JP5669349B2 - Method for evaluating relaxation / induction suppression effect of tension state in vivo, and screening method for substance - Google Patents

Description

The present invention relates to a method Evaluation of the relaxation-induced suppression of in vivo tension, and relates to the screening how materials, and more particularly, specific in a body fluid collected from animals stressed with administering the test substance Using the marker substance of the present invention as an index, an evaluation method for the relaxation / induction suppression effect of the in vivo tension state for evaluating the relaxation effect or induction suppression effect of the in vivo tension state of the test substance, and in vivo tension using the evaluation method It relates to a substance having a relaxing effect or inductive effect of suppressing state in screening how the screening substance.

  Symptoms and diseases caused by stress are increasing, which is a big problem in modern society. In general, the term “stress” is used in two ways. One is the meaning mainly at the living body level, and refers to the “in vivo tension state” that reacts to an induced adverse effect factor (stresser) in the living body. The other is mainly at the cellular level and refers to the agent itself (eg high temperature stress, oxidative stress).

  It is useful for the prevention and treatment of diseases caused by stress to alleviate in-vivo tension (stress in the living body) or to suppress its induction. For this reason, attention has been focused on biomarkers that reflect the in-vivo tension state. That is, by using such a biomarker, it becomes possible to easily examine whether or not a test substance has an effect of alleviating in vivo tension or suppressing induction, and is useful for screening food materials and the like. It is. And it becomes possible to avoid a useless in-vivo tension state by daily ingesting the food material selected by such an evaluation and screening system.

Glutathione and cortisol have been known for a long time as markers in blood that reflect the tension in the body. However, since these are low molecular weight compounds, they are rapidly metabolized, and the blood concentration varies greatly in the absence of stress. Therefore, protein markers with smaller fluctuations in a stress-free state have been searched, and several proteinaceous stress markers have been found (Patent Documents 1 and 2).
JP 7-181180 A JP 2007-225606 A

  The present invention provides a series of techniques for newly identifying a biomarker that reflects the in-vivo tension state and evaluating the relaxation effect or induction suppression effect of the in-vivo strain state of the test substance using the biomarker. It is intended to do.

The related invention for solving the above-mentioned problem is to compare at least one concentration of the following marker substances (M1) to (M8) in a body fluid collected from a stressed animal while administering a test substance with a reference value. By this, it is the evaluation method of the relaxation / induction suppression effect of the in-vivo tension state characterized by evaluating the relaxation effect or induction suppression effect of the in-vivo tension state which the said test substance has.
(M1) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 6020 when subjected to mass spectrometry;
(M2) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 9310 when subjected to mass spectrometry;
(M3) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 13700 when subjected to mass spectrometry;
(M4) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 13800 when subjected to mass spectrometry;
(M5) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 17700 when subjected to mass spectrometry;
(M6) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 19600 when subjected to mass spectrometry;
(M7) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 39900 when subjected to mass spectrometry;
(M8) A protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 77000 when subjected to mass spectrometry.

  The present invention relates to a method for evaluating the relaxation / induction suppression effect of in vivo tension, and includes eight kinds of (M1) to (M8) in body fluids collected from a stressed animal while administering a test substance. The concentration of at least one marker substance is compared with a reference value, and the effect of alleviating or suppressing the in vivo tension state of the test substance is evaluated. Each of the marker substances (M1) to (M9) is a protein present in a body fluid and reflects the in-vivo tension state. ADVANTAGE OF THE INVENTION According to this invention, the relaxation effect or induction | guidance | derivation suppression effect of the in-vivo tension state which a test substance has can be evaluated easily and with high precision. The “animal” includes humans as well as animals such as mice.

  Here, the “relaxing effect of the in-vivo tension state” refers to an effect of relieving, reducing, suppressing, etc. the tension of the living body in the tension state, and the living body is directed to the direction of being released from the tension state by the effect. Further, the “inhibition effect of inducing tension in the living body” refers to an effect of suppressing or preventing the tension state from being induced in a living body in a calm state. Due to the effect, the living body is directed to maintain a calm state. In the present invention, the “relaxing effect of the in vivo tension state” or “induction suppressing effect of the in vivo tension state” of the test substance is evaluated.

  Here, the values of “about 6020”, “about 17700”, “about 77000”, etc. of the mass / charge ratio (hereinafter sometimes abbreviated as “m / z”) in each marker substance are This is a value that takes into account the error range of the measured value, and has a width of approximately ± 0.2%. That is, about 6020 represents about 6020 ± 0.2%, about 17700 represents about 17700 ± 0.2%, and about 77000 represents about 77000 ± 0.2%. The other mass / charge ratios have a width of approximately ± 0.2% in a similar manner. In addition, these marker substances are mainly proteins present in blood. When the test substance has a relaxation effect or induction suppression effect on the tension state in the living body, the concentrations of the marker substances (M3) and (M4) in the body fluid of the animal show lower values, and the marker substances (M1) and (M2) , (M5), (M6), (M7), and (M8) have higher concentrations.

A related invention is the above-described method for evaluating the relaxation / induction suppression effect of the in-vivo tension state characterized by satisfying at least one of the following (1) to (5).
(1) The marker substance (M1) is serum albumin or a modified form thereof.
(2) The marker substance (M2) is proapolipoprotein A2 or a modified form thereof.
(3) The marker substance (M3) is transthyretin or a modified form thereof.
(4) The marker substance (M4) is transthyretin or a modified form thereof.
(5) The marker substance (M8) is transferrin or a modified product thereof.

  Serum albumin, pro-apolipoprotein A2, transthyretin, and transferrin are all well known for their physicochemical properties. Analysis of the substances (M1), (M2), (M3), (M4), and (M8) is easy.

  A representative example of “modified protein” is a protein in which at least one of the amino acid residues constituting the protein is modified. “Modification” includes not only addition of a compound or functional group (eg, phosphorylation) but also elimination (eg, dephosphorylation). The “protein or a modified form thereof” includes an isoform of the protein. Further, “protein or a modified form thereof” includes substantially the same protein in which several amino acid residues are deleted, substituted or added in the primary structure of the protein. Furthermore, “protein or a modified product thereof” includes a protein fragment derived from the protein that has been cleaved by a protease. In the case of a complex protein, the “protein or a modified form thereof” includes its subunits.

A related invention for solving the same problem is based on at least one concentration belonging to any one of the following marker substances (A1) to (A4) in a body fluid collected from an animal given a stress while administering a test substance: It is an evaluation method of the relaxation / induction suppression effect of the in vivo tension state characterized by evaluating the relaxation effect or induction suppression effect of the in vivo tension state of the test substance by comparing with the value.
(A1) serum albumin or a modified product thereof,
(A2) Proapolipoprotein A2 or a modified product thereof,
(A3) transthyretin or a modified form thereof,
(A4) Transferrin or a modified product thereof.

  The present invention also relates to a method for evaluating the effect of mitigating / inducing in vivo tension, and any of the above (A1) to (A4) in a body fluid collected from a stressed animal while administering a test substance At least one concentration of the marker substance to which it belongs is compared with a reference value, and the relaxation effect or induction suppression effect of the in vivo tension state of the test substance is evaluated. The marker substances belonging to the above (A1) to (A4) are all proteins present in the body fluid and reflect the in vivo tension state. ADVANTAGE OF THE INVENTION According to this invention, the relaxation effect or induction | guidance | derivation suppression effect of the in-vivo tension state which a test substance has can be evaluated easily and with high precision. In particular, serum albumin, proapolipoprotein A2, transthyretin, and transferrin are all well known for their physicochemical properties and are therefore easy to analyze. In the present invention, the term “animal” includes humans as well as animals such as mice.

  In the present invention, a representative example of a “modified protein” is a protein in which at least one of the amino acid residues constituting the protein is modified. The “modification” includes not only addition of a compound or functional group but also desorption. Separation is also included. In addition, “protein or a modified form thereof” includes an isoform of the protein, a substantially identical protein in which several amino acid residues are deleted, substituted or added in the primary structure of the protein, and Protein fragments derived from the protein that have been cleaved by a protease are included. Furthermore, in the case of a complex protein, the “protein or a modified form thereof” includes its subunits.

According to the first aspect of the present invention, the concentration of the following marker substance (M1) in a body fluid collected from an animal given a test substance and stressed in a living body is compared with a reference value, and the concentration is higher. This is a method for evaluating the relaxation / induction suppression effect of the in vivo tension state , wherein the relaxation effect or induction suppression effect of the in vivo tension state of the test substance is evaluated.
(M1) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 6020 when subjected to mass spectrometry;
further,
The marker substance (M1) includes the amino acid sequence represented by SEQ ID NO: 8 or 9.

