NZ626975B2 - Hydroxymethylfurfural derivative - Google Patents

Abstract

Disclosed is a hydroxymethylfurfural derivative represented by general formula (1) where R is (I). Also disclose are methods of producing the compound (5-formylfuran-2-yl)methyl (2R)-5-oxopyrrolidine-2-carboxylate) comprising the step of heat treating an asparagus stem with hot water. The compound is suitable as a heat shock protein inducer, anti-stress agent, or autonomic nerve regulator. s suitable as a heat shock protein inducer, anti-stress agent, or autonomic nerve regulator.

Description

DESCRIPTION Title of Invention HYDROXYMETHYLFURFURAL DERIVATIVE Technical Field The present disclosure relates to hydroxymethylfiirfiiral tives, pharmaceuticals, heat shock protein inducers, anti—stress , autonomic nerve regulators, foods and drinks, and methods of producing the hydroxymethylfirrfiiral derivative.

Background Art People nowadays are d to long working hours, s physical and mental stress environment, or the like; and many of those people complain physical symptoms such as loss of appetite, sleep disruption, dizziness, or cold sweat, or mental symptoms such as a hatred or distrust of other people, emotional ility, state of being in'itated, or sed mood, even though no abnormalities are found when examinations such as physical checkup. Symptoms associated with such unidentified complaints are often diagnosed as dysautonomia. In the present circumstances, such a dysautonomia is usually treated with a drug therapy such as a minor tranquilizer or hormonal agent, treatment by diet, lifestyle changes for the better such as exercise, or the like.

[0003] It has been known that autonomic ers including dysautonomia described above are induced by excessive loads of stress. In the autonomic disorder, disturbance in the balance between the sympathetic nerve and the parasympathetic nerve (the balance of the autonomic nerve), lowered ty of the autonomic nerve, or the like is observed. The disturbance in the balance of the autonomic nerve means a state with increased activity of the hetic nerve or a state with increased activity of the parasympathetic nerve. Further, it has been known that the capacity to deal with stress is decreased by a lowered activity ofthe autonomic nerve. For instance, because ons of the gastrointestinal tract are mainly innervated by the parasympathetic nerve, prolonged tonus of the sympathetic nerve due to loads of stress suppresses the fimction of the gastrointestinal tract, resulting in gastrointestinal disorders such as loss of appetite or constipation. In addition, it is thought that when the parasympathetic nerve does not function well because of loads of stress and the activity ofthe sympathetic nerve remains ed, sleep disruption is brought about.

While there is, as described above, a close onship between stresses and autonomic disorders, there are also autonomic disorders that are not caused by loads of stress. In addition, the load of stress does not necessarily bring about the autonomic disorder and may in some cases induce other physical symptoms.

As one of the proteins called stress proteins, there are heat shock proteins (hereinafter, referred to as HSPS). HSPs are proteins with a molecular weight of about several tens of thousands to one d fifty thousand and are divided into several families on the basis of the molecular weight thereof (HSP10, HSP27, HSP40, HSP60, HSP70, HSP90, HSP110, and the like). HSPs are a group of proteins that are induced within cells when living organisms are put under physical, chemical, physiological, or mental stress. To be specific, HSPs have a role of protecting cells, exhibiting increased expression when living organism are d to various conditions ing heat, bacterial infection, inflammation, ve oxygen species, iolet rays, starvation, and hypoxia. In addition, HSPs also have ons as molecular chaperones including control of protein folding and inhibition of aggregation of abnormal proteins.

Among HSPs, HSP70 has been actively studied in particular, and has been reported to be constitutively sed in many internal organs including the gastrointestinal tract and skin. Recently, HSP70'S antiapoptotic action and anti-inflammatory action have been recognized and HSP70's cell protection effect against various stresses has been reported (Non Patent tures l to 4). Due to this, s have come to be conducted, attempting to apply materials with HSP70 inducing activities in pharmaceuticals, cosmetics, or the like. When it comes to l product-derived materials with the HSP70 inducing activity, paeoniflorin which is a major component of Paeoniae radix has been ed (Non Patent Literature 5).

Asparagus is a vegetable that is cultivated and harvested in various regions including Hokkaido in Japan. It has been found that asparagus has various biological activities. Patent Literature 1 describes that an asparagus stem extract has preventive and ative effects for various types of fatigue (such as physical fatigue or fatigue due to mental stress). Further, Patent Literature 2 describes that an asparagus stem extract has an effect of ing brain functions. Further, Patent Literature 3 describes that an of controlling the mic nerve. asparagus pseudo leaf extract has an effect Patent Literature Patent Literature 1: ined Japanese Patent Application Kokai Publication No. 2007-45750 Patent Literature 2: Unexamined Japanese Patent Application Kokai Publication No. 2007—230870 Patent Literature 3: Unexamined Japanese Patent Application Kokai Publication No. 2011-153125 Non Patent Literature Non Patent Literature 1: Xiao-Rong Chang et al, World J Gastroenterol; 13(32):4355-4359(2007) Non Patent Literature 2: Sarah Met al, FASEB J. 22, 3836-3845 (2008) Non Patent ture 3: Hirata I et al, Digestion; 79(4):243-50 (2009) Non Patent Literature 4: Tadashi Nishida et al, Journal of clinical biochemistry and nutrition; 46(1):43-51 (2010) Non Patent ture 5: Dai Yan et al, Cell Stress & Chaperones; 9(4), 378-389 (2004) [0009A] In this specification, references to prior art are not intended to acknowledge or t that such prior art is widely known or forms part ofthe common general dge in the field either in New Zealand or ere.

] In this specification, the term ‘comprises’ and its ts are not intended to exclude the presence ofother integers, ents or steps.

Summary of Invention Technical Problem r, there has thus far been no report concerning components in asparagus extracts or products obtained by processing asparagus, which components are involved in an anti-stress effect and autonomic nerve regulatory effect. [001 1] The present inventors discovered a novel hydroxymethylfurfural derivative that was derived from a product obtained by heat-treating asparagus with hot water, and found out that such a hydroxymethylfiirfural derivative has an excellent HSP inducing activity, anti-stress effect, and autonomic nerve regulatory effect, thereby completing the present disclosure. An objective of the disclosure is to provide a novel hydroxymethylfurfural derivative, a highly effective pharmaceutical, HSP inducer, anti-stress agent, and autonomic nerve regulator. Further, it is an objective to provide foods and drinks having an excellent HSP inducing activity, anti-stress , and autonomic nerve regulatory effect. Further, it is an objective to e a method of producing a hydroxymethylfurfural derivative that can reduce the cost and is simple and convenient. on to Problem In order to attain the above object, a hydroxymethylfurfiiral derivative in accordance with a first viewpoint of the present disclosure is represented by the general formula [FORMULA 1] [FORMULA 2] 0.1%! Yfl‘ (I) (II) HOOCCHZCOCO- and (III) HOOCCH2CH2COCO-).

The above-mentioned hydroxymethylfurfural derivative may be ed by heat-treating an asparagus stem with hot water.

A pharmaceutical in accordance with the invention has the above-mentioned hydroxymethylfurfural derivative as an active ent.

[0015] A heat shock protein inducer in accordance with the invention has the mentioned hydroxymethylfiirfiiral derivative as an active component.

An anti-stress agent in accordance with the invention has the above-mentioned hydroxymethylfurfiiral derivative as an active component.

An autonomic nerve regulator in accordance with the invention has the above-mentioned hydroxymethylfiirfural derivative as an active component.

In a second aspect the invention provides a method of producing a hydroxymethylfurfural derivative represented by the general formula: [FORMULA 1] \0 (>40 in, R is selected from the group consisting of the following a (I), [FORMULA 2] N “PAl 0:2:- (I ) (H) HOOCCH2C0C0-, (Ill) ZCH2C0C0-, and (IV) a hydrogen atom), comprising the step of heat-treating an asparagus stem with hot water.

The mentioned method uction may comprise the step ofan enzyme treatment.

A heat shock protein inducer in accordance with the invention has a product obtained by the method of the second aspect, as an active component.

An anti-stress agent in accordance with a ninth viewpoint of the present disclosure has a product obtained by the method of the second aspect, as an active component.

An autonomic nerve regulator in accordance with a tenth viewpoint of the present disclosure has a product obtained by the method of the second aspect, as an active component.

[0023] In a third aspect the invention provides a method for induction of a heat shock protein, anti-stress or regulation of an autonomic nerve in a non-human patient which method comprises administering a hydroxymethylfiirfiiral derivative represented by the general formula: [FORMULA 1] (wherein, R is selected from the group consisting of the ing a (I), [FORMULA 2] H f i 022.:— (I) ~ r ‘t __J (11) HOOCCH2C0C0-, (111) ZCHZCOCO-, and (IV) a hydrogen atom). [0023A] The method according to the third , wherein the hydroxymethylfiirfural derivative is obtained by heat-treating an asparagus stem with hot water. [0023B] The method according to the third aspect, wherein the hydroxymethylfurfilral derivative is contained in a product obtained by reating an asparagus stem with hot water. [0023C] In a fourth aspect the invention es the use of a hydroxymethylfurfural derivative represented by the general formula: [FORMULA 1] (wherein, R is selected from the group consisting of the following formula (I), [FORMULA 2] H H N A! KT", \ O ‘—‘:z-;;./ (I) i.“ 'l K J ‘_~_._ in the (11) HOOCCHZCOCO-, (III) HOOCCH2CH2C0CO-, and (IV) a hydrogen atom) of an preparation of a medicament to induce heat shock protein, anti-stress or regulation autonomic nerve in a patient, wherein a condition to be treated is load of stress, autonomic disorder or mic disorder by load of stress.

