JP4995440B2 - Composition for preventing and / or treating tumor containing garlic treatment product - Google Patents

Description

  The present invention relates to a composition for preventing and / or treating a tumor containing a processed garlic product, a method for producing the same, and a food and feed containing the composition.

  Since tumors or cancers still have the highest mortality among lifestyle-related diseases and there are no specific drugs, research on their prevention and treatment is more active than ever before. Drugs currently used for tumor patients include alkylating agents such as nitrogen mustard and cyclophosphamide, purine antimetabolites such as 6-mercaptopurine and azathioprine, and 5-fluorouracil and silabitan. Such as pyrimidine antimetabolite, antifolate antimetabolite such as methotrexate and aminopterin, and antitumor antibiotics such as mitomycin C and bleomycin. Toxic to the side, causing side effects such as impaired hematopoiesis. In addition, as foods with antitumor activity, health foods containing many polysaccharides having immunostimulatory effects such as Agaricus koji and Meshimakob are commercially available, and the public is highly conscious about prevention and treatment of tumors, Development of pharmaceuticals and health foods having high antitumor activity is desired.

  On the other hand, garlic has been used as a seasoning and spice since ancient times, but in recent years, it has become clear that various physiologically active ingredients are contained therein, and is widely used as health foods and pharmaceuticals. For example, Patent Document 1 reports that allicin isolated and purified from garlic is effective in treating or preventing prostate cancer and bladder cancer. However, it has not been known that garlic-derived oligosaccharides have antitumor activity.

JP 2001-302531 A

  An object of this invention is to provide the composition for preventing and / or treating a tumor, the foodstuff and feed containing this, and its manufacturing method.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that the garlic-derived oligosaccharide has an antitumor activity, and that the oligosaccharide is combined with the garlic-derived lectin to improve the synergistic effect. As a result, the present invention has been completed.

That is, the present invention includes the following inventions.
(1) A composition for preventing and / or treating a tumor, comprising a processed garlic product containing garlic oligosaccharide as an active ingredient.
(2) The composition according to (1), wherein the processed garlic contains 5% by mass or more of garlic oligosaccharides based on the solid component.
(3) The composition according to (1) or (2), wherein the processed garlic is a garlic extract.
(4) The composition according to any one of (1) to (3), wherein the processed garlic further contains garlic lectin.
(5) The composition according to (4), wherein the processed garlic contains 2% by mass or more of garlic lectin with respect to the solid component.
(6) Add water to garlic, grind, filter and heat the filtrate, cool, centrifuge and salt out the supernatant, dissolve the resulting precipitate in water or buffer to remove insolubles The composition for preventing and / or treating the tumor obtained by this.
(7) A food for treating and / or preventing a tumor, comprising the composition according to any one of (1) to (6).
(8) A feed for treating and / or preventing a tumor, comprising the composition according to any one of (1) to (6).
(9) A method for producing a composition for preventing and / or treating a tumor, comprising adding water to garlic, crushing and filtering, heating the filtrate, cooling and then centrifuging the supernatant to salt And a step of centrifuging and dissolving the resulting precipitate in water or a buffer to remove insoluble matters.

  The present invention provides a composition for preventing and / or treating a tumor, a food and feed containing the composition, and a method for producing the same.

  In the present invention, garlic has a meaning usually used in the art, that is, Allium sativum L. belonging to the genus Liliaceae and Allium.

  In the present invention, the garlic oligosaccharide means an oligosaccharide derived from garlic, and includes an oligosaccharide contained in garlic, an oligosaccharide obtained from garlic, an oligosaccharide extracted from garlic, and the like. In the present invention, the garlic oligosaccharide has a molecular weight of usually 1500 to 2000, preferably 1700 to 1900, more preferably about 1800. In the present invention, the garlic oligosaccharide is usually composed of 10 to 12 monosaccharides, preferably 11 and is composed of glucose and fructose. The molar ratio of glucose: fructose is usually 1: 0.4 to 1:15, preferably 1: 8 to 1:12, and preferably 1: 9 to 1:11. Furthermore, in the present invention, the garlic oligosaccharide is preferably glucose at its non-reducing end.