The invention according to claim 2 compares the concentration of the following marker substance (M1) in a body fluid collected from an animal to which a test substance is administered and stressed in a living body with a reference value, and the concentration is higher. This is a method for evaluating the relaxation / induction suppression effect of the in vivo tension state , wherein the relaxation effect or induction suppression effect of the in vivo tension state of the test substance is evaluated.
(M1) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 6020 when subjected to mass spectrometry;
further,
The marker substance (M1) is serum albumin or a modified form thereof.

The invention according to claim 3 compares the concentration of the following marker substance (A1) in a body fluid collected from an animal given a test substance and stressed in the living body with a reference value, and the concentration is higher. This is a method for evaluating the relaxation / induction suppression effect of the in vivo tension state , wherein the relaxation effect or induction suppression effect of the in vivo tension state of the test substance is evaluated.
(A1) Serum albumin or a modified product thereof.

The invention according to claim 4 further compares the concentration of the following marker substance (M3) or (M4) with a reference value, and the in vivo tension according to any one of claims 1 to 3 This is a method for evaluating the effect of mitigating and suppressing the state.
(M3) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 13700 when subjected to mass spectrometry;
(M4) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 13800 when subjected to mass spectrometry;
further,
The marker substances (M3) and (M4) are transthyretin or a modified form thereof.

The invention according to claim 5 further compares the concentration of the following marker substance (A3) with a reference value, and alleviates the in vivo tension state according to any one of claims 1 to 3. This is a method for evaluating the induction suppression effect.
(A3) Transthyretin or a modified product thereof.

  The animal is preferably an animal that has been subjected to stress in a living body after administration of the test substance (claim 6).

  According to a seventh aspect of the present invention, the reference value is determined by administering a known substance that does not have a relaxation effect or induction suppression effect on the tension state in the living body, and the marker substance in the body fluid of an animal subjected to stress in the living body. It is a density | concentration, It is an evaluation method of the relaxation / induction suppression effect of the in-vivo tension state of any one of Claims 1-6 characterized by the above-mentioned.

  With this configuration, it is possible to more easily and accurately evaluate the relaxation effect or induction suppression effect of the in vivo tension state of the test substance.

  The invention according to claim 8 is the method for evaluating the effect of mitigating / suppressing induction of an in vivo tension state according to any one of claims 1 to 7, wherein the body fluid is blood.

  With this configuration, it is possible to easily collect a body fluid as a measurement sample, and it is possible to evaluate the relaxation effect or induction suppression effect of the in vivo tension state of the test substance more simply and quickly.

  The invention according to claim 9 is characterized in that the test substance is a food material or a pharmaceutical material, and the effect of mitigating / suppressing induction of an in vivo tension state according to any one of claims 1 to 8. It is an evaluation method.

  With such a configuration, for the purpose of developing functional foods or pharmaceuticals, it is possible to evaluate the relaxation effect or induction suppression effect of the in vivo tension state.

  In the invention according to claim 10, the body fluid or the body fluid component is brought into contact with a carrier on which a substance having affinity for the marker substance is immobilized, and the marker substance in the body fluid is captured on the carrier and captured. The concentration of the marker substance in a body fluid is calculated based on the amount of the marker substance, and the evaluation of the relaxation / induction suppression effect of the in vivo tension state according to any one of claims 1 to 9 Is the method.

  In the method for evaluating the effect of mitigating / inducing in vivo tension in the present invention, a carrier on which a substance having affinity for a marker substance is immobilized is used. Then, the body fluid or body fluid component is brought into contact with the carrier, and the marker substance contained in the body fluid or body fluid component is captured on the carrier via a substance having affinity for the marker substance, and the amount of the marker substance captured is increased. Based on this, the concentration of the marker substance in the body fluid is calculated. According to the present invention, since the marker substance captured on the carrier is the measurement target, the influence of the contaminant substance contained in the measurement sample can be reduced, and the concentration of the marker substance can be increased with higher sensitivity and higher accuracy. Can be measured. Examples of the body fluid component include serum or plasma when the body fluid is blood.

  The invention according to claim 11 is characterized in that the carrier has a flat portion, and the substance having affinity for the marker substance is immobilized on a part of the flat portion. It is the evaluation method of the relief / induction suppression effect of the described in-vivo tension state.

  In the method for evaluating the effect of mitigating / inducing in vivo tension in the present invention, a carrier having a planar portion is used, and a substance having affinity for the marker substance is immobilized on a part of the planar portion. With this configuration, a substance having affinity for the marker substance can be spot-fixed at a plurality of locations on the carrier. As a result, it is possible to simultaneously process a plurality of measurement samples with one carrier, and simultaneously measure the concentrations of a plurality of marker substances with one carrier, and work efficiency is improved. Furthermore, by reducing the area of each spot, the concentration of the marker substance can be measured even from a very small amount of measurement sample. An example of the carrier having a planar portion is a substrate such as a chip.

  The invention according to claim 12 is characterized in that the substance having affinity for the marker substance is an ion exchanger or an antibody, and the effect of mitigating / inducing in vivo tension state according to claim 10 or 11 This is an evaluation method.

  In the method for evaluating the effect of mitigating / inducing suppression of in vivo tension state of the present invention, an ion exchanger or an antibody is used as a substance having affinity for a marker substance, and the marker substance in the measurement sample is passed through the ion exchanger or antibody. Is captured on a carrier. When the substance is an ion exchanger, various substances are easily available, and a carrier for capturing the marker substance can be easily prepared. Further, when the substance is an antibody, the marker substance can be captured more specifically. Examples of the method for measuring the amount of the captured marker substance include mass spectrometry and immunoassay (in the case of an antibody).

  A related invention is a substance characterized in that a test substance is evaluated by the above-described evaluation method for relaxation / induction suppression effect of in vivo tension state, and a substance having a relaxation effect or induction suppression effect of in vivo tension state is screened Screening method.

  The present invention relates to a method for screening a substance, wherein at least one concentration of each of the marker substances (M1) to (M8) and each marker substance belonging to (A1) to (A4) in a body fluid of an animal is used as a reference value. In comparison, a substance having a relaxing effect or an induction suppressing effect on the in-vivo tension state is screened. Each of the marker substances (M1) to (M8) and the marker substances belonging to the above (A1) to (A4) are proteins present in the body fluid and reflect the in-vivo tension state. According to the screening method for a substance of the present invention, a substance having a relaxing effect or an induction suppressing effect on the in-vivo tension state can be easily and accurately screened. For example, when the test substance is a food material, it is possible to screen for a food material useful for the development of a functional food having a relaxing effect on the in vivo tension state or an induction suppressing effect.

  The invention according to claim 13 evaluates the test substance by the method for evaluating the relaxation / induction suppression effect of the in vivo tension state according to any one of claims 1 to 12, and the relaxation effect of the in vivo tension state or A screening method for a substance characterized by screening a substance having an induction suppressing effect.

  A related invention is a kit for use in the above-described method for evaluating the effect of mitigating / inducing suppression of tension in a living body, characterized by comprising a carrier on which a substance having affinity for the marker substance is immobilized. This is a kit for evaluating the effect of mitigating / suppressing induction of in vivo tension.

  The kit for evaluating the effect of relieving / suppressing induction of the in vivo tension state of the present invention includes a carrier on which a substance having affinity for a marker substance is immobilized. With this configuration, it is not necessary to prepare the carrier separately when measuring the concentration of the marker substance, and the concentration of the marker substance can be measured very simply.

  The related invention is the evaluation kit for evaluating the effect of mitigating / inducing in vivo tension, wherein the substance having affinity for the marker substance is an ion exchanger.

  With this configuration, the marker substance can be more reliably captured on the carrier.

  In the kit for evaluating the relaxation / induction suppression effect of the in vivo tension state, a configuration used for screening a substance having a relaxation effect or induction suppression effect on the in vivo tension state is recommended.

  According to the evaluation method of the relaxation / induction suppression effect of the in vivo tension state of the present invention, the relaxation effect or induction suppression effect of the in vivo tension state of the test substance can be evaluated easily and with high accuracy.

  According to the screening method for a substance of the present invention, a substance having a relaxing effect or an induction suppressing effect on the in-vivo tension state can be easily and accurately screened.

  According to the kit for evaluating the relaxation effect or induction suppression effect of the in vivo tension state, it is not necessary to prepare the carrier separately when measuring the concentration of the marker substance, and the concentration of the marker substance can be measured very simply. .

