JP5592100B2 - Anti-stress composition comprising lily root extract fraction - Google Patents

Description

  The present invention relates to an anti-stress composition useful for prevention and treatment of depression, for example.

  According to WHO statistics, it is estimated that there are more than 3 million “depressed” patients in Japan, which is becoming a major social problem. In addition, the number of cases of suicide due to depression has exceeded 20,000 in recent years, reaching more than three times the number of deaths in traffic accidents.

  Depression is a complex disease that should be called the fate of modern people who are exposed to mental stress, and there is a strong need for effective detection and treatment. However, there is currently no diagnosis of depression other than counseling based on the individual skill of doctors, and the development of effective markers for quantitatively detecting mental stress and depression is awaited. The development of new antidepressants and antistress foods that are used is eagerly desired.

  Non-specific reactions that occur when a living body is exposed to harmful environments and factors are defined as "stress". To date, stress-induced factors have been identified in a number of diseases, and the relationship between the factors and the pathology has been pointed out, but the exact level of mental stress and depression, which is the main factor, is still accurate. A method for evaluation has not been established. This is because mental stress requires evaluation from various viewpoints such as clinical psychology, sociology, physiology, and clinical medicine, and each evaluation method lacks a reliable index. Therefore, due to its nature, evaluation of mental stress has been considered particularly difficult.

  Patent Document 1 discloses an antidepressant food containing a powder or extract such as a bulb of a lily family. In addition, as a method for obtaining the extract, it is also described that lily bulbs and the like are extracted with a solvent such as water or ethanol.

JP 2002-58450 A

  Regarding the marker effective for the evaluation of the above mental stress, the present inventor recently found that some proteins in the serum increase due to the stress of the mental stress, and the mental stress suppressor or the mental using the protein A patent application was filed for an invention such as a screening method for stress enhancing substances (Japanese Patent Application No. 2009-031326).

  If these proteins are used as markers of mental stress, the accumulation of mental stress including depression is measured by measuring the increase or decrease of those proteins present in the serum when the test substance is administered. Can be found very easily.

  Therefore, the present invention provides a composition containing an effective component for the prevention and treatment of stress-related diseases including depression, a method for extracting the effective component, and the like using the invention relating to the prior application. Objective.

As a result of screening many substances using the invention according to Japanese Patent Application No. 2009-031326, the present inventors have found an extremely effective anti-stress component and a method for extracting the same and completed the present invention.
The present invention includes the following inventions.

(1) Antistress composition containing one or more fractions of any of the following (i) to (iv):
(I) Solid fraction obtained by fractionating lily root (ii) Liquid fraction obtained by fractionating lily root is mixed with a hydrophilic organic solvent to form a precipitate, and the mixture The soluble fraction (iii) obtained by dissolving a part of the precipitate is mixed with an acid by lowering the concentration of the hydrophilic organic solvent in the mixed solution obtained by An acid-soluble fraction obtained by mixing an acid-insoluble fraction obtained by mixing the acid-soluble fraction (iv) fraction (i) with an acid with an aqueous alkali solution.

  (2) The anti-stress property according to (1), characterized in that as a fractionation operation to be performed when obtaining the fraction (i) or (ii), a lily root grinding treatment, centrifugation and solvent extraction are performed. Composition.

(3) The anti-stress composition according to (1) or (2), wherein the lily is a white lily (white silver), a white lily (Yaezaki), or a lily of the tree.
(4) The concentration of the hydrophilic organic solvent in the liquid mixture obtained by mixing the liquid fraction and the hydrophilic organic solvent is 40 to 90% by weight (1) to ( The anti-stress composition according to any one of 3).

(5) The antistress composition according to (4), wherein the concentration of the hydrophilic organic solvent in the mixed solution is lowered to 20 to 50% by weight.
(6) The antistress composition according to any one of (1) to (5), wherein the hydrophilic organic solvent is ethanol.

(7) The antistress composition according to any one of (1) to (6), which contains a fraction (ii).
(8) Step 1 of fractionating lily roots by grinding, centrifugation and solvent extraction of the lily roots, and mixing the liquid fraction obtained in step 1 with a hydrophilic organic solvent to have a precipitate Step 2 for obtaining a mixed solution, and a step for obtaining a soluble fraction containing a stress suppressing component by dissolving a part of the precipitate in the mixed solution by lowering the concentration of the hydrophilic organic solvent in the mixed solution. 3. A method for extracting a stress-suppressing component, comprising:

(9) The method for extracting a stress-suppressing component according to (8), wherein the lily is a white-necked lily (white silver), a white-necked lily (Yaezaki), or a lily of the tree.
(10) The method for extracting a stress-suppressing component according to (8) or (9), wherein the concentration of the hydrophilic organic solvent in the mixed solution obtained in the step 2 is 40 to 90% by weight. .

  (11) The extraction of the stress-suppressing component according to any one of (8) to (10), wherein the concentration of the hydrophilic organic solvent in the mixed solution is lowered to 20 to 50% by weight in the step 3 Method.

(12) The method for extracting a stress suppressing component according to any one of (8) to (11), wherein the hydrophilic organic solvent is ethanol.
(13) A stress disease inhibitor comprising the anti-stress composition according to any one of (1) to (7).

(14) A food and drink for stress suppression containing the anti-stress composition according to any one of (1) to (7).
(15) A food / beverage product for preventing and / or improving stress-related diseases comprising the anti-stress composition according to any one of (1) to (7).

  According to the present invention, there are provided an anti-stress composition containing a specific fraction obtained by fractionating a lily root, an agent for suppressing stress-related diseases such as depression, and the like.

