JP2016150904A - Antitumor agent - Google Patents
Antitumor agent Download PDFInfo
- Publication number
- JP2016150904A JP2016150904A JP2015027582A JP2015027582A JP2016150904A JP 2016150904 A JP2016150904 A JP 2016150904A JP 2015027582 A JP2015027582 A JP 2015027582A JP 2015027582 A JP2015027582 A JP 2015027582A JP 2016150904 A JP2016150904 A JP 2016150904A
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- JP
- Japan
- Prior art keywords
- cells
- lactic acid
- sulforaphane
- acid bacteria
- tumor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
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Abstract
Description
本発明は、抗腫瘍剤、アポトーシス促進剤、および抗腫瘍効果を有する組成物に関する。詳細には、乳酸菌とスルフォラファンとの組み合わせを含む、抗腫瘍剤、アポトーシス促進剤、および抗腫瘍効果を有する組成物に関する。 The present invention relates to an antitumor agent, an apoptosis promoter, and a composition having an antitumor effect. Specifically, the present invention relates to an antitumor agent, a proapoptotic agent, and a composition having an antitumor effect, comprising a combination of lactic acid bacteria and sulforaphane.
乳酸菌は、生体の免疫反応を活性化する作用、すなわち免疫賦活作用を示すことが知られている。たとえば、ラクトバチルス・ペントーサス（Lactobacillus pentosus）Ｓ−ＰＴ８４株は、免疫担当細胞に作用して、IL-12の発現を誘導する効果を有することが報告されている（非特許文献１、特許文献１、特許文献２）。また、ラクトバチルス・サケイ（Lactobacillus sakei）はTNF-αの発現を誘導する効果を有することが報告されている（非特許文献２）
一方、ブロッコリーのようなアブラナ科の植物に含まれるスルフォラファン（sulforaphane（SFN））は、生体の免疫反応を抑制する作用、すなわち免疫抑制作用を有することが知られている。たとえば、LPS刺激によるNF-κBの活性化をスルフォラファンが抑制することで、IFN-γ、IL-2、IL-17の産生が抑制された（非特許文献３）。また、関節炎誘発モデルマウスの脾細胞、リンパ節細胞にスルフォラファンを添加した結果、IFN-γ、TNF-α、IL-6の発現抑制効果を認め、さらに、免疫抑制型のIL-10の発現誘導効果も認めた（非特許文献４）。さらに、スルフォラファンがNrf2を活性化することで、LPS刺激によるTNF-α、IL-1βの産生が抑制された（非特許文献５）。
Lactic acid bacteria are known to exhibit an action that activates an immune response in a living body, that is, an immunostimulatory action. For example, Lactobacillus pentosus S-PT84 strain has been reported to act on immunocompetent cells to induce the expression of IL-12 (Non-patent Document 1, Patent Document 1). Patent Document 2). Further, Lactobacillus sakei has been reported to have an effect of inducing the expression of TNF-α (Non-patent Document 2).
On the other hand, sulforaphane (SFN) contained in cruciferous plants such as broccoli is known to have an action of suppressing an immune response of a living body, that is, an immunosuppressive action. For example, the production of IFN-γ, IL-2, and IL-17 was suppressed by inhibiting sulforaphane activation of NF-κB by LPS stimulation (Non-patent Document 3). In addition, sulforaphane was added to spleen cells and lymph node cells of arthritis-induced model mice. As a result, IFN-γ, TNF-α and IL-6 expression was suppressed, and immunosuppressive IL-10 expression was induced. The effect was also recognized (nonpatent literature 4). Furthermore, when sulforaphane activated Nrf2, production of TNF-α and IL-1β by LPS stimulation was suppressed (Non-patent Document 5).
スルフォラファンはがん予防効果が期待されている食品由来成分としても知られている（非特許文献６）。たとえば、スルフォラファンが、前立腺癌細胞株において、Bcl-2やcIAP-1, 2とXIAPの発現を減少させることでアポトーシスを誘導したり（非特許文献７）、肝癌細胞株においてBaxの増加、Bcl-2、Bcl-xLの減少を介してアポトーシスを引き起こす（非特許文献８）ことが報告されている。 Sulforaphane is also known as a food-derived component that is expected to have a cancer prevention effect (Non-patent Document 6). For example, sulforaphane induces apoptosis by decreasing the expression of Bcl-2, cIAP-1, 2 and XIAP in prostate cancer cell lines (Non-Patent Document 7), increases in Bax in liver cancer cell lines, Bcl -2, It is reported that apoptosis is caused through a decrease in Bcl-xL (Non-patent Document 8).
本発明は、高い抗腫瘍効果または腫瘍細胞に対する高いアポトーシス促進効果を有する抗腫瘍剤、腫瘍細胞に対するアポトーシス促進剤、および組成物を提供することを目的とする。 An object of the present invention is to provide an antitumor agent having a high antitumor effect or a high apoptosis promoting effect on tumor cells, an apoptosis promoting agent for tumor cells, and a composition.
