式（Ｉ）におけるＲ 1 が炭素原子数１７の飽和又は不飽和のアルキル基であるアルキルレゾルシノールを１．０〜２０．０質量％、３）下記一般式（Ｉ）におけるＲ 1 が炭素原子数１９の飽和又は不飽和のアルキル基であるアルキルレゾルシノールを２５．０〜４０．０質量％、４）下記一般式（Ｉ）におけるＲ 1 が炭素原子数２１の飽和又は不飽和のアルキル基であるアルキルレゾルシノールを４０．０〜５５．０質量％、５）下記一般式（Ｉ）におけるＲ 1 が炭素原子数２３の飽和又は不飽和のアルキル基であるアルキルレゾルシノールを１．０〜１５．０質量％、及び６）下記一般式（Ｉ）におけるＲ 1 が炭素原子数２５の飽和又は不飽和のアルキル基であるアルキルレゾルシノールを０〜５．０質量％含有する炎症予防剤。
The specific alkyl resorcinol mixture is 1) 0.1 to 10.0% by mass of alkyl resorcinol in which R 1 in the following general formula (I) is a saturated or unsaturated alkyl group having 15 carbon atoms, and 2) 1.0 to 20.0% by mass of alkylresorcinol in which R 1 in formula (I) is a saturated or unsaturated alkyl group having 17 carbon atoms, 3) R 1 in the following general formula (I) is the number of carbon atoms 25.0 to 40.0% by weight of alkyl resorcinol 19 is a saturated or unsaturated alkyl group of, 4) R 1 in the general formula (I) below is a saturated or unsaturated alkyl group having a carbon number of 21 4) 5 to 55.0% by mass of alkylresorcinol, 5) an alkylresorcinol in which R 1 in the following general formula (I) is a saturated or unsaturated alkyl group having 23 carbon atoms 1.0 to 15.0% by mass, and 6) inflammation containing 0 to 5.0% by mass of alkylresorcinol in which R 1 in the following general formula (I) is a saturated or unsaturated alkyl group having 25 carbon atoms Preventive agent.
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