FIG. 1 shows the detection of three stress markers (haptoglobin α chain, serum amyloid P, serum amyloid A1) and a known marker (haptoglobin β chain) used in the invention of Japanese Patent Application No. 2009-031326 in Test Example 1 by Western blotting. Bands and their stress dependence. Three mice were bred each with or without stress load, and here, serum from each one was analyzed as a representative example. FIG. 2 shows the results of examining whether or not the increase in serum haptoglobin β chain and serum amyloid P due to stress load on mice for 12 hours per day for 3 days is suppressed by lily roots in Test Example 1. Mouse groups A to F were each composed of 3 mice, and the same stress was applied to all groups. Groups A to C were bred with normal food only, and D to F were pre-administered with raw lily roots from the day before stress loading, and were given raw lily roots only for half a day during the stress loading period and thereafter. Each mouse received an average of 3.3 g of fresh lily roots per day. Blood collection is performed on the first day (groups A and D), the second day (groups B and E), and the fourth day (groups C and F) from the end of stress loading, and haptoglobin β chain in the serum of each mouse The presence of serum amyloid P was verified. In group A, blood could not be collected from one animal, and only two animals were obtained. The left panel of FIG. 3 shows the results of evaluating the behavioral changes of the stressed mouse groups A and B by the light / dark search method in Test Example 2. Group A was bred only with normal food, and Group B was pre-administered with raw lily roots from the day before stress load, and was given raw lily roots for half a day during the stress load period and thereafter. The behavior test was performed on the day before the start of stress loading, and on the first and third days of loading. Each group consisted of 3 mice, and each mouse was tested for 5 minutes and showed the mean and standard deviation of staying time in the light room. The right panel of FIG. 3 shows the results of evaluating three types of stress markers for mouse groups A and B that have completed three stress loads and subsequent behavioral tests. Group C is a control without stress loading. FIG. 4 shows the relationship between the intake of raw lily root and the anti-stress effect in Test Example 3. Five groups of mice (three each) were stressed, and during that time period, each group was given the amount shown in the figure, that is, 0-2.4 g / unit of live lily root. After repeating the stress load of 12 hours per day for 3 days, the amounts of the three markers Hpβ, SAP and SAA1 in the serum were evaluated. The results are shown as the mean and standard deviation of 3 mice. FIG. 5 shows the results of separating lily root components into a water-soluble fraction and an insoluble fraction in Example 1 and evaluating the anti-stress function of each fraction using two types of markers Hpβ and SAP. For the marker amount, the average value of three animals when fed only with normal food was taken as 100, and the relative value was plotted. Also shown is the standard deviation of the values for the three animals. FIG. 6 shows that the water-soluble fraction obtained in Example 1 was first precipitated by adding ethanol until the ethanol concentration reached 80% by weight, and then water was added to the liquid to reduce the ethanol concentration. It is a figure which shows the result of having tried re-dissolution of the functional component contained in the said precipitation, reducing in steps, 40, 20, and 0% (Example 2). Three mice were bred under each condition for the ethanol concentration, and the mean values and standard deviations for the two markers Hpβ and SAP were plotted. FIG. 7 is a diagram showing the results of evaluating the stress-suppressing function of components solubilized with acid by adding markers to the water-insoluble fraction obtained in Example 3 using markers Hpβ and SAP. Acid-insoluble components are solubilized in 0.1M NaOH, and the remaining components are evaluated as stress-inhibiting functions of those fractions (alkali-soluble fraction and acid / alkali-insoluble fraction) as insoluble fractions. did. These results are also shown in FIG. In addition, each ingested fraction is shown in the graph. The marker amount was shown as a relative value, with the marker amount of a mouse bred only with normal food as 100. The number of mice to be measured was 3 for each fraction, and the standard deviation for each mouse is also shown in FIG. FIG. 8 is a diagram showing the results of preparing a 40% ethanol fraction from six varieties of lily roots and orally administering to a mouse the amount obtained from 3.3 g of raw lily roots in Example 4. Anti-stress function was evaluated using two markers, Hpβ and SAP. The relative amount of each lily species was shown as the average value and standard deviation of three mice, assuming that the amount of marker when water was administered in the same volume as the 40% ethanol fraction was 100.

Hereinafter, the present invention will be described in detail. The present invention has been completed using the invention according to Japanese Patent Application No. 2009-031326.
The invention according to Japanese Patent Application No. 2009-031326 is the analysis of proteins expressed in blood by two-dimensional electrophoresis before and after applying a mental stress to mice. The expression was confirmed, and among these spots, three spots with particularly large increase due to the stress of mental stress and excellent reproducibility were obtained.
It is based on the fact that it was found in the vicinity of ˜6.4 and molecular weight of 9˜27 kDa and identified as serum amyloid P, serum amyloid A1, and haptoglobin α chain.

That is, the invention described above refers to blood, serum, or serum amyloid P in a mammal that has been subjected to mental stress and to which a test substance has been administered before the mental stress has been applied. Quantify any one or more of the three proteins of serum amyloid A1 and haptoglobin α chain,
The test substance is evaluated by comparing it with the amount of the three proteins in blood, serum or plasma derived from a mammal that has not been administered the test substance and is loaded with the mental stress. This is a screening method for a physical stress suppressor or a mental stress enhancer. In evaluating each fraction of lily roots contained in the anti-stress composition of the present invention, instead of haptoglobin α-chain, it is similarly loaded with mental stress, The increased amount of haptoglobin β chain was used as a stress marker.

[Anti-stress composition of the present invention]
The antistress composition of the present invention contains any one or more of the above-mentioned lily root-derived fractions (i) to (iv). Lily root has been used as a traditional Chinese medicine and is said to have a sedative effect, but there has been no scientific evidence of its effect.

  Patent Document 1 describes that lily bulb has an antidepressant action, which is based on the result that a sedative effect was observed in an experiment using rats. However, this sedative effect lacks any numerical support. In addition, the present inventor has newly found that by extracting lily roots, a plurality of fractions exhibiting a depressive effect higher than that of lily roots can be obtained. Furthermore, since the anti-stress composition of the present invention contains a component derived from a natural food material that has been used safely for a long time as an active ingredient, the composition is safe with a low possibility of causing side effects.

  Furthermore, although there are many types of lilies, as will be apparent from the examples described later, the tiger lily (white silver), the tiger lily (Yaezaki), and the lily lily are extremely excellent in stress-suppressing action.

In obtaining the predetermined fraction contained in the anti-stress composition of the present invention, for example, fractionation is performed by fractionating the lily root in a state of being pulverized to an appropriate size, and A solid fraction is obtained. Both of these fractions have a stress-inhibiting effect, but when these are further extracted, some fractional components obtained are fractionated into those that have a stress-inhibiting effect and those that are not particularly effective. The fractional component is part of it. Hereinafter, these fractionation methods and extraction fractions will be described.