本発明者らは、免疫賦活作用を有することが知られる乳酸菌であるラクトバチルス・ペントーサスと、それとは逆の作用である免疫抑制作用を有することが知られるスルフォラファンとの組み合わせを、ヒト末梢血単核細胞（PBMC）及びヒト大腸癌細胞の共培養系に添加したところ、まったく予想もしていなかったことに、ラクトバチルス・ペントーサスの免疫賦活作用が増強され、その結果、大腸癌細胞に対して、相加効果を超える相乗的なアポトーシス誘導活性が示されることを見出した。つまり、ラクトバチルス・ペントーサスと、スルフォラファンとの組み合わせが、ラクトバチルス・ペントーサスの免疫賦活作用を増強させ、高い抗腫瘍効果または腫瘍細胞に対する高いアポトーシス促進効果を示すことを見出し、本発明を完成させた。 The inventors of the present invention used a combination of Lactobacillus pentosus, a lactic acid bacterium known to have an immunostimulatory effect, and sulforaphane, which is known to have an immunosuppressive effect, which is the opposite action, to human peripheral blood unit. When added to the co-culture system of nuclear cells (PBMC) and human colon cancer cells, the immunostimulatory action of Lactobacillus pentosas was enhanced, which was not expected at all, and as a result, against colon cancer cells, It was found that a synergistic apoptosis-inducing activity exceeding the additive effect is shown. That is, the inventors found that the combination of Lactobacillus pentosasus and sulforaphane enhances the immunostimulatory action of Lactobacillus pentosasus and exhibits a high antitumor effect or a high apoptosis promoting effect on tumor cells, and completed the present invention. .
すなわち、これに限定されるものではないが、本発明は以下の態様の発明を包含する。
［１］ 乳酸菌とスルフォラファンとを含む、抗腫瘍剤。
［２］ 乳酸菌がラクトバチルス・ペントーサスである、［１］に記載の抗腫瘍剤。
［３］ ラクトバチルス・ペントーサスが、ラクトバチルス・ペントーサスＳ−ＰＴ８４株（受託番号：ＦＥＲＭ ＢＰ−１００２８）である、［２］に記載の抗腫瘍剤。
［４］ 乳酸菌が生菌体、死菌体、または菌体処理物の形態で含まれる、［１］〜［３］のいずれかに記載の抗腫瘍剤。
［５］ 腫瘍が悪性腫瘍である、［１］〜［４］のいずれかに記載の抗腫瘍剤。
［６］ 乳酸菌とスルフォラファンとを含む、腫瘍細胞に対するアポトーシス促進剤。
［７］ 乳酸菌がラクトバチルス・ペントーサスである、［６］に記載のアポトーシス促進剤。
［８］ ラクトバチルス・ペントーサスが、ラクトバチルス・ペントーサスＳ−ＰＴ８４株（受託番号：ＦＥＲＭ ＢＰ−１００２８）である、［７］に記載のアポトーシス促進剤。
［９］ 乳酸菌が生菌体、死菌体、または菌体処理物の形態で含まれる、［６］〜［８］のいずれかに記載のアポトーシス促進剤。
［１０］ 腫瘍が悪性腫瘍である、［６］〜［９］のいずれかに記載のアポトーシス促進剤。
［１１］ 乳酸菌とスルフォラファンとを含む、組成物。
［１２］ 乳酸菌がラクトバチルス・ペントーサスである、［１１］に記載の組成物。
［１３］ ラクトバチルス・ペントーサスが、ラクトバチルス・ペントーサスＳ−ＰＴ８４株（受託番号：ＦＥＲＭ ＢＰ−１００２８）である、［１２］に記載の組成物。
［１４］ 腫瘍細胞の増殖を阻害するための、［１０］〜［１３］のいずれかに記載の組成物。
［１５］ 腫瘍が悪性腫瘍である、［１４］に記載の組成物。
［１６］ 飲食品である、［１１］〜［１５］のいずれかに記載の組成物。
［１７］ 乳酸菌およびスルフォラファンを保健機能成分（関与成分）とする機能表示を付した、［１６］に記載の組成物。
That is, although not limited to this, this invention includes invention of the following aspects.
[1] An antitumor agent comprising lactic acid bacteria and sulforaphane.
[2] The antitumor agent according to [1], wherein the lactic acid bacterium is Lactobacillus pentosus.
[3] The antitumor agent according to [2], wherein the Lactobacillus pentosas is Lactobacillus pentosas S-PT84 strain (Accession number: FERM BP-10028).
[4] The antitumor agent according to any one of [1] to [3], wherein the lactic acid bacteria are contained in the form of live cells, dead cells, or processed cells.
[5] The antitumor agent according to any one of [1] to [4], wherein the tumor is a malignant tumor.
[6] An apoptosis promoter for tumor cells, comprising lactic acid bacteria and sulforaphane.
[7] The apoptosis promoter according to [6], wherein the lactic acid bacterium is Lactobacillus pentosus.
[8] The apoptosis promoter according to [7], wherein the Lactobacillus pentosas is Lactobacillus pentosas S-PT84 strain (Accession number: FERM BP-10028).
[9] The apoptosis promoter according to any one of [6] to [8], wherein the lactic acid bacteria are contained in the form of live cells, dead cells, or processed cells.
[10] The apoptosis promoting agent according to any one of [6] to [9], wherein the tumor is a malignant tumor.
[11] A composition comprising lactic acid bacteria and sulforaphane.
[12] The composition according to [11], wherein the lactic acid bacterium is Lactobacillus pentosus.
[13] The composition according to [12], wherein the Lactobacillus pentosas is Lactobacillus pentosas S-PT84 strain (Accession number: FERM BP-10028).
[14] The composition according to any one of [10] to [13] for inhibiting the growth of tumor cells.
[15] The composition according to [14], wherein the tumor is a malignant tumor.
[16] The composition according to any one of [11] to [15], which is a food or drink.
[17] The composition according to [16], which is provided with a function indication having lactic acid bacteria and sulforaphane as health function components (participating components).
