JP4783550B2 - Colon cancer inhibitor and food containing the same - Google Patents

Description

  The present invention relates to a colorectal cancer suppressant, and more particularly to an agent effective in suppressing the onset of colorectal cancer and preventing colorectal cancer. The present invention further relates to a food containing such a colon cancer inhibitor.

In recent years, bioactive functions of natural ingredients have attracted attention. Among them, an interesting physiological activity has been found in sphingolipids (a general term for lipids having sphingoid bases), and improvements in moisture retention and skin beautifying effects have been reported and are attracting attention as functional lipids. Sphingolipids are ubiquitous in most eukaryotes and are used as cosmetic and pharmaceutical ingredients. Until recently, the main source of sphingolipids was the cranial nerve tissue of animals such as cattle and sheep. However, when it is used as a food or pharmaceutical raw material, there is a problem in its safety.
On the other hand, in experiments using colon cancer cells in vitro, it has been reported that sphingoid bases present in wheat induce colon cancer cell death (see Non-Patent Document 1 and Non-Patent Document 2). In addition, it has been reported that animal-derived sphingolipids containing sphingoid bases mainly composed of 4-sphingenin obtained from milk suppress colon adenoma (aberrant crypt foci (ACF)) in mice (non-patented). Reference 3).
Currently, while functional foods are attracting attention, there is a demand for active ingredients that can be safely ingested and exhibit excellent effects.

Sugawara, T., Kinoshita, M., Ohnishi, M., Miyazawa, T., Apoptosis induction by wheat-fluor sphingoid bases in DLD-1 human colon cancer cells, Biosci. Biotechnol. Biochem., 66 (10), 2228 -2231, 2002 Aida, K., Takakuwa, N., Kinoshita, M., Sugawara, T., Imai, H., Ono, J., Ohnishi, M., Properties and physiological effects of plant cerebroside species as functional lipids, N. Murata et al. (eds.), Advanced Research on Plant lipids, 233-236, 2003 Schmelz et al., J. Nutr., 130, 522-527, 2000

  An object of the present invention is to provide an active ingredient that is highly safe and capable of exhibiting an excellent bioactive effect when administered to the human body, and to provide a food containing such an active ingredient.

As a result of repeated research to find a natural ingredient that can be safely ingested and exhibit an excellent physiologically active effect, the present inventors have found that glucosylceramide, a sphingolipid obtained from a plant, develops colon cancer in an actual living body. It has been found that it exhibits an inhibitory effect. Specifically, using a chemical carcinogenesis model system using mice, the glucosylceramide fraction extracted and separated and purified from the plant was mixed with the feed and administered, resulting in the first stage of colon cancer onset. It was found that the occurrence of adenoma (pre-cancerous lesion (Aberrant Crypt Foci: ACF)) was significantly reduced compared with the non-administration group (control). From this result, the plant-derived glucosylceramide fraction can be expected to prevent colon cancer onset.
Therefore, this invention is a colon cancer inhibitor which contains the glucosylceramide fraction extracted from a plant as an active ingredient. In a specific embodiment of the present invention, cereals can be mentioned as an example of a plant that is an extraction raw material. The glucosylceramide fraction specifically includes glucosylceramide containing 4-trans, 8-cis-sphingadienin as a sphingoid base site, and 4-trans, 8-trans-sphingadienin as a sphingoid base site. Containing glucosylceramide, glucosylceramide containing 8-cis-sphingenin as sphingoid base site, glucosylceramide containing 8-trans-sphingenin as sphingoid base site, containing 4-hydroxy-8-cis-sphingenin as sphingoid base site It contains at least one selected from glucosylceramide and glucosylceramide containing 4-hydroxy-8-trans-sphingenin as a sphingoid base moiety.
The present invention is also directed to foods containing the aforementioned colon cancer inhibitor.

