JP5126644B2 - Evaluation method of traditional Chinese medicine using gene expression profile - Google Patents

Description

  The present invention relates to a technique for evaluating the activity of a herbal medicine, a drug containing the active ingredient, a food, or the like using a gene expression profile.

Traditional Chinese medicine has played a role as a medicine or health food by decocting and extracting medicinal herbs etc. by various methods based on many years of experience and ingesting the active ingredients contained in the body efficiently and effectively. However, the effect is affected by various factors such as the number, amount, and shape of active ingredients (including those due to chemical modifications and changes in chemical structure due to metabolism), or the mixing ratio of ingredients that increase or decrease the effectiveness of active ingredients. Therefore, there was no effective activity search and activity evaluation and quality control method. In particular, component analysis by HPLC, TLC, mass spectrometry, or the like is not a method for evaluating activity or effect, and therefore has a drawback in that the effect of components that increase or decrease the effect of active ingredients cannot be evaluated. There are also methods for measuring the effect of components of Chinese herbal medicine on the activity of a specific enzyme (see Patent Document 1) and evaluating the effect by an assay based on animal experiments. However, since these methods are time-consuming and expensive and are not highly accurate, better techniques have been demanded. In particular, licorice is a material (or herbal medicine) used in many herbal medicines and is also used in health foods, and its activity is known to be affected by the climate, production area, preservation method and processing method. . Such various factors have been included, making it difficult to evaluate activity and quality control.

  There are various effective activities of Kampo medicines, for example, estrogenic activity. Estrogen, a type of female hormone, has been conventionally used as an estrogen replacement therapy to reduce symptoms of hot flashes and genital atrophy, and to prevent postmenopausal osteoporosis. It has also been reported that estrogen lowers cholesterol, increases collagen, prevents cranial nerve aging, and has therapeutic effects on neurological diseases such as Alzheimer's. However, administration of estrogen also has a risk of inducing endometriosis, malignant tumors, and the like. Plants also contain trace amounts of substances that have an estrogenic effect called plant estrogens. These substances bind to estrogen receptors in mammals and exert estrogenic effects. In particular, it is known that such a substance is present in many traditional Chinese medicines such as Amakusa. Compared with estrogen replacement therapy in which estrogen is administered, mild treatment is possible and there is less risk of causing the above-mentioned side effects. In addition, various health foods that are said to contain phytoestrogens are also commercially available, focusing on the effects of phytoestrogens. However, some of these foods actually contain little phytoestrogens. It was not always easy to accurately evaluate the estrogenic activity of these foods.

On the other hand, a method for evaluating the activity of various active compounds by measuring the influence on gene expression profile using a DNA microarray has been reported. This included a method for evaluating the activity of estrogens and substances similar in action to estrogen using estrogen-responsive genes whose expression varies depending on estrogen or a chemical substance having estrogen-like activity (Patent Documents 2 and 2). (See 3 etc.).

JP 2003-70496 A Japanese Patent No.3713461 JP 2005-323600 A

An object of the present invention is to provide a method for evaluating the effect of a Chinese medicine by examining changes in the gene expression profile caused by the Chinese medicine for a specific gene, and a reagent such as a DNA microarray for evaluation.

The present invention relates to licorice that is a herbal medicine using a DNA chip (gene names are summarized in FIG. 1) developed to solve the problem (its components and pharmaceuticals / food / reagents containing the components and their components). This is a method for activity search and evaluation (including processed products) and quality control thereof (hereinafter abbreviated as “method of licorice activity evaluation”). The feature of this method is that the biological and physiological activity of licorice is evaluated by using the expression variation of cellular genes as an index. For example, as shown in the examples described later, it has a characteristic gene expression profile of estrogen. A method for evaluating the activity of licorice based on the homology as an index and the similarity between the homology and the homology according to gene function. Therefore, the method for obtaining the gene expression profile may not be a DNA chip, and the genes to be used need not be all of the genes shown in FIG. 1, and a plurality of genes may be used. . In addition, an expression profile of licorice can be obtained. For example, if a correlation analysis can be performed in comparison with an expression profile by estrogen, such a gene group can be used.

That is, the present invention is as follows.
[1] A method for evaluating the activity of a herbal medicine or an active ingredient thereof, or a plant, extract or composition containing the active ingredient, using a gene whose expression varies when the active ingredient is applied to a living body, Obtained when the expression profile of the gene obtained by applying the herbal medicine or its active ingredient to be evaluated, or a plant, extract or composition containing the active ingredient to the living body and the active ingredient applied to the living body A method for evaluating the activity of a herbal medicine or an active ingredient thereof, or a plant, extract or composition containing the active ingredient, which comprises comparing the expression profile of the gene.
[2] The method for evaluating the activity of the Chinese medicine of [1] or an active ingredient thereof, or a plant, extract or composition containing the active ingredient, wherein the Chinese medicine is a Chinese medicine containing licorice.
[3] The active ingredient is an ingredient having estrogenic activity or estrogen-like activity, and the gene whose expression varies when the active ingredient is applied to a living body is an estrogen responsive gene [1] or [2] Chinese medicine or its activity A method for evaluating the activity of an ingredient, or a plant, extract or composition containing the active ingredient.
[4] The application of the active ingredient to the living body is performed by culturing cells in a medium containing the active ingredient, including any of the Chinese medicines or active ingredients thereof according to [1] to [3], or the active ingredients thereof A method for evaluating the activity of a plant, extract or composition.

[5] The method for evaluating the activity of a Chinese medicine or an active ingredient thereof according to [4], wherein the cells are MCF-7 cells, or a plant, extract or composition containing the active ingredient.
[6] A gene profile is obtained by using a DNA microarray in which a gene whose expression changes when an active ingredient is applied to a living body or a fragment thereof is immobilized, or a herbal medicine of any one of [1] to [5] A method for evaluating the activity of an active ingredient or a plant, extract or composition containing the active ingredient.
[7] A Chinese medicine or active ingredient thereof according to any one of [1] to [5], wherein the gene profile is obtained by quantitative PCR targeting a gene whose expression varies when the active ingredient is applied to a living body or a fragment thereof, Or a method for evaluating the activity of a plant, extract or composition containing the active ingredient.
[8] The evaluation of the activity of a herbal medicine or its active ingredient, or a plant, extract or composition containing the active ingredient is effective in the herbal medicine or its active ingredient, or a plant, extract or composition containing the active ingredient Exploration or evaluation of activity, or quality control of Chinese medicine or a composition containing an active ingredient thereof [1] to [7] any Chinese medicine or its active ingredient, or a plant, extract or composition containing the active ingredient A method for evaluating the activity of objects.

[9] The gene whose expression fluctuates when the active ingredient of Chinese medicine is applied to the living body is shown in FIG.
A method for evaluating the activity of a herbal medicine or active ingredient thereof according to any one of [1] to [8], which is at least one of the estrogen-responsive genes, or a plant, extract or composition containing the active ingredient.
[10] The Kampo medicine according to any one of [1] to [8], wherein the gene whose expression varies when the active ingredient of Kampo medicine is applied to a living body is at least 5 of the 115 estrogen response genes shown in FIG. A method for evaluating the activity of an active ingredient or a plant, extract or composition containing the active ingredient.
[11] A herbal medicine or an active ingredient thereof, or a plant, extract or composition containing the active ingredient, comprising all or part of the base sequence of at least one gene of 115 estrogen responsive genes shown in FIG. Reagent for evaluating activity.
[12] Of a herbal medicine or an active ingredient thereof, or a plant, extract or composition containing the active ingredient, comprising all or part of the base sequence of at least 5 genes of 115 estrogen responsive genes shown in FIG. Reagent for evaluating activity.

[13] A herbal medicine of [11] or [12] or an active ingredient thereof, or a plant, extract or composition containing the active ingredient, which is a DNA microarray in which all or part of the base sequence of the gene is immobilized Reagent for evaluating activity.
[14] A primer set for quantitative PCR consisting of all or part of the base sequence of a gene, [11] or [12] Chinese herbal medicine or an active ingredient thereof, or a plant, extract or composition containing the active ingredient Reagent for evaluating activity.
[15] The activity of a herbal medicine or its active ingredient according to [14], which is a primer set for quantitative PCR comprising all or part of the base sequence of the following gene, or a plant, extract or composition containing the active ingredient Reagent for evaluation.
16. cathepsin D (lysosomal aspartyl peptidase) (CTSD) [NM_001909]
25. Ribosomal protein S6 kinase, 90kDa, polypeptide 3 (RPS6KA3) [NM_004586]
26. protein kinase C, delta (PRKCD) [NM_006254]
30. cyclin-dependent kinase inhibitor 1A (p21, Cip1) (CDKN1A) [NM_000389]
38. Rho GDP dissociation inhibitor (GDI) alpha (ARHGDIA) [NM_004309]
48. trefoil factor 1 (breast cancer, estrogen-inducible sequence expressed in) (TFF1) [NM_003225]
67. tumor protein p53 inducible protein 11 (TP53I11) [NM_006034]
76. nuclear receptor interacting protein 1 (NRIP1) [NM_003489]
[16] A reagent for evaluating the activity of a Chinese medicine or an active ingredient thereof according to [15] containing the primer set forth in SEQ ID NOs: 1 to 16, or a plant, extract or composition containing the active ingredient.

As shown in the Examples, in order to demonstrate the effectiveness of this method, the gene expression profile analysis using a DNA chip (FIG. 3) and the gene found in the licorice extract (the assay concentration is shown in FIG. 2) It was reproduced by statistical analysis incorporating functions (Fig. 4). Moreover, it was shown that it differs from the profile which Glycyrrhizin (glycyrrhizin) which is a licorice main ingredient considered to be an active ingredient conventionally. Furthermore, the differences in profiles are not biologically and physiologically meaningless differences, but are based on differences in biological and physiological functions. Real-time quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method Analysis (FIG. 5), analysis of the phosphorylation reaction of signal transduction protein Erk (FIG. 6), and analysis of the ability of rat PC12 cells to form neural dendrites (FIG. 7).

  These results show that by using the method of the present invention, the useful biological / physiological activity of an herbal medicine or an active ingredient thereof, or a plant, extract or composition containing the active ingredient in animals such as humans can be evaluated. Show what you can do. The method of the present invention can be used to search for useful biological / physiological activity, useful biological / physiological activity magnitude, useful biological / biological activity for a herbal medicine or an active ingredient thereof, or a plant, extract or composition containing the active ingredient. Allows quality control in physiological activity.

  The method of the present invention is a method for evaluating a herbal medicine or an active ingredient thereof, or a plant, extract or composition containing the active ingredient. Herbal medicine includes herbal medicine. The active ingredient of Kampo medicine refers to an active ingredient contained in Kampo medicine that has a beneficial biological / physiological function and brings certain beneficial physiological effects to animals such as humans. A composition containing an active ingredient of traditional Chinese medicine includes foods, pharmaceuticals, quasi-drugs, reagents, etc. that contain an active ingredient of traditional Chinese medicine and have a beneficial effect on animals due to the biological and physiological activities exhibited by the active ingredient. Good, including herbal medicine and processed products of plants such as medicinal herbs that can be used as raw materials for the herbal medicine. The food includes health food, food for specified health use, nutritional functional food, and the like, and food includes beverages. Here, the food for specified health refers to food that is ingested for the purpose of specific health in the diet and displays that the purpose of the health can be expected by the intake. In the present invention, evaluation refers to evaluation of effective biological / physiological activity based on the above active ingredient, and the herbal medicine or its active ingredient to be evaluated, or the plant, extract or composition containing the active ingredient is Search for activity to test whether it has activity, evaluation, evaluation of Chinese medicine or its active ingredient to be evaluated, or how much activity a plant, extract or composition containing the active ingredient has This includes quality control of the target Chinese herbal medicine or its active ingredient, or a plant, extract or composition containing the active ingredient. Quality control is particularly useful for testing whether or not a composition such as a pharmaceutical, quasi-drug, reagent, food, etc. that is indicated as having an activity has a predetermined activity. In the manufacturing process of a product, it is useful for testing whether or not a composition as a product or an intermediate has a predetermined activity. As used herein, “effective activity” refers to how much beneficial effect is obtained when a herbal medicine or an active ingredient thereof, or a plant, extract or composition containing the active ingredient has any beneficial effect on an animal. This refers to the activity that can be determined using this as an index. Depending on the Kampo medicine, the active ingredient that brings about its medicinal effect may not be specified. In such a case, the herbal medicine or its active ingredient, or the plant, extract or composition containing the active ingredient may be evaluated. Have difficulty. Since the method of the present invention can grasp the activity by using the variation of gene expression due to the active ingredient as an index even if the active ingredient is not specified, it contains the Chinese medicine or its active ingredient, or its active ingredient An accurate assessment of the plant, extract or composition can be made. The magnitude of the effective activity is usually correlated with the amount of the active ingredient in the traditional Chinese medicine or its active ingredient, or the plant, extract or composition containing the active ingredient. However, the effectiveness of the active activity may be reduced by other components contained in the composition, and in such a case, the amount of the active component does not correlate with the magnitude of the effective activity. In the present invention, rather than measuring the physicochemical amount of the active ingredient, the magnitude of the effective activity for animals in the herbal medicine or its active ingredient to be evaluated, or the plant, extract or composition containing the active ingredient. Since it is possible to measure the thickness, it is possible to make an evaluation in consideration of the influence of other components.