  Hereinafter, the best mode for carrying out the present invention will be described in detail.

The evaluation method of the relaxation / induction suppression effect of the in vivo tension state of the present invention includes two aspects. In one aspect, the test substance has a test substance administered by comparing at least one concentration of the following marker substances (M1) to (M8) in a body fluid collected from a stressed animal with a reference value: Evaluate the relaxation effect or induction suppression effect of the in vivo tension state.
(M1) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 6020 when subjected to mass spectrometry;
(M2) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 9310 when subjected to mass spectrometry;
(M3) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 13700 when subjected to mass spectrometry;
(M4) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 13800 when subjected to mass spectrometry;
(M5) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 17700 when subjected to mass spectrometry;
(M6) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 19600 when subjected to mass spectrometry;
(M7) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 39900 when subjected to mass spectrometry;
(M8) A protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 77000 when subjected to mass spectrometry.

  All of these marker substances are proteins mainly present in blood and reflect the in-vivo tension state. The marker substances (M3) and (M4) (hereinafter, a group consisting of these marker substances may be referred to as “group 1”) shows a higher value when the living body is in a tension state. Therefore, when the test substance has a relaxation effect or induction suppression effect on the in vivo tension state, it shows a lower value in the animal administered the test substance. On the other hand, each marker substance of (M1), (M2), (M5), (M6), (M7), and (M8) (hereinafter, a group consisting of these marker substances may be referred to as “group 2”). .) Shows a lower value when the living body is in a tense state, and therefore has a higher value in the animal to which the test substance is administered when it has a relaxing effect or an induction suppressing effect on the in vivo tense state. .

According to certain conditions of peptide mapping, the marker substance (M1) can be identified as serum albumin, the marker substances (M3) and (M4) as transthyretin, and the marker substance (M5) as transferrin. In addition, the marker substance (M2) can be identified as proapolipoprotein A2 from peptide mapping under certain conditions, information on molecular weight, and isoelectric point. That is, in an embodiment, at least one of the following (1) to (5) is satisfied.
(1) The marker substance (M1) is serum albumin or a modified form thereof.
(2) The marker substance (M2) is proapolipoprotein A2 or a modified form thereof.
(3) The marker substance (M3) is transthyretin or a modified form thereof.
(4) The marker substance (M4) is transthyretin or a modified form thereof.
(5) The marker substance (M8) is transferrin or a modified product thereof.

In another aspect of the method for evaluating the effect of mitigating / suppressing induction of an in vivo tension state according to the present invention, any of the following marker substances (A1) to (A4) in a body fluid collected from a stressed animal while administering a test substance: By comparing at least one concentration belonging to the reference value with a reference value, the relaxation effect or induction suppression effect of the in vivo tension state of the test substance is evaluated.
(A1) serum albumin or a modified product thereof,
(A2) Proapolipoprotein A2 or a modified product thereof,
(A3) transthyretin or a modified form thereof,
(A4) Transferrin or a modified product thereof.

  The amino acid sequences of mouse serum albumin, proapolipoprotein A2, transthyretin, and transferrin are as shown in SEQ ID NOs: 1 to 4, respectively.

  Examples of protein modifications include methylation, acetylation, adenylylation, myristylation, etc. of N-terminal α-amino group and lysine ε-amino group; addition of sugar or sugar chain to serine / threonine / asparagine; serine / threonine / Examples include phosphorylation of tyrosine, arginine, histidine; cysteine cysteinylation, homocysteinylation, sulfonylation, etc .; γ-carboxylation of glutamic acid; conversion of N-terminal glutamic acid to pyroglutamic acid, and the like. Further, elimination of these modifications (demethylation, elimination of sugars or sugar chains, dephosphorylation, etc.) is also included in “modification”.

  The “protein or a modified form thereof” includes an isoform of the protein. Isoforms include proteins generated by alternative splicing in addition to the various modifications described above. Furthermore, the “protein or a modified form thereof” includes substantially the same protein in which several amino acid residues are deleted, substituted or added in the primary structure of the protein. Furthermore, “protein or a modified product thereof” includes a protein fragment derived from the protein that has been cleaved by a protease. For example, a protein fragment having a length that can be recognized as derived from the protein, for example, a protein fragment composed of 20 or more amino acid residues, a protein fragment having a molecular weight of 2,000 or more, and the like can be mentioned. In the case of a complex protein, the “protein or a modified product thereof” includes its subunits.

  For example, a protein fragment (SEQ ID NO: 5) consisting of 55 amino acids corresponding to amino acid numbers 553 (Ala) to 608 (Ala) of serum albumin (SEQ ID NO: 1) is included in the “modified form of serum albumin”.

  A protein fragment (SEQ ID NO: 6) lacking the C-terminal amino acid residue (Lys) of proapolipoprotein A2 (SEQ ID NO: 2) is included in the “modified product of proapolipoprotein A2”.

  Transthyretin (also called prealbumin) is a complex protein consisting of four subunits with a molecular weight of about 14,000. For transthyretin, various modified transthyretins in which only the Cys residue is modified have been found (Amareth Lim et al., J. Biol. Chem., Vol. 258, No. 50). , 2003). As shown in Example 4 described later, S-sulfonated transthyretin obtained by adding a sulfonic acid to the Cys residue of mouse-derived transthyretin (SEQ ID NO: 3) is included in the “modified form of transthyretin”. .

  In the evaluation method of the relaxation effect or induction suppression effect of the in vivo tension state of the present invention, each marker substance of (M1) to (M8) and only one of each marker substance belonging to (A1) to (A4) are used. Alternatively, a plurality of them may be used in combination. There is no particular limitation on the combination method in the case of using a plurality, but for example, the marker substance (s) selected from group 1 and the marker substance (s) selected from group 2 can be combined. .

  In a preferred embodiment of the method for evaluating the relaxation / induction suppression effect of the in vivo tension state of the present invention, the reference value is “a known substance having no relaxation effect or induction suppression effect on the in vivo tension state” (hereinafter, “ The concentration of the marker substance in the bodily fluid of the animal that is administered and stressed is used. That is, when an ineffective known substance is administered to an animal and stress is applied, the concentration of the marker substance in the body fluid becomes an “abnormal value”. Then, the value (measured value) in the animal administered the test substance is compared with the reference value (abnormal value), and the measured value is significantly different from the reference value and is on the normal side (maintained on the normal side) The test substance can be evaluated as having a relaxing effect on the in vivo tension state or an induction suppressing effect. Specifically, when the marker substance belonging to group 1 is used as an index, when the measured value is significantly lower than the reference value, on the other hand, when the marker substance belonging to group 2 is used as an index, the measured value Is significantly higher than the reference value, it can be evaluated that the test substance has a relaxing effect or an induction suppressing effect on the in vivo tension state.

  Furthermore, there may be a plurality of reference values. For example, in addition to the abnormal value described above, a value in an animal not subjected to stress (normal value, negative control) can be added to the reference value. Specifically, (1) a group in which a known substance or test substance having no effect is administered and stress is not given (group showing normal values), (2) a group in which a known substance having no effect is administered and stress is given ( A total of three groups are set up: an abnormal value group), and (3) a group to which a test substance is administered and stress is applied, and animals are bred. And the said marker substance in the bodily fluid of each animal is measured, and each measured value is compared. At this time, there is a significant difference between (1) and (2), there is a significant difference between (3) and (2), and (3) is closer to the normal side ((1) than (2)) Side) (when maintained on the normal side), it can be evaluated that the test substance has a relaxing effect or an induction suppressing effect on the in vivo tension state.

  Furthermore, as a reference value, a value (positive control) in (4) a group in which “a known substance having an alleviation effect or induction suppression effect on in vivo tension state” (effective known substance) is administered and stress is applied (positive control) is added. You can also. Specifically, in addition to (1) to (3) above, the group (4) above is set and animals are raised. At this time, there is a significant difference between (1) and (2), there is a significant difference between (3) and (2), and (3) is normal ((1) and (2) compared to (2). 4), the test substance can be evaluated as having a relaxing effect or an induction suppressing effect on the in vivo tension state. That is, such a test substance exhibits the same behavior as the known substance adopted in (4) and can be said to have a similar action.

  Here, as an example of a material containing “a known substance having a relaxing effect or an induction suppressing effect on an in-vivo tension state” (a known substance having an effect), there is Ketsumeishi. It is known that Ketsumeishi has an effect of increasing blood glutathione concentration (for example, JP-A-2004-2231). In addition, it is known that blood glutathione concentration decreases due to restraint stress (Koichi Doichi et al., “Responsibility of glutathione in the brain and blood due to restraint stress”, Journal of Occupational Health, Japan Society for Occupational Health , 2002, 44, 262).

