JP7565055B2 - Defensin and interleukin production inducer composition, medicines, quasi-drugs, cosmetics, foods, beverages - Google Patents
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Description
本発明は、ディフェンシン及びインターロイキンの産生誘導剤組成物、医薬品、医薬部外品、化粧料、食品、飲料に関する。 The present invention relates to a defensin and interleukin production inducer composition, a pharmaceutical product, a quasi-drug product, a cosmetic product, a food product, and a beverage.
ヒトを含めた哺乳類、植物、昆虫、両性類等の生物に生来備わっている生体防御機構（自然免疫）の１つとして、生体内の抗菌物質、特に抗菌ペプチドの存在が知られている。哺乳類における抗菌ペプチドは、ディフェンシンと総称され、真性細菌、真菌類、ウィルス等に対して抗菌活性を有している。哺乳類のディフェンシンは、α－ディフェンシン、β－ディフェンシン、θ－ディフェンシンの３つのファミリーからなる。この中でも皮膚、肺、器官、腎臓、生殖器当の粘膜上皮に発現するβ－ディフェンシンは、感染防御や獲得免疫に関与するだけでなく、腫瘍免疫を誘導して、抗腫瘍効果を発揮することやがん細胞の増殖抑制作用があることも解明されている。 The presence of antibacterial substances, particularly antibacterial peptides, in the body is known as one of the biological defense mechanisms (natural immunity) that are innate to organisms such as mammals (including humans), plants, insects, and amphibians. Antibacterial peptides in mammals are collectively called defensins, and have antibacterial activity against bacteria, fungi, viruses, etc. Mammalian defensins consist of three families: α-defensin, β-defensin, and θ-defensin. Among these, β-defensin, which is expressed in the mucosal epithelium of the skin, lungs, organs, kidneys, and reproductive organs, is not only involved in infection defense and acquired immunity, but has also been elucidated to induce tumor immunity, exert an anti-tumor effect, and inhibit the proliferation of cancer cells.
また、感染や疾患に対する身体の自然な反応を改善する物質として、サイトカイン(生物学的反応修飾物質)が知られている。サイトカインの一種であるインターロイキンは、腫瘍細胞を攻撃する免疫系の機能を高める作用があるほか、腫瘍への血流を妨げる可能性もある。この種のサイトカインは通常は体内で作られている。このインターロイキンの中でも、インターロイキン－１２（IL-12）は、ナチュラルキラー細胞（ＮＫ細胞）を活性化してＩＦＮ－γの産生を促進するとともに、Ｔｈ１細胞の分化を誘導することから、感染防御や抗がん療法、免疫不全症の改善における臨床応用が期待されている。 Cytokines (biological response modifiers) are also known to improve the body's natural response to infection and disease. Interleukins, a type of cytokine, enhance the immune system's ability to attack tumor cells and may also impede blood flow to tumors. This type of cytokine is normally produced in the body. Among these interleukins, interleukin-12 (IL-12) activates natural killer cells (NK cells) to promote the production of IFN-γ and induces differentiation of Th1 cells, making it promising for clinical applications in infection defense, anti-cancer therapy, and the improvement of immunodeficiency.
免疫の仕組みとしては、まず、ディフェンシンが、鼻や口、腸などの粘膜から病原体が体内に侵入するのを抑制する。病原体が体内に侵入した場合には、インターロイキンが誘導され、病原体を攻撃する。したがって、ディフェンシンやサイトカインの直接的な抗菌活性や免疫機能を強化し、さらには抗腫瘍効果やがん細胞の増殖抑制作用をより強化するべく、生体内でのディフェンシンやサイトカインの産生を促進することができる技術の開発が切望されている。 In terms of immune system mechanisms, first, defensins inhibit pathogens from entering the body through mucous membranes such as the nose, mouth, and intestines. When pathogens enter the body, interleukins are induced to attack the pathogens. Therefore, there is a strong need for the development of technology that can promote the production of defensins and cytokines in the body, in order to strengthen the direct antibacterial activity and immune function of defensins and cytokines, and further enhance their antitumor effects and their ability to inhibit the proliferation of cancer cells.
ところで、甘酒の抽出物、焼酎醪、米抽出物の発酵物等を有効成分とする組成物を用いてヒトβ－ディフェンシンの産生を促進する技術が開示されている（例えば、特許文献１、２参照）。また、乳酸菌を用いてインターロイキンの産生を促進する技術が開示されている（例えば、特許文献３、４参照）。 Incidentally, a technique for promoting the production of human β-defensin using a composition containing as active ingredients an extract of sweet sake, shochu mash, a fermented product of rice extract, etc. has been disclosed (see, for example, Patent Documents 1 and 2). Also, a technique for promoting the production of interleukin using lactic acid bacteria has been disclosed (see, for example, Patent Documents 3 and 4).
しかしながら、上記従来技術は、ディフェンシン又はインターロイキンの何れか一方の産生を促進する技術であり、双方の産生を促進する技術は未だ開示がなく、抗腫瘍効果やがん細胞の増殖抑制作用において、必ずしも満足し得るものではなかった。 However, the above-mentioned conventional technologies promote the production of either defensins or interleukins, and no technology has been disclosed that promotes the production of both, and therefore the antitumor effects and cancer cell proliferation inhibitory effects have not been entirely satisfactory.
本発明は、上記の事情に鑑みて為されたもので、ディフェンシン及びインターロイキンの産生を、より効果的に誘導することを目的とする。 The present invention was made in consideration of the above circumstances, and aims to more effectively induce the production of defensins and interleukins.
本発明者らは、上記の目的を達成するため鋭意研究を重ねた結果、ヒト由来の上皮細胞に酒かすを添加することで、ディフェンシン及びインターロイキン、特にヒトβ－ディフェンシン－２及びヒトインターロイキン－１２の産生を誘導できることを知見し、本発明を完成するに至った。 As a result of extensive research to achieve the above object, the inventors discovered that the addition of sake lees to human-derived epithelial cells can induce the production of defensins and interleukins, particularly human β-defensin-2 and human interleukin-12, and thus completed the present invention.
即ち、本発明は、次の（１）～（４）に示す通りである。
（１）酒かす、酒かす粉末又はこれらの抽出物を有効成分とする、ディフェンシン及びインターロイキンの産生誘導剤組成物。
（２）ディフェンシンが、ヒトβ－ディフェンシン－２であり、インターロイキンがヒトインターロイキン－１２である、前記（１）に記載のディフェンシン及びインターロイキンの産生誘導剤組成物。
（３）酒かす、酒かす粉末又は抽出物が、加熱殺菌されたものである、前記（１）又は（２）に記載の産生誘導剤組成物。
（４）前記（１）～（３）のいずれかに記載の産生誘導剤組成物を含有する、ディフェンシン及びインターロイキンの産生誘導用の医薬品、医薬部外品、化粧料、食品又は飲料。
（５）がん抑制用、抗感染症用、又は免疫不全改善用である、前記（４）に記載の医薬品、医薬部外品、化粧料、食品又は飲料化粧料。
That is, the present invention is as shown in the following (1) to (4).
