JP4399142B2 - Cancer cell infiltration preventive or inhibitor and health food - Google Patents

Description

【００２５】
・６，６’−ジヒドロキシチオビヌファリジン（４２）:無色油状、[α]_D ²⁵＋７８．０°（ｃ＝１．５、ＣＨ₂Ｃｌ₂）、EI-MS m/z: ４９０ （M−２H₂O）⁺
【００２６】
【数２】

【００２７】
・６−ヒドロキシチオヌフルチンＢ（５０）:無色油状、[α]_D ²⁵−３７．２°（ｃ＝１．６、ＣＨＣｌ₃）、EI-MS m/z: ４９２ （M−H₂O）⁺
【００２８】
【数３】

【００２９】
・６’−ヒドロキシチオヌフルチンＢ（５１）:無色油状、[α]_D ²⁵＋５５．２°（ｃ＝１．６、ＣＨＣｌ₃）、EI-MS m/z: ４９２ （M−H₂O）⁺
【００３０】
【数４】

【００３１】
また、上記化合物の¹³Ｃ−ＮＭＲデータを以下に示す。
【００３２】
【数５】

【００３３】
（２）高転移Ｂ１６細胞の選別・作成
理研細胞バンクから購入したＢ１６−４Ａ５メラノーマ細胞を１０％ウシ子牛血清（ＦＣＳ）、１００単位／ｍｌペニシリン、１００μｇ／ｍｌストレプトマイシン（ライフ・テクノロジー社）含有のDulbecco modified Eagle's medium（ＤＭＥＭ、シグマ社）で培養（５％ＣＯ₂、３７℃）した。ｄｄＹ系雌性マウス（約２２ｇ、紀和実験動物）へＢ１６−４Ａ５メラノーマ細胞を５×１０⁵セル／２００μｌ）腹腔内投与し、２週間後に解剖し、背部筋肉部位に転移の認められたマウスから癌巣を無菌的に摘出し、癌細胞を切り、ミンチ状にした。トリプシン２５０（０．２５％、日本ベクトン・ディッキンソン社）処理を行い、ＤＭＥＭを添加して滅菌ガーゼでろ過した。１０分間振動した後、転移メラノーマを採取し、ＤＭＥＭにて継代・培養を行った。同様の操作をｄｄｙ系雌性マウスに繰り返し、６代目を得て、高転移株とした。
【００３４】
（３）癌転移浸潤阻害作用の検討
上記方法により選別したＢ１６メラノーマ細胞を１０％ウシ子牛血清（ＦＣＳ）、１００単位／ｍｌペニシリン、１００μｇ／ｍｌストレプトマイシン（ライフ・テクノロジー社）含有のDulbecco modified Eagle's medium（ＤＭＥＭ、シグマ社）で培養（５％ＣＯ₂、３７℃）した。
【００３５】
次に、２４ウェル平底マイクロプレートにcell culture insertsを取り付け、その中に豚腱製由来コラーゲンタイプＩ溶液（１００μｌ／セル、日本ハム社）でメンブレンをコートした。２時間放置後、ＰＢＳとＤＭＥＭでメンブレンを洗浄し、下層２４ウェルプレートにＤＭＥＭ（７００マイクロｌ／ウェル）を分注し、２０分間予備加熱（５％、ＣＯ₂、３７℃）した。その後、１〜０．００１μｇ／ｍｌの種々の濃度で抽出物、各分画及び各化合物を加えて（ＤＭＳＯ終濃度０．１％）、２４時間（５％、ＣＯ₂、３７℃）培養した。ＤＭＥＭを除去した後、２５％グルタールアルデヒド溶液（７５μｌ／ウェル和光純薬社）で細胞を固定し、クリスタルバイオレット（３００μｌ／ウェル、和光純薬社）で核染色し、顕微鏡下に１ウェルごとにカウントした。
【００３６】
得られた抽出物及び各分画の結果から、ＩＣ₅₀を算出した。その結果を表１に示す。
【００３７】
【表１】

【００３８】
表１によれば、特に、メタノール抽出物及び酢酸エチル分画で強力な癌細胞浸潤抑制作用が認められた。
【００３９】
また、酢酸エチル分画から得られた各化合物について癌細胞浸潤に対する抑制作用を測定した。その結果を表２に示す。
【表２】

