JP2006056854A - Immunostimulating agent, antineoplastic agent, antiinflammatory agent, antiaging agent and skin cosmetic - Google Patents

Immunostimulating agent, antineoplastic agent, antiinflammatory agent, antiaging agent and skin cosmetic Download PDF

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JP2006056854A
JP2006056854A JP2004243001A JP2004243001A JP2006056854A JP 2006056854 A JP2006056854 A JP 2006056854A JP 2004243001 A JP2004243001 A JP 2004243001A JP 2004243001 A JP2004243001 A JP 2004243001A JP 2006056854 A JP2006056854 A JP 2006056854A
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JP5132030B2 (en
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Akinori Kiso
昭典 木曽
Toshiyuki Murakami
敏之 村上
Yoko Ito
洋子 伊藤
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Maruzen Pharmaceutical Co Ltd
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Abstract

<P>PROBLEM TO BE SOLVED: To find a natural product having TNF-α production-promoting action, platelet aggregation-suppressing action, hexosaminidase isolation-suppressing action, collagen production-promoting action, fibroblast growth-promoting action and epidermal corneocyte growth-promoting action from natural products having high safety and to provide an immunostimulating agent, an antineoplastic agent, a TNF-α production promoter, an antiinflammatory agent, an antiaging agent and a skin cosmetic each containing the natural product as an active ingredient. <P>SOLUTION: The immunostimulating agent, the antineoplastic agent, the TNF-α production promoter, the antiinflammatory agent, the antiaging agent and the skin cosmetic each comprises an extract from Bolbostemma paniculata (Maxim.) Franq. as an active ingredient. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

本発明は、安全性の高い植物抽出物を有効成分として含有する免疫賦活剤、抗腫瘍剤、抗炎症剤、抗老化剤及び皮膚化粧料に関する。   The present invention relates to an immunostimulant, an antitumor agent, an anti-inflammatory agent, an anti-aging agent, and a skin cosmetic containing a highly safe plant extract as an active ingredient.

近年、消費者の健康に対する意識はますます高まりを見せている。一方で、現代社会には、不規則な生活習慣、食事の偏り、精神的ストレス等、免疫機構にダメージを与える要因が氾濫している。このようにして免疫力が低下することにより、癌、感染症、アレルギー症状等の各種疾患を誘発することが知られており、逆に免疫力が賦活されると、発癌抑制、制癌作用、抗感染症、抗アレルギー作用、さらには体調リズムの回復・恒常性維持などの様々な効果が期待できる。   In recent years, consumers' awareness of health has increased. On the other hand, in modern society, factors that damage the immune system, such as irregular lifestyles, dietary bias, and mental stress, are inundated. It is known that immunity is reduced in this way, thereby inducing various diseases such as cancer, infectious diseases, allergic symptoms, and conversely, when immunity is activated, carcinogenesis suppression, anticancer action, Various effects such as anti-infection, antiallergic action, recovery of physical condition rhythm and maintenance of homeostasis can be expected.

免疫機構には、多くの種類の細胞が関与しているが、特に白血球の役割は大きく、なかでもマクロファージは全動物に普遍的に存在しており、免疫応答の特に初期段階での働きを含め、あらゆる段階に関与している重要な白血球の一種である。近年、白血球の働きが物質レベルで解明されてきており、白血球の機能や細胞間相互作用は、白血球が分泌する微量タンパク質(サイトカイン)によって担われていることがわかってきた。   Many types of cells are involved in the immune mechanism, but the role of leukocytes is particularly large, and macrophages are ubiquitous in all animals, including the actions at the early stages of the immune response. It is a kind of important white blood cell that is involved in every stage. In recent years, the function of leukocytes has been elucidated at the substance level, and it has been found that the functions and intercellular interactions of leukocytes are borne by trace proteins (cytokines) secreted by leukocytes.

サイトカインには多くの種類があり、なかでも腫瘍壊死因子(TNF)やインターロイキン類が注目されている。それらのなかで、TNF-αに代表される炎症性サイトカインは、主にマクロファージから放出され、最終的には抗腫瘍作用等を示すことが報告されている。したがって、TNF−αの産生機能を亢進させることにより、免疫機能を賦活させ、悪性腫瘍の増殖を抑制できるものと考えられる。このような考えに基づき、TNF−α産生促進剤として、ユキノシタ科スグリ属に属する植物からの抽出物(特許文献1参照)等が提案されている。   There are many types of cytokines, among which tumor necrosis factor (TNF) and interleukins are attracting attention. Among them, inflammatory cytokines typified by TNF-α are reported to be released mainly from macrophages and finally to exhibit antitumor effects and the like. Therefore, it is considered that by enhancing the production function of TNF-α, the immune function is activated and the growth of malignant tumor can be suppressed. Based on such an idea, as a TNF-α production promoter, an extract from a plant belonging to the genus Curranta (see Patent Document 1) and the like have been proposed.

炎症性の疾患、例えば接触性皮膚炎(かぶれ)、乾癬、尋常性天疱瘡、その他肌荒れに伴う各種皮膚疾患等の原因や発症機構は多種多様であるが、その原因として、ホスホリパーゼAの活性化、血小板凝集、ヒスタミン遊離によるものが知られている。 Inflammatory diseases such as contact dermatitis (rash), psoriasis, pemphigus vulgaris, although other causes and pathogenic mechanisms of various skin disorders associated with skin irritation are manifold, as its cause, the activity of phospholipase A 2 It is known to be due to crystallization, platelet aggregation and histamine release.

血小板は、凝集して活性化することにより、生理的には止血、病理的には血栓形成を生じる他、血小板の凝集は、動脈硬化症の進展、ガン転移、炎症等に関与していると考えられている。したがって、血小板の凝集を抑制することにより、上記疾患の予防、治療又は改善をすることができるものと考えられる。このような考えに基づき、血小板凝集抑制剤として、コウサンフウ抽出物(特許文献2参照)等が提案されている。   Platelet aggregates and activates to cause hemostasis physiologically and pathologically cause thrombus formation, and platelet aggregation is involved in the progression of arteriosclerosis, cancer metastasis, inflammation, etc. It is considered. Therefore, it is considered that the above-mentioned diseases can be prevented, treated or ameliorated by suppressing platelet aggregation. Based on such an idea, as a platelet aggregation inhibitor, a Kangsanfu extract (see Patent Document 2) and the like have been proposed.

また、ヒスタミンは、マストセル内に存在し、脱顆粒反応により放出されたものが起炎物質として作用する。細胞内のヒスタミンが遊離されると同時にヘキソサミニダーゼも遊離されることから、ヘキソサミニダーゼの遊離を指標にヒスタミン遊離抑制作用を評価することができる。したがって、ヘキソサミニダーゼの遊離を抑制することにより、同時にヒスタミンの遊離も抑制でき、これにより炎症性疾患等の予防、治療又は改善に効果があるものと考えられる。このような考えに基づき、ヘキソサミニダーゼ遊離抑制剤として、紅雪茶抽出物(特許文献3参照)等が提案されている。   Histamine is present in the mast cell, and the substance released by the degranulation reaction acts as a flammable substance. Since histamine in the cells is released and hexosaminidase is released at the same time, histamine release inhibitory action can be evaluated using hexosaminidase release as an index. Therefore, it is considered that by suppressing the release of hexosaminidase, the release of histamine can also be suppressed at the same time, which is effective for preventing, treating or improving inflammatory diseases. Based on this idea, red snow tea extract (see Patent Document 3) and the like have been proposed as a hexosaminidase release inhibitor.

皮膚の表皮及び真皮は、表皮細胞、線維芽細胞、及びこれらの細胞の外にあって皮膚構造を支持するコラーゲン等の細胞外マトリックスにより構成されている。若い皮膚においては線維芽細胞の増殖は活発であり、線維芽細胞、コラーゲン等の皮膚組織の相互作用により水分保持、柔軟性、弾力性等が確保され、皮膚は外見的にも張りや艶があってみずみずしい状態に維持される。   The epidermis and dermis of the skin are composed of epidermal cells, fibroblasts, and an extracellular matrix such as collagen that is outside these cells and supports the skin structure. In young skin, fibroblasts are actively proliferating, and skin retention such as fibroblasts and collagen ensures moisture retention, flexibility, elasticity, etc. It is kept fresh and fresh.