  In the present invention, “animal administered with test substance and stressed” is used, but the order of administration of test substance and application of stress is not particularly limited. For example, stress may be applied after administration of the test substance, or the test substance may be administered after applying stress first. Stress may be applied in parallel with the administration of the test substance. The administration route of the test substance is typically oral administration, but may be administered by other routes.

  There are no particular limitations on the types of stress (acting factors) given to animals, for example, physical stress such as restraint, behavioral restrictions, noise, cold, chemical stress such as drug administration, organisms such as inflammation, infection, etc. Mental stress, anxiety, and psychological stress such as anger.

There is no particular limitation on the type of animal in “animal administered with test substance and stressed”, and for example, mouse, rat, rabbit, pig and the like can be employed. Furthermore, humans can be employed as animals. In this case, the effect of the test substance is evaluated based on the result of the clinical test.

  Blood is preferably used as the animal body fluid used in the method for evaluating the effect of mitigating / suppressing induction of the in vivo tension state of the present invention. In particular, it is preferable to use serum or plasma (body fluid component) prepared from blood as a measurement sample. Serum or plasma can be prepared from blood by a known method such as centrifugation.

  Examples of the test substance in the method for evaluating the effect of mitigating / inducing in vivo tension in the present invention include food materials and pharmaceutical materials. For example, when a food material is an evaluation target, it can be used for development of a functional food.

  In the method for evaluating the effect of mitigating / inducing in vivo tension in the present invention, the method for measuring the concentration of a marker substance is generally used for protein quantification as long as the method can specifically measure the concentration of the marker substance. Can be used as it is. For example, various immunoassays, mass spectrometry (MS), chromatography, electrophoresis and the like can be used.

  According to the immunoassay, the concentration of the marker substance can be accurately measured even with a sample having a lot of contaminants. Examples of immunoassays include classical methods such as precipitation, agglutination, and hemolysis, which directly or indirectly measure antigen-antibody conjugates, and enzyme immunoassays (EIA) with increased detection sensitivity in combination with labeling methods. , Radioimmunoassay (RIA), fluorescent immunoassay (FIA) and the like. The antibody specific for the marker substance used in these immunoassays may be monoclonal or polyclonal.

  According to mass spectrometry, an ion peak derived from each marker substance can be specified, and the amount (concentration) of each marker substance can be measured with the ion peak intensity. As the ionization method for measuring the concentration of the marker substance by mass spectrometry, any of matrix-assisted laser desorption / ionization (MALDI) and electrospray ionization (ESI) can be applied. However, MALDI is preferred because it produces less multivalent ions. In particular, according to MALDI-TOF-MS combined with a time-of-flight mass spectrometer (TOF), an ion peak derived from a marker substance can be identified more accurately.

  When measuring the concentration of the marker substance by electrophoresis, for example, the test material is subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to separate the target marker substance, and the gel is prepared with an appropriate dye or fluorescent substance. It is only necessary to stain and measure the intensity and fluorescence intensity of the band corresponding to the target marker substance. When the separation of the marker substance is insufficient by SDS-PAGE alone, two-dimensional electrophoresis combined with isoelectric focusing (IEF) can also be used. Furthermore, instead of detecting directly from the gel, Western blotting can also be performed to measure the amount of marker substance on the membrane.

  When measuring the concentration of the marker substance by chromatography, for example, a method by liquid high performance chromatography (HPLC) can be used. That is, the concentration of the marker substance in the sample can be measured by subjecting the sample to HPLC to separate the target marker substance and measuring the peak area of the chromatogram.

  In a preferred embodiment, a marker substance is captured on a carrier, and the captured marker substance is a measurement target. That is, a substance having affinity for the marker substance is immobilized on the carrier, and the marker substance is captured on the carrier via the substance having the affinity. Then, the concentration of the marker substance in the body fluid is calculated based on the amount of the captured marker substance. According to this embodiment, it is possible to reduce the influence of contaminants contained in the sample, and to measure the concentration of the marker substance with higher sensitivity and higher accuracy. As an example of the carrier that can be used in the present embodiment, a carrier having a flat portion such as a substrate can be used in addition to a general one such as beads, metal, glass, resin, and the like. In the case of using a substrate, it is preferable to immobilize a substance having affinity for the marker substance on a part of the planar portion. As an example, there may be mentioned a carrier in which a chip is used as a substrate and a substance having affinity for a marker substance is spot-fixed at a plurality of spots on the surface. Examples of “affinity” include ion binding, affinity between metal chelate and histidine residue in protein, antigen and antibody, enzyme and substrate, bioaffinity such as hormone and receptor, and hydrophobicity. Chemical interactions such as interactions.

  When capturing the marker substance on the carrier by ionic bonding, the ion exchanger is immobilized on the carrier. In this case, either a cation exchanger or an anion exchanger can be used as the ion exchanger, and further, a strong cation exchanger, a weak cation exchanger, a strong anion exchanger, and a weak anion exchanger. Either of these can be used, but a strong anion exchanger and a weak cation exchanger are preferably used. Examples of strong anion exchangers include quaternary ammonium (trimethylaminomethyl) (QA), quaternary aminoethyl (diethyl, mono-2-hydroxybutylaminoethyl) (QAE), quaternary ammonium (trimethylammonium) ( And those having a strong anion exchange group such as QMA). Examples of weak cation exchangers include those having weak cation exchange groups such as carboxymethyl (CM). Examples of strong cation exchangers include those having a strong cation exchange group such as sulfopropyl (SP). Furthermore, examples of the weak anion exchanger include those having a weak anion exchange group such as dimethylaminoethyl (DE) and diethylaminoethyl (DEAE).

When capturing a marker substance via a metal chelate, for example, a metal chelate such as Cu ²⁺ , Zn ²⁺ , Ni ²⁺ , Co ²⁺ , Al ³⁺ , Fe ³⁺ , Ga ³⁺ is fixed. A fluorinated carrier can be used.

  When a marker substance is captured on a carrier by an antibody, an antibody specific for the marker substance may be immobilized on the carrier.

  When a marker substance is captured on a carrier by hydrophobic interaction, a substance having a hydrophobic group is immobilized on the carrier. Examples of hydrophobic groups include C4-C20 alkyl groups, phenyl groups, and the like.

  In this embodiment, when an immunoassay is used as a method for measuring a marker substance, it is preferable to use a carrier on which an antibody is immobilized. In this way, an immunoassay system using the antibody immobilized on the carrier as the primary antibody can be easily constructed. For example, two types of antibodies specific to a marker substance and having different epitopes are prepared, one is immobilized on a carrier as a primary antibody, and the other is enzyme-labeled as a secondary antibody to construct a sandwich EIA system. . In addition, immunoassay systems based on binding inhibition methods and competitive methods can be constructed. Further, when a substrate is used as a carrier, immunoassay using an antibody chip is possible. According to the antibody chip, the concentration of a plurality of marker substances can be measured simultaneously, and rapid measurement is possible.

  On the other hand, when mass spectrometry is used in the present embodiment, for example, in addition to antibodies, ion exchangers, metal chelates, or carriers on which hydrophobic groups are immobilized can be used. Since binding by these substances is not as specific as bioaffinity such as antigens and antibodies, when using a carrier on which these substances are immobilized, substances other than the marker substance can also be captured on the carrier. According to the analysis, there is no problem because it is quantified by the mass spectrometer spectrum reflecting the molecular weight. In particular, using a substrate as a carrier, surface-enhanced laser desorption / ionization-time-of-flight mass spectrometry (hereinafter referred to as "SELDI-TOF-MS") ), The concentration of the marker substance can be measured more accurately. As the types of substrates that can be used, cation exchange substrates, anion exchange substrates, normal phase substrates, reverse phase substrates, metal ion substrates, antibody substrates, etc. can be used, but cation exchange substrates, particularly weak cation exchanges. A substrate and an anion exchange substrate, particularly a strong anion exchange substrate are preferably used.

  The substance screening method of the present invention evaluates a test substance by the method for evaluating the relaxation / induction suppression effect of the in vivo tension state of the present invention, and screens for a substance having a relaxation effect or induction suppression effect of the in vivo tension state. It is. In the method for screening a substance of the present invention, the same embodiment as the above-described method for evaluating the effect of mitigating / inducing suppression of in vivo tension state of the present invention can be employed.

