JP2014208702A - Composition for controlling autonomic nerve activity, and method for controlling autonomic nerve - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for controlling autonomic nerve activities, especially a method of suppressing the increase of sympathetic nerve activities.SOLUTION: The method for controlling autonomic nerve activities is characterized by administration of Val-Pro-Pro or a salt thereof.

Description

  The present invention relates to a composition for regulating autonomic nerve activity and a method for regulating autonomic nerve activity. In particular, the present invention relates to a composition for suppressing an increase in sympathetic nerve activity and / or a composition for promoting parasympathetic nerve activity. The present invention also relates to a method for suppressing an increase in sympathetic nerve activity and a method for promoting parasympathetic nerve activity.

The autonomic nervous system is a part of the nervous system that is not regulated by consciousness. Unlike the somatic motor nervous system that mainly controls skeletal muscle, the autonomic nervous system controls the heart muscle, smooth muscle, and glandular tissue. ing. Also, the autonomic nervous system is structurally different from the motor nervous system and is composed of two nerve cells called the prenodal nerve and the retronodal nerve. The autonomic nervous system is roughly divided into two types, the sympathetic nervous system and the parasympathetic nervous system, and their functions are different. Sympathetic activity generally causes an increase in heart rate, increased blood pressure, pupil enlargement, blood glucose release, etc., and parasympathetic activity generally stimulates an increase in gastrointestinal motility and secretion of digestive juices. It is known to decrease. The two work together to play an important role in maintaining homeostasis.
As a report of directly measuring the effect on autonomic nerve activity with respect to peptides or peptide derivatives, the autonomic nerve regulating action of L-carnosine has been reported. Oral administration of low-dose L-carnosine suppresses sympathetic nerve activity and exhibits parasympathetic nerve activity, and also exhibits hypertension and hyperglycemia due to increased sympathetic nerve activity. It has been reported that the effect of improving is also shown (Patent Document 1). However, this effect was measured by intravenous administration, and the effect by oral administration was unknown. Furthermore, there is no report that directly measured the effect on autonomic nerve activity for the tripeptide Val-Pro-Pro (VPP) and its analogs.
In addition, as a report showing amnesia-improving effects on peptides or peptide derivatives, it is possible to obtain an improvement effect on scopolamine-induced amnesia by intraventricular or oral administration of 0.5 to 2.3 mg / kg of VPDPR, PDPR, CPR, and cyclo DP. It has been known that the possibility of via a mu opioid receptor is suggested as one of the mechanisms (Patent Document 2). Moreover, it has been shown that these peptides also have an effect of lowering serum cholesterol by oral administration (Patent Document 2). Furthermore, it has been reported that XPLPR (X is L, I, M, F, W) shows an improvement effect on scopolamine-induced amnesia by intraventricular or oral administration of 300 mg / kg. It has been suggested that acetylcholine is released by C3a receptors in the brain (Patent Document 3). However, none of these actions showed an action through regulation of autonomic nervous activity, and the tripeptide Val-Pro-Pro (VPP) and its analogs were directly measured for their effects on amnesia prevention effects. There is no.

  There are also several reports on the function of the tripeptide Val-Pro-Pro (VPP) and its analogs. For example, an angiotensin converting enzyme inhibitor containing Val-Pro-Pro as an active ingredient is known, Val-Pro-Pro shows ACE inhibitory activity, and the blood pressure-lowering action of Val-Pro-Pro by oral administration is known. It has been reported that fermented milk containing Val-Pro-Pro also has a blood pressure lowering effect (Patent Document 4). Moreover, oral administration of Val-Pro-Pro and Ile-Pro-Pro (IPP) suppresses blood pressure fluctuations due to cold stress, and at the same time, changes in blood amino acid balance (Fischer ratio), thymus and spleen It is known that atrophy is observed and an inhibitory effect is exhibited against a decrease in immune function index such as a decrease in spleen cell reactivity (Patent Document 5). In addition, it is known that Val-Pro-Pro is applied to the skin to improve skin smears. As a mechanism, substances with angiotensin-converting enzyme inhibitory activity (such as VPP) have an effect of smooth muscle expansion. It has been suggested that blood circulation is promoted by suppressing the degradation of some bradykinin (Patent Document 6). However, none of these reports have directly measured the effect of peptides on autonomic nerve activity, and no specific reports have focused on their regulatory effects.