ところが、紫外線、空気の著しい乾燥、過度の皮膚洗浄等、ある種の外的因子の影響があったり、加齢が進んだりすると、細胞外マトリックスの主要構成成分であるコラーゲンの産生量が減少すると共に架橋による弾力性低下を引き起こす。また、線維芽細胞の増殖が遅くなり皮膚の保湿機能や弾力性が低下する。その結果、皮膚は保湿機能や弾力性が低下し、角質は異常剥離を始めるため、肌は張りや艶を失い、肌荒れ、シワ等の老化現象を呈するようになる。そのため、真皮層線維芽細胞におけるコラーゲンの産生を促進することにより、また、線維芽細胞の増殖を促進することにより皮膚の老化を防止又は改善することができると考えられる。このような考えに基づき、コラーゲン産生促進剤として、五斂子からの抽出物(特許文献4参照)等が、線維芽細胞増殖促進剤として、クスノハガシワ抽出物(特許文献5参照)等が提案されている。   However, the production of collagen, which is a major component of the extracellular matrix, decreases when it is affected by certain external factors such as ultraviolet rays, drastic drying of air, excessive skin washing, and so on. At the same time, the elasticity is lowered by cross-linking. In addition, the proliferation of fibroblasts is slowed, and the moisture retaining function and elasticity of the skin are reduced. As a result, the skin retains its moisturizing function and elasticity, and the keratin begins to exfoliate abnormally. Therefore, the skin loses its tension and gloss, and exhibits an aging phenomenon such as rough skin and wrinkles. Therefore, it is considered that aging of the skin can be prevented or improved by promoting the production of collagen in dermal fibroblasts and by promoting the proliferation of fibroblasts. Based on this idea, an extract from pentagon (see Patent Document 4) and the like are proposed as a collagen production promoter, and a Kusunohagashi extract (see Patent Document 5) and the like as a fibroblast proliferation promoter. ing.

表皮は、最下層である基底層から始まって、有棘層、顆粒層、角質層へと連なる4層構造からなるが、各層に存在する大部分の細胞は、基底層から分化した角化細胞である。通常、角化細胞は基底層で産生され、徐々に上層に分化しながら移動して角質細胞となって角質層を構成し、最終的に垢として角質層から脱落していく。   The epidermis has a four-layer structure that starts with the basal layer, the lowest layer, and continues to the spiny layer, the granule layer, and the stratum corneum. Most of the cells in each layer are keratinocytes differentiated from the basal layer. It is. Normally, keratinocytes are produced in the basal layer, move while gradually differentiated into the upper layer, become keratinocytes, constitute the horny layer, and finally fall off from the horny layer as plaque.

上記角質層は皮膚の最外殻に存在しており、外界からの刺激に対する物理的なバリアーとしての役割を果たしている。皮膚ではこのバリアー機能を持たせるため、角化細胞が基底層で産生されてから垢となって剥がれ落ちるまでのサイクル(角化)を通常4週間の周期で繰り返し、表皮の新陳代謝を行っている。しかしながら、この角質層も加齢によって新陳代謝機能が衰え、こじわ、くすみ、色素沈着、肌荒れ等の皮膚トラブルを発生することになる。そのため、角化細胞の増殖を促進し、肌の新陳代謝機能を回復させることにより、こじわ、くすみ、色素沈着等の皮膚の老化を改善できるものと考えられる。このような考えに基づき、表皮角化細胞増殖促進剤として、ハス胚芽抽出物(特許文献6参照)等が提案されている。
特開2004−107660号公報 特開2002−53477号公報 特開2003−212789号公報 特開2002−226323号公報 特開2003−146837号公報 特開2002−68993号公報
The stratum corneum is present in the outermost shell of the skin, and serves as a physical barrier against irritation from the outside. In order to have this barrier function in the skin, the cycle (keratinization) from the time when keratinocytes are produced in the basal layer until they become plaque and peel off is repeated in a cycle of usually 4 weeks to perform epidermal metabolism. . However, the metabolic function of this stratum corneum also deteriorates with aging, and skin troubles such as wrinkles, dullness, pigmentation, and rough skin occur. Therefore, it is considered that skin aging such as wrinkles, dullness, and pigmentation can be improved by promoting the proliferation of keratinocytes and restoring the metabolic function of the skin. Based on this idea, a lotus germ extract (see Patent Document 6) and the like have been proposed as an epidermal keratinocyte proliferation promoter.
JP 2004-107660 A JP 2002-53477 A JP 2003-212789 A JP 2002-226323 A JP 2003-146837 A JP 2002-68993 A

本発明は、安全性の高い天然物の中から、TNF−α産生促進作用、血小板凝集抑制作用、ヘキソサミニダーゼ遊離抑制作用、コラーゲン産生促進作用、線維芽細胞増殖促進作用又は表皮角化細胞増殖促進作用を有するものを見出し、当該天然物からの抽出物を有効成分として含有する免疫賦活剤、抗腫瘍剤、TNF−α産生促進剤、抗炎症剤及び抗老化剤、並びに当該天然物からの抽出物を配合した皮膚化粧料を提供することを目的とする。   The present invention includes TNF-α production promoting action, platelet aggregation inhibiting action, hexosaminidase release inhibiting action, collagen production promoting action, fibroblast proliferation promoting action or epidermal keratinocytes among highly safe natural products. An immunostimulant, an antitumor agent, a TNF-α production promoter, an anti-inflammatory agent and an anti-aging agent containing an extract from the natural product as an active ingredient, which has a growth promoting action, and the natural product It aims at providing the skin cosmetics which mix | blended this extract.

本発明の免疫賦活剤、抗腫瘍剤、TNF−α産生促進剤、抗炎症剤又は抗老化剤は、土貝母(Bolbostemma paniculata(Maxim.)Franq.)からの抽出物を有効成分として含有することを特徴とし、本発明の皮膚化粧料は、土貝母(Bolbostemma paniculata(Maxim.)Franq.)からの抽出物又は土貝母(Bolbostemma paniculata(Maxim.)Franq.)の鱗片からの抽出物を配合したことを特徴とする。   The immunostimulant, the antitumor agent, the TNF-α production promoter, the anti-inflammatory agent or the anti-aging agent of the present invention contains an extract from a shellfish mother (Bolbostemma paniculata (Maxim.) Franq.) As an active ingredient. The skin cosmetic of the present invention is characterized in that an extract from a shellfish mother (Bolbostemma paniculata (Maxim.) Franq.) Or an extract from a scale of a shellfish mother (Bolbostemma paniculata (Maxim.) Franq.) It is characterized by blending.

また、本発明の抗炎症剤においては、前記抽出物が、血小板凝集抑制作用及び/又はヘキソサミニダーゼ遊離抑制作用を有することが好ましく、本発明の抗老化剤においては、前記抽出物が、コラーゲン産生促進作用、線維芽細胞増殖促進作用及び表皮角化細胞増殖促進作用の群から選ばれた1種又は2種以上の作用を有することが好ましい。   In the anti-inflammatory agent of the present invention, the extract preferably has a platelet aggregation inhibitory action and / or hexosaminidase release inhibitory action. In the anti-aging agent of the invention, the extract contains: It is preferable to have one or more actions selected from the group of collagen production promoting action, fibroblast proliferation promoting action and epidermal keratinocyte proliferation promoting action.

本発明によれば、土貝母からの抽出物が有するTNF−α産生促進作用、血小板凝集抑制作用、ヘキソサミニダーゼ遊離抑制作用、コラーゲン産生促進作用、線維芽細胞増殖促進作用又は表皮角化細胞増殖促進作用に基づく免疫賦活効果、抗炎症効果又は抗老化効果に優れ、安全性の高い免疫賦活剤、抗腫瘍剤、TNF−α産生促進剤、抗炎症剤、抗老化剤及び皮膚化粧料を提供することができる。   According to the present invention, the TNF-α production promoting action, platelet aggregation inhibiting action, hexosaminidase release inhibiting action, collagen production promoting action, fibroblast proliferation promoting action or epidermal keratinization of the extract from the shellfish mother Highly safe immunostimulator, antitumor agent, TNF-α production promoter, anti-inflammatory agent, anti-aging agent, and skin cosmetics that are excellent in immunostimulatory effect, anti-inflammatory effect, or anti-aging effect based on cell growth promoting action Can be provided.

以下、本発明について説明する。
〔免疫賦活剤,抗腫瘍剤,TNF−α産生促進剤,抗炎症剤,抗老化剤〕
本発明の免疫賦活剤、抗腫瘍剤、TNF−α産生促進剤、抗炎症剤又は抗老化剤は、土貝母(学名:Bolbostemma paniculata(Maxim.)Franq.)からの抽出物を有効成分として含有する。
The present invention will be described below.
[Immune stimulant, antitumor agent, TNF-α production promoter, anti-inflammatory agent, anti-aging agent]
The immunostimulant, antitumor agent, TNF-α production promoter, anti-inflammatory agent, or anti-aging agent of the present invention is an extract from a mussel mother (scientific name: Bolbostemma paniculata (Maxim.) Franq.) As an active ingredient. contains.

