JP2007232574A - Screening method of sympathetic nerve activator - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of retrieving a matter for activating the peripheral sympathetic nervous system without promoting the secretion of adrenalin and noradrenalin from the adrenal medulla. <P>SOLUTION: A specimen having activity for promoting or adjusting the activation of VRI is prepared, the specimen is perorally administered to an inspected animal, and a material that does not promote the secretion of adrenalin and noradrenalin originated in the adrenal is selected, thereby screening the sympathetic nervous system activator of peripheral tissue. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for screening a sympathetic nerve activator having a novel mechanism of action.

  With the recent health boom, the needs for diet foods are increasing day by day. Diet foods include those that suppress calorie absorption such as lipase inhibition and those that promote energy consumption through sympathetic activation. Among them, diet foods that promote energy consumption include substances that promote adrenaline secretion and act on the cardiovascular system, such as ephedra, and their sale is prohibited due to side effects such as sudden death and arrhythmia. There is a concern about the danger of activating the sympathetic nervous system uniformly (Non-patent Document 1).

  On the other hand, chili peppers have long been known to contain compounds having a sympathetic nervous system activation effect. Among them, capsaicin is well known as a main component of pungent taste, and is also known to have analgesic action and anti-inflammatory action. In 1997, vanilloid receptor subtype 1 (hereinafter referred to as “VR1”) was cloned as a receptor on which capsaicin acts (Non-patent Document 2). Capsaicin activates peripheral nociceptors by binding to VR1 and activates the sympathetic nervous system, and thus has been applied to diet foods for the purpose of suppressing body fat accumulation. However, the capsaicin-activated organ contains the adrenal medulla and stimulates adrenal secretion from the adrenal medulla, and capsaicin is absorbed into the body after oral ingestion and non-specific nociceptors distributed in the periphery Since it can be activated and affect blood pressure and heart rate, side effects are a concern for ingestion in humans with obesity who are at risk for heart disease.

  Similar to capsaicin, capsiate (4-hydroxy-3-methoxybenzyl (E) -8-methyl-6-nonenoate) and dihydrocapsiate (4-hydroxy-3-methoxybenzyl 8-methylnonanoate) contained in peppers that are not spicy Capsinoids such as are also known. Capsinoids, which are also called capsaicinoid-like substances, have recently attracted attention as diet ingredients that have a similar structure to capsaicin having a pungent taste but have a VR1 binding ability and do not exhibit a pungent taste and have a metabolic enhancing action, similar to capsaicin. (Patent Document 1). However, the mechanism of action of capsinoids is rapidly hydrolyzed in blood and in aqueous solutions at neutral pH, so there are still many unclear points about where they are received and how they increase metabolism after ingestion. .

It is also known that administration of capsinoid in blood promotes adrenal secretion in the adrenal gland (Non-patent Document 3). However, it is not known that when the capsinoid is orally administered, the peripheral sympathetic nervous system is activated without promoting the secretion of adrenaline and noradrenaline from the adrenal medulla.
JP-A-11-246478 Shekelle PG, et al., JAMA. 2003 Mar 26; 289 (12): 1537-45 Michael J. Caterina, et al., Nature, 1997; 389, 816-824 kazuo Iwai, et al., Proc. Japan Acad. 2003; 79 (7), 207-212.

An object of the present invention is to provide a method for searching for a substance that activates the peripheral sympathetic nervous system without promoting secretion of adrenaline and noradrenaline from the adrenal medulla.

  The present inventors compared the physiological effects of capsaicinoid, which is a spicy capsicum component known to have an energy consumption promoting action, and capsinoid having a structure similar to that of capsaicin but having no pungency. However, among VR1 receptor stimuli that mediate sympathetic nerve activation, capsinoids do not promote adrenal secretion from adrenal medulla and promote noradrenaline metabolism from peripheral sympathetic nerve endings in peripheral tissues such as muscles. I found out. That is, among the compounds having the same VR1 receptor stimulating activity, the presence of a compound having specific activation activity in the peripheral sympathetic nervous system that has not been confirmed so far has been confirmed, and such a compound is used. Thus, it was found that activation stimulation specific to the peripheral sympathetic nervous system governing peripheral tissues such as muscle is possible without using adrenaline derived from adrenal medulla. Furthermore, by selecting a compound that is difficult to be absorbed in the gastrointestinal tract, the VR1 receptor distributed in the gastrointestinal tract is specifically stimulated without non-specific stimulation of nociceptors in the living body, and the above-mentioned characteristics are achieved. It was also found that it can be retained. That is, the present invention relating to a method for screening a safe and effective sympathetic nerve activator having a novel mechanism of action was completed by applying the knowledge on the physiological action mechanism of such capsinoids.