  In this invention, the active ingredient of a Chinese medicine and a Chinese medicine is not limited, The effective activity based on the active ingredient of a known or unknown Chinese medicine can be evaluated using the method of this invention. For example, a Chinese medicine having estrogenic activity can be mentioned. Chinese medicines having estrogenic activity are not limited, but Chinese medicines containing so-called phytoestrogens that bind to human estrogen receptors and have an action equivalent to estrogen on humans are targeted. Such herbal medicines include herbal medicines made from licorice, glaze, peony and the like.

  “Estrogen-like activity” as used herein means an activity that exhibits an effect similar to or similar to that of the female hormone estrogen. Estrogen is a variety of hormones, growth factors, and various physiological factors such as growth and immune responses of individuals. Since direct or indirect interaction is also performed with chemical substances involved in the action, it is difficult to clearly detect and evaluate the influence. In particular, in the case of Kampo, various components affect each other in a complicated manner, so that they may not be detected or evaluated as an effect equivalent to or similar to that of estrogen. In addition, when the activity other than the estrogen-like activity is large, the estrogen-like activity may not be clearly detected. However, the effect of estrogen-like activity on cells is thought to be common to some chemical substances with estrogen-like activity, although it is considered to be common, and the effect can be measured by gene expression. is there. Therefore, in this method, not only chemical substances that can detect and evaluate a clear estrogen-like activity by other methods, but also other methods cannot detect and evaluate a clear estrogen-like activity. The activity can also be evaluated for chemical substances that have the same or similar effects on estrogen as gene expression. That is, one embodiment of the method of the present invention is based on the homology with the characteristic gene expression profile of estrogen as an index, and licorice etc. based on the high homology and similarity of homology by gene function. It is a method to evaluate the activity of traditional Chinese medicine.

  In the method of the present invention, the expression of a gene whose expression is known to vary when an active ingredient of Chinese herbal medicine or a component having an active ingredient-like activity thereof is applied to a living body such as an animal, animal tissue or animal cell. A herbal medicine or an active ingredient thereof, or a plant, extract or composition containing the active ingredient is evaluated using the expression fluctuation as an index. The component having an active ingredient-like activity of Chinese herbal medicine refers to, for example, an animal estrogen or a substance having an activity similar to that of an estrogen with respect to the above-mentioned component having an estrogenic activity of a plant. In this case, a gene group whose expression varies with estrogen in an animal is specified in advance. Next, the variation of the gene group is measured when the Chinese medicine to be evaluated or its active ingredient, or a plant, extract or composition containing the active ingredient is applied to a living body such as an animal, animal tissue or animal cell. By doing so, evaluation can be performed. Here, applying Chinese medicine or an active ingredient thereof, or a plant, extract or composition containing the active ingredient to a living body such as an animal, an animal tissue or an animal cell, administration to an animal, administration to a tissue, This refers to contact with cells. Preferably, it is contacted with an animal cell such as a human cell in vitro. Contacting means culturing animal cells for a certain period of time in the presence of the above-mentioned Chinese medicine or an active ingredient thereof, or a plant, extract or composition containing the active ingredient. The cells to be used are not particularly limited, but cells collected from tissues of animals such as humans such as mammary gland, uterus, liver, brain and stomach, or cell lines derived from animals such as humans can be used.

  After applying a herbal medicine or an active ingredient thereof, or a plant, extract or composition containing the active ingredient to a living body such as an animal, animal tissue or animal cell, the animal, animal tissue or animal cell Variations in gene expression can be measured by obtaining a gene expression profile. The gene expression profile can be obtained by measuring the expression level of the identified gene group. The expression level may be measured by measuring a gene transcription product, ie, mRNA, or by measuring a gene translation product, ie, protein. Preferably, it is carried out by measuring a gene transcription product. A gene transcription product also includes cDNA obtained by reverse transcription from mRNA.

In the present invention, the expression profile can be obtained by, for example, a method using a DNA chip, a method using a DNA microarray, a cDNA subtraction method, a differential display method, a real-time quantitative PCR method, or the like. Real-time quantitative PCR includes SAGE (serial analysis of gene expression), ATAC-PCR method (Kato, K. et al., Nucl. Acids Res., 25, 4694-4696, 1997), Taqman PCR method (SYBR (
(Registered trademark) Green method) (Schmittgen TD, Methods 25, 383-385, 2001), Body Map method (Gene, 174, 151-158 (1996)), Serial analysis of gene expression (SAGE) method (US Patent No. 527,154, No. 544,861, European Patent Publication No. 0761822), and MAGE (Micro-analysis of Gene Expression) (Japanese Patent Laid-Open No. 2000-232888).

  In the present invention, a specific gene whose expression varies when an active ingredient having an effective activity to be evaluated is applied to a living body such as an animal, an animal tissue, or an animal cell is selected.

Such a gene selection method is preferably performed using a DNA chip or a DNA microarray in terms of comprehensive expression analysis. A DNA chip can be produced by spotting genetic DNA or EST of which the sequence is known in advance using a pin head or an ink jet on a substrate, or by synthesizing it on a suitable substrate by optical lithography. At this time, DNA sequence information can be obtained from a DNA database such as GenBank. DNA microarrays obtain cDNA clones from human cells, amplify the cDNA by PCR,
It can be produced by being attached on a suitable substrate.

  Analysis by these methods should be performed according to the description of “DNA microarray and latest PCR method”, separate volume of cell engineering, Genome Science Series 1, supervised by Masaaki Muramatsu, Hiroyuki Nami, published on March 16, 2000, Shujunsha etc. Can do.

Here, the DNA microarray is a generic term for devices in which DNA is aligned (arrayed) and immobilized on a substrate. Depending on the size of the substrate and the density of the DNA to be loaded, it can be divided into DNA macroarrays and DNA microarrays. In the present invention, both DNA macroarrays and DNA microarrays are called DNA microarrays. Further, the number of DNAs to be aligned is not limited, and the DNAs are included in the DNA microarray as long as at least one DNA is spotted on the substrate. Furthermore, in a DNA microarray, DNA spotted on a substrate is hybridized with a labeled sample DNA, but this labeling method is not limited, and includes fluorescent labeling, labeling with a radioisotope, and the like.

The specific gene for evaluating a herbal medicine or its active ingredient used in the present invention, or a plant, extract or composition containing the active ingredient is selected using a cDNA microarray as follows, for example. can do. First, human EST or full-length cDNA is obtained. A cDNA library can be prepared from human cells, or a commercially available gene set may be used. The obtained cDNA and EST are used as probe DNA and spotted on a slide glass using a commercially available array machine. As the slide glass to be used, for example, a glass treated with poly-L-lysine is used. For example, DNA used for immobilization is dissolved in 3 × SSC and spotted. The concentration is not limited, but a concentration of 0.5 μg / μl to 1 μg / μl or more is desirable. For example, if a glass slide of 4 cm ² is used, spotting at a diameter of about 100 to 400 μm can immobilize 10000 or more kinds of DNA per sheet. After immobilization, post-treatment is performed by rehydration, blocking with succinic acid, and the like. In the present invention, the term “gene” includes EST.

Apply Chinese herbal medicine or its active ingredient, or a substance having an effective activity to evaluate a plant, extract or composition containing the active ingredient to a living body such as an animal, animal tissue or animal cell, and stimulate To do. Here, a herbal medicine or an active ingredient thereof, or a substance having an effective activity to be evaluated for a plant, extract or composition containing the active ingredient is a specific ingredient of a traditional Chinese medicine or a specific ingredient of a traditional Chinese medicine. An extract containing the same, a composition containing the specific component of Chinese medicine, a substance having an activity equivalent to that of the specific component of Chinese medicine, and the like. Examples of substances having an activity equivalent to that of the specific component of Chinese medicine include analogs and derivatives of the specific component. Total mRNA is extracted from cells of animals stimulated with these substances and unstimulated cells. mRNA is prepared by extracting total RNA by the guanidine thiocyanate / cesium chloride method, etc., and then using an affinity column method such as oligo dT-cellulose or poly U-sepharose, or a batch method to obtain poly (A) + RNA (mRNA ). The cDNA may be labeled when a single-stranded cDNA is synthesized using the thus obtained mRNA as a template using an oligo dT primer and reverse transcriptase. The labeling is performed with a fluorescent dye, and the cDNA obtained from cells stimulated with the substance and the cDNA obtained from unstimulated cells may be labeled with different fluorescent dyes. Examples of fluorescent dyes include Cy3 (red) and Cy5 (green) from Amersham Pharmacia Biotech.

CDNA labeled with each fluorescent dye in a suitable hybridization solution is hybridized with the DNA spotted on the microarray. The hybridization conditions at this time can be appropriately selected by changing the salt concentration of the hybridization solution or changing the reaction temperature according to the desired sensitivity.

After hybridization, by observing or measuring the fluorescence pattern on the slide glass with a fluorescence microscope, fluorescence detector (for example, CHIP READER (trademark) (manufactured by VERTEK)), etc. The expression profile of can be analyzed. For example, when cDNA obtained from cells stimulated with the above substances is labeled with red Cy3 and cDNA obtained from non-stimulated cells is labeled with green Cy5, a gene that is expressed only in the stimulated cells is immobilized. The spot on the microarray emits red fluorescence, the spot on the microarray in which genes that are expressed only in unstimulated cells are immobilized emits green fluorescence, and the spot on the microarray in which the genes that are expressed in both cells are immobilized is red and green The synthetic color fluorescence is emitted. DNA immobilized on a spot showing a result that the intensity of Cy3 fluorescence (red fluorescence) when stimulated with the above substances is greater than the intensity of Cy5 fluorescence (green fluorescence) when not stimulated is Chinese medicine or its active ingredient, or its activity It is a gene whose expression is increased by a plant, extract or composition containing components, and the DNA immobilized on a spot showing a result that is less than the fluorescence intensity of Cy5 when stimulated when the fluorescence intensity of Cy3 is not stimulated is a Chinese medicine or It is a gene whose expression is suppressed by the active ingredient, or a plant, extract or composition containing the active ingredient. Measure Cy3 fluorescence intensity and Cy5 fluorescence intensity of each spot on the DNA microarray, calculate the Cy3 / Cy5 ratio, for example, and use these ratios as indicators to evaluate the effects of Kampo medicines or their components . At this time, an internal standard gene is spotted on the DNA microarray. Hybridization is performed by adding DNA that can hybridize with the internal standard gene and labeled with Cy3 or Cy5 to the sample. When calculating the Cy3 / Cy5 ratio on each spot, the Cy3 / Cy5 ratio of each spot can be corrected by normalizing with the Cy3 / Cy5 ratio of the internal standard gene. At this time, genes whose expression varies can be analyzed by hierarchical cluster analysis or the like. Hierarchical cluster analysis can be performed using commercially available software.

For example, when the active ingredient of Chinese medicine is an ingredient having an estrogenic activity, an estrogen responsive gene whose expression varies depending on an estrogen or a chemical substance having an estrogen-like activity is selected. As the estrogen responsive gene, genes described in Japanese Patent No. 3371461, Japanese Patent Application Laid-Open No. 2002-360249, Japanese Patent Application Laid-Open No. 2003-169682, and Japanese Patent Application Laid-Open No. 2005-312456 can be used. In addition, 115 estrogen or estrogen responsive genes whose expression varies with chemical substances having estrogen-like activity shown in FIG. 1 are selected, and at least one of these genes, preferably 5 or more, more preferably 10 or more. More preferably, 20 or more, more preferably 30 or more, more preferably 40 or more, more preferably 50 or more, more preferably 60 or more, more preferably 70 or more, more preferably 80 or more, More preferably 90 or more, more preferably 100 or more, more preferably 110 or more, more preferably 115 using herbal medicine or an active ingredient thereof, or the effect of a plant, extract or composition containing the active ingredient Can be evaluated. The estrogen responsive gene includes a gene whose expression is enhanced by a chemical substance having estrogen or an estrogen-like activity and a gene whose expression is attenuated.