【００４０】
これらの化合物においては、いずれも有意に癌細胞浸潤に対する抑制作用を示した。これらの化合物は、すでに癌細胞移転抑制効果が知られているクルクミン（culcumin、ＩＣ₅₀：２０μｍ）よりも非常に強い効果であった。
【００４１】
（４）マウス肺癌転移抑制作用の検討
ｄｄｙ系雌性マウス（約２２ｇ）へ上記方法により選別したＢ１６メラノーマ細胞を、ＰＢＳで懸濁し、静脈注射（５×１０⁵セル／２００μｌＰＢＳ）した。その後、酢酸エチル分画及び化合物（４１）を、種々の濃度で１０日間、１日１回１１時ごろに経口投与した。
【００４２】
１０日後に開腹し、肺を摘出し、表面に発現した肺癌転移数を肉眼でカウントし、コントロールに対する阻害率を算出した。
なお、比較物質としてクルクミンを用いた。
その結果を表３に示す。
【００４３】
【表３】

表３から、酢酸エチル抽出物及びその中に含まれる化合物（４１）は、いずれも強力な癌転移抑制効果を示した。その効果は、クルクミンよりも強力であった。
【００４４】
（５）毒性
得られた抽出物及び化合物についてＭＭＴアッセイ法により、毒性を検討したところ、表２に示すように、化合物（４１）では１０μＭにて、化合物（４２）では１００μＭ付近から毒性が認められた。また、化合物（４４）及び（４５）にも毒性は認められたが、化合物（４１）及び（４２）の毒性よりも弱かった。さらに、化合物（５１）及び（５４）では、毒性がほとんど認められなかった。
【図面の簡単な説明】
【図１】本発明のスイレン科のコウホネ属の植物の抽出方法を説明するための概略図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a preventive or suppressant for cancer cell invasion and a health food, and more particularly to a preventive or suppressant for cancer cell infiltration using a plant of the genus Nuphar of Nymphaeaceae and a health food.
[0002]
[Prior art and problems to be solved by the invention]
Traditionally, the rhizomes of the water lily family and nemuro kone are also called “river bones” in Japan and “Rhizome root” in China, and have been used as edible or home remedies.
[0003]
Kouhone is mainly effective for diuresis and water use, and is used for edema and bruises, as a blood purification agent for prenatal and postpartum, menstrual irregularities, bloody thrombosis, etc. It was applied to. Furthermore, it has been applied to colds as a fatigue recovery and sweating agent, and to gastrointestinal diseases as a healthy stomach medicine.
[0004]
However, the medicinal properties of sexeterpene alkaloids contained in corn are still unclear, and further research is required.
[0005]
[Means for Solving the Problems]
According to the present invention, there is provided a preventive or suppressant for cancer cell invasion comprising a powder of a plant of the genus Nuphar of Nymphaeaceae or an extract of a lower aliphatic alcohol or a hydrous lower aliphatic alcohol. . Moreover, the health food containing said powder and / or an extract is provided.
[0006]
DETAILED DESCRIPTION OF THE INVENTION
Plants that can be used to obtain the preventive or suppressive agent for cancer cell invasion according to the present invention are plants belonging to the genus Nuphar in the family Nymphaeaceae, such as Nuphar japonicum DC. (Nuphar pumilum (TIMM.) DC.), Nuphar luteum, N. japonicum DC. From a fubrotinctum (CASP.) Kitam, N. subintegerrimum Makino, N. pumilum DC. Var. ozeense (MIKI) Hara) and N. oguraense Miki. This plant may be grown in any of Honshu, Shikoku, Kyushu, Gunma, Niigata / Tohoku, eastern Hokkaido, Russia, Europe, and the like in Japan. This plant can use any part of roots, rhizomes, stems, leaves, seeds, etc., but usually rhizomes and stems are preferably used. When used as a powder, the collected plant is dried or freeze-dried as it is and pulverized with a pulverizer or the like to obtain a powder of 100 to 1000 mesh, preferably 200 to 500 mesh.
[0007]
In addition, when used as an extract, such a plant is directly dried or pulverized to obtain a lower aliphatic alcohol (methanol, ethanol, butanol, etc.), a hydrous lower aliphatic alcohol, or a weakly acidic aqueous solution (dilute hydrochloric acid, (Citric acid, oxalic acid aqueous solution). The solvent is suitably used in an amount of about 2 to 10 times, preferably about 2 to 5 times the weight of the plant. Alternatively, the extract may be prepared by immersing for about 1 to 10 days at a temperature of about 10 to 35 ° C. for about 1 to 10 hours or about 10 to 35 ° C. with or without shaking. it can.
[0008]