  In the present invention, garlic lectin means a lectin derived from garlic, and includes lectins contained in garlic, lectins obtained from garlic, lectins extracted from garlic, and the like. In the present invention, the molecular weight of garlic lectin is usually 20000-25000 Da, preferably 21000-24000 Da, more preferably about 23000 Da. In the present invention, garlic lectin is generally considered to have a dimer structure composed of subunits of 11000 to 12000 Da. In the present invention, the garlic lectin preferably has the amino acid structure shown in Table 4. The amino acid sequence near the N-terminus (1 to 36 amino acids) is usually RNILMNGEGLYAGELSLDVEPYHFIMQDDCNLVLYXH (SEQ ID NO: 1).

  In the present invention, the processed garlic product is not particularly limited as long as it is produced from garlic and contains garlic oligosaccharide. In the present invention, the processed garlic includes dry garlic powder, garlic extract, dry powder of garlic extract, and the like. In the present invention, the garlic processed product is preferably derived from the bulb portion of garlic.

  The dried garlic powder can be produced by methods commonly used in the art, such as sun drying, heating or drying in a heating chamber, hot air drying, freeze drying, and the like.

  The processing method for obtaining a garlic extract can use the method normally used in this technical field. Extraction of garlic is usually performed at a temperature of 0 to 70 ° C., preferably 5 to 60 ° C., more preferably 10 to 50 ° C., usually for 1 minute or more, preferably 5 minutes to 24 hours. When performing in a short time, you may extract under pressure. As a solvent used for extraction, water, 35% or less alcohol, pH 4.5 to 10 or H4.5 to 7.5 buffer or the like can be used. As the water, any of distilled water, purified water, ion exchange water, tap water and the like can be used. The solvent is used in an amount of 0.1 to 10 parts by weight, preferably 0.5 to 5 parts by weight, more preferably 1 part by weight, based on 1 part by weight of raw garlic. In extraction, it is necessary to grind garlic, and it is preferable to grind garlic before heating.

  The obtained garlic extract can be used directly as it is, or it can be further concentrated and used as a powder by a conventional drying means such as fluid extract or concentrated drying, spray drying, vacuum drying or freeze drying. .

  In the present invention, the garlic-treated product is preferably prepared by the following method. Add water to garlic and grind it with a mixer. At this time, water is preferably added in the same amount as the mass of garlic. This is filtered with gauze, filter paper, cloth, etc., and the filtrate is usually 30 to 100 ° C., preferably 60 to 100 ° C., more preferably 80 to 100 ° C., usually 5 minutes to 10 hours, preferably 10 minutes to 1 Heat for a time, more preferably 20-40 minutes. After cooling, it is centrifuged to obtain a supernatant. The conditions for centrifugation are usually 3000-15000 rpm and about 5-15 minutes. This is salted out and left at 0 to 30 ° C., preferably 0 to 10 ° C. Salting out can be carried out, for example, by adding ammonium sulfate. Thereafter, the mixture is centrifuged again under the same conditions to obtain a precipitate. This precipitate is usually dissolved in water or a buffer of pH 4.5 to 10 or pH 4.5 to 7.5, and insoluble matters are removed by centrifugation. As the buffer solution, for example, acetate buffer solution, phosphate buffer solution and the like can be used.

  Preferably, the obtained processed product is further applied to a column equilibrated with the same buffer, subjected to gel filtration, the absorbance at 280 nm is measured, and the second peak portion is collected.

  In the present invention, the garlic processed product is usually 5% by mass or more, preferably 10% by mass or more, more preferably 20 to 90% by mass, and even more preferably 30 to 80% by mass with respect to the solid component of garlic oligosaccharide. The most preferable content is 50 to 70% by mass. In the present invention, the processed garlic preferably further contains garlic lectin. The garlic lectin is usually 2% by mass or more, preferably 3 to 45% by mass, more preferably 10 to 40% by mass with respect to the solid component.

  When the processed garlic product contains garlic lectin, the ratio of garlic oligosaccharide to garlic lectin is not particularly limited, but is usually 10: 1 to 5: 4, preferably 5: 1 to 5: 3.

  In the present invention, the processed garlic includes purified garlic oligosaccharide. The purity of the purified garlic oligosaccharide is usually 80 to 100% by mass, preferably 90 to 100% by mass, and more preferably 95 to 100% by mass. In order to obtain a purified product, a method usually used in this technical field such as a precipitation method, a chromatographic method, and a gel filtration method can be used.

  For example, the product obtained by collecting the second peak by gel filtration as described above is concentrated, and the column equilibrated with phosphate buffer is eluted with a gradient of saline using the same buffer. Purified garlic oligosaccharides can be obtained by collecting portions and dialyzing against water.