FIG. 1 shows the UniGene name and Accession No. of the gene used in the GenBank database. Moreover, the group name of the gene function used for classification of cluster analysis was shown.

The estrogen responsive genes shown in FIG. 1 can be classified according to gene function. That is, a gene encoding an enzyme (group name: enzyme, gene numbers 1 to 24), a gene encoding a kinase (group name: kinase, gene numbers 25 to 31), a gene encoding a receptor (group name: receptor, Gene numbers 32 and 33), genes involved in signal transduction (group name: signaling, gene numbers 34-50), genes involved in apoptosis (group name: apoptosis, gene numbers 51 and 52), genes involved in the cell cycle (Group name: cell cycle, gene numbers 53 and 54), genes involved in differentiation (group name: differentiation, gene numbers 55 and 56), genes involved in cell proliferation (group name: proliferation, gene numbers 57-68) , Genes involved in transcription (group name: transcription, gene numbers 69-79), calci Gene on no bond (
Group name: calcium, gene number 80-83), genes involved in processing (group name: processing, gene numbers 84-86), genes involved in translation (group name: translation, gene numbers 87 and 88), transport Genes involved (group name: transport, gene numbers 89-97), genes involved in membranes (group name: membrane, gene numbers 98 and 99), genes involved in protein folding (group name: protein folding, gene number) 100 to 102), 17 groups of genes involved in the structure (group name: structure, gene numbers 103 and 104) and genes of unknown function (group name: unknown, gene numbers 105 to 115).

In FIG. 1, the name of the gene is indicated by UniGene Symbol, and the description of each gene is as follows. In the following, genes are shown together with gene numbers for each of the 17 groups. [] Indicates the GenBank registration number. For these genes, sequence information can be obtained from a database based on the GenBank registration number shown in FIG. Furthermore, these genes may have variants, and the genes used in the present invention include variants of the above genes. In addition, a gene fragment can be used for the gene, and fragments of a plurality of sites of the same gene may be used. In general, it is known that the reliability of a DNA chip is increased by using a plurality of sites as probes even in the same gene. Which part is suitable as a probe depends on the gene. Therefore, a sufficiently reliable result can be obtained even if one site is used as a probe for each gene. However, depending on the gene, the reliability of the results can be further increased by using a plurality of sites as probes. Can be expected. The base length of the fragment is 10 to 200 bp, preferably 10 to 100 bp, more preferably 10 to 50 bp, more preferably 15 to 25 bp. For example, among the 115 genes in FIG. 1, the gene number 34 gene (insulin-like growth factor binding protein 5 (IGFBP5) [NM_000599]) may use three fragments of different parts of the same gene. In addition, the gene number 57 gene (amphiregulin (schwannoma-derived growth factor) (AREG) [NM_001657]) may use two fragments of different parts of the same gene. Similarly, the gene number 77 gene (transcription elongation factor A (SII), 1 (TCEA1) [NM — 006756]) may also use two fragments of different parts of the same gene. Furthermore, the gene No. 109 gene (SH3 domain binding glutamic acid-rich protein (SH3BGR) [NM — 007341]) may also use two fragments of different parts of the same gene. In this case, 120 kinds of genes or fragments thereof (probes) can be used in the present invention.

Group name: enzyme
1. phosphoenolpyruvate carboxykinase 2 (mitochondrial) (PCK2) [NM_004563]
2. phosphoserine aminotransferase 1 (PSAT1) [NM_058179]
3. asparagine synthetase (ASNS) [NM_133436]
4. phosphoglycerate dehydrogenase (PHGDH) [NM_006623]
5. serine hydroxymethyltransferase 2 (mitochondrial) (SHMT2) [NM_005412]
6. 24-dehydrocholesterol reductase (DHCR24) [NM_014762]
7. tryptophanyl-tRNA synthetase (WARS) [NM_004184]
8. fucosyltransferase 8 (alpha (1,6) fucosyltransferase) (FUT8) [NM_178155]
9. argininosuccinate synthetase (ASS) [NM_054012]
10. enolase 2 (gamma, neuronal) (ENO2) [NM_001975]
11. enolase 3 (beta, muscle) (ENO3) [NM_001976]
12. fructose-1,6-bisphosphatase 1 (FBP1) [NM_000507]
13. methylenetetrahydrofolate dehydrogenase (NADP + dependent) 2, methenyltetrahydrofolate cyclohydrolase (MTHFD2) [NM_006636]
14. carnitine palmitoyltransferase 1A (liver) (CPT1A) [NM_001876]
15. peptidase (mitochondrial processing) alpha (PMPCA) [NM_015160]
16. cathepsin D (lysosomal aspartyl peptidase) (CTSD) [NM_001909]
17. glutamic-oxaloacetic transaminase 1, soluble (aspartate aminotransferase 1) (GOT1) [NM_002079]
18. sorbitol dehydrogenase (SORD) [NM_003104]
19. stearoyl-CoA desaturase (delta-9-desaturase) (SCD) [NM_005063]
20. surfactant, pulmonary-associated protein B (SFTPB) [NM_000542]
21. serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 3 (SERPINA3) [NM_001085]
22. aconitase 2, mitochondrial (ACO2) [NM_001098]
23. glutamine-fructose-6-phosphate transaminase 1 (GFPT1) [NM_002056]
24. CDP-diacylglycerol--inositol 3-phosphatidyltransferase (phosphatidylinositol
synthase) (CDIPT) [NM_006319]

Group name: kinase
25. Ribosomal protein S6 kinase, 90kDa, polypeptide 3 (RPS6KA3) [NM_004586]
26. protein kinase C, delta (PRKCD) [NM_006254]
27. unc-51-like kinase 1 (C. elegans) (ULK1) [NM_003565]
28. integrin-linked kinase (ILK) [NM_004517]
29. protein kinase C substrate 80K-H (PRKCSH) [NM_002743]
30. cyclin-dependent kinase inhibitor 1A (p21, Cip1) (CDKN1A) [NM_000389]
31. phosphoinositide-3-kinase, class 3 (PIK3C3) [NM_002647]

Group name: receptor
32. poliovirus receptor (PVR) [NM_006505]
33. progesterone receptor (PGR) [NM_000926]

Group name: signaling
34. Insulin-like growth factor binding protein 5 (IGFBP5) [NM_000599]
35. catenin (cadherin-associated protein), delta 2 (neural plakophilin-related arm-repeat protein) (CTNND2) [NM_001332]
36. neuropeptide Y receptor Y1 (NPY1R) [NM_000909]
37. protein tyrosine phosphatase, non-receptor type 18 (brain-derived) (PTPN18) [NM_014369]
38. Rho GDP dissociation inhibitor (GDI) alpha (ARHGDIA) [NM_004309]
39. endothelin 2 (EDN2) [NM_001956]
40. stanniocalcin 2 (STC2) [NM_003714]
41.lectin, galactoside-binding, soluble, 3 binding protein (LGALS3BP) [NM_00556
7]
42. angiotensin II receptor, type 1 (AGTR1) [NM_000685]
43. proprotein convertase subtilisin / kexin type 6 (PCSK6) [NM_002570]
44. SH3-domain binding protein 5 (BTK-associated) (SH3BP5) [NM_004844]
45. growth differentiation factor 15 (GDF15) [NM_004864]
46. ras homolog gene family, member C (RHOC) [NM_175744]
47. estrogen receptor 1 (ESR1) [NM_000125]
48. trefoil factor 1 (breast cancer, estrogen-inducible sequence expressed in) (TFF1) [NM_003225]
49. aryl-hydrocarbon receptor nuclear translocator 2 (ARNT2) [NM_014862]
50. v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro / glioblastoma derived oncogene homolog (avian) (ERBB2) [NM_004448]

Group name: apoptosis
51. tribbles homolog 3 (Drosophila) (TRIB3) [NM_021158]
52. tyrosyl-tRNA synthetase (YARS) [NM_003680]
Group name: cell cycle
53. quiescin Q6 (QSCN6) [NM_002826]
54. cyclin A1 (CCNA1) [NM_003914]

Group name: differentiation
55. interferon-related developmental regulator 1 (IFRD1) [NM_001550]
56. matrix Gla protein (MGP) [NM_000900]

Group name: proliferation
57. amphiregulin (schwannoma-derived growth factor) (AREG) [NM_001657]
58. insulin-like growth factor binding protein 4 (IGFBP4) [NM_001552]
59. Kruppel-like factor 10 (KLF10) [NM_005655]
60. calpain, small subunit 1 (CAPNS1) [NM_001749]
61. interferon stimulated exonuclease gene 20kDa (ISG20) [NM_002201]
62. tumor-associated calcium signal transducer 2 (TACSTD2) [NM_002353]
63. PDZ domain containing 1 (PDZK1) [NM_002614]
64. ferritin, heavy polypeptide 1 (FTH1) [NM_002032]
65. peripheral myelin protein 22 (PMP22) [NM_000304]
66. lysosomal-associated membrane protein 3 (LAMP3) [NM_014398]
67. tumor protein p53 inducible protein 11 (TP53I11) [NM_006034]
68. tetraspanin 1 (TSPAN1) [NM_005727]

Group name: transcription
69. CCAAT / enhancer binding protein (C / EBP), beta (CEBPB) [NM_005194]
70. activating transcription factor 3 (ATF3) [NM_001674]
71.enolase 1, (alpha) (ENO1) [NM_001428]
72. activating transcription factor 3 (ATF3) [NM_004024]
73. early growth response 3 (EGR3) [NM_004430]
74.runt-related transcription factor 1 (acute myeloid leukemia 1; aml1 oncogene) (RUNX1) [NM_001001890]
75. v-fos FBJ murine osteosarcoma viral oncogene homolog (FOS) [NM_005252]
76. nuclear receptor interacting protein 1 (NRIP1) [NM_003489]
77. transcription elongation factor A (SII), 1 (TCEA1) [NM_006756]
78. general transcription factor II, i (GTF2I) [NM_032999]
79. TAF9 RNA polymerase II, TATA box binding protein (TBP) -associated factor, 32kDa (TAF9) [NM_003187]

Group name: calcium
80. EGF-containing fibulin-like extracellular matrix protein 1 (EFEMP1) [NM_004105]
81. reticulocalbin 1, EF-hand calcium binding domain (RCN1) [NM_002901]
82.cadherin 18, type 2 (CDH18) [NM_004934]
83. S100 calcium binding protein P (S100P) [NM_005980]

Group name: processing
84. chromosome 1 open reading frame 19 (C1orf19) [NM_052965]
85. IMP4, U3 small nucleolar ribonucleoprotein, homolog (yeast) (IMP4) [NM_033416]
86.elongation factor Tu GTP binding domain containing 2 (EFTUD2) (U5-116KD) [NM_004247]

Group name: translation
87. glycyl-tRNA synthetase (GARS) [NM_002047]
88. eukaryotic translation initiation factor 3, subunit 9 eta, 116kDa (EIF3S9) [NM_003751]

Group name: transport
89.solute carrier family 12 (sodium / potassium / chloride transporters), member 2 (SLC12A2) [NM_001046]
90.solute carrier family 7 (cationic amino acid transporter, y + system), member
5 (SLC7A5) [NM_003486]
91.solute carrier family 1 (glutamate / neutral amino acid transporter), member 4
(SLC1A4) [NM_003038]
92.solute carrier family 7, (cationic amino acid transporter, y + system) member
11 (SLC7A11) [NM_014331]
93. lipocalin 2 (oncogene 24p3) (LCN2) [NM_005564]
94. exportin, tRNA (nuclear export receptor for tRNAs) (XPOT) [NM_007235]
95.solute carrier family 1 (neutral amino acid transporter), member 5 (SLC1A5) [NM_005628]
96. chloride intracellular channel 4 (CLIC4) [NM_013943]
97. transcobalamin I (vitamin B12 binding protein, R binder family) (TCN1) [NM_001062]