<Fractionation of stress inhibitory components from lily roots>
By separating the lily root, it is separated into a liquid fraction and a solid fraction. In addition, in order to improve the extraction efficiency in the extraction operation described later, an appropriate process can be performed on the lily root sample in advance. An example of such treatment is degreasing.

  In the fractionation operation, the lily root is usually first pulverized so that it can be easily separated into a liquid fraction and a solid fraction. The pulverization process can be easily performed by a food processor or the like. In the pulverization process, for example, a lily root sample is pulverized to a size of about 2 mm.

Next, the liquid component in the pulverized product can be separated from the solid content (residue) to some extent by squeezing, crushing, or lowering the pulverized product.
Since the obtained liquid component contains a slight solid content, the mixed solid content can be separated as a precipitate by centrifuging the liquid component. Although there is no restriction | limiting in particular in the conditions of centrifugation, Usually, it is about 5 to 20 minutes at 5000-20000xg. Centrifugation may be further repeated for the liquid component obtained by centrifugation. By repeating the centrifugation operation, a cleaner liquid fraction can be obtained. The precipitate thus obtained is combined with the above residue and used to obtain a solid fraction.

  On the other hand, the liquid component is slightly mixed in the solid content (residue) obtained by squeezing the pulverized product. Therefore, for example, a liquid component contained in the solid content is extracted by adding a solvent such as water to the solid content. As described above, a precipitate generated in the process of obtaining the liquid fraction may be added to this residue. The extraction operation method is not particularly limited, and may be performed according to a conventional method. In order to improve extraction efficiency, you may perform heating, stirring, shaking, etc. during extraction operation. Centrifugation may also be performed. Centrifugation is preferable because the liquid component and the solid content are separated cleanly. Note that the centrifugation conditions at this time are the same as in the above case. A solid fraction is obtained by the above operation.

The solvent used for the extraction may be water, a hydrophilic organic solvent, or a mixture of two or more thereof. Also, extraction using a supercritical fluid is possible.
The type of water is not particularly limited, and may be pure water, tap water or the like. In addition, water may be subjected to usual treatments such as purification, sterilization, sterilization, filtration, osmotic pressure adjustment, buffering, for example, physiological saline and buffer solutions such as phosphate buffer It can be used as an extraction solvent. As the extraction solvent, purified water, physiological saline and buffer are preferable, and purified water is particularly preferable.

  Examples of the hydrophilic organic solvent include alcohols such as methanol, ethanol, butanol, glycerin, propylene glycol and 1,3-butylene glycol, acetone, tetrahydrofuran, acetonitrile, 1,4-dioxane, pyridine, dimethyl sulfoxide, N, N- Examples include dimethylformamide and acetic acid.

  In this way, lily roots are fractionated to obtain a liquid fraction and a solid fraction. As will be apparent from the examples below, both of these fractions are surprisingly more stress-inhibiting than the lily root itself when considered on the basis of the dose required to achieve the same effect.

The reason for this is that, as will be discussed in the examples below, many components are mixed in the lily root, the component having a stress suppressing action is not a kind, and there is also a component that inhibits the effect of the active component, It is thought that these are separated into a combination of an active ingredient and an inhibitory ingredient so that the stress suppressing effect is higher than that of the lily root itself by the above fractionation operation.
In the present invention, various treatments may be applied to the solid fraction. For example, what dried this fraction is also contained in the solid fraction in this invention.

<Extraction of stress inhibitory components from liquid fraction>
When the liquid fraction obtained by the extraction is mixed with a hydrophilic organic solvent, the solubility of various components (including stress-suppressing components) in the liquid fraction is reduced, and a precipitate is generated. And when the density | concentration of the said hydrophilic organic solvent in this liquid mixture is lowered | hung, the produced | generated precipitation redissolves. A soluble fraction that is thought to have a very low content of contaminants that are thought to re-dissolve the stress-suppressing component and inhibit the activity of the stress-suppressing component by appropriately reducing the concentration of this hydrophilic organic solvent. A can be obtained. As will be apparent from the examples described later, this soluble fraction A has a much higher stress suppressing effect than the lily root itself than the liquid fraction and the solid fraction.

  As the hydrophilic organic solvent, those similar to the hydrophilic organic solvent described in the description of “fractionation of stress-suppressing components from lily root” can be used, but a viewpoint of obtaining a fraction having a high stress-suppressing effect. From the above, alcohol is preferable, and ethanol is particularly preferable.

  The concentration of the hydrophilic organic solvent in the mixed solution obtained by mixing with the liquid fraction is usually 30 to 95% by weight, preferably 40 to 90% by weight. Thus, by increasing the concentration of the hydrophilic organic solvent in the mixed solution, the solubility of various components including the stress suppressing component is lowered.

  Next, there is no particular limitation on the method of lowering the concentration of the hydrophilic organic solvent in the mixed solution, but typically, the solvent (for example, water) used for the extraction of the liquid component from the solid content is mixed. By adding to the liquid, the concentration of the hydrophilic organic solvent in the mixed liquid is lowered.

  By such a method, the concentration of the hydrophilic organic solvent in the mixed liquid is usually lowered to a concentration of 10 to 60% by weight, preferably 20 to 50% by weight. When the concentration of the hydrophilic organic solvent is lowered to the above concentration, the precipitate in the mixed solution is redissolved. It should be noted that if the concentration of the hydrophilic organic solvent is lowered too much, impurities that are considered to inhibit the activity of the stress suppressing component other than the stress suppressing component are also dissolved.

  By carrying out precipitation and re-dissolution with a hydrophilic organic solvent as described above, it is possible that the content of a contaminant that is thought to contain a stress-suppressing component and inhibit the activity of the stress-suppressing component is very low. Soluble fraction A can be obtained.

  In the present invention, the soluble fraction A may be subjected to various treatments. For example, the soluble fraction A obtained by drying the soluble fraction A and volatilizing the extraction solvent used for the re-dissolution, or the soluble fraction A diluted with a solvent such as water is also used in the present invention. include.