  The colorectal cancer inhibitor of the present invention is highly safe for ingestion by the human body and is useful for preventing the onset of colorectal cancer. The colon cancer inhibitor of the present invention can be taken on a daily basis. Moreover, the colon cancer inhibitor of this invention can be used in order to prevent a metastasis during the treatment of colon cancer or after the treatment of colon cancer. The food containing the colorectal cancer inhibitor of the present invention is advantageous for easily taking the active ingredient of the colorectal cancer inhibitor.

The plant used as a raw material which extracts the glucosylceramide fraction used by this invention is not specifically limited, The plant part used as an extraction raw material is not specifically limited. Among plants, cereals are preferably used, and grains such as corn, rice, wheat, barley, rye, soybean, rice bran, millet, barnyard millet, pigeon, and oats can be used as an extraction raw material. More specifically, any part of such grains, such as whole grains, germ, bran, bran, and endosperm can be used. As the extraction raw material, a pulverized product of these materials can be preferably used.
Examples of the extraction solvent to be used include an organic solvent and / or a mixture of an organic solvent and water. Specifically, ethanol, hydrous ethanol, methanol, hydrous methanol, hexane, acetone, chloroform, chloroform-methanol mixture, benzene, There is isopropanol. Among them, ethanol and hydrous ethanol (ethanol content is about 70 to 99% by volume) are preferable. Two or more kinds can be used as the extraction solvent.

In the extraction, the extraction solvent is generally used in an amount of 2 to 50 times (volume), preferably 3 to 10 times the amount of the extraction solvent relative to the plant extraction raw material. As the extraction method, stirring extraction, reflux extraction, immersion extraction, shaking extraction, ultrasonic extraction and the like can be employed, and the extraction temperature may be room temperature, generally 20 to 100 ° C., preferably 50 to 70 ° C. The extraction time is suitably 30 minutes to 24 hours, preferably about 30 minutes to 4 hours.
The extraction operation is not limited to one time, and a fresh solvent can be added again to the extraction residue, and the extraction operation can be repeated. The continuous extraction may be performed up to about 10 times, preferably up to 3 times. .

The extract thus obtained can be concentrated by an appropriate concentration operation, for example, by a vacuum concentrator such as an evaporator, or by removing the solvent by heating, to obtain a concentrated solution. Further, it can be concentrated and dried to obtain a concentrated dried product. The concentrated dried product thus obtained is usually in the form of a yellow solid fat, and the glucosylceramide fraction can be obtained from the extract thus obtained by an appropriate purification means. For example, the extract can be purified several times using chloroform-methanol on a silica gel column to obtain a solid oily glucosylceramide fraction.
In order to quantify glucosylceramide in the glucosylceramide fraction thus obtained, for example, an HPLC-light scattering detector can be used. Actually, a glucosylceramide fraction containing about 80 to 99% by mass of glucosylceramide can be obtained by the above extraction operation.

Plant-derived glucosylceramide consists of a glucose moiety, a 2-hydroxy fatty acid residue, and a sphingoid base moiety. Among the glucosylceramides extracted from plants, the main component of rice and corn is 4-trans, 8-cis-sphingadienin in sphingobase and 4-trans, 8-trans in soybean. Sphingadienin, wheat and rye are of 8-cis-sphingenin. 4-trans, 8-cis-sphingadienin and 8-cis-sphingenin are known to be sphingoid bases characteristic of plants. In addition, 4-trans, 8-trans-sphingadienin is known to be a sphingoid base characteristic of plants and fungi.
As an example of molecular species of plant-derived glucosylceramide, a 2-hydroxy fatty acid residue is a saturated fatty acid having 20 carbon atoms, and a sphingoid base site is from 4-trans, 8-cis-sphingadienin. This shows the structure of glucosylceramide. This structure is
“20h: 0-d18: 2 ^{4t, 8c} -Glc”.

The composition of the ceramide portion of glucosylceramide in the glucosylceramide fraction obtained by extraction and purification from plants as described above is obtained by adding the obtained glucosylceramide to hydrous methanolic 1N hydrochloric acid, methanolic 5% hydrochloric acid or dioxane. It can be determined by analyzing the composition of the constituents (sphingoid base, fatty acid and sugar) obtained by decomposition with 5% barium hydroxide using gas chromatography. Table 1 below shows an example of the analysis result of the constituent components of the ceramide site in glucosylceramide extracted from corn seed, rice bran, soybean seed, wheat grain, rye grain, and milk.