Group name: membrane
98. synaptogyrin 2 (SYNGR2) [NM_004710]
99.transmembrane 4 L six family member 1 (TM4SF1) [NM_014220]

Group name: protein folding
100. heat shock 70kDa protein 1A (HSPA1A) [NM_005345]
101. heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa) (HSPA5) [NM_005347]
102. heat shock protein 90kDa beta (Grp94), member 1 (HSP90B1) (TRA1) [NM_003299]

Group name: structure
103.chromobox homolog 1 (HP1 beta homolog Drosophila) (CBX1) [NM_006807]
104. H3 histone, family 3B (H3.3B) (H3F3B) [NM_005324]

Group name: unknown
105. retinoblastoma binding protein 8 (RBBP8) [NM_002894]
106. tumor protein D52-like 1 (TPD52L1) [NM_003287]
107. absent in melanoma 1 (AIM1) [NM_001624]
108. chromosome 19 open reading frame 21 (C19orf21) [NM_173481]
109. SH3 domain binding glutamic acid-rich protein (SH3BGR) [NM_007341]
110. selenium binding protein 1 (SELENBP1) [NM_003944]
111. KIAA0196 (KIAA0196) [NM_014846]
112. LOC401397 [BC065765]
113. phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1) [NM_021127]
114. PREDICTED: paternally expressed 10 (PEG10) [XM_496907]
115. DAZ associated protein 2 (DAZAP2) [NM_014764]

For the evaluation of the Chinese medicine of the present invention or an active ingredient thereof, or a plant, extract or composition containing the active ingredient, at least one gene of each group may be selected, and the genes selected for each group may be used in combination. Good. In this case, at least one gene may be selected from all 17 groups. Alternatively, at least one group may be selected from the 17 groups, and at least one gene may be selected from the selected groups and used in combination. For example, at least one of the above 17 groups of enzyme, kinase, receptor, signaling, apoptosis, cell cycle, differentiation, proliferation, transcription, calcium, processing, translation, transport, membrane, protein folding, structure and unknown , Preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 These groups may be selected, and at least one gene may be selected from a plurality of gene groups belonging to the selected group and combined. When n groups are selected from 17 groups and m genes are selected from each group, the total number of genes used is n × m. The number of genes selected from each group need not be the same.

  In addition, the above functional groups may be grouped into related groups, for example, the enzyme, kinase, receptor and signaling groups are combined into one group, and the apoptosis, cell cycle, differentiation and proliferation groups are combined into one group. In summary, calcium, processing, translation, and transport groups may be combined into one group, membrane, protein folding, structure, and unknown may be combined into one group, and genes may be selected as described above for six groups.

The genes thus obtained are spotted on a slide glass to prepare a DNA microarray.

In the present invention, the above gene includes not only the above gene but also a DNA that hybridizes under stringent conditions with a DNA having a base sequence complementary to the base sequence of the above gene. Here, stringent conditions refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, DNAs having high homology, that is, DNAs having a homology of 60% or more, preferably 80% or more hybridize, whereby nucleic acids having low homology do not hybridize. More specifically, the sodium concentration is 150 to 900 mM, preferably 600 to 900 mM, and the temperature is 60 to 68 ° C, preferably 65 ° C.

  Furthermore, the full length of the above-mentioned gene may be used, and a partial fragment may be used. When using a partial fragment, the position of the partial sequence is not limited, and a continuous partial sequence of each gene may be used. The length of the fragment is not limited, but is 10 to 200 bp, preferably 10 to 100 bp, more preferably 10 to 50 bp, and still more preferably 15 to 25 bp.

  By immobilizing the full-length sequence or a partial sequence of the above-mentioned gene on an appropriate substrate, a DNA microarray for use in evaluating a herbal medicine or an active ingredient thereof, or a plant, extract or composition containing the active ingredient Can be produced.

Examples of the fixed substrate include a glass plate, a quartz plate, and a silicon wafer. Examples of the substrate size include 3.5 mm x 5.5 mm, 18 mm x 18 mm, and 22 mm x 75 mm, but this can be set according to the number of DNA probe spots on the substrate and the size of the spots. can do. As a DNA immobilization method, an appropriate method is selected according to the type of DNA and the type of carrier. For example, when the DNA to be immobilized is a cDNA or PCR product, it can be electrostatically bound to a solid support surface treated with a polycation such as polylysine, polyethyleneimine, or polyalkylamine using the charge of the DNA. A DNA into which a functional group such as an amino group, an aldehyde group, an SH group, or biotin is introduced can be covalently bound to a solid phase surface into which a functional group such as a group, an aldehyde group, or an epoxy group is introduced. Immobilization may be performed using an array machine as described above. In this case, the full length may be used as the gene to be immobilized, or a partial fragment may be used. When using a partial fragment, the position of the partial sequence is not limited, and a continuous partial sequence of each gene may be used. The length of the fragment is not limited, but is 10 to 200 bp, preferably 10 to 100 bp, more preferably 10 to 50 bp, and still more preferably 15 to 25 bp.

  Evaluation of a Chinese medicine or an active ingredient thereof, or a plant, extract or composition containing the active ingredient is carried out by the method described below.

For example, when using a DNA microarray, it is performed as follows using a DNA microarray in which the above-mentioned gene or a partial fragment thereof is immobilized. An internal standard gene is immobilized on the DNA microarray to be used, if necessary. The above-described cells are stimulated with Chinese medicine or an active ingredient thereof, or a plant, extract or composition containing the active ingredient. Stimulation can be performed by culturing cells in an appropriate medium, adding Chinese herbal medicine or an active ingredient thereof, or a plant, extract or composition containing the active ingredient to the medium, and further culturing. The concentration to be added is not limited. Next, mRNA is extracted from the cultured cells,
MRNA labeled with a fluorescent dye or cDNA labeled with a fluorescent dye is prepared. By hybridizing the labeled mRNA or cDNA with a microarray and measuring the fluorescence intensity on each spot, the expression level of the gene on each spot is known and which gene is suppressed or induced. Expression profiles can be obtained. Chinese herbal medicine to be evaluated or its active ingredient, or a plant containing the active ingredient, an expression profile obtained from the extract or composition, a specific ingredient of Chinese medicine, an extract containing the specific ingredient of Chinese medicine, the identification of Chinese medicine A composition containing an ingredient, a herbal medicine or an active ingredient thereof, or a plant, an extract or a composition containing the active ingredient by comparing the expression profile obtained with a substance having an activity equivalent to that of the specific ingredient of the Kampo medicine Can be evaluated.

  For example, in the case where the active ingredient of Chinese medicine is an ingredient having estrogenic activity, the gene shown in FIG. 1 is used to determine the Chinese medicine to be evaluated or its active ingredient, or a plant, extract or composition containing the active ingredient. What is necessary is just to compare the obtained expression profile with the expression profile obtained by the substance which has estrogen or an estrogen-like activity.

The expression profile can also be obtained by quantitative PCR such as real-time PCR targeting the gene or a fragment thereof. For example, the analysis by real-time PCR can be performed with an apparatus in which a thermal cycler and a spectrofluorometer are integrated, for example, LightCycler (trademark) manufactured by Roche. Real-time PCR makes it possible to detect and analyze the production process of PCR amplification products in real time. Since the PCR product is labeled with a fluorescent dye, accurate quantification can be performed, which is suitable for the method of the present invention.

The procedure using LightCycler (Roche Diagnostics) and a hot start reaction solution kit LightCycler-FastStart DNA Master SYBR Green I (Roche Diagnostics) will be described below. First, the primer mixture and the template cDNA solution are heat-treated at 82 ° C. for 5 minutes and then rapidly cooled in ice. Next, 12.6 mL of H ₂ O, 2.4 mL of MgCl ₂ , 2.0 mL of 10 × Light Cycler Mix, and 1.0 mL of template cDNA are mixed to prepare a mixed solution. Dispense 19 mL of the prepared mixed solution into the capillary, add 1 mL of the primer mixture (10 pmol / mL) to it, and cap the capillary. Centrifuge at 1900 rpm for 10-15 seconds with the capillary on the centrifuge adapter. Set this in the LightCycler carousel and perform real-time PCR. Perform these operations on ice or on a centrifuge adapter stored at 4 ° C as much as possible. The analysis can be performed using LightCycler Software Ver.3.3 (Roche Diagnostics).

  A herbal medicine to be evaluated or an active ingredient thereof, or a plant containing the active ingredient, an extract or a composition obtained by using a gene expression profile in advance is a specific ingredient of a traditional Chinese medicine, an extract containing the specific ingredient of a traditional Chinese medicine, If there is a homology with a gene expression profile obtained using a composition containing the specific component of the Chinese herbal medicine, a substance having an activity equivalent to that of the specific component of the Chinese herbal medicine, that is, if it is similar, An ingredient, or a plant, extract or composition containing the active ingredient can be evaluated as containing an active active ingredient, containing a useful amount of the active active ingredient, or excellent in quality. In addition, if the genes used can be grouped by function, compare the expression profiles for each group by gene function, determine the homology for each group, and evaluate the Chinese medicine to be evaluated based on the similarity of the homology for each group Alternatively, the active ingredient, or a plant, extract or composition containing the active ingredient can be evaluated. At this time, for example, a determination criterion may be set for the expression level of each gene of the gene.

For example, when the active active ingredient is an estrogen-like active ingredient, for at least one gene of the 115 estrogen responsive genes shown in FIG. 1, a Chinese medicine to be evaluated or an active ingredient thereof, or a plant containing the active ingredient, An expression profile is obtained using the extract or composition and compared to a gene expression profile previously obtained using estrogen. If both expression profiles are similar, the herbal medicine to be evaluated or its active ingredient, or the plant, extract or composition containing the active ingredient has an estrogenic active ingredient or has an estrogenic active ingredient. It can be assessed that it contains a dose or is of good quality.

  The homology (similarity) of the expression profile is, for example, a substance having an effective activity to evaluate a herbal medicine or an active ingredient thereof, or a plant, extract or composition containing the active ingredient for a selected gene. Measure the expression level of each gene when using, and the expression level of each gene when using the herbal medicine or plant, extract or composition containing the active ingredient to be evaluated. It can be done by taking a correlation at the gene level for each and comparing. For example, the comparison is made as described in the examples described later.

In addition, the gene expression profile is recorded as an image in which the pattern of an expression signal such as fluorescence intensity is a digital value or a color. Comparison of gene expression profiles can be performed using, for example, pattern comparison software, and Cox hazard analysis, discriminant analysis, or the like can be used. Establishing a discriminant analysis model for evaluating in advance the Kampo medicine or its active ingredient, or a plant, extract or composition containing the active ingredient, and the Kampo medicine or its active ingredient to be evaluated in the discriminant analysis model, or its activity Data relating to gene expression profiles obtained from plants, extracts or compositions containing components can also be input for evaluation. For example, a discriminant is obtained by discriminant analysis, the fluorescence intensity is associated with the effectiveness of the active ingredient concentration, activity, etc., and the herbal medicine or its active ingredient, or the plant, extract or composition containing the active ingredient in the discriminant It can be evaluated by substituting the expression signal value.

  The present invention relates to a herbal medicine or an active ingredient thereof, or a plant, extract or composition containing the active ingredient, according to a method of evaluating a plant, extract or composition containing the active ingredient. Includes a system to evaluate

A system for evaluating the herbal medicine of the present invention or an active ingredient thereof, or a plant, extract or composition containing the active ingredient,
(a) Means for inputting data relating to gene expression profile when using Chinese herbal medicine to be evaluated or its active ingredient, or plant, extract or composition containing the active ingredient, relating to gene expression profile inputted here The data is, for example, data indicating the expression level of each gene such as a signal value in each gene;
(b) storage means for storing the constructed discrimination model;
(c) Applying the data input using the input means of (a) to the discrimination model stored in the storage means of (b), the herbal medicine or its active ingredient, or a plant or extract containing the active ingredient Or data processing means for evaluating the composition, and
(d) an output means for outputting an evaluation of the obtained Chinese herbal medicine or an active ingredient thereof, or a plant, extract or composition containing the active ingredient;
It is a system including.

The means for inputting data in (a) includes a keyboard or an external storage device storing data. The storage means (b) includes a hard disk or the like. The data processing means receives the discrimination model from the storage means, processes the input data, sends the processing result to the output means, and the processing result is displayed by the output means. The data processing means includes a central processing unit (CPU) that performs arithmetic processing on data. The output means includes a monitor and a printer for displaying the result.