International Publication No. 2002/076455 Japanese Patent No. 3660978 Japanese Patent No. 3898389 Japanese Patent No. 2782142 Japanese Patent Laid-Open No. 11-100328 JP 2002-284668 A

The present invention provides an orally ingestible composition for modulating autonomic nerve activity. The present invention also provides a method for regulating autonomic nerve activity.
In particular, the present invention provides an orally ingestible composition for suppressing an increase in sympathetic nerve activity and / or an ingestible composition for promoting parasympathetic nerve activity.

(1) The present invention is a composition for regulating autonomic nervous activity comprising Val-Pro-Pro or a salt thereof as an active ingredient.
(2) The present invention is also a composition for promoting parasympathetic nerve activity, comprising Val-Pro-Pro or a salt thereof as an active ingredient.
(3) The present invention is a composition for suppressing enhancement of sympathetic nerve activity, comprising Val-Pro-Pro or a salt thereof as an active ingredient.
(4) Moreover, this invention is a composition in any one of (1)-(3) characterized by preventing, improving or relieving the disorder of an autonomic nerve, and the symptom resulting from it.
(5) The present invention is the composition according to (2) or (3), which has an amnesia-preventing action, a blood flow promoting action or an anxiolytic action, comprising Val-Pro-Pro or a salt thereof as an active ingredient.
(6) The present invention is also an amnesia-preventing composition comprising Val-Pro-Pro or a salt thereof as an active ingredient.
(7) The present invention is also a composition for promoting blood flow comprising Val-Pro-Pro or a salt thereof as an active ingredient.
(8) The present invention is also an anxiety-suppressing composition comprising Val-Pro-Pro or a salt thereof as an active ingredient.
(9) The present invention is also the composition according to any one of (1) to (8), which is for oral intake.
(10) The present invention also relates to a method for regulating autonomic nerve activity in an animal comprising administering Val-Pro-Pro or a salt thereof to the animal (excluding medical practice for humans).
(11) The present invention is also a method for promoting parasympathetic nerve activity in an animal, comprising administering Val-Pro-Pro or a salt thereof to the animal (excluding medical practice for humans).
(12) The present invention is also a method for suppressing an increase in sympathetic nerve activity in an animal, comprising administering Val-Pro-Pro or a salt thereof to the animal (excluding medical practice for humans).
(13) The present invention is also a method according to (10) to (12), which prevents, ameliorates or alleviates autonomic nerve disturbances and symptoms caused thereby in animals (excluding medical practices for humans).
(14) The present invention is also a method according to (11) or (12), which prevents amnesia, promotes blood flow, or suppresses anxiety in animals (excluding medical practices for humans).
(15) The present invention is also a method for preventing amnesia in an animal, including administering Val-Pro-Pro or a salt thereof to the animal (excluding medical practice for humans).
(16) The present invention is also a method for promoting blood flow in an animal, including administering Val-Pro-Pro or a salt thereof to the animal (excluding medical practice for humans).
(17) The present invention is also a method for suppressing anxiety in an animal comprising administering Val-Pro-Pro or a salt thereof to the animal (excluding medical practice for humans).
(18) The present invention is also the method according to any one of (10) to (17), wherein the administration is oral administration.