That is, the present invention is as follows.
(1) A method of screening a peripheral tissue sympathetic nervous system activator,
A test substance having an activity of promoting or regulating VR1 activation is prepared, the test substance is orally administered to a test animal, and a substance that does not promote secretion of adrenal and noradrenaline derived from the adrenal gland is selected. how to.
(2) The method as described above, further comprising the step of contacting the selected substance with a digestive tract model system to select a compound having low absorbability from the digestive tract.
(3) The method as described above, wherein the substance that does not promote secretion of adrenaline and noradrenaline is selected by selecting a substance that promotes secretion of 3-methoxy-4-hydroxyphenylglycol to peripheral blood.
(4) Selection of a substance that promotes secretion of 3-methoxy-4-hydroxyphenyl glycol into the peripheral blood,
The test substance having an activity of promoting or regulating the activation of VR1 is administered to a vagus nerve-cutting animal and a normal animal, and the vagus nerve-cutting animal is more sensitive to peripheral blood than normal animals. The method as described above, which is carried out by selecting a substance having little secretion of hydroxyphenyl glycol.
(5) Preparation of a test substance having an activity of promoting or regulating the VR1 activation is conducted by contacting a candidate substance or a candidate substance with a VR1 agonist in a cell line expressing VR1, and measuring VR1 activation. And selecting the substance having the activity of promoting or regulating the activation of VR1.
(6) The method as described above, wherein the digestive tract model system is selected from the group consisting of a digestive tract-derived cell line, an animal-derived digestive tract specimen, and an animal-derived digestive tract lumen.

  According to the present invention, it is possible to search for a substance that activates the peripheral sympathetic nervous system without promoting the secretion of adrenaline and noradrenaline from the adrenal medulla. In addition, a promising diet food can be provided by the present invention.

The method of the present invention is a method for screening a sympathetic nervous system activator in peripheral tissues, comprising preparing a test substance having an activity of promoting or regulating the activation of VR1, and orally administering the test substance to a test animal. Administration, and screening for substances that do not promote secretion of adrenal and noradrenaline derived from the adrenal gland. The peripheral tissue sympathetic nervous system activator which is a screening target of the present invention is a substance having the activity of promoting or regulating the activation of VR1 and, when orally administered, secretes adrenal and noradrenaline derived from the adrenal gland. It is a substance that does not promote.

VR1 is also called a capsaicin receptor or Transient Receptor Potential Vanilloid Receptor 1 (TRPV1).
For the test substance having the activity of promoting or regulating the activation of VR1, for example, a candidate substance is brought into contact with a cell line expressing VR1, the activation of VR1 is measured, and the substance that activates VR1 is selected. Can be obtained.

  The cell lines expressing VR1 are, for example, various cell lines such as vectors containing the gene encoding VR1, such as Xenopus oocytes, Chinese hamster ovary cells (CHO), baby hamster kidney (BHK) cells, human It can be obtained by transforming embryonic kidney (HEK) cells, Sf-9 insect cells, PC12 cells, CACO-2 cells, etc. (Non-patent Document 2). In addition, when DNA encoding VR1 is incorporated into chromosomal DNA and VR1 is expressed permanently, cells other than Xenopus oocytes can be used. A known method can be used as a method for introducing DNA encoding VR1 into these cells. Techniques necessary for operations such as introduction of DNA into cells are described in Sambrook, J., Fritsch, EF, and Maniatis, T., "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, (1989) Etc. are described.

  As long as VR1 accepts capsaicin or capsinoid, and induces a current change such as calcium and sodium and a change in membrane potential, mammals such as humans, monkeys, rats, mice, dogs, cows, rabbits, birds, It may be a protein derived from fish or any other animal, and may be a variant thereof. The amino acid sequence of VR1 is registered in GenBank as Accetion no: CAB89866 (human), NP_058903 (rat). The base sequence of the gene encoding VR1 is registered in GenBank as Accetion no: AJ272063 (human) and NM_017207 (rat). The nucleotide sequence encoding human VR1 is shown in SEQ ID NO: 1, and the amino acid sequence of VR1 is shown in SEQ ID NO: 2.