  In order to simply perform the method for evaluating the relaxation / induction suppression effect of the in vivo tension state of the present invention, an evaluation kit can be constructed by collecting necessary reagents. Examples of the evaluation kit include those containing a carrier on which a substance having affinity for a marker substance is immobilized. In particular, according to the evaluation kit including a substrate on which a weak cation exchanger such as CM or a strong anion exchanger such as QA or QAE is immobilized as a carrier, SELDI-TOF-MS or the like is easily performed. be able to. The kit may contain other reagents such as standard substances and various pretreatment buffers. In addition, this kit can be used also as a kit for screening the substance which has the relaxation effect or induction | guidance | derivation suppression effect of a tension state in a living body. Examples of the configuration of the kit of the present invention are given below.

[Example of kit configuration]
(1) Weak cation exchange substrate: 1 sheet (2) Strong anion exchange substrate: 1 sheet (3) Substrate cleaning buffer A (pH 3.0): appropriate amount (4) Substrate cleaning buffer B (pH 9.0) : Appropriate amount (5) Standard product of each marker substance:

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

1. Animal Breeding and Preparation of Body Fluid Samples C57BL / 6 mice (male, 6 weeks old) that had finished pre-breeding were divided into the following 4 groups that differed in the combination of the type of drinking water and whether or not restraint stress was applied. The number of individuals in each group was 8 or more.

Group 1: Normal drinking (1 week) + no restraint stress (17 hours)
Group 2: Normal drinking (1 week) + Restraint stress (17 hours)
Third group: Ketsumeishi extract drinking water (1 week) + restraint stress load (17 hours)
Group 4: Ketsumeishi extract drinking water (1 week) + no restraint stress load (17 hours)
That is, the first group corresponds to a group in which symptoms due to stress do not develop, the second group corresponds to a group in which symptoms due to stress develop, and the third group corresponds to a group in which symptoms due to stress are suppressed. The fourth group is a group for verifying the action of beetles.

  For each group, normal drinking (Group 1 and Group 2) or ketsumeishi extract drinking water (Group 3 and Group 4) was given for 1 week, followed by fasting and drinking for 17 hours. Only restrained stress load. Ingestion amount of group 3 and group 4 was 2 mg (0.1 g / kg / day) per animal per day. After 17 hours of restraint stress application or non-application, 8 mice each for groups 1 and 4 and 9 mice each for groups 2 and 3 were sacrificed. Blood was collected from each mouse and a plasma sample was prepared.

2. Protein chip analysis and ion peak selection To 20 μL of each collected plasma sample, 30 μL of denaturation buffer (9 M urea, 2% CHAPS, 50 mM Tris-HCl (pH 9.0)) is added and pretreated to denature the protein. It was. Next, each pretreated body fluid sample was applied to a strong anion exchange resin column (Q-Sepharose, GE Healthcare). Next, a pH 9.0 buffer solution (50 mM Tris-HCl (pH 9.0), 0.1% (w / v) 1-o-N-octyl-β-D-glucopyranoside (hereinafter referred to as “OGP”). )), PH 5.0 buffer (100 mM sodium acetate (pH 5.0), 0.1% (w / v) OGP), pH 3.0 buffer (50 mM sodium citrate (pH 3.0), 0. 1% (w / v) OGP) and organic solvent (mixed solution consisting of 0.1% trifluoroacetic acid, 50.0% acetonitrile) were eluted in order with 200 μL each, and fraction 1 (eluted at pH 9.0, passed) ), Fraction 2 (eluted at pH 5.0), fraction 3 (eluted at pH 3.0), and fraction 4 (eluted with an organic solvent).

  10 μL of each of the obtained fractions was diluted 10-fold with a pH 3.0 protein chip binding buffer (50 mM sodium citrate), and then added to a weak cation exchange chip CM10 (BioRad). Similarly, 10 μL of each obtained fraction was diluted 10-fold with a pH 9.0 protein chip binding buffer (50 mM Tris-HCl (pH 9.0)) and then added to a strong anion exchange chip Q10 (Bio-Rad). did. Each chip was washed 3 times with each binding buffer, then once with deionized water and dried. Next, sinapinic acid (SPA-H, SPA-L) or α-cyano-4-hydroxycinnamic acid (CHCA), which is an energy absorbing molecule, is added and a protein chip reader Model PBS IIc (Bio-Rad) is used. Then, SELDI-TOF-MS was performed. In addition, the measurement molecular weight range (m / z) was performed in the range of 3000-200000. Moreover, the measurement was performed in duplicate and the average value of m / z was calculated. Data analysis was performed using Protein Chip Software, Ciphergen Express Data Manager, and Biomarker Patterns Software (all from Bio-Rad). Specifically, after performing baseline correction, molecular weight calibration, and spectrum normalization processing, single marker analysis and multiflow analysis combining several markers were performed. As a result, a large number of peaks were detected depending on the combination of the type of crude fraction, the type of protein chip, the washing conditions of the chip, and the like. For each peak, a p-value (Mann-Whitney test method), ROC area, and ion peak intensity were calculated, and eight candidate peaks were selected using the following conditions (1) to (3) as indices.

(1) Candidate peak search (basic)
There is a significant difference in ionic strength between the first group and the second group (p <0.05).
(2) Ketsumeishi effect verification There is a significant difference in ionic strength between the second group and the third group (p <0.05 or 0.1), and the value of the third group is the value of the second group Is closer to the value of the first group (the value of the third group has returned to the first group side).
(3) Increase / decrease pattern analysis Only the second group shows a high value or a low value, and there is not much difference among the first group, the third group, and the fourth group. In addition, even if there is a difference between the third group and the fourth group, if the value of the fourth group is farther from the second group than the value of the third group, this condition is satisfied. handle.

3. Identification of marker substance (M1) Fraction 3 (pH 3.0) is brought into contact with a weak cation exchange chip CM10, washed with a protein chip binding buffer of pH 3.0, and SELDI-TOF-MS (EAM: SPA-H) , An ion peak with a mass / charge ratio of 6020 (average value) was detected. As a result of the increase / decrease pattern analysis, this peak showed a high value in the first group, the third group, and the fourth group, and a low value in the second group. FIG. 1 shows a box diagram when the peak intensity of each peak is plotted for each group. In the figure, the upper and lower edges of the bag are the maximum and minimum values, respectively, the upper and lower sides of the box are the third quartile (75th percentile) and the first quartile (25th percentile), respectively, and the line in the box is the center Value (the same applies to FIG. 2 and subsequent figures). Each ROC area and each p value were as follows.

ROC area (first group vs second group): 0.712
p value (first group vs second group): 0.0324
ROC area (second group vs. third group): 0.744
p value (second group vs. third group): 0.0086

  From the above, it was found that a protein (marker substance (M1)) that produces a peak with a mass / charge ratio of about 6020 when subjected to SELDI-TOF-MS can be a marker reflecting the in-vivo tension state. Thereby, using the concentration of the marker substance (M1) in the body fluid of the “animal administered with the test substance and stressed” as an index, evaluation of the relaxation effect or induction suppression effect of the in vivo tension state of the test substance, and It was shown that screening for substances having this effect can be performed. For example, when a body fluid sample is prepared by performing the same animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a protein that produces a peak with a mass / charge ratio of about 6020 When the concentration of is maintained at a normal value, the test substance can be evaluated as having a relaxing effect or an induction suppressing effect on the in vivo tension state.

4). Identification of marker substance (M2) Fraction 1 (pH 9.0) is contacted with strong anion exchange chip Q10, washed with a protein chip binding buffer of pH 9.0, and SELDI-TOF-MS (EAM: SPA-L) , An ion peak with a mass / charge ratio of 9312 (average value) was detected. As a result of the increase / decrease pattern analysis, this peak showed a high value in the first group, the third group, and the fourth group, and a low value in the second group. FIG. 2 shows a box diagram when the peak intensity of this peak is plotted for each group. Each ROC area and each p value were as follows.

ROC area (first group vs second group): 0.771
p value (first group vs second group): 0.0058
ROC area (second group vs. third group): 0.701
p value (second group vs. third group): 0.0462

  From the above, it was found that a protein (marker substance (M2)) that produces a peak with a mass / charge ratio of about 9310 when subjected to SELDI-TOF-MS can be a marker reflecting the in-vivo tension state. Thereby, using the concentration of the marker substance (M2) in the body fluid of the “animal to which the test substance is administered and stressed” as an index, the evaluation of the relaxation effect or induction suppression effect of the in vivo tension state of the test substance, and It was shown that screening for substances having this effect can be performed. For example, a protein that produces a peak with a mass / charge ratio of about 9310 when a body fluid sample is prepared by performing the same animal experiment using a desired test substance and SELDI-TOF-MS is performed according to the same procedure. When the concentration of is maintained at a normal value, the test substance can be evaluated as having a relaxing effect or an induction suppressing effect on the in vivo tension state.