ここで、本発明において、「土貝母からの抽出物」には、土貝母を抽出原料として得られる抽出液、該抽出液の希釈液若しくは濃縮液、該抽出液を乾燥して得られる乾燥物、又はこれらの粗精製物若しくは精製物のいずれもが含まれる。   Here, in the present invention, “the extract from the shellfish mother” is obtained by drying the extract obtained by using the shellfish mother as an extraction raw material, a diluted or concentrated solution of the extract, and the extract. A dried product, or any of these roughly purified products or purified products is included.

本発明において、抽出原料として使用する植物は、土貝母(学名:Bolbostemma paniculata(Maxim.)Franq.)である。土貝母は、ウリ科の多年生のよじのぼり性草本であり、その鱗茎は数個から十数個の鱗片からなる。土貝母は、中国の遼寧省、河北省、河南省、山東省、山西省等に分布しており、これらの地域から容易に入手することができる。   In the present invention, a plant used as an extraction raw material is a mussel mother (scientific name: Bolbostemma paniculata (Maxim.) Franq.). The mussel mother is a perennial climbing herb of the Cucurbitaceae family, and its bulb consists of several to a dozen scales. The mussels are distributed in Liaoning, Hebei, Henan, Shandong, Shanxi, etc. in China and can be easily obtained from these areas.

抽出原料として使用し得る土貝母の構成部位としては、全草を用いることができ、例えば、葉部、花部、茎部、種子、鱗茎(鱗片)、根部又はこれらの混合物を用いることができる。これらのうち、鱗茎(鱗片)を抽出原料として用いるのが好ましい。   Whole plant can be used as a constituent part of the shellfish mother that can be used as an extraction raw material. For example, a leaf part, a flower part, a stem part, a seed, a bulb (scale), a root part, or a mixture thereof can be used. it can. Among these, it is preferable to use bulbs (scale pieces) as an extraction raw material.

TNF−α産生促進作用、血小板凝集抑制作用、ヘキソサミニダーゼ遊離抑制作用、コラーゲン産生促進作用、線維芽細胞増殖促進作用又は表皮角化細胞増殖促進作用を有する土貝母からの抽出物は、植物の抽出に一般に用いられている抽出方法によって得ることができる。例えば、抽出原料を乾燥した後、そのまま、又は粉砕機を用いて粉砕し、抽出溶媒による抽出に供することにより得ることができる。この際、抽出原料の乾燥は天日で行ってもよいし、通常用いられる乾燥機を使用して行ってもよい。また、土貝母は、ヘキサン等の非極性溶媒によって脱脂等の前処理を施してから抽出原料として使用してもよい。脱脂等の前処理を行うことにより、土貝母の極性溶媒による抽出処理を効率よく行うことができる。   TNF-α production promoting action, platelet aggregation inhibiting action, hexosaminidase release inhibiting action, collagen production promoting action, fibroblast proliferation promoting action or epidermal keratinocyte proliferation promoting action, It can be obtained by an extraction method generally used for plant extraction. For example, it can be obtained by drying the raw material for extraction and pulverizing it as it is or using a pulverizer and subjecting it to extraction with an extraction solvent. At this time, the extraction raw material may be dried in the sun or using a commonly used dryer. In addition, the mussel mother may be used as an extraction raw material after pretreatment such as degreasing with a nonpolar solvent such as hexane. By performing pretreatment such as degreasing, the extraction treatment of the shellfish mother with the polar solvent can be performed efficiently.

抽出溶媒としては、極性溶媒を使用することが好ましく、水若しくは親水性有機溶媒又はこれらの混合液を室温又は溶媒の沸点以下の温度で使用することが特に好ましい。   As the extraction solvent, it is preferable to use a polar solvent, and it is particularly preferable to use water, a hydrophilic organic solvent, or a mixture thereof at room temperature or a temperature not higher than the boiling point of the solvent.

抽出溶媒として使用し得る水は、純水、水道水、井戸水、鉱泉水、鉱水、温泉水、湧水、淡水等の他、これらに各種処理を施したものが含まれる。水に施す処理としては、例えば、精製、加熱、殺菌、滅菌、濾過、イオン交換、浸透圧の調整、緩衝化等が含まれる。したがって、本発明において抽出溶媒として使用し得る水には、精製水、熱水、イオン交換水、生理食塩水、リン酸緩衝液、リン酸緩衝生理食塩水等も含まれる。   The water that can be used as the extraction solvent includes pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, and the like, as well as those subjected to various treatments. Examples of the treatment applied to water include purification, heating, sterilization, sterilization, filtration, ion exchange, adjustment of osmotic pressure, buffering, and the like. Therefore, the water that can be used as the extraction solvent in the present invention includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate buffered saline, and the like.

抽出溶媒として使用し得る親水性有機溶媒としては、例えば、メタノール、エタノール、プロピルアルコール、イソプロピルアルコール等の炭素数1〜5の低級脂肪族アルコール;アセトン、メチルエチルケトン等の低級脂肪族ケトン;1,3−ブチレングリコール、グリセリン、プロピレングリコール等の炭素数2〜5の多価アルコールが挙げられる。   Examples of the hydrophilic organic solvent that can be used as the extraction solvent include lower aliphatic alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol, and isopropyl alcohol; lower aliphatic ketones such as acetone and methyl ethyl ketone; -C2-C5 polyhydric alcohols, such as butylene glycol, glycerol, propylene glycol, are mentioned.

2種以上の極性溶媒の混合液を抽出溶媒として使用する場合、その混合比は適宜調整することができる。例えば、水と低級脂肪族アルコールとの混合液を使用する場合には、水10質量部に対して低級脂肪族アルコール1〜90質量部を混合することが好ましく、水と低級脂肪族ケトンとの混合液を使用する場合には、水10質量部に対して低級脂肪族ケトン1〜40質量部を混合することが好ましく、水と多価アルコールとの混合液を使用する場合には、水10質量部に対して多価アルコール1〜90質量部を混合することが好ましい。   When using the liquid mixture of 2 or more types of polar solvents as an extraction solvent, the mixing ratio can be adjusted suitably. For example, when using a liquid mixture of water and a lower aliphatic alcohol, it is preferable to mix 1 to 90 parts by weight of a lower aliphatic alcohol with respect to 10 parts by weight of water. When using a mixed solution, it is preferable to mix 1 to 40 parts by mass of a lower aliphatic ketone with 10 parts by mass of water, and when using a mixed solution of water and a polyhydric alcohol, water 10 It is preferable to mix 1-90 mass parts of polyhydric alcohol with respect to mass parts.

抽出処理は、土貝母に含有される可溶性成分を抽出溶媒に溶出させ得る限り特に限定されるものではなく、常法に従って行うことができる。抽出処理の際には、特殊な抽出方法を採用する必要はなく、室温又は還流加熱下において任意の装置を使用することができる。   The extraction treatment is not particularly limited as long as the soluble component contained in the shellfish mother can be eluted in the extraction solvent, and can be performed according to a conventional method. In the extraction process, it is not necessary to adopt a special extraction method, and any apparatus can be used at room temperature or under reflux heating.

具体的には、抽出溶媒を満たした処理槽に抽出原料を投入し、必要に応じて時々撹拌しながら、通常30分〜4時間静止して可溶性成分を溶出した後、濾過又は遠心分離により固形物を除去し、抽出液を得る。抽出溶媒量は、通常、抽出原料の5〜15倍量(質量比)である。抽出条件は、抽出溶媒として水を用いた場合には、通常50〜95℃で1〜4時間程度であり、抽出溶媒として水とエタノールとの混合溶媒を用いた場合には、通常40〜80℃で30分〜4時間程度である。   Specifically, the extraction raw material is put into a treatment tank filled with the extraction solvent, and after standing to eluate the soluble components usually for 30 minutes to 4 hours with occasional stirring as necessary, solids are obtained by filtration or centrifugation. Things are removed and an extract is obtained. The amount of extraction solvent is usually 5 to 15 times the mass of the extraction raw material (mass ratio). The extraction conditions are usually about 1 to 4 hours at 50 to 95 ° C. when water is used as the extraction solvent, and usually 40 to 80 when a mixed solvent of water and ethanol is used as the extraction solvent. It is about 30 minutes to 4 hours at ° C.

得られた抽出液から溶媒を留去するとペースト状の濃縮物が得られ、この濃縮物をさらに乾燥すれば、固形の抽出物が得られる。ただし、土貝母からの抽出物は、固形の抽出物にしたものである必要はなく、上記抽出液又はその濃縮液の状態であっても構わない。これら土貝母からの抽出物は、その生理活性の低下を招かない範囲で、活性炭処理、吸着樹脂処理、イオン交換樹脂、液−液向流分配等の方法により精製してから用いてもよい。   When the solvent is distilled off from the obtained extract, a paste-like concentrate is obtained, and when this concentrate is further dried, a solid extract is obtained. However, the extract from the shellfish mother does not need to be a solid extract, and may be in the state of the above extract or its concentrated solution. Extracts from these shellfish mothers may be used after being purified by a method such as activated carbon treatment, adsorption resin treatment, ion exchange resin, liquid-liquid countercurrent distribution, etc., as long as the physiological activity is not reduced. .