本発明の抗腫瘍剤、アポトーシス促進剤、および組成物は、乳酸菌とスルフォラファンとの組み合わせを含むことにより、高い抗腫瘍効果または腫瘍細胞に対する高いアポトーシス促進効果を示す。 The antitumor agent, apoptosis promoting agent, and composition of the present invention exhibit a high antitumor effect or a high apoptosis promoting effect on tumor cells by containing a combination of lactic acid bacteria and sulforaphane.
＜乳酸菌＞
本発明に用いる乳酸菌は、生体の免疫反応を活性化する作用、すなわち免疫賦活作用を示す乳酸菌であればいずれを用いてもよいが、たとえばラクトバチルス・ペントーサスを好適に用いることができる。本発明に用いるラクトバチルス・ペントーサスは、ラクトバチルス・ペントーサス（Lactobacillus pentosus）に属し、かつ、グリセロールの資化性が無いまたは弱いものであればよい。
<Lactic acid bacteria>
Any lactic acid bacterium may be used as long as the lactic acid bacterium used in the present invention activates the immune response of a living body, that is, an immunostimulatory effect. For example, Lactobacillus pentosas can be preferably used. The Lactobacillus pentosus used in the present invention may belong to Lactobacillus pentosus and has no or weak glycerol utilization.
ラクトバチルス・ペントーサスは、さらに、細胞外多糖（Extracellular Polysaccharide：以下「EPS」と略記する）産生株であることが好ましい。ラクトバチルス・ペントーサスのEPSは、菌体表面にとどまる莢膜多糖であり、墨汁染色により容易に確認できる。 Lactobacillus pentosas is further preferably an extracellular polysaccharide (hereinafter abbreviated as “EPS”) producing strain. The Lactobacillus pentosasus EPS is a capsular polysaccharide that remains on the surface of the cell, and can be easily confirmed by ink stain.
本発明に用いるラクトバチルス・ペントーサスの代表的なものとして、ラクトバチルス・ペントーサスＳ−ＰＴ８４株を挙げることができる。本株は独立行政法人産業技術総合研究所、特許生物寄託センターに寄託されており、その受託番号はＦＥＲＭ ＢＰ−１００２８である。 Lactobacillus pentosasus S-PT84 strain can be mentioned as a representative example of Lactobacillus pentosasus used in the present invention. This stock has been deposited at the National Institute of Advanced Industrial Science and Technology, the Patent Biological Depositary Center, and the deposit number is FERM BP-1000028.
本発明に用いる乳酸菌は、生菌体、死菌体、または菌体処理物のいずれの形態であってもよい。菌体は培養上清を除いて精製したものでも、固体培地や液体培地等の夾雑物が含まれるものでもよく、湿潤な状態または乾燥した状態のいずれの状態でもよい。湿潤な状態の菌体は、たとえば乳酸菌の菌体と固体培地または液体培地とを含む培養物をそのまま、あるいは培養物を濃縮した濃縮物を用いることができる。乾燥した状態の菌体は、噴霧乾燥、凍結乾燥、真空乾燥等の方法によって乾燥した菌体をそのまま、あるいは粉末化、顆粒化したものを用いることができる。死菌体は、乳酸菌の培養物、培養物の濃縮物、精製した菌体、または乾燥した菌体を加熱処理や放射線処理等によって殺菌処理して得られる。菌体処理物とは、超音波破砕や物理破砕等の破砕処理または粉砕処理によって得られる菌体破砕物や菌体粉砕物、細胞壁分解酵素等の酵素処理によって得られる菌体溶解物、菌体を固体担体やマイクロカプセル等に固定化した固定化菌体等や菌体培養液上清のように菌体を除去した後の培養物を意味する。
＜スルフォラファン＞
スルフォラファンは以下の構造を有する化合物である。
The lactic acid bacteria used in the present invention may be in any form of live cells, dead cells, or processed cells. The bacterial cells may be purified by removing the culture supernatant, may contain impurities such as a solid medium or a liquid medium, and may be in a wet state or a dry state. As the wet cells, for example, a culture containing lactic acid bacteria and a solid medium or a liquid medium can be used as it is, or a concentrate obtained by concentrating the culture can be used. As the dried cells, those obtained by spraying, freeze-drying, vacuum drying or the like can be used as they are, or powdered and granulated. A dead cell is obtained by sterilizing a culture of lactic acid bacteria, a concentrate of the culture, a purified cell, or a dried cell by heat treatment, radiation treatment, or the like. The treated cell product is a lysed cell lysate obtained by enzymatic treatment such as crushed cell material, crushed cell material, cell wall degrading enzyme, etc. Means a culture after removing the microbial cells such as immobilized microbial cells immobilized on a solid carrier, microcapsules, etc. or microbial cell culture supernatant.
<Sulforafan>
Sulforaphane is a compound having the following structure.
スルフォラファンはアブラナ科の植物、たとえばブロッコリー、ブロッコリーの芽、キャベツ、芽キャベツ、カリフラワー、カリフラワーの芽、チンゲンサイ等に含まれ、これらの植物から公知の方法によって抽出・精製することができる。また、市販のスルフォラファンを用いてもよい。本発明において、スルフォラファンはＤ体、Ｌ体、またはこれらの混合物のいずれを用いてもよい。 Sulforaphane is contained in cruciferous plants such as broccoli, broccoli sprouts, cabbage, brussels sprouts, cauliflower, cauliflower sprouts, chingensai, and the like, and can be extracted and purified from these plants by known methods. Commercially available sulforaphanes may also be used. In the present invention, sulforaphane may be any of D-form, L-form, or a mixture thereof.