  The system of the present invention can be constructed using a commercially available personal computer or the like.

  The present invention further includes a nucleotide comprising a base sequence of a gene selected for evaluation of a herbal medicine or an active ingredient thereof, or a plant, extract or composition containing the active ingredient, or a partial sequence thereof. Or a reagent or kit for evaluating a herbal medicine or an active ingredient thereof, or a plant, extract or composition containing the active ingredient. The reagent is a reagent comprising a nucleotide comprising the nucleotide sequence of the gene or a nucleotide comprising a partial sequence thereof as a probe or primer, and a nucleotide comprising the nucleotide sequence of the gene or a nucleotide comprising a partial sequence thereof is solid-phased. It is a reagent containing a substrate such as a microarray.

For example, when the active ingredient is an estrogen-like active ingredient, the reagent or kit is at least one, preferably 5 or more, more preferably 10 or more, more preferably 20 of the 115 estrogen responsive genes shown in FIG. More than 30, more preferably 30 or more, more preferably 40 or more, more preferably 50 or more, more preferably 60 or more, more preferably 70 or more, more preferably 80 or more, more preferably 90 More preferably, it contains 100 or more, more preferably 110 or more, and even more preferably 115.

As such a DNA microarray, for example, EstrArray (Infogenes, Inoue A.,
et al., Journal of molecular endocrinology. 2002 Oct; 29 (2): 175-192). In EstrArray, about 200 estrogen-responsive genes including the above 115 genes or a fragment consisting of a partial sequence of the base sequence of the gene is arranged.

  The present invention further includes a medicinal herb medicine of the present invention or an active ingredient thereof, or a medicinal product containing an active ingredient of a Chinese herb medicine by a method for evaluating the activity of a plant, extract or composition containing the active ingredient, and having an effective biological / physiological activity, It also includes a method for producing a composition such as a food or research reagent, and a composition such as a pharmaceutical, food or research reagent produced by the method.

  The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.

(1) Genes for evaluating estrogenic activity 115 types of genes shown in FIG. 1 were used as estrogen-responsive genes.
In addition, the gene number 34 gene (insulin-like growth factor binding protein 5 (IGFBP5) [NM_000599]) used three fragments of different parts of the same gene. Gene number 5
The 7 gene (amphiregulin (schwannoma-derived growth factor) (AREG) [NM_001657]) uses two fragments of another part of the same gene. Similarly, the gene number 77 (transcription elongation factor A (SII), 1 (TCEA1) [NM_006756]) also used two fragments of different parts of the same gene. Furthermore, the gene No. 109 gene (SH3 domain binding glutamic acid-rich protein (SH3BGR) [NM_007341]) also used two fragments of different parts of the same gene. Therefore, 120 genes or fragments were used.

(2) Cell proliferation test for DNA chip condition study (Figure 2)
Cell proliferation ability at each concentration of licorice extract and Glycyrrhizin was assayed by SRB (Sulforhodamine B) test (FIG. 2A) and reporter gene test using estrogen response factor (FIG. 2B). As a control, the result in the case of adding only vehicle (Control) and adding estrogen (E ₂ : 10 nM 17β-estradiol) is shown. Both test results show that licorice extract (containing about 10% Glycyrrhizin) has a clear cell proliferative activity similar to estrogen, but Glycyrrhizin shows little activity. As the licorice extract, a commercially available licorice extract (purchased from Alps Pharmaceutical Co., Ltd.) was diluted 1:10 in pure water, autoclaved and used after sterilization. Concentration (μg / ml) was calculated using the weight of the extract.

A reporter gene test using an estrogen response factor was performed as follows.
A reporter vector constructed by inserting an ERα gene promoter region (site from −838 bases to +214 bases from the cap site) into the KpnI / HindIII site of the vector (ProGL pGL3Basic vector) was used. The assay used Lipofectamine 2000 (Invitrogen) to transform the reporter vector into MCF-7 cells. Estrogen and licorice extract were added to MCF-7 cell medium and cultured for 24 hours, and then luciferase activity was measured. This reporter assay was performed by the method described in a paper (Inoue A., Journal of Pharmacological and Toxicological Methods, 2002, May-Jun; 47 (3): 129-135).

(3) Licorice extract and Glycyrrhizin DNA chip analysis (Figure 3)
Using a DNA chip (EstrArray, Infogenes), the influence of licorice extract, Glycyrrhizin, and estrogen (E ₂ : 10 nM 17β-estradiol) on human MCF-7 cells was evaluated and 115 cells shown in FIG. Gene expression profiles were obtained for these genes. Furthermore, the results were examined by cluster analysis. Genes were classified by function (shown in FIG. 1 as a group name), and the increase in gene expression was shown in dark color, and the decrease in expression was shown in light color (the relative value of increase / decrease was shown in the lower left on a gray scale).

The impact evaluation was performed by the following method.
The closeness of the profile between Glycyrrhizin, licorice extract, and estrogen was analyzed by cluster analysis. Cluster analysis was performed by the method described in a paper (Terasaka S., et al., Toxicology Letters 2006 May 25; 163 (2): 130-141).

(4) Evaluation of DNA chip analysis results using gene function (Figure 4)
Gene expression profiles obtained using DNA chips (EstrArray, Infogenes) for estrogen and licorice extract (Fig. 4A), estrogen and Glycyrrhizin (Fig. 4B), licorice extract and Glycyrrhizin (Fig. 4C), respectively Each gene or gene fragment (probe) (indicated as “120 genes” in the figure) or gene function was compared, and the correlation coefficient values based on linear approximation were displayed in graphs.

Here, regarding 115 genes (120 probes) or genes belonging to the functional group shown in FIG. 1, the expression profile obtained using the DNA chip was subjected to correlation analysis between the combinations of the respective samples. The correlation coefficient obtained was displayed on a graph. The correlation coefficient was calculated using statistical analysis software SPSS 12.0J for Windows (SPSS Japan Inc.).

As for the classification of genes by function, 115 genes are classified into “enzyme”, “signaling” (a group of gene groups belonging to “kinase”, “signaling”, “receptor” in FIG. 1), “proliferation” (in FIG. 1). "A group of gene groups belonging to" apoptosis "," cell cycle "," differentiation "," proliferation ")," transcription "," transport "
(A group of gene groups belonging to “calcium”, “transport”, “processing”, “translation” in FIG. 1), “others” (“membrane”, “structure”, “protein folding”, “ The gene groups belonging to “unknown” were reclassified into 6 groups). There is no functional group between estrogen and Glycyrrhizin (Fig. 4B) that shows not only a statistically significant correlation, but also between estrogen and licorice extract (Fig. 4A). , “Proliferation” and “transport” were statistically significant and higher than the comparison of all genes. In addition, licorice extract and Glycyrrhizin (FIG. 4C) were statistically significant only for genes classified as “signaling” and showed a higher correlation than the comparison of all genes. This is because licorice extract induces general gene responses that are characteristic of estrogen responses in MCF-7 cells, and is particularly common to genes involved in signal transduction, cell proliferation, and intracellular substance transfer. However, between licorice extract and Glycyrrhizin, it affects genes that are involved in signal transduction common to MCF-7 cells, which may be different from the response to estrogen.

(5) Estrogen-like response evaluation of licorice extract by estrogen response gene (Fig. 5)
Transcription of estrogen-responsive genes (eight of those mounted on EstrArray, TFF1, CDKN1A, RPS6KA3, NRIP1, CTSD, TP53I11, PRKCD, ARHGDIA) for each sample (estrogen, licorice extract and Glycyrrhizin) The fluctuation of the reaction was analyzed using a real-time quantitative RT-PCR method, and was expressed as a relative expression fluctuation value (Relative expression index) with respect to the case where only vehicle was added (Control).

Real-time quantitative RT-PCR was performed by the following method.
Previous studies have shown that TFF1, CDKN1A, RPS6KA3, NRIP1, CTSD, TP53I11, PRKCD, and ARHGDIA genes respond to estrogen and phytoestrogens (examples: Ise R., et al., FEBS Letters, 2005, Mar 14; 579 (7): 1732-1740). Real-time quantitative RT-PCR was performed using Invitrogen's Two-Step qRT-PCR kit using RNA extracted from each treated MCF-7 cell. The following primers were used: TFF1, 5'-CACCATGGAGAACAAGGTGA-3 '(SEQ ID NO: 1) and 5'-CTAGAACAGCACCTGGCACA-3' (SEQ ID NO: 2); CDKN1A, 5'-CCTTTCCCTTCAGTACCCTCTCA-3 '(SEQ ID NO: 1) 3) and 5′-ACTAGGGTGCCCTTCTTCTTGTG-3 ′ (SEQ ID NO: 4); RPS6KA3, 5′-CAAAGGACCTGGTGTCAAAGATG-3 ′ (SEQ ID NO: 5) and 5′-AGATGTGGTGCATCCTGTCTGTT-3 ′ (SEQ ID NO: 6); NRIP1, 5'-TGCATTAGAACCATCTGGAGCA-3 '(SEQ ID NO: 7) and 5'-CAGATGGAAGATTCCAGGTCCA-3' (SEQ ID NO: 8); CTSD, 5'-AGGCCATTGTGGACAAGGCA-3 '(SEQ ID NO: 9) and 5'-CACGGTCAAACACAGTGTAGTAG- Three
'(SEQ ID NO: 10); TP53I11, 5'-CACGGACAAAGGGACACACA-3' (SEQ ID NO: 11) and 5'-GTACATGCCAAGGGGCAAGA-3 '(SEQ ID NO: 12); PRKCD, 5'-GACCTCAAACTGGACAATGTGCT-3' (SEQ ID NO: 13) ) And 5′-ATCTCAGGGGCGATATAGTCAGG-3 ′ (SEQ ID NO: 14); ARHGDIA, 5′-CCTCACTAGCCTCTACTCCCTGT-3 ′ (SEQ ID NO: 15) and 5′-ACTGAGGTGACTTGAGTGTTGG-3 ′ (SEQ ID NO: 16); β-Actin 5′-CAAGAGATGGCCACGGCTGCT-3 ′ (SEQ ID NO: 17) and 5′-TCCTTCTGCATCCTGTCGGCA-3 ′ (SEQ ID NO: 18). Real-time quantitative RT-PCR reaction was performed using LightCycler (Roch). Β-Actin was used for data correction. Calculation of Relative expression index was performed as follows.

Relative expression index = 2- ^{(DCt- DCt ')} .
However, for each sample,
ΔCt = Ct _Gene -Ct _Actin ,
As Control
ΔCt '= Ct' _Gene -Ct ' _Actin
(Ct indicates threshold cycle).

Each shows Log ₂ conversion value. Error bars indicate the deviation (SD) of three tests. The reliability of each value (risk rate assuming no change) is indicated by "*" (p <0.05) or "**" (p <0.01). The gene name is indicated by the UniGene name registered in the GenBank database operated by NCBI (National Center for Biotechnology Information) of NIH, USA. The obtained results showed that the licorice extract showed the same fluctuation (increase or decrease in gene expression) as that of estrogen, but Glycyrrhizin did not show any clear fluctuation in any gene. From this result, it was shown that the results obtained with the DNA chip were also reproduced by the real-time quantitative RT-PCR method.

(6) Evaluation of estrogen-like response of licorice extract by signal transduction protein (Fig. 6)
Using the phosphorylation of Erk1 / 2, a signal transduction protein, as an index, the response of MCF-7 cells to each sample (estrogens, licorice extract, and Glycyrrhizin) was phosphorylated Erk1 / 2 protein (P-Erk1 / 2) and Western blotting using antibodies against the total Erk1 / 2 protein (T-Erk1 / 2). When the change in the passage after treatment was examined (FIG. 6A), the amount of phosphorylated Erk1 / 2 protein (P-Erk1 / 2) was increased 5 to 30 minutes after treatment. Western blotting was performed by the following method.

MCF-7 cells were cultured in Phenol red free PRMI1640 medium (containing 10% DCC-FBS) for 48 hours, and then further cultured in PRMI1640 medium not containing FBS for 36 hours. Then, after adding each sample, the change over time of Erk1 / 2 protein phosphorylation was analyzed. 30 μg of each protein sample extracted using SDS sample buffer was subjected to SDS-PAGE using 12.5% gel and subjected to Western blot analysis. Western blot analysis uses rabbit anti-phosphorylated Erk1 / 2 and anti-Total-Erk1 / 2 antibodies as the primary antibody, anti-rabbit Ig antibody horse dysperperosidase complex as the secondary antibody, and ECL plus Western detection A chemiluminescence detector Cool Saver AE-6955 (Ato) was used with a Blotting Detection System (Amsham).