<Extraction of stress-suppressing components from solid fraction>
By mixing the solid fraction obtained by fractionating the lily root with an acid, an acid-soluble fraction and an acid-insoluble fraction can be obtained.

(Acid soluble fraction)
As the acid, either an inorganic acid or an organic acid can be used. Examples of acids include hydrochloric acid, sulfuric acid and acetic acid. Among these, hydrochloric acid is preferable from the viewpoint of obtaining a fraction having a high stress suppressing effect.

The concentration of the acid is usually set to 0.02 to 1M so that sufficient acidity is expressed and the stress suppressing component is not destroyed.
The acid-soluble fraction thus obtained has a high stress suppressing effect, as will be apparent from the examples described later. The acid-soluble fraction may be neutralized with an alkali, or a hydrophilic organic solvent such as ethanol may be added to reduce the solubility of the stress-suppressing component, and the stress-suppressing component may be recovered as a precipitate. Good. In the present invention, such precipitation itself is also included in the acid-soluble fraction. The acid-soluble fraction may be used after diluting with a solvent such as water.

  Moreover, after mixing the solid fraction and the acid, the acid-soluble fraction and the acid-insoluble fraction can be separated to a higher degree by centrifuging. Centrifugation is usually 5000 to 20000 × g for 5 to 20 minutes.

(Acid-insoluble fraction)
On the other hand, the acid-insoluble fraction also contains a stress suppressing component. This stress suppressing component can be extracted by mixing the acid-insoluble fraction with an alkaline aqueous solution. By mixing the acid-insoluble fraction with an alkaline aqueous solution, an alkali-soluble fraction and an alkali-insoluble fraction are obtained.

  Examples of the alkaline aqueous solution include an aqueous sodium hydroxide solution and an aqueous potassium hydroxide solution. Among these, an aqueous sodium hydroxide solution is preferable from the viewpoint of obtaining a fraction having a high stress suppressing effect.

The concentration of the alkaline aqueous solution is usually set to 0.05 to 1M so that sufficient alkalinity is expressed and the stress suppressing component is not destroyed.
If the acid is removed from the acid-insoluble fraction, the amount of the alkaline aqueous solution used can be reduced.

  The alkali-soluble fraction thus obtained has a stress suppressing effect, although not as much as the acid-soluble fraction. The alkali-soluble fraction may be neutralized with an acid, or a hydrophilic organic solvent such as ethanol may be added to reduce the solubility of the stress-suppressing component, and the stress-suppressing component may be recovered as a precipitate. Good. In the present invention, such precipitation itself is also included in the alkali-soluble fraction. The alkali-soluble fraction may be diluted with a solvent such as water.

<Anti-stress composition>
The anti-stress composition of the present invention contains any one or more of the fractions having a stress-suppressing effect described above, that is, the following fractions (i) to (iv).

(I) Solid fraction obtained by fractionating lily root (ii) Liquid fraction obtained by fractionating lily root is mixed with a hydrophilic organic solvent to form a precipitate, and the mixture The soluble fraction (iii) obtained by dissolving a part of the precipitate is mixed with an acid by lowering the concentration of the hydrophilic organic solvent in the mixed solution obtained by Acid-soluble fraction (iv) An alkali-soluble fraction obtained by mixing the acid-insoluble fraction obtained by mixing the fraction (i) with an acid with an aqueous alkali solution.

It should be noted that all fractions except fraction (iv) have a higher effect than lily root itself when considered at the dose required to show the same stress-inhibiting action. is there. In particular, the effect of fraction (ii) is remarkable.

  The reason why many fractions have a higher stress suppressing effect than the lily root itself is that, as described above, the stress suppressing component mixed in the lily root and the component that inhibits the effect of the effective component are It is thought that this is because the fractionation operation separates the active ingredient / inhibitory ingredient combination so that the stress suppression effect is higher than the lily root itself.

  Such knowledge about the lily root has been obtained for the first time by the present inventor, and in Patent Document 1, there are several fractions having a higher stress effect than the lily root itself by such fractionation operation. There is no description or suggestion about what is obtained.

[Stressed disease inhibitor of the present invention]
The anti-stress composition of the present invention can be formulated by a conventional method. The dosage form of the preparation is not particularly limited and is appropriately selected as necessary.Generally, tablets, capsules, granules, fine granules, powders, solutions, syrups, suspensions, emulsions, Oral agents such as elixirs, or parenteral agents such as injections, drops, suppositories, inhalants, transdermal absorption agents, transmucosal absorption agents, patches, ointments and the like.

  The oral preparation can be produced according to a conventional method using the antistress composition of the present invention and excipients such as starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, inorganic salts and the like.

In addition to the above-mentioned excipients, this type of preparation can appropriately contain a binder, a disintegrant, a surfactant, a lubricant, a fluidity promoter, a corrigent, a colorant, a fragrance and the like.
Specific examples of the binder include crystalline cellulose, crystalline cellulose / carmellose sodium, methylcellulose, hydroxypropylcellulose, low-substituted hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, carmellose Sodium, ethylcellulose, carboxymethylethylcellulose, hydroxyethylcellulose, wheat starch, rice starch, corn starch, potato starch, dextrin, pregelatinized starch, partially pregelatinized starch, hydroxypropyl starch, pullulan, polyvinylpyrrolidone, aminoalkyl methacrylate copolymer E, amino Alkylme Chestnut Rate copolymer RS, methacrylic acid copolymer L, methacrylic acid copolymer, polyvinyl acetal diethylamino acetate, polyvinyl alcohol, gum arabic, gum arabic powder, agar, gelatin, white shellac, tragacanth, purified sucrose, macrogol.

  Specific examples of the disintegrant include crystalline cellulose, methylcellulose, low-substituted hydroxypropylcellulose, carmellose, carmellose calcium, carmellose sodium, croscarmellose sodium, wheat starch, rice starch, corn starch, potato starch, and partial alpha. And modified starch, hydroxypropyl starch, sodium carboxymethyl starch and tragacanth.