N.D.：検出せず （単位：質量％）
上記表中、 “24h:1”は炭素原子数２４で炭素-炭素二重結合を１つ有することを表す。

ND: Not detected (Unit: mass%)
In the above table, “24h: 1” represents 24 carbon atoms and one carbon-carbon double bond.

The glucosylceramide fraction from the plant obtained as described above is extremely low in toxicity. The colon cancer inhibitor of the present invention containing these as active ingredients is suitably taken by oral administration. The glucosylceramide fraction can be ingested as it is. The colorectal cancer inhibitor of the present invention may contain additives in addition to the above active ingredients.
Moreover, the glucosylceramide fraction which is an active ingredient can be formulated into arbitrary forms with a suitable adjuvant, and it can be set as the colon cancer inhibitor which can be administered orally. Examples of such dosage forms include tablets, soft / hard capsules, granules, fine granules, powders (powder), liquids (aqueous solutions, oil preparations, syrups, dry syrups, etc.), gums and the like. In addition to oral dosage forms, it can be formulated into suppositories, ointments, creams, and the like.
The intake of the colorectal cancer inhibitor of the present invention may vary depending on age, medical condition, general condition, etc., but as an active ingredient, about 10 to 10,000 mg / day is appropriate for adults, preferably 10 to 5 1,000 mg / day. The content of the glucosylceramide fraction, which is an active ingredient in the colorectal cancer inhibitor of the present invention, can be appropriately changed according to the dosage form and the like, and is not particularly limited, but is usually 0.1 to 50. 0% by mass is appropriate.

In formulation, in order to give stability and acid resistance that can withstand long-term storage to the active ingredient and to maintain the drug effect completely, if the formulation is applied with a pharmaceutically acceptable coating, excellent stability It can be used as a colorectal cancer inhibitor.
Examples of surfactants, excipients, lubricants, adjuvants and pharmaceutically acceptable film-forming substances used for the preparation of the colon cancer inhibitor of the present invention include the following.
In order to improve the dissolution and dissolution of the preparation, surfactants such as alcohols, esters, polyethylene glycol derivatives, sorbitan fatty acid esters, sulfated fatty alcohols and the like may be added. it can.
Examples of excipients include sucrose, lactose, starch, crystalline cellulose, mannitol, light anhydrous silicic acid, magnesium aluminate, magnesium aluminate metasilicate, synthetic aluminum silicate, calcium carbonate, sodium bicarbonate, calcium hydrogen phosphate, 1 type, or 2 or more types, such as carboxymethylcellulose calcium, can be added and combined.

As the lubricant, for example, magnesium stearate, talc, hydrogenated oil or the like can be added, or as a flavoring agent and flavoring agent, salt, saccharin, sugar, mannitol, orange oil, Sweeteners such as licorice extract, citric acid, glucose, menthol, eucalyptus oil, malic acid, flavoring agents, coloring agents, storage amount, and the like may be included.
As adjuvants such as suspending agents and wetting agents, for example, coconut oil, olive oil, sesame oil, peanut oil, calcium lactate, safflower oil, soybean phospholipid and the like can be contained. As film-forming substances, cellulose derivatives such as cellulose and saccharides, cellulose acetate phthalate (CAP), acrylic acid copolymers, and polyvinyl derivatives such as dibasic acid monoesters such as methyl acrylate / methacrylic acid Examples thereof include a polymer and a methyl methacrylate / methacrylic acid copolymer.
In addition, when coating the film-forming substance, the properties of the film-forming agent can be improved by adding various commonly used coating agents such as plasticizers, as well as various additives for preventing mutual adhesion during the coating operation. It can be improved and the coating operation can be made easier.