] The use according to the fourth aspect, wherein the hydroxymethylfurfiiral derivative is obtained by heat-treating an asparagus stem with hot water. [0023 E] The use according to the fourth aspect, wherein the hydroxymethylfurfural derivative is contained in a product obtained by heat-treating an asparagus stem with hot water.

Advantageous Effects of Invention According to the present disclosure, a novel hydroxymethylfiirfiiral derivative, a highly effective pharmaceutical, HSP inducer, anti-stress agent, and autonomic nerve regulator can be provided. In addition, foods and drinks having an excellent HSP inducing activity, anti-stress effect, and autonomic nerve regulatory effect can be provided. Further, a method ofproducing a hydroxymethylfurfural derivative that can reduce the cost and is simple and convenient can be ed.

Brief ption of Drawings is a figure showing the HSP70 mRNA expression inducing activity by a hydroxymethylfurfural tive; is a figure showing the HSP70 mRNA expression inducing activities by hydroxymethylfurfural, a product obtained by heat-treating an asparagus stem with hot water, and a product obtained by heat-treating an asparagus stem with hot water and enzyme treatment; is a figure showing the HSP70 protein expression inducing activities by hydroxymethylfurfiiral, a t obtained by heat-treating an asparagus stem with hot water, and a product obtained by reating an asparagus stem with hot water and enzyme treatment; is a figure showing changes in lipid peroxide levels in the blood serum by administration of a product obtained by heat-treating an asparagus stem with hot water in a mouse model of sleep deprivation; is a figure g s in corticosterone concentration in the blood by administration of a product obtained by heat-treating an asparagus stem with hot water in a mouse model of sleep deprivation; is a figure showing s in an incidence rate of hair loss by administration of a product obtained by heat-treating an gus stem with hot water a mouse model of sleep deprivation; is a figure showing changes in the expression level of HSP70 protein by administration of a product obtained by heat-treating an asparagus stem with hot water the stomach of a mouse model of sleep deprivation; is a figure showing changes in the expression level of HSP70 protein by administration of a product obtained by heat-treating an gus stem with hot water in the liver of a mouse model of sleep deprivation; is a figure showing changes in the expression level of HSP70 protein by administration of a product ed by heat-treating an asparagus stem with hot water the kidney of a mouse model of sleep deprivation; is a figure showing changes in the expression level of HSP70 mRNA by administration of a product obtained by heat-treating an asparagus stem with hot water human; is a figure showing changes in autonomic nervous balance by administration of a product obtained by heat-treating an asparagus stem with hot water human; is a figure showing s in autonomic nervous activity by administration of a product obtained by heat-treating an asparagus stem with hot water human; is a figure showing changes in the sion level of HSP70 mRNA by administration of a t ed by heat-treating an asparagus stem with hot water and enzyme treatment in human; is a figure showing changes in autonomic nervous balance by administration of a product obtained by heat-treating an asparagus stem with hot water and enzyme treatment in human; and is a figure showing changes in an autonomic nervous activity by administration of a product obtained by heat—treating an asparagus stem with hot water and enzyme treatment in human.

Description of Embodiments Embodiments ofthe present disclosure will be described in detail below. (l. Hydroxymethylfiirfural derivative) The hydroxymethylfurfural derivative according to the present sure is represented by the following general formula.

[FORMULA 3] \O O O ' H In the above general a, R selected from the group consisting of the ing formula (I), [FORMULA 4] oafi/ “we“ (I) “—4 (II) HOOCCH2C0C0-, and (III) HOOCCHzCHZCOCO-, and (IV) a hydrogen atom.

In cases where R is the above formula (I), the hydroxymethylfurfilral derivative is ented by the following structural formula.

[FORMULA 5] In cases where R is the above formula (I), the hydroxymethylfurfural derivative includes the following two stereo isomers (R form and S form).

[FORMULA 6] 0 o N H 0 *.“"“K | / l / H H R form 8 form In cases where R is the above (IV) a hydrogen atom, the hydroxymethylfurfural derivative is represented by the following structural formula (name of compound: hydroxymethylfurfiiral).

[FORMULA 7] The hydroxymethylfiirfural tive according to the present disclosure an asparagus stem with hot water. may be, as shown below, obtained by heat-treating The hydroxymethylfurfural derivative according to the present disclosure, as shown below, has an excellent heat shock protein inducing activity, anti-stress effect, autonomic nerve regulatory effect. (2. Method of producing a ymethylfurfiiral derivative) The method of producing a ymethylfiirfural tive according to the t disclosure comprises the step of heat-treating an asparagus stem with hot water.

In the present specification, the phrase treating with hot water" means reating in hot water. As an asparagus stem used in the present disclosure, the stem portion of, for example, green asparagus, white asparagus, purple asparagus, or the like can be used. Further, the origin of asparagus is not particularly restricted; and asparagus that is domestically produced may be used or asparagus that is imported may be used.

As long as asparagus is one that brings out effects of the present disclosure, asparagus may be selected as riate.

The step of reating an asparagus stem with hot water is carried out by, for example, adding 1 to 50 volumes of water to an asparagus stem and heating in hot water for 20 to 180 minutes. A temperature on this occasion is, preferably, 50 to 300°C.

In cases where the heat treatment is carried out under atmospheric pressure, it is preferred to be, for example, a temperature of 100°C, or more. It is to be noted that the heat ent with hot water may be carried out under pressure being applied and the pressure is preferably, for example, 0.1 to 0.2 MPa (for e, 012 MPa in cases where an autoclave is used).

As described above, the hydroxymethylfiirfural derivative according to the present disclosure is obtained by heat-treating an asparagus stem with hot water. Thus, the method of producing a hydroxymethylfurfural derivative according to the present disclosure ses the step of heat-treating an asparagus stem with hot water. In such a method of production, because the stem portion of asparagus which is widely distributed as a vegetable is used, the hydroxymethylfiirfiiral derivative can be produced at low cost. In addition, the hydroxymethylfiirfiiral tive can be simply and conveniently produced by heat-treating asparagus with hot water without using sophisticated techniques, special devices, or the like. Further, e asparagus which is a food material is subjected to the heat treatment with hot water, the hydroxymethylfurfural derivative obtained by such a method of production can be said to because of heating with hot be high in safety; and the asparagus stem can be sterilized with hot water is one water. It is to be noted that as long as a method of heat treatment that brings out effects of the present disclosure, the method may be selected as appropriate. of heat treatment For the purpose of increasing the efficiency of the step the method of with hot water to ntly produce hydroxymethylfiirfiiral derivatives, disclosure may producing a hydroxymethylfurfiiral derivative according to the present comprise additional steps illustrated below.

Examples of the above additional step may include the of finely step The asparagus stem chopping asparagus stems before the heat treatment with hot water. 0.5 to 10 cm in size. The fine chopping may be can finely d into pieces of about Or a machine such manually carried out using, for example, a knife, cutter, or the like. used. As long as a method of finely chopping is as a chopping machine or mill may be one that brings out s of the present disclosure, the method may be selected appropriate. of compressing Examples of the above additional step may include the step heat treatment with hot water. The asparagus stem can be gus stems before the compressed using, for example, a compressor. As long as a method of compressing is the method may be as one that brings out effects of the present disclosure, selected appropriate. of breaking Examples of the above additional step include, for the purpose before or after the step of down plant s or the like, the step of an enzyme treatment the heat treatment with hot water. The enzyme treatment increases the efficiency of the which allows hydroxymethylfurfural derivatives step of the heat treatment with hot water, to be more efficiently produced. For ce, an enzyme such as cellulase, of 2, 3, or more of hemicellulase, ase, amylase, or pullulanase; or a combination down fibers, pectin, these enzymes is suitably used for the e of efficiently breaking or the like in the asparagus stem. As long as an enzyme is one that brings out effects of the t disclosure, the enzyme may be selected as appropriate. In the step of the enzyme treatment, an amount to be added, temperature, and reaction time that are most appropriate for the enzyme to be used may be selected. In cases where cellulase is used, the enzyme treatment can be d out, for example, at an amount of cellulase added of 0.1 to 5%(w/w) at a temperature of 30 to 60°C, for 1 to 72 hours. The step of the enzyme treatment may be d out before the step of the heat treatment with hot water or may be afier the step of the heat ent with hot water. It is to be noted that, from the viewpoint of efficiently breaking down cellulose in the asparagus stem to more efficiently produce the hydroxymethylfurfural derivative, it is preferred to carry out the enzyme treatment by cellulase after the step of the heat treatment with hot water. As long as a method of enzyme treatment is one that brings out effects of the present disclosure, the method may be selected as appropriate.

Examples of the above additional step e the step of ically grinding residues afier the heat treatment with hot water. In the grinding, a machine such as a mill or blender may, for example, be used. As long as a method of grinding is one that brings out effects of the present disclosure, the method may be selected as appropriate.