The concentration of garlic oligosaccharide and / or garlic lectin relative to the solid component in the processed garlic can be measured by a method commonly used in this technical field. For example, chromatography, TOF-MS, etc. may be used.
Specifically, it can be measured by the following procedure.

  Chromatography is performed by elution with distilled water using a Bio-Gel P-2 (BIO-RAD) column. In TOF-MS, a mass spectrometer is used, Matrix Assisted Laser Deposition Ionization is used as an ionization method, and the wavelength of laser light is 337 nm (nitrogen laser).

  The composition for preventing and / or treating a tumor of the present invention contains the above-mentioned garlic-treated product as an active ingredient. The composition of the present invention can be used for the purpose of preventing and / or treating tumors, particularly malignant tumors. The composition of the present invention is effective for the prevention and / or treatment of hematopoietic tumors, tumors composed of epithelial cells, and sarcomas composed of non-epithelial cells. Examples of hematopoietic tumors include leukemia, malignant lymphoma, and myeloma. Tumors composed of epithelial cells include lung cancer, breast cancer, stomach cancer, colon cancer, colon adenoma cancer, uterine cancer, ovarian cancer, pharyngeal cancer, tongue cancer. Examples of the sarcoma include osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, fibrosarcoma, liposarcoma and the like. The composition of the present invention is effective for the prevention and / or treatment of hematopoietic tumors, particularly lymphomas, tumors composed of epithelial cells, particularly gastric cancer and colon adenoma cancer.

  Garlic which is an active ingredient of the composition of the present invention is usually used for food and generally has low toxicity. Therefore, the composition of the present invention is considered to be highly safe. Furthermore, since garlic as a raw material can be obtained at low cost, a composition having antitumor activity can be produced at low cost.

  The composition of the present invention can be made into foods or feeds by conventional methods, or can be made into pharmaceuticals of various dosage forms together with pharmaceutically acceptable carriers. Among these, when preparing an oral solid preparation, after adding an excipient, if necessary, a binder, a disintegrant, a lubricant, a coloring agent, a corrigent, a corrigent, etc. to the composition of the present invention, Tablets, coated tablets, granules, powders, capsules and the like can be produced by conventional methods. Such additives may be those commonly used in the art. For example, excipients include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, silicic acid As a binder, water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl starch, methylcellulose, ethylcellulose, shellac, calcium phosphate, polyvinylpyrrolidone, etc. As disintegrants, dried starch, carmellose calcium, sodium alginate, agar powder, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, lactose, etc. are purified as lubricants. Torque, stearate, borax, polyethylene glycol, sucrose as a flavoring agent, orange peel, citric acid, can be exemplified tartaric acid.

  When preparing an oral liquid preparation, a liquid preparation, a syrup, an elixir, etc. can be manufactured by a conventional method by adding a corrigent, a buffer, a stabilizer, a corrigent, etc. to the processed garlic. In this case, the flavoring agents may be those listed above, examples of the buffer include sodium citrate, and examples of the stabilizer include tragacanth, gum arabic, and gelatin.

  The dose and administration method of the composition of the present invention can be appropriately determined depending on age, weight, symptoms, etc., but usually 10 to 100 mg in terms of garlic oligosaccharide per day or once per adult. It is preferable to administer them separately.

(Example 1) Preparation of garlic oligosaccharide The same amount of water was added to garlic, applied to a mixer for 2 minutes, filtered through gauze, and the filtrate was heated at 90 ° C. for 30 minutes in a thermostatic bath. After cooling, the mixture was centrifuged at 8000 rpm for 10 minutes to obtain a supernatant. Ammonium sulfate was added thereto so as to be 80% saturation, and the mixture was allowed to stand at 5 ° C. for 30 minutes, and then centrifuged again at 8000 rpm for 10 minutes to obtain a precipitate. This precipitate was dissolved in 0.02 M acetate buffer (pH 5.0), and insoluble matters were removed by centrifugation at 8000 rpm for 10 minutes. This was applied to a Sephadex G-75 (Pharmacia) column equilibrated with the same buffer solution and subjected to gel filtration. The absorbance at 280 nm of each fraction was measured, the second peak was collected, concentrated with an Amicon ultrafilter, and DEAE-Toyopearl 650M (Tosoh) equilibrated with 0.02 M phosphate buffer (pH 6.0). Were eluted with a gradient of 0-2.5 M saline in the same buffer. The absorbance at 490 nm is a single peak, and this portion was collected, dialyzed against water, and then lyophilized.