Specific examples of the surfactant include soybean lecithin, sucrose fatty acid ester, polyoxyl stearate, polyoxyethylene hydrogenated castor oil, polyoxyethylene polyoxypropylene glycol, sorbitan sesquioleate, sorbitan trioleate, monostearic acid Examples include sorbitan, sorbitan monopalmitate, sorbitan monolaurate, polysorbate, glyceryl monostearate, sodium lauryl sulfate, and lauromacrogol.

  Specific examples of the lubricant include wheat starch, rice starch, corn starch, stearic acid, calcium stearate, magnesium stearate, hydrous silicon dioxide, light anhydrous silicic acid, synthetic aluminum silicate, dry aluminum hydroxide gel, talc , Magnesium aluminate metasilicate, calcium hydrogen phosphate, anhydrous calcium hydrogen phosphate, sucrose fatty acid ester, waxes, hydrogenated vegetable oil, polyethylene glycol.

Specific examples of the fluidity promoter include hydrous silicon dioxide, light anhydrous silicic acid, dry aluminum hydroxide gel, synthetic aluminum silicate, and magnesium silicate.
As described above, the stress disease inhibitor of the present invention, which can be produced by a conventional method using the anti-stress composition of the present invention and additives such as excipients, suppresses the stress of the anti-stress composition. Based on the action, it can be used for prevention, treatment or amelioration of various stress diseases.

  Examples of the stress disease include depression, stress-induced gastric ulcer, duodenal ulcer, insomnia and the like. Among these, the stress disease inhibitor of the present invention can be suitably used for depressed patients.

  In addition, the active ingredient of the stress-related disease inhibitor of the present invention is derived from lily root, and since lily root has been widely used as a natural food material in the past, it has almost no side effects and is very Expected to be safe.

  The dose of the stress disorder inhibitor of the present invention is usually 10 g to 1000 g per day, and usually 1 to 3 times a day, depending on which fraction described above contains the fraction. Be administered. In addition, the dosage and the number of administrations can be appropriately changed in consideration of the patient's age, sex, weight, symptoms and the like.

[The food and drink for stress suppression and the food and drink for preventing and improving stress-related diseases of the present invention]
As described above, the anti-stress composition of the present invention contains a component derived from a lily root that has been conventionally used as a natural food material as a stress-suppressing component. Therefore, it is expected that the anti-stress composition of the present invention can be safely applied to food and drink. That is, the antistress composition of the present invention can be prepared in the form of a food or drink by a conventional method.

  The “food and beverage” includes, for example, tablets (tablets) and sugar-coated tablets including candy, troches and the like, granules, powdered drinks, powdered soups and the like, biscuits and other block confectionery forms, capsules, It may be in any form such as a jelly form, a paste form such as a jam, a gum form such as a chewing gum, or a supplement form, and may be a food for specific health use (for example, a food for preventing depression) . Moreover, as a form of a drink, the soft drink containing tea, an alcoholic drink, a lactic acid bacteria drink, a coffee drink etc. are mentioned.

  The stress-suppressing food or drink of the present invention that can take such various forms can be used for the prevention and improvement of various stress-related diseases based on the stress-suppressing effect of the contained anti-stress composition. . Examples of the stress disease are as described above.

  In the present invention, the stress-suppressing food and drink and the stress-related disease prevention and improvement food and drink may be blended with various other ingredients that are usually used as raw materials for food and drink, as long as the effects of the present invention are not impaired. it can.

  Examples of the other components include water, alcohols, sweeteners, acidulants, colorants, preservatives, fragrances, excipients, stabilizers, pH adjusters, sugars, various vitamins, minerals, and antioxidants. , Solubilizers, binders, lubricants, suspending agents, wetting agents, film-forming substances, flavoring agents, flavoring agents, surfactants, fluidity promoters and the like. These components can be used alone or in combination.

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

[Test Example 1]
[Verification of anti-stress function of fresh lily root]
(Obtain lily root)
There are many varieties of lily having an anti-stress function in the rhizome part, but the strength varies among varieties (see Example 4 described later). Since high activity is observed in the white silver of “Koonyuri” which is most widely distributed for food use, the present application mainly shows the results obtained with this variety. Silver from Hokkaido, such as Furano Town and Yuni Town, was obtained from autumn to winter after harvest and stored at 4 ° C until spring. It is desirable to avoid further storage because the roots grow and the bulbs discolor. Moreover, the thing preserved for a long time showed the tendency for the sweetness of a large outside bulb to fall and the intake of a mouse | mouth to decrease. In that case, a small scale close to the inner core was used.

(Mouse breeding, stress load, lily root administration, and blood sampling)
Anti-stress activity of lily roots was evaluated by giving lily roots to stressed mice. The mice used were 5-week-old CD1 (ICR) male mice (26-31 g body weight), and 3 mice per cage were preliminarily raised for 7 days. Place a flooring in the rearing cage (28cm x 17cm)
Normal chow and water were freely consumed.

Stress was applied from the 8th to the 10th day of breeding. In this case, the cage was filled with shallow water (25 ° C.) having a depth of about 2 mm instead of flooring, and stress was applied by transferring three mice to the cage for 10-12 hours only during the day or at night. According to the present inventors' studies so far, it has been obtained that the nighttime is slightly more stressful. For the control mice that did not stress, normal breeding was continued as it was.

Mice subjected to the lily root function test were given lily roots only during the stress-loaded time period and their anti-stress function was evaluated. Lily roots were ingested freely by placing scales on a wire mesh instead of regular chow. The lily root intake was determined from the reduced weight of the lily root. If no restriction is applied, 2.3 to 3.3 g of lily root per mouse will be consumed in 12 hours. For statistical and control evaluations, serum evaluation of 3 groups of mice (stress unloaded / normal diet, stress loaded / normal diet, stress loaded / lily root + normal diet) consisting of at least 3 animals in each group is necessary.

Whole blood was collected from the heart under ether anesthesia the day after the 3-day stress load on the mice (10-12 hours each) was completed. If minimally invasive blood collection is required, a small amount of blood can be collected from the tail vein. The obtained blood was allowed to stand at room temperature for 1 hour and then centrifuged (11,000 × g, 10 minutes), and the supernatant was stored as serum at −80 ° C.