Examples of the above additional step include the step of centrifugation or filtration after the heat treatment with hot water. Further inclusion of these steps enables the residue to be efficiently removed to obtain a heat treatment liquid. The centrifugation may be d out, for example, at a revolution of 3,000 to 7,000 rpm at 4 to 50°C. In the filtration, a commercially available filter paper, filter cloth, or the like may, for e, be used. As long as a method of centrifugation or a method of filtration is one that brings out effects of the present disclosure, the method may be selected as appropriate.

Examples of the above additional step include the step of concentrating the with hot obtained heat treatment liquid under reduced pressure after the heat treatment like. water. The concentration can be carried out, for example, by an evaporator or the As long effects of the as a method of concentration is one that brings out present disclosure, the method may be ed as riate.

Examples of the above additional step include the step of spray drying or The spray freeze drying the heat treatment liquid after the heat treatment with hot water. and a drying may be carried out, for example, at an exhaust air temperature of 70 to 90°C, chamber temperature of 80 to 100°C. As long as a method of spray drying or a method of freeze drying is one that brings out effects of the present disclosure, the method may be selected as appropriate.

By further including the additional step illustrated above in the step of heat-treating an asparagus stem with hot water, the ymethylfurfural derivative can be more efficiently produced. As long as an additional step is one that brings out effects ofthe present disclosure, the step may be selected as appropriate.

[0049] The step of heat-treating an asparagus stem with hot water will be illustrated below. The asparagus stem is finely chopped into pieces of about 0.5 to 10 cm and added with l to 50 volumes of water. The heat treatment with hot water is canied out at 50 to 100°C, or at 121°C under pressure being applied, for 20 to 180 minutes. The resultant was, after allowed to cool, added with ase at 0.1 to 5%(w/w); and enzyme treatment is carried out at 30 to 60°C, for l to 72 hours. Subsequently, es are then mechanically ground and fuged at a revolution of 3,000 to 7,000 rpm at 4 to 50°C, to spray drying to obtain a supernatant. Such a supernatant is then subjected at an exhaust air temperature of 70 to 90°C, and a chamber temperature of 80 to 100°C.

In the present specification, a "product obtained by heat-treating an refers to one ed by reating an asparagus stem asparagus stem with hot water" followed in hot water, and then removing residues by centrifugation, filtration, or the like, obtained by concentration. Further, in the present specification, a "product by heat-treating an asparagus stem with hot water and an enzyme treatment" means a product obtained by reating an gus stem with hot water that is obtained by going through the step of the enzyme treatment as described above before or alter the step of the heat treatment with hot water. In the t obtained by heat-treating an asparagus stem with hot water and the product obtained by heat-treating an asparagus stem with hot water and enzyme treatment, the aforementioned hydroxymethylfiirfural derivative is contained, for example, at least 0.05% or more as an active component.

The obtained hydroxymethylfurfiiral derivative by the method of production according to the present disclosure can be fractionated by, for example, dissolving the product ed by heat-treating an asparagus stem with hot water in water or an organic solvent (such as methanol) and subjecting to open column chromatography with a reverse phase carrier (for example, DIAION HP-20 (product name) (manufactured by Mitsubishi Chemical Corporation) or the like). It also can, for e, be fractionated by a carrier for gel filtration (for example, Sephadex LH-20 (product name) (manufactured by Pharrnacia Fine Chemicals) or the like). Further, a predetermined fraction that is eluted by the above-mentioned method can be ed, for example, by isolating by high performance liquid chromatography .

Without wishing to be bound by a particular theory, it is thought that, by heat-treating the asparagus stem with hot water as described above, an organic acid and sugars derived from the asparagus stem are d at high temperatures, thereby obtaining the hydroxymethylfurfural derivative according to the present disclosure.

Examples of the organic acid include pyroglutamic acid, u—ketoglutaric acid, and oxaloacetic acid. ile, examples of the sugar include se, glucose, e, or mannose.

[0053] For instance, it is thought that, in the step of heat-treating an asparagus stem with hot water, pyroglutamic acid and fructose which are derived from the asparagus stem are reacted at high atures, thereby obtaining the following compound.

[FORMULA 8] In the method of producing a hydroxymethylfiirfiiral derivative according to acid the present disclosure, plants other than asparagus, the plant containing an organic and sugar illustrated above, can be used as appropriate. Vegetables that n utamic acid and fructose can, for example, preferably be used. Vegetables such or carrot may, for example, be suitably used. as cabbage, broccoli, pumpkin, onion, garlic, the plant may be As long as a plant is one that brings out effects of the present disclosure, selected as appropriate.

[0055] (3. HSP inducer, anti-stress agent, and autonomic nerve regulator) autonomic nerve By the present disclosure, an HSP inducer, anti-stress agent, and regulator that contain the hydroxymethylfurfiiral tive according to the present disclosure as an active component are provided.

The HSP inducer according to the t disclosure may be used in order to induce HSP that is present in vivo or in who HSP used here is, for example, HSP70, HSP10, HSP27, HSP40, HSP60, HSP90, HSPl 10, or the like, with HSP70 being cells preferred. An HSP inducing activity can be evaluated, for example, by culturing with such a HSP inducer being added and measuring, by a known method, an the like. mRNA expression ng activity, HSP protein expression inducing activity, or As long as a method of evaluation is one that brings out effects of the present disclosure, the method may be selected as appropriate.

The tress agent according to the present disclosure can be stered The anti-stress effect can be to a living organism to y obtain an anti-stress effect. evaluated, for example, by administrating such an anti-stress agent to mammals and ing an oxidative stress index, stress hormone concentration in the blood, or the like before and after the administration. As long as a method of evaluation is one that brings out effects of the present disclosure, the method may be selected as appropriate.

The autonomic nerve tor according to the present disclosure can be administered to living organisms to thereby obtain an autonomic nerve regulatory effect.

The autonomic nerve regulatory effect can be evaluated, for example, by administrating such an autonomic nerve regulator to mammals and measuring an autonomic s balance, an autonomic nervous ty, or the like before and after the administration.

As long as it is a method of evaluation is one that brings out s of the present disclosure, the method may be selected as appropriate. r, by the present disclosure, an HSP inducer, anti-stress agent, and autonomic nerve regulator that n a product obtained by heat-treating an asparagus stem with hot water as an active component are also provided. Such a product obtained by heat-treating an asparagus stem with hot water is, as described above, obtained by heat-treating an asparagus stem with hot water. Therefore, the hydroxymethylfurfural derivative according to the t sure is contained in such a product obtained by reating an asparagus stem with hot water. (4. Foods and drinks, and pharmaceuticals) By the present disclosure, foods and drinks that contain the HSP inducer, anti-stress agent, and autonomic nerve regulator according to the present disclosure are provided. Such foods and drinks can be processed, by a conventional method, into a form suitable to eat and drink including, for example, a form of granule, a form of particulate, tablets, capsules, a form of gel, a form of cream, a form of paste, a form of suspension, a form of s solution, a form of emulsion, and a form of powder. In addition, excipients, binders, lubricants, coloring agents, disintegrants, thickeners, preservatives, stabilizers, pH ers, or the like, which are usually used in foods and drinks, can be added. Further, for the purpose of improving the quality of taste, sugars, amino acids, organic acids, glycerin, or the like can sugar alcohols, salts, fats and oils, is not ed. It is to be added in a range where the effects of the present disclosure noted that in cases where the HSP r, anti-stress agent, and autonomic nerve foods and drinks and regulator according to the present disclosure are added to existing base foods and drinks as used, any foods and drinks may be selected as appropriate as ofthe present disclosure. long as the foods and drinks are ones that bring out effects In cases where the HSP inducer, anti-stress agent, and autonomic nerve food or drink, the food or drink regulator according to the present disclosure are used as a and preferably 100 mg to 1000 can be taken at, for example, 50 mg to 2000 mg/day, stem with hot water mg/day, in terms of a product obtained by heat-treating an asparagus with hot water and enzyme (or a product ed by heat-treating an asparagus stem thereby obtain desired HSP inducing activity, anti-stress effect, and treatment) to a taken may be selected as autonomic nerve regulatory effect. The intake amount the like. appropriate on the basis of an object of intake, a form of food or drink, or The foods and drinks according to the present disclosure have both an anti-stress effect and autonomic nerve regulatory . Therefore, it is expected that effect synergistically act to bring out the anti-stress effect and mic nerve regulatory loads of stress. In addition, it is expected to a higher effect on autonomic ers by for autonomic disorders that are not bring out an autonomic nerve regulatory effect also caused by loads of . and autonomic nerve regulator The HSP inducer, anti-stress agent, according can used to the present disclosure be as a pharmaceutical. The pharmaceutical ing to the aforementioned present sure contains the active In this case, the ymethylfurfural derivative as an component. in a dosage form including, pharmaceutical can be prepared, by a conventional method, powders, capsules, syrups, and injection ons. for example, tablets, granules, Further, excipients, binders, lubricants, coloring agents, disintegrants, thickeners, preservatives, stabilizers, pH adjusters, or the like, which are usually used in pharmaceuticals, can be added. A method of administration may be selected as riate in a range where effects of the present sure is brought out, which method includes oral administration, intravenous administration, intraperitoneal administration, intraderrnal administration, and gual administration.

In cases where the HSP inducer, anti-stress agent, and mic nerve regulator according to the present disclosure are used as a pharmaceutical, the pharmaceutical can be administrated at, for example, 50 mg to 2000 , and preferably 100 mg to 1000 mg/day, in terms of a product obtained by reating an asparagus stem with hot water (or a product obtained by heat-treating an asparagus stem with hot water and enzyme treatment) to thereby obtain a d HSP inducing activity, anti-stress effect, and autonomic nerve tory effect. The dosage may be selected as appropriate on the basis of an object of administration, dosage form, patients' age and body weight, or the like.