(Example 2) Composition analysis of freeze-dried product The carbohydrate content, protein content and water content of the obtained freeze-dried product were measured. The sugar content was measured by the phenol / sulfuric acid method using glucose as a standard substance. The protein content was measured by the Lowry method using bovine serum albumin as a standard substance. The water content was measured by a normal pressure heating drying method (105 ° C.).

  The results are shown in Table 1 below.

  From the results in Table 1, it was revealed that the obtained lyophilized product contained almost no protein and that saccharides were the main component.

Example 3 Measurement of Sugar Purity and Molecular Weight The sugar purity and molecular weight contained in the lyophilized product obtained in Example 1 were measured by gel filtration and MALDI-TOF / MS.

(1) Gel filtration method Purity was measured by elution with distilled water using a Bio-Gel P-2 (BIO-RAD) column (2.0 × 64.1 cm). The molecular weight was determined using D-glucose (molecular weight 180), sucrose (molecular weight 342), raffinol (molecular weight 505), stachyose (molecular weight 649) and maltoheptaose (molecular weight 1090) as standard substances.

(2) MALDI-TOF / MS method 2,5-dihydroxybenzoic acid was dissolved in a matrix solvent in which distilled water and ethanol were mixed at 1: 4 to prepare a 10 mg / ml solution. Next, the sample and the matrix solution were mixed 1: 1, and 1.5 μl was dropped on the target and air-dried.
The mass spectrometer used BIFLEX III (BRUKER), the ionization method used Matrix Assisted Laser Desorption Ionization, and the wavelength of the laser beam used was 337 nm (nitrogen laser).

(3) Results In the results of Bio-Gel P-2 column chromatography, the peak at 490 nm showed a single peak, indicating that this carbohydrate was pure. The molecular weight of this carbohydrate was about 1800. MALDI-TOF / MS results also showed that the carbohydrate molecular weight was about 1800.

  From the above, it was revealed that the saccharide contained in the lyophilized product obtained in Example 1 was an oligosaccharide having a molecular weight of about 1800.

(Example 4) Analysis of oligosaccharide constituent monosaccharides The oligosaccharide constituent units were analyzed by HPLC using a post-label fluorescence detection method. For hydrolysis, 2M trifluoroacetic acid was used and heated at 100 ° C. for 6 hours, and for fructose analysis, 0.1M HCl was used and heated at 60 ° C. for 30 minutes.

  The HPLC column used was TSK-gel Sugar AXG (Tosoh) 4.6 mm × 15 cm, and 0.5 M potassium borate buffer (pH 8.7) was used.

  As a result, it was found that the oligosaccharide obtained in Example 1 was composed of glucose and fructose, and the molar ratio was 1.5: 1. From the above, it was shown that this oligosaccharide is composed of 11 hexose sugars and is composed of 7 glucose and 4 fructose, or 6 glucose and 5 fructose.

(Example 5) Methylation analysis of oligosaccharide Complete methylation of oligosaccharide was performed by the Hakomori method. Hydrolyzed purified methylated oligosaccharide was decomposed into monosaccharides, then reduced acetylated and partially methylated. GC and GC / MS measurements were performed in the form of an acetyl derivative of sugar alcohol (partially methylated alditol acetate).

Since fructose was contained, hydrolysis was performed under weak conditions, that is, using 0.02M HCl at 60 ° C. for 30 minutes. Reductive methylation sugar was performed using NaBD _4.

  For GC measurement, Hewlett-Packard HP5890A was used as an apparatus, and SPB-5 (Spelco Japan) 0.25 mm × 25 m was used as a column.

  The GC / MS was measured using a JMS DX-303 mass spectrometer and a Hewlett-Packard gas chromatograph.

  The results are shown in Table 2 below.

  The non-reducing end of this oligosaccharide is fructose, 1 → 6 linked fructose, 1 → 1 linked fructose or 1 → 2 linked glucose, 1 → 6 linked glucose, 1 → 2, 1 → 6 linked glucose It was shown that they are linked by various bonding modes.