When the influence of antagonists on estrogen was examined (FIG. 6B), phosphorylation was suppressed for tamoxifen (OHT), but licorice extract showed inhibition for ICI 182,780 (ICI). However, in the case of Glycyrrhizin treatment, the degree of suppression was low. This suggests that there is a difference between the licorice extract and Glycyrrhizin in terms of the signal transduction mechanism. Since Glycyrrhizin promotes the phosphorylation of Erk1 / 2 protein like licorice extract, a signal transduction system common to both can be considered. This is DNA
It explains well the homology of gene expression profile between licorice extract and Glycyrrhizin obtained by analysis result of chip. In addition, Erk1 / 2 protein is involved in various receptor responses and activation / inactivation of transcription factors. The increase in phosphorylation of Erk1 / 2 protein is a signal response obtained by DNA chip analysis. The results showing gene variation are also well explained.

(7) Evaluation of estrogen-like response of licorice extract by PC12 neural dendrite formation ability (Fig. 7)
PC12 cells derived from rat adrenal medulla show dendritic formation upon stimulation with nerve growth factor NGF, but in the presence of low levels of NGF, dendrites can also be induced against estrogen stimulation in the presence of estrogen receptor. Shows the formation of. This is because estrogen receptor (ER) -dependent signal transduction occurs through other signal transduction cascades even in PC12 cells where estrogen receptor expression is not observed and estrogen-responsive signal transduction is thought to be nonfunctional It shows that. The cells were examined for their ability to form dendrites after the addition of each sample (estrogen (E ₂ : 17β-estradiol), licorice extract, and Glycyrrhizin) in the presence of NGF, and estrogen and licorice extraction were performed. In the presence of the substance, estrogen receptor-dependent dendrite formation was observed, but in the case of Glycyrrhizin, dendrite formation was not observed. The results are shown in FIG. 7A is untreated cells, FIG. 7B is added ₂ NGF and E, Fig. 7C ER and NGF added, Figure 7D ER, NGF
And E ₂ addition, Figure 7E shows ER, NGF and licorice extract added, FIG. 7F ER, the results of NGF and Glycyrrhizin added. As a result, licorice extract has dendritic ability similar to estrogen (FIGS. 7D and 7E), but Glycyrrhizin has no activity (FIG. 7F).

It is a figure which shows the gene list used in order to evaluate an estrogen-like activity. It is a figure which shows the result of the cell proliferation test for DNA chip condition examination. It is a figure which shows the result of a DNA chip analysis of a licorice extract and Glycyrrhizin. It is a figure which shows the evaluation of the DNA chip analysis result using a gene function. It is a figure which shows the result of the estrogen-like response evaluation of a licorice extract by an estrogen response gene. It is a figure which shows the result of the estrogen-like response evaluation of a licorice extract by a signal transduction system protein. It is a figure which shows the result of the estrogen-like response evaluation of a licorice extract by PC12 neural dendrite formation ability.

Claims (14)