  VR1 may be a protein containing one or more amino acid deletions, substitutions, insertions or additions at one or more positions, so long as its activity is not lost. The number of “plurality” amino acids depends on the type of amino acid or the position of amino acid residues in the three-dimensional structure of the protein, but the number of “plurality” amino acids is 2-50, preferably 2-25, more preferably 2 to 10, particularly preferably 2 to 5. A DNA encoding a protein substantially the same as VR1 can be obtained, for example, by modifying a nucleotide sequence encoding VR1, for example, SEQ ID NO: 1. Modification of the amino acid sequence can use, for example, site-directed mutagenesis, so that deletion, substitution, insertion or addition of amino acid residue (s) is performed at a specific site. Such modified DNA can be obtained by conventional methods, for example by treatment with mutation-generating reagents or other conditions. Such methods include treatment of DNA encoding VR1 with hydroxyamine, or UV irradiation, or bacteria that retain the DNA with reagents such as N-methyl-N′-nitro-N-nitrosoguanidine or nitrous acid. Can be mentioned.

The DNA encoding VR1 may be a homologue of a non-human animal. Such a VR1 homolog can be obtained by isolating a DNA that hybridizes with SEQ ID NO: 1 or a partial nucleotide sequence thereof under stringent conditions and encodes a protein having VR1 activity. The term “stringent conditions” encompasses conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, as stringent conditions, highly homologous DNAs, for example, 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more, are hybridized to each other. The conditions to do are mentioned. Alternatively, stringent conditions include normal washing conditions for Southern hybridization, such as conditions containing 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS. Can be mentioned. A partial sequence of the nucleotide sequence of SEQ ID NO: 1 can also be used as a probe. Such a probe can be prepared by PCR using an oligonucleotide prepared based on the nucleotide sequence of SEQ ID NO: 1 as a primer and a DNA fragment containing the nucleotide sequence of SEQ ID NO: 1 as a template. When a DNA fragment having a length of about 300 bp is used as a probe, hybridization washing conditions are, for example, 50 ° C., 2 × SSC, and 0.1% SDS.

  A test substance having an activity of promoting or regulating the activation of VR1 is obtained by bringing the candidate substance into contact with a cell line that expresses VR1 as described above, measuring the activation of VR1, and adding a substance that activates VR1. It can be obtained by selecting. In the present invention, the “candidate substance” is a candidate for obtaining a substance having an activity for promoting or regulating the activation of VR1 as described above, and the “test substance” is for promoting the activation of VR1 or It is a substance that has been confirmed to have a modulating activity.

  Candidate substances may be any known compounds and novel compounds. For example, nucleic acid, carbohydrate, lipid, protein, peptide, organic low molecular weight compound, compound library prepared using combinatorial chemistry technology, solid phase synthesis And random peptide libraries prepared by the phage display method, or natural components derived from microorganisms, animals and plants, marine organisms, and the like. The candidate substance may be a single substance or a mixture of a plurality of substances.

  The substance that activates VR1 is a VR1 agonist or modulator that is a VR1 activity promoter, and the modulator includes a substance that functions to expand the agonist activity of VR1. In the present invention, the test substance having the activity of promoting or regulating the activation of VR1 may be a VR1 agonist or a modulator of VR. The activation of VR1 can be measured, for example, by contacting a candidate substance with VR1 and measuring a change in membrane potential caused by binding of the candidate substance to VR1. In addition, VR1 is contacted with a candidate substance and a VR1 agonist, or VR1 is combined with a VR1 agonist and a candidate substance, and the membrane potential generated by binding of VR1 agonist to VR1 is measured. The activation of VR1 can also be measured by measuring. Here, the VR1 agonist includes a VR1 ligand.

  Alternatively, instead of detecting a second messenger, a labeled VR1 agonist is used, and the binding between the labeled agonist and VR1 is measured to detect the inhibition of the binding by the candidate substance, thereby activating VR1. Can be obtained.

Examples of VR1 agonists include capsaicin, olbanil, and capsinoid. Examples of capsinoids include capsiate, dihydrocapsiate, nordihydrocapsiate, and various straight chain chains having the same fatty acid chain length as capsiate and nordihydrocapsiate, such as vanillyl decanoate, vanillyl nonanoate, and vanillyl octanoate. Examples include fatty acid esters of chain or branched chain fatty acids and vanillyl alcohol. Capsiate (4-hydoxy-3-methoxybenzyl (E) -8-methyl-6-nonenoate; hereinafter abbreviated as “CST”), dihydrocapsiate (4-hydroxy-3-methoxybenzyl 8-methylnonanoate. "DCT" may be abbreviated), nordihydrocapsiate (4-hydroxy-3-meet)
hoxybenzyl 7-methyl-octanoate. Hereinafter, “NDCT” may be abbreviated as follows.