FIG. 1 shows the effect of peptide Val-Pro-Pro (VPP) on gastric parasympathetic activity. Administer water (control) or Val-Pro-Pro 0.09mg / kg body weight, 0.15mg / kg body weight or 15mg / kg body weight to rats (n = 3) and determine the action potential of the parasympathetic (vagus) nerve that governs the stomach Measured over time. The vertical axis shows the relative action potential of each experimental group as a percentage when the value before administration (0 minute value) is 100 (GVNA: gastric vagus nerve activity). The horizontal axis represents the time (minutes) after sample administration. The significant difference test with the water-administered control group was performed by analysis of variance (ANOVA) with a value of 5 to 60 minutes after administration as a group. FIG. 2 shows the effect of peptide Val-Pro-Pro (VPP) on adrenal sympathetic nerve activity. Water (control) or Val-Pro-Pro 15 mg / kg body weight was administered to rats (n = 3), and the action potential of the sympathetic nerve governing the adrenal gland was measured over time. The vertical axis shows the relative action potential of each experimental group as a percentage when the value before administration (0 minute value) is 100 (ASNA: adrenal sympathetic nerve activity). The horizontal axis represents the time (minutes) after sample administration. The significant difference test with the water-administered control group was performed by analysis of variance (ANOVA) with the relative action potential value at 5 to 60 minutes after administration as a group. FIG. 3 shows the effect of peptide Val-Pro-Pro (VPP) on cutaneous arterial sympathetic nerve activity. Water (control) or Val-Pro-Pro 0.15 mg / kg body weight or 15 mg / kg body weight was administered to rats (n = 3), and action potentials of cutaneous arterial sympathetic nerves were measured over time. The vertical axis shows the relative action potential of each experimental group as a percentage when the value before administration (0 minute value) is 100 (CASNA: cutaneous artery sympathetic nerve activity). The horizontal axis represents the time (minutes) after sample administration. The significant difference test with the water-administered control group was performed by analysis of variance (ANOVA) with the relative action potential value at 5 to 60 minutes after administration as a group. FIG. 4 shows the scopolamine-induced amnesia-preventing effect of peptide Val-Pro-Pro (VPP). Water (control) or scopolamine alone, or Val-Pro-Pro 0.64 mg / kg body weight or 6.4 mg / kg body weight or 10 mg / kg body weight was administered to mice together with scopolamine, and amnesia was obtained by the method described in Example 4, respectively. The preventive effect was evaluated. The vertical axis in FIG. 4 indicates the voluntary alternation behavior change rate. In order to confirm whether forgetfulness was induced, a significant difference test between the water-administered control group and the scopolamine control group administered scopolamine alone was performed by Student's t-test. ** indicates P <0.01 with respect to the water-administered control group. The significant difference test between the VPP administration group and the scopolamine control group was performed by Student's t-test. † indicates P <0.1 and ## indicates P <0.01 with respect to the scopolamine single administration group. FIG. 5 shows the blood flow promoting effect of the peptide Val-Pro-Pro (VPP). Water or Val-Pro-Pro 20 mg / kg body weight was administered to mice, and blood flow was measured in the skin 3 cm above the ridge on the midline of the back. The vertical axis shows the relative blood flow as a percentage when the value before administration (0 minute value) is 100. The horizontal axis represents the time (minutes) after sample administration. The significant difference test between the VPP-administered group and the water-administered control group was performed by analysis of variance (ANOVA) with the relative action potential values at 5 to 60 minutes after administration as a group. FIG. 6 shows the anxiolytic effect of the peptide Val-Pro-Pro (VPP). Water or Val-Pro-Pro 0.001, 0.1, 10, 100 mg / kg was administered to mice (n = 15), and the open arm invasion frequency ratio was measured. The vertical axis shows the open arm intrusion frequency ratio of the control group given only water and the Val-Pro-Pro administration group. Data show mean ± standard error.

The composition of the present invention contains the tripeptide Val-Pro-Pro as an active ingredient. The peptide Val-Pro-Pro which is an active ingredient may be a peptide synthesized organically or may be a peptide derived from a natural product. As an organic chemical synthesis method for these peptides, a general method such as a solid phase method (Boc method, Fmoc method) or a liquid phase method can be used. For example, a peptide synthesizer manufactured by Shimadzu Corporation (PSSM-8) Type) and a peptide synthesized using an automatic peptide synthesizer. About the reaction conditions of peptide synthesis, based on the common general knowledge of those skilled in the art, the synthesis method to be selected, appropriate reaction conditions, and the like can be arbitrarily set. Methods for purifying chemically synthesized peptides are also well known to those skilled in the art.
In this specification, when referring to the peptide Val-Pro-Pro, “Val-Pro-Pro” and “Peptide Val-Pro-Pro” unless otherwise stated and apparently excluded from context. "Includes salts thereof. Such salts include salts that may exist under physiological conditions such as sodium salts, potassium salts and the like. The composition of the present invention may contain other peptides and free amino acids or salts thereof in addition to the peptide, Val-Pro-Pro, which is an active ingredient of the composition of the present invention. In the context of the present invention, the three-letter code and single-letter code for amino acids and the code for peptides follow general rules well known to those skilled in the art.