The obtained extract may be used after being concentrated, may be in the form of powder, or may be a lyophilized product. Concentration is preferably performed under low temperature and low pressure. Further, this concentration may be performed until it is solidified. In addition, you may filter before concentrating and may concentrate a filtrate. A method known in the art can be used as the method for preparing the powder and lyophilized product.
[0009]
The obtained extract may be subjected to a purification treatment. Examples of the purification treatment method include a method using a chromatographic method, an elution method using an ion exchange resin, a partition extraction with a solvent, or the like alone or in combination.
[0010]
For example, examples of the chromatographic method include normal phase chromatography, reverse phase chromatography, high performance liquid chromatography, centrifugal liquid chromatography, column chromatography, thin layer chromatography and the like, or a combination thereof. In this case, purification conditions such as a carrier and an elution solvent can be appropriately selected according to various chromatographies. For example, a chloroform-methanol solvent can be used for normal phase chromatography, and a water-methanol solvent can be used for reverse phase chromatography.
[0011]
As an elution method using an ion exchange resin, the obtained extract is diluted / dissolved in water or a lower alcohol, and this solution is brought into contact with the ion exchange resin to be adsorbed and then eluted with the lower alcohol or water. A method is mentioned. The lower alcohol used in this case is as described above, and methanol is particularly preferable. The ion exchange resin is not particularly limited as long as it is usually used for purification treatment in the field. For example, a porous crosslinked polystyrene-based resin having a large network structure, urbanite, cellulose, etc. Is mentioned.
[0012]
Moreover, the partition extraction method by a solvent can be performed by a well-known method in the said field | area using suitably acidic or alkaline aqueous solution, an organic solvent, etc.
[0013]
The extract of the present invention consists of 6-hydroxythiovinufaridine, 6,6′-dihydroxythiobinufaridine, 6-hydroxythionuflutin B, 6′-hydroxythionuflutin B, and nuphalidine. Contains one or more compounds selected from the group (Masayuki Yoshikawa et. Al., Heterocycles, vol.46, p301-308 (1997) and Heterocycles, vol.45, p1815-1824 (1997) ), These compounds may be isolated from the extract and used. The isolation method can be performed by a method known in the art.
[0014]
The powder and / or extract of the plant of the genus Lilyaceae is diluted as it is or with an appropriate medium, and is used in the form of a powder, granule, tablet, capsule or It can be used in various forms such as a liquid agent.
[0015]
Suitable media include pharmaceutically acceptable salts, excipients, preservatives, colorants, flavoring agents, etc., specifically lactose, starch, sugar, mannitol, dextrin, gelatin, calcium phosphate, Calcium carbonate, synthetic and natural aluminum silicate, magnesium oxide, dry aluminum hydroxide, magnesium stearate, sodium bicarbonate, dry yeast, zinc oxide, talc, kaolin, boric acid powder, zinc stearate, magnesium stearate, magnesium carbonate, Precipitated calcium carbonate, bismuth hyposilicate, potassium aluminum sulfate powder, alginic acid, alginate, glycerin, propylene glycol, fatty oil, ethylene glycol, polyethylene glycol, sorbitol, polyvinyl pyrrolidone, surfactants and the like.
[0016]