(Example 6) Preparation of garlic lectin The same amount of water was added to garlic, applied to a mixer for 2 minutes, filtered through gauze, and the filtrate was heated in a thermostat at 90 ° C for 30 minutes. After cooling, the mixture was centrifuged at 8000 rpm for 10 minutes to obtain a supernatant. Ammonium sulfate was added thereto so as to be 80% saturation, and the mixture was allowed to stand at 5 ° C. for 30 minutes and then centrifuged again at 8000 rpm for 10 minutes to obtain a precipitate. This precipitate was dissolved in 0.02 M acetate buffer (pH 5.0), and insoluble matters were removed by centrifugation at 8000 rpm for 10 minutes. This was applied to a Sephadex G-75 (Pharmacia) column equilibrated with the same buffer solution and subjected to gel filtration. The absorbance at 280 nm of each fraction was measured, the second peak was collected, concentrated with an Amicon ultrafilter, and DEAE-Toyopearl 650M (Tosoh) equilibrated with 0.02 M phosphate buffer (pH 6.0). Were eluted with a gradient of 0-2.5 M saline in the same buffer. The absorbance peak at 280 nm was collected, dialyzed against water, and then lyophilized.

(Example 7) Composition analysis of freeze-dried product About the freeze-dried product obtained in Example 6, the carbohydrate content and protein content were measured by the same method as in Example 2. The results are shown in Table 3 below.

  The carbohydrate obtained here is considered to be bound to the protein by a covalent bond. In addition, as a result of polyacrylamide gel electrophoresis, a single band was shown.

(Example 8) Molecular weight measurement and amino acid analysis The purified product (protein) obtained in Example 6 was subjected to molecular weight measurement and amino acid analysis. The molecular weight was measured by gel filtration using a Sephadex G-75 column (2.0 × 49 cm) equilibrated with 0.02 M acetate buffer (pH 5.0). In gel filtration, the molecular weight of the obtained protein was 23000 Da.

  The amino acid analysis was performed using a Hitachi 835 type amino acid analyzer. The results are shown in Table 4 below.

  The primary structure of the protein was measured using an Applied Biosystem 477A protein sequencer. As a result, an amino acid sequence near the N-terminus (1 to 36 amino acids), RNILMNGEGLYAGELSLDVEPYHFIMQDDCNLVLYXH (SEQ ID NO: 1) was obtained.

(Example 9) Test of antitumor activity against lymphoma cells Using human lymphoma cells (U937) as tumor cells, the anti-tumor activity of garlic oligosaccharide obtained in Example 1 and garlic lectin obtained in Example 6 Tested for tumor activity.

The method for culturing U937 cells and seeding on microplates are as follows (reference: Food Research International 34 (2001) 7-13).
(1) A cell culture solution was prepared by adding 10% fetal bovine serum FBS (manufactured by Gibco Laboratories) and 50 g / ml of the antibiotic gendamicin to RPMI 1640 (manufactured by Sigma Chemical Co.).
(2) The number of cells was diluted to 10 ⁵ cells / ml, and 0.1 ml (10 ⁴ cells) was added to each well of a 96-well microplate.
(3) After 5 to 6 hours, garlic oligosaccharide (lyophilized product obtained in Example 1), lectin (lyophilized product obtained in Example 6), or a mixture of garlic oligosaccharide and garlic lectin 0.1 ml of the above cell culture solution in which each was dissolved at the indicated concentration was added to each well. In Table 7, (lectin + oligosaccharide) 10 mg / ml means that gel filtration is performed on a Sephadex G-75 (Pharmacia) column in Example 1, and the absorbance at 280 nm of each fraction. Garlic processed product A containing garlic lectin and garlic oligosaccharide, obtained by dialysis and lyophilization after collecting the second peak portion. Table 9 also has the same meaning. In this processed garlic product A, the composition ratio of garlic lectin and garlic oligosaccharide is garlic lectin: garlic oligosaccharide = 1: 2 in terms of mass ratio.
(4) Cells were cultured in a CO ₂ incubator at 37 ° C., and the number of cells was counted under a microscope with an appropriate time passage.

  The results are shown in Tables 5-7 below.

  From the above results, it has been clarified that garlic oligosaccharide has antitumor activity against lymphoma cells and that an excellent antitumor activity can be obtained by the synergistic effect of garlic oligosaccharide and garlic lectin.