A method for evaluating the effective activity of an active ingredient of Chinese herbal medicine (including herbal medicine) having estrogenic activity,
In this method, the estrogen responsive genes used in the evaluation are selected from 115 estrogen responsive genes shown in 1. to 115 below and grouped into a plurality of functional groups. It is a method for evaluating estrogen activity by creating an expression profile of at least 10 estrogen responsive genes for each and determining the statistical significance of homology for each functional group between test subjects,
Expression profiles of estrogen-responsive genes for each functional group obtained when the above-mentioned Kampo medicine is applied to the living body and estrogen-responsive gene expression profiles for each functional group obtained when the estrogen is applied to the living body. The step of judging the statistical significance of homology between the two compared to each group, and the expression of the estrogen response gene for each functional group obtained when the active ingredient of the Chinese medicine to be evaluated is applied to the living body Comparing the profile and the expression profile of the estrogen response gene for each functional group in the herbal medicine for each functional group to determine the statistical significance of homology between the two, Said method ;
1． phosphoenolpyruvate carboxykinase 2 (mitochondrial) (PCK2) [NM_004563]
2． phosphoserine aminotransferase 1 (PSAT1) [NM_058179]
3． asparagine synthetase (ASNS) [NM_133436]
Four. phosphoglycerate dehydrogenase (PHGDH) [NM_006623]
Five. serine hydroxymethyltransferase 2 (mitochondrial) (SHMT2) [NM_005412]
6. 24-dehydrocholesterol reductase (DHCR24) [NM_014762]
7． tryptophanyl-tRNA synthetase (WARS) [NM_004184]
8． fucosyltransferase 8 (alpha (1,6) fucosyltransferase) (FUT8) [NM_178155]
9． argininosuccinate synthetase (ASS) [NM_054012]
Ten. enolase 2 (gamma, neuronal) (ENO2) [NM_001975]
11． enolase 3 (beta, muscle) (ENO3) [NM_001976]
12． fructose-1,6-bisphosphatase 1 (FBP1) [NM_000507]
13. methylenetetrahydrofolate dehydrogenase (NADP + dependent) 2, methenyl tetrahydrofolate cyclohydrolase (MTHFD2) [NM_006636]
14. carnitine palmitoyltransferase 1A (liver) (CPT1A) [NM_001876]
15． peptidase (mitochondrial processing) alpha (PMPCA) [NM_015160]
16． cathepsin D (lysosomal aspartyl peptidase) (CTSD) [NM_001909]
17． glutamic-oxaloacetic transaminase 1, soluble (aspartate aminotransferase 1) (GOT1) [NM_002079]
18． sorbitol dehydrogenase (SORD) [NM_003104]
19. stearoyl-CoA desaturase (delta-9-desaturase) (SCD) [NM_005063]
20． surfactant, pulmonary-associated protein B (SFTPB) [NM_000542]
twenty one. serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 3 (SERPINA3) [NM_001085]
twenty two. aconitase 2, mitochondrial (ACO2) [NM_001098]
twenty three. glutamine-fructose-6-phosphate transaminase 1 (GFPT1) [NM_002056]
twenty four. CDP-diacylglycerol-inositol 3-phosphatidyltransferase (phosphatidylinositol synthase) (CDIPT) [NM_006319]
twenty five. ribosomal protein S6 kinase, 90kDa, polypeptide 3 (RPS6KA3) [NM_004586]
26. protein kinase C, delta (PRKCD) [NM_006254] 27. unc-51-like kinase 1 (C. elegans) (ULK1) [NM_003565]
28. integrin-linked kinase (ILK) [NM_004517] 29. protein kinase C substrate 80K-H (PRKCSH) [NM_002743]
30. cyclin-dependent kinase inhibitor 1A (p21, Cip1) (CDKN1A) [NM_000389]
31. phosphoinositide-3-kinase, class 3 (PIK3C3) [NM_002647]
32. poliovirus receptor (PVR) [NM_006505]
33. progesterone receptor (PGR) [NM_000926]
34. insulin-like growth factor binding protein 5 (IGFBP5) [NM_000599]
35. catenin (cadherin-associated protein), delta 2 (neural
plakophilin-related arm-repeat protein) (CTNND2) [NM_001332]
36. neuropeptide Y receptor Y1 (NPY1R) [NM_000909]
37. protein tyrosine phosphatase, non-receptor type 18 (brain-derived) (PTPN18) [NM_014369]
38. Rho GDP dissociation inhibitor (GDI) alpha (ARHGDIA) [NM_004309]
39. endothelin 2 (EDN2) [NM_001956]
40. stanniocalcin 2 (STC2) [NM_003714]
41. lectin, galactoside-binding, soluble, 3 binding protein (LGALS3BP) [NM_005567]
42. angiotensin II receptor, type 1 (AGTR1) [NM_000685]
43. proprotein convertase subtilisin / kexin type 6 (PCSK6) [NM_002570]
44. SH3-domain binding protein 5 (BTK-associated) (SH3BP5) [NM_004844]
45. growth differentiation factor 15 (GDF15) [NM_004864]
46. ras homolog gene family, member C (RHOC) [NM_175744]
47. estrogen receptor 1 (ESR1) [NM_000125]
48. trefoil factor 1 (breast cancer, estrogen-inducible sequence expressed in) (TFF1) [NM_003225]
49. aryl-hydrocarbon receptor nuclear translocator 2 (ARNT2) [NM_014862]
50. v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro / glioblastoma derived oncogene homolog (avian) (ERBB2) [NM_004448]
51. tribbles homolog 3 (Drosophila) (TRIB3) [NM_021158]
52. tyrosyl-tRNA synthetase (YARS) [NM_003680]
53. quiescin Q6 (QSCN6) [NM_002826]
54. cyclin A1 (CCNA1) [NM_003914]
55. interferon-related developmental regulator 1 (IFRD1) [NM_001550]
56. matrix Gla protein (MGP) [NM_000900]
57. amphiregulin (schwannoma-derived growth factor) (AREG) [NM_001657]
58. insulin-like growth factor binding protein 4 (IGFBP4) [NM_001552]
59. Kruppel-like factor 10 (KLF10) [NM_005655]
60. calpain, small subunit 1 (CAPNS1) [NM_001749]
61. interferon stimulated exonuclease gene 20kDa (ISG20) [NM_002201]
62. tumor-associated calcium signal transducer 2 (TACSTD2) [NM_002353]
63. PDZ domain containing 1 (PDZK1) [NM_002614]
64. ferritin, heavy polypeptide 1 (FTH1) [NM_002032]
65. peripheral myelin protein 22 (PMP22) [NM_000304]
66. lysosomal-associated membrane protein 3 (LAMP3) [NM_014398]
67. tumor protein p53 inducible protein 11 (TP53I11) [NM_006034]
68. tetraspanin 1 (TSPAN1) [NM_005727]
69. CCAAT / enhancer binding protein (C / EBP), beta (CEBPB) [NM_005194]
70. activating transcription factor 3 (ATF3) [NM_001674]
71. enolase 1, (alpha) (ENO1) [NM_001428]
72. activating transcription factor 3 (ATF3) [NM_004024]
73. early growth response 3 (EGR3) [NM_004430]
74. runt-related transcription factor 1 (acute myeloid leukemia 1; aml1 oncogene) (RUNX1) [NM_001001890]
75. v-fos FBJ murine osteosarcoma viral oncogene homolog (FOS) [NM_005252]
76. nuclear receptor interacting protein 1 (NRIP1) [NM_003489]
77. transcription elongation factor A (SII), 1 (TCEA1) [NM_006756]
78. general transcription factor II, i (GTF2I) [NM_032999]
79. TAF9 RNA polymerase II, TATA box binding protein (TBP) -associated factor, 32kDa (TAF9) [NM_003187]
80. EGF-containing fibulin-like extracellular matrix protein 1 (EFEMP1) [NM_004105]
81. reticulocalbin 1, EF-hand calcium binding domain (RCN1) [NM_002901]
82. cadherin 18, type 2 (CDH18) [NM_004934]
83. S100 calcium binding protein P (S100P) [NM_005980]
84. chromosome 1 open reading frame 19 (C1orf19) [NM_052965]
85. IMP4, U3 small nucleolar ribonucleoprotein, homolog (yeast) (IMP4) [NM_033416]
86. elongation factor Tu GTP binding domain containing 2 (EFTUD2) (U5-116KD) [NM_004247]
87. glycyl-tRNA synthetase (GARS) [NM_002047]
88. eukaryotic translation initiation factor 3, subunit 9 eta, 116kDa (EIF3S9) [NM_003751]
89. solute carrier family 12 (sodium / potassium / chloride transporters), member 2 (SLC12A2) [NM_001046]
90. solute carrier family 7 (cationic amino acid transporter, y + system), member 5 (SLC7A5) [NM_003486]
91. solute carrier family 1 (glutamate / neutral amino acid transporter), member 4 (SLC1A4) [NM_003038]
92. solute carrier family 7, (cationic amino acid transporter, y + system) member 11 (SLC7A11) [NM_014331]
93. lipocalin 2 (oncogene 24p3) (LCN2) [NM_005564]
94. exportin, tRNA (nuclear export receptor for tRNAs) (XPOT) [NM_007235]
95. solute carrier family 1 (neutral amino acid transporter), member 5 (SLC1A5) [NM_005628]
96. chloride intracellular channel 4 (CLIC4) [NM_013943]
97. transcobalamin I (vitamin B12 binding protein, R binder family) (TCN1) (NM_0
01062]
98. synaptogyrin 2 (SYNGR2) [NM_004710]
99. transmembrane 4 L six family member 1 (TM4SF1) [NM_014220]
100. heat shock 70kDa protein 1A (HSPA1A) [NM_005345]
101. heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa) (HSPA5) [NM_005347]
102. heat shock protein 90kDa beta (Grp94), member 1 (HSP90B1) (TRA1) [NM_003299]
103. chromobox homolog 1 (HP1 beta homolog Drosophila) (CBX1) [NM_006807]
104. H3 histone, family 3B (H3.3B) (H3F3B) [NM_005324]
105. retinoblastoma binding protein 8 (RBBP8) [NM_002894]
106. tumor protein D52-like 1 (TPD52L1) [NM_003287]
107. absent in melanoma 1 (AIM1) [NM_001624]
108. chromosome 19 open reading frame 21 (C19orf21) [NM_173481]
109. SH3 domain binding glutamic acid-rich protein (SH3BGR) [NM_007341]
110. selenium binding protein 1 (SELENBP1) [NM_003944]
111. KIAA0196 (KIAA0196) [NM_014846]
112. LOC401397 [BC065765]
113. phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1) [NM_021127]
114. PREDICTED: paternally expressed 10 (PEG10) [XM_496907]
115. DAZ associated protein 2 (DAZAP2) [NM_014764].   Furthermore, the expression profile for each functional group of the estrogen response gene in the active ingredient of the Chinese herbal medicine and the expression profile for each functional group of the estrogen response gene by the estrogen are compared for each functional group. The method of claim 1, comprising the step of determining the statistical significance of homology.   The function-specific group is selected from an "enzyme" group, a "signaling" group, a "proliferation" group, a "transcription" group, a "transport" group, and an "others" group. The method described in 1. Among the 115 estrogen responsive genes shown in 1. to 115. below, the functional group “enzyme” group (1 to 24.), “signaling” group (25. to 50. ) "Proliferation" group (51.-68.) "Transcription" group (69.-79.) "Transport" group (80.-97.) "Others" group (98.-115.) 4. The method of claim 3, wherein at least 10 genes are selected from
1． phosphoenolpyruvate carboxykinase 2 (mitochondrial) (PCK2) [NM_004563]
2． phosphoserine aminotransferase 1 (PSAT1) [NM_058179]
3． asparagine synthetase (ASNS) [NM_133436]
Four. phosphoglycerate dehydrogenase (PHGDH) [NM_006623]
Five. serine hydroxymethyltransferase 2 (mitochondrial) (SHMT2) [NM_005412]
6. 24-dehydrocholesterol reductase (DHCR24) [NM_014762]
7． tryptophanyl-tRNA synthetase (WARS) [NM_004184]
8． fucosyltransferase 8 (alpha (1,6) fucosyltransferase) (FUT8) [NM_178155]
9． argininosuccinate synthetase (ASS) [NM_054012]
Ten. enolase 2 (gamma, neuronal) (ENO2) [NM_001975]
11． enolase 3 (beta, muscle) (ENO3) [NM_001976]
12． fructose-1,6-bisphosphatase 1 (FBP1) [NM_000507]
13. methylenetetrahydrofolate dehydrogenase (NADP + dependent) 2, methenyl tetrahydrofolate cyclohydrolase (MTHFD2) [NM_006636]
14. carnitine palmitoyltransferase 1A (liver) (CPT1A) [NM_001876]
15． peptidase (mitochondrial processing) alpha (PMPCA) [NM_015160]
16． cathepsin D (lysosomal aspartyl peptidase) (CTSD) [NM_001909]
17． glutamic-oxaloacetic transaminase 1, soluble (aspartate aminotransferase 1) (GOT1) [NM_002079]
18． sorbitol dehydrogenase (SORD) [NM_003104]
19. stearoyl-CoA desaturase (delta-9-desaturase) (SCD) [NM_005063]
20． surfactant, pulmonary-associated protein B (SFTPB) [NM_000542]
twenty one. serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 3 (SERPINA3) [NM_001085]
twenty two. aconitase 2, mitochondrial (ACO2) [NM_001098]
twenty three. glutamine-fructose-6-phosphate transaminase 1 (GFPT1) [NM_002056]
twenty four. CDP-diacylglycerol-inositol 3-phosphatidyltransferase (phosphatidylinositol synthase) (CDIPT) [NM_006319]
twenty five. ribosomal protein S6 kinase, 90kDa, polypeptide 3 (RPS6KA3) [NM_004586]
26. protein kinase C, delta (PRKCD) [NM_006254]
27. unc-51-like kinase 1 (C. elegans) (ULK1) [NM_003565]
28. integrin-linked kinase (ILK) [NM_004517]
29. protein kinase C substrate 80K-H (PRKCSH) [NM_002743]
30. cyclin-dependent kinase inhibitor 1A (p21, Cip1) (CDKN1A) [NM_000389]
31. phosphoinositide-3-kinase, class 3 (PIK3C3) [NM_002647]
32. poliovirus receptor (PVR) [NM_006505]
33. progesterone receptor (PGR) [NM_000926]
34. insulin-like growth factor binding protein 5 (IGFBP5) [NM_000599]
35. catenin (cadherin-associated protein), delta 2 (neural plakophilin-related arm-repeat protein) (CTNND2) [NM_001332]
36. neuropeptide Y receptor Y1 (NPY1R) [NM_000909]
37. protein tyrosine phosphatase, non-receptor type 18 (brain-derived) (PTPN18) [NM_014369]
38. Rho GDP dissociation inhibitor (GDI) alpha (ARHGDIA) [NM_004309]
39. endothelin 2 (EDN2) [NM_001956]
40. stanniocalcin 2 (STC2) [NM_003714]
41. lectin, galactoside-binding, soluble, 3 binding protein (LGALS3BP) [NM_005567]
42. angiotensin II receptor, type 1 (AGTR1) [NM_000685]
43. proprotein convertase subtilisin / kexin type 6 (PCSK6) [NM_002570]
44. SH3-domain binding protein 5 (BTK-associated) (SH3BP5) [NM_004844]
45. growth differentiation factor 15 (GDF15) [NM_004864]
46. ras homolog gene family, member C (RHOC) [NM_175744]
47. estrogen receptor 1 (ESR1) [NM_000125]
48. trefoil factor 1 (breast cancer, estrogen-inducible sequence expressed in) (TFF1) [NM_003225]
49. aryl-hydrocarbon receptor nuclear translocator 2 (ARNT2) [NM_014862]
50. v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro / glioblastoma derived oncogene homolog (avian) (ERBB2) [NM_004448]
51. tribbles homolog 3 (Drosophila) (TRIB3) [NM_021158]
52. tyrosyl-tRNA synthetase (YARS) [NM_003680]
53. quiescin Q6 (QSCN6) [NM_002826]
54. cyclin A1 (CCNA1) [NM_003914]
55. interferon-related developmental regulator 1 (IFRD1) [NM_001550]
56. matrix Gla protein (MGP) [NM_000900]
57. amphiregulin (schwannoma-derived growth factor) (AREG) [NM_001657]
58. insulin-like growth factor binding protein 4 (IGFBP4) [NM_001552]
59. Kruppel-like factor 10 (KLF10) [NM_005655]
60. calpain, small subunit 1 (CAPNS1) [NM_001749]
61. interferon stimulated exonuclease gene 20kDa (ISG20) [NM_002201]
62. tumor-associated calcium signal transducer 2 (TACSTD2) [NM_002353]
63. PDZ domain containing 1 (PDZK1) [NM_002614]
64. ferritin, heavy polypeptide 1 (FTH1) [NM_002032]
65. peripheral myelin protein 22 (PMP22) [NM_000304]
66. lysosomal-associated membrane protein 3 (LAMP3) [NM_014398]