土貝母からの抽出物は、好ましくない臭いもなく、抽出物特有の色調も有してないため、そのままでも免疫賦活剤、抗腫瘍剤、TNF−α産生促進剤、抗炎症剤又は抗老化剤として利用可能であるが、濃縮・乾燥した剤形にして使用することもできるし、必要ならば活性の向上や脱色・脱臭を目的とする精製を施したり、任意の助剤と混合して製剤化したりして使用することもできる。   Since the extract from the shellfish mother does not have an unpleasant odor and does not have a color tone peculiar to the extract, as it is, an immunostimulant, an antitumor agent, a TNF-α production promoter, an anti-inflammatory agent or an anti-aging agent Although it can be used as an agent, it can also be used in a concentrated and dried dosage form. If necessary, it can be purified for the purpose of improving its activity, decolorizing or deodorizing, and mixing with any auxiliary agent. It can also be used as a formulation.

以上のようにして得られる土貝母からの抽出物は、TNF−α産生促進作用、血小板凝集抑制作用、ヘキソサミニダーゼ遊離抑制作用、コラーゲン産生促進作用、線維芽細胞増殖促進作用又は表皮角化細胞増殖促進作用を有しているため、それらの作用を利用して免疫賦活剤、抗腫瘍剤、TNF−α産生促進剤、抗炎症剤又は抗老化剤の有効成分として用いることができるとともにと、血小板凝集抑制剤、ヘキソサミニダーゼ遊離抑制剤、コラーゲン産生促進剤、線維芽細胞増殖促進剤又は表皮角化細胞増殖促進剤の有効成分としても用いることができる。   The extract from the shellfish mother obtained as described above has TNF-α production promoting action, platelet aggregation inhibiting action, hexosaminidase release inhibiting action, collagen production promoting action, fibroblast proliferation promoting action or epidermal angle. Since it has an action to promote cell growth, it can be used as an active ingredient of an immunostimulant, an antitumor agent, a TNF-α production promoter, an anti-inflammatory agent, or an anti-aging agent by utilizing these actions. And an active ingredient of a platelet aggregation inhibitor, a hexosaminidase release inhibitor, a collagen production promoter, a fibroblast proliferation promoter or an epidermal keratinocyte proliferation promoter.

本発明の免疫賦活剤、抗腫瘍剤、TNF−α産生促進剤、抗炎症剤又は抗老化剤は、上記土貝母からの抽出物のみからなるものでもよいし、上記土貝母からの抽出物を製剤化したものでもよい。   The immunostimulant, antitumor agent, TNF-α production promoter, anti-inflammatory agent or anti-aging agent of the present invention may consist of only an extract from the above-mentioned shellfish mother, or may be extracted from the above-mentioned shellfish mother. A product may be formulated.

上記土貝母からの抽出物は、デキストリン、シクロデキストリン等の薬学的に許容し得るキャリアーその他任意の助剤を用いて、常法に従い、粉末状、顆粒状、液状等の任意の剤形に製剤化して提供することができ、軟膏剤、外用液剤、貼付剤等として使用することができる。この際、助剤としては、例えば、賦形剤、結合剤、崩壊剤、滑沢剤、安定剤、矯臭剤等を用いることができる。   The extract from the above-mentioned shellfish mother is used in any dosage form such as powder, granule, liquid, etc. according to a conventional method using a pharmaceutically acceptable carrier such as dextrin and cyclodextrin and other optional auxiliaries. It can be provided in the form of a preparation and can be used as an ointment, a liquid for external use, a patch and the like. In this case, as an auxiliary agent, for example, an excipient, a binder, a disintegrant, a lubricant, a stabilizer, a flavoring agent and the like can be used.

なお、本発明の免疫賦活剤、抗腫瘍剤、TNF−α産生促進剤、抗炎症剤又は抗老化剤は、必要に応じて、TNF−α産生促進作用、血小板凝集抑制作用、ヘキソサミニダーゼ遊離抑制作用、コラーゲン産生促進作用、線維芽細胞増殖促進作用又は表皮角化細胞増殖促進作用を有する他の天然抽出物を配合して有効成分として用いることができる。   In addition, the immunostimulant, antitumor agent, TNF-α production promoter, anti-inflammatory agent or anti-aging agent of the present invention may be a TNF-α production promoting action, a platelet aggregation inhibiting action, hexosaminidase, if necessary. Other natural extracts having a release inhibiting action, a collagen production promoting action, a fibroblast proliferation promoting action or an epidermal keratinocyte proliferation promoting action can be blended and used as an active ingredient.

本発明の免疫賦活剤は、有効成分である土貝母からの抽出物が有するTNF−α産生促進作用を通じて、免疫機能を賦活させ、免疫機能の低下等に伴う各種疾病を予防、治療又は改善することができる。ただし、本発明の免疫賦活剤は、これらの用途以外にも、TNF−α産生促進作用を発揮することに意義のあるすべての用途に用いることができる。   The immunostimulant of the present invention activates the immune function through the TNF-α production promoting action of the extract from the shellfish mother, which is an active ingredient, and prevents, treats or improves various diseases associated with a decrease in the immune function. can do. However, the immunostimulant of this invention can be used for all the uses meaningful in exhibiting a TNF- (alpha) production promotion effect besides these uses.

本発明の抗腫瘍剤は、有効成分である土貝母からの抽出物が有するTNF−α産生促進作用を通じて、悪性腫瘍の増殖を抑制することができる。ただし、本発明の抗腫瘍剤は、これらの用途以外にも、TNF−α産生促進作用を発揮することに意義のあるすべての用途に用いることができる。   The antitumor agent of the present invention can suppress the growth of malignant tumors through the TNF-α production promoting action of the extract from the shellfish mother, which is an active ingredient. However, the antitumor agent of the present invention can be used for all uses other than these uses, which are meaningful for exerting a TNF-α production promoting action.

本発明のTNF−α産生促進剤は、有効成分である土貝母からの抽出物が有するTNF−α産生促進作用を通じて、TNF−αの産生を促進することができる。ただし、本発明のTNF−α産生促進剤は、これらの用途以外にも、TNF−α産生促進作用を発揮することに意義のあるすべての用途に用いることができる。   The TNF-α production promoter of the present invention can promote the production of TNF-α through the TNF-α production promoting action of the extract from the shellfish mother, which is an active ingredient. However, the TNF-α production promoter of the present invention can be used for all uses other than these uses, which are meaningful for exerting a TNF-α production promoting action.

本発明の抗炎症剤は、有効成分である土貝母からの抽出物が有する血小板凝集抑制作用及び/又はヘキソサミニダーゼ遊離抑制作用を通じて、炎症性疾患等を抑制することができる。ただし、本発明の抗炎症剤は、これらの用途以外にも、血小板凝集抑制作用及び/又はヘキソサミニダーゼ遊離抑制作用を発揮することに意義のあるすべての用途に用いることができる。   The anti-inflammatory agent of the present invention can suppress inflammatory diseases and the like through the platelet aggregation inhibitory action and / or hexosaminidase release inhibitory action of the extract from the shellfish mother, which is an active ingredient. However, the anti-inflammatory agent of the present invention can be used for all uses other than these uses, which are meaningful for exhibiting the platelet aggregation inhibitory action and / or hexosaminidase release inhibitory action.

本発明の抗老化剤は、有効成分である土貝母からの抽出物が有するコラーゲン産生促進作用、線維芽細胞増殖促進作用及び表皮角化細胞増殖促進作用の群から選ばれた1種又は2種以上の作用を通じて、皮膚の老化症状の予防、治療又は改善をすることができる。ただし、本発明の抗老化剤は、これらの用途以外にも、コラーゲン産生促進作用、線維芽細胞増殖促進作用及び表皮角化細胞増殖促進作用の群から選ばれた1種又は2種以上の作用を発揮することに意義のあるすべての用途に用いることができる。   The anti-aging agent of the present invention is one or two selected from the group of collagen production promoting action, fibroblast growth promoting action and epidermal keratinocyte growth promoting action of an extract from a shellfish mother as an active ingredient Through the action of more than one species, skin aging symptoms can be prevented, treated or ameliorated. However, the anti-aging agent of the present invention has one or more actions selected from the group of collagen production promoting action, fibroblast proliferation promoting action and epidermal keratinocyte proliferation promoting action in addition to these uses. It can be used for all purposes that are meaningful for exhibiting.