(Evaluation of anti-stress function of lily root by stress marker)
In Japanese Patent Application No. 2009-031326, the present inventor showed that haptoglobin α chain (hereinafter Hpα), serum amyloid P (hereinafter SAP), or serum amyloid A1 (hereinafter SAA1) in mouse serum can be used as a stress biomarker. (The amount in these sera increases with stress loading). As will be described below, haptoglobin β chain (hereinafter Hpβ) can also be used as a stress biomarker. Here, the result of verifying the anti-stress function of lily root using Hpβ, SAP, and SAA1 is shown.

Proteins contained in 1.1 mL of serum from mice loaded with stress and mice not loaded with stress were separated by SDS-polyacrylamide electrophoresis (12.5% acrylamide). The gel after electrophoresis is overlaid on the nitrocellulose membrane, and in Protein Transfer Buffer (25 mM Tris, 192 mM Glycine, 20% methanol), a voltage of 30 V is applied perpendicularly to the gel surface, and the protein is placed on the membrane by electrophoresis for 13 hours. (Blotting).

The blotted nitrocellulose membrane is blocked by immersing it in a blocking solution (TBS Buffer (50 mM Tris-HCl pH7.6, 150 mM NaCl) containing 2% skim milk) for 1 hour, and then a blocking solution containing the antibody at 1: 1000. The reaction was allowed to proceed for 1 hour. Anti-haptoglobin β chain antibody (chicken polyclonal IgY, Abcam), antiserum amyloid P antibody (goat polyclonal IgG, Santa Cruz) or antiserum amyloid A1 antibody (goat polyclonal IgG, R & D Systems) Company).

  After washing the membrane reacted with the antibody, anti-IgY antibody (goat polyclonal, Santa Cruz) labeled with HRP (horseradish peroxidase) for the antibody consisting of IgY, and HRP labeling for the antibody consisting of IgG The protein G was reacted in a blocking solution containing 1: 1000 each for 45 minutes, and then washed with TBS Buffer containing 0.1% Tween20.

The location where HRP is present on the membrane (which means that Hpβ, SAP or SAA1 is present) was detected by luminescence using a chemiluminescent reagent ECL (GE Life Science) utilizing HRP activity. Light emission is high sensitivity camera ARGUS (Hamamatsu Photonics) by photon counting
(FIG. 1).

FIG. 1 shows that the amounts of Hpα, Hpβ, SAP and SAA1 in serum increase due to stress, and thus can be used as stress biomarkers.
By applying this result, the anti-stress function of the lily root was verified (FIG. 2). In this functional test, raw lily roots were pre-administered from the day before stress load, and raw lily roots were administered for half a day during the stress load period and thereafter. Each mouse ingests approximately 3.3g of lily root per day. In the lily root administration group (right panel), the concentration of the band corresponding to Hpβ and SAP is low, but in the non-lily root administration group (left panel), the band remains without disappearing until 4 days after the end of stress loading. I understand. From this result, it was verified that the lily root has a component having an action to relieve stress and accelerate recovery.

[Test Example 2]
[Confirmation of anti-stress function of lily root based on mouse behavior test]
In Test Example 1, the anti-stress function of lily root was verified by a biochemical evaluation method using a stress biomarker developed by the present inventors. However, the evaluation of stress accumulation has so far been based on mouse behavioral tests. Therefore, the effects of mouse behavior before and after stress load and the presence or absence of lily root administration on mouse behavior were examined, and the consistency between the evaluation method using the marker in Test Example 1 and the evaluation method based on the behavior test was verified.

Various mouse behavioral tests that are thought to reflect mental stress are known. Of these, the one with the fewest individual differences was the “light / dark search test”. In this method, the mouse is placed in a box divided into a light room and a dark room (each vertical 20x horizontal 30x height 20cm), and there is a passage (4cmx4cm) through which the mouse can pass between the two rooms. This is a test that uses the behavior that mice spend less time in the light room (200 lux) to escape to a dark place (Taku Yamaguchi, Mitsuhiro Yoshioka. Nihon Pharmacology, 2007; 130: 105-111).

  The conditions of stress load (nighttime) and lily root administration are the same as in Test Example 1. The first behavior test is 16:00 the day before stress load, the second test is 16:00 corresponding to 5 hours after the end of stress load on the first day, and the third test is after the stress load on the third day is completed. 5 hours later. After the third behavioral test, blood was collected and the markers were analyzed.

FIG. 3 shows the results. It can be seen that in the lily root administration group (group B, 3 animals), the staying time in the bright room is remarkably increased as compared to the non-administration group (group A, 3 animals) (left in FIG. 3). Therefore, it was clarified that the lily root has an anti-stress function also by the stress evaluation by the conventional method. The result of detecting three types of stress markers in each mouse is a photograph in the right panel of FIG. For the No. 3 mouse in the lily root administration group B, Hpβ and SAP among the three markers
The band is still in a level that cannot be ignored, and the cause of this is thought to be a delay in recovery from stress. This was well reflected in behavioral tests, and only this mouse out of the three had a short stay in the light room, which resulted in a large standard deviation in stay time.

[Test Example 3]
[Neutral root liquor intake necessary for anti-stress effect]
In this test example, in order to know the amount of intake necessary for the raw lily root to exhibit the stress-suppressing effect, a test was conducted in which the amount of intake was controlled in 5 stages from 0 to 2.4 g / day / animal. After the mouse ingested a predetermined amount of fresh lily root, it was switched to a normal chow diet. FIG. 4 shows the result. Although there is a range of lily root inhibitory effects depending on each marker, an increase in the amount of marker was suppressed to 20% or less with intake of approximately 1.5 to 2.4 g / day / animal.

Further, as shown in Test Example 1, when the intake amount is increased to 3.3 g, both markers are suppressed to below the detection limit.
[Example 1]
[Extraction of anti-stress components (1): Fractionation into soluble and insoluble components in water]
A crushed 1.7 kg of fresh lily root with a food processor was squeezed with double gauze and separated into liquid components and residues.