本明細書において、用語「スルフォラファン」は、上記構造を有するスルフォラファン、生体内でスルフォラファンに変換されるスルフォラファン誘導体、およびスルフォラファンの塩を含むものとする。 In this specification, the term “sulforaphane” includes a sulforaphane having the above structure, a sulforaphane derivative converted into sulforaphane in vivo, and a salt of sulforaphane.
生体内でスルフォラファンに変換されるスルフォラファン誘導体の例としては、たとえばスルフォラファングルコシネートが挙げられるが、これに限定されない。スルフォラファングルコシネートは、ブロッコリーの芽やカリフラワーの芽に多く含まれる。 Examples of sulforaphane derivatives that are converted into sulforaphane in vivo include, but are not limited to, sulforaphane glucosinate. Sulforaphane glucosinate is abundant in broccoli buds and cauliflower buds.
スルフォラファンの塩の例としては、たとえば酢酸塩、アジピン酸塩、アルギン酸塩、アスパラギン酸塩、安息香酸塩、ベンゼンスルホン酸塩、重硫酸塩、酪酸塩、クエン酸塩、ショウノウ酸塩、カンファースルホン酸塩、シクロペンタンプロピオン酸塩、ジグルコン酸塩、ドデシル硫酸塩、エタンスルホン酸塩、フマル酸塩、グルコヘプタン酸塩、グリセロリン酸塩、ヘミ硫酸塩、ヘプタン酸塩、ヘキサン酸塩、塩酸塩、臭化水素酸塩、ヨウ化水素酸塩、２−ヒドロキシエタンスルホン酸塩、乳酸塩、マレイン酸塩、メタンスルホン酸塩、２−ナフタレンスルホン酸塩、ニコチン酸塩、シュウ酸塩、パモ酸塩、ペクチン酸塩、過硫酸塩、３−フェニルスルホン酸塩、ピクリン酸塩、ピバル酸塩、プロピオン酸塩、コハク酸塩、酒石酸塩、チオシアン酸塩、トシラート、及びウンデカン酸塩のような酸付加塩；並びに、アンモニウム塩、ナトリウム及びカリウム塩のようなアルカリ金属塩、カルシウム及びマグネシウム塩のようなアルカリ土類金属塩、ジシクロヘキシルアミン塩、Ｎ−メチル−Ｄ−グルカミンのような有機塩基との塩、及びアルギニン及びリジンのようなアミノ酸との塩といった塩基性塩が挙げられるが、これらに限定されない。
＜抗腫瘍剤、アポトーシス促進剤＞
本発明は、乳酸菌とスルフォラファンとを含む、抗腫瘍剤に関する。
Examples of salts of sulforaphane include, for example, acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorsulfonate, camphorsulfonate Salt, cyclopentanepropionate, digluconate, dodecyl sulfate, ethane sulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, odor Hydrohalide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, Pectate, persulfate, 3-phenylsulfonate, picrate, pivalate, propionate, succinate, tartrate, Acid addition salts such as cyanate, tosylate and undecanoate; and alkali metal salts such as ammonium, sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, dicyclohexylamine salts, Examples include, but are not limited to, salts with organic bases such as N-methyl-D-glucamine and salts with amino acids such as arginine and lysine.
<Anti-tumor agent, apoptosis promoter>
The present invention relates to an antitumor agent comprising lactic acid bacteria and sulforaphane.
本明細書において「腫瘍」とは、遺伝子変異によって自律的で制御されない増殖を行うようになった細胞集団をいい、良性腫瘍および悪性腫瘍を含む。本明細書において、用語「悪性腫瘍」は用語「がん」と交換可能に用いられる。用語「がん」は、上皮由来の癌腫である癌、肉腫、および白血病等の血液悪性腫瘍も含む広い意味で用いられる。上皮由来の癌腫である癌の例としては、胃癌、大腸癌、胆嚢癌、胆管癌、膵臓癌、十二指腸癌、腎臓癌、前立腺癌、卵巣癌、子宮癌、乳癌、皮膚癌、肝細胞癌、舌癌、食道癌、咽頭癌等が挙げられるが、これらに限定されない。肉腫の例としては、線維肉腫、悪性線維性組織球腫、皮膚線維肉腫、脂肪肉腫、筋肉腫、血管肉腫、カポジ肉腫、リンパ管肉腫、滑膜肉腫、骨肉腫等が挙げられるが、これらに限定されない。血液悪性腫瘍の例としては、白血病、悪性リンパ腫、多発性骨髄腫等が挙げられるが、これらに限定されない。 As used herein, the term “tumor” refers to a cell population that has grown autonomously and uncontrolled by gene mutation, and includes benign tumors and malignant tumors. As used herein, the term “malignant tumor” is used interchangeably with the term “cancer”. The term “cancer” is used in a broad sense, including hematologic malignancies such as carcinoma, sarcoma, and leukemia, which are carcinomas derived from the epithelium. Examples of cancers that are epithelial carcinomas include stomach cancer, colon cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, duodenal cancer, kidney cancer, prostate cancer, ovarian cancer, uterine cancer, breast cancer, skin cancer, hepatocellular carcinoma, Examples include, but are not limited to, tongue cancer, esophageal cancer, and pharyngeal cancer. Examples of sarcomas include fibrosarcoma, malignant fibrous histiocytoma, cutaneous fibrosarcoma, liposarcoma, myoma, angiosarcoma, Kaposi sarcoma, lymphangiosarcoma, synovial sarcoma, osteosarcoma, etc. It is not limited. Examples of hematological malignancies include, but are not limited to, leukemia, malignant lymphoma, multiple myeloma and the like.