(Example 10) Test of antitumor activity against colon adenoma cancer cells WiDr cells (human colon adenoma cancer cells) were used as tumor cells, and garlic oligosaccharide obtained in Example 1 and obtained in Example 6 The antitumor activity of garlic lectin as well as the antitumor activity of krestin, known as an anticancer agent, was tested. WiDr cells were obtained from the Human Science Research Resource Bank.

(1) A culture medium (DMEM: Dulbecco's modified Eagle Medium, manufactured by Nacalai Tesque Co., Ltd.) plus 10% FBS and an antibiotic gentamicin (manufactured by Gibco Co., Ltd.) 50 μg / ml was used as a cell culture medium. The cell number was diluted to 2 × 10 ⁴ cells / ml, and 0.1 ml (2 × 10 ³ cells) was added to each well of a 96-well microplate.
(2) After one night, 0.1 ml of the cell culture solution in which a reagent such as lectin or oligosaccharide was dissolved was added to each well.
(3) The cells were cultured in a carbon dioxide incubator at 37 ° C.
(4) The number of cells was measured every 24 hours with a cell counting kit-8 (manufactured by Doujin Chemical Laboratory Co., Ltd.). That is, 10 μl of cell counting kit-8 was added to each well. The color reaction was carried out for 1 to 4 hours in a carbon dioxide incubator. Absorbance at 450 nm was measured using a microreader.

  The results are shown in Tables 8-12 below.

  From the above results, it has been clarified that garlic oligosaccharide has antitumor activity against colon adenoma cells, and that excellent antitumor activity can be obtained by the synergistic effect of garlic oligosaccharide and garlic lectin.

(Example 11) Test of antitumor activity against gastric cancer cells As tumor cells, KATOIII cells (human gastric cancer cells) were used, and the garlic oligosaccharide obtained in Example 1 and the garlic lectin obtained in Example 6 were used. Antitumor activity was tested for antitumor activity. KATOIII cells were obtained from the Human Science Research Resource Bank.

(1) A cell culture solution was prepared by adding 10% FBS and an antibiotic gentamicin (manufactured by Gibco) 50 μg / ml to a culture solution (RPMI 1640 medium and Eagle MEM mixed 1: 1).
(2) The number of cells was diluted to 2 × 10 ⁴ cells / ml, and 0.1 ml (2 × 10 ³ cells) was added to each well of a 96-well microplate.
(3) After one night, 0.1 ml of the cell culture solution in which a reagent such as lectin or oligosaccharide was dissolved was added to each well.
(4) The cells were cultured in a carbon dioxide incubator at 37 ° C.
(5) The number of cells was measured every 24 hours with cell counting kit-8 (manufactured by Doujin Chemical Laboratory Co., Ltd.). That is, 10 μl of cell counting kit-8 was added to each well. The color reaction was carried out for 1 to 4 hours in a carbon dioxide incubator. Absorbance at 450 nm was measured using a microreader.

  The results are shown in Tables 13 and 14 below.

  From the above results, it was revealed that garlic oligosaccharide has an antitumor activity against gastric cancer cells and that an excellent antitumor activity can be obtained by the synergistic effect of garlic oligosaccharide and garlic lectin.

(Example 12) Preparation of processed garlic B 210 g of garlic and 210 ml of distilled water were applied to a mixer and then filtered through gauze. The filtrate was heat treated at 90 ° C. for 30 minutes. It cooled and centrifuged (4 degreeC, 10000 rpm, 10 minutes). Ammonium sulfate was added to the supernatant to 80% saturation, and after dissolution, it was cooled at 5 ° C. for 15 minutes or more. Centrifugation (4 ° C., 10000 rpm, 10 minutes) was performed, and the precipitate was dissolved in 100 ml of distilled water and centrifuged again (4 ° C., 10000 rpm, 10 minutes). The obtained supernatant was dialyzed against water, desalted and lyophilized to obtain a garlic processed product B. When the composition of the obtained lyophilized product was analyzed, it contained 47% by mass of garlic oligosaccharide and 23% by mass of garlic lectin.

(Example 13) Transplantation of processed garlic / cancer cell suspension into mice
13-1. Cancer cell culture Colon-26 cancer cells were used as cancer cells. Mouse-derived colon-26 cancer cells were provided by Kanazawa University Cancer Institute. The cancer cells are shown in the following table.