The liquid components were left overnight at 4 ° C., and then separated into a soluble fraction (top) and an insoluble fraction (precipitate) by centrifugation (10,000 × g, 10 minutes). In order to collect soluble components that did not enter the supernatant and remained in the insoluble fraction, the precipitate obtained here and the residue obtained above were suspended and mixed in 200 mL of water, and then centrifuged again ( It was divided into a soluble fraction (supernatant) and an insoluble fraction (precipitate and residue) by 10,000 × g for 10 minutes. Finally, a soluble fraction (liquid fraction 372 g) and an insoluble fraction (solid fraction) 1510 g were recovered.

Therefore, it is estimated that 1.7 kg of fresh lily roots consist of about 1.5 kg of residue and about 0.17 kg of liquid components (about 0.37 kg when including water used for extraction).
The soluble fraction was diluted with water and allowed to freely ingest mice with a water supply bottle during the stress load period (12 hours). Since one mouse drinks 3.3 mL of water on average for half a day, the soluble fraction was diluted to include 1.5 g of the soluble fraction derived from 3.3 g of fresh lily roots per 3.3 mL.

The effect of the soluble fraction administration on the stress biomarker in stressed mouse serum is shown in FIG. The soluble fraction had a stronger effect on SAP than Hpβ, but it showed an inhibitory effect of 80% or more by administration of 0.6 to 0.8 g (including water for dilution) per day for both.

In Test Example 3, a stress-suppressing effect was observed by administration of 1.5 to 2.4 g of raw lily roots per day, but the soluble fraction was based on the dose required to exert the same effect as lily root administration. It can be seen that the stress suppression effect is increased at least 2 to 4 times (see also [Summary of Lily Root Component Extraction Results] described later).

On the other hand, for the insoluble fraction, another 2.8 kg of raw lily roots is ground using a mixer with a high recovery rate, and then the soluble components are extracted with about 800 mL of water and the supernatant is centrifuged. Freshly prepared by removal. As a result, 2.58 kg insoluble fraction was obtained from 2.8 kg raw lily root.

  7.5 g insoluble fraction (amount obtained from about 8.5 g of fresh lily root) per cage (3 animals) was placed in a powder feeder and given to mice. The amount of intake was adjusted in two stages, and the effect on the stress biomarker in the stressed mouse serum by administering the insoluble fraction was examined.

The results are shown in FIG. In both markers, suppression of over 80% was observed by insoluble fraction intake of about 1.1 g per day. According to Test Example 3, inoculation with 1.5 to 2.4 g of raw lily roots per day showed 80% or more stress suppression function, but compared with this, the stress-inhibitory effect was about 1.5 to 2.2 in the insoluble fraction. You can see that it has doubled.

[Example 2]
[Anti-stress component extraction (2): ethanol fractionation of liquid fraction]
As described in Example 1, the soluble fraction has a stronger function of suppressing SAP increase than the function of suppressing Hpβ (upper part of FIG. 5). The components contained in the soluble fraction can be precipitated with ethanol, but the precipitate is added to the mixture containing the precipitate by adding water to 40, 20, about 0.
Redissolve by decreasing the ethanol concentration in steps of wt%, and when the ethanol concentration reaches each concentration, the soluble fraction is separated from the precipitate (thus, three soluble fractions were obtained) It was investigated whether the fraction contains an anti-stress component. The results are shown in FIG.

As a result of oral administration of 0.15 mL (about 0.15 g including water as a solvent) to mice once each day, each fraction in which the components contained in the soluble fraction were redissolved during stress loading It was revealed that many functional components were present in the 40% ethanol-soluble fraction (see FIG. 6).

Compared with Test Example 3, this fraction exhibits 0.15 mL of Hpβ inhibitory action equivalent to approximately 0.7 g of raw lily root (50% inhibition) (approximately 0.15 g including water as a solvent). It is estimated that approximately five times the effect has been achieved up to this stage. On the other hand, with respect to the SAP inhibitory action, this fraction 0.15 g has a function equivalent to 2.4 g of raw lily root, which is 16 times as effective (see also [Summary of Lily Root Component Extraction Results] described later).

  Note that the fraction administered to the mouse here is mostly the weight of water added as a solvent, and considering only the lily root component (0.023 g), the stress inhibitory action is about 30 for the Hpβ inhibitory action. The SAP suppression effect is about 100 times.

[Example 3]
[Extraction of anti-stress components (3): acid / alkali extraction of water-insoluble fraction]
In the same manner as in Example 1, 80 g of lily root was pulverized, 20 ml of water was added, and water was sufficiently extracted. Then, 22 g of liquid component and 77 g of solid content were obtained by centrifugation (10000 xg, 10 minutes). . The stress suppressing activity of these liquid components and solids is as described in Example 1.

Ethanol was added to the liquid component, water was added to the solution containing the precipitate obtained when the ethanol concentration in the solution reached 80% by weight, the ethanol concentration was lowered to 40% by weight, and the precipitate was dissolved again. The resulting liquid portion was evaporated to dryness to obtain 7 g of a solid content. The stress suppression activity of this ethanol fraction is as described in Example 2.

On the other hand, 74 mL of 0.1 M HCl was added to the above 77 g of the solid content, and the mixture was stirred on ice for 30 minutes. The acid solubilized component was recovered as a supernatant (supernatant) by centrifugation (15,000 × g, 4 ° C., 15 minutes) and neutralized with 1M NaOH. The functional component was recovered as an 80% ethanol precipitate (13 g) to obtain a test material.

The acid-insoluble fraction was acid-removed with water, suspended and stirred in 74 mL of 0.1M NaOH, and the solubilized component was recovered as an 80% ethanol precipitate (7 g). The last remaining 40 g of acid / alkali insoluble was also used as a test material.

Each fraction is equivalent to the amount obtained from 3.3 g of raw lily root per animal, that is, 0.53 g (3.3 g / 80 g × 13 g) for the acid-soluble fraction and 0.3 g for the alkali-soluble fraction ( 3.3g / 80g × 7
g) In the insoluble fraction, 1.7 g (3.3 g / 80 g × 40 g) was usually mixed with 1 g of solid feed and crushed into a feeder. This diet was given to mice during the stress period, and only normal diet was given during the normal breeding period.