Examples By way of the examples, the present disclosure will be concretely described below. However, the present disclosure is by no means limited to these examples.

(Example 1) (Production of hydroxymethylfilrfiiral by heat-treating an asparagus stem with hot water) Water 1.5 L was added to green asparagus stem (fresh weight 1.5 kg); and the mixture was, for the purpose of heat treatment with hot water, autoclaved (121°C, for 20 s) using an autoclave and filtered with a filter cloth. The obtained liquid was concentrated under reduced pressure in an evaporator to obtain a heat treatment product.

The obtained heat treatment product was onated by column chromatography (product name: DIAION HP-20, manufactured by Mitsubishi Chemical Corporation; 500 1.7 g. Next, the obtained mL, elution; H20, 50% methanol, 100% methanol) to obtain Hitachi L-7100, fractionation product was purified by preparative HPLC (product name: manufactured by Hitachi Ltd.) to obtain the compound (X) (5.0 mg). A column whose product name: CAPCELL PAK C18 UG 120, 20 (p X 250 mm (manufactured in Table 1 (A: H20, B: Shiseido Co., Ltd.) was used; and the mobile phase was as shown carried methanol). The flow rate of preparative HPLC was 8 mL/min and ion was absorbance detector. out at a detection ngth of280 nm by an ultraviolet [Table 1] Time A(%) B(%) (minutes) 0 80 20 80 20 50 50 40 50 50 41 80 20 45 80 20 above are shown The NMR data of the compound (X) obtained as described below. (400 MHz, DMSO-dé) 4.49 (1 H,dd,J=5.2Hz) .59 (1H,dt,J=5.6Hz) 6.55 (1H,d,J=3.6Hz) 7.48 (1 H,d,J=3.6Hz) 9.52 (1H,s) ”C—NMRO 00 MHz, DMSO-dé) a 55.9 109.7 124.5 151.7 162.1 178.0 In order to determine the structure of the compound (X) obtained above, the NMR data of a commercially ble product of hydroxymethylfiirfural (product name: -Hydroxymethylfuraldehyde, Tokyo Chemical Hanbai Co., Ltd.) was ed with that of the compound (X). The NMR data ofthe commercially available product of hydroxymethylfurfural is shown below. lH-NMR(400 MHz, DMSO'dfi) 4.51 (1H,dd,J=5.2Hz) .59 (1H,dt,J=5.6Hz) 6.60 (1H,d,J=3.6Hz) 7.49 (1H,d,J=3.6HZ) 9.56 (1 H,s) '3C-NMR(100 MHz, DMSO-dé) 6 55.9 109.7 124.4 151.7 162.1 177.9

[0073] Because the NMR data of the compound (X) obtained above matched that ofthe commercially available t, it was proven that the compound (X) was hydroxymethylfilrfural (the following structural formula). From the above, it became apparent that hydroxymethylfurfilral was contained in the t obtained by heat-treating an asparagus stem with hot water.

[FORMULA 9] Her/”\l'f, 0 O (Example 2) (Production of novel hydroxymethylfurfiiral derivative by heat-treating an asparagus stem with hot water) Water 1 L was added to green asparagus stem (fresh weight 1.0 kg); and the e was, for the purpose of heat ent with hot water, autoclaved (121°C, for 20 minutes) using an autoclave and filtered with a filter cloth. The obtained liquid was concentrated under reduced pressure in an evaporator to obtain a product obtained by heat-treating an asparagus stem with hot water. The ed heat treatment product was fractionated by column chromatography (product name: DIAION HP-20, manufactured by Mitsubishi Chemical Corporation; 500 mL, elution; H20, 30% methanol, 100% methanol). Next, the obtained fractionation product 723.8 mg was purified by column chromatography (product name: Sephadex LH-20, manufactured by cia Fine Chemicalsg250 mL, elution; H20). Further, the ed fractionation product 12.6 mg was purified by preparative HPLC (product name: Hitachi , manufactured by Hitachi Ltd.) to obtain the compound (Y) (2.0 mg). The conditions for the preparative HPLC were the same as those in Example 1.

The compound (Y) obtained above was subjected to HPLC analysis (product name: Hitachi L-7100, manufactured by Hitachi Ltd.) and, as a , the retention time was found to be 22.99 minutes. In this analytical HPLC, a column whose product name: CAPCELL PAK C18 UG 120, 4.6 (p X 250 mm (manufactured by do Co., Ltd.) was used; and the mobile phase was as shown in Table 2 (C: 20 mM sodium phosphate buffer (pH 2.3), D: acetonitrile). The flow rate of analytical HPLC was 1 mL/min and detection was carried out at a detection wavelength of280 nm by an ultraviolet absorbance detector.

[Table 2] ‘% ‘%’ (minutes) The LC/TofMS analysis data ofthe compound (Y) obtained above are shown below.

Found m/z 10([M+H]+); C11H|2N05 tical value m/z 238.07] 5([M+H]+); C1 lleNOS From the above, it became apparent that the compound (Y) obtained above has a molecular formula of C1 1H1 .NOs.

The lH-NMR data ofthe compound (Y) obtained above are shown below 'H—NMR(400 MHz, CD3OD) 5 2.00 (4H,m) 4.20 (1H,dd,J=3 .9,9.2Hz) .20 (2H,s) 6.55 (1H,d,J=3.6Hz) 7.37 (1H,d,J=3.6Hz) 9.45 (1 H,s) (Example 3) (Synthesis of novel hydroxymethylfurfurai derivative) It was estimated that the compound (Y) (C1 .H1 .NOS) obtained in Example 2 was generated by reacting utamic acid (C5H7N03) and fructose (C6H1206) d from the asparagus stem under heating. In order to verify this, a compound was synthesized by the following method using pyroglutamic acid and fructose. It is to be noted that because stereo isomers were, as described below, thought to exist for the compound (Y), acid as a starting an S(L) form and R(D) form were synthesized with L-pyroglutamic material and with D-pyroglutamic acid as a ng material, respectively.

[FORMULA 10] O O

[0081] L—pyroglutamic acid (product name: L-pyroglutamic acid, Tokyo Chemical Hanbai Co., Ltd.) 3.0 g and D-fructose (product name: D(-)-fructose, Junsei for 20 Co., Ltd.) 1.5 g were mixed in an Erlenmeyer flask and autoclaved (121°C, minutes) using an autoclave. The obtained reaction product was fractionated by column Chemical chromatography (product name: DIAION HP-20, manufactured by Mitsubishi Corporation; 150 mL, elution; H20, 30% methanol, 100% methanol). Further, a 100% methanol fraction was purified by preparative HPLC using a column ct name: CAPCELL PAK C18 UG 120, 20 (p X 250 mm, manufactured by Shiseido Co., Ltd.) to obtain S form compound (Z) (24.4 mg). The ions for the preparative HPLC were the same as those in Example 1.

[0082] D-pyroglutamic acid (product name: D-pyroglutamic acid, Tokyo Chemical Chemical Hanbai Co., Ltd.) 2.0 g and D-fi'uctose (product name: D(-)-fructose, Junsei for 20 Co., Ltd.) 1.0 g were mixed in an Erlenmeyer flask and autoclaved (121°C, s) using an autoclave. The ed on product was fractionated by column chromatography (product name: DIAION HP-20, manufactured by Mitsubishi Chemical Corporation; 100 mL, elution; H20, 30% methanol, 60% methanol). A 60% methanol fraction was concentrated to about 50 mL under reduced pressure by an evaporator then separated with ethyl acetate (50 mL X 5). An ethyl acetate layer was concentrated under reduced pressure by an evaporator and onated by column chromatography ct name: DIAION HP—20, manufactured by Mitsubishi Chemical Corporation; 10 mL, elution; H20, 30% methanol, 60% methanol). A 60% methanol fraction was concentrated under reduced pressure by an evaporator to obtain R form compound (Z) (24.6 mg).

The S form and R form compounds (Z) obtained above were subjected to HPLC analysis (product name: Hitachi L-7100, manufactured by i Ltd.) and, as a result, the retention time was found to be 22.89 minutes for each. The conditions for the analytical HPLC were the same as those in Example 2.

With regard to the S form and R form compounds (Z) ed above, MS data and NMR analysis data are shown below.

EI-MS :m/z 237 EI-HR-MS:m/z 237.0612; CHHHNOS (400 MHz, CD3OD)I S form 6 2.33 (4H,m) 4.34 (1H,dd,J=3.9,9.0Hz) .51 (2H,s) 6.73 (1H,d,J=3.4Hz) 7.38 (1H,d,J=3.4Hz) 9.57 (1 HS)

[0086] 13C—NMR(100 MHz, CD30D): s form 25.8 .2 57.0 59.6 1 14.0 124.0 154.5 156.7 173.4 179.6 181.1

[0087] lH-NMR(400 MHz, c0300): R form 8 2.33 (4H,m) 4.34 (1H,dd,J=3.9,9.0Hz) .27 (2H,s) 6.73 (1H,d,J=3.6Hz) 7.38 (1H,d,J=3.6Hz) 9.57 (1H,s) l3c-NMR(100 MHz, CD3OD): R form 8 25.8 .3 57.0 59.6 1 13.7 124.0 154.5 156.8 173.4 179.6 181.1 obtained in In order to ine the absolute structure of the compound (Y) and R Example 2, the compound (Y) obtained in e 2, the S form compound (Z) HPLC form compound (Z), both of which were obtained above, were subjected to chiral analysis (product name: Hitachi L-7100, manufactured by Hitachi Ltd.) using a column (product name: CHIRAL PAK IA, 4.6 (p X 150 mm, ctured by Daicel Corporation). The conditions for the analytical HPLC were the same as those in Example 2 except that the different column was used. As a result, the retention time of the S form compound (Z) was found to be 18.92 minutes and the retention time of the R form compound (Z) was found to be 20.57 s. Because the retention time of the compound (Y) obtained in Example 2 was 19.06 minutes, it became apparent that the compound (Y) obtained in Example 2 was the S form.