Capsinoids are abundant in plants belonging to the genus Capsicum, and therefore are prepared by purifying and separating from plants and / or fruits of the plant belonging to the genus Capsicum (hereinafter referred to as “capsicum”). Can do. Capsicum used for purification may be derived from capsicum varieties having capsioids, such as `` Nikko '', `` Five colors '', etc., but any kind of capsicum can be used. Can also be used. Among them, the traditional spicy varieties of chili pepper typified by “CH-19 Amane”, “Manganji”, “Fushimi Amami”, Shishito, green pepper, etc. contain many capsinoids and should be used appropriately. Can do. Furthermore, it is particularly preferable to use “CH-19 sweet” which is a spicy-tasting variety because of the high content of the component. Here, the term “CH-19 sweet” includes “CH-19 sweet” and progeny related varieties derived from “CH-19 sweet”. Is used to mean all of these.

“CH-19 Sweet” is a non-pungency fixed variety of red pepper selected and fixed by Yazawa et al. As a low-pungency red pepper. Capsaicinoid compounds (capsaicin, dihydrocapsaicin, etc.) that have the pungency and invasiveness of common red peppers ) (Hereinafter sometimes simply referred to as “capsaicinoid”) is hardly contained, and is therefore particularly preferable as a raw material for an agent for improving dysphagia for oral intake. “CH-19 sweet” is also preferable from the viewpoint of containing a large amount of CST, ECT and NDCT (Japanese Patent Laid-Open No. 11-246478).
In addition, it has been confirmed that capsinoids are also present in other plants belonging to the genus Capsicum (Horticulture Society Journal 58, 601-607).

  For purification and separation of capsinoids from plants belonging to the genus Capsicum, various methods such as solvent extraction, silica gel chromatography, and high-performance liquid chromatography for preparation are used singly or as appropriate. It can be done by combining. Specifically, for example, capsicum plants can be extracted with ethanol under acidic conditions, adsorbed on an adsorbent, eluted with ethanol containing acidic substances, and separated and purified through vacuum concentration (Japanese Patent Application Laid-Open No. 2004-18428). Issue gazette). Specifically, for example, dried capsicum fruits such as “CH-19 sweet”, extracted using various organic solvents such as ethyl acetate and hexane, and molecular distillation of the resulting extracted oil, capsinoids were obtained. A containing fraction can be obtained.

  Also, for example, as described in JP-A No. 11-246478, it can be synthesized by a transesterification reaction using a corresponding fatty acid ester and vanillyl alcohol as starting materials. Alternatively, it can be synthesized based on the structural formula by other reaction methods well known to those skilled in the art. Furthermore, capsinoids can also be easily prepared by a synthetic method using an enzyme. That is, for example, according to the methods described in JP-A No. 2000-31598 and Kobata et al. (Biosci. Biotechnol. Biochem., 66 (2), 319-327, 2002), esters of fatty acids corresponding to the desired compound and / or Alternatively, a desired capsinoid compound can be obtained by utilizing a reverse reaction of lipase using a compound such as triglyceride having the fatty acid and vanillyl alcohol as a substrate. Further, the quantitative analysis of capsinoid can be appropriately performed using high performance liquid chromatography (HPLC). Various conditions for HPLC can be easily set by those skilled in the art, but for example, it can be carried out by the method described in JP-A-2004-18428.

A test substance having the activity of promoting or regulating the activation of VR1 obtained as described above is orally administered to a test animal, and a substance that does not promote secretion of adrenal and noradrenaline derived from the adrenal gland is selected. The target substance can be obtained, for example, by measuring the concentrations of adrenaline and noradrenaline in the blood of an animal to which the test substance has been administered, and selecting substances that do not change in concentration before and after administration. Specifically, for example, a catheter is placed in advance in the vein of an animal, a test substance is orally administered in an unanesthetized and unrestrained state, blood is collected before and after administration, and is collected in serum or plasma. The concentration of catecholamine is measured using high performance liquid chromatography. Examples of test animals include monkeys, rats, mice, dogs, cows, rabbits, and the like.