(1) Preparation of medium RPMI 1640 medium powder was measured, dissolved in ultrapure water, and sterilized by filtration. 100 ml of 10% NaHCO ₃ solution was prepared and sterilized by autoclave. In RPMI 1640 medium, FBS (Fetal Bovine Serum, fetal bovine serum) becomes 10% so that NaHCO ₃ solution becomes 2 mg / ml, penicillin / streptomycin solution becomes 100 units / ml, 100 μg / ml. Each was added to prepare a medium. Specifically, 86 ml of RPMI 1640, 2 ml of 10% NaHCO _{3 and} 2 ml of penicillin / streptomycin solution were mixed, and after confirming the color change, 10 ml of FBS was added.

(2) Cell culture The culture medium and Hanks solution were warmed in a 37 ° C, 5% CO ₂ incubator, and frozen colon-26 cancer cells were warmed in a 37 ° C thermostat. While still a little frozen, it was transferred to a Falcon tube containing Hanks solution. Pipetting and centrifugation (1000 rpm, 5 minutes) were performed, and the supernatant was removed by suction with a Pasteur. 1 ml of Hanks solution was added and pipetting was performed. 30 μl of cell suspension and 30 μl of trypan blue were collected in an Eppendorf tube. The number of cells was counted using a hemocytometer and a microscope, and seeded in a 100 mm dish at 3 × 10 ⁵ cells / dish. Medium was added at 10 ml / dish. Cells were cultured in a 37 ° C., 5% CO ₂ incubator.

(3) Cell Passage It was confirmed with a microscope whether the cells had grown to sub-confluence. If they had grown, the cells were passaged by the following procedure. The medium was removed from the dish using a Pasteur pipette, washed with 5 ml of Hanks solution, and then removed with a Pasteur pipette. 3 ml of trypsin / EDTA solution was added and left in an incubator at 37 ° C. for about 4 minutes. The bottom of the dish was flicked with a finger and it was confirmed that it was peeled off with a microscope. If peeled off, 7 ml of medium was immediately added to stop the trypsin action, and after stirring, collected in a falcon tube with a measuring pipette. The dish was washed with 2 ml of Hanks solution, which was also collected with a pipette. Subsequently, the operation after centrifugation in the above cell culture procedure was performed.

13-2. Preparation of Garlic Treatment / Cancer Cell Suspension A garlic treatment / cancer cell suspension was prepared in order to transplant the garlic treatment and cancer cells to mice in the manner shown in the following table. As the garlic processed product, the garlic processed product B prepared in Example 12 was used.

(1) 1 ml of garlic solution was prepared using PBS (Phosphate Buffered Saline, phosphate buffered saline) so that the concentration of each target garlic product B was doubled.
(2) The cultured colon-26 cancer cells were treated with trypsin, transferred to a falcon tube, and centrifuged (1000 rpm, 5 minutes).
(3) After removing the supernatant, a medium was added, pipetted, and the number of cells was counted using trypan blue and a hemocytometer.
(4) After counting, centrifugation (1000 rpm, 5 minutes) was performed, the supernatant was removed, and a cell suspension was prepared with PBS so that the cell concentration was 1 × 10 ⁷ cells / ml.
(5) The garlic processed product B solution prepared in (1) and the cell suspension prepared in (4) were mixed at a ratio of 1: 1 (1 ml each) to a total of 2 ml.

13-3. BALB / c mice (male) were used for transplantation of garlic processed product / cancer cell suspension to mice.
Prior to transplantation, mice were weighed. A garlic processed product / cell suspension containing garlic processed product B at each concentration was prepared as described above, and allowed to stand for 10 minutes. In 5 groups of mice consisting of 7 mice, 0.2 ml (1 × 10 ⁶ cells) of garlic treatment product / cell suspension containing garlic treatment product B at each concentration was implanted subcutaneously on the right flank (1 × 10 ⁶ cells). This day was designated Day 0).

Seven days after the tumor became prominent, measurement of the body weight and the area of the tumor [mm ² ] (= longest diameter of tumor × shortest diameter of tumor) was started. Thereafter, transplantation was performed every 4 days, and measurement was similarly performed until the 19th day. On the 15th and 19th days, the tumor volume was also determined by measuring the height of the tumor (tumor volume [mm ³ ] = (longest tumor diameter) × (shortest tumor diameter) × (tumor height). Sa)).

  For statistical analysis, t-test was performed using Microsoft Excel. The results are shown in Table 17 below and FIG.

  From the above results, it was demonstrated that the garlic-treated product containing the garlic oligosaccharide of the present invention has anticancer activity in vivo against cancer.