67. tumor protein p53 inducible protein 11 (TP53I11) [NM_006034]
68. tetraspanin 1 (TSPAN1) [NM_005727]
69. CCAAT / enhancer binding protein (C / EBP), beta (CEBPB) [NM_005194]
70. activating transcription factor 3 (ATF3) [NM_001674]
71. enolase 1, (alpha) (ENO1) [NM_001428]
72. activating transcription factor 3 (ATF3) [NM_004024]
73. early growth response 3 (EGR3) [NM_004430]
74. runt-related transcription factor 1 (acute myeloid leukemia 1; aml1 oncogene) (RUNX1) [NM_001001890]
75. v-fos FBJ murine osteosarcoma viral oncogene homolog (FOS) [NM_005252]
76. nuclear receptor interacting protein 1 (NRIP1) [NM_003489]
77. transcription elongation factor A (SII), 1 (TCEA1) [NM_006756]
78. general transcription factor II, i (GTF2I) [NM_032999]
79. TAF9 RNA polymerase II, TATA box binding protein (TBP) -associated factor, 32kDa (TAF9) [NM_003187]
80. EGF-containing fibulin-like extracellular matrix protein 1 (EFEMP1) [NM_004105]
81. reticulocalbin 1, EF-hand calcium binding domain (RCN1) [NM_002901]
82. cadherin 18, type 2 (CDH18) [NM_004934]
83. S100 calcium binding protein P (S100P) [NM_005980]
84. chromosome 1 open reading frame 19 (C1orf19) [NM_052965]
85. IMP4, U3 small nucleolar ribonucleoprotein, homolog (yeast) (IMP4) [NM_033416]
86. elongation factor Tu GTP binding domain containing 2 (EFTUD2) (U5-116KD) [NM_004247]
87. glycyl-tRNA synthetase (GARS) [NM_002047]
88. eukaryotic translation initiation factor 3, subunit 9 eta, 116kDa (EIF3S9) [NM_003751]
89. solute carrier family 12 (sodium / potassium / chloride transporters), member 2 (SLC12A2) [NM_001046]
90. solute carrier family 7 (cationic amino acid transporter, y + system), member 5 (SLC7A5) [NM_003486]
91. solute carrier family 1 (glutamate / neutral amino acid transporter), member 4 (SLC1A4) [NM_003038]
92. solute carrier family 7, (cationic amino acid transporter, y + system) member 11 (SLC7A11) [NM_014331]
93. lipocalin 2 (oncogene 24p3) (LCN2) [NM_005564]
94. exportin, tRNA (nuclear export receptor for tRNAs) (XPOT) [NM_007235]
95. solute carrier family 1 (neutral amino acid transporter), member 5 (SLC1A5) [NM_005628]
96. chloride intracellular channel 4 (CLIC4) [NM_013943]
97. transcobalamin I (vitamin B12 binding protein, R binder family) (TCN1) (NM_0
01062]
98. synaptogyrin 2 (SYNGR2) [NM_004710]
99. transmembrane 4 L six family member 1 (TM4SF1) [NM_014220]
100. heat shock 70kDa protein 1A (HSPA1A) [NM_005345]
101. heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa) (HSPA5) [NM_005347]
102. heat shock protein 90kDa beta (Grp94), member 1 (HSP90B1) (TRA1) [NM_003299]
103. chromobox homolog 1 (HP1 beta homolog Drosophila) (CBX1) [NM_006807]
104. H3 histone, family 3B (H3.3B) (H3F3B) [NM_005324]
105. retinoblastoma binding protein 8 (RBBP8) [NM_002894]
106. tumor protein D52-like 1 (TPD52L1) [NM_003287]
107. absent in melanoma 1 (AIM1) [NM_001624]
108. chromosome 19 open reading frame 21 (C19orf21) [NM_173481]
109. SH3 domain binding glutamic acid-rich protein (SH3BGR) [NM_007341]
110. selenium binding protein 1 (SELENBP1) [NM_003944]
111. KIAA0196 (KIAA0196) [NM_014846]
112. LOC401397 [BC065765]
113. phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1) [NM_021127]
114. PREDICTED: paternally expressed 10 (PEG10) [XM_496907]
115. DAZ associated protein 2 (DAZAP2) [NM_014764].   5. The method of claim 4, wherein the estrogen responsive gene comprises all of the 115 estrogen responsive genes of claim 4.   The method according to any one of claims 1 to 5, wherein the application of the traditional Chinese medicine or an active ingredient thereof or estrogen to a living body is performed by culturing cells in a medium containing the traditional Chinese medicine or an active ingredient thereof or estrogen.   The method according to claim 6, wherein the cells are MCF-7 cells.   The method according to any one of claims 1 to 7, wherein a gene profile is obtained using a DNA microarray on which the estrogen-responsive gene or a fragment thereof is immobilized.   The method according to any one of claims 1 to 7, wherein a gene profile is obtained by quantitative PCR targeting the estrogen-responsive gene or a fragment thereof.   The evaluation of the effective activity of the active ingredient of the Kampo medicine having estrogenic activity is the search or evaluation of the effective activity in the Kampo medicine or its active ingredient, or the plant, extract or composition containing the active ingredient, or the Kampo medicine or its The method according to claim 1, which is an evaluation for quality control of a composition containing an active ingredient. A reagent for use in a method for evaluating the effective activity of an active ingredient of a traditional Chinese medicine (including traditional Chinese medicine) having an estrogenic activity according to any one of claims 1 to 10,
The reagent, following 1.～115 become. 115 pieces of all or partial sequence thereof consisting of contiguous 10~200bp nucleotide sequence of genes selected from the estrogen-responsive genes shown in the including DNA,
The estrogen responsive gene is expressed in the “enzyme” group (1.-24.), The “signaling” group (25.-50.), The “proliferation” group (51.-68.), The “transcription” group (69.-79. ) Check that at least 10 genes have been selected from one of the groups grouped by function in the “transport” group (80.-97.) And “others” group (98.-115.). Features, reagents;
1． phosphoenolpyruvate carboxykinase 2 (mitochondrial) (PCK2) [NM_004563]
2． phosphoserine aminotransferase 1 (PSAT1) [NM_058179]
3． asparagine synthetase (ASNS) [NM_133436]
Four. phosphoglycerate dehydrogenase (PHGDH) [NM_006623]
Five. serine hydroxymethyltransferase 2 (mitochondrial) (SHMT2) [NM_005412]
6. 24-dehydrocholesterol reductase (DHCR24) [NM_014762]
7． tryptophanyl-tRNA synthetase (WARS) [NM_004184]
8． fucosyltransferase 8 (alpha (1,6) fucosyltransferase) (FUT8) [NM_178155]
9． argininosuccinate synthetase (ASS) [NM_054012]
Ten. enolase 2 (gamma, neuronal) (ENO2) [NM_001975]
11． enolase 3 (beta, muscle) (ENO3) [NM_001976]
12． fructose-1,6-bisphosphatase 1 (FBP1) [NM_000507]
13. methylenetetrahydrofolate dehydrogenase (NADP + dependent) 2, methenyl
tetrahydrofolate cyclohydrolase (MTHFD2) [NM_006636]
14. carnitine palmitoyltransferase 1A (liver) (CPT1A) [NM_001876]
15． peptidase (mitochondrial processing) alpha (PMPCA) [NM_015160]
16． cathepsin D (lysosomal aspartyl peptidase) (CTSD) [NM_001909]
17． glutamic-oxaloacetic transaminase 1, soluble (aspartate aminotransferase 1) (GOT1) [NM_002079]
18． sorbitol dehydrogenase (SORD) [NM_003104]
19. stearoyl-CoA desaturase (delta-9-desaturase) (SCD) [NM_005063]
20． surfactant, pulmonary-associated protein B (SFTPB) [NM_000542]
twenty one. serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 3 (SERPINA3) [NM_001085]
twenty two. aconitase 2, mitochondrial (ACO2) [NM_001098]
twenty three. glutamine-fructose-6-phosphate transaminase 1 (GFPT1) [NM_002056]
twenty four. CDP-diacylglycerol-inositol 3-phosphatidyltransferase (phosphatidylinositol synthase) (CDIPT) [NM_006319]
twenty five. ribosomal protein S6 kinase, 90kDa, polypeptide 3 (RPS6KA3) [NM_004586]
26. protein kinase C, delta (PRKCD) [NM_006254]
27. unc-51-like kinase 1 (C. elegans) (ULK1) [NM_003565]
28. integrin-linked kinase (ILK) [NM_004517]
29. protein kinase C substrate 80K-H (PRKCSH) [NM_002743]
30. cyclin-dependent kinase inhibitor 1A (p21, Cip1) (CDKN1A) [NM_000389]
31. phosphoinositide-3-kinase, class 3 (PIK3C3) [NM_002647]
32. poliovirus receptor (PVR) [NM_006505]
33. progesterone receptor (PGR) [NM_000926]
34. insulin-like growth factor binding protein 5 (IGFBP5) [NM_000599]
35. catenin (cadherin-associated protein), delta 2 (neural plakophilin-related arm-repeat protein) (CTNND2) [NM_001332]
36. neuropeptide Y receptor Y1 (NPY1R) [NM_000909]
37. protein tyrosine phosphatase, non-receptor type 18 (brain-derived) (PTPN18) [NM_014369]
38. Rho GDP dissociation inhibitor (GDI) alpha (ARHGDIA) [NM_004309]
39. endothelin 2 (EDN2) [NM_001956]
40. stanniocalcin 2 (STC2) [NM_003714]
41. lectin, galactoside-binding, soluble, 3 binding protein (LGALS3BP) [NM_005567]
42. angiotensin II receptor, type 1 (AGTR1) [NM_000685]
43. proprotein convertase subtilisin / kexin type 6 (PCSK6) [NM_002570]
44. SH3-domain binding protein 5 (BTK-associated) (SH3BP5) [NM_004844]
45. growth differentiation factor 15 (GDF15) [NM_004864]
46. ras homolog gene family, member C (RHOC) [NM_175744]
47. estrogen receptor 1 (ESR1) [NM_000125]
48. trefoil factor 1 (breast cancer, estrogen-inducible sequence expressed in) (TFF1) [NM_003225]
49. aryl-hydrocarbon receptor nuclear translocator 2 (ARNT2) [NM_014862]
50. v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro / glioblastoma derived oncogene homolog (avian) (ERBB2) [NM_004448]
51. tribbles homolog 3 (Drosophila) (TRIB3) [NM_021158]
52. tyrosyl-tRNA synthetase (YARS) [NM_003680]
53. quiescin Q6 (QSCN6) [NM_002826]
54. cyclin A1 (CCNA1) [NM_003914]
55. interferon-related developmental regulator 1 (IFRD1) [NM_001550]
56. matrix Gla protein (MGP) [NM_000900]
57. amphiregulin (schwannoma-derived growth factor) (AREG) [NM_001657]
58. insulin-like growth factor binding protein 4 (IGFBP4) [NM_001552]
59. Kruppel-like factor 10 (KLF10) [NM_005655]
60. calpain, small subunit 1 (CAPNS1) [NM_001749]
61. interferon stimulated exonuclease gene 20kDa (ISG20) [NM_002201]
62. tumor-associated calcium signal transducer 2 (TACSTD2) [NM_002353]
63. PDZ domain containing 1 (PDZK1) [NM_002614]
64. ferritin, heavy polypeptide 1 (FTH1) [NM_002032]
65. peripheral myelin protein 22 (PMP22) [NM_000304]
66. lysosomal-associated membrane protein 3 (LAMP3) [NM_014398]
67. tumor protein p53 inducible protein 11 (TP53I11) [NM_006034]
68. tetraspanin 1 (TSPAN1) [NM_005727]
69. CCAAT / enhancer binding protein (C / EBP), beta (CEBPB) [NM_005194]
70. activating transcription factor 3 (ATF3) [NM_001674]
71. enolase 1, (alpha) (ENO1) [NM_001428]
72. activating transcription factor 3 (ATF3) [NM_004024]
73. early growth response 3 (EGR3) [NM_004430]
74. runt-related transcription factor 1 (acute myeloid leukemia 1; aml1 oncogene) (RUNX1) [NM_001001890]
75. v-fos FBJ murine osteosarcoma viral oncogene homolog (FOS) [NM_005252]
76. nuclear receptor interacting protein 1 (NRIP1) [NM_003489]
77. transcription elongation factor A (SII), 1 (TCEA1) [NM_006756]
78. general transcription factor II, i (GTF2I) [NM_032999]
79. TAF9 RNA polymerase II, TATA box binding protein (TBP) -associated factor, 32kDa (TAF9) [NM_003187]
80. EGF-containing fibulin-like extracellular matrix protein 1 (EFEMP1) [NM_004105]
81. reticulocalbin 1, EF-hand calcium binding domain (RCN1) [NM_002901]
82. cadherin 18, type 2 (CDH18) [NM_004934]
83. S100 calcium binding protein P (S100P) [NM_005980]
84. chromosome 1 open reading frame 19 (C1orf19) [NM_052965]
85. IMP4, U3 small nucleolar ribonucleoprotein, homolog (yeast) (IMP4) [NM_033416]
86. elongation factor Tu GTP binding domain containing 2 (EFTUD2) (U5-116KD) [NM_004247]
87. glycyl-tRNA synthetase (GARS) [NM_002047]
88. eukaryotic translation initiation factor 3, subunit 9 eta, 116kDa (EIF3S9) [NM_003751]
89. solute carrier family 12 (sodium / potassium / chloride transporters), member 2 (SLC12A2) [NM_001046]
90. solute carrier family 7 (cationic amino acid transporter, y + system), member 5 (SLC7A5) [NM_003486]
91. solute carrier family 1 (glutamate / neutral amino acid transporter), member 4 (SLC1A4) [NM_003038]
92. solute carrier family 7, (cationic amino acid transporter, y + system) member 11 (SLC7A11) [NM_014331]
93. lipocalin 2 (oncogene 24p3) (LCN2) [NM_005564]
94. exportin, tRNA (nuclear export receptor for tRNAs) (XPOT) [NM_007235]
95. solute carrier family 1 (neutral amino acid transporter), member 5 (SLC1A5) [NM_005628]
96. chloride intracellular channel 4 (CLIC4) [NM_013943]
97. transcobalamin I (vitamin B12 binding protein, R binder family) (TCN1) (NM_0
01062]
98. synaptogyrin 2 (SYNGR2) [NM_004710]
99. transmembrane 4 L six family member 1 (TM4SF1) [NM_014220]
100. heat shock 70kDa protein 1A (HSPA1A) [NM_005345]
101. heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa) (HSPA5) [NM_005347]
102. heat shock protein 90kDa beta (Grp94), member 1 (HSP90B1) (TRA1) [NM_003299]
103. chromobox homolog 1 (HP1 beta homolog Drosophila) (CBX1) [NM_006807]
104. H3 histone, family 3B (H3.3B) (H3F3B) [NM_005324]
105. retinoblastoma binding protein 8 (RBBP8) [NM_002894]
106. tumor protein D52-like 1 (TPD52L1) [NM_003287]
107. absent in melanoma 1 (AIM1) [NM_001624]
108. chromosome 19 open reading frame 21 (C19orf21) [NM_173481]
109. SH3 domain binding glutamic acid-rich protein (SH3BGR) [NM_007341]
110. selenium binding protein 1 (SELENBP1) [NM_003944]
111. KIAA0196 (KIAA0196) [NM_014846]
112. LOC401397 [BC065765]
113. phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1) [NM_021127]
114. PREDICTED: paternally expressed 10 (PEG10) [XM_496907]
115. DAZ associated protein 2 (DAZAP2) [NM_014764]. A partial sequence consisting of all or a continuous 10~200bp the base sequence of the gene including DNA is immobilized DNA microarray, reagent according to claim 11. In whole or quantitative PCR primer set consisting of a partial sequence consisting of the consecutive 10~200bp from including DNA base sequence of the gene, reagent according to claim 11. A partial sequence consisting of all or 10~200bp its continuous nucleotide sequence of the following genes is quantitative PCR primer set consisting of including DNA, reagent according to claim 13;
16． cathepsin D (lysosomal aspartyl peptidase) (CTSD) [NM_001909]
twenty five. ribosomal protein S6 kinase, 90kDa, polypeptide 3 (RPS6KA3) [NM_004586]
26. protein kinase C, delta (PRKCD) [NM_006254]
30. cyclin-dependent kinase inhibitor 1A (p21, Cip1) (CDKN1A) [NM_000389]
38. Rho GDP dissociation inhibitor (GDI) alpha (ARHGDIA) [NM_004309]
48. trefoil factor 1 (breast cancer, estrogen-inducible sequence expressed in) (TFF1) [NM_003225]
67. tumor protein p53 inducible protein 11 (TP53I11) [NM_006034]
76. nuclear receptor interacting protein 1 (NRIP1) [NM_003489].

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