  In addition, selection of substances that do not promote the secretion of adrenaline and noradrenaline is performed by measuring the concentration of 3-methyl-4-hydroxyphenylglycol (MHPG) in the blood of animals administered with the test substance. However, it can also be performed by selecting a substance that increases the MHPG concentration. In an animal in a normal state, MHPG in blood is below the detection limit. Therefore, if a significant concentration of MHPG is detected, it can be determined that the MHPG concentration has increased.

  Alternatively, the test substance may be administered to a vagus nerve-cutting animal and a normal animal, and the vagus nerve-cutting animal may select a substance having a lower blood MHPG concentration than the normal animal.

  Furthermore, it is preferable that the substance selected as described above is brought into contact with the digestive tract model system to select a compound having low absorbability from the digestive tract. Compounds with low absorbability from the gastrointestinal tract can prevent nonspecific stimulation of nociceptors in vivo. Examples of the digestive tract model system include a digestive tract-derived cell line, an animal-derived digestive tract specimen, and an animal-derived digestive tract lumen. Specifically, the gastrointestinal permeability is determined by, for example, administering a test substance into the digestive tract of an animal such as a rat, collecting portal vein blood or venous blood over time, and measuring the concentration of the test substance. Or by measuring the concentration of the test substance on the serosa side after exposing the test substance to the luminal side using an intestinal epithelial layer reconstructed in a test tube or an intestinal tract collected from an animal can do.

  Among the substances known as VR1 ligands, capsaicin binds to VR1 and activates the sympathetic nervous system by exciting nociceptors. As a result, adrenaline and noradrenaline are secreted from the adrenal medulla, and the heart rate and blood pressure rise. Similarly, adrenaline and noradrenaline are secreted from the adrenal medulla when capsinoids are administered in the blood. On the other hand, when capsinoid is administered orally, secretion of adrenaline and noradrenaline from the adrenal medulla is not promoted, and the MHPG concentration in the blood increases. Noradrenaline released from peripheral sympathetic nerve endings is taken up again into nerve tissue, and 3,4-dihydroxyphenylglycol (DHPG) released as a metabolite is further metabolized in the target tissue (muscles, etc.), and MHPG as the final product Is known to be released into the blood. MHPG has been reported to be mostly derived from muscle tissue in the blood (Lambert GW, et al., Journal of the Autonomic Nervous System. 1995. 55: 169-78). It is known that MHPG hardly increases even when noradrenaline is injected (Eisenhofer G, et al., Journal of the Autonomic Nervous System. 1994. 50: 93-107). Thus, blood MHPG concentration can be an indicator of peripheral sympathetic nerve activity.

  As shown in Examples, the increase in blood MHPG concentration by oral administration of capsiate was suppressed by vagus nerve cutting. Furthermore, removal of the adrenal medulla had no effect on the blood MHPG concentration after administration of capsiate. These results indicate that capsaicin enhances adrenal medulla adrenaline secretion, whereas capsiate activates the sympathetic nervous system that governs peripheral tissues such as muscle without stimulating adrenal medulla adrenaline secretion, resulting in noradrenaline metabolism. Indicates to promote. In addition, since the above-described effects of capsiate are mediated through the vagus nerve, it is suggested that capsiate is received in the gastrointestinal tract and exerts the above-described action through the ascending nerve pathway. It has not been known so far that there is a selective activation pathway of the sympathetic nervous system via the nerve pathway from the vagus nerve, and capsinoids are safer and more effective as substances acting on this pathway. This suggests that it can be used as a sympathetic nerve activator.

  The substance selected by the method of the present invention can enhance energy metabolism and suppress body fat accumulation by specifically activating peripheral sympathetic nerves that govern peripheral tissues such as muscles. Furthermore, by selecting a compound having low absorbability into the living body, it is possible to avoid the influence on blood pressure and heart rate caused by nonspecific activation of nociceptors distributed in the periphery.

  Substances obtained by administering the substance obtained according to the present invention to animals and confirmed to suppress body fat accumulation are supplements, medicines, foods and drinks or veterinary medicines for body fat reduction, obesity prevention or obesity improvement It can be used for animal nutritional supplements.

