JP2009227633A - Anti-cancer compound derived from allium fistulosum to which alien chromosome is added, and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a compound produced by an offspring plant derived from interspecies crossing of a plant of genus Allium, and having growth inhibitory effects on cancer cells, and to provide a method for producing the compound. <P>SOLUTION: The compound is characterized by having one chromosome 1 and/or one chromosome 8 of a plant of the Allium cepa, derived from a plant of non-Allium cepa (a plant belonging to genus Allium without belonging to the cepa species) in which the nuclear genome exhibits diploidy, and having solubility in ketonic organic solvents and the growth inhibitory effects on the cancer cells in order to solve the problems. The method for producing the compound is provided. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

The present invention relates to a compound derived from a useful leek genus plant produced using a heterologous chromosome addition technique and having anticancer properties. More specifically, the present invention relates to one chromosome 1 of a cepa species Allium cepa. And / or derived from a non-sepa species of genus Allium (defined as a plant belonging to the genus and not belonging to Sepa species, the same applies hereinafter) having one chromosome 8 and having a nuclear diploidy in the nuclear genome. The present invention relates to an anticancer compound and a method for producing the same.
In the present specification and claims, the Sepa species Allium plant is the following characteristics of the plant belonging to the genus Allium, that is, the basic number of chromosomes is 8, has a tubular leaf blade part, and a bulb is developed. It has a short stem in the vertical direction (Subgenus Cepa), the flower color is greenish white, the leaves begin to appear flat or semi-cylindrical, mainly 4-9 pieces, and a wide flower cover It has a green fiber running along the middle of the flower piece, the honey secretion gland is pocket-shaped, and the lower half of the flower stem has a balloon-like bulge (Cepa alliance). It refers to plants (Non-patent Document 1).
The non-sepa species Allium plant is a generic term for allium plants that do not have the above characteristics among the Allium plants.

  The onion plant (Allium) is a group of monocotyledonous plants including onions, leeks, garlic, rakkyo and the like, and has been used as an edible crop for a long time. Many plants of the genus Allium contain a lot of useful substances such as quercetin, polyphenol, and alliin (converted into allicin) in the cells, and are attracting attention as crops containing a lot of functional components. As an invention for utilizing a useful substance derived from a genus Allium plant or enhancing a useful component, a method for increasing the flavonoid content of a plant by treating the plant with a growth-regulating acylcyclohexanedione has been hitherto (patent document). 1) Health supplements from which carbohydrates are removed and active ingredients such as quercetin and pectin are high in concentration, a method for producing the same (Patent Document 2), and the like are disclosed.

In allium plants, it has been pointed out that the allyl sulfide component containing sulfur has functions such as thrombus dissolution and anti-cancer activity. Drugs and nutritional supplements using functionality are also disclosed. Examples of these include the use of lectins in the manufacture of drugs for the treatment of metabolic disorders (Patent Document 3), nutritional supplements that treat malignant diseases and viral infections and improve immune function (Patent Document 4), edible plant extracts In the past, selective COX-2 inhibition by a product (Patent Document 5), an alliin fatty acid conjugate (Patent Document 6) that exhibits an excellent neutral fat-reducing action with weak side effects have been disclosed.
However, the substances having anticancer activity derived from the plants of the genus Allium so far have been obtained from plants of the original species regardless of wild species or cultivated species. Since allium plants are easy to intercross, development of new anticancer components utilizing the effect of combination has been awaited in progeny plants derived from hybrid plants and hybrid plants.
Method for increasing the content of plant flavonoids and phenolic components Patent application title: Processed onion food and method for producing the same Special table 2001-510447 lectin compound and use thereof JP 2003-504341 A method for treating malignant diseases and viral infections using nutritional supplements and improving immune function Special Table 2004-532811 Selective COX-2 Inhibition by Edible Plant Extract Special table 2006-502712 Extraction, purification and conversion of plant biomass-derived flavonoids Fritsch R.D. M.M. & Friesen N. 2002. Evolution, domestication and taxonomy. in: Allium Crop Science: Regent Advances. 5-30. CAB International. Shigyo M. et al. et al. (1996): Genes Genet. Syst. 71: 363-371. Masuzaki S. et al. Shigyo M .; & Yamauchi N. (2006): Theor. Appl. Genet. 112: 607-617.

  In view of the above situation, the present invention aims to provide a compound produced by a progeny plant derived from cross-species crossing of an allium plant and having a growth inhibitory effect on cancer cells, and a method for producing the compound. .