〔皮膚化粧料〕
上記土貝母からの抽出物は、皮膚に適用した場合の使用感と安全性に優れているため、皮膚化粧料に配合するのに好適である。この場合、土貝母からの抽出物又は土貝母の鱗片からの抽出物をそのまま配合してもよいし、土貝母からの抽出物から製剤化した免疫賦活剤、抗腫瘍剤、TNF−α産生促進剤、抗炎症剤又は抗老化剤を配合してもよい。上記土貝母からの抽出物又は土貝母の鱗片からの抽出物を皮膚化粧料に配合することによって、皮膚化粧料にTNF−α産生促進作用、血小板凝集抑制作用、ヘキソサミニダーゼ遊離抑制作用、コラーゲン産生促進作用、線維芽細胞増殖促進作用又は表皮角化細胞増殖促進作用を付与することができる。
[Skin cosmetic]
Since the extract from the above-mentioned shellfish mother is excellent in the feeling of use and safety when applied to the skin, it is suitable for blending into skin cosmetics. In this case, an extract from the shellfish mother or an extract from the scale of the shellfish mother may be added as it is, or an immunostimulant, an antitumor agent, TNF- formulated from the extract from the shellfish mother. You may mix | blend alpha production promoter, an anti-inflammatory agent, or an anti-aging agent. By blending the extract from the above-mentioned shellfish mother or the scale from the shellfish mother scale into the skin cosmetics, TNF-α production promoting action, platelet aggregation inhibitory action, hexosaminidase release inhibition to the skin cosmetics Action, collagen production promoting action, fibroblast proliferation promoting action or epidermal keratinocyte proliferation promoting action can be imparted.

上記土貝母からの抽出物又は土貝母の鱗片からの抽出物を配合し得る皮膚化粧料としては、特に限定されるものではなく、例えば、軟膏、クリーム、乳液、ローション、パック、リップ、入浴剤、ヘアートニック、ヘアーローション、石鹸、ボディーシャンプー等が挙げられる。   The skin cosmetic that can be blended with the extract from the shellfish mother or the shellfish mother scale is not particularly limited, for example, ointment, cream, emulsion, lotion, pack, lip, Examples include bath salts, hair nicks, hair lotions, soaps, body shampoos and the like.

上記土貝母からの抽出物又は土貝母の鱗片からの抽出物を皮膚化粧料に配合する場合、その配合量は、皮膚化粧料の種類等によって適宜調整することができるが、好適な配合率は、標準的な抽出物に換算して約0.0001〜10質量%である。   When the extract from the shellfish mother or the extract from the scale of the shellfish mother is blended in the skin cosmetic, the blending amount can be appropriately adjusted depending on the type of the skin cosmetic, etc. The rate is about 0.0001 to 10% by weight in terms of standard extract.

本発明の皮膚化粧料は、土貝母からの抽出物又は土貝母の鱗片からの抽出物が有するTNF−α産生促進作用、血小板凝集抑制作用、ヘキソサミニダーゼ遊離抑制作用、コラーゲン産生促進作用、線維芽細胞増殖促進作用又は表皮角化細胞増殖促進作用を妨げない限り、通常の皮膚化粧料の製造に用いられる主剤、助剤又はその他の成分、例えば、収斂剤、殺菌・抗菌剤、美白剤、紫外線吸収剤、保湿剤、細胞賦活剤、消炎・抗アレルギー剤、抗酸化・活性酸素除去剤、油脂類、ロウ類、炭化水素類、脂肪酸類、アルコール類、エステル類、界面活性剤、香料等を併用することができる。このように併用することにより、より一般性のある製品となり、また、それにより、併用された他の有効成分との間の相乗作用が通常期待される以上の優れた効果をもたらすことがある。   The skin cosmetics of the present invention have a TNF-α production promoting action, platelet aggregation inhibiting action, hexosaminidase release inhibiting action, collagen production promoting possessed by an extract from a shellfish mother or an extract from a scale of a shellfish mother. As long as it does not interfere with the action, the fibroblast proliferation promoting action or the epidermal keratinocyte proliferation promoting action, the main ingredients, auxiliaries or other components used in the production of normal skin cosmetics, such as astringents, bactericidal / antibacterial agents, Whitening agent, UV absorber, moisturizer, cell activator, anti-inflammatory / antiallergic agent, antioxidant / active oxygen remover, fats and oils, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants A fragrance or the like can be used in combination. By using in combination, it becomes a more general product, and thereby a synergistic effect with other active ingredients used in combination may lead to a superior effect than would normally be expected.

なお、本発明の免疫賦活剤、抗腫瘍剤、TNF−α産生促進剤、抗炎症剤、抗老化剤又は皮膚化粧料は、ヒトに対して好適に適用されるものであるが、それぞれの作用効果が奏される限り、ヒト以外の動物に対して適用することもできる。   Note that the immunostimulant, antitumor agent, TNF-α production promoter, anti-inflammatory agent, anti-aging agent or skin cosmetic of the present invention is preferably applied to humans. As long as the effect is exhibited, it can be applied to animals other than humans.

以下、製造例及び試験例を示し、本発明を具体的に説明するが、本発明は下記の各例に何ら制限されるものではない。   Hereinafter, although a manufacture example and a test example are shown and this invention is demonstrated concretely, this invention is not restrict | limited to each following example at all.

〔製造例1〕土貝母抽出物の製造
細切りにした土貝母の鱗片200gに抽出溶媒2000Lを加え、還流抽出器で80℃の温度条件下にて2時間加熱抽出し、熱時濾過した。残渣についてさらに同様の抽出処理を行った。得られた抽出液を合わせて、減圧下に濃縮し、乾燥して土貝母抽出物を得た。抽出溶媒として、水、50質量%エタノール(水とエタノールとの質量比=1:1)、80質量%エタノール(水とエタノールとの質量比=1:4)を用いたときの各抽出物の収率を表1に示す。
[Production Example 1] Manufacture of mussel mother extract To 200 g of shredded mussel mother scale, 2000 L of extraction solvent was added, and the mixture was heated and extracted with a reflux extractor at 80 ° C for 2 hours, and filtered while hot. . The same extraction process was further performed on the residue. The obtained extracts were combined, concentrated under reduced pressure, and dried to obtain a shellfish mother extract. As the extraction solvent, water, 50% by mass ethanol (mass ratio of water and ethanol = 1: 1), 80% by mass ethanol (mass ratio of water and ethanol = 1: 4) The yield is shown in Table 1.

[表1]
試 料 抽出溶媒 抽出物収率(%)
1 水 52.3
2 50%エタノール 49.5
3 80%エタノール 40.1
[Table 1]
Sample extraction solvent extract yield (%)
1 Water 52.3
2 50% ethanol 49.5
3 80% ethanol 40.1

〔試験例1〕TNF−α産生促進作用試験
製造例1で得られた土貝母抽出物(試料1〜3)について、以下のようにしてTNF−α産生促進作用を試験した。
[Test Example 1] Test for promoting TNF-α production The TNF-α production promoting action was tested for the shellfish mother extracts (Samples 1 to 3) obtained in Production Example 1 as follows.

マウスマクロファージ細胞(RAW264.7)を、10%FBS含有DMEM培地を用いて培養した後、セルスクレーパーにより細胞を回収した。回収した細胞を1.0×10cells/mLの細胞密度になるように10%FBS含有DMEMで希釈した後、96ウェルプレートに1ウェルあたり100μLずつ播種し、4時間培養した。 Mouse macrophage cells (RAW264.7) were cultured using 10% FBS-containing DMEM medium, and then the cells were collected with a cell scraper. The collected cells were diluted with 10% FBS-containing DMEM to a cell density of 1.0 × 10 6 cells / mL, then seeded at 100 μL per well in a 96-well plate, and cultured for 4 hours.

培養終了後、培地を抜き、終濃度1%のDMSOを含む10%FBS含有DMEMで試料を溶解した試料溶液(試料濃度:50μg/mL)を各ウェルに200μLずつ添加し、24時間培養した。培養終了後、各ウェルの培養上清中のTNF−α量をサンドイッチELISA法により測定した。同様に、試料溶液を添加せずに培養した培養上清中のTNF−α量についても測定した。得られた結果から、以下の式により、試料溶液添加時のTNF−α産生促進率を算出した。   After completion of the culture, the medium was removed, and 200 μL of a sample solution (sample concentration: 50 μg / mL) in which the sample was dissolved in 10% FBS-containing DMEM containing DMSO having a final concentration of 1% was added to each well and cultured for 24 hours. After completion of the culture, the amount of TNF-α in the culture supernatant of each well was measured by a sandwich ELISA method. Similarly, the amount of TNF-α in the culture supernatant cultured without adding the sample solution was also measured. From the obtained results, the TNF-α production promotion rate at the time of adding the sample solution was calculated by the following formula.