FIG. 7 summarizes the results of evaluation of the antistress function of these three fractions using markers. It was revealed that the acid-soluble fraction effectively suppresses increases in both Hpβ and SAP markers.

First, regarding Hpβ inhibitory action, 0.53 g of the acid-soluble fraction was suppressed almost 100% (left of FIG. 7).
From the ratio of 3.3g of raw lily roots (see mouse group D in Fig. 2), which shows the same effect, it was found that the acid-soluble fraction increased the stress-inhibitory effect about 6 times that of raw lily roots. It was.

On the other hand, about SAP, about 80% was suppressed by 0.53 g of the acid-soluble fraction (FIG. 7 right). From the ratio of 2.4 g of raw lily roots (see Fig. 4), which shows the same effect, an effect increase of about 4.5 times that of lily roots was observed by using an acid-soluble fraction.

Although the stress-inhibiting function still remains in the alkali-soluble fraction, it was mainly an action to suppress the increase in Hpβ, and the inhibitory effect of SAP was not noticeable. In addition, the acid / alkali insoluble fraction did not have a stress suppressing function.

[Summary of lily root component extraction results]
The extraction results of lily root components having a stress suppressing function in Examples 1 to 3 are shown step by step, and Table 1 below summarizes these results focusing on the stress suppressing effect of each fraction. .

What has become clear during the extraction process is that at least two types of stress-inhibiting components (components soluble or insoluble in water) that are chemically different exist in the lily root. As can be seen from the comparison of the total activity in Table 1, these components are almost equally contained in the water extract of 2 and the extract of ethanol / hydrochloric acid in 3.

Further noteworthy is that the sum of the activities of each of the two fractions greatly exceeds the activity of the original raw lily root (set to 1 in this table). The reason for this is considered to be that raw lily roots, which contain many ingredients, also have components that have an inhibitory effect on the active ingredients. Indicates importance.

In addition, the markers Hpβ and SAP are not necessarily suppressed to the same extent in each fraction, but one of them may be more effectively suppressed. That is, each marker is not induced by a single mechanism due to stress, but it is considered that different pathways are involved in the induction of each marker.

Therefore, Table 1 summarizes the inhibitory activity for each marker. As a result, it was found that the fraction obtained by extraction with ethanol or hydrochloric acid had an effect of suppressing stress by about 4.7 to 16.7 times that of the lily root itself. However, for the 40% ethanol soluble fraction, most of the ingested amount of 0.15 g is water added to redissolve the once solidified component (0.023 g as described above). Considering this point, the substantial effect is 30 to 100 times.

[Example 4]
[Comparison of functionality among lily root cultivars]
Currently, “Shirogane” belonging to Kouniyuri is mainly distributed as edible lily.

In the examples in the present specification, the results using white silver have been shown. However, in addition to taste, it is unclear whether silver is superior to other varieties in terms of functionality. In addition, other varieties may be superior in terms of ease of cultivation or cost. Therefore, the stress-inhibiting action (inhibiting action of Hpβ and SAP) was examined for several varieties including those for flower buds in the same manner as in Example 2, and the effects were compared. In the case of flower buds, since lily roots often have astringency, the effect of suppressing stress was compared only with a 40% ethanol fraction with a weak astringency. The results are shown in FIG.

  The edible varieties Coonyuri (Shirogane) and Oniyuri (Yaezaki) have high anti-stress function, but the edible varieties Coonyuri (Tango) and Yamayuri did not show the function. On the other hand, the anti-stress function of the silver lily for flower buds was almost the same as that of white silver. However, the parent varieties Takasago Lily showed no activity, suggesting the importance of variety selection.

Claims (4)

An inhibitor of a stress-related disease selected from depression, stress-induced gastric ulcer, duodenal ulcer and insomnia, which contains one or more fractions of any of the following (i) to (iv):
(I) Solid fraction obtained by fractionation operation using lily root of white lily (white silver), white lily (Yaezaki), or lily of Shinteppou lily by pulverization, centrifugation and solvent extraction of the lily root (ii) white lily (Silver), using Tiger (double flower) or Shin lily lily root, pulverization of the lily root, with the liquid fraction obtained by fractionated is mixed with ethanol by centrifugation and solvent extraction, ethanol after concentration to produce a precipitate in a mixture of 40 to 90 wt%, the concentration of ethanol in the mixture obtained by the mixing to lower up to 20 to 50 wt%, a portion of the precipitate Soluble fraction (iii) obtained by dissolving lysate and acid-insoluble fraction obtained by mixing fraction (i) with acid and acid-soluble fraction (iv) obtained by mixing fraction (i) with acid Mix the fraction with aqueous alkaline solution. Alkali soluble fraction obtained. The inhibitor of stress-related diseases according to claim 1 , comprising a fraction (ii). Extraction of a stress disease inhibitor selected from depression, stress-induced gastric ulcer, duodenal ulcer and insomnia, characterized by comprising one or more of the following steps [I] to [IV] Method:
[I] A step of obtaining a solid fraction (i) by fractionating the lily root by crushing, centrifugation and solvent extraction using the lily root of the white lily (white silver), the lily of the white lily (Yasaki), or the lily of the genus Cyprus;
[II] Step 1 of obtaining liquor fraction (ii) by fractionating the lily root by crushing, centrifugation and solvent extraction using lily root of white lily (white silver), white lily (Yasaki), or lily of the sea urchin When,
Step 2 of mixing the liquid fraction obtained in Step 1 with ethanol to obtain a mixed solution having a concentration of ethanol of 40 to 90% by weight and having a precipitate;
By lowering the concentration of the ethanol in the mixture in up to 20 to 50 wt%, dissolve some of the precipitated in the mixture to obtain a soluble fraction containing the suppressed component of the stress disorder Step 3 ;
[III] A step of mixing the solid fraction (i) with an acid to obtain an acid-soluble fraction (iii);
[IV] A step of mixing the acid-insoluble fraction obtained by mixing the solid fraction (i) with an acid with an aqueous alkali solution to obtain an alkali-soluble fraction (iv). It has process [II], The extraction method of Claim 3 characterized by the above-mentioned.