  In order to solve the above-mentioned problems, the present inventors performed cross-breeding using a leek (Allium fistulosum) and a charlotte (A. cepa) in a leek genus plant. Chromosome-added "leek lines have been created for chromosomes 1 to 8 (Non-Patent Documents 2 and 3). In the leek line with single heterogeneous chromosomes, it has been found that ascorbic acid content and polysaccharide content show different traits from both leek and charlotte, and has already been proposed in patent applications and the like. Furthermore, as a result of examining the new utility for these single heterologous chromosome-added leek lines, the present inventors surprisingly have one Charlotte first chromosome and / or one eighth chromosome, Acetone extract obtained from a line of a non-sepa genus plant with diploid nuclear genome (here, leeks, i.e., fistulosum species) is a strong growth inhibitory effect against cancer cells, which is not found in normal leeks or charlottes The present invention was completed by finding that it has an action.

  That is, in the first aspect of the present invention, a non-sepa scallion having one chromosome 1 and / or one chromosome 8 of a plant belonging to the genus Sepha species (Allium cepa) and having a diploid nuclear genome. A compound derived from a genus plant (a plant belonging to the genus Allium and not belonging to Sepa), soluble in ketone-based organic solvents, and having a cancer cell growth inhibitory effect is provided. To do.

  According to a second aspect of the present invention, there is provided a compound according to the first aspect, wherein the non-sepa species Allium plant is Allium fistulosum.

  According to a third aspect of the present invention, there is provided the compound according to the first or second aspect, wherein the Sepa species Allium plant is an allium cepa aggregatum group.

  According to a fourth aspect of the present invention, there is provided the compound according to any one of the first to third aspects, wherein the ketone-based organic solvent is acetone.

According to a fifth aspect of the present invention, there is provided a method for producing a compound according to any one of the first to fourth aspects, characterized by comprising the following steps.
(A) A step of mating a non-sepa genus plant and a Sepa plant (B) a step of doubling the chromosome of the hybrid plant cell obtained in step (A) (C) a chromosome obtained in step (B) Step of cultivating doubling cells and obtaining a regenerated plant (D) Crossing the regenerated plant obtained in step (C) with the same type of non-sepa genus plant used in step (A) Step of obtaining a triploid plant (E) A progeny obtained by crossing an allogeneous triploid plant obtained in step (D) with a non-sepa genus plant belonging to the same species as used in step (A) A step of investigating a chromosome of a plant cell and selecting a heterologous chromosome-added plant having one chromosome 1 and / or one chromosome 8 of a Sepa species plant (F) The heterogeneity obtained in step (E) Crushing a chromosome-added plant and adding a ketone organic solvent to extract

  According to the present invention, it is possible to use a compound having anticancer activity derived from a genus Allium plant born by a mating technique different from the conventional mating technique, regardless of genetic recombination. This compound can be used as a pharmaceutical compound for the treatment and prevention of cancer, and in daily eating habits, it has one chromosome 1 and / or chromosome 8 of the Sepa genus plant developed by the present inventors. It is also possible to use it as a food compound for reducing cancer risk, for example, by eating a non-sepa species of genus Allium that has one and has a nuclear genome that is doubled.

  The best mode for carrying out the present invention will be described below. The first aspect of the present invention is a non-sepa scallion that has one chromosome 1 and / or one chromosome 8 of the plant belonging to the genus Sepa spp. (Allium cepa) and has a nuclear genome that is diploid. It is derived from a genus plant (defined as a plant belonging to the genus Allium and not belonging to the Sepa species. The same shall apply hereinafter), is soluble in ketone-based organic solvents, and has an effect of inhibiting the growth of cancer cells. A featured compound is provided. The compound of this embodiment may be a single compound or a plurality of compounds including a main component derivative and the like. The plant material that produces this compound is a non-sepa diploid plant belonging to the genus Allium, which is a first and / or eighth chromosome of the Sepa species, in particular one chromosome 1 or one chromosome 8 Plants having this are preferred. Examples of non-sepa genus genus include leeks (Allium fistulosum), garlic (Allium sativum), leek (Allium tuberosum), raccoon (Allium chinese), chili (Allium schoenopurasum), umtsuno sill (um. An edible genus plant selected from (Allium grayi), Gyoja garlic (Allium victoralis), Leek (Allium ampeloprasum) and the like is preferable, and as shown in the examples below, leeks (A. fistulosum) are more preferable. .

In addition, the Sepa seed onion genus plant in the present invention includes any varieties of so-called onions that are cultivated in various parts of the world. ) Is a more preferred example.
Furthermore, the ketone organic solvent in the present invention includes any compound that has a basic structure of ketone and can be used as an organic solvent. Examples thereof include acetone, diethyl ketone, methyl ethyl ketone, methyl isobutyl ketone, diisobutyl ketone, cyclohexanone, Difernyl ketone is preferred, and among these, acetone used for organic solvent extraction is a more preferred example.