When the HPLC analysis data, LC/TofMS analysis data, and IH-NMR data in Example 2 were compared with the mentioned analysis data in this Example, the compound (Y) obtained by Example 2 and the compound (Z) obtained by this Example were shown to be identical compounds. Therefore, it became apparent that the product obtained by heat-treating an asparagus stem with hot water, which product was obtained in Example 2, at least contained the S form of hydroxymethylfinfural derivative having the following structural formula.

[0091] [FORMULA l l] Oirk 0'Y0 (Example 4) (Evaluation of HSP70 mRNA expression inducing activity) With regard to a commercially available product oxymethylfurfural (the same as 11 Example 1) (hereinafter, ed to as sample 1), the S form of hydroxymethylfurfural derivative synthesized in Example 3 (hereinafter, ed to as sample 2-8), and the R form of hydroxymethylfurfural derivative sized in Example 3 (hereinafter, referred to as sample 2-R), and a product obtained by heat-treating an the following method, asparagus stem with hot water, which product was ed by (hereinafter, referred to as sample 3), and a product obtained by heat-treating an referred to as sample asparagus stem with hot water and enzyme treatment (hereinafter, level 4), an HSP70 inducing activity was evaluated by ing the mRNA expression of HSP70.

A method of producing sample 3 is shown below. To green asparagus for the stems (fresh weight 6.63 kg), water 28.5 L was added; and the mixture was, autoclaved (121°C, 20 s). After cooling, purpose of heat treatment with hot water, filtration was carried out with a filter paper (product name: Toyo Roshi No. 5A, manufactured by Toyo Roshi Kaisha, Ltd); and tration was carried out by an evaporator. To about 10 L of the concentrated liquid, an excipient (product name: Pinedex, ctured by Matsutani Chemical Industry Co., Ltd.) 275.4 g was added and the resulting mixture was freeze-dried to obtain 542.4 g er containing a t obtained by heat-treating an asparagus stem with hot water (in this, the product obtained by heat-treating an gus stem with hot water derived from the solid content the excipient accounted for 275.4 g). asparagus stem accounted for 2670 g and A method ofproducing sample 4 is shown below. To green asparagus for the purpose stems (fresh weight 12 kg), water 24 L was added; and the mixture was, ofheat treatment with hot water, autoclaved (121°C, 20 minutes). Afier allowed to cool to 45°C, the resultant was added with sucrase C (product name) (manufactured by Mitsubishi-Kagaku Foods Corporation) 20 g and Macerozyme A ct name) (manufactured by Yakult Pharmaceutical Industry Co., Ltd.) 20 g; and enzyme treatment was carried out at 45°C, for three days. Subsequently, autoclave (121°C, 20 minutes) cloth to collect a filtrate 35 L. was d out and filtration was carried out with a filter Concentration was carried out by an evaporator until the volume reached 9 L. To this trated liquid, an excipient (product name: Pinedex, manufactured by Matsutani Chemical Industry Co., Ltd.) 1.20 kg was added; and the resulting mixture was again autoclaved (121°C, 20 s). Subsequently, freeze drying was carried out to obtain 2.12 kg of a powder containing a t obtained by heat-treating an asparagus stem with hot water and enzyme treatment (in this, the product obtained by heat-treating an asparagus stem with hot water and enzyme treatment derived from the solid content of asparagus stem accounted for 0.92 kg and the excipient accounted for 1.20 kg).

First, with regard to the sample 1, sample 2-8, and sample 2-R, an HSP70 mRNA expression level was evaluated using human promyelocytic leukemia cells (HL-60 cells).

A method of evaluating the HSP70 mRNA expression level using human promyelocytic leukemia cells (HL-60 cells) is shown below. HL-60 (source: Dainippon Pharmaceutical Co., Ltd.) was suspended in RPMIl640 medium (product name: RPM11640 medium "NISSUI" (2) powder, manufactured by Nissui Pharmaceutical Co., Ltd.) supplemented with 10% fetal bovine serum (FBS) (product name: MultiSer, manufactured by Thenno Trace) and was transferred into a 1.5 mL sampling tube (500,000 l mL/tube). At the same time, 0.1 mL ofeach of the samples (sample 1, sample 2-8, and sample 2-R) that had been prepared with ion-exchanged water was added such that the final concentration was 1 mg/mL. To a control, ion-exchanged water 0.1 mL was added. After cultured at 37°C, in the presence of5% C02 for four hours, cells were harvested at 3,000 rpm and subjected to mRNA detection. From this, total RNA was extracted using TRIzol reagent (manufactured by Life Technologies Corporation) and the concentration thereof was measured by Nanodrop (manufactured by Thermo Fisher Scientific K.K.). Using a cDNA synthesis kit (product name: ReverTra Ace qPCR RT Master Mix with gDNA Remover, manufactured by Toyobo Life Science), cDNA was sized. A on liquid after the reverse transcription was d with Nuclease-free water so as to have a concentration of 3 ng/uL to be used as a template for real-time PCR.

In PCR, HSP70 d primer (SEQ ID NO: 1) and HSP70 reverse primer (SEQ ID NO: 2) were used as primers. Beta 2 microglobulin gene was used as an internal control gene for correction of the HSP70 gene expression; and beta 2 microglobulin forward primer (SEQ ID NO: 3) and beta 2 microglobulin reverse primer (SEQ ID NO: 4) were used as primers therefor. Real—time PCR was carried out using a reaction kit (product name: SsoAdvanced SYBR Green Supermix, manufactured by Bio-Rad Laboratories, Inc.) by a real time PCR analysis system (product name: Connect, manufactured by Bio-Rad Laboratories, Inc.). A total of 10 uL ofPCR reaction liquid was subjected to a three—minute incubation at 95°C, (initial ration) followed by repetition of 40 cycles, each cycle ofwhich sed denaturation at 95°C, for 1 second and annealing at 59°C, for 10 seconds. ratio Using Cq value obtained by the above real time PCR is system, a of expression level of HSP70 gene was calculated on the basis of the following calculation formula (AACt method). It is to be noted that the Cq value represents the certain number of reaction cycles at the time when the level of amplified gene reaches a ermined level in the amplification reaction of the gene.

Cq value of control HSP70: A Cq value of control B2M: B Cq value of sample HSP70: C Cq value of sample BZM: D ACq(control)= A-B ACq(sample)= C-D A(ACq): mple)—ACq(control) Ratio of the expression level: 243““) The results are shown in In the HSP70 mRNA expression ratio inducing activity of the sample 1, sample 2-S, and sample 2-R was expressed as a (%) to that of the control. As compared with the control, the samples 1, 2-S, and 2-R 2-S and 2-R, exhibited an about 3- to 9-fold increased HSP70 mRNA sion (samples **p < 0.01 vs. control; sample 1, *p=0.069 vs. control). From this, it became apparent that hydroxymethylfurfural and the S form and R form ofhydroxymethylfurfitral tives having the following structural formula had the HSP70 inducing activity at an mRNA expression level.

[FORMULA 12] l / Next, with regard to the sample 1, sample 3, and sample 4, the HSP70 mRNA expression level was evaluated using human uterine cervical cancer cells (HeLa cells).