Hereinafter, the present invention will be described more specifically with reference to examples.
In the following examples, capsaicin manufactured by Wako Pure Chemical Industries, Ltd. was used. In Examples 1 and 2, the capsinoid was obtained by circulating capsaicin in hydrous methanolic hydrochloric acid according to the method described in Kobata et al. (Biosci. Biotechnol. Biochem., 66 (2), 319-327, 2002). Enzymatically synthesized fatty acid methyl and vanillyl alcohol were used as starting materials. Specifically, the molar ratio of fatty acid methyl to vanillyl alcohol was 1: 5, the enzyme used was an immobilized lipase (product name: Novozyme 435, manufactured by Novozyme), and the reaction conditions were 25 ° C. and 45 hours. The purity was 88% as capsinoid and the ratio of capsiate to dihydrocapsiate was 2.15: 1.

[Example 1]
A catheter was placed in the jugular vein of an 8-week-old male Wistar rat acclimated to a 12-hour light-dark cycle, fasted for 20 hours after recovery after surgery, and the following experiment was started 3 hours after the start of the dark period. When using a vagus nerve-cutting animal, open an 8-week-old male Wistar rat, cut the vagus nerve under the diaphragm, and recover it for at least one week after suturing, and then place an intravenous catheter in the same manner. It used for experiment. When using sham-operated animals, the same treatment was performed except that the vagus nerve was not removed.

Solvent (3% ethanol / 10% Tween80 / 87% physiological saline) or capsaicin solution (dose 15 mg / kg body weight) dissolved in the same solvent, or capsiate solution (dose 50 mg / kg) dissolved in the solvent (Body weight or 100 mg / kg body weight) was administered intragastrically with an oral sonde. Before and after administration, 800 μl of blood was collected over time from the catheter, and immediately 400 μl of red blood cells suspended in physiological saline were transfused. To the collected blood, EDTA and Na ₂ S ₂ O ₅ as an antioxidant were added, and plasma was separated and stored at −80 ° C. until measurement. After extracting the plasma with alumina, the concentration of catecholamines was measured using a high performance liquid chromatograph and an electrochemical detector (HPLC-EC). The conditions for alumina extraction and HPLC-EC are shown below.

<Alumina extraction conditions>
Add the internal standard (3,4-dihydroxybenzylamine) to 300 μl of plasma, stir, add 10 mg of physiological saline and activated alumina equivalent to plasma, and 100 μl of Tris-HCl buffer (pH 8.6) well. Stir. After centrifugation, the supernatant is removed, and the precipitate is washed twice with 200 μl of methanol and twice with distilled water, then added with 100 μl of 0.4N HClO ₄ and stirred, and the supernatant is collected and analyzed by HPLC-EC did.

<HPLC-EC analysis conditions>
Analytical column: Cosmosil 5C18-AR-II (Nacalai Tesque, φ4.6 × 250mm)
Guard column: Cosmosil Guard column 5C18-AR-II (Nacalai Tesque, φ4.6 × 10mm)
Column temperature: 30 ° C
Eluent: 8% methanol / 50 mM K-phosphate buffer (100 mg / l SOS (Sodium 1-octanesulfo
nate), 0.01mM EDTA)
Flow rate: 1.0ml / min
Detection voltage: 0, 300mV
Injection volume: 20μl

<Result>
The results are shown in FIGS.
15 minutes after administration, blood adrenaline and noradrenaline concentrations in the capsaicin-administered group were significantly higher than those in the solvent-administered group (FIG. 1). On the other hand, in the capsiate administration group, the blood adrenaline and noradrenaline concentrations were not different from those in the solvent administration group.

  In addition, as shown in FIG. 2, only in the capsiate administration group, an increase in MHPG, which is a major noradrenaline metabolite in rats, was observed 180 minutes after administration (FIG. 2-a). It was suppressed (FIG. 2-b. Capsiate dose 100 mg / kg). The blood MHPG concentration in the solvent administration group and capsaicin administration group was below the detection limit.

  These results indicate that capsaicin increases adrenal medulla adrenaline secretion, whereas capsiate activates the sympathetic nervous system that governs peripheral tissues such as muscle without stimulating adrenal medulla adrenaline secretion, resulting in noradrenaline metabolism. To promote. Moreover, since the above-mentioned effect by capsiate is mediated by the vagus nerve, it was suggested that capsiate is received in the digestive tract and exerts the above-mentioned action via the ascending nerve pathway. It has not been known so far that there is a selective activation pathway of the sympathetic nervous system via a nerve pathway from the vagus nerve, and capsiate is safer and more effective as a substance acting on this pathway. This suggests that it can be used as a sympathetic nerve activator.