5. Identification of marker substance (M3) Fraction 2 (pH 5.0) is brought into contact with weak cation exchange chip CM10, washed with a protein chip binding buffer of pH 3.0, and SELDI-TOF-MS (EAM: CHCA) is performed. In this case, an ion peak having a mass / charge ratio of 13664 (average value) was detected. As a result of the increase / decrease pattern analysis, this peak showed a low value in the first group, the third group, and the fourth group, and a high value in the second group. FIG. 3 shows a box diagram when the peak intensity of this peak is plotted for each group. Each ROC area and each p value were as follows.

ROC area (first group vs second group): 0.819
p value (first group vs second group): 0.0013
ROC area (second group vs. third group): 0.744
p value (second group vs. third group): 0.0072

  From the above, it was found that a protein (marker substance (M3)) that produces a peak with a mass / charge ratio of about 13700 when subjected to SELDI-TOF-MS can be a marker reflecting the in vivo tension state. Thereby, using the concentration of the marker substance (M3) in the body fluid of the “animal to which the test substance is administered and stressed” as an index, evaluation of the relaxation effect or induction suppression effect of the in vivo tension state of the test substance, and It was shown that screening for substances having this effect can be performed. For example, when a body fluid sample is prepared by performing a similar animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a protein that produces a peak with a mass / charge ratio of about 13700 When the concentration of is maintained at a normal value, the test substance can be evaluated as having a relaxing effect or an induction suppressing effect on the in vivo tension state.

6). Identification of marker substance (M4) Fraction 2 (pH 5.0) is brought into contact with a weak cation exchange chip CM10, washed with a protein chip binding buffer of pH 3.0, and SELDI-TOF-MS (EAM: CHCA) is performed. In this case, an ion peak having a mass / charge ratio of 13762 (average value) was detected. As a result of the increase / decrease pattern analysis, this peak showed a low value in the first group, the third group, and the fourth group, and a high value in the second group. FIG. 4 shows a box diagram when the peak intensity of this peak is plotted for each group. Each ROC area and each p value were as follows.

ROC area (first group vs second group): 0.778
p value (first group vs second group): 0.0058
ROC area (second group vs. third group): 0.701
p value (second group vs third group): 0.0290

  From the above, it was found that a protein (marker substance (M4)) that produces a peak with a mass / charge ratio of about 13800 when subjected to SELDI-TOF-MS can serve as a marker reflecting the in-vivo tension state. Thereby, using the concentration of the marker substance (M4) in the body fluid of the “animal to which the test substance is administered and stressed” as an index, evaluation of the relaxation effect or induction suppression effect of the in vivo tension state of the test substance, and It was shown that screening for substances having this effect can be performed. For example, a protein that produces a peak with a mass / charge ratio of about 13800 when a body fluid sample is prepared by performing the same animal experiment using the desired test substance and SELDI-TOF-MS is performed in the same procedure. When the concentration of is maintained at a normal value, the test substance can be evaluated as having a relaxing effect or an induction suppressing effect on the in vivo tension state.

7). Identification of marker substance (M5) Fraction 3 (pH 3.0) is contacted with a weak cation exchange chip CM10, washed with a protein chip binding buffer at pH 3.0, and SELDI-TOF-MS (EAM: SPA-H) , An ion peak with a mass / charge ratio of 17724 (average value) was detected. As a result of the increase / decrease pattern analysis, this peak showed a high value in the first group, the third group, and the fourth group, and a low value in the second group. FIG. 5 shows a box diagram when the peak intensity of this peak is plotted for each group. Each ROC area and each p value were as follows.

ROC area (first group vs second group): 0.792
p value (first group vs second group): 0.0052
ROC area (second group vs. third group): 0.701
p value (second group vs. third group): 0.0268

  From the above, it was found that when subjected to SELDI-TOF-MS, a protein (marker substance (M5)) that produces a peak with a mass / charge ratio of about 17700 can serve as a marker reflecting the in vivo tension state. Thereby, using the concentration of the marker substance (M5) in the body fluid of the “animal to which the test substance is administered and stressed” as an index, the evaluation of the relaxation effect or induction suppression effect of the in vivo tension state of the test substance, and It was shown that screening for substances having this effect can be performed. For example, when a body fluid sample is prepared by performing the same animal experiment using a desired test substance and SELDI-TOF-MS is performed by the same procedure, a protein that produces a peak with a mass / charge ratio of about 17700 When the concentration of is maintained at a normal value, the test substance can be evaluated as having a relaxing effect or an induction suppressing effect on the in vivo tension state.

8). Identification of marker substance (M6) Fraction 4 (organic solvent) is brought into contact with a weak cation exchange chip CM10, washed with a protein chip binding buffer of pH 3.0, and SELDI-TOF-MS (EAM: SPA-H) is obtained. When performed, an ion peak with a mass / charge ratio of 19632 (average value) was detected. As a result of the increase / decrease pattern analysis, this peak showed a high value in the first group, the third group, and the fourth group, and a low value in the second group. FIG. 6 shows a box diagram when the peak intensity of this peak is plotted for each group. Each ROC area and each p value were as follows.

ROC area (first group vs second group): 0.753
p value (first group vs second group): 0.0071
ROC area (second group vs. third group): 0.701
p value (second group vs third group): 0.0290

  From the above, it was found that a protein (marker substance (M6)) that produces a peak with a mass / charge ratio of about 19600 when subjected to SELDI-TOF-MS can serve as a marker reflecting the in vivo tension state. Thereby, using the concentration of the marker substance (M6) in the body fluid of the “animal to which the test substance is administered and stressed” as an index, evaluation of the relaxation effect or induction suppression effect of the in vivo tension state of the test substance, and It was shown that screening for substances having this effect can be performed. For example, when a body fluid sample is prepared by performing the same animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a protein that produces a peak with a mass / charge ratio of about 19600 When the concentration of is maintained at a normal value, the test substance can be evaluated as having a relaxing effect or an induction suppressing effect on the in vivo tension state.

9. Identification of marker substance (M7) Fraction 2 (pH 5.0) is brought into contact with a weak cation exchange chip CM10, washed with a protein chip binding buffer of pH 3.0, and SELDI-TOF-MS (EAM: SPA-H). , An ion peak with a mass / charge ratio of 39922 (average value) was detected. As a result of the increase / decrease pattern analysis, this peak showed a high value in the first group, the third group, and the fourth group, and a low value in the second group. FIG. 7 shows a box diagram when the peak intensity of this peak is plotted for each group. Each ROC area and each p value were as follows.

ROC area (first group vs second group): 0.736
p value (first group vs second group): 0.0228
ROC area (second group vs. third group): 0.667
p value (second group vs. third group): 0.0619

  From the above, it was found that a protein (marker substance (M7)) that produces a peak with a mass / charge ratio of about 39900 when subjected to SELDI-TOF-MS can be a marker reflecting the in vivo tension state. Thereby, using the concentration of the marker substance (M7) in the body fluid of the “animal to which the test substance is administered and stressed” as an index, the evaluation of the relaxation effect or induction suppression effect of the in vivo tension state of the test substance, and It was shown that screening for substances having this effect can be performed. For example, a protein that produces a peak with a mass / charge ratio of about 39,900 when a body fluid sample is prepared by performing a similar animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure. When the concentration of is maintained at a normal value, the test substance can be evaluated as having a relaxing effect or an induction suppressing effect on the in vivo tension state.

10. Identification of marker substance (M8) Fraction 1 (pH 9.0) is brought into contact with a weak cation exchange chip CM10, washed with a protein chip binding buffer of pH 3.0, and then SELDI-TOF-MS (EAM: SPA-H). , An ion peak with a mass / charge ratio of 76959 (average value) was detected. As a result of the increase / decrease pattern analysis, this peak showed a high value in the first group, the third group, and the fourth group, and a low value in the second group. FIG. 8 shows a box diagram when the peak intensity of this peak is plotted for each group. Each ROC area and each p value were as follows.