The colon cancer inhibitor containing a plant-derived glucosylceramide fraction as an active ingredient can also be added to foods and health foods, and can also be used as a component of food additives. The amount of the active ingredient in the food is generally in the range of 0.001 to 25% by mass, preferably 0.01 to 10% by mass.
Any kind of food may be incorporated into the colorectal cancer inhibitor of the present invention, for example, foods mainly composed of flour or starch such as bread, noodles, cereals, confectionery, biscuits, hot cakes, and tablets, chocolate , Jelly, gummy, butter, margarine, jam, cheese, yogurt, ice cream, milk beverage, juice, dressing, drink, health food and the like. As a method of blending the glucosylceramide fraction into food, it may be blended at an appropriate stage in the production process according to various foods.

[Extraction and purification of glucosylceramide fraction from corn]
From 100 kg of pulverized corn germ, stirring extraction was performed with 600 liters of ethanol at 70 ° C. for 3 hours, and the resulting extract was dried under reduced pressure. The obtained extract was purified 5 times with a silica gel column (manufactured by Fuji Silysia Chemical Ltd., trade name: BW-820MH) with chloroform-methanol to obtain 9.6 g of a glucosylceramide fraction. This was a white solid oil.

In the obtained glucosylceramide fraction, glucosylceramide was quantified using an HPLC-evaporative light scattering detector (manufactured by Shimadzu Corporation, LC-VP evaporative light scattering detection system, evaporative light scattering detector (ELSD-LT)). Used.
As the mobile phase, the following gradient solvent system was set using two liquids of liquid A: chloroform, liquid B: methanol-water (95: 5; v / v).

カラムはシリカカラム（Ｉｎｅｒｔｓｉｌ ＳＩＬ １００−５；４．６φ×１５０ｍｍ；ＧＬサイエンス製）を用い、カラムオーブン：３５℃、流速；１ｍｌ／ｍｉｎにて、蒸発光散乱検出器：ＥＬＳＤ（エバポレーター温度：７０℃、ガス流量：３５０ＭＰａ）で行った。グルコシルセラミド標準品は、トウモロコシ胚芽より抽出し、分取用薄層クロマトグラフにてグルコシルセラミドを単一に精製して調製したものを用いた。
その結果、上記グルコシルセラミド画分においてグルコシルセラミドの含量が９５％以上であることが判った。
このグルコシルセラミド画分を用いて、以下の試験で使用するグルコシルセラミド食群に与える試験食を調製した。

A silica column (Inertsil SIL 100-5; 4.6φ × 150 mm; manufactured by GL Science) was used as a column, column oven: 35 ° C., flow rate: 1 ml / min, evaporative light scattering detector: ELSD (evaporator temperature: 70) C., gas flow rate: 350 MPa). The standard glucosylceramide was extracted from corn germ and prepared by single purification of glucosylceramide using a preparative thin layer chromatograph.
As a result, it was found that the content of glucosylceramide in the glucosylceramide fraction was 95% or more.
Using this glucosylceramide fraction, a test meal to be given to the glucosylceramide meal group used in the following test was prepared.

[In vivo study of the effect of inhibiting the development of colorectal adenoma]
40 BALB / c male mice of 4 weeks old (Clea Japan Co., Ltd.) were used and divided into 4 groups: blank group, control group, glucosylceramide 0.1% diet group, glucosylceramide 0.5% diet group, There were 10 animals in each group.
After purchase, mice were preliminarily raised for 10 days in a commercial solid feed (Nosan Experimental Animal Feed; Lab MR Stock: Nippon Agricultural Industry Co., Ltd.), and then AIN-76A (American Institute of Nutrition, J Nutr. 107: 1340-1348 (1977)), the test meal was administered by pair feeding (the amount of the test meal was 4.4 g / animal / day by pair feeding). Drinking water was ad libitum for each group. The composition (mass%) of each test meal is shown in the following table.

＊上記のトウモロコシから抽出、精製して得たグルコシルセラミド画分。

* Glucosylceramide fraction obtained by extraction and purification from the above corn.