  In some embodiments of the present invention, the peptide Val-Pro-Pro is a raw material comprising a protein containing the Val-Pro-Pro sequence (eg, milk, horse milk, goat milk or sheep milk, and their nonfat dry milk or soy , Wheat, etc.) may be derived from a fermented product obtained by fermenting with bacteria belonging to the species Lactobacillus helveticus. The content of the protein raw material during fermentation is not particularly limited, but generally 1 to 19% by weight will be preferable. Such a fermented product can be used as it is or after appropriate treatment such as centrifugation, filtration, chromatogram, and drying to obtain the composition of the present invention.

  In another embodiment of the present invention, the peptide Val-Pro-Pro may be derived from a hydrolyzate of a protein containing Val-Pro-Pro. Such proteins include animal milk casein, such as casein such as cow's milk, horse milk, goat milk, sheep milk, or concentrates thereof. Moreover, you may originate in the fermented material obtained by fermenting the foodstuff raw material containing animal milk casein with fungi, such as a gonococcus and a lactic acid bacterium. Although the casein concentration at the time of hydrolyzing or fermenting animal milk casein is not particularly limited, it is generally preferably 1 to 19% by weight. In the case of using a commercially available enzyme group, the optimum conditions are usually set. However, depending on the enzyme group used, the conditions such as the amount of enzyme used and the reaction time are used so that the casein hydrolyzate can be obtained. Can be changed as appropriate. For example, in an aqueous solution in which animal milk casein is dissolved, the enzyme group / animal milk casein has a weight ratio of 1/1000 or more, preferably 1/100 to 1/10, particularly preferably 1/40 to 1/10. Degrading enzymes can be added. Although reaction conditions can be suitably selected according to the enzyme group to be used, it is usually 25 to 60 ° C., preferably 45 to 55 ° C., pH 3 to 11, and preferably 5 to 9. The reaction time can be 2 to 48 hours, preferably 7 to 15 hours. Such a protein hydrolyzate, in particular casein hydrolyzate, may be used as it is, or may be subjected to treatment such as centrifugation, filtration, chromatogram, and drying to obtain the composition of the present invention.

The composition of the present invention has an action of regulating autonomic nerve activity. Although autonomic nerve activity includes sympathetic nerve activity and parasympathetic nerve activity, the composition of the present invention has an action of suppressing an increase in sympathetic nerve activity and an action of promoting parasympathetic nerve activity. In particular, the composition of the present invention has the effect of preventing amnesia, promoting blood flow, and anxiolytic effects as a result of suppressing the increase in sympathetic nerve activity or promoting parasympathetic nerve activity. Further, the composition of the present invention has a blood flow promoting action, and particularly has a skin blood flow promoting action by suppressing cutaneous arterial sympathetic nerve activity. Furthermore, since the composition of the present invention has an autonomic nerve regulating action, it has broadly a heart rate regulating action, a blood sugar regulating action, a gastric secretion regulating action, a body temperature regulating action, a blood pressure regulating action, a stress relieving action and the like.
The action of the composition of the present invention and its active ingredient peptide Val-Pro-Pro to regulate the autonomic nervous activity, particularly the action to suppress the increase of sympathetic nerve activity and the action to promote parasympathetic nerve activity are confirmed as follows, for example. can do.

The gastric parasympathetic nerve activity promoting action of the composition of the present invention is, for example, by administering this composition to a laboratory animal such as a rat or a mouse, and measuring the action potential of the parasympathetic (vagus) nerve governing the stomach over time, This can be confirmed by an increase in action potential. In addition, the composition of the present invention is administered to rats or mice, etc., and after a certain period of time (for example, after 15 to 30 minutes), a dye such as phenol red is introduced into the stomach of rats or mice, etc. For example, after 20 minutes, the rat or mouse stomach is removed and the amount of residual pigment is measured to enhance gastric emptying ability, that is, the activity of the stomach is activated. Can also be confirmed.
The effect of the composition of the present invention or the peptide Val-Pro-Pro on the adrenal sympathetic nerve activity is obtained by administering the composition to rats or mice and measuring the action potential of the parasympathetic nerve governing the adrenal gland over time. Can be confirmed. If the action potential decreases, it can be confirmed that the adrenal sympathetic nerve activity is suppressed.