本発明の抗腫瘍剤は、腫瘍の増殖を抑制することによって、抗腫瘍効果を発揮する。 The antitumor agent of the present invention exerts an antitumor effect by suppressing tumor growth.
抗腫瘍効果は、固形の腫瘍の場合は、腫瘍の大きさが小さくなること、または腫瘍がなくなることを指標に評価することができる。また、血液悪性腫瘍の場合は、血液中の悪性腫瘍細胞の数が少なくなること、またはなくなることを指標に評価することができる。 In the case of a solid tumor, the antitumor effect can be evaluated by using as an index the decrease in the size of the tumor or the disappearance of the tumor. In the case of a hematological malignancy, it can be evaluated using an index that the number of malignant tumor cells in the blood decreases or disappears.
本発明はまた、乳酸菌とスルフォラファンとを含む、腫瘍細胞に対するアポトーシス促進剤にも関する。 The present invention also relates to an apoptosis promoting agent for tumor cells, comprising lactic acid bacteria and sulforaphane.
腫瘍細胞に対するアポトーシス促進効果は、目的の腫瘍細胞に誘導されたアポトーシスを検出することによって評価することができる。アポトーシスの誘導は、細胞膜の変化を利用した検出法、アポトーシス特有の酵素活性を利用した検出法、核の断片化を利用した検出法等の公知の手法によって検出することができる。具体的には、実地例に示したように、アポトーシス誘導によってDNAの断片化・分解が生じた結果、G1期の細胞よりDNA含量が少ない細胞の分画（sub-G1細胞）を、フローサイトメトリー等によって検出することができる
乳酸菌とスルフォラファンとの組み合わせが高い抗腫瘍効果または腫瘍細胞に対する高いアポトーシス促進効果を示すメカニズムは、理論に拘束されるものではないが、たとえば以下のようなものが考えられる。すなわち、スルフォラファンによって、乳酸菌の免疫賦活作用が増強され、免疫担当細胞が活性化されることにより、腫瘍細胞がアポトーシスを引き起こすことが考えられる。
The effect of promoting apoptosis on tumor cells can be evaluated by detecting apoptosis induced in the target tumor cells. Induction of apoptosis can be detected by a known method such as a detection method using changes in cell membranes, a detection method using enzyme activity peculiar to apoptosis, or a detection method using fragmentation of the nucleus. Specifically, as shown in the actual example, as a result of DNA fragmentation / degradation due to induction of apoptosis, a fraction of cells (sub-G1 cells) having a lower DNA content than cells in the G1 phase is flow-sited. The mechanism by which the combination of lactic acid bacteria and sulforaphane, which can be detected by a metric method, shows a high antitumor effect or a high apoptosis promoting effect on tumor cells, is not limited by theory. For example, the following may be considered: It is done. That is, it is considered that sulforaphane enhances the immunostimulatory action of lactic acid bacteria and activates immunocompetent cells, thereby causing apoptosis of tumor cells.
本発明では、乳酸菌とスルフォラファンとの組み合わせをそのまま抗腫瘍剤またはアポトーシス促進剤として用いてよい。また、乳酸菌とスルフォラファンとの組み合わせに薬理学的に許容可能な担体や賦形剤を添加したものを、抗腫瘍剤またはアポトーシス促進剤として用いることもできる。担体の例としては、培地、水、生理食塩水、エタノール、プロピレングリコール、グリセリン等が挙げられる。賦形剤の例としては、ブドウ糖、ショ糖、乳糖、デキストリン、シクロデキストリン、キサンタンガム、グァーガム、アラビアガム、トラガントガム、ジェランガム、ローカストビーンガム、カラギーナン、ペクチン、寒天等が挙げられる。また、製剤化において一般的に使用される乳化剤、緊張化剤（等張化剤）、緩衝剤、溶解補助剤、防腐剤、安定化剤、抗酸化剤等を適宜配合することもできる。 In the present invention, a combination of lactic acid bacteria and sulforaphane may be used as it is as an antitumor agent or an apoptosis promoter. A combination of lactic acid bacteria and sulforaphane to which a pharmacologically acceptable carrier or excipient is added can also be used as an antitumor agent or an apoptosis promoter. Examples of the carrier include a medium, water, physiological saline, ethanol, propylene glycol, glycerin and the like. Examples of excipients include glucose, sucrose, lactose, dextrin, cyclodextrin, xanthan gum, guar gum, gum arabic, tragacanth gum, gellan gum, locust bean gum, carrageenan, pectin, agar and the like. In addition, emulsifiers, tonicity agents (isotonic agents), buffers, solubilizers, preservatives, stabilizers, antioxidants, and the like that are generally used in formulation can be appropriately blended.
本発明の抗腫瘍剤またはアポトーシス促進剤の有効成分である乳酸菌の含量、およびスルフォラファンの含量は、効果面を考慮して任意に決定することができる。 The content of lactic acid bacteria that are the active ingredients of the antitumor agent or apoptosis promoter of the present invention and the content of sulforaphane can be arbitrarily determined in view of the effect.