  The suppressive effect on cancer cells exhibited by the compound of the present invention is an effect of suppressing the proliferation or survival of cancerous cells, and the type or the like does not limit the present invention. Breast cancer, uterine cancer, ovarian cancer, esophageal cancer, stomach cancer, colon cancer, liver cancer, gallbladder cancer, pancreatic cancer, gastrointestinal stromal tumor, mesothelioma, laryngeal cancer, oral cancer It is considered effective against cancer cells selected from thyroid cancer, kidney cancer, lung cancer, prostate cancer, bladder cancer, and skin cancer. As described above, it has an inhibitory effect on bladder cancer cells, which is preferable.

The compound of the present invention is present in an Allium plant satisfying the above conditions, and a method for producing a heterologous chromosome-added Allium strain can be selected as appropriate. Preferably, the following steps, namely (A ) A step of mating a non-sepa genus plant and a cepa plant, (B) a step of doubling the chromosome of the hybrid plant cell obtained in step (A), and (C) a chromosome obtained in step (B). Culturing doubling cells to obtain a regenerated plant body, (D) crossing the regenerated plant body obtained in step (C) with the same non-sepa genus plant used in step (A), A step of obtaining an allotriploid plant; (E) a cross between the allotriploid plant obtained in step (D) and the same kind of non-sepa genus Allium plant used in step (A); The chromosomes of the progeny plant cells, one chromosome 1 and / or chromosome 8 of the Sepa species plant A method of selecting a heterologous chromosome-added plant having one of the above, (F) crushing the heterologous chromosome-added plant obtained in step (E), and adding a ketone-based organic solvent to perform extraction. ing.
Among the above steps, the steps (A) to (E) relate to a method for producing an Allium plant capable of producing the compound provided by the present invention. Combining the production of the body and backcrossing, one chromosome 1 and / or one chromosome 8, more preferably one chromosome 1 of the plant of the genus Sepa, This is a process for obtaining a diploid non-sepa genus plant having one chromosome 8. The method of cross-breeding, the method of chromosome doubling, etc. may be appropriately selected from methods usually used in the field of breeding and is not intended to limit the present invention. For example, leeks from which cocoons have been removed before maturation (seed parent) ) Artificially pollinate pollen collected from Shallot (pollen parent), cut out the growth point of the obtained hybrid seed progeny plant, and doubled the chromosome by colchicine treatment, and this was treated with LS (Linsmeier-Skoog) medium or MS ( A regenerated plant body is obtained by culturing on an appropriate medium such as Murashige-Skoog) medium, and a leek (pollen parent) is crossed with the grown regenerated plant to obtain an allogeneic triploid. (Pollen parent) are crossed to induce unequal distribution of the charlotte chromosome at the time of meiosis, and cells are collected from the obtained progeny and observed under a microscope to obtain a Charlotte dye. Identify the chromosome of the charlott present in the genome, such as by performing morphological identification of the body or extracting genomic DNA from its offspring and amplifying the gene that is a marker of a specific chromosome by PCR etc. A process for selecting a target plant is suitable.

The method for extracting the compound provided by the present invention from the plant of the genus Allium thus prepared may be appropriately selected from methods for extraction with an organic solvent, preferably a ketone organic solvent, and the present invention is limited. Although it is not a thing, for example, the leaf sheath part of the genus Allium plant produced in the above process is collected and freeze-dried, and then crushed and added with the ketone organic solvent, preferably acetone, and extracted. Can be given. The practitioner may appropriately select the composition, amount, extraction time, extraction temperature, and the like of the solvent.
Examples of the present invention will be described below, but the present invention is not limited to the examples.

  (Production of Shallot Chromosome Added Leek) A diploid leek line having one Shalot 1 or 8 chromosome is produced by the method described in Non-Patent Document 2 as a plant material that produces the anticancer component of the present invention. did. The outline of the production method is shown in Table 1 below. First, as a step (A), a green onion 'Kujo S-3' line (Allium fistulosum; non-sepa species; genome symbol FF) is used as a seed parent, and a Charlotte 'Mynanmar 17-2' line (A. cepa aggregatum group; Sepa Species; genome symbol AA) was crossed as pollen parent to obtain hybrid F1 (AF). At the time of mating, males were emasculated so that self-fertilization with leek pollen did not occur, and a small amount of Charlotte pollen was manually pollinated by leek to obtain F1. Mature seeds were collected from the fruits obtained by these crossings. A part of the collected seeds was placed on a petri dish with moistened filter paper and germinated at 25 ° C. under dark conditions. After germination, the plant was greened under illumination at 25 ° C., grown to a certain extent, acclimatized, transplanted to culture soil, and cultivated in a greenhouse. The same method was used for subsequent mating.