(Example 14) Oral administration of processed garlic to mice transplanted with cancer cells
14-1. Preparation of cancer cell suspension Colon-26 cancer cells cultured in Example 13 were trypsinized and then centrifuged (1000 rpm, 5 minutes). After removing the supernatant, a medium was added, pipetted, and the number of cells was counted using trypan blue and hemocytometer. After counting, centrifugation (1000 rpm, 5 minutes) was performed, and a cell suspension was prepared with PBS so that the cell concentration was 5 × 10 ⁶ cells / ml.

14-2. BALB / c mice (male) were used as transplanted cancer cell suspensions and treated mice treated with garlic . Mice were weighed prior to cancer cell suspension transplantation and garlic treatment administration. The above cancer cell suspension was transplanted subcutaneously in the right flank of 0.2 ml (1 × 10 ⁶ cells) of 4 groups of 8 mice each (this day was designated as day 0).

Garlic treatment B solution containing 10 mg / ml, 1 mg / ml and 0.1 mg / ml of garlic treatment B was prepared using PBS, and 0.5 ml was added to each mouse group every 2 days. Administered. PBS was administered to the control group. Starting 10 days after the tumor became prominent, measurement of body weight and tumor area [mm ² ] using a caliper (= longest diameter of tumor × shortest diameter of tumor) was started, and thereafter, cancer was measured every 3 days. The measurement was performed in the same manner until day 28 after transplanting the cells. On the 16th and 22nd days, the tumor volume was also determined by measuring the height of the tumor (tumor volume [mm ³ ] = (longest diameter of tumor) × (shortest diameter of tumor) × (tumor height Sa)).

  For statistical analysis, t-test was performed using Microsoft Excel. The results are shown in Table 18 below and FIG.

  From the above results, it was demonstrated that a cancer-suppressing effect can be obtained in vivo by orally ingesting a garlic-treated product containing the garlic oligosaccharide of the present invention.

(Example 15) Anticancer activity test of colon-26 cancer cells treated with garlic (1) A culture medium (RPMI1640: manufactured by Nacalai Tesque Co., Ltd.) plus 10% FBS was used as a cell culture medium. Since it is a monolayer culture (adherent) cell, peel off the cell from the flask with 0.25% trypsin + 1 mM EDTA, dilute the cell number to 1 × 10 ⁵ cells / ml, and add 0.1 ml (1 × 10 ⁴ cells). Added to each well of a 96-well microplate.
(2) After one night, 0.1 ml of the cell culture solution prepared by dissolving garlic processed product B prepared in Example 12 at various concentrations was added to each well.
(3) The cells were cultured in a carbon dioxide incubator at 37 ° C.
(4) The number of cells was measured every 24 hours with a cell counting kit-8 (manufactured by Doujin Chemical Laboratory Co., Ltd.). That is, 10 μl of cell counting kit-8 was added to each well. The color reaction was carried out for 1 to 4 hours in a carbon dioxide incubator. The number of cells was measured based on the absorbance at 450 nm using a microreader. The results are shown in Table 19 below.

  From the above, it was confirmed that the garlic-treated product containing the garlic oligosaccharide of the present invention exhibited anticancer activity even in vitro against colon-26 cancer cells. Moreover, it was demonstrated that the anticancer activity in vitro has a correlation with the anticancer activity in vivo.

(Example 16) Analysis of garlic oligosaccharide by NMR Various two-dimensional NMR (DEPT, DQF-COSY, TOCSY, ROESY, HSQC, HMBC) was measured for garlic oligosaccharide. Furthermore, ¹ H NMR and ¹³ C NMR spectra of inulin were measured and analyzed.

  As a result, the main component of garlic oligosaccharide was estimated to be the following β-2,1-fructan having glucose at the non-reducing end.

  Therefore, the ratio of glucose to fructose is estimated to be 1:10.

The change of the tumor area when the garlic treatment product / cancer cell suspension containing the garlic treatment product at various concentrations is transplanted into a mouse is shown. The change of a tumor area when the garlic processed product solution which contains a garlic processed product in various density | concentrations is orally ingested to the mouse | mouth which transplanted the cancer cell is shown.

Claims (1)

The following formula (I) extracted and purified from garlic :

A medicament for preventing and / or treating a tumor, which contains a garlic oligosaccharide represented by the formula:

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