たとえば乳酸菌としてラクトバチルス・ペントーサスの菌体を用いる場合は、菌体の乾燥重量として抗腫瘍剤またはアポトーシス促進剤中に０．００００１〜９０重量％含まれることが好ましく、０．０００５〜５０重量％含まれることがより好ましく、０．００１〜１０重量％含まれることがさらに好ましい。あるいは、菌体数として、１．０×１０２〜１．０×１０１２個／ｇの範囲内であることが好ましく、１．０×１０６〜１．０×１０１２個／ｇの範囲内であることがより好ましい。また、ラクトバチルス・ペントーサスの菌体処理物を用いる場合は、固形成分重量として抗腫瘍剤またはアポトーシス促進剤中に０．００００１〜９０重量％含まれることが好ましく、０．０００５〜５０重量％含まれることがより好ましく、０．０００１〜１０重量％含まれることがさらに好ましい。また、ラクトバチルス・ペントーサスの菌体と菌体処理物とを組み合わせて用いる場合は、菌体の乾燥重量と菌体処理物の固形成分重量とを合わせて、抗腫瘍剤またはアポトーシス促進剤中に０．００００１〜９０重量％含まれることが好ましく、０．０００５〜５０重量％含まれることがより好ましく、０．００１〜１０重量％含まれることがさらに好ましい。 For example, when Lactobacillus pentosus cells are used as lactic acid bacteria, the dry weight of the cells is preferably 0.00001 to 90% by weight, preferably 0.0005 to 50% by weight, in the antitumor agent or apoptosis promoter. More preferably, it is more preferably 0.001 to 10% by weight. Alternatively, the number of cells is preferably in the range of 1.0 × 10 2 to 1.0 × 10 12 cells / g, and in the range of 1.0 × 10 6 to 1.0 × 10 12 cells / g. More preferably, it is within. Moreover, when using the Lactobacillus pentosasus cell processed material, it is preferable that 0.00001-90 weight% is contained in an antitumor agent or an apoptosis promoter as a solid component weight, and 0.0005-50 weight% is contained. More preferably, the content is 0.0001 to 10% by weight. In addition, when using a combination of Lactobacillus pentosasus cells and a treated product, the dry weight of the cells and the solid component weight of the treated product are combined in the antitumor agent or apoptosis promoter. It is preferably contained in 0.00001 to 90% by weight, more preferably contained in 0.0005 to 50% by weight, and further preferably contained in 0.001 to 10% by weight.
また、スルフォラファンは、抗腫瘍剤またはアポトーシス促進剤中に０．０００１〜５０重量％含まれることが好ましく、０．００１〜１０重量％含まれることがより好ましく、０．０１〜１重量％含まれることがさらに好ましい。
＜組成物＞
本発明はまた、乳酸菌とスルフォラファンとを含む、組成物にも関する。
Also, sulforaphane is preferably contained in the antitumor agent or apoptosis promoter in an amount of 0.0001 to 50% by weight, more preferably 0.001 to 10% by weight, and 0.01 to 1% by weight. More preferably.
<Composition>
The present invention also relates to a composition comprising lactic acid bacteria and sulforaphane.
本発明の組成物は、高い抗腫瘍効果または腫瘍細胞に対する高いアポトーシス促進効果を有するため、腫瘍細胞の増殖を阻害する目的に好適に用いることができる。 Since the composition of the present invention has a high antitumor effect or a high apoptosis promoting effect on tumor cells, it can be suitably used for the purpose of inhibiting the growth of tumor cells.
本発明では、乳酸菌とスルフォラファンとの組み合わせをそのまま本発明の組成物として用いてよい。また、乳酸菌とスルフォラファンとの組み合わせに薬理学的に許容可能な担体や賦形剤を添加したものを、本発明の組成物として用いることもできる。担体の例としては、培地、水、生理食塩水、エタノール、プロピレングリコール、グリセリン等が挙げられる。賦形剤の例としては、ブドウ糖、ショ糖、乳糖、デキストリン、シクロデキストリン、キサンタンガム、グァーガム、アラビアガム、トラガントガム、ジェランガム、ローカストビーンガム、カラギーナン、ペクチン、寒天等が挙げられる。また、製剤化において一般的に使用される乳化剤、緊張化剤（等張化剤）、緩衝剤、溶解補助剤、防腐剤、安定化剤、抗酸化剤等を適宜配合することもできる。 In the present invention, a combination of lactic acid bacteria and sulforaphane may be used as it is as the composition of the present invention. Moreover, what added the pharmacologically acceptable support | carrier and excipient | filler to the combination of lactic acid bacteria and sulforaphane can also be used as a composition of this invention. Examples of the carrier include a medium, water, physiological saline, ethanol, propylene glycol, glycerin and the like. Examples of excipients include glucose, sucrose, lactose, dextrin, cyclodextrin, xanthan gum, guar gum, gum arabic, tragacanth gum, gellan gum, locust bean gum, carrageenan, pectin, agar and the like. In addition, emulsifiers, tonicity agents (isotonic agents), buffers, solubilizers, preservatives, stabilizers, antioxidants, and the like that are generally used in formulation can be appropriately blended.
本発明の組成物の利用形態は特に制限されない。カプセル剤、キャンデー、ドロップ、錠剤、顆粒剤、散剤、粉末剤、カプセル剤等の固形剤や、通常液剤、懸濁剤、乳剤等の液剤等に製剤化することもできる。また、カプセル剤の内部組成物としても利用することができる。 The usage form of the composition of the present invention is not particularly limited. It can also be formulated into solid preparations such as capsules, candy, drops, tablets, granules, powders, powders, capsules, etc., or liquids such as normal solutions, suspensions, emulsions, and the like. It can also be used as an internal composition of a capsule.