ROC area (first group vs second group): 0.795
p value (first group vs second group): 0.0034
ROC area (second group vs. third group): 0.657
p value (second group vs. third group): 0.0665

  From the above, it was found that a protein (marker substance (M8)) that produces a peak having a mass / charge ratio of about 77000 when subjected to SELDI-TOF-MS can serve as a marker reflecting the in vivo tension state. Thereby, using the concentration of the marker substance (M8) in the body fluid of the “animal to which the test substance is administered and stressed” as an index, evaluation of the relaxation effect or induction suppression effect of the in vivo tension state of the test substance, and It was shown that screening for substances having this effect can be performed. For example, a protein that produces a peak with a mass / charge ratio of about 77000 when a body fluid sample is prepared by performing the same animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure. When the concentration of is maintained at a normal value, the test substance can be evaluated as having a relaxing effect or an induction suppressing effect on the in vivo tension state.

  In addition, when a protein equivalent to the marker substances (M1) to (M8) is also present in human body fluid, there is a possibility that the tension state in the living body can be diagnosed using the concentration of the protein in the human body fluid as an index. Also suggested.

Purification and identification of marker substance (M1) A column (1 mL) packed with strong anion exchange resin Q-Sepharose HP (GE Healthcare) was equilibrated with buffer (50 mM Tris-HCl (pH 9.0), 1 M urea, 0). .22% CHAPS). Denaturation buffer (50 mM Tris-HCl (pH 9.0), 9 M urea, 2% CHAPS) 750 μL was added to 500 μL of ICR mouse heparin plasma (Shimizu Experimental Materials Co., Ltd.) and then added to the equilibrated column. did. After sequentially washing with 2.5 column volumes (CV) of 50 mM Tris-HCl (pH 9.0), 5 CV of 100 mM sodium acetate buffer (pH 5.0), and 5 CV of 50 mM sodium citrate buffer (pH 3.0), Elute with 5 CV 33.3% isopropanol / 16.7 acetonitrile / 0.1% TFA. SELDI-TOF-MS analysis was performed on the collected elution fractions, and a peak showing a mass / charge ratio equivalent to that of the marker substance (M1) was confirmed.

  A weak cation exchange column HiTrap CM FF 1 mL (GE Healthcare) was equilibrated with 50 mM sodium acetate buffer (pH 5.0), and the elution fraction was added thereto. The column was washed with 5 CV of 50 mM sodium acetate buffer (pH 5.0), and eluted with 5 CV of 50 mM sodium acetate buffer (pH 5.0) / 100 mM NaCl to collect the peak fraction. SELDI-TOF-MS analysis was performed on the collected fraction, and a peak showing a mass / charge ratio equivalent to that of the marker substance (M1) was confirmed.

  A part of the collected fraction was subjected to acetone precipitation, and then subjected to SDS-PAGE using a polypeptide separating gel (15-20%) NTH-5A2T gel (DRC) (FIG. 9). In FIG. 9, lanes 1 to 3 are all recovered fractions, and M is a molecular weight marker. The separated band of about 6.0 kDa (arrow in FIG. 9) was cut out, protein was extracted, SELDI-TOF-MS analysis was performed, and a peak showing a mass / charge ratio equivalent to that of the marker substance (M1) was confirmed. (Arrows in FIG. 10).

  The same SDS-PAGE was performed again to cut out a band of about 6.0 kDa, and after reduction alkylation treatment, 0.01 μg / μL trypsin solution (dissolved in 50 mM ammonium bicarbonate (pH 8.0)) was allowed to act. Digested in the gel. 1 μL of the digested sample was dropped on a metal plate, 0.4 μL of saturated CHCA solution was further dropped and dried, and then measured with a mass spectrometer Proteomics Analyzer 4700 (Applied Biosystems). At least two peaks Were detected and their exact masses were calculated as “1850.84” and “1981.99”. Based on these data, a known protein was searched by Mascot database (Matrix Science), and peptide mass fingerprinting was performed. These peptides were consistent with peptides having amino acid sequences represented by SEQ ID NOs: 8 and 9, respectively. The target protein was identified as “serum albumin” with a probability of 99% or more. Table 1 shows the correspondence between the exact mass, amino acid sequence, and SEQ ID NO of each peptide. The amino acid sequence of previously reported serum albumin is shown in SEQ ID NO: 1. Furthermore, the predicted isoelectric point of the protein fragment (SEQ ID NO: 5) corresponding to 55 amino acids on the N-terminal side of serum albumin (SEQ ID NO: 1) is 5.65, the molecular weight is 6019, and these values are marker substances ( It was in good agreement with the physical properties of M1). Thus, the marker substance (M1) was finally identified as a serum albumin fragment (SEQ ID NO: 5).

Purification and identification of marker substance (M2) Spin column (1 mL) packed with strong anion exchange resin Q-Ceramic Hyper DF (Nippon Pole) was equilibrated with buffer (50 mM Tris-HCl (pH 9.0), 1 M urea, 0.22% CHAPS). Denaturation buffer (50 mM Tris-HCl (pH 9.0), 9 M urea, 2% CHAPS) was added to 450 μL of mouse standard plasma (Pel-Freez) to 300 μL for denaturation treatment and added to the equilibrated column. The flow-through fraction was collected. The collected fraction was subjected to SELDI-TOF-MS analysis, and a peak showing a mass / charge ratio equivalent to that of the marker substance (M2) was confirmed.

  A portion of the collected fraction was subjected to acetone precipitation treatment, and then two-dimensional electrophoresis (first dimension: Immobiline DryStrip pH6-11, 7 cm (GE Healthcare)), second dimension: polypeptide separation gel (15-20) %) NTH-5A2T (DRC)) (FIG. 11). A spot (mass: about 9.3 kDa, isoelectric point: about 5.8) indicated by an arrow is cut out, protein is extracted, SELDI-TOF-MS analysis is performed, and mass / charge equivalent to that of the marker substance (M2) A peak indicating the ratio was confirmed (arrow in FIG. 12).

  The same two-dimensional electrophoresis was performed again to cut out the same spot, and in-gel digestion and mass spectrometry were performed in the same manner as in Example 2. As a result, at least three peaks were detected. .66 "," 165.79 ", and" 18331.97 ". Based on these data, peptide mass fingerprinting was performed in the same manner as in Example 2. As a result, these peptides coincided with peptides having the amino acid sequences represented by SEQ ID NOs: 10 to 12, respectively, and the target protein was 99% or more. Was identified as “apolipoprotein A2”. Table 2 shows the correspondence between the exact mass, amino acid sequence, and SEQ ID NO of each peptide. The amino acid sequence of the previously reported apolipoprotein A2 is shown in SEQ ID NO: 7.

  The mature form of apolipoprotein A2 (SEQ ID NO: 7, molecular weight: 8736, isoelectric point: 4.94) is cleaved from the propeptide (ALVKR) consisting of 5 amino acids on the N-terminal side of proapolipoprotein A2 (SEQ ID NO: 2). Generated. It is also known that Lys at the C-terminus of proapolipoprotein A2 can be cleaved (Yamagishi et al., Nucleic Acids Research, vol. 14, No. 14, 1986). The predicted isoelectric point of the protein fragment (SEQ ID NO: 6; modified product of proapolipoprotein A2) from which the C-terminal Lys of proapolipoprotein A2 has been dropped is 5.76, the molecular weight is 9304, and these values are markers This was in good agreement with the physical properties of the substance (M2). Thereby, the marker substance (M2) was finally identified as a fragment of the proapolipoprotein A2 (SEQ ID NO: 6).

Purification and identification of marker substances (M3) and (M4) Strong anion exchange column HiTrap Q HP 1 mL (GE Healthcare) was equilibrated with buffer (50 mM Tris-HCl (pH 9.0), 1 M urea, 0.22%) CHAPS). Denaturation treatment (1.5 mM of denaturation buffer (50 mM Tris-HCl (pH 9.0), 9 M urea, 2% CHAPS)) was added to 1 mL of mouse standard plasma (Pel-Freez) and added to the equilibrated column. did. After sequentially washing with 50 mM Tris-HCl (pH 9.0) and 50 mM Tris-HCl (pH 7.0), elution was performed with 50 mM Tris-HCl (pH 7.0) / 50 mM NaCl. SELDI-TOF-MS analysis was performed on the eluted fraction, and each peak showing a mass / charge ratio equivalent to that of the marker substances (M3) and (M4) was confirmed.

  A part of the collected fraction was subjected to acetone precipitation, and then subjected to SDS-PAGE using a polypeptide separating gel (15-20%) NTH-5AOT gel (DRC) (FIG. 13). In FIG. 13, lanes 1 to 6 are all collected fractions, and M is a molecular weight marker. The separated band of about 13.8 kDa (arrow in FIG. 13) is cut out, protein is extracted, SELDI-TOF-MS analysis is performed, and a peak showing a mass / charge ratio equivalent to that of the marker substance (M3) (FIG. 14). And a peak (arrow b in FIG. 14) showing a mass / charge ratio equivalent to that of the marker substance (M4).