In addition, the powder and / or extract of a plant belonging to the genus Lilyaceae can be used as a health food. Health food means a food that is more active than ordinary food, and is intended for health, health maintenance and promotion, for example, liquid or semi-solid, solid products such as cookies, Examples include confectionery such as rice crackers, jelly, yokan, yogurt and manju, soft drinks, nutritional drinks, soups and the like. To these foods, components that are usually blended in health foods such as garlic, yellow egg oil, and various vitamins may be further added. Moreover, it may be decocted as it is to make a tea preparation. In the production process of these foods or to the final product, the above powder, extract and the like can be added by mixing, coating, spraying or the like to obtain a health food.
[0017]
The amount of powder and / or extract of the plant belonging to the genus Lilyaceae is appropriately determined depending on the degree of concentration, purification, etc., the purpose of use, the target disease for treatment or prevention, the degree of the disease, body weight, age, symptoms, etc. For example, about 50 to 1000 mg, preferably about 50 to 300 mg for powder per adult, and about 10 to 200 mg, preferably about 10 to 50 mg, depending on the degree of purification, moisture content, etc. for the extract. It is desirable to take 3 times a day about 30 minutes before a meal. In addition, when used as a health food, it is appropriate to use a powder or extract in an amount of about 10 to 1000 mg per 1 kg of the target food, for example, in an amount that does not adversely affect the taste and appearance of the food. .
[0018]
Examples of the preventive or suppressive agent for cancer cell infiltration according to the present invention will be described below.
(1) Extraction of plants belonging to the genus Lilyaceae An outline of a method for extracting plants belonging to the genus Lilyaceae is shown in FIG.
[0019]
7.9 kg of rhizome of Nuphar pumilum (TIMM.) DC. Collected from Russia is immersed in 15 liters of methanol (99.9%) and extracted for 3 hours under heating at 80 ° C. did. This was repeated three times, and the resulting methanol was combined and the methanol was evaporated to obtain a methanol extract (811 g).
[0020]
Next, 10 l of 1N HCl / chloroform (1: 1) was added to 811 g of the obtained methanol extract at room temperature and shaken and mixed, and the resulting chloroform layer and aqueous layer were separated and collected. This operation was repeated three times, the chloroform layers were combined, the solvent was removed, and 110 g of a chloroform fraction was obtained.
[0021]
Further, to the obtained aqueous layer, aqueous ammonia was added at room temperature to make it alkaline (pH 1), 5 liters of ethyl acetate was added and shaken, and the ethyl acetate soluble part and the water soluble part thus obtained were respectively separated. Liquid separation was collected. This operation was repeated three times. The solvent was evaporated from the ethyl acetate soluble part to obtain an ethyl acetate fraction (16.3 g).
[0022]
The chloroform fraction and the ethyl acetate fraction were purified, respectively, and as shown in FIG. 1, dihydrodehydrodiconiferyl alcohol (58) and 6-hydroxythiobinufaridine (41), 6,6′-dihydroxythiobi Nufaridin (42), Anti-6'-hydroxythiobinufaridine sulfoxide (43), Anti-thiobinufaridine sulfoxide (44), Syn-thiobinufaridine sulfoxide (45), Anti-1 -Epi-1'-epi-thiobinufaridine sulfoxide (46), syn-1-epi-1'-epi-thiobinufaridine sulfoxide (47), neothiobinufaridine (48), neothiobi Nufaridin β-sulfoxide (49), 6-hydroxythionuflutin B (50), 6′-hydroxythionuflutin B (51), Onufurtin B β-sulfoxide (52), 6′-hydroxythionuflutin B β-sulfoxide (53), nuphalidine (54), deoxynufaridine (55), 7-epideoxynufaridine (56), nuphaloltin ( 57) was isolated.
[0023]
6-hydroxythiobinufaridine (41): colorless oil, [α] _D ²⁵ + 39.2 ° (c = 3.0, CH ₂ Cl ₂ ), EI-MS m / z: 492 (M−H ₂ O) ⁺
[0024]
[Expression 1]