(1) A composition for inducing the production of defensins and interleukins, comprising sake lees, sake lees powder, or extracts thereof as active ingredients.
(2) The defensin and interleukin production inducer composition described in (1) above, wherein the defensin is human β-defensin-2 and the interleukin is human interleukin-12.
(3) The production inducer composition according to (1) or (2), wherein the sake lees, sake lees powder or extract is heat-sterilized.
(4) A pharmaceutical, quasi-drug, cosmetic, food, or beverage for inducing the production of defensins and interleukins, comprising the production-inducing composition according to any one of (1) to (3) above.
(5) The pharmaceutical, quasi-drug, cosmetic, food or beverage cosmetic according to (4) above, which is for use in suppressing cancer, combating infectious diseases, or improving immunodeficiency.
本発明によれば、酒かす、酒かす粉末又はこれらの抽出物によって、ディフェンシンｍＲＮＡ及びインターロイキンｍＲＮＡの発現が誘導される。その結果、ディフェンシン及びインターロイキンの産生を、より効果的に誘導することができる。 According to the present invention, the expression of defensin mRNA and interleukin mRNA is induced by sake lees, sake lees powder, or extracts thereof. As a result, the production of defensins and interleukins can be more effectively induced.
以下、本発明の一実施の形態について、詳細に説明する。本実施の形態であるディフェンシン及びインターロイキンの産生誘導剤組成物は、酒かす、酒かす粉末又はこれらの抽出物を有効成分とする。また、本実施の形態であるディフェンシン及びインターロイキンの産生誘導用の医薬品、医薬部外品、化粧料、食品又は飲料は、本実施の形態である産生誘導剤組成物を含有する。 One embodiment of the present invention will be described in detail below. The defensin and interleukin production inducer composition of this embodiment contains sake lees, sake lees powder, or an extract of these as an active ingredient. Furthermore, pharmaceuticals, quasi-drugs, cosmetics, foods, or beverages for inducing the production of defensins and interleukins of this embodiment contain the production inducer composition of this embodiment.
酒かす（酒粕、酒糟）とは、米、麹、水を発酵させた日本酒などの醪、又は米、麹、水で発酵させた醪に対して、醸造アルコール、糖類酸味料などを添加した後、圧搾機などで搾った後の残渣であり、本明細書では、水分を含んでウェットな状態のものをいう。このような酒かすとしては、醪を圧搾した後に残る固形物（板かす）であってもよいし、醪を袋絞りした後に残る液体物であってもよい。 Sake lees (sake lees, sake lees) refers to the residue left over from mash such as sake lees made by fermenting rice, koji, and water, or from mash fermented with rice, koji, and water to which brewer's alcohol, sugars, acidulants, etc. have been added, and then squeezed out with a press, etc. In this specification, it refers to the wet state containing moisture. Such sake lees may be the solid matter (slabs of sake lees) that remains after the mash is squeezed, or the liquid matter that remains after the mash is squeezed out of a bag.
酒かすは、水分及びアルコール分（液体成分）を含有している。液体成分の含有量としては、固形物（板粕）の場合は、例えば３０％～６０％であり、液体物の場合は、例えば５０％～８０％であるが、この範囲に限定されることはない。酒かすのアルコール度数は、日本酒の造り方や蔵元によって異なり、一般的には６％～１４％（６度～１４度）であるが、この範囲に限定されることはない。酒かすには、水分、アルコール分のほかに、炭水化物、蛋白質、脂質、灰分、ペプチド、アミノ酸、ビタミン、ミネラル、酵母などの各種栄養素が含有されている。なお、日本酒の醸造工程で、微量の乳酸菌が混入する場合があるが、ほとんどはアルコール分で死滅するため、酒かす中には乳酸菌は殆ど含有されない。通常の酒かす中の乳酸菌の含有量は、３００個／ｇ以下が好ましく、３０個／ｇ以下がより好ましい。これに対して、いわゆる「生もと造り」、「山廃造り」のような伝統的な方法で造られた日本酒では、雑菌を除去すべく、蔵付乳酸菌を増殖させた造りや乳酸菌を添加した造りをしていることから、その酒かすには乳酸菌が比較的多く含まれている。本実施の形態では、「生もと造り」、「山廃造り」の酒かすを用いてもよいが、これらの方法ではない、通常の方法で作られた日本酒（吟醸酒、純米酒、醸造酒等）の酒かすを好適に用いることができる。「純米酒」は、白米、米こうじ及び水のみを原料として製造した清酒である。「醸造酒」は、純米酒に醸造アルコールを加えて製造した清酒である。「吟醸酒」は、純米酒及び醸造酒の中で、米を６０％以下に精米して低温発酵する吟醸造りという手法で製造した清酒である。「普通酒」は、純米酒、本醸造酒、吟醸酒等の特定名称酒以外の清酒である。 Sake lees contain water and alcohol (liquid components). The content of the liquid components is, for example, 30% to 60% in the case of solids (sheet lees) and 50% to 80% in the case of liquids, but is not limited to these ranges. The alcohol content of sake lees varies depending on the sake brewing method and the brewery, and is generally 6% to 14% (6% to 14%), but is not limited to this range. In addition to water and alcohol, sake lees contain various nutrients such as carbohydrates, proteins, lipids, ash, peptides, amino acids, vitamins, minerals, and yeast. Although a small amount of lactic acid bacteria may be mixed in during the sake brewing process, most of them are killed by the alcohol, so there is almost no lactic acid bacteria in the sake lees. The content of lactic acid bacteria in normal sake lees is preferably 300 cells/g or less, and more preferably 30 cells/g or less. In contrast, sake made using traditional methods such as the so-called "kimoto" and "yamahai" methods is made by multiplying brewery-specific lactic acid bacteria or adding lactic acid bacteria to remove unwanted bacteria, so the sake lees contain a relatively large amount of lactic acid bacteria. In this embodiment, sake lees from "kimoto" and "yamahai" methods may be used, but sake lees from sake made using normal methods other than these (ginjo sake, junmai sake, brewed sake, etc.) can be used preferably. "Junmai sake" is sake made using only white rice, rice malt, and water as ingredients. "Brewed sake" is sake made by adding brewing alcohol to junmai sake. "Ginjo sake" is sake made using the ginjo brewing method, which is a type of junmai sake and brewed sake, in which rice is polished to 60% or less and fermented at a low temperature. "Ordinary sake" is sake other than specified name sake such as junmai sake, honjozo sake, and ginjo sake.