  As the step (B), the chromosome of the hybrid seed progeny obtained in the step (A) was doubled. The shoot apex tissue was cut out from F1, placed on Linsmeier-Skoog (LS) solid medium containing 0.1% colchicine, and cultured under dark conditions for 4 days. Thereafter, the tissue was transferred onto an LS-free medium and cultured for 2 months. The resulting regenerated plant was stained with foilgen on the chromosome of the root tip cell, and the chromosome was examined under a microscope. The shape and size of the onion and charlotte chromosomes were examined in advance under a microscope, and the chromosomal image of the regenerated plant body was morphologically compared to determine which chromosomes had and how much. Chromosomes were observed by the following method. Secondary roots extending to 5-10 mm were collected and fixed overnight in a refrigerator in an acetic acid-ethanol mixture (1: 3). The fixed roots were treated with 1N hydrochloric acid at 60 ° C. for 6 minutes, dissociated and hydrolyzed, and then subjected to foilen staining with basic fuchsin. One drop of 45% acetic acid was dropped on the slide glass, the dyed root tip was put in it, the cover glass was placed, the sample was hit from the cover glass with a pointed stick, and the cover glass was pressed. By this operation, cells and chromosomes were dispersed and the chromosomes were arranged on a single plane, and then the chromosomes were observed under an optical microscope. Based on this observation, those having two pairs of each of a leek chromosome and a charlott chromosome, that is, a double diploid (AAFF: 2n = 32) were selected from the regenerated plants and used for the next generation of crosses.

As the step (C), the double diploid obtained in the step (B) is used as a seed parent, the back onion 'Kujo 4-2' line is used as a pollen parent, and backcrossing is performed, so that allotriploid (BC ₁₎ ). The method of mating is as described above. Of the obtained BC _1, the '4042' line with good growth is subjected to the second backcross as a step (D) with the leek 'Kujo 4-2' line as the pollen parent, and the seeds obtained by this crossing are collected. Then, as a step (E), the presence or absence of the Charlotte chromosome is confirmed by the above-described method, and additional chromosomes are identified using a molecular marker specific to each chromosome as an index (see Non-Patent Document 1). The “single heterologous chromosome-added” leek line having one Charlotte chromosome was selected for each chromosome (FF + 1A to FF + 8A). The molecular markers specific to each chromosome are shown in Table 2 below.

  (Acetone extraction from plant material) As a step (F) of the present invention, for each line from FF + 1A to FF + 8A planted in the field, the leaves are collected at a stage where the height grows to about 30 cm, and the sheath is peeled off. Immediately after lyophilization, 80% acetone was added as a ketone organic solvent so that the final concentration of the lyophilized sample was 250 mg / ml, and the mixture was crushed on ice for extraction. Extraction was also carried out in the same manner from the parent strains FF, AA, and heterologous triploid FFA.

(Assay of Extract Using Cultured Cancer Cells) Anticancer activity assay using cultured cancer cells for the acetone extract of the heterologous chromosome-added leek system obtained by the steps (A) to (F). Went. As a cultured cancer cell, UMUC3 strain which is a human bladder cancer-derived cell line was used. Culture conditions were 10% fetal bovine serum, 100 units / ml penicillin, 100 μg / ml streptomycin-containing RPMI1640 medium, and cultured at 37 ° C. and 5% CO ₂ .
UMUC3 cells were seeded in a 96-well microplate (manufactured by Nunc) so that the number of cells per well was 1 × 10 ⁴ cells, and cultured under conditions of 37 ° C. and 5% CO ₂ for 24 hours. Thereafter, 2 μl of acetone extract was added to each well so that the final concentrations were 2.5, 5, and 25 mg / ml, and the culture was continued under the same conditions. For the effect of the extract, MTS [3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium], which is a reagent for measuring cell proliferation, is used. The percentage of living cells that had incorporated MTS was evaluated by absorbance (using CellTiter 96 AQueous One Solution Cell Proliferation Assay manufactured by Promega). 20 μl of MTS was added to each well and the culture was further continued for 4 hours, and then the absorbance at 490 nm was measured using a microplate reader. For comparison of the growth inhibitory effect, a control group to which only 80% acetone as a solvent was added instead of the acetone extract was prepared, and the value was compared between the plant materials from the absorbance of the treatment group relative to the absorbance of this control group. went. The repetition was performed twice, and the average value was obtained.