[0102] Human e cervical cancer cells (HeLa cells) (source: Incorporated administrative agency RIKEN, the Institute of Physical and Chemical ch, BioResource Center) were suspended in Dulbecco's Modified Eagle’s Medium (DMEM) (product name: Dulbecco's Modified Eagle's Medium "Nissui" 2 powder, manufactured by Nihon Pharmaceutical Co., Ltd.) supplemented with 10% fetal bovine serum (FBS) (product name: er, manufactured by Thenno Trace) and seeded a six-well plate (200,000 cells/2 mL/well). On the following day, the medium was replaced with fresh DMEM (1.8 mL); and 0.2 mL of each of the samples was added, which samples had been each prepared with ion-exchanged water so as to have a final concentration of 1 mg/mL (with regard to the samples 3 and 4, a final concentration of 1 mg/mL in terms of a t obtained by heat-treating an asparagus stem with hot water and a product obtained by reating an asparagus stem with hot water and enzyme treatment d from the solid content of asparagus stem). To a control, ion-exchanged water 0.2 mL was added. After cultured for 22 hours, cells were scraped off with a cell scraper and subjected to mRNA detection. From this, total RNA was extracted using an RNA extraction kit (product name: Fast Pure RNA kit, ctured by Takara Bio Inc.), d 100 fold with DEPC treated water; and the absorbance (wavelength 260 nm) was measured by a spectrophotometer. The RNA concentration was calculated using the calculation formula: absorbance ength 260 nm) x 40 X dilution factor = RNA concentration (ng/uL). An RNA solution was diluted to an optional concentration with kit ct name: a TE buffer; and cDNA was sized using a cDNA sis Prime Script 1st Strand cDNA synthesis kit, manufactured by Takara Bio Inc.) Oligo dT primer (product name) (manufactured by Takara Bio Inc.) was used as a primer. have reaction liquid after the reverse ription was diluted with the TE buffer so as to for PCR. a tration of 10 ng/uL to be used as a template In PCR, HSP70 forward primer (SEQ ID NO: 5) and HSP70 reverse primer (SEQ ID NO: 6) were used as primers. Beta 2 microglobulin gene was used as an internal control gene for correction of the HSP70 gene expression; and beta 2 microglobulin forward primer (SEQ ID NO: 3) and beta 2 microglobulin reverse primer (SEQ ID NO: 4) were used as primers therefor. For PCR, a PCR enzyme (product was used. A total of 20 uL name: TaKaRa Ex Taq, manufactured by Takara Bio Inc.) of PCR reaction liquid was subjected to a one-minute incubation at 94°C, (initial denaturation), followed by repetition of 32 cycles (HSP70) or 24 cycle (beta 2 microglobulin), each cycle of which comprised denaturation at 94°C, for 30 seconds, annealing at 57°C, (HSP70) or 59°C, (beta 2 microglobulin) for 30 seconds, and elongation at 72°C, for 30 seconds. An elongation reaction at 72°C, for 30 seconds was performed to end all PCRs. The PCR reaction liquid was electrophoresed by a conventional method; and staining with ethidium bromide was carried out.

[0104] By measuring the fluorescence intensity under ultraviolet irradiation in HSP70 AlphaView (product name) (manufactured by Alpha Innotech Corporation), the On this occasion, a value ed by being gene the expression level was measured. corrected with the expression level of the internal control gene was regarded as the expression level of HSP70 gene.

The s are shown in In the HSP70 mRNA expression inducing activity of the samples 1, 3, and 4 was expressed as a ratio (%) to that of the control. As compared with the control, the samples 1, 3, and 4 exhibited an about 1.2- to 1.5-fold increased HSP70 mRNA expression (samples 1 and 4, **p < 0.01 vs. control; sample 3, *p < 0.05 vs. control). From this, it became apparent that hydroxymethylfilrfiiral, and the product obtained by heat-treating an asparagus stem with hot water, and the product obtained by heat-treating an asparagus stem with hot water and enzyme treatment by this Example had the HSP70 inducing activity at an mRNA sion level. {0106] (Example 5) (Evaluation ofHSP70 protein expression inducing ty) The HSP70 inducing activity ofthe s 1, 3, and 4 which were the same ones as used in Example 4 was evaluated by ing an HSP70 n expression level.

In the same manner as described in Example 4, HeLa cells that had been ded in DMEM (added with 10% FBS) were seeded in a 12-well plate (100,000 cells/mL/well). On the following day, the medium was replaced with fresh DMEM (0.9 mL); and 0.1 mL ofeach ofthe samples was added, which samples had been each prepared with ion-exchanged water so as to have a final concentration of 1 mg/mL (with regard to the samples 3 and 4, a final concentration of 1 mg/mL in terms of a product obtained by heat-treating an asparagus stem with hot water and a product obtained by heat-treating an gus stem with hot water and enzyme treatment derived from the solid content of asparagus stem). To a control, ion-exchanged water 0.1 mL was added.

After culturing for 24 hours, a culture supernatant was removed and cells were washed with PBS(—) (phosphate buffered logical saline, pH 7.2). A part of the cells were then scraped off with a cell scraper, collected into a 1.5 mL sample tube, and subjected to HSP70 n quantification and total protein quantification.

The quantification ofHSP70 protein was carried out using HSP70 ELISA kit (product name) (manufactured by Enzo); and the quantification of total protein was carried out using Micro BCA Protein Assay Reagent kit (product name) (manufactured by PIERCE Biotechnology). With regard to the ing cells, an effect on cell proliferation was evaluated by 3-(4,5-dimethyl thialyl)—2,5-diphenyltetrazalium bromide (MTT) method. Subsequently, a value corrected with the amount of total ns and the number of viable cells was regarded as the amount of HSP70 protein.

The results are shown in In the HSP70 protein expression inducing activity of the samples 1, 3, and 4 was expressed as a ratio (%) to that of the control. As compared with the control, the samples 1, 3, and 4 exhibited an about 13-fold sed HSP70 protein expression (*p < 0.05 vs. control). From this, it became apparent that hydroxymethylfurfilral, the product ed by heat-treating an obtained by heat-treating an asparagus asparagus stem with hot water, and the product stem with hot water and enzyme treatment by this Example had the HSP70 inducing activity at a protein expression level.

From the above, it was demonstrated that ymethylfurfural, the product obtained by heat-treating an asparagus stem with hot water, and the product obtained by heat-treating an gus stem with hot water and enzyme treatment by this Example had an excellent HSP70 inducing activity. [01 1 1] (Example 6) (Evaluation of anti-stress effect in mouse model of sleep deprivation) The anti-stress effect ofthe product obtained by heat-treating an asparagus stem with hot water which was obtained in Example 4 (the "sample 3" in Example 4), was evaluated using a mouse model of sleep deprivation.

Thirty two male SlczddY mice at 6 weeks of age (manufactured by CLEA Japan, Inc.) were d into 4 groups (8 mice in each group). Each of the groups was designated as a normal group, a control group, a group with a low dose of the product obtained by reating an asparagus stem with hot water (hereinafter, referred to as a low dose group), and a group with a high dose of the product obtained by heat-treating an gus stem with hot water (hereinafier, referred to as a high dose group). Starting seven days before mice were put under the stress of sleep deprivation, a powder containing the product obtained by heat-treating an asparagus stem with hot water was added to regular mash feed (product name: CE-2, ctured by CLEA Japan, Inc.) and fed everyday to mice in the low dose group at a dose of 200 mg/kg (in terms of the product obtained by heat-treating an asparagus stem with hot water derived from the solid content of asparagus stem) and to mice in the high dose group at a dose of 1000 mg/kg (in terms of the same). The normal group and l group were fed the regular mash feed. In the control group, low dose group, and high dose group, mice were immersed in water for 12 hours (8:00 to 20:00) per day to apply the stress of sleep deprivation over 3 days. To the normal group, the stress of sleep deprivation was not applied. [01 13] The anti-stress effect was evaluated in mice on the day following the last load of the stress of sleep deprivation by measuring ( 1) lipid peroxide levels in the blood serum (a ratio of the amount of lipid peroxide (LPO) to the amount of triglyceride (TG) in the blood serum (LPO/TG)) as an oxidative stress index, (2) measuring the blood concentration of corticosterone which is known as a stress hormone, and (3) evaluating the incidence rate of mice with hair loss. [01 14] shows the result of ement of the lipid de level in the blood serum. In the figure, a higher value of LPO/TG tes a state of a higher oxidative stress in the blood. The value of LPO/TG was high in the control group whereas the value of LPO/TG decreased to the almost same level as the normal group in the low dose group and high dose group (*p < 0.05 vs. control). From this, it was shown that when the product obtained by heat-treating an asparagus stem with hot water by this Example was taken by mice with a state where the oxidative stress in the blood was high e of load of the stress of sleep deprivation, the oxidative stress in the blood was reduced to the level at which the stress of sleep deprivation was not d. shows the result ofmeasurement of the corticosterone concentration in the blood. In the figure, a higher value of corticosterone concentration indicates a higher stress state. The value of corticosterone concentration was high in the control group whereas the value of osterone concentration decreased to the almost same level as the normal group in the low dose group and the value of osterone concentration was reduced to the same or lower level than the normal group in the high dose group (**p < 0.01 vs. control, *p < 0.05 vs. control). From this, it was shown that when the product obtained by heat-treating an asparagus stem with hot water by this Example was taken by mice that were in a higher stress state because of load of the stress of sleep deprivation, the higher stress state was reduced to the level at which the stress of sleep deprivation was not applied and, in the high dose group, reduced r to the same or lower level than the stress of sleep deprivation was not applied. [01 16] shows the incidence rate of mice with hair loss. In the figure, a higher incidence rate of hair loss tes a higher stress state. The incidence rate of hair loss in the normal group, control group, low dose group, and high dose group was 0%, 75.0%, 37.5%, and 12.5%, respectively; and the incidence rate of hair loss was lower in the low dose group and high dose group, as compared with that of the control group.

From this, it was shown that when the product obtained by heat-treating an asparagus stem with hot water by this Example was taken by mice that were in a higher stress state because of load of the stress of sleep deprivation, the higher stress state was reduced.

From the above, it was demonstrated that the product obtained by heat-treating an asparagus stem with hot water ning the hydroxymethylfurfural derivative by this Example had an excellent anti-stress effect.