[Example 2]
The adrenal gland of a 6-week-old male Wistar rat acclimated to a 12-hour light-dark cycle was removed under ether anesthesia, fasted for 20 hours after a 1-week recovery period, and the following experiment was started 3 hours after the start of the dark period. did.

Solvent (3% ethanol / 10% Tween80 / 87% physiological saline), capsaicin solution (dose 3 mg / kg body weight or 15 mg / kg body weight) dissolved in the same solvent, or capsiate solution dissolved in the solvent ( A dose of 15 mg / kg body weight, 50 mg / kg body weight or 100 mg / kg body weight) was intragastrically administered with an oral sonde. 180 minutes after administration, 1 ml of blood was collected from the subclavian vein under ether anesthesia, EDTA and Na ₂ S ₂ O ₅ were added as antioxidants, and plasma was separated and stored at −80 ° C. until measurement. After extracting the plasma with alumina, the concentration of MHPG was measured using a high performance liquid chromatograph and an electrochemical detector (HPLC-EC). The conditions for alumina extraction and HPLC-EC were the same as in Example 1.

<Result>
The results are shown in Table 1 and FIG.
The blood MHPG concentration was below the detection limit in the solvent administration group and the capsaicin administration group, whereas a dose-dependent increase was observed in the capsiate administration group (Table 1). In addition, removal of adrenal medulla that secreted adrenaline and noradrenaline into the blood did not affect the blood MHPG concentration after administration of capsiate (FIG. 3. Capsiate dose 100 mg / kg).

  These results indicate that orally administered capsiate promotes MHPG secretion in a dose-dependent manner, and capsaicin has no such effect. In addition, since adrenalectomy did not affect the MHPG concentration, it was shown that capsiate promoted MHPG secretion without adrenal or noradrenaline derived from the adrenal gland.

  Conventionally, it is difficult to increase MHPG even if adrenaline or noradrenaline is injected into blood (Eisenhofer G, et. Al., Journal of the Autonomic Nervous System. 1994. 50: 93-107). Almost all MHPGs present have been reported to be derived from muscle tissue (Lambert GW, et. Al., Journal of the Autonomic Nervous System. 1995. 55: 169-78). Among the nerves, the possibility of activating sympathetic nerves that control muscles in particular was considered.

Example 3
The fruit of “CH-19 sweet” was pulverized, freeze-dried, and then the oil content was extracted with a Soxhlet extractor using 3 volumes of hexane. Thereafter, the solvent was evaporated to dryness to obtain an extraction composition. 25% by weight of tricaprylin (M-2 manufactured by Riken Vitamin Co., Ltd.) was added to the extracted composition, and a flow-down thin film molecular distillation apparatus (evaporation heating area 0.024m ² 0.0088 m ² ), molecular distillation was performed under the conditions of an evaporation heating temperature of 180 ° C., a degree of vacuum of 12 to 14 Pa, and a fat feed amount of 1.1 g / min. To obtain an extracted oil having a capsinoid content of about 7.7%. The components and ratios of capsinoids contained in the extracted oil were capsiate 5.6%, dihydrocapsiate 1.5%, nordihydrocapsiate 0.6%.
Male SD rats at 7 weeks of age were orally administered as a capsinoid at a dose of 10 mg / kg body weight and 100 mg / kg body weight as a capsinoid in a single dose under fasting. Blood was collected from the portal vein and inferior aorta at 5, 15, 30 minutes after administration and 1, 2, 4 hours later (3 animals / time point / dose). The obtained blood was immediately collected into a tube to which 0.5 mg / L citrate buffer (pH 4.3) containing 20 mg / mL dichlorvos was added and transferred to ice. Plasma was then separated by centrifugation, and after pretreatment, plasma concentrations of various capsinoids (capsiate, dihydrocapsiate, and nordihydrocapsiate) were measured using high performance liquid chromatography and tandem mass spectrometry (LC / MS / MS). . The pretreatment method, analysis conditions and results are shown below.

<Pretreatment conditions>
After adding an internal standard to plasma, acetonitrile was added and the mixture was centrifuged to obtain a supernatant. A 20 mmol / L phosphate buffer (pH 3.0) was added, followed by solid phase extraction, followed by concentration to dryness under a nitrogen gas stream. 20 mmol / L phosphate buffer solution (pH 3.0) / acetonitrile = 50/50 (v / v) was added and dissolved, and subjected to liquid chromatography / tandem mass spectrometry.