  Prior to verifying the effect of an extract from the genus Allium plant, in order to confirm whether addition of acetone alone is appropriate as a control group, an acetone added group and an unadded group were provided in the above UMUC3 strain culture system. MTS assays were performed over time to compare absorbance at 490 nm. FIG. 1 shows the result. The vertical axis of the graph represents absorbance at 490 nm, and the horizontal axis represents no addition (first time, second time) and 80% acetone addition (first time, second time). As shown in the graph, the 80% acetone added group showed the same cell growth as the non-added group, indicating that the acetone added group is suitable as a control. In subsequent experiments, the 80% acetone addition group was used as a control group.

FIG. 2 shows the effect of each extract on cultured cancer cells. The vertical axis of the graph shows the growth of cells 24 hours after the addition of the extract by comparison with the control group (% of control), and the horizontal axis shows the final concentration of each extract (2.5, 5, 25 mg / ml). To express. Graph ... ◆ ... is onion FF, ... ■ ... is charlotte AA, ... ○ ... is the effect of an extract from allogeneic triploid FFA,-▲-is FF + 1A,-*-is FF + 2A,-○-is FF + 3A , − + − Represents FF + 4A, −−− represents FF + 5A, − ◇ − represents FF + 6A, −Δ− represents FF + 7A, and − ■ − represents the effect of the extract from FF + 8A.
As shown by the results at 2.5 mg / ml in the graph, at this concentration, most extracts did not show an inhibitory effect on cancer cells, but only FF + 1A and FF + 8A showed an inhibitory effect on cancer cell proliferation. . The effect on the control group was 88.1% (11.9% suppression) with FF + 1A and 60.3% (39.7% suppression) with FF + 8A.

The results at 5 mg / ml in the graph also showed the same tendency as 2.5 mg / ml, and the inhibitory effect was not observed in most extracts, whereas a remarkable inhibitory effect was observed in FF + 1A and FF + 8A. The effects on the control group were 55.6% (44.4% inhibition) with FF + 1A and 46.9% (53.1% inhibition) with FF + 8A, showing strong cancer cell growth inhibition effects.
Addition at a concentration of 25 mg / ml showed an effect of 53.7% (46.3% suppression) with Charlotte AA compared to the control group, and also had a constant effect with FF + 3A, FF + 6A, and FF + 7A. . However, the suppression effect was not seen with FF. Even at this concentration, the effects of FF + 1A (suppression of 71.2%) and FF + 8A (suppression of 91.1%) are remarkably high. In particular, FF + 8A suppresses 90% or more of the control and has a very strong cancer cell proliferation inhibitory effect. It was observed.
From these results, it was shown that the compound provided by the present invention has an inhibitory effect on the growth of cancer cells.

  By using the compound provided by the present invention, it is possible to produce a novel anticancer component derived from a genus Allium plant. The compound provided by the present invention can be used as a raw material for medicines for the production of anticancer agents and the like, and since the leek plant producing the compound is produced without genetic recombination, the normal diet It can also be used as a food compound in the form of preventing cancer.

The influence of 80% acetone addition on the growth of cultured cancer cells is shown. The influence of the compound provided by the present invention on the growth of cultured cancer cells is shown.

Claims (5)

  It is derived from a non-sepa plant belonging to the genus Genus, which has one chromosome 1 and / or one chromosome 8 of an allium cepa plant and has a nuclear genome diploid. A compound characterized by being soluble in and having a cancer cell growth inhibitory effect.   The compound according to claim 1, wherein the non-sepa species Allium plant is Allium fistulosum.   3. A compound according to claim 1 or claim 2, wherein the Sepa scallion plant is Shallot (Allium cepa aggregatum group).   The compound according to any one of claims 1 to 3, wherein the ketone organic solvent is acetone. The method for producing a compound according to any one of claims 1 to 4, which comprises the following steps.
(A) A step of mating a non-sepa genus plant and a Sepa plant (B) a step of doubling the chromosome of the hybrid plant cell obtained in step (A) (C) a chromosome obtained in step (B) Step of cultivating doubling cells and obtaining a regenerated plant (D) Crossing the regenerated plant obtained in step (C) with the same type of non-sepa genus plant used in step (A) Step of obtaining a triploid plant (E) A progeny obtained by crossing an allogeneous triploid plant obtained in step (D) with a non-sepa genus plant belonging to the same species as used in step (A) A step of investigating a chromosome of a plant cell and selecting a heterologous chromosome-added plant having one chromosome 1 and / or one chromosome 8 of a Sepa species plant (F) The heterogeneity obtained in step (E) Crushing a chromosome-added plant and adding a ketone organic solvent to extract

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