(Example 7) (Evaluation of HSP70 protein expression inducing activity in a mouse model of sleep deprivation) Using the mice used in Example 6, the HSP70 inducing activity of the product obtained by reating an asparagus stem with hot water which was obtained in Example 4 (the "sample 3" in Example 4) was evaluated by measuring the HSP70 protein expression level in the h, liver, and kidney. [01 19] On the last day ofthe study in Example 6, the mice in each of the groups were sacrificed; and the stomach, liver, and kidney were each harvested. Each of the organs (50 mg) was placed in a 1.5 mL sample tube; and 500 {L of the extraction reagent ofHSP70 ELISA kit (product name) (manufactured by Enzo) added with Protease inhibitor cocktail (product name) (manufactured by Sigma) at 0.2% (v/v) was added o. Each of the organ was then mashed on ice using a pestle rod and centrifuged at 4°C, at 1,500 rpm for 30 minutes, and the atant was collected. This supernatant was subjected to the quantification of HSP70 protein and the quantification of total proteins.

[0120] In the same manner as described in Example 5, the quantification of HSP70 protein was carried out using HSP70 ELISA kit (product name) (manufactured by Enzo) and the quantification of total proteins was carried out using Micro BCA Protein Assay Reagent kit ct name) (manufactured by PIERCE Biotechnology). A value corrected with the amount of total proteins was ed as the amount of HSP70 protein.

[0121] FIGS. 7 to 9 show the expression level of the HSP70 protein in the stomach, liver, and kidney. In FIGS. 7 to 9, the HSP70 protein expression inducing activity of the control group, low dose group, and high dose group was expressed as a ratio (%) to that of the normal group. In the stomach ( and liver (, the expression level of HSP70 n decreased in the l group as compared with the normal group; whereas the HSP70 protein expression increased in the low dose group and high dose group to the same or higher level than that in the normal group (*p < 0.05 vs. control).

In the kidney (, the HSP70 protein expression increased in the low dose group and high dose group, as compared with the control group (**p < 0.01 vs. control).

From the above, it became apparent that the product obtained by heat-treating an asparagus stem with hot water containing the hydroxymethylfurfural excellent HSP70 derivative by this Example had, even when administrated to animals, an inducing ty at a protein expression level. Further, it was suggested that one of the the HSP70 mechanisms ofaction of the tress effect shown in Example 6 was stem expression inducing activity of the product obtained by reating an asparagus this Example. with hot water containing the hydroxymethylfilrfural tive by (Example 8) (Evaluation of HSP70 mRNA expression inducing activity in human) with hot water Using the product obtained by heat-treating an asparagus stem HSP70 inducing which was obtained in Example 4 (the e 3" in Example 4), an HSP70 mRNA activity in human white blood cells was evaluated by measuring an expression level.

[0124] Three volunteers who arily expressed intent to participate were and subject 3). A employed as subjects (hereinafter, referred to as subject 1, subject 2, with hot water powder containing product obtained by heat-treating an asparagus stem for 3 days, by the subject 1 at a dose of 200 was taken twice a day (morning and evening) dose of 800 mg/day, mg/day, the subject 2 at a dose of 400 mg/day, and the subject 3 at a of such a powder of (in 200 mg ct 1), 400 mg (subject 2), and 800 mg (subject 3) with hot water, 98 mg, 197 mg, the t obtained by heat-treating an asparagus stem with hot water and 394 mg ofthe product obtained by heat-treating an asparagus stem derived from the solid content of asparagus stem were ned, respectively). blood was Before the start of the intake and on the last day of the intake, the cells was measured. drawn and the expression level of HSP70 mRNA in white blood M ammonium The blood 1 mL was mixed with 10 mL ofACK buffer solution (0.15 37°C, and kept chloride, 1.0 mM potassium onate, 0.1 mM EDTA-ZNa, pH 7.2) at at 37°C, for 10 minutes. The resultant was then centrifuged at 3,000 rpm for 5 s and the supernatant was removed. The precipitated white blood cells were again added with 10 mL ofACK buffer solution to be suspended. The same procedure was repeated three times; and Trizol reagent ct name) (Life Technologies) 1.5 mL was added to the precipitated white blood cells to extract total RNA. Procedures subsequent to this, including a PCR reaction, were carried out in the same manner as described in Example 4 (the evaluation in HeLa cells); and the expression amount of HSP70 mRNA was evaluated.

The results are shown in . In , a ratio (%) of the expression level of HSP70 mRNA in the white blood cells after the completion of the intake to that before the start of the intake is presented. The HSP70 mRNA expression in the white blood cells after the completion of the intake increased about 2.5 to 3.5 fold in a fashion dependent on the dose of the product obtained by heat-treating an asparagus stem with hot water, as compared with that before the start of the intake.

[0127] From the above, it became apparent that the product obtained by heat-treating an asparagus stem with hot water containing the ymethylfiirfural tive by this Example had, even when administrated in human, an excellent HSP70 inducing ty at an mRNA expression level.

(Example 9) 2O (Clinical evaluation of autonomic nerve regulatory effect of a product obtained by heat-treating an asparagus stem with hot water) Using the product obtained by heat-treating an asparagus stem with hot water which was obtained in Example 4 (the "sample 3" in Example 4), an mic nerve regulatory effect in human was evaluated.

[0129] Thirty eers who voluntarily expressed intent to participate were employed as subjects to carry out a randomized placebo controlled double blind study.

The ts were allocated, by using a lottery, to a o group (hereinafter, referred to as P group) 15 subjects, or a group with the t obtained by heat-treating subjects. Over asparagus stem with hot water (hereinafter, referred to as A group) four weeks during the study period, the ts in the P group took an excipient (product name: Pinedex, manufactured by Matsutani Chemical Industry Co., Ltd.) (400 mg/day) whereas the subjects in the A group took a powder containing the product obtained by heat-treating an asparagus stem with hot water (400 mg/day) twice a day (morning and evening) everyday (in 400 mg of such a powder of the product obtained by heat-treating an asparagus an asparagus stem with hot water, the product ed by heat-treating stem with hot water derived from the solid content of asparagus stem accounted for mg and the remaining 203 mg was the excipient (same as above)).

Before the start of the study and on the last day of the study, an autonomic acceleration nervous balance and mic nervous activity were evaluated using an pulse wave inspection system (product name; Pulse analyzer plus TAS-9, manufactured by YKC Corporation). Such a system is a system in which acceleration pulse waves are measured at the fingertip to thereby detect subtle changes in the heart rate (Heart Rate ility: HRV) and evaluate autonomic nerve functions. HRV is expressed as clinical consequences for various influences that the autonomic nerve brings about to the heart rate. The mic nervous balance was ted by plotting an index for sympathetic nerve activity (Low Frequency: LF) on the X axis and an index for parasympathetic nerve activity (High Frequency: HF) on the Y axis, which activities a distance were given by such a system, to prepare a two-dimensional graph, and using between the point at which the autonomic nervous balance was best and the point at a measured value on such a graph. Meanwhile, with regard to the autonomic nervous activity, cal values representing the activity of the autonomic nerve that was given by such a system (the numerical values being ated using LF, HF, and the like by such a system) were used. shows changes in the autonomic s balance. In the figure, a closer numerical value to zero tes a better balance ofthe autonomic nerve. In the P group (the o group), the balance of the autonomic nerve on the last day of the study ed, as compared with that before the start of the study. On the other hand, in the A group (the group with the product obtained by heat-treating an gus stem with hot water), the balance of the autonomic nerve on the last day of the study significantly improved, as compared with that before the start ofthe study (*p < 0.05 vs. before the start of the study, p < 0.01 vs. the placebo group). From this, it was demonstrated that, by taking the product obtained by heat-treating an asparagus stem with hot water by this e, the balance of the autonomic nerve improved.

[0132] shows changes in the autonomic nervous activity. In the figure, a higher numerical value indicates a higher mic nervous activity. In the P group (the o group), a decreased autonomic nervous activity was found on the last day of the study, as compared with that before the start of the study. On the other hand, in the A group (the group with the product obtained by heat-treating an asparagus stem with hot water), an elevated autonomic nervous activity was found on the last day of the study, as compared with that before the start ofthe study. From this, it was demonstrated that, by taking the product obtained by heat~treating an asparagus stem with hot water by this Example, the autonomic nervous activity improved.

From the above, it was demonstrated that the product obtained by heat-treating an asparagus stem with hot water ning the hydroxymethylfurfiiral tive by this Example had an excellent autonomic nerve regulatory effect.

(Example 10) (Evaluation 0 mRNA expression inducing activity of a product obtained by heat-treating an asparagus stem with hot water and enzyme treatment in human and clinical evaluation of autonomic nerve regulatory effect thereof) Using the obtained capsule filled with the product obtained by heat-treating an asparagus stem with hot water and enzyme treatment, evaluation of an HSP70 mRNA expression ng activity in human and clinical tion of an autonomic nerve regulatory effect were carried out as described below.