The effect of the composition of the present invention or the peptide Val-Pro-Pro on the cutaneous arterial sympathetic nerve activity is obtained by, for example, orally administering the composition of the present invention or Val-Pro-Pro to a mouse or a rat, etc. This can be confirmed by measuring the action potential over time. In addition, the blood flow promoting action of the composition of the invention or the peptide Val-Pro-Pro can be achieved by administering the composition of the present invention or Val-Pro-Pro to a mouse or rat, etc., and using a contact laser Doppler blood flow meter or the like. This can be confirmed by measuring the skin blood flow on the midline of the back. The blood flow can be measured, for example, on the back midline.
The amnesia-preventing action of the composition of the present invention or the peptide Val-Pro-Pro can be confirmed using, for example, a system according to an evaluation system for Alzheimer's disease therapeutic drugs using a Y-shaped maze. Specifically, an agent that induces amnesia such as scopolamine or a composition of the present invention or Val-Pro-Pro is administered to a rat or mouse simultaneously with such an agent, or prior to administration of such an agent. The composition of the present invention was administered using the composition of the present invention or Val-Pro-Pro and the rate of spontaneous alternation behavior to different arms and the total number of times of entering the maze in the test using the Y-shaped maze. Can prevent amnesia.
In these tests, as a negative control, for example, animals administered with water alone can be used. In the experiment for confirming the preventive action of Val-Pro-Pro against amnesia induced by a drug, an animal administered only with a drug that induces amnesia such as scopolamine can be added as a control.

  The anxiolytic effect of the composition of the present invention or the peptide Val-Pro-Pro can be confirmed, for example, by an elevated plus maze test in mice. Specifically, mice are administered water or a composition of the invention or peptide Val-Pro-Pro, the mice are placed on the platform at the center of the maze at a high altitude, and open arms (arms without walls) within a certain time. ) And closed arms (arms with walls), and the time spent staying was measured, and the ratio of the number of times of intrusion into the open arm or the staying time ratio increased compared to the control group administered with water alone. This can be confirmed as an anxiolytic effect.

The active ingredient of the composition of the present invention is Val-Pro-Pro, and the above-mentioned desired effect can be achieved by oral administration or ingestion. The administration or ingestion period of the composition of the present invention can be variously adjusted in consideration of the age of the subject to be administered or ingested, for example, the age of the human or non-human animal, the health condition of the subject, and the like. Non-human animals include non-human higher vertebrates, particularly non-human mammals, including but not limited to pets such as dogs and cats, and domestic animals such as cows, horses, pigs and sheep. The effect of the composition of the present invention can be seen even after one administration, but a continuous effect can be expected by continuously ingesting once or more a day. When the composition of the present invention is used as a medicine, it can be in the form of a preparation for oral administration. Examples include tablets, pills, hard capsules, soft capsules, microcapsules, powders, granules, liquids and the like. In the case of producing as a pharmaceutical, for example, a pharmaceutically acceptable carrier, excipient, excipient, preservative, stabilizer, binder, pH adjuster, buffer, thickener, if necessary. Can be manufactured in unit dosage forms required for generally accepted formulation administration, using agents, gelling agents, preservatives, antioxidants, and the like.
The composition of the present invention can also be used as a material for food and drink or an animal feed material. For example, the peptide of the present invention or the peptide Val-Pro-Pro, which is an active ingredient of the composition of the present invention, promotes gastrointestinal motility. Functional foods such as foods for specified health use that have effects such as action, anti-anxiety action, blood flow promotion action, amnesia prevention, heart rate regulation action, blood glucose regulation action, gastric secretion regulation action, body temperature regulation action, stress relaxation action, etc. It can be.
The administration or intake of the present composition or peptide Val-Pro-Pro for obtaining a desired effect is generally 0.001 mg / kg body weight to 100 mg / kg as the amount of peptide Val-Pro-Pro which is an active ingredient per time. About weight. In particular, when promotion of parasympathetic nerve activity is mainly desired, the intake of the composition of the present invention is preferably 0.09 mg / kg body weight to 15 mg / kg body weight as the amount of Val-Pro-Pro. When an inhibitory effect on sympathetic nerve activity is desired, the amount of Val-Pro-Pro is preferably 0.15 mg / kg body weight to 20 mg / kg body weight, particularly 0.15 mg / kg when an effect on cutaneous artery sympathetic nerve activity is mainly desired. kg body weight to 1.5 mg / kg body weight is preferred. When blood flow promoting action is mainly desired, about 20 mg / kg is preferable. When an anxiolytic effect is mainly desired, the amount of Val-Pro-Pro is particularly preferably 0.001 mg / kg body weight to 100 mg / kg body weight, and when an amnesia-preventing effect is mainly desired, the amount of Val-Pro-Pro is 0.64 mg / kg body weight to 10 mg / kg body weight is preferred. Depending on the number of intakes per day, it is possible to further reduce the intake amount per time in foods such as functional foods. The appropriate intake can be further adjusted taking into account various factors as described above.