酒かす粉末（酒かすパウダー）は、酒かすを各乾燥装置（スプレードライ、フリーズドライ、ダブルドラム）を用いて、適切な温度（－３０℃から１５０℃）で乾燥させた乾燥物であり、本乾燥物を微粉砕した粉末である。酒かす粉末の水分量は、１０．０％以下が好ましく、５．０％以下がより好ましいが、この範囲に限定されることはない。また、酒かす粉末のアルコール度数は、１．０％以下が好ましく、０．１％以下がより好ましいが、この範囲に限定されることはない。また、酒かす粉末の製造に使用する酒かすは、普通酒、純米酒、本醸造酒、吟醸酒等の種類に限定されることはなく、いずれを使用してもよい。 Sake lees powder is a dried product made by drying sake lees at an appropriate temperature (-30°C to 150°C) using various drying devices (spray drying, freeze drying, double drum), and then pulverizing this dried product into a powder. The moisture content of the sake lees powder is preferably 10.0% or less, and more preferably 5.0% or less, but is not limited to this range. The alcohol content of the sake lees powder is preferably 1.0% or less, and more preferably 0.1% or less, but is not limited to this range. The sake lees used to produce the sake lees powder are not limited to the type of sake, such as ordinary sake, junmai sake, honjozo sake, or ginjo sake, and any type may be used.
酒かす又は酒かす粉末の抽出物としては、例えば、酒化し又は酒かす粉末を、精製水に懸濁し、遠心することによって得られる上清（抽出液）、又はその分画等が挙げられるが、これに限定されることはない。また、上記酒かす、酒かす粉末、抽出物は、非加熱であってもよいし、加熱殺菌されたものであってもよい。加熱殺菌されたものでは、経口摂取する製品、特に食品や飲料に好適に用いることができる。また、加熱殺菌されたものでは、アルコールがほぼ除去されるとともに、乳酸菌等がほぼ死滅する。このため、本実施形態の産生誘導剤組成物によるディフェンシン及びインターロイキンの産生誘導効果に、乳酸菌が影響することはない。 Examples of extracts of sake lees or sake lees powder include, but are not limited to, the supernatant (extract) obtained by suspending sake or sake lees powder in purified water and centrifuging, or a fraction thereof. The sake lees, sake lees powder, and extracts may be unheated or heat-sterilized. Heat-sterilized products are suitable for use in products to be taken orally, particularly foods and beverages. Heat-sterilized products have almost all of the alcohol removed and the lactic acid bacteria and the like almost killed. For this reason, the lactic acid bacteria do not affect the defensin and interleukin production-inducing effect of the production inducer composition of this embodiment.
ディフェンシンとしては、特に限定されることはなく、α－ディフェンシン、β－ディフェンシン、θ－ディフェンシンのいずれのファミリーに属するものであってもよい。この中でも、本実施の形態は、ヒトβ－ディフェンシンの産生の誘導、及びヒトβディフェンシンｍＲＮＡの発現の誘導に特に好適であり、更にはヒトβ－ディフェンシン－２の産生の誘導、及びヒトディフェンシン－２ｍＲＮＡの発現の誘導に最適である。 The defensin is not particularly limited, and may belong to any of the α-defensin, β-defensin, and θ-defensin families. Among these, this embodiment is particularly suitable for inducing the production of human β-defensin and the expression of human β-defensin mRNA, and is further optimal for inducing the production of human β-defensin-2 and the expression of human defensin-2 mRNA.
インターロイキンとしては、特に限定されることはないが、本実施の形態は、ＮＫ細胞を活性化してIFN-γの産生を促進し、かつナイーブＴ細胞のＴｈ１細胞への分化を誘導することで、感染防御や抗がん療法、免疫不全症の改善における臨床応用に期待されることから、ヒトインターロイキン－１２の産生の誘導、及びヒトインターロイキン－１２ｍＲＮＡの発現の誘導に特に好適である。 The interleukin is not particularly limited, but this embodiment is particularly suitable for inducing the production of human interleukin-12 and the expression of human interleukin-12 mRNA, since it is expected to be used in clinical applications in infection prevention, anti-cancer therapy, and improvement of immunodeficiency by activating NK cells to promote the production of IFN-γ and inducing the differentiation of naive T cells into Th1 cells.
また、酒かす、酒かす粉末又はこれらの抽出物を主成分とする産生誘導剤組成物によって刺激される細胞としては、特に限定されることはなく、ディフェンシン及びインターロイキンを分泌する細胞であればよい。より具体的には、例えば、ヒト上皮細胞が好ましく、この中でも、免疫細胞が多く存在し、免疫において極めて重要な機能を有している点で、ヒト小腸上皮細胞を刺激することがより好ましい。この場合、酒かす、酒かす粉末又はこれらの抽出物を、終濃度０．１～１．０mg/mLとなるように５％ＦＢＳ（ウシ胎児血清：Fetal Bovine Serum）等で調整し、その調整物を細胞に添加することが好ましい。 The cells stimulated by the production inducer composition containing sake lees, sake lees powder, or an extract thereof as the main component are not particularly limited, and may be any cells that secrete defensins and interleukins. More specifically, for example, human epithelial cells are preferred, and among these, it is more preferred to stimulate human small intestinal epithelial cells, since they contain many immune cells and have extremely important immune functions. In this case, it is preferred to adjust the sake lees, sake lees powder, or an extract thereof with 5% FBS (fetal bovine serum) or the like to a final concentration of 0.1 to 1.0 mg/mL, and add the adjusted product to the cells.
本実施の形態におけるディフェンシン及びインターロイキンの発現レベルを定量するため、酒かす、酒かす粉末又はこれらの抽出物を添加する細胞として、培養細胞を用いることができる。このような培養細胞は、ＡＴＣＣなどの細胞バンクに保管されている既知の培養細胞又はその突然変異株を用いることができる。また、培養細胞として、例えば、ヒト大腸がん細胞であるＣａｃｏ２を培養し、分化させることによって作製したヒト小腸上皮細胞を好適に用いることができる。 In order to quantify the expression levels of defensins and interleukins in this embodiment, cultured cells can be used as cells to which sake lees, sake lees powder, or extracts thereof are added. As such cultured cells, known cultured cells stored in a cell bank such as ATCC or mutant strains thereof can be used. In addition, as cultured cells, for example, human small intestinal epithelial cells prepared by culturing and differentiating Caco2, which is a human colon cancer cell, can be preferably used.
本実施の形態においては、上記のような培養細胞に、酒かす、酒かす粉末又はこれらの抽出物を添加し、ディフェンシン及びインターロイキンの発現レベルの変化を、ＲＴ－ＰＣＲ法によって測定することで、ディフェンシンｍＲＮＡ及びインターロイキンｍＲＮＡの発現が、効果的に誘導されているか否かを検出することができる。 In this embodiment, sake lees, sake lees powder, or extracts thereof are added to the cultured cells as described above, and changes in the expression levels of defensins and interleukins are measured by RT-PCR, making it possible to detect whether the expression of defensin mRNA and interleukin mRNA has been effectively induced.