TNF−α産生促進率(%)=A/B×100
ただし、上記式において、Aは「試料溶液添加時のTNF−α量」を、Bは「試料溶液無添加時のTNF−α量」を示す。
上記試験の結果を、表2に示す。
TNF-α production promotion rate (%) = A / B × 100
In the above formula, A indicates “TNF-α amount when sample solution is added” and B indicates “TNF-α amount when no sample solution is added”.
The results of the above test are shown in Table 2.

[表2]
試 料 抽出溶媒 TNF−α産生促進率(%)
1 水 142.1±5.5
2 50%エタノール 163.5±8.9
3 80%エタノール 132.2±9.4
[Table 2]
Sample extraction solvent TNF-α production promotion rate (%)
1 Water 142.1 ± 5.5
2 50% ethanol 163.5 ± 8.9
3 80% ethanol 132.2 ± 9.4

表2に示すように、土貝母抽出物は、優れたTNF−α産生促進作用を有することが確認された。   As shown in Table 2, it was confirmed that the shellfish mother extract has an excellent TNF-α production promoting action.

〔試験例2〕血小板凝集抑制作用試験
製造例1で得られた土貝母抽出物(試料1〜3)について、以下のようにして血小板凝集抑制作用を試験した。
[Test Example 2] Platelet aggregation inhibitory action test The shellfish mother extract (samples 1 to 3) obtained in Production Example 1 was tested for platelet aggregation inhibitory action as follows.

(1)血小板浮遊液の調製
採血したウサギの血液に77mmol/LのEDTA(pH7.4)を1/10量加えて、遠心(180×g,10分,室温)して血小板浮遊液を得た。さらに遠心(810×g,10分,4℃)して、上清を除去して血小板を得た。これを血小板洗浄液(0.15mol/Lの塩化ナトリウム,0.15mol/Lのトリス−塩酸緩衝液(pH7.4)及び77mmol/LのEDTA溶液(pH7.4)を90:8:2で混合)に浮遊させ、上記と同様に遠心して、得られた血小板を145mmol/Lの塩化ナトリウム、5mmol/Lの塩化カリウム及び5.5mmol/Lのグルコースを含む10mmol/LのHEPES緩衝液(pH7.4)に浮遊させて血小板数を調整(3.0×10cells/μL)し、洗浄血小板浮遊液とした。
(1) Preparation of platelet suspension 1/10 amount of 77 mmol / L EDTA (pH 7.4) was added to the collected rabbit blood and centrifuged (180 × g, 10 minutes, room temperature) to obtain a platelet suspension. It was. Further, centrifugation (810 × g, 10 minutes, 4 ° C.) was performed, and the supernatant was removed to obtain platelets. This was mixed with platelet washing solution (0.15 mol / L sodium chloride, 0.15 mol / L Tris-HCl buffer (pH 7.4) and 77 mmol / L EDTA solution (pH 7.4) at 90: 8: 2. The resulting platelets were centrifuged in the same manner as described above, and the obtained platelets were mixed with 10 mmol / L HEPES buffer (pH 7. 5) containing 145 mmol / L sodium chloride, 5 mmol / L potassium chloride and 5.5 mmol / L glucose. 4), the number of platelets was adjusted (3.0 × 10 5 cells / μL) to obtain a washed platelet suspension.

(2)血小板凝集抑制作用試験
上記洗浄血小板浮遊液223μLに200mmol/Lの塩化カルシウム溶液1μLを加え、37℃の温度条件下で1分間反応させた。この反応溶液に試料溶液1μLを加え、さらに2分間反応させ、攪拌子を入れて1分間攪拌した後、コラーゲン溶液を25μL添加して、37℃の温度条件下で10分間血小板凝集率を測定した。また、コントロールとして試料溶液を添加しない以外は同様にして血小板凝集率を測定した。得られた結果から、下記の式により血小板凝集抑制率(%)を算出した。
(2) Test for inhibiting platelet aggregation 1 μL of a 200 mmol / L calcium chloride solution was added to 223 μL of the washed platelet suspension and allowed to react at 37 ° C. for 1 minute. Add 1 μL of the sample solution to this reaction solution, react for another 2 minutes, add a stir bar and stir for 1 minute, add 25 μL of collagen solution, and measure the platelet aggregation rate for 10 minutes at 37 ° C. . As a control, the platelet aggregation rate was measured in the same manner except that no sample solution was added. From the obtained results, the platelet aggregation inhibition rate (%) was calculated by the following formula.

血小板凝集抑制率(%)=(A−B)/A×100
ただし、上記式において、Aは「試料無添加時の凝集率」を、Bは「試料添加時の凝集率」を示す。
Platelet aggregation inhibition rate (%) = (A−B) / A × 100
In the above formula, A indicates “aggregation rate when no sample is added” and B indicates “aggregation rate when a sample is added”.

試料溶液の濃度を段階的に減少させて、上記血小板凝集抑制率を算出し、血小板凝集抑制率が50%になる試料濃度IC50(μg/mL;ppm)の値を内挿法により算出した。
上記試験の結果を表3に示す。
The concentration of the sample solution was decreased stepwise to calculate the platelet aggregation inhibition rate, and the value of sample concentration IC 50 (μg / mL; ppm) at which the platelet aggregation inhibition rate was 50% was calculated by interpolation. .
The results of the above test are shown in Table 3.

[表3]
試 料 抽出溶媒 IC 50 (μg/mL)
1 水 395
2 50%エタノール 388
3 80%エタノール 400
[Table 3]
Sample extraction solvent IC 50 (μg / mL)
1 water 395
2 50% ethanol 388
3 80% ethanol 400

表3に示すように、土貝母抽出物は、優れた血小板凝集抑制作用を有することが確認された。   As shown in Table 3, it was confirmed that the shellfish mother extract has an excellent platelet aggregation inhibitory action.

〔試験例3〕ヘキソサミニダーゼ遊離抑制作用試験
製造例1で得られた土貝母抽出物(試料1〜3)について、以下のようにしてヘキソサミニダーゼ遊離抑制作用を試験した。
[Test Example 3] Hexosaminidase release inhibitory action test With respect to the shellfish mother extract (Samples 1 to 3) obtained in Production Example 1, the hexosaminidase release inhibitory action was tested as follows.

ラット好塩基球白血病細胞(RBL−2H3)を15%FBS含有S−MEM培地を用いて培養した後、トリプシン処理により細胞を回収した。回収した細胞を4.0×10cells/mLの細胞密度になるように希釈し、DNP-specific IgEの終濃度が0.5μg/mLとなるようにDNP-specific IgEを添加した後、96ウェルプレートに1ウェルあたり100μLずつ播種し、一晩培養した。培養終了後、培地を抜き、シラガニアン緩衝液500μLにて洗浄を2回行った。 Rat basophil leukemia cells (RBL-2H3) were cultured in S-MEM medium containing 15% FBS, and then cells were collected by trypsin treatment. The collected cells are diluted to a cell density of 4.0 × 10 5 cells / mL, and after adding DNP-specific IgE so that the final concentration of DNP-specific IgE is 0.5 μg / mL, 96 100 μL per well was seeded on a well plate and cultured overnight. After completion of the culture, the medium was removed and washing was performed twice with 500 μL of Silaganian buffer.

次に、シラガニアン緩衝液30μL及び同緩衝液に溶解した試料溶液10μLを加え、37℃の温度条件下にて10分間静置した。その後、100ng/mLのDNP−BSA溶液10μLを加え、37℃の温度条件下にて15分間静置し、ヘキソサミニダーゼを遊離させた。その後、96ウェルプレートを氷上に静置することによりヘキソサミニダーゼの遊離を停止させた。各ウェルの細胞上清10μL及び1mmol/Lのp−ニトロフェニル−N−アセチル−α−D−グルコサミニド(p−NAG)溶液10μLを、新たな96ウェルプレートに添加し、37℃の温度条件下で1時間反応させた。反応終了後、各ウェルに0.1mol/LのNaCO/NaHCO250μLを加え、波長415nmにおける吸光度を測定した。同様にして試料を添加せずに、細胞上清10μLと0.1mol/LのNaCO/NaHCO250μLとの混合液の吸光度を測定した。また、同様にして試料を添加しp−NAGを添加せずに吸光度を測定した。得られた結果から、下記の式によりヘキソサミニダーゼ遊離抑制率(%)を算出した。 Next, 30 μL of the Silaganian buffer and 10 μL of the sample solution dissolved in the same buffer were added, and the mixture was allowed to stand at 37 ° C. for 10 minutes. Thereafter, 10 μL of a 100 ng / mL DNP-BSA solution was added, and the mixture was allowed to stand at 37 ° C. for 15 minutes to release hexosaminidase. Thereafter, the release of hexosaminidase was stopped by allowing the 96-well plate to stand on ice. 10 μL of cell supernatant in each well and 10 μL of 1 mmol / L p-nitrophenyl-N-acetyl-α-D-glucosaminide (p-NAG) solution were added to a new 96-well plate, and the temperature was maintained at 37 ° C. For 1 hour. After completion of the reaction, 250 μL of 0.1 mol / L Na 2 CO 3 / NaHCO 3 was added to each well, and the absorbance at a wavelength of 415 nm was measured. Similarly, the absorbance of a mixed solution of 10 μL of cell supernatant and 250 μL of 0.1 mol / L Na 2 CO 3 / NaHCO 3 was measured without adding a sample. Similarly, the sample was added and the absorbance was measured without adding p-NAG. From the obtained results, the hexosaminidase release inhibition rate (%) was calculated by the following formula.