本発明の組成物の有効成分である乳酸菌の含量、およびスルフォラファンの含量は、効果面を考慮して任意に決定することができる。 The content of lactic acid bacteria, which are active ingredients of the composition of the present invention, and the content of sulforaphane can be arbitrarily determined in view of the effect.
たとえば乳酸菌としてラクトバチルス・ペントーサスの菌体を用いる場合は、菌体の乾燥重量として組成物中に０．００００１〜９０重量％含まれることが好ましく、０．０００５〜５０重量％含まれることがより好ましく、０．００１〜１０重量％含まれることがさらに好ましい。あるいは、菌体数として、１．０×１０２〜１．０×１０１２個／ｇの範囲内であることが好ましく、１．０×１０６〜１．０×１０１２個／ｇの範囲内であることがより好ましい。また、ラクトバチルス・ペントーサスの菌体処理物を用いる場合は、固形成分重量として組成物中に０．００００１〜９０重量％含まれることが好ましく、０．０００５〜５０重量％含まれることがより好ましく、０．００１〜１０重量％含まれることがさらに好ましい。また、ラクトバチルス・ペントーサスの菌体と菌体処理物とを組み合わせて用いる場合は、菌体の乾燥重量と菌体処理物の固形成分重量とを合わせて、組成物中に０．００００１〜９０重量％含まれることが好ましく、０．０００５〜５０重量％含まれることがより好ましく、０．００１〜１０重量％含まれることがさらに好ましい。 For example, when Lactobacillus pentosus cells are used as lactic acid bacteria, the dry weight of the cells is preferably 0.00001 to 90% by weight, more preferably 0.0005 to 50% by weight. Preferably, 0.001 to 10% by weight is contained. Alternatively, the number of cells is preferably in the range of 1.0 × 10 2 to 1.0 × 10 12 cells / g, and in the range of 1.0 × 10 6 to 1.0 × 10 12 cells / g. More preferably, it is within. Moreover, when using the microbial cell processed material of Lactobacillus pentosus, it is preferable that 0.00001-90 weight% is contained in a composition as a solid component weight, and it is more preferable that 0.0005-50 weight% is contained. More preferably, 0.001 to 10% by weight is contained. Moreover, when using combining the microbial cell of Lactobacillus pentosasus, and a microbial cell processed material, it combines 0.00001-90 in a composition combining the dry weight of microbial cells, and the solid component weight of a microbial cell processed material. It is preferably contained in an amount of 0.005 to 50% by weight, more preferably 0.001 to 10% by weight.
また、スルフォラファンは、組成物中に０．０００１〜５０重量％含まれることが好ましく、０．００１〜１０重量％含まれることがより好ましく、０．０１〜１重量％含まれることがさらに好ましい。 Further, the sulforaphane is preferably contained in the composition in an amount of 0.0001 to 50% by weight, more preferably 0.001 to 10% by weight, and further preferably 0.01 to 1% by weight.
本発明の組成物は、乳酸菌およびスルフォラファンが有する各種の保健機能（又は生理作用）を発揮することが期待されるため、例えば、健康食品、機能性食品（特定保健用食品、条件付き特定保健用食品、栄養機能食品が含まれる）、特別用途食品、健康補助食品等として好適に利用できる。 Since the composition of the present invention is expected to exhibit various health functions (or physiological actions) possessed by lactic acid bacteria and sulforaphane, for example, health food, functional food (food for specified health use, food for specified health use, conditional specified health use) Foods and functional nutritional foods), special-purpose foods, health supplements, and the like.
本発明の組成物は、その成分である乳酸菌およびスルフォラファンなどの保健機能成分（関与成分）に起因して、種々の保健機能（又は生理作用）を発揮する。ここで、保健機能成分（関与成分）とは、一定量摂取することで健康の維持増進に役立つ成分をいう。従って、本発明の組成物は、そのような保健機能成分に起因して発揮される機能に基づく用途に用いられ得る。保健機能成分である乳酸菌およびスルフォラファンに起因して発揮される機能に基づく用途の例としては、限定はされないが、腸内環境の改善、免疫賦活化、免疫活性化、腫瘍細胞の増殖抑制、および寿命の延長のための用途等が挙げられる。 The composition of the present invention exhibits various health functions (or physiological effects) due to the health function components (participating components) such as lactic acid bacteria and sulforaphane, which are the components. Here, the health functional component (participating component) refers to a component that is useful for maintaining and promoting health by ingesting a certain amount. Therefore, the composition of this invention can be used for the use based on the function exhibited due to such a health functional ingredient. Examples of applications based on the functions exerted by the lactic acid bacteria and sulforaphane that are health functional components include, but are not limited to, improvement of the intestinal environment, immune activation, immune activation, tumor cell growth inhibition, and Applications for extending the service life are listed.
たとえば、本発明の組成物は、乳酸菌およびスルフォラファンに起因して発揮される機能に基づく用途に用いるための、乳酸菌およびスルフォラファンを含む組成物、特に飲食品であることができる。 For example, the composition of the present invention can be a composition containing lactic acid bacteria and sulforaphane, particularly a food or drink, for use in applications based on the functions exhibited by lactic acid bacteria and sulforaphanes.