また、本実施の形態の医薬品、医薬部外品、化粧料、食品又は飲料は、上述した本実施の形態のディフェンシン及びインターロイキンの産生誘導剤組成物を含有し、ディフェンシン及びインターロイキンの産生を効果的に誘導する。このような優れた効果を奏することで、抗感染症用、がん抑制用、又は免疫不全症改善用の医薬品、医薬部外品、化粧料、食品、飲料として特に好適である。 The pharmaceuticals, quasi-drugs, cosmetics, food, or beverages of this embodiment contain the defensin and interleukin production inducer composition of this embodiment described above, and effectively induce the production of defensins and interleukins. Because of such excellent effects, they are particularly suitable as pharmaceuticals, quasi-drugs, cosmetics, foods, or beverages for anti-infectious diseases, cancer suppression, or improvement of immunodeficiency.
本実施の形態の医薬品、医薬部外品、化粧料、食品、飲料は、経口用（内用）又は非経口用（外用）の両形態をとることができる。経口用の医薬品、医薬部外品としては、例えば、産生誘導剤組成物以外にも、各種担体や添加剤、他の薬効成分等を含有してもよく、錠剤、散剤、顆粒剤、乳剤、シロップ剤、ドリンク剤、ハード又はソフトカプセル剤等の形態をとることができる。また、経口用の食品、飲料としては、例えば、産生誘導剤組成物に加え、他の食品原料や添加剤等を含有するチョコレート菓子、クッキー等の焼菓子、キャンディー、ドロップ、錠菓、チューイングガム、カプセル、清涼飲料水、ドリンク、スープ等の形態をとることができる。また、非経口用の医薬品、医薬部外品、化粧料の場合には、例えば、液体状、ジェル状、クリーム状、軟膏等の形態をとることができる。これら医薬品、医薬部外品、化粧料、食品、飲料への産生誘導剤組成物の配合量としては、特に限定されることはなく、ディフェンシン及びインターロイキンの産生誘導効果を奏する範囲内で含有させればよい。 The pharmaceuticals, quasi-drugs, cosmetics, foods, and beverages of this embodiment can be in the form of either oral (internal) or non-oral (external). Oral pharmaceuticals and quasi-drugs may contain, for example, various carriers, additives, other medicinal ingredients, etc., in addition to the production inducer composition, and can be in the form of tablets, powders, granules, emulsions, syrups, drinks, hard or soft capsules, etc. Oral foods and beverages can be in the form of, for example, chocolate confectionery, baked confectionery such as cookies, candies, drops, tablets, chewing gum, capsules, soft drinks, drinks, soups, etc., which contain, in addition to the production inducer composition, other food ingredients and additives, etc. In addition, in the case of non-oral pharmaceuticals, quasi-drugs, and cosmetics, they can be in the form of, for example, liquids, gels, creams, ointments, etc. There are no particular limitations on the amount of the production inducer composition that can be added to these pharmaceuticals, quasi-drugs, cosmetics, foods, and beverages, and it is sufficient that it is contained within a range that exerts the effect of inducing the production of defensins and interleukins.
以上、本実施の形態によれば、酒かす、酒かす粉末又はこれらの抽出物を有効成分とすることで、ディフェンシン及びインターロイキンの産生を効果的に誘導可能な産生誘導剤組成物、この産生誘導剤組成物を含有する医薬品、医薬部外品、化粧料、食品又は飲料を提供することができる。また、酒かす、酒かす粉末又はこれらの抽出物が、加熱殺菌されたものであれば、乳酸菌等を除去し、これらによる影響を抑制できる。また、加熱殺菌により、医薬品、医薬部外品、化粧料、食品、飲料に好適に用いられ、経口用のこれらにより好適に用いられ、食品、飲料に特に好適に用いられる。 As described above, according to this embodiment, by using sake lees, sake lees powder, or an extract thereof as an active ingredient, it is possible to provide a production inducer composition that can effectively induce the production of defensins and interleukins, and a medicine, quasi-drug, cosmetic, food, or beverage containing this production inducer composition. Furthermore, if the sake lees, sake lees powder, or an extract thereof is heat-sterilized, lactic acid bacteria and the like can be removed and the effects of these can be suppressed. Furthermore, by heat sterilization, it is suitable for use in medicines, quasi-drugs, cosmetics, foods, and beverages, and is particularly suitable for use in oral preparations, and is particularly suitable for use in foods and beverages.
また、本実施の形態の産生誘導剤組成物は、ディフェンシンｍＲＮＡ及びインターロイキンｍＲＮＡの発現誘導方法に適用できる。この発現誘導方法の工程としては、例えば、酒かす、酒かす粉末又はこれらの抽出物を培養細胞に添加する工程を有してなる。この発現誘導方法により、ディフェンシンｍＲＮＡ及びインターロイキンｍＲＮＡの発現を効果的に誘導可能な方法を提供することができる。この結果、ディフェンシン及びインターロイキンの産生を効果的に誘導することができる。 The production inducer composition of this embodiment can also be applied to a method for inducing the expression of defensin mRNA and interleukin mRNA. The steps of this expression induction method include, for example, adding sake lees, sake lees powder, or an extract thereof to cultured cells. This expression induction method can provide a method that can effectively induce the expression of defensin mRNA and interleukin mRNA. As a result, the production of defensin and interleukin can be effectively induced.
また、本実施の形態の産生誘導剤組成物は、ヒトβ－ディフェンシン－２及びヒトインターロイキン－１２の産生の誘導及びこれらのｍＲＮＡの発現の誘導に最適である。この結果、感染予防だけでなく、優れた抗腫瘍効果やがん細胞の増殖抑制作用を得ることが可能となり、感染防御や抗がん療法、免疫不全症の改善における臨床への応用が可能となる。また、本実施の形態の産生誘導剤組成物を含有することで、感染防御効果に優れる抗感染症用、がん細胞の増殖抑制等の効果に優れるがん治抑制用、免疫不全症の改善効果に優れる免疫不全症改善用の医薬品、医薬部外品、化粧料、食品、飲料を提供することができる。 The production inducer composition of this embodiment is also optimal for inducing the production of human β-defensin-2 and human interleukin-12 and inducing the expression of their mRNAs. As a result, it is possible to obtain not only infection prevention but also excellent antitumor effects and cancer cell proliferation inhibitory effects, enabling clinical applications in infection prevention, anticancer therapy, and improvement of immunodeficiency. Furthermore, by containing the production inducer composition of this embodiment, it is possible to provide pharmaceuticals, quasi-drugs, cosmetics, foods, and beverages for anti-infectious diseases with excellent infection prevention effects, for cancer treatment inhibition with excellent effects such as inhibition of cancer cell proliferation, and for improving immunodeficiency with excellent effects of improving immunodeficiency.
また、酒かす、酒かす粉末又はこれらの抽出物を、ヒト小腸上皮細胞に添加することで、ヒト小腸上皮細胞におけるディフェンシンｍＲＮＡ及びインターロイキンｍＲＮＡの発現を効果的に誘導することができる。この結果、ディフェンシン及びインターロイキンの産生を効果的に誘導することができ、小腸からの病原体等の侵入の防御、がん細胞の増殖抑制など、生体防御機構を高めることができる。 In addition, by adding sake lees, sake lees powder, or extracts thereof to human small intestinal epithelial cells, it is possible to effectively induce the expression of defensin mRNA and interleukin mRNA in human small intestinal epithelial cells. As a result, it is possible to effectively induce the production of defensins and interleukins, and it is possible to enhance the body's defense mechanisms, such as preventing the invasion of pathogens from the small intestine and suppressing the proliferation of cancer cells.
本発明を、実施例（実験例）によってさらに詳細に説明するが、本実施例は発明の具体的な説明のためのものであって、本発明が実施例によって限定されるものではない。
（実施例１）
＜ヒト小腸上皮細胞の調製＞
１．６×１０6cell/mlのＣａｃｏ２細胞（ヒト大腸がん細胞）を、５％ＦＢＳ（ウシ胎児血清：Fetal Bovine Serum）を含むＤＭＥＭ培地に懸濁し、３７℃で２４時間、５％ＣＯ2雰囲気下で培養を行った。さらに、培地を５ｍＭの酪酸を添加したＤＭＥＭ培地に交換して、３７℃で４日間、培養し、ヒト小腸上皮細胞に分化させた。分化終了後、５％ＦＢＳを含むＤＭＥＭ培地に交換した。
The present invention will be described in more detail with reference to examples (experimental examples). However, these examples are for the purpose of specifically explaining the invention, and the present invention is not limited to these examples.
Example 1
<Preparation of human small intestinal epithelial cells>
1.6 x 106 cells/ml of Caco2 cells (human colon cancer cells) were suspended in DMEM medium containing 5% FBS (Fetal Bovine Serum) and cultured at 37°C for 24 hours in a 5% CO2 atmosphere . The medium was then replaced with DMEM medium containing 5 mM butyric acid and cultured at 37°C for 4 days to differentiate into human small intestinal epithelial cells. After differentiation, the medium was replaced with DMEM medium containing 5% FBS.
＜酒かすサンプルの添加＞
この実施例１（実施例１－１～１－７）では、酒かすサンプルとして、下記表１に示す酒かす１～７を、それぞれＰＢＳに溶かして凍結乾燥し、粉末にしたものを使用した。なお、下記酒かす１～７の乳酸菌含有量は、３００個／ｇ以下であった。酒かす２～５として、出願人である秋田銘醸が醸造した普通酒の中から、ロットが異なる（つまり、製造時期、製造環境等が異なる）４つの普通酒１～４由来の酒かすを使用した。同様に酒かす６、７として、ロットの異なる２つの純米酒１、２由来の酒かすを使用した。
<Addition of sake lees sample>
In this Example 1 (Examples 1-1 to 1-7), sake lees samples were used which were prepared by dissolving sake lees 1 to 7 shown in Table 1 below in PBS, freeze-drying, and powdering them. The lactic acid bacteria content of sake lees 1 to 7 below was 300 cells/g or less. As sake lees 2 to 5, sake lees derived from four types of ordinary sake 1 to 4, which were different lots (i.e., different production times, production environments, etc.), were used from ordinary sake brewed by the applicant, Akita Meijo. Similarly, as sake lees 6 and 7, sake lees derived from two different lots of junmai sake 1 and 2 were used.
上記凍結乾燥し粉末化した酒かす１～７のサンプルを、終濃度０．１mg/mLになるように、ＰＢＳで調整した。調整した酒かす１～７の各サンプルを、上記＜ヒト小腸上皮細胞の調製＞で調整したヒト小腸上皮細胞に添加して、３７℃で３時間、５％ＣＯ2雰囲気下で培養した。また、同量のＰＢＳをヒト小腸上皮細胞に添加し、コントロール（比較例）とした。 The freeze-dried and powdered sake lees samples 1 to 7 were adjusted with PBS to a final concentration of 0.1 mg/mL. Each adjusted sake lees sample 1 to 7 was added to the human small intestinal epithelial cells prepared in the above <Preparation of human small intestinal epithelial cells> and cultured at 37°C for 3 hours in a 5% CO2 atmosphere. The same amount of PBS was also added to the human small intestinal epithelial cells to serve as a control (comparative example).
＜ＲＮＡ回収＞
各サンプルの培地から、培養したヒト小腸上皮細胞を回収し、回収したヒト小腸上皮細胞からRNeasy Mini Kit（QUIAGEN社製）を用いてトータルＲＮＡ（総ＲＮＡ）を抽出した。次いで、Oligo-dtcelluloseカラムを用いて、ｍＲＮＡを抽出した。トータルＲＮＡの抽出の詳細な手順を、以下に示す。
<RNA recovery>
Cultured human small intestinal epithelial cells were collected from the medium of each sample, and total RNA was extracted from the collected human small intestinal epithelial cells using RNeasy Mini Kit (QIAGEN). Then, mRNA was extracted using an Oligo-dtcellulose column. The detailed procedure for extracting total RNA is shown below.
（ａ）培地を除去後、トリプシンを５００μl加えて洗浄
（ｂ）再びトリプシンを５００μｌ加えて３７℃で５分培養
（ｃ）培養物を、２，０００ｒｐｍで３分間遠心してヒト小腸上皮細胞を回収
（ｄ）回収したヒト小腸上皮細胞にRTL bufferを６００μｌ加えて懸濁
（ｅ）２１Ｇシリンジで１０回ストローク
（ｆ）７０％エタノールを６００μｌ加えてパイペティング
（ｇ）RNeasyスピンカラムに、（ｆ）のサンプルを６００μｌ加えて、１０，０００ｒｐｍで１５秒遠心
（ｈ）濾液を除去後、残っているサンプル６００μｌをRNeasyスピンカラムに加えて、１０，０００ｒｐｍで１５秒遠心
（ｉ）濾液を除去後、RW1 buffer７００μｌをRNeasyスピンカラムに加えて同様に遠心
（ｊ）濾液を除去後、RPE buffer５００μｌをRNeasyスピンカラムに加えて同様に遠心
（ｋ）濾液を除去後、RPE buffer５００μｌをRNeasyスピンカラムに加えて１０，０００ｒｐｍで２分遠心
（ｌ）新しいコレクトチューブにRNeasyスピンカラムを移し、１５，０００ｒｐｍで１分遠心
（ｍ）１．５ｍｌのコレクトチューブにRNeasyスピンカラムを移し、Rnase free water を３０μｌ加えて１０，０００ｒｐｍで１分遠心してＲＮＡを溶出
（ｎ）－３０℃のフリーザーで保存
(a) After removing the medium, 500 μl of trypsin was added and washed. (b) 500 μl of trypsin was added again and incubated at 37°C for 5 minutes. (c) The culture was centrifuged at 2,000 rpm for 3 minutes to recover the human small intestinal epithelial cells. (d) 600 μl of RTL buffer was added to the recovered human small intestinal epithelial cells and suspended. (e) 10 strokes with a 21G syringe. (f) 600 μl of 70% ethanol was added and pipetted. (g) 600 μl of the sample from (f) was added to the RNeasy spin column and centrifuged at 10,000 rpm for 15 seconds. (h) After removing the filtrate, 600 μl of the remaining sample was added to the RNeasy spin column and centrifuged at 10,000 rpm for 15 seconds. (i) After removing the filtrate, 700 μl of RW1 buffer was added to the RNeasy spin column and centrifuged in the same manner. (j) After removing the filtrate, RPE Add 500 μl of buffer to the RNeasy spin column and centrifuge in the same manner (k). After removing the filtrate, add 500 μl of RPE buffer to the RNeasy spin column and centrifuge at 10,000 rpm for 2 minutes (l). Transfer the RNeasy spin column to a new collect tube and centrifuge at 15,000 rpm for 1 minute (m). Transfer the RNeasy spin column to a 1.5 ml collect tube, add 30 μl of RNase free water, and centrifuge at 10,000 rpm for 1 minute to elute the RNA (n). Store in a -30°C freezer.
＜ｃＤＮＡへの変換＞
Super Script III First strand kit（Invitrogen社製）を用いて、上記で抽出したｍＲＮＡからｃＤＮＡを合成した。詳細な手順を以下に示す。
Conversion to cDNA
Using a Super Script III First strand kit (Invitrogen), cDNA was synthesized from the mRNA extracted above. The detailed procedure is shown below.
（ａ）下記表２に示す組成で溶液を調製
（ｂ）６５℃で５分培養
（ｃ）４℃で１分培養
（ｄ）下記表２に示す組成の溶液を添加
（ｅ－１）５０℃、５０分
（ｅ－２）８５℃、５分
（ｅ－３）４℃、１分
（ｆ）ＰＣＲ終了後、氷上に移し、RNase Hを１μＬ加えて、３７℃で２０分培養
(a) Prepare a solution having the composition shown in Table 2 below.
(b) Incubation at 65°C for 5 minutes; (c) Incubation at 4°C for 1 minute; (d) Addition of a solution having the composition shown in Table 2 below.
＜ｃＤＮＡの増幅＞
上記で合成したｃＤＮＡを、下記表４に示す組成でＰＣＲチューブに調整する。
The cDNA synthesized above is prepared in a PCR tube with the composition shown in Table 4 below.
ヒトインターロイキン－１２（IL-12 p40）、β－アクチン（β-actin）及びヒトβ－ディフェンシン－２（ｈBD-2）の各々の遺伝子プライマー（Fw：フォワード、Rv：リバース）の塩基配列及びバンドサイズは、以下のとおりである。
・IL-12 p40
IL-12 p40 Fwプライマー：ACCTGACCCACCCAAGAACTT（配列番号１）
IL-12 p40 Rvプライマー：TGGACCTGAACGCAGAA（配列番号２)
バンドサイズ：１３２ｂｐ
・β-actin
β-actin Fwプライマー：GCTCGTCGTCGACAACGGCTC（配列番号３）
β-actin Rvプライマー：CAAACATGATCTGGGTCATCTTCTC（配列番号４)
バンドサイズ：３２０ｂｐ
・hBD-2
hBD-2 Fwプライマー：TGATGAGGGAGCCCTTTCTG（配列番号５）
hBD-2 Rvプライマー：TGATGCCTCTTCCAGGTGTT（配列番号６）
バンドサイズ：２０６ｂｐ
The base sequences and band sizes of the gene primers (Fw: forward, Rv: reverse) for human interleukin-12 (IL-12 p40), β-actin, and human β-defensin-2 (hBD-2) are as follows:
・IL-12 p40
IL-12 p40 Fw primer: ACCTGACCCACCCAAGAACTT (SEQ ID NO: 1)
IL-12 p40 Rv primer: TGGACCTGAACGCAGAA (SEQ ID NO: 2)
Band size: 132bp
・β-actin
β-actin Fw primer: GCTCGTCGTCGACAACGGCTC (SEQ ID NO: 3)
β-actin Rv primer: CAAACATGATCTGGGTCATCTTCTC (SEQ ID NO: 4)
Band size: 320bp
・hBD-2
hBD-2 Fw primer: TGATGAGGGAGCCCTTTCTG (SEQ ID NO: 5)
hBD-2 Rv primer: TGATGCCTCTTCCAGGTGTT (SEQ ID NO: 6)
Band size: 206bp
以下で説明するＰＣＲ条件で、ＲＴ－ＰＣＲを行った。IL-12 p40及びβ-actinは、逆転写反応を９４℃で１５秒、６０℃で３０秒、７２℃で３０秒の条件で、３７サイクル行った。hBD-2は、逆転写反応を９５℃で３０秒、６１℃で３０秒、７２℃で３０秒の条件で、４０サイクル行った。 RT-PCR was performed under the PCR conditions described below. For IL-12 p40 and β-actin, the reverse transcription reaction was performed under the conditions of 94°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, for 37 cycles. For hBD-2, the reverse transcription reaction was performed under the conditions of 95°C for 30 seconds, 61°C for 30 seconds, and 72°C for 30 seconds, for 40 cycles.
ＲＴ－ＰＣＲ終了後、各サンプルの反応液に、６×ＳＢをそれぞれ４μＬずつ加え、よく懸濁した。その後、１％アガロースゲルを用いて、１００Ｖ、１５μL/wellで電気泳動を行った。電気泳動を行った後、ＵＶ照射下で観察し、Image Jで解析を行った。 After RT-PCR was completed, 4 μL of 6xSB was added to each sample reaction solution and suspended thoroughly. Then, electrophoresis was performed at 100 V and 15 μL/well using 1% agarose gel. After electrophoresis, the samples were observed under UV irradiation and analyzed using Image J.
＜結果＞
図１は、酒かす１～７により刺激した、ヒト小腸上皮細胞におけるヒトインターロイキン－１２の産生誘導効果を示すグラフである。このグラフは、酒かす１～７により刺激したヒト小腸上皮細胞におけるIL-12をＵＶ照射下で観察し、得られた蛍光を、β-actinを基準として数値化したものである。左から順に、ＰＢＳ、酒かす１、酒かす２、酒かす３、酒かす４、酒かす５、酒かす６、酒かす７の数値を示す。
<Results>
1 is a graph showing the effect of inducing human interleukin-12 production in human small intestinal epithelial cells stimulated with sake lees 1 to 7. This graph shows IL-12 in human small intestinal epithelial cells stimulated with sake lees 1 to 7 observed under UV irradiation, and the resulting fluorescence quantified using β-actin as the standard. From the left, the values shown are for PBS, sake lees 1, sake lees 2, sake lees 3, sake lees 4, sake lees 5, sake lees 6, and sake lees 7.
この図１のグラフによれば、コントロール（比較例）であるＰＢＳと比較して、実施例１－１～１－７の酒かす１～７のサンプルを添加したヒト小腸上皮細胞はIL-12のｍＲＮＡの発現レベルが大きく、IL-12の産生が誘導されていることがわかる。図１及び後述の図２～図４中の「*」は、ｔ検定（有意水準５％以下）の結果、コントロールと比較して非常に有意差（ｐ＜０．０１）があった旨を示し、「**」は、コントロールと比較して有意差（ｐ＜０．０５）があった旨を示す。 The graph in Figure 1 shows that, compared to the control (comparison example) PBS, human small intestinal epithelial cells to which sake lees samples 1 to 7 from Examples 1-1 to 1-7 were added had high IL-12 mRNA expression levels, indicating that IL-12 production was induced. In Figure 1 and Figures 2 to 4 described below, "*" indicates that there was a highly significant difference (p<0.01) compared to the control as a result of the t-test (significance level 5% or less), and "**" indicates that there was a significant difference (p<0.05) compared to the control.
図２は、酒かす１～７により刺激した、ヒト小腸上皮細胞におけるヒトβ－ディフェンシン－２の産生誘導効果を示すグラフである。このグラフもIL-12の場合と同様に、酒かす１～７により刺激したヒト小腸上皮細胞におけるhBD-2をＵＶ照射下で観察し、得られた蛍光を、β-actinを基準として数値化したものである。左から順に、ＰＢＳ、酒かす１、酒かす２、酒かす３、酒かす４、酒かす５、酒かす６、酒かす７の数値を示す。 Figure 2 is a graph showing the effect of inducing the production of human β-defensin-2 in human small intestinal epithelial cells stimulated with sake lees 1 to 7. As in the case of IL-12, this graph was also obtained by observing hBD-2 in human small intestinal epithelial cells stimulated with sake lees 1 to 7 under UV irradiation, and quantifying the resulting fluorescence using β-actin as the standard. From left to right, the values are shown for PBS, sake lees 1, sake lees 2, sake lees 3, sake lees 4, sake lees 5, sake lees 6, and sake lees 7.
この図２のグラフによれば、コントロール（比較例）であるＰＢＳと比較して、実施例１－１～１－７の酒かす１～７のサンプルを添加したヒト小腸上皮細胞はhBD-2のｍＲＮＡの発現レベルが著しく大きく、hBD-2の産生が誘導されていることがわかる。 The graph in Figure 2 shows that, compared to the control (comparison example) PBS, human small intestinal epithelial cells to which sake lees samples 1 to 7 from Examples 1-1 to 1-7 were added showed significantly higher hBD-2 mRNA expression levels, indicating that hBD-2 production was induced.
（実施例２）
＜ヒト小腸上皮細胞の調製＞
上記実施例１と同様の手順で、ヒト小腸上皮細胞を調整した。
Example 2
<Preparation of human small intestinal epithelial cells>
Human small intestinal epithelial cells were prepared in the same manner as in Example 1 above.
＜酒かすサンプルの添加＞
下記の表５に、実施例２（実施例２－１、２－２）で使用した製造ロットの異なる酒かす粉末のサンプルを示した。なお、実施例２（実施例２－１、２－２）で使用する酒かす粉末Ｉ、ＩＩのサンプルは、普通酒の酒かす８０％、賦形剤２０％を配合し、加熱乾燥機（温度１００－１５０℃）で加熱乾燥し粉末化した粉末（酒かす含有量８０％）である。さらに、下記酒かす粉末Ｉ、ＩＩの乳酸菌含有量は０個／ｇであった。
<Addition of sake lees sample>
Table 5 below shows sake lees powder samples from different production lots used in Example 2 (Examples 2-1 and 2-2). The sake lees powder samples I and II used in Example 2 (Examples 2-1 and 2-2) were made by blending 80% sake lees from ordinary sake and 20% excipient, drying the mixture in a heating dryer (temperature 100-150°C), and pulverizing it (sake lees content 80%). Furthermore, the lactic acid bacteria content of the sake lees powders I and II below was 0 cells/g.
上記の加熱乾燥機で粉末化した製造ロットの異なる酒かす粉末Ｉ，ＩＩのサンプルを、終濃度０．１２５mg/mL（酒かす粉末の酒粕含有量８０％から実施例１の添加終濃度に対して１．２５倍の濃度を添加量とした。）になるように、ＰＢＳで調整した。調整した酒かす粉末Ｉ、ＩＩの各サンプルを、ヒト小腸上皮細胞に添加して、３７℃で３時間、５％ＣＯ2雰囲気下で培養した。また、同量のＰＢＳをヒト小腸上皮細胞に添加し、コントロール（比較例）とした。 Samples of sake lees powder I and II from different production lots powdered using the above-mentioned heated dryer were adjusted with PBS to a final concentration of 0.125 mg/mL (the amount added was 1.25 times the final concentration added in Example 1, since the sake lees content in the sake lees powder was 80%). Each of the adjusted sake lees powder samples I and II was added to human small intestinal epithelial cells and cultured at 37°C for 3 hours in a 5% CO2 atmosphere. The same amount of PBS was also added to the human small intestinal epithelial cells to serve as a control (comparative example).
＜ＲＮＡ回収＞
上記実施例１と同様の手順で、各ヒト小腸上皮細胞からＲＮＡを回収（抽出）した。
＜ｃＤＮＡへの変換＞
上記実施例１と同様の手順で、上記で抽出したｍＲＮＡからｃＤＮＡを合成した。
＜ｃＤＮＡの増幅＞
上記実施例１と同様の手順で、上記で合成したｃＤＮＡを増幅した。
<RNA recovery>
RNA was recovered (extracted) from each of the human small intestinal epithelial cells in the same manner as in Example 1 above.
Conversion to cDNA
Using the same procedure as in Example 1 above, cDNA was synthesized from the mRNA extracted above.
<Amplification of cDNA>
The cDNA synthesized above was amplified in the same manner as in Example 1 above.
ＲＴ－ＰＣＲ終了後、実施例１と同様に、各サンプルの反応液に、６×ＳＢをそれぞれ４μＬずつ加え、よく懸濁した。その後、１％アガロースゲルを用いて、１００Ｖ、１５μL/wellで電気泳動を行った。電気泳動を行った後、ＵＶ照射下で観察し、Image Jで解析を行った。 After RT-PCR, 4 μL of 6xSB was added to each sample reaction solution and suspended thoroughly, as in Example 1. Then, electrophoresis was performed at 100 V and 15 μL/well using 1% agarose gel. After electrophoresis, the samples were observed under UV irradiation and analyzed using Image J.
＜結果＞
図３は、酒かす粉末Ｉ、ＩＩにより刺激した、ヒト小腸上皮細胞におけるヒトインターロイキン－１２の産生誘導効果を示すグラフである。このグラフは、酒かすＩ、ＩＩにより刺激したヒト小腸上皮細胞におけるIL-12をＵＶ照射下で観察し、得られた蛍光を、β-actinを基準として数値化したものである。左から順に、ＰＢＳ、酒かす粉末Ｉ、酒かす粉末ＩＩの数値を示す。
<Results>
3 is a graph showing the effect of inducing human interleukin-12 production in human small intestinal epithelial cells stimulated with sake lees powder I and II. This graph shows IL-12 in human small intestinal epithelial cells stimulated with sake lees I and II observed under UV irradiation, and the resulting fluorescence was quantified using β-actin as the standard. From the left, the values are shown for PBS, sake lees powder I, and sake lees powder II.
この図３のグラフによれば、コントロールで（比較例）であるＰＢＳと比較して、実施例２－１、２－２の酒かすＩ、ＩＩのサンプルを添加したヒト小腸上皮細胞はIL-12のｍＲＮＡの発現レベルが大きく、IL-12の産生が誘導されていることがわかる。 The graph in Figure 3 shows that, compared to the control (comparative example) PBS, human small intestinal epithelial cells to which sake lees I and II samples from Examples 2-1 and 2-2 were added showed high levels of IL-12 mRNA expression, indicating that IL-12 production was induced.
図４は、酒かす粉末Ｉ、ＩＩにより刺激した、ヒト小腸上皮細胞におけるヒトβ－ディフェンシン－２の産生誘導効果を示すグラフである。このグラフもIL-12の場合と同様に、酒かす粉末Ｉ、ＩＩにより刺激したヒト小腸上皮細胞におけるhBD-2をＵＶ照射下で観察し、得られた蛍光を、β-actinを基準として数値化したものである。左から順に、ＰＢＳ、酒かすＩ、酒かすＩＩの数値を示す。 Figure 4 is a graph showing the effect of inducing the production of human β-defensin-2 in human small intestinal epithelial cells stimulated with sake lees powder I and II. As with the case of IL-12, this graph was obtained by observing hBD-2 in human small intestinal epithelial cells stimulated with sake lees powder I and II under UV irradiation, and quantifying the resulting fluorescence using β-actin as the standard. From the left, the values are shown for PBS, sake lees I, and sake lees II.
この図４のグラフによれば、コントロール（比較例）であるＰＢＳと比較して、実施例２－１、２－２の酒かす粉末Ｉ、ＩＩのサンプルを添加したヒト小腸上皮細胞はhBD-2のｍＲＮＡの発現レベルが著しく大きく、hBD-2の産生が誘導されていることがわかる。また、酒かす粉末Ｉ、ＩＩは、加熱乾燥機で加熱乾燥し粉末化することで乳酸菌を除去したものであるため、図４、図５に示すIL-12の産生誘導効果及びhBD-2の産生誘導効果への乳酸菌の影響によるものではないことは明らかである。 The graph in Figure 4 shows that, compared to the control (comparative example) PBS, human small intestinal epithelial cells to which samples of sake lees powder I and II from Examples 2-1 and 2-2 were added had significantly higher hBD-2 mRNA expression levels, indicating that hBD-2 production was induced. Furthermore, sake lees powder I and II were obtained by removing the lactic acid bacteria through heating and drying in a heating dryer and powdering, so it is clear that the IL-12 production induction effect and hBD-2 production induction effect shown in Figures 4 and 5 are not due to the influence of lactic acid bacteria.
以上、本発明の実施形態及び実施例を詳述してきたが、上記各実施形態及び実施例は本発明の例示にしか過ぎないものであり、本発明は上記各実施形態及び実施例の構成にのみ限定されるものではない。本発明の要旨を逸脱しない範囲の設計の変更等があっても、本発明に含まれることは勿論である。 Although the embodiments and examples of the present invention have been described in detail above, the above-mentioned embodiments and examples are merely examples of the present invention, and the present invention is not limited to the configurations of the above-mentioned embodiments and examples. Naturally, any design changes that do not deviate from the gist of the present invention are also included in the present invention.
以上説明したように、本発明は、酒かす、酒かす粉末又はこれらの抽出物を用いることで、ディフェンシン及びインターロイキンの産生を効果的に誘導することができるため、一般の飲食品や飲料品、健康食品、保健機能食品、保健機能飲料、栄養補助食品、栄養補助飲料、医薬部外品、医薬品等に適用することができる。また、感染防御や抗がん療法、免疫不全症の改善における臨床への応用が期待できる。 As described above, the present invention can effectively induce the production of defensins and interleukins by using sake lees, sake lees powder, or extracts thereof, and can therefore be applied to general food and beverage products, health foods, health functional foods, health functional drinks, nutritional supplements, quasi-drugs, pharmaceuticals, etc. Furthermore, it is expected to be applied clinically in infection prevention, anti-cancer therapy, and improvement of immunodeficiency.
Claims (4)
前記酒かす又は前記酒かす粉末は、終濃度が０．１～１．０ｍｇ／ｍＬとなるようにＰＢＳで調整されているだけであり、アルカリ抽出、熱水抽出、エタノール抽出及び水抽出を含む抽出処理がされていない、ヒトβ－ディフェンシン－２及びヒトインターロイキン－１２の産生誘導剤組成物。 The active ingredient is sake lees containing sake lees derived from honjozo sake, ordinary sake or junmai sake brewed by Akita Meijo Brewery, or sake lees powder containing sake lees powder derived from honjozo sake, ordinary sake or junmai sake brewed by Akita Meijo Brewery , and the number of lactic acid bacteria is 30/g or less,
The sake lees or the sake lees powder are simply adjusted with PBS to a final concentration of 0.1 to 1.0 mg/mL, and have not been subjected to any extraction treatment including alkaline extraction, hot water extraction, ethanol extraction, or water extraction in this human β-defensin-2 and human interleukin-12 production inducer composition.
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| KR101646002B1 (en) * | 2014-08-01 | 2016-08-08 | 안동대학교 산학협력단 | Pharmaceutical composition for prevention or treatment of colorectal cancer comprising ethylacetate fraction of jubak ethanol extract as an effective component and health functional food comprising the same |
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| JP2005325100A (en) | 2004-04-12 | 2005-11-24 | Hakutsuru Shuzo Kk | Dna synthase inhibitor, dna topoisomerase inhibitor, human neovascularization inhibitor, human cancer cell proliferation inhibitor, and health food and cosmetic |
| JP2009292785A (en) | 2008-06-06 | 2009-12-17 | Gekkeikan Sake Co Ltd | Immunomodulator and antiallergic agent containing the same |
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