A food comprising the composition of the present invention or its active ingredient peptide Val-Pro-Pro, for example, a functional food, is a protein degradation product containing Val-Pro-Pro obtained as described above or a concentrate thereof. It can be produced by adding a raw material fermented product containing a protein containing Val-Pro-Pro, or a fermented product in powder form or granular form to various foods. If necessary, other ingredients used in foods, such as sugars, proteins, lipids, vitamins, minerals, flavors, such as various carbohydrates, lipids, vitamins, minerals, sweeteners, fragrances, pigments, texture improvers Etc. or a mixture thereof may be added to improve nutritional balance, flavor, and the like. Animal feed containing the composition of the present invention or the peptide Val-Pro-Pro which is an active ingredient thereof can also be prepared in the same manner as human food.
For example, the functional food described above can be in any form of solid, gel, and liquid, for example, fermented milk products such as lactic acid bacteria beverages, various processed foods and drinks, dry powders, tablets, capsules, Examples thereof include granules, and various beverages, yogurt, liquid food, jelly, candy, retort food, tablet confectionery, cookies, castella, bread, biscuits, chocolate and the like.
When producing a functional food such as a food for specified health use containing the composition of the present invention, depending on the form of addition or product, the inclusion of the peptide Val-Pro-Pro which is an active ingredient contained in the final product The amount is adjusted to 0.00001% by mass to 10% by mass, preferably 0.00003% by mass to 3% by mass, and more preferably 0.0001% by mass to 1% by mass.
EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, the scope of the present invention is not restricted to an Example.

Effect of Val-Pro-Pro on gastric parasympathetic nerve activity Wistar male rats (about 9 weeks old) (n = 3) weighing about 300 g were used, and food and water were ad libitum. In order to examine autonomic nerve activity, a polyethylene tube for oral administration was inserted into the oral cavity under urethane anesthesia in the middle of the light period after fasting for 3 hours. Thereafter, the parasympathetic (vagus) nerve that controls the stomach was lifted with a silver electrode, and the electrical activity of the nerve was measured. The tube was inserted into the trachea from the start of surgery to the end of measurement to secure the airway, and the body temperature (rat rectal temperature) was maintained at 35.0 ± 0.5 ° C. with a heat retaining device. When the nerve activity settled, each Val-Pro-Pro peptide aqueous solution sample was orally administered in a 1 ml polyethylene tube, and the change in nerve activity was measured. Samples were Val-Pro-Pro 0.09 mg / kg body weight, 0.15 mg / kg body weight or 15 mg / kg body weight. As a control, 1 ml of solvent water was orally administered. The data of gastric parasympathetic nerve activity is analyzed by the average value of the firing frequency (pulse / 5 s) per 5 seconds every 5 minutes, and the value before the start of stimulation (0 minute value) is expressed as a percentage. The average value ± standard error is shown. The significant difference test with the control group was carried out by analysis of variance (ANOVA) with the relative action potential value at 5 to 60 minutes after administration as a group. The results are shown in FIG.
Compared to the water-administered group, the gastric parasympathetic nerve activity after administration was significantly increased in any of the Val-Pro-Pro 0.09 mg / kg body weight, 0.15 mg / kg body weight, and 15 mg / kg body weight groups (FIG. 1). . It was shown that oral administration of Val-Pro-Pro promotes gastric parasympathetic nerve activity in the range of 0.09 mg / kg body weight to 15 mg / kg body weight.