ヘキソサミニダーゼ遊離抑制率(%)={1−(B−C)/A}×100
ただし、上記式において、Aは「試料無添加時の吸光度」を、Bは「試料添加時の吸光度」を、Cは「試料添加・p−NAG無添加時の吸光度」を示す。
Inhibition rate of hexosaminidase release (%) = {1− (B−C) / A} × 100
In the above formula, A indicates “absorbance when no sample is added”, B indicates “absorbance when sample is added”, and C indicates “absorbance when sample is added and p-NAG is not added”.

試料溶液の濃度を段階的に減少させて、上記ヘキソサミニダーゼ遊離抑制率を算出し、ヘキソサミニダーゼ遊離抑制率が50%になる試料濃度IC50(μg/mL;ppm)の値を内挿法により算出した。
上記試験の結果を表4に示す。
The concentration of the sample solution is decreased stepwise to calculate the hexosaminidase release inhibition rate, and the sample concentration IC 50 (μg / mL; ppm) at which the hexosaminidase release inhibition rate is 50% is calculated. Calculated by interpolation.
Table 4 shows the results of the above test.

[表4]
試 料 抽出溶媒 IC 50 (μg/mL)
1 水 225
2 50%エタノール 198
3 80%エタノール 210
[Table 4]
Sample extraction solvent IC 50 (μg / mL)
1 water 225
2 50% ethanol 198
3 80% ethanol 210

表4に示すように、土貝母抽出物は、優れたヘキソサミニダーゼ遊離抑制作用を有することが確認された。   As shown in Table 4, it was confirmed that the shellfish mother extract has an excellent hexosaminidase release inhibitory action.

〔試験例4〕コラーゲン産生促進作用試験
製造例1で得られた土貝母抽出物(試料1〜3)について、以下のようにしてコラーゲン産生促進作用を試験した。
[Test Example 4] Collagen production promoting action test The shellfish mother extract obtained in Production Example 1 (Samples 1 to 3) was tested for collagen production promoting action as follows.

ヒト正常線維芽細胞(Detroit 551)を10%FBS、1%NEAA及び1mmol/Lのピルビン酸ナトリウムを含有するMEM培地を用いて培養した後、トリプシン処理により細胞を回収した。回収した細胞を2×10cells/mLの細胞密度になるように上記MEM培地で希釈した後、96ウェルマイクロプレートに1ウェルあたり100μLずつ播種し、一晩培養した。培養終了後、培地を抜き、0.5%FBS含有MEM培地に試料を溶解した試料溶液(試料濃度:50μg/mL)を各ウェルに150μLずつ添加し、3日間培養した。培養終了後、各ウェルの培地中のコラーゲン量をELISA法により測定した。同様にして試料溶液を添加せずにコラーゲン量を測定した。得られた結果から、下記の式により、試料溶液添加時のコラーゲン産生促進率を算出した。 Human normal fibroblasts (Detroit 551) were cultured in MEM medium containing 10% FBS, 1% NEAA and 1 mmol / L sodium pyruvate, and then cells were collected by trypsin treatment. The collected cells were diluted with the above-mentioned MEM medium so that the cell density was 2 × 10 5 cells / mL, then seeded at 100 μL per well in a 96-well microplate, and cultured overnight. After completion of the culture, the medium was removed, and 150 μL of a sample solution (sample concentration: 50 μg / mL) in which the sample was dissolved in a 0.5% FBS-containing MEM medium was added to each well and cultured for 3 days. After completion of the culture, the amount of collagen in the medium of each well was measured by ELISA. Similarly, the amount of collagen was measured without adding the sample solution. From the obtained results, the collagen production promotion rate when the sample solution was added was calculated by the following formula.

コラーゲン産生促進率(%)=A/B×100
ただし、上記式において、Aは「試料溶液添加時のコラーゲン量」を、Bは「試料溶液無添加時のコラーゲン量」を示す。
上記試験の結果を表5に示す。
Collagen production promotion rate (%) = A / B × 100
In the above formula, A represents “collagen amount when sample solution is added”, and B represents “collagen amount when sample solution is not added”.
The results of the above test are shown in Table 5.

[表5]
試 料 抽出溶媒 コラーゲン産生促進率(%)
1 水 122.2±8.2
2 50%エタノール 153.6±10.5
3 80%エタノール 145.9±4.6
[Table 5]
Sample extraction solvent Collagen production promotion rate (%)
1 Water 122.2 ± 8.2
2 50% ethanol 153.6 ± 10.5
3 80% ethanol 145.9 ± 4.6

表5に示すように、土貝母抽出物は、優れたコラーゲン産生促進作用を有することが確認された。   As shown in Table 5, it was confirmed that the shellfish mother extract has an excellent collagen production promoting action.

〔試験例5〕線維芽細胞増殖促進作用試験
製造例1で得られた土貝母抽出物(試料1〜3)について、以下のようにして線維芽細胞増殖促進作用を試験した。
[Test Example 5] Fibroblast proliferation promoting action test The shellfish mother extract (Samples 1 to 3) obtained in Production Example 1 was tested for the fibroblast proliferation promoting action as follows.

ヒト正常皮膚線維芽細胞(NB1RGB)を10%FBS含有α−MEMを用いて培養した後、トリプシン処理により細胞を回収した。回収した細胞を7.0×10cells/mLの細胞密度になるように、5%FBS含有α―MEMで希釈した後、96ウェルプレートに1ウェルあたり100μLずつ播種し、一晩培養した。培養終了後、5%FBS含有α−MEMで試料を溶解した試料溶液(試料濃度:12.5μg/mL)を各ウェルに100μL添加し、3日間培養した。 Human normal skin fibroblasts (NB1RGB) were cultured using α-MEM containing 10% FBS, and then cells were collected by trypsin treatment. The collected cells were diluted with α-MEM containing 5% FBS so that the cell density would be 7.0 × 10 4 cells / mL, then seeded at 100 μL per well in a 96-well plate and cultured overnight. After completion of the culture, 100 μL of a sample solution (sample concentration: 12.5 μg / mL) in which the sample was dissolved with 5% FBS-containing α-MEM was added to each well and cultured for 3 days.

線維芽細胞増殖作用は、MTTアッセイ法を用いて測定した。培養終了後、各ウェルから100μLずつ培地を抜き、終濃度5mg/mLでPBS(−)に溶解したMTTを各ウェルに20μLずつ添加した。4.5時間培養した後に、10%SDSを溶解した0.01mol/mLの塩酸溶液を各ウェルに100μLずつ添加し、一晩培養した後、波長570nmにおける吸光度を測定した。同様にして波長650nmにおける吸光度を測定し、両吸光度の差をもってブルーホルマザン生成量とした。また、同様の方法で空試験を行い補正した。補正後の各吸光度から、下記の式により線維芽細胞増殖促進率(%)を算出した。   Fibroblast proliferation was measured using the MTT assay. After completion of the culture, 100 μL of the medium was removed from each well, and 20 μL of MTT dissolved in PBS (−) at a final concentration of 5 mg / mL was added to each well. After culturing for 4.5 hours, 100 μL of a 0.01 mol / mL hydrochloric acid solution in which 10% SDS was dissolved was added to each well and incubated overnight, and then the absorbance at a wavelength of 570 nm was measured. Similarly, the absorbance at a wavelength of 650 nm was measured, and the difference between both absorbances was defined as the amount of blue formazan produced. In addition, a blank test was performed and corrected in the same manner. From each corrected absorbance, the fibroblast proliferation promotion rate (%) was calculated by the following formula.