<Analysis conditions>
(LC condition)
HPLC system: Agilent 1100 Series (Agilent Technologies)
Analytical column: Inertsil Ph-3, 4.6 mm id x 150 mm, 3 μm (GL Sciences)
Guard column: SUMIPAX Filter PG-ODS, 4.0 mm id x 4 mm, 3 μm (SCAS)
Mobile phase: Liquid A / Liquid B: 35:65 (v / v), Liquid A: H ₂ OB liquid: Methanol flow rate: 0.8 mL / min
Autosampler tray temperature: 4 ° C
Column temperature: 50 ° C
Valve switching: 0.0 to 7.0 min was discarded and 7.0 to 18.0 min was subjected to mass spectrometry.
Sample injection volume: 20μL
Analysis time: 19min

(MS / MS conditions)
MS / MS equipment: API2000 (AB / MDS SCIEX)
Ionization method: Atmospheric pressure chemical ionization (APCI), positive
MS / MS mode: Multiple Reaction Monitoring (MRM)
Probe temperature: 450 ° C
Quantitative range: 0.0500 μg / mL to 1.00 μg / mL
Monitor ion: Table 2

<Result>
Capsinoid portal vein and lower aortic plasma concentrations were below the limit of quantification at all time points in the 10 mg / kg and 100 mg / kg groups. This result suggests that capsinoid is difficult to be absorbed into the living body after ingestion. VR1 exists widely in the living body, and the living body causes various noxious stimulus responses by the action of capsaicins on these. Capsinoids are considered to be highly safe substances compared to capsaicin that is easily absorbed.

  The present invention can be used for searching for new drug candidate substances, diet food material candidate substances, pet supplements, pet foods, and the like.

The figure which shows transition of the density | concentration (average value +/- standard deviation) of blood adrenaline (a) and noradrenaline (b) after capsaicin (CAP) or capsiate (CST) administration. “*” Indicates that there is a statistically significant difference at a risk rate of 0.05 or less as compared with the solvent administration group (Veh). The figure which shows the blood MHPG density | concentration after capsaiate (CST) administration. a): Transition of blood MHPG concentration (mean ± standard deviation) after administration of capsiate 50 mg / kg, 100 mg / kg (n = 7 for each group). b): A graph showing the blood MHPG concentration (mean ± standard deviation) 180 minutes after administration of capsiate 100 mg / kg in vagus nerve-cut animals. “*” Indicates that there is a statistically significant difference with a risk rate of 0.05 or less compared to the sham operation group. The figure which shows the blood MHPG density | concentration (average value +/- standard deviation) after capsiate 100mg / kg administration of an adrenalectomy animal and a sham operation animal.

Claims (6)

A method for screening a peripheral tissue sympathetic nervous system activator,
A test substance having an activity of promoting or regulating VR1 activation is prepared, the test substance is orally administered to a test animal, and a substance that does not promote secretion of adrenal and noradrenaline derived from the adrenal gland is selected. how to.   The method according to claim 1, further comprising the step of contacting the selected substance with a digestive tract model system to select a compound having low absorbability from the digestive tract.   The method according to claim 1 or 2, wherein the substance that does not promote secretion of adrenaline and noradrenaline is selected by selecting a substance that promotes secretion of 3-methoxy-4-hydroxyphenylglycol to peripheral blood. Selecting a substance that promotes the secretion of 3-methoxy-4-hydroxyphenyl glycol into the peripheral blood,
The test substance having an activity of promoting or regulating the activation of VR1 is administered to a vagus nerve-cutting animal and a normal animal, and the vagus nerve-cutting animal is more sensitive to peripheral blood than normal animals. The method according to claim 3, wherein the method is carried out by selecting a substance having low secretion of hydroxyphenyl glycol.   The preparation of the test substance having the activity of promoting or regulating the VR1 activation is conducted by contacting the candidate substance or the candidate substance with a VR1 agonist in a cell line expressing VR1, and measuring the VR1 activation. The method as described in any one of Claims 1-4 performed by selecting the substance which has the activity which accelerates | stimulates or regulates activation.   3. The method of claim 2, wherein the gastrointestinal model system is selected from the group consisting of a gastrointestinal tract-derived cell line, an animal-derived gastrointestinal specimen, and an animal-derived gastrointestinal lumen.

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