Effect of Val-Pro-Pro on adrenal sympathetic nerve activity Wistar male rats (about 9 weeks old) (n = 3) weighing about 300 g were used, and food and water were ad libitum. In order to examine autonomic nerve activity, a polyethylene tube for oral administration was inserted into the oral cavity under urethane anesthesia in the middle of the light period after fasting for 3 hours. Thereafter, the sympathetic nerve that controls the adrenal gland was lifted with a silver electrode, and the electrical activity of the nerve was measured. The tube was inserted into the trachea from the start of surgery to the end of measurement to secure the airway, and the body temperature (rat rectal temperature) was maintained at 35.0 ± 0.5 ° C. with a heat retaining device. When the nerve activity settled, 1 ml of Val-Pro-Pro peptide aqueous solution sample was orally administered in a polyethylene tube, and the change in nerve activity was measured. The sample was Val-Pro-Pro 15 mg / kg body weight. As a control, 1 ml of solvent water was orally administered. Data on the activity of the adrenal sympathetic nerve is analyzed by the average value of the firing frequency (pulse / 5 s) per 5 seconds every 5 minutes and expressed as a percentage with the value before the start of stimulation (0 minute value) as 100%. The average value ± standard error is shown. The significant difference test with the control group was carried out by analysis of variance (ANOVA) with the relative action potential value at 5 to 60 minutes after administration as a group. The results are shown in FIG.
Compared to the water-administered group, administration of Val-Pro-Pro 15 mg / kg body weight significantly decreased the adrenal sympathetic nerve activity (FIG. 2). Val-Pro-Pro was shown to suppress adrenal sympathetic nerve activity at 15 mg / kg body weight.

Effect of Val-Pro-Pro on cutaneous arterial sympathetic nerve activity Wistar male rats (about 9 weeks old) (n = 3) weighing about 300 g were used, and food and water were ingested freely. In order to examine autonomic nerve activity, a polyethylene tube for oral administration was inserted into the oral cavity under urethane anesthesia in the middle of the light period after fasting for 3 hours. Then, the tail cutaneous artery sympathetic nerve was lifted with a silver electrode, and the electrical activity of the nerve was measured. The tube was inserted into the trachea from the start of surgery to the end of measurement to secure the airway, and the body temperature (rat rectal temperature) was maintained at 35.0 ± 0.5 ° C. with a heat retaining device. When these nerve activities settled, 1 ml of each Val-Pro-Pro peptide aqueous solution sample was orally administered through a polyethylene tube, and changes in these nerve activities were measured. The sample was Val-Pro-Pro 0.15 mg / kg body weight or 1.5 mg / kg body weight. As a control, 1 ml of solvent water was orally administered. Cutaneous arterial sympathetic nerve activity data is analyzed with the average value of the firing frequency (pulse / 5 s) per 5 seconds every 5 minutes, and the percentage before the start of stimulation (0 minute value) is 100%. Expressed as an average value ± standard error. The significant difference test with the control group was carried out by analysis of variance (ANOVA) with the relative action potential value at 5 to 60 minutes after administration as a group. The results are shown in FIG.
Cutaneous arterial sympathetic nerve activity after administration was significantly reduced in the Val-Pro-Pro 1.5 mg / kg body weight and 0.15 mg / kg body weight groups as compared to the water administration group (FIG. 3). Oral administration of Val-Pro-Pro was shown to suppress cutaneous arterial sympathetic nerve activity in the range of 0.15 mg / kg body weight to 1.5 mg / kg body weight.