[0025]
6,6′-dihydroxythiobinufaridine (42): colorless oil, [α] _D ²⁵ + 78.0 ° (c = 1.5, CH ₂ Cl ₂ ), EI-MS m / z: 490 ( M-2H ₂ O) ⁺
[0026]
[Expression 2]

[0027]
- 6-hydroxy-thio j Furuchin B (50): colorless ^{_{oil, [α] D 25 -37.2 °}} (c = 1.6, CHCl 3), EI-MS m / z: 492 (M-H 2 O ) ⁺
[0028]
[Equation 3]

[0029]
6′-hydroxythionuflutin B (51): colorless oil, [α] _D ²⁵ + 55.2 ° (c = 1.6, CHCl ₃ ), EI-MS m / z: 492 (M−H ₂ O ) ⁺
[0030]
[Expression 4]

[0031]
In addition, ¹³ C-NMR data of the above compound is shown below.
[0032]
[Equation 5]

[0033]
(2) Selection and preparation of high metastasis B16 cells B16-4A5 melanoma cells purchased from RIKEN Cell Bank contain 10% calf serum (FCS), 100 units / ml penicillin, 100 μg / ml streptomycin (Life Technology) (DMEM, Sigma) (5% CO ₂ , 37 ° C.). B16-4A5 melanoma cells (5 × 10 ⁵ cells / 200 μl) were intraperitoneally administered to ddY female mice (approx. 22 g, Kiwa experimental animals), dissected after 2 weeks, and cancer was observed from mice in which metastasis was observed at the back muscle site. The nest was aseptically removed and the cancer cells were cut and minced. Trypsin 250 (0.25%, Nippon Becton Dickinson) was treated, DMEM was added, and the mixture was filtered through sterile gauze. After shaking for 10 minutes, metastatic melanoma was collected and subcultured and cultured in DMEM. The same operation was repeated for ddy female mice to obtain the sixth generation, and a high metastatic strain was obtained.
[0034]
(3) Examination of inhibition of cancer metastasis invasion B16 melanoma cells selected by the above method are Dulbecco modified Eagle's containing 10% calf serum (FCS), 100 units / ml penicillin, 100 μg / ml streptomycin (Life Technologies). Cultured in medium (DMEM, Sigma) (5% CO ₂ , 37 ° C.).
[0035]
Next, cell culture inserts were attached to a 24-well flat bottom microplate, and a membrane was coated with a collagen type I solution (100 μl / cell, Nippon Ham Co., Ltd.) derived from porcine tendon. After standing for 2 hours, the membrane was washed with PBS and DMEM, and DMEM (700 microliters / well) was dispensed into the lower 24-well plate and preheated (5%, CO ₂ , 37 ° C.) for 20 minutes. Thereafter, the extract, each fraction and each compound were added at various concentrations of 1 to 0.001 μg / ml (DMSO final concentration 0.1%) and cultured for 24 hours (5%, CO ₂ , 37 ° C.). . After removing DMEM, the cells were fixed with a 25% glutaraldehyde solution (75 μl / well Wako Pure Chemical Industries), stained with crystal violet (300 μl / well, Wako Pure Chemical Industries), and each well under a microscope. Counted.
[0036]
IC ₅₀ was calculated from the obtained extract and the result of each fraction. The results are shown in Table 1.
[0037]
[Table 1]

[0038]
According to Table 1, a strong cancer cell invasion inhibitory action was observed particularly in the methanol extract and the ethyl acetate fraction.
[0039]
Moreover, the inhibitory effect with respect to cancer cell invasion was measured about each compound obtained from the ethyl acetate fraction. The results are shown in Table 2.
[Table 2]

[0040]
All of these compounds showed a significant inhibitory effect on cancer cell invasion. These compounds had a much stronger effect than curcumin (IC ₅₀ : 20 μm), which is already known to have a cancer cell migration inhibitory effect.
[0041]
(4) Examination of inhibitory effect on mouse lung cancer metastasis B16 melanoma cells selected by the above method in ddy female mice (about 22 g) were suspended in PBS and intravenously injected (5 × 10 ⁵ cells / 200 μl PBS). Thereafter, the ethyl acetate fraction and the compound (41) were orally administered once a day at around 11:00 at various concentrations for 10 days.
[0042]
Ten days later, the abdomen was opened, the lungs were removed, the number of lung cancer metastases expressed on the surface was counted with the naked eye, and the inhibition rate relative to the control was calculated.
Curcumin was used as a comparative substance.
The results are shown in Table 3.
[0043]
[Table 3]

From Table 3, the ethyl acetate extract and the compound (41) contained therein all showed a strong cancer metastasis inhibitory effect. The effect was stronger than curcumin.
[0044]
(5) Toxicity The obtained extract and compound were examined for toxicity by the MMT assay. As shown in Table 2, the compound (41) was found to be toxic at 10 μM, and the compound (42) was toxic from around 100 μM. It was. Moreover, although toxicity was recognized also to compound (44) and (45), it was weaker than the toxicity of compound (41) and (42). Further, in the compounds (51) and (54), almost no toxicity was observed.
[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a schematic diagram for explaining a method for extracting a plant belonging to the genus Lilyaceae of the present invention.

Claims (4)

  An extract of a plant of the genus Nuphar in the family Nymphaeaceae is an extract of methanol or ethanol, or an extract of methanol or ethanol that is further soluble in ethyl acetate under ammonia alkaline conditions A preventive or suppressant for cancer cell invasion comprising a substance.   The prophylactic or inhibitory agent for cancer cell invasion according to claim 1, wherein the plant belonging to the genus Komone of the water lily family is Nuphar japonicum DC., Nuphar pumilum (TIMM.) DC. Or Nuphar luteum.   The extract is selected from the group consisting of 6-hydroxythiobinufaridine, 6,6'-dihydroxythiobinufaridine, 6-hydroxythionuflutin B, 6'-hydroxythionuflutin B, and nuphalidine. The preventive or inhibitory agent for cancer cell invasion according to claim 1 or 2, comprising one or more compounds as active ingredients.   One or two selected from the group consisting of 6-hydroxythiobinufaridine, 6,6′-dihydroxythiobinufaridine, 6-hydroxythionuflutin B, 6′-hydroxythionuflutin B and nufaridine A cancer cell infiltration suppressor containing at least one kind of compound as an active ingredient.

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