(Method of producing a capsule filled with a product obtained by heat-treating an asparagus stem with hot water and enzyme ent) A method for ing a capsule filled with a product obtained by heat-treating intake is shown an asparagus stem with hot water and enzyme treatment for human below. To green asparagus stems (fresh weight 130 kg), water 170 L was added; and the mixture was, for the purpose of heat treatment with hot water, subjected to heat sterilization (100°C, 45 minutes). After allowed to cool to 45°C, the resultant was added with 3.0 kg of enzymes (Sumizyme C and Sumizyme MC; ctured by Yakult Pharmaceutical Industry Co., Ltd.) and stirred at 45°C, for 24 hours. The enzymes were then deactivated (100°C, 20 minutes); and centrifugation was carried out. The resultant name: was concentrated by an evaporator, added with 9.0 kg of ent (product Pinedex, manufactured by Matsutani Chemical Industry Co., Ltd.), and aved (121°C, 45 minutes). Subsequently, by spray drying, a powder containing the product obtained by heat-treating an asparagus stem with hot water and enzyme treatment 16.0 kg (in this, the product obtained by heat-treating an asparagus stem with hot water and of asparagus stem accounted for 7.0 kg enzyme treatment derived from the solid content and the excipient accounted for 9.0 kg) was obtained. This powder containing the product obtained by heat-treating an asparagus stem with hot water and enzyme treatment 8.50 kg was mixed with an anticaking agent (product name: Calcium stearate, manufactured by Sun Ace Corporation) 1.86 kg and cellulose (product name: Ceolus, manufactured by Asahi Kasei Corp.) 8.20 kg to prepare a powder for es containing % the product obtained by heat-treating an asparagus stem with hot water and enzyme treatment d from the solid t of gus stem. One obtained by filling this powder for capsules in No.1 capsule at 280 mg per capsule was used as to a capsule filled with the product obtained by heat-treating an asparagus stem with hot water and enzyme treatment.

(Method of evaluation) Twenty volunteers who voluntarily expressed intent to participate were employed as subjects; and a short term randomized placebo controlled double blind study was canied out at a low dose. The subjects were randomly allocated to a placebo group (hereinafter, referred to as P group) 10 subjects or a group with a product obtained by heat-treating an asparagus stem with hot water and enzyme treatment (hereinafter, referred to as E group) 10 subjects. Over one week during the study period, the subjects in the P group took a placebo capsule (a total of 840 mg of e of product name: Pinedex (manufactured by Matsutani Chemical Industry Co., Ltd.) 699.9 mg and product name: Malt extract (Oriental Kogyo) 140.] mg (3 capsules)/day) ay whereas the subjects in the B group took the capsule filled with the product obtained by heat-treating an asparagus stem with hot water and enzyme treatment (840 mg (3 capsules)/day) after dinner every day (in 840 mg of such a capsule filled with t obtained by heat-treating an asparagus stem with hot water and enzyme treatment, the product ed by reating an asparagus stem with hot water and enzyme treatment derived from the solid content of asparagus stem accounted for 168 mg and the remaining 672 mg was Pinedex, calcium stearate, and Ceolus). The expression amount of HSP70 mRNA in white blood cells, autonomic nervous balance, and mic nervous activity are employed as endpoints.

(HSP70 mRNA expression inducing activity evaluation) First, the sion level of HSP70 mRNA in white blood cells was ed.

Before the start of the study and on the last day of the study, the blood was drawn; and total RNA was extracted from 400 uL of the blood using an RNA extraction kit (product name: Nucleo Spin RNA Blood, manufactured by Takara Bio Inc.). The method of cDNA synthesis and PCR conformed to the method described in Example 8.

The results are shown in . In , a ratio (%) of the expression level of HSP70 mRNA in the white blood cells after the completion of the intake to that before the start of the intake is presented. The expression level of HSP70 mRNA in the P group (the placebo group) was, on the basis of the average value, 175% ofthat before the start of the . On the other hand, the expression level of HSP70 mRNA in the B with hot group (the group with the product obtained by heat—treating an asparagus stem water and enzyme treatment) was, on the basis ofthe average value, 278% of that before the start of the intake; and the expression increased (*p=0.098 vs. the P group). From this, it was demonstrated that, by taking the product obtained by heat-treating an asparagus stem with hot water and enzyme treatment by this Example, the expression amount of HSP70 mRNA increased markedly. cal evaluation on autonomic nerve regulatory effect) Next, the autonomic nervous balance and autonomic nervous activity were evaluated using an acceleration pulse wave inspection system (product name; Pulse analyzer plus TAS-9, manufactured by YKC Corporation) before the start of the study and on the last day of the study. The details of measurement were the same as described in Example 9. shows changes in the autonomic nervous balance. In the P group (the placebo group), the autonomic s balance on the last day of the study significantly worsened, as compared with that before the start ofthe study (*p < 0.05 vs. before the start of the study). On the other hand, in the B group (the group with the product obtained by heat-treating an asparagus stem with hot water and enzyme treatment), the balance ofthe autonomic nerve on the last day ofthe study improved, as ed with that before the start of the study. From this, it was trated that, by taking the t ed by heat-treating an asparagus stem with hot water and enzyme treatment by this e, the balance of the autonomic nerve improved. shows changes in the autonomic s activity. In the P group (the placebo group), the autonomic nervous activity on the last day of the study significantly ed, as compared with that before the start of the study (**p < 0.01 vs. before the start of the study). 011 the other hand, in the B group (the group with the product obtained by heat-treating an asparagus stem with hot water and enzyme treatment), the mic nervous activity on the last day of the study improved, as compared with that before the start ofthe study. From this, it was trated that, by taking the product obtained by heat-treating an asparagus stem with hot water and enzyme ent by this Example, deterioration of the autonomic nervous activity was prevented. [0142} As described above, according to the present disclosure, a novel hydroxymethylfurfural tive, a highly effective pharmaceutical, HSP inducer, anti-stress agent, and autonomic nerve regulator can be provided. In addition, foods and drinks having an excellent HSP inducing activity, anti-stress effect, and autonomic nerve regulatory effect can be provided. Further, a method of producing a hydroxymethylfurfural tive that can reduce the cost and is simple and convenient can be provided.

It is to be noted that various embodiments and modifications are le in the present disclosure without departing from the broad spirit and scope of the present disclosure. Further, the above-mentioned embodiments are intended to illustrate the t disclosure and are not ed to limit the scope of the present disclosure. That is, the scope of the present disclosure is indicated by the claims rather than by the embodiments. And various modifications which come within the claims and within the meaning of invention equivalent to the claims are deemed to be within the scope of the present disclosure.

The present disclosure is based on Japanese Patent Application No. 201 1-277926 filed on December 20, 201 1. The description, , and drawings of Japanese Patent Application No. 2011-277926 are incorporated into the present specification by reference in their entirety.

Claims (17)

The claims defining the invention are as follows:

1. A hydroxymethylfiufural derivative represented by the general formula: [FORMULA 1] \o o o 5 (wherein, R is selected from the group ting of the following formula (I), [FORMULA 2] H H N H oag/ we“ (I) (11) HOOCCHZCOCO- and (111) HOOCCHzCH2COCO-). 10

2. The hydroxymethylfiirfiiral derivative according to Claim 1, that is obtained by heat-treating an gus stem with hot water.

3. A pharmaceutical comprising the hydroxymethylfurfural derivative according to Claim 1 or claim 2, as an active component.

4. A heat shock protein inducer comprising the hydroxymethylfurfural derivative according to Claim 1 or claim 2, as an active component.

5. An anti-stress agent sing the hydroxymethylfiufiiral derivative 20 according to Claim 1 or claim 2, as an active ent.

6. An autonomic nerve regulator comprising the hydroxymethylfurfural derivative according to Claim 1 or claim 2, as an active component.

7. A method ofproducing a hydroxymethylfiirfural derivative represented by the general formula: [FORMULA 1] \O O O 5 (wherein, R is selected from the group consisting of the following formula (I), [FORMULA 2] N ill! O 32:— \\f”//\ (I) 5“ J} (11) HOOCCH2COCO-, (III) ZCH2C0C0-, and (IV) a hydrogen atom), sing the step of heat-treating an asparagus stem with hot water.

8. The method ofproducing the hydroxymethylfiirfural derivative according to Claim 7, further sing the step of an enzyme treatment.

9. A heat shock protein inducer comprising a product obtained by the method 15 of claim 7 or claim 8, as an active component.

10. An anti-stress agent comprising a product obtained by the method of claim 7 or claim 8, as an active component. 20

l 1. An autonomic nerve regulator comprising a product obtained by the method of claim 7 or claim 8, as an active ent.

12. A method for induction of a heat shock protein, anti-stress or regulation of an autonomic nerve in a non-human patient which method comprises administering a hydroxymethylfilrfiiral derivative represented by the l formula: LA 1] (wherein, R is ed from the group consisting of the following formula (I), 5 [FORMULA 2] Orfi/ 4”“ (I) (II) HOOCCH2C0C0-, (111) HOOCCHZCH2C0C0-, and (IV) a hydrogen atom).

13. The method according to Claim 12, wherein the hydroxymethylfurfiiral 10 derivative is obtained by heat-treating an asparagus stem with hot water.

14. The method according to Claim 12, wherein the hydroxymethylfurfural derivative is contained in a product obtained by heat-treating an asparagus stem with hot water.

15. Use of a hydroxymethylfurfural derivative represented by the general 15 formula: [FORMULA 1] (wherein, R is selected from the group consisting of the following formula (I), [FORMULA 2] 0.3% \r’“ to (II) HOOCCHzCOCO-, (III) HOOCCHzCHzCOCO—, and (IV) a hydrogen atom) in the preparation of a medicament to induce heat shock n, anti-stress or regulation of an autonomic nerve in a patient, wherein a condition to be treated is load of stress, autonomic er or autonomic disorder by load of .

16. The use according to Claim 15, wherein the hydroxymethylfurfiiral derivative is obtained by heat—treating an asparagus stem with hot water.

17. The use according to Claim 15, wherein the hydroxymethylfurfural 10 derivative is contained in a product obtained by heat-treating an asparagus stem with hot water.