Amnesia preventive action of Val-Pro-Pro
ddY male mice (approximately 7 weeks of age) were used (n = 15), and food and water were ad libitum. As test substances, Val-Pro-Pro 0.64 mg / kg body weight, 6.4 mg / kg body weight, and 10 mg / kg body weight were used. Each dose of Val-Pro-Pro was administered orally once to mice 60 minutes before the Y-maze test to evaluate spontaneous alternation behavior.
In addition, 30 minutes before the Y-maze test, scopolamine was subcutaneously administered to the back so as to achieve 1 mg / kg body weight in order to induce memory impairment and / or cognitive impairment in mice. In the Y-maze test, one arm is 40 cm long, the wall height is 12 cm, the floor width is 3 cm, the top width is 10 cm, and the three arms are connected at an angle of 120 degrees each. The character maze was used as an experimental device. The mouse was placed on the tip of any arm of the Y-shaped maze and allowed to freely explore the maze for 8 minutes, and the arm to which the mouse moved was recorded in order. Count the number of times the mouse has moved to each arm within the measurement time, and use this as the total number of intrusions. In this combination, select three different arms in succession (for example, three arms A, B, When the order of the arms entered was ABCBACACB, it was counted as 4 including duplication), and this number was taken as the number of voluntary replacement actions. The number of voluntary alternations was divided by the total number of ingresses minus 2, and the value obtained by multiplying it by 100 was the voluntary alternation behavior change rate, which was used as an index of voluntary alternation behavior. The measured values were expressed as mean ± standard error for each group. The significant difference test with the control group or the scopolamine control group was performed by Student's t-test. The results are shown in FIG. Val-Pro-Pro was shown to exhibit amnesia-preventing action in the range of 0.64 mg / kg body weight to 10 mg / kg body weight.

Peripheral blood flow promoting action of Val-Pro-Pro
Six-week-old female Hos: HR-1 hairless mice were purchased from Japan SLC Co., Ltd. and subjected to the following test after acclimation breeding for one week. The age at the time of the test was in the range of 7 to 15 weeks. After measuring the body weight, gas anesthesia with isoflurane (Abbott Japan) was performed. During introduction, anesthesia was performed for 1 minute under conditions of a concentration of 4% and a flow rate of 1 L / min. Maintenance anesthesia was performed using a mouthpiece under conditions of a concentration of 1% and a flow rate of 1 L / min. After the start of maintenance anesthesia, the mouse was placed on a hot plate (manufactured by OMRON) set at 37 ° C. and kept warm. A piece of Kim Towel was placed between the plate and the mouse. After 30 minutes after the induction of anesthesia, each sample was orally administered once using a sonde when the previous blood flow was stable for 10 minutes or more. Samples were administered at 10 ml / kg body weight such that the Val-Pro-Pro administered was 20 mg / kg. As a control, 10 ml / kg body weight of solvent was orally administered. A contact type laser Doppler blood flow meter manufactured by Advance was used for blood flow measurement. The contact type probe was attached to the skin 3 cm above the ridge on the midline of the back using vinyl tape. Using the blood flow rate (Flow) as an evaluation index, an average value was calculated at intervals of 1 minute after administration, and expressed as a relative value with the average value for 1 minute before administration being 100. Comparison between the control group and the control group was performed by ANOVA analysis with the measured value of 5 to 60 minutes after administration as a group. The results are shown in FIG. Val-Pro-Pro was shown to have a blood flow promoting effect when administered at 20 mg / kg body weight.

Evaluation of anxiolytic activity of Val-Pro-Pro by elevated plus maze test
Using ddY male mice (approximately 6 weeks old) (n = 15), after preliminary breeding, Val-Pro-Pro (0.001 mg / kg body weight, 0.1 mg / kg body weight, 10 mg / kg body weight or 100 mg / kg body weight) Were administered orally once, and 30 minutes after administration, the anxiolytic effect was examined by an elevated plus maze test. The elevated cross maze is formed by crossing two vinyl chloride plates with a length of 65 cm and a width of 5 cm in a cross shape, and around the single passage, except for the central intersection, A 50cm wall (black acrylic resin) is attached. That is, the cross maze is composed of two arms with no walls (open arms), two arms with walls (closed arms), and a central intersection. In the elevated cross maze test, place the mouse on the arm with one wall at the center of the maze, let it freely explore the maze for 5 minutes, observe its behavior, and enter the open arm Was measured. The ratio of the number of open arm intrusions during the 5-minute test was determined from the measured values, and this was used as an anxiolytic action index. In the control group, distilled water was orally administered once. Each measured value was expressed as an average value ± standard error for each group. The results are shown in FIG. Val-Pro-Pro had an anti-anxiety action in the range of 0.001 mg / kg body weight to 100 mg / kg body weight, with a higher value of the open arm invasion frequency ratio than the control group.

Claims (2)

  A method for preventing amnesia, including administering Val-Pro-Pro or a salt thereof (excluding treatment for humans).   The method of claim 1, wherein the administration is oral administration.

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