  The same SDS-PAGE was performed again to cut out a band of about 13.8 kDa, and when digestion in the gel and mass spectrometry were performed in the same manner as in Example 2, at least 8 peaks were detected. “1510.75”, “1382.65”, “15887.79”, “2438.25”, “869.46”, “155.85”, “2517.28”, and “2597.38” are calculated. It was done. Based on these data, peptide mass fingerprinting was performed in the same manner as in Example 2. As a result, these peptides coincided with the peptides of the amino acid sequences represented by SEQ ID NOs: 13 to 20, respectively, and the target protein was 99% or more. Was identified as “transthyretin”. Table 3 shows the correspondence between the exact mass, amino acid sequence, and SEQ ID NO of each peptide. The amino acid sequence of the previously reported transthyretin is shown in SEQ ID NO: 3.

  As described above, various modified transthyretins in which only one Cys residue is modified have been found for transthyretin. From the charge / mass ratio, it was considered that the marker substance (M3) was unmodified transthyretin and the marker substance (M4) was S-sulfonated transthyretin.

Purification and identification of marker substance (M8) A column (80 μL) packed with strong anion exchange resin Q-Sepharose HP (GE Healthcare) was equilibrated with buffer (50 mM Tris-HCl (pH 9.0), 1M urea, 0). .22% CHAPS). Denaturation buffer (50 mM Tris-HCl (pH 9.0), 9M urea, 2% CHAPS) 60 μL was added to 40 μL of mouse standard plasma (Pel-Freez) and then added to the equilibrated column. . The flow-through fraction was collected. SELDI-TOF-MS analysis was performed on the collected elution fractions, and a peak showing a mass / charge ratio equivalent to that of the marker substance (M8) was confirmed.

  A portion of the eluted fraction was subjected to acetone precipitation, and then subjected to SDS-PAGE using a 7.5% polyacrylamide gel (FIG. 15). In FIG. 15, lanes 1 and 2 are both elution fractions, and M is a molecular weight marker. The isolated band of about 77 kDa (arrow in FIG. 15) was cut out, protein was extracted, and SELDI-TOF-MS analysis was performed to confirm a peak showing a mass / charge ratio equivalent to that of the marker substance (M1) (FIG. 16 arrows).

  The same SDS-PAGE was performed again to cut out a band of about 77 kDa, and in-gel digestion and mass spectrometry were performed in the same manner as in Example 2. As a result, at least 8 peaks were detected. .48 "," 951.53 "," 1171.58 "," 1419.79 "," 1577.92 "," 1639.78 "," 2008.00 ", and" 2741.22 " . Based on these data, peptide mass fingerprinting was carried out in the same manner as in Example 2. As a result, these peptides coincided with the peptides having the amino acid sequences represented by SEQ ID NOs: 21 to 28, respectively, and the target protein was 99% or more. Was identified as “transferrin”. Table 4 shows the correspondence between the exact mass, amino acid sequence, and SEQ ID NO of each peptide. The amino acid sequence of the previously reported transthyretin is shown in SEQ ID NO: 4.

It is a box diagram about the ion peak whose mass / charge ratio is 6020 (average value). It is a box diagram about the ion peak whose mass / charge ratio is 9312 (average value). It is a box diagram about the ion peak whose mass / charge ratio is 13664 (average value). It is a box diagram about the ion peak whose mass / charge ratio is 13762 (average value). It is a box diagram about an ion peak whose mass / charge ratio is 17724 (average value). It is a box diagram about the ion peak whose mass / charge ratio is 19632 (average value). It is a box diagram about an ion peak whose mass / charge ratio is 39922 (average value). It is a box diagram about the ion peak whose mass / charge ratio is 76959 (average value). 6 is a photograph showing the results of SDS-PAGE performed in Example 2. FIG. 4 is an ion peak diagram showing the results of SELDI-TOF-MS analysis performed in Example 2. FIG. 6 is a photograph showing the results of SDS-PAGE performed in Example 3. FIG. 6 is an ion peak diagram showing the results of SELDI-TOF-MS analysis performed in Example 3. FIG. It is a photograph which shows the result of SDS-PAGE performed in Example 4. It is an ion peak figure which shows the result of the SELDI-TOF-MS analysis performed in Example 4. 6 is a photograph showing the results of SDS-PAGE performed in Example 5. FIG. It is an ion peak figure which shows the result of the SELDI-TOF-MS analysis performed in Example 6.

Claims (13)

By comparing the concentration of the following marker substance (M1) in a body fluid collected from an animal that has been administered a test substance and stressed in the living body with a reference value, and that the concentration is higher, the test A method for evaluating a relaxation / induction suppression effect of an in vivo tension state, wherein the relaxation effect or induction suppression effect of the in vivo tension state of a substance is evaluated.
(M1) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 6020 when subjected to mass spectrometry;
further,
The marker substance (M1) includes the amino acid sequence represented by SEQ ID NO: 8 or 9. By comparing the concentration of the following marker substance (M1) in a body fluid collected from an animal that has been administered a test substance and stressed in the living body with a reference value, and that the concentration is higher, the test A method for evaluating a relaxation / induction suppression effect of an in vivo tension state, wherein the relaxation effect or induction suppression effect of the in vivo tension state of a substance is evaluated.
(M1) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 6020 when subjected to mass spectrometry;
further,
The marker substance (M1) is serum albumin or a modified form thereof. By comparing the concentration of the following marker substance (A1) in a body fluid collected from an animal that has been administered a test substance with stress in a living body with a reference value, and that the concentration shows a higher value, the test A method for evaluating a relaxation / induction suppression effect of an in vivo tension state, wherein the relaxation effect or induction suppression effect of the in vivo tension state of a substance is evaluated.
(A1) Serum albumin or a modified product thereof. Furthermore, the concentration of the following marker substance (M3) or (M4) is compared with a reference value, The evaluation of the relaxation / induction suppression effect of in vivo tension state according to any one of claims 1 to 3 Method.
(M3) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 13700 when subjected to mass spectrometry;
(M4) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 13800 when subjected to mass spectrometry;
further,
The marker substances (M3) and (M4) are transthyretin or a modified form thereof. Furthermore, the evaluation method of the relaxation / induction suppression effect of the in vivo tension state according to any one of claims 1 to 3, wherein the concentration of the marker substance (A3) below is compared with a reference value.
(A3) Transthyretin or a modified product thereof.   The said animal is an animal which gave the stress in a living body after administering a test substance, The evaluation method of the relaxation / induction | guidance | derivation suppression effect of the in vivo tension state of any one of Claims 1-5 characterized by the above-mentioned. .   The reference value is a concentration of the marker substance in a body fluid of an animal to which a known substance having no relaxation effect or induction suppression effect of in vivo tension is administered and stressed in the living body. Item 7. The method for evaluating the relaxation / induction suppression effect of the in vivo tension state according to any one of Items 1-6.   The said body fluid is blood, The evaluation method of the relaxation / induction | guidance | derivation suppression effect of the in-vivo tension state of any one of Claims 1-7 characterized by the above-mentioned.   The said test substance is a foodstuff raw material or a pharmaceutical raw material, The evaluation method of the relaxation / induction | guidance | derivation suppression effect of the in-vivo tension state of any one of Claims 1-8 characterized by the above-mentioned.   The bodily fluid or bodily fluid component is brought into contact with a carrier on which a substance having affinity for the marker substance is immobilized, the marker substance in the bodily fluid is captured on the carrier, and based on the amount of the captured marker substance The method for evaluating the effect of mitigating / suppressing induction of an in vivo tension state according to any one of claims 1 to 9, wherein the concentration of the marker substance in body fluid is calculated.   The in-vivo strained condition according to claim 10, wherein the carrier has a planar portion, and the substance having affinity for the marker substance is immobilized on a part of the planar portion. Evaluation method of induction suppression effect.   The method for evaluating the effect of mitigating / inducing in vivo tension state according to claim 10 or 11, wherein the substance having affinity for the marker substance is an ion exchanger or an antibody.   The test substance is evaluated by the method for evaluating the relaxation / induction suppression effect of the in vivo tension state according to any one of claims 1 to 12, and a substance having a relaxation effect or induction suppression effect on the in vivo tension state is screened. A screening method for a substance characterized by the above.