また、本発明の組成物は、乳酸菌およびスルフォラファンに起因して発揮される機能に基づく用途に用いられるものである旨の表示を付した、乳酸菌およびスルフォラファンを含む組成物、特に飲食品であることができる。本願明細書において、当該表示及び機能表示のような表示は、組成物自体に付されてもよいし、組成物の容器又は包装に付されていてもよい。 In addition, the composition of the present invention is a composition containing lactic acid bacteria and sulforaphane, particularly a food and drink, with an indication that the composition is used for an application based on a function exhibited by lactic acid bacteria and sulforaphane. Can do. In the specification of the present application, the display such as the display and the function display may be attached to the composition itself, or may be attached to a container or a package of the composition.
以下に本発明の実施例を示すが、本発明を制限するものではない。 Examples of the present invention are shown below, but are not intended to limit the present invention.
（１）材料と方法
＜１日目＞
ヒト大腸癌細胞であるHT-29細胞およびHCT116細胞（以下、単に大腸癌細胞と記載する）は、American Type Culture Collectionより入手した。
(1) Materials and methods <Day 1>
Human colon cancer cells, HT-29 cells and HCT116 cells (hereinafter simply referred to as colon cancer cells), were obtained from the American Type Culture Collection.
大腸癌細胞は、共培養用の12穴 プレートに3 × 104 細胞/1 ml/穴となるように播種し、DMEM培地（日水製薬株式会社）を用いて、37 ℃、5 % CO2の条件下で培養した。 Colorectal cancer cells are seeded in a 12-well plate for co-culture so that 3 × 10 4 cells / 1 ml / well, and using DMEM medium (Nissui Pharmaceutical Co., Ltd.) at 37 ° C., 5% CO 2. The culture was performed under the following conditions.
健常者の静脈より採取した血液を、血液分離溶液Lymphoprep（リンホプレップ）（商標）（Axis-Shield社）を用いた比重遠心に供し、末梢血単核細胞（PBMC）を分離した。得られたPBMCを、RPMI培地（日水製薬株式会社）を用いて3 × 106 細胞/ml の濃度に調整し、ラクトバチルス・ペントーサスS-PT84株(10 μg/ml)を添加した。
＜２日目＞
ラクトバチルス・ペントーサスS-PT84株刺激の24時間後に、BD Falcon 0.4μｍ セルカルチャーインサート（コーニングインターナショナル株式会社）を用い、PBMCと大腸癌細胞との共培養を開始した。ここでスルフォラファン(5 μM, 20 μM)も添加した。
＜４日目＞
共培養開始から48時間後にセルカルチャーインサートを除去した。上清中の大腸癌細胞を回収し、さらにtrypsin処理によって付着した大腸癌細胞をはがして回収した。回収した大腸癌細胞をPBSで洗浄した後、PI-PBS (0.1% Triton-X, 10 μg/ml propidium iodide)で懸濁した。FACSCalibur（日本ベクトン・ディッキンソン株式会社）を用いてフローサイトメトリーを施行し、FL-2Aを測定した。Sub-G1の細胞の割合をCell Quest（日本ベクトン・ディッキンソン株式会社）を用いて解析した。
（２）結果
結果を図１Ａ（HT-29細胞）および図１Ｂ（HCT116細胞）に示す。
Blood collected from a healthy person's vein was subjected to specific gravity centrifugation using a blood separation solution Lymphoprep (trademark) (Axis-Shield) to separate peripheral blood mononuclear cells (PBMC). The obtained PBMC was adjusted to a concentration of 3 × 10 6 cells / ml using RPMI medium (Nissui Pharmaceutical Co., Ltd.), and Lactobacillus pentosas S-PT84 strain (10 μg / ml) was added.
<Day 2>
After 24 hours of stimulation with Lactobacillus pentosas S-PT84, co-culture of PBMC and colon cancer cells was started using a BD Falcon 0.4 μm cell culture insert (Corning International Inc.). Here, sulforaphane (5 μM, 20 μM) was also added.
<Day 4>
The cell culture insert was removed 48 hours after the start of co-culture. The colon cancer cells in the supernatant were collected, and the colon cancer cells attached by trypsin treatment were further removed and collected. The collected colon cancer cells were washed with PBS and then suspended in PI-PBS (0.1% Triton-X, 10 μg / ml propidium iodide). Flow cytometry was performed using FACSCalibur (Nippon Becton Dickinson Co., Ltd.) to measure FL-2A. The ratio of Sub-G1 cells was analyzed using Cell Quest (Nippon Becton Dickinson Co., Ltd.).
(2) Results The results are shown in FIG. 1A (HT-29 cells) and FIG. 1B (HCT116 cells).
ラクトバチルス・ペントーサスS-PT84株およびスルフォラファンの組み合わせを、大腸癌細胞とPBMCとの共培養系に添加した場合、ラクトバチルス・ペントーサスS-PT84株のみ、またはスルフォラファンのみを大腸癌細胞とPBMCとの共培養系に添加した場合と比較して、有意に大腸癌細胞のアポトーシス誘導効果が増加することが示された。 When the combination of Lactobacillus pentosas S-PT84 and sulforaphane is added to the co-culture system of colon cancer cells and PBMC, only Lactobacillus pentosas S-PT84 strain or only sulforaphane between colon cancer cells and PBMC It was shown that the apoptosis-inducing effect of colon cancer cells was significantly increased as compared with the case where it was added to the co-culture system.
Claims (17)
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| CANCER BIOLOGY AND THERAPY, vol. 15, no. 9, JPN6018042586, 2014, pages 1219 - 1225, ISSN: 0004026610 * |
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