<Evaluation by ACF measurement method>
One week after administration of the test meal, DMH (1,2-dimethylhydrazine dihydrochloride, a colorectal cancer inducer) was intraperitoneally administered once a week at 30 mg / kg body weight. After breeding on a test meal for 10 weeks, after decapitation for 24 hours, the mice were decapitated and the large intestine (liver, blood) was removed. The liver was weighed and stored at -80 ° C.
The excised large intestine contents were washed out with sterilized physiological saline, then the large intestine was cut open, and the width and length were measured. Thereafter, the cells were fixed with 4% formalin, stained with 0.3% methylene blue, and the number of colorectal adenomas (Aberrant Crypt Foci: ACF) was counted under a microscope. The significant difference was evaluated by one-way analysis of variance followed by Scheffe's multiple comparison test.
The results are shown in Table 4 below.

※ACF数の各値はmeans±SD。
ACF数はグルコシルセラミド摂取群でコントロール群に比べて、有意に減少した。なお、体重変化に有意差がなく、肝臓重量にも有意差がなかった。
以下に試験食投与前の体重と最終体重とを、各群１匹当たりの数値（means±SD）で示す。

* Each value of ACF number is means ± SD.
The number of ACF was significantly decreased in the glucosylceramide intake group compared to the control group. There was no significant difference in body weight change, and there was no significant difference in liver weight.
Below, the body weight before administration of the test meal and the final body weight are shown as numerical values (means ± SD) per animal in each group.

Examples of preparations and foods using the corn-derived glucosylceramide fraction obtained as described above will be given below.
[Example 1]
Manufacture of tablets and tablets Eggshell calcium (108 g), ferric pyrophosphate (2 g), ascorbic acid (40 g), microcrystalline cellulose (40 g), reduced maltose (285 g) and glucosylceramide (25 g) were mixed by a conventional method using a mixer, and then tableted. Tablets were manufactured.
[Example 2]
Production of biscuits 120 g of wheat flour, 2.4 g of glucosylceramide, 35 g of sugar, 15 g of shortening, 1.5 g of whole egg powder, 1 g of sodium chloride, 0.6 g of sodium hydrogen carbonate, 0.75 g of ammonium carbonate, and 20 g of water in a conventional manner A dough was made, molded and roasted to produce biscuits.

[Example 3]
Manufacture of bread Using 3 kg of flour, 3 g of glucosylceramide, 60 g of yeast, 3 g of yeast food, 150 g of sugar, 60 g of salt, 150 g of shortening, 60 g of skimmed milk powder and 2070 g of water, a dough is prepared in a conventional manner, molded and roasted. Bread was produced.
[Example 4]
Manufacture of Chinese noodles After adding 12 parts by weight of glucosylceramide, 34 parts by weight of water, 1 part by weight of sodium chloride and 1 part by weight of kanpaku to 100 parts by weight of semi-strong wheat flour, The noodle strip was rolled and formed several times in a noodle machine to obtain a raw noodle strip of Chinese noodles and a raw noodle strand.

Claims (3)

  A method for producing a food selected from bread, Chinese noodles and biscuits, comprising blending a dough with a glucosylceramide fraction obtained by extraction from corn seeds.   The food selected from bread, Chinese noodles and biscuits according to claim 1, wherein the glucosylceramide fraction is blended into the dough so that the glucosylceramide fraction is contained in the food in a range of 0.001 to 25% by mass. Production method.   A glucosylceramide fraction containing 4-trans, 8-cis-sphingadienin as a sphingoid base moiety, glucosylceramide containing 4-trans, 8-trans-sphingadienin as a sphingoid base moiety, And glucosylceramide containing 8-cis-sphingenin as a sphingoid base site, glucosylceramide containing 8-trans-sphingenin as a sphingoid base site, glucosylceramide containing 4-hydroxy-8-cis-sphingenin as a sphingoid base site, A method for producing a food selected from bread, Chinese noodles and biscuits according to claim 1 or 2, comprising at least one selected from glucosylceramide containing 4-hydroxy-8-trans-sphingenin as a sphingoid base moiety.