線維芽細胞増殖促進率(%)=(St−Sb)/(Ct−Cb)×100
ただし、上記式において、Stは「試料溶液添加・細胞添加時の吸光度」を示し、Sbは「試料溶液添加・細胞無添加時の吸光度」を示し、Ctは「試料溶液無添加・細胞添加時の吸光度」を示し、Cbは「試料溶液無添加・細胞無添加時の吸光度」を示す。
上記試験の結果を表6に示す。
Fibroblast proliferation promotion rate (%) = (St−Sb) / (Ct−Cb) × 100
However, in the above formula, St indicates “absorbance when sample solution is added / cells are added”, Sb indicates “absorbance when sample solution is added / no cells are added”, and Ct is “when sample solution is not added / cells are added” "Cb" indicates "absorbance when no sample solution is added and cells are not added".
The results of the above test are shown in Table 6.

[表6]
試 料 抽出溶媒 線維芽細胞増殖促進率(%)
1 水 108.8±6.3
2 50%エタノール 112.5±2.5
3 80%エタノール 102.5±7.2
[Table 6]
Sample extraction solvent Fibroblast growth promotion rate (%)
1 water 108.8 ± 6.3
2 50% ethanol 112.5 ± 2.5
3 80% ethanol 102.5 ± 7.2

表6に示すように、土貝母抽出物は、優れた線維芽細胞増殖促進作用を有することが確認された。   As shown in Table 6, it was confirmed that the shellfish mother extract has an excellent fibroblast proliferation promoting action.

〔試験例6〕表皮角化細胞増殖促進作用試験
製造例1で得られた土貝母抽出物(試料1〜3)について、以下のようにして表皮角化細胞増殖促進作用を試験した。
[Test Example 6] Epidermal keratinocyte proliferation promoting action test The shellfish mother extract (Samples 1 to 3) obtained in Production Example 1 was tested for epidermal keratinocyte proliferation promoting action as follows.

正常ヒト皮膚表皮角化細胞(NHEK)を、正常ヒト表皮角化細胞用培地(KGM)を用いて培養した後、トリプシン処理により細胞を回収した。回収した細胞を1.5×10cells/mLの細胞密度になるようにKGMで希釈した後、96ウェルプレートに1ウェルあたり200μLずつ播種し、一晩培養した。培養終了後、培地を抜き、KGMで試料を溶解した試料溶液(試料濃度:3.125μg/mL)を各ウェルに200μL添加し、3日間培養した。 Normal human skin epidermal keratinocytes (NHEK) were cultured using normal human epidermal keratinocyte medium (KGM), and then cells were collected by trypsin treatment. The collected cells were diluted with KGM to a cell density of 1.5 × 10 4 cells / mL, and then seeded at 200 μL per well in a 96-well plate and cultured overnight. After completion of the culture, 200 μL of a sample solution (sample concentration: 3.125 μg / mL) in which the sample was dissolved with KGM was added to each well and cultured for 3 days.

表皮角化細胞増殖作用は、MTTアッセイ法を用いて測定した。培養終了後、培地を抜き、終濃度0.4mg/mLでPBS(−)に溶解したMTTを各ウェルに200μLずつ添加した。2時間培養した後に、細胞内に生成したブルーホルマザンを2−プロパノール200μLで抽出した。抽出後、波長570nmにおける吸光度を測定した。同時に濁度として波長650nmにおける吸光度を測定し、両者の差をもってブルーホルマザン生成量とした。測定された各吸光度から、下記の式により表皮角化細胞増殖促進率(%)を算出した。   Epidermal keratinocyte proliferation was measured using the MTT assay. After completion of the culture, the medium was removed, and 200 μL of MTT dissolved in PBS (−) at a final concentration of 0.4 mg / mL was added to each well. After culturing for 2 hours, blue formazan produced in the cells was extracted with 200 μL of 2-propanol. After extraction, the absorbance at a wavelength of 570 nm was measured. At the same time, the absorbance at a wavelength of 650 nm was measured as turbidity, and the difference between the two was used as the amount of blue formazan produced. From each measured absorbance, the epidermal keratinocyte proliferation promotion rate (%) was calculated by the following formula.

表皮角化細胞増殖促進率(%)=St/Ct×100
ただし、上記式において、Stは「試料溶液添加時の吸光度」を、Ctは「試料溶液無添加時の吸光度」を示す。
上記試験の結果を、表7に示す。
Epidermal keratinocyte proliferation promotion rate (%) = St / Ct × 100
In the above formula, St represents “absorbance when the sample solution is added”, and Ct represents “absorbance when no sample solution is added”.
The results of the above test are shown in Table 7.

[表7]
試 料 抽出溶媒 表皮角化細胞増殖促進率(%)
1 水 115.7±3.6
2 50%エタノール 122.2±0.8
3 80%エタノール 111.6±5.1
[Table 7]
Sample extraction solvent Epidermal keratinocyte growth promotion rate (%)
1 water 115.7 ± 3.6
2 50% ethanol 122.2 ± 0.8
3 80% ethanol 111.6 ± 5.1

表7に示すように、土貝母からの抽出物は、優れた表皮角化細胞増殖促進作用を有することが確認された。   As shown in Table 7, it was confirmed that the extract from the shellfish mother has an excellent epidermal keratinocyte proliferation promoting action.

本発明の免疫賦活剤、抗腫瘍剤及びTNF−α産生促進剤は、悪性腫瘍細胞の増殖の抑制に、本発明の抗炎症剤は、炎症性疾患等の予防、治療又は改善に、本発明の抗老化剤は、皮膚の老化症状等の予防、治療又は改善に有用である。
The immunostimulant, antitumor agent and TNF-α production promoter of the present invention are used to suppress the growth of malignant tumor cells, and the anti-inflammatory agent of the present invention is used to prevent, treat or improve inflammatory diseases. This anti-aging agent is useful for the prevention, treatment or improvement of skin aging symptoms and the like.

Claims (9)

土貝母(Bolbostemma paniculata(Maxim.)Franq.)からの抽出物を有効成分として含有することを特徴とする免疫賦活剤。   An immunostimulant characterized by containing an extract from a soil mother (Bolbostemma paniculata (Maxim.) Franq.) As an active ingredient. 土貝母(Bolbostemma paniculata(Maxim.)Franq.)からの抽出物を有効成分として含有することを特徴とする抗腫瘍剤。   An antitumor agent comprising, as an active ingredient, an extract from earthworm mother (Bolbostemma paniculata (Maxim.) Franq.). 土貝母(Bolbostemma paniculata(Maxim.)Franq.)からの抽出物を有効成分として含有することを特徴とするTNF−α産生促進剤。   A TNF-α production promoter characterized by containing, as an active ingredient, an extract from earthworm mother (Bolbostemma paniculata (Maxim.) Franq.). 土貝母(Bolbostemma paniculata(Maxim.)Franq.)からの抽出物を有効成分として含有することを特徴とする抗炎症剤。   An anti-inflammatory agent characterized by containing an extract from a soil mother (Bolbostemma paniculata (Maxim.) Franq.) As an active ingredient. 前記抽出物が、血小板凝集抑制作用及び/又はヘキソサミニダーゼ遊離抑制作用を有することを特徴とする請求項4に記載の抗炎症剤。   The anti-inflammatory agent according to claim 4, wherein the extract has a platelet aggregation inhibitory action and / or a hexosaminidase release inhibitory action. 土貝母(Bolbostemma paniculata(Maxim.)Franq.)からの抽出物を有効成分として含有することを特徴とする抗老化剤。   An anti-aging agent characterized by containing an extract from a soil mother (Bolbostemma paniculata (Maxim.) Franq.) As an active ingredient. 前記抽出物が、コラーゲン産生促進作用、線維芽細胞増殖促進作用及び表皮角化細胞増殖促進作用の群から選ばれた1種又は2種以上の作用を有することを特徴とする請求項6に記載の抗老化剤。   The said extract has 1 type, or 2 or more types of effects chosen from the group of collagen production promotion effect | action, a fibroblast growth promotion effect | action, and an epidermal keratinocyte growth promotion effect | action. Anti-aging agent. 土貝母(Bolbostemma paniculata(Maxim.)Franq.)からの抽出物を配合したことを特徴とする皮膚化粧料。   Skin cosmetic characterized by blending an extract from Dombostemma paniculata (Maxim.) Franq. 土貝母(Bolbostemma paniculata(Maxim.)Franq.)の鱗片からの抽出物を配合したことを特徴とする皮膚化粧料。
A skin cosmetic characterized by containing an extract from scales of a shellfish mother (Bolbostemma paniculata (Maxim.) Franq.).
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JP2016193857A (en) * 2015-03-31 2016-11-17 丸善製薬株式会社 Skin cosmetic, and food and drink
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CN109864911A (en) * 2017-12-01 2019-06-11 伽蓝(集团)股份有限公司 A kind